Dairy Science & Technology · Dairy Microbiology · also Dairy Starter Cultures, Microbiology of Starters
The lactic acid bacteria and other organisms used as starters: their metabolism, genetics, production, failure and control.
In the exams: ICAR AICE subject 36 Unit 5; ICAR AIEEA-PG group 16 (classification, genetic aspects and carbohydrate metabolism of LAB; propagation and inhibition of starters)
Starter cultures are the microorganisms deliberately added to milk to make cheese, yoghurt, cultured butter and other fermented products. Most are lactic acid bacteria, which convert lactose to lactic acid, lowering the pH, coagulating or firming the casein, preserving the product and producing flavour compounds; others, such as propionibacteria, moulds and smear organisms, are added to give particular cheeses their eyes, rinds and aromas. This subject covers the lactic acid bacteria and their classification, their carbohydrate, citrate and protein metabolism, the kinds of starter and how they are grown, delivered and preserved, the bacteriophages and inhibitors that make starters fail, and the genetics that underlie their properties and their improvement.
Starter performance decides the economics of fermented dairy products. A starter that acidifies on schedule gives cheese with the right moisture, texture and flavour; one that is slowed by phage, antibiotic residues or sanitisers gives a dead vat, a defective product and a lost day. Modern plants depend on culture companies for concentrated direct-vat-set cultures and on strict phage control, while traditional producers still rely on back-slopping and natural whey cultures. Understanding starters is therefore central to dairy microbiology at every level.
The chemistry of fermentation and flavour compounds is described in Chemistry of Dairy Products; the technology of yoghurt, dahi, kefir, buttermilk and probiotic fermented milks in Fermented Milks Technology; the role of starters in cheesemaking in Cheese Science and Technology; and general microbiology in the foundation subjects and Food Technology's Food Microbiology. This manual refers to those subjects and concentrates on the organisms themselves. It is examined as Unit 5 of ICAR AICE-JRF/SRF subject 36 (Dairy Microbiology) and in group 16 of ICAR AIEEA-PG, which names the classification, genetic aspects and carbohydrate metabolism of lactic acid bacteria and the propagation and inhibition of starters. Expect questions on taxonomy and nomenclature, metabolic pathways and their products, culture types, propagation, preservation, phage biology and control, inhibitors, and plasmid-borne traits, with calculations of inoculum, phage titres and product yields.
Lactic acid bacteria (LAB) are a functional group of Gram-positive, catalase-negative, non-spore-forming, non-motile cocci and rods that produce lactic acid as the main end product of carbohydrate fermentation. They are aerotolerant anaerobes: they lack a respiratory chain under normal conditions and obtain energy by fermentation, but they tolerate oxygen. They have complex nutritional needs for amino acids, vitamins and nucleotides, which is why they are found in nutrient-rich environments such as milk, plants, meat and the intestines of animals, and why milk, which contains few free amino acids, selects for strains with efficient proteolytic systems. They tolerate acid well and so dominate spontaneous fermentations. Taxonomically, most LAB belong to the order Lactobacillales in the phylum Bacillota (formerly Firmicutes), with a low guanine plus cytosine content in their DNA. Bifidobacteria, often grouped with LAB because they are used in probiotic products and produce lactic and acetic acids, belong to a different phylum, Actinomycetota, with a high guanine plus cytosine content and a different metabolic pathway. Many LAB species have a long history of safe use in food; in the European Union they are assessed under the Qualified Presumption of Safety approach, and in the United States many are generally recognised as safe for their intended uses. Classification once relied on cell shape, growth temperature, salt and pH tolerance, the configuration of lactic acid formed and sugar fermentation; it now rests on DNA sequences, especially 16S ribosomal RNA genes and whole genomes.
The dairy LAB belong mainly to a few genera. Lactococcus contains the mesophilic cocci Lactococcus lactis subspecies lactis, its citrate-fermenting biovar diacetylactis, and the organism long called Lactococcus lactis subspecies cremoris, which has been raised to the rank of species as Lactococcus cremoris. Streptococcus thermophilus is the only dairy streptococcus and the main thermophilic coccus. Leuconostoc species, such as Leuconostoc mesenteroides subspecies cremoris and Leuconostoc lactis, are heterofermentative cocci used for aroma. Lactobacillus formerly included more than 250 species of very different biology; in 2020 it was divided into 25 genera on the basis of genome analysis. The main dairy thermophiles, Lactobacillus delbrueckii subspecies bulgaricus and subspecies lactis, Lactobacillus helveticus and Lactobacillus acidophilus, stay in Lactobacillus, but many other familiar species were renamed: Lactobacillus casei, paracasei and rhamnosus became Lacticaseibacillus; Lactobacillus plantarum became Lactiplantibacillus plantarum; Lactobacillus reuteri and fermentum became Limosilactobacillus; Lactobacillus brevis became Levilactobacillus brevis; and Lactobacillus buchneri became Lentilactobacillus buchneri. Older names remain in textbooks, standards and labels, so both should be recognised. Other LAB genera in dairy products include Pediococcus, Enterococcus (present in many traditional cheeses, with safety questions about some strains), Weissella and Carnobacterium.
Lactic acid bacteria ferment hexoses by one of two main pathways. Homofermentative LAB use the Embden-Meyerhof-Parnas pathway (glycolysis), in which fructose 1,6-bisphosphate is split by the enzyme aldolase into two triose phosphates, each converted to pyruvate, which lactate dehydrogenase reduces to lactate to regenerate NAD+. The result is two molecules of lactic acid and a net gain of two ATP per molecule of glucose, with lactic acid typically making up more than 85 per cent of the end products. Lactococci, Streptococcus thermophilus, Lactobacillus delbrueckii, L. helveticus and pediococci are homofermentative. Heterofermentative LAB lack aldolase and use the phosphoketolase pathway (also called the 6-phosphogluconate or pentose phosphate-phosphoketolase pathway): glucose 6-phosphate is oxidised and decarboxylated to a pentose phosphate, releasing carbon dioxide, and phosphoketolase splits the pentose phosphate into glyceraldehyde 3-phosphate, which becomes lactate, and acetyl phosphate, which becomes ethanol or, if an extra electron acceptor such as oxygen or fructose is available, acetate with extra ATP. The products per glucose are one lactic acid, one carbon dioxide and one ethanol or acetate, with a net gain of one ATP. Leuconostoc species, Limosilactobacillus fermentum and Levilactobacillus brevis are obligately heterofermentative. Facultatively heterofermentative organisms, such as Lacticaseibacillus casei and Lactiplantibacillus plantarum, ferment hexoses homofermentatively but pentoses heterofermentatively.
The type of fermentation decides the products. Homofermentative starters give fast, efficient acidification and a clean lactic flavour, which suits cheese and yoghurt, where speed of acid production governs whey drainage, texture and safety. Heterofermentative organisms produce carbon dioxide, which forms small openings in some cheeses and gives effervescence in kefir, and ethanol and acetate, which contribute to flavour; they also grow more slowly and acidify less. The optical form of lactic acid differs between organisms because they carry lactate dehydrogenases of different specificity. Lactococci and Streptococcus thermophilus produce L(+)-lactic acid; Lactobacillus delbrueckii subspecies bulgaricus and leuconostocs produce D(-)-lactic acid; Lactobacillus helveticus produces a mixture of both forms. Yoghurt therefore contains both forms. The stoichiometry of homolactic fermentation of lactose, with one lactose and one water giving four lactic acid, means that each gram of lactose fermented gives up to about 1.05 grams of lactic acid, as calculated in Lactose, Milk Salts and Vitamins; in practice only a fraction of milk lactose is fermented before the low pH stops growth. Some LAB also have additional pathways that affect flavour and texture: pyruvate can be diverted to formate, acetate and ethanol when sugar is limiting, to diacetyl and acetoin from citrate, as described in chapter 3, and some strains produce exopolysaccharides from sugar nucleotides that increase the viscosity of fermented milks.
Starter bacteria take up lactose in two different ways, described in Chemistry of Dairy Products. Lactococci use the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS), in which lactose is phosphorylated as it crosses the membrane; the resulting lactose 6-phosphate is split by phospho-beta-galactosidase into glucose and galactose 6-phosphate. Glucose enters glycolysis, and galactose 6-phosphate is metabolised through the tagatose 6-phosphate pathway, so lactococci use both halves of lactose. In many lactococci the genes for the lactose PTS and the tagatose pathway are carried on plasmids, so strains that lose the plasmid cannot ferment lactose (chapter 12). Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus take up lactose by a permease (LacS in S. thermophilus) that exchanges it for galactose, and hydrolyse it inside the cell with beta-galactosidase. Most dairy strains of these species use only the glucose half and excrete galactose, which accumulates in yoghurt and in Mozzarella and can cause browning on baking pizza; galactose-positive strains, which use the Leloir pathway, are selected to reduce it. Galactose excretion is also a reason why lactose is never completely used up in yoghurt. Sugar metabolism is regulated by catabolite repression, so organisms use their preferred sugars first, and the activity of these transport systems decides how fast a starter acidifies milk.
Milk contains about 1.7 to 1.8 grams of citrate per litre, which certain starter organisms use to produce flavour and carbon dioxide. Lactococcus lactis biovar diacetylactis takes up citrate through a citrate permease, CitP, encoded on a plasmid, and Leuconostoc species metabolise citrate mainly when lactose is also available and the pH has fallen, typically below about 6. Citrate is split by citrate lyase into acetate and oxaloacetate, and oxaloacetate is decarboxylated to pyruvate and carbon dioxide. Because citrate provides pyruvate without the NADH produced in glycolysis, there is an excess of pyruvate, which is diverted by alpha-acetolactate synthase into alpha-acetolactate. Alpha-acetolactate is unstable: it is decarboxylated either enzymatically to acetoin, which has little flavour, or chemically by oxidative decarboxylation to diacetyl, the key buttery flavour compound, a reaction favoured by low pH and oxygen. Diacetyl can be reduced by diacetyl reductase to acetoin and then 2,3-butanediol, so diacetyl levels fall if cultures are held too long, as described in Chemistry of Dairy Products. The carbon dioxide from citrate metabolism forms the small round eyes of Dutch-type cheeses and gives openness to cottage cheese and buttermilk. Mutant strains lacking alpha-acetolactate decarboxylase accumulate more alpha-acetolactate and therefore more diacetyl, and cultures with high diacetyl production are used in cultured butter and cream.
Milk contains only small amounts of free amino acids and peptides, far less than starter bacteria need for growth to high numbers, so they obtain most of their amino acids by breaking down casein. The proteolytic system of lactic acid bacteria has three parts: a cell-envelope proteinase (CEP) that hydrolyses casein outside the cell to oligopeptides; transport systems that carry peptides and amino acids into the cell; and a battery of intracellular peptidases that break the peptides into amino acids. The CEP is anchored to the cell wall. In lactococci it is called PrtP, in Streptococcus thermophilus PrtS and in Lactobacillus delbrueckii subspecies bulgaricus PrtB. Lactococcal proteinases were classified by their specificity: type PI enzymes degrade mainly beta-casein, while type PIII enzymes degrade alpha-s1-, beta- and kappa-casein; many intermediate types exist. In lactococci, the gene for PrtP is commonly carried on a plasmid, so proteinase-negative (Prt-) variants arise by plasmid loss. Prt- strains grow poorly in milk on their own because they depend on the small amount of free nitrogen, but they grow well in mixtures with Prt+ strains, which supply peptides. Commercial starters often contain both, and proteinase-negative strains have been used to reduce bitterness in some cheeses. In yoghurt, the strong proteinase of L. bulgaricus supplies peptides to the weakly proteolytic S. thermophilus, part of their protocooperation.
Oligopeptides produced by the CEP are taken into the cell mainly by the oligopeptide permease (Opp), an ATP-binding cassette transporter that can carry peptides of up to about 18 amino acids; di- and tripeptides enter through separate transporters (DtpT and Dpp), and free amino acids through amino acid permeases. Inside the cell, peptidases with different specificities complete the breakdown. They include general aminopeptidases such as PepN and PepC, which remove N-terminal amino acids; the X-prolyl dipeptidyl aminopeptidase PepX, which removes dipeptides with proline in the second position, important because casein is rich in proline; endopeptidases such as PepO and PepF, which cut within peptides; proline-specific peptidases such as PepI, PepR and PepQ; and di- and tripeptidases such as PepV and PepT. Peptidases are released into cheese when starter cells lyse during ripening and contribute to the breakdown of bitter peptides and the release of free amino acids, which are converted to flavour compounds, as described in Chemistry of Dairy Products. The balance between CEP activity, which forms peptides, and peptidase activity, which removes them, determines whether bitter hydrophobic peptides accumulate. Strains with high autolysis rates release peptidases early and are used to accelerate ripening and reduce bitterness. The proteolytic activity of starters is measured by the increase in free amino groups in milk, for example with o-phthaldialdehyde or trinitrobenzenesulfonic acid, and by electrophoresis of the caseins.
Starters are divided by their temperature range. Mesophilic starters have optimum growth temperatures of about 25 to 30 degrees Celsius and do not grow well above about 40 degrees Celsius. They consist of Lactococcus lactis, Lactococcus cremoris, Lactococcus lactis biovar diacetylactis and Leuconostoc species, and are used for Cheddar, Gouda, Edam, cottage cheese, quarg, cultured butter, cultured buttermilk, sour cream and many fresh cheeses, where the curd is not heated above about 40 degrees Celsius. Classical phenotypic tests distinguished the lactococci: Lactococcus lactis subspecies lactis grows at 40 degrees Celsius, in 4 per cent salt and at pH 9.2 and produces ammonia from arginine, whereas strains of cremoris do not; cremoris strains are often preferred in cheese for their milder flavour and lower bitterness, though they are less robust. Biovar diacetylactis is recognised by its ability to metabolise citrate. Leuconostoc species are heterofermentative, form D-lactate, grow slowly in milk on their own and are used in combination with lactococci for aroma and carbon dioxide. Mesophilic mixed starters are classified by their aroma-producing components into types: O (no citrate fermenters), D (with biovar diacetylactis), L (with Leuconostoc) and DL or LD (with both). These types determine the flavour and openness of the product.
Thermophilic starters have optimum growth temperatures of about 37 to 45 degrees Celsius and tolerate the cooking temperatures of about 50 to 55 degrees Celsius used for Swiss-type and Italian cheeses. They consist of Streptococcus thermophilus with Lactobacillus delbrueckii subspecies bulgaricus or subspecies lactis and Lactobacillus helveticus. Streptococcus thermophilus grows at 45 degrees Celsius but not at 10 degrees Celsius, survives 60 degrees Celsius for 30 minutes, and is sensitive to antibiotics and to sodium chloride above a few per cent; it is used alone or with lactobacilli in yoghurt, Mozzarella, Swiss-type cheeses, and increasingly in Cheddar-type cheeses as an adjunct to speed acidification. Lactobacillus delbrueckii subspecies bulgaricus is the partner of S. thermophilus in yoghurt and produces most of its acetaldehyde, as described in Chemistry of Dairy Products; Lactobacillus helveticus is strongly proteolytic and is used in Swiss-type and Italian hard cheeses and as an adjunct to accelerate ripening and reduce bitterness, and it is known for releasing ACE-inhibitory peptides in fermented milks. The yoghurt cultures grow together in protocooperation, each stimulating the other, and the ratio between cocci and rods, which changes during fermentation and with temperature, affects acidity and flavour; the details are described in Fermented Milks Technology. Thermophilic natural whey cultures, made by incubating whey from the previous day's cheese at high temperature, are the traditional starters of Parmigiano Reggiano, Grana Padano and many other Italian cheeses.
Starters differ in how many strains they contain and how well those strains are known. A single-strain starter contains one strain; it gives very reproducible performance but is vulnerable, because one phage able to attack that strain can stop acid production completely. Multiple-strain starters contain a small number of selected, characterised strains, commonly two to six, combined so that if one is attacked by phage the others keep acidifying; such cultures are defined, because each strain is known. Mixed-strain starters contain an unknown number of strains of one or more species, often derived from traditional cultures maintained in dairies for decades; they are undefined, because their exact composition is not known and can change with propagation. Undefined mixed-strain mesophilic cultures, used especially in Dutch and other European cheese production, are robust against phage because they contain many strains with different phage sensitivities and because they coexist with phage populations in a balance that keeps them active. Their disadvantages are variability in performance and flavour and the difficulty of controlling their composition. Defined strain systems, developed especially in New Zealand and Australia for Cheddar production, use selected phage-resistant strains with phage monitoring and rotation, giving consistent performance and allowing strain improvement. Commercial culture suppliers now offer both kinds as direct-vat-set cultures (chapter 8).
Many traditional products are made without commercial starters. In back-slopping, part of a previous batch of fermented milk, whey or cheese is used to inoculate the next, a practice used for dahi in South Asian households and many traditional fermented milks; it maintains a complex, adapted community but varies with handling and can carry spoilage organisms or pathogens if hygiene is poor. Natural whey cultures are made by incubating the whey from the previous day's cheesemaking under controlled conditions, so that heat-tolerant, acid-tolerant thermophilic LAB are selected; they are required by the specifications of some protected-designation cheeses. Natural milk cultures are made by allowing raw milk, sometimes heat-treated mildly, to ferment spontaneously. These cultures are sources of diversity, including strains with valuable flavour or phage resistance, and culture companies and research collections isolate strains from them. The choice between defined and undefined, commercial and artisanal cultures balances consistency, phage robustness, flavour complexity and tradition. Kefir grains are a special case: they are a symbiotic matrix of polysaccharide (kefiran) and protein containing lactic acid bacteria, acetic acid bacteria and yeasts, which are recovered and reused after each fermentation, as described in Fermented Milks Technology. Whatever the culture, regular checks of activity, purity and phage contamination are needed to keep it reliable.
Secondary cultures are organisms added for a specific function other than acidification, usually during ripening. Propionibacterium freudenreichii, the classical propionic acid bacterium, is added to Swiss-type cheeses such as Emmental and Gruyère. During warm-room ripening it ferments lactate to propionate, acetate and carbon dioxide; the carbon dioxide forms the large eyes and propionate contributes the sweet, nutty flavour, as described in Chemistry of Dairy Products. It also produces vitamin B12. Its growth depends on salt content, pH and temperature, and on the absence of competing organisms. Moulds are used for surface-ripened and blue cheeses. Penicillium camemberti, used for Camembert and Brie, forms the white felt-like rind; it oxidises lactate and degrades protein, raising the pH of the surface and softening the cheese inward. Penicillium roqueforti is used for blue cheeses such as Roquefort, Gorgonzola and Stilton; its spores are added to the milk or curd, and the cheese is pierced so that air enters and the mould grows in the openings, where its lipases and the conversion of fatty acids to methyl ketones produce the typical flavour. Geotrichum candidum, a yeast-like fungus, grows on the surface of many soft and semi-soft cheeses, deacidifies the surface and contributes to rind formation and flavour. Mould cultures are supplied as spore suspensions or freeze-dried preparations.
Smear-ripened (washed-rind) cheeses such as Limburger, Münster, Tilsit and Appenzeller develop a sticky, orange to red-brown surface flora. The smear develops in a sequence. First, salt-tolerant yeasts such as Debaryomyces hansenii grow on the acid surface and metabolise lactate, raising the pH. Then salt-tolerant, acid-sensitive coryneform bacteria grow: Brevibacterium (especially Brevibacterium linens and related species, which give the orange pigment), Corynebacterium, Arthrobacter and Brachybacterium, together with staphylococci such as Staphylococcus equorum. These organisms degrade protein and produce volatile sulphur compounds such as methanethiol, giving the strong aroma. The surface is washed repeatedly with brine, sometimes inoculated with a smear culture or with smear from older cheeses (old-young smearing, which risks transferring Listeria). Defined smear cultures are increasingly used to improve consistency and safety. Adjunct cultures are non-starter organisms added to cheese milk to improve or accelerate flavour development without contributing much acid. They include strains of Lacticaseibacillus paracasei, Lacticaseibacillus casei, Lactiplantibacillus plantarum and Lactobacillus helveticus, sometimes attenuated by heat, freezing or other treatment so that they release enzymes without producing acid. Adjuncts help control the non-starter lactic acid bacteria that dominate mature Cheddar, accelerate ripening and reduce bitterness. Protective cultures, which inhibit spoilage organisms or pathogens, and probiotic cultures, described in Fermented Milks Technology, are other kinds of added culture.
In the traditional system, a stock culture is propagated through a series of transfers: mother culture, intermediate (feeder) culture and bulk starter, each grown in heat-treated milk or medium and used to inoculate the next. The bulk starter is added to the cheese vat or fermentation tank at typically about 0.5 to 2 per cent; for a 5,000-litre vat at 1 per cent, 50 litres of bulk starter are needed. The medium for bulk starter is commonly skim milk or reconstituted skim milk heated at about 90 to 95 degrees Celsius for 30 to 60 minutes, which kills competing organisms and phages, removes oxygen and denatures inhibitory proteins; whey-based media are also used. Phage-inhibitory media contain phosphates or citrates that bind calcium, which many phages need for adsorption, and so reduce phage replication in the bulk starter. In pH-controlled bulk starter systems, alkali such as ammonia or sodium hydroxide is added during growth to keep the pH near 6, preventing acid from stopping growth and giving higher cell numbers and more active cultures; internally pH-controlled media use buffering salts for the same purpose. Bulk starter must be produced in a separate, hygienic starter room with filtered air under positive pressure, aseptic transfers, dedicated equipment and strict separation from whey and cheesemaking areas, because phage contamination at this stage spreads to every vat.
Direct-vat-set (DVS) or direct-vat-inoculation (DVI) cultures are highly concentrated frozen or freeze-dried cultures, containing of the order of ten billion to a hundred billion colony-forming units per gram, which are added directly to the milk in the vat without intermediate propagation. They are dosed in units defined by the supplier per volume of milk; if a culture is used at 50 units per 1,000 litres, a 5,000-litre vat needs 250 units. DVS cultures remove the need for a starter room, reduce the risk of phage build-up during propagation, give consistent strain composition and allow planned rotation of strains, and have become the dominant form in industrialised dairying. Their disadvantages are higher cost per vat, dependence on suppliers and cold-chain delivery, and a longer lag before acid production, because the cells must recover from freezing or drying; semi-direct systems that use a concentrate to make bulk starter are a compromise. Whatever the system, the activity of starters must be checked. Activity tests measure the acid produced in a standard time and temperature in heat-treated skim milk, often simulating the temperature profile of the cheesemaking process; the pH or titratable acidity reached is compared with the expected value. Vat records of the time taken to reach set pH values at each stage are the most sensitive daily indicator of starter problems, revealing slow vats caused by phage, inhibitors or culture defects before they become dead vats. Purity is checked by microscopy and plating, and phage by the tests described in chapter 10.
Starter cultures must be preserved without loss of viability, activity or strain balance. Repeated subculturing in milk, the oldest method, risks contamination, phage infection, loss of plasmids and drift in the proportions of strains in mixed cultures, so it is now used only for short-term maintenance; refrigerated storage of coagulated cultures keeps them for days to a few weeks. Freezing is the main method for long-term storage of stock cultures and for commercial frozen concentrates. Cells are grown, usually in pH-controlled fermenters, harvested in the late exponential or early stationary phase, when they survive freezing best, concentrated by centrifugation or membrane filtration, mixed with cryoprotectants and frozen. Cryoprotectants such as glycerol, sucrose, lactose, trehalose, skim milk and glutamate reduce damage from ice crystals and from the concentration of solutes during freezing. Freezing in liquid nitrogen at minus 196 degrees Celsius, often as small pellets, and storage at minus 45 degrees Celsius or below preserve viability for months; storage at domestic freezer temperatures of about minus 20 degrees Celsius gives much shorter life because slow changes continue. Frozen cultures must be kept frozen throughout distribution and thawed only immediately before use, by adding the pellets directly to milk.
Freeze-drying (lyophilisation) removes water from frozen cultures by sublimation under vacuum, producing a dry powder that can be stored and shipped more easily than frozen cultures. Before drying, cells are mixed with protective agents such as skim milk, sucrose, trehalose, lactose and sodium glutamate, which protect membranes and proteins during freezing and drying and replace water around biological molecules. The dried culture is packed in moisture-proof containers, often under nitrogen, and stored cold, commonly at refrigeration or freezer temperatures, to maintain viability. Freeze-drying causes some loss of viability, and survivors may have damaged membranes, giving a longer lag phase after inoculation; species and strains differ in their tolerance, with lactococci and S. thermophilus generally surviving well and some lactobacilli less well. Spray drying is cheaper and faster but exposes cells to high temperatures and usually gives lower survival, so it is used for some robust strains and probiotics. Fluidised-bed drying and vacuum drying are other options. For research and culture collections, freezing at minus 80 degrees Celsius with glycerol and storage in liquid nitrogen are standard, together with freeze-dried ampoules. Quality control of preserved cultures includes viable counts, activity tests, checks on strain composition by molecular methods, absence of contaminants and phages, and stability testing during storage.
Bacteriophages (phages) are viruses that infect bacteria, and they are the most common cause of starter failure in dairy fermentations. Dairy phages have double-stranded DNA genomes and tails. Virulent (lytic) phages infect a cell, take over its machinery and produce new phage particles, which are released when the cell bursts; temperate phages can either do this or integrate their DNA into the bacterial chromosome as a prophage, which is passed on to daughter cells (lysogeny) until it is induced to enter the lytic cycle. The lytic cycle consists of adsorption of the phage to specific receptors on the cell surface, for which many lactococcal phages require calcium ions, injection of DNA, replication and assembly, and lysis; the latent period is commonly tens of minutes, and each infected cell releases a burst of tens to hundreds of new phages. Because each burst infects further cells, a few phages can multiply to enormous numbers during a cheesemaking: if a million cells are infected and each releases 100 phages, 100 million phages result. The most frequently isolated lactococcal phages belong to the 936 group (now the genus Skunavirus), followed by the c2 group (Ceduovirus) and the P335 group, which includes temperate phages; Streptococcus thermophilus phages and Lactobacillus phages form other groups. Phage attack causes slow or stopped acid production (slow and dead vats), defective cheese with high moisture and poor flavour, and economic loss.
Phages enter dairies mainly with raw milk, in which they are commonly present at low numbers, and multiply in the vats; whey from infected vats can contain very high titres, of the order of a billion plaque-forming units per millilitre, and spreads them through aerosols, equipment, whey processing and personnel. Detection uses plaque assays, in which dilutions of whey are mixed with a sensitive host strain in a soft agar overlay; each phage forms a clear plaque in the bacterial lawn, and the titre in plaque-forming units per millilitre equals the number of plaques divided by the volume plated times the dilution, so 45 plaques from 0.1 millilitre of a 1 in a million dilution indicate 450 million per millilitre. Activity tests compare acid production by the starter with and without added whey filtrate, and molecular methods such as PCR identify phage groups. Control combines several measures. Hygiene and plant design separate whey and cheesemaking areas from starter preparation, use closed vats and filtered air, and clean and sanitise equipment with agents effective against phages, such as peracetic acid and chlorine. Starter management uses rotation of strains with different phage sensitivities, phage-resistant strains, including bacteriophage-insensitive mutants selected after phage challenge, and DVS cultures that avoid in-plant propagation. Phage-inhibitory media protect bulk starter. Heat treatment of whey before reuse and of milk reduces phage numbers, though some phages survive pasteurisation. Bacteria themselves resist phage by blocking adsorption, blocking DNA injection, restriction-modification systems, abortive infection systems and CRISPR-Cas, an adaptive immune system first shown experimentally in Streptococcus thermophilus in 2007, in which the cell stores fragments of phage DNA and uses them to destroy matching phage DNA on later infection.
Apart from phage, the main causes of slow starter activity are inhibitory substances in milk. Antibiotic residues, mainly from cows treated for mastitis whose milk is not withheld for the withdrawal period, inhibit starter bacteria at very low concentrations. Starter organisms differ in sensitivity: Streptococcus thermophilus and other thermophiles are especially sensitive to penicillin and other beta-lactams, and yoghurt cultures can be inhibited at levels close to the legal maximum residue limits, while lactococci are somewhat less sensitive. Effects range from slow acid development to complete failure, with weak curd, high moisture, poor flavour and possible growth of undesirable organisms that are not inhibited. Prevention relies on farm management and residue testing of milk, described in Dairy Analysis and Instrumental Methods. Residues of sanitisers and detergents from inadequately rinsed equipment also inhibit starters: quaternary ammonium compounds are particularly inhibitory and persistent on surfaces, and iodophors and chlorine compounds inhibit at higher concentrations. Correct concentrations, rinsing where required, draining and the use of sanitisers compatible with fermentation are the remedies. Natural inhibitors in milk, including the lactoperoxidase system, agglutinins that clump starter cells and cause uneven distribution, lysozyme and lactoferrin in colostrum, free fatty acids from lipolysis, and leucocytes in mastitic milk, can also slow starters, especially in milk from late-lactation or mastitic cows, as described in Microbiology of Raw and Market Milk.
Bacteriocins are antimicrobial peptides or proteins produced by bacteria that inhibit closely related bacteria. Many lactic acid bacteria produce them, which is one reason LAB dominate fermentations. Nisin, produced by some strains of Lactococcus lactis, is the best-known example. It is a lantibiotic, a peptide containing unusual amino acids such as lanthionine formed by post-translational modification, and it acts on the membranes of Gram-positive bacteria by binding to lipid II, a cell wall precursor, forming pores and blocking cell wall synthesis. Nisin is permitted in many countries as a food preservative, where it is used particularly in processed cheese to inhibit the outgrowth of clostridial spores, and in some other dairy products. Other LAB bacteriocins include lacticin 3147 from lactococci and pediocin from pediococci, which is active against Listeria. Bacteriocin production by starter strains can be useful, for example in protective cultures that inhibit Listeria or clostridia in cheese, but it can also cause problems when one strain in a mixed or multiple-strain starter produces a bacteriocin that inhibits the others, upsetting the balance of the culture or slowing acidification. Nisin-producing strains carry immunity genes that protect them from their own bacteriocin, and the genes for nisin production and immunity, together with sucrose metabolism genes, lie on a conjugative transposon. Bacteriocins are inactivated by proteases, which distinguishes them from many other inhibitors, and they are generally not effective against Gram-negative bacteria unless the outer membrane is damaged.
Many technologically important traits of lactococci are carried on plasmids, extrachromosomal DNA molecules that replicate independently: lactose fermentation (the lactose PTS and tagatose pathway genes), the cell-envelope proteinase, citrate permease, bacteriophage resistance mechanisms, bacteriocin production and immunity, and exopolysaccharide production. Industrial lactococcal strains commonly carry several plasmids. Because plasmids can be lost, especially during growth at high temperatures or in the presence of curing agents, cultures can give rise to lactose-negative and proteinase-negative variants that acidify milk slowly, which is one reason why culture stocks must be maintained carefully and checked. Genes move between strains by three natural mechanisms: conjugation, in which plasmids or transposons are transferred by cell-to-cell contact; transduction, in which DNA is carried by phage particles; and transformation, the uptake of free DNA, which in the laboratory is achieved by electroporation. Conjugation was used to construct improved strains by transferring natural phage-resistance plasmids, such as plasmids encoding abortive infection systems, into otherwise good industrial strains; because the DNA comes from lactococci and is transferred by a natural process, such strains are not regarded as genetically modified organisms in many jurisdictions. Streptococcus thermophilus carries few plasmids, and its genome shows evidence of adaptation to milk through loss of genes for functions not needed there.
The genome of Lactococcus lactis strain IL1403, published in 2001, was the first complete genome of a lactic acid bacterium; genomes of all the main starter species and of many strains are now known, revealing the genes for metabolism, proteolysis, stress responses and phage defence, and the prophages and CRISPR systems present. Strain improvement uses several approaches. Natural selection and classical methods include screening of wild isolates from traditional products, selection of spontaneous mutants with desired properties, such as bacteriophage-insensitive mutants obtained by challenging a strain with phage, and random mutagenesis followed by screening, for example for galactose-positive or low-post-acidification strains. Natural gene transfer by conjugation adds traits such as phage resistance. CRISPR-based selection, in which strains are exposed to phages and those that acquire new phage DNA spacers are selected, produces phage-resistant derivatives without genetic engineering and has been used commercially for Streptococcus thermophilus. Genetic engineering, using food-grade vectors without antibiotic resistance markers, self-cloning and inducible expression systems such as the nisin-controlled expression system, has produced strains with altered metabolism, enzyme overproduction and other properties, but the use of genetically modified starters in food is restricted by regulation and consumer acceptance in many countries, notably in the European Union. Genome editing raises the same regulatory questions. Safety assessment of starter strains includes checking for transferable antibiotic resistance genes and harmful metabolites such as biogenic amines.
A student uses this subject in dairy microbiology and fermented products practicals. You will maintain and propagate mesophilic and thermophilic cultures, prepare bulk starter, run activity tests, make Gram stains and catalase tests, enumerate starter bacteria on selective media, detect phage by plaque assay and activity tests, and test milk for inhibitory substances. Learn the organisms and their current names, the metabolic pathways and their products, culture types and the causes of starter failure, because examiners ask them repeatedly and because every fermented product practical depends on them.
Five years into work in a cheese or yoghurt plant, you choose and rotate cultures, keep vat acidification records and recognise a phage attack from a slow vat, sample whey for phage testing, decide whether to switch to a phage-resistant culture or change rotation, check milk for antibiotic and sanitiser residues when cultures slow, manage the starter room or DVS stocks and cold chain, and work with culture suppliers on flavour, texture and post-acidification problems. You also train operators to recognise early signs of culture trouble and to keep the records that make investigations possible.
A senior technologist with twenty-five years of experience sets culture strategy for a group of plants: defined versus undefined cultures, in-house propagation versus DVS, phage control programmes and plant layout, culture budgets and supplier contracts, and the introduction of adjunct, protective and probiotic cultures. Such a person also handles regulatory questions on culture safety and labelling, and decides when production problems justify a change of culture or process. Such a person balances cost, consistency, product character and risk across plants and markets with different traditions and regulations.
A researcher with long experience works on the biology of starter organisms: genomics and metabolic engineering, phage-host interactions and new defence systems, mechanisms of flavour formation, stress tolerance during drying and freezing, exopolysaccharide production, and the microbial ecology of traditional and artisanal cultures. Such a person develops new strains by natural or regulated methods and advises industry and regulators on their safe use. The work is judged by whether new strains perform reliably in commercial vats, not only in the laboratory, so collaboration with plants is essential.
This manual covers the organisms, metabolism, technology and genetics of starters; the manufacture of specific fermented milks and cheeses is in Fermented Milks Technology and Cheese Science and Technology, the chemistry of flavour compounds in Chemistry of Dairy Products, and probiotics and their health effects in the fermented milks and probiotics subjects. Taxonomic names follow the 2020 reclassification of Lactobacillus and later changes; names continue to change and older names are still used in standards. Phage taxonomy is given only for the main groups. Specific inoculation rates, activity test protocols and culture units vary by supplier and process and are given as examples. Detailed molecular genetics, regulatory procedures for genetically modified cultures in each country, and starter cultures for non-dairy fermentations are not covered.
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