Structural and physical-chemical properties of milk fat globules fractionated by a series of silicon carbide membranes.

Driven by the acknowledged health and functional properties of milk fat globules (MFGs), there is a growing interest to develop gentle methodologies for separation of fat from milk. In this study, separation of fat from raw milk and fractionation in streams containing MFGs of different size was achieved using a series of two silicon carbide ceramic membranes. A first step consisting of a 1.4 µm membrane aimed to concentrate the bulk of the fat, i.e. the larger MFGs (D[4,3] âˆ¼ 4 µm) followed by a 0.5 µm fractionation aimed to concentrate the residual milk fat in the permeate, i.e. fraction with the smaller MFGs (D[4,3] âˆ¼ 1.8-2.4 µm. The fat separation performance showed a yield of 92 % for the 1.4 µm membrane and 97 % for the 0.5 µm membrane. Both fat enriched retentates showed, by the confocal laser scanning microscopy, intact MFGs with limited damage in the MFG membrane. The fatty acid profile analysis and SAXS showed minor differences in fat acid composition and the crystallization behavior was related to differences in the fat content. The 0.5 µm permeate containing the smallest MFGs however showed larger aggregates and a trinomial particle size distribution, due to probably pore pressure induced coalescences. The series of silicon carbide membranes showed potential to concentrate some of MFGM proteins such as Periodic Schiff base 3/4 and cluster of differentiation 36 especially in the 0.5 µm retentates. A shift in casein to whey protein ratio from 80:20 (milk) to 50:50 was obtained in the final 0.5 µm permeate, which opens new opportunities for product development.

Isolation and Characterization of Milk Exosomes for Use in Advanced Therapies.

Exosomes are cell-derived extracellular vesicles (EVs) with diameters between 30 and 120 nm. In recent years, several studies have evaluated the therapeutic potential of exosomes derived from different fluids due to their low immunogenicity and high biocompatibility. However, producing exosomes on a large scale is still challenging. One of the fluids from which they could be isolated in large quantities is milk. Moreover, regeneration is a well-known property of milk. The present work seeks to optimize a method for isolating exosomes from bovine and human milk, comparing different storage conditions and different extraction protocols. We found differences in the yield extraction associated with pre-storage milk conditions and observed some differences according to the processing agent. When we removed milk fat globules and added rennet before freezing, we obtained a cleaner final fraction. In summary, we attempted to optimize a rennet-based new milk-exosome isolation method and concluded that pre-treatment, followed by freezing of samples, yielded the best exosome population.

Ultrasound modification on milk fat globule membrane and soy lecithin to improve the physicochemical properties, microstructure and stability of mimicking human milk fat emulsions.

Starting from the consideration of the structure of human milk fat globule (MFG), this study aimed to investigate the effects of ultrasonic treatment on milk fat globule membrane (MFGM) and soy lecithin (SL) complexes and their role in mimicking human MFG emulsions. Ultrasonic power significantly affected the structure of the MFGM-SL complex, further promoting the unfolding of the molecular structure of the protein, and then increased solubility and surface hydrophobicity. Furthermore, the microstructure of mimicking MFG emulsions without sonication was unevenly distributed, and the average droplet diameter was large. After ultrasonic treatment, the droplets of the emulsion were more uniformly dispersed, the particle size was smaller, and the emulsification properties and stability were improved to varying degrees. Especially when the ultrasonic power was 300 W, the mimicking MFG emulsion had the highest encapsulation rate and emulsion activity index and emulsion stability index were increased by 60.88 % and 117.74 %, respectively. From the microstructure, it was observed that the spherical droplets of the mimicking MFG emulsion after appropriate ultrasonic treatment remain well separated without obvious flocculation. This study can provide a reference for the screening of milk fat globules mimicking membrane materials and the further utilization and development of ultrasound in infant formula.

Comparative study of interfacial properties and thermal behaviour of milk fat globules and membrane prepared from ultrasonicated bovine milk.

Milk fat globules or milk fat globule membranes (MFGs/MFGM) have been added to the infant formula to fortify the phospholipids and narrow the nutritional gap from breast milk. The main aim of this study was to profile the interfacial and thermal properties of MFGs/MFGM prepared from ultrasonicated bovine milk. Bovine milk was sonicated at ultrasonic intensities of 20 kHz and 40 kHz independently or synchronously with the duration time of 0 min (control), 5 min, 10 min, and 15 min (work/rest cycles = 5 s: 3 s). Ultrasonic treatments at 20 kHz/ 5 min and 20 + 40 kHz/ 5 min improved the volume density (%) of smaller particles (1-10 µm) while significantly decreasing the surface hydrophobicity (H0) (p < 0.05). 40 kHz/5 min samples showed significantly higher ζ- potential than the other samples (p < 0.05), which might be because more negative charges were detected. In comparison with control samples, ultrasonic treatments decreased the interfacial tension (π) between the air and MFGs/MFGM liquid phase. 20 kHz ultra-sonicated treatments decreased the diffusion rate (k diff) of MFGs/MFGM interfacial compositions significantly as the duration prolonged from 5 min to 15 min (p < 0.05) but did not affect the adsorption or penetration rate (k a) (p > 0.05). X-ray diffraction (XRD) results showed that α-crystal peaks only existed in control and ultrasonicated 5 min samples but disappeared in all 15 min samples. According to the different scanning calorimetry (DSC), one or two new exothermic events (in the range of 17.29 - 18.81 â„ƒ and 22.14 - 25.21 â„ƒ) appeared after ultrasonic treatments, which, however, were not found in control samples. Ultrasonic treatments resulted in the low-melting fractions (LMF) (TM1) peaks undetectable in MFGs/MFGM samples in which only peaks of medium-melting fractions (MMF) (TM2) and high-melting fractions (HMF) (TM3) were detected. Compared with the control, both enthalpies of crystallisation (ΔHC) and melting (ΔHM) decreased in ultrasonicated samples. In conclusion, ultrasonic treatment affects the interfacial and thermal properties of MFGs/MFGM.

A study of the effect of hypothyroidism during pregnancy on human milk quality based on rheological properties.

Hypothyroidism has been found to have an effect on the nutritional composition of human milk during pregnancy. This study aims to explore the combined influence of rheological properties, macronutrient content, particle size, and the zeta potential of milk fat globules, as well as the composition of milk fat globule membrane (MFGM) proteins on the quality of human milk in gestational hypothyroidism. The study revealed that human milk from the group with hypothyroidism during pregnancy (AHM) was less viscoelastic and stable when compared with normal pregnancy group human milk (NHM). Furthermore, the particle size and macronutrient content of NHM were found to be larger than that of AHM. In contrast, the zeta potential of AHM was greater than that of NHM. The sodium dodecyl sulfate-PAGE results disclosed that the composition of MFGM proteins in these 2 groups were generally the same, but the content of AHM was lower than that of NHM. In conclusion, this study confirms that hypothyroidism during pregnancy can have a significant effect on the quality of human milk.

Changes in Milk Fat Globules and Membrane Proteins Prepared from pH-Adjusted Bovine Raw Milk.

Milk fat globules (MFGs) have tri-layer biological membrane structures, and their compositions are gaining more interest for their physiological benefits. In this study, the changes in MFGs and milk fat globule membrane (MFGM) proteins after cream separation from different pH bovine raw milk were investigated. Raw milk samples were adjusted to pH 5.30 and 6.30 using citric acid at 25 °C. The effect of pH and centrifugation on the structure of MFGs was evaluated by means of particle size, zeta potential and confocal laser scanning microscopy (CLSM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the proteins in the obtained fractions. It was found that both pH and centrifugation could affect the particle size of all samples. As the volume distribution (Dv; Dv (10), Dv(50)and Dv (90)) decreased, the corresponding specific surface area (SSA) increased, and span and uniformity values showed the same trend. The decrease in the zeta potential of MFG correlated with the Dv(50), which was further confirmed by CLSM observation. More butyrophilin (BTN) and periodic acid Schiff 6/7 (PAS 6/7) were lost in cream samples at pH 5.30. The findings could provide valuable knowledge for the application of MFGs ingredient in the food industry since their structures and compositions could affect their potential functional and physiological properties.

Functional Significance of Different Milk Constituents in Modulating the Gut Microbiome and Infant Health.

Human milk, the gold standard for optimal nourishment, controls the microbial composition of infants by either enhancing or limiting bacterial growth. The milk fat globule membrane has gained interest in gut-related functions and cognitive development. The membrane proteins can directly interact with probiotic bacteria, influencing their survival and adhesion through gastrointestinal transit, whereas membrane phospholipids increase the residence time of probiotic bacteria in the gut. The commensal bacteria in milk act as the initial inoculum in building up the gut colonization of an infant, whereas oligosaccharides promote proliferation of beneficial microorganisms. Interestingly, milk extracellular vesicles are also involved in influencing the microbiota composition but are not well-explored. This review highlights the contribution of different milk components in modulating the infant gut microbiota, particularly the fat globule membrane, and the complex interplay between host- and brain-gut microbiota signaling affecting infant and adult health positively.

Composition and interfacial properties play key roles in different lipid digestion between goat and cow milk fat globules in vitro.

The different TAG, interfacial properties and digestion rate between goat and cow milk fat globules were investigated. The mechanism of their different lipid digestion was also elucidated. Raw goat milk fat globules had smaller size, less large molecular weight and unsaturated TAG, larger liquid-ordered region and fewer glycoproteins, which contributed to the higher digestion rate than cow milk. After homogenization, the goat lipids also had higher digestion rate that was attributed to the special structure of easy-to-digest TAG and less glycosylated molecules not globule size. More integrated phospholipid layers and glycosylated molecules of HTST milk fat globules resulted in a lower lipid digestion rate than other processed milks. No difference in digestion rate between pasteurized goat and cow milk fat globules might be explained by the more denatured proteins and glycosylated molecules, respectively. Therefore, the TAG and interfacial properties contributed to different digestion between goat and cow milk fat globules.

Comparative Structural and Compositional Analyses of Cow, Buffalo, Goat and Sheep Cream.

Factors affecting milk and milk fraction composition, such as cream, are poorly understood, with most research and human health application associated with cow cream. In this study, proteomic and lipidomic analyses were performed on cow, goat, sheep and Bubalus bubalis (from now on referred to as buffalo), bulk milk cream samples. Confocal laser scanning microscopy was used to determine the composition, including protein, lipid and their glycoconjugates, and the structure of the milk fat globules. BLAST2GO was used to annotate functional indicators of cream protein. Functional annotation of protein highlighted a broad level of similarity between species. However, investigation of specific biological process terms revealed distinct differences in antigen processing and presentation, activation, and production of molecular mediators of the immune response. Lipid analyses revealed that saturated fatty acids were lowest in sheep cream and similar in the cream of the other species. Palmitic acid was highest in cow and lowest in sheep cream. Cow and sheep milk fat globules were associated with thick patches of protein on the surface, while buffalo and goat milk fat globules were associated with larger areas of aggregated protein and significant surface adsorbed protein, respectively. This study highlights the differences between cow, goat, sheep, and buffalo milk cream, which can be used to support their potential application in functional foods such as infant milk formula.

A Comprehensive Review of the Composition, Nutritional Value, and Functional Properties of Camel Milk Fat.

Recently, camel milk (CM) has been considered as a health-promoting icon due to its medicinal and nutritional benefits. CM fat globule membrane has numerous health-promoting properties, such as anti-adhesion and anti-bacterial properties, which are suitable for people who are allergic to cow's milk. CM contains milk fat globules with a small size, which accounts for their rapid digestion. Moreover, it also comprises lower amounts of cholesterol and saturated fatty acids concurrent with higher levels of essential fatty acids than cow milk, with an improved lipid profile manifested by reducing cholesterol levels in the blood. In addition, it is rich in phospholipids, especially plasmalogens and sphingomyelin, suggesting that CM fat may meet the daily nutritional requirements of adults and infants. Thus, CM and its dairy products have become more attractive for consumers. In view of this, we performed a comprehensive review of CM fat's composition and nutritional properties. The overall goal is to increase knowledge related to CM fat characteristics and modify its unfavorable perception. Future studies are expected to be directed toward a better understanding of CM fat, which appears to be promising in the design and formulation of new products with significant health-promoting benefits.

Effects of Human, Caprine, and Bovine Milk Fat Globules on Microbiota Adhesion and Gut Microecology.

Milk fat is an essential nutrient for infant development. The effects and mechanisms of human, caprine, and bovine milk fat globules (MFGs) on the gut microbiota were investigated in this study. Human MFGs enhance the efficacy of probiotics by inhibiting pathogen function. Akkermansia and Bifidobacterium were identified as the dominant microbiota by human MFGs. Mucin and complement inhibitory proteins in human MFGs were found to inhibit different pathogens. Caprine MFGs directly promoted the colonization of probiotics and the emergence of the biomarker Allobaculum. Mucin 1 in caprine MFGs was primarily responsible for inducing probiotic adhesion. Bovine MFGs increased the abundance of Oscillospira, which reduces the risk of obesity. Due to the enrichment of cell-cell junction proteins and the lack of mucin, the regulation of gut microecology by bovine MFGs was not readily apparent. In short, this study paves the way for the development of functional infant formula.

A novel approach for the reconstitution of bovine milk fat globules with different-melting-temperature triacylglycerol cores.

Reconstructing milk fat globules (MFG) with different-melting-temperature triacylglycerols (TAG) to improve its nutritional and functional properties has great potential for expanding industrial applications. Butterfat was fractionated by stepwise crystallization at 30, 20 and 15 °C to yield six fractions (30S, 30L, 20S, 20L, 15S and 15L). Fractions were analyzed for thermal properties and fatty acid composition. An efficient method for analyzing TAG was established using HPLC-ESI-Q-TOF-MS/MS combined with principal component analysis, and total 146 TAGs in butterfat and its fractions were identified. The melting enthalpy, melting temperature, and long-chain saturated TAG content of 30S fraction were 71.5 J/g, 42.1 °C, and 19.3%, respectively, while that of 15L fraction corresponded to 11.9 J/g, 17.1 °C and 0.1%, indicating that the butterfat was effectively separated. Then MFG were reconstituted with milk fat globule membrane and different-melting-temperature TAG cores from obtained fractions, and reconstituted MFG gave excellent microstructural stability and emulsifying activity.

Comparative Proteomics of Milk Fat Globule Membrane (MFGM) Proteome across Species and Lactation Stages and the Potentials of MFGM Fractions in Infant Formula Preparation.

Milk is a lipid-in-water emulsion with a primary role in the nutrition of newborns. Milk fat globules (MFGs) are a mixture of proteins and lipids with nutraceutical properties related to the milk fat globule membrane (MFGM), which protects them, thus preventing their coalescence. Human and bovine MFGM proteomes have been extensively characterized in terms of their formation, maturation, and composition. Here, we review the most recent comparative proteomic analyses of MFGM proteome, above all from humans and bovines, but also from other species. The major MFGM proteins are found in all the MFGM proteomes of the different species, although there are variations in protein expression levels and molecular functions across species and lactation stages. Given the similarities between the human and bovine MFGM and the bioactive properties of MFGM components, several attempts have been made to supplement infant formulas (IFs), mainly with polar lipid fractions of bovine MFGM and to a lesser extent with protein fractions. The aim is thus to narrow the gap between human breast milk and cow-based IFs. Despite the few attempts made to date, supplementation with MFGM proteins seems promising as MFGM lipid supplementation. A deeper understanding of MFGM proteomes should lead to better results.

Physico-chemical behaviors of human and bovine milk membrane extracts and their influence on gastric lipase adsorption.

Milk fat globule membrane conditions the reactivity and enzymatic susceptibility of milk lipids. The use of bovine membrane extracts to make infant formulas more biomimetic of human milk has been suggested recently. A comparison of the physico-chemical behavior of human and bovine milk membrane extracts and their interaction with gastric lipase is here undertaken using biophysical tools. Milk membrane extracts (70% of polar lipids) were obtained either pooling of mature human milk (n = 5) or bovine buttermilk. Human extract contained more anionic glycerophospholipids, less phosphatidylethanolamine and more unsaturated fatty acids (57% versus 46%) than bovine extract. Human extract presented a higher compressibility, with slower increase of surface pressure, than bovine extract. Micronic liquid condensed (LC) domains were evidenced in both extracts at 10 mN/m, but the evolution differs upon compression. Upon gastric lipase addition, an adsorption preference for liquid expanded phase (LE) was observed for both extracts. However, insertion was more homogeneous in terms of height level in human extract and impacted less its lipid lateral organization than in bovine extract. Both membrane extracts share close physico-chemical properties, however human membrane higher compressibility may favour gastric lipase insertion and higher interfacial reactivity in gastric conditions.

Morphological Analysis of Human Milk Membrane Enclosed Structures Reveals Diverse Cells and Cell-like Milk Fat Globules.

Over the past decade, the cellular content of human milk has been a focus in lactation research due to the benefit a potential non-invasive stem cell compartment could provide either to the infant or for therapeutic applications. Despite an increase in the number of studies in this field, fundamental knowledge in regard to milk cell identification and characterisation is still lacking. In this project, we investigated the nature, morphology and content of membrane enclosed structures (MESs) and explored different methods to enrich human milk cells (HMCs) whilst reducing milk fat globule (MFG) content. Using both flow cytometry and immunofluorescence imaging, we confirmed previous reports and showed that nucleated HMCs make up a minority of milk-isolated MESs and are indistinguishable from MFGs without the use of a nuclear stain. HMC heterogeneity was demonstrated by differential uptake of nuclear stains Hoechst 33258 and DRAQ5™ using a novel technique of imaging milk MESs (by embedding them in agar), that enabled examination of both extracellular and intracellular markers. We found that MESs often contain multiple lipid droplets of various sizes and for the first time report that late post-partum human milk contains secretory luminal binucleated cells found across a number of participants. After investigation of different techniques, we found that viably freezing milk cells is an easy and effective method to substantially reduce MFG content of samples. Alternatively, milk MESs can be filtered using a MACS® filter and return a highly viable, though reduced population of milk cells. Using the techniques and findings we've developed in this study; future research may focus on further characterising HMCs and the functional secretory mammary epithelium during lactation.

Selective enrichment of milk fat globules using functionalized polyvinylidene fluoride membrane.

We report on the development of a functionalized membrane-based technology for selective enrichment of milk fat globules from raw bovine milk. Functionalization was conducted by in situ polymerization of acrylic acid within a polyvinylidene fluoride membrane, followed by the electrostatic attachment of a cationic polymer to impart a net positive charge. The functionalized membrane-based technology enabled a one-step method of selective separation of globules directly from milk-based on size and charge. The presence of globules in the eluate was confirmed by fluorescence microscopy. Quantification of the extracted phospholipids from globules in the eluant revealed a significantly higher amount of polar lipids than the permeate. Our study describes a comprehensive analysis of selective enrichment of fat globules using a functionalized membrane and demonstrates the beneficial effect of extracted phospholipids from enriched fat globules.

Real-time measurements of milk fat globule membrane modulation during simulated intestinal digestion using electron paramagnetic resonance spectroscopy.

Milk Fat Globules with their unique interfacial structure and membrane composition are a key nutritional source for mammalian infants, however, there is a limited understanding of the dynamics of fat digestion in these structures. Lipid digestion is an interfacial process involving interactions of enzymes and bile salts with the interface of suspended lipid droplets in an aqueous environment. In this study, we have developed an electron paramagnetic resonance spectroscopy approach to evaluate real time dynamics of milk fat globules interfacial structure during simulated intestinal digestion. To measure these dynamics, natural milk fat globule membrane was labeled with EPR-active probe, partitioning of EPR probes into MFGs membrane was validated using saturation-recovery measurements and calculation of the depth parameter Φ. After validation, the selected spin probe was used to evaluate the membrane's fluidity as a measure of the interface's modulation in the presence of bile salts and pancreatic lipase. Independently, bile salts were found to have a rigidifying effect on the spin probed MFGM, while pancreatic lipase resulted in an increase in membrane fluidity. When combined, the effect of lipase appears to be diminished in the presence of bile salts. These results indicate the efficacy of EPR in providing an insight into small time scale molecular dynamics of phospholipid interfaces in milk fat globules. Understanding interfacial dynamics of naturally occurring complex structures can significantly aid in understanding the role of interfacial composition and structural complexity in delivery of nutrients during digestion.

Complex lipid globules in early-life nutrition improve long-term metabolic phenotype in intra-uterine growth-restricted rats.

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.

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence.

Enterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness.

Influence of Homogenization and Thermal Processing on the Gastrointestinal Fate of Bovine Milk Fat: In Vitro Digestion Study.

Dairy lipids are an important source of energy and nutrients for infants and adults. The dimensions, aggregation state, and interfacial properties of fat globules in raw milk are changed by dairy processing operations, such as homogenization and thermal processing. These changes influence the behavior of fat globules within the human gastrointestinal tract (GIT). The gastrointestinal fate of raw milk, homogenized milk, high temperature short time (HTST) pasteurized milk, and ultrahigh temperature (UHT) pasteurized milk samples was therefore determined using a simulated GIT. The properties of particles in different regions of the GIT depended on the degree of milk processing. Homogenization increased the initial lipid digestion rate but did not influence the final digestion extent. Thermal processing of homogenized milk decreased the initial rate and final extent of lipid digestion, which was attributed to changes in interfacial structure. These results provide insights into the impact of dairy processing on the gastrointestinal fate of milk fat.