Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Hepatology and Nutrition
New Prebiotic Blend of Polydextrose and Galacto-oligosaccharides Has a Bifidogenic Effect in Young Infants
Scalabrin, Deolinda M.F.*; Mitmesser, Susan H.*; Welling, Gjalt W.†; Harris, Cheryl L.*; Marunycz, John D.*; Walker, D. Carey‡; Bos, Nico A.§; Tölkkö, Satu||; Salminen, Seppo||; Vanderhoof, Jon A.*
*Department of Medical Affairs, Mead Johnson Nutrition, Evansville, IN
†Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
‡Department of Global Scientific Affairs, Mead Johnson Nutrition, Evansville, IN
§Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
||Functional Foods Forum, University of Turku, Turku, Finland.
Address correspondence and reprint requests to Susan H. Mitmesser, PhD, Department of Medical Affairs, Mead Johnson Nutrition, 2400 W Lloyd Expressway, Evansville, IN 47721 (e-mail: firstname.lastname@example.org).
Received 16 June, 2010
Accepted 6 September, 2011
The present study was funded by Mead Johnson Nutrition.
D.M.S., S.H.M., C.L.H., J.D.M., J.A.V., and D.C.W. are employees of Mead Johnson Nutrition. The other authors report no conflicts of interest.
www.clinicaltrials.gov registration number: NCT00503789.
Objective: The aim of the study was to evaluate the effect of infant formula with polydextrose (PDX) and galacto-oligosaccharides (GOS) on fecal microbiota and secretory IgA (sIgA).
Materials and Methods: In the present double-blind, randomized study, term infants received control (Enfamil Lipil) or the same formula with PDX/GOS (4 g/L, 1:1 ratio; PDX/GOS) for 60 days; a reference breast-fed group was included. Formula intake, tolerance, and stool characteristics were collected via electronic diary and analyzed by repeated measures analysis of variance. Anthropometric measurements and stool samples were obtained at baseline and after 30 and 60 days of feeding. Fecal sIgA was measured by enzyme-linked immunosorbent assay and fecal bacteria by fluorescent in situ hybridization and quantitative real-time polymerase chain reaction (qPCR); both were analyzed by Wilcoxon rank sum test.
Results: Two hundred thirty infants completed the study. Infants consuming PDX/GOS had softer stools than control at all times (P < 0.001). Using qPCR, counts in PDX/GOS were closer to the breast-fed group, tended to be higher than control for total bifidobacteria (P = 0.069) and Bifidobacterium longum (P = 0.057) at 30 days, and were significantly higher for total bifidobacteria and B longum at 60 days and B infantis at 30 days (P = 0.002). No significant differences were detected between PDX/GOS and control in changes from baseline to 30 or 60 days for sIgA or total bifidobacteria by fluorescent in situ hybridization or qPCR; however, significantly higher changes from baseline were detected between PDX/GOS and control for B infantis at 30 days and B longum at 60 days (P ≤ 0.035).
Conclusions: Infant formula with PDX/GOS produces soft stools and a bifidogenic effect closer to breast milk than formula without PDX/GOS.
The human large intestine harbors approximately 1014 CFU of >1000 different species of bacteria, some potentially pathogenic but the majority benign (1). Colonization of the human gut starts soon after birth and continues during the first year of life, depending on multiple factors such as mode of delivery, and dietary, environmental, and host factors (2–4). After the first inoculation, the gut microbiota changes rapidly and, in a few weeks, is dominated by bifidobacteria, according to most (2–5) but not all of the studies (6). The gut microbiota plays a critical role in stimulating maturation of the immune system in early infancy (7,8) and differs in breast-fed (BF) and formula-fed infants (2,9). The gut microbiota of BF infants has been associated with a lower number of intestinal pathogens and less infectious diarrhea (4,5,10). Human milk oligosaccharides (HMOs), the third largest component of human milk (5–10 g/L in mature milk) after lactose and fat (10), are one of the factors responsible for the specific gut microbiota composition of BF infants and have demonstrated bifidogenic activity by increasing the number of bifidobacteria in the gut (2,4). Recent genomic analyses of Bifidobacterium spp, such as B longum and B infantis, indicated the presence of particular genes related to use by HMOs (11).
Prebiotics are nondigestible or partially digestible carbohydrates similar to HMOs. They can selectively stimulate the growth and/or activity of beneficial bacteria in the colon (12), thus potentially promoting health benefits. Infant formulas supplemented with galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) have been shown to promote an increase in fecal bifidobacteria in term (13,14) and preterm infants (15,16). Bifidobacteria levels, stool consistency, and fecal pH were closer to those of BF infants (13,15,17). GOS and FOS have also been associated with higher levels of fecal secretory IgA (sIgA) (18,19), reduction in the incidence of atopic dermatitis (14,20), recurrent wheezing and urticaria (20), acute diarrhea (21), and respiratory and overall infections (20). Increased fecal sIgA has been associated with prebiotic exposure caused by either increased secretion (7,22,23) or lower numbers of Clostridium(16,19), which may degrade sIgA in the gut (24). Polydextrose (PDX) is a prebiotic that has been shown to stimulate bifidobacteria and lactobacilli and inhibit Bacteroides and Clostridium growth (25–27), and may stimulate sIgA (28).
In the present study, we evaluated a new prebiotic blend of GOS (a mixture of rapidly fermented oligosaccharides) and PDX (a mixture of complex, slowly fermented polysaccharides). In combination, these 2 prebiotics cover the molecular weight range of most HMOs. This blend was added to a cow's-milk–based infant formula in a 1:1 ratio at a level of 4 g/L, which was previously demonstrated to be well tolerated and associated with normal growth in infants, while promoting a stool consistency closer to that of BF infants than infants fed an unsupplemented formula (29). Based on the described effects of prebiotics on the gut and immune response, the effect of this PDX/GOS blend on stool microbiota and fecal sIgA was evaluated during a 60-day feeding period.
Healthy 21- to 30-day-old infants were recruited at 27 sites in the United States. Eligible infants were vaginally delivered at 37 to 42 weeks of gestational age with birth weight ≥2500 g and receiving only 1 form of nutrition (infant formula or human milk) for at least 7 consecutive days before study enrollment. Exclusion criteria included history of underlying disease or congenital malformation likely to interfere with normal growth; acute infection, gastroenteritis, feeding difficulties, or formula intolerance at study enrollment; consumption of supplemental foods or formula supplemented with probiotics; or use of oral or intravenous antibiotics or steroids by the infant or by the infant's mother within 15 days before delivery or while breast-feeding.
The institutional review board at each study site reviewed and approved the protocol. A parent or guardian of each infant provided written informed consent.
In the present double-blind, randomized controlled study, the formula-fed participants were randomly assigned to receive 1 of the 2 study formulas for a 60-day feeding period: control (Enfamil Lipil, Mead Johnson Nutrition, Evansville, IN) or the same formula supplemented with 4 g/L (1:1 ratio) of a blend of PDX (Litesse Two Polydextrose; Danisco, Beaminster, UK) and GOS (Vivinal GOS; Friesland Foods Domo, Zwolle, The Netherlands) (PDX/GOS). BF infants formed a reference group (BF). Anthropometric measures (body weight, length, and head circumference) were recorded at baseline and after 30 and 60 days of feeding. Parents completed a daily electronic diary (SymPro, SYMFO USA, Boston, MA) on intake, tolerance, and stool characteristics throughout the study. Responses were graded from 1 to 5 for stool consistency (hard, formed, soft, loose, watery), 0 to 3 for amount of gas (none, slight amount, moderate amount, excessive amount), and 0 to 4 for fussiness (not fussy, slightly fussy, moderately fussy, very fussy, extremely fussy).
All of the adverse events were recorded by the investigators throughout the study. An adverse event was defined as “any unfavorable and unintended sign, symptom, or disease (including abnormal laboratory findings), temporally associated with participation in the study whether related to the study product or not.” Any preexisting medical conditions were reported as adverse events if the condition worsened during the course of the study.
Quantification of fecal bacteria by fluorescent in situ hybridization (FISH) (log10 CFU/g stool) was the primary outcome of the study; secondary outcomes included quantification of fecal bacteria by quantitative real-time polymerase chain reaction (qPCR), stool characteristics, sIgA in feces, anthropometric measures, formula tolerance, and incidence of adverse events.
Fecal Collection and Homogenate Preparation
Infant stool specimens collected at home using chemical-free diapers (Tushies, TenderCare International, Eau Claire, WI) were frozen and subsequently shipped to a central collection site (BioStorage Technologies, Indianapolis, IN) for storage at −80°C and then processed at a research facility (Functional Foods Research Unit, University of Turku, Turku, Finland; Department of Medical Microbiology, University Medical Center Groningen, Groningen, The Netherlands) at baseline and after 30 and 60 days of feeding. Fecal supernatants were prepared as previously described (30).
Analysis of Fecal Microbiota by FISH
Fecal supernatants were fixed overnight at 4°C with 4% (wt/vol) fresh paraformaldehyde solution prepared in phosphate buffered saline (PBS) (1:4 dilution), and diluted in PBS based on most probable number. They were subjected to FISH (30) using the following 4 probes: Eub338 for total bacteria (31), the genus-specific Bif164 probe (32), Clit135 specific for the C lituseburense group (Clostridium cluster XI), and Chis150 specific for the C histolyticum group (Clostridium clusters I and II) (30). Using an epifluorescence microscope (model DMRA2; Leica Microsystems, Wetzlar, Germany), digital images of the fluorescent cells were captured and counted either visually (1000×, average number in 10–25 fields of 30–100 positive objects) or automatically (630×) using Quantimet HR600 image analysis software (Leica).
Analysis of Fecal Microbiota by qPCR
DNA was extracted and the qPCRs were conducted as described previously (33). Sequences of primers and appropriate annealing temperatures are listed in Table 1. PCR amplification and detection were performed with an ABI PRISM 7300-PCR sequence detection system (Applied Biosystems, Foster City, CA).
Analysis of Fecal sIgA by Enzyme-linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA) plates (96-well microplates; Nunc-Immuno Maxisorp, Roskilde, Denmark) were sensitized with mouse anti-human secretory component clone GA-1 (1:10,000 in PBS; Sigma, Zwijndrecht, The Netherlands) for 1 hour at 37°C or overnight at 4°C. Sensitized plates were incubated at room temperature with: fecal homogenate supernatants (1:1000 in PBS-bovine serum albumin [BSA]), purified human sIgA (positive control; P020 Nordic Immunology, Tiburg, The Netherlands), or human standard serum with assigned values for IgA subclasses (specificity control; NOR-04 Nordic Immunology); biotin-conjugated mouse anti-human IgA1/2 monoclonal antibody clone G20–359 (Pharmingen, Alphen aan den Rijn, The Netherlands; 0.25 μg/mL PBS-BSA); and streptavidin-conjugated horseradish peroxidase (Sigma S2438; 1:10,000 in PBS-BSA).
A comparison of study formula groups’ change in fecal bifidobacteria from baseline to 60 days of feeding quantified by FISH (log10 CFU/g stool) was the primary outcome of the present study. A sample size of 64 infants per group was required to detect an effect size of 0.5 (assuming a standard deviation of 0.6 , 80% power, α = 0.05). The Wilcoxon rank sum test was performed on FISH, qPCR, and ELISA data to compare changes from baseline between formula groups. The Kruskal-Wallis test was performed on these data to compare absolute counts among all of the study groups. If groups were determined to be significantly different, then pairwise comparisons were performed using the Wilcoxon test. The Cochran-Mantel-Haenszel row mean scores test was performed to compare the change in the number of bifidobacteria species from baseline between formula groups. Diary entries for formula intake, number of bowel movements, stool consistency, amount of gas, and level of fussiness were averaged during study periods and mean values were analyzed using repeated measures analysis of variance. Anthropometric measurements were analyzed by analysis of variance. Race, ethnicity, sex, adverse events, and study discontinuation were analyzed by Fisher exact test. Analyses were performed by using SAS version 9 (SAS Institute Inc, Cary, NC). All of the testing was conducted at an α level of 0.05.
Growth, Stool Characteristics, Formula Tolerance, and Adverse Events
Of the 289 participants enrolled, 230 completed the study (control 81, PDX/GOS 78, BF 71) (Fig. 1). Participants were stratified by duration of breast-feeding to ensure an even distribution of breast-feeding among groups. Demographics and baseline anthropometric characteristics of participants were similar for study groups at enrollment (Table 2). No statistically significant differences among groups were detected in study discontinuation rates or in the number of infants who discontinued because of study formula (control 8, PDX/GOS 7). Mean formula intake (ounces per day) was significantly higher in the PDX/GOS versus the control group at 16 to 30 days (29.3 vs 26.6 ± 0.8; P = 0.025), 31 to 45 days (30.8 vs 27.9 ± 0.9; P = 0.016), and ≥46 days (33.2 vs 29.7 ± 0.9; P = 0.006). No differences in weight growth rate from baseline to 30 or 60 days were detected between formula groups (data not shown). Mean weight growth rate (grams per day) at 60 days in the BF group (27.4 ± 0.8) was significantly lower than either the control (31.7 ± 0.8; P < 0.001) or PDX/GOS groups (30.1 ± 0.8; P = 0.025). No group differences in length or head circumference growth rates or achieved weight, length, or head circumference were detected at any time point.
Stool characteristics and tolerance measures are reported in Table 3. During all of the study periods, stool frequency was significantly higher in the BF group than in both formula groups, and there were no differences between formula groups. Stool consistency scores were significantly different among all of the groups, with the BF group being highest (range of means 4.0–4.1 ± 0.1), followed by PDX/GOS (3.3–3.5 ± 0.1) and control (2.9–3.1 ± 0.1). Means of parental-reported amount of gas were low (≤1.4, closest to “slight amount” of gas on the gassiness scale) in all of the groups throughout the study; however, it was significantly lower in the BF than in both formula groups in all of the study periods, with no differences between formula groups. Mean levels of fussiness were also low (≤1.2, closest to “slightly fussy” on the fussiness scale) during all of the study periods, and no significant differences were detected among groups.
There were no differences among study groups in the number of participants who experienced 1 or more adverse events. Also, there were no differences in the number of participants who experienced adverse events when analyzed by type of event, with 2 exceptions: differences were detected among groups in the number of participants with nasal/tear duct obstruction (control 2, PDX/GOS 1, BF 8; P = 0.045) and respiratory syncytial virus infection (control or PDX/GOS 0, BF 3; P = 0.028).
Quantification of Fecal Bacteria
FISH and qPCR were used to quantify fecal bifidobacteria and clostridia. Absolute counts of Bifidobacterium spp by FISH or qPCR are presented as box plots for comparison of results by these analytical methods (Fig. 2). By FISH, counts (log10 CFU/g stool) were significantly lower in the BF versus the control or PDX/GOS groups (P = 0.021 and P = 0.003, respectively) at baseline. No differences between groups were detected at 30 days. At 60 days, counts for the BF (median 9.86) were significantly higher than the counts for the control (9.36; P = 0.007) and PDX/GOS (9.58; P = 0.049) groups and were higher for the PDX/GOS versus the control group but did not reach statistical significance (P = 0.084). By qPCR, significant differences detected in Bifidobacterium spp among control, PDX/GOS, and BF groups demonstrated a time-dependent population shift from baseline to 30 and 60 days. At baseline, counts were significantly higher for the BF versus the control (P = 0.033) or PDX/GOS (P = 0.025) groups but not different between the control and PDX/GOS groups. At 30 days, a shift toward higher counts was observed for the PDX/GOS group. By 60 days, a pattern of higher counts for PDX/GOS was established: PDX/GOS counts, like BF counts, were significantly higher versus the control group (P = 0.002 and P = 0.001, respectively).
Across all of the fecal samples analyzed by qPCR, B longum was the most frequently detected species (71% of fecal samples from all of the study groups and time points), followed by B breve (33%), B animalis (28%), B bifidum (26%), B catenulatum (20%), B infantis (16%), and B adolescentis (4%). No group differences for B breve, B bifidum, or B adolescentis were detected at any measured time point (data not shown). Absolute counts of Bifidobacterium spp that demonstrated different time-dependent population shifts among the study groups are reported in Table 4. B longum closely paralleled the pattern demonstrated in total Bifidobacterium spp from baseline to 30 and 60 days and was similar to the BF group. No differences between groups were detected for counts of B animalis, B catenulatum, or B infantis at baseline or 60 days; however, at 30 days, counts of B animalis were similar between the BF and PDX/GOS but significantly higher in the BF versus the control group (P = 0.001), and B catenulatum was significantly higher in the PDX/GOS versus the BF group (P = 0.012). At 30 days, B infantis was significantly lower in the control versus the BF (P = 0.026) or PDX/GOS (P = 0.002) groups.
By FISH, absolute C lituseburense/C histolyticum counts (log10 CFU/g stool) at baseline, 30 days, and 60 days in the BF group (medians below detection limit at all time points) were significantly lower than the control (medians: 7.52, baseline; 7.13, 30 days; 7.34, 60 days; P < 0.001) and PDX/GOS (medians: 7.59, baseline; 7.16, 30 days; 7.40, 60 days; P < 0.001) groups. By qPCR, organisms in the C coccoides group were detected in 78% and C difficile in only 23% of the total fecal samples including the 3 study groups at all of the time points. Absolute counts of Clostridium spp are reported in Table 5. Counts of C coccoides group were lower in the BF versus both formula groups at all of the measured time points (P ≤ 0.002); formula groups were significantly different only at 60 days (P = 0.005). No differences were detected among groups for C difficile at baseline or between control and PDX/GOS groups at 30 or 60 days. Counts in the BF group were significantly lower versus PDX/GOS group at 30 days (P = 0.003) and versus control and PDX/GOS groups at 60 days (P = 0.013 and P = 0.003, respectively). Throughout the 60-day feeding period, however, few counts of C difficile or the C coccoides group reached a level >10 log10 CFU/g stool, and C difficile was below the detection limit by qPCR in >75% of infants from all of the groups at all of the time points.
Change From Baseline in Fecal Bacteria in Study Formula Groups
By FISH, no significant differences between formula groups in total Bifidobacterium spp or combined C lituseburense/C histolyticum as change from baseline to 30 or 60 days were detected (data not shown). Similarly, by qPCR, no significant differences between formula groups in change from baseline to 30 or 60 days were detected in total Bifidobacterium spp, B bifidum, B breve, B animalis, B adolescentis, C coccoides, or C difficile (data not shown). The change from baseline was significantly higher in PDX/GOS than in the control group for B catenulatum (percentage of infants with an increase: 27% vs 14%; P = 0.004) and B infantis (19% vs 2%; P = 0.024) at 30 days and for B longum (72% vs 59%; P = 0.035) at 60 days. Furthermore, a significantly larger change in the number of Bifidobacterium spp was detected in the PDX/GOS compared with the control group at both 30 (mean 0.82 ± 0.14 vs 0.31 ± 0.12; P = 0.008) and 60 days (mean 1.20 ± 0.19 vs 0.59 ± 0.18; P = 0.021) of feeding (Fig. 3).
Quantification of Fecal sIgA
The BF group showed significantly higher levels of fecal sIgA by ELISA (milligram per gram of stool) than control or PDX/GOS groups at baseline (medians 3.07, 0.48, 0.45; P < 0.001), 30 (medians 3.42, 0.86, 1.21; P < 0.001), and 60 days (medians 3.38, 1.09, 1.26; P < 0.001) of feeding. Levels of fecal sIgA at any time point or change of sIgA from baseline to 30 or 60 days of feeding were not significantly different between formula groups (data not shown).
The intestinal microbiota of BF infants is a model for early nutritional intervention, such as supplementation of infant formula with PDX and GOS. This supplementation may stimulate the microbiota at a stage in which immune maturation and consequent resistance to infections and allergic diseases can be influenced (7,14,20,21). Although levels of beneficial bifidobacteria tend to be higher in BF infants when compared with formula-fed infants (2,4,9), a bifidogenic effect can be achieved in infants receiving formula supplemented with prebiotics in amounts up to 10 g/day (13,15,16). We previously demonstrated that supplementation of PDX and GOS (4 g/L; 1:1 ratio) to routine cow's-milk–based formula is well tolerated, safe, and promotes normal growth (29,34). In the present study, infant formula added with this prebiotic blend promoted an increase in members of the Bifidobacterium genus (specifically, B infantis, B longum, and B catenulatum) in healthy term infants during a 60-day feeding period.
Bifidogenic effects for PDX and GOS in isolation have been demonstrated in adults and infants (25,35,36). In these studies, bifidobacteria were enumerated using traditional culture-based techniques, which can elicit lower counts than probe hybridization techniques (32). In particular, fecal anaerobes can be damaged if anaerobic incubation is not performed immediately after defecation (30,32). Bifidobacteria may also be overestimated in culture-dependent methods because total culturable counts are only a fraction of the total intestinal microbiota (32), whereas molecular methods more accurately quantify bacterial populations (37).
In the present study, 2 quantitative molecular culture-independent techniques, FISH and qPCR, were used to analyze potential shifts in the infant fecal microbiota. These methods enable detection of less abundant organisms and allow species-level discrimination (37). Although no difference in change from baseline of total bifidobacteria between groups was observed in the present study, significantly higher changes from baseline of B longum and B infantis were observed in the PDX/GOS compared with the control group. Along with B breve, B longum and B infantis are the most common Bifidobacterium spp found in infants and they predominate in the intestinal microbiota of BF infants (38). Higher absolute counts of total bifidobacteria and B longum in the PDX/GOS compared with the control group observed at 30 days became statistically significant by 60 days of feeding. Furthermore, the control group but not the PDX/GOS group had significantly lower counts of total bifidobacteria, B longum, B infantis, and B animalis than the BF group, corroborating that the prebiotic supplementation may have modified the gut microbiota toward a profile observed in BF infants.
Subtle differences in the microbiota of BF infants and formula-fed infants may occur at the species level. Prebiotic intervention has been linked to a shift in the microbiota of formula-fed infants toward a greater number of Bifidobacterium spp, making it closer to that of BF infants (17). A higher number of bifidobacterial species may be necessary for postnatal maturation of a normal immune system, which requires constant microbial stimulation by the developing intestinal microbiota (1,8). In the present study, the PDX/GOS group developed a higher number of Bifidobacterium spp compared with the control group. We also observed a time-dependent shift toward higher counts of total bifidobacteria and B longum during the 60-day feeding period, possibly reflecting maturation of the gut microbiota. A similar shift in infants fed infant formula supplemented with GOS/FOS has been described by others (39).
Although some previous studies have reported a potentially beneficial prebiotic effect of lowering pathogenic species, including C difficile(16,19), this effect was not observed in other studies (9,13,40), including our study. Bifidobacteria are among the initial and most prevalent colonizers of the gastrointestinal tract. In contrast, clostridia, including members of the C coccoides group, appear later in the colonization process, as demonstrated by their relatively low levels in infants (6,41). In vitro experiments suggest that the suppression of C difficile by some prebiotics is not linked to a competition with bifidobacteria, but likely involves multiple barrier factors that inhibit pathogen growth (42). In the present study, clostridia appeared at a much lower level than bifidobacteria, yet increased in all of the 3 groups during the 60-day study period, possibly reflecting normal maturation toward higher, typical adult-like numbers (41).
In agreement with several previous reports (13,14,29,34,36), we demonstrated a stool-softening effect with PDX/GOS, which was closer to that of the BF than the control group. This effect is potentially beneficial to help manage the hard stools or constipation that may affect formula-fed infants (43). Transition from breast to bottle has been shown to occur during the 3 months preceding onset of constipation in nearly 25% of constipated children in the first 2 years of life (44). Harder stools, typical of formula-fed infants, have less water-soluble and complex carbohydrates when compared with stools of BF infants (43). The protective effect of fiber against constipation is the result of the osmotic stimulation caused by the short-chain fatty acids generated during fermentation of fiber by colonic bacteria, in addition to the water-holding capacity of undigested components of fiber (45).
Infant growth in the present study was similar among groups, with the exception of a lower weight growth rate at 60 but not 30 days in the BF group. No differences in the incidence of adverse events were observed between the prebiotic-supplemented and the unsupplemented formula groups, and the only significantly higher incidence of adverse events occurred in the BF group (nasal/tear duct obstruction and respiratory syncytial virus infection).
One potential concern linked to supplementation of infant formula with prebiotics is excessive gas formation. The addition of PDX/GOS to infant formula had no negative effect on gassiness, which is consistent with previous studies (29,34). This indicates that the amount of prebiotics in the supplemented formula, 4 g/L, achieves a bifidogenic effect and at the same time promotes a health benefit of stool softening without excessive gas production. The induction of more watery stools is recognized by the European Society for Gastroenterology, Hepatology, and Nutrition as a relevant benefit of prebiotics in infants experiencing constipation (46). Collectively, the stimulation of beneficial bacteria and the benefit of softer stools indicate that the PDX/GOS blend indeed meets the European Society for Gastroenterology, Hepatology, and Nutrition's and the Food and Agriculture Organization of the United Nations's definition of a prebiotic (ie, it confers a health benefit on the host associated with modulation of the microbiota) (46,47).
Formula-fed infants who do not benefit from the protective sIgA transferred from breast milk may benefit from measures aimed to increase production of endogenous sIgA. Increased sIgA has been associated with the feeding of PDX to an animal model (28) and GOS in combination with FOS to infants (18,19). In the present study, the BF group had consistently higher sIgA than the formula groups, but no statistically significant differences in sIgA were observed between formula groups. In another study, although the addition of prebiotics to infant formula elicited no increase in total sIgA, a positive correlation between anti-poliovirus sIgA and B longum/B infantis species after vaccination was observed (48). Therefore, such a potential effect of infant formula with PDX/GOS on sIgA warrants further evaluation.
Not only the type and amount of prebiotics but also the analytical methods used to identify and quantify bacteria can influence fecal microbiota results (9,32,49). The 2 molecular techniques chosen in our study corroborated our microbiota results with complementary analyses. The high detection limit of FISH (which does not allow quantification of extremely low bacterial numbers in fecal samples) (37) may have been responsible for the absence of statistically significant differences between formula groups that were detected by qPCR. Differently from conventional PCR, qPCR monitors complete DNA amplification at all of the stages, not just the plateau phase, and consequently detects decreasing differences in PCR product abundance, which is appropriate for accurate bacteria quantification (6). In addition, qPCR provides high specificity by using specific primers and probes (17,49).
When the criterion standard of infant nutrition, breast milk, is not available, supplementation of infant formula with prebiotics early in life may offer an opportunity to affect microbiota development (34). The results of the present study demonstrate that early nutritional intervention of infant formula with 4 g/L of a PDX/GOS 1:1 blend has a bifidogenic effect and promotes softer stools that are closer to those of BF infants than infants fed an unsupplemented formula. As such, the PDX/GOS–supplemented formula may be helpful to manage or prevent early constipation in formula-fed infants.
We thank study investigators and their staff for their cooperation. The participation of parents and infants in the present study is gratefully acknowledged. Additionally, we thank Jennifer L. Wampler, PhD, for contributions to manuscript development, and Laura B. Beck, Rebecca A. Hirsch, and Bryan Liu, MD, for management of study sites.
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