This document discusses milk fat synthesis, including its components, precursors, and synthesis process. Milk fat is composed of fatty acids derived from de novo synthesis in the mammary gland or from uptake of preformed fatty acids from the blood. Roughly 50% come from each source. Fat precursors include acetate, B-hydroxybutyrate, and circulating long-chain fatty acids. The fat synthesis process involves fatty acid uptake and triglyceride assembly in mammary epithelial cells.

1. Milk Fat Synthesis
Eng. Mohammad AlSaleh
info@technicaljo.com
Technical Company for Industry and Trade

2. OUTLINE
• Introduction.
• Objective.
• Fat components.
• Fat precursors.
• Fat synthesis.
• References

3. INTRODUCTION
Milk composition is economically important to both milk
producers and milk processors.
Milk consists of approximately 13% solids and 87% water
and it is the concentration of solids that determines the
economic value of milk.
A high proportion of total milk production is used for
product manufacture.

4. The consumers interest in milk composition arises mainly from a
nutrition point of view.
For many years fat was the most valuable component of milk and
increasing it was a major focus of breeding programmers.
Bovine milk fat has unique functional and nutritional properties
which became persistent need to understanding how milk fat is
synthesized.

5. OBJECTIVE
Highlight about the component, precursor and
synthesis of milk fat.

6. MILK FAT
COMPONENTS
Milk fatty acid (FA) are derived from two sources:
1. De novo synthesis.
2. Uptake of preformed FA.
De novo synthesis substrates are mainly acetate and B-
hydroxybutyrate derived from rumen organic matter
fermentation.
They are used by the mammary epithelial cells to synthesize
short- and medium-chain fatty acids (C4:0 to C14:0) plus a
portion of the 16-carbon FA.

7. The second source of FA in milk is the mammary uptake of
circulating long-chain FA.
This source provides a portion of the 16-carbon and all of the
long-chain FA (≥ C14:0) originated from the intestinal
absorption of dietary and microbial lipids and also from the
mobilization of body fat reserves.
Under normal conditions, about 50% of the FA in milk originate
from de novo synthesis in the mammary gland, while the other
50% originate from the uptake of preformed FA.
In this situation the mobilization of body fat reserves accounts
for less than 10 % of the FA in milk fat.

8. LIPIDS OR FATS
. % of lipids in Milk .
Lipid Cow Human Rat
Triglyceride 97-98 98.2 87.5
Diglyceride 0.25-0.48 0.7 2.9
Monoglyceride 0.02-0.04 T 0.4
Free fatty acids 0.1-0.4 0.4 3.1
Phospholipids 0.6 – 1 0.25 0.7
Cholesterol 0.2 - 0.4 0.25 1.6

9. FA carbon FA common name
4:0 Butyric
6:0 Caproic
8:0 Caprylic
10:0 Capric
12:0 Lauric
14:0 Myristic
15:0 Pentadecanoic
16:0 Palmitic
16:1 Palmitoleic
17:0 Margaric
18:0 Stearic
18:12 Oleic
18:2 Linoleic
18:3 Linolenic

10. MILK FATTY ACIDS-COW
% From De novo % From VLDL
Fatty acid synthesis fatty acids
C4 - C10 100 0
C12 80 - 90 10 - 20
C14 30 - 40 60 - 70
C16 20 - 30 70 - 80
C18 0 100

11. DIETARY LIPID
Lipids are substances which are water insoluble, but are soluble in
organic solvents (ether, chloroform, hexane, etc.).
Usually, the diet eaten by cows contains only 2 - 4% lipids.
However, lipids are an important part of the ration of dairy cows
because they contribute directly to about 50% of the fat in milk
and they are the most concentrated source of energy in feed.

12. TYPES OF LIPID
1. Triglycerides are found primarily in cereal grains, oilseeds
and animal fats.
The basic structure of triglycerides consist of one unit of
glycerol (a 3 carbon sugar) and three units of fatty acid.
2. Glycolipids form a second class of lipids found primarily
in forage (grasses and legumes).
These compounds have a structure similar to the triglycerides
except that one of the three fatty acid has been replaced by a
sugar (usually galactose).

13. 3. Phospholipid are minor components in feedstuffs, but they
are found in a high concentration in ruminal bacteria .
When one of the fatty acids is replaced by a phosphate bound to
another complex structure the lipid is referred to as
phospholipid.
4. Free fatty acids are not attached to a glycerol molecule.
Fatty acids in feeds consist of a hydrocarbon chain ranging
in length from 14 to 18 carbons.
Melting point is influenced primarily by the degree of
saturation and to a lesser extent by the length of the carbon
chain.

14. Triglyceride
Glycerol + 3 Fatty Acids Triglyceride
Connected by an ester bond
Fatty acids can be the same or mixed

16. Plant lipids typically contain 70 to 80% unsaturated fatty
acids and they tend to remain in the liquid state (oils).
On the other hand, animal fats contain 40 to 50%
saturated fatty acids and they tend to remain in the solid
state (fats).
The degree of unsaturation has a marked effect on how
well it is digested by an animal and, in the case of
ruminants, whether or not it interferes with the
fermentation of carbohydrates in the rumen.

17. Common Name Structure Abbreviation Melting point °C
................................................ Saturated acids ..............................................................................
Myristic CH3-(CH2)12-COOH (C14:0) 54
Palmitic CH3-(CH2)14-COOH (C16:0) 63
Stearic CH3-(CH2)16-COOH (C18:0) 70
.............................................. Unsaturated acids ............................................................................
Palmitoleic CH3-(CH2)5-CH=CH-(CH2)7-COOH (C16:1) 61
Oleic CH3-(CH2)7-CH=CH-(CH2)7-COOH (C18:1) 13
Linoleic CH3-(CH2)4-CH=CH-CH2-CH=CH-(CH2)7-COOH (C18:2) - 5
Linolenic CH3-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)7-COOH (C18:3) -11
* The first number denotes the total number of carbons and the second number denotes the
number of double bonds in the molecule.
Common fatty acid found in
dairy diet

18. LIPID METABOLISM IN RUMEN
1. Lipid Hydrolysis
In the rumen, the majority of the lipids are hydrolyzed.
Some microbes help with fat hydrolysis in the rumen such as A.
lipolytic bacteria, Protozoal lipase, galactosidase, phospholipase ,
Microbes also ferment glycerol .
The bonds between the glycerol and the fatty acids are broken
down to give rise to glycerol and three fatty acids. Glycerol is
fermented rapidly into volatile fatty acids .
Some fatty acids are used by bacteria for the synthesis of
phospholipids that are needed to build cell membranes.
Another important action of ruminal microbes is to hydrogenate
unsaturated fatty acids.

19. 2. Isomerization
The second step in the transformation of dietary lipids in the
rumen is isomerization, comprising positional change in the
double bond configuration. The change from cis to trans
configuration depends on bacterial isomerase activity.
The trans configuration means that the hydrogen atoms are
located on the opposite side of the double bond. In the FA
formula syntax, the double bond configurations of trans and cis
are abbreviated as t and c, respectively, followed by the number
indicating where the double bond is located in the FA. The
isomerized double bond is separated by one single bond, and
occurs in a conjugated configuration.
Therefore, the FA resulting from the isomerization of linoleic
acid is called conjugated linoleic acid (CLA), and a conjugated
triene occurs for linolenic acid.

20. The most common CLA isomer is C18:2c9t11, rumenic acid
Rumenic acid is predominant in ruminal outflow as compared
to other conjugated isomers.
Dietary changes and changes in rumen environment may shift
isomerization pathways, resulting in the appearance of other
intermediates.
Increased amounts of the CLA isomer C18:2t10c12 are found in
rumen fluid and milk fat of cows showing milk fat depression.
And its associated with low rumen pH, which typically occurs
when cows are fed diets with high amounts of rapidly
fermentable carbohydrates.

22. 3. Biohydrogenation
Unsaturated FA are reported to be toxic for many rumen
bacteria, and interfere in rumen metabolism.
A strategy to reduce these toxic effects is the hydrogenation of
the double bond of unsaturated FA by rumen microbes to
saturate these FA.
This process is known as ruminal BH, which is the final step of
the ruminal FA transformation.
Major BH pathways for LA and LNA including several
intermediates and the end product C18:0 stearic acid (SA)

25. Free fatty acids in the rumen tend to attach to feed and
microbial particles and impede normal fermentation, especially
of fibrous carbohydrates.
However, lipids may be "protected" to slow down the rate of
hydrolysis and make them more "inert" in the rumen.
Also, industrial treatments that usually involve the formation of
soaps (calcium salts of fatty acids) make the fatty acids insoluble
and thus inert in the rumen.
Microbial phospholipids make up 10 to 15% of the lipids
leaving the rumen, the remaining 85 to 90% are saturated free
fatty acids found primarily in the form of palmitic and stearic
acids bound to feed and microbial particles.

27. INTESTINALABSORPTION
OF LIPIDS
Microbial phospholipids are digested in the small intestine and
contribute to the pool of fatty acids that are processed and
absorbed through the intestinal wall.
The bile secreted by the liver and the pancreatic juice (rich in
enzymes and bicarbonate) are mixed with the contents of the
small intestine.
These secretions are essential to prepare the lipids for absorption
by forming water miscible particles called micelles that can enter
the intestinal cells.
In the intestinal cells, a major portion of fatty acids are bound to
glycerol (coming from blood glucose) to form triglycerides.

28. Triglycerides, some free fatty acids, cholesterol and other lipid-
like substances are coated with protein to form triglyceride-rich
lipoproteins (TG-rich LP) also called chylomicrons or very low
density lipoproteins.
The TG-rich LP enter lymph vessels and flow to the thoracic duct
(the junction of the lymphatic system with the blood system)
where they enter the blood system.
In contrast to most nutrients absorbed from the gastro intestinal
tract, the absorbed lipids enter the general circulation directly
and are used by all body tissues without a preliminary processing
by the liver .

30. FAT PRECURSORS
Precursors taken up via basal membrane.
Milk fat triglycerides are synthesized in the mammary
epithelial cells.
Fatty acids used to synthesize milk triglycerides may arise
from two sources:
1. Breakdown of blood lipids (Preformed fatty acids,
glycerol, and monoacylglycerid).
2. De novo synthesis within the mammary epithelial
cells(Acetate and β-hydroxybutyrate)

31. Basal membrane ER membrane Luminal membrane
LPL
FAS
ACC
de novo FA synthesis (C4 - C16)
TAG
synthesis
Glucose
SFA (C16 - C18)
Synthesis Secretion
Acetate
ßHBA
TAG
Glucose
Circulation Translocation
UFA
FABP
NEFA
+
Glycerol
MFGM
Glycerol
Glycerol-P

32. BLOOD LIPIDS
About 40 to 60% of milk fatty acids come from blood mostly from
very low density lipoproteins (VLDL) which synthesized in
intestines and liver.
VLDL are 90 to 95% lipid on inside and 5 to 10% protein on outer
surface. And triglycerides in the VLDL are hydrolyzed in the
mammary capillaries by lipoprotein lipase (LPL).
LPL can hydrolyze off one, two or all three of the fatty acids from
the glycerol backbone which results in free fatty acids plus
diacylglycerides, monoacylglycerides, or glycerol.

33. Non-esterified fatty acid Released from adipose tissue by
hormone-sensitive lipase during periods of energy shortage
travel in blood via albumin and only significant during first
month of lactation its activated to fatty acyl-CoA.
Free fatty acids, monacylglycerides, diacylgyceridesand glycerol
can be taken up by epithelial cells and be reused for triglyceride
synthesis.
Blood lipids: long chains FA from diet and adipose tissue.
• lipoproteins (liver).
• chylomicrons (gut).
• free fatty acids / ketones.

35. DE NOVO SYNTHESIS
Fat synthesis within the mammary gland:
• Acetate is the main carbon source.
• β-hydroxybutyrate(BHBA) can also serve as the initial 4
carbons.
Both absorbed through cell’s basal membrane. Fatty acids are
built 2 carbons at a time 16 carbon limit.
Synthesis of short and medium chain fatty acids in the mammary
gland occurs in the cytoplasm of the mammary epithelial cell.
In ruminants, the carbon sources used for FA synthesis are acetate
and BHBA. And glucose is the carbon source for FA synthesis in
non-ruminants.

36. FATTY ACID SYNTHESIS PATHWAY
The Fatty Acid Synthesis Pathway involves the following steps :
• Activation -acetyl-CoA carboxylation.
The acetyl group from acetyl-CoA is transferred to ketoacyl
acyl carrier protein (ACP) synthase.
This reaction is catalyzed by acetyl- CoA transacetylase.
• Elongation -the malonyl-CoA pathway.
The malonyl-CoA pathway occurs with the growing FA chain
esterified to an acyl carrier protein. Each cycle through the
malonyl-CoA pathway results in two carbons being added to the
FA chain. Total reaction is (e.g. palmitate; C16):
Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH2are catalyzed by
Fatty Acid Synthetase to yield = Palmitate + 7 CO2+ 14 NADP + 8
CoA.

38. • Condensation step.
Condensation of the activated acetyl and malonyl groups takes
place to form Acetoacetyl-ACP.
The reaction is catalyzed by β- ketoacyl-ACP synthase.
• Reduction step.
The Acetoacetyl- ACP is reduced to β-hydroxybutyryl-ACP,
catalyzed by β-ketoacyl- ACP reductase.
NADPH + H+ are required
• Dehydration step.
Dehydration yields a double bond in the product, trans-Δ2-
butenoyl-ACP. Reaction is catalyzed by β-hydroxybutyryl-ACP
dehydratase.
• Another reduction step.
Reduction of the double bond takes place to form butyryl-ACP.
Reaction is catalyzed by enoyl-reductase.
Another NADPH dependent reaction The cycle is then repeated

42. • The cycle is then repeated
Butryl-ACP condenses with another malonyl-CoA to start the
second cycle.
Even though malonyl-CoA is a three carbon primer, one carbon
is lost in the condensation step and therefore only two carbons
are added to the growing fatty acid chain at each round
Seven cycles of condensation and reduction produce the
16-carbon saturated palmitoyl group still bound to ACP.
Chain elongation usually stops at this point and free
palmitate is released from the ACP molecule by hydrolytic
activity in the synthase complex.
Smaller amounts of longer fatty acids such as stearate
(18:0) are also formed.
In mammary gland, there is a separate Thioesterase specific
for acyl residues of C8, C10 or C12, which are subsequently
found in milk lipids.

44. Fatty acid synthetase is a large complex of enzymatic activities
which are responsible for the reactions of FA synthesis.
Acylthioesterases cleave off the growing FA chain from the acyl
carrier protein once it has reached a certain chain length.
Medium chain acylthioesterase cleaves off the growing FA chain at
or before it reaches C16.
In nonruminants, medium chain acylthioesteraseis cytoplasmic
and cleaves off free Fas.
In ruminants, medium chain acylthioesteraseis associated with the
fatty acid synthetase complex and releases acyl-CoA thioesters
ENZYMES IN FATTY ACID
SYNTHESIS PATHWAY

45. FATTY ACID SYNTHESIS
Acetate carbons come in twice as a source of acetyl-CoA to enter
the malonyl-CoA pathway that adds the two carbons to each
cycle of the FA synthetase.
Conversion of acetyl-CoA to malonyl-CoA is the rate limiting
step in FA synthesis.
Reaction is catalyzed by acetyl-CoA carboxylase which regulated
by lactogenic hormones and is one of the enzymes up-regulated
during the first stage of lactogenesis

46. ß-HYDROXYBUTYRATE
Acetate and β-hydroxybutyrate are primers and cannot be used in
fatty acid synthesis at later stages.
Contributes up to 50% of the first 4 carbons and cannot be split
into acetate in the cytosol, but can be converted to 2 acetyl-CoA's
in the mitochondria
If Can’t leave the mitochondria its lead to not available for FA
synthesis.
Glucose does not contribute to carbons of fatty acids in ruminants
because of Lack citrate lyase

47. Acetyl-CoA carboxylase is rate limiting enzyme for the fatty acid
synthesis pathway.
Fatty acid synthetase is large complex of enzyme activities
responsible for the chain elongation of the fatty acid.
Fatty acyl deacylase In liver and adipose tissue, fatty acid
synthesis is terminated when there are > 16 carbons by a
thioesterase I

48. Occurs in cytoplasm and intermediates are linked to acyl carrier
protein.
Enzymes of fatty acid synthesis are linked in a complex.
Elongation occurs by 2 carbons/cycle and the source of 2-carbon
units is acetyl-CoA via MalonylCoA which actually contributes the
carbons each pass through the cycle and required reducing agent
NADPH2 and elongation stops at C16.
De novo fatty acid synthesis Required:
• Carbon source (acetyl-CoA).
• Source of reducing equivalents (NADPH2).
• Proper enzymes (Acetyl CoA carboxylase/fatty acid synthetase).
FATTY ACID SYNTHESIS
SUMMARY

51. Milk fat triglycerides synthesized in cytoplasmic surface of
smooth endoplasmic reticulum and form small droplets
Small droplets fuse and moves toward apical membrane and
pinches off to alveolar lumen
Thus, inside cell nonmembrane-bound lipid droplet but in
alveolus lumen, milk fat globule surrounded by membrane
Secretion of fat to lumen

53. References
• Palmquist D. L. 2006. Milk fat: Origin of fatty acids and influence of nutritional
factors thereon. Pages 43–92 in Advanced Dairy Chemistry. Vol. 2. Lipids. 3rd ed.
• Sterk, A. 2011. Altering rumen biohydrogenation to improve milk fatty acid profile of
dairy cows. Ruminal fatty acid metabolism. ISBN 978-94-6173-020-6.
• Murphy J.J. 2000. Synthesis of milk fat and opportunities for nutritional manipulation.
British Society of Animal Science.
• Conte G., Serra A., Mele M. 2017. Dairy Cow Breeding and Feeding on the Milk Fatty
Acid Pattern. Chapter 2. Pages 19–41 in Nutrients in Dairy and Their Implications
for Health and Disease.
• Institute B. 1999. Dairy Essentials. ISBN 1592150535. 3th edition.
• McManaman J. L. 2009. Formation of milk lipids: a molecular perspective. Clin
Lipidol. 4(3): 391–401.