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[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 48h at 5±1°C, brine was added to the muscle, which was then canned raw (live larvae) or heated (90°C, 30min) (dead larvae) and treated at 113°C for 60min or at 115°C for 90min. 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., This work has been financed by the Spanish project PlanNacional de I + D + i AGL2009-12485-C03-01/02/03 (ANIDET)and FP7-312068 EU PARASITE.

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Heat treatments (40 to 94C, 30 s to 60 min) were applied to different batches of Anisakis simplex L3 larvae isolated from hake ovaries and viscera to study the effect of heat on the viability of the larvae measured as mobility, emission of fluorescence under UV light, and changes in color after staining with specific dyes, and on A. simplex antigenic proteins. The aim was to determine the lowest time–temperature conditions needed to kill the larvae to avoid anisakiasis in consumers, and to evaluate whether high temperature modifies the antigenicity of A. simplex extracts. Heating at 60uC for 10 min (recommended by some authors) was considered unsafe, as differences in viability between batches were found, with some larvae presenting spontaneous movements in one batch. At higher temperatures (70 C for 1 min), no movement of the larvae was observed. Antigenic protein Ani s 4 and A. simplex crude antigens were detected in the larvae heated at 94 6 1uC for 3 min. This indicates that allergic symptoms could be provoked in previously sensitized consumers, even if the larvae were killed by heat treatment.

High pressure (HP) ranging from 100 to 350 MPa (1–15 min) was applied to Anisakis simplex larvae and parasitised hake (Merluccius merluccius) muscle. The aim of the study was to kill the larvae to prevent human anisakidosis, to evaluate the effect on A. simplex allergens and to minimally alter fish muscle quality. The larvae were killed at pressures ≥200 MPa and times ≥1 min, producing alterations in the larva body and ruptures in the cuticle when observed by scanning electron microscopy. Nevertheless, Ani s 4 and A. simplex crude antigens were recognised by immunoblotting and immunohistochemistry at all HPs assayed. Small changes in colour and texture were observed in fishmuscle under all pressure/time conditions. Major changes were observed visually at 300 MPa, where the muscle appeared as slightly cooked. Apparent viscosity of muscle homogenates decreased significantly at longer times or higher applied pressure. No changes were detected at 200 MPa in the electrophoretic pattern of proteins treated with or without β-mercaptoethanol, suggesting that disulfide bonds were not formed. Application of HP at 200 MPa for up to 5min would kill A. simplex larvae, avoiding infestation of the consumer and causing small changes in the hakemuscle perceived sensorially. However, HP-treated A. simplex-parasitised fishwould still be a potential hazard for consumers allergic to the larvae.

Some Anisakis simplex allergens, Ani s 4 among them, are reported to be resistant to freezing, heat and pepsin.However, the effect of conventional and microwave heating on live and frozen larvae, common conditions for fish preparation, and consecutive pepsin treatment have not been studied previously. In this study, live and frozen/thawed A. simplex larvae were subjected to conventional or microwave heating during time–temperature sufficient to kill live larvae, and digestedwith pepsin in the strong conditions used in fish inspection. The antigenicity of A. simplex in the larvae extracts and in the incubation media filtrates after all treatments was studied. The immunoblotting assay showed the presence of Ani s 4 in all the larvae extracts and all the incubation media filtrates. A. simplex crude antigens were detected in all conditions ; nevertheless, differences were observed among treatments, with lower values detected in the filtrates obtained after the strong acidic pepsin treatment. The results indicate that ingestion of A. simplex larvae can cause allergy in consumers already sensitised to this allergen, even if the parasitised fish is consumed well-cooked and after freezing in the recommended conditions selected for killing the larvae to avoid human anisakiasis.

tesis que para obtener el grado de Doctor en Derecho, presenta Margarita María Guerra y Tejada ; asesor Ricardo Franco Guzmán, Emma C. Mendoza Bremauntz, Elssie Núñez Carpizo. VI, 284 páginas : ilustraciones. Doctorado en Derecho UNAM, Facultad de Derecho, 2009