Food Biochemistry and Food Processing (Simpson/Food Biochemistry and Food Processing) || Chemistry and Biochemistry of Milk Constituents

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24Chemistry and Biochemistry

of Milk ConstituentsP.F. Fox and A.L. Kelly

IntroductionSaccharides

LactoseIntroductionChemical and Physico-Chemical Properties of Lactose

Food Applications of LactoseLactose DerivativesNutritional Aspects of LactoseLactose in Fermented Dairy ProductsOligosaccharides

Milk LipidsDefinition and VariabilityFatty Acid ProfileConjugated Linoleic AcidStructure of Milk TriglyceridesRheological Properties of Milk FatMilk Fat as an EmulsionStability of Milk Fat GlobulesCreamingHomogenisation of MilkLipid OxidationFat-Soluble Vitamins

Milk ProteinsIntroductionHeterogeneity of Milk ProteinsMolecular Properties of Milk ProteinsInterspecies Comparison of Milk ProteinsCasein MicellesMinor Proteins

ImmunoglobulinsBlood Serum AlbuminMetal-Binding Proteinsβ2-MicroglobulinOsteopontinProteose Peptone 3Vitamin-Binding ProteinsAngiogeninsKininogen

GlycoproteinsProteins in the Milk Fat Globule MembraneGrowth FactorsMilk Protein-Derived Bioactive PeptidesIndigenous Milk Enzymes

Nutritional and ProtectiveTechnologicalIndices of Milk Quality and HistoryAntibacterial

Milk SaltsVitaminsSummaryReferences

Abstract: Mammalian milk is a highly complex physicochemicalsystem, containing colloidal proteins (the casein micelle) and emul-sified lipids, as well as dissolved lactose, minerals, vitamins andminerals. The properties of milk, and the products made or isolatedfrom milk, are very much determined by the properties of its con-stituents. These properties are particularly relevant when milk isprocessed, for example through denaturation of proteins, oxidation,hydrolysis of proteins and lipids, or Maillard reactions involvinglactose. In this chapter, the principal families of milk constituents,and their most significant characteristics, are described.

INTRODUCTIONMilk is a fluid secreted by female mammals, of which there areapproximately 4500 species, to meet the complete nutritional,and some of the physiological, requirements of the neonate of thespecies. Because nutritional requirements are species-specificand change as the neonate matures, it is not surprising thatthe composition of milk shows very large interspecies differ-ences, for example the concentrations of fat, protein and lactoserange from 1% to 50%, 1% to 20% and 0% to 10%, respec-tively. Interspecies differences in the concentrations of many of

Food Biochemistry and Food Processing, Second Edition. Edited by Benjamin K. Simpson, Leo M.L. Nollet, Fidel Toldra, Soottawat Benjakul, Gopinadhan Paliyath and Y.H. Hui.C© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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24 Chemistry and Biochemistry of Milk Constituents 443

the minor constituents of milk are even greater than those ofthe macro constituents. The composition of milk also changesmarkedly during lactation, reflecting the changing nutritional re-quirements of the neonate and during mastitis and physiologicalstress. In this chapter, the typical characteristics of the princi-pal, and some of the minor, constituents of bovine milk willbe described. The milk of the principal dairying species, cow,buffalo, sheep and goat are generally similar but differ in detail.The milks of several non-bovine species are described by Parkand Haenlein (2006) and Fuquay et al. (2011); the latter includesarticles on monotremes and marsupials, marine mammals andprimates.

Sheep and goats were domesticated around 8000 bc and theirmilk has been used by humans since. However, cattle, espe-cially breeds of Bos taurus, are now the dominant dairy animals.Total recorded world milk production is approximately 600 ×106 tonnes/annum, of which approximately 85% is bovine,11% is buffalo and 2% each is from sheep and goats. Camels,mares, reindeer and yaks are important dairy animals in lim-ited geographical regions with specific cultural and/or climaticconditions.

Milk is a very flexible raw material; several thousand dairyproducts are produced around the world in a great diversityof flavours and forms, including about 1400 varieties/variantsof cheese. The principal dairy products and the percentage ofmilk used in their production are: liquid (beverage) milk, ap-proximately 40%; cheese, approximately 35%; butter, approxi-mately 32%; whole milk powder, approximately 6%; skimmedmilk powder, approximately 9%; concentrated milk products ap-proximately 2%; fermented milk products, approximately 2%;casein, approximately 2% and infant formulae, approximately0.3%. The flexibility of milk as a raw material is a result of theproperties, many of them unique, of its principal constituents;many of these are very easily isolated, permitting the productionof valuable food ingredients. Milk is free of off-flavours, pig-ments and toxins, which greatly facilitates its use as a food or asa raw material for food production.

The processability and functionality of milk and milk productsare determined by the chemical and physicochemical propertiesof its principal constituents, that is lactose, lipids, proteins, andsalts, which will be described in this chapter. The exploitationand significance of the chemical and physico-chemical prop-erties of milk constituents in the production and properties ofthe principal groups of dairy foods, that is liquid milk products,cheese, butter, fermented milks, functional milk proteins andlactose will be described in Chapter 25. Many of the principalproblems encountered during the processing of milk are causedby variability in the concentrations and properties of the principalconstituents arising from several factors, including breed, indi-viduality of the animal (i.e., genetic factors), stage of lactation,health of the animal, especially mastitis, and nutritional status.Synchronised calving, as practised in New Zealand, Australiaand Ireland, to avail of cheap grass as the principal componentof the cow’s diet, has a very marked effect on the compositionand properties of milk (see O’Brien et al. 1999a, b, c, Mehraet al. 1999). However, much of the variability can be offset bystandardising the composition of milk using various methods

(e.g., centrifugation, ultrafiltration or supplementation) or bymodifying the process technology.

The chemical and physico-chemical properties of the princi-pal constituents of milk are well characterised and described.The very extensive literature includes the following textbooks:Walstra and Jenness (1984), Wong et al. (1988), Fox (1992,1995, 1997, 2003a), Fox and McSweeney (1998, 2003, 2006),Walstra et al. (1999, 2005) and McSweeney and Fox (2009).

SACCHARIDESLactose

Introduction

Lactose is a reducing disaccharide comprising glucose andgalactose, linked by a β1–4-O-glycosidic bond (Fig. 24.1). Itis unique to milk and is synthesised in the mammary glandfrom glucose transported from the blood; one molecule of glu-cose is epimerised to galactose, as UDP-galactose (Gal), via theLeloir pathway and is condensed with a second molecule ofglucose by a two-component enzyme, lactose synthetase. Com-ponent A is a general UDP-galactosyl transferase (UDP-GT; EC2.4.1.2.2), which transfers galactose from UDP-Gal to a rangeof sugars, peptides or lipids. Component B is the whey pro-tein, α-lactalbumin (α-La), in the presence of which, the KM

of UDP-GT for glucose is reduced 1000-fold and lactose is theprincipal product synthesised. There is a good correlation be-tween the concentrations of lactose and α-La in milk. Lactoseis responsible for approximately 50% of the osmotic pressure ofmilk, which is equal to that of blood and varies little; therefore,the concentration of lactose in milk is tightly controlled and isindependent of breed, individuality and nutritional factors, butdecreases as lactation advances and especially during mastitis,in both cases due to the influx of NaCl from the blood. Thephysiological function of α-La is probably to control the syn-thesis of lactose, and thus maintain the osmotic pressure of milkrelatively constant.

The concentration of lactose in milk ranges from approxi-mately 0, for some species of seal, to approximately 10% inthe milk of some monkeys. The concentration of lactose in themilk of the principal dairy species is quite similar (cow, 4.8%;buffalo, 4.3%; sheep, 4.6%; goat, 4.9%; camel, 5.1%), excep-tions are the horse (6.1%), donkey (6.9%) and reindeer (2.5%);human milk contains about 7.0% lactose. The lactose content ofbulk herd milk from randomly calved cows varies little through-out the year but differences can be quite large when calving ofcows is synchronised, for example in Ireland, the level of lac-tose in creamery milk varies from approximately 4.8% in Mayto approximately 4.2% in October.

Chemical and Physico-Chemical Properties of Lactose

Among sugars, lactose has a number of distinctive character-istics, some of which cause problems in milk products duringprocessing and storage; however, some of its characteristics areexploited to advantage.

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444 Part 4: Milk

ββ

α

β (1→ 4)GlucoseGalactose

LactoseO-β-D-Galactopyranosyl-(1→4)-α-D-Glucopyranose: α-Lactose

O-β-D-Galactopyranosyl-(1→4)-β-D-Glucopyranose: β-Lactose

β

Anomeric carbon

1

2

βα4 1

6

5

3

4 1

CH2OH

H

OHO

HOH

H

H

OH

H

CH2OH

HO

H H

OH

OH

OH

OOH

H

(1→4)6

5

O

O

O

OH

O

O

O OH

2 3

OH

H

H

OH

2

Figure 24.1. Structures of lactose.

The functional aldehyde group at the C-1 position of the glu-cose moiety exists mainly in the hemiacetal form, forming acyclic structure and, consequently, C-1 is a chiral, asymmet-ric, carbon. Therefore, like all reducing sugars, lactose can ex-ist as two anomers, α and β, which have markedly differentproperties. From a functional viewpoint, the most important ofthese properties are differences in solubility and crystallisationcharacteristics between the isomers; α-lactose crystallises as amonohydrate, while crystals of β-lactose are anhydrous. Sincecrystalline α-lactose contains 5% H2O, the yield of this anomeris higher than that of β-lactose and this must be considered whenexpressing the concentration of lactose. The solubility of α- andβ-lactose in water at 20◦C is approximately 7 g/100 mL and50 g/100 mL, respectively. However, the solubility of α-lactoseis much more temperature-dependent than that of β-lactose andthe solubility curves intersect at approximately 93.5◦C (see Foxand McSweeney 1998).

At equilibrium in aqueous solution, lactose exists as a mixtureof α and β anomers in the approximate ratio of 37:63. When anexcess of α-lactose is added to water, approximately 7 g/100 mLdissolve immediately, some of which mutarotates to give an α:βratio of 37:63, leaving the solution unsaturated with respect toboth α- and β-lactose. Further α-lactose then dissolves, some ofwhich mutarotates to β-lactose. Solubilisation and mutarotationcontinue until two conditions exist, that is approximately 7 gof dissolved α-lactose/100 mL of water and an α :β ratio of37:63, giving a final solubility of approximately 18.2 g/100 mL.When β-lactose is added to water, approximately 50 g/100 mLdissolve initially but approximately 18.5 g of this mutarotatesto α-lactose, which then exceeds its solubility and some lactosecrystallises. This upsets the α:β ratio and more β-lactose mutaro-tates to α-lactose, which crystallises. Mutarotation of β-lactoseand crystallisation of α-lactose continue until approximately 7 gand 11.2 g of α- and β-lactose, respectively, are in solution.

Although lactose has low solubility in comparison withother sugars, once dissolved, it crystallises with difficulty andforms supersaturated solutions. Highly supersaturated solutions(greater than twofold saturated) crystallise spontaneously butif the solution is only slightly supersaturated (one to twofold),lactose crystallises slowly and forms large, sharp, tomahawk-shaped crystals of α-lactose. If the dimensions of the crystalsexceed approximately 15 µm, they are detectable on the tongueand palate as a sandy texture. Crystals of β-lactose are smallerand monoclinical in shape. In the metastable zone, crystallisationof lactose is induced by seeding with finely powdered lactose(Fig. 24.2). Since the solubility of α-lactose is lower than that ofthe β anomer below 93.5◦C, α-lactose is the normal commercialform.

When concentrated milk is spray-dried, the lactose does nothave sufficient time to crystallise during drying and an amor-phous glass is formed. If the moisture content of the powderis kept low (<4%), the lactose glass is stable, but if the mois-ture content increases to about 6%, for example on exposureof the powder to a high-humidity atmosphere, the lactose willcrystallise as α-lactose monohydrate. If extensive crystallisationoccurs, an inter-locking mass of crystals is formed, resulting in‘caking’ of the powder, which is a particularly serious problemin whey powders owing to the high content of lactose ∼70%).The problem is avoided by pre-crystallising, as much as possi-ble, of the lactose before drying, which is achieved by seedingthe concentrated solution with finely powdered lactose.

Controlled agglomeration (caking) is used to improve the wet-tability of spray-dried milk powder This product has poor wet-tability because the small particles swell on contact with water,thereby blocking the channels between the particles (see Kellyet al. 2003 for review). The wettability (often incorrectly re-ferred to as ‘solubility’) of spray-dried milk powder may beimproved by controlling the drying process to produce milk

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05

10

Sol

ubili

ty (

gala

ctos

e/10

0 g

wat

er)

20

40

100

200

20 40

Temperature (°C)

60

2.1

Labile

Intermediate

Metastable

Not saturated

1.6 1

β

α

80 100

Figure 24.2. Initial solubility of α- and β-lactose, final solubility atequilibrium (line 1) and supersaturation by a factor of 1.6 and 2.1(α-lactose excluding water of crystallisation) (Fox and McSweeney1998).

powder with coarser, more easily wetted particles; such pow-ders are said to be ‘instantised’ and are produced by agglom-erating the fine powder particles, in effect by controlling thecaking process. In the case of whole milk powder, instantisa-tion processes must also overcome the intrinsic hydrophobicnature of milk fat; this is normally achieved by adding the am-phiphilic agent, lecithin. Although lactose is hygroscopic whenit crystallises, properly crystallised lactose has very low hygro-scopicity and, consequently, is a very effective component oficing sugar.

The crystallisation of lactose in frozen milk products resultsin destabilisation of the casein, which aggregates when the prod-uct is thawed. In this case, the effect of lactose is indirect; whenmilk is frozen, pure water freezes and the concentration of so-lutes in the unfrozen water is increased. Since milk is super-saturated with calcium phosphate (∼66% and ∼57% of the Caand PO4, respectively, are insoluble and occur in the casein mi-celles, known as colloidal calcium phosphate (CCP); see Section‘Milk Salts’), when the amount of water becomes limiting, sol-uble Ca(H2PO4)2 and CaHPO4 crystallise as Ca3(PO4)2, withthe concomitant release of H+ and a decrease in pH to ap-proximately 5.8. During frozen storage, lactose crystallises asα-lactose monohydrate, thus, reducing the amount of solventwater and aggravating the problems of calcium phosphate solu-bility and pH decline. Thorough crystallisation of lactose beforefreezing alleviates, but does not eliminate, this problem. Pre-heating milk prior to freezing also alleviates the problem, butpre-hydrolysis of lactose to the more soluble sugars, glucose andgalactose, using β-galactosidase, appears to be the best solution.

Lactose has low sweetness (16% as sweet as sucrose in a 1%solution). This limits its usefulness as a sweetener (the principalfunction of sugars in foods) but makes it is a very useful diluent,for example for food colours, flavours or enzymes, when a highlevel of sweetness is undesirable.

Being a reducing sugar, lactose can participate in the Mail-lard (non-enzymatic) browning reaction, with very undesirableconsequences in all dairy products, for example brown colour,off-flavours, reduced solubility and reduced nutritional value.The Maillard reaction, and factors that affect it, has been studiedextensively for around 100 years and the relevant literature hasbeen reviewed frequently (see O’Brien 2009, Nursten 2011).

Food Applications of Lactose

Total milk production (∼600 × 106 tonnes/annum) contains∼30 × 106 tonnes of lactose. Most of this lactose is consumedas a constituent of milk but whey, a by-product of the man-ufacture of cheese and, to a lesser extent, of casein, contains8–9 × 106 tonnes of lactose. About 400,000 tonnes of lac-tose are isolated/prepared per annum. A number of high-lactosefood products are also produced, for example approximately2,000,000 tonnes of whey powder, electrodialysed whey pow-der and whey permeate powder; these serve as crude sourcesof lactose for several food products, including infant formulae.Thus, most available lactose is now utilised in some form andlittle is wasted. The production of lactose is basically similarto that for other sugars, involving concentration, crystallisation,recovery, washing and drying of the crystals (see Paterson 2009,2011).

Although some of its properties, especially its low sweetnessand low solubility, limit the usefulness of lactose as a sugar,other properties, that is very low hygroscopicity if properly crys-tallised, low sweetness and reducing properties, make it a valu-able ingredient for the food and pharmaceutical industries. In thepharmaceutical industry, lactose is widely used as a diluent inpelleting operations. The principal application of lactose in thefood industry is in the humanisation of infant formulae; humanmilk contains approximately 7% lactose, compared to approxi-mately 4.8% in bovine milk. For this application, demineralisedwhey is widely used; it is cheaper and more suitable than puri-fied lactose because it also supplies whey proteins, which helpbring the casein:whey protein ratio of the formulae closer tothe value 40:60, found in human milk, compared with 80:20in bovine milk. It is necessary to demineralise the whey be-cause bovine milk contains approximately 4 times as much inor-ganic salts as human milk. Demineralisation is accomplished byelectrodialysis, ion exchange or nanofiltration or combinationsof these.

Lactose is also used as an agglomerating/free-flowing agentin foods (e.g., butter powders), in the confectionery industryto improve the functionality of shortenings, as an anti-cakingagent in icing mixtures at high humidity, or as a reducing sugarif Maillard browning is desired. The low sweetness of lactoselimits its widespread use as a sugar but is advantageous in manyapplications. Lactose absorbs compounds and may be used as adiluent for food flavours and colours or to trap flavours.

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Lactose Derivatives

A number of more useful and more valuable products may beproduced from lactose (see Playne and Crittenden 2009). Themost significant are the following:

� Lactulose (galactosyl β-1–4 fructose): A sugar not foundin nature, which is produced from lactose by heating, es-pecially under slightly alkaline conditions. The concentra-tion of lactulose in milk is a useful index of the severity ofthe heat treatment to which milk is subjected, for examplein-container sterilisation > indirect ultra-high tempera-ture (UHT) > direct UHT > high-temperature short-time(HTST) pasteurisation. Lactulose is not hydrolysed by in-testinal β-galactosidase and enters the large intestine, whereit promotes the growth of Bifidobacterium spp. It also hasa laxative effect and is widely used for this purpose; morethan 20,000 tonnes are produced annually.

� Glucose-galactose syrups, produced by acid or enzymatic(β-galactosidase) hydrolysis (see Chapter 25): The tech-nology for the production of such hydrolysates has beendeveloped but the product is not cost-competitive with othersugars (sucrose, glucose, glucose–fructose).

� Tagatose: Is the keto analogue of galactose. It occurs at alow level in the gum of the evergreen tree, Sterculia setig-era, and in severely heated milk and stored milk powder.It can be produced by treating β-galactosidase–hydrolysedlactose with a weak alkali, for example Ca(OH)2, whichconverts the galactose to tagatose, which can be purified bydemineralisation and chromatography. Tagatose is nearlyas sweet as sucrose, has a good quality taste and enhancesthe flavour of other sweeteners. It is absorbed poorly fromthe small intestine, serves as a probiotic and has little effecton blood glucose; it is fermented in the lower intestine tovolatile short-chain acids that can be absorbed but provideonly approximately 35% of the energy derived from sug-ars catabolised via the normal route. Tagatose has GRASstatus and is produced commercially by SweetGredients, acompany formed by Arla Foods and Nordzuker (Denmark).

� Galacto-oligsaccharides: β-Galactosidase has transferaseas well as hydrolytic activity and under certain condi-tions, the former predominates, leading to the formationof galacto-oligosaccharides containing up to six monosac-charides linked by glycosidic bonds that are not hydrolysedby the enzymes secreted by the human small intestine. Theundigested oligosaccharides enter the large intestine, wherethey have bifidogenic properties and are considered to havepromising food applications. These oligosaccharides arequite distinct from the naturally occurring oligosaccharidesreferred to in Section ‘Oligosaccharides’(see Ganzle 2011).

� Ethanol: Is produced commercially by the fermentation oflactose by Kluyveromyces lactis. Depending on local legis-lation, the ethanol may be used in alcoholic drinks, whichare profitable. The current interest in renewable energysources has created vast opportunities for lactose-derivedethanol but its commercial success will depend on localtaxation policy.

Other derivatives that have limited but potentially importantapplications include lactitol, lactobionic acid, lactic acid, aceticacid, propionic acid, lactosyl urea and single-cell proteins. Mostof these derivatives can be produced by fermentation of su-crose, which is cheaper than lactose, or by chemical synthesis.However, lactitol and lactobionic acids are derived specificallyfrom lactose and may have economic potential. Lactitol is a syn-thetic sugar alcohol produced by reduction of lactose; it is notmetabolised by higher animals but is relatively sweet, and hencehas potential for use as a non-calorific sweetener. It has alsobeen reported that lactitol reduces blood cholesterol level, re-duces sucrose absorption and is anti-carcinogenic. Lactobionicacid has a sweet taste, which is unusual for an acid and thereforeshould have some interesting applications.

Nutritional Aspects of Lactose

Lactose is responsible for two enzyme deficiency syndromes:lactose intolerance and galactosemia. The former is due to adeficiency of intestinal β-galactosidase, which is rare in infantsbut common in adults, except North-Western Europeans and afew African tribes. Since humans are unable to absorb disaccha-rides, including lactose, from the small intestine, unhydrolysedlactose enters the large intestine where it is fermented by bac-teria, leading to flatulence and cramp, and to the absorption ofwater from the intestinal mucosa, causing diarrhoea. These con-ditions cause discomfort and perhaps death. Lactose intolerancehas been studied extensively since its discovery in 1959 and theliterature has been reviewed regularly (see Ingram and Swallow2009).

The problems caused by lactose intolerance can beavoided by:

� excluding lactose-containing products from the diet, whichis the normal practice in regions of the world where lactoseintolerance is widespread;

� removing lactose from milk, for example by ultrafiltration;� hydrolysis of the lactose by adding β-galactosidase at the

factory or in the home. The technology for the produc-tion of lactose-hydrolysed milk and dairy products is welldeveloped but is of commercial interest mainly for lactose-intolerant individuals in Europe or North America. Becausethe consumption of milk is very limited in South-East Asia,the use of β-galactosidase is of little interest, although lac-tose intolerance is widespread.

Galactosemia is caused by the inability to catabolise galactose,owing to a deficiency of either of two enzymes, galactokinase orgalactose-1P uridyltransferase (see Flynn 2003). A deficiencyof galactokinase leads to the accumulation of galactose that iscatabolised via alternative routes, one of which leads to the accu-mulation of galactitol in various tissues, including the eye, whereit causes cataracts over a period of about 20 years. A deficiencyof galactose-1P uridyltransferase leads to abnormalities in mem-branes of the brain and to mental retardation unless galactose isexcluded from the diet within a few weeks of birth. Both formsof galactosemia occur at a frequency of 1 per approximately50,000 births.

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Lactose in Fermented Dairy Products

The fermentation of lactose to lactic acid, by lactic acid bacte-ria (LAB) is a critical step in the manufacture of all fermenteddairy products (cheese, fermented milks and lactic butter). Thefermentation pathways are well established (see Cogan and Hill1993, Poolman 2002). Lactose is not a limiting factor in the man-ufacture of fermented dairy products; only approximately 20%of the lactose is fermented in the production of these products.Individuals suffering from lactose intolerance may be able toconsume fermented milks without ill-effects, possibly becauseLAB produce β-galactosidase and emptying of the stomach isslower than that for fresh milk products, thus, releasing lactosemore slowly into the intestine.

In the manufacture of cheese, most (96–98%) of the lactoseis removed in the whey. The concentration of lactose in freshcurd depends on its concentration in the milk and on the mois-ture content of the curd and varies from approximately 1.7%,w/w, in fresh Cheddar curd to approximately 2.4%, w/w, in freshCamembert. The metabolism of residual lactose in the curd tolactic acid has a major effect on the quality of mature cheese(Fox et al. 1990, 2000). The resultant lactic acid may remain es-sentially unchanged in the cheese during ripening (e.g., Cheddarcheese) or may be catabolised to other compounds, for exampleCO2 and H2O, by surface mould in Camembert, or to propionicacid, acetic acid, H2O and CO2 in Emmental-type cheeses. Ex-cessive lactic acid in cheese curd may lead to a low pH and anumber of defects, such as a strong, acid, harsh taste, an increasein brittleness and a decrease in firmness. The pH of full-fat Ched-dar is inversely related to the lactose/lactic acid content of thecurd. Excess residual lactose may also be fermented by hetero-fermentative lactobacilli, with the production of CO2 leading toan open texture.

In the manufacture of some cheese varieties, for exampleDutch cheese, the curds are washed to reduce the lactose contentand thereby regulate the pH of the pressed curd at approximately5.3. For Emmental, the curd-whey mixture is diluted with waterby approximately 20%, again to reduce the lactose content ofthe curd, maintain the pH at approximately 5.3, and keep thecalcium concentration high, which is important for the texturalproperties of this cheese. For Cheddar, the level of lactose, andhence lactic acid, in the curd is not controlled. Hence, changesin the concentration of lactose in milk, such as those occurringthroughout lactation, can result in marked changes in the qualityof such cheeses. To overcome seasonal variations in the lactosecontent of milk, the level of wash water used for Dutch-typecheeses is related to the concentrations of lactose and caseinin the milk. Ideally, the lactose-to-protein ratio in any particularvariety should be standardised, for example by washing the curd,to minimise variations in the concentration of lactic acid, pH andthe quality of cheese.

Oligosaccharides

Lactose is the principal sugar in milk but the milk of most, if notall, species also contains oligosaccharides, up to hexasaccha-rides, derived from lactose (the reducing end of the oligosaccha-

rides is lactose and many contain fucose and N-acetylneuraminicacid). About 130 oligosaccharides have been identified in hu-man milk; the milk of elephant, bears and marsupials also con-tains high levels of oligosaccharides. The oligosaccharides areconsidered to be important sources of certain monosaccharides,especially fucose and N-acetylglucosamine, for neonatal devel-opment, especially of the brain (Urashima et al. 2001, 2009,2011).

MILK LIPIDSDefinition and Variability

The lipid fraction of milk is defined as those compounds that aresoluble in non-polar solvents (ethyl/petroleum ether or chloro-form/methanol) and is comprised mainly of triglycerides (98%),with approximately 1% phospholipids and small amounts ofdiglycerides, monoglycerides, cholesterol, cholesterol esters andtraces of fat-soluble vitamins and other lipids. The lipids occuras globules, 0.1–20 µm in diameter, each surrounded by a mem-brane, the milk fat globule membrane (MFGM), which servesas an emulsifier. The concentration of total and individual lipidsvaries with breed, individual animal, stage of lactation, mastiticinfection, plane of nutrition, interval between milking and pointduring milking when the sample is taken. Among the principaldairy breeds, Friesian/Holsteins produce milk with the lowest fatcontent (∼3.5%) and Jersey/Guernsey the highest (∼6%). Thefat content varies considerably throughout lactation; when syn-chronised calving is practised, the fat content of bulk Friesianmilk varies from approximately 3% in early lactation to >4.5%in late lactation. Such large variations in lipid content obviouslyaffect the economics of milk production and the composition ofmilk products, but can be modified readily by natural cream-ing, centrifugal separation or addition of cream, and hence neednot affect product quality. Milk lipids also exhibit variabilityin fatty acid composition and in the size and stability of theglobules. These variations, especially fatty acid profile, are es-sentially impossible to standardise and hence are responsiblefor considerable variations in the rheological properties, colour,chemical stability and nutritional properties of fat-containingdairy products.

Fatty Acid Profile

Ruminant milk fat contains a wider range of fatty acids than anyother lipid system – up to 400 fatty acids have been reportedin bovine milk fat; the principal fatty acids are the homologousseries of saturated fatty acids with an even number of C-atoms,C4:0–C18:0, and C18:1. The outstanding features of the fatty acidprofile of bovine milk fat are a high concentration of short- andmedium -chain acids (ruminant milk fats are the only naturallipids that contain butanoic acid, C4:0) and a low concentrationof polyunsaturated fatty acids.

In ruminants, the fatty acids for the synthesis of milk lipidsare obtained from triglycerides in chylomicrons in the bloodor synthesised de novo in the mammary gland from acetate orβ-hydroxybutyrate produced in the rumen. The triglycerides in

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chylomicrons are derived from the animal’s feed or synthesisedin the liver. Butanoic acid (C4:0) is produced by the reduction ofβ-hydroxybutyrate, which is synthesised from dietary roughageby bacteria in the rumen and therefore varies substantially withthe animal’s diet. All C6:0–C14:0 and 50% of C16:0 are synthe-sised in the mammary gland via the malonylCoA pathway fromacetylCoA produced from acetate synthesised in the rumen.Essentially 100% of C18:0, C18:1, C18:2 and C18:3 and 50% ofC16::0 are derived from blood lipids (chylomicrons) and repre-sent approximately 50% of total fatty acids in ruminant milkfat. Unsaturated fatty acids in the animal’s diet are saturated bybacteria in the rumen unless they are protected, for example byencapsulation.

Mainly due to saturation in the rumen, ruminant milk fats arequite saturated, approximately 65% of the fatty acids in bovinemilk fat are saturated, and are considered nutritionally undesir-able although not all to an equal extent. However, according toParodi (2009), the case against saturated fatty acids as causativefactors for coronary heart disease is not proven and further re-search is required.

When milk production is seasonal, for example Australia,New Zealand and Ireland, very significant changes occur in thefatty acid profile of milk fat throughout the production season(see Fox 1995, Fox and McSweeney 1998, 2006). These varia-tions are reflected in the hardness of butter produced from suchmilk; the spreadability of butter produced in winter is muchlower than that of summer butter. Owing to the lower degreeof unsaturation, winter butter should be less susceptible to lipidoxidation than the more unsaturated summer product but thereverse appears to be the case, probably owing to higher levelsof pro-oxidants, for example Cu and Fe, in winter milk.

Although a ruminant’s diet, especially if grass-based, is richin polyunsaturated fatty acids (PUFA), these are hydrogenatedby bacteria in the rumen and, consequently, ruminant milk fatcontains very low levels of PUFAs, for example bovine milk fatcontains approximately 2.4% C18:2 compared to approximately13% in human or porcine milk fat. PUFAs are considered to benutritionally desirable and consequently there has been interestin increasing the PUFA content of bovine milk fat. This canbe done by feeding encapsulated PUFA-rich lipids or crushedPUFA-rich oil seed to the animal. Increasing the PUFA contentalso reduces the melting point (MP) of the fat and makes butterproduced from it more spreadable. However, the lower MP fatmay have undesirable effects on the rheological properties ofcheese, and PUFA-rich dairy products are very susceptible tolipid oxidation. Although the technical feasibility of increasingthe PUFA content of milk fat by feeding protected PUFA-richlipids to the cow has been demonstrated, it is not economicalto do so in most cases. Blending milk fat with PUFA-rich orC18:1-rich vegetable oil appears to be much more viable and isnow widely practised commercially.

Conjugated Linoleic Acid

Linoleic acid (cis, cis 9, 12-octadecadienoic acid) is the princi-pal essential fatty acid and has been the focus of nutritional re-search for many years. However, conjugated isomers of linoleic

acid (CLA) have attracted very considerable attention recently(for review, see Bauman and Lock 2006). CLA is a mixture ofeight positional and geometric isomers of linoleic acid, whichhave a number of health-promoting properties, including anti-carcinogenic and anti-atherogenic activities, reduction of thecatabolic effects of immune stimulation and the ability to en-hance growth and reduce body fat (see Parodi 1999, Yuraweczet al. 1999). Of the eight isomers of CLA, only the cis-9, trans-11 isomer is biologically active. This compound is effective atvery low concentrations, 0.1 g/100 g diet.

Fat-containing foods of ruminant origin, especially milk anddairy products, are the principal sources of dietary CLA, whichis produced as an intermediate during the biohydrogenation oflinoleic acid by the rumen bacterium, Butyrivibrio fibrisolvens.Since CLA is formed from linoleic acid, it is not surprising thatthe CLA content of milk is affected by diet and season, beinghighest in summer when cows are on fresh pasture rich in PUFAs(Lock and Garnsworthy 2000, Lawless et al. 2000) and is higherin the fat of milk from cows on mountain pasture than on lowlandpasture (Collomb et al. 2002). The concentration of CLA in milkfat can be increased five to seven folds by increasing the levelof dietary linoleic acid, for example by duodenal infusion (Kraftet al. 2000) or by feeding a linoleic acid-rich oil, for examplesunflower oil (Kelly et al. 1998)

A number of other lipids may have anticarcinogenic activity,for example sphingomyelin, butanoic acid and ether lipids, butlittle information is available on these to date (Parodi 1997,1999)

Structure of Milk Triglycerides

Glycerol for milk lipid synthesis is obtained in part from hy-drolysed blood lipids (free glycerol and monoglycerides), partlyfrom glucose and a little from free blood glycerol. Synthesisof triglycerides within the cell is catalysed by enzymes locatedon the endoplasmic reticulum. Esterification of fatty acids is notrandom (Table 24.1). The concentrations of C4:0 and C18:1 appearto be rate-limiting because of the need to keep the lipid liquid atbody temperature. Some notable features of the structure are asfollows:

� Butanoic and hexanoic acids are esterified almost entirely,and octanoic and decanoic acids predominantly, at the sn-3position.

� As the chain-length increases up to C16:0, an increasingproportion is esterified at the sn-2 position; this is moremarked for human than for bovine milk fat, especially inthe case of palmitic acid (C16:0).

� Stearic acid (C18:0) is esterified mainly at sn-1.� Unsaturated fatty acids are esterified mainly at the sn-1 and

sn-3 positions, in roughly equal proportions.

The fatty acid distribution is significant from two viewpoints:

1. It affects the MP and hardness of the fat, which can bereduced by randomising the fatty acid distribution. Trans-esterification can be performed by treatment with SnCl2or enzymatically under certain conditions: increasing

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Table 24.1. Composition of Fatty Acids (mol% of theTotal) Esterified to Each Position of theTriacyl-sn-Glycerols in Bovine or Human Milk

Cow HumanFattyAcid sn-1 sn-2 sn-3 sn-1 sn-2 sn-3

4:0 – – 35.4 – – –6:0 – 0.9 12.9 – – –8:0 1.4 0.7 3.6 – – –

10:0 1.9 3.0 6.2 0.2 0.2 1.112:0 4.9 6.2 0.6 1.3 2.1 5.614:0 9.7 17.5 6.4 3.2 7.3 6.916:0 34.0 32.3 5.4 16.1 58.2 5.516:1 2.8 3.6 1.4 3.6 4.7 7.618:0 10.3 9.5 1.2 15.0 3.3 1.818:1 30.0 18.9 23.1 46.1 12.7 50.418:2 1.7 3.5 2.3 11.0 7.3 15.018:3 – – – 0.4 0.6 1.7

attention is being focused on the latter as an acceptablemeans of modifying the harness of butter.

2. Pancreatic and many other lipases are specific for the fattyacids at the sn-1 and sn-3 positions. Therefore, C4:0–C8:0

are released rapidly from milk fat; these are water-solubleand are readily absorbed from the intestine. Medium-and long-chain acids are absorbed more effectively as 2-monoglycerides than as fatty acids; this appears to be quiteimportant for the digestion of lipids by human infants whohave limited ability to digest lipids due to the absence ofbile salts. Infants metabolise human milk fat more effi-ciently than bovine milk fat, apparently due to the veryhigh proportion of C16:0 esterified at sn-2 in the former.The effect of trans-esterification on the digestibility ofmilk fat by infants merits investigation.

Short-chain fatty acids (C4:0–C10:0) have a strong aroma andflavour and their release by indigenous lipoprotein lipase (LPL)and microbial lipases cause off-flavours in milk and many dairyproducts, referred to as hydrolytic rancidity.

Rheological Properties of Milk Fat

The melting characteristics of ruminant milk fat are such that, atlow temperatures (e.g., ex-refrigerator), it contains a high pro-portion of solid fat and has poor spreadability. The rheologicalproperties of milk lipids may be modified by fractional crystalli-sation, for example an effective treatment involves removing themiddle MP fraction and blending high- and low-MP fractions.Fractional crystallisation is expensive and is practised in industryto only a limited extent; in particular, securing profitable outletsfor the middle-MP fraction is a major economic problem.

Alternatively, the rheological properties of milk fat may bemodified by increasing the level of PUFAs through feeding cowswith protected PUFA-rich lipids, but this practise is also expen-sive. The melting characteristics of blends of milk fat and veg-

etable oils can be easily varied by changing the proportions ofthe different fats and oils in the blend. This procedure is eco-nomical and is widely practised commercially; blending alsoincreases the level of nutritionally desirable PUFAs. The rheo-logical properties of milk fat-based spreads can also be improvedby increasing the moisture content of the product; obviously,this is economical and nutritionally desirable in the sense thatthe caloric value is reduced, but the resultant product is lessmicrobiologically stable than butter.

The melting characteristics and rheological properties ofmilk fat can also be modified by inter- and trans-esterification.Chemically-catalysed inter- and trans-esterification are not per-mitted in the food industry but enzymatic catalysis may be ac-ceptable. Lipases capable of such modifications on a commer-cial scale are available but their use is rather limited. Enzymatictrans-esterification allows modification of the nutritional as wellas the rheological properties of lipids. The nutritional and rheo-logical properties of lipids can also be modified by the use of adesaturase that converts C18:0 to C18:1 (these enzymes are a sub-ject of ongoing research, see hppt://bioinfo.pbi.nrc.ca/covello/r-fattyacid.html; and Meesapyodsuk et al. 2000). However, thistype of enzyme does not seem to be available commercially yet.

Milk Fat as an Emulsion

An emulsion consists of two immiscible, mutually insoluble liq-uids, usually referred to as oil and water, in which one of theliquids is dispersed as small droplets (globules; the dispersedphase) in the other (the continuous phase). If the oil is the dis-persed phase, the emulsion is referred to as an oil-in-water (O/W)emulsion; if water is the dispersed phase, the emulsion is referredto as a water-in-oil (W/O) emulsion. The dispersed phase is usu-ally, but not necessarily, the phase present in the smaller amount.An emulsion is prepared by dispersing one phase into the other.Since the liquids are immiscible, they will remain discrete andseparate if they differ in density, as is the case with lipids andwater, the density of which are 0.9 and 1.0, respectively; thelipid globules will float to the surface and coalesce. Coales-cence is prevented by adding a compound which reduces theinterfacial tension, γ , between the phases. Compounds capableof doing this have an amphipathic structure, that is hydropho-bic and hydrophilic regions, for example phospholipids, mono-glycerides, diglycerides, proteins, soaps and numerous syntheticcompounds, and are known as emulsifiers or detergents. Theemulsifier forms a layer on the surface of the globules with itshydrophobic region penetrating the oil phase and its hydrophilicregion in the aqueous phase. An emulsion thus stabilised willcream if left undisturbed, but the globules remain discrete andcan be redispersed readily by gentle agitation.

In milk, the lipids exist as an O/W emulsion in which the glob-ules range in size from approximately 0.1 to 20 µm, with a meanof 3–4 µm. The mean size of the fat globules is higher in high-fat milk than in low-fat milk, for example Jersey compared toFriesian, and decreases with advancing lactation. Consequently,the separation of fat from milk is less efficient in winter thanin summer, especially when milk production is seasonal, and it

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450 Part 4: Milk

may not be possible to meet the upper limit for fat content insome products, for example casein, during certain periods.

Stability of Milk Fat Globules

In milk, the emulsifier is the MFGM. On the inner side of theMFGM is a layer of unstructured lipoproteins, acquired withinthe secretory cells as the triglycerides move from the site of syn-thesis in the rough endoplasmic reticulum (RER) in the basalregion of the cell towards the apical membrane. The fat glob-ules are excreted from the cells by exocytosis, that is they arepushed through and become surrounded by the apical cell mem-brane. Milk proteins and lactose are excreted from the cell bythe reverse process: the proteins are synthesised in the RER andare transported to the Golgi region, where the synthesis of lac-tose occurs under the control of α-La. The milk proteins andlactose are encapsulated in Golgi membrane; the vesicles movetowards, and fuse with, the apical cell membrane, open and dis-charge their contents into the alveolar lumen, leaving the vesicle(Golgi) membrane as part of the apical membrane, thereby re-placing the membrane lost on the excretion of fat globules. Thus,the outer layer of the MFGM is composed of a trilaminar mem-brane, consisting of phospholipids and proteins, with a fluidmosaic structure.

Many of the proteins of the MFGM are strongly hydrophobicand difficult to isolate and characterise. Modern proteomic meth-ods have shown that the MFGM contains about 100 proteins, ofwhich the following are the principal and have been isolatedand characterised: butyrophilin (BTN), xanthine dehydrogenase(XDH), acidophilin, PAS (periodic acid Schiff staining) 6/7, CD(cluster of differentiation) 36, fatty acid-binding protein, mucins1 and 15 (MUC). BTN is a trans-membrane protein that com-plexes with XDH (located on the inner face of the membrane)that initiates the blebbing of the fat globule through the api-cal membrane of the cell (in this role, XDH does not act asan enzyme). MUC1, MUC 15, CD 36 and PAS6/7 are heav-ily glycosylated and are located mainly on the outer surface ofthe membrane, increasing its hydrophilicity. Very considerableprogress has been made on the proteins during the past 10 years,which has been reviewed by Mather (2000, 2011) and Keenanand Mather (2006). In human and equine milk, the MUC formlong (up to 50 µm) filaments that are lost easily; the filamentsprobably retard the passage of lipids through the small intes-tine, thereby improving digestibility, and prevent the adhesionof pathogens.

The MFGM contains many enzymes that originate mainlyfrom the Golgi apparatus: in fact, most of the indigenous en-zymes in milk are concentrated in the MFGM, notable excep-tions being plasmin and LPL that are associated with the caseinmicelles. The trilaminar membrane is unstable and is shed dur-ing storage, and especially during agitation, into the aqueousphase, where it forms microsomes.

The stability of the MFGM is critical for many aspects of themilk fat system:

� The existence of milk as an emulsion depends on the effec-tiveness of the MFGM.

� Damage to the MFGM leads to the formation of non-globular (free) fat, which may be evident as ‘oiling-off’on tea or coffee, cream plug or age thickening. An elevatedlevel of free fat in whole milk powder reduces its wettabil-ity. Problems related to, or arising from, free fat are moreserious in winter than in summer, probably due to the re-duced stability of the MFGM. Homogenisation, which re-places the natural MFGM by a layer of proteins from theskim milk phase, principally caseins, eliminates problemscaused by free fat.

� The MFGM protects the lipids in the core of the globuleagainst lipolysis by LPL in the skim milk (adsorbed on thecasein micelles). The MFGM may be damaged by agitation,foaming, freezing, for example on bulk tank walls, and es-pecially by homogenization, allowing access for LPL to thecore lipids and leading to lipolysis and hydrolytic rancidity.This is potentially a major problem in the dairy industryunless milking machines, especially pipeline milking instal-lations, are properly installed and serviced.

� The MFGM appears to be less stable in winter/late lactationthan in summer/mid lactation; therefore, hydrolytic rancid-ity is more likely to be a problem in winter than in summer.An aggravating factor is that less milk is usually producedin winter than in summer, especially in seasonal milk pro-duction systems, which leads to greater agitation and airincorporation during milking and, consequently, a greaterrisk of damage to the MFGM.

Creaming

Since the specific gravity of lipids and skim milk is 0.9 and1.036, respectively, the fat globules in milk held under quiescentconditions will rise to the surface under the influence of gravity,a process referred to as creaming. The rate of creaming, V , offat globules is given by Stoke’s equation:

V =2r2(ρ1 − ρ2)g

9η

wherer = radius of the fat globulesρ1 = specific gravity of skim milkρ2 = specific gravity of the fat globulesg = acceleration due to gravityη = viscosity of milk

The typical values of r, ρ1, ρ2 and η suggest that a creamlayer should form in milk after approximately 60 hours but milkcreams in approximately 30 minutes. The rapid rate of creamingis due to the strong tendency of the fat globules to agglutinate(stick together) due to the action of indigenous immunoglobulin(Ig) M, which precipitates onto the fat globules when milk iscooled (hence, they are called cryoglobulins). Considering theeffect of globule size (r) on the rate of creaming, large glob-ules rise faster than smaller ones and collide with, and adhereto, smaller globules, an effect promoted by cryoglobulins. Ow-ing to the larger value of r, the clusters of globules rise fasterthan individual globules, and therefore the creaming process

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accelerates as the globules rise and clump. Ovine, caprine andbuffalo milk do not contain cryoglobulins and therefore creammuch more slowly than bovine milk.

In the past, creaming was a very important physicochemicalproperty of milk:

� The cream layer served as an index of fat content and henceof quality to the consumer.

� Creaming was the traditional method for preparing fat(cream) from milk for use in the manufacture of butter. Itssignificance in this respect declined after the developmentof the mechanical separator by Gustav de Laval in 1878but natural creaming is still used to adjust the fat contentof milk for some cheese varieties, for example Parmigiano-Reggiano. A high proportion (∼90%) of the bacteria inmilk become occluded in the clusters of fat globules.

Homogenisation of Milk

Today, creaming is of little general significance. In most cases,its effect is negative and for most dairy products, milk is ho-mogenised, that is subjected to a high-shear pressure that re-duces the size of the fat globules (average diameter <1 µm),increases the fat surface area (four to six fold), replaces the nat-ural MFGM by a layer of caseins and denatures cryoglobulins,hence preventing the agglutination of globules.

Homogenization of milk is usually performed using a valvehomogeniser (developed by August Gaulin in 1899), in whichmilk is forced at a pressure of approximately 20 MPa againsta spring-loaded valve. The residence time in the valve is veryshort and many newly formed globules share emulsifier (proba-bly casein micelles), leading to the formation of clusters that willcream rapidly and increase the viscosity of the system. The clus-ters are dispersed by a second homogenisation at approximately3.5 MPa. Recently, a high-pressure homogeniser, operating ata pressure up to 300 MPa, has been developed. This type ofhomogeniser gives a very narrow distribution of fat globules,resulting in improved product quality.

Homogenisation has several very significant effects on theproperties of milk:

� If properly executed, creaming is delayed indefinitely dueto the reduced size of the fat globules, the denaturation ofcryoglobulins and the increased density of the fat globules,due to the layer of adsorbed casein on the surface.

� Susceptibility to hydrolytic rancidity is markedly increasedbecause indigenous LPL has ready access to the triglyc-erides; consequently, milk must be heated under conditionssufficiently severe to inactivate LPL before (usually) orimmediately after homogenisation.

� Susceptibility to oxidative rancidity is reduced because pro-oxidants in the MFGM, for example metals and xanthineoxidase, are distributed throughout the milk.

� The whiteness of milk is increased, due to the greater num-ber of light-scattering particles.

� The strength and syneretic properties of rennet-coagulatedmilk gels for cheese manufacture are reduced; hence,cheese with a higher moisture content is obtained. Con-

sequently, milk for cheese manufacture is not normallyhomogenised; an exception is reduced-fat cheese, in whicha higher moisture content improves texture.

� The heat stability of whole milk and cream is reduced, themagnitude of the effect increasing directly with fat contentand homogenisation pressure; homogenisation has no effecton the heat stability of skimmed milk.

� The viscosity of whole milk and cream is increased bysingle-stage homogenisation due to the clustering ofnewly formed fat globules, caused by sharing of caseinmicelles; the clumps of globules are dispersed by a secondhomogenisation stage at a lower pressure, which may beomitted if an increased viscosity is desired.

Lipid Oxidation

The chemical oxidation of lipids is a major cause of instabil-ity in dairy products (and many other foods). Lipid oxidationis a free radical, autocatalytic process principally involving themethylene group between a pair of double bonds in PUFAs;oxygen is a primary reactant (see O’Brien and O’Connor 2011).The process is initiated and/or catalysed by polyvalent metals,especially Cu and Fe, ultraviolet (UV) light, ionising radiationor enzymes, such as lipoxygenase in the case of plant oils, andxanthine oxidase, which is a major component of the MFGM,in milk. The principal end-products are unsaturated carbonyls,which cause major flavour defects; the reaction intermediates,that is fatty acid free radicals, peroxy free radicals and hydroper-oxides, have no flavour. Polymerisation of free radicals and otherspecies leads to the formation of pigmented products and to anincrease in viscosity but it is unlikely that polymerisation-relatedproblems occur to a significant extent in dairy products.

Lipid oxidation can be prevented or controlled by thefollowing:

� Avoiding metal contamination at all stages of processingthrough the use of stainless steel equipment.

� Avoiding exposure to UV light by using opaque packing(foil or paper).

� Packaging under an inert atmosphere, usually N2.� Use of O2 or free-radical scavengers, for example glucose

oxidase and superoxide dismutase (an indigenous enzymein milk), respectively.

� Use of antioxidants which break the free radical chain re-action; synthetic antioxidants are not permitted in dairyproducts but the level of natural antioxidants, for exam-ple tocopherols (vitamin E), in milk may be increased bysupplementing the animal’s feed. Polyphenols are very ef-fective antioxidants; their direct addition to dairy productsis not permitted but it may also be possible to increase theirconcentration in milk by supplementing the animal’s feed.Antioxidants are compounds which readily supply a H· tofatty acid and peroxy radicals, leaving a stable oxidised rad-ical. Many antioxidants are polyphenols, which give up a H·

and are converted to a quinone; examples are tocopherols(vitamin E) and catechins. At low concentrations, ascor-bic acid is a good antioxidant but at high concentrations it

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452 Part 4: Milk

functions as a pro-oxidant, apparently as a complex withCu. Sulphydryl groups of proteins are also effective antiox-idants; cream for butter making is usually heated to a hightemperature to denature proteins and expose and activatesulphydryl groups.

Fat-Soluble Vitamins

Since the fat-soluble vitamins (A, D, E and K) in milk are derivedfrom the animal’s diet, large seasonal variations can be expectedin their concentration in milk. The breed of cow also has asignificant effect on the concentration of fat-soluble vitamins inmilk; high-fat milk (Jersey and Guernsey) has a higher contentof these vitamins than Friesian or Holstein milk. Variations inthe concentrations of fat-soluble vitamins in milk have a numberof consequences:

� Nutritionally, milk contributes a substantial portion of theRDA (recommended daily allowance) for these vitaminsto Western diets; it is common practice to fortify milk andbutter with vitamins A and D.

� The yellow-orange colour of high-fat dairy products de-pends on the concentrations of carotenoids and vitamin Apresent, and hence on the diet of the animal. New Zealandbutter is much more highly coloured than Irish butter,which in turn is much more yellow than American or Ger-man products. The differences are due in part to the greaterdependence of milk production in New Zealand and Irelandon pasture and to the higher proportion of carotenoid-richclover in New Zealand pasture and the higher proportion ofJersey cows in New Zealand herds.

� Goats, sheep and buffalo do not transfer carotenoids totheir milk, which is, consequently, whiter than bovine milk.Products produced from these milks are whiter than cor-responding products made from bovine milk. The darkercolour of the latter may be unattractive to consumers accus-tomed to caprine or ovine milk products. If necessary, thecarotenoids in bovine milk may be bleached (by benzoylperoxide) or masked (by chlorophyll or TiO2).

� Vitamin E (tocopherols) is a potent antioxidant and con-tributes to the oxidative stability of dairy products. The to-copherol content of milk and meat can be readily increasedby supplementing the animal’s diet with tocopherols, whichis sometimes practised.

MILK PROTEINSIntroduction

Technologically, the proteins of milk are its most importantconstituents. They play important, even essential, roles in alldairy products except butter and anhydrous milk fat. The rolesplayed by milk proteins include the following:

� Nutritional: All milk proteins.� Physiological: Igs, lactoferrin (Lf), lactoperoxidase,

vitamin-binding proteins, protein-derived biologically-active peptides.

� Physico-chemical:� Gelation. Enzymatically, acid- or thermally induced

gelation in all cheeses, fermented milks, whey proteinconcentrates and isolates.

� Heat stability: All thermally processed dairy products,� Surface activity: Caseinates, whey protein concentrates

and isolates.� Rheological: All protein-containing dairy products.� Water sorption: Most dairy products, comminuted meat

products.

Milk proteins have been studied extensively and are very wellcharacterised at molecular and functional levels (for reviews, seeFox and McSweeney 2003).

Heterogeneity of Milk Proteins

It has been known since 1830 that milk contains two types ofprotein which can be separated by acidification, to what we nowknow is pH 4.6. The proteins insoluble at pH 4.6 are calledcaseins and represent approximately 78% of the total nitrogenin bovine milk; the soluble proteins are called whey or serumproteins. As early as 1885, it was shown that there are two typesof whey protein, globulins and albumins, which were thoughtto be transferred directly from the blood (the proteins of bloodand whey have generally similar physico-chemical propertiesand are classified as albumins and globulins). Initially, the termcasein was not restricted to the acid-insoluble proteins in milkbut was used to describe all acid-insoluble proteins; however,it was recognised at an early stage that the caseins are uniquemilk-specific proteins.

The casein fraction of milk protein was considered initiallyto be homogeneous but from 1918 onwards, evidence began toaccumulate that it is heterogeneous. Through the application offree boundary electrophoresis (FBE) in the 1930s and especiallyzone electrophoresis in starch or polyacrylamide gels (SGE,PAGE) containing urea and a reducing agent in the 1960s, it hasbeen shown that casein is in fact very heterogeneous. Bovinecasein consists of four families of caseins: αs1-, αs2- β- and κ-,which represent about 38%, 10%, 36% and 12%, respectively,of whole casein. Urea-PAGE showed that each of the caseinfamilies exhibits micro-heterogeneity due to:

� genetic polymorphism, usually involving substitution ofone or two amino acids;

� variations in the degree of phosphorylation;� variations in the degree of glycosylation of κ-casein;� inter-molecular disulphide bond formation in αs2- and

κ-caseins;� limited proteolysis, especially of β- and αs2-caseins, by

plasmin; the resulting peptides include the γ - and λ-caseinsand proteose peptones.

In the 1930s, FBE showed that both the globulin and albuminfractions of whey protein are heterogeneous and, in the 1950s,the principal constituents were isolated and characterised. It isnow known that the whey protein fraction of bovine milk com-prises four main proteins: β-lactoglobulin (β-Lg), α-La, Igs and

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blood serum albumin (BSA), which represent about 40%, 20%,10% and 10%, respectively, of the total whey protein in maturemilk. The remaining 10% is mainly non-protein nitrogen andtrace amounts of several proteins, including ca. 60 indigenous.Sixty indigenous enzymes. About 1% of total milk protein ispart of the MFGM, including many enzymes. β-Lg and α-Laare synthesised in the mammary gland and are milk-specific;they exhibit genetic polymorphism (in fact, the genetic poly-morphism of milk proteins was first demonstrated for β-Lg in1956). BSA, most of the Ig (i.e., IgG) and most of the minorproteins are transferred from the blood.

Methods for the isolation of the individual proteins were de-veloped and gradually improved during the period 1950–1970,so that by around 1970 all the principal milk proteins had beenpurified to homogeneity.

The concentration of total protein in milk is affected by mostof the factors that affect the concentration of fat, that is breed,individuality, nutritional status, health and stage of lactation but,with the exception of the last, the magnitude of most effects isless than in the case of milk fat. The concentration of proteinin milk decreases very markedly during the first few days post-partum, mainly due to the decrease in Ig from approximately10% in the first colostrum to 0.1% within about 1 week. Theconcentration of total protein continues to decline more slowlythereafter, to a minimum after about 4 weeks and then increasesuntil the end of lactation. Data on variations in the groups of pro-teins throughout lactation have been published (see Mehra et al.1999) but there are few data on variations in the concentrationsof the individual principal proteins.

Molecular Properties of Milk Proteins

The six principal milk-specific proteins have been isolated andare very well characterised at the molecular level; their chemicalcomposition is summarised in Table 24.2. The most notablefeatures of the principal milk-specific proteins are discussedlater.

They are quite small molecules, approximately 15–25 kDa.All the caseins are phosphorylated but to different and vari-able degrees, 1–13 mol P/mol protein; the phosphate groups areesterified as monoesters of serine residues. The primary struc-

tures of the caseins have a rather uneven distribution of polarand non-polar residues along their sequences. Clustering of thephosphoseryl residues is particularly marked, resulting in stronganionic patches in αs1-, αs2- and β-caseins. κ-Casein does nothave a phosphoseryl cluster but the N-terminal two-thirds of themolecule is quite hydrophobic while its C-terminal is relativelyhydrophilic – it contains no aromatic and no cationic residues.These features give the caseins an amphipathic structure, makingthem very surface-active, with good emulsifying and foamingproperties. The amphipathic structure of κ-casein is particularlysignificant and is largely responsible for its micelle-stabilisingproperties. The distribution of amino acids in β-Lg and α-La isquite random.

The two principal caseins, αs1- and β-caseins, are devoidof cysteine or cystine residues; the two minor caseins, αs2-and κ-caseins, contain two inter-molecular disulphide bonds.β-Lg contains two intra-molecular disulphide bonds and onesulphydryl group that is buried and unreactive in the native pro-tein but becomes exposed and reactive when the molecule isdenatured; it reacts via sulphydryl-disulphide interactions withother proteins, especially κ-casein, with major consequencesfor many important properties of the milk protein system, espe-cially heat stability and cheese making properties. α-La has fourintra-molecular disulphide bonds.

All the caseins, especially β-casein, contain a high level ofproline (in β-casein, 17 of the 209 residues are proline), whichdisrupts α- and β-structures; consequently, the caseins are ratherunstructured molecules and are readily susceptible to proteol-ysis, which is as would be expected for proteins the putativefunction of which is as a source of amino acids for the neonate.However, theoretical calculations suggest that the caseins mayhave a considerable level of secondary and tertiary structure;to explain the differences between the experimental and theo-retical indices of higher structures, it has been suggested thatthe caseins are very mobile, flexible, rheomorphic molecules(i.e., the casein molecules, in solution, are sufficiently flexibleto adopt structures that are dictated by their environment; Holtand Sawyer 1993). In contrast, the whey proteins are highly com-pact and structured, with high levels of α-helices, β-sheets andβ-turns. In β-Lg, the β-sheets are in an anti-parallel arrangementand form a β-barrel calyx.

Table 24.2. Characteristics of the Principal Proteins in Bovine Milk

Amino Acid Residues

ProteinMolecular

Weight Total Proline CysteinePhosphate

GroupsConcentration

(g/L) Genetic Variants

αs1-casein 23,164 199 17 0 8 10.0 A, B, C, D, E, F, G, Hαs2-casein 25,388 207 10 2 10–13 2.6 A, B, C, Dβ-casein 23,983 209 35 0 5 9.3 A1, A2. A3, B, C, D, E, F, Gκ-casein 19,038 169 20 2 1 3.3 A, B, C, D, E, FS, FI, GS, H, I, Jβ-lactoglobulin 18,277 162 8 5 0 3.2 A, B, C, D, E, F, G, H, I, Jα-lactalbumin 14,175 123 2 8 0 1.2 A, B, C

Source: Adapted from Fox 2003.

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454 Part 4: Milk

The caseins are often regarded as very hydrophobic proteinsbut they are not particularly so; however, they do have a highsurface hydrophobicity, owing to their open structures. Becauseof their hydrophobic patches they have a high propensity to yieldbitter hydrolysates, even in cheese that undergoes relatively littleproteolysis. In contrast, the highly structured whey proteins arevery resistant to proteolysis in the native state and may traversethe intestinal tract of the neonate intact.

About 60% of κ-casein molecules are glycosylated. Thesugar moieties present are galactose, galactosamine and N-acetylneuraminic acid (sialic acid), which occur as trisaccha-rides or tetrasaccharides. The oligosaccharides are attached tothe polypeptide via threonine residues in the C-terminal regionof the molecule and vary from 0 to 4 mol/mol of protein.

Probably because of their rather open structures, the caseinsare extremely heat stable, for example sodium caseinate can beheated at 140◦C for 1 hour without obvious physical effects. Themore highly structured whey proteins are comparatively heat-labile, although compared to many other globular proteins, theyare quite heat-stable; they are completely denatured on heatingat 90◦C for 10 minutes.

Under the ionic conditions in milk, α-La exists as monomersof molecular weight (MW) approximately 14.7 kDa. β-Lg existsas dimers (MW ∼36 kDa) in the pH range 5.5–7.5; at pH values<3.5 or >7.5, it exists as monomers, while at pH 3.5–5.5, itexists as octamers. The caseins have a very strong tendency toassociate. Even in sodium caseinate, the most soluble form ofwhole casein, the proteins exist mainly as decamers or larger ag-gregates at 20◦C. At low concentrations, for example <0.6%, at4◦C, β-casein exists as monomers but associates to form micellesif the concentration or temperature is increased. In the presenceof Ca, for example, calcium caseinate, casein forms large ag-gregates of several million Daltons. In milk, the caseins exist asvery complex structures, known as casein micelles, which aredescribed in Section ‘Casein Micelles’.

The function of the caseins appears to be to supply aminoacids to the neonate. They have no biological function strictosensu but their Ca-binding properties enable a high concentra-tion of calcium phosphate to be carried in milk in a ‘soluble’form; the concentration of calcium phosphate far exceeds its sol-ubility and, without the ‘solubilising’ influence of casein, wouldprecipitate in the ducts of the mammary gland and cause atopicmilk stones.

β-Lg has a hydrophobic pocket within which it can bindsmall hydrophobic molecules. It binds and protects retinol invitro and perhaps functions as a retinol carrier in vivo. In theintestine, it exchanges retinol with a retinol-binding protein. Italso binds fatty acids and thereby stimulates lipase; this is per-haps its principal biological function. β-Lg is a member of thelipocalin family, which now includes 14 proteins (for review seeAkerstrom et al. 2000); all members of the lipocalin family havesome form of binding function.

α-La is a metalloprotein; it binds one Ca atom per molecule ina peptide loop containing four aspartic acid residues. The apo-protein is quite heat-labile but the metalloprotein is rather heat-stable; when studied by differential scanning calorimetry, α-Lais in fact observed to be quite heat-labile. The metallo-protein

renatures on cooling, whereas the apo-protein does not. The dif-ference in heat stability between the halo- and apo-protein maybe exploited in the isolation of α-La on a large (i.e., potentiallyindustrial) scale.

As discussed in Section ‘Lactose’, α-La is an enzyme mod-ifier protein in the lactose synthesis pathway; it makes UDP-galactosyl transferase highly specific for glucose as an acceptorfor galactose, resulting in the synthesis of lactose.

Interspecies Comparison of Milk Proteins

The milk of all species that have been studies contain caseinsand whey proteins, but the protein systems differ in detail:

� The total concentration of protein ranges from approxi-mately 1% in human milk and that of the other great apes toapproximately 20% for lagomorphs; the protein content isdirectly related to the growth rate of the neonate.

� The ratio of casein to whey proteins ranges from approxi-mately 80:20 in bovine, buffalo, ovine and caprine milk toapproximately 30:70 for human milk.

� The types and proportions of the caseins and whey proteinsshow considerable inter-species differences. The milk ofall species studied seems to contain α-, β- and κ- caseinsbut human milk and that of some other primates containsvery little α-casein and equine milk contains little κ-casein.The milk of humans and that of many others, but not all,primates and rodents lacks β-Lg. The principal protein inhuman milk is α-La but the milk of some seals lacks thisprotein. The milk of some species contains a protein calledacid whey protein, which is absent from the milk of manyspecies, including cow, buffalo, sheep and goat. There areparticularly large inter-species differences with respect tomany minor proteins, including enzymes.

� All individual milk proteins show inter-species differenceswith respect to amino acid composition and perhaps otherfeatures. An interspecies comparison of the principal lacto-proteins of several species has been made by Martin et al.(2003, 2011).

Casein Micelles

Three of the caseins, αs1-, αs2- and β-, which together representapproximately 85% of total casein, are precipitated by calciumat concentrations >6 mM at temperatures >20◦C. Since milkcontains approximately 30 mmol/L Ca, it would be expectedthat most of the caseins would precipitate in milk. However, κ-casein is soluble at high concentrations of Ca and it reacts withand stabilises the Ca-sensitive caseins through the formation ofcasein micelles.

Since the stability, or perhaps instability, of the casein micellesis responsible for many of the unique properties and processingcharacteristics of milk, their structure and properties have beenstudied intensively. It has been known for nearly 200 years thatthe casein in milk exists as large colloidal particles which are re-tained by porcelain-Chamberlain filters. During the early part ofthe twentieth century, there was interest in explaining the rennet

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coagulation of milk and hence in the structure and properties ofthe Ca-caseinate particles (for review, see Fox and Kelly 2003,Fox and McSweeney 2003). The term casein micelle appears tohave been first used by Beau (1921). The idea that the rennet-induced coagulation of milk is due to the destruction of a pro-tective colloid (Schutz colloid) dates from the 1920s; initially, itwas suggested that the whey proteins were the ‘protective col-loid’. The true nature of the protective colloid, the structure ofthe casein micelle and the mechanism of rennet coagulation didnot become apparent until the pioneering work on the identifi-cation, isolation and characterisation of κ-casein by Waugh andvon Hippel (1956). Since then, the structure and properties ofthe casein micelle have been studied intensively. The evolutionof views on the structure of the casein micelle was described byFox and Kelly (2003), Horne (2003, 2011) and Fox and Brod-korb (2008). Current knowledge on the composition, structureand properties of the casein micelle and the key features thereofare summarised later.

The micelles are spherical colloidal particles, with a meandiameter of approximately 120 nm (range, 50–600 nm). Theyhave a mean particle mass of approximately 108 Da, that is thereare about 5000 casein molecules (20,000–25,000 Da each) inan average micelle. On a dry weight basis, the micelles containapproximately 94% protein and approximately 6% non-proteinspecies, mainly calcium and phosphate, with smaller amountsof Mg and citrate and traces of other metals; these are collec-tively called colloidal (or micellar) calcium phosphate (CCPor MCP). Under the conditions that exist in milk, the micellesare hydrated to the extent of approximately 2 g H2O/g protein.There are approximately 1015 micelles/mL milk, with a total sur-face area of approximately 5 × 104 cm2; the micelles are about240 nm apart. Owing to their very large surface area, the surfaceproperties of the micelles are of major significance and becausethey are quite closely packed, even in unconcentrated milk, theycollide frequently due to Brownian, thermal and mechanicalmotion.

The casein micelles scatter light; the white appearance ofmilk is due mainly to light scattering by the micelles, witha contribution from the fat globules. The micelles are gener-ally stable to most processes and conditions to which milk isnormally subjected and may be reconstituted from spray-driedor freeze-dried milk without major changes in their properties.Freezing milk destabilises the micelles, due to a decrease in pHand an increase in [Ca2+]. On very severe heating, for example at140◦C, the micelles shatter initially, then aggregate and eventu-ally, after approximately 20 minutes, the system coagulates (seeChapter 25)

The micelles can be sedimented by centrifugation; approxi-mately 50% are sedimented by centrifugation at 20,000 × g for30 minute and approximately 95% by 100,000 × g for 60 minute.The pelleted micelles can be redispersed by agitation, for exam-ple by ultrasonication, in natural or synthetic milk ultrafiltrate;the properties of the redispersed micelles are not significantlydifferent from those of native micelles.

The casein micelles are not affected by regular (∼20 MPa)or high-pressure (up to 250 MPa) homogenisation (Hayes andKelly 2003). The average size is increased by high-pressure

treatment at 200 MPa, but they are disrupted (i.e., average sizereduced by ∼50%) by treatment at a somewhat higher pressure,that is ≥400 MPa (Huppertz et al. 2002, 2004).

The micro-structure of the casein micelle has been the subjectof considerable research during the past 40 years, that is sincethe discovery and isolation of the micelle-stabilising protein,κ-casein; however, there is still a lack of general consensus.Numerous models have been proposed, the most widely sup-ported initially was the sub-micelle model, first proposed byMorr (1967) and refined several times since (Fox and Kelly2003, Horne 2003, 2011, Fox and Brodkorb 2008). Essentially,this model proposes that the micelle is built up from sub-micelles(MW ∼5×106 Da) held together by CCP and surrounded andstabilised by a surface layer, approximately 7 nm thick, rich in κ-casein but also containing some of the other caseins (Fig. 24.3A).It is proposed that the hydrophilic C-terminal region of κ-caseinprotrudes from the surface, creating a hairy layer around themicelle and stabilising it through a zeta potential of ca −20 mVand by steric stabilisation. The principal direct experimental ev-idence for this model is provided by electron microscopy, whichindicates a non-uniform electron density; this has been inter-preted as indicating sub-micelles.

However, several authors have expressed reservations aboutthe sub-unit model and three alternative models have been pro-posed. Visser (1992) suggested that the micelles are sphericalaggregates of casein molecules randomly aggregated and heldtogether partly by salt bridges in the form of amorphous calciumphosphate and partly by other forces, for example hydrophobicinteractions, with a surface layer of κ-casein. Holt (1992) pro-posed that the Ca-sensitive caseins are linked by micro-crystalsof CCP and surrounded by a layer of κ-casein, with its C-terminal region protruding from the surface (Fig. 24.3B). In thedual-binding model of Horne (2003, 2011), it is proposed thatindividual casein molecules interact via hydrophobic regions intheir primary structures, leaving the hydrophilic regions free andwith the hydrophilic C-terminal region of κ-casein protrudinginto the aqueous phase (Fig. 24.3C). Thus, the key structuralfeatures of the sub-micelle model are retained in the three alter-natives, that is the integrating role of CCP and a κ-casein-richsurface layer.

The micelles disintegrate:

� when the CCP is removed, for example by acidificationto pH 4.6 and dialysis in the cold against bulk milk, or byaddition of trisodium citrate, also followed by dialysis;removal of >60% of the CCP results in disintegration of themicelles;

� on raising the pH to approximately 9.0, which does notsolubilise the CCP and presumably causes disintegration byincreasing the net negative charge;

� on adding urea to >5 M, which suggests that hydro-gen and/or hydrophobic bonds are important for micelleintegrity.

The micelles are precipitated by ethanol or other low MW al-cohols at levels of ≥ approximately 35% at 20◦C. However, if thetemperature is increased to ≥70◦C, surprisingly, the precipitatedcasein dissolves and the solution becomes quite clear, indicating

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456 Part 4: Milk

(A)

(C)

(B)

Figure 24.3. Models of casein micelle structure: (A) the submicelle model of Walstra (1999); (B) the Holt model (from Fox and McSweeney1998); (C) the dual binding model, showing interactions between αs1-, β- and κ-caseins (Horne 2003).

dissociation of the micelles (O’Connell et al. 2001a, b). Micelle-like particles reform on cooling and form a gel at ∼4◦C. It isnot known if the sub-particles formed by any of these treatmentscorrespond to casein sub-micelles.

The turbidity of skim milk increases on addition of SDS upto 21mM, at which concentration a gel is formed; however, at≥28 mM (∼0.8%, w/v) the micelles disperse (see Lefebvre-Cases et al. 2001a, b).

There have been few studies on variations in micelle sizethroughout lactation and these have failed to show consistenttrends. No studies on variability in the micro-structure of thecasein micelle have been published. We are not aware of studieson the effect of the nutritional or health status of the animal onthe structure of the casein micelles, although their stability andbehaviour are strongly dependent on pH, milk salts and whey

proteins, all of which are affected by the health and nutritionalstatus of the cow.

The milk of all species that have been studied is white, pre-sumably due to light scattering by the casein micelles. However,the micelles of only a few species have been studied in detail;electron microscopy indicates that the micelles of all species arespherical and range in size from approximately 50 nm in humanmilk to ∼600 nm in equine milk. Some of the large micelles inequine milk settle out under gravity or low centrifugal forces;the large micelles reflect the low level of κ-casein in equinemilk. The milk of most species is supersaturated with calciumphosphate and the insoluble part (CCP) is present in the caseinmicelles, in which it acts as a cementing material; the micellesdisintegrate when the CCP is removed. Human milk lacks CCPand its micelles have a rather porous structure.

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Minor Proteins

In addition to the caseins and the two principal whey proteins,milk contains several proteins at low or trace levels. Many ofthese minor proteins are biologically active (see Schrezenmeiret al. 2000); some are regarded as highly significant and haveattracted considerable attention as nutraceuticals. When ways ofincreasing the value of milk proteins are discussed, the focusis usually on these minor proteins but they are, in fact, of littleeconomic value to the overall dairy industry. They are foundmainly in the whey but some are also located in the fat globulemembrane. Reviews on the minor proteins include Fox and Flynn(1992), Haggarty (2003) and Wynn (2011).

Immunoglobulins

Bovine colostrum contains approximately 10% (w/v) Igs, butthis level declines rapidly to approximately 0.1% about 5 dayspost-partum. IgG1 is the principal Ig in bovine, caprine or ovinemilk, with lesser amounts of IgG2, IgA and IgM; IgA is theprincipal Ig in human milk. The cow, sheep and goat do nottransfer Ig to the foetus in utero and the neonate is born withoutIg in its blood serum; consequently, it is very susceptible tobacterial infection, with a very high risk of mortality. The youngof these species can absorb Ig from the intestine for severaldays after birth and thereby acquire passive immunity until theysynthesise its own Ig, within a few weeks of birth. The humanmother transfers Ig in utero and the offspring is born with abroad spectrum of antibodies. Although the human baby cannotabsorb Ig from the intestine, the ingestion of colostrum is stillvery important because the Igs it contains prevent intestinalinfection. Some species, for example the horse, transfer Ig bothin utero and via colostrum.

The modern dairy cow produces colostrum far in excess ofthe requirements of her calf. Therefore, surplus colostrum isavailable for the recovery of Ig and other nutraceuticals (Paaka-nen and Aalto 1997, Marnila and Korhonen 2011). There is alsoconsiderable interest in hyper-immunising cows against certainhuman pathogens, for example rota virus, for the production ofantibody-rich milk for human consumption, especially by in-fants; the Ig could be isolated from the milk and presented as a‘pharmaceutical’ or consumed directly in the milk.

Blood Serum Albumin

About 1–2% of the protein in bovine milk is BSA, which entersfrom the blood by leakage through inter-cellular junctions. Asbefits its physiological importance, BSA is very well charac-terised (see Carter and Ho 1994). BSA has no known biologicalfunction in milk and, considering its very low concentration, itprobably has no technological significance either.

Metal-Binding Proteins

Milk contains several metal-binding proteins: the caseins (whichbind Ca, Mg, PO4) are quantitatively the most important; othersare α-La (Ca), xanthine oxidase (Fe, Mo), alkaline phosphatase

(Zn, Mg), lactoperoxidase (Fe), catalase (Fe), ceruloplasmin(Cu), glutathione peroxidase (Se), Lf (Fe) and seroferrin (Fe).

Lf, a non-haem iron-binding glycoprotein (see Lonnerdal2003, Korhonen and Marnila 2011), is a member of a family ofiron-binding proteins, which includes seroferrin and ovotrans-ferrin (conalbumin). It is present in several body fluids, includingsaliva, tears, sweat and semen. Lf has several potential biologi-cal functions; it improves the bioavailability of Fe, is bacterio-static (by sequestering Fe and making it unavailable to intestinalbacteria), and has antioxidant, antiviral, anti-inflammatory, im-munomodulatory and anti-carcinogenic activity. Human milkcontains a much higher level of Lf (∼20% of total N) thanbovine milk and therefore there is interest in fortifying bovinemilk-based infant formula with Lf. The pH of Lf is ∼9.0, that isit is cationic at the pH of milk, whereas most milk proteins areanionic, and can be isolated on an industrial scale by adsorptionon a cation-exchange resin. Hydrolysis of Lf by pepsin yieldspeptides called lactoferricins, which are more bacteriostatic thanLf and their activity is independent of iron status. Bovine milkalso contains a low level of serum transferrin.

Milk contains a copper-binding glycoprotein, ceruloplasmin,also known as ferroxidase (EC 1.16.3.1) (see Wooten et al. 1996).Ceruloplasmin is an α2-globulin with a MW of ∼126,000 Da; itbinds six atoms of copper per molecule and may play a role indelivering essential copper to the neonate.

β2-Microglobulin

β2-Microglobulin, initially called lactollin, was first isolatedfrom acid-precipitated bovine casein by Groves et al. (1963).Lactollin, with a MW of 43, 000 Da, is a tetramer of β2-microglobulin, which consists of 98 amino acids, with a cal-culated MW of 11, 636 Da. β2-Microglobulin, a component ofthe immune system (see Groves and Greenberg 1982), is prob-ably produced by proteolysis of a larger protein, mainly withinthe mammary gland; it has no known significance in milk.

Osteopontin

Osteopontin (OPN) is a highly phosphorylated acidic glycopro-tein, consisting of 261 amino acid residues with a calculatedMW of 29,283 (total MW of the glycoprotein, ∼60, 000 Da).OPN has 50 potential calcium-binding sites, about half of whichare saturated under normal physiological concentrations of cal-cium and magnesium. OPN occurs in bone (it is one of themajor non-collagenous proteins in bone), in many other normaland malignant tissues, in milk and urine and can bind to manycell types. It is believed to have a diverse range of functions(Denhardt and Guo 1993, Bayless et al. 1997), but its role inmilk is not clear.

Proteose Peptone 3

Bovine proteose peptone 3 (PP3) is a heat-stable phospho-glycoprotein that was first identified in the proteose-peptone(heat-stable, acid-soluble) fraction of milk. Unlike the otherpeptides in this fraction, which are proteolytic products of the

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caseins, PP3 is an indigenous milk protein, synthesised in themammary gland. Bovine PP3 is a polypeptide of 135 aminoacids, with five phosphorylation and three glycosylation sites.When isolated from milk, the PP3 fraction contains at leastthree components of MW ∼28, 18 and 11 kDa; the largestof these is PP3, while the smaller components are fragmentsthereof generated by plasmin (see Girardet and Linden 1996).PP3 is present mainly in acid whey but some is present in theMFGM also. Girardet and Linden (1996) proposed that its nameshould be changed to lactophorin or lactoglycoporin. PP3 hasalso been referred to as the hydrophobic fraction of proteosepeptone.

Owing to its strong surfactant properties (Campagna et al.1998), PP3 can prevent contact between milk lipase and itssubstrates, thus preventing spontaneous lipolysis. Although itsamino acid composition suggests that PP3 is not a hydrophobicprotein, it behaves hydrophobically, possibly owing to the for-mation of an amphiphilic α-helix, one side of which containshydrophilic residues while the other side is hydrophobic. Thebiological role of PP3 is unknown.

Vitamin-Binding Proteins

Milk contains binding proteins for at least the following vi-tamins: retinol (vitamin A, i.e., β-Lg, see Section ‘MolecularProperties of Milk Proteins’), biotin, folic acid and cobalamine(vitamin B12). The precise role of these proteins is not clear butthey may improve the absorption of vitamins from the intestineor act as antibacterial agents by rendering vitamins unavailableto bacteria. The concentration of these proteins varies duringlactation, but the influence of other factors such as individu-ality, breed and nutritional status is not known. The activity ofthese proteins is reduced or destroyed on heating at temperaturessomewhat higher than HTST pasteurisation.

Angiogenins

Angiogenins induce the growth of new blood vessels, that isangiogenesis. They have high sequence homology with mem-bers of the RNase A superfamily of proteins and have RNaseactivity. Angiogenesis is a complex biological process of whichthe ribonuclease activity of angiogenins is one of a number ofessential biochemical steps that lead to the formation of newblood vessels (Strydom 1998).

Two angiogenins (ANG-1 and ANG-2) have been identifiedin bovine milk and blood serum. Both strongly promote thegrowth of new blood vessels in a chicken membrane assay.Bovine ANG-1 has 64% sequence identity with human angio-genin and 34% identity with bovine RNase A. The amino acidsequence of bovine ANG-2 has 57% identity with that of bovineANG-1; ANG-2 has lower RNase activity than ANG-1. Thefunction(s) of the angiogenins in milk is unknown; they maybe part of a repair system to protect either the mammary glandor the intestine of the neonate and/or part of the host-defencesystem.

Kininogen

Two forms of kininogen have been identified in bovine milk, ahigh (>68 kDa) and a low (16–17 kDa) MW form (Wilson et al.1989). Bradykinin, a biologically-active peptide containing nineamino acids that is released from the high MW kininogen bythe action of the enzyme, kallikrein, has been detected in themammary gland, and is secreted into milk, from which it hasbeen isolated. Plasma kininogen is an inhibitor of thiol proteasesand has an important role in blood coagulation. Bradykinin af-fects smooth muscle contraction, induces hypertension and isinvolved in natriuresis and diuresis. The biological significanceof bradykinin and kininogen in milk is unknown.

Glycoproteins

Many of the minor proteins discussed previously are glycopro-teins; in addition, several other minor glycoproteins have beenfound in milk and colostrum but their identity and function havenot been elucidated fully. Some of these glycoproteins belong toa family of closely related, highly acidic glycoproteins, calledM-1 glycoproteins. Some glycoproteins stimulate the growth ofbifidobacteria, presumably via their amino sugars. One of the M-1 glycoproteins in colostrum, but which has not been detectedin milk, is orosomucoid (α1-acid glycoprotein), a member ofthe lipocalin family, which is thought to modulate the immunesystem.

One of the high MW glycoproteins in bovine milk is pros-aposin, a neurotrophic factor that plays an important role inthe development, repair and maintenance of the nervous system(Patton et al. 1997). It is a precursor of saposins A, B, C andD, which are sphingolipid-activator proteins, but saposins havenot been detected in milk. The physiological role of prosaposinin milk is not known, although the potent biological activityof saposin C, released by digestion, could be important for thegrowth and development of the young.

Proteins in the Milk Fat Globule Membrane

About 1% of the total protein in milk is in the MFGM. Most of theproteins are present at trace levels, including many of the indige-nous enzymes in milk. The principal proteins in the MFGM in-clude MUC1 and MUC15, adipophilin, BTN and XDH (Keenanand Mather 2003, 2003, 2006). BTN is a very hydrophobic pro-tein and has similarities to the Igs (for review see Mather 2000,2011).

Growth Factors

A great diversity of protein growth factors (hormones), includingepidermal growth factor, insulin, insulin-like growth factors 1and 2, three human milk growth factors (α1, α2 and β), twomammary-derived growth factors (I and II), colony-stimulatingfactor, nerve growth factor, platelet-derived growth factor andbombasin, are present in milk. It is not clear whether thesefactors play a role in the development of the neonate or in the

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development and functioning of the mammary gland, or both(Fox and Flynn 1992, Pihlanto-Leppala 2011, Wynn 2011).

Milk Protein-Derived Bioactive Peptides

The six lacto-proteins contain sequences, which when releasedon proteolysis display various biological activities (Pihlanto-Leppala 2011, Fosset and Tome 2011). Some of these peptidesare formed in the GIT in vivo but it not known whether or notthey are active in vivo.

Indigenous Milk Enzymes

Milk contains about 60 indigenous enzymes, which represent aminor but very important part of the milk protein system (forreview, see Fox and Kelly 2006, Fox 2003b). The enzymes orig-inate from the secretory cells or the blood. Many of the indige-nous enzymes are concentrated in the MFGM and originate inthe Golgi membranes of the cell or the cell cytoplasm, someof which occasionally becomes entrapped as crescents insidethe encircling membrane during exocytosis. Plasmin and LPLare associated with the casein micelles and several enzymes arepresent in the milk serum, many of which are derived from theMFGM, which is shed during the storage of milk.

There has been interest in indigenous milk enzymes sincelactoperoxidase was discovered in 1881. Although present atrelatively low levels, the indigenous enzymes are significant forseveral reasons:

Nutritional and Protective Some indigenous enzymes havea protective effect on the neonate or perhaps on the mammarygland of the mother, for example lysozyme, lactoperoxidase,superoxide dismutase and xanthine oxidoreductase. Many ofthe enzymes have the potential to participate in digestion butprobably the only enzyme significant in this regard is bile saltsstimulated lipase (carboxyl ester hydrolase, CEH) which is be-lieved to make a significant contribution to lipolysis; human milkis particularly rich in CEH but it is also present in the milk ofseveral other species.

Technological

� Plasmin causes proteolysis in milk and some dairy prod-ucts; it may be responsible for age gelation in UHT milkand contributes to proteolysis in cheese during ripening,especially in varieties that are cooked at a high temperature,in which the coagulant is extensively or completely dena-tured, for example Emmental, Parmesan and Mozzarella.

� LPL may cause hydrolytic rancidity in milk and butter butcontributes positively to cheese ripening, especially thatmade from raw milk.

� Acid phosphatase can dephosphorylate casein and mod-ify its functional properties; it may contribute to cheeseripening.

� Xanthine oxidoreductase is a very potent pro-oxidant andmay cause oxidative rancidity in milk; it reduces nitrate,

used to control the growth of clostridia in several cheesevarieties, to nitrite.

� Lactoperoxidase is a very effective bacteriocidal agent inthe presence of a low level of H2O2 and SCN− and is ex-ploited for the cold-sterilisation of milk.

Indices of Milk Quality and History

� The standard assay to assess the adequacy of HTST pas-teurisation is the inactivation of alkaline phosphatase. Pro-posed assays for super-pasteurisation of milk are based onthe inactivation of γ -glutamyltranspeptidase or lactoperoxi-dase.

� The concentration/activity of several enzymes in milkincreases during mastitic infection and some have beenused as indices of this condition, for example catalase, acidphosphatase and especially N-acetylglucosaminidase.

Antibacterial

� Milk contains several bactericidal agents, two of which arethe enzymes, lysozyme and lactoperoxidase.

MILK SALTSMilk contains six inorganic elements, Ca, Mg, Na, K, Cl and P, atsubstantial concentrations (macroelements) and about 20 moreat trace levels (microelements). These elements are referred to asmilk salts or, incorrectly, as minerals. In addition, milk containscitrate that interacts with and affects the state of inorganic salts.Although they are relatively minor constituents, the milk saltsof milk have major effects on its technological and biologicalproperties and there have been several reviews, including Holt(1997) and Lucey and Horne (2009).

When milk is heated in a muffle furnace at 500◦C for approx-imately 5 hours, an ash derived mainly from the inorganic saltsof milk and representing approximately 0.7%, w/w, of the milk,remains. However, the elements are changed from their origi-nal forms to oxides or carbonates and the ash contains P andS derived from caseins, lipids, sugar phosphates or high-energyphosphates. The organic salts, the most important of which iscitrate, are oxidised and lost during ashing; some volatile met-als, for example sodium, are partially lost. Thus, ash does notaccurately represent the salts of milk. However, the principalinorganic and organic ions in milk can be determined directlyby potentiometric, spectrophotometric or other methods. Thetypical concentrations of the principal elements, often referredto as macro-elements, are shown in Table 24.3. Considerablevariability occurs, due, in part, to use of poor analytical methodsand/or to samples from cows in very early or late lactation orsuffering from mastitis. The micro-elements are very importantfrom a nutritional viewpoint, some, for example Fe and Cu, arevery potent lipid pro-oxidants and some, for example Fe, Mo,Zn, are enzyme co-factors.

Some of the salts in milk are fully soluble but others, especiallycalcium phosphate, exceed their solubility under the conditionsin milk and occur partly in the colloidal state, associated with the

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460 Part 4: Milk

Table 24.3. Distribution of Organic and Inorganic Ions Between Soluble and ColloidalPhases of Bovine Milk

Soluble

SpeciesConcentration

(mg/L) % Form Colloidal (%)

Sodium 500 92 Ionised 8Potassium 1450 92 Ionised 8Chloride 1200 100 Ionised _Sulphate 100 100 Ionised _Phosphate 750 43 10% bound to Ca+2 and Mg+2 57

51% H2PO4−

39% HPO42−

Citrate 1750 94 84% bound to Ca and Mg 614% Citr3−

1% HCitr2−

Calcium 1200 34 35% Ca2+ 6655% bound to citrate10% bound to phosphate

Magnesium 130 67 Probably similar to calcium 33

Source: Modified from Fox and McSweeney 1998.

casein micelles; these salts are referred to as CCP, although somemagnesium, citrate and traces of other elements are also presentin the micelles. The typical distribution of the principal organicand inorganic ions between the soluble and colloidal phasesis summarised in Table 24.3. The actual form of the principalspecies can be determined or calculated after making certainassumptions; typical values are shown in Table 24.3. Many ofthe micro-elements are bound by proteins, including enzymes,some of which are present in the MFGM.

The precise nature and structure of CCP are uncertain. It isassociated with the caseins, probably via the casein phosphateresidues; it probably exists as nanocrystals that include PO4

residues of casein. The simplest stoichiometry is Ca3(PO4)2 butspectroscopic data suggest that CaHPO4 is the most likely form.CCP plays a major integrating role in the casein micelles, asdiscussed earlier.

The solubility and ionisation status of many of the principalionic species are interrelated, especially H+, Ca2+, PO4

3− andcitrate3−. These relationships have major effects on the stabil-ity of the caseinate system and consequently on the processingproperties of milk. The status of various species in milk can bemodified by adding certain salts to milk, for example [Ca2+] byPO4

3− or citrate3−; addition of CaCl2 to milk affects the distri-bution and ionisation status of calcium and phosphate and thepH of milk.

The distribution of species between the soluble and colloidalphases is strongly affected by pH and temperature. As the pHis reduced, CCP dissolves and is completely soluble < ca. pH4.9; the reverse occurs when the pH is increased. These pH-dependent shifts mean that acid-precipitated products, for ex-ample acid casein and acid-coagulated cheeses, have a very lowconcentration of Ca.

The solubility of calcium phosphate decreases as the temper-ature is increased. Consequently, soluble calcium phosphate istransferred to the colloidal phase, with the release of H+ and adecrease in pH:

CaHPO4/Ca(H2PO4)2 ↔ Ca3(PO4)2 + 3H+

These changes are quite substantial, but are at least partiallyreversible on cooling.

Since milk is supersaturated with calcium phosphate, concen-tration of milk by evaporation of water increases the degree ofsuper-saturation and the transfer of soluble calcium phosphate tothe colloidal state, with the concomitant release of H+. Dilutionhas the opposite effect. The concentration of salts in milk maybe modified by electrodialysis, ion exchangers or addition ofchelators or selected salts; such operations are practised widely,for example for sterilised milks, processed cheese or nutritionalproducts, such as infant formulae.

Milk salts equilibria are also shifted on freezing; as pure waterfreezes, the concentrations of solutes in unfrozen liquid are in-creased. Soluble calcium phosphate precipitates as Ca3(PO4)2,releasing H+ (the pH may decrease to 5.8). The crystallisationof lactose as a monohydrate aggravates the situation by reducingthe amount of solvent water.

There are substantial changes in the concentrations of themacroelements in milk during lactation, especially at the begin-ning and end of lactation and during mastitic infection (see Whiteand Davies 1958, Keogh et al. 1982, O’Keeffe 1984, O’Brienet al. 1999a). Changes in the concentration of some of the saltsin milk, especially calcium phosphate and citrate, have majoreffects on the physico-chemical properties of the casein systemand on the processability of milk, especially rennet coagulabilityand related properties and heat stability.

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Table 24.4. Typical Concentrations of Vitamins in Milkand Proportion of Recommended Daily Allowance(RDA) Supplied by 1 L Milk

VitaminAverage Level

in 1 L MilkRDA (%)

in 1 L Milk

A 380 rational equivalents 38B1 (thiamine) 0.4 mg 33B2 (riboflavin) 1.8 mg 139B3 (niacin) 9.0 niacin equivalents 53B5 (pantothenic acid) 3.5 mg 70B6 (pyridoxine) 0.5 mg 39B7 (biotin) 30 g 100B11 (folic acid) 50 g 13B12 (cobalamin) 4 g 167C 15 mg 25D 0.5 g 10E 1 mg 10K 35 mg 44

Source: Adapted from Schaafma 2002.

VITAMINSMilk contains all the vitamins in sufficient quantities to allownormal growth and maintenance of the neonate. Cow’s milk isa very significant source of vitamins, especially biotin (B7), ri-boflavin (B2) and cobalamine (B12), in the human diet. Typicalconcentrations of all the vitamins and the percentage of recom-mended daily allowance supplied by 1 L of milk are shown inTable 24.4. For specific aspects of vitamins in relation to milkand dairy products, including stability during processing andstorage, the reader is referred to a set of articles in Roginskiet al. (2002) or Fuquay et al. (2011).

In addition to their nutritional significance, four vitamins areof significance for other reasons, which have been discussed inSection ‘Fat-Soluble Vitamins’:

� Vitamin A (retinol), and especially carotenoids, are re-sponsible for the yellow-orange colour of fat-containingproducts made from cows’ milk.

� Vitamin E (tocopherols) is a potent antioxidant.� Vitamin C (ascorbic acid) may act as an antioxidant or pro-

oxidant, depending on its concentration.� Vitamin B2 (riboflavin), which is greenish-yellow, is re-

sponsible for the colour of whey or UF permeate. It co-crystallises with lactose and is responsible for its yellow-ish colour, which may be removed by recrystallisation orbleached by oxidation. Riboflavin acts as a photocatalyst inthe development of light-oxidised flavour in milk, which isdue to the oxidation of methionine (not to the oxidation oflipids).

SUMMARYMilk is a very complex fluid. It contains several hundred molecu-lar species, mostly at trace levels. Most of the micro-constituents

are derived from blood or mammary tissue but most of the macro-constituents are synthesised in the mammary gland and are milk-specific. The constituents of milk may be in true aqueous solution(e.g., lactose and most inorganic salts) or as a colloidal solu-tion (proteins, which may be present as individual molecules oras large aggregates of several thousand molecules, called mi-celles) or as an emulsion (lipids). The macro-constituents canbe fractionated readily and are used as food ingredients. Thenatural function of milk is to supply the neonate with its com-plete nutritional requirements for a period (sometimes, severalmonths) after birth and with many physiologically importantmolecules, including carrier proteins, protective proteins andhormones.

The properties of milk lipids and proteins may be modifiedreadily by biological, biochemical, chemical or physical meansand thus converted into a wide range of dairy products. In thischapter, the chemical and physico-chemical properties of milksugar (lactose), lipids, proteins and inorganic salts are discussed.The technology used to convert milk into a range of food prod-ucts is described in Chapter 25.

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Food Biochemistry and Food Processing (Simpson/Food Biochemistry and Food Processing) || Chemistry and Biochemistry of Milk Constituents