Dairy Science & Technology · Foundations · also General Biochemistry, Principles of Biochemistry
Carbohydrates, proteins, lipids, enzymes and metabolism at the level a dairy chemist and microbiologist needs before studying milk itself.
In the exams: Background for ICAR AICE subject 34 Units 1–3 and GATE XL-Q/XL-U Section 1
Biochemistry is the chemistry of living things: the structures of carbohydrates, proteins, lipids and nucleic acids, the enzymes that transform them, and the metabolic pathways by which cells obtain energy and build their own material. For a dairy scientist it is the foundation of two whole branches of the degree. Dairy chemistry studies lactose, the caseins and whey proteins, milk fat and the native enzymes of milk; dairy microbiology studies the lactic acid bacteria, yeasts and moulds that ferment lactose, break down proteins and fat during ripening, and sometimes spoil the product. Neither can be understood without knowing how a glycosidic bond forms and breaks, why a protein unfolds when heated, how an enzyme's rate depends on substrate concentration, and how a bacterium turns one molecule of lactose into lactic acid.
This manual covers the general biochemistry that dairy science and food technology programmes teach before milk itself. It starts with water, pH and buffers, because milk is an aqueous buffered system and acidity is the commonest measurement in a dairy. It then treats the four classes of biomolecules in turn, the classification and kinetics of enzymes and how their activity is inhibited and regulated, the central pathways of energy metabolism, the metabolism of lipids and amino acids, the nucleic acids and protein synthesis, and the vitamins and minerals that act as coenzymes and cofactors. Throughout, the dairy example sits next to the general principle.
Postgraduate entrance and doctoral fellowship examinations in dairy chemistry and food technology assume this background and ask it directly: buffer calculations, the structure of lactose, essential amino acids, the iodine and saponification values, Michaelis–Menten constants, types of inhibition, ATP yields and the coenzyme forms of the B vitamins.
Water makes up about 87% of cow milk and most of every living cell. Its properties come from the shape of the molecule: the oxygen atom carries a partial negative charge and the two hydrogens partial positive charges, so water is a polar molecule. Neighbouring molecules attract each other by hydrogen bonds, each molecule able to form up to four. Hydrogen bonding gives water a high specific heat (about 4.18 kJ per kg per kelvin), a high latent heat of vaporisation, and a high boiling point for so small a molecule. These properties matter in every dairy process: milk heats and cools slowly, evaporation needs a great deal of energy, and freezing releases large amounts of heat that a hardening room must remove.
Water is an excellent solvent for ions and polar molecules, which become surrounded by a shell of oriented water molecules. Lactose, mineral salts and whey proteins are dissolved in the aqueous phase of milk. Non-polar molecules, such as triglycerides, cannot form hydrogen bonds with water; they are pushed together by the hydrophobic effect, which is why milk fat exists as separate globules. The same effect drives the folding of proteins, which bury their non-polar side chains away from water, and the formation of biological membranes.
Water ionises slightly into hydrogen ions (more exactly hydronium ions) and hydroxide ions. The ion product of water, Kw = [H⁺][OH⁻], is 1.0 × 10⁻¹⁴ at 25 °C. The pH is the negative base-10 logarithm of the hydrogen-ion concentration: pH = −log[H⁺]. Pure water at 25 °C has pH 7; acid solutions are below 7 and alkaline solutions above. Because the scale is logarithmic, a fall of one pH unit means a tenfold increase in hydrogen-ion concentration. Kw rises with temperature, so the neutral pH of water falls slightly as it is heated, and the pH of milk measured hot differs from that measured cold.
A Brønsted acid donates a proton and a base accepts one. A weak acid, HA, dissociates only partly, and its acid dissociation constant Ka = [H⁺][A⁻]/[HA] measures its strength; pKa = −log Ka. The smaller the pKa, the stronger the acid. Lactic acid, the main product of lactic fermentation, has a pKa of about 3.86. Acetic acid has a pKa of about 4.76. Polyprotic acids, such as phosphoric acid (pKa values about 2.1, 7.2 and 12.3) and citric acid (about 3.1, 4.8 and 6.4), lose their protons in steps.
Fresh cow milk has a pH of about 6.6 to 6.8. Dairy plants also measure titratable acidity, the volume of standard alkali needed to bring a sample to the phenolphthalein end point, expressed as percentage lactic acid or in degrees of one of several national scales. Titratable acidity is not the same as pH: fresh milk has an apparent titratable acidity of roughly 0.13 to 0.17% expressed as lactic acid, even though it contains almost no lactic acid, because its proteins, phosphates and citrates take up alkali.
A buffer is a solution that resists changes in pH when small amounts of acid or alkali are added. It contains a weak acid and its conjugate base (or a weak base and its conjugate acid) in comparable amounts. The Henderson–Hasselbalch equation relates pH to their ratio: pH = pKa + log([A⁻]/[HA]). When the concentrations of acid and conjugate base are equal, pH = pKa. Buffering capacity, the amount of acid or base needed to change the pH by one unit, is greatest at pH = pKa and becomes small more than about one unit away.
Milk is a well-buffered system. Its main buffers are the phosphates (inorganic and the phosphoserine groups of casein), citrate, the proteins with their many ionisable side chains, and carbon dioxide and bicarbonate. When milk is plotted as buffering capacity against pH, maxima appear around pH 5 to 6, where phosphate, citrate and protein groups act together and where colloidal calcium phosphate dissolves as milk is acidified. This buffering is why starter cultures must produce a substantial amount of lactic acid before the pH of milk falls toward the isoelectric point of casein, about 4.6, at which casein coagulates in yoghurt and acid-curd cheeses.
Buffers are central to laboratory work as well. A worked example: an acetate buffer containing 0.10 mol/L acetic acid and 0.20 mol/L sodium acetate has pH = 4.76 + log 2 = 5.06.
Carbohydrates are polyhydroxy aldehydes or ketones and the compounds that yield them on hydrolysis. Monosaccharides are classified by the number of carbon atoms (trioses, pentoses, hexoses) and by the carbonyl group: an aldose has an aldehyde group and a ketose a ketone group. Glucose and galactose are aldohexoses; fructose is a ketohexose; ribose and deoxyribose are aldopentoses found in nucleic acids.
Most monosaccharides have several chiral carbon atoms. The D and L designations refer to the configuration of the chiral carbon farthest from the carbonyl group, compared with D-glyceraldehyde; nearly all natural sugars are D sugars. Two sugars that differ in configuration at only one carbon are epimers: galactose is the C-4 epimer of glucose, and mannose its C-2 epimer.
In solution, pentoses and hexoses exist mainly as rings formed when a hydroxyl group reacts with the carbonyl group to make a hemiacetal. Glucose forms a six-membered pyranose ring. Ring closure creates a new chiral centre at the former carbonyl carbon, the anomeric carbon, giving two anomers, α and β. In α-D-glucopyranose the hydroxyl on the anomeric carbon is on the opposite side of the ring from the CH₂OH group; in the β anomer it is on the same side. In solution the two anomers interconvert through the open-chain form, a process called mutarotation, until an equilibrium is reached; for glucose it is about one third α and two thirds β. Because the open-chain form carries a free carbonyl group, such sugars reduce mild oxidising agents and are called reducing sugars.
Two monosaccharides join through a glycosidic bond, formed between the anomeric carbon of one and a hydroxyl group of the other with loss of water. The bond is described by the anomeric configuration and the carbons joined. Maltose is glucose α(1→4) glucose. Sucrose is glucose α(1→2)β fructose; because both anomeric carbons are tied up in the bond, sucrose is a non-reducing sugar.
Lactose, the sugar of milk, is β-D-galactopyranosyl-(1→4)-D-glucopyranose: galactose linked through its β anomeric carbon to carbon 4 of glucose. The glucose unit keeps a free anomeric carbon, so lactose is a reducing sugar and exists as α and β anomers that mutarotate. α-Lactose crystallises from solution below about 93.5 °C as the monohydrate, the hard, sandy crystals of defective sweetened condensed milk and ice cream; β-lactose crystallises above that temperature as an anhydrous form. Lactose is much less soluble and much less sweet than sucrose. It is hydrolysed by the enzyme β-galactosidase (lactase) into glucose and galactose, the basis of lactose-free milk and of lactose digestion in the human gut.
Polysaccharides are long chains of monosaccharides. Starch, the plant storage carbohydrate, consists of amylose, a mostly linear chain of glucose units linked α(1→4), and amylopectin, which also has α(1→6) branch points roughly every 24 to 30 residues. Glycogen, the animal storage polysaccharide, is like amylopectin but more highly branched. Cellulose is a linear chain of glucose linked β(1→4); humans lack an enzyme to digest it. Starch, pectin, carrageenan, alginate, guar and locust bean gum and other polysaccharides are used as thickeners and stabilisers in dairy desserts, and starch is an adulterant detected in milk by the iodine test.
Several reactions of carbohydrates are important in dairy products. Oxidation of the free carbonyl group by alkaline copper(II) reagents, as in Fehling's and Benedict's tests, is the classical test for reducing sugars; the copper is reduced to red copper(I) oxide. Iodine forms a blue complex with the helix of amylose, which is why starch gives a blue colour with iodine. Reduction of the carbonyl group gives sugar alcohols: glucose gives sorbitol, and lactose gives lactitol, a low-calorie bulk sweetener.
Under alkaline conditions or on heating, aldoses isomerise to ketoses through an enediol intermediate, the Lobry de Bruyn–Alberda van Ekenstein transformation. In milk, lactose isomerises to lactulose (galactose linked to fructose), which is formed in measurable amounts during in-container sterilisation and much less during pasteurisation; its concentration is therefore used to distinguish heat treatments. Lactulose is not digested in the human small intestine and acts as a prebiotic and laxative.
The Maillard reaction is a set of reactions between a reducing sugar and a free amino group, mainly the ε-amino group of lysine in proteins. It begins with condensation to a Schiff base, which rearranges to an Amadori product (lactulosyl-lysine in milk), and proceeds through many intermediates to brown polymers called melanoidins and to flavour compounds. It gives the colour and flavour of dulce de leche, khoa-based sweets and caramelised milk products, but in milk powders and UHT milk it causes browning, stale flavours and a loss of available lysine. Its rate rises with temperature, pH and intermediate water activity. Caramelisation is a different reaction, the browning of sugars heated alone at high temperature without amino compounds.
Proteins are polymers of amino acids. Each standard amino acid has a central α-carbon bonded to an amino group, a carboxyl group, a hydrogen atom and a side chain, the R group, which gives each amino acid its character. Twenty amino acids are coded by the standard genetic code. Except for glycine, whose R group is a hydrogen atom, they are chiral, and those in proteins are of the L configuration.
The side chains are grouped by their properties. Non-polar, hydrophobic side chains belong to alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline. Polar uncharged side chains belong to serine, threonine, cysteine, asparagine, glutamine and tyrosine. Acidic side chains, negatively charged at neutral pH, belong to aspartate and glutamate; basic side chains, positively charged, belong to lysine, arginine and histidine. Cysteine can form disulfide bonds with another cysteine. Proline has its side chain bonded back to the amino nitrogen, which makes it a helix breaker; caseins are rich in proline, one reason why they have little ordered secondary structure.
In solution an amino acid exists mainly as a zwitterion, with a positively charged amino group and a negatively charged carboxyl group. The isoelectric point, pI, is the pH at which the net charge is zero; for an amino acid with no ionisable side chain it is the average of the two pKa values. Proteins have isoelectric points too: about 4.6 for casein and about 5.2 for β-lactoglobulin. Nine amino acids are essential for adult humans because the body cannot synthesise them: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. Milk proteins supply all of them in good proportions.
Amino acids join by peptide bonds, formed between the carboxyl group of one and the amino group of the next with loss of water. A chain of a few amino acids is a peptide; a long chain is a polypeptide. The chain has an amino terminus (N-terminus) and a carboxyl terminus (C-terminus), and sequences are written from N to C. The peptide bond has partial double-bond character, so it is planar and rigid, usually in the trans configuration; rotation is possible only about the bonds on either side of the α-carbon.
Peptides released from milk proteins by digestion, fermentation or cheese ripening have important effects. Small hydrophobic peptides from casein are a main cause of bitterness in cheese when proteolysis is unbalanced. Some peptides are biologically active: casein phosphopeptides bind calcium, and certain tripeptides from fermented milks inhibit the angiotensin-converting enzyme in laboratory studies. Glycomacropeptide (caseinomacropeptide) is the C-terminal fragment of κ-casein released by chymosin during cheesemaking, and it passes into the whey. Hydrolysates of whey and casein proteins, broken into small peptides, are used in infant formulas intended to reduce allergenicity.
Proteins and peptides are measured in several ways. The Kjeldahl method measures total nitrogen, which is converted to protein with a factor, 6.38 for milk proteins, because they contain about 15.7% nitrogen. The biuret reaction detects peptide bonds with copper in alkali; the Lowry and bicinchoninic acid methods are more sensitive; and dye-binding methods use the binding of acid dyes to basic groups. Free amino groups react with ninhydrin to give a purple colour and with o-phthaldialdehyde, methods used to follow proteolysis.
Protein structure is described at four levels. The primary structure is the amino acid sequence. The secondary structure is the local regular folding of the backbone held by hydrogen bonds between backbone groups: the α-helix, a right-handed coil with 3.6 residues per turn, and the β-pleated sheet, formed of extended strands lying side by side. The tertiary structure is the overall three-dimensional fold of one chain, stabilised by hydrophobic interactions, hydrogen bonds, ionic bonds and disulfide bonds. The quaternary structure is the association of several chains; β-lactoglobulin, for example, is a dimer at the natural pH of milk.
Globular whey proteins, such as β-lactoglobulin and α-lactalbumin, have compact, well-defined folds. β-Lactoglobulin contains two disulfide bonds and one free thiol group, which becomes reactive when the protein unfolds. The caseins are very different: they are rich in proline and phosphoserine, have little stable secondary or tertiary structure, and are often described as rheomorphic or intrinsically disordered. That open structure makes them resistant to heat, easy for proteolytic enzymes to attack, and able to bind calcium phosphate in the casein micelle.
Denaturation is any change in secondary, tertiary or quaternary structure without breaking peptide bonds. Heat, extremes of pH, high pressure, organic solvents, detergents and some salts denature proteins. Denaturation is often followed by aggregation through hydrophobic contacts and thiol–disulfide exchange. In milk, heating above about 70 °C denatures β-lactoglobulin, which then attaches to κ-casein on the micelle surface. That reaction raises the water-holding capacity and firmness of yoghurt made from heated milk, but slows rennet coagulation of cheese milk and contributes to fouling of heat exchangers.
Lipids are a diverse group of compounds that are insoluble in water and soluble in organic solvents. Fatty acids are their basic building blocks: carboxylic acids with a hydrocarbon chain, usually unbranched and with an even number of carbons in nature. A fatty acid is written as number of carbons : number of double bonds, so stearic acid is 18:0, oleic acid 18:1 and linoleic acid 18:2.
Saturated fatty acids have no carbon–carbon double bonds. Unsaturated fatty acids have one (monounsaturated) or more (polyunsaturated). The positions of the double bonds are given either from the carboxyl carbon (Δ numbering, as in cis-Δ9) or from the methyl end (the n or omega numbering). Linoleic acid, 18:2 n-6, and α-linolenic acid, 18:3 n-3, are essential fatty acids for humans, because the body cannot insert double bonds beyond carbon 9 from the carboxyl end. Most natural double bonds are cis, which puts a bend in the chain. Trans double bonds keep the chain nearly straight; they arise from partial hydrogenation of vegetable oils and, naturally, from biohydrogenation by rumen bacteria, so ruminant milk fat contains a few per cent of trans fatty acids, chiefly vaccenic acid (trans-11 18:1), and conjugated linoleic acid, mainly rumenic acid (cis-9, trans-11 18:2).
Melting point rises with chain length and falls with unsaturation; cis double bonds lower it much more than trans. This is why milk fat, a mixture of hundreds of fatty acids, melts over a wide range from about −40 to +40 °C. Milk fat of ruminants is unusual in containing short-chain fatty acids, especially butyric acid (4:0), which come from de novo synthesis in the mammary gland. Free butyric and caproic acids released by lipases give the rancid flavour of hydrolysed milk and the characteristic notes of some cheeses.
Triacylglycerols (triglycerides) are esters of glycerol with three fatty acids. They are the main storage lipid in animals and plants, and make up about 98% of milk fat. The three positions on the glycerol are numbered sn-1, sn-2 and sn-3, and the fatty acids are not distributed at random: in cow milk fat butyric and caproic acids are found almost entirely at sn-3, and palmitic acid is concentrated at sn-2 in human milk fat, which aids its absorption by infants. Mono- and diacylglycerols occur in small amounts and are used industrially as emulsifiers.
Phospholipids have two fatty acids esterified to glycerol and a phosphate group linked to a polar head group such as choline (phosphatidylcholine), ethanolamine or serine. Sphingomyelin is built on sphingosine instead of glycerol. Because they have both polar and non-polar parts, phospholipids are amphipathic; they form bilayers in cell membranes. In milk they are concentrated in the milk fat globule membrane, which surrounds each fat globule, and in buttermilk, which is rich in them.
Sterols have a four-ring steroid nucleus. Cholesterol is the main sterol of animal fats, including milk fat; plant oils contain phytosterols such as β-sitosterol instead. This difference is the basis of tests for the adulteration of ghee and butter with vegetable oils, through detection of phytosterols or their acetates. Other lipid-soluble components of milk include the fat-soluble vitamins A, D, E and K and carotenoids, which give butter its yellow colour in cows fed on fresh pasture; buffalo and goat milk fat is whiter because those animals convert carotene to vitamin A more completely.
Classical constants characterise fats and detect adulteration. The saponification value is the milligrams of potassium hydroxide needed to saponify one gram of fat; it is high when the average chain length is short, so milk fat, with its short-chain acids, has a high saponification value of roughly 210 to 235 compared with about 190 to 200 for most vegetable oils. The iodine value is the grams of iodine taken up by 100 g of fat and measures unsaturation; milk fat is about 25 to 42, much lower than sunflower or soybean oil. The Reichert–Meissl value measures volatile, water-soluble fatty acids, mainly butyric and caproic acids, and is characteristically high for milk fat; the Polenske value measures volatile, water-insoluble acids. The acid value measures free fatty acids. These values vary with species, breed, feed and season, so standards are set nationally and should be read from the relevant regulation.
Lipids deteriorate in two main ways. Hydrolytic rancidity is the release of free fatty acids from triacylglycerols by lipases, either native lipoprotein lipase in milk, activated when the globule membrane is damaged by agitation, foaming or homogenisation of raw milk, or bacterial lipases from psychrotrophs, which survive pasteurisation. Oxidative rancidity is the reaction of unsaturated fatty acids with oxygen through a free-radical chain reaction of initiation, propagation and termination. Hydroperoxides form first and break down into aldehydes and ketones with very low flavour thresholds, giving cardboard, metallic, oily or fishy off-flavours. Copper and iron ions and light catalyse it, which is why dairy equipment is made of stainless steel rather than copper and why milk is packed in light-protective containers. Antioxidants, such as tocopherols, interrupt the chain.
Enzymes are biological catalysts, almost all of them proteins. Like any catalyst they speed up a reaction by lowering its activation energy without changing its equilibrium, and they are not consumed. They are highly specific for their substrates and work under mild conditions of temperature and pH. Many need a non-protein helper: a cofactor, which may be a metal ion such as zinc, magnesium or iron, or a coenzyme, an organic molecule often derived from a vitamin. A coenzyme bound tightly is called a prosthetic group. The protein part without its cofactor is the apoenzyme, and the complete active enzyme is the holoenzyme.
The International Union of Biochemistry and Molecular Biology classifies enzymes by the reaction catalysed and gives each an EC number of four parts. The main classes are: EC 1, oxidoreductases (oxidation–reduction, such as lactate dehydrogenase and lactoperoxidase); EC 2, transferases (transfer of groups, such as transaminases and kinases); EC 3, hydrolases (hydrolysis, such as lipases, proteases, β-galactosidase and alkaline phosphatase); EC 4, lyases (removal or addition of groups to double bonds, such as decarboxylases and aldolase); EC 5, isomerases (such as glucose isomerase); and EC 6, ligases (joining with ATP use, such as DNA ligase). A seventh class, translocases, was added in 2018 for enzymes that move ions or molecules across membranes.
Many enzymes matter in dairying. Chymosin, an aspartic protease, cleaves κ-casein to coagulate milk. Plasmin, a serine protease native to milk, breaks down β-casein during storage of UHT milk and cheese ripening. Lipoprotein lipase causes hydrolytic rancidity. Alkaline phosphatase is inactivated by pasteurisation and is used to check that milk has been adequately heated. Lactoperoxidase, a heat-stable enzyme, is the basis of the lactoperoxidase system for preserving raw milk where cooling is not available.
The rate of an enzyme reaction depends on substrate concentration. At low substrate concentration the rate rises almost in proportion to it; at high concentration the enzyme becomes saturated and the rate approaches a maximum, Vmax. Michaelis and Menten explained this by a model in which enzyme E binds substrate S to form a complex ES, which then forms product P and releases the enzyme: E + S ⇌ ES → E + P.
The resulting Michaelis–Menten equation is v = Vmax[S]/(Km + [S]), where v is the initial rate. The Michaelis constant, Km, is the substrate concentration at which the rate is half of Vmax. It has units of concentration and is, under some conditions, an inverse measure of the enzyme's affinity for the substrate: a low Km means the enzyme reaches half its maximum rate at a low substrate concentration. Vmax equals kcat[E]total, where kcat, the turnover number, is the number of substrate molecules converted per active site per second. The ratio kcat/Km measures catalytic efficiency.
Two worked results are common in examinations. When [S] equals Km, v is exactly half of Vmax. When [S] is nine times Km, v = 9/10 of Vmax, so a large excess of substrate is needed to approach the maximum rate. Enzyme activity is expressed in units: one international unit (U) is the amount of enzyme that converts one micromole of substrate per minute under stated conditions, and the SI unit, the katal, is one mole per second. Assays measure initial rates, before substrate depletion and product accumulation slow the reaction.
Before computers made non-linear fitting easy, Km and Vmax were found from linear transformations. The Lineweaver–Burk, or double-reciprocal, plot graphs 1/v against 1/[S]: the line has slope Km/Vmax, intercept on the y-axis 1/Vmax and intercept on the x-axis −1/Km. It is easy to read but gives undue weight to the least reliable points at low substrate concentration. The Eadie–Hofstee plot (v against v/[S]) and the Hanes–Woolf plot ([S]/v against [S]) spread the error more evenly. Today Km and Vmax are best estimated by non-linear regression on the untransformed data, but the Lineweaver–Burk plot remains the standard way to show the type of inhibition.
Temperature affects enzyme activity in two opposing ways. Raising temperature increases the rate of the catalysed reaction, roughly doubling it for every 10 °C over the useful range, but it also increases the rate of denaturation. The apparent optimum temperature therefore depends on how long the enzyme is exposed. Heat inactivation of enzymes follows first-order kinetics and is described by D and z values, exactly as for micro-organisms. Some milk enzymes and bacterial enzymes are notably heat-stable: bacterial proteases and lipases from psychrotrophic pseudomonads can survive UHT treatment and cause gelation and off-flavours in stored UHT milk.
Each enzyme also has an optimum pH, set by the ionisation of groups at its active site and on the substrate. Chymosin acts best on κ-casein at the acid pH of cheese milk; pepsin works at the low pH of the stomach; alkaline phosphatase, as its name says, prefers alkaline conditions; and plasmin is most active near neutral to slightly alkaline pH.
An inhibitor is a substance that reduces the rate of an enzyme reaction. Reversible inhibitors bind non-covalently and can be removed, for example by dilution or dialysis. They are distinguished by their effect on Km and Vmax, which is shown most clearly on a Lineweaver–Burk plot.
A competitive inhibitor resembles the substrate and binds to the active site, competing with it. Its effect can be overcome by a high enough substrate concentration, so Vmax is unchanged while the apparent Km rises. On the double-reciprocal plot the lines for different inhibitor concentrations meet on the y-axis. A classic example is malonate inhibiting succinate dehydrogenase; galactose is a competitive inhibitor of some β-galactosidases, which is why lactose hydrolysis slows as galactose accumulates.
A non-competitive inhibitor (in the pure sense) binds to a site other than the active site, equally well whether or not substrate is bound. It reduces the amount of functional enzyme, so Vmax falls while Km is unchanged; the lines meet on the x-axis. An uncompetitive inhibitor binds only to the enzyme–substrate complex; both Vmax and the apparent Km fall by the same factor, and the double-reciprocal lines are parallel. Mixed inhibition, in which the inhibitor binds both free enzyme and the complex with different affinities, changes both parameters and gives lines meeting to the left of the y-axis away from either axis.
The inhibition constant, Ki, is the dissociation constant of the enzyme–inhibitor complex. For a competitive inhibitor, the apparent Km equals Km(1 + [I]/Ki); if [I] equals Ki, the apparent Km doubles.
An irreversible inhibitor binds covalently or so tightly that it does not dissociate, permanently inactivating the enzyme molecule. Organophosphorus insecticides and nerve agents, for example, react with the serine in the active site of acetylcholinesterase, which is why they are so toxic; the same chemistry makes diisopropyl fluorophosphate and related compounds inhibit serine proteases such as plasmin and trypsin in the laboratory. Heavy metal ions such as mercury and lead react with thiol groups of cysteine. Penicillin acylates the transpeptidase enzymes that build bacterial cell walls, which is why it kills growing bacteria.
Irreversible inhibition has two direct uses in dairy science. First, inhibition of enzymes is the principle of several residue tests: some rapid tests for antibiotic residues in milk detect β-lactam antibiotics because these drugs inhibit the growth of a sensitive test organism or bind to a specific receptor protein. Antibiotic residues are also important because they inhibit starter cultures, causing slow acid development in yoghurt and cheese. Second, inhibitors are tools for identifying enzymes: if a milk protease is inhibited by reagents specific for serine proteases, it is a serine protease.
Unlike reversible inhibition, irreversible inhibition cannot be overcome by adding more substrate, and the remaining activity depends on the time of exposure as well as on inhibitor concentration.
Cells control their metabolism by regulating key enzymes, usually those that catalyse an early, irreversible step of a pathway. Several mechanisms are used.
Allosteric regulation occurs when a molecule binds at a site other than the active site and changes the enzyme's shape and activity. Allosteric enzymes are often made of several subunits and show cooperative binding, so that a plot of rate against substrate concentration is sigmoidal rather than hyperbolic. Cooperativity is measured by the Hill coefficient: values above one indicate positive cooperativity. Activators shift the curve to the left and inhibitors to the right. Phosphofructokinase-1, the key control point of glycolysis, is inhibited allosterically by ATP and citrate and activated by AMP and fructose 2,6-bisphosphate. Feedback inhibition is a form of allosteric control in which the end product of a pathway inhibits the first committed enzyme, so that the cell stops making what it already has in plenty.
Covalent modification switches enzymes on or off by attaching or removing a group, most often phosphate, added by kinases and removed by phosphatases. Glycogen phosphorylase and glycogen synthase are regulated this way in opposite directions. Proteolytic activation turns an inactive precursor, a zymogen or proenzyme, into the active enzyme by cutting the chain. Prochymosin is activated to chymosin in the acid of the calf stomach; plasminogen, present in milk at higher concentration than plasmin, is converted to plasmin by plasminogen activators, a process that continues during storage of milk and cheese. Cells also regulate the amount of enzyme by controlling gene expression, a slower but more lasting form of control; the lac operon of Escherichia coli, induced by lactose, is the textbook example.
Glycolysis, the Embden–Meyerhof–Parnas pathway, is the sequence of ten reactions in the cytoplasm that splits one molecule of glucose into two molecules of pyruvate. In the first, preparatory phase two ATP are used: glucose is phosphorylated by hexokinase to glucose 6-phosphate, isomerised to fructose 6-phosphate, and phosphorylated again by phosphofructokinase-1 to fructose 1,6-bisphosphate, which aldolase splits into two three-carbon triose phosphates. In the second, pay-off phase each triose phosphate is oxidised by glyceraldehyde 3-phosphate dehydrogenase, reducing NAD⁺ to NADH, and four ATP in all are made by substrate-level phosphorylation, the last step catalysed by pyruvate kinase. The net yield per glucose is 2 ATP, 2 NADH and 2 pyruvate. The irreversible steps catalysed by hexokinase, phosphofructokinase-1 and pyruvate kinase are the points of regulation.
Glycolysis can continue only if NAD⁺ is regenerated from NADH. With oxygen and mitochondria, NADH is reoxidised by the respiratory chain. Without them, cells regenerate NAD⁺ by reducing pyruvate. Lactate dehydrogenase reduces pyruvate to lactate; this is homolactic fermentation, carried out by the lactococci, Streptococcus thermophilus and the homofermentative lactobacilli of dairy starters, and by human muscle during intense exercise. Yeasts decarboxylate pyruvate to acetaldehyde and reduce it to ethanol, as in kefir and koumiss. Heterofermentative lactic acid bacteria, such as Leuconostoc species, use the phosphoketolase pathway and produce lactate, ethanol or acetate, and carbon dioxide, which is why they give gas holes and flavour in some cheeses and cultured butters.
Lactose enters these pathways after hydrolysis. Many lactic acid bacteria take lactose up and split it with β-galactosidase; lactococci take it up as lactose phosphate through a phosphotransferase system, split it with phospho-β-galactosidase, and metabolise the galactose 6-phosphate by the tagatose pathway. Galactose itself is converted to glucose 1-phosphate by the Leloir pathway.
In aerobic cells pyruvate enters the mitochondria and is oxidatively decarboxylated by the pyruvate dehydrogenase complex to acetyl-coenzyme A, releasing one CO₂ and reducing one NAD⁺. The complex needs five coenzymes derived from vitamins and related compounds: thiamine pyrophosphate, lipoic acid, coenzyme A, FAD and NAD⁺.
The tricarboxylic acid (TCA) cycle, also called the citric acid cycle or Krebs cycle, oxidises the two-carbon acetyl group completely to CO₂. Acetyl-CoA combines with oxaloacetate (four carbons) to form citrate (six carbons), catalysed by citrate synthase. Citrate is isomerised to isocitrate, which is oxidatively decarboxylated to α-ketoglutarate; α-ketoglutarate is decarboxylated to succinyl-CoA; succinyl-CoA yields succinate with formation of one GTP or ATP; succinate is oxidised to fumarate by succinate dehydrogenase, reducing FAD; fumarate is hydrated to malate; and malate is oxidised to regenerate oxaloacetate. Each turn of the cycle therefore yields 3 NADH, 1 FADH₂, 1 GTP and 2 CO₂.
The cycle is amphibolic: it serves both breakdown and synthesis. Its intermediates are drawn off to make amino acids (α-ketoglutarate to glutamate, oxaloacetate to aspartate) and are replenished by anaplerotic reactions, such as the carboxylation of pyruvate to oxaloacetate. Citrate is a key milk constituent: in the mammary gland it is exported from mitochondria and is both secreted into milk, where it binds calcium, and cleaved to provide acetyl-CoA for fatty acid synthesis. Some lactic acid bacteria, such as Lactococcus lactis biovar diacetylactis and Leuconostoc species, ferment citrate to diacetyl, the buttery aroma of cultured butter and buttermilk.
The NADH and FADH₂ produced by glycolysis, pyruvate oxidation and the TCA cycle carry electrons to the respiratory chain in the inner mitochondrial membrane of eukaryotes, or in the cell membrane of respiring bacteria. The chain consists of four complexes and two mobile carriers. Complex I (NADH dehydrogenase) accepts electrons from NADH; complex II (succinate dehydrogenase) from FADH₂; ubiquinone carries them to complex III; cytochrome c carries them to complex IV, cytochrome oxidase, which reduces oxygen to water. As electrons flow, complexes I, III and IV pump protons out across the membrane, building a proton-motive force.
According to Mitchell's chemiosmotic theory, the protons flow back through ATP synthase, a rotary enzyme, and the energy released drives the synthesis of ATP from ADP and phosphate. This is oxidative phosphorylation. Uncouplers, such as 2,4-dinitrophenol, let protons leak back without making ATP, so energy is released as heat; brown adipose tissue uses a natural uncoupling protein for heat production. Cyanide and carbon monoxide block cytochrome oxidase.
Modern estimates give about 2.5 ATP per NADH and 1.5 ATP per FADH₂ oxidised, so the complete oxidation of one glucose yields about 30 to 32 ATP, depending on how cytoplasmic NADH enters the mitochondria. Older textbooks, using whole-number ratios of 3 and 2, give 36 to 38; examinations may use either convention, so the basis should be stated. The contrast with fermentation is the key point: homolactic fermentation yields only 2 ATP per glucose, so lactic acid bacteria must ferment large amounts of sugar and produce much acid to grow, which is exactly what makes them useful in dairy fermentations.
Triacylglycerols are hydrolysed by lipases to glycerol and fatty acids. Glycerol enters glycolysis as dihydroxyacetone phosphate. Fatty acids are activated in the cytoplasm to fatty acyl-CoA at the cost of the equivalent of two ATP, carried into the mitochondria by the carnitine shuttle, and broken down by β-oxidation. Each round of β-oxidation removes two carbons as acetyl-CoA through four steps: oxidation by an FAD-linked dehydrogenase, hydration, oxidation by an NAD-linked dehydrogenase, and thiolytic cleavage. Each round thus yields one FADH₂, one NADH and one acetyl-CoA.
Palmitic acid (16:0) passes through seven rounds, producing 8 acetyl-CoA, 7 FADH₂ and 7 NADH. With 10 ATP per acetyl-CoA oxidised in the TCA cycle, 1.5 per FADH₂ and 2.5 per NADH, the total is 80 + 10.5 + 17.5 = 108 ATP, less 2 for activation, giving a net 106 ATP; older ratios give 129. Unsaturated fatty acids need extra isomerase and reductase steps, and odd-chain fatty acids yield propionyl-CoA, which is converted to succinyl-CoA in a vitamin B12-dependent reaction.
When acetyl-CoA is produced faster than the TCA cycle can use it, as in starvation or when oxaloacetate is scarce, the liver converts it to ketone bodies: acetoacetate, β-hydroxybutyrate and acetone. High-yielding dairy cows in early lactation often mobilise body fat faster than they can oxidise it and develop ketosis, with lowered milk yield and a high fat-to-protein ratio in milk. In ruminants β-hydroxybutyrate is also made from butyrate in the rumen wall and is a normal precursor of milk fat.
Fatty acid synthesis takes place in the cytoplasm and is not simply β-oxidation in reverse. Acetyl-CoA is first carboxylated to malonyl-CoA by acetyl-CoA carboxylase, a biotin-dependent enzyme and the main control point of the pathway. The fatty acid synthase complex then builds the chain two carbons at a time, using malonyl-CoA as the donor and NADPH as the reductant; the NADPH comes mainly from the pentose phosphate pathway. In most tissues the product is palmitic acid, which can be lengthened by elongases and desaturated by desaturases such as stearoyl-CoA desaturase, which converts stearic acid to oleic acid.
The mammary gland of ruminants is a remarkable fat factory. Ruminants absorb little glucose; their main carbon sources are acetate and butyrate from rumen fermentation, carried as acetate and β-hydroxybutyrate in the blood. The mammary gland uses these to synthesise fatty acids from 4 to 14 carbons and about half of the palmitic acid de novo, and the mammary fatty acid synthase releases the short and medium chains that give ruminant milk fat its character. Fatty acids of 18 carbons and the rest of the palmitic acid come preformed from the blood, from the diet and from body fat. Stearoyl-CoA desaturase in the gland converts stearic acid to oleic acid and vaccenic acid to rumenic acid, the main conjugated linoleic acid of milk.
This biochemistry explains how feeding changes milk fat. Diets low in fibre or high in readily fermentable starch, or supplemented with unsaturated oils, lead to trans fatty acid intermediates from incomplete rumen biohydrogenation, some of which, notably trans-10, cis-12 conjugated linoleic acid, inhibit mammary fat synthesis and cause milk fat depression.
Amino acids are used to build proteins and many other molecules, and those in excess are broken down. The first step in the breakdown of most amino acids is transamination: the amino group is transferred to α-ketoglutarate, forming glutamate and the corresponding α-keto acid. Aminotransferases (transaminases) need pyridoxal phosphate, the coenzyme form of vitamin B6. Glutamate is then oxidatively deaminated by glutamate dehydrogenase, releasing ammonia. In mammals ammonia is converted to urea in the liver by the urea cycle, which consumes the equivalent of four high-energy phosphate bonds per urea; urea passes into the blood and, at low concentration, into milk, where its level is used to judge the protein-energy balance of a herd's diet. Added urea in milk is an adulterant that some national tests look for.
The carbon skeletons of amino acids are converted to intermediates of central metabolism. Glucogenic amino acids give pyruvate or TCA cycle intermediates and can be used to make glucose; ketogenic amino acids, leucine and lysine alone in the strict sense, give acetyl-CoA or acetoacetate.
In dairy products microbial amino acid metabolism matters for flavour and safety. During cheese ripening, proteolysis releases free amino acids, which starter and non-starter bacteria convert by transamination, decarboxylation, deamination and elimination into flavour compounds: methional and methanethiol from methionine, aldehydes and alcohols from the branched-chain amino acids, and phenolic compounds from aromatic amino acids. Decarboxylation of amino acids gives biogenic amines, such as histamine from histidine and tyramine from tyrosine, which can reach high concentrations in long-ripened cheese and cause adverse reactions in sensitive consumers. The Strecker degradation of amino acids by dicarbonyl compounds from the Maillard reaction produces aldehydes that contribute to cooked and stale flavours in heated milk products.
Nucleic acids store and express genetic information. Their units are nucleotides, each made of a five-carbon sugar, a phosphate group and a nitrogenous base. The bases are purines, adenine (A) and guanine (G), and pyrimidines, cytosine (C), thymine (T) and uracil (U). DNA contains deoxyribose and the bases A, G, C and T; RNA contains ribose and A, G, C and U. Nucleotides are linked by phosphodiester bonds between the 3′ carbon of one sugar and the 5′ carbon of the next, giving each strand a direction from 5′ to 3′.
DNA, as described by Watson and Crick in 1953, is a double helix of two antiparallel strands held together by hydrogen bonds between complementary bases: adenine pairs with thymine by two hydrogen bonds, and guanine with cytosine by three. Chargaff's rules follow: in double-stranded DNA the amount of A equals T and G equals C. DNA rich in G–C pairs needs a higher temperature to separate its strands, a property used in classifying bacteria by their G + C content; lactic acid bacteria are a low G + C group, while bifidobacteria have high G + C DNA.
RNA is usually single-stranded. Messenger RNA (mRNA) carries the code for a protein; transfer RNA (tRNA) brings amino acids to the ribosome; ribosomal RNA (rRNA) forms the structure and catalytic core of the ribosome. The gene for the small-subunit ribosomal RNA, 16S rRNA in bacteria, has regions conserved across all bacteria and regions that vary between species, which makes it the standard target for identifying bacteria by sequencing and for profiling the microbial communities of milk and cheese.
DNA replication is semi-conservative: each new double helix contains one old and one new strand, as Meselson and Stahl showed. Replication begins at origins, where helicase unwinds the strands; DNA polymerase adds nucleotides only to a free 3′ end, so one strand (the leading strand) is copied continuously and the other (the lagging strand) in short Okazaki fragments joined by DNA ligase. Proofreading by the polymerase keeps errors very rare. The polymerase chain reaction (PCR) copies a chosen stretch of DNA in a tube by cycles of heat denaturation, primer annealing and extension with a heat-stable polymerase, and underlies most rapid detection and identification methods in dairy microbiology.
Transcription copies a gene into RNA. RNA polymerase binds to a promoter and synthesises RNA from 5′ to 3′, using one DNA strand as the template. In bacteria the mRNA is used directly, often while still being made; in eukaryotes the initial transcript is processed by adding a 5′ cap and a poly(A) tail and by splicing out introns.
The genetic code relates the sequence of mRNA to the sequence of amino acids. Each codon is a group of three bases, giving 64 codons: 61 specify amino acids and 3 (UAA, UAG and UGA) are stop signals. AUG codes for methionine and is the usual start codon. The code is degenerate, because most amino acids have more than one codon, and it is nearly universal across living things. A change of one base, a point mutation, may leave the protein unchanged, substitute one amino acid, or create a stop codon. Milk protein genetic variants arise this way: β-casein variants A1 and A2 differ by one amino acid at position 67, histidine in A1 and proline in A2.
Translation is the synthesis of a polypeptide on the ribosome. Bacterial ribosomes are 70S particles made of 30S and 50S subunits; eukaryotic cytoplasmic ribosomes are 80S, made of 40S and 60S subunits. The difference is the basis of the selective action of antibiotics such as tetracyclines, aminoglycosides and macrolides, which block bacterial ribosomes. Each amino acid is attached to its tRNA by a specific aminoacyl-tRNA synthetase, using ATP. The anticodon of the tRNA pairs with the codon of the mRNA, and the ribosome joins the amino acids by peptide bonds in the order the mRNA dictates. Translation has three stages, initiation, elongation and termination, and ends when a stop codon is reached and a release factor frees the chain.
Proteins destined for secretion, such as the milk proteins, are made on ribosomes bound to the rough endoplasmic reticulum. A signal peptide at the N-terminus directs the growing chain into the reticulum and is then cut off. The proteins pass to the Golgi apparatus, where the caseins are phosphorylated on serine residues by a casein kinase and κ-casein is glycosylated. In the Golgi and secretory vesicles the phosphorylated caseins bind calcium phosphate and assemble into casein micelles, which are released into the alveolar lumen by exocytosis. α-Lactalbumin, also made in the mammary cell, joins galactosyltransferase in the Golgi to form lactose synthase, so the cell makes lactose; the lactose draws water osmotically into the vesicles and so largely controls the volume of milk produced.
Mammary gene expression is controlled by hormones, especially prolactin, together with glucocorticoids and insulin. The casein genes lie close together in a cluster on one chromosome in cattle, and their variants are inherited together.
Vitamins are organic compounds needed in small amounts that the body cannot make in sufficient quantity. Most water-soluble vitamins act as precursors of coenzymes. Thiamine (vitamin B1) forms thiamine pyrophosphate, the coenzyme of oxidative decarboxylations such as pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and of transketolase. Riboflavin (vitamin B2) forms flavin mononucleotide and flavin adenine dinucleotide, the carriers of hydrogen in many oxidation–reduction reactions. Niacin forms NAD⁺ and NADP⁺. Pantothenic acid is part of coenzyme A, the carrier of acyl groups. Pyridoxine (vitamin B6) forms pyridoxal phosphate, the coenzyme of transamination and decarboxylation of amino acids. Biotin is the coenzyme of carboxylation reactions such as those of acetyl-CoA carboxylase and pyruvate carboxylase. Folate forms tetrahydrofolate, which carries one-carbon units for the synthesis of nucleotides. Cobalamin (vitamin B12) is the coenzyme of methylmalonyl-CoA mutase and of methionine synthase. Vitamin C, ascorbic acid, is a reducing agent and the cofactor of hydroxylases, including those that make collagen.
Milk is a good source of riboflavin and vitamin B12 and a moderate source of several other B vitamins, but a poor source of vitamin C, which is also largely lost during processing and storage. Riboflavin is sensitive to light: in milk exposed to sunlight or fluorescent light it acts as a photosensitiser, giving the light-induced off-flavour through the oxidation of methionine to methional and of lipids, and is itself destroyed. Some lactic acid bacteria and propionibacteria synthesise B vitamins, so fermented milks and Swiss-type cheeses can be richer in folate or B12 than the milk they came from; others consume them.
The fat-soluble vitamins are A, D, E and K. They are absorbed with dietary fat, stored in the liver and fatty tissues, and can accumulate to toxic levels if taken in large excess, especially vitamins A and D.
Vitamin A, retinol, is formed from β-carotene and other provitamin A carotenoids. Its aldehyde, retinal, is the light-sensitive part of the visual pigment rhodopsin, and retinoic acid regulates gene expression in growth and immunity. Deficiency causes night blindness and, in severe cases, damage to the eye and increased susceptibility to infection; it remains a public-health problem in parts of Africa and Asia. Milk fat contains both retinol and carotene, so skim milk loses most of its vitamin A and is often fortified.
Vitamin D (cholecalciferol, D3, from animals and from the skin under ultraviolet light; ergocalciferol, D2, from fungi) is converted in the liver to 25-hydroxyvitamin D and in the kidney to the active hormone 1,25-dihydroxyvitamin D. It increases the absorption of calcium and phosphate from the gut and is essential for bone mineralisation; deficiency causes rickets in children and osteomalacia in adults. Milk contains little vitamin D naturally, and many countries fortify milk with it, at levels set by national rules.
Vitamin E is a group of tocopherols and tocotrienols, of which α-tocopherol is the most active. It is the main lipid-soluble antioxidant, protecting membrane lipids from peroxidation, and its presence in milk fat helps to protect against oxidised flavour. Vitamin K is needed for the γ-carboxylation of glutamate residues in blood-clotting factors and in bone proteins; milk is a minor source.
Minerals are inorganic elements needed in the diet. The macrominerals, needed in amounts of more than about 100 mg per day by adults, are calcium, phosphorus, magnesium, sodium, potassium and chloride. Calcium and phosphorus form hydroxyapatite in bone and teeth; calcium also acts in muscle contraction, nerve transmission and blood clotting, and as a second messenger in cells. Magnesium is the cofactor of every enzyme that uses ATP, because the true substrate is the magnesium–ATP complex. Sodium and potassium maintain membrane potentials and fluid balance, and chloride balances their charges.
Milk is one of the richest dietary sources of calcium, about 1.2 g per litre in cow milk, much of it held with phosphate in the colloidal calcium phosphate of the casein micelles. It is also rich in phosphorus and potassium. Milk is, however, poor in iron, and infants fed unmodified cow milk are at risk of iron deficiency, which is why infant formulas are fortified with iron.
Trace elements are needed in milligram or microgram amounts and often act as enzyme cofactors. Iron is part of haemoglobin, myoglobin and cytochromes. Zinc is the cofactor of more than three hundred enzymes, including alkaline phosphatase and carbonic anhydrase. Copper is the cofactor of cytochrome oxidase and superoxide dismutase; in milk products, even traces of copper strongly catalyse lipid oxidation. Selenium is part of glutathione peroxidase, an antioxidant enzyme; the selenium content of milk varies widely with that of the soil and feed. Iodine is part of the thyroid hormones; milk iodine depends on feed supplements and on the use of iodine-based teat dips and sanitisers, which can raise it markedly. Molybdenum is the cofactor of xanthine oxidase, an enzyme abundant in the milk fat globule membrane.
Draw the structures that recur in every paper until you can do so without notes: α- and β-lactose, a triacylglycerol, a phospholipid, the zwitterion of an amino acid and a peptide bond. Learn the Lineweaver–Burk patterns for the three types of reversible inhibition as pictures, and practise Henderson–Hasselbalch and Michaelis–Menten calculations. In the laboratory, carry out the reducing-sugar, biuret, ninhydrin and saponification tests yourself, because examiners and employers expect you to know what a positive result looks like and why it appears.
A dairy chemist or microbiologist five years into the job uses this biochemistry to explain problems: why stored UHT milk gels (heat-stable bacterial proteases, plasmin), why a cheese is bitter (hydrophobic peptides), why a powder browns (Maillard reaction at intermediate water activity), why milk tastes oxidised after display under lights (riboflavin photosensitisation), and why a starter is slow (antibiotic residues or phage). Enzyme kinetics guide the dosing of lactase for lactose-free milk and the choice of time and temperature for its action.
A technical or quality director with 25 years' experience relies on biochemistry when approving new processes and ingredients: lactose hydrolysis and galacto-oligosaccharide production, enzyme-modified cheese flavours, protein hydrolysates for infant nutrition, and the stability of vitamin-fortified products through shelf life. Questions from regulators and customers about allergenic proteins, biogenic amines, trans fatty acids and A1/A2 milk need answers grounded in the underlying chemistry rather than marketing claims. Decisions on enzyme suppliers, processing aids and their labelling also rest on knowing what each enzyme does and when it is inactivated.
A professor or research leader works at the molecular level: protein structure–function relations under processing, enzyme engineering of lactases and proteases, the genomics and metabolic pathways of starter bacteria, and the regulation of mammary synthesis of milk components. This biochemistry is the language of the field's journals and research proposals, and teaching it well, with dairy examples rather than generic ones, is what prepares students for the advanced chemistry and microbiology subjects that follow. Setting examination papers and reviewing manuscripts call for the same precision.
Detailed mechanisms of individual glycolytic and TCA enzymes, gluconeogenesis, glycogen metabolism, the pentose phosphate pathway, nucleotide metabolism and membrane transport are only named or summarised. Recommended intakes of vitamins and minerals and fortification levels are not quoted because they differ by country and age group; see human-nutrition-dairy. The detailed chemistry of milk proteins, lipids, lactose and native enzymes is left to the dairy chemistry subjects, and the metabolism of starter bacteria to starter-cultures-lab and fermentation-dairy-biotechnology.
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