Microbiome Taxonomic and Functional Differences in C3H/HeJ Mice Fed a Long-Term High-Fat Diet with Casein Protein ± Ammonium Hydroxide Supplementation

(1) Background: Acidogenic Western-style diets disrupt gut bacteria promoting obesity-related diseases. Here, we investigated whether long-term feeding of alkalinized dietary casein as a protein source (ammonium hydroxide enhancement, AHE) modulates microbiome structure/functions under high-fat conditions, and normal diets, and whether these responses are sex-dimorphic. (2) Methods: C3H/HeJ mice (N = 256; equal sex distribution) received either control casein (CC), AHE casein (CCN), high-fat casein (HFC), or AHE high-fat casein (HFCN) diets from 6 to 18 months. Body mass and survival were tracked; fecal samples collected at 16 months were sequenced and underwent shotgun metagenomics. (3) Results: Diet and sex jointly shaped host metrics. AHE diets taxonomically showed an abundance of Verrucomicrobiota phyla predominating in most cohorts, notably Akkermansia muciniphila. Within Pseudomonadota, Christensenella was identified, along with other taxa associated with beneficial health outcomes, including Lactococcus lactis, Lactococcus cremoris, Pediococcus acidilactici, and families Lachnospiraceae/Oscillospiraceae. Additionally, sex- and diet-dependent advantageous enriched functions associated with AHE that enhanced electron transport, B-vitamin cofactor pathways, and mucosal/redox support were observed. (4) Conclusions: In the long term, pH-directed protein chemistry is a tractable lever for gut ecology during high-fat feeding, enriching and promoting the balance of beneficial taxa, providing a mechanistic bridge between dietary acid load and microbiome remodeling.