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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
142
3. Results
143
Table 1
Main Raman peak assignments.a
Lipid
Raman shift
(cm1 )
Structural
11251133
1270
1303
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
144
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).
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
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
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
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
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
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
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
148
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.
149
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
151