ABSTRACT
The present manuscript is the expansion of the method Modelling of freezing time described in a previous paper. This modeling was used for simulating freezing times required for the inactivation of anisakids. The method described here can also be used for a number of other applications where the time or the temperature of the food needs to be modeled. In general, when a food is brought from room temperature to temperatures below - 5 ∘ C , the temperature kinetics follow three different parts which include cooling from initial temperature to the initial freezing point, freezing from the freezing point to - 5 ∘ C in the center of the food, and cooling from - 5 ∘ C to the final temperature in the center of the food. The present customized procedure is mainly based upon established estimation procedures. Following the description of the methods, an example of the calculation for freezing hake (Merluccius merluccius) mince muscle is provided for each of the phases. The method consists in the following:â¢Calculation of the pre freezing and sub freezing times with a similar procedure but separately since the sample has different thermo physical properties in each stage (cooling).â¢Calculation of the freezing time first for an infinite flat plate, and then a correction is applied for the actual geometry (finite cylinder).â¢The total freezing time is the sum of the three separate parts of the freezing process.
ABSTRACT
L3 larvae of anisakid nematodes are an important problem for the fisheries industry and pose a potential risk for human health by acting as infectious agents causing allergies and as potential vectors of pathogens and microrganisms. In spite of the close bacteria-nematode relationship very little is known of the anisakids microbiota. Fresh fish could be contaminated by bacteria vectored in the cuticle or in the intestine of anisakids when the L3 larvae migrate through the muscles. As a consequence, the bacterial inoculum will be spread, with potential effects on the quality of the fish, and possible clinical effects cannot be discarded. A total of 2,689,113 16S rRNA gene sequences from a total of 113 L3 individuals obtained from fish captured along the FAO 27 fishing area were studied. Bacteria were taxonomically characterized through 1803 representative operational taxonomic units (OTUs) sequences. Fourteen phyla, 31 classes, 52 orders, 129 families and 187 genera were unambiguously identified. We have found as part of microbiome an average of 123 OTUs per L3 individual. Diversity indices (Shannon and Simpson) indicate an extraordinary diversity of bacteria at an OTU level. There are clusters of anisakids individuals (samples) defined by the associated bacteria which, however, are not significantly related to fish hosts or anisakid taxa. This suggests that association or relationship among bacteria in anisakids, exists without the influence of fishes or nematodes. The lack of relationships with hosts of anisakids taxa has to be expressed by the association among bacterial OTUs or other taxonomical levels which range from OTUs to the phylum level. There are significant biological structural associations of microbiota in anisakid nematodes which manifest in clusters of bacteria ranging from phylum to genus level, which could also be an indicator of fish contamination or the geographic zone of fish capture. Actinobacteria, Aquificae, Firmicutes, and Proteobacteria are the phyla whose abundance value discriminate for defining such structures.
ABSTRACT
This work studied the performance of the artificial digestion method in terms of recovery and viability of Anisakis simplex third-stage larvae (L3) when previous treatments given to the infected fish muscle may accidentally render viable larvae. For that: a) hake mince was spiked with 10 L3/75g mince, frozen at -10, -15, -20, and -30 °C and immediately thawed, or stored for 12 or 24 h, and subjected to pepsin digestion; b) the mince was spiked under the same conditions, frozen at the above temperatures and thawed immediately. After manual recovery, L3 were assessed for viability, used to spike again the minced fish and subjected to pepsin digestion; c) the mince was spiked with 10 L3 which were: i) living (i.e. chilled), ii) freeze-surviving (live L3 had been previously recovered after freezing at -10 °C), or iii) dead (frozen at -30 °C or - 80 °C), and then subjected to pepsin digestion. Results showed that the artificial digestion method kills a significant number of larvae that may have survived freezing and thus may underestimate the number of viable larvae in a given batch. The method may also underestimate the infection level of fish batches containing dead larvae. It is suggested to take these limitations into account when designing digestion protocols for specific applications, especially when there is a risk of insufficiently treated or cooked fish batches or ready-to-eat foods.
ABSTRACT
BACKGROUND: Freezing is considered the most suitable technological treatment to avoid Anisakis infection from eating raw or undercooked fish but modifications of their cuticles upon freezing may reduce their resistance to gastric fluids, provoking a greater release of allergens. This work aimed to study the relationship between freezing-induced modifications of Anisakis simplex s.l., antigen recognition, and resistance to oral and gastric digestion in spiked fish mince. RESULTS: (i) Differences between non-treated larvae and larvae that survived freezing / thawing were studied in terms of respiratory capacity, survival in simulated gastric fluid (SGF), recognition of antigens and allergens. (ii) Untreated (i.e. chilled) mince containing live larvae, mince frozen at two freezing rates, with a negative (uninfected) mince and a positive mince (infected with broken larvae) as controls, were subjected to the oral and gastric phases of a simulated digestion process. Anisakis able to survive freezing showed lower resistance to gastric fluid (i.e. faster mortality as compared to controls). Untreated larvae released significantly more antigens than freeze-surviving larvae but only after 96 h in SGF. In treatments rendering complete larvae mortality, the highest loss of larvae integrity was found upon fast freezing. There was a positive correlation between antigen release and the number of ruptures of larvae after the oral digestion phase, whereas a more complex trend was observed after oral plus gastric digestion phases. CONCLUSION: These results suggest a new factor to consider for sensitized patients and suggest that the numbers of L3 should be reduced before industrial freezing to minimize risk. © 2020 Society of Chemical Industry.
Subject(s)
Anisakiasis/metabolism , Anisakis/metabolism , Antigens, Helminth/metabolism , Food Contamination/analysis , Gadiformes/parasitology , Gastric Juice/enzymology , Animals , Anisakiasis/parasitology , Anisakis/classification , Anisakis/genetics , Anisakis/immunology , Antigens, Helminth/analysis , Food Handling , Freezing , Humans , Larva/classification , Larva/genetics , Larva/immunology , Larva/metabolism , Models, BiologicalABSTRACT
This review presents some applications of proteomics and selected spectroscopic methods to validate certain aspects of seafood traceability. After a general introduction to traceability and the initial applications of proteomics to authenticate traceability information, it addresses the application of proteomics to trace seafood exposure to some increasingly abundant emergent health hazards with the potential to indicate the geographic/environmental origin, such as microplastics, triclosan and human medicinal and recreational drugs. Thereafter, it shows the application of vibrational spectroscopy (Fourier-Transform Infrared Spectroscopy (FTIR) and Fourier-Transform Raman Spectroscopy (FT Raman)) and Low Field Nuclear Magnetic Resonance (LF-NMR) relaxometry to discriminate frozen fish from thawed fish and to estimate the time and temperature history of frozen fillets by monitoring protein modifications induced by processing and storage. The review concludes indicating near future trends in the application of these techniques to ensure seafood safety and traceability.
ABSTRACT
The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes' biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomarkers among A. simplex, A. pegreffii and their hybrid genotype; 19 of these biomarkers, encoded by ten loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12, and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen. The rest of the markers comprise four unknown or non-characterized proteins; five different proteins (leucine) related to innate immunity, four proteolytic proteins (metalloendopeptidases), a lipase, a mitochondrial translocase protein, a neurotransmitter, a thyroxine transporter, and a structural collagen protein. The proposed methodology (proteomics and statistical) solidly characterize a set of proteins that are susceptible to take advantage of the new targeted proteomics.
Subject(s)
Anisakis/metabolism , Genotype , Hybridization, Genetic , Proteome , Proteomics , Animals , Anisakis/classification , Anisakis/genetics , Biomarkers , Chromatography, Liquid , DNA Barcoding, Taxonomic , Gene Regulatory Networks , Mass Spectrometry , Proteomics/methodsABSTRACT
BACKGROUND: Anisakis spp. are nematode parasites found in a wide range of marine organisms. Human beings may accidentally become infected, showing the symptoms of anisakiasis and allergic responses. There has been evidence of increased intestinal permeability in A. simplex-sensitized subjects and that specific IgE titres increase in some allergic patients when fishery products are re-introduced into their diet. The aims of this work were to study the effect of A. simplex crude extract on the intestinal integrity and permeability by using Caco-2 cell monolayer. To analyse the capacity of Ani s 4 allergen to cross the epithelial barrier. METHODOLOGY/PRINCIPAL FINDINGS: Cellular bioenergetics, transepithelial electrical resistance, viability, permeability, reactive oxygen species generation and immunofluorescent staining of tight junction proteins were analysed. A. simplex crude extract compromises the Caco-2 cell monolayer integrity in a dose-dependent manner. This effect is detected at 1 hour of culture and integrity is recovered after 24 hours of culture. The epithelial barrier disruption is accompanied by an increase in paracellular permeability and reactive oxygen species production and by a delocalization of occludin and zonula occludens-1. Finally, Ani s 4, a thermostable and resistant to digestion allergen with cystatin activity, is able to cross the epithelial barrier in Caco-2 monolayer and reach a cumulative mean percentage of 22.7% of total concentration in the basolateral side after 24 hours of culture. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that A. simplex induces an early and reversible alteration of integrity and permeability of Caco-2 cell monolayer and that an underlying mechanism of this effect would involve the oxidative stress and disruption of epithelial tight junctions. Additionally, it has been shown that Ani s 4 allergen is able to cross the epithelial barrier. These findings could explain the increased intestinal permeability observed in Anisakis-sensitized patients, the changes over time in IgE sensitization to A. simplex allergens, and the specific IgE persistence in Anisakis allergy.
Subject(s)
Anisakis/chemistry , Intestinal Mucosa/drug effects , Occludin/metabolism , Zonula Occludens-1 Protein/metabolism , Animals , Caco-2 Cells , Cell Survival , Humans , Mitochondria/drug effects , Oxidative Stress , Oxygen Consumption , Protein Transport , Reactive Oxygen Species/metabolism , Tissue ExtractsABSTRACT
In plant and animal nematode parasites, proteins derived from esophageal gland cells have been shown to be important in the host-nematodes relationship but little is known about the allergenic potential of these proteins in the genus Anisakis. Taking into account the increase of anisakiasis and allergies related to these nematodes, immunoreactive properties of gland cell proteins were investigated. Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s. to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining which revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp.-allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, with 82 spots revealed in 2D. The corresponding protein bands and spots were excised from the silver-stained gel and protein assignation was made by MALDI-TOF/TOF. A total of 13 (including proteoforms) were unambiguously identified. The majority of these proteins are known to be secreted by nematodes into the external environment, of which three are described as being major allergens in other organisms with different phylogenetic origin and one is an Anisakis simplex allergen.
Subject(s)
Anisakiasis/immunology , Anisakis/immunology , Fish Diseases/immunology , Host-Parasite Interactions/immunology , Allergens/immunology , Allergens/isolation & purification , Animals , Anisakiasis/genetics , Anisakiasis/parasitology , Anisakis/pathogenicity , Esophagus/immunology , Esophagus/parasitology , Fish Diseases/genetics , Fish Diseases/parasitology , Humans , Larva/genetics , Larva/immunology , Larva/pathogenicity , Phylogeny , Proteins/immunology , Seafood/parasitology , Species Specificity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Human infection due to eating fish parasitized by live Anisakis larvae in the third stage is considered an important health problem, and the application of treatments to ensure their mortality in the fish products is crucial to prevent the risk of infection. Mobility is used to assess viability, but mobile larvae may not always be infective and immobile larvae may be erroneously considered as non-viable. The objective was to establish whether the analysis of respiratory activity by means of the oxygen consumption rate (OCR) of Anisakis could be used to identify subtle differences between larvae that were still considered viable in terms of their mobility but had been subjected to thermal and/or chemical stress. The metabolic modulators FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone] and sodium azide were used and the basal, maximum, spare and residual respiration rates calculated. Results showed that maximum respiratory capacity of larvae subjected to freezing significantly decreased immediately after thawing, but after some acclimatization, they recovered their capacity fully. However, when these larvae were stored at 4.6 °C, their mitochondria became dysfunctional faster than those of untreated larvae. OCR also showed that mitochondria of larvae were affected by incubation at 37 °C in NaCl or gastric juice. To conclude, OCR of Anisakis in the presence of metabolic modulators can help to identify subtle changes that occur in the larva. These measurements could be used to characterize larvae subjected to various stresses so that a broader picture of Anisakis pathogenic potential can be gained.
Subject(s)
Anisakis/metabolism , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Enzyme Inhibitors/pharmacology , Larva/metabolism , Mitochondria/metabolism , Oxygen Consumption/physiology , Sodium Azide/pharmacology , Acclimatization/physiology , Animals , Anisakiasis/veterinary , Anisakis/embryology , Fish Diseases/parasitology , Fishes/parasitology , Humans , Seafood/parasitology , Sodium Chloride/pharmacologyABSTRACT
Low-Field Nuclear Magnetic Resonance of proton transverse relaxation signal (T2) was monitored in hake (Merluccius merluccius) mince with different thermal histories (fresh, frozen, cooked) as affected by pH, water and NaCl addition, and it was related to water holding capacity (WHC). Modifications of T2 signals were found in terms of changes in relaxation times and relative abundance of the relaxation components. The relaxation rate of the major component (1/T21) increased significantly upon frozen storage or pH increase, whereas water or NaCl addition had the opposite effect. WHC decreased with freezing or water addition and increased with NaCl or basic pH; thus, T2 and WHC best correlated when each factor was analysed separately. Linearity found for pH and protein concentration with 1/T21 was consistent with chemical exchange being responsible for these changes. The significance of these results for technological situations where compositional and biochemical changes are occurring is discussed.
Subject(s)
Cooking , Freezing , Gadiformes , Proton Magnetic Resonance Spectroscopy , Sodium Chloride/chemistry , Water/chemistry , Animals , Food Handling , Hydrogen-Ion ConcentrationABSTRACT
A general overview, focusing on new trends in the different techniques used in restructured seafood product processing has been described in this work. Heat-induced gelation has been more widely studied in scientific literature than cold gelation technology. This latter technology includes the use of hydrocolloids (alginates and glucomannan) or enzymes (microbial transglutaminase) for making both raw and cooked restructured products. In restructuration processes, fortification processing with some functional ingredients is studied, giving as a result extra value to the products as well as increasing the variety of new seafood products. The process of alleviating heavy metals and organic pollutants from the raw material used has also been reviewed in the present paper.
Subject(s)
Food Technology , Meat Products/analysis , Seafood/analysis , Animals , Cooking , Hot TemperatureABSTRACT
Low field NMR T2 transverse relaxation measurements were performed on muscle samples from sixty hake (Merluccius merluccius, L.). Fish fillets from hake stored in ice for 3 and 14 days were subjected to different freezing methods (air blast, liquid nitrogen or walk-in freezer) and storage conditions (-20 and -10°C for 5 days, 8 and 18 weeks). Distribution analysis of T2 data of unfrozen muscle displayed a major band (T21), accounting for 90-92% of the total signal, with a relaxation time centred at 47-60 ms and a broad band with protons of higher mobility between 300 and 800 ms, accounting for 3-5% of the signal. Upon freezing, T21 became wider and an extra band appeared within the range 120-360 ms. Whereas no changes were detected at -20°C, the T21 time constant decreased during frozen storage at -10°C in a similar way for all three freezing methods. The relative abundance of T21 declined with storage time but differences were found as a function of freezing. Results are discussed in the light of morphological alterations and protein denaturation, and it is concluded that LF NMR relaxometry is sensitive to different freezing and frozen storage conditions which can have important implications for the quality of fish muscle.
Subject(s)
Food Preservation/methods , Food Storage/methods , Muscle, Skeletal/chemistry , Seafood/analysis , Animals , Cold Temperature , Gadiformes/physiology , Magnetic Resonance Spectroscopy , Muscle Relaxation , Muscle, Skeletal/physiologyABSTRACT
The potential of low field NMR (LF NMR) as a fast monitoring technique to estimate the quality of hake (Merluccius merluccius) frozen stored at -10°C for up to 6months was evaluated. LF NMR clearly detected three populations of water: water strongly bound to macromolecules (T(2b)), trapped water (T(21)) and free water (T(22)). As storage time increased, and concomitant with an increase in the T(22) and a decrease in the T(21) water populations, the water holding capacity (WHC) and apparent viscosity values decreased and the shear strength increased, reflecting the characteristic loss of juiciness and tougher texture developed by hake during frozen storage. Two mathematical models were constructed: a simple regression using the biexponential analysis of the relaxation times (T(21), T(22)) and amplitudes (A(21), A(22)) and a partial least square regression (PLS) of CONTIN analysis. Both models seemed suitable to estimate the quality of the product.
Subject(s)
Gadiformes , Magnetic Resonance Spectroscopy/methods , Meat/analysis , Seafood/analysis , Animals , Cold Temperature , Food Preservation , Meat/standards , Quality Control , Seafood/standards , Time Factors , Water/analysisABSTRACT
Vibrational spectroscopy (mid FTIR and FT-Raman) was used to monitor lipids extracted from hake fillets during frozen storage. Kramer shear resistance was used as a marker of texture changes and lipid damage was also investigated by following the development of conjugated dienes and free fatty acids by spectrophotometric methods. Results show that the intensity of the free fatty acid carboxylic ν(CO) band measured by ATR-FTIR spectroscopy can be used for monitoring the development of lipid hydrolysis in hake lipids. Changes in the Raman ν(CC) stretching region (1658cm(-1) band), partially attributed to conjugated dienes development, were the only observed spectroscopic alterations related to lipid oxidation of hake lipids during frozen storage at -10°C. The high correlation of free fatty acids with instrumental texture and the disappearance of the νas(PO2(-)) band are consistent with membrane lipid hydrolysis being one of the factors directly related with toughening of lean fish flesh.
Subject(s)
Frozen Foods/analysis , Gadiformes/metabolism , Spectroscopy, Fourier Transform Infrared/methods , Spectrum Analysis, Raman/methods , Animals , Fishes , Food Storage , Lipid Metabolism , Lipolysis , Oxidation-Reduction , VibrationABSTRACT
First processing steps of fish are species-dependent and have common practices for wild and for farmed fish. Fish farming does, however, have certain advantages over traditional fisheries in that the processor can influence postmortem biochemistry and various quality parameters. This review summarizes information about the primary processing of fish based on the influence of catching, slaughtering, bleeding, gutting, washing, and filleting. Recommendations are given for the correct primary processing of fish.
Subject(s)
Fishes , Food Handling/methods , Seafood/analysis , Animals , Aquaculture , Fisheries , Quality Control , Stress, PhysiologicalABSTRACT
The effectiveness of a white grape dietary fiber concentrate (WGDF) against hemoglobin-mediated oxidation of washed cod mince, with and without 10% added herring oil, was evaluated during ice storage. WGDF was added at two different levels: 2 and 4% based on final weight. An ethanol extract with the ethanol extractable polyphenols (EPP) and the ethanol-extracted grape dietary fiber residue were also tested as antioxidants in the washed cod mince. The addition of WGDF to the model system completely and significantly (p Subject(s)
Dietary Fiber/pharmacology
, Gadus morhua
, Hemoglobins/metabolism
, Lipid Peroxidation
, Seafood/analysis
, Vitis
, Animals
, Food Preservation/methods
, Food, Fortified/analysis
, Lipid Peroxidation/drug effects
, Lipids/analysis
, Oxidation-Reduction
, Plant Extracts/pharmacology