Optimizing Lipid Removal and Protein Digestion in Human Milk for Microplastic Analysis using Candida rugosa Lipase, KOH Digestion, and Hydrogen Peroxide Oxidation

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Abstract
This study successfully developed and evaluated a multi-step digestion protocol for the effective extraction and identification of microplastics (MPs) from human breast milk (HBM). The method combined hexane-mediated lipid removal, enzymatic digestion using Candida rugosa lipase at a milk-to-enzyme ratio of 20:1, protein digestion with potassium hydroxide (KOH) at 1:3 ratio, and oxidative treatment with hydrogen peroxide (H₂O₂) at 10% concentration. Ethanol was found essential for reducing sample viscosity, particularly post-saponification, thereby facilitating efficient filtration. Results showed that extended incubation times during enzymatic digestion notably enhanced lipid removal and improved clarity of the digestate, contributing to higher extraction efficiency. This protocol establishes a reproducible framework for reliable isolation and downstream spectroscopic identification of MPs in breast milk. Despite these advances, improvements in mechanical handling and contamination control are necessary to further enhance accuracy and consistency. The present findings contribute important methodological advancements to the field and support the urgent need for standardised, scalable protocols in monitoring neonatal exposure to environmental microplastic contaminants. Future work should expand on validation across diverse sample sets and explore correlations between MP presence and neonatal health outcomes.

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