Optimization of the pepsin digestion method for anisakids inspection in the fishing industry

Abstract

During the last 50 years human anisakiasis has been rising while parasites have increased their prevalence at determined fisheries becoming an emergent major public health problem. Although artificial enzymatic digestion procedure by CODEX (STAN 244-2004: standard for salted Atlantic herring and salted sprat) is the recommended protocol for anisakids inspection, no international agreement has been achieved in veterinary and scientific digestion protocols to regulate this growing source of biological hazard in fish products. The aim of this work was to optimize the current artificial digestion protocol by CODEX with the purpose of offering a faster, more useful and safer procedure for factories workers, than the current one for anisakids detection. To achieve these objectives, the existing pepsin chemicals and the conditions of the digestion method were evaluated and assayed in fresh and frozen samples, both in lean and fatty fish species. Results showed that the new digestion procedure considerably reduces the assay time, and it is more handy and efficient (the quantity of the resulting residue was considerably lower after less time) than the widely used CODEX procedure. In conclusion, the new digestion method herein proposed based on liquid pepsin format is an accurate reproducible and user-friendly off-site tool, that can be useful in the implementation of screening programs for the prevention of human anisakiasis (and associated gastroallergic disorders) due to the consumption of raw or undercooked contaminated seafood products.

Recovery of Anisakid larvae by means of chloro-peptic digestion and proposal of the method for the official control

“…In the European Union the official method for larvae detection is visual inspection and candling of fish fillets indicated in Regulation EC 2074/2005 but it is known that this methods not detect all the larvae present in the flesh of fish (Levsen et al 2005) or in seafood preparations unlike other more accurate and efficient methods as, in particularly, artificial digestion. Several digestion methods have been proposed in the literature (Jackson et al 1981) and recent studies have shown that pepsin and HCl conentration may be different according to the material to be digested (Llarena-Reino et. al.…”

Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758), and twaite shad, Alosa fallax (Lacépède, 1803), from Western Iberian Peninsula Rivers: zoonotic and ecological implications

“…The artificial digestion of the flesh and visceral cavity of shad was carried out on the basis of an optimized artificial digestion protocol (Llarena-Reino et al 2013a). The flesh was digested at 37-40°C during approximately 3-4 h (3 h for visceral cavity material) in an ACM-11806 Magnetic Stirrer Multiplate, using a weight/volume pepsin ratio of 1:20, understanding that ratio as 20 ml of a 0.5 % pepsin solution in HCl 0.063 M (pH 1.5) for 1 g of flesh.…”

Proficiency testing carried out by the European Union Reference Laboratory for Parasites

“…Anisakiasis can be prevented by freezing fish (at -20°C or below for 7 days, or at -35°C or below for 15 h). At present, detection of Anisakidae nematodes in fishery products is performed by different methods based on visual inspection (candling), light microscopy, UV examination of fish fillets after pressing and freezing, molecular analysis and chlorine-peptic digestion [10]. In the European Union, the official method for larva detection is the direct observation [11], sometimes coupled with the more accurate digestion method [10].…”