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a
Institute of Hygiene and Toxicology, Federal Research Centre for Nutrition, Engesserstr. 20, D-76131 Karlsruhe, Germany
Department of the Science of Food of Animal Origin, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80175, Yalelaan 2,
NL-3508 TD Utrecht, The Netherlands
Abstract
Scientific developments in recent years have opened new frontiers and enable a better understanding of the gastrointestinal tract (GIT) as a complex and delicately balanced ecosystem. This paper focuses on more recent information related
to the microbial population of the GIT and its functional role in human physiology and health. Special attention is also given
to modern approaches for improving or stabilising the intestinal system and its functioning by the deliberate application of
viable microbial cultures, so-called probiotics, selected for special functional properties. 1998 Elsevier Science B.V.
Keywords: Bifidobacteria; Functional properties; Gastro-intestinal tract; Lactobacilli; Probiotics
1. Introduction
There is general agreement on the important role
of the gastro-intestinal (GI) microflora in the health
status of men and animals. The importance of
lactobacilli for human health and longevity was first
hypothesized by Metchnikoff at the beginning of this
century. He, however, considered the gut microbes in
total as detrimental rather than beneficial, and suggested that desirable effects might only be expected
from their substitution by yogurt bacteria. Since then,
*Corresponding author. IHT / BFE, Engesserstr. 20, D-76131
Karlsruhe, Germany. Tel.: 1 49 721 6625 115; fax: 1 49 721
6625 142; e-mail: ob07@rz.uni-karlsruhe.de
86
intestinal microflora, but also to microbial communities at other sites of the body;
the probiotic might consist of more than one
bacterial species; and
it can be applied to both man and animals.
Several studies have focused on the pharmacokinetics of different probiotics in humans, and
our knowledge, especially of the complex mechanisms behind these effects, is constantly increasing.
Today we know that certain lactic acid bacteria
(LAB) can induce specific immune regulators as a
result of interaction with mononuclear phagocytes
and endothelial cells of the host (Brassart and
Schiffrin, 1997). Furthermore, it has been observed
that particular strains of LAB showed adjuvant
properties by stimulation of a specific antibody
response after infection with (attenuated) pathogenic
microorganisms (Pouwels et al., 1996). Another
intriguing development is the observation that certain
LAB can strengthen the gut mucosal barrier and
thereby influence gut mucosal permeability and
possible diarrhoea. This paper will evaluate the
present status of probiotics and discuss recent developments in this area.
Fig. 1. Increase in the mucosal surface by folding (modified after Waldeck, 1990).
87
88
Table 1
Factors affecting the microflora of the gastro-intestinal tract
89
90
Table 2
Microorganisms applied in probiotic products
Lactobacillus species
L. acidophilus
L. casei
L. crispatus
L. gallinarum a
L. gasseri
L. johnsonii
(L. paracasei)
L. plantarum
L. reuteri
L. rhamnosus
a
Bifidobacterium species
B.
B.
B.
B.
B.
B.
B.
adolescentis
animalis
bifidum
breve
infantis
lactis b
longum
Other LAB
Non-lactics
a
Ent. faecalis
Ent. faecium
Lactoc. lactis c
Leuc. mesenteroides c
Ped. acidilactici c
Sporolactobacillus inulinus a
Strep. thermophilus
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Table 3
Probiotic lactobacilli detected in novel-type yoghurts (data combined from own results and from Reuter, 1997a)
Manufacturer
Origin
Lactobacillus viable
counts (log CFU g 21 )
Lactobacilli indicated by
the manufacturer
Lactobacilli identified by
DNADNA hybridization
A
B
C
Germany
Germany
Germany
L. casei Shirota
L. acidophilus LA-1
Lactobacillus casei GG
D
E
F
Germany
Germany
Germany
G
H
I
J
K
L
M
Germany
Germany
Germany
Germany
Germany
Germany
Germany
L. paracasei (casei) b
L. johnsonii
L. rhamnosus
L. acidophilus
L. acidophilus
L. paracasei (casei)
L. acidophilus
L. paracasei (casei)
L. acidophilus
L. acidophilus
L. acidophilus
ND
L. johnsonii
L. paracasei (casei)
ND
Netherlands
N
O
P
Sweden
Germany
Switzerland
Q
R
Switzerland
France
7.98.9
6.48.1
8.08.4
6.37.8
5.46.4
7.48.1
6.88.2
6.27.8
3.95.7
4.76.2
5.25.9
5.56.8
7.17.8
8.68.7
8.18.4
4.75.3
6.8 a
6.4 a
9.2
7.3
7.6 a
9.0 a
5.2 a
8.3 a
8.3 a
BactoLab cultures
L. casei Actimell
L. acidophilus
L. casei
L. acidophilus LA-7
L. acidophilus LA7
Not indicated
L. acidophilus
BIOGARDE cultures
L. casei
L. acidophilus LA-H3
L. casei LC-H2
L. acidophilus Gilliland
L. casei
L. acidophilus
L.
L.
L.
L.
L.
acidophilus
casei
reuteri
casei
casei
Table 4
Autochthonous lactobacilli associated with the human host (according to Reuter, 1997b)
I. Homofermentative a
L. acidophilus
L. acidophilus sensu stricto
L. gasseri
L. crispatus
L. johnsonii
L. salivarius
L. salivarius
subsp. salivarius
subsp. salicinius
L. casei
L. casei
subsp. casei
subsp. tolerans
L. rhamnosus
II. Heterofermentative b
L. reuteri (formerly L. fermentum II) including L. oris and L. vaginalis
a
L.
L.
L.
L.
L.
L.
L.
L.
L.
crispatus
paracasei (casei) a
acidophilus
acidophilus a
acidophilus a
paracasei (casei) a
reuteri a
rhamnosus a
paracasei (casei) a
92
Table 5
Typical features of the species of the acidophilus-group (modified after Mitsuoka, 1992; Reuter, personal communication)
Species
L. acidophilus
L. amylovorus
L. crispatus
L. gallinarum
L. gasseri
L. johnsonii
a
Habitat a
All?
P/ C
H/W
W
H/C
H / P/ W
3237
40
3538
3336
3335
3238
I,II
IV (III)
III
Ia
Ib
Ic
Id
IIa
IIb
A-1
A-3
A-2
A-4
B-1
B-2
I
I,II
93
Table 6
Successful probiotic bacteria and their reported effects (as reported by Salminen et al., 1996)
Strain
Immune enhancer, adjuvant, adherent to human intestinal cells, balances intestinal microflora
Lowering faecal enzyme activity, decreased faecal mutagenicity, prevention of radiotherapy related diarrhoea, treatment of constipation
Prevention of antibiotic associated diarrhoea, treatment and prevention of rotavirus diarrhoea, treatment of relapsing Clostridium difficile
diarrhoea, prevention of acute diarrhoea, Crohns disease, antagonistic against cariogenic bacteria, vaccine adjuvant
Prevention of intestinal disturbances, treatment of rotavirus diarrhoea, balancing intestinal bacteria, lowering faecal enzyme activities,
positive effects in the treatment of superficial bladder cancer, immune enhancer
in early colon cancer, immune enhancement
No effect on rotavirus diarrhoea, no immune enhancing effect during rotavirus diarrhoea, no effect on faecal enzymes
Streptococcus thermophilus;
Lactobacillus bulgaricus
Bifidobacterium bifidum
Lactobacillus gasseri (ADH)
Lactobacillus reuteri
94
5. Future developments
In spite of considerable progress in probiotic
research over the last five years, not all probiotic
bacteria available on the market have a solid sci-
95
96
bacterium (Angert et al., 1993). Difficulties in culturing techniques have led to the development of novel
molecular methods based on DNA technology by
which bacteria can be identified without cultivation
(Amann et al., 1995).
Presently, ribosomal RNA (rRNA) is commonly
used as a tool to study microbial populations. During
the last two decades the gene encoding for 16S
rRNA of Escherichia coli was sequenced, and many
more 16S rRNA sequences of other bacteria have
been determined. The polymerase chain reaction
(PCR) and improved sequencing technology have
greatly facilitated retrieval of new sequences. At
present, several thousands of 16S rRNA sequences
of different bacteria are available in genetic databases, even from intestinal bacterial species that
cannot be cultured in vitro (Angert et al., 1993; Snel
et al., 1994). The 16S rRNA gene is approximately
1540 bases long, and includes several variable
regions while the overall structure is highly conserved. This makes it possible to use this gene to
study phylogenetic relationships between microorganisms (Woese, 1987).
An approach to analyse the genetic diversity of
complex microbial populations is denaturing gradient
gel electrophoresis (DGGE) or temperature gradient
gel electrophoresis (TGGE). This technique is based
on the separation of PCR amplified fragments of
genes coding for 16S rRNA, all having the same
length (Muyzer et al., 1993). This results in unique
separation patterns for different microbial populations, and will contribute to the description of
changes or differences in the microflora composition
of (uncharacterised) microbial populations.
From sequence data, rRNA-targeted oligonucleotide probes can be developed that are species- or
group-specific. For analysis of intestinal microflora,
such probes labeled with a radio-active marker have
been used for the first time to study ruminal microbial ecology (Stahl et al., 1988). In this study,
hybridisation was quantified in a dot-blot procedure.
Later, for similar studies, oligonucleotide probes
were labeled with radio-active or fluorescent markers
and used for in situ hybridisation, which enables
specific identification of individual cells (DeLong et
al., 1989; Amann et al., 1990, 1995). Techniques
based on 16S rRNA detection have recently been
used to detect probiotic Bifidobacterium species in
infant faeces (Kok et al., 1996).
97
6. Safety
The use of LAB in food products has a long safety
record. Strains used in probiotic dairy products are
also considered to be non-pathogenic for humans. In
several clinical and epidemiological studies LAB and
bifidobacteria have been examined for their role in
health (Donohue and Salminen, 1996). In addition,
while it has been suggested that probiotic strains
have been implicated in some cases of patients with
bacteraemia, it is important to note that none of the
pathogenic strains isolated in such cases was related
to lactobacilli used in probiotic food products
(Donohue and Salminen, 1996; Saxelin et al., 1996).
98
7. Conclusions
Increasing knowledge underlines the important
role of the intestinal flora for maintaining health and
in the prevention of disease. Evidence is emerging
that the intestinal flora does not exist as an entity by
itself, but is constantly interacting (communicating)
with the environment, the central nervous system, the
endocrine system and the immune system (Bry et al.,
1996; Shanahan, 1997; Umesaki et al., 1997; Wang
et al., 1997). Disturbance of this delicate balance
may lead to other disorders and thus facilitate
establishing a state of disease.
Probiotics offer dietary means to support the
balance of intestinal flora. They may be used to
counteract local immunological dysfunctions, to
stabilise the gut mucosal barrier function, to prevent
infectious succession of pathogenic microorganisms
or to influence intestinal metabolism. Many of the
proposed mechanisms have to be validated in controlled clinical trials in humans. Novel methods are
urgently needed to monitor changes in the composition of the intestinal flora and their mutual
interaction with the hosts immunological functions
and metabolism.
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