The Journal of Nutrition

Nutritional Immunology

Fecal Secretory Immunoglobulin A Is Increased

in Healthy Infants Who Receive a Formula with

Short-Chain Galacto-Oligosaccharides and

Long-Chain Fructo-Oligosaccharides1,2

Petra A. M. J. Scholtens,3* Philippe Alliet,4 Marc Raes,4 Martine S. Alles,3 Hilde Kroes,3

Guenther Boehm,3,5 Leon M. J. Knippels,3 Jan Knol,3 and Yvan Vandenplas6

3Numico Research, Wageningen 6700 CA, The Netherlands; 4Virga Jesse Hospital, Hasselt 3500, Belgium; 5Sophia Children’s Hospital,

Rotterdam 3015 GJ, The Netherlands; and 6University Hospital Brussel, Brussels 1090, Belgium

Abstract

In this double-blind, randomized, placebo-controlled study, we investigated the effect of an infant milk formula with 6 g/L

short-chain galacto- and long-chain fructo-oligosaccharides [(scGOS/lcFOS) ratio 9:1] on the development of the fecal

secretory immunoglobulin A (sIgA) response and on the composition of the intestinal microbiota in 215 healthy infants

during the first 26 wk of life. The infants received breast milk or were randomized to receive an infant milk formula with or

without scGOS/lcFOS. Stool samples were collected after 8 and 26 wk of intervention. The concentration of fecal sIgA

was determined by ELISA, and the composition of the intestinal microbiota was determined by quantitative fluorescent in

situ hybridization. The scGOS/lcFOS group and the control group were compared in the statistical analysis. A breast fed

group was included as a reference. In total, 187 infants completed the study. After 26 wk of intervention, in infants that

were exclusively formula fed, the concentration of sIgA was higher (P, 0.001) in the scGOS/lcFOS group (719 mg/g) than

in the control group (263 mg/g). In addition, the percentages of bifidobacteria were higher in the scGOS/lcFOS group

(60.4%) than in the control group (52.6%, P ¼ 0.04). The percentages of Clostridium spp. were 0.0 and 3.27%, respec-

tively (P ¼ 0.006). In conclusion, an infant milk formula with 6 g/L scGOS/lcFOS results in higher concentrations of fecal

sIgA, suggesting a positive effect on mucosal immunity. J. Nutr. 138: 1141–1147, 2008.

Introduction

Prebiotic oligosaccharides are specific types of dietary fiber thatcannot be hydrolyzed by the digestive enzymes in the gastroin-testinal tract. Hence, they enter the colon intact, where they canbe fermented by bacteria of the intestinal microbiota (1). Theintestinal microbiota consists of large numbers of different typesof bacteria, such as bifidobacteria, lactobacilli, bacteroides, andClostridium spp. Development of the intestinal microbiota isdifferent in infants that received breast milk or formula milk (2).The intestinal microbiota of breast fed infants mainly containsbifidobacteria and lactobacilli, whereas the intestinal microbiotaof formula fed infants contains more variable types of bacteria,such as bifidobacteria, lactobacilli, bacteroides, enterobacteria-ceae, and Clostridium spp. (2). An important determinant forthe development of a bifidogenic microbiota are the human milkoligosaccharides (3). Human milk oligosaccharides are prebioticoligosaccharides that are fermented by beneficial bacteria in the

gastrointestinal tract. Due to the selective fermentation, the growthof beneficial bacteria is favored over the growth of potentiallypathogenic bacteria.

Several studies have investigated the effect of a specificmixture of prebiotic short-chain galacto-oligosaccharides andlong-chain fructo-oligosaccharides [(scGOS/lcFOS)7 also namedIMMUNOFORTIS] on the composition of the intestinal micro-biota in preterm, term, and weaning infants (4–8). These studiesshowed that scGOS/lcFOS induces an intestinal microbiota withhigh percentages of bifidobacteria (4–8), and a metabolic ac-tivity of the microbiota that is similar to that of breast fed infants(7). Infant milk formulas with scGOS/lcFOS have also beenshown to have beneficial effects on the development of the im-mune system. In a double-blind, randomized, controlled clinicaltrial performed byMoro et al. (9,10), 206 infants at risk of atopycompleted a 6-mo intervention period in which they received ahypoallergenic infant milk formula with 8 g/L scGOS/lcFOS or acontrol hypoallergenic formula without scGOS/lcFOS. The cu-mulative incidence of atopic dermatitis was significantly lower

7 Abbreviations used: BM group, breast milk group; BSA, bovine serum albumin;

Ig, immunoglobulin; scGOS/lcFOS, short-chain galacto-oligosaccharides and long-

chain fructo-oligosaccharides; sIgA, secretory immunoglobulin A; Th, T-helper.

1 Supported by Numico Research, Wageningen, The Netherlands.2 Author disclosures: P. A. M. J. Scholtens, M. S. Alles, H. Kroes, G. Boehm,

L. M. J. Knippels, and J. Knol are employees of Numico Research. P. Alliet,

M. Raes, and Y. Vandenplas, no conflicts of interest.

* To whom correspondence should be addressed. E-mail: petra.scholtens@

numico-research.nl.

0022-3166/08 $8.00 ª 2008 American Society for Nutrition. 1141Manuscript received 21 December 2007. Initial review completed 22 January 2008. Revision accepted 27 February 2008.

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in the infants that received scGOS/lcFOS (9,10). These infantsalso had a lower incidence of infections (9,10). In another study,326 healthy infants were randomized to receive an infant milkformula with 4 g/L scGOS/lcFOS or a control formula withoutscGOS/lcFOS (11). This open, prospective, randomized, placebo-controlled study also showed that infants that received scGOS/lcFOS had a lower incidence of infections (11). The exactmechanism behind the immune modulatory effects of scGOS/lcFOS is not yet fully understood, but the effects may be medi-ated by the composition of the intestinal microbiota. Newborninfants are mainly dependent on passive immune responses, suchas the maternal immunoglobulin (Ig) G that was transported tothe fetus during pregnancy, and the maternal secretory immu-noglobulin A (sIgA), derived from the mother via human milk(12). Besides the passive transfer of sIgA via breast milk, theinfant’s acquired immune system develops gradually. The intes-tinal microbiota may play an important role in the developmentof the acquired immune system of infants, especially in thedevelopment of the mucosal immune system and the productionof endogenous sIgA (13). In a study by Bakker-Zierikzee et al.(14), a group of 38 healthy, formula fed infants was randomizedto receive an infant milk formula with 6 g/L scGOS/lcFOS or acontrol formula without scGOS/lcFOS from birth until 32 wk ofage. Infants that received scGOS/lcFOS had a significantly higherconcentration of fecal sIgA after 16 wk of intervention thaninfants in the control group. Our study evaluated the effect of aninfant milk formula with 6 g/L scGOS/lcFOS on the develop-ment of the fecal sIgA response and on the composition of theintestinal microbiota in healthy infants during the first 26 wk oflife. An important difference between this study and that ofBakker-Zierikzee et al. is that the infants in our study wereallowed to start with breast milk and to switch to 1 of the studyformulas after cessation of breast feeding.

Materials and Methods

Study design. The study was a double-blind, randomized, placebo-

controlled intervention trial, with a 6-mo intervention period. The

results presented in this article are part of a larger trial (15). The study

was approved by the medical ethics committee of the Virga Jesse hospital

in Hasselt, Belgium.

Subjects. Pregnant women were recruited via pediatricians in the Virga

Jesse hospital in Hasselt, Belgium and the University Hospital in

Brussels, Belgium in the last 4 mo before their estimated delivery date.

Infant eligibility was verified by the pediatrician during its stay in the

hospital after birth. The study population consisted of healthy, term

infants, with a normal birth weight (between the 5th and 95th

percentiles). Infants with a double heredity for atopic diseases, congen-

ital abnormality or disease, gastrointestinal disease, a strongly suspected

cow’s milk intolerance, or with regular use of products with probiotic

bacteria were excluded from the study. Participation in the study was

voluntary, and written informed consent was obtained before the start of

the study.

Nutritional intervention. The feeding intention of the mother was

assessed before delivery. If the mother decided to start with formula, she

was randomized to receive 1 of 2 types of infant milk formula: an infant

milk formula with 6 g/L scGOS/lcFOS in a ratio of 9:1, or a control

infant milk formula with the same composition, but without scGOS/

lcFOS (provided by Numico Research). Both formulas were identical in

appearance and taste. Mothers with the intention to start with breast

milk were encouraged to continue breast feeding during the course of the

study, and they were randomized to receive 1 of the 2 formulas at the

time they decided to start with formula. Eachmother/infant pair received

a sequential number at study entry and kept that number throughout the

study. The study products were coded on randomization number. A

randomization number was allocated for those infants that started with

formula, by assigning the next blank number of the randomization list. A

randomization list was generated prior to the study, using a 2 block

design, by the logistics manager of Numico Research, who was not in-

volved in the study. Stratification was applied according to the duration

of exclusive breast feeding, ,3 mo or .3 mo.

Data collection. Breast milk or formula intake was recorded in a daily

diary by the parents, and adverse events were recorded by the pedia-

trician in a case report form. The parents were asked to collect a fecal

sample from their infant before their visits to the pediatrician at the age

of 8 and 26 wk. They were carefully instructed on the method for the

collection of the samples before the study. Fresh fecal samples were

collected in plastic feces containers, without a medium or buffer (Greiner

Bio-One), frozen immediately after collection by the parents, and stored

at 220�C until transport to the pediatrician. Fecal samples were

transported to the pediatrician in a cooling bag with ice packs, without

unfreezing the samples. At the end of the study period, the frozen fecal

samples were transported to Numico Research.

Fecal homogenates. The frozen fecal samples were defrosted on ice,

and 10% wt:vol fecal homogenates were made by weighing 1 g of feces,

adding 9 mL of PBS (pH 7.4) and homogenizing this suspension for 10

min in the stomacher (IUL Instruments). One milliliter of the homog-

enized fecal suspension was directly fixed in 3 mL freshly prepared 4%

(wt:vol) paraformaldehyde in PBS and incubated overnight at 40�C.

Fixed samples were separated into aliquots and stored at 220�C to be

used for the microbiota analysis. The remainders of the homogenates

were aliquoted and stored at 280�C until used for the analysis sIgA.

Measurement of sIgA response by ELISA. sIgAwas determined from

the single feces samples that were collected at the age of 8 and 26 wk.

The analysis of sIgA was performed as described before, with minor

modifications (14). NUNC 96-well Immuno-maxisorp plates were

coated overnight at 4�C with mouse a human secretory component

(Sigma, clone GA-1), 1:10.000 in PBS. After thoroughly washing with

wash buffer (0.005% Tween-20 in PBS), the plates were incubated for

1 h at room temperature with PBS containing 1% of bovine serum

albumin (BSA) [PBS/BSA (Sigma, BSA Fraction V)] to block nonspecific

protein binding sites. Supernatants of the fecal homogenates (defrosted

on ice, vortexed, and centrifuged at 16,000 3 g for 5 min at 4�C) were

diluted in PBS/BSA between 2,500 and 125,000 times. Purified human

IgA isolated from Colostrum (Sigma, I-1010) was used as a standard

calibration curve and serial diluted in PBS/BSA starting from 80 mg/L.

After blocking, the plates were washed and the samples and standards

were incubated for 2 h at room temperature. Plates were then washed

and biotin conjugated mouse a human IgA1/IgA2 monoclonal antibody

(Pharmingen, clone G20–359) was added to the plates at a concentration

of 1 mg/L PBS/BSA. After 1 h of incubation at room temperature, the

plates were washed again and finally incubated with Streptavidin

conjugated horseradish peroxidase (CLB, M2051), 1:10.000 diluted in

PBS/BSA for 30 min at room temperature. Plates were washed and

incubated with 1-step Ultra TMB substrate solution (Pierce, 34028), 2

times diluted with 0.1 mol/L sodium acetate, pH 5.5 (Merck, 1.06268).

The enzymatic color development was stopped by adding stop solution

(1.8 mol/L H2SO4), and then the absorbance was measured at 450 nm in

a plate reader. sIgA concentrations of the fecal samples were calculated

from a calibration curve. Samples were assayed at 2 successive days in

duplicate in 2 different dilutions.

Analysis of intestinal microbiota. The composition of the intestinal

microbiota was analyzed with quantitative fluorescent in situ hybridi-

zation, as described previously (2,16,17). Fecal samples were applied

to gelatin-coated glass slides [8-well object slides with square-shaped

wells (1 cm2/well); CBN Labsuppliers], air dried, and hybridized with

10 ng/KL Cy3-labeled Bifidobacterium specific probe Bif164 (16),

Cy3-labeled Clostridium histolyticum/Clostridium lituseburense specific

probe Chis150/Clit135, or Cy3 labeled Escherichia coli–specific probe

1142 Scholtens et al.

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Ec1531, or incubated with 0.25 ng in 1 mL 4#,6-diamidino-2-phenyl-

indole for total cell counts. Slides were automatically counted using an

Olympus AX70 epifluorescence microscope and image analysis soft-

ware. The percentage of bifidobacteria per sample was determined by

analyzing 25 randomly chosen microscopic positions. At each position,

the number of bifidobacteria was determined by counting all cells with

a 4#,6-diamidino-2-phenylindole filter set and counting all bifidobac-

teria using a Cy3 filter set (SP100 and 41007, respectively; Chroma

Technology).

Data analysis. With a sample size of 65 infants per formula group, it

was possible to detect a difference in the percentage of bifidobacteria

between the groups of 22%, with a 2-sided test, assuming a standard

deviation of 20% (our unpublished results), and a ¼ 0.05 and a power

of 80%.

Data were checked for normality by visual inspection of the normal

probability curves, and with the Shapiro-Wilk test of normality. In the

statistical analysis, infants that received the formula with added scGOS/

lcFOS were compared with infants that received the control formula. A

study group with infants that received at least 97.5% of the total number

of feedings as breast milk was included as a reference (BM group). The

2 formula fed groups contained both infants that started with breast

milk and changed to formula during the study period, and infants that

received formula feeding for the whole 26-wk period (whole group

analysis). To assess the differences between the 2 exclusively formula

groups, a subgroup analysis was performed in which only children that

received 97.5% of the total number of feedings as formula feeding were

included. All analyses were performed on an intention-to-treat basis.

Due to the skewed distribution of the data, nonparametric Mann-

Whitney U tests were performed to assess differences among the groups.

The breast fed group was not included in the pairwise comparisons, as

these infants could not be randomized, and therefore deviated from the

double-blind randomized design. The results are presented as medians,

with the 10th and the 90th percentiles, unless noted otherwise.

Results

Subject characteristics. 215 infants were included in the study(Fig. 1). In total, 202 infants completed the first 8 wk of thestudy, of which 21, 25, and 90 exclusively received a formula

with scGOS/lcFOS, a control formula, or breast milk, respec-tively (66 infants started with breast milk, and transferred to 1 ofthe 2 formulas before the age of 8 wk). 187 Infants completedthe entire 26-wk study period, of which 22, 24, and 31 exclu-sively received a formula with scGOS/lcFOS, a control formula,or breast milk, respectively (110 infants started with breast milkand transferred to 1 of the 2 formulas between the age of 8 and26 wk). The baseline and subject characteristics were compa-rable for the groups (Table 1).

Fecal sIgA. After 26 wk of intervention, in the whole group ofinfants, the concentration of sIgA in feces was significantlyhigher in the scGOS/lcFOS group (729 mg/g) than in the controlgroup (377 mg/g, Fig. 2). The results of the subgroup of infantsthat were exclusively formula fed were comparable to those inthe whole group, with concentrations of 719 mg/g in the scGOS/lcFOS group and 263 mg/g in control group (P , 0.001).

Intestinal microbiota. After 8 wk of intervention, the for-mula groups did not differ with respect to the percentages ofbifidobacteria and Clostridium spp. (Table 2). In the subgroupof infants that were exclusively formula fed, the percentage ofE. coli was lower in the scGOS/lcFOS group than in the controlgroup. At the end of the study period, after 26 wk of inter-vention, in the whole group the percentage of bifidobacteria inthe scGOS/lcFOS group was higher than in the control group.Furthermore, the percentage of Clostridium spp. was lower inthe scGOS/lcFOS group than in the control group. The results ofthe subgroup of infants that were exclusively formula fed werecomparable to those in the whole group.

The pH was lower in the scGOS/lcFOS group than in thecontrol group after 8 and 26 wk of intervention in the wholegroup of infants (Table 2).

In total, 96 subjects reported adverse events during the study,of which 40 (of 85 infants) were in the scGOS/lcFOS group, 40(of 89 infants) were in the control group, and 16 (of 38 infants)were in the breast fed reference group.

FIGURE 1 Flow-chart of the study population after 8 and 26 wk of intervention.

Effect of scGOS/lcFOS on fecal secretory IgA 1143

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Discussion

In this study, infants who received an infant milk formula withadded scGOS/lcFOS had significantly higher concentrations ofsIgA than infants who received a control infant milk formula.This study is, to our knowledge, the first to demonstrate theeffect of supplementation with scGOS/lcFOS on fecal sIgA con-centrations after 26 wk of intervention.

Fecal sIgA. The fecal concentration of sIgA after 26 wk ofintervention was significantly higher in the infants that receivedan infant milk formula with added scGOS/lcFOS. Concentra-tions of sIgA in the group of exclusively formula fed infantsreached 719 mg/g in the scGOS/lcFOS group, 263 mg/g in the

control group, and 584 mg/g in the breast fed group after 26 wkof intervention. As the concentration of sIgA was determined insingle feces samples, no information is available on the dailyexcretion of sIgA. Similar results were observed in a study byBakker-Zierikzee et al. (14), in which healthy newborns wererandomized to receive an infant milk formula with 6 g/L scGOS/lcFOS, or a control infant milk formula, for a period of 4 mo. Abreast fed group was included as a reference. Concentrations ofsIgA in feces in these infants reached 654mg/g, 449mg/g, and 767mg/g in the scGOS/lcFOS, control, and breast fed groups,respectively, after 24 wk of intervention, but concentrationsdid not differ significantly at this time point. With samples sizesof 13, 13, and 12 infants in the scGOS/lcFOS, control, and breastfed groups, the groups may have been too small, and thevariation may have been too high to detect statistically signif-icant differences. An important difference between the study ofBakker-Zierikzee et al. (14) and this study is the comparison ofthe effect of scGOS/lcFOS in a group of infants that started withbreast milk and were randomized to receive an infant milkformula after cessation of breast feeding, and in a group ofinfants that were exclusively formula fed from birth. Breast fedinfants are provided with sIgA via human milk during the firstfew months of life. This was clearly observed in our study, withhigher concentrations of sIgA in the breast fed reference group inthe week 8 of intervention. The difference in the concentration offecal sIgA between breast fed infants and formula fed infants wassmaller after 26 wk of intervention, possibly indicating thedecreased provision of sIgA via breast milk (18). In addition tothe provision of sIgA, breast milk also contains other factors,such as sIgA-stimulating cytokines, that stimulate the infants’own immune system (12,19). Kuitunen and Savilahti (20)compared the concentration of fecal sIgA in term and preterminfants that received breast milk or formula. The concentrationof sIgA was higher in breast fed infants throughout the studyperiod. Probiotics also increase concentrations of fecal sIgA (21).In an unblinded, nonplacebo-controlled study performed byFukushima et al. (21), an increase in concentrations of fecal sIgAwas observed in infants that received an infant milk formula withBifidobacterium lactis Bb-12. In a study performed by Mullieet al. (22), an infant formula fermented with Bifidobacteriumbreve strain C50 and Streptococcus thermophilus failed toincrease the concentration of total fecal sIgA in healthy infants.

TABLE 1 Baseline and subject characteristics in the whole group and in the subgroups of exclusively formula fed infants whoreceived a formula with scGOS/lcFOS or a control formula with the group classification after 26 wk of intervention1

scGOS/lcFOS Control

Whole group (n) Subgroup (n) Whole group (n) Subgroup (n) BM reference (n)

Gender, % male 57.0 (86) 66.7 (27) 54.4 (90) 55.2 (29) 53.8 (39)

Type of delivery, % caesarian 25.9 (85) 22.2 (27) 22.2 (90) 27.6 (29) 15.4 (39)

Group B Streptococcus, % positive 15.1 (86) 22.2 (27) 26.7 (90) 20.7 (29) 12.8 (39)

Birth weight, g 3510.7 6 436.7 (86) 3610.0 6 448.7 (27) 3340.3 6 453.8 (90) 3312.1 6 434.1 (29) 3400.0 6 393.8 (39)

Birth length, cm 50.8 6 1.9 (84) 51.2 6 1.9 (26) 50.3 6 2.2 (88) 49.9 6 1.9 (28) 50.3 6 1.9 (39)

Head circumference, cm 34.6 6 1.4 (82) 35.0 6 1.5 (25) 34.4 6 1.4 (87) 34.4 6 1.3 (28) 34.7 6 1.5 (38)

Apgar score, 1 min 8.7 6 0.9 (83) 8.5 6 1.1 (27) 8.6 6 0.7 (82) 8.6 6 0.5 (28) 8.6 6 0.5 (38)

Apgar score, 5 min 9.5 6 0.7 (83) 9.4 6 0.8 (27) 9.4 6 0.9 (81) 9.5 6 0.7 (28) 9.6 6 0.5 (38)

Formula feedings, % of total 61.3 6 34.8 (86) 99.0 6 4.4 (27) 64.5 6 32.5 (90) 99.8 6 0.4 (29) 0.4 6 0.7 (39)

Breast feedings, % of total 38.7 6 34.8 (86) 1.0 6 4.4 (27) 35.5 6 32.5 (90) 0.2 6 0.4 (29) 99.6 6 0.7 (39)

Duration of breast feeding,2 wk 11.5 6 10.0 (75) 0.3 6 0.7 (22) 10.3 6 9.0 (81) 0.1 6 0.3 (24) 26.0 6 0.0 (31)

Parental allergy (single heredity), % yes 33.7 (86) 33.3 (27) 38.2 (89) 31.0 (29) 57.9 (38)

1 Values are means 6 SD or percentages.2 Excluding drop outs.

FIGURE 2 Concentration of fecal sIgA after 8 and 26 wk of inter-

vention in infants in the whole group and in the subgroups of

exclusively formula fed infants who received a formula with scGOS/

lcFOS or a control formula. The results represent the median concen-

tration of fecal sIgA with the 10th and the 90th percentiles. *Different

from control, P , 0.05.

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Viljanen et al. (23) randomized infants with atopic eczema/dermatitis syndrome to receive a supplement with Lactobacillusrhamnosus GG, a supplement with a mixture of 4 types ofprobiotics, or a placebo supplement. Fecal concentrations ofsIgA tended to be higher in the infants that received 1 of the 2probiotics supplements than in infants that received the placebosupplement. This difference was mainly due to a decrease in con-centrations of fecal sIgA in the placebo group during the studyperiod. These studies indicate that the concentration of fecal sIgAin breast fed infants is higher than that of formula fed infants, butthat it is not always possible to detect differences in the effects ofinfant milk formulas on concentrations of fecal sIgA.

The main function of fecal sIgA is to agglutinate microorgan-isms and to prevent the adherence of pathogenic bacteria andviruses to the mucosal surface (12,13,24–26). Another importantfunction of sIgA is the maintenance of the intestinal microbialhomeostasis. Animal studies have shown a substantial increase inanaerobic bacteria in the small intestine of mice in the absence ofnormal sIgA, whereas normalization of the production of sIgAresulted in a recovery of the regular composition of the intestinalmicrobiota (27). So on one hand, the composition of the intestinalmicrobiota regulates the production of sIgA, but on the otherhand, sIgA regulates the composition of the intestinal microbiotato prevent an overstimulation of the immune system. At the sametime, the continuous presence of commensal bacteria favors aconstant stimulation of the immune system without invokinginflammatory responses. An interesting finding by Liepke et al.(28) was that the secretory component derived from human milkmay have bifidogenic properties, which can further enhance thegrowth of beneficial bacteria in the gastrointestinal lumen, andmay thus also play a role in the microbial homeostasis. Thesestudies indicate that there is a strong link between the compositionof the intestinal microbiota and the mucosal immune system.

Composition of the intestinal microbiota. Previous studies(4–8) have shown that the prebiotic mixture with scGOS/lcFOS

in a ratio of 9:1 induces an intestinal microbiota with higherpercentages of bifidobacteria, and an intestinal metabolicactivity similar to that of breast fed infants in preterm, term,and weaning infants. Similar results were observed in this study,with higher percentages of bifidobacteria and lower percentagesof Clostridium spp. in the scGOS/lcFOS group than in thecontrol group after 26 wk of intervention in the subgroup and inthe whole group of infants. All groups have a microbiota that isdominated by bifidobacteria, but surprisingly, the percentages ofbifidobacteria appeared to be low in the breast fed group, whencompared with both formula groups. In the subgroup ofexclusively formula fed infants, percentages of 53.5 and 40.2were observed in the scGOS/lcFOS and the control groups,respectively. In the breast fed reference group, a percentage of33.2 bifidobacteria was observed in week 8 of intervention.Similar low percentages of bifidobacteria in breast fed infantswere observed in a study of Bakker-Zierikzee et al. (29). In arecent study by Penders et al. (30), counts of bifidobacteria inbreast fed infants equaled those in formula fed infants. Althoughthe results of our study were unexpected, they may indicateregional differences in the composition of breast milk or in thecomposition of the intestinal microbiota. For instance, compar-ative studies in adults have shown differences in percentages ofbifidobacteria in several countries (31). In this study, thepercentage of Clostridium spp. was significantly lower in thescGOS/lcFOS group than in the control group after 26 wk ofintervention. Percentages of E. coli were lower in the scGOS/lcFOS group after 8 wk of intervention, but the groups did notdiffer after 26 wk of intervention. The presence of E. coli andClostridium spp. indicates the development of a more maturemicrobiota. TheClostridium spp. probes used in our study coverpathogenic species (Clostridium perfringens and Clostridiumdifficile), and the observed lower percentage thus indicates lowerpercentages of pathogens.

The composition of the intestinal microbiota is important inthe skewing of the immune response in young infants (13). The

TABLE 2 Intestinal microbiota and pH in the whole group and in the subgroup of exclusively formulafed infants, receiving an infant formula with scGOS/lcFOS or a control infant formula1

Week scGOS/lcFOS (n) Control (n) BM reference (n)

Bifidobacteria, %

Whole group 8 49.5 [0.0, 83.6] (41) 40.4 [0.0, 75.0] (46) 33.2 [0.0, 74.0] (49)

26 59.8 [37.1, 88.0]* (66) 47.2 [12.1, 64.0] (74) 63.9 [20.1, 103.2] (30)

Subgroup 8 53.4 [0.0, 79.1] (16) 40.2 [0.0, 75.2] (24) 33.2 [0.0, 74.0] (49)

26 60.4 [37.8, 93.3]* (19) 52.6 [1.3, 68.7] (21) 63.9 [20.1, 103.2] (30)

Clostridium spp., %

Whole group 8 0.0 [0.0, 1.6] (42) 0.0 [0.0, 13.8] (47) 0.0 [0.0, 16.2] (50)

26 1.1 [0.0, 8.0]* (68) 3.9 [0.0, 10.4] (74) 0.0 [0.0, 6.1] (30)

Subgroup 8 0.0 [0.0, 2.9] (17) 0.0 [0.0, 15.5] (24) 0.0 [0.0, 16.2] (50)

26 0.0 [0.0, 3.6]* (20) 3.3 [0.0, 9.6] (21) 0.0 [0.0, 6.1] (30)

E. coli, %

Whole group 8 2.8 [0.0, 13.0] (42) 3.7 [0.0, 21.0] (47) 1.0 [0.0, 19.7] (50)

26 1.8 [0.0, 6.2] (68) 0.5 [0.0, 14.5] (74) 2.6 [0.0, 11.1] (30)

Subgroup 8 1.7 [0.0, 8.3]* (17) 5.8 [1.1, 25.3] (24) 1.0 [0.0, 19.7] (50)

26 0.8 [0.0, 4.8] (20) 0.5 [0.0, 11.2] (21) 2.6 [0.0, 11.1] (30)

pH

Whole group 8 5.9 [4.9, 7.5]* (47) 6.8 [5.7, 7.9] (60) 5.7 [4.8, 7.3] (83)

26 6.2 [5.4, 7.3]* (71) 6.5 [5.4, 7.7] (82) 6.1 [5.0, 7.6] (32)

Subgroup 8 6.3 [5.2, 7.8]* (19) 7.0 [5.6, 8.0] (26) 5.7 [4.8, 7.3] (83)

26 6.2 [5.4, 7.3] (21) 6.3 [5.6, 7.7] (24) 6.1 [5.0, 7.6] (32)

1 Values are medians [10th, 90th percentiles]. *Different from control, P , 0.05.

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immune system of pregnant women is skewed toward T-helper(Th)-2 responses, to avoid rejection of the fetus by the mother(32,33). Due to this preserved skewness, newborn infants areborn with a skewed immune system toward Th2 responses (34).As Th2 responses have been shown to be involved in the onsetof allergic disease, regulation of this skewed Th2 response isfavorable. In the hygiene hypothesis, it was suggested thatmicrobial stimulation was necessary to skew the immune systemmore toward Th1 responses (35,36). Regulatory T cells havealso been suggested to play an important role in this process, byregulating both Th1 and Th2 responses (36). The developmentof a healthy microbiota may favor the production of regulatoryT-cells, which in turn may regulate Th1 or Th2 responses (35–37). sIgA can be produced in response to pathogenic bacteria viaa Th2 response, or in response to commensal bacteria via aregulatory T-cell response. As sIgA plays an important role in thedefense against all types of pathogens in the gastrointestinallumen (25,26), an increased production induced by a stimula-tion of commensal bacteria favors the defense against pathogensin the gastrointestinal tract. We cannot conclude from our studywhether the increased concentration of fecal sIgAwas due to anincreased level of commensal bacteria, such as bifidobacteria, ordue to an increased level of pathogenic bacteria. However, as thepercentages of Clostridium spp. and E. coli tended to be lowerin the infants that received the scGOS/lcFOS formula, we canspeculate that the increased percentages of bifidobacteria mayhave been involved in the stimulation of the production of sIgA.Animal experiments have shown that supplementation withscGOS/lcFOS can also induce systemic immune effects (38,39).Although lymphocytes that were activated in the gut associatedlymphoid tissue by commensal or pathogenic bacteria have beenshown to be destined to return to mucosal surfaces, withoutcross-talk with the systemic circulation (40), it can be hypoth-esized that specific Th1, Th2, or regulatory T cells cytokines aretransported to the systemic circulation via mesenteric lymphnodes, thereby also inducing systemic effects. In addition,scGOS/lcFOS may have direct effects on the immune system.For instance, a study performed by Roller et al. (41), showedthat prebiotic inulin can directly stimulate the production ofregulatory cytokines, such as interleukin 10, and protectivecytokines, such as interferon-g. Infant milk formulas withscGOS/lcFOS have also been shown to have beneficial effects onthe development of the immune system (9–11). Hence, theprovision of scGOS/lcFOS in an infant’s diet has more functionaleffects than the stimulation of sIgA. However, this article focusesonly on the production of sIgA, and further studies are needed toevaluate other functional effects.

In conclusion, an infant milk formula with 6 g/L scGOS/lcFOS in a ratio of 9:1 results in higher concentrations of fecalsIgA, suggesting a positive effect on mucosal immunity.

Acknowledgments

We thank Marie-Paule Verjans for her assistance in the practicalexecution of the study, Inge Peeters for data entry, and RobSlump and Esmeralda van der Linde for the quantitative fluo-rescent in situ hybridization analyses.

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