- Email: [email protected]
MICROORGANISMS ASSOCIATED WITH MILK
MICROORGANISMS ASSOCIATED WITH MILK A N Hassan and J F Frank, South Dakota State University, Brookings, SD, USA, University of Georgia, Athens, GA, USA ª 2011 Elsevier Ltd. All rights reserved.
Introduction Milk is a good growth medium for many microorganisms because of its near-neutral pH, complex biochemical composition, and high water content. Although in the absence of mastitis it is secreted free of microorganisms, it is subject to microbial contamination from a number of sources (Figure 1). Some microorganisms move up the teat canal causing aseptically drawn milk to be contaminated. These contaminants, called udder commensals, are present in small numbers and are mainly lactic acid bacteria. Their numbers are limited by the animal’s immune system and antimicrobial agents secreted into the milk. Therefore, contaminants external to the udder from locations such as udder skin, hide, milking equipment, and utensils form the vast majority of microorganisms in raw milk. Although technology has allowed the production of milk with very low microbial levels, product quality still depends on controlling entry and growth of microorganisms in milk from farm to the consumer. The number and types of microorganisms present in milk are influenced by season, farm hygiene, feed, and efficiency of cooling. The number of bacteria in milk ranges from a few hundred to thousands per milliliter of freshly drawn milk from healthy cows. Four physiological groups of spoilage bacteria are commonly found in raw milk: producers of lactic acid, propionic acid, butyric acid, and degradative enzymes (primarily proteases and lipases). In addition, raw milk may contain pathogens whose multiplication depends mainly on the temperature and competing microflora. The main criteria for highquality raw milk are the presence of a low number of spoilage microorganisms and the absence of animal pathogens. The sources of microorganisms found in milk, their characteristics, and their growth are discussed below.
Microorganisms Found in Milk The microorganisms found in milk can be divided into three groups: animal pathogens and toxin producers, spoilage agents (saprophytes), and microorganisms used to produce fermented products. Some overlap among these groups occurs; for example, Bacillus cereus is a toxin
producer and is also involved in spoilage, and lactic acid bacteria can cause spoilage and be used in fermentations. Figure 2 shows the different morphological groups commonly found in raw milk.
Pathogenic Microorganisms Pathogenic bacteria are those capable of causing infection or intoxication. Milk is a good growth and protective medium for most pathogens. The growth of pathogens in milk is inhibited by cooling and the growth of nonpathogenic competing microorganisms. Many pathogens, such as Mycobacterium tuberculosis and Brucella species, grow very slowly in milk, and others, such as viruses, do not multiply at all. Therefore, the threat associated with their presence in milk will depend on the initial load of milk contamination. Although the potential hazard of pathogens has been minimized by modern milk production and technology practices, disease outbreaks caused by Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, and Escherichia coli O157:H7 have occurred in recent years.
Staphylococcus spp. Staphylococcus spp. are nonmotile, non-spore-forming, Gram-positive, catalase-positive cocci. They grow well in media containing 10% NaCl and produce proteases, lipases, and esterases. Staphylococcus aureus is the most important species of this genus associated with milk. It produces heat-stable enterotoxins that are responsible for food poisoning. Its natural habitat includes the skin and mucous membranes of mammals. Most contamination occurs during milking and originates with the cow. About 40% of bulk tank milk from a nationally representative sample of 542 dairies tested positive for Staph. aureus. It has been reported that 16 and 18% of the cows from organic and conventional farms, respectively, harbored oxacillin-resistant staphylococci, which indicated the presence of the methicillin-resistant gene (mecA). Although the presence of Staph. aureus in milk is difficult to prevent, appropriate cleaning and sanitation of the cow and milking equipment controls initial numbers and
447
448 Microorganisms Associated with Milk
Figure 1 Sources of milk contamination at the dairy farm.
Figure 2 Different morphological groups of microorganisms commonly found in raw milk.
Microorganisms Associated with Milk
cooling the milk prevents growth, toxin production, and spreading of bacteria to humans via the milk chain. Staphylococcus hyicus has been isolated from the skin of cows and from the milk of cows with mastitis. Some strains are enterotoxigenic.
Streptococcus spp. Streptococcus pyogenes is pathogenic to humans and some animals. It resists phagocytosis and produces an erythrogenic toxin that causes scarlet fever. Streptococcus agalactiae belongs to group B streptococci (GBS) and is an important agent of bovine mastitis in some countries and a cause of diseases in humans. The gastrointestinal tract is the main human reservoir. Streptococcus dysgalactiae belongs to Lancefield group C streptococci. It is found mainly in mastitic bovine udders. Streptococcus uberis is found in the lips and skin of cows, udder tissue, and milk. It is the main cause of mastitis in some countries, especially during the winter. The only phenotypic criterion that differentiates between Sc. uberis and Sc. parauberis is the ability of the former to produce -D-glucuronidase. Streptococcus equi subsp. equi and Sc. equi subsp. zooepidemicus are -hemolytic group C Streptococcus species that infect animals and humans, respectively.
Campylobacter jejuni Campylobacter jejuni is a common cause of human gastroenteritis. It is a Gram-negative, curved, slender, motile rod and is found in food, feces, and water. Although it does not grow in milk, the consumption of fresh raw milk has caused numerous outbreaks of gastroenteritis (see Pathogens in Milk: Campylobacter spp.). Results from NAHMS Dairy 2002 indicated that the percentage of cows shedding Campylobacter spp. was 51.4%, while the percentage of dairy herds with at least one cow shedding Campylobacter was 97.9%.
Yersinia spp. The genus Yersinia belongs to the family Enterobacteriaceae. Members of this genus are Gram-negative, motile rods, facultatively anaerobic at 28–29 C but strictly aerobic at 37 C, which can grow at 2–4 C. Yersinia enterocolitica is the only species of importance in milk. The most common source of contamination is the rinsing water used at the farm. Yersinia species grow well in milk and cause an enteric infection that mimics appendicitis (see Pathogens in Milk: Yersinia enterocolitica).
449
Salmonella spp. The genus Salmonella (family Enterobacteriaceae) consists of small, Gram-negative, non-spore-forming rods. Most strains are motile. The primary habitat is the intestinal tract. Cows suffering from salmonellosis can excrete large numbers of salmonellae in their feces and occasionally directly into milk. Milk can be contaminated with Salmonella from feces, livestock feed, humans, water, and dust. Direct contact from other herds can result in the introduction of Salmonella spp. into dairy farms. Many outbreaks of salmonellosis have been attributed to milk and milk products. Unlike staphylococcal food poisoning, the ingestion of viable cells of Salmonella is necessary for salmonellosis. Raw milk or dairy products made from raw milk have been the cause of outbreaks of salmonellosis in the Western world. Nontyphoid Salmonella enterica infects an estimated 2–4 million people every year in the United States. Growth of Salmonella in milk is not necessary as ingestion of only a few cells can cause the disease (see Pathogens in Milk: Salmonella spp.).
Escherichia coli Escherichia coli is a member of the family Enterobacteriaceae. It is a facultatively anaerobic, Gram-negative rod, and ferments lactose. Four categories of pathogenic E. coli are recognized: enteropathogenic, enterotoxigenic, enteroinvasive, and enterohemorrhagic. Enterotoxigenic E. coli produces enterotoxins that cause diarrhea. Humans are the main reservoir. To cause diarrhea, sufficient numbers of toxigenic E. coli must be ingested and they must adhere to the small intestine. In recent years, enterohemorrhagic E. coli, mainly E. coli O157:H7, have caused milk-borne illness. These pathogens cause hemorrhagic colitis (bloody diarrhea), an illness that can lead to renal failure in children. The dairy cow gut is a reservoir for E. coli O157:H7. Hemorrhagic strains of E. coli do not ferment sorbitol and the incubation temperature used to detect fecal coliforms (44–45 C) will not allow E. coli O157:H7 to grow (see Pathogens in Milk: Escherichia coli). Non-O157 shiga toxin-producing E. coli (STEC) was also associated with raw milk and raw milk cheese.
Listeria spp. Within the genus Listeria, only Li. monocytogenes and Li. ivanovii are considered virulent, with Li. monocytogenes being the only species of public health concern. Listeria innocua is the most frequently encountered nonpathogenic Listeria species. Listeria monocytogenes is a Gram-positive, short rod with rounded ends that occurs singly, in parallel,
450 Microorganisms Associated with Milk
or in V shapes. It grows slowly in milk at refrigeration temperature. Listeria monocytogenes causes mastitis and abortion in animals. In humans, it causes miscarriages and meningitis in its severe form and flu-like symptoms in its mild form. Raw milk can be contaminated with Li. monocytogenes from feces, cows suffering from mastitis, poor-quality silage, and milking equipment (see Pathogens in Milk: Listeria monocytogenes).
Mycobacterium spp. Mycobacterium avium subsp. paratuberculosis (MAP) is one of the most heat-resistant pathogenic microorganisms likely to be present in milk. Mycobacterium tuberculosis may gain access to milk from infected animals through milk secretion and fecal contamination or from milkers and other environmental sources such as cow alleyways, manure storage, calving area, sick cow pen, water runoff, and postweaned calves areas. The organism does not multiply in milk but survives in unpasteurized milk and milk products. MAP causes the infection commonly known as Johne’s disease. Detectable numbers of this organism have been reported in the milk of both clinically affected and asymptomatic cattle. Pasteurization inactivates MAP when present at levels associated with milk from asymptomatic cattle. It is unknown whether this microorganism is pathogenic for man. Mycobacterium bovis causes infection in animals and humans. Compared with M. tuberculosis, growth of M. bovis is less luxuriant and it adapts to aerobic growth after repeated subculturing. Contaminated milk, feces, soil, and water are the main sources of infection (see Diseases of Dairy Animals: Infectious Diseases: Johne’s Disease; Infectious Diseases: Tuberculosis).
Brucella spp. Brucella species are Gram-negative, aerobic, short rods that do not ferment carbohydrates. They survive well in milk and dairy products and are pathogenic to humans and animals. The most important causes of brucellosis in milk are Br. abortus and Br. melitensis (see Pathogens in Milk: Brucella spp.).
Coxiella burnetii Coxiella burnetii is a rickettsia (family Rickettsiaceae), which causes Q fever in humans. Rickettsiae are obligate intracellular parasites. Coxiella burnetii is a small pleomorphic, sometimes filamentous, nonmotile rod that
produces a capsule-like structure. It is highly resistant to chemical and physical disinfectants and is the most heatresistant pathogen found in milk (see Pathogens in Milk: Coxiella burnetii).
Aeromonas spp. Aeromonas species are facultatively anaerobic, Gramnegative, motile cocci or rods that grow in media containing up to 5% salt. Aeromonas hydrophila is an opportunistic pathogen found in the feces of healthy animals.
Bacillus spp. Bacillus species are Gram-positive, aerobic, sporeforming rods belonging to the family Bacillaceae. Their spores make them resistant to heat and other destructive agents. Strains can be psychrotrophic, mesophilic, or thermophilic. Bacillus cereus is a common soil bacterium that is often found in raw milk. It is capable of producing enterotoxins (especially in starchy foods), which cause food poisoning. Bacillus anthracis causes anthrax, which is a deadly acute infectious disease. Purposeful contamination of foods by B. anthracis was considered a potential biological weapon. The presence of Bacillus in raw milk shows marked seasonal variation. Soil, feces, bedding, air, silage (possibly via feces), and the milking equipment are the main sources of Bacillus in milk (see Pathogens in Milk: Bacillus cereus).
Clostridium spp. Clostridia are Gram-positive, spore-forming rods, which are usually found in sediments, the intestinal tract of humans and animals, soil, dust, feces, and silage. They are anaerobic and often poisoned by atmospheric oxygen unless in the spore form. Clostridium perfringens causes food poisoning and gas gangrene in humans and a variety of diseases in animals. Milk and milk products provide a ready medium for transmission of Cl. perfringens. Vegetative cells sporulate in the gut and release exterotoxin, which is responsible for symptoms such as profuse diarrhea, sudden acute inflammation of the abdomen, and severe abdominal pain. Large numbers of cells must be consumed for illness to occur. Botulism is an intoxication attacking the nervous system caused by ingestion of toxins produced by Cl. botulinum. Very low levels (<1 spore ml1) of these organisms are common in milk. They do not germinate or grow in raw or pasteurized milk, but cheese and cheese
Microorganisms Associated with Milk
products can support growth if they are not sufficiently acidic (see Pathogens in Milk: Clostridium spp.).
Xanthomonas maltophilia Xanthomonas maltophilia, previously known as Pseudomonas maltophilia, is an aerobic rod of the family Pseudomonadaceae. It is one of several pseudomonad-like organisms in hospital laboratories associated with serious infections. It has been isolated from milk and water.
Klebsiella pneumoniae Klebsiella pneumoniae belongs to the family Enterobacteriaceae and is an agent for pneumonia, urinary tract infection, and gastroenteritis and mastitis. It is present in animal bedding materials and water. Fecal shedding of Kl. pneumoniae is considered one of the sources of this microorganism in milk. Inorganic bedding such as sand reduces its occurrence.
Serratia spp. Serratia species are Gram-negative, facultative, anaerobic rods of the family Enterobacteriaceae. They are distributed in soil, air, water, and plants. Some strains produce hemolysins and infected animals produce contaminated milk. Their presence in pasteurized dairy products indicates poor sanitation practices since they are heat sensitive. Serratia marcescens is the most important species and causes mastitis.
Proteus spp. Proteus species are Gram-negative, non-spore-forming rods belonging to the family Enterobacteriaceae. They are commonly found in the feces of humans and animals, soil, and plants. They frequently cause a variety of infections in humans, especially urinary tract infections. Proteus mirabilis is the most common species followed by Pr. vulgaris and Pr. penneri.
Enterobacter sakazakii Enterobacter sakazakii belongs to the family Enterobacteriaceae. Although rarely found, it can cause life-threatening neonatal meningitis. It does not survive pasteurization but is found in ultra-high temperature (UHT) milk and milk powder that have been subjected to postprocessing contamination. Its natural habitat is not known.
451
Hafnia alvei Hafnia alvei, which belongs to the family Enterobacteriaceae, resembles Salmonella. Some strains cause mild gastroenteritis. It is found in sewage, soil, water, and feces. Hafnia species can grow on the surface of Camembert cheese (see Pathogens in Milk: Enterobacteriaceae).
Actinomyces spp. Actinomyces species are irregular, non-spore-forming, Gram-positive rods that occur mainly in the oral cavity and on the mucous membranes of warm-blooded vertebrates. They cause pyogenic infections. Actinomyces bovis and Ac. pyogenes are the important species in milk.
Leptospira spp. Species of the genus Leptospira (family Leptospiraceae) are Gram-negative, flexible, helicoidal rods. Leptospira interrogans causes leptospirosis in animals and humans. The kidney is the natural habitat and contaminated urine is the main source of contamination.
Molds and Yeasts Many different genera of molds such as Aspergillus, Penicillium, and Fusarium produce mycotoxins in milk and dairy products. Mycotoxins are secondary metabolites that are acutely toxic, carcinogenic, emetic, estrogenic, hallucinogenic, mutagenic, or teratogenic. Aspergillus flavus and some other Aspergillus species produce aflatoxins when growing on cheeses. Some strains of Geotrichum might cause infection in humans. Candida albicans is pathogenic to humans and animals (see Yeasts and Molds: Spoilage Molds in Dairy Products; Yeasts and Molds: Yeasts in Milk and Dairy Products).
Viruses Viruses are genetic elements that replicate inside cells and also have an extracellular nonreplicating state. Replication of the virus often destroys the host cell causing illness. The extracellular form of the virus is a submicroscopic particle and can be transmitted between hosts. Viruses do not multiply in milk but some may survive for long periods. Viruses can be infective at very low doses and most that are found in milk produce gastroenteritis and originate with the cow. Viruses in milk are inactivated by pasteurization.
452 Microorganisms Associated with Milk
Rotaviruses can produce severe gastrointestinal illnesses. Milk may be a vector for this virus but detection in raw milk is prevented by the presence of antiviral antibodies (immunoglobulins; IgG). The antibody/virus complex is dissociated in the highly acidic conditions of the stomach. Hepatitis A virus enters milk through fecal contamination or polluted water. This virus causes symptoms of fever, vomiting, nausea, and abdominal discomfort. The virus eventually affects the liver, causing enlargement and jaundice. Poliovirus causes the acute infectious disease poliomyelitis, which is characterized by fever, vomiting, and headache. The poliovirus might infect the central nervous system and cause paralysis. It can be transmitted to milk through fecal contamination. Milk can serve as a vector for the spread of the footand-mouth disease virus. This virus, which is nonenveloped, belongs to the Picornaviridae family, which also includes rhinoviruses and enteroviruses.
Protozoa Toxoplasma gondii is a parasite of humans and many warmblooded animals, especially dairy goats. Infection occurs as a result of ingestion of foods or water contaminated with oocysts. Contaminated milk is considered a potential source of human toxoplasmosis. Cryptosporidiosis is an infection caused by Cryptosporidium muris and Cr. parvum. Diarrhea is the primary symptom of infection. Cows can shed Cr. muris oocysts in their feces, which may lead to contamination of milk. Drinking water is the most common vehicle for transmission of this disease. Entamoeba histolytica is a protozoan that causes amoebiasis, the third most common cause of death by parasites in the world. Transmission occurs via ingestion of cysts in contaminated foods or water or through poor personal hygiene. Giardia intestinalis is a flagellated protozoan that inhabits the intestinal tracts of humans and animals. Cysts survive well in the environment and are transmitted via contaminated hands, drinking water, and food contaminated with feces.
Psychrotrophic Bacteria Most psychrotrophic bacteria present in raw milk belong to the family Pseudomonadaceae, which includes Gram-negative, non-spore-forming, nonfermentative, aerobic rods. Pseudomonas fluorescens is the species most commonly isolated from milk, with Ps. fragi and Ps. putida being of less importance. Soil, water, animals, and plant material are the natural habitat of psychrotrophic bacteria found in milk. Contaminated milk equipment and air provide an additional source of contamination, especially after processing. As little as 1 cfu ml1 of raw milk may be enough to spoil milk during cold storage within 5 days. Some Pseudomonas spp. grow at cold and warm temperatures, and others found in milk cannot propagate at temperatures greater than 25 C. Defects associated with the growth of these organisms in milk include development of bitter and fruity offflavors. Although psychrotrophic, Gram-negative bacteria are not heat resistant, they produce heat-stable extracellular proteolytic and lipolytic enzymes that can spoil heat-treated products. However, not all Gram-negative isolates from raw milk display proteolytic activities at 7 or 20 C. Other spoilage Gramnegative rods include Acinetobacter, Psychrobacter, Flavobacterium, Shewanella putrefaciens, and Alcaligenes spp. Cold-tolerant endospore-forming bacteria such as Bacillus and Paenibacillus spp. in raw milk are considered a major cause of spoilage of pasteurized milk.
Family Moraxellaceae Moraxellaceae are Gram-negative, psychrotrophic, nonpigmented, nonmotile, aerobic coccobacilli. There are three genera: Acinetobacter are nonmotile, oxidase-negative organisms, Moraxella are similar but oxidase-positive, and the third genus is Psychrobacter. Moraxellaceae seldom spoil milk because they lack sufficient biochemical activities such as proteolysis and lipolysis that cause off-odors. In addition, they are overgrown by Pseudomonas during cold storage.
Spoilage Microorganisms Spoilage of milk and dairy products is manifest as offflavors and odors and changes in texture and appearance. The most important spoilage microorganisms of milk and dairy products are the Gram-negative, rod-shaped bacteria (e.g., Pseudomonas and coliforms), Gram-positive, sporeforming bacteria (Bacillus and Clostridium), lactic acid bacteria, members of coryneform group, yeasts, and molds.
Shewanella putrefaciens Shewanella putrefaciens, previously known as Pseudomonas putrefaciens or Alteromonas putrefaciens, is a Gram-negative rod. It is found in water and soil and contaminates milk and milk products causing spoilage such as surface taint in butter.
Microorganisms Associated with Milk
Flavobacterium spp. Flavobacterium species are Gram-negative organisms found in water, soil, and milk. They hydrolyze casein and cause spoilage in milk and dairy products during cold storage. Important species are Fb. maloloris, Fb. aquatile, and Fb. lutensis.
Alcaligenes spp Alcaligenes species are Gram-negative, short, motile rods. They are aerobic and do not use lactose. They are found in water, soil, and milk. Alcaligenes faecalis produces the exopolysaccharide curdlan and is a potential contaminant of dairy products. Alcaligenes can grow under refrigeration conditions, producing off-flavors and reducing the shelf life of milk.
Coliforms Coliforms are Gram-negative, oxidase-negative, nonspore-forming rods that grow aerobically or facultatively anaerobically in the presence of bile salts. They also ferment lactose to produce acid and gas within 48 h at 37 C. Coliforms include the genera Escherichia, Enterobacter, Klebsiella, Proteus, Serratia, and Citrobacter. They do not survive pasteurization. Gas production may result in early blowing of hard cheese and a poor curd structure in Cottage cheese. Approximately 107 cfu g1 of coliforms is needed for a gassy defect to be noticed. Other types of spoilage include acid production, slime production in Cottage cheese, bitter flavors, and grassy, unclean, medicinal, or fecal odors. Cheese varieties in which acid production is delayed are more susceptible to coliform growth. Klebsiella (Kl. pneumoniae and Kl. oxytoca) and Enterobacter aerogenes are responsible for gas formation in cheese, which can lead to swelling of the package. Serratia marcescens produces proteolytic enzymes that might cause gelation of UHT milk. In addition, they produce methanethiol in Cheddar cheese. Soiled udders and teats are common sources of coliforms. Coliforms can also grow and contaminate milk from poorly sanitized milk contact surfaces and equipment.
453
habitat is soil, but they can also be found in water, air, fodder, and feed. New subtyping methods for DNA sequencing of the rpoB gene have reclassified some Bacillus into new genera such as Paenibacillus and Alicyclobacillus. Bacillus cereus causes defects in high-temperaturetreated milk and cream including sweet curdling (curd formation with no acid production). It also produces lecithinase, an enzyme that degrades fat globule membranes resulting in fat aggregation in cream (bitty cream). Bacillus subtilis survives pasteurization, and milk concentration and perhaps UHT treatment and produces a variety of enzymes that are capable of hydrolyzing casein and polysaccharides. Bacillus licheniformis is a common cause of spoilage in UHT and pasteurized milk. Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) is extremely heat resistant; it is more resistant than Cl. botulinum spores. It can survive canning and can cause flat sour spoilage and sweet curdling defects with little or no gas formation. This microorganism is also used to detect antibiotics in milk through growth inhibition assays. Bacillus coagulans has also been implicated in the spoilage of UHT and concentrated milk; it also produces chymosin-like enzymes, which cause sweet curdling. Bacillus circulans is a psychrotrophic bacterium, which causes an acid defect in aseptically packaged heat-treated milk. Bacillus macerans causes a flat sour defect in canned condensed milk. Clostridium species are important spoilage microorganisms in cheese and canned milk products. Late blowing of some cheese varieties that have a relatively high pH, a high moisture content, and a low interior salt content, such as Emmental, Gouda, and Edam, is caused by these microorganisms. Clostridium tyrobutyricum and, occasionally, Cl. sporogenes and Cl. butyricum are involved. These clostridia produce carbon dioxide, hydrogen gas, and butyric and acetic acids.
Lactic Acid Bacteria and Related Organisms Many species of lactic acid bacteria cause souring, offflavors, and texture defects (ropiness, curdling) in milk and dairy products. The general characteristics of lactic acid bacteria will be discussed later.
Spore-Forming, Gram-Positive Rods
Lactobacillus spp.
Two genera in this group are associated with milk: Bacillus and Clostridium. They are unique spoilage microorganisms because of their ability to form heat- and chemical-resistant spores and their ability to grow over a wide temperature range (>0–75 C). Their main
Heterofermentative lactobacilli such as Lb. brevis and Lb. casei subsp. pseudoplantarum cause an open texture in Cheddar and Mozzarella cheeses due to gas production. Lactobacilli also convert L(þ)-lactate to D()-lactate, which reacts with calcium to form calcium lactate,
454 Microorganisms Associated with Milk
which appears as white insoluble crystals in ripened Cheddar and other cheeses. Salt-tolerant lactobacilli produce phenolic and putrid sulfide-like flavors during ripening. Lactobacillus casei subsp. casei causes a soft body in Mozzarella cheese due to proteolysis. Lactobacillus delbrueckii subsp. bulgaricus can produce a pink discoloration in Italian cheese. Lactobacillus casei species sometimes produce a phenolic flavor in Cheddar cheese.
Lactococcus spp. Lactococcus lactis is commonly used as a starter culture for cheese and fermented milks. These strains are carefully selected so that they do not produce off-flavors or other defects. Wild strains of lactococci often contaminate raw milk, and if the milk is not kept sufficiently cold they will produce sourness and other off-flavors and odors. One variant, Lc. lactis subsp. lactis var. maltigenes, produces a malty flavor in fluid milk due to the production of 3-methylbutanal. Wild strains of lactococci may also contaminate cheeses and grow during manufacture and aging. Some of these strains produce fruity flavors due to the production of esters such as ethyl hexanoate and ethyl butyrate.
Propionibacterium spp. Pigmented strains of Propionibacterium cause pink spots in Swiss cheese. Split formation is influenced by Prop. freudenreichii subsp. shermanii strains used in making Swiss cheese.
Enterococcus spp. Enterococcus faecalis, Ec. faecium, and Ec. durans are part of the normal microflora of many cheeses and are sometimes used as starter cultures. Some strains produce undesirable flavors and high levels of amines during cheese ripening. Some enterococci are also considered probiotic. Two species, Ec. faecalis and Ec. faecium, were formerly classified as fecal streptococci (see Lactic Acid Bacteria: Enterococcus in Milk and Dairy Products).
flavor, depending on the cheese variety and strains involved.
Yeasts The presence of large numbers of yeasts on a cheese surface may result in a slimy rind, discolored appearance, and undesirable flavors. Non-lactose-fermenting Saccharomyces produce gas in fruit yogurt. Their source is usually a contaminated processing plant environment or unpasteurized fruits. Saccharomyces cerevisiae can spoil sweetened and acid dairy products. Kluyveromyces marxianus var. lactis, K. marxianus var. marxianus, Debaryomyces hansenii, and Yarrowia lipolytica spoil acidified refrigerated dairy products such as yogurt. Yarrowia lipolytica does not utilize lactose but metabolizes fat, protein, and organic acids, allowing it to spoil high-fat products such as butter and cream. It liberates amino and fatty acids and usually causes off-odors and softening of cheese curd. In addition, it produces melanin pigment from tyrosine and causes discoloration of cheese surfaces. Candida species are potential dairy spoilage organisms because of their ability to digest casein and fat, grow at storage temperatures, and ferment lactose and sucrose. They form surface slime on cream cheese. In yogurt, they cause yeasty and bitter flavors and a gassy texture. The important spoilage species in cheese is Can. famata and in yogurt Can. famata, Can. versatilis, and Can. lusitaniae.
Molds Mucor, Rhizopus, Penicillium, and Aspergillus grow at the yogurt–air interface of undisturbed packages. One mold spore can spoil a carton of yogurt by producing a visible colony. Molds also produce off-flavors in contaminated dairy products. Scopulariopsis brevicaulis causes odor defects in mold-ripened cheese. Sporendonema sebi grows on sweet condensed milk forming discrete colonies (mold buttons). Geotrichum grows on most dairy products, especially cheese and butter. Cheese varieties with low salt content are often spoiled by Mucor species.
Beneficial Microorganisms in the Dairy Industry Micrococcus spp. Some strains of Micrococcus species are able to survive pasteurization, but spoilage of heat-treated products is also caused by postprocessing contamination. Micrococcus species can produce swelling in UHT milk packs and have the ability to grow on some cheese varieties. Their growth may be either beneficial or detrimental to cheese
Selected lactic acid bacteria and other microorganisms are used by the dairy industry to produce fermented products such as yogurt and cheese. Commercially available starter cultures have been selected for a variety of desirable properties such as flavor production, lack of associated off-flavors, fast acid production, bacteriophage resistance, salt tolerance, exopolysaccharide production,
Microorganisms Associated with Milk
bacteriocin production, and heat sensitivity. These microbial preparations may consist of different species of lactic acid bacteria, propionibacteria, surface-ripening bacteria, yeasts, and molds.
Lactococcus spp. Lactococci are the major acid-producing microorganisms used for dairy fermentations, Lc. lactis being the main species of importance. Lactococcus lactis is homofermentative and weakly proteolytic. There are two subspecies: Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris. Citratepositive strains of Lc. lactis (Lc. lactis subsp. lactis biovar diacetylactis) utilize citrate to produce diacetyl, carbon dioxide, and other compounds in cheeses and cultured milks. Some strains are highly ropy.
Streptococcus spp. Streptococcus thermophilus is used to produce acid in dairy products subjected to high temperatures during fermentation (yogurt and Mozzarella cheese). It is weakly proteolytic and some strains produce extracellular polysaccharides.
Leuconostoc spp. Leuconostoc species are heterofermentative, Gram-positive cocci. They produce flavor compounds such as diacetyl and acetoin, and some strains produce exopolysaccharides. Leuconostoc mesenteroides subsp. cremoris is used in the production of cottage and cream cheese and cultured milks. Leuconostoc mesenteroides subsp. mesenteroides and Ln. mesenteroides subsp. dextranicum often produce slime (glucan, formerly known as dextran) from sucrose. Leuconostoc paramesenteroides does not produce glucan and is used in making brined cheese. Leuconostoc lactis is also found in milk and dairy products. Leuconostoc species reduce an undesirable ‘green’ flavor in cultured dairy products by converting acetaldehyde to diacetyl, but grow only slowly in milk.
Lactobacillus spp. Lactobacillus species are a genetically and physiologically diverse group of rod-shaped lactic acid bacteria. They are the most acid tolerant of the lactic acid bacteria and produce numerous proteolytic enzymes. There are three groups based on the type of fermentation end products: homofermentative (Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis, Lb. acidophilus, Lb. helveticus, and
455
Lb. helveticus subsp. jugurti), facultatively heterofermentative (Lb. casei, Lb. curvatus, and Lb. plantarum), and obligately heterofermentative (Lb. kefir, Lb. fermentum, and Lb. brevis). Some lactobacilli, such as Lb. acidophilus, are claimed to confer health benefits. Homofermentative lactobacilli are usually thermophilic and are used to produce fermented milk and some cheese varieties that require the use of high temperature during manufacturing. Lactobacillus casei and Lb. plantarum cannot grow at high temperatures (at 45 C). They are involved in the ripening of Cheddar and other cheese varieties.
Propionibacterium spp. Propionibacteria produce propionic and acetic acids and carbon dioxide from sugars and lactic acid. They are responsible for eye formation in Swiss cheese. Propionibacterium freudenreichii, Prop. jensenii, Prop. thoenii, and Prop. acidipropionici are the species commonly found in cheese.
Coryneform Bacteria Coryneform bacteria are a group of irregular, non-sporeforming, Gram-positive rods, often found growing on the surface of surface-ripened cheese. From a taxonomic viewpoint, dairy species differ from the reference pathogenic species. Cheese smear coryneform bacteria are mostly psychrotrophic and do not grow at 37 C, whereas pathogenic coryneform bacteria are facultative anaerobes and grow at 37 C. The cheese smear group includes Brevibacterium, Arthrobacter, Microbacterium, Aureobacterium, Brachybacterium, Rhodococcus, and Corynebacterium. These microorganisms are responsible for the viscous, red-orange surface (smear) on surface-ripened cheese varieties. They survive high salt concentrations and produce various proteolytic enzymes. Their presence in large numbers causes a slimy rind, discolored appearance, and undesirable flavors in cheese. Currently, the genus Brevibacterium is restricted to four species: Brev. linens, Brev. iodinum, Brev. casei, and Brev. epidermidis. Arthrobacter species include Ab. globiformis, Ab. nicotianae, Ab. protophormiae, Ab. agilis, Ab. sulphurous, and Ab. citreus. The genus Microbacterium contains Microb. lacticum, Microb. laevaniformans, Microb. imperiale, and Microb. arborescens. The genus Aureobacterium contains Au. liquefaciens and Au. testaceum. Brachybacterium alimentarium and Brach. tyrofermentans are two new species of coryneform bacteria isolated from the surface of Gruye`re and Beaufort cheeses and they tolerate up to 20% salt. Corynebacterium species found in dairy products include C. ammoniagenes and C. variabilis. Rhodococcus fascians was also isolated
456 Microorganisms Associated with Milk
from cheese. The habitats of coryneform bacteria are cheese and human skin (see Bacteria, Beneficial: Brevibacterium Linens, Brevibacterium Aurantiacum and Other Smear Microorganisms).
Bifidobacterium spp. Bifidobacterium species are inhabitants of the intestinal tract, which are added to dairy products for probiotic applications. The species most often used are Bif. longum, Bif. bifidum, and Bif. animalis. Bifidobacterium species grow poorly in milk (see Bacteria, Beneficial: Bifidobacterium spp.: Applications in Fermented Milks; Bifidobacterium spp.: Morphology and Physiology).
Pediococcus spp. Pediococcus species are salt-tolerant members of the family Streptococcaceae, which usually grow as tetrads of cells. Most species can grow at 4–6.5% NaCl. They produce acetic acid and small amounts of lactic acid when growing aerobically and mostly lactic acid when growing anaerobically. The most important species used in the dairy industry are Pc. acidilactici and Pc. pentosaceus (see Lactic Acid Bacteria: Pediococcus spp.).
Micrococcus spp. Micrococcus species are the predominant microorganisms found in raw milk drawn aseptically from the udder. They are the constituents of the natural microflora of teat skin and have also been isolated from soil, water, and dust. Based on phylogenetic and chemotaxonomic analysis, many micrococci (except Mc. luteus and Mc. lylae) have been renamed. The new names are Kocuria varians, Kc. roseus, Kc. kristinae, Dermacoccus nishinomiyaensis, Kytococcus sedentarius, Ab. agilis, and Nesterenkonia halobia. Micrococcus species have proteolytic, lipolytic, and esterase activities, which make them important in the ripening of various cheeses, especially those that are surface-ripened.
Molds Penicillium species are widely distributed in the environment. This genus includes four subgenera. The surface of soft white cheese can be colonized by P. camemberti Thom to produce many different cheese varieties such as Brie and Camembert. This mold produces a white layer of the mycelium on the cheese surface, while spores (candida) are embedded in the curd. The mold metabolizes some of lactic acid thereby raising the pH, which allows growth of
Brev. linens and other ripening microflora. In blue-veined cheese varieties, P. roqueforti (blue mold) is used in cheese ripening. Penicillium roqueforti has a relatively low oxygen requirement for growth (<4.2%) and tolerates 6–10% salt. Both P. camemberti and P. roqueforti produce lipolytic and proteolytic enzymes. They produce additional compounds that give cheese its distinctive flavor and aroma. Penicillium casei is similar to P. roqueforti and is found in Swiss cheese. Penicillium nalgiovense (Lava), another white mold, tolerates up to 8% salt and is used in some cheese varieties. Aspergillus niger and A. awamori produce lipase and contribute to the flavor of some cheese varieties. Geotrichum candidum is often found in raw milk, on milking and dairy processing equipment, and on the surface of ripened cheese. It can be used in starter cultures for Brie and Camembert cheese when combined with Penicillium species. Rhizomucor miehei and Rm. pusillus produce chymosin-like enzymes, which are used in cheese manufacture.
Yeasts Saccharomyces species are ascosporogenous yeasts with a capability for vigorous anaerobic or semianaerobic fermentation of sugar to produce ethanol and carbon dioxide. None of them utilizes lactose. Saccharomyces cerevisiae is the most important species and can be found on the surface of mold-ripened cheeses. Saccharomyces cerevisiae metabolizes hexoses, lactic acid, and other organic acids. The optimum pH for the growth of Saccharomyces species is 4.5–6.5. Oxygen is important to maintain viability but they survive under microaerophilic conditions. Kluyveromyces marxianus and K. lactis can ferment lactose and produce various hydrolytic enzymes. Kluyveromyces species are often isolated from Nordic fermented milks. They produce lactase, which can be used to reduce the lactose content of milk and assist in the digestion of milk by lactose maldigesters. Debaryomyces species are isolated from soil, water, and plants. Debaryomyces hansenii is involved in the ripening of mold-ripened soft cheeses. It tolerates high salt concentrations, utilizes lactic acid, produces protease and lipase, and grows well at low temperature. Candida kefyr is present in the microflora of the fermented milk kefir.
Microbial Population Dynamics in Milk Microbial populations in milk are continually changing and interacting with the milk components and with each other. The Pasteurized Milk Ordinance in
Microorganisms Associated with Milk
the United States requires grade A milk to have a total bacterial count of less than 100 000 cfu ml1. The initial microbial population of raw milk is affected by factors such as equipment and animal cleanliness, season, feed, and cow health. The initial microflora of good quality milk mainly consists of organisms found within the udder and the normal teat skin flora. These microorganisms include nonthermoduric micrococci and streptococci, which do not grow well at low temperatures. Pathogens will predominate in milk from mastitic cows. Seasonal factors include feed source and housing, both of which influence the level of bacterial spores in milk. Psychrotrophic bacteria (those that grow at low temperatures) are at low or even undetectable levels in the best quality fresh raw milk. They originate from the water and milking equipment. After collection, milk is cooled, which initiates a change in microbial population from Gram-positive rods and cocci, which do not grow, to Gram-negative rods, which can grow slowly at low temperatures. These Gram-negative rods are primarily Pseudomonas spp. and are capable of causing various off-flavors when populations exceed 106 cells ml1. When raw milk is not kept cold, lactic acid bacteria will grow. The resulting increase in acidity will inhibit the growth of many other microorganisms including sporeforming Bacillus and Clostridium species and Gram-negative Pseudomonas species and coliforms. Growth of lactic acid bacteria is rapid at 20 C and slows as the temperature decreases. Many strains of lactic acid bacteria produce bacteriocins, which inhibit other lactic acid bacteria as well as some pathogens such as Listeria and E. coli. Both the lactic acid bacteria and the psychrotrophic bacteria that grow in raw milk are heat sensitive, so when milk is pasteurized, microorganisms that have previously grown in the product are greatly reduced in number. The surviving microflora mainly consists of spore-forming bacteria, micrococci, and lactobacilli. Pasteurized milk that is not recontaminated with microorganisms will often keep at refrigeration temperature for 3 weeks or longer, because these surviving microorganisms grow slowly or not at all in the cold milk. Spoilage may occur more rapidly if significant numbers of psychrotrophic B. cereus are present in the raw milk. Most pasteurized milk is recontaminated with low numbers of psychrotrophic bacteria, resulting in a shorter shelf life. See also: Bacteria, Beneficial: Bifidobacterium spp.: Applications in Fermented Milks; Bifidobacterium spp.:
457
Morphology and Physiology; Brevibacterium linens, Brevibacterium Aurantiacum and Other Smear Microorganisms. Diseases of Dairy Animals: Infectious Diseases: Johne’s Disease; Infectious Diseases: Tuberculosis. Lactic Acid Bacteria: Enterococcus in Milk and Dairy Products; Pediococcus spp.. Liquid Milk Products: Pasteurization of Liquid Milk Products: Principles, Public Health Aspects. Pathogens in Milk: Bacillus cereus; Brucella spp.; Campylobacter spp.; Clostridium spp.; Coxiella burnetii; Enterobacteriaceae; Escherichia coli; Listeria monocytogenes; Salmonella spp.; Yersinia enterocolitica. Yeasts and Molds: Spoilage Molds in Dairy Products; Yeasts in Milk and Dairy Products.
Further Reading Boor KJ (1997) Pathogenic microorganisms of concern to the dairy industry. Dairy, Food and Environmental Sanitation 17: 714–717. Davies FL and Law BA (1984) Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk. London: Elsevier Applied Science. Doyle MP, Beuchat LR, and Montville TJ (2001) Food Microbiology: Fundamentals and Frontiers. Washington, DC: ASM Press. Durak MZ, Fromm HI, Huck JR, Zadoks RN, and Boor KJ (2006) Development of molecular typing methods for Bacillus ssp. and Paenibacillus ssp. isolated from fluid milk products. Journal of Food Science 71: 50–56. Frank JF and Hassan AN (1998) Starter cultures and their use. In: Marth EH and Steele JL (eds.) Applied Dairy Microbiology, pp. 131–172. New York: Marcel Dekker. Holt JG, Krieg NR, Sneath PH, Stanley JT, and Williams ST (eds.) (1994) Bergey’s Manual of Determinative Bacteriology, 2nd edn. Baltimore, MD: Williams & Wilkins. Hui YH (1993) Dairy Science and Technology Handbook. New York: VCH. IDF (1993) Indigenous antimicrobial agents of milk. Proceedings of the International Dairy Federation Seminar. Uppsala, Sweden, September. IDF (1994) The Significance of Pathogenic Microorganisms in Raw Milk. Brussels, Belgium: IDF. IDF (1996) Bacteriological quality of raw milk. Proceedings of the International Dairy Federation Symposium. Wolfpassing, Austria, March. Robinson RK (1990) Dairy Microbiology: The Microbiology of Milk, Vol. 1. London: Elsevier Applied Science. Robinson RK, Batt CA, and Patel PD (eds.) (2000) Encyclopedia of Food Microbiology. London: Academic Press. Smith DE and Vasavada PC (1988) Problems of pathogenic bacteria in the dairy industry. Journal of Dairy Science 71: 2807–2808. Stiles ME and Holzapfel WH (1997) Lactic acid bacteria of foods and their current taxonomy. International Journal of Food Microbiology 36: 1–29. Varnam AH and Sutherland JP (1994) Milk and Milk Products, Technology, Chemistry and Microbiology. London; New York: Chapman & Hall. Vasavada PC (1988) Pathogenic bacteria in milk: A review. Journal of Dairy Science 71: 2809–2816. Wiedmann M (2003) ADSA Foundation Scholar Award – an integrated science-based approach to dairy food safety: Listeria monocytogenes as a model system. Journal of Dairy Science 86: 1865–1875.
Introduction Milk is a good growth medium for many microorganisms because of its near-neutral pH, complex biochemical composition, and high water content. Although in the absence of mastitis it is secreted free of microorganisms, it is subject to microbial contamination from a number of sources (Figure 1). Some microorganisms move up the teat canal causing aseptically drawn milk to be contaminated. These contaminants, called udder commensals, are present in small numbers and are mainly lactic acid bacteria. Their numbers are limited by the animal’s immune system and antimicrobial agents secreted into the milk. Therefore, contaminants external to the udder from locations such as udder skin, hide, milking equipment, and utensils form the vast majority of microorganisms in raw milk. Although technology has allowed the production of milk with very low microbial levels, product quality still depends on controlling entry and growth of microorganisms in milk from farm to the consumer. The number and types of microorganisms present in milk are influenced by season, farm hygiene, feed, and efficiency of cooling. The number of bacteria in milk ranges from a few hundred to thousands per milliliter of freshly drawn milk from healthy cows. Four physiological groups of spoilage bacteria are commonly found in raw milk: producers of lactic acid, propionic acid, butyric acid, and degradative enzymes (primarily proteases and lipases). In addition, raw milk may contain pathogens whose multiplication depends mainly on the temperature and competing microflora. The main criteria for highquality raw milk are the presence of a low number of spoilage microorganisms and the absence of animal pathogens. The sources of microorganisms found in milk, their characteristics, and their growth are discussed below.
Microorganisms Found in Milk The microorganisms found in milk can be divided into three groups: animal pathogens and toxin producers, spoilage agents (saprophytes), and microorganisms used to produce fermented products. Some overlap among these groups occurs; for example, Bacillus cereus is a toxin
producer and is also involved in spoilage, and lactic acid bacteria can cause spoilage and be used in fermentations. Figure 2 shows the different morphological groups commonly found in raw milk.
Pathogenic Microorganisms Pathogenic bacteria are those capable of causing infection or intoxication. Milk is a good growth and protective medium for most pathogens. The growth of pathogens in milk is inhibited by cooling and the growth of nonpathogenic competing microorganisms. Many pathogens, such as Mycobacterium tuberculosis and Brucella species, grow very slowly in milk, and others, such as viruses, do not multiply at all. Therefore, the threat associated with their presence in milk will depend on the initial load of milk contamination. Although the potential hazard of pathogens has been minimized by modern milk production and technology practices, disease outbreaks caused by Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, and Escherichia coli O157:H7 have occurred in recent years.
Staphylococcus spp. Staphylococcus spp. are nonmotile, non-spore-forming, Gram-positive, catalase-positive cocci. They grow well in media containing 10% NaCl and produce proteases, lipases, and esterases. Staphylococcus aureus is the most important species of this genus associated with milk. It produces heat-stable enterotoxins that are responsible for food poisoning. Its natural habitat includes the skin and mucous membranes of mammals. Most contamination occurs during milking and originates with the cow. About 40% of bulk tank milk from a nationally representative sample of 542 dairies tested positive for Staph. aureus. It has been reported that 16 and 18% of the cows from organic and conventional farms, respectively, harbored oxacillin-resistant staphylococci, which indicated the presence of the methicillin-resistant gene (mecA). Although the presence of Staph. aureus in milk is difficult to prevent, appropriate cleaning and sanitation of the cow and milking equipment controls initial numbers and
447
448 Microorganisms Associated with Milk
Figure 1 Sources of milk contamination at the dairy farm.
Figure 2 Different morphological groups of microorganisms commonly found in raw milk.
Microorganisms Associated with Milk
cooling the milk prevents growth, toxin production, and spreading of bacteria to humans via the milk chain. Staphylococcus hyicus has been isolated from the skin of cows and from the milk of cows with mastitis. Some strains are enterotoxigenic.
Streptococcus spp. Streptococcus pyogenes is pathogenic to humans and some animals. It resists phagocytosis and produces an erythrogenic toxin that causes scarlet fever. Streptococcus agalactiae belongs to group B streptococci (GBS) and is an important agent of bovine mastitis in some countries and a cause of diseases in humans. The gastrointestinal tract is the main human reservoir. Streptococcus dysgalactiae belongs to Lancefield group C streptococci. It is found mainly in mastitic bovine udders. Streptococcus uberis is found in the lips and skin of cows, udder tissue, and milk. It is the main cause of mastitis in some countries, especially during the winter. The only phenotypic criterion that differentiates between Sc. uberis and Sc. parauberis is the ability of the former to produce -D-glucuronidase. Streptococcus equi subsp. equi and Sc. equi subsp. zooepidemicus are -hemolytic group C Streptococcus species that infect animals and humans, respectively.
Campylobacter jejuni Campylobacter jejuni is a common cause of human gastroenteritis. It is a Gram-negative, curved, slender, motile rod and is found in food, feces, and water. Although it does not grow in milk, the consumption of fresh raw milk has caused numerous outbreaks of gastroenteritis (see Pathogens in Milk: Campylobacter spp.). Results from NAHMS Dairy 2002 indicated that the percentage of cows shedding Campylobacter spp. was 51.4%, while the percentage of dairy herds with at least one cow shedding Campylobacter was 97.9%.
Yersinia spp. The genus Yersinia belongs to the family Enterobacteriaceae. Members of this genus are Gram-negative, motile rods, facultatively anaerobic at 28–29 C but strictly aerobic at 37 C, which can grow at 2–4 C. Yersinia enterocolitica is the only species of importance in milk. The most common source of contamination is the rinsing water used at the farm. Yersinia species grow well in milk and cause an enteric infection that mimics appendicitis (see Pathogens in Milk: Yersinia enterocolitica).
449
Salmonella spp. The genus Salmonella (family Enterobacteriaceae) consists of small, Gram-negative, non-spore-forming rods. Most strains are motile. The primary habitat is the intestinal tract. Cows suffering from salmonellosis can excrete large numbers of salmonellae in their feces and occasionally directly into milk. Milk can be contaminated with Salmonella from feces, livestock feed, humans, water, and dust. Direct contact from other herds can result in the introduction of Salmonella spp. into dairy farms. Many outbreaks of salmonellosis have been attributed to milk and milk products. Unlike staphylococcal food poisoning, the ingestion of viable cells of Salmonella is necessary for salmonellosis. Raw milk or dairy products made from raw milk have been the cause of outbreaks of salmonellosis in the Western world. Nontyphoid Salmonella enterica infects an estimated 2–4 million people every year in the United States. Growth of Salmonella in milk is not necessary as ingestion of only a few cells can cause the disease (see Pathogens in Milk: Salmonella spp.).
Escherichia coli Escherichia coli is a member of the family Enterobacteriaceae. It is a facultatively anaerobic, Gram-negative rod, and ferments lactose. Four categories of pathogenic E. coli are recognized: enteropathogenic, enterotoxigenic, enteroinvasive, and enterohemorrhagic. Enterotoxigenic E. coli produces enterotoxins that cause diarrhea. Humans are the main reservoir. To cause diarrhea, sufficient numbers of toxigenic E. coli must be ingested and they must adhere to the small intestine. In recent years, enterohemorrhagic E. coli, mainly E. coli O157:H7, have caused milk-borne illness. These pathogens cause hemorrhagic colitis (bloody diarrhea), an illness that can lead to renal failure in children. The dairy cow gut is a reservoir for E. coli O157:H7. Hemorrhagic strains of E. coli do not ferment sorbitol and the incubation temperature used to detect fecal coliforms (44–45 C) will not allow E. coli O157:H7 to grow (see Pathogens in Milk: Escherichia coli). Non-O157 shiga toxin-producing E. coli (STEC) was also associated with raw milk and raw milk cheese.
Listeria spp. Within the genus Listeria, only Li. monocytogenes and Li. ivanovii are considered virulent, with Li. monocytogenes being the only species of public health concern. Listeria innocua is the most frequently encountered nonpathogenic Listeria species. Listeria monocytogenes is a Gram-positive, short rod with rounded ends that occurs singly, in parallel,
450 Microorganisms Associated with Milk
or in V shapes. It grows slowly in milk at refrigeration temperature. Listeria monocytogenes causes mastitis and abortion in animals. In humans, it causes miscarriages and meningitis in its severe form and flu-like symptoms in its mild form. Raw milk can be contaminated with Li. monocytogenes from feces, cows suffering from mastitis, poor-quality silage, and milking equipment (see Pathogens in Milk: Listeria monocytogenes).
Mycobacterium spp. Mycobacterium avium subsp. paratuberculosis (MAP) is one of the most heat-resistant pathogenic microorganisms likely to be present in milk. Mycobacterium tuberculosis may gain access to milk from infected animals through milk secretion and fecal contamination or from milkers and other environmental sources such as cow alleyways, manure storage, calving area, sick cow pen, water runoff, and postweaned calves areas. The organism does not multiply in milk but survives in unpasteurized milk and milk products. MAP causes the infection commonly known as Johne’s disease. Detectable numbers of this organism have been reported in the milk of both clinically affected and asymptomatic cattle. Pasteurization inactivates MAP when present at levels associated with milk from asymptomatic cattle. It is unknown whether this microorganism is pathogenic for man. Mycobacterium bovis causes infection in animals and humans. Compared with M. tuberculosis, growth of M. bovis is less luxuriant and it adapts to aerobic growth after repeated subculturing. Contaminated milk, feces, soil, and water are the main sources of infection (see Diseases of Dairy Animals: Infectious Diseases: Johne’s Disease; Infectious Diseases: Tuberculosis).
Brucella spp. Brucella species are Gram-negative, aerobic, short rods that do not ferment carbohydrates. They survive well in milk and dairy products and are pathogenic to humans and animals. The most important causes of brucellosis in milk are Br. abortus and Br. melitensis (see Pathogens in Milk: Brucella spp.).
Coxiella burnetii Coxiella burnetii is a rickettsia (family Rickettsiaceae), which causes Q fever in humans. Rickettsiae are obligate intracellular parasites. Coxiella burnetii is a small pleomorphic, sometimes filamentous, nonmotile rod that
produces a capsule-like structure. It is highly resistant to chemical and physical disinfectants and is the most heatresistant pathogen found in milk (see Pathogens in Milk: Coxiella burnetii).
Aeromonas spp. Aeromonas species are facultatively anaerobic, Gramnegative, motile cocci or rods that grow in media containing up to 5% salt. Aeromonas hydrophila is an opportunistic pathogen found in the feces of healthy animals.
Bacillus spp. Bacillus species are Gram-positive, aerobic, sporeforming rods belonging to the family Bacillaceae. Their spores make them resistant to heat and other destructive agents. Strains can be psychrotrophic, mesophilic, or thermophilic. Bacillus cereus is a common soil bacterium that is often found in raw milk. It is capable of producing enterotoxins (especially in starchy foods), which cause food poisoning. Bacillus anthracis causes anthrax, which is a deadly acute infectious disease. Purposeful contamination of foods by B. anthracis was considered a potential biological weapon. The presence of Bacillus in raw milk shows marked seasonal variation. Soil, feces, bedding, air, silage (possibly via feces), and the milking equipment are the main sources of Bacillus in milk (see Pathogens in Milk: Bacillus cereus).
Clostridium spp. Clostridia are Gram-positive, spore-forming rods, which are usually found in sediments, the intestinal tract of humans and animals, soil, dust, feces, and silage. They are anaerobic and often poisoned by atmospheric oxygen unless in the spore form. Clostridium perfringens causes food poisoning and gas gangrene in humans and a variety of diseases in animals. Milk and milk products provide a ready medium for transmission of Cl. perfringens. Vegetative cells sporulate in the gut and release exterotoxin, which is responsible for symptoms such as profuse diarrhea, sudden acute inflammation of the abdomen, and severe abdominal pain. Large numbers of cells must be consumed for illness to occur. Botulism is an intoxication attacking the nervous system caused by ingestion of toxins produced by Cl. botulinum. Very low levels (<1 spore ml1) of these organisms are common in milk. They do not germinate or grow in raw or pasteurized milk, but cheese and cheese
Microorganisms Associated with Milk
products can support growth if they are not sufficiently acidic (see Pathogens in Milk: Clostridium spp.).
Xanthomonas maltophilia Xanthomonas maltophilia, previously known as Pseudomonas maltophilia, is an aerobic rod of the family Pseudomonadaceae. It is one of several pseudomonad-like organisms in hospital laboratories associated with serious infections. It has been isolated from milk and water.
Klebsiella pneumoniae Klebsiella pneumoniae belongs to the family Enterobacteriaceae and is an agent for pneumonia, urinary tract infection, and gastroenteritis and mastitis. It is present in animal bedding materials and water. Fecal shedding of Kl. pneumoniae is considered one of the sources of this microorganism in milk. Inorganic bedding such as sand reduces its occurrence.
Serratia spp. Serratia species are Gram-negative, facultative, anaerobic rods of the family Enterobacteriaceae. They are distributed in soil, air, water, and plants. Some strains produce hemolysins and infected animals produce contaminated milk. Their presence in pasteurized dairy products indicates poor sanitation practices since they are heat sensitive. Serratia marcescens is the most important species and causes mastitis.
Proteus spp. Proteus species are Gram-negative, non-spore-forming rods belonging to the family Enterobacteriaceae. They are commonly found in the feces of humans and animals, soil, and plants. They frequently cause a variety of infections in humans, especially urinary tract infections. Proteus mirabilis is the most common species followed by Pr. vulgaris and Pr. penneri.
Enterobacter sakazakii Enterobacter sakazakii belongs to the family Enterobacteriaceae. Although rarely found, it can cause life-threatening neonatal meningitis. It does not survive pasteurization but is found in ultra-high temperature (UHT) milk and milk powder that have been subjected to postprocessing contamination. Its natural habitat is not known.
451
Hafnia alvei Hafnia alvei, which belongs to the family Enterobacteriaceae, resembles Salmonella. Some strains cause mild gastroenteritis. It is found in sewage, soil, water, and feces. Hafnia species can grow on the surface of Camembert cheese (see Pathogens in Milk: Enterobacteriaceae).
Actinomyces spp. Actinomyces species are irregular, non-spore-forming, Gram-positive rods that occur mainly in the oral cavity and on the mucous membranes of warm-blooded vertebrates. They cause pyogenic infections. Actinomyces bovis and Ac. pyogenes are the important species in milk.
Leptospira spp. Species of the genus Leptospira (family Leptospiraceae) are Gram-negative, flexible, helicoidal rods. Leptospira interrogans causes leptospirosis in animals and humans. The kidney is the natural habitat and contaminated urine is the main source of contamination.
Molds and Yeasts Many different genera of molds such as Aspergillus, Penicillium, and Fusarium produce mycotoxins in milk and dairy products. Mycotoxins are secondary metabolites that are acutely toxic, carcinogenic, emetic, estrogenic, hallucinogenic, mutagenic, or teratogenic. Aspergillus flavus and some other Aspergillus species produce aflatoxins when growing on cheeses. Some strains of Geotrichum might cause infection in humans. Candida albicans is pathogenic to humans and animals (see Yeasts and Molds: Spoilage Molds in Dairy Products; Yeasts and Molds: Yeasts in Milk and Dairy Products).
Viruses Viruses are genetic elements that replicate inside cells and also have an extracellular nonreplicating state. Replication of the virus often destroys the host cell causing illness. The extracellular form of the virus is a submicroscopic particle and can be transmitted between hosts. Viruses do not multiply in milk but some may survive for long periods. Viruses can be infective at very low doses and most that are found in milk produce gastroenteritis and originate with the cow. Viruses in milk are inactivated by pasteurization.
452 Microorganisms Associated with Milk
Rotaviruses can produce severe gastrointestinal illnesses. Milk may be a vector for this virus but detection in raw milk is prevented by the presence of antiviral antibodies (immunoglobulins; IgG). The antibody/virus complex is dissociated in the highly acidic conditions of the stomach. Hepatitis A virus enters milk through fecal contamination or polluted water. This virus causes symptoms of fever, vomiting, nausea, and abdominal discomfort. The virus eventually affects the liver, causing enlargement and jaundice. Poliovirus causes the acute infectious disease poliomyelitis, which is characterized by fever, vomiting, and headache. The poliovirus might infect the central nervous system and cause paralysis. It can be transmitted to milk through fecal contamination. Milk can serve as a vector for the spread of the footand-mouth disease virus. This virus, which is nonenveloped, belongs to the Picornaviridae family, which also includes rhinoviruses and enteroviruses.
Protozoa Toxoplasma gondii is a parasite of humans and many warmblooded animals, especially dairy goats. Infection occurs as a result of ingestion of foods or water contaminated with oocysts. Contaminated milk is considered a potential source of human toxoplasmosis. Cryptosporidiosis is an infection caused by Cryptosporidium muris and Cr. parvum. Diarrhea is the primary symptom of infection. Cows can shed Cr. muris oocysts in their feces, which may lead to contamination of milk. Drinking water is the most common vehicle for transmission of this disease. Entamoeba histolytica is a protozoan that causes amoebiasis, the third most common cause of death by parasites in the world. Transmission occurs via ingestion of cysts in contaminated foods or water or through poor personal hygiene. Giardia intestinalis is a flagellated protozoan that inhabits the intestinal tracts of humans and animals. Cysts survive well in the environment and are transmitted via contaminated hands, drinking water, and food contaminated with feces.
Psychrotrophic Bacteria Most psychrotrophic bacteria present in raw milk belong to the family Pseudomonadaceae, which includes Gram-negative, non-spore-forming, nonfermentative, aerobic rods. Pseudomonas fluorescens is the species most commonly isolated from milk, with Ps. fragi and Ps. putida being of less importance. Soil, water, animals, and plant material are the natural habitat of psychrotrophic bacteria found in milk. Contaminated milk equipment and air provide an additional source of contamination, especially after processing. As little as 1 cfu ml1 of raw milk may be enough to spoil milk during cold storage within 5 days. Some Pseudomonas spp. grow at cold and warm temperatures, and others found in milk cannot propagate at temperatures greater than 25 C. Defects associated with the growth of these organisms in milk include development of bitter and fruity offflavors. Although psychrotrophic, Gram-negative bacteria are not heat resistant, they produce heat-stable extracellular proteolytic and lipolytic enzymes that can spoil heat-treated products. However, not all Gram-negative isolates from raw milk display proteolytic activities at 7 or 20 C. Other spoilage Gramnegative rods include Acinetobacter, Psychrobacter, Flavobacterium, Shewanella putrefaciens, and Alcaligenes spp. Cold-tolerant endospore-forming bacteria such as Bacillus and Paenibacillus spp. in raw milk are considered a major cause of spoilage of pasteurized milk.
Family Moraxellaceae Moraxellaceae are Gram-negative, psychrotrophic, nonpigmented, nonmotile, aerobic coccobacilli. There are three genera: Acinetobacter are nonmotile, oxidase-negative organisms, Moraxella are similar but oxidase-positive, and the third genus is Psychrobacter. Moraxellaceae seldom spoil milk because they lack sufficient biochemical activities such as proteolysis and lipolysis that cause off-odors. In addition, they are overgrown by Pseudomonas during cold storage.
Spoilage Microorganisms Spoilage of milk and dairy products is manifest as offflavors and odors and changes in texture and appearance. The most important spoilage microorganisms of milk and dairy products are the Gram-negative, rod-shaped bacteria (e.g., Pseudomonas and coliforms), Gram-positive, sporeforming bacteria (Bacillus and Clostridium), lactic acid bacteria, members of coryneform group, yeasts, and molds.
Shewanella putrefaciens Shewanella putrefaciens, previously known as Pseudomonas putrefaciens or Alteromonas putrefaciens, is a Gram-negative rod. It is found in water and soil and contaminates milk and milk products causing spoilage such as surface taint in butter.
Microorganisms Associated with Milk
Flavobacterium spp. Flavobacterium species are Gram-negative organisms found in water, soil, and milk. They hydrolyze casein and cause spoilage in milk and dairy products during cold storage. Important species are Fb. maloloris, Fb. aquatile, and Fb. lutensis.
Alcaligenes spp Alcaligenes species are Gram-negative, short, motile rods. They are aerobic and do not use lactose. They are found in water, soil, and milk. Alcaligenes faecalis produces the exopolysaccharide curdlan and is a potential contaminant of dairy products. Alcaligenes can grow under refrigeration conditions, producing off-flavors and reducing the shelf life of milk.
Coliforms Coliforms are Gram-negative, oxidase-negative, nonspore-forming rods that grow aerobically or facultatively anaerobically in the presence of bile salts. They also ferment lactose to produce acid and gas within 48 h at 37 C. Coliforms include the genera Escherichia, Enterobacter, Klebsiella, Proteus, Serratia, and Citrobacter. They do not survive pasteurization. Gas production may result in early blowing of hard cheese and a poor curd structure in Cottage cheese. Approximately 107 cfu g1 of coliforms is needed for a gassy defect to be noticed. Other types of spoilage include acid production, slime production in Cottage cheese, bitter flavors, and grassy, unclean, medicinal, or fecal odors. Cheese varieties in which acid production is delayed are more susceptible to coliform growth. Klebsiella (Kl. pneumoniae and Kl. oxytoca) and Enterobacter aerogenes are responsible for gas formation in cheese, which can lead to swelling of the package. Serratia marcescens produces proteolytic enzymes that might cause gelation of UHT milk. In addition, they produce methanethiol in Cheddar cheese. Soiled udders and teats are common sources of coliforms. Coliforms can also grow and contaminate milk from poorly sanitized milk contact surfaces and equipment.
453
habitat is soil, but they can also be found in water, air, fodder, and feed. New subtyping methods for DNA sequencing of the rpoB gene have reclassified some Bacillus into new genera such as Paenibacillus and Alicyclobacillus. Bacillus cereus causes defects in high-temperaturetreated milk and cream including sweet curdling (curd formation with no acid production). It also produces lecithinase, an enzyme that degrades fat globule membranes resulting in fat aggregation in cream (bitty cream). Bacillus subtilis survives pasteurization, and milk concentration and perhaps UHT treatment and produces a variety of enzymes that are capable of hydrolyzing casein and polysaccharides. Bacillus licheniformis is a common cause of spoilage in UHT and pasteurized milk. Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) is extremely heat resistant; it is more resistant than Cl. botulinum spores. It can survive canning and can cause flat sour spoilage and sweet curdling defects with little or no gas formation. This microorganism is also used to detect antibiotics in milk through growth inhibition assays. Bacillus coagulans has also been implicated in the spoilage of UHT and concentrated milk; it also produces chymosin-like enzymes, which cause sweet curdling. Bacillus circulans is a psychrotrophic bacterium, which causes an acid defect in aseptically packaged heat-treated milk. Bacillus macerans causes a flat sour defect in canned condensed milk. Clostridium species are important spoilage microorganisms in cheese and canned milk products. Late blowing of some cheese varieties that have a relatively high pH, a high moisture content, and a low interior salt content, such as Emmental, Gouda, and Edam, is caused by these microorganisms. Clostridium tyrobutyricum and, occasionally, Cl. sporogenes and Cl. butyricum are involved. These clostridia produce carbon dioxide, hydrogen gas, and butyric and acetic acids.
Lactic Acid Bacteria and Related Organisms Many species of lactic acid bacteria cause souring, offflavors, and texture defects (ropiness, curdling) in milk and dairy products. The general characteristics of lactic acid bacteria will be discussed later.
Spore-Forming, Gram-Positive Rods
Lactobacillus spp.
Two genera in this group are associated with milk: Bacillus and Clostridium. They are unique spoilage microorganisms because of their ability to form heat- and chemical-resistant spores and their ability to grow over a wide temperature range (>0–75 C). Their main
Heterofermentative lactobacilli such as Lb. brevis and Lb. casei subsp. pseudoplantarum cause an open texture in Cheddar and Mozzarella cheeses due to gas production. Lactobacilli also convert L(þ)-lactate to D()-lactate, which reacts with calcium to form calcium lactate,
454 Microorganisms Associated with Milk
which appears as white insoluble crystals in ripened Cheddar and other cheeses. Salt-tolerant lactobacilli produce phenolic and putrid sulfide-like flavors during ripening. Lactobacillus casei subsp. casei causes a soft body in Mozzarella cheese due to proteolysis. Lactobacillus delbrueckii subsp. bulgaricus can produce a pink discoloration in Italian cheese. Lactobacillus casei species sometimes produce a phenolic flavor in Cheddar cheese.
Lactococcus spp. Lactococcus lactis is commonly used as a starter culture for cheese and fermented milks. These strains are carefully selected so that they do not produce off-flavors or other defects. Wild strains of lactococci often contaminate raw milk, and if the milk is not kept sufficiently cold they will produce sourness and other off-flavors and odors. One variant, Lc. lactis subsp. lactis var. maltigenes, produces a malty flavor in fluid milk due to the production of 3-methylbutanal. Wild strains of lactococci may also contaminate cheeses and grow during manufacture and aging. Some of these strains produce fruity flavors due to the production of esters such as ethyl hexanoate and ethyl butyrate.
Propionibacterium spp. Pigmented strains of Propionibacterium cause pink spots in Swiss cheese. Split formation is influenced by Prop. freudenreichii subsp. shermanii strains used in making Swiss cheese.
Enterococcus spp. Enterococcus faecalis, Ec. faecium, and Ec. durans are part of the normal microflora of many cheeses and are sometimes used as starter cultures. Some strains produce undesirable flavors and high levels of amines during cheese ripening. Some enterococci are also considered probiotic. Two species, Ec. faecalis and Ec. faecium, were formerly classified as fecal streptococci (see Lactic Acid Bacteria: Enterococcus in Milk and Dairy Products).
flavor, depending on the cheese variety and strains involved.
Yeasts The presence of large numbers of yeasts on a cheese surface may result in a slimy rind, discolored appearance, and undesirable flavors. Non-lactose-fermenting Saccharomyces produce gas in fruit yogurt. Their source is usually a contaminated processing plant environment or unpasteurized fruits. Saccharomyces cerevisiae can spoil sweetened and acid dairy products. Kluyveromyces marxianus var. lactis, K. marxianus var. marxianus, Debaryomyces hansenii, and Yarrowia lipolytica spoil acidified refrigerated dairy products such as yogurt. Yarrowia lipolytica does not utilize lactose but metabolizes fat, protein, and organic acids, allowing it to spoil high-fat products such as butter and cream. It liberates amino and fatty acids and usually causes off-odors and softening of cheese curd. In addition, it produces melanin pigment from tyrosine and causes discoloration of cheese surfaces. Candida species are potential dairy spoilage organisms because of their ability to digest casein and fat, grow at storage temperatures, and ferment lactose and sucrose. They form surface slime on cream cheese. In yogurt, they cause yeasty and bitter flavors and a gassy texture. The important spoilage species in cheese is Can. famata and in yogurt Can. famata, Can. versatilis, and Can. lusitaniae.
Molds Mucor, Rhizopus, Penicillium, and Aspergillus grow at the yogurt–air interface of undisturbed packages. One mold spore can spoil a carton of yogurt by producing a visible colony. Molds also produce off-flavors in contaminated dairy products. Scopulariopsis brevicaulis causes odor defects in mold-ripened cheese. Sporendonema sebi grows on sweet condensed milk forming discrete colonies (mold buttons). Geotrichum grows on most dairy products, especially cheese and butter. Cheese varieties with low salt content are often spoiled by Mucor species.
Beneficial Microorganisms in the Dairy Industry Micrococcus spp. Some strains of Micrococcus species are able to survive pasteurization, but spoilage of heat-treated products is also caused by postprocessing contamination. Micrococcus species can produce swelling in UHT milk packs and have the ability to grow on some cheese varieties. Their growth may be either beneficial or detrimental to cheese
Selected lactic acid bacteria and other microorganisms are used by the dairy industry to produce fermented products such as yogurt and cheese. Commercially available starter cultures have been selected for a variety of desirable properties such as flavor production, lack of associated off-flavors, fast acid production, bacteriophage resistance, salt tolerance, exopolysaccharide production,
Microorganisms Associated with Milk
bacteriocin production, and heat sensitivity. These microbial preparations may consist of different species of lactic acid bacteria, propionibacteria, surface-ripening bacteria, yeasts, and molds.
Lactococcus spp. Lactococci are the major acid-producing microorganisms used for dairy fermentations, Lc. lactis being the main species of importance. Lactococcus lactis is homofermentative and weakly proteolytic. There are two subspecies: Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris. Citratepositive strains of Lc. lactis (Lc. lactis subsp. lactis biovar diacetylactis) utilize citrate to produce diacetyl, carbon dioxide, and other compounds in cheeses and cultured milks. Some strains are highly ropy.
Streptococcus spp. Streptococcus thermophilus is used to produce acid in dairy products subjected to high temperatures during fermentation (yogurt and Mozzarella cheese). It is weakly proteolytic and some strains produce extracellular polysaccharides.
Leuconostoc spp. Leuconostoc species are heterofermentative, Gram-positive cocci. They produce flavor compounds such as diacetyl and acetoin, and some strains produce exopolysaccharides. Leuconostoc mesenteroides subsp. cremoris is used in the production of cottage and cream cheese and cultured milks. Leuconostoc mesenteroides subsp. mesenteroides and Ln. mesenteroides subsp. dextranicum often produce slime (glucan, formerly known as dextran) from sucrose. Leuconostoc paramesenteroides does not produce glucan and is used in making brined cheese. Leuconostoc lactis is also found in milk and dairy products. Leuconostoc species reduce an undesirable ‘green’ flavor in cultured dairy products by converting acetaldehyde to diacetyl, but grow only slowly in milk.
Lactobacillus spp. Lactobacillus species are a genetically and physiologically diverse group of rod-shaped lactic acid bacteria. They are the most acid tolerant of the lactic acid bacteria and produce numerous proteolytic enzymes. There are three groups based on the type of fermentation end products: homofermentative (Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis, Lb. acidophilus, Lb. helveticus, and
455
Lb. helveticus subsp. jugurti), facultatively heterofermentative (Lb. casei, Lb. curvatus, and Lb. plantarum), and obligately heterofermentative (Lb. kefir, Lb. fermentum, and Lb. brevis). Some lactobacilli, such as Lb. acidophilus, are claimed to confer health benefits. Homofermentative lactobacilli are usually thermophilic and are used to produce fermented milk and some cheese varieties that require the use of high temperature during manufacturing. Lactobacillus casei and Lb. plantarum cannot grow at high temperatures (at 45 C). They are involved in the ripening of Cheddar and other cheese varieties.
Propionibacterium spp. Propionibacteria produce propionic and acetic acids and carbon dioxide from sugars and lactic acid. They are responsible for eye formation in Swiss cheese. Propionibacterium freudenreichii, Prop. jensenii, Prop. thoenii, and Prop. acidipropionici are the species commonly found in cheese.
Coryneform Bacteria Coryneform bacteria are a group of irregular, non-sporeforming, Gram-positive rods, often found growing on the surface of surface-ripened cheese. From a taxonomic viewpoint, dairy species differ from the reference pathogenic species. Cheese smear coryneform bacteria are mostly psychrotrophic and do not grow at 37 C, whereas pathogenic coryneform bacteria are facultative anaerobes and grow at 37 C. The cheese smear group includes Brevibacterium, Arthrobacter, Microbacterium, Aureobacterium, Brachybacterium, Rhodococcus, and Corynebacterium. These microorganisms are responsible for the viscous, red-orange surface (smear) on surface-ripened cheese varieties. They survive high salt concentrations and produce various proteolytic enzymes. Their presence in large numbers causes a slimy rind, discolored appearance, and undesirable flavors in cheese. Currently, the genus Brevibacterium is restricted to four species: Brev. linens, Brev. iodinum, Brev. casei, and Brev. epidermidis. Arthrobacter species include Ab. globiformis, Ab. nicotianae, Ab. protophormiae, Ab. agilis, Ab. sulphurous, and Ab. citreus. The genus Microbacterium contains Microb. lacticum, Microb. laevaniformans, Microb. imperiale, and Microb. arborescens. The genus Aureobacterium contains Au. liquefaciens and Au. testaceum. Brachybacterium alimentarium and Brach. tyrofermentans are two new species of coryneform bacteria isolated from the surface of Gruye`re and Beaufort cheeses and they tolerate up to 20% salt. Corynebacterium species found in dairy products include C. ammoniagenes and C. variabilis. Rhodococcus fascians was also isolated
456 Microorganisms Associated with Milk
from cheese. The habitats of coryneform bacteria are cheese and human skin (see Bacteria, Beneficial: Brevibacterium Linens, Brevibacterium Aurantiacum and Other Smear Microorganisms).
Bifidobacterium spp. Bifidobacterium species are inhabitants of the intestinal tract, which are added to dairy products for probiotic applications. The species most often used are Bif. longum, Bif. bifidum, and Bif. animalis. Bifidobacterium species grow poorly in milk (see Bacteria, Beneficial: Bifidobacterium spp.: Applications in Fermented Milks; Bifidobacterium spp.: Morphology and Physiology).
Pediococcus spp. Pediococcus species are salt-tolerant members of the family Streptococcaceae, which usually grow as tetrads of cells. Most species can grow at 4–6.5% NaCl. They produce acetic acid and small amounts of lactic acid when growing aerobically and mostly lactic acid when growing anaerobically. The most important species used in the dairy industry are Pc. acidilactici and Pc. pentosaceus (see Lactic Acid Bacteria: Pediococcus spp.).
Micrococcus spp. Micrococcus species are the predominant microorganisms found in raw milk drawn aseptically from the udder. They are the constituents of the natural microflora of teat skin and have also been isolated from soil, water, and dust. Based on phylogenetic and chemotaxonomic analysis, many micrococci (except Mc. luteus and Mc. lylae) have been renamed. The new names are Kocuria varians, Kc. roseus, Kc. kristinae, Dermacoccus nishinomiyaensis, Kytococcus sedentarius, Ab. agilis, and Nesterenkonia halobia. Micrococcus species have proteolytic, lipolytic, and esterase activities, which make them important in the ripening of various cheeses, especially those that are surface-ripened.
Molds Penicillium species are widely distributed in the environment. This genus includes four subgenera. The surface of soft white cheese can be colonized by P. camemberti Thom to produce many different cheese varieties such as Brie and Camembert. This mold produces a white layer of the mycelium on the cheese surface, while spores (candida) are embedded in the curd. The mold metabolizes some of lactic acid thereby raising the pH, which allows growth of
Brev. linens and other ripening microflora. In blue-veined cheese varieties, P. roqueforti (blue mold) is used in cheese ripening. Penicillium roqueforti has a relatively low oxygen requirement for growth (<4.2%) and tolerates 6–10% salt. Both P. camemberti and P. roqueforti produce lipolytic and proteolytic enzymes. They produce additional compounds that give cheese its distinctive flavor and aroma. Penicillium casei is similar to P. roqueforti and is found in Swiss cheese. Penicillium nalgiovense (Lava), another white mold, tolerates up to 8% salt and is used in some cheese varieties. Aspergillus niger and A. awamori produce lipase and contribute to the flavor of some cheese varieties. Geotrichum candidum is often found in raw milk, on milking and dairy processing equipment, and on the surface of ripened cheese. It can be used in starter cultures for Brie and Camembert cheese when combined with Penicillium species. Rhizomucor miehei and Rm. pusillus produce chymosin-like enzymes, which are used in cheese manufacture.
Yeasts Saccharomyces species are ascosporogenous yeasts with a capability for vigorous anaerobic or semianaerobic fermentation of sugar to produce ethanol and carbon dioxide. None of them utilizes lactose. Saccharomyces cerevisiae is the most important species and can be found on the surface of mold-ripened cheeses. Saccharomyces cerevisiae metabolizes hexoses, lactic acid, and other organic acids. The optimum pH for the growth of Saccharomyces species is 4.5–6.5. Oxygen is important to maintain viability but they survive under microaerophilic conditions. Kluyveromyces marxianus and K. lactis can ferment lactose and produce various hydrolytic enzymes. Kluyveromyces species are often isolated from Nordic fermented milks. They produce lactase, which can be used to reduce the lactose content of milk and assist in the digestion of milk by lactose maldigesters. Debaryomyces species are isolated from soil, water, and plants. Debaryomyces hansenii is involved in the ripening of mold-ripened soft cheeses. It tolerates high salt concentrations, utilizes lactic acid, produces protease and lipase, and grows well at low temperature. Candida kefyr is present in the microflora of the fermented milk kefir.
Microbial Population Dynamics in Milk Microbial populations in milk are continually changing and interacting with the milk components and with each other. The Pasteurized Milk Ordinance in
Microorganisms Associated with Milk
the United States requires grade A milk to have a total bacterial count of less than 100 000 cfu ml1. The initial microbial population of raw milk is affected by factors such as equipment and animal cleanliness, season, feed, and cow health. The initial microflora of good quality milk mainly consists of organisms found within the udder and the normal teat skin flora. These microorganisms include nonthermoduric micrococci and streptococci, which do not grow well at low temperatures. Pathogens will predominate in milk from mastitic cows. Seasonal factors include feed source and housing, both of which influence the level of bacterial spores in milk. Psychrotrophic bacteria (those that grow at low temperatures) are at low or even undetectable levels in the best quality fresh raw milk. They originate from the water and milking equipment. After collection, milk is cooled, which initiates a change in microbial population from Gram-positive rods and cocci, which do not grow, to Gram-negative rods, which can grow slowly at low temperatures. These Gram-negative rods are primarily Pseudomonas spp. and are capable of causing various off-flavors when populations exceed 106 cells ml1. When raw milk is not kept cold, lactic acid bacteria will grow. The resulting increase in acidity will inhibit the growth of many other microorganisms including sporeforming Bacillus and Clostridium species and Gram-negative Pseudomonas species and coliforms. Growth of lactic acid bacteria is rapid at 20 C and slows as the temperature decreases. Many strains of lactic acid bacteria produce bacteriocins, which inhibit other lactic acid bacteria as well as some pathogens such as Listeria and E. coli. Both the lactic acid bacteria and the psychrotrophic bacteria that grow in raw milk are heat sensitive, so when milk is pasteurized, microorganisms that have previously grown in the product are greatly reduced in number. The surviving microflora mainly consists of spore-forming bacteria, micrococci, and lactobacilli. Pasteurized milk that is not recontaminated with microorganisms will often keep at refrigeration temperature for 3 weeks or longer, because these surviving microorganisms grow slowly or not at all in the cold milk. Spoilage may occur more rapidly if significant numbers of psychrotrophic B. cereus are present in the raw milk. Most pasteurized milk is recontaminated with low numbers of psychrotrophic bacteria, resulting in a shorter shelf life. See also: Bacteria, Beneficial: Bifidobacterium spp.: Applications in Fermented Milks; Bifidobacterium spp.:
457
Morphology and Physiology; Brevibacterium linens, Brevibacterium Aurantiacum and Other Smear Microorganisms. Diseases of Dairy Animals: Infectious Diseases: Johne’s Disease; Infectious Diseases: Tuberculosis. Lactic Acid Bacteria: Enterococcus in Milk and Dairy Products; Pediococcus spp.. Liquid Milk Products: Pasteurization of Liquid Milk Products: Principles, Public Health Aspects. Pathogens in Milk: Bacillus cereus; Brucella spp.; Campylobacter spp.; Clostridium spp.; Coxiella burnetii; Enterobacteriaceae; Escherichia coli; Listeria monocytogenes; Salmonella spp.; Yersinia enterocolitica. Yeasts and Molds: Spoilage Molds in Dairy Products; Yeasts in Milk and Dairy Products.
Further Reading Boor KJ (1997) Pathogenic microorganisms of concern to the dairy industry. Dairy, Food and Environmental Sanitation 17: 714–717. Davies FL and Law BA (1984) Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk. London: Elsevier Applied Science. Doyle MP, Beuchat LR, and Montville TJ (2001) Food Microbiology: Fundamentals and Frontiers. Washington, DC: ASM Press. Durak MZ, Fromm HI, Huck JR, Zadoks RN, and Boor KJ (2006) Development of molecular typing methods for Bacillus ssp. and Paenibacillus ssp. isolated from fluid milk products. Journal of Food Science 71: 50–56. Frank JF and Hassan AN (1998) Starter cultures and their use. In: Marth EH and Steele JL (eds.) Applied Dairy Microbiology, pp. 131–172. New York: Marcel Dekker. Holt JG, Krieg NR, Sneath PH, Stanley JT, and Williams ST (eds.) (1994) Bergey’s Manual of Determinative Bacteriology, 2nd edn. Baltimore, MD: Williams & Wilkins. Hui YH (1993) Dairy Science and Technology Handbook. New York: VCH. IDF (1993) Indigenous antimicrobial agents of milk. Proceedings of the International Dairy Federation Seminar. Uppsala, Sweden, September. IDF (1994) The Significance of Pathogenic Microorganisms in Raw Milk. Brussels, Belgium: IDF. IDF (1996) Bacteriological quality of raw milk. Proceedings of the International Dairy Federation Symposium. Wolfpassing, Austria, March. Robinson RK (1990) Dairy Microbiology: The Microbiology of Milk, Vol. 1. London: Elsevier Applied Science. Robinson RK, Batt CA, and Patel PD (eds.) (2000) Encyclopedia of Food Microbiology. London: Academic Press. Smith DE and Vasavada PC (1988) Problems of pathogenic bacteria in the dairy industry. Journal of Dairy Science 71: 2807–2808. Stiles ME and Holzapfel WH (1997) Lactic acid bacteria of foods and their current taxonomy. International Journal of Food Microbiology 36: 1–29. Varnam AH and Sutherland JP (1994) Milk and Milk Products, Technology, Chemistry and Microbiology. London; New York: Chapman & Hall. Vasavada PC (1988) Pathogenic bacteria in milk: A review. Journal of Dairy Science 71: 2809–2816. Wiedmann M (2003) ADSA Foundation Scholar Award – an integrated science-based approach to dairy food safety: Listeria monocytogenes as a model system. Journal of Dairy Science 86: 1865–1875.