ISO 23036-2:2021

Overview

ISO 23036-2:2021 - Microbiology of the food chain - Methods for the detection of Anisakidae L3 larvae in fish and fishery products - Part 2: Artificial digestion method.
This international standard specifies an enzymatic artificial digestion method (pepsin–HCl) for detecting and quantifying Anisakidae third-stage (L3) larvae in fresh, frozen and lightly processed fish (e.g., marinated, salted, smoked). It is also suitable for visceral organs and as a confirmatory method following visual inspection. The method detects larvae and, for non-frozen fresh/lightly processed samples, can assess viability of larvae; species/genotype identification requires separate morphological or molecular tests.

Key topics and technical requirements

• Principle: Enzymatic digestion of fish musculature using a pepsin–HCl digest fluid followed by filtration and microscopic examination.
• Sample types: Fresh, frozen, lightly processed fish and visceral organs (confirmatory use).
• Digestion conditions: Maintain temperature at around 37 °C ± 2 °C; recommended meat-to-digest fluid ratio up to 1:20. Typical digestion time 15–30 minutes, not exceeding 45 minutes unless validated.
• Filtration: Digest fluid is filtered through a sieve (mesh typically in the range indicated in the standard) and retained larvae are rinsed and examined under stereo-microscope.
• Reagents & equipment: Certified pepsin (activity-equivalent formulations acceptable), hydrochloric acid, appropriate sieves, glass beakers, magnetic stirrer with heating, calibrated thermometer, stereo-microscope, PPE.
• Limitations: Method does not identify species/genotype. Viability assessment is only valid for samples that have not been frozen prior to processing.
• Quality & reporting: Includes procedure steps, expression of results, performance characteristics, and test reporting; no internal QC control is defined in-method.
• Safety: Handling pepsin powder may pose allergen/inhalation risk - use protective equipment and consider liquid pepsin.

Applications

• Routine food safety testing in fish processing and inspection to estimate parasitic infestation levels.
• Confirmatory testing after visual inspection or UV-press screening to verify presence/number of Anisakidae L3 larvae.
• Risk-based sampling for public health surveillance and regulatory compliance in seafood supply chains.
• Determination of larval viability in fresh/lightly processed products to assess consumer risk from raw/undercooked seafood.

Who should use this standard

• National and local food inspection authorities and laboratories
• Accredited microbiology testing laboratories and reference labs
• Seafood processors and quality assurance teams
• Public health agencies and research laboratories studying fish-borne parasites

Related standards

• ISO 23036 series (other parts) and cross-reference to ISO 7218 (general microbiology laboratory requirements).
• Final species identification requires morphological and/or molecular standards and reference methods.