Buttermilk: Much more than a source of milk phospholipids

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1 Buttermilk: Much more than a source of milk phospholipids V. Conway,* S.F.Gauthier,* and Y. Pouliot* *STELA Dairy Research Center, Institute of Nutrition and Functional Food (INAF), Université Laval, Québec, Canada, G1V 0A6 Research Center on Aging, Health, and Social Services Center, University Institute of Geriatrics of Sherbrooke, Sherbrooke, Canada, J1H 4C4 Key words: buttermilk, cholesterol-lowering activity, milk fat globule membrane, minor lipids, sphingomyelin Introduction The minor components of the milk fat globule membrane (MFGM) have been associated with various health benefits. As concluded in previous reviews (Dewettinck et al., 2008; El-Loly, 2011; Vanderghem et al., 2011; Contarini and Povolo, 2013), there is much evidence in support of cholesterol-lowering, anti-inflammatory, chemotherapeutic and anti-neurodegenerative effects of MFGM lipids, mainly through the action of the polar lipid portion (i.e., phospholipids). Not only lipids, but also minor proteins associated with the MFGM are thought to be important bioactive components. Sweet buttermilk, the co-product of butter making, is particularly rich in MFGM components. Processes of isolating and purifying buttermilk MFGM components have been examined in several studies over the past decade (Astaire et al., 2003; Morin et al., 2007; Morin et al., 2006; Rombaut et al., 2006). The number of scientific publications on the subject of buttermilk components has quadrupled over the past 20 years (PubMed). So far, the vast majority of these studies have focused on either fractionating or concentrating various MFGM components, primarily minor lipids. Meanwhile, the biological effects of the whole sweet buttermilk matrix remain poorly understood. A few well-controlled clinical trials provide support for the buttermilk health-benefits hypothesis (Baumgartner Conway, Gauthier, and Pouliot. doi: /af Implications Resulting from churning of cream, sweet buttermilk is a source of unique bioactive molecules capable of modulating cell signaling, lipid transport, metabolism, and immunity. Several in vitro and in vivo results provide support for the claim of the cholesterol-lowering action of buttermilk components. Clinical evidence now confirms the cardiovascular health benefits of short-term consumption of whole buttermilk, due most likely to its phospholipid content. Buttermilk consumption represents a natural way to manage blood pressure and blood lipids among healthy subjects. Peptides and polar lipids originating from the milk fat globule membrane are most likely responsible for the improved blood pressure and blood chemistry resulting from buttermilk consumption. et al., 2013; Conway et al., 2013, 2014). The purpose of this review is to present recent advances in sweet buttermilk utilization and examine its status as a high-value by-product of dairy processing. The health benefits of sweet buttermilk MFGM components will be discussed in view of recent examples from in vitro, in vivo, and clinical studies. The Churning Process and its Consequences: The Unique Composition of Buttermilk Buttermilk can be obtained through acidification of cream (i.e., cultured buttermilk) or by the churning of cream into butter (i.e., sweet buttermilk). Cultured buttermilk, the commercially available form of buttermilk, will not be discussed in this review. Sweet buttermilk, referred simply as buttermilk in the following discussion, is the aqueous fraction resulting when cream is churned to make butter. Churning causes the separation of cream (an oil-in water emulsion) into two distinct phases, an aqueous phase called buttermilk, and an oily phase or dairy fat concentrate (i.e., butterfat). This separation happens as a result of mechanical destabilization of the initial emulsion. Contact with air and repetitive physical collisions disrupt the thin membrane that surrounds and stabilizes the triglyceride globules, causing globule coalescence (i.e., aggregation) and ultimately, formation of a solid phase (i.e., butter) from which buttermilk is separated simply by draining. Buttermilk is very similar to skim milk in many ways. For example, more than 80% of its proteins are major milk proteins, namely caseins and whey proteins. However, its fat composition differs substantially from that of skim milk, in amount and composition, due to the presence of MFGM-derived substances. Close to 20% of buttermilk proteins are of MFGM origin. These proteins are solubilized during churning, as illustrated schematically in Figure 1. In fact, any treatment causing disruption or breakdown of the MFGM will affect the distribution of the minor components of milk-origin in the final matrix. For example, polar lipids account for about 0.9% of the total fat content of cream; they make up more than 4.5% of buttermilk fat, but only 0.2% of butterfat (Contarini and Povolo, 2013). The MFGM residues in buttermilk are responsible for the unique nutritional and technological properties of this dairy co-product. Table 1 shows the ratio of polar lipids to total fat in various dairy products. The MFGM is a thin structure (10 to 50 nm) but also a complex biophysical system that represents 2 to 6% of the milk fat globule total mass (Lopez, 2011). In whole milk, fat globules are dispersed throughout the continuous serum phase due to the emulsifying capacity of the MFGM components, and coalescence is prevented through electrostatic and steric repulsions (Lopez, 2011). To fulfill the function of ensuring the physicochemical stability of milk, the MFGM is composed of a highly complex mixture of proteins and polar and non-polar lipids, which represent more than 90% of its dry 44 Animal Frontiers

2 Figure 1. Schematic representation of the different processing steps used in the manufacturing of butter, with their impact on milk matrix structure. (I) Pasteurized cream is churned in order to induce a phase inversion; (II) the milk fat globule membrane (MFGM) is ruptured; (III) the expelled liquid (i.e., sweet buttermilk) resulting from churning is drained, and butter grains are further processed. mass (Spitsberg, 2005). These constituents are arranged in a heterogeneous tri-layer structure secreted by the epithelial cells of the mammary glands of cattle. The MFGM thus consists principally of 1) a single inner layer of polar lipids, 2) a dense protein coat, and 3) an outer bilayer of polar lipids and embedded specific proteins (Vanderghem et al., 2010). Proteins are reported to represent 25 to 70% of the MFGM total mass, but those minor MFGM proteins account only for 1 to 4% of all milk proteins (El-Loly, 2011). As noted by authors of other reviews, this high variability is due primarily to the method used for membrane isolation and analysis. More than 130 specific proteins have now been identified using contemporary proteomic approaches (Affolter et al., 2010). However, resolution by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) reveals a small number of major bands. These correspond to the major MFGM proteins, namely butyrophilin (BTN), xanthine oxidase/dehydrogenase (XO/XDH), mucin-like glycoproteins MUC1 and MUC15, cluster of differentiation 36 (CD36), lactadherin (PAS6/7), adipophilin (ADPH), and fatty-acid-binding protein (FABP). Buttermilk is particularly rich in BTN, XO/XDH, PAS6/7, ADPH, and FABP (Affolter et al., 2010). BTN is by far the most abundant, making up about 40% of MFGM protein (Lopez, 2011). Figure 2A provides an example of the major bands resolved by SDS-PAGE, while Figure 2B illustrates the heterogeneous distribution of proteins in the MFGM tri-layer structure. Due to their low abundance in the whole milk matrix, MFGM proteins are interesting from a bio-functional perspective rather than for their basic nutritional value. Although the primary function of milk is nutritional, important bioactive molecules are delivered to the newborn at the same time to promote healthy growth. It is not surprising that MFGM acts as a vehicle for the transport of important signaling lipids and proteins to the gastrointestinal tract of newborns, to promote the development of their immune and nervous systems as well as proper development of their intestinal and metabolic functions (Lopez, 2011). The biological activities associated with the major MFGM proteins have been reviewed recently (Dewettinck et al., 2008) and will not be discussed further in the present paper. The MFGM lipids are a complex mixture containing around 70% neutral lipids (primarily triglycerides, di-glycerides, mono-glycerides, cholesterol esters, and free cholesterol) and 26 to 30% polar lipids, as presented in Table 2. The MFGM contains about 60 to 70% of all milk polar lipids (Vanderghem et al., 2010). These are largely responsible for the stability of fat globules in the milk oil/water emulsion, due to their amphiphilic nature. Milk polar lipids are divided into two major classes, namely Table 1. Phospholipid contents of different dairy products. Adapted from MacGibbon and Taylor, Dairy product Composition (%, wt/wt) Whole milk Skim milk Cream Buttermilk Lipids (a) Phospholipids (b) Ratio [(b/a) X 100] Apr. 2014, Vol. 4, No. 2 45

3 Figure 2. (A) Separation of buttermilk proteins (six samples) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (gel concentration 13.5%, STD = molecular weight standard; Conway et al., 2010). The identified proteins are (top to bottom): mucin-like glycoprotein 1 (MUC1), xanthine oxidase/dehydrogenase (XO/XDH), cluster of differentiation 36 (CD36), butyrophilin (BTN), lactadherin (PAS6/7), caseins (CN), lactoglobulin (LB), and lactalbumin (LA). (B) Schematic representation of the multilayer heterogeneous structure of the milk fat globule membrane, according to Lopez (2010, 2011) glycerophospholipids and sphingolipids. Glycerophospholipids are composed of a polar head (e.g., ethanolamine, choline, serine, or inositol attached to a phosphate group) and a glycerol backbone to which C14 C24 fatty acid chains are esterified to form the hydrophobic tail of the molecule (Figure 3A). Milk fat is typically composed of short-to-medium-length fatty acid (C4 C14), mostly saturated. However, these fatty acids (C4 C14) are fairly absent in milk glycerophospholipids, which are primarily composed of medium-to-long unsaturated fatty acids (C18:1, C18:2, and C18:3) (Dewettinck et al., 2008). MFGM matrix fluidity is due to these longer unsaturated fatty acids (Lopez, 2010). Like glycerophospholipids, Table 2. Average lipid composition of bovine MFGM. Based on Keenan and Mather, 2006; Lopez, 2010; and MacGibbon and Taylor, Lipid class Total lipids (%) Neutral Triglycerides 62 Di-glycerides 9 Mono-glycerides Trace Sterols 0.2 to 2.0 Esters 0.1 to 0.3 Free fatty acids 0.6 to 6.0 Polar Phospholipids 26 to 31 - Phosphatidylethanolamine 31.1 to 42.0* - Phosphatidylcholine 19.2 to 34.5* - Sphingomyelin 17.9 to 34.5* - Phosphatidylinositol 4.7 to 6.2* - Phosphatidylserine 2.8 to 8.5* * % of total phospholipids. sphingolipids are composed of a backbone (i.e., sphingosine) to which a long saturated fatty acid chain is attached to form a molecule called a ceramide. Different units are added to ceramides, namely a sugar unit to form a cerebroside, an oligosaccharide residue to form a ganglioside, or a phosphate group to form sphingomyelin, as shown in Figure 3B (MacGibbon and Taylor, 2006). It is interesting that about 97% of all MFGM sphingomyelins are composed of medium-chain or long-chain saturated fatty acids (i.e., C16:0, C22:0, C23:0, and C24:0) with high melting temperatures. More rarely, 17% or more of milk sphingomyelins contain C23:0 fatty acids (Dewettinck et al., 2008). This uncommon characteristic of milk sphingomyelins suggests a specific function, more likely biological, well beyond its simple structural role in the MFGM. Sphingomyelin is the only phosphorus-containing sphingolipid in milk. Thus, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin are the major milk phospholipids, representing about 90% of all MFGM polar lipids (Table 2). The MFGM sphingomyelins are now known to interact with cholesterol to form organized rigid domains called lipid rafts, which float in a disorganized fluid matrix of glycerophospholipids (Lopez, 2010). This ability to gather cholesterol into ordered domains depends greatly on the saturation of the fatty acid tails. Lipid rafts have important functions in cell signaling, transduction, and intracellular trafficking (Küllenberg et al., 2012). Modification of MFGM lipid composition may therefore greatly affect the biological activity of buttermilk. Differences in the size and pattern of sphingomyelin-containing lipid rafts have been reported (Lopez, 2011). The biological significance of milk phospholipids will be discussed in detail in the following section. Health Benefits Associated with Minor Lipids in Milk Health benefits of milk phospholipids have been suggested in association with cardiovascular disease, inflammation and cancer since the early 1900s (Contarini and Povolo, 2013). A recent review examines more than 46 Animal Frontiers

4 Figure 3. Structure of milk phospholipids: (A) Glycerophospholipids and (B) sphingomyelin. 100 papers dealing with the health benefits of dietary phospholipids, primarily glycerophospholipids (Küllenberg et al., 2012). However, sphingolipids are also recognized as highly bioactive compounds capable of exerting their benefits at low concentrations (El-Loly, 2011). Their metabolites (i.e., ceramides or sphingosine-1-phosphate) are important lipid messengers controlling cell growth and proliferation, apoptosis, and angiogenesis as well as immune function (Contarini and Povolo, 2013). First discovered in 1884 par J.L.W. Thudichum in studies of the brain, sphingolipids are important structural lipids found in small amounts in various food products including dairy, meat, eggs, and vegetables (Vesper et al., 1999). However, the nature of the sphingolipid backbone, attached fatty acids, and head groups varies considerably depending on the food source. For example, plant sphingolipids are composed mainly of cerebrosides, while eggs contain almost no sphingolipid, and MFGM sphingolipids are represented almost entirely by sphingomyelin (Vesper et al., 1999; Lopez, 2010). Of all MFGM constituents, sphingomyelin is by far the most interesting bioactive component and the most studied. As mentioned, sphingomyelin is unusually rich in very long saturated fatty acids compared with glycerophospholipids, in which fatty acids over 20 carbons long are almost absent. It is this characteristic that allows its close interaction with cholesterol. The lipid rafts thus formed may provide more fragile points in the MFGM structure (Figure 4). These weakened domains may contribute to the biological functions of MFGM during digestion (Lopez, 2010), possibly by providing binding sites for digestive enzymes and facilitating lipid transport. They might also provide docking sites on microorganisms and abnormal cells, thus promoting phagocytosis and apoptosis, and facilitate the delivery of important fatty acids (e.g., omega-3 fatty acids) to cell-signaling pathways. In newborns, absorbed phospholipids may be incorporated into cell membranes, all over the body, thus modifying their composition and the structure of their lipid rafts, resulting in modulation of various metabolic pathways. As noted in recent reviews (Küllenberg et al., 2012; Contarini and Povolo, 2013), in vitro and in vivo studies suggest a wide range of biological activities of milk phospholipids, including anti-cancer and anti-stress, as well as potential for disease prevention. An exhaustive description of the health benefits attributed to MFGM polar lipids will not be provided here. Furthermore, most studies of milk minor lipids have focused on specific purified components rather than on the whole MFGM fraction, and these will not be described in depth either. In the section below, we discuss studies published over the past five years on the positive actions of bovine MFGM, particularly on neurological and immune functions, the proliferation of cancerous cells, and cardiovascular risk, with emphasis on whole buttermilk fraction. From In Vitro Data to Clinical Studies Sphingomyelin plays an important structural role in all cellular membranes, but particularly in the brain cells. In view of their functions in brain myelination, dietary phospholipids have been studied as effective carriers of essential fatty acids to promote brain health (Küllenberg et al., 2012). For example, a commercially available fraction of bovine MFGM phospholipids (sphingomyelin 8.4%, phosphatidylethanolamine 8.3%, and phosphatidylcholine 1.9%) has been shown to reduce endoplasmicreticulum-stress-induced cell death in vitro (Nagai, 2012). Endoplasmic reticulum stress has been linked to many neurodegenerative disorders, including Alzheimer s disease. The protective action was attributed to modulation of cell signaling, measured as protein kinase C activation, as well as stimulation of autophagocytosis. Moreover, bovine MFGM sphingomyelin has been shown to promote neurobehavioral development in premature babies in an eight-week clinical trial (n = 24) using either milk sphingolipid or a control treatment with egg sphingomyelin (Tanaka et al., Apr. 2014, Vol. 4, No. 2 47

5 Figure 4. Representation of sphingomyelin (SM)-cholesterol lipid rafts in the milk fat globule membrane and their biological significance. Adapted from Lopez (2010). 2013). In this study, serum levels of sphingomyelin, and all measured neurodevelopment parameters, were positively associated with the MFGM sphingomyelin treatment. The products of sphingomyelin digestion are also known to affect cell proliferation, apoptosis, and senescence, based mostly on in vitro studies. Native MFGM isolates from raw milk have been shown to inhibit the proliferation of HT-29 colon cancer cells in vitro (Zanabria et al., 2013). In view of these effects, the anti-cancer potential of MFGM concentrate obtained from buttermilk ultrafiltration has been investigated in vivo (Snow et al., 2010). In this study, rats (n = 16 or 17 per group) were fed diets containing either 0.03% (wt/wt) sphingomyelin in corn oil, 0.03% (wt/wt) sphingomyelin in anhydrous milk fat, or MFGM from buttermilk (0.11% sphingomyelin, wt/wt). Using the aberrant crypt foci (ACF) model, the buttermilk product was found to protect against colon cancer. The authors suggested, based on evidence in the literature, that sphingomyelin was specifically responsible for the reduction in the number of apoptosis-resistant cells, predictive of colon cancer. The mechanism proposed was related to sphingomyelin cell-signaling functions, particularly in cell growth, development, and differentiation. To the best of our knowledge, no clinical evidence of the anticancer potential of buttermilk consumption is actually available in literature, due likely to the difficulty of carrying out long-term cohort studies on humans. Protective effects of dietary sphingolipids against toxins and microorganisms have been proposed in the literature, the active mechanism most often suggested being competition for binding sites on intestinal mucosal cells. An in vitro study of the anti-infective action of buttermilk and whey cream MFGM concentrates, obtained by microfiltration, has been published recently (Fuller et al., 2013). The buttermilk concentrate was found to be the more potent inhibitor of infection of monkey kidney cells by rotavirus. The authors attributed this anti-infective difference between buttermilk and whey cream to variation of their MFGM lipid composition. As discussed earlier in this paper, sphingomyelin-cholesterol lipid rafts might 48 Animal Frontiers constitute major binding sites for rotaviruses and other microorganisms, and any factor that influences milk fat globule composition (e.g., processing, cow diet, breed, and stage of lactation) might also affect the composition, size, and number of these rafts (Lopez, 2011) and thus, their bioactivities. Clinical evidence supporting a positive action of MFGM-enriched milk on immunity in healthy children (n = 182) has been published by Veereman-Wauters et al. (2012). Using a validated questionnaire, the authors found a significant reduction in short ( < 3 days) febrile episodes and days of fever, in association with 4-month consumption of MFGM-enriched milk. Using the Achenbach System of Empirically Based Assessment (i.e., standardized questionnaires evaluating anxiety, depression, somatic complaints, social problems, cognitive problems, attention difficulties, rule-breaking behavior, and aggressive behavior), positive changes in behavior among the treatment group were also noted. Although no precise mechanisms were proposed, the authors concluded that dietary MFGM supplementation may provide clinically significant improvement of both immune and central nervous system functions. The cholesterol-lowering action of milk phospholipids is one of the most studied health benefits related to buttermilk minor components consumption. A paper published in 1979 (Howard and Marks, 1979) was the first to report a lower concentration of cholesterol in plasma of subjects consuming cream compared with those consuming butter. This fascinating observation led to further in vivo research, which linked the effect first observed by Howard and Marks to milk sphingolipids. Indeed, as shown schematically in Figure 5A, the absorption of cholesterol depends almost entirely on its incorporation into and dissolution in mixed micelles (Ros, 2000). Furthermore, to be absorbed by intestinal enterocytes, cholesterol must be free for desorption from these micelles. The solubility of cholesterol in mixed micelles depends greatly on the concentration and nature of the phospholipids present, as well as on the availability of bile salts (Ros, 2000). This dependence on micellar solubility explains the cholesterol-lowering action of sphingolipids. For example, through interaction with long saturated fatty acid chains, cholesterol forms molecular complexes with sphingomyelin in the intestinal lumen, resulting in a mutual inhibition of their absorption (Figure 5B). Slow and incomplete digestion of sphingomyelin in the proximal segment of the intestine, an important site for lipid absorption, enhances formation of complexes and reduces cholesterol availability (Eckhardt et al., 2002). The effect of raw-cream buttermilk and pasteurized-cream buttermilk concentrates, as obtained by microfiltration, on the micellar solubility of cholesterol in vitro has been investigated (Conway et al., 2010). These authors observed a significant reduction ( 57.1%) of cholesterol solubility in the presence of buttermilk compared with control. Although the bioactive component was not clearly identified in this study, the formation of sphingomyelin-cholesterol complexes was proposed as an important mechanism modulating cholesterol solubility. It is interesting that the effect was much smaller (-17.0%) with pasteurized-cream buttermilk. Processing of cream is known to cause the aggregation of multiple proteins on the MFGM surface, as well as modification of MFGM composition (Lopez, 2011). Processing of raw cream or buttermilk (e.g.,

6 pumping, air inclusion, homogenization, and temperature changes) likely affects the ability of the MFGM surface to interact with enzymes, proteins, lipids, or microorganisms. The authors also observed unexpectedly a greater decrease in cholesterol solubility when the buttermilk was not fractionated or concentrated through microfiltration. The effect on solubility was greater for whole buttermilk, followed by buttermilk microfiltration retentate, and finally microfiltration permeate. In an in vitro study of the antioxidant properties of buttermilk, similar losses due to fractionation were observed (Conway et al., 2012). Clinical evidences of the cholesterol-lowering properties of buttermilk consumption have been published recently (Baumgartner et al., 2013; Conway et al., 2013). Short-term consumption significantly reduced plasma cholesterol and triglyceride concentrations in a double-blind, randomized, placebo-controlled crossover study on healthy subjects (n = 34) with mild-hypercholesterolemia (Conway et al., 2013). Participants underwent both treatments consecutively in random order, consuming either 45 g of buttermilk (sphingomyelin = 0.6% of total fat) or 45 g of a macronutrient-matched placebo (sphingomyelin < 0.1% of total fat) daily for 4 weeks. The participant then switched to the other treatment (i.e., buttermilk or placebo) for an additional 4-week period. A reduction in serum LDL-cholesterol ( 5.6% compared with placebo) was observed in subjects with greater LDL-cholesterol at screening. The authors attributed this improvement in blood lipid profile to the inhibitory action of sphingomyelin on intestinal absorption of cholesterol. The highly significant reduction in blood triglycerides observed in the buttermilk group (-10.7% compared with placebo) could be attributed to a reduction of triglyceride hepatic synthesis, resulting from the increase in polar lipids due to buttermilk consumption, as proposed in a rodent model by Reis et al. (2013). Baumgartner et al. (2013) studied the effect of the food matrix on cholesterol absorption in a 12-week placebo-controlled study (n = 97). Subjects were assigned to a control group, a group that consumed one egg per day, or a group that consumed one egg yolk in 100 ml of buttermilk per day. Among women, daily consumption of one egg resulted in increased plasma cholesterol and LDL-cholesterol compared with the control group, while no difference was seen between the control and egg yolk in buttermilk groups. The authors suggested that buttermilk components, mainly sphingomyelin, interfered with intestinal absorption of cholesterol. These two studies are, to the best of our knowledge, the only available clinical investigations of the impact of whole buttermilk on plasma lipids. Clinical trials conducted using purified sources of sphingolipids have been published in recent years. An investigation of the acute (n = 29) and long-term (n = 20) benefits of sphingolipid-enriched buttermilk on the blood chemistry of healthy humans indicated no significant effect on fasting and postprandial plasma lipid concentrations (Ohlsson et al., 2009, 2010). In a randomized crossover study of the cholesterol-reducing potential of sphingomyelin (of unspecified source) in 10 healthy subjects, the supplement did not affect blood lipid profile or cholesterol absorption and synthesis (Ramprasath et al., 2013). Studies involving less than 30 subjects with relatively normal LDL-cholesterol concentrations likely limit the possibility of observing significant effects. Furthermore, in Ohlsson et al. (2009, 2010) studies, the beverages used were sterilized at 143 C, which very likely affected the composition of the MFGM components. Although the exact nature of the compound responsible was not identified in earlier clinical studies on cholesterol-reducing action of whole buttermilk fraction (Baumgartner et al., 2013; Conway et al., 2013), its MFGM origin appears likely. Supporting this assumption, Thompson et Figure 5. (A) The different steps of lipid digestion in the intestinal lumen (adapted from Ros, 2000): (1) Arrival of the initial lipid emulsion (i.e., gastric chyme) in the duodenum; (2) hydrolysis in the duodenum by pancreatic enzymes; (3) formation of mixed micelles (i.e., multi-molecular soluble aggregates) and transport of digested lipids to the intestinal microvilli to be further absorbed as monomers; (4) in the absence of adequate amounts of bile salt, digested lipids form large vesicles that are poorly absorbed (PL = phospholipid, TG = triglyceride, MG = mono-glyceride, FA = fatty acid, DG = di-glyceride) (B) Complex formed between the OH group of the cholesterol molecule (i.e., polar head) and the amine group of sphingomyelin (SM) and through hydrophobic interaction. This complex inhibits the absorption of free cholesterol. al. (1982) reported no modification of blood lipid levels on consumption of cultured buttermilk for 3 weeks in a small group (n = 11) of healthy subjects. As explained previously, unlike buttermilk obtained from the churning of cream (i.e., sweet buttermilk), cultured buttermilk is obtained through acidification. Thus, cultured buttermilk is not enriched in MFGM components like sweet buttermilk. Even if MFGM polar lipids are usually considered as the active components in many studies, the in vivo production of bioactive peptides by digestion of MFGM material cannot be ruled out as a contributor to the health benefits observed on buttermilk consumption. For example, it was found through secondary analysis of data published originally by Conway et al. (2013) that consumption of buttermilk for 4 weeks significantly reduced blood pressure in normotensive subjects compared with control patients (Conway et al., 2014). Although the exact nature of the blood-pressurereducing principle was not identified, the authors suggested that peptides Apr. 2014, Vol. 4, No. 2 49

7 Sweet buttermilk is the co-product resulting from the churning of cream into butter. This unique dairy product in enriched in high-value components from the milk fat globule membrane (MFGM). Courtesy of Valérie Conway. There is solid evidence in support of the high value of buttermilk minor components in disease prevention. Published clinical studies nevertheless remain rare. Larger and longer successful clinical trials are still needed before the dairy industry can make health claims for buttermilk. The largest body of evidence so far has been gathered for its cholesterolreducing activity. However, the clinical studies available have involved primarily healthy patients, who generally are less responsive to dietary interventions, and the benefits might therefore be underestimated. Clinical trials with patients diagnosed with metabolic syndrome, moderate hypercholesterolemia, or elevated blood pressure would give a broader view of the potential of buttermilk for providing cardiovascular health benefits. In addition, more research is needed to identify the exact nature of the components responsible for the health benefits observed (e.g., cholesterol-reducing, blood-pressure-reducing, anti-cancer, and antiviral) and the underlying mechanisms. As mentioned in this review, depending on the processing history of the raw cream, the nutritional and functional properties of the resulting buttermilk may vary considerably. In addition, the origin (e.g., whey buttermilk, serum-butter, or sweet buttermilk) of MFGM components and matrix surrounding them both seem to affect their biological activity. For this reason, future research should view buttermilk as a functional food rather than as a source of ingredients needing concentration, isolation, or purification by industrial separation processes. Since separation methods could not achieve enrichment of buttermilk polar lipids without modification of its bioactivity, the production of enriched phospholipids milk through modifications of cow diets must be further investigated as an innovative way to improve buttermilk composition. From a product development point of view, it could be of a great interest to conduct clinical investigations on the synergic action of other well-known cholesterol-reducing or triglyceride-reducing molecules, such as phytosterols and omega-3 fatty acids, in buttermilkbased drinks. This could lead to the development of new and innovative functional foods to prevent cardiovascular diseases through improvement in blood chemistry, anti-oxidative stress reduction, and blood pressure management. Functional food is a growing and lucrative market. Thus, buttermilk-based bioactive drinks may represent a solution to the disposal of buttermilk, still seen as a waste effluent of butter making by dairy industrials. originating from the digestion of MFGM components could be responsible. In support of this hypothesis, the authors noted a significant reduction ( 10.9%) in the plasma concentrations of angiotensin-i-converting enzyme (ACE) compared with the placebo condition. This enzyme plays a wellknown and important role in blood pressure regulation, and inhibition of its activity by various peptides is well documented. It should be noted that commercial purified sphingolipids do not contain any peptides. Unresolved Questions and Prospects for Future Research Literature Cited Affolter, M., L. Grass, F. Vanrobaeys, B. Casado, and M. Kussmann Qualitative and quantitative profiling of the bovine milk fat globule membrane proteome. J. Proteomics 73: Astaire, J.C., R. Ward, J.B. 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Blecker Study on the susceptibility of the bovine milk fat globule membrane proteins to enzymatic hydrolysis and organization of some of the proteins. Int. Dairy J. 21: Veereman-Wauters, G.S. Staelens, R. Rombaut, K. Dewettinck, D. Deboutte, R.- J. Brummer, M. Boone, and P. Le Ruyet Milk fat globule membrane (INPULSE) enriched formula milk decreases febrile episodes and may improve behavioral regulation in young children. Nutrition 28: Vesper, H., E.-M. Schmelz, M.N. Nikolova-Karakashian, D.L. Dillehay, D.V. Lynch, and A.H. Merrill, Jr Sphingolipids in food and the emerging importance of sphingolipids to nutrition. J. Nutr. 129: Zanabria, R., A.M. Tellez, M. Griffiths, and M. Corredig Milk fat globule membrane isolate induces apoptosis in HT-29 human colon cancer cells. Food Funct. 4: About the Authors Dr. Valérie Conway has a Ph.D. in Food Science and is currently a Postdoctoral Fellow in Medicine at the Université de Sherbrooke (Sherbrooke, Canada). During her Ph.D., under the supervision of Dr. Yves Pouliot, she specialized in the valorization of buttermilk component, mainly its lipids fraction, in the prevention of cardiovascular diseases. From 2007 to 2013, she worked at the Dairy Science and Technology Research Centre (STELA) at Université Laval (Quebec, Canada). She is currently doing research in nutrigenomics, under the supervision of Dr. Mélanie Plourde, at the Research Center on Aging. Her interest is prevention of age-related neurological impairments through omega-3 fatty acid supplementation. Dr. Sylvie Gauthier is professor at the department of Food Science and Nutrition at Université Laval. She has a strong background in biochemistry and nutrition. Her initial expertise was on the comparative digestion profile of various food proteins in model digestion system. Over the years, she became recognized for her practical expertise in enzymatic hydrolysis of proteins, production and characterization of bioactive peptides from food proteins. Dr Gauthier has been involved in the development and scale-up of bioactive peptide fractions and milk growth factors from dairy products. Dr Gauthier is member of the STELA Dairy research Center at the Institute of Nutrition and Functional Foods (INAF) from Université Laval. Dr. Yvse Pouliot is professor at the department of Food Science and Nutrition at Université Laval. He graduated from University Laval in 1987 with a PhD in Food Science and Technology. From the very beginning of his career at the STE- LA Dairy research Center, he focused at membrane separation processes applied to dairy fluids. The main focus has been the production/separation of whey bioactive peptides, buttermilk minor components in the development of nutraceuticals from whey and buttermilk. Dr Pouliot has been involved in a large number of university-industry research projects. He is currently Director of the Institute of Nutrition and Functional Foods (INAF) from Université Laval. Correspondence: yves.pouliot@fsaa.ulaval.ca Apr. 2014, Vol. 4, No. 2 51