A simplified protocol for fatty acid profiling of milk fat without lipid extraction

Abstract

Determination of the fatty acid profile of milk fat generally involves a protocol that comprises total lipid extraction, transesterification and GC analysis. The lipid extraction step is time-consuming and often employs toxic solvents such as chloroform. A novel protocol is presented here that skips the lipid extraction step and allows the determination of fatty acid composition via direct methylation of milk fat isolated after centrifugation of raw milk. This new method is reliable for relative quantification of fatty acids in raw milk fat and offers a much higher throughput compared with the classical methods.

Introduction

Fatty acid (FA) composition is one of the most important indicators of nutritive quality and physicochemical properties of milk fat and can also be used to reflect the health status of cows (Chouinard et al., 1998, Liu et al., 2018). FA composition of bovine milk is known to be influenced by animal genetics, diets and stage of lactation (Palmquist, Beaulieu, & Barbano, 1993). As a result, analysis of FA composition is required in various research projects related to dairy research.

Generally, FA composition of milk fat is determined by gas chromatography-flame ionisation detector (GC-FID) or gas chromatography-mass spectrometry (GC–MS) as FA methyl esters (FAMEs) after acid- or alkaline-catalysed transesterification (Jensen, 2002, Kramer et al., 1997). Prior to the methylation step, milk lipids are usually extracted by chloroform-based solvent systems (Bligh and Dyer, 1955, Folch et al., 1957), and 2–3 cycles of extractions are often needed to achieve a good recovery of all lipid classes. Consequently, the lipid extraction step is not only hazardous and expensive, but also time-consuming, which contributes to a low throughput in FA profiling of milk samples.

With the aim to increase the throughput and reduce the cost of FA profiling of milk samples, in the first instance we have compared different methylation methods and validated a simple one-step protocol that combines the use of low concentration of KOH, a short reaction time and a mild reaction temperature (Liu, Ezernieks, Rochfort, & Cocks, 2018). The aim of this study was to evaluate the feasibility of performing methylation of milk fat isolated by a simple step of centrifugation of raw milk (i.e., skipping the lipid extraction step with organic solvents).