Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, it contains anti-nutritional compounds including phenolics (large and small molecular weight), which must be addressed to increase uptake by the industry. Microbial fermentation is currently used as a strategy to decrease larger molecular weight poly-phenolics, but results in the undesirable accumulation of small mono-phenolics. Here, we investigate cell-free biocatalytic reduction of phenolic content in faba bean ( Vicia faba L.) meal. A representative phenolic ring-breaking catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Initially, large-scale fermentative recombinant production and purification of BLC23O was carried out, with functionality validated by in vitro kinetic analysis. When applied to faba bean meal, BLC23O yielded greatest reductions in phenolic content in a coarse air classified fraction (high carbohydrate), compared to either a fine fraction (high protein) or the original unfractionated meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (e.g. tannins, or complexes with proteins and carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Consistent with this, when applied to a selection of commercially available purified phenolic compounds, known to occur in faba bean, BLC23O was found to have high activity against monophenolic acids and little if any detectable activity against larger molecular weight compounds. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry. Graphical Abstract