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WANG et al .
to standardized Illumina protocols (Edgar, 2010). OTUs were aligned using PyNAST and taxonomy was assigned using BLAST against the SILVA SSURef database release v123.
LPN1115 group and 12 subjects in control group) and 18 months + subgroup ( n = 38, 20 subjects in LPN1115 group and 18 subjects in control group). No adverse events associated with the consumption of the tested LP N1115 were reported. Stool consistency was evaluated using the Bristol stool scale (BSS). Only stool frequency and flatulence data could be analyzed, while the data for pain and bloating were considered negligible as it were not possible to conduct meaningful data analysis for these vari - ables as variance. At the aspects of both stool consistency and flat - ulence, no significant differences were found between two groups ( p > .05), and no changes for the entire sample overtime ( p > .05). So were them in the 6–18 months subgroup ( p > .05) (Figure 2b,c). However, the pattern of stool data showed that the LP N1115 group moved from soft to normal and the control group remained at soft. And it also showed the LP N1115 group remained stable while the control group experienced a spike in flatulence incidence in weeks 3–4, before stabilizing to a normal level similar to weeks 0–1.
2.6 | SCFAs analysis
Fecal sample was diluted in 2 × wt/vol of sterile Dulbecco's Phosphate buffer saline. The filter was removed before storing the fecal water at −20 ℃ until gas chromatography mass spectrometry (GC-MS). Fecal waters were sent to MS-OMICS for targeted SCFAs analysis containing 10 different compounds (acetic acid, formic acid, propa - noic acid, 2-methylpropanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, 4-methylpentatoic acid, hexanoic acid, and heptanoic acid).
2.7 | Statistical analysis
3.2 | Salivary cortisol
The study was analyzed according to the intention to treat, which comprised all randomized subjects who have taken at least one dose of treatment. In case of missing data, excepting those related to the daily diary, last observation carried forward method was ap - plied to conduct the statistical analysis. Descriptive analyses and mixed analysis of variance models (ANOVAs) with Tukey post hoc analyses were conducted as appropriate for all primary and second - ary efficacy variables including flatulence, bloating, abdominal pain, SCFAs, and salivary cortisol. Where assumptions of mixed ANOVAs were breached, a mixed ANOVA using 20% trimmed means was performed. Fecal microbiota composition was analyzed using the web application MicrobiomeAnalyst (available at: http://www.micro biomeanalyst.ca) (Dhariwal et al., 2017), and raw OTU sequence counts were subjected to cumulative sum scaling normalization to account for the differences in sequencing depth across the samples. In addition, taxa with less than 0.01% relative abundance across all samples were excluded. All statistical analyses were calculated using SPSS v.24 and R Studio v.1.1.456. A p -value of ≤.05 was considered a significant difference.
Cortisol levels were assessed at baseline, week 4, and week 8 (Figure 3). Taking into account the whole populations, there were no changes in the entire sample overtime ( p > .05) and no significant difference between two groups ( p > .05). Likewise, there were no significant differences in the 6–18 months subgroup ( p > .05).
3.3 | Fecal microbiota composition
As microbial changes in the infant gut are subject to variation and maturation overtime, age was stratified according to 6–18 and > 18 months to illustrate the impact on microbial community and di - versity across the time points (weeks 0, 4, and 8). Upon examination of the fecal microbiota, it was clear that age-related differences have a strong influence on the separation of samples during the study. A principal coordinate analysis (PCoA) plot based on Bray–Curtis dis - similarity matrix at OTU level revealed a distinct separation between the samples (PERMANOVA R 2 = .07, p < .001) (Figure S1). When considering LP N1115 and placebo groups overtime, beta-diversity was measured based on the Bray–Curtis dissimilarity matrix at OTU level and no visible separation or clustering of samples was evident (PERMANOVA R 2 = .01, p = .998) (Figure 4a). There were significant effects between two groups across the time points ( p ≤ .01, p ≤ .01) by inverse Simpson and Chao-1 alpha-diversity measures (Figure 4b). A hierarchical clustering heatmap was used to investigate the relationships among age, treatment, and differentially abundant genera across all time points (Figure 5). The upper and mid sections on the right-hand side of the heatmap show greater abundances of Bifidobacterium , Klebsiella , Veillonella , Enterococcus , Rhodococcus , Actinomyces , Citrobacter , Blautia , and Clostridium_sensu_stricto_1 present in LP N1115 group and subjects aged 6–18 months. Bacteria such as these typically represent the core gut bacteria of C-section
3 | RESULTS
3.1 | Demographics and intestinal movements
Between October 2017 and March 2018, a total of 61 subjects were screened for eligibility, of which one subject was lost to follow-up and, therefore, not randomized. The remaining 60 subjects were randomly assigned to the LP N1115 group ( n = 30) and the control group ( n = 30), respectively (Figure 1). During the study, two sub - jects withdrew before completion of the trial due to vomiting and abdominal pain. Randomization was well balanced and the anthro - pometrics were presented (Figure 2a). Besides, two subgroups of age were divided to 6–18 months subgroup ( n = 22, 10 subjects in
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