Dairy Science & Technology · Dairy Chemistry · also Chemistry of Milk Fat, Dairy Chemistry III
Milk fat composition, the fat globule, crystallisation, oxidation and lipolysis: the chemistry behind butter, ghee, cream and off-flavours.
In the exams: ICAR AICE subject 34 Unit 3; ICAR AIEEA-PG group 16 (spoilage of ghee and antioxidants)
Milk fat is the most valuable and the most variable major constituent of milk. It carries flavour, the fat-soluble vitamins and much of the energy of milk; it determines the price paid to farmers in most payment schemes; and its chemistry decides how butter spreads, how ghee keeps, how cream whips and why milk turns rancid or tastes of cardboard. This subject covers the lipid classes of milk, the fatty acid profile and what changes it, how the mammary gland makes fat, conjugated linoleic acid, the fat globule membrane, the crystallisation, polymorphism, melting and fractionation of milk fat, auto-oxidation and the antioxidants that slow it, lipolysis, cholesterol, and the fat-soluble vitamins.
Gross fat content, fat globule size and the physical properties of milk are treated in the subject Milk Composition and Physical Properties, and the proteins of the fat globule membrane and the enzyme lipoprotein lipase in Milk Proteins and Enzymes; this manual refers to those subjects rather than repeating them. Values quoted are typical textbook figures for cow milk fat. Fatty acid composition varies with diet, breed, season and stage of lactation, and legal standards for butter and ghee, such as Reichert-Meissl value or permitted antioxidants, differ between countries and are given here only in principle.
The subject is examined as Unit 3 of ICAR AICE-JRF/SRF subject 34 (Dairy Chemistry) and within group 16 of ICAR AIEEA-PG, which asks specifically about the spoilage of ghee and antioxidants. ASRB NET Dairy Chemistry, food analyst examinations (which test the constants of ghee and butter and the detection of adulteration with other fats) and GATE food technology (lipid chemistry and oxidation) cover parts of it. Expect recall of fatty acid figures and lipid constants, mechanisms of oxidation and lipolysis, polymorphic forms, and calculations of peroxide value, saponification value and vitamin A activity.
Milk lipids are the substances extracted from milk by fat solvents such as diethyl ether and petroleum ether, as in the Röse-Gottlieb method. In cow milk about 97 to 98 per cent of the lipid is triglyceride (triacylglycerol), three fatty acids esterified to glycerol. The rest is diglycerides (about 0.3 to 0.6 per cent of the lipid), monoglycerides (a trace), free fatty acids (about 0.1 to 0.4 per cent in fresh milk), phospholipids (about 0.8 per cent) and sterols (about 0.3 per cent), with small amounts of carotenoids, fat-soluble vitamins, hydrocarbons and flavour compounds such as lactones and methyl ketones. Partial glycerides and free fatty acids rise when lipolysis occurs, so their level is an index of freshness. Because milk fat contains several hundred different fatty acids, the number of possible triglycerides is enormous, and no single triglyceride makes up more than a few per cent of the total. The distribution of fatty acids among the three positions of glycerol is not random. Short-chain acids, butyric and caproic, are esterified almost entirely at the sn-3 position; palmitic acid is concentrated at sn-1 and sn-2; and stearic and oleic acids are mainly at sn-1 and sn-3. This positional distribution matters for melting behaviour, for the release of short-chain acids by lipases that attack the outer positions, and for digestion, because palmitic acid at sn-2 is absorbed well as a monoglyceride. Milk fat has a lower average triglyceride molecular mass than most vegetable oils because of its short-chain acids, which gives it a high saponification value.
Phospholipids are polar lipids containing phosphate. In cow milk the main ones are phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, each roughly a quarter to a third of the total, with smaller amounts of phosphatidylserine and phosphatidylinositol; glycolipids such as cerebrosides and gangliosides are also present. About 60 per cent or more of the phospholipid of milk is in the fat globule membrane and the rest in membrane fragments in the skim milk. Because phospholipids are amphiphilic, they are surface active and help stabilise the fat emulsion; because they are rich in polyunsaturated fatty acids, they are the first lipids to oxidise in milk, which is why oxidised flavour starts in the membrane. Processes that disrupt the membrane, such as churning and the separation of butter oil, concentrate phospholipids in buttermilk and butter serum, which are used as ingredients rich in milk polar lipids. Sterols in milk fat are mainly cholesterol, about 95 per cent of the sterol fraction, present mostly in the free form and partly as cholesteryl esters; milk contains roughly 10 to 15 milligrams of cholesterol per 100 grams, and butter roughly 200 to 250 milligrams per 100 grams, varying with fat content. Plant sterols such as beta-sitosterol are absent or present only in traces in milk fat but are abundant in vegetable oils, so the detection of phytosterols, for example as phytosterol acetates, is a classic test for adulteration of ghee and butter with vegetable fat.
More than 400 fatty acids have been identified in milk fat, but about a dozen make up most of it. Typical proportions by weight in cow milk fat are palmitic acid (C16:0) about 25 to 30 per cent, oleic acid (C18:1) about 20 to 25 per cent, myristic acid (C14:0) and stearic acid (C18:0) about 10 to 12 per cent each, lauric acid (C12:0) about 3 per cent, the short-chain acids butyric (C4:0) about 3 to 4 per cent, caproic (C6:0) about 2 per cent, caprylic (C8:0) about 1 per cent and capric (C10:0) about 2 to 3 per cent, and the polyunsaturated linoleic (C18:2) and alpha-linolenic (C18:3) acids together only about 2 to 4 per cent. Milk fat is therefore highly saturated, about 65 to 70 per cent saturated fatty acids, 25 to 30 per cent monounsaturated and only a few per cent polyunsaturated. Several features are typical of ruminant milk fat. Butyric acid occurs in no other common edible fat, and because butyric acid is volatile and water soluble when liberated, the Reichert-Meissl value, which measures steam-volatile water-soluble fatty acids, is high for milk fat and low for other fats; the Polenske value measures steam-volatile insoluble acids, mainly caprylic and capric, and is high in coconut oil. Trans fatty acids, about 2 to 5 per cent, are formed by incomplete biohydrogenation in the rumen, the main one being vaccenic acid (trans-11 C18:1). Odd-chain and branched-chain fatty acids (pentadecanoic, heptadecanoic and iso and anteiso forms) come from rumen bacteria. The iodine value of milk fat, a measure of unsaturation, is commonly about 25 to 40, far below that of most vegetable oils.
Diet has the largest effect. Fresh pasture is rich in alpha-linolenic acid, and cows grazing it produce milk fat with more unsaturated fatty acids, more vaccenic and conjugated linoleic acid and more carotene, so pasture butter is softer and more yellow than butter from cows fed hay and concentrates. Feeding oilseeds or oils increases unsaturated C18 acids and decreases the short and medium-chain acids made in the gland, while rumen-protected fats, coated or calcium soaps, allow more unsaturated fatty acids to reach the udder without disturbing the rumen. Diets low in fibre or high in rapidly fermentable starch cause milk fat depression, with a fall in total fat and a shift in its composition, linked to particular biohydrogenation intermediates as described in chapter 4. Stage of lactation matters: in early lactation, when the cow is in negative energy balance and mobilises body fat, milk fat contains more long-chain acids, especially oleic and stearic acid, and fewer short-chain acids; the proportion of de novo synthesised acids rises as lactation proceeds. Season acts mainly through feed. Breed and individual genetics affect the activity of enzymes such as the delta-9 desaturase, so the ratio of oleic to stearic acid differs between cows. Species differ too: goat and sheep milk fat contain more caproic, caprylic and capric acids, and buffalo milk fat has more palmitic and fewer polyunsaturated acids than cow milk fat. These differences change the hardness and spreadability of butter, the grain of ghee, the oxidative stability of the fat and the flavour of products.
The fatty acids of milk fat have two origins. About half, by weight, are synthesised in the mammary epithelial cells (de novo synthesis), and the rest are taken up preformed from the blood. In ruminants the carbon sources for de novo synthesis are acetate and beta-hydroxybutyrate, both produced by fermentation of fibre in the rumen; glucose is used little for fatty acid synthesis in ruminants because the pathway that converts glucose-derived citrate into cytosolic acetyl-CoA is not active, but glucose supplies glycerol-3-phosphate for esterification and NADPH through the pentose phosphate pathway. Acetyl-CoA carboxylase converts acetyl-CoA to malonyl-CoA, the committed step, and fatty acid synthase lengthens the chain by two carbons at a time. Mammary fatty acid synthase releases chains of 4 to 16 carbons, rather than only 16 as in liver, so all of the short and medium-chain acids, C4 to C14, and roughly half of the palmitic acid are made in the gland. The fatty acids of 18 carbons and the other half of palmitic acid come from the blood: from triglycerides of chylomicrons and very-low-density lipoproteins, hydrolysed at the capillary wall by lipoprotein lipase, and from non-esterified fatty acids released from body fat, especially in early lactation. This explains why fibre-rich diets that produce acetate support short-chain acid synthesis, and why dietary oils and body fat mobilisation raise long-chain acids.
Within the mammary cell, the enzyme stearoyl-CoA desaturase, also called delta-9 desaturase, inserts a cis double bond at the ninth carbon of saturated and trans fatty acids. It converts much of the stearic acid taken up from blood into oleic acid, which keeps milk fat liquid at body temperature, and converts vaccenic acid into rumenic acid, the main conjugated linoleic acid of milk. Fatty acids are then esterified to glycerol-3-phosphate on the endoplasmic reticulum through the glycerol phosphate pathway; the choice of acyltransferases gives the non-random positional distribution described in chapter 1. The triglycerides accumulate between the leaflets of the endoplasmic reticulum membrane and bud off as small lipid droplets coated with a monolayer of phospholipid and protein. These droplets grow by fusion as they move towards the apical surface of the cell, where they are progressively enveloped by the apical plasma membrane and pinched off into the alveolar lumen. The globule therefore leaves the cell surrounded by an inner monolayer derived from the endoplasmic reticulum and an outer bilayer derived from the plasma membrane, together forming the tri-layer milk fat globule membrane described in chapter 5, sometimes with a crescent of cytoplasm trapped between them. Fat secretion is regulated by nutrition and hormones; in milk fat depression, mammary expression of the enzymes of de novo synthesis and desaturation falls.
Conjugated linoleic acid (CLA) is a collective name for positional and geometric isomers of octadecadienoic acid (C18:2) in which the two double bonds are conjugated, separated by one single bond, instead of being separated by a methylene group as in ordinary linoleic acid. Ruminant milk and meat fats are the richest natural dietary sources. In milk fat, the cis-9, trans-11 isomer, called rumenic acid, makes up most of the CLA, commonly 75 to 90 per cent. Total CLA in cow milk fat is commonly about 0.3 to 1 per cent and can reach about 2 per cent on fresh pasture or with oilseed supplements. CLA arises in two ways. In the rumen, bacteria biohydrogenate dietary linoleic acid, isomerising it first to rumenic acid, then reducing it to vaccenic acid (trans-11 C18:1) and finally to stearic acid; some rumenic acid and much vaccenic acid escape complete hydrogenation and reach the duodenum. In the mammary gland, the delta-9 desaturase converts vaccenic acid into rumenic acid, and this endogenous synthesis supplies most of the rumenic acid in milk. CLA content therefore rises with any feeding practice that increases dietary linoleic and linolenic acid and the escape of vaccenic acid from the rumen, such as pasture, linseed, sunflower or soybean oil, and with genetics that favour high desaturase activity. Processing has little effect: pasteurisation, cheesemaking and butter making carry CLA into products in proportion to their fat.
Not all CLA isomers behave alike. The trans-10, cis-12 isomer, formed in the rumen when diets low in fibre or high in starch and unsaturated oils shift the pathway of biohydrogenation, is a potent inhibitor of milk fat synthesis: very small amounts reaching the mammary gland reduce the expression of the enzymes of de novo synthesis and desaturation and cause milk fat depression. This explains why rumen conditions, not just the amount of fat fed, govern milk fat yield. Interest in CLA for human health began when animal and cell studies showed that some isomers inhibited chemically induced tumours, and later reported effects on body fat, atherosclerosis and immune function. Results in human trials have been inconsistent, and effects on body composition with supplements have been small; synthetic supplements also contain large amounts of the trans-10, cis-12 isomer, which is not the main form in milk. Regulatory reviews, including those of the European Food Safety Authority, have not accepted health claims for CLA. A dairy technologist should be able to explain how CLA is formed, which isomer predominates in milk, how feeding raises it, and why the health evidence is regarded as unproven. CLA-enriched milk and products are marketed in some countries on the basis of their composition rather than proven health effects.
The size distribution, number and creaming of fat globules are covered in Milk Composition and Physical Properties; this chapter deals with the membrane. The milk fat globule membrane (MFGM) is a thin layer, about 10 to 20 nanometres thick, that makes up roughly 2 to 6 per cent of the mass of the globule. It has the tri-layer structure described in chapter 3: an inner monolayer of phospholipid and protein derived from the endoplasmic reticulum, covering the triglyceride core, and an outer bilayer derived from the apical plasma membrane. The membrane is rich in phospholipids, especially sphingomyelin in the outer bilayer, and in cholesterol, glycolipids and glycoproteins, and it carries the enzymes and proteins listed in Milk Proteins and Enzymes, including butyrophilin, xanthine oxidoreductase, mucins and alkaline phosphatase. In addition, a layer of high-melting triglycerides is thought to lie at the inner surface. The membrane gives the globule a negative surface charge and steric stability, keeps lipoprotein lipase away from the fat, and lowers interfacial tension so that globules do not coalesce. It is also the site of copper binding and of the polyunsaturated phospholipids that oxidise first, which links membrane chemistry directly to flavour defects. Because the outer bilayer is a true cell membrane, the MFGM is the only part of milk that carries such membrane components in quantity, which is why buttermilk and butter serum are studied as nutritional ingredients.
The native membrane is fragile, and much of dairy processing either damages it deliberately or must avoid damaging it. Agitation with air, pumping, foaming and turbulence strip parts of the outer bilayer; cooling and crystallisation of fat inside the globule can make crystals protrude through the membrane; freezing and thawing rupture it. Damage has three main consequences. First, the fat becomes accessible to lipoprotein lipase, causing induced lipolysis and rancid flavour. Second, globules lose stability and undergo coalescence (merging into larger globules) or partial coalescence (clumping held together by fat crystals, without full merging). Partial coalescence is wanted in some products: it builds the fat network that stabilises whipped cream and ice cream, and in churning it leads to butter grains and phase inversion. Third, free fat appears, oiling off in cream liqueurs, coffee cream and powders. Homogenisation replaces the native membrane on the new, much larger surface with adsorbed casein micelles, casein fragments and whey proteins, producing a recombined membrane that is more resistant to coalescence but that also makes fat globules behave like large protein particles, joining the casein network in cheese and yoghurt. Because homogenisation of raw milk exposes fat to lipoprotein lipase, milk is homogenised hot and pasteurised immediately, as noted in Milk Proteins and Enzymes. Measuring free fat, the fat extractable by solvent without disrupting globules, is a routine check on membrane damage in cream and powders.
Milk fat crystallises over a wide temperature range because it is a mixture of thousands of triglycerides with melting points from about minus 40 to plus 40 degrees Celsius. Crystallisation needs nucleation, the formation of a stable cluster of molecules, followed by growth of the crystals. In bulk fat, a single nucleus can seed crystallisation of the whole mass, but in milk and cream the fat is divided into billions of globules, and each globule must nucleate independently. Because few globules contain a catalytic impurity, emulsified fat supercools substantially and crystallises more slowly and at lower temperatures than the same fat in bulk. Rapid cooling produces many small crystals; slow cooling produces fewer, larger crystals, and holding at an intermediate temperature lets high-melting triglycerides crystallise first. Triglycerides crystallise together into mixed crystals (compound crystals or solid solutions) rather than separately, so the crystal composition depends on the cooling history. These principles are used in the physical ripening of cream before churning: by controlling the temperature programme, the manufacturer controls how much fat is solid and in what form, and so controls the hardness and spreadability of butter. The Alnarp method, for example, uses a cold-warm-cold sequence to produce fewer, larger crystals and more liquid fat in the globules, giving softer butter from hard winter fat; the opposite sequence is used for soft summer fat. Ghee develops its granular texture when high-melting triglycerides crystallise slowly during cooling.
Triglycerides can crystallise in more than one crystal form, each with a distinct packing of the fatty acid chains, a property called polymorphism. The three main forms are alpha, beta prime and beta. The alpha form has hexagonal packing of the chains, is the least stable, has the lowest melting point and forms first on rapid cooling. The beta prime form has orthorhombic perpendicular packing, intermediate stability and melting point, and gives small, fine crystals and a smooth texture. The beta form has triclinic parallel packing, is the most stable with the highest melting point, and tends to form large, coarse crystals. Transitions go from less stable to more stable forms (alpha to beta prime to beta), never in reverse except through melting. In milk fat, the alpha form appears on rapid cooling and transforms within minutes to hours into beta prime, which is the predominant stable form in butter and cream. Milk fat rarely transforms to the beta form, because the great diversity of its triglycerides hinders the regular packing that beta requires; this is why milk fat is valued for smooth textures and why it can delay the beta-form transitions that cause bloom in chocolate. Polymorphic forms are identified by X-ray diffraction, using the short spacings characteristic of each subcell, and by differential scanning calorimetry, which shows the melting of each form. In practice, cooling rate, tempering and the presence of high-melting fractions determine which forms appear and how the product feels in the mouth.
The melting behaviour of milk fat is described by its solid fat content (SFC) at different temperatures, measured by pulsed nuclear magnetic resonance, or by differential scanning calorimetry (DSC), which records the heat absorbed as the fat melts. Anhydrous milk fat stabilised at low temperature is roughly half solid at about 0 to 5 degrees Celsius, about a fifth solid near 20 degrees Celsius, only a few per cent solid near 30 degrees Celsius, and fully liquid by about 37 to 40 degrees Celsius; exact values depend on composition and thermal history. A DSC melting curve of milk fat typically shows three broad overlapping peaks, often called the low-, middle- and high-melting fractions. This melting range has practical consequences. Butter taken from a refrigerator is hard because much of its fat is solid; it softens near room temperature and melts completely in the mouth, giving a clean flavour release without waxiness, because milk fat is liquid at body temperature. Winter butter, from fat richer in saturated long-chain acids, is harder than summer pasture butter. Cream at churning temperature must contain the right ratio of solid to liquid fat for efficient butter formation. The melting profile also matters in ice cream, where fat partially solid at freezing temperatures forms a network, and in confectionery and bakery products. Other measurements include the slip melting point, the temperature at which a column of fat in a capillary rises, and the dropping point.
Because milk fat is a mixture, it can be separated into fractions with different melting properties, widening its use. In dry fractionation, the most common commercial method, anhydrous milk fat is melted, cooled slowly to a chosen temperature and held so that high-melting triglycerides crystallise; the crystals (the stearin, or solid fraction) are separated from the liquid (the olein) by vacuum filtration or membrane filter presses. Repeating the process at lower temperatures gives several fractions. No chemicals are added, which keeps the products natural dairy fat. Other methods include solvent fractionation with acetone or hexane, which gives sharper separation but leaves solvent to remove, fractionation with supercritical carbon dioxide, and detergent-assisted fractionation. High-melting fractions are used in puff pastry and laminated doughs, where a firm, plastic fat is needed, and in chocolate, where they improve bloom resistance. Low-melting fractions are used in spreadable butter, which remains spreadable from the refrigerator, and in cold-spread products and as a source of more liquid fat. Fractionation also redistributes cholesterol and flavour: the olein tends to carry more cholesterol and more of the flavour compounds. Milk fat can also be modified by blending with vegetable oils (in dairy blends where law permits), by interesterification, which rearranges fatty acids among triglycerides chemically or with lipases, and by controlled crystallisation and texturisation in butter manufacture. Labelling law restricts what can be called butter, ghee or dairy spread.
Auto-oxidation is the reaction of unsaturated lipids with atmospheric oxygen through a free-radical chain. In initiation, a hydrogen atom is removed from a carbon next to a double bond of an unsaturated fatty acid (RH), forming a lipid radical (R•). Initiation is catalysed by heat, light, traces of metals such as copper and iron, existing hydroperoxides, and singlet oxygen formed by photosensitisers such as riboflavin and chlorophyll. In propagation, the lipid radical reacts very fast with oxygen to form a peroxyl radical (ROO•), which removes hydrogen from another fatty acid, producing a lipid hydroperoxide (ROOH) and a new lipid radical, so one initiation event can oxidise many molecules. Termination occurs when radicals combine into non-radical products. Hydroperoxides are tasteless, but they decompose, especially in the presence of metals, into secondary products: aldehydes such as hexanal, alkenals and alkadienals, ketones, alcohols and hydrocarbons. These have very low flavour thresholds and give the defects recognised in dairy products: oxidised, cardboard or metallic flavour in milk, tallowy flavour in butter and ghee, and fishy flavour in butter, associated with oxidation of phospholipids. The rate of oxidation rises steeply with the number of double bonds: linoleic acid oxidises many times faster than oleic acid and linolenic faster still. In milk, oxidation starts in the polyunsaturated phospholipids of the fat globule membrane, where copper is also bound.
The factors that promote oxidation are oxygen, unsaturation, light, metals, temperature and, in some products, low pH and salt. Copper is the most active metal catalyst in milk, effective at a fraction of a milligram per kilogram, which is why copper and brass were removed from dairy equipment in favour of stainless steel; iron is less active. Light acts through riboflavin-sensitised reactions. Higher temperatures speed oxidation, but frozen products still oxidise slowly. Water activity has a complex effect: oxidation is slowest at intermediate water activity, roughly around the monolayer value, and faster in very dry and in moister foods, which matters for milk powders. Heat treatment that denatures whey proteins and exposes sulfhydryl groups lowers the redox potential and protects milk against oxidation, as noted in Milk Composition and Physical Properties. Oxidation is measured in several ways. The peroxide value (PV), determined by iodometric titration, gives milliequivalents of active oxygen per kilogram of fat and measures primary products: PV equals (sample titre minus blank titre) times normality of thiosulphate times 1,000, divided by grams of fat. If 5 grams of fat need 2.4 millilitres of 0.01 normal thiosulphate and the blank 0.1 millilitre, PV is 4.6 milliequivalents per kilogram. Because hydroperoxides decompose, PV can fall in advanced oxidation, so secondary products are also measured: the thiobarbituric acid (TBA) test for malonaldehyde and related compounds, the anisidine value for aldehydes, conjugated dienes by ultraviolet absorption near 232 nanometres, and hexanal by headspace gas chromatography. Accelerated tests such as the Schaal oven test and instruments that measure the induction period at high temperature with air flow estimate stability. Sensory evaluation remains essential.
Antioxidants delay oxidation in two main ways. Chain-breaking (primary) antioxidants donate a hydrogen atom to peroxyl radicals, converting them to hydroperoxides and forming a stabilised antioxidant radical that does not continue the chain; phenolic compounds such as tocopherols act this way. Preventive (secondary) antioxidants reduce the rate of initiation: metal chelators such as citric acid and phosphates bind copper and iron; singlet-oxygen quenchers such as carotenoids deactivate excited oxygen; and oxygen scavengers and reducing agents remove oxygen. Synergists enhance the effect of primary antioxidants, as citric acid does with phenolic antioxidants and ascorbate does by regenerating tocopherol. Milk contains several natural antioxidants. Alpha-tocopherol (vitamin E) in the fat and membrane is the main chain-breaking antioxidant. Beta-carotene quenches singlet oxygen. Ascorbic acid can act as an antioxidant or, at certain concentrations with copper, as a pro-oxidant. Sulfhydryl groups exposed by heating and products of the Maillard reaction formed during intense heat treatment also protect. Phospholipids can act as synergists in ghee, although they are themselves oxidisable in milk. Feeding cows vitamin E reduces the susceptibility of milk to oxidised flavour, while feeding polyunsaturated oils raises it; this is why the balance between unsaturation and antioxidant status matters when milk fat is modified by diet.
Ghee, the clarified butter fat of South Asia, contains very little moisture, commonly below about 0.5 per cent, so microbial spoilage is rare and its deterioration is mainly chemical: oxidative rancidity giving a tallowy, oxidised flavour, and, if moisture or residual enzymes remain, hydrolytic rancidity. Its keeping quality depends on the method and temperature of clarification, the amount of ghee residue (the browned non-fat solids), contamination with copper and iron, exposure to light and air, storage temperature and packaging. Ghee made at a higher clarification temperature keeps better, because more phospholipids pass into the fat and Maillard reaction products formed in the residue have antioxidant activity; ghee residue itself is a source of natural antioxidants. Packaging in containers that exclude light and oxygen, filling with little headspace or under nitrogen, and avoiding metal contamination all extend shelf life. Synthetic antioxidants are phenolic compounds added in very small amounts: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ) and propyl gallate, often with citric acid as a synergist. Which of these may be used, in which dairy products and at what maximum level, is set by national food law and Codex standards and differs between countries; India's food regulations, for example, permit specified antioxidants in ghee and butter oil within stated limits, while many countries do not permit synthetic antioxidants in butter. Natural tocopherol and rosemary extracts are used where clean labels are wanted. Antioxidants slow oxidation but cannot reverse it, so they must be added to fresh fat.
Lipolysis is the hydrolysis of triglycerides by lipases, releasing free fatty acids and partial glycerides. In milk it is caused by the indigenous lipoprotein lipase, whose properties and activation are described in Milk Proteins and Enzymes, and by microbial lipases. Lipoprotein lipase acts preferentially at the sn-1 and sn-3 positions, so it releases short-chain fatty acids, which are concentrated at sn-3, more than their overall proportion would suggest. Short-chain fatty acids, especially butyric acid, have low flavour thresholds and give a rancid, butyric, soapy or bitter flavour; caproic, caprylic and capric acids give goaty and soapy notes. Free fatty acids also lower the surface tension of milk and impair foaming, which affects milk used for frothed coffee drinks. Microbial lipases come mainly from psychrotrophic bacteria such as Pseudomonas species that grow during cold storage of raw milk. Many of these lipases are very heat-stable and survive pasteurisation and even UHT treatment, so they cause slow lipolysis and rancidity in UHT milk, cream and milk powder during storage although the bacteria themselves are killed. Limiting the growth of psychrotrophs by rapid cooling, clean equipment and short raw-milk storage is therefore the main control. Lipolysis is measured as free fatty acids, by titration of extracted fat (the acid degree value), by copper-soap colorimetric methods or by gas chromatography of individual acids; results are often expressed as per cent oleic acid or as millimoles per 100 grams of fat.
Lipolysis in raw milk is classified by its cause. Spontaneous lipolysis develops in the milk of some cows simply on cooling, without mechanical treatment; it is more frequent in late lactation, in low-yielding animals and on poor-quality feed, and blending such milk with normal milk dilutes it. Induced lipolysis follows damage to the fat globule membrane or activation of the enzyme: agitation and foaming, especially from air leaks in milking pipelines, excessive pumping and turbulence; homogenisation of raw milk; temperature activation, in which milk cooled to about 5 degrees Celsius is warmed to about 30 degrees Celsius and cooled again; freezing and thawing; and mixing raw with homogenised milk. Prevention combines good milking-system design, avoidance of air admission and foaming, fast cooling without repeated warming, and pasteurisation immediately after homogenisation. Lipolysis is not always a defect. In many cheeses, controlled lipolysis provides characteristic flavour: pregastric esterases from kid, lamb or calf glands are used in Italian cheeses such as Romano and Provolone to release short-chain acids for a sharp, piquant flavour; Penicillium roqueforti lipases in blue cheeses release fatty acids that are converted to methyl ketones, the key flavour compounds; and lipolysis contributes to the flavour of Feta and some hard cheeses. Enzyme-modified cheese and lipolysed butter oil, made by deliberate lipase treatment, are flavour ingredients. In milk, cream and butter, however, free fatty acids above a low threshold are a defect, and in ghee they reduce keeping quality and contribute to rancidity.
Cholesterol is the main sterol of milk, located mostly in the fat globule membrane and the fat, and mostly in the free rather than esterified form. Its content in a dairy product rises with fat: roughly 10 to 15 milligrams per 100 grams in whole milk, much less in skim milk, more in cream and cheese, and roughly 200 to 250 milligrams per 100 grams in butter, with ghee somewhat higher on a fat basis. Because public health advice for many years linked dietary cholesterol and saturated fat with heart disease, methods were developed to remove cholesterol from milk fat. Beta-cyclodextrin, a ring of seven glucose units with a hydrophobic cavity, binds cholesterol selectively when mixed with milk, cream or melted butter oil; the complex is removed by centrifugation, and removal of the order of 90 per cent is possible. Supercritical carbon dioxide extraction, steam stripping or short-path distillation of butter oil, adsorption on saponins or other adsorbents, and enzymatic oxidation or reduction of cholesterol have also been used. Each method affects flavour, cost and other components, and few cholesterol-reduced products have stayed on the market. Heating and storage of ghee, butter oil and milk powders in air can oxidise cholesterol to cholesterol oxidation products, which are of concern for health; their formation is limited by the same measures that limit lipid oxidation. Dietary guidelines in several countries have moved away from fixed limits on cholesterol intake towards attention to overall diet, so the commercial interest in cholesterol reduction has declined.
Vitamins A, D, E and K are dissolved in milk fat, so their content in dairy products follows the fat. Vitamin A is present as retinol and its esters and, in cow milk, also as beta-carotene; buffalo, goat and sheep milks contain retinol but little carotene. Vitamin A activity is expressed as retinol equivalents (RE), in which 1 microgram of retinol counts as 1 and, by the older convention, 6 micrograms of beta-carotene count as 1 microgram of retinol; newer conventions use 12 micrograms of beta-carotene per retinol activity equivalent, so the basis must be stated. Under the RE convention, a sample with 30 micrograms of retinol and 60 micrograms of beta-carotene supplies 40 micrograms RE. One international unit of vitamin A equals 0.3 micrograms of retinol. Milk and especially butter and ghee are important sources of vitamin A; content is higher with green feed and lower on dry rations. Milk is naturally low in vitamin D, which is why many countries fortify milk with vitamins D and A, and why skimmed and low-fat milks, which lose these vitamins with the fat, are fortified where regulations require or allow. Alpha-tocopherol (vitamin E) is present at low levels and protects the fat. Vitamin K is present in small amounts as phylloquinone, with menaquinones (vitamin K2) formed by bacteria in some cheeses. Processing has modest effects: pasteurisation and UHT cause little loss of fat-soluble vitamins, but exposure to light and oxygen destroys vitamin A and carotene, and long storage and high clarification temperatures reduce vitamin A and carotene in ghee. Opaque packaging protects them.
A student uses this subject in dairy chemistry practicals and in product courses on cream, butter, ghee and ice cream. You will extract milk fat, determine Reichert-Meissl, Polenske, iodine and saponification values and the butyro-refractometer reading, measure peroxide value and free fatty acids, and run the phytosterol acetate test. Learn the major fatty acids and their proportions, the origin of short-chain acids, the polymorphic forms, the free-radical mechanism of oxidation and the causes of lipolysis, because they recur in entrance and PG papers and explain the defects you will meet in the plant.
Five years into work in a butter, ghee or cream plant or a food laboratory, you trace rancid flavour in raw milk to air leaks in a milking pipeline, set cream-ripening temperature programmes to make butter spreadable in winter, choose packaging and antioxidants to extend ghee shelf life, interpret peroxide and free fatty acid results against specifications, and test suspected ghee for vegetable fat or animal body fat adulteration using fat constants and sterol tests. You also train shift staff to recognise oxidised and rancid notes at reception.
A senior manager or consultant with twenty-five years of experience decides on milk fat strategy: whether to fractionate anhydrous milk fat for bakery and confectionery customers, how to manage seasonal changes in fat hardness across a product range, how to specify raw milk quality to limit psychrotroph lipases in UHT and powder plants, and which antioxidant and packaging systems meet export rules in different markets. Such a person also handles customer complaints of oxidised or rancid flavour and defends product claims about CLA, cholesterol and vitamins.
A researcher with long experience works on the open questions of milk fat: how feeding and genetics control fatty acid composition and CLA, the structure of the fat globule membrane and its nutritional role, crystallisation in emulsions and its effect on texture, the mechanisms of oxidation in powders and infant formula, heat-stable microbial lipases in UHT products, and the health effects of dairy fat. Methods include gas chromatography and mass spectrometry of lipids, NMR and X-ray scattering of crystals, DSC, microscopy and controlled feeding and human studies.
This manual gives typical fatty acid figures and principles; it does not tabulate national legal limits for Reichert-Meissl, Polenske, butyro-refractometer or iodine values of ghee and butter, nor the permitted antioxidants and their maximum levels, which differ by country and change; read them from current regulations. Detailed analytical procedures belong to the analysis subject. The technology of cream, butter, ghee and anhydrous milk fat is covered in the fat-rich products subject, and lipoprotein lipase and the membrane proteins in Milk Proteins and Enzymes. Exact solid fat content curves and DSC peak temperatures vary with composition and thermal history and are given only approximately. Human health evidence on dairy fat, CLA and cholesterol is summarised in outline and is changing.
Original practice questions written to the syllabus and pattern of the exams named in each question's examTags. They are not past papers and do not reproduce any board's questions.
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