Tamaño de Gñobulo de Bufalo Etc 2010

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Buffalo vs. cow milk fat globules: Size distribution, zeta-potential, compositionsin total fatty acids and in polar lipids from the milk fat globule membrane

Olivia Ménard, Sarfraz Ahmad, Florence Rousseau, Valérie Briard-Bion,Frédéric Gaucheron, Christelle Lopez *

INRA, AGROCAMPUS OUEST, UMR 1253 Science et Technologie du Lait et de l’Oeuf, 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 impor-

tance 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 significantly lower amount of polar lipids

expressed per gram of lipids (0.26% vs. 0.36%), but significantly 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 constitu-

ents. A significant 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 con-

tribute 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. Buf-

falo 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 cow’s 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 physi-

cal 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, Hofi, and

Mahran (1974), fat content of buffalo milks ranged from 6.9% to

8.5%. Varrichio, Di francia, Masucci, Romano, and Proto (2007) re-

ported that the fat content in buffalo milks averages 8.3% but can

reach 15% under favourable conditions. From a technological point

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

butter oil (clarified butter or ghee), soft and hard cheeses, con-

densed 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, moz-

zarella and ricotta in Italy, gemir in Iraq, paneer in India, domiati in

Egypt, pecorino in Bulgaria, and pickled cheeses from the Middle-

Eastern countries. An increase in the knowledge of the specific

physicochemical properties of buffalo milk fat globules will permit

dairy plants to improve their technological processes and to pro-

duce high quality products.

Fat is dispersed in milk in the form of spherical droplets calledmilk 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 sat-

urated 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 lacta-

tion (Arumughan & Narayanan, 1981), the season (Talpur, Bhanger,

Khooharo, & Zuhra Memon, 2008), and animal diet (Patiño et al.,

2008). Milk fat globules are surrounded by a biological membrane,

which results from the mechanisms of secretion of fat globules

0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2009.10.053

* Corresponding author. Tel.: +33 2 23 48 56 17; fax: +33 2 23 48 53 50.

E-mail address: [email protected] (C. Lopez).

Food Chemistry 120 (2010) 544–551

Contents lists available at ScienceDirect

Food Chemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m


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from the epithelial cells of the mammary gland (Lopez et al., 2008).

The milk fat globule membrane (MFGM) is rich in proteins, glyco-

proteins, glycerophospholipids, sphingolipids (mainly sphingomy-

elin), cholesterol, enzymes and other minor components (Keenan &

Patton, 1995). Several components of bovine MFGM have been re-

lated to health-enhancing functions, particularly the glycerophos-

pholipids and sphingolipids (Parodi, 1997; Spitsberg, 2005).

Glycerophospholipids comprise about 33% of bovine MFGM. Theyare composed of a glycerol backbone on which two acylglycerols

are esterified 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 esterified with phosphorylcholine group.

The five major classes of polar lipids in cow milk fat are phospha-

tidylcholine (PC; 35%), phosphatidylethanolamine (PE; 30%),

sphingomyelin (SM; 25%), phosphatidylinositol (PI; 5%) and phos-

phatidylserine (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 pre-

vents 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 glob-

ules 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 15–21 buffaloes of Mediterranean breed of Bubalus bubalis

(three out of 21 buffaloes were primiparas). Cow milk corre-

sponded 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 per-

form 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., Coopérative de Bufflonnes (Mau-

rs, Cantal region, France), at the end of August until mid-Septem-

ber. 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 zeta-

potential and particle-size measurements, as well as for micros-

copy observations. They were then stored at 20 C until further

analysis.

2.2. Chemical analysis

The chemical composition of the milks was determined as inAhmad et al. (2008). Fat and lactose contents of whole buffalo

and cow milks were determined by using an infra-red spectropho-

tometer (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 per-

formed 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, respec-

tively, and 1.33 for water. The absorption coefficient at both wave-

lengths was 0.0001. The experiments were performed at roomtemperature. Depending on both fat content and size of milk fat

globules, about 50–150 ll of whole milks and about 10–30 ll of

creams were introduced into the measurement cell of the appara-

tus, which contained 100 ml of water, in order to reach 10% obscu-

ration (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 mea-

surement cell to disrupt the casein micelles. All analyses were per-

formed in triplicate. Standard parameters were calculated by the

software: the volume-weighted average diameter d43, defined as

Rnidi4/Rnidi

3 (where ni is the number of fat globules of diameterdi); the volume–surface average diameter d3,2, defined as Rnidi

3/

Rnidi2; the modal diameter that corresponds to the population of fat globules the most important in volume; the specific surface

area defined as: S ¼ 6:u=d3;2 where u is the volume fraction of

milk fat; and the size distribution width, defined as:

Span ¼ ðdv;0:9 dv;0:1Þ=dv;0:5 where dv,0.9 is the diameter below

which lies 90% of the globule volume, and likewise 10% for dv,0.1and 50% for dv,0.5.

2.5. Apparent zeta-potential

Milk fat globule electrophoretic mobility was measured by elec-

trophoretic 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 Henry’s equation:

f ¼ 3gl2e f ðjaÞ

where g and e are the viscosity and dielectric constant of the solu-

tion 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 3–5 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 per-formed three times for each sample.

O. Ménard et al. / Food Chemistry 120 (2010) 544–551 545


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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 (pur-

ity > 98%) were purchased from Sigma–Aldrich (Saint QuentinFallavier, France). The phospholipid standards were also supplied

by Sigma–Aldrich: 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-glyce-

ro-3 phosphoserine, purity 98%), PC (1,2-dipalmitoyl-sn-glycero-3-

phosphocholine, purity 99%) and sphingomyelin (SM from bovine

brain, purity 99%). For gas chromatography (GC) analysis, methy-

lene chloride and hexane were provided by Carlo Erba Reagents.

Sodium methoxide 0.5 M and 10% BF3-methanol were provided

by Sigma–Aldrich. 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 Sigma–Aldrich.

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 to-

tal 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 coefficient of variation


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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 inmilks 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 significantly 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 glob-

ules: 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 linearand positive relationship which was found in the milks between

fat globule size and fat content was the following:

diameter ðlmÞ¼0:0335fat content ðg=kgÞþ2:37 ðr 2¼0:8537Þ

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 produc-

tion 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 glob-ules in high-fat content milks such as buffalo milk.

From a physical point of view, the sensitivity of milk fat glob-

ules to disruption during processing of milk depends on their sizeand 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 ¼ 4c=d

where c is the interfacial tension and d the diameter of the particles.

If c is 1–2 mN/m, as assumed for native milk fat globules ( Phipps &

Temple, 1982), the Laplace pressure is in the order of 0.8–1.6 kPa for

buffalo milk fat globules and in the order of 1.1–2.2 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 buf-

falo milk performed using plate separators may be more efficient

than the skimming of cows’ milk. Such considerations can be help-

ful to better understand the physical instability of fat globules dur-

ing the manufacture of dairy products.

3.3. Fatty acid compositions

Table 3 shows the comparative analysis of the fatty acid compo-

sitions of buffalo and cow milks. For both milks, the major fattyacids were palmitic acid (C16:0), oleic acid (C18:1c9), myristic acid

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 zeta-

potential of milk fat globules (mean ± standard deviation).

Size distribution parameters Milk Statisticsa Cream Statisticsa

Buffalo Cow Buffalo Cow

Mode (lm) 4.93 ± 0.04 3.56 ± 0.15 *** 5.13 ± 0.09 3.57 ± 0.15 ***

d32 (lm) 3.65 ± 0.03 3.31 ± 0.13 *** 3.99 ± 0.08 3.35 ± 0.13 ***

d43 (lm) 5.18 ± 0.04 3.88 ± 0.18 *** 5.46 ± 0.12 3.89 ± 0.18 ***

Span 1.37 ± 0.02 1.11 ± 0.03 *** 1.36 ± 0.02 1.06 ± 0.06 ***

Specific surface area (m2 per gram of fat) 1.78 ± 0.02 1.97 ± 0.08 *** 1.63 ± 0.04 1.95 ± 0.07 ***

Apparent zeta-potential (mV) 11.0 ± 0.7 9.4 ± 0.6 ***

a 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.

O. Ménard et al. / Food Chemistry 120 (2010) 544–551 547


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(C14:0) and stearic acid (C18:0). Although both milk fats contained

about 70% saturated fatty acids (Table 3), buffalo milks contained

significantly ( 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 significantly

different between buffalo and cow milks (Table 3). Buffalo milks

contained significantly ( p < 0.0001) lower amounts of medium-chain fatty acids (C8:0 to C12:0). Regarding the long-chain fatty

acids, buffalo milks contained significantly 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 significantly lower amounts of oleic acid (C18:1 c9; p < 0.05) and significantly higher ( p < 0.0001) amounts of C18:1trans 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 ru-

men of the animals (Griinari & Bauman, 1999). As vaccenic acid is

an intermediate in the biohydrogenation which leads to the forma-

tion of stearic acid, the higher amount of vaccenic acid and thelower 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 significantly ( p < 0.0001) higher

amount of rumenic acid (C18:2 c9 tr11, the main conjugated lino-

leic 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 consump-

tion, as CLA isomers are regarded as anticarcinogenic, antiathero-

genic, antiobesity and antidiabetic components (Parodi, 1999).Patiño 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

significantly ( 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 recom-

mended intakes (Aro, 2006).

Buffalo milks contained significantly lower amounts of polyun-

saturated fatty acids. Considering the individual polyunsaturated

fatty acids, buffalo milks contained significantly ( p < 0.0001) loweramounts of linoleic acid (C18:2, x6) and significantly higher

0

2

4

6

8

10

12

14

1001010.1

Size ( m)

V o l u m

e ( % )

Milk_buffalo

Milk_cow

Cream_buffalo

Cream_cow

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14 16 18 20

Size ( m)

V o l u m e ( % )

Milk_buffalo

Milk_cow

Cream_buffalo

Cream_cow

A

B

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

548 O. Ménard et al. / Food Chemistry 120 (2010) 544–551


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( 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 significantly ( p < 0.0001) higher for cow milks. Since the rec-

ommended x6/x3 ratio is estimated to be 4–5 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 re-

gard 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 charac-terised in this study are due to the genetics of the two animal spe-

cies 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 triacylglyce-

rols) were first 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 phospho-lipids 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 inter-

preted as the absence or the presence of an extra hydroxyl group,

whereas Fong, Norris and MacGibbon (2007) reported 3 peaks cor-

responding to various sphingoloid bases and fatty acids. Lopez

et al. (2008) reported 3 peaks for SM in milks from cows fed a con-

trol diet or a diet enriched in unsaturated fatty acids.

The sum of glycerophospholipids (PE, PI, PS, PC) and SM concen-tration 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 significantly ( 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 glob-

ules and the amount of polar lipids, per gram of fat (r 2 = 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 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


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

lipids per surface area of MFGM: 1840 ± 312 lg of polar lipids perm2 MFGM for cow milk and 1470 ± 286 lg of polar lipids per m2

MFGM for buffalo milk. Thus, the MFGM seems to be more com-

pact 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 re-

ported in the literature for milk: PE (19.8–42.0%, w/w), PC (19.2–

37.3%, w/w), PS (1.9–10.5%, w/w), PI (0.6–11.8%, w/w) and SM

(18.0–34.1%, 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 (20–25%) 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 chromatogra-phy (Morrison, 1968; Kuchroo & Narayanan, 1981; Beri, Sharma, &

Singh, 1984). Buffalo milk contained a significant ( p < 0.01) higher

percentage of PC and a significant ( p < 0.001) lower percentage of

SM, whereas no significant 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 re-

ported 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 thatSM represents only 18–20% of the total phospholipids in cow milk

Buffalo

Cow

0

50

100

150

200

250

300

350

18.5 19.5 20.5 21.5 22.5

Retention time (minute)

E L S D o u t p u t ( m V )

1

2

3

4

PC

1

2

3

SM

0

100

200

300

400

500

600

700

800


E L S D o u t p u t

( m V )

Buffalo

Cow

PE

PI

PS

PC

SM

Neutral

lipids

0

100

200

300

400

500

600

700

800

0 5 10 15 20 250 5 10 15 20 25


E L S D o u t p u t

( m V )

Buffalo

Cow

PE

PI

PS

PC

SM

Neutral

lipids

A

B

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 figure. 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.

550 O. Ménard et al. / Food Chemistry 120 (2010) 544–551


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(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) re-

cently 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 significantly ( 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 define

the structural properties of membranes and lipoproteins and con-

tribute to the interesting nutritional value of buffalo milk. Particu-

larly, sphingolipids are involved in the intestinal uptake of

cholesterol. In several experiments, SM was found to significantly

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 anti-

carcinogenic 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 emul-

sifying properties.

Acknowledgements

The authors thank Joel Guillemin (Coopérative de Bufflonnes,

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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