You are on page 1of 19

Journal of Biotechnology 84 (2000) 197 215

Review article

Probiotic bacteria: safety, functional and technological

Maria Saarela a, Gunnar Mogensen b, Rangne Fonden c, Jaana Matto a,
Tiina Mattila-Sandholm a,*

VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland

GM-Consult, Thorsmose6ej 16, 3200 Helsinge, Denmark
Arla Foods, 10546 Stockholm, Sweden

Received 19 July 2000; received in revised form 21 September 2000; accepted 4 October 2000

During the past two decades probiotic (health promoting) micro-organisms have been increasingly included in
various types of food products, especially in fermented milks. Several aspects, including safety, functional and
technological characteristics, have to be taken into consideration in the selection process of probiotic micro-organisms. Safety aspects include specifications such as origin (healthy human GI-tract), non-pathogenicity and antibiotic
resistance characteristics. Functional aspects include viability and persistence in the GI-tract, immunomodulation,
antagonistic and antimutagenic properties. Before probiotic strains, chosen on the basis of their good safety and
functional characteristics, can benefit the consumer, they must first be able to be manufactured under industrial
conditions. Furthermore, they have to survive and retain their functionality during storage, and also in the foods into
which they are incorporated without producing off-flavours. Factors related to the technological and sensory aspects
of probiotic food production are of utmost importance since only by satisfying the demands of the consumer can the
food industry succeed in promoting the consumption of functional probiotic products in the future. 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Probiotic bacteria; Health; Safety

1. Introduction

* Corresponding author. Fax: +358-9-4552103.

E-mail address: (T. MattilaSandholm).

Probiotics are live microbial food supplements

which benefit the health of consumers by maintaining or improving their intestinal microbial
balance (Fuller, 1989). Due to their perceived
health benefits probiotic bacteria have been in-

0168-1656/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 3 7 5 - 8


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

creasingly included in yoghurts and fermented

milks during the past two decades. Most commonly they have been lactobacilli such as Lactobacillus acidophilus, and bifidobacteria often
referred to as bifidus (Daly and Davis, 1998). A
major development in functional foods pertain to
foods containing probiotics and prebiotics which
enhance health promoting microbial flora in the
intestine. There is growing scientific evidence to
support the concept that the maintenance of
healthy gut microflora may provide protection
against gastrointestinal disorders including gastrointestinal infections, inflammatory bowel diseases, and even cancer (Haenel and Bendig, 1975;
Mitsuoka, 1982; Salminen et al., 1998a). The use
of probiotic bacterial cultures stimulates the
growth of preferred micro-organisms, crowds out
potentially harmful bacteria, and reinforces the
bodys natural defence mechanisms. Today,
plenty of evidence exists on the positive effects of
probiotics on human health. However, this has
usually been demonstrated in diseased human
populations only (Salminen et al., 1998a). Thus
there is an urgent need for evidence for probiotic
health benefits in average (generally healthy)
Before a probiotic can benefit human health it
must fulfil several criteria: It must have good
technological properties so that it can be manufactured and incorporated into food products
without loosing viability and functionality or creating unpleasant flavours or textures; it must survive passage through the upper gastrointestinal
(GI) tract and arrive alive at its site of action; and
it must be able to function in the gut environment. To study the probiotic strain in the GItract, molecular techniques must be established
for distinguishing the ingested probiotic strain
from the potentially thousands of other bacterial
strains that make up the gastrointestinal ecosystem. Additionally, techniques are required to establish the effect of the probiotic strain on other
members of the intestinal microbiota and importantly on the host. This includes not only positive
health benefits, but also demonstration that probiotic strains do not have any deleterious effects.
Armed with this knowledge, the probiotics can
then enter human pilot studies that attempt to

assess their health benefits to consumers (MattilaSandholm and Salminen, 1998; Mattila-Sandholm
et al., 1999).

2. Selecting probiotic strains: important aspects

The theoretical basis for the selection of probiotic micro-organisms including safety, functional
and technological aspects is illustrated in Fig. 1.
Current safety criteria for successful probiotics
have been defined in several reviews (Lee and
Salminen, 1995; Donohue and Salminen, 1996;
Salminen et al., 1996b, 1998b; Adams, 1999). The
significance of human origin has been debated
recently, but most current successful strains are
indicated to be of human origin. It can also be
argued that a probiotic strain can function better
in a similar environment (e.g. human GI-tract) to
where it was originally isolated from. Safety aspects include the following specifications:
1. Strains for human use are preferably of human

Fig. 1. The theoretical basis for selection of probiotic microorganisms includes safety, functional (survival, adherence,
colonisation, antimicrobial production, immune stimulation,
antigenotoxic activity and prevention of pathogens) and technological aspects (growth in milk, sensory properties, stability,
phage resistance, viability in processes).

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

2. They are isolated from healthy human GItract.

3. They have a history of being non-pathogenic.
4. They have no history of association with diseases such as infective endocarditis or GIdisorders.
5. They do not deconjugate bile salts (bile salt
deconjucation or dehydroxylation would be a
negative trait in the small bowel; Marteau et
al., 1995).
6. They do not carry transmissible antibiotic resistance genes.
The functional requirements of probiotics
should be established by using in vitro methods
and the results of these studies should be reflected
in controlled human studies. While selecting a
preferable probiotic strain several aspects of functionality have to be considered:
1. Acid tolerance and tolerance to human gastric
2. Bile tolerance (an important property for survival in the small bowel).
3. Adherence to epithelial surfaces and persistence in the human GI-tract.
4. Immunostimulation, but no proinflammatory
5. Antagonistic activity against pathogens such
as Helicobacter pylori, Salmonella sp., Listeria
monocytogenes and Clostridium difficile.
6. Antimutagenic
Feeding trials with different probiotic strains
have shown that the probiotic strain usually disappears from the GI-tract within a couple of
weeks after the ingestion is discontinued
(Fukushima et al., 1998; Johansson et al., 1998;
Alander et al., 1999; Donnet-Hughes et al., 1999).
The role of the probiotic persistence in the human
GI-tract has therefore been questioned. However,
even temporary persistence, which has been noted
for several ingested probiotic strains, may enhance their chances for beneficial functions in the
GI-tract, and is therefore considered a desirable
Even though a probiotic strain fulfils the necessary safety and functional criteria the aspects
related to probiotic production and processing are
also of utmost importance. Several technological


Fig. 2. Safety aspects of probiotics.

aspects have to be considered in probiotic selection. These include the following:

1. Good sensory properties.
2. Phage resistance.
3. Viability during processing.
4. Stability in the product and during storage.
Good viability and activity of probiotics are
considered prerequisites for optimal functionality.
However, several studies have shown that non-viable probiotics can have beneficial effects such as
immune modulation and carcinogen binding in
the host (for a review see Ouwehand and Salminen, 1998; Salminen et al., 1999). Thus, for certain
probiotic strains it might be sufficient that they
grow well during initial production steps (to obtain high enough numbers in the product) but
they do not necessarily need to retain good viability during storage.
Safety, functional and technological aspects of
probiotics are discussed in more detail below. The
review is limited to Lactobacillus and Bifidobacterium, since these genera are by far the most
important with probiotic strains for human use.

3. Safety aspects of probiotics

The safety of probiotic strains is of prime importance and guidelines for the safety assessment
can be found in several articles (Lee and Salminen, 1995; Donohue and Salminen, 1996; Salminen et al., 1996b, 1998b; Adams, 1999) (Fig. 2).
Several approaches are possible in the assessment
of the probiotic safety: (a) studies on the instrinsic
properties of the probiotic strain; (b) studies on


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

the pharmacokinetics of the probiotic strain; and

(c) studies on interactions between the probiotic
strain and the host.
Knowledge on survival of the probiotics within
the GI-tract, their translocation and colonization
properties, and the fate of probiotic-derived active
components is important for the evaluation of
possible positive and negative effects of probiotic
consumption. The survival of different probiotic
strains in different parts of the GI-tract varies:
Some strains are rapidly killed in the stomach
while others can pass through the whole gut in
high numbers (Marteau et al., 1993). The pharmacokinetics of probiotics has been studied in vivo
using intubation, perfusion and biopsy techniques
(Pochart et al., 1992; Johansson et al., 1993;
Nielsen et al., 1994; Alander et al., 1997; Marteau
et al., 1997). Some enzymatic properties such as
excessive deconjugation of bile salts or degradation of mucus has been postulated to be potentially detrimental (Marteau et al., 1995;
Ruseler-van Embden et al., 1995). Such properties
can be studied in vitro. Platelet aggregation properties (Korpela et al., 1997), enzymes which seem
to favour cardiac valve colonization (Pelletier et
al., 1996) and formation of unwanted metabolites
can also be studied in vitro.
Assessing the risks of probiotic consumption
could be a very expensive and time-consuming
task. A low risk may have to be accepted when
recommended to immunocompromised individuals, but the risk to benefit ratio needs to be clearly
established in such cases. This requires relevant
information on the efficacy and safety of the
products. At present, the available information on
current probiotic lactic acid bacteria provides
good safety back-up. To our knowledge there are
up to date two reports of probiotic bacterium
causing infection. A L. rhamnosus strain indistinguishable from L. rhamnosus GG has been isolated from a liver abscess from an elderly lady
with a history of hypertension and diabetes mellitus (Rautio et al., 1999). In another case a probiotic L. rhamnosus strain (strain or product
specifications were not given) was suggested to
have caused endocarditis in an elderly male
(Mackay et al., 1999). However, unlike in the liver
abscess case where strain identification was based

on thorough genomic fingerprinting of the L.

rhamnosus isolates, the endocarditis case study
relied on conventional phenotypic strain identification methods and on pyrolysis mass spectrometry identification. The discriminatory power of
conventional phenotypic identification methods in
strain differentiation is usually poor and there is
no data on the ability of pyrolysis mass spectrometry to differentiate between different lactobacillar strains. Therefore the data on the
endocarditis case must be considered insufficient
for proving that the ingested probiotic L. rhamnosus strain and not one of the indogenous L.
rhamnosus strains is the causative agent.
These two findings (one proved and one yet
presumptive) indicate that although probiotic
products have been safely consumed in large
quantities over the years throughout Europe and
Japan, occasional severe infections, especially in
immunocompromised patients, may occur.

3.1. Safety considerations of antibiotic resistances

in lactobacilli and bifidobacteria
Due to the indiscriminate use of antibiotics in
human and veterinary medicine and in animal
growth promoters antibiotic resistance has become an increasingly common characteristic in
micro-organisms (Austin et al., 1999; Robredo et
al., 2000), thus causing serious problems in treatment of microbial infections. Antibiotic resistance
in bacteria may be intrinsic or acquired. Intrinsic
resistance is a naturally occurring trait and may
be considered as a species characteristic, whereas
acquired resistance derives either from genetic
mutations or acquisition of foreign DNA from
other bacteria.
Lactobacilli display a wide range of antibiotic
resistances naturally (Charteris et al., 1998b), but
in most cases antibiotic resistance is not of the
transmissible type. Lactobacillus strains with nontransmissible antibiotic resistances do not usually
form a safety concern. Several species of lactobacilli including L. rhamnosus and L. casei are
intrinsically resistant to vancomycin (Nicas et al.,
1989; Swenson et al., 1990; Charteris et al.,
1998b). These species have peptidoglycan precursors terminating with D-lactate instead of the

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

target precursor for vancomycin activity terminating with D-alanine (Billot-Klein et al., 1994).
Many intrinsically vancomycin resistant strains of
lactobacilli have a long history of safe use as
probiotics and there is no indication that vancomycin resistant lactobacilli could transfer the
resistance to other bacteria. Tynkkynen et al.
(1998) have demonstrated that the vancomycin
resistance factor of the probiotic strain L. rhamnosus GG is not closely related to those of enterococci, and they could not observe the transfer of
antibiotic resistances between L. rhamnosus GG
and enterococci.
Although plasmid-linked antibiotic resistances
are not very common among lactobacilli, they do
occur (Ishiwa and Iwata, 1980; Vescovo et al.,
1982; Rinckel and Savage, 1990), and their safety
implications should be taken into consideration.
Since transfer of antibiotic resistant genes may
occur between phylogenetically distant bacteria
(Courvalin, 1994), strains harbouring mobile elements carrying resistance genes should not be
used either as human or animal probiotics.
Checking the ability of a proposed probiotic
strain to act as a donor of conjugative antibiotic
resistance genes may be a prudent precaution
especially when probiotics are administered during antibiotic therapy, and at least in the case of
animal feeding, where the use of antibiotics as
growth promoters apparently creates selective advantage for spreading of the resistance factors.
Antibiotic susceptibility patterns vary greatly between different species of lactobacilli, and strains
with atypical resistance to some clinically important antibiotics have been detected among lactobacilli (Charteris et al., 1998b; Felten et al., 1999),
indicating the necessity for susceptibility testing of
each probiotic strain.
Most bifidobacteria are intrinsically resistant to
kanamycin, gentamycin, streptomycin and
metronidazole (Miller and Finegold, 1967; Matteuzzi et al., 1983; Charteris et al., 1998a). In
earlier studies vancomycin has been found highly
inhibitory against bifidobacteria (Miller and Finegold, 1967; Lim et al., 1993), whereas in a recent
study by Charteris et al. (1998a) vancomycin resistance was suggested to be a general characteris-


tic of bifidobacteria. However, differences in the

techniques used in susceptibility testing hinders
the comparison of data. Suppression of fecal
counts of bifidobacteria during vancomycin therapy would suggest susceptibility of intestinal
bifidobacteria to this agent (Edlund et al., 1997).
Studies on the genetics of the antibiotic resistance
of bifidobacteria are warranted for understanding
of the mechanisms of resistance in this genus.

4. Functional aspects of probiotics

Clinical effects of some probiotic strains in
humans are shown in Table 1. These include, for
example, immunomodulation, modulation of intestinal flora, prevention of diarrhoeas, and lowering of fecal enzyme activities. Some of the
functional aspects of probiotics are discussed in
more detail below.

4.1. Adhesion properties

Adhesion of probiotic strains to the intestinal
surface and the subsequent colonization of the
human GI-tract has been suggested as an important prerequisite for probiotic action. Adherent
strains of probiotic bacteria are likely to persist
longer in the intestinal tract and thus have better
possibilities of showing metabolic and immunomodulatory effects than non-adhering
strains. Adhesion provides an interaction with the
mucosal surface facilitating the contact with gut
associated lymphoid tissue mediating local and
systemic immune effects. Thus, only adherent probiotics have been thought to effectively induce
immune effects and to stabilise the intestinal mucosal barrier (Salminen et al., 1996a). Adhesion
may also provide means of competitive exclusion
of pathogenic bacteria from the intestinal epithelium: Exclusion of pathogens by lactic acid bacteria and bifidobacteria has been shown in vitro
using Caco-2 and HT-29-MTX cell lines (Bernet
et al., 1993, 1994; Coconnier et al., 1993a,b). In
the inhibition of pathogen adhesion in vitro both
living and heat-killed L. acidophilus cells have
been effective (Coconnier et al., 1993a).

Lowering faecal enzyme activities, reduction of

antibiotic-associated diarrhoea in children,
treatment and prevention of rotavirus and acute
diarrhoea in children, treatment of relapsing
Clostridium difficile diarrhoea, immune response
modulation, alleviation of atopic dermatitis
symptoms in children

Modulation of intestinal flora, immune

enhancement, adjuvant in Helicobacter pylori
Prevention of travellers diarrhoea, treatment of
viral diarrhoea including rotavirus diarrhoea,
modulation of intestinal flora, improvement of
constipation, modulation of immune response,
alleviation of atopic dermatitis symptoms in
Shortening of rotavirus diarrhoea in children,
treatment of acute diarrhoea in children, safe
and well-tolerated in HIV-positive adult subjects
Modulation of intestinal flora, lowering faecal
enzyme activities, positive effects on superficial
bladder cancer and cervical cancer, no influence
on the immune system of healthy subjects
Modulation of intestinal flora, increase in faecal
short-chain fatty acid content
Prevention of antibiotic-associated diarrhoea,
treatment of Clostridium difficile colitis,
prevention of diarrhoea in critically ill tube-fed
No effect on rotavirus diarrhoea, no immune
enhancing effect during rotavirus diarrhoea, no
effect on faecal enzymes, weak effect on
respiratory burst activity of blood leuckocytes
but not on overall phagocytic activity in healthy

Lactobacillus rhamnosus GG (ATCC 53103)

Lactobacillus johnsonii (acidophilus) LJ-1 (La1)

Yoghurt strains (Streptococcus thermophilus and/or L.

delbrueckii subsp. bulgaricus)

Saccharomyces boulardii

Lactobacillus plantarum DSM9843 (299v)

Lactobacillus casei Shirota

Lactobacillus reuteri (BioGaia Biologics)

Bifidobacterium lactis Bb-12

Clinical effects in humans


Table 1
Clinical effects of some probiotic strains and yoghurt strains

Goldin et al. (1992), Majamaa et al. (1995),

Donnet-Hughes et al. (1999)

Surawicz et al. (1989), Buts et al. (1993),

McFarland et al. (1994), Bleichner et al. (1997)

Johansson et al. (1993, 1998)

Aso and Akazan (1992), Okawa et al. (1993),

Tanaka and Ohwaki (1994), Aso et al. (1995),
Spanhaak et al. (1998)

Wolf et al. (1995, 1998), Shornikova et al.


Siitonen et al. (1990), Goldin et al. (1992), Kaila

et al. (1992), Hosoda et al. (1994), Isolauri et al.
(1991, 1994), Majamaa et al. (1995), Raza et al.
(1995), Sepp et al. (1995), Bennett et al. (1996),
Malin et al. (1996), Hilton et al. (1997),
Majamaa and Isolauri (1997), Shornikova et al.
(1997c), Alander et al. (1997, 1999),
Kankaanpaa et al. (1998), Pelto et al. (1998),
Rautanen et al. (1998), Arvola et al. (1999)
Link-Amster et al. (1994), Schiffrin et al. (1995),
Marteau et al. (1997), Michetti et al. (1999),
Donnet-Hughes et al. (1999)
Black et al. (1989, 1991), Marteau et al. (1990),
Alm et al. (1993), Link-Amster et al. (1994),
Saavedra et al. (1994), Schiffrin et al. (1995),
Kankaanpaa et al. (1998), Fukushima et al.


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

Controlled comparable studies on in vitro

model systems, such as the human colon carcinoma cell lines HT-29 and Caco-2, are important in the assessment of adhesion properties
(Chauviere et al., 1992; Lehto and Salminen,
1997; Tuomola and Salminen, 1998). HT-29 and
Caco-2 cell lines differentiate into enterocytes and
can thus be used as a model for the small intestine
epithelium. A mucus (gel covering the intestinal
epithelial surface) secreting variant of the HT-29
cell line, HT-29-MTX, has also been used in
adhesion studies (Tuomola, 1999). In vitro adhesion experiments have indicated differences in the
adhesion potential of different probiotic strains
(Lehto and Salminen, 1997; Tuomola and Salminen, 1998). Lately, glycoproteins obtained from
human ileostomy effluent or from faecal material
have been used as a model for small intestinal
mucus in probiotic adhesion assays (Kirjavainen
et al., 1998; Tuomola et al., 1999). The mucus
layer is the first contact between ingested bacteria
and intestinal mucous membrane, and therefore
important additional data on probiotic adhesion
properties can be obtained by supplementing the
data obtained with cell lines without mucus-secreting ability (Caco-2 and HT-29) with results of
mucus adhesion assays. Already it has been
shown that probiotic strains vary in their ability
to adhere to mucus, and also that strains adhering
to Caco-2 cells do not necessarily adhere to mucus
as efficiently (Tuomola et al., 1999). Furthermore,
it has been shown that bifidobacteria adhere differently to mucus isolated from subjects representing different age groups (being poorest to mucus
isolated from elderly) (Ouwehand et al., 1999).
In vivo human adhesion of probiotic strains
can be studied by obtaining biopsy material from
a subject after a period of probiotic consumption.
Since biopsy sampling raises ethical issues, sampling is usually limited to subjects going through
routine diagnostic colonoscopy. From these subjects tissue samples can be obtained, not only
from the rectal-sigmoidal region, but also from
other parts of large intestine (ascending, transverse, and descending colon). The preferential adhesion of a commercial probiotic strain (L.
rhamnosus GG) to the descending part of large
colon was detected by using biopsy material


(Alander et al., 1997). Johansson et al. (1993)

have also demonstrated the adhesion of different
Lactobacillus strains to rectal mucosal biopsy
samples obtained from volunteers who had consumed fermented oatmeal soup. Biopsy sampling
probably gives the most accurate information on
the adhesion ability of probiotic strains. However,
there are severe limitations in the technique: first
and most importantly, ethical considerations limit
the use of the technique. Secondly, the technique
is very laborious and therefore only few individuals can be included in trials. Thirdly, the evacuation of the colon prior to colonoscopy probably
leads to a loss of large number of adhering bacteria, leaving only the bacteria with the strongest
adhering ability attached.
Although a lot of research effort has been
expended on probiotic adhesion studies, the role
of adhesion in successful probiotic function remains speculative. It could also be argued that
strong adhesion ability may increase the risk of
infection in the host. Some probiotic strains are
poorly adhering in vitro and/or in vivo and still
they can show positive effects in the hosts. In
addition, the reproducibility of in vitro adhesion
studies can be poor (especially between different
laboratories), which also complicates the interpretation of the results.

4.2. Immunomodulatory properties

Gut associated lymphoid tissue may have contact with adhesive probiotic strains and their components and therefore adhesion is one way of
provoking immune effects. Human studies have
shown that probiotic bacteria can have positive
effects on the immune system of their host. However, differences between probiotic bacteria in respect to their immunomodulatory effects have
been observed (Isolauri et al., 1999). In two separate trials, L. johnsonii LJ-1 (previously L.
acidophilus LA1) and L. sali6arius UCC 118 stimulated a mucosal IgA response and increased
phagocytic activity. The immunomodulation mediated by these strains was not linked to an
inflammatory response or general modification of
immune responsiveness that could potentially
have harmful effects, but was rather associated


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

with transient alterations beneficial to the consumer (Schiffrin et al., 1997; Mattila-Sandholm
and Kauppila, 1998). L. rhamnosus GG and
Bifidobacterium lactis Bb-12 derived extracts have
been shown to suppress lymphocyte proliferation
in vitro (Mattila-Sandholm and Kauppila, 1998).
Further evidence for immunomodulation by these
two strains was provided by a trial involving
children with severe atopic eczema resulting from
food allergy. Children fed L. rhamnosus GG and
B. lactis Bb-12 showed a significant improvement
in clinical symptoms compared to the placebo
group (Mattila-Sandholm and Kauppila, 1998;
Alander and Mattila-Sandholm, 2000). L. rhamnosus GG has been shown to down-regulate the
milk-induced inflammatory response in milk-hypersensitive subjects, whereas it had an immunostimulatory effect in healthy subjects (Pelto et al.,
1998). Furthermore, L. rhamnosus GG was shown
to promote IgA immune response in Crohns disease patients (Malin et al., 1996) and enhanced
the circulating antibody secreting cell response in
children with rotavirus diarrhoea (Kaila et al.,

4.3. Antagonistic properties

To have an impact on the colonic flora it is
important for probiotic strains to show antagonism against pathogenic bacteria via antimicrobial
substance production or competitive exclusion.
Enormous research efforts have focused on bacteriocin research. Although probiotic strains may
produce bacteriocins, their role in the pathogen
inhibition in vivo can only be limited, since traditional bacteriocins have an inhibitory effect only
against closely related species such as other Lactobacillus or on sporefomers such as Bacillus or
Clostridium (Holzapfel et al., 1995). However, low
molecular weight metabolites (such as hydrogen
peroxide, lactic and acetic acid, and other aroma
compounds) and secondary metabolites may be
more important since they show wide inhibitory
spectrum against many harmful organism like
Salmonella, Escherichia coli, Clostridium, and Helicobacter (Skytta et al., 1992; Helander et al.,
1997; Niku-Paavola et al., 1999). L. rhamnosus
strain GG produces in vitro low molecular weight

antimicrobial(s), possibly short chain fatty acid(s)

but distinct from lactic and acetic acid, with inhibitory activity against anaerobes such as
Clostridium, Bacteroides and Bifidobacterium,
against Enterobacteriaceae, Pseudomonas, Staphylococcus and Streptococcus, but not against other
lactobacilli (Silva et al., 1987). The antagonistic
activity of L. rhamnosus GG against enteropathogenic bacteria has also been shown in vivo in S.
typhimurium infected mice (Hudault et al., 1997).
The spent culture supernatant (SCS) of L.
acidophilus strain LB decreased the viability of
Staphylococcus aureus, Listeria monocytogenes, S.
typhimurium, Shigella flexneri, Escherichia coli,
Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, and Enterobacter spp. in
vitro. The unidentified low molecular weight antimicrobial substance(s) was independent of lactic
acid production and did not effect Lactobacillus
or Bifidobacterium strains tested. The antibacterial
activity of L. acidophilus LB SCS towards S.
typhimurium was also maintained in vivo in the
infected-mouse model (Coconnier et al., 1997). L.
acidophilus ( johnsonii ) strain LA1 (LJ-1) produces nonbacteriocin antibacterial substance(s)
(unidentified but distinct from lactic acid) that
inhibits in vitro a wide range of gram-negative
and gram-positive pathogens, such as S. aureus,
L. monocytogenes, S. typhimurium, S. flexneri, K.
pneumoniae, P. aeruginosa and Enterobacter cloacae. However, inhibition of lactobacilli and
bifidobacteria could not be detected. Inhibitory
activity of the strain LA1 towards S. typhimurium
was also shown in vivo in mouse model (BernetCamard et al., 1997). Furthermore, L. acidophilus
LA1 shows antimicrobial effect against H. pylori,
both in vitro and in humans (Michetti et al.,

4.4. Antimutagenic and anticarcinogenic properties

Antimutagenic and anticarcinogenic properties
of bacteria ingested in foods and/or representing
the microflora of gastrointestinal tract have been
widely studies for a number of years. Bacterial
anticarcinogenic properties are considered to represent one or more of the following types: binding
and degradation of (pro)carcinogens, production

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

of antimutagenic compounds, modulation of procarcinogenic enzymes in the gut, and suppression

of tumours by an immune response mechanism
(Fernandes et al., 1987; Lidbeck et al., 1992a;
Hirayama and Rafter, 1999). Special emphasis has
been put on in vitro antimutagenic (especially
mutagen binding) and anticarcinogenic properties
of lactic acid bacterial strains (representing Lactobacillus, Lactococcus, Leuconostoc, Enterococcus,
and Pediococcus species) isolated from fermented
dairy and non-dairy products (Hosono et al.,
1986, 1990a,b; Fernandes and Shahani, 1990;
Zhang et al., 1990; Thyagaraja and Hosono, 1993,
1994; El-Nezami et al., 1998a,b). However, according to the current criteria, only few of these
strains can be considered to represent probiotics.
In addition, antimutagenic property is by no
means typical for lactic acid bacteria (or probiotics) only: This property seems to be distributed
among bacteria representing both gram-positive
and gram-negative species (for a review see Fernandes and Shahani, 1990). A wide range of
bacteria (and also yeasts) are capable of mutagen
binding in vitro (Morotomi and Mutai, 1986;
Zhang and Ohta, 1993; Orrhage et al., 1994).
Furthermore, also non-viable bacterial cells are
able to bind mutagens and carcinogens (Zhang et
al., 1990; Thyagaraja and Hosono, 1994; ElNezami et al., 1998a). In addition to in vitro
studies antigenotoxic and antitumor activities of
some Bifidobacterium and Lactobacillus strains
has also been shown using rat and mice models
(Reddy and Rivenson, 1993; Pool-Zobel et al.,
1993, 1996; Singh et al., 1997; Takagi et al., 1999).
In human trials probiotic strains have been
associated with the reduction of fecal mutagenicity or fecal enzymatic activities involved in mutagen or carcinogen activation. Faecal enzymes such
as nitroreductase and b-glucuronidase have the
ability to convert procarcinogens to carcinogens
in the colon. In human studies L. acidophilus
NCFB 1748 consumption was shown to decrease
fecal and urinary mutagenicity (Lidbeck et al.,
1992b). Suppression of urinary mutagenicity has
also been shown after the consumption of L. casei
strain Shirota (Hayatsu and Hayatsu, 1993). L.
rhamnosus GG supplementation decreased fecal
b-glucuronidase, nitroreductase and glycocholic


acid hydrolase activities in healthy females (Ling

et al., 1994). Reduction of faecal enzyme activities
has also been shown after L. gasseri strain ADH
and L. casei strain Shirota consumption in humans (Pedrosa et al., 1995; Spanhaak et al., 1998).
L. casei strain Shirota consumption has further
proved beneficial for some cancer patients by
reducing the recurrence of superficial bladder cancer and by prolonging survival and relapse-free
interval in cervical cancer (Aso and Akazan, 1992;
Aso et al., 1995; Okawa et al., 1993). However,
although there is evidence that probiotic bacteria
may show antimutagenic and anticarcinogenic
properties in vitro and in animal models, the
possible role of probiotics in the cancer prevention in humans still remains highly controversial.

5. Technological aspects of probiotics

Functional foods with probiotics are now well
established on the European market. Starting
about 20 years ago this product range has increased (Young, 1998) and is presently known to
most consumers. To succeed in promoting the
consumption of functional probiotic products the
food industry has to satisfy the demands of the
consumer. All probiotic foods should be safe and
have good sensory properties. The probiotic foods
should also include specific probiotic strains at a
suitable level during the storage time. By examining existing products it has been suggested that
this is not always the case (Hamilton-Miller et al.,
Before probiotic strains can be delivered to
consumers, they must first be able to be manufactured under industrial conditions, and then survive and retain their functionality during storage
as frozen or freeze dried cultures, and also in the
food products into which they are finally formulated. Additionally, they must be able to be incorporated into foods without producing off-flavours
or textures. The packaging materials used and the
storage conditions under which the products are
stored, are important for the quality of products
containing probiotic bacteria.
Foods used for dissemination of probiotics are
usually fermented foods even if probiotics also


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

could be present in infant formula, fruit drinks,

whey drinks and sweet milk. Fermented milk and
cheese are the most common foods with probiotics (Svensson, 1999). Fermented foods are produced by a microbial fermentation in which
fermentable carbohydrates are transformed into
ethanol and/or organic acids mainly acetic, lactic
and propionic acid. Yeast and lactic acid bacteria
are the microbes commonly used in food fermentation. In fermented dairy products mainly Lactobacillus and Lactococcus species, but also yeast
and propionic acid bacteria, are used (Driessen
and Loones, 1992).

5.1. Manufacturing processes for fermented

probiotic products
Most leading starter culture manufacturers today produce common GI lactic acid bacteria and
Mogensen and Friis, 1997). Commercially available probiotic cultures may consist of a single
strain or a mixture of several strains. In most
cases the probiotic properties are affected by the
way of which the strain or culture has been produced (for a review see German et al., 1999).
Therefore specific information on strain-specific
properties should be available for the process
optimisation. Probiotic cultures may be used in
special formulations like capsules or tablets, or
they may be used in the production of a large
variety of fermented food products. In some cases
the cultures may be added to a food to contribute
specific probiotic or functional properties.
Most commercial probiotic culture preparations are supplied in highly concentrated form,
and most of them are constructed for DVS (direct
vat set) application (Honer, 1995). Use of these
highly concentrated DVS cultures is common due
to the difficulties involved in propagating probiotic micro-organisms at the production site. The
DVS cultures are supplied either as highly concentrated frozen cultures or as freeze-dried cultures.
The cultures should be filled in gas and light proof
containers to protect the cultures against light and
humidity. Most often alu-foil coated cartons or
pouches are used. As the cultures are sensitive, it
is important to handle and store the cultures

according to the manufacturers instructions.

Usually deep-frozen cultures contain more than
1010 cfu g 1, whereas freeze-dried cultures typically contain more than 1011 cfu g 1 (Oberman
and Libudzisz, 1998). The cell concentration per
gram of product varies with the culture and the
type of organisms used.
In fermented probiotic products it is important
that the probiotic culture used contributes to
good sensory properties. Therefore it is quite
common to use probiotic bacteria mixed together
with other types of bacteria suited for the fermentation of the specific product. For milk-based
products the probiotic strains are often mixed
with S. thermophilus and L. delbrueckii to achieve
the desired flavour and texture. The main flavour
components of species often used in probiotic
formulations are as shown in the Table 2. In
many cases the consumers find products fermented with L. delbrueckii too acidic and with too
heavy acetaldehyde flavour (yoghurt flavour).
Therefore probiotic cultures have been developed
to bring out the preferred flavours in the products
in which they are used. Examples of such cultures
are the so called ABT cultures (ABT standing for
L. acidophilus, Bifidobacterium and S. thermophilus). The manufacturing technology for producing
Bifidobacterium strains has been extensively reviewed by Tamime et al. (1995).
In selecting starter micro-organisms reliable
acid-forming ability is the most important characteristics. However, when selecting probiotics the
Table 2
Main flavour characteristics of some strains commonly used in
probiotic mixtures

Main flavour

Lactobacillus acidophilus a
Bifidobacterium spp.a
Streptococcus thermophilus b

Lactate (DL)
Lactate (L+), acetate
Lactate (L+),
acetaldehyde, diacetyl
Lactate (D),
acetaldehyde, diacetyl

Lactobacillus delbrueckii subsp.

bulgaricus b

Probiotic strains.
Non-probiotic strains.

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

criteria should be connected to the impact on

human health and well-being. As the environment
within the GI-tract and within the food might be
quite different the probiotic is often not suitable
as a starter organism (Oberman and Libudzisz,
1998; German et al., 1999). The growth rate might
be too slow and they might give off-flavours
(Svensson, 1999). This could partly be overcome
by using specific aseptic processes as used in producing fermented acidophilus milk with levels of
probiotic reaching 109 cfu g 1 (Fonden, 1989).
Another possibility is to improve the suitability of
the food as a substrate for the probiotic by adding
energy sources (e.g. glucose), growth factors (e.g.
yeast extract and protein hydrolysates) or suitable
antioxidants, minerals or vitamins (Kurmann,
1988; Ishibashi and Shimamura, 1993; Dave and
Shah, 1998; Gomes et al., 1998). However, even if
such an adjustment may improve the performance
of the probiotic as a starter, it is often not
enough. By use of a help starter in addition to a
probiotic preparation this problem can usually be
solved (Fonden et al., 2000).
When producing probiotic-containing fermented products a fermentation temperature of
37 40C is usually recommended since these temperatures span the range in which most probiotic
strains multiply well. Probiotic strains can be an
integral part of the starter and grow in a symbiotic relation with the other strains composing the
culture (Saxelin et al., 2000). There are examples
where the probiotic strain or strains are added to
the fermented milk after fermentation (e.g. the
Danish product Cultura). In special milk products (e.g. sweet products) probiotics are added to
the product in a such a way that they will retain
their viability but are prevented from multiplying
by cooling the product.

5.1.1. Probiotic interaction with starter bacteria

The interactions between probiotic and starter
might have an impact on the quality of the
product. It has been shown that it is possible to
produce fermented dairy products with excellent
sensory properties and good survival of the bacteria by using starter and probiotic organisms together (Fonden et al., 2000). Even if several
negative interactions have been proposed it seems


to be possible to find a suitable starter for most

probiotics by testing the presently available
starters. Suitable starters might be S. thermophilus, yoghurt cultures and mesophilic starters
with different combinations of Lactococcus
strains. The most suitable combination of starter
and a specific probiotic bacteria has to be determined by a screening process evaluating the impact of different starters on the sensory properties
and on the survival of the probiotic strain
(Ishibashi and Shimamura, 1993; Samona et al.,
In this screening process some general principles should be followed. If possible, the probiotic
should be able to grow during the fermentation. It
will increase the total number of the probiotic
resulting in a lower process cost and increased
adaptation of the probiotic to the fermented food.
As most probiotics grow well at 37C a thermophilic starter might be preferable to a
mesophilic one (Svensson, 1999). The growth rate
of the starter should be moderate allowing some
growth of the probiotic bacteria. It is also important to add the probiotic before or at the same
time as the starter (Reddy, 1989; Kailasapathy
and Rybka, 1997). Addition of the probiotic after
fermentation does not allow any growth but instead might result to a reduced viability as shown
when L. acidophilus was mixed with yoghurt (Hull
et al., 1984). The starter might improve the
growth conditions of probiotic by producing substances favourable to the growth of the probiotic
or by reducing the oxygen pressure. Bifidobacteria
are quite sensitive to oxygen but by selection of
specific strains of S. thermophilus it was possible
to increase survival of B. longum (Ishibashi and
Shimamura, 1993). L. delbrueckii subsp. bulgaricus strains might also increase growth of
bifidobacteria by their proteolytic activity resulting in increased availability of valine, glycine and
histidine (Misra and Kulia, 1994).
In selecting a suitable starter the negative impact on probiotic survival in vitro and in vivo
should also be taken in consideration. The survival of the probiotic bacteria might be influenced
by the metabolites formed by the starter such as
lactic acid, hydrogen peroxide and bacteriocins.
L. delbrueckii subsp. bulgaricus, a part of an


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

was ten times higher with the sweet milk (Pettersson et al., 1983).

Fig. 3. Changes in CFU (10log) of seven probiotic strains

(AG) in fermented milk products after 14 days storage at
+4C. Milks were fermented either with the probiotic strain
only (None), or together with a supporter culture St 20 (S.
thermophilus), YC 380 (S. thermophilus and L. delbrueckii
subsp. bulgaricus) or YC 280 (S. thermophilus and L. delbrueckii subsp. bulgaricus). The figure shows the strain to
strain variation between probiotics in regard to their survival
in the presence of different supporter cultures.

ordinary yoghurt culture, produces high levels of

acid also when the product is stored after
fermentation at low temperature. The impact of
D-lactate on the probiotic varies from strain to
strain (Dave and Shah, 1997; Kailasapathy and
Rybka, 1997; Rybka and Fleet, 1997). Samona et
al. (1996) showed that bifidobacterial strains
could not grow in the presence of youghurt cultures but by choosing the right combination of
probiotic and starter strains the decline in
bifidobacterial counts could be prevented. Starters
with only S. thermophilus might function better
together with probiotics sensitive to acids and low
pH. They are forming less acid both during fermentation and storage (Okongi et al., 1984;
Ishibashi and Shimamura, 1993) (Fig. 3).
The impact of the metabolites formed by the
starter might be enhanced in vivo. When the
functional food reaches the stomach the pH decreases and the level of undissociated lactic acid
increases. As the concentration of the unionised
acid has the greatest impact on survival of the
probiotic strain, the viability might be influenced
by the amount of hydrochloric acid from the
gastric juice and the amount of lactic acid from
the product. The importance of this is indicated
by the difference in survival of L. acidophilus
NCFB 1748 through the GI-tract when comparing sweet milk with fermented milk. Survival rate

5.1.2. Interactions between probiotics and

Functional foods with both probiotics and prebiotics are called synbiotics (Roberfroid, 1998).
Most prebiotics as known today are fermentable,
non-digestible carbohydrates with different number of sugar moieties from two up to several
hundreds. Some examples are lactulose, galactoand fructooligosaccharides and resistant starch.
As the concept of synbiotics is quite new there are
not many specific studies of interactions between
pro- and prebiotics. Due to their general properties prebiotics might influence the growth and
survival of the probiotic by influencing the growth
and metabolites of both the probiotic and the
starter. This has to be kept in mind while considering interactions between probiotics and starters.
Interaction in vivo might be favoured by an
adaptation of the probiotic to the prebiotic. By
adapting its metabolism to a substrate given
simultaneously with the probiotic might result in
a competitive advantage for the probiotic. However, as far as we know most studies have been
done with probiotic grown without such an
5.2. Manufacturing of non-dairy probiotic foods
Application of probiotic cultures in non-dairy
products and environments represents a challenge
(Andersen, 1998). Probiotic viability in the food
matrix depends on factors such as pH, storage
temperature, presence of competing micro-organisms and inhibitors (e.g. NaCl). In products like
probiotic-containing baby foods or confectionery
(e.g. chocolate) it is important that the formulation maintains the activity and viability of the
probiotic for extended periods of time. Since the
probiotic cultures are added as additives to these
kind of products, they do not usually multiply,
which sets great demands for the probiotic stability. Factors like water activity, oxygen tension
and temperature becomes increasingly important
when dealing with these kinds of products. Storage at room temperature, which is common for

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

several types of non-dairy products such as cereal

products, drinks, chocolate etc. can create an
overwhelming challenge for probiotic stability.
This problem can sometimes be solved by using
probiotic encapsulation technology to ensure the
viability and stability of probiotic cultures (Myllarinen et al., 1998). Stable probiotic-containing
baby food formulations and confectioneries have
been developed and are currently on the market
(Langhendries et al., 1995; Fukushima et al.,

6. Future trends
Diet is a major focus of public health strategy
aimed at maintaining optimum health throughout
life, preventing early onset of chronic diseases
such as gastrointestinal disorders, cardiovascular
disease, cancer, osteoporosis, as well as promoting
healthier ageing. Although the highly complex
relationship of food and health is still poorly
understood, recent research advances in different
disciplines provide promising new approaches to
improve our understanding. The growing demand
for healthy foods is stimulating innovation and
new product development in the food industry
internationally. The food industry has a central
role in facilitating healthier eating practices
through the provision and promotion of healthy
Continuously increasing consumer health consciousness and expenditure are socio-economic
factors responsible for the expanding European
and world-wide interest in functional foods. Considerable confusion and scepticism, however, exists between consumers, consumer organisations,
scientific communities and media about the claims
associated with probiotic products. Recent EU
projects have demonstrated that, with co-ordinated efforts towards communication and a scientific approach to selecting and applying
probiotics, functional food products can be developed with measurable health benefits for consumers. Probiotic strains can be successfully
manufactured and incorporated into highly acceptable food products where they can retain their
viability and functionality. There are many strain


to strain variations, not only in their technological

properties, but also in their effects on human
health (Alander and Mattila-Sandholm, 2000).
The probiotic concept is today widely spread in
the scientific and industrial fields. However, further scientific input is required. Important target
research areas, including GI-tract diagnostics and
immunology, methodology, biomarkers, and
functionality, will lead to tools and scientifically
sound methods for well-designed informative human studies. Controlled human studies are essential for the socio-economic success of probiotic
functional foods, and they should be tailored for
specific population groups such as the elderly and
babies. Future research on probiotic bacteria will
centre on selecting new and more specific strains
for the well-being of the host (age groups, healthy
populations, disease specific).
The future scientific and technological research
trends will be:
to inter-link the expertise and scientific knowledge on food, GI-tract functionality and human health.
to study the mechanisms of action of probiotics
in the GI-tract, and develop diagnostic tools
and biomarkers for their assessment.
to evaluate the role of probiotics in healthy
consumer groups.
to examine the effects of probiotics on GI-diseases, GI-infections, and allergies.
to address the consumer aspects and trade-offs.
to ensure the stability and viability of probiotic
products by developing technologies (e.g.
to develop technology for non-dairy probiotic
applications (i.e. cereal based materials).

This work was supported by European Concerted Action PL98 4230.

Adams, M.R., 1999. Safety of industrial lactic acid bacteria. J.
Biotechnol. 68, 171 178.


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

Alander, M., Mattila-Sandholm, T. (Eds.), 2000. Functional

foods for EU-Health in 2000, 4th Workshop, FAIR CT961028, PROBDEMO, VTT Symposium 198, Rovaniemi,
Alander, M., Korpela, R., Saxelin, M., Vilpponen-Salmela, T.,
Mattila-Sandholm, T., von Wright, A., 1997. Recovery of
Lactobacillus rhamnosus GG from human colonic biopsies.
Lett. Appl. Microbiol. 24, 361364.
Alander, M., Satokari, R., Korpela, R., Saxelin, M., Vilpponen-Salmela, T., Mattila-Sandholm, T., von Wright, A.,
1999. Persistence of colonization of human colonic mucosa
by a probiotic strain, Lactobacillus rhamnosus GG, after
oral consumption. Appl. Environ. Microbiol. 65, 351354.
Alm, L., Ryd-Kjellen, E., Setterberg, G., Blomquist, L., 1993.
Effect of a new fermented milk product Cultura on
constipation in geriatric patients. 1st Lactic Acid Bacteria
Computer Conference.
Andersen, L., 1998. Pre- and probiotics sausage science.
Functional Foods June, 2629.
Arvola, T., Laiho, K., Torkkeli, S., Mykkanen, H., Salminen,
S., Maunula, L., Isolauri, E., 1999. Prophylactic Lactobacillus GG reduces antibiotic-associated diarrhea in children with respiratory infections: a randomized study.
Pediatrics 104, e64.
Aso, Y., Akazan, H., 1992. Prophylactic effect of a Lactobacillus casei preparation on the recurrence of superficial bladder cancer. Urol. Int. 49, 125129.
Aso, Y., Akazan, H., Kotake, T., Tsukamoto, T., Imai, K.,
Naito, S., 1995. Preventive effect of a Lactobacillus casei
preparation on the recurrence of superficial bladder cancer
in a double-blind trial. Eur. Urol. 27, 104109.
Austin, D.J., Kristinsson, K.G., Anderson, R.M., 1999. The
relationship between the volume of antimicrobial consumption in human communities and the frequency of
resistance. PNAS 96, 11521156.
Bennett, R.G., Gorbach, S.L., Goldin, B.R., Chang, T.-W.,
Laughon, B.E., Greenough, W.B., Bartlett, J.G., 1996.
Treatment of relapsing Clostridium difficile diarrhea with
Lactobacillus GG. Nutr. Today 31 (Suppl.), 35S38S.
Bernet, M.-F., Brassart, D., Neeser, J.-R., Servin, A.L., 1993.
Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen cell interactions. Appl. Environ. Microbiol.
59, 4121 4128.
Bernet, M.-F., Brassart, D., Neeser, J.-R., Servin, A.L., 1994.
Lactobacillus acidophilus LA 1 binds to cultured human
intestinal cell lines and inhibits cell attachment and cell
invasion by enterovirulent bacteria. Gut 35, 483489.
Bernet-Camard, M.-F., Lievin, V., Brassart, D., Neeser, J.-R.,
Servin, A.L., Hudault, S., 1997. The human Lactobacillus
acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo. Appl. Environ.
Microbiol. 63, 2747 2753.
Billot-Klein, D., Gutmann, L., Sable, S., Guittet, E., van
Heijenoort, J., 1994. Modification of peptidoglucan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally

glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum. J. Bacteriol. 176, 2398 2405.
Black, F., Einarsson, K., Lidbeck, A., Orrhage, K., Nord,
C.E., 1991. Effect of lactic acid producing bacteria on the
human intestinal microflora during ampicillin treatment.
Scand. J. Infect. Dis. 23, 247 254.
Black, F.T., Andersen, P.L., Orskov, J., Orskov, F., Gaarslev,
K., Laulund, S., 1989. Prophylactic efficacy of lactobacilli
on travellers diarrhoea. Travel Med. 333 335.
Bleichner, G., Blehaut, H., Mentec, H., Moyse, D., 1997.
Saccharomyces boulardii prevents diarrhea in critically ill
tube-fed patients. A multicenter, randomized, double-blind
placebo-controlled trial. Intensive Care Med. 23, 517 523.
Buts, J.P., Corthier, G., Delmee, M., 1993. Saccharomyces
boulardii for Clostridium difficile-associated enteropathies
in infants. J. Pediatr. Gastroenterol. Nutr. 16, 419 425.
Charteris, W.P., Kelly, P.M., Morelli, L., Collins, J.K., 1998a.
Antibiotic susceptibility of potentially probiotic Bifidobacterium isolates from the human gastrointestinal tract. Lett.
Appl. Microbiol. 26, 333 337.
Charteris, W.P., Kelly, P.M., Morelli, L., Collins, J.K., 1998b.
Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Food Prot. 61, 1636 1643.
Chauviere, G., Cocoinnier, M.-H., Kerneis, S., Fourniat, J.,
Servin, A.L., 1992. Adhesion of human Lactobacillus
acidophilus strain LB to human enterocyte-like Caco-2
cells. J. Gen. Microbiol. 138, 1689 1696.
Coconnier, M.-H., Bernet, M.-F., Chauviere, G., Servin, A.L.,
1993a. Adhering heat-killed human Lactobacillus
acidophilus, strain LB, inhibits the process of pathogenicity
of diarrhoeagenic bacteria in cultured human intestinal
cells. J. Diarrh. Dis. 11, 235 242.
Coconnier, M.-H., Bernet, M.-F., Kerneis, S., Chauviere, G.,
Fourniat, J., Servin, A.L., 1993b. Inhibition of adhesion of
enteroinvasive pathogens to human intestinal Caco-2 cells
by Lactobacillus acidophilus strain LB decreases bacterial
invasion. FEMS Microbiol. Lett. 110, 299 306.
Coconnier, M.-H., Lievin, V., Bernet-Camard, M.-F., Hudault, S., Servin, A.L., 1997. Antibacterial effect of the
adhering human Lactobacillus acidophilus strain LB. Antimicrob. Agents Chemother. 41, 1046 1052.
Courvalin, P., 1994. Transfer of antibiotic resistance genes
between gram-positive and gram-negative bacteria. Antimicrob. Agents Chemother. 38, 1447 1451.
Daly, C., Davis, R., 1998. The biotechnology of lactic acid
bacteria with emphasis on applications in food safety and
human health. Agric. Food Sci. Finland 7, 219 250.
Dave, R., Shah, N., 1997. Viability of yoghurt and bacteria in
yoghurts made from commercial starter cultures. Int.
Dairy J. 7, 31 41.
Dave, R.I., Shah, N.P., 1998. Ingredient supplementation effects on viability of probiotic bacteria in yougurt. J. Dairy
Sci. 81, 2804 2816.
Donnet-Hughes, A., Rochat, F., Serrant, P., Aeschlimann,
J.M., Schiffrin, J.E., 1999. Modulation of nonspecific
mechanisms of defense by lactic acid bacteria: effective
dose. J. Dairy Sci. 82, 863 869.

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

Donohue, D.C., Salminen, S.J., 1996. Safety of probiotic
bacteria. Asia Pacific J. Clin. Nutr. 5, 2528.
Driessen, F.M., Loones, A., 1992. Developments in the fermentation process. In: New Technologies for Fermented
Milks, IDF Bullentin 277. IDF, Brussels, pp. 2840.
Edlund, C., Barkholt, L., Olsson-Liljequist, B., Nord, C.E.,
1997. Effect of vancomycin on intestinal flora of patients
who previously received antimicrobial therapy. Clin. Infect. Dis. 25, 729 732.
El-Nezami, H., Kankaanpaa, P., Salminen, S., Ahokas, J.,
1998a. Ability of dairy strains of lactic acid bacteria to
bind a common food carcinogen, aflatoxin B1. Food
Chem. Toxicol. 36, 321326.
El-Nezami, H., Kankaanpaa, P., Salminen, S., Ahokas, J.,
1998b. Physicochemical alterations enhance the ability of
dairy strains of lactic acid bacteria to remove aflatoxin
from contaminated media. J. Food Prot. 61, 466468.
Felten, A., Barreau, C., Bixet, C., Lagrange, H., Philippon, A.,
1999. Lactobacillus species identification, H2O2 production,
and antibiotic resistance and correlation with human clinical status. J. Clin. Microbiol. 37, 729733.
Fernandes, C.F., Shahani, K.M., 1990. Anticarcinogenic and
immunological properties of dietary lactobacilli. J. Food
Prot. 53, 704 710.
Fernandes, C.F., Shahani, K.M., Amer, M.A., 1987. Therapeutic role of dietary lactobacilli and lactobacillic fermented dairy products. FEMS Microbiol. Rev. 46,
343 356.
Fonden, R., 1989. Development of new fermented milk products. Food Technol. Int. Eur. 158160.
Fonden, R., Grenov, B., Reniero, R., Saxelin, M., Birkeland,
S.-E., 2000. Industrial panel statements: technological aspect. In: Alander, M., Mattila-Sandholm, T. (Eds.), Functional Foods for EU-Health in 2000, 4th Workshop, FAIR
CT96-1028, PROBDEMO, VTT Symposium 198.
Rovaniemi, Finland, pp. 4350.
Fukushima, Y., Kawata, Y., Hara, H., Terada, A., Mitsuoka,
T., 1998. Effect of a probiotic formula on intestinal immunoglobulin A production in healthy children. Int. J.
Food Microbiol. 42, 3944.
Fukushima, Y., Shou-Tou, L., Hara, H., Terada, A., Mitsuoka, T., 1997. Effect of follow-up formula containing
bifidobacteria (NAN BF) on fecal flora and fecal metabolites in healthy children. Biosci. Microflora 6, 6572.
Fuller, R., 1989. Probiotics in man and animals. J. Appl. Bact.
66, 365 378.
German, B., Schiffrin, E.J., Reniero, R., Mollet, B., Pfeifer,
A., Neeser, J.-R., 1999. The development of functional
foods: lessons from the gut. TIBTECH 17, 492499.
Goldin, B., Gorbach, S.L., Saxelin, M., Barakat, S., Gualtieri,
L., Salminen, S., 1992. Survival of Lactobacillus species
(strain GG) in human gastrointestinal tract. Dig. Dis. Sci.
37, 121 128.
Gomes, A.M., Malcata, F.X., Klaver, F.A., 1998. Growth
enhancement of Bifidobacterium lactis Bo and Lactobacillus
acidophilus Ki by milk hydrolyzates. J. Dairy Sci. 81,
2817 2825.


Haenel, H., Bendig, J., 1975. Intestinal flora in health and

disease. Progr. Food Nutr. Sci. 1, 21 64.
Hamilton-Miller, J., Shah, S., Winkler, J., 1999. Public health
issues arising from microbiological and labelling quality of
foods and supplements containing probiotic microrganisms. Publ. Hlth. Nutr. 2, 223 229.
Hayatsu, H., Hayatsu, T., 1993. Suppressing effect of Lactobacillus casei administration on the urinary mutagenicity
arising from ingestion of fried ground beef in human.
Cancer Lett. 73, 173 179.
Helander, I., von Wright, A., Mattila-Sandholm, T., 1997.
Potential of lactic acid bacteria and novel antimicrobials
against gram-negative bacteria. Trends Food Sci. Technol.
8, 146 150.
Hilton, E., Kolakowski, P., Singer, C., Smith, M., 1997.
Efficacy of Lactobacillus GG as a diarrhea preventative. J.
Travel Med. 4, 41 43.
Hirayama, K., Rafter, J., 1999. The role of lactic acid bacteria
in colon cancer prevention: mechanistic considerations.
Antonie van Leeuwenhoek 76, 391 394.
Holzapfel, W.H., Geisen, R., Schillinger, G.U., 1995. Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes. Int. J. Food
Microbiol. 24, 343 362.
Honer, C., 1995. Culture shift. Dairy Field 178, 54 58.
Hosoda, M., He, F., Hiramatu, M., Hashimoto, H., Benno,
Y., 1994. Effects of Lactobacillus GG intake on fecal
microflora and defecation in healthy volunteers. Bifidus 8,
21 28.
Hosono, A, Kashina, T., Kada, T., 1986. Antimutagenic properties of lactic acid cultured milk on chemical and fecal
mutagens. J. Dairy Sci. 69, 2237 2242.
Hosono, A., Tanabe, T., Otani, H., 1990a. Binding properties
of lactic acid bacteria isolated from kefir milk with mutagenic amino acid pyrolyzates. Milchwissenschaft 45, 647
Hosono, A., Wardojo, R., Otani, H., 1990b. Binding of amino
acid pyrolyzates by lactic acid bacterial isolated from
Dahih. Lebensm. Wiss. Technol. 23, 149 153.
Hudault, S., Lievin, V., Bernet-Camard, M.-F., Servin, A.L.,
1997. Antagonistic activity exerted in vitro and in vivo by
Lactobacillus casei (strain GG) against Salmonella typhimurium infection. Appl. Environ. Microbiol. 63, 513
Hull, R., Roberts, V., Mayes, J., 1984. Survival of L.
acidophilus in yoghurt. Aust. J. Dairy Technol. 39, 164
Ishibashi, N., Shimamura, S., 1993. Bifidobacteria: research
and development in Japan. Food Technol. June, 126 135.
Ishiwa, H., Iwata, M., 1980. Drug resistance plasmids in
Lactobacillus fermentum. J. Gen. Appl. Microbiol. 26, 71
Isolauri, E., Salminen, S., Mattila-Sandholm, T., 1999. New
functional foods in the treatment of food allergy. Ann.
Med. 31, 299 302.
Isolauri, E., Juntunen, M., Rautanen, T., Sillanaukee, P.,
Koivula, T., 1991. A human Lactobacillus strain (Lacto-


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

bacillus casei sp. strain GG) promotes recovery from acute

diarrhea in children. Pediatrics 88, 9097.
Isolauri, E., Kaila, M., Mykkanen, H., Ling, W.H., Salminen,
S., 1994. Oral bacteriotherapy for viral gastroenteritis. Dig.
Dis. Sci. 39, 2595 2600.
Johansson, M.-L., Molin, G., Jeppson, B., Nobaek, S., Ahrne,
S., Bengmark, S., 1993. Administration of different Lactobacillus strains in fermented oatmeal soup. In vivo colonization of human intestinal mucosa and effect on the
indigenous flora. Appl. Environ. Microbiol. 59, 1520.
Johansson, M.-L., Nobaek, S., Berggren, A., Nyman, M.,
Bjorck, I., Ahrne, S., Jeppsson, B., Molin, G., 1998. Survival of Lactobacillus plantarum DSM 9843 (299v), and
effect on the short-chain fatty acid content of faeces after
ingestion of a rose-hip drink with fermented oats. Int. J.
Food Microbiol. 42, 2938.
Kaila, M., Isolauri, E., Soppi, E., Virtanen, E., Laine, S.,
Arvilommi, H., 1992. Enhancement of the circulating antibody secreting cell response in human diarrhea by a human
Lactobacillus strain. Pediatr. Res. 32, 141144.
Kailasapathy, K., Rybka, S., 1997. L. acidophilus and
Bifidobacterium ssp their theurapeutic potential and
survival in yoghurt. Aust. J. Dairy Technol. 52, 2835.
Kankaanpaa, P., Sutas, Y., Salminen, S., Isolauri, E., 1998.
Results on clinical demonstration of probiotics in children.
In: Mattila-Sandholm, T., Kauppila, T. (Eds.), Functional
Food Research in Europe, 3rd Workshop, FAIR CT961028, PROBDEMO, VTT Symposium 187. Haikko, Finland, pp. 38 39.
Kirjavainen, P.V., Ouwehand, A.C., Isolauri, E., Salminen,
S.J., 1998. The ability of probiotic bacteria to bind human
intestinal mucus. FEMS Microbiol. Lett. 15, 185189.
Korpela, R., Moilanen, E., Saxelin, M., Vapaasalo, H., 1997.
Lactobacillus rhamnosus GG (ATCC 53013) and platelet
aggregation in vitro. Int. J. Food Microbiol. 37, 8386.
Kurmann, J.A., 1988. Starters for fermented milks. In: Fermented Milks Science and Technology, IDF Bulletin
227. IDF, Brussels, pp. 4155.
Langhendries, J.P., Detry, J., Van Hees, J., Lamboray, J.M.,
Darimont, J., Mozin, M.J., Secretin, M.C., Senterre, J.,
1995. Effect of a fermented infant formula containing
viable bifidobacteria on the fecal flora composition and pH
of healthy full-term infants. J. Pediat. Gastroenterol. Nutr.
21, 177 181.
Lee, Y.-K., Salminen, S., 1995. The coming of age of probiotics. Trends Food Sci. Technol. 6, 241245.
Lehto, E.M., Salminen, S., 1997. Adhesion of two Lactobacillus strains, one Lactococcus and one Propionibacterium
strain to cultured intestinal Caco-2 cell line. Biosci. Microflora 16, 13 17.
Lidbeck, A., Nord, C.E., Gustafsson, J.-A., Rafter, J., 1992a.
Lactobacilli, anticarcinogenic activities and human intestinal microflora. Eur. J. Cancer Prev. 1, 341353.
Lidbeck, A., O8 vervik, E., Rafter, J., Nord, C.E., Gustafsson,
J.-A., 1992b. Effect of Lactobacillus acidophilus supplements on mutagen excretion in faeces and urine in humans.
Microb. Ecol. Health Dis. 5, 5967.

Lim, K.S., Huh, C.S., Baek, Y.J., 1993. Antimicrobial susceptibility of bifidobacteria. J. Dairy Sci. 76, 2168 2174.
Ling, W.H., Korpela, R., Mykkanen, H., Salminen, S., Hanninen, O., 1994. Lactobacillus strain GG supplementation
decreases colonic hydrolytic and reductive enzyme activities in healthy female adults. J. Nutr. 124, 18 23.
Link-Amster, H., Rochat, F., Saudan, K.Y., Mignot, O.,
Aeschlimann, J.M., 1994. Modulation of a specific humoral immune response and changes in intestinal flora
mediated through fermented milk intake. FEMS Immunol.
Med. Microbiol. 10, 55 63.
Mackay, A.D., Taylor, M.B., Kibbler, C.C., Hamilton-Miller,
J.M.T., 1999. Lactobacillus endocarditis caused by a probiotic organism. Clin. Microbiol. Infect. 5, 290 292.
Majamaa, H., Isolauri, E., 1997. Probiotics: a novel approach
in the management of food allergy. J. Allergy Clin. Immunol. 99, 179 185.
Majamaa, H., Isolauri, E., Saxelin, M., Vesikari, T., 1995.
Lactic acid bacteria in the treatment of acute rotavirus
gastroenteritis. J. Pediatr. Gastroenterol. Nutr. 20, 333
Malin, M., Suomalainen, H., Saxelin, M., Isolauri, E., 1996.
Promotion of IgA immune response in patients with
Crohns disease by oral bacteriotherapy with Lactobacillus
GG. Ann. Nutr. Metab. 40, 137 145.
Marteau, P., Pochart, P., Bouhnik, Y., Rambaud, J.C., 1993.
Fate and effects of some transiting micro-organisms in the
human gastrointestinal tract. World Rev. Nutr. Diet. 74,
1 21.
Marteau, P., Gerhardt, M.F., Myara, A., Bouvier, E., Trivin,
F., Rambaud, J.C., 1995. Metabolism of bile salts by
alimentary bacteria during transit in the human small
intestine. Microb. Ecol. Health Dis. 8, 151 157.
Marteau, P., Pochart, P., Flourie, B., Pellier, P., Santos, L.,
Desjeux, J.F., Rambaud, J.C., 1990. Effect of chronic
ingestion of a fermented dairy product containing Lactobacillus acidophilus and Bifidobacterium bifidum on
metabolic activities of the colonic flora in humans. Am. J.
Clin. Nutr. 52, 685 688.
Marteau, P., Vaerman, J.P., Dehennin, J.P., Bord, S., Brassart, D., Pochart, P., Desjeux, J.F., Rambaud, J.C., 1997.
Effects of intrajejunal perfusion and chronic ingestion of
Lactobacillus johnsonii strain La1 on serum concentrations
and jejunal secretions of immunoglobulins and serum
proteins in healthy humans. Gastroenterol. Clin. Biol. 21,
293 298.
Matteuzzi, D., Crociani, F., Brigidi, P., 1983. Antimicrobial
susceptibility of Bifidobacterium. Ann. Microbiol. (Paris)
134A, 339 349.
Mattila-Sandholm, T., Kauppila, T. (Eds.), 1998. Functional
Food Research in Europe 3rd Workshop, FAIR CT961028, PROBDEMO, VTT Symposium 187. Haikko, Finland, p. 125.
Mattila-Sandholm, T., Salminen, S., 1998. Up-to-date on probiotics in Europe. Gastroenterol. Int. 11, 8 16.
Mattila-Sandholm, T., Matto, J., Saarela, M., 1999. Lactic
acid bacteria with health claims interference and interactions with gastrointestinal flora. Int. Dairy J. 9, 25 35.

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

McFarland, L.V., Surawicz, C.M., Greenberg, R.N., 1994. A
randomised placebo-controlled trial of Saccharomyces boulardii in combination with standard antibiotics for
Clostridium difficile disease. JAMA 271, 19131918.
Michetti, P., Dorta, G., Wiesel, P.H., Brassart, D., Verdu, E.,
Herranz, M., Felley, C., Porta, N., Rouvet, M., Blum,
A.L., Corthesy-Theulaz, I., 1999. Effect of whey-based
( johnsonii ) La1 on Helicobacter pylori infection in humans. Digestion 60, 203209.
Miller, L.G., Finegold, S.M., 1967. Antimicrobial sensitivity of
Bifidobacterium (Lactobacillus bifidus). J. Bacteriol. 93,
125 130.
Misra, A., Kulia, R., 1994. Use of Bifidobacterium bifidum for
the manufacturing of bio-yoghurt and fruit bio-yoghurt.
Indian J. Dairy Sci. 47, 192197.
Mitsuoka, T., 1982. Recent trends in research on intestinal
flora. Bifidobacteria Microflora 1, 324.
Mogensen, G., Friis, M., 1997. L. casei 431 a strategic
probiotic strain from Chr. Hansen A/S. The World of
Ingredients. October, 4142.
Morotomi, M., Mutai, M., 1986. In vitro binding of potent
mutagenic pyrolyzates to intestinal bacteria. J. Natl. Cancer Inst. 77, 195 201.
Myllarinen, P., Forssell, P., von Wright, A., Alander, M.,
Mattila-Sandholm, T., 1998. The use of starch as a capsulation material for lactic acid bacteria. In: Mattila-Sandholm, T., Kauppila, T. (Eds.), Functional Food Research
in Europe, 3rd Workshop, FAIR CT96-1028, PROBDEMO, VTT Symposium 187. Haikko, Finland, p. 91.
Nicas, T.I., Cole, C.T., Preston, D.A., Schabel, A.A., Nagarajan, R., 1989. Activity of glycopeptides against vancomycin-resistant gram-positive bacteria. Antimicrob.
Agents Chemother. 33, 14771481.
Nielsen, O.H., Jorgensen, S., Pedersen, K., Justesen, T., 1994.
Microbiological evaluation of jejunal aspirates and faecal
samples after oral administration of bifidobacteria and
lactic acid bacteria. J. Appl. Bacteriol. 76, 469474.
Niku-Paavola, M.-L., Latva-Kala, K., Laitila, A., MattilaSandholm, T., Haikara, A., 1999. New types of antimicrobial compounds produced by Lactobacillus plantarum. J.
Appl. Microbiol. 86, 2935.
Oberman, H., Libudzisz, Z., 1998. Fermented milks. In:
Wood, B.J.B. (Ed.), Microbiology of Fermented Foods.
Blackie Academic & Professional, London, pp. 308350.
Okawa, T., Niibe, H., Arai, T., Sekiba, K., Noda, K.,
Takeuchi, S., Hashimoto, S., Ogawa, N., 1993. Effect of
LC9018 combined with radiation therapy on carcinoma of
the uterine cervix. A phase III, multicenter, randomized,
controlled study. Cancer 72, 19491954.
Okongi, S., Ono, J., Kudo, T., Hiramatsu, A., Teraguchi, S.,
1984. Jap. Patent c J59085288-A.
Orrhage, K., Sillerstrom, E., Gustafsson, J.A., Nord, C.E.,
Rafter, J., 1994. Binding of mutagenic heterocyclic amines
by intestinal and lactic acid bacteria. Mut. Res. 311, 239


Ouwehand, A.C., Salminen, S.J., 1998. The health effects of

cultured milk products with viable and non-viable bacteria.
Int. Dairy J. 8, 749 758.
Ouwehand, A.C., Isolauri, E., Kirjavainen, P.V., Salminen,
S.J., 1999. Adhesion of four Bifidobacterium strains to
human intestinal mucus from subjects in different age
groups. FEMS Microbiol. Lett. 172, 61 64.
Pedrosa, M.C., Golner, B.B., Goldin, B.R., Barakat, S., Dallal, G.E., Russell, R.M., 1995. Survival of yougurt-containing organisms and Lactobacillus gasseri (ADH) and their
effect on bacterial enzyme activity in the gastrointestinal
tract of healthy and hypochlorhydric elderly subjects. Am.
J. Clin. Nutr. 61, 353 359.
Pelletier, C., Bouley, C., Bourliov, P., Carbon, C., 1996.
Evaluation of safety properties of Lactobacillus strains by
using an experimental model of endocarditis in rabbit
(abstr.) SOMED Meeting, Paris, France.
Pelto, L., Isolauri, E., Lilius, E.M., Nuutila, J., Salminen, S.,
1998. Probiotic bacteria down-regulate the milk-induced
inflammatory response in milk-hypersensitive subjects but
have an immunostimulatory effect in healthy subjects.
Clin. Exp. Allergy. 28, 1474 1479.
Pettersson, L., Graf, W., Sewelin, U., 1983. Survival of Lactobacillus acidophilus NCDO 1748 in human gastrointestinal
tract. 2. Ability to pass the stomach and intestine in vivo.
In: Hallgren, B. (Ed.), Nutrition and the Intestinal Flora.
XV Symp. Swed. Nutr. Found. Almqvist and Wicksell,
Uppsala, pp. 127 130.
Pochart, P., Marteau, P., Bouhnik, Y., Goderel, I., Bourlioux,
P., Rambaud, J.-C., 1992. Survival of bifidobacteria ingested via fermented milk during their passage through the
human small intestine: an in vivo study using intestinal
perfusion. Am. J. Clin. Nutr. 55, 78 80.
Pool-Zobel, B.L., Bertram, B., Knoll, M., Lamertz, R., Neudecker, C., Schillinger, U., Schmezer, P., Holzapfel, W.H.,
1993. Antigenotoxic properties of lactic acid bacteria in
vivo in the gastrointestinal tract of rats. Nutr. Cancer 20,
271 281.
Pool-Zobel, B.L., Neudecker, C., Domizlaff, I., Ji, S.,
Schillinger, U., Rumney, C., Moretti, M., Vilarini, I.,
Scassellati-Sforzolini, R., Rowland, I., 1996. Lactobacillusand Bifidobacterium-mediated antigenotoxicity in the colon
of rats. Nutr. Cancer 26, 365 380.
Rautanen, T., Isolauri, E., Salo, E., Vesikari, T., 1998. Management of acute diarrhoea with low osmolarity oral rehydration solution and Lactobacillus strain GG. Arch. Dis.
Child 79, 157 160.
Rautio, M., Jousimies-Somer, H., Kauma, H., Pietarinen, I.,
Saxelin, M., Tynkkynen, S., Koskela, M., 1999. Liver
abscess due to a Lactobacillus rhamnosus strain indistinguishable from L. rhamnosus strain GG. Clin. Infect. Dis.
28, 1160 1161.
Raza, S., Graham, S.M., Allen, S.J., Sultana, S., Cuevas, L.,
Hart, C.A., 1995. Lactobacillus GG promotes recovery
from acute nonbloody diarrhea in Pakistan. Pediatr. Infect.
Dis. J. 14, 107 111.


M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

Reddy, M.S., 1989. Enhancement of L. acidophilus viability in

yoghurt and other cultured products. US Patent 4.797.289.
Reddy, B.S., Rivenson, A., 1993. Inhibitory effect of
Bifidobacterium longum on colon, mammary, and liver
carcinogenesis induced by 2-amino-3-methylimidazo[4,5f]quinoline, a food mutagen. Cancer Res. 53, 39143918.
Rinckel, L.A., Savage, D.C., 1990. Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33. Plasmid 23, 119125.
Roberfroid, M.B., 1998. Prebiotics and synbiotics: concepts
and nutritional properties. Br. J. Nutr. 80, S197S202.
Robredo, B., Singh, K.V., Baquero, R., Murray, B.E., Torres,
C., 2000. Vancomycin-resistant enterococci isolated from
animals and food. Int. J. Food Microbiol. 54, 197204.
Ruseler-van Embden, J.G.H., van Lieshout, L.M.C., Marteau,
P., 1995. No degradation of intestinal mucus glycoproteins
by Lactobacillus casei strain GG. Microecol. Ther. 25,
304 309.
Rybka, S., Fleet, G., 1997. Populations of L. delbrueckii ssp
bulgaricus, S. thermophilus, L. acidophilus and Bifidobacterium species in Australian yoghurts. Food Austr. 49,
471 475.
Saavedra, J.M., Bauman, N.A, Oung, I., Perman, J.A.,
Yolken, R.H., 1994. Feeding of Bifidobacterium bifidum
and Streptococcus thermophilus to infants in hospital for
prevention of diarrhea and shedding of rotavirus. Lancet
344, 1046 1049.
Salminen, S., Isolauri, E., Salminen, E., 1996a. Probiotics and
stabilisation of the gut mucosal barrier. Asia Pacific J.
Clin. Nutr. 5, 53 56.
Salminen, S., Ouwehand, A.C., Isolauri, E., 1998a. Clinical
applications of probiotic bacteria. Int. Dairy J. 8, 563572.
Salminen, S., Ouwehand, A., Benno, Y., Lee, Y.K., 1999.
Probiotics: how should they be defined? Trends Food Sci.
Technol. 10, 107 110.
Salminen, S., von Wright, A., Laine, M., Vuopio-Varkila, J.,
Korhonen, T., Mattila-Sandholm, T., 1996b. Development
of selection criteria for probiotic strains to assess their
potential in functional foods: a Nordic and European
approach. Biosci. Microbiol. 15, 6167.
Salminen, S., von Wright, A., Morelli, L., Marteau, P., Brassart, D., de Vos, W.M., Fonden, R., Saxelin, M., Collins,
K., Mogensen, G., Birkeland, S.-E., Mattila-Sandholm, T.,
1998b. Demonstration of safety of probiotics a review.
Int. J. Food Microbiol. 44, 93106.
Samona, A., Robinson, R.K., Marakis, S., 1996. Acid production by bifidobacteria and yoghurt bacteria during fermentation and storage of milk. Food Microbiol. 13, 275280.
Saxelin, M., Grenov, B., Svensson, U., Fonden, R., Reniero,
R., Mattila-Sandholm, T., 2000. The technology of probiotics. Trends Food Sci. Technol. 10, 387392.
Schiffrin, E., Brassart, D., Servin, A.L., Rochat, F., DonnetHughes, A., 1997. Immune modulation of blood leukocytes
in humans by lactic acid bacteria: criteria for strain selection. Am. J. Clin. Nutr. , S 66, 15S20.
Schiffrin, E.J., Rochat, F., Link-Angler, H., Acschlimann,
J.M., Donnet-Hughes, A., 1995. Immuno-modulation of

human blood cells following the ingestion of lactic acid

bacteria. J. Dairy Sci. 78, 491 497.
Sepp, E., Tamm, E., Torm, S., Lutsar, I., Mikelsaar, M.,
Salminen, S., 1995. Impact of a Lactobacillus probiotic on
the faecal microflora in children with shigellosis. Microecol. Ther. 23, 74 80.
Shornikova, A.V., Casas, I., Isolauri, E., Mykkanen, H.,
Vesikari, T., 1997a. Lactobacillus reuteri as a therapeutic
agent in acute diarrhea in young children. J. Pediatr.
Gastroenterol. Nutr. 24, 399 404.
Shornikova, A.V., Casas, I.A., Mykkanen, H., Vesikari, T.,
1997b. Bacteriotherapy with Lactobacillus reuteri in rotavirus gastroenteritis. Pediatr. Infect. Dis. 16, 1103 1107.
Shornikova, A.V., Isolauri, E., Burkanova, L., Lukovnikova,
S., Vesikari, T., 1997c. A trial in Karelian republic of oral
rehydration and Lactobacillus GG for treatment of acute
diarrhoea. Acta Paediatr. 86, 460 465.
Siitonen, S., Vapaatalo, H., Salminen, S., Gordin, A., Saxelin,
M., Wikberg, R., Kirkkola, A.L., 1990. Effect of Lactobacillus GG yoghurt in prevention of antibiotic associated
diarrhea. Ann. Med. 22, 57 59.
Silva, M., Jacobus, N.V., Deneke, C., Gorbach, S.L., 1987.
Antimicrobial substance from a human Lactobacillus
strain. Antimicrob. Agents Chemother. 31, 1231 1233.
Singh, J., Riveson, A., Tomita, M., Shimamura, S., Ishibashgi,
N., Reddy, B.S., 1997. Bifidobacterium longum, a lactic acid
producing intestinal bacterium, inhibits colon cancer and
modulates the intermediate biomarkers of colon carcinogenesis. Carcinogenesis 18, 833 841.
Skytta, E., Haikara, A., Mattila-Sandholm, T., 1992. Production and characterization of antibacterial compounds produced by Pediococcus damnosus and Pediococcus
pentosaceus. J. Appl. Bacteriol. 72, 134 142.
Spanhaak, S., Havenaar, R., Schaafsma, G., 1998. The effect
of consumption of milk fermented by Lactobacillus casei
strain Shirota on the intestinal microflora and immune
parameters in humans. Eur. J. Clin. Nutr. 52, 899 907.
Spork, A.C., 1994. New trends for probiotic cultures. Eur.
Dairy Mag. 3076 3077.
Surawicz, C.M., Elmer, G.W., Speelman, P., McFarland, L.,
Chinn, J., van Belle, G., 1989. Prevention of antibiotic-associated diarrhea by Saccharomyces boulardii. A prospective study. Gastroenterology 84, 981 988.
Svensson, U., 1999. Industrial perspectives. In: Tannock,
G.W. (Ed.), Probiotics: A Critical Review. Horizon Scientific Press, Wymondham, pp. 57 64.
Swenson, J.M., Facklam, R.R., Thornsberry, C., 1990. Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus, and Lactobacillus species.
Antimicrobiol. Agents Chemother. 34, 543 549.
Takagi, A., Matsuzaki, T., Sato, M., Nomoto, K., Morotomi,
M., Yokokura, T., 1999. Inhibitory effect of oral administration of Lactobacillus casei on 3-methycholanthrene-induced carcinogenesis in mice. Med. Microbiol. Immunol.
(Berl.) 188, 111 116.
Tamime, A.Y., Marshall, V.M.E., Robinson, R.K., 1995. Microbiological and technological aspects of milks fermented
by bifidobacteria. J. Dairy Res. 62, 151 187.

M. Saarela et al. / Journal of Biotechnology 84 (2000) 197215

Tanaka, R., Ohwaki, M.A., 1994. Controlled study on the
ingestion of Lactobacillus casei fermented milk on the
intestinal microflora, its microbiology and immune system
in healthy adults. In: Proceedings of XII Riken Symposium
on Intestinal Flora. Tokyo, Japan, pp. 85104.
Thyagaraja, N., Hosono, A., 1993. Antimutagenicity of lactic
acid bacteria from Idly against food-related mutagens. J.
Food Prot. 56, 1061 1066.
Thyagaraja, N., Hosono, A., 1994. Binding properties of lactic
acid bacteria from Idly towards food-borne mutagens.
Food Chem. Toxic. 32, 805809.
Tuomola, E., 1999. In vitro adhesion of probiotic lactic acid
bacteria. Ph.D. thesis, Turku, Finland.
Tuomola, E.M., Ouwehand, A.C., Salminen, S., 1999. Human
ileostomy glycoproteins as a model for small intestinal
mucus to investigate adhesion of probiotics. Lett. Appl.
Microbiol. 28, 159 163.
Tuomola, E.M., Salminen, S.J., 1998. Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. Int. J. Food Microbiol. 41, 4551.
Tynkkynen, S., Singh, K.V., Varmanen, P., 1998. Vancomycin
resistance factor of Lactobacillus rhamnosus GG in relation


to enterococcal vancomycin resistance (6an) genes. Int. J.

Food Microbiol. 41, 195 204.
Vescovo, M., Morelli, L., Bottazzi, V., 1982. Drug resistance
plasmids in Lactobacillus acidophilus and Lactobacillus
reuteri. Appl. Environ. Microbiol. 43, 50 56.
Wolf, B.W., Garleb, D.G., Casas, I., 1995. Safety and tolerance of Lactobacillus reuteri in healthy adult male subjects.
Microb. Ecol. Health Dis. 8, 41 50.
Wolf, B.W., Wheeler, K.B., Ataya, D.G., Garleb, K.A., 1998.
Safety and tolerance of Lactobacillus reuteri supplementation to a population infected with human immunodeficiency virus. Food Chem. Toxicol. 36, 1085 1094.
Young, J., 1998. European market developments in prebioticand probiotic containing foodstuffs. Br. J. Nutr. 80, S231
Zhang, X.B., Ohta, Y., 1993. Antimutagenicity of cell fractions of microorganisms of potent mutagenic pyrolysates.
Mut. Res. 298, 247 253.
Zhang, X.B., Ohta, Y., Hosono, A., 1990. Antimutagenicity
and binding of lactic acid bacteria from a Chinese cheese
to mutagenic pyrolyzates. J. Dairy Sci. 73, 2702 2710.