Application of phage therapy during bivalve depuration improves Escherichia coli decontamination.

The present study investigated the potential application of the bacteriophage (or phage) phT4A, ECA2 and the phage cocktail phT4A/ECA2 to decrease the concentration of Escherichia coli during the depuration of natural and artificially contaminated cockles. Depuration in static seawater at multiplicity of infection (MOI) of 1 with single phage suspensions of phT4A and ECA2 was the best condition, as it decreased by ∼2.0 log CFU/g the concentration of E. coli in artificially contaminated cockles after a 4 h of treatment. When naturally contaminated cockles were treated in static seawater with single phage suspensions and the phage cocktail, similar decreases in the concentration of E. coli (∼0.7 log CFU/g) were achieved. However, when employing the phage cocktail, a longer treatment time was required to obtain comparable results to those achieved when using single phage suspensions. When naturally contaminated cockles were depurated with phage phT4A in a recirculated seawater system (mimicking industrial depuration conditions), a 0.6 log CFU/g reduction of E. coli was achieved after a 2 h of treatment. When the depuration process was performed without phage addition, a 4 h treatment was necessary to obtain a similar decrease. By combining phage therapy and depuration procedures, a reduction in bivalves depuration period can be achieved for, thus decreasing the cost associated with this procedure and even enhance the quality and safety of depurated bivalves destined for human consumption.

Towards a deeper understanding of fatty acid bioaccessibility and its dependence on culinary treatment and lipid class: a case study of gilthead seabream (Sparus aurata).

The bioaccessibility of total lipids and fatty acids (FA) in raw and grilled gilthead seabream (Sparus aurata) was determined using an in vitro digestion model. The particular impact of grilling on the FA profile of seabream was also studied. In addition, the influence of lipid class on the bioaccessibility of each FA was analysed. Grilling did not change the relative FA profile, and only the absolute values were altered. However, the relative FA profile varied across lipid classes, being more dissimilar between TAG and phospholipids. Long-chain SFA and PUFA seemed to be less bioaccessible. Moreover, grilling reduced bioaccessibility of protein, fat and many FA, with the highest reductions found in PUFA such as the DHA. Strong evidence supporting a predominantly regioselective action of lipase during in vitro digestion was found, and the impact of this phenomenon on FA bioaccessibility was assessed.

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.

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.

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.

Elemental composition of four farmed fish produced in Portugal.

Farmed gilthead sea bream (Sparus aurata), European sea bass (Dicentrarchus labrax), rainbow trout (Oncorhynchus mykiss) and turbot (Psetta maxima) produced in Portugal were analysed in order to characterize their elemental composition. Atomic absorption (flame and cold vapour) and molecular absorption spectrometry techniques were used to determine all the studied elements. Similar patterns of macro, trace and ultra trace elements were observed for all fish species. The main elements were potassium (K), sodium (Na), phosphorus (P), magnesium (Mg) and calcium (Ca), followed by zinc (Zn), iron (Fe), copper (Cu), chromium (Cr), manganese (Mn) and nickel (Ni). Cadmium (Cd), mercury (Hg) and lead (Pb) concentrations, obtained in this study, allow concluding that these species do not present a hazard for human consumption. In addition, they contain almost all essential elements at concentrations sufficient to suit the dietary reference intake. Nevertheless, P. maxima nutritious trace element content is relatively low compared with the other three species.

Biofortified Diets Containing Algae and Selenised Yeast: Effects on Growth Performance, Nutrient Utilization, and Tissue Composition of Gilthead Seabream (Sparus aurata).

Efforts have been made to find natural, highly nutritious alternatives to replace fish meal (FM) and fish oil (FO), which can simultaneously promote fish health and improve the nutritional quality of filets for human consumption. This study evaluated the impact of biofortified diets containing microalgae (as replacement for FM and FO), macroalgae (as natural source of iodine) and selenised yeast (organic source of selenium) on gilthead seabream growth, nutrient utilization, tissue composition and gene expression. A control diet (CTRL) with 15% FM and 5.5% FO was compared with three experimental diets (AD1, AD2, and AD3), where a microalgae blend (Chlorella sp., Tetraselmis sp., and DHA-rich Schizochytrium sp.) replaced 33% of FM. Diet AD1 contained 20% less FO. Diets were supplemented with Laminaria digitata (0.4% AD1 and AD2; 0.8% AD3) and selenised yeast (0.02% AD1 and AD2; 0.04% AD3). After feeding the experimental diets for 12 weeks, growth was similar in fish fed AD1, AD2, and CTRL, indicating that microalgae meal can partially replace both FM and FO in diets for seabream. But AD3 suppressed fish growth, suggesting that L. digitata and selenised yeast supplementation should be kept under 0.8 and 0.04%, respectively. Despite lower lipid intake and decreased PUFAs bioavailability in fish fed AD3, compared to CTRL, hepatic elovl5 was upregulated resulting in a significant increase of muscle EPA + DHA. Indeed, filets of fish fed AD2 and AD3 provided the highest EPA + DHA contents (0.7 g 100 g-1), that are well above the minimum recommended values for human consumption. Fish consuming the AD diets had a higher retention and gain of selenium, while iodine gain remained similar among diets. Upregulation of selenoproteins (gpx1, selk, and dio2) was observed in liver of fish fed AD1, but diets had limited impact on fish antioxidant status. Overall, results indicate that the tested microalgae are good sources of protein and lipids, with their LC-PUFAs being effectively accumulated in seabream muscle. Selenised yeast is a good fortification vehicle to increase selenium levels in fish, but efforts should be placed to find new strategies to fortify fish in iodine.

Diets supplemented with Saccharina latissima influence the expression of genes related to lipid metabolism and oxidative stress modulating rainbow trout (Oncorhynchus mykiss) fillet composition.

This study aimed to evaluate the impact of diets including increasing amounts (1, 2 and 4%) of an iodine-rich macroalgae, Saccharina latissima, on gene expression and fillet composition of commercial-sized rainbow trout. Liver and muscle expression of genes related to growth, iodine, oxidative stress, and lipid metabolism, and the fillet content of fatty acids, cholesterol, and vitamin D3 were assessed. The highest kelp inclusion led to lower final body weight and HSI, without significant differences in mRNA transcription of genes involved in growth (ghr1, ghr2 and igf1) or iodine metabolism (dio1, thra, and thrb). A significant downregulation of an oxidative stress marker, gpx1b2, was observed in fish fed 2% S. latissima, which might suggest the need for less endogenous antioxidants. Dietary inclusion of kelp impacted lipid metabolism, with a downregulation of fatty acid synthase, accompanied by a general decrease of fatty acids in fillet. The present study demonstrated that supplementation of diets with 1 or 2% S. latissima can be achieved without detrimental effects on rainbow trout final weight. Evidence suggest a lipid-lowering effect of diets that did not compromise fillet EPA and DHA concentrations, being 3.7 times above the recommended levels for human consumption.

Freshness and quality criteria of iced farmed Senegalese sole (Solea senegalensis).

Senegalese sole (Solea senegalensis) is a high-value commercial species with increasing importance in aquaculture. The aim of this work was to study the quality changes of this species during chilled storage under refrigeration, through sensory and chemical methods. In particular, the optimization of a quality index method (QIM) scheme was proposed as well as the definition of sensory and chemical quality criteria. A shelf life of 15 days was reported, and a QIM scheme based on a total of 22 demerit points (dp) was proposed. Biogenic amines were never detected, and the usual spoilage indicators, such as TVB-N and TMA-N, were not significantly produced during the period of sensory-acceptable quality (15 days). On the basis of the significant correlations (p<0.001) between sensory data and Ki values, a quality index (QI) 19 dp and a Ki>40% indicate unacceptable quality of iced Senegalese sole.

Characterization and in vitro evaluation of new bacteriophages for the biocontrol of Escherichia coli.

In the present study two new phages (phT4A and ECA2) were characterized and their efficacy was evaluated separately and in cocktail (phT4A/ECA2) to control Escherichia coli. The isolated phages, phT4A and ECA2, belonged to the Myoviridae and Podoviridae family, respectively and both are safe (no integrase and toxin codifying genes) to be used in bacterial control. In general, the increase of multiplicity of infection (MOI) from 1 to 100 promoted a significant increase in the efficiency of phage phT4A and phage cocktail phT4A/ECA2. Both phages were effective against E. coli, but phage phT4A (reduction of 5.8 log CFU/mL after 8h treatment) was more effective than phage ECA2 phage (reduction of 4.7 log CFU/mL after 8h treatment). The use of a cocktail phT4A/ECA2 was significantly more effective (reductions of 6.2 log CFU/mL after 6h treatment) than the use single phage suspensions of phT4A and ECA2 (reductions 5.3 log CFU/mL and 4.9 log CFU/mL, respectively, after 6h treatment). The rate of emergence of phage-resistant mutants was lower for phage phT4A when compared with phage ECA2 and phage cocktail phT4A/ECA2.The results indicate that in addition to the efficacy, the potential development of phage-resistant mutants must also be considered in the design of phage cocktails.

Bacteriophages with potential to inactivate Salmonella Typhimurium: Use of single phage suspensions and phage cocktails.

The aim of this study was to compare the dynamics of three previously isolated bacteriophages (or phages) individually (phSE-1, phSE-2 and phSE-5) or combined in cocktails of two or three phages (phSE-1/phSE-2, phSE-1/phSE-5, phSE-2/phSE-5 and phSE-1/phSE-2/phSE-5) to control Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in order to evaluate their potential application during depuration. Phages were assigned to the family Siphoviridae and revealed identical restriction digest profiles, although they showed a different phage adsorption, host range, burst size, explosion time and survival in seawater. The three phages were effective against S. Typhimurium (reduction of ∼2.0 log CFU/mL after 4h treatment). The use of cocktails was not significantly more effective than the use of single phages. A big fraction of the remained bacteria are phage-resistant mutants (frequency of phage-resistant mutants 9.19×10(-5)-5.11×10(-4)) but phage- resistant bacterial mutants was lower for the cocktail phages than for the single phage suspensions and the phage phSE-1 presented the highest rate of resistance and phage phSE-5 the lowest one. The spectral changes of S. Typhimurium resistant and phage-sensitive cells were compared and revealed relevant differences for peaks associated to amide I (1620cm(-1)) and amide II (1515cm(-1)) from proteins and from carbohydrates and phosphates region (1080-1000cm(-1)). Despite the similar efficiency of individual phages, the development of lower resistance indicates that phage cocktails might be the most promising choice to be used during the bivalve depuration to control the transmission of salmonellosis.

Application of bacteriophages during depuration reduces the load of Salmonella Typhimurium in cockles.

As bivalve molluscs are filter feeder, often consumed raw or lightly cooked and are frequently cultivated in contaminated waters, they are implicated in food-borne disease transmission to human. The present study investigated the potential application of bacteriophage (or phage) phSE-2, phage phSE-5 and phage cocktail phSE-2/phSE-5 to decrease the concentration of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) during the depuration of natural and artificially contaminated cockles (Cerastoderma edule). Cockles were artificially infected with 105 and 106colony-forming units (CFU)/mL of S. Typhimurium in static seawater and infected group were treated with phages at four different MOI values: 0.1, 1, 10 and 100. Depuration in static seawater at multiplicity of infection (MOI) of 0.1 with single phage suspensions of phSE-2 and phSE-5 provided the best results, as it decreased by ~1.3 and 1.7 logCFU/g, respectively, the concentration of Salmonella spp. after a 4h treatment. At a MOI of 0.1, the rate of inactivation with single phage suspensions was higher when compared with the results obtained using the phage cocktail. However, in naturally contaminated cockles treated in static seawater with single phage suspensions and phage cocktail phSE-2/phSE-5, similar decreases in cultivable bacteria concentration (~0.7-0.9 logCFU/g) were achieved after 6h of treatment. When artificially contaminated cockles were depurated with phage phSE-5 in a recirculated seawater system (mimicking industrial depuration conditions), a 0.9 and 2.0 logCFU/g reduction of Salmonella spp. was reached after 4 and 6h treatment. Once the depuration process was performed without phage, a 6h treatment was needed to obtain a 1.1 logCFU/g reduction of Salmonella spp. Results indicated that combining phage biocontrol with depuration procedures enhance bivalve microbial safety for human consumption by improving decontamination efficiency, proving that this technology can be transposed to the bivalves industry. Moreover, this approach also displays the advantage of reducing the time required for depuration and consequently its associated costs.

Fractionation of protein hydrolysates of fish and chicken using membrane ultrafiltration: investigation of antioxidant activity.

In this work, chicken and fish peptides were obtained using the proteolytic enzymes α-Chymotrypsin and Flavourzyme. The muscle was hydrolyzed for 4 h, and the resulting peptides were evaluated. Hydrolysates were produced from Argentine croaker (Umbrina canosai) with a degree of hydrolysis (DH) of 25.9 and 27.6% and from chicken (Gallus domesticus) with DH of 17.8 and 20.6% for Flavourzyme and α-Chymotrypsin, respectively. Membrane ultrafiltration was used to separate fish and chicken hydrolysates from Flavourzyme and α-Chymotrypsin based on molecular weight cutoff of >1,000, <1,000 and >500, and <500 Da, to produce fractions (F1,000, F1,000-500, and F500) with antioxidant activity. Fish hydrolysates produced with Flavourzyme (FHF) and α-Chymotrypsin showed 60.8 and 50.9% of peptides with a molecular weight of <3 kDa in its composition, respectively. To chicken hydrolysates produced with Flavourzyme and α-Chymotrypsin (CHC) was observed 83 and 92.4% of peptides with a molecular weight of <3 kDa. The fraction that showed, in general, higher antioxidant potential was F1,000 from FHF. When added 40 mg/mL of FHF and CHC, 93 and 80% of lipid oxidation in ground beef homogenates was inhibited, respectively. The composition of amino acids indicated higher amino acids hydrophobic content and amino acids containing sulfuric residues for FHF, which showed antioxidant potential.