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Milk chemistry—An introduction

Physical status of milk

Ionic solutions
Molecular solutions

pH and acidity

Buffer solutions

Milk constituents

Milk fat
Milk proteins
Milk carbohydrates
Minor milk constituents

Physical status of milk

About 87% of milk is water, in which the other constituents are distributed in various forms. We distinguish among several kinds of distribution according to the type and size of particle present in the liquid.

Kind of solution

Particle diameter (nm)

Ionic solution


Molecular solution


Colloid (fine dispersion)


Coarse dispersion
(suspension or emulsion)


In milk we find examples of emulsions, colloids, molecular and ionic solutions.

Ionic solutions

An ionic solution is obtained when the forces that hold the ions together in a solid salt are overcome. The dissolved salt breaks up into ions which float freely in the solvent. Thus when common salt—sodium chloride—is dissolved in water it becomes an ionic solution of free sodium and chloride ions. Ionic solutions are largely of inorganic compounds.

Molecular solutions

In a molecular solution the molecules are only partly, if at all, dissociated into ions. The degree of dissociation represents an equilibrium which is influenced by other substances in the solution and by the pH (or hydrogen ion concentration) of the solution. Molecular solutions are usually of organic compounds


In a colloid, one substance is dispersed in another in a finer state than an emulsion but the particle size is larger than that in a true solution. Colloidal systems are classified according to the physical state of the two phases. In a colloid, solid particles consisting of groups of molecules float freely. The particles in a colloid are much smaller than those in a suspension and a colloid is much more stable.


An emulsion consists of one immiscible liquid dispersed in another in the form of droplets—the disperse phase. The other phase is referred to as the continuous phase. The systems have minimal stability and require the presence of a surface-active or emulsifying agent for stability. In foods, emulsions usually contain oil and water. If water is the continuous phase and oil the disperse phase, it is an oil-in-water (o/w) emulsion, e.g. milk or cream. In the reverse case the emulsion is a water-in-oil (w/o) type, e.g. butter. In summary, an emulsion consists of three elements, the continuous phase, the disperse phase and the emulsifying agent.


A dispersion is obtained when particles of a substance are dispersed in a liquid. A suspension consists of solid particles dispersed in a liquid, and the force of gravity can cause them to sink to the bottom or float to the top. For example, fine sand, dispersed in water, soon settles out.

pH and acidity

An acid is a substance which dissociates to produce hydrogen ions in solution. A base (alkaline) is a substance which produces hydroxyl ions in solution. It can equally be stated that an acid is a substance which donates a proton and a base is a substance which accepts a proton.

The symbol pH is used to denote acidity; it is inversely related to hydrogen ion concentration.

Neutrality is pH 7
Acidity is less than pH 7
Alkalinity is more than pH 7

Fresh milk has a pH of 6.7 and is therefore slightly acidic.

When an acid is mixed with a base, neutralisation takes place; similarly a base will be neutralised by an acid.

Buffer solutions

Buffers are defined as materials that resist a change in pH on addition of acid or alkali. Characteristically they consist of a weak acid or a weak base and its salt. Milk contains a large number of these substances and consequently behaves as a buffer solution. Fresh cows milk has a pH of between 6.7 and 6.5. Values higher than 6.7 denote mastitic milk and values below pH 6.5 denote the presence of colostrum or bacterial deterioration. Because milk is a buffer solution, considerable acid development may occur before the pH changes. A pH lower than 6.5 therefore indicates that considerable acid development has taken place. This is normally due to bacterial activity.

Litmus test papers, which indicate pH, are used to test milk activity; pH measurements are often used as acceptance tests for milk.

Measuring milk acidity is an important test used to determine milk quality. Acidity measurements are also used to monitor processes such as cheese-making and yoghurt-making. The titratable acidity of fresh milk is expressed in terms of percentage lactic acid, because lactic acid is the principal acid produced by fermentation after milk is drawn from the udder and fresh milk contains only traces of lactic acid. However, due to the buffering capacity of the proteins and milk salts, fresh milk normally exhibits an initial acidity of 0.14 to 0.16% when titrated using sodium hydroxide to a phenolphthalein end-point.

Milk constituents

The quantities of the main milk constituents can vary considerably depending on the individual animal, its breed, stage of lactation, age and health status. Herd management practices and environmental conditions also influence milk composition. The average composition of cows milk is shown in Table 1.

Table 1. Composition of cows milk

Main constituent




85.5 – 89.5


Total solids

10.5 – 14.5



2.5 – 6.0



2.9 – 5.0



3.6 – 5.5



0.6 – 0.9


Water is the main constituent of milk and much milk processing is designed to remove water from milk or reduce the moisture content of the product.

Milk fat

If milk is left to stand, a layer of cream forms on the surface. The cream differs considerably in appearance from the lower layer of skim milk.

Under the microscope cream can be seen to consist of a large number of spheres of varying sizes floating in the milk. Each sphere is surrounded by a thin skin—the fat globule membrane—which acts as the emulsifying agent for the fat suspended in milk (Figure 3).The membrane protects the fat from enzymes and prevents the globules coalescing into butter grains. The fat is present as an oil-in-water emulsion: this emulsion can be broken by mechanical action such as shaking.

Figure 3. Fat globules in milk.

Fats are partly solid at room temperature. The term oil is reserved for fats that are completely liquid at room temperature. Fats and oils are soluble in non-polar solvents, e.g. ether.

About 98% of milk fat is a mixture of triacyl glycerides. There are also neutral lipids, fat-soluble vitamins and pigments (e.g. carotene, which gives butter its yellow colour), sterols and waxes. Fats supply the body with a concentrated source of energy: oxidation of fat in the body yields 9 calories/g. Milk fat acts as a solvent for the fat-soluble vitamins A, D, E and K and also supplies essential fatty acids (linoleic, linolenic and arachidonic).

A fatty-acid molecule comprises a hydrocarbon chain and a carboxyl group (-COOH). In saturated fatty acids the carbon atoms are linked in a chain by single bonds. In unsaturated fatty acids there is one double bond and in poly-unsaturated fatty acids there is more than one double bond. Examples of each type of fatty acid are shown in Figure 4.

Figure 4. Structural formulae of four 18-carbon fatty acids varying in degree of saturation.

Fatty acids vary in chain length from 4 carbon atoms, as in butyric acid (found only in butterfat), to 20 carbon atoms, as in arachidonic acid. Nearly all the fatty acids in milk contain an even number of carbon atoms.

Fatty acids can also vary in degree of unsaturation, e.g. C18:0 stearic (saturated), C18:1 oleic (one double bond), C18:2 linoleic (two double bonds), C18:3 linolenic (three double bonds).

The most important fatty acids found in milk triglycerides are shown in Table 2. Fatty acids are esterified with glycerol as follows:

Glycerol + fatty acids →  triglyceride (fat) + water

Table 2. Principal fatty acids found in milk triglycerides.


Molecular formula

Chain length

Melting point







































C18: 1




C18: 2




C18: 3


The melting point and hardness of the fatty acid is affected by:

As chain length increases, melting point increases. As the degree of unsaturation increases, the melting point decreases.

Fats composed of short-chain, unsaturated fatty acids have low melting points and are liquid at room temperature, i.e. oils. Fats high in long-chain saturated fatty acids have high melting points and are solid at room temperature. Butterfat is a mixture of fatty acids with different melting points, and therefore does not have a distinct melting point. Since butterfat melts gradually over the temperature range of 0–40°C, some of the fat is liquid and some solid at temperatures between 16 and 25°C. The ratio of solid to liquid fat at the time of churning influences the rate of churning and the yield and quality of butter.

Fats readily absorb flavours. For example, butter made in a smoked gourd has a smokey flavour.

Fats in foods are subject to two types of deterioration that affect the flavour of food products.

  1. Hydrolytic rancidity: In hydrolytic rancidity, fatty acids are broken off from the glycerol molecule by lipase enzymes produced by milk bacteria. The resulting free fatty acids are volatile and contribute significantly to the flavour of the product.
  2. Oxidative rancidity: Oxidative rancidity occurs when fatty acids are oxidised. In milk products it causes tallowy flavours. Oxidative rancidity of dry butterfat causes off-flavours in recombined milk.

Milk proteins

Proteins are an extremely important class of naturally occurring compounds that are essential to all life processes. They perform a variety of functions in living organisms ranging from providing structure to reproduction. Milk proteins represent one of the greatest contributions of milk to human nutrition. Proteins are polymers of amino acids. Only 20 different amino acids occur, regularly in proteins. They have the general structure:

R represents the organic radical. Each amino acid has a different radical and this affects the properties of the acid. The content and sequence of amino acids in a protein therefore affect its properties. Some proteins contain substances other than amino acids, e.g. lipoproteins contain fat and protein. Such proteins are called conjugated proteins:

Phosphoproteins: Phosphate is linked chemically to these proteins—examples include casein in milk and phosphoproteins in egg yolk.

Lipoproteins: These combinations of lipid and protein are excellent emulsifying agents. Lipoproteins are found in milk and egg yolk.

Chromoproteins: These are proteins with a coloured prosthetic group and include haemoglobin and myoglobin.


Casein was first separated from milk in 1830, by adding acid to milk, thus establishing its existence as a distinct protein. In 1895 the whey proteins were separated into globulin and albumin fractions.

It was subsequently shown that casein is made up of a number of fractions and is therefore heterogeneous. The whey proteins are also made up of a number of distinct proteins as shown in the scheme in Figure 5.

Figure 5. Milk protein fractions.

Casein is easily separated from milk, either by acid precipitation or by adding rennin. In cheese-making most of the casein is recovered with the milk fat. Casein can also be recovered from skim milk as a separate product.

Casein is dispersed in milk in the form of micelles. The micelles are stabilised by the Κ-casein. Caseins are hydrophobic but Κ-casein contains a hydrophilic portion known as the glycomacropeptide and it is this that stabilises the micelles. The structure of the micelles is not fully understood.

When the pH of milk is changed, the acidic or basic groups of the proteins will be neutralised. At the pH at which the positive charge on a protein equals exactly the negative charge, the net total charge of the protein is zero. This pH is called the isoelectric point of the protein (pH 4.6 for casein). If an acid is added to milk, or if acid-producing bacteria are allowed to grow in milk, the pH falls. As the pH falls the charge on casein falls and it precipitates. Hence milk curdles as it sours, or the casein precipitates more completely at low pH.

Whey proteins

After the fat and casein have been removed from milk, one is left with whey, which contains the soluble milk salts, milk sugar and the remainder of the milk proteins. Like the proteins in eggs, whey proteins can be coagulated by heat. When coagulated, they can be recovered with caseins in the manufacture of acid-type cheeses. The whey proteins are made up of a number of distinct proteins, the most important of which are b-lactoglobulin and lactoglobulin. b-lactoglobulin accounts for about 50% of the whey proteins, and has a high content of essential amino acids. It forms a complex with Κ-casein when milk is heated to more than 75°C, and this complex affects the functional properties of milk. Denaturation of b-lactoglobulin causes the cooked flavour of heated milk.

Other milk proteins

In addition to the major protein fractions outlined, milk contains a number of enzymes. The main enzymes present are lipases, which cause rancidity, particularly in homogenised milk, and phosphatase enzymes, which catalyse the hydrolysis of organic phosphates. Measuring the inactivation of alkaline phosphatase is a method of testing the effectiveness of pasteurisation of milk.

Peroxidase enzymes, which catalyse the breakdown of hydrogen peroxide to water and oxygen, are also present. Lactoperoxidase can be activated and use is made of this for milk preservation.

Milk also contains protease enzymes, which catalyse the hydrolysis of proteins, and lactalbumin, bovine serum albumin, the immune globulins and lactoferrin, which protect the young calf against infection.

Milk carbohydrates

Lactose is the major carbohydrate fraction in milk. It is made up of two sugars, glucose and galactose (Figure 6). The average lactose content of milk varies between 4.7 and 4.9%, though milk from individual cows may vary more. Mastitis reduces lactose secretion.

Figure 6. Structure of a lactose molecule.

Lactose is a source of energy for the young calf, and provides 4 calories/g of lactose metabolised. It is less soluble in water than sucrose and is also less sweet. It can be broken down to glucose and galactose by bacteria that have the enzyme b-galactosidase. The glucose and galactose can then be fermented to lactic acid. This occurs when milk goes sour. Under controlled conditions they can also be fermented to other acids to give a desired flavour, such as propionic acid fermentation in Swiss-cheese manufacture.

Lactose is present in milk in molecular solution. In cheese-making lactose remains in the whey fraction. It has been recovered from whey for use in the pharmaceutical industry, where its low solubility in water makes it suitable for coating tablets. It is used to fortify baby-food formula. Lactose can be sprayed on silage to increase the rate of acid development in silage fermentation. It can be converted into ethanol using certain strains of yeast, and the yeast biomass recovered and used as animal feed. However, these processes are expensive and a large throughput is necessary for them to be profitable. For smallholders, whey is best used as a food without any further processing.

Heating milk to above 100oC causes lactose to combine irreversibly with the milk proteins. This reduces the nutritional value of the milk and also turns it brown.

Because lactose is not as soluble in water as sucrose, adding sucrose to milk forces lactose out of solution and it crystallises. This causes sandiness in such products as ice cream. Special processing is required to crystallise lactose when manufacturing products such as instant skim milk powders.

Some people are unable to metabolise lactose and suffer from an allergy as a result. Pre-treatment of milk with lactase enzyme breaks down the lactose and helps overcome this difficulty.

In addition to lactose, milk contains traces of glucose and galactose. Carbohydrates are also present in association with protein. Κ-casein, which stabilises the casein system, is a carbohydrate-containing protein.

Minor milk constituents

In addition to the major constituents discussed above, milk also contains a number of organic and inorganic compounds in small or trace amounts, some of which affect both the processing and nutritional properties of milk.

Milk salts

Milk salts are mainly chlorides, phosphates and citrates of sodium, calcium and magnesium. Although salts comprise less than 1 % of the milk they influence its rate of coagulation and other functional properties. Some salts are present in true solution. The physical state of other salts is not fully understood. Calcium, magnesium, phosphorous and citrate are distributed between the soluble and colloidal phases (Table 3). Their equilibria are altered by heating, cooling and by a change in pH.

Table 3. Distribution of milk salts between the soluble and colloidal phases.





(mg/100 ml of milk)









Total phosphorus








In addition to the major salts, milk also contains trace elements. Some elements come to the milk from feeds, but milking utensils and equipment are important sources of such elements as copper, iron, nickel and zinc.

Milk vitamins

Milk contains the fat-soluble vitamins A, D, E and K in association with the fat fraction and water-soluble vitamins B complex and C in association with the water phase. Vitamins are unstable and processing can therefore reduce the effective vitamin content of milk.

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