What question did this study set out to answer?

The research aims to understand how high-fat diets and ammonium hydroxide-enhanced proteins affect gut microbiomes in C3H/HeJ mice.

February 28, 2026Dietetics0 citationsOpen Access

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

BMBrayan Montoya-Torres AKAmandeep Kaur BBBenjamin Barr

Key Points

The research aims to understand how high-fat diets and ammonium hydroxide-enhanced proteins affect gut microbiomes in C3H/HeJ mice.
C3H/HeJ mice were fed four different diets over 12 months.
Diets included control casein, AHE casein, high-fat casein, and AHE high-fat casein.
Body mass and survival were assessed, and fecal samples were collected and sequenced.
AHE diets showed increased abundance of beneficial Verrucomicrobiota, particularly Akkermansia muciniphila.
Identified beneficial taxa included Lactococcus and Pediococcus species.
Diet and sex influenced health outcomes, highlighting the role of protein chemistry in gut ecology.

Abstract

(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.

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