Trends in Food Science & Technology 15 (2004) 67–78
Review
Lactic acid bacteria
as functional starter
cultures for the
food fermentation
industry
Frédéric Leroy and
Luc De Vuyst*
Research Group of Industrial Microbiology,
Fermentation Technology and Downstream
Processing (IMDO), Department of Applied
Biological Sciences, Vrije Universiteit Brussel (VUB),
Pleinlaan 2, B-1050 Brussels, Belgium
(tel.: +32-2-6293245; fax: +32-2-6292720;
e-mail: ldvuyst@vub.ac.be)
The production of fermented foods is based on the use of
starter cultures, for instance lactic acid bacteria that initiate
rapid acidification of the raw material. Recently, new starter
cultures of lactic acid bacteria with an industrially impor-
tant functionality are being developed. The latter can con-
tribute to the microbial safety or offer one or more
organoleptic, technological, nutritional, or health advan-
tages. Examples are lactic acid bacteria that produce anti-
microbial substances, sugar polymers, sweeteners, aromatic
compounds, vitamins, or useful enzymes, or that have
probiotic properties.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
A starter culture can be defined as a microbial pre-
paration of large numbers of cells of at least one
microorganism to be added to a raw material to
* Corresponding author.
0924-2244/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tifs.2003.09.004
produce a fermented food by accelerating and steering
its fermentation process. The group of lactic acid bac-
teria (LAB) occupies a central role in these processes,
and has a long and safe history of application and con-
sumption in the production of fermented foods and
beverages (Caplice & Fitzgerald, 1999; Ray, 1992;
Wood, 1997; Wood & Holzapfel, 1995) (Table 1). They
cause rapid acidification of the raw material through the
production of organic acids, mainly lactic acid. Also,
their production of acetic acid, ethanol, aroma com-
pounds, bacteriocins, exopolysaccharides, and several
enzymes is of importance. In this way they enhance shelf
life and microbial safety, improve texture, and contribute
to the pleasant sensory profile of the end product.
The earliest production of fermented foods was based
on spontaneous fermentation due to the development of
the microflora naturally present in the raw material. The
quality of the end product was dependent on the
microbial load and spectrum of the raw material.
Spontaneous fermentation was optimised through
backslopping, i.e., inoculation of the raw material with
a small quantity of a previously performed successful
fermentation. Hence, backslopping results in dominance
of the best adapted strains. It represents a way, be it
unconsciously, of using a selected starter culture to
shorten the fermentation process and to reduce the risk
of fermentation failure. Backslopping is still in use, for
instance in the production of sauerkraut and sour-
dough, and particularly for products for which the
microbial ecology and the precise role of successions in
microbial population are not well known (Harris, 1998).
Today, the production of fermented foods and bev-
erages through spontaneous fermentation and back-
slopping represents a cheap and reliable preservation
method in less developed countries, whereas in Western
countries the large-scale production of fermented foods
has become an important branch of the food industry.
Moreover, the Western consumer appreciates traditionally
fermented products for their outstanding gastronomic
qualities.
The direct addition of selected starter cultures to raw
materials has been a breakthrough in the processing of
fermented foods, resulting in a high degree of control
over the fermentation process and standardisation of
the end product. Strains with the proper physiological
and metabolic features were isolated from natural habi-
tats or from successfully fermented products (Oberman
68 F. Leroy, L. De Vuyst / Trends in Food Science & Technology 15 (2004) 67–78
Table 1. Fermented foods and beverages and their associated lactic acid bacteria
Type of fermented product
Dairy products
-Hard cheeses without eyes
-Cheeses with small eyes
-Swiss- and Italian-type cheeses
-Butter and buttermilk
-Yoghurt
-Fermented, probiotic milk
-Kefir
Fermented meats
-Fermented sausage (Europe)
-Fermented sausage (USA)
Fermented fish products
Fermented vegetables
-Sauerkraut
-Pickles
-Fermented olives
-Fermented vegetables
Soy sauce
Fermented cereals
-Sourdough
Alcoholic beverages
-Wine (malolactic fermentation)
-Rice wine
a
B.=Bifidobacterium, C.=Carnobacterium, L.=Lactococcus, Lb.=Lactobacillus, Leuc.=Leuconostoc, O.=Oenococcus, P.=Pediococcus,
S.=Streptococcus, T.=Tetragenococcus, W.=Weissella.
& Libudzisz, 1998). However, some disadvantages have
to be considered. In general, the initial selection of
commercial starter cultures did not occur in a rational
way, but was based on rapid acidification and phage
resistance. These starters are not very flexible with
regard to the desired properties and functionality of the
end product. Originally, industrial starter cultures were
maintained by daily propagation. Later, they became
available as frozen concentrates and dried or lyophilised
preparations, produced on an industrial scale, some of
them allowing direct vat inoculation (Sandine, 1996).
Because the original starter cultures were mixtures of
several undefined microbes, the daily propagation
probably led to shifts of the ecosystem resulting in the
disappearance of certain strains. Moreover, some
important metabolic traits in LAB are plasmid-encoded
and there is a risk that they are lost during propagation.
It is further likely that loss of genetic material occurred
due to adaptation to the food matrix. The biodiversity
of commercial starters has therefore become limited.
This often leads to a loss of the uniqueness of the origi-
nal product and the loss of the characteristics that have
made the product popular (Caplice & Fitzgerald, 1999).
In contrast, the fermentation of traditional fermented
foods is frequently caused by natural, wild-type LAB
Lactic acid bacteriaa
L. lactis subsp. lactis, L. lactis subsp. cremoris
L. lactis subsp. lactis, L. lactis subsp. lactis var. diacetylactis,
L. lactis subsp. cremoris, Leuc. mesenteroides subsp. cremoris
Lb. delbrueckii subsp. lactis, Lb. helveticus, Lb. casei,
Lb. delbrueckii subsp. bulgaricus, S. thermophilus
L. lactis subsp. lactis, L. lactis subsp. lactis var. diacetylactis,
L. lactis subsp. cremoris, Leuc. mesenteroides subsp. cremoris
Lb. delbrueckii subsp. bulgaricus, S. thermophilus
Lb. casei, Lb. acidophilus, Lb. rhamnosus, Lb. johnsonii,
B. lactis, B. bifidum, B. breve
Lb. kefir, Lb. kefiranofacies, Lb. brevis
Lb. sakei, Lb. curvatus
P. acidilactici, P. pentosaceus
Lb. alimentarius, C. piscicola
Leuc. mesenteroides, Lb. plantarum, P. acidilactici
Leuc. mesenteroides, P. cerevisiae, Lb. brevis, Lb. plantarum
Leuc. mesenteroides, Lb. pentosus, Lb. plantarum,
P. acidilactici, P. pentosaceus, Lb. plantarum, Lb. fermentum
T. halophilus
Lb. sanfransiscensis, Lb. farciminis, Lb. fermentum, Lb. brevis,
Lb. plantarum, Lb. amylovorus, Lb. reuteri, Lb. pontis, Lb. panis,
Lb. alimentarius, W. cibaria
O. oeni
Lb. sakei
that originate from the raw material, the process appa-
ratus, or the environment, and that initiate the fermen-
tation process in the absence of an added commercial
starter (Böcker, Stolz, & Hammes, 1995; Weerkamp,
Klijn, Neeter, & Smit, 1996). Moreover, many tradi-
tional products obtain their flavour intensity from the
non-starter lactic acid bacteria (NSLAB), which are not
part of the normal starter flora but develop in the pro-
duct, particularly during maturation, as a secondary
flora (Beresford, Fitzsimons, Brennan, & Cogan, 2001).
Pure cultures isolated from complex ecosystems of tra-
ditionally fermented foods exhibit a diversity of meta-
bolic activities that diverge strongly from the ones of
comparable strains used as industrial bulk starters
(Klijn, Weerkamp, & de Vos, 1995). These include dif-
ferences in growth rate and competitive growth beha-
viour in mixed cultures, adaptation to a particular
substrate or raw material, antimicrobial properties, and
flavour, aroma, and quality attributes. Wild strains need
to withstand the competition of other microorganisms
to survive in their hostile natural environment, so that
they often produce antimicrobials such as bacteriocins
(Ayad, Verheul, Wouters, & Smit, 2002). In addition,
they are more dependent on their own biosynthetic
capacity than industrial strains and harbour more
 F. Leroy, L. De Vuyst / Trends in Food Science & Technology 15 (2004) 67–78
amino acid converting enzymes that play a key role in
flavour formation. Such findings underline the impor-
tance of the Designation of Protected Origin (DPO) of
many of these products, which is crucial from an eco-
nomical point of view since they contribute to the sur-
vival of small-scale fermentation plants in a world of
ongoing globalisation. A recent trend exists in the iso-
lation of wild-type strains from traditional products to
be used as starter cultures in food fermentation (Beukes,
Bester, & Mostert, 2001; De Vuyst et al., 2002; Hébert,
Raya, Tailliez, & de Giori, 2000).
Lactic acid bacteria as functional starter cultures
Definition
Nowadays, the consumer pays a lot of attention to the
relation between food and health. As a consequence, the
market for foods with health-promoting properties, so-
called functional foods, has shown a remarkable growth
over the last few years (Nutrition Business Journal,
2002). Also, the use of food additives is regarded as
unnatural and unsafe (Ray, 1992). Yet, additives are
needed to preserve food products from spoilage and to
improve the organoleptic properties. The demand for a
reduced use of additives and processing seems contra-
dictory with the market preference for products that are
fresh, safe, tasty, low in sugar, fat, and salt, and easy to
prepare. In cheese-making, for instance, the use of raw
milk permits the manufacture of high-value traditional
artisan varieties but brings about safety risks, e.g. the
development of Listeria monocytogenes. On the other
hand, pasteurisation of the milk results in loss of flavour
and gives end products that are perceived by the con-
sumer as ‘‘boring’’ (Law, 2001). These market trends
put the food industry under pressure to look for alter-
natives. In food fermentation, one of the key points for
intervention seems to be on the level of the starter cul-
ture. Unfortunately, industrial starter cultures lack the
necessary characteristics for product diversification, and
the commercial availability of new interesting starter
cultures is limited. The increased understanding of the
genomics and metabolomics of food microbes opens
perspectives for starter improvement. Through mole-
cular biology it is now possible to express desirable and
suppress undesirable properties of starter cultures (Del-
cour, De Vuyst, & Shortt, 1999; Law, 2001; Mogensen,
1993).
Recently, the use of functional starter cultures in the
food fermentation industry is being explored (De Vuyst,
2000). Functional starter cultures are starters that pos-
sess at least one inherent functional property. The latter
can contribute to food safety and/or offer one or more
organoleptic, technological, nutritional, or health
advantages (Table 2). The implementation of carefully
selected strains as starter cultures or co-cultures in fer-
mentation processes can help to achieve in situ expres-
sion of the desired property, maintaining a perfectly
69
natural and healthy product. Examples are LAB that
are able to produce antimicrobial substances, sugar
polymers, sweeteners, aromatic compounds, useful
enzymes, or nutraceuticals, or LAB with health-pro-
moting properties, so called probiotic strains. This
represents a way of replacing chemical additives by
natural compounds, at the same time providing the
consumer with new, attractive food products. It also
leads to a wider application area and higher flexibility of
starter cultures. Although probiotic strains may also be
classified as functional starter or co-cultures for food
fermentations, they will not be dealt with in this paper
(Chandan, 1999; Erkkilä et al., 2001; Jahreis et al., 2002;
Pidcock, Heard, & Henriksson, 2002; Ross, Stanton,
Hill, Fitzgerald, & Coffey, 2000).
Application of functional starter cultures in food
fermentations
Food preservation and safety
Chemical food additives such as nitrite, sulphite, pro-
pionic acid, sorbic acid, and benzoic acid are commonly
applied in food preservation technology (Smith, 1993).
As an alternative, the antimicrobial activity displayed
by LAB strains may help to combat microbial con-
tamination (Holzapfel, Geisen, & Schillinger, 1995;
Lücke, 2000). LAB produce several natural anti-
microbials, including organic acids (lactic acid, acetic
acid, formic acid, phenyllactic acid, caproic acid),
carbon dioxide, hydrogen peroxide, diacetyl, ethanol,
bacteriocins, reuterin, and reutericyclin. Acetic acid, for
instance, contributes to the aroma and prevents mould
spoilage in sourdough (Messens & De Vuyst, 2002).
Bacteriocins from LAB are low-molecular-mass pep-
tides or proteins with an antibacterial mode of action
restricted to related Gram-positive bacteria. Bacter-
iocin-producing LAB can be applied for food preserva-
tion because of their microbiological, physiological and
technological advantages (Cleveland, Montville, Nes, &
Chikindas, 2001; De Vuyst & Vandamme, 1994; Nettles
& Barefoot, 1993; Ray & Daeschel, 1992; Smith, 1993).
The in situ production of bacteriocins may increase the
competitiveness of the producer strain in the food
matrix and contribute to the prevention of food spoilage
(Hugas, Garriga, Aymerich, & Monfort, 1995; Ross et
al., 2000; Ruiz-Barba, Cathcart, Warner, & Jiménez-
Diaz, 1994; Vogel, Pohle, Tichaczek, & Hammes,
1993). For instance, bacteriocin-producing LAB can be
used as an alternative to potassium nitrate to prevent
late loss of cheese due to contamination by clostridia
(Thomas, Clarkson, & Delves-Broughton, 2000).
Another example is the suppression of flavour-disturb-
ing contaminating microbes, e.g., certain strains of
L. lactis that produce off-flavours in dairy products
(Stanley, 1998). In addition, many bacteriocins are
active towards foodborne pathogens such as Clos-
tridium botulinum, Staphylococcus aureus, and Listeria
70 F. Leroy, L. De Vuyst / Trends in Food Science & Technology 15 (2004) 67–78
Table 2. Typical examples of functional starter cultures or co-cultures and their advantages for the food industry
Advantage Functionality
Food preservation Bacteriocin production
-Dairy products
-Fermented meats
-Fermented olives
-Fermented vegetables
Organoleptic Production of exopolysaccharides
Production of amylase
Aroma generation
Enhanced sweetness
-Homoalanine-fermenting starters
-Galactose-positive/
glucose-negative starters
malolactic fermentation
Technological Bacteriophage resistance
Prevention of overacidification in
yoghurt
Autolysing starters
-Phage-mediated
-Bacteriocin-induced
Nutritional Production of nutraceuticals
-Low-calorie sugars
(e.g., sorbitol and mannitol)
-Production of oligosaccharides
-Production of B-group vitamins
(e.g., folic acid)
-Release of bioactive peptides
Reduction of toxic and
anti-nutritional compounds
-Production of l(+)-lactic acid
isomer
-Removal of lactose and galactose
-Removal of raffinose in soy
-Reduction of phytic acid content,
amylase inhibitors,
and polyphenolic compounds
-Decreased production of biogenic
amines
a
E.=Enterococcus, L.=Lactococcus, Lb.=Lactobacillus, O.=Oenococcus, P.=Pediococcus, S.=Streptococcus.
b
Recombinant strain.
monocytogenes (Nettles & Barefoot, 1993). Several
studies have indicated that LAB starter strains are
able to produce their bacteriocins in food matrices and
consequently display inhibitory activity towards sen-
sitive food spoilage or pathogenic bacterial strains. The
latter has been documented for fermented sausage
(Callewaert, Hugas, & De Vuyst, 2000; Foegeding,
Thomas, Pilkington, & Klaenhammer, 1992; Hugas et al.,
1995; Vogel et al., 1993), fermented vegetables and
olives (Harris, 1998; Harris, Fleming, & Klaenhammer,
1992; Ruiz-Barba et al., 1994), and dairy products
Lactic acid bacteriaa Relevant references
L. lactis subsp. lactis Maisnier-Patin et al. (1992),
Roberts et al. (1992)
Enterococcus spp. Giraffa (1995)
Lb. curvatus Vogel et al. (1993)
Lb. sakei Hugas et al. (1995)
P. acidilactici Foegeding et al. (1992)
E. faecium Callewaert et al. (2000)
L. plantarum Ruiz-Barba et al. (1994)
L. lactis Harris et al. (1992)
Several lactobacilli and De Vuyst & Degeest (1999)
streptococci De Vuyst & Marshall (2001)
De Vuyst et al. (2001)
Several lactobacilli Mogensen (1993)
Several strains Marshall (1987)
Demeyer et al. (2000)
Kleerebezem et al. (2000)
L. lactisb Hols et al. (1999)
Lb. delbrueckii subsp. under development
bulgaricus, S. thermophilus
O. oeni Lonvaud-Funel (1999)
Several strains Forde & Fitzgerald (1999)
lactose-negative Mollet (1996)
Lb. delbrueckii subsp. bulgaricus
L. lactis subsp. lactis Crow et al. (1996)
L. lactis Morgan et al. (1997)
Lb. plantarum Wisselink et al. (2002)
L. lactis
L. lactis Ruas-Madiedo et al. (2002)
Lb. delbrueckii subsp. Hugenholtz & Kleerebezem (1999)
bulgaricus, L. lactis, S. thermophilus Wouters et al. (2002)
Several strains Meisel & Bockelman (1999)
l(+)-lactic acid-producing Wouters et al. (2002)
strains Holzapfel (2002)
S. thermophilus Wouters et al. (2002)
Several strains Scalabrini et al. (1998)
Lb. plantarum Sharma & Kapoor (1996)
Lb. acidophilus
E. faecalis Joosten et al. (1995)
(Benkerroum et al., 2002; Buyong, Kok & Luchansky,
1998; Foulquié Moreno, Rea, Cogan, & De Vuyst,
2003; Giraffa, 1995; Maisnier-Patin, Deschamps, Tatini,
& Richard, 1992; McAuliffe, Hill, & Ross, 1999;
Roberts, Zottola, & Mckay, 1992; Rodriguez, Gaya,
Nuñez, & Medina, 1998).
Reuterin (b-hydroxypropionaldehyde) produced by
Lb. reuteri is active towards a wide spectrum of bac-
teria, moulds and yeasts (Talarico & Dobrogosz, 1989),
but is not formed in sufficient amounts in the presence
of sugars. Reutericyclin, a tetramic acid antibiotic with
 F. Leroy, L. De Vuyst / Trends in Food Science & Technology 15 (2004) 67–78
broad antimicrobial activity produced by Lb. reuteri
(Gänzle, Höltzel, Walter, Jung, & Hammes, 2000;
Gänzle & Vogel, 2003), is believed to be responsible for
the stability of certain German sourdoughs (Messens &
De Vuyst, 2002).
Recently, it has been shown that the production of
phenyllactic and 4-hydroxy-phenyllactic acids by Lb.
plantarum strains is responsible for a broad antifungal
activity in sourdough (Lavermicocca et al., 2000).
Besides the production of phenyllactic acid, Lb. plan-
tarum MiLAB 393 displays antifungal activity due to
the production of cyclic dipeptides (Ström, Sjögren,
Broberg, & Schnürer, 2002). Also, caproic acid pro-
duced, among other acids, by Lb. sanfranciscensis CB1
plays a key role in inhibiting mould growth (Corsetti,
Gobbetti, Rossi, & Damiani, 1998).
In addition to using selected natural strains, geneti-
cally engineered microorganisms may find applications.
The heterologous production of bacteriocins is well
known (Rodrı́guez, Martı́nez, Horn, & Dodd, 2002). A
Lb. curvatus strain, harbouring a gene for the expression
of the lytic enzyme lysostaphin, was shown to produce it
in sufficient quantities to inactivate St. aureus during
sausage fermentation (Cavadini, Hertel, & Hammes,
1998).
Functional starters for a more appealing product
Improvement of texture
To give a desired texture and mouthfeel to yoghurt,
skim-milk powder or whey is frequently added to the
milk. Although the consumer does not consider this as
unnatural, it represents an extra cost for the producer.
In some countries, however, gelatine or plant (e.g.,
starch, pectin, guar gum, and alginate) and microbial
polysaccharides (e.g., xanthan and gellan) are added.
Polysaccharides increase the viscosity and firmness,
improve the texture, reduce susceptibility to syneresis,
and contribute to the mouthfeel of low-fat products.
Some polysaccharides, e.g., plant carbohydrates, xan-
than and gellan, have the additional advantage of being
suitable to chemical modification to improve their
rheological properties (Harvey & McNeil, 1998). How-
ever, the modified molecules are perceived by the con-
sumer as unnatural and the food regulation of several
European countries forbids the use of additives in, for
instance, yoghurt.
Recently, the in situ production of natural texture-
improving sugar polymers (exopolysaccharides) pro-
duced by LAB for the manufacturing of yoghurts (De
Vuyst & Degeest, 1999, De Vuyst, De Vin, Vaningel-
gem, & Degeest, 2001), of sour cream and whipped
toppings (Duboc & Mollet, 2001), of ice cream (Chris-
tiansen, Madeira, & Edelsten, 1999), and of low-fat
Mozzarella (Broadbent, McMahon, Oberg, & Welker,
2001; Low et al., 1998) is being explored. Functional,
71
exopolysaccharide-producing starters of Lb. delbrueckii
subsp. bulgaricus or S. thermophilus are promising. It is
not likely that these exopolysaccharides will be utilised
in other areas than the dairy industry since they would
have to compete with established gums (Harvey &
McNeil, 1998). However, they can be produced directly
in the food matrix through the in situ use of functional
starter cultures (De Vuyst et al., 2001; De Vuyst &
Marshall, 2001). Another application can be found in
the bakery industry for a beneficial effect on bread
volume and staling (Tieking, Korakli, Ehrmann, Gän-
zle, & Vogel, 2003). Current research is investigating
the biodiversity of exopolysaccharides produced by
LAB from artisan yoghurts, fermented milks, vege-
tables, and cereals, the conditions for optimal produc-
tion, and their technological implementation in the
industrial production of fermented foods (De Vuyst &
Degeest, 1999; De Vuyst et al., 2001; Marshall et al.,
2001).
Another example of texture improvement of foods
through functional starter cultures is the use of amylase-
producing LAB. LAB producing thermostable amylases
have potential in cereal fermentations, in particular
in sourdough technology for the natural inhibition of
staling in bread (Mogensen, 1993).
Production of aroma and flavour
LAB contribute to the aroma and flavour of fer-
mented products. They acidify the food, resulting in a
tangy lactic acid taste, frequently exert proteolytic and
lipolytic activities, and produce aromatic compounds
from, for instance, amino acids upon further bio-
conversion (van Kranenburg et al., 2002; Williams,
Noble, & Banks, 2001; Yvon & Rijnen, 2001). Control
over the activities of peptidases from LAB is a key tar-
get of cheese ripening technology (Law, 2001). As an
example, overexpression of certain peptidases of L. lac-
tis subsp. cremoris improved the sensory quality of
cheese (Guldfeldt et al., 2001).
Wild strain starter cultures and NSLAB play an
important role in flavour formation because they have a
high biosynthetic capacity and produce interesting aro-
matic compounds (Ayad, Verheul, de Jong, Wouters, &
Smit, 1999; Bouton, Guyot, & Grappin, 1998; Weer-
kamp et al., 1996). For instance, Italian ewe milk
cheeses are characterised by a very heterogeneous
NSLAB flora which is influenced by geographical and
technological factors, and which could be responsible
for cheese diversity (De Angelis et al., 2001). Because
such strains offer an important base for product inno-
vation, research is going on to study their application in
the food fermentation industry. The addition of
NSLAB as adjunct cultures for cheese manufacturing
increases the level of free amino acids, peptides, and free
fatty acids, leading to flavour intensity and accelerated
cheese ripening (Crow, Curry, & Hayes, 2001).
72 F. Leroy, L. De Vuyst / Trends in Food Science & Technology 15 (2004) 67–78
Furthermore, they help to reproduce the flavour of raw
milk cheeses when pasteurised milk is used (De Angelis
et al., 2001).
Homofermentative LAB convert the available energy
source (sugar) almost completely into lactic acid via
pyruvate to produce energy and to equilibrate the redox
balance. However, pyruvate can lead to the generation
of many other metabolites such as acetate, ethanol,
diacetyl, and acetaldehyde. In this way, LAB produce
volatile substances that contribute to the typical flavour
of certain fermented products, such as sourdough
(determined by the lactate/acetate ratio), kefir and
koumiss (ethanol), butter and buttermilk (diacetyl),
and yoghurt (acetaldehyde). Optimal fermentation
control leads to improved production of some of these
volatiles whereas metabolic engineering focuses on the
steering of the metabolic flux in a well-defined direc-
tion. Strategies aiming at a direct modification of the
redox balance have led to overproduction of the
desired metabolites mentioned above (Kleerebezem,
Hols, & Hugenholtz, 2000). Examples include the
enhancement of diacetyl production by L. lactis subsp.
lactis biovar. diacetylactis in buttermilk by redirection
of pyruvate catabolism (Henriksen, Nilsson, Hansen, &
Johansen, 1999; Hugenholtz et al., 2000), and meta-
bolic engineering of acetaldehyde production by S.
thermophilus in fermented dairy products (Chaves et
al., 2002).
Alternatively, the introduction of novel enzymatic
activities into LAB may lead to cells that produce
interesting metabolites from the supplemented sugar.
Overproduction of alanine dehydrogenase in suitable L.
lactis cells has lead to a homofermentative, stereo-
specific production of l-alanine from pyruvate (Hols et
al., 1999). l-Alanine is used as a sweetener in the food
industry and its in situ production can lead to dairy
products with an intrinsic sweetness.
Functional starters with a technological advantage
Phage-resistant starters for the dairy industry
Bacteriophages pose a serious problem to the dairy
industry. In addition to strict sanitary conditions, the
use of appropriate media, the rotation of starter cultures
and the use of phage-resistant starter cultures offers a
solution. Phage resistance may be caused by natural
resistance mechanisms (restriction and modification
enzymes), prevention of intracellular phage develop-
ment through phage adsorption and abortive phage
infection, or by intracellular defence strategies (Forde
& Fitzgerald, 1999). Strains that have acquired natural
mechanisms of phage resistance, e.g. through in vivo
recombination (conjugation) or in vitro self-cloning,
are currently applied on a large scale in the dairy
industry (Daly, Fitzgerald, & Davis, 1996; Moineau,
1999).
Lactose-negative starters for the production of mild
yoghurt
In yoghurt production, lactose is converted by a
yoghurt culture into lactic acid until a final pH of 4.2–
4.5 is achieved. Upon storage, the pH can decrease
below 4.0. This undesirable post-acidification, ascribed
to Lb. delbrueckii subsp. bulgaricus, leads to an acid and
bitter taste. Lactose-negative mutants of Lb. delbrueckii
subsp. bulgaricus enable production of mild yoghurts
since such cells can, given their protocooperation, only
grow in the presence of actively lactose fermenting S.
thermophilus cells (Mollet, 1996).
Acceleration of the maturation process of cheese
During cheese maturation, several aromatic com-
pounds are generated due to the action of endogenous
milk enzymes as well as the proteolytic and lipolytic
activities of LAB present in the cheese. The maturation
stage is time- and space-consuming so that accelerated
maturation techniques are being sought. Besides
rational selection of the LAB starter and co-cultures,
and the application of process conditions for optimal
activity of the endogenous enzymes, the addition of
exogenous enzymes (enzyme-modified cheese) as well as
the increased in situ autolysis of the LAB, represent
alternative solutions (Fox et al., 1996). Autolysis of the
starter cells is followed by the release of intracellular
peptidases in the curd. Bacteriocins (see above) may
cause bacteriolysis, for example by inducing autolysins
or by deregulating enzyme action due to energetic
deficiency, which will result in degradation of the cell
wall and consequent cell lysis (Morgan, Ross, & Hill,
1997; Martı́nez-Cuesta, Fernández de Palencia,
Requena, & Paláez, 1998; Martı́nez-Cuesta, Requena,
& Peláez, 2001). Furthermore, genetic strategies exist
for the induction of phage holins and lysins to pro-
mote lysis of the starter cells during maturation
(Crow, Martley, Coolbear, & Roundhill, 1996;
Gasson, 1996).
Functional starters with a health advantage
Production of nutraceuticals
Nutraceuticals are food components that, through
specific physiological action, contribute to the health of
the consumer (Andlauer & Fürst, 2002). Several nutra-
ceuticals from bacterial origin have been added to food
products (Hugenholtz et al., 2002). Through strain
selection and process optimisation, the activity of LAB
can be modified to increase the content of nutraceuticals
in fermented foods such as fermented dairy products.
As an example, fermented milks can be produced
with LAB starter strains that produce high amounts of
low-calorie polyols so as to reduce the sugar content
(Wisselink, Weusthuis, Eggink, Hugenholtz, &
Grobben, 2002).
 F. Leroy, L. De Vuyst / Trends in Food Science & Technology 15 (2004) 67–78
Also, the use of oligosaccharide-producing LAB that
produce sugar polymers with a controlled structure and
chain length (and hence molecular mass) may yield fer-
mented products with health applications (Ruas-
Madiedo, Hugenholtz, & Zoon, 2002). Health effects of
such oligosaccharides are ascribed to their low-calorie
character, their fibre-like nature, and their bifidogenic
effect (Voragen, 1998; Wang & Gibson, 1993).
Certain LAB, such as the yoghurt bacteria Lb. del-
brueckii subsp. bulgaricus and S. thermophilus, are able
to produce vitamins such as folate. A controlled use of
these bacteria may lead to dairy products with increased
folate content (Crittenden, Martinez, & Playne, 2002;
Hugenholtz & Kleerebezem, 1999; Lin & Young,
2000a,b; Wouters, Ayad, Hugenholtz, & Smit, 2002).
The proteolytic system of LAB can contribute to the
liberation of health-enhancing bioactive peptides from
milk (Meisel & Bockelmann, 1999; Wouters et al., 2002).
The latter may improve absorption in the intestinal tract,
stimulate the immune system, exert antihypertensive or
antithrombotic effects, display antimicrobial activity, or
function as carriers for minerals, especially calcium.
Reduction of toxic or antinutritive factors
The fermentative action of specific LAB stains may
lead to the removal of toxic or antinutritive factors,
such as lactose and galactose from fermented milks to
prevent lactose intolerance and accumulation of galac-
tose (Wouters et al., 2002). Other examples are the
removal of raffinose, stachyose, and verbascose from
soy to prevent flatulence and intestinal cramps (Hol-
zapfel, 1997, 2002; Hou, Yu & Chou, 2000; Scalabrini,
Rossi, Spettoli, & Matteuzzi, 1998), proteinase inhibi-
tors from legumes and cereals to prevent maldigestion
(Holzapfel, 2002), phytic acid and tannins from cereals
and legumes to increase mineral bioavailability (Hol-
zapfel, 1997, 2002; Sharma & Kapoor, 1996), and nat-
ural toxins such as cyanogenic glucosides from cassava
(Holzapfel, 2002; Kimaryo, Massawe, Olasupod, &
Holzapfel, 2000) as well as biogenic amines from tradi-
tional fermented foods (Holzapfel, 2002).
Functional starters for the industry: drawbacks and
perspectives
Selection and construction of suitable strains
Selecting for strains with interesting properties to be
used as new, functional starter cultures may lead to an
improved fermentation process and an enhanced quality
of the end product. However, as it has been shown for
bacteriocin-producing LAB, the success of using func-
tional starter cultures in a particular food is strongly
strain dependent (Leroy, Verluyten, Messens, & De
Vuyst, 2002). The kinetics of the applied strains have to
be adapted to the process conditions and the intrinsic
factors prevailing in the food. Rational selection of
appropriate strains is crucial.
73
Because of legislation and marketing reasons, the
industrial application of carefully selected natural food
isolates with functional properties seems attractive. In
contrast, the use of genetically modified organisms is
hindered by the hostility of the consumer (Grunert,
Bech-Larsen, & Bredahl, 2000). Nevertheless, molecular
biology offers immense perspectives for strain improve-
ment and the construction of tailor-made starters or co-
cultures (Law, 2001). Molecular tools and gene transfer
systems to support self-cloning are useful in the
improvement of starter functionality (Klaenhammer &
Kullen, 1999; Ross et al., 2000). Also, bioinformatics
and comparative genomics approaches can provide
strategies that lead to an improved functionality of
food-grade microorganisms (de Vos, 2001; van
Kranenburg et al., 2002).
Of course, selection of starter cultures must not only
aim at expressing functional properties, but also at
eliminating undesirable side effects, such as the forma-
tion of d-lactic acid or a racemate of lactic acid (dl), or
the formation of biogenic amines. For instance, Joosten,
Gaya, and Nuñez (1995) have isolated tyrosine dec-
arboxylase-less mutants of a bacteriocin-producing E.
faecalis strain to be applied in cheese. Moreover, only a
limited number of NSLAB can be successfully applied
as adjunct cultures in cheese making, since many may
induce possible defects or spoilage (Wouters et al.,
2002).
Mechanism and stability of functionality
Research to unravel the mechanism of functionality
will contribute to a better control of the expression of
the desired properties. As an example, the specific
mechanisms by which flavour is generated are not fully
understood (Demeyer et al., 2000; Law, 2001; Weimer,
Seefeldt, & Dias, 1999). Knowledge of the proteolytic
system of LAB may allow the construction of tailor-
made starter cultures that generate desirable flavour
characteristics. One of the challenges of using wild-type
strains will be to allow large-scale production of fer-
mented foods without loosing their unique flavour and
other traits (Caplice & Fitzgerald, 1999). In this matter,
the flavour-forming abilities of several wild lactococcal
strains to be used for cheese manufacture were found to
be stable during subcultivation, making them suitable as
starter cultures. Moreover, population dynamics
revealed that, when used in Gouda-type cheese-making,
lactococcal strains from natural niches were generally
more stable than strains from industrial starter cultures
(Ayad, Verheul, de Jong, Wouters, & Smit, 2001).
Adaptation to the existing process technology
In the new generation of fermented foods, LAB with
diverse physiological and metabolic traits are combined.
Their metabolic and technological properties are often
different from those of traditional starter cultures, so
74 F. Leroy, L. De Vuyst / Trends in Food Science & Technology 15 (2004) 67–78
that appropriate production processes must be devel-
oped (Oberman & Libudzisz, 1998). The positive results
observed with functional starters under optimal labora-
tory conditions are not necessarily obtained in practice,
i.e. in the actual food matrix. For instance, some LAB
produce bacteriocins that are active towards food spoi-
lage organisms and/or pathogenic bacteria when they
are cultivated in complex media (in vitro), but fail to
show inhibitory activity when they are grown in a food
environment (in situ). This has been ascribed to a lim-
ited diffusion of the bacteriocin molecules in the food
matrix and their inhibition or inactivation by certain
food components. In meat, adsorption of bacteriocin
molecules to proteins or fat particles and inactivation by
endogenous enzymes are major factors thought to be
responsible for the loss of bioactivity (Stiles & Hastings,
1991). Similar problems have been observed for the in
situ production of exopolysaccharides. Hetero-
polysaccharide production in milk is around 100 milli-
grams per litre, whereas in complex media more than
one gram per litre may be obtained (Degeest, Van de
Ven, & De Vuyst, 1999). Furthermore, interactions with
the food matrix and the applied process technology
have an important effect on the exopolysaccharide
levels.
Limited quantitative information is available on the
influence of food technology on microbial functionality
and on the interaction between microbial systems and
the wide variety of food components, including probio-
tics (Knorr, 1998). The use of mathematical modelling
may help to get a better understanding of these rela-
tionships (Leroy, Degeest, & De Vuyst, 2002). In this
matter, the influence of process parameters on the
functionality of the bacteriocin producers Lb. sakei
CTC 494 (Leroy & De Vuyst, 1999a,b), Lb. curvatus
LTH 1174 (Messens, Verluyten, Leroy, & De Vuyst,
2003), Lb. amylovorus DCE 471 (De Vuyst, Callewaert,
& Crabbé, 1996; De Vuyst, Callewaert, & Pot, 1996,
Messens, Neysens, Vansieleghem, Vanderhoeven, & De
Vuyst, 2002), and E. faecium RZS C5 (Leroy & De
Vuyst, 2002; Foulquié Moreno et al., 2003; Leroy,
Vankrunkelsven, De Greef, & De Vuyst, 2003), and on
the exopolysaccharide producer S. thermophilus LY03
(Degeest & De Vuyst, 1999) has been studied by the
authors of this paper.
Conclusion
Novel insights into the metabolism of LAB offer per-
spectives for the application of a new generation of
starter cultures. Functional LAB starter cultures may
offer several health, marketing, and technological
advantages. They may be obtained by genetic engineer-
ing or as wild-type organisms. Bioinformatics will be
available soon to search in genomes for specific genes,
gene clusters or functionalities. However, fundamental
and applied research is still needed to optimally
implement functional starter cultures in the existing
production technology and to obtain quantitative data.
Mathematical analysis of the biokinetics of functional
starter cultures may yield precious information about
the relationship between the food environment and
bacterial functionality, and may contribute to optimal
strain selection and process design. This may result in
better process control, enhanced food safety and qual-
ity, and reduction of economic losses. It may further
contribute to the development of small and medium sized
enterprises on the one hand, and product diversification
of large companies on the other hand.
Acknowledgements
The authors acknowledge their financial support from
the Research Council of the Vrije Universiteit Brussel,
the Fund for Scientific Research-Flanders (FWO), the
Institute for the Promotion of Innovation by Science
and Technology in Flanders (IWT), in particular the
STWW project ‘Functionality of novel starter cultures
in traditional fermentation processes’’ and the GBOU
project ‘Development of a fast, non-invasive technolo-
gical tool to investigate the functionality and effective-
ness of pro- and prebiotics in normal healthy humans:
the use of a labelled biomarker’, the Brussels Capital
Region (LINK Action), the European Commission
(grants FAIR-CT97-3078, FAIR-CT97-3227, FAIR-
CT97-5013, FAIR-CT98-4267, IC15-CT98-0905), and
from different food companies. F.L. was supported by a
grant of the IWT (PhD bursary) and the FWO (post-
doctoral fellowship).
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