**2. Extraction, concentration, and analysis of phospholipids from the MFGM of buttermilk**

Dairy glycerophospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol are the phospholipids commonly found in buttermilk. Of the class of sphingolipids, the main ones, found in buttermilk, are sphingomyelin, glucosylceramide, and lactosylceramide. The most commonly used phospholipid concentration processes are microfiltration (MF) and ultrafiltration (UF) [8]. The importance of extracting and concentrating these phospholipids is summarized by their bioactivity and their ability to act as emulsifiers.

 MF has some limitations due to the size of casein fragments and MFGM phospholipids, which are very similar. Regarding the optimization of UF, studies performed pretreatments for the elimination of casein, predicting UF, precipitating it with acid, as well as the addition of agents that dissociate casein micelles such as citrate [8].

 Isolation of MFGM has been hampered by the interaction of its components with other proteins during processing. The formation of the aggregates begins by ionic attraction between positively charged amino acid residues of the protein and the polar grouping of the lipids [9]. Researchers found that spraying, aiming at the drying of buttermilk, may induce the formation of MFGM protein and phospholipid complexes, making extraction of phospholipids difficult. It was further verified that in pasteurized buttermilk, there was an almost three times increase in the amount of MFGM-bound proteins compared to raw buttermilk. In addition, there was a high incorporation of β-lactoglobulin (β-LG) in MFGM isolated after heat treatment, and β-LG constitutes the major MFGM protein separated from pasteurized cream [10].

 According to previous studies, the whey proteins of cheese may undergo thermal aggregation in the presence of concentrated buttermilk. In conclusion, its study showed that the phospholipids of the buttermilk membrane contributed to the formation of heat-induced aggregates with whey proteins [9]. Other analyzes have reported that most mechanisms of protein aggregation depend on nucleation and, as a consequence, the onset is done through the formation of an aggregation nucleus. These protein aggregates can be used as fat mimics in low-fat cheese or as yoghurt texture modifiers [11].

*Technological and Biological Properties of Buttermilk: A Minireview DOI: http://dx.doi.org/10.5772/intechopen.80921* 

 A study developed a gradient solvent system for high-performance liquid chromatography (HPLC) coupled to the ELSD detector (evaporative detector with light scattering) to separate the lipids present in buttermilk. The ELSD detector is sensitive only to the mass of the vaporized analyte, not being limited by solvent flow or ambient temperature, thus allowing better analysis time and adequate level of sensitivity. Among the standards used for analysis in HPLC were cholesterol, monostearin, diolein, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and other neutral lipids. In this study, separation with high reproducibility and accuracy of all classes of lipids present in buttermilk powder, including phospholipids, was observed, with no previous fractionation stage. The total concentration of polar lipids found in buttermilk was about 30%, the predominant phospholipid being phosphatidylcholine, followed by phosphatidine ethanolamine and phosphatidylserine, and with a lower concentration of sphingomyelin [12].
