Milk chemistry and composition - Basics for Dairy processing

INTRODUCTION TO MILK

Dairy processing

Definition, Composition, physical and chemical properties

Topics

Milk: Definition

Composition

Physical and chemical properties

National standards - India

Definition (legal)

Whole, fresh, clean, lacteal secretions, obtained by complete

milking of one or more healthy milch animals

Excluding that obtained 15 days before or 5 days after or

such periods as may be necessary to render the milk

practically colostrum free

Containing legally prescribed minimum percentage of fat and

Solids-not-fat (SNF).

Definition (FDA)

Lacteal secretion, practically free from colostrum, obtained by

the complete milking of one or more healthy cows,

May be clarified and may be adjusted by separating part of

the fat there from; concentrated milk, reconstituted milk, and

dry whole milk.

Water, in a sufficient quantity to reconstitute concentrated and

dry forms, may be added

Definition (Chemical)

Complex chemical substance in which:

Fat is present in the form of an emulsion

Protein and some mineral matter in the colloidal state

Lactose with some minerals and soluble proteins in the form of

true solution

Description of Milk

Emulsion of milk fat globules which contain the milk lipids, fat

soluble vitamins, and the components of the FGM

Colloidal suspension of casein micelles (which contain casein

proteins, calcium, phosphate, citrate and water), globular

proteins, and lipoprotein particles

Solution of lactose, soluble proteins, minerals, vitamins, acids,

enzymes, and other components

Milk secretion

All species of mammals secrete milk

To provide nutrients required for the optimum growth of the

new born

Immunity, protecting it from some of the common diseases.

The development of the young one in all species of mammals is

not uniform as such

composition of the milk vary depending up on the nutritional

needs of the young one

Differences in the composition of milk

from various species

Buffalo and Sheep milk - maximum fat

Fat percent in goat milk is much similar to cow milk

Variation among the protein percent less

Human milk:

Higher percent of lactose and fat

Lesser percent of protein and ash content compared with

other species.

Energy supplied - highest in buffalo and sheep milk

Difference less between the milk from the remaining species

Legal standards for various classes of milk

(FSSAI, 2006)

Legal standards for various classes of milk

Legal standards for various classes of milk

Colostrum

First mammary gland fluid secreted by mammals during the

first 5-7 days after calving

Composition is similar to that of blood and differs

significantly from milk.

Nutrients (proteins, fats, lactose, essential fatty acids and

amino acids)

Non-nutrients (biologically active substances)

First food for neonates after the parturition that provides

them with all necessary nutrients

Contains more protein - serum protein

Colostrum

Growth factors

Insulin-like growth hormone (IGF) – IGF-I and IGF-II,

Transforming growth factor (TGF)

Epidermal growth factor (EGF)

Control the growth and development of gastrointestinal

tract

For the functional maturation of the organism during the

first days after birth

Colostrum

Immune factors (Immunoglobulins, Ig, Lactoferrin)

For the passive immunization of the newborn

Antibacterial factors passes in the offspring

Supports their protection against infections during the first

days after birth

Colostrum composition and its biological value affected by:

Rearing technology

Nutrition during the pregnancy

Health status of cows impact

Milk composition

Relative composition of Dried milk

Factors affecting Milk composition

Animal factor

Genetic, Species, Breed, Individual cow, Lactation period

Age & Genetic factors

Breed, species, feed and individuality

Stage of lactation, pregnancy, nutritional balance

Health status of the cow

Oestrus, gestation, presence of mastitic infection

Environmental factors

Extreme climates, stress, exhaustion, housing

Milking technique and milking frequency and stage

Composition(%) of Milk from Dairying species

Water

Principal constituent in milk

79% to 90%, depending on the species

It encompasses all other constituents of milk (total solids) that

are either dissolved or suspended in it

Small amounts of water are hydrated or bound chemically to

lactose, salt, or protein

The water activity in milk is relatively high, 0.993

Removal of water increases shelf life – powdered milk

Regulations prohibit the addition of water to raw milk

Milk Fat

In fat globules - protected by a membrane (FGM)

Fat globules range from 1 to 20 μm in diameter

Made up of app.

98% triglycerides

0.2% to 1% phospholipids

0.2% to 0.4% sterols

Phospholipids and proteins mostly associate with the fat

globule membrane.

Milk fat

Contain traces of fatty acids; vitamins A, D, E, and K; and

enzymes.

More than 400 different fatty acids

Predominant fatty acids in bovine milk

Myristic acid (C14:0 )

Palmitic acid (C16:0 )

Stearic acid (C18:0 )

Oleic acid (C18:1)

Milk fat

Milk fat

Lipids, lipoproteins, cerebrosides, nucleic acids, enzymes, trace

elements (minerals), and some bound water molecules

stabilize and prevent the fat globules from coalescence

during milk processing and handling.

FGM prevents attack from lipases (lipolysis)

Or increase in the amount of diglycerides, monoglycerides,

and free fatty acids in milk

Free fatty acids are fairly water - soluble and are situated in

milk plasma and fat

Milk fat

Short free fatty acids situated in the milk plasma are ionized

and more water-soluble than long free fatty acids ( >C14)

found in fat and at the oil - water interface.

Minerals associated with the fat globule membrane are

copper (5-25%) and iron (30-60%)

Other minerals include cobalt, calcium, sodium, potassium,

magnesium, manganese, molybdenum, and zinc.

Milk fat

Compound lipids also occur in milk such as phospholipids and

phosphatides that are situated mainly in the fat globule

membranes but also in the milk plasma, lipoproteins, and milk

microsomes.

Phospholipids and phosphatides are highly surface active and

polar, and dissolve poorly in both water and oil.

Lipids can be crystallized, which affects the fat structure,

melting range, and rheological properties of milk.

Milk fat

Furthermore, autoxidation of the double fatty acid bonds or

residues can occur, leading to off flavors.

Whole milk contains 10 to 20mg/100g cholesterol (3.3% fat).

The amount of cholesterol is positively correlated with the

amount of fat in the product.

Cholesterol is located in the fat globule membrane, and

approximately 10% of the cholesterol is esterified.

Milk Proteins

Proteins are made up of amino acids with specific properties

that are determined by the side chains of the amino acids in

the polypeptide chain.

The conformation of the protein depends on the hydrogen

bonds, hydrophobic interactions, and salt bridges formed

between the peptide chains.

Regular arrangements include β-sheets and α-helices.

Milk Proteins

Temperature, ionic strength, and pH affect protein

conformation.

Major classes – Casein, Whey or serum proteins

The proteins are synthesized in the mammary gland,

Derived genetically

Protein content of milk

remains constant

with a concentration range of 30 to 35g/kg.

influenced by the lactation stage of the cow.

Casein Proteins

Four main types of casein have genetic variants:

αS1-casein, αS2-casein, β- casein, and κ-casein.

They are phosphorylated and hydrophobic and associate

with themselves and each other.

They represent 38%, 10%, 36%, and 13% of whole casein

Overall, 21 variants of casein have been identified that occur

by genetic mutations.

Casein Proteins

Caseins have distinct disordered molecular structures that lack

disulfide bridges

Very heat stable, withstanding temperatures above 140 ° C

before dissociating.

Their hydrophobicity is due to the

high ratios of apolar amino acids

including valine, leucine, isoleucine, phenylalanine, and

proline (between 35% and 45%).

Casein Proteins

Hydrophobicity - counteracted by the

high phosphate content

low concentrations of sulphur - containing amino acids such

as methionine and cysteine that allow the caseins to be

reasonably water soluble.

Their susceptibility to proteolysis is due to the

lack of secondary and tertiary structures (αS1-casein and β-

casein)

α-Caseins

199 amino acids and is app. 23.6 kDa.

highest charge of all the casein molecules.

Consists of at least eight phosphoserine units.

It has 17 proline residues that ultimately disrupt the formation of secondary structures, such as α - helices and β - sheets.

207 amino acids and is app

25.4 kDa.

least hydrophobic casein

molecule.

Several genetic variants,

contain between 10 and 13

phosphoserine units.

Contains two cysteine

residues.

It exists as a dimer in milk.

αS1-casein αS2 - casein

β-Casein

β-casein consists of 209 amino acids and is approximately 24

kDa.

It is the most hydrophobic casein molecule.

There are six known genetic variants that contain between zero

and five phosphoserine units.

Similar to αS1-casein, β-casein has few secondary structures

due to the presence of 35 proline residues.

γ-Casein

γ-casein is derived by hydrolysis of β-casein by the enzyme

plasmin.

Three variants have been identified near the C - terminal end

of the β-casein molecule.

Κ - Casein

κ - casein consists of 169 amino acids and is approximately 19 kDa, and it contains both glycosylated and phosphorylated residues.

It can exist as a dimer up to a decamer with the subunits held together by disulfide linkages.

Unlike the other caseins, it is not sensitive to calcium and surrounds the micelles, keeping them intact.

It usually contains one phosphoserine unit;

however, genetic variants containing two or three phosphoserine units have been identified.

Further, nine variants have been identified that demonstrate different degrees of glycosylation.

Casein micelles

Casein micelles consist of a large portion (approximately

95%) of casein proteins that interact with each other and

calcium.

They vary in size from 80 to 1,000 nm with an average

diameter of 150 nm in bovine milk.

The micelle sizes of sheep and goat milk are different, and the

caprine micelles are less heat stable than bovine milk micelles

Casein micelles

Bovine casein micelles contain

water, protein (about 94%),

salts (about 6%), including calcium, phosphorus, magnesium,

citrate - colloidal calcium phosphate [CCP]

other traces of metals, enzymes (lipases, esterases,

proteases), and milk serum.

Casein micelles contain submicelles that range from 12 to 15

nm in diameter

Contain approximately 20 to 25 casein molecules and water

(2 to 5 g/protein)

with some submicelles containing K - casein.

Whey proteins

Whey proteins are hydrophobic, globular, highly ordered

proteins that contain disulfide linkages.

Whey proteins have well - developed secondary, tertiary, and

quaternary structures

Poorer heat stability

denature at temperatures greater than 75 ° C.

Whey proteins

The two principal whey proteins in milk

α - lactalbumin and β - lactoglobulin

Synthesized in the mammary gland.

They constitute approximately 20% and 40% of total whey

protein in bovine milk

Other whey proteins are

Proteose peptones

Immunoglobulins

Serum albumin

α-Lactalbumin

α-lactalbumin is a spherical, glycosylated compactly folded

calcium metalloprotein

Consists of approximately 142 amino acids and is

approximately 14 kDa.

It is synthesized and secreted by the mammary gland

Contains four disulfide bonds and eight cysteine residues, and

is rich in tryptophan

Three genetic variants have been identified

It is the principal protein in human milk.

β-Lactoglobulin

β-lactoglobulin consists of 178 amino acids with an

approximate molecular weight of 18 kDa.

It exists in both the monomeric and dimeric form at equilibrium

in bovine milk;

Its association depends on temperature, pH, protein

concentration, and ionic conditions.

The hydrophobic dimeric form linked by one to three disulfide

bonds is approximately 36 kDa.

β-Lactoglobulin

Higher concentrations of β - lactoglobulin are present in

bovine milk when compared with human milk.

Better heat stability than α - lactalbumin due to the

presence of one free sulphohydryl unit.

It contains an open β - barrel enclosing a hydrophobic

cleft and a single three - turn α - helix.

It binds to several hydrophobic molecules including retinol and

fatty acids via the hydrophobic cleft, which in turn stimulates

lipase activity.

Immunoglobulin

Immunoglobulins are antibodies that are synthesized in

response to specific antigens.

They are large, heterogeneous molecules found in the blood.

The main immunoglobulins in milk are IgG, IgG2, IgA, and IgM.

They provide offspring with protection against pathogenic

microorganisms and their toxins, and the mammary gland

against infection.

Immunoglobulin

Approximately 0.7 to 1mg/ml is present in bovine milk.

The basic structural unit of the immunoglobulins is similar,

consisting of two heavy and two light chains joined together by

disulfide bonds.

IgG is the main immunoglobulin in milk.

Bovine serum albumin

Bovine serum albumin consists of 582 amino acids

Longest protein.

It is approximately 66 kDa

Predominantly composed of α - helices.

It makes up approximately 1% to 5% of total whey protein.

It is synthesized in the liver and enters the milk via secretory

cells.

Proteose peptones

Derived from the hydrolysis of β - casein.

Considered whey proteins

Because elute in the whey fraction when isolated from milk

Heat stable

Acid - soluble proteins

Mainly responsible for the foaming of skim milk

Inhibit rancidity

Have an immunological role

Lactoferrin

Globular glycoprotein

74 kDa and binds to iron (Fe) as it contains two metal binding sites

Bovine milk contains app. 20 to 200 mg/L

Human milk contains 2 g/L.

Other whey proteins

Other minor whey proteins includes

growth factors

Vitamin - binding proteins

Folate, vitamin D, riboflavin, and vitamin B 12

Angiogenins

Osteopontin

Non - protein nitrogen compounds:

Urea, uric acid, creatine, creatinine, and hippuric acid

In trace amounts

Chemical components of

Bovine milk proteins

Carbohydrates

Several carbohydrates in milk:

Lactose

Glucose

Galactose

Glycoconjugates (oligosaccharides, glycoproteins, and

glycoaminoglycans)

Main carbohydrate – lactose

4-5% of total milk content

Glucose, galactose, Oligosaccharides - app. 1 mg/ml

Lactose

Disaccharide comprised of α / β-D – glucose and β - D -

galactose that are linked by a β 1-4 - O - glycosidic bond.

Depends on the milk yield and lactation stage of the cow

Amount of lactose decreases as the lactation stage advances

Lactose exists in three forms:

α - lactose monohydrate

β - lactose

Anhydrous α - lactose.

Lactose

β - lactose form has the greatest solubility and is sweeter

than the α - lactose forms.

Major food source for bacteria during the fermentation of

milk.

The bacteria hydrolyse the milk into glucose and galactose

to produce lactic acid, which inhibits the growth of most

other microorganisms.

Minerals and Salts

Milk contains all minerals - essential for human nutrition

Including potassium (K), sodium (Na), calcium (Ca), magnesium

(Mg), chloride (Cl), and phosphate esters

Make up between 0.7% and 0.8% of total milk content.

Sodium, potassium, and chloride as free ions

readily diffusible

Their concentrations are negatively correlated with lactose

Minerals

Concentrations of calcium, magnesium, ionized phosphate, and

citrate

Depend on the casein content in the milk

Concentration of citrate varies depending on season and diet

of the cow

Affect the soluble calcium content and milk stability

Equilibrium between colloidal dispersion and salts

Salt

If the colloidal equilibrium is destabilized,

Concentration of minerals in milk may affect the processing

Require the addition of anions to bind to ionic calcium that

would restabilize the caseins against aggregation.

Salt is added as an additive in certain dairy products

The concentration of calcium in milk is relatively high

Milk - considered to be an important source of calcium.

Salt

The primary salts in milk are phosphates, citrates, chlorides,

sulphates, carbonates and bicarbonates of

sodium, potassium, calcium and magnesium.

Since milk contains organic and inorganic salts,

Level of salts is not equivalent to the level of mineral

substances

Level of salts is by no means equivalent to the ash content.

Factors influencing salt composition:

Species, Breed of species

Stage of lactation and feed

Milk Enzymes

In cow’s milk, ∼20 enzymes have been characterized

Another 40 enzymes - demonstrated via their activity.

Indigenous milk enzymes are found in, or associated with

Casein micelles

Milk fat globule membrane

Milk serum or somatic cells

May originate from blood, somatic cells, the MFGM or the cell

cytoplasm.

Milk enzymes

Used as indices of

Animal health

Thermal history of the milk

Functions in milk and dairy products:

Result in quality deterioration

Induce desirable changes

May also offer protective effects

Milk enzymes

Enzymes of known or potential technological significance

Plasmin

Predominant indigenous proteinase in milk.

Optimally active at pH ∼7.5 and ∼37◦C

Plasmin and plasminogen originate from the mammal’s

blood and are predominantly associated with the casein

micelle in milk

Cheese ripening and the stability of casein micelles in UHT

milk

Milk enzymes

Lipoprotein lipase

Optimal activity at pH 9.2 and 37◦C, relatively heat-labile

enzyme

Initial digestion and absorption of milk lipids in the intestinal

tract and flavor development in certain cheeses from raw

milk

Lipolysis - development of hydrolytic rancidity in milk

Milk enzymes

Alkaline phosphatase

Optimum activity at pH 9.0–10.5 and ∼37◦ C

ALP is relatively heat-sensitive and its thermal stability is

only slightly higher than that of non-spore forming

pathogenic bacteria

Indicator of pasteurization efficiency

The enzyme's heat-stability profile closely follows that

necessary for adequate pasteurization

Milk enzymes

Sulfhydryl oxidase

Catalyzes the oxidation of thiols and the formation of

disulfide bonds in proteins and peptides.

Sulfhydryl oxidase- treated UHT milk may have longer

flavor stability due to reduced lipid oxidation

Milk enzymes

Lactoperoxidase

relatively heat-stable milk enzyme; heating up to 80◦C

appears required to ensure thermal inactivation.

anti bacterial agent

pH optimum of ∼8.0, and exists primarily in the milk serum

Milk enzymes

N-acetyl-p-D-glucosaminidase - activity diagnosed for mastitis

test

Catalase – ass. with somatic cell membrane

Xanthine oxidase - contains all of the molybdenum in milk

Superoxide dismutase - Protective effect on lipid oxidation

γ- Glutamyltransferase - Transport of amino acids into

mammary gland

Lactose synthase – synthesis of Lactose