Food Chemistry 120 (2010) 544551

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Buffalo vs. cow milk fat globules: Size distribution, zeta-potential, compositions
in total fatty acids and in polar lipids from the milk fat globule membrane
Olivia Mnard, Sarfraz Ahmad, Florence Rousseau, Valrie Briard-Bion,
Frdric Gaucheron, Christelle Lopez *
INRA, AGROCAMPUS OUEST, UMR 1253 Science et Technologie du Lait et de lOeuf, F-35042 Rennes, France

a r t i c l e

i n f o

Article history:
Received 22 May 2009
Received in revised form 3 September 2009
Accepted 20 October 2009

Keywords:
Milk fat globule membrane
Sphingomyelin
Phospholipids
Fatty acid composition
Buffalo milk

a b s t r a c t
Although buffalo milk is the second most produced milk in the world, and of primary nutritional importance in various parts of the world, few studies have focused on the physicochemical properties of buffalo
milk fat globules. This study is a comparative analysis of buffalo and cow milk fat globules. The larger size
of buffalo fat globules, 5 vs. 3.5 lm, was related to the higher amount of fat in the buffalo milks: 73.4 9.9
vs. 41.3 3.7 g/kg for cow milk. Buffalo milks contained signicantly lower amount of polar lipids
expressed per gram of lipids (0.26% vs. 0.36%), but signicantly higher amount of polar lipids per litre
of milk (+26%). Buffalo and cow milk fat globule membranes contain the same classes of polar lipids;
phosphatidylethanolamine, sphingomyelin (SM) and phosphatidylcholine (PC) being the main constituents. A signicant higher percentage of PC and lower percentage of SM were found for buffalo milks. The
fatty acid analysis revealed that saturated fatty acids, mainly palmitic acid, trans fatty acids, linolenic acid
(x3) and conjugated linolenic acid were higher in buffalo milk than in cow milk. Such results will contribute to the improvement of the quality of buffalo milk-based dairy products.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Numerous studies have focused on cow milk, although milks
from other animal species, such buffaloes, sheep, goats and camels
are essential to the human diet in various parts of the world. Buffalo milk is the second most produced milk in the world with 82
billion litres produced each year (12.5% of milk produced in the
world), after cows milk (84% with 551 billion litres) (IDF,
2007). More than 91% of buffalo milk is produced in India (60%)
and Pakistan (31%). Buffalo milk is also one of the richest milks
from a compositional point of view. Particularly, fat constitutes
the main fraction of buffalo milk and is responsible for its high
energetic and nutritive value. Thus, buffalo milk is important from
a nutritional point of view in the countries that breed buffaloes.
However, information about the chemical composition and physical characteristics of buffalo milk fat is scarce, compared to cow
milk.
Fat content was found to be higher in buffalo milk, compared to
cow milk. In the study performed by Asker, Ahmed, Ho, and
Mahran (1974), fat content of buffalo milks ranged from 6.9% to
8.5%. Varrichio, Di francia, Masucci, Romano, and Proto (2007) reported that the fat content in buffalo milks averages 8.3% but can
reach 15% under favourable conditions. From a technological point
* Corresponding author. Tel.: +33 2 23 48 56 17; fax: +33 2 23 48 53 50.
E-mail address: Christelle.Lopez@rennes.inra.fr (C. Lopez).
0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.10.053

of view, buffalo milk can provide a wide variety of products: butter,

butter oil (claried butter or ghee), soft and hard cheeses, condensed or evaporated milks, ice cream, yogurt, and buttermilk.
The high-fat content of buffalo milk makes it highly suitable for
processing. For example, the production of 1 kg of butter requires
14 kg of cow milk against 10 kg of buffalo milk. In many countries,
buffalo milk is used to make traditional cheeses, for example, mozzarella and ricotta in Italy, gemir in Iraq, paneer in India, domiati in
Egypt, pecorino in Bulgaria, and pickled cheeses from the MiddleEastern countries. An increase in the knowledge of the specic
physicochemical properties of buffalo milk fat globules will permit
dairy plants to improve their technological processes and to produce high quality products.
Fat is dispersed in milk in the form of spherical droplets called
milk fat globules, the size distribution of which can vary between
milk species (Mehaia, 1995; El-Zeini, 2006). Fat globules are
mainly composed of triacylglycerols (TG: 98% of milk lipids) which
are esters of glycerol and fatty acids. Milk fat contains mainly saturated fatty acids (about 70%) with various chain lengths, and low
amounts of unsaturated fatty acids (about 30%). Authors reported
changes in the fatty acid composition of buffalo milks as a function
of the breed (Talpur, Memon, & Bhanger, 2007), the stage of lactation (Arumughan & Narayanan, 1981), the season (Talpur, Bhanger,
Khooharo, & Zuhra Memon, 2008), and animal diet (Patio et al.,
2008). Milk fat globules are surrounded by a biological membrane,
which results from the mechanisms of secretion of fat globules

O. Mnard et al. / Food Chemistry 120 (2010) 544551

from the epithelial cells of the mammary gland (Lopez et al., 2008).
The milk fat globule membrane (MFGM) is rich in proteins, glycoproteins, glycerophospholipids, sphingolipids (mainly sphingomyelin), cholesterol, enzymes and other minor components (Keenan &
Patton, 1995). Several components of bovine MFGM have been related to health-enhancing functions, particularly the glycerophospholipids and sphingolipids (Parodi, 1997; Spitsberg, 2005).
Glycerophospholipids comprise about 33% of bovine MFGM. They
are composed of a glycerol backbone on which two acylglycerols
are esteried in positions sn-1 and sn-2 and a phosphate residue
with different organic groups (i.e., ethanolamine, choline, serine,
or inositol) is located in the sn-3 position. Sphingomyelin, which
is the main sphingolipid in milk, is characterised by a sphingoid
base (sphingosine), in which the amino group is linked with a
fatty acid and which is esteried with phosphorylcholine group.
The ve major classes of polar lipids in cow milk fat are phosphatidylcholine (PC; 35%), phosphatidylethanolamine (PE; 30%),
sphingomyelin (SM; 25%), phosphatidylinositol (PI; 5%) and phosphatidylserine (PS; 3 %) (Christie, Noble, & Davies, 1987).
The physicochemical properties of the MFGM are of primary
importance regarding the physical stability of fat globules in milk.
Phospholipids are excellent emulsifying agents and the MFGM prevents fat globules from their aggregation and coalescence. The
MFGM is also a physical barrier against the hydrolysis of TG by
lipolytic enzymes. Hamzawi (1990) revealed that phospholipids
from the MFGM possess antioxidant activity in buffalo butter and
showed that milk phospholipids are important for delaying the
deterioration of ghee which is a very common product of India
and Pakistan.
Analysis of the literature showed that numerous studies have
focused on the characteristics of cow milk fat globules and MFGM
composition and revealed that information about buffalo milk fat
globules needs to be improved. The objective of this study was to
investigate the physicochemical properties of buffalo milk fat globules and to perform a comparative analysis with cow milk fat
globules.
2. Materials and methods
2.1. Whole milks and creams
Buffalo milks corresponded to a mixture of the milks produced
by 1521 buffaloes of Mediterranean breed of Bubalus bubalis
(three out of 21 buffaloes were primiparas). Cow milk corresponded to a mixture of the milks produced by 34 cows, of which
24 were Holstein breed of Bos taurus and 10 were Brown Swiss
breed of Bos taurus (nine out of 34 cows were primiparas). To perform a comparative analysis of the fatty acid composition of the
milks, cows and buffaloes were kept under identical conditions
of feeding and management. Whole buffalo and cow milks were
collected from the same farm, i.e., Cooprative de Bufonnes (Maurs, Cantal region, France), at the end of August until mid-September. Both buffalo and cow milks were skimmed by centrifugation
with a plate separator (Elecrem, Vanves, France) to concentrate
milk fat globules in creams. NaN3 (0.02%) was added to the milks
and creams to prevent the growth of bacteria. The samples of milks
and creams were stored at ambient temperature for apparent zetapotential and particle-size measurements, as well as for microscopy observations. They were then stored at 20 C until further
analysis.
2.2. Chemical analysis
The chemical composition of the milks was determined as in
Ahmad et al. (2008). Fat and lactose contents of whole buffalo

545

and cow milks were determined by using an infra-red spectrophotometer (Lactoscope, Delta Instruments, Laboratoire Humeau,
France). The nitrogen content of the milks was determined by
the Kjeldahl method. Nitrogen content was converted into protein
content using 6.38 as conversion factor.

2.3. Microstructural analysis

The microstructural analysis of buffalo and cow milks was performed at room temperature using an inverted microscope, NIKON
Eclipse-TE2000-C1si (NIKON, Champigny sur Marne, France), with
differential interferential contrast (DIC).

2.4. Fat globule size measurements

The fat globule size distributions were measured in the whole
milks and creams by laser light scattering, using a Mastersizer
2000 (Malvern, UK) with two laser sources. The refractive indexes
used were 1.458 and 1.460 for milk fat at 633 and 466 nm, respectively, and 1.33 for water. The absorption coefcient at both wavelengths was 0.0001. The experiments were performed at room
temperature. Depending on both fat content and size of milk fat
globules, about 50150 ll of whole milks and about 1030 ll of
creams were introduced into the measurement cell of the apparatus, which contained 100 ml of water, in order to reach 10% obscuration (optimal conditions for particle-size measurements with
this apparatus). To determine the fat globule size distribution,
1 ml of 35 mM EDTA/NaOH pH 7.0 buffer (>98% disodium salt2
H2O, Prolabo, Fortenay-sous-Bois, France) was added to the measurement cell to disrupt the casein micelles. All analyses were performed in triplicate. Standard parameters were calculated by the
software: the volume-weighted average diameter d43, dened as
Rnidi4/Rnidi3 (where ni is the number of fat globules of diameter
di); the volumesurface average diameter d3,2, dened as Rnidi3/
Rnidi2; the modal diameter that corresponds to the population of
fat globules the most important in volume; the specic surface
area dened as: S 6:u=d3;2 where u is the volume fraction of
milk fat; and the size distribution width, dened as:
Span dv;0:9  dv;0:1 =dv;0:5 where dv,0.9 is the diameter below
which lies 90% of the globule volume, and likewise 10% for dv,0.1
and 50% for dv,0.5.

2.5. Apparent zeta-potential

Milk fat globule electrophoretic mobility was measured by electrophoretic light scattering at 25 C, using a Zetasizer 3000 HS
(Malvern Instruments) equipped with palladium electrodes and
an avalanche photodiode detector. The apparent zeta-potential of
milk fat globules was calculated from its electrophoretic mobility,
l, according to Henrys equation:

3gl
2ef ja

where g and e are the viscosity and dielectric constant of the solution respectively, at the temperature of the measurement. 1/j is the
Debye length and a is the radius of the particle. The Smoluchowski
approximation, assuming f(ja) = 1.5, was used. In the experiments,
the viscosity was 0.89 cp, the dielectric constant was 79. Samples
were prepared by suspending 35 ll milk in 10 ml buffer (20 mM
imidazole, 50 mM NaCl, 5 mM CaCl2, pH 7.0), which was introduced
into the capillary tube for measurement. All analyses were performed three times for each sample.

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O. Mnard et al. / Food Chemistry 120 (2010) 544551

2.6. Lipid analysis

2.6.1. Chemicals and reagents for extraction and analysis of the lipids
For high-performance liquid chromatography (HPLC) analysis,
chloroform stabilised with ethanol (for analysis) and methanol
(HPLC grade) were purchased from Carlo Erba Reagents (Val de
Reuil, France). Triethylamine (purity > 99%) and formic acid (purity > 98%) were purchased from SigmaAldrich (Saint Quentin
Fallavier, France). The phospholipid standards were also supplied
by SigmaAldrich: PE (L-a-phosphatidylethanolamine dipalmitoyl,
N,N-dimethyl (C16:0), purity 99%), PI (L-a phosphatidylinositol
ammonium salt from soybean, purity 98%), PS (1,2-diacyl-sn-glycero-3 phosphoserine, purity 98%), PC (1,2-dipalmitoyl-sn-glycero-3phosphocholine, purity 99%) and sphingomyelin (SM from bovine
brain, purity 99%). For gas chromatography (GC) analysis, methylene chloride and hexane were provided by Carlo Erba Reagents.
Sodium methoxide 0.5 M and 10% BF3-methanol were provided
by SigmaAldrich. Retention times were determined by injection
of commercial mixes of fatty acid methyl esters standards: from
C4:0 to C24:0 (FAME Mix C4-C24), from C14:0 to C22:0 (FAME
Mix C14-C22) and trans fatty acids purchased from SigmaAldrich.
2.6.2. Extraction of total lipids
An adapted protocol of the cold extraction procedure developed
by Folch, Lees, and Stanley (1957) was used for the extraction of total lipids from the creams, as detailed in Lopez et al. (2008). The
extraction of total lipids was performed in duplicate or in triplicate
to obtain a coefcient of variation <5%. Total lipids extracted were
stored at 20 C until further HPLC and GC analysis.
2.6.3. Analysis of polar lipids: glycerophospholipids and sphingolipids
2.6.3.1. Concentration and individual classes of polar lipids. The quantication of the glycerophospholipids and sphingolipids and the
determination of the polar lipid classes were performed using
HPLC combined with an evaporative light-scattering detector
(ELSD). The chromatographic method used for the separation of
the polar lipids extracted from the creams was adapted from the
method of Rombaut, Camp, and Dewettinck (2005), and detailed
in Lopez et al. (2008).
2.6.3.2. HPLC/ELSD calibration. The identication of the glycerophospholipids and the sphingomyelin was carried out by a comparison with the retention times of pure standards. To obtain a
quantitative evaluation of the glycerophospholipids and sphingomyelin, ve calibration curves were determined from the area values obtained by injecting 10 ll of chloroform:methanol (88:12, v/v)
serially diluted solutions containing 0.25 to 2 lg of PE, 0.5 to 2.5 lg
of PC, 0.25 to 2 lg of PS, 0.25 to 2 lg of PI and 0.5 to 2.5 lg of SM.
Each solution was prepared and injected in triplicate. Calibration
curves were calculated by applying the equations of the power
model to the area and concentration values, PE: y =
2492x1.791 (r2 = 0.995); PI: y = 2341x1.787 (r2 = 0.994); PS: y =
1406x1.756 (r2 = 0.994); PC: y = 2016x1.773 (r2 = 0.985); SM:
y = 1427x1.748 (r2 = 0.982). The sum of glycerophospholipids (PE,
PI, PS, PC) and sphingomyelin concentration was regarded as total
phospholipid concentration.
2.6.4. Total fatty acid analysis
Methyl esters of fatty acids were prepared from total lipids,
according to an adapted method (Park & Goins, 1994), as described
in Lopez et al. (2008). Fatty acid methyl esters were measured on a
Varian gas chromatograph (Model 3800, Varian, Walnut Creek, CA)
equipped with a ame ionisation detector, a programmed temperature injector and two capillary columns (both 50 m  0.32 mm;
lm thickness 0.25 lm) coated with 70% dicyanopropyl 30%
dimethyl polysilphenylene-siloxane (BPX-70, SGE, Ringwood,

Australia) mounted in series. Experimental conditions were previously detailed in Lopez et al. (2008). The GC analysis was performed in triplicate for each sample.
2.7. Statistical analysis
Analyses of variance (ANOVA) were performed using the
General Linear Model procedure of Statgraphics Plus, Version 5
(Statistical Graphics Corp., Englewood Cliffs, NJ). Differences between the treatment means were compared at the 5% level of signicance, using Fishers least signicance difference (LSD) test.
3. Results and discussion
3.1. Chemical composition of the buffalo and cow milks
The comparative analysis of the chemical composition of buffalo and cow milks revealed signicant (p < 0.0001) differences
for fat, protein and lactose contents (Table 1). Similar results have
been previously described in the literature (Ahmad et al., 2008).
3.2. Size distribution and apparent zeta-potential of fat globules
Both the fat globule size distributions and apparent zeta-potentials were determined, in order to characterise the physicochemical properties of fat globules dispersed in buffalo and cow milks.
The apparent zeta-potential of fat globules was signicantly
(p < 0.0001) higher in absolute value for buffalo milks compared
to cow milks (Table 2). This could be explained by differences in
the mineral composition of the aqueous phase, which can affect
the ionic strength of milk (e.g., amount of calcium), and/or by differences in the MFGM composition.
Fig. 1 shows the size distribution and circular shape of fat globules characterised in both buffalo and cow milks using optical
microscopy. Fig. 1 also reveals the higher fat content in buffalo
milk compared to cow milk, as reported in Table 1. Fig. 2 shows
the size distributions of fat globules in the milks and creams from
cow and buffalo species. The parameters calculated from the size
distributions of fat globules are presented in Table 2. The fat globule size distributions were similar for the milks and the corresponding creams in both animal species. This shows that the
smallest fat globules which were present in buffalo milks,
2.7 0.4% of fat globules with d < 1 lm, were not lost in the
skimmed milk after the concentration of fat globules in the creams.
The size distributions of fat globules span from 1.1 to 10 lm for
cow milks and creams and from 0.4 to 15 lm for buffalo milks
and creams. The fat globule size distribution was characterised
by a signicantly (p < 0.0001) greater width for buffalo compared
to cow milk (Fig. 2, Table 2). The mean diameter of fat globules
was signicantly (p < 0.0001) higher in the samples originating
from buffalo than from cow (Table 2). As a result, the specic surface area was signicantly (p < 0.0001) higher for cow fat globules
than for buffalo fat globules. The most abundant population of fat
globules in buffalo milk corresponded to a mean diameter of about

Table 1
Chemical analysis of buffalo and cow milks (mean standard deviation).

pH
Fat (g/kg)
Protein (g/kg)
Lactose (g/kg)

Buffalo milk

Cow milk

Statisticsa

6.74 0.09
73.4 9.9
45.7 1.2
55.8 0.9

6.76 0.07
41.3 3.7
33.6 1.7
48.7 1.6

NS

NS: no signicant difference.

a
Results of the analysis of variance.
***
Probability of F-test: p < 0.0001.

***
***
***

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O. Mnard et al. / Food Chemistry 120 (2010) 544551

Table 2
Physicochemical characteristics of buffalo and cow milk fat globules. Parameters calculated from the size distributions of fat globules in milks and creams and apparent zetapotential of milk fat globules (mean standard deviation).
Size distribution parameters

Mode (lm)
d32 (lm)
d43 (lm)
Span
Specic surface area (m2 per gram of fat)
Apparent zeta-potential (mV)
a
***

Statisticsa

Milk
Buffalo

Cow

4.93 0.04
3.65 0.03
5.18 0.04
1.37 0.02
1.78 0.02
11.0 0.7

3.56 0.15
3.31 0.13
3.88 0.18
1.11 0.03
1.97 0.08
9.4 0.6

***
***
***
***
***

Statisticsa

Cream
Buffalo

Cow

5.13 0.09
3.99 0.08
5.46 0.12
1.36 0.02
1.63 0.04

3.57 0.15
3.35 0.13
3.89 0.18
1.06 0.06
1.95 0.07

***
***
***
***
***

***

Results of the analysis of variance.

Probability of F-test: p < 0.0001.

Fig. 1. Micrographs of: (A) buffalo and (B) cow milks taken by optical microscopy and showing the differences in fat content and fat globule size distribution. Scale
bar = 20 lm.

5 lm, whereas it was about 3.5 lm in cow milk (Table 2, Fig. 2).
Even if the size distribution and mean diameter of fat globules in
milks from individual cows and buffaloes may be weakly affected
by milk production, fat production and genetic factors, the size
parameters of fat globules were not signicantly different within
the milks from the same species collected for the experiments
(Fig. 2). Our results are in agreement with the literature. Authors
reported differences in the size distribution of buffalo fat globules
compared to cow fat globules. Using scanning electron microscopy,
El-Zeini (2006) reported a larger diameter for buffalo milk fat globules: 8.7 vs. 3.95 lm for cow milk fat globules. Moreover, El-Zeini
(2006) reported a lower amount of small fat globules (d < 4 lm)
and higher amount of large fat globules (>8 lm) for buffalo milks
compared to other species, e.g., goats, camels, cows, and sheep.
The larger size of fat globules in buffalo milk compared to cow
milk was related with the higher fat content (Table 1). The linear
and positive relationship which was found in the milks between
fat globule size and fat content was the following:

diameter lm 0:0335  fat content g=kg 2:37 r 2 0:8537

These results are in agreement with El-Zeini (2006) who reported
that milk with a high-fat content, such as that of buffaloes, usually
contains larger fat globules than milks with a lower fat content. The
possible explanation for the formation of larger fat globules when
the synthesis of milk fat is important is the limitation in the production of the MFGM when fat globules are enveloped during their
secretion from the epithelial cells of the mammary gland, as already
discussed by Wiking, Stagsted, Bjorck, and Nielsen (2004). Thus, the
MFGM could be a limiting factor in the formation of small fat globules in high-fat content milks such as buffalo milk.

From a physical point of view, the sensitivity of milk fat globules to disruption during processing of milk depends on their size
and is increased for larger fat globules, according to the Laplace
equation. The Laplace equation states that a pressure difference,
DP, exists between the two sides of a curved surface:

DP 4c=d
where c is the interfacial tension and d the diameter of the particles.
If c is 12 mN/m, as assumed for native milk fat globules (Phipps &
Temple, 1982), the Laplace pressure is in the order of 0.81.6 kPa for
buffalo milk fat globules and in the order of 1.12.2 kPa for cow
milk fat globules. The large buffalo milk fat globules may have a
lower stability against rupture of the MFGM and a lower resistance
to deformation and coalescence under mechanical pressure than
cow fat globules. Hence, the larger size of buffalo milk fat globules
may facilitate their disruption during the churning of cream for the
manufacture of butter and ghee. Moreover, large fat globules have
the capacity to rapidly move up and separate from the aqueous
phase to form a cream layer at the surface of milk, according to
the Stokes equation. As a consequence, the skimming of whole buffalo milk performed using plate separators may be more efcient
than the skimming of cows milk. Such considerations can be helpful to better understand the physical instability of fat globules during the manufacture of dairy products.
3.3. Fatty acid compositions
Table 3 shows the comparative analysis of the fatty acid compositions of buffalo and cow milks. For both milks, the major fatty
acids were palmitic acid (C16:0), oleic acid (C18:1c9), myristic acid

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O. Mnard et al. / Food Chemistry 120 (2010) 544551

A 14

Milk_buffalo
Milk_cow
Cream_buffalo
Cream_cow

12

Volume (%)

10
8
6
4
2
0
0.1

10

Size (m)

100

B 14
Milk_buffalo
Milk_cow
Cream_buffalo
Cream_cow

12

Volume (%)

10
8
6
4
2
0

10

12

14

16

18

20

Size (m)
Fig. 2. Fat globule size distribution in the milks and creams obtained from buffaloes and cows (A) exponential x-axis, (B) linear x-axis.

(C14:0) and stearic acid (C18:0). Although both milk fats contained
about 70% saturated fatty acids (Table 3), buffalo milks contained
signicantly (p < 0.05) higher amounts of saturated fatty acids
and lower amounts of unsaturated fatty acids than cow milks.
These results are in accordance with previous studies (Varrichio
et al., 2007; Blasi et al., 2008). However, Haggag, Hamzawi, and
Shahin (1987) reported higher levels of unsaturated fatty acids
for Egyptian buffalo milks (23%), compared to Egyptian cow milks
(18.4%). The short-chain fatty acid contents were not signicantly
different between buffalo and cow milks (Table 3). Buffalo milks
contained signicantly (p < 0.0001) lower amounts of mediumchain fatty acids (C8:0 to C12:0). Regarding the long-chain fatty
acids, buffalo milks contained signicantly higher contents of
myristic acid (C14:0; p < 0.0001) and palmitic acid (C16:0;
p < 0.01) and lower content of stearic acid (C18:0; p < 0.01) than
cow milks.
Considering the monounsaturated fatty acids, buffalo milks
contained signicantly lower amounts of oleic acid (C18:1 c9;
p < 0.05) and signicantly higher (p < 0.0001) amounts of C18:1
trans fatty acids, mainly vaccenic acid (C18:1 tr11; p < 0.0001).
Vaccenic acid, which is the main C18:1 trans fatty acid found in
milks, originates from the biohydrogenation mechanisms in the rumen of the animals (Griinari & Bauman, 1999). As vaccenic acid is
an intermediate in the biohydrogenation which leads to the formation of stearic acid, the higher amount of vaccenic acid and the
lower amount of stearic acid found in buffalo milks could be ex-

plained by a lower activity in the rumen of buffaloes compared

to cows (Griinari & Bauman, 1999).
Buffalo milks contained a signicantly (p < 0.0001) higher
amount of rumenic acid (C18:2 c9 tr11, the main conjugated linoleic acid; CLA) than cow milks. This is not surprising since C18:1
tr11 is the precursor of the C18:2 c9 tr11, formed in the mammary
gland by delta-9 desaturase. Varrichio et al. (2007) also reported
that the average content in CLA is higher in buffalo milks compared
to cow milks. Such results are important for buffalo milk consumption, as CLA isomers are regarded as anticarcinogenic, antiatherogenic, antiobesity and antidiabetic components (Parodi, 1999).
Patio et al. (2008) recently reported a positive correlation
(r = 0.87) between the content in C18:1 tr11 and the content in
CLA in buffalo milks from Argentina.
The total trans fatty acids (C18:1 trans + C18:2 c9 tr11) were
signicantly (p < 0.0001) higher in buffalo milks than in cow milks
(Table 3). Trans fatty acids are minor dietary components but the
foods that contain these fatty acids affect the balance between
the soft (cis-monounsaturated fatty acids + polyunsaturated fatty
acids) and hard (saturated fatty acids + trans fatty acids) fats in
the diet, which is 2/3 and 1/3 respectively, according to recommended intakes (Aro, 2006).
Buffalo milks contained signicantly lower amounts of polyunsaturated fatty acids. Considering the individual polyunsaturated
fatty acids, buffalo milks contained signicantly (p < 0.0001) lower
amounts of linoleic acid (C18:2, x6) and signicantly higher

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O. Mnard et al. / Food Chemistry 120 (2010) 544551

Table 3
Comparative analysis of the fatty acid composition of buffalo and cow milk fat
globules (% weight of total methyl esters; mean standard deviation).
Fatty acidsa

C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C14:1
C15:0
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:1
C18:1
C18:1
C18:1
C18:2
C18:3
C20:0
C18:2

c9
c10
c9
c10
t6 + t7 + t8 + t9
t10
t11
t12
c9
c9, c12 (x6)
c9, c12, c15 (x3)
c9, t11 (main CLA)

Saturated FA
Unsaturated FA
x6/x3
C18:1 trans
Total trans (C18:1 trans + CLA)

Milk from different species

Buffalo

Cow

2.80 0.49
1.85 0.32
1.08 0.18
1.80 0.21
2.30 0.15
11.77 0.15
0.73 0.01
1.74 0.08
0.36 0.00
36.02 1.24
1.91 0.06
0.84 0.02
0.30 0.02
9.85 0.17
0.40 0.02
0.17 0.05
2.00 0.08
0.14 0.03
20.25 0.65
0.93 0.12
0.72 0.17
0.22 0.03
0.90 0.06

2.53 0.49
2.10 0.44
1.35 0.23
2.52 0.33
2.87 0.16
11.14 0.44
1.05 0.15
1.16 0.03
0.27 0.00
33.80 0.93
1.59 0.05
0.61 0.05
0.24 0.03
11.07 0.85
0.31 0.17
0.18 0.07
1.43 0.06
0.17 0.09
22.12 1.65
1.34 0.07
0.61 0.03
0.17 0.06
0.70 0.02

70.8 0.8
29.2 0.8
1.3 0.1
2.70 0.08
3.61 0.13

69.6 1.7
30.4 1.7
2.2 0.2
2.09 0.31
2.79 0.33

Statsb

NS
NS
**
***
***
***
***
***
***
**
***
***
**
**
*

NS
***

NS
*
***
*
*
***
*
*
***
***
***

NS: no signicant difference.

a
Abbreviations: fatty acids: FA; conjugated linoleic acid: CLA.
b
Results of the analysis of variance.
Probability of F-test:
*
p < 0.05.
**
0.0001 6 p < 0.01.
***
p < 0.0001.

(p < 0.05) amounts of linolenic acid (C18:3, x3). These fatty acids
cannot be formed de novo in humans and are essential for health.
Therefore, they need to be ingested from the diet. The x6/x3 ratio,
was signicantly (p < 0.0001) higher for cow milks. Since the recommended x6/x3 ratio is estimated to be 45 while the current
dietary x6/x3 ratio is about 10 (Kris-Etherton et al., 2000), the
lower x6/x3 ratio found for buffalo milks is interesting with regard to improving human nutrition. Blasi et al. (2008) reported
higher x6/x3 ratios of 10 for buffalo milk and 9.5 for cow milk.
These higher ratios were explained by a higher amount of linoleic
acid and a lower amount of linolenic acid, in contrast to our results.
As a conclusion, and considering the identical conditions of
feeding and management used for the experiments, the differences
in the fatty acid compositions of the buffalo and cow milks characterised in this study are due to the genetics of the two animal species considered.
3.4. Glycerophospholipids and sphingolipids
The comparative analysis of the lipid composition of the MFGM
from buffalo and cow milks was performed. The chromatograms
presented in Fig. 3 show that neutral lipids (mainly triacylglycerols) were rst eluted and did not interfere with the separation
of the glycerophospholipids, i.e., PE, PI, PS, PC and of the main milk
sphingolipid, SM. The same classes of polar lipids were detected in
buffalo and cow milks (Fig. 3). PC eluted as 4 peaks and SM eluted
as 3 peaks because of the partial separation of molecular species
(Fig. 3B). As in all the natural samples, each class of milk phospholipids is a mixture of various molecular species differing in acyl

chain composition. Christie, Noble, and Davis (1987) and Rombaut

and Dewettinck (2006) reported 2 peaks for SM that were interpreted as the absence or the presence of an extra hydroxyl group,
whereas Fong, Norris and MacGibbon (2007) reported 3 peaks corresponding to various sphingoloid bases and fatty acids. Lopez
et al. (2008) reported 3 peaks for SM in milks from cows fed a control diet or a diet enriched in unsaturated fatty acids.
The sum of glycerophospholipids (PE, PI, PS, PC) and SM concentration corresponded to total polar lipids concentration (Table 4).
Per gram of total lipids, the polar lipids corresponded to
2.6 0.5 mg in buffalo milks and to 3.6 0.7 mg for cow milks.
Our results are in accordance with Asker et al. (1974) who reported
0.26% of phospholipids, calculated as percent of fat in buffalo milks.
The amount of polar lipids was signicantly (p < 0.001) higher in
the milks originating from cows by about 28%. This was related
to the smaller size of fat globules in cows milks (Fig. 2), and then
to the greater surface area covered by the MFGM (Table 2). A linear
and positive correlation was found between the surface of fat globules and the amount of polar lipids, per gram of fat (r2 = 0.71).
According to the data found in the literature, fat-rich products
such as cream obtained by concentration of fat globules from milk,
have a polar lipid content of <1% (w/w) of total lipids. More precisely, authors found in cream 0.250.53 g of polar lipids per
100 g of total lipids (Rombaut et al., 2005; Avalli & Contarini,
2005; Lopez et al., 2008; Rombaut & Dewettinck, 2006). Variations
in the polar lipid content of raw milk can be ascribed to differences
in the methods of preparation and analysis of the samples. Variations in the phospholipid content of buffalo milks were reported
as a function of the stage of lactation, seasonal variations, feeding
of the animals and milk processing (Mahran, Askar, & Ho, 1973;
Ho, Mahran, & Askar, 1973; Asker, Hamzawi, Hagrass, & Abd el
Hamid, 1978).
The concentrations of all the individual phospholipids were signicantly higher in cow milk compared to buffalo milk (expressed
per gram of total lipids): PE + 27% (p < 0.01), PI + 27% (p < 0.001),
PS + 33% (p < 0.01), PC + 20% (p < 0.001) and SM + 33% (p < 0.0001)
(Table 4). Thus, the amount of the choline-containing phospholipids (sum of SM and PC) quantied per gram of total lipids is significantly higher in cow milk compared to buffalo milk. The increase
in individual polar lipids content was homogeneous compared to
the increase of total polar lipids.
Considering the amount of polar lipids per gram of fat (Table 4)
and the surface of the MFGM determined per gram of fat using laser light scattering measurements (Table 2), we calculated that

Table 4
Concentration of polar lipids (glycerophospholipids and sphingolipids) in the milk fat
globule membrane of buffalo and cow milk fat globules and relative proportion of
each class of polar lipids (mean standard deviation).
Polar
lipidsa

PE
PI
PS
PC
SM
Polar lipids

Concentration in polar lipids

(lg polar lipids per gram total
lipids)

Relative proportion of polar

lipids
(% of polar lipids)

Buffalo

Cow

Statsb

Buffalo

Cow

Statsb

777 175
276 46
298 73
624 84
652 132
2627 496

1069 269
377 68
442 101
784 106
970 151
3641 748

**

29.4 1.4
10.6 0.5
11.3 1.3
24.0 1.7
24.8 0.7

29.1 1.6
10.4 0.6
11.8 2.0
21.8 1.7
26.9 1.6

NS
NS
NS

***
**
***
****

**
***

***

NS: no signicant difference p > 0.05.

a
PE: phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine;
PC: phosphatidylcholine; SM: sphingomyelin.
b
Results of the analysis of variance.
Probability of F-test:
**
0.001 < p < 0.01.
***
p < 0.001.
****
p < 0.0001.

550

O. Mnard et al. / Food Chemistry 120 (2010) 544551

800

PE

Neutral
lipids

700

ELSD output (mV)

600
500

PC

PI

400

PS

300

SM
Buffalo

200
100

Cow
0
0

B 350

10

15

20

Retention time (minute)

25

PC
2

300

SM
ELSD output (mV)

250

200

Buffalo

150

100

50

Cow
0
18.5

19.5

20.5

21.5

22.5

Retention time (minute)

Fig. 3. Normal-phase liquid chromatography (LC) evaporative light-scattering detector (ELSD) chromatograms of (A) the total lipid fraction obtained from buffalo and cow
milks, as indicated in the gure. These two chromatograms are representative of the chromatograms obtained for all the milks (n = 12; two independent milks, two
extractions of fat, three injections). The polar lipids from the milk fat globule membrane are phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine
(PS), phosphatidylcholine (PC) and sphingomyelin (SM). (B) Enlarged part of the chromatograms showing 4 peaks for PC and 3 peaks for SM.

cow milk contained signicantly (p < 0.01) higher amounts of polar

lipids per surface area of MFGM: 1840 312 lg of polar lipids per
m2 MFGM for cow milk and 1470 286 lg of polar lipids per m2
MFGM for buffalo milk. Thus, the MFGM seems to be more compact in cow milk compared to buffalo milk. This could be explained
by the curvature of the interface which is higher for cow milk fat
globules, as a result of their smaller size compared to buffalo milk
fat globules.
Regarding the percentage of each class of polar lipids in the
MFGM (Table 4), our results are in accordance with the data reported in the literature for milk: PE (19.842.0%, w/w), PC (19.2
37.3%, w/w), PS (1.910.5%, w/w), PI (0.611.8%, w/w) and SM
(18.034.1%, w/w) (Bitman & Wood, 1990; Rombaut et al., 2005;
Avalli & Contarini, 2005; Christie et al., 1987; Rombaut &
Dewettinck, 2006). The main polar lipids characterised for buffalo
and cow milks were PE (about 29%), PC (2025%) and SM (24
28%) (Table 4). Authors found that PC, PE and SM are the most pre-

valent classes of polar lipids in buffalo milk, using 31P-NMR,

(Andreotti, Trivellone, & Motta, 2006) and thin layer chromatography (Morrison, 1968; Kuchroo & Narayanan, 1981; Beri, Sharma, &
Singh, 1984). Buffalo milk contained a signicant (p < 0.01) higher
percentage of PC and a signicant (p < 0.001) lower percentage of
SM, whereas no signicant differences were observed for PE, PI
and PS (Table 4). Authors reported a slightly higher SM content
in buffalo MFGM in comparison with cow MFGM (Kuchroo &
Narayanan, 1981; Beri, Sharma, & Singh, 1984). The differences reported in the literature may originate from the preparation of the
samples (fat globules or MFGM) and to the analytical methods
used to quantify the individual classes of phospholipids (thin layer
chromatography, HPLC). Focusing on SM, authors reported that it
contributes approximately one-quarter to one-third of the
phospholipid portion (Parodi, 1997; Bitman & Wood, 1990; Lopez
et al., 2008), although more recent reports have indicated that
SM represents only 1820% of the total phospholipids in cow milk

O. Mnard et al. / Food Chemistry 120 (2010) 544551

(Rombaut et al., 2005; Avalli & Contarini, 2005). Regarding the

fatty acid composition of the MFGM polar lipids, Kuchroo and
Narayanan (1981) did not reveal any differences between buffalo
and cow milks, the main ones being long-chain saturated fatty
acids (C16:0, C18:0, C20:0, C23:0 and C24:0) and unsaturated fatty
acids (C18:1, C18:2 and C18:3). However, Lopez et al. (2008) recently reported that the fatty acid composition of milk polar lipids
can change as a function of the diet.
Taking into account their higher amount of fat (Table 1), buffalo
milks contained signicantly (p < 0.0001) higher amounts of polar
lipids (+26%): 189 9 mg/l of milk vs. 140 20 mg/l of milk for cow
milks. These polar lipids are bioactive compounds, which dene
the structural properties of membranes and lipoproteins and contribute to the interesting nutritional value of buffalo milk. Particularly, sphingolipids are involved in the intestinal uptake of
cholesterol. In several experiments, SM was found to signicantly
lower cholesterol absorption in rats and this decrease was found
to be higher for SM from milk than from other sources (Noh &
Koo, 2004). Moreover, some studies have demonstrated the anticarcinogenic potential of phospholipids, especially the role of SM
against colon cancer (Berra, Colombo, Sottocornola, & Giacosa,
2002). Thus, the buffalo buttermilk produced during the
manufacture of ghee is an important source of polar lipids, which
could be concentrated and used as nutraceuticals or for their emulsifying properties.
Acknowledgements
The authors thank Joel Guillemin (Cooprative de Bufonnes,
Maurs, France) for providing buffalo and cow milks, as well as Eric
Beaucher and Benoit Robert (INRA-STLO, Rennes, France) for the
transportation of the milks.
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