Dairy Science & Technology · Dairy Microbiology · also Industrial Microbiology, Biotechnology for Dairy Applications
Bioreactors and fermentation processes, recombinant enzymes, precision fermentation and the biotechnology now reshaping dairy ingredients.
In the exams: ICAR AICE subject 36 Units 2 and 7 and subject 37 Unit 7
Fermentation technology and dairy biotechnology deal with the use of microorganisms and their enzymes, grown under controlled conditions in fermenters, to make products and ingredients for the dairy industry and from dairy raw materials. The subject spans the engineering of fermenters and the kinetics of microbial growth, the recovery and purification of products, the industrial production of organic acids and enzymes from whey and other substrates, the coagulants and lactases that the dairy industry buys in large amounts, immobilised enzymes and continuous processes, enzyme-modified cheese, the genetic modification and genome editing of dairy microbes, the production of milk proteins by precision fermentation, and the conversion of whey into biomass, ethanol and other products.
It matters because a large part of modern dairy processing depends on products of fermentation made outside the dairy: almost all cheese is now made with coagulants produced by microorganisms, lactose-free milk depends on industrially produced lactase, and flavour ingredients such as enzyme-modified cheese are made in enzyme reactors. Fermentation also offers routes to add value to whey and permeate, and precision fermentation is beginning to produce milk proteins without cows, raising questions of technology, regulation, labelling and the future of the dairy industry.
This manual builds on Starter Cultures and Lactic Acid Bacteria, which covers the metabolism, genetics and phage biology of starters, on Whey and Dairy By-product Utilisation, which covers whey composition, disposal and fermentation routes in outline, and on Milk Proteins and Enzymes and Cheese Science and Technology, which cover chymosin action and coagulation. Advanced Dairy Microbiology covers genomics and CRISPR biology in more depth. It is examined in doctoral and research-fellowship entrance papers in dairy microbiology and dairy technology, in graduate engineering and food technology aptitude papers, and in agricultural research service and lectureship eligibility tests at Master's level. Expect questions on fermenter types, growth kinetics and chemostat calculations, scale-up criteria, downstream steps, lactic acid production, enzyme sources and properties, coagulants, immobilisation methods, lactose hydrolysis, enzyme-modified cheese, genetic tools and regulation, precision fermentation and whey utilisation.
A fermenter, or bioreactor, provides a controlled, contained environment in which a chosen microorganism grows or makes a product without contamination. The most common industrial design is the stirred-tank reactor: a cylindrical stainless steel vessel with a height-to-diameter ratio of about 2 to 3 to 1, an agitator shaft carrying one or more impellers, usually four baffles fixed to the wall to prevent vortex formation, a sparger that introduces sterile air at the bottom for aerobic processes, a jacket or internal coils for heating and cooling, and ports for probes measuring temperature, pH, dissolved oxygen, foam and pressure. Radial-flow disc turbines (Rushton turbines) give high shear and good gas dispersion; axial-flow hydrofoil impellers circulate liquid with lower power. Airlift and bubble-column fermenters mix the broth by the rising air alone, with no moving parts, which suits shear-sensitive cells and very large vessels. Packed-bed and fluidised-bed reactors hold immobilised cells or enzymes, and membrane bioreactors retain cells while removing product. Fermenters are designed hygienically, as described in the subject on cleaning-in-place and hygienic design, with polished surfaces, steam-sealed valves, sterile air filters, cleaning-in-place and sterilisation-in-place, because a single contaminating organism can outgrow the production strain. Media are sterilised in the vessel at 121 degrees Celsius or continuously at higher temperatures for shorter times in heat exchangers, which reduces damage to nutrients.
Fermentations are classified in several ways. By the physical state of the substrate, submerged fermentation grows organisms in liquid medium and is used for most dairy-related products, while solid-state fermentation grows organisms, usually fungi, on moist solid substrates such as wheat bran, with little free water; it is used for some fungal enzymes and traditional foods. By mode of operation, batch fermentation adds all nutrients at the start and harvests at the end; fed-batch fermentation adds substrate during the run to keep its concentration low, avoiding substrate inhibition and catabolite repression and reaching high cell densities, and is the dominant mode for industrial enzymes and recombinant proteins; continuous fermentation adds fresh medium and removes broth at the same rate, holding the culture in a steady state, as in the chemostat described in chapter 2. By oxygen requirement, aerobic fermentations, such as those making fungal enzymes and yeast biomass, need intensive aeration, while anaerobic or microaerophilic fermentations, such as lactic acid production and the growth of most starter cultures, need no aeration and simpler vessels. By the relation of product to growth, products may be growth-associated, such as lactic acid and biomass, non-growth-associated, such as many secondary metabolites and some enzymes, or mixed. In the dairy industry, the production of concentrated starter cultures is itself a fermentation: cultures are grown in pH-controlled batch fermenters on milk-based or whey-based media, concentrated by centrifugation, and frozen or freeze-dried, as described in Starter Cultures and Lactic Acid Bacteria.
In the exponential phase of a batch culture, biomass increases at a rate proportional to its concentration: dX/dt equals mu times X, so X at time t equals X0 times e to the power mu t, and the doubling time is ln 2 divided by mu. A culture with a specific growth rate of 0.5 per hour doubles every 1.39 hours, and 0.1 gram per litre of cells becomes about 2.0 grams per litre in 6 hours. The Monod equation relates the specific growth rate to the concentration of a limiting substrate: mu equals mu max times S divided by Ks plus S, where Ks, the saturation constant, is the substrate concentration at which mu is half of mu max. The biomass yield coefficient, Yx/s, is the mass of cells formed per mass of substrate used. Product formation is described by the Luedeking-Piret equation, in which the rate of product formation equals alpha times the growth rate plus beta times the biomass: alpha is the growth-associated and beta the non-growth-associated term; lactic acid production has both. In a chemostat, the dilution rate D is the flow rate divided by the culture volume. At steady state the specific growth rate equals D, and the residual substrate concentration is Ks times D divided by mu max minus D. With mu max 0.6 per hour, Ks 0.2 grams per litre, D 0.3 per hour, feed substrate 20 grams per litre and yield 0.5, the residual substrate is 0.2 grams per litre, the biomass is 9.9 grams per litre and the biomass productivity is D times X, 2.97 grams per litre per hour. If D exceeds about mu max times S0 divided by Ks plus S0, 0.594 per hour here, the cells are washed out.
In aerobic fermentations, oxygen is often the limiting nutrient, because it dissolves poorly in water, about 7 milligrams per litre at 30 degrees Celsius in equilibrium with air. The oxygen transfer rate equals kLa times the difference between the saturation concentration C star and the actual dissolved oxygen concentration CL, where kLa is the volumetric mass transfer coefficient; a kLa of 100 per hour with C star 7 and CL 2 milligrams per litre gives an oxygen transfer rate of 500 milligrams per litre per hour. kLa increases with agitation power per unit volume and with air flow, and is measured by methods such as dynamic gassing-out. Scale-up is the transfer of a process from laboratory to pilot and production fermenters, typically by factors of ten at each stage, while keeping the conditions that matter for the organism. Because not all conditions can be held constant when vessel size increases, engineers choose a scale-up criterion: constant power per unit volume, constant kLa, constant impeller tip speed (which governs shear), constant mixing time, or constant dissolved oxygen. For geometrically similar vessels, power is proportional to the cube of impeller speed times the fifth power of impeller diameter, so keeping power per unit volume constant requires the impeller speed to fall as the diameter ratio to the power two thirds: a tenfold increase in diameter needs about 0.22 times the speed, while constant tip speed needs one tenth. Large vessels have longer mixing times and gradients of substrate, oxygen and pH, which can reduce yields; scale-down experiments in the laboratory simulate these gradients to predict performance.
Downstream processing comprises all the steps that recover and purify a product from the fermentation broth, and it often accounts for half or more of the total production cost, especially for high-purity proteins. The sequence depends on whether the product is the cells themselves, inside the cells or secreted into the medium, but generally follows four stages. Removal of insolubles separates cells and debris from the liquid, by centrifugation with disc-stack separators, by filtration on rotary vacuum filters, or by microfiltration with cross-flow membranes, often after flocculation. If the product is intracellular, cells are disrupted by high-pressure homogenisers, bead mills, enzymatic lysis or chemical treatment, and the debris removed. Isolation or concentration reduces the volume and removes much of the water, using ultrafiltration, evaporation, precipitation with salts such as ammonium sulphate or with solvents, or extraction. Purification removes closely related impurities, mainly by chromatography: ion-exchange chromatography separates by charge, hydrophobic interaction chromatography by surface hydrophobicity, gel filtration by size, and affinity chromatography by specific binding, for example of a tagged protein. Polishing and formulation give the final form: crystallisation, sterile filtration, spray drying or freeze-drying, and addition of stabilisers, carriers and preservatives. Many of these unit operations, such as membranes, evaporators and dryers, are the same as those in dairy plants, described in Membrane Processing and Dairy Ingredients and Evaporation and Drying of Dairy Products.
The required purity drives the cost of recovery. Dairy enzymes such as coagulants and lactases are sold as concentrated liquid or powder preparations of specified activity, standardised, stabilised and free from production organisms; they are not crystalline proteins. Food enzyme preparations must meet specifications such as those of the Joint FAO/WHO Expert Committee on Food Additives and the Food Chemicals Codex, including limits on heavy metals, absence of Salmonella and of antibiotic activity, and, for preparations from genetically modified organisms, absence of viable production organisms and, in many jurisdictions, of recombinant DNA. For organic acids such as lactic acid, the traditional recovery route is precipitation as a calcium salt, described in chapter 4, while newer routes use electrodialysis, ion exchange, extraction and esterification with distillation. For biomass products such as starter cultures, probiotics and single-cell protein, recovery is mainly centrifugation, washing and drying, and survival of the cells may be critical, so cryoprotectants and gentle drying are used. Downstream processing must be designed together with the fermentation: for example, using a defined medium without complex proteins, or a host that secretes the product with few other proteins, simplifies purification. In situ product removal, in which the product is taken out continuously during fermentation by extraction, adsorption or membranes, relieves product inhibition, which is important for lactic acid. Mass balances across each step allow overall recovery to be calculated as the product of step yields: four steps of 90 per cent each give an overall yield of about 66 per cent.
Lactic acid is used as an acidulant and preservative in foods, in pharmaceuticals and cosmetics, and increasingly as the monomer for poly-lactic acid, a biodegradable plastic. Most of the world's lactic acid is now made by fermentation, because chemical synthesis from acetaldehyde and hydrogen cyanide gives a racemic mixture of the D and L forms, while fermentation can give optically pure L- or D-lactic acid, which is needed for poly-lactic acid with controlled properties. Production organisms include homofermentative lactic acid bacteria such as Lacticaseibacillus casei and rhamnosus for L-lactic acid and Lactobacillus delbrueckii for D-lactic acid, thermophilic Bacillus-related species such as Heyndrickxia coagulans growing at about 50 degrees Celsius, where contamination is less likely, and engineered acid-tolerant yeasts that can produce at low pH. Substrates include glucose from starch, sucrose and, in the dairy context, whey and whey permeate, whose lactose is fermented by lactose-positive strains. Whey permeate must be supplemented with nitrogen and growth factors, such as yeast extract or corn steep liquor, because lactic acid bacteria are nutritionally demanding. The theoretical yield for homolactic fermentation of lactose is 4 moles of lactic acid per mole, or 360 grams from 342 grams, 1.05 grams per gram, and practical yields are around 0.9 grams per gram. Lactic acid inhibits its producers, so the pH is held at about 5.5 to 6.5 by adding calcium carbonate, calcium hydroxide, ammonia or sodium hydroxide, which forms lactate salts.
In the classical recovery process, the fermentation is neutralised with calcium carbonate or hydroxide to give calcium lactate; the broth is filtered, the calcium lactate is acidified with sulphuric acid to release lactic acid and precipitate calcium sulphate (gypsum), and the lactic acid is purified by carbon treatment, ion exchange and evaporation. About one tonne of gypsum is produced per tonne of lactic acid, which is a disposal problem and has stimulated alternatives: fermentation at low pH with acid-tolerant organisms, electrodialysis, solvent extraction, adsorption, and esterification with methanol or ethanol followed by distillation and hydrolysis to give high-purity acid. Other organic acids relevant to dairy can also be made by fermentation. Propionic acid and propionates, used as mould inhibitors in bakery products, can be made from whey by propionibacteria such as Acidipropionibacterium acidipropionici, though most propionic acid is still made petrochemically; fermented whey or skim milk containing propionate and bacteriocins is sold as a clean-label preservative ingredient. Acetic acid is made by acetic acid bacteria from ethanol, which can itself come from whey. Citric acid is made by Aspergillus niger, mostly from sucrose and starch hydrolysates. Vitamin B12 is made commercially by fermentation, and Propionibacterium freudenreichii, a cheese organism, is one of the producers. Lactobionic acid, made by oxidising lactose, and galacto-oligosaccharides are other value-added products from lactose, discussed in Whey and Dairy By-product Utilisation and Probiotics, Prebiotics and Functional Dairy Foods.
Industrial enzymes are produced mainly by submerged fed-batch fermentation of a small number of safe, well-characterised production organisms, often called production hosts or platforms: the filamentous fungi Aspergillus niger, Aspergillus oryzae and Trichoderma reesei; the yeasts Kluyveromyces lactis and Komagataella phaffii (formerly Pichia pastoris); and the bacteria Bacillus subtilis and Bacillus licheniformis. Most industrial enzymes are secreted into the medium, which makes recovery easier, and many are now produced by recombinant strains carrying the gene for the enzyme of interest, giving higher yields and more consistent products. Beta-galactosidase, or lactase, hydrolyses lactose to glucose and galactose and, at high lactose concentrations, transfers galactose to other sugars to form galacto-oligosaccharides. Its properties depend on the source. Yeast lactases from Kluyveromyces lactis and Kluyveromyces marxianus are intracellular enzymes with a neutral pH optimum of about 6.5 to 7, suited to milk; they need potassium or magnesium ions and are relatively heat-labile. Fungal lactases from Aspergillus oryzae and Aspergillus niger are extracellular with acid pH optima of about 3.5 to 5, suited to acid whey and pharmaceutical tablets taken by lactose-intolerant people. Bacterial lactases, such as that of Bacillus circulans, are used mainly for making galacto-oligosaccharides because of their strong transgalactosylation activity. Lactase preparations for milk must be free of proteolytic side activities, which would cause bitterness and gelation in long-life milk.
Proteases are used in the dairy industry as coagulants (chapter 6), to accelerate cheese ripening, to make enzyme-modified cheese (chapter 9), and to make protein hydrolysates for infant formula, sports nutrition and clinical nutrition. Bacterial proteases from Bacillus licheniformis and Bacillus subtilis, such as the alkaline serine protease subtilisin and neutral metalloproteases, and fungal proteases from Aspergillus species, are commonly used, often together with peptidases, which remove amino acids from the ends of peptides and reduce the bitterness caused by hydrophobic peptides. Hydrolysates are characterised by the degree of hydrolysis, the percentage of peptide bonds cleaved, which is related to functional properties, bitterness and allergenicity: extensively hydrolysed whey or casein is used in formulas for infants with cow's milk protein allergy. Lipases hydrolyse triglycerides into free fatty acids and partial glycerides and are used to develop flavour in cheese, enzyme-modified cheese and butter flavours. Their specificity matters: pregastric esterases from the tongue glands of calves, kids and lambs preferentially release short-chain fatty acids such as butyric acid and give the piquant flavour of Italian cheeses such as Provolone and Pecorino, while microbial lipases from Rhizomucor miehei, Penicillium roqueforti and Candida species give different fatty acid profiles. Enzymes for food use are regulated as processing aids or food enzymes: in the European Union they are being placed on a positive list under Regulation (EC) No 1332/2008 after evaluation by the European Food Safety Authority, while in the United States they are generally recognised as safe or approved as additives.
Calf rennet, extracted from the fourth stomach (abomasum) of milk-fed calves, contains chymosin as its main enzyme, together with some pepsin. Chymosin is an aspartic protease that cleaves the Phe105-Met106 bond of kappa-casein very specifically, destabilising the casein micelles and allowing coagulation, as described in Milk Proteins and Enzymes and Cheese Science and Technology; its low general proteolytic activity gives a high ratio of clotting to proteolytic activity, good cheese yield and little bitterness. From the 1960s, the growth of cheese production and a shortage of calf stomachs led to substitutes. Microbial coagulants are aspartic proteases secreted by fungi: Rhizomucor miehei protease, the most widely used, Rhizomucor pusillus protease, and Cryphonectria parasitica (formerly Endothia parasitica) protease. Compared with chymosin, they have higher general proteolytic activity, which can reduce yield through loss of protein and fat into whey and cause bitterness in long-ripened cheeses, and some are more heat-stable, so that residual activity in whey can hydrolyse proteins in whey products. To address this, Rhizomucor miehei coagulant is chemically modified, for example by oxidation, to make it more heat-labile. Cryphonectria parasitica protease is highly proteolytic but heat-labile, so it is used for cheeses with high cooking temperatures such as Swiss-type cheeses, where it is inactivated. Microbial coagulants are accepted for vegetarian cheese and are less expensive than animal rennet.
Fermentation-produced chymosin is calf chymosin made by microorganisms that carry a copy of the calf prochymosin gene. Chymosin produced in Escherichia coli K-12 was the first recombinant enzyme approved for food use in the United States, in 1990, and the enzyme is also produced in Kluyveromyces lactis and Aspergillus niger. The enzyme is identical in amino acid sequence to calf chymosin B or A and so has the same specificity and cheese-making performance, while being purer, free of pepsin and more consistent and abundant than animal rennet. It is regarded as acceptable for vegetarian cheese, but some organic, protected-origin and religious certification schemes have specific rules about it. Fermentation-produced chymosin is used for most cheese made in the United States and a large share elsewhere. A second generation of recombinant coagulant is camel chymosin, produced in Aspergillus niger, which has a markedly higher ratio of clotting to general proteolytic activity than bovine chymosin, giving slightly higher yields and less bitterness; it is used particularly in low-fat and long-ripened cheeses. Coagulant strength is expressed in international milk clotting units (IMCU), measured under standard conditions by comparison with a reference standard according to ISO 11815, and the composition of rennets, as the proportions of chymosin and pepsin, is determined by chromatography according to ISO 15163. Recombinant coagulants are an example of how biotechnology was introduced into dairy processing with little consumer controversy, because the enzyme ends up mostly in whey and is a processing aid.
An immobilised enzyme is physically confined or attached to a support while keeping its catalytic activity, so that it can be reused and used in continuous reactors. Four main methods are used. Adsorption binds the enzyme to a carrier such as ion-exchange resin, porous glass or charcoal by weak forces; it is simple and cheap and preserves activity, but the enzyme may leach off with changes in pH or ionic strength. Covalent binding attaches the enzyme through amino or carboxyl groups to activated supports, often with glutaraldehyde or carbodiimide chemistry; it is stable but can reduce activity if the active site is affected. Entrapment encloses the enzyme or whole cells in a polymer gel such as calcium alginate, kappa-carrageenan or polyacrylamide, or within fibres; it is gentle but limits diffusion of large substrates. Cross-linking joins enzyme molecules to each other with bifunctional reagents such as glutaraldehyde, giving cross-linked enzyme crystals or aggregates without a carrier. Encapsulation within semi-permeable membranes, including enzyme membrane reactors that keep a soluble enzyme behind an ultrafiltration membrane, is a related approach. The effects of immobilisation include improved stability to heat and pH, changes in apparent pH optimum and kinetic constants because of the microenvironment of the support, and mass-transfer limitations: substrate must diffuse to the enzyme, so apparent Km often increases. Whole-cell immobilisation is also used, for example to retain lactic acid bacteria at high density in continuous fermentation of milk or whey, giving high productivity in pre-fermentation of milk for cheese or fermented milks.
Immobilised enzymes are used in packed-bed reactors, in which substrate flows through a column of enzyme particles in plug flow; in fluidised-bed reactors, in which upward flow keeps particles suspended and reduces blockage by particles in the feed; and in stirred-tank reactors with retained enzyme. Continuous processes give constant product quality, compact equipment and lower enzyme cost per unit of product, but they must be protected from microbial contamination, which is a serious problem with milk and whey because these are excellent growth media; reactors are therefore run at low temperatures or at pH values that limit growth, and are sanitised regularly, which can inactivate the enzyme. Fouling of supports by milk proteins and fat, loss of enzyme activity over time, and the cost of supports are other limitations. The largest industrial use of immobilised enzymes in food is glucose isomerase for high-fructose syrup, outside dairy. In dairy, continuous lactose hydrolysis of whey and whey permeate with immobilised fungal lactase, which works at acid pH where contamination is less of a problem, has been used commercially, and processes for milk with immobilised yeast or fungal lactase have been operated since the 1970s. Immobilised lactase is also used in the continuous production of galacto-oligosaccharides from concentrated lactose solutions, and immobilised proteases and lipases have been studied for hydrolysates and flavours. Enzyme activity in continuous reactors declines with time, so flow rate or temperature is adjusted to keep conversion constant, and the enzyme is replaced when activity falls to a set fraction of the initial value.
Lactose is hydrolysed in milk and dairy products for several reasons. The main one is to make lactose-free or lactose-reduced products for people with lactose malabsorption and intolerance, which affects a majority of adults worldwide, with large differences between populations, as described in Lactose, Salts and Vitamins. Other reasons are to increase sweetness, because a mixture of glucose and galactose is sweeter than lactose, allowing sugar reduction in flavoured milks and ice cream; to prevent lactose crystallisation and sandiness in ice cream, sweetened condensed milk and concentrated whey products; to speed up acid production in fermented milks; and to make whey syrups. In batch hydrolysis, neutral yeast lactase is added to milk in a tank, either at about 4 to 8 degrees Celsius overnight or at 35 to 40 degrees Celsius for two to four hours, after which the product is pasteurised or UHT-treated, which also inactivates the enzyme. For UHT milk, a sterile-filtered lactase can instead be dosed aseptically into the milk after heat treatment, so that hydrolysis takes place in the package during the first days of storage; this uses much less enzyme but requires a lactase free of proteolytic side activities, which would otherwise cause bitterness and age gelation in storage. Labelling rules differ: lactose-free milk usually contains less than 0.1 gram of lactose per 100 grams or millilitres in European practice, and less than 0.01 per cent in some countries, while lactose-reduced products have defined minimum reductions.
Continuous lactose hydrolysis uses immobilised lactase in packed-bed or fluidised-bed reactors, or soluble enzyme retained in an enzyme membrane reactor, as described in chapter 7. It is most practical for acid whey and permeate, where fungal lactase works at an acid pH that limits microbial growth, while continuous treatment of milk faces greater contamination and fouling problems. Modern lactose-free milk production often combines membrane filtration with hydrolysis: ultrafiltration and nanofiltration remove part of the lactose before the remainder is hydrolysed, so that the product has the sweetness and composition of ordinary milk rather than the extra sweetness of fully hydrolysed milk. Hydrolysis changes several properties. Hydrolysing the 4.8 grams of lactose in 100 grams of milk adds a molecule of water and gives about 5.05 grams of glucose and galactose, doubling the number of sugar molecules; this lowers the freezing point by about a further 0.25 to 0.3 degrees Celsius, which is used to follow the degree of hydrolysis with a cryoscope, and raises osmotic pressure. Glucose and galactose are reducing sugars that react faster than lactose in the Maillard reaction, so hydrolysed UHT milk and milk powders brown more readily. During hydrolysis, especially at high lactose concentrations, lactase forms galacto-oligosaccharides by transgalactosylation, which persist in the product and are not digested by lactase-deficient people, though their amounts in milk are small. The degree of hydrolysis is measured by cryoscopy, enzymatic assays or chromatography, and lactose-free claims require sensitive methods such as high-performance anion-exchange chromatography.
Enzyme-modified cheese is a concentrated cheese flavour ingredient made by treating young cheese curd with enzymes under controlled conditions so that, in a few days, it develops flavour compounds similar to those formed during months of natural ripening. Its flavour intensity is commonly 15 to 30 times that of natural cheese, so small amounts give strong flavour at lower cost than aged cheese. In a typical process, young cheese or curd of the chosen type, sometimes with added milk fat or butter, is mixed with water, salt and emulsifying salts to form a homogeneous slurry or paste with a solids content of about 40 to 50 per cent. The slurry is pasteurised to destroy contaminating organisms and native enzymes, cooled to the incubation temperature, often 30 to 40 degrees Celsius, and dosed with a combination of enzymes: proteases and peptidases to produce peptides, amino acids and savoury and brothy flavours; lipases or pregastric esterases to release free fatty acids; and sometimes cultures such as lactic acid bacteria or Penicillium roqueforti spores. After incubation for one to five days, during which the slurry is mixed and its pH and flavour monitored, the enzymes are inactivated by heat, and the product is homogenised and sold as a paste or spray-dried powder. Enzyme-modified cheese is used in processed cheese and cheese spreads, sauces, dips, soups, snacks, crackers, salad dressings and ready meals, where it supplies cheese character while reducing the amount of natural cheese required.
The characteristic flavour of each enzyme-modified cheese depends on the balance of proteolysis and lipolysis. Excessive or unbalanced proteolysis produces hydrophobic bitter peptides, so peptidases and controlled protease doses are used to degrade them into amino acids and small peptides, which contribute savoury and sweet notes and are precursors of further flavour compounds. The choice of lipase determines the pattern of free fatty acids: pregastric esterases preferentially release butyric, caproic and other short-chain acids, giving the sharp, piquant flavour of Italian-type cheese, while other lipases release more medium- and long-chain acids, giving Cheddar-like or soapy notes if overdone. Blue cheese flavours are made by fungal lipolysis followed by conversion of fatty acids to methyl ketones such as 2-heptanone by Penicillium roqueforti, and Cheddar flavour relies on a balance of free fatty acids, peptides, amino acids and sulphur compounds. Because the product is incubated at warm temperatures, microbiological safety depends on the initial pasteurisation, on hygienic handling, and on the final heat treatment; the slurry contains salt and emulsifying salts that limit some organisms, but spore-formers such as Clostridium and Bacillus must be controlled, and the risk of Staphylococcus aureus toxin formation during incubation must be considered in hazard analysis. Quality control includes sensory evaluation against standards, measurement of free fatty acids and degree of proteolysis, and microbiological testing. Enzyme-modified cheese is labelled as cheese or as an ingredient depending on national rules. Natural cheese ripening, which enzyme-modified cheese imitates, is described in Cheese Science and Technology.
Recombinant DNA technology allows genes to be isolated, modified and introduced into organisms. Its basic tools are restriction enzymes, which cut DNA at specific sequences; DNA ligase, which joins fragments; plasmid vectors carrying origins of replication and selection markers; transformation, usually by electroporation for lactic acid bacteria; and the polymerase chain reaction. Classical gene transfer in lactic acid bacteria, by conjugation and transduction and the properties encoded on plasmids, is described in Starter Cultures and Lactic Acid Bacteria. Lactococcus lactis became the model organism: strain IL1403 was the first lactic acid bacterium to have its genome sequenced, in 2001, and strain MG1363, cured of its plasmids, is the standard laboratory host. Food-grade genetic systems avoid antibiotic resistance markers and DNA from non-food organisms. They use food-grade selection, such as complementation of a lactose-metabolism gene in a lactose-negative host, or resistance to nisin, and food-grade expression systems such as the nisin-controlled gene expression (NICE) system, in which the nisA promoter is switched on by adding sub-inhibitory nisin through the NisR-NisK two-component regulatory system. Self-cloning, in which only DNA from the same species is reintroduced, is treated less strictly in some regulations. Engineered L. lactis strains have been developed for overproduction of diacetyl, vitamins such as folate and riboflavin, low-calorie sugars such as L-alanine and mannitol, and for delivery of therapeutic proteins to the gut, such as an interleukin-10-secreting strain whose thymidylate synthase gene was replaced to prevent its survival outside the host, a strategy called biological containment.
Genome editing makes targeted changes to an organism's own genome. In lactic acid bacteria, recombineering uses single-stranded DNA oligonucleotides with a phage recombinase, such as RecT, to introduce point mutations at high frequency, and CRISPR-Cas9 is used to kill unedited cells, selecting for those carrying the desired change, so that markerless deletions, insertions and point mutations can be made efficiently. Many lactic acid bacteria have their own CRISPR-Cas systems, and these endogenous systems can be repurposed for editing by supplying a programmed CRISPR array and a repair template. Catalytically inactive Cas9 (dCas9) can repress genes without changing DNA, a method called CRISPR interference, and base editors change single bases without double-strand breaks. Not all strain improvement uses genetic engineering: classical methods such as random mutagenesis, adaptive laboratory evolution, selection of spontaneous mutants and natural phage-resistant derivatives remain important because their products are not regulated as genetically modified. Regulation is a decisive constraint. In the European Union, genetically modified microorganisms intended for food require authorisation under Regulations (EC) No 1829/2003 and 1830/2003, with traceability and labelling, so live genetically modified starter cultures are not used commercially; Directive 2009/41/EC excludes self-cloned non-pathogenic organisms from its contained-use rules. The EU's 2023 proposal on new genomic techniques covers plants, not microorganisms, and EFSA has considered microorganisms separately. In the United States, food uses of engineered microorganisms are assessed case by case, often through generally recognised as safe notifications. Products made with genetically modified organisms but free of them, such as fermentation-produced chymosin, are treated differently from live genetically modified cultures in most jurisdictions.
Precision fermentation uses microorganisms engineered with genes for specific proteins to produce those proteins, which are then separated from the organism and used as ingredients. It is distinguished from traditional fermentation, in which microorganisms transform a food, and from biomass fermentation, in which the microbial cells themselves are the product. The technology is not new: fermentation-produced chymosin, insulin and many food enzymes are made this way. What is new is its use to make milk proteins for dairy-like foods without animals. Whey proteins, particularly beta-lactoglobulin, have been the first targets because they are soluble globular proteins that are secreted efficiently by fungal hosts such as Trichoderma reesei and Aspergillus species and by yeasts such as Komagataella phaffii; in 2020 the US Food and Drug Administration issued a no-questions letter on a generally recognised as safe notice for non-animal beta-lactoglobulin produced in Trichoderma reesei, and products such as ice cream and cream cheese followed. Other targets include alpha-lactalbumin, lactoferrin and osteopontin for infant and medical nutrition. The process follows the pattern of enzyme production: fed-batch aerobic fermentation of a sugar-based medium, removal of biomass, concentration and purification of the secreted protein by membranes and chromatography, and drying. Production cost depends on titre, productivity and recovery: a 100 cubic metre fermentation with a titre of 10 grams per litre and 80 per cent recovery yields 800 kilograms of protein, and achieving costs close to those of dairy proteins requires high titres, very large vessels and cheap sugar.
Caseins are harder to produce. In milk they are phosphorylated by a Golgi kinase (FAM20C) at specific serine residues, kappa-casein is glycosylated, and the caseins assemble with calcium phosphate into micelles that give milk its white appearance, heat stability and ability to form curds and cheese. Microorganisms do not phosphorylate caseins in the same way, so recombinant caseins must either be modified after production, co-expressed with a kinase, or engineered with substitutions that mimic phosphorylation, and micelles must be reassembled from the purified proteins with calcium and phosphate. Achieving the melting and stretching of real cheese has therefore been slower than making whey-protein products. Human milk oligosaccharides such as 2'-fucosyllactose, made by precision fermentation in engineered Escherichia coli and authorised as novel foods in the European Union since about 2016 and as generally recognised as safe in the United States, show that such ingredients can reach mass markets, in this case infant formula. Regulatory and labelling questions are significant. Precision-fermented proteins are evaluated as novel foods in the European Union under Regulation (EU) 2015/2283 and by national authorities elsewhere; they must be shown to be free of the production organism and its DNA, or the absence of risk shown; and because they are identical or nearly identical to milk proteins, they are allergenic to people with milk allergy and must be labelled accordingly, even if the product is described as animal-free. Whether such products may use dairy terms such as milk or cheese depends on national rules on dairy names, as described in Dairy Standards and Law Worldwide. Claims about environmental benefits depend on life-cycle analyses that are sensitive to energy sources and sugar supply.
Single-cell protein is the protein-rich biomass of microorganisms, such as yeasts, bacteria, fungi and algae, grown for food or feed. Whey and whey permeate are suitable substrates because they contain about 4.5 to 5 per cent lactose together with minerals and vitamins, and because converting them into biomass also reduces their high polluting load, described in Whey and Dairy By-product Utilisation. The usual organism is the lactose-fermenting yeast Kluyveromyces marxianus, which grows rapidly at temperatures up to about 45 degrees Celsius, tolerates low pH, and has a long history of safe use. Aerobic fermentation in continuous or fed-batch fermenters, with intensive aeration and supplementation with ammonia or ammonium salts as a nitrogen source, gives biomass yields of about 0.5 grams of dry biomass per gram of lactose: a whey containing 48 grams of lactose per litre can produce about 24 grams of dry yeast per litre, containing roughly 45 to 55 per cent protein. The biomass is recovered by centrifugation and dried. Yeast single-cell protein contains about 6 to 12 per cent nucleic acid, mostly RNA, which in humans is broken down to uric acid; daily intake from microbial sources is limited to about 2 grams to avoid gout and kidney stones, so biomass intended for food undergoes heat shock or alkaline treatment to reduce its RNA content, while feed uses do not require this. Whole fermented whey, containing yeast biomass and whey proteins, has been used as animal feed, and yeast extracts made from whey-grown yeasts are used as flavour ingredients.
Ethanol is made from whey permeate by fermentation with Kluyveromyces marxianus or with engineered Saccharomyces cerevisiae strains able to use lactose. The theoretical yield is 4 moles of ethanol per mole of lactose, or 0.538 grams per gram; a permeate with 48 grams of lactose per litre can therefore give at most about 25.8 grams of ethanol per litre, or 3.3 per cent by volume, which is low compared with grain fermentations and makes distillation costly, so permeate is often concentrated by reverse osmosis or evaporation before fermentation. Whey ethanol has been produced commercially for potable spirits, such as vodkas and liqueurs, and for fuel and industrial use. Anaerobic digestion converts whey and dairy wastewater to biogas, mainly methane and carbon dioxide, recovering energy while reducing chemical oxygen demand; it is described in Whey and Dairy By-product Utilisation and in the subject on dairy plant utilities and energy. Other fermentation products from whey include lactic and propionic acids (chapter 4), bacterial cellulose, polyhydroxyalkanoate bioplastics, biosurfactants, exopolysaccharides, bacteriocins such as nisin, which is produced commercially by fermentation of milk-based media by Lactococcus lactis, and starter and probiotic cultures. Modern thinking treats a dairy plant as a biorefinery, in which whey proteins are recovered first as high-value ingredients, lactose is crystallised or converted into galacto-oligosaccharides, lactulose or lactobionic acid, and the remaining permeate and mother liquor are fermented into products of lower value or into energy. The best route depends on the scale of whey production, distance to markets, energy prices and the value of the products relative to the cost of processing.
A student meets this subject in practicals and projects on fermenters, kinetics and enzymes: measuring growth curves and fitting Monod constants, running a lactic acid fermentation on whey permeate, assaying lactase and coagulant activity, immobilising enzymes in alginate, and following lactose hydrolysis by cryoscopy. Learn the kinetic equations and scale-up rules, the sources and properties of dairy enzymes, the logic of recombinant and food-grade genetic tools, and the regulation of products made with genetically modified organisms, because examiners combine calculations and concepts.
Five years into work in a dairy plant, an enzyme or culture company, or a whey processing business, you choose and dose coagulants and lactases, run lactose-free milk processes, troubleshoot bitterness and gelation, operate starter culture or ingredient fermentations, evaluate whey valorisation projects, and work with suppliers on enzyme specifications and regulatory documents. You use kinetic and mass-balance calculations to size equipment and estimate yields, and you explain to marketing teams what claims are allowed for lactose-free and animal-free products.
A senior technical director with twenty-five years of experience decides on investments in fermentation and enzyme technologies, such as lactose-free lines, whey biorefineries, culture production plants or precision fermentation partnerships; negotiates with suppliers of coagulants and enzymes; manages the regulatory and reputational aspects of biotechnology, including labelling and consumer acceptance; and positions the company for competition and collaboration with animal-free dairy producers. Such a person also builds the scientific team, sets the research agenda with universities and institutes, and judges which new technologies are ready for industrial use.
A researcher with long experience engineers production strains and enzymes, develops food-grade genetic tools and genome editing for lactic acid bacteria, optimises fermentation and downstream processes for milk proteins, studies casein phosphorylation and micelle assembly, models scale-up, and evaluates the techno-economics and life-cycle impacts of new dairy biotechnologies. Such a person also advises regulators on the safety assessment of products from engineered organisms, trains the next generation of bioprocess scientists, and publishes the independent data that industry and policy makers need to weigh promises against evidence.
This manual gives principles and representative figures; titres, yields, enzyme dosages and costs vary widely between processes and suppliers and are often confidential. Regulatory approvals for precision-fermented proteins are changing quickly and differ by country, so the current status must be checked. Commercial enzyme preparations, culture and fermentation companies are not named. Detailed bioreactor engineering, sterilisation kinetics and process control are introduced only to the depth needed for dairy applications. Starter genetics and phage are covered in Starter Cultures and Lactic Acid Bacteria and in Advanced Dairy Microbiology, and whey processing in Whey and Dairy By-product Utilisation.
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