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Small Ruminant Research 109 (2013) 141151

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Small Ruminant Research


journal homepage: www.elsevier.com/locate/smallrumres

Characterization of size and composition of milk fat globules from


Sarda and Saanen dairy goats
S. Pisanu a , G. Marogna b , D. Pagnozzi a , M. Piccinini a , G. Leo a , A. Tanca a , A.M. Roggio a ,
T. Roggio a , S. Uzzau a,c , M.F. Addis a,
a
b
c

Porto Conte Ricerche Srl, Tramariglio, Alghero (SS), Italy


Istituto Zooprolattico della Sardegna G. Pegref, Sassari, Italy
Dipartimento di Scienze Biomediche, Universit degli Studi di Sassari, Italy

a r t i c l e

i n f o

Article history:
Received 28 October 2011
Received in revised form 8 May 2012
Accepted 30 July 2012
Available online 25 August 2012
Keywords:
Raman spectroscopy
Tandem mass spectrometry
Proteomics
Biodiversity

a b s t r a c t
The small size of goat milk fat globules (MFGs) is one of the factors contributing to the higher
digestibility of goat milk compared to other milks. In this study, size, protein composition
and lipid distribution of MFGs were evaluated comparatively in a popular dairy breed, Saanen, and in a minor breed, Sarda. MFGs were found to be signicantly smaller in Sarda
compared to Saanen goats, with average diameters of 2.73 0.15 m and 3.63 0.27 m,
respectively. Raman spectroscopy revealed differences in the lipid proles of differently
sized MFGs within each breed, with MFGs of the same size class having comparable proles between breeds. Proteomic characterization by SDS-PAGE followed by tandem mass
spectrometry (GeLCMS/MS) and label-free differential quantication highlighted signicant differences in expression levels of MFG proteins from the two breeds, with a higher
abundance of cytoplasmic proteins in Sarda MFGs and of membrane proteins in Saanen
MFGs. Moreover, differences in the relative abundance of several major MFG proteins were
observed for the two breeds. In conclusion, this study demonstrates the existence of breeddependent differences in the lipid and protein makeup of goat MFGs, likely related to their
different size distribution. This highlights once again the importance of investigating biodiversity in autochthonous and neglected breeds, which often possess valuable attributes
that might be lost as a consequence of the widespread diffusion of highly productive, but
more homogeneous, dairy breeds.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Goat milk is receiving growing interest and consumer
appreciation in view of its higher digestibility and tolerability compared to other types of milk for human
consumption. In fact, goat milk contains a higher percentage of short- and medium-chain fatty acids, facilitating
degradation by lipases, and has lower levels of alpha-S1

Corresponding author at: Porto Conte Ricerche Srl, S.P. 55 Porto


Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero (SS), Italy.
Tel.: +39 079 998 526; fax: +39 079 998 567.
E-mail address: addis@portocontericerche.it (M.F. Addis).
0921-4488/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.smallrumres.2012.07.024

casein leading to generation of a less compact and more


digestible curd compared to cow milk (Haenlein, 2004;
Silanikove et al., 2010). Higher digestibility is also conferred
by the small size of fat globules, allowing for a greater surface to volume ratio for enzymatic attack (Jenness, 1980).
This renders goat milk excellent for direct human consumption, although it should be considered that processes
such as homogenization and pasteurization can have an
impact on MFG size and structure. Milk fat globules (MFGs),
small membrane-coated vesicles released from lactating
cells, maintain fats in a dispersed state in milk, avoiding
their coalescence and separation from the liquid phase.
MFGs are composed by a triglyceride core enveloped by
a three-layered phospholipid membrane structure, arising

142

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151

from the endoplasmic reticulum and the apical membrane


of lactocytes as a result of secretion (Heid and Keenan,
2005). The role played by MFGs in determining the quality
of dairy products is known, and several MFG components
have been recently identied as being benecial for human
health (Dewettinck et al., 2008). This has led to an increased
awareness of the MFG as a food ingredient with functional
properties and health benets.
One of the most widespread and productive dairy
breeds used for producing milk for direct human consumption is the Saanen. The Saanen is considered the Holstein
of dairy goats for its superior milk production, and is
also one of the largest dairy goat breeds. However, less
widespread and minor breeds may have preserved interesting attributes in nutritional terms, which could be more
prominent compared to those belonging to homogeneous,
highly selected, and highly productive goat breeds. Indeed,
there is a growing interest toward the characterization of
minor breeds for the discovery and exploitation of unique
and peculiar attributes useful for improving quality and
nutritional value of milk and dairy products. In this respect,
autochthonous breeds are of particular interest, since these
are the result of a prolonged selective pressure imposed
by the specic requirements of the breeder or of the specic climatic, geographical, and pasture conditions of the
farming land, all facilitated and maintained by genetic and
geographical isolation. This has often led to a signicant
divergence of local breeds in rural, arid, or particularly
isolated geographical contexts, with the development or
the maintenance of peculiar and potentially interesting
traits.
The Sarda is an autochthonous breed from the Island of
Sardinia, where it has been the main goat breed for a long
time (Brandano and Piras, 1978). Sarda goats are mediumsmall sized animals, sturdy, frugal, and fully adapted to the
exploitation of low-potential rural areas and wastelands
of Sardinia. The phenotypic characteristics of Sarda goats,
such as coat color, udder shape, horn shape, and ear shape
and size, were described as non-homogeneous, leading to
classication into three sub-populations (Brandano and
Piras, 1978). The Sarda breed is characterized by a higher
genetic diversity compared to other goat breeds present
in Italy; in particular, a considerable genetic distance
between the Alpine breed Saanen and the Mediterranean
Sarda has been demonstrated (Ajmone-Marsan et al.,
2001).
Several preliminary and anecdotic observations have
been made in the past concerning the size of MFGs
in Sarda goats, suggested as being among the smallest
found in dairy goat breeds. Since size and composition
of MFGs provide extremely interesting attributes to milk,
both in terms of digestibility, tolerability, and technological attributes of dairy products, we felt that a study
was required in order to gather information on size and
composition of MFGs from this breed, and to compare
them to one of the most productive and widespread goat
breeds. For this purpose, a combined approach based on
optical microscopy, Raman spectroscopy and GeLCMS/MS
was used for characterization of MFG size, lipid, and
protein composition, respectively, in Saanen and Sarda
goats.

2. Materials and methods


2.1. Collection of milk and preparation of goat milk fat globules (MFGs)
Sarda and Saanen goats included in this study belonged to pure breed
herds composed only by animals registered in the respective purebred
herd book. Sarda and Saanen herds were farmed in the same geographical
area in Northern Sardinia, and both were kept with a semi/free grazing style integrated with commercial feeds (less than 400 g/day) during
the two daily milkings. The two herds used for this study were under
a monitoring program by the Regional Health Institution (IZS, Istituto
Zooprolattico Sperimentale della Sardegna). All animals included in this
study were subjected to clinical examination and to microbiological culture of milk (Marogna et al., 2012). Goats selected for sampling were
clinically healthy, negative to microbial culture of milk, multiparous, and
in mid-lactation (7080 DIM). In order to collect a homogeneous milk
sample, collection was performed from 10 individuals of each breed 2 h
after the morning milking into sterile 50 mL tubes. Goat milk was brought
to the laboratory in refrigerated conditions within few hours.
2.2. Raman spectroscopy analysis
Whole goat milk samples were centrifuged at 5000 g for 15 min at
4 C, and the milk fat ring was recovered and resuspended in phosphate
buffered saline (0.01 M phosphate buffer, 0.0027 M potassium chloride,
0.137 M sodium chloride, pH 7.4) containing 35 mM ethylenediamminetetra acetic acid (PBSEDTA). The resuspended fat ring was again centrifuged
at 5000 g for 15 min at 4 C, and washed MFGs were recovered and
resuspended in PBSEDTA to prevent aggregation of caseins (Argov et al.,
2008). Raman spectra were acquired with a Bruker Senterra dispersive
Raman microscope using an excitation wavelength of 532 nm at 25 mW
of power, with a 400 groove per millimeter grating in the 754400 cm1
spectral range and an Olympus 50 objective (numerical aperture 0.5,
working distance 23.5 mm). Each spectrum was the co-addition of thirty
1 s exposures and was recorded at room temperature. Peak intensities
were measured after averaging the spectra from 4 globules of the same
size. Bruker OPUS 6.5 software was used for controlling the microscope
and for spectra acquisition. For Raman measurements, diluted cream samples were deposited inside a cell for infrared spectroscopy transmission
measurements of liquid samples with a 25 m thick spacer and BaF2 windows, which enabled to achieve a quiescent sample condition. BaF2 is
transparent to visible light and has no Raman bands in the analyzed spectral ranges (10001800 cm1 and 27003000 cm1 ), so the MFG Raman
spectra were totally unaffected by the presence of the cell windows. For
sample observation and image acquisition, the Olympus BX microscope
mounted on the Bruker Senterra system was used, and diluted cream
samples were deposited onto a concave microscope slide. MFG size was
determined by manual counting and measuring of all MFGs in the snapshot, for a total of 20 snapshots (5 per sample) for each breed. Statistic
calculations and plots were performed with Microsoft Excel .
2.3. Extraction and SDS-PAGE of milk fat globule membrane proteins
Extraction of MFG proteins (MFGPs) from goat milk was carried out
as described previously (Pisanu et al., 2011a, 2011b). Briey, milk samples were centrifuged three times at 5000 g at 4 C for 15 min, and the
fat layer was separated and washed by gentle rocking at 37 C for 5 min,
twice in PBS and once in tridistilled water. All 20 washed fat ring samples were weighed, and weight was related to 100 mL of milk. MFGPs
were then extracted from the washed fat ring with a three phase process
including overnight cold crystallization, mechanical homogenization with
a TissueLyser (Qiagen, Hilden, Germany), and warming at 45 C for 30 min.
MFGPs were then recovered by centrifugation at 10,000 g for 15 min at
4 C, and protein concentration was determined using the 2D Quant Kit
(GE Healthcare, Uppsala, Sweden). MFGPs were subjected to SDS-PAGE in
a Mini PROTEAN Tetra Cell slab gel apparatus (Bio-Rad Laboratories, Hercules, CA, USA) using 12% polyacrylamide gels, by loading 25 g of MFGPs
resuspended in Laemmli buffer (Laemmli, 1970). For illustrative purposes,
an SDS-PAGE experiment was also carried out by loading 10 g aliquots
of the same samples subjected to proteomic characterization. After the
runs, gels were stained by Colloidal Coomassie Blue G-250 (Candiano et al.,
2004). Molecular masses were determined by running standard protein
markers (Precision Plus Protein All Blue Standards, Bio-Rad Laboratories)
covering the range of 10250 kDa.

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151


2.4. Tandem mass spectrometry analysis
Gel electrophoresis followed by liquid chromatographytandem mass
spectrometry (GeLCMS/MS) analysis was performed as described previously (Addis et al., 2009; Pisanu et al., 2011a) on 2 Saanen MFGP samples
and 2 Sarda MFGP samples. For each sample, 25 g of MFGPs were separated by SDS-PAGE and stained as described above; then, each lane was cut
into 32 slices that were subjected to bleaching, reduction with dithiothreitol, alkylation with iodoacetamide, and digestion with trypsin. Following
extraction, the peptide extracts were evaporated and resuspended in
0.02% formic acid. LCMS/MS analyses were performed on a Q-TOF hybrid
mass spectrometer equipped with a nano lock Z-spray source and coupled on-line with a capillary chromatography system CapLC (Waters), as
described previously (Addis et al., 2009). Briey, the peptide mixture was
concentrated and washed onto a RP pre-column, and fractionated onto
a C18 RP capillary column. The mass spectrometer was set up in a datadependent MS/MS mode where a full-scan spectrum was followed by a
tandem mass spectrum, selecting peptide ions as the three most intense
peaks of the previous scan. Argon was used as the collision gas, and a
collision energy depending on mass and charge of the precursor ion was
applied. ProteinLynx software (Version 2.2.5), was used for analysis of raw
MS and MS/MS spectra. The peak lists corresponding to gel slices belonging to the same sample were merged into a single MGF le, which was
analyzed by Proteome Discoverer (version 1.3, Thermo Scientic) using an
in-house Mascot server (version 2.3, Matrix Science) for protein identication according to the following criteria: Database UniProtKB/Swiss-Prot
(release 2011 08), enzyme trypsin, maximum missed cleavage sites 2,
taxonomy mammalia, precursor mass tolerance 30 ppm, fragment mass
tolerance 0.4 Da, cysteine carbamidomethylation as static modication,
N-terminal glutamine conversion to pyro-glutammic acid and methionine oxidation as dynamic modications. The percolator algorithm was
used for peptide validation (peptide condence: q-value < 0.05), and only
rank 1 peptides were considered. Peptide and protein grouping according to the Proteome Discoverers algorithm were allowed, applying strict
maximum parsimony principle. Trypsin and skin keratins were excluded
from the nal protein list, while in case of multiple homology identication (i.e. when matching with homologous protein sequences from
two or more species different from Capra hircus) the protein sequence
with the higher number of peptide matches was selected, and the total
number of spectral counts (SpCs) was calculated by summing the SpCs
of the selected protein sequence to the unique SpCs of the remaining
matched protein sequences. Protein annotation for cellular localization
and biological process was performed exploiting Proteome Discoverers
annotations. Semi-quantitative analysis for estimating protein abundance
was performed using the Normalized Spectral Abundance Factor (NSAF)
(Zhang et al., 2010). A two-tailed t-test was applied using Microsoft Excel
in order to evaluate the statistical signicance of differences between
groups.

3. Results

143

to be signicantly lower (P = 0.004) than Saanen MFGs,


being of 2.73 0.15 m vs. 3.63 0.27 m, respectively.
Fig. 1 (upper panel) shows two representative images of
washed milk fat globules from both breeds. Differences in
MFGs between the two breeds were also clearly evident
in terms of size distribution, expressed as relative abundance of the diameter classes. In fact, as shown in Fig. 1
(lower panel), the diameter class distribution revealed that
72% of Sarda MFGs measured 23 m in diameter, compared to only 37% of Saanen MFGs. On the contrary, larger
MFGs in the 1020 m diameter range accounted to 7%
in Saanen and only to 0.6% in Sarda milk. The difference in MFG size distribution was statistically signicant
(P < 0.05). In this respect, however, it should be considered
that MFGs below 1 m in size were not included in the
measurements because of methodological and technical
constraints.
3.2. Lipid composition by Raman spectroscopy
Raman spectroscopy is ideally suited to the characterization of differently sized MFGs, since it enables to study
samples in situ and in vivo without requiring sample prefractionation or chemical labeling (Krafft et al., 2005). In the
past, its application to MFGs has been successfully demonstrated by other authors on human and cow milk (Argov
et al., 2008; Forrest, 1978; Gallier et al., 2011). Therefore,
Raman spectroscopy combined with optical microscopy
was applied here with the aim of comparing MFG lipid proles in the two breeds and among different size classes.
Since the signals in the Raman spectrum arise mainly from
the vibrational levels of carbon bonds in lipid molecules,
these can be assigned to known lipid vibrations by comparison with published data (Faiman and Larsson, 1976;
Gallier et al., 2011). In order to gain insights on the lipid
composition, the main peaks were assigned to the major
lipid classes (Table 1), and the relative peak intensities
were used for comparing the different MFG proles. Specifically, Raman spectra were generated and interpreted for
three different size classes (Fig. 2B and D), enabling the
comparison of the chemical signature of lipids in MFGs of
15, 10, and 15 m from the two goat breeds. As a result,
the Raman spectra appeared to be comparable for MFGs
falling within the same size classes; moreover, Raman

3.1. Size distribution of milk fat globules in Sarda and


Saanen goats
The weight of the washed fat ring was evaluated on all
20 Sarda and Saanen goat milk samples. As a result, the
average weight of the fat ring for 100 mL of whole milk was
8.42 1.22 g and 7.34 0.96 g in Sarda and Saanen goats,
respectively. However, this difference was not statistically
signicant, probably due to the higher variability observed
for Sarda goats (t-test, P > 0.05).
In order to evaluate the size distribution of MFGs in
Sarda and Saanen dairy goats, 5 random snapshots of
washed MFG samples (n = 4 milk samples per breed, n = 20
snapshots per breed) were taken with an Olympus BX series
optical microscope, and all MFGs which were enclosed in
the snapshots (348 42) were measured and compared.
As a result, the average size of Sarda MFGs was found

Table 1
Main Raman peak assignments.a
Lipid

Raman shift
(cm1 )

Structural

Saturated fatty acid


Unsaturated fatty acid
Unsaturated fatty acid

11251133
1270
1303

Saturated fatty acid


Unsaturated fatty acid
Triacylglycerol
Acyl chains
Acyl chains
Acyl chains

1443
1654
1744
2726
2850
2885

(C C)
C C cis unsaturation
(CH2 ) twisting
unsaturation
(CH2 ) scissoring
(C C) cis unsaturation
Ester (C O)
(C H)
CH2 symmetric stretching
CH2 anti-symmetric
stretching

Adapted from Gallier et al. (2011).

144

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151

Fig. 1. Raman images and size distribution of milk fat globules in Sarda and Saanen goats. Upper panel. Optical microscopy images of washed Sarda (A)
and Saanen (B) MFGs, 50 magnication. Lower panel. Size distribution of MFGs in Sarda and Saanen milk (C).

spectra obtained for MFGs of the same size were similar


between the two breeds. Conversely, several differences
could be observed when comparing spectra generated by
MFGs of different sizes, both within and among breeds.
Using the same peak attributions, the level of unsaturation
was also determined on the three MFG size classes for both
breeds. According to the method proposed by Gallier et al.
(2011), it was calculated as the ratio of the band intensity
at 1654 (unsaturated fatty acids) versus either of the two
band intensities at 1443 and 1744 cm1 (corresponding to
the CH2 scissoring mode of saturated fatty acids and to
the triglyceride band, respectively). As a result, a decrease
in the level of unsaturation was observed at increasing
globule size, in agreement with a higher contribution of
the triglyceride core in larger MFGs. In fact, as shown in
Fig. 2, both parameters used for evaluating the degree of
unsaturation demonstrated a decreasing trend with larger
diameters.

3.3. Proteomic characterization by SDS-PAGE followed


by liquid chromatographytandem mass spectrometry
(GeLCMS/MS)
In order to perform a differential characterization of the
MFG protein makeup in the two goat breeds, MFG proteins
(MFGPs) were extracted and analyzed by GeLCMS/MS.
This approach entails protein separation by SDS-PAGE,
slicing of the whole electrophoretic lane, in-gel digestion of proteins from each slice, and peptide analysis by
LCMS/MS, followed by merging and analysis of proteomic
data. GeLCMS/MS is a powerful and robust approach for
studying the whole proteome; moreover, being based on
denaturing electrophoresis in presence of strong detergents for sample prefractionation, it is particularly suited
for the analysis of MFGPs, where a considerable portion
of membrane and highly hydrophobic proteins are present
(Pisanu et al., 2011a).

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151

145

Table 2
List of identied proteins in alphabetical order and relative abundance by the normalized spectral abundance factor (Acc. N., UniProtKB accession number;
MW, molecular weight in kDa; NSAF, normalized spectral abundance factor; Saa, Saanen; Sar, Sarda).
Acc. N.

Protein ID

Species

MW

P61981
P63103
P56937
P63325
Q3T0X6
Q3T0R1
P46791
P55832
Q5E988
P05388
Q9XSI3
Q3T087
P79324
Q2YGT9
O95336
A7YY28
P11497
P48975
Q5E9B5
O46373
P84080
Q3SZF2
P62330
Q3SXC5
Q5EA19
Q3ZCJ2
P12814
P04653
P04654
P54920
P46193
P04272
Q3SWX7
P13214
Q4R4H7
Q4GZT4
P11839
P02756
P18892
Q17QE5
P30932
Q96AQ7
O60543
Q2KJ93
P49454
A7YH32
P18760
Q4V8B5
Q0VCU1
Q96LJ7
A6QPY0
A2Q0Z0
Q717R8
A0SXL6
P27105
Q95135
Q5BK32
Q71SP7
Q4TZH2
Q28372
P68265
P81447
A6H7F6
P01112
Q07983
Q3T054
P04897

14-3-3 protein gamma


14-3-3 protein zeta/delta
3-Keto-steroid reductase
40S ribosomal protein S10
40S ribosomal protein S16
40S ribosomal protein S18
40S ribosomal protein S2 (Fragment)
40S ribosomal protein S4 (Fragment)
40S ribosomal protein S5
60S acidic ribosomal protein P0
60S ribosomal protein L10
60S ribosomal protein L11
60S ribosomal protein L15 (Fragment)
60S ribosomal protein L6
6-Phosphogluconolactonase
Abhydrolase domain-containing protein 14B
Acetyl-CoA carboxylase 1
Actin, cytoplasmic 1
Actin, gamma-enteric smooth muscle
ADP/ATP translocase 1
ADP-ribosylation factor 1
ADP-ribosylation factor 4
ADP-ribosylation factor 6
ADP-ribosylation factor-like protein 14
ADP-ribosylation factor-like protein 15
Alcohol dehydrogenase [NADP+]
Alpha-actinin-1
Alpha-S1-casein
Alpha-S2-casein
Alpha-soluble NSF attachment protein
Annexin A1
Annexin A2
Annexin A3
Annexin A4
Annexin A5
ATP-binding cassette sub-family G member 2
Beta-casein
Beta-lactoglobulin
Butyrophilin subfamily 1 member A1
Calcium and integrin-binding protein 1
CD9 antigen
Cell death activator CIDE-3
Cell death activator CIDE-A
Cell division control protein 42 homolog
Centromere protein F
Cingulin
Colin-1
Coiled-coil domain-containing protein 116
Cytoplasmic aconitate hydratase
Dehydrogenase/reductase SDR family member 1
Dolichyl-diphosphooligosaccharide-glycosyltransf.
Elongation factor 1-alpha 1
Elongation factor 1-delta
Elongation factor 2
Erythrocyte band 7 integral membrane protein
Excitatory amino acid transporter 3
FAS-associated factor 2
Fatty acid synthase
Fatty acid-binding protein, heart
Gelsolin
Glycolipid transfer protein
Glycosylation-dependent cell adhesion molecule 1
Golgi phosphoprotein 3-like
GTPase HRas
GTPase KRas
GTP-binding nuclear protein Ran
GTP-binding protein G(i) subunit alpha-2

Homo sapiens
Bos taurus
Homo sapiens
Mus musculus
Bos taurus
Bos taurus
Cricetulus griseus
Equus caballus
Bos taurus
Homo sapiens
Bos taurus
Bos taurus
Sus scrofa
Sus scrofa
Homo sapiens
Bos taurus
Rattus norvegicus
Cricetulus griseus
Bos taurus
Oryctolagus cuniculus
Bos taurus
Bos taurus
Homo sapiens
Mus musculus
Bos taurus
Bos taurus
Homo sapiens
Ovis aries
Ovis aries
Homo sapiens
Bos taurus
Bos taurus
Bos taurus
Bos taurus
Macaca fascicularis
Bos taurus
Ovis aries
Capra hircus
Bos taurus
Bos taurus
Bos taurus
Homo sapiens
Homo sapiens
Bos taurus
Homo sapiens
Canis familiaris
Mus musculus
Rattus norvegicus
Bos taurus
Homo sapiens
Bos taurus
Equus caballus
Ovis aries
Callithrix jacchus
Homo sapiens
Bos taurus
Rattus norvegicus
Bos taurus
Bos mutus grunniens
Equus caballus
Bos taurus
Capra hircus
Bos taurus
Homo sapiens
Monodelphis domestica
Bos taurus
Rattus norvegicus

28
28
38
19
16
18
22
22
23
34
25
20
18
32
28
22
265
42
42
33
21
21
20
22
23
37
103
24
26
33
39
39
36
36
36
73
25
20
59
22
25
27
25
21
368
135
19
60
98
34
49
50
31
95
32
57
41
274
15
81
24
17
33
21
21
24
40

NSAF
Saa1
8
6
1
0
0
0
2
0
2
0
0
0
3
0
0
0
1
10
6
0
0
5
0
24
9
3
0
53
14
2
7
6
0
1
4
32
17
5
374
5
11
14
28
23
0
2
6
5
0
6
0
6
0
1
82
1
0
9
33
0
0
0
2
13
12
2
4

Saa2
6
4
0
0
0
0
0
0
0
0
0
0
3
0
0
3
0
8
7
0
0
3
0
8
5
3
0
19
33
0
5
8
2
7
10
54
12
0
346
0
12
4
19
17
0
0
0
2
0
12
0
9
0
1
72
0
0
4
4
0
2
28
0
14
19
0
1

Sar1
2
0
0
0
0
4
0
6
5
0
0
3
0
2
2
0
0
30
21
2
24
24
0
29
0
2
1
15
26
0
5
10
2
9
3
75
23
0
317
11
12
7
71
21
1
0
17
7
1
11
1
19
0
0
100
1
2
16
8
2
0
7
0
3
6
0
5

Sar2
6
6
0
3
3
3
0
2
5
2
2
0
0
0
0
0
0
32
23
0
26
15
5
19
0
3
1
17
32
3
7
5
0
7
3
58
19
5
325
17
6
6
73
12
0
0
9
16
1
8
0
14
3
0
94
0
1
9
7
0
0
12
0
2
2
0
8

146

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151

Table 2 (Continued)
Acc. N.

Protein ID

Species

MW

P62871
P11017
P08753
P04896
Q3T0D7
Q3T0T7
P27601
O95837
A2Q0Z1
Q3T149
P02252
Q96KK5
P16104
O60814
Q32L48
P68432
P62803
Q13325
O75874
P02670
Q95114
Q29477
P48449
Q9D1G5
Q5E9X4
Q9JID6
Q865V6
P08582
Q8WML4
Q8MI01
Q29RW1
P07514
A3KN22
B5T255
P62935
P23284
Q9TUM6
Q5E947
Q5RC63
P00558
Q9NRY7
P26201
Q15102
Q3T067
P02584
Q3UUY6
Q5PQS4
P82197
P62998
P24409
P62490
P61106
P35293
P62822
Q06AU7
E2RQ15
P05712
Q15286
O95716
P18066
P61020
Q58DS9
P09527
P11233
P61223
Q06AU2
D3Z8L7
P62070
Q99P72

GTP-binding protein G(I)/G(S)/G(T) subunit beta-1


GTP-binding protein G(I)/G(S)/G(T) subunit beta-2
GTP-binding protein G(k) subunit alpha
GTP-binding protein G(s) subunit alpha isoforms
GTP-binding protein SAR1a
GTP-binding protein SAR1b
Guanine nucleotide-binding protein subunit alpha-13
Guanine nucleotide-binding protein subunit alpha-14
Heat shock cognate 71 kDa protein
Heat shock protein beta-1
Histone H1.4 (Fragment)
Histone H2A type 1-H
Histone H2A.x
Histone H2B type 1-K
Histone H2B type 1-N
Histone H3.1
Histone H4
IFN-induced protein with tetratricopeptide repeats 5
Isocitrate dehydrogenase [NADP] cytoplasmic
Kappa-casein
Lactadherin
Lactotransferrin
Lanosterol synthase
Leucine-rich repeat-containing protein 57
Leucine-rich repeat-containing protein 59
Long-chain-fatty-acid CoA ligase 1
Macrophage-capping protein
Melanotransferrin
Mucin-1
Mucin-15
Myosin-4
NADH-cytochrome b5 reductase 3
NEDD8-conjugating enzyme Ubc12
Peptidoglycan recognition protein 1
Peptidyl-prolyl cis-trans isomerase A
Peptidyl-prolyl cis-trans isomerase B
Perilipin-2
Peroxiredoxin-1
Peroxiredoxin-2
Phosphoglycerate kinase 1
Phospholipid scramblase 2
Platelet glycoprotein 4
PLT-act. factor acetylhydrolase IB subunit gamma
Probable saccharopine dehydrogenase
Prolin-1
Prominin-2
PTB domain-containing engulfment adapter protein 1
Pyridoxal kinase
Ras-related C3 botulinum toxin substrate 1
Ras-related protein Rab-10
Ras-related protein Rab-11A
Ras-related protein Rab-14
Ras-related protein Rab-18
Ras-related protein Rab-1A
Ras-related protein Rab-1B
Ras-related protein Rab-25
Ras-related protein Rab-2A
Ras-related protein Rab-35
Ras-related protein Rab-3D
Ras-related protein Rab-5A
Ras-related protein Rab-5B
Ras-related protein Rab-5C
Ras-related protein Rab-7a
Ras-related protein Ral-A
Ras-related protein Rap-1b
Ras-related protein Rap-2a
Ras-related protein R-Ras
Ras-related protein R-Ras2
Reticulon-4

Bos taurus
Bos taurus
Rattus norvegicus
Bos taurus
Bos taurus
Bos taurus
Mus musculus
Homo sapiens
Equus caballus
Bos taurus
Oryctolagus cuniculus
Homo sapiens
Homo sapiens
Homo sapiens
Bos taurus
Bos taurus
Bos taurus
Homo sapiens
Homo sapiens
Capra hircus
Bos taurus
Capra hircus
Homo sapiens
Mus musculus
Bos taurus
Cavia porcellus
Bos taurus
Homo sapiens
Bos taurus
Bos taurus
Rattus norvegicus
Bos taurus
Bos taurus
Bos indicus
Bos taurus
Homo sapiens
Bos taurus
Bos taurus
Pongo abelii
Homo sapiens
Homo sapiens
Bos taurus
Homo sapiens
Bos taurus
Bos taurus
Mus musculus
Rattus norvegicus
Ovis aries
Bos taurus
Canis familiaris
Canis familiaris
Homo sapiens
Mus musculus
Canis familiaris
Sus scrofa
Canis familiaris
Rattus norvegicus
Homo sapiens
Homo sapiens
Canis familiaris
Homo sapiens
Bos taurus
Rattus norvegicus
Homo sapiens
Bos taurus
Sus scrofa
Rattus norvegicus
Homo sapiens
Mus musculus

37
37
40
46
22
22
44
42
71
22
7
14
15
14
14
15
11
56
47
21
47
77
83
27
35
78
39
80
58
36
223
34
21
21
18
24
49
22
19
45
26
53
26
47
15
93
34
35
21
23
24
24
23
23
22
24
24
23
24
24
24
23
23
24
21
20
24
23
127

NSAF
Saa1
11
13
5
6
10
10
4
1
0
0
7
8
4
4
4
0
42
0
0
32
35
4
0
4
0
2
0
2
1
7
0
0
3
8
21
0
187
5
3
0
0
14
2
0
0
1
2
2
37
19
4
2
63
33
22
5
0
14
7
27
29
30
14
7
13
0
4
5
0

Saa2
20
14
1
1
24
18
0
0
0
3
8
8
4
25
17
4
16
1
0
14
55
1
0
7
0
1
0
7
0
5
0
10
0
3
10
0
205
8
0
1
2
10
0
5
0
0
0
0
33
26
17
2
77
39
27
2
7
18
0
12
17
23
30
17
25
6
2
0
0

Sar1
8
5
6
1
17
6
0
0
2
3
0
0
0
13
9
8
5
1
0
17
25
2
0
0
2
5
0
2
2
0
0
5
0
0
0
3
193
3
0
0
0
5
2
3
4
0
0
2
17
17
13
0
32
25
17
3
3
14
3
13
18
21
8
8
6
0
3
0
0

Sar2
13
13
8
2
24
21
4
1
1
2
0
4
3
15
11
3
9
0
3
17
25
3
1
2
0
5
1
4
1
0
0
3
0
0
12
0
218
17
0
0
0
10
0
2
4
0
0
0
10
21
7
0
25
33
24
2
2
16
4
11
18
14
16
2
18
0
0
0
0

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151

147

Table 2 (Continued)
Acc. N.

Protein ID

Species

MW

P19803
Q61085
Q3ZBW5
Q1RMJ6
Q9HBH0
Q2KJ32
P42819
Q53HV7
Q27960
P19623
Q3ZBE9
Q99536
Q4R6M4
Q2LGB5
B2LT62
P61585
P40142
P49020
O35587
P07948
Q865C5
P30085
O35156
P80457
Q91W10

Rho GDP-dissociation inhibitor 1


Rhophilin-1
Rho-related GTP-binding protein RhoB
Rho-related GTP-binding protein RhoC
Rho-related GTP-binding protein RhoF
Selenium-binding protein 1
Serum amyloid A protein
SS selective monofunctional uracil DNA glycosylase
Sodium-dependent phosphate transport protein 2B
Spermidine synthase
Sterol-4-alpha-carboxylate 3-dehydrogenase
Synaptic vesicle membrane protein VAT-1 homolog
Tetratricopeptide repeat protein 29
Toll-interacting protein
Toll-like receptor 2
Transforming protein RhoA
Transketolase
Transmembrane emp24 domain-containing protein 2
Transmembrane protein Tmp21
Tyrosine-protein kinase Lyn
Ubiquitin
UMP-CMP kinase
UTPglucose-1-phosphate uridylyltransferase
Xanthine dehydrogenase/oxidase
Zinc transporter ZIP8

Bos taurus
Mus musculus
Bos taurus
Bos taurus
Homo sapiens
Bos taurus
Ovis aries
Homo sapiens
Bos taurus
Homo sapiens
Bos taurus
Homo sapiens
Macaca fascicularis
Bos taurus
Capra ibex
Bos taurus
Mus musculus
Cricetulus griseus
Mesocricetus auratus
Homo sapiens
Camelus dromedarius
Homo sapiens
Cricetulus griseus
Bos taurus
Mus musculus

23
71
22
22
24
53
13
30
76
34
40
42
55
30
90
22
68
22
25
59
9
22
57
147
50

NSAF
Saa1

Fig. 3 displays the electrophoretic prole obtained for


Sarda and Saanen MFGPs. In spite of a generally comparable protein prole for the two goat breeds, inter-breed
differences could be appreciated in the abundance of some
bands. Mass spectrometry analysis led to identication of
161 unique proteins (Table 2). To enable evaluation of
inter-breed differences in expression levels, proteins were
classied according to cellular localization and normalized
spectral abundance (Pham et al., 2010) (Table S1 reports
all the details concerning protein identications for each
sample examined, and Fig. S1 illustrates the total protein
proles of the gels used for GeLCMS/MS). As shown in
Fig. 4, there was a signicantly higher expression of cytoplasmic proteins in Sarda MFGs goats to Saanen MFGs,
with an inverse behavior for membrane proteins (Fig. 4A).
Accordingly, in terms of single proteins (Fig. 4B), it was
interesting to observe the higher abundance of typical
proteins of the MFG surface, such as butyrophilin (BTN),
lactadherin and platelet glycoprotein 4 (PAS-4), in Saanen goats, as opposed to the higher abundance of proteins
from the internal MFG membranes and cytoplasm, such
as xanthine dehydrogenase (XO), perilipin-2 (adipophilin,
ADP), stomatin, actin, and fatty acid synthase (FAS), in Sarda
goats. Interestingly, the BTN/XO ratio was signicantly different for the two breeds: 1.56 for Saanen and 1.04 for
Sarda.
4. Discussion
Goat milk is an important nutritional resource in view of
its high digestibility and tolerability, and its physicochemical characteristics make it one of the preferred human
milk substitutes (Haenlein, 2004). Currently, one of the
most popular dairy goat breeds is the Saanen, a large and

7
0
5
19
9
0
0
0
1
2
1
0
1
0
1
47
1
0
0
2
19
2
0
225
2

Saa2
3
0
8
32
0
0
0
0
5
0
0
1
0
0
1
35
0
0
0
0
21
0
0
238
0

Sar1
3
0
6
17
5
0
0
2
2
0
5
1
0
0
0
34
0
6
5
0
29
0
0
338
0

Sar2
0
1
5
14
4
3
4
0
2
0
1
4
0
2
0
17
0
2
2
0
25
0
2
282
2

highly productive animal. However, in spite of a lower


productivity, milk from minor goat breeds should not be
underestimated in terms of specic nutritional attributes
or preferable technological features. Among other milk
components, MFG size and composition, both in terms
of proteins and lipids, can impact on both aspects considerably (Dewettinck et al., 2008; Michalski et al., 2002;
Michalski and Januel, 2006), since differently sized MFGs
have different physicochemical properties and different
lipid compositions (Michalski et al., 2004). As far as direct
consumption is concerned, smaller MFGs make milk more
digestible and tolerated, being more accessible to digestive
enzymes and containing a lower proportion of saturated
fatty acids (Haenlein, 2004; Jenness, 1980), although pasteurization and homogenization can have an impact on
these aspects. Concerning dairy products, milk is strongly
conditioned by the size of MFGs. Smaller globules require
more pressure and time to be broken down during churning
(Couvreur and Hurtaud, 2007); therefore, butter made from
smaller globules contains more unbroken MFG and more
water, and is therefore more spreadable. The same applies
to cheese (Goudedranche et al., 2000), making cheese produced from milk with smaller MFGs softer than cheese
made from milk with larger MFGs. Moreover, small globules are more accessible to bacterial enzymes during milk
fermentation and cheese ripening, impacting avor and
texture of the nal product. On the other hand, larger MFGs
make milk more suitable for creaming, since small MFGs
will have a reduced ability to be whipped compared to
larger ones (Walstra et al., 1999).
Upon comparison of MFGs in Saanen and Sarda goats,
the rst and most relevant observation emerged in this
study is the signicant difference in the average sizes of
MFGs between the two breeds. In fact, MFGs of Sarda

148

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151

Fig. 2. Qualitative evaluation of milk fat globule composition by Raman spectrometry. Upper panel: Raman spectra of milk fat globules measuring < 5 (15),
10, and 15 m. Lower panel: Level of unsaturation in MFGs measuring < 5 (15), 10, and 15 m. A, B: Sarda; C, D: Saanen.

goats are considerably smaller compared to Saanen goats


(2.73 0.15 m vs. 3.63 0.27 m), with a higher uniformity of MFG average sizes in the range between 2 and 3 m,
in contrast to Saanen goats where size distribution is more
heterogeneous. A number of studies carried out on milk
of other goat breeds showed breed-dependent differences
in MFG size, and an average size of 2.76 m and 3.07 m
was reported in French-Alpine goats (Attaie and Richter,
2000) and Murciana-Granadina goats (Zamora et al., 2009),
respectively. With their mean diameter of 2.73 m, MFGs
of Sarda goats are the smallest reported so far.
To understand how the variation in the average size of
MFGs is reected in their relative chemical composition,
an approach based on Raman spectroscopy was used. In
fact, by combining microscopy and Raman spectroscopy
techniques, milk fat globules of different size ranges can
be proled without altering the sample to attain their
physical separation, which would be required for other
more detailed characterization approaches such as GCMS.
Since our aim was to assess whether general differences in
lipid composition were present among MFG size classes
in the two breeds, the choice fell on this non-destructive,

explorative technique. According to the studies of Forrest


(1978) and the more recent application by Gallier et al.
(2011) on cow milk, it is possible to associate specic peaks
in the Raman spectrum, as well as their ratios, to signals generated by saturated and unsaturated fatty acids.
Based on their results, we assessed how these signals
changed according to MFG size and goat breed, observing that the lipid composition of MFGs is subjected to
changes which might be more size-dependent, rather than
breed-dependent. In fact, when MFGs of different sizes are
compared within the same breed, differences in the Raman
spectrum can be appreciated, while spectra belonging to
MFGs of similar sizes appear to be highly similar. Moreover,
a concurrent decrease in the degree of unsaturation was
observed at increasing MFG sizes in both breeds. This can be
explained with the higher contribution of the triglyceride
core versus the external phospholipid bilayer and internal
monolayer components, mostly composed by unsaturated
fatty acids. These observations are in agreement with the
study of Gallier et al. (2011), where the same trend was
observed for MFGs from Friesian and FriesianJersey cows.
In fact, smaller globules have a higher membrane to core

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151

149

Fig. 4. Classication of protein abundance by localization and relative


abundance of milk fat globule proteins in Saanen and Sarda goats. (A) Cellular localization and (B) relative abundance of the main MFGPs. Averaged
values (AVG) and SD are shown for the samples subjected to GeLCMS/MS.

Fig. 3. Representative SDS-PAGE prole of Saanen and Sarda milk fat


globule proteins. Molecular weight markers are reported on the left. Ten
micrograms of protein was loaded in each lane.

ratio, and are therefore composed by a higher amount of


unsaturated fatty acids.
Goat MFGPs were also extensively characterized by
label-free shotgun proteomics to attain a quantitative estimate of the relative MFGP composition in the two breeds,
and to investigate whether their different MFG sizes distribution could be associated with differences in relative
expression of crucial proteins. A signicantly higher abundance of cytoplasmic proteins was observed in Sarda MFGs
compared to Saanen MFGs, with a concurrent higher abundance of membrane proteins in Saanen MFGs. Taking
into account the physiology of milk secretion, this nding might be explained with differences in the dynamics
of MFG production and release from the lactocyte. MFGs
originate as lipid droplets in the endoplasmic reticulum,
surrounded by a monolayer of proteins and polar lipids
originating from the ER membrane. During formation, the
droplets grow in size and migrate at the apical region of the

mammary epithelial cells, where they are further surrounded by the outer cell membrane and released in milk.
During this process, inclusion of cytoplasmic materials
(known as cytoplasmic crescents) may occur within the
MFG membranes; the extent of crescent inclusion in MFGs
is reported to vary from species to species, and may also
vary among different breeds within a species (Heid and
Keenan, 2005). The reason of such variability is still unclear,
although an association with genetic traits, such as the
alpha-S1-casein genotype, has been reported (Cebo et al.,
2010; Neveu et al., 2002).
The comparison of the expression levels of typical
MGFPs highlighted differences in the expression levels of
two proteins which are crucial for MFG secretion, BTN and
XO. Specically, these two proteins are thought to mediate
docking of cytoplasmic droplets on the cell membrane by
interacting with perilipins (also known as adipophilin and
TIP 47) located on surface of the fat droplets, and to mediate
their release from the cell by means of a zipper mechanism (Heid and Keenan, 2005). Other authors have reported
BTNBTN interactions occurring between the cytoplasmic
membrane and the lipid core membrane as the main drive
for MFG secretion (Robenek et al., 2006). These two fundamental MFG proteins are present in different amounts

150

S. Pisanu et al. / Small Ruminant Research 109 (2013) 141151

in Sarda and Saanen; specically, BTN is more abundant


in Saanen, while XO is more abundant in Sarda, leading
to a different ratio between the two (1.56 vs. 1.04, respectively). Differences in the BTN/XO amount and ratio and
their correlations with MFG secretion and diameter have
been discussed by several authors (Mondy and Keenan,
1993; Huston and Patton, 1990; Zamora et al., 2009), and
expression of the XO gene is known to be tightly linked with
MFG secretion (Mc Manaman et al., 2002). Moreover, it has
been reported that abundance and distribution of BTN and
XO are also important factors in globule crescent formation (Huston and Patton, 1990). It is tempting to speculate
that a breed-dependent genetic variability in the relative
expression levels of these two key proteins might be a crucial factor in determining the nal size distribution and the
structural differences observed in MFGs of the two breeds
under study.
The heterogeneity in MFG size depends on the genetic
variability inherent in different goat breeds, although MFG
size might also be affected by environmental factors that
inuence physical and chemical properties of milk and its
production. The two breeds evaluated in this study belong
to two extremes within the species. In fact, Saanen and
Sarda goats have been subjected to considerably different
selective pressures, dictated by the different availability
of resources and environmental conditions of the farming
lands: one advantaging size, milk productivity, disposition
for packing, docility, uniformity, and elevated condence
to man, versus the other advantaging frugality, sturdiness,
elevated roaming attitude, high resistance to drought, high
independence, and reduced condence to man. Concurrently, sensorial and technological traits of milk have been
subjected to selection, such as curd texture and amount of
fat. Together with the nutritional and technological implications, it would be interesting to investigate further on
the impact of this millennial selection operated by man
for milk quality and productivity on the efciency of the
milk fat globule secretion mechanism, especially when
considering that milk fat globules from cows, the most
specialized and selected milk factory, show the highest
triglyceride core/phospholipid ratio, as well as the highest
membrane/cytoplasmic protein ratio, and the highest size
and composition homogeneity, of all dairy species. Interestingly, a recent study compared the MFGs of Chianina
and Holstein cows, a typical meat and dairy breed, respectively, reporting differences in their proteomic makeup
(Murgiano et al., 2009). It may be hypothesized that highly
selected dairy species, and highly selected dairy breeds
within one species, might have more efcient milk fat
globule secretion mechanisms, as opposed to species and
breeds not exposed to a selective pressure for the dairy
attitude, which show a higher heterogeneity in size and
composition, as well as higher proportions of cytoplasmic
materials in their MFGs.
5. Conclusions
MFGs in Sarda milk are signicantly smaller than in
Saanen milk. However, when taken individually, globules
of the same size class show a very high inter-breed level
of similarity. On a general scale, MFGPs show signicant

differences in expression levels between the two breeds,


with membrane proteins and MFGM surface proteins being
more represented in Saanen, and cytoplasmic and internal
MFGM proteins being more represented in Sarda. Therefore, the different MFG size distribution is most likely
responsible for a different total MFG protein and lipid composition in milk and milk products from the two breeds.
The genetic or environmental drives for such differences
remain to be elucidated.
Acknowledgement
This work was supported by funding from Regione
Autonoma della Sardegna Progetto Cluster Proteomica.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.smallrumres.2012.07.024.
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