An Exploratory Study on Spoilage Bacteria and Listeria monocytogenes in Fresh Salmon: Extending Shelf-Life Using Vacuum and Seasonings as Natural Preservatives.

A growing population increases the demand for food, but short shelf-lives and microbial hazards reduce supply and increase food waste. Fresh fish is highly perishable and may be consumed raw, such as salmon in sushi. This work aims to identify strategies to improve the shelf-life and safety of fresh salmon, using available methods (i.e., vacuum) and exploring the use of natural preservatives (i.e., seasonings). Vacuum packaging and good hygiene practices (which reduce initial flora) extended shelf-life up to 20 days. Carnobacterium maltaromaticum was dominant in vacuum packaging conditions and showed potential for inhibiting Listeria monocytogenes. For natural preservatives, L. monocytogenes required higher inhibitory concentrations in vitro when compared to the 10 spoilage bacteria isolated from fresh salmon fillets, presenting a minimum inhibitory concentration (MIC) of 0.13% for oregano essential oil (OEO), 10% for lemon juice, 50 mg mL-1 for garlic powder, and >10% for NaCl. A good bacteriostatic and bactericidal effect was observed for a mixture containing 5% NaCl, 0.002% OEO, 2.5% lemon juice, and 0.08 mg mL-1 garlic powder. Finally, using the salmon medium showed an adequate correlation with the commercial culture medium.

Combined Effects of Temperature and Dietary Lipid Level on Body Composition, Growth, and Freshness Profile in European Seabass, Dicentrarchus labrax.

A fish trial was carried out to evaluate the combined effects of temperature and dietary lipid level on the body composition, growth performance, and freshness profile of the European seabass (Dicentrarchus labrax). Fish were kept for 56 days at 20 °C and 24 °C and fed on two diets, with 16% and 20% lipid. At the end of the trial, fish were euthanized at two temperature conditions (0.6 °C or -0.6 °C) and kept on ice for 10 days at 4 °C to evaluate their freshness condition. Findings demonstrated that fish reared at 24 °C presented a lower lipid level and a higher daily growth index than those at 20 °C. Additionally, sensory analysis (Quality Index Method-QIM) and microbiological analysis revealed that fish reared at 24 °C showed better freshness conditions than those at 20 °C. However, the 16S rRNA metabarcoding analyses revealed a higher proliferation of genera associated with fish-spoiling bacteria in the skin microbiome of fish reared at 24 °C, i.e., Vibrio and Acinetobacter, which was not observed in the skin microbiome of fish reared at 20 °C. Nevertheless, the dietary lipid level did not have any influence on fish freshness. Therefore, our data suggest that the increase in temperature to 24 °C is beneficial for the growth and freshness profile (lower QIM and lower CFUs/cm2) of this particular species. Additionally, the lower euthanasia temperature (-0.6 °C) seems to lead to higher fish freshness than the normal temperature (0.6 °C).

Gulls in Porto Coastline as Reservoirs for Salmonella spp.: Findings from 2008 and 2023.

Gulls act as intermediaries in the exchange of microorganisms between the environment and human settlements, including Salmonella spp. This study assessed the antimicrobial resistance and molecular profiles of Salmonella spp. isolates obtained from fecal samples of gulls in the city of Porto, Portugal, in 2008 and 2023 and from water samples in 2023. Antimicrobial susceptibility profiling revealed an improvement in the prevalence (71% to 17%) and antimicrobial resistance between the two collection dates. Two isolate collections from both 2008 and 2023 underwent serotyping and whole-genome sequencing, revealing genotypic changes, including an increased frequency in the monophasic variant of S. Typhimurium. qacE was identified in 2008 and 2023 in both water and fecal samples, with most isolates exhibiting an MDR profile. The most frequently observed plasmid types were IncF in 2008 (23%), while IncQ1 predominated in 2023 (43%). Findings suggest that Salmonella spp. circulate between humans, animals, and the environment. However, the genetic heterogeneity among the isolates from the gulls' feces and the surface water may indicate a complex ecological and evolutionary dynamic shaped by changing conditions. The observed improvements are likely due to measures to reduce biological contamination and antimicrobial resistance. Nevertheless, additional strategies must be implemented to reduce the public health risk modeled by the dissemination of pathogens by gulls.

Improved approach based on MALDI-TOF MS for establishment of the fish mucus protein pattern for geographic discrimination of Sparus aurata.

Food fraud is still a recurrent practice throughout food supply chains. In the case of seafood, misidentification of species and products repackaging constitute the most common frauds. Therefore, the development of appropriate analytical approaches to be used against food fraud is necessary. The present study goal is to explore for the first time, the possibility to differentiate between Sparus aurata from two different mariculture farms located in Madeira Island (Caniçal and Ribeira Brava), using the mass fingerprint of fish mucus obtained from MALDI-TOF MS and analyzed using Mass-UP software for multivariate statistical analysis and biomarker identification. It was possible to establish, from the mucus protein fraction, a set of potential biomarkers for each location in a total of 35 peaks, being 17 peaks specific to Caniçal located farm and 18 to Ribeira Brava. The proposed analytical approach revealed a useful strategy providing accurate and fast results for fish geographical origin discrimination.

Seasonal Evaluation of Freshness Profile of Commercially Important Fish Species.

Freshness is considered one of the most important parameters to judge the quality of most fish products. In the current study, the seasonality effect on the freshness profile of different economic fish species was evaluated for the first time, using three different approaches (sensory: Quality Index Method (QIM) and European (EC) Scheme; physical: Torrymeter (TRM) values; and microbiological analyses: Total Viable Counts (TVC) and degradative bacteria). Over a year, individuals of farmed fish Sparus aurata and Dicentrarchus labrax, as well as the wild fish Trachurus trachurus, Scomber colias, and Sardina pilchardus, were sampled seasonally for the evaluation of their freshness profile over 10 days on ice. In general, data showed an increase in QIM values, a decline in TRM, and an increase of spoilage bacteria throughout the storage time, revealing a clear temporal degradation of the quality of the fish. Additionally, some signs of seasonality effect could only be observed for some species. For example, the seabass D. labrax showed lower numbers of degradative bacteria in winter than in the other seasons, suggesting a high potential to be marketed in a fresher condition, especially during that season. On the other hand, S. colias showed higher freshness scores (i.e., higher TRM values in spring and autumn and lower numbers of bacteria in summer) from spring to autumn. However, from the five studied species, S. colias presented the lowest freshness values, indicating a higher fragility of this species. This information is extremely relevant for consumers and retailers that want to invest in higher quality products, as they would thus be able to choose certain species in detriment of others. Additionally, obtained data showed that farmed species reached day 10 of storage time with lower values of QIM and microbial counts (cfu), as well as higher values of TRM, in relation to wild species. These results reinforce the idea that farmed fish can, under proper conditions, present high quality/freshness profile.

Multi-elemental composition of white and dark muscles in swordfish.

Concentrations of 16 elements (K, Na, Mg, Ca, Fe, Zn, Hg, Se, As, Cu, Cd, Mn, Ni, Cr, Pb and Co) were determined in dorsal white and dark muscle of Xiphias gladius, sampled at various positions of a single swordfish and at the same position of eight specimens. Hg was quantified by thermal decomposition atomic absorption spectrometry (LECO AMA-254) and the rest of the elements by inductively coupled plasma optical emission spectrometry (ICP OES) or mass spectrometry (ICP MS). Element partitioning differed in dark and white muscle. Dark muscle was particularly enriched in Fe (median 13 times) and Cu (9) and moderately enhanced in Se, Mn, Zn and Cd (2.8-4.0). Dark:white ratios of the potentially toxic elements (As, Cr, Hg, Ni and Pb) varied from 0.9 to 1.4, pointing to a similar distribution between the two muscles types and indicating no additional risk in the consumption of dark muscle.

A Systematic AQbD Approach for Optimization of the Most Influential Experimental Parameters on Analysis of Fish Spoilage-Related Volatile Amines.

The volatile amines trimethylamine (TMA) and dimethylamine (DMA) could be used as important spoilage indices for seafood products, assisting in the determination of the rejection period. In the present study, a systematic analytical duality-by-design (AQbD) approach was used as a powerful strategy to optimize the most important experimental parameters of headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) conditions for the quantification of TMA and DMA in Sparus aurata. This optimization enabled the selection of the best points in the method operable design region for HS-SPME extraction (30 min; 35 °C; NaOH 15 M and NaCl 35%, w/v) and GC-MS analysis (80 °C; gradient 50 °C/min; flow rate 1 mL/min and splitless mode). The rejection day, estimated through the TMA concentration (>12 mg/100 g, at days 9-10), was compared with sensory (quality index method: day 7-8), physical (Torrymeter: day 8-9), and microbial (day 9-10) analysis, corroborating the suitability of the proposed approach for estimating the period for which they will retain an acceptable level of eating quality from a safety and sensory perspective.

Freshness Assessment and Shelf-Life Prediction for Seriola dumerili from Aquaculture Based on the Quality Index Method.

Fish and fish-based products are easily perishable foods due to different factors, including fragile organization, abundant endo-enzymes, psychrophilic bacteria, and impact of pre-harvest operations, that contribute to reducing its value. Therefore, a timely effective method for fish freshness and shelf-life evaluation is important. In this context, this study aimed to develop a sensory scheme based on the Quality Index Method (QIM) (sensory table and point system) for freshness monitorization and shelf-life prediction for Seriola dumerili from aquaculture in Madeira Island. Evaluation of appearance, texture, eyes, and gills was performed during 20 days of storage on ice (0 ± 1 °C). The shelf-life prediction was supported by the analysis of microorganisms (total viable colonies, TVC, counts), texture (Torrymeter), and production of trimethylamine (TMA), evaluated by HS-SPME-GC-MS and validated according to Association of Official Analytical Chemists AOAC guidelines. The result is a QIM scheme with 25 demerit points, where zero indicates total freshness. From the integration of sensory analysis, microbial growth at the time of rejection (TVC, 108 cfu/cm2 and H2S producers, 107 cfu/cm2), texture (Torrymeter value < 8), and TMA analyses (>12.5 mg/100 g), shelf-life was estimated as 12 days (±0.5 days). The obtained results show the high-throughput potential of the developed method for fish freshness assessment and shelf-life prediction. This QIM scheme is a secure way to measure quality and provide users with a reliable standardized fish freshness measure.

Elemental composition and bioaccessibility of farmed oysters (Crassostrea gigas) fed different ratios of dietary seaweed and microalgae during broodstock conditioning.

The Pacific oyster (Crassostrea gigas) culture has been expanding, thereby leading to a greater importance of hatcheries. Broodstock conditioning is very important in the hatchery process, in which diet composition may have a strong influence on the offspring production and quality. Therefore, the current study evaluated elemental composition and bioaccessibility of oysters fed different ratios of dietary seaweed (SW) and microalgae. The dietary conditioning consisted of direct replacement of microalgae by SW at four substitution levels (0%, 25%, 50%, and 100% diet). It was observed that oysters fed 100% SW had the highest levels of Be, Cu, Zn, Sr, and Cd. The most important trend was a concentration decline of most elements with progressively lower levels of SW substitution for microalgae in the feeds. No Cd or Pb hazard (contents below 1.0 mg/kg for Cd and 1.5 mg/kg for Pb) was found in oyster meat. Regarding elemental bioaccessibility, values were similar, near 100% in the cases of Cu, Br, and I. Only for Mn and Pb, bioaccessibility percentages deviated more from 100%. Indeed, the value for Pb was 50% ± 7% (initial group), and for Mn, all values were equal or lower than 29% ± 2% (final group of oysters fed microalgae). It was observed that Mn, Cd, and Pb bioaccessibility increased with a growing share of microalgal biomass in the feed. Therefore, this study showed that SW incorporation into the feed influences elemental composition and bioaccessibility of the oysters.

Fatty Acid Profile of Pacific Oyster, Crassostrea gigas, Fed Different Ratios of Dietary Seaweed and Microalgae during Broodstock Conditioning.

The fatty acid (FA) profile of oysters generally reflects the dietary FA composition. Moreover, incorporation of FA into tissues is modulated by various metabolic factors, and final composition will depend upon the dietary sources, cumulative intake, and oysters' development stage. Thus, the aim of this study was to assess the impact of dietary incorporation of seaweed (SW) Ulva rigida, in replacement of traditional microalgae diet, on the FA composition of Pacific oysters Crassostrea gigas, during broodstock conditioning. The dietary conditioning consisted of direct replacement of microalgae (33% Tisochrysis lutea, 50.25% Skeletonema costatum, and 16.75% Chaetoceros calcitrans) by SW at four different substitution levels (0%, 25%, 50%, and 100% diet). The dietary docosahexaenoic acid (DHA) (22:6n-3) and eicosapentaenoic acid (EPA) (20:5n-3) contents showed a positive correlation with the dietary microalgae level. During the trial, oysters fed with higher percentages of microalgae revealed a depletion of DHA and accumulation of EPA. The 100% SW caused a significant reduction in oxygen consumption and, consequently, in the standard metabolic rate. Based on these results, a partial substitution of up to 25% of dietary microalgae seems to be a suitable alternative, because it elicited similar results to the commercial 100% microalgae diet.

Genetic diversity and population structure of the blue jack mackerel Trachurus picturatus across its western distribution.

Blue jack mackerel Trachurus picturatus collected at six sampling locations of the north-east Atlantic Ocean (Azores, Madeira, Canaries, and Matosinhos, Peniche and Portimão, mainland Portugal) and one location in the Mediterranean (Sicily), were used to examine the genetic structure of this species. Three mitochondrial gene regions (cytochrome c oxidase subunit I, cytochrome b and control region) were used to study the genetic structure of the species in Macaronesia, as well as to compare the genetic diversity of this region with published results from its eastern distribution. All markers indicated the absence of genetic structure among populations, with high indices of genetic diversity. These results suggest that the species went through a bottleneck event, followed by a recent population expansion. Moreover, the comparison with previously published results from the T. picturatus Mediterranean distribution suggests the existence of a single panmictic population throughout the species' full range. This was, however, an unexpected result since other methodologies have shown the presence of, at least, three different population-units in the NE Atlantic Ocean.

River water analysis using a multiparametric approach: Portuguese river as a case study.

The Ave River in northern Portugal has a history of riverbanks and water quality degradation. The river water quality was assessed by physicochemical, biological (macroinvertebrates) and microbiological (Enterococcus spp. and Escherichia coli) parameters in six locations (A-F, point A being the nearest to the source) throughout its course during a year. Epilithic biofilms were studied through polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Antimicrobial susceptibility testing helped with selecting isolates (n = 149 E. coli and n = 86 enterococci) for further genetic characterization. Pursuant to physicochemical and macroinvertebrates-based parameters, the river water was of reasonable quality according to European legislation (Directive 2000/60/EC). However, the microbiological analysis showed increased fecal contamination downstream from point C. At point D, four carbapenem-resistant E. coli isolates were recovered. Paradoxically, point D was classified as a point of 'Good Water Quality' according to macroinvertebrates results. Point F presented the highest contamination level and incidence of multidrug-resistant (MDR) isolates in the water column (13 MDR enterococci out of 39 and 33 MDR E. coli out of 97). Epilithic biofilms showed higher diversity in pristine points (A and B). Thus, biological and microbiological parameters used to assess the water quality led to divergent results; an outcome that reinforces the need for a holistic evaluation.

Viability of dietary substitution of live microalgae with dry Ulva rigida in broodstock conditioning of the Pacific oyster (Crassostrea gigas).

The current study evaluated the microalgae replacement by dry macroalgae (Ulva rigida) in the reproductive success and biochemical composition of the Pacific oyster (Crassostrea gigas) during broodstock conditioning. Five nutritional regimes were tested: 100% macroalgae (diet 1), 50% macroalgae+50% microalgae (diet 2), 25% macroalgae+75% microalgae (diet 3) and 100% microalgae (diet 4). An unfed group was used as a negative control. The microalgae blend was composed of 33% Isochrysis galbana and 67% diatoms (75% Skeletonema costatum+25% Chaetoceros calcitrans). Gonadal maturation was reflected in the physiological condition of the individuals. All treatments, except diet 1, showed an increase in condition index and were fully matured at the end of the trial, with the best physiological condition observed in oysters fed diet 3 and diet 4. Protein and total lipid content increased during the conditioning period, whereas glycogen content decreased. Oysters conditioned with diet 3 had higher protein and total lipid content and lower glycogen content than the other treatments. In addition, diet 3 showed the highest percentage of viable veliger larvae. The current study demonstrated that it is possible to replace 25% of microalgae with macroalgae in the broodstock conditioning, minimizing the operative cost in bivalve hatcheries.This article has an associated First Person interview with the first author of the paper.

Influence of temperature and fish thickness on the mass transfer kinetics during the cod (Gadus morhua) desalting process.

BACKGROUND: The aim of this study was to analyse the influence of desalting temperature, fish thickness and desalting time on the mass transfer kinetics during the cod desalting process by physico-chemical analyses. RESULTS: Both water uptake and salt loss increased with increasing temperature (15 °C > 10 °C > 5 °C) up to 24 h in 'thicker' pieces. The equilibrium achievement was faster in 'thinner' pieces and also with increasing temperature. Longer desalting times at 10 °C can be a good practice to be used during cod desalting at an industrial scale in order to obtain commercial products with higher yields. The faster mass transfer during desalting of 'thinner' pieces appears to follow three periods as a result of diffusion of the components (water, NaCl, and soluble proteins) because of the concentration differences, and pressure gradients due to expansion/shrinkage of the protein matrix, which is dependent on the NaCl content. The refractive index can be used by industry as an indirect measurement to determine the moment at which the 'thicker' samples are near the Z(NaCl) = Y(NaCl) equilibrium. CONCLUSION: Optimum combinations between the process variables analysed are essential in order to speed up the mass transfer kinetics during cod desalting at an industrial scale. © 2016 Society of Chemical Industry.

MULTIDRUG RESISTANCE IN WILD BIRD POPULATIONS: IMPORTANCE OF THE FOOD CHAIN.

The presence of multidrug-resistant (MDR) Escherichia coli has recently been reported in wild birds (gulls and birds of prey) that had no apparent exposure to antimicrobials. Little work has been done to assess the role of the food chain in the emergence and spread of MDR E. coli . In this study, we evaluated the presence of MDR E. coli in 29 fecal samples collected from wild birds living in a rehabilitation center (the center receives injured animals found in their natural habitat) and in eight feed samples. In total, 166 E. coli isolates were obtained: 129 from cloacal swabs and 37 from raw feed samples. The antimicrobial resistance profile of these isolates was determined, and we found that 75 isolates showed resistance to five or more drugs, resulting in a total of 38 different antimicrobial resistance patterns. Subsequently, the molecular characterization of 36 isolates, performed by pulsed-field gel electrophoresis, revealed a great similarity between isolates collected from various species of birds and also between these last ones and the ones found in their feed samples.

Quality changes during cod (Gadus morhua) desalting at different temperatures.

BACKGROUND: To advise consumers and manufacturers regarding decreasing the time needed to desalt salted cod (a time-consuming process), there is a need to develop knowledge about quality changes at different desalting temperatures. The objective of this work was to evaluate the physico-chemical, microbiological and sensory quality changes and their causes during cod desalting at 5, 10 and 15 °C, using a cod/water ratio of 1:9 without water changes. The influence of slices with different thickness and different desalting times was also evaluated. RESULTS: Desalting promoted a decrease in the levels of total volatile basic nitrogen, thiobarbituric acid reactive substances and free amino acids nitrogen (FAA-N). The highest FAA-N values were found in 'thicker' samples desalted at 15 °C due to the higher proteolytic bacteria number observed in these samples, which activity compensated the leaching of soluble components to the desalting solution. The water uptake and the salt leaching out of the muscle found during the processes created conditions for the bacterial growth, contributing to the spoilage at 15 °C. Based on fresh odour and 'off'-flavours results, 'thicker' samples desalted at 15 °C after 72 h were close to spoilage and on microbial levels were spoiled. CONCLUSION: In order to extend the shelf life and safety of cod desalted products, desalting at temperatures above 10 °C is not advisable. © 2014 Society of Chemical Industry.

High prevalence of multidrug-resistant Escherichia coli and Enterococcus spp. in river water, upstream and downstream of a wastewater treatment plant.

In this study, microbial quality and antimicrobial resistance of faecal bacteria from a Portuguese river were assessed. River water samples collected upstream and downstream of a wastewater treatment plant, throughout a 3-month period, were used for the enumeration of Escherichia coli and Enterococcus spp. The highest numbers found for E. coli and enterococci were 1.1 × 104 and 1.2 × 104 colony forming units (CFU)/100 ml, respectively. In total, 144 isolates of E. coli and 144 of enterococci were recovered and tested for antimicrobial susceptibility; 104 E. coli and 78 Enterococcus spp. showed resistance to one or more antimicrobial drugs. Overall, 70 and 32 different resistance patterns were found for E. coli and enterococci, respectively. One E. coli showed resistance to imipenem and 29 isolates were extended spectrum ß-lactamase-producers. Multidrug-resistant E. coli belonged mostly to groups A, B1 and group D. Enterococcus spp. were mostly resistant to rifampicin, tetracycline, azithromycin and erythromycin; six isolates showed resistance to vancomycin, presenting the VanA phenotype. The high levels of E. coli and enterococci and the remarkable variety of antimicrobial resistance profiles, reinforces the theory that these river waters can be a pool of antimicrobial resistance determinants, which can be easily spread among different bacteria and reach other environments and hosts.

Effect of seasonal changes on the gelling properties of farmed sea bass (Dicentrarchus labrax).

The effect of seasonal changes (summer versus winter) upon the quality of heat-induced gel products from farmed sea bass (Dicentrarchus labrax) was studied. Moreover, the effect of microbial transglutaminase addition (0.5%, w/w) on the quality was assessed. Fat content of gel products attained from sea bass grown during summer was higher (12.0% versus 7.6%). Textural properties of the gels from the summer fish were higher. Protein of gels from winter fish was less soluble in SDS + DTT (sodium dodecyl sulphate, SDS; dithiothreitol, DTT). Contrary to some literature, higher fat level may have played a protective role during processing. Accordingly, season via fat content variation had a strong effect on the gelling ability of heat-induced gels prepared from sea bass.

Changes in antimicrobial resistance among faecal enterococci isolated from growing broilers prophylactically medicated with three commercial antimicrobials.

A controlled environment poultry house (1600m(2)) was divided into two separated compartments. Three hundred twenty chick containers (each containing 100 chicks) were randomly allocated to each compartment. Antimicrobial products were provided (via drinking water) to one broiler group in three different growth phases for prophylactic purposes: lincomycin associated with spectinomycin from day 1 to day 3, trimethoprim associated with sulfamethoxazole from day 19 to day 21, and tylosin from day 26 to day 28. Feed and faecal samples from both groups were obtained on days 0, 4, 9, 14, 18, 22, 25, 29 and 33. One sample of the drinking water and samples from the broiler house environment were obtained one day before bird placement. A maximum of 16 enterococci isolated from Kanamycin Aesculin Azide Agar were tested for resistance using the disk diffusion method. A high prevalence of resistance to drugs such as tetracycline, erythromycin and nitrofurantoin was detected in enterococcal isolates from medicated and unmedicated flocks. Resistance occurrence in the non-medicated group revealed a shift in resistance profiles with the gradual change of the initial strains, isolated from the day-old chicks' cloacal swabs, by others displaying a different phenotype. Resistance phenotypes obtained from faecal strains were correlated with those isolated from the farm environment and feeds.

Effect of season on the chemical composition and nutritional quality of the edible crab Cancer pagurus.

Cancer pagurus is most appreciated in southern Europe for its muscle and brown meat content. In Portugal, consumption occurs mostly in summer and at Christmas. In this study the seasonal nutritional quality of edible tissues of female and male C. pagurus was determined. Tissue composition was affected by season and sex. All tissues had a well-balanced essential amino acid composition. Muscle and gonads of females had n-3/n-6 ratios in the range of the usual recommended values, and all tissues had PUFA/SFA above the recommended level. Autumn was the season with the highest brown meat yield, total essential amino acids (muscle), taurine (all tissues), EPA (male gonads), and n-3/n-6 ratio (gonads). However, it was also the season with the highest fat content and cholesterol concentration in ovaries. Therefore, people with restricted diets should moderate the consumption of ovaries in autumn. The remaining tissues pose no risks with respect to their proximate chemical composition.