BIOSYNTHESIS OF MILK COMPONENTS

Dairy Cattle Production 342-450A

Milk Biosynthesis

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Department of Animal Science

BIOSYNTHESIS OF MILK COMPONENTS



Milk Biosynthesis

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Introduction: Precursors of milk come from the bloodstream. It is estimated that theproduction of 1 liter of milk requires 500 liter of blood moving through the mammarygland to provide the milk precursors.

Some materials in the milk come unchanged from blood. These include minerals, somehormones and some proteins (e.g. immunoglobulins). Only precursors of milk protein andcarbohydrates are present in blood. The primary substrates extracted from blood by thelactating mammary gland include glucose, amino acids, fatty acids, β-hydroxybutyrate,and salt.

Biosynthesis of Milk FatBackgroundCow's milk contains 3.5 to 5% fat. About 97 to 98 of the fat is triglycerides (also knownas triacylglycerols or triacylglycerides) and Phospholipids constitute about 1% (Table 1).Palmitic (C16:0) and oleic (C18:1) acid are the main fatty acid in milk fat (Table 2).Milk fat contains low levels short chain fatty acid (C12 and less).

Table 1. Milk lipid composition of dairy cowsLipid class % of total lipidsTriglycerides 95.81,2 diglycerides 2.3Phospholipids 1.1Cholesterol 0.5Free fatty acids 0.3

Table 2. Fatty acid composition of milk.Fatty acid % weightC4:0 3.6C6:0 2.2C8:0 1.2C10:0 2.5C12:0 2.8C14:0 10.1C15:0 1.1C16:0 25.0C16:1 2.6C17:0 0.9C18:0 12.1C18:1 27.1C18:2 2.4C18:3 2.1Other 2.4

Biosynthesis of Milk Lipids (Triglycerides)


Milk Biosynthesis

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• Milk fat triglycerides are synthesized in the cytoplasm surface of the smoothendoplasmic reticulum of mammary epithelial cells. Milk lipids (triglycerides) aresynthesized from fatty acids and glycerol through the αα-glycerol phosphate pathway(Figure). Acetyl CoA carboxylase is the key milk biosynthesis enzyme and itsactivity increases considerably during lactogenesis (copious milk secretion).

• Two acyl CoA molecules react with α-glycerol-3-phosphate to form phosphatidicacid, which upon removal of the phosphate, leaves a 1,2 diacylglycerol. Anadditional long chain acyl CoA adds the final fatty acid, with the formation oftriacylglycerol and CoA.

Sources of Milk Fatty Acids• The fatty acids used to synthesize milk fat (triglycerides) come from two sources:

1- Blood lipids2- De novo synthesis within the mammary epithelial cells (synthesis of new moleculesof fatty acids from precursors absorbed from the blood)

1- Blood lipids: Derived from digestion and absorption of dietary fat and frommobilization of fatty acids from adipose tissue. Most of the fatty acids derived fromblood plasma are of dietary origin (> 80%). This amount could differ according to stageof lactation and milk yield. Blood lipids are the source of all of the C18 and most of theC16 fatty acids in milk. One third of the C16 and most of the C18 fatty acids in the milkare of dietary origin. In total, about 1/2 of the milk fatty acids are derived from the bloodplasma lipids.

B i o s y n t h e s i s o f M ilk T r ig lycer ides

B l o o d t r ig lycer ides

M o n o g l y c e r i d e s F r e e F A G lycero l

F a t ty A cy l C o A

M ilk t r ig lycer ides

αα -g l y c e r o l - P

A c e t a t e ββ -h y d r o x y b u t y r a t e

A c e t a t e ββ -h y d r o x y b u t y r a t e

C 4-C 1 2

M a m m a ry ce l l

B a s a l m em b r a n e

C a p i l l a r y w a ll

B l o o d

L i p o p r o t e i n l i p a s eL i p o p r o t e i n l i p a s e


Milk Biosynthesis

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• In dairy cow diets, dietary fats consist mainly of long chain fatty acids (palmitic, C16;stearic, C18:0; oleic, C18:1, linoleic, C18:2; linolenic, C18:3). Dietary fatty acids arebiohydrogenated (saturated) in the rumen by ruminal microbes. Therefore fatty acidsin adipose tissue and in milk of dairy cows are more saturated in nature than those ofthe diet. Intestinal and mammary epithelia of ruminants contain an active desaturaseenzyme that converts saturated fatty acids to mono-saturated fatty acids (mainlystearic to oleic acid). However, most of the desaturation takes place in the mammarygland rather than in the small intestine. The desaturation of fatty acids helps to offsetthe extensive biohydrogenation that occur in the rumen and reduces the ratio ofstearic:oleic acids in cow’s milk. This also ensures sufficient fluidity of milk fat forefficient secretion of milk from the mammary gland.

2- De novo synthesis: Acetate and β-hydroxy-butyrate are the major carbon sources offatty acid biosynthesis in the mammary gland. Almost all C4 to C14 fatty acids (short andmedium-chain fatty acids) are synthesized de novo. Short chain fatty acids of variouslengths are synthesized by the step-wise addition of acetate to β-hydroxy-butyrate. Extraβ-hydroxy-butyrate will be converted to acetate. The mammary gland of the dairy cowutilizes acetate for short-chain fatty acid biosynthesis and also as an energy source.

The Good & the Bad

• Despite the fact that the cholesterol level in milk is low, milk fat is consideredhypercholesterolemic. This is mainly because of its high-saturated fatty acid content(60 to 65%). Palmitic (C16:0) and C14:0 acids have been shown to behypercholesterolemic while shorter fatty acids (C4-C10) are neutral. Stearic, C18:1and C18:2 acids are hypocholesterolemic.

• Bovine milk fat contains significant amounts of short chain fatty acids and relativelylower concentrations of C18 fatty acids than are found in other sources of animal fatsuch as beef or pork. Bovine milk is also a poor source of polyunsaturated fatty acids.Milk fatty acid are derived in part from dietary long chain fatty acids, microbialsynthesis of fatty acids and body stores of fat with the remainder coming from denovo synthesis in the mammary gland. Manipulating the diet of the dairy cow cansubstantially alter the balance between mammary de novo synthesis of short andmedium chain fatty acids, and dietary long chain fatty acids presented to themammary gland.


Milk Biosynthesis

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Biosynthesis of Milk Proteins

Milk Protein Fractions

• The nitrogen content milk is distributed among three major groups:1- Caseins (76% of total milk nitrogen)2- Whey protein (18% of total milk nitrogen)3- Non-protein nitrogen (6% of total milk nitrogen)

True proteins (i.e. excluding NPN) are classified into three fractions: caseins present inmicelles, whey proteins present in solution and fat globule membrane proteins on thesurface of fat globules.

• Milk proteins contain more amino acids than any other natural food. The major milkproteins are only found in milk. These include:

Casein proteins; αα-, ββ-, and κκ-caseinLactoglobulin; ββ-lactoglobulin (~50% of whey proteins)Lactalbumin. αα-lactalbumin (~25% of whey protein)

A second group blood proteins (e.g. immunoglobulins) and some proteins synthesized inthe plasma cell adjacent to the secretory epithelium, enter the mammary gland and appearin the milk unchanged

Steps of Protein Biosynthesis

Milk proteins are synthesized from amino acids present in the mammary secretory cell.The biosynthesis takes place in the ribosome, which is attached to the rough endoplasmicreticulum. Steps of biosynthesis are similar to those of any other protein:

1- Transcription: A strand of messenger RNA (mRNA) is formed from DNA. Itcarries the code of a specific protein. The mRNA is located in theribosome, which is attached to the rough endoplasmic reticulum.

2- Activation: Amino acids in the cytoplasm are activated by reaction with ATPand attachment to transfer RNA (tRNA). The tRNAs are specificfor each amino acid.

3- Translation: Takes place in the ribosome. The mRNA contains codes for aminoacids. Each code consists of three nucleotides and is known as acodon. Located in the tRNA a trinucleotide anticodon thatrecognizes it. As each codon in the mRNA comes in position, theappropriate amino acid-tRNA complex moves in the amino acidjoined the previous one in the chain.


Milk Biosynthesis

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In the synthesis of export proteins(milk proteins) in the RER,ribosomes are attached to the RERand the protein polypeptide issynthesized with an additionalinitial chain of 10-20 amino acids.The initial signal peptide sequencemediates the passage of the start ofthe amino acid chain through themembrane of the RER into the innerpassages and is clipped off in theprocess. The polypeptide chaininserted into the RER folds up in aconfiguration as a function of thephysical forces in the amino acidsequence. Depending on theprotein, certain other materials (e.g.phosphate) may be added later. Thefinal three-dimensional structure of the protein determines its function and structure.

Biological Importance of Milk Proteins

• Milk proteins are only present in milk. They are the main source of amino acid for thenewborn. Casein micelles also provide Ca and P for skeletal development. Caseinmicelles are high digestible by the proteolytic enzymes of the newborn

• Some milk proteins have intracellular functions. For instance, α-lactalbumin forms apart of the enzyme lactose snynthase.

• Milk contains proteins such as lactoferrin and lysozyme. The antibacterial propertiesof these materials, lysozyme digesting bacterial polysaccharides and lactoferrinsequestering iron required by bacteria emphasize their importance in reducingmastitis infections. Lactoferrin concentration is high in the dry bovine mammarygland.

• In many mammalian species including bovine colostrum is a vital way of transferringpassive immunity from the mother to the newborn


Milk Biosynthesis

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Biosynthesis of Milk Carbohydrates (Lactose)

Background• Lactose is the most constant constituent in bovine milk (about 4.5%). The main

function of lactose is to maintain the osmolality of milk during the formation andsecretion process. Glucose is essential for lactose synthesis and cannot be replacedby any other sugar. About 45-60% of blood glucose in ruminants is synthesized inthe liver from propionate by a process known as gluconeogenesis. Blood glucoselevels in ruminants are about half those found in non-ruminants.

Site and Steps in Lactose Biosynthesis• The site of lactose synthesis is the membranes of the Golgi apparatus. Glucose is the

only precursor and two molecules of glucose are required for each molecule oflactose. One molecule of glucose is converted to galactose. The enzyme catalyzingthis conversion appears just before parturition and its activity increase dramatically atthe onset of milk synthesis in lactation (Lactogenesis).

• The condensation of glucose and galactose involves the enzyme lactose synthase.The enzyme composed of two proteins (galactocyl trnasferase and α-lactalbumin)that must be together for lactose biosynthesis to take place. Therefore, the rate oflactose biosynthesis is greatly influenced by the availability of αα-lactalbumin fromthe rough endoplasmic reticulum.

• Lactose is a nonpermeable disaccharide, which cannot diffuse out the Golgimembrane or out of the secretory vesicles' membrane. This is important for milksynthesis because it is the synthesis of the nondiffusible lactose, which results inwater being drawn into the Golgi. Water is osmotically drawn into the Golgi to try todilute the lactose.


Milk Biosynthesis

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Secretion of Milk Constituents

• The individual component of milk are kept separate inside the secretory cell andtherefore, milk is not formed until the individual components reached the lumenwhere they are mixed together.

• Milk protein synthesized in the Rough-Endoplasmic Reticulum where it incorporatedinto the Golgi vesicles (vacuoles). Other non-fat components including lactose andsalts are also incorporated into the Golgi vesicles. The secretory vesicles separatefrom the Golgi apparatus and move towards the apical region of the cell where themembrane surrounding each vesicle fuses with the plasma membrane and the contentis discharged into the lumen.

• Milk fat or lipids take a separate secretion pathway than that taken by non-fat milkcomponents (i.e. protein & lactose). Lipid molecules increase in size as they movefrom the endoplasmic reticulum towards the apical membrane where they pushthrough and break away as globules engulfed in an envelope made of apical plasmamembrane. The apical membrane in composed of lipids, which come from the wallsof the secretory vesicles carrying the non-fat components of milk to the apicalmembrane. The milk fat globule is membrane-surrounded and has a number ofmembrane associated proteins, These proteins and others trapped during the processof cream separation are important for the whipping properties of cream


Milk Biosynthesis

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Rate of Milk Secretion

The period after milking is characterized by low intra-alveolar pressure, facilitates thetransport of newly synthesized milk into alveolar lumen. As secretion continues betweenmilkings, backpressure is exerted ontothe secretory process by the alveolarluminal contents. At some point theluminal pressure exceeds the force ofsecretion as the alveolar enlargementreaches its limit. The distension ofpressure of the lumen exceeds thestrength of secretory mechanismsneeded to push the newly formed milkout of the cell. In turn, the buildup ofnewly formed milk in the cells reducesthe uptake of milk precursors bychemical (a negative feedbackmechanism) and / or mechanicalprocess. The mechanical factors are aresult of a distended alveoli partiallyreplacing all other intramammary compartments including the blood vessels.

After 10 hours from the last milking, the average secretion rate begins to decrease andsecretion stops after 35 hours. The increase in udder pressure per unit of newly formedmilk varies according to

1- Level of milk production. The pressure per unit of newly formed milk is lower forhigh-producing cows than for low producing cows.

2- Age of the cow. Pressure is lower for older than younger cows.3- Stage of lactation. Pressure is lower in early lactation than in late lactation

Udder pressure & secretion rate

0102030405060

0 10 20 30 40

hours since last milking

Udder Pressure (mm

Hg) (----)

Secretion Rate

(kg/hr) (____ )

1.5

1.0

0.5

0

BIOSYNTHESIS OF MILK COMPONENTS

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