Download Role of Lactobacilli in Gastrointestinal Ecosystem

63
Bulgarian Journal of Agricultural Science, 12 (2006), 63-114
National Centre for Agrarian Sciences
Role of Lactobacilli in Gastrointestinal Ecosystem
S. A. DENEV
Trakia University Department of Biology, Section of Microbiology, Agricultural
Faculty, BG – 6000 Stara Zagora, Bulgaria
Abstract
DENEV, S. A., 2006. Role of Lactobacilli in gastrointestinal ecosystem. Bulg. J. Agric.
Sci., 12: 63-114
Lactobacilli are normal and important inhabitants of the gastrointestinal tract of human and
animals. They constitute an integral part of healthy gastrointestinal microecology and have
received considerable scientific attention. The role that lactobacilli play in the ecology of the
gastrointestinal tracts of humans and animals may be of major importance, but it is as yet poorly
understood.
The present review summarizes the current state of knowledge regarding the role of lactobacilli in gastrointestinal ecosystem. Topics covered include: nutritional and therapeutic benefits
of lactobacilli; deconjugation of bile acids; neutralization of toxins and other intestinal reactions; protection against intestinal pathogens by production of antibacterial substances such
as organic acids, hydrogen peroxide, bacteriocins and etc.; stabilization of intestinal microflora,
excluding colonization of enteropathogenic bacteria by adhesion to the intestinal wall and competition for nutrients; reduction of the cholesterol level in blood serum by cholesterol assimilation; decrease risk of colon cancer by detoxification of toxic, mutagenic and carcinogenic substances, and modulation of fecal procarcinogenic enzymes such as b-Glucuronidase, azoreductase
and nitroreductase; tumor suppression by stimulation of non-specific and specific immune
response.
Key words: Lactobacilli, gastrointestinal microflora, gastrointestinal ecosystem, antagonistic, antimutagenic, anticarcinigenic, immunological effects
Abbreviations: GIT - Gastrointestinal tract; GIE - Gastrointestinal ecosystem; GIM
- Gastrointestinal microflora; CE - Competitive exclusion; IT – Intestinal tract; LAB
- Lactic acid bacteria; BSH - Bile salt hydrolase
Introduction
Gastrointestinal ecosystems (GE) of
humans and animals are complex, open,
integrated, interactive units containing
E-mail: [email protected]
many microbial species, which are divided
in two major groups: autochthonous (indigenous) which exist in close association
with the gut epithelium and allochthonous
(non-indigenous) which occurs free in the
64
gut lumen. It is, however, difficult to make
a clear distinction between the two groups.
In regard to autochthonous microorganisms, Savage (1977, 2001) has suggested
a useful definition. According to Savage
the autochthonous microflora must:
• Be capable of growing anaerobically;
• Be always present in the gut of normal adult
animals;
• Colonize the various parts of the digestive
tract;
• Establish their own habitats during succession (i.e., sequential establishment of the various
microorganisms) in infant animals;
• Maintain a stable population in normal adults;
Associate intimately with the epithelial
mucosa of the tract of the gut they colonize.
Its high population density, wide diversity, and complexity of interactions characterize the microbial community inhabiting the gastrointestinal tract (GIT). It has
been estimated that there are in the gut
1014 bacterial cells whereas the number
of eukaryotic cells comprising the human
body amounts to only 1013 (Luckey, 1972).
Within this population there are about 500
different bacterial species (Savage, 1984;
Ewing and Cole, 1994; Bengmark, 1998;
Bird et al., 2000; Miguel, 2001; Cole et al.
2003), although 40 types comprise 99%
of the microflora (Tannock, 1988). The
human body contains 10 times more protective indigenous flora than do eukaryotic cells (Tancrede, 1992). The unique
biotic and abiotic components of the GIT
(specialized chemical environment and appropriately adapted microbes) permit the
definition of the “gastrointestinal ecosystem” (GIE) (Savage, 1977, 2001). The
systems are established during postnatal
life, when various microbial species find
their way into the intestinal tract. When
S. A. Denev
germfree animals are conventionalized, i.e.
colonized with conventional flora, similar
ecosystems are established. Results of
studies on ex-germfree animals have demonstrated that some microbial species may
act synergistically and others may act antagonistically with each other. The exact
mechanisms involved in these interactions,
however, have not been elucidated
(Ducluzeau et al., 1984; Midtvedt et al.,
1987; Savage, 2001).
The numerically dominant inhabitants
of the GIE are obligate anaerobes. In the
neonatal mammal, such as the human infant, piglet and calf the intestinal tract is
sterile at birth (Haenel, 1970). It is rapidly
colonized from the mouth and rectum, usually initially with the mother’s vaginal and
perianal flora. In detailed of wide variety
of young animals showed that at birth the
GIT becomes flooded with multiplying
bacteria: coliforms, bifidobacteria, streptococci, including enterococci, clostridia,
bacteroides, and other anaerobes, followed
rapidly by lactobacilli.
The metabolic activities of the gastrointestinal microflora (GIM) are extremely complex and diverse. Many investigations have shown that intestinal bacteria play a major part in the enzyme activities of intestine; production of b-glucuronidase (Hawksworth et al., 1971),
azoreductase (Brown, 1981; Raffii et al.,
1990), nitroreductase (Zachariah and
Juchau, 1974), 7a-dehydroxylase (Hirano
et al., 1981), 7a-dehydrogenase (Wilkins
and Tassell, 1983), toxic, carcinogenic or
mutagenic metabolites from diet (Rowland
et al., 1985; Goldin et al., 1996);
deconjugation of bile acids (Aries and Hill,
1970; Floch et al., 1971, 1972; Gilliland and
Speck, 1977a; Tannock et al.,1994); detoxification of dietary toxicants; enterohepatic
circulation of drugs, food additives and ste-
Role of Lactobacilli in Gastrointestinal Ecosystem
65
roids; alteration in susceptibility of host
tumor induction etc. For review see Drasar
and Hill (1974), Yuguchi et al., (1992),
Denev (1996, 1997); Bird et al. (2000);
Denev et al. (2000); Savage (2001).
The composition and metabolic activities of GIM is very complex and strictly
determined by the local environmental
conditions (Croucher et al., 1983; Edwards
et al., 1985; Seddon, 1989), physiological
interactions (Floch et al., 1971, 1972), diet
(Winitz et al., 1970; Maier et al., 1974;
Moore and Holdeman, 1975, Denev,
1993a; Savage, 2001) and another factors
(Hill et al., 1982; Kim, 1989; Seddon, 1989;
Mitsuoka, 1990, Denev, 1996, 1997). The
size of the microbial population in different regions of the tract also varies between
animal species too (Savage, 1977;
Tannock, 1984, 2004).
Microbiological interest in the role of
lactobacilli inhabiting the GIT dates, from
the time of Metchnikoff (1903, 1907) when
it was observed that the natural fermentation of milk by lactic acid bacteria (LAB)
prevented the growth of non-acid-tolerant
types of microbe. It was proposed that, if
lactic fermentation prevented the putrefaction of milk, it should have a similar
effect in the digestive tract. The implantation of lactobacilli in the GIT would suppress the growth of “putrefactive” bacteria, thus reducing the amount of toxic substances generated in the digestive tract.
Although Metchnikoff’s hypothesis was
popular at the beginning of that century it
was unsupported by experimental or clinical proof, partly due to the lack of adequate
experimental techniques at that time. However, the remarkable developments in techniques for microbiological research and for
raising germ-free animals in recent years,
have produced data showing the important role played by intestinal bacteria in
animals, leading to a fresh reassessment
of Metchnikoff’s hypothesis.
The balance of the GIM is then controlled by acid secretion in the stomach (although this is buffered to some extent by
the milk imbibed) and ingestion of immunoglobulins and other protective factors in
the mother’s milk, so that lactobacilli become the dominant organisms and other
groups rapidly decline (Reiter, 1978). Lactobacilli predominate in the stomach and
small intestine, but in the jejunum and large
intestine strict anaerobes such as bacteroides are the majority flora and lactobacilli
constitute only 0.07-1.0% of the total flora.
This desirable predominance of lactobacilli in the upper intestine, which is established on suckling, help to prevent the potentially lethal diarrhea or scouring that occurs in young animals when enteropathogenic coliforms proliferate in the upper
GIT. The mechanisms by which lactobacilli suppress the rest of the bacterial flora
is not fully understood but is probably partly
due to lactic acid production and partly to
inhibitory systems present in the raw milk,
including the lactoperoxidase system,
which can be activated by H2O2 -producing lactobacilli (Reiter, 1978). In the young
animals, lactobacilli usually become the
principal component of stomach and small
intestine, developing from about 104 to
108/g intestinal content, highest numbers
being obtained in the lower part of the
small intestine (Smith, 1971).
Lactobacillus spp. are important in creating a balanced microbial population in
GIE (Sandine et al., 1972; Shahani and
Ayebo, 1980; Bianchi-Salvadory, 1986;
Denev et al., 2000; Varnam, 2002;
Tannock, 2004). The interest in their existence in different areas of the body and
their role in the host and microflora-related
physiochemical conditions in the organism
66
has become one of the subjects of a comparatively new research field - microbial
ecology (Haenel et al., 1957, 1957a,
Haenel, 1980; Dubos, 1966; Reuter, 1975;
Goldin and Gorbach, 1984; Gorbach, 1990;
Prins, 1996; Mackie et al., 1999).
The therapeutic and nutritive attributes
of LAB particularly the lactobacilli have
been actively researched since then
(Speck, 1976; Sandine et al., 1972;
Sandine, 1979; Shahani and Ayebo, 1980;
Garg and Mital, 1992). Thereafter, many
investigations have shown that the presence of lactobacilli in the intestinal tract
(IT) is helpful in maintaining good health,
restoring body vigor, stimulating host immune mechanisms, productivity, and combating intestinal and other disease disorders (Rettger et al., 1935; Gilliland and
Speck, 1977; Gilliland, 1979; Friend and
Shahani, 1984; Perdigon et al., 1987;
Denev, 1993; Denev et al., 2000;
Morishita, 2003; Sandholm and M.
Saarela, 2003).
Subsequent research has emphasized
that the lactobacilli to be used as dietary
adjunct must be able to survive the hostile
environment in the GIT and proliferate
(Hawley et al., 1959; Gilliland et al., 1978;
Hargrove and Alford, 1978). Several investigations have shown that Lactobacillus acidophilus is not only capable of establishing in the complex ecosystem of the
gut (Kopeloff, 1926; Myers, 1931; Stark
et al., 1934; Rettger et al., 1935) but also
of imparting many health benefits
(Gilliland, 1989; Mital and Garg, 1992;
Sellars, 1992; Mital et al., 1995). These
include stabilization of intestinal microflora,
antagonistic action toward intestinal and
food-borne pathogens, control of serum
cholesterol, prevention of colon cancer,
and enhanced availability of nutrients
(Shah, 2001).
S. A. Denev
There are many other reports, beginning with Metchnikoff suggesting that
LAB particularly the lactobacilli are a numerically dominant and important group of
microorganisms colonizing the GIT and of
the female reproductive tract of humans
(Drasar and Hill, 1974; Mitsuoka et al.,
1975; Conway, 1989; Mikelsaar and
Mandar, 1993; Varnam, 2002) and animals
(Roach et al., 1977; Savage, 1975, 1977;
Sharpe, 1981; Worthington and Fulghum,
1988; Tannock et al., 1990; Denev et al.
2000; Tannock, 1992, 2004).
Beyond these roles, some Lactobacillus species have been exploited as “live
microbial supplements”, intended to beneficially affect the host by balancing the
natural microflora of the GIT. Probiotics,
literally meaning “for life” (Fuller, 1992)
have been the subject of considerable scientific and commercial attention over the
past two decades (for review see Denev,
1996; 1997; Fuller, 1989, 1997;
Klaenhammer, 1995, 1997, 1997a,b; Denev
et al., 2000; Shah, 2001; Tannock, 2002,
2004).
Due to their many beneficial properties lactobacilli have attracted the greatest attention of researchers. In addition,
the mechanisms involved in the expression
of these activities are not clearly understood. Therefore, an attempt has been
made to review the pertinent literature on
these aspects critically.
MAJOR CHARACTERISTICS OF THE
LACTOBACILLI
Lactobacilli are members of the LAB,
a broadly defined group characterized by
the formation of lactic acid as a sole or
main end product of carbohydrate metabolism (Tannock, 2004). There are 44 species of lactobacilli listed in Bergey’s
Manual of Systematic Bacteriology
Role of Lactobacilli in Gastrointestinal Ecosystem
67
(Kandler and Weiss, 1986). Eighty species of lactobacilli are recognized at present
(Satokari et al., 2003). Many Lactobacillus spp. have been detected in GT of vertebrate animals and humans (Table 1.).
Members of the genus Lactobacillus
constitute an extremely diverse group of
bacteria that provide considerable benefits
to humans and animals as natural inhabitants of the IT. They are Gram-positive,
non-sporeforming, rods or coccobacilli
with a G+C content usually below 50
mol%. The mol % G+C of the DNA
ranges from 32-53. They are strictly fermentative, aerotolerant (microaerophilic)
or anaerobic and have complex nutritional
requirements (carbohydrates, amino acids,
peptides, fatty acid esters, salts, nucleic
acid derivatives, vitamins). Various compounds (e.g. citrate, malate, tartarate,
quinolate, nitrate, nitrite, etc.) may be metabolized and used as energy source
(Kandler and Weiss, 1986; Hammes and
Vogel, 1995). Using glucose as a carbon
source, lactobacilli may be either homofermentative (producing more than 85%
of fermentative products as lactic acid) or
heterofermentative (producing lactic acid,
carbon dioxide, ethanol, and/or acetic acid
in equimolar amounts). The nutritional requirements of lactobacilli are reflected in
their habitats, which are rich in carbohydrate-containing substrates: they are found
on plants or material of plant origin, in fermented or spoiled food, or in association
with the bodies of animals. Lactobacilli are
catalase and cytochrome negative, usually
non-motile, that does not usually reduce
nitrate. Gelatin not liquefied. Casein not
digested but small amounts of soluble nitrogen produced by most strains. Indole
and H2S2 not produced. Lactobacilli are
aciduric with optimal pH 5.5-6.2. Growth
temperature range of Lactobacilli are 2-
53 o C - optimum generally 30-40 o C
(Hammes and Vogel, 1995). The classification of lactobacilli relies on biochemical-physiological criteria include three
groups (Table 2).
Recently, Hammes et al. (1991), Pot
et al. (1994), Gasson and Vos (1994),
Klaenhammer (1995), Hammes and Vogel
(1995), Denev et al.(2000), Tannock
(2002) reviewed lactobacilli. Venema et
al. (1996) in the former publications a
thorough survey of the ecophysiology,
genetics, metabolism, biotechnology and
application of the organisms was presented. For the role of lactobacilli in health
and disease Wood (1992) and Denev et
al. (2000) provide an excellent survey. For
microecology of lactobacilli inhabiting the
GIT see Ahrne et al. (1998), Tannock
(1988a, 1990, 1995, 2002).
COLONIZATION OF LACTOBACILLI TO
EPITHELIAL SURFACES
In recent years much attention has
been given to the study of the administration of living microbial preparations to farm
animals (probiotics), which would to assure improvement in nutrition and in the
animal’-s resistance to disease. Since the
report of Metchnikoff (1907) many reports
have indicated that Lactobacillus strains
are important in creating balanced microbial populations in the IT. Colonization of
intestinal tissue by lactobacilli is a natural
phenomenon which arises earlier and more
intensely in animals reared on traditional
farms than those in factory-farms, where
colonization may be detained or upset by
acceleration of the rearing cycles, and
above all by the often indiscriminate use
of antibiotics at subtherapeutic levels.
For the administration of Lactobacillus culture to animals to be of practical
68
S. A. Denev
Table 1
Gastrointestinal Species of Lactobacilli
Species
References
L. acidophilus
Axelsson and Lindgren (1987); Tannock et al. (1990);
Arihara et al.(1996); Holzapfel et al. (2001)
Baele et al. (2001)
Roach et al. (1977)
Fujisawa et al. (1984)
Gusils et al. (1999)
L. agilis
L. animalis
L. aviarius
L. aviarius subsp. aviaries
L. aviarius subsp. araffinosus
L. brevis
L. casei
L. crispatus
L. delbruecki
L. coryniformis
L. curvatus
L. fermentum subsp. cellobiosus
L. animalis
L. gallinarum
L. johnsonii
L. gasseri
L. fermentum
L. intestinalis
Lactobacillus gastricus
Lactobacillus antri
Lactobacillus kalixensis
Lactobacillus ultunensis
L. murinus
L. plantarum
L. rhamnosus
L. reuteri
L. ruminis
L. salivarius
Lactobacillus thermotolerans
L. vitulinus
Russell (1979)
Kandler and Weiss (1986); Salminen et al. (1993);
Ahrne et al. (1998); Holzapfel et al (2001)
Tannock et al. (1990); Du Toit et al. (2001)
Russell (1979)
Lin and Savage (1984)
Gusils et al. (1999)
Fujisawa et al. (1992)
Klein et al. (1998); Fujiwara et al. (2001)
Fuller et al. (1978); Barrow et al. (1980); Morishita (1982);
Hautefort et al. (1996)
Du Toit et al. (2001)
Roos et al. (2005)
Ma et al. (1990)
Russell (1979); Bengmark (1998); Du Toit et al. (2001)
Holzapfel et al. (2001)
Tannock et al. (1982); Sara et al. (1985);
Molin et al. (1992, 1992a);
Holzapfel et al. (2001)
Sharpe et al. (1973)
Fuller (1973); Barrow et al. (1980); Sara et al. (1985);
Rada and Vorisek (1996); Du Toit et al. (2001); Holzapfel
et al. (2001)
Niamsup et al. (2003)
Sharpe et al. (1973); Tannock et al. (1982)
Role of Lactobacilli in Gastrointestinal Ecosystem
69
Table 2
Clasification of Lactobacillus spp.
Group I:
Obligately homofermentative (15 species).
Hexoses are almost exclusively ( <85%)
fermented to lactic acid by the EmbdenMeyerhov-Parnas (EMP) pathway. The organisms
possess fructose 1,6-bisphos-phate-aldolase but
lack phospho- ketolase, and therefore, neither
gluconate nor pentoses are fermente
Group II:
Facultative heterofermentative (11 species)
Hexoses are almost exclusively fermented to
lactic acid by the RMP pathway. The organisms
possess both aldolase and phosphoketolase, and
therefore, not only ferment hexose but also
pentoses (and often gluconate). In the presence of
glucose, the enzyme
Group III:
Obligately heterofermentative (18 species)
Hexoses are fermented by the phosphogluconate
pathway yielding lactate, ethanol (acetic acid) and
CO2 in equimolar amounts. Pentoses enter this
pathway and may be fermented.
* Kandler and Weiss (1986); Du Toit et al. (2001)
use, the following conditions are essential:
a) host specificity of the strains used, and
b) their ability to form stable populations
in the GIT. Attachment of the strain to the
intestinal mucosa is one of the selection
criteria for probiotic microorganisms. Adhesion or close association of potentially
probiotic LAB, including lactobacilli to epithelial cells may further contribute to competitive exclusion (CE) (Gusils et al.,
2002a).
Some strains of lactobacilli inhabiting
the GIT of animals exhibit a particularly
intimate association with their hosts. The
lactobacilli colonize the proximal region of
the human small intestine at population levels that are minor (<108 g-1) when compared to the colonic flora (1010 g-1) where
bifidobacteria are predominant members
(109 g-1). Tissue-associated lactobacilli
have been found on the gastrointestinal surfaces of many animal species. Lactobacilli adhere to and multiply on the stratified squamous epithelial cells of the proximal small intestine in animals (rodents, pigs
and fowl), and are continually shed into
the lower regions of the tract (Pedersen
and Tannock, 1989; Tannock, 1990). In
mice, for example, lactobacilli inhabit the
tissue surface the esophagus and forestomach (Tannock, 1992). In rats, lactobacilli colonize the fore-stomach
(Brownlee and Moss 1961, Savage, 1977).
In fowl, they inhabit the epithelial surface
of the crop (Fuller and Turvey, 1971; Fuller,
1973, 1975, 1978). In pigs, lactobacilli colonize the epithelial surface of the esophagus and of a small area of tissue in the
70
S. A. Denev
stomach - pars esophagea (Fuller et al.,
1978; Barrow et al., 1980). Under natural
conditions, lactobacilli colonize epithelial
surfaces in mice, rats, fowl and pigs soon
after birth (hatching). Lactobacilli attain
high population levels on the epithelial surface (of the order of 108 bacteria per gram
of sample) and a layer of lactobacilli,
sometimes several cells thick, is present
on the tissue surface throughout the host’s
life. The lactobacilli are shed from the colonized surface and can be detected throughout the remainder of the animal’s digestive tract (Tannock, 1990).
Specificity of colonization. There are
several indications in the literature that the
colonization ability of lactobacilli is host
specific (Lin and Savage, 1984; Tannock
and Archibald, 1984; Conway and
Kjelleberger, 1989; Gusils et al., 2002).
The host specificity of epithelium-associating strains has been demonstrated in both
in vitro and in vivo experiments (Fuller,
1973; Suegara et al., 1975; Kotarski and
Savage, 1979; Wesney and Tannock, 1979;
Barrow et al., 1980; Fuller and Brooker,
1980; Tannock et. al., 1982). Mitsuoka
(1969) observed differences between the
adhesion characteristics of L. acidophilus strains isolated from man and animals.
Morishita et al. (1971) found that L. acidophilus isolated from a human IT could
not be implanted in the IT of a chick.
Gilliland et al. (1980) compared two L.
acidophilus strains for their use as a dietary adjunct for young calves. They found
that the L. acidophilus strain of calf origin was more effective as a dietary adjunct in calf than the strain of human origin. According to Tannock (1990), several
factors are likely to be involved in this phenomenon:
• Lactobacilli isolated from fowl adhere only to
crop epithelial cells, not to cells from the rat forestomach in in vitro experiments in which the bacteria have been cultivated in a standard laboratory
medium. Rat isolates of lactobacilli cultured in the
same standard medium adhere only to epithelial
cells from rats. Bacterial strains from different hosts
cultured under the same physiological conditions
thus exhibit host specificity, which suggests that
interactions occur between specific adhesins and
receptors on bacterial and host cells.
• The stratified, squamous epithelia lining the
proximal digestive tracts of fowl, pigs, and rodents
differ in chemical composition. The murine forestomach, for example, has a keratinized epithelium, whereas the chicken crop does not. Lactobacilli may have to be nutritionally adapted to inhabit certain types of epithelial surfaces.
• Physiological conditions, including the nature of the diet, may influence the colonization of
epithelial surfaces by lactobacilli. Although clearly
marked differences in diet affect the composition
of the normal microflora of the gastrointestinal tract,
investigation of subtle physiological differences on
intestinal microbes has proved technologically difficult.
This indicates that a Lactobacillus
strain isolated from indigenous microflora
of one animal species will not necessarily
colonize the same site in another animal
species. Beside the animal species specificity for adhesion, one can also recognize
strain specificity within the bacterial species. Barrow et al. (1980) demonstrated
that the degree of adherence to squamous
epithelial cells of the stomach of pigs was
different for various strains within the same
species of lactobacilli. This may be caused
by the fact that some Lactobacillus spp.
do not consist of genetically homologous
group. For example, according to DNAhybridization techniques, L. acidophilus
forms a heterogeneous group of bacteria
(Johnson et al., 1987). Host-species specificity of the intestinal microflora has also
Role of Lactobacilli in Gastrointestinal Ecosystem
71
become clear from characterization studies (differences in biotypes, serotypes and
plasmid patterns). In a study by Convay
et al. (1987), the tested lactic acid bacteria showed comparable adhesion patterns
for human and pig ileal cells. On the other
hand, it is not surprising that MayraMakinen et al. (1983) showed that lactobacilli from plant and cultured milk and
cheese did not adhere in vitro to epithelial
cells of pigs and calves.
Although many strains of lactobacilli do
not associate with epithelial surfaces and
many animal species do not demonstrate
the phenomenon of epithelial colonization
by microbes, the tissue-associating lactobacilli provide a useful focus for further
discussion of the microecology of gastrointestinal lactobacilli.
surfaces by adsorption to gastric epithelial cells (Fuller, 1973; Savage, 1979;
Kotarski and Savage, 1979). This physical association with epithelial surfaces is
an important criterion in determining the
ability of the organism to colonize the gut.
Many investigations have reported the
presence of a surface layer in certain lactobacilli exterior to the cell wall and its involvement in the adherence of these organisms to the gastrointestinal tract
(Brooker and Fuller, 1975; Masuda and
Kawata, 1980, 1981; Hood and Zottola,
1987). Subsequent research has shown
that this layer is composed of protein submits that bind to the neutral polysaccharide moiety of the wall, other than peptidoglycan or teichoic acid, probably through
H-bonding (Masuda and Kawata, 1980,
1981; Sleytr and Messner, 1983; Hood and
Zottola, 1987). Being on the surface, these
proteins are presumably directly involved
in the interactions between the cell and its
environment.
Bhowmik et al. (1985) demonstrated
the presence of a surface protein layer in
some L. acidophilus strains and partially
characterized it. The surface protein was
resistant to hydrolysis by several proteolytic enzymes, suggesting that the layer
may have a protective function. L. acidophilus exhibited in high degree of hydrophobicity, and this property was depending on the surface protein. Therefore, they
theorized that the surface layer might have
a role in the attachment of these organisms to the intestinal mucosa. Schneitz et
al. (1993) observed that the surface layer
formed the outermost part of the cell wall
in strongly adherent L. acidophilus
strains, whereas this layer was covered
with polymerized material or was absent
in strains that lacked the ability to adhere
or those with reduced adherence.
Mechanisms of colonization. In recent
years there has been increasing interest
in the phenomenon of microbial attachment to surfaces as an ecological determinant (Tannock, 1990; Salminen et al.,
1996). Adhesion of lactobacilli to epithelial surfaces in the beginning of the colonization process for some strains of lactobacilli in the GIT. Anchored to the surface of an epithelium, these lactobacilli are
able to resist the flushing action of the relatively rapid flow of digest passing through
the proximal digestive tract. Successful
colonization by any strain of lactobacillus,
however, depends on the metabolic ability
of the microbe to function in the GE
(Tannock, 1990, 2002).
Adhesion can be non-specific, based
on physicochemical factors, or specific involving adhesin molecules on the surfaces
of adhesive bacteria and receptor molecules on epithelial cells (Salminen et al.,
1996). Earlier studies using animal systems
shown that lactobacilli colonize intestinal
72
Fuller and Brooker (1980) suggested
that the adherence of lactobacilli to epithelial cells is a multistage process from
the initial phase of electrostatic attraction
to the formation of fibrils and microcapsules. They theorized that the outermost
surface layer is a potential mediator of the
initial steps in adherence. Further, adherence is not only a mechanical tie between
epithelial cells and bacteria but are also
an active process that probably elicits a
metabolic response in both (Hoepelman
and Tuomanen, 1992).
Coconnier et al. (1992) proposed a
model for the adherence of L. acidophilus BG2 F04 to human intestinal cells. The
mechanism involves two factors: the bacterial cell-wall factor and the adhesion-promoting factor. The bacterial cell-wall factor is polysaccharide in nature and is responsible for the interaction between the
bacterium and the extracellular adhesionpromoting factor. Earlier, Brooker and
Fuller (1975) had also reported that the
surface layer was rich in carbohydrates.
Later, Hood and Zottola (1987) observed
that the adherence was mediated by a
polysaccharide located at the bacterial surface. The second factor, adhesion-promoting factor, is an extracellular proteinaceous
component produced by adhering lactobacilli and provides a divalent bridge that links
the bacteria to the intestinal enterocyte.
This classic work has provided conclusive
evidence about the factors involved in the
adherence of lactobacilli to the IT and their
chemical nature. For more information
about adhesion ability of lactobacilli see
Tuomola and Salminen (1998); Tuomola
et al. (2001); Tannock (2002); Gusils et
al. (2002).
S. A. Denev
FUNCTIONS OF LACTOBACILLI IN THE
GASTROINTESTINAL TRACT
The activities of LAB, especially lactobacilli in the GT have interested microbiologists for at least 90 years, since the
time of Metchnikoff (1903, 1907). Since
then, there has been much interest and
research activity focused on the functions
of lactobacilli in the GIT, and on their potential health and nutritional benefits. Several reviews and books related to this topic
have been published (Gilliland, 1979, 1990;
Sandine, 1979; Shahani and Ayebo, 1980,
Nakazawa and Hosono, 1992; Mital and
Gard, 1995; Tonila, 1996); Tannock, 1995,
2002). The most important nutritional and
therapeutic effects of lactobacilli (Gurr,
1977; Sandine, 1979; Shahani and Auebo,
1980; Fernandes et al., 1987; Gilliland,
1990; Mital and Garg, 1995; Denev, 1996,
1997; Denev et al., 2000; Tannock, 2002)
can be summarized as follows:
• Stimulation of the overall metabolism by producing vitamins (e.g., folic acid) and enzymes (e.g.
lactase);
• Stabilization of the intestinal microflora, excluding colonization by pathogenic bacteria such
as Staphylococcus aureus, Salmonella spp., Shigella spp. and enteropathogenic E. coli strains, via
adhesion to the intestinal wall and competition for
nutrients;
• Control of growth of undesirable organisms
in the intestinal tract and protection against intestinal infections by production of antibacterial substances and competitive exclusion;
• Deconjugation of bile acids;
• Reduction of the cholesterol level in blood
serum by cholesterol assimilation, bile salt hydrolysis and/or modulation of the ratio of high-density
to low-density lipoproteins/cholesterol;
Role of Lactobacilli in Gastrointestinal Ecosystem
73
• Decreased risk of colon cancer by detoxification of toxic, mutagenic and carcinogenic compounds and modulation of fecal procarcinogenic
enzymes such as b-glucuronidase, azoreductase and
nitroreductase;
• Tumor suppression via aspecific stimulation
of the immune system.
et al., 1989), E. coli, Salmonella spp.
(Drago et al., 1997), and others (Coconnier
et al., 1997).
Gilliland and Speck, 1977 were among
the first investigators reporting on antibacterial activity associated with Lactobacillus spp. Numerous reviews and books addressing this field have recently been published (McCormick and Savage, 1983;
Barefoot and Klaenhammer, 1983, 1984;
Reddy et al., 1984; Joerger and
Klaenhammer, 1986; Muriana and
Klaenhammer, 1987; Wong and Chen,
1988; Daeschel, 1989; Geis, 1989;
Klaenhammer, 1988, 1993; James et al.,
1992; Juven et al., 1992; Piard and
Desmazeaud, 1992; Ray and Daeschel,
1992; Hoover and Steenson, 1993; Nettles
and Barefoot, 1993; Sudirman et al., 1993;
Benoit et al., 1994; De Vuyst and
Vandamine, 1994; Dodd and Gasson, 1994;
Desmazeaud, 1994; Larpent, 1994;
Kanatani et al., 1995; De Vuyst et al.,
1996; Tahara et al., 1996; Takatoshi et al.,
1996; Thanaruttikannont and Rengpipat,
1996; Van der Vossen et al., 1996;
Ouwehand, 1998; Guilliams,1999; Denev,
1996, 1997; Denev et al., 2000; Dunne et
al., 2001). This antibacterial activity may
result not only from decreasing the pH and
redox potential, competition for nutrients
and adhesion sites, but also because of the
formation of specific antimicrobial compounds against Gram-negative and Grampositive organisms.
The intestinal lactobacilli play a major
role in the regulation of bacterial population and the control of various enteropathogens in the GE. They suppress the
multiplication of the pathogenic and putrefactive bacteria that cause intestinal problems, diarrhea and digestive upsets, etc.,
and contribute to the maintenance of
health. Lactobacilli are consumed by hu-
Control of Growth of Undesirable
Organisms in the Intestinal Tract
Antagonistic Effect
It is well known that the presence of
lactobacilli is important for the maintenance of the intestinal microbial ecosystem (Denev et al., 2000). The potential
control of intestinal pathogens by LAB has
received much attention for the past 90
years. Since Metchnikoff (1903, 1907)
proposed a role of lactobacilli in suppressing undesirable intestinal microflora, numerous researchers have investigated the
antagonistic activities of lactobacilli (reviewed by Lindgren and Dobrogosz, 1990;
Klaenhammer, 1988, 1993; Boris et al.,
2001; Bird et al., 2002).
Several investigations have shown that
Lactobacillus spp. exerts antagonistic action against intestinal and food-borne
pathogens and other related organisms
(Grossowics et. al, 1947; Wager, 1948;
Wheater et al., 1951, 1952; Polonskaya,
1952; Vincent et al., 1959. De Klerrk and
Coetzee, 1961; Tramer, 1966; Reddy and
Shahani, 1971; Hamdan and Mikolajcik,
1973; Mikolajcik and Hamdan, 1975; Tagg
et al., 1976; Shahani et al., 1976, 1977;
Lascar, 1995; Singh et al., 1995; Denev et
al., 2000; Bird et al., 2002). They have
been shown to possess inhibitory activity
toward the growth of pathogenic bacteria
such as Listeria monocytogenes (Harris
74
mans and fed to animals of commercial
value in efforts to control a variety of diseases and to maintain a balanced microflora by reduction of potential pathogens
residing in the gut.
It is not the intent of this paper to review all of the literature on control of intestinal pathogens by lactobacilli however
a few examples will be given. Certain Lactobacillus strains inhibit E.coli adhesion
to porcine enterocytes and attachment to
porcine mucus (Ouwehand and Conway,
1996). Furthemore, metabolic end products of lactobacilli present in cell-free culture supernatant fluids inhibit adhesion or
invasion by pathogenic bacteria. Probiotic
Lactobacillus strains also inhibit mucosal
adherence of EPEC and S. typhimurium
in vitro (Bernet et al., 1994). In addition
to prevent colonization by pathogens, Lactobacilli may strengthen the epithelial barrier, thereby preventing pathogenic translocation of the epithelium by promoting accelerated epithelial repair. For example,
oral administration of Lactobacillus acidophilus can ameliorate diarrhea in patients undergoing pelvic radiotherapy, perhaps by preventing radiation-induced damage to the intestinal epithelium (Kaila et
al., 1995). Furthermore, enhanced macromolecule intestinal permeability and
translocation of intact proteins induced by
cow milk was reversed in suckling rats fed
milk supplemented with L. casei GG
(Salminen et al., 1988).
The exact mechanism whereby lactobacilli may inhibit intestinal pathogens is
not clear. Antagonistic factors that may
contribute to colonization and continuous
presence of lactobacilli in the digestive
tract of animals are the presence of specific antimicrobial compounds. The antagonism could be due to the production of
inhibitory compounds such as organic ac-
S. A. Denev
ids, hydrogen peroxide, bacteriocins and
broad-range antibiotic-like substances, or
to competitive adhesion to the epithelium
and etc.
Bacteriocins and bacteriocin-like
substances. The discovery of bacteriocins dates back to 1925, when Gratia observed the inhibition of E. coli by E. coli
V. The antibacterial substances produced
by E. coli were named colicins (Fredericq,
1948). The first report of production of antagonistic substances other than metabolic
end products by LAB was made in 1928
by Rogers. He observed antagonistic activity for L. lactis subsp. lactis against L.
delbrueckii subsp. bulgaricus. The substance was determined to be a polypeptide (Whitehead, 1933) and subsequently
named nisin (Mattick and Hirsch, 1947).
De Klerk and Coetzee (1961) first isolated
bactericidal substances from 11 homofermentative strains and one heterofermentative strain of Lactobacillus, that were
only active against other Lactobacillus
spp. and thus could be defined as bacteriocins. Among the Gram-positive bacteria, the lactobacilli in particular produce a
wide variety of antimicrobial proteins including peptide antibiotics, antibiotic-like
substances, bacteriocins and bacteriocinlike substances.
Bacteriocins, as defined by Tagg et al.
(1976), are proteinaceous compounds produced by bacteria that have bactericidal
activity against closely related species.
They form a heterogeneous group with
respect to producing bacterial species,
molecular size, physical and chemical properties, stability, antimicrobial spectrum,
mode of action, and so on.
Bacteriocins of LAB have been the
subject of many studies over the past 10
years (De Vuyst and Vandamine, 1994;
Role of Lactobacilli in Gastrointestinal Ecosystem
75
De Vuyst, 1994, 1996; Cenatiempo et al.,
1996). Investigations on bacteriocins have
historically focused on four general areas
that include: bacteriocin expression, production and mode of action; characterization of plasmids encoding bacteriocin production and immunity and their use as vehicles for gene transfer; identification and
description of novel bacteriocins produced
by bacteria not previously recognized as
bacteriocinogenic; and taxonomic characterization of bacteria based on bacteriocin
production or susceptibility (Klaenhammer,
1988).
Since the late 1920s and early 1930s,
when the discovery of nisin initiated the
investigation of proteinaceous antimicrobial compounds from LAB, a large number of chemically diverse bacteriocins have
been identified and characterized, particularly in recent years. Nonetheless, we can
observe common traits that justify their
classification into just a few classes
(Klaenhammer, 1993). On a sound scientific basis, three defined classes of bacteriocins in LAB have been established,
class I: the lantibiotics; class II: the small
heat-stable non-lantibiotics and class III:
large heat-labile bacteriocins (Nes et al.,
1996). In the past decades a large number of reports have been published describing Lactobacillus spp. that is claimed to
produce bacteriocin and bacteriocin-like
components. A selection is listed in Table 3.
Bacteriocins and bacteriocin-like compounds play an important role in controlling the composition of indigenous microflora and the regulation of population dynamics in various bacterial ecosystems.
Since, bacteriocins usually are only active
against organisms closely related to the
producer, their role in inhibiting intestinal
pathogens may be limited. Bacteriocins of
Gram-positive organisms usually possess
a somewhat broader inhibitory spectrum
and are also active against other Grampositive species. However, they may be
very important in enabling a selected strain
of Lactobacillus in competing with other
LAB in the intestines. Some bacteriocins
as acidolin, acidophilin, bulgarican and
reuterin have been reported to possess a
much broader inhibitory spectrum: many
Gram-positive and Gram-negative bacteria were said to be inhibited by the purified products (Shahani et al.,1977; Fernandes et al., 1987; Talarico et al., 1988;
Axelsson, 1989; Chung et al., 1989; Talarico and Dobrogosz, 1989). Certainly these
types of bacteriocins may be very effective in helping to control the growth of intestinal pathogens (Gilliland, 1990). For
more information about general characteristics, mechanisms of action, genetics
and biosynthesis of bacteriocins see Fuller
(1992), Klaenhammer (1993), De Vuyst
and Vandamme (1994), Mishra and Lambert (1996), Nes et al. (1996), Tannock
(2002).
Recently, bacteriocins have been studied intensively from every possible scientific angle: microbiology, biochemistry, molecular biology and food technology.
Knowledge, especially about bacteriocins
from lactobacilli, is accumulating very rapidly. Future investigations of bacteriocins
have provided new information in each of
these areas.
Other antagonistic compounds. Antagonism by lactobacilli has also been associated with major end products of their
metabolism. Several by-products of lactobacillus metabolism are capable of antagonism in vitro and in vivo. The best
knows of these compounds are lactic and
acetic acids, hydrogen peroxide, diacetyl
and etc.
Organic acids such as lactic and ace-
76
S. A. Denev
Table 3
Bacteriocins and bacteriocin-like substances of some intestinal Lactobacillus species
Species
Bacteriocin
References
L. acidophilus
Lactocidin
Acidophilin
Acidolin
Lactacin B
Lactacin F
Vincent et al. (1959)
Vakil and Shahani (1965)
Hamdan and Mikolajcik (1973)
Barefoot and Klaenhammer (1983)
Muriana and Klaenhammer(1987, 1991)
Klaenhammer et al. (1993)
Bacteriocin M46
Acidophilucin A
Acidocin A
Ten Brink et al. (1990)
Toba et al. (1991)
Kanatani et al. (1992, 1995)
L. brevis
Lactobrevin
Brevicin
Kavasnikov and Sudenko (1967)
Rammelsberg and Radler (1990)
L. casei
Caseicin 80
Rammelsberg et al. (1990)
Disks et al. (1992)
L. delbrueckii subsp. lactis UO004 Bacteriocin UO004
Boris et al. (2001)
L. fermentum
Bacteriocin 466
De Klerk and Coetzee (1961)
L. gasseri
Gassericin A
Toba et al. (1991a)
Takatoshi et al. (1996)
L. plantarum
Lactolin
Plantacin A
Plantacin B
Plantacin S
Plantacin D
Plantacin T
Plantacin Y
Kodama (1952)
Daeschel et al. (1990)
West and Warner (1988)
Jumenez-Diaz et al. (1993)
Aymerich et al. (2000)
Jumenez-Diaz et al. (1993)
Chin et al. (2001)
L. reuteri
Reutericin 6
Toba et al. (1991b)
L. salivarius
Salivaricin B
Ten Bring and Holo (1996)
tic which are responsible for significant pH
changes in gut-sufficient to antagonize
many microorganisms, including pathogenic Gram-negative organisms (Adams
and Hall, 1988). While pH per se is a factor in such inhibitions, lower pH values also
potentate the activities of these acids because undissociated forms are more bactericidal than dissociated ones. This phe-
nomenon may be attributed to the ability
of undissociated acids to penetrate the
bacterial cell (Kashet, 1987). Further, the
volatile acids are especially antimicrobial
under the low oxidation-reduction potentials that lactobacilli help maintain in the
intestine (Goepfert and Hicks, 1969;
Tannock, 2002). Organic acids decrease
or kill harmful bacteria, and as a result sup-
Role of Lactobacilli in Gastrointestinal Ecosystem
77
press the production of harmful substances
such as amines, phenols, indole, scatole
and hydrogen sulphide which are produced
by these harmful bacteria. Because many
of these harmful bacteria have a neutral
optimum pH and are not acid-tolerant, the
lowering of pH by organic acid metabolites of lactobacilli, such as lactic acid, has
a bacteriostatic or bactericidal effect on
these acid-sensitive bacteria (Fuller, 1992).
Hydrogen peroxide (H2O2) is one of
the primary metabolites produced by lactobacilli. The lactobacilli have the ability
to generate hydrogen peroxide during
growth by several different mechanisms
(Table 4.).
The accumulation of hydrogen peroxide in growth media is shown in occurs
because lactobacilli do not possess the
catalase enzyme (Kandler and Weiss,
1986). The antimicrobial activity of hydrogen peroxide is well recognized and documented. This compound is cytotoxic because of its capacity to generate reactive
cytotoxic radicals that are strongly oxidative (Halliwell, 1978). Lactobacilli usually
produce hydrogen peroxide by direct reduction of oxygen. Aside from the generation of hydroxyl radicals, the in vivo
bactericidal effects of hydrogen peroxide
may be relevant to the activation of a peroxidase-hydrogen peroxide system, commonly referred to as lactoperoxidase system. A well-studied lactoperoxidase system is described in milk, where bactericidal activity against enteric Gram-negative bacteria is associated with thiocyanate (Reiter and Harnulv, 1984). This
mechanism may also occur in the intestines (Retire et al., 1980; Fervidness et al.,
1987). Lactobacilli stimulated the lactoperoxidase thiocyanate system in the intestines, which reduced the ability of E. coli
and other enteropathogens to survive in
the gut (Fernandes and Shahani, 1989).
Hydrogen peroxide production by lactobacilli will usually occur by direct reduction of atmospheric oxygen catalysed by
Table 4
Substrates and reactions that might lead to production of H2O2 by Lactobacilli
Substrates, reaction
Catalyzed by
End Products
References
Gotz et al. (1980)
Acetyl phosphate +
Murphy and Cobdon
CO2 + H2O2
(1984)
Pyruvate + O2 + phosphate
Pyruvate oxidase
Lactate + O2
L-lactate oxidase
Pyruvate + H2O2
Kandler (1983)
Lactate + O2
NAD-independent
D-lactate dehydrogenase
Pyruvate + H2O2
Sedewitz et al. (1983)
Fatty acyl- CoA
H2O2
De Kairuz et al. (1988)
Saturated fatty acids + O2
α-Glycerophosphate + O2
α -Glycerophosphate
oxidase
Dihydroxyacetone
Condon (1987)
phosphate + H2O2
78
either pyruvate-, NADH, alpha-glycerophosphate-oxidase (Condon, 1987) or lactate-oxidase. In phosphate buffer with glucose and pyruvate added, resting cell of a
strain of L. acidophilus, isolated from the
chicken GT, produced 280 nmol hydrogen
peroxide per hour per milligram of cell dry
weight. Concentrations of oxygen high
enough for significant amounts of hydrogen peroxide to be produced are likely to
exist only in the upper portions of the GIT
of live animals. That might be one of the
explanations for the antagonistic effects
of lactobacilli towards salmonellas observed in the crop of chicks whereas none
or very limited antagonism was found in
their caeca (Juven et al., 1991). For more
information abou antimicrobial compounds
produced by lactobacilli and their structures and properties see Yang (2000).
COMPETITIVE EXCLUSION OF PATHOGENIC BACTERIA BY LACTOBACILLI
One of the physiological effects of lactobacilli in the host has proposed to be the
antagonism against pathogens. Competitive exclusion (CE) by the lactobacilli is
another mechanism that has been suggested as being important in controlling intestinal infections (Watkins and Miller,
1983a; Edens et al., 1997; Edelman, 2005).
CE involves the ability of the lactobacilli
to occupy binding sites on the intestinal
wall, thereby preventing attachment and
growth of enteric pathogens.
Nurmi and Rantala (1973) first recognized the importance of early establishment
of protective microflora in young chicks.
Originally, these workers treated chicks
with a suspension of crop and intestinal
tract materials obtained from adult birds;
in subsequent studies, cecal content or feces cultures anaerobically in a liquid me-
S. A. Denev
dium were used for treatment. These
preparations of unknown bacterial composition were administered orally to dayold chicks, which were challenged 24 or
48 h later with a standardized dose of Salmonella. Results showed that such treated
chicks were more resistant to infection
than control birds. This prophylactic approach, known as the “Nurmi concept” or
“competitive exclusion”, opened new horizons for the control of Salmonella in
poultry.
The advent of CE technologies for the
control of pathogenic enteric bacteria in
poultry as well as humans has received
considerable interest during the past two
decades. Intensive efforts by groups of
USDA scientists (Bailey, 1988; Ziprin et
al. 1991; Corrier et al., 1994, 1995a, 1995b;
Hollister et al., 1995) signal the importance
of this technology and its application to the
poultry industry. The mechanisms used by
one species of bacteria to exclude or reduce the growth of another species are
varied, but Rolfe (1991) determined that
there are at least four major mechanisms
of CE. These mechanisms are: 1) creation
of microecology that is hostile to other bacterial species; 2) elimination of available
bacterial receptor sites; 3) production and
secretion of antimicrobial metabolites, and
4) selective and competitive depletion of
essential nutrients.
The effect of CE lactobacillus cultures
on the growth of undesirable organisms in
the IT has been well documented the last
decade. Watkins et al. (1979, 1982) reported that broiler chicks inoculated with
L. acidophilus were more resistant to the
pathogenic effect of E. coli. The addition
of L. acidophilus acidified the crop,
cecum and colon of the inoculated chicks,
and this acidification appeared to increase
the competitiveness of the L. acidophi-
Role of Lactobacilli in Gastrointestinal Ecosystem
79
lus against the other intestinal microflora.
Watkins and Miller (1983a) suggested that
L. acidophilus increases competitive gut
exclusion against harmful organisms (S.
typhimurium, Staphylococcus aureus,
and E. coli) in the intestinal tract. Watkins
and Miller (1983b) demonstrated that L.
acidophilus colonized the epithelial cells
of the GIT through a close relationship and
through actual physical attachment.
With the discovery that L. reuteri is a
major component of the intestinal microflora of humans (Gibson et al., 1997),
chicks and other animals (Sarra et al.,
1985; Dobrogosz et al., 1991; Edens et al.,
1993; 1997) is appeared that the population dynamics of L. reuteri might be involved in establishing a favorable microecology in the IT via its ability to secrete
lactic and acetic acid, and reuterin, which
act as modulators of intestinal tract microbial populations (Dobrogosz et al.,
1991).
According to Soerjadi et al. (1981a,
1981b) members of the Lactobacillaceae
family are responsible for CE of pathogens from the gut of chicks. Schneitz et
al. (1993) demonstrated the importance of
lactobacilli with the ability to adhere as CE
microorganisms in poultry. Mulder et al.,
(1997) also reported that Lactobacillus
species are very important in establishing
a stable GIM which is able to prevent colonization of potentially pathogenic microorganisms. Therefore, the use of probiotics
and other CE products containing intestinal Lactobacillus spp. may be also exert
a positive effect against this colonization.
Lactobacilli have been shown to affect
the prevalence of several intestinal pathogens in vitro and in vivo (Coconnier et
al., 1998; Jin et al., 1996; Pascual et al.,
1999; Mack et al., 1999; La Ragione et
al., 2004). The detailed molecular mecha-
nisms underlying this phenomenon remain
poorly characterized, and the reports vary
in degrees of successful inhibition depending on the strains used as CE agents, the
pathogens as well as the methods of assessment. The importance of adhesion in
pathogen exclusion at chicken ileal epithelia
has been shown in vitro with L. acidophilus against Salmonella pullorum (Jin
et al., 1996) but L. acidophilus failed to
exclude Salmonella enterica serovar
Enteritidis and Salmonella enterica
serovar typhimurium and APEC (Jin et
al., 1996, 1998). Also, exclusion of E. coli
and salmonalla by lactobacilli in human and
piglet mucus have been reported (Lee et
al., 2003), but lactobacilli failed to inhibit
the adhesion of Salmonella ssp. to chicken
mucus (Gusils et al., 2003). L. crispatus
and its collagen binding S-layer protein inhibit the adherence of E. coli to the components of basement membrane (Horie et
al., 2002) and a 29-kDa surface protein
from L. fermentum inhibited adhesion of
uropathogenic Enterococcus faecalis to
polystyrene (Heinemann et al., 2000). For
more information about CE properties of
lactobacilli see (Edens et al., 1997; Mulder
et al., 1997; Edelman, 2005). Improving
our understanding of the development and
the regulatory mechanisms of the lactobacillus flora in GE is essential for the development of effective CE products for
humans and animals.
NEUTRALIZATION OF TOXINS AND
OTHER INTESTINAL REACTIONS
Among the intestinal bacteria there
are some which act on food components
and digestive secretions like bile acids to
produce harmful putrefactive products
such as enterotoxins, ammonia, amines,
phenols, indole and hydrogen sulphide, or
80
other toxic and carcinogenic substances.
If these harmful metabolites are produced
in large quantities, they promote a decrease
in liver function and the development of
cancer.
Intestinal lactobacilli have been shown
to inhibit the growth of pathogenic bacteria, decrease the metabolic activity of
harmful bacteria, including production of
enzymes which contribute to the production of toxic and cancer-related substances
(Mitsuoka, 1984; Honma, 1986). Intestinal lactobacilli can also suppress the production (Honma et al., 1987) and breakdown of nitrozamines, which have been
shown to be connected with the development of stomach cancer and cancer of the
small intestine (Kanbe, 1992).
Investigations of L. bulgaricus in pigs
showed that the organism produces a metabolite thought to neutralize the effect of
enterotoxin released from coliforms. Piglets fed a L. bulgaricus culture, rendered
non-viable with lactic acid, and artificially
infected with E. coli, grew faster and suffered less diarrhea compared with control
animals. These studies showed that some
lactobacilli especially L. bulgaricus was
able to neutralize E. coli enterotoxin
(Mitchel and Kenworthy,1976). Although
the neutralizing substance has yet to be
identified, further support for antienterotoxic activity has also been obtained
from experiments with rats and calves. Cell
free extracts of L. casei and L. acidophilus have also been shown in vitro to
inhibit the growth of E. coli. Scheline
(1972) tested a number of different substrates and intestinal microorganisms to
determine the type of reactions specific
Lactobacillus species catalyze. In the
study cited above, these investigators found
that a Lactobacillus species could reduce
the double bond in hydroxycinnamic acid,
S. A. Denev
reduce the nitro group of 4-nitrobenzoic
acid and reduce the azo bond found in
methyl red and acid yellow. In more recent publication Pradham and Majumdar
(1986) reported that L. acidophilus
cleaves the azo bond of sulfasalazine, also
known as azulfidine, a drug used to treat
patients with ulcerative colitis. These investigators also found that L. acidophilus degraded 17.6% of the antimicrobial
agent phthalylsulfathiazole and 8% of the
antibiotic chloramphenocol palmitate.
These investigators also confirmed the
previous study and demonstrated that L.
acidophilus could rapidly hydrolyze the
azo bound of tartrazine and methyl red.
L. helveticus and L. salivarius also had
similar but lower activity when compared
to L. acidophilus. Lactobacilli have also
been shown to reduce the double bond of
3-hydroxy cinnamic acid and cinnamic acid
(Whiting and Carr, 1970) to produce respectively 3-hydroxy phenylproponic acid
and phenylproponic acid. A Lactobacillus species has been shown to be capable
of amino acids (Melnoykowycz and
Johansson, 1955). A L. acidophilus isolates from the stomach of rat was shown
to exhibit histidine decarboxylase activity
(Horakova et al., 1971).
Nitrosamines have been shown to be
potent carcinogens (Magee, 1971). The nitrosamines found in foods are dimethylnitrosarnine, diethylnitrosamine, Nnitrosopyrrolidine, and N-nitro-sopiperidine. These are formed when nitrites
react with secondary amines in an acidic
medium (Hawksworth and Hill, 1971).
Nitrites are derived from nitrates, which
are found in green plants, vegetables, cured
meats, and some cheese products (Ender
and Ceh, 1968). Nitrates may also be
present in drinking water in higher concentrations (Correa et al., 1970). A rela-
Role of Lactobacilli in Gastrointestinal Ecosystem
81
tionship between gastric cancer and high
nitrate intake has also been suggested
(Correa et al., 1970). Lactobacillus species could degrade nitrosamines and thus
mitigate their toxical and carcinogenic effect (Rowland and Grasso, 1975; Kanbe,
1992).
Some lactobacilli (L. acidophilus)
have been shown to suppress the production of harmful substances such as ammonia, indole and hydrogen sulfide, which
are hazardious to the body. This suppressing effect may help to decrease the amount
of toxins going to the liver and injuring it
(Yamamoto et al., 1986; Yamashita et al.,
1987).
Lactobacilli reduced activities of fecal
bacterial enzyme b-glucuronidase, nitroreductase, and azoreductase (Goldin and
Gorbach, 1984) that are involved in procarcinogen activation and reduced excretion of mutagens in feces and urine
(Lidbeck et al., 1992).
These studies demonstrated the potential of lactobacilli as toxin neutralizers, inhibitors of cancer promoting enzyme activity, destructors of fecal nitrosamines and
other drugs, harmful, mutagenic and carcinogenic substances in the GIT.
spread among members of the autochthonous gastrointestinal microflora (Hayakawa, 1973; Midtvedt, 1974), including species of the genera Bacteroides (Masuda,
1981), Bifidobacterium (Ferrari et al.,
1980; Grill et al., 1995), Clostridium
(Masuda, 1981), Lactobacillus (Gilliland
and Speck, 1977a; Christiaens et al, 1992;
Tannock et al., 1994), Fusobacterium
(Shimada et al., 1969), Streptococcus
(Kobashi et al., 1978) and others species
(Midtedt, 1974). The reaction is catalyzed
by the enzyme bile salt hydrolase (BSH)
(Lundeen and Savage, 1990).
BSH catalyze the cleavage of an amino
acid from the steroid nucleus of conjugated
bile salts. It is not clear why lactobacilli
produce an enzyme with this property, because they would not gain energetically
from the deconjugation process, but it may
be an essential property enabling the bacteria to survive transit through the small
bowel, into which relatively high concentrations of conjugated bile acids are released (De Boever and Verstraete. 1999).
The deconjugating activity of the lactobacilli could be important to the host, because
deconjugated bile salts are less effective
in emulsification of dietary lipids and micelle formation. Thus, the BSH activity of
lactobacilli in the small bowel could impair
lipid digestion and absorption by the host
and could have implications in the poultry
and pig industries, where rapid growth and
efficient feed conversion are required for
profitability (Tannock, 2004).
BSH has been purified from three organisms, Bacteroides fragilis (Stellwag
and Hylemon, 1976), Clostridium
perfringens (Gopal-Srivastava and
Hylemon, 1988; Coleman and Hudson,
1995) and Lactobacillus ssp. (Lundeen and
Savage, 1990, 1992). Lactobacilli have
been shown to be the predominant pro-
DECONJUGATION OF BILE ACIDS
Bile acids are synthesized from cholesterol in the liver, conjugated to either
glycine or taurine, and then released into
the intestines, where they facilitate fat absorption by the epithelial cells (Hofmann
and Mehjian, 1973). These acids are
deconjugated, dehydroxylated, dehydrogenated, and desulfated in the intestines by
microbial enzymes (Hylemon and Glass,
1983). The capacity to deconjugate bile
acids (hydrolyze the amide bond between
the steroid nucleus and amino acid) is wide-
82
ducers of BSH activity in the mouse gut.
Gastric lactobacilli contribute approximately 86% of the total BSH activity and
the 74% in the cecum of mice (Tannock
et al., 1989). However, the enzymes catalyzing the reaction in these bacteria have
not been extensively studied (Tannock,
2004). Hill and Drasar (1968) and Aries
et al. (1969) reported that Lactobacillus
ssp. isolated from human feces were unable to deconjugate taurocholic acid.
Midtvedt and Norman (1968) reported that
L. arabinosus deconjugated both taurocholic and glycocholic acids, whereas
L. brevis deconjugated only glycocholic
acid. L. acidophilus,L casei and L.
delbrueckii did not deconjugate either of
the two bile acids. Floch et al. (1972) isolated Lactobacillus species from human
feces, which deconjugated glycocholic,
glycodeoxycholic, and glycochenodeoxycholic acids. The species of lactobacillus were not identified.
Gilliland and Speck (1977a) examined
a number of lactobacilli from human feces for their ability to deconjugate bile
acids. They found that all of them possessed this capacity but acted on either a
tauro- or glycoconjugate. However, only
L. buchneri among the lactobacilli tested
acted upon both.
The bile acids excreted in feces are entirely deconjugated. They represent about
5% of the enterohepatically circulated
pool and consist of a variety of secondary bile acids in addition to small quantities of primary bile acids (Mallory et al.,
1973). The deconjugated bile acids exert
a greater inhibitory action against bacteria than conjugated forms (Dunne et al.,
2001).
Deconjugated bile acids, particularly
dehydroxy acids (deoxycholic and chenodeoxycholic acids) inhibit the growth of a
S. A. Denev
variety of intestinal bacteria (Binder et al.,
1975). Further, dihydroxy bile acids are
more inhibitory than trihydroxy one- cholic
acids (Floch et al., 1972). Percy-Robb and
Collee (1972) observed that the in vitro
antibacterial actions of bile acids were
strongly pH dependent. They suggested
that bile acids might be involved in a selfregulatory mechanism that prevented microbial colonization of the upper bowel.
Bacterial metabolism of bile acids has
been postulated to play an important role
in etiology of colon cancer (Hill, 1986). It
is suggested that the secondary bile acids
(i.e. those resulting from bacterial metabolism) can act as promoters of the tumorogenic process in the colon. In addition,
Hill has proposed that dehydrogenation of
the steroid nucleus to produce delta 1 and
delta 4 bonds in association with 3-keto
groups is particularly important in relation
to colon cancer. Certain strains can perform this reaction in vitro although the significance of the reaction in vivo is questionable (Lombardi et al., 1978).
Deconjugated bile acids by lactobacilli
play a significant role in maintaining ecological equilibrium in the intestine (Hoch
et al., 1972; Mital and Garg, 1995;
Tannock, 2004) They are more inhibitory
to bacteria than conjugated ones (PercyRobb and Collee, 1972); therefore,
deconjugation of bile acids may enhance
antagonistic actions of intestinal lactobacilli toward intestinal pathogens, thus helping to maintain a favorable balance among
species present in the intestinal tract. Several reports have been published on this
topic during the last years (Charteris et
al., 1998; Boever and Verstaete, 1999; Grill
et al., 2000; Shinoda et al., 2001; Tannock,
2004). Understanding of the structural,
enzymatic, and regulatory properties of the
hydrolases from lactobacilli will be impor-
Role of Lactobacilli in Gastrointestinal Ecosystem
83
tant to understanding of the role of these
organisms in bile acid transformations in
the GIT.
Much attention has recently been paid
to the phylogeny of the gut microbiota, but
little has been paid to the microbial physiology of complex bacterial communities or
their individual components (Lu et al.
2003). It is time that this imbalance was
rectified. Lactobacilli could provide model
bacteria for such physiological studies because their relationship with the farm animal host (chickens, pigs) is much better
defined than that of other members of the
microbiota.
uct of other intestinal bacteria, a result of
a diet, or a metabolite derived from the
host. Further, it has been shown that cellfree extracts of bacteroides can produce
the mutagen in the presence of bile. The
enzyme system involved is not oxygen
sensitive (Mital and Gard, 1995). However,
the mutagen is only produced anaerobically (Wilkins et al., 1981).
It is well known that carcinogenesis is
initiated by mutation induced in animal and
human cells with carcinogen. Several compounds displaying mutagenic properties
have been identified in numerous foods
(Hosono, 1992; Thyagaraja and Hosono,
1993).According to McCann et al. (1975)
mutagenicity and carcinogenicity show
good correlation.
Recently, attention has been focused
on the biological transformation of substances to carcinogens by intestinal microflora. Studies on the conditions that enhance or decrease the activation or inactivation of environmental mutagens are important for the control of their risks to human (Thyagaraja and Hosono, 1994).
There is evidence that LAB, especially lactobacilli, influence the mutagenicity of intestinal content and the levels of fecal microbial enzymes. The some strain lactobacilli have been shown to significantly decrease mutagenicity in healtly volunteers
consuming fried ground beef. They decreased fecal E.coli levels in colon cancer patients and reduced the fecal enzymes
(Goldin et al., 1992; Ling et al., 1994;
Morotomi, 1996).
Hosono et al. (1986a) were the first to
report that milks fermented with L.delbrueckii subsp. bulgaricus IFO 3533, L.
lactis subsp. lactis IFO 12546 and Enterococcus faecalis IFO 12964 exhibited
antimutagenic activity against 4-nitroquinoline-N-oxide (4NQO). Hosono et
ANTIMUTAGENIC ACTIVITY
Bruce et al. (1977) was the first to report the presence of mutagenic compounds
in human feces. Since then, many researchers have investigated the occurrence and levels of fecal mutagens in different populations (Ehrich et al., 1979;
Reddy et al., 1980; Ferguson et al., 1985).
Mutagenic activity in feces has been reported to be promoted by bile, enhanced
by anaerobiosis, and inhibited by exposure
to oxygen (Lederman et al., 1980; Van
Tassell et al., 1982a).
Studies with pure cultures have shown
that Bacteroides fragilis, B. ovatus, B.
uniformis, and B. thetaiotaomicron are
capable of producing mutagens (Van
Tassell et al., 1982b). The fact that these
organisms are common inhabitants of the
intestinal tract and that only a relatively
small percentage of individuals produce the
mutagen indicates that a precursor is the
determining factor in their production. It
has been demonstrated that bacteria from
feces that do not contain the mutagen are
capable of producing it in the presence of
a precursor. The precursor is either a prod-
84
al.(1986b) have also described the antimutagenic activity of milks fermented
with Lactobacillus delbrueckii subsp.
bul-garicus and Streptococcus thermophilus again all the mutagens.
Nishioka et al. (1989) have reported
that with respect to 49 strains of Lactobacillus three exhibited strong antimutagenic activity against 4NQO and 2-(2furyl)-3-(5-nitro-2- furyl)acryl-amide (AF2).
Hosoda et al. (1996) investigated the
antimutagenic effect of milk fermented with
L.acidophilus. They concluded that L.
acidophilus decreased fecal mutagenicity in the human intestine.
For more information about antimutagenic properties of LAB, including lactobacilli see Hosono (1988, 1992), Renner
and Munzner (1991), Hosoda et al.
(1992a,b, 1996), Thyagaraja et al. (1992),
Thyagaraja and Hosono (1993, 1994),
Aldelali et al. (1995), Hosanna et al. (1990,
1997), Shah (2001), Wollowski et al.
(2001).
HYPOCHOLESTEROLEMIC EFFECT
High-serum cholesterol levels are considered to be a major risk factor for coronary heart disease, and also a factor inducing colon cancer in addition to high dietary fat and low fiber (Pekkanen et al.,
1990; Lichtenstein and Goldin, 1993; Law
et al., 1994). Several reports have indicated that serum cholesterol levels can be
reduced by the consumption of certain cultured dairy products (Suzuki, 1995). In addition, intestinal microflora has been implicated in influencing the serum cholesterol level. Germ-free animals on an elevated cholesterol diet accumulate approximately twice as much cholesterol in
blood as do conventional animals on a simi-
S. A. Denev
lar diet (Eyssen, 1973). The latter excrete
much higher levels of cholesterol in the
feces than germ-free animals. This suggests that the intestinal microflora interfere with the cholesterol absorption from
the intestines.
Several studies have suggested that lactobacilli and especially intestinal strains of
L. acidophilus have the potential of decreasing serum cholesterol levels (Danielson et al., 1989; Walker and Gilliland, 1993;
De Rodas et. al., 1996). However, not all
strains of L. acidophilus were active in
this regard (Gilliland et al., 1985; Lin et
al., 1989). On the other hand L. acidophilus had the ability to assimilate cholesterol in in vitro studies, but only in the presence of bile and under anaerobic conditions.
Gilliland et al. (1985) found that some
strains of L. acidophilus could remove
cholesterol from the laboratory medium
only in the presence of bile and under
anaerobic conditions. Because these conditions also occur in the intestine, they
tested the manifestation of such an action
in vitro using young pigs and L. acidophilus strains of pig origin. The results
showed that the feeding of pigs with L.
acidophilus possessing the ability to grow
in the presence of bile and to remove cholesterol from laboratory medium, significantly inhibited the increase in their serum cholesterol levels on a high cholesterol diet.
The lactobacilli are the primary contributors to bile salt hydrolase activity in
the ileum and cecum of the mouse (Tannock et al., 1989). If strains of Lactobacillu sssp.exhibiting a high degree of bile
salt hydrolase activity could be incorporated into the intestinal tract, it is possible
that the amount of deconjugation activity
in the intestine could be increased.
Role of Lactobacilli in Gastrointestinal Ecosystem
85
Some species of lactobacilli that are
present in the intestinal tract can
deconjugate taurocholic and glycocholic
acids in anaerobic conditions Gilliland and
Speck (1977a). This deconjugation activity is important because deconjugated bile
acids do not function as well as conjugated
bile acids in the intestinal absorption of cholesterol (Eyssen, 1973). Increased
deconjugation of bile acids could also result in greater excretion of bile acids from
the intestinal tract because free bile acids
are less likely to be reabsorbed in the intestines (Chickai et al., 1987). Increased
excretion of bile acids stimulates the synthesis of replacement bile acids from cholesterol, thus providing the potential to reduce cholesterol levels in the body (Gilliland, 1990). The synthesis of bile acids is
homeostatically regulated by the amount
of bile acids returning to the liver (Danielsson and Sjovall, 1975). Also, cholesterol
absorption into the blood from the intestines is not supported as well by deconjugated bile acids as it is by conjugated
bile acids.
According to Walker and Gilliland
(1993) there is no correlation between bile
tolerance and cholesterol uptake ability of
the L. acidophilus.They also have evaluated four strains of L. casei and observed
considerable variation among strains in
their ability to assimilate cholesterol. Additionally they have shown that L.
plantarum and bifidobacteria possess the
ability to assimilate cholesterol. Thus, it
may be possible to use other of intestinal
lactobacilli or related organisms in exerting beneficial influences on serum cholesterol levels. Additional research will be
required to confirm whether or not this is
a possibility.
Imaizumi et al. (1992) found many strain
differences and that which microbial could
draw no clear conclusions on the general
mechanism activity within the gut might
regulate serum cholesterol levels.
Bile tolerance, the ability to deconjugate
bile salts, and the ability to assimilate cholesterol are factors that may affect the
potential of some strains of L. acidophilus to help control human serum cholesterol concentration when used as dietary
adjuncts (Lys and Gilliland, 1994). Future
research is needed to determine the
mechanism of cholesterol uptake and to
determine whether or not the ingestion of
cells of a selected strains of lactobacilli
and especially L. acidophilus could decrease serum cholesterol levels in adult humans with primary hypercholesterolemia.
For more information about hypocholesterolemic effect of LAB, including lactobacilli see Denev et al. (2000), Shah
(2001), Lovegrove and Jackson (2003).
ANTICARCINOGENIC EFFECTS
Since the first observation by Bogdanov
et al. (1962) that L. bulgaricus produced
substances, which were active against tumor development there, have been a number of reports in the same vein. During
the last two decades, several experiments
and reviews indicating that LAB play a
significant role in suppressing carcinogenesis have been published (Sandine, 1979;
Friend et al., 19S2; Reddy et al., 1983;
Deeth, 1984; Friend and Shahani, 1984a;
Gurr, 1987; Fernandes et al., 1987, 1988;
Hosono, 1988a; Kanbe, 1988; Gilliland,
1989; Wada and Hayakawa, 1990;
Guilliams, 1999; Wollowski et al., 2001;
Mercenier et al., 2002).
Epidemiological evidence and dietary
studies have shown that consumption of
dairy products fermented by lactobacilli
may reduce the risk of colon cancer in both
86
animals and humans. Numerous studies
have suggested that intestinal and other
lactobacilli possess anticarcinogenic activity (Mitsuoka, 1981; Goldin and Gorbach,
1980, 1983; Shahani et al., 1983; Friend
and Shahani, 1984; Shimizu et al., 1987;
Fernandes and Shahani, 1990; Adachi,
1992; Salminen, 1996).
Parenteral administration of some
strains lactobacilli, especially Lactobacillus casei Shirota strain has had antitumor
and immunostimulating activities on experimentally implanted tumors (Morotomi,
1996; Miguel, 2001). Similar effects have
been verified by oral administration. In a
recent study Lactobacillus GG was
shown to cause a dramatic reduction in
DMH induced tumor formation in rats. It
was reported that Lactobacillus GG inhibited the initiation and early phase promotion on the tumorigenesis process
(Goldin et al., 1996). However, it was reported that the organism was not able to
prevent the growth of tumors once they
had been established (Goldin et al., 1996).
The fact that Lactobacillus GG is an organism of human origin capable of surviving in the GT and that it inhibits tumor
initiation or early promotion in the colon
provides a basis for further studies of these
factors in humans. In a similar manner, L.
casei Shirota strain has been shown to
have inhibitory properties on chemically
induced tumors in animals (Kato et al.,
1994; Morotomi, 1996). In a clinical study,
prophylactic effects of oral administration
of L. casei Shirota strain on initiation or
early promotion in the colon provides a
basis for further studies of these factors
in humans. In a similar manner, L. casei
Shirota strain has been shown to have inhibitory properties on chemically induced
tumors in animals (Kato et al., 1994;
Morotomi, 1996). In a clinical study, pro-
S. A. Denev
phylactic effects of oral administration of
L. casei Shirota strain on the recurrence
of superficial bladder cancer has been reported in two Japanese studies (Aso and
Akazan, 1992; Aso et al, 1995). LAB has
also been indicated in colonic fermentation producing butyrate or butyric acid in
the colon. This alteration in intestinal metabolism may be due to changes in the
intestinal microecology following probiotic
intake or the direct metabolism of slowly
absorbable components by orally administered colonizing LAB (Salminen et al.,
1996). Butyrate has been shown in vitro
studies to slow the growth of cultured colon cancer cells. Young (1996) has proposed that butyrate may be a diet-regulated, natural antitumor compounds at least
partly responsible for the antitumor effect
of dietary components and also dietary
probiotics. When these studies are related
to animal data on several Lactobacillus
strains and colon cancer related parameters, it is important to assess the potential of probiotic lactobacilli for cancer
chemoprevention (Salminen, 1996;
Wollowski et al., 2001).
Since intestinal microorganisms have
been postulated to play an important role
in conversion of chemical procarcinogens
to carcinogens in the gut, the activity of
certain fecal enzymes (azoreductase,
nitroreductase, β-Glucuronidase β-Glucosidase and etc.) has been used to monitor colon carcinogenesis (Golden and Gorbach, 1976; Wollowski et al., 2001).
β-Glucuronidase plays a role in the generation of carcinogenic aglycones, such as
3-hydroxybenzopyrene from 3-hydroxybenzopyrene-p-glucuronide (Kinoshita
and Gelboin, 1978) in colon, from glucuronides formed in the liver. Nitroreductase
and azoreductase are related to the formation of aromatic amines which can be
Role of Lactobacilli in Gastrointestinal Ecosystem
87
converted to nitroso- and N-hydroxycompounds in many tissues. These products are known carcinogens (Cerniglia et
al., 1982; Manning et al., 1985). Steroid7-dehydroxylase is responsible for the
formation of lithocholic acid from chenodeoxycholic acid which is primary bile acid
formed from cholesterol in the liver. Lithocholic acid, a secondary bile acid
formed by the bacterial enzyme in the colon, is a co- carcinogenic substance (Carey,
1973).
Experiments performed in animal and
human models showed that some lactobacilli could decrease the levels of enzymes
azoreductase, nitroreductase, β-Glucuronidase and β−Glucosidase, responsible
for activation of some procarcinogens, and
decrease the risk or the speed of tumor
development (Goldin and Gorbach, 1992).
The results of some studies are shown in
Table 5.
Several investigations have shown an
influence of the intake of LAB and especially Lactobacillus ssp. fermented-milk
products on the gut flora, enzyme activities and colon carcinogenesis (Wollowski
et al., 2001). According to Friend and
Shahani (1984) the lactobacilli inhibit carcinogenesis by (a) inactivating or inhibiting formation of carcinogenic compounds
in the gastrointestinal tract; and (b) suppressing promotion of cancer through
stimulation or enhancement of the immune
properties of the host. Fernandes and
Shahani (1990) observed that LAB, including lactobacilli, inhibits carcinogenesis by
(a) prevention of cancer initiation by means
Table 5
d*
d
d
d
d
d
d
d
d
β -Glucosidase
β -Glucuronidase
L. acidophilus
L. acidophilus
L. acidophilus
L. acidophilus
L. acidophilus
L. acidophilus
L. acidophilus
L. acidophilus (NCFM)
L. casei strain GG
L acidophilus
L. reuteri
L. casei Shirota
L. johnsonii
L rhamnosus GG
(d)* Decrease
Nitroreductase
Species
Azoreductase
Effect of Lactobacilli on fecal bacterial enzymes
d
d
d
d
d
d
d
d
d
d
d
d
Reference
Goldin and Gorbach (1977, 1984)
Goldin et al. (1980)
Auebo et al (1980)
Pedrosa et al. (1990)
Marteau et al. (1990)
Lidbeck et al. (1991)
Bouhnik et al. (1996)
Cole et al. (1989)
Goldin et al. (1992)
Spanhaak et al. (1998)
Haberer et al. (1995)
Ling et al. (1994)
88
of direct and indirect reduction of pro-carcinogens and (b) suppression of initiated
cancer.
Another possible explanation for the
preventive effect of lactobacilli on carcinogenesis is their effect on other bacteria in
the intestine. Lactobacilli might suppress
the growth of bacteria that convert
procarcinogens into carcinogens, thereby
reducing the amount of carcinogens in the
intestine. The activity of the enzymes that
convert procarcinogens into carcinogens
is often used as an indicator of the effect
of probiotics on the intestinal microflora
(Roos and Katan, 2000).
The mechanism of anticarcinogenic action has not yet been fully elucidated. Several studies have suggested that anti-carcinogenic activity of Lactobacillus spp.
may be attributed to:
• Activation of the host’s immune system to
antitumourigenesis (McIntosh, 1996);
• Improvement (quantitatively/qualitatively) of
the intestinal microflora, reducing the putative producers of carcinogens and cancer promoters (improvement of the intestinal microecology: e.g. more
bile-acid degrading bacteria; less bacteria producing the enzymes azoreductase, nitroreduc-tase,
beta-glucuronidase, beta-glucosidase, etc.
(Mercenier et al., 2002);
• Production of compounds that inhibit the
proliferation of tumor cells (Reddy et al., 1973);
• Antagonistic action against the organisms
that convert procarcinogens to carcinogens by enzyme activity (Auebo et al., 1980; McIntosh, 1996;
Roos and Katan, 2000);
• Bind to mutagenic compounds in the intestine and decreasing the absorbtion of these mutagens (Orrhage et al., 1994; Young, 1996;
Guilliams, 1999; Roos and Katan, 2000);
• Direct supprecion of the carcinogens and/or
procarcinogens by binding, blocking, remowing
(McIntosh, 1996);
• Degradation of the carcinogens formed
S. A. Denev
(Rowland and Grasso, 1975);
• Alteration of colonic motility and transit
time (McIntosh, 1996).
During the last five years an impresive
and useful reviews in the field of anti-carcinogenic properties of lactobacilli are provided by Wollowski et al. (2001); Herich
et al. (2002); Norat and Riboli (2003); Gill
and Rowland (2003).
IMMUNE SYSTEM STIMULATION
The immune system consist of a number of organs and several different cell
types that recognize accurately and specifically foreign antigens on microorganisms and thereby, eliminate those organisms. The organs of the immune system
are bone marrow, thymus, spleen, Peyer’s
patches, and lymph nodes. The cells of this
system are the leukocytes, or white blood
cells. There are two categories of leukocytes; 1) phagocytes, including neutrophils,
monocytes, and macrophages, which form
part of the innate immune system and provide nonspecific immunity, and 2) the lymphocytes, which mediate adaptive, or specific immunity. The specific immune response can produce cellular immunity
mediated by specifically sensitive immune
cells and the humoral immune response
mediated by the antibody production
(Perdigon et al., 1995; Cebra, 1999).
A large proportion of the immune cells
of the body are associated with the gut. In
the healthy host, the presence of the
microbiota is tolerated by the immune system, although the mechanisms involved are
not precisely known. Nevertheless, it can
be inferred that tolerance toward the
microbiota exists, because human patients
with inflammatory bowel diseases and experimental animals with dysfunctional im-
Role of Lactobacilli in Gastrointestinal Ecosystem
89
mune systems suffer from chronic, immune-mediated inflammation of the bowel
mucosa (Podolsky, 2002). Much evidence
points to the presence of the microbiota as
the fuel for this smoldering inflammation.
The autochthonous microbe-immune system relationship in healthy animals must
therefore be one of tolerance and requires
mechanistic investigation. The allochthonous microbe-immune system relationship
is presumably quite different, at least initially, because the immune system will experience novel antigenic complexes with
each encounter with a different bacterial
strain. Continuous close encounters with
the same strain, either serendipitous (food
microbiota) or intentional (probiotic), could,
one supposes, eventually engender tolerance (Tannock, 2004).
The gut microflora is an important constituent in the intestine’s defense barrier,
as shown by increased antigen transport
across the gut mucosa in the absence of
an intestinal microflora. This notion further supported by a demonstration that the
gut microflora elicit specific immune responses at a local and a systemic level
(Kailua et al., 1992; Isolauri et al., 2001;
Perdigon et al., 2001). It has been hypothesized that LAB may have some potential
role in augmenting the immune system of
the host (Turjman et al., 1982). This hypothesis was proposed on the observation
that LAB suppressed peritoneally and subcutaneously implanted tumors in mice in
short-term studies. LAB did not directly
suppress the tumor, but the suppression
appeared to be mediated indirectly through
the host’s immune system.
The effects of LAB on the immune system could have theoretical applications for
tumor suppression, and anti-infective activity. None of these potential effects has
been in man, and LAB do not yet have
a place among human cancer therapies
(Marteau and Rambaud, 1993). However,
very active and interesting research is developing in this field.
During the past ten years, interactions
between lactobacilli and immunocompetence have been studied extensively in
vitro and in vivo. These investigations
have revealed that lactobacilli not only constitute an integral part of the healthy gastrointestinal microecology, but are also involved in the host’s protective mechanisms
by increasing its specific and non-specific
immune mechanisms (Renner, 1995). The
intestinal microflora has a profound influence on the immunological state of the
host. Live indigenous bacteria or their antigens can, in fact, penetrate the epithelial
barrier of the intestine and directly stimulate the immunocompetent cells. The
germs present in the intestinal lumen favour the production of suppressor cells, the
lymphocytic differentiation or even the
“helper” cell activity.
The intestinal lactobacilli can stimulate
the immune system in two ways. They can
either migrate thought the gut wall as viable cells and multiply to a limited extent
or antigens released by the dead organisms can be absorbed and stimulate the
immune system directly. A third school of
thought suggests that the lactobacilli are
acting indirectly through an effect on the
other components of the gut flora. It is a
product of this change that induces the
immune response. At present, which of
these methods is, being used is not certain, but there appears to be some relationship between the ability of a strain to
translocate and the ability to be immunogenic.
The improved immunity induced by lactobacilli may be manifested in three ways:
• Increased macrophage activity shown by the
90
enhanced ability to phagocytose microorganisms
or carbon particle;
• Increased production of systemic antibody
usually of the immunoglobulin classes IgG, IgM
and interferon;
• Increased local antibody at mucosal surfaces
such as the gut wall. These are usually IgA.
The first (and possibly the only) contact of lactobacilli with the immune system occurs in the gut-associated lymphoid
tissue. Perdigon et al. (1990) showed that
oral administration of L. casei, L. acidophilus, L. bulgaricus and S. thermophilus increased the levels of immunoglobulins in the intestinal fluid. However, only
L. casei pretreated mice had increased secretion of specific antibodies against Salmonellae when challenged with Salmonellae. De Simone et al. (1987a, 1988) reported that yogurt or heated yogurt administered to mice increased the percentage
of B-lymphocytes in the Peyer’s patches,
and the proliferative responses of these
cells to stimulants. When those mice challenged with Salmonellae, antibacterial activity of the Peyer’s patche lymphocytes
and thus resistance to infection was increased by living yogurt.
A large number of experiments have
demonstrated that LAB, especially lactobacilli could exert immunostimulatory effects (Adachi, 1992; Tomioka and Saito,
1992; Malin et. al., 1996; Perdigon et al.,
1999, 2001; Koop-Hoolihan, 2001) and enhance host resistance against experimental infections (Perdigon and Alvarez, 1992).
The effects seem mainly due to the activation of the macrophages and non-specific immunity by cell wall components of
the lactobacilli (Kato et al., 1984; Sato,
1984; Yokokura et al., 1986; Sato et al.,
1988a, 1988b; Ohashi et al., 1989; Moineau
and Goulet, 1991; Miettinen et al., 1996;
S. A. Denev
Fuller and Perdigon, 2000; Perdigon et al.,
2001).
The non-specific defence mechanisms
of the host include phagocytosis, which is
effected by macrophages, polymorphonuclear leucocytes, histiocytes and monocytic cells that are part of the mononuclear
phagocytic system, which removes foreign
antigens.In this system, the most important cells are macrophages. The state of
activation of these is a measure of the nonspecific immune response of the host
(Perdigon and Alvarez, 1992)
Changes in monocytes’ and lymphocytes’ activities, a measure of cell-mediated immunity, have also been studied
following short-therm feeding of viable
LAB and yogurt to mice. Mice were fed
viable L. acidophilus, L. casei subsp.
casei, L. delbrueckii subsp. bulgaricus
and S. thermophilus, and changes in
macrophage enzymes and in vivo phagocytic function of reticuloendothelial system were measured (Perdigon et al.,
1986a). Feeding of L acidophilus and L.
casei subsp. casei increased all three enzymatic activities (Perdigon et al., 1986b,
1987). The feeding of L delbrueckii
subsp. bulgaricus also increased β-glucuronidase and β -galactosidase activities.
(Perdigon et al., 1986a, 1987).
The effect of lactobacilli on the cellular and the humoral immune response has
been investigated by Perdigon et al.(1986c,
1988a, b). They found that the oral administration of L. casei, L. acidophilus
and L. delbrueckii subsp. bulgaricus
enhanced both the cellular and the humoral
immune response. L. casei and L. acidophilus were tested singly and together by
Perdigon (1986b) for stimulation of phagocytic activity. It was found that the mixture was more effective than the strains
given singly. These suggest that a mixture
Role of Lactobacilli in Gastrointestinal Ecosystem
91
of lactobacilli was more effective and the
individual effects of the component strains
may be additive.
In feeding experiments with mice
where 1.2 x 109 cells of LAB per day
were administered, the following results
were obtained (Perdigon et al., 1993): L
acidophilus increased IgA production
only temporarily, but increased IgG production thoughout the feeding period.; L.
casei enhanced the proliferation of IgAproducing cells; yoghurt feeding increased
the number of plasma cells, lymphocytes
and macrophages. L. casei feeding given
to mal-nourished animals slightly increased
the number of circulating leukocytes and
the phagocytic activity of peritoneal macrophages. This feeding also restored the
strict anaerobic population in the small intestine, but failed to prevent bacterial
translocation. L. casei induced an increase
in the number of IgA-producing cells as
well as the level of IgM (Perdigon et al.,
1995). The above results suggested that:
(a) L. casei,L. acidophilus, L. delbrueckii subsp. bulgaricus and certain
doses of S. thermophilus are capable of
activating the cells involved in the non-specific immune response; (b) the LAB capable of survival and growth in the intestinal tract, such as L. casei and L. acidophilus, are more efficient in the activation.
De Simone et al (1986, 1987b) showed
that several LAB including yogurt bacteria stimulated in vitro production of interferon-g (IFN-g) by human blood lymphocytes. Several in vivo studies in man
showed then that ingested yogurt bacteria
at last in very large doses (1011 and 3x1012
day-1) stimulated IFN-g production (De
Simone et al., 1991; SoIis and Lemonnier,
1991). Kishi et al. (1996) observed that L.
brevis subsp.coagulans significantly in-
creased a-interferon production. A beneficial consequence of interferon stimulation could be an increase in resistance to
some infections. However, interferon can
exert both beneficial and detrimental effects to man (Murray, 1988). More experiments, including dose-response studies, and trials with clinical end-points are
needed.
Perdigon et al. (1993) present the following hypothesis for the immunomodulating effects of LAB: not all lactobacilli
induce synthesis of IgA; for those that do,
the dose administered plays a critical role;
the first effect of lactobacilli on the immune system occurs at the local level on
the gut-associated lymphoreticular tissue,
which may or may not send signals that
would permit the activation of the immune
system in general. Many other studies
have shown that oral administration of lactobacilli could influence parameters of the
systemic immunity (Morissete et al., 1991;
Perdigon and Alvarez, 1992; Marteau,
1995; Sotirov et al., 2000, 2001; Isolauri et
al., 2001). Although many of these results
were obtained using mice as experimental models, they are not easily transferred
to human beings. For the use of lactobacilli as immunomodulatory agents, experimental models are absolutely necessary
to determine that effects of lactobacilli on
the host are innocuous and to select those
bacteria that most effectively enhance the
immune response. However, the exploitation of that knowledge for therapeutic purposes is still limited (Perdigon et al., 1995).
For more information about immunologic
effects of lactobacilli see Guilliams (1999),
Matsuzaki and Chin (2000), Isolauri et al.
(2001); Perdigon et al. (2001); Kaur et al.
(2002).
Lactobacilli clearly offer microbiologists exciting research prospects, both for
92
biomedical applications and for acquiring
fundamental knowledge of how bacterial
cells function in the gut ecosystem. As
model gut bacteria, they may provide lessons in the molecular mechanisms that define autochthony as well as in understanding bacterial physiology in relation to host
welfare. For these reasons, lactobacilli are
set to remain the fond favorites of many
microbiologists.
Acknowledgements
This work was supported by Post-Doctoral
Research Fellowship Grant from the Japan International Science & Technology Exchange Center,
Japan, and EU Grant (University of Groningen),
The Netherlands. The financial assistance is highly
appreciated. The author is indebted to all partners
of the Research Project “Role of Lactobacilli in
Gastrointestinal Ecosystem” for the excellent working conditions were provided. Gratitude is expressed to I. Suzuki, H. Kimoto, H. Nakai, and W.
Konnings, for their research support and collaboration. The contributions of many universities, laboratories and investigators who provided publications are gratefully acknowledged. The author
would like to thank many colleagues who kindly
supplied published information for this manuscript:
R. Fuller, G. Tannock, T. R. Klaenhammer, S. E.
Gilliland, D.S. Savage, T. Mitsuoka, A. Hosono,
G. Perdigon, C. F. Fernandes, K. M. Shahani, E.
Nurmi, P. G. Sara, W. E. Sandine, F. Edens, and etc.
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Received November, 10, 2005; accepted January, 8, 2006.