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Section Nutrition & Health, NIZO Food Research, 6710 BA Ede, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • calcium phosphate • infection • salmonella • lactobacilli • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Considering the increased fecal excretion of several bacterial mass
markers, it was noticed that dietary CaPi had a trophic
effect on the intestinal microflora (Bovee-Oudenhoven et al. 1997b
).
Stimulation of bacterial growth may be of relevance because the
endogenous microflora is an extremely important determinant of host
defense against intestinal pathogens (Sarker and Gyr 1992
, Vollaard and Clasener 1994
, Wells et al. 1988
). Besides an appreciation of the
protective endogenous gut flora in general, several investigations have
shown that orally administered lactobacilli exert antagonistic action
against intestinal and food-borne pathogens (for review see Mital and
Garg 1995
). Competition for luminal nutrients and adhesion sites on the
intestinal epithelium and the production of antimicrobial compounds,
such as organic acids and hydrogen peroxide, may account for this
inhibitory action (Mital and Garg 1995
). Many important intestinal
pathogens, for instance salmonella, campylobacter and Escherichia
coli, mainly produce their noxious effects in the small intestine
(Salyers and Whitt 1994
). The predominance of lactobacilli in this
relatively sparsely populated region of the intestinal tract might
explain their functionality in combating intruding microbes (Drasar 1988
). Supplemental dietary CaPi reduces the cytotoxicity
of intestinal contents by precipitating luminal surfactants, such as
bile acids and fatty acids (Govers and Van der Meer 1993
, Govers et al. 1996
). A less harsh environment might favor growth of the resident
microflora and improve their antagonistic action towards pathogenic
bacteria. It is conceivable that lactobacilli and other Gram-positive
bacteria, lacking the outer-membrane lipopolysaccharide screen, are
particularly vulnerable to the intestinal surfactant concentration.
Therefore, they might benefit most by the lowered luminal cytotoxicity
induced by dietary CaPi. The main purpose of the present study was to test the hypothesis that the inhibitory effects of dietary CaPi on salmonella colonization and translocation coincide with a stimulation of the endogenous lactobacilli. Therefore, we first performed an in vitro study to compare the bactericidal activity of bile acids and fatty acids to Salmonella enteritidis and Lactobacillus acidophilus. Additionally, the proposed stimulating effect of supplemental CaPi on the endogenous lactobacilli and the subsequent resistance to salmonella were verified in a strictly controlled infection experiment with rats on either a low- or a high-CaPi purified diet. Special attention was paid to the composition of the microflora in the ileum because this is the most relevant part of the intestinal tract in this infection model, as mentioned above.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S. enteritidis (clinical isolate, phage type 1) and L.
acidophilus (NIZO B224, collection of our institute) were cultured
and stored as described earlier (Bovee-Oudenhoven et al. 1997a
,
Oudenhoven et al. 1994
). Precultures of S. enteritidis and
L. acidophilus were prepared by inoculating a few colonies
from appropriate agar plates in Brain Heart Infusion broth (BHI Difco,
Detroit, MI) and de Man, Rogosa, and Sharpe broth (MRS; Merck,
Darmstadt, Germany), respectively, followed by overnight aerobic
incubation at 37°C. The next day, bacteria were collected by
centrifugation (20 min at 3,500 x g; Heraeus, Sepatech
GmbH, Osterode, Germany), washed once,and resuspended in sterile saline
to prepare stock suspensions. The bactericidal activity of conjugated
bile acids (taurodeoxycholic acid and taurochenodeoxycholic acid 1:2
mol/mol, sodium salts; Sigma Chemical Co., St. Louis, MO), deconjugated
bile acids (deoxycholic acid and chenodeoxycholic acid 1:2 mol/mol,
sodium salts; Sigma), and the fatty acid lauric acid (C12:0; Fluka
Chemie AG, Buchs, Switzerland) in a buffer (250 mmol
3-N-morpholinopropanesulfonic acid/L, adjusted to physiological pH and
ionic strength) was determined by inoculating the sterilized test media
with the stock suspensions (final ~1010 viable bacteria/L
test medium), followed by a 4-h aerobic incubation at 37°C. The bile
acids, as well as lauric acid, were tested in the physiologically
relevant concentration range of 0–4 mmol/L (Lapré et al. 1993
).
Preferably, the bactericidal activity of palmitic acid, oleic acid or
linoleic acid was tested because these are the main fatty acids of the
triglycerides provided by palm and corn oil in the diets. However,
these fatty acids are insoluble in water in the desired concentration
range, whereas lauric acid is not. Immediately after inoculation of the
test media with bacteria, and after 4 h at 37°C, small samples
were taken from the incubates, serially diluted in saline and plated on
Brilliant Green Agar (BGA; Oxoid, Basingstoke, England) and MRS Agar
(Merck) to determine the viable counts of S. enteritidis and
L. acidophilus, respectively. The BGA plates were incubated
aerobically for 1 d at 37°C and the MRS plates for 2 d at
37°C in an anaerobic cabinet (Coy Laboratory products Inc., Ann
Arbor, MI). The whole experiment was performed in triplicate.
Animals, diets and infection.
The experimental protocol was approved by the animal welfare officer of
the Agricultural University, Wageningen, The Netherlands. Four groups
of specific pathogen-free male Wistar rats (WU, Harlan, Zeist, The
Netherlands; n = 8 per group), 8 wk old, were housed
individually in metabolic cages in a room with controlled temperature
(22–24°C), relative humidity (50–60%), and light/dark cycle
(lights on from 06.00 to 18.00). The rats were fed purified diets
differing only in CaPi content. The control diet contained
20 mmol and the supplemented diet 180 mmol CaHPO4 ·
2H2O/kg diet. After preparation, the calcium content of the
diets was checked in dry ashed samples using an atomic absorption
spectrophotometer (model 1100; Perkin Elmer Corp., Norwalk, CT). The
calcium content of the control and CaPi-supplemented diet
was 21 and 170 mmol/kg, respectively. The exact composition of the
diets is provided in Table 1
. Compared with diets prescribed for rodents (AIN 1977
), the two diets
of the present study had a high fat content, and the control diet had a
low calcium content to mimic the composition of a Western human diet
(Van der Meer et al. 1990
). The animals had free access to
demineralized drinking water and daily, freshly prepared porridges
(demineralized water was added and mixed with the dry diets to obtain a
final dry matter content of 680 g/kg). The diets were administered as
porridges to prevent spilling and subsequent contamination of the feces
and urine by food. Food intake was recorded every day and body weight
every 2–3 d.
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). In addition, oral
administration of about 108 viable S.
enteritidis to rats results in a well quantifiable, systemic
infection without excessive diarrhea and loss of body weight
(Oudenhoven et al. 1994
). The virulence of the salmonella strain used
is sustained by routine oral passage in Wistar rats, followed by
isolation of translocated salmonella from liver or spleen. Microbiological analyses and dissection of the animals.
Immediately before and 1, 3 and 6 d after infection, fresh fecal
samples were collected directly from the anus of the animals and
analyzed for viable salmonella as described earlier (Bovee-Oudenhoven et al. 1997a
). Briefly, 10-fold dilutions of the feces in saline were
plated on Modified Brilliant Green Agar (Oxoid) containing
sulphamandelate (Oxoid) and incubated overnight at 37°C. The
detection limit of this method was 102 colony-forming units
of salmonella/g feces.
After collection of the fecal samples at Day 6 after infection, the rats were killed by carbon dioxide inhalation. The distal ileum (defined as the last 12 cm of the small intestine proximal to the cecum) was pinched off and excised. Sterile saline (500 µL) was injected into the ileal lumen and mixed with the ileal contents by turning the ileum upside down five times. The total numbers of salmonella in the ileal lavages were determined with the same microbiological technique as described for feces. Subsequently, the ilea were freed of remaining contents by rinsing with 5 mL of sterile saline, followed by longitudinal dissection. Using a sterile spatula, the ileal mucosa was scraped off to collect salmonella adhering to the ileal epithelium. Scrapings were suspended in sterile saline, homogenized (Ultraturrax Pro200, Pro Scientific Inc., Monroe, CT) and serially diluted in saline. Viable salmonella were quantified as described for feces. However, to compare the number of salmonella in the ileal lavages with their number found in the ileal scrapings, results were expressed as total numbers instead of colony-forming units per g ileal lavage or g ileal scraping.
Also at d 6, the viable lactobacilli counts in feces, ileal lavages and
ileal scrapings of noninfected rats were determined. Handling of the
rats, the procedures to collect biological material for microbiological
analysis and plating techniques were as described for salmonella
determination, except that Rogosa Agar (Oxoid) plates were used for the
quantification of lactobacilli (Mathew et al. 1996
). The Rogosa plates
were incubated for 3 d at 37 °C in an anaerobic cabinet (Coy
Laboratory products).
Chemical analyses of feces and ileal contents.
All feces produced by each animal at d 3, 4 and 5 after infection were
quantitatively collected and pooled per rat for chemical analyses. At
the same time, this was also done for the noninfected animals. Feces
were freeze-dried and subsequently ground to obtain homogeneous
samples. Direct determination of the water content of feces by
freeze-drying underestimates the water content due to drying up of
fecal pellets during collection in the metabolic cages, especially
feces in diarrheal states. Therefore, the sodium, potassium, and
ammonia concentrations in feces were used to calculate the water
content, assuming that these electrolytes are the main cations in
feces, and intestinal contents have an osmolarity of 300 mOsmol/L, even
when diarrhea is present (Fine et al. 1993
). Sodium and potassium were
extracted from freeze-dried feces with 50 g trichloroacetic acid/L
(1:25 wt/v) and analyzed by atomic emission spectrophotometry (Model
1100, Perkin Elmer) as described earlier (Bovee-Oudenhoven et al.
1997). Fecal ammonia was determined using a colorimetric kit as
described earlier (Bovee-Oudenhoven et al. 1997a
). Fecal L- and
D-lactic acid were determined with a colorimetric enzymatic assay as
described earlier (Bovee-Oudenhoven et al. 1997a
).
Reconstituted feces were prepared by adding double-distilled water to
freeze-dried feces to obtain 30% dry weight, which reflects the dry
weight percentage of colonic contents (Govers et al. 1993
).
Subsequently fecal water was prepared by centrifuging reconstituted
feces, followed by collection of the water phase as described earlier
(Bovee-Oudenhoven et al. 1997a
). Fecal water was stored at -20°C
until further use. To remove insoluble material, ileal contents were
centrifuged (5 min at 15,000 x g; Eppendorf 5415,
Merck), and the supernatants were collected and stored at -20°C. The
cytotoxicity of fecal water (40 µL) was determined with an
erythrocyte assay as described previously (Bovee-Oudenhoven et al. 1996
) and validated elsewhere (Lapré et al. 1992
). This bio-assay
estimates the total cytolytic potential of intestinal contents or
feces. As shown earlier (Govers et al. 1993
), intestinal surfactants,
such as bile acids and fatty acids, are major determinants of the
cytotoxicity of intestinal contents or feces. The same bio-assay was
used to determine the cytotoxicity of the ileal contents (10 µL). The
total bile acid concentration of fecal water was determined with a
fluorimetric assay as described earlier (Bovee-Oudenhoven et al. 1997a
). The composition of bile acids in ileal lavages was determined
by high performance liquid chromatography (HPLC) as described earlier
(Dekker et al. 1991
), but preceded by some purification. Before HPLC
analyses, the ileal lavages were diluted with double-distilled water
and cleared from hydrophilic substances on octadecyl columns (500 mg;
IST, Mid-Glamorgan, UK). The water eluates were discarded, whereas the
methanol eluates were sampled and analyzed for bile acid composition.
The synthetic steroid 5ß-cholanic acid-7
,12-diol (Steraloids
Inc., Wilton, NH) was used as internal standard. Using this procedure,
recoveries of taurodeoxycholic acid and cholic acid (both Sigma) added
to the ileal lavages always exceeded 93%. The total concentration of
ileal bile acids was calculated by adding up the concentrations of all
individual bile acids appearing in the chromatogram. The conjugated or
deconjugated nature of the unknown bile acids was determined by using
bile salt hydrolase as described elsewhere (Setchell et al. 1983
). The
concentration of fatty acids in ileal lavages and fecal water was
determined as described earlier (Govers et al. 1993
), with some
modification. For extraction of fatty acids, fecal water and ileal
contents were acidified with HCl (final concentration 1 and 4 mol/L for
fecal water and ileal contents, respectively). To improve the recovery,
methanol (final 20%) was added to the acidified ileal contents,
although this was not necessary for fecal water. Subsequently fatty
acids were extracted three times with 10 volumes of diethyl ether.
After evaporation of diethyl ether, fatty acids were resolubilized in
ethanol and quantified using a colorimetric enzymatic assay (NEFA-C,
Wako Chemicals, Neuss, Germany). Addition of a mixture of lauric acid
and palmitic acid to fecal water and ileal contents resulted in >93%
and >85% recovery, respectively.
Analysis of NOx in urine.
Complete 24-h urine samples were collected for 7 d starting 1 d before infection. Oxytetracycline (approximately 100 times the
minimal inhibitory concentration for most aerobes; Sigma) was added to
the urine collection vessels of the metabolic cages to prevent
bacterial deterioration. The concentration of NOx (nitrate
and nitrite) was determined by automated flow injection analysis.
Briefly, diluted urine was passed over a cadmium column to reduce
nitrate to nitrite, followed by reaction of nitrite with Griess reagent
(Green et al. 1982
). The purple-colored product was measured
spectrophotometrically at 538 nm. Recovery of nitrate added to rat
urines always exceeded 95%. Daily urinary NOx excretion
was calculated by multiplication of the NOx concentration
by the urine volume produced in 24 h.
Statistics.
Results are expressed as mean values ± SEM(n = 8). Student's t-test (one-sided) was used to identify significant differences between noninfected rats consuming the control and the CaPi-supplemented diet. The same was done for the infected rats. Statistical significance was declared when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the absence of bile acids and fatty acids (control incubation), the
viability of S. enteritidis and L. acidophilus
did not change during the 4-h aerobic incubation. S.
enteritidis was practically insensitive to bile acids, even at
high concentrations (Fig. 1A
). In contrast, bile acids, especially unconjugated bile acids, were
strongly bactericidal for L. acidophilus. Similar effects
were observed with lauric acid: S. enteritidis was
completely unaffected, whereas 2 mmol of this fatty acid/L was
sufficient to kill all L. acidophilus present in the
incubates (Fig. 1
B). The bactericidal activity of bile acids
and fatty acids for the lactobacillus seemed to be an immediate effect
because very similar effects of these surfactants on the viability of
this strain were obtained when the incubation time was shorter (0 or
2 h; data not shown).
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At the start of the experiment, mean body weight of the rats was 252 g. Dietary CaPi did not affect body weight gain of noninfected rats (5.2 g/d), whereas salmonella infection reduced growth of the rats in both diet groups (4.3 g/d). Food intake (mean 18 g/d dry matter) was not affected by dietary CaPi or the infection. CaPi had no effect on the calculated water content of feces (37%) of noninfected rats. After infection, the water content of feces of the control group and CaPi group was 45 ± 2% and 39 ± 1%, respectively (P < 0.05). In noninfected rats, CaPi supplementation significantly stimulated total fecal lactic acid output (control 1.91 ± 0.31 and CaPi 12.19 ± 2.72 µmol/d). L-Lactic acid comprised 53 ± 2% of total lactic acid in the control group and 67 ± 2% in the CaPi group. The infection did not significantly affect fecal lactic acid excretion (data not shown).
Cytotoxicity and chemical composition of ileal contents and fecal water.
The cytotoxicity of ileal lavages was significantly lower in the
CaPi-supplemented group (Fig. 2
). Dietary CaPi approximately halved the total bile acid and
fatty acid concentration of ileal lavages (P < 0.05,
Fig. 2
). In addition, major differences were observed in the
composition of ileal bile acids because of CaPi
supplementation (Table 2
). Dietary CaPi significantly decreased the total
concentration of unconjugated bile acids (control 1.43 ± 0.54 and
CaPi 0.34 ± 0.08 mmol/L). The molar ratio of known
secondary to primary bile acids in the CaPi group was only
45% of the ratio observed in the control group (P < 0.05).
Substantial amounts of four unknown bile acids were detected in the
ileal lavages (Table 2)
. The in vitro incubation experiments with bile
salt hydrolase resulted in a complete disappearance of peaks B, C and D
in the chromatogram and a concomitant increase in free ß-muricholic
acid and peak A. This indicates that peaks B, C and D are taurine or
glycine conjugates of ß-muricholic acid and the unidentified bile
salt A. Additional analyses are being performed to identify the unknown
bile acids. CaPi supplementation also reduced the total
concentration of bile acids and fatty acids in ileal lavages of
infected rats (P < 0.05; data not shown). Noninfected rats
fed the CaPi-supplemented diet had a significantly lower
cytotoxicity of fecal water and concomitantly decreased concentration
of bile acids and fatty acids in fecal water (Fig. 2)
. These effects of
dietary CaPi were also observed in salmonella-infected rats
(data not shown).
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After oral infection, the fecal salmonella excretion of the
CaPi group was only 1–10% of that observed in the control
group (P < 0.05, Fig. 3A
). In accordance, significantly fewer viable salmonella were measured in
ileal lavages and ileal scrapings of the CaPi group
(Table 3
). The improved colonization resistance of rats fed the
CaPi-supplemented diet coincided with a significantly
reduced translocation of S. enteritidis, as measured by the
lower infection-induced urinary NOx excretion (Fig. 3
B).
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Dietary CaPi stimulated the intestinal lactobacilli as
judged by significantly more viable counts of these lactic acid
bacteria in ileal contents, ileal scrapings and feces of noninfected
animals (Table 3)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the colonization resistance to salmonella was greatly improved
by dietary CaPi, because fecal shedding of this pathogen
was significantly reduced. We now show that this coincides with a
diminished colonization of the ileal epithelium by this pathogen.
Viable salmonella numbers detected in scrapings of the ileal mucosa and
in ileal lavages of the CaPi group were only 10% of those
observed in the control group. The observation that salmonella was
still present in the ileum 6 d after the oral introduction of this
pathogen indicates that salmonella did not simply pass, but truly
colonized the ileal epithelium. The ileum is the initial intestinal
region exploited by salmonella to gain access to deeper host tissues,
resulting in a systemic infection (Clark et al. 1994
). Considering the
significantly reduced urinary NOx excretion after
infection, the CaPi-mediated reduction in colonization of
the ileal mucosa by salmonella resulted in a decreased translocation of
this pathogen. Nitric oxide (NO) is aspecifically produced by host
phagocytes, such as macrophages and Kupffer cells, when activated by
bacteria or bacterial components. NO is rapidly oxidized to nitrite and
nitrate and excreted in urine (Fang 1997
). We have shown previously
that urinary NOx excretion is a more valid and quantitative
biomarker to assess total intestinal bacterial translocation than
classical microbiological organ cultures (Bovee-Oudenhoven et al. 1997b
, Oudenhoven et al. 1994
). Though obvious signs of diarrhea were
absent, the more severe infection of the control group was also
reflected in a significantly greater fecal wet weight percentage, as
calculated by the concentration of fecal electrolytes. In contrast, the
wet weight percentage of feces of the CaPi-supplemented
group after infection did not differ from that of noninfected rats.
The most interesting finding of the present study is the modulation of
the intestinal microflora by dietary CaPi, which might be
the mechanism responsible for the observed protective effects of
dietary CaPi. The endogenous microflora is an extremely
important determinant of host defense against intruding pathogenic
bacteria (Sarker and Gyr 1992
, Vollaard and Clasener 1994
). Elimination
of parts of the protective intestinal microflora by use of antibiotics
in humans (Bartlett 1992
, Neal et al. 1994
) and animals (Wells et al. 1988
) is frequently accompanied by opportunistic infections. An optimal
endogenous microflora outcompetes invading pathogens for adhesion sites
on the intestinal mucosa and for luminal nutrients. In addition, many
products of the active bacterial metabolism in the intestine, for
instance lactic acid and short-chain fatty acids, can damage pathogens
and prevent them from colonizing the epithelium (Bovee-Oudenhoven et al. 1997a
, Sarker and Gyr 1992
). In a former study, we showed that
dietary CaPi has a trophic effect on the rat intestinal
microflora in general. CaPi-supplementation increased fecal
excretion of dry weight, nitrogen, phospholipids, and organic
phosphate, which are supposed to be markers of bacterial mass
(Bovee-Oudenhoven et al. 1997b
). A similar effect of dietary calcium
was found in humans (Govers et al. 1996
). This phenomenon is now
extended because the present study clearly shows that dietary
CaPi changed the composition of the endogenous microflora
and stimulated the lactobacilli. Besides an increased excretion of
these lactic acid bacteria in feces, significantly greater numbers were
found in the ileal lumen and adhering to the ileal epithelium. Rather
suggestive is the observation that the extent of the rise of intestinal
lactobacilli and the increase of fecal lactic acid excretion caused by
CaPi supplementation were roughly comparable. As mentioned
above, translocation of salmonella mainly takes place in the ileum
(Clark et al. 1994
). Because lactobacilli are the predominant species
in this region of the gastro-intestinal tract of humans and animals
(Drasar 1988
), they might play an important role in the resistance to
colonization and translocation of food-borne pathogens. Though it is
not yet proved that autochthonous lactobacilli are antagonistic towards
invading bacteria, orally administered lactobacilli are reported to be
protective. For instance, Hudault et al. (1997)
showed that
administration of L. acidophilus to mice improved their
colonization- and translocation-resistance to Salmonella
typhimurium. Additionally in a number of clinical investigations,
ingestion of lactic acid bacteria resulted in shortening of the
duration of antibiotic-associated diarrhea (Siitonen et al. 1990
), the
prevention of recurrent relapses of Clostridium difficile
colitis (Biller et al. 1995
) and amelioration of acute rotavirus
enteritis in children (Kaila et al. 1996
).
A possible mechanism by which dietary CaPi
strengthens the intestinal lactobacillus flora might involve bile acids
and fatty acids. The present in vitro experiment showed that L.
acidophilus was much more sensitive than S. enteritidis
to the surface-active properties of bile acids and fatty acids. In line
with results reported earlier (Floch et al. 1971
, Van der Meer et al. 1991
), the more hydrophobic, unconjugated bile acids are more strongly
bactericidal than their conjugated counterparts. The range of
surfactant concentrations used in the in vitro experiment was
physiologically relevant because comparable concentrations were
determined in ileal contents and fecal water. Moreover, to obtain
sufficient material from the ileal lumen for analyses, the ileal
contents were diluted (approximately three-fold) with saline. So, the
reported bile acid and fatty acid concentration in ileal lavages will,
if anything, underestimate the in vivo situation. In agreement with
results reported earlier (Govers and Van der Meer 1993
, Govers et al. 1996
, Van der Meer et al. 1991
), bile acids and fatty acids are
strongly adsorbed to and precipitated by intestinal CaPi,
resulting in a significantly lower concentration of these surfactants
in fecal water. CaPi also reduced the bile acid and fatty
acid concentration of the water phase of ileal contents. In the small
intestine, the majority of the bile acids is still conjugated with
either taurine or glycine because extensive bacterial deconjugation of
bile acids takes place only in the colon (Eyssen and Caenepeel 1988
).
In general, unconjugated bile acids and glycine-conjugated bile acids
were more strongly precipitated by CaPi than
taurine-conjugated bile acids, in agreement with our earlier study
(Govers et al. 1994
). The present study clearly shows that dietary
CaPi significantly affected the composition of ileal bile
acids. The total concentration of unconjugated bile acids, as well as
the ratio of secondary to primary bile acids, was significantly lower
in rats fed CaPi. The cytotoxicity or lytic activity of
bile acids increases with increasing hydrophobicity (Lapré et al. 1992
). Unconjugated bile acids and secondary (dehydroxylated) bile
acids are more hydrophobic and cytotoxic than conjugated bile acids and
primary bile acids, respectively. Therefore, the above mentioned change
in the composition of ileal bile acids indicates a
CaPi-mediated shift to a less cytotoxic bile acid pool.
This might also have favored growth of the endogenous lactobacilli.
Extrapolation to the human situation is probably permissible. As shown
earlier in a human intervention trial, dietary calcium significantly
decreased the ratio of dihydroxy bile acids to trihydroxy bile acids
and thus decreased the cytotoxicity of the duodenal bile acid pool (Van der Meer et al. 1990
). However, no direct evidence is yet available
demonstrating that dietary calcium stimulates the endogenous
lactobacilli in humans.
In conclusion, by precipitating fatty acids and bile acids in the
intestinal lumen and changing the composition of ileal bile acids into
a less cytotoxic one, supplemental CaPi probably creates a
less aggressive environment for the endogenous lactobacilli and
stimulates their growth. The increased numbers of intestinal
lactobacilli might have improved the resistance to colonization and
translocation of salmonella in rats. The growth-promoting activity of
dietary CaPi for the endogenous lactobacilli is probably
also relevant for the functionality of probiotic strains used in foods.
It is generally accepted that lactobacilli used as a dietary adjunct
must be able to survive the hostile environment in the
gastro-intestinal tract and proliferate (Mital and Garg 1995
). That aim
might be achieved better when an adequate CaPi intake is
ensured.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
3 Abbreviations used: BGA, brilliant green agar;
CaPi, dietary calcium phosphate; HPLC, high performance
liquid chromatography; MRS, de Man, Rogosa, and Sharpe broth; NO,
Nitric oxide; NOx, nitrite and nitrate.
Manuscript received May 15, 1998. Initial review completed July 7, 1998. Revision accepted November 19, 1998.
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