Milk derived ingredients from the production of cheese and butter can be used as vehicles for nutrients. Buttermilk is a nutritious product of milk that comes from the churning of cream into butter. One of the advantages of buttermilk is that it is enriched in milk fat globule components, such as phospholipids and forms emulsions with fat when treated with high shear. The objective of this work was to explore the effects of shear on regular buttermilk and whey buttermilk in terms of liposomal aggregate size and chemical composition. The effects of microfluidization at 2000 psi and homogenization at 2000 psi/500 psi on the particle size distribution of liposomal aggregates between whey buttermilk (WBM) at pH 4.6 and 6.8 and commercial sweet buttermilk (SBM) at pH 4.60 were compared with whey protein isolate (WPI) at pH 4.6. At pH 6.80, WPI and SBM are too soluble in water to measure particle size but WBM is not as soluble. From this investigation, the mean particle diameter of the SBM aggregates at pH 4.6 decreased after the first pass through the microfluidizer and the same is true, after homogenization. SBM aggregates at pH 4.6 had a significantly larger mean particle diameter before treatments in both shear processes compared to WPI at pH 4.6 and WBM at pH 4.6 and WBM at pH 6.8 (p < 0.0001). WPI at pH 4.6 and WBM at both pH showed no significant differences in their mean particle size in both homogenized and microfluidized treatments. WPI and SBM samples resulted in significant particle diameter differences vi from before to after homogenizing at pH 4.6. SBM at pH 4.6 had significantly larger average particle diameter than WBM at pH 4.6 (p < 0.0002), WPI at pH 4.6 (p < 0.0002) and WBM at pH 6.8 (p < 0.0045) before microfluidization at pass 0. WBM and WPI across all treatments showed very similar tendencies in small particle size attributes and some similarities in protein composition. In addition, the small aggregate size of WBM is suggested to be influenced by the presence of phospholipids and thus, creating significantly smaller mean particles compared to SBM even before inducing high shear. In contrast, treated and untreated SBM differed from WBM in phospholipid composition in both homogenization and microfluidization techniques. WBM samples contained more phospholipids than SBM, whereas WPI samples contained very low concentrations of phospholipids. Through HPLC analysis, WPI, SBM, and WBM showed different profiling of the phospholipid classes. These differences may be due structural changes of the aggregates from shearing, initial thermal treatments or hydrophobic and/or protein-phospholipid interactions between the aggregates. SBM samples also exhibited different protein profiling than WBM and WPI samples. This study suggests that high shear and presence of phospholipids impact the size distribution of liposomal aggregates through structural alterations. The aggregates can be utilized as a novel ingredient and in the processing of dairy foods to deliver nutrition.

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

The milk fat globule membrane (MFGM) obtained from bovine milk is a source of bioactive compounds. In this research, the hypothesis that the MFGM possesses anticarcinogenic capacity and immune modulatory properties was tested, along with the hypothesis that processing history of the fat globules will affect the bio-functionality of the MFGM. Throughout all experiments, lipopolysaccharide (LPS) was minimized by extracting the milk using a catheter, working under aseptic conditions and avoiding contamination via reagents. To study the anticarcinogenic capacity, native MFGM was tested in vitro using two colon adenocarcinoma cell lines (HT-29, Caco-2). Stimulated cells showed a dose-dependent decrease in cell proliferation, starting at very low concentrations (1 μg/mL based on protein) and its efficiency was comparable or even superior to the commercial anticarcinogenic drugs melphalan and C2-ceramide. Cytotoxicity and apoptosis were found to be mechanisms responsible for this bioactivity, as shown by the lactate dehydrogenase enzyme activity and the increase in caspase-3 production. MFGM- immune modulatory capacity was also tested by studying its effects on splenocyte proliferation, apoptosis and cytokine production. While splenocyte proliferation was not affected when the MFGM isolate was used alone, it suppressed cell division in the presence of polyclonal activators (LPS, Concanavalin A) used to simulate inflammatory conditions. Cytokine production suggests inhibition of the splenocytes’ activation process as the mechanism behind its bioactivity. Milk heating caused a significant decrease in bioactivity, indicating that protein interactions and denaturation as well as the structural changes caused by this treatment, directly affect the MFGM bio-functionality. Partial hydrolysis (by trypsin and phospholipase-A2) caused a similar effect, suggesting that not only the phospholipids are involved in the anticarcinogenic capacity, but also, the complex mixture of proteins which form part of the MFGM complete system. In summary, the bioactivity of the MFGM extracted from milk is not limited to its anticarcinogenic but also to its immune regulatory ability, by helping control the response of the immune system when inflammatory conditions arise. However, pre-treatment of the raw material greatly affects this bioactivity, suggesting that greater care may be needed in processing of fat globules to maintain such important characteristics of their components. / Canadian Dairy Council (CDC), Ontario Dairy Council (ODC), Natural Sciences and Engineering Research Council of Canada (NSERC).

Background Observational studies have shown that early nutrition influences short- and long-term health of infants. Formula-fed infants have higher protein and energy intakes and lower intakes of several biologically active components present in human milk. Some of these are present in the milk fat globule membrane (MFGM). The aim of the present study was to examine the effects of feeding term infants an experimental low energy low protein formula supplemented with bovine milk fat globule membranes. Our hypothesis was that infants fed experimental formula (EF), compared to infants fed standard formula (SF), would have outcomes more similar to a breast-fed reference (BFR) group. Methods In a double-blinded randomized controlled trial, 160 exclusively formula-fed, healthy, term infants were randomized to receive EF or SF from &lt;2 to 6 months of age. A BFR group consisted of 80 breast-fed infants. Measurements were made at baseline, 4, 6 and 12 months of age. The EF had lower energy (60 vs. 66 kcal/100 mL) and protein (1.20 vs. 1.27 g/100 mL) concentrations, and was supplemented with a bovine MFGM concentrate. Results At 12 months of age, the EF group performed better than the SF group in the cognitive domain of Bayley Scales of Infant Development, 3rd Ed. During the intervention, the EF group had a lower incidence of acute otitis media than the SF group, less use of antipyretics and the EF and SF groups differed in concentrations of s-IgG against pneumococci. The formula-fed infants regulated their intakes by increasing meal volumes. Thus, there were no differences between the EF and SF groups in energy or protein intakes, blood urea nitrogen, insulin or growth including body fat percent until 12 months of age. Pressure-to-eat score at 12 months of age was reported lower by parents of formula-fed infants than by parents of breast-fed infants, indicating a low level of parental control of feeding in the formula-fed groups. Neither high pressure-to-eat score nor high restrictive score was associated with formula feeding. During the intervention, the EF group gradually reached higher serum cholesterol concentrations than the SF group, and closer to the BFR group. At 4 months of age, there was no significant difference in the prevalence of lactobacilli in saliva between the EF and SF groups. Conclusions Supplementation of infant formula with a bovine MFGM fraction enhanced both cognitive and immunological development in formula-fed infants. Further, the intervention narrowed the gap in serum cholesterol concentrations between formula-fed and breast-fed infants. The lower energy and protein concentrations of the EF were totally compensated for by a high level of self-regulation of intake which might, at least partly, be explained by a low level of parental control of feeding in the study population. The findings are of importance for further development of infant formulas and may contribute to improved short- and long-term health outcomes for formula-fed infants.

Konsumtion av kosttillskott i samband med träning är något som ökat i Sverige de senaste åren. Det är främst produkter med högt proteininnehåll som konsumeras, även om behovet av extra protein är under diskussion. Men det kan finnas andra ämnen med positiv påverkan på träning. Exempelvis mjölkfettkulemembran (MFGM) som är en viktig del av bröstmjölk då det bland annat verkar bidra till spädbarnets kognitiva utveckling. Senare studier på möss har visat att MFGM tycks ha positiva effekter på träning. Syftet med denna litteraturstudie var därför att undersöka om MFGM även har positiva effekter på människor i samband med träning. Artiklar togs fram genom sökning i databasen PubMed. Artiklarna skulle undersökt effekten av MFGM på människors träning för att inkluderas, samt vara randomiserade, dubbelblindade och placebokontrollerade. Totalt inkluderades fem studier. Samtliga studier visade på positiva effekter av MFGM i samband med träning, dock skiljde sig resultaten mellan studierna beroende på vilken effekt MFGM hade. Om MFGM har någon effekt på styrka är fortfarande oklart då resultaten är oeniga, däremot kan det tänkas att rörlighet och neuromuskulär utveckling förbättras av ett tillskott av MFGM, men ytterligare studier behövs för att bekräfta detta. / Consumption of dietary supplements associated with exercise has increased in Sweden the last couple of years. It is mainly products with high protein that are consumed, although the need for additional protein is questionable. However, there may be other substances with positive effects on exercise. For example milk fat globule membrane (MFGM) which is an important part of breast milk that seems to contribute to infant's cognitive development. Studies on mice have shown that MFGM seems to have positive effects on exercise. The purpose of this literature study was therefore to investigate whether MFGM also has positive effects on humans in combination with exercise. Articles for this study was selected by searching the PubMed database. The articles were included if they were studying the effect of MFGM on exercise on humans, they also had to be randomized, double blinded and placebo-controlled. A total of five studies were included. All studies showed positive effects of MFGM combined with exercise, however, the results differed between studies in terms of what kind of effect. If MFGM has any effect on strength is still unclear since the results varies. Agility and neuromuscular development may be improved by a dietary supplement of MFGM, but further studies are needed to confirm this.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19365号 / 医博第4042号 / 新制||医||1011(附属図書館) / 32379 / 新制||医||1011 / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 佐藤 俊哉, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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ABSTRACT Effects of Milk Processing on the Milk Fat Globule Membrane Constituents Xiomara E. Elías-Argote The milk fat globule membrane (MFGM) is avidly studied by many groups of scientists around the world due to its unprecedented nutritional and functional properties; however, limited research has been performed on the effects of milk processing on the chemical changes of the MFGM. Thus, this study highlights the changes that lipids and proteins undergo from the time milk leaves cow’s udders. Cooling (4 °C) was included along with subsequent pasteurization by different traditional thermal processes and cold pasteurization by pulse light ultra violet treatment. Cooling milk to 4 °C had a measureable effect on the MFGM composition, resulting in protein alterations, particularly to butyrophilin and adipophilin. Thermal treatments disturbed the native structures of molecules and increased the adsorption of milk and whey proteins on the globules, especially a-lactalbumin and b-lactoglobulin. As the heat intensity increased, the aggregation of fat globules became more pronounced due to protein interactions. Intrinsic MFGM proteins also varied in relative abundance during the processing steps. The concentrations of polar lipids did not change during processing, with the exception of phosphatidylserine, which decreased during the cooling and thermal treatments. Cold pasteurization (UV treatment) had a minimal effect on fat globules and MFGM proteins. Since the MFGM promises to deliver nutritional effects and more when included in food products, currently HTST pasteurization was shown to be the best method to process milk and obtain MFGM isolates for further supplementation.

Milk fat globule membrane surrounds the fat droplets of milk. It is a biopolymer containing primarily membrane glycoproteins and polar lipids which contribute to its properties as a possible neutraceutical. The aims of the studies were to determine if dietary milk fat globule membrane: (1) confers protection against colon carcinogenesis; and (2) promotes gut mucosal integrity while decreasing inflammation compared to diets containing corn oil or anhydrous milk fat. Aim 1. Three dietary treatments differing only in the fat source were formulated: (1) AIN-76A, corn oil; (2) AIN-76A, anhydrous milk fat; and (3) AIN-76A, 50% milk fat globule membrane, 50% anhydrous milk fat. Each diet was formulated to contain 50 g/kg diet of fat and to be identical in macro and micro nutrient content. To assess protection against colon carcinogenesis, male, weanling Fischer-344 rats were randomly assigned to one of the three dietary treatments. Animals were injected with 1,2-dimethylhydrazine once per week at weeks 3 and 4. After 13 weeks animals were sacrificed, colons were removed, and aberrant crypt foci were counted by microscopy. Rats fed the milk fat globule membrane diet (n = 16) had significantly fewer aberrant crypt foci (20.9 ± 5.7) compared to rats fed corn oil (n = 17) or anhydrous milk fat (n = 16) diets (31.3 ± 9.5 and 29.8 ± 11.4 respectively; P < 0.05). Aim 2. Male BALB/c mice were randomly assigned to one of two diets: AIN- 76A, corn oil or AIN-76A, 50% milk fat globule membrane, 50% anhydrous milk fat. After 5 weeks mice were injected with saline vehicle control or lipopolysaccharide and gavaged with dextran-FITC. To assess gut mucosal integrity and inflammation, serum samples were assayed for dextran-FITC 24 and 48 hours after gavage, and a panel of 16 cytokine concentrations was analyzed. Serum concentrations of IL-6, IL-10, IL-17, MCP-1, IFNγ, and TNFα decreased and gut permeability decreased 45% in lipopolysaccharide challenged mice fed milk fat globule membrane diet compared to control diet at 24 hours (P < 0.05). Overall, the results of these aims suggest that diets containing milk fat globule membrane are protective against colon carcinogenesis, inhibit the inflammatory response, and protect against gastrointestinal stress.