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 Lactose in Fermented Dairy Products The fermentation of lactose to lactic acid, by lactic acid bacteria (LAB) is a critical step in the manufacture of all fermented dairy products (cheese, fermented milks and lactic butter) The fermentation pathways are well established (see Cogan and Hill 1993, Poolman 2002) Lactose is not a limiting factor in the manufacture 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 to consume fermented milks without ill-effects, possibly because LAB produce β-galactosidase and emptying of the stomach is slower than that for fresh milk products, thus, releasing lactose more slowly into the intestine In the manufacture of cheese, most (96–98%) of the lactose is removed in the whey The concentration of lactose in fresh curd depends on its concentration in the milk and on the moisture content of the curd and varies from approximately 1.7%, w/w, in fresh Cheddar curd to approximately 2.4%, w/w, in fresh Camembert The metabolism of residual lactose in the curd to lactic acid has a major effect on the quality of mature cheese (Fox et al 1990, 2000) The resultant lactic acid may remain essentially unchanged in the cheese during ripening (e.g., Cheddar cheese) or may be catabolised to other compounds, for example CO2 and H2 O, by surface mould in Camembert, or to propionic acid, acetic acid, H2 O and CO2 in Emmental-type cheeses Excessive lactic acid in cheese curd may lead to a low pH and a number of defects, such as a strong, acid, harsh taste, an increase in brittleness and a decrease in firmness The pH of full-fat Cheddar is inversely related to the lactose/lactic acid content of the curd Excess residual lactose may also be fermented by heterofermentative lactobacilli, with the production of CO2 leading to an open texture In the manufacture of some cheese varieties, for example Dutch cheese, the curds are washed to reduce the lactose content and thereby regulate the pH of the pressed curd at approximately 5.3 For Emmental, the curd-whey mixture is diluted with water by approximately 20%, again to reduce the lactose content of the curd, maintain the pH at approximately 5.3, and keep the calcium concentration high, which is important for the textural properties of this cheese For Cheddar, the level of lactose, and hence lactic acid, in the curd is not controlled Hence, changes in the concentration of lactose in milk, such as those occurring throughout lactation, can result in marked changes in the quality of such cheeses To overcome seasonal variations in the lactose content of milk, the level of wash water used for Dutch-type cheeses is related to the concentrations of lactose and casein in the milk Ideally, the lactose-to-protein ratio in any particular variety should be standardised, for example by washing the curd, to minimise variations in the concentration of lactic acid, pH and the quality of cheese Oligosaccharides Lactose is the principal sugar in milk but the milk of most, if not all, species also contains oligosaccharides, up to hexasaccharides, derived from lactose (the reducing end of the oligosaccha- 447 rides is lactose and many contain fucose and N-acetylneuraminic acid) About 130 oligosaccharides have been identified in human milk; the milk of elephant, bears and marsupials also contains high levels of oligosaccharides The oligosaccharides are considered to be important sources of certain monosaccharides, especially fucose and N-acetylglucosamine, for neonatal development, especially of the brain (Urashima et al 2001, 2009, 2011) MILK LIPIDS Definition and Variability The lipid fraction of milk is defined as those compounds that are soluble in non-polar solvents (ethyl/petroleum ether or chloroform/methanol) and is comprised mainly of triglycerides (98%), with approximately 1% phospholipids and small amounts of diglycerides, monoglycerides, cholesterol, cholesterol esters and traces of fat-soluble vitamins and other lipids The lipids occur as globules, 0.1–20 µm in diameter, each surrounded by a membrane, the milk fat globule membrane (MFGM), which serves as an emulsifier The concentration of total and individual lipids varies with breed, individual animal, stage of lactation, mastitic infection, plane of nutrition, interval between milking and point during milking when the sample is taken Among the principal dairy breeds, Friesian/Holsteins produce milk with the lowest fat content (∼3.5%) and Jersey/Guernsey the highest (∼6%) The fat content varies considerably throughout lactation; when synchronised calving is practised, the fat content of bulk Friesian milk varies from approximately 3% in early lactation to >4.5% in late lactation Such large variations in lipid content obviously affect the economics of milk production and the composition of milk products, but can be modified readily by natural creaming, centrifugal separation or addition of cream, and hence need not affect product quality Milk lipids also exhibit variability in fatty acid composition and in the size and stability of the globules These variations, especially fatty acid profile, are essentially impossible to standardise and hence are responsible for considerable variations in the rheological properties, colour, chemical stability and nutritional properties of fat-containing dairy products Fatty Acid Profile Ruminant milk fat contains a wider range of fatty acids than any other lipid system – up to 400 fatty acids have been reported in bovine milk fat; the principal fatty acids are the homologous series of saturated fatty acids with an even number of C-atoms, C4:0 –C18:0 , and C18:1 The outstanding features of the fatty acid profile of bovine milk fat are a high concentration of short- and medium -chain acids (ruminant milk fats are the only natural lipids that contain butanoic acid, C4:0 ) and a low concentration of polyunsaturated fatty acids In ruminants, the fatty acids for the synthesis of milk lipids are obtained from triglycerides in chylomicrons in the blood or synthesised de novo in the mammary gland from acetate or β-hydroxybutyrate produced in the rumen The triglycerides in P1: SFK/UKS BLBS102-c24 P2: SFK BLBS102-Simpson March 21, 2012 13:47 448 Trim: 276mm X 219mm Printer Name: Yet to Come Part 4: Milk chylomicrons are derived from the animal’s feed or synthesised in the liver Butanoic acid (C4:0 ) is produced by the reduction of β-hydroxybutyrate, which is synthesised from dietary roughage by bacteria in the rumen and therefore varies substantially with the animal’s diet All C6:0 –C14:0 and 50% of C16:0 are synthesised in the mammary gland via the malonylCoA pathway from acetylCoA produced from acetate synthesised in the rumen Essentially 100% of C18:0 , C18:1 , C18:2 and C18:3 and 50% of C16::0 are derived from blood lipids (chylomicrons) and represent approximately 50% of total fatty acids in ruminant milk fat Unsaturated fatty acids in the animal’s diet are saturated by bacteria in the rumen unless they are protected, for example by encapsulation Mainly due to saturation in the rumen, ruminant milk fats are quite saturated, approximately 65% of the fatty acids in bovine milk fat are saturated, and are considered nutritionally undesirable although not all to an equal extent However, according to Parodi (2009), the case against saturated fatty acids as causative factors for coronary heart disease is not proven and further research is required When milk production is seasonal, for example Australia, New Zealand and Ireland, very significant changes occur in the fatty acid profile of milk fat throughout the production season (see Fox 1995, Fox and McSweeney 1998, 2006) These variations are reflected in the hardness of butter produced from such milk; the spreadability of butter produced in winter is much lower than that of summer butter Owing to the lower degree of unsaturation, winter butter should be less susceptible to lipid oxidation than the more unsaturated summer product but the reverse appears to be the case, probably owing to higher levels of pro-oxidants, for example Cu and Fe, in winter milk Although a ruminant’s diet, especially if grass-based, is rich in polyunsaturated fatty acids (PUFA), these are hydrogenated by bacteria in the rumen and, consequently, ruminant milk fat contains very low levels of PUFAs, for example bovine milk fat contains approximately 2.4% C18:2 compared to approximately 13% in human or porcine milk fat PUFAs are considered to be nutritionally desirable and consequently there has been interest in increasing the PUFA content of bovine milk fat This can be done by feeding encapsulated PUFA-rich lipids or crushed PUFA-rich oil seed to the animal Increasing the PUFA content also reduces the melting point (MP) of the fat and makes butter produced from it more spreadable However, the lower MP fat may have undesirable effects on the rheological properties of cheese, and PUFA-rich dairy products are very susceptible to lipid oxidation Although the technical feasibility of increasing the PUFA content of milk fat by feeding protected PUFA-rich lipids to the cow has been demonstrated, it is not economical to so in most cases Blending milk fat with PUFA-rich or C18:1 -rich vegetable oil appears to be much more viable and is now widely practised commercially acid (CLA) have attracted very considerable attention recently (for review, see Bauman and Lock 2006) CLA is a mixture of eight positional and geometric isomers of linoleic acid, which have a number of health-promoting properties, including anticarcinogenic and anti-atherogenic activities, reduction of the catabolic effects of immune stimulation and the ability to enhance growth and reduce body fat (see Parodi 1999, Yurawecz et al 1999) Of the eight isomers of CLA, only the cis-9, trans11 isomer is biologically active This compound is effective at very low concentrations, 0.1 g/100 g diet Fat-containing foods of ruminant origin, especially milk and dairy products, are the principal sources of dietary CLA, which is produced as an intermediate during the biohydrogenation of linoleic acid by the rumen bacterium, Butyrivibrio fibrisolvens Since CLA is formed from linoleic acid, it is not surprising that the CLA content of milk is affected by diet and season, being highest in summer when cows are on fresh pasture rich in PUFAs (Lock and Garnsworthy 2000, Lawless et al 2000) and is higher in the fat of milk from cows on mountain pasture than on lowland pasture (Collomb et al 2002) The concentration of CLA in milk fat can be increased five to seven folds by increasing the level of dietary linoleic acid, for example by duodenal infusion (Kraft et al 2000) or by feeding a linoleic acid-rich oil, for example sunflower oil (Kelly et al 1998) A number of other lipids may have anticarcinogenic activity, for example sphingomyelin, butanoic acid and ether lipids, but little information is available on these to date (Parodi 1997, 1999) Structure of Milk Triglycerides Glycerol for milk lipid synthesis is obtained in part from hydrolysed blood lipids (free glycerol and monoglycerides), partly from glucose and a little from free blood glycerol Synthesis of triglycerides within the cell is catalysed by enzymes located on the endoplasmic reticulum Esterification of fatty acids is not random (Table 24.1) The concentrations of C4:0 and C18:1 appear to be rate-limiting because of the need to keep the lipid liquid at body temperature Some notable features of the structure are as follows: r Butanoic and hexanoic acids are esterified almost entirely, and octanoic and decanoic acids predominantly, at the sn-3 position r As the chain-length increases up to C16:0 , an increasing proportion is esterified at the sn-2 position; this is more marked for human than for bovine milk fat, especially in the case of palmitic acid (C16:0 ) r Stearic acid (C18:0 ) is esterified mainly at sn-1 r 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: Conjugated Linoleic Acid Linoleic acid (cis, cis 9, 12-octadecadienoic acid) is the principal essential fatty acid and has been the focus of nutritional research for many years However, conjugated isomers of linoleic It affects the MP and hardness of the fat, which can be reduced by randomising the fatty acid distribution Transesterification can be performed by treatment with SnCl2 or enzymatically under certain conditions: increasing 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 Table 24.1 Composition of Fatty Acids (mol% of the Total) Esterified to Each Position of the Triacyl-sn-Glycerols in Bovine or Human Milk Cow Human Fatty Acid sn-1 sn-2 sn-3 sn-1 sn-2 sn-3 4:0 6:0 8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 – – 1.4 1.9 4.9 9.7 34.0 2.8 10.3 30.0 1.7 – – 0.9 0.7 3.0 6.2 17.5 32.3 3.6 9.5 18.9 3.5 – 35.4 12.9 3.6 6.2 0.6 6.4 5.4 1.4 1.2 23.1 2.3 – – – – 0.2 1.3 3.2 16.1 3.6 15.0 46.1 11.0 0.4 – – – 0.2 2.1 7.3 58.2 4.7 3.3 12.7 7.3 0.6 – – – 1.1 5.6 6.9 5.5 7.6 1.8 50.4 15.0 1.7 attention is being focused on the latter as an acceptable means of modifying the harness of butter Pancreatic and many other lipases are specific for the fatty acids at the sn-1 and sn-3 positions Therefore, C4:0 –C8:0 are released rapidly from milk fat; these are water-soluble and are readily absorbed from the intestine Mediumand long-chain acids are absorbed more effectively as 2monoglycerides than as fatty acids; this appears to be quite important for the digestion of lipids by human infants who have limited ability to digest lipids due to the absence of bile salts Infants metabolise human milk fat more efficiently than bovine milk fat, apparently due to the very high proportion of C16:0 esterified at sn-2 in the former The effect of trans-esterification on the digestibility of milk fat by infants merits investigation Short-chain fatty acids (C4:0 –C10:0 ) have a strong aroma and flavour and their release by indigenous lipoprotein lipase (LPL) and microbial lipases cause off-flavours in milk and many dairy products, referred to as hydrolytic rancidity Rheological Properties of Milk Fat The melting characteristics of ruminant milk fat are such that, at low temperatures (e.g., ex-refrigerator), it contains a high proportion of solid fat and has poor spreadability The rheological properties of milk lipids may be modified by fractional crystallisation, for example an effective treatment involves removing the middle MP fraction and blending high- and low-MP fractions Fractional crystallisation is expensive and is practised in industry to only a limited extent; in particular, securing profitable outlets for the middle-MP fraction is a major economic problem Alternatively, the rheological properties of milk fat may be modified by increasing the level of PUFAs through feeding cows with protected PUFA-rich lipids, but this practise is also expensive The melting characteristics of blends of milk fat and veg- 449 etable oils can be easily varied by changing the proportions of the different fats and oils in the blend This procedure is economical and is widely practised commercially; blending also increases the level of nutritionally desirable PUFAs The rheological properties of milk fat-based spreads can also be improved by increasing the moisture content of the product; obviously, this is economical and nutritionally desirable in the sense that the caloric value is reduced, but the resultant product is less microbiologically stable than butter The melting characteristics and rheological properties of milk fat can also be modified by inter- and trans-esterification Chemically-catalysed inter- and trans-esterification are not permitted in the food industry but enzymatic catalysis may be acceptable Lipases capable of such modifications on a commercial scale are available but their use is rather limited Enzymatic trans-esterification allows modification of the nutritional as well as the rheological properties of lipids The nutritional and rheological properties of lipids can also be modified by the use of a desaturase that converts C18:0 to C18:1 (these enzymes are a subject of ongoing research, see hppt://bioinfo.pbi.nrc.ca/covello/rfattyacid.html; and Meesapyodsuk et al 2000) However, this type of enzyme does not seem to be available commercially yet Milk Fat as an Emulsion An emulsion consists of two immiscible, mutually insoluble liquids, usually referred to as oil and water, in which one of the liquids is dispersed as small droplets (globules; the dispersed phase) in the other (the continuous phase) If the oil is the dispersed phase, the emulsion is referred to as an oil-in-water (O/W) emulsion; if water is the dispersed phase, the emulsion is referred to as a water-in-oil (W/O) emulsion The dispersed phase is usually, 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 and separate if they differ in density, as is the case with lipids and water, the density of which are 0.9 and 1.0, respectively; the lipid globules will float to the surface and coalesce Coalescence is prevented by adding a compound which reduces the interfacial tension, γ , between the phases Compounds capable of doing this have an amphipathic structure, that is hydrophobic and hydrophilic regions, for example phospholipids, monoglycerides, diglycerides, proteins, soaps and numerous synthetic compounds, and are known as emulsifiers or detergents The emulsifier forms a layer on the surface of the globules with its hydrophobic region penetrating the oil phase and its hydrophilic region in the aqueous phase An emulsion thus stabilised will cream if left undisturbed, but the globules remain discrete and can be redispersed readily by gentle agitation In milk, the lipids exist as an O/W emulsion in which the globules range in size from approximately 0.1 to 20 µm, with a mean of 3–4 µm The mean size of the fat globules is higher in highfat milk than in low-fat milk, for example Jersey compared to Friesian, and decreases with advancing lactation Consequently, the separation of fat from milk is less efficient in winter than in summer, especially when milk production is seasonal, and it P1: SFK/UKS BLBS102-c24 P2: SFK BLBS102-Simpson March 21, 2012 13:47 Trim: 276mm X 219mm 450 Printer Name: Yet to Come Part 4: Milk may not be possible to meet the upper limit for fat content in some products, for example casein, during certain periods Stability of Milk Fat Globules In milk, the emulsifier is the MFGM On the inner side of the MFGM is a layer of unstructured lipoproteins, acquired within the secretory cells as the triglycerides move from the site of synthesis in the rough endoplasmic reticulum (RER) in the basal region of the cell towards the apical membrane The fat globules are excreted from the cells by exocytosis, that is they are pushed through and become surrounded by the apical cell membrane Milk proteins and lactose are excreted from the cell by the reverse process: the proteins are synthesised in the RER and are transported to the Golgi region, where the synthesis of lactose occurs under the control of α-La The milk proteins and lactose are encapsulated in Golgi membrane; the vesicles move towards, and fuse with, the apical cell membrane, open and discharge their contents into the alveolar lumen, leaving the vesicle (Golgi) membrane as part of the apical membrane, thereby replacing the membrane lost on the excretion of fat globules Thus, the outer layer of the MFGM is composed of a trilaminar membrane, consisting of phospholipids and proteins, with a fluid mosaic structure Many of the proteins of the MFGM are strongly hydrophobic and difficult to isolate and characterise Modern proteomic methods have shown that the MFGM contains about 100 proteins, of which the following are the principal and have been isolated and characterised: butyrophilin (BTN), xanthine dehydrogenase (XDH), acidophilin, PAS (periodic acid Schiff staining) 6/7, CD (cluster of differentiation) 36, fatty acid-binding protein, mucins and 15 (MUC) BTN is a trans-membrane protein that complexes with XDH (located on the inner face of the membrane) that initiates the blebbing of the fat globule through the apical membrane of the cell (in this role, XDH does not act as an enzyme) MUC1, MUC 15, CD 36 and PAS6/7 are heavily glycosylated and are located mainly on the outer surface of the membrane, increasing its hydrophilicity Very considerable progress has been made on the proteins during the past 10 years, which has been reviewed by Mather (2000, 2011) and Keenan and Mather (2006) In human and equine milk, the MUC form long (up to 50 µm) filaments that are lost easily; the filaments probably retard the passage of lipids through the small intestine, thereby improving digestibility, and prevent the adhesion of pathogens The MFGM contains many enzymes that originate mainly from the Golgi apparatus: in fact, most of the indigenous enzymes in milk are concentrated in the MFGM, notable exceptions being plasmin and LPL that are associated with the casein micelles The trilaminar membrane is unstable and is shed during storage, and especially during agitation, into the aqueous phase, where it forms microsomes The stability of the MFGM is critical for many aspects of the milk fat system: r The existence of milk as an emulsion depends on the effectiveness of the MFGM r Damage to the MFGM leads to the formation of nonglobular (free) fat, which may be evident as ‘oiling-off’ on tea or coffee, cream plug or age thickening An elevated level of free fat in whole milk powder reduces its wettability Problems related to, or arising from, free fat are more serious in winter than in summer, probably due to the reduced stability of the MFGM Homogenisation, which replaces the natural MFGM by a layer of proteins from the skim milk phase, principally caseins, eliminates problems caused by free fat r The MFGM protects the lipids in the core of the globule against lipolysis by LPL in the skim milk (adsorbed on the casein micelles) The MFGM may be damaged by agitation, foaming, freezing, for example on bulk tank walls, and especially by homogenization, allowing access for LPL to the core lipids and leading to lipolysis and hydrolytic rancidity This is potentially a major problem in the dairy industry unless milking machines, especially pipeline milking installations, are properly installed and serviced r The MFGM appears to be less stable in winter/late lactation than in summer/mid lactation; therefore, hydrolytic rancidity is more likely to be a problem in winter than in summer An aggravating factor is that less milk is usually produced in winter than in summer, especially in seasonal milk production systems, which leads to greater agitation and air incorporation during milking and, consequently, a greater risk of damage to the MFGM Creaming Since the specific gravity of lipids and skim milk is 0.9 and 1.036, respectively, the fat globules in milk held under quiescent conditions will rise to the surface under the influence of gravity, a process referred to as creaming The rate of creaming, V, of fat globules is given by Stoke’s equation: V = 2r (ρ − ρ )g 9η where r = radius of the fat globules ρ = specific gravity of skim milk ρ = specific gravity of the fat globules g = acceleration due to gravity η = viscosity of milk The typical values of r, ρ , ρ and η suggest that a cream layer should form in milk after approximately 60 hours but milk creams in approximately 30 minutes The rapid rate of creaming is 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 is cooled (hence, they are called cryoglobulins) Considering the effect of globule size (r) on the rate of creaming, large globules rise faster than smaller ones and collide with, and adhere to, smaller globules, an effect promoted by cryoglobulins Owing to the larger value of r, the clusters of globules rise faster than individual globules, and therefore the creaming process 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 accelerates as the globules rise and clump Ovine, caprine and buffalo milk not contain cryoglobulins and therefore cream much more slowly than bovine milk In the past, creaming was a very important physicochemical property of milk: r The cream layer served as an index of fat content and hence of quality to the consumer r Creaming was the traditional method for preparing fat (cream) from milk for use in the manufacture of butter Its significance in this respect declined after the development of the mechanical separator by Gustav de Laval in 1878 but natural creaming is still used to adjust the fat content of milk for some cheese varieties, for example ParmigianoReggiano A high proportion (∼90%) of the bacteria in milk 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 homogenised, that is subjected to a high-shear pressure that reduces the size of the fat globules (average diameter