Triacylglycerolsq

Alastair KH MacGibbon and George Thomas Fuller, Fonterra Research and Development Centre, Palmerston North, New Zealand
© 2022 Elsevier Ltd. All rights reserved.
This is an update of M.W. Taylor, A.K.H. MacGibbon, Milk Lipids j Triacylglycerols, In Encyclopedia of Dairy Sciences, Second Edition, edited by John
W. Fuquay, Elsevier Ltd, 2011, https://doi.org/10.1016/B978-0-12-374407-4.00334-4.

Introduction 849
Structure of Triacylglycerols 850
Composition of Triacylglycerols 851
Crystallization and Melting Behavior of Triacylglycerols 853
References 853
Further Reading 853

Introduction

Bovine milk fat consists of a complex mixture of triacylglycerols, which vary considerably in molecular weight and degree of unsa-
turation. Despite the complexity, the major triacylglycerols can be broadly categorized into two structural types, those containing
combinations of long-chain fatty acids and those containing two long-chain fatty acids and a short-chain fatty acid esterified at posi-
tion sn-3. The latter type is found in significant quantities only in ruminant milk fats. The melting properties of milk fat are deter-
mined largely by the fatty acid composition of its constituent triacylglycerols.
The most important class of compounds in bovine milk fat, from a quantitative viewpoint, are by far triacylglycerols, accounting
for 98% of the total fat. Triacylglycerols are relatively simple, nonpolar molecules, consisting of a glycerol backbone to which are
esterified three fatty acid molecules (Fig. 1). Although triacylglycerols are not highly reactive, they do undergo two important chem-
ical reactions, namely hydrolysis and oxidation, which cause deterioration in the quality of food.
The complex nature of triacylglycerols of bovine milk fat is a direct result of the large number and wide variety of fatty acids that
make up the triacylglycerols. Although the distribution of the fatty acids on the triacylglycerol molecules is not entirely random, the
number of distinct triacylglycerols arising from the combination of a large number of fatty acids is in the order of several hundred.
For example, if only the 14 acids that are present at concentrations above 1% are considered and if the position on the triacylglycerol
molecule is ignored, then there are a possible 560 compositionally different triacylglycerols (this figure is derived from the equation
N ¼ (n (n þ 1)  (n þ 2)/6), where N is the number of triacylglycerols and n is the number of fatty acids). Because of this
complexity, the determination of individual triacylglycerols is a very difficult task.
The general composition of milk triacylglycerols, determined by capillary gas chromatography, is presented in Table 1. The
triacylglycerols show a wide range of molecular weight (from carbon number 28 to 56), which arises from the large difference
in the chain length of the constituent fatty acids (from C4 to C18). Interestingly, the range of values for the different carbon
numbers is considerable, indicating that there is significant variation in triacylglycerol composition throughout the dairying
season.
It should be noted that there are a number of individual triacylglycerols having the same carbon number; for example, C50 could
possibly include the triacylglycerols 16:0, 16:0, 18:0; 14:0, 18:0, 18:0; 16:0, 16:0, 18:1; and 14:0, 18:0, 18:1.

Fig. 1 Triacylglycerol molecule.

q
Change History: September 2020. Revision to text and references. No change in figures but figure 2 has new file. Same tablesdupdated values.

Encyclopedia of Dairy Sciences, 3rd edition, Volume 3 https://doi.org/10.1016/B978-0-12-818766-1.00319-6 849

850 Triacylglycerols

Table 1 Composition of triacylglycerols of milk fat

Triacylglycerol composition (mol%)

Carbon number a Typical Range b

C28 0.8 0.5–1.1

C30 1.5 1.0–1.8
C32 3.1 2.3–3.6
C34 6.8 5.3–7.5
C36 12.5 10.6–13.3
C38 14.5 13.2–15.9
C40 11.2 10.2–12.6
C42 7.4 6.6–7.9
C44 6.5 5.4–7.0
C46 6.7 5.2–7.2
C48 7.7 6.2–9.0
C50 9.3 8.4–11.4
C52 7.9 6.4–10.9
C54 3.8 2.7–5.9
C56 0.4 0.3–0.5

A.K.H. MacGibbon (personal communication), Fonterra Research Centre seasonal survey.

a
Carbon number is the sum of the acyl carbons on the triacylglycerol molecule.
b
Range of values over a dairying season.

Structure of Triacylglycerols

An initial examination of the glycerol molecule may suggest that positions sn-1 and sn-3 are equivalent (Fig. 2). However, further
examination of configurations A and B shows that when the three-dimensional arrangement of the bonds involving the central
carbon is taken into account, the two configurations cannot be superimposed. Thus the carbon atom at position sn-1, in relation
to the other groups attached to the central carbon, is different from the carbon atom at position sn-3.
Triacylglycerols are synthesized in the mammary gland by enzymatic reactions that normally involve multi-point attachment,
and consequently the spatial arrangement of neighboring groups is critical. This means that it is possible for enzymes to distinguish
between positions sn-1 and sn-3 and the enzymic mechanisms involved in the biosynthesis of triacylglycerols exert some selectivity
over the placement of fatty acids at the different positions.
Stereospecific analytical procedures have been developed that have permitted the determination of the positional distribution of
fatty acids on the triacylglycerols. Results obtained by several workers have shown that there is a general pattern of stereospecific
distribution of fatty acids in the triacylglycerols of bovine milk fat (Table 2). For cows fed a normal diet, the fatty acids 4:0 and
6:0 are esterified almost entirely at position sn-3. In contrast, 12:0 and 14:0 are esterified preferentially at position sn-2 while
16:0 is incorporated preferentially at positions sn-1 and sn-2. 18:0 is esterified preferentially at position sn-1 and 18:1 shows a pref-
erence for positions sn-1 and sn-3. This overall pattern of fatty acid distribution does not change significantly either throughout the
dairying season or between countries. This indicates that in normal milk fats, in which fatty acids vary to some extent as a result of
dietary and lactational effects, the overall pattern of positional specificity of fatty acids is relatively constant.
When a cow is placed on a restricted diet, there is a decrease in the fatty acids that are synthesized de novo in the mammary gland
(4:0 to 16:0) and an increase in the fatty acids 18:0 and 18:1 as a result of the mobilization of body reserves. The net effect is
a marked increase in the proportion of 18:1 at position sn-3 from 17.1% to 47.6%, which is accompanied by reductions in the
proportions of 4:0 to 16:0 at this position (Table 2). Despite these marked changes caused by the restricted diet, the overall pattern
of stereospecific distribution of fatty acids is altered only slightly.

sn-1 sn-1

sn-2 sn-2

sn-3 sn-3

Configuration A Configuration B
Fig. 2 Fischer projection diagrams of glycerol using the stereospecific numbering (sn-) convention. Configuration B is obtained from configuration
A by flipping the molecule through 180 .
 Triacylglycerols 851

Table 2 Positional distribution of fatty acids in the triacylglycerols of milk fat

Fatty acid composition (mol%)

Normal diet Restricted diet
Fatty acid sn-1 sn-2 sn-3 sn-1 sn-2 sn-3

4:0 0.4 30.6 0.4 26.8

6:0 0.7 13.8 0.7 0.4 9.2
8:0 0.3 3.5 4.2 0.7 2.6 1.8
10:0 1.4 8.1 7.5 1.1 5.6 2.4
12:0 3.5 9.5 4.5 2.1 6.7
14:0 13.1 25.6 6.9 7.4 17.4
16:0 43.8 38.9 9.3 32.6 40.7 2.1
18:0 17.6 4.6 6.0 20.9 5.6 10.1
18:1 19.7 8.4 17.1 34.0 21.1 47.6

Calculated from the data of Parodi (1979).

Composition of Triacylglycerols

Although milk fat contains a very complex mixture of triacylglycerols, it is possible to fractionate these triacylglycerols into different
classes according to their molecular weight and degree of unsaturation using a combination of chromatographic methods. This
approach has enabled a detailed analysis of the different classes of triacylglycerols in milk fat.
Milk fats from different countries have been separated in this manner and the proportions of the different fractions are given in
Table 3. The proportion of saturated triacylglycerols ranges from 33% to 39% and is high relative to other fats and oils. For example,
many vegetable oils contain only very small amounts of saturated triacylglycerols, and even palm oil, which contains about 40%
16:0, has only 8%–10% saturated triacylglycerols. A further feature of bovine milk fat is the high proportion of low molecular-
weight triacylglycerols (31%–44%). These triacylglycerols largely consist of two long-chain fatty acids in combination with
a short-chain fatty acid and are found in significant quantities only in ruminant milk fats.

Table 3 Proportions of triacylglycerol classes in bovine milk fat from different countries

Nature of triacylglycerols Proportions of triacylglycerols (percentage in milk fat)

a
Molecular weight Level of unsaturation New Zealand milk fat Australian milk fat Canadian milk fat

High Saturated 11.4 12.7 6.4

Monoene 15.6 19.8 14.4
Diene 8.7 10.1 10.8
Triene 4.2 3.7 5.0
Polyene 2.4
Total 39.9 46.3 39.0
Medium Saturated 7.7 9.3 6.6
Monoene 6.3 8.6 6.6
Diene 1.7 2.5 2.5
Triene 1.2 2.3 1.5
Polyene
Total 16.9 22.7 17.2
Low Saturated 20.1 14.5 19.7
Monoene 16.1 12.0 16.7
Diene 4.3 2.8 5.1
Triene 2.7 1.7 2.3
Polyene
Total 43.2 31.0 43.8
Milk fat Saturated 39.2 36.5 32.7
Monoene 38.0 40.4 37.7
Diene 14.7 15.4 18.4
Triene 8.1 7.7 8.8
Polyene 2.4
Total 100.0 100.0 100.0

a
Saturated, monoene, diene, and triene triacylglycerols contain zero, one, two, and three carbon–carbon double bonds, respectively.
Data from Hawke and Taylor (1995).
852 Triacylglycerols

The proportions of corresponding classes (i.e., similar molecular weight and the same degree of unsaturation) in the three milk
fats are broadly similar, with saturated and monoene triacylglycerols being the dominant classes. One interesting exception is the
lower amount of high-molecular-weight saturated triacylglycerols reported in Canadian milk fat.
Stereospecific analyses of the different triacylglycerol classes have shown that the general pattern of positional distribu-
tion of fatty acids of chain length C4–C16 in these classes is similar to that in normal milk fat. However, the pattern of
distribution of 18:0 and 18:1 varies according to the molecular weight of the triacylglycerols; these fatty acids tend to be
esterified preferentially at positions sn-1 and sn-3 in triacylglycerols of high molecular weight and concentrated at position
sn-1 in triacylglycerols of medium and low molecular weight where 4:0 and 6:0 occupy much of the sn-3 position. A further
interesting point is that the sn-3 position in unsaturated triacylglycerols of low molecular weight consists largely of short-
chain fatty acids and 18:1. The sn-3 position is the last to be acylated during triacylglycerol biosynthesis and it has been
postulated that the placement of these fatty acids at this position may well be to keep the fat liquid at physiological
temperatures.
In recent times, the use of combinations of chromatographic techniques has been refined further to permit the separation and
identification of many of the triacylglycerols in milk fat. Complex mixtures of triacylglycerols are subdivided into much simpler
mixtures prior to the separation and identification of individual triacylglycerols.
In one study (Kalo et al., 2009), milk fat was separated by normal-phase high-performance liquid chromatography and electro-
spray ionization mass spectrometry (HPLC-ESI-MS) of ammonium ion adducts to enable the identification of over 400 triacylgly-
cerols species in 184 quantified peaks. This exhaustive study included the use of enzymatic sn-2 analysis, thin-layer chromatography,
and solid-phase extraction (and gas chromatography analysis) to isolate a wide range of fractions for analysis by HPLC-ESI-MS. This
allowed their proportion in milk fat, and in many cases the stereoisomeric positions, to be determined. The vast majority of these
triacylglycerols were present at very low levels, that is, less than 0.2 mol%. However, a few were present at surprisingly high
amounts: for example, in the order of increasing acyl carbon number from quantified peaks 16:0/14:0/4:0 þ 18:0/12:0/4:0
(3.4%),16:0/16:0/4:0 þ 18:0/12:0/6:0(5.2%), 18:1/16:0/4:0 (4.1%), 18:0/16:0/4:0 (2.9%), 18:1/14:0/16:0 þ 16:0/16:1/16:0
(3.1%), 16:0/16:0/16:0 þ 18:0/14:1/16:0 (2.4%), 18:1/16:0/18:1 þ 18:2/18:0/16:0 (3.7%). These species account for approxi-
mately 25% of the milk fat sample analyzed. In an extensive study, Liu et al. (2020) detected 3454 different triacylglycerol species
in their study of Australian commercial milk.
These sophisticated separation techniques, in combination with stereospecific analyses, have enabled the major triacylgly-
cerols and many of the minor triacylglycerols in milk fat to be determined. The major triacylglycerols are presented in a some-
what simplistic manner in Fig. 3. The major high-molecular-weight triacylglycerols comprise combinations of four long-chain
fatty acids (14:0, 16:0, 18:0, and 18:1), whereas the major triacylglycerols of medium and low molecular weight contain two of
these four long-chain fatty acids and a short-chain fatty acid (either 4:0 or 6:0) esterified at position sn-3 (Taylor and Hawke,
1975).
Although these painstaking studies have identified a large number of triacylglycerols in milk fat, the time-consuming nature of
these investigations means that it is currently impractical to measure the level of individual triacylglycerols in milk fat on a routine
basis. However, simple separation of milk fat triacylglycerol species by carbon number on gas chromatography have proved an effec-
tive method of detecting milk fat adulteration due to the fact that it is a combination of the fatty acids involved in the triacylglycerol
and the way the fatty acids are combined (Molkentin et al., 2019).

Fig. 3 Major triacylglycerols of milk fat.

 Triacylglycerols 853

Crystallization and Melting Behavior of Triacylglycerols

The crystallization and melting characteristics of triacylglycerols of milk fat have a marked effect on the functional properties of
dairy products, particularly the consistency of butter. These characteristics are influenced to a large degree by the fatty acid compo-
sition of the triacylglycerols and to a lesser extent by the physical processes of polymorphism and mixed crystal formation.
Triacylglycerols form crystals when they solidify. Generally, triacylglycerols crystallize in layered structures, with glycerol groups
and methylene groups in one plane and the axes of the hydrocarbon chains straight and parallel to each other within a layer. The
attractive forces acting between molecules are lateral van der Waals’ interactions between hydrocarbon chains, van der Waals’ inter-
actions of methyl end groups, and polar interactions of ester groups. The melting points of triacylglycerols are determined largely by
the packing density of the hydrocarbon chains in the crystal lattice; the greater the packing density, the closer the hydrocarbon
chains and the greater the attractive van der Waals’ forces, and hence the higher the melting point.
The fatty acid composition of triacylglycerols has a marked effect on their molecular arrangement in the crystal lattice, which in
turn affects their packing density. Triacylglycerols consisting of three long, “straight-chain”, saturated fatty acids have a closely
packed lattice structure and a high melting point. The replacement of a saturated fatty acid with a “bent-chain” cis-unsaturated fatty
acid has a large effect on the molecular arrangement of triacylglycerols in the lattice, which in turn reduces the packing density of
hydrocarbon chains and hence lowers the melting point of cis-unsaturated triacylglycerols compared to saturated triacylglycerols. In
a similar manner, the inclusion of short- and medium-chain fatty acids lowers the melting points of low-molecular-weight triacyl-
glycerols compared to high-molecular-weight triacylglycerols.
As noted previously, milk fat triacylglycerols vary considerably in both molecular weight and degree of unsaturation and as
a result they melt over a wide range of temperature. Indeed, milk fat has a very broad melting range, from 40 to þ40  C. A further
consequence of the above trend in melting points is that the higher the proportion of low-molecular-weight and unsaturated tri-
acylglycerols in milk fat, the greater the proportion of low-melting triglycerides and hence the softer the milk fat. New Zealand
spring and summer milk fat shows this trend. Spring milk fat contains greater proportions of triacylglycerols containing 4:0 and
18:1 than summer milk fat, which leads to a higher proportion of low-melting triacylglycerols in the former. As a consequence,
spring butter is softer than summer butter. Similarly, in Europe, summer butter contains greater proportions of triacylglycerols con-
taining 18:1 than winter butter and consequently is softer.

See Also: Fatty Acids; Lipid Oxidation; Lipolysis and Hydrolytic Rancidity; Milk Lipids: Rheological Properties and Their Modification

References

Hawke, J.C., Taylor, M.W., 1995. Influence of nutritional factors on the yield, composition and physical properties of milk fat. In: Fox, P.F. (Ed.), Advanced Dairy Chemistry, Lipids,
second ed., vol. 2. Chapman & Hall, London, pp. 37–88.
Kalo, P., Kemppinen, A., Ollilainen, V., 2009. Determination of triacylglycerols in butterfat by normal-phase HPLC and electrospray-tandem mass spectrometry. Lipids 44, 169–195.
Liu, Z., Li, C., Pryce, J., Rochfort, S., 2020. Comprehensive Characterization of Bovine Milk Lipids: Triglycerides. ACS Omega 5 (21), 12573–12582.
Molkentin, J., Golay, P.A., Contarini, G., Johnson, R., MacGibbon, A.K.,H., 2019. Guidance on the Practical Application of IDF/ISO Standard on the Determination of Milkfat Purity,
Brussels. Bulletin of the IDF No. 499/2019.
Parodi, P.W., 1979. Stereospecific distribution of fatty acids in the bovine milk fat triglycerides. J. Dairy Res. 46, 75–81.
Taylor, M.W., Hawke, J.C., 1975. The triacylglycerol compositions of bovine milkfats. N. Z. J. Dairy Sci. Technol. 10, 40–48.

Further Reading

Breckenridge, W.C., 1978. Stereospecific analysis of triacylglycerols. In: Kuksis, A. (Ed.), Handbook of Lipid Research Fatty Acids and Glycerides, vol. 1. Plenum Press, New York,
pp. 197–232.
Gresti, J., Bugaut, M., Maniongui, C., Bezard, J., 1993. Composition of molecular species of triacylglycerols in bovine milk fat. J. Dairy Sci. 76, 1850–1869.
Janssen, P.W.M., MacGibbon, A.K.H., 2007. Non-isothermal crystallization of bovine milk fat. J. Am. Oil Chem. Soc. 84, 871–875.
Lopez, C., 2018. Crystallization properties of milk fats. In: Sato, K. (Ed.), Crystallization of Lipids. Wiley, New York, pp. 283–321.
MacGibbon, A.K.H., Taylor, M.W., 2006. Composition and structure of bovine milk lipids. In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry, Lipids, third ed., vol. 2.
Springer, New York, pp. 1–42.
Robinson, N.P., MacGibbon, A.K.H., 1998. The composition of New Zealand milk fat triacylglycerols by reverse-phase high-performance liquid chromatography. J. Am. Oil Chem.
Soc. 75, 993–999.
Walstra, P., Jenness, R., 1984. Dairy Chemistry and Physics. John Wiley, New York.
Wright, A.J., Marangoni, A.G., 2006. Crystallization and rheological properties of milk fat. In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry, Lipids, third ed., vol.
2. Springer, New York, pp. 245–291.
Xu, S.L., Wei, F., Xie, Y., et al., 2018. Research advances based on mass spectrometry for profiling of triacylglycerols in oils and fats and their applications. Electrophoresis 39,
1558–1568.