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Comparative evaluation of enzymatic, acidic, and alkaline digestion protocols for organic matrix removal from lyophilized and frozen mussel samples (Mytilus galloprovincialis)
Summary
Scientists tested different ways to break down mussel tissue in order to accurately count the tiny plastic particles (microplastics) hiding inside — a key step since mussels are a popular seafood that can absorb plastic pollution from the ocean. They found that using a gentle enzyme-based method (similar to a digestive enzyme) removed the tissue effectively without damaging or destroying the plastic pieces, unlike harsher acid treatments that could distort some plastic types. This matters because getting an accurate microplastic count in seafood is essential for understanding how much plastic pollution we might be consuming when we eat shellfish.
Efficient removal of organic matter from biological matrices is a critical step in the analysis of microplastics in marine organisms. In this study, enzymatic (Kreon®25,000), acidic (HNO3 + H2O2), and alkaline (KOH + H2O2) digestion protocols were comparatively evaluated for the treatment of lyophilized and frozen Mytilus galloprovincialis samples. Digestion efficiency was assessed gravimetrically, while the effects of the protocols on polymer integrity were examined using ATR-FTIR, HQI analysis, and microscopic observations on representative polymers (HDPE, PA, PET, PVC). All three methods demonstrated high digestion efficiencies (>96%). Acidic digestion provided rapid and stable removal of organic matter within 20 min, whereas enzymatic digestion required longer incubation times (2–24 h) but exerted the least impact on polymer integrity. Frozen samples consistently showed slightly higher digestion efficiencies compared to lyophilized ones, likely due to preserved tissue hydration facilitating reagent penetration. Microscopic and spectroscopic analyses revealed that HDPE and PET maintained their structural and chemical integrity under all treatments, whereas PA and PVC exhibited surface alterations after acidic digestion. Enzymatic and alkaline protocols did not produce visible or spectral changes in any polymer type. Based on these findings, the enzymatic protocol was selected for recovery experiments. Mass-corrected recovery values ranged from 92.87% to 95.36% for PA, PET, and PVC, and 75.69% for HDPE, indicating that the method allows effective isolation of most polymers while preserving their integrity. The results demonstrate that although all digestion methods are efficient in removing organic matter, enzymatic digestion provides the most reliable approach for microplastic analysis in Mytilus galloprovincialis, ensuring both high digestion efficiency and preservation of polymer characteristics.