Fig 1.
Study design and analysis scheme.
(A) Study design. Group 1, A1/A2 milk consumption followed by A2 milk consumption (A1/A2 → A2); Group 2, A2 milk consumption followed by A1/A2 milk consumption (A2 → A1/A2). (B) Analysis scheme. Gut microbiome data obtained from fecal samples of 35 participants were analyzed as follows: first, a comparison before and after consumption of A1/A2 milk and A2 milk; second, a comparison between A1/A2 milk and A2 milk after consumption.
Fig 2.
Beta diversity of gut microbiota. Sample clustering by Generalized UniFrac-based PCoA at the species level.
(A) PCoA plot comparing samples before and after A1/A2 milk consumption. (B) PCoA plot comparing samples before and after A2 milk consumption. (C) PCoA plot comparing A2 milk and A1/A2 milk before consumption. (D) PCoA plot comparing A2 milk and A1/A2 milk after consumption. Significance for similarity of bacterial population clustering was analyzed by PERMANOVA. *, p < 0.05, n.s., no significance. The clustering of each group is marked with a different color: A1/A2 before, yellowish green ellipse; A1/A2 after, green ellipse; A2 before, pink ellipse; A2 after, red ellipse. PCoA, principal coordinates analysis; PERMANOMA, permutational multivariate analysis of variance.
Fig 3.
Alpha diversity of gut microbiota.
(A) Observed OTU count, (B) ACE, (C) Chao1, (D) Jackknife, (E) Shannon, (F) NPShannon, (G) Simpson, (H) Phylogenetic Diversity, (I) Good’s coverage of library. Statistical analysis for (A-I) was performed using a generalized linear model (GLM). No significant p-values for p_treatment (A2 treatment effect), p_period (period effect), and p_group (group effect) were observed, and thus, they are not displayed above the graphs.
Fig 4.
Distribution of gut microbiome before and after consumption of A1/A2 and A2 milk.
(A-C) Gut microbiota compositions at the phylum (A), family (B), and genus (C) levels. (D-H) Box plots for relative taxonomic abundance of Firmicutes (D), Bacteroidetes (E), Actinobacteria (F), Verrucomicrobia (G), and Proteobacteria (H). Statistical analysis for (D-H) was performed using a generalized linear model (GLM). Significant p-values for p_treatment (A2 treatment effect), p_period (period effect), and p_group (group effect) are displayed above the graphs. Only p_period effects were significant, and these are indicated in the figures.
Fig 5.
Identification of taxonomic biomarkers in the A2 milk group.
(A-B) Linear discriminant analysis (LDA) effect size (LEfSe) analysis before and after consumption of A2 milk (A), and after consumption of A2 milk compared to A1/A2 milk (B). (C-I) Box plots for relative taxonomic abundance of genus Bifidobacterium (C), species Bifidobacterium longum (D), genus Blautia (E), species Blautia wexlerae (F), genus Anaerostipes (G), species Anaerostipes hadrus group (H), and genus Dorea (I). Statistical analysis for (C-I) was performed using a generalized linear model (GLM). Significant p-values for p_treatment (A2 treatment effect), p_period (period effect), and p_group (group effect) are displayed above the graphs.
Fig 6.
Identification of functional biomarkers in the A2 milk group.
(A-B) Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis before and after consumption of A2 milk (A), and after consumption of A2 milk compared to A1/A2 milk (B). The p-values were calculated for comparison difference between independent two groups.
Fig 7.
Relationships and predicted functions of gut microbiota.
(A-B) Spearman’s correlation analysis between significant taxonomic and functional biomarkers before and after consumption of A2 milk (A), and after A2 milk consumption compared to A1/A2 milk consumption (B). Spearman’s rho values are indicated beneath each graph box. n.a., not assigned. The p-values were calculated for comparison difference between independent two groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 8.
Correlation between calprotectin and gut microbiota.
(A-C) Spearman’s correlation analysis between Calprotectin and Bifidobacterium longum group (A), Bifidobacterium genus (B), and Blautia genus (C). Spearman’s rho values and p-values are displayed above the graphs.