Published December 22, 2014
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
Implications
• R
esulting from churning of cream, sweet buttermilk is a source of
unique bioactive molecules capable of modulating cell signaling,
lipid transport, metabolism, and immunity.
• S
everal in vitro and in vivo results provide support for the claim of
the cholesterol-lowering action of buttermilk components.
• C
linical evidence now confirms the cardiovascular health benefits of
short-term consumption of whole buttermilk, due most likely to its
phospholipid content.
• B
uttermilk consumption represents a natural way to manage blood
pressure and blood lipids among healthy subjects.
• P
eptides 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.
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:10.2527/af.2014-0014
44 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
Animal Frontiers
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, im-
portant 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, 2006.
Composition (%, wt/wt)
Lipids (a)
Phospholipids (b)
Ratio [(b/a) X 100]
Apr. 2014, Vol. 4, No. 2
Whole
milk
4
0.035
0.9
Dairy product
Skim
milk
Cream
0.06
40
0.015
0.21
25
0.5
Buttermilk
0.6
0.13
22
45
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 , 2006.
Lipid class
Total lipids (%)
Neutral
Triglycerides
Di-glycerides
Mono-glycerides
Sterols
Esters
Free fatty acids
62
9
Trace
0.2 to 2.0
0.1 to 0.3
0.6 to 6.0
Polar
Phospholipids
- Phosphatidylethanolamine
- Phosphatidylcholine
- Sphingomyelin
- Phosphatidylinositol
- Phosphatidylserine
* % of total phospholipids.
46 26 to 31
31.1 to 42.0*
19.2 to 34.5*
17.9 to 34.5*
4.7 to 6.2*
2.8 to 8.5*
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
Animal Frontiers
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
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 imFigure 4. Representation of sphingomyelin (SM)-cholesterol lipid rafts in the milk fat globule
mune and central nervous system functions.
membrane and their biological significance. Adapted from Lopez (2010).
The cholesterol-lowering action of milk phospholipids is one of the most studied health benefits related to
2013). In this study, serum levels of sphingomyelin, and all measured neubuttermilk minor components consumption. A paper published in 1979
rodevelopment parameters, were positively associated with the MFGM
(Howard and Marks, 1979) was the first to report a lower concentration
sphingomyelin treatment.
of cholesterol in plasma of subjects consuming cream compared with
The products of sphingomyelin digestion are also known to affect cell
those consuming butter. This fascinating observation led to further in vivo
proliferation, apoptosis, and senescence, based mostly on in vitro studies.
research, which linked the effect first observed by Howard and Marks
Native MFGM isolates from raw milk have been shown to inhibit the proto milk sphingolipids. Indeed, as shown schematically in Figure 5A, the
liferation of HT-29 colon cancer cells in vitro (Zanabria et al., 2013). In
absorption of cholesterol depends almost entirely on its incorporation
view of these effects, the anti-cancer potential of MFGM concentrate obinto and dissolution in mixed micelles (Ros, 2000). Furthermore, to be
tained from buttermilk ultrafiltration has been investigated in vivo (Snow
absorbed by intestinal enterocytes, cholesterol must be free for desorpet al., 2010). In this study, rats (n = 16 or 17 per group) were fed diets
tion from these micelles. The solubility of cholesterol in mixed micelles
containing either 0.03% (wt/wt) sphingomyelin in corn oil, 0.03% (wt/wt)
depends greatly on the concentration and nature of the phospholipids pressphingomyelin in anhydrous milk fat, or MFGM from buttermilk (0.11%
ent, as well as on the availability of bile salts (Ros, 2000). This depensphingomyelin, wt/wt). Using the aberrant crypt foci (ACF) model, the
dence on micellar solubility explains the cholesterol-lowering action of
buttermilk product was found to protect against colon cancer. The ausphingolipids. For example, through interaction with long saturated fatty
thors suggested, based on evidence in the literature, that sphingomyelin
acid chains, cholesterol forms molecular complexes with sphingomyelin
was specifically responsible for the reduction in the number of apoptoin the intestinal lumen, resulting in a mutual inhibition of their absorpsis-resistant cells, predictive of colon cancer. The mechanism proposed
tion (Figure 5B). Slow and incomplete digestion of sphingomyelin in the
was related to sphingomyelin cell-signaling functions, particularly in cell
proximal segment of the intestine, an important site for lipid absorption,
growth, development, and differentiation. To the best of our knowledge,
enhances formation of complexes and reduces cholesterol availability
no clinical evidence of the anticancer potential of buttermilk consumption
(Eckhardt et al., 2002). The effect of raw-cream buttermilk and pasteuris actually available in literature, due likely to the difficulty of carrying out
ized-cream buttermilk concentrates, as obtained by microfiltration, on the
long-term cohort studies on humans.
micellar solubility of cholesterol in vitro has been investigated (Conway
Protective effects of dietary sphingolipids against toxins and microoret al., 2010). These authors observed a significant reduction (–57.1%) of
ganisms have been proposed in the literature, the active mechanism most
cholesterol solubility in the presence of buttermilk compared with control.
often suggested being competition for binding sites on intestinal mucosal
Although the bioactive component was not clearly identified in this study,
cells. An in vitro study of the anti-infective action of buttermilk and whey
the formation of sphingomyelin-cholesterol complexes was proposed as
cream MFGM concentrates, obtained by microfiltration, has been puban important mechanism modulating cholesterol solubility. It is interestlished recently (Fuller et al., 2013). The buttermilk concentrate was found
ing that the effect was much smaller (-17.0%) with pasteurized-cream butto be the more potent inhibitor of infection of monkey kidney cells by
termilk. Processing of cream is known to cause the aggregation of mulrotavirus. The authors attributed this anti-infective difference between buttiple proteins on the MFGM surface, as well as modification of MFGM
termilk and whey cream to variation of their MFGM lipid composition. As
composition (Lopez, 2011). Processing of raw cream or buttermilk (e.g.,
discussed earlier in this paper, sphingomyelin-cholesterol lipid rafts might
48 Animal Frontiers
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
ponents 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
Sweet buttermilk is the co-product resulting from the churning of cream into butter. This
drinks may represent a solution to the disposal of butunique dairy product in enriched in high-value components from the milk fat globule membrane
termilk, still seen as a waste effluent of butter making
(MFGM). Courtesy of Valérie Conway.
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
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 com-
50 Literature Cited
Affolter, M., L. Grass, F. Vanrobaeys, B. Casado, and M. Kussmann. 2010. Qualitative and quantitative profiling of the bovine milk fat globule membrane proteome. J. Proteomics 73:1079–1088.
Astaire, J.C., R. Ward, J.B. German, and R. Jiménez-Flores. 2003. Concentration
of polar MFGM lipids from buttermilk by microfiltration and supercritical fluid
extraction. J. Dairy Sci. 86:2297–2307.
Baumgartner, S., E.R. Kelly, S. van der Made, T.T. Berendschot, C. Husche, D.
Lütjohann, and J. Plat. 2013. The influence of consuming an egg or an egg-yolk
buttermilk drink for 12 weeks on serum lipids, inflammation, and liver function
markers in human volunteers. Nutrition 29:1237–1244.
Contarini, G., and M. Povolo. 2013. Phospholipids in milk fat: Composition, biological and technological significance, and analytical strategies. Int. J. Mol. Sci.
14:2808–2831.
Conway, V., P. Couture, S.F. Gauthier, Y. Pouliot, and B. Lamarche. 2013. Impact of
buttermilk consumption on plasma lipids and surrogate markers of cholesterol
homeostasis in men and women. Nutr. Metab. Cardiovasc. Dis. 23:1255–1262.
Conway, V., P. Couture, S.F. Gauthier, Y. Pouliot, and B. Lamarche. 2014. Effect of
buttermilk consumption on blood pressure in moderately hypercholesterolemic
men and women. Nutrition 30:116–119.
Conway, V., S.F. Gauthier, and Y. Pouliot. 2010. Effect of cream pasteurization, microfiltration and enzymatic proteolysis on in vitro cholesterol-lowering activity
of buttermilk solids. Dairy Sci. Technol. 90:449–460.
Conway, V., S.F. Gauthier, and Y. Pouliot. 2012. Antioxidant activities of buttermilk proteins, whey proteins and their enzymatic hydrolyzates. J. Agric. Food
Chem. 61:364–372.
Dewettinck, K., R. Rombaut, N. Thienpont, T.T. Le, K. Messens, and J. Van Camp.
2008. Nutritional and technological aspects of milk fat globule membrane material. Int. Dairy J. 18:436–457.
Eckhardt, E.R.M., D.Q.H. Wang, J.M. Donovan, and M.C. Carey. 2002. Dietary
sphingomyelin suppresses intestinal cholesterol absorption by decreasing thermodynamic activity of cholesterol monomers. Gastroenterology 122:948–956.
El-Loly, M. 2011. Composition, properties and nutritional aspects of milk fat globule membrane- a review. Pol. J. Food Nutr. Sci. 61:7–32.
Animal Frontiers
Fuller, K.L., T.B. Kuhlenschmidt, M.S. Kuhlenschmidt, R. Jiménez-Flores, and
S.M. Donovan. 2013. Milk fat globule membrane isolated from buttermilk or
whey cream and their lipid components inhibit infectivity of rotavirus in vitro.
J. Dairy Sci. 96:3488–3497.
Howard, A.H., and J. Marks. 1979. Effect of milk product on serum-cholesterol.
Lancet 2:957.
Keenan, T.W., and I.H. Mather. 2006. Intracellular origin of milk fat globules and the
nature of the milk fat globule membrane. In: P. F. Fox and P. L. H. McSweeney, editors, Advanced dairy chemistry: Volume 2. Lipids. Springer, New York. p. 137–171.
Küllenberg, D., L. Taylor, M. Schneider, and U. Massing. 2012. Health effects of
dietary phospholipids. Lipids Health Dis. 11:1–16.
Lopez, C. 2010. Lipid domains in the milk fat globule membrane: Specific role of
sphingomyelin. Lipid Technol. 22:175–178.
Lopez, C. 2011. Milk fat globules enveloped by their biological membrane: Unique
colloidal assemblies with a specific composition and structure. Curr. Opin. Colloid Interface Sci. 16:391–404.
MacGibbon, A.K.H., and M.W. Taylor. 2006. Composition and structure of bovine
milk lipids. In: P. F. Fox and P. L. H. McSweeney, editors, Advanced dairy
chemistry: Volume 2. Lipids. Springer, New York. p. 1–43.
Morin, P., R. Jiménez-Flores, and Y. Pouliot. 2007. Effect of processing on the composition and microstructure of buttermilk and its milk fat globule membranes.
Int. Dairy J. 17:1179–1187.
Morin, P., Y. Pouliot, and R. Jiménez-Flores. 2006. A comparative study of the
fractionation of regular buttermilk and whey buttermilk by microfiltration. J.
Food Eng. 77:521–528.
Nagai, K. 2012. Bovine milk phospholipid fraction protects Neuro2a cells from
endoplasmic reticulum stress via PKC activation and autophagy. J. Biosci. Bioeng. 114:466–471.
Ohlsson, L., H. Burling, R.D. Duan, and A. Nilsson. 2010. Effects of a sphingolipid-enriched dairy formulation on postprandial lipid concentrations. Eur. J. Clin.
Nutr. 64:1344–1349.
Ohlsson, L., H. Burling, and Å. Nilsson. 2009. Long term effects on human plasma
lipoproteins of a formulation enriched in butter milk polar lipid. Lipids Health
Dis. 8:1–12.
Ramprasath, V., P. Jones, D. Buckley, L. Woollett, and J. Heubi. 2013. Effect of
dietary sphingomyelin on absorption and fractional synthetic rate of cholesterol
and serum lipid profile in humans. Lipids Health Dis. 12:125.
Reis, M.G., N.C. Roy, E.N. Bermingham, L. Ryan, R. Bibiloni, W. Young, L.
Krause, B. Berger, M. North, K. Stelwagen, and M.M. Reis. 2013. Impact of
dietary dairy polar lipids on lipid metabolism of mice fed a high-fat diet. J.
Agric. Food Chem. 61:2729–2738.
Rombaut, R., J.V. Camp, and K. Dewettinck. 2006. Phospho- and sphingolipid distribution during processing of milk, butter and whey. Int. J. Food Sci. Technol. 41:435–443.
Ros, E. 2000. Intestinal absorption of triglyceride and cholesterol. Dietary and pharmacological inhibition to reduce cardiovascular risk. Atherosclerosis 151:357–379.
Snow, D.R., R. Jimenez-Flores, R.E. Ward, J. Cambell, M.J. Young, I. Nemere, and
K.J. Hintze. 2010. Dietary milk fat globule membrane reduces the incidence of
aberrant crypt foci in Fischer-344 rats. J. Agric. Food Chem. 58:2157–2163.
Spitsberg, V.L. 2005. Invited review: Bovine milk fat globule membrane as a potential nutraceutical. J. Dairy Sci. 88:2289–2294.
Tanaka, K., M. Hosozawa, N. Kudo, N. Yoshikawa, K. Hisata, H. Shoji, K. Shinohara, and T. Shimizu. 2013. The pilot study: Sphingomyelin-fortified milk has
a positive association with the neurobehavioural development of very low birth
weight infants during infancy, randomized control trial. Brain Dev. 35:45–52.
Thompson, L.U., D.J. Jenkins, M.A. Amer, R. Reichert, A. Jenkins, and J. Kamulsky. 1982. The effect of fermented and unfermented milks on serum cholesterol.
Am. J. Clin. Nutr. 36:1106–1111.
Vanderghem, C., P. Bodson, S. Danthine, M. Paquot, C. Deroanne, and C. Blecker.
2010. Milk fat globule membrane and buttermilks: From composition to valorization. Biotechnol. Agron. Soc. Environ. 14:485–500.
Vanderghem, C., F. Francis, S. Danthine, C. Deroanne, M. Paquot, E. De Pauw,
and C. Blecker. 2011. 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. 21:312–318.
Veereman-Wauters, G.S. Staelens, R. Rombaut, K. Dewettinck, D. Deboutte, R.J. Brummer, M. Boone, and P. Le Ruyet. 2012. Milk fat globule membrane
(INPULSE) enriched formula milk decreases febrile episodes and may improve
behavioral regulation in young children. Nutrition 28:749–752.
Vesper, H., E.-M. Schmelz, M.N. Nikolova-Karakashian, D.L. Dillehay, D.V.
Lynch, and A.H. Merrill, Jr. 1999. Sphingolipids in food and the emerging importance of sphingolipids to nutrition. J. Nutr. 129:1239–1250.
Zanabria, R., A.M. Tellez, M. Griffiths, and M. Corredig. 2013. Milk fat globule
membrane isolate induces apoptosis in HT-29 human colon cancer cells. Food
Funct. 4:222–230.
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 STELA 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: [email protected]
Apr. 2014, Vol. 4, No. 2
51