Dairy Science & Technology · Dairy Microbiology · also Dairy Microbiology I, Microbiology of Milk
Where milk bacteria come from, how they grow and spoil milk, and how processing changes the count.
In the exams: ICAR AICE subject 36 Unit 3; ICAR AIEEA-PG group 16 (classification by growth temperature, spoilage, grading of raw milk, bacteriological aspects of processing)
Milk leaves a healthy udder almost free of bacteria and becomes, within hours, one of the best growth media in nature. Microbiology of Raw and Market Milk explains where the bacteria in milk come from, which groups matter at each temperature, how they spoil milk, how milk's own antimicrobial systems slow them, how raw milk is graded, and what chilling, heat treatment, bactofugation and microfiltration do to the microbial population. It ends with the two problems that most often decide the shelf life of market milk: biofilms in equipment and contamination after pasteurisation.
The subject matters because the microbial quality of raw milk sets the limits of everything made from it. A tanker of milk with a high psychrotroph count may pass every test at reception and still produce UHT milk that gels in three months, cheese with poor yield, or powder with rancid flavour, because the bacteria's heat-stable enzymes survive processing. A pinhole in a pasteuriser plate or a dirty filler can halve the shelf life of an otherwise excellent product. Dairy microbiologists therefore work across the whole chain, from the cow's teats to the retail chiller.
General food microbiology, the factors controlling microbial growth, food-borne pathogens, indicator organisms, rapid methods and microbiological criteria are covered in Food Technology's Food Microbiology subject; the chemistry of the lactoperoxidase system, indigenous enzymes and heat indicators in Milk Proteins and Enzymes; and pasteurisation, UHT, bactofugation and ESL equipment in Market Milk Processing. This manual refers to those subjects and concentrates on the microbiology of milk itself. It is examined in Unit 3 of ICAR AICE-JRF/SRF subject 36 (Dairy Microbiology) and in group 16 of ICAR AIEEA-PG, which names classification by growth temperature, spoilage, grading of raw milk and the bacteriology of thermisation, pasteurisation, boiling, sterilisation, UHT, bactofugation and membrane filtration. Food safety officer and analyst papers ask about grading tests and hygiene. Expect calculations of plate counts, generation times, log reductions and D and z values.
Milk inside the udder of a healthy animal is close to sterile, but milk drawn aseptically from a healthy quarter still contains a small number of bacteria, typically up to a few hundred per millilitre, from the teat canal and lower teat cistern: mainly micrococci, coagulase-negative staphylococci and coryneform bacteria such as Corynebacterium bovis. These udder commensals contribute little to the count of well-produced milk. Infected quarters are a different matter: mastitis can add large numbers of pathogens, as described in chapter 6. The outside of the animal is the main source of the bacteria that enter milk during milking. Teat skin carries organisms from soil, bedding, faeces and feed: coliforms and other Enterobacteriaceae, enterococci, psychrotrophic Gram-negative rods, and the spores of Bacillus and Clostridium species. Silage is an important source of clostridial spores, especially Clostridium tyrobutyricum, which survive pasteurisation and cause late blowing in hard cheeses, as described in Chemistry of Dairy Products. Wet, dirty teats and udders can contribute thousands of bacteria per millilitre. The milker's hands and clothing add organisms, including potential pathogens such as Staphylococcus aureus. Good udder preparation, with fore-stripping, cleaning and drying of teats (often with a pre-milking disinfectant dip), clean housing and bedding, clipped udders and clean hands, keeps this contribution low.
Inadequately cleaned equipment is usually the largest single source of high bacterial counts in raw milk. Milk residues left in milking machines, pipelines, rubber liners and tubes, cans, filters and bulk tanks support growth between milkings, and the bacteria are washed into the next milk. Rubber parts that are cracked or perished, dead ends in pipework, and deposits of milkstone (calcium phosphate and protein) shelter bacteria from cleaning solutions. Organisms from equipment are often psychrotrophs, thermodurics and biofilm formers, as described in chapters 2 and 11. Water used for rinsing equipment and udders can contribute Pseudomonas, coliforms and other organisms, so it must be of potable quality or treated. Air contributes relatively few bacteria to milk compared with other sources, though dusty feeding during milking and poorly ventilated milking parlours increase airborne spores and moulds. Flies, cans left uncovered and unclean collection points add contamination in smallholder systems, where milk may be carried for hours in warm conditions before cooling. The combined effect is that the count of freshly drawn bulk milk can range from below a thousand to hundreds of thousands per millilitre, depending almost entirely on hygiene. Legal limits for raw milk, such as the European Union limit of 100,000 colony-forming units per millilitre at 30 degrees Celsius (as a rolling geometric mean) and the United States limit of 100,000 per millilitre for an individual producer's milk, are achievable only with clean equipment and prompt cooling.
Bacteria in milk are grouped by their response to temperature, because temperature decides which of them grow during storage and which survive heat treatment. Mesophiles grow best at about 20 to 40 degrees Celsius; they include the lactic acid bacteria such as Lactococcus lactis and enterococci, coliforms and many others, and they dominate in milk that is not cooled, where they cause rapid souring. Psychrotrophs are organisms able to grow at 7 degrees Celsius or below, whatever their optimum temperature, which is usually higher, at about 20 to 30 degrees Celsius; they are distinguished from true psychrophiles, whose optimum is 15 degrees Celsius or below and which are rare in milk. Psychrotrophs become dominant in refrigerated raw milk; the most important are Gram-negative rods of the genus Pseudomonas, especially P. fluorescens and P. fragi, with Acinetobacter, Aeromonas, Serratia, Enterobacter and others, and some Gram-positive spore-formers such as psychrotrophic Bacillus cereus group strains and Paenibacillus. Thermophiles grow at high temperatures, with optima of about 50 to 60 degrees Celsius; they include Geobacillus stearothermophilus and Anoxybacillus flavithermus, which grow in the warm sections of evaporators and heat exchangers during long production runs, and Streptococcus thermophilus, used as a starter. Psychrotrophic counts are estimated by incubating plates at about 7 degrees Celsius for 10 days or by faster equivalent procedures.
Thermodurics are bacteria that survive pasteurisation, though they need not grow at high temperatures. They include Micrococcus, Microbacterium, some enterococci and coryneform bacteria, Streptococcus thermophilus, and the spores of Bacillus and Clostridium. Their numbers in raw milk are estimated by the laboratory pasteurisation count: the sample is heated at about 62.8 degrees Celsius for 30 minutes, cooled and plated. High thermoduric counts usually indicate inadequately cleaned equipment, especially where milk residues have been heated, as in poorly cleaned milking systems with hot water used incorrectly, or deposits in heated equipment. Thermodurics matter because they pass into pasteurised milk and products: although most do not grow rapidly at refrigeration temperatures, they raise the plate count of pasteurised milk, and spore-formers such as Bacillus cereus and B. licheniformis can grow in pasteurised milk and cause sweet curdling and bitty cream, described in chapter 4. Spore counts are made by heating samples at 80 degrees Celsius for 10 minutes (to kill vegetative cells) before plating, with separate counts for highly heat-resistant spores after more intense heating, which are relevant to UHT and powder production. For each group, knowing the source helps control it: mesophiles by prompt cooling, psychrotrophs by short cold storage and clean equipment, thermodurics by effective cleaning, thermophiles by limiting the run time of heated equipment, and spores by farm hygiene, especially in feeding and bedding.
The main technological harm done by psychrotrophs is not their numbers in the final product, because pasteurisation and UHT kill them, but the enzymes they secrete into milk before processing. Pseudomonas species, especially P. fluorescens, produce an extracellular alkaline metalloprotease (often called AprX), extracellular lipases and phospholipases; other psychrotrophs such as Serratia and psychrotrophic Bacillus also produce such enzymes. Most enzyme production occurs in the late exponential and stationary phases of growth, when counts reach roughly a million to ten million colony-forming units per millilitre or more, so milk held long enough for psychrotrophs to reach these levels accumulates enzyme even if it is then processed promptly. The protease hydrolyses casein, with kappa-casein often attacked first, causing bitterness, loss of cheese yield, destabilisation of micelles and gelation. The lipase hydrolyses triglycerides, releasing free fatty acids that give rancid and soapy flavours, especially in products with long shelf lives. Phospholipases (lecithinases) attack the phospholipids of the fat globule membrane, making the fat more accessible to lipases. Because milk is held for longer and longer in modern collection systems, with every-other-day collection and several days in silos before processing, the control of psychrotroph growth in raw milk has become one of the main quality issues for UHT milk, powders and cheese.
Many of these enzymes are extraordinarily heat-stable. Their decimal reduction times at UHT temperatures are measured in minutes, so a UHT treatment of a few seconds at about 140 degrees Celsius destroys only part of the activity, and pasteurisation destroys little of it. The enzymes therefore remain active throughout the shelf life of UHT milk, cream and concentrated products stored at ambient temperature, where months of slow action produce bitterness, gelation and rancidity, as described for age gelation in Chemistry of Dairy Products. Some of the proteases show an unusual behaviour called low-temperature inactivation: they are inactivated more by holding at about 55 to 60 degrees Celsius than by brief heating at higher temperatures, apparently by self-digestion when partly unfolded, and this has been suggested as a deliberate treatment, though it is not widely used. In milk powder, enzymes carried over from the raw milk act after reconstitution and in the recombined product. In cheese, psychrotroph proteases in the milk cause yield losses because casein fragments are lost in whey, and lipases cause rancid flavour, although most of the enzyme partitions into the whey. The practical defence is to prevent psychrotroph growth: clean equipment, rapid and deep cooling, short storage times before processing, and, where long storage is unavoidable, thermisation of raw milk on receipt, as described in chapter 9. Rapid methods for detecting heat-stable proteases in raw milk help processors reserve the best milk for long-life products.
The type of spoilage that develops in milk depends on which organisms are present and at what temperature the milk is held. In uncooled milk at about 20 to 30 degrees Celsius, mesophilic lactic acid bacteria, mainly Lactococcus lactis, ferment lactose to lactic acid and the milk sours and curdles when the pH approaches the isoelectric point of casein; enterococci and coliforms contribute at warmer temperatures. Souring was the normal fate of raw milk before refrigeration and is still common where milk is not cooled. Coliforms and some other Enterobacteriaceae produce acid and gas, giving a gassy curd with an unclean, faecal flavour; gas is also produced by yeasts and by heterofermentative lactic bacteria. Sweet curdling is coagulation without acidity, caused by rennet-like proteases of bacteria, especially Bacillus species such as B. cereus, in pasteurised milk stored at refrigeration temperature or slightly above. Bitty cream is a related defect of pasteurised milk caused by lecithinase of B. cereus, which damages the fat globule membrane so that fat particles aggregate and float as flecks in hot drinks. In refrigerated raw and pasteurised milk, Gram-negative psychrotrophs dominate and spoilage is more often proteolytic and lipolytic than acid, with bitter, putrid, fruity or rancid flavours before any curdling occurs. The general principles of food spoilage and of the factors that select organisms are covered in Food Technology's Food Microbiology subject.
Ropiness, a slimy, stringy texture, is caused by organisms that produce exopolysaccharides, such as Alcaligenes and Klebsiella species and some lactic acid bacteria; it usually traces to contaminated equipment or water. Ropy strains of lactic bacteria are, however, deliberately used in some Nordic fermented milks. Proteolysis by Pseudomonas, Bacillus and other organisms produces bitter flavours and, in advanced stages, putrid odours and clearing of the milk as casein is digested. Lipolysis by microbial lipases produces rancid and soapy flavours; fruity flavours from ethyl esters formed by Pseudomonas fragi are another typical defect of milk held long in the cold. Colour defects are rarer: blue milk has been associated with Pseudomonas syncyanea, red milk with Serratia marcescens, and yellow discolouration with pigmented Pseudomonas and Flavobacterium strains. A malty flavour is produced by a variant of Lactococcus lactis that forms 3-methylbutanal from leucine. Cheesy, yeasty, bitter and unclean flavours also arise from mixed microbial growth. Identifying the defect helps locate its cause: acid curd points to delayed cooling, sweet curd and bitty cream to spore-formers that survived pasteurisation, rancid and fruity flavours to long cold storage of raw milk, ropiness to contaminated equipment, and off-flavours appearing early in pasteurised milk to post-pasteurisation contamination, described in chapter 12.
Raw milk contains several systems that inhibit bacteria, protecting the calf and, to a limited extent, the milk. Their chemistry is described in Milk Proteins and Enzymes; here the concern is their microbiological effect. The lactoperoxidase system, consisting of lactoperoxidase, thiocyanate and hydrogen peroxide, produces hypothiocyanite, which is bacteriostatic to many Gram-positive organisms such as streptococci and lactococci and bactericidal to some Gram-negative organisms, including coliforms and pseudomonads. Its activity in normal milk is limited by the low concentration of hydrogen peroxide. Lactoferrin withholds iron from bacteria that need it and, after digestion, releases antimicrobial peptides; its effect is limited in cow milk because its concentration is low and citrate competes for iron. Lysozyme attacks the cell walls of some Gram-positive bacteria but is present only in traces in cow milk. Immunoglobulins and agglutinins bind and clump bacteria; agglutinins can clump lactic starter organisms and affect their distribution in cheese curd. Leucocytes, especially in milk from infected quarters, can phagocytose bacteria while the milk is warm. Vitamin-binding proteins for folate and vitamin B12 bind these vitamins so that some bacteria cannot use them, and free fatty acids and other minor components also have antibacterial activity.
Together, these systems give freshly drawn milk a short period, often called the germicidal or bacteriostatic phase, during which bacterial numbers increase little or even fall. The phase is short at warm temperatures and much longer at low temperatures, and it is one reason why prompt cooling is so effective. It does not replace cooling, because its effect is limited and varies with the organisms present. Where cooling is not possible, as in many smallholder systems in hot countries, the lactoperoxidase system can be activated by adding small amounts of thiocyanate and a source of hydrogen peroxide, such as sodium percarbonate, according to the Codex guideline for the preservation of raw milk by the lactoperoxidase system. The activated system delays the growth of bacteria in raw milk for several hours, longer at lower temperatures, which can allow milk to reach a cooling or processing centre in acceptable condition. Its use is restricted to situations where refrigeration is not available, it must not be used to hide poor hygiene or replace cooling, and it is not permitted in all countries or for milk intended for some markets. The treated milk must be processed normally, including pasteurisation. Milk containing an activated lactoperoxidase system can be slower to acidify with some starters, so cheese and fermented milk makers need to know whether it has been used.
Mastitis, inflammation of the mammary gland, is usually caused by bacterial infection and changes both the composition of milk, as described in Milk Composition and Physical Properties, and its microbial content. Mastitis pathogens are commonly grouped by their reservoir. Contagious pathogens live mainly in infected udders and spread from cow to cow during milking, through milking units, milkers' hands and cloths: Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma species (especially M. bovis) and Corynebacterium bovis. Environmental pathogens live in the cow's surroundings, especially bedding, soil and faeces, and infect the udder between milkings or when teat ends are exposed: Escherichia coli, Klebsiella species and other coliforms, Streptococcus uberis, and others. Streptococcus dysgalactiae shows features of both groups. Coagulase-negative staphylococci, now often called non-aureus staphylococci, are common causes of mild, subclinical infections. Less common agents include Trueperella pyogenes, Pseudomonas aeruginosa, Bacillus cereus, Nocardia, yeasts, and the alga Prototheca. Contagious pathogens are controlled by milking hygiene, post-milking teat disinfection, treatment of dry cows and culling of chronically infected cows; environmental pathogens by clean, dry housing and bedding and good teat preparation.
Mastitic milk affects the dairy in several ways. Infected quarters can shed very large numbers of bacteria; Streptococcus agalactiae in particular can raise the bulk tank count markedly, so a high count with a high somatic cell count points to mastitis rather than equipment. Mastitic milk has a high somatic cell count, more plasmin and leucocyte enzymes and altered composition, which shorten the shelf life of pasteurised and UHT milk and reduce cheese yield. Some mastitis pathogens are also hazards to people. Staphylococcus aureus can form heat-stable enterotoxins in raw milk and raw-milk cheese if allowed to grow, and toxin survives pasteurisation even though the organism does not. Streptococcus agalactiae, Listeria monocytogenes (which can be shed in milk from infected udders), toxin-producing E. coli and, historically, Mycobacterium bovis and Brucella reach people through raw milk and inadequately processed products, as covered in Food Technology's Food Microbiology subject. Milk from animals treated with antibiotics for mastitis carries residues during the withdrawal period, which inhibit starter cultures and must be kept out of supply. Monitoring of bulk milk somatic cell counts, routine individual cow testing, culture of milk samples from clinical and high-cell-count cows, and the use of bulk tank culture to identify contagious pathogens are part of herd health programmes. Pasteurisation of all milk for sale, together with veterinary control of mastitis and zoonoses, is the main protection for consumers.
Raw milk is graded and paid for partly on its microbial quality. The standard plate count (SPC), also called the total viable count or aerobic plate count, is the reference method: decimal dilutions of milk are mixed with plate count agar (containing tryptone, yeast extract and glucose, and in some standards skim milk) in Petri dishes and incubated aerobically, for example at 30 degrees Celsius for 72 hours under the international standard or at 32 degrees Celsius for 48 hours under United States methods, and the colonies are counted. Only plates with a countable number of colonies, commonly about 25 to 250 or 30 to 300 depending on the standard, are used. The count per millilitre equals the number of colonies divided by the volume plated times the dilution: 156 colonies on a plate of 1 millilitre of a 1 in 1,000 dilution means 156,000 colony-forming units per millilitre. The result counts colony-forming units, not individual bacteria, because clumps and chains form single colonies. The direct microscopic count (Breed smear) spreads 0.01 millilitre of milk over 1 square centimetre on a slide, stains it, and counts bacteria or clumps in microscope fields; the count per millilitre is the average per field times the microscopic factor. It is quick and shows the types of organisms, but counts dead cells too. Modern payment laboratories use automated flow cytometry, which stains bacterial DNA and counts individual bacteria within minutes, with results converted to equivalent plate counts. Other counts used for investigation include the coliform count, the psychrotrophic count, the laboratory pasteurisation count and spore counts.
Dye-reduction tests estimate the microbial activity of raw milk from the rate at which bacteria lower its oxidation-reduction potential, as explained in Milk Composition and Physical Properties. In the methylene blue reduction test (MBRT), a standard methylene blue solution is added to milk in a sterile tube, which is incubated at 37 degrees Celsius and examined at intervals; the time taken for the blue colour to disappear is the reduction time. Milk with many active bacteria decolourises quickly; good milk takes hours. National standards, including Indian standards, give tables grading milk from very good to poor by reduction time, longer times indicating better quality. In the resazurin test, resazurin is reduced first to pink resorufin and then to colourless dihydroresorufin; the colour after a fixed time, commonly 10 minutes or 1 hour at 37 degrees Celsius, is compared with standard colour discs numbered from blue to colourless. The 10-minute test is used as a rapid platform test and the 1-hour test for grading; resazurin is also reduced by leucocytes, so mastitic milk can give a poor result. Dye-reduction tests are cheap and need no incubator for plates, but they reflect metabolic activity rather than numbers: they are insensitive to psychrotrophs, which reduce dyes slowly at 37 degrees Celsius after cold storage, so refrigerated milk with a high psychrotroph count can give a good reduction time. For this reason, plate counts and automated counts have replaced dye-reduction tests for payment in many countries, although the tests remain in use at collection centres.
Cooling is the most effective single measure for preserving raw milk quality. Lowering the temperature lengthens the generation time of bacteria: mesophiles that double in less than an hour at 30 degrees Celsius barely grow below about 10 degrees Celsius. Milk is therefore cooled as soon as possible after milking, by bulk tank coolers on the farm, by bulk milk coolers at village collection centres, or by plate or tubular coolers before storage. Regulations set cooling requirements, for example in the European Union milk must be cooled immediately to 8 degrees Celsius or below if collected daily and to 6 degrees Celsius or below if not, and in the United States to 10 degrees Celsius or less within 4 hours of the start of the first milking and to 7 degrees Celsius or less within 2 hours after milking ends; requirements in other countries differ. Bacterial growth can be calculated from the generation time: the number after time t equals the initial number times 2 raised to t divided by the generation time. A milk with 10,000 colony-forming units per millilitre whose dominant organisms double every 8 hours reaches about 640,000 after 48 hours, while a 6-hour generation time gives about 2.6 million. In smallholder systems in hot countries, the time between milking and cooling is often the main determinant of quality, so collection schedules, the location of coolers and the use of clean cans matter as much as the coolers themselves.
Cooling does not stop all growth; it changes which organisms grow. In milk held at about 4 to 7 degrees Celsius, mesophilic lactic acid bacteria are suppressed, so the milk does not sour, but psychrotrophic Gram-negative rods continue to grow after a lag and become dominant within a few days. Pseudomonas species typically make up the large majority of the flora of raw milk held for several days in the cold, with Acinetobacter, Enterobacteriaceae and others. Their growth is slower than that of mesophiles at warm temperatures but steady, so counts that are low at collection can rise by several orders of magnitude during extended storage in farm tanks, tankers and plant silos, and once they pass about a million per millilitre, heat-stable enzymes accumulate, as described in chapter 3. The growth of psychrotrophs depends on the initial number, which reflects equipment hygiene, on the storage temperature, with even 2 to 3 degrees Celsius making a large difference, and on the total storage time. Good practice therefore combines low initial counts, rapid cooling to below about 4 degrees Celsius, short storage before processing and clean storage tanks. Where milk must be held for more than a day or two at the plant, thermisation (chapter 9) reduces the psychrotroph population and its future enzyme production. Other measures studied include the addition of carbon dioxide to raw milk, which inhibits psychrotrophs, and activation of the lactoperoxidase system where cooling is impossible. Processors increasingly measure psychrotroph counts or enzyme activity, not only total counts, for milk destined for long-life products.
The killing of microorganisms by heat follows, to a first approximation, first-order kinetics: in each equal time interval at a fixed temperature, the same fraction of the surviving population is killed. The decimal reduction time, D, is the time at a stated temperature to reduce the population by 90 per cent, one log cycle; the number of survivors after time t equals the initial number times 10 raised to minus t divided by D. If an organism has a D value of 1.5 seconds at 72 degrees Celsius, a 15-second treatment gives 10 log reductions. The z value is the temperature increase that reduces D tenfold; with a z of 5 degrees Celsius, an organism whose D is 1 second at 72 degrees Celsius has a D of 100 seconds at 62 degrees Celsius. Vegetative bacteria have low D values and small z values, while bacterial spores have far higher D values and larger z values. Thermisation is a mild heat treatment of raw milk, commonly about 63 to 65 degrees Celsius for about 15 seconds, applied on receipt when milk must be stored for some time before processing. It kills most Gram-negative psychrotrophs, typically by several log cycles, and so limits their growth and enzyme production during storage, but it leaves the milk phosphatase-positive and does not make it safe; thermised milk must be cooled quickly and later pasteurised or used for products whose regulations permit it. Thermisation does not inactivate enzymes already produced.
Pasteurisation, at 63 degrees Celsius for 30 minutes or 72 degrees Celsius for 15 seconds, or equivalent combinations, is designed to destroy the vegetative pathogens of milk. The original target organism was Mycobacterium tuberculosis; the 72-degree standard was later confirmed to inactivate Coxiella burnetii, the agent of Q fever, which is somewhat more heat-resistant. Pasteurisation kills nearly all vegetative bacteria, including Listeria monocytogenes, Salmonella, Campylobacter, E. coli and Staphylococcus aureus, but not spores or some thermodurics, and not heat-stable toxins and enzymes. Its adequacy is checked by the alkaline phosphatase test described in Milk Proteins and Enzymes. Boiling milk at home kills vegetative bacteria but not all spores, and spores of Bacillus cereus can germinate and grow if boiled milk is kept warm. In-container sterilisation, at about 110 to 120 degrees Celsius for 10 to 20 minutes, and UHT treatment, at 135 to 150 degrees Celsius for a few seconds, aim at commercial sterility: absence of organisms able to grow in the product under normal storage. UHT treatment is described by two indices. The bacteriological effect B* equals 1 for a treatment that gives 9 log reductions of thermophilic spores, defined as 10.1 seconds at 135 degrees Celsius with a z of 10.5 degrees Celsius; the chemical effect C* equals 1 for 3 per cent loss of thiamine, 30.5 seconds at 135 degrees Celsius with a z of 31.4 degrees Celsius. A good UHT process has B* above 1 and C* below 1. Highly heat-resistant spores of Bacillus sporothermodurans and Geobacillus can survive UHT and cause non-sterility. Equipment and processes are described in Market Milk Processing.
Bactofugation removes bacteria, and especially bacterial spores, from milk by centrifugation. A bactofuge is a high-speed centrifugal separator designed so that particles denser than milk, including bacterial cells and spores, are thrown to the periphery of the bowl and discharged in a small stream called the bactofugate, usually a few per cent of the feed. Spores are removed particularly well because they are denser than vegetative cells; a single-stage bactofuge typically removes of the order of 90 per cent or more of spores, and two stages in series remove more. If milk containing 2,000 spores per millilitre is treated with 98 per cent removal, about 40 spores per millilitre remain. The bactofugate is either discarded or, more often, sterilised by UHT heating and returned to the milk, so that its protein is not lost. The main application is in milk for hard and semi-hard cheeses, where removing spores of Clostridium tyrobutyricum prevents late blowing without, or with less, use of nitrate or lysozyme. Bactofugation is also used to improve the keeping quality of pasteurised milk, to reduce spores in milk for powders and infant formula, and in combination with heat treatment for extended shelf life milk. It is applied at about 55 to 60 degrees Celsius, at which milk viscosity is low, and is usually combined in the same line with separation and standardisation, as described in Market Milk Processing.
Microfiltration removes bacteria and spores by passing skim milk through porous ceramic membranes with pores of about 0.8 to 1.4 micrometres, large enough to let casein micelles (typically 0.1 to 0.3 micrometres) and whey proteins pass with the permeate but small enough to retain most bacteria and spores. Fat globules are similar in size to bacteria and would also be retained, so whole milk is first separated; the cream is heat-treated separately and recombined with the microfiltered skim milk. Crossflow operation, with the milk flowing at high velocity along the membrane surface, limits fouling, and systems with uniform transmembrane pressure along the membrane allow long runs. Microfiltration typically reduces bacterial counts by about 3 to 4 log cycles or more, so a skim milk with 50,000 colony-forming units per millilitre would retain only about 16 after a 3.5 log reduction. The small retentate stream, containing the bacteria and spores, is heat-treated at high temperature and may be returned. Microfiltered milk, combined with mild pasteurisation and hygienic filling, gives extended shelf life milk with a fresh flavour, and microfiltration is also used for cheese milk, for milk for powders, and for producing casein- and whey protein-enriched streams with different pore sizes. Because microfiltration and bactofugation remove microorganisms without the flavour changes of high heat, they are attractive for products where fresh taste matters, but neither replaces pasteurisation as the legal safety step, and both are followed by heat treatment for market milk.
A biofilm is a community of microorganisms attached to a surface and embedded in a matrix of extracellular polymeric substances, mainly polysaccharides, proteins and DNA, that they produce. In dairy equipment, biofilm formation begins when milk proteins and other components adsorb to stainless steel, rubber or plastic surfaces, forming a conditioning film. Bacteria attach reversibly and then irreversibly, multiply into microcolonies, produce matrix and develop a mature three-dimensional structure with channels, from which cells and fragments detach and contaminate the milk. Biofilms form readily on surfaces that are not effectively cleaned, especially gaskets, rubber liners, valves, dead ends, joints, pump seals, bulk tank outlets and areas of low flow, and on worn or scratched surfaces. Important dairy biofilm formers include Pseudomonas and other Gram-negative psychrotrophs in milking systems, tanks and pasteurised milk lines; Listeria monocytogenes in drains, floors, conveyors and filling areas; spore-forming Bacillus species; thermophilic Geobacillus and Anoxybacillus species in the warm sections of evaporators and plate heat exchangers, where long production runs allow them to grow and release spores into milk powder; and Streptococcus thermophilus in the cooling sections of pasteurisers handling yoghurt milk. General principles of biofilms and their role in food safety are also covered in Food Technology's Food Microbiology subject.
Bacteria in biofilms are much more resistant to cleaning chemicals and sanitisers than free cells, often by orders of magnitude, because the matrix limits penetration and because cells in biofilms are physiologically different. A biofilm is therefore a persistent source of contamination that survives routine cleaning and seeds every batch, causing high counts in raw milk, post-pasteurisation contamination, spore contamination of powders and, with Listeria, a serious safety hazard. Control rests on preventing establishment rather than removing mature biofilms. Hygienic design of equipment, following guidelines such as those of 3-A Sanitary Standards and the European Hygienic Engineering and Design Group, eliminates crevices, dead ends and rough surfaces and ensures drainability. Clean-in-place systems must deliver the right detergent concentrations, temperatures, contact times and turbulent flow, typically with velocities of at least about 1.5 metres per second in pipes, with an alkaline step to remove protein and fat, an acid step to remove mineral deposits, and a sanitising step. Production run times in evaporators and heat exchangers are limited to prevent thermophile build-up, and rubber parts are inspected and replaced on schedule. Enzyme-based cleaners and alternating sanitisers help with resistant biofilms. Verification uses ATP bioluminescence swabs, which detect residues of organic matter and organisms within minutes, microbiological swabs and contact plates, inspection of hard-to-clean parts, and environmental monitoring programmes for Listeria in processing areas.
Post-pasteurisation contamination (PPC) is contamination of milk after it has passed the pasteuriser holding tube, and it is the main factor limiting the refrigerated shelf life of conventional pasteurised milk. Pasteurisation kills Gram-negative psychrotrophs, so their presence in the packaged product almost always indicates recontamination. The main sources are biofilms and residues in pipes, valves and tanks downstream of the pasteuriser, the filling machine and its environment, packaging materials, and air. Leakage of raw milk into pasteurised milk through pinholes or cracked gaskets in the regeneration section of plate heat exchangers is a particular hazard; it is prevented by keeping the pasteurised milk at a higher pressure than the raw milk, using booster pumps and pressure differential controls, and by regular plate inspection, as described in Market Milk Processing. Failures of the flow diversion valve allow under-heated milk to pass. Even a very small number of contaminating psychrotrophs can spoil milk in storage: one cell per millilitre doubling every 12 hours reaches about a million per millilitre in 10 days, enough to cause off-flavours. Contamination of pasteurised products with Listeria monocytogenes from the processing environment has caused serious outbreaks, which is why environmental monitoring and hygiene zoning in filling areas are central to food safety plans.
Post-pasteurisation contamination is detected by testing pasteurised milk for organisms that cannot survive pasteurisation. Coliforms and Enterobacteriaceae are the usual indicators; their presence in pasteurised milk shows recontamination or under-processing. In the European Union, for example, the process hygiene criterion for pasteurised milk sets a limit for Enterobacteriaceae of fewer than 10 colony-forming units per millilitre, and United States standards set limits for coliforms. Because initial contamination is often below the detection limit of direct plating, preliminary incubation tests are used: samples are held at a raised temperature, for example 21 degrees Celsius for about 25 hours or at refrigeration temperature for several days, so that any contaminants grow before counting, and the result predicts keeping quality. Shelf-life testing of packaged products under realistic storage conditions, with sensory evaluation and counts at intervals, is the ultimate check. Control combines hygienic design and effective cleaning of all post-pasteurisation equipment, aseptic or ultra-clean filling for extended shelf life milk, protection of the filling area by positive-pressure filtered air and hygiene zoning, decontamination of packaging, monitoring of heat exchanger integrity and of the flow diversion system, and storage of the product at 4 degrees Celsius or below throughout distribution. With these measures, conventional pasteurised milk commonly achieves a shelf life of one to two weeks or more under good refrigeration, and extended shelf life systems considerably longer, though actual shelf life depends on storage temperature as much as on processing.
A student uses this subject in dairy microbiology practicals and plant training. You will take aseptic samples, prepare dilutions and plate standard, coliform, psychrotrophic, thermoduric and spore counts, run the methylene blue and resazurin tests, examine Breed smears, and test pasteurised milk for post-pasteurisation contamination. Learn the groups of milk bacteria by temperature and their sources, the main spoilage defects and their causes, and the heat-kinetic calculations, because examiners ask them directly and plant work depends on them. Keep careful records of every count, because results that cannot be traced are worthless.
Five years into work as a quality or microbiology manager, you trace a high bulk milk count to a perished liner or a cooling failure, decide whether milk with a high psychrotroph count should go to UHT or to quick-turnover products, investigate short shelf life in pasteurised milk by sampling along the line after the holding tube, check pasteuriser plates for pinholes, set run times for evaporators to control thermophiles, and run environmental Listeria monitoring. You also advise farmers on teat preparation, cleaning and cooling.
A senior quality director with twenty-five years of experience designs the microbiological control of a whole milk supply: raw milk quality schemes and payment incentives, collection schedules and cooling infrastructure, storage limits before processing, thermisation policy, choice of bactofugation or microfiltration for cheese and ESL lines, hygienic design standards for new plant, and shelf-life claims. Such a person also leads the response to contamination incidents and recalls, deals with regulators and auditors, and decides when evidence justifies changing a process or a supplier.
A researcher with long experience studies the ecology of milk microbiota from farm to factory using culture and sequencing methods, the regulation and heat stability of psychrotroph enzymes, spore formers in powder and UHT production, biofilm formation and control, and predictive models of milk shelf life. Such a person develops rapid detection methods for spoilage enzymes and organisms and advises regulators on microbiological criteria. The work links farm practice, process engineering and product quality, and its results are judged by whether they reduce spoilage and risk in real supply chains.
This manual covers the microbiology of raw and market milk; food-borne pathogens, indicator organisms, rapid and molecular methods and microbiological criteria in general are covered in Food Technology's Food Microbiology subject, and process equipment in Market Milk Processing. Grading tables for MBRT and resazurin and national microbiological standards are given only in principle or as examples because they differ by country and change. D and z values for individual organisms vary with strain and medium and are not tabulated. Microbiology of fermented milks, cheese and other products, starter cultures and probiotics are treated in their own subjects. Culture-independent methods for studying milk microbiota are mentioned only briefly.
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