P1: SFK/UKS BLBS102-c24 P2: SFK BLBS102-Simpson March 21, 2012 13:47 Trim: 276mm X 219mm Printer Name: Yet to Come 24 Chemistry and Biochemistry of Milk Constituents P.F Fox and A.L Kelly Introduction Saccharides Lactose Introduction Chemical and Physico-Chemical Properties of Lactose Food Applications of Lactose Lactose Derivatives Nutritional Aspects of Lactose Lactose in Fermented Dairy Products Oligosaccharides Milk Lipids Definition and Variability Fatty Acid Profile Conjugated Linoleic Acid Structure of Milk Triglycerides Rheological Properties of Milk Fat Milk Fat as an Emulsion Stability of Milk Fat Globules Creaming Homogenisation of Milk Lipid Oxidation Fat-Soluble Vitamins Milk Proteins Introduction Heterogeneity of Milk Proteins Molecular Properties of Milk Proteins Interspecies Comparison of Milk Proteins Casein Micelles Minor Proteins Immunoglobulins Blood Serum Albumin Metal-Binding Proteins β2 -Microglobulin Osteopontin Proteose Peptone Vitamin-Binding Proteins Angiogenins Kininogen Glycoproteins Proteins in the Milk Fat Globule Membrane Growth Factors Milk Protein-Derived Bioactive Peptides Indigenous Milk Enzymes Nutritional and Protective Technological Indices of Milk Quality and History Antibacterial Milk Salts Vitamins Summary References Abstract: Mammalian milk is a highly complex physicochemical system, containing colloidal proteins (the casein micelle) and emulsified lipids, as well as dissolved lactose, minerals, vitamins and minerals The properties of milk, and the products made or isolated from milk, are very much determined by the properties of its constituents These properties are particularly relevant when milk is processed, for example through denaturation of proteins, oxidation, hydrolysis of proteins and lipids, or Maillard reactions involving lactose In this chapter, the principal families of milk constituents, and their most significant characteristics, are described INTRODUCTION Milk is a fluid secreted by female mammals, of which there are approximately 4500 species, to meet the complete nutritional, and some of the physiological, requirements of the neonate of the species Because nutritional requirements are species-specific and change as the neonate matures, it is not surprising that the composition of milk shows very large interspecies differences, for example the concentrations of fat, protein and lactose range from 1% to 50%, 1% to 20% and 0% to 10%, respectively 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 Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H Hui C 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc 442 P1: SFK/UKS BLBS102-c24 P2: SFK BLBS102-Simpson March 21, 2012 13:47 Trim: 276mm X 219mm Printer Name: Yet to Come 24 Chemistry and Biochemistry of Milk Constituents the minor constituents of milk are even greater than those of the macro constituents The composition of milk also changes markedly during lactation, reflecting the changing nutritional requirements of the neonate and during mastitis and physiological stress In this chapter, the typical characteristics of the principal, and some of the minor, constituents of bovine milk will be 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 Park and Haenlein (2006) and Fuquay et al (2011); the latter includes articles on monotremes and marsupials, marine mammals and primates Sheep and goats were domesticated around 8000 bc and their milk has been used by humans since However, cattle, especially 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 limited geographical regions with specific cultural and/or climatic conditions Milk is a very flexible raw material; several thousand dairy products are produced around the world in a great diversity of flavours and forms, including about 1400 varieties/variants of cheese The principal dairy products and the percentage of milk used in their production are: liquid (beverage) milk, approximately 40%; cheese, approximately 35%; butter, approximately 32%; whole milk powder, approximately 6%; skimmed milk powder, approximately 9%; concentrated milk products approximately 2%; fermented milk products, approximately 2%; casein, approximately 2% and infant formulae, approximately 0.3% The flexibility of milk as a raw material is a result of the properties, many of them unique, of its principal constituents; many of these are very easily isolated, permitting the production of valuable food ingredients Milk is free of off-flavours, pigments and toxins, which greatly facilitates its use as a food or as a raw material for food production The processability and functionality of milk and milk products are determined by the chemical and physicochemical properties of its principal constituents, that is lactose, lipids, proteins, and salts, which will be described in this chapter The exploitation and significance of the chemical and physico-chemical properties of milk constituents in the production and properties of the principal groups of dairy foods, that is liquid milk products, cheese, butter, fermented milks, functional milk proteins and lactose will be described in Chapter 25 Many of the principal problems encountered during the processing of milk are caused by variability in the concentrations and properties of the principal constituents arising from several factors, including breed, individuality 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, Australia and Ireland, to avail of cheap grass as the principal component of the cow’s diet, has a very marked effect on the composition and properties of milk (see O’Brien et al 1999a, b, c, Mehra et al 1999) However, much of the variability can be offset by standardising the composition of milk using various methods 443 (e.g., centrifugation, ultrafiltration or supplementation) or by modifying the process technology The chemical and physico-chemical properties of the principal 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) SACCHARIDES Lactose Introduction Lactose is a reducing disaccharide comprising glucose and galactose, linked by a β1–4-O-glycosidic bond (Fig 24.1) It is unique to milk and is synthesised in the mammary gland from glucose transported from the blood; one molecule of glucose is epimerised to galactose, as UDP-galactose (Gal), via the Leloir pathway and is condensed with a second molecule of glucose by a two-component enzyme, lactose synthetase Component A is a general UDP-galactosyl transferase (UDP-GT; EC 2.4.1.2.2), which transfers galactose from UDP-Gal to a range of sugars, peptides or lipids Component B is the whey protein, α-lactalbumin (α-La), in the presence of which, the K M of UDP-GT for glucose is reduced 1000-fold and lactose is the principal product synthesised There is a good correlation between the concentrations of lactose and α-La in milk Lactose is responsible for approximately 50% of the osmotic pressure of milk, which is equal to that of blood and varies little; therefore, the concentration of lactose in milk is tightly controlled and is independent of breed, individuality and nutritional factors, but decreases as lactation advances and especially during mastitis, in both cases due to the influx of NaCl from the blood The physiological function of α-La is probably to control the synthesis of lactose, and thus maintain the osmotic pressure of milk relatively constant The concentration of lactose in milk ranges from approximately 0, for some species of seal, to approximately 10% in the milk of some monkeys The concentration of lactose in the milk of the principal dairy species is quite similar (cow, 4.8%; buffalo, 4.3%; sheep, 4.6%; goat, 4.9%; camel, 5.1%), exceptions are the horse (6.1%), donkey (6.9%) and reindeer (2.5%); human milk contains about 7.0% lactose The lactose content of bulk herd milk from randomly calved cows varies little throughout the year but differences can be quite large when calving of cows is synchronised, for example in Ireland, the level of lactose in creamery milk varies from approximately 4.8% in May to approximately 4.2% in October Chemical and Physico-Chemical Properties of Lactose Among sugars, lactose has a number of distinctive characteristics, some of which cause problems in milk products during processing and storage; however, some of its characteristics are exploited to advantage P1: SFK/UKS BLBS102-c24 P2: SFK BLBS102-Simpson March 21, 2012 13:47 Trim: 276mm X 219mm Printer Name: Yet to Come 444 Part 4: Milk (1→ 4) CH2OH HO OH H H β H CH2OH O H H O O H OH H OH Anomeric carbon H O H OH H α OH H OH OH Lactose β H O O-β-D-Galactopyranosyl-(1→4)-α-D-Glucopyranose: α-Lactose O α O OH Galactose β (1→ 4) Glucose O OH O O O-β-D-Galactopyranosyl-(1→4)-β-D-Glucopyranose: β-Lactose β Figure 24.1 Structures of lactose The functional aldehyde group at the C-1 position of the glucose moiety exists mainly in the hemiacetal form, forming a cyclic structure and, consequently, C-1 is a chiral, asymmetric, carbon Therefore, like all reducing sugars, lactose can exist as two anomers, α and β, which have markedly different properties From a functional viewpoint, the most important of these properties are differences in solubility and crystallisation characteristics between the isomers; α-lactose crystallises as a monohydrate, while crystals of β-lactose are anhydrous Since crystalline α-lactose contains 5% H2 O, the yield of this anomer is higher than that of β-lactose and this must be considered when expressing the concentration of lactose The solubility of α- and β-lactose in water at 20◦ C is approximately g/100 mL and 50 g/100 mL, respectively However, the solubility of α-lactose is much more temperature-dependent than that of β-lactose and the solubility curves intersect at approximately 93.5◦ C (see Fox and McSweeney 1998) At equilibrium in aqueous solution, lactose exists as a mixture of α and β anomers in the approximate ratio of 37:63 When an excess of α-lactose is added to water, approximately g/100 mL dissolve immediately, some of which mutarotates to give an α:β ratio of 37:63, leaving the solution unsaturated with respect to both α- and β-lactose Further α-lactose then dissolves, some of which mutarotates to β-lactose Solubilisation and mutarotation continue until two conditions exist, that is approximately g of dissolved α-lactose/100 mL of water and an α :β ratio of 37:63, giving a final solubility of approximately 18.2 g/100 mL When β-lactose is added to water, approximately 50 g/100 mL dissolve initially but approximately 18.5 g of this mutarotates to α-lactose, which then exceeds its solubility and some lactose crystallises This upsets the α:β ratio and more β-lactose mutarotates to α-lactose, which crystallises Mutarotation of β-lactose and crystallisation of α-lactose continue until approximately g and 11.2 g of α- and β-lactose, respectively, are in solution Although lactose has low solubility in comparison with other sugars, once dissolved, it crystallises with difficulty and forms supersaturated solutions Highly supersaturated solutions (greater than twofold saturated) crystallise spontaneously but if the solution is only slightly supersaturated (one to twofold), lactose crystallises slowly and forms large, sharp, tomahawkshaped crystals of α-lactose If the dimensions of the crystals exceed approximately 15 µm, they are detectable on the tongue and palate as a sandy texture Crystals of β-lactose are smaller and monoclinical in shape In the metastable zone, crystallisation of lactose is induced by seeding with finely powdered lactose (Fig 24.2) Since the solubility of α-lactose is lower than that of the β anomer below 93.5◦ C, α-lactose is the normal commercial form When concentrated milk is spray-dried, the lactose does not have sufficient time to crystallise during drying and an amorphous glass is formed If the moisture content of the powder is kept low ( indirect ultra-high temperature (UHT) > direct UHT > high-temperature short-time (HTST) pasteurisation Lactulose is not hydrolysed by intestinal β-galactosidase and enters the large intestine, where it promotes the growth of Bifidobacterium spp It also has a laxative effect and is widely used for this purpose; more than 20,000 tonnes are produced annually r Glucose-galactose syrups, produced by acid or enzymatic (β-galactosidase) hydrolysis (see Chapter 25): The technology for the production of such hydrolysates has been developed but the product is not cost-competitive with other sugars (sucrose, glucose, glucose–fructose) r Tagatose: Is the keto analogue of galactose It occurs at a low level in the gum of the evergreen tree, Sterculia setigera, and in severely heated milk and stored milk powder It can be produced by treating β-galactosidase–hydrolysed lactose with a weak alkali, for example Ca(OH)2 , which converts the galactose to tagatose, which can be purified by demineralisation and chromatography Tagatose is nearly as sweet as sucrose, has a good quality taste and enhances the flavour of other sweeteners It is absorbed poorly from the small intestine, serves as a probiotic and has little effect on blood glucose; it is fermented in the lower intestine to volatile short-chain acids that can be absorbed but provide only approximately 35% of the energy derived from sugars catabolised via the normal route Tagatose has GRAS status and is produced commercially by SweetGredients, a company formed by Arla Foods and Nordzuker (Denmark) r Galacto-oligsaccharides: β-Galactosidase has transferase as well as hydrolytic activity and under certain conditions, the former predominates, leading to the formation of galacto-oligosaccharides containing up to six monosaccharides linked by glycosidic bonds that are not hydrolysed by the enzymes secreted by the human small intestine The undigested oligosaccharides enter the large intestine, where they have bifidogenic properties and are considered to have promising food applications These oligosaccharides are quite distinct from the naturally occurring oligosaccharides referred to in Section ‘Oligosaccharides’(see Ganzle 2011) r Ethanol: Is produced commercially by the fermentation of lactose by Kluyveromyces lactis Depending on local legislation, the ethanol may be used in alcoholic drinks, which are profitable The current interest in renewable energy sources has created vast opportunities for lactose-derived ethanol but its commercial success will depend on local taxation policy Other derivatives that have limited but potentially important applications include lactitol, lactobionic acid, lactic acid, acetic acid, propionic acid, lactosyl urea and single-cell proteins Most of these derivatives can be produced by fermentation of sucrose, which is cheaper than lactose, or by chemical synthesis However, lactitol and lactobionic acids are derived specifically from lactose and may have economic potential Lactitol is a synthetic sugar alcohol produced by reduction of lactose; it is not metabolised by higher animals but is relatively sweet, and hence has potential for use as a non-calorific sweetener It has also been reported that lactitol reduces blood cholesterol level, reduces sucrose absorption and is anti-carcinogenic Lactobionic acid has a sweet taste, which is unusual for an acid and therefore should 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 a deficiency of intestinal β-galactosidase, which is rare in infants but common in adults, except North-Western Europeans and a few African tribes Since humans are unable to absorb disaccharides, including lactose, from the small intestine, unhydrolysed lactose enters the large intestine where it is fermented by bacteria, leading to flatulence and cramp, and to the absorption of water from the intestinal mucosa, causing diarrhoea These conditions cause discomfort and perhaps death Lactose intolerance has been studied extensively since its discovery in 1959 and the literature has been reviewed regularly (see Ingram and Swallow 2009) The problems caused by lactose intolerance can be avoided by: r excluding lactose-containing products from the diet, which is the normal practice in regions of the world where lactose intolerance is widespread; r removing lactose from milk, for example by ultrafiltration; r hydrolysis of the lactose by adding β-galactosidase at the factory or in the home The technology for the production of lactose-hydrolysed milk and dairy products is well developed but is of commercial interest mainly for lactoseintolerant individuals in Europe or North America Because the consumption of milk is very limited in South-East Asia, the use of β-galactosidase is of little interest, although lactose intolerance is widespread Galactosemia is caused by the inability to catabolise galactose, owing to a deficiency of either of two enzymes, galactokinase or galactose-1P uridyltransferase (see Flynn 2003) A deficiency of galactokinase leads to the accumulation of galactose that is catabolised via alternative routes, one of which leads to the accumulation of galactitol in various tissues, including the eye, where it causes cataracts over a period of about 20 years A deficiency of galactose-1P uridyltransferase leads to abnormalities in membranes of the brain and to mental retardation unless galactose is excluded from the diet within a few weeks of birth Both forms of galactosemia occur at a frequency of per approximately 50,000 births ... textbooks: Walstra and Jenness (1 984 ), Wong et al (1 988 ), Fox (1992, 1995, 1997, 2003a), Fox and McSweeney (19 98, 2003, 2006), Walstra et al (1999, 2005) and McSweeney and Fox (2009) SACCHARIDES Lactose... 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 limited geographical regions with specific cultural and/ or... of valuable food ingredients Milk is free of off-flavours, pigments and toxins, which greatly facilitates its use as a food or as a raw material for food production The processability and functionality