Fat Globule Membrane Research Articles

A comparison of milk fat globule membranes and whey proteomes: New insight into variation nutrient differences between Buffalo, Cow, Goat, and Yak.

In this study, the whey and milk fat globule membrane (MFGM) proteomes of buffalo, cow, goat, and yak milk were analyzed using label-free proteomic technology. Totally, 1,292 MFGM proteins and 686 whey proteins were identified from these four species, and GO analysis revealed there were specific proteins with different functions in both whey (376) and MFGM (982) proteomes. The principal component analysis showed that ALB, TF, CSN1S1, and GLYCAM1 are characteristic markers of the milk for each of the four species. Furthermore, the conserved and differential in the expression of whey and MFGM proteins across the four species were identified by limma, and subsequent KEGG pathway analysis showed that immune-related proteins are both conserved and species-specific in the four species. These results provide a deepening of the understanding of the characteristics of proteins in whey and MFGMs from these four common dairy animals and new insight into developing dairy production.

Protein Stability in Pasteurized Cow and Goat Milk using Protein Gel Electrophoresis

Milk is a nutritious beverage that contains whey, casein, and milk fat globule membrane (MFGM) proteins. Pasteurization kills pathogenic microorganisms in milk, making it safe to drink. A high heat load during processing can affect the protein quality of milk. In this study, protein gel electrophoresis (Native and SDS-PAGE) was used to view the protein profiles of retail cow and goat milk samples pasteurized at different temperatures (145, 165, or 280 °F) compared to commercially spray dried milk (356–482 °F). SDS-PAGE provided better resolution of the milk proteins compared to Native-PAGE. There were clear species differences in the MFGM proteins. This could be used to spot adulteration of goat milk with cow milk, however none of the samples showed signs of adulteration. There were no overt patterns of protein degradation with increasing pasteurization temperature. However drying reduced band intensity, especially MFGM and whey proteins in the goat milk sample. Whereas most samples came from different herds where genetics and environmental conditions varied, two of the goat milk samples were from the same herd, one fresh and one spray dried. It was evident from these two samples that spray drying can alter proteins in goat milk. Even so, if fresh milk is unavailable or unaffordable, dry milk remains a valuable protein source.

Milk fat globule membrane modulates inflammatory pathways in human monocytes: A crossover human intervention study

BackgroundIntake of high-fat foods raises postprandial plasma triglycerides and inflammatory markers, which may depend on the type of fat ingested. Dairy products are commonly consumed, but not much is known about the impact of milk fat and the milk fat globule membrane on postprandial inflammation. Here, we aimed to study the effect of milk fat with and without milk fat globule membrane and vegetable blend fat on post-prandial inflammation, with a focus on blood monocyte gene expression. MethodsWe performed a randomized, double-blind cross-over trial in 37 middle-aged healthy male and female volunteers (BMI 22-27 kg/m2). The participants consumed a meal shake containing 95.5 g of fat consisting of either a vegetable fat blend (VEGE), anhydrous milk fat (AMF, without milk fat globule membrane), or cream (CREAM, containing milk fat globule membrane). Blood monocytes were collected at 0 hours and 6 hours postprandially and used for bulk RNA sequencing and ex vivo stimulation with LPS. ResultsConsumption of all three shakes significantly decreased the percentage of classical monocytes and increased the percentages of intermediate monocytes and non-classical monocytes. No differences in these measures were observed between shakes. Using a threshold of P<0.01, 787 genes were differentially regulated postprandially between the three shakes. 89 genes were differentially regulated postprandially between AMF and VEGE, 373 genes between AMF and CREAM, and 667 genes between VEGE and CREAM, indicating that the effect of CREAM on monocyte gene expression was distinct from AMF and VEGE. Pathway analyses showed that VEGE significantly increased the expression of genes involved in inflammatory pathways, whereas this was less pronounced after AMF and not observed after CREAM. In addition, CREAM significantly down-regulated the expression of genes involved in energy metabolism-related pathways, such as glycolysis, TCA cycle, and oxidative phosphorylation, as well as HIF1 signaling. ConclusionCompared to the consumption of an anhydrous milk fat without milk fat globule membrane and a vegetable fat blend, the consumption of cream with milk fat globule membrane downregulated inflammatory pathways in blood monocytes, thus suggesting a potential inflammation inhibitory effect of milk fat globule membrane.

Milk fat globule membrane promotes brain development in piglets by enhancing the connection of white matter fiber trace

IntroductionBrain development during infancy is crucial for later health and development. Although Milk Fat Globule Membrane (MFGM) has been demonstrated to enhance brain development, further investigation is needed to determine the optimal dose.MethodsIn this study, 80 piglets aged 2 days were randomly assigned to four groups: Control group, MFGM-L (1.74 g MFGM per 100 g diet), MFGM-M (4.64 g MFGM per 100 g diet), and MFGM-H (6.09 g MFGM per 100 g diet). Daily body weight and milk intake of the piglets were recorded until 31 days postnatal. Learning and memory abilities were evaluated using the spatial T-maze test on day 15. MRI analysis was conducted to assess functional and structural changes in brain tissues. Additionally, mRNA and protein expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NTF-3) in the hippocampus and prefrontal cortex were evaluated.ResultsThe results indicated that the MFGM supplemented diet significantly improved the accuracy of the piglets in the T-maze test, with the MFGM-L group exhibiting the best performance. MRI showed no volumetric differences in the gray and white matter between the groups. However, the fractional anisotropy in the left and right hippocampus of piglets in the MFGM-L group was significantly higher than in the other three groups. Furthermore, there was a strong correlation between the accuracy of the T-maze test and hippocampal fractional anisotropy.DiscussionThe MFGM supplemented diet also increased the expression of BDNF in the cerebral cortex. However, the changes in BDNF were not consistent with the results of the T-maze test. In conclusion, adding 1.74 g MFGM per 100 g diet can significantly improve neonatal piglets’ learning and memory abilities, potentially by enhancing the connection of white matter fiber bundles in the brain.

PSVI-13 Dietary Milk Fat Globule Membrane Improves Neonatal Piglet Intestinal Architecture and Enhances Mucosal Diamine Oxidase Activity Following Lipopolysaccharide Challenge

Abstract Early life stressors alter the trajectory of gut development and impair nutrient utilization, barrier function, and animal performance throughout life. Gastrointestinal (GI) maladies increase morbidity and mortality in neonates, and dietary nutrients are essential in supporting GI function. Although maternal milk is the gold-standard for yielding developmental benefits to offspring, genetic selection in the modern sow has resulted in large litter size and milk yield potentially insufficient to achieve the high biological growth potential in sow-reared piglets. Research into milk quality has led to the identification of many bioactive nutrients important in neonatal development. Components of the milk fat globule membrane (MFGM) contribute immensely to neonatal nutrition and gut health. However, mechanisms of actions are still being defined. The objective is to define the effect of MFGM supplementation on neonatal piglet intestinal health. We hypothesize dietary MFGM will enhance piglet intestinal architecture and tight junction (TJ) protein abundance, attenuating systemic lipopolysaccharide (LPS)-induced intestinal barrier disruption. One-day-old piglets (n = 32) were assigned to soy (CON) or MFGM phospholipid supplemented diet (0.75% wt/wt) ± LPS (100 µg/kg body weight) challenge in a 2 × 2 factorial design (n = 8/diet/challenge). Piglets were fed for 21 d and 8 animals/diet received saline or LPS injections 4 h before euthanasia. Piglet weights and feed intake were recorded daily to calculate performance. Intestinal tissues were collected to assess villus height (VH) and crypt depth (CD) by hematoxylin and eosin staining. Two biomarkers of intestinal integrity, intestinal fatty acid-binding protein (I-FABP) and diamine oxidase (DAO), were measured in serum or tissue. Protein abundance of claudin-1 and occludin in ileal and colonic mucosa were detected by Western blot. Data were analyzed by 2 × 2 factorial arrangement of traetments using the PROC MIXED. Dietary MFGM had no adverse impact on growth performance or TJ proteins (P &amp;gt; 0.05). Dietary MFGM increased VH (CON: 348.8 and MFGM: 427.0 ± 26.79 µm) and decreased CD (CON: 166.3 and MFGM: 147.4 ± 8.42 µm) in ileum, thereby increasing the VH: CD ratio (CON: 2.2; MFGM: 3.1 ± 0.13; P &amp;lt; 0.05). Further, LPS treatment increased serum I-FABP by 4.4-fold and decreased jejunal mucosal DAO by 0.5-fold compared with saline treatment (P &amp;lt; 0.05). Additionally, there was a 93.1 % increase in colon DAO activity with MFGM diet compared with soy-fed pigs, regardless of LPS challenge (P &amp;lt; 0.05). Dietary MFGM improved neonatal piglet ileum architecture and increased colon DAO activity, potentially attenuating the intestinal barrier disruption following LPS challenge.

Identification and comparison of milk fat globule membrane and whey proteins from Selle Français, Welsh pony, and Tieling Draft horse mare’s milk

Horse’s milk, with a high nutritional value and few allergenic proteins, could substitute cow’s milk for infant consumption. Herein, a label-free, proteomic method was used to identify and compare milk fat globule membrane (MFGM) and whey proteins from three different horse breeds: Selle Français (SF), Welsh pony (WP), and Tieling Draft Horse (TDH). In MFGMs, 16 (SF), 66 (WP), and 45 (TDH) unique proteins were identified, which are involved in the endocytosis, ribosome, and staphylococcus aureus infection pathways, respectively. In whey, 31 (SF), 75 (WP), and 23 (TDH) unique proteins were identified, which are involved in the autophagy–animal, phenylalanine metabolism, and Vasopressin-regulated water reabsorption pathways, respectively. SF contained the lowest concentration of β-lactoglobulin, which can cause allergic reactions in humans. Our findings describe the nutritional differences and functional diversities of MFGM and whey proteins in different horse breeds, which could support developing formula more suitable for human infants.

Milk fat globule membrane: formation and transformation.

The milk fat globule membrane (MFGM) is formed by complex cell biological processes in the lactating mammary epithelial cell which result in the release of the milk fat globule (MFG) into the secretory alveolus. The MFG is bounded by a continuous unit membrane (UM), separated from the MFG lipid by a thin layer of cytoplasm. This unique apocrine secretion process has been shown in all of the mammary species so far investigated. Once the MFG is released into the alveolus there is a considerable transformation of the UM with its attached cytoplasm. This is the MFGM. The transformation is stable and expressed milk shows the same transformed MFGM structure. Again, this transformation of structure is common to all mammalian species so far investigated. However, the explanation of the transformation very much depends on the method of investigation. Transmission electron microscope (TEM) studies suggest a literal breakdown to a discontinuous UM plus cytoplasm in patches and strands, whereas more recent confocal laser scanning light microscopy (CLSM) studies indicate a separation, in a continuous UM, of two phases, one liquid ordered and the other liquid disordered. This review is designed to show that the TEM and CLSM results show different views of the same structures once certain deficiencies in techniques are factored in.

Label-free quantitative proteomic analysis of milk fat globule membrane proteins in porcine colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are nutritional components with various biological functions. This study aimed to analyze and compare MFGM proteins in porcine colostrum (PC) and porcine mature milk (PM), via label-free quantitative proteomics. In total, 3917 and 3966 MFGM proteins were identified in PC and PM milk, respectively. A total of 3807 common MFGM proteins were found in both groups, including 303 significant differentially expressed MFGM proteins. Gene Ontology (GO) analysis revealed that the differentially expressed MFGM proteins were mainly related to the cellular process, cell, and binding. The dominant pathway of the differentially expressed MFGM proteins was related to the phagosome according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. These results reveal crucial insights into the functional diversity of MFGM proteins in porcine milk during lactation and provide theoretical guidance for the development of MFGM proteins in the future.

Effects of processing on component interactions and peptide profiles of preterm infant formula based on peptidomics by Q Exactive plus analysis

The study aimed to investigate the effects of processing on the component interactions, the peptide profile as well as the bioactive peptides of preterm infant formula after digestion through EASY-nLC1200 Q Exactive Plus system. Results indicated that whey proteins and caseins transferred from serum and pellet fractions to fat fraction to recombine the milk fat globule membrane destroyed after homogenization. Protein interactions caused the changes of nitrogen distribution, protein profiles, and morphology after pasteurization and spray drying. Peptidomics analysis demonstrated that the component interactions of preterm infant formula affected the peptide profiles after digestion shown by the different peptide intensities of certain amino acid compositions and the totally different abundance of the peptides in heatmap. The hydrolysates of five groups exhibited 334, 274, 421, 214, and 265 unique peptides belonging to 144, 116, 185, 101, and 231 master accession proteins respectively. The top 8 abundant proteins identified were β-casein, Lipocln, αS1-casein, αS2-casein, κ-casein, bovine Glycosylation-dependent cell adhesion molecule, α-lactalbumin, and Albumin. The number of bioactive peptides increased after processing but were different from each other. In total, component interactions during processing of preterm infant formula could change the peptide profile and bioactive peptides after in vitro digestion.

The effect of freezing and storage temperature on the stability of the fat emulsion in cream

The article presents the results of a study of frozen cream, the purpose of which was to assess the feasibility of using various freezing and storage modes to obtain a product with a stable fat phase. The objects of the study were cream samples of various fat contents (30%, 40% and 50%). Freezing was carried out at three temperature conditions (–50 °C, —25 °C and –18 °C) followed by storage for 4 months in refrigerators maintaining temperatures of –18 °C, —25 °C and –50 °C. Before and after low-temperature treatment, the state of the fat phase of the cream and the size of ice crystals were assessed using a microscopic method. Destabilization of fat dispersion was determined by the content of destabilized fat. It has been confirmed that with increasing mass fraction of fat in cream, the degree of destabilization increases. The amount of destabilized fat in cream samples with 30, 40 and 50% fat content before freezing was 14.3%, 20.0% and 32.0%, respectively. According to the research results, it has been revealed that when the freezing temperature decreases from –18 °C to –50 °C, there is no noticeable reduction in the amount of destabilized fat. The degree of destabilization decreased when cream was moved from lower to more gentle temperature conditions. At a storage temperature of –18 °C for cream previously frozen at –50 °C and –25 °C, the amount of destabilized fat in the cream was lower by 8.0–14.0% and 20.0–25.0%, respectively, in comparison with samples frozen and stored at the same temperature. When conducting microstructural studies, it has been revealed that freezing at a lower temperature with a change in storage temperature allows obtaining ice crystals with a smoother surface, which cause less damage to the membranes of fat globules.

Bioactive Functions of Lipids in the Milk Fat Globule Membrane: A Comprehensive Review.

The milk fat globule membrane (MFGM) is a complex tri-layer membrane that wraps droplets of lipids in milk. In recent years, it has attracted widespread attention due to its excellent bioactive functions and nutritional value. MFGM contains a diverse array of bioactive lipids, including cholesterol, phospholipids, and sphingolipids, which play pivotal roles in mediating the bioactivity of the MFGM. We sequentially summarize the main lipid types in the MFGM in this comprehensive review and outline the characterization methods used to employ them. In this comprehensive review, we sequentially describe the types of major lipids found in the MFGM and outline the characterization methods employed to study them. Additionally, we compare the structural disparities among glycerophospholipids, sphingolipids, and gangliosides, while introducing the formation of lipid rafts facilitated by cholesterol. The focus of this review revolves around an extensive evaluation of the current research on lipid isolates from the MFGM, as well as products containing MFGM lipids, with respect to their impact on human health. Notably, we emphasize the clinical trials encompassing a large number of participants. The summarized bioactive functions of MFGM lipids encompass the regulation of human growth and development, influence on intestinal health, inhibition of cholesterol absorption, enhancement of exercise capacity, and anticancer effects. By offering a comprehensive overview, the aim of this review is to provide valuable insights into the diverse biologically active functions exhibited by lipids in the MFGM.

Milk fat globule membrane regulates the physicochemical properties and surface composition of infant formula powders by improving the stability of the emulsion

The milk fat globules in infant formula (IF) are encapsulated by a component known as milk fat globule membrane (MFGM). However, it is currently unclear whether the improved emulsion stability of MFGM can have a profound effect on the finished IF. Therefore, this study investigated the effects of MFGM on the particle size, stability, rheology, and microstructure of emulsions prepared by dairy ingredients via wet mixing. Further, IF were processed using such emulsions, the physicochemical properties, surface composition of the powders were examined. The results showed that MFGM reduced the particle size of the emulsion, increased the viscosity, and improved the microstructure of the MFGM. Furthermore, MFGM reduced the moisture content of the powder, increased the glass transition temperature, and reduced the presence of surface fat. In conclusion, the addition of MFGM enhance the finished powder stability by improving the emulsion stability prepared during IF manufacturing.

Improving the Physicochemical Stability of Soy Phospholipid-Stabilized Emulsions Loaded with Lutein by the Addition of Sphingomyelin and Cholesterol: Inspired by a Milk Fat Globule Membrane.

The emulsifying performance of glycerophospholipids alone is inferior to proteins, etc., while the sphingomyelin (SM) and cholesterol (Chol) naturally existing in biological membranes could interact with glycerophospholipids to influence the polar lipid arrangement. Inspired by the natural membranes, the effect of SM and Chol on the physicochemical stability of soy phospholipid (SPL)-stabilized emulsions during storage or under environmental stresses was determined. The results indicated that the addition of SM and/or Chol could improve the storage stability of the emulsions and protective effect on lutein significantly (p < 0.05). Except for UV irradiation, the addition of Chol significantly improved the stability of the emulsions against acid, salt, and heat. The strong intermolecular hydrogen bonds and condensed assembly formed by SM and Chol contributed to the best stability of SPL + SM + Chol-stabilized emulsions. The results gave insight into improving the emulsifying properties of glycerophospholipids with SM and Chol.

Bovine Milk Proteome: Milk Fat Globule Membrane Protein Is the Most Sensitive Fraction in Response to High Somatic Cell Count.

The impacts of high milk somatic cell count (SCC) on different milk fractions are not well understood. In this study, proteins in milk exosomes, milk fat globule membrane (MFGM), and whey from cows with low (<105 cells/mL, CG) and high SCC (>5 × 105 cells/mL, HSG) were identified using a tandem mass tag proteomic approach. In total, 1568, 2160, and 1002 proteins were identified, with 65, 552, and 98 proteins being altered by high SCC in exosomes, MFGM, and whey, respectively. With high SCC, the exosome marker (ACTB) was increased in the exosomes of HSG. The main MFGM proteins (BTN1A1, PLIN3, FABP3, and MFGE8) and functional proteins (MUC1, IGSF5, TLR5, and CD36/14) were decreased, while the lipid/energy metabolism-related proteins were increased in the MFGM of HSG. The glycolysis-related proteins were increased in the whey of HSG. Also, the host defense/inflammation-related proteins were changed in three fractions under high SCCs. MFGM was the most sensitive fraction to a high SCC, followed by whey. These findings provide guidance for the early detection of unhealthy mammary glands.

Effects of dietary forage-to-concentrate ratio and forage type on milk phospholipids and fatty acid composition of polar lipids

The effects of grass silage and red clover silage on milk fatty acid (FA) composition are extensively studied, but little is known of their effects on minor lipid constituents of milk fat globule membrane. We investigated the effects of forage:concentrate (FC) ratio in grass silage-based diets and forage type (grass silage vs. red clover silage) on selected molecular species of milk phospholipids (PL) and the FA composition of PL. Ten multiparous Nordic Red cows were offered following dietary treatments: grass silage-based diets containing 70:30 (HG) or 30:70 (LG) FC ratio or a red clover silage-based diet (RC) comprising 50:50 FC ratio on a dry matter basis. The most abundant molecular species within the phosphatidylcholines was 16:0-18:1 phosphatidylcholine that was increased by 18% in HG compared with LG milk. Dietary treatments did not affect the relative proportion of 18:1-18:1+18:0-18:2 phosphatidylethanolamine that was the most prevalent species (ca. 44%-45%) in that class. We identified the d18:1-22:0 sphingomyelin as the most abundant sphingomyelin species that tended to increase in HG milk compared with LG. The FC ratio did not affect the relative proportions of saturated FA nor monounsaturated FA in PL, but the proportion of cis-9 18:1 was elevated in HG versus LG milk, whereas the proportion of 18:2n-6 was 50% higher in LG versus HG milk. The RC diet increased monounsaturated FA and 18:3n-3 levels in PL compared with grass silage-based diets and decreased the relative proportion of saturated FA. However, the RC diet did not affect the relative proportion of polyunsaturated FA in PL, although red clover silage typically increases the proportion of polyunsaturated FA in milk fat. This study provides valuable knowledge of the minor lipid components in milk on species level in relation to common feeding strategies in high-forage systems.

Why breast milk matters

Midwives understand the benefits of breastfeeding, both for the infant and mother. However, the biochemical and physiological reasons for the superiority of breast milk are less widely known. This is, in part, because of the extraordinary complexity of breast milk's composition. Its key components include nucleotides, which benefit gut and immune development, human milk oligosaccharides, which promote an optimal gut biome, lipids in the milk fat globule membrane, which promote gut health and brain development, immunoglobulins, which modulate the infant's immune system, and an optimum protein content, which is high in the first 2 weeks after birth but decreases thereafter. A greater awareness and understanding of the mechanisms behind the benefits of breastfeeding could help midwives to have informed discussions with parents and potentially contribute to improving the UK's breastfeeding rates. Growing understanding of breast milk's unique composition may also help infant formula manufacturers drive innovation and improve the formulation of their products.

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.

Relative quantitative proteomic profiling of bovine colostrum and transition milk at onset of lactation

Colostrum, the first postpartum secretion of the mammary gland, plays a vital role in the nutrition, development, and immunity of a newborn calf. We characterised the protein profile as milk transitions from colostrum to transition milk over the first 5 days of lactation, collecting samples on d0, d2 and d5 after calving. Protein concentration decreased significantly after d0 (colostrum) before stabilising. Principal component analysis revealed samples clustered by day postpartum, with a limited effect from parity. In total, 471 proteins were identified across all samples; 199 protein groups showed altered relative abundance by day of lactation (fold change ≥2, P < 0.05), but none were altered significantly by parity. Higher relative levels of immune proteins were found in colostrum, and milk fat globule membrane proteins were found to increase over early lactation. Our findings provide a better understanding of the dynamics of the protein profile of bovine milk in early lactation.

The impact of dairy matrix structure on postprandial lipid responses.

This review presents evidence related to the postprandial responses after consumption of dairy products focusing on the effect of the dairy matrix and lipid response, which was also presented as part of a speech at the Nutrition Society Winter Conference, January 2023. The key findings are that the dairy product(s) that differentiate from others in the postprandial TAG response are products with a semi-solid structure. There were no differences in lipid responses between cheese and butter. The main factors viscosity, fat globule size and milk fat globule membrane do not seem to explain the effect of the dairy matrix in the acute postprandial response. In summary, it is very difficult to investigate the effects of the dairy matrix per see and with the few studies conducted to date, no clear cause and effect can be established. Future research should focus on the semi-solid dairy matrix, and studies investigating specifically the yoghurt matrix are warranted.

Milk fat globule membrane protects Bifidobacterium longum ssp. infantis ATCC 15697 against bile stress by modifying global transcriptional responses

The milk fat globule membrane (MFGM) can protect probiotic bacteria from bile stress. However, its potential mechanism has not been reported. In this study, the viability, morphology and gene transcriptional response of Bifidobacterium longum ssp. infantis ATCC 15697 (BI_15697) stressed by bile salts with or without MFGM were investigated. It was shown that MFGM alleviated the reduction in BI_15697 population induced by 0.2% porcine bile stress and restored the population to the control levels. MFGM ameliorated the shrunken, fragmented appearance and irregular morphology of BI_15697 and maintained cell integrity disrupted by bile stress. RNA-sequencing results showed that MFGM increased transport of glucose and raffinose and decreased that of branched-chain amino acids (BCAA) in the presence of bile salts. MFGM stimulated the expression of genes involved in the synthesis of raffinose in galactose metabolism and the metabolism of BCAA, suggesting that MFGM stimulated the accumulation of raffinose and BCAA in the presence of bile. In addition, MFGM stimulated the expression of 2 bile efflux transporters under bile stress. Together, the multifactorial response helps BI_15697 excrete bile salts and maintain cellular integrity in response to bile stress. This study proposes a mechanism for the protection of BI_15697 against bile salt stress by MFGM, thereby providing a molecular basis for its application in incorporation of probiotics.