Download Cheese Flavor and the Genomics of Lactic Acid Bacteria

Cheese Flavor and the Genomics
of Lactic Acid Bacteria
Genomics and molecular biology are valuable in helping to define how
these bacteria contribute to the flavor and texture of cheeses
Jeffery R. Broadbent and James L. Steele
Cheese—milk’s leap toward immortality
-Clifton Paul Fadiman
umans place great value on technologies to improve the keeping qualities of foods, and one of the most
ancient of these practices depends on
lactic acid bacteria (LAB) to ferment
milk. Because these bacteria are constituents of
raw milk, cheese and other fermented milk foods
have likely been part of the diet since humans first
collected milk and held it in crude containers.
Over the centuries, these “accidental” fermentations were controlled and molded into the more
than 1,000 unique cheeses, yogurts, and fermented milks that are available today.
Because fermented dairy foods developed before the emergence of microbiology as a science,
manufacturing processes for all varieties long
relied upon naturally occurring LAB to acidify
milk. It was not until discovery of the lactic acid
fermentation by Pasteur in 1857, and development of pure LAB starter cultures later that
century, that the door to industrialized cheese
and milk fermentations opened. Since then, production of fermented milk and especially cheese
have undergone dramatic, sustained growth. In
the United States alone, for example, cheese
production increased more than 200% in the
last quarter century, and total worldwide production now runs approximately 13 million
tons per year.
To sustain such growth and productivity, the
dairy industry has evolved into a leader in
starter microbiology and fermentation technology. Decades of experience have proved that
large-scale production of uniform, high-quality
H
cheese is facilitated by the use of thoroughly
characterized starter bacteria. Thus, even
though some traditional cheese fermentations
rely on the natural souring of raw milk, the great
majority of industrialized processes use starter
cultures. Since future growth and economic vitality of the cheese industry depends on starter
cultures with known, predictable, and stable
characteristics, fundamental understanding of
LAB genetics and physiology holds enormous
value globally.
Genome Studies in Dairy
Lactic Acid Bacteria
LAB are a relatively heterogeneous group of
gram-positive cocci, coccobacilli, and bacilli
that inhabit a broad range of ecological niches,
yet share several defining characteristics, including: (i) low (⬍55 mol%) G ⫹ C content; (ii) high
acid tolerance; (iii) non-spore forming; (iv) nutritionally fastidious; (v) aerotolerant but not
aerobic; (vi) unable to synthesize porphyrins;
and (vii) strictly fermentative metabolism with
lactic acid as the major metabolic end product.
Included within this group are several species of
Lactobacillus, Lactococcus, Leuconostoc, and
Streptococcus that serve as starter cultures for
the commercial manufacture of cheese and fermented milks.
Genetics research in “food-grade” LAB began
about 35 years ago, during which period four
basic types of genetic elements were characterized in dairy LAB: plasmid DNA, transposable
elements, bacteriophages, and complete chromosomes. Representatives from all four of these
genetic elements affect milk fermentation. However, detailed knowledge of LAB chromosome
Jeffery R. Broadbent is a professor
of food science in
the Department of
Nutrition and Food
Sciences and Western Dairy Center,
Utah State University, Logan, and
James L. Steele is
a professor of food
science in the Department of Food
Science, University
of Wisconsin,
Madison.
Volume 71, Number 3, 2005 / ASM News Y 121
Table 1. Genome sequencing projects for dairy-related lactic acid bacteria and other species
Species
Strain
Genome
size
(MBp)
Lactobacillus acidophilus
L. brevis
L. casei
L. casei
L. delbrueckii subsp. bulgaricus
L. delbrueckii subsp. bulgaricus
L. delbrueckii subsp. bulgaricus
L.gasseri
L. helveticus
L.helveticus
L. johnsonii
L. plantarum
L. rhamnosus
Lactococcus lactis subsp.
cremoris
L.lactis subsp. cremoris
L. lactis subsp. lactis
Leuconostoc mesenteroides
Pediococcus pentosaceus
Streptococcus thermophilus
S. thermophilus
S. thermophilus
Bifidobacterium longum
B. longum
B. breve
Brevibacterium linens
Propionibacterium freundenreichii
ATCC700396
ATCC 367
ATCC 334
BL23
ATCCBAA-365
ATCC11842
DN-100107
ATCC 33323
CNRZ32
DPC 4571
NCC533
WCFS1
HN001
SK11
2.0
2.0
2.9
2.6
2.3
2.3
2.1
2.0
2.4
NRc
2.0
3.3
2.4
2.3
Dairy Management, Inc. and Rhodia, Inc. (U.S.)
JGI-LABGCb (U.S.)
JGI-LABGC (U.S.)
INRA (France)
JGI-LABGC (U.S.)
INRA and Genoscope (France)
Danone Vitapole (France)
JGI-LABGC (U.S.)
Dairy Management, Inc. and Chr. Hansen, Inc.(U.S.)
Teagasc and University College, Cork (Ireland)
Nestlé (Switzerland)
Wageningen Centre for Food Sciences (Netherlands)
Fonterra Research Center (New Zealand)
JGI-LABGC (U.S.)
No
Yes
Yes
No
Yes
No
No
Yes
No
No
Yes
Yes
No
Yes
MG1363
IL1403
ATCC 8293
ATCC 25745
LMD-9
LMG18311
CNRZ1066
NCC2705
DJ010A
NCIMB8807
ATCC9174
ATCC6207
2.6
2.3
2.0
2.0
1.8
1.9
1.8
2.3
2.1
2.4
3.0
2.6
Univ. Groningen (Ne); INRA (France)
INRA and Genoscope (France)
JGI-LABGC (U.S.)
JGI-LABGC (U.S.)
JGI-LABGC (U.S.)
Univ. Catholique de Louvain (Belgium)
INRA (France)
Nestlé (Switzerland)
JGI-LABGC (U.S.)
University College, Cork (Ireland)
JGI-LABGC (U.S.)
DSM Food Specialties (Netherlands)
No
Yes
Yes
Yes
Yes
No
No
Yes
Yes
No
Yes
No
Project sponsora
Public
a
As of 1 January 2005.
JGI-LABGC, Department of Energy Joint Genome Institute and Lactic Acid Bacteria Genomics Consortium
c
NR, not reported.
b
structure and organization is of particular value
because genes for all the essential housekeeping,
catabolic, and biosynthetic activities of the cell
are located in the chromosome.
As with many microorganisms, efforts to
characterize LAB chromosomes began in earnest with the advent of pulsed field gel electrophoresis (PFGE) technology during the early
1980s. Researchers quickly learned that LAB
have a single, relatively small (1.8 to 3.4 Mbp),
circular chromosome, and that genome size and
organization differ among individual species
and strains. Although PFGE is still useful in
chromosome studies, the most exciting and innovative research in this realm of microbiology
is now fueled by genomic nucleotide sequence
analysis.
Genome sequence information for the first of
several industrially important LAB starter species appeared in 2001, when Sorokin and co-
122 Y ASM News / Volume 71, Number 3, 2005
workers released the genomic DNA sequence
for Lactococcus lactis IL1403 (Table 1). Genome sequence information for several other
important dairy LAB is also now available, and
additional sequencing projects are under way
and, indeed, sequence information is being gathered for more than one strain of a particular
species (Table 1). The latter development could
provide insight to the molecular basis for commercially significant strain-dependent properties, such as the ability to produce specific flavors, propensity for autolysis, acidification
rates, and cell vitality in frozen or lyophilized
starter concentrates, which are commonly encountered in dairy LAB.
Because of their economic relevance, many of
these sequences are being mined for intellectual
property and are not yet available to the general
scientific community. Nonetheless, nucleotide
sequence data is publicly available for more than
Broadbent Finds Pleasure in Studying Lactobacilli, Watching Raptors
Jeffery Broadbent finds pleasure
studying and teaching about bacteria that, instead of being harmful, do much good. In particular,
lactic acid bacteria (LAB) provide
a central service worldwide to the
fermentation and bioprocessing
industries—helping, for instance,
to produce 13 million tons of
cheese annually. The many foods
produced with these bacteria “remain steeped in artisan tradition
and thus provide fertile opportunity to researchers like myself
who are intrigued by microbial
ecology and physiology in complex environments,” he says.
“How many other foods do we
knowingly consume that contain
millions of live bacteria?” Broadbent continues. “One of the most
basic goals of science is to teach us
to view seemingly common objects with renewed curiosity and
appreciation. I hope the next time
people find themselves enjoying a
good piece of cheese, they pause
to savor the microbiological marvel it represents.”
Broadbent, 43, is professor of
dairy microbiology in the department of nutrition and food sciences at Utah State University in
Logan City, where he focuses on
this diverse group of gram-positive cocci, coccobacilli, and bacilli. “The future of LAB research
is bright with promise; with genome sequences and molecular biology tools now available for several key species, opportunities to
investigate LAB evolution, genetics, physiology, and metabolism
have never been greater,” he says.
His interest in science, however, did not originate with
cheese-making bacteria, but instead flourished from a “lifelong
enchantment with wild fauna and
flora, particularly birds,” he says.
“Some of my earliest memories
are rooted in these experiences—
catching frogs in the Utah mountains with my grandfather, being
spellbound as a young boy by the
dazzling cacophony of color and
sound in cage-loads of exotic
birds at an open air market.”
A native of Utah, Broadbent
moved with his family as a young
child to places that further fueled
these interests. For example, his
family spent five years in Sao
Paulo, Brazil, “where my fascination with birds and other wildlife
became indelibly imprinted into
my psyche, and also where I
learned to feed my curiosity
through a fairly voracious reading
habit,” he says.
By the time Broadbent began
high school, in Tempe, Ariz., he
had blossomed into a competent
amateur ornithologist, and was
particularly interested in birds of
prey. Determined then to pursue a
career in wildlife biology, he became involved in several research
projects in his biology and zoology courses, and eventually spent
the summer before his senior year
as a volunteer ornithologist doing
field research on a peregrine falcon project in the Gila National
Forest of New Mexico.
Broadbent enrolled at Utah
State University in the fall of
1979, but his undergraduate studies were disrupted several times
due to financial problems, keeping him from completing his
bachelor of sciences degree until
1987. By then, his coursework focus had shifted from wildlife research to microbial biotechnology, a field he found equally
fascinating.
“After graduation, I had the re-
markable good fortune of landing
a research tech position in the laboratory of Dr. Jeffrey Kondo, a
world-renowned geneticist in the
lactic acid bacteria community,”
Broadbent says. “Jeff’s thorough
and articulate research opened my
eyes to the elegance of lactic acid
bacteria and introduced me to a
field that, to this day, is bright
with opportunity.”
He became a graduate fellow
and, while still under Kondo’s supervision, received his Ph.D. in
nutrition and food sciences in
1992. Later, he accepted a position on the faculty at Utah State.
While Broadbent’s research
and academic interests shifted
from wildlife to lactic acid bacteria, he continues to pursue those
early passions. He and his wife—a
high school special education
teacher—and their two daughters, 12 and 11, maintain “what
sometimes feels like a small zoo”
in their home, he says. It includes
a falcon, four dogs, a cat, a hedgehog, fish, a loft of homing pigeons, and three egg-laying chickens. “As you might expect,
leaving town for a family vacation
requires some planning,” he says.
Broadbent also is a practicing
falconer, having engaged in the
sport since high school. With his
falcon and two of the dogs, both
English Setters, he pursues ducks,
pheasant, grouse, and partridge.
“I am still enchanted by raptors,
and falconry provides the connection to wildlife I have sought
throughout my life,” he says.
Marlene Cimons
Marlene Cimons is a freelance writer
who lives in Bethesda, Md.
Volume 71, Number 3, 2005 / ASM News Y 123
TABLE 2. Components of the Lactobacillus helveticus CNRZ32
proteolytic enzyme system isolated before and after genome
sequence determination
Genes isolated prior to
sequencing project
(1990 –2001)
Proteinases:
prtH
Endopeptidases:
pepE, pepO, pepO2
New genes from genome annotation
(2001–2004)
prtH2 plus 9 additional proteases
pepE2, pepF, pepO3, plus 2
glycoprotein endopeptidases
Aminopeptidases:
pepC, pepN, pepX
pepC2 plus 7 additional
aminopeptidases
Di-Tripeptidases:
pepD, pepI, pepQ, pepR
Oligo- and di-tripeptide transport
systems:
None
pepD2, pepD3, pepD4, pepQ2, pepT1,
and pepT2
oppA,oppA2, oppB-D, oppF, and dtpA,
dtpA2, and dtpT
Multiple amino acid transporters
half of the sequenced LAB strains (Table 1), and
10 of the 14 publicly accessible sequences were
contributed as part of a joint venture between
the Department of Energy Joint Genome Institute and the U.S.-based Lactic Acid Bacteria
Genomics Consortium (LABGC). The LABGC
mission is to advance academic and industrial
research on LAB through release of genome sequence information for microorganisms prominently associated with the fermented foods industry. (For additional information on the LABGC
effort, see http://wineserver.ucdavis.edu/people
/Faculty/mills/LABGC/lab.htm).
Cheese Flavor Basics: Add
Lactic Acid Bacteria
Converting bland and rubbery fresh curds into a
delicious mature cheese is a complex and dynamic process whose intricacies are dictated by
the type and composition of milk being used, the
cultures and enzymes that are added, and the
specific manufacturing and ripening regimens
that are applied. Many cheese types are stored at
low temperature for months or years to attain
their characteristic flavor and body attributes.
During storage, the microorganisms and enzymes that are trapped in the cheese matrix act
on carbohydrates, citrate, proteins, and lipids in
a manner that is heavily influenced by the curd
microenvironment and which ultimately yields
distinct types of cheeses.
124 Y ASM News / Volume 71, Number 3, 2005
Although a link between LAB and cheese
flavor was first postulated more than 100
years ago, complexities in microbiology, enzymology, and cheese microenvironments
confounded early efforts to establish a definitive role for these bacteria affecting flavors.
However, in the late 1950s, Elisabeth Sharpe
and coworkers at the Institute for Food Research in Shinfield, England, developed technologies to manufacture cheeses aseptically,
enabling researchers to prove LAB are essential for flavor development in Cheddar and
other cheeses.
LAB that contribute to this process may
include deliberately added starters, adjunct
bacteria (select strains that intensify or accelerate flavor development), and adventitious
species, called nonstarter lactic acid bacteria
(NSLAB), that enter curd from the processing environment. While many different LAB
species may affect cheese flavor, research in
this area mainly focuses on Lactococcus lactis, which serves as the starter bacterium for
Cheddar, Gouda, and many other cheeses, and
on dairy-related species of Lactobacillus (Fig.
1). Interest in lactobacilli such as Lactobacillus
helveticus and Lactobacillus delbrueckii subsp.
bulgaricus stems from their widespread use as
both starter and adjunct cultures, and because
NSLAB populations are almost always dominated by facultatively heterofermentative Lactobacillus sp. such as Lactobacillus casei.
Because of the role of LAB in developing
flavor, efforts to define its biochemical basis in
cheese focus on the physiology of these microorganisms. The numbers of starter bacteria
commonly exceed 109 CFU per gram of cheese
when ripening begins (Fig. 2), but the microenvironment of ripening cheese is harsh. For instance, it is typified by an absence of residual
lactose, high levels of NaCl, low pH, and low
temperature. Those conditions extract a toll on
starter viability, and, typically, a sizable fraction
of the starter cells undergo autolysis, which releases intracellular enzymes and other cellular
components into the cheese matrix where they,
too, can influence ripening.
Meanwhile, NSLAB populations, whose initial numbers are typically below 102 CFU/g,
begin to grow and eventually plateau at cell
densities of 107-109 CFU/g after 3–9 months of
aging (Fig. 2). Depending on the species that is
used and whether a particular strain can grow in
FIGURE 1
Colored scanning electron micrographs of representative cheese starter (A-C) and nonstarter (D-F) lactic acid bacteria (LAB). Species shown
include Lactobacillus helveticus (A), Lb. delbrueckii subsp. bulgaricus (B), Lactococcus lactis (C), Lb. casei (D), Pediococcus pentosaceus (E),
and Lb. brevis (F). Images provided by B. McManus and J. Broadbent.
ripening cheese, populations of adjunct bacteria
may mirror those of the starter or NSLAB fractions.
Key Modes of Microbial Action
in Cheese Ripening
Starter, adjunct, and NSLAB collectively influence flavor development through several basic
mechanisms that include fermenting lactose,
converting milk proteins (primarily caseins) into
peptides and free amino acids, and breaking
down citrate, lipids, esters, and amino acids into
volatile aroma compounds.
Fermenting lactose into L-lactic acid is a primary function of any starter culture in cheese
manufacture. Acid productivity is critical for
controlling cheese quality because the culture
determines the final pH and mineral content of
the curd, which affects the protein structure and
amount of residual coagulant in the curd, and,
thus, texture and flavor properties. Lactate itself
is also a component of cheese flavor and in
Swiss-type cheeses serves as a key nutrient for
propionibacteria. They convert it into propionic
acid, which is another important flavor component, and carbon dioxide, which gives the cheese
its “eyes.” If starter bacteria rapidly deplete
residual milk sugar in the curd, they can help to
prevent its use as a substrate for undesirable
adventitious bacteria, such as heterofermentative Lactobacillus brevis, that can produce serious flavor and texture defects.
Proteolysis and its secondary reactions also
play a major role in bacterially ripened cheeses,
making casein hydrolysis and its relationship to
flavor development an area of intense research
interest for decades. The hydrolysis of intact
caseins is almost exclusively catalyzed by the
coagulant and endogenous milk proteinases
(e.g., plasmin), while LAB proteinases and pep-
Volume 71, Number 3, 2005 / ASM News Y 125
FIGURE 2
10
Non-starter lactics
Log 10 CFU/g cheese
8
6
4
Lactococcal starter
2
0
Ripening time
Microbiology of ripening Cheddar cheese.
tidases are responsible for producing water-soluble peptides and free amino acids.
Together, primary and secondary proteolysis
of caseins influences cheese flavor in at least
three significant ways. First, casein network
breakdown softens cheese texture, which facilitates the release of flavor compounds when the
cheese is consumed. Second, some of the lowmolecular-weight peptides produced in these reactions directly affect flavor, but this consequence is generally negative since these peptides
impart bitterness. Third, the free amino acids
that are liberated can also directly affect flavor.
For instance, glutamate and aspartate residues
enhance flavors.
More commonly, released amino acids are
precursors for a broad range of potent aroma
compounds. These reactions are of particular
interest because a growing body of evidence
indicates that LAB’s converting of free amino
acids into aroma compounds is the rate-limiting
step in the development of mature cheese aromas. The products of amino acid catabolism,
which may arise via decarboxylation, deamination, transamination, desulfuration, or side
chain removal, can impart desirable or undesirable flavor attributes.
Much of the research on amino acid catabolism by LAB has been directed toward the fates
of aromatic, sulfur-containing, and branchedchain classes of amino acids because of their key
126 Y ASM News / Volume 71, Number 3, 2005
role in aroma. For example, converting methionine into volatile sulfur
compounds such as methanethiol,
hydrogen sulfide, dimethyl sulfide,
and dimethyl trisulfide is thought to
contribute desirable “sulfur” flavors
to many cheese types, whereas
breaking down leucine is the likely
source of a desirable nutty flavor
note in Cheddar cheese. In contrast,
breaking down aromatic amino acids contributes several undesirable
“off-flavors” to cheese, including derivatives such as indole, skatole,
[para]-cresol, and phenyl acetaldehyde.
Free fatty acids formed by lipase
or esterase activity on milk fat also
directly affect cheese flavor, and can
have further effects by serving as precursors for esters and other flavor
compounds. Moreover, esterases and lipases
catalyze the hydrolysis or synthesis of esters,
depending on cheese water activity and levels of
other available fatty acids and alcohols. Enzymes involved in these reactions may come
from rennet pastes, from milk itself, and from
starter and nonstarter LAB.
It is well established, for example, that pregastric lipases and esterases from ruminants are
responsible for the sharp, fatty acid-based flavors that characterize some Italian cheeses. In
cheeses such as Parmesan that do not use pregastric lipases and esterases, however, flavor
notes associated with lipolysis are probably due
to indigenous milk enzymes and microbial enzymes. Most LAB lack lipolytic activity and
have very low esterolytic activity, but in cheese
with long ripening times these cells can generate
enough free fatty acids and esters to impact
flavor.
Finally, LAB use citrate to produce succinate
or diacetyl. Succinate, a compound with
monosodium glutamate-like flavor-enhancing
properties, can be isolated from several cheese
varieties, and sensory studies suggest it contributes savory flavor to Swiss-type cheese and to a
full, aged flavor in Cheddar. In Swiss and other
cheeses where Propionibacterium freudenreichii
subsp. shermanii attain high numbers, succinate
production is attributed to aspartic acid catabolism by the propionibacteria. In Cheddar and
other varieties, however, NSLAB produce succinate from citrate via the reductive tricarboxylic
acid pathway.
The other important citrate-derived flavor
component, diacetyl, imparts a “buttery” note
whose importance in butter, buttermilk, and
some cheese types has been recognized for decades. Diacetyl is formed by oxidative decomposition of ␣-acetolactate, an intermediate in the
pathways for pyruvate metabolism and amino
acid biosynthesis. In recent years, detailed
knowledge of citrate metabolism and diacetyl
production has yielded effective strategies for
engineering L. lactis strains to enhance diacetyl
production.
Genomics Will Propel Further
Advancements
Though great progress has been made toward
understanding LAB physiology and the processes that drive cheese flavor development,
much remains to be learned about these reactions. Currently, significant research advances
depend on recombinant DNA technology. The
complexity of the peptidase enzyme system in
LAB, for example, confounded earlier efforts to
establish the role of individual enzymes in casein
hydrolysis and cheese ripening. However, tools
for constructing isogenic strains that differ in the
activity of only single peptidases now are providing researchers with an effective approach to
determine how individual enzymes contribute to
cell growth and cheese properties.
By combining molecular tools with genomics,
researchers in industry and academia are creating even greater opportunities to investigate the
means by which LAB act within and respond to
cheese and milk microenvironments. Hence, research to better define the relationship between
LAB physiology and flavor development should,
whenever possible, focus on strains that: (i) possess established flavor-producing capabilities;
(ii) are amenable to genetic manipulation; and
(iii) are analyzed at the genome sequence level.
One such candidate strain for forthcoming
study is Lactobacillus helveticus CNRZ32, a
commercial cheese flavor adjunct that can reduce bitterness and intensify flavor development. We recently assembled a draft (fourfold
coverage) genome sequence for CNRZ32, and
are currently using that sequence to investigate
mechanisms by which this strain affects cheese
flavor.
For example, because proteolysis plays such a
critical part in cheese ripening, one of us (Steele
and his collaborators) spent more than a decade
cloning and characterizing CNRZ32 genes that
encode proteolytic enzymes (Table 1), developing gene transfer systems, and constructing a
series of single and multiple deletion mutants
lacking functional genes for many of those enzymes. Despite such concerted efforts, initial
annotation of the genome sequence revealed a
large number of additional genes in CNRZ32
whose products are predicted to contribute to
the proteolytic enzyme system of this bacterium.
From our perspective, such data underscore
both the power of genome sequence information
for applied bacteriology, and the challenges one
must face in interpreting and applying that information. Although sequencing efforts expanded the genetic database for the CNRZ32
proteolytic enzyme system by about fivefold,
efforts to confirm and characterize all the new
gene assignments will require more time and
resources. Nonetheless, functional analysis of
the newly discovered endopeptidase genes has
already identified enzymes with important roles
in the hydrolysis of bitter peptides in cheese.
Functional genomics is also being used to
investigate pathways for amino acid biosynthesis and catabolism in Lactobacillus helveticus
CNRZ32. In cheese, converting amino acids
into volatile cheese flavor compounds may occur directly or through interactions among
starter, adjunct, and NSLAB components. Some
strains can independently convert amino acids
into aroma compounds, while others may produce or degrade only one or more metabolic
intermediates.
The basis for this phenomenon has not been
determined, but all LAB are auxotrophic for one
or more amino acids, and the primary mechanism for amino acid breakdown by LAB involves the reversible action of enzymes involved
in biosynthetic pathways. Thus, much of the
interplay that occurs between LAB in amino
acid catabolism probably reflects the nature of
amino acid auxotrophies among the different
bacteria in cheese. Since the primary sequences
of most enzymes involved in these reactions are
relatively well-conserved, access to genome sequence information should dramatically enhance our ability to predict—and test—how in-
Volume 71, Number 3, 2005 / ASM News Y 127
dividual organisms contribute to amino acid
catabolism in cheese.
Dairy technologists and microbiologists have
identified many of the fundamental mechanisms
by which LAB affect flavor, and this knowledge
is facilitating industry efforts to accelerate and
intensify flavors. There is still a great deal to be
learned, however, and the combined strengths
of genomics and molecular biology tools are
certain to play a leading role in research to
define the molecular dynamics of LAB in producing fine cheeses.
SUGGESTED READING
Beresford, T. P., N. A. Fitzsimons, N. L. Brennan, and T. M. Cogan. 2001. Recent advances in cheese microbiology. Int. Dairy
J. 11:259 –274.
Broadbent, J. R. 2001. Genetics of lactic acid bacteria, pp. 243–299. In J. L. Steele and E. H. Marth (eds.), Applied Dairy
Microbiology, 2nd ed. Marcel Dekker, Inc., New York.
Davidson, B. E., N. Kordias, M. Dobos, and A. J. Hillier. 1996. Genomic organization in lactic acid bacteria. Antonie van
Leeuwenhoek 70:161–183.
Klaenhammer, T., E. Altermann, F. Arigoni, A. Bolotin, F. Breidt, J. Broadbent, R. Cano, S. Chaillou, J. Deutscher, M.
Gasson, M. van de Guchte, J. Guzzo, T. Hawkins, P. Hols, R. Hutkins, M. Kleerebezem, J. Kok, O. Kuipers, M. Lubbers, E.
Maguin, L. McKay, D. Mills, A. Nauta, R. Overbeek, H. Pel, D. Pridmore, M. Saier, D. van Sinderen, A. Sorokin, J. Steele, D.
O’Sullivan, W. de Vos, B. Weimer, M. Zagorec, and R. Siezen. 2002. Discovering lactic acid bacteria by genomics. Antonie
van Leeuwenhoek 82:29 –58.
Olson, N. F. 1990. The impact of lactic acid bacteria in cheese flavor. FEMS Microbiol. Rev. 87:131–148.
Yvon, M., and L. Rijnen. 2001. Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11:185–202.
128 Y ASM News / Volume 71, Number 3, 2005