Unveiling the culprit halotolerant tetragenococcus in multi-amplicon microbiome analysis of defective dry cured hams and pangenomic insights

Abstract

The production of dry-cured ham, with roots tracing back to ancient recipes, grapples with persistent technological challenges, resulting in defects and financial setbacks. Traditionally, the identification of microbial defects relies on the "probe and sniff technique" conducted approximately between the 10th and 12th month of aging. These potential defects, broadly classified into deep spoilage and surface defects, pose a significant hurdle for the ham industry.In this comprehensive study, we sampled and analyzed three defective dry-cured hams, exhibiting noticeable defects, and three normal hams carefully chosen from a producer in Andalusia, Spain. The objective was to scrutinize the microbiome of these samples to understand whether the observed defects were associated with shifts in the microbial population.16s multi-amplicon sequencing was performed using the Ion 16S Metagenomics Kit and Ion Torrent Sequencing Platform. Raw reads were then analysed using QIIME2 for data processing. Alpha-diversity and beta-diversity indexes, ANCOM test, Linear Discriminant Analysis (LEfSe) and co-occurrence network analysis were computed and visualized using the “microeco” R package.A pangenomic analysis and functional enrichment analysis of genomes available in the NCBI database was conducted.The analysis unveiled a distinctive association between Tetragenococcus, a genus recognized for its prevalence in fermented fish and vegetable products, as well as in cheese microbiota, and defective dry-cured hams. Both the LEfSe and ANCOM tests concurred in highlighting this taxon as a potential culprit.Subsequent scrutiny of Tetragenococcus genomes highlighted disparities between the two species T. koreensis and T. halophilus in terms of metabolic activity. Notably, T. koreensis manifested a distinctive set of genes encoding enzymes for carbohydrate metabolism. This study not only provided the opportunity to address the producer´s inquiry regarding the potential role of a specific microbial population in causing the defect, but also indicated the likelihood of a halotolerant microorganism being the culprit.While previous research has linked halotolerant microorganisms to defects in dry-cured meat, this represents the first time Tetragenococcus has been linked to such cases. This result sheds light on Tetragenococcus´ involvement in defected dry-cured hams.Ongoing investigations will focus on identifying the specific Tetragenococcus strains responsible for these defects, helping to refine our knowledge of their impact on dry-cured ham production.