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Research in Microbiology 161 (2010) 480e487

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Importance of lactobacilli in food and feed biotechnology


Giorgio Giraffa a,*, Nina Chanishvili b, Yantyati Widyastuti c
a

Agricultural Research Council, Research Centre for Forage and Dairy Productions (CRA-FLC), Via Lombardo 11, 26900 Lodi, Italy
b
The G. Eliava Institute of Bacteriophage, Microbiology and Virology, 0160 Tbilisi, Georgia
c
Research Center for Biotechnology, Indonesian Institute of Sciences, 16911 Cibinong, Indonesia
Received 14 October 2009; accepted 2 March 2010
Available online 17 March 2010

Abstract
The genus Lactobacillus is a heterogeneous group of lactic acid bacteria (LAB) with important implications in food fermentation. The ability
to colonize a variety of habitats is a direct consequence of the wide metabolic versatility of this group of LAB. Consequently, lactobacilli have
been used for decades in food preservation, as starters for dairy products, fermented vegetables, fish and sausages as well as silage inoculants.
Lactobacilli have also been proposed as probiotics and microbial cell factories for the production of nutraceuticals. However, a wide range of
applications of lactobacilli in food biotechnology remains potential, whereas a number of important strains still need to be discovered and
characterized. This article provides an overview of the taxonomy of lactobacilli and describes four of the most significant case studies on the
application of this group of LAB in food and feed biotechnology, including their use as probiotics, dairy starters, silage inoculants, and microbial
cell factories. The importance of access to and exchange of biological material within and between different strain collections as a crucial step in
expanding the range of different biotechnological applications of lactobacilli is also emphasized.
2010 Elsevier Masson SAS. All rights reserved.
Keywords: Lactobacillus spp.; Probiotic lactobacilli; Dairy starter cultures; Silage inoculants; Microbial cell factories

1. Introduction
The genus Lactobacillus belongs to the large group of lactic
acid bacteria (LAB) which are all Gram-positive organisms
which produce lactic acid by fermentation. Genera of LAB
include, among others, Lactococcus, Enterococcus, Oenococcus, Pediococcus, Streptococcus, Leuconostoc and Lactobacillus (Kandler and Weiss, 1986). With over 100 species and
subspecies, the genus Lactobacillus represents the largest group
within the family Lactobacillaceae. Members of the genus are
rod-shaped, often organized in chains. They are strictly
fermentative and aerotolerant, but grow well under anaerobic
conditions. There are two groups of species depending on the
ability to ferment sugars: homofermentative species, converting
sugars mostly into lactic acid, and heterofermentative species,

converting sugars into lactic acid, acetic acid, ethanol and CO2.
Because the main catabolite is lactic acid, lactobacilli prefer
relatively acidic conditions (pH 5.5e6.5).
Bacteria belonging to the genus Lactobacillus (lactobacilli)
can be found in a variety of ecological niches such as plants,
animals and raw milk (Hammes and Vogel, 1995). In addition,
lactobacilli can be found in insects. The ability to colonize
such a variety of habitats is a direct consequence of the wide
metabolic versatility of this group of LAB. Hence, it is not
unexpected that lactobacilli have been used for decades in
food preservation, as starters for dairy products, fermented
vegetables, fish and sausages as well as silage inoculants.
Because of their potential therapeutic and prophylactic attributes, lactobacilli have also been proposed as probiotics.
1.1. Taxonomy and phylogeny

* Corresponding author.
E-mail addresses: giorgio.giraffa@entecra.it (G. Giraffa), n_chanish.
ibmv@caucasus.net (N. Chanishvili), yantyatiwidyastuti@yahoo.com (Y.
Widyastuti).
0923-2508/$ - see front matter 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.resmic.2010.03.001

In addition to their ecological versatility, the diversity of


Lactobacillus is reflected in the considerable phenotypic and
genotypic variation within the genus. Comparative analysis of

G. Giraffa et al. / Research in Microbiology 161 (2010) 480e487

16S/23S rRNA gene sequences revealed phylogenetic relationships among lactobacilli. Collins et al. (1991) originally
distinguished three phylogenetic groups, the Lactobacillus
caseiePediococcus, Lactobacillus delbrueckii, and Leuconostoc group. Later, the L. delbrueckii group was renamed as
the Lactobacillus acidophilus group (Schleifer and Ludwig,
1995) and the L. caseiePediococcus group was split into the
L. casei group, the Lactobacillus plantarum group, the
Lactobacillus reuteri group, the Lactobacillus buchneri group
and the Lactobacillus salivarius group. At present, the
phylogenetic structure of the genus Lactobacillus also
includes the Lactobacillus perolens group, the Pediococcus
group, and the Lactobacillus vitulinusecatenaformis group
(Pot and Tsakalidou, 2009).
The L. acidophilus group contains almost exclusively
obligately homofermentative lactobacilli. Besides L. acidophilus, many other species of industrial interest such as
Lactobacillus amylovorus, Lactobacillus crispatus, L. delbrueckii with the subspecies delbrueckii, lactis, bulgaricus,
and indicus, Lactobacillus johnsonii, Lactobacillus helveticus,
and Lactobacillus gasseri are included. L. helveticus and L.
delbrueckii play an acknowledged role as starters in dairy and
vegetable fermentation, respectively, whereas some strains of
the species L. acidophilus have long been known to play a key
role in human health and nutrition by their positive influence
on the intestinal flora (Pot, 2008).
The L. casei group contains the most well-known species
Lactobacillus rhamnosus, Lactobacillus paracasei, L. casei, and
Lactobacillus zeae. This subgroup has been subjected to extensive taxonomic revision in the past, leading to a provisional
rejection of the species name L. paracasei. At the moment, these
species are still considered separate species within the genus
(Dellaglio et al., 2002; Desai et al., 2006; De Vos et al., 2005;
Pot, 2008). L. paracasei and L. rhamnosus are the most
common foodborne isolates within the L. casei group of lactobacilli, especially from cheese. L. paracasei and L. casei are also
found in silage and are common inhabitants of the animal/human
gastrointestinal tract (Pot and Tsakalidou, 2009).
The L. plantarum group encompasses the taxa L. plantarum
subsp. plantarum, L. plantarum subsp. argentoratensis, Lactobacillus paraplantarum, and Lactobacillus pentosus. L. plantarum is widely found in a range of food such as dairy, meat, and
vegetable products. It is commonly found in the human
gastrointestinal tract as a consequence of its proven ability to
survive gastric transit and colonize the gut. L. plantarum is
considered a food-grade microorganism because of its long and
documented history of safe use in fermented foods (de Vries
et al., 2006).
The L. reuteri group contains at least six species exclusively
isolated from sourdough. It should be noted that L. reuteri
strains may also be dominant members of sourdough populations (Del Bello et al., 2005). Lactobacillus fermentum, one
of the best-known species of this group, has been isolated from
vegetable and dairy fermentation (Pot, 2008; Pot and
Tsakalidou, 2009).
The L. buchneri group is a large and heterogeneous group
of lactobacilli mostly linked to food fermentations. To this

481

group belong species isolated from vegetable fermentation


(L. buchneri, Lactobacillus brevis, Lactobacillus hilgardii),
sourdough (Lactobacillus sanfranciscensis) and kefir grains
(Lactobacillus kefiri and Lactobacillus parakefiri).
The L. salivarius group is a very heterogeneous group. L.
salivarius is a homofermentative Lactobacillus isolated from
the oral cavity of man. Lactobacillus algidus was isolated as
part of the predominant psychrophilic flora from vacuumpackaged beef. Lactobacillus agilis was shown to be the
main component of the pigeon crop flora (Pot, 2008; Pot and
Tsakalidou, 2009).
2. Application of lactobacilli in food and feed
biotechnology
Lactobacilli are associated with food production because of
the preservative action due to acidification, and/or enhancement of flavor, texture and nutrition. Lactobacilli are used as
starters or complementary cultures for several varieties of
cheese, fermented plant foods, fermented meats, in wine and
beer production, sourdough bread and silage. They cause rapid
pH decrease in the raw material through the production of
lactic acid as the main catabolic product. In addition, the
proteolytic activity and production of aroma compounds,
bacteriocins and exopolysaccharides are relevant for the
quality and nutritional value of the end product and expand the
spectrum of biotechnological applications of this important
group of LAB (Leroy and De Vuyst, 2004). Some lactobacilli
of the gastrointestinal tract (GIT) have also been associated
with health benefits, which have given rise to their designation
as probiotics. At present, the consumer is paying considerable
attention to the relationship between food and health. As
a consequence, the demand for functional foods, i.e. foods
claimed to possess health-promoting properties beyond basic
nutrition, has dramatically increased over the last years
(Keohane et al., 2009; OMay and MacFarlane, 2005;
Ouwehand et al., 2002).
Bacteriocin and exopolysaccharide production are two
meaningful examples of functional applications of lactobacilli in
the food industry. In the last two decades, several studies have
demonstrated the potential of bacteriocins to control growth of
pathogenic microorganisms in food products. Bacteriocinproducing lactobacilli have been suggested as protective
cultures in fermented meats, fermented olives, and dairy products (Hammes and Vogel, 1995; Leroy and De Vuyst, 2004).
Plant or microbial polysaccharides are widely used in the food
industry. They are known to increase viscosity and firmness,
improve the texture and contribute to the taste of low-fat products. The in situ production of exopolysaccharides has been
reported in several lactobacilli (Leroy and De Vuyst, 2004).
These few examples suggest that lactobacilli are polyfunctional bacteria with industrially important implications.
The following paragraphs will provide more detailed insight
through four case studies on the most significant applications
of lactobacilli in food and food biotechnology: lactobacilli as
probiotics; lactobacilli as dairy starters; lactobacilli as silage
inoculants; and lactobacilli as microbial cell factories.

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G. Giraffa et al. / Research in Microbiology 161 (2010) 480e487

2.1. Lactobacilli as probiotics


2.1.1. Definition and mechanism of action of probiotics
Probiotics are defined by FAO/WHO (2002) as live
microorganisms which, when administered in adequate
amounts, confer health benefit on the host. There is solid
scientific evidence to support the concept that the maintenance
of healthy gut microflora may provide protection against
gastrointestinal disorders, including gastrointestinal infections and inflammatory bowel diseases. Many studies have
also demonstrated the efficiency of probiotics at offering an
appropriate alternative to the use of antibiotics in the treatment of enteric infection (Marteau et al., 2001) or to reduce
the symptoms of antibiotic-associated diarrhea (Rastall et al.,
2005). Probiotic bacterial cultures modulate the growth of
intestinal microbiota, suppress potentially harmful bacteria
and reinforce the bodys natural defense mechanisms.
Currently, much evidence exists on the positive effects of
probiotics on human health (Mattila-Sandholm et al., 2002;
Millette et al., 2008; OMay and MacFarlane, 2005; Parvez
et al., 2006).
2.1.2. Selection and application
Lactobacilli are the most extensively studied and widely
used probiotics within the LAB. Most Lactobacillus strains
belong to the L. acidophilus group. L. (para)casei, L. plantarum,
L. reuteri, and L. salivarius, which represent the respective
phylogenetic groups, are known to contain probiotic strains.
In order for a probiotic to be of benefit to human health, it
must fulfill several criteria. It must survive passage through the
upper GIT and reach its site of action alive, and it must be able
to function in the gut environment. The functional requirements of probiotics include tolerance to human gastric juice
and bile, adherence to epithelial surfaces, persistence in the
human GIT, immune stimulation, antagonistic activity toward
intestinal pathogens (such as Helicobacter pylori, Salmonella
spp., Listeria monocytogenes, and Clostridium difficile), and
the capacity to stabilize and modulate the intestinal microbiota
(Mattila-Sandholm et al., 2002; Millette et al., 2008; OMay
and MacFarlane, 2005). Several technological aspects have
to be considered in probiotic selection. These include good
sensory properties, phage resistance, viability during processing and stability in production and during storage. Probiotics must be non-pathogenic and non-toxic and should not
carry acquired, genetically exchangeable antibiotic resistance
(Mattila-Sandholm et al., 2002; Ouwehand et al., 2002).
2.1.3. Overview of probiotic food products containing
lactobacilli
Dairy products are, however, the most widely used food
carriers for delivering probiotics. The probiotic microorganisms
most often belong to L. acidophilus, L. gasseri, L. helveticus,
L. johnsonii, L. (para)casei, L. reuteri, L. plantarum, L. rhamnosus, and L. fermentum (Table 1) (for a review, see Tamime
et al., 2005). A wide range of probiotic dairy products is
available in different markets; typical examples include
pasteurized milk, ice cream, fermented milks, cheeses, and

baby feed milk powder. Yoghurt is the classical example of


probiotic fermented milk. The manufacturing stages of probiotic yoghurt are very similar to classical yoghurt, but the
fermentation time is slightly longer compared with the classical product (Tamime et al., 2005).
Due to its limited acidity, low oxygen level, high lipid
content and low storage temperature, cheese also appears to be
a suitable carrier for delivering live probiotic bacteria
(Grattepanche et al., 2008). Probiotic L. paracasei and L.
rhamnosus GG have been used in cheddar and cottage cheese
manufacturing. Usually, probiotic bacteria are introduced into
cheese as adjunct cultures along with the lactic starter cultures
(Tamime et al., 2005).
2.1.4. Research prospects and future applications
Although it has been extensively demonstrated that dairy
fermented products are the best matrices for delivering probiotics, there is growing evidence of the possibility of obtaining
probiotic foods from non-dairy matrices. Several raw materials
(such as cereals, fruits, and vegetables) have recently been
investigated to determine their suitability for designing new,
non-dairy probiotic foods (for a review, see Rivera-Espinoza and
Gallardo-Navarro, 2010).
Most presently available probiotics belong to the genus
Lactobacillus. However, few strains are commercially available for probiotic application (Table 1). Gene technology and
comparative genomics will play a role in rapid searching and
developing of new strains, with gene sequencing allowing for
an increased understanding of mechanisms and the functionality of probiotics (Reid, 2008).

Table 1
The most commonly used species of lactobacilli in probiotic preparations
(especially in dairy products) (Keohane et al., 2009; Ouwehand et al., 2002;
Parvez et al., 2006; Rastall et al., 2005).
Species

Main commercially
used strain(s) (when available)
with documented health benefits

Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus

acidophilus
johnsonii
(para)casei
rhamnosus

Lactobacillus
Lactobacillus
bulgaricus
Lactobacillus
lactis
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus

plantarum
delbrueckii subsp.

LA1; LA5
La1; NCFM; DDS-1; SBT-2062
F19; CRL 431; Immunitass; Shirota
GG; LB21; 271; GR-1;
VTT E-97800
299v; Lp01
Lb12

delbrueckii subsp.

Lla

cellobiosus
curvatus
fermentum
reuteri
brevis
salivarius
helveticus
amylovorus
crispatus
gallinarum
gasseri

RC-14
MM2
UCC118
B02

LG21

G. Giraffa et al. / Research in Microbiology 161 (2010) 480e487

2.2. Lactobacilli as dairy starters


2.2.1. Role and use
Starter cultures are microorganisms that are intentionally
added to raw material to create a desired outcome in the final
product, most often through their metabolic activities. The
most common use of starter cultures is for production of lactic
acid from lactose (milk sugar), which in most cases causes or
assists in the coagulation of milk protein by lowering its pH
value.
Certain starter organisms are added specifically for their
ability to produce flavor compounds such as diacetyl. Starter
organisms can also influence flavor and texture of cultured
and/or aged products through the breakdown of proteins, fats
and other milk constituents in addition to the pH effect. A
decreased pH of cultured products can be inhibitory to certain
spoilage organisms, although inhibition is also associated with
other byproducts such as hydrogen peroxide (Chamba, 2008).
The starter cultures used today in the dairy industry are
composed of selected strains of LAB, which were originally
present as part of the contaminating microflora of milk
(Chamba, 2008; Pot, 2008; Pot and Tsakalidou, 2009).
Modern starter cultures have developed from the practice of
retaining small quantities of whey or cream from the
successful manufacture of a fermented product on a previous
day and using this as the inoculum or starter for the next days
production. In another type of fermentation, designated as
controlled or pure culture fermentation, the microorganisms are first purified from the original food product, identified, and maintained in the laboratory. When required for
fermentation, selected strains are grown in high volumes and
added to the raw material (e.g. milk) in very high numbers.
These cultures, when used in controlled fermentation, are
referred to as starter cultures (Parente and Cogan, 2004).
Traditional cultures are of significant scientific and technological interest. Natural whey starters, despite their unpredictable performance, are still used extensively, for example, in the
manufacture of mozzarella cheese using milk obtained from
water buffaloes (Bubalus arnee) in southern Italy (Parente and
Cogan, 2004). Natural starters used for preparation of Matsoni (a yogurt-like product produced in the Caucasian region)
are known to contain lactobacilli, streptococci, sometimes
lactococci and small amounts of yeasts (Brusetti et al., 2008;
Merabishvili et al., 1999; Uchida et al., 2007).
2.2.2. Selection and application
The Lactobacillus genus is one of the most important in
development of dairy starters, which play a crucial role in souring
raw milk and in the production of fermented dairy products such
as cheeses and fermented milks (including probiotics) (Leroy
and De Vuyst, 2004). Lactobacilli are used as starters in the
manufacturing of yoghurt and mozzarella cheese. They are also
used as starter adjuncts to promote faster ripening of cheddar and
similar cheeses, to reduce the incidence of bitterness and as
probiotics in yoghurt-type products (Parente and Cogan, 2004).
L. delbrueckii subsp. bulgaricus is widely used along with
Streptococcus thermophilus as a starter in yoghurt

483

manufacturing. L. casei is used as a probiotic, but it is also found


in some starter cultures and commonly belongs to the non-starter
lactic acid bacteria (NSLAB) found in cheddar cheese. L. helveticus is frequently used along with other thermophilic LAB in
the manufacturing of a range of cheeses including emmental,
Grana Padano, Parmigiano Reggiano and mozzarella.
2.2.3. Starter performance and activity
Starter activity can be influenced by a number of factors,
including the age of the culture, handling and storage practices, incubation temperature, the quality of the raw milk,
bacteriophage attack and the presence of inhibitors such as
drugs or sanitizers. High product loss, especially in cheese
manufacturing practice, is usually associated with bacteriophages (Parente and Cogan, 2004). Since phages are strainspecific, a culture rotation strategy is necessary to control
phage multiplication. Phages can enter the dairy plant through
the raw milk supply, although some lysogenic strains themselves are phage carriers. Problems with dead vats due to
phages can often be linked to phages in the plant environment
(e.g. poor plant hygiene, residual culture). Stringent culture
handling and plant sanitation programs are essential in preventing phage problems (Kutter and Sulakvelidze, 2004).
2.3. Lactobacilli as silage inoculants
2.3.1. Silage making
Silage is an important feed for livestock during food shortages, not only in winter in cold and temperate regions, but also
during the dry season in the tropics (Mannetje, 1999). The
purpose of silage making is to preserve fresh forages while
minimizing loss of nutrients and avoiding adverse changes in
the chemical composition of the ensiled forages. It is therefore
a suitable feed for maintaining the productivity of the livestock.
Silage is a product of fermentation which is carried out by LAB
under anaerobic conditions. In such an environment, LAB
convert water-soluble carbohydrates (WSCs) of the forages into
organic acids, mainly lactic acid. The production of these acids
reduces the pH of the ensiled forages and inhibits growth of
aerobic spoilage microorganisms (McDonald et al., 1991).
The fermentation of silage requires several weeks for
completion, with a critical time normally taking only a few
hours (Oude Elferink et al., 1999). Silage fermentation is
a complex and sometimes unpredictable process because it
involves many species of LAB where interactions among them
may occur (Table 2). It is commonly recommended to add
bacterial inoculants, especially those based on lactobacilli, to
ensure the fermentation process. Fig. 1 reports different steps in
silage preparation and fermentation, which is usually applied in
plastic bags.
2.3.2. The role of inoculants
Silage inoculants are selected lactobacilli added to ensiled
forage to dominate or outnumber the naturally epiphytic LAB
present in the forage. Both homofermentative and heterofermentative lactobacilli have potential advantages as silage
inoculants. At the beginning of fermentation, production of

G. Giraffa et al. / Research in Microbiology 161 (2010) 480e487

484
Table 2
Lactic acid bacteria in silage.
Species

Forage

Reference

Lactobacillus plantarum
Lactococcus lactis
Leuconostoc
pseudomesenteroides
Pediococcus acidilactici
Lactobacillus brevis
Enterococcus faecalis
Weissella kimchii
Pediococcus pentosaceus
Lactobacillus plantarum
Pediococcus pentosaceus
Lactobacillus homohiochii
Lactobacillus brevis
Lactobacillus gasseri
Lactobacillus plantarum
Lactobacillus coryniformis
Leuconostoc spp.
Enterococcus faecium
Enterococcus faecalis
Pediococcus acidilactici
Pediococcus pentosaceus
Pediococcus spp.
Lactobacillus brevis
Lactobacillus fermentum
Leuconostoc mesenteroides

Paddy rice
(Oryza sativa)

Ennahar
et al., 2003

Alfalfa
(Medicago sativa L.)

Lin
et al., 1992

Pangola (Digitaria
decumbens), Setaria
(Setaria sphacelata) and
Hamil (Panicum maximum)

Tjandraatmadja
et al., 1994a,b

lactic acid by homofermentative lactobacilli is preferred to


reduce pH faster, which may inhibit growth of undesirable
microorganisms and improve fermentation quality (Cai et al.,
1999). Good aerobic stability is then controlled by the heterofermentative lactobacilli, since the activity of yeast is
impaired due to acetic acid produced (Driehuis et al., 2001;
Filya, 2003). Combining homofermentative and heterofermentative inoculants has become popular and has been used
for various forages (Filya, 2003; Weinberg et al., 1999; Zhang
et al., 2009).
2.3.3. Selection and application
There are many commercial inoculants available on the
market. They may vary and be based on several requirements
such as their ability to dominate the natural population of
microorganisms in the forages and to produce lactic acid
rapidly as a result of effective fermentation, which leads to
a drastic drop in pH. It is also essential that inoculants be
stable during storage so that the correct bacterial concentration
is applied to the ensiled forage. Several strains of L. plantarum
(homofermentative) and L. buchneri (heterofermentative) have
been selected and developed as silage inoculants.
Ideally, the inoculants should provide 106 CFU per g of
fresh crop forages (McDonald et al., 1991). This concentration

Fig. 1. Silage preparation using lactobacilli as inoculants. A: chopping grass; B: adding rice bran and inoculants as additives; C: silage inoculants; D: silage
fermentation in plastic bags.

G. Giraffa et al. / Research in Microbiology 161 (2010) 480e487

may decrease, since their viability declines with increasing


temperature. Higher temperature results in even greater losses
in viability for some inoculants. Mulrooney and Kung (2008)
reported that L. plantarum MTD/1 was the most thermotolerant inoculant. Selection of thermotolerant silage inoculants
is necessary for the best performance of this practice in the
tropics. L. plantarum CA28 grew well during the ensilage of
perennial ryegrass at 25  C (Zhang et al., 2000).
2.3.4. Future prospects
Although silage making has been practiced since ancient
times, research to improve the quality of inoculants for silage
has been intensively carried out. The aim is to find strains with
improved properties such as antibacterial activity and probiotic potential. Among available silage inoculants, L. plantarum MTD1 (Ecosyl), and L. plantarum (Agri-King) showed
strong antibacterial activity against Micrococcus luteus, while
L. buchneri (both of Biotal and Pioneer Hi-Bred International)
was effective against Pseudomonas aeruginosa (Gollop et al.,
2005). Several studies of Weinberg et al. (1999, 2003,
2004a,b) showed that some lactobacilli as silage inoculants
were able to survive in the rumen fluid. Their survival is
a good indication that they might have probiotic effects upon
the host animals.
The development of genetically modified L. plantarum as
a silage inoculant expressing cellulases and xylanases has
proven to be attractive as an alternative to the direct addition
of hydrolytic enzymes during ensiling. Cellulolytic recombinant L. plantarum modified by integration of celA gene
encoding an alkaline endo-1,4-b glucanase from Bacillus sp.
in their chromosome showed improved acidification ability for
alfalfa (Rossi et al., 2001).
2.4. Lactobacilli as microbial cell factories
Through their metabolic activity, lactobacilli may act as
cell factories for the de novo generation of bioactivities from
a range of food protein sources. Biological activities associated
with such peptides include immunomodulating, antibacterial,
antihypertensive and opioid-like properties. Milk proteins are
recognized as a primary source of bioactive peptides, which can
be encrypted within the amino acid sequence of dairy proteins,
requiring proteolysis for release and activation. These observations have highlighted interest in developing value-added
fermented foods that are selectively enriched with compounds
known to deliver a particular health benefit to the consumer,
such as the antihypertensive angiotensin-converting enzyme
(ACE) inhibitors, which are produced by lactobacilli through
their proteolytic system (Broadbent, 2008; Hayes et al., 2007).
2.4.1. Metabolic engineering
Metabolic engineering may be an alternative tool when
searching for new strains with improved biotechnological
characteristics, e.g. for the production of nutraceuticals. Metabolic engineering strategies of LAB may lead to the efficient
rerouting of sugar metabolism to nutritional end-products other
than lactic acid, such as aroma compounds, low-calories sugars

485

and natural sweeteners (Hugenholtz et al., 2002). Different


Lactobacillus species have been metabolically engineered for
production of L() lactic acid, mannitol, pyruvate and L-ribulose
(Aarnikunnas et al., 2003; Helanto et al., 2007; Nikkila et al.,
2000).
2.4.2. Lactobacilli as live vaccine carriers
Similarly to other LAB, lactobacilli can also be efficient oral
vaccine carriers. Most of our current knowledge of the use of
lactobacilli for vaccination purposes has been obtained with
tetanus toxin fragment C as the model antigen. This knowledge,
together with our increasing understanding of the immune
system and recent advancements in cloning and expression
techniques, will make development of lactobacilli as delivery
systems for live vaccines a realistic perspective (Seegers, 2002).
3. Concluding remarks
The genus Lactobacillus is a heterogeneous group of LAB
with important implications in food and feed fermentation.
Lactobacilli are currently used as probiotics, silage inoculants
and as starters in fermented food. However, a wide range of
applications for lactobacilli in food biotechnology is possible,
and a number of important strains need to be discovered and
characterized. In this respect, access and exchange of biological
material within and between different strain collections will be
crucial for expanding the range of different biotechnological
applications of lactobacilli. This would enable us to dispose of
new isolates from different countries and geographic areas
which hopefully would carry new and interesting metabolic
traits. With reference to the case studies covered here, the
availability of new strains could help, for example, to design
new probiotic products or starter cultures for the food industry
or as inoculants in silage.
Undoubtedly, common sharing of genomic information is
helping to develop biotechnological-related applications of
lactobacilli. The present availability of 25 LAB genome
sequences (including 15 Lactobacillus spp.) from public databases is providing an expanded view of genetic and metabolic
capacities of these bacteria and enabling researchers to perform
functional and comparative genomics studies. Genome
sequencing and functional genomics studies of a variety of LAB,
including lactobacilli, are now rapidly providing insight into
their diversity and evolution and revealing the molecular basis
for important traits such as flavor formation, sugar metabolism,
stress response, adaptation and interactions (Siezen et al., 2004).
For example, bioinformatics tools can be used, in a genomemining approach, to search genomes for essential components
such as proteinases, peptidases, aminotransferases, enzymes for
amino acid biosynthesis and transport systems for peptides and
amino acids which may be involved in flavor-forming reactions
in LAB (van Kranenburg et al., 2002). Expanding this approach
to other genomes, combined with experimental verification of
the predicted substrate specificities, will lead to the design of
probes for high-throughput screening and Lactobacillus spp.
strain selection in the future.

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