Although milk polar lipids such as phospholipids and sphingolipids located in the milk fat globule membrane constitute 0.1 to 1% of the total milk fat, those lipid fractions are gaining increasing interest because of their potential beneficial effects on human health and technological properties. In this context, the accurate quantification of the milk polar lipids is crucial for comparison of different milk species, products, or dairy treatments. Although the official International Organization for Standardization-International Dairy Federation method for milk lipid extraction gives satisfactory results for neutral lipids, it has important disadvantages in terms of polar lipid losses. Other methods using mixtures of solvents such as chloroform:methanol are highly efficient for extracting polar lipids but are also associated with low sample throughput, long time, and large solvent consumption. As an alternative, we have optimized the milk fat extraction yield by using a pressurized liquid extraction (PLE) method at different temperatures and times in comparison with those traditional lipid extraction procedures using 2:1 chloroform:methanol as a mixture of solvents. Comparison of classical extraction methods with the developed PLE procedure were carried out using raw whole milk from different species (cows, ewes, and goats) and considering fat yield, fatty acid methyl ester composition, triacylglyceride species, cholesterol content, and lipid class compositions, with special attention to polar lipids such as phospholipids and sphingolipids. The developed PLE procedure was validated for milk fat extraction and the results show that this method performs a complete or close to complete extraction of all lipid classes and in less time than the official and Folch methods. In conclusion, the PLE method optimized in this study could be an alternative to carry out milk fat extraction as a routine method.

SUMMARY Conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLNAs) have gained significant attention due to their anticarcinogenic and lipid/energy metabolism-modulatory effects. However, their concentration in foodstuffs is insufficient for any therapeutic application to be implemented. From a biotechnological standpoint, microbial production of these conjugated fatty acids (CFAs) has been explored as an alternative, and strains of the genera Propionibacterium, Lactobacillus, and Bifidobacterium have shown promising producing capacities. Current screening research works are generally based on direct analytical determination of production capacity (e.g., trial and error), representing an important bottleneck in these studies. This review aims to summarize the available information regarding identified genes and proteins involved in CLA/CLNA production by these groups of bacteria and, consequently, the possible enzymatic reactions behind such metabolic processes. Linoleate isomerase (LAI) was the first enzyme to be described to be involved in the microbiological transformation of linoleic acids (LAs) and linolenic acids (LNAs) into CFA isomers. Thus, the availability of lai gene sequences has allowed the development of genetic screening tools. Nevertheless, several studies have reported that LAIs have significant homology with myosin-cross-reactive antigen (MCRA) proteins, which are involved in the synthesis of hydroxy fatty acids, as shown by hydratase activity. Furthermore, it has been suggested that CLA and/or CLNA production results from a stress response performed by the activation of more than one gene in a multiple-step reaction. Studies on CFA biochemical pathways are essential to understand and characterize the metabolic mechanism behind this process, unraveling all the gene products that may be involved. As some of these bacteria have shown modulation of lipid metabolism in vivo, further research to be focused on this topic may help us to understand the role of the gut microbiota in human health.