Impacts of Age, Snack Food, and Whole and Refined Wheat on the Rat Fecal Microbiome
Authors: Cotton Christopher , Westerman Kenneth , Van Roosebeke Delphine , Kane Anne V , Tai Albert , Blumberg Jeffrey B , Chen C‐Y. Oliver , Obin Martin S 
Published: June 2021

Early exposures that promote gut microbiome dysbiosis are associated with childhood and adult disease. The ability of whole (vs refined) grains to enhance gut microbiome composition and function and host immunometabolic health remains controversial. We assessed the impact of whole vs refined wheat on gut microbiome maturation in Sprague Dawley rats fed a snack food‐based diet.Individually‐housed male Sprague Dawley rats were weaned at 4 weeks onto either: 1) AIN‐93G (control), a standard casein‐, cornstarch‐ and soy oil‐based diet; 2, 3) AIN‐93G containing 18% (wt) refined wheat (RW) rusk or whole wheat (WW) rusk; 4,5) A 65% snack (S) mixture of chips, crackers, candy, etc. supplemented with either 18% refined wheat (SRW) or whole wheat (SWW) rusk. Diets were balanced to match protein and micronutrient content. Oral glucose tolerance tests were performed at 20 weeks. Fecal samples were collected at weaning and after 24 weeks and frozen. DNA extraction, V4 region amplification of 16S rRNA, 250 bp paired‐ end sequencing and downstream processing were performed as described (PLoS One. 2015 Aug 18;10(8):e0135758.) Closed reference OTUs (99% threshold), OTU tables, heat‐maps, diversity measures and phylogenetic distances were obtained (QIIMEv1.8 and R). PCoA and jackknifed UPGMA clustering were performed. Defining OTUs were identified with LDA (Linear Discriminant Analysis) Effect Size (Lefse) and OTU relationship to glucose (in)tolerance were assessed by Multivariate Association with Linear Models (MaAsLin).210 samples generated 12.2 million quality reads and 738 OTU designations. Alpha diversity (Shannon) was greater at 24 weeks (p = 0.04), coincident with decreased Bacteroidetes and increased Firmicutes across all diets. 4 and 24 week samples were separable by PCoA and UPGMA (p < 1010). Distinguishing OTUs at 4 weeks included B. fragilis, A. muciniphila, P. gordoni and C. perfringens; mature microbiomes were distinguished by Coprococcus, Allobaculum and the Bacteroidales lineage S24‐7. Alpha diversity was comparable among the 5 diets and C, WW and RW diets were taxonomically indistinguishable. SWW or SRW microbiomes were distinct from C, WW and RW microbiomes (UPGMA, p< 107), coincident with impaired glucose tolerance in snack‐based vs wheat‐supplemented groups (p<0.05). Defining OTUs of snack‐based diets were S24‐7, C. areofaciens, and Oscillospora, Lachnospira, Clostridium and Holdemania; non‐snack diets were distinguished by A. muciniphila, C. eutactus, L. salivarius and Adlercreutzia. Microbiomes of SWW and SRW diets were indistinguishable.Maturation per se most significantly impacted the adult fecal microbiome in this study. A diet high in sucrose and fat and lower in fiber (S) was associated with dysbiosis and impaired glucose tolerance. Supplementation of this diet with whole (WW) or refined (RW) wheat protected glucose tolerance but did not detectably alter fecal microbial community structure.Support or Funding InformationSupported by Mondelez International, Inc and USDA ARS Cooperative Agreement no. 58‐1950‐7‐707.