3 Milk lipids
3.1 Introduction
The milks of all mammals contain lipids but the concentration varies widely
between species from c. 2% to greater than 50% (Table 3.1). The principal
function of dietary lipids is to serve as a source of energy for the neonate
and the fat content in milk largely reflects the energy requirements of the
species, e.g. land animals indigenous to cold environments and marine
mammals secrete high levels of lipids in their milks.
Milk lipids are also important:
1. as a source of essential fatty acids (i.e. fatty acids which cannot be
synthesized by higher animals, especially linoleic acid, &) and fat-
soluble vitamins (A, D, E, K); and
2. for the flavour and rheological properties of dairy products and foods in
which they are used.
Because of its wide range of fatty acids, the flavour of milk fat is superior to
that of other fats. In certain products and after certain processes, fatty acids
serve as precursors of very flavourful compounds such as methyl ketones
and lactones. Unfortunately, lipids also serve as precursors of compounds
Table 3.1 The fat content of milks from various species (g I-')
Species Fat content Species Fat content
cow
Buffalo
Sheep
Goat
Dall-sheep
Moose
Antelope
Elephant
Human
Horse
Monkeys
Lemurs
Pig
Musk-ox
33-47
47
40-99
41 -45
109
32-206
39-105
93
85-190
38
19
10-51
8-33
68
Marmoset
Rabbit
Guinea-pig
Snowshoe hare
Muskrat
Mink
Chinchilla
Rat
Red kangaroo
Dolphin
Manatee
Pygmy sperm whale
Harp seal
Bear (four species)
77
183
39
71
110
134
117
103
9-119
62-330
55-215
502- 5 32
108-331
153
From Christie (1995).
68 DAIRY CHEMISTRY AND BIOCHEMISTRY
that cause off-flavour defects (hydrolytic and oxidative rancidity) and as
solvents for compounds in the environment which may cause off-flavours.
For many years, the economic value of milk was based mainly or totally
on its fat content, which is still true in some cases. This practice was
satisfactory when milk was used mainly or solely for butter production.
Possibly, the origin of paying for milk on the basis of its fat content, apart
from its value for butter production, lies in the fact that relatively simple
quantitative analytical methods were developed for fat earlier than for
protein or lactose. Because of its economic value, there has long been
commercial pressure to increase the yield of milk fat per cow by nutritional
or genetic means.
To facilitate the reader, the nomenclature, structure and properties of the
principal fatty acids and of the principal lipid classes are summarized in
Appendices 3A, 3B and 3C. The structure and properties of the fat-soluble
vitamins, A, D, E and K, are discussed in Chapter 6.
3.2 Factors that affect the fat content of bovine milk
Bovine milk typically contains c. 3.5% fat but the level varies widely,
depending on several factors. including: breed, individuality of the animal,
stage of lactation, season, nutritional status, type of feed, health and age of
the animal, interval between milkings and the point during milking when
the sample is taken.
Of the common European breeds, milk from Jersey cows contains the
highest level of fat and that from Holstein/Friesians the lowest (Figure 3.1).
The data in Figure 3.1 also show the very wide range of fat content in
individual-cow samples.
The fat content of milk decreases during the first 4-6 weeks after
parturition and then increases steadily throughout the remainder of lacta-
tion, especially toward the end (Figure 3.2). For any particular population,
fat content is highest in winter and lowest in summer, due partly to the effect
of environmental temperature. Production of creamery (manufacturing)
milk in Ireland, New Zealand and parts of Australia is very seasonal;
lactational, seasonal and possibly nutritional effects coincide, leading to
large seasonal changes in the fat content of milk (Figure 3.3), and also in
the levels of protein and lactose.
For any individual animal, fat content decreases slightly during success-
ive lactations, by c. 0.2% over a typical productive lifetime (about five
lactations). In practice, this factor usually has no overall effect on the fat
content of a bulk milk supply because herds normally include cows of
various ages. The concentration of fat (and of all other milk-specific
constituents) decreases markedly on mastitic infection due to impaired
MILK LIPIDS
I
35
-
30 -
B
f - 25-
0
%
3
-
S
s 20-
r
0
15-
2
10
-
5-
69
Percentage fat
Figure 3.1 Range of fat content in the milk of individual cows of four breeds (from Jenness and
Patton, 1959).
synthesizing ability of the mammary tissue; the effect is clear-cut in the case
of clinical mastitis but is less so for subclinical infection.
Milk yield is reduced by underfeeding but the concentration of fat usually
increases, with little effect on the amount of fat produced. Diets low in
roughage have a marked depressing effect on the fat content of milk, with
little effect on milk yield. Ruminants synthesize milk fat mainly from
carbohydrate-derived precursors; addition of fat to the diet usually causes
slight increases in the yield of both milk and fat, with little effect on fat
content of milk. Feeding of some fish oils (e.g. cod liver oil, in an effort to
increase the concentrations of vitamins A and D in milk) has a very marked
(c. 25%) depressing effect on the fat content of milk, apparently due to the
high level of polyunsaturated fatty acids (the effect is eliminated by hydro-
genation), although oils from some fish species do not cause this effect.
70 DAIRY CHEMISTRY AND BIOCHEMISTRY
5.0
-
0
L.
2
4.0
3.0
0 10 20 30 40 50
Week of lactation
Figure 3.2 Typical changes in the concentrations of fat (O), protein (m) and
bovine milk during lactation.
4.6 -
4.4 -
4.2 -
4.0 -
3.8 -
3.6 -
3.4 -
lactose (0) in
JFMAMJJASOND
Month
Figure 3.3 Seasonal changes in the fat content of bovine milk in some European countries:
(Denmark (O), Netherlands (O), United Kingdom (O), France (U), Germany (A), Ireland (A)
(From An Foras Taluntais, 1981.)
MILK LIPIDS 71
The quarters of a cow’s udder are anatomically separate and secrete milk
of markedly different composition. The fat content of milk increases continu-
ously throughout the milking process while the concentrations of the
various non-fat constituents show no change; fat globules appear to be
partially trapped in the alveoli and their passage is hindered. If a cow is
incompletely milked, the fat content of the milk obtained at that milking will
be reduced; the ‘trapped’ fat will be expressed at the subsequent milking,
giving an artificially high value for fat content.
If the intervals between milkings are unequal (as they usually are in
commercial farming), the yield of milk is higher and its fat content lower
after the longer interval; the content of non-fat solids is not influenced by
milking interval.
3.3 Classes of lipids in milk
Triacylglycerols (triglycerides) represent 97-98% of the total lipids in the
milks of most species (Table 3.2). The diglycerides probably represent
incompletely synthesized lipids in most cases, although the value for the rat
probably also includes partially hydrolysed triglycerides, as indicated by the
high concentration of free fatty acids, suggesting damage to the milk fat
globule membrane (MFGM) during milking and storage. The very high
level of phospholipids in mink milk probably indicates the presence of
mammary cell membranes.
Although phospholipids represent less than 1% of total lipid, they play a
particularly important role, being present mainly in the MFGM and other
membraneous material in milk. The principal phospholipids are phos-
phatidylcholine, phosphatidylethanolamine and sphingomyelin (Table 3.3).
Trace amounts of other polar lipids, including ceramides, cerobrosides and
gangliosides, are also present. Phospholipids represent a considerable pro-
portion of the total lipid of buttermilk and skim milk (Table 3.4), reflecting
Table 3.2
species (weight YO of the total lipids)
Composition of individual simple lipids and total phospholipids in milks of some
Lipid class
Triacylgl ycerols
Diacylglycerols
Monoacylgl ycerols
Cholesteryl esters
Cholesterol
Free fatty acids
Phospholipids
cow Buffalo Human Pig Rat Mink
97.5
0.36
0.027
T
0.31
0.027
0.6
98.6 98.2
0.7
T
0.1 T
0.3 0.25
0.5 0.4
0.5 0.26
96.8
0.7
0.1
0.06
0.6
0.2
1.6
87.5
2.9
0.4
1.6
3.1
0.7
-
81.3
1.7
T
T
T
1.3
15.3
From Christie (1995). T, Trace.
Table 3.3 Composition of the phospholipids in milk from various species (expressed as mol YO of total lipid phosphorus)
Phosphatidyl- Phosphatidyl- Phosphatidyl- Phosphatidyl- Lysophospho-
Species choline ethanolamine serine inositol Sphingomyelin lipids“
cow
Sheep
Buffalo
Goat
Camel
Ass
Pig
Human
Cat
Rat
Guinea-pig
Rabbit
Mouse‘
Mink
34.5
29.2
27.8
25.7
24.0
26.3
21.6
27.9
25.8
38.0
35.7
32.6
32.8
52.8
31.8
36.0
29.6
33.2
35.9
32.1
36.8
25.9
22.0
31.6
38.0
30.0
39.8
10.0
3.1
3.1
3.9
6.9
4.9
3.1
3.4
5.8
2.7
3.2
3.2
5.2
10.8
3.6
4.1
3.4
4.2
5Ab
5.9
3.8
3.3
4.2
7.8b
4.9
7.1b
5.8’
3.6
6.6
25.2
28.3
32.1
21.9
28.3
34.1
34.9
31.1
31.9
19.2
11.0
24.9
12.5
15.3
0.8
2.4
0.5
1 .o
5.1
3.4
3.1
2.0
0.4
8.3
“Mainly lysophosphatidylcholine but also lysophosphatidylethanolamine.
bAlso contains lysophosphatidylethanolamine.
‘Analysis of milk fat globule membrane phospholipids.
From Christie (1995).
MILK LIPIDS 73
Table 3.4 Total fat and phospholipid content of some milk products
Total lipid Phospholipids Phospholipid as
Product (%. WIV) (%, WIV) YO, w/w, of total lipids
Whole milk 3-5
Cream 10-50
Butter 81-82
Butter oil - 100
Skim milk 0.03-0.1
Buttermilk 2
0.02-0.04 0.6- 1 .O
0.07-0. I8 0.3-0.4
0.14-0.25 0.16-0.29
0.02-0.08 0.02-0.08
0.01-0.06 17-30
0.03 -0.18 10
the presence of proportionately larger amounts of membrane material in
these products.
Cholesterol (Appendix 3C) is the principal sterol in milk (> 95% of total
sterols); the level (-O.3%, w/w, of total lipids) is low compared with many
other foods. Most of the cholesterol is in the free form, with less than 10%
as cholesteryl esters. Several other sterols, including steroid hormones, occur
at trace levels.
Several hydrocarbons occur in milk in trace amounts. Of these, caro-
tenoids are the most significant. In quantitative terms, carotenes occur at
only trace levels in milk (typically -2OOpg1-') but they contribute
10-50% of the vitamin A activity in milk (Table 3.5) and are responsible for
the yellow colour of milk fat. The carotenoid content of milk varies with
breed (milk from Channel Island breeds contains 2-3 times as much
p-carotene as milk from other breeds) and very markedly with season
(Figure 3.4). The latter reflects differences in the carotenoid content of the
diet (since they are totally derived from the diet); fresh pasture, especially if
it is rich in clover and alfalfa, is much richer in carotenoids than hay or
silage (due to oxidation on conservation) or cereal-based concentrates. The
higher the carotenoid content of the diet, the more yellow will be the colour
of milk and milk fat, e.g. butter from cows on pasture is yellower than that
Table 3.5 Vitamin A activity and P-carotene in milk of different breeds of cows
~ ~ ~ ~~ ~~~~~
Channel Island breeds Non-Channel Island breeds
Summer Winter Summer Winter
Retinol (pl 1- ') 649 265 619 412
j-Carotene (pl I-') 1143 266 315 105
Retinollb-carotene ratio 0.6 11.0 2.0 4.0
Contribution (%) of 46.8 33.4 20.3 11.4
p-carotene to vitamin A
activity
Modified from Cremin and Power (1985).
4 P
e,w gih rnE
Ob
-.
"I
-
Vitamin
A
(mg/100
g
butter)
-3
Y
Tocopherol
(&g
fat)
'<
-. P 2. 2
Carotene
(pg/100
ml
milk)
Vitamin
D
(IUA
milk)
P
MILK LIPIDS 75
peroxides, e.g. H,O, or benzoyl peroxide, or masked, e.g. with chlorophyll
or titanium oxide).
Milk contains significant concentrations of fat-soluble vitamins (Table
3.5, Figure 3.4) and milk and dairy products make a significant contribution
to the dietary requirements for these vitamins in Western countries. The
actual form of the fat-soluble vitamins in milk appears to be uncertain and
their concentration varies widely with breed of animal, feed and stage of
lactation, e.g. the vitamin A activity of colostrum is c. 30 times higher than
that of mature milk.
Several prostaglandins occur in milk but it is not known whether they
play a physiological role; they may not survive storage and processing in a
biologically active form. Human milk contains prostaglandins E and F at
concentrations 100-fold higher than human plasma and these may have a
physiological function, e.g. gut motility.
3.4 Fatty acid profile of milk lipids
Milk fats, especially ruminant fats, contain a very wide range of fatty acids:
more than 400 and 184 distinct acids have been detected in bovine and
human milk fats, respectively (Christie, 1995). However, the vast majority of
these occur at only trace concentrations. The concentrations of the principal
fatty acids in milk fats from a range of species are shown in Table 3.6.
Notable features of the fatty acid profiles of milk lipids include:
1. Ruminant milk fats contain a high level of butanoic acid (C4:o) and other
short-chain fatty acids. The method of expressing the results in Table 3.6
(Yo, w/w) under-represents the proportion of short-chain acids - if ex-
pressed as mol %, butanoic acid represents c. 10% of all fatty acids (up
to 15% in some samples), i.e. there could be a butyrate residue in c. 30%
of all triglyceride molecules. The high concentration of butyric (butanoic)
acid in ruminant milk fats arises from the direct incorporation of
P-hydroxybutyrate (which is produced by micro-organisms in the rumen
from carbohydrate and transported via the blood to the mammary gland
where it is reduced to butanoic acid). Non-ruminant milk fats contain no
butanoic or other short-chain acids; the low concentrations of butyrate
in milk fats of some monkeys and the brown bear require confirmation.
The concentration of butanoic acid in milk fat is the principle of the
widely used criterion for the detection and quantitation of adulteration
of butter with other fats, i.e. Reichert Meissl and Polenski numbers,
which are measures of the volatile water-soluble and volatile water-
insoluble fatty acids, respectively.
Short-chain fatty acids have strong, characteristic flavours and
aromas. When these acids are released by the action of lipases in milk or
Table 3.6 Principal fatty acids (wt YO of total) in milk triacylglycerols or total lipids from various species
Species 4:O 6:O 8:O 1O:O 12:O 14:O 16:O 16.1 18:O 18.1 18:2 18:3 C,,-C2,
cow
Buffalo
Sheep
Goat
Dall-sheep
Moose
Blackbuck antelope
Elephant
Human
Monkey (mean of six
species
Baboon
Lemur macaco
Horse
Pig
Rat
Guinea-pig
Marmoset
Rabbit
Cottontail rabbit
European hare
Mink
Chinchilla
Red kangaroo
Platypus
Numbat
Bottle-nosed dolphin
Manatee
Pygmy sperm whale
Harp seal
Northern elephant seal
Polar bear
Grizzly bear
Musk-ox
1.6
1.6
2.8
2.9
0.9
0.3
T
6.0
T
0.6
0.4
T
-
-
-
-
T
T
T
-
-
-
-
-
-
-
-
-
-
-
-
T
T
1.3
1.1
2.7
2.7
1.9
0.2
8.4
2.7
0.3
T
5.9
5.1
0.2
1.8
1.1
-
-
-
22.4
9.6
10.9
-
-
-
~
-
-
0.6
-
-
-
-
-
3 .O
1.9
9.0
8.4
4.7
4.9
5.5
6.5
29.4
1.3
11.0
7.9
1.9
5.1
0.7
7.0
8.0
20.1
14.3
17.7
-
-
-
-
-
-
3.5
-
-
-
T
-
3.1
2.0
5.4
3.3
2.3
1.8
0.6
3.5
18.3
3.1
4.4
2.3
10.5
6.2
0.5
7.5
8.5
2.9
3.8
5.5
0.5
T
0.1
0.1
0.3
4.0
-
-
-
-
-
0.5
0.1
9.5
8.7
11.8
10.3
6.2
10.6
2.0
11.5
5.3
5.1
2.8
1.3
15.0
5.7
4.0
8.2
2.6
7.7
1.7
2.0
5.3
3.3
3.0
2.7
1.6
0.9
3.2
6.3
3.6
5.3
2.6
3.9
2.1
26.3 2.3
30.4 3.4
25.4 3.4
24.6 2.2
19.5 1.7
23.0 2.4
28.4 4.3
39.3 5.7
12.6 3 .O
20.2 5.7
21.4 6.7
16.5 1.2
27.1 9.6
23.8 7.8
32.9 11.3
22.6 1.9
31.3 2.4
18.1 5.5
14.2 2.0
18.7 1 .o
24.8 5.0
26.1 5.2
30.0 -
31.2 6.8
19.8 13.9
14.1 3.4
21.1 13.3
20.2 11.6
27.6 9.1
13.6 17.4
14.2 5.7
18.5 16.8
16.4 3.2
14.6
10.1
9.0
12.5
23.0
15.5
4.5
5.5
0.5
5.9
4.9
4.2
1 .o
2.3
3.5
6.5
2.9
3.4
3.8
3.0
2.9
10.9
6.3
3.9
7.0
3.3
0.5
7.4
4.9
3.6
13.9
20.4
-
29.8
28.7
20.0
28.5
27.2
23.1
21.2
19.2
17.3
46.4
26.0
22.7
25.7
20.9
35.2
26.7
33.6
29.6
13.6
12.7
14.4
36.1
35.2
37.2
22.7
57.7
23.1
47.0
46.6
21.5
41.6
30.1
30.2
2.4
2.5
2.1
2.2
2.1
4.0
20.2
3.3
3.0
13.0
14.5
37.6
6.6
14.9
11.9
16.3
18.4
10.9
14.0
24.7
10.6
14.9
26.8
10.4
5.4
7.9
1.2
1.8
0.6
1.2
1.9
1.2
5.6
0.8
2.5
1.4
3.0
4.1
3.7
0.7
1.4
1.3
0.6
0.5
12.6
0.7
0.8
5.7
0.9
4.4
9.8
1.7
1.5
2.9
2.1
7.6
0.1
0.2
2.2
0.6
0.9
0.4
2.3
-
-
-
T
T
-
-
0.4
2.6
-
-
-
T
-
-
-
-
-
1.1
T
7.0
T
0.4
T
-
-
0.1
12.2
0.2
17.3
0.4
4.5
31.2
29.3
11.3
9.5
From Christie (1995).
MILK LIPIDS 77
dairy products, they impart strong flavours which are undesirable in milk
or butter (they cause hydrolytic rancidity) but they contribute to the
desirable flavour of some cheeses, e.g. Blue, Romano, Parmesan.
2. Ruminant milk fats contain low levels of polyunsaturated fatty acids
(PUFAs) in comparison with monogastric milk fats. This is because a
high proportion of the fatty acids in monogastric milk fats are derived
from dietary lipids (following digestion and absorption) via blood.
Unsaturated fatty acids in the diet of ruminants (grass contains con-
siderable levels of PUFAs) are hydrogenated by rumen micro-organisms
unless protected by encapsulation (section 3.16.1). The low level of
PUFAs in bovine milk fat is considered to be nutritionally undesir-
able.
3. The milk fats from marine mammals contain high levels of long-chain,
highly unsaturated fatty acids, presumably reflecting the requirement that
the lipids of these species remain liquid at the low temperatures of their
environments.
4. Ruminant milk fats are also rich in medium-chain fatty acids. These are
synthesized in the mammary gland via the usual malonyl CoA pathway
(section 3.5) and are released from the synthesizing enzyme complex by
thioacylases; presumably, the higher levels of medium chain acids in
ruminant milk fats compared with those of monogastric animals reflect
higher thioacylase activity in the mammary tissue of the former.
5. The fatty acid profile of bovine milk fat shows a marked seasonal pattern,
especially when cows are fed on pasture in summer. Data for Irish milk
fat are shown in Figure 3.5; the changes are particularly marked for C,:,,
C,,:, and c18:I. These changes affect the Reichert Meissl, Polenski and
iodine (a measure of unsaturation) (Figure 3.6) numbers and the melting
point and hardness (spreadability) of butter made from these milks:
winter butter, with low levels of C4:o and c18:] and a high level of C,,:, is
much harder than summer butter (Figure 3.7).
6. Unsaturated fatty acids may occur as cis or trans isomers; trans isomers,
which have higher melting points than the corresponding cis isomers, are
considered to be nutritionally undesirable. Bovine milk fat contains a low
level (5%) of trans fatty acids in comparison with chemically hydro-
genated (hardened) vegetable oils, in which the value may be 50% due to
non-stereospecific hydrogenation.
Bovine milk fat contains low concentrations of keto and hydroxy acids
(each at c. 0.3% of total fatty acids). The keto acids may have the carbonyl
group (C=O) at various positions. The 3-keto acids give rise to methyl
ketones (R-C-CH,) on heating (high concentrations of methyl ketones
are produced in blue cheeses through the oxidative activity of Penicilliurn
roqueforti). The position of the hydroxy group on the hydroxy acids also
0
//
78 DAIRY CHEMISTRY AND BIOCHEMISTRY
4-
3-
2-
. . . I I . I-,.,.,.,.
MJJASONDJFMA
13 i
12-
0
0
8
3 11-
g lo-
W
.-
cd
LL
9-
MJJASONDJFMAMJJA
Month
(b)
MILK LIPIDS 79
42 -
h Y
d? 40-
2
M
0
!l 38-
-
on
v
8
36-
$
2
5
0 34 -
MJJASONDJF
Month
Figure 3.6 Seasonal changes in the iodine number of Irish bovine milk fat (from Cullinane et
al., 1984a).
MJJASONDJFMAMJJASO
Month
Figure 3.7 Seasonal variations in the mean firmness of Irish butter at 4°C (@) or 15°C (0)
(from Cullinane et al., 1984b).
Table 3.7 The fatty acid composition of cholesteryl esters, phosphatidylcholine and phosphatidylethanolamine in the milks of some species
Fatty acid composition (wt% of the total)
Fatty acid
12:o
14:O
16:O
16: 1
18:O
18: 1
18:2
18:3
20: 3
20:4
22:6
cow Human Pig
CE PC PE CE PC PE PC PE
0.2 0.3
0.1 3.2 -
2.3 7.1 1 .o 4.8 4.5 1.1 1.8 0.4
23.1 32.2 11.4 23.8 33.7 8.5 39.9 12.4
8.8 3.4 2.7 1.5 1.7 2.4 6.3 7.3
10.6 7.5 10.3 8.0 23.1 29.1 10.3 12.3
17.1 30.1 47.0 45.7 14.0 15.8 21.8 36.2
27.1 8.9 13.5 12.4 15.6 17.7 15.9 17.8
4.2 1.4 2.3 T 1.3 4.1 1.5 1.9
0.7 1 .o 1.7 2.1 3.4 0.3 0.7
1.4 1.2 2.7 T 3.3 12.5 1.3 6.6
0.1 0.4 2.6 0.2 1.6
~
-
- - -
Mink Mouse
CE PC PE PC PE
0.3
1.1 1.3 0.8
25.4 26.4 20.6
4.4 1.1 1.2
14.7 20.8 29.3
35.7 31.7 27.8
13.5 17.4 19.1
2.6 2.2 0.5
- -
- - -
- -
- 4.5
20.3 8.9
-
2.7
30.0 18.0
13.9 19.8
22.8 17.2
- -
8.9 20.0
1.8 6.3
Abbreviations: CE, cholesteryl esters; PC, phosphatidylcholine; PE, phosphatidylethanolamine; T, trace amount. From Christie (1995).
MILK LIPIDS 81
varies; some can form lactones, e.g. the 4- and 5-hydroxy acids can form
y- and 8-lactones, respectively.
C ‘2
I
R
A 6-lactone
Lactones have strong flavours; traces of S-lactones are found in fresh milk
and contribute to the flavour of milk fat, but higher concentrations may
occur in dried milk or butter oil as a result of heating or prolonged storage
and may cause atypical flavours.
The fatty acids in the various polar lipids and cholesteryl esters are
long-chain, saturated or unsaturated acids, with little or no acids of less than
C,,:, (Table 3.7; for further details see Christie, 1995).
3.5 Synthesis of fatty acids in milk fat
In non-ruminants, blood glucose is the principal precursor of fatty acids in
milk fat; the glucose is converted to acetyl CoA in the mammary gland. In
ruminants, acetate and P-hydroxybutyrate, produced by micro-organisms in
the rumen and transported to the blood, are the principal precursors; in fact,
ruminant mammary tissue has little ‘ATP citrate lyase’ activity which is
required for fat synthesis from glucose. Blood glucose is low in ruminants
and is conserved for lactose synthesis. The differences in fatty acid precur-
sors are reflected in marked interspecies differences in milk fatty acid
profiles. Restriction of roughage in the diet of ruminants leads to sup-
pression of milk fat synthesis, possibly through a reduction in the available
concentration of acetate and P-hydroxybutyrate.
In all species, the principal precursor for fatty acid synthesis is acetyl
CoA, derived in non-ruminants from glucose and in ruminants from acetate
or oxidation of 8-hydroxybutyrate. Acetyl CoA is first converted, in the
cytoplasm, to malonyl CoA:
82 DAIRY CHEMISTRY AND BIOCHEMISTRY
0
&-OH
0 I
I
b CH2 t ADP + Pi CH,&-S-C~A + co2 + ATP
Mn2+
Acetyl CoA
Acetyl CoA C-S-CoA
carboxylase d
Malonyl CoA
Reduced bicarbonate supply (source of CO,) depresses fatty acid synthesis.
Some P-hydroxybutyrate is reduced to butyrate and incorporated directly
into milk fat; hence, the high level of this acid in ruminant milk fat.
In non-ruminants, the malonyl CoA is combined with an ‘acyl carrier
protein’ (ACP) which is part of a six-enzyme complex (molecular weight
c. 500 kDa) located in the cytoplasm. All subsequent steps in fatty acid
synthesis occur attached to this complex; through a series of steps and
repeated cycles, the fatty acid is elongated by two carbon units per cycle
(Figure 3.8, see also Lehninger, Nelson and Cox, 1993).
The net equation for the synthesis of a fatty acid is:
n Acetyl CoA + 2(n - 1)NADPH + 2(n - 1)H’ + (n - 1)ATP
0
il
+ (n - 1)C02 -+ CH,CH,(CH~CH,),-~CH,C-COA + (n - 1)CoA
+ (n - 1)ADP + (n - 1)Pi + 2(n - 1)NADP + (n - 1)COl
The large supply of NADPH required for the above reactions is ob-
tained through the metabolism of glucose-6-phosphate via the pentose
pathway.
In ruminants, P-hydroxybutyrate is the preferred chain initiator (labelled
P-hydroxybutyrate appears as the terminal four carbons of short- to
medium-chain acids), i.e. the first cycle in fatty acid synthesis commences at
P-hydroxybut yryl-S- ACP.
Synthesis of fatty acids via the malonyl CoA pathway does not proceed
beyond palmitic acid (C,,,,) and mammary tissue contains an enzyme,
thioacylase, capable of releasing the acyl fatty acid from the carrier protein
at any stage between C, and c16. Probable interspecies differences in the
activity of thioacylase may account for some of the interspecies differences in
milk fatty acid profiles.
The malonyl CoA pathway appears to account for 100% of the C,,, C,,
and C14, and c. 50% of the C,,:, acids in ruminant milk fat, as indicated by
labelling experiments (Figure 3.9). However, C,, c6 and C, are synthesized
MILK LIPIDS
Acyl Carrier Protein (ACP)
83
SH3 I
Acetyl-S-ACP I
ACP-SC=O + CH,
CoA-S-C=O
Malonyl-SACP
y3
c=o
ACP-S-C=O CH2 I / + CO, + CoA-SH
bketobutyryl-S-ACP c=o
KR r""' NADP' + H+
7H3
HC-OH
I
I
CH2
I
ACP-SC=O
CH2
I
ACP-S-C=O
khy droxybutyryl-S-ACP
,733 7%
CH2
I
HD kHZ0
CH II ** 'HZ I
CH
ACP-S-C=O
I
ACP-S-C=O NADPH + H+ NADP
2, J-butenoyl-S-ACP
butyryl-S-ACP
CoA-S-C=O
Malonyl CoA
Figure 3.8 One complete cycle and the first step in the next cycle of the events during the
synthesis of fatty acids. ACP = acyl carrier protein, a complex of six enzymes: i.e. acetyl
CoA-ACP transacetylase (AT); malonyl CoA-ACP transferase (MT); B-keto-ACP synthase
(KS); 8-ketoacyl-ACP reductase (KR); p- hydroxyacyl-ACP-dehydrase (HD); enoyl-ACP
reductase (ER).
84 DAIRY CHEMISTRY AND BIOCHEMISTRY
Source Fatry acids
1
Acetate ., 4: 0
to
14: 0
hutyrate
\
d
TGs of
hlood plasma
16 :O
18: 0
18: 1
18: 2
Figure 3.9 Sources of the fatty acids in bovine milk fat; TG, triglyceride (from Hawke and
Taylor, 1995).
Blood Endothelial cell
D-hvdroxv
..
Alveolar cell
Lumen
- TGs
Figure 3.10 Uptake of blood constituents by the mammary gland; CoA, coenzyme A; G-3-P,
glycerol-3-phosphate; FFA, free fatty acid; FA, fatty acid; TG, triglyceride, VLDL, very low
density lipoprotein (from Hawke and Taylor, 1995).
MILK LIPIDS 85
from P-hydroxybutyrate and acetate mainly via two other pathways not
involving malonyl CoA.
In the mammary gland, essentially 100% of C,8:o, C,,:, and c. 50% of C,,
are derived from blood lipids (chylomicrons, free triglycerides, free fatty
acids, cholesteryl esters). The blood lipids are hydrolysed by lipoprotein
lipase which is present in the alveolar blood capillaries, the activity of which
increases eightfold on initiation of lactation. The resulting monoglycerides,
free fatty acids and some glycerol are transported across the basal cell
membrane and re-incorporated into triglycerides inside the mammary cell
(Figure 3.10).
In blood, lipids exist as lipoprotein particles, the main function of which
is to transport lipids to and from various tissues and organs of the body.
There is considerable interest in blood lipoproteins from the viewpoint of
human health, especially obesity and cardiovascular diseases. Lipoproteins
are classified into four groups on the basis of density, which is essentially a
function of their triglyceride content, i.e. chylomicrons, very low density
lipoprotein particles (VLDL), low density lipoprotein (LDL) particles and
high density lipoprotein (HDL) particles, containing c. 98, 90, 77 and 45%
total lipid, respectively (Figure 3.1 1).
Lipoproteins, especially chylomicrons, are at an elevated level in the
blood after eating, especially after high-fat meals, and give blood serum a
milky appearance. They are also elevated during or after tension (so-called
Chylomicron
VIDL
HDL
LDL
0 Proteins Triacylglycerols Cholesterol Phospholipids
Figure 3.11 Composition (%) of human serum lipoproteins; VLDL, very low density lipo-
proteins; LDL, low density lipoproteins; HDL, high density lipoproteins.
86 DAIRY CHEMISTRY AND BIOCHEMISTRY
Palmitic Acid
'almitoleic acid Stcaric acid
9 -Cm1
Vaccenic acid
11 - CI,:,
Oleic acid
ClOl
Jy
5,8,11- C,, 15 - C,,,
Eicosatrienoic Nervonic
acid acid
up Series
9.12 - c,,;, 9.12.15 - c,,;,
Linoleic acid Linolenic acid
K
6.9,12,15 - C,,;,
11.14 - C,, #q
Eicosadienoic acid 6,9,12-cl,!
Linolenic acid
+cll
8,11,14 - c,,
-2Hl
5,s. 11,14 - Cm,
Arachidonic acid
w series
(b)
8,11,14,17 - Cm.
5,8,11,14,17 - C,
1.c'
7,10,13,16,19 - Ca5
1 -2H
P.7.10,13,16,19 - c,,:,
Docosahexenoic acid
0, series
Figure 3.12 Elongation and/or desaturation of fatty acids in the mammary gland.
MILK LIPIDS 87
racing driver syndrome). Chylomicrons, which are formed in the intestinal
mucosa, are secreted into the lymph and enter the blood via the thoracic
duct. VLDL lipoproteins are synthesized in intestinal mucosa and liver.
LDL lipoproteins are formed at various sites, including mammary gland, by
removing of triglycerides from VLDL.
Since about 50% of c16:o and 100% of C,,:,, C,,:, and C18:2 are derived
from blood lipids, about 50% of the total fatty acids in ruminant milk fat
originate from the blood via diet or other organs.
In liver mitochondria, palmitic acid, as its CoA ester, is lengthened by
successive additions of acetyl CoA. There is also a liver microsomal enzyme
capable of elongating saturated and unsaturated fatty acids by addition of
acetyl CoA or malonyl CoA.
The principal monoenoic acids, oleic (C18:J and palmitoleic (Cl6:1), are
derived from blood lipids but about 30% of these acids are produced by
microsomal enzymes (in the endoplasmic reticulum) in the secretory cells by
desaturation of stearic and palmitic acids, respectively:
Stearyl CoA + NADPH + 0, - oleoyl CoA + NADP' + 2H,O
Shorter chain unsaturated acids (Clo:l to C14: ,) are probably also produced
by the same enzyme.
Linoleic (&) and linolenic (c1@3) acids cannot be synthesized by
mammals and must be supplied in the diet, i.e. they are essential fatty acids
(linoleic is the only true essential acid). These two polyenoic acids may then
be elongated and/or further desaturated by mechanisms similar to stearic +
oleic, to provide a full range of polyenoic acids. A summary of these
reactions is given in Figure 3.12a, b.
b-Hydroxy acids are produced by &oxidation of fatty acids and p-keto
acids may arise from incomplete syntheses or via P-oxidation.
desaturase
3.6 Structure of milk lipids
Glycerol for milk lipid synthesis is obtained in part from hydrolysed blood
lipids (free glycerol and monoglycerides), partly from glucose and a little
from free blood glycerol. Synthesis of triglycerides within the cell is catalysed
by enzymes located on the endoplasmic reticulum, as shown in Figure 3.13.
Esterification of fatty acids is not random: c,,-c16 are esterified
principally at the sn-2 position while C, and (26 are esterified principally at
the sn-3 position (Table 3.8). The concentrations of C, and C,, appear to
be rate-limiting because of the need to keep the lipid liquid at body
temperature. Some features of the structures are notable:
0 Butanoic and hexanoic acids are esterified almost entirely, and octanoic
and decanoic acids predominantly, at the sn-3 position.
CH2OH c--. Glucose
CH20H
I 1
CHOH
CH,O -Pe OH
I 0
I
I
c=o
I 0
CH,-0 -(Lo"
Glycerol t ATP ___t Glycerol-3-P 0 t ADP 4 Dihydroxy 6- acetone P
glycerokinase
Phosphatidic acid
*P Phosphatase
H,C-O-C-R 4-
I *P
HC-0 - C - R
I
H2C - OH
Diglyceride
H,C -0 -C 1 R
I .P
I s.0
HC-0 -C - R
H2C-0 -C- R
RC-S-CoA
Triglyceride
NADPH + H,
0.0
2 RC-S-CoA
acyl hansferase
H,C-o -c-R
I 9
I ..o
HC-0 -C - R
HZC-0 - P -OH
'0
Phosphatidic acid
Figure 3.13 Biosynthesis of triglycerides in the mammary gland
Table 3.8 Composition of fatty acids (mol% of the total) esterified to each position of the triacyl-sn-glycerols in the milks of various species
cow Human Rat Pig Rabbit Seal Echidna
Fatty
acid sn-1 sn-2 SIT-3 sn-l sn-2 sn-3 sn-l sn-2 sn-3 sn-l sn-2 sn-3 sn-l sn-2 sn-3 sn-l sn-2 sn-3 sn-1 sn-2 sn-3
~~
4:O -
6:O - 0.9
8:O 1.4 0.7
lo:o 1.9 3.0
12:o 4.9 6.2
14:O 9.7 17.5
16:O 34.0 32.3
16: 1 2.8 3.6
18:O 10.3 9.5
18: 1 30.0 18.9
18:2 1.7 3.5
18:3 - -
-
c20-c22
- -
35.4 - -
12.9 - -
- - - - - - - - -
- - - - - - - - -
3.6 - - 3.7 5.7 10.0 - - 19.2
6.2 0.2 0.2 1.1 10.1 20.0 26.0 - - - - 22.5
0.6 1.3 2.1 5.6 10.4 15.9 15.1 - - 3.5
6.4 3.2 7.3 6.9 9.6 17.8 8.9 2.4 6.8 3.7 2.7 2.1
5.4 16.1 58.2 5.5 20.2 28.7 12.6 21.8 57.6 15.4 24.1 12.7
1.4 3.6 4.7 7.6 1.8 2.1 1.8 6.6 11.2 10.4 4.1 1.3
1.2 15.0 3.3 1.8 4.9 0.8 1.5 6.9 1.1 5.5 6.9 3.5
23.1 46.1 12.7 50.4 24.2 3.3 11.8 49.6 13.9 51.7 40.8 16.6
2.3 11.0 7.3 15.0 14.1 5.2 11.6 11.3 8.4 11.5 15.6 15.1
- - -
- -
- 0.4 0.6 1.7 1.2 0.5 0.7 1.4 1.0 1.8 3.4 3.5
- - - - - - - - - - - -
- - - - --
- - - - - --
- - --
33.7 38.9 -
22.5 26.1 - --
- -
--
2.8 1.8 0.3 0.2 -
2.6 0.7 23.6 3.8 1.7 0.9 0.4
23.8 6.1 31.0 1.0 31.5 9.0 27.9
0.9 1.9 0.7 1.0 16.8 2.1 14.3
3.8 11.4 19.4 45.4 33.1 57.6 39.8
6.4 9.7 2.3 2.8 4.1 18.3 4.9
2.0 2.3 0.5 0.7 1.0 2.9 2.0
--
1.5 1.1 16.8 14.1 -
- --
-- 0.8 28.7 -
From Christie (1995).
90 DAIRY CHEMISTRY AND BIOCHEMISTRY
0 As the chain length increases up to c,6,0, an increasing proportion is
esterified at the sn-2 position; this is more marked for human than for
bovine milk fat, especially in the case of palmitic acid (CI6:,J,
0 Stearic acid (C,8:o) is esterified mainly at sn-1.
0 Unsaturated fatty acids are esterified mainly at the sn-1 and sn-3 posi-
Fatty acid distribution is significant from two viewpoints:
0 It affects the melting point and hardness of the fat, which can be reduced
by randomizing the fatty acid distribution. Transesterification can be
performed by treatment with SnCl, or enzymatically under certain
conditions; increasing attention is being focused on the latter as an
acceptable means of modifying the hardness of butter.
0 Pancreatic lipase is specific for the fatty acids at the sn-1 and sn-3
positions. Therefore, C4:o to C8:o are released rapidly from milk fat; these
are water-soluble and are readily absorbed from the intestine. Medium-
and long-chain acids are absorbed more effectively as 2-monoglycerides
than as free acids; this appears to be quite important for the digestion of
lipids by human infants who have limited ability to digest lipids due to
the absence of bile salts. Infants metabolize human milk fat more
efficiently than bovine milk fat, apparently owing to the very high
proportion of C,6:o esterified at sn-2 in the former. The effect of transes-
terification on the digestibility of milk fat by infants merits investigation.
tions, in roughly equal proportions.
3.7 Milk fat as an emulsion
In 1674, Van Leeuwenhoek reported that the fat in milk exists as micro-
scopic globules. Milk is an oil-in-water emulsion, the properties of which
have a marked influence on many properties of milk, e.g. colour, mouthfeel,
viscosity. The globules range in diameter from approximately 0.1 to 20 pm,
with a mean of about 3.5pm (the range and mean vary with breed and
health of the cow, stage of lactation, etc.). The size and size distribution of
fat globules in milk may be determined by light microscopy, light scattering
(e.g. using the Malvern Mastersizer) or electronic counting devices (such as
the Coulter counter). The frequency distribution of globule number and
volume as a function of diameter for bovine milk are summarized in Figure
3.14. Although small globules are very numerous (c. 75% of all globules
have diameters < 1 pm), they represent only a small proportion of total fat
volume or mass. The number average diameter of the globules in milk is
only c. 0.8 pm. The mean fat globule size in milk from Channel Island breeds
(Jersey and Guernsey) is larger than that in milk from other breeds (the fat
content of the former milks is also higher) and the mean globule diameter
decreases throughout lactation (Figure 3.1 5).
MILK LIPIDS 91
I I I I
v N;/Ad
\
-30
-
-20
-lo
Figure 3.14 Number (NJAd) and volume (% of fat) frequency of the fat globules in bovine
milk (from Walstra and Jenness, 1984).
01
IIIIIIIII
10 20 30 40 50
Weeks of lactation
Figure 3.15 Average diameter of the fat globules in milk of Guernsey or Friesian cows
throughout lactation (from Walstra and Jenness, 1984).
92 DAIRY CHEMISTRY AND BIOCHEMISTRY
Milk contains - 15 x lo9 globules ml-', with a total interfacial area of
1.2-2.5 m2 per g fat.
Example.
of 3 pm.
Assume a fat content Of 4.O%, w/v, with a mean globule diameter
4
3
Volume of typical globule = - 71r3
4 22 (3)3
= - x - x - pm3
37 2
- 14pm3.
1 ml milk contains: 0.04g fat
= 4.4 x 10" pm3.
1 ml milk contains: 4'4 1010-3.14 x lo9 globules.
14
Surface area of a typical globule = 471r2
22 9
74
= 4 x - x -pmZ
= 28.3 pm2.
Interfacial area per ml milk = 28.3 x (3.14 x 10') pm2
= 88.9 x 109pm2
= 889 cm2 % 0.09 rn2.
1
x - m2
0.04
Interfacial area per g fat = 88.9 x
= 2.22mZ.
3.8 Milk fat globule membrane
Lipids are insoluble in water and an interfacial tension therefore exists
between the phases when lipids are dispersed (emulsified) in water (or vice
versa). This tension in toto is very large, considering the very large interfacial
area in a typical emulsion (section 3.7). Owing to the interfacial tension, the
oil and water phases would quickly coalesce and separate. However,
coalescence (but not creaming) is prevented by the use of emulsifiers (surface
active agents) which form a film around each fat globule (or each water
MILK LIPIDS 93
droplet in the case of water-in-oil emulsions) and reduce interfacial tension.
In the case of unprocessed milk, the emulsifying film is much more complex
than that in ‘artificial’ emulsions, and is referred to as the milk fat globule
membrane (MFGM).
In 1840, Ascherson observed an emulsion-stabilizing membrane sur-
rounding the fat globules in milk and suggested that the membrane was
‘condensed’ albumin (from the skim-milk phase) aggregated at the fat/
plasma interface. Babcock, in the 188Os, also felt that the milk fat emulsifier
was adsorbed serum protein. Histological staining and light microscopy
were employed around the turn of the century to identify the nature of the
membrane material but it was early recognized that contamination of fat
globules by skim-milk components presented a major problem. By analysing
washed globules, it was shown that the MFGM contained phospholipids
and protein which differed from the skim-milk proteins (see Brunner (1974)
for historical review).
3.8. I Isolation of the fat globule membrane
The definition of what precisely constitutes the membrane leads to consider-
able difficulty and uncertainty. The outer boundary is assumed to constitute
everything that travels with the fat globule when it moves slowly through
milk; however, the outer regions of the membrane are loosely attached and
some or all may be lost, depending on the extent of mechanical damage the
globule suffers. The inner boundary is ill-defined and depends on the method
of preparation; there is considerable discussion as to whether a layer of high
melting point triglyceride, immediately inside the membrane, is part of the
membrane or not. Some hydrophobic constituents of the membrane prob-
ably diffuse into the core of the globules while components of the plasma
may adsorb at the outer surface. Since the membrane contains numerous
enzymes, enzymatic changes may occur.
Several methods are available for isolating all or part of the membrane.
The usual initial step involves separating a cream from milk by mechanical
centrifugation (which may cause some damage) or by gravity. The cream is
washed repeatedly (3-6 times) with water or dilute buffer by dilution and
gravity separation; soluble salts and other small molecules are probably lost
into the serum. Mechanical damage may remove the loosely bound outer
layers and may even cause some homogenization and adsorption of serum
constituents; small globules are lost during each washing cycle.
The washed cream is destabilized by churning or freezing; then the fat
(mainly triglycerides) is melted and separated from the membrane material
by centrifugation. Cross-contamination of membrane with core material
may be considerable, and methods must be carefully standardized. An
elaborate scheme for the isolation and fractionation of the MFGM was
developed by Brunner and co-workers (Brunner, 1974).
94 DAIRY CHEMISTRY AND BIOCHEMISTRY
Treatment of washed cream with surfactants, usually sodium de-
oxycholate, releases part of the membrane, assumed to represent only the
outer layer. Unless the treatment is carefully controlled, some inner material
will be released also.
3.8.2
Yields of 0.5-1.5g MFGM per lOOg fat have been reported; the range
reflects variations in temperature history, washing technique, age, agitation,
etc. The gross chemical composition of the membrane is reasonably well
established and the relatively small differences reported are normally at-
tributed to different methods used to isolate and fractionate the membrane
material. The data in Table 3.9, from Mulder and Walstra (1974) and based
on the investigations of many workers, give a reasonable estimate of the
gross composition of the MFGM. A more detailed compositional analysis
is provided by Keenan et al. (1983) (Table 3.10). Brunner (1965, 1974),
Mulder and Walstra (1974), Patton and Keenan (1975), Keenan et al. (1983)
and Keenan and Dylewski (1995) should be consulted for more detailed
compositional data.
Gross chemical composition of MFGM
3.8.3 The protein fraction
Depending on the preparative method used, the membrane may or may not
contain skim-milk proteins (i.e. caseins and whey proteins); if the membrane
has been damaged prior to isolation, it may contain considerable amounts
of these proteins. The membrane contains unique proteins which do not
occur in the skim-milk phase. Many of the proteins are glycoproteins and
contain a considerable amount of carbohydrate (hexose, 2.8-4.1 5%;
hexosamine, 2.5-4.2%; and sialic acid, 1.3-1.8%).
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
PAGE), with silver staining of the gels, resolves MFGM proteins into as
many as 60 discrete bands, ranging in molecular mass from 11 to 250 kDa
(Keenan and Dylewski, 1995). Most of these proteins are present at very low
concentrations (many are detectable only when gels are stained with silver
but not with Coomassie blue). Some of these proteins may be genetic
variants and, since the MFGM contains a plasmin-like proteinase, some of
the smaller polypeptides may be fragments of larger proteins. The three
principal proteins, with molecular masses (by SDS-PAGE) of 155, 67 and
48 kDa, are xanthine oxidase, butyrophilin and glycoprotein B, respectively;
five or six glycoproteins have been detected by staining with SchifYs reagent.
Xanthine oxidase, which requires Fe, Mo and flavin adenine dinucleotide
(FAD) as co-factors, is capable of oxidizing lipids via the production of
superoxide radicals. It represents about 20% of the MFGM protein and
part is readily lost from the membrane, e.g. on cooling; isoelectric focusing
MILK LIPIDS 95
indicates at least four variants with isoelectric points (pl) in the range
Butyrophilin, the principal MFGM protein and so named because of its
high affinity for milk lipids, is a very hydrophobic, difficult to solubilize
(insoluble or only sparingly soluble in most protein solvents, including
detergents) glycoprotein. Isoelectric focusing indicates at least four variants
(pls 5.2-5.3). The amino acid sequence of butyrophilin has been determined
and its gene has been cloned, which indicates that butyrophilin is syn-
thesized with a leader sequence; it consists of 526 amino acids and has a
molecular mass, without carbohydrate, of 56 460 Da. It binds phospholipids
tenaciously and perhaps even contains covalently bound fatty acids. It is
located only at the apical cell surface of the mammary epithelial cells,
suggesting a role in membrane envelopment of fat globules.
Several of the minor proteins of the MFGM have been isolated and
partially characterized (Keenan and Dylewski, 1995). A systematic nomen-
clature has not been developed for the MFGM proteins and most are
referred to by their relative electrophoretic mobility on SDS-PAGE and
whether or not they are glycoproteins. The proteins of the MFGM represent
approximately 1% of the total proteins in milk.
7.0-7.5.
3.8.4 The lipidfvaction
The membrane contains 0.5-1.0% of the total lipid in milk and is composed
principally of phospholipids and neutral lipids in the approximate ratio 2 : 1,
with lesser amounts of other lipids (Tables 3.9 and 3.10); contamination with
core lipid is a major problem. The phospholipids are principally phos-
phatidylcholine, phosphatidylethanolamine and sphingomyelin in the ap-
proximate ratio 2 : 2 : 1. The principal fatty acids and their approximate
percentages in the phospholipids are C14:o (5%), C,,:, (25%), c,&o (14%),
CI,:, (25%) C,,:, YO), C,,:, (3%) and C24:o (3%). Thus, the membrane
contains a significantly higher level of polyunsaturated fatty acids than milk
Table 3.9 Gross composition of the milk fat globule membrane
~ ~~~~
mg 100 g-' mg m-' fat Yo (w/w) of
Component fat globule globule surface total membrane
Protein 900 4.5 41
Phospholipid 600 3.0 27
Cerebrosides 80 0.4 3
Cholesterol 40 0.2 2
Neutral glycerides 300 1.5 14
Water 280 1.4 13
Total 2200 11.0 100
From Mulder and Walstra (1974).
96 DAIRY CHEMISTRY AND BIOCHEMISTRY
Table 3.10 Composition of bovine milk fat globule membranes
Constituent class Amount
Protein
Total lipid
Phospholipid
Phosphatidyl choline
Phosphatidylethanolamine
Sphingomyelin
Phosphatidylinositol
Phosphatidylserine
Hydrocarbons
Sterols
Sterol esters
Glycerides
Free fatty acids
Cerebrosides
Gangliosides
Total sialic acids
Hexoses
Hexosamines
Cytochrome b, + P420
Uronic acids
RNA
Neutral lipid
25-60% of dry weight
0.5- 1.2 mg per mg protein
0.13-0.34 mg per mg protein
34% of total lipid phosphorus
28% of total lipid phosphorus
22% of total lipid phosphorus
10% of total lipid phosphorus
6% of total lipid phosphorus
56-80% of total lipid
1.2% of total lipid
0.2-5.2% of total lipid
0.1-0.8% of total lipid
53-74% of total lipid
0.6-6.3% of total lipid
3.5 nmoles per mg protein
6-7.4 nmoles sialic acid per mg protein
63 nmoles per mg protein
0.6 pmoles per mg protein
0.3 pmoles per mg protein
30 pmoles per mg protein
99 ng per mg protein
20 pg per mg protein
From Keenan et a/. (1983).
Table 3.1 1 Structures of glycosphingolipids of bovine milk fat globule membrane
GI ycosphingolipid Structure
Glucosyl cerarnide
Lactosyl ceramide
GM, (hematoside)
GM,
GM,
GD, (disialohematoside)
GD,
GD,,
p-Glucosyl-(1 -+ 1)-ceramide
fl-Glucosyl-( 1 -+ 4)-/3-glucosyl-( 1 -+ 1)-ceramide
NeuraminosyL(2 + 3)-galactosyl-glucosyl-ceramide
N- Acetylgalactosamin yl-( neuraminosy1)-galactosyl-glucosyl-
Galactosyl-N-acetylgalactosaminyl-(neuraminosyl)-galactosyl-
Neuraminosyl-(2 -+ 8)-neuraminosyl-(2 -+ 3)-galactosyl-
N-Acetylgalactosaminyl-(neuraminosyl-neuraminosyl)-
Galactosyl-N-acetylgalactosaminyl-(neuraminosyl-
ceramide
glucosyl-ceramide
glucosyl-ceramide
galactosyl-glucosyl-ceramide
neuraminosy1)-galactosyl-glucosyl-ceramide
From Keenan et al. (1983).
MILK LIPIDS 97
fat generally and is, therefore, more susceptible to oxidation. The cerebro-
sides are rich in very long chain fatty acids which possibly contribute to
membrane stability. The membrane contains several glycolipids (Table
3.11).
The amount and nature of the neutral lipid present in the MFGM is
uncertain because of the difficulty in defining precisely the inner limits of the
membrane. It is generally considered to consist of 83-88% triglyceride,
514% diglyceride and 1-5% free fatty acids. The level of diglyceride is
considerably higher than in milk fat as a whole; diglycerides are relatively
polar and are, therefore, surface-active. The fatty acids of the neutral lipid
fraction are longer-chained than in milk fat as a whole and in order of
proportion present are palmitic, stearic, myristic, oleic and lauric.
Most of the sterols and sterol esters, vitamin A, carotenoids and squalene
in milk are dissolved in the core of the fat globules but some are probably
present in the membrane.
3.8.5 Other membrane components
Trace metals. The membrane contains 525% of the indigenous Cu and
30-60% of the indigenous Fe of milk as well as several other elements, e.g.
Co, Ca, Na, K, Mg, Mn, Mo, Zn, at trace levels; Mo is a constituent of
xanthine oxidase.
Enzymes. The MFGM contains many enzymes (Table 3.12). These en-
zymes originate from the cytoplasm and membranes of the secretory cell and
are present in the MFGM due to the mechanism of globule excretion from
the cells.
3.8.6 Membrane structure
Several early attempts to describe the structure of the MFGM included
King (1955), Hayashi and Smith (1965), Peereboom (1969), Prentice (1969)
and Wooding (1971). Although the structures proposed by these workers
were inaccurate, they stimulated thinking on the subject. Keenan and
Dylewski (1995) and Keenan and Patton (1995) should be consulted for
recent reviews.
Understanding of the structure of the MFGM requires understanding
three processes: the formation of lipid droplets from triglycerides synthesized
in or on the endoplasmic reticulum at the base of the cell, movement of the
droplets (globules) through the cell and excretion of the globules from the
cell into the lumen of the alveolus.
The MFGM originates from regions of apical plasma membrane, and
also from endoplasmic reticulum (ER) and perhaps other intracellular
compartments. That portion of the MFGM derived from apical plasma
98 DAIRY CHEMISTRY AND BIOCHEMISTRY
Table 3.12 Enzymatic activities detected in bovine milk fat
globule membrane preparations
Enzyme EC number
Lipoamide dehydrogenase
Xanthine oxidase
Thiol oxidase
NADH oxidase
NADPH oxidase
Catalase
y-Glutamyl transpeptidase
Galactosyl transferase
Alkaline phosphatase
Acid phosphatase
N -Nucleotidase
Phosphodiesterase I
Inorganic pyrophosphatase
Nucleotide pyrophosphatase
Phosphatidic acid phosphatase
Adenosine triphosphatase
Cholinesterase
UDP-glycosyl hydrolase
Glucose-6-phosphatase
Plasmin
P-Glucosidase
P-Galactosidase
Ribonuclease I
Aldolase
Acetyl-CoA carboxylase
1.6.4.3
1.2.3.2
1.8.3.2
1.6.99.3
1.6.99.1
1.1 1.1.6
2.3.2.1
2.4.1 -
3.1.3.1
3.1.3.2
3.1.3.5
3.1.4.1
3.6.1.1
3.6.1.9
3.1.3.4
3.6.1.15
3.1.1.8
3.1.3.9
3.4.21.7
3.2.1.21
3.2.1-
3.2.1.23
3.1.4.22
4.1.2.1 3
6.4.1.2
From Keenan and Dylewski (1995).
membrane, termed the primary membrane, has a typical bilayer membrane
appearance, with electron-dense material on the inner membrane face. The
components derived from ER appear to be a monolayer of proteins and
polar lipids which covers the triacylglycerol-rich core lipids of the globule
before its secretion. This monolayer or coat material compartmentalizes the
core lipid within the cell and participates in intracellular fusions through
which droplets grow in volume. Constituents of this coat also may be
involved in interaction of droplets with the plasma membrane.
Milk lipid globules originate as small lipid droplets in the ER. Lipids,
presumed to be primarily triacylglycerols, appear to accumulate at focal
points on or in the ER membrane. This accumulation of lipids may be due
to localized synthesis at these focal points, or to accretion from dispersed or
uniformly distributed biosynthetic sites. It has been suggested that triacyl-
glycerols accumulate between the halves of the bilayer membrane and are
released from the ER into the cytoplasm as droplets coated with the outer
or cytoplasmic half of the ER membrane. A cell-free system has been
developed in which ER isolated from lactating mammary gland can be
induced to release lipid droplets which resemble closely droplets formed in
situ in both morphology and composition. In this cell-free system, lipid
MILK LIPIDS 99
droplets were formed only when a fraction of cytosol with a molecular mass
greater than 10 kDa was included in the incubation mixture, suggesting that
cytosolic factors are involved in droplet formation or release from ER.
By whatever mechanism they are formed, on or in, and released from the
ER, milk lipid globule precursors first appear in the cytoplasm as droplets
with diameters of less than 0.5 pm, with a triglyceride-rich core surrounded
by a granular coat material that lacks bilayer membrane structure, but
which appears to be thickened, with tripartite-like structure, in some
regions. These small droplets, named microlipid droplets, appear to grow in
volume by fusing with each other. Fusions give rise to larger droplets, called
cytoplasmic lipid droplets, with diameters of greater than 1 pm.
Droplets of different density and lipid : protein ratios ranging from about
1.5 : 1 to 40 : 1 have been isolated from bovine mammary gland. Triglycerides
are the major lipid class in droplets of all sizes and represent increasingly
greater proportions of total droplet mass in increasingly less dense droplet
preparations. Surface coat material of droplets contains cholesterol and the
major phospholipid classes found in milk, i.e. sphingomyelin, phosphatidyl-
choline, phosphatidylethanolamine, phosphatidylinositol and phosphatidyl-
serine.
SDS-PAGE shows that micro- and cytoplasmic lipid droplets have
complex and similar polypeptide patterns. Many polypeptides with elec-
trophoretic mobilities in common with those of intracellular lipid droplets
are present also in milk lipid globules. Some polypeptides of MFGM and
intracellular lipid droplets share antigenic reactivity. Taken together, current
information suggests that lipid droplet precursors of milk lipid globules
originate in the ER and retain at least part of the surface material of droplets
during their secretion as milk fat globules. The protein and polar lipid coat
on the surface of lipid droplets stabilizes the triglyceride-rich droplet core,
preventing coalescence in the cytoplasm. Beyond a stabilization role, consti-
tuents of the coat material may participate also in droplet fusions and in
droplet-plasma membrane interactions. If elements of the cytoskeleton
function in guiding lipid droplets from their sites of origin to their sites of
secretion from the cell, coat constituents may participate in interaction with
filamentous or tubular cytoskeletal elements.
Within mammary epithelial cells, one mechanism by which lipid droplets
can grow is by fusion of microlipid droplets. Microlipid droplets can also
fuse with cytoplasmic lipid droplets, providing triacylglycerols for continued
growth of larger droplets. The size range of lipid globules in milk can be
accounted for, at least in part, by a droplet fusion-based growth process.
Small milk fat globules probably arise from secretion of microlipid droplets
which have undergone no or a few fusions while larger droplets can be
formed by continued fusions with microlipid droplets.
While accumulated evidence favours the view that lipid droplets grow by
fusion, there is no evidence as to how this process is regulated to control the
ultimate size distribution of milk lipid globules. The possibility that fusion
100 DAIRY CHEMISTRY AND BIOCHEMISTRY
is purely a random event, regulated only by probability of droplet-droplet
contact before secretion, cannot be ruled out. Insufficient evidence is
available to conclude that fusion of droplets is the sole or major mechanism
by which droplets grow. Other possible mechanisms for growth, e.g. lipid
transfer proteins which convey triglycerides from their site of synthesis to
growing lipid droplets, cannot be excluded.
Available evidence indicates that lipid droplets migrate from their sites of
origin, primarily in basal regions of the cell, through the cytoplasm to apical
cell regions. This process appears to be unique to the mammary gland and
in distinct contrast to lipid transit in other cell types, where triacylglycerols
are sequestered within ER and the Golgi apparatus and are secreted as
lipoproteins or chylomicrons that are conveyed to the cell surface via
secretory vesicles.
Mechanisms which guide unidirectional transport of lipid droplets are
not yet understood. Evidence for possible involvement of microtubules and
microfilaments, elements of the cytoskeletal system, in guiding this transit
has been obtained, but this evidence is weak and is contradictory in some
cases. Cytoplasmic microtubules are numerous in milk-secreting cells and
the tubulin content of mammary gland increases substantially prior to milk
secretion. A general role for microtubules in the cytoplasm, and the
association of proteins with force-producing properties with microtubules,
provide a plausible basis for assuming the microtubules may be involved in
lipid droplet translocation. Microfilaments, which are abundant in milk-
secreting cells, appear to be concentrated in apical regions.
3.8.7 Secretion of milk lipid globules
The mechanism by which lipid droplets are secreted from the mammocyte
was first described in 1959 by Bargmann and Knoop and has been
confirmed by several investigators since (Keenan and Dylewski, 1995). The
lipid droplets are pushed through and become enveloped progressively by
Figure 3.16 Schematic representation of the excretion of a fat globule through the apical
membrane of the mammary cell.
MILK LIPIDS 101
the apical membrane up to the point where they are dissociated from the
cell, surrounded entirely by apical membrane (Figure 3.16). Current con-
cepts of the pathway by which lipid droplets originate, grow and are
secreted are summarized diagrammatically in Figure 3.17.
Lipid droplets associate with regions of the plasma membrane that are
characterized by the appearance of electron-dense material on the cytoplas-
mic face of the membrane. Droplet surfaces do not contact the plasma
Figure 3.17 The roles of components of the endo-membrane system of mammary epithelial cells
in the synthesis and secretion of the constituents of milk. Intracellular lipid globules (LG-1,
LG-2, LG-3) are discharged from the cell by progressive envelopment in regions of apical
plasma membrane. MFG denotes a lipid globule being enveloped in plasma membrane. Milk
proteins (MP) are synthesized on polysomes of endoplasmic reticulum and are transported,
perhaps in small vesicles which bleb from endoplasmic reticulum, to dictyosomes (D1, D,, D3)
of the Golgi apparatus. These small vesicles may fuse to form the proximal cisterna of Golgi
apparatus dictyosomes. Milk proteins are incorporated into secretory vesicles formed from
cisternal membranes on the distal face of dictyosomes. Lactose is synthesized within cisternal
luminae of the Golgi apparatus and is incorporated into secretory vesicles. Certain ions of milk
are also present in secretory vesicles. Three different mechanisms for exocytotic interaction of
secretory vesicle with apical plasma membrane have been described: (1) through the formation
of a chain of fused vesicles (V-I); (2) by fusion of individual vesicles with apical plasma
membrane (V-2), with integration of vesicle membrane into plasma membrane; (3) by direct
envelopment of secretory vesicles in apical plasma membrane (V-3). Lysosomes (LY) may
function in the degradation of excess secretory vesicle membrane (from Keenan, Mather and
Dylewski, 1988).
102 DAIRY CHEMISTRY AND BIOCHEMISTRY
membrane directly but rather the electron-dense cytoplasmic face material;
which constituents of the latter recognize and interact with constituents on
the droplet surface are not known. Immunological and biochemical studies
have shown that butyrophilin and xanthine oxidase, two of the principal
proteins in the MFGM, are major constituents of the electron-dense
material on the cytoplasmic face of apical plasma membrane. Butyrophilin,
a hydrophobic, transmembrane glycoprotein that is characteristic of milk-
secreting cells, is concentrated highly at the apical surface of these cells; it
binds phospholipids tightly, and is believed to be involved in mediating
interaction between lipid droplets and apical plasma membrane. Xanthine
oxidase is distributed throughout the cytoplasm, but appears to be enriched
at the apical cell surface.
In the secretion process, milk fat globules usually are enveloped compact-
ly by apical plasma membrane, but closure of the membrane behind the
projecting fat droplet occasionally entrains some cytoplasm as a so-called
crescent or signet between the membrane and the droplet surface. These
crescents can vary from thin slivers of cellular material to situations in which
the crescent represents a greater volume than does the globule core lipid.
Except for nuclei, cytoplasmic crescents contain nearly all membranes and
organelles of the milk-secreting cell. Globule populations with a high
proportion of crescents exhibit a more complex pattern of proteins by
SDS-PAGE than low-crescent populations. Presumably, the many addi-
tional minor bands arise from cytoplasmic components in crescents. Cres-
cents have been identified in association with the milk fat globules of all
species examined to date, but the proportion of globules with crescents
varies between and within species; about 1% of globules in bovine milk
contain crescents.
Thus, the fat globules are surrounded, at least initially, by a membrane
typical of eukaryotic cells. Membranes are a conspicuous feature of all cells
and may represent 80% of the dry weight of some cells. They serve as
barriers separating aqueous compartments with different solute composition
and as the structural base on which many enzymes and transport systems
are located. Although there is considerable variation, the typical composi-
tion of membranes is about 40% lipid and 60% protein. The lipids are
mostly polar (nearly all the polar lipids in cells are located in the mem-
branes), principally phospholipids and cholesterol in varying proportions.
Membranes contain several proteins, perhaps up to 100 in complex mem-
branes. Some of the proteins, referred to as extrinsic or peripheral, are
loosely attached to the membrane surface and are easily removed by mild
extraction procedures. The intrinsic or integral proteins, about 70% of the
total protein, are tightly bound to the lipid portion and are removed only
by severe treatment, e.g. by SDS or urea.
Electron microscopy shows that membranes are 79 nm thick, with a
trilaminar structure (a light, electron-sparse layer, sandwiched between two
MILK LIPIDS 103
Figure 3.18 Schematic representation of a trilaminar cell membrane which is derived from the
apical membrane of the mammary cell and forms the outer layer of the milk fat globule
membrane following expression from the mammary cell, but which is more or less extensively
lost on ageing. 1, phospholipid/glycolipid; 2, protein; 3, glycoprotein.
dark, electron-dense layers). The phospholipid molecules are arranged in a
bilayer structure (Figure 3.1 8); the non-polar hydrocarbon chains are
orientated inward where they ‘wriggle’ freely and form a continuous hydro-
carbon base; the hydrophilic regions are orientated outward and are
relatively rigid. In this bilayer, individual lipid molecules can move laterally,
endowing the bilayer with fluidity, flexibility, high electrical resistance and
low permeability to polar molecules. Some of the globular membrane
proteins are partially embedded in the membrane, penetrating into the lipid
phase from either side, others are completely buried within it, while others
transverse the membrane. The extent to which a protein penetrates into the
lipid phase is determined by its amino acid composition, sequence, second-
ary and tertiary structure. Thus, membrane proteins form a mosaic-like
structure in an otherwise fluid phospholipid bilayer, i.e. the fluid-mosaic
model (Figure 3.18).
Thus, the milk fat globules are surrounded and stabilized by a structure
which includes the trilaminar apical membrane (which is replaced by Golgi
membranes on secretion of proteins and lactose). The inner face of the
membrane has a dense proteinaceous layer, 10-50 nm thick, probably
acquired within the secretory cell during movement of the globule from the
rough endoplasmic reticulum at the base of the cell, where the triglycerides
are synthesized, to the apex of the cell. A layer of high melting triglycerides
may be present inside this proteinaceous layer. Much of the trilaminar
membrane is lost on ageing of the milk, especially if it is agitated; the
membrane thus shed is present in the skim milk as vesicles (or microsomes),
which explains the high proportion of phospholipids in skim milk.
McPherson and Kitchen (1983) proposed a detailed structural model of
the MFGM, which appears rather speculative. Keenan et aI. (1983), Keenan
and Dylewski (1995) and Keenan and Patton (1995) describe the current
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