Abstract
Fats and oils pose unique challenges for microbiological analysis due to their hydrophobic nature, yet the accurate detection and enumeration of microorganisms in these matrices is essential for food safety and quality. This study evaluated and validated sample preparation protocols for the microbiological examination of a diverse range of edible oils and fats, including sunflower oil, olive oil, cocoa butter, poultry fat, beef tallow, and Medium-Chain Triglycerides (MCT) oil. Different approaches, such as filtration, centrifugation, direct plating, and dilution using emulsifiers were compared for their suitability in both pathogen detection and enumeration of hygiene indicators and probiotics. Filtration and centrifugation proved impractical due to filter clogging and handling difficulties, respectively, while direct plating lacked applicability across all matrix types. A 1:10 dilution using pre-warmed buffered peptone water (BPW) supplemented with 1 % polysorbate 80 w/w was identified as an effective horizontal protocol for oils and fats with melting points below 50 °C. This protocol was successfully validated for five oils and fats using both qualitative and quantitative methods. For a high-melting-point fat, a two-step procedure was developed and validated in which the sample is first pre-diluted with pre-warmed oil before dilution in pre-warmed BPW with polysorbate 80. This procedure extends the current ISO 6887 series that describe horizontal methods for the preparation of test samples by addressing the gap for high-melting-point fats.