# Galacto-oligosaccharide preconditioning improves metabolic activity and engraftment of Limosilactobacillus reuteri and stimulates osteoblastogenesis ex vivo

## TL;DR

Preconditioning a probiotic with galacto-oligosaccharides boosts its activity and improves bone cell development in lab experiments.

## Contribution

This study introduces preconditioning as a novel method to enhance probiotic function and bone-related outcomes.

## Key findings

- Preconditioning L. reuteri with GOS increases short-chain fatty acid production and microbial engraftment.
- Preconditioned L. reuteri supernatants stimulate osteoblast differentiation and bone mineralization.
- GOS addition and preconditioning affect different scales but both improve ex vivo metabolic and osteogenic outcomes.

## Abstract

A probiotic-related benefit for the host is inherently linked to metabolic activity and integration in the gut ecosystem. To facilitate these, probiotics are often combined with specific prebiotics in a synbiotic formulation. Here, we propose an approach for improving probiotic metabolic activity and engraftment. By cultivating the probiotic strain in the presence of a specific prebiotic (preconditioning), the bacterial enzymatic machinery is geared towards prebiotic consumption. Today, it is not known if preconditioning constitutes an advantage for the synbiotic concept. Therefore, we assessed the effects galacto-oligosaccharide (GOS) addition and preconditioning on GOS of Limosilactobacillus reuteri DSM 17938 on ex vivo colonic metabolic profiles, microbial community dynamics, and osteoblastogenesis. We show that adding GOS and preconditioning L. reuteri DSM 17938 act on different scales, yet both increase ex vivo short-chain fatty acid (SCFA) production and engraftment within the microbial community. Furthermore, preconditioned supernatants or SCFA cocktails mirroring these profiles decrease the migration speed of MC3T3-E1 osteoblasts, increase several osteogenic differentiation markers, and stimulate bone mineralization. Thus, our results demonstrate that preconditioning of L. reuteri with GOS may represent an incremental advantage for synbiotics by optimizing metabolite production, microbial engraftment, microbiome profile, and increased osteoblastogenesis.