Bioinformatic analysis was performed in 28 children in the synbiotic group and 26 children in the placebo group. Alpha diversity (within-sample species diversity) was evaluated with Chao1 (a measure of community richness), observed ASVs, Shannon (a measure of richness and evenness or entropy) and Simpson indices, which were used to measure species richness and evenness (similar abundance) in the groups. While there was no difference in the Shannon (which measures richness) and Simpson indices (data not shown) in the synbiotic group at the beginning and at the end of the 12th week, the observed ASVs and Chao1 indices were found to be lower at the 12th weeks compared to the initial period (p < 0.001) (Supplementary Fig. 3). There was no difference between theASVs, Chao-1, Simpson (data not shown), and Shannon indices observed at the beginning of the placebo group and at the end of the 12th week (p > 0.05) (Supplementary Figs. 2 and 3). At the 12 weeks of intervention, the Chao1 index was found to be lower in the synbiotic group than in the placebo group, but there was no significant difference (Supplementary Fig. 2). Bray–Curtis dissimilarity was used to compare the abundance of each ASV between the synbiotic and placebo groups. The β-diversity (between-sample dissimilarity) weighted UniFrac distance of ASVs (Bray-Curtis) revealed no statistically significant clustering (p > 0.05) (data not shown).

At the phylum level, the intestinal microbiota composition of the study groups was similar at baseline. In the synbiotic group, the major phyla were Firmicutes (66.7%), Bacteroidetes (18.8%), Actinobacteria (7.6%), Proteobacteria (3.3%) and Verrucomicrobia (2.93%). In the synbiotic group, 12 weeks of intervention, at the phylum level, Firmicutes (66.0%), Bacteroidetes (24.0%), Actinobacteria (6.2%), Proteobacteria (2.0%) and Verrucomicrobia (1.22%) were observed. In the synbiotic group, the Bacteroidetes phylum was higher at 12 weeks of intervention than at baseline (24.0% vs. 18.8%, p < 0.01). In the placebo group, at baseline, the major phyla were Firmicutes (72.3%), Bacteroidetes (15.4%), Actinobacteria (8.7%), Proteobacteria (1.56%) and Verrucomicrobia (0.91%), and at 12 weeks of intervention, the major phyla were Firmicutes (69.2%), Bacteroidetes (22.6%), Actinobacteria (5.73%), Proteobacteria (1.8%) and Verrucomicrobia (0.59%). There was no difference between baseline and the 12th week of intervention in the placebo group (p > 0.05). There was also no difference between the synbiotic and placebo groups at the phylum level after 12 weeks of intervention (p > 0.05). In the synbiotic group, the Firmicutes/Bacteroidetes ratio was 3.54 at baseline and 2.75 at 12 weeks of intervention (p < 0.05). In the placebo group, the Firmicutes/Bacteroidetes ratio was 4.70 at baseline and 3.54 at 12 weeks of intervention (p < 0.05). After 12 weeks of intervention, the Firmicutes/Bacteroidetes ratio was also lower in the synbiotic group than in the placebo group (p < 0.05).

The genus level comparisons of the intestinal microbiota compositions of the synbiotic group and placebo group at baseline and at week 12 and among themselves at baseline and at week 12 are shown in Figs. 1 and 2.

The distribution and comparison of the dominant microorganisms in the intestinal microbiota composition at baseline and at the 12th week of treatment in the synbiotic group at the genus level. Comparing the baseline, we observed a statistically significant increase in the genera Prevotella (5.28–14.4%, p < 0.001) and Dialister (9.68–13.4%; p < 0.05)

The distribution and comparison of the dominant microorganisms in the intestinal microbiota composition at baseline and at the 12th week of treatment in the placebo group at the genus level. Comparing the baseline, we observed a statistically significant increase in the genera Prevotella (6.4–12.4%, p < 0.01) and Oscillospira (4.95% vs. 5.70%, p < 0.001)

In the synbiotic group, the most abundant genera were Faecalibacterium (20.5%), Bacteroides (16.3%), Dialister (9.68%), Bifidobacterium (9.55%), Blautia (6.62%), Prevotella (5.28%), Gemmiger (4.66%), Akkermansia (4.33%), Ruminococcus (4.14%), Oscillospira (3.91%), Streptooccus (2.27%), and Lactobacillus (1.76%). Twelve weeks of intervention, the most abundant genera were Faecalibacterium (18.7%), Prevotella (14.4%), Bacteroides (13.5%), Dialister (13.4%), Bifidobacterium (7.78%), Blautia (4.92%), Oscillospira (4.58%), Ruminococcus (4.03%), Gemmiger (2.52%), Akkermansia (1.77%), Streptooccus (1.01%), and Lactobacillus (0.37%) (Fig. 1). Comparing the baseline, we observed a statistically significant increase in the genera Prevotella (5.28–14.4%, p < 0.001) and Dialister (9.68–13.4%; p < 0.05) (Fig. 1).

In the placebo group, the most abundant genera were Faecalibacterium (23.2%), Bacteroides (11.4%), Bifidobacterium (10.9%), Dialister (8.72%), Prevotella (6.4%), Ruminococcus (6.07%), Blautia (5.74%), Oscillospira (4.95%), Gemmiger (4.6%), Akkermansia (4.33%), and Lactobacillus (2%). After 12 weeks of intervention, the most abundant genera were Faecalibacterium (22.0%), Prevotella (12.4%), Bacteroides (14.6%), Dialister (11.9%), Bifidobacterium (6.44%), Blautia (5.06%), Oscillospira (5.70%), Ruminococcus (3.77%), Gemmiger (3.02%), Akkermansia (0.92%), and Lactobacillus (0.76%). Comparing the baseline, we observed a statistically significant increase in the genera Prevotella (6.4–12.4%, p < 0.01) and Oscillospira (4.95% vs. 5.70%, p < 0.001) (Fig. 2).

At baseline and 12 weeks of intervention, there were no statistically significant differences in genera between the synbiotic and placebo groups (Figs. 3 and 4). Faecalibacterium prausnitzii is the most abundant strain in both groups at baseline and 12 weeks of intervention for synbiotic and placebo groups. There are no difference for the presence of Faecalibacterium prausnitzii at baseline and 12 weeks of intervention in the synbiotic group (35.6% and 32.9%, consecutively) and in the placebo group (23.2% and 22.0%) (p > 0.05).

The distribution and comparison of the dominant microorganisms in the intestinal microbiota composition at baseline in the synbiotic and placebo groups at the genus level

The distribution and comparison of the dominant microorganisms in the intestinal microbiota composition at the 12th week of treatment in the synbiotic and placebo groups at the genus level

Microbiota elements with an LDA score of > 2 were determined between the groups to show statistically significant taxonomies by LEFSe analysis in the study groups. At the beginning of the study, there was no significant difference between the synbiotic and placebo groups. In the placebo group, after 12 weeks of follow-up, an increase in the Bacteroidetes phylum, Oscillospira genus and Oscillospira guillermondi species was detected compared to the baseline period. In the synbiotic group, after 12 weeks of follow-up, an increase was detected in the Bacteroides phylum, Prevotella, Coprococcus genus and Prevotella copri, Coprococcus eutactus, Ruminococcus albus, Ruminococcus flavefacines species compared to the baseline period. In the synbiotic group, a decrease was detected in Lactobacillus and Erysiplerotrichhaceae_Clostridium genera and Lactobacillus ruminis, Clostridium ramosum, Eubacterium dolichum, Clostridium spiroforme and Bulleidia moorei species compared to the baseline period (Fig. 5).

LEfSe analysis of stool samples at baseline and 3 months in the synbiotic group. Horizontal bars represent the log 10 converted LDA score, indicated by vertical dotted lines. Treatment initiation (green) 3 months (red). p—phylum, c-class, o—order; f—family, g—genus, s—species

At the end of the 12th week of the study, when the synbiotic and placebo groups were compared, Bacteroides eggerthi species were dominant in the placebo group, while Collinsella stercoris species were dominant in the synbiotic group.