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1;
*
Institut Paul Lambin and
Université Catholique de Louvain, B1200 Brussels, Belgium
1To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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n 60) yields an oligofructose preparation that
is composed of both GFn-type and Fn-type oligosaccharides (2
n 7; 2 m 7), where G is
glucose, F is fructose, and n is the number of ß(2
1) bound
fructose moieties. Human studies have shown that feeding GFn-type
oligomers significantly modifies the composition of the fecal
microflora especially by increasing the number of bifidobacteria. The
experiments reported here were used to test the hypothesis that the
Fn-type molecules have the same property. During a controlled
feeding study, 8 volunteers (5 females and 3 males) consumed 8 g/d of
an Fn-rich product for up to 5 wk. Fecal samples were collected and
analyzed for total anaerobes, bifidobacteria, lactobacilli,
bacteroides, coliforms and Clostridium perfringens. Both
2 and 5 wk of oligofructose feeding resulted in a selective increase in
bifidobacteria (P < 0.01). In addition, a daily
intake of 8 g of the Fn-type oligofructose preparation reduced
fecal pH and caused little intestinal discomfort.
KEY WORDS: • prebiotics • inulin • oligofructose • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). Proliferation of bifidobacteria in
fecal microflora, a surrogate marker for the colonic microbiota, has
been associated with several beneficial effects. A dietary approach
aimed at improving the composition of the fecal microflora by supplying
substrates that allow selective proliferation of such indigenous
bacteria, the prebiotic approach, has been proposed (Gibson and Roberfroid 1995
) and validated in different human studies using
different nondigestible oligosaccharides (Gibson et al. 1999
). In particular, it has been shown that the consumption of
chicory inulin or its partial hydrolysate (oligofructose), a mixture of
ß(21) bound GFn-type (glucosyl-[fructosyl]n-1-fructose) and
ß(21) bound Fn-type ([fructosyl]n-1-fructose) species
(De Leenheer and Hoebregs 1994
), significantly modifies
the composition of the human fecal flora in such a way that
bifidobacteria become numerically predominant (Roberfroid et al. 1998
, Van Loo et al. 1999
). Native chicory
inulin is composed of >99% of the GFn-type species (2 n 60), but the oligofructose preparation, which is
produced from inulin by partial enzymatic hydrolysis, is a mixture of
both
GFn2
(2 n 7) and Fn (2 n
7)-type molecules [where G is glucose, F is fructose and
n is the number of ß(21) bound fructose
moieties] which also occur naturally in plant foods such as
banana, garlic, onion, salsify, asparagus, leek, wheat, chicory, etc.
(Van Loo et al. 1995
).
The objective of the present study was to test the hypothesis that,
like the GFn-type, the Fn-type chicory oligofructose
preparation selectively stimulates the growth of fecal bifidobacteria
in humans. The protocol for the human study was very similar to
recently published studies in terms of number of volunteers (8–12),
protocol and bacteriological methodologies employed (Buddington et al. 1996
, Gibson et al. 1995
, Kleessen et al. 1997
, Williams et al. 1994
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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All chemicals used in this study were of the purest grade available and were purchased from Merck (Darmstadt, Germany); Oxoid (Basingstoke, United Kingdom) or Sigma (St. Louis, MO).
Study food.
The Fn-type-rich chicory oligofructose preparation was provided by ORAFTI (Tienen, Belgium) as Raftilose® L60, which is produced by partial enzymatic hydrolysis of a refined hot-water extract of chicory roots (i.e., inulin). It is available as an aqueous syrup containing 750 g/kg dry matter composed of 75 g (10%) glucose + fructose, 225 g (30%) sucrose and 450 g (60%) oligofructose [with 45 g (10%) GFn-type and 405 g (90%) Fn-type]. The product used in the experiments was of food-grade quality.
Volunteers.
The study protocol was approved by the ad hoc ethical committee of the University (UCL-Brussels, Belgium) and complies with the Helsinki declaration of 1975 as revised in 1983. No history of gastrointestinal disease and no use of gastrointestinal or antibiotic medications for at least 3 mo prior to and during the trials were the inclusion criteria. Human subjects who participated in the trial were five women and three men aged between 20 and 50 y having a body mass index between 19 and 25 kg/m2, and between 18 and 24 kg/m2, respectively. Subjects gave written consent to participate in the study.
Protocol for the human study.
The eight volunteers participated in the experiment, which lasted for 7 wk divided into three successive periods: i) control, a period of 2 wk, during which the volunteers were all given a controlled diet without any addition of oligofructose; ii) treatment 1, a first treatment period of 2 wk, during which the controlled diet was supplemented with 8 g/d of chicory oligofructose; iii) treatment 2, a second treatment period of 3 wk, during which the volunteers consumed their usual home-cooked diet to which they added 8 g/d of chicory oligofructose. The chicory oligofructose (Raftilose® L60) composed of 90% Fn-type and 10% GFn-type molecules was incorporated into orange juice, various desserts (puddings, creams and fruit mousses), cakes and biscuits that were part of the food consumed by the volunteers during the day, in such quantities as to provide a total daily intake of 8 g of chicory oligofructose, of which 90% (7.2 g) was pure Fn-type.
Feeding a controlled diet during periods 1 and 2 was intended to minimize the interindividual variations in food intakes that could have influenced the composition of the fecal microflora independent of oligofructose intake.
During these two periods, the volunteers were required to visit a
central restaurant, where they had access to a buffet (breakfast and
lunch) and were given a vacuum-sealed dinner to consume at home.
These meals were prepared so as to minimize the consumption of
naturally oligofructose/inulin-rich products (Van Loo et al. 1995
) like onions, leeks, bananas, artichokes and wheat, as
well as yogurts and fermented milk products. During these two periods,
the foods given to the volunteers were very similar, except for the
intake of chicory oligofructose (8 g/d) during period 2. During period
3, the volunteers were asked to consume their usual home-cooked
meals but still excluding oligofructose/inulin-rich food products and
fermented dairy products.
As in other studies on the bifidogenic effect of fructans
(Buddington et al. 1996
, Gibson et al. 1995
, Kleessen et al. 1997
, Williams et al. 1994
), each volunteer acted as his/her own control and no
separate placebo group was included. Using such a protocol avoids a
cross-over design in which the length of the wash-out interval
is often difficult to evaluate precisely.
Sample collection.
Fresh stools were collected: sample 1 (last day of wk 2) at the end of the control period; sample 2 (last day of wk 4) at the end of the treatment 1 period; and sample 3 (last day of wk 7) at the end of the treatment 2 period.
During both the control and treatment 1 periods, the volunteers were requested to complete a daily well-being questionnaire, providing information about possible digestive discomfort (cramps, bloating, flatulence, soft stools or diarrhea) as well as frequency and appearance of stools.
Protocol for bacteriological analyses (Beerens 1991
, Gibson et al. 1995
).
All stool samples (minimum weight 20 g) were processed anaerobically (desk-type home-made anaerobic glove-box containing an atmosphere of H2, CO2 and N2, 10:10:80) within 60 min after defecation. Samples were weighed and then homogenized in 0.1 mol/L (pH 7) phosphate buffer to obtain a 100 g/L fecal suspension. Serial dilutions were prepared using half-strength Peptone water (Oxoid), the samples (0.1 mL) were inoculated onto agar medium specific for the growth of total anaerobes (Wilkins-Chalgren anaerobic agar), bifidobacteria (Clostridia agar supplemented with 0.0125 g/L iodoacetic acid, 0.02 g/L nalidixic acid, 0.05 g/L kanamycin, 0.009 g/L polymyxin, 0.025 g/L triphenyltetrazolium chloride), lactobacilli (Rogosa), coliforms (MacConkey #3), bacteroides (BMS supplemented with 5 g/L glucose, 0.5 g/L ammonium sulfate, 0.01g/L nalidixic acid and 0.003 g/L vancomycin) and Clostridium perfringens (Tryptose Sulfite Cycloserine Agar Base or TSC supplemented with fluorcult).
Anaerobic incubations (in duplicate) for colony development took place
in anaerobic jars containing Anaerocult A (Merck). For each fecal
sample, a count was made of viable colony-forming units (cfu) of
total anaerobes after incubation at 37°C for 4 d, bifidobacteria
(4 d), bacteroides (4 d), lactobacilli (3 d), coliforms (1 d) and
clostridia (1 d). After incubation, individual colonies were removed
from the plates and subcultured into peptone/yeast/glucose broth.
Bacteria were characterized to genus level on the basis of colony
appearance, Gram’s reaction and cell morphology. Presumptive culture
identities were confirmed through colony morphotype, microscopic
characteristics and limited biochemical tests (Gibson et al. 1995
).
Statistical analysis.
The nonparametric Friedman test was used after logarithmic transformation of the data. This test, made by order of rank (rank averages) was chosen because it permits comparison of several mean values of nonindependent observations, which is the case in this study, where comparable samples were all taken from the same volunteers but during different feeding periods. Results were statistically analyzed on the basis of: i) a global comparison of mean values to identify differences between the three feeding periods and ii) paired comparisons to search for differences between periods. The significance threshold was set at 5% (P < 0.05).
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RESULTS AND DISCUSSION |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
,
Gibson et al. 1999
, Roberfroid et al. 1998
).
Table 1
reports the values of the total numbers of cfu (expressed as
log10 cfu/g of feces) for the various bacteria
analyzed in the feces of the eight volunteers fed a diet with and
without chicory oligofructose. A global analysis of the different
values reveals that the daily intake of 8 g of oligosaccharides
did not significantly (P > 0.05) modify the counts of
total anaerobes, lactobacilli, bacteroides, coliforms or C.
perfringens, but it did significantly (P < 0.01)
increase the counts of bifidobacteria.
|
These data thus demonstrate that, as is the case with GFn-type
oligofructose (Gibson et al. 1995
, Roberfroid et al. 1998
, Van Loo et al. 1999
), a preparation of
chicory oligofructose containing 90% of Fn-type molecules
selectively stimulates the growth of colonic bifidobacteria in human
volunteers, as evidenced by the increase in fecal number. Furthermore,
the data demonstrate the selectivity of that stimulation of growth,
thus confirming the prebiotic nature of chicory Fn-type
oligofructose.
At the end of the treatment 1 and treatment 2 periods, the fecal pH in
all the volunteers had dropped by ~1 pH unit compared to the end of
the control period. Such an effect is best explained by a change in
colonic fermentation and confirms previous observations both in vitro
(Wang and Gibson 1993
) and in vivo (Gibson et al. 1995
, Kleessen et al. 1997
). The present study
was not specifically designed to quantify changes in gut function
parameters. However, when analyzing answers to the well-being
questionnaires recorded during the control period vs. the treatment 1
period, changes in stool frequency (+ 12%) as well as in the
appearance (softer) and the amount (evaluated qualitatively as "more
than usual") of stools showed a tendency to confirm the bulking
effect reported by Gibson et al. (1995)
and by
Den Hond et al. (1997). Moreover, an analysis of the
intestinal side-effects associated with the meals during the
periods of chicory oligofructose intake, as reported on the
acceptability forms, revealed that from a total of 224 meals (8
volunteers receiving 2 meals/day for 2 wk), only six "mild"
complaints were reported. These included one case of increased
flatulence, three cases of intestinal distension and two cases of
cramps in the intestine. It can be stated that the consumption of 8 g/d
chicory oligofructose (of which 7.2 g was Fn-type molecules)
is therefore not likely to cause significant intestinal discomfort.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1)
fructose moieties; Fn, F is fructose, and n is number of ß(21)
fructose moieties. 
Manuscript received September 9, 1999. Revision accepted January 10, 2000.
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REFERENCES |
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|---|
1. Beerens H. An elective and selective isolation medium for bifidobacterium spp. Lett. Appl. Microbiol. 1991;11:155-157
2.
Buddington R. K., Williams C. H., Chen S. C., Witherly S. A. Dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. Am. J. Clin. Nutr. 1996;63:709-716
3. Cummings, J. H. (1997) The large intestine in nutrition and disease. Danone Chair Monograph, Institut Danone, Brussels, Belgium.
4. De Leenheer L., Hoebregs H. Progress in the elucidation of the composition of chicory inulin. Starch 1994;46:193-196
5. Den Hond E. M., Geypens B. J., Ghoos Y. F. Long chain chicory inulin positively affects bowel habit in healthy subjects with a low stool frequency. Nutr. Res. 2000;108:975-982in press
6. Gibson G. R., Beatty E. R., Wang X., Cummings J. H. Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology 1995;108:975-982[Medline]
7. Gibson G. R., Rastall R. A., Roberfroid M. B. Prebiotics. Gibson G. R. Roberfroid M. B. eds. Colonic Microbiota, Nutrition and Health 1999:101-124 Kluwer Academic Press Dordrecht, The Netherlands.
8. Gibson G. R., Roberfroid M. B. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 1995;125:1401-1412
9.
Kleessen B., Sykura B., Zunft H. J., Blaut M. Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons. Am. J. Clin. Nutr. 1997;65:1397-1402
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Roberfroid M. B., Van Loo J.A.E., Gibson G. R. A review of the bifidogenic nature of chicory inulin and its hydrolysis products. J. Nutr. 1998;128:11-19
11. Van Loo J., Coussement P., De Leenheer L., Hoebregs H., Smits G. On the presence of inulin and oligofructose as natural ingredients in the western diet. CRC Crit. Rev. Food Sci. Nutr. 1995;35:525-552
12. Van Loo J, Cummings J. H., Delzenne N., Englyst H. N., Franck A., Hopkins M. J., Kok N., Macfarlane G. T., Newton D. F., Quigley M. E., Roberfroid M. R., van Vliet T., Van den Heuvel E.G.H.M. Functional food properties of nondigestible oligosaccharides: a consensus report from the ENDO project (DGXII AIRII-CT94–1095). Br. J. Nutr. 1999;81:121-132[Medline]
13. Wang X., Gibson G. R. Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the large intestine. J. Appl. Bacteriol. 1993;75:373-380[Medline]
14. Williams C. H., Witherly S. A., Buddington R. K. Influence of dietary Neosugar on selected bacterial groups of the human fecal microbiota. Microbial Ecol. Health Dis. 1994;7:91-97
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