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Prebiotic Effect of an Infant Formula Supplemented with Galacto-Oligosaccharides: Randomized Multicenter Trial a
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Marcello Giovannini MD , Elvira Verduci MD , Dario Gregori PhD , Simonetta Ballali BA , Sara c
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Soldi PhD , Diana Ghisleni MD , Enrica Riva MD & for the PLAGOS Trial Study Group a
Department of Pediatrics, San Paolo Hospital, University of Milan, Milan, ITALY
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Unit of Biostatics, Epidemiology and Public Health, Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Padova, Italy. c
AAT-Advanced Analytical Technologies S.r.l., Piacenza, Italy. Published online: 10 Oct 2014.
Click for updates To cite this article: Marcello Giovannini MD, Elvira Verduci MD, Dario Gregori PhD, Simonetta Ballali BA, Sara Soldi PhD, Diana Ghisleni MD, Enrica Riva MD & for the PLAGOS Trial Study Group (2014) Prebiotic Effect of an Infant Formula Supplemented with Galacto-Oligosaccharides: Randomized Multicenter Trial, Journal of the American College of Nutrition, 33:5, 385-393, DOI: 10.1080/07315724.2013.878232 To link to this article: http://dx.doi.org/10.1080/07315724.2013.878232
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Original Research
Prebiotic Effect of an Infant Formula Supplemented with Galacto-Oligosaccharides: Randomized Multicenter Trial Marcello Giovannini, MD, Elvira Verduci, MD, Dario Gregori, PhD, Simonetta Ballali, BA, Sara Soldi, PhD, Diana Ghisleni, MD, Enrica Riva, MD, for the PLAGOS Trial Study Group Department of Pediatrics, San Paolo Hospital, University of Milan, Milan, ITALY (M.G., E.V., D.G., E.R.), and Unit of Biostatistics, Epidemiology and Public Health, Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Padova, ITALY (D.G., S.B.), AAT-Advanced Analytical Technologies S.r.l, Piacenza, ITALY (S.S.)
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Keywords: galacto-oligosaccharides, infant formula, prebiotic, Clostridium, bifidogenic activity Objective: The objective of the study was to investigate the effects of a galacto-oligosaccharides (GOS)supplemented formula on the intestinal microbiota in healthy term infants, with a specific consideration for gastrointestinal symptoms as colic, stool frequency and consistency, regurgitation. Methods: This was a randomized, double-blind, controlled, parallel-group clinical trial performed simultaneously by 6 centers in Italy. Three groups were considered: breastfed, formula-fed, and GOS-supplemented formula-fed infants. Formula-fed infants were randomized to receive either the control or the study formula and consume the assigned formula exclusively until the introduction of complementary feeding. The nutritional composition of the 2 formulas were identical, apart from the supplemented GOS (0.4 g/100 mL) in the study formula. Four different types of bacteria were evaluated in order to assess the efficacy of GOS-supplemented formula on infants: Bifidobacterium, Lactobacillus, and Clostridium, Escherichia coli. Results: A total of 199 breastfed infants and 163 formula-fed infants were recruited. When considering stool frequency and consistency, GOS-supplemented formula presented normal and soft stools in the majority of episodes (89%). In the supplemented group the incidence of colic was lower with respect to the control group. A significantly lower count of Clostridium and a higher count of Bifidobacterium were found when comparing study formula and control formula in infants with colic. In children with colic the ratio between Clostridium count and Bifidobacterium and Lactobacillus count was in favor of the latter two when considering the GOS-supplemented formula group with respect to the control one. Conclusions: The prebiotic-supplemented formula mimicked the effect of human milk in promoting Bifidobacterium and Lactobacillus growth and in inhibiting Clostridium growth, resulting in a significantly lower presence of colic.
INTRODUCTION
formulas have been developed with the specific purpose of mimicking human milk effects on both growth and gastrointestinal (GI) bacteria colonization [4]. The gastrointestinal microbiota of infants are highly influenced by the type of feeding they receive [5]; the stools of breastfed infants are dominated by Bifidobacterium and, to a lesser degree, by Lactobacillus [6]. The colonizing Bifidobacterium and Lactobacillus may inhibit the growth of pathogenic microorganisms through the production of lactic, acetic, and other organic acids, with a
Exclusive breastfeeding is widely recognized as the gold standard for infant nutrition in the first 6 months of life [1,2]. If, for whatever reason, human milk becomes unavailable to an infant, milk formulas are the only alternatives to breast milk. Their aim is to provide the best alternative to human milk for infants who cannot be breastfed and to approach the structural and functional effects observed in breastfed infants [3]; therefore, milk
Address correspondence to: Marcello Giovannini, MD, Professor of Medicine, Chief of the Department of Pediatrics, San Paolo Hospital, University of Milan, Via Antonio di Rudin´ı n. 8, 20142 Milan, ITALY. E-mail: [email protected] PLAGOS Trial Study Group: Giuseppe Banderali, Diana Ghisleni (Department of Pediatrics, San Paolo Hospital, University of Milan, Milan, ITALY); GianVincenzo Zuccotti, Laura Pogliani (Department of Paediatrics, Luigi Sacco Hospital, Universit`a di Milano, Milan, ITALY); Giacomo Biasucci, Cristina Cicero (Department of Paediatrics and Neonatology, Guglielmo da Saliceto Hospital, Piacenza, ITALY); Alberto Podest`a, Monica Tonella (Department of Pediatrics and Neonatology, San Carlo Borromeo Hospital, Milan, ITALY); Amilcare Rottoli, Tiziana Bonabitacola (Unit of Pediatrics and Neonatology, Presidio Ospedaliere di Cernusco sul Naviglio and Presidio Ospedaliero di Melzo, Cernusco sul Naviglio and Melzo, ITALY)
Journal of the American College of Nutrition, Vol. 33, No. 5, 385–393 (2014) C American College of Nutrition Published by Taylor & Francis Group, LLC 385
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Galacto-Oligosaccharide and Prebiotic Effect consequent decrease in intraluminal pH that inhibits the growth of some bacterial pathogens. Moreover, Bifidobacterium and Lactobacillus compete with potentially pathogenic bacteria for nutrients and epithelial adhesion sides [7]. The effects of breastfeeding on the intestinal microbiota cannot be attributed to a single compound, but there is accumulating evidence that human milk oligosaccharides play a crucial role [8]. Therefore, the addition of prebiotic oligosaccharides to infant formulas has grown in diffusion in the latest years, mainly focusing on supplementation of a mixture of fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) [9]. Several studies have been published, specifically investigating the effect of prebiotics on microbiota growth in the infant’s gastrointestinal tract. Results significantly vary. In their study, Brunser et al. evaluated whether supplementation of milk formulas with prebiotic FOS or a probiotic could modulate the composition of the fecal microbiota of formula-fed infants compared to breastfed infants [10]. Their results confirmed the presence of higher counts of Bifidobacterium and Lactobacillus in the microbiota of breastfed infants compared to formula-fed infants before dietary diversification. Similar results were found by Harmsen et al. [11], Guaraldi and Salvatori [12], and Bezirtzoglou et al. [13]. On the other hand, Knol and colleagues found that the addition of the prebiotic GOS/FOS mixture to an infant formula had a stimulating effect on the growth of Bifidobacterium and on the metabolic activity of the total intestinal flora of their considered sample [14]. Bakker-Zierikzee et al. also compared the effects of infant formula containing a mixture of galactoand fructo-oligosaccharides or viable Bifidobacterium animalis on the composition and metabolic activity of the intestinal microflora, concluding that feeding infants GOS/FOS formula resulted in a similar effect on metabolic activity of the flora as in breastfed infants [15]. Their conclusions were subsequently backed by Miniello et al.’s analysis of the composition of the intestinal bacterial populations; it was concluded that the prebiotic induced a significantly higher colonization of Bifidobacterium and Lactobacillus [16]. Similar conclusions were drawn by Fanaro and Vigi [17]. Their research highlighted that a mixture of oligosaccharides showed its efficacy in stimulating the establishment of a “bifidus flora,” with stools closer to those found in breastfed infants. Despite this, the Committee on Nutrition of the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition recently concluded that there is still a lack of data on the long-term effects of the administration of formulas supplemented with prebiotics [18]. When considering supplemented infant formulas, adverse events are specifically considered. From a microbiological point of view, colicky infants are usually associated with lower Lactobacillus and Bifidobacterium colonies and increased anaerobic Clostridium species in place of anaerobic bacteroides species and Bifidobacterium species [19]. Our study evaluated the effect of a GOS-supplemented infant milk formula (0.4 g/100 mL)
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on beneficial bacteria, such as Bifidobacterium and Lactobacillus, as well as on other bacteria, in particular, Clostridium and Escherichia coli. Results were also evaluated in order to assess the functional effect on digestive issues, such as colic or regurgitation. This article is the first to present results from a sample receiving an exclusive GOS-supplemented formula. Previous studies have considered the safety of FOS-GOS formulas [15] or GOS formulas [20], showing specific effects on beneficial bacteria. The present results furthermore investigated the specific role of supplemented formula in decreasing total pathogen count and colonization.
MATERIALS AND METHODS Study Design This was a randomized, double-blind, controlled, parallelgroup clinical trial performed simultaneously by 6 centers in Italy from February 2006 to October 2011. The study was conducted in compliance with the Declaration of Helsinki (revised Tokyo 2004 version) and Good Clinical Practices (CPMP/ICH/135/95) and in accordance with national regulatory requirements and laws. All relevant study documents were approved by the Ethics Committees of each participating center. All patients provided signed informed consent and could withdraw from the study at any time. This trial is registered with ClinicalTrials.gov, number NCT00486148.
Subjects In this study, 362 infants who met the following inclusion criteria were enrolled by the 15th day of life: Caucasian origin; healthy infants of both sexes; natural or Caesarian delivery; single birth; gestational age from 37 to 42 completed weeks; birth weight ≥ 2500 g; 5-min APGAR score > 7; exclusive breastfeeding or formula feeding at the moment of enrollment; and parents’ full understanding of the protocol. Infants were excluded if they met even only one of the following criteria: bearing congenital malformation; being diagnosed with hereditary diseases, chronic diseases, or neonatal illnesses requiring more than 7 days of hospitalization; presence of prenatal pathologies undermining intrauterine growth; infant’s mother affected by dismetabolic or chronic diseases; unknown father; uncertainty of parents’ compliance toward protocol and regular visits; or infant recruited or selected for other clinical studies. Three groups were considered according to the type of feeding: breast milk, GOS-supplemented infant milk formula (study formula), and standard infant milk formula (control formula). The breast milk group was considered as the reference group; therefore, no randomization was performed. A total of 199 breastfed infants and 163 formula-fed infants (80 in the control and 83 in the study group) were recruited.
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Galacto-Oligosaccharide and Prebiotic Effect Diets
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Formula-fed infants were randomized to receive either the control or the study formula and were to consume the assigned formula exclusively until the introduction of complementary feeding. The compositions of the 2 formulas were identical, with the exception of the supplemented oligosaccharides. The study formula was supplemented with 0.4 g/100 mL of GOS (Vivinal GOS Galactooligosaccharide; Friesland Foods Domo, Amersfoort, The Netherlands). Study and control formulas were supplied by Heinz Italia S.p.A. (Latina, Italy). The 2 formulas were provided in powdered form and could not be differentiated by smell, consistency, or any other characteristics. Neither the investigators nor the parents knew which product the infant was receiving.
Visits Anthropometric parameters and any adverse events (AEs) were recorded at 6 different time points: baseline visit (B) within 15 days after birth, visit 1 (V1; 30 ± 10 days of life), visit 2 (V2; 60 ± 10 days of life), visit 3 (V3; 90 ± 10 days of life), visit 4 (V4; 120 ± 10 days of life), and final visit (Vf), performed just before the introduction of complementary feeding. A fecal sample was collected at B, V1, V2, and Vf. In the presence of adverse effects, an extra visit was planned and a fecal sample was collected. At B, parents were given a diary to record daily stool frequency and consistency and adverse events. Infants’ growth was assessed through anthropometric measurements of weight (g), length [21], and occipito-frontal head circumference [21] and z-scores were calculated.
Adverse Effects In order to evaluate the safety of the study formula anthropometric data, incidence of AEs and infants’ acceptance of the formulas were considered. AEs were defined as undesired, unplanned clinical events, in the form of signs and symptoms or disease occurring in an infant participating in a clinical study, regardless of causal relationship, like diarrhea, infantile colic, prolonged crying, regurgitation, vomiting, cutaneous manifestations, and fever.
Bacterial Analysis Bacterial analysis was performed at the Advanced Analytical Technologies Center in Piacenza by the assigned researchers. Four different types of bacteria were evaluated in order to assess the efficacy of GOS-supplemented formula on infants: Bifidobacterium, Lactobacillus, Clostridium, and Escherichia coli.
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Samples collected at B, V1, and V2 and derived from a subpopulation of 75 naturally delivered infants who had not taken antibiotics or probiotics during the study period were analyzed. Analyses included (1) quantification of Lactobacillus, Bifidobacterium, and coliforms through real-time polymerase chain reaction (PCR) and (2) spore counts by means of inclusion in selective medium for Clostridium. For biomolecular quantification of the indicated genera, total bacterial DNA was extracted from fecal samples by means of mechanical cell disruption using a Fast Prep Instrument (MP Biomedical, Santa Ana, CA, USA) and the related FastDNA Spin Kit. DNA concentrations were determined using a NanoDrop (Thermo Scientific, Rodano, Milan, Italy). All fecal DNA samples were adjusted to equal concentrations for subsequent qPCR analysis. An amplification mixture was prepared using a RealMasterMix Sybr Rox 2.5X (Eppendorf AG, Hamburg, Germany) with primers that amplify the genes encoding 16S rRNA from the 3 specific bacterial groups, according to supplier’s instruction and following, as a reference, scientific papers appropriate for the purpose of the analysis. Different protocols and primer sequences were applied for quantification of the 3 bacterial groups Bifidobacterium, coliforms, and Lactobacillus [22–24] and optimized thermal cycles were followed. Each PCR was carried out in a final volume of 25 µl and each plate included duplicate reactions per DNA sample, a no-template negative control, and the appropriate set of standards. The latter were generated by the dilution of reference strain pure DNAs from Internationally Recognized Collections. All assays were conducted in 96-well plates on an Eppendorf Realplex2 mastercycler thermocycler (Eppendorf). To achieve Clostridium genus quantification, fecal samples were suspended 1/10 (w/v) in Ringer solution and an aliquot of this first suspension was then used for further decimal dilutions. For each fecal sample the appropriate dilutions were included in Reinforced Clostrium Medium (RCM) Agar (Oxoid Ltd., Altrincham, Cheshire, England) after thermal pasteurization at 75◦ C for 30 min. Plates were then incubated in anaerobic conditions for 48 h at 37◦ C. All colonies encompassing different morphologies were counted on these plates. Though other methods can be used for Clostridium count, RCM implemented with the thermization process is a recognized technique [25,26].
Sample Size Estimation A total of 160 formula-fed infants (80 receiving the control formula and 80 receiving the study formula) would give the power to detect a 24% reduction in adverse events for the GOSsupplemented formula compared to the control, at 80% power and a 5% significance level, assuming a 20% drop-out rate. Recruiting 200 breastfed infants, assuming a 20% drop-out rate and a breastfeeding rate at 6 months at about 50% would ensure a control group of 64 exclusively breastfed infants at 6 months.
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Galacto-Oligosaccharide and Prebiotic Effect Statistical Method
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Data have been presented using median (I and III quartile as measures of dispersion) for quantitative variables and as percentages (absolute numbers are presented in brackets) for categorical variables. Tests for the difference of main variables at each visit were performed using a test statistic based on the chi-square test and on Wilcoxon or Kruskal-Wallis test using the F distribution, whenever appropriate. Risk rates of adverse events were based on a Poisson log-linear model, treating child-days of exposure as an offset in the model. In case of multiple assessments per subject, p values were derived by the estimation of a robustified version of the above-mentioned statistics, using the Huber-White method [27]. The ratio between bacterial classes was performed on total bacterial count. All analyses were performed using R [28].
RESULTS A total of 199 breastfed infants and 163 formula-fed infants (80 in the control group and 83 in the study group) were recruited, with a drop-out rate of 39% and 26%, respectively. Voluntary interruption was the most common reason for dropout in all groups, 58% in the breastfed group and 86% in the formula-fed group. Table 1 shows the general characteristics of the sample, based on the type of delivery. Female and male infants were equally represented in both groups, and the majority of the infants (53%, 187 infants) were first-born children. Maternal choice was the main reason to opt for infant formula instead of breast milk (48% of overall cases), and agalactia was the second most frequent reason (26%). When considering the general data, a significant association (p < 0.001) was found between natural delivery and the choice to breastfeed. A separate evaluation of descriptive variables was performed, considering the 3 different groups divided by feeding choice.
Table 1. Demographics and Birth Characteristics
Gender (male) Parity: 1 2 3 Human milk Control formula Study formula Reason for not breastfeeding: Agalactia Breast refusal Hypogalactia Previous breast cancer Maternal choice
Caesarean Delivery (N = 86)
Natural Delivery (N = 276)
51 (44) 52 (45) 43 (37) 5 (4) 29 (25) 49 (30) 51 (31) 22 (12) 0 (0) 30 (16) 2 (1) 46 (25)
50 (137) 53 (142) 40 (108) 6 (17) 63 (174)1 49 (50) 51 (52) 29 (24) 1 (1) 18 (15) 2 (2) 49 (41)
All data, if not differently indicated, are reported as relative frequencies (%) with absolute values in brackets (N). 1 Significant association (p < 0.05) is indicated when found.
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All 3 groups showed appropriate physical growth throughout the study period (results on safety evaluation not shown in this article). Risk rates of adverse events were evaluated, considering in the model total episodes and treating child-days of exposure. When considering diarrhea episodes, human milk and the study formula showed comparable results. Stool frequency was also assessed. A significant difference was observed when comparing human milk and the study formula and when comparing the study formula and control formula, in both cases showing a lower occurrence in the study formula group. A significant difference in stool frequency was also observed between the human milk group and study formula group. When considering stool consistency, the study group presented normal and soft stools in the majority of episodes (89%). A significant difference was observed when considering watery consistency, showing a significant lower risk rate in the study formula group. Infantile colic showed a lower risk rate in the study formula group compared to both other groups (p < 0.05). Regurgitation risk rates were significantly different when comparing the study formula group and control formula group. The most frequent cutaneous manifestations in all groups were erythema and dry skin. All collected data referring to stool frequency and consistency, diarrhea episodes, and gastrointestinal effects are presented in Table 2. Table 2. Gastrointestinal Symptoms in the Follow-up Period Human Milk (17,548) Diarrhea episodes Frequency Stool frequency Consistency: Watery Semisolid Hard Normal Soft Infantile colic Regurgitation Cutaneous manifestations: No Erythema Blemishes Dry skin Localization: Arms Arms and legs Arms and trunk Arms and face Arms, face, and legs
Control Formula (8692)
Study Formula (7328)
2 (260) 3 (2–6) 9 (7–11) 26 (3431) 1 (191) 0 (24) 45 (5875) 28 (3677) 15 (2631) 37 (6381)
1 (112) 7 (7–7) 3 (3–7)1,2 4 (256)1 9 (645) 4 (277) 57 (3942) 26 (1841) 16 (1399) 33 (2868)1
2 (116) Not applicable 3 (3–7)3 4 (209)3 4 (197) 3 (186) 51 (2737) 38 (2005) 10 (751)2 3 24 (1714)2
89 (15,303) 5 (862) 1 (171) 5 (860) 0 (5) 2 (31) 0 (2) 0 (0) 0 (4)
91 (7914) 3 (242) 1 (45) 5 (473) 3 (19)1,2 0 (3) 0 (0) 0 (0) 0 (0)
93 (6748) 3 (249)3 1 (107) 2 (114)3 4 (22)3 1 (7) 0 (2) 1 (3) 0 (0)
All qualitative variables are reported with as risk rates and absolute number of events (in parentheses). Denominators (column headings) are expressed as childdays of exposure. Quantitative data are presented as median value, with 5th and 95th percentiles in brackets. 1 Significant difference (p < 0.05) when considering human milk vs control formula. 2 Significant difference (p < 0.05) when considering study formula vs control formula. 3 Significant difference (p < 0.05) when considering human milk vs study formula.
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Galacto-Oligosaccharide and Prebiotic Effect Table 3. Comparison between Different Formulas and Bacterial Colonization
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N Entire follow up Clostridium Bifidobacterium Escherichia coli Lactobacillus group Clostridium/ (Lactobacillus Baseline Clostridium Bifidobacterium Escherichia coli Lactobacillus group Clostridium/ (Lactobacillus V1 Clostridium Bifidobacterium Escherichia coli Lactobacillus group Clostridium/ (Lactobacillus V2 Clostridium Bifidobacterium Escherichia coli Lactobacillus group Clostridium/ (Lactobacillus
+ Bifidobacterium)
220 220 209 219 217
+ Bifidobacterium)
74 74 69 74 73 73 73 68 73
+ Bifidobacterium) 73 73 72 72 + Bifidobacterium)
Control Formula
Study Formula
(79) 3.9E04 (0.1E04–41E04) 4.1E09 (1.2E09–12E09) 0.3E06 (0.0–12.0E06) 5.1E05 (0.2E05–504.6E05) 9.0E-6 (3.4E-07–1.9E-04) (79) 0.8E04 (0.0–74E04) 6.0E09 (1.1E09–15.7E09) 0.01E06 (0.0–6.4E06) 5.1E05 (0.5E05–55.4E05) 1.3E-06 (0.0–5.2E-04) (80) 1.9E04 (0.5E04–28.2E04) 2.7E09 (0.5E09–7.2E09) 0.3E06 (0.0–9.0E06) 3.1E05 (0.2E05–527.7E05) 9.0E-06 (1.3E-06–1.1E-04) (80) 7.8E04 (1.8E04–3.1E05) 4.7E09 (2.0E09–12.53E09) 0.3E06 (0.0–39.1E06) 6.7E05 (1.0E05–576.1E05) 1.1E-05 (3.5E-06–9.8E-05)
(83) 0.9E04 (0–9.8E04) 4.8E09 (1.9E09–100E09) 0.6E06 (0–77.3E06) 6.4E05 (0.6E05–43.0E05) 1.4E-06 (1.7E-10–2.8E-05)1 (83) 0.65E04 (0.005E04–16.6E04) 7.4E09 (1.6E09–79–9E09) 0.5E06 (0.001E06–71.6E06) 5.2E05 (0.5E05–76E05) 1.4E-06(3.6E-09–1.1E-04) (83) 0.6E04 (0.0–8.3E04) 3.6E09 (1.9E09–55.9E09) 0.6E06 (0.04E06–37.7E06) 3.4E05 (0.3E05–87.6E05) 1.3E-06 (1.7E-10–2.7E-05) (83) 1.2E04 (0.0–3.7E04)1 5.3E09 (1.8E09–159.7E09) 0.8E06 (0.02E06–348.7E06) 11.6E05 (3.1E05–2.45E05) 9.9E-07 (7.0E-11–1.6E-05)1
V1 = visit 1, V2 = visit 2. All data are presented as median values, with 25th and 75th percentiles in brackets. The letter E indicates a power of 10. 1 Significant difference (p < 0.05) when considering study formula vs control formula.
Bacterial colonization was considered in terms of proliferation of Bifidobacterium, Lactobacillus, E. coli, and sporigen behavior of Clostridium. Total counts of the selected bacteria for visits B, V1, and V2 were performed. In addition, an analysis of the whole period (from B to V2) was performed. All data are presented in Table 3, referring to the control group and GOS formula. Bacterial colonization showed comparable figures at baseline and at V1, and at V2 Clostridium count showed a significant difference when comparing the study formula and control formula (p < 0.05). When considering the entire follow-up, no significant difference in bacterial count was observed. In order to better establish the proportion of Clostridium with respect to other bacteria, an additional evaluation was carried out as suggested by the authors of the present article, showing a significant difference over the entire follow-up and at V2 in the ratio between Clostridium and other bacteria (Clostridium/ Bifidobacterium + Lactobacillus), with p values of 0.02 and 0.021, respectively. Clostridium was specifically considered, due to the well-known association between infant colic and Clostridium colonization [29,30]. In colicky infants higher numbers of colonic bacteria-producing gas have been detected, concomitant with fewer bacteria that do not produce gas (e.g., Lactobacillus and Bifidobacterium) [31,32]. To compare the effect of different formula compositions with respect to the occurrence of infantile colic and bacterial colonization, a subgroup analysis was performed in the formula groups focusing on the period from baseline to the 70th day of life. Data
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showed that in infants with colic, the study formula resulted in a better bacterial profile, showing a significantly lower Clostridium count (p < 0.05) and stimulating both Bifidobacterium, and showed a significantly different bacterial count between the 2 groups (p < 0.05) and Lactobacillus growth. In the absence of colic, the 2 groups showed similar bacterial colonization when considering Bifidobacterium, E. coli, and Lactobacillus, whereas Clostridium count showed a significant difference between the 2 groups (p < 0.05). All data are presented in Table 4. When considering the breast milk group, a difference between human milk and control formula patterns was observed: the first had a significantly better microbiological pattern, as expected from the literature [1].
DISCUSSION The results of our study confirmed the initial hypothesis that the GOS-supplemented formula had an efficient performance on gastrointestinal microbiota, mimicking human milk promotion of Bifidobacterium growth and reduction of Clostridium after an initial period of one month. When comparing the study formula with the control formula, the GOS-supplemented formula showed a significant effect in lowering risk rates of colic and regurgitation, with a boosting effect on the beneficial bacterial flora and an inhibiting effect on Clostridium growth. Clostridium growth, inducing gas production, appears to be linked to infant
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Galacto-Oligosaccharide and Prebiotic Effect Table 4. Bacterial Colonization and Infantile Colic Occurrences in the Formula Randomized Groups from Baseline up to the 70th Day
Presence of colic Clostridium Bifidobacterium Escherichia coli Lactobacillus Absence of colic Clostridium Bifidobacterium Escherichia coli Lactobacillus
N
Control Formula
Study Formula
28 28 25 28
2.4E04 (0.4E04–47E04) 2.9E09 (1.6E09–10.8E09) 0.3E06 (0E06–13.4E06) 0.1E06 (0.03E06–37.8E06)
0E04 (0E04–0.4E4)1 1.2E11(8.3E10–1.7E11)1 0.2E06 (0.1E06–20.1E06) 0.10E06 (0.04E06–1.8E06)
81 81 79 80
6.1E04 (0.6E04–23.8E04) 4.1E09 (0.08E9–9.4E09) 0.3E06 (0E06–39.2E06) 0.6E06 (0.02E06–105E06)
0.1E04 (0E04–4.2E04) 1 6.3E09 (1.6E09–171E09) 0.3E06 (0E06–124.8E06) 0.6E06 (0.1E06–3.1E06)
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All qualitative variables are reported with absolute number of studied infants in brackets, (N) referring to the bacterial count from baseline to visit 2. Continuous data are presented as median values, with 25th and 75th percentiles in brackets. The letter E indicates a power of 10. 1 Significant difference (p < 0.05) when considering study formula vs control formula.
colic, from a physiological perspective. This consideration was taken into account in order to better establish the impact of the new formula on the growth of non-gas-producing bacteria and gas-producing bacteria, in order to better assess the efficacy of the new formula on gastrointestinal symptoms. The predominance of beneficial bacteria in the gut microbiota of breastfed infants is thought to result from the fermentation of oligosaccharides; that is, nondigestible carbohydrates consisting of 3 to 10 linked monosaccharides. Human milk contains more than 130 different oligosaccharides at a concentration of 15– 23 g/L in colostrum and 8–12 g/L in transitional and mature milk [7]. The GOS-supplemented formula used in the present study contained 0.4 g of galactooligosaccharides per 100 mL of reconstituted product. Numerous studies have reported that, since the very beginning of life gut microbiota play an important role in immune response and the growth [33]. In the present study, after an initial phase, the GOS-supplemented formula group showed the same trends of Bifidobacterium and Lactobacillus bacterial development as the breastfed group. This result is in line with Xia et al.’s study, which showed that the milk-based formula used in this study supported Bifidobacterium and Lactobacillus populations comparable to those in human milk [34]. A systematic review of trials evaluating the effect infant milk formulas supplemented with prebiotics (GOS-FOS, long-chain FOS [lcFOS], polydextrose-GOS-lactulose [PDX-GOS-LOS], or GOS) in full-term neonates has shown that doses ranging from 0.15 to 0.8 g/dL appear to achieve a maximum bifidogenic effect with minimal intolerance in the form of flatulence, abdominal distention, colic, etc. [35]. The stools of breastfed infants are dominated by Bifidobacterium and, to a lesser degree, by Lactobacillus [6]; thus, the goal of a cow milk–based formula should be to mimic the flora established due to human milk, reducing the GI colonization from potentially detrimental bacteria such as Clostridium [36]. Knol and colleagues compared a standard formula with a formula containing both short-chain GOS and inulin in healthy term
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newborns exclusively formula-fed [14,37] and they found that the prebiotic formula has a stimulating effect on the growth of and on the metabolic activity of the total intestinal flora. In addition, the proportion of E. coli and Clostridium species was lower in the prebiotic group. The bifidogenic effect of the supplementation of an infant formula with GOS was evaluated by Fanaro and colleagues, who concluded that it contributes to a favorable intestinal microbiota; formula’s effect on pathogens was not considered in the study [6]. Similar results were found by Costalos and colleagues, whose study presented a trend toward a reduction in pathogenic bacteria in the prebiotic-supplemented group (0.4 g/100 ml GOS and lcFOS, ratio 90:10) [38]. In this prospective double-blind study, Costalos and colleagues concluded that an infant formula containing prebiotic oligosaccharides was well tolerated and suppressed the numbers of Clostridium in the feces with a trend for a higher percentage of stool Bifidobacterium and lower percentage of E. coli. A reduction in pathogens was found also in the study performed by Alliet et al. with a GOSFOS mixture formula (0.6 g/100 mL of GOS/lcFOS, in a ratio of 9:1) [39]. To our knowledge, this is the first study that analyzed the efficacy of an infant formula supplemented with 0.4 g/100 mL of only GOS and showed similar effect in Bifidobacterium and Lactobacillus group development [40], as well as exerted an inhibiting function on the growth of sporulating Clostridium and a reduction in colic and regurgitations. Savino et al.’s observational study investigated the effect of FOS/GOS-supplemented formula on colic, regurgitation, and constipation, concluding that within 2 weeks of feeding with that new formula minor gastrointestinal symptoms improved [41]. In a subsequent study in 2005, Savino and colleagues confirmed previous findings, observing that a prebiotic mixture of FOS/GOS with a high beta-palmitic acid content reduced digestive problems and improved intestinal tolerance in infants during the first months of life [42]. In order to assess the performance of the study formula with respect to the control formula, in the present study bacterial counts were analyzed in children both with or without
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Galacto-Oligosaccharide and Prebiotic Effect presence of colic, from baseline to the 70th day of life. The bacterial profile was more favorable in infants who received GOS-supplemented formula, with respect to the control group. Infants fed the GOS-supplemented formula showed a significantly lower presence of Clostridium, when considering children with colic and without colic, and a significantly higher number of Bifidobacterium in children with colic. Therefore, in addition to the bifidogenic effect, the GOS-supplemented formula used in this study contributed to a decreased count of potentially pathogenic enteric bacteria and softer stools, without adversely affecting weight gain, as recently published [20]. In the present study, a reduction in colic and regurgitation frequency was recorded, even if not statistically associated with Clostridium and E. coli reduction. The association between microbiota patterns and infant colic has recently been investigated, highlighting specific conditions such as gastrointestinal colonization from anaerobic bacteria [31]. In particular, it is highlighted that infants with colic are most often colonized by gram-negative anaerobic bacteria and less frequently by Bifidobacterium and Lactobacillus, with differences in the patterns of the same intestinal Lactobacillus, L. lactis and L. brevis, producers of ethyl alcohol and carbon dioxide, while L. acidophilus has been shown only in healthy infants [42]. This pattern could be influencing gastrointestinal regulation, leading to an increase in gas production, with the consequent onset of the typical symptoms of the disorder [43]. Further studies need to be performed in order to assess the functional effect of GOS-supplemented formula. In this study, Bifidobacterium count was at higher median levels than Lactobacillus in the fecal samples over the whole study period in all 3 groups. This is in accordance with other studies [33] showing that Bifidobacterium dominate over Lactobacillus in this age group [27]. Several studies have reported a geographical behavior in Lactobacillus development, stressing how they appear to be lower in Western countries [44]. In this study, Lactobacillus in all fecal samples could be detected and quantified by real-time PCR and from B to V2 counts, and the quantification performed on the entire follow-up showed the same median values in feces from breastfed infants and infants fed the GOS-supplemented formula. Breastfed infants presented a GI flora dominated by Bifidobacterium and Lactobacillus, not only in the first days of life but also in the following weeks. Our study showed that GOSsupplemented formula performed in the same way, promoting an appropriate level of both bacteria in the gastrointestinal tract of the studied infants. When considering the quantification of potentially noxious bacteria as, for instance, many components of the wide Clostridium genus, their presence was lower in the study group with respect to those fed the control formula. In order to evaluate the progression of flora development, which at these early stages is considerably complex [45], the ratio between Clostridium count and Bifidobacterium and Lactobacillus was considered. A significantly lower ratio, synonymous with a higher presence of the latter desirable bacteria, was
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found when considering both the entire follow-up and the retrieval after 2 months of life (V2), confirming the efficacy of the GOS-supplemented formula with respect to the control formula in favoring the growth of positive flora, as commonly found in breastfed children [45,46].
CONCLUSION Thus, the initial hypothesis of the bifidogenic activity of the GOS-supplemented formula can be confirmed. Moreover, we have demonstrated an inhibitory effect on the development of gastrointestinal Clostridium in infants born at term who were fed the GOS-supplemented formula. The association between microbial gut composition and functional effects and improvement exerted by the administration of enriched formulas needs to be investigated in depth by following studies on subjects receiving infant formula supplemented exclusively with GOS.
FUNDING This project has been funded by Heinz Italia SpA (Latina, Italy).
REFERENCES 1. Section on Breastfeeding. Breastfeeding and the use of human milk. Pediatrics 129:e827–e841, 2012. 2. World Health Organization: “The Optimal Duration of Exclusive Breastfeeding. Report of an Expert Consultation.” Geneva: World Health Organization, March 28–30, 2001. 3. D’Auria E, Agostoni C, Giovannini M, Riva E, Zetterstrom R, Fortin R, Greppi GF, Bonizzi L, Roncada P: Proteomic evaluation of milk from different mammalian species as a substitute for breast milk. Acta Paediatr 94:1708–1713, 2005. 4. Koletzko B, Baker S, Cleghorn G, Neto UF, Gopalan S, Hernell O, Hock QS, Jirapinyo P, Lonnerdal B, Pencharz P, Pzyrembel H, Ramirez-Mayans J, Shamir R, Turck D, Yamashiro Y, Zong-Yi D: Global standard for the composition of infant formula: recommendations of an ESPGHAN coordinated international expert group. J Pediatr Gastroenterol Nutr 41:584–599, 2005. 5. Noverr MC, Huffnagle GB: The “microflora hypothesis” of allergic diseases. Clin Exp Allergy 35:1511–1520, 2005. 6. Fanaro S, Marten B, Bagna R, Vigi V, Fabris C, Pena-Quintana L, Arguelles F, Scholz-Ahrens KE, Sawatzki G, Zelenka R, Schrezenmeir J, de Vrese M, Bertino E: Galacto-oligosaccharides are bifidogenic and safe at weaning: a double-blind randomized multicenter study. J Pediatr Gastroenterol Nutr 48:82–88, 2009. 7. Agostoni C, Axelsson I, Goulet O, Koletzko B, Michaelsen KF, Puntis JW, Rigo J, Shamir R, Szajewska H, Turck D: Prebiotic oligosaccharides in dietetic products for infants: a commentary by
391
Galacto-Oligosaccharide and Prebiotic Effect
8. 9.
10.
Downloaded by [University of Louisville] at 12:44 01 January 2015
11.
12.
13.
14.
15.
16. 17.
18.
19.
20.
21. 22.
392
the ESPGHAN Committee on Nutrition. J Pediatr Gastroenterol Nutr 39:465–473, 2004. Boehm G, Moro G: Structural and functional aspects of prebiotics used in infant nutrition. J Nutr 138:1818S–1828S, 2008. Knol J, Boehm G, Lidestri M, Negretti F, Jelinek J, Agosti M, Stahl B, Marini A, Mosca F: Increase of faecal bifidobacteria due to dietary oligosaccharides induces a reduction of clinically relevant pathogen germs in the faeces of formula-fed preterm infants. Acta Paediatr 94:31–33, 2005. Brunser O, Figueroa G, Gotteland M, Haschke-Becher E, Magliola C, Rochat F, Cruchet S, Palframan R, Gibson G, Chauffard F, Haschke F: Effects of probiotic or prebiotic supplemented milk formulas on fecal microbiota composition of infants. Asia Pac J Clin Nutr 15:368–376, 2006. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp AA, Klijn N, Bindels JG, Welling GW: Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 30:61–67, 2000. Guaraldi F, Salvatori G: Effect of breast and formula feeding on gut microbiota shaping in newborns. Front Cell Infect Microbiol 2:94, 2012. Bezirtzoglou E, Tsiotsias A, Welling GW: Microbiota profile in feces of breast- and formula-fed newborns by using fluorescence in situ hybridization (FISH). Anaerobe 17:478–482, 2011. Knol J, Scholtens P, Kafka C, Steenbakkers J, Gro S, Helm K, Klarczyk M, Sch¨opfer H, B¨ockler HM, Wells J: Colon microflora in infants fed formula with galacto- and fructo-oligosaccharides: more like breast-fed infants. J Pediatr Gastroenterol Nutr 40:36–42, 2005. Bakker-Zierikzee AM, Alles MS, Knol J, Kok FJ, Tolboom JJ, Bindels JG: Effects of infant formula containing a mixture of galacto- and fructo-oligosaccharides or viable Bifidobacterium animalis on the intestinal microflora during the first 4 months of life. Br J Nutr 94:783–790, 2005. Miniello VL, Moro GE, Armenio L: Prebiotics in infant milk formulas: new perspectives. Acta Paediatr 91:68–76, 2003. Fanaro S, Vigi V: Infant formulas supplemented with prebiotics: intestinal microbiota and immune responses. Minerva Pediatr 60:327– 335, 2008. Braegger C, Chmielewska A, Decsi T, Kolacek S, Mihatsch W, Moreno L, Piescik M, Puntis J, Shamir R, Szajewska H, Turck D, van Goudoever J: Supplementation of infant formula with probiotics and/or prebiotics: a systematic review and comment by the ESPGHAN committee on nutrition. J Pediatr Gastroenterol Nutr 52:238–250, 2011. Lin HC: Small intestinal bacterial overgrowth: a framework for understanding irritable bowel syndrome. JAMA 292:852–858, 2004. Ashley C, Johnston WH, Harris CL, Stolz SI, Wampler JL, Berseth CL: Growth and tolerance of infants fed formula supplemented with polydextrose (PDX) and/or galactooligosaccharides (GOS): doubleblind, randomized, controlled trial. Nutr J 11:38, 2012. McMahon E, Reguly T: Canada’s approach to functional foods. Health Law Can 32:29–34, 2011. Huijsdens XW, Linskens RK, Mak M, Meuwissen SG, Vandenbroucke-Grauls CM, Savelkoul PH: Quantification of bac-
23.
24.
25.
26. 27.
28. 29. 30.
31.
32.
33. 34.
35.
36. 37.
38.
39.
40.
teria adherent to gastrointestinal mucosa by real-time PCR. J Clin Microbiol 40:4423–4427, 2002. Haarman M, Knol J: Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula. Appl Environ Microbiol 71:2318–2324, 2005. Malinen E, Rinttila T, Kajander K, Matto J, Kassinen A, Krogius L, Saarela M, Korpela R, Palva A: Analysis of the fecal microbiota of irritable bowel syndrome patients and healthy controls with realtime PCR. Am J Gastroenterol 100:373–382, 2005. Willey SH, Bartlett JG: Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin. J Clin Microbiol 10:880–884, 1979. de Jong AE, Beumer RR, Rombouts FM: Optimizing sporulation of Clostridium perfringens. J Food Prot 65:1457–1462, 2002. Ouwehand AC, Salminen S, Arvola T, Ruuska T, Isolauri E: Microbiota composition of the intestinal mucosa: association with fecal microbiota? Microbiol Immunol 48:497–500, 2004. R Core Team: “R: A Language and Environment for Statistical Computing.” 2012. Accessed at: http://www.R-project.org/. Lifschitz C: Prevention of excessive crying by intestinal microbiota programming. J Pediatr 163:1250–1252, 2013. Lehtonen L, Korvenranta H, Eerola E: Intestinal microflora in colicky and noncolicky infants: bacterial cultures and gas–liquid chromatography. J Pediatr Gastroenterol Nutr 19:310–314, 1994. Savino F, Cresi F, Pautasso S, Palumeri E, Tullio V, Roana J, Silvestro L, et al.: Intestinal microflora in breastfed colicky and non-colicky infants. Acta Paediatr 93:825–829, 2004. Rhoads JM, Fatheree NY, Norori J, Liu Y, Lucke JF, Tyson JE, Ferris MJ: Altered fecal microflora and increased fecal calprotectin in infants with colic. J Pediatr 155:823–828, 2009. Chen J, Cai W, Feng Y: Development of intestinal bifidobacteria and lactobacilli in breast-fed neonates. Clin Nutr 26:559–566, 2007. Xia Q, Williams T, Hustead D, Price P, Morrison M, Yu Z: Quantitative analysis of intestinal bacterial populations from term infants fed formula supplemented with fructo-oligosaccharides. J Pediatr Gastroenterol Nutr 55:314–320, 2012. Rao S, Srinivasjois R, Patole S: Prebiotic supplementation in fullterm neonates: a systematic review of randomized controlled trials. Arch Pediatr Adolesc Med 163:755–764, 2009. Gibson GR, Wang X: Regulatory effects of bifidobacteria on the growth of other colonic bacteria. J Appl Bacteriol 77:412–420, 1994. Sherman PM, Cabana M, Gibson GR, Koletzko BV, Neu J, Veereman-Wauters G, Ziegler EE, Walker WA: Potential roles and clinical utility of prebiotics in newborns, infants, and children: proceedings from a global prebiotic summit meeting, New York City, June 27–28, 2008. J Pediatr 155:S61–S70, 2009. Costalos C, Kapiki A, Apostolou M, Papathoma E: The effect of a prebiotic supplemented formula on growth and stool microbiology of term infants. Early Hum Dev 84:45–49, 2008. Alliet P, Scholtens P, Raes M, Hensen K, Jongen H, Rummens JL, Boehm G, Vandenplas Y: Effect of prebiotic galactooligosaccharide, long-chain fructo-oligosaccharide infant formula on serum cholesterol and triacylglycerol levels. Nutrition 23:719– 723, 2007. Ben XM, Zhou XY, Zhao WH, Yu WL, Pan W, Zhang WL, Wu SM, Van Beusekom CM, Schaafsma A: Supplementation of milk
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Galacto-Oligosaccharide and Prebiotic Effect gas-producing coliforms isolated from colicky infants. BMC Microbiol 11:157, 2011. 44. Reuter G: The Lactobacillus and Bifidobacterium microflora of the human intestine: composition and succession. Curr Issues Intest Microbiol 2:43–53, 2001. 45. Fanaro S, Chierici R, Guerrini P, Vigi V: Intestinal microflora in early infancy: composition and development. Acta Paediatr 91:48– 55, 2003. 46. Mariat D, Firmesse O, Levenez F, Guimaraes V, Sokol H, Dore J, Corthier G, Furet JP: The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age. BMC Microbiol 9:123, 2009.
Received September 17, 2013; revision accepted December 18, 2013.
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formula with galacto-oligosaccharides improves intestinal microflora and fermentation in term infants. Chin Med J (Engl) 117:927– 931, 2004. 41. Savino F, Cresi F, Maccario S, Cavallo F, Dalmasso P, Fanaro S, Oggero R, Vigi V, Silvestro L: “Minor” feeding problems during the first months of life: effect of a partially hydrolysed milk formula containing fructo- and galacto-oligosaccharides. Acta Paediatr 91:86–90, 2003. 42. Savino F, Maccario S, Castagno E, Cresi F, Cavallo F, Dalmasso P, Fanaro S, Oggero R, Silvestro L: Advances in the management of digestive problems during the first months of life. Acta Paediatr 94:120–14, 2005. 43. Savino F, Cordisco L, Tarasco V, Locatelli E, Di Gioia D, Oggero R, Matteuzzi D: Antagonistic effect of Lactobacillus strains against
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Journal of the American College of Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uacn20
Prebiotic Effect of an Infant Formula Supplemented with Galacto-Oligosaccharides: Randomized Multicenter Trial a
a
a
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Marcello Giovannini MD , Elvira Verduci MD , Dario Gregori PhD , Simonetta Ballali BA , Sara c
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Soldi PhD , Diana Ghisleni MD , Enrica Riva MD & for the PLAGOS Trial Study Group a
Department of Pediatrics, San Paolo Hospital, University of Milan, Milan, ITALY
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Unit of Biostatics, Epidemiology and Public Health, Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Padova, Italy. c
AAT-Advanced Analytical Technologies S.r.l., Piacenza, Italy. Published online: 10 Oct 2014.
Click for updates To cite this article: Marcello Giovannini MD, Elvira Verduci MD, Dario Gregori PhD, Simonetta Ballali BA, Sara Soldi PhD, Diana Ghisleni MD, Enrica Riva MD & for the PLAGOS Trial Study Group (2014) Prebiotic Effect of an Infant Formula Supplemented with Galacto-Oligosaccharides: Randomized Multicenter Trial, Journal of the American College of Nutrition, 33:5, 385-393, DOI: 10.1080/07315724.2013.878232 To link to this article: http://dx.doi.org/10.1080/07315724.2013.878232
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Original Research
Prebiotic Effect of an Infant Formula Supplemented with Galacto-Oligosaccharides: Randomized Multicenter Trial Marcello Giovannini, MD, Elvira Verduci, MD, Dario Gregori, PhD, Simonetta Ballali, BA, Sara Soldi, PhD, Diana Ghisleni, MD, Enrica Riva, MD, for the PLAGOS Trial Study Group Department of Pediatrics, San Paolo Hospital, University of Milan, Milan, ITALY (M.G., E.V., D.G., E.R.), and Unit of Biostatistics, Epidemiology and Public Health, Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Padova, ITALY (D.G., S.B.), AAT-Advanced Analytical Technologies S.r.l, Piacenza, ITALY (S.S.)
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Keywords: galacto-oligosaccharides, infant formula, prebiotic, Clostridium, bifidogenic activity Objective: The objective of the study was to investigate the effects of a galacto-oligosaccharides (GOS)supplemented formula on the intestinal microbiota in healthy term infants, with a specific consideration for gastrointestinal symptoms as colic, stool frequency and consistency, regurgitation. Methods: This was a randomized, double-blind, controlled, parallel-group clinical trial performed simultaneously by 6 centers in Italy. Three groups were considered: breastfed, formula-fed, and GOS-supplemented formula-fed infants. Formula-fed infants were randomized to receive either the control or the study formula and consume the assigned formula exclusively until the introduction of complementary feeding. The nutritional composition of the 2 formulas were identical, apart from the supplemented GOS (0.4 g/100 mL) in the study formula. Four different types of bacteria were evaluated in order to assess the efficacy of GOS-supplemented formula on infants: Bifidobacterium, Lactobacillus, and Clostridium, Escherichia coli. Results: A total of 199 breastfed infants and 163 formula-fed infants were recruited. When considering stool frequency and consistency, GOS-supplemented formula presented normal and soft stools in the majority of episodes (89%). In the supplemented group the incidence of colic was lower with respect to the control group. A significantly lower count of Clostridium and a higher count of Bifidobacterium were found when comparing study formula and control formula in infants with colic. In children with colic the ratio between Clostridium count and Bifidobacterium and Lactobacillus count was in favor of the latter two when considering the GOS-supplemented formula group with respect to the control one. Conclusions: The prebiotic-supplemented formula mimicked the effect of human milk in promoting Bifidobacterium and Lactobacillus growth and in inhibiting Clostridium growth, resulting in a significantly lower presence of colic.
INTRODUCTION
formulas have been developed with the specific purpose of mimicking human milk effects on both growth and gastrointestinal (GI) bacteria colonization [4]. The gastrointestinal microbiota of infants are highly influenced by the type of feeding they receive [5]; the stools of breastfed infants are dominated by Bifidobacterium and, to a lesser degree, by Lactobacillus [6]. The colonizing Bifidobacterium and Lactobacillus may inhibit the growth of pathogenic microorganisms through the production of lactic, acetic, and other organic acids, with a
Exclusive breastfeeding is widely recognized as the gold standard for infant nutrition in the first 6 months of life [1,2]. If, for whatever reason, human milk becomes unavailable to an infant, milk formulas are the only alternatives to breast milk. Their aim is to provide the best alternative to human milk for infants who cannot be breastfed and to approach the structural and functional effects observed in breastfed infants [3]; therefore, milk
Address correspondence to: Marcello Giovannini, MD, Professor of Medicine, Chief of the Department of Pediatrics, San Paolo Hospital, University of Milan, Via Antonio di Rudin´ı n. 8, 20142 Milan, ITALY. E-mail: [email protected] PLAGOS Trial Study Group: Giuseppe Banderali, Diana Ghisleni (Department of Pediatrics, San Paolo Hospital, University of Milan, Milan, ITALY); GianVincenzo Zuccotti, Laura Pogliani (Department of Paediatrics, Luigi Sacco Hospital, Universit`a di Milano, Milan, ITALY); Giacomo Biasucci, Cristina Cicero (Department of Paediatrics and Neonatology, Guglielmo da Saliceto Hospital, Piacenza, ITALY); Alberto Podest`a, Monica Tonella (Department of Pediatrics and Neonatology, San Carlo Borromeo Hospital, Milan, ITALY); Amilcare Rottoli, Tiziana Bonabitacola (Unit of Pediatrics and Neonatology, Presidio Ospedaliere di Cernusco sul Naviglio and Presidio Ospedaliero di Melzo, Cernusco sul Naviglio and Melzo, ITALY)
Journal of the American College of Nutrition, Vol. 33, No. 5, 385–393 (2014) C American College of Nutrition Published by Taylor & Francis Group, LLC 385
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Galacto-Oligosaccharide and Prebiotic Effect consequent decrease in intraluminal pH that inhibits the growth of some bacterial pathogens. Moreover, Bifidobacterium and Lactobacillus compete with potentially pathogenic bacteria for nutrients and epithelial adhesion sides [7]. The effects of breastfeeding on the intestinal microbiota cannot be attributed to a single compound, but there is accumulating evidence that human milk oligosaccharides play a crucial role [8]. Therefore, the addition of prebiotic oligosaccharides to infant formulas has grown in diffusion in the latest years, mainly focusing on supplementation of a mixture of fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) [9]. Several studies have been published, specifically investigating the effect of prebiotics on microbiota growth in the infant’s gastrointestinal tract. Results significantly vary. In their study, Brunser et al. evaluated whether supplementation of milk formulas with prebiotic FOS or a probiotic could modulate the composition of the fecal microbiota of formula-fed infants compared to breastfed infants [10]. Their results confirmed the presence of higher counts of Bifidobacterium and Lactobacillus in the microbiota of breastfed infants compared to formula-fed infants before dietary diversification. Similar results were found by Harmsen et al. [11], Guaraldi and Salvatori [12], and Bezirtzoglou et al. [13]. On the other hand, Knol and colleagues found that the addition of the prebiotic GOS/FOS mixture to an infant formula had a stimulating effect on the growth of Bifidobacterium and on the metabolic activity of the total intestinal flora of their considered sample [14]. Bakker-Zierikzee et al. also compared the effects of infant formula containing a mixture of galactoand fructo-oligosaccharides or viable Bifidobacterium animalis on the composition and metabolic activity of the intestinal microflora, concluding that feeding infants GOS/FOS formula resulted in a similar effect on metabolic activity of the flora as in breastfed infants [15]. Their conclusions were subsequently backed by Miniello et al.’s analysis of the composition of the intestinal bacterial populations; it was concluded that the prebiotic induced a significantly higher colonization of Bifidobacterium and Lactobacillus [16]. Similar conclusions were drawn by Fanaro and Vigi [17]. Their research highlighted that a mixture of oligosaccharides showed its efficacy in stimulating the establishment of a “bifidus flora,” with stools closer to those found in breastfed infants. Despite this, the Committee on Nutrition of the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition recently concluded that there is still a lack of data on the long-term effects of the administration of formulas supplemented with prebiotics [18]. When considering supplemented infant formulas, adverse events are specifically considered. From a microbiological point of view, colicky infants are usually associated with lower Lactobacillus and Bifidobacterium colonies and increased anaerobic Clostridium species in place of anaerobic bacteroides species and Bifidobacterium species [19]. Our study evaluated the effect of a GOS-supplemented infant milk formula (0.4 g/100 mL)
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on beneficial bacteria, such as Bifidobacterium and Lactobacillus, as well as on other bacteria, in particular, Clostridium and Escherichia coli. Results were also evaluated in order to assess the functional effect on digestive issues, such as colic or regurgitation. This article is the first to present results from a sample receiving an exclusive GOS-supplemented formula. Previous studies have considered the safety of FOS-GOS formulas [15] or GOS formulas [20], showing specific effects on beneficial bacteria. The present results furthermore investigated the specific role of supplemented formula in decreasing total pathogen count and colonization.
MATERIALS AND METHODS Study Design This was a randomized, double-blind, controlled, parallelgroup clinical trial performed simultaneously by 6 centers in Italy from February 2006 to October 2011. The study was conducted in compliance with the Declaration of Helsinki (revised Tokyo 2004 version) and Good Clinical Practices (CPMP/ICH/135/95) and in accordance with national regulatory requirements and laws. All relevant study documents were approved by the Ethics Committees of each participating center. All patients provided signed informed consent and could withdraw from the study at any time. This trial is registered with ClinicalTrials.gov, number NCT00486148.
Subjects In this study, 362 infants who met the following inclusion criteria were enrolled by the 15th day of life: Caucasian origin; healthy infants of both sexes; natural or Caesarian delivery; single birth; gestational age from 37 to 42 completed weeks; birth weight ≥ 2500 g; 5-min APGAR score > 7; exclusive breastfeeding or formula feeding at the moment of enrollment; and parents’ full understanding of the protocol. Infants were excluded if they met even only one of the following criteria: bearing congenital malformation; being diagnosed with hereditary diseases, chronic diseases, or neonatal illnesses requiring more than 7 days of hospitalization; presence of prenatal pathologies undermining intrauterine growth; infant’s mother affected by dismetabolic or chronic diseases; unknown father; uncertainty of parents’ compliance toward protocol and regular visits; or infant recruited or selected for other clinical studies. Three groups were considered according to the type of feeding: breast milk, GOS-supplemented infant milk formula (study formula), and standard infant milk formula (control formula). The breast milk group was considered as the reference group; therefore, no randomization was performed. A total of 199 breastfed infants and 163 formula-fed infants (80 in the control and 83 in the study group) were recruited.
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Galacto-Oligosaccharide and Prebiotic Effect Diets
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Formula-fed infants were randomized to receive either the control or the study formula and were to consume the assigned formula exclusively until the introduction of complementary feeding. The compositions of the 2 formulas were identical, with the exception of the supplemented oligosaccharides. The study formula was supplemented with 0.4 g/100 mL of GOS (Vivinal GOS Galactooligosaccharide; Friesland Foods Domo, Amersfoort, The Netherlands). Study and control formulas were supplied by Heinz Italia S.p.A. (Latina, Italy). The 2 formulas were provided in powdered form and could not be differentiated by smell, consistency, or any other characteristics. Neither the investigators nor the parents knew which product the infant was receiving.
Visits Anthropometric parameters and any adverse events (AEs) were recorded at 6 different time points: baseline visit (B) within 15 days after birth, visit 1 (V1; 30 ± 10 days of life), visit 2 (V2; 60 ± 10 days of life), visit 3 (V3; 90 ± 10 days of life), visit 4 (V4; 120 ± 10 days of life), and final visit (Vf), performed just before the introduction of complementary feeding. A fecal sample was collected at B, V1, V2, and Vf. In the presence of adverse effects, an extra visit was planned and a fecal sample was collected. At B, parents were given a diary to record daily stool frequency and consistency and adverse events. Infants’ growth was assessed through anthropometric measurements of weight (g), length [21], and occipito-frontal head circumference [21] and z-scores were calculated.
Adverse Effects In order to evaluate the safety of the study formula anthropometric data, incidence of AEs and infants’ acceptance of the formulas were considered. AEs were defined as undesired, unplanned clinical events, in the form of signs and symptoms or disease occurring in an infant participating in a clinical study, regardless of causal relationship, like diarrhea, infantile colic, prolonged crying, regurgitation, vomiting, cutaneous manifestations, and fever.
Bacterial Analysis Bacterial analysis was performed at the Advanced Analytical Technologies Center in Piacenza by the assigned researchers. Four different types of bacteria were evaluated in order to assess the efficacy of GOS-supplemented formula on infants: Bifidobacterium, Lactobacillus, Clostridium, and Escherichia coli.
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Samples collected at B, V1, and V2 and derived from a subpopulation of 75 naturally delivered infants who had not taken antibiotics or probiotics during the study period were analyzed. Analyses included (1) quantification of Lactobacillus, Bifidobacterium, and coliforms through real-time polymerase chain reaction (PCR) and (2) spore counts by means of inclusion in selective medium for Clostridium. For biomolecular quantification of the indicated genera, total bacterial DNA was extracted from fecal samples by means of mechanical cell disruption using a Fast Prep Instrument (MP Biomedical, Santa Ana, CA, USA) and the related FastDNA Spin Kit. DNA concentrations were determined using a NanoDrop (Thermo Scientific, Rodano, Milan, Italy). All fecal DNA samples were adjusted to equal concentrations for subsequent qPCR analysis. An amplification mixture was prepared using a RealMasterMix Sybr Rox 2.5X (Eppendorf AG, Hamburg, Germany) with primers that amplify the genes encoding 16S rRNA from the 3 specific bacterial groups, according to supplier’s instruction and following, as a reference, scientific papers appropriate for the purpose of the analysis. Different protocols and primer sequences were applied for quantification of the 3 bacterial groups Bifidobacterium, coliforms, and Lactobacillus [22–24] and optimized thermal cycles were followed. Each PCR was carried out in a final volume of 25 µl and each plate included duplicate reactions per DNA sample, a no-template negative control, and the appropriate set of standards. The latter were generated by the dilution of reference strain pure DNAs from Internationally Recognized Collections. All assays were conducted in 96-well plates on an Eppendorf Realplex2 mastercycler thermocycler (Eppendorf). To achieve Clostridium genus quantification, fecal samples were suspended 1/10 (w/v) in Ringer solution and an aliquot of this first suspension was then used for further decimal dilutions. For each fecal sample the appropriate dilutions were included in Reinforced Clostrium Medium (RCM) Agar (Oxoid Ltd., Altrincham, Cheshire, England) after thermal pasteurization at 75◦ C for 30 min. Plates were then incubated in anaerobic conditions for 48 h at 37◦ C. All colonies encompassing different morphologies were counted on these plates. Though other methods can be used for Clostridium count, RCM implemented with the thermization process is a recognized technique [25,26].
Sample Size Estimation A total of 160 formula-fed infants (80 receiving the control formula and 80 receiving the study formula) would give the power to detect a 24% reduction in adverse events for the GOSsupplemented formula compared to the control, at 80% power and a 5% significance level, assuming a 20% drop-out rate. Recruiting 200 breastfed infants, assuming a 20% drop-out rate and a breastfeeding rate at 6 months at about 50% would ensure a control group of 64 exclusively breastfed infants at 6 months.
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Galacto-Oligosaccharide and Prebiotic Effect Statistical Method
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Data have been presented using median (I and III quartile as measures of dispersion) for quantitative variables and as percentages (absolute numbers are presented in brackets) for categorical variables. Tests for the difference of main variables at each visit were performed using a test statistic based on the chi-square test and on Wilcoxon or Kruskal-Wallis test using the F distribution, whenever appropriate. Risk rates of adverse events were based on a Poisson log-linear model, treating child-days of exposure as an offset in the model. In case of multiple assessments per subject, p values were derived by the estimation of a robustified version of the above-mentioned statistics, using the Huber-White method [27]. The ratio between bacterial classes was performed on total bacterial count. All analyses were performed using R [28].
RESULTS A total of 199 breastfed infants and 163 formula-fed infants (80 in the control group and 83 in the study group) were recruited, with a drop-out rate of 39% and 26%, respectively. Voluntary interruption was the most common reason for dropout in all groups, 58% in the breastfed group and 86% in the formula-fed group. Table 1 shows the general characteristics of the sample, based on the type of delivery. Female and male infants were equally represented in both groups, and the majority of the infants (53%, 187 infants) were first-born children. Maternal choice was the main reason to opt for infant formula instead of breast milk (48% of overall cases), and agalactia was the second most frequent reason (26%). When considering the general data, a significant association (p < 0.001) was found between natural delivery and the choice to breastfeed. A separate evaluation of descriptive variables was performed, considering the 3 different groups divided by feeding choice.
Table 1. Demographics and Birth Characteristics
Gender (male) Parity: 1 2 3 Human milk Control formula Study formula Reason for not breastfeeding: Agalactia Breast refusal Hypogalactia Previous breast cancer Maternal choice
Caesarean Delivery (N = 86)
Natural Delivery (N = 276)
51 (44) 52 (45) 43 (37) 5 (4) 29 (25) 49 (30) 51 (31) 22 (12) 0 (0) 30 (16) 2 (1) 46 (25)
50 (137) 53 (142) 40 (108) 6 (17) 63 (174)1 49 (50) 51 (52) 29 (24) 1 (1) 18 (15) 2 (2) 49 (41)
All data, if not differently indicated, are reported as relative frequencies (%) with absolute values in brackets (N). 1 Significant association (p < 0.05) is indicated when found.
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All 3 groups showed appropriate physical growth throughout the study period (results on safety evaluation not shown in this article). Risk rates of adverse events were evaluated, considering in the model total episodes and treating child-days of exposure. When considering diarrhea episodes, human milk and the study formula showed comparable results. Stool frequency was also assessed. A significant difference was observed when comparing human milk and the study formula and when comparing the study formula and control formula, in both cases showing a lower occurrence in the study formula group. A significant difference in stool frequency was also observed between the human milk group and study formula group. When considering stool consistency, the study group presented normal and soft stools in the majority of episodes (89%). A significant difference was observed when considering watery consistency, showing a significant lower risk rate in the study formula group. Infantile colic showed a lower risk rate in the study formula group compared to both other groups (p < 0.05). Regurgitation risk rates were significantly different when comparing the study formula group and control formula group. The most frequent cutaneous manifestations in all groups were erythema and dry skin. All collected data referring to stool frequency and consistency, diarrhea episodes, and gastrointestinal effects are presented in Table 2. Table 2. Gastrointestinal Symptoms in the Follow-up Period Human Milk (17,548) Diarrhea episodes Frequency Stool frequency Consistency: Watery Semisolid Hard Normal Soft Infantile colic Regurgitation Cutaneous manifestations: No Erythema Blemishes Dry skin Localization: Arms Arms and legs Arms and trunk Arms and face Arms, face, and legs
Control Formula (8692)
Study Formula (7328)
2 (260) 3 (2–6) 9 (7–11) 26 (3431) 1 (191) 0 (24) 45 (5875) 28 (3677) 15 (2631) 37 (6381)
1 (112) 7 (7–7) 3 (3–7)1,2 4 (256)1 9 (645) 4 (277) 57 (3942) 26 (1841) 16 (1399) 33 (2868)1
2 (116) Not applicable 3 (3–7)3 4 (209)3 4 (197) 3 (186) 51 (2737) 38 (2005) 10 (751)2 3 24 (1714)2
89 (15,303) 5 (862) 1 (171) 5 (860) 0 (5) 2 (31) 0 (2) 0 (0) 0 (4)
91 (7914) 3 (242) 1 (45) 5 (473) 3 (19)1,2 0 (3) 0 (0) 0 (0) 0 (0)
93 (6748) 3 (249)3 1 (107) 2 (114)3 4 (22)3 1 (7) 0 (2) 1 (3) 0 (0)
All qualitative variables are reported with as risk rates and absolute number of events (in parentheses). Denominators (column headings) are expressed as childdays of exposure. Quantitative data are presented as median value, with 5th and 95th percentiles in brackets. 1 Significant difference (p < 0.05) when considering human milk vs control formula. 2 Significant difference (p < 0.05) when considering study formula vs control formula. 3 Significant difference (p < 0.05) when considering human milk vs study formula.
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Galacto-Oligosaccharide and Prebiotic Effect Table 3. Comparison between Different Formulas and Bacterial Colonization
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N Entire follow up Clostridium Bifidobacterium Escherichia coli Lactobacillus group Clostridium/ (Lactobacillus Baseline Clostridium Bifidobacterium Escherichia coli Lactobacillus group Clostridium/ (Lactobacillus V1 Clostridium Bifidobacterium Escherichia coli Lactobacillus group Clostridium/ (Lactobacillus V2 Clostridium Bifidobacterium Escherichia coli Lactobacillus group Clostridium/ (Lactobacillus
+ Bifidobacterium)
220 220 209 219 217
+ Bifidobacterium)
74 74 69 74 73 73 73 68 73
+ Bifidobacterium) 73 73 72 72 + Bifidobacterium)
Control Formula
Study Formula
(79) 3.9E04 (0.1E04–41E04) 4.1E09 (1.2E09–12E09) 0.3E06 (0.0–12.0E06) 5.1E05 (0.2E05–504.6E05) 9.0E-6 (3.4E-07–1.9E-04) (79) 0.8E04 (0.0–74E04) 6.0E09 (1.1E09–15.7E09) 0.01E06 (0.0–6.4E06) 5.1E05 (0.5E05–55.4E05) 1.3E-06 (0.0–5.2E-04) (80) 1.9E04 (0.5E04–28.2E04) 2.7E09 (0.5E09–7.2E09) 0.3E06 (0.0–9.0E06) 3.1E05 (0.2E05–527.7E05) 9.0E-06 (1.3E-06–1.1E-04) (80) 7.8E04 (1.8E04–3.1E05) 4.7E09 (2.0E09–12.53E09) 0.3E06 (0.0–39.1E06) 6.7E05 (1.0E05–576.1E05) 1.1E-05 (3.5E-06–9.8E-05)
(83) 0.9E04 (0–9.8E04) 4.8E09 (1.9E09–100E09) 0.6E06 (0–77.3E06) 6.4E05 (0.6E05–43.0E05) 1.4E-06 (1.7E-10–2.8E-05)1 (83) 0.65E04 (0.005E04–16.6E04) 7.4E09 (1.6E09–79–9E09) 0.5E06 (0.001E06–71.6E06) 5.2E05 (0.5E05–76E05) 1.4E-06(3.6E-09–1.1E-04) (83) 0.6E04 (0.0–8.3E04) 3.6E09 (1.9E09–55.9E09) 0.6E06 (0.04E06–37.7E06) 3.4E05 (0.3E05–87.6E05) 1.3E-06 (1.7E-10–2.7E-05) (83) 1.2E04 (0.0–3.7E04)1 5.3E09 (1.8E09–159.7E09) 0.8E06 (0.02E06–348.7E06) 11.6E05 (3.1E05–2.45E05) 9.9E-07 (7.0E-11–1.6E-05)1
V1 = visit 1, V2 = visit 2. All data are presented as median values, with 25th and 75th percentiles in brackets. The letter E indicates a power of 10. 1 Significant difference (p < 0.05) when considering study formula vs control formula.
Bacterial colonization was considered in terms of proliferation of Bifidobacterium, Lactobacillus, E. coli, and sporigen behavior of Clostridium. Total counts of the selected bacteria for visits B, V1, and V2 were performed. In addition, an analysis of the whole period (from B to V2) was performed. All data are presented in Table 3, referring to the control group and GOS formula. Bacterial colonization showed comparable figures at baseline and at V1, and at V2 Clostridium count showed a significant difference when comparing the study formula and control formula (p < 0.05). When considering the entire follow-up, no significant difference in bacterial count was observed. In order to better establish the proportion of Clostridium with respect to other bacteria, an additional evaluation was carried out as suggested by the authors of the present article, showing a significant difference over the entire follow-up and at V2 in the ratio between Clostridium and other bacteria (Clostridium/ Bifidobacterium + Lactobacillus), with p values of 0.02 and 0.021, respectively. Clostridium was specifically considered, due to the well-known association between infant colic and Clostridium colonization [29,30]. In colicky infants higher numbers of colonic bacteria-producing gas have been detected, concomitant with fewer bacteria that do not produce gas (e.g., Lactobacillus and Bifidobacterium) [31,32]. To compare the effect of different formula compositions with respect to the occurrence of infantile colic and bacterial colonization, a subgroup analysis was performed in the formula groups focusing on the period from baseline to the 70th day of life. Data
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showed that in infants with colic, the study formula resulted in a better bacterial profile, showing a significantly lower Clostridium count (p < 0.05) and stimulating both Bifidobacterium, and showed a significantly different bacterial count between the 2 groups (p < 0.05) and Lactobacillus growth. In the absence of colic, the 2 groups showed similar bacterial colonization when considering Bifidobacterium, E. coli, and Lactobacillus, whereas Clostridium count showed a significant difference between the 2 groups (p < 0.05). All data are presented in Table 4. When considering the breast milk group, a difference between human milk and control formula patterns was observed: the first had a significantly better microbiological pattern, as expected from the literature [1].
DISCUSSION The results of our study confirmed the initial hypothesis that the GOS-supplemented formula had an efficient performance on gastrointestinal microbiota, mimicking human milk promotion of Bifidobacterium growth and reduction of Clostridium after an initial period of one month. When comparing the study formula with the control formula, the GOS-supplemented formula showed a significant effect in lowering risk rates of colic and regurgitation, with a boosting effect on the beneficial bacterial flora and an inhibiting effect on Clostridium growth. Clostridium growth, inducing gas production, appears to be linked to infant
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Galacto-Oligosaccharide and Prebiotic Effect Table 4. Bacterial Colonization and Infantile Colic Occurrences in the Formula Randomized Groups from Baseline up to the 70th Day
Presence of colic Clostridium Bifidobacterium Escherichia coli Lactobacillus Absence of colic Clostridium Bifidobacterium Escherichia coli Lactobacillus
N
Control Formula
Study Formula
28 28 25 28
2.4E04 (0.4E04–47E04) 2.9E09 (1.6E09–10.8E09) 0.3E06 (0E06–13.4E06) 0.1E06 (0.03E06–37.8E06)
0E04 (0E04–0.4E4)1 1.2E11(8.3E10–1.7E11)1 0.2E06 (0.1E06–20.1E06) 0.10E06 (0.04E06–1.8E06)
81 81 79 80
6.1E04 (0.6E04–23.8E04) 4.1E09 (0.08E9–9.4E09) 0.3E06 (0E06–39.2E06) 0.6E06 (0.02E06–105E06)
0.1E04 (0E04–4.2E04) 1 6.3E09 (1.6E09–171E09) 0.3E06 (0E06–124.8E06) 0.6E06 (0.1E06–3.1E06)
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All qualitative variables are reported with absolute number of studied infants in brackets, (N) referring to the bacterial count from baseline to visit 2. Continuous data are presented as median values, with 25th and 75th percentiles in brackets. The letter E indicates a power of 10. 1 Significant difference (p < 0.05) when considering study formula vs control formula.
colic, from a physiological perspective. This consideration was taken into account in order to better establish the impact of the new formula on the growth of non-gas-producing bacteria and gas-producing bacteria, in order to better assess the efficacy of the new formula on gastrointestinal symptoms. The predominance of beneficial bacteria in the gut microbiota of breastfed infants is thought to result from the fermentation of oligosaccharides; that is, nondigestible carbohydrates consisting of 3 to 10 linked monosaccharides. Human milk contains more than 130 different oligosaccharides at a concentration of 15– 23 g/L in colostrum and 8–12 g/L in transitional and mature milk [7]. The GOS-supplemented formula used in the present study contained 0.4 g of galactooligosaccharides per 100 mL of reconstituted product. Numerous studies have reported that, since the very beginning of life gut microbiota play an important role in immune response and the growth [33]. In the present study, after an initial phase, the GOS-supplemented formula group showed the same trends of Bifidobacterium and Lactobacillus bacterial development as the breastfed group. This result is in line with Xia et al.’s study, which showed that the milk-based formula used in this study supported Bifidobacterium and Lactobacillus populations comparable to those in human milk [34]. A systematic review of trials evaluating the effect infant milk formulas supplemented with prebiotics (GOS-FOS, long-chain FOS [lcFOS], polydextrose-GOS-lactulose [PDX-GOS-LOS], or GOS) in full-term neonates has shown that doses ranging from 0.15 to 0.8 g/dL appear to achieve a maximum bifidogenic effect with minimal intolerance in the form of flatulence, abdominal distention, colic, etc. [35]. The stools of breastfed infants are dominated by Bifidobacterium and, to a lesser degree, by Lactobacillus [6]; thus, the goal of a cow milk–based formula should be to mimic the flora established due to human milk, reducing the GI colonization from potentially detrimental bacteria such as Clostridium [36]. Knol and colleagues compared a standard formula with a formula containing both short-chain GOS and inulin in healthy term
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newborns exclusively formula-fed [14,37] and they found that the prebiotic formula has a stimulating effect on the growth of and on the metabolic activity of the total intestinal flora. In addition, the proportion of E. coli and Clostridium species was lower in the prebiotic group. The bifidogenic effect of the supplementation of an infant formula with GOS was evaluated by Fanaro and colleagues, who concluded that it contributes to a favorable intestinal microbiota; formula’s effect on pathogens was not considered in the study [6]. Similar results were found by Costalos and colleagues, whose study presented a trend toward a reduction in pathogenic bacteria in the prebiotic-supplemented group (0.4 g/100 ml GOS and lcFOS, ratio 90:10) [38]. In this prospective double-blind study, Costalos and colleagues concluded that an infant formula containing prebiotic oligosaccharides was well tolerated and suppressed the numbers of Clostridium in the feces with a trend for a higher percentage of stool Bifidobacterium and lower percentage of E. coli. A reduction in pathogens was found also in the study performed by Alliet et al. with a GOSFOS mixture formula (0.6 g/100 mL of GOS/lcFOS, in a ratio of 9:1) [39]. To our knowledge, this is the first study that analyzed the efficacy of an infant formula supplemented with 0.4 g/100 mL of only GOS and showed similar effect in Bifidobacterium and Lactobacillus group development [40], as well as exerted an inhibiting function on the growth of sporulating Clostridium and a reduction in colic and regurgitations. Savino et al.’s observational study investigated the effect of FOS/GOS-supplemented formula on colic, regurgitation, and constipation, concluding that within 2 weeks of feeding with that new formula minor gastrointestinal symptoms improved [41]. In a subsequent study in 2005, Savino and colleagues confirmed previous findings, observing that a prebiotic mixture of FOS/GOS with a high beta-palmitic acid content reduced digestive problems and improved intestinal tolerance in infants during the first months of life [42]. In order to assess the performance of the study formula with respect to the control formula, in the present study bacterial counts were analyzed in children both with or without
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Galacto-Oligosaccharide and Prebiotic Effect presence of colic, from baseline to the 70th day of life. The bacterial profile was more favorable in infants who received GOS-supplemented formula, with respect to the control group. Infants fed the GOS-supplemented formula showed a significantly lower presence of Clostridium, when considering children with colic and without colic, and a significantly higher number of Bifidobacterium in children with colic. Therefore, in addition to the bifidogenic effect, the GOS-supplemented formula used in this study contributed to a decreased count of potentially pathogenic enteric bacteria and softer stools, without adversely affecting weight gain, as recently published [20]. In the present study, a reduction in colic and regurgitation frequency was recorded, even if not statistically associated with Clostridium and E. coli reduction. The association between microbiota patterns and infant colic has recently been investigated, highlighting specific conditions such as gastrointestinal colonization from anaerobic bacteria [31]. In particular, it is highlighted that infants with colic are most often colonized by gram-negative anaerobic bacteria and less frequently by Bifidobacterium and Lactobacillus, with differences in the patterns of the same intestinal Lactobacillus, L. lactis and L. brevis, producers of ethyl alcohol and carbon dioxide, while L. acidophilus has been shown only in healthy infants [42]. This pattern could be influencing gastrointestinal regulation, leading to an increase in gas production, with the consequent onset of the typical symptoms of the disorder [43]. Further studies need to be performed in order to assess the functional effect of GOS-supplemented formula. In this study, Bifidobacterium count was at higher median levels than Lactobacillus in the fecal samples over the whole study period in all 3 groups. This is in accordance with other studies [33] showing that Bifidobacterium dominate over Lactobacillus in this age group [27]. Several studies have reported a geographical behavior in Lactobacillus development, stressing how they appear to be lower in Western countries [44]. In this study, Lactobacillus in all fecal samples could be detected and quantified by real-time PCR and from B to V2 counts, and the quantification performed on the entire follow-up showed the same median values in feces from breastfed infants and infants fed the GOS-supplemented formula. Breastfed infants presented a GI flora dominated by Bifidobacterium and Lactobacillus, not only in the first days of life but also in the following weeks. Our study showed that GOSsupplemented formula performed in the same way, promoting an appropriate level of both bacteria in the gastrointestinal tract of the studied infants. When considering the quantification of potentially noxious bacteria as, for instance, many components of the wide Clostridium genus, their presence was lower in the study group with respect to those fed the control formula. In order to evaluate the progression of flora development, which at these early stages is considerably complex [45], the ratio between Clostridium count and Bifidobacterium and Lactobacillus was considered. A significantly lower ratio, synonymous with a higher presence of the latter desirable bacteria, was
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found when considering both the entire follow-up and the retrieval after 2 months of life (V2), confirming the efficacy of the GOS-supplemented formula with respect to the control formula in favoring the growth of positive flora, as commonly found in breastfed children [45,46].
CONCLUSION Thus, the initial hypothesis of the bifidogenic activity of the GOS-supplemented formula can be confirmed. Moreover, we have demonstrated an inhibitory effect on the development of gastrointestinal Clostridium in infants born at term who were fed the GOS-supplemented formula. The association between microbial gut composition and functional effects and improvement exerted by the administration of enriched formulas needs to be investigated in depth by following studies on subjects receiving infant formula supplemented exclusively with GOS.
FUNDING This project has been funded by Heinz Italia SpA (Latina, Italy).
REFERENCES 1. Section on Breastfeeding. Breastfeeding and the use of human milk. Pediatrics 129:e827–e841, 2012. 2. World Health Organization: “The Optimal Duration of Exclusive Breastfeeding. Report of an Expert Consultation.” Geneva: World Health Organization, March 28–30, 2001. 3. D’Auria E, Agostoni C, Giovannini M, Riva E, Zetterstrom R, Fortin R, Greppi GF, Bonizzi L, Roncada P: Proteomic evaluation of milk from different mammalian species as a substitute for breast milk. Acta Paediatr 94:1708–1713, 2005. 4. Koletzko B, Baker S, Cleghorn G, Neto UF, Gopalan S, Hernell O, Hock QS, Jirapinyo P, Lonnerdal B, Pencharz P, Pzyrembel H, Ramirez-Mayans J, Shamir R, Turck D, Yamashiro Y, Zong-Yi D: Global standard for the composition of infant formula: recommendations of an ESPGHAN coordinated international expert group. J Pediatr Gastroenterol Nutr 41:584–599, 2005. 5. Noverr MC, Huffnagle GB: The “microflora hypothesis” of allergic diseases. Clin Exp Allergy 35:1511–1520, 2005. 6. Fanaro S, Marten B, Bagna R, Vigi V, Fabris C, Pena-Quintana L, Arguelles F, Scholz-Ahrens KE, Sawatzki G, Zelenka R, Schrezenmeir J, de Vrese M, Bertino E: Galacto-oligosaccharides are bifidogenic and safe at weaning: a double-blind randomized multicenter study. J Pediatr Gastroenterol Nutr 48:82–88, 2009. 7. Agostoni C, Axelsson I, Goulet O, Koletzko B, Michaelsen KF, Puntis JW, Rigo J, Shamir R, Szajewska H, Turck D: Prebiotic oligosaccharides in dietetic products for infants: a commentary by
391
Galacto-Oligosaccharide and Prebiotic Effect
8. 9.
10.
Downloaded by [University of Louisville] at 12:44 01 January 2015
11.
12.
13.
14.
15.
16. 17.
18.
19.
20.
21. 22.
392
the ESPGHAN Committee on Nutrition. J Pediatr Gastroenterol Nutr 39:465–473, 2004. Boehm G, Moro G: Structural and functional aspects of prebiotics used in infant nutrition. J Nutr 138:1818S–1828S, 2008. Knol J, Boehm G, Lidestri M, Negretti F, Jelinek J, Agosti M, Stahl B, Marini A, Mosca F: Increase of faecal bifidobacteria due to dietary oligosaccharides induces a reduction of clinically relevant pathogen germs in the faeces of formula-fed preterm infants. Acta Paediatr 94:31–33, 2005. Brunser O, Figueroa G, Gotteland M, Haschke-Becher E, Magliola C, Rochat F, Cruchet S, Palframan R, Gibson G, Chauffard F, Haschke F: Effects of probiotic or prebiotic supplemented milk formulas on fecal microbiota composition of infants. Asia Pac J Clin Nutr 15:368–376, 2006. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp AA, Klijn N, Bindels JG, Welling GW: Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 30:61–67, 2000. Guaraldi F, Salvatori G: Effect of breast and formula feeding on gut microbiota shaping in newborns. Front Cell Infect Microbiol 2:94, 2012. Bezirtzoglou E, Tsiotsias A, Welling GW: Microbiota profile in feces of breast- and formula-fed newborns by using fluorescence in situ hybridization (FISH). Anaerobe 17:478–482, 2011. Knol J, Scholtens P, Kafka C, Steenbakkers J, Gro S, Helm K, Klarczyk M, Sch¨opfer H, B¨ockler HM, Wells J: Colon microflora in infants fed formula with galacto- and fructo-oligosaccharides: more like breast-fed infants. J Pediatr Gastroenterol Nutr 40:36–42, 2005. Bakker-Zierikzee AM, Alles MS, Knol J, Kok FJ, Tolboom JJ, Bindels JG: Effects of infant formula containing a mixture of galacto- and fructo-oligosaccharides or viable Bifidobacterium animalis on the intestinal microflora during the first 4 months of life. Br J Nutr 94:783–790, 2005. Miniello VL, Moro GE, Armenio L: Prebiotics in infant milk formulas: new perspectives. Acta Paediatr 91:68–76, 2003. Fanaro S, Vigi V: Infant formulas supplemented with prebiotics: intestinal microbiota and immune responses. Minerva Pediatr 60:327– 335, 2008. Braegger C, Chmielewska A, Decsi T, Kolacek S, Mihatsch W, Moreno L, Piescik M, Puntis J, Shamir R, Szajewska H, Turck D, van Goudoever J: Supplementation of infant formula with probiotics and/or prebiotics: a systematic review and comment by the ESPGHAN committee on nutrition. J Pediatr Gastroenterol Nutr 52:238–250, 2011. Lin HC: Small intestinal bacterial overgrowth: a framework for understanding irritable bowel syndrome. JAMA 292:852–858, 2004. Ashley C, Johnston WH, Harris CL, Stolz SI, Wampler JL, Berseth CL: Growth and tolerance of infants fed formula supplemented with polydextrose (PDX) and/or galactooligosaccharides (GOS): doubleblind, randomized, controlled trial. Nutr J 11:38, 2012. McMahon E, Reguly T: Canada’s approach to functional foods. Health Law Can 32:29–34, 2011. Huijsdens XW, Linskens RK, Mak M, Meuwissen SG, Vandenbroucke-Grauls CM, Savelkoul PH: Quantification of bac-
23.
24.
25.
26. 27.
28. 29. 30.
31.
32.
33. 34.
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36. 37.
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39.
40.
teria adherent to gastrointestinal mucosa by real-time PCR. J Clin Microbiol 40:4423–4427, 2002. Haarman M, Knol J: Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula. Appl Environ Microbiol 71:2318–2324, 2005. Malinen E, Rinttila T, Kajander K, Matto J, Kassinen A, Krogius L, Saarela M, Korpela R, Palva A: Analysis of the fecal microbiota of irritable bowel syndrome patients and healthy controls with realtime PCR. Am J Gastroenterol 100:373–382, 2005. Willey SH, Bartlett JG: Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin. J Clin Microbiol 10:880–884, 1979. de Jong AE, Beumer RR, Rombouts FM: Optimizing sporulation of Clostridium perfringens. J Food Prot 65:1457–1462, 2002. Ouwehand AC, Salminen S, Arvola T, Ruuska T, Isolauri E: Microbiota composition of the intestinal mucosa: association with fecal microbiota? Microbiol Immunol 48:497–500, 2004. R Core Team: “R: A Language and Environment for Statistical Computing.” 2012. Accessed at: http://www.R-project.org/. Lifschitz C: Prevention of excessive crying by intestinal microbiota programming. J Pediatr 163:1250–1252, 2013. Lehtonen L, Korvenranta H, Eerola E: Intestinal microflora in colicky and noncolicky infants: bacterial cultures and gas–liquid chromatography. J Pediatr Gastroenterol Nutr 19:310–314, 1994. Savino F, Cresi F, Pautasso S, Palumeri E, Tullio V, Roana J, Silvestro L, et al.: Intestinal microflora in breastfed colicky and non-colicky infants. Acta Paediatr 93:825–829, 2004. Rhoads JM, Fatheree NY, Norori J, Liu Y, Lucke JF, Tyson JE, Ferris MJ: Altered fecal microflora and increased fecal calprotectin in infants with colic. J Pediatr 155:823–828, 2009. Chen J, Cai W, Feng Y: Development of intestinal bifidobacteria and lactobacilli in breast-fed neonates. Clin Nutr 26:559–566, 2007. Xia Q, Williams T, Hustead D, Price P, Morrison M, Yu Z: Quantitative analysis of intestinal bacterial populations from term infants fed formula supplemented with fructo-oligosaccharides. J Pediatr Gastroenterol Nutr 55:314–320, 2012. Rao S, Srinivasjois R, Patole S: Prebiotic supplementation in fullterm neonates: a systematic review of randomized controlled trials. Arch Pediatr Adolesc Med 163:755–764, 2009. Gibson GR, Wang X: Regulatory effects of bifidobacteria on the growth of other colonic bacteria. J Appl Bacteriol 77:412–420, 1994. Sherman PM, Cabana M, Gibson GR, Koletzko BV, Neu J, Veereman-Wauters G, Ziegler EE, Walker WA: Potential roles and clinical utility of prebiotics in newborns, infants, and children: proceedings from a global prebiotic summit meeting, New York City, June 27–28, 2008. J Pediatr 155:S61–S70, 2009. Costalos C, Kapiki A, Apostolou M, Papathoma E: The effect of a prebiotic supplemented formula on growth and stool microbiology of term infants. Early Hum Dev 84:45–49, 2008. Alliet P, Scholtens P, Raes M, Hensen K, Jongen H, Rummens JL, Boehm G, Vandenplas Y: Effect of prebiotic galactooligosaccharide, long-chain fructo-oligosaccharide infant formula on serum cholesterol and triacylglycerol levels. Nutrition 23:719– 723, 2007. Ben XM, Zhou XY, Zhao WH, Yu WL, Pan W, Zhang WL, Wu SM, Van Beusekom CM, Schaafsma A: Supplementation of milk
VOL. 33, NO. 5
Galacto-Oligosaccharide and Prebiotic Effect gas-producing coliforms isolated from colicky infants. BMC Microbiol 11:157, 2011. 44. Reuter G: The Lactobacillus and Bifidobacterium microflora of the human intestine: composition and succession. Curr Issues Intest Microbiol 2:43–53, 2001. 45. Fanaro S, Chierici R, Guerrini P, Vigi V: Intestinal microflora in early infancy: composition and development. Acta Paediatr 91:48– 55, 2003. 46. Mariat D, Firmesse O, Levenez F, Guimaraes V, Sokol H, Dore J, Corthier G, Furet JP: The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age. BMC Microbiol 9:123, 2009.
Received September 17, 2013; revision accepted December 18, 2013.
Downloaded by [University of Louisville] at 12:44 01 January 2015
formula with galacto-oligosaccharides improves intestinal microflora and fermentation in term infants. Chin Med J (Engl) 117:927– 931, 2004. 41. Savino F, Cresi F, Maccario S, Cavallo F, Dalmasso P, Fanaro S, Oggero R, Vigi V, Silvestro L: “Minor” feeding problems during the first months of life: effect of a partially hydrolysed milk formula containing fructo- and galacto-oligosaccharides. Acta Paediatr 91:86–90, 2003. 42. Savino F, Maccario S, Castagno E, Cresi F, Cavallo F, Dalmasso P, Fanaro S, Oggero R, Silvestro L: Advances in the management of digestive problems during the first months of life. Acta Paediatr 94:120–14, 2005. 43. Savino F, Cordisco L, Tarasco V, Locatelli E, Di Gioia D, Oggero R, Matteuzzi D: Antagonistic effect of Lactobacillus strains against
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