The effect of neutral and acidic oligosaccharides on stool viscosity, stool frequency and stool pH in preterm infants

REGULAR ARTICLE

The effect of neutral and acidic oligosaccharides on stool viscosity, stoolfrequency and stool pH in preterm infantsEAM Westerbeek1, RL Hensgens1, WA Mihatsch2, G Boehm3,4, HN Lafeber1, RM van Elburg ([email protected]) 1

1.Department of Neonatology, VU University Medical Center, Amsterdam, the Netherlands2.Department of Paediatrics, Deaconry Hospital Schwaebisch Hall, Germany3.Danone Research, Centre for Specialised Nutrition, Friedrichsdorf, Germany4.Sophia Children’s Hospital, Erasmus University, Rotterdam, The Netherlands

KeywordsNutrition, Prebiotics, Preterm infants, Stoolcharacteristics

CorrespondenceRM van Elburg, Division of Neonatology, Departmentof Paediatrics, VU University Medical Center,De Boelelaan 1117, 1081 HV Amsterdam,The Netherlands.Tel: +31-20-444-2413 |Fax: +31-20-444-3045 |Email: [email protected]

Received4 December 2010; revised 23 February 2011;accepted 24 March 2011.

DOI:10.1111/j.1651-2227.2011.02295.x

Changes made after first online publication25/04/2011. Figure 2 changed to correct version.27/06/2011.

ABSTRACTAim: To determine the effect of neutral oligosaccharides [small-chain galacto-oligo-

saccharides ⁄ long-chain fructo-oligosaccharides (scGOS ⁄ lcFOS)] in combination with acidic

oligosaccharides (pAOS) on stool viscosity, stool frequency and stool pH in preterm infants.Methods: In this explorative RCT, preterm infants with gestational age <32 weeks

and ⁄ or birth weight <1500 g received enteral supplementation with scGOS ⁄ lcFOS ⁄ pAOS

or placebo (maltodextrin) between days 3 and 30 of life. Stool samples were collected at

day 30 after birth.Results: In total, 113 infants were included. Baseline and nutritional characteristics

were not different between both groups. Stool viscosity at day 30 was lower in the

prebiotics group (16.8N) (3.9–67.8) compared with the placebo group (26.3N)

(1.3–148.0) (p = 0.03; 95% CI )0.80 to 0.03). There was a trend towards higher stool

frequency in the prebiotics group (3.1 ± 0.8) compared with the placebo group

(2.8 ± 0.7) (p = 0.15; 95% CI )0.08 to 0.52). Stool pH at day 30 was lower in the in the

prebiotics group (5.9 ± 0.6) compared with the placebo group (6.2 ± 0.3) (p = 0.009;

95% CI 0.08 to 0.53).Conclusions: Enteral supplementation of a prebiotic mixture consisting of neutral

(scGOS ⁄ lcFOS) and acidic oligosaccharides (pAOS) decreases stool viscosity and stool pH

with a trend towards increased stool frequency in preterm infants. The inclusion of pAOS in

a formula containing a mixture of scGOS ⁄ lcFOS does not add specific advantages to the

formula in terms of stool viscosity, frequency, pH as well as feeding tolerance.

INTRODUCTIONPreterm infants have an immature gastrointestinal tract,and increasing enteral feeding volumes is often restricted.However, enteral nutrition is essential for maturation of thegastrointestinal tract (1). Furthermore, adequate nutrition isnecessary for optimal growth and neurodevelopment (2,3).Breast milk is often not available in preterm infants. For-mula feeding is commonly associated with hard stools,delayed gastrointestinal transport and constipation (4). Asthese problems considerably hamper the tolerance of ent-eral feeding, (nutritional) interventions to attain reductionin stool viscosity and acceleration in gastrointestinal trans-port are warranted (4).

Preterm infants fed with mother’s milk have lower stoolconsistency and higher stool frequency than formula-fedinfants (5). These effects can partially be attributed to thelarge amount of prebiotic oligosaccharides in human milk.Over 200 human milk oligosaccharides have been identifiedwith significant variability between individuals over time(6). Nonhuman milk oligosaccharides, such as neutralsmall-chain galacto-oligosaccharides (scGOS) and long-

chain fructo-oligosaccharides (lcFOS), have been devel-oped to mimic the prebiotic function of human milk oligo-saccharides (7).

Recent studies on the supplementation of neutraloligosaccharides show reduction in stool viscosity, increasein stool frequency and acceleration in gastrointestinal trans-port in preterm infants (4,5,8,9). This was accompaniedwith reduced gastric emptying and total gastrointestinaltransit time (10). Human milk contains neutral

Key notes• Preterm infants have an immature gastrointestinal tract

and often have feeding intolerance. Improving stool char-acteristics may increase enteral feeding tolerance. In arandomized controlled trial in preterm infants £32 weeksor £1500 g, enteral supplementation of a prebiotic mix-ture of neutral and acidic oligosaccharides in the firstmonth of life decreased stool viscosity and decreasedstool pH with a trend towards increased stool frequency.

Acta Pædiatrica ISSN 0803–5253

1426 ª2011 The Author(s)/Acta Pædiatrica ª2011 Foundation Acta Pædiatrica 2011 100, pp. 1426–1431

(approximately 80%) and acidic (up to 20%) oligosaccha-rides. More recently, nonhuman pectin-derived acidic oligo-saccharides (pAOS) have been developed as supportagainst intestinal infections. Nonhuman milk acidic oligo-saccharides derived from pectin (pAOS) are able to interactwith the epithelial surface and are known to inhibit theadhesion of pathogens on the epithelial surface (11,12).Because pAOS interact directly with the epithelial surface,it might be influencing the effect of the neutral oligosaccha-rides. In the initial study, we found that there was a trend, ifgiven in sufficient amounts, towards a lower incidence ofendogenous infections after enteral supplementation of aprebiotic mixture consisting of neutral and acidic oligosac-charides (13). One of the mechanisms underlying this bene-ficial effect of prebiotics may be the effect on stool viscosity,stool frequency and stool pH. For the neutral mixture ofscGOS ⁄ lcFOS (ratio: 9:1), it has been demonstrated thatthey stimulate the growth of a ‘bifidogenic’ flora, resulting inincreased small-chain fatty acids (SCFA) and decreased pH(12). SCFA affect gastrointestinal motility, (12,14,15) whichmay increase stool frequency. The neutral prebiotics mightalso contribute to the osmotic pressure of the faecal mass,resulting in the increase in the faecal water content and fae-cal bulking (4,14). Because the mechanism of action ofacidic pAOS might be different from neutral prebiotics, itwas the aim of the study to measure the effect of a combina-tion of neutral and pAOS on stool viscosity, frequency andfaecal pH. We hypothesize that adding the pAOS to thescGOS ⁄ lcFOS mixture in an amount similar to the quantityof acidic human milk oligosaccharides will not negativelyinfluence the physiological effects of the neutral prebioticmixture.

Therefore, the aims of this trial were to determine theeffects of enteral supplementation of a prebiotic mixtureconsisting of neutral and acidic oligosaccharides on stoolviscosity, stool frequency and stool pH in preterm infantsand furthermore to determine the effect of stool viscosity onfeeding tolerance and stool frequency.

MATERIALS AND METHODSSubjectsInfants with a gestational age (GA) <32 weeks and ⁄ or birthweight (BW) <1500 g, admitted to the level III NICU of theVU University Medical Center, Amsterdam, were eligiblefor participation in the study. Exclusion criteria were infantswith a GA ‡33 weeks, major congenital or chromosomalanomalies, death within 48 h after birth and transfer toanother hospital within 48 h after birth. The medical ethicalreview board of our hospital approved the study protocol.Written informed consent was obtained from all parents.

Randomization, blinding and treatmentTo have the same BW distribution in both groups, theinfants were stratified in three BW groups (£799, 800–1199,‡1200 g) and randomly allocated within 48 h after birth toreceive either enteral 80% scGOS ⁄ lcFOS (9:1) and 20%AOS or placebo (maltodextrin). An independent researcher

used a computer-generated randomization table (providedby Danone Research, Friedrichsdorf, Germany) to assigninfants to treatment with prebiotics or placebo. Investiga-tors, parents, medical and nursing staff were blinded. Therandomization code was broken after data analysis was per-formed. Prebiotics and the placebo powder (maltodextrin)were prepared and packed sterile under nitrogen environ-ment in sachets. (Danone Research). The two powders wereindistinguishable by appearance, colour and smell. Duringthe study period, prebiotics and placebo powder were moni-tored for stability and microbiological contamination.

The supplementation of prebiotics or placebo was admin-istered in increasing doses between days 3 and 30 of life to amaximum of 1.5 g ⁄ kg ⁄ day to breast milk or preterm formulain the intervention group. Infants received breast milk orpreterm formula according to the parents’ choice. Twomembers of the nursing staff added the daily supplement tobreast milk or to preterm formula (Nenatal Start�). Per100 mL, the preterm formula provided 80 kcal, 2.4 g pro-tein (casein ⁄ whey protein ratio 40:60), 4.4 g fat and 7.8 gcarbohydrate. The preterm formula did not contain oligo-saccharides. When infants were transferred to another hos-pital before the end of the study, the protocol wascontinued under supervision of the principal investigator(EW).

Analysis of stool viscosityStool samples were collected in sterile syringes at day 30 oflife and stored at )20�C until analysis and thawed justbefore measuring. Stool viscosity was determined by the useof high-pressure capillary rheometry (viscosimetry) asrecently described by Mihatsch et al. (4) Viscometry mea-sures the mean force (extrusion force) needed to press stoolat constant flow (0.19 mL ⁄ sec) out of the extrusion cell[low resistance medical 10-mL syringes (inner diameter of15.5 mm)] through an 83-mm steel capillary (inner diame-ter 1.05 mm). The mean of the measured force itself wastaken as a measure of viscosity. Measurements were per-formed using a TA-XT2i ⁄ 25 Texture Analyser (Stable MicroSystems, Surrey GU7 1Yl, UK): load cell TA ⁄ CEL25, forcerange )25 to 250 N, force resolution 0.01 N, speed range0.01–10 mm ⁄ sec and speed accuracy 0.01%. Calibrationwas performed using a standardized 5 kg calibration weight.An independent investigator, unaware of treatment alloca-tion, did the assessment of stool viscosity.

pH measurementsStool samples were collected in sterile tubes at day 30 of lifeand stored at )20�C until analysis and thawed just beforemeasuring. The pH was measured directly using a HandylabpH meter (Schott Glas, Mainz, Germany) equipped with anInlab 423 pH electrode (Mettler-Toledo, Columbus, OH,USA).

Feeding tolerance and stool frequencyTo determine feeding tolerance, the following patient andnutritional characteristics were determined: time to full ent-eral feeding, defined as >120 mL ⁄ kg ⁄ day, age of finishing

Westerbeek et al. Prebiotics in preterm infants

ª2011 The Author(s)/Acta Pædiatrica ª2011 Foundation Acta Pædiatrica 2011 100, pp. 1426–1431 1427

parenteral nutrition, total days of minimal enteral feeding,defined as 12–24 mL ⁄ kg ⁄ day, necrotizing enterocolitis (16)and growth (17).

Infants suspected for a serious infection received antibi-otic treatment. If cultures were negative, antibiotics werediscontinued.

Stool frequency was determined as the number of dailystools during a study period of 30 days. If >50% of the dataduring the study period were not available, infants were notincluded in the analysis of stool frequency.

Nutritional supportProtocol guidelines for the introduction of parenteral andenteral nutrition followed current practice at our NICU.Nutritional support was administered as previouslydescribed (13,18). For each infant in the study, a feedingschedule was proposed based on BW and the guidelinesas previously described (13,18). However, the medicalstaff of our NICU and the responsible doctors in theregional hospitals had final responsibility for the adminis-tration of parenteral nutrition and advancement of ent-eral nutrition. After discharge, all infants received breastmilk or preterm formula (Nenatal Start� without oligo-saccharides) and a postdischarge formula (Nenatal 1�

without oligosaccharides) until the corrected age of6 months.

Statistical analysisThe sample size of 113 infants was based on the samplesize calculation for the primary outcome of the main trial(serious infectious morbidity) (13,18). Normally distrib-uted and nonparametric data are presented asmeans ± standard deviations and medians (ranges),respectively. Patient and nutritional characteristics wereanalysed with Student’s t-test, chi-square test or Fisher’sexact test for continuous normally distributed and dichot-omous data, respectively. If parameters had a skewed dis-tribution, a natural logarithmic transformation wasperformed before analysis. The effect of prebiotics onstool viscosity, stool frequency and stool pH comparedwith the placebo group were analysed with Student’st-test. In an additional linear regression analysis on theeffect of prebiotics on stool viscosity, stool frequency andstool pH, a correction was made for the type of feeding.Linear regression analysis was performed to determinethe influence of stool viscosity on time to full enteralfeeding, age of finishing parenteral nutrition, total days ofminimal enteral feeding, growth and stool frequency.Logistic regression analysis was performed to determinethe influence of stool viscosity on necrotizing enterocoli-tis. Multiple linear regression analysis was performed todetermine host- and treatment-related factors (GA, BW,mode of delivery, Apgar score <6 at 5 min and chorioam-nionitis) influencing stool viscosity, stool pH and stoolfrequency. All statistical analyses were performed on anintention to treat basis. A two-tailed p value of <0.05was considered significant. We used SPSS 17.0 (SPSSInc, Chicago, IL, USA) for data analysis.

RESULTSBetween May 2007 and November 2008, 113 of 208 eligiblepreterm infants entered the study. Reasons for not partici-pating in the study were no informed consent (n = 45), par-ticipation in another trial (n = 7), transfer to a regionalhospital within 48 h (n = 12), death within 48 h (n = 5)and severe congenital malformations (n = 12). After ran-domization, one infant in the placebo group was excluded,because of strong suspicion of a syndrome. Baseline patientand nutritional characteristics were not different in prebiot-ics (n = 55) and placebo group (n = 58), (Table 1). Stoolsamples for analysis for stool viscosity and stool pH werecollected from the same diaper at a mean age of29.8 ± 1.9 days. Of these 113 infants, 66 stool samples, 31in the prebiotics group and 35 stool samples in the placebogroup, were available for analysis of stool viscosity. Missingstool samples were mainly because of stool samples lackingenough biomass (n = 39). Stool viscosity at day 30 waslower in the prebiotics group (16.8N) (3.9–67.8) comparedwith the placebo group (26.3N) (1.3–148.0) (p = 0.03; 95%confidence interval (CI) )0.80 to 0.03) (Fig. 1).

Table 1 Baseline and nutritional characteristics**.

Prebiotic mixture(N = 55) Placebo (N = 58)

Baseline characteristics

Chorioamnionitis (%) 11 (20) 13 (22)

PE, E or HELLP (%) 16 (31) 18 (31)

Placental insufficiency (%) 4 (7) 3 (5)

Antenatal antibiotics (%) 11 (20) 16 (28)

Antenatal corticosteroids (%) 30 (56) 32 (56)

Multiple birth (%) 9 (16) 13 (22)

Vaginal delivery (%) 31 (56) 32 (55)

Gestational age (weeks) 29.9 ± 1.9 29.3 ± 2.1

Birth weight (kg) 1.3 ± 0.4 1.2 ± 0.3

Birth weight <10th

percentile (%)

12 (22) 8 (14)

Sex, male (%) 31 (56) 36 (62)

Apgar at 5 min <6 (%) 9 (16) 5 (9)

Antibiotics at birth (%) 41 (75) 44 (76)

Nutritional characteristics

Age at start of study

supplementation (days)

2.1 (1.5–5.3) 2.1 (1.5–3.3)

Time to full supplementation

dose (days)

11 (4–28) 11 (5–27)

Mean supplementation

dose during study

period (g ⁄ kg ⁄ days)

1.30 (0.1–1.6) 1.27 (0.2–1.8)

Age at advancement

of enteral nutrition (days)

2.8 (0.6–27.5) 2.5 (0.3–18.0

Exclusive breast milk (%)* 38 (69%) 33 (57%)

Mixed breast milk and

formula feeding (%)*

12 (21%) 11 (20%)

PE, preeclampsia; E, eclampsia; HELLP, syndrome of haemolysis, elevated

liver enzymes and low platelets; PIVH, periventricular–intraventricular haemor-

rhage.

*During 30 day study period.

Values are expressed as mean ± standard deviation or median and range.

**There were no statistically differences (p < 0.05) between both groups.

Prebiotics in preterm infants Westerbeek et al.


Stool frequency was determined from 47 infants in theprebiotics group and 52 infants in the placebo group. Miss-ing data was mainly due <50% of the data available for anal-ysis. (n = 14) There was a trend towards higher stoolfrequency in the prebiotics group (3.1 ± 0.8) compared withthe placebo group (2.8 ± 0.7) (p = 0.15; 95% CI )0.08 to0.52) (Fig. 2).

For analysis of stool pH, 51 stool samples in the prebiot-ics group and 51 stool samples in the placebo group wereavailable. Missing stool samples were mainly because ofdeath or exclusion before day 30 of life (n = 8). Stool pH atday 30 was lower in the prebiotics group (5.9 ± 0.6) com-pared with the placebo group (6.2 ± 0.3) (p = 0.009; 95%CI 0.08 to 0.53) (Fig. 3). Correction for type of feeding didnot influence the results of the primary analysis on stool vis-cosity, stool frequency or stool pH. The incidence of

necrotizing enterocolitis was not different between the pre-biotics and placebo group, 10 ⁄ 55 (18%) and 6 ⁄ 58 (10%),respectively.

Stool viscosity did not influence feeding tolerance(Table 2). In a multiple regression analysis, low GAincreased stool viscosity [beta 0.36 95% CI (0.04 to 0.24)(p = 0.008)] and was not related to stool frequency andstool pH. Other perinatal factors (BW, mode of delivery,Apgar score <6 at 5 min and chorioamnionitis) had noinfluence on stool viscosity, stool frequency and stool pH.

DISCUSSIONIn this study, we found that enteral supplementation of aprebiotic mixture consisting of neutral and acidic oligosac-charides decreases stool viscosity as measured by high-pres-sure capillary rheometry. This is in line with a previousstudy with only neutral oligosaccharides in preterm infants(4) and in previous studies in term infants on visual assess-ment of stool viscosity (5,19,20). Nondigestible prebiotic ol-igosaccharides have been referred to as soluble dietaryfibres and are known to increase stool volume. SCFA pro-duced by colonic fermentation of prebiotic oligosaccharides

Figure 1 Stool viscosity at day 30. The boxplot shows the median (central hori-zontal line), the 10th centile (lower box border) and 90th centile (upper box bor-der). The upper and lower horizontal line refer to the 95th and 5th centile,respectively. p = 0.03 between prebiotic mixture group (n = 31) and placebogroup (n = 35).

Figure 2 Stool frequency first 30 days of life. The boxplot shows the median(central horizontal line), the 10th centile (lower box border) and 90th centile(upper box border). The upper and lower horizontal line refer to the 95th and5th centile, respectively. p = 0.15 between prebiotic mixture group (n = 47) andplacebo group (n = 52).

Figure 3 Stool pH at day 30. The boxplot shows the median (central horizontalline), the 10th centile (lower box border) and 90th centile (upper box border).The upper and lower horizontal line refer to the 95th and 5th centile, respec-tively. p = 0.009 between prebiotic mixture group (n = 51) and placebo group(n = 51).

Table 2 Influence of stool viscosity on feeding tolerance and stool frequency

Regression coefficient p

Time to full enteral

feeding (days)

0.005 ()0.005 to 0.005) 0.97

Age at finishing parenteral

feeding (days)

0.06 ()0.004 to 0.006) 0.67

Total days of minimal enteral

feeding (days)

)0.40 ()0.009 to 0.006) 0.75

Necrotizing enterocolitis* 0.99 (0.94 to 1.03) 0.52

Growth 0.06 ()0.01 to 0.01) 0.67

Stool frequency )0.10 ()0.10 to 0.005) 0.45

Values are expressed as beta or *odds ratio (95% confidence interval).



are thought to increase stool water content and subse-quently reduction in stool viscosity (14). Human volunteersshowed an excess of wet stool mass of approximately 1.5 gper g of fructo-oligosaccharides consumed (21,22). It isunknown whether intestinal microbiota (i.e. higher bifido-bacteria and lactobacillus) additionally affects stool viscos-ity. An extensive review on the effect of prebiotics in infantsand adults found that prebiotic supplementation increasesthe faecal concentration of bifidobacteria, which concomi-tantly improves stool quality (23). Decreased stool viscosityin preterm infants can lead to shorter duration for luminalnutrients remaining in the intestine which may prevent theinflammation cascade and reduce the risk of developingnecrotizing enterocolitis (24). The same mechanism mayapply to the increased risk to (endogenous) infections inpreterm infants. We previously found that enteral supple-mentation of prebiotics, if given in sufficient amounts,decreases the risk of serious endogenous infections in pre-term infants (13). However, in an additional analysis in thepresent study, we did not find a (direct) effect of stool vis-cosity on the incidence of serious (endogenous) infections.

Bacterial fermentation of nondigestible carbohydratesand proteins in the colon results in the production of SCFAand lactate and decrease in the luminal pH (12). In thisstudy, we found a decrease in stool pH at day 30 after ent-eral supplementation of a prebiotic mixture consisting ofneutral and acidic oligosaccharides. One of the mechanismsof the decreased stool pH may be a ‘bifidogenic’ effect onthe intestinal microbiota resulting in the production ofSCFA. The production of SCFA in combination with alower pH may decrease the growth of other groups of bacte-ria, including potentially pathogenic microorganisms(12,25). Moreover, AOS are able to act as receptor ana-logues and are known to inhibit the adhesion of pathogenson the epithelial surface (12).

There was a trend towards increased stool frequency afterenteral supplementation of prebiotics. This is in line withprevious studies with neutral oligosaccharides in pretermand term infants (4,8,19,20,26,27). Mihatsch et al. (4)found, beside increased stool viscosity, accelerated gastroin-testinal transit time as measured with carmine red. Thiseffect may be mediated by SCFA (15).

Improving feeding tolerance is important to reach opti-mal growth and neurodevelopmental outcome. In arecent study, Ehrenkranz et al. (28) found that as therate of weight gain increased from 12.0 to 21.2 g ⁄ kg perday, neurodevelopmental outcome significantly improved.In the previous study, we found no difference in feedingtolerance (e.g. time to full enteral feeding, total days ofparenteral feeding and total day of minimal enteral feed-ing) (13), and in the present study, we found no effect ofthe increased stool viscosity on feeding tolerance. How-ever, in a recent study, Modi et al. found an improvedenteral tolerance in preterm infants <29 weeks after sup-plementation with neutral oligosaccharides. They con-cluded that prebiotic supplementation appears safe andmay benefit enteral tolerance in the most immatureinfants (29). We hypothesize that beside an increased

stool viscosity and pH, other mechanisms may play arole in the effect of prebiotics on feeding tolerance.

Some remarks may be formulated with regard to themethodology of our study. First, the sample size calculatedwas based on the primary outcome of the main trial. There-fore, the sample size may have been insufficient to detect asignificant difference in stool frequency and to detect aneffect of stool viscosity on feeding tolerance. Second, breastmilk itself contains neutral and acidic oligosaccharides.Therefore, the effect of enteral supplementation of prebiot-ics may be less pronounced in preterm infants who exclu-sively received breast milk. However, in an additionalanalysis, we did not find an effect of type of feeding on stoolviscosity, stool frequency or stool pH. As breast milk isstrongly promoted at our NICU, most infants receivedbreast milk (>60%), and relatively few received exclusivelyformula feeding (20%).

In conclusion, enteral supplementation of a prebioticmixture consisting of neutral (scGOS ⁄ lcFOS) and acidic ol-igosaccharides (pAOS) decreases stool viscosity and stoolpH with a trend towards increased stool frequency in pre-term infants. The inclusion of pAOS in a formula containinga mixture of scGOS ⁄ lcFOS does not add specific advanta-ges to the formula in terms of stool viscosity, frequency, pHas well as feeding tolerance.

ACKNOWLEDGEMENTSStudy supplementation (prebiotics and maltodextrin) andpreterm formula (Nenatal Start�) and postdischarge for-mula (Nenatal 1�) for the present study was provided byDanone Research, Friedrichsdorf, Germany. We acknowl-edge the parents for allowing their infants to participate inthe study, the medical and nursing staff of the neonatalintensive care unit of the VU University Medical Center andall participating hospitals. The funding source was notinvolved in the analysis of the data or the interpretation ofthe results. All authors approved the final version of themanuscript.

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The effect of neutral and acidic oligosaccharides on stool viscosity, stool frequency and stool pH in preterm infants