Food Science & Technology · Food Science Core · also Food Biotechnology, Industrial Microbiology, Fermented Foods, Fermentation Technology
Microorganisms and enzymes put to work: starter cultures, the microbiology of fermented foods, industrial fermenter design and operation, downstream recovery, and the modern biotechnology (recombinant enzymes, precision fermentation, single-cell protein) that is reshaping ingredients.
In the exams: GATE XE-G and XL-U Section 2 (fermented foods and beverages); ICAR AICE 35.a Unit 6 (food microbiology and biotechnology)
Fermentation is the oldest food biotechnology and one of the newest. For thousands of years people on every continent have used lactic acid bacteria, yeasts and moulds to turn milk into yoghurt and cheese, cabbage into sauerkraut and kimchi, soybeans into soy sauce, miso and tempeh, rice and pulses into idli and dosa, maize and teff into fermented porridges and flatbreads, meat into salami and fish into fish sauce. Fermentation preserves food by acid, alcohol and competition, makes it more digestible, removes some anti-nutrients and creates flavours that cannot be made any other way. Today the same organisms, and engineered ones, are grown in stainless-steel fermenters of hundreds of cubic metres to make enzymes, citric acid, amino acids, flavours, vitamins, thickeners, single-cell protein and, by precision fermentation, milk and egg proteins without animals.
This subject covers both sides. The first half deals with the microbes of food fermentation and their starter cultures, including protection against bacteriophage, and the microbiology of fermented dairy, vegetable, cereal and legume, meat and fish foods and vinegar. The second half deals with industrial fermentation: fermenter design, aeration and agitation; the kinetics of batch, fed-batch and continuous culture; downstream recovery and purification; the production of food enzymes, organic acids, amino acids, flavours, single-cell protein and single-cell oil; recombinant DNA technology and genetically modified foods; and precision fermentation and how it is regulated. General microbiology and food microbiology, which cover microbial physiology, spoilage and pathogens, come before it.
Fermentation and biotechnology are examined in graduate entrance examinations in food technology, in the food microbiology sections that ask about fermented foods and beverages; in postgraduate and research-fellowship examinations, which add fermenter design, kinetics and biotechnology; and in food technologist licensure and technical qualification examinations. The practice questions follow those patterns, with full working for the kinetic and engineering calculations.
A starter culture is a preparation of live microorganisms added to a food to start and steer its fermentation. The most important group is the lactic acid bacteria: Gram-positive, catalase-negative, non-spore-forming, aerotolerant anaerobes that ferment sugars mainly to lactic acid and tolerate the acid they make. The main food genera are Lactococcus, Streptococcus (S. thermophilus is the only food species of that genus), Leuconostoc, Pediococcus, Weissella, Oenococcus and the lactobacilli. In 2020 the old genus Lactobacillus was split into 25 genera on genomic evidence, so many familiar species have new names: Lactobacillus delbrueckii subsp. bulgaricus and L. helveticus keep the old genus, but L. plantarum is now Lactiplantibacillus plantarum, L. casei and L. rhamnosus are in Lacticaseibacillus, L. reuteri and L. fermentum in Limosilactobacillus, L. brevis in Levilactobacillus, and L. sakei in Latilactobacillus.
Lactic acid bacteria are classed by how they ferment glucose. Homofermentative species use the Embden-Meyerhof-Parnas (glycolytic) pathway and convert more than 85 per cent of the sugar to lactic acid, with 2 ATP per glucose. Heterofermentative species use the phosphoketolase pathway and produce lactic acid, carbon dioxide and ethanol or acetic acid, with 1 ATP per glucose; Leuconostoc and Levilactobacillus brevis are examples, and their carbon dioxide gives the openness of some cheeses and the leavening of some batters. Starters are also classed by temperature: mesophilic cultures (Lactococcus lactis, Leuconostoc) grow best around 20 to 30 °C and are used for many cheeses, cultured butter and fermented milks of temperate and subtropical regions; thermophilic cultures (S. thermophilus, L. delbrueckii subsp. bulgaricus, L. helveticus) grow at 37 to 45 °C and are used for yoghurt and hard cooked cheeses. Besides acid, lactic acid bacteria contribute flavour (diacetyl from citrate by some Lactococcus and Leuconostoc strains, acetaldehyde in yoghurt), texture (exopolysaccharides) and protection (bacteriocins). Nisin, produced by some Lactococcus lactis strains, is a permitted food preservative in many jurisdictions and acts against Gram-positive bacteria, including spore-formers.
Yeasts are unicellular fungi. Saccharomyces cerevisiae leavens bread and ferments ale, wine and many spirits; lager is fermented by S. pastorianus at lower temperatures. S. cerevisiae ferments sugar to ethanol even in the presence of oxygen when sugar is plentiful (the Crabtree effect), which matters in the design of baker's yeast production (chapter 9). Other food yeasts include Zygosaccharomyces rouxii, which tolerates high salt and sugar and contributes to soy sauce and miso; Debaryomyces hansenii, on the surfaces of cheeses and dry sausages; Kluyveromyces marxianus and K. lactis, which ferment lactose; and the yeasts of sourdough and kefir.
Moulds are filamentous fungi. Aspergillus oryzae and A. sojae grown on steamed rice, barley or soybean form koji, the enzyme-rich starter of soy sauce, miso and rice wines in East Asia; Rhizopus oligosporus binds soybeans into tempeh in Southeast Asia; Penicillium roqueforti gives blue cheeses their veins and flavour and P. camemberti the white rind of soft cheeses; Monascus purpureus colours red yeast rice. Because some moulds can produce mycotoxins and some bacteria carry transferable antibiotic resistance genes, starter strains are chosen and documented for safety. Regulators use different approaches: the European Food Safety Authority keeps a list of microorganisms with a qualified presumption of safety (QPS), and in the United States many cultures are marketed as generally recognised as safe (GRAS); elsewhere national authorities and Codex principles apply. A probiotic, in the definition used by FAO and WHO, is a live microorganism that, when administered in adequate amounts, confers a health benefit on the host; that benefit must be shown for the specific strain, so not every fermented food is a probiotic food.
Traditional fermentations relied on spontaneous fermentation or on back-slopping, inoculating a new batch with part of the previous one, and some products, especially artisanal cheeses, still use undefined natural whey or milk cultures that contain many strains. Industrial practice moved first to propagation in the plant, a chain of mother culture, intermediate culture and bulk starter grown in heat-treated milk or a special medium, and then largely to direct vat set (direct vat inoculation) cultures: concentrated, frozen or freeze-dried cultures from a specialist supplier, added straight to the process vat. Direct vat set cultures remove the in-plant propagation steps, the most common route by which phage and contaminants entered, and give consistent activity, at a higher cost per tonne.
Culture manufacture is itself a fermentation. Selected strains are grown in a rich medium in a fermenter, with the pH held near neutral by adding alkali because acid would otherwise stop growth, to cell densities far above those reached in milk. The cells are harvested by centrifugation, mixed with cryoprotectants such as glycerol, sucrose, trehalose or skim-milk solids, and either frozen as pellets in liquid nitrogen and stored at about −45 °C or colder, or freeze-dried. Frozen concentrates usually keep higher activity; freeze-dried cultures are easier to ship and store. Cultures are described as defined (known strains in known proportions) or mixed and undefined. Each lot is tested for activity (the rate of acid production in a standard milk test), cell count, purity and absence of contaminants.
Bacteriophages are viruses that infect bacteria, and they are the main cause of failed or slow fermentations in dairy plants. A lytic phage injects its DNA, takes over the cell, makes new phage particles and bursts the cell, releasing typically tens to hundreds of new phages that infect neighbouring cells; in a vat of milk, phage numbers can rise by many orders of magnitude within one fermentation, so acid production slows or stops ('dead vats'), leaving milk that can no longer be made into the intended product and that may allow pathogens to grow. Temperate phages can integrate into the host chromosome as prophages and later be induced. Whey is a major reservoir because phages are released into it in vast numbers, and aerosols from whey handling spread them through the plant.
Control relies on several measures at once. Hygiene and plant design separate starter handling from whey and raw milk, use closed vats, filtered air and effective cleaning and sanitising (phages are inactivated by many sanitisers, though some resist heat better than vegetative bacteria). Culture rotation alternates strains that are not attacked by the same phages, so a phage population that builds up on one strain cannot grow on the next. Phage-resistant strains are selected, including strains carrying natural resistance mechanisms such as blocking phage adsorption, restriction-modification systems and abortive infection. The CRISPR-Cas system, now famous as a genome-editing tool, was first shown to be an adaptive immune system against phage in Streptococcus thermophilus, a dairy starter, and phage-resistant strains made by natural CRISPR acquisition are used commercially. Phage-inhibitory media for bulk starters contain phosphates or citrates that bind calcium, which many lactococcal phages need to adsorb and replicate. Monitoring uses activity tests, in which a slowed starter in whey-inoculated milk signals phage, and plaque assays on lawns of the host strain. Direct vat set cultures reduce exposure but do not remove the need for rotation and hygiene, because phage can still build up in the plant from the process vats.
Fermented milks are made in almost every dairy region of the world: curd (dahi) in South Asia, yoghurt across West Asia, Europe and the Americas, laban, ayran, skyr, filmjölk, kefir in the Caucasus and koumiss from mare's milk in Central Asia, among many others. Their common principle is that lactic acid bacteria convert part of the lactose to lactic acid, lowering the pH to about 4.6 or below, at which the casein micelles lose their charge and calcium phosphate and aggregate into a gel. Curd is traditionally set with a mesophilic mixed culture carried over from the previous batch; the result varies with the culture and the temperature. Kefir is fermented with kefir grains, gelatinous masses of polysaccharide (kefiran) carrying lactic acid bacteria, yeasts and acetic acid bacteria, and contains a little ethanol and carbon dioxide.
Yoghurt is defined in the Codex standard for fermented milks (CXS 243-2003) as a product fermented by symbiotic cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, which must remain viable and abundant until the end of shelf life, at least 10⁷ colony-forming units per gram in total for the starter organisms. The two species stimulate each other (protocooperation): S. thermophilus grows first, producing formic acid, carbon dioxide and pyruvate that stimulate L. bulgaricus, while L. bulgaricus, which is more proteolytic, releases peptides and amino acids that S. thermophilus needs. The manufacturing steps are standardisation of fat and solids (often fortified with milk powder or concentrated by membrane filtration), homogenisation, heat treatment much more severe than pasteurisation (for example 85 °C for 30 minutes or 90 to 95 °C for 5 to 10 minutes), which denatures whey proteins so that they bind to casein and give a firmer gel with less whey separation (syneresis), cooling to about 42 to 45 °C, inoculation, incubation for 3 to 6 hours to pH about 4.5 to 4.6, and cooling. Set yoghurt is incubated in the retail pot; stirred yoghurt is fermented in a tank and the gel broken before filling. Acetaldehyde, mostly from L. bulgaricus, gives the characteristic yoghurt flavour; continued acid production in the cold (post-acidification) is a quality problem that strain choice reduces.
Cheese making concentrates the casein and fat of milk by coagulating it and removing the whey. Starter lactic acid bacteria acidify the milk and later the curd; coagulation is by rennet (in most cheeses) or by acid (in fresh cheeses such as cottage cheese, quark and paneer, where heat and acid are used). The milk-clotting enzyme of rennet is chymosin, which specifically cleaves κ-casein at the Phe105-Met106 bond, removing the hydrophilic caseinomacropeptide that stabilises the micelle; the remaining para-κ-casein micelles then aggregate in the presence of calcium. Calf rennet has been largely replaced in many markets by microbial coagulants from Rhizomucor species and by fermentation-produced chymosin made by genetically modified fungi and yeasts (chapter 12). After coagulation the curd is cut, stirred, cooked (heated), drained, often textured (cheddaring), salted and moulded or pressed.
Ripening turns bland curd into cheese over weeks to years. The chief processes are glycolysis of residual lactose and metabolism of lactate; lipolysis of fat into free fatty acids; and proteolysis, first by residual rennet and the milk enzyme plasmin, then by peptidases from the starter and from non-starter lactic acid bacteria that grow in the cheese, producing peptides, amino acids and, from amino acids, many flavour compounds. Secondary microflora give each variety its identity: Propionibacterium freudenreichii ferments lactate to propionate, acetate and carbon dioxide, forming the eyes and sweet nutty flavour of Swiss-type cheeses; Penicillium roqueforti in blue cheeses produces methyl ketones from fatty acids; P. camemberti on mould-ripened soft cheeses deacidifies the surface and softens the paste; and the orange smear of washed-rind cheeses comes from coryneform bacteria and yeasts. The main microbial defect of hard and semi-hard cheeses is late blowing by Clostridium tyrobutyricum spores from silage-fed milk, which ferment lactate to butyric acid, hydrogen and carbon dioxide, cracking the cheese weeks later. It is prevented by milk-production hygiene, removal of spores by bactofugation or microfiltration, lysozyme, or, where permitted, nitrate. Faulty acidification, from phage or inactive starter, allows coliforms and Staphylococcus aureus to grow and is a food-safety as well as a quality failure.
Vegetables are fermented by the lactic acid bacteria already present on them, in a process controlled by salt, anaerobic conditions and temperature. Salt draws water and nutrients out of the tissue to form a brine, suppresses many Gram-negative spoilage bacteria and pathogens while the salt-tolerant lactic acid bacteria grow, and helps keep the tissue firm. Excluding air prevents the growth of moulds and film yeasts, which would consume the acid. As the bacteria lower the pH to about 4 or below, the product becomes microbiologically stable, especially when kept cool and protected from air.
Sauerkraut, the fermented cabbage of Central and Eastern Europe, shows the classic microbial succession. Shredded cabbage is mixed with about 2 to 3 per cent salt by weight (2.25 per cent is a common target) and packed tightly to exclude air. Heterofermentative Leuconostoc mesenteroides starts the fermentation quickly at moderate temperature and produces lactic acid, acetic acid, carbon dioxide (which flushes out oxygen), and mannitol and esters that contribute flavour; as acidity rises, more acid-tolerant homofermentative species, such as Lactiplantibacillus plantarum, Levilactobacillus brevis and Pediococcus, take over and carry the acidity to about 1.5 to 2 per cent as lactic acid and the pH to about 3.5. Too little or unevenly distributed salt gives soft kraut; too much salt or too high a temperature favours homofermentative organisms too early, giving a sharp, less aromatic product; surface yeasts can produce pink kraut.
Kimchi, the national dish of Korea and eaten across East Asia, is made from salted napa cabbage or radish mixed with garlic, ginger, red pepper powder, spring onion and often fish sauce or salted seafood. It is fermented at low temperatures, often near or below 10 °C, and is eaten at a range of acidities; Leuconostoc and Weissella species dominate the early fermentation, and Lactiplantibacillus plantarum and other acid-tolerant species become more prominent as it ages and sours. Its many ingredients add their own microbes and flavour precursors, and the low fermentation temperature gives a carbonated, fresh taste.
Cucumber pickles are fermented in brines whose salt content varies by tradition and process, commonly several per cent, with Lactiplantibacillus plantarum and Pediococcus as the main fermenting organisms; some processors add a starter culture and buffer the brine to obtain a more complete fermentation. Two characteristic defects show the biochemistry at work. Bloaters, cucumbers with hollow centres, form when carbon dioxide from heterofermentative bacteria, yeasts and the cucumber's own respiration accumulates inside the fruit; purging the brine with nitrogen or air removes the gas. Softening is caused by pectinolytic enzymes from moulds on flowers and from the cucumber itself, controlled by removing flowers, keeping brine surfaces free of air, and adding calcium. Many pickles elsewhere, such as the oil and spice pickles of South Asia, are preserved mainly by salt, acid, oil and spices with little or no lactic fermentation.
Table olives must lose their bitterness, caused by the phenolic glucoside oleuropein, before they are eaten. In the Spanish (Sevillian) style, green olives are treated with dilute lye, which hydrolyses oleuropein, washed and then fermented in brine by lactic acid bacteria; in the Greek style, naturally black olives are fermented in brine without lye, a slower process in which yeasts are prominent and enzymes and diffusion remove the bitterness. Across all fermented vegetables, the safety record is good when acidification is prompt and the pH falls to the intended value, but contamination after fermentation and inadequate acidification can allow pathogens to survive, and biogenic amines can accumulate in some products.
Fermentation of cereals and legumes is the basis of many staple foods: sourdough breads in Europe and the Americas, injera from teff in the Horn of Africa, ogi, kenkey and other fermented maize and sorghum products in West Africa, idli and dosa in South Asia, and tempeh, natto, miso and soy sauce in East and Southeast Asia. Fermentation improves these foods in several ways: it acidifies and so protects them; it leavens batters and doughs by carbon dioxide; it hydrolyses phytate (releasing minerals), oligosaccharides such as raffinose and stachyose (reducing flatulence) and some protease inhibitors; it can raise B-vitamin content; and it develops flavour and texture.
Idli (steamed cakes) and dosa (thin crêpes) are made from a batter of soaked and ground rice and black gram (Vigna mungo, urad dal), fermented overnight at ambient temperature. Leuconostoc mesenteroides, a heterofermenter, grows rapidly and its carbon dioxide raises the batter, held by the mucilaginous proteins and polysaccharides of black gram; Enterococcus faecalis and Pediococcus also acidify, and yeasts contribute in some batters. The pH falls to about 4.5 or lower overnight, and the batter roughly doubles in volume. Tempeh is made in Indonesia and now worldwide from dehulled soybeans that are soaked (during which lactic acid bacteria acidify the beans, which helps suppress pathogens), partly cooked, cooled, inoculated with Rhizopus oligosporus and incubated at about 30 °C for one to two days. The mould's mycelium knits the beans into a firm white cake and its proteases and lipases soften and flavour them. Vitamin B12 found in some traditional tempeh comes from contaminating bacteria, not from the mould. Natto, a Japanese product, uses Bacillus subtilis (natto strains), which forms sticky poly-γ-glutamic acid and is notably rich in vitamin K2; related Bacillus fermentations of African locust bean and other seeds give the savoury condiments dawadawa and iru of West Africa.
Miso and soy sauce depend on koji, made by growing Aspergillus oryzae or A. sojae on a solid substrate for two to three days. The mould secretes amylases, proteases and peptidases that later break down starch into sugars and proteins into peptides and amino acids, especially glutamic acid, the source of the savoury taste. Koji making is a solid-state fermentation, controlled for moisture, temperature and aeration on trays or in automated beds; strains used are non-toxigenic.
Miso is made by mixing koji (from rice, barley or soybean) with cooked soybeans and salt and fermenting the paste for weeks to years; salt-tolerant microbes, especially the lactic acid bacterium Tetragenococcus halophilus and the yeast Zygosaccharomyces rouxii, grow slowly and add acid, alcohol and aroma, and Maillard reactions darken long-aged types. Soy sauce is made from a koji of steamed soybeans (or defatted soybean meal) and roasted, crushed wheat. The koji is mixed with brine of about 18 per cent salt to form a mash called moromi, which is fermented for several months: first T. halophilus produces lactic acid and lowers the pH, then Z. rouxii ferments sugars to ethanol and forms aroma compounds, and Candida species contribute further aroma during ageing. The mash is pressed, and the raw sauce is pasteurised, which also develops colour and aroma, and filtered. Similar fermented soybean sauces and pastes are made across East and Southeast Asia, such as Korean doenjang and gochujang and Indonesian kecap. Brewed soy sauce must be distinguished from acid-hydrolysed vegetable protein sauces, made quickly by hydrolysing protein with hydrochloric acid; these can contain the chloropropanols 3-MCPD and 1,3-DCP, for which Codex and many countries set limits. Fermented cereal and legume foods made at home or in small enterprises can carry risks when acidification is slow, and contamination by toxigenic moulds or Bacillus cereus must be controlled.
Fermented sausages, such as the salami of Southern Europe, the dry sausages of Central Europe, sucuk in West Asia, pepperoni and summer sausage in North America and nem chua in Southeast Asia, are made from minced meat and fat with salt (often about 2.5 to 3 per cent), curing agents (nitrite and/or nitrate), sugars, spices and, in industrial production, a starter culture. The mixture is stuffed into casings, fermented for one to several days at a controlled temperature and humidity, and then dried for weeks. Lactic acid bacteria, either natural ones such as Latilactobacillus sakei or added starters such as Pediococcus acidilactici, P. pentosaceus and Lactiplantibacillus plantarum, ferment the added sugar to lactic acid, lowering the pH to about 4.8 to 5.3. The fall in pH towards the isoelectric point of meat proteins, together with salt, causes the proteins to gel and release water, so the sausage becomes firm and sliceable and dries faster. Catalase-positive cocci, Staphylococcus carnosus, S. xylosus and Kocuria varians, are added in many starters: they reduce nitrate to nitrite (which forms nitric oxide and the cured colour), decompose peroxides with catalase, so protecting colour and fat, and generate flavour through proteolysis and lipolysis. Mould-ripened types are covered with Penicillium nalgiovense or similar non-toxigenic moulds, which protect the surface, regulate drying and add flavour.
The safety of fermented sausages comes from a sequence of hurdles: nitrite, salt and low water activity at the start; competitive lactic acid bacteria; low pH after fermentation; and falling water activity during drying. The early hours are critical: if acidification is slow, Staphylococcus aureus can multiply and form heat-stable enterotoxin. Outbreaks of illness from Shiga toxin-producing Escherichia coli and Salmonella in dry sausages showed that fermentation and drying alone may not destroy pathogens from raw meat, so some jurisdictions require processes validated to achieve a specified log reduction, or a heat step. Listeria monocytogenes and the formation of biogenic amines such as tyramine and histamine are also controlled through raw-material quality, starter choice and process control.
Fish is fermented in two broad ways. In the first, fish is mixed with a high concentration of salt and left for months while enzymes from the fish's own tissues and gut, with a small contribution from halophilic bacteria, hydrolyse the proteins to peptides and amino acids. Fish sauce is the liquid that drains from such a mixture, commonly in a ratio of about three parts fish to one part salt by weight, after fermenting for many months in Southeast Asia (nuoc mam, nam pla, patis and others); fermented fish and shrimp pastes, such as bagoong and belacan, are made similarly with less liquid. The high salt prevents spoilage and pathogens, and the hydrolysis gives intense savoury taste. The main hazard is histamine, formed by bacterial decarboxylation of histidine in fish such as anchovy, mackerel and sardine before salting; it is not destroyed by later processing, so freshness and chilling before fermentation are critical, and Codex sets histamine limits for fish sauce.
In the second way, fish is fermented with a carbohydrate source, usually cooked rice, which lactic acid bacteria ferment to lower the pH with less salt. Examples include narezushi in Japan (the ancestor of sushi), plaa-som and burong isda in Southeast Asia, and the fermented fish of parts of Africa. Northern European traditions include surströmming, Baltic herring fermented in brine in Sweden, and rakfisk, trout fermented in Norway, which rely on low temperature as well as salt. Products fermented at low salt and without prompt acidification carry a recognised risk of botulism, particularly from Clostridium botulinum type E, which is associated with fish and grows at low temperatures; outbreaks linked to traditional fermented fish and marine-mammal products have been recorded in northern regions. Good practice therefore combines fresh raw material, correct salt levels, active acidification and cool storage.
Vinegar is the product of two successive fermentations: an alcoholic fermentation of a sugar source by yeast, and an acetic fermentation in which acetic acid bacteria oxidise the ethanol to acetic acid. Almost any sugary or starchy material can be used, and vinegars are named by their source: wine, cider, malt, rice, cane, coconut, date, fruit and spirit (distilled alcohol) vinegars, among others. Starchy materials such as rice and malted barley must first be saccharified by enzymes. Codex and national standards define vinegar by its origin and minimum acidity; a minimum of about 4 g of acetic acid per 100 mL is common for table vinegars, but values differ by jurisdiction and type.
Acetic acid bacteria are Gram-negative, obligately aerobic rods of the genera Acetobacter, Gluconobacter, Gluconacetobacter and Komagataeibacter. They oxidise ethanol in two steps, catalysed by membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase: ethanol to acetaldehyde, then acetaldehyde to acetic acid. The overall reaction, C₂H₅OH + O₂ → CH₃COOH + H₂O, needs one mole of oxygen per mole of ethanol and releases considerable heat, so industrial acetators need both intensive aeration and cooling. From the molar masses (46.07 and 60.05 g/mol), 1 g of ethanol can give at most 1.30 g of acetic acid; real yields are a little lower because some ethanol evaporates or is over-oxidised. Some Acetobacter strains will over-oxidise acetic acid to carbon dioxide and water when ethanol runs out, which destroys the product; industrial vinegar strains of Komagataeibacter (such as K. europaeus) tolerate high acidity and are less prone to this. Komagataeibacter xylinus produces bacterial cellulose, which forms the gelatinous 'mother of vinegar' and is grown deliberately to make nata de coco in Southeast Asia.
Three methods have been used historically and are still found. In the surface (Orleans) method, the slowest, wine is held in partly filled barrels with access to air, and a film of acetic acid bacteria at the surface converts it to vinegar over weeks to months, some of the vinegar being withdrawn and replaced with new wine; it produces high-quality speciality vinegars. In the trickling generator method, developed in the nineteenth century, the alcoholic liquid is sprayed repeatedly over a packing of beech-wood shavings or similar material carrying the bacteria, with air drawn upwards, so that conversion takes days instead of weeks. The submerged method, now dominant for industrial production, grows the bacteria freely suspended in the liquid in a stirred and vigorously aerated fermenter (acetator) at about 30 °C. Because the bacteria are very sensitive to oxygen interruption at high acidity, even a brief failure of aeration can kill the culture. The process is semi-continuous: when the ethanol content falls to a low set point, part of the vinegar is withdrawn and replaced with fresh mash, and each cycle takes about a day. Submerged processes reach acidities well above those of traditional methods, and concentrated vinegars are diluted for sale.
After fermentation, vinegar is clarified, filtered, sometimes aged in wood, and often pasteurised or sterile-filtered and bottled. Defects and contaminants include vinegar eels (the nematode Turbatrix aceti), which are controlled by filtration and pasteurisation, and slime formed by cellulose-producing bacteria. Speciality vinegars show how much the process affects quality. Traditional balsamic vinegar is made from cooked grape must that undergoes simultaneous yeast and acetic fermentation and is then aged for many years in a series of progressively smaller wooden barrels.
A fermenter (bioreactor) provides a controlled, contamination-free environment in which microorganisms grow and make products. The workhorse of industry is the stirred-tank reactor: a stainless-steel cylindrical vessel with a height-to-diameter ratio commonly of about 2 to 3, fitted with one or more impellers on a central shaft, four baffles (each about one-tenth of the tank diameter wide) to prevent vortex formation and improve mixing, a sparger at the bottom to introduce sterile air, a jacket or internal coils for heating and cooling, and ports for addition, sampling and harvest. Instruments measure and control temperature, pH (with acid and base addition), dissolved oxygen, foam (with mechanical breakers or antifoam addition), pressure and air flow, and many plants measure off-gas oxygen and carbon dioxide to calculate the microbial oxygen uptake rate. The vessel, pipework and valves are designed for steam sterilisation in place, typically at about 121 °C, and for cleaning in place, with polished surfaces, drainable lines and steam-sealed valves; air is sterilised by filtration through membrane or depth filters rated at about 0.2 µm, and the medium is sterilised in the vessel (batch) or continuously in a heat exchanger, the continuous method giving less heat damage to the nutrients.
Other designs suit particular needs. Airlift fermenters circulate the broth by the density difference between an aerated riser and an unaerated downcomer, without mechanical agitation, so they have low shear and low power demand for a given oxygen transfer, and were used for very large single-cell protein plants and for shear-sensitive cultures. Bubble columns are simpler still. Packed-bed and immobilised-cell reactors hold cells on a support, as in vinegar generators. Solid-state fermentation grows moulds on moist solid substrates, in trays, packed beds or rotating drums, as in koji making and some enzyme production; it needs little water and gives concentrated products but is hard to control because heat removal and moisture control are difficult.
Oxygen is often the limiting nutrient in aerobic fermentations because it is poorly soluble: water saturated with air holds only about 8 mg of oxygen per litre at 25 °C, less in warm or concentrated broths, while a dense culture can consume that in seconds. The oxygen transfer rate from bubbles to liquid is OTR = kLa(C* − C_L), where kLa is the volumetric mass-transfer coefficient, C* the saturation concentration and C_L the actual dissolved-oxygen concentration. At steady state it must equal the oxygen uptake rate of the culture, OUR = q_O₂·X, the specific uptake rate times the biomass concentration. The dissolved oxygen must be kept above the critical concentration below which the organism's respiration slows. kLa is raised by increasing agitation power per unit volume (which breaks bubbles into smaller ones and increases interfacial area), increasing air flow (superficial gas velocity), using enriched oxygen or raising back-pressure (which raises C*); empirical correlations of the form kLa = K(P/V)^α·(v_s)^β describe it for a given system. kLa is measured by the dynamic method (stopping aeration, then following the re-oxygenation curve) or by the older sulphite-oxidation method.
Agitation is described by the power drawn: P = N_p·ρ·N³·D⁵, where N_p is the power number (about 5 to 6 for a six-bladed Rushton disc turbine in turbulent flow), ρ the density, N the rotational speed and D the impeller diameter. Doubling the speed therefore raises power eightfold, and doubling the impeller diameter raises it thirty-two-fold. Aeration lowers the power drawn because gas collects behind the blades. Rushton turbines disperse gas well; axial-flow hydrofoil impellers mix with less power and shear. Heat must also be removed: the metabolic heat of aerobic growth is roughly proportional to oxygen consumed, about 460 kJ per mole of oxygen, and is added to the heat of agitation, so large fermenters need large cooling areas.
Scale-up from laboratory to production is difficult because the physical parameters cannot all be kept constant at once. Engineers choose a criterion, commonly constant power per unit volume, constant kLa, constant impeller tip speed (πND, related to shear) or constant mixing time, and accept changes in the others. Large vessels have longer mixing and circulation times, so cells pass through zones of different substrate and oxygen concentrations; scale-down experiments in the laboratory mimic these gradients to test how the organism responds.
In batch culture all the nutrients are present at the start and nothing is added except air and pH control agents. A batch culture passes through the lag phase (adaptation), the exponential phase, the stationary phase (when a nutrient runs out or inhibitors accumulate) and the death phase. In the exponential phase the growth rate is proportional to the biomass, dX/dt = μX, so X = X₀e^(μt), where μ is the specific growth rate; the doubling time is t_d = ln 2/μ = 0.693/μ. A culture with μ = 0.35 per hour doubles in about 2 hours and in 10 hours grows by a factor of e^3.5, about 33.
The Monod equation relates the specific growth rate to the concentration S of the limiting substrate: μ = μ_max·S/(K_s + S), where μ_max is the maximum specific growth rate and K_s the saturation constant, the substrate concentration at which μ = μ_max/2; it has the same form as the Michaelis-Menten equation. K_s is usually small, so in a batch culture μ stays near μ_max until the substrate is nearly exhausted. The yield coefficient Y_X/S = ΔX/(−ΔS) gives the biomass formed per unit of substrate used; aerobic growth of yeast on glucose gives about 0.5 g dry biomass per gram of glucose, whereas fermentative growth gives far less, because most of the sugar becomes ethanol.
Products are classed by their relation to growth, as described by the Luedeking-Piret equation, dP/dt = α·dX/dt + β·X. Growth-associated products (β = 0), such as ethanol and lactic acid in primary metabolism, are formed in proportion to growth; non-growth-associated products (α = 0), such as many secondary metabolites, antibiotics and some enzymes and pigments, are formed mainly in the stationary phase; mixed products have both terms. This classification decides the best operating strategy: growth-associated products favour fast growth or continuous culture, non-growth-associated products a growth phase followed by a long production phase.
In fed-batch culture, nutrients are added during the fermentation, usually without removing broth, so the volume rises. Feeding keeps the substrate concentration low and controlled, which avoids substrate inhibition, avoids catabolite repression of enzyme synthesis, and avoids overflow metabolism. Baker's yeast production is the standard example: Saccharomyces cerevisiae produces ethanol even aerobically if glucose is in excess (the Crabtree effect), wasting carbon, so molasses is fed at a rate that keeps glucose very low, often following the exponential growth of the culture, and the respiratory quotient is monitored, a rise above about 1 signalling ethanol formation and the need to slow the feed. Fed-batch is also the usual mode for producing enzymes, recombinant proteins and many amino acids, reaching very high cell densities.
In continuous culture, fresh medium is fed at flow rate F and broth removed at the same rate, so the volume V stays constant. In a chemostat, the dilution rate D = F/V sets the growth rate: at steady state μ = D. From the Monod equation, the steady-state substrate concentration is S = K_s·D/(μ_max − D), and the biomass concentration is X = Y_X/S(S_R − S), where S_R is the substrate concentration in the feed. If D exceeds the maximum growth rate attainable at the feed concentration, μ_max·S_R/(K_s + S_R), essentially μ_max, the cells are removed faster than they grow and wash out. Biomass productivity, D·X, rises with D and peaks just below washout. For example, with μ_max = 0.5 per hour, K_s = 0.2 g/L, S_R = 10 g/L and Y = 0.5, a dilution rate of 0.25 per hour gives S = 0.2 × 0.25/0.25 = 0.2 g/L and X = 0.5 × (10 − 0.2) = 4.9 g/L. A turbidostat instead adjusts the feed to hold biomass constant. Continuous culture gives high productivity and steady product quality and is used for some single-cell protein and mycoprotein, ethanol and vinegar processes, and for research on microbial physiology. Its limits are contamination over long runs, the selection of faster-growing but less productive mutants, and the difficulty of matching continuous production to market demand, so most food fermentations remain batch or fed-batch.
Downstream processing turns a dilute, complex fermentation broth into a product of the required purity, concentration and form. It often accounts for a large share of total production cost, and for high-purity products frequently more than half, so it is planned together with the fermentation. A useful framework is the sequence removal of insolubles, isolation (primary recovery and concentration), purification and polishing (final formulation), with the number and severity of steps set by the purity needed: a fermented food ingredient may need only cell removal and drying, an enzyme for food use moderate purification, and a pharmaceutical protein extensive chromatography.
The first step separates cells from the broth. Centrifugation in disc-stack centrifuges suits yeasts and bacteria; decanter centrifuges handle concentrated slurries and mycelia; rotary vacuum filters, often with a filter aid, handle filamentous moulds; and cross-flow microfiltration through membranes is increasingly used because it gives a clear filtrate without filter aid. If the product is inside the cells, as for many recombinant proteins in bacteria and some yeast products, the cells must be disrupted: high-pressure homogenisers (passing the suspension through a narrow valve at high pressure) and bead mills are the industrial methods, with enzymatic lysis (lysozyme for some bacteria), chemical permeabilisation and ultrasonication more common at small scale. Disruption releases cell debris and nucleic acids that make the broth viscous, so nuclease treatment or further clarification follows. Secreted products, such as most fungal enzymes, citric acid and amino acids, avoid this step, which is one reason secretion hosts are preferred in food biotechnology.
Products are concentrated by evaporation (for heat-stable products such as organic acids and amino acids), by ultrafiltration (for proteins, which are retained by membranes of suitable molecular-weight cut-off while water and small molecules pass), by precipitation (with ammonium sulphate or organic solvents for proteins, with calcium salts for organic acids), by adsorption onto resins, or by extraction into a solvent or into one phase of an aqueous two-phase system of polymers and salts. Distillation recovers ethanol and other volatile products. Chromatography gives the highest resolution: ion-exchange chromatography separates by charge, gel filtration (size exclusion) by molecular size, hydrophobic-interaction chromatography by surface hydrophobicity, and affinity chromatography by specific binding to a ligand, for example a metal-chelate column binding a histidine tag engineered onto a protein. Industrial food enzymes are usually purified only partly, by clarification, ultrafiltration and perhaps precipitation, and standardised to a declared activity.
Classic recovery of citric acid illustrates the chemistry of downstream processing. After the mycelium is filtered off, calcium hydroxide (lime) is added to precipitate calcium citrate, which is filtered and washed; sulphuric acid then releases citric acid into solution and precipitates calcium sulphate (gypsum), and the citric acid solution is treated with activated carbon and ion exchange, evaporated and crystallised. Lactic acid was traditionally recovered the same way via calcium lactate; both routes produce large amounts of gypsum, so newer processes use solvent extraction, electrodialysis or membrane methods. Final finishing includes crystallisation, drying (spray drying for enzymes and yeast products, freeze drying for sensitive cultures and proteins, drum or fluid-bed drying for some), granulation or encapsulation to reduce dust and improve stability, and formulation with stabilisers and preservatives for liquid enzyme products. Throughout, the product must meet specifications for identity, purity, activity, microbial limits and contaminants, such as the specifications for food enzymes set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), and, for products from genetically modified organisms, the absence of viable production organisms or recombinant DNA where the regulator requires it.
Most food enzymes are now made by microbial fermentation. Bacillus species produce heat-stable α-amylases for starch liquefaction and proteases; Aspergillus niger produces glucoamylase, pectinases, glucose oxidase and catalase; A. oryzae produces fungal α-amylase for baking and lactase; Kluyveromyces lactis produces lactase; immobilised glucose isomerase from Streptomyces converts glucose to fructose for high-fructose syrups; Rhizomucor miehei produces a microbial milk coagulant; Streptomyces mobaraensis produces transglutaminase for protein cross-linking; and genetically modified strains of Aspergillus, Kluyveromyces and other hosts produce chymosin and many other enzymes (chapter 12). Enzymes are made by submerged fed-batch fermentation, or by solid-state fermentation for some fungal enzymes, and are secreted into the medium.
Citric acid, the most widely used food acidulant, is made almost entirely by Aspergillus niger in submerged culture on molasses or glucose syrups. High yields need high sugar concentration, low pH (about 2, which also suppresses contaminants and the formation of gluconic and oxalic acids), intense aeration, and limitation of trace metals, especially manganese; under these conditions the fungus overflows the TCA cycle and excretes citrate. Lactic acid is made by homofermentative lactic acid bacteria, or by engineered yeasts, from glucose or sucrose; optically pure L-lactic acid is needed for polylactic acid plastics. Gluconic acid is made by A. niger's glucose oxidase. Amino acids are produced in large tonnages: glutamic acid, sold as monosodium glutamate, by Corynebacterium glutamicum, which excretes it when its membrane is made leaky by limiting biotin or by adding penicillin or surfactants; and lysine, chiefly for animal feed, by C. glutamicum mutants whose aspartate kinase is no longer subject to feedback inhibition. Fermentation also makes polysaccharides (xanthan gum by Xanthomonas campestris, gellan by Sphingomonas elodea, dextran by Leuconostoc), vitamins (riboflavin by Ashbya gossypii or Bacillus subtilis, vitamin B12 by Propionibacterium or Pseudomonas denitrificans), carotenoids (β-carotene by the fungus Blakeslea trispora) and the flavour nucleotides IMP and GMP. Flavour compounds made by microorganisms or enzymes from natural precursors, such as γ-decalactone (peach) by Yarrowia lipolytica from ricinoleic acid, vanillin from ferulic acid, and diacetyl, can be labelled as natural flavourings in several jurisdictions, which is a major reason for their production.
Single-cell protein (SCP) is the dried biomass of microorganisms grown for food or feed. Microbes grow far faster than plants or animals, with doubling times of hours, on land-independent substrates, and their biomass is rich in protein: bacteria often contain 50 to 80 per cent protein on a dry basis, yeasts about 45 to 55 per cent, filamentous fungi and microalgae somewhat less to similar amounts depending on species and conditions. Substrates have included methanol (the bacterium Methylophilus methylotrophus in very large airlift fermenters in the 1970s and 1980s), methane (Methylococcus capsulatus, now used for fish and animal feed), whey (Kluyveromyces), sulphite liquor and molasses (Candida utilis, the fodder yeast), starch and glucose (the fungus Fusarium venenatum, grown continuously to make mycoprotein), and carbon dioxide with light (the microalgae Arthrospira, sold as spirulina, and Chlorella) or with hydrogen (hydrogen-oxidising bacteria). The main nutritional limitation is nucleic acid: bacterial biomass may contain around 8 to 16 per cent RNA and DNA, and yeast somewhat less. Purines from nucleic acids are metabolised to uric acid, and humans, lacking uricase, risk gout and kidney stones from high intakes; an expert recommendation of the former UN Protein Advisory Group limited nucleic acid from SCP to about 2 g per day for adults. For food use, RNA is therefore reduced, for example by holding mycoprotein at around 64 to 68 °C, which activates the organism's own ribonucleases. Other requirements are safety (absence of toxins and pathogens), digestibility, acceptable flavour and texture, and economics against soybean and fish meal.
Single-cell oil is lipid made by oleaginous microorganisms, defined as those accumulating more than about 20 per cent of their dry weight as lipid. Accumulation is triggered when the nitrogen source runs out while carbon remains: growth stops but carbon continues to be converted into triacylglycerol. Oleaginous yeasts include Yarrowia lipolytica, Rhodotorula and Lipomyces species; the fungus Mortierella alpina produces oil rich in arachidonic acid, and the marine microorganisms Crypthecodinium cohnii and Schizochytrium (thraustochytrids) produce oils rich in docosahexaenoic acid. These microbial arachidonic and docosahexaenoic acid oils are used in infant formula and supplements and as a vegetarian alternative to fish oil.
Recombinant DNA technology joins DNA from different sources and introduces it into a host organism. Its tools are restriction endonucleases, which cut DNA at specific short recognition sequences, often palindromic (EcoRI cuts G^AATTC, leaving sticky ends); DNA ligase, which joins fragments; vectors, such as plasmids with an origin of replication, a selectable marker and a multiple cloning site; methods of introducing DNA into cells (transformation, electroporation, and, for plants, Agrobacterium tumefaciens carrying a modified Ti plasmid or particle bombardment); the polymerase chain reaction to amplify genes; and expression signals (promoters, terminators, secretion signals) that make the host produce the encoded protein. Common hosts for food enzymes and ingredients are Escherichia coli, Bacillus subtilis, the yeasts Saccharomyces cerevisiae, Kluyveromyces lactis and Komagataella phaffii (formerly Pichia pastoris), and the fungi Aspergillus niger, A. oryzae and Trichoderma reesei. Genome editing, especially CRISPR-Cas9, uses a guide RNA to direct a nuclease to a chosen site, where the cell's repair of the double-strand break can produce small changes without inserted foreign DNA, or insert a template.
Genetically modified (GM) crops have been grown commercially since the mid-1990s; the first GM whole food on sale was a tomato with delayed softening (1994). The most widespread traits are herbicide tolerance (for example a bacterial EPSPS enzyme insensitive to glyphosate) and insect resistance (Cry proteins from Bacillus thuringiensis), mainly in soybean, maize, cotton and canola; nutritional traits include Golden Rice, engineered to make β-carotene, and high-oleic soybean oil. Safety assessment follows the Codex principles for foods derived from modern biotechnology (CAC/GL 44-2003) and guidelines for recombinant-DNA plants and microorganisms (CAC/GL 45-2003 and 46-2003). It is comparative: the GM food is compared with a conventional counterpart with a history of safe use for composition, nutrition, toxicity and allergenicity of new proteins (assessed by sequence similarity to known allergens and resistance to digestion), and unintended effects. The Cartagena Protocol on Biosafety governs the transboundary movement of living modified organisms. Approval, labelling and coexistence rules differ widely: many jurisdictions require labelling above a threshold of adventitious presence (0.9 per cent in the European Union), some use disclosure standards, and others label only where composition differs. Regulators also differ on genome-edited organisms without foreign DNA: several countries in the Americas and Asia-Pacific treat some of them like conventionally bred organisms, while the European Union has applied its GMO rules to them since a 2018 court ruling and has been revising this approach. Detection of GM material in food uses PCR, from screening for common elements such as the cauliflower mosaic virus 35S promoter and the nos terminator to event-specific quantitative methods.
Fermentation for food ingredients is now often described in three categories: traditional fermentation, which uses microbes to transform a food (yoghurt, tempeh); biomass fermentation, which grows microbes as the food itself (mycoprotein, SCP); and precision fermentation, which uses engineered microorganisms as factories for a specific ingredient that is then purified from the broth. Fermentation-produced chymosin, first approved in the United States in 1990 and now used for a large share of the world's cheese, was the first major precision-fermentation food ingredient. Others include enzymes of many kinds, the sweetener components rebaudioside M and other steviol glycosides made by engineered yeast, soy leghaemoglobin made in Komagataella phaffii to give plant-based burgers a meat-like colour and flavour, human-milk oligosaccharides such as 2′-fucosyllactose made by engineered E. coli for infant formula, and animal proteins such as β-lactoglobulin from milk and ovalbumin from egg, made by fungi or yeasts for 'animal-free' dairy and egg products. The process is a fed-batch fermentation with secretion where possible, followed by downstream purification; the purified ingredient is chemically identical or very similar to its natural counterpart.
Regulation focuses on the ingredient, the production strain and the process. Regulators ask whether the production organism is safe and well characterised, whether it or its recombinant DNA remains in the final product, whether the ingredient is identical to the counterpart or differs (for example in glycosylation), whether it is toxic or allergenic, what impurities and by-products are present, and what the intended uses and intakes are. Routes differ: in the United States many such ingredients are self-affirmed or notified as generally recognised as safe or approved as additives; the European Union assesses them as novel foods, food enzymes or additives, with GMO rules applying if viable organisms or recombinant DNA remain; Singapore, Australia and New Zealand, Canada, Israel and other jurisdictions have their own novel-food procedures, and Codex principles underlie many of them. Labelling raises its own questions: a milk protein made without cows is still a milk allergen and must be declared as such, and terms such as 'animal-free', 'dairy' or 'natural' are interpreted differently by jurisdiction.
A student uses this subject in microbiology and processing laboratories: making yoghurt, sauerkraut and vinegar and following pH and acidity; enumerating lactic acid bacteria; running a shake-flask or small-fermenter culture and calculating μ, doubling time and yield; and measuring kLa by gassing out. In examinations the high-value items are the organisms of each fermented food, the Monod and chemostat relations, the vinegar yield, and the chemistry of cheese coagulation and citric acid production.
Five years into a dairy, beverage, bakery or ingredient plant, a technologist manages starter cultures and phage risk, investigates slow vats, sets fermentation temperature and salt for vegetable and meat products, validates the lethality of fermented sausage processes, scales up a fermentation from pilot to production, and troubleshoots oxygen limitation, foaming and contamination in fermenters. Ingredient technologists also assess suppliers' fermentation-derived enzymes and additives and their regulatory status.
A senior manager with twenty-five years' experience decides on technology and investment: direct-vat-set versus in-plant cultures, building or contracting fermentation capacity, entering precision-fermented ingredients, and the regulatory and labelling strategy for GM-derived and novel foods in each market. They balance yield, cost of downstream processing and energy, and manage risk around phage, contamination and product recalls.
A researcher at this level works on strain development and physiology (metabolic engineering, CRISPR-based tools, phage resistance), microbial ecology of traditional fermentations using sequencing, bioprocess engineering and scale-down studies, techno-economic and life-cycle analysis of precision and biomass fermentation, and the scientific basis of safety assessments for new strains and ingredients.
This manual covers fermented foods and food biotechnology at degree level. It does not cover the brewing, winemaking and distilling of beverages in detail (beverage-technology), bread fermentation (bakery-pasta-snacks), probiotic health evidence (functional-foods-nutraceuticals) or general microbial physiology (general-microbiology). Process parameters (salt levels, temperatures, times, yields) are typical ranges; they vary by product, tradition and plant. Regulation of GM, genome-edited and precision-fermented foods is changing quickly and differs by jurisdiction, so only the common principles are given; current national rules must be checked.
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