02 the chemistry of milk

8/12/2019 02 the Chemistry of Milk

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Dairy Processing Handbook/Chapter 2 17

The chemistry of milkThe principal constituents of milk are water, fat, proteins, lactose (milk

sugar) and minerals (salts). Milk also contains trace amounts of othersubstances such as pigments, enzymes, vitamins, phospholipids(substances with fatlike properties), and gases.

The residue left when water and gases are removed is called the dry matter(DM)or total solidscontent of the milk.

Milk is a very complex product. In order to describe the variousconstituents of milk and how they are affected by the various stages oftreatment in the dairy, it is necessary to resort to chemical terminology. Thischapter on the chemistry of milk therefore begins with a brief review ofsome basic chemical concepts.


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Dairy Processing Handbook/Chapter 218

Basic chemical concepts

AtomsThe atom is the smallest building block of all matter in nature and cannot bedivided chemically. A substance in which all the atoms are of the same kindis called an element. More than 100 elements are known today. Examplesare oxygen, carbon, copper, hydrogen and iron. However, most naturally-occurring substances are composed of several different elements. Air, forexample, is a mixture of oxygen, nitrogen, carbon dioxide and rare gases,while water is a chemical compound of the elements hydrogen and oxygen.

The nucleus of the atom consists of protons and neutrons, Figure 2.1.The protons carry a positive unit charge, while the neutrons are electricallyneutral. The electrons, which orbit the nucleus, carry a negative chargeequal and opposite to the unit charge of the protons.

An atom contains equal numbers of protons and electrons with an equalnumber of positive and negative charges. The atom is therefore electricallyneutral.

An atom is very small, Figure 2.2. There are about as many atoms in a

small copper coin as there are seconds in a thousand million million years!Even so, an atom consists mostly of empty space. If we call the diameter ofthe nucleus one, the diameter of the whole atom is about 10 000.

IonsAn atom may lose or gain one or more electrons. Such an atom is no longerelectrically neutral. It is called an ion. If the ion contains more electrons thanprotons it is negatively charged, but if it has lost one or more electrons it ispositively charged.

Positive and negative ions are always present at the same time;i.e.insolutions as cations (positive charge) and anions (negative charge) or insolid form as salts. Common salt consists of sodium (Na) and chlorine (Cl)ions and has the formula NaCl (sodium chloride).

MoleculesAtoms of the same element or of different elements can combine into largerunits which are called molecules. The molecules can then form solidsubstances, e.g.iron (Fe) or siliceous sand (SiO

2), liquids, e.g.water (H

2O),

or gases, e.g.hydrogen (H2). If the molecule consists mainly of carbon (C),

hydrogen (H2) and oxygen (O

2) atoms, the compound formed is said to be

organic,i.e.produced from organic cells. An example is lactic acid (C3H

60

3).

The formula means that the molecule is made up of three carbon atoms, sixhydrogen atoms and three oxygenatoms.

The number of atoms in a moleculecan vary enormously. There aremolecules which consist of two linkedatoms, and others composed ofhundreds of atoms.

Chemical symbols of somecommon elements in organicmatter:

Fig. 2.1The nucleus of the atomconsists of protons and neutrons.

Electrons orbit the nucleus.

Fig 2.2The nucleus is so small in rela-

tion to the atom that if it were enlargedto the size of a tennis ball, the outer

electron shell would be 325 metres from

the centre.Fig 2.3Three ways of symbolising a

water molecule.Fig 2.4Three ways of symbolisingan ethyl alcohol molecule.

H

H HO

H2O

O

H

Molecular formula Structural formula

H

C2H5OH

H H

H H

C C O

H

HH

H

HH

H

C C

O

Molecular formula

Structural formula

Electron

Atomicnucleus

Diameter 1

Diameter 10 000

Electron

NeutronProton

Electron

NeutronProton

C CarbonCl ChlorineH HydrogenI IodineK Potassium

N NitrogenNa SodiumO OxygenP PhosphorusS Sulphur


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Basic physical-chemicalproperties of cows milkCows milk consists of about 87 % water and 13 % dry substance. The drysubstance is suspended or dissolved in the water. Depending on the type ofsolids, there are different distribution systems of them in the water phase.

Fig 2.5When milk and creamturn to butter, there is a phase

inversion from an oil-in-wateremulsion to a water-in-oil emulsion.

Table 2.2Relative sizes of particles in milk.

Size (mm) Type of particles

102to 103 Fat globules104to 105 Casein-calcium phosphates105to 106 Whey proteins106to 107 Lactose, salts and other substances in true solutions

Ref.A Dictionary of Dairyingby J G Davis

DefinitionsEmulsion:a suspension of droplets of one liquid in another. Milk is anemulsion of oil in water (o/w), butter an emulsion of water in oil (w/o). Thefinely divided liquid is known as the dispersed phase and the other as thecontinuous phase.Collodial solution:when matter exists in a state of division intermediate totrue solution (e.g.sugar in water) and suspension (e.g.chalk in water) it issaid to be in colloidal solution or colloidal suspension. The typicalcharacteristics of a colloid are: Small particle size Electrical charge and Affinity of the particles for water molecules

Substances such as salts destabilise colloidal systems by changing thewater binding and thereby reducing protein solubility, and factors such asheat, causing unfolding of the whey proteins and increased interactionbetween the proteins, or alcohol which may act by dehydrating theparticles.

Organiccompounds containmainly carbon, oxygen andhydrogen.

Inorganiccompounds containmainly other atoms.

Table 2.1

Physical-chemical status of cows milk.

Average Emulsion Collodial True

composition type oil/water solution/ solution% suspension

Moisture 87,0Fat 4,0 X Proteins 3,5 X Lactose 4,7 X Ash 0,8 X

Butter

Butte

r

Milk

1LITRE

In milk the whey proteins are in colloidal solutionand the casein in colloidal suspension.

Fig 2.6Milk proteins can be madevisible by an electron microscope.

Ref. Miloslav Kalb, Food structure, 1993.

True solutions:Matter which, when mixed with water or other liquids,forms true solutions, is divided into: Non-ionic solutions.When lactose is dissolved in water,

no important changes occur in the molecular structure ofthe lactose.


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Ionic solutions.When common salt is dissolved in water,cations ( Na+) and anions (Cl) are dispersed in the water,forming an electrolyte.

Acidity of solutionsWhen an acid (e.g.hydrochloric acid, HCl) is mixed with water it releaseshydrogen ions (protons) with a positive charge (H+). These quickly attach

themselves to water molecules, forming hydrogen (H30+

) ions.When a base (a metal oxide or hydroxide) is added to water, it forms a

basic or alkaline solution. When the base dissolves it releases hydroxide(OH) ions. A solution that contains equal numbers of hydroxide and

hydrogen ions is neutral. Figure 2.8. A solution that contains more hydroxide ions than hydrogen

ions is alkaline. Figure 2.9. A solution that contains more hydrogen ions than hydroxide

ions is acid. Figure 2.10.

pHThe acidity of a solution is determined as the concentration of hydrogen

ions. However, this varies a great deal from one solution to another. Thesymbol pH is used to denote the hydrogen ion concentration.

Mathematically, pH is defined as the negative logarithm to the base 10 ofthe hydrogen ion concentration expressed in molarity,i.e.pH = log [H+].

This results in the following scale at 25 C:

Na+

Cl-Na+

Na+

Cl-

Cl-

Fig 2.7Ionic solution.

OH- H+ OH-

OH-H+

H+

H+

H+

Fig 2.10Acid

solution with pHless than 7.

pH > 7 alkaline solution

pH = 7 neutral solution

pH < 7 acid solution

NeutralisationWhen an acid is mixed with an alkali the hydrogen and hydroxide ions reactwith each other to form water. If the acid and alkali are mixed in certainproportions, the resulting mixture will be neutral, with no excess of eitherhydrogen or hydroxide ions and with a pH of 7. This operation is calledneutralisation and the chemical formula:

H30++ OHresults in H

20 + H

20

Neutralisation results in the formation of a salt. When hydrochloric acid (HCl)is mixed with sodium hydroxide (NaOH), the two react to form sodiumchloride (NaCl) and water (H

2

0). The salts of hydrochloric acid are called

chlorides, and other salts are similarly named after the acids from whichthey are formed: citric acid forms citrates, nitric acid forms nitrates, and soon.

DiffusionThe particles present in a solution ions, molecules or colloids areinfluenced by forces which cause them to migrate (diffuse) from areas ofhigh concentration to areas of low concentration. The diffusion processcontinues until the whole solution is homogeneous, with the sameconcentration throughout.

Sugar dissolving in a cup of coffee is an example of diffusion. The sugardissolves quickly in the hot drink, and the sugar molecules diffuse until they

are uniformly distributed in the drink.The rate of diffusion depends on particle velocity, which in turn depends

on the temperature, the size of the particles, and the difference inconcentration between various parts of the solution.

H+ OH- H+

H+

OH- OH-

OH-OH-

Fig 2.9Alkaline

solution with pHhigher than 7.

OH- H+H+

H+H+

OH- OH-

OH-

Fig 2.8Neutralsolution with pH 7.


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Figure 2.11 illustrates the principle of the diffusionprocess. The U-tube is divided into two compartmentsby apermeable membrane. The left leg is then filledwith water and the right with a sugar solution whosemolecules can pass through the membrane. After awhile, through diffusion, the concentration is equalisedon both sides of the membrane.

OsmosisOsmosis is the term used to describe the spontaneousflow of pure water into an aqueous solution, or from aless to a more concentrated solution, when separatedby a suitable membrane. The phenomenon of osmosiscan be illustrated by the example shown in Figure2.12. The U-tubes are divided in two compartments byasemi-permeablemembrane. The left leg is filled withwater and the right with a sugar solution whosemolecules cannot pass through the membrane. Nowthe water molecules will diffuse through the membraneinto the sugar solution and dilute it to a lower

concentration. This process is called osmosis.The volume of the sugar solution increases when it

is diluted. The surface of the solution rises as shown inFigure 2.12, and the hydrostatic pressure, a, of thesolution on the membrane becomes higher than thepressure of the water on the other side. In this state ofimbalance, water molecules begin to diffuse back inthe opposite direction under the influence of the higherhydrostatic pressure in the solution. When the diffusionof water in both directions is equal, the system is inequilibrium.

If hydrostatic pressure is initially applied to the sugarsolution, the intake of water through the membrane

can be reduced. The hydrostatic pressure necessaryto prevent equalisation of the concentration bydiffusion of water into the sugar solution is called theosmotic pressure of the solution.

Reverse osmosisIf a pressure higher than the osmotic pressure isapplied to the sugar solution, water molecules can bemade to diffuse from the solution to the water, therebyincreasing the concentration of the solution. Thisprocess illustrated in Figure 2.13 is used commerciallyto concentrate solutions and is termed ReverseOsmosis(RO).

DialysisDialysis is a technique employing the difference inconcentration as a driving force to separate largeparticles from small ones in a solution, for exampleproteins from salts. The solution to be treated is placedon one side of a membrane, and a solvent (water) onthe other side. The membrane has pores of a diameterwhich allows the small salt molecules to pass through,but is too small for the protein molecules to pass, seeFigure 2.14.

The rate of diffusion varies with the difference in

concentration, so dialysis can be speeded up if thesolvent on the other side of the membrane is changedoften.

Water

Permeablemembrane

Sugarmolecules

Permeablemembrane

Phase 1 Phase 2

Fig. 2.12The sugar molecules are too large to diffuse throughthe semi-permeable membrane. Only the small water molecules

can diffuse to equalise the concentration. a is the osmotic

pressure of the solution.

{a

Semi-permeablemembrane

Water

Semi-permeablemembrane

Sugarmolecules

Phase 1 Phase 2

{a

Counter pressure

higher than a

Phase 1 Phase 2

Plunger

Fig 2.14Diluting the solution on one side of the membraneconcentrates the large molecules as small molecules pass

throught it.

Water

Permeablemembrane

Salt

Protein

Fig. 2.13If a pressure higher than the osmotic pressure is

applied to the sugar solution, water molecules diffuse and thesolution becomes more concentrated.

Fig 2.11The sugar molecules diffuse through the permeable

membrane and the water molecules diffuse in the oppositedirection in order to equalise the concentration of the solution.


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Composition of cows milkThe quantities of the various main constituents of milk can vary considerablybetween cows of different breeds and between individual cows of the samebreed. Therefore only limit values can be stated for the variations. Thenumbers in Table 2.3 are simply examples.

Besides total solids, the term solids-non-fat (SNF) is used in discussingthe composition of milk. SNF is the total solids content less the fat content.

The mean SNF content according to Table 2:3 is consequently 13,0 3,9 =9,1 %. The pH of normal milk generally lies between 6,5 and 6,7, with 6,6as the most common value. This value is true for pH measurement of milkof approximately 25 C.

Fig 2.16 The composition of milk fat.

Size 0,1 20 m. Average size 3 4 m.

Skim milk

Fat globule

Fig 2.15A look into milk.

Fig 2.17 If milk is left to stand for a whilein a vessel, the fat will rise and form a

layer of cream on the surface.

PhospholipidsLipoproteins

GlyceridesCerebrosides

ProteinsNucleic acids

Enzymes

MetalsWater

TriglyceridesDiglyceridesFatty Acids

SterolsCarotenoids

Vitamins: A, D, E, K

Cream layer

Skim milk

Table 2.3

Quantitative composition of milk

Main constituent Limits of variation Mean value

Water 85,5 89,5 87,5Total solids 10,5 14,5 13,0

Fat 2,5 6,0 3,9Proteins 2,9 5,0 3,4Lactose 3,6 5,5 4,8Minerals 0,6 0,9 0,8

Milk fatMilk and cream are examples of fat-in-water(or oil-in-water) emulsions. Themilk fat exists as small globules or droplets dispersed in the milk serum,Figure 2.15. Their diameters range from 0,1 to 20 m (1 m = 0,001 mm).The average size is 3 4 m and there are some 15 billion globules per ml.

The emulsion is stabilised by a very thin membrane only 5 10 nm thick

(1 nm = 109 m ) which surrounds the globules and has a complicatedcomposition.

Milk fat consists of triglycerides (the dominating components), di- andmonoglycerides, fatty acids, sterols, carotenoids (giving the yellow colour ofthe fat) and vitamins (A, D, E, and K). Trace elements, are minorcomponents. The composition of a milk fat globule is outlined in Figure2.16.

The membrane consists of phospholipids, lipoproteins, cerebrosides,proteins, nucleic acids, enzymes, trace elements (metals) and bound water.It should be noted that the composition and thickness of the membrane arenot constant, because components are constantly being exchanged withthe surrounding milk serum.

As the fat globules are not only the largest particles in the milk but also

the lightest (density at 15,5 C = 0,93 g/cm3), they tend to rise to thesurface when milk is left to stand in a vessel for a while, Figure 2.17.

The rate of rise follows Stokes Law, but the small size of the fat globulesmakes creaming a slow process. Cream separation can, however, beaccelerated by aggregation of fat globules under the influence of a proteincalledagglutinin.These aggregates rise much faster than individual fatglobules. The aggregates are easily broken up by heating or mechanicaltreatment. Agglutinin is denatured at time-temperature combinations suchas 65 C/10 min or 75 C/2 min and the possibility of aggregationdisappears.

Chemical structure of milk fat

Milk fat is liquid when milk leaves the udder at 37 C. This means that thefat globules can easily change their shape when exposed to moderatemechanical treatment pumping and flowing in pipes for instance withoutbeing released from their membranes.


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All fats belong to a group of chemical substances called esters,which are compounds of alcohols and acids. Milk fat is a mixture ofdifferent fatty-acid esters called triglycerides, which are composed of analcohol called glycerol and various fatty acids. Fatty acids make upabout 90 % of milk fat.

A fatty-acid molecule is composed of a hydrocarbon chain and acarboxyl group (formula RCOOH). In saturated fatty acids, the carbonatoms are linked together in a chain by single bonds, while inunsaturated fatty acids there are one or more double bonds in thehydrocarbon chain. Each glycerol molecule can bind three fatty-acidmolecules, and as the three need not necessarily be of the same kind, thenumber of different glycerides in milk is extremely large.

Table 2.4 lists the most important fatty acids in milk fat triglycerides.Milk fat is characterised by the presence of relatively large amounts of

butyric and caproic acid.

Fig 2.18Sectional view of a fat globule.

GLYCEROL

GLYCEROL

GLYCEROL

BUTYRIC ACID

BUTYRIC ACID

BUTYRIC ACID

BUTYRIC ACID

STEARIC ACID

OLEIC ACID

FATTY ACID

FATTY ACID

FATTY ACID

Fig 2.19 Milk fat is a mixture of differentfatty acids and glycerol.

Fig 2.20 Molecular and structural formulae of stearic and oleic acids.

CH3(CH2)16COOH

CH3(CH2)7CH=CH(CH2)7COOH

H H H H H H H H H H H H H H H H

H3C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C

O

OHH H H H H H H H H H H H H H H H

| | | | | | | | | | | | | | | |

| | | | | | | | | | | | | | | |

H H H H H H H H H H H H H H H H

H3C-C-C-C-C-C-C-C-C=C-C-C-C-C-C-C-C-C

O

OHH H H H H H H H H H H H H H

| | | | | | | | | | | | | | | |

| | | | | | | | | | | | | |

Molecular formula of stearic acid

Molecular formula of oleic acid

Structral formula of stearic acid

Structural formula of oleic acid

Double bond

Liquid fat

Solid,crystalised fatwith variousmelting points

Melting point of fatTable 2.4 shows that the four most abundant fatty acids in milk are myristic,palmitic, stearic and oleic acids.

The first three are solid and the last is liquid at room temperature. As thequoted figures indicate, the relative amounts of the different fatty acids canvary considerably. This variation affects the hardness of the fat. Fat with ahigh content of high-melting fatty acids, such as palmitic acid, will be hard;but on the other hand, fat with a high content of low-melting oleic acidmakes soft butter.

Determining the quantities of individual fatty acids is a matter of purelyscientific interest. For practical purposes, it is sufficient to determine one ormore constants or indices which provide certain information concerning thecomposition of the fat.

Iodine valueFatty acids with the same numbers of C and H atoms but with differentnumbers of single and double bonds have completely different charac-

teristics. The most important and most widely used method of indicating

Table 2.4

Principal fatty acids in milk fat

Fatty acid % of total fatty- Melting point Number of atoms

acid content C H C O

SaturatedButyric acid 3,0 4,5 7,9 8 4 2Caproic acid 1,3 2,2 1,5 12 6 2Caprylic acid 0,8 2,5 +16,5 16 8 2

Capric acid 1,8 3,8 +31,4 20 10 2Lauric acid 2,0 5,0 +43,6 24 12 2Myristic acid 7,0 11,0 +53,8 28 14 2Palmitic acid 25,0 29,0 +62,6 32 16 2Stearic acid 7,0 3,0 +69,3 36 18 2

UnsaturatedOleic acid 30,0 40,0 +14,0 34 18 2Linoleic acid 2,0 3,0 5,0 32 18 2Linolenic acid up to 1,0 5,0 30 18 2Arachidonic acid up to 1,0 49,5 32 20 2

Liquid atroom temp-erature

Solid atroomtemp-erature

Liquid atroom temp-erature


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their specific characteristics is to measure theiodine value(IV) of the fat.The iodine value states the percentage of iodine that the fat can bind. Iodineis taken up by the double bonds of the unsaturated fatty acids. Since oleicacid is by far the most abundant of the unsaturated fatty acids, which areliquid at room temperature, the iodine value is largely a measure of theoleic-acid content and thereby of the softness of the fat.

The iodine value of butterfat normally varies between 24 and 46. Thevariations are determined by what the cows eat. Green pasture in thesummer promotes a high content of oleic acid, so that summer milk fat issoft (high iodine value). Certain fodder concentrates, such as sunflowercake and linseed cake, also produce soft fat, while types of fodder such ascoconut and palm oil cake and root vegetable tops produce hard fat. It istherefore possible to influence the consistency of milk fat by choosing asuitable diet for the cows. For butter of optimum consistency, the iodinevalue should be between 32 and 37.

Figure 2.21 shows an example of how the iodine value of milk fat canvary in the course of a year (Sweden).

Refractive indexThe amount of different fatty acids in fat also affects the way it refracts light.

It is therefore common practice to determine therefractive indexof fat,which can then be used to calculate the iodine value. This is a quickmethod of assessing the hardness of the fat. The refractive index normallyvaries between 40 and 46.

Nuclear Magnetic Resonance (NMR)Instead of analysing the iodine value or refractive index, the ratio of satura-ted fat to unsaturated fat can be determined by pulsed NMR. A conversionfactor can be used to transform the NMR value into a corresponding iodinevalue if desired.

The NMR method can also be utilised to find out the degree of fatcrystallisation as a function of the time of crystallisation. Trials made at theSMR laboratory in Malm, Sweden, 1979 to 1981, show that fat

crystallisation takes a long time in a 40 % cream cooled from 60 C to 5 C.A crystallisation time of at least two hours was needed, and the proportionof crystallised fat was 65 % of the total.

It was also noted that only 15 to 20 % of the fat was crystallised twominutes after 5 C was reached. The NMR value of butterfat normally variesbetween 30 and 41.

Fat crystallisationDuring the crystallisation process, the fat globules are in a very sensitivestate and are easily broken opened up even by moderate mechanicaltreatment.

39

37

35

33

31

29

J F M A M J J A S O N D

IV

Month

Fig 2.21Iodine value at different times ofthe year. The iodine value is a measure

of the oleic acid content of the fat.

10

20

30

40

50

60

70

5 10 15 20 25 30 35 40 45 50 55 60 120 min

Cryst. fat %

Exothermic reaction*

Cooling

* Exothermic = a chemical reaction accompanied by development of heat. (Heat of fusion)

C

Fig 2.22Milk fat crystallisation is an exothermic

reaction, which means that the chemical reaction isaccompanied by evolution of heat. The crystallisa-

tion curve is based on analysis made by the NMR

method.

Fat with a high content of high-melting fatty acids is hard.

Fat with a high content of low-melting fatty acids is soft.


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Electron microscope studies have shown that fat crystallises inmonomolecular spheres, see Figure 2.22. At the same time fractionationtakes place, so that the triglycerides with the highest melting points formthe outer spheres. Because crystallised fat has a lower specific volume thanliquid fat, tensions arise inside the globules, making them particularlyunstable and susceptible to breakage during the crystallisation period. Theresult is that liquid fat is released into the milk serum, causing formation oflumps where the free fat glues the unbroken globules together (the samephenomenon that occurs in butter production). Crystallisation of fatgenerates fusion heat, which raises the temperature somewhat (40% creamcooled from 60 C to 7 8 C grows 3 4 C warmer during thecrystallisation period).

It is important to bear this important property of milk fat in mind inproduction of cream for various purposes.

Proteins in milkProteins are an essential part of our diet. The proteins we eat are brokendown into simpler compounds in the digestive system and in the liver.These compounds are then conveyed to the cells of the body where theyare used as construction material for building the bodys own protein. The

great majority of the chemical reactions that occur in the organism arecontrolled by certain active proteins, the enzymes.

Proteins are giant molecules built up of smaller units called amino acids,Figure 2.23. A protein molecule consists of one or more interlinked chains ofamino acids, where the amino acids are arranged in a specific order. Aprotein molecule usually contains around 100 200 linked amino acids, butboth smaller and much larger numbers are known to constitute a proteinmolecule.

Amino acidsThe amino acids in Figure 2.24 are the building blocks forming the protein,and they are distinguished by the simultaneous presence of one aminogroup (NH

2) and one carboxyl group (COOH) in the molecule. The

proteins are formed from a specific kind of amino acids, amino acids,i.e.those which have both an amino group and a carboxyl group bound to thesame carbon atom, the -carbon.

The amino acids belong to a group of chemical compounds which canemit hydrogen ions in alkaline solutions and absorb hydrogen ions in acidsolutions. Such compounds are called amphotery electrolytes orampholytes. The amino acids can thus appear in three states:1 Negatively charged in alkaline solutions2 Neutral at equal + and charges3 Positively charged in acid solutionsProteins are built from a supply of approx. 20 amino acids,18 of which are found in milk proteins.

An important fact with regard to nutrition is that eight (nine for infants) of

the 20 amino acids cannot be synthesized by the human organism. As theyare necessary for maintaining a proper metabolism, they have to besupplied with the food. They are called essential amino acids, and all ofthem are present in milk protein.

The type and the order of the amino acids in the protein moleculedetermine the nature of the protein. Any change of amino acids regardingtype or place in the molecular chain may result in a protein with differentproperties.

As the possible number of combinations of 18 amino acids in a chaincontaining 100 200 amino acids is almost unlimited, the number ofproteins with different properties is also almost unlimited. Figure 2.24 showsa model of an amino acid. As mentioned before, amino acids contain both aslightly basic amino group (NH

2

) and a slightly acid carboxyl group(COOH). These groups are connected to a side chain, (R).

If the side chain is polar, the water-attracting properties of the basic andacid groups, in addition to the polar side chain, will normally dominate and

NH2 COOH

Amino acid

Amino group Carboxyl group

Fig 2.23 Model of a protein molecule

chain of amino acids, the amino and

carboxyl groups.

CR C

NH2

H O

O

Fig 2.24 The structure of a generalamino acid. R in the figure stands fororganic material bound to the central

carbon atom.


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Fig 2.25A protein molecule at pH 6,6has a net negative charge.

the whole amino acid will attract water and dissolve readily in water. Suchan amino acid is namedhydrophilic (water-loving).

If on the other hand the side chain is of hydrocarbon which does notcontain hydrophilic radicals, the properties of the hydrocarbon chain willdominate. A long hydrocarbon chain repels water and makes the aminoacid less soluble or compatible with water. Such an amino acid is calledhydrophobic (water-repellent).

If there are certain radicals such as hydroxyl (OH) or amino groups(NH

2) in the hydrocarbon chain, its hydrophobic properties will be modified

towards more hydrophilic. If hydrophobic amino acids are predominant inone part of a protein molecule, that part will have hydrophobic properties.An aggregation of hydrophilic amino acids in another part of the moleculewill, likewise, give that part hydrophilic properties. A protein molecule maytherefore be either hydrophilic, hydrophobic, intermediate or locallyhydrophilic and hydrophobic.

Some milk proteins demonstrate very great differences within themolecules with regard to water compitability, and some very importantproperties of the proteins depend on such differences.

Hydroxyl groups in the chains of some amino acids in casein may beesterified with phosphoric acid. Such groups enable casein to bind calcium

ions or colloidal calcium hydroxyphosphate, forming strong bridges bet-ween or within the molecules.

The electrical status of milk proteinsThe side chains of some amino acids in milk proteins carry an electriccharge which is determined by the pH of the milk. When the pH of milk ischanged by addition of an acid or a base, the charge distribution of theproteins is also changed. The electrical status of the milk proteins and theresulting properties are illustrated in the Figures 2.25 to 2.28.

At the normal pH of milk, (6,6) a protein molecule has a net negativecharge, Figure 2.25. The protein molecules remain separated becauseidentical charges repel each other.

If hydrogen ions are added, Figure 2.26, they are adsorbed by theprotein molecules. At a pH value where the positive charge of the protein is

equal to the negative charge,i.e.where the numbers of NH3+and COOgroups on the side chains are equal, the net total charge of the protein iszero. The protein molecules no longer repel each other, but the positivecharges on one molecule link up with negative charges on the neighbouringmolecules and large protein clusters are formed. The protein is thenprecipitated from the solution. The pH at which this happens is called theisoelectric pointof the protein.

In the presence of an excess of hydrogen ions, the molecules acquire anet positive charge as shown in Figure 2.27. Then they repel each otheronce more and therefore remain in solution.

If, on the other hand, a strong alkaline solution (NaOH) is added, allproteins acquire negative charges and dissolve.

Classes of milk proteinsMilk contains hundreds of types of protein, most of them in very smallamounts. The proteins can be classified in various ways according to their

Fig 2.26Protein molecules at pH 4,6,the isoelectric point.

Fig 2.28Protein moleculesat pH 14

Fig 2.27 Protein molecules

at pH 1


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Table 2.5

Concentration of proteins in milk

Conc. in milk % of totalg/kg protein

w/w

Casein

s1-casein*) 10,0 30,6

s2-

casein*) 2,6 8,0

-casein**) 10,1 30,8-casein 3,3 10,1Total Casein 26,0 79,5

Whey Proteins-lactalbumin 1,2 3,7-lactoglobulin 3,2 9,8Blood Serum Albumin 0,4 1,2Immunoglobulins 0,7 2,1Miscellaneous (includingProteose-Peptone) 0,8 2,4

Total Whey Proteins 6,3 19,3

Fat Globule Membrane Proteins 0,4 1,2

Total Protein 32,7 100

*) Henceforth called s-casein

**) Including -casein

Ref: Walstra & Jennis

Fig 2.29Structure of a casein sub-micelle.

-casein molecules

Hydrophobic core

PO3-

group

Protrudingchains

4

present in whey in lower concentrations than in the original milk. This is dueto heat denaturation during pasteurisation of the milk prior to cheese-making. The three main groups of proteins in milk are distinguished by theirwidely different behaviour and form of existence. The caseins are easilyprecipitated from milk in a variety of ways, while the serum proteins usually

remain in solution. The fat-globule membrane proteins adhere, as the nameimplies, to the surface of the fat globules and are only released bymechanical action, e.g.by churning cream into butter.

CaseinCasein is a group name for the dominant class of proteins in milk. Thecaseins easily form polymers containing several identical or different typesof molecules. Due to the abundance of ionisable groups and hydrophobicand hydrophilic sites in the casein molecule, the molecular polymers formedby the caseins are very special. The polymers are built up of hundreds andthousands of individual molecules and form a colloidal solution, which iswhat gives skim milk its whitish-blue tinge. These molecular complexes areknown as casein micelles. Such micelles may be as large as 0,4 microns,

and can only be seen under an electron microscope.

chemical or physical properties and their biological functions. The old wayof grouping milk proteins into casein, albumin and globulin has given way toa more adequate classification system. Table 2.5 shows an abridged list ofmilk proteins according to a modern system. Minor protein groups havebeen excluded for the sake of simplicity.

Whey protein is a term often used as a synonym for milk-serum proteins,but it should be reserved for the proteins in whey from the cheesemakingprocess. In addition to milk-serum proteins, whey protein also containsfragments of casein molecules. Some of the milk-serum proteins are also


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Fig 2.30Buildup and stabilisation of

casein micelles.

Ref: A digest of models by Slattery and Evard (1973),

Schmidt (1982) and Walstra (1990) according to Rollema

(1992). Rollema H.S. (1992) Casein Association and Micelle

Formation p 63-111. Elsevier Science Publications Ltd.

Submicelle

Protrudingchain

-casein

Hydrophoricinteractions(PO4) groups

Calcium

phosphate

Casein micellesCasein micelles are fairly dense aggregates of sub-micelles with small

regions of calcium phosphate, which links the sub-micelles together,giving the micelles an open, porous structure. Removal of calcium

phosphate (CCP colloidal calcium phosphate), e.g.byacidification or addition of EDTA or citrates, leads to

disintegration of the micelles. Disintegration also occurs whenpH becomes greater than 9.

There are three main subgroups of casein, s-casein,

-casein and -casein, which are all heterogeneousand consist of 2 8 genetic variants. Genetic variants

of a protein differ from each other only by a fewamino acids.

Casein micelles, shown in Figure 2.30, consistof a complex of sub-micelles, Figure 2.29, of a

diameter of 10 to 15 nm (nanometer = 109m). Thecontent of

s-, - and -caseins is heterogeneously

distributed in the different sub-micelles. The - and -caseins are mainly concentrated in the middle of the

sub-micelles, while -casein predominates on the

surface. It has been suggested that the hydrophilicprotruding chain of the -casein protrudes from the surfaceof the sub-micelles forming a hairy layer ( 5 10 nm).

However, a few s- and - caseins can also be found at

the surface. The current view is that the -casein content of thesub-micelles varies. The -casein-deficient sub-micelles are

mainly located in the centre of the micelle, whereas the -casein-richsub-micelles predominate on the surface, giving the whole micelle a hairysurface layer. The hairy layer of the -caseins protruding chain is partiallyresponsible for the micelles stability through a major contribution to thenegative charge of the micelles ( 20 mV) and to their steric stabilisation. Ifthe hairy layer is removed, e.g.by ethanol addition or rennet inducedhydrolysis, the colloidal stability of the micelle is destroyed and the micelles

coagulate or precipitate.Calcium salts of s-casein and -casein are almost insoluble in water,

while those of -casein are readily soluble. Due to the dominant localisationof -casein at the surface of the micelles, the solubility of calcium -caseinate prevails over the insolubility of the other two caseins in themicelles, and the whole micelle is soluble as a colloid.

According to Rollema (1992), a combination of the models of Slattery &Evard (1973), Schmidt (1982) and Walstra (1990) gives the best availableillustration of how the casein micelles are built up and stabilised.

The calcium phosphate and hydrophobic interactions between sub-micelles are responsible for the integrity of the casein micelles. Adding anexcess of Ca and phosphate results in aggregation of sub-micelles intolarger units micelles. The reason for this aggregation is presumably the

deposition of Ca-phosphate in the sub-micelles, which lowers their electriccharge and makes them more compact. They attract each other, causingaggregation. If two sub-micelles approach each other and the protrudingpeptide chains of at least one of them are in the way, they cannot agglom-erate. This means that agglomeration goes on until roughly sphericalparticles are formed, fully covered by a more or less continuous layer of -casein with protruding chains that together form the hairy layer. Micelles arenot uniform and vary in size. When measuring the size of casein particles, agreat number of very small particles can be found. These are loose sub-micelles, but these normally form only a minor part of the total caseincontent.

A casein micelle and its surroundings keep exchanging components, seeFigure 2.31. The size of a micelle depends very much on the calcium ion

(Ca++

) content. If calcium leaves the micelle, for instance by dialysis, themicelle will disintegrate into sub-micelles. A medium-sized micelle consistsof about 400 to 500 sub-micelles, which are bound together as describedabove.

Fig 2.31A casein micelle.


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If the hydrophilic protruding chain end of -casein on the surfaces ofmicelles is split, e.g.by rennet, the micelles will lose their solubility and startto aggregate and form casein curd. In an intact micelle there is a surplus ofnegative charges, therefore they repel each other. Water molecules held bythe hydrophilic sites of -casein form an important part of this balance. Ifthe hydrophilic sites are removed, water will start to leave the structure. Thisgives the attracting forces room to act. New bonds are formed, one of thesalt type, where calcium is active, and the second of the hydrophobic type.These bonds will then enhance the expulsion of water and the structure willfinally collapse into a dense curd.

The micelles are adversely affected by low temperature, at which the -casein chains start to dissociate and the CCP leaves the micelle structure,where it existed in colloidal form, and goes into solution. The explanation ofthis phenomenon is that -casein is the most hydrophobic casein, and thatthe hydrophobic interactions are weakened when the temperature islowered. Micelles appear to disintegrate into smaller ones, and the volumeof the casein micelles increases. The loss of CCP causes a weakerattraction between sub-micelles and individual casein molecules in the sub-micelles. These changes make the milk less suitable for cheesemaking, asthey result in longer renneting time and a softer curd.

-casein is then also more easily hydrolysed by various proteases in themilk after leaving the micelle. Hydrolysis of -casein to -casein andproteose-peptones means lower yield at cheese production because theproteose-peptone fractions are lost in the whey. The breakdown of -caseinmay also result in formation of bitter peptides, causing off-flavour problemsin the dairy products. The line graph in Figure 2.32 shows the approximateamount of -casein (in %) that leaves a micelle at +5 C during 20 hoursstoring time.

In this context it should also be mentioned that when raw or pasteurisedchill-stored milk is heated to 62 65 C for about 20 seconds, the -caseinand calcium phosphate will revert to the micelle, thereby at least partlyrestoring the original properties of the milk.

On increasing the temperature, the micelles shrink somewhat and the

amount of CCP increases. Serum proteins become largely associated withcasein micelles during their heat denaturation bound to the micelle surface.One example is the association of -lactoglobulin and -casein. Most ofthese associations cannot be reversed by cooling.

Precipitation of caseinOne characteristic property of casein is its ability to precipitate. Due to thecomplex nature of the casein molecules, and that of the micelles formedfrom them, precipitation can be caused by many different agents. It shouldbe observed that there is a great difference between the optimumprecipitation conditions for casein in micellar and non-micellar form,e.g.assodium caseinate. The following description refers mainly to precipitation ofmicellar casein.

Precipitation by acidThe pH will drop if an acid is added to milk or if acid-producing bacteria areallowed to grow in milk. This will change the environment of the caseinmicelles in two ways. The course of events are illustrated in Figure 2.33.Firstly colloidal calcium hydroxyphosphate, present in the casein micelle, willdissolve and form ionised calcium, which will penetrate the micelle structureand create strong internal calcium bonds. Secondly the pH of the solutionwill approach the isoelectric points of the individual casein species.

Both methods of action initiate a change within the micelles, starting withgrowth of the micelles through aggregation and ending with a more or lessdense coagulum. Depending on the final value of the pH, this coagulum willeither contain casein in the casein salt form, or casein in its isoelectric state,

or both.The isoelectric points of the casein components depend on the ions of

other kinds present in the solution. Theoretical values, valid under certain

Note:If a large excess of acid isadded to a given coagulum, thecasein will redissolve, forming asalt with the acid. If hydrochloricacid is used, the solution willcontain casein hydrochloride,partly dissociated into ions.

200 10

0,5

1,0

%

h

Fig 2.32-casein in milk serum at +5 C.

Ref: Dr B Lindquist (1980), Arla Stockholm, Sweden.


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There are two ways to makecaseinate particles flocculateand coagulate: Precipitation by acid

Precipitation by enzymes

conditions, are pH 5,1 to 5,3. In salt solutions, similar to the condition ofmilk, the range for optimum precipitation is pH 4,5 to 4,9. A practical valuefor precipitation of casein from milk is pH 4,6.

If a large excess ofsodium hydroxideis added to the precipitated iso-electric casein, the redissolved casein will be converted into sodiumcaseinate, partly dissociated into ions. The pH of cultured milk products isusually in the range of 3,9 4,5, which is on the acid side of the isoelectricpoints. In the manufacture of casein from skim milk by the addition ofsulphuric or hydrochloric acid, the pH chosen is often 4,6.

Precipitation by enzymesThe amino-acid chain forming the -casein molecule consists of 169 aminoacids. From an enzymatic point of view the bond between amino acids 105(phenylalanin) and 106 (methionin) is easily accessible to many proteolyticenzymes.

Some proteolytic enzymes will attack this bond and split the chain. Thesoluble amino end contains amino acids 106 to 169, which are dominatedby polar amino acids and the carbohydrate, which give this sequencehydrophilic properties. This part of the -casein molecule is called theglycomacro-peptide and is released into the whey in cheesemaking.

The remaining part of the -casein, consisting of amino acids 1 to 105, isinsoluble and remains in the curd together with

s- and -casein. This part

is called para--casein. Formerly, all the curd was said to consist of para--casein.

The formation of the curd is due to the sudden removal of the hydrophilicmacropeptides and the imbalance in intermolecular forces caused thereby.Bonds between hydrophobic sites start to develop and are enforced bycalcium bonds which develop as the water molecules in the micelles start toleave the structure. This process is usually referred to as the phase of co-agulation and syneresis.

The splitting of the 105 106 bond in the -casein molecule is oftencalled the primary phase of the rennet action, while the phase ofcoagulation and syneresis is referred to as the secondary phase. There isalso a tertiary phase of rennet action, where the rennet attacks the caseincomponents in a more general way. This occurs during cheese ripening.

The durations of the three phases are determined mainly by pH andtemperature. In addition the secondary phase is strongly affected by thecalcium ion concentration and by the condition of micelles with regard toabsence or presence of denatured milk serum proteins on the surfaces ofthe micelles.

0 4 5 7 144,6

pH

Dehydratisation

Increase of particle size

Destabilisation

Hydratisation

Decrease of particle size

Stabilisation

Lowest solubility

Precipitation

Isoelectric casein

Hydratisation

Decrease of particle size

Partial dissociationinto ions

Stabilisation

Neutralisation

Increase of particle size

Dissociation of Ca fromthe micellar complex

Destabilisation

The isoelectricpoint

Casein salts (Ex: Casein chloride) Caseinates (Ex: Sodium caseinate)

The pH of normal milk, pH 6,6 6,8

Fig. 2.33Three simplified stages of influence on casein by an acid and alkali

respectively.


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Whey proteinsWhey protein is the name commonly applied to milk serum proteins.

If the casein is removed from skim milk by some precipitation method,such as the addition of mineral acid, there remains in solution a group ofproteins which are called milk serum proteins.

As long as they are not denatured by heat, they are not precipitated attheir isoelectric points. They are, however, usually precipitated by

polyelectrolytes such as carboxymethyl cellulose. Technical processes forrecovery of whey proteins often make use of such substances or of acombination of heat and pH adjustment.

When milk is heated, some of the whey proteins denature and formcomplexes with casein, thereby decreasing the ability of the casein to beattacked by rennet and to bind calcium. Curd from milk heated to a hightemperature will not release whey as ordinary cheese curd does, due to thesmaller number of casein bridges within and between the casein molecules.

Whey proteins in general, and -lactalbumin in particular, have very highnutritional values. Their amino acid composition is very close to that whichis regarded as a biological optimum. Whey protein derivatives are widelyused in the food industry.

-lactalbuminThis protein may be considered to be the typical whey protein. It is presentin milk from all mammals and plays a significant part in the synthesis oflactose in the udder.

-lactoglobulinThis protein is found only in ungulates and is the major whey proteincomponent of milk from cows. If milk is heated to over 60 C, denaturationis initiated where the reactivity of the sulphur-amino acid of -lactoglobulinplays a prominent part. Sulphur bridges start to form between the -lactoglobulin molecules, between one -lactoglobulin molecule and a -casein molecule and between -lactoglobulin and -lactalbumin. At hightemperatures, sulphurous compounds such as hydrogen sulphide are

gradually released. These sulphurous compounds are responsible for thecooked flavour of heat treated milk.

Immunoglobulins and related minor proteinsThis protein group is extremely heterogeneous, and few of its membershave been studied in detail, Figure 2.34.

Immunoglobulins are antibodies synthesised in response to stimulationby specific antigens. They are specifically present in blood. Their content incows milk is low, but some of them are present in higher levels in colostrumand human milk. They can also act against particles such as bacteria,viruses and even fat globules, and flocculate them, a reaction calledagglutination. In this way, bacteria can also be flocculated on fat globulesand accumulate in the cream layer. When micro-organisms are flocculated,

their growth and action can be significantly inhibited.The agglutination reaction is specific with respect to a particular antigen.

However, some of the agglutinates are non-specific, especially in the caseof so called cryoprecipitation agglutination, which takes place in cold milkat temperatures below 37 C. The proteins involved are called cryo-globulines. The agglutinins are inactivated by heat treatment and theirpossibility to flocculate particles disappears. Because of that, agglutinationdoes not occur in pasteurised milk.

In the future, many substances of importance will probably be isolatedon a commercial scale from milk serum or whey. Lactoferrin andlactoperoxidase are substances of possible use in the pharmaceutical andfood industries, and are now isolated from whey by a commercial process.Lactoferrin is also an inhibitor of bacteria including B.stearothermophilus

and B.subtilis. The inhibition is caused by removal of iron from their serum.

The whey proteins are:

-lactalbumin-lactoglobulin

Fig. 2.34Schematic shape of twoimmunoglobulins.

Ref. P.F. Fox and P.L.H. McSweeney, Dairy Chemistry andBiochemistry, 1998.


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Membrane proteinsMembrane proteins are a group of proteins that form a protective layeraround fat globules to stabilise the emulsion, Figure 2.35. Their consistencyranges from soft and jelly-like, in some of the membrane proteins, to rathertough and firm in others. Some of the proteins contain lipid residues and arecalled lipoproteins. The lipids and the hydrophobic amino acids of thoseproteins make the molecules direct their hydrophobic sites towards the fat

surface, while the less hydrophobic parts are oriented towards the water.Weak hydrophobic membrane proteins attack these protein layers in thesame way, forming a gradient of hydrophobia from fat surface to water.

The gradient of hydrophobia in such a membrane makes it an ideal placefor adsorption for molecules of all degrees of hydrophobia. Phospholipidsand lipolytic enzymes in particular are adsorbed within the membranestructure. No reactions occur between the enzymes and their substrate aslong as the structure is intact, but as soon as the structure is destroyed theenzymes have an opportunity to find their substrate and start reactions.

An example of enzymatic reaction is the lipolytic liberation of fatty acidswhen milk has been pumped cold with a faulty pump, or afterhomogenisation of cold milk without pasteurisation following immediately.The fatty acids and some other products of this enzymatic reaction give a

rancid flavour to the product.

Denatured proteinsAs long as proteins exist in an environment with a temperature and pHwithin their limits of tolerance, they retain their biological functions. But ifthey are heated to temperatures above a certain maximum their structure isaltered. They are said to be denatured, Figure 2.37. The same thinghappens if proteins are exposed to acids or bases, to radiation or to violentagitation. The proteins are denatured and lose their original solubility.

Fig 2.38If an alkali is added to milk thepH changes very slowly there is a

considerable buffering action in milk.

Fig 2.37If an alkali is added to acid thepH of the solution rises immediately

there is no buffering action.

Nobufferingaction

Acid

Addition of alkali

pH

Strongbufferingaction

Milk

Addition of alkali

pH

SH

SHSH

Fig 2.36Part of a whey protein in native (left) and denatured state.

When proteins are denatured, their biological activity ceases. Enzymes, aclass of proteins whose function is to catalyse reactions, lose this abilitywhen denatured. The reason is that certain bonds in the molecule arebroken, changing the structure of the protein. After a weak denaturation,proteins can sometimes revert to their original state, with restoration of theirbiological functions.

In many cases, however, denaturation is irreversible. The proteins in aboiled egg, for example, cannot be restored to the raw state.

Milk is a buffer solutionMilk contains a large number of substances which can act either as weakacids or as weak bases, e.g.lactic acid, citric acid and phosphoric acid andtheir respective salts: lactates, citrates and phosphates. In chemistry, sucha system is called a buffer solution because, within certain limits, the pHvalue remains constant when acids or bases are added. This effect can beexplained by the characteristic qualities of the proteins.

When milk is acidified, a large number of hydrogen ions (H+) are added.These ions are almost all bound to the amino groups in the side chains ofthe amino acids, forming NH

3+ ions. The pH value, however, is hardly

affected at all, as the increase in the concentration of free hydrogen ions isvery small.

When a base is added to milk, the hydrogen ions (H+) in the COOH

Fig 2.35Membrane proteins cover the

surface of the fat globule.1 Phospholipid

2 Protein3 Glycoprotein

2

3

2

1


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groups of the side chains are released, forming a COOgroup. Because ofthis, the pH value remains more or less constant. The more base that isadded, the greater the number of hydrogen ions released.

Other milk constituents also have this ability to bind or release ions, andthe pH value therefore changes very slowly when acids or bases are added.

Almost all of the buffering capacity is utilised in milk that is already aciddue to long storage at high temperatures. In such a case it takes only asmall addition of acid to change the pH value.

Enzymes in milkEnzymes are proteins having the ability to trigger chemical reactions and toaffect the course and speed of such reactions. Enzymes do this withoutbeing consumed. They are therefore sometimes calledbiocatalysts. Thefunctioning of an enzyme is illustrated in Figure 2.39.

The action of enzymes is specific; each type of enzyme catalyses onlyone type of reaction.

Two factors which strongly influence enzymatic action are temperatureand pH. As a rule, enzymes are most active in an optimum temperaturerange between 25 and 50 C. Their activity drops if the temperature isincreased beyond optimum, ceasing altogether somewhere between 50

and 120 C. At these temperatures the enzymes are more or lesscompletely denatured (inactivated). The temperature of inactivation variesfrom one type of enzyme to another a fact which has been widely utilisedfor the purpose of determining the degree of pasteurisation of milk.Enzymes also have their optimum pH ranges; some function best in acidsolutions, others in an alkaline environment.

The enzymes in milk come either from the cows udder or from bacteria.The former are normal constituents of milk and are called original enzymes.The latter,bacterial enzymes, vary in type and abundance according to thenature and size of the bacterial population. Several of the enzymes in milkare utilised for quality testing and control. Among the more important onesare peroxidase, catalase, phosphatase and lipase.

PeroxidasePeroxidase transfers oxygen from hydrogen peroxide (H

2O

2) to other readily

oxidisable substances. This enzyme is inactivated if the milk is heated to80C for a few seconds, a fact which can be used to prove the presenceor absence of peroxidase in milk and thereby check whether or not apasteurisation temperature above 80 C has been reached. This test iscalled Storchs peroxidase test.

CatalaseCatalase splits hydrogen peroxide into water and free oxygen. Bydetermining the amount of oxygen that the enzyme can release in milk, it ispossible to estimate the catalase content of the milk and learn whether or

not the milk has come from an animal with a healthy udder. Milk fromdiseased udders has a high catalase content, while fresh milk from ahealthy udder contains only an insignificant amount. There are, however,many bacteria which produce this kind of enzyme. Catalase is destroyed byheating at 75 C for 60 seconds.

PhosphatasePhosphatasehas the property of being able to split certain phosphoric-acidesters into phosphoric acid and the corresponding alcohols. The presenceof phosphatase in milk can be detected by adding a phosphoric-acid esterand a reagent that changes colour when it reacts with the liberated alcohol.A change in colour reveals that the milk contains phosphatase.

Phosphatase is destroyed by ordinary pasteurisation (72 C for 15 20

seconds), so the phosphatase test can be used to determine whether thepasteurisation temperature has actually been attained. The routine test usedin dairies is called the phosphatasetest according to Scharer.

The enzyme fits into a particular spotin the molecule chain, where it weak-

ens the bond.

Fig 2.39A given enzyme will onlysplit certain molecules, and only at

certain bonds.

The molecule splits. The enzyme is

now free to attack and split anothermolecule in the same way.


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The phosphatase test should preferably be performed immediately afterheat treatment. Failing that, the milk must be chilled to below + 5 C andkept at that temperature until analysed. The analysis should be carried outthe same day, otherwise a phenomenon known as reactivation may occur,i.e.an inactivated enzyme becomes active again and gives a positive testreading. Cream is particularly susceptible in this respect.

LipaseLipase splits fat into glycerol and free fatty acids. Excess freefatty acids in milk and milk products result in a rancid taste.

The action of this enzyme seems, in most cases, to be veryweak, though the milk from certain cows may show stronglipase activity. The quantity of lipase in milk is believed toincrease towards the end of the lactation cycle. Lipase is, to agreat extent, inactivated by pasteurisation, but highertemperatures are required for total inactivation. Many micro-

organisms produce lipase. This can cause serious problems, as theenzyme is very resistant to heat.

Lactose in milkLactose is a sugar found only in milk; it belongs to the group of organicchemical compounds called carbohydrates.

Carbohydrates are the most important energy source in our diet. Breadand potatoes, for example, are rich in carbohydrates, and provide areservoir of nourishment. They break down into high-energy compoundsthat can take part in all biochemical reactions, where they provide thenecessary energy. Carbohydrates also supply material for the synthesis ofsome important chemical compounds in the body. They are present inmuscles as muscle glycogen and in the liver as liver glycogen.

Glycogen is an example of a carbohydrate with a very large molecularweight. Other examples are starch and cellulose. Such compositecarbohydrates are called polysaccharides and have giant molecules madeup of many glucose molecules. In glycogen and starch the molecules are

often branched, while in cellulose they are in the form of long, straightchains.

Figure 2.41 shows some disaccharides,i.e.carbohydrates composed oftwo types of sugar molecules. The molecules of sucrose (ordinary cane orbeet sugar) consist of two simple sugars (monosaccharides), fructose andglucose. Lactose (milk sugar) is a disaccharide, with a molecule containingthe monosaccharides glucose and galactose.

Table 2.3 shows that the lactose content of milk varies between 3,6 and5,5 %. Figure 2.42 shows what happens when lactose is attacked by lacticacid bacteria. These bacteria produce an enzyme called lactase, whichattacks lactose, splitting its molecules into glucose and galactose. Otherenzymes from the lactic-acid bacteria then attack the glucose andgalactose, which are converted via complicated intermediary reactions into

mainly lactic acid. The enzymes involved in these reactions act in a certainorder. This is what happens when milk goes sour; lactose is fermented tolactic acid. Other micro-organisms in the milk generate other breakdownproducts.

If milk is heated to a high temperature, and is kept at that temperature, itturns brown and acquires a caramel taste. This process is calledcaramelisation and is the result of a chemical reaction between lactose andproteins called the Maillard reaction.

Maillard reactions are just initiated by heat treatment and will continueduring storage of the product. The reaction kinetics is directly dependent onfactors such as heat load and storage temperature.

Lactose is water soluble, occurring as a molecular solution in milk. Incheesemaking, most of the lactose remains dissolved in the whey.Evaporation of whey in the manufacture of whey cheese increases thelactose concentration further. Lactose is not as sweet as other sugars; it isabout 30 times less sweet than cane sugar, for example.

GalactoseGlucose

Lactase

Lactose

Lactic acid

GlucoseGalactose

Bacterial enzymes

GLYCEROL

FATTY ACID

Free

Free

FATTY ACID

FATTY ACID

Fig 2.41Lactose and sucrose are split

to galactose, glucose and fructose.

Fructose Glucose Galactose

Sucrose Lactose

Fig 2.40Schematic picture of fat split-ting by lipase enzyme.

Fig 2.42Breakdown of lactose by enzy-matic action and formation of lactic acid.


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Vitamins in milkVitamins are organic substances that occur in very small concentrations inboth plants and animals. They are essential to normal life processes, butcannot be synthesised by the body. The chemical composition of vitamins isusually very complex, but that of most vitamins is now known. The variousvitamins are designated by capital letters, sometimes followed by numericalsubscripts, e.g.A, B

1and B

2.

Milk is a good source of vitamins, which are present in varying amounts.Among the best known are A, the vitamin B group, vitamin C and D.Vitamins A and D are soluble in fat, or fat solvents, while the others aresoluble in water.

In terms of fat-soluble vitamins, A and D are the most important. Theyaffect eyesight and skin. For natural reasons, low-fat milk products containless of these vitamins. In many countries this deficiency in low-fat milk iscompensated for by enrichment with vitamins A and D, in order to achievethe same vitamin level as whole milk.

Table 2.6 lists the amounts of the different vitamins in one litre of marketmilk and the daily vitamin requirement of an adult person. The table showsthat milk is a good source of vitamins. Lack of vitamins can result indeficiency diseases, see Table 2.7.

Table 2.6

Vitamins in milk and daily requirements

Amount in Adult daily

1 litre of requirement

Vitamin milk, mg mg

A 0,2 2 1 2B

10,4 1 2

B2

1,7 2 4C 5 20 30 100

D 0,002 0,01

Table 2.7

Vitamins deficiencies and corresponding diseases

Vitamin A deficiency Night blindness, impaired resistanceto infectious diseases

Vitamin B1deficiency Stunted growth

Vitamin B2deficiency Loss of appetite, indigestion

Vitamin C deficiency Fatigue, pyorrhoea, susceptibilityto infection (scurvy)

Vitamin D deficiency Skeletal deformation (rickets)

Minerals and salts in milkMilk contains a number of minerals. The total concentration is less than1 %. Mineral salts occur in solution in milk serum or in casein compounds.The most important salts are those of calcium, sodium, potassium andmagnesium. They occur as phosphates, chlorides, citrates and caseinates.Potassium and calcium salts are the most abundant in normal milk. Theamounts of salts present are not constant. Towards the end of lactation,and even more so in the case of udder disease, the sodium chloridecontent increases and gives the milk a salty taste, while the amounts ofother salts are correspondingly reduced.


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Other constituents of milkMilk always containssomatic cells (white blood corpuscles or leucocytes).The content is low in milk from a healthy udder, but increases if the udder isdiseased, usually in proportion to the severity of the disease. The somaticcell content of milk from healthy animals is as a rule lower than 200 000cells/ml, but counts of up to 400 000 cells/ml can be accepted.

Milk also containsgases, some 5 6 % by volume in milk fresh from the

udder, but on arrival at the dairy, the gas content may be as high as 10 %by volume. The gases consist mostly of carbon dioxide, nitrogen andoxygen. They exist in the milk in three states:1 Dissolved in the milk2 Bound and non-separable from the milk3 Dispersed in the milkDispersed and dissolved gases are a serious problem in the processing ofmilk, which is liable to burn on to heating surfaces if it contains too muchgas.

Changes in milk and its constituents

Changes during storageThe fat and protein in milk may undergo chemical changes during storage.These changes are normally of two kinds: oxidation and lipolysis. Theresulting reaction products can cause off-flavours, principally in milk andbutter.

Oxidation of fatOxidation of fat results in ametallic flavour, whilst it gives butter an oily,tallowy taste. Oxidation occurs at the double bonds of the unsaturated fattyacids, those of lecithin being the most susceptible to attack. The presenceof iron and copper salts accelerates the onset of auto-oxidation and devel-opment of metallic flavour, as does the presence of dissolved oxygen andexposure to light, especially direct sunlight or light from fluorescent tubes.

Oxidation of fat can be partly counteracted by micro-organisms in themilk, by pasteurisation at a temperature above 80 C, or by antioxidantadditives (reducing agents) such as DGA, dodecyl gallate. The maximumDGA dosage is 0,00005 %. Micro-organisms such as lactic-acid bacteriaconsume oxygen and have a reducing effect. Oxidation off-flavour is moreliable to occur at low temperatures, because these bacteria are less activethen. The solubility of oxygen in milk is also higher at low temperatures.High-temperature pasteurisation helps, as reducing compounds, (SH)groups, are formed when milk is heated.

The metallic oxidation off-flavour is more common in winter than insummer. This is partly due to the lower ambient temperature and partly todifferences in the cows diet. Summer feed is richer in vitamins A and C,which increase the amount of reducing substances in the milk.

In the presence of light and/or heavy metal ions, the fatty acids are

further broken down in steps into aldehydes and ketones, which give rise tooff-flavours such as oxidation rancidity in fat dairy products.

The above strongly simplified course of events at oxidation (really auto-

It generally is assumed that oxygen molecules in singlet state (1O2)can oxidise a CH-group directly, while shifting the double bondand forming a hydroperoxide according the formula:

1O2+ CH = CH CH

2 CHOOH CH = CH


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oxidation) of unsaturated fatty acids is taken from "Dairy Chemistry andPhysics" by P. Walstra and R. Jennis.

Oxidation of proteinWhen exposed to light, the amino acid methionine is degraded to methionalby a complicated participation of riboflavin ( Vitamin B

2) and ascorbic acid

(Vitamin C). Methional or 3-mercapto-methylpropionaldehyde is the

principal contributor tosunlight flavour,as this particular flavour is called.Since methionine does not exist as such in milk, but as one of thecomponents of the milk proteins, fragmentation of the proteins must occurincidental to development of the off-flavour.

Factors related to sunlight flavour development are: Intensity of light (sunlight and/or artificial light, especially from

fluorescent tubes). Duration of exposure. Certain properties of the milk homogenised milk has turned out

to be more sensitive than non-homogenised milk. Nature of package opaque packages such as plastic and

paper give good protection under normal conditions.See also Chapter 8 concerning maintenance of the quality of pasteurised

milk.

LipolysisThe breakdown of fat into glycerol and free fatty acids is calledlipolysis.Lipolysed fat has a rancid taste and smell, caused by the presence of low-molecular free fatty acids (butyric and caproic acid).

Lipolysis is caused by the action of lipases and is encouraged by highstorage temperatures. But lipase cannot act unless the fat globules havebeen damaged so that the fat is exposed. Only then can the lipase attackand hydrolyse the fat molecules. In normal dairying routine, there are manyopportunities for the fat globules to be damaged, e.g.by pumping, stirringand splashing. Undue agitation of unpasteurised milk should therefore beavoided, as this may involve the risk of widespread lipase action with the

liberation of fatty acids that make the milk taste rancid. Lipase must beinactivated by high-temperature pasteurisation, to prevent it from degradingthe fat. This completely destroys the original enzymes. Bacterial enzymesare more resistant. Not even UHT treatment can destroy them entirely. (UHT= Ultra High Temperature,i.e.heating to 135 150 C or more for a fewseconds.)

Effects of heat treatmentMilk is heat treated at the dairy to kill any pathogenic micro-organisms thatmay be present. Heat treatment also causes changes in the constituents ofthe milk. The higher the temperature and the longer the exposure to heat,

the greater the changes. Within certain limits, time and temperature can bebalanced against each other. Brief heating to a high temperature can havethe same effect as longer exposure to a lower temperature. Both time andtemperature must therefore always be considered in connection with heattreatment.

FatIt has been shown (Thom et al, Milchwissenschaft 13, 115, 1958) thatwhen milk is pasteurised at 70 80 C for 15 seconds, the cream plugphenomenon is already evident at 74 C (see Figure 2.44). Various theorieshave been discussed, but it appears that liberated free fat cements the fatglobules when they collide. Homogenisation is recommended to avoidcream plug formation.

A. Fink and H.G. Kessler (Milchwissenschaft 40, 6-7, 1985) have shownthat free fat leaks out of the globules in cream with 30 % fat, unhomogen-ised as well as homogenised, when it is heated to temperatures between

FATTYACID

FATTYACID

FATTY

ACID

GLYCEROL

Fig 2.43 When fat globule membranesare damaged, lipolysis can release fatty

acids.

Membrane intact.No Lipolysis.

Damagedmembrane.Lipolysis of fat

releasesfatty acids.


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105 and 135 C. This is believed to be caused by destabilisation of theglobule membranes resulting in increased permeability, as a result of whichthe extractable free fat acts as a cement between colliding fat globules andproduces stable clusters.

Above 135 C, the proteins deposited on the fat globule membrane forma network which makes the membrane denser and less permeable.Homogenisation downstream of the steriliser is therefore recommended inUHT treatment of products with a high fat content.

ProteinThe major protein, casein, is not considered denaturable by heat withinnormal ranges of pH, salt and protein content.

Whey proteins, on the other hand, particularly-lactoglobulin whichmakes up about 50 % of the whey proteins, are fairly heat sensitive.Denaturation begins at 65C and is almost total when whey proteins areheated to 90 C for five minutes.

Whey protein heat denaturation is an irreversible reaction. The randomlycoiled proteins "open up", and-lactoglobulin in particular is bound to the-casein fraction by sulphur bridges. The strongly generalisedtransformation is shown in Figure 2.45.

Blockage of a large proportion of the-casein interferes with therenneting ability of the milk, because the rennet used in cheesemakingassists in splitting the casein micelles at the -casein locations. The higherthe pasteurisation temperature at constant holding time, the softer thecoagulum; this is an undesirable phenomenon in production of semi-hardand hard types of cheese. Milk intended for cheesemaking should thereforenot be pasteurised, or at any rate not at higher temperatures than 72 C for15 20 seconds.

In milk intended for cultured milk products (yoghurt, etc.), the wheyprotein denaturation and interaction with casein obtained at 90 95 C for3 5 minutes will contribute to improved quality in the form of reducedsyneresis and improved viscosity.

Milk heated at 75 C for 20 60 seconds will start to smell and taste

cooked. This is due to release of sulphurous compounds from-lactoglobulin and other sulphur-containing proteins, inactive lipoproteins.

EnzymesEnzymes can be inactivated by heating. The temperature of inactivationvaries according to the type of enzyme.

There are some bacteria, Pseudomonas spp, (spp = species) nowadaysvery often cited among the spoilage flora of both raw cold-stored milk andheat-treated milk products, that have extremely heat-resistant proteolyticand lipolytic enzymes. Only a fraction of their activity is inhibited bypasteurisation or UHT treatment of the milk.

Fig. 2.45During denaturation -casein adheres to -lactoglobulin.

SH

SH

SH

SH

SH

SS

-casein

SH

SH

SH

SH

Casein micelles

Denaturated(-lactoglobulin)

Sulphurbridges

Whey proteins(-lactoglobulin)

Denaturation

Fig. 2.44Cream plug formation in milk

as a function of pasteurisation tempera-ture. Scale from 0 (no effect) to 4 (solid

cream plug). All pasteurisation was

short-time (about 15 s).Ref: Thom & al.

4

3

2

1

0 70 75 80

C

Cream plug

Average of some practical experiments

Tests in a laboratory pasteuriser


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LactoseLactose undergoes changes more readily in milk than in the dry state. Attemperatures above 100 C, a reaction takes place between lactose andprotein, resulting in a brownish colour. The series of reactions, occuringbetween amino groups of amino acid residues and aldehyde groups frommilk carbohydrates, is called the Maillard reaction or browning reaction. Itresults in a browning of the product and a change of flavour as well as loss

in nutritional value, particularly loss of lysine, one of the essential aminoacids.It appears that pasteurised, UHT and sterilised milks can be differen-

tiated by their lactulose content. Lactulose is an epimer of lactose formed inheated milks (Adachi, 1958). It is thought to be formed by the free aminogroups of casein (Adachi & Patton, 1961; Richards & Chandrasekhara,1960) Martinez Castro & Olano, 1982, and Geier & Klostermeyer, 1983,showed that pasteurised, UHT and sterilised milks contain different levels oflactulose. The lactulose content thus increases with increased intensity ofthe heat treatment.

VitaminsMilk is an important source of A, D and group B vitamins. The fat-soluble

vitamins are very thermostable and their level is not lowered by heattreatment. However, when milk is fortified with vitamin A, the relative lossseems to increase.

Losses of vitamins mainly concern vitamin C and some of the group Bvitamins. The loss of vitamin C as such is generally of minor importance, asmilk is not an important source of this vitamin, but it may influence thenutritional value anyway. The breakdown of vitamin C is connected with thatof vitamin B

12and protects folic acid from oxidation.

The degradation of vitamins is not only related to the heat treatment, butalso to storage of the final product. The loss of vitamins during storage canlargely be avoided if oxygen and light penetration are excluded. Vitamin Cand B

9may completely disappear within a few days if a high level of oxygen

is present. The reaction is catalysed by riboflavin (vitamin B2) and

accelerated by exposure to light. Most of the riboflavin disappears afterlong-term exposure to light.

Losses of some vitamins due to different treatments are presented inTable 2.8.

At temperatures above 100 C,a reaction takes place betweenlactose and protein, resulting ina brownish colour.

Table 2.8

Reduction of important vitamins in milk processed and stored in different conditions

Pasteurised milk UHT milk Vitamin Heat treatment Chilled storage Heat treatment Ambient storage

Ascorbic Acid, C 0 10 % 10 100 % 0 100 % up to 100 %

Thiamin, B1 < 10 % not investigated enough < 10 % no further loss*Riboflavin, B

2not significant not significant* < 10 % no further loss*

Pyridoxine, B6

< 10 % not investigated enough < 10 % up to 50 %Folic acid, B

9not significant not investigated enough 10 20 % up to 100 %**

Cobulamin, B12

< 10 % not significant 0 30 % up to 100 %Retinol, B-carotene, A not significant*** not significant*** not significant*** not significant***Calciferols, D not significant not significant not significant not significant

* When not exposed to light ** Dependent on oxygen content *** If not fortified

MineralsThe solubility of calcium phosphate is very temperature-dependent. Unlikemost compounds, the solubility of calcium phosphate decreases withtemperature. This means that heating causes precipitation of calcium


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phosphate in the form of CCP in the micelle, while cooling increases theconcentration of soluble calcium phosphate. After cooling, the reaction isreadily reversible, but after heating to high temperatures, the reversibility ismore sluggish and incomplete.

The changes at high temperature imply that the milk becomes more acidand the pH drops as described in Table 2.9 below. The changes in pH canbe explained as follows:The shift in pH increases when milk is concentrated.

Physical properties of milk

AppearanceThe opacity of milk is due to its content of suspended particles of fat,proteins and certain minerals. The colour varies from white to yellow,according to the coloration (carotene content) of the fat. Skim milk is moretransparent, with a slightly bluish tinge.

Heating

Cooling3 Ca2++ 2 HPO4

2- Ca3(PO4)2 + 2H+

Table 2.9

Effect of temperature on the pH of milk

Temperature, C pH

20 6,6430 6,5540 6,45

50 6,3460 6,23

Source: P.F.Fox and P.L.H. McSweeney: Dairy Chemistry and Biochemistry (1998)

DensityThe density of cows milk normally varies between 1,028 and 1,038 g/cm3,depending on the composition.

F = % fatSNF = % Solids Non FatWater % = 100 F SNF

100

F SNF

0,93 1,608+

g/cm3d15,5C

=

+ Water

100

3,2

0,93 1,608

d15,5C = 8,5+

Example: Milk of 3,2 % fat and 8,5 % SNF

+ (100 3,2 8,5)= 1,0306 g/cm

3

The density of milk at 15,5 C can be calculated according to thefollowing formula on previous page and the example below.


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Osmotic pressureOsmotic pressure is controlled by thenumber of molecules or particles, notthe weight of solute; thus 100 molecules of size 10 will have 10 times theosmotic pressure of 10 molecules of size 100.

It follows that for a given weight, the smaller the molecules the higher theosmotic pressure.

Milk is formed from blood, the two being separated by a permeable

membrane, hence they have the same osmotic pressure, or in other words,milk isisotonic with blood. The osmotic pressure of blood is remarkablyconstant although the composition, as far as pigment, protein etc., areconcerned, may vary. The same condition applies to milk, the total osmoticpressure being made up as in Table 2.10.

Table 2.10

Osmotic pressure in milk

Constituent Molecular Normal Osmotic D % of totalweight conc. pressure C osmotic

% atm pressure

Lactose 342 4,7 3,03 0,25 46Chlorides, NaCl 58,5 0,1 1,33 0,11 19Other salts, etc. 2,42 0,20 35Total 6,78 0,560 100

Ref: A Dictionary of Daiyring, J.G. Davis.

Freezing pointThe freezing point of milk is the only reliable parameter to check foradulteration with water. The freezing point of milk from individual cows hasbeen found to vary from 0,54 to 0,59 0C.

In this context it should also be mentioned that when milk is exposed tohigh temperature treatment (UHT treatment or sterilisation), precipitation ofsome phosphates will cause the freezing point to rise.

The internal or osmotic pressure also defines the difference in freezingpoint between the solution and the solvent (water) so that the freezing-pointdepression (D in Table 2.10) is a measure of this osmotic pressure. Whenthe composition of milk alters due to physiological or pathological causes(e.g.late lactation and mastitis respectively), it is termed abnormal milk, butthe osmotic pressure and hence the freezing-point remains constant. Themost important change is a fall in lactose content and a rise in chloridecontent.

AcidityThe acidity of a solution depends on the concentration of hydrogen ions[H+] in it. When the concentrations of [H+] and [OH] (hydroxyl) ions areequal, the solution is called neutral. In a neutral solution the numberof [H+] per liter of the solution is 1:10 000 000 g or 107.

pH represents the hydrogen ion concentration of a solution and canmathematically be defined as the negative logarithm of the hydrogen ion[H+] concentration.

Sodium hydroxidesolution (NaOH)

Concentration N/10(0,1N). The amount ofN/10 NaOH added isread when the samplechanges from colour-less to red.

10 ml milk sample

20 ml distilled water

5 drops of phenol-phtalien (5%).

Fig 2.46Determination of acidity in

Thrner degrees, Th.

pH = log [H+]

Applied to the example above, the pH is pH = log 107= 7 which is thetypical value of a neutral solution. When [H+] is 1:100 000 g/l or 106, the pHis 6 and the solution is acid. Thus the lower the exponent, the higher theacidity.


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ThepH value of a solution or product represents thepresent (true)acidity.Normal milk is a slightly acid solution with a pH falling between 6,6 6,8 with 6,6 the most usual value, at a measurement about 25 C. The pHis checked with a pH-meter.

Titratable acidityAcidity can also be expressed as the titratable acidity.The titratable acidity

of milk is the amount of a hydroxyl ion (OH

) solution of a given strengthneeded to increase the pH of a given amount of milk to a pH of about 8,4,the pH at which the most commonly used indicator,phenolphtalein,changes colour from colourless to pink. What this test really does is to findout how much alkali is needed to change the pH from 6,6 to 8,4.

If milk sours on account of bacterial activity, an increased quantity ofalkali is required, and so the acidity or titration value of the milk increases.

The titratable acidity can be expressed in a variety of units basically as aresult of the strength of the sodium hydroxide (NaOH) needed at titration.

SH= Soxhlet Henkel degrees, obtained by titrating 100 ml of milk withN/4 NaOH , using phenolphtalein as the indicator. Normal milks give valuesabout 7. This method is mostly used in Central Europe.

Th= Thrner degrees, obtained by titrating 100 ml of milk, thinned with

2 parts of distilled water, with N/10 NaOH, using phenolphtalein as theindicator. Normal milks give values about 17. Mostly used in Sweden.

D= Dornic degrees, obtained by titrating 100 ml of milk with N/9NaOH, using phenolphtalein as the indicator. Normal milks give valuesabout 15. Mostly used in the Netherlands and France.

% l.a.= per cent lactic acid, obtained as D with the result divided by100. Frequently used in the UK, USA, Canada, Australia and New Zealand.

In Table 2.11 the various expressions for the titratable acidity arecombined. The determination of acidity according to Thrner degrees isvisualised in Figure 2.47.

Fig 2.47Changes in the composition ofcows milk after parturition.

12

10

8

6

4

2

00 1 2 3 4 5 6 7

% Composition by weight

Days after parturition

Lactose

Fat

Casein

Whey protein

Ash

Table 2.11

Acidity is often expressed in

one of these ways

SH Th D % l.a.

1 2,5 2,25 0,02250,4 1 0,9 0,0094/9 10/9 1 0,01

Example:

1,7 ml of N/10 NaOH are required for titration of a 10 ml sample of milk.10 x 1,7 = 17 ml would therefore be needed for 100 ml, and the acidityof the milk is consequently 17 Th.

ColostrumThe first milk that a cow produces after calving is called colostrum. It differsgreatly from normal milk in composition and properties. One highlydistinctive characteristic is the high content of whey proteins about 11 %compared to about 0,65 % in normal milk, as shown in Figure 2.47. Thisresults in colostrum coagulating when heated. A fairly large proportion of

whey protein is immunoglobulins (Ig G, dominating in colostrum), whichprotect the

02 the chemistry of milk

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