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Istituto Nazionale della Nutrizione, 00178 Rome, Italy;
*
Istituto di Microbiologia Agraria e Tecnica, Bologna, Italy; and
Istituto Ematologia-Oncologia Medica, Policlinico S. Orsola, Bologna, Italy.
1To whom correspondence should be addressed at Istituto Nazionale della Nutrizione, via Ardeatina 546, 00178, Roma, Italy. e-mail: mengheri@inn.ingrm.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • Bifidobacterium animalis • zinc deficiency • intestine • rats • epithelial proliferation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Fuller 1989
,
Salminen et al. 1996
, Sanders 1993
,
Walker and Duffy 1998
).
Probiotics have been used in both humans and experimental animals in
the treatment or prevention of gastrointestinal disease
(Bengmark 1996
, Macfarlane and Cummings 1999
, Salminen et al. 1996
). However, the
results are not sufficiently clear. In addition, only a few strains
with beneficial effects have been identified. A study of a gnotobiotic
murine model suggested that oral inoculation of Lactobacillus
salivarius prevented Helicobacter pylori
infection (Kabir et al. 1997
). The exogenous
administration of Lactobacillus reuteri was shown to
prevent the development (and enhance the healing) of acetic
acid–induced colitis (Fabia et al. 1993
).
Lactobacillus reuteri were also demonstrated to prevent
bacteremia in a rat model of peritonitis (Bengmark 1996
). Other strains, such as Bifidobacterium
bifidum and Streptococcus thermophilus, have
been used for the prevention of diarrhea and shedding of rotavirus in
hospitalized infants (Saavedra et al. 1994
,
Sanders 1993
).
In previous studies, we have reported that ulcerations, edema,
inflammatory cell infiltration and dilatation of blood vessels
developed in the small intestine of zinc-deficient
(ZD)2
rats, depending on the severity of the deficiency (Nobili et al. 1997
, Virgili et al. 1999
). In this study, we
have investigated the potential beneficial effect of
Bifidobacterium animalis on intestinal damage by using
ZD rats as a model in which intestinal alterations of different
severity may be induced. Valuable selection criteria for probiotic
bacteria are resistance to gastric acidity, adhesion to enterocytes or
proliferation in large number (Bengmark 1996
,
Schiffrin et al. 1997
). It has already been shown that
different strains of bifidobacteria can survive the passage through the
stomach and small intestine of humans (Biavati et al. 1995
, Marteau et al. 1990
, Pochart et al. 1992
). In this study, we used B.
animalis as a species able to survive and proliferate
through the gastrointestinal tract (Biavati et al. 1995
). This species is ingested by large numbers of consumers
of fermented milk containing bifidobacteria (Biavati et al. 1992
).
|
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bifidobacterium animalis, strain MB5, was grown for
24 h at 37°C in TPY (tryptone-phytone-yeast extract) broth
medium, in anaerobic conditions as previously reported (Biavati et al. 1991
). Escherichia coli strain K 12,
ATCC14948 (American Type Culture Collection, Rockville, MD),
was cultured in LB broth medium (10 g tryptone, 5 g yeast extract
and 5 g NaCl/L), and incubated overnight at 37°C in moderate
rotation. The cells were collected by centrifugation at 12000 x g for 10 min, resuspended in sucrose solution (120 g/L)
and lyophilized.
Animals.
Male Sprague-Dawley rats (Charles River, Como, Italy) weighing 150–160 g were housed in stainless steel cages and maintained at 23°C with a 12-h light:dark cycle. Zinc-deficient (ZD) rats (n = 70) were fed a purified diet containing 1 mg Zn/kg diet. They were divided in ZD20 and ZD40 groups and fed the ZD diet for 20 or 40 d, respectively, to induce intestinal damage of different severity. Fourteen rats remained in each group; the other rats were divided in three subgroups. The subgroups ZD20 + B and ZD40 + B (n = 14/group) received orally, by gavage, a suspension of pure viable B. animalis in saline, at the dose of 3.5 x 108 colony forming units (cfu), daily for the last 10 d of the dietary treatment (i.e., after 10 or 30 d of zinc deficiency, respectively). The subgroup ZD40 + B + 7 d (n = 6) was treated as the subgroup ZD40 + B and then fed the ZD diet for another 7 d after the ingestion period of B. animalis. As negative controls, two other subgroups (n = 4/group) of the ZD40 group were used. The subgroup ZD40 + E received a suspension of E. coli (3.5 x 108 cfu) daily for the last 10 d of the experiment. The subgroup ZD40 + Zn received by gavage, for the last 10 d, 5 µg zinc as zinc carbonate dissolved in deionized, distilled water, corresponding to the amount of zinc present in the suspension of bifidobacteria, as determined by flame atomic absorption spectrophotometry after a complete bacterial lysis. The control (C) group (n = 14) was fed the ZD diet supplemented with zinc carbonate to have an adequate level of zinc (50 mg Zn/kg).
The rats had free access to food. The water was deionized, distilled
and freely available to the rats. The diets were purchased from Ditta
Rieper (Vandoies, Italy). The composition of the diet has been
described previously (Nobili et al. 1997
). At the end of
the experimental period, rats were anesthetized with an intraperitoneal
injection of pentobarbital (10 mg/kg) before collection of blood and
intestine.
All experiments were approved by the Animal Care and Ethics Committee
of the Istituto Nazionale della Nutrizione, Rome, Italy and conformed
to published guidelines (National Health and Medical Research Council 1985
).
Histology.
Pieces of duodenum, jejunum and ileum (~2.5 cm) were immersed in Bouin’s fixative for 12 h, washed in PBS for 24 h, embedded in paraffin at 58°C and sectioned at 7 µm. After Mallory staining, the intestinal sections were examined without knowledge of origin under a light microscope. The variables selected to establish the extent of the most evident tissue modifications were ulcerations, edema, inflammatory cell infiltration (ICI) and dilatation of blood vessels. A score from 0 to 4 was assigned to the histologic variables in relation to the severity of alterations as follows: 0 = normal; 1 = mild; 2 = moderate; 3 = marked; 4 = severe.
Fecal analysis.
Feces of the C, ZD40, ZD40 + B and
ZD40 + B + 7d rats were collected on the last day of their
experimental periods. Samples were serially diluted and plated onto
selective agar medium (Biavati et al. 1991
). After
incubation in anaerobic jars for 3–4 d at 37°C, the colonies were
counted and checked for cell morphology. Some colonies were transferred
into TPY broth medium, and B. animalis
was identified by morphology, electrophoretic pattern of total cellular
proteins, presence of fructose-6-phosphate-phosphoketolase
(Biavati et al. 1991
) and DNA homology with MB5 by
DNA-DNA hybridization as previously described (Biavati et al. 1992
).
Scanning electron microscopy.
Pieces of colon from the C, ZD40 and ZD40 + B rats were fixed in 50 g/L glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.2, for 15 min and dehydrated in graded ethanol series for 15 min at 5°C. Samples were "critical point" dried, coated with gold-palladium film and observed under a Philips 515 scanning electron microscope (Milan, Italy) at 9–10 kV.
Hydrophobicity assay.
The hydrophobicity of B. animalis was
assayed by the salt aggregation test (SAT), as described by
Lindahl et al. (1981)
; the SAT is based on precipitation
of cells by salts, that is, the higher the hydrophobicity of the
proteins, the lower the salt concentration required to aggregate the
cells.
Assessment of epithelial cell proliferation.
Epithelial cell proliferation was expressed as a labeling index (LI). Rats were injected intraperitoneally with vincristine (1 mg/kg) to arrest cells in metaphase. After 6 h, the intestine was removed and sections of duodenum, jejunum and ileum were processed as described above for histology. The LI was estimated in 20 crypts under light microscope by the mean number of nuclei arrested in metaphase and was calculated by the following formula: number of arrested metaphases x 100/number of cells per crypt column. Crypt column was defined by the number of epithelial nuclei in the left column of the crypt.
Enzyme and protein assays.
Sucrase and maltase activities were assayed in the three intestinal
regions by the method of Messer and Dahlqvist (1966)
.
Protein content was measured by the method of Lowry et al. (1951)
.
Zinc analysis.
Zinc concentration in rat serum and suspension of bifidobacteria, previously mineralized with nitric acid in the presence of hydrogen peroxide, was determined by flame atomic absorption spectrophotometry using a SpectrAA-400 model atomic absorption spectrometer (Varian, Sunnyvale, Melbourne, Australia).
Statistical analysis.
Data are the mean ± SEM of at least six experiments.
The significance of the difference was evaluated by one-way ANOVA
followed by Fisher’s test (Winer 1971
). Differences
with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The body weights of both the ZD20 and
ZD40 rats were dramatically lower than that of
the C rats (P < 0.05; Table 1
). The serum zinc concentration was similarly lower in the
ZD20 and ZD40 rats compared
with the C rats. The body weights and serum zinc concentrations of the
ZD20 + B and ZD40 + B rats
did not differ from those of the ZD20 and
ZD40 rats, respectively.
|
As previously reported (Nobili et al. 1997
,
Virgili et al. 1999
), zinc deficiency induced several
alterations in the morphology of the three intestinal regions
(Fig. 1
). The intestine of the ZD20 rats had a few
ulcerations in duodenum and jejunum, and a marked edema and moderate
ICI in all three regions (score: from 1 to 2). The intestine of the
ZD40 rats had developed widespread ulcerations,
severe edema, dense ICI and severe dilatation of blood vessels (score:
from 2 to 4). The most severely damaged region was the jejunum, with a
score of 4, whereas the ileum was less affected, with a score of 2. We
had also made a histologic examination of intestine at the beginning of
each treatment with bifidobacteria (i.e., after 10 or 30 d of zinc
deficiency). After 10 of zinc deficiency the mucosa was normal, whereas
after 30 d, the duodenum and ileum developed a few ulcerations,
moderate edema and ICI, and the jejunum appeared already severely
damaged (not shown). After B. animalis
administration, the morphologic alterations induced by zinc deficiency
did not develop or were notably less. In fact, the mucosa of
ZD20 + B rats was well preserved, although mild
edema in jejunum and mild ICI in the three regions were seen (score: 
1). In addition, the intestine of the ZD40 + B
rats had developed only very rare ulcerations and moderate edema in
jejunum, and mild ICI in the three regions (score from 1 to
2). The beneficial effect of B. animalis
persisted after the end of the ingestion period because the mucosa of
the ZD40 + B + 7 rats did not differ from that of
the ZD40 + B rats. Histologic scores are
presented in Table 2
. Treatment with either E. coli or zinc solution did not
induce any effect because the mucosa of the ZD + E and ZD + Zn rats did
not differ from that of the ZD40 rats (not
shown). Thus, the results presented highlight that B.
animalis can protect the intestine from the morphologic
damage induced by zinc deficiency.
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Light microscope observation and assay of
fructose-6-phosphate-phosphoketolase identified several isolates
ascribable to Bididobacterium spp. in the feces of the
ZD40 + B rats but not in the feces of either the
C or ZD rats. Gel electrophoresis of cellular proteins from
bifidobacteria isolated from the ZD40 + B rats
showed a protein pattern that corresponded to that of B.
animalis strain MB5 (not shown). The same identification was
obtained by DNA-DNA hybridization. The homology of the isolates
with MB5 was always close to 100%. The recovery of B.
animalis in the ZD40 + B rats at the
end of treatment was 10.3 x 109 cfu
(Table 3
), a value even higher than the total amount of bifidobacteria ingested
for the 10 d of treatment (3.5 x 109
cfu). The concentration of B. animalis was not
reduced 7 d after ingestion stopped. Thus, these data indicate
that B. animalis survived and proliferated along
the digestive tract.
|
The electron microscope scanning of cecum (Fig. 2
) confirmed the presence of numerous bifidobacterium-like bacteria
in the cecum of the ZD40 + B but not in the cecum
of the ZD40 rats.
|
The SAT revealed that B. animalis aggregated at
low salt concentrations (i.e., 0.85 mol/L ammonium sulfate). It has
been shown that bacterial aggregation caused by low salt concentrations
(from 0.01 to 2.0 mol/L) is an index of high surface hydrophobicity
(Lindahl et al. 1981
). Therefore, our results indicate
high hydrophobicity of B. animalis; according to
some authors (Weerkamp et al. 1985
), this suggests
adhesion capacity of this bacterium (see also Discussion).
Epithelial cell proliferation.
As shown in a previous study (Nobili et al. 1997
), the
LI was markedly affected by 40 d of zinc deficiency (Fig. 3
). In this study, we also measured the epithelial cell proliferation
after a less severe zinc deficiency. No difference was seen in LI among
the ZD20 + B, ZD20 and C
rats. On the other hand, the LI of the ZD40 + B
rats was higher than that of the ZD40 rats in all
intestinal regions, and was not different from the other rats. Thus,
these data indicate that treatment with B. animalis
normalized the reduced crypt cell proliferation induced by severe zinc
deficiency.
|
We confirmed previous results that the activities of sucrase and
maltase were significantly affected by 40 d of zinc deficiency
(Fig. 4
; Nobili et al. 1997
). In addition, the disaccharidase
activities of the ZD20 rats did not differ from
those of the C rats. After the administration of B.
animalis, the disaccharidase activities of the
ZD20 + B rats did not differ from those of the C
and ZD20 rats. On the other hand, both the
sucrase and maltase activities of the ZD40 + B
rats were significantly greater than those of the
ZD40 rats. The jejunal maltase of the
ZD40 + B rats was significantly higher than that
of the C rats. Thus, the results indicate that B.
animalis can also affect some functional activities of
intestine.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 1998
, Salminen et al. 1996
). In this study,
using ZD rats as a model for intestinal damage, we showed that
exogenous administration of B. animalis, strain
MB5, may have beneficial effects by either preventing or normalizing
intestinal alterations. In fact, we found that this treatment almost
completely prevented the development of alterations in the
ZD20 rats. Moreover, most of the ulcerations,
inflammation and edema that appeared between 20 and 40 d of zinc
deficiency were prevented or normalized by treatment with B.
animalis.
Crucial for the successful treatment with probiotic bacteria is its
survival in the gastrointestinal tract, resistance to gastric acidity,
capacity to adhere to mucosal surface and transient colonization above
a critical level. It has been shown that some species of probiotics
survive the passage through the gastrointestinal tract, whereas others
do not (Pedrosa et al. 1995
). Bouhnik et al. (1992)
showed fecal recovery in humans of viable
Bifidobacterium sp. ingested in fermented milk. Other
authors found that Lactobacillus acidophilus, Lactobacillus
bulgaricus and B. infantilis were usually
but not always recovered in the small intestine for 3 h after
their ingestion (Clements et al. 1983
). A recent study
by Biavati et al. (1995)
reported that B.
animalis survived passage through the gastrointestinal tract
when administered to humans. That study was conducted among healthy
individuals. Thus, the possibility that B.
animalis did not survive transit through the damaged
intestine of the ZD rats could not be excluded. However, our data on
cecal and fecal bifidobacteria clearly indicate that B.
animalis survived the gastronintestinal transit of the ZD
rats and colonized. The survival and resulting high fecal
concentrations of B. animalis are compatible with
potential probiotic activity (Bouhnik et al. 1992
). This
bacterium was able to proliferate 7 d after the end of the
ingestion period. It is interesting that after this period, the
intestine was still protected from the ZD-induced morphologic
alterations. Prolonging the postingestion period decreased the recovery
of B. animalis. We had done both fecal analysis
and histology of small intestine 14 and 21 d after the end of the
ingestion period (data not shown). At 21 d postingestion, when
bacteria were no longer detectable in feces, all morphologic damage was
again present in the small intestine. Thus, the beneficial effect
appeared to be dependent on the presence of viable bifidobacteria. Our
results are in agreement with several studies showing that the numbers
of fecal bifidobacteria and lactobacilli remain high for a certain
period after the end of exogenous administration of such bacteria
(Biavati et al. 1995
, Goldin et al. 1992
,
Wagner et al. 1997
).
A certain number of bifidobacteria possibly could not survive gastrointestinal passage, thus releasing their zinc. Consequently, the beneficial effect observed after the administration of bifidobacteria could be due to the bacterial zinc. However, both serum zinc concentration and body weight did not increase after the administration of B. animalis, as would be expected after a zinc supplementation derived from bacterial lysis. In addition, the concentration of zinc in the B. animalis suspension was very low. Despite these considerations, we treated the ZD rats with a solution of zinc sulfate containing an amount of zinc equivalent to that measured in the bacterial suspension. We also tested the treatment of the ZD rats with another bacterium, by administrating E. coli at the same concentration as that of B. animalis and for the same period of time. Neither the zinc solution nor E. coli showed any beneficial effect, indicating the specificity of the activity of viable B. animalis in the intestine of ZD rats.
It has been suggested that the ability of probiotic bacteria to adhere
to intestinal epithelial cells may be important for colonization in the
gastrointestinal tract and optimal function (Bengmark 1996
, Fuller 1989
). Some strains of
bifidobacteria are able to adhere to intestinal cells, whereas others,
such as Bifidobacterium sp., are not or are only weakly
adhesive in vitro (Bernet et al. 1993
, Perez et al. 1998
, Wagner et al. 1997
). A recent study
showed that B. bifidum with agglutinating
capacity can adhere to epithelial cells in vitro (Perez et al. 1998
). This study also indicated that high hydrophobicity was
necessary for adhesion and autoagglutination, in agreement
with the findings of other investigators (Weerkamp et al. 1985
). On the basis of these data, our results of the high
surface hydrophobicity of B. animalis suggest
that this bacterial strain has the capacity for cell adhesion. In
addition, a previous study has shown adhesion of B.
animalis to the keratinized stomach of congenitally
immunodeficient mice (Wagner et al. 1997
).
Regardless of the capacity to adhere, several effects have been
reported concerning different bifidobacterial species. Schiffrin et al. (1997)
, using L. acidophilus and
B. bifidum for their high adhesion and
colonization capacity, respectively, demonstrated an immune modulation
of blood leukocytes by both lactic acid bacteria. Thoreux et al. (1995)
reported that fermented milk with different strains of
lactobacilli and Bifidobacterium sp. modulates cell
proliferation and differentiation of IEC-6 intestinal cells,
independently of the capacity of adhesion. On the contrary,
Bernet et al. (1993)
demonstrated an inhibition in vitro
of enteropathogen-cell interaction by those bifidobacterial
strains, such as B. breve and B.
infantis, which showed high adhesivness to intestinal
epithelial cells. The results of this study indicate the ability of
another bacterial strain of the Bifiodbacterium species (one
that likely has adhesion capacity) to affect epithelial intestinal
cells in a beneficial way.
Rat enterocytes are continuously exfoliated from the tips of the villi
and replaced with new cells migrating out of the crypts to reach the
villous apex in 2–3 d (Leblond 1981
). In this study, we
showed for the first time to our knowledge, that B.
animalis can normalize reduced crypt cell proliferation. Few
studies have shown that lactobacilli and bifidobacteria can regulate
cell proliferation under certain conditions. Some authors reported that
lactic acid bacterial cell wall components, peptidoglucans, have the
unique ability to both inhibit the growth of tumor cells and stimulate
the proliferation of normal cells (Bengmark 1998
). Other
authors have found, both in vivo and in vitro, that intestinal
epithelial cells are stimulated to proliferate after treatment with
lactobacilli or bifidobacteria (Thoreux et al. 1995
and 1998
). In this study, the epithelial cell proliferation of the
ZD20 rats was unaffected by treatment with
bifidobacteria (ZD20 + B rats), whereas it was
stimulated in the ZD40 + B rats. Considering that
diffusely severe necrotic mucosa was generated in the
ZD40 and not in the ZD20
rats, it is likely that a significant enhancement of epithelial cell
proliferation by bifidobacteria was generated only in a damaged tissue
in which where a great replacement with new cells was required.
The disaccharidase activities were affected by 40 d of zinc
deficiency. Treatment with bifidobacteria effectively increased both
sucrase and maltase activities. At the end of treatment with
bifidobacteria, the intestinal mucosa of the ZD40
+ B rats was almost completely normalized morphologically; the higher
sucrase and maltase activities may be a consequence of this
normalization rather than a direct effect of B.
animalis. However, the disaccharidase activities
of the ZD40 + B rats tended to be greater than
those of the C rats, suggesting that B. animalis
can have a specific effect on these enzymes. Consistent with our data,
some authors have demonstrated that treatment with Saccharomyces
boulardii (Buts et al. 1994
) or milk fermented by
L. casei (Thoreux et al. 1998
)
enhanced brush border enzyme activities of rats.
The mechanisms by which B. animalis exerts its beneficial
effects must be clarified. In previous studies, we suggested that
inflammation plays an important part in intestinal damage induced by
zinc deficiency; treatment with dexamethasone, thyroxine or cyclosporin
A may counteract the inflammation (Nobili et al. 1997
,
Virgili et al. 1999
). Moreover, we found that the
expression of proinflammatory interleukin-1ß reverted to the normal
level after treatment of ZD rats with cyclosporin A (Vignolini et al.1998
). Considering that probiotic species are able to
stimulate the immune system and cytokine production (Marin et al. 1996
, Schiffrin et al. 1997
), we
hypothesized that the beneficial effect of B.
animalis results from its ability to exert an
anti-inflammatory activity by modulating cytokine expression. We
are now analyzing the proinflammatory cytokine expression in intestine
of ZD rats treated with B. animalis; preliminary
results indicate that the level of interleukin-1ß and interleukin-8
are greater in ZD rats and are decreased after treatment with B.
animalis. Thus, among other potential mechanisms of action, an
anti-inflammatory activity may be fundamental to the protective
function of B. animalis. The increased cell
proliferation and disaccharidase activities may be secondary to the
reconstituted integrity of intestine that follows the reduced
inflammation, although a specific effect on these enzymes seems to be
exerted by B. animalis.
In conclusion, the results presented in this study indicate that B. animalis exerts beneficial effects by either preventing or normalizing the intestinal alterations induced by zinc deficiency. Such protection of B. animalis supports the concept that bifidobacteria play a role in the intestinal mucosal defense and gives new information on the probiotic properties of B. animalis. Better understanding of the mechanisms by which bifidobacteria exert the beneficial effects is a necessary step in clarifying their role in zinc deficiency and may provide insights into their role in treating gastrointestinal diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
|---|
Manuscript received March 11, 1999. Initial review completed May 11, 1999. Revision accepted August 9, 1999.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. Ballongue J. Bifidobacteria and probiotic action. Salminen A. Von Wreight A. eds. Lactic Acid Bacteria 1993:357-428 Marcel Dekker New York, NY.
2. Bengmark S. Econutrition and health maintenance—a new concept to prevent GI inflammation, ulcerations and sepsis. Clin. Nutr. 1996;15:1-10
3. Bengmark S. Probiotics and prebiotics in prevention and treatment of gastrointestinal diseases. Gastroenterol. Int. 1998;11(suppl. 1):4-7
4.
Bernet M. F., Brassart J. R., Servin A. Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions. Appl. Environ. Microbiol. 1993;59:4121-4128
5. Biavati B., Mattarelli P., Alessandrini A., Crociani F., Guerrini M. Survival in fermented milk products of B. animalis and its recovery in human faeces. Microecol. Ther. 1995;25:231-235
6. Biavati B., Mattarelli P., Crociani F. Identification of bifidobacteria from fermented milk products. Microbiologica 1992;15:7-13[Medline]
7. Biavati B., Sgorbati B., Scardovi V. The genus Bifidobacterium. Balows A. Trüper H.G. eds. The Prokaryotes 1991:816-833 Springer-Verlag New York, NY.
8. Bouhnik Y., Pochart P., Marteau P., Arlet G., Goderel I., Rambaud J. C. Fecal recovery in humans of viable Bifidobacterium sp. in fermented milk. Gastroenterology 1992;102:875-878[Medline]
9. Buts J. P., De Keyser N., De Raedemaeker L. Saccharomyces boulardii enhances rat intestinal enzyme expression by endoluminal release of polyamines. Pediatr. Res. 1994;36:522-527[Medline]
10. Clements M. L., Levine M. M., Ristiano P. A., Daya V. E., Hughes T. P. Exogenous Lactobacilli fed to man. Their fate and ability to prevent diarrheal disease. Prog. Food Nutr. 1983;7:29-37
11. Fabia R., Ar’Rajab A., Johansson M. L., Willen R., Andersson R., Molin G., Bengmark S. The effect of exogenous administration of Lactobacillus reuteri R2LC and oat fiber on acetic acid-induced colitis in the rat. Scand. J. Gastroenterol. 1993;28:155-162[Medline]
12. Fuller R. Probiotics in man and animals. J. Appl. Bacteriol. 1989;66:365-378[Medline]
13. Goldin B. R., Gorbach S. L., Saxelin M., Barakat S., Gualtieri L. Survival of Lactobacillus species (strain GG) in the human gastrointestinal tract. Dig. Dis. Sci. 1992;37:121-128[Medline]
14.
Kabir A. M., Aiba Y., Takagi A., Kamiya S., Miwa T., Koga Y. Prevention of Helicobacter pylori infection by lactobacilli in a gnotobiotic murine model. Gut 1997;41:49-55
15. Leblond C. P. The life history of cells in renewing system. Am. J. Anat. 1981;160:114-158[Medline]
16. Lindahl M., Faris A., Wadström T., Hjertén S. A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells. Biochim. Biophys. Acta 1981;677:471-476[Medline]
17.
Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with Folin phenol reaction. J. Biol. Chem. 1951;193:265-271
18.
Macfarlane G. T., Cummings J. H. Probiotics and prebiotics: can regulating the activities of intestinal bacteria benefit health?. Br. Med. J. 1999;318:999-1003
19. Marin M. L., Lee J. H., Murtha J., Ustunol Z., Pestka J. J. Differential cytokine production in clonal macrophage and T-cell lines cultured with bifidobacteria. J. Dairy Sci. 1996;80:2713-2720
20.
Marteau P., Pochart P., Flourié B., Pellier P., Santos L., Desjeux J. F. Effects of chronic ingestion of a fermented diary product containing Lactobacillus acidofilus and Bifidobacterium bifidum on metabolic activities on the colonic flora in humans. Am. J. Clin. Nutr. 1990;52:685-688
21. Messer M., Dahlqvist A. One-step ultramicro method for the assay of intestinal disaccharidases. Anal. Biochem. 1966;14:376-392[Medline]
22. National Research Council (1985) Guide for the Care and Use of Laboratory Animals. Publ. No. 85–23, National Institute of Health, Bethesda, MD.
23.
Nobili F., Vignolini F., Figus E., Mengheri E. Treatment of rats with dexamethasone and thyroxine protects against zinc deficiency-induced intestinal damage. J. Nutr. 1997;127:1807-1813
24.
Pedrosa M. C., Golner B. B., Goldin B. R., Barakat S., Dallal G. E. Survival of yogurt-containing organisms and Lactobacillus gasseri (ADH) and their effect on bacterial enzyme activity in the gastrointestinal tract of healthy and hypochlorhydric elderly subjects. Am. J. Clin. Nutr. 1995;61:353-358
25.
Perez P. F., Minnaard Y., Disalvo E. A., De Antoni G. L. Surface properties of bifidobacterial strains of human origin. Appl. Environ. Microbiol. 1998;64:21-26
26.
Pochart P., Marteau P., Bouhnik Y., Goderel I., Bourlioux P., Rambaud J. C. Survival of bifidobacteria ingested in a fermented milk during their passage in the upper gastrointestinal tract: an in vivo study using an intestinal perfusion technique in man. Am. J. Clin. Nutr. 1992;55:78-80
27. Saavedra J. M., Bauman N., Oung I., Perman J., Yolken R. Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhea and shedding of rotavirus. Lancet 1994;344:1046-1049[Medline]
28. Salminen S., Isolauri E., Salminen E. Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenge. Antonie Leeuwenhoek 1996;70:347-358
29. Sanders M. E. Effect of consumption of lactic cultures on human health. Adv. Food Nutr. Res. 1993;37:67-130[Medline]
30.
Schiffrin E. J., Brassart J. R., Servin A. L., Rochat F., Donnet-Hughes A. Immune modulation of blood leukocytes in humans by lactic acid bacteria: criteria for strain selection. Am. J. Clin. Nutr. 1997;66:515S-520S
31. Thoreux K., Balas D., Bouley C., Senegas-Balas F. Diet supplemented with yoghurt or milk fermented by Lactobacillus casei DN-114001 stimulates growth and brush-border enzyme activities in mouse small intestine. Digestion 1998;59:349-359[Medline]
32. Thoreux K., Senegas-Balas F., Bernard-Perrone F., Giannarelli S., Denariaz G., Bouley C., Balas D. Modulation of proliferation, second messenger levels, and morphotype expression of the rat intestinal epithelial cell line IEC-6 by fermented milk. J. Dairy Sci. 1995;79:33-43
33. Vignolini F., Nobili F., Mengheri E. Involvement of interleukin-1ß in zinc deficiency-induced intestinal damage and beneficial effect of cyclosporin A. Life Sci 1998;62:131-141[Medline]
34. Virgili F, Canali R., Figus E., Vignolini F., Nobili F., Mengheri E. Intestinal damage induced by zinc deficiency is associated with enhanced CuZn superoxide dismutase activity in rats. Effect of dexamethasone or thyroxine treatment. Free Radic. Biol. Med. 1999;26:1194-1201[Medline]
35. Wagner R. D., Warner T., Roberts L., Farmer J., Balish E. Colonization of congenitally immunodeficient mice with probiotic bacteria. Infect. Immun. 1997;65:3345-3351[Abstract]
36. Walker W., Duffy L. C. Diet and bacterial colonization: role of probiotics and prebiotics. J. Nutr. Biochem. 1998;9:668-675
37.
Weerkamp A. H., Van der Mei H. C., Busscher H. J. The surface free energy of oral streptococci after being coated with saliva and its relation to adhesion in the mouth. J. Dent. Res. 1985;64:1204-1210
38. Winer B. J. Statistical Principles in Experimental Design 2nd ed. 1971:198-201 McGraw-Hill New York, NY.
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6. Within each
region, values with different letters are significantly different. P
