# Development of novel microbial synthetic consortia for the production of antimicrobial fermentates containing caproate

## TL;DR

This study designed a microbial consortium to produce caproate, a natural antimicrobial, which showed strong activity against bacteria, yeast, and mold in food applications.

## Contribution

A novel multi-kingdom microbial consortium was developed to produce caproate-containing fermentates with antimicrobial properties.

## Key findings

- Caproate levels reached 63.9 mM in a 22-day bioprocess with ethanol addition.
- Fermentates inhibited bacteria, yeast, and mold growth, especially at pH 4.5.
- Lactate and caproate acted synergistically in minced meat to enhance antimicrobial activity.

## Abstract

Short- and medium chain carboxylic acids (SCCA/MCCA) are natural antimicrobials produced by fermentation and chain elongation, but currently only a few SCCA/MCCA are used in food-related applications. With the aim to diversify the SCCA/MCCA profile of fermentates for biopreservation, we designed bioprocesses employing bacterial or multi-kingdom consortia to produce caproate-containing fermentates using a targeted cross-feeding strategy. We combined Limosilactobacillus reuteri, Clostridium kluyveri, and Saccharomyces cerevisiae, and quantified substrates utilization and metabolites. The antimicrobial activity of SCCA/MCCA and fermentates was analysed in vitro and in a meat model system. In a first bioprocess, the addition of ethanol (EtOH) initiated caproate formation by the bacterial consortium with levels of 63.9 mM (7.4 g L−1) after 22 days. Next, we run two shorter bioprocesses (12 days) and more caproate (28.9 mM, 3.4 g L−1) was produced if EtOH was regularly added than without EtOH supplementation (21.7 mM, 2.5 g L−1). When S. cerevisiae was included, 37.9 ± 11.4 mM (4.4 g L−1) caproate was formed without EtOH addition. Beyond the intended cross-feeding activity, consortia produced and re-metabolised mannitol and glycerol. Lactate and caproate were the major carboxylic acids in fermentates. Caproate inhibited bacteria, yeast and molds at pH 4.5 and 6.5 in vitro, while lactate and caproate acted synergistically in minced meat. Fermentates conferred antimicrobial activity against indicator microbes mostly at pH 4.5 and to a lesser extent at pH 6.5. In this study we successfully designed a consortium to produce caproate. We provide evidence that caproate-containing fermentates had strong antimicrobial activity, and our results indicate the complexity of metabolic interactions that occurred within consortium members.

• Design of a self-contained bioprocess with multi-kingdom consortium.

• Cross-feeding of bacteria and yeast to produce caproate fermentates.

• Fermentates containing caproate inhibited bacteria, yeast and mold growth.

The online version contains supplementary material available at 10.1007/s00253-026-13718-z.