Taxonomy, Sequence Variance and Functional Profiling of the Microbial Community of Long-Ripened Cheddar Cheese Using Shotgun Metagenomics

Abstract

Shotgun metagenomic sequencing was used to investigate the diversity of the microbial community of Cheddar cheese ripened over 32 months. The changes in taxa abundance were compared from assembly-based, non-assembly-based, and mOTUs2 sequencing pipelines to delineate the community profile for each age group. Metagenomic assembled genomes (MAGs) passing the quality threshold were obtained for 11 species from 58 samples. Although Lactococcus cremoris and Lacticaseibacillus paracasei were dominant across the shotgun samples, other species were identified using MG-RAST. NMDS analysis of the beta diversity of the microbial community revealed the similarity of the cheeses in older age groups (7 months to 32 months). As expected, the abundance of Lactococcus cremoris consistently decreased over ripening, while the proportion of permeable cells increased. Over the ripening period, the relative abundance of viable Lacticaseibacillus paracasei progressively increased, but at a variable rate among trials. Reads attributed to Siphoviridae and Ascomycota remained below 1% relative abundance. The functional profiles of PMA-treated cheeses differed from those of non-PMA-treated cheeses. Starter rotation was reflected in the single nucleotide variant profiles of Lactococcus cremoris (SNVs of this species using mOTUs2), while the incoming milk was the leading factor in discriminating Lacticaseibacillus paracasei/casei SNV profiles. The relative abundance estimates from Kraken2, non-assembly-based (MG-RAST) and marker gene clusters (mOTUs2) were consistent across age groups for the two dominant taxa. Metagenomics enabled sequence variant analysis below the bacterial species level and functional profiling that may affect the metabolic interactions between subpopulations in cheese during ripening, which could help explain the overall flavour development of cheese. Future work will integrate microbial variants with volatile profiles to associate the development of compounds related to cheese flavour at each ripening stage.

Metagenomic insight into taxonomic composition, environmental filtering and functional redundancy for shaping worldwide modern non-lithifying microbial mats

“… Taxonomic and functional annotations were done using the Lowest Common Ancestor algorithm ( Wood & Salzberg, 2014 ) utilizing the RefSeq and KO (Kyoto Encyclopedia of Genes and Genomes Orthology) as reference databases. To define true hits, we used thresholds similar to previous comparative short-read shotgun metagenomics studies but with a longer minimal alignment ( Koo et al, 2016 ; Rabelo-Fernandez et al, 2018 ; Angeli et al, 2019 ; Kibegwa et al, 2020 ; Akinola, Ayangbenro & Babalola, 2021 ; Mohamed et al, 2023 ); hits of >25 bases and >65% similarity to a taxonomic group or a function with an E-value of ≤10−5 were included in this study. …”