Chapter 7

Milk, Fermentation, and Fermented

and Nonfermented Dairy Products

Although this chapter is devoted to milk and other dairy products, it begins with a discussion of
fermentation because of the importance of this process to dairy products.

FERMENTATION

Background

Numerous food products owe their production and characteristics to the fermentative activities of
microorganisms. Many foods such as ripened cheeses, pickles, sauerkraut, and fermented sausages are
preserved products in that their shelf life is extended considerably over that of the raw materials from
which they are made. In addition to being made more shelf stable, all fermented foods have aroma
and flavor characteristics that result directly or indirectly from the fermenting organisms. In some
instances, the vitamin content of the fermented food is increased along with an increased digestibility
of the raw materials. The fermentation process reduces the toxicity of some foods (for example, gari
and peujeum), whereas others may become extremely toxic during fermentation (as in the case of
bongkrek). From all indications, no other single group or category of foods or food products is as
important as these are and have been relative to nutritional well-being throughout the world.
The microbial ecology of food and related fermentations has been studied for many years in the
case of ripened cheeses, sauerkraut, wines, and so on, and the activities of the fermenting organisms
are dependent on the intrinsic and extrinsic parameters of growth discussed in Chapter 3. For example,
when the natural raw materials are acidic and contain free sugars, yeasts grow readily, and the alcohol
they produce restricts the activities of most other naturally contaminating organisms. If, on the other
hand, the acidity of a plant product permits good bacterial growth and at the same time the product is
high in simple sugars, lactic acid bacteria may be expected to grow, and the addition of low levels of
NaCl will ensure their growth preferential to yeasts (as in sauerkraut fermentation).
Products that contain polysaccharides but no significant levels of simple sugars are normally stable
to the activities of yeasts and lactic acid bacteria due to the lack of amylase in most of these organisms.
To effect fermentation, an exogenous source of saccharifying enzymes must be supplied. The use of
barley malt in the brewing and distilling industries is an example of this. The fermentation of sugars

149
150 Modern Food Microbiology

to ethanol that results from malting is then carried out by yeasts. The use of koji in the fermentation
of soybean products is another example of the way in which alcoholic and lactic acid fermentations
may be carried out on products that have low levels of sugars but high levels of starches and proteins.
Whereas the saccharifying enzymes of barley malt arise from germinating barley, the enzymes of koji
are produced by Aspergillus oryzae growing on soaked or steamed rice or other cereals (the commercial
product takadiastase is prepared by growing A. oryzae on wheat bran). The koji hydrolysates may be
fermented by lactic acid bacteria and yeasts, as is the case for soy sauce, or the koji enzymes may act
directly on soybeans in the production of products such as Japanese miso.

Defined and Characterized

The word fermentation has had many shades of meaning in the past. According to one dictionary
definition, it is “a process of chemical change with effervescence . . . a state of agitation or unrest . . . any
of various transformations of organic substances.” The word came into use before Pasteur’s studies on
wines. Prescott and Dunn56 and Doelle12 have discussed the history of the concept of fermentation,
and the former authors note that in the broad sense in which the term is commonly used, it is “a process
in which chemical changes are brought about in an organic substrate through the action of enzymes
elaborated by microorganisms.” It is in this broad context that the term is used in this chapter. In the
brewing industry, a top fermentation refers to the use of a yeast strain that carries out its activity at the
upper parts of a large vat, such as in the production of ale; a bottom fermentation requires the use of
a yeast strain that will act in lower parts of the vat, such as in the production of lager beer.
Biochemically, fermentation is the metabolic process in which carbohydrates and related compounds
are partially oxidized with the release of energy in the absence of any external electron acceptors.
The final electron acceptors are organic compounds produced directly from the breakdown of the
carbohydrates. Consequently, incomplete oxidation of the parent compound occurs, and only a small
amount of energy is released during the process. The products of fermentation consist of some organic
compounds that are more reduced than others.

The Lactic Acid Bacteria

This group is composed of 13 genera of Gram-positive bacteria at this time:

Carnobacterium Oenococcus
Enterococcus Pediococcus
Lactococcus Paralactobacillus
Lactobacillus Streptococcus
Lactosphaera Tetragenococcus
Leuconostoc Vagococcus
Weissella

With the enterococci and lactococci having been removed from the genus Streptococcus, the member
of this genus of most importance in foods is S. salivarius subsp. thermophilus. S. diacetilactis has
been reclassified as a citrate-utilizing strain of Lactococcus lactis subsp. lactis.
Related to the lactic acid bacteria but not considered to fit the group are genera such as Aerococcus,
Microbacterium, and Propionibacterium, among others. The last genus has been reduced by the transfer
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 151

of some of its species to the new genus Propioniferax, which produces propionic acid as its principal
carboxylic acid from glucose.80
The history of our knowledge of the lactic streptococci and their ecology has been reviewed by
Sandine et al.63 These authors believe that plant matter is the natural habitat of this group, but they
note the lack of proof of a plant origin for Lactococcus cremoris. It has been suggested that plant
streptococci may be the ancestral pool from which other species and strains developed.47
Although the lactic acid group is loosely defined with no precise boundaries, all members share
the property of producing lactic acid from hexoses. As fermenting organisms, they lack functional
heme-linked electron transport systems or cytochromes, and they obtain their energy by substrate-level
phosphorylation while oxidizing carbohydrates; they do not have a functional Krebs cycle.
Kluyver divided the lactic acid bacteria into two groups based on end products of glucose
metabolism. Those that produce lactic acid as the major or sole product of glucose fermentation
are designated homofermentative (Figure 7–1(A)). The homolactics are able to extract about twice as
much energy from a given quantity of glucose as are the heterolactics. The homofermentative pat-
tern is observed when glucose is metabolized but not necessarily when pentoses are metabolized, for
some homolactics produce acetic and lactic acids when utilizing pentoses. Also the homofermentative

Figure 7–1 Generalized pathways for the production of some fermentation products from glucose by various
organisms. (A) Homofermentative lactics; (B) heterofermentative lactics; (C) and (D) Propionibacterium (see
Figure 7–3); (E) Saccharomyces spp.; (F) Acetobacter spp.; and (G) Acetobacter “overoxidizers.”
152 Modern Food Microbiology

Table 7–1 Some of the Homo- and Heterofermentative Lactic

Acid Bacteria

Homofermentative Heterofermentative
Lactobacillus Lactobacillus
L. acetotolerans L. brevis
L. acidipiscis L. buchneri
L. acidophilus L. cellobiosus
L. alimentarius L. coprophilus
L. casei L. fermentum
L. hilgardii
L. coryniformis L. sanfranciscensis
L. curvatus L. trichoides
subsp. curvatus L. pontis
subsp. melibiosus L. fructivorans
L. delbrueckii L. kimchii
subsp. bulgaricus L. paralimentarius
subsp. delbrueckii L. panis
subsp.lactis L. sakei
L. fuchuensis subsp.sakei
L. helveticus subsp.carnosus
L. jugurti Leuconostoc
L. jensenii L. argentinum
L. kefiranofaciens L. citreus
subsp. kefiranofaciens L. fallax
subsp. kefirgranum L. carnosum
L. leichmannii L. gelidum
L. mindensis L. inhae
L. plantarum L. kimchii
L. salivarius L. lactis
Lactococcus L. mesenteroides
L. lactis subsp. cremoris
subsp. lactis subsp. dextranicum
subsp. cremoris subsp. mesenteroides
subsp. diacetylactis Carnobacterium
subsp. hordniae C. divergens
L. garvieae C. gallinarum
L. plantarum C. mobile
L. raffinolactis C. piscicola
Paralactobacillus C. viridans
P. selangorensis Oenococcus
Pediococcus O. oeni
P. acidilactici Weissella
P. claussenii W. cibaria
P. pentosaceus W. confusa
P. damnosus W. hellenica
P. dextrinicus W. halotolerans
P. inopinatus W. kandleri
P. parvulus W. kimchii
(continued)
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 153

Table 7–1 (continued)

Streptococcus W. minor
S. bovis W. thialandensis
S. salivarius W. paramesenteroides
subsp. salivarius W. viridescens
subsp. thermophilus W. koreensis
Tetragenococcus
T. halophilus
T. muriaticus
Vagococcus
V. fluvialis
V. salmoninarum

character of homolactics may be shifted for some strains by altering growth conditions such as glucose
concentration, pH, and nutrient limitation.8,42
Those lactics that produce equal molar amounts of lactate, carbon dioxide, and ethanol from hexoses
are designated heterofermentative (Figure 7–1(B)). All members of the genera Pediococcus, Strep-
tococcus, Lactococcus, and Vagococcus are homofermenters, along with some of the lactobacilli.
Heterofermenters consist of Leuconostoc, Oenococcus, Weissella, Carnobacterium, Lactosphaera,
and some lactobacilli (Table 7–1). The heterolactics are more important than the homolactics in pro-
ducing flavor and aroma components such as acetylaldehyde and diacetyl (Figure 7–2).
The genus Lactobacillus was subdivided historically into three subgenera: Betabacterium, Strepto-
bacterium, and Thermobacterium. All of the heterolactic lactobacilli in Table 7–1 are betabacteria. The
streptobacteria (for example, L. casei and. plantarum) produce up to 1.5% lactic acid with an optimal
growth temperature of 30◦ C, whereas the thermobacteria (such as L. acidophilus and L. delbrueckii
subsp. bulgaricus) can produce up to 3% lactic acid and have an optimal temperature of 40◦ C.43
More recently, the genus Lactobacillus has been arranged into three groups based primarily on
fermentative features.70 Group 1 includes obligate homofermentative species (L. acidophilus, L. del-
brueckii subsp. bulgaricus, etc.). These are the thermobacteria, and they do not ferment pentoses.
Group 2 consists of facultative heterofermentative species (L. casei, L. plantarum, L. sakei; etc.).
Members of this group ferment pentoses. Group 3 consists of the obligate heterofermentative species,
and it includes L. fermentum, L. brevis, L. reuteri, L. sanfranciscensis, and others. They produce CO2
from glucose. The lactobacilli can produce a pH of 4.0 in foods that contain a fermentable carbohydrate,
and they can grow up to a pH of about 7.1.70
In terms of their growth requirements, the lactic acid bacteria require preformed amino acids,
B vitamins, and purine and pyrimidine bases—hence their use in microbiological assays for these
compounds. Although they are mesophilic, some can grow below 5◦ C and some as high as 45◦ C. With
respect to growth pH, some can grow as low as 3.2, some as high as 9.6, and most grow in the pH
range 4.0–4.5. The lactic acid bacteria are only weakly proteolytic and lipolytic.69
The cell mucopeptides of lactics and other bacteria have been reviewed by Schleifer and Kandler.64
Although there appear to be wide variations within most of the lactic acid genera, the homofermentative
lactobacilli of the subgenus Thermobacterium appear to be the most homogeneous in this regard in
having l-lysine in the peptidoglycan peptide chain and d-aspartic acid as the interbridge peptide. The
lactococci have similar wall mucopeptides.
154 Modern Food Microbiology

Figure 7–2 The general pathway by which acetoin and diacetyl are produced from citrate by group N lactococci
and Leuconostoc spp. Pyruvate may be produced from lactate, and acetyl coenzyme A (CoA) from acetate.

Molecular genetics have been employed by McKay and co-workers to stabilize lactose fermentation
by L. lactis. The genes responsible for lactose fermentation by some lactic cocci are plasmidborne, and
loss of the plasmid results in the loss of lactose fermentation. In an effort to make lactose fermentation
more stable, lac+ genes from L. lactis were cloned into a cloning vector, which was incorporated into
a Streptococcus sanguis strain.28 Thus, the lac genes from L. lactis were transformed into S. sanguis
via a vector plasmid, or transformation could be effected by use of appropriate fragments of DNA
through which the genes were integrated into the chromosome of the host cells.29 In the latter state,
lactose fermentation would be a more stable property than when the lac genes are plasmidborne.

Metabolic Pathways and Molar Growth Yields

The end-product differences between homo- and heterofermenters when glucose is attacked are a re-
sult of basic genetic and physiological differences (Figure 7–1). The homolactics possess the enzymes
aldolase and hexose isomerase but lack phosphoketolase (Figure 7–1(A)). They use the Embden–
Meyerhof–Parnas (EMP) pathway toward their production of two lactates/glucose molecule. The
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 155

heterolactics, on the other hand, have phosphoketolase but do not possess aldolase and hexose iso-
merase, and instead of the EMP pathway for glucose degradation, these organisms use the hexose
monophosphate or pentose pathway (Figure 7–1(B)).
The measurement of molar growth yields provides information on fermenting organisms relative
to their fermentation substrates and pathways. By this concept, the microgram dry weight of cells
produced per micromole of substrate fermented is determined as the molar yield constant, indicated
by Y . It is tacitly assumed that essentially none of the substrate carbon is used for cell biosynthesis,
that oxygen does not serve as an electron or hydrogen acceptor, and that all of the energy derived from
the metabolism of the substrate is coupled to cell biosynthesis.25 When the substrate is glucose, for
example, the molar yield constant for glucose, YG , is determined by

g dry weight of cells

YG =
moles glucose fermented

If the adenosine triphosphate (ATP) yield or moles of ATP produced per mole of substrate used is
known for a given substrate, the amount of dry weight of cells produced per mole of ATP formed can
be determined by

g dry weight of cells/moles ATP formed

YAT P =
moles substrate fermented

A large number of fermenting organisms has been examined during growth and found to have
YATP = 10.5 or close thereto. This value is assumed to be a constant, so that an organism that ferments
glucose by the EMP pathway to produce 2 ATP/mole of glucose fermented should have YG = 21 (i.e.,
it should produce 21 g of cells dry weight/mole of glucose). This has been verified for E. faecalis,
Saccharomyces cerevisiae, Saccharomyces rosei, and L. plantarum on glucose (all YG = 21, YATP =
10.5, within experimental error). A study by Brown and Collins8 indicates that YG and YATP values
for Lactococcus lactis subsp. lactis biovar diacetylactis and Lactococcus lactis subsp. cremoris differ
when cells are grown aerobically on a partially defined medium with low and higher levels of glucose,
and further when grown on a complex medium. On a partially defined medium with low glucose levels
(1–7 µmol/ml), values for L. lactis subsp. lactis biovar diacetylactis were YG = 35.3 and YATP = 15.6,
whereas for L. lactis subsp. cremoris, YG = 31.4 and YATP = 13.9. On the same medium with higher
glucose levels (1–15 µmol/ml), YG for L. lactis subsp. lactis biovar diacetylactis was 21, YATP values
for these two organisms on the complex medium with glucose 2 µmol/ml were 21.5 and 18.9 for L.
lactis subsp. lactis biovar diacetylactis and L. lactis subsp. cremoris, respectively. Anaerobic molar
growth yields for enterococcal species on low levels of glucose have been studied by Johnson and
Collins.36 Zymomonas mobilis utilizes the Entner–Doudoroff pathway to produce only 1 ATP/mole of
glucose fermented (YG = 8.3, YATP = 8.3). If and when the produced lactate is metabolized further,
the molar growth yield would be higher. Bifidobacterium bifidum produces 2.5–3 ATP/mole of glucose
fermented resulting in YG = and YATP = 13.71

ACETIC ACID BACTERIA

These Gram-negative bacteria belong to the family Acetobacteriaceae, and to the alpha-subclass
of Proteobacteria. The recognized genera are: Acetobacter, Asaia, Acidomonas, Gluconobacter, Glu-
conacetobacter, and Kozakia.79 With the exception of Asaia, they produce large quantities of acetic
acid from ethanol, and can grow in the presence of 0.35% acetic acid. The metabolic pathway employed
156 Modern Food Microbiology

by the acetic acid producing strains is shown in Figure 7–1(F) and (G). Asaia, on the other hand, pro-
duces little or no acetic acid from ethanol, and its species do not grow in the presence of 0.35% acetic
acid.79 The three recognized species oxidize acetate and lactate to CO2 and water.

DAIRY PRODUCTS

Milk

Milk is used throughout the world as a human food in at least one form, and from at least one
of a number of different mammals. Bovine milk is typical of other milk types and it is the basis of
the discussion that follows. Many of the aspects of milk microbiology not covered below have been
presented or reviewed by Frank17 and Murphy and Boor.50

Composition
From the general chemical composition of cow’s milk in Table 7–2, some differences between this
product and red meats in Table 4–9 are readily evident. The protein content of milk is considerably
lower (3.5 vs. 18.0%) while the carbohydrate content is considerably higher (14.9 vs. ca. 1.0%). The
higher structural protein content of red meats enables these products to exist as solids. Although the
average water content near the surface of fresh meats of ca. 75.5% is lower than the average of 87%
for milk, the aw of both products is near 1.0. The milk of goats and sheep is similar in composition to
that of cows.
Milk protein consists mainly of casein, and it exists in several classes: α, β, etc. If milk pH falls
below 4.6, the casein precipitates. Although casein represents 80–85% of total milk protein, when
precipitation occurs, the liquid portion is referred to as whey. The remaining proteins are found in
whey and they include serum albumin, immunoglobulins, α-lactalbumin, etc. Milk carbohydrate is
principally lactose and its content is fairly consistent among breeds of milk cows at around 5.0%.
Although lactose is the main sugar, smaller quantities of glucose and citric acid exist. The fat content
varies between ca. 3.5 and 5.0% depending upon cattle breed, and it consists mainly of triglycerides
composed of C14 , C16 , C18 , and C18:1 fatty acids. Smaller quantities of diglycerides and phospholipids
occur. Milk lipids exist largely in the form of fat globules that are surrounded by a phospholipid layer.
The ash content of around 0.7% consists of a relatively high level of Ca2+ and a lower level of Fe2+ .
Overall, the nonfat solids in cow’s milk average ca. 9.0% while total solids range between 12.5 and
14.5%, and average ca. 12.9% depending upon breed.

Table 7–2 Average Chemical Composition (%) of Whole

Bovine Milk (Summarized from the Literature)

Water 87.0
Protein 3.5
Fat 3.9
Carbohydrate 4.9
Ash 0.7
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 157

The pH of fresh whole milk is around 6.6 but it may reach ca. 6.8 from a cow that has mastitis.
Mastitis is an infection of the udder that is most often caused by Streptococcus agalactiae and S. uberis
but sometimes by Staphylococcus aureus or Streptococcus dysgalactiae. Fresh milk from a mastitic
cow typically contains leucocytes (white blood cells) >106 /ml in contrast to nonmastitic milk that
contains leucocytes around 70,000/ml.
Milk contains a very adequate supply of B vitamins with pantothenic acid and riboflavin being the
two most abundant. Vitamins A and D are added for human consumption, and their presence has no
known effect on the activity of microorganisms.
Overall, the chemical composition of whole cow’s milk makes it an ideal growth medium for
heterotrophic microorganisms, including the nutritionally fastidious Gram-positive lactic acid bacteria.
How the milk microbiota utilize these constituents and bring about its spoilage is covered below under
spoilage.

Processing

Milk is processed in a number of ways to produce a variety of products such as cream, cheese, and
butter. Whole fresh milk is processed to produce a number of fluid products. Skim milk (0.5% fat)
or reduced fat milk (up to 2.0% fat) is produced by high-speed centrifugation following heating to
ca. 100◦ F to remove butter fat as cream, or by use of skim milk to which the desired fat content is
added. The latter is pasteurized either at 150–155◦ F (65.5–68.3◦ C) for 30 minutes or at 166–175◦ F
(74.4–79.4◦ C) for 15 sec prior to cooling to around 4◦ C.78
Evaporated milk is produced by the removal of about 60% water from whole milk which results
in the lactose content being about 11.5%. Sweetened condensed milk is produced by the addition
of sucrose or glucose before evaporation. This leads to a product with a sugar content of about 54%
or >64% in solution.
In the United States, grade A raw milk that is to be pasteurized should not have an APC that exceeds
300,000 cfu/ml for commingled or blended milk, or should not exceed 100,000/ml for milk from an
individual producer. After pasteurization, the APC should not exceed 20,000 cfu/ml, and the coliform
count should not exceed 10/ml.15 Raw milk should not be held longer than 5 days at 40◦ F (4.4◦ C)
prior to pasteurization.
Chocolate milk is processed at a slightly higher temperature than unflavored milk (75◦ C for 15 sec
rather than 72◦ C). A study of chocolate milk from four plants revealed that the APC was higher at
14 days post-processing than unflavored milk even though the initial numbers for both types were
essentially the same.13 On day 14, 76.1% of unflavored and 91.6% of chocolate milk had APCs
>20,000 cfu/ml with 26.1% of the former and 53.7% of the latter products having APCs >106 cfu/ml.
These investigators suggested that the chocolate flavor powder contributed to increased growth.13 The
higher numbers were not due to higher numbers in the chocolate powder per se.

Pasteurization

The objective of milk pasteurization is the destruction of all disease-causing microorganisms. En-
dospores of pathogens such as Clostridium botulinum and spoilage organisms such as Clostridium
tyrobutyricum, C. sporogenes, or Bacillus cereus are not destroyed. Although pathogens can be de-
stroyed by nonthermal means, milk pasteurization is achieved solely by heating.
158 Modern Food Microbiology

The low temperature-long time (LTLT) method consists of heating the coolest part to 145◦ F (63◦ C)
for 30 minutes. This is referred to as the batch method. The other more widely used method is the high
temperature-short time (HTST) method, and it consists of heating to 161◦ F (72◦ C) for 15 sec. This is
the flash method, and it is inherently less destructive than the batch method. The basis for the heating
time and temperature is the thermal death time (TDT) of the most heat-resistant non-sporeforming
milk-borne pathogens. Prior to 1950, the LTLT method involved heating at 143◦ F for 30 minutes,
which was the TDT of Mycobacterium tuberculosis. However, after the discovery of the Q fever agent
(Coxiella burnetti) and the determination of its presence in bovine, goat, and sheep milk, the LTLT
method was changed so that it involved heating at 145◦ F for 30 minutes to correspond to the TDT of
this pathogen. In properly pasteurized milk, the naturally occurring enzyme alkaline phosphatase is
destroyed.76
UHT (ultra-high temperature) is another thermal treatment that destroys non-sporeforming
pathogens in milk, but in addition some sporeformers are severally reduced in numbers. The UHT
treatment is achieved by heating at temperatures of 275–284◦ F (135–140◦ C) for a few sec (the mini-
mum treatment is 130◦ C for 1 sec). UHT-treated milk is commercially sterile with a shelf life of 40–45
days at 40◦ F when aseptically packaged in sterile containers.7 UHT-treated whole milk is said to be
more flavorful, due apparently to formation of some Maillard products.
Although pasteurized milk is free of non-sporeforming pathogens, it is not sterile. The efficacy of
either LTLT or HTST to destroy the mycobacterial subspecies that is associated with Crohn’s disease in
humans has been called in to question, and this is discussed further below under milk-borne diseases.
Most if not all Gram-negative bacteria (especially psychrotrophs) are destroyed along with many
Gram positives. Thermoduric Gram positives belonging to the genera Enterococcus, Streptococcus
(especially Streptococcus salivarius subsp. thermophilus), Microbacterium, Lactobacillus, Mycobac-
terium, Corynebacterium, and most if not all sporeformers survive. Among the survivors are a number
of psychrotrophic species of the genus Bacillus.45

General Microbiota of Milk

Theoretically, milk that is secreted to the udder of a healthy cow should be free of microorganisms.
However, freshly drawn milk is generally not free of microorganisms. Numbers of several hundred to
several thousand cfu/ml are often found in freshly drawn milk, and they represent the movement up
the teat canal of some and the presence of others at the lower ends of teats. Although the APC of milk
from healthy cows is generally <103 cfu/ml, numbers of 104 /ml are not uncommon.50

Milk-Borne Pathogens

Since it is such an excellent nutrient source and because milk-producing animals may harbor
organisms that cause human diseases, it is not surprising that raw milk can be a source of diseases.
Some of the most obvious are the animal diseases below to which humans are susceptible and which
may occur in milk of cows:
Brucellosis Anthrax
Tuberculosis Listeriosis
Salmonellosis Q fever
Campylobacteriosis Crohn’s disease (?)
Enterohemorrhagic colitis Staph./Strep. Mastitis
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 159

Prior to the general use of mechanical milking devices, raw milk was the source of both human
respiratory diseases (e.g., diphtheria) as well as enteric infections (e.g., typhoid fever). When milking
a cow by hand where the milk is collected in an open pail, an infected person (or carrier) may
contaminate the milk by coughing or by hand contact. It was largely the connection between raw milk
and cases of human scarlet fever, diphtheria, and typhoid fever in the early 1900s that led the New
York City Board of Health to require milk pasteurization in 1910. The adoption of this law followed a
large outbreak of typhoid fever in New York City in the previous year, and it was traced to a common
milk supply where a chronic typhoid carrier worked.58 The etiologic agents of all of the above-listed
diseases are destroyed by the milk pasteurization process.
In spite of the widespread use of pasteurization, milk continues to be a vehicle for some diseases. In
the case of campylobacteriosis, it is not surprising that its etiologic agent can be found in milk since
the organism exists in cow feces. In a survey of 108 samples from bulk tanks of raw milk in Wisconsin,
only 1 was positive for C. jejuni, whereas the feces of 64% of the cows in a grade A herd were C. jejuni
positive.14 In the Netherlands, 22% of 904 cow fecal and 4.5% of 904 raw milk samples contained
C. jejuni.6 In regard to Helicobacter pylori, it was not detected in 120 raw bovine milk samples, and
when it was added to sterile milk and refrigerated at 4◦ C, it could not be detected after 6 days.35 An
outbreak of 75 cases of campylobacteriosis traced to raw milk occurred in Wisconsin in 2001.10 The
milk was from a grade A organic farm.
For the years 1973–1992, 46 outbreaks and 1,733 cases of human gastroenteritis were traced to raw
milk and reported to the Centers for Disease Control and Prevention in the United States. Of the 1,733
cases, 57% were caused by Campylobacter, 26% by Salmonella, and 2% by E. coli 0157:H7.31 Of
the 50 states in the United States, 28 allow the sale of raw milk but <1% of the milk-borne outbreaks
during the period noted were traced to raw milk. The interstate shipment/sale of raw milk was banned
in the United States in 1987. Some of the earliest outbreaks of human listeriosis were traced to milk
(see Chapter 25). Although the outbreaks traced to dairy products may be presumed to result from the
shedding of virulent strains into milk, this is not always confirmed. In one study, L. monocytogenes
was found to be shed in milk from the left forequarter of a mastitic cow, but milk from the other
quarters was uninfected.20 When bulk tank milk from 474 dairy herds in the Pacific northwest of the
United States were examined in 2000, 4.9% were positive for L. monocytogenes and in 2001, 7.0%
were positive with serotype 1/2a being the most common each year.48
Yersinia enterocolitica has been found in several studies on raw milk samples. The first documented
outbreak of this organism in the United States was traced to chocolate milk, and this outbreak is further
described in Chapter 28. Twelve of 100 raw milk samples in the state of Wisconsin were positive for
Y. enterocolitica but only 1 pasteurized sample was positive.46 Of 219 raw milk samples tested in
Brazil, 37 (16.9%) contained Listeria spp. and 32.4% were Y. enterocolitica.74 Of 280 pasteurized
milk samples, 13.7% were positive for Yersinia spp. with 41.5% being Y. enterocolitica. The latter
species was the most common in raw milk while Y. frederiksenii at 56.1% was the most prevalent in
pasteurized milk.74 The two largest human outbreaks of salmonellosis to occur in the United States
involved milk and ice cream, and they are described in Chapter 26.
In regard to aflatoxin M1 in milk, a study of 290 2-liter samples of pasteurized and ultrapasteurized
milk was carried out in Mexico on the seven most widely used brands with varying fat content. Forty
percent contained AFM1 at levels ≥0.05 ppm and 9.7% contained ≥0.5 ppm, and the range was 0–8.35
ppm in 40% of those with the lower concentration and in 10% of those with the higher concentration.9
Milk with the highest fat content had a slightly higher probability of containing AFM1 .9
A study of the prevalence of E. coli 0157:H7 in fecal samples from cull cows and bulk tank milk
in east Tennessee found 8 of 415 (2%) fecal samples and 2 of 268 (0.7%) bulk tank milk samples
positive.49 At least one large outbreak of E. coli 0157:H7 was traced to raw milk (see Chapter 27).
160 Modern Food Microbiology

A bacterium of continuing concern in milk is the etiologic agent of Johne’s disease of cattle, which
appears to play some role in Crohn’s disease of humans. The organisms in question are classified as
follows: Mycobacterium avium subsp. avium causes tuberculosis in birds and it is infectious for AIDS
patients. M. avium subsp. paratuberculosis is an obligate pathogen of ruminants and it is thought to
be involved in the etiology of Crohn’s disease. M. avium subsp. silvaticum is an obligate pathogen
of animals where it causes paratuberculosis in mammals and tuberculosis in birds.73 M. avium subsp.
paratuberculosis is the strain of interest in cow’s milk because of its possible role in Crohn’s disease.
Of primary concern is whether the pasteurization methods in use are adequate to destroy this organism.
In one study, neither the HTST nor the LTLT method destroyed 103 –104 cfu/ml in all milk samples,24
but in another study, up to 106 cfu/ml were destroyed by HTST carried out at 72◦ C for 15 sec.68
Crohn’s disease is an inflammatory bowel disease (regional ileitis), a condition wherein the terminal
ileum and sometimes the cecum and ascending colon are thickened and ulcerated. The lumen of the
affected region is much narrowed, resulting in intestinal obstruction.
In a survey of 814 bovine milk samples in the United Kingdom over a 17-month period in 1999–
2000, a mean of 7.8% of raw and 11.8% of pasteurized samples were positive for M. avium subsp.
paratuberculosis DNA.22 Culture confirmation was achieved in 1.6% of the raw and 1.8% of the
pasteurized samples. The pasteurized milk samples in this study were phosphatase negative. In another
study of raw and pasteurized cows’ milk in the United Kingdom, M. avium subsp. paratuberculosis
was found in 4 of 40 (6.7%) raw and 10 of 144 (6.9%) pasteurized milk samples.23 The investigators
found viable organisms in milk samples processed by four different treatments including heating at
73◦ C for 25 sec.
The fate of this organism in the ripening of cheese has been investigated.72 According to the FDA,
two options are available for producing safe cheese: (1) use only pasteurized milk, or (2) hold finished
cheese for at least 60 days at 2◦ C. In the study noted above, a soft white cheese using pasteurized
milk that was spiked with ca. 106 M. avium subsp. paratuberculosis was prepared by varying pH and
NaCl content and tested for viable cells after storage (ripening). It was found that cheese made from
HTST-pasteurized milk, pH 6.0, 2% NaCl, and cured for 60 days resulted in ca. log-3 reduction of M.
avium subsp. paratuberculosis cells.72 In a review of Crohn’s disease and the role of M. avium subsp.
paratuberculosis, Harris and Lammerding30 concluded that the evidence for cause and effect is not
conclusive.

Spoilage

As the only natural source of the disaccharide lactose, milk undergoes microbial spoilage in a way
that is unique. Only a relatively small number of milk-borne bacteria can obtain energy from this sugar
(especially at refrigerator temperatures) in contrast to the disaccharides sucrose and maltose, and the
lactic acid bacteria are well suited to this task. The coliform bacteria are the most conspicuous utilizers
of lactose among Gram-negative bacteria. Thus, the bacterial spoilage of either raw or pasteurized
milk is conspicuous by the production of lactic acid by lactose users, with the normal pH of around
6.6 being reduced to 4.5 or so that leads to the precipitation of casein (curdling). The thermoduric
Streptococcus salivarius subsp. thermophilus strains preferentially use the glucose moiety of lactose
and excrete galactose, which is a ready substrate for nonlactose users.
The spoilage of UHT milk is caused by Bacillus spp. that survive the UHT process. Anaerobic
spores appear not to be a problem because of the relatively high Eh of milk. Among the Bacillus
species that have been recovered from spoiled products are B. cereus, B. licheniformis, B. badius,
and B. sporothermodurans.55 Paenibacillus spp. have been isolated also from UHT-treated products.
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 161

During cold storage of pasteurized milk, psychrotrophic Bacillus weihenstephanensis causes “sweet
curdling” due to its production of proteases and peptidases. The spoilage of UHT-treated milk can
result from the actions of heat-resistant proteases and lipases that are produced by some psychrotrophs
in raw milk, and more on this can be found in reference 17.
Ropiness is a condition sometimes seen in raw milk that is caused by Alcaligenes viscolactis.
Its growth is favored by low-temperature maintenance of raw milk for several days. The “rope”
consists of a slime-layer material produced by the bacterial cells, and it gives the product a stringy
consistency.

PROBIOTICS AND PREBIOTICS

The definition of a probiotic that dates back to the mid-1960s has been modified during the past
decade. In general, it is a consumable product that contains live organisms that are or are believed to
be beneficial to the consumer. The ingestion of live organisms such as those in yogurt or fermented
milk is critical to the original concept. The term has been applied to consumable products that produce
a number of clinical benefits, but which do not contain viable cells. One example of this is the easing
of symptoms of lactose intolerance by the ingestion of heated yogurt (for a review, see reference 53).
A definition of probiotic that encompasses the above has been proposed by Salminen et al.61 In the
absence of viable organisms, the term “abiotics” has been suggested.37,67 To make the definition of a
probiotic even wider, the term has been applied to bacteria that act as control agents in an aquaculture
environment.77 For a more detailed review of probiotics, see reference 37.
Yogurt appears to be the most widely consumed of probiotic products (especially in the United
States) and while it is consumed by some because it is a fermented dairy product, it is consumed by
others because of its real or presumed health benefits. Although the typical starter cultures for yogurt are
Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, some
preparations are made by the addition of bifidobacteria. Regarding the number of viable cells that
should exist in probiotic and probiotic-like products, the Swiss Food Regulation and the International
Standard of FIL/IDF require that such products contain at least 106 cfu/ml while the Fermented Milks
and Lactic Acid Beverages Association in Japan requires at least 107 cfu/ml of viable bifidobacteria
(see reference 66). It is unclear whether viable cell numbers can be of both lactic acid bacteria and
bifidobacteria, or only one of these groups. When bifidobacteria are involved in the yogurt fermentation,
they tend to die out during storage. These organisms are anaerobes that require negative Eh conditions
and a pH near neutrality.66 The effect of a whey protein hydrolysate on some probiotic bacteria in milk
was investigated and while the hydrolysate initially increased the growth of Bifidobacterium longum
along with two lactobacilli, after 28 days at refrigerator temperatures, the probiotic organisms were at
about the same level as in the control milk.44
Live Bacillus spp. spores are used as probiotics and the three species most often used are B. clausii,
B. pumilus, and B. cereus at levels of ca. 109 /g. Positive effects have been demonstrated and they
appear to be due to the immunogenic properties of the spores, not the vegetative cells.
The effect of three starter cultures on the survival of Yersinia enterocolitica in yogurt is shown
in Figure 7–3 where the rapid acid-producing starter effected a log-5.0 reduction in 72 hours, and a
slow acid-producing strain effected a log-5.6 reduction in 96 hours.7 Inhibitory efforts of this type
by probiotic-type products against foodborne pathogens have been demonstrated for a number of
pathogens, and while pH reduction is one factor in the inhibition, factors such as bacteriocins and
organic acid toxicity are undoubtedly involved, and this is discussed further in Chapter 13. The cause
of lactose intolerance and its detection are described below.
162 Modern Food Microbiology

Figure 7–3 Survival of Yersinia enterocolitica in yogurt during fermentation at 44◦ C and storage at 4◦ C. Yogurt
was prepared using slow (YC 180) and rapid (YC 470) acid-producing starter cultures and a “home-use” starter
culture. Error bars represent standard errors of the mean,7 copyright

c 1998, used with permission from Institute
of Food Technologists

Prebiotics are not microorganisms; they are substrates for the indigenous probiotic-type bacteria
that reside in the colon. These substrates are nondigestible as they pass through the small intestine, and
they consist of oligosaccharides such as fructooligosaccharides of which inulin is an example. They
are metabolized by the bidifobacteria and the anaerobic lactobacilli (both of which are indigenous
to the colon) where the Eh favors their growth and activity, which results in an environment that is
antagonistic to aerobic pathogens. Unlike probiotics, these substrates can be added to a number of
food types that do not support cell viability over long periods of time. Their use obviates the need for
cultures that can persist in the small intestines.

Lactose Intolerance

Lactose intolerance (lactose malabsorption, intestinal hypolactemia) is the normal state for adult
mammals, including most adult humans, and many more groups are intolerant to lactose than are
tolerant.40 Among the relatively few groups that have a majority of adults who tolerate lactose are
northern Europeans, white Americans, and members of two nomadic pastoral tribes in Africa.40 When
lactose malabsorbers consume certain quantities of milk or ice cream, they immediately experience
flatulence and diarrhea. The condition is due to the absence or reduced amounts of intestinal lactase,
and this allows the bacteria in the colon to utilize lactose with the production of gases. The breath
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 163

Table 7–3 Summary of Some of the Many Claimed and Presumed Human Benefits from
Probiotics (Summarized from references 37, 53, 61, and 62)

Benefits Comments

Lactose intolerance Well established benefits (see text)

Acute gastroenteritis Incidence/duration often reduced
Serum cholestrol reduction Positive in vitro results; mixed in vivo results
Tumor size reduction/survival rate Limited studies
Reducing secondary tumor growth Positive results from limited studies
Production of IFNα,β Positive results from limited studies
Vaginal candidiases Mixed results
Antihypertensive effects Positive results shown
Anticolon cancer Some positive effects shown
Helicobacter pylori infection Inhibitors are produced
Enteric pathogen resistance Some positive effects demonstrated
Travelers’diarrhea Positive effects reported
Attenuation of colonic hyperplasia Positive results in mice
Increase in human longevity Not yet proven

hydrogen test for lactose intolerance is based on the increased levels of H2 produced by anaerobic and
facultatively anaerobic bacteria utilizing the nonabsorbed lactose.
“Sweet” acidophilus milk has been reported by some to prevent symptoms of lactose intolerance,
whereas others have found this product to be ineffective. Developed by M.L. Speck and co-workers,
it consists of normal pasteurized milk to which is added large numbers of viable L. acidophilus
cells as frozen concentrates. As long as the milk remains under refrigeration, the organisms do not
grow, but when it is drunk, the consumer gets the benefit of viable L. acidophilus cells. It is “sweet”
because it lacks the tartness of traditional acidophilus milk. When 18 lactase-deficient patients ingested
unaltered milk for 1 week, followed by “sweet” acidophilus milk for an additional week, they were as
intolerant to the latter product as to the unaltered milk.51 Lactose-free milk is available in Germany
that is pretreated with β-galactosidase. In one study with rats, the yogurt bacteria had little effect in
preventing the malabsorption of lactose. The indigenous lactics in the gut tended to be suppressed by
yogurt, and the rat lactobacillus biota changed from one that was predominantly heterofermentative
to one that was predominantly homofermentative. A number of other possible health benefits from
both viable and nonviable probiotic bacteria have been studied, and an extensive review has been
provided.62 Summaries of some health benefits are presented in Table 7–3.

STARTER CULTURES, FERMENTED PRODUCTS

The products discussed in this subsection require the use of an appropriate starter culture. A lactic
starter is a basic starter culture with widespread use in the dairy industry. For cheese making of all
kinds, lactic acid production is essential, and the lactic starter is employed for this purpose. Lactic
starters are also used for preparing butter, cultured buttermilk, cottage cheese, and cultured sour cream
and are often referred to by product (butter starter, buttermilk starter, and so on). Lactic starters always
164 Modern Food Microbiology

Figure 7–4 Reactions of the propionic acid fermentation and the formation of acetate, CO2 , propionate, and
ATP. Me-malonyl-CoA is methylmalonyl-CoA and (a) and (b) are the two isomers. FP is flavoprotein, and FPH2 is
reduced flavoprotein. Summary: 1.5 glucose + 6 Pi + 6 ADF → 6 ATP + 2H2 O + CO2 + acetate + 2 propionate.
Source: Allen et al.,3 copyright

c 1964 by American Society for Microbiology.

include bacteria that convert lactose to lactic acid, usually L. lactis subsp. lactis, L. lactis subsp.
cremoris, or L. lactis subsp. lactis biovar diacetylactis. Where flavor and aroma compounds such as
diacetyl are desired, the lactic starter will include a heterolactic such as Leuconostoc mesenteroides
subsp. cremoris, L. lactis subsp. lactis biovar diacetylactis, or Leuconostoc mesenteroides subsp.
dextranicum (for biosynthetic pathways, see Figure 7–4). Starter cultures may consist of single or
mixed strains. They may be produced in bulk and preserved by freezing in liquid nitrogen19 or by
freeze drying. The lactococci generally make up around 90% of a mixed dairy starter population, and
a good starter culture can convert most of the lactose to lactic acid. The titratable acidity may increase
to 0.8–1.0%, calculated as lactic acid, and the pH usually drops to 4.3–4.5.16

Fermented Products

Butter, buttermilk, and sour cream are produced generally by inoculating pasteurized cream or milk
with a lactic starter culture and holding until the desired amount of acidity is attained. In the case of
butter, where cream is inoculated, the acidified cream is then churned to yield butter, which is washed,
salted, and packaged.54 Buttermilk, as the name suggests, is the milk that remains after cream is
churned for the production of butter. The commercial product is usually prepared by inoculating skim
milk with a lactic or buttermilk starter culture and holding until souring occurs. The resulting curd is
broken up into fine particles by agitation, and this product is termed cultured buttermilk. Cultured sour
cream is produced generally by fermenting pasteurized and homogenized light cream with a lactic
starter. These products owe their tart flavor to lactic acid and their buttery aroma and taste to diacetyl.
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 165

An outbreak of Campylobacter enteritis in the state of Louisiana in 1995 where garlic butter was
incriminated led to studies on the fate of foodborne pathogens in butter with or without garlic. In one
study, garlic butter was inoculated with ca. 104 and 106 cfu/g of C. jejuni and held at 5 or 21◦ C. At 5◦ C,
the pathogen decreased to <10 cfu/g within 3 hours for two batches and within 24 hours for a third
batch.81 In butter with no garlic, C. jejuni survived for 13 days at 5◦ C. At 21◦ C, the pathogen decreased
to <10 cfu/g within 5 hours for two preparations and to 50 cfu/g in 5 hours for the third.81 Overall,
up to 105 cells were killed within a few hours in butter with garlic, but the organism could survive for
days in refrigerated butter. In another study, Salmonella, E. coli 0157:H7, and L. monocytogenes were
found not to grow in unsalted butter with or without 20% garlic when held at 4.4, 21, or 37◦ C for up
to 48 hours.1
Yogurt (yoghurt) is produced with a yogurt starter, which is a mixed culture of S. salivarius subsp.
thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in a 1:1 ratio. The coccus grows faster
than the rod and is primarily responsible for initial acid production at a higher rate than that produced
by either when growing alone, and more acetaldehyde (the chief volatile flavor component of yogurt)
is produced by L. delbrueckii subsp. bulgaricus when growing in association with S. salivarius subsp.
thermophilus (see reference 57). The coccus can produce about 0.5% lactic acid and the rod about
0.6–0.8% (pH of 4.2–4.5). However, if incubation is extended, pH can decrease to about 3.5 with lactic
acid increasing to about 2%.32
The product is prepared either by reducing the water content of either whole or skim milk by at least
one-fourth (may be done in a vacuum pan following sterilization of milk), or by adding about 5% milk
solids followed by water reduction (condensing). The concentrated milk is then heated to 82–93◦ C
for 30–60 minutes and cooled to around 45◦ C.54 The yogurt starter is now added at a level of around
2% by volume and incubated at 45◦ C for 3–5 hours followed by cooling to 5◦ C. The titratable acidity
of a good finished product is around 0.85–0.90%, and to get this amount of acidity the fermenting
product should be removed from 45◦ C when the titratable acidity is around 0.65–0.70%.11 Good
yogurt keeps well at 5◦ C for 1–2 weeks. The coccus grows first during the fermentation followed by
the rod, so that after around 3 hours, the numbers of the two organisms should be approximately equal.
Higher amounts of acidity, such as 4%, can be achieved by allowing the product to ferment longer,
with the effect that the rods will exceed the cocci in number. The streptococci tend to be inhibited at
yogurt pH values of 4.2–4.4, whereas the lactobacilli can tolerate pH values in the 3.5–3.8 range. The
lactic acid of yogurt is produced more from the glucose moiety of lactose than the galactose moiety.
Goodenough and Kleyn21 found only a trace of glucose throughout yogurt fermentation, whereas
galactose increased from an initial trace to 1.2%. Samples of commercial yogurts showed only traces
of glucose, but galactose varied from around 1.5% to 2.5%.
Freshly produced yogurt typically contains around 109 organisms/g, but during storage, numbers
may decrease to 106 /g, especially when stored at 5◦ C for up to 60 days.27 The rod generally decreases
more rapidly than the coccus. The addition of fruits to yogurt does not appear to affect the numbers
of fermenting organisms.27 The International Dairy Federation norm for yogurt is 107 /g or above. In
one study, E. coli 0157:H7 did not survive in skim milk at pH 3.8, and the organism was inactivated
in yogurt, sour cream, and buttermilk similarly.26
The antimicrobial qualities of yogurt, buttermilk, sour cream, and cottage cheese have been examined
by inoculating Enterobacter aerogenes and Escherichia coli separately into commercial products and
studying the fate of these organisms when the products were stored at 7.2◦ C. A sharp decline of
both coliforms was noted in yogurt and buttermilk after 24 hours. Neither could be found in yogurt
generally beyond 3 days. Although the numbers of coliforms were reduced also in sour cream, they
were not reduced as rapidly as in yogurt. Some cottage cheese samples actually supported an increase
in coliform numbers, probably because the products had higher pH values. The initial pH ranges for
166 Modern Food Microbiology

the products studied by these workers were as follows: 3.65–4.40 for yogurts, 4.1–4.9 for buttermilk,
4.18–4.70 for sour creams, and 4.80–5.10 for cottage cheese samples. In another study, commercially
produced yogurts in Ontario were found to contain the desired 1:1 ratio of coccus to rod in only 15%
of 152 products examined.5 Staphylococci were found in 27.6% and coliforms in around 14% of these
yogurts. Twenty-six percent of the samples had yeast counts more than 1,000/g and almost 12% had
psychrotroph counts more than 1,000/g. In his study of commercial unflavored yogurt in Great Britain,
Davis11 found counts of the two starters to range from a low of around 82 million to a high of over
1 billion/g, and the final pH to range from 3.75 to 4.20. The antimicrobial activities of lactic acid
bacteria are discussed further in Chapters 3 and 13.
Kefir is prepared by the use of kefir grains, which contain one or more bacterial species of the
genera Acetobacter, Lactobacillus, Lactococcus, Leuconostoc, and one or more yeast species of the
genera Candida, Kluyveromyces, and Saccharomyces. These symbionts are held together by coagulated
protein.18 The important Lactobacillus spp. in kefir are: L. kefiri, L. parakefiri, L. kefiranofaciens
subsp. kefiranofaciens, and L. kefiranofaciens subsp. kefirgranum.75 The last two are responsible for
the production of kefiran (a water-soluble polysaccharide), which accounts for about 24% of kefir
grains.75 Kumiss is similar to kefir except that mare’s milk is used, the culture organisms do not form
grains, and the alcohol may reach 2%.
Acidophilus milk is produced by the inoculation of an intestinal implantable strain of L. acidophilus
into sterile skim milk. The inoculum of 1–2% is added, followed by holding the product at 37◦ C until
a smooth curd develops. A popular variant of this product that is produced commercially in the United
States consists of adding a concentrated implantable strain culture of L. acidophilus to a pasteurized
and cold vat of whole milk (or skim or 2% milk), and it is bottled immediately. It has the pH of normal
milk and is more palatable than the more acidic product. The numbers of L. acidophilus should be
in the 107 –108 /ml range.32 Bulgarian buttermilk is produced in a similar manner by the use of L.
bulgaricus as the inoculum or starter, but unlike L. acidophilus, L. bulgaricus is not implantable in
the human intestines. A summary of fermented milk is presented in Table 7–4.
Butter contains around 15% water, 81% fat, and generally less than 0.5% carbohydrate and protein.
Although it is not a highly perishable product, it does undergo spoilage by bacteria and molds. The main
source of microorganisms for butter is cream, whether sweet or sour, pasteurized or nonpasteurized.
The biota of whole milk may be expected to be found in cream because as the fat droplets rise
to the surface of milk, they carry up microorganisms. The processing of both raw and pasteurized
creams to yield butter brings about a reduction in the numbers of all microorganisms, with values
for finished cream ranging from several hundred to over 100,000/g having been reported for finished
salted butter. Salted butter may contain up to 2% salt, and this means that water droplets throughout
may contain an effective level of about 10%, thus making this product even more inhibitory to bacterial
spoilage.32
Bacteria cause two principal types of spoilage in butter. The first is a condition known as “surface
taint” or putridity. This condition is caused by Pseudomonas putrefaciens as a result of its growth on
the surface of finished butter. It develops at temperatures within the range 4–7◦ C and may become
apparent within 7–10 days. The odor of this condition is apparently due to certain organic acids,
especially isovaleric acid. Surface taint along with an apple odor is caused also by Chryseobacterium
joostei.34 The second most common bacterial spoilage condition of butter is rancidity. This condition
is caused by the hydrolysis of butterfat with the liberation of free fatty acids. Lipase from sources other
than microorganisms can cause the effect. The causative organism is Pseudomonas fragi, although P.
fluorescens is sometimes found. Bacteria may cause three other less common spoilage conditions in
butter. Malty flavor is reported to be due to the growth of Lactococcus lactis var. maltigenes. Skunklike
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 167

Table 7–4 Some Fermented Milk Products

Foods and Products Raw Ingredients Fermenting Organisms Where Produced

Acidophilus milk Milk Lactobacillus acidophilus Many countries

Bulgarian buttermilk L. delbrueckii subsp. Balkans, other areas
bulgaricus
Cheeses (ripened) Milk curd Lactic starters Worldwide
Kefir Milk Lactococcus lactis, L. Southwestern Asia
delbrueckii subsp.
bulgaricus, “Torula”
spp.
Kumiss Raw mare’s milk Lactobacillus leichmannii, Russia
L. delbrueckii subsp.
bulgaricus, “Torula” spp.
Taette Milk S. lactis var. taette Scandinavian
peninsula
Tarhana∗ Wheat meal and Lactics Turkey
yogurt
Yogurt† Milk, milk solids L. delbrueckii subsp. Worldwide
bulgaricus, S. salivarius
subsp. thermophilus
Bioghurt Milk, milk solids L. acidophilus, Lactococcus Worldwide
lactis
∗ Similar to Kishk in Syria and Kushuk in Iran.
† Also yoghurt (matzoon in Armenia; leben in Egypt; naja in Bulgaria; gioddu in Italy; dadhi in India).

odor is reported to be caused by Pseudomonas mephitica; black discolorations of butter have been
reported to be caused by P. nigrifaciens.
Butter undergoes fungal spoilage rather commonly by species of Cladosporium, Alternaria, As-
pergillus, Mucor, Rhizopus, Penicillium, and Geotrichum, especially G. candidum (Oospora lactis).
These organisms can be seen growing on the surface of butter, where they produce colorations refer-
able to their particular spore colors. Black yeasts of the genus Torula also have been reported to
cause discolorations on butter. The microscopic examination of moldy butter reveals the presence of
mold mycelia some distances from the visible growth. The generally high lipid content and low water
content make butter more susceptible to spoilage by molds than by bacteria.
Cottage cheese undergoes spoilage by bacteria, yeasts, and molds. The most common spoilage
pattern displayed by bacteria is a condition known as slimy curd. Alcaligenes spp. have been reported
to be among the most frequent causative organisms, although Pseudomonas, Proteus, Enterobacter,
and Acinetobacter spp. have been implicated. Penicillium, Mucor, Alternaria, and Geotrichum all
grow well on cottage cheese, to which they impart stale, musty, moldy, and yeasty flavors. The shelf
life of commercially produced cottage cheese in Alberta, Canada was found to be limited by yeasts and
molds.59 Although 48% of fresh samples contained coliforms, these organisms did not increase upon
storage in cottage cheese at 40◦ F for 16 days. For more on fermented dairy products, see references
52, 54.
168 Modern Food Microbiology

Cheeses

Most but not all cheeses result from a lactic fermentation of milk. In general, the process of
manufacture consists of two important steps:

1. Milk is prepared and inoculated with an appropriate lactic starter. The starter produces lactic acid,
which, with added rennin, gives rise to curd formation. The starter for cheese production may
differ depending on the amount of heat applied to the curds. S. salivarius subsp. thermophilus is
employed for acid production in cooked curds (up to 60◦ C) because it is more heat tolerant than
either of the other more commonly used lactic starters; or a combination of S. salivarius subsp.
thermophilus and L. lactis subsp. lactis is employed for curds that receive an intermediate cook.
2. The curd is shrunk and pressed, followed by salting, and, in the case of ripened cheeses, allowed
to ripen under conditions appropriate to the cheese in question.

Although most ripened cheeses are the product of metabolic activities of the lactic acid bacteria, several
well-known cheeses owe their particular character to other related organisms. In the case of Swiss
cheese, a mixed culture of L. delbrueckii subsp. bulgaricus and S. salivarius subsp. thermophilus is
usually employed along with a culture of Propionibacterium shermanii or P. freundenreichii added
to function during the ripening process in flavor development and eye formation. (See Figure 7–1(C)
and (D)) for a summary of propionibacteria pathways and Figure 7–4 for pathway in detail.) These
organisms have been reviewed extensively by Hettinga and Reinbold.33 For blue cheeses such as
Roquefort, the curd is inoculated with spores of Penicillium roqueforti, which effect ripening and
impart the blue-veined appearance characteristic of this type of cheese. In a similar fashion, either the
milk or the surface of Camembert cheese is inoculated with spores of Penicillium camemberti.
Two coryneform bacteria of the genus Brachybacterium have been recovered from the surfaces
of French Gruyère and Beaufort cheeses65 but the role these organisms play in the ripening process
is unclear. In a study of L. monocytogenes in European red smear cheese (soft, semisoft, and hard),
5.8% of 329 test samples contained Listeria spp. with 6.4% being L. monocytogenes and 10.6% L.
innocua.60 Eight samples contained >100 L. monocytogenes/cm2 ; and two samples contained 104
cfu/cm2 .
There are over 400 varieties of cheeses representing fewer than 20 distinct types, and these are
grouped or classified according to texture or moisture content, whether ripened or unripened, and if
ripened, whether by bacteria or molds. The three textural classes of cheeses are hard, semihard, and
soft. Examples of hard cheeses are all cheddar, Provolone, Romano, Parmesan, Gruyère, Emmental,
and Edam. All hard cheeses are ripened by bacteria over periods ranging from 2 to 16 months. Semihard
cheeses include Muenster, Roquefort, Limburger, and Gouda and are ripened by bacteria over periods
of 1–8 months. Blue and Roquefort are two examples of semihard cheeses that are mold ripened for
2–12 months. Limburger is an example of a soft bacteria-ripened cheese, and Brie and Camembert are
examples of soft mold-ripened cheeses. Among unripened cheeses are cottage, cream, Mozzarella,
and Neufchatel.
The low moisture content of hard and semihard ripened cheeses makes them insusceptible to
spoilage by most organisms, although molds can and do grow on these products as would be expected.
Some ripened cheeses have sufficiently low oxidation–reduction potentials to support the growth
of anaerobes. It is not surprising to find that anaerobic bacteria sometimes cause the spoilage of
these products when aw (water activity) permits growth to occur. Clostridium spp., especially C.
pasteurianum, C. butyricum, C. sporogenes, and C. tyrobutyricum, have been reported to cause late
gassiness of cheeses. One of these (C. tyrobutyricum) is well established as the cause of a butyric acid
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 169

Figure 7–5 Inhibition of Clostridium tyrobutyricum in processed cheese spread (cheese blend B) by 0.5% and
1.0% of HBS polyphosphate. Reprinted with permission from J. Food Protect., (c) held by the Int. Assoc. Food
Protect., Des Moines, IA, USA. Source: Loessner et al.41

fermentation or the late-blowing defect in cheeses such as Gouda and Emmentaler.39 With 0.5% of a
long-chain polyphosphate mixture, growth of C. tyrobutyricum was inhibited for at least 8 days and
could not be detected after 16–50 days (see Figure 7–5). Growth was completely inhibited by 1.0%
polyphosphate,41 and it was due to the sequestration of Ca2+ /Mg2+ by poly-P, which led to filamentous
cells and lysis. An aerobic sporeformer, Paenibacillus polymyxa, has been reported to cause gassiness.
This condition is the result of CO2 being produced from lactic acid.
For the years 1973–1992, there were 32 cheese-associated disease outbreaks in the United States
with 1,700 cases and 58 deaths with 52 of the latter caused by L. monocytogenes in the 1985 California
outbreak.4 The most common vehicle was soft cheeses, and improper pasteurization was common.

DISEASES CAUSED BY LACTIC ACID BACTERIA

Although the beneficial aspects of the lactic acid bacteria to human and animal health are unques-
tioned, some of these bacteria are associated with human illness. This subject has been reviewed by
Aguirre and Collins,2 who noted that around 68 reports of involvement of lactobacilli in human clinical
illness were made over about a 50-year period. Several species of leuconostocs were implicated in
about 27 reports in 7 years, the pediococci in 18 reports over 3 years, and the enterococci in numerous
reports. The enterococci are the third leading cause of nosocomial (hospital acquired) infections, with
E. faecalis and E. faecium being the two most common species. It appears that lactic acid bacteria are
opportunists that are not capable of initiating infection in normal healthy individuals. To determine
whether vancomycin-resistant enterococci (VRE) existed in ground beef and pork in Germany, 555
samples were examined for VRE, and overall their incidence in ground beef was too low to be a
significant source of nosocomial infections.38
170 Modern Food Microbiology

REFERENCES

1. Adler, B.B., and L.R. Beuchat. 2002. Death of Salmonella, Escherichia coli 0157:H7, and Listeria monocytogenes in garlic
butter as affected by storage temperature. J. Food Protect. 65:1976–1980.
2. Aguirre, M., and M.D. Collins. 1993. Lactic acid bacteria and human clinical infection. J. Appl. Bacteriol. 75:95–107.
3. Allen, S.H.G., R.W. Killermeyer, R.L. Stjernholm, and H.G. Wood. 1964. Purification and properties of enzymes involved
in the propionic acid fermentation. J. Bacteriol. 87:171–187.
4. Altekruse, S.F., B.B. Timbo, J.C. Mobray, N.H. Bean, and M.E. Potter. 1998. Cheese-associated outbreaks of human
illness in the United States, 1973 to 1992: Sanitary manufacturing practices protect consumers. J. Food Protect. 61:1405–
1407.
5. Arnott, D.R., C.L. Duitschaever, and D.H. Bullock. 1974. Microbiological evaluation of yogurt produced commercially in
Ontario. J. Milk Food Technol. 37:11–13.
6. Beumer, R.R., J.J.M. Cruysen, and I.R.K. Birtantie. 1988. The occurrence of Campylobacter jejuni in raw cows’ milk. J.
Appl. Bacteriol. 65:93–96.
7. Bodnaruk, P.W., R.G. Williams, and D.A. Golden. 1998. Survival ofYersinia enterocolitica during fermentation and storage
of yogurt. J. Food Sci. 63:535–537.
8. Brown, W.V., and E.B. Collins. 1977. End products and fermentation balances for lactic streptococci grown aerobically on
low concentrations of glucose. Appl. Environ. Microbiol. 33:38–42.
9. Carvajal, M., A. Bolanos, F. Rojo, and I. Méndez. 2003. Aflatoxin M1 in pasteurized and ultrapasteurized milk with different
fat content in Mexico. J. Food Protect. 66:1885–1892.
10. Centers for Disease Control and Prevention. 2002. Outbreak of Campylobacter jejuni infections associated with drinking
unpasteurized milk procured through a cow-leasing program—Wisconsin, 2001. Morb. Mort. Wkly. Rept. 51:548–549.
11. Davis, J.G. 1975. The microbiology of yoghurt. In Lactic Acid Bacteria in Beverages and Food, ed. J.G. Carr, C.V. Cutting,
and G.C. Whiting, 245–263. New York: Academic Press.
12. Doelle, H.A. 1975. Bacterial Metabolism. New York: Academic Press.
13. Douglas, S.A., M.J. Gray, A.D. Crandall, and K.J. Boor. 2000. Characterization of chocolate milk spoilage patterns. J.
Food Protect. 63:516–521.
14. Doyle, M.P., and D.J. Roman. 1982. Prevalence and survival of Campylobacter jejuni in unpasteurized milk. Appl. Environ.
Microbiol. 44:1154–1158
15. Food and Drug Administration, United States. 1995. Grade A pasteurized milk ordinance. Washington, D.C.: U.S. Depart-
ment of Health and Human Services, Public Health Service.
16. Foster, E.M., F.E. Nelson, M.L. Speck, R.N. Doetsch, and J.C. Olson. 1957. Dairy Microbiology. Englewood Cliffs, N.J.:
Prentice-Hall.
17. Frank, J.F. 2001. Milk and dairy products, In Food Microbiology: Fundamentals and Frontiers, 2nd ed., ed. M.P. Doyle,
L.R. Beuchat, and T.J. Montville, 111–126. Washington, DC: ASM Press.
18. Garrote, G.L., A.G. Abraham, and G.L. de Antoni. 2000. Inhibitory power of kefir: The role of organic acids. J. Food
Protect. 63:364–369.
19. Gilliland, S.E., and M.L. Speck. 1974. Frozen concentrated cultures of lactic starter bacteria: A review. J. Milk Food
Technol. 37:107–111.
20. Gitter, M., R. Bradley, and P.H. Blampied. 1980. Listeria monocytogenes infection in bovine mastitis. Vet. Rec. 107:390–393.
21. Goodenough, E.R., and D.H. Kleyn. 1976. Qualitative and quantitative changes in carbohydrates during the manufacture
of yoghurt. J. Dairy Sci. 59:45–47.
22. Grant, I.R., H.J. Ball, and M.T. Rowe. 2002a. Incidence of Mycobacterium paratuberculosis in bulk raw and commercially
pasteurized cows’ milk from approved dairy processing establishments in the United Kingdom. Appl. Environ. Microbiol
68:2428–2435.
23. Grant, I.R., E.I. Hitchings, A. McCartney, F. Ferguson, and M.T. Rowe. 2002b. Effect of commercial-scale high-temperature,
short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows’ milk. Appl.
Environ. Microbiol. 68:602–607.
24. Grant, I.R., H.J. Ball, S.D. Neill, and M.T. Rowe. 1996. Inactivation of Mycobacterium paratuberculosis in cows’ milk at
pasteurization temperatures. Appl. Environ. Microbiol. 62:631–636.
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 171

25. Gunsalus, I.C., and C.W. Shuster. 1961. Energy yielding metabolism in bacteria. In The Bacteria, ed. I.C. Gunsalus and
R.Y. Stanier, vol. 2, 1–58. New York: Academic Press.
26. Guraya, R., J.F. Frank, and A.N. Hassan. 1998. Effectiveness of salt, pH, and diacetyl as inhibitors of Escherichia coli
0157:H7 in dairy foods stored at refrigeration temperatures. J. Food Protect. 61:1098–1102.
27. Hamann, W.T., and E.H. Marth. 1984. Survival of Streptococcus thermophilus and Lactobacillus bulgaricus in commercial
and experimental yogurts. J. Food Protect. 47:781–786.
28. Harlander, S.K., and L.L. McKay. 1984. Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid
DNA. Appl. Environ. Microbiol. 48:342–346.
29. Harlander, S.K., L.L. McKay, and C.F. Schachtels. 1984. Molecular cloning of the lactose-metabolizing genes from
Streptococcus lactis. Appl. Environ. Microbiol. 48:347–351.
30. Harris, J.E., and A.M. Lammerding. 2001. Crohn’s disease and Mycobacterium avium subsp. paratuberculosis: Current
issues. J. Food Protect. 64:2103–2110.
31. Headrick, M.L., S. Korangy, N.H. Bean, F.J. Angulo, S.F. Altekruse, M.E. Potter, and K.C. Klontz. 1998. The epidemiology
of raw milk-associated foodborne disease outbreaks reported in the United States, 1973 through 1992. J. Amer. Public Health
88:1219–1221.
32. Henning, D.R. 1999. Personal communication.
33. Hettinga, D.H., and G.W. Reinbold. 1972. The propionic-acid bacteria—A review. J. Milk Food Technol. 35:295–301,
358–372, 436–447.
34. Hugo, C.J., P. Segers, B. Hoste, M. Vancanneyt, and K. Kersters. 2003. Chryseobacterium joostei sp. nov., isolated from
the dairy environment. Int. J. Syst. Evol. Microbiol. 53:771–777.
35. Jiang, X., and M.P. Doyle. 2002. Optimizing enrichment culture conditions for detecting Helicobacter pylori in foods. J.
Food Protect. 65:1949–1954.
36. Johnson, M.G., and E.B. Collins. 1973. Synthesis of lipoic acid by Streptococcus faecalis 10C1 and end-products produced
anaerobically from low concentrations of glucose. J. Gen. Microbiol. 78:47–55.
37. Klaenhammer, T.R. 2001. Probiotics and prebiotics. In Food Microbiology: Fundamentals and Frontiers, 2nd ed., ed. M.P.
Doyle, L.R. Beuchat, and T.J. Montville, 97–811. Washington, D.C.: ASM Press.
38. Klein, G., A. Pack, and G. Reuter. 1998. Antibiotic resistance patterns of enterococci and occurrence of vancomycin-resistant
enterococci in raw minced beef and pork in Germany. Appl. Environ. Microbiol. 64:1825–1830.
39. Klijn, N., F.F.J. Nieuwenhof, J.D. Hoolwerf, C.B. van der Waals, and A.H. Weerkamp. 1995. Identification of Clostridium
tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification. Appl. Environ.
Microbiol. 61:2919–2924.
40. Kretchmer, N. 1972. Lactose and lactase. Sci. Am. 227(10):71–78.
41. Loessner, M.J., S.K. Maier, P. Schiwek, and S. Scherer. 1997. Long-chain polyphosphates inhibit growth of Clostridium
tyrobutyricum in processed cheese spreads. J. Food Protect. 60:493–498.
42. London, J. 1976. The ecology and taxonomic status of the lactobacilli. Ann. Rev. Microbiol. 30:279–301.
43. Marth, E.H. 1974. Fermentations. In Fundamentals of Dairy Chemistry, ed. B.H. Webb, A.H. Johnson, and J.A. Alford,
chap. 13. Westport, C.T.: AVI.
44. McComas, K.A., Jr., and S.E. Gilliland. 2003. Growth of probiotic and traditional yogurt cultures in milk supplemented
with whey protein hydrolysate. J. Food Sci. 68:2090–2095.
45. Meer, R.R., J. Baker, F.W. Bodyfelt, and M.W. Griffiths. 1991. Psychrotrophic Bacillus spp. in fluid milk products: A
review. J. Food Protect. 54:969–979.
46. Moustafa, M.K., A.A.-H. Admed, and E.H. Marth. 1983. Occurrence of Yersinia enterocolitica in raw and pasteurized
milk. J. Food Protect. 46:276–278.
47. Mundt, J.O. 1975. Unidentified streptococci from plants. Int. J. Syst. Bacteriol. 25:281–285.
48. Muraoka, W., C. Gay, D. Knowles, and M. Borucki. 2003. Prevalence of Listeria monocytogenes subtypes in bulk milk of
the Pacific Northwest. J. Food Protect. 66:1413–1419.
49. Murinda, S.E., K.T. Nguyen, S.J. Ivey, B.E. Gillespie, R.A., Almeida, F.A. Draughon, and S.P. Oliver. 2002. Prevalence and
molecular characterization of Escherichia coli 0157:H7 in bulk tank milk and fecal samples from cull cows: A 12-month
survey of dairy farms in east Tennessee. J. Food Protect. 65:752–759.
50. Murphy, S.C., and K.J. Boor. 2000. Trouble-shooting sources and causes of high bacteria counts in raw milk. Dairy Fd.
Environ. Sanit. 20:606–611.
172 Modern Food Microbiology

51. Newcomer, A.D., H.S. Park, P.C. O’Brien, and D.B. McGill. 1983. Response of patients with irritable bowel syndrome
and lactase deficiency using unfermented acidophilus milk. Am. J. Clin. Nutr. 38:257–263.
52. National Academy of Science, USA. 1992. Applications of Biotechnology to Traditional Fermented Foods. Washington,
D.C.: National Academy Press.
53. Ouwehand, A.C., and S.J. Salminen. 1998. The health effects of cultured milk products with viable and non-viable bacteria.
Int. Dairy J. 8:749–758.
54. Pederson, C.S. 1979. Microbiology of Food Fermentations, 2nd ed. Westport, C.T.: AVI.
55. Pettersson, B., F. Lembke, P. Hammer, E. Stackebrandt, and F.G. Priest. 1996. Bacillus sporothermodurans, a new species
producing highly heat-resistant endospores. Int. J. System. Bacteriol. 46:759–764.
56. Prescott, S.C., and C.G. Dunn. 1957. Industrial Microbiology. New York: McGraw-Hill.
57. Radke-Mitchell, L., and W.E. Sandine. 1984. Associative growth and differential enumeration of Streptococcus ther-
mophilus and Lactobacillus bulgaricus: A review. J. Food Protect. 47:245–248.
58. Rosen, G. 1958. A History of Public Health, 358–360. New York: MD Publications.
59. Roth, L.A., L.F.L. Clegg, and M.E. Stiles. 1971. Coliforms and shelf life of commercially produced cottage cheese. Can.
Inst. Food Technol. J. 4:107–111.
60. Rudolf, M., and S. Scherer. 2001. High incidence of Listeria monocytogenes in European red smear cheese. Int. J. Food
Microbiol. 63:91–98.
61. Salminen, S., A. Ouwehand, Y.H. Benno, and Y.K. Lee. 1999. Probiotics: How should they be defined? Trends Food. Sci.
Technol. 10:107–110.
62. Sanders, M.E. 1999. Probiotics. Food Technol. 53(11):67–77.
63. Sandine, W.E., P.C. Radich, and P.R. Elliker. 1972. Ecology of the lactic streptococci: A review. J. Milk Food Technol.
35:176–185.
64. Schleifer, K.H., and O. Kandler. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications.
Bacteriol. Rev. 36:407–477.
65. Schubert, K., W. Ludwig, N. Springer, R.M. Kroppenstedt, J.-P. Accolas, and F. Fiedler. 1996. Two coryneform bacte-
ria isolated from the surface of French Gruyère and Beaufort cheeses are new species of the genus Brachybacterium:
Brachybacterium alimentarium sp. nov. and Brachybacterium tyrofermentans sp. nov. Int. J. Syst. Bacteriol. 46:81–
87.
66. Shin, M.-S., J.-H. Lee, J.J. Pestka, and Z. Ustunol. 2000. Viability of bifidobacteria in commercial dairy products during
refrigerated storage. J. Food Protect. 63:327–331.
67. Shortt, C. 1998. The probiotic century: Historical and current perspectives. Trends Food Sci. Technol. 10:411–417.
68. Stabel, J.R., E.M. Steadham, and C.A. Bolin. 1997. Heat inactivation of Mycobacterium paratuberculosis in raw milk: Are
current pasteurization conditions effective? Appl. Environ. Microbiol. 63:4975–4977.
69. Stamer, J.R. 1976. Lactic acid bacteria. In Food Microbiology: Public Health and Spoilage Aspects, ed. M.P. deFigueiredo
and D.F. Splittstoesser, 404–426. New York: Kluwer Academic Publishers.
70. Stiles, M.E., and W.H. Holzapfel. 1997. Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol.
36:1–29.
71. Stouthamer, A.H. 1969. Determination and significance of molar growth yields. Methods Microbiol. 1:629–663.
72. Sung, N., and M.T. Collins. 2000. Effect of three factors in cheese production (pH, salt, and heat) on Mycobacterium avium
subsp. paratuberculosis viability. Appl. Environ. Microbiol. 66:1334–1339.
73. Thorel, M.-F., M. Krichevsky, and V.V. Levy-Frébault. 1990. Numerical taxonomy of mycobactin-dependent mycobacteria,
emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., and
Mycobacterium avium subsp. silvaticum subsp. nov. Int. J. Syst. Bacteriol. 40:254–260.
74. Tibana, A., M.B. Warnken, M.P. Nunes, I.D. Ricciaradi, and A.L.S. Noleto. 1987. Occurrence of Yersinia species in raw
and pasteurized milk in Rio de Janeiro, Brazil. J. Food Protect. 50:580–583.
75. Vancanneyt, M., J. Mengaud, I. Cleenwerck, K. Vanhonacker, H. Hoste, P. Dawyndt, M.C. Degivry, D. Ringuet, D. Janssens,
and J. Swings. 2004. Reclassification of Lactobacillus kefirgranum Takizawa et al. 1994 Lactobacillus kefiranofaciens subsp.
kefirgranum subsp. nov. and emended description of L. kefiranofaciens Fujisawa et al. 1988. Int. J. Syst. Evol. Microbiol.
54:551–556.
76. Vaclavik, V.A., and E.W. Christian. 2003. Essentials of Food Science, 2nd ed. New York: Springer.
 Milk, Fermentation, and Fermented and Nonfermented Dairy Products 173

77. Verschuere, L., G. Rombaut, P. Sorgeloos, and W. Verstraete. 2000. Probiotic bacteria as biological control agents in
aquaculture. Microbiol. Mol. Biol. Rev. 64:655–671.
78. Vieira, E.R. 1996. Elementary Food Science, 4th ed., chap. 15. New York: Kluwer/Plenum Publishing, Inc.
79. Yukphan, P., W. Potacharoen, S. Tanasupawat, M. Tanticharoen, and Y. Yamada. 2004. Asaia krungthepensis sp. nov., an
acetic acid bacterium in the alpha-Proteobacteria. Int. J. Syst. Evol. Microbiol. 54:313–316.
80. Yokota, A., T. Tamura, M. Takeuchi, N. Weiss, and E. Stackebrandt. 1994. Transfer of Propionibacterium innocuum Pitcher
and Collins 1991 to Propioniferax gen. nov. as Propioniferax innocua comb. nov. Int. J. Syst. Bacteriol. 44:579–582.
81. Zhao, T., M.P. Doyle, and D.E. Berg. 2000. Fate of Campylobacter jejuni in butter. J. Food Protect. 63:120–122.