Fat Globule Membrane Research Articles

Milk fat globule membrane attenuates acute Salmonella typhimurium infection induced mouse model of colitis and ameliorates intestinal immunity

Research investigating the effects of milk fat globule membranes (MFGM) on Salmonella typhimurium-induced colitis, intestinal barrier integrity, and gut-associated immune responses remains scarce. This study aimed to investigate the potential of dietary MFGM to alleviate S. typhimurium-induced colitis in mice. Male C57BL/6J mice were administered S. typhimurium to induce colitis and were then subjected to dietary interventions with either bovine MFGM (BMFGM) or goat MFGM (GMFGM). Both BMFGM and GMFGM mitigated S. typhimurium-induced colitis, as evidenced by increased body weight, decreased mRNA levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) in the colon, reduced intestinal permeability, and increased mRNA expression of tight junction-associated genes (Claudin-1 and ZO-1). Additionally, there was a reduction in the number of S. typhimurium in the mesenteric lymph nodes and an improvement in CD4 T cell activation status. Furthermore, liver and spleen injury was reduced, and serum levels of pro-inflammatory cytokines (IFN-γ, IL-1β, IL-4, IL-2, IL-6, and TNF-α) were decreased by both type of MFGM. Overall, BMFGM and GMFGM effectively alleviated S. typhimurium-induced colitis and associated hepatic and splenic injuries.

Emulsion Structural Remodeling in Milk and Its Gelling Products: A Review.

The fat covered by fat globule membrane is scattered in a water phase rich in lactose and milky protein, forming the original emulsion structure of milk. In order to develop low-fat milk products with good performance or dairy products with nutritional reinforcement, the original emulsion structure of milk can be restructured. According to the type of lipid and emulsion structure in milk, the remolded emulsion structure can be divided into three types: restructured single emulsion structure, mixed emulsion structure, and double emulsion structure. The restructured single emulsion structure refers to the introduction of another kind of lipid to skim milk, and the mixed emulsion structure refers to adding another type of oil or oil-in-water (O/W) emulsion to milk containing certain levels of milk fat, whose final emulsion structure is still O/W emulsion. In contrast, the double emulsion structure of milk is a more complicated structural remodeling method, which is usually performed by introducing W/O emulsion into skim milk (W2) to obtain milk containing (water-in-oil-in-water) W1/O/W2 emulsion structure in order to encapsulate more diverse nutrients. Causal statistical analysis was used in this review, based on previous studies on remodeling the emulsion structures in milk and its gelling products. In addition, some common processing technologies (including heat treatment, high-pressure treatment, homogenization, ultrasonic treatment, micro-fluidization, freezing and membrane emulsification) may also have a certain impact on the microstructure and properties of milk and its gelling products with four different emulsion structures. These processing technologies can change the size of the dispersed phase of milk, the composition and structure of the interfacial layer, and the composition and morphology of the aqueous phase substance, so as to regulate the shelf-life, stability, and sensory properties of the final milk products. This research on the restructuring of the emulsion structure of milk is not only a cutting-edge topic in the field of food science, but also a powerful driving force in promoting the transformation and upgrading of the dairy industry to achieve high-quality and multi-functional dairy products, in order to meet the diversified needs of consumers for health and taste.

Microbiota, metabolic profiles and immune biomarkers in infants receiving formula with added bovine milk fat globule membrane: a randomized, controlled trial.

Few studies have evaluated the effects of milk fat globule membrane (MFGM) on microbiota and immune markers in early infant nutrition. In this double-blind randomized study, infants (7-18 days of age) received either bovine milk-based infant formula (Control) or similar formula with an added source (5 g/L) of bovine MFGM (INV-MFGM) for 60 days. A reference group received mother's own human milk over the same period (HM). Oral and stool samples were collected (Baseline and Day 60) to evaluate microbiota, immune markers, and metabolites. At Day 60, stool bacterial diversity and richness were higher in formula groups vs HM, as were Bifidobacterium bifidum and B. catenulatum abundance. Compared to HM, stool pH was higher in Control, while acetate, propionate, isovalerate, and total short- and branched-chain fatty acids were higher in INV-MFGM. Butyrate and lactate increased for INV-MFGM from baseline to Day 60. No group differences in oral microbiota or immune markers (α- and β-defensin, calprotectin, or sIgA) were detected, although sIgA increased over time in all study groups. Added bovine MFGM in infant formula modulated stool microbiota and short- and branched-chain fatty acids compared to human milk; changes were modest relative to control formula. Overall, distinct patterns of stool metabolites and microbiota development were observed based on early nutrition. ClinicalTrials.gov, identifier NCT04059666.

Lactation stage-specific variations in health and lipid-associated milk fat globule membrane proteins in Holstein Friesian cow and Murrah buffalo

Bovine milk fat globule membrane (MFGM) proteins are crucial to calf health and human nutrition, but a knowledge gap exists for its lactation stage-specific variations in cows and buffaloes. This study employed mass spectrometry 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, besides possessing 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.

Size-dependent composition and in-situ structure analysis of the milk fat globule membrane in buffalo milk

This study provides insights into the composition and in-situ structures of the milk fat globule membrane (MFGM) in buffalo milk with different fat globule sizes (0.55 μm and 8.04 μm). Small fat globules contained a higher amount of MFGM proteins and polar lipids, while the content of MFGM components (except for ADPH) was greater per unit membrane area in large fat globules. A total of 386 differentially expressed proteins were identified, with many of the most differentially expressed proteins being associated with human diseases, such as cancer and elderly diseases. The coverage of sphingomyelin and certain MFGM proteins (BTN and PAS 6/7) on large fat globules was higher, potentially linked to the presence of larger, irregular lipid rafts and patch-shaped glycocalyxes, respectively. These findings suggest that small fat globules are suitable for nutritional fortification of buffalo milk, while large fat globules are appropriate for the preparation of functional dairy-based ingredients.

Neuroprotective effects of whey and buttermilk-based formulas on a DSS-induced colitis murine model.

Inflammatory bowel disease is a gut-brain axis disorder that comprises chronic inflammatory conditions affecting the gastrointestinal tract, where alterations in the mood of patients are common. Gut-brain axis is a bidirectional communication that link gut and brain. The close association between inflammatory bowel disease and neuroinflammation has far-reaching implications, as is increasingly recognized as a contributing factor to neuropsychiatric and neurodegenerative diseases. The increasing prevalence and high economic cost, together with the loss of life quality of people suffering from these diseases, point to the need to find alternatives to alleviate them. Exploring new therapeutic avenues prompts us to consider the potential benefits of milk fractions, taking advantage of the use of dairy by-products, such as whey and buttermilk. This study examines the impact of cow's whey- and buttermilk-based formulas supplemented with bovine lactoferrin and milk fat globule membrane on the expression of cytokines, as well as on the components of immune and serotonergic system of the brain in a murine model of DSS-induced colitis. Our results show the potential of these dairy by-products, especially whey, as functional foods in ameliorating neuroinflammation and safeguarding the central nervous system function amid the neurological complications induced or concomitant with intestinal inflammatory processes.

An absolute quantification method for selected MFGM proteins in infant formula using targeted proteomics

Milk fat globule membrane (MFGM) proteins are receiving increased attention due to their reported benefits for human health, particularly in infant populations. Challenges exist in MFGM protein quantification due to their low quantities, complex chemistry, interactions with other matrix components, and the high matrix complexity. In this study, a subset of four MFGM proteins were selected as relevant targets for identification and quantification in an infant formula (IF) matrix: butyrophilin, mucin 1, xanthine dehydrogenase/oxidase, and perilipin 2. An analytical protocol for absolute quantification of these four MFGM proteins in IF was successfully developed and validated. Additionally, the feasibility to apply the method in selected MFGM ingredients was also demonstrated. The method demonstrated good performance; high accuracy and robustness, low LOQ and uncertainty, while controlling the matrix effect. This study represents a report of an analytical method to quantify selected MFGM proteins in IF.

Buttermilk treatment strategies to obtain MFGM-enriched isolates and comprehensive characterization of their lipid and protein components

The extraction of Milk Fat Globule Membrane (MFGM) from dairy by-products, particularly buttermilk, involves a highly intricate procedures to obtain a bioactive isolate with multiple health benefits. This study delves into a laboratory ultrafiltration-based method enabling the acquisition of a concentrated MFGM isolate. Pre-ultrafiltration treatments, designed to clarify the sample, such as casein coagulation and the addition of sodium citrate, were examined to evaluate their impact on the lipid and protein components of the MFGM. Treatments combining rennet coagulation with centrifugation steps, as well as those combining rennet with sodium citrate, yielded highly pure MFGM isolates exhibiting a substantial concentration of polar lipids and a rich content of MFGM proteins. This laboratory investigation offers valuable insights into the potential industrial-scale applications of MFGM isolation and purification.

An innovative method for fractionating the milk fat globule membrane and the major buttermilk proteins

Buttermilk (BM) is a by-product of butter manufacturing that shares a similar composition to skim milk but contains a higher concentration of phospholipids due to milk fat globule membrane (MFGM) fragments released during cream churning. Despite its high added-value components, MFGM is underutilized mainly due to the challenge in separating its components from the other constituents of buttermilk. Our work has focused on the use of calcium phosphate particles, called hydroxyapatite (HA), to extract and separate the different components from buttermilk. This study investigated the impact of various physicochemical parameters (temperature, ionic strength, and pH) on the adsorption of a mixture of micellar casein (CM), whey proteins, and MFGM from BM onto HA surfaces. A box-Behnken design was utilized to predict optimal physicochemical conditions for selectively separating components in buttermilk. Ionic strength (IS) played a crucial role, with low IS having a minimal positive influence and high IS negatively influencing adsorption. pH influenced adsorption by altering protein and HA surface charges. The optimal conditions for adsorption were an IS of 100 mM for a pH of 7, for 90 % of CM, 37 % of β-lactoglobulin, 11 % of α-lactalbumin and 7 % of MFGM. Desorption experiments using citrate and alkaline pH showed promising results for CM recovery, with citrate efficiently releasing the adsorbed CM. Additionally, MFGM were separated from whey proteins through selective precipitation at a pH of 4.0, allowing sedimentation of MFGM (100 %) while keeping whey proteins soluble. The presence of β-lactoglobulin in the protein fraction of MFGM (31 %) and CM (37 %) was attributed to pasteurization, which denatured a portion of β-lactoglobulin and led to its attachment to MFGM surfaces and interaction with CM. This protocol produces a nearly pure fraction of MFGM with a simple method which is suitable for industrial production, representing significant valorization for the dairy sector.

Comparative analysis of milk fat globule membrane proteins between porcine and human colostrum by a label‐free proteomics approach

This study aimed to investigate the similarities and differences in milk fat globule membrane (MFGM) proteins from porcine colostrum (PC) and human colostrum (HC) based on a label‐free proteomics approach. In total, 3901 and 4027 MFGM proteins were identified in PC and HC, respectively, including 3452 common proteins. Similar biological functions and pathways were found in two groups, whereas the quantities and types of MFGM proteins involved were different. This study compared the differences between MFGM proteins in PC and HC, effectively expanding the MFGM proteome and its bioinformatic database and providing new insights for the exploration of porcine milk.

Effect of phosphatidylcholine regioisomerism on lateral segregation of milk sphingomyelin in bilayer membranes

Milk fat globule membrane (MFGM) promotes the lateral phase separation of milk lipids and stabilizes the fat globules in milk. The composition and structures of lipids have a significant impact on physicochemical properties of MFGM, which in turn influences the digestion and absorption of milk lipids. Phospholipids (PL), sphingolipids, and cholesterol are the major lipid constituents of MFGM. While the effects of the head-group and structure of the fatty acids (FAs) on membrane properties are commonly studied, little is known on the impact of PL regioisomerism. The present study investigated the impact of phosphatidylcholine (PC) regioisomerism on lateral segregation of milk-sphingomyelin (milk-SM) as well as the influence on the interaction of milk-SM with ceramide and cholesterol in simulated membrane systems. The regioisomer pairs of four molecular species PC 16:0/18:1n-9, PC 16:0/18:2n-6, PC 16:0/18:3n-3, and PC 16:0/20:4n-6 were included in this study. The lateral segregation was determined using lifetime analysis of trans-parinaric acid (tPA) fluorescence. Thermostability of the domains was detected using steady-state anisotropy of tPA. Our results demonstrated a clear impact of PC regioisomerism on membrane properties. PC regioisomers having the unsaturated FAs at the sn-2 position enhanced the lateral segregation of milk-SM with and without the presence of ceramide and cholesterol compared to the regioiosmers having 16:0 at the sn-2 position. Furthermore, the characteristics i. e. the acyl chain length and degree of unsaturatin of sn-2 FA of the PCs had a major impact on the milk-SM gel phase and the intermolecular forces between milk-SM and ceramide/cholesterol. This work is the first investigation showing the effect of PL regioisomerism on milk-SM domains, which might have significant influence on functional properties of MFGM.

Human milk fat globule delivers entrapped probiotics to the infant's gut and acts synergistically to ameliorate oxidative and pathogenic stress

The human milk fat globule membrane (hMFGM) and Lactobacillus modulate the infant's gut and benefit health. Hence, the current study assesses the probiotic potential of Lactiplantibacillus plantarum (MRK3), Limosilactobacillus ferementum (MK1) isolated from infant feces, and its interaction with hMFGM during conditions mimicking infant digestive tract. Both strains showed high tolerance to gastrointestinal conditions, cell surface hydrophobicity, and strong anti-pathogen activity against Staphylococcus aureus. During digestion, hMFGM significantly exhibited xanthine oxidase activity, membrane roughness, and surface topography. In the presence of hMFGM, survival of MRK3 was higher than MK1, and electron microscopic observation revealed successful entrapment of MRK3 in the membrane matrix throughout digestion. Interestingly, probiotic-membrane matrix interaction showed significant synergy to alleviate oxidative stress and damage induced by cell-free supernatant of Escherichia coli in Caco-2 cells. Our results show that a probiotic-encapsulated membrane matrix potentially opens the functional infant formula development pathway.

Milk Fat Globule Membrane Is Associated with Lower Blood Lipid Levels in Adults: A Meta-Analysis of Randomized Controlled Trials.

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide, with dyslipidemia being a significant risk factor. This meta-analysis provides a comprehensive evaluation of the impact of bovine dairy-derived milk fat globule membrane (MFGM) supplementation on blood lipid profiles in adults. A systematic search was conducted across various databases up until March 2024, resulting in the inclusion of 6 trials with a total of 464 participants. The findings indicated that MFGM phospholipid supplementation may significantly reduce total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol levels. A combined analysis of the effects on TC, LDL, and triglycerides (TG) revealed a significant overall reduction in these markers. However, no significant increase or reduction was observed on high-density lipoprotein (HDL) and TG levels. Overall, MFGM phospholipid intake may significantly decrease the level of TC and LDL, while no significant changes in TG and HDL were observed. These results suggest that MFGM supplementation could be a promising dietary intervention for improving lipid profiles in adults. Nonetheless, further research is warranted to confirm these results and to better understand the potential variability in the impact of MFGM on blood lipid levels.

Improvement in probiotic intestinal survival by electrospun milk fat globule membrane-pullulan nanofibers: Fabrication and structural characterization

Studies have demonstrated the protective effect of milk fat globule membrane (MFGM) on probiotics in harsh environments. However, currently, there are no reports on the encapsulation of probiotics using MFGM. In this study, MFGM and pullulan (PUL) polysaccharide fibers were prepared by electrostatic spinning and used to encapsulate probiotics, with whey protein isolates (WPI)/PUL as the control. The morphology, physical properties, mechanical properties, survival, and stability of the encapsulated Lacticaseibacillus rhamnosus GG (LGG) were studied. The results showed that the MFGM/PUL solution had significant effects on pH, viscosity, conductivity, and stability. Electrostatic spinning improved the mechanical properties and encapsulation ability of the polymer formed by MFGM/PUL. LGG encapsulated in MFGM/PUL nanofibers survived rate was higher than WPI/PUL nanofibers in mimic intestinal juice, which could be attributed to the phospholipid content contained in MFGM. These results demonstrate that MFGM is a promising material for probiotic encapsulation, providing an important basis for the potential use of MFGM/PUL nanofibers as a robust encapsulation matrix.

The Role of Food Matrices Supplemented with Milk Fat Globule Membrane in the Bioaccessibility of Lipid Components and Adaptation of Cellular Lipid Metabolism of Caco-2 Cells.

This study aimed to evaluate the digestive efficiency of food matrices supplemented with milk fat globule membrane isolated from buttermilk (BM-MFGM), using the INFOGEST in vitro digestion protocol hyphenated with the assessment of the digested material on the lipid profile of the Caco-2 cell culture model. First, we examined lipid profiles in food matrices supplemented with BM-MFGM and their subsequent digestion. The results showed distinct lipid profiles in different food matrices and micellar fractions. The presence of BM-MFGM lipids changed the cellular lipid profiles in Caco-2 cell cultures, with diverging contents in cholesteryl esters, triacylglycerides, and neutral lipids depending on the micellar food matrix factor. Hierarchical clustering analysis revealed patterns in cellular lipid responses to micellar stimuli, while volcano plots highlighted significant changes in cellular lipid profiles post-treatment. Thus, this study underscores the importance of in vitro digestion protocols in guiding food matrix selection for bioactive ingredient supplementation, elucidating intestinal epithelium responses to digested food stimuli.

Vitamin D3 formation in milk by UV treatment – Novel insights into a rediscovered process

Vitamin D3 is essential for several functions in the human body and the demand is usually covered by natural reactions in skin with UV radiation delivered by the sun. But living beyond a latitude of 35° can lead to a lack of sufficient exposition to the deciding wavelength. Here, many countries fortify their milk prophylactically with artificial vitamin D3. However, the precursor molecule of vitamin D3 (7-deydrocholesterol) is already naturally located in the milk fat globule membrane. Thus, this study deals with the transformation of the naturally occurring 7-dehydrocholesterol into vitamin D3 through UV treatment of the milk - a mechanism that was observed a century ago only indirectly. Different parameters such as temperature (10 - 50°C), fluid flow regimen (turbulent vs. laminar thin film, i.e., 0.6 mm) and wavelength (254, 280 and 313 nm) were investigated in this study for their efficiencies. The UV dose of each experiment was measured with chemical actinometry delivering the actually applied dose reaching the milk. Thus, the connection between applied UV dose and generated vitamin D3 content in the milk measured quantitively with LC-MS/MS was evaluated here that both were not possible a hundred years ago. The experimental results revealed that temperature generally promotes the vitamin D3 formation at 254 nm. Further, a turbulent flow is not as efficiently treated as a laminar thin film flow that is as narrow as 0.6 mm. As expected from absorbance spectra of the precursor molecule 7-dehydrocholesterol, 280 nm turned out to be the most efficient wavelength, followed by intermediate success through irradiation with 254 nm and almost no effect by 313 nm. Generally, it was shown that vitamin D3 concentration of milk was easily increased by UV treatment with today's technologies and that adjustment of certain physical parameters have a significant effect on the efficiency.

Gestational Diabetes Mellitus-Induced Milk Fat Globule Membrane Protein Changes of Human Mature Milk Based on TMT Proteomic Analysis

Breastfeeding by mothers with gestational diabetes mellitus (GDM) has been shown to reduce maternal insulin demands and diminish the risks of diabetes in infants, leading to improved long-term health outcomes. Milk fat globule membrane (MFGM) proteins play a crucial role in influencing the immunity and cognitive development of infants. Understanding the alterations in MFGM proteins in breastmilk from mothers with GDM is essential for enhancing their self-efficacy and increase breastfeeding rates. The objective of this study is to investigate and compare MFGM proteins in milk from mothers with GDM and without based on tandem mass tag (TMT) labeling and liquid chromatography tandem mass spectrometry (LC-MS) techniques. A total of 5402 proteins were identified, including 4 upregulated proteins and 24 downregulated proteins. These significantly altered proteins were found to be associated with human diseases, cellular processes, and metabolism pathways. Additionally, the oxidative phosphorylation pathway emerged as the predominant pathway through Gene Set Enrichment Analysis (GSEA) involving all genes.

Bovine Milk Fat Globule Membrane Supplementation and Neurocognitive Development: A Systematic Review and Meta-Analysis.

Given the limited evidence, there is no conclusive proof of the neurocognitive benefits of bovine milk fat globule membrane supplementation in infant formula. This study evaluates the neurocognitive benefits of bovine milk fat globule membrane supplementation in formula, comparing it to standard formula and assessing its noninferiority to breast milk. Data were sourced from studies published between January 2000 and March 2024 from PubMed, Cochrane Library, Web of Science, and Embase. Eight randomized controlled trials involving 1352 healthy term neonates, infants, and children up to 2 years old were included. Bovine milk fat globule membrane supplementation was significantly associated with improved cognitive development (mean difference: 3.29, 95% CI: 1.65 to 4.93, p < 0.001) and demonstrated minimal heterogeneity (I2 = 0%, p = 0.564). It showed significant improvement in executive function but not in language, motor, or social-emotional development. In non-inferiority analysis, there was no significant difference compared to breast milk regarding cognitive development. These findings support bovine milk fat globule membrane as a valuable addition to infant formula for cognitive benefits.

Milk Fat Globule Membrane-Containing Protein Powder Promotes Fitness in Caenorhabditis elegans.

Milk-derived peptides and milk fat globule membrane (MFGM) have gained interest as health-promoting food ingredients. However, the mechanisms by which these nutraceuticals modulate the function of biological systems often remain unclear. We utilized Caenorhabditis elegans to elucidate how MFGM-containing protein powder (MProPow), previously used in a clinical trial, affect the physiology of this model organism. Our results demonstrate that MProPow does not affect lifespan but promotes the fitness of the animals. Surprisingly, gene expression analysis revealed that MProPow decreases the expression of genes functioning on innate immunity, which also translates into reduced survival on pathogenic bacteria. One of the innate immunity-associated genes showing reduced expression upon MProPow supplementation is cpr-3, the homolog of human cathepsin B. Interestingly, knockdown of cpr-3 enhances fitness, but not in MProPow-treated animals, suggesting that MProPow contributes to fitness by downregulating the expression of this gene. In summary, this research highlights the value of C. elegans in testing the biological activity of food supplements and nutraceuticals. Furthermore, this study should encourage investigations into whether milk-derived peptides and MFGM mediate their beneficial effects through the modulation of cathepsin B expression in humans.

Regulating interfacial structure of fat globules based on milk fat globule membrane with milk phospholipids to improve physicochemical properties and fat digestion of infant formula emulsions

Modulation of a suitable proportion of milk fat globule membrane (MFGM) and milk phospholipid (MPL) at the lipid droplet interface could be an effective strategy to improve the physicochemical properties and digestibility of infant formula emulsion (FE) systems that contain milk protein. The results showed that the distribution of lipid droplets became more uniform, and the adsorption of MFGM and MPL with different thicknesses was observed at the interface by adding different ratios of MFGM and MPL. Meantime, the stability of the FE was significantly increased than FE prepared by only protein-coated oil core, especially FE4 prepared by adding MFGM and MPL (1:3, w/w) as membrane components, the emulsifying activity index (48.28 m2/g) and emulsion stability index (73.08 min) reached the maximum. The emulsion prepared by phospholipid (PL) encapsulation oil core (FE5) exhibited lipid droplet aggregation and poor emulsion dispersion. The results of lipid digestion indicated that FE obtained with MFGM and MPL encapsulated lipid droplets maintained more PL ring structure similar to human milk structure during gastric digestion, which in turn promotes intestinal digestion of lipids by preventing the formation of protein aggregation. At the end of intestinal digestion, FE4 exhibited the highest lipolysis rate (86.48%) and release of free fatty acids (92.05 μmol/mL). Overall, the addition of a suitable ratio of MFGM to MPL, especially MFGM/MPL (1:3, w/w), contributes to the creation of a thin PL interface structure similar to that of human milk. This could facilitate fat degradation and fatty acid release by regulating the lipid droplet interface.