Dietary prebiotic oligosaccharides are detectable in the faeces
of formula-fed infants
GUIDO E. MORO1, BERND STAHL2, SILVIA FANARO4, JU¨RGEN JELINEK2,
GU¨NTHER BOEHM1,2& GIOVANNI V. COPPA3
1Centre for Infant Nutrition, Macedonio Melloni Maternity Hospital, Milan, Italy,2Numico Research Germany,
Friedrichsdorf, Germany,3Department of Paediatrics, University of Ancona Institute of Paediatrics, Ancona, Italy, and
4Department of Clinical and Experimental Medicine, Division of Neonatology, University of Ferrara, Ferrara, Italy
Human milk oligosaccharides are not digested during intestinal passage and can be detected in stools. In this study it was
fructo-oligosaccharides (FOS) can be detected in stool samples of formula-fed infants. The test formula was supplemented
with 0.8 g/dl oligosaccharides (GOS+FOS). In the control formula, maltodextrins were used as placebo. Fecal flora was
assessed at the beginning (day 1) and at the end of a 28-d feeding period (day 2). At day 2 the content of galacto- and fructo-
oligosaccharides in the stool samples were measured. On study day 1, the number of bifidobacteria was not different among
the groups (supplemented group: 7.7 (6.2) CFU/g; placebo group: 8.0 (6.0) CFU/g). At the end of the 28-d feeding period,
the number of bifidobacteria was significantly higher in the group fed the supplemented formula when compared to placebo
(supplemented group: 9.8 (0.7) CFU/g stool; placebo group: 7.1 (4.7) CFU/g stool; p50.001). In all infants fed the sup-
plemented formula, GOS and FOS could be identified in the stool samples. That was not the case in infants fed the non-
Conclusion: The present data confirm the bifidogenicity of oligosaccharides and indicate that dietary galacto-
oligosaccharides and long chain fructo-oligosaccharides remain during the whole passage in the lumen of the gastrointestinal
tract, similarly to human milk oligosaccharides.
Key Words: Galacto-oligosaccharides, long chain fructo-oligosaccharides, digestibility, infants
In breastfed infants the intestinal microflora is domi-
nated by bifidobacteria and lactobacilli , and this
microbial pattern beneficially affects the intestinal
function and the development of the immune system
[2,3]. Although the mechanisms of these effects are
very complex and not fully understood, dietary inter-
ventions to establish an intestinal microflora rich in
bifidobacteria and lactobacilli are recommended [4,5].
The effect of human milk on the intestinal flora is
caused by its content of selective agents which can
stimulate the growth of bifidobacteria and lactobacilli.
Oligosaccharides, which are a major component of
factor of human milk [6–8].
The composition of neutral human milk oligo-
saccharides is very complex . Due to their
complexity, it is not possible to reproduce in infant
human milk. To mimic the prebiotic effect of human
milk oligosaccharides, a mixture of 90% galacto-
oligosaccharides (GOS) (derived from lactose ) and
10% fructo-oligosaccharides (FOS) (high-molecular-
weight fraction of inulin extracted from chicory roots
) has been used. The mixture was designed to have
a molecular size distribution similar to that of neutral
human milk oligosaccharides .
Human milk oligosaccharides can be detected in the
faeces of breastfed infants . More recently, it has
been shown that human milk oligosaccharides are
resistant to enzymatic digestion in the upper gastro-
intestinal tract . Non-digestibility and selective
fermentation in the colon by potentially beneficial
bacteria are prerequisites for the prebiotic effect of
these dietary ingredients .
identicalto those of
Correspondence: Bernd Stahl, Numico Research Germany, Bahnstr. 14–30, 61381 Friedrichsdorf/Ts, Germany. Tel: +49 6172 991496. Fax: +49 6172
991862. E-mail: firstname.lastname@example.org
Acta Pædiatrica, 2005; 94(Suppl 449): 27–30
ISSN 0803-5326 print/ISSN 1651-2227 online#2005 Taylor & Francis Group Ltd
Thus, the aim of the present study was to investigate
whether the mixture of galacto- and fructo-oligo-
fed an infant formula supplemented with the prebiotic
mixture of GOS and FOS.
Patients and methods
The study protocol was approved by the two ethical
committees of the hospitals, and informed parental
in the study.
Thirty-two term infants, appropriate for gestational
age, were randomly selected from a cohort of the
population of a study designed to investigate the dose
dependence of the bifidogenic effect of the prebiotic
mixture . In all infants, enteral nutrition was star-
ted with breast milk according to the practice of the
hospital. Only when the mother was unable or decided
not to breastfeed was the infant randomly assigned to
one of three formula groups. The composition of the
two formulas was identical, except for the supple-
mented oligosaccharides. The active formula was
supplemented with 0.8 g/dl of the oligosaccharides
mixture, and the control formula was supplemented
with maltodextrines as placebo (Table I).
The most relevant clinical data of the formula-fed
infants under study are summarized in Table II.
For microbiological analysis, stool samples were
collected at the beginning when formula feeding star-
ted (study day 1) and 28 d after (study day 2). The
stool samples of study day 2 were analysed for
the presence of components of the oligosaccharides
A quantity of 0.2 g of fresh faecal sample was
homogenized in a cryo-protective glycerol transport
medium (glycerol 10 ml, oxoid 0.1 g, H2O ad 100 ml)
and immediately frozen at 780?C. The samples were
transported on dry ice. For the identification of bifi-
dobacteria and lactobacilli, selective media were used
(bifidobacteria: DIC medium (Bonaparte 1997);
lactobacilli: Rogosa) as described previously .
The components of the oligosaccharide mixture
were analysed using high-performance anion-exchange
chromatography. A faecal sample (200 mg) from each
infantwas storedin2.0 mlofculturemedium (equalto
1:10, w/v dilution). The sample was further diluted
with deionized water (1:1, v/v) and stored overnight at
4?C. The sample was centrifuged at 5000 rpm
for 15 min. The supernatant was filtered through a
0.22-mm membrane (Millipore, Bedford, MA, USA).
The sample was further diluted (1:200, v/v) with
deionized water for chromatographic analyses.
High-performance anion-exchange chromatography
(HPAEC) was performed on a HPAEC-system AI
450 equipped with a CarboPac PA-1 precolumn
(4?25 mm) and a CarboPac PA-1 column (4?
250 mm). A 25-ml fecal solution sample was injected,
by means of an AMS autosampler. The detector was a
pulsed amperometric detector PAD II (all: Dionex,
Sunnyvale, CA, USA). Two different methods were
used to detect low-molecular-weight oligosaccharides
and high-molecular-weight oligosaccharides .
Anthropometric data are given as means+standard
deviation (SD). The respective homogeneity of groups
was tested by one-way analysis of variance (ANOVA).
To account for data not normally distributed, the
data on the microflora were described as medians and
interquartile ranges (IQR, 25th–75th percentile). As a
consequence, the influence of the feeding regimens
on these parameters was investigated using non-
parametric tests. For an overall group effect, the
Kruskal-Wallis test was used.
All tests were performed on an alpha-level of 5%.
P-values 50.05 were considered significant. The
software StatView 5.0 (SAS Institute Inc.) was used.
Results and discussion
On study day 1, the number of bifidobacteria was not
different among the groups (supplemented group: 7.7
Table I. Composition of the two studied formulas per decilitre.
- Lactose (g)
- GOS/FOS mixture (g)
- Maltodextrines (g)
Energy content (kcal)
Table II. Clinical data of the infants enrolled in the study.
Gestational age (wk)
Weight at birth (g)
Length at birth (cm)
Age at study entry (d)
Feeding volume (ml/kg?d)
Weight gain during study
Length gain during study
G. E. Moro et al.
(6.2) CFU/g; placebo group: 8.0 (6.0) CFU/g). At the
end of the 28-d feeding period, the number of bifido-
bacteria was significantly higher in the group fed the
supplemented formula when compared to the placebo
group (supplemented group: 9.8 (0.7) CFU/g stool;
placebo group: 7.1 (4.7) CFU/g stool; p50.001).
In all infants fed the supplemented formula, GOS
and FOS could be identified in the respective
stool samples (Figure 1A). That was not the case
in all infants fed the non-supplemented formula
The data demonstrate clearly that GOS as well as
FOS are present during the whole passage in the lumen
of the gastrointestinal tract. The same effect has been
shown for human milk oligosaccharides using the same
methodology as in the present study .
theprebiotic function ofan ingredient. The bifidogenic
effect ofthe GOS/FOS mixture hasbeen demonstrated
in several studies, which is in line with the present
The core structure of human milk oligosaccharides
consists of monosaccharides in b-glycosidic linkage.
This is also the distinctive feature of the galacto-
oligosaccharides and long-chain fructo-oligosacchar-
ides . The enzymes of the human gastrointestinal
tract are not able to hydrolyse these glycosidic bonds;
this holds especially true for the brush border glyco-
hydrolases of the small intestine . This is one of the
prerequisites of a prebiotic compound to be available
for the beneficial bacteria of the human intestine. In
addition to that, b-linked galactose is the key structural
element of the GOS representing the majority of
molecules in the GOS/FOS mixture, but also an
important characteristic of all human milk oligo-
In conclusion, the data of the present study
demonstrate that dietary GOS as well as long chain
FOS are present during the whole gastrointestinal
passage, and this underlines their capability to act as
prebiotic ingredients for infant formulas.
 Harmsen HJM, Wildeboer-Veloo ACM, Raangs GC, Wagen-
dorp AA, Klijn N, Bindels JG, et al. Analysis of intestinal flora
development in breast-fed and formula fed infants by using
molecular identification and detection methods. J Pediatr
Gastroenterol Nutr 2000;30:61–7.
 Hanson LA,TelemoE,Wiedermann U,Dahlman-Hoglund A,
Ludin S, Friman V, et al. Immunological mechanisms of the
gut. Pediatr Allergy Immunol 1995;6 Suppl 8:7–12.
 Gro ¨nlund MM, Arvilommi H, Kero P, Lethonen OP, Isolauri
E. Importance of intestinal colonisation in the maturation of
humoral immunity in early infancy: a prospective follow up
study of healthy infants aged 0–6 months. Arch Dis Child Fetal
Neonatal Ed 2000;83:F186–92.
 Gibson GR, Roberfroid MB. Dietary modulation of the human
colonic microbiota: introducing the concept of probiotics.
J Nutr 1995;125:1401–12.
 Fooks lJ, Fuller R, Gibson GR. Prebiotics, probiotics and
human gut microbiology. Intern Diary J 1999;9:53–61.
 Boehm G, Stahl B. Oligosaccharides. In: Mattila-Sandholm T,
editor. Functional dairy products. Cambridge: Woodhead
Publishing; 2003. p. 203–43.
 Newburg DS. Oligosaccharides in human milk and bacterial
colonisation. J Pediatr Gastroenterol Nutr 2000;30:S8–17.
 Kunz C, Rudloff S. Biological functions of oligosaccharides in
human milk. Acta Paediatr 1993;82:903–12.
 Zarate S. & Lopez-Leiva M.H. Oligosaccharide formation
during enzymatic lactose hydrolysis: a literature review. J Food
 Roberfroid MB, Van Loo JAE, Gibson GR. The bifidogenic
nature of chicory inulin and its hydrolysis products. J Nutr
 Boehm G, Fanaro S, Jelinek J, Stahl B, Marini A. Prebiotic
concept for infant nutrition. Acta Paediatr 2003;91 Suppl
 Coppa GV, Pierani P, Zampini L, Bruni S, Carloni I, Gabrielli
O. Characterization of oligosaccharides in milk and feces
of breast fed infants by high-performance anion-exchange
chromatography. Adv Exp Med Biol 2001;501:307–14.
 Engfer MB, Stahl B, Finke B, Sawatzki G, Daniel H. Human
milk Oligosaccharides are resistant to enzymatic hydrolysis
in the upper gastrointestinal tract. Am J Clin Nutr 2000;71:
 Moro G, Minoli I, Mosca F, Jelinek J, Stahl B, Boehm G.
Dosage effect of oligosaccharides on faecal flora and stool
characteristics in preterm infants. J Pediatr Gastroenterol Nutr
 Fanaro S, Vigi V, Chierci R, Boehm G. Fecal flora measure-
ments of breast fed infants using an integrated transport and
culturing system. Acta Paediatr 2003;92:634–5.
 Coppa GV, Bruni S, Zampini L, Galeazzi T, Gabrielli O.
Prebiotics in infant formulas: biochemical characterization by
Retention time (min)
PED response (µC)
PED response (µC)
Figure 1. HPAE chromatography according to  of diluted
human faecal samples. (A) Chromatogram of a faecal sample from a
GOS/FOS-fed infant. (B) Chromatogram of a faecal sample from a
standard formula (i.e. non-supplemented)-fed infant. The peaks
eluting between 25 min and 35 min represent individual structures
of galacto-oligosaccharides (GOS). The peaks eluting between
45 min and 53 min represent individual structures of fructo-
Digestibility of prebiotic oligosaccharides
thin layer chromatography and high performance anion
exchange chromatography. Dig Liver Dis 2002;34:S124–8.
 Boehm G, Lidestri M, Casetta P, Jelinek J, Negretti F, Stahl B,
et al. Supplementation of an oligosaccharide mixture to a
bovine milk formula increases counts of faecal bifidobacteria in
 Schmelzle H, Wirth S, Skopnik H, Radke M, Knol J, Bo ¨ckler
H, et al. Randomised, double-blind study to investigate the
nutritional efficacy and bifidogenic characteristics of a new
infant formula. J Ped Gastroenterol 2003;36:343–51.
 Boehm G. Funktionelle Komponenten fu ¨r Sa ¨uglingsnahrun-
gen. In: Erbersdobler HF, Meyer AH, editors. Praxishandbuch
functional food. Hamburg: Behr’s Verlag; 2003. p. 1–24.
 VanBeersEH,Bu ¨llerHA,GrandRJ,EinerhandAWC,Dekker
J. Intestinal brush border glycohydrolases: structure, function
and development. Crit Rev Biochem Mol Biol 1995;30:
G. E. Moro et al.
Page 5 Download full-text