Milk Fat Globule Membrane Proteins Research Articles

Proteomic Analysis of Milk Fat Globule Membrane Protein Modulation of Differently Expressed Proteins in Lactobacillus plantarum under Bile Salt Stress.

Tolerance to bile stress is a crucial property for lactic acid bacteria (LAB) to survive in the gastrointestinal tract and exert their beneficial effects. Whey powder enriched with milk fat globule membrane proteins (M-WPI) as a functional component is protective for strains under stress conditions. The current study investigated the key mechanisms of action involved in Lactobacillus plantarum (L. plantarum) CGMCC 23701 survival in the presence of bile and the protective mechanism of M-WPI. According to proteomic analysis (proteomics), there could be several reasons for the greater protective effect of M-WPI. These include promoting the synthesis of fatty acids and peptidoglycans to repair the structure of the cell surface, regulating the metabolism of carbohydrates and amino acids to release energy and produce a range of precursors, enabling the expression of the repair system to repair damaged DNA, and promoting the expression of proteins associated with the multidrug efflux pump, which facilitates the exocytosis of intracellular bile salts. This study helps us to better understand the changes in proteome of L. plantarum CGMCC 23701 under bile salt stress and M-WPI protection, which will provide a new method for the protection and development of functional LAB.

New insights into the destabilization of fat globules in ultra-instantaneous UHT milk induced by added plasmin: Molecular mechanisms and the effect of membrane structure on plasmin action

Residual plasmin activity in whole ultra-instantaneous UHT (UI-UHT) milk causes rapid fat rise during storage, seriously affecting consumers' purchase intentions. In this work, the molecular mechanisms underlying fat destabilization in whole UI-UHT milk by added plasmin were investigated based on the hydrolysis behavior of interfacial proteins. By using SDS-PAGE and peptidomic analysis, we found that the hydrolysis of interfacial proteins by plasmin led to a decrease in the amount and coverage of interfacial proteins and an increase in zeta-potential value, causing the flocculation and coalescence of fat globules. Moreover, the hydrolysis pattern varied in different categories of interfacial proteins by plasmin. In total, 125 peptides in all samples were identified. Plasmin tended to hydrolyze most major milk fat globule membrane (MFGM) proteins into protein fragments (>10 kDa) rather than peptides (<10 kDa). In contrast, peptides derived from caseins were more preferentially identified within a relatively short incubation time. It was the co-hydrolysis of caseins and some major MFGM proteins as anchors that destroyed the stability of MFGM. Furthermore, studies on the effect of trilayer membrane structure remaining at the interface on the hydrolysis rate of major MFGM proteins by plasmin revealed that ADPH and BTN were very sensitive to plasmin action, while PAS 7 was very resistant to plasmin action. Overall, membrane structure reduced the susceptibility of some major MFGM proteins to plasmin and provided protective effects. Therefore, this study provided important insights into the hydrolysis behavior of interfacial proteins in whole UI-UHT milk induced by plasmin.

Differences in proteomic profiles and immunomodulatory activity of goat and cow milk fat globule membrane

This study aimed to clarify the composition and bioactivity differences between goat and cow milk fat globule membrane (MFGM) protein by proteomic, and the immunomodulatory activity of MFGM proteins was further evaluated by using mouse splenic lymphocytes in vitro. A total of 257 MFGM proteins showed significant differences between goat and cow milk. The upregulated and unique MFGM proteins in goat milk were significantly enriched in the positive regulation of immune response, negative regulation of Interleukin-5 (IL-5) secretion, and involved in nucleotide-binding oligomerization domain (NOD)-like receptor signaling. The contents of IL-2 and Interferon-γ in the supernatant of spleen lymphocytes treated with goat MFGM proteins were much higher than those of IL-4 and IL-5, suggesting a Th1-skewed immune response. These results revealed that goat MFGM proteins could possess better immunomodulatory effects as compared to cow milk. Our findings may provide new insights to elucidate the physiological functions and nutritional of goat milk.

Changes in milk fat globule membrane proteins along lactation stage of Laoshan dairy goat

The milk fat globule membrane (MFGM) is a complex structure with numerous functions, and its composition is affected by many factors. There have been few systematic investigations on goat MFGM proteome profiling during lactation. Individual milk samples from 15 healthy dairy goats were obtained at six lactation time points for investigation of the MFGM proteome using both data-independent acquisition (DIA) and data-dependent acquisition (DDA) proteomics techniques combined with multivariate statistical analysis. Using the DIA method, 890 variably abundant MFGM proteins were discovered throughout the lactation cycle. From 1 to 240 d, butyrophilin subfamily 1 member A1, lipoprotein lipase, perilipin-2, and adipose triglyceride lipase were upregulated, while APOE, complement C3, clusterin, and IgG were downregulated. Furthermore, from 1 to 90 d, annexin A1, annexin A2, and antithrombin-III were downregulated, then upregulated by d 240. Albumin had a high degree of connectedness, indicating that it was a key protein, according to protein–protein interaction research. Overall, our findings gave new insights into the biological features of MFGM protein in goat milk throughout lactation, which may aid in the creation of specialized MFGM products and infant formula.

Phosphoproteomics Revealed Differentially Expressed Sites and Function of the Bovine Milk Fat Globule Membrane in Colostrum and Mature Milk.

One type of large and intricate post-translational modification of milk proteins that has significant biological implications is phosphorylation. The characterization of phosphoproteins found in the bovine milk fat globule membrane (MFGM) is still mostly unknown. Here, label-free phosphoproteomics was used to identify 94 phosphorylation sites from 54 MFGM phosphoproteins in bovine colostrum (BC) and 136 phosphorylation sites from 91 MFGM phosphoproteins in bovine mature milk (BM). αs1-Casein and β-casein were the most phosphorylated proteins in bovine colostrum. In bovine mature milk, perilipin-2 was the protein with the greatest number of phosphorylation sites. The results show that bovine colostrum MFGM phosphoproteins were mainly involved in immune function, whereas bovine mature MFGM phosphoproteins were mainly involved in metabolic function. Plasminogen and osteopontin were the most strongly interacting proteins in colostrum, whereas perilipin-2 was the most strongly interacting protein in bovine mature milk. This work demonstrates the unique alterations in the phosphorylation manner of the bovine MFGM protein during lactation and further expands our knowledge of the site characteristics of bovine MFGM phosphoproteins. This result confirms the value of MFGM as a reference ingredient for infant formula during different stages.

Common proteins analysis of different mammals' mature milk by 4D-Label-Free

The milk proteins from samples of 13 different animals were identified utilizing 4D-Label-Free proteomics technology, leading to the identification of a substantial number of proteins. Among the various samples, Chinese people (CHP) milk proteins exhibited the highest count, with 1149 distinct proteins. Simultaneously, we identified common proteins present in these animal milk. It's notable presence in goat milk contributes to enhancing infant infection resistance, showcasing the beneficial role of lactoperoxidase. Galectin-3 binding protein (Gal-3BP) and tetraspanin in human milk are significantly higher than those in other animals, which determine the prominent antiviral effect of human milk and the important processes related to cell transduction. Furthermore, human milk, camel milk, goat milk and sheep milk proved to be rich sources of milk fat globule membrane (MFGM) proteins. The insights obtained from this study can serve as a foundational framework for exploring the role of different animal milk proteins in disease treatment and the composition of infant formula.

Formation by high power ultrasound of mimicking human fat emulsions stabilized with milk fat globule membrane

Ultrasound (US) is an innovative food processing technique that has gained significant interest for its role in modifying proteins. Our study focused on how different US time exposure influence the stability, interfacial structure, and rheological characteristics of whey protein infused with milk fat globule membrane (MFGM). Results indicated that a 9-min ultrasound treatment (US-9) of protein, yielding an emulsion with particle size of 338.3 nm and ζ-potential of −48 mV, was optimal for enhancing functional properties of the emulsion. The US-9 emulsion exhibited different surface rheological properties compared to other emulsions, revealing a heterogeneous MFGM stabilization interface predominantly made of MFGM fragments, free phospholipids, and proteins. The US-9 emulsion surface covered more phospholipids, indicating improved emulsion persistence, based on interfacial protein and phospholipid content calculations and microstructure observation. While the US-9 emulsion did not affect fat digestion, the US-6 min treatment resulted in an optimal fatty acid release rate. Further experimentation may be necessary to elucidate the intricate relationship between ultrasound duration and fat digestion. These findings have leaded to a better understanding of the emulsification capabilities of MFGM materials, as well as a foundation to improve infant formula quality.

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.

Identification and anti-oxidative potential of milk fat globule membrane (MFGM)-derived bioactive peptides released through in vitro gastrointestinal digestion

This study investigated the stability of milk fat globule membrane (MFGM) protein under simulated gastrointestinal conditions using an in vitro enzymatic digestion method. The optimal hydrolysis conditions were determined by monitoring the changes in particle size and zeta-potential of MFGM protein hydrolysates over time. Furthermore, the distribution of small molecular weight peptides with antioxidant activity was explored through DEAE-52 combined with in vitro cell experiments. Two novel antioxidant peptides (TGIIT and IITQ) were identified based on molecular docking technology and evaluated their potential scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS+) radicals. TGIIT and IITQ also demonstrated remarkable abilities in promoting mitochondrial biogenesis and activating Keap1/Nrf2 signaling pathway, which can effectively counteract skeletal muscle dysfunction induced by oxidative stress. Thus, MFGM-derived antioxidant peptides have the potential to be employed in food to regulate muscle protein metabolism and alleviate sarcopenia.

Белки мембраны молочной жировой глобулы. 8. Белок, связывающий жирные кислоты (FABP)

This article continues a series of reviews about the structure and functions of milk fat globule membrane (MFGM) proteins in cow’s milk. This installment introduces publications on the biochemistry of fatty acid binding protein (FABP), which is a polypeptide component of MFGM. The article gives a brief history of its discovery, as well as profiles the proteins that bind hydrophobic ligands and describes the methods of FABP identification in MFGM preparations. Other aspects of FABP studies include: isoforms and modern nomenclature; amino acid sequence and reactions to post-translational modification; spatial organization; mechanisms for binding fatty acids to lipids; well-investigated and hypothesized physiological functions; the effect of haplotypes on milk fatty acids, yield, and protein content.

Proteomic analysis of milk fat globule membrane proteins in mature human milk of women with and without gestational diabetes mellitus

Milk fat globule membrane proteins (MFGMP) in human milks have positive effects on infant’s health. As gestational diabetes mellitus (GDM) causes variations in MFGMP, it is essential to understand the effects of GDMon MFGMP. This study aims to investigate and compare the MFGMP (>3 months postpartum) of GDM and non-GDM (NGDM) women using four-dimensional-data-independent-acquisition proteomics technology. Principal component analysis shows significant differences in the MFGMP of GDM and NGDM women. A total of 4747 MFGMP were identified in maturehuman milk of GDM and NGDM women. Among these proteins, 174 differentially expressed proteins (DEPs) were identified in MFGM of GDM and NGDM women. Albumin (FC = 7.96) and transthyretin (FC = 2.57) which are related to insulin resistance and involved in thyroid hormone synthesis, are significantly up-regulated in MFGMP of GDM mothers indicating insulin resistance, imbalance of glucose homeostasis and poor glucose metabolism might persist in postpartum period.

Characterization and Comparison Analysis of Milk Fat Globule Membrane Proteins between Human and Porcine Milk.

This study aimed to explore the differences in milk fat globule membrane (MFGM) proteins between human milk (HM) and porcine milk (PM) using a label-free quantitative proteomic approach. A total of 3920 and 4001 MFGM proteins were identified between PM and HM, respectively. Among them, 3520 common MFGM proteins were detected, including 956 significant differentially expressed MFGM proteins (DEPs). Gene ontology (GO) enrichment analysis showed that the DEPs were highly enriched in the lipid metabolic process and intrinsic component of membrane. Kyoto Encyclopedia of Genes and Genomes pathways suggested that protein processing in the endoplasmic reticulum was the most highly enriched pathway, followed by peroxisome, complement, and coagulation cascades. This study reflects the difference in the composition of MFGM proteins between HM and PM and provides a scientific and systematic reference for the development of MFGM protein nutrition.

Comparative Site-Specific O-Glycosylation Analysis of the Milk Fat Globule Membrane Proteome in Donkey Colostrum and Mature Milk.

Donkey milk fat globule membrane (MFGM) proteins are a class of membrane-bound secreted proteins with broad-spectrum biofunctional activities; however, their site-specific O-glycosylation landscapes have not been systematically mapped. In this study, an in-depth MFGM O-glycoproteome profile of donkey milk during lactation was constructed based on an intact glycopeptide-centered, label-free glycoproteomics pipeline, with 2137 site-specific O-glycans from 1121 MFGM glycoproteins and 619 site-specific O-glycans from 217 MFGM glycoproteins identified in donkey colostrum and donkey mature milk, respectively. As lactation progressed, the number of site-specific O-glycans from three glycoproteins significantly increased, whereas that of 11 site-specific O-glycans from five glycoproteins significantly decreased. Furthermore, donkey MFGM O-glycoproteins with core-1 and core-2 core structures and Lewis and sialylated branch structures may be involved in regulating apoptosis. The findings of this study reveal the differences in the composition of donkey MFGM O-glycoproteins and their site-specific O-glycosylation modification dynamic change rules during lactation, providing a molecular basis for understanding the complexity and biological functions of donkey MFGM protein O-glycosylation.

Does lactoferrin, free, encapsulated or in dairy matrices, maintain its antibacterial activity after in vitro digestion?

Milk is a source of active compounds with defensive properties, such as lactoferrin and milk fat globule membrane proteins. These proteins generate bioactive peptides in the gastrointestinal tract and it is known that industrial processing can modify their susceptibility to digestion. However, does lactoferrin maintain its antibacterial activity after passing through the gastrointestinal tract? The aim of this study was to evaluate the effect of technological treatments, encapsulation and in vitro digestion on lactoferrin antibacterial activity against Listeria monocytogenes. The results showed that the gastric digest of free lactoferrin presented greater effect against L. monocytogenes than the intestinal digest; although less than undigested lactoferrin. Alginate-lactoferrin microbeads allowed the release of lactoferrin into the intestine, protecting it from pepsin, although it was not sufficient to maintain its antibacterial activity to the intestine. However, homogenization and pasteurization favoured the antibacterial activity of dairy formulas supplemented with lactoferrin and of their digests.

Proteomic Comparisons of Caprine Milk Whole Cream Buttermilk Whey and Cheese Whey Cream Buttermilk.

Buttermilk, a potential material used to produce milk fat globule membrane (MFGM), is obtained as a byproduct of butter making from milk whole cream and cheese whey cream. This study investigated the effects of rennet and acid coagulation on the protein profiles of buttermilk rennet-coagulated whey (BRW) and buttermilk acid-coagulated whey (BAW). They were compared to those of whey cream buttermilk (WCB). Rennet coagulation was more efficient in removing casein, while retaining more IgG and lactoferrin than acid coagulation. BRW had more MFGM than BAW. Butyrophilin, xanthine dehydrogenase, and mucin1 were significantly higher (P < 0.05) in BRW, while fatty acid-binding protein 3 was enriched in BAW. KEGG analysis showed that complement and coagulation cascades had the greatest differences, and the abundance of proteins involved in this signaling pathway in BRW and BAW was higher, suggesting their potential anticoagulant and anti-inflammatory activity. BAW had higher apolipoprotein A4 and transcobalamin 2, which are essential carriers for transporting long-chain fatty acids and vitamin B12 from the intestine to the blood. Therefore, BAW intake might improve lipids and vitamin B12 absorption. This study can help deepen the understanding of protein composition of MFGM-enriched whey and facilitate the production of MFGM proteins for infants and old-aged populations.

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.

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

Hypothyroidism has been found to have an impact 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 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. On the other hand, the zeta-potential of AHM was greater than that of NHM. The SDS-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 impact on the quality of human milk.

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.

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.

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.