# Elucidation of Bifidobacterium isolates from human milk and feces: investigating their anti-inflammatory effects on raw 264.7 via NF-κB signaling pathway

## TL;DR

This study identifies Bifidobacterium strains with strong anti-inflammatory properties, particularly B. longum AC15, which could help reduce inflammation via the NF-κB pathway.

## Contribution

The study introduces a comprehensive evaluation of Bifidobacterium isolates for anti-inflammatory effects, focusing on their survival, adhesion, and impact on macrophage inflammation.

## Key findings

- B. longum AC15 showed superior adhesion to enterocytes and acid/bile tolerance.
- Heat-inactivated Bifidobacterium reduced inflammation by suppressing NF-κB signaling in macrophages.
- Strains exhibited strong antimicrobial activity against both Gram-positive and Gram-negative bacteria.

## Abstract

This study aimed to identify potent strains from seven Bifidobacterium isolates by evaluating their ability to counteract inflammation triggered by Staphylococcus aureus-derived extracellular vesicles (SA-EVs). Characteristics such as tolerance to simulated gastric and intestinal juice, adhesion/competitive adhesion, antimicrobial activity, and anti-inflammatory effects were investigated. Generation time of tested isolates ranged from 112.27 to 135.24 min, except for Bifidobacterium adolescentis AC19 (64.56 min) and Bifidobacterium longum AC18 (208.20 min). Seven strains, except B. longum CICC6186, remained viable at pH 3.0 (maximal reduction from 108 to 107 CFU/mL), yet were unable to withstand 3.0 g/L bile salts for 3 h (reduced from 108 to 0 CFU/mL). They demonstrated good competitive adhesion ability against Staphylococcus aureus and Escherichia coli on Caco-2 cells with an average of 55.86% and 57.41% reduction of bacteria/cell, respectively, and showed strong antimicrobial ability against both Gram-positive and Gram-negative bacteria, possibly via production of acid and bacteriocins. Heat-inactivated Bifidobacterium (106 CFU/mL) inhibited the inflammation triggered by SA-EVs, most likely by suppressing RAW-BLUE™ proliferation into M1 and TNF-α secretion, in turn affecting the activation of the NF-κB signaling pathway. B. longum AC15 showed overall outstanding capabilities amongst all, especially the adhesion ability to enterocytes, tolerance to acid and bile salt, and anti-inflammatory ability on macrophages.

A scientific infographic outlining the comprehensive workflow for Bifidobacterium research. The process begins with the isolation and identification of strains from human breast milk and fecal samples. Subsequent panels detail the functional characterization of these strains, including their gastrointestinal survival, colonization capacity, antimicrobial efficacy, and SCFAs production. The final stages illustrate the isolation of extracellular vesicles and the evaluation of their anti-inflammatory properties through cell-based models and associated molecular signaling pathways.