Fecal Metaproteomic Analysis Reveals Unique Changes of the Gut Microbiome Functions After Consumption of Sourdough Carasau Bread

Abstract

Sourdough-leavened bread (SB) is acknowledged for its great variety of valuable effects on consumer's metabolism and health, including a low glycemic index and a reduced content of the possible carcinogen acrylamide. Here, we aimed to investigate how these effects influence the gut microbiota composition and functions. Therefore, we subjected rats to a diet supplemented with SB, baker's yeast leavened bread (BB), or unsupplemented diet (chow), and, after 4 weeks of treatment, their gut microbiota was analyzed using a metaproteogenomic approach. As a result, diet supplementation with SB led to a reduction of specific members of the intestinal microbiota previously associated to low protein diets, namely Alistipes and Mucispirillum, or known as intestinal pathobionts, i.e., Mycoplasma. Concerning functions, asparaginases expressed by Bacteroides were observed as more abundant in SB-fed rats, leading to hypothesize that in their colonic microbiota the enzyme substrate, asparagine, was available in higher amounts than in BB- and chow-fed rats. Another group of protein families, expressed by Clostridium, was detected as more abundant in animal fed SB-supplemented diet. Of these, manganese catalase, small acid-soluble proteins (SASP), Ser/Thr kinase PrkA, and V-ATPase proteolipid subunit have been all reported to take part in Clostridium sporulation, strongly suggesting that the diet supplementation with SB might promote environmental conditions inducing metabolic dormancy of Clostridium spp. within the gut microbiota. In conclusion, our data describe the effects of SB consumption on the intestinal microbiota taxonomy and functions in rats. Moreover, our results suggest that a metaproteogenomic approach can provide evidence of the interplay between metabolites deriving from bread digestion and microbial metabolism.

Keywords: diet; food processes; gut microbiota; metagenomics; metaproteomics; sourdough.

Figures

Figure 1

Relative abundance of the genus Mycoplasma in stool and colon content samples. Each dot represents a different rat, with dots with same shape and color being referred to the same rat. For stool samples, both 16S rRNA gene sequencing (16S) and metaproteomic (MP) data are shown. BB, rats fed chow supplemented with baker's yeast leavened bread (light blue); SB, rats fed chow supplemented with sourdough leavened bread (orange); chow, rats fed chow only (green). Statistically significant differences between groups (according to edgeR test followed by SGoF adjustment) are indicated with asterisks (* = adjusted p-value < 0.05; ** = adjusted p-value < 0.01; *** = adjusted p-value < 0.00001).

Figure 2

Differential family-specific microbial functions in rats fed chow supplemented with bread leavened with baker's yeast (BB) vs. sourdough (SB). In each line, a dot represents a single animal, with its color intensity being proportional to the relative abundance of that given microbial protein in that subject, according to the scale depicted in the bottom-right corner. Missing values (function not identified in that animal) are in white; features with missing values in the most abundant group were filtered out. The upper part of the heatmap lists functions with higher abundance in the fecal microbiota of SB-fed animals, while the lower part lists those with higher abundance in the fecal microbiota of BB-fed animals. Functions are ordered based on the Cluster of Orthologous Groups (COG) category to which they belong (C, Energy production and conversion; E, Amino acid transport and metabolism; G, Carbohydrate transport and metabolism; J, Translation, ribosomal structure and biogenesis; M, Cell wall/membrane/envelop biogenesis; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism), and then in alphabetical order.

Figure 3

Differential family-specific microbial functions in rats fed chow supplemented with bread leavened with sourdough (SB) vs. chow only. In each line, a dot represents a single animal, with its color intensity being proportional to the relative abundance of that given microbial protein in that subject, according to the scale depicted in the bottom-right corner. Missing values (function not identified in that animal) are in white; features with missing values in the most abundant group were filtered out. The upper part of the heatmap lists functions with higher abundance in the fecal microbiota of SB-fed animals, while the lower part lists those with higher abundance in the fecal microbiota of chow-fed animals. Functions are ordered based on the Cluster of Orthologous Groups (COG) category to which they belong (C, Energy production and conversion; E, Amino acid transport and metabolism; G, Carbohydrate transport and metabolism; I, Lipid metabolism; J, Translation, ribosomal structure and biogenesis; M, Cell wall/membrane/envelop biogenesis; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; T, Signal transduction mechanisms; V, Defense mechanisms), and then in alphabetical order.