J. Dairy Sci.
93:4435–4454
doi:10.3168/jds.2010-3327
© American Dairy Science Association®, 2010.
Invited review: Lactobacillus helveticus—A thermophilic
dairy starter related to gut bacteria
L. Slattery,* J. O’Callaghan,* G. F. Fitzgerald,† T. Beresford,*1 and R. P. Ross*‡
*Teagasc, Moorepark Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
†Microbiology Department, University College Cork, Ireland
‡Alimentary Pharmabiotic Centre, Cork, Ireland
ABSTRACT
The strain Lactobacillus helveticus DPC4571 has
emerged as a promising flavor adjunct culture for
Cheddar cheese given that it is consistently associated
with improved flavor. The availability of the complete
genome sequence of Lb. helveticus DPC4571 has en-
abled the search for the presence or absence of specific
genes on the genome, in particular those of techno-
logical interest. Indeed, this analysis has facilitated a
greater understanding into the functioning of lactic
acid bacteria as a whole. The biochemical pathways
of Lb. helveticus responsible for producing flavor com-
pounds during cheese ripening are poorly understood
but now with the availability of a complete genomic
sequence are ripe for exploitation. Bioinformatic analy-
sis of the genome of Lb. helveticus DPC4571 has re-
vealed a plethora of genes with industrial potential
including those responsible for key metabolic functions
that contribute to cheese flavor development such as
proteolysis, lipolysis, and cell lysis. In addition, it has
been demonstrated that Lb. helveticus has the potential
to produce bioactive peptides such as angiotensin con-
verting enzyme inhibitory activity in fermented dairy
products, demonstrating the therapeutic value of this
species. A most intriguing feature of the genome of
Lb. helveticus DPC4571 is the remarkable similarity in
gene content with many intestinal lactobacilli, although
originating from considerably different environments.
Bioinformatic analysis demonstrated that 65 to 75% of
genes were conserved between the commensal and dairy
lactobacilli, which allowed key niche-specific gene sets
to be described. This review focuses on the isolation,
characterization, and exploitation of the Lb. helveticus
species with particular emphesis on taking into consid-
eration recent genome sequence data for Lb. helveticus
and other Lactobacillus species.
Received April 8, 2010.
Accepted May 22, 2010.
1
Corresponding author: tom.beresford@teagasc.ie
4435
Key words: Lactobacillus helveticus, starter culture,
flavor adjunct, bioinformatics
INTRODUCTION
Lactobacillus helveticus belongs to a group of organ-
isms collectively known as lactic acid bacteria (LAB),
which are so named because they produce lactic acid
as a major product of their metabolism. These organ-
isms occupy a diverse set of ecological niches ranging
from foods, such as fermented dairy products, wine,
and sourdough, to more diverse environments includ-
ing soil, plants, and the human gastrointestinal (GI)
tract. Lactobacillus helveticus was first described by
Orla-Jensen in 1919 as an isolate from Emmental
cheese (Naser et al., 2006). The species is character-
ized as having the ability to grow at a relatively high
temperature (~55°C) and being proteolytic. It is an
important food-associated species traditionally used in
the manufacture of Swiss-type cheeses and long-ripened
Italian cheeses such as Emmental, Gruyere, and Provo-
lone (Stiles and Holzapfel, 1997; Giraffa et al., 2000).
In addition, Lb. helveticus is used in the production
of fermented drinks such as Evolus (Valio Ltd., Valio,
Finland) and Calpis (Calpis Food Industry Co. Ltd.,
Tokyo, Japan), which have properties associated with
reduction of blood pressure following ingestion. This
is due to the production of significant levels of bioac-
tive tripeptides that are known to inhibit the enzyme
angiotension converting enzyme (ACE). Given the
commercial importance of the species, the genome of
a representative dairy isolate, Lb. helveticus DPC4571,
was recently sequenced to completion and compared
with other genomically characterized lactobacilli (Fig-
ure 1). Remarkably, this analysis demonstrated that its
nearest comparator is the gut organism Lb. acidophilus
NCFM, which suggests that the difference between the
dairy and gut species is caused by a relatively small but
highly specific gene set (Callanan et al., 2008).
This review concerns the isolation, characterization,
and exploitation of the Lb. helveticus species especially
taking into consideration recent genome sequence data
for Lb. helveticus and other Lactobacillus species. The
4436
Figure 1. Completed sequence of Lactobacillus helveticus DPC 4571(Callanan et al., 2008). ORF = open reading frame; IS = insertion se-
quence; GC = guanine-cytosine.
Lb. helveticus DPC4571 strain, which was isolated from
cheese whey (Callanan et al., 2008), was selected for
genome sequence analysis because it produced cheese
of consistently high quality when used as an adjunct
starter culture, which was attributed to its characteris-
tics of rapid lysis and high proteolytic activity (Kiernan
et al., 2000; Hannon et al., 2003; Kenny et al., 2003;
Hickey et al., 2006). Genome analysis has demonstrated
that although Lb. helveticus DPC4571 and Lb. acido-
philus NCFM share 98.4% identity, they have adapted
to grow in very different environments (i.e., dairy prod-
ucts and the human gut, respectively). These strains
represent the most closely related dairy and GI tract
bacteria studied to date. Comparative analysis of their
complete genome sequences provides important insights
into the evolution of dairy cultures and GI tract bac-
teria. Lactobacillus helveticus DPC4571 possesses many
nonfunctioning gut-specific genes resulting from dele-
tions, nonsense mutations, and truncations. This selec-
tive loss of gene function from the DPC4571 genome
points to the role of those genes in the gut environment
and consequently their relative unimportance in dairy
fermentations (Callanan et al., 2008).
TAXONOMY AND BIOCHEMICAL CHARACTERISTICS
Lactobacillus is a diverse genus that has been well
characterized and comprises at least 87 species (http://
Journal of Dairy Science Vol. 93 No. 10, 2010
SLATTERY ET AL.
www.ncbi.nlm.nih.gov/genomes/lproks.cgi). The lac-
tobacilli form part of a group of bacteria commonly
referred to as LAB that also includes the core genera
Leuconostoc, Pediococcus, Lactococcus, and Streptococ-
cus as well as the more peripheral genera Aerococcus,
Carnobacterium, Enterococcus, Oenococcus, Terageno-
coccus, and Weisella. In general, hexose fermentation by
the LAB occurs via 2 main pathways. Under conditions
of excess glucose and limited oxygen, homolactic LAB
catabolize 1 mole of glucose via the Embden-Meyerhof
pathway (glycolysis) to yield 2 moles of pyruvate. The
cellular redox balance is maintained through the oxida-
tion of NADH, concomitant with reduction of pyruvate
to lactic acid, yielding 2 moles of ATP per mole of
glucose consumed. In contrast, heterofermentative LAB
utilize the pentose phosphoketolase pathway. In this
case, 1 mole of glucose-6-phosphate is dehydrogenated
to 6-phosphogluconate and subsequently decarboxy-
lated to yield 1 mole of CO2. The resulting pentose-
5-phosphate is cleaved into 1 mole of glyceraldehyde
phosphate and 1 mole of acetyl phosphate. Then, glyc-
eraldehyde phosphate is further metabolized to lactate
as in the homofermentative pathway, with the acetyl
phosphate reduced to ethanol via acetyl-CoA and acet-
aldehyde intermediates. The net yield of ATP from the
heterofermentative metabolism of glucose is 1 mole of
ATP per mole of glucose consumed. Theoretically, all
the end-products are produced in equimolar quantities
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
from the catabolism of 1 mole of glucose (Sneath et al.,
2009).
Lactobacillus is so named because its members are
bacilliform cells that convert lactose and other sugars
to lactic acid as a major metabolic end-product. They
belong to the phylum Firmicutes, class Bacillus, order
Lactobacillales. Members of the Lactobacillus genus
are rod-shaped, gram-positive, catalase-negative, acid-
tolerant, and nonsporulating anaerobic bacteria with a
low GC content in their DNA, generally about 40 mol%
(Holt et al., 1994). Traditionally, the genus is divided
into 3 main groups based on fermentation patterns:
obligatory homofermentative, obligatory heterofermen-
tative, and facultatively heterofermentative species.
Lactobacillus helveticus is a member of the homofer-
mentative group and displayes thermophilic tendencies
with an optimum growth temperature of 42 to 45°C.
The growth of Lb. helveticus is optimum at pH 5.5 to
5.8 and the species is regarded as fastidious in that it
has complex nutritional requirements for amino acids,
peptides, nucleotide bases, vitamins, minerals, fatty
acids, and carbohydrates. Unlike other lactobacilli,
the species is auxotrophic for many amino acids. For
example, Lb. plantarum is prototropic for most amino
acids. Indeed, it has been established that Lb. helveti-
cus CNRZ32 requires 14 amino acids for growth and
that this level of auxotrophy is primarily due to gene
absence rather than loss of gene function due to point
mutations, insertions, or deletions. This prediction was
made using a 9-fold draft-quality genome sequence
for Lb. helveticus CNRZ32 to reconstruct amino acid
biosynthetic pathways in this organism. The use of
single-amino-acid omission studies in a chemically de-
fined medium determined that Lb. helveticus CNRZ32
was auxotrophic for Arg, Glu, His, Ile, Leu, Lys, Met,
Phe, Pro, Thr, Trp, Tyr, Val, and either Asp or Asn
(Christiansen et al., 2008). Lactic acid produced by Lb.
helveticus is an equal mixture of l-(+)- and d-(−)- iso-
mers. For food applications, l-(+)- lactic acid is consid-
ered the ideal isomer because d-(−)- lactic acid is not
metabolized in humans; thus, there is much ongoing
research to optimize the production of the l-(+)- lactic
acid isomer solely (Kyla-Nikkila et al., 2000).
Lactobacillus helveticus is closely related to Lactoba-
cillus acidophilus with respect to DNA-DNA hybridiza-
tion, biochemical features, and 16S rRNA gene sequenc-
es. Indeed, Lb. acidophilus, Lactobacillus gallinarum,
and Lactobacillus crispatus together form a cluster of
closely related species. Interestingly, Lb. helveticus and
Lb. acidophilus branch together with other gut bacteria
in terms of phylogenetic relatedness (O’Sullivan et al.,
2009; Figure 2). The recent sequencing of Lb. helveti-
cus DPC4571 enabled comparison with Lb. acidophilus
NCFM, which demonstrated that gene-gene DNA simi-
4437
Figure 2. Phylogenetic supertree of the 10 selected lactic acid
bacteria of Lactobacillus (L) and Streptococcus (S) genera and Bacillus
subtilus. The supertree was calculated from 47 individual ribosomal
protein trees. All branches are supported at >75% bootstrap values
(O’Sullivan et al., 2009).
larity is approximately 80 to 85% between the dairy
and gut organisms (Callanan et al., 2008).
LB. HELVETICUS GENOME ANALYSIS
The complete genome sequence of Lb helveticus
DPC4571 along with a large number of other LAB
genomes are now available (Bolotin et al., 1999; Kleer-
ebezem et al., 2003; Pridmore et al., 2004; Altermann
et al., 2005; Chaillou et al., 2005; Claesson et al., 2006;
Makarova et al., 2006; van de Guchte et al., 2006; Weg-
mann et al., 2007; Callanan et al., 2008). To date, the
data from these studies demonstrate that all have com-
paratively small genomes (~2.4 Mb) and share several
common metabolic pathways. Despite this phylogenetic
similarity, they occupy a diverse set of environmental
niches, which suggests that considerable genetic adap-
tation has occurred during their evolution. The use of
comparative genomics has allowed the identification
of key gene sets that facilitate a variety of lifestyles
including adaptation to the gut or dairy environment
(O’Sullivan et al., 2009; Table 1).
Currently (May 2010), 21 complete Lactobacil-
lus genome sequences exist, with 78 projects ongoing
(http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi).
This abundance of sequence data has allowed investi-
gation of the genetic basis of adaptation to a specific
niche. Lactobacillus delbrueckii ssp. bulgaricus and Lb.
helveticus belong to the dairy niche, whereas Lb. acido-
philus, Lactobacillus gasseri, and Lactobacillus johnsonii
belong to the gut niche. Others, such as Lactobacillus
brevis, Lb. plantarum, Lactobacillus salivarius, and Lac-
tobacillus sakei, are multi-niche bacteria, which thus
Journal of Dairy Science Vol. 93 No. 10, 2010
4438 SLATTERY ET AL.

possess a more elaborate metabolic, regulatory, and

Human gastrointestinal (GI) tract

transport machinery compared with the niche-specific
species (Callanan et al., 2008; O’Sullivan et al., 2009;
Figure 2).
The genome data have provided insights into the

Environmental niche
different sets of metabolic capabilities necessary for

Human GI tract

Human GI tract
Human GI tract
each Lactobacillus species to evolve to occupy a spe-
cific environmental niche. The main driving forces for

Multi-niche
Multi-niche

Multi-niche

Multi-niche
niche adaptation are environmental stress and nutrient
availability. The presence and diversity of certain genes

Dairy

Dairy

Dairy
involved in sugar metabolism appears to be one of the
main determinants of the particular niche that a spe-
cies can inhabit, whether it is gut, dairy, or multi-niche.

Terminal ileum of human

In dairy microorganisms, the main sugar encountered
is lactose, whereas gut microorganisms are exposed to

Human GI tract
Human GI tract
Fermented meat
a wider variety of sugars derived from complex carbo-

Human saliva
Isolated from

Human feces
hydrates present in food. Some examples of this are

Infant feces
the presence of numerous 6-phospho-β glucosidase

Cheese

Yogurt

Yogurt
Silage
gene types that may be involved in the hydrolysis of
disaccharides in gut or multi-niche strains compared
with fewer copies present in the dairy strains. Also,

Pseudogenes, n
α-1,6-glucosidase enzymes involved in the breakdown
of oligosaccharides released from starch by amylases are

217
0
0
30
49
533
39
180
49
48
49
more numerous in multi-niche Lb. plantarum and gut
strains, with most common probiotic strains containing
2 copies. Lactobacillus helveticus DPC 4571 is the only
dairy strain to possess this type of enzyme (1 copy), and
Genes,

1,618
1,864
1,821
1,884
1,765
1,562
3,051
1,890
2,314
1,898
2,314
Table 1. General genome features of selected lactic acid bacteria (O’Sullivan et al., 2009)

detailed sugar utilization of Lb. helveticus DPC 4571 is

n

shown in Figure 3. Maltose is the least abundant disac-

charide in the environment and is usually only present
content, %
Guanine-
cytosine

in locations, such as the gut, where starch breakdown

37.1
34.7
34.6
41.3

49.7
44.4
39.1
46.1
35.3
33

46
products are present. Maltose-6-phosphate glycosidase
is found predominantly in gut microorganisims and is
even absent from the multi-niche lactobacilli. It ap-
size, kbp
Genome

pears that for adaptation to the gut niche there is a

3.34

2.35
2.1

1.9
2.1
1.9

1.8

1.9
22.9
2
2

strong association with the presence of higher numbers

of glycosidase genes. In the case of amino acid synthe-
sis, Lb. helveticus DPC4571, like other dairy species,
is very fastidious unlike the multi-niche species such
Lactobacillus delbrueckii ssp. bulgaricus ATCC11842

as Lb. plantarum, which is prototrophic for 18 or 19 of

the amino acids necessary for protein synthesis. This
reflects adaptation of the dairy strain to the peptide-
rich milk environment where transport and hydrolysis
Streptococcus thermophilus LMG18311

Lactobacillus reuteri F275 JCM 1112

of peptides fulfills the amino acid requirements of the

Lactobacillus helveticus DPC4571

Lactobacillus gasseri ATCC33323

Lactobacillus plantarum WCFS1

cell (O’Sullivan et al., 2009).

Lactobacillus acidophilus NCFM
Lactobacillus johnsonii NCC533

Lactobacillus salvarius UCC118

Lactobacillus brevis ATCC3567

Lactobacillus helveticus DPC4571 shares 98.4% iden-

tity at the amino acid level with Lb. acidophilus NCFM
Lactobacillus sakei 23K

(Callanan et al., 2008) although Lb. helveticus DPC4571

is a dairy strain and occupies a different environmental
niche than the gut organism NCFM of the acidophilus
group. Lactobacillus helveticus DPC4571 has adapted
extensively to the dairy environment niche possessing
Species

nonfunctioning gut-specific genes resulting from dele-

tions, nonsense mutations, and truncations. A promi-
Journal of Dairy Science Vol. 93 No. 10, 2010
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
Figure 3. Sugar utilization of Lactobacillus helveticus DPC 4571. Gal = galactose; gal-P = galactose-phosphate; UDP-gal = uridine
diphosphate-galactose; UDP-glu = uridine diphosphate-glucose; glu-6-p = glucose-6-phosphate; mannose PTS = mannose phosphotransferase
system.
nent example of this is the presence of a frame-shifted,
nonfunctioning bile salt hydrolase gene in Lb. helveticus
DPC4571. A functioning bile salt hydrolase gene (bsh)
would be necessary for survival in the gastrointestinal
tract and thus the presence of an ancestral bsh gene in
Lb. helveticus DPC4571 suggests that Lb. helveticus has
only recently lost gut-related functions. The extraordi-
nary degree to which gene synteny (75%) and homology
(98.4%) have been maintained between Lb. acidophilus
NCFM and Lb. helveticus DPC4571 indicates that the
gene loss involved in the reductive evolution of the dairy
strain occurred in a tightly controlled manner. It is
tempting to speculate that the fastidious metabolism of
Lb. acidophilus (relative to multi-niche species such as
Lb. plantarum) indicates that Lb. acidophilus possesses
the minimum complement of genes necessary for the
gut environment. Comparative analysis of a range of
Lactobacillus genomes has allowed the linkage of their
genetic adaptation to their chosen environmental niche
to be established. The presence and absence of certain
genes involved in sugar metabolism, lipid metabolism,
and the proteolytic system, as well as several restriction
enyme and mucin-binding genes, enabled the niche of
individual strains to be predicted (O’Sullivan et al.,
2009).
4439
LB. HELVETICUS DPC4571 IN CHEESE
MANUFACTURE AND RIPENING
Given that Lb. helveticus is commonly used in the
production of Italian-type cheeses or as a flavor adjunct
for Cheddar cheeses, several studies have characterized
the organism in terms of this role. Lactobacillus helve-
ticus is mainly used as starter in Swiss-type cheeses. It
has been shown to lyse early in Swiss cheeses at maxi-
mal growth, releasing its intracellular peptides, which
remain active for several weeks in the curd (Valence et
al., 2000; Deutsch et al., 2003). For example, previous
work by Hickey et al. (2007) and Hannon et al. (2003)
has shown that Lb. helveticus DCP4571 could be used
alone as a novel starter for Cheddar cheese production
to produce a strongly flavored cheese within a shorter
ripening period (2 mo) than cheese made with conven-
tional starter systems. In addition, the use of the Lb.
helveticus DPC4571 strain as an adjunct to traditional
starter cultures was examined in cheese ripening. One of
the main features of this strain is its ability to autolyze
during cheese ripening as evidenced by the release of
intracellular enzymes such as d-lactate dehydrogenase
and prolinase (0.25 and 0.93 μmol/min per mL of cheese
juice, respectively), which were not detected in control
cheeses. The release of another intracellular enzyme,
Journal of Dairy Science Vol. 93 No. 10, 2010
4440 SLATTERY ET AL.
proline iminopeptidase, was observed to be 2.3-fold
higher in the cheese made with DPC4571 compared
with the control cheeses. This was also demonstrated
when cheese was made commercially with Lb. helveticus
DPC4571; proteolysis was increased and the cheese had
improved flavor compared with control cheese, suggest-
ing a correlation between autolysis (with the resulting
increase in proteolysis) and improved flavor (Kiernan
et al., 2000). In addition, Lb. helveticus DPC4571 also
produced the highest levels of free fatty acids (P <
0.05) during ripening in Cheddar cheese compared with
cheeses made with single starter stains Lactococcus lac-
tis ssp. lactis 303, Lc. lactis ssp. cremoris HP, and Lc.
lactis ssp. cremoris AM2 (Hickey et al., 2007).
In a study that compared the effects of various Lb.
helveticus strains, it was found that there is not an
absolute relationship between degrees of autolysis and
flavor development (Kenny et al., 2006). Indeed, the lat-
ter study found that a strain with intermediate levels of
autolysis, Lb. helveticus DPC5364, resulted in the best-
flavored cheese. The conclusion drawn from this study
was that although starter autolysis is vital for good
flavor development, it only serves to enhance the effect
of metabolic pathways already present in the starters
and, in the absence of high lipolytic and proteolytic
activity autolysis, will not, in itself, result in enhanced
flavor development (Kenny et al., 2006). In a further
study (Fenelon et al., 2002), a range of different culture
systems containing lactococci plus adjunct cultures, all
of which included different Lb. helveticus strains, were
demonstrated to produce novel flavors and improve
the acceptability of reduced-fat Cheddar cheese over
that of the control cheeses containing only mesophilic
starter lactococci. In this study, the use of Lb. helveticus
DPC4571 as the sole adjunct culture resulted in cheeses
with increased concentrations of low-molecular-mass
peptides (<0.5kDa) and free amino acids and flavor
scores at 90 and 180 d (Fenelon et al., 2002). This study
indicated that the use of Lb. helveticus adjuncts may
be important in the manufacture of low-fat cheeses
with more acceptable flavor to consumers, which is an
important application of these adjuncts because poor
flavor development is a problem in low-fat cheese.
GENETICS, ENZYMOLOGY, AND GENOMICS
OF CHEESE FLAVOR
As previously discussed, the primary function of LAB
in fermented dairy products is acid production during
the fermentation process; however, LAB also contribute
to the maturation of products such as cheese, in which
their enzymes are involved in lipolysis, proteolysis, and
conversion of amino acids into flavor compounds (Fox
and Wallace, 1997). The importance of such enzymes
Journal of Dairy Science Vol. 93 No. 10, 2010
is demonstrated by the fact that proteinase, peptidase,
and lipase enzymes are often added to cheese to ac-
celerate ripening or to bring about flavor diversification
(Kilcawley et al., 2002). Although proteinase enzymes
of LAB are normally located outside the cell, pepti-
dases and lipases are generally located intracellularly;
thus, to play an active role in a cheese system they
have to be released into the cheese matrix. Autolysis
is the spontaneous disintegration of bacterial cells that
results in the liberation of the cytoplasmic contents of
the cell, including the intracellular enzymes (Lortal and
Chapot-Chartier, 2005). As discussed above, autolytic
starters including some strains of Lb. helveticus are
often applied during cheese manufacture to augment
proteolysis and lipolysis and enhance flavor develop-
ment during ripening (Gonzales and Robert-Baudouy,
1996).
In milk, the level of free amino acids and peptides
is too low to support significant growth of LAB. Thus
LAB, including Lb. helveticus, depend on their prote-
olytic systems to release sufficient petides and amino
acids to support growth in this environment. The struc-
tural components of the proteolytic systems of LAB
can be divided into 3 groups based on their function:
(1) proteinases that break down caseins to peptides,
(2) transport systems that translocate the breakdown
products across the cytoplasmic membrane, and (3)
peptidases that degrade peptides (Kunji et al., 1996).
THE PROTEOLYTIC SYSTEM
The proteolytic system of LAB usually consists of a
cell wall-associated serine proteinase, transport systems
specific for di-, tri-, and oligopeptides, and a multitude
of intracellular peptidases (Kok, 1990; Figure 4). It has
emerged that the proteolytic systems of lactococci and
lactobacilli are similar in their components and modes
of action in terms of converting milk proteins (primar-
ily caseins) into free peptides and free amino acids. The
degradation of caseins plays a crucial role in the devel-
opment of texture and flavor, as well as being essential
for cell growth (Kunji et al., 1996). Certain peptidases
contribute to the formation of flavor by production
of free amino acids or degradation of bitter peptides,
whereas others may produce undesirable bitter-tasting
peptides that can lead to off-flavors.
Lactobacillus helveticus is among the most nutrition-
ally fastidious LAB, requiring 14 exogenous amino
acids, as stated above (Morishita et al., 1981; Chopin,
1993). The sequenced strain Lb. helveticus DPC4571 has
previously been reported to have a potent proteolytic
system consisting of at least 24 intracellular peptidases
(Callanan et al., 2008). Growth phase and growth me-
dium were demonstrated to affect the relative activity
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
Figure 4. The structural components of the proteolytic system of
lactic acid system consisting of the proteinases that break down casein
to peptides, the transport systems that translocate the breakdown
products across the cytoplasmic membrane, and the peptidases that
degrade the peptides. Opp = oligopeptide transport protein; DtpT =
di- and tripeptide transport protein for hydrophilic substrates; DtpP
= di- and tripeptide transport protein for hydrophobic substrates;
PrtP = proteinase; Pep = peptidase.
of these peptidases within the cell (Kenny et al., 2003).
Many peptidases have been characterized in various
lactobacilli either biochemically or genetically. These
include the general aminopeptidases, aminopeptidase C
(Vesanto et al., 1994), aminopeptidase N (Christensen,
Lin et al., 1995), and proline-specific peptidases such
as X-prolyl dipeptidyl aminopeptidase (Vesanto et al.,
1994), proline iminopeptidase (Varmanen et al., 1996),
or proline-specific aminopeptidase (Shao et al., 1997).
A number of endopeptidase enzymes including endo-
peptidase E (Fenster et al., 1997), endopeptidase E2
(Sridhar et al., 2005), endopeptidase F (Sridhar et al.,
2005), endopeptidase O (Chen and Steele, 1998), endo-
peptidase O2 (Chen et al., 2003), and endopeptidase
O3 (Sridhar et al., 2005) have been characterized, in
addition to dipeptidase A (Dudley et al., 1996) and
a tripeptidase T (Savijoki and Palva, 2000). Much of
the research in this area was conducted by Steele and
coworkers who have had a huge impact on this area
through their characterization of a large number of pro-
teolytic enzymes from Lb. helveticus CNRZ32 (Vesanto
et al., 1994; Christensen et al., 1995; Vesanto et al.,
1995; Dudley et al., 1996; Fenster et al., 1997; Shao et
al., 1997; Chen and Steele, 1998; Pederson et al., 1999;
Chen et al., 2003; Sridhar et al., 2005) described in
detail below.
Proteinases
The cell wall proteinase (PrtP) of LAB plays a cru-
cial role in the initial degradation of casein. This was
4441
initially shown in lactococci (Exterkate and De Veer,
1987; Monnet et al., 1987; Bockelmann et al., 1989;
Vos et al., 1989; Visser et al., 1991; Coolbear et al.,
1992) where 2 specificity-class enzymes, PI and PIII,
were identified.
Compared with the lactococci, less is known about
the cell envelope-associated proteinase of lactobacilli.
However, proteinase genes have been characterized
from Lb. helveticus, Lb. casei ssp. casei, Lb. bulgaricus,
Lb. paracasei ssp. paracasei, and Lb. delbrueckii ssp.
bulgaricus (de Palencia et al., 1997; Pederson et al.,
1999; Bernasconi et al., 2002; Shin et al., 2004; Genay
et al., 2009). It has been shown that the specificity of
the cell envelope proteinase varies in different strains of
Lb. helveticus where 2 types of proteinases have been
identified, PrtH and PrtH2. In a study that compared
29 different strains, it was found that all possessed the
prtH2 gene, whereas prtH was strain dependent (Genay
et al., 2009). Lactobacillus helveticus DPC4571 is the
only complete genome available from this species and,
like the others, it was found to contain a serine protease
gene (prtH2) lhv_1641, but unusually it is annotated as
a pseudogene (Callanan et al., 2008). Numerous stud-
ies have focused on the cloning and characterization
of proteinases from Lb. helveticus. Initially, a putative
extracellular proteinase gene was cloned from Lb. hel-
veticus CP790 in Escherichia coli. The transformant
expressed a protein that reacted with monoclonal
antibodies specific to the proteinase and appeared to
be a pre-proteinase but had no proteolytic activity
(Yamamoto et al., 2000). Further research revealed the
presence of a cell envelope-associated proteinase (PrtH)
in Lb. helveticus CNRZ32 and, interestingly, demon-
strated that it was not required for rapid growth or
fast acid production in milk by that strain (Pederson et
al., 1999). A comparison of CNRZ32 and its prtH dele-
tion mutant indicates that CNRZ32 has at least 2 cell
surface proteinases that differ in substrate specificity
(Pederson et al., 1999). In another study, an intracel-
lular proteinase was purified and characterized with
optimum enzyme activity at pH 6.5 and temperature
37°C, whose activity increased in the presence of Ca2+,
Mn2+, and Co2+ and was inhibited by Cu2+, Mg2+, and
Zn2+ (Shinet al., 2004).
Amino Acid and Peptide Transport
Amino acids and peptides are transported across the
cytoplasmic membrane by specific transport proteins.
Separate transport systems for amino acids and di-,
tri-, and oligopeptides are present in lactococci (Kunji
et al., 1996) and lactobacilli (Nakajima et al., 1998).
The recent sequencing of Lb. helveticus DPC4571
has allowed the identification of an amino acid trans-
Journal of Dairy Science Vol. 93 No. 10, 2010
4442 SLATTERY ET AL.
porter and an oligopeptide transporter (lhv_1028 and
lhv_2931, respectively) in this particular strain (Cal-
lanan et al., 2008).
The uptake of 13 amino acids reported to be essential
or growth stimulating in Lb. helveticus was examined by
Nakajima et al. (1998). Ten of the 13 amino acids were
accumulated by Lb. helveticus NCDO2712, of which
5 were transported by a proton motive force coupled
system. Indeed, the amino acid and the di- and tripep-
tide transport systems of Lb. helveticus were similar to
those found in Lc. lactis with regard to the mechanism
of uptake and the substrate specificity of the trans-
port systems, which have a preference for hydrophilic
peptides (Nakajima et al., 1998). Several studies have
focused on the peptide uptake systems of Lb. helve-
ticus. In one such study (Nakajima et al., 1998), the
gene encoding the di- and tripeptide transport protein
(DtpT) of Lb. helveticus (DtpTLH) was cloned and
used to complement a dipeptide transport-deficient
and proline-auxotrophic E. coli E1772. Interestingly,
this DtpTLH gene was located downstream of the gene
encoding the general aminopeptidase N. The cloning of
the DtpTLH gene and the subsequent characterization
of DtpTLH established that this system is specific for
transport of di- and tripeptides. Functional expression
of the peptide transporter was shown by the uptake
of prolyl-[14C] alanine in whole cells and membrane
vesicles. Indeed the transporter was shown to have
specificity for di- and tripeptides but not for amino
acids or tetrapeptides. Substrate specificity studies sug-
gested that DtpTLH has a preference for more hydro-
philic peptides. In this respect, dipeptides containing
hydrophobic amino acids, such as Phe-Val and Leu-Val,
inhibited Pro-[14C] Ala uptake only slightly, although
the hydrophobic peptides Leu-Leu and Leu-Leu-Leu
were quite effective inhibitors of Pro-Ala transport.
In addition, peptide transport via DtpT in membrane
vesicles was shown to be energy dependent and driven
by the proton motive force (Nakajima et al., 1997). In
Lb. helveticus DPC4571 the di-/tripeptide transporter
(lhv_1885) is frameshifted, and the frameshift is about
a quarter of theway along the sequence so the protein
is unlikely to be functional; therefore, it is annotated as
a pseudogene. There is a functional oligopeptide trans-
porter lhv_2931 that is probably fulfilling the peptide
transport role, whereas lhv_1028 and lhv_1375 act as
amino acid transporters (Callanan et al., 2008).
Peptidases
Peptides generated from casein by the action of
proteinases are further degraded by peptidases into
smaller peptides and amino acids. The majority of
peptidases reported for lactobacilli are located intracel-
Journal of Dairy Science Vol. 93 No. 10, 2010
lularly (Vesanto et al., 1994; Christensen et al., 1995;
Dudley et al., 1996; Varmanen et al., 1996; Fenster et
al., 1997; Shao et al., 1997; Chen and Steele, 1998; Savi-
joki and Palva, 2000; Chen et al., 2003; Sridhar et al.,
2005). The major catalytic types are serine-, cysteine-,
aspartic- and metallo-peptidases (Kunji et al., 1996).
Peptidases are generally classified into endopeptidases,
aminopeptidases, and carboxypeptidases. Endopepti-
dases are enzymes that cleave internal peptide bonds,
aminopeptidases catalyze release of N-terminal amino
acids, and carboxypeptidases catalyze release of C-
terminal amino acids. Peptidase enzymes are classified
depending upon the chemical nature of the group re-
sponsible for catalysis.
Aminopeptidases
The availability of the fully sequenced Lb. helveticus
DPC4571 genome has allowed for the screening of pep-
tidase genes and has revealed that this strain contains 8
putative aminopeptidase genes (Callanan et al., 2008).
One of these encodes aminopeptidase N (PepN), a
general aminopeptidase with broad substrate specificity
that has been characterized in several lactobacilli. The
pepN of Lb. helveticus CNRZ32 was cloned into Lc. lac-
tis LM0230 resulting in a >180-fold increase in general
aminopeptidase activity using l-lysine-ρ-nitroanilide as
a substrate. Southern hybridization was conducted to
determine the distribution of homology to the CNRZ32
pepN gene among other LAB, which confirmed it was
found in different strains of lactobacilli, pediococci,
leuconostoc, streptococci, and lactococci (Christensen
et al., 1995).
Aminopeptidase C (PepC) is a general aminopepti-
dase with a specificity profile that overlaps that of PepN
(Luoma et al., 2001). It is a cysteine-peptidase whose
activity is inhibited by N-ethylmaleimide (NEM). In
general, PepC is highly conserved among dairy LAB.
Originally, the gene encoding aminopeptidase C was
cloned from an industrially important Lb. helveticus
strain. The deduced amino acid sequence of open read-
ing frame (ORF) 1 was shown to share 48.3 and 98%
identity with PepC proteins from Lc. lactis and Lb.
helveticus CNRZ32, respectively. In bioreactor studies,
expression of the gene was found to be almost constant
throughout the stationary growth phase (Vesanto et
al., 1994). This PepC aminopeptidase is also present
on Lb. helveticus DPC4571 and is shown to share 99%
identity with PepC protein of Lb. helveticus CNRZ32
(Callanan et al., 2008).
Aminopeptidase A (PepA), also known as glutamyl
aminopeptidase, has specificity for N-terminal glutamyl
and aspartyl residues. This metallo-enzyme has been
purified and characterized from Lc. lactis (Niven,
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
1991). It is reported to liberate N-terminal glutamyl
and aspartyl residues from di-, tri-, and oligopeptides
consisting of up to 10 amino acid residues. The gene
encoding this enzyme has been cloned and sequenced
from Lc. lactis, but as yet has not been purified from
lactobacilli (l’Anson et al., 1995).
The genome of DPC4571 reveals that other more
unusual aminopeptidases may be present, at least in
this strain. For example, activity detected in cell-free
extracts of Lb. helveticus DPC4571 against methionine-
7-amino-4-methylcoumarin (Met-AMC) was previously
attributed to the general aminopeptidases PepN and
PepC. However, Christensen and Steele (2003) reported
Met-ρ-NA activity in Lb. helveticus CNRZ32 that was
independent of the activity of PepN and PepC. Inter-
estingly, the genome of DPC4571 contains a putative
methionine aminopeptide with >50% identity at the
amino acid level to the E. coli enzyme (Ben-Bassatet
al., 1987). The DPC4571 PepM also encodes the cobalt-
binding signature sequence of methionine aminopepti-
dases, further supporting the hypothesis that it encodes
a methionine aminopeptidase. Another example is pyr-
rolidonyl carboxyl peptidase (Pcp) which is a narrow-
specificity aminopeptidase that cleaves N-terminal
pyroglutamyl residues. Pyrrolidonyl carboxyl peptidase
has been purified and characterized from lactococci,
but to date has not been characterized in lactobacilli. A
putative pcp gene, however, is found on the genome of
Lb. helveticus DPC4571 that has significant homology
to the characterized Pcp (60% at the amino acid level)
of Lc. lactis (Curley and van Sinderen, 2000). Activ-
ity of Pcp is of particular significance in thermophilic
lactobacilli because significant levels of pyroglutamic
acid are only found in cheeses manufactured with ther-
mophilic starters (Mucchetti et al., 2002). Low levels of
Pcp activity against pyrrolidone carboxylyl peptidase-
7-amino-4-methylcoumarin (Pyr-AMC) have been
observed in cheeses manufactured with L. helveticus
DPC4571 (Kenny et al., 2003).
Endopeptidases
The genome of Lb. helvticus DPC4571 contains 7 en-
dopeptidase genes in total: pepO, pepO2, pepO3, pepE2,
pepF, gcp, and ydiC, whereas various other endopepti-
dases have been found in other LAB (Callanan et al.,
2008). To date, 2 thiol-dependent endopeptidases, PepE
(Fenster et al., 1997) and PepE2 (Sridhar et al., 2005),
and 3 metalloendopeptidases, PepO (Chen and Steele
1998), PepO2 (Chen et al., 2003), and PepO3 (Sridhar
et al., 2005), have been characterized in lactobacilli.
An endopeptidase gene (PepE) was isolated from a
genomic DNA library of Lb. helveticus CNRZ32. Signifi-
cant identity was found between the deduced amino acid
4443
sequence of pepE and the sequences for aminopeptidase
C from Lb. delbrueckii ssp. lactis DSM7290, Lb. helve-
ticus CNRZ32, Streptococcus thermophilus CNRZ302,
and Lc. lactis ssp. cremoris AM2, suggesting that they
evolved from the same ancestral proteolytic enzyme. A
highly conserved substrate binding and catalysis motif
was identified in PepE, suggesting that the enzyme is
a cysteine proteinase with a mechanism of catalytic
action similar to those of other cysteine proteinases.
Characterization of a purified recombinant His-tagged
PepE fusion revealed that it was a thiol-dependent pro-
tease and had significant activity under conditions that
simulate those of ripening cheese. Endopeptidase E has
the ability to hydrolyze internal peptide bonds in small
peptides such Met-enkephalin and bradykinin but not
intact α-, β-, and κ-caseins, indicating that PepE is
an endopeptidase and cannot hydrolyze intact proteins
(Fenster et al., 1997).
Endopeptidase F (PepF) is an endopeptidase report-
ed to hydrolyze peptides between 7 and 17 amino acids
in length with a rather wide specificity. It has been
characterized as a metalloenzyme, in that it contains
a typical Zn-binding site. This endopeptidase was ob-
served to cleave Phe-X bonds with a Pro in position 2
of the cleaved bond. The purified enzyme from Lc. lac-
tis hydrolyzed bradykinin at the Phe5–Ser6 bond. The
equivalent gene (pepF) from Lb. helveticus CNRZ32 has
been cloned and sequenced (Sridhar et al., 2005). In
that study, a range of different endopeptidases (PepE,
PepE2, PepF, PepO, PepO2, and PepO3) were cloned
from Lb. helveticus CNRZ32 into E. coli and were shown
to hydrolyze the model bitter peptides, β-casein (β-CN)
(f193–209) and αS1-casein (αS1-CN) (f1–9), under cheese-
ripening conditions. Bitterness is a major flavor defect
in Cheddar and Gouda cheeses. This defect is primarily
caused by the accumulation of hydrophobic peptides
such as (αS1-CN) (f1–9) and (β-CN) (f193–209) to con-
centrations greater than their respective taste thresh-
olds. The reduction of bitterness in cheese is believed to
be the result of preferential hydrolysis of bitter peptides
to nonbitter hydrolysis products by specific peptidases
from starter or nonstarter bacteria. In this context,
PepE2 and PepO of Lb. helveticus CNRZ32 did not
hydrolyze either bitter peptide under conditions that
simulated cheese ripening. The PepO3 from CNRZ32
was determined to be a functional paralog of PepO2
and hydrolyzed both peptides, whereas PepE and PepF
had unique specificities toward (αS1-CN) (f1–9) and
(β-CN) (f193–209), respectively. To demonstrate the
utility of these peptidases in cheese, PepE, PepO2, and
PepO3 were expressed in a Lc. lactis cheese starter. The
resultant cell-free extracts of Lc. lactis derivatives ex-
pressing these peptidases were used to hydrolyze (β-CN)
(f193–209) and (αS1-CN) (f1–9) under cheese-ripening
Journal of Dairy Science Vol. 93 No. 10, 2010
4444 SLATTERY ET AL.
conditions in single-peptide reactions, in a defined pep-
tide mix and in Cheddar cheese serum. In all systems
examined, PepO2 and PepO3 had the highest common
activity with (β-CN) (f193–209) and (αS1-CN) (f1–9).
Importantly, this indicated that Lb. helveticus CNRZ32
endopeptidases PepO2 and PepO3 are likely to play a
key role in this strain’s ability to reduce bitterness in
cheese (Sridhar et al., 2005).
Endopeptidase PepO from Lb. helveticus CNRZ32 is
40% identical to endopeptidase O from Lc. lactis P8–2-
47 and has been cloned in E. coli (Chen and Steele,
1998). Enzyme assays revealed that the PepO-deficient
mutants of Lb. helveticus CNRZ32 had significantly
reduced endopeptidase activity (79 and >94%) with
the substrates N-benzoyl-Phe-Val-Arg-pNA and N-
benzoyl-Val-Gly-Arg-pNA, respectively, allowing the
use of these substrates to accurately quantify PepO
activity. However, growth studies indicated that PepO
deficiency has no detectable effect on growth rate or
acid production in amino acid-defined or skim milk
medium. These results demonstrate that hydrolysis of
milk-derived peptides by PepO is not growth-rate limit-
ing in this strain (Chen and Steele, 1998).
Another endopeptidase commonly found in LAB
is post-proline endopeptidase (PepO2), which was
initially identified in cell extracts prepared from a
genomic library of Lb. helveticus CNRZ32 using the
synthetic substrate N-acetyl-β-casein- (f203–209)-ρ-
nitroanilide in a coupled reaction with aminopeptidase
N. Analysis of the predicted peptide sequence revealed
that Lb. helveticus Pep02 contained the zinc-dependent
metalloprotease motif HEXXH and exhibited levels of
amino acid sequence similarity of 72, 61, 59, and 53% to
Lb. helveticus PepO, Lc. lactis PepO2, Lc. lactis PepO,
and Lb. rhamnosus PepO, respectively. The activity of
chymosin on β-casein results in the production of the
peptide β-casein (f193–209) and has been implicated in
the development of bitterness in cheese. The ability of
an endopeptidase to hydrolyse β-casein (f193–209) was
examined by the above group. This work revealed that
only PepO2 possessed the ability to hydrolyze β-casein
(f193–209), suggesting that this enzyme may play a
central role in the hydrolysis of casein-derived bitter
peptides (Chen et al., 2003).
Di- and Tripeptidases
As outlined above, bioinformatic analysis of the
genome of Lb. helveticus DPC4571 suggests that this
strain contains 4 dipeptidases: pepD, pepA, pepD3, and
pepD4, and 1 tripeptidase: pepT (Callanan et al., 2008).
Peptidase D is a dipeptidase that, like PepV, cleaves
Met-Ala; unlike PepV, it also acts solely as a dipepti-
dase. A general dipeptidase, pepDA, was isolated from
Journal of Dairy Science Vol. 93 No. 10, 2010
a gene library of Lb. helveticus CNRZ32. Southern hy-
bridization studies with a pepDA probe indicated that
the nucleotide sequence for pepDA is not well conserved
among a variety of LAB. Growth studies indicated that
a pepDA deletion had no detectable effect on growth
rate or acid production by Lb. helveticus CNRZ32 in
milk. Further studies indicated no difference in total
cellular dipeptidase activity between a mutant and a
wild-type strain during logarithmic growth in de Man,
Rogosa, and Sharpe medium. Thus, PepDA is not es-
sential for the liberation of amino acids from casein-
derived peptides (Dudley et al., 1996).
A tripeptidase (PepT) from Lb. helveticus has also
been purified and biochemically characterized. This
work was part of a larger project that focused on the
characterization of the proteolytic system from the
industrial Lb. helveticus strain 53/7. Peptidase T ap-
peared to be a trimeric metallopeptidase exhibiting
maximum activity against hydrophobic tripeptides,
with the highest activity for Met-Gly-Gly. Some of the
hydrophobic dipeptides were slowly hydrolyzed, distin-
guishing the Lactobacillus PepT from its counterpart in
mesophilic Lc. lactis. Interestingly, no activity against
tetrapeptides or amino acid ρ-nitroanilide derivatives
was observed with this enzyme (Savijoki and Palva,
2000).
Proline-Specific Peptidases
The availability of the genome data of Lb. helveti-
cus DPC4571 has allowed identification of 5 putative
genes encoding proline-specific peptidases (Callanan et
al., 2008). In general, LAB are dependent on proline-
specific peptidases to hydrolyze peptides with proline
in the second position because the action of general
aminopeptidases or broad specificity di- and tripepti-
dases in isolation cannot hydrolyze these peptides as
they are unable to cleave the imido bond. The key
peptidases involved in degrading proline-rich peptides
are aminopeptidase P, proline iminopeptidase, proline
aminodipeptidase, and X-prolyl dipeptidyl amniopepti-
dase (Kunji et al., 1996).
Aminopeptidase P (PepP) has been purified and
characterized from Lc. lactis ssp. cremoris (Mc Donnell
et al., 1997). Peptidase P removes the N-terminal amino
acid from peptides containing Pro in the penultimate
position from the N terminus. Dipeptides were not hy-
drolyzed even if proline was present at the C-terminus.
Peptidase P activity has been shown to be present in
lactobacilli (Hickey et al., 1983); however, it has not yet
been purified.
Proline iminopeptidase (PepI), also known as prolyl
iminopeptidase, catalyzes the removal of N-terminal Pro
residues from peptides with Pro-X N-terminal sequenc-
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
es. A proline iminopeptidase gene from an industrial
Lb. helveticus strain was cloned and found to be located
in an operon-like structure of 3 ORF (ORF1, ORF2,
and ORF3; Varmanen et al., 1996). It was reported
that the ORF3-encoded PepI protein displayed 65%
identity with the PepI proteins from Lb. delbrueckii ssp.
bulgaricus and Lb. delbrueckii ssp. lactis. The ORF1-
encoded protein had significant homology with several
members of the ABC transporter family of proteins
and contained distinct putative ATP-binding sites; and
ORF2 encoded a putative integral membrane protein
also characteristic of the ABC transporter family. The
recombinant PepI hydrolyzed only di- and tripeptides
with Pro in the first position. Peptidase I was shown to
be a metal-independent serine peptidase having thiol
groups at or near the active site (Varmanen et al.,
1996).
X-Prolyl dipeptidyl aminopeptidase (PepX) cleaves
N-terminal dipeptides sequentially from polypeptides
with unsubstituted N termini of the type X-Pro-Y- or
X-Ala-Y-, provided that the penultimate residue is Pro
or Ala. The peptide bond is cleaved at the carboxyl
side of the Pro or Ala residue. The X-prolyl dipeptidyl
aminopeptidase gene (pepX) of an industrially impor-
tant Lb. helveticus strain was detected by nucleic acid
hybridization and subsequently cloned, characterized,
and sequenced. The deduced amino acid sequence of
PepX protein showed 49.3, 49.4, and 77.7% identity
with the PepX from Lc. lactis ssp. lactis, Lc. lactis ssp.
cremoris, and Lb. delbrueckii ssp. lactis, respectively
(Vesanto et al., 1995). The pepX gene was cloned and
expressed at a high level in E. coli and purified to ho-
mogeneity. The enzyme was inactivated by heavy metal
ions such as Cu2+, Cd2+, and Zn2+. It was also shown
to be a metal-independent serine peptidase having
functional sulfhydryl groups at or near the active site
(Vesanto et al., 1994, 1995).
A further dipeptidase is proline iminodipeptidase
(PepR), which catalyzes the cleavage of dipeptides in
the form Pro-X with proline or hydroxyproline at the
N-terminal position. Generally, prolinases have specific-
ity for dipeptides containing an amino-terminal prolyl
residue. Peptidase R was purified to homogeneity and
found to hydrolyze Pro-Met, Thr-Leu, and Ser-Phe
as well as dipeptides containing neutral, nonpolar
amino acid residues at the amino terminus (Shao et al.,
1997). Site-directed mutagenesis indicated that PepR
is a serine-dependent protease. Gene replacement was
employed to construct a PepR-deficient derivative of
CNRZ32 that did not differ from the wild-type strain
in its ability to acidify milk (Shao et al., 1997). This
suggests that PepR is not involved in the hydrolysis
of milk-derived peptides, or that other peptidases pos-
sess overlapping specificities with PepR. Interestingly,
4445
however, the PepR-deficient construct was determined
to have reduced dipeptidase activity compared with the
wild-type strain with all dipeptide substrates examined
(Shao et al., 1997).
Imidodipeptidase (PepQ) is a dipeptidase specific
for dipeptides containing Pro at the carboxy-terminus
position. It has a strict dipeptidase activity on X-Pro
dipeptides except Gly-Pro and Pro-Pro. It is a metal-
loenzyme that is inhibited by EDTA. Peptidase Q is of
particular interest in dairy starter cultures because of
the relatively high number of proline residues in casein.
The PepQ homolog (PepQ2) of Lb. helveticus DPC4571
has homology to the Xaa-Pro-specific PepP of Lc. lactis
(35% identity at the amino acid level) and encodes the
PROSITE PS00491 proline dipeptidase motif, which
might indicate that the gene encodes an additional
proline-specific enzyme (Kunji et al., 1996).
Carboxypeptidases are enzymes that cleave C-termi-
nal amino acids from peptides. One carboxypeptidase
(lhv_1706) is present in the genome of Lb. helveticus
DPC4571. No carboxylpeptidases have yet been puri-
fied or characterized in LAB, and substrates for the
detection of carboxypeptidase activity were not hydro-
lyzed by Lb. helveticus or by nonstarter lactobacilli iso-
lated from Cheddar cheese (Williams and Banks, 1997),
which may indicate that this enzyme does not have a
significant role in cheese ripening.
LIPOLYTIC ACTIVITY IN LB. HELVETICUS
Importance of Lipolysis
The breakdown of milk fat by lipases and esterases is
one of the main biochemical events that occur during
the ripeing of some cheese varieties, where it is known
to be a major contributor to flavor development. Li-
polysis involves the release of free fatty acids, major
flavor compounds that directly affect cheese flavor, by
imparting specific fatty acid flavor notes (Hickey et al.,
2007). The characteristic flavor development of some
Italian cheeses is thought to be due to the release of
short-chain fatty acids such as butanoate, hexanoate,
and octanoate.
Mechanism of Lipolysis
Lipolytic enzymes are hydrolases that cleave the
ester linkage between a fatty acid and the glycerol
backbone of triacylglycerol to produce FFA and di- and
mono-acylglycerols (Holland et al., 2005). The major
FFA in cheese have straight-chain carbons of C2 to C18
in length and have high flavor thresholds, imparting
rancid, cheesy, pungent, soapy, or waxy flavor notes.
Lipolytic enzymes are generally classed as esterases or
Journal of Dairy Science Vol. 93 No. 10, 2010
4446 SLATTERY ET AL.
lipases. Esterases hydrolyze acyl ester chains between 2
and 8 carbon atoms in length, whereas lipases hydrolyze
acyl ester chains of 10 or more carbon atoms in length
(Holland et al., 2005). Lipases are defined as acting
on lipids that are water insoluble, whereas esterases
generally act on shorter lipid substrates that are water
soluble to a limited extent. They can be further dif-
ferentiated by the kinetics of the respective reactions;
lipases exhibit interfacial Michaelis-Menten activation,
a phenomenon in which only substrate at the interface
of the aqueous and lipid phases is hydrolysed, whereas
esterases exhibit the classical Michaelis-Menten type
kinetics (Chich et al., 1997; Collins, 2003b).
Characterized Esterase Genes
Compared with organisms such as Pseudomonas, Fla-
vobacterium, Acinetobacter, and propionic acid bacteria
(Stadhouders and Veringa, 1974; Chich et al., 1997,
Collins et al., 2003a), the LAB are generally considered
to be weakly lipolytic; however, it should be emphasized
that LAB-derived enzymes are thought to be the main
contributors to lipolysis during ripening of Cheddar
cheese (Hickey et al., 2006). Esterase enzymes have been
identified, purified, and characterized from several LAB
including Lc. lactis (Tsakalidou and Kalantzopoulous,
1992; Holland and Coolbear, 1996; Chich et al., 1997;
Fernandez et al., 2000; Macedo et al., 2003), Lb. casei
(Lee and Lee, 1990; Castillo et al., 1999; Fenster et al.,
2000, 2003a,b), Lb. fermentum (Gobbetti et al., 1997b),
Lb. plantarum (Gobbetti et al., 1997a), Lb. helveticus
(Williams and Banks, 1997), Lb. rhamnosus (Holland et
al., 2002), and Strep. thermophilus (Liu et al., 2003).
The sequenced genome of Lb. helveticus DPC4571
revealed 6 esterase-like genes consisting of 1 ary-
lesterase, 3 putative esterases, and 2 putative acyl-like
thioesterases, which belong to the esterase family and
exhibit esterase activity, in addition to 1 putative lipase
gene. Bioinformatic analysis demonstrated that 2 of the
esterase genes, EstA and Lhv1434, had 99 and 30%
homology, respectively, to the already characterized
esterase EstA from Lb. helveticus CNRZ32 (Fenster et
al., 2000) and esterase EstB from Lb. casei (Fenster et
al., 2003a).
The serine-dependent arylesterase from Lb. helveticus
CNRZ32 and an arylesterase from Lb. casei LILA have
been sequenced, purified, and characterized (Fenster et
al., 2003a,b). Both were observed to have significant
activity under conditions simulating ripening cheese
(pH 5.1, 10°C, 4% NaCl) and their selectivity for short
n-chain fatty acid esters suggested that these enzymes
could play an important role in cheese flavor develop-
ment. These enzymes were also shown to mediate the
accumulation of short-chain ethyl esters in a model sys-
Journal of Dairy Science Vol. 93 No. 10, 2010
tem simulating Parmesan cheese ripening. Site-directed
mutagenesis on 6 serines, 3 histidines, and 1 cysteine
in the EstA protein identified a serine located in the
esterase/lipase-associated GDSI motif and a histidine
located in the esterase/lipase-associated GXH motif
that were both essential for EstA activity. Esterase B
also contains the characteristic GXSXG active-site ser-
ine motif identified in most lipases and esterases.
CELL LYSIS OF LB. HELVETICUS
The Importance of Lysis in Cheese
Flavor Development
Cheese flavor development has long been thought to
be linked to the release of intracellular enzymes into
the cheese matrix, which is inherently dependent on the
ability of the starter strains to lyse during fermentation
or during the ripening process. Lysis of the starter cells
leads to the release of, inter alia, peptidases and es-
terases that, as already discussed, convert milk peptides
and fats into desirable flavor compounds (Pillidge et
al., 2002). For example, the reduction of bitterness and
overall acceptable flavor development in cheese corre-
lates with the liberation of intracellular peptidases and
their hydrolysis of bitter peptides (Courtin et al., 2002).
Hickey et al. (2006) reported that individual starter
strains lyse at different rates beyond the influence of the
cheese manufacturer and because ripening of cheese can
be both a slow and a costly process, controlling the rate
and level of lysis would be of immense benefit to the
cheese manufacturer (Hickey et al., 2006). Lactobacillus
helveticus DPC4571 is a highly autolytic strain (Figure
3) and its genome sequence revealed the presence of 8
putative lysin genes including autolysin amidases, en-
terolysins, and n-acetylmuramidases. Lactococcus lactis
is considered a model organism in the study of dairy
fermentations and a positive effect on cheese flavor
has been associated with lactococcal lysis. Lysis of Lc.
lactis has been shown to be highly strain dependent in
Cheddar cheese, just as the lysis of Lb. helveticus has
been demonstrated to be strain dependent in Swiss and
Cheddar cheeses (Hannon et al., 2006).
Mechanism of Lysis
The mechanism of cell lysis has been identified as
occurring from both within and without the cell. Au-
tolysis is the result of the action of endogenous enzymes
(autolysins) or exogenous enzymes such as bacterio-
cins and phage lysins (Nilsen et al., 2003; Lortal and
Chapot-Chartier, 2005).
Autolysins hydrolyze specific bonds in the protec-
tive and shape-maintaining cell wall peptidoglycan, a
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
Figure 5. Peptidoglycan structure of lactic acid bacteria and pep-
tidoglycan hydrolase specificities for the cleavage of certain bonds.
Peptidoglycan is a unique 3-dimensional structure containing linear
chains of polysaccharides consisting of N-acetyl-muramic acid (Mur-
NAc) and N-acetyl-glucosamine (GlcNAc) (Lortal and Chapot-
Chartier, 2005).
function that is necessary to facilitate growth of the
bacterial cell including cell separation after division,
cell wall turnover, and cell wall expansion. Several key
enzymes are involved including N-acetylmuramidase,
N-acetylmuramyl-l-alanine amidase, and endopeptidase
(Lortal and Chapot-Chartier, 2005; Figure 5). Loss of
control of any of these enzymes is potentially lethal to
the cell and leads to cell lysis or autolysis (Rice and
Bayles, 2008).
Exogenous enzymes involved in cell lysis include
bacteriocins, which are ribosomally synthesized an-
timicrobial peptides produced by some bacteria that
provide them with a selective advantage in complex
environmental systems by killing closely related com-
petitors. Another example of an exogenous enzyme
involved in autolysis is bacteriophage lysins. These are
encoded by bacteriophage DNA and are synthesized
during the bacteriophage lytic cycle. Bacteriophages
utilize holin protein(s), which mediate the transport of
the lysin across the cytoplasmic membrane, resulting in
the degradation of the bacterial cell wall (Djorjevic and
Klaenhammer, 1997). Released lysin is then free to act
on neighboring cells.
The autolysis of 14 Lb. helveticus strains grown in
complex broth (de Man, Rogosa, and Sharpe) growth
medium has been compared with that seen for the same
strains during Cheddar cheese ripening (Kenny et al.,
2005). This allowed the assessment of the relationship
between autolysis in vitro and flavor development in
4447
cheese by specific Lb. helveticus adjuncts with dif-
fering autolytic properties. The results of this study
revealed that although the autolysis of adjunct strains
is of importance to cheese flavor it is not the only
contributing factor because some nonautolytic strains
were determined to produce high-quality cheese. It was
also noted that there was not full correlation between
the observed autolysis in growth media and cheese. For
example, of 4 strains exhibiting high autolytic potential
in growth media, only one, DPC4571, showed a signifi-
cant decrease in cell numbers during cheese ripening.
The other 3 strains did not undergo a decrease in cell
numbers during cheese ripening and resulted in low lev-
els of d-lactate dehydrogenase release into the cheese
matrix, did not lead to high levels of secondary pro-
teolysis in the cheese as measured by phosphotungstic
acid-soluble nitrogen, and had lower scores for flavor
intensity compared with cheese manufactured with the
highly lytic DPC4571 strain. No correlation could be
found between the autolysis that occurs in vitro to that
which occurs in the cheese. These results demonstrated
that making cheese is probably the best approach to
screening for autolytic strains of lactobacilli and that
autolysis that occurs in vitro could not be compared
with that in cheese trials (Kenny et al., 2005).
Characterized Lysin Genes
Endopeptidases. Analysis of the genome sequence
of Lb. helveticus DPC4571 revealed the presence of 2
putative enterolysin A genes (lhv_1295 and lhv_1307),
which exhibit identity (37 and 40%, respectively) to
that of the characterized enterolysin A from Enterococ-
cus faecalis. The enterolysin A endopeptidases have 2
different specificities in which they can cleave the bond
between d-glutamyl-m-DAP and α-amino group of
l-lysine and that between l-lysine and d-alanine. En-
terolysin A was first described by Nilsen et al. (2003),
who reported the characterization of a novel type III
bacteriocin produced by E. faecalis LMG 2333, which
degrades the cell wall of sensitive bacteria. However, a
more recent classification of bacteriocins by Cotter et
al. (2005) places enterolysins in a new group of bacte-
riocidal proteins called the bacteriolysins because their
mode of action differs from “true” bacteriocins, which
are generally small peptides that form pores or inhibit
cell wall synthesis (Cotter et al., 2005). In contrast,
the bacteriolysin group consists of large heat-labile pro-
teins and includes the staphylococcal lysin lysostaphin,
a metalloendopeptidase produced by a single strain
of Staphylococcus simulans (NRRL-B2628) that lyses
other Staphylococcus strains (Schindler and Schuhardt,
1964; Recsei et al., 1987). Sequence analysis indicated
that enterolysin A consists of 2 domains: an N-terminal
Journal of Dairy Science Vol. 93 No. 10, 2010
4448 SLATTERY ET AL.
region of the protein contained a domain found in
the M37 family of metallopeptidases, whereas the C-
terminal part showed similarity to different muralytic
bacteriophage proteins (Nilsen et al., 2003). Sequences
homologous to the N-terminal region of the enterolysin
A were lhv_1295 and lhv_1307 (Callanan et al., 2008),
lysostaphin (Recsei et al., 1987), and zoocin (Sim-
monds et al., 1997), and the C-terminal part to LytM
(Ramadurai and Jayaswal, 1997) and ALE-1 (Sugai et
al., 1997).
Autolysin Amidases. Lactobacillus helveticus
DPC4571 contains one known autolysin amidase gene
(lhv_0191) in its genome sequence; amidases have the
ability to cleave the bond that separates the peptide
components from the amino sugar chains in peptidogly-
can. They specifically cleave the amide bond between
the lactyl group of muramic acid and the α-amino
group of l-alanine (Lortal and Chapot-Chartier, 2005).
Interestingly, the genome of this strain, unlike many
other LAB strains, does not contain a prophage, sug-
gesting that the associated autolytic properties are
most likely due to the effects of some or all of the puta-
tive lysins (Kenny et al., 2005; Callanan et al., 2008).
This autolysin, amidase (lhv_0191) from Lb. helveticus
DPC4571, has identity to the Atl-like staphyloccal
autolysins and in particular to the characterized AtlL
from Staphylococcus lugdunensis and to the autolytic
amidase of Listeria monocytogenes EDG (36 and 47%,
respectively; McLaughlan and Foster, 1998; Bourgeois
et al., 2009). The staphylococcal autolysins are bi-
functional enzymes with N-acetylmuramoyl-l-alainine
amidase and N-acetylglucosaminidase cleavage activity
on the peptidoglycan. To date, no characterized Lac-
tobacillus autolysin amidases homologous to lhv_0191
from Lb. helveticus DPC4571 have been identified.
Endolysin Muramidases. Lactobacillus helveti-
cus DPC4571 contains 5 putative N-acetyl murami-
dases (lhv_0190, lhv_0549, lhv_1059, lhv_1433, and
lhv_2053), which cleave the β-1,4-glycosidic bond be-
tween N-acetyl-muramic acid and N-acetyl-glucosamine
(Lortal and Chapot-Chartier, 2005). The endolysin mu-
ramidases of Lb. helveticus DPC4571 exhibit a degree
of identity (15–50%) with that of well-characterized Lb.
helveticus CNRZ 303 muramidase. Significant homol-
ogy with the N termini of known muramidases suggests
that Lb. gasseri Φadh (Henrich et al., 1995), Lc. lactis
AM2 ΦLC3 (Birkeland, 1994; Lepeuple et al., 1998), Lb.
helveticus Φ303 (Deutsch et al., 2004), Lb. delbrueckii
ssp. bulgaricus (Boizet et al., 1990), and Lb. johnsonii
Lj965 and Lj928 (Desiere et al., 2000) lysins act by
means of a similar catalytic mechanism.
Lortal et al. (2005) identified and partially charac-
terized 7 autolysins present in Lb. helveticus ISLC5,
Journal of Dairy Science Vol. 93 No. 10, 2010
and further analysis of the isolated endolysin from the
temperate bacteriophage Mur-LH from Lb. helveticus
CNRZ303 showed similarity with numerous endolysins
of other bacteriophages. Phage Φ-0303 is a temperate
phage of Lb. helveticus CNRZ 303, which is induced
after mitomycin C treatment of the strain or dur-
ing the manufacture of Swiss-type cheese. It belongs
to the Myoviridae family and possesses an isometric
head. Lytic activity was detected against Lb. helveticus
CNRZ 892 as a substrate, and hydrolysis of cell walls
of Lb. helveticus CNRZ 303 by the endolysin proved
that it was in fact N-acetylmuramidase activity. In
addition, Mur-LH demonstrated N-acetylmuramidase
activity with a broad spectrum of lytic activity, which
included not only thermophilic lactobacilli, its normal
target, but also lactococci, pediococci, Bacillus subtilis,
Brevibacterium linens, and E. faecium (Deutsch et al.,
2004).
BACTERIOCINS OF LB. HELVETICUS
Bacteriocins are proteinaceous substances exhibit-
ing bactericidal activity against closely related species.
They are of interest because of their inhibitory activity
against food spoilage and food-borne pathogenic bacte-
ria (Riley and Wertz, 2002). Bacteriocins have a nar-
row host range and are most effective against related
bacteria competing for the same resources or ecological
niche. Bacteriocins produced by LAB are of most inter-
est to the food industry, because they have potential
use as food biopreservatives to control spoilage and
pathogenic bacteria (Venema et al., 1995). The main
targets of LAB bacteriocins are the cell membrane and
cell wall but they can work through many mechanisms
to exert an antimicrobial effect (Deegan et al., 2006).
Lactobacillus helveticus 481 produces a class III bac-
teriocin known as helveticin J, which inhibits growth
of Lactobacillus species. Joerger and Klaenhammer
(1990) cloned helveticin J from Lb. helveticus 481 and
expressed it in Lactococcus. The expressed protein was
heat labile and demonstrated the expected spectrum of
inhibition. Several Lactobacillus strains have been iden-
tified as bacteriocin-producing, including Lb. plantarum
(plantaricin LC74, Rekhif et al., 1994; plantaricin W,
Holo et al., 2001; plantaricin NC8, Maldonado et al.,
2004), Lb. acidophilus (acidocin B10, ten Brink et al.,
1994; lactacin F, Muriana and Klaenhammer, 1991), Lb.
salivarius (ABP-118, Flynn et al., 2002; salivaricin P,
Barrett et al., 2007), Lb. delbrueckii ssp. lactis (UO004,
Boris et al., 2001), Lb. reuteri (reutericyclin, Ganzle,
2004), and Lb. sake (sakacin B, Urso et al., 2006). Two
previous papers report bacteriocins in Lb. helveticus:
Joerger and Klaenhammer (1986) and Vaughan et al.
(1992).
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
Lactobacillus helveticus DPC4571 contains 2 putative
helveticin genes (lhv_0086 and lhv_1632) with homol-
ogy to helveticin J from Lb. helveticus 481 (Callanan et
al., 2008). However, although both have approximately
44% similarity to helveticin J, the predicted amino
acid sequences of the proteins encoded by lhv_0086
and lhv_1632 are only approximely 44% similar to
each other. It is interesting to note that homologs of
helveticin are present in the genomes of Lb. gasseri,
Lb. johnsonii, Lb. crispatus, Lb. ultunensis, and Lb. aci-
dophilus NCFM. Although the bacteriocin production
status of these strains is unknown, it may be concluded
that helveticin production is widespread in lactobacilli.
The helveticin J bacteriocin, reported by Joerger and
Klaenhammer (1986) in Lb. helveticus 481, is a narrow-
spectrum bacteriocin that inhibits growth of a small
number of Lactobacillus species, is a large heat-labile
bacteriocin, and has been classified as a class III bac-
teriocin (Cotter et al., 2005) because it is neither a
lantibiotic nor a small heat-stable peptide bacteriocin.
Joerger and Klaenhammer (1990) cloned helveticin J
from Lb. helveticus 481 and expressed the protein in Lb.
acidophilus. The expressed protein was heat labile and
demonstrated the expected spectrum of inhibition. Jo-
erger and Klaenhammer (1986) reported the existence
of a second open reading frame that appeared to be
co-transcribed with the helveticin ORF; the DPC4571
lhv_0068 helveticin gene has a similar ORF (lhv_0085)
associated with it. Analysis by PCR of helveticin-
producing resistant and sensitive strains has indicated
that in DPC4571, the protein encoded by lhv_0086 is
responsible for the observed antimicrobial activity. The
helveticin V-1829 reported by Vaughan et al. (1992),
although being heat labile and having a similar narrow
spectrum of inhibition, did not appear to be similar to
helveticin J because DNA probes specific to helveticin
J did not hybridize to the producing strain of V-1829.
No sequence data are available for helveticin V-1829,
and comparison of the nucleotide sequences of different
helveticin ORF shows little similarity in the nucleotide
sequences. It is therefore possible that V-1829 is a
helveticin J type protein as suggested by its physical
characteristics and inhibitory spectrum.
In conclusion, there is no evidence to suggest that
Lb. helveticus produces a broad-spectrum bacteriocin
although the narrow-spectrum helveticin type bacterio-
cins are widespread.
BACTERIOPHAGE OF LB. HELVETICUS
Bacteriophages are a constant threat to the dairy in-
dustry because phage attack of starter cultures during
milk fermentation can result in huge economic losses.
It is a persistent problem in the cheese industry, where
4449
cheese manufacturing processes involve the use of high
populations of starter cells in an intensive production
system that produces ideal conditions for the multiplica-
tion of phage specific to the starter cultures, which can
result in failed fermentation. Birkeland (1994) cloned,
expressed, and characterized genes encoding lysin pro-
teins of Lc. lactis bacteriophage phi LC3 into E. coli.
Both LysA and LysB shared significant similarity to
lysins from Streptococcus pneumoniae phage, possess-
ing a similar holin-lysin structure of endolysin phages.
Unlike the lactococci, in which phage biology has been
intensively studied for many years, much less is known
about the bacteriophages of Lb. helveticus. However,
although phage attack is not a recognized problem in
thermophilic starters, Lb. helveticus phages have been
isolated and characterized from Emmental starters
(Sozzi and Maret, 1975). A comparative study of Lb.
helveticus bacteriophages identified 23 phages isolated
from cheese whey and 12 temperate phages induced
with mitomycin from their lysogenic host strains in
French factories (Sechaud et al., 1992). Further study
demonstrated the inactivation of Lb. helveticus phage
by thermal and chemical treatments (Quiberoni et al.,
1999). Lactobacillus helveticus phages have been iso-
lated from natural whey starters (Zago et al., 2005),
and recently a new PCR protocol for the detection of
Lb. helveticus bacteriophages was optimized (Zago et
al., 2008).
LB. HELVETICUS APPLICATIONS
IN FOOD AND HEALTH
Research into the benefits of different therapies has
indicated that consumption of milk fermented with
lactobacilli and other LAB may have positive effects
on cardiovascular health by reducing blood pressure.
As discussed above, Lb. helveticus has a suite of pep-
tides and proteinases that can degrade milk protein
to discrete peptides. For this reason, Lb. helveticus in
particular is very effective in the production of bioac-
tive peptides that have possible therapeutic values from
milk. The main beneficial effect demonstrated to date of
such products is the production of angiotensin convert-
ing enzyme (ACE) inhibitory peptides. These peptides
have been shown in clinical trials (Jauhiainen et al.,
2005) to lower blood pressure. They act by inhibiting
the action of the ACE enzyme, which is the activator for
angiotensin, a molecule that promotes vasoconstriction
with cocomitant increase in blood pressure. Products
such as Calpis and Evolus are already on the market
and the health benefits for both are due to the actions
of Lb. helveticus. Calpis, a Japanese soft drink, is fer-
mented by Lb. helveticus and Saccharomyces cerevisiae
and contains 2 known ACE-inhibitory peptides. The ac-
Journal of Dairy Science Vol. 93 No. 10, 2010
4450 SLATTERY ET AL.
tion of these antihypertensive peptides has been shown
to decrease the systolic blood pressure in hypertensive
rats after 4 to 8 h of consumption (Nakamura et al.,
1995). The effect of Evolus, a Lb. helveticus fermented
milk produced in Finland, has also been demonstrated
in several studies to lower blood pressure in hyperten-
sive subjects (Jauhiainen et al., 2005, 2007).
It has been demonstrated that consumption of Lb.
helveticus-fermented milk results in increased calcium
absorption compared with ordinary sour milk. Lactoba-
cillus helveticus-fermented milk increases bone mineral
density and bone mineral content in relation to body
weight in long-term feeding of growing rats (Narva et
al., 2004). In another study, animal feeding trials using
rats demonstrated that consumption of cheese produced
using Lb. helveticus cultures resulted in the suppression
of abdominal adipose tissue accumulation compared
with rats given an isocaloric feed prepared using butter
oil and casein. The cheese diet led to a 1.5-fold reduc-
tion in the production of adiponectin from abdominal
adipose tissue. The amounts of serum cholesterol (al-
most halved in value), triglyceride (decreased by a few
mg/dL), very low density lipoprotein, and low-density
lipoprotein were lower in the rats receiving the cheese
diet (Higurashi et al., 2007). These data demonstrate
that consumption of cheese manufactured using Lb.
helveticus strains might have a beneficial suppressive
effect on abdominal adipose accumulation and prevent
the development of metabolic syndrome.
Treatment for Hypertension
Hypertension, or high blood pressure, is a medical
condition in which the blood pressure is chronically
elevated, which, as mentioned above, has become an
increasingly important issue in the modern lifestyle
(Jauhiainen et al., 2007). The means by which ACE-
inhibitory substances are proposed to lower blood pres-
sure is by their ability to catalyze the degradation of
bradykinin, a vasodilating peptide, and inhibition of the
production of angiotension II, a potent vasoconstrictor.
Angiostension II also induces the release of aldosterone,
which causes the retention of sodium ions by the kidney
and elevated blood volume and thus, increased blood
pressure. Milk proteins contain encrypted peptidic
angiotension I-converting enzyme inhibitors, which can
be released by proteolysis during milk fermentation
by some strains of Lb. helveticus (Leclerc et al., 2002).
Leclerc et al. (2002) demonstrated that the antihyper-
tensive activity of caseinate-enriched milk appears to
reflect the effect of digestive enzymes on the release of
ACE-inhibitory peptides from caseins and their blood
pressure-lowering effect.
Journal of Dairy Science Vol. 93 No. 10, 2010
Jauhiainen et al. (2005) studied the effect of Lb.
helveticus-fermented milk containing the tripeptides
isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline
(VPP) on the ambulatory arterial stiffness index by us-
ing ambulatory 24-h blood pressure registration. There
was a mean difference of −4.1 ± 0.9 mm Hg in systolic
(P = 0.001) and a −1.8 ± 0.7 mm Hg in diastolic blood
pressure (P = 0.048) between the Lb. helveticus group
and the control group. Daily consumption of Lb. hel-
veticus LBK-16H fermented milk containing bioactive
peptides has a proven blood pressure-lowering effect in
hypertensive subjects and thus is a potential dietary
treatment of hypertension (Jauhiainen et al., 2005).
Probiotic Applications of Lb. helveticus
Even though Lb. helveticus is not a gut microbe, it
is intriguing that it is closely related to commensal
GI lactobacilli. Consequently, one might expect that
it could have some probiotic potential when ingested
orally. Bacterial infections of the GI tract represent a
major global health problem, even in the presence of
normally effective mucosal immune mechanisms, and
are important targets for vaccine development (Saito,
2004). Many probiotics have been reported to be useful
in the treatment of disturbed intestinal microflora and
diarrheal diseases (Vinderola et al., 2007). Vinderola
et al. (2007) examined whether the production of me-
tabolites during fermentation by Lb. helveticus R389
could confer enhanced protection against Salmonella
typhimurium infection and whether potentially bioac-
tive metabolites produced in fermented milk contrib-
uted to any protection observed in BALB/c mice. They
observed that both the milk fermented by Lb. helveticus
R389 and the nonbacterial fermented milk fraction
conferred protection. It was seen that both the milk
fermented by Lb. helveticus R389 and the fermented
milk with no bacteria exhibited the probiotic effect
and therefore involved not only the probiotic bacteria
but also the biological metabolities produced during
fermentation of milk. The only differences that were
seen between the 2 were in the production of specific
secretory IgA and in the number of macrophage inflam-
matory protein-1α–producing cells. It was also demon-
strated that the mucosal immune response was involved
in the protection observed and that it was not limited
to competitive interactions between Lb. helveticus R389
and S. typhimurium (Vinderola et al., 2007).
SUMMARY
Lactobacillus helveticus DPC4571 is a commercial
Swiss cheese isolate that has been shown to demon-
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
strate several desirable traits including rapid autolysis,
reduced bitterness, and increased flavor (Kiernan et
al., 2000; Hannon et al., 2003). The recent complete
sequencing of the Lb. helveticus DPC571 genome (Cal-
lanan et al., 2008) has revealed a plethora of genes with
industrial potential including those responsible for pro-
teolysis, lipolysis, and cell lysis. These groups of genes
and their derived enzymes can facilitate the production
of cheese and cheese derivatives with potential for use
as ingredients in consumer foods. There is increasing
interest in enzyme-modified cheeses in the dairy sector
because they are cost effective and can increase the
efficiency of manufacturing processes. Opportunities
for the use of novel enzyme technology in cheese have
now come to the forefront with the increased knowledge
available from of the sequenced genomes of LAB. The
opportunity now exists for the application of starter
culture-derived recombinant enzymes in cheese manu-
facture and ripening. The ability to form a bank of
recombinant enzymes that will produce specific flavors
and textures in cheese in a controlled and reproduc-
ible manner will be of enormous benefit to the dairy
industry. The close relationship between Lb. helveticus
DPC4571 and Lb. acidophilus NCFM is intriguing and
points to a common ancestral organism, which may have
evolved in tandem with the evolution of mammals.
ACKNOWLEDGMENTS
This research has been supported by the Department
of Agriculture (Department of Agriculture, Food and
Forestry of Ireland) under the Food Institutional Re-
search Measure (04/RandD/TD/311).
REFERENCES
Altermann, E., W. M. Russell, M. A. Azcarate-Peril, R. Barrangou,
B. L. Buck, O. McAuliffe, N. Souther, A. Dobson, T. Duong, M.
Callanan, S. Lick, A. Hamrick, R. Cano, and T. R. Klaenham-
mer. 2005. Complete genome sequence of the probiotic lactic acid
bacterium Lactobacillus acidophilus NCFM. Proc. Natl. Acad. Sci.
USA 102:3906–3912.
Barrett, E., M. Hayes, P. O’Connor, G. Gardiner, G. F. Fitzgerald,
C. Stanton, R. P. Ross, and C. Hill. 2007. Salivaricin P, one of
a family of two-component antilisterial bacteriocins produced by
intestinal isolates of Lactobacillus salivarius. Appl. Environ. Mi-
crobiol. 73:3719–3723.
Ben-Bassat, A., K. Bauer, S. Y. Chang, K. Myambo, A. Boosman,
and S. Chang. 1987. Processing of the initiation methionine from
proteins: Properties of the Escherichia coli methionine aminopepti-
dase and its gene structure. J. Bacteriol. 169:751–757.
Bernasconi, E., J. E. Germond, M. Delley, R. Fritsche, and B. Cor-
thesy. 2002. Lactobacillus bulgaricus proteinase expressed in Lac-
tococcus lactis is a powerful carrier for cell wall-associated and
secreted bovine beta-lactoglobulin fusion proteins. Appl. Environ.
Microbiol. 68:2917–2923.
Birkeland, N. K. 1994. Cloning, molecular characterization, and ex-
pression of the genes encoding the lytic functions of lactococcal
bacteriophage phi LC3: A dual lysis system of modular design.
Can. J. Microbiol. 40:658–665.
4451
Bockelmann, W., V. Monnet, A. Geis, M. Teuber, and J.-C. Gripon.
1989. Comparison of cell wall proteinases from Lactococcus lactis
ssp. cremoris AC1 and Lactococcus lactis ssp. lactis NCDO 763.
Appl. Environ. Microbiol. 31:278–282.
Boizet, B., Y. Lahbib-Mansais, L. Dupont, P. Ritzenthaler, and M.
Mata. 1990. Cloning, expression and sequence analysis of an endo-
lysin-encoding gene of Lactobacillus bulgaricus bacteriophage mv1.
Gene 94:61–67.
Bolotin, A., S. Mauger, K. Malarme, S. D. Ehrlich, and A. Sorokin.
1999. Low-redundancy sequencing of the entire Lactococcus lactis
IL1403 genome. Antonie van Leeuwenhoek 76:27–76.
Boris, S., R. Jimenez-Diaz, J. L. Caso, and C. Barbes. 2001. Par-
tial characterization of a bacteriocin produced by Lactobacillus
delbrueckii ssp. lactis UO004, an intestinal isolate with probiotic
potential. J. Appl. Microbiol. 91:328–333.
Bourgeois, I., E. Camiade, R. Biswas, P. Courtin, L. Gibert, F. Götz,
M. P. Chapot-Chartier, J. L. Pons, and M. Pestel-Caron. 2009.
Characterization of AtlL, a bifunctional autolysin of Staphylo-
coccus lugdunensis with N-acetylglucosaminidase and N-acetyl-
muramoyl-l-alanine amidase activities. FEMS Microbiol. Lett.
290:105–113.
Callanan, M., P. Kaleta, J. O’Callaghan, O. O’Sullivan, K. Jordan, O.
McAuliffe, A. Sangrador-Vegas, L. Slattery, G. F. Fitzgerald, T.
Beresford, and R. P. Ross. 2008. Genome sequence of Lactobacillus
helveticus, an organism distinguished by selective gene loss and
insertion sequence element expansion. J. Bacteriol. 190:727–735.
Castillo, I., T. Requena, P. Ferandez de Palencia, J. Fontecha, and M.
Gobbetti. 1999. Isolation and characterisation of an intercellular
esterase from Lactobacillus casei ssp. casei IFPL731. J. Appl. Mi-
crobiol. 86:653–659.
Chaillou, S., M. C. Champomier-Verges, M. Cornet, A. M. Crutz-Le
Coq, A. M. Dudez, V. Martin, S. Beaufils, E. Darbon-Rongère, R.
Bossy, V. Loux, and M. Zagorec. 2005. The complete genome se-
quence of the meat-borne lactic acid bacterium Lactobacillus sakei
23K. Nat. Biotechnol. 23:1527–1533.
Chen, Y. S., J. E. Christensen, J. R. Broadbent, and J. L. Steele.
2003. Identification and characterization of Lactobacillus helveti-
cus PepO2, an endopeptidase with post-proline specificity. Appl.
Environ. Microbiol. 69:1276–1282.
Chen, Y. S., and J. L. Steele. 1998. Genetic characterization and
physiological role of endopeptidase O from Lactobacillus helveticus
CNRZ32. Appl. Environ. Microbiol. 64:3411–3415.
Chich, J. F., K. Marchesseau, and J. C. Gripon. 1997. Intracellular
esterase from Lactococcus lactis ssp. lactis NCD0 763: Purification
and characterisation. Int. Dairy J. 7:169–174.
Chopin, A. 1993. Organization and regulation of genes for amino
acid biosynthesis in lactic acid bacteria. FEMS Microbiol. Rev.
12:21–37.
Christensen, J. E., D. L. Lin, A. Palva, and J. L. Steele. 1995. Se-
quence analysis, distribution and expression of an aminopeptidase
N-encoding gene from Lactobacillus helveticus CNRZ32. Gene
164:189–190. [Erratum to Gene 155:89–93]
Christensen, J. E., and J. L. Steele. 2003. Impaired growth rates in
milk of Lactobacillus helveticus peptidase mutants can be overcome
by use of amino acid supplements. J. Bacteriol. 185:3297–3306.
Christiansen, J. K., J. E. Hughes, D. L. Welker, B. T. Rodriguez, J. L.
Steele, and J. R. Broadbent. 2008. Phenotypic and genotypic anal-
ysis of amino acid auxotrophy in Lactobacillus helveticus CNRZ 32.
Appl. Environ. Microbiol. 74:416–423.
Claesson, M. J., Y. Li, S. Leahy, C. Canchaya, J. P. van Pijkeren,
A. M. Cerdeño-Tárraga, J. Parkhill, S. Flynn, G. C. O’Sullivan,
J. K. Collins, D. Higgins, F. Shanahan, G. F. Fitzgerald, D. van
Sinderen, and P. W. O’Toole. 2006. Multireplicon genome archi-
tecture of Lactobacillus salivarius. Proc. Natl. Acad. Sci. USA
103:6718–6723.
Collins, Y. F., P. L. McSweeney, and M. G. Wilkinson. 2003a. Evidence
of a relationship between autolysis of starter bacteria and lipolysis
in cheddar cheese during ripening. J. Dairy Res. 70:105–113.
Collins, Y. F., P. L. H. McSweeney, and M. G. Wilkinson. 2003b.
Lipolysis and free fatty acid catabolism in cheese: A review of cur-
rent knowledge. Int. Dairy J. 13:841–866.
Journal of Dairy Science Vol. 93 No. 10, 2010
4452 SLATTERY ET AL.
Coolbear, T., J. R. Reid, and G. G. Pritchard. 1992. Stability and
specificity of the cell wall-associated proteinase from Lactococcus
lactis ssp. cremoris H2 released by treatment with lysozyme in
the presence of calcium ions. Appl. Environ. Microbiol. 58:3263–
3270.
Cotter, P. D., C. Hill, and R. P. Ross. 2005. Bacteriocins: Developing
innate immunity for food. Nat. Rev. Microbiol. 3:777–788.
Courtin, P., M. Nardi, U. Wegmann, V. Joutsjoki, J. C. Ogier, J. C.
Gripon, A. Palva, B. Henrich, and V. Monnet. 2002. Accelerating
cheese proteolysis by enriching Lactococcus lactis proteolytic sys-
tem with lactobacilli peptidases. Int. Dairy J. 12:447–454.
Curley, P., and D. van Sinderen. 2000. Identification and characterisa-
tion of a gene encoding aminoacylase activity from Lactococcus
lactis MG1363. FEMS Microbiol. Lett. 183:177–182.
de Palencia, P. F., C. Pelaez, C. Romero, and M. C. Martin-Hernandez.
1997. Purification and characterization of the cell wall proteinase
of Lactobacillus casei ssp. casei IFPL 731 isolated from raw goat’s
milk cheese. J. Agric. Food Chem. 45:3401–3405.
Deegan, L. H., P. D. Cotter, C. Hill, and P. Ross. 2006. Bacteriocins:
Biological tools for bio-preservation and shelf-life extension. Int.
Dairy J. 16:1058–1071.
Desiere, F., R. D. Pridmore, and H. Brussow. 2000. Comparative ge-
nomics of the late gene cluster from Lactobacillus phages. Virology
275:294–305.
Deutsch, S. M., S. Guezenec, M. Piot, S. Foster, and S. Lortal. 2004.
Mur-LH, the broad-spectrum endolysin of Lactobacillus helveti-
cus temperate bacteriophage phi-0303. Appl. Environ. Microbiol.
70:96–103.
Deutsch, S. M., A. Neveu, S. Guezenec, P. Ritzenthaler, and S. Lortal.
2003. Early lysis of Lactobacillus helveticus CNRZ 303 in Swiss
cheese is not prophage-related. Int. J. Food Microbiol. 81:147–
157.
Djorjevic, G. M., and T. R. Klaenhammer. 1997. Genes and gene ex-
pression in Lactococcus bacteriophages. Int. Dairy J. 7:489–508.
Dudley, E. G., A. C. Husgen, W. He, and J. L. Steele. 1996. Se-
quencing, distribution, and inactivation of the dipeptidase A gene
(pepDA) from Lactobacillus helveticus CNRZ32. J. Bacteriol.
178:701–704.
Exterkate, F. A., and G. J. C. M. De Veer. 1987. Complexity of the na-
tive cell wall proteinase of Lactococcus lactis ssp. cremoris HP and
purification of the enzyme. Syst. Appl. Microbiol. 9:183–191.
Fenelon, M. A., T. P. Beresford, and T. P. Guinee. 2002. Comparison
of different bacterial culture systems for the production of reduced-
fat Cheddar cheese. Int. J. Dairy Technol. 55:194–203.
Fenster, K. M., K. L. Parkin, and J. L. Steele. 1997. Characterization
of a thiol-dependent endopeptidase from Lactobacillus helveticus
CNRZ32. J. Bacteriol. 179:2529–2533.
Fenster, K. M., K. L. Parkin, and J. L. Steele. 2000. Characterization
of an arylesterase from Lactobacillus helveticus CNRZ32. J. Appl.
Microbiol. 88:572–583.
Fenster, K. M., K. L. Parkin, and J. L. Steele. 2003a. Nucleotide se-
quencing, purification, and biochemical properties of an arylesterase
from Lactobacillus casei LILA. J. Dairy Sci. 86:2547–2557.
Fenster, K. M., K. L. Parkin, and J. L. Steels. 2003b. Intracellular
esterase from Lactobacillus casei LILA: Nucleotide sequencing, pu-
rification, and characterization. J. Dairy Sci. 86:1118–1129.
Fernandez, L., M. M. Beerthuyzen, J. Brown, R. J. Siezen, T. Cool-
bear, R. Holland, and O. P. Kuipers. 2000. Cloning, character-
ization, controlled overexpression, and inactivation of the major
tributyrin esterase gene of Lactococcus lactis. Appl. Environ. Mi-
crobiol. 66:1360–1368.
Flynn, S., D. van Sinderen, G. M. Thornton, H. Holo, I. F. Nes, and
J. K. Collins. 2002. Characterization of the genetic locus respon-
sible for the production of ABP-118, a novel bacteriocin produced
by the probiotic bacterium Lactobacillus salivarius ssp. salivarius
UCC118. Microbiology 148:973–984.
Fox, P. F., and J. M. Wallace. 1997. Formation of flavor compounds in
cheese. Adv. Appl. Microbiol. 45:17–85.
Ganzle, M. G. 2004. Reutericyclin: Biological activity, mode of ac-
tion, and potential applications. Appl. Microbiol. Biotechnol.
64:326–332.
Journal of Dairy Science Vol. 93 No. 10, 2010
Genay, M., L. Sadat, V. Gagnaire, and S. Lortal. 2009. prtH2 and not
prtH is the ubiquitous cell-wall proteinase gene in Lactobacillus
helveticus. Appl. Environ Microbiol. 19:89–95.
Giraffa, G., M. Gatti, L. Rossetti, L. Senini, and E. Neviani. 2000.
Molecular diversity within Lactobacillus helveticus as revealed by
genotypic characterization. Appl. Environ. Microbiol. 66:1259–
1265.
Gobbetti, M., P. F. Fox, and L. Stepaniak. 1997a. Isolation and char-
acterization of a tributyrin esterase from Lactobacillus plantarum
2739. J. Dairy Sci. 80:3099–3106.
Gobbetti, M., E. Smacchi, and A. Corestti. 1997b. Purification and
characterisation of a cell surface-associated esterase from Lactoba-
cillus fermentarum DT41. Int. Dairy J. 7:13–21.
Gonzales, T., and J. Robert-Baudouy. 1996. Bacterial aminopeptidas-
es: Properties and functions. FEMS Microbiol. Rev. 18:319–344.
Hannon, J. A., K. N. Kilcawley, M. G. Wilkinson, C. M. Delahunty,
and T. P. Beresford. 2006. Flavor precursor development in Ched-
dar cheese due to lactococcal starters and the presence and lysis of
Lactobacillus helveticus. Int. Dairy J. 17:316–327.
Hannon, J. A., M. G. Wilkinson, C. M. Delahunty, J. M. Wallace, P.
A. Morrissey, and T. P. Beresford. 2003. Use of autolytic starter
systems to accelerate the ripening of Cheddar cheese. Int. Dairy
J. 13:313–323.
Henrich, B., B. Binishofer, and U. Blasi. 1995. Primary structure and
functional analysis of the lysis genes of Lactobacillus gasseri bacte-
riophage phi adh. J. Bacteriol. 177:723–732.
Hickey, D. K., K. N. Kilcawley, T. P. Beresford, E. M. Sheehan, and
M. G. Wilkinson. 2007. Starter strain related effects on the bio-
chemical and sensory properties of Cheddar cheese. J. Dairy Res.
74:9–17.
Hickey, D. K., K. N. Kilcawley, T. P. Beresford, and M. G. Wilkinson.
2006. Starter bacteria are the prime agents of lipolysis in cheddar
cheese. J. Agric. Food Chem. 54:8229–8235.
Hickey, M. W., A. J. Hillier, and J. R. Jago. 1983. Peptidase activities
in lactobacilli. Aust. J. Dairy Technol. 38:118–127.
Higurashi, S., Y. Kunieda, H. Matsuyama, and H. Kawakami. 2007.
Effects of cheese consumption on the accumulation of abdominal
adipose and decrease in serum adiponectin levels in rats fed a calo-
rie dense diet. Int. Dairy J. 17:1224–1231.
Holland, R., and T. Coolbear. 1996. Purification of tributyrin esterase
from Lactococcus lactis ssp. cremoris E8. J. Dairy Res. 63:131–
140.
Holland, R., S.-Q. Liu, V. L. Crow, M.-L. Delabre, M. W. Lubbers, M.
Bennett, and G. Norris. 2005. Esterases of lactic acid bacteria and
cheese flavor: Milk fat hyrolysis, alcoholsis and esterfication. Int.
Dairy J. 15:711–718.
Holland, R., S.-Q. Liu, T. Wang, M. Bennett, G. Norris, M.-L. Dela-
bre, M. W. Lubbers, J. M. Dekker, and V. L. Crow. 2002. Esterase
of lactic acid bacteria. Aust. J. Dairy Technol. 57:116.
Holo, H., Z. Jeknic, M. Daeschel, S. Stevanovic, and I. F. Nes. 2001.
Plantaricin W from Lactobacillus plantarum belongs to a new fam-
ily of two-peptide lantibiotics. Microbiology 147:643–651.
Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley and S. T. Wil-
liams 1994. Section 19. Regular, non sporing gram-positive rods.
Bergey’s Manual of Determinative Bacteriology. 9th ed. Williams
and Wilkins, Baltimore, MD.
Jauhiainen, T., M. Ronnback, H. Vapaatalo, K. Wuolle, H. Kauti-
ainen, and R. Korpela. 2007. Lactobacillus helveticus fermented
milk reduces arterial stiffness in hypertensive subjects. Int. Dairy
J. 17:1209–1211.
Jauhiainen, T., H. Vapaatalo, T. Poussa, S. Kyronpalo, M. Rasmus-
sen, and R. Korpela. 2005. Lactobacillus helveticus fermented milk
lowers blood pressure in hypertensive subjects in 24-h ambulatory
blood pressure measurement. Am. J. Hypertens. 18:1600–1605.
Joerger, M. C., and T. R. Klaenhammer. 1986. Characterization and
purification of helveticin J and evidence for a chromosomally de-
termined bacteriocin produced by Lactobacillus helveticus 481. J.
Bacteriol. 167:439–446.
Joerger, M. C., and T. R. Klaenhammer. 1990. Cloning, expression,
and nucleotide sequence of the Lactobacillus helveticus 481 gene en-
coding the bacteriocin helveticin J. J. Bacteriol. 172:6339–6347.
 INVITED REVIEW: LACTOBACILLUS HELVETICUS
Kenny, O. M., R. J. Fitzgerald, G. O’Cuinn, T. P. Beresford, and K.
N. Jordan. 2003. Growth phase and growth medium effects on
the peptidase activities of Lactobacillus helveticus. Int. Dairy J.
13:509–516.
Kenny, O. M., R. J. Fitzgerald, G. O’Cuinn, T. P. Beresford, and K.
N. Jordan. 2005. Comparative analysis of the autolytic potential
of Lactobacillus helveticus strains during Cheddar cheese ripening.
Int. Dairy J. 58:207–213.
Kenny, O. M., R. J. Fitzgerald, G. O’Cuinn, T. P. Beresford, and
K. N. Jordan. 2006. Autolysis of selected Lactobacillus helveticus
adjunct strains during Cheddar cheese ripening. Int. Dairy J.
6:797–804.
Kiernan, R. C., T. P. Beresford, G. O’Cuinn, and K. N. Jordan. 2000.
Autolysis of lactobacilli during Cheddar cheese ripening. Ir. J.
Agric. Food Res. 39:95–106.
Kilcawley, K. N., M. G. Wilkinson, and P. F. Fox. 2002. Determina-
tion of key enzyme activities in commercial peptidase and lipase
preparations from microbial or animal sources. Enzyme Microb.
Technol. 31:310–320.
Kleerebezem, M., J. Boekhorst, R. van Kranenburg, D. Molenaar, O.
P. Kuipers, R. Leer, R. Tarchini, S. A. Peters, H. M. Sandbrink,
M. W. E. J. Fiers, W. Stiekema, R. M. Klein Lankhorst, P. A.
Bron, S. M. Hoffer, M. N. Nierop Groot, R. Kerkhoven, M. de
Vries, B. Ursing, W. M. de Vos, and R. J. Siezen. 2003. Complete
genome sequence of Lactobacillus plantarum WCFS1. Proc. Natl.
Acad. Sci. USA 100:1990–1995.
Kok, J. 1990. Genetics of the proteolytic system of lactic acid bacteria.
FEMS Microbiol. Rev. 7:15–42.
Kunji, E. R., I. Mierau, A. Hagting, B. Poolman, and W. N. Konings.
1996. The proteolytic systems of lactic acid bacteria. Antonie van
Leeuwenhoek 70:187–221.
Kyla-Nikkila, K., M. Hujanen, M. Leisola, and A. Palva. 2000. Meta-
bolic engineering of Lactobacillus helveticus CNRZ32 for produc-
tion of pure L-(+)-lactic acid. Appl. Environ. Microbiol. 66:3835–
3841.
l’Anson, K. J., S. Movahedi, H. G. Griffin, M. J. Gasson, and F.
Mulholland. 1995. A non-essential glutamyl aminopeptidase is re-
quired for optimal growth of Lactococcus lactis MG1363 in milk.
Microbiology 141:2873–2881.
Leclerc, P.-L., S. F. Gauthier, H. Bachelard, M. Santure, and D. Roy.
2002. Antihypertensive activity of casein-enriched milk fermented
by Lactobacillus helveticus. Int. Dairy J. 12:995–1004.
Lee, S. Y., and B. H. Lee. 1990. Esterolytic and lipolytic activities of
Lactobacillus casei subsp casei LLG. J. Food Sci. 55:119–126.
Lepeuple, A. S., E. Van Gemert, and M. P. Chapot-Chartier. 1998.
Analysis of the bacteriolytic enzymes of the autolytic Lactococ-
cus lactis ssp. cremoris strain AM2 by renaturing polyacrylamide
gel electrophoresis: Identification of a prophage-encoded enzyme.
Appl. Environ. Microbiol. 64:4142–4148.
Liu, S. Q., R. Holland, and V. L. Crow. 2003. Ester synthesis in an
aqueous environment by Streptococcus thermophilus and other dairy
lactic acid bacteria. Appl. Microbiol. Biotechnol. 63:81–88.
Lortal, S., and M.-P. Chapot-Chartier. 2005. Role, mechanisms and
control of lactic acid bacteria lysis in cheese. Int. Dairy J. 15:857–
871.
Luoma, S., K. Peltoniemi, V. Joutsjoki, T. Rantanen, M. Tamminen,
I. Heikkinen, and A. Palva. 2001. Expression of six peptidases
from Lactobacillus helveticus in Lactococcus lactis. Appl. Environ.
Microbiol. 67:1232–1238.
Macedo, A. C., T. G. Tavares, and F. X. Malcata. 2003. Esterase activ-
ities of intracellular extracts of wild strains of lactic acid bacteria
isolated from Serra da Estrela cheese. Food Chem. 81:379–381.
Makarova, K., A. Slesarev, Y. Wolf, A. Sorokin, B. Mirkin, E. Koonin,
A. Pavlov, N. Pavlova, V. Karamychev, N. Polouchine, V. Shak-
hova, I. Grigoriev, Y. Lou, D. Rohksar, S. Lucas, K. Huang, D. M.
Goodstein, T. Hawkins, V. Plengvidhya, D. Welker, J. Hughes, Y.
Goh, A. Benson, K. Baldwin, J. H. Lee, I. Díaz-Muñiz, B. Dosti,
V. Smeianov, W. Wechter, R. Barabote, G. Lorca, E. Altermann,
R. Barrangou, B. Ganesan, Y. Xie, H. Rawsthorne, D. Tamir, C.
Parker, F. Breidt, J. Broadbent, R. Hutkins, D. O’Sullivan, J.
Steele, G. Unlu, M. Saier, T. R. Klaenhammer, P. Richardson,
4453
S. Kozyavkin, B. Weimer, and D. Mills. 2006. Comparative ge-
nomics of the lactic acid bacteria. Proc. Natl. Acad. Sci. USA
103:15611–15616.
Maldonado, A., R. Jimenez-Diaz, and J. L. Ruiz-Barba. 2004. Induc-
tion of plantaricin production in Lactobacillus plantarum NC8 af-
ter coculture with specific gram-positive bacteria is mediated by
an autoinduction mechanism. J. Bacteriol. 186:1556–1564.
McDonnell, M., R. Fitzgerald, I. N. Fhaolain, P. V. Jennings, and
G. O’Cuinn. 1997. Purification and characterization of amino-
peptidase P from Lactococcus lactis ssp. cremoris. J. Dairy Res.
64:399–407.
McLaughlan, A. M., and S. J. Foster. 1998. Molecular characterization
of an autolytic amidase of Listeria monocytogenes EGD. Microbi-
ology 144:1359–1367.
Monnet, V., D. LeBars, and J.-C. Gripon. 1987. Purification and
characterization of a cell wall proteinase from Streptococcus lactis
NCDO 763. J. Dairy Res. 54:247–255.
Morishita, T., Y. Deguchi, M. Yajima, T. Sakurai, and T. Yura. 1981.
Multiple nutritional requirements of lactobacilli: Genetic lesions
affecting amino acid biosynthetic pathways. J. Bacteriol. 148:64–
71.
Mucchetti, G., F. Locci, P. Massara, R. Vitale, and E. Neviani. 2002.
Production of pyroglutamic acid by thermophilic lactic acid bacte-
ria in hard-cooked mini-cheeses. J. Dairy Sci. 85:2489–2496.
Muriana, P. M., and T. R. Klaenhammer. 1991. Purification and partial
characterization of lactacin F, a bacteriocin produced by Lactoba-
cillus acidophilus 11088. Appl. Environ. Microbiol. 57:114–121.
Nakajima, H., A. Hagting, E. R. Kunji, B. Poolman, and W. N. Kon-
ings. 1997. Cloning and functional expression in Escherichia coli of
the gene encoding the di- and tripeptide transport protein of Lac-
tobacillus helveticus. Appl. Environ. Microbiol. 63:2213–2217.
Nakajima, H., E. R. S. Kunji, B. Poolman, and W. N. Konings. 1998.
Amino acid transport in Lactobacillus helveticus. FEMS Micro-
biol. Lett. 158:249–253.
Nakamura, Y., N. Yamamoto, K. Sakai, A. Okubo, S. Yamazaki, and
T. Takano. 1995. Purification and characterization of angiotensin
I-converting enzyme inhibitors from sour milk. J. Dairy Sci.
78:777–783.
Narva, M., M. Collin, C. Lamberg-Allardt, M. Karkkainen, T. Poussa,
H. Vapaatalo, and R. Korpela. 2004. Effects of long-term interven-
tion with Lactobacillus helveticus-fermented milk on bone mineral
density and bone mineral content in growing rats. Ann. Nutr.
Metab. 48:228–234.
Naser, S. M., K. E. Hagen, M. Vancanneyt, I. Cleenwerck, J. Swings,
and T. A. Tompkins. 2006. Lactobacillus suntoryeus Cachat and
Priest 2005 is a later synonym of Lactobacillus helveticus (Orla-
Jensen 1919) Bergey et al. 1925 (Approved Lists 1980). Int. J.
Syst. Evol. Microbiol. 56:355–360.
Nilsen, T., I. F. Nes, and H. Holo. 2003. Enterolysin A, a cell wall-de-
grading bacteriocin from Enterococcus faecalis LMG 2333. Appl.
Environ. Microbiol. 69:2975–2984.
Niven, G. W. 1991. Purification and characterization of amino-pep-
tidase A from Lactococcus lactis ssp. lactis NCDO 712 . J. Gen.
Microbiol. 137:1207–1212.
O’Sullivan, O., J. O’Callaghan, A. Sangrador-Vegas, O. McAuliffe, L.
Slattery, P. Kaleta, M. Callanan, G. F. Fitzgerald, R. P. Ross, and
T. Beresford. 2009. Comparative genomics of lactic acid bacteria
reveals a niche-specific gene set. BMC Microbiol. 9:50.
Pederson, J. A., G. J. Mileski, B. C. Weimer, and J. L. Steele. 1999.
Genetic characterization of a cell envelope-associated proteinase
from Lactobacillus helveticus CNRZ32. J. Bacteriol. 181:4592–
4597.
Pillidge, C. J., P. S. V. S. Rallabhandi, X.-Z. Tong, P. K. Gopal, P. C.
Farley, and P. A. Sullivan. 2002. Autolysis of Lactococcus lactis.
Int. Dairy J. 12:133–144.
Pridmore, R. D., B. Berger, F. Desiere, D. Vilanova, C. Barretto,
A. C. Pittet, M. C. Zwahlen, M. Rouvet, E. Altermann, R. Bar-
rangou, B. Mollet, A. Mercenier, T. R. Klaenhammer, F. Arigoni,
and M. A. Schell. 2004. The genome sequence of the probiotic
intestinal bacterium Lactobacillus johnsonii NCC 533. Proc. Natl.
Acad. Sci. USA 101:2512–2517.
Journal of Dairy Science Vol. 93 No. 10, 2010
4454 SLATTERY ET AL.
Quiberoni, A., V. B. Suarez, and J. A. Reinheimer. 1999. Inactivation
of Lactobacillus helveticus bacteriophages by thermal and chemi-
cal treatments. J. Food Prot. 62:894–898.
Ramadurai, L., and R. K. Jayaswal. 1997. Molecular cloning, sequenc-
ing, and expression of lytM, a unique autolytic gene of Staphylococ-
cus aureus. J. Bacteriol. 179:3625–3631.
Recsei, P. A., A. D. Gruss, and R. P. Novick. 1987. Cloning, sequence,
and expression of the lysostaphin gene from Staphylococcus simu-
lans. Proc. Natl. Acad. Sci. USA 84:1127–1131.
Rekhif, N., A. Atrih, and G. Lefebvre. 1994. Characterization and
partial purification of plantaricin LC74, a bacteriocin produced by
Lactobacillus plantarum LC74. Biotechnol. Lett. 16:771–776.
Rice, K. C., and K. W. Bayles. 2008. Molecular control of bacterial
death and lysis. Microbiol. Mol. Biol. Rev. 72:85–109.
Riley, M. A., and J. E. Wertz. 2002. Bacteriocins: Evolution, ecology,
and application. Annu. Rev. Microbiol. 56:117–137.
Saito, T. 2004. Selection of useful probiotic lactic acid bacteria from
the Lactobacillus acidophilus group and their applications to func-
tional foods. Anim. Sci. J. 75:1–13.
Savijoki, K., and A. Palva. 2000. Purification and molecular charac-
terization of a tripeptidase (PepT) from Lactobacillus helveticus.
Appl. Environ. Microbiol. 66:794–800.
Schindler, C. A., and V. T. Schuhardt. 1964. Lysostaphin: A new
bacteriolytic agent for the Staphylococcus. Proc. Natl. Acad. Sci.
USA 51:414–421.
Sechaud, L., M. Rousseau, B. Fayard, M. L. Callegari, P. Quenee, and
J. P. Accolas. 1992. Comparative study of 35 bacteriophages of
Lactobacillus helveticus: Morphology and host range. Appl. Envi-
ron. Microbiol. 58:1011–1018.
Shao, W., G. U. Yuksel, E. G. Dudley, K. L. Parkin, and J. L. Steele.
1997. Biochemical and molecular characterization of PepR, a di-
peptidase, from Lactobacillus helveticus CNRZ32. Appl. Environ.
Microbiol. 63:3438–3443.
Shin, J. Y., W. M. Jeon, G. B. Kim, and B. H. Lee. 2004. Purification
and characterization of intracellular proteinase from Lactobacillus
casei ssp. casei LLG. J. Dairy Sci. 87:4097–4103.
Simmonds, R. S., W. J. Simpson, and J. R. Tagg. 1997. Cloning and
sequence analysis of zooA, a Streptococcus zooepidemicus gene en-
coding a bacteriocin-like inhibitory substance having a domain
structure similar to that of lysostaphin. Gene 189:255–261.
Sneath, P. H. A., N. S. Mair, and E. Sharpe. 2009. Pages 1208–1234 in
Bergey’s Manual of Systematic Bacteriology 3. 2nd ed. P. deVos,
G. M. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey,
K.-H. Schleifer, and W. B. Whitman, ed. Springer, London, UK.
Sozzi, T., and R. Maret. 1975. Isolation and characteristics of Strepto-
coccus thermophilus and Lactobacillus helveticus phages from Em-
mental starters. Lait 55:269–288.
Sridhar, V. R., J. E. Hughes, D. L. Welker, J. R. Broadbent, and J.
L. Steele. 2005. Identification of endopeptidase genes from the ge-
nomic sequence of Lactobacillus helveticus CNRZ32 and the role of
these genes in hydrolysis of model bitter peptides. Appl. Environ.
Microbiol. 71:3025–3032.
Stadhouders, J., and H. A. Veringa. 1974. Fat hydrolysis by lactic acid
bacteria. Neth. Milk Dairy J. 27:77–91.
Stiles, M. E., and W. H. Holzapfel. 1997. Lactic acid bacteria of foods
and their current taxonomy. Int. J. Food Microbiol. 36:1–29.
Sugai, M., T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S.
Inoue, and H. Suginaka. 1997. Purification and molecular charac-
terization of glycylglycine endopeptidase produced by Staphylococ-
cus capitis EPK1. J. Bacteriol. 179:1193–1202.
ten Brink, B., M. Minekus, J. M. van der Vossen, R. J. Leer, and J. H.
Huis in’t Veld. 1994. Antimicrobial activity of lactobacilli: Prelimi-
nary characterization and optimization of production of acidocin
B, a novel bacteriocin produced by Lactobacillus acidophilus M46.
J. Appl. Bacteriol. 77:140–148.
Tsakalidou, E., and G. Kalantzopoulous. 1992. Purification and par-
tial characterisation of an esterase from Lactococcus lactis ssp. lac-
tis strain ACA-DC 127. Lait 72:533–543.
Journal of Dairy Science Vol. 93 No. 10, 2010
Urso, R., K. Rantsiou, C. Cantoni, G. Comi, and L. Cocolin. 2006.
Technological characterization of a bacteriocin-producing Lactoba-
cillus sakei and its use in fermented sausages production. Int. J.
Food Microbiol. 110:232–239.
Valence, F., S. M. Deutsch, R. Richoux, V. Gagnaire, and S. Lortal.
2000. Autolysis and related proteolysis in Swiss cheese for two
Lactobacillus helveticus strains. J. Dairy Res. 67:261–271.
van de Guchte, M., S. Penaud, C. Grimaldi, V. Barbe, K. Bryson,
P. Nicolas, C. Robert, S. Oztas, S. Mangenot, A. Couloux, V.
Loux, R. Dervyn, R. Bossy, A. Bolotin, J. M. Batto, T. Walunas,
J. F. Gibrat, P. Bessières, J. Weissenbach, S. D. Ehrlich, and E.
Maguin. 2006. The complete genome sequence of Lactobacillus bul-
garicus reveals extensive and ongoing reductive evolution. Proc.
Natl. Acad. Sci. USA 103:9274–9279.
Varmanen, P., T. Rantanen, and A. Palva. 1996. An operon from
Lactobacillus helveticus composed of a proline iminopeptidase gene
(pepI) and two genes coding for putative members of the ABC
transporter family of proteins. Microbiology 142:3459–3468.
Vaughan, E. E., C. Daly, and G. F. Fitzgerald. 1992. Identification and
characterization of helveticin V-1829, a bacteriocin produced by
Lactobacillus helveticus 1829. J. Appl. Bacteriol. 73:299–308.
Venema, K., G. Venema, and J. Kok. 1995. Lactococcal bacteriocins:
Mode of action and immunity. Trends Microbiol. 3:299–304.
Vesanto, E., K. Savijoki, T. Rantanen, J. L. Steele, and A. Palva. 1995.
An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactoba-
cillus helveticus. Microbiology 141:3067–3075.
Vesanto, E., P. Varmanen, J. L. Steele, and A. Palva. 1994. Charac-
terization and expression of the Lactobacillus helveticus pepC gene
encoding a general aminopeptidase. Eur. J. Biochem. 224:991–
997.
Vinderola, G., C. Matar, and G. Perdigon. 2007. Milk fermented by
Lactobacillus helveticus R389 and its non-bacterial fraction confer
enhanced protection against Salmonella enteritidis serovar Typh-
imurium infection in mice. Immunobiology 212:107–118.
Visser, S., A. J. Robben, and C. J. Slangen. 1991. Specificity of a
cell-envelope-located proteinase (PIII-type) from Lactococcus lac-
tis ssp. cremoris AM1 in its action on bovine beta-casein. Appl.
Microbiol. Biotechnol. 35:477–483.
Vos, P., M. van Asseldonk, F. van Jeveren, R. Siezen, G. Simons,
and W. M. de Vos. 1989. A maturation protein is essential for
production of active forms of Lactococcus lactis SK11 serine pro-
teinase located in or secreted from the cell envelope. J. Bacteriol.
171:2795–2802.
Wegmann, U., M. O’Connell-Motherway, A. Zomer, G. Buist, C. Shear-
man, C. Canchaya, M. Ventura, A. Goesmann, M. J. Gasson, O.
P. Kuipers, D. van Sinderen, and J. Kok. 2007. Complete genome
sequence of the prototype lactic acid bacterium Lactococcus lactis
ssp. cremoris MG1363. J. Bacteriol. 189:3256–3270.
Williams, A. G., and J. M. Banks. 1997. Proteolysis and other hydro-
lytic enzyme activities in non-starter lactic acid bacteria (NSLAB)
isolated from Cheddar cheese manufactured in the United King-
dom. Int. Dairy J. 7:763–774.
Yamamoto, N., T. Shinoda, and T. Takano. 2000. Molecular cloning
and sequence analysis of a gene encoding an extracellular pro-
teinase from Lactobacillus helveticus CP790. Biosci. Biotechnol.
Biochem. 64:1217–1222.
Zago, M., L. Comaschi, E. Neviani, and D. Carminati. 2005. Investi-
gation on the presence of bacteriophages in natural whey starters
used for the production of Italian long-ripened cheeses. Milchwis-
senschaft 60:171–174.
Zago, M., L. Rossetti, J. Reinheimer, D. Carminati, and G. Giraffa.
2008. Detection and identification of Lactobacillus helveticus bac-
teriophages by PCR. J. Dairy Res. 75:196–201.