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No data analysis was performed for incidences of abdominal bloating and pain as minimal variance was reported in both groups. Moreover, this result may further strengthen the safety associated with LP N1115. Flatulence scores were similar between LP N1115 and placebo groups, albeit a trend toward significance was observed in LP N1115 group overtime. When analyzing the 6–18 months sub - group, a similar result was observed to that seen in the stool consis - tency data. Although not statistically significant, a large effect size was found and the pattern of data showed LP N1115 group remain - ing stable overtime, with the placebo group experiencing a spike in flatulence incidence by week 4, before decreasing to the same low level as baseline. Once again, these results should be interpreted cautiously considering both populations had low samples sizes. Consistent with other studies, the gut microbial consortia at 6–18 months consisted of Klebsiella , Veillonella , Citrob acter, Enterobacter , Enterococcus Clostridium, and Streptococcus, whereas subjects older than 18 months were colonized by taxa such as Dialister , Lachnospiraceae , Ruminococcaceae, and Christensenellaceae (Azad et al., 2014; Bäckhed et al., 2015; Dominguez-Bello et al., 2010; Fouhy et al., 2019; Hill et al., 2017). LP N1115 had no impact on the alpha- and beta-diversity measures overtime or in comparison to the placebo at each time point. Conversely, there was a strong separa - tion of the gut microbiota based on the age of the subjects when grouped as 6–18 and 18 + months. The only consistent change in relation to the gut microbiota overtime was a significant increase in Lactobacillus abundance at weeks 4 and 8 in LP N1115 group. This observation most likely reflects the transient colonization of LP N1115 within the gut microbiota of these subjects. Our results indi - cate that this increase in Lactobacillus is mainly due to the probiotic itself in the fecal samples. A similar result was also found in a study by Laursen et al. (2017), where two probiotics Bifidobacterium anima- lis subsp. lactis (BB-12 ® ) and Lactobacillus rhamnosus (LGG ® ) adminis - tered to infants aged 8–14 months for a period of 6 months did not significantly alter the gut microbial community and diversity com - pared to placebo, despite their detection in infant feces. Moreover, this further illustrates the importance of demonstrating efficacy of individual or multispecies probiotics in robust clinical trials. Short-chain fatty acids are end products produced from the fermentation of nondigestible dietary fibers and play fundamental roles in providing energy for colonocytes, acting as signaling mole - cules between the gut microbiota and host metabolism, and regulate immune function (den Besten et al., 2013). SCFAs such as acetic, propanoic, and butanoic acids were prevalent in both LP N1115 and placebo groups at baseline and postintervention (week 8). However, concentrations were subject to variation between the treatment groups and overtime. This may be partially explained by a number of factors. For example, information relating to the subjects' diet was not collected over the 8-week intervention, thus, we cannot correlate any dietary variables to the levels of SCFAs seen in both groups. It is likely that LP N1115 intake for 8 weeks was not suffi - cient to alter the SCFA profiles in these infants/children. It is also important to note cross-feeding interactions between preferential SCFA-producing bacteria may have led to an accumulation of various
products in vivo (Ríos-Covián et al., 2016). In addition, SCFA concen - trations were correlated with the most abundant fecal microbiota postintervention. SCFA-producing bacteria including Akkermansia and Alistipes were positively correlated with 2-methylpropanoic acid, pentanoic acid, and 3-methylbutanoic acid, whereas Veillonella , Bifidobacterium , Blautia , Lachnoclostridium, and other genera were negatively associated with these SCFAs. Recent longitudinal studies have provided strong evidence of an increase in fecal butyrate con - centration in infants between the ages of 3 and 12 months (Mueller et al., 2021; Nilsen et al., 2020). Interestingly, the former study ob - served a significant increase in butyrate production at 3 months but not at 12 months with respect to C-section delivery. In light of the above, this study has some limitations. Firstly, due to the exploratory nature of this study, sample size was limited and not statistically powered to conclude on any clinical health-related end points. Secondly, a washout period and a longer follow-up would have been useful to evaluate the differences between the treatment groups more closely. Thirdly, additional factors such as aspects of genetic influence, lifestyle, antibiotic usage, siblings, or household pets which may have an effect on gut microbiota were not studied. In addition, the preliminary findings of this study may warrant a larger clinical trial to elucidate the possible role of LP N1115 admin - istration with regards to a younger cohort. ACKNOWLEDGMENTS The study was carried out and finished by Atlantia Food Clinical Trials Ltd., APC Microbiome Institute, and Teagasc Food Research Centre. We wish to acknowledge the Teagasc Sequencing Facility, Dr. Fiona Crispie, Dr. Paul Cotter, and Ms. Laura Finnegan for their technical assistance with the 16S rRNA MiSeq sequencing, and Fiona Fouhy for assistance with the bioinformatics pipeline and generation of 16S raw data files. And we would like to extend our most grateful thanks to all the families who agreed to take part in the study.
CONFLICT OF INTEREST No conflict of interest stated from authors.
AUTHOR CONTRIBUTION Shijie Wang: Conceptualization (equal); Project administration (equal); Writing-original draft (equal). Yiping Xun: Conceptualization (equal); Writing-original draft (equal). Grace Ahern: Investigation (equal); Writing-original draft (equal). Lili Feng: Project adminis - tration (equal); Validation (equal). Dong Zhang: Software (equal); Visualization (equal). Yuling Xue: Project administration (equal); Validation (equal). Reynolds Paul Ross: Investigation (equal); Methodology (equal). Andrea Doolan: Project administration (equal). Catherine Stanton: Methodology (equal); Validation (equal). Hong Zhu: Conceptualization (equal); Project administration (equal). ETHICAL APPROVAL The study's protocols and procedures were ethically reviewed and approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals (CREC, Ireland), and written, informed consent
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