Human Milk Fat Globule Membrane Research Articles

Infant formula ingredients: Updates for clinicians.

Take Away Points All infant formulas meet specific nutritional standards, but there are now numerous versions of formula with newer ingredients that more closely approximate breast milk. Select infant formulas include ingredients such as human milk oligosaccharides, lactoferrin, or milk fat globule membranes, which may have additional clinical benefits. These ingredients are considered safe in infants but there is not dosing standardization across formula companies, and they may increase cost.

Effects of maternal and perinatal factors on human milk fat globule membrane proteome: A data independent acquisition approach

Milk fat globule membrane proteins (MFGMP) are essential for supporting infants’ growth and development. The composition of MFGMP may vary depending on maternal and perinatal factors. However, there is a lack of research on the effects of parity, maternal age, and mode of delivery on MFGMP. This study attempts to address this knowledge gap by utilizing a data-independent acquisition (DIA) approach to perform proteomics comparisons of human MFGMP. Mature human milk (MHM) was collected from Chinese women with different parities (multiparous vs. primiparous, MP vs. PP), maternal reproductive ages (age ≥30 vs. age <30, MA1 vs. MA2), and delivery modes (cesarean delivery vs. vaginal delivery, CD vs. VD). We found variations in MFGMP between different cohorts. Differentially expressed proteins (DEP) in MHM of primiparous and multiparous women were mostly related to innate immune response which provides the first line of defense against pathogens. Higher abundances of these DEP were found in the MHM of multiparous women. Meanwhile, DEP in MHM of women with different delivery modes were primarily related to inflammatory response and innate immune response. We postulate that upregulation of the defense response-related DEP in MHM of women who had cesarean delivery was in relation to modulation of the cesarean delivery-induced inflammatory response and to strengthen the immune defense of newborns.

Lipidomics insight on differences between human MFGM and dietary-derived lipids.

Milk fat globule membrane (MFGM) contains lipids, which are essential for promoting infant brain development and improving cognition. In this study, the lipid differences between human MFGM and four dietary lipid sources (cow MFGM, soybean, krill, and yolk) were compared using the UHPLC-Q-Exactive MS-based lipidomics techniques. A total of 45 lipid classes and 5048 lipid species were detected. The analysis of phospholipid classes revealed that the lipid composition of human MFGM and cow MFGM was more similar than the other dietary-derived lipids. Additionally, the human MFGM lipid species were compared with cow MFGM, soybean, krill, and yolk, and 401, 416, 494, and 444 significantly different lipids were identified, respectively. Through lipid metabolic pathway analysis, differential lipids were mainly involved in the glycerophospholipid metabolic pathway. Overall, these results will provide a rationale for the future addition of lipids to infant formula to more closely approximate human MFGM lipid profiles.

Characterization and comparison site-specific N-glycosylation profiling of milk fat globule membrane proteome in donkey and human colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are highly glycosylated and involved in various biological processes within the body. However, information on site-specific N-glycosylation of MFGM glycoproteins in donkey and human milk remains limited. This study aimed to map the most comprehensive site-specific N-glycosylation fingerprinting of donkey and human MFGM glycoproteins using a site-specific glycoproteomics strategy. We identified 1,360, 457, 2,617, and 986 site-specific N-glycans from 296, 77, 214, and 196 N-glycoproteins in donkey colostrum (DC), donkey mature milk (DM), human colostrum (HC), and human mature milk (HM), respectively. Bioinformatics was used to describe the structure-activity relationships of DC, DM, HC, and HM MFGM N-glycoproteins. The results revealed differences in the molecular composition of donkey and human MFGM N-glycoproteins and the dynamic changes to site-specific N-glycosylation of donkey and human MFGM glycoproteins during lactation, deepening our understanding of the composition of donkey and human MFGM N-glycoproteins and their potential physiological roles.

Effect of cold storage on human milk fat globule membrane: Microstructure and proteomic analysis

We investigated the changes in microstructure and composition of milk fat globule membrane (MFGM) in fresh human milk under different storage temperatures (-18, -3, -0.5, and 4 °C). Freezing at -18 °C resulted in the formation of ice crystals that ruptured the MFGM, leading to aggregation and increased particle size, which could potentially impact lipid digestion and absorption in infants. Conversely, storage at -3, -0.5, or 4 °C were more favorable for maintaining the stability of MFGM structure without ice crystals formation. Notably, -3 °C exhibited the highest preservation of MFGM integrity. Label-free proteomics analysis identified a total of 1525 different MFGM proteins in the initial sample (0 day) and samples preserved at varying temperatures (-18, -3, -0.5, and 4 °C) for a duration of 4 days. Principal component analysis and hierarchical clustering analysis demonstrated two distinct clusters of MFGM proteins across the different storage temperatures. Specifically, MFGM proteomic characteristics at -18 and -3 °C showed more similarities and clustered together with the 0-day samples, while -0.5 and 4 °C formed a separate cluster. Comparative analysis with the control sample (day 0) highlighted significant changes in 202 proteins, most of which played essential roles in binding, catalytic activities, and pathways related to protein, lipid, and fatty acid metabolism. Our findings highlight the superior ability of -3 °C to inhibit bacterial growth compared to 4 and -0.5 °C, and to preserve the microstructure of milk fat globules compared to -18 °C. Thus, for household storage up to 4 days, we recommend a temperature of -3±1 °C, considering temperature fluctuations in domestic refrigerators.

Quantitative label-free site-specific glycoproteomic analysis of the milk fat globule membrane protein in human colostrum and mature milk

Human milk fat globule membrane (MFGM) proteins, which are N-glycosylated, play essential roles in neonatal development and physiological health. However, the profiles and landscape changes in the site-specific N-glycosylation of human MFGM proteins during lactation remain unclear. Therefore, in this study, based on an intact glycopeptide-centred strategy, 2617 unique site-specific N-glycans of 221 MFGM glycoproteins in human colostrum and 986 unique site-specific N-glycans of 200 MFGM glycoproteins in mature milk were characterised and quantified using label-free glycoproteomics. With milk maturation, 33 site-specific N-glycans on 10 N-glycoproteins increased significantly, and 113 site-specific N-glycans on 25 N-glycoproteins decreased significantly. Moreover, human MFGM glycoproteins with core-α1,6-fucosylated structures and Lewis and sialylated branching structures play a role in the biological processes of antigen processing and presentation. This study reveals the dynamic changes in human MFGM protein N-glycosylation patterns during lactation. Meanwhile, the study deepens our understanding of site-specific N-glycosylation of human MFGM glycoproteins. The results of the study provide a background reference for the development of infant formulas.

Lectin microarray profiling demonstrates equivalent global glycosylation for whey protein ingredients enriched with α-lactalbumin and milk fat globule membrane

Human milk fat globule membrane (MFGM) and whey proteins are nutritionally and functionally valuable, with many beneficial bioactivities associated with their glycosylation. However glycosylation of milk components other than free milk oligosaccharides are underinvestigated. Whey protein concentrate (WPC) ingredients with various enrichments or depletions are used in infant formula (IF) formulations to contribute to human milk equivalence and bioactivity benefits, but their overall or global glycosylation has not been compared. We compared the global glycosylation of commercial WPC ingredients for use in various IF formulations; two MFGM-enriched WPC ingredients (high fat HF1 and lower fat HF2), an α-lactalbumin-enriched WPC (WPC Lac) which has α-lactalbumin concentration closer to human milk and significantly less β-lactoglobulin which is not present in human milk, and two base WPC ingredients (WPC 80 and WPC 35) using lectin microarray profiling. WPC Lac and WPC HF1 glycosylation were highly similar to each other and both somewhat similar to WPC 35, while WPC HF2 was more similar to the base WPC 80 ingredient. N-linked glycosylation analysis demonstrated that WPC HF1 and WPC Lac were qualitatively most similar to one another, with WPC 80 and WPC 35 having similar structures, confirming lectin microarray profiling as a valuable method to compare global glycosylation. Thus WPC Lac may be a valuable ingredient for providing equivalent glycosylation to MFGM supplementation.

The Immunological Role of Milk Fat Globule Membrane.

Human milk is the ideal food for newborns until the age of six months. Human milk can be defined as a dynamic living tissue, containing immunological molecules, such as immunoglobulins, supra-molecular structures, such as the milk fat globule membrane (MFGM), and even entire cells, such as the milk microbiota. The milk composition changes throughout lactation to fulfill the infant's requirements and reflect the healthy/disease status of the lactating mother. Many bioactive milk components are either soluble or bound to the MFGM. In this work, we focus on the peculiar role of the MFGM components, from their structural organization in fat globules to their route into the gastrointestinal tract. Immunometabolic differences between human and bovine MFGM components are reported and the advantages of supplementing infant formula with the MFGM are highlighted.

Unveiling the influence of ultrasonic-assisted lipolysis: A pilot study of short-chain fatty acid profiling in human milk based on mass spectrometry

Short-chain fatty acids (SCFAs) were recently emerging components in human milk (HM), which might have positive effect on the growth and development of infants. Thus, rapid monitoring of SCFAs in HM, as well as their physiological roles, was vitally important. In this study, the complete profiling of HM SCFA was established in order to further understand the potential association of SCFA with human milk fat globule (HMFG) membranes. A new liquid-liquid extraction coupled with crucial pretreatment of ultrasonic-assisted lipolysis was used to break HMFG membranes efficiently, releasing valeric acid (VA) and hexanoic acid (HA) from their lipid-binding forms, while unchanged acetic acid (AA), propionic acid (PA) and butyric acid (BA). Moreover, an inter-platform comparison for the rapid semi-quantification of HM SCFAs, based on gas chromatography and liquid chromatography-mass spectrometry (GC–MS/LC-MS) was also performed. Our observations linked certain features of AA and HA, such as higher intensity under electron impact ionization than under electrospray ionization, to method performance characteristics. The concentrations of most SCFAs, obtained by GC-MS and LC-MS, showed consistent trends among individuals (p < 0.0001). This study was the first report on the comparative analysis of HM SCFA profiling, with all detectable components including AA, PA, BA, VA, and HA.

Profiling of phospholipid classes and molecular species in human milk, bovine milk, and goat milk by UHPLC-Q-TOF-MS

This study aimed to identify differences of lipidome and fatty acid composition among human, bovine and caprine milk fat globule membrane (MFGM) using a lipidomics approach. In total, 11 lipid classes were determined, including PC, PE, SM, PG, PI, PS, Cer, PA, HexCer, Hex2Cer and CL. Using positive and negative ionization modes, 73 and 156 common significantly differential lipid species were identified in three typical MFGM. The oleic acid (C18:1) was the dominant fatty acid in human (28.29 ± 0.04 g/100 g) and caprine (35.71 ± 0.02 g/100 g) MFGM, whereas Palmitic acid (C16:0, 29.13 ± 0.07 g/100 g) was the dominant fatty acid in bovine MFGM. In addition, there was no significant difference in long chain fatty acids between human MFGM and caprine milk. Overall, the results provide essential insights into differential lipids characterization in MFGM, along with a theoretical basis for the design of new infant formula.

Brain-immune-gut benefits with early life supplementation of milk fat globule membrane.

The milk fat globule membrane (MFGM) has been recognized as a milk component for more than 60 years, but its exact benefits remain unknown. Research on human MFGM has revealed that the membrane holds a host of bioactive components with potential benefits for the brain–immune–gut (BiG) axis in early life. Gangliosides and sphingomyelin, components within the MFGM, have been included in infant formulas for many years. Recent advancements in dairy milk processing have allowed the successful separation of MFGM from bovine milk, enabling it to be used for supplementing infant formulas. Evidence indicates the potential benefits of MFGM in early life supplementation, including better cognitive development, reduction of infection risks, and modulation of the gut microbiome. However, larger and more robust randomized trials are needed, in addition to long‐term outcome data beyond the infancy period.

Changes in Human Milk Fat Globule Composition Throughout Lactation: A Review.

There has been a growing interest in understanding how the relative levels of human milk fat globule (MFG) components change over the course of lactation, how they differ between populations, and implications of these changes for the health of the infant. In this article, we describe studies published over the last 30 years which have investigated components of the MFG in term milk, focusing on changes over the course of lactation and highlighting infant and maternal factors that may influence these changes. We then consider how the potential health benefits of some of the milk fat globule membrane (MFGM) components and derived ingredients relate to compositional and functional aspects and how these change throughout lactation. The results show that the concentrations of phospholipids, gangliosides, cholesterol, fatty acids and proteins vary throughout lactation, and such changes are likely to reflect the changing requirements of the growing infant. There is a lack of consistent trends for changes in phospholipids and gangliosides across lactation which may reflect different methodological approaches. Other factors such as maternal diet and geographical location have been shown to influence human MFGM composition. The majority of research on the health benefits of MFGM have been conducted using MFGM ingredients derived from bovine milk, and using animal models which have clearly demonstrated the role of the MFGM in supporting cognitive and immune health of infants at different stages of growth and development.

Neurodevelopmental outcomes of healthy Chinese term infants fed infant formula enriched in bovine milk fat globule membrane for 12 months - A randomized controlled trial.

Human milk fat globule membrane (MFGM) has multifunctional health benefits. We evaluated neurodevelopment and growth of healthy term infants fed bovine milk-derived MFGM-enriched formula (MF) over 12 months. A prospective, multi-center, double-blind, randomized trial was conducted in Fuzhou, China. Healthy term infants (n=212), aged <14 days, were assigned randomly to be fed MF or a standard formula (SF) for 6 months and then switched to stage 2 MF and SF formula until 12 months. A reference group (n=206) contained healthy breastfed infants (BFR). Neurodevelopment was assessed with Bayley-III Scales. At 12 months, the composite social emotional (+3.5) and general adaptive behaviour (+5.62) scores were significantly higher in MF than SF (95% CIs 0.03 to 6.79 and 1.78 to 9.38; p=0.048 and 0.004, respectively). Mean cognitive (+2.86, 95% CIs -1.10 to 6.80, p=0.08), language (+0.39, 95% CIs -2.53 to 3.30, p=0.87) and motor (+0.90, 95% CIs -2.32 to 4.13, p=0.49) scores tended to be higher in MF than SF, but the differences between the two groups were not significant. BFR scored higher on Bayley-III than either MF or SF at 6 and 12 months. Cognitive scores were significantly higher in BFR than SF (95% CI 0.05 to 7.20; p=0.045), but not MF (p=0.74) at 6 months. Short-term memory was significantly higher in MF than SF at 12 months (95% CI 1.40 to 12.33; p=0.002). At 4 months, serum gangliosides were significantly higher in MF and BFR than SF (95% CI 0.64 to 13.02; p=0.025). Milk intake, linear growth, body mass and head circumference were not significantly different between formula-fed groups. MFGM supplementation in early life supports adequate growth, increased serum gangliosides concentration and improves some measures of cognitive development in Chinese infants.

Healthy Breastfeeding Infants Consume Different Quantities of Milk Fat Globule Membrane Lipids.

The human milk fat globule membrane (MFGM) contains important lipids for growing infants. Anthropometric measurements, milk samples, and infant milk intake were collected in a cohort of eleven healthy mother–infant dyads during exclusive breastfeeding from birth to six months. One hundred and sixty-six MFGM lipids were analysed using liquid chromatography-mass spectrometry, and the infant intake was calculated. The concentrations and intake were compared and associations between infant intake and growth characteristics explored. The lipid concentrations and infant intake varied widely between mother–infant dyads and between months one and three. The infant intake for many species displayed positive correlations with infant growth, particularly phospholipid species. The high variation in lipid intake is likely an important factor in infant growth, with strong correlations identified between the intake of many MFGM lipids and infant head circumference and weight. This study highlights the need for intake measurements and inclusion in cohort studies to elucidate the role of the human milk lipidome in infant growth and development.

Infant Formulas: A Long Story

AbstractThe ideal feeding for infants is the breast milk because it has a balanced nutritional composition, which includes all essential nutrients, in addition to many bioactive factors that contribute to the growth and development of the child, as well as to the maturation of the gastrointestinal tract. Among them are immunological factors, antimicrobials and anti-inflammatory components, digestive enzymes, various types of hormones, and growth factors. If human milk is not available, there is an indication of infant formulas that should follow the recommendations of the Codex Alimentarius of the Food and Agriculture Organization/World Health Organization (WHO). In a century of history, infant formulas have gone from a simple combination of cow milk (evaporated or condensed) and water to highly sophisticated products, elaborated by very refined technological processes to produce lactose-free, antiregurgitation, based on soy protein, hydrolyzed protein in various grades, and only amino acids formulas. The major milestones in the modification of infant formulas were the incorporation of nutrients/ingredients such as: iron, nucleotides, alpha lactalbumin, long-chain polyunsaturated fatty acids, prebiotics, probiotics, postbiotics, oligosaccharides similar to human milk, lactoferrin, and milk fat globule membrane. Many of these ingredients have shown benefits on the immunological system. Despite the technological advances, breast milk remains irreplaceable, being the gold standard for infant feeding.

Cohousing-mediated microbiota transfer from milk bioactive components-dosed mice ameliorate colitis by remodeling colonic mucus barrier and lamina propria macrophages

ABSTRACT Human milk oligosaccharides (HMOs) and milk fat globule membrane (MFGM) are highly abundant in breast milk, and have been shown to exhibit potent immunomodulatory effects. Yet, their role in the gut microbiota modulation in relation to colitis remains understudied. Since the mixtures of fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) perfectly mimic the properties and functions of HMOs, the combination of MFGM, FOS, and GOS (CMFG) has therefore been developed and used in this study. Here, CMFG were pre-fed to mice for three weeks to investigate its preventive effect on dextran sodium sulfate (DSS) induced colitis. Moreover, CMFG-treated and vehicle-treated mice were cohoused to further elucidate the preventive role of the gut microbiota transfer in colitis. At the end of the study, 16S rDNA gene amplicon sequencing, short-chain fatty acids (SCFAs) profiling, transcriptome sequencing, histological analysis, immunofluorescence staining and flow cytometry analysis were conducted. Our results showed that CMFG pre-supplementation alleviated DSS-induced colitis as evidenced by decreased disease activity index (DAI) score, reduced body weight loss, increased colon length and mucin secretion, and ameliorated intestinal damage. Moreover, CMFG reduced macrophages in the colon, resulting in decreased levels of IL-1β, IL-6, IL-8, TNF-α, and MPO in the colon and circulation. Furthermore, CMFG altered the gut microbiota composition and promoted SCFAs production in DSS-induced colitis. Markedly, the cohousing study revealed that transfer of gut microbiota from CMFG-treated mice largely improved the DSS-induced colitis as evidenced by reduced intestinal damage and decreased macrophages infiltration in the colon. Moreover, transfer of the gut microbiota from CMFG-treated mice protected against DSS-induced gut microbiota dysbiosis and promotes SCFAs production, which showed to be associated with colitis amelioration. Collectively, these findings demonstrate the beneficial role of CMFG in the gastrointestinal diseases, and further provide evidence for the rational design of effective prophylactic functional diets in both animals and humans.

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.

Qualitative and Quantitative Study of Glycosphingolipids in Human Milk and Bovine Milk Using High Performance Liquid Chromatography-Data-Dependent Acquisition-Mass Spectrometry.

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography–tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9–17.4 µg Crb/mL and 1.3–3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8–12.0 and 14.3–16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

Quantitative Phosphoproteomics of Milk Fat Globule Membrane in Human Colostrum and Mature Milk: New Insights into Changes in Protein Phosphorylation during Lactation.

Phosphorylation is a widespread posttranslational protein modification and is important in various biological processes. However, milk fat globule membrane (MFGM) phosphoproteins have not been explored systematically in human milk. Here, we used quantitative phosphoproteomics to analyze phosphorylation sites in human MFGM proteins and their differences at different stages of lactation; 305 phosphorylation sites on 170 proteins and 269 phosphorylation sites on 170 proteins were identified in colostrum and mature MFGM, respectively. Among these, 71 phosphorylation sites on 48 proteins were differentially expressed between the different stages of lactation. Osteopontin in human MFGM was the most heavily phosphorylated protein, with a total of 39 identified phosphorylation sites. Our results shed light on phosphorylation sites, composition, and biological functions of MFGM phosphoproteins in human colostrum and mature milk, and provide novel insights into the crucial roles of protein phosphorylation during infant development.

Changes in milk fat globule membrane proteome after pasteurization in human, bovine and caprine species

Milk fat globule membrane (MFGM) proteins have been shown to be very sensitive to processing. This study aims to investigate the thermos-stability of human, bovine, and caprine MFGM proteins after pasteurization. The milk fat globule size was determined using confocal laser scanning microscopy. The MFGM proteins were characterized using SDS-PAGE and label-free proteomic techniques. The results indicated that pasteurization didn’t influence the MFGs size distributions in these three species. A total of 1104, 632, and 137 proteins were identified in human, bovine, and caprine MFGM, respectively. The significantly changed proteins after pasteurization were mainly involved in lipid syntheses and secretion as well as immune response. In addition, the changes of these proteins also differed among species, and a higher thermo-sensitivity was observed in human and caprine MFGM proteins than bovine MFGM proteins. In sum, pasteurization affected MFGM protein composition in different extent among three species.