Dairy Science & Technology · Dairy Chemistry · also Dairy Chemistry II, Chemistry of Milk Proteins
Caseins and the casein micelle, whey proteins, minor proteins, genetic variants, heat denaturation, and the indigenous milk enzymes used as process indicators.
In the exams: ICAR AICE subject 34 Unit 2; ICAR AIEEA-PG group 16; ASRB NET Dairy Chemistry
Milk proteins are the reason milk can be made into cheese, yoghurt, paneer, casein, whey protein concentrate and infant formula, and the indigenous enzymes of milk are the reason a laboratory can tell, from a few drops, whether milk has been properly pasteurised. This subject covers the classification of milk proteins and the nitrogen fractions used to measure them; the four caseins and the casein micelle they form; what keeps the micelle stable and what destroys that stability; genetic variants of the proteins, including the A1 and A2 forms of beta-casein; the whey proteins and the minor protective proteins; the proteins of the fat globule membrane; heat denaturation of whey proteins and their reactions with casein; the indigenous enzymes and their use as indicators of heat treatment; bioactive peptides and hydrolysates; and the allergenicity of milk proteins.
The subject matters in practice because protein chemistry explains most of what happens in a dairy plant. Rennet coagulation depends on one bond in kappa-casein. The firmness of yoghurt depends on how much whey protein was denatured during heating. The heat stability of evaporated milk depends on micellar calcium phosphate and kappa-casein. Age gelation of UHT milk and bitterness in cheese come from a heat-stable indigenous protease. The alkaline phosphatase test, run every day in every pasteurising plant, depends on an enzyme being destroyed at a temperature just above that needed to kill the most heat-resistant vegetative pathogens of raw milk. Protein figures given here are typical textbook values for cow milk; they vary with breed, genetics, lactation stage and udder health.
The subject is examined as Unit 2 of ICAR AICE-JRF/SRF subject 34 (Dairy Chemistry), within group 16 of ICAR AIEEA-PG, and in ASRB NET Dairy Chemistry. GATE food technology asks about food proteins and enzymes in general, and food analyst papers ask about the phosphatase and peroxidase tests. Expect recall of residue numbers, phosphorylation and cysteine content of the caseins, the chymosin-sensitive bond, micelle models, variant differences, whey protein properties, enzyme heat stabilities and test principles, and calculations of protein fractions from nitrogen data.
Cow milk contains about 3.2 to 3.5 per cent protein. The classical division, based on behaviour at pH 4.6 and 20 degrees Celsius, separates casein, which precipitates at that pH, from the whey or serum proteins, which stay in solution. Casein forms about 80 per cent of cow milk protein and comprises four gene products: alpha-s1-, alpha-s2-, beta- and kappa-casein, present in roughly the proportions 4 : 1 : 4 : 1.3, together with fragments formed from them by proteolysis, the gamma-caseins. Whey proteins form about 20 per cent; the main ones are beta-lactoglobulin (about half of whey protein in cow milk), alpha-lactalbumin (about a fifth), serum albumin, immunoglobulins and the proteose-peptone fraction. Minor proteins include lactoferrin, transferrin, the milk fat globule membrane proteins, many enzymes, and protein hormones and growth factors. The caseins are phosphoproteins with little ordered secondary structure, rich in proline, and stable to heat; they exist in micelles with calcium phosphate. The whey proteins are compact globular proteins with disulphide bonds, sensitive to heat but stable at pH 4.6 in the native state. This difference in structure explains why casein can be precipitated by acid or rennet, while whey proteins can be precipitated by heat.
Proteins are measured through nitrogen, usually by the Kjeldahl method. Total nitrogen (TN) is determined on milk. The milk is then acidified to pH 4.6 with acetic acid and sodium acetate, the casein is filtered off, and the nitrogen in the filtrate is the non-casein nitrogen (NCN). A separate portion is treated with trichloroacetic acid (TCA) to a final concentration of 12 per cent, which precipitates all proteins; the nitrogen in this filtrate is the non-protein nitrogen (NPN), comprising urea, peptides, amino acids, creatine, orotic acid and other small molecules, about 5 per cent of TN in cow milk. From these, casein nitrogen equals TN minus NCN, whey protein nitrogen equals NCN minus NPN, and true protein nitrogen equals TN minus NPN; each is multiplied by 6.38 to give protein. For milk with TN 0.52, NCN 0.12 and NPN 0.03 per cent, casein is 0.40 x 6.38 = 2.55 per cent, whey protein 0.09 x 6.38 = 0.57 per cent, true protein 3.13 per cent and crude protein 3.32 per cent. The casein number, casein as a percentage of true protein, is about 82 per cent in this example; expressed on crude protein it would be about 77 per cent, so the basis must be stated. Payment schemes and labels increasingly use true protein because urea, which varies with feeding, carries no processing value. Proteose-peptones are defined operationally as the proteins that stay soluble after heating milk at 95 degrees Celsius for 20 minutes and acidifying to pH 4.6, but precipitate in 12 per cent TCA.
The caseins are synthesised in the mammary gland and phosphorylated on serine residues, mostly in clusters of three phosphoserines followed by two glutamic acid residues. These phosphoserine clusters bind calcium and calcium phosphate strongly, which is the chemical basis of the micelle. Alpha-s1-casein, the most abundant, has 199 amino acid residues, a molecular mass near 23.6 kilodaltons and usually 8 or 9 phosphate groups. It contains no cysteine. It is strongly charged, with hydrophobic regions, and is precipitated by calcium at milk concentrations. Alpha-s2-casein has 207 residues, the highest phosphorylation of the caseins (10 to 13 phosphate groups) and two cysteine residues, usually linked as an intramolecular or intermolecular disulphide; it is the most calcium-sensitive and the most hydrophilic of the caseins. Beta-casein has 209 residues, a molecular mass near 24 kilodaltons and 5 phosphate groups clustered in its N-terminal region, which is highly charged, while the rest of the molecule is very hydrophobic, so beta-casein is amphiphilic, like a soap. It contains no cysteine. Its association is strongly temperature dependent: at low temperatures, around 4 degrees Celsius, hydrophobic interactions weaken and part of the beta-casein leaves the micelles for the serum, which is reversed on warming. Plasmin hydrolysis of beta-casein produces the gamma-caseins (gamma-1, gamma-2 and gamma-3) and the corresponding N-terminal proteose-peptone fragments.
Kappa-casein has 169 residues, a molecular mass near 19 kilodaltons, usually one phosphate group (sometimes more) and two cysteine residues, through which it forms disulphide-linked polymers. It is the only major casein that is glycosylated: its C-terminal region carries chains containing galactose, N-acetylgalactosamine and N-acetylneuraminic (sialic) acid, which vary in number between molecules. Kappa-casein is not precipitated by calcium and protects the calcium-sensitive caseins from precipitation, which is why it is found mainly on the surface of the micelles. The bond between phenylalanine 105 and methionine 106 is unusually susceptible to chymosin and other acid proteases. Cleavage splits kappa-casein into para-kappa-casein (residues 1 to 105), which stays with the micelle, and the caseinomacropeptide (residues 106 to 169), which is hydrophilic and passes into the whey; this is the primary phase of rennet coagulation. Common to all caseins are a high content of proline, which prevents compact folding, little ordered alpha-helix or beta-sheet structure in solution, and few or no disulphide bonds. They are therefore described as rheomorphic or intrinsically disordered proteins. This open structure makes caseins very heat-stable, readily hydrolysed by proteases, good emulsifiers and easily digested. Caseins have an isoelectric point near pH 4.6 and are precipitated there; acid casein and caseinates for food use are made by exploiting this precipitation and then neutralising with sodium, potassium or calcium hydroxide.
The casein micelle is a highly hydrated, roughly spherical particle of casein and calcium phosphate. On a dry basis it is about 94 per cent protein and about 6 per cent inorganic material, mainly calcium phosphate together with magnesium and citrate, described as colloidal or micellar calcium phosphate (CCP). Diameters range from about 50 to 500 nanometres, with an average near 150 to 200 nanometres, and a typical micelle contains thousands of casein molecules. Micelles hold a large amount of water, about 3 to 4 grams per gram of protein, giving a voluminosity of about 4 millilitres per gram, so they occupy roughly a tenth of the volume of milk although casein is under 3 per cent by weight. The surface carries a layer of kappa-casein whose hydrophilic, negatively charged C-terminal segments protrude into the serum as a hairy brush several nanometres thick. Micelles are not fixed particles: they exchange beta-casein and calcium phosphate with the serum when temperature, pH and calcium activity change, and they can be disrupted by chelating agents that remove calcium or by agents that break hydrophobic bonds. They are seen in the electron microscope as particles with an irregular, open surface. All models of the micelle must explain this composition, the surface location of kappa-casein, the role of CCP and the dynamic exchange with the serum.
Three families of models are taught. The submicelle model, developed by Slattery and Evard and by Schmidt and others, proposed that micelles are built from smaller units, submicelles about 10 to 20 nanometres across, each with a hydrophobic core and a hydrophilic surface, linked together by CCP. Submicelles poor in kappa-casein were thought to lie inside and those rich in kappa-casein on the surface, explaining surface kappa-casein. The model explained much early data but is no longer favoured, because high-resolution electron microscopy and scattering studies have not shown discrete submicelles. In the nanocluster model developed by Holt and co-workers, the calcium-sensitive caseins are cross-linked by very small clusters of calcium phosphate, a few nanometres across, each stabilised by the phosphoserine clusters of the caseins that bind to it, forming a protein matrix in which the nanoclusters are distributed evenly; kappa-casein, with too few phosphoserines to bind nanoclusters, ends up at the surface and limits growth. The dual-binding model proposed by Horne emphasises that two kinds of interaction hold the micelle together: hydrophobic interactions between caseins and bridging of phosphoserine clusters by calcium phosphate. A casein molecule can bind through both, and kappa-casein, which has one hydrophobic region but no phosphoserine cluster, acts as a chain terminator at the surface. Current understanding draws on both: CCP nanoclusters bound by phosphoserine clusters, a casein network held by hydrophobic and electrostatic interactions, and a kappa-casein brush on the surface. The models agree on the practical consequences: removing CCP or kappa-casein, or neutralising charge, destabilises the micelle.
In normal milk, casein micelles do not aggregate, even though they collide constantly and are heated, pumped and stored. Two mechanisms keep them apart. The first is steric stabilisation by the kappa-casein brush: when two micelles approach, the protruding hydrophilic chains overlap, and the loss of freedom and increase in local concentration create a repulsion. The second is electrostatic repulsion: at the natural pH of milk the micelle carries a net negative charge, with a zeta potential of about minus 20 millivolts, much of it on the brush. Internally, the micelle is held together by CCP and hydrophobic interactions, so the integrity of the micelle depends on CCP while its colloidal stability depends mainly on the surface brush and charge. Calcium activity matters in both directions: added calcium reduces charge and bridges micelles, lowering stability, while removing calcium with phosphates, citrates or chelators dissolves CCP and eventually disrupts the micelles. Milk also has a natural salt balance between calcium and magnesium on one side and phosphate and citrate on the other; shifting it, for example by adding stabilising salts to evaporated milk, alters stability. The temperature, pH and ionic strength at which milk is held all change these balances.
Each major dairy process destabilises micelles in a particular way. Rennet removes the stabilising brush: chymosin cleaves kappa-casein at Phe105-Met106, the caseinomacropeptide is released, steric and electrostatic repulsion fall, and when most of the kappa-casein (commonly quoted near 85 per cent) has been hydrolysed, the para-casein micelles aggregate in the secondary phase, which requires calcium ions and does not occur below about 15 to 20 degrees Celsius. Acid removes the charge and the CCP: as pH falls, CCP dissolves progressively and is almost entirely dissolved by about pH 5, and at pH 4.6 the caseins reach their isoelectric point and aggregate into an acid gel or precipitate. Ethanol lowers the dielectric constant of the solvent and collapses the brush; the alcohol stability of milk, the ethanol concentration that causes flocculation, is a reception test and a guide to heat stability. Heat at very high temperatures eventually coagulates milk; the heat coagulation time (HCT) at 140 degrees Celsius is used to describe heat stability. Most cow milks show a type A HCT-pH profile, with a maximum near pH 6.7 and a minimum near pH 6.9, linked to dissociation of kappa-casein from the micelles at higher pH; type B milks show HCT rising steadily with pH. Cold storage releases beta-casein and some calcium phosphate into the serum, which slows rennet coagulation until the milk is warmed. High pressure above a few hundred megapascals disrupts micelles into smaller particles, and freezing concentrated milk can destabilise casein.
All major milk proteins occur as genetic variants: forms that differ by one or a few amino acids because of differences in the genes inherited from the sire and dam. Variants are named by the protein followed by a letter, for example beta-lactoglobulin A and B or kappa-casein A and B, following a nomenclature maintained by a committee of the American Dairy Science Association. Variants were discovered by electrophoresis, because a substitution that changes charge alters mobility, and are now identified by isoelectric focusing, chromatography, mass spectrometry and DNA genotyping. Because they are inherited codominantly, an animal can be homozygous (AA, BB) or heterozygous (AB), and both variants appear in its milk. Some variants change not only the protein but also its amount in milk or its phosphorylation and glycosylation. The important variants for technology are those of kappa-casein, beta-lactoglobulin and beta-casein in cattle and alpha-s1-casein in goats. Beta-lactoglobulin A and B differ at positions 64 (aspartic acid in A, glycine in B) and 118 (valine in A, alanine in B); milk from cows with the A variant tends to contain more beta-lactoglobulin, while BB milk contains a higher proportion of casein and gives a higher cheese yield. Kappa-casein A and B differ at positions 136 and 148; the B variant is associated with more kappa-casein, smaller micelles, faster rennet coagulation, firmer curd and higher cheese yield, so some breeding programmes for cheese milk select for it. In goats, the alpha-s1-casein gene has strong, intermediate, weak and null alleles that change the amount of this casein secreted several-fold, with large effects on cheese yield and curd firmness.
Beta-casein occurs in several variants, of which A1 and A2 are the most common in cattle. They differ at position 67: A2, considered the ancestral form, has proline, whereas A1 (and the less common B and C variants) has histidine. The difference matters for digestion. In A1 beta-casein, the bond on the N-terminal side of histidine 67 is cleaved more readily by digestive enzymes, releasing beta-casomorphin-7 (BCM-7, residues 60 to 66 of beta-casein), an opioid peptide; proline at position 67 in A2 beta-casein makes this cleavage much less likely. Studies have proposed links between A1 beta-casein or BCM-7 and conditions such as type 1 diabetes, heart disease and digestive discomfort. These claims are contested. A review of the scientific literature for the European Food Safety Authority in 2009 concluded that a cause-and-effect relationship between BCM-7 or related peptides and non-communicable diseases could not be established. Some later human trials have reported fewer digestive symptoms with A2-only milk in self-reported intolerant people, but the evidence is limited and debated. The frequency of the A1 and A2 alleles differs between breeds and populations: A1 is common in many northern European breeds such as Holstein-Friesian, while A2 predominates in Guernsey cattle, in many Bos indicus breeds and in buffalo, whose beta-casein has proline at the corresponding position. Milk from herds genotyped as A2A2 is marketed as A2 milk in several countries. A dairy technologist should be able to state the molecular difference precisely and to describe the health evidence as uncertain rather than settled.
Beta-lactoglobulin is the main whey protein of the milk of cows, buffaloes, goats and sheep, at about 3 grams per litre in cow milk, but it is absent from human, camel and rodent milk. It has 162 amino acid residues and a molecular mass near 18.3 kilodaltons. At the pH of milk it exists mainly as a dimer; it dissociates to monomers at acid pH below about 3.5 and at high temperature. Its structure is a beta-barrel of eight antiparallel strands with one alpha-helix, the typical fold of the lipocalin family of proteins, which form a hydrophobic pocket that can bind retinol, fatty acids and other small hydrophobic molecules; its biological function remains uncertain. It has five cysteine residues: four form two disulphide bonds, and one, cysteine 121, carries a free sulfhydryl group buried in the native protein. Its isoelectric point is near pH 5.2. Native beta-lactoglobulin is resistant to pepsin, so it passes the stomach largely intact, which is one reason it is a major allergen. When heated above about 70 degrees Celsius, it unfolds, exposing the free sulfhydryl group, which then reacts with disulphide bonds of other proteins; this reaction underlies cooked flavour, heat-induced complexes with kappa-casein, fouling of heat exchangers and the gelling and foaming of whey protein products. Beta-lactoglobulin is the main determinant of the functional behaviour of whey protein concentrates and isolates.
Alpha-lactalbumin is present in the milk of all mammals that secrete lactose, at about 1.2 grams per litre in cow milk; it is a major protein of human milk. It has 123 residues, a molecular mass near 14.2 kilodaltons, four disulphide bonds and no free sulfhydryl group, and an isoelectric point near pH 4.8. It binds one calcium ion strongly, which stabilises its structure; the calcium-free (apo) form is much less stable. Its biological role is as the regulatory subunit of lactose synthase: on its own, the enzyme galactosyltransferase transfers galactose to N-acetylglucosamine, but in the presence of alpha-lactalbumin its specificity changes so that it transfers galactose to glucose, forming lactose. The amount of alpha-lactalbumin therefore affects how much lactose, and hence how much milk, is secreted. Alpha-lactalbumin is homologous in sequence and structure with the C-type lysozymes, from which it evolved, but has little or no lytic activity. Its thermal unfolding begins at a lower temperature than that of beta-lactoglobulin, but much of the unfolding is reversible on cooling when calcium is bound, so in milk it is the most resistant of the whey proteins to irreversible heat denaturation; it lacks a free thiol and joins heat-induced aggregates mainly through reactions with beta-lactoglobulin. Because its amino acid profile is rich in tryptophan and close to the needs of the human infant, whey protein fractions enriched in alpha-lactalbumin are used in infant formula to bring the protein closer to that of human milk.
Immunoglobulins are antibodies. In cow milk they are present at roughly 0.6 to 1 gram per litre, and at many times that concentration in colostrum. The main class in bovine milk and colostrum is IgG1, transported from blood by receptors in the mammary epithelium; IgG2, IgA and IgM are present in smaller amounts. In human milk the main class is secretory IgA. Immunoglobulins are the most heat-sensitive of the whey proteins, which matters for products made to preserve them, such as colostrum supplements and immune milk preparations, and for the cold agglutination of fat globules, which depends on IgM and is lost on heating. Bovine serum albumin, at about 0.4 grams per litre, comes from blood; it has 582 residues, binds fatty acids and other ligands, and its level rises in mastitic milk. Lactoferrin is an iron-binding glycoprotein of about 80 kilodaltons with two iron-binding sites. Bovine milk contains only about 0.1 gram per litre, colostrum more, while human milk contains far more, about 1 to 2 grams per litre in mature milk. By binding iron, lactoferrin withholds it from bacteria that need it, giving a bacteriostatic effect; pepsin digestion releases lactoferricin, a cationic peptide with direct antimicrobial activity. Lactoferrin is strongly basic, with an isoelectric point near pH 8 to 9, so it is isolated from whey by cation-exchange chromatography and is added to some infant formulas and supplements.
Lysozyme hydrolyses the beta-1,4 bonds between N-acetylmuramic acid and N-acetylglucosamine in bacterial cell walls and is most active against Gram-positive bacteria. Human milk contains thousands of times more lysozyme than cow milk, in which it is only a trace. Lactoperoxidase is a haem glycoprotein of about 78 kilodaltons and is the most abundant enzyme in cow milk, at about 30 milligrams per litre. With hydrogen peroxide it oxidises thiocyanate ions, which come from the diet and from detoxification in the animal, to hypothiocyanite, which inhibits many bacteria by oxidising their sulfhydryl groups. This lactoperoxidase system can be activated deliberately by adding thiocyanate and a source of hydrogen peroxide; a Codex guideline describes its use to preserve raw milk for some hours where cooling is not available, and it is not a substitute for refrigeration where cooling is possible. Lactoperoxidase is relatively heat-stable, surviving normal pasteurisation, which makes it useful as an indicator of more intense heating. The milk fat globule membrane carries its own proteins, about 1 to 2 per cent of total milk protein. The major ones are butyrophilin, the most abundant membrane protein in cow milk; xanthine oxidoreductase, a molybdenum-containing enzyme; adipophilin; the mucins MUC1 and MUC15; lactadherin (PAS 6/7); and CD36. Together with alkaline phosphatase and other membrane enzymes, they enter buttermilk and cream products, and MFGM-enriched ingredients are studied for infant nutrition.
Denaturation is the loss of the native folded structure of a protein without breaking peptide bonds. For whey proteins, heat denaturation proceeds in two stages: unfolding, which is often reversible, followed by aggregation, which is not. When beta-lactoglobulin unfolds, its buried free sulfhydryl group becomes reactive and attacks disulphide bonds of other molecules in thiol-disulphide interchange reactions, while exposed hydrophobic patches promote non-covalent association. The whey proteins differ in their sensitivity to irreversible denaturation in milk; the usual order from most to least sensitive is immunoglobulins, serum albumin, beta-lactoglobulin and alpha-lactalbumin. Denaturation depends on temperature, time, pH, concentration and the presence of calcium, lactose and other solutes; lactose and sugars protect proteins, and higher protein concentrations speed aggregation. The kinetics of beta-lactoglobulin denaturation in milk are commonly described by a reaction order near 1.5, with an Arrhenius plot that shows a break near 90 degrees Celsius: below it the rate is controlled by unfolding and the activation energy is very high, above it by aggregation and the activation energy is much lower. Denaturation is measured by the loss of protein soluble at pH 4.6, by chromatographic or electrophoretic determination of native whey proteins, or by immunological methods. In industry, milk powders are classified by the whey protein nitrogen index (WPNI), milligrams of undenatured whey protein nitrogen per gram of powder: by the classification widely used for skim milk powder, low-heat powder has 6.0 or more, medium-heat 1.51 to 5.99 and high-heat 1.5 or less.
When milk is heated above about 70 degrees Celsius, denatured beta-lactoglobulin reacts with kappa-casein, and to some extent with alpha-s2-casein, through thiol-disulphide interchange, forming complexes. At the natural pH of milk and below, much of this complex forms on the surface of the casein micelles; at pH above about 6.7, more kappa-casein dissociates from the micelles and the complexes are found in the serum. These reactions have large technological effects. In cheesemaking, the complex on the micelle surface hinders chymosin access and aggregation, so milk heated well above pasteurisation coagulates slowly and gives a weak curd; this is why cheese milk is normally pasteurised at minimum conditions. In yoghurt making, the same complex is wanted: milk is heated typically at 85 degrees Celsius for about 30 minutes or 90 to 95 degrees Celsius for about 5 to 10 minutes, so that denatured whey protein joins the casein network, raising the gelation pH, increasing gel firmness and reducing syneresis. In evaporated and UHT milk, the complex influences heat stability and the tendency to gel. Denatured whey proteins and the exposed sulfhydryl groups cause cooked flavour through volatile sulphur compounds such as hydrogen sulphide, and lower the redox potential, which protects against oxidised flavour. Whey protein deposits are a major part of the fouling of heat exchangers at temperatures roughly between 75 and 110 degrees Celsius, while mineral-rich deposits dominate at higher temperatures. Pasteurisation denatures only a few per cent of the whey protein, whereas UHT treatment denatures a large fraction, more with indirect than direct heating.
Milk contains about sixty indigenous enzymes, secreted by the mammary gland, released from somatic cells or derived from blood. They are distinguished from microbial enzymes, which come from bacteria growing in milk. A few are technologically important. Alkaline phosphatase hydrolyses phosphate monoesters with an optimum near pH 10; in milk it is associated mainly with the fat globule membrane and with lipoprotein particles in the serum, so cream contains more than skim milk. Its thermal inactivation is slightly more demanding than the destruction of the most heat-resistant vegetative pathogens that historically set pasteurisation standards, Mycobacterium tuberculosis and later Coxiella burnetii, so its absence shows that milk has received at least minimum pasteurisation. Lactoperoxidase, described in chapter 7, is more heat-stable; it largely survives minimum pasteurisation and is inactivated by heating to about 80 degrees Celsius for a few seconds. Its activity is detected by adding hydrogen peroxide and a chromogen, as in the Storch test with p-phenylenediamine, which turns blue when the enzyme is active.
Lipoprotein lipase hydrolyses triglycerides to free fatty acids and partial glycerides. Cow milk contains about 1 to 2 milligrams per litre, enough in principle to cause rancidity within minutes if it had free access to the fat, but most of it is bound to casein micelles while the fat is protected by the globule membrane, so little lipolysis occurs in milk handled carefully. Lipolysis is induced when the membrane is damaged or the enzyme is activated: by agitation and foaming, particularly with air leaks in milking systems and pumps; by homogenising raw milk, which creates new fat surfaces not covered by native membrane; by temperature cycling, such as cooling to about 5 degrees Celsius, warming to about 30 degrees Celsius and cooling again; and by mixing raw and homogenised milk. Spontaneous lipolysis, without such treatment, occurs in the milk of some cows, especially in late lactation and on poor feed. Short-chain fatty acids such as butyric acid released from milk fat give a rancid, soapy or bitter flavour at low concentrations. Lipoprotein lipase is largely inactivated by pasteurisation, so raw milk should be pasteurised before or immediately after homogenisation. Free fatty acid content is measured as the acid degree value or by titration of extracted fat.
Plasmin is the main indigenous protease of milk. It is a serine protease with trypsin-like specificity, cleaving after lysine and arginine residues, and it originates from blood plasminogen. Milk contains a complete system: plasminogen, plasmin, plasminogen activators that convert one to the other, and inhibitors of both. Plasmin and plasminogen are bound to casein micelles, whereas many of the inhibitors are in the serum and are heat-labile. Plasmin is very heat-stable: it survives pasteurisation and partly survives UHT treatment, and because pasteurisation destroys inhibitors of plasminogen activation, plasmin activity can even increase in pasteurised milk during storage. Its preferred substrate is beta-casein, which it cleaves to the gamma-caseins and proteose-peptones; it also hydrolyses alpha-s2- and alpha-s1-casein. Plasmin activity is higher in late-lactation and mastitic milk. Its effects include bitterness and age gelation in UHT milk, loss of casein and so of cheese yield, and a contribution to proteolysis during the ripening of cheeses cooked at high temperatures, such as Swiss types. Other indigenous enzymes include cathepsin D, an acid protease from somatic cells that cleaves alpha-s1-casein at sites similar to chymosin; xanthine oxidoreductase in the fat globule membrane, which can generate superoxide and hydrogen peroxide; catalase and N-acetyl-beta-D-glucosaminidase, which rise with somatic cells and serve as mastitis indicators; gamma-glutamyl transferase; superoxide dismutase; sulfhydryl oxidase; and an acid phosphatase that is much more heat-stable than the alkaline enzyme.
The alkaline phosphatase (ALP) test is the standard check that milk has been pasteurised and has not been contaminated afterwards with raw milk. The principle is simple: a phosphate ester substrate is added to milk, the sample is incubated, and the product released by any active enzyme is measured. In the Aschaffenburg-Mullen method, disodium p-nitrophenyl phosphate is hydrolysed to yellow p-nitrophenol, read against standards. In the Scharer method, phenyl phosphate releases phenol, which is reacted with a chromogen. The current reference method, ISO 11816-1, is fluorimetric and very sensitive. Both the European Union and the United States Pasteurized Milk Ordinance use a limit of 350 milliunits per litre by the fluorimetric method for pasteurised cow milk, and the test can detect contamination of pasteurised milk with a small fraction of a per cent of raw milk. Results need careful interpretation. ALP can reactivate in products given high-temperature treatment, such as UHT cream, when they are stored warm; reactivated enzyme is stimulated by magnesium ions, which distinguishes it from residual native enzyme. Heat-stable phosphatases from some bacteria can give false positives. Fat protects the enzyme, so cream needs more intense heating than milk to reach a negative result. Goat and sheep milks have different natural ALP activities, so limits for them are set separately or under review.
The lactoperoxidase test separates milk heated at minimum pasteurisation conditions from milk heated more intensively. Because lactoperoxidase survives minimum pasteurisation but is destroyed at about 80 degrees Celsius for a few seconds, pasteurised milk is expected to be phosphatase-negative and peroxidase-positive, while milk that is phosphatase-negative and peroxidase-negative has been heated to at least about 80 degrees Celsius. Some regulations have used a negative peroxidase test to define high-temperature pasteurised milk. Gamma-glutamyl transferase, with an inactivation temperature between those of ALP and lactoperoxidase, has been proposed as an indicator for heat treatments in that intermediate range. For more severe treatments, enzymes are of little use because all are inactivated, and chemical indicators take over: lactulose, formed by isomerisation of lactose during heating, distinguishes UHT milk from in-container sterilised milk; furosine, from the early Maillard reaction, indicates heat load and the use of reconstituted powder; and the amount of undenatured whey protein, including acid-soluble beta-lactoglobulin, distinguishes pasteurised, high-pasteurised and UHT milk. The Aschaffenburg turbidity test identifies sterilised milk: after the casein and denatured protein are removed with ammonium sulphate, a filtrate that stays clear on boiling contains no remaining native whey protein. Together, these indicators let regulators check that a product labelled pasteurised, UHT or sterilised has received the heat treatment claimed and no more than is appropriate.
Bioactive peptides are fragments of proteins that, once released, have a physiological effect beyond nutrition. They are inactive within the parent sequence and are released by digestion in the gut, by proteolysis during fermentation by lactic acid bacteria, or by commercial enzymatic hydrolysis. Milk proteins are the best-studied source. Caseinophosphopeptides (CPPs), released from the phosphoserine-rich regions of the caseins, bind calcium and keep it soluble, and are used in products intended to support mineral uptake and in dental care products to help remineralise enamel. Angiotensin-converting enzyme (ACE) inhibitory peptides lower the formation of angiotensin II in vitro; the best known are the lactotripeptides valine-proline-proline and isoleucine-proline-proline, released from beta-casein (and, for IPP, also from kappa-casein) by Lactobacillus helveticus fermentation. Opioid peptides include the beta-casomorphins from beta-casein, discussed with A1 and A2 variants, and alpha- and beta-lactorphins from the whey proteins. Lactoferricin from lactoferrin is antimicrobial. The caseinomacropeptide released by chymosin, also called glycomacropeptide when glycosylated, lacks the aromatic amino acids phenylalanine, tryptophan and tyrosine, which makes it a protein source for people with phenylketonuria once the missing essential amino acids are added. Many effects shown in cell or animal studies have not been confirmed in humans, and regulators such as the European Food Safety Authority have not accepted most proposed health claims for milk peptides, so a technologist should separate demonstrated activity from marketing.
Protein hydrolysates are produced by controlled enzymatic hydrolysis of caseins or whey proteins using proteases such as trypsin, pepsin, chymotrypsin, papain and bacterial subtilisin-type enzymes. The extent of hydrolysis is expressed as the degree of hydrolysis (DH), the percentage of peptide bonds cleaved: DH equals h divided by h-total, multiplied by 100, where h is the number of bonds cleaved, in milliequivalents per gram of protein, and h-total is the total number of peptide bonds, taken as about 8.2 milliequivalents per gram for casein and 8.8 for whey protein. If 1.0 milliequivalent per gram of bonds is cleaved in whey protein, DH is 11.4 per cent. Hydrolysis is followed by the pH-stat method, by measuring free amino groups with reagents such as trinitrobenzenesulfonic acid or o-phthaldialdehyde, or by osmometry. Hydrolysis changes function: solubility over a wide pH range and heat stability improve, gelling is lost, emulsifying and foaming change, and bitterness appears as hydrophobic peptides are released, which is controlled by choice of enzyme, limited DH and debittering treatments. The most important use is infant formula for infants with cow's milk protein allergy: extensively hydrolysed formulas, in which most peptides are too small to cross-link IgE, are standard first-line products, while partially hydrolysed formulas are not suitable for treating allergy. Hydrolysates are also used in sports and clinical nutrition because small peptides are absorbed quickly, and as flavour and functional ingredients.
Cow's milk protein allergy (CMPA) is an adverse immune reaction to milk proteins. It must be distinguished from lactose intolerance, which is a non-immune inability to digest lactose because of low intestinal lactase. CMPA is among the most common food allergies of infancy. Older estimates put it at about 2 to 3 per cent of infants; studies using controlled oral food challenges have found lower figures, and most children outgrow it by school age. Reactions are of two broad types. IgE-mediated allergy produces symptoms within minutes to about two hours of exposure, including urticaria, angioedema, vomiting, wheezing and, rarely, anaphylaxis. Non-IgE-mediated allergy produces delayed, mainly gastrointestinal symptoms such as proctocolitis, enteropathy and food protein-induced enterocolitis syndrome. Diagnosis combines history with skin-prick testing or specific IgE measurement for the IgE type, elimination of milk, and an oral food challenge, the double-blind placebo-controlled challenge being the reference standard. The main allergens are the caseins (collectively designated Bos d 8), beta-lactoglobulin (Bos d 5), alpha-lactalbumin (Bos d 4), serum albumin (Bos d 6), immunoglobulin G (Bos d 7) and lactoferrin. Most allergic patients react to several proteins. Casein epitopes are largely sequential (linear) and survive heating, whereas many whey protein epitopes are conformational and are reduced by heat denaturation, which is one reason why many allergic children tolerate milk baked into foods while reacting to fresh milk.
Milk proteins of goats and sheep have amino acid sequences very similar to those of cow milk, and most people with CMPA react to them, so goat and sheep milks are not safe substitutes. Camel, mare and donkey milks are less closely related and cross-react less, but are not recommended without specialist supervision. Processing changes allergenicity in different ways. Heating reduces many conformational epitopes but can create new ones through Maillard reactions or aggregation; fermentation and enzymatic hydrolysis reduce allergenicity in proportion to how far the proteins are broken down; and none of these makes ordinary dairy products safe for allergic people. For infants with CMPA who are not breastfed, guidelines recommend an extensively hydrolysed formula, and an amino acid-based formula for those who react to hydrolysates or have severe disease; an extensively hydrolysed formula is expected to be tolerated by at least 90 per cent of infants with confirmed allergy. Partially hydrolysed formulas and formulas based on goat or sheep milk are not suitable. Because milk proteins are used as ingredients in many foods, labelling law treats milk as a major allergen that must always be declared: the Codex General Standard for the Labelling of Prepackaged Foods, the European Union's food information regulation (Annex II of Regulation 1169/2011) and the United States food allergen labelling law all list it. Dairy plants manage allergen risk by segregation, cleaning validation and testing for casein or beta-lactoglobulin with immunoassays or mass spectrometry when producing milk-free products on shared lines.
A student uses this subject to understand why milk behaves as it does in every product course that follows. In practicals you will fractionate milk nitrogen into casein, whey protein and NPN, precipitate casein at pH 4.6, run SDS-PAGE of milk proteins, and perform the phosphatase and peroxidase tests on raw, pasteurised and boiled milk. Learn the four caseins with their residue numbers, phosphate groups and cysteines, the chymosin-sensitive bond, the micelle models, the whey proteins and their heat sensitivity, and the heat stabilities of the main enzymes, because examiners ask them repeatedly and later courses assume them.
Five years into work in quality assurance, production or product development, you use protein chemistry to solve problems. You read a positive phosphatase result and decide whether it means under-pasteurisation, raw-milk contamination or reactivation; you trace bitterness and gelation in stored UHT milk to plasmin in late-lactation milk; you adjust yoghurt heat treatment to get firmness without syneresis; you choose low-heat or high-heat powder by WPNI for a customer's application; and you manage allergen controls when a line also runs milk-free products.
A senior technologist or plant manager with twenty-five years of experience makes decisions that depend on protein behaviour at scale: specifying milk quality for cheese plants, including somatic cell and plasmin-related limits; choosing heat-treatment regimes for UHT and extended-shelf-life lines; deciding whether to market A2 or protein-standardised products and what claims can be defended; investing in whey fractionation for alpha-lactalbumin, lactoferrin or hydrolysates; and setting allergen management policy across sites. Such a person also answers to regulators and customers when a phosphatase failure or an undeclared-allergen complaint reaches the market.
A researcher with long experience works on open questions: the fine structure of the casein micelle and the arrangement of calcium phosphate nanoclusters, the mechanisms of heat-induced interactions and age gelation, the activity of indigenous enzymes in new processes such as high pressure and microfiltration, genetic selection for protein variants that improve processing, the human evidence for bioactive peptides and A1/A2 claims, and the reduction of allergenicity. Methods include scattering techniques, cryo-electron microscopy, mass-spectrometric proteomics, kinetic modelling and controlled human trials.
This manual does not give full amino acid sequences, detailed three-dimensional structures or the complete list of variants of each protein; see the ADSA nomenclature reviews. Rennet coagulation is introduced only as it concerns kappa-casein and micelle stability; its kinetics and cheese applications belong to the cheese subject. Exact heat-inactivation constants (D and z values) for each enzyme, legal ALP limits for goat, sheep and buffalo milk, and the lactulose and furosine limits proposed by different bodies are not tabulated because they differ between sources and are under review. Protein concentrations in non-bovine milks are given only in outline. Evidence on A1 and A2 beta-casein and on bioactive peptide health effects is changing; the manual reports the position of the cited reviews and should be updated as new human trials appear. Clinical management of allergy is summarised, not prescribed.
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.
These notes are free to read here, with no account.
What the Laisha app adds:
Free for ten days, then a subscription. We would rather write that here than have you find it later.