Anisakis simplex (s.l.) resistance to the action of gastric enzymes depends upon previous treatments applied to infected fish mince and affects antigen release.

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.

Respiratory analysis as a tool to detect physiological changes in Anisakis larvae subjected to stress.

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.

Ani s 11-Like Protein Is a Pepsin- and Heat-Resistant Major Allergen of Anisakis spp. and a Valuable Tool for Anisakis Allergy Component-Resolved Diagnosis.

BACKGROUND: Anisakis simplex is a fish parasite responsible for gastrointestinal and allergic symptoms in humans. The Ani s 11-like protein has been proposed as an Anisakis allergen because its primary structure is similar to that of Ani s 11. The aims of this work were to analyse the frequency of detection of the Ani s 11-like protein and assess its diagnostic value. METHODS: rAni s 11-like protein, rAni s 5 and rAni s 4 were expressed in Escherichia coli and rAni s 1 was produced in Pichia pastoris. Recombinant allergen detection patterns in 37 Anisakis-sensitised patients were determined. The stability to pepsin digestion and heat treatment of rAni s 11-like protein was also analysed by IgE immunoblotting. RESULTS: Ani s 11-like protein is a major allergen detected by 78% of Anisakis-allergic patients, and 13.5% of patients detect only the rAni s 11-like allergen. This allergen is heat stable because it retains its capability of binding IgE after boiling for 30 min and it is resistant to pepsin digestion for 120 min. CONCLUSIONS: These data indicate that the Ani s 11-like protein is a pepsin- and heat-resistant major allergen (Ani s 11.0201) of Anisakis spp. and a valuable tool for Anisakis allergy component-resolved diagnosis.

Removal of Anisakis simplex allergens from infected fish during the washing step of surimi production.

BACKGOUND: The washing operation of fish muscle is one of the key steps in the production of surimi. The aim of this study was to assess in parasitised minced fish the effect of the washing steps on the allergen removal of Anisakis simplex and on protein yield during surimi processing. Experimentally infected hake (Merluccius merluccius) (50 Anisakis simplex s.s L3 larvae per 100 g of muscle) underwent three successive washing steps with water, phosphate buffer (20 mmol L(-1) ), sodium bicarbonate (60 mmol L(-1) ), or sodium hypochlorite (0.27 mmol L(-1) ) in the surimi processing (4 kg muscle, 1:4 w/v for each solution). Total protein concentration and A. simplex antigens and allergens were evaluated in each waste fraction. RESULTS: The highest removal of Ani s 4 and A. simplex antigens was achieved by using phosphate buffer, together with a good protein yield in the raw surimi. Decrease of the concentration of allergens and antigens as a function of the washing steps rendered a linear trend (R(2) = 0.95 and 0.98 for Ani s 4 and A. simplex antigens, respectively). CONCLUSION: The conditions for an optimal removal of Anisakis allergens can be established and calculated as a function of the washing steps. This approach opens a line to utilise parasitised fish in a safer way. © 2014 Society of Chemical Industry.

Antigenicity of Anisakis simplex s.s. L3 in parasitized fish after heating conditions used in the canning processing.

BACKGROUND: Some technological and food processing treatments applied to parasitized fish kill the Anisakis larvae and prevent infection and sensitization of consumers. However, residual allergenic activity of parasite allergens has been shown. The aim here was to study the effect of different heat treatments used in the fish canning processing industry on the antigen recognition of Anisakis L3. Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) were experimentally infected with live L3 Anisakis. After 48 h at 5 ± 1 °C, brine was added to the muscle, which was then canned raw (live larvae) or heated (90 °C, 30 min) (dead larvae) and treated at 113 °C for 60 min or at 115 °C for 90 min. Anisakis antigens and Ani s 4 were detected with anti-crude extract and anti-Ani s 4 antisera respectively. RESULTS: Ani s 4 decreased in all lots, but the muscle retained part of the allergenicity irrespective of the canning method, as observed by immunohistochemistry. Dot blot analysis showed a high loss of Ani s 4 recognition after canning, but residual antigenicity was present. CONCLUSION: The results indicate that heat treatment for sterilization under the conditions studied produces a decrease in Ani s 4 and suggest a potential exposure risk for Anisakis-sensitized patients.

Re-evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1-3.

Surveillance data published since 2010, although limited, showed that there is no evidence of zoonotic parasite infection in market quality Atlantic salmon, marine rainbow trout, gilthead seabream, turbot, meagre, Atlantic halibut, common carp and European catfish. No studies were found for greater amberjack, brown trout, African catfish, European eel and pikeperch. Anisakis pegreffii, A. simplex (s. s.) and Cryptocotyle lingua were found in European seabass, Atlantic bluefin tuna and/or cod, and Pseudamphistomum truncatum and Paracoenogonimus ovatus in tench, produced in open offshore cages or flow-through ponds or tanks. It is almost certain that fish produced in closed recirculating aquaculture systems (RAS) or flow-through facilities with filtered water intake and exclusively fed heat-treated feed are free of zoonotic parasites. Since the last EFSA opinion, the UV-press and artificial digestion methods have been developed into ISO standards to detect parasites in fish, while new UV-scanning, optical, molecular and OMICs technologies and methodologies have been developed for the detection, visualisation, isolation and/or identification of zoonotic parasites in fish. Freezing and heating continue to be the most efficient methods to kill parasites in fishery products. High-pressure processing may be suitable for some specific products. Pulsed electric field is a promising technology although further development is needed. Ultrasound treatments were not effective. Traditional dry salting of anchovies successfully inactivated Anisakis. Studies on other traditional processes - air-drying and double salting (brine salting plus dry salting) - suggest that anisakids are successfully inactivated, but more data covering these and other parasites in more fish species and products is required to determine if these processes are always effective. Marinade combinations with anchovies have not effectively inactivated anisakids. Natural products, essential oils and plant extracts, may kill parasites but safety and organoleptic data are lacking. Advanced processing techniques for intelligent gutting and trimming are being developed to remove parasites from fish.

Calculation of full process freezing time in minced fish muscle.

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:•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).•Calculation of the freezing time first for an infinite flat plate, and then a correction is applied for the actual geometry (finite cylinder).•The total freezing time is the sum of the three separate parts of the freezing process.

The artificial digestion method underestimates the viability of Anisakis simplex (s.l.) L3 present in processed fish products.

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.

Metagenomics Analysis Reveals an Extraordinary Inner Bacterial Diversity in Anisakids (Nematoda: Anisakidae) L3 Larvae.

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.

Protein Signatures to Trace Seafood Contamination and Processing.

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.

Quantitative Proteomics Comparison of Total Expressed Proteomes of Anisakis simplex Sensu Stricto, A. pegreffii, and Their Hybrid Genotype.

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.

Anisakis simplex products impair intestinal epithelial barrier function and occludin and zonula occludens-1 localisation in differentiated Caco-2 cells.

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.

Immunoreactive Proteins in the Esophageal Gland Cells of Anisakis Simplex Sensu Stricto Detected by MALDI-TOF/TOF Analysis.

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.

LF 1H NMR T2 relaxation rate as affected by water addition, NaCl and pH in fresh, frozen and cooked minced hake.

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.

Changes over Time in IgE Sensitization to Allergens of the Fish Parasite Anisakis spp.

BACKGROUND: Sensitization to Anisakis spp. can produce allergic reactions after eating raw or undercooked parasitized fish. Specific IgE is detected long after the onset of symptoms, but the changes in specific IgE levels over a long follow-up period are unknown; furthermore, the influence of Anisakis spp. allergen exposure through consumption of fishery products is also unknown. OBJECTIVE: To analyse the changes in IgE sensitization to Anisakis spp. allergens over several years of follow-up and the influence of the consumption of fishery products in IgE sensitization. METHODS: Total IgE, Anisakis spp.-specific IgE, anti-Ani s 1 and anti-Ani s 4 IgE were repeatedly measured over a median follow-up duration of 49 months in 17 sensitized patients. RESULTS: Anisakis spp.-specific IgE was detected in 16/17 patients throughout the follow-up period. The comparison between baseline and last visit measurements showed significant decreases in both total IgE and specific IgE. The specific IgE values had an exponential or polynomial decay trend in 13/17 patients. In 4/17 patients, an increase in specific IgE level with the introduction of fish to the diet was observed. Three patients reported symptoms after eating aquaculture or previously frozen fish, and in two of those patients, symptom presentation was coincident with an increase in specific IgE level. CONCLUSIONS: IgE sensitization to Anisakis spp. allergens lasts for many years since specific IgE was detectable in some patients after more than 8 years from the allergic episode. Specific IgE monitoring showed that specific IgE titres increase in some allergic patients and that allergen contamination of fishery products can account for the observed increase in Anisakis spp.-specific IgE level. CLINICAL RELEVANCE: Following sensitization to Anisakis spp. allergens, the absence of additional exposure to those allergens does not result in the loss of IgE sensitization. Exposure to Anisakis spp. allergens in fishery products can increase the specific IgE level in some sensitized patients.

Raman spectroscopic study of structural changes in Hake (Merluccius merluccius L.) muscle proteins during frozen storage.

This paper examines changes in the structure and functionality of fish muscle proteins at frozen storage temperatures known to render very different practical storage lives (-10 and -30 degrees C). Apparent viscosity and dimethylamine (DMA) content showed drastic temperature-related differences during storage. Raman spectroscopy revealed the occurrence of some structural changes involving secondary and tertiary protein structures. The changes in secondary structure were quantified, showing an increase of beta-sheet at the expense of alpha-helix structure. The nuC-H stretching band near 2935 cm(-)(1) increased in intensity, indicating denaturation of the muscle proteins through the exposure of aliphatic hydrophobic groups to the solvent. These structural changes were more pronounced at -10 degrees C but occurred at both storage temperatures, whereas changes in apparent viscosity and DMA only occurred in storage at -10 degrees C. The possible utility of these structural changes for quality assessment is discussed.

Influence of chilling methods on the quality of sardines (Sardina pilchardus).

The aim of this study was to determine for sardines (Sardina pilchardus) the effect of (i) chilling in ice and water in small expanded polystyrene boxes during distribution and in ice thereafter and (ii) chilling in ice and water for the entire storage period. These storage methods were compared with storage of the fish in ice in wooden boxes or in expanded polystyrene boxes. Three storage experiments were performed to take into account the variability in handling conditions and seasonality. There were significant (P < 0.05) differences in the results of some of the sensory, physical, chemical, and microbiological analyses performed that showed that the fish were of better quality when preserved in water and ice than when preserved in ice alone. The effects of dewatering of the fish after transport and subsequent storage in ice were also significantly different (P < 0.05) from those of storage in ice, with the former storage method resulting in better sensory and microbial indices. However, oxidative rancidity, as measured by thiobarbituric acid-reactive substances, was higher for the dewatered fish than for fish preserved in water and ice throughout the storage period. It is concluded that for sardines, storage in water and ice, either only during transport or throughout the storage period, can be useful as an alternative preservation method during distribution and retail sale.

Anchovy shelf life as affected by different chilling methods during distribution.

Anchovies are a very labile fish and deteriorate fast under chilling conditions. In the South of Spain, fishing boats land their catches in wooden boxes with ice (12 to 14 kg). For some years now, fish processors have prepared this species for market distribution by placing about 7 kg fish in expanded polystyrene (EPS) boxes containing water and ice. Then, in the distribution market, boxes are dewatered and re-iced. Transportation of the fish in EPS boxes containing water and ice was recently forbidden on the grounds that boxes for transportation of fish in ice must have holes to let melted ice drain away. In this paper, the effect of preserving the anchovy in water and ice from landing to the distribution market was studied and compared with the more traditional methods of storing the fish in ice in either wooden or EPS boxes. Physical, chemical, microbiological, and sensory analyses were carried out over three different storage trials to account for the effect of seasonality. Little differences were found among lots, but some of the parameters showed that fish transported in water and ice did present less spoilage than fish stored in ice, especially when compared to the wooden boxes. According to these results, chilling of this fish in water and ice can be used as an alternative preserving method during transport.

Low field nuclear magnetic resonance (LF-NMR) relaxometry in hake (Merluccius merluccius, L.) muscle after different freezing and storage conditions.

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.

Identification of autoclave-resistant Anisakis simplex allergens.

Anisakis simplex is a fish parasite able to induce allergic reactions in humans infected when eating raw or undercooked fish parasitized with viable third-stage larvae. Some authors claim that exposure to nonviable Anisakis material can result in allergic symptoms in previously sensitized patients, indicating that parasite allergens are resistant to the thermal treatments of usual cooking procedures. Furthermore, some patients report symptoms after eating canned fish. The aim of this work was the analysis of parasite allergen stability in heating to 121 °C in an autoclave to simulate the thermal process applied to canned fish. Third-stage larvae were subjected to autoclaving for 20, 40, and 80 min, and parasite crude extracts were analyzed by electrophoresis, immunoblotting, and a flow-cytometric basophil activation test. Allergens resistant to autoclaving were separated by reversed-phase high-performance liquid chromatography and identified by ion trap mass spectrometry. Protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that autoclaving considerably reduced the number and intensity of identifiable protein bands in a time-dependent manner. Several allergens were detected by immunoblotting with a pool of A. simplex allergic patients' sera after autoclaving. Allergens of 9 and 14 kDa resistant to autoclaving were identified as Ani s 4 and Ani s 1 allergens, respectively. Functional analysis showed that allergens retain their capacity to activate basophils even after autoclaving for 80 min. In conclusion, some relevant A. simplex allergens retain their capacity to bind immunoglobulin E and activate basophils after being subjected to autoclaving, which is a method equivalent to that used in industrial canning processes.