Wheat bran extract (WBE), a food-grade, fiber-rich, water-soluble preparation that is produced by enzymatic extraction from wheat bran, is highly enriched in arabinoxylan-oligosaccharides (AXOS). AXOS consist of a backbone of β-1,4-linked D-xylopyranosyl residues (xylose), some of which are mono- or disubstituted at the C(O)2 and/or C(O)3 position with α-L-arabinofuranosyl residues (arabinose) (1). Some of the xylose units in the backbone of AXOS carry glucuronic acid at the C(O)2 position, whereas some of the arabinose units are ester linked at the C(O)5 position with ferulic acid (1). The AXOS in WBE form a heterogeneous mixture of oligosaccharides differing in degree of polymerization and degree of substitution of the xylan backbone. Besides AXOS, WBE contains up to 15% glucans (mainly β-D-(1,3)(1,4)-linked glucan oligomers) and low levels (<2%) of proteins, minerals, and monosaccharides (2).
AXOS are nondigestible fermentable prebiotic oligosaccharides with bifidogenic activity, as demonstrated in in vitro studies (3), animal studies [chicken (4,5), rats (6)], and clinical trials with healthy adults (7–9). The evidence for AXOS having prebiotic activity has been reviewed (1). In addition, AXOS consumption decreases the excretion of urinary and fecal p-cresol, a marker of intestinal protein fermentation (7,8,10). Colonic protein fermentation is often regarded as detrimental to host health, in particular with respect to colon toxicity, mutagenicity, and carcinogenicity (11). The proteolytic fermentation leads to the production of toxic compounds such as phenolic compounds, sulfur-containing compounds, amines, and ammonia (12–14). The toxicity of these protein fermentation metabolites has mainly been established in in vitro studies (15–17) and animal studies (18,19).
Until this writing, the gastrointestinal (GI) effects of and tolerance effect to WBE in humans have only been investigated in adult volunteers. It is known that the composition of gut microbiota in preadolescent and adolescent children differs from that of adults (20,21), with notably a higher abundance of bifidobacteria in teenage children versus adults (21). The purpose of this study, therefore, was to evaluate the effect of intake of 5 g/day WBE on GI health parameters in healthy preadolescent children ages between 8 and 12 years. The effect of WBE administration on colonic carbohydrate fermentation was investigated through measurement of fecal levels of short-chain fatty acids (SCFAs), and the effect on colonic protein fermentation was analyzed through measurement of fecal levels of isovaleric acid and isobutyric acid. Because WBE is intended to be added to food products, including food products for children, it is also important to assess tolerance to the product and its safety profile. Tolerance to WBE was assessed through self-reported scoring by the children of distress severity of the following 3 surveyed GI symptoms: flatulence, abdominal pain/cramps, and urge to vomit. Safety was evaluated by assessing the occurrence of adverse events (AEs).
Figure 1 presents a schematic overview of the randomized placebo-controlled double-blind crossover study. The study started with a 1-week run-in (RI) period, followed by two 3-week treatment periods, during which the children received placebo or 5 g/day WBE, with a 2-week washout (WO) period in between the treatment periods. The WBE dose of 5 g/day was half the WBE dose that was shown in an earlier trial performed on adults to raise the levels of bifidobacteria (8), taking into consideration the lower average body weight of preadolescent children versus adults. WBE and placebo were administered as noncarbonated soft drinks of which the volunteer drank daily 70 mL after breakfast and 70 mL after dinner (140 mL/day in total). The WBE-containing soft drinks contained sucrose, colorant, flavor, citric acid, and potassium sorbate. The placebo soft drink had the same composition as the WBE-containing soft drink, except that WBE was omitted and that 0.25 g/L tricalcium phosphate was added to mimic the turbidity of the WBE-containing soft drinks. Subjects were randomly assigned to 1 of 2 randomization groups, differing in the treatment sequence by which the 2 types of drinks were to be consumed. The investigators who had direct contact with the subjects were blinded to the treatment because they were unaware of the randomization groups to which the subjects were assigned. Moreover, the appearance and the taste of the different soft drinks were nearly identical, and the 2 types of soft drinks could not be discriminated from each other without a careful side-by-side comparison. The side-by-side comparison of the drinks consumed by the volunteers was not possible because only 1 type of drink was supplied before each treatment period.
A total of 29 healthy children (11 girls and 18 boys, all of white ethnicity) participated. Exclusion criteria were extreme dietary habits in the 6 weeks before the start of the trial, intake of antibiotics in the 3 months before the start of the trial, intake of medication or dietary supplements influencing GI tract processes in the 2 weeks before the start of the trial, abdominal surgery in the past (with exception of appendectomy), chronic diseases/conditions, serious illness in the 3 months before the start of the trial, complete anesthesia in the month before the start of the trial, and history of chronic GI conditions such as inflammatory bowel disease and irritable bowel syndrome, allergy to wheat products, and celiac disease.
During the study, the intake of food substances containing probiotics and/or prebiotics was forbidden. The children and their parents were asked to read food labels carefully to check for absence of pro- and/or prebiotics. This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the ethics committee of the University Hospital UZ Leuven (Belgium) under approval number ML5282. A written informed consent was obtained from all of the children and their parents.
WBE (Brana Vita 200) was produced from wheat bran by FUGEIA NV (Leuven, Belgium), using a procedure based on that described earlier (22). WBE was analyzed for AXOS content, AXOS average degree of polymerization, arabinose/xylose ratio, bound ferulic acid and glucuronic acid, glucose as part of poly-/oligosaccharides, mannose as part of poly-/oligosaccharides, galactose as part of poly-/oligosaccharides, free monosaccharides, moisture, protein, and ash by analytical procedures outlined (2). Table 1 shows the composition of the WBE preparation used in the present study. It consisted of 79% AXOS (on dry matter basis), and the AXOS had an average degree of polymerization of 5 and an arabinose/xylose ratio of 0.19.
Biochemical and Microbiological Analyses of Fecal Samples
On the evening of day 5 or during day 6 of the RI period and on the evening of day 19 or during day 20 of each treatment period, 1 single stool was collected by each child. Fecal samples were analyzed for microbiota composition using fluorescence in situ hybridization analysis. The processing of paraformaldehyde-fixed samples and fluorescence in situ hybridization analysis to quantify bifidobacteria, the Lactobacillus/Enterococcus group, the Clostridium histolyticum/lituseburense group, the Faecalibacterium prausnitzii group, and the Roseburia/Eubacterium rectale group was performed as described (8). Concentrations of the SCFAs acetate, propionate, butyrate, and the branched-chain fatty acids (BCFAs) isovaleric acid and isobutyric acid were determined as described (23), using 2-methylhexanoic acid as internal standard. To determine the fecal pH, an aliquot of approximately 1 g feces was homogenized by mixing with demineralized water (final concentration 10% [w/w]) (24). The pH was measured immediately upon homogenization. Ammonia levels were measured on the same fecal slurries as used for pH determination, following the procedure described (25).
Recording of GI Symptoms and Stool Parameters
GI symptoms were monitored daily during the RI period and the last week of each treatment period. The volunteers were asked to grade the distress severity of abdominal pain/cramps, flatulence, and urge to vomit using a 5-step scale ranging from no (0), minimal (1), mild (2), moderate (3) to severe (4) distress (7). During the RI period and the last week of each treatment period, the number of stools and stool consistency according to the Bristol Stool Form Scale (26) were recorded daily. The average stool frequency was calculated as the number of stools divided by the number of days of diary recording, the average stool consistency as the sum of Bristol Stool Form Scale scores divided by the number of stools, and the composite parameter of stool frequency and consistency (called the Bristol composite measure) as the sum of Bristol Stool Form Scale scores divided by the number of days of diary recording (8,27).
Recording of AEs and Treatment Compliance
At the end of the RI period and after each intake period, the children were asked to record whether they had experienced a medical condition, had received medication, or had incidentally taken pre- or probiotics. Additionally, at each clinic visit, the children were asked these questions. This information was recorded in the appropriate section of the case report form. Treatment compliance was defined as the number of times per treatment period that a serving of soft drink was not consumed. During each intake period, the children had to report daily whether they consumed the soft drinks after breakfast and after dinner.
Statistical Analyses of Efficacy Variables
To test for differences at baseline, the treatment sequence groups were compared with respect to age, sex, fecal bifidobacteria content, and stool frequency. The comparison of the groups was based on a 1-way analysis of variance and a χ2 test in case of sex. The Fisher exact test was used if the χ2 test was judged to be inappropriate because of small cell sizes. When 1-way analysis of variance analysis could not be used, the groups were compared using the nonparameteric Kruskal-Wallis test and pairwise using the Mann-Whitney U test.
For each efficacy variable, the difference between both treatments was analyzed within the statistical model. A mixed model was used to capture the intravolunteer correlation as a result of the repeated measurements for each volunteer. The treatment effect for the different parameters was analyzed using a linear mixed model (28). The models were estimated using the linear and nonlinear mixed-effects model package from R's base distribution (R Development Core Team, University of Auckland, New Zealand). The fitted model included subject as a random effect and contained terms for treatment and treatment sequence. Additionally, the results of the RI period were included as covariate in the linear mixed model. All tests of significance were performed at α = 0.05 and were 2-sided, unless otherwise stated. Assumptions of normality of residuals were investigated for each variable using the Shapiro-Wilk test (30). When the data were normally distributed, linear mixed models were applied to the raw data as such, except for the microbiota data, which were log transformed before analysis. When the distribution of the data was not approximated by a normal curve, values were ranked before analysis and the linear mixed model was performed on the rank-transformed data (31). Ties occurring during the rank transformation were replaced by their average rank. The data to estimate the fixed effect parameter for the RI of the response remained unranked.
Evaluations of the effects of treatment on the efficacy variables were completed on an efficacy evaluable (EE) population, defined as all randomized subjects who received placebo and at least 1 serving of a WBE-containing soft drink and who provided at least 1 postrandomization outcome data point during each of the 2 treatment phases. The per-protocol (PP) population was defined as the subset of EE subjects who completed the study, did not take excluded medications (eg, antibiotics) or products, and had no major protocol violations. In the present study, the PP population coincided with the EE population, and statistical analyses were not repeated for the PP population.
Treatment effects and treatment by treatment sequence interaction effects were tested with linear mixed models using conditional F and t tests (28) (significance at α = 0.1). The single-step Tukey post-hoc multiple comparison procedure was used for the pairwise comparisons of the treatments, using R's multcomp package (32). In case no significant interactions were found, treatment differences were evaluated based on the main effect model. In case of significant interactions, treatment differences were evaluated within each treatment sequence group (results not shown). Next to that, the overall differences were also analyzed by aggregating over the interaction effects, which was performed by setting up a linear combination of the treatment differences for each treatment sequence group, giving equal weights to both treatment sequence groups. For the distress severity of the 3 GI symptoms, an analysis was performed using binary data. Although the symptoms were scored daily during 1 week on an ordinal scale and were subsequently aggregated (averaged) during the 7 days, the lack of variation in the symptom scores obliged us to use binary response models. In this case, all volunteers who indicated for an aggregated GI symptom score “no distress” were regarded as “0.” All of the people who indicated an aggregated distress severity score differing from “no distress” were regarded as “1.” The level of the C histolyticum/lituseburense group was in a large proportion of the volunteers below the detection limit (log10 5.65/g wet feces), leading to a binary distribution of this dataset (“1” = content of Clostridium group ≥log10 5.65/g wet feces; “0” = content of Clostridium group <log10 5.65/g wet feces). For the analysis of binary data, a generalized linear mixed-effects model was used as described (8).
Statistical Analysis of Safety Parameters and Treatment Compliance
The safety population was defined as all randomized subjects who received at least 1 serving of WBE. Safety was analyzed using the emergent AEs in the safety population. An AE was attributed to the treatment period during which the AE started. An AE that started during a WO period was attributed to the treatment preceding the specific WO period. The McNemar test was used to compare differences in AE frequencies between the 2 treatments (α = 0.025, Sidak correction for 2 comparisons) (33). A statistical analysis of the number of nonconsumed study product servings was performed using a Poisson mixed-effects model with similar fixed effects as for the efficacy variables but excluding the RI period because there were no consumptions at the RI. For the analysis of Poisson data, a generalized linear mixed-effects model (34) with the log-link function was used with equal random and fixed effects as in the linear mixed model. For the Poisson data, a similar approach as for the continuous data was used starting from a basic model that contained the main effects: the value at baseline, treatment, and treatment sequence. The test for a significant interaction between treatment and treatment sequence was done based on a likelihood ratio test (34). P values were obtained similarly as described for the models of the efficacy variables. The evaluation of a significant treatment effect was done by comparing the basic model to a model containing only treatment sequence and RI. Adding and evaluating the interaction effects were done stepwise by adding treatment sequence based on the most significant likelihood ratio test. In case of insufficient data in each interaction cell (<4), the final model to evaluate the treatment was the basic model and interactions were disregarded. For models with interaction effects and inflated standard errors of the treatment differences, the model containing only main effects was referred to.
The disposition of all of the study participants is presented in Figure 2. A total of 30 children were screened and 29 were randomized to the 2 different randomization groups. Because all of the children received WBE, the 29 children were included in the safety population. Of these, 1 child was excluded from the EE population because no data points were obtained from this volunteer during the placebo treatment. Hence, the EE population consisted of 28 children. Because none of them had received antibiotics and all of the children were compliant, the PP population was same as that of the EE population.
Baseline characteristics for the EE/PP population are presented for both randomization groups in Table 2. No significant differences could be observed at baseline between both randomization groups with respect to sex, age, stool frequency, and fecal bifidobacteria level.
The number of times per treatment period that the volunteers of the PP population did not receive a serving of soft drink was on average low (2.0% during the placebo-intake period and 1.8% during the WBE-intake period), indicating a good compliance with the study based on self-reporting by the volunteer. No statistically significant differences (P > 0.1) were observed between the treatments. None of the volunteers reported that they had incidentally received pre- or probiotics during the study. Hence, overall compliance with the study was considered to be good.
AEs were categorized into 6 categories according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0) before the unblinding of the study. During the RI period, placebo-intake period, and WBE intake period, 4, 7, and 5 AEs occurred, respectively. The statistical analysis of the AEs in the safety population revealed no difference between the placebo and WBE treatment in frequency of any of the different AE categories (P > 0.1).
Analysis of Efficacy Variables
Conditional F tests showed overall WBE-related significant treatment effects for 5 parameters (Table 3): fecal levels of bifidobacteria, percentage of bifidobacteria in feces, fecal levels of isobutyric acid, fecal levels of isovaleric acid, and fecal levels of total BCFAs (P < 0.1). The main results of the subsequent pairwise comparisons of these parameters will be discussed in the following section.
Levels of Fecal Microbiota
In the PP population, WBE intake selectively increased bifidobacteria levels in the feces (Table 3). Intake of 5 g/day WBE tended to increase the levels of bifidobacteria in the feces relative to placebo intake by 0.19 log units (P = 0.069). The percentage of bifidobacteria relative to the total bacterial content in feces upon 5 g/day WBE intake increased by 1.7-fold relative to placebo intake (P = 0.002). The fecal levels of the other bacterial groups analyzed, the Lactobacillus/Enterococcus group, the C histolyticum/lituseburense group, the F prausnitzii group, and the R rectale/E rectale group, remained unchanged after WBE intake.
Biochemical Parameters in Feces
The intake of 5 g/day WBE decreased the level of total fecal BCFAs and the levels of isobutyric acid and isovaleric acid by approximately 28% relative to placebo intake (P < 0.05) (Table 3). WBE intake did not affect the percentage moisture in feces, nor did it influence fecal ammonia levels, fecal SCFAs levels, and fecal pH (P > 0.1).
Bowel Habits: Defecation Frequency and Stool Consistency
WBE intake did not influence the number of bowel movements per day, nor did it modulate stool consistency as measured using the Bristol Stool Form Scale (P > 0.1).
Analysis of Tolerance Variables
Tolerability was assessed through self-reported scoring by the children of the distress severity of flatulence, urge to vomit, and abdominal pain/cramps, using a 5-step scale ranging from no (0), minimal (1), mild (2), moderate (3) to severe and (4) distress. In Table 4, an overview of the scoring of the distress severity of the 3 surveyed GI symptoms can be found. The statistical analysis of the distress severity of the 3 surveyed GI symptoms in the PP population demonstrated no difference between placebo and WBE treatment (P > 0.1, binary mixed model).
This study investigated, for the first time, the effect of WBE consumption in healthy preadolescent children (ages 8–12 years). The effects of WBE consumption at a dose of 5 g/day were analyzed on the following GI parameters: fecal levels of microbiota, SCFAs, BCFAs, ammonia, fecal pH, and fecal moisture. In addition, using self-reported scoring of the distress severity of 3 surveyed GI symptoms, tolerance to WBE was assessed in children.
WBE consumption by healthy children during 3 weeks at a daily dosage of 5 g led to an increase in fecal bifidobacteria levels, expressed as percentage of total microbiota, relative to placebo intake. These data extend earlier studies evaluating the effect of WBE and WBE-like material on fecal microbiota in healthy adult volunteers (7–9,35), despite the fact that the relative level of fecal bifidobacteria in preadolescent children is higher than that in adults (24). As was also observed in adult volunteers, the intake of WBE by children only modulated fecal levels of bifidobacteria. The levels of the other bacterial groups analyzed, that is, the Lactobacillus/Enterococcus group, the C histolyticum/lituseburense group, the F prausnitzii group, and the R rectale/E rectale group, were not modulated upon WBE intake. This points to a selective increase in fecal bifidobacteria levels relative to total fecal microbiota, upon WBE intake by healthy children.
Beneficial effects of bifidobacteria on host health have been demonstrated through placebo-controlled clinical trials involving direct oral supplementation with viable bifidobacteria. For instance, oral intake of bifidobacteria by healthy infants was shown to lower the risk of experiencing respiratory infections (36). In addition, studies performed on patients experiencing mild-to-moderate irritable bowel syndrome showed that supplementation with bifidobacteria improves symptoms of abdominal pain/discomfort, distension/bloating, and bowel movement difficulty (37,38). The mechanisms responsible for such effects have not been fully elucidated, yet may involve modification of the gut microbiota, competitive adherence to the intestinal mucosa and epithelium, strengthening of the gut epithelial barrier, and/or modulation of the immune system through interaction with pattern recognition receptors on gut epithelial cells (39).
The intake of 5 g/day WBE resulted in a marked reduction of the fecal levels of BCFAs isobutyric acid and isovaleric acid by 28% as compared with the fecal BCFAs levels after placebo intake. BCFAs are not produced by human enzymes and are therefore unique bacterial metabolites. Isobutyric acid and isovaleric acid are produced from the fermentation of valine and leucine, respectively (40). As a consequence, excretion of BCFAs is often considered a marker for the degree of protein fermentation in the colon (41). The reduction in protein fermentation observed in this study confirms previous results in adults, which showed a reduction in urinary and fecal p-cresol (7,8,10) or a beneficial modulation of the colonic ammonia metabolism (both protein fermentation metabolites) (42) after the intake of WBE or WBE-like material. Colonic fermentation of proteins results in the formation of ammonia, nitrosamines, thiols, and phenolic compounds, which are generally believed to be harmful. Hence, the reduction of colonic protein fermentation is believed to be beneficial to human health (43).
Consumption of 5 g/day WBE did not result in increased fecal levels of the carbohydrate fermentation products acetate, propionate, and butyrate. François et al showed increased fecal levels of these SCFAs after intake of 10 g/day WBE in healthy adults, but not after intake of 3 g/day WBE (8). The fact that an increase in fecal SCFA levels was not observed could be because of the intake of a WBE dose that was extremely low to modulate fecal SCFA levels; however, it should be kept in mind that fecal SCFA levels are the result of both colonic SCFAs production and mucosal absorption of these SCFAs (44). As such, the absence of increased fecal SCFA levels does not exclude an increased colonic fermentation following WBE fermentation.
The intake of 5 g/day WBE by healthy children did not affect stool frequency, stool consistency, or the composite Bristol measure. In addition, 5 g/day WBE consumption did not have an effect on any of the 3 surveyed GI symptoms: flatulence, abdominal pain/cramps, and urge to vomit. In healthy adult volunteers, a mild increase of flatulence was observed at 10 g/day WBE intake (11). A mild-to-moderate flatulence is observed in studies with other prebiotic compounds such as inulin and fructo-oligosaccharides, which is caused by the production of gases upon fermentation of the prebiotic compound (45–49).
The low incidence of GI complaints together with the absence of a difference between the placebo and WBE treatment in occurrence frequency of the AE categories provides evidence for the excellent tolerability and safety of WBE in children. This is important because addition of WBE to food products intended for children may be a way to increase the fiber intake by these children. Indeed, intake data from the United States indicate that dietary fiber consumption is inadequate in most children, especially from low-income and minority backgrounds (50). In this respect, it is also important to note that WBE is water soluble and does not have a pronounced taste, which makes it easy to formulate in food products without affecting their texture or taste. Other fiber sources, such as insoluble fibers, can disturb the taste or texture of a food product and thereby make it less attractive for consumption, in particular by children.
In conclusion, intake of 5 g/day WBE exerts beneficial effects on gut parameters, in particular reduction of colonic protein fermentation and increase in relative fecal bifidobacteria levels. Moreover, WBE is well tolerated and does not cause adverse effects at up to 5 g/day in healthy children.
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