Biosynthesis Of Milk Components

Dairy Cattle Production 342-450A Milk Biosynthesis

Department of Animal Science

BIOSYNTHESIS OF MILK COMPONENTS

Dairy Cattle Production 342-450A

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Dairy Cattle Production 342-450A Milk Biosynthesis

Introduction: Precursors of milk come from the bloodstream. It is estimated that the production of 1 liter of milk requires 500 liter of blood moving through the mammary gland to provide the milk precursors. Some materials in the milk come unchanged from blood. These include minerals, some hormones and some proteins (e.g. immunoglobulins). Only precursors of milk protein and carbohydrates are present in blood. The primary substrates extracted from blood by the lactating mammary gland include glucose, amino acids, fatty acids, β-hydroxybutyrate, and salt.

Biosynthesis of Milk Fat Background Cow's milk contains 3.5 to 5% fat. About 97 to 98 of the fat is triglycerides (also known as 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 cows Lipid class % of total lipids Triglycerides 95.8 1,2 diglycerides 2.3 Phospholipids 1.1 Cholesterol 0.5 Free fatty acids 0.3 Table 2. Fatty acid composition of milk. Fatty acid % weight C4:0 3.6 C6:0 2.2 C8:0 1.2 C10:0 2.5 C12:0 2.8 C14:0 10.1 C15:0 1.1 C16:0 25.0 C16:1 2.6 C17:0 0.9 C18:0 12.1 C18:1 27.1 C18:2 2.4 C18:3 2.1 Other 2.4

Biosynthesis of Milk Lipids (Triglycerides)

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Dairy Cattle Production 342-450A Milk Biosynthesis

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

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

B i o s y n t h e s i s o f M ilk T r i g l y c e r i d e s B lood

B lood triglycerides

β -hydroxybutyrate

A cetate

C a p i l l a r y w a ll B a s a l m em b r a n e

L ipoprotein lipase M onoglycerides

M a m m a ry cell A cetate

β -h y d r o x y b u t y r a t e

Free FA

F a tty A cyl C o A

G lycerol

α -g lycerol-P

M ilk triglycerides

C 4-C 1 2

Sources of Milk Fatty Acids • The fatty acids used to synthesize milk fat (triglycerides) come from two sources: 1- Blood lipids 2- De novo synthesis within the mammary epithelial cells (synthesis of new molecules of fatty acids from precursors absorbed from the blood) 1- Blood lipids: Derived from digestion and absorption of dietary fat and from mobilization of fatty acids from adipose tissue. Most of the fatty acids derived from blood plasma are of dietary origin (> 80%). This amount could differ according to stage of lactation and milk yield. Blood lipids are the source of all of the C18 and most of the C16 fatty acids in milk. One third of the C16 and most of the C18 fatty acids in the milk are of dietary origin. In total, about 1/2 of the milk fatty acids are derived from the blood plasma lipids.

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Dairy Cattle Production 342-450A 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 are biohydrogenated (saturated) in the rumen by ruminal microbes. Therefore fatty acids in adipose tissue and in milk of dairy cows are more saturated in nature than those of the diet. Intestinal and mammary epithelia of ruminants contain an active desaturase enzyme that converts saturated fatty acids to mono-saturated fatty acids (mainly stearic to oleic acid). However, most of the desaturation takes place in the mammary gland rather than in the small intestine. The desaturation of fatty acids helps to offset the extensive biohydrogenation that occur in the rumen and reduces the ratio of stearic:oleic acids in cow’s milk. This also ensures sufficient fluidity of milk fat for efficient secretion of milk from the mammary gland.

2- De novo synthesis: Acetate and β-hydroxy-butyrate are the major carbon sources of fatty acid biosynthesis in the mammary gland. Almost all C4 to C14 fatty acids (short and medium-chain fatty acids) are synthesized de novo. Short chain fatty acids of various lengths are synthesized by the step-wise addition of acetate to β-hydroxy-butyrate. Extra β-hydroxy-butyrate will be converted to acetate. The mammary gland of the dairy cow utilizes 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 considered hypercholesterolemic. 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 be hypercholesterolemic while shorter fatty acids (C4-C10) are neutral. Stearic, C18:1 and C18:2 acids are hypocholesterolemic.

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Bovine milk fat contains significant amounts of short chain fatty acids and relatively lower concentrations of C18 fatty acids than are found in other sources of animal fat such 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, microbial synthesis of fatty acids and body stores of fat with the remainder coming from de novo synthesis in the mammary gland. Manipulating the diet of the dairy cow can substantially alter the balance between mammary de novo synthesis of short and medium chain fatty acids, and dietary long chain fatty acids presented to the mammary gland.

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Dairy Cattle Production 342-450A Milk Biosynthesis

Biosynthesis of Milk Proteins Milk Protein Fractions • 123-

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

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

Milk proteins contain more amino acids than any other natural food. The major milk proteins are only found in milk. These include: Casein proteins; α-, β -, and κ-casein Lactoglobulin; β -lactoglobulin (~50% of whey proteins) Lactalbumin. α-lactalbumin (~25% of whey protein) A second group blood proteins (e.g. immunoglobulins) and some proteins synthesized in the plasma cell adjacent to the secretory epithelium, enter the mammary gland and appear in 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 endoplasmic reticulum. Steps of biosynthesis are similar to those of any other protein: 1- Transcription:

A strand of messenger RNA (mRNA) is formed from DNA. It carries the code of a specific protein. The mRNA is located in the ribosome, which is attached to the rough endoplasmic reticulum.

2- Activation:

Amino acids in the cytoplasm are activated by reaction with ATP and attachment to transfer RNA (tRNA). The tRNAs are specific for each amino acid.

3- Translation:

Takes place in the ribosome. The mRNA contains codes for amino acids. Each code consists of three nucleotides and is known as a codon. Located in the tRNA a trinucleotide anticodon that recognizes it. As each codon in the mRNA comes in position, the appropriate amino acid-tRNA complex moves in the amino acid joined the previous one in the chain.

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Dairy Cattle Production 342-450A Milk Biosynthesis

In the synthesis of export proteins (milk proteins) in the RER, ribosomes are attached to the RER and the protein polypeptide is synthesized with an additional initial chain of 10-20 amino acids. The initial signal peptide sequence mediates the passage of the start of the amino acid chain through the membrane of the RER into the inner passages and is clipped off in the process. The polypeptide chain inserted into the RER folds up in a configuration as a function of the physical forces in the amino acid sequence. Depending on the protein, certain other materials (e.g. phosphate) may be added later. The final 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 the newborn. Casein micelles also provide Ca and P for skeletal development. Casein micelles are high digestible by the proteolytic enzymes of the newborn

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

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Milk contains proteins such as lactoferrin and lysozyme. The antibacterial properties of these materials, lysozyme digesting bacterial polysaccharides and lactoferrin sequestering iron required by bacteria emphasize their importance in reducing mastitis infections. Lactoferrin concentration is high in the dry bovine mammary gland.

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

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Dairy Cattle Production 342-450A Milk Biosynthesis

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 and secretion process. Glucose is essential for lactose synthesis and cannot be replaced by any other sugar. About 45-60% of blood glucose in ruminants is synthesized in the liver from propionate by a process known as gluconeogenesis. Blood glucose levels 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 of lactose. One molecule of glucose is converted to galactose. The enzyme catalyzing this conversion appears just before parturition and its activity increase dramatically at the 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 of lactose biosynthesis is greatly influenced by the availability of α-lactalbumin from the rough endoplasmic reticulum.

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Lactose is a nonpermeable disaccharide, which cannot diffuse out the Golgi membrane or out of the secretory vesicles' membrane. This is important for milk synthesis because it is the synthesis of the nondiffusible lactose, which results in water being drawn into the Golgi. Water is osmotically drawn into the Golgi to try to dilute the lactose.

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Dairy Cattle Production 342-450A Milk Biosynthesis

Secretion of Milk Constituents •

The individual component of milk are kept separate inside the secretory cell and therefore, milk is not formed until the individual components reached the lumen where they are mixed together.

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

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

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Dairy Cattle Production 342-450A Milk Biosynthesis

Rate of Milk Secretion

After 10 hours from the last milking, the average secretion rate begins to decrease and secretion stops after 35 hours. The increase in udder pressure per unit of newly formed milk varies according to 1- Level of milk production. The pressure per unit of newly formed milk is lower for high-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

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Secretion Rate (kg/hr) (____)

Udder Pressure (mm Hg) (----)

The period after milking is characterized by low intra-alveolar pressure, facilitates the transport of newly synthesized milk into alveolar lumen. As secretion continues between milkings, backpressure is exerted onto the secretory process by the alveolar Udder pressure & secretion rate luminal contents. At some point the luminal pressure exceeds the force of secretion as the alveolar enlargement 1.5 60 reaches its limit. The distension of 50 pressure of the lumen exceeds the 1.0 40 strength of secretory mechanisms 30 needed to push the newly formed milk 20 0.5 out of the cell. In turn, the buildup of 10 newly formed milk in the cells reduces 0 0 the uptake of milk precursors by 0 10 20 30 40 chemical (a negative feedback mechanism) and / or mechanical hours since last milking process. The mechanical factors are a result of a distended alveoli partially replacing all other intramammary compartments including the blood vessels.