Brevibacterium from Austrian hard cheese harbor a putative histamine catabolism pathway and a plasmid for adaptation to the cheese environment

Justin M Anast,Martin Wagner,Stephan Schmitz-Esser,Monika Dzieciol,Dylan L Schultz,Evelyne Mann

doi:10.1038/s41598-019-42525-y

Justin M Anast, Martin Wagner + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-42525-y

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Abstract

The genus Brevibacterium harbors many members important for cheese ripening. We performed real-time quantitative PCR (qPCR) to determine the abundance of Brevibacterium on rinds of Vorarlberger Bergkäse, an Austrian artisanal washed-rind hard cheese, over 160 days of ripening. Our results show that Brevibacterium are abundant on Vorarlberger Bergkäse rinds throughout the ripening time. To elucidate the impact of Brevibacterium on cheese production, we analysed the genomes of three cheese rind isolates, L261, S111, and S22. L261 belongs to Brevibacterium aurantiacum, whereas S111 and S22 represent novel species within the genus Brevibacterium based on 16S rRNA gene similarity and average nucleotide identity. Our comparative genomic analysis showed that important cheese ripening enzymes are conserved among the genus Brevibacterium. Strain S22 harbors a 22 kb circular plasmid which encodes putative iron and hydroxymethylpyrimidine/thiamine transporters. Histamine formation in fermented foods can cause histamine intoxication. We revealed the presence of a putative metabolic pathway for histamine degradation. Growth experiments showed that the three Brevibacterium strains can utilize histamine as the sole carbon source. The capability to utilize histamine, possibly encoded by the putative histamine degradation pathway, highlights the importance of Brevibacterium as key cheese ripening cultures beyond their contribution to cheese flavor production.

Highlights

Production of cheese has been documented in many different world cultures dating back more than 7500 years[1,2]
Brevibacterium is abundant on Vorarlberger Bergkäse (VB) cheese rinds already at the first day of production; this suggests that Brevibacterium might originate in high numbers from the raw milk used for production of VB
S22 showed less than 98% identity to Brevibacterium Operational Taxonomic Units (OTUs) from[42] and 99.8% identity to OTU18 from[6]. These results suggest that different Brevibacterium strains or species can be found in the VB cheese rind communities and in the VB production environment for over a time period of several years, it should be noted that, based on 16S rRNA similarity alone, a differentiation of strains is not possible; for this, approaches with higher taxonomic resolution such as Pulsed-Field Gel Electrophoresis (PFGE) or Multi Locus Sequence Typing (MLST) would be needed

Summary

Introduction

Production of cheese has been documented in many different world cultures dating back more than 7500 years[1,2]. In long-ripened cheeses, the microorganisms on the cheese surface contribute significantly to flavor production. The second study analyzed 23 Brevibacterium isolate genomes, 12 of which were isolated from cheese, and found many putative genes involved in iron acquisition and bacteriocin production. This study aimed to characterize the abundance of Brevibacterium on VB cheese rinds using qPCR and to analyze the contribution of Brevibacterium to cheese ripening based on draft genome sequences for three Brevibacterium isolates from VB: L261, S22, and S111. Their genetic potential in regards to cheese ripening was compared to other cheese-associated Brevibacterium strains. We hypothesize that the VB Brevibacterium isolates are habituated for the competitive environment of the cheese rind throughout the ripening time and contribute to the texture, color, and aroma of VB

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