SEPARATION AND CHARACTERIZATION OF BOVINE MILK FAT GLOBULES

Abstract

Bovine milk is a complex fluid comprising diverse proteins, lipids, carbohydrates, and vitamins. It is a crucial nutritional source for newborns and adults of all ages. The fat in milk exists as small spherical droplets, called milk fat globules (MFG), ranging in size from 0.15 to 15 μm and surrounded by a tri-layered membrane known as the milk fat globule membrane (MFGM). Despite the abundance of bioactive components with known health benefits, there is a knowledge gap in characterizing, quantifying, and profiling across the lactation stages and in the efficient separation of bovine milk fat globules and their components. To this end, a study was designed by HPTLC to quantify two major lipid classes present in the MFG of cow, goat, and water buffalo and establish a correlation with the globule size. This study used high-performance thin-layer chromatography to identify and quantify five major polar lipids(PL) and three neutral lipids (NL) from the MFG of cow, goat, and water buffalo. Optimal separation was achieved for PLs using chloroform: methanol: water (65:25:4) and hexane: diethyl ether: acetic acid (70:30:1) for NLs. The lower detectable (0.12 to 1.53 μg/mL) and quantification (0.12 to 1.53 μg/mL) limits indicated the high sensitivity of the method. Quantification at 540 nm showed the highest abundance of phosphatidylethanolamine and triglycerides. Fat globules were further characterized for size and microstructural properties, which revealed smaller globules in goats (0.99 ± 0.04 μm) than in cows (1.85 ± 0.03 μm) and water buffaloes (2.91 ± 0.08 μm), indicating a negative correlation with PL but a positive correlation with NL. The variation in lipid quantity among different animal species suggests more research is needed to support their selection as a suitable source for developing functional food that will impact human health positively. Milk microfiltration has gained attention in the dairy industry due to its ability to selectively separate and concentrate bioactive components from milk and its further application in food fortification. The MFG undergoes structural and functional damage, membrane phospholipid loss, longer processing time, and higher recovery cost due to its current multi-step separation process. Therefore, a single-step approach was employed based on size for the separation of smaller-size milk fat globules from cow and buffalo milk using a porous polysulfone membrane through a cross-flow microfiltration system. An asymmetric polysulfone membrane with an average pore size of 0.8 ± 0.03 μm was synthesized via phase inversion and subsequently characterized for surface morphology, hydrophilicity, porosity, thermo-mechanical properties, and water contact angle. The membrane demonstrated good performance, with initial milk permeateflux ranging from 296 to 613 L/m2h at different transmembrane pressures (e.g., 0.2, 0.4, 0.6 bar) for both animal groups, with a gradual decline due to concentration polarization, and eventually reaching a steady state. Notably, at lower transmembrane pressure, the membrane exhibited excellent antifouling properties (flux recovery of 81.56–83 %) and reusability over three cycles. Microscopic examination of milk fat globules confirmed the successful size-based separation with intact globule membrane, high phospholipid yield, and mass balance. The results, to the best of our knowledge, demonstrate for the first time an in-depth analysis of membrane fouling and reusability for selective isolation of milk fat globules. Bovine MFGMproteins are crucial to human and calf health and nutrition, but a knowledge gap exists for its lactation stage-specific variations in cows and buffaloes. Therefore, mass spectrometry was performed to identify the inter- and intra-lactation stage-specific MFGM proteins in Murrah buffalo (Mu) and Holstein Friesian cow (HF). Mu exhibited higher proteins (n=264) than HF (n=250); utilizing multivariate analysis, differentially abundant proteins (n=78 in HF, n=31 in Mu) were identified specific to lactation stages. The MFGM proteins were categorized into health-associated (47.1%), lipid-associated proteins (44.1%), and shared proteins (8.8%). HF milk contained all health-associated proteins detected in Mu, and it possessed unique proteins (e.g., BTN1A1, SAA3, and ENPP3), including lipid-associated proteins that contributed to improved calf immunity. These results suggest HF milk is more suitable for calf health and dairy product development, including expanding our understanding of lactation stage-specific MFGM proteins and highlighting their potential health benefits.