Milk fat is a high-value component of the U.S. dairy market. It is the major energy component of milk and is responsible for many organoleptic and technological characteristics of milk and dairy products. In addition, milk fat is unquestionably distinctive among all dietary fats that humans consume, as it is not only comprised of several hundred different fatty acids (FAs) but also contains a wide and unique array of bioactive lipids. Milk fat is dispersed in milk primarily in the form of fat globules. These cytoplasmic lipid droplets originate from mammary epithelial cells (MECs) and are secreted into the alveolar lumen surrounded by a membrane. Many advances in our knowledge of specific enzymes involved in milk lipid synthesis, the selectivity of the triacylglyceride (TAG) synthesis enzymes for specific FAs, the molecular mechanisms behind the uptake of long-chain FAs into the cells and the milk lipid secretion process have led to an improved understanding of the biology of milk fat synthesis. However, research to provide deeper insights into the mechanism of lipid synthesis in MECs is warranted and might lead to novel strategies to alter milk fat content and quality to benefit the dairy industry and meet dietary recommendations and consumer demands for foods that positively impact health. In this review, we aimed to provide a general overview of our current knowledge of the molecular aspects of milk lipid synthesis in MECs, from the uptake of blood-derived precursors to the intracellular formation of TAG-rich fat droplets secreted into milk as milk fat globules. We also highlight some current gaps in the knowledge that warrant further exploration. Given the importance of dairy food in the human diet, a better understanding of these processes could help develop novel strategies to alter milk fat composition in ways that benefit both human health and dairy producers.

Mammary lipid secretion is generally held to be unique and remarkably uniform between the many different orders of mammals. It produces a unit membrane-bounded milk fat globule (MFG). The unit membrane is separated from the lipoprotein boundary of what was the cytoplasmic lipid droplet (CLD) boundary by a uniform layer of cytoplasmic proteins. In 3–8% of the MFG in all species examined this cytoplasmic layer widens to include cytoplasmic organelles which are referred to as ‘crescents’. This defines the MFG secretion as apocrine indicating a closely regulated process which minimises the loss of mammary epithelial cell (MEC) cytoplasm. The apocrine nature of the secretion might be expected since the evolution of the mammary gland is considered to be from an apocrine secreting skin gland. This short Research Reflection review is designed to investigate the exact cytoplasmic interactions which allow such efficient lipid secretion. There are two main scenarios: one which assumes that the observed close association between CLD and GV results in the CLD being released as a consequence of sequential exocytosis of the content of the associated GV. The second assumes that the CLD and the MEC apical plasmalemma interact in some way which causes the CLD to rise out of the cytoplasm enveloped in the plasmalemma. Here I present the evidence for the two possibilities. The first scenario is favoured, but the second cannot be ruled out.

Intra-uterine growth restriction (IUGR) is associated with adverse metabolic outcome later in life. Healthy mice challenged with a Western-style diet (WSD) accumulated less body fat when previously fed a diet containing large lipid globules (complex lipid matrix (CLM)). This study was designed to clarify whether an early-life CLM diet mitigates ‘programmed’ visceral adiposity and associated metabolic sequelae after IUGR. In rats, IUGR was induced either by bilateral uterine vessel ligation (LIG) or sham operation (i.e. intra-uterine stress) of the dam on gestational day 19. Offspring from non-operated (NOP) dams served as controls. Male offspring of all groups were either fed CLM or ‘normal matrix’ control diet (CTRL) from postnatal days (PND) 15 to 42. Thereafter, animals were challenged with a mild WSD until dissection (PND 98). Fat mass (micro computer-tomograph scan; weight of fat compartments), circulating metabolic markers and expression of ‘metabolic’ genes (quantitative real-time PCR) were assessed. CLM diet significantly reduced visceral fat mass in LIG at PND 40. At dissection, visceral fat mass, fasted blood glucose, TAG and leptin concentrations were significantly increased in LIG-CTRL v. NOP-CTRL, and significantly decreased in LIG-CLM v. LIG-CTRL. Gene expression levels of leptin (mesenteric fat) and insulin-like growth factor 1 (liver) were significantly reduced in LIG-CLM v. LIG-CTRL. In conclusion, early-life CLM diet mitigated the adverse metabolic phenotype after utero-placental insufficiency. The supramolecular structure of dietary lipids may be a novel aspect of nutrient quality that has to be considered in the context of primary prevention of obesity and metabolic disease in at-risk populations.

Many studies have shown that milk fat globule (MFG) diameter varies in dairy cows in relation to diet and/or breed. However, the mechanisms governing the size of the fat globules remain hypothetical. Our objective was to determine the variable biochemical characteristics (fat, protein, fatty acids (FA), casein and calcium (Ca) contents) between individual milk which differed in both MFG diameter and membrane content, in order to speculate about the links between milk synthesis and MFG secretion. With this aim, we built five databases of individual milk samples from 21 experiments performed between 2003 and 2011. Three of them grouped data from trials dealing with breed/diet effects and included information about: (i) MFG size/membrane, fat and protein contents (n=982), (ii) previous parameters plus FA profile (n=529) and (iii) previous parameters plus true protein composition and calcium contents (n=101). A hierarchical clustering analysis performed on these three databases yielded four groups differing in the MFG characteristics. We observed significant differences among groups for the following parameters: (i) fat content and fat : protein ratio; (ii) de novo and polyunsaturated FA contents; (iii) Ca contents. These relationships could result from potential process regulating the synthesis and secretion of MFG: (i) the apical membrane turnover for MFG secretion and (ii) cytoplasmic lipid droplet formation in the lactocyte during its migration from the basal to the apical pole. The two other databases grouped data from trials dealing with milking frequency (n=211), milking kinetics and milk type (residual v. cisternal) (n=224). They were used to study the relationships between the size of the MFG and milk composition for high native fat contents (from 60 up to 100 g/kg in residual milks). We observed curvilinear relationships between the size of the MFG and fat content, as well as with the fat : protein ratio. This result suggests that MFG diameter reaches a threshold but mechanisms are still unknown.

Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells.

Lactation represents an important element of the life history strategies of all mammals, whether monotreme, marsupial, or eutherian. Milk originated as a glandular skin secretion in synapsids (the lineage ancestral to mammals), perhaps as early as the Pennsylvanian period, that is, approximately 310 million years ago (mya). Early synapsids laid eggs with parchment-like shells intolerant of desiccation and apparently dependent on glandular skin secretions for moisture. Mammary glands probably evolved from apocrine-like glands that combined multiple modes of secretion and developed in association with hair follicles. Comparative analyses of the evolutionary origin of milk constituents support a scenario in which these secretions evolved into a nutrient-rich milk long before mammals arose. A variety of antimicrobial and secretory constituents were co-opted into novel roles related to nutrition of the young. Secretory calcium-binding phosphoproteins may originally have had a role in calcium delivery to eggs; however, by evolving into large, complex casein micelles, they took on an important role in transport of amino acids, calcium and phosphorus. Several proteins involved in immunity, including an ancestral butyrophilin and xanthine oxidoreductase, were incorporated into a novel membrane-bound lipid droplet (the milk fat globule) that became a primary mode of energy transfer. An ancestral c-lysozyme lost its lytic functions in favor of a role as α-lactalbumin, which modifies a galactosyltransferase to recognize glucose as an acceptor, leading to the synthesis of novel milk sugars, of which free oligosaccharides may have predated free lactose. An ancestral lipocalin and an ancestral whey acidic protein four-disulphide core protein apparently lost their original transport and antimicrobial functions when they became the whey proteins β-lactoglobulin and whey acidic protein, which with α-lactalbumin provide limiting sulfur amino acids to the young. By the late Triassic period (ca 210 mya), mammaliaforms (mammalian ancestors) were endothermic (requiring fluid to replace incubatory water losses of eggs), very small in size (making large eggs impossible), and had rapid growth and limited tooth replacement (indicating delayed onset of feeding and reliance on milk). Thus, milk had already supplanted egg yolk as the primary nutrient source, and by the Jurassic period (ca 170 mya) vitellogenin genes were being lost. All primary milk constituents evolved before the appearance of mammals, and some constituents may have origins that predate the split of the synapsids from sauropsids (the lineage leading to ‘reptiles’ and birds). Thus, the modern dairy industry is built upon a very old foundation, the cornerstones of which were laid even before dinosaurs ruled the earth in the Jurassic and Cretaceous periods.

Cooling strategies for pumping of raw milk were evaluated. Milk was pumped for 450 s at 31 °C, or pumped after cooling to 4 °C and subsequently subjected to various incubation times. Two types of milk were used; i.e. milk from cows fed a diet high in saturated fat supplements resulting in significantly larger milk fat globules than the other type of milk which comes from cows fed a low-fat diet that stimulates high de novo fat synthesis. The content of liquid fat was determined by low-field 1H NMR, which showed that milk from cows given the saturated fat diet also contained less liquid fat at both 4 ° and 31 °C than the other type of milk. This can be ascribed to the differences in the fatty acid composition of the milk as a result of the fatty acid composition of the diets. After pumping of the milk at 31 °C, measurement of fat globule size distribution revealed a significant coalescence of milk fat globules in the milk obtained from the saturated fat diet due to pumping. Pumping at 4 °C or pumping the other type of milk did not result in coalescence of milk fat globules. Formation of free fatty acids increased significantly in both types of milk by pumping at 31 °C. Cooling the milk to 4 °C immediately before pumping inhibited an increased content of free fatty acids. However, when the milk was incubated at 4 °C for 60 min after cooling and then subjected to pumping, a significant increase in the formation of free fatty acids was observed in both types of milk. It is suggested that this increase in free fatty acids is caused by transition of polymorphic crystal forms or higher level of attached lipoprotein lipases to the milk fat globule before pumping.

A novel 2-stage gravity separation scheme was developed for fractionation of raw, whole bovine milk into fractions enriched in small (SFG) or large (LFG) fat globules. The volume mean diameter of fat globules in SFG, LFG or control (CTRL) milk was 3·45, 4·68 and 3·58 μm, respectively. The maximum in storage modulus (index of firmness) decreased with increasing fat globule size for rennet-induced gels formed from SFG, LFG or CTRL milks. Miniature (20 g) Cheddar cheeses were manufactured using each of the 3 milks. There were no significant (P>0·05) differences in the pH, moisture and fat in dry matter levels between cheeses made using any of the 3 milks, however, the fat content of the cheese made using SFG milk was ~1% lower than that of cheese made using LFG or CTRL milk in each of the 2 trials. Image analysis of confocal scanning laser micrographs of the cheeses illustrated that the star volume of fat globules in the cheeses decreased significantly (P[les ]0·05) as the size of fat globules in the milks used for cheesemaking was reduced. This indicates that it is possible to manipulate the size distribution of fat globules in Cheddar cheese by adjusting the fat globule size distribution of the milk used for cheesemaking. The concentration of free fatty acids (FFA) increased in all cheeses during ripening. At 120 d of ripening, the concentration of FFA varied significantly (P[les ]0·05 and P[les ]0·001 for trials 1 and 2, respectively) with fat globule size, with cheeses made in trial 2 from LFG, SFG or CTRL milks having total FFA levels of 3391, 2820 and 2612 mg/kg cheese, respectively.