Chapter 5

Bacteriology of Cheese Milk

Summary Milk is a highly nutritious medium which can be easily contaminated

by microorganisms. In this chapter, the sources of microorganisms, particularly
bedding, faeces, the milking machine and milk storage tank are considered as is the
role of disease, particularly mastitis. The most important source of contamination is
the milking machine and bulk storage tank and the most important bacteria are psy-
chrotrophs, i.e., bacteria that can grow relatively rapidly at <7 °C. Pseudomonas
spp. are the dominant psychrotrophs. Pathogens in milk, the role of pasteurisation in
controlling them and the alternatives to pasteurisation, e.g., bactofugation and
microfiltration, are also considered. The production of cheese from raw milk and
standards for production of milk hygienically are also examined.

Keywords Contamination of milk • Mastistis • Psychrotrophs • Pathogens in raw

milk • Standards for raw milk

5.1 Introduction

Milk is highly nutritious, containing fat, protein and sugar, as well as significant
amounts of the minerals and vitamins required for the growth of calves and humans.
It is also an excellent medium for the growth of many bacteria. The temperature of
milk in the udder is ~38.5 °C, which is ideal for bacterial growth. However, growth
does not usually occur because milk in the udder is sterile, unless it becomes infected.
During milking, the milk becomes contaminated with microorganisms and will, if
maintained at a temperature >15 °C for several hours, coagulate due to growth and
acid production by adventitious bacteria like lactic acid bacteria (LAB) and coliforms.
Therefore, great care must be taken to ensure that milk is produced hygienically.

5.2 Sources of Microorganisms

The sources of microorganisms in milk are summarised in Fig. 5.1. Materials such
as air, bedding, faeces, feed residues, soil, water used to wash the cows’ teats and
water residues left on the milking equipment, mastitis, the teat surfaces and the

© Springer New York 2017 105

P.F. Fox et al., Fundamentals of Cheese Science,
DOI 10.1007/978-1-4899-7681-9_5
106 5 Bacteriology of Cheese Milk

Air

Bedding
Teats
Faeces

Feed residues
MILK
Soil Milking Machine
&
Mastitis
Water Bulk Tank

Increasing importance of contamination

Fig. 5.1 Sources of contamination of raw milk. The most important sources are milking machines
and bulk tanks

milking and bulk milk storage equipment are the main sources of contamination.
The first six of these are probably the least important on well-run farms; the most
important are the milking machine and bulk milk storage tank. Extremely dirty
udders may contaminate milk with up to 105 cfu/mL. Dirty udders are more likely
to occur in the winter, when cows are housed, than in summer, when cows are out
on pasture. Where cows are milked by hand, the milker sometimes wets his hands
with a few squirts of milk before starting to milk. Drops of contaminated milk drip-
ping from the hands of the milker into the milk bucket can be a potent source of
contamination in hand-milking operations.
Cows’ faeces contain high numbers of bacteria and, in the past, were considered
to be a major source of bacteria in milk. The cow lay on her own or another cow’s
faeces and so contaminated the udder and the teats. One of the reasons why teats are
washed before milking is to reduce contamination from any faeces that may be pres-
ent on the teats; another reason is to stimulate oxytocin production and milk let-­
down. It is also becoming more common for farmers to dip teats in disinfectants
before putting on the teat cups of the milking machine as a mastitis control measure.
A recent detailed study by Kagli et al. (2007) showed that contamination of milk by
coliforms and enterococci present in faeces did not occur, at least when milking
machines were used for milking. Numbers of coliforms in the faeces ranged from
20 to 4 × 106 cfu/g and were two orders of magnitude greater than those of
­enterococci which ranged from 10 to 32,000 cfu/g. The medium used to isolate the
enterococci was Kanamycin Aesculin Agar and the enterococci in the faeces were
identified mainly as Aerococcus viridans and small numbers of Ec. faecium, while
those in the milk were identified as a single clone of Ec. faecalis. All the coliforms
in the faeces were identified as Escherichia coli, while those in the milk were identi-
fied as Enterobacter amnigenus, Hafnia alvei, Serratia liquefaciens and Yersinia
enterocolitica. Thus, no enterococcus or coliform in the faeces was subsequently
recovered from the milk. In a similar study on another farm, all the enterocooci in
5.3 Mastitis and Other Diseases 107

the milk were identified as Ec. casseliflavus while those in the faeces were identified
mainly as Aerococcus viridians, with small numbers of Ec. hirae.
Hay rather than silage is fed in the winter-time to cows, the milk of which is used
to produce Comté, a French cheese, made from raw milk; silage is not fed because of
the danger of contaminating the milk with clostridial spores, which could germinate
and cause late gas formation during subsequent ripening of the cheese. Vacheyrou
et al. (2011) evaluated the contamination of milk in winter from air, dust from hay and
the teat surface as sources of contamination of milk in winter, on 16 farms producing
the raw milk cheese, Comté, and found that useful cheesemaking bacteria, e.g.,
­lactobacilli and propionic acid bacteria, were frequently identified on the teats and
milk used to make the cheese, and that most of the fungi and other bacteria were also
found in the stable and the milking parlour, indicating a large transfer of microorgan-
isms from the stable to the milking parlour and then to milk. Not all the microorgan-
isms were of environmental origin as 19 species present in the milk were not found in
any environmental sample. They concluded that contamination of the milk from the
stable environment was considerable even when a milking machine was used.
A recent study (Verdier-Metz et al. 2012) showed that the skin of cows’ teats was
a potent source of bacteria. Forty six different genera or species were identified
among the 309 isolates, including Leuconostoc mesenteroides, and several species
of staphylococci and actinobacteria found on the surface of smear-ripened cheeses,
implying that teat surfaces could be potent sources of these bacteria in raw milk
cheeses. In another study (Verdier-Metz et al. 2009), a link was found between
milking practices and bacterial diversity in raw milk. Group A milks were charac-
terised by a majority of corynebacteria and micrococci and a high level of milking
hygiene. Groups B and C milks had less intensive hygienic practices with Group B
milks being dominated by Gram-negative bacteria and the starter organism,
Lactococcus lactis and Group C milks by Leuc. mesenteroides, which can be pres-
ent in some mixed-starter cultures and Brevibacterium linens, which is a cheese
surface ripening organism (Chap. 11).
Monthly removal of hair surrounding the teats of dairy cows did not result in an
improvement in milk quality in a dairy herd over a 10 month period (Silk et al. 2003).

5.3 Mastitis and Other Diseases

Milk within the udder of healthy cows is sterile but small numbers of
­microorganisms can enter the teat canal of healthy animals from the outside of the
teat through the teat orifice and are generally washed out in the first squirts of milk.
These include streptococci, staphylococci, micrococci, corynebacteria and coliform
bacteria. Less than 200 cfu/mL are probably added to the milk from the teat canal
during milking.
Cows suffering from diseases like salmonellosis, tuberculosis (TB) or brucel-
losis may shed the causative bacteria, Salmonella enterica, Mycobacterium bovis
and Brucella abortus, in their milk, potentially causing disease, when the milk is
108 5 Bacteriology of Cheese Milk

consumed subsequently. Shedding into milk normally occurs only in extreme cases
of the diseases or when the udder is directly infected, which generally occurs late
in the course of the disease. Therefore, milk from cows suffering from these dis-
eases is not a major source of these bacteria in raw milk. Inhalation is the normal
route for transmission of TB among cows but infection by ingestion of contami-
nated drinking water also occurs in countries where TB in cows is endemic; how-
ever, a large infective dose is required. TB is endemic in badgers and this is a major
source of TB in cows in Ireland and the UK. M. bovis also causes TB in humans.
Mastitis, an inflammatory infection of the mammary gland, is common in dairy
cows; for a review, see Hillerton and Berry (2005). It can occur in sub-clinical (the
majority of outbreaks) and clinical forms and is caused mainly by Staphylococcus
aureus and Streptococcus agalactiae although Streptococcus pyogenes, Escherichia
coli and Corynebacterium bovis may also be responsible. S. aureus is a Gram-­
positive coccus and many strains produce heat-stable toxins, called enterotoxins,
which can cause food poisoning. Generally, growth to ~106 cfu/mL is necessary
before sufficient toxin is produced to cause food poisoning. In sub-clinical mastitis,
no physical change or abnormality is evident in the milk whereas in clinical masti-
tis, large clots consisting of a mixture of milk constituents, somatic cells and bacte-
ria are produced in the milk. Sub-clinical mastitis is generally manifested by
increased numbers of somatic cells in the milk and these are routinely checked for
in herd samples at factories. High numbers of somatic cells (~106/mL) reduce
cheese yield because they reduce the levels of fat and casein in the milk and increase
the losses of these components in the whey.
The number of mastitic bacteria shed in the milk depends on the stage of the
mastitis development and particularly on whether phagocytosis, i.e., engulfment of
the bacteria by polymorphonuclear leucocytes and macrophages, has occurred or
not. At the beginning of infection, before phagocytosis has occurred, several million
bacteria/mL of milk may be present but as phagocytosis develops the numbers of
bacteria added to the milk from the infected quarter will decrease rapidly to perhaps
<1000/mL; in other words, the number of bacteria in the milk will be high at the
beginning of infection and will decrease to low numbers as the infection progresses.
Milk from cows with sub-clinical mastitis may contain 1000–10,000 of the caus-
ative bacterium/mL.
The treatment of mastitis in dairy cows generally involves the use of antibiotics,
particularly penicillin or its various derivatives. This will result in contamination of
the milk with antibiotics and a consequent decrease in the ability of the starter cul-
tures to produce acid (Chap. 6). Therefore, milk from the treated quarter should be
withheld from the rest of the milk for the prescribed period of time.
Antibiotic treatment of mastitis is being challenged on the basis that it results in
the development of antibiotic-resistant microorganisms in man, which may limit the
use of antibiotics in treating human disease. Despite this, antibiotic treatment is still
the most effective method for treating mastitis in cows. Two important components
of treatment are the use of a long-acting antibiotic on all quarters of cows at the end
of their lactation to prevent new infection and the dipping of all teats of cows in an
effective disinfectant after every milking (Hillerton and Berry 2005).
5.4 Milking Machines and Bulk Tanks 109

5.4 Milking Machines and Bulk Tanks

The major source of contamination of raw milk is improperly cleaned milking equip-
ment. For this reason, considerable emphasis is placed on the satisfactory cleaning
of the milking machine, the rubber hoses and associated pipework, and the bulk
storage tank. The machine should be cleaned after each milking and the bulk ­storage
tank after it is emptied. Hot and cold detergent washes are used and generally a hot
acid rinse is given once a week to prevent the build-up of ‘milk scale’ which can
harbour bacteria and make the equipment difficult to clean. Milk scale is composed
mainly of calcium phosphate but sufficient nutrients may be present to allow signifi-
cant microbial growth between milkings, if the ambient temperature is high (>15 °C).
Heavily contaminated milking equipment is needed to cause a marked increase in
the bacterial content of the raw milk. For example, to increase the bacterial count in
1000 L of milk by 1 bacterium/mL requires 1 million organisms; so, to increase the
count by 10,000/mL would require the addition of 1010 bacteria. The milking
machine and its associated pipe lines and rubber hoses have a large surface area and
may contain such large numbers of bacteria, if they are not adequately cleaned.
Immediately after milking, good quality milk produced using a properly cleaned
milking machine and bulk storage tank should have a plate count of <2000 cfu/mL.
Gram-positive bacteria (e.g., Micrococcus, Corynebacterium, Microbacterium,
Lactobacillus, Lactococcus, Enterococcus, etc) and Gram-negative bacteria
(Pseudomonas, Achromobacter, Enterobacter, Escherichia, Flavobacterium, etc)
are found in milk immediately after milking. Many of the Gram-positive genera
include bacteria that are used as cheese starters. In the past, milk was either not
cooled at all or cooled to ambient temperature with water. Under these conditions,
growth of some of the Gram-positive bacteria, particularly LAB, e.g., Lactobacillus,
Lactococcus, Enterococcus and Streptococcus spp. was quite rapid. Nowadays,
milk is normally cooled to less <5 °C within 1–2 h of milking and the flora has
changed from one dominated by Gram-positive bacteria to one dominated by
Gram-­
­ negative, psychrotrophic bacteria, particularly Pseudomonas and
Achromobacter species. Psychrotrophic bacteria are those bacteria capable of
growth at <7 °C and are normally determined by incubating plates at 7 °C for 10
days. Psychrotrophic bacteria grow faster than Gram-positive bacteria at both 2 and
6 °C (Griffiths et al. 1987).
In modern milk production, the milk is held in the bulk tank at 4 °C for several
days. Although, cooling significantly slows down the rate of multiplication of bac-
teria in raw milk (Fig. 5.2), slow growth, particularly of psychrotrophs, still occurs
at 4 °C and significant numbers, e.g., 106 or 107 cfu/mL can be reached in 3 or 4
days. Raw milk may also be stored in silos for 1 or 2 days at the factory before use,
during which further growth of psychrotrophs will occur. It is important to remem-
ber that rapid cooling of milk is no substitute for improperly cleaned milking
machines and storage tanks, either on the farm or at the factory.
The growth of bacteria in four samples of raw milks during storage at 5 °C is
shown in Fig. 5.3. Little or no growth occurred during the first 2 days of storage,
after which two milks showed a significant increase in bacterial numbers while the
110 5 Bacteriology of Cheese Milk

10,000,000
20 C

1,000,000 15 C
10 C

100,000 5C
Cfu/ml

10,000

1,000

100
0 5 10 15
Time, h

Fig. 5.2 Effect of temperature on the growth of bacteria in a sample of raw milk

8
A
7 C
D
6 F
Log cfu/ml

3
0 1 2 3 4
Time, days

Fig. 5.3 Growth of bacteria in four samples of raw milk, coded A, C, D and F, incubated at 5 °C.
(Modified from Bramley & McKinnon, 1993)

other two did not (Bramley and McKinnon 1990). This was probably due to differ-
ences in the species of organisms present and their ability to grow at 5 °C. The ini-
tial level of contamination had little effect on the subsequent rates of bacterial
growth. After 4 days at 5 °C, counts in some cases were >1 × 107 cfu/mL. Bacterial
5.6 Pathogens in Raw Milk 111

counts of this magnitude are totally unacceptable in milk for cheesemaking or,
indeed, for making any dairy product. A linear correlation has been found between
the initial psychrotrophic count of raw milk and its shelf-life, defined as the number
of days to reach 108 cfu/mL (Griffiths et al. 1987).
Raw milk is often stored in large silos for perhaps 24 h or longer and, therefore,
further growth and contamination from improperly cleaned silos can occur so that
milk before pasteurisation for cheesemaking, may have a count in excess of 105 cfu/
mL. Such counts, while high, will not have a major effect on cheese quality.
However, counts >106 cfu/mL in the milk before pasteurisation could affect cheese
quality because many psychrotrophs, especially Pseudomonas spp., produce heat-­
stable lipases and proteinases, which withstand heating to 100 °C for 30 min, even
though the bacteria that produce these enzymes are killed. These enzymes may be
retained in the curd during cheesemaking and cause off-flavours to develop during
ripening, especially in semi-hard and hard varieties which are ripened for a long
time, e.g., Cheddar, Gouda, Comté, etc.

5.5 Natural Inhibitors

Raw milk contains several natural inhibitors of bacterial growth, e.g., lactoperoxi-
dase, immunoglobulins, lysozyme and lactoferrin but they have limited roles in pro-
longing the shelf-life of raw milk. Lactoperoxidase requires H2O2 and SCN− for
maximum inhibitory activity (see below) while the immunoglobulins act by aggluti-
nating bacteria. H2O2 can be produced by starter bacteria and SCN− arises from plants
that the cows eat. Lysozyme cleaves bonds in the peptidoglycan of bacterial cell
walls and is more active on Gram-positive than on Gram-negative bacteria because of
the high levels of peptidoglycans in the cell walls of Gram-positive bacteria.
Lactoferrin acts by chelating the Fe necessary for the growth of some bacteria.

5.6 Pathogens in Raw Milk

Cows suffering from severe forms of TB, brucellosis and salmonellosis may shed
the causative organisms, M. bovis, Brucella abortus and Salmonella enteric respec-
tively, in their milk and thus these organisms may get into cheese. It is difficult to get
information on the levels of M. bovis and B. abortus in milk. This may be due to the
difficulty in isolating them, e.g., 6 weeks incubation is needed to enumerate M. bovis
because it grows so slowly. Another possibility is that milk is no longer a source of
these organisms due to the almost total eradication of these two diseases in dairy
cows in developed countries. However, an outbreak of salmonellosis in cheese in
Canada was traced to a single cow which was shedding 200 cfu salmonella/mL in
her milk (D’Aoust et al. 1985). A cow has also been shown to shed 280 L. monocy-
togenes/ml of milk from one quarter without any signs of clinical disease in the cow
or abnormality in the milk (Hunt et al. 2012). In addition, mastitis caused by
112 5 Bacteriology of Cheese Milk

S. aureus can result in the presence of this organism in the raw milk. Cows’ faeces
can contain large numbers of salmonella and E. coli but, as already stated, the likeli-
hood of these organisms gaining entry to milk is very low in modern milking
practice.
Analysis of US records by Headrick et al. (1998) for the period 1973–1992
showed that there were 46 outbreaks of foodborne disease associated with the con-
sumption of raw milk, which were caused mainly by Campylobacter and Salmonella
spp. Outbreaks of foodborne disease, due to milk and milk products, over periods of
5–10 years, in the USA, Finland, The Netherlands, England and Wales, Germany,
Poland and France were low, and ranged from 2.2 % in the USA to 6.1 % of all
foodborne outbreaks in France (De Buyser et al. 2001).
E. coli 0157 and Listeria monocytogenes have become important causes of
­foodborne illness in recent years and both can occur in raw milk. E. coli 0157 causes
bloody diarrhoea, haemolytic uremic syndrome and kidney failure while L. mono-
cytogenes causes listeriosis in pregnant women and immune-compromised people;
both organisms can cause death. Listeriosis may lead to meningitis and bacteremia.
Oliver et al. (2005) have summarized surveys of the incidence of Campylobacter
jejuni, enteropathogenic E. coli, L. monocytogenes and Salmonella spp. in bulk tank
milk. The results (Table 5.1) show that significant variation occurs for each of the
four pathogens examined. There have been three recent surveys of raw milk in the
US, two of which involved milk samples from artisanal, raw milk, cheese producers
and the third, a nation-wide survey. Each showed that the raw milk was of high
microbiological quality. The first one (D’Amico et al. 2008) comprised milk from
11 artisanal cheese producers (5 from cow, 4 from goat and 2 from sheep milk)
which were sampled weekly from June to September, 2006. S. aureus was found in
46 of the 133 samples (average level 250 cfu/mL), L. monocytogenes in three sam-
ples (two of which were from the same farm), E. coli 0157:H7 in one sample and
salmonella in no sample; 61 % of samples contained <10 coliforms/mL. The second
survey (D’Amico and Donnelly 2010) comprised milk samples from 21 artisanal
cheese operations (12 from cow, 5 from goat and 4 from sheep milk) which were
sampled weekly from July to September, 2008. No L. monocytogenes, E. coli
O157:H7 or salmonella were recovered from any sample. Fourteen of the farm
milks contained an average of 20 cfu of S. aureus/mL. Eighty-six % of samples had
plate counts <10,000/mL and 42 % <1000/mL. The third survey (Jackson et al.
2012) was US wide and comprised 214 silo milk samples. Several different methods
were used for each organism. By direct plating, E. coli O157:H7 was found in 1.1 %
of samples, Salmonella in 13.6 % of samples, and L. monocytogenes in 12.5 % of

Table 5.1 Isolation rates of various pathogens in bulk tank milks1

Pathogen No. of surveys Range of isolation rates (%)
C. jejuni 7 0.4–12.3
Verotoxigenic E. coli 3 0.8–3.8
L. monocytogenes 13 1.0–12.6
Salmonella 8 0.2–8.9
1
From Oliver et al. (2005)
5.8 Pasteurisation 113

samples. In the case of salmonella, there were significant differences between the
four media used to detect them. Total counts ranged from 7.3 × 102 to >4.7 × 103 cfu/
mL, coliform, E. coli counts from 10 to 4.9 × 104/mL and S. aureus counts from < 10
to 1.5 × 104/mL. The average log-transformed counts of total viable bacteria were
slightly lower in samples containing no pathogens.
An organism gaining increasing importance in raw milk is M. avium subsp. para-
tuberculosis (MAP). It causes Johne’s disease in cattle, which is characterised by
reduced milk yield and weight loss in cows and has been associated with Crohn’s
disease, which is a chronic inflammation of the bowel in humans. Levels reported in
milk are low and whether it is inactivated by pasteurisation is not clear. Quantifying
the numbers of MAP in milk samples is not possible because very few articles pro-
vide quantitative information.

5.7 Raw Milk Cheeses

Many cheeses, including such famous varieties as Emmental, Gruyère, Comté,

Parmigiano Reggiano, Reblochon and Roquefort are produced from raw milk. One
of the important safety factors in these cheeses is that most of them (Reblochon and
Roquefort are exceptions) are cooked to a high temperature (>50 °C) for up to 1 h
during manufacture which kills many of the bacteria that may be present in the raw
milk. Many countries require that cheese be (1) made from pasteurized milk or (2)
aged for 60 days, during which food-poisoning or pathogenic bacteria die, or (3) the
cheese itself be pasteurized, i.e., converted to processed cheese. The production of
cheese from raw milk has been questioned because pathogens which might be pres-
ent in the milk will also grow during manufacture. This is still a contentious issue
but most pathogens will die off during ripening or will be inactivated by high cook-
ing temperatures. Donnelly (2001) critically examined food-poisoning outbreaks
due to raw milk cheeses and concluded that confounding parameters other than the
use of raw milk contributed to the presence of bacterial pathogens in the majority of
the outbreaks. These aspects are discussed more fully in Chap. 19.

5.8 Pasteurisation

Pasteurisation of cheese milk became widespread about 1940, primarily for public
health reasons but also to provide a milk supply of more uniform bacteriological
quality and improve its keeping quality. Prior to 1950, TB was prevalent in both
cows and humans and the organism causing tuberculosis in cows, M. bovis, can also
cause the disease in man. M. tuberculosis is still the major cause of TB in man. The
minimum time/temperature combination required to kill M. tuberculosis, the most
heat-resistant bacterial pathogen likely to be present in milk at that time, was the
primary driving force for the development of pasteurisation. Batch pasteurisation
(low temperature-long time, LTLT; 63–65 °C × 30 min) was used initially but was
114 5 Bacteriology of Cheese Milk

replaced by continuous, high temperature-short time (HTST) pasteurisation

(72 °C × 15 s) when plate heat exchangers were introduced in the 1930s. For a
review of the history of pasteurisation see Westhoff (1978).
Most (>99.9 %) of the bacteria found in raw milk are heat labile and are killed by
pasteurisation at 72 °C for 15 s and most milk for cheesemaking is given this heat
treatment. Pasteurisation kills all potential pathogens which are likely to be present in
milk but spores of Clostridium and Bacillus are not killed by this treatment. Whether
vegetative cells of sporeformers are inactivated by pasteurisation is not clear. Recently,
Pearce et al (2012) undertook a thorough study of the heat resistance of several patho-
gens. Approximately 30 strains each of E. coli, L. monocytogenes, S. aureus, Yersinia
enterocolitica, Cronobacter sakazakii (formerly Enterobacter sakazakii) and various
Salmonella species were screened initially and then the most heat-resistant strain of
each species was selected for detailed study in a pilot-scale pasteuriser. The mean
log10 reductions and temperatures of inactivation of the 6 pathogens during a 15 sec
treatment in UHT milk were: >6.7 at 66.5 °C for S. aureus, >6.8 at 62.5 °C for
Y. enterocolitica, >6.8 at 65 °C for E. coli, >6.7 at 67.5 °C for C. sakazakii, >6.9 at
66.5 °C for L. monocytogenes and >6.9 at 61.5 °C for Salmonella serotype
Typhimurium. This data implies that pasteurisation of milk for 15 sec at 72 °C has a
significant safety margin in inactivating pathogens in raw milk.
Micrococcus, Microbacterium, Enterococcus, Arthrobacter and Lactobacillus
spp., as well as Bacillus and Clostridium spores, which withstand HTST pasteurisa-
tion, are found in raw milk. These are called thermoduric bacteria and invariably
come from improperly cleaned equipment (Hull et al. 1992). Generally, thermoduric
bacteria grow only slowly, if at all, in raw milk so that counts of thermoduric bacteria,
even within 24 h of milking are a useful indicator of how well the milking equipment
and bulk storage tanks have been cleaned. These bacteria are enumerated by heat-
treating the milk at 63 °C for 30 min before plating on Plate Count Agar PCA).
Thermophilic starters (see Chap. 6) can also be considered to be thermoduric but their
incidence in raw milk is low and they grow poorly on Plate Count Agar, the medium
used to estimate the bacteriological quality of milk. In cheese factories, Streptococcus
thermophilus, a starter bacterium for many cheeses, has been shown to grow as a
biofilm in the regeneration section of the pasteuriser during long pasteurization runs.
Pasteurisation also inactivates several enzymes in milk, including lipase and
alkaline phosphatase. Lack of alkaline phosphatase activity in pasteurised milk is
used as an index that raw milk has been properly pasteurised while milk lipase
activity causes rancidity development in raw milk due to the production of free fatty
acids, particularly butyric, when the milk is excessively agitated. Whether lipase
activity plays a role in rancidity development in raw milk cheeses is not clear.
In some countries, e.g. Canada, significant amounts of cheese are made from milk
heat-treated to time/temperatures lower than those of HTST pasteurisation. This heat
treatment is called thermisation and generally involves heating the milk to 63 °C for
10–15 s. This treatment results in less inactivation of enzymes and non-­starter lactic
acid bacteria, which may be important in developing cheese flavour. It also kills only
some pathogenic and food-poisoning microorganisms and the milk must be subse-
quently fully pasteurized to meet public health regulations. The purpose of thermiza-
tion is to kill psychrotrophs, which dominate the microflora of refrigerated milk, and
5.9 Alternatives to Heat Treatment 115

which excrete potent proteinases and lipases, which may cause flavour and textural
defects in cheese. Such treatments are also used in Europe to partially inactivate the
microflora as the raw milk is delivered to the factory, and increase the keeping quality
of the raw milk; in this case, the milk is usually pasteurised before cheesemaking.
While pasteurisation reduces the risk of producing poor quality cheese due to the
growth of undesirable bacteria and destroys food-poisoning microorganisms, pas-
teurisation of cheese milk may damage the cheesemaking properties of milk if the
heat treatment is too severe (due to heat denaturation of the whey proteins and their
interaction with κ-casein; see Chap. 7). The extent of this damage is negligible
under HTST pasteurisation conditions; some manufacturers heat-treat the cheese
milk at sub-HTST conditions. The flavour of cheese made from pasteurised milk
develops more slowly and is less intense than that made from raw milk, apparently
because certain components of the microflora of raw milk contribute positively to
cheese flavour. To overcome this deficiency, adjunct cultures of selected NSLAB
are being recommended for use in the manufacture of long-ripened, low-moisture
cheese made from pasteurised milk (see Chap. 11).

5.9 Alternatives to Heat Treatment

There are at least four alternatives to heat treatment which reduce the numbers of bac-
teria in cheesemilk: (1) treatment with hydrogen peroxide (H2O2), (2) activation of the
lactoperoxidase-H2O2-thiocyanate system, (3) bactofugation and (4) microfiltration.

5.9.1 Treatment with Hydrogen Peroxide

Hydrogen peroxide (H2O2) is an effective bactericidal agent, the use of which is per-
mitted in cheesemaking in some countries, including the USA. The excess H2O2 is
usually destroyed by adding catalase. The use of H2O2 to treat cheese milk has received
little attention recently and its use in developed dairying countries is very limited.

5.9.2 Lactoperoxidase-H2O2-Thiocyanate

Lactoperoxidase (LPO), an indigenous enzyme in milk, reduces H2O2 in the pres-

ence of a suitable reducing agent:

H 2 O2 + 2HA ® 2H 2 O + 2 A

One such reducing agent is the thiocyanate anion, SCN−, which is oxidized to various
short-lived but highly active inhibitory ions, particularly hypothiocyanite, OSCN−,
which are strongly bactericidal. The level of indigenous SCN− in milk is low and
varies significantly (0.02–0.25 mM) and 8–25 mM is needed for optimum activity.
116 5 Bacteriology of Cheese Milk

SCN− arises from the catabolism of glucosinolates, which are present in plants of the
Cruciferae or cabbage family, by bacteria in the rumen. Milk contains no H2O2 which
must either be deliberately added or produced in situ via oxidation of glucose by
glucose oxidase, from xanthine by xanthine oxidase or by starter cultures grown in
the presence of O2. For the full inhibitory effect about 0.3 μM H2O2 is required.
The LPO system is very effective for the “cold-pasteurization” of milk. The pro-
cess has been patented but has attracted very limited, if any, interest in developed
dairy countries, possibly for economic reasons. It is practiced to a small extent in
developing countries.

5.9.3 Bactofugation

A high percentage (98–99 %) of somatic and bacterial cells and bacterial spores in
milk can be removed by centrifugation at high gravitational forces in a b­ actofuge.
The cells and spores are heavier than the milk constituents and are concentrated in
the sludge during bactofugation. The sludge may be sterilised subsequently to kill
the spores and bacteria and added back to the milk to increase cheese yield.
Bactofugation is not widely used in general cheesemaking but is commonly used to
remove Clostridium tyrobutyricum from milk intended for Dutch- and Swiss-type
cheeses which undergo a propionic acid fermentation (PAF). Cheeses which
undergo a PAF are generally ripened at <13 °C for a few weeks after which the
temperature is increased to ~22 °C for 3–4 weeks to promote the PAF. Clostridia,
especially Cl. tyrobutyricum, can also grow under these circumstances and produce
late gas (see Chap. 11) but bactofugation is very effective in eliminating spores of
Cl. tyrobutyricum from the milk.
Cl. tyrobutyricum is an obligate anaerobe, which can ferment lactate, the major
acid in cheese, and the anaerobic cheese environment is ideal for its growth. The
major source of the organism in raw milk is improperly fermented silage. For this
reason, feeding of silage to cows is prohibited in the areas of Switzerland where
Emmental cheese is made. Contamination with spores is much greater in the winter-
time, when the cows are fed indoors with silage, than in the summertime when they
are out on pasture. The vegetative cells are probably killed by pasteurisation (though
scientific proof of this appears to be lacking), but the spores are heat resistant,
requiring several minutes at 100 °C to kill them.
When silage contaminated with clostridia is eaten by cows, the spores pass
through their gastrointestinal tract. Cows lying in their own or other cows dung,
pick up faecal material containing clostridial spores on their teats and udders from
which contamination of the milk with clostridia occurs. Proper cleaning of the teats
and udders will reduce contamination from this source. Less than 10 spores/100 mL
of milk is sufficient to cause late gas in Dutch-type cheeses. A lower number is
required in cheeses which undergo a PAF because of the higher temperature used in
ripening these cheeses.
The design of the bactofuge is essentially similar to that of separators used to sepa-
rate the fat from milk but is modified such that only the bacteria and spores, which are
more dense than skim milk, are forced outwards and move down along the lower side
5.9 Alternatives to Heat Treatment 117

of the upper of a pair of discs and eventually through orifices in the bowl of the cen-
trifuge as a bacterial concentrate (bactofugate) representing ~3 % of feed volume.
Some large, dense, casein micelles are also removed by this process, perhaps as much
as 6 % of the total casein. This loss of casein will cause a decrease in cheese yield
which may be avoided by heat-sterilizing the bactofugate and returning it to the milk
or by otherwise supplementing the casein content, e.g., by adding UF retentate.

5.9.4 Microfiltration

Microfiltration is a membrane separation process, in principle like reverse osmosis,

nanofiltration or ultrafiltration, except that large pore size (0.8–1.4 μm) membranes
are used. The semi-permeable membranes used retain bacteria but allow milk con-
stituents, including most of the casein micelles, to pass through into the permeate.
The process can be applied only to skim milk as the fat globules in whole milk block
the pores of the membrane and reduce its efficiency. Therefore, the cream must be
separated from the milk, pasteurised and added back to the microfiltered skim milk
before cheesemaking.
Microfiltration is very efficient at removing bacterial cells (>99 %) and is being
used increasingly in the dairy industry, e.g., in the production of extra-long life pas-
teurized milk. It is not yet widely used for cheese milk, except for the removal of
spores from milk for Swiss and similar cheeses. The technique has been very useful
in studying the effect of the indigenous raw milk microflora and of enzymes
­inactivated by pasteurisation on cheese flavour. The quality of Cheddar and Comté
cheeses made from microfiltered milk is similar to that made from pasteurized milk
and different from that made from raw milk which indicates that the differences in
flavour between raw and pasteurized milk cheeses are due to the indigenous microor-
ganisms which are efficiently removed by microfiltration or killed by pasteurization
rather than to the inactivation of indigenous enzymes or other heat-induced change.
A microfiltration system known as the “Bactocatch System” has been developed
by the Alfa Laval Company for the decontamination of milk as an alternative to
pasteurization.

5.9.5 Pre-Maturation

In some countries, particularly France, the starter may be added to the raw milk
which is then incubated at 8–10 °C for 12–15 h (overnight). This process is called
pre-maturation. The rationale is that sufficient growth of the starter (and a decrease
in pH of 0.1 units) occurs during the overnight incubation to bring it out of the lag
phase of growth so that exponential growth and acid production begin as soon as the
temperature is brought up to the renneting temperature (~30 °C). Pre-maturation is
believed to suppress the growth of psychrotrophs. Problem-causing bacteria may also
grow during pre-maturation so, sometimes, the milk is repasteurised before more starter
is added to inactivate them and avoid subsequent potential defects in the cheese.
118 5 Bacteriology of Cheese Milk

5.10 Standards for Raw Milk

Microbiological standards for raw milk have evolved over the years as scientific
research has shown what can be accomplished by relatively simple procedures. The
European Union (EU) operates an integrated approach to food safety through assuring
a high level of animal health, animal welfare and food safety through coherent farm-to-
table measures and adequate monitoring. This ensures a high level of consumer protec-
tion with regard to food safety. Three recent documents (Anon 2004a, b, 2005) have
consolidated various EU regulations on the general hygiene of food, specific rules on
the hygiene of food and microbiological criteria for food. These lay down the respon-
sibilities of the primary producer and the processor to ensure the safety of the food.
Regarding raw milk, the regulation (Anon 2004b) covers the health requirements
for milk production, hygiene in milk production units, and the microbiological stan-
dards and temperature to which raw milk should be cooled. According to the regula-
tion, raw milk should have a plate count of <100,000 cfu/mL and a somatic cell
count of <400,000/mL when received at the manufacturing plant and should be
cooled quickly to not more than 6 °C until it is processed (Anon 2004b). Raw milk
can be kept at a higher temperature if processing begins immediately or within 4 h
of acceptance at the processing establishment. Testing to ensure compliance is con-
ducted once or twice a month. These criteria are very easy to meet in practice and
to-day it is easy to produce milk with <2000 cfu/mL, whereas 40 years ago it was
difficult to produce it with <500,000 cfu/mL. These improvements in the microbial
quality of raw milk are due to better hygiene during milking, improved design of
milking equipment making it easier to clean, and cooling to and storage of milk at
6 °C within a few hours of production in easily cleaned, stainless steel, bulk storage
tanks until it is collected and transported to the factory.
Traditionally culture-dependent methods based on plating decimal dilutions of
raw milk on suitable non-selective or selective media have been used to determine
the levels of microbial contamination of raw milk with microorganisms. More
recently, DNA based technologies including culture-dependent methods, e.g.,
Randomly Amplified Polymorphic DNA (RAPD), and Restriction Fragment Length
polymorphism (RFLP), and culture-independent methods, e.g., Real Time PCR,
Single Stranded Conformation Polymorphism (SSCP), Denaturing gradient Gel
Electrophoresis (DGGE) and Fluorescence in situ Hybridisation (FISH) and high-
throughput DNA sequencing, have been used (for reviews see Quigley et al. 2011
and 2013). Such methods have confirmed that psychrotrophs are the important con-
taminants in raw milk but they have also identified bacteria which are usually not
associated with milk, e.g., Bacteroides, Faecalibacterium, Prevotella and
Catenibacterium species. Many of these methods do not distinguish between live
and dead cells but this disadvantage can be overcome by treating the sample with
stains, e.g., ethidium bromide or propidium monoazide, which are able to penetrate
dead cells and interact with their DNA so that the DNA is not amenable to amplifi-
cation by the Polymerase Chain Reaction.
A French survey (Desmasures et al. 1997) of milks in Normandy, around the area
where raw milk Camembert cheese is made, showed that the microbiological quality
Suggested Reading 119

Table 5.2 Microbiological quality (cfu/mL) of milk produced in Normandy, France1

Winter Spring/Summer
n n
Total count 39 71,000 ± 27,000 30 86000 ± 21,000
Enterococci 37 74 ± 150 25 79 ± 400
Coliform 29 57 ± 2400 19 77 ± 5000
S. aureus 25 450 ± 1700 18 350 ± 280
L. monocytogenes 39 4 positive samples 30 No positive samples
Salmonella 39 1 positive sample 30 1 positive sample
Y. entercoitica2 39 19 positive samples 30 6 positive samples
Campylobacter 39 1 positive sample 30 No positive sample
1
From Desmasures et al. (1997)
2
Only 1 of 61 isolates was a potential pathogen

of the milk was generally very good; 69 milks were sampled and 83 % of them had
a total bacterial count <20,000 cfu/mL and an average somatic cell count of 176,000/
mL (Table 5.2). The average numbers of coliforms, enterococci and S. aureus were
77, 79 and 350/mL, respectively, in milk produced in summer and 57, 74 and 450/
mL in milk produced in winter, respectively. However, the incidence of Yersinia
entecolitica was relatively high and 14 of the 43 milks examined did not meet the EU
criteria for S. aureus in milk (<500/mL) destined for cheesemaking from raw milk.
Despite this, these data suggest that hygiene and milk cooling were effective.
Application of Hazard Analysis and Critical Control Points (HAACP) can be
applied to cheese production units but is not generally feasible at milk production
level (Anon 2004a). Nevertheless, guides to good practice should be developed to
encourage the use of appropriate hygiene practices at farm level. Included in these
should be details of how udders and cows’ teats should be cleaned before milking and
how milking machines and bulk storage tanks should be cleaned and disinfected.

Suggested Reading

De Buyser ML, Dufour B, Maire M et al (2001) Implication of milk and milk products in food-
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Donnelly CW (2001) Factors associated with hygienic control and quality of cheeses prepared
from raw milk: a review. Bulletin 369. International Dairy Federation, Brussels, pp 16–27
Hillerton JE, Berry EA (2005) Treating mastitis in the cow – a tradition or an archaism. J Appl
Microbiol 98:1250–1255
Oliver SP, Jayarao BM, Almeida RA (2005) Foodborne pathogens in milk and the dairy farm envi-
ronment: food safety and public health implications. Foodborne Pathog Dis 2:115–129
Vacheyrou M, Normand AC, Guyot P et al (2011) Cultivable microbial communities in raw cow
milk and potential transfers from stables of sixteen French farms. Int J Food Microbiol
148:253–262
Verdier-Metz I, Gagne G, Bornes S et al (2012) Cow teat skin, a potential source of diverse popula-
tions for cheese production. Appl Environ Microbiol 78:326–333
120 5 Bacteriology of Cheese Milk

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