In vitro–in vivo Validation of Stimulatory Effect of Oat In…

Pathogens 2021 , 10 , 235

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that OTU. To identify related bacterial species, the obtained sequences were matched using the SeqMatch tool in the Ribosomal Database Project software (Michigan State University, East Lansing, MI, USA) [64]. Results were presented as proportional values versus the total amount of sequences within each sample. Combining the proportional values of the 16S-targeted Illumina together with the enumeration of the total cell count obtained through flow cytometry, as was previously described by Vandeputte et al. [65], provided quantitative abundances of the different taxonomic entities inside the colonic reactors. These quantitative abundances were obtained by multiplying the proportional values of the 16S-targeted Illumina with the total log cell count obtained by flow cytometry. With respect to microbial parameters during the in vitro SHIME ® study, consistent findings were made for the three different donors in response to the treatment with the different oat products so that the averages of all parameters over the three donors were cal- culated per colon compartment for optimal visualisation of donor-independent treatment effects. Statistical analyses were carried out using Statistical Analysis Software version 9.4 (SAS Institute, Cary, NC, USA). Data were analysed by comparing the averages over the three donors for the control period as compared to the treatment period, and for the differences between the test product during the treatment period and this for both PC and DC separately, using Bonferroni correction. For 16S-targeted Illumina sequence data, statistical analysis was only performed for those bacterial families with complete data from at least two donors to capture both the noise within each donor and variability between donors. The significance level for all statistical tests was set at α = 0.05. Shapiro–Wilk tests were conducted to assess the normality of the data. Log-transformation was applied where appropriate. For the in vivo study, outcomes were analysed on the per-protocol population (PP), i.e., all subjects with complete data and at least 80% compliance in terms of the amount of product consumed. Statistical analysis was performed using the SPSS Statistics software (IBM, Armonk, NY, USA), version 25. Normality of data and equality of the variances were determined with a Shapiro–Wilk test and a Levene’s test, respectively. For normally distributed data with equal variances, a one-way ANOVA with a Bonferroni post hoc test was used. For normally distributed data with unequal variances, a Welch test with a Games–Howell post hoc test was conducted. For non-normally distributed data, a Kruskal– Wallis one-way ANOVA test with multiple post hoc pairwise comparisons were performed. In terms of statistics, the differences indicated by ‘ p < 0.05’ were significant. All data were normalised to the DNA concentration of the extracts obtained from the faecal samples. Supplementary Materials: The following are available online at 7/10/2/235/s1, Figure S1: Cross-over study design. Author Contributions: Conceptualisation, P.V.d.A., M.M. (Massimo Marzorati), P.D.C., S.A.J., A.K., Y.C., and L.F.; methodology, P.V.d.A., P.D.C., and S.A.J.; formal analysis, C.D., W.B., O.O., P.D.C., K.Q., S.A.J., M.M. (Mike Murphy), G.D., and T.G.D.; investigation, C.D., W.B., O.O., P.D.C., S.A.J., M.M. (Mike Murphy), G.D., and T.G.D.; data curation, C.D., P.J.D.C., Y.C., W.B., O.O., P.D.C., S.A.J., M.M. (Mike Murphy), G.D., and T.G.D.; writing—original draft preparation, C.D. and P.V.d.A.; writing—review and editing, C.D., P.V.d.A., A.K., L.F., P.D.C., S.A.J., P.J.D.C., and G.D.; supervision, P.V.d.A., M.M. (Massimo Marzorati), and Y.C.; project administration, M.M. (Massimo Marzorati), A.K., and T.G.D.; funding acquisition, A.K., Y.C., and L.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of University College Cork, Ireland (study code AFCRO-083, approved on and 17/01/2018). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data available on request due to restrictions.

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