New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data

Abstract

Cheese microbiota and metabolites and their inter-relationships that underpin specific cheese quality attributes remain poorly understood. Here we report that multi-omics and integrative data analysis (multiple co-inertia analysis, MciA) can be used to gain deeper insights into these relationships and identify microbiota and metabolite fingerprints that could be used to monitor product quality and authenticity. Our study into different brands of artisanal and industrial cheddar cheeses showed that Streptococcus, Lactococcus and Lactobacillus were the dominant taxa with overall microbial community structures differing not only between industrial and artisanal cheeses but also among different cheese brands. Metabolome analysis also revealed qualitative and semi-quantitative differences in metabolites between different cheeses. This also included the presence of two compounds (3-hydroxy propanoic acid and o-methoxycatechol-o-sulphate) in artisanal cheese that have not been previously reported in any type of cheese. integrative analysis of multi-omics datasets revealed that highly similar cheeses, identical in age and appearance, could be distinctively clustered according to cheese type and brand. furthermore, the analysis detected strong relationships, some previously unknown, which existed between the cheese microbiota and metabolome, and uncovered specific taxa and metabolites that contributed to these relationships. these results highlight the potential of this approach for identifying product specific microbe/metabolite signatures that could be used to monitor and control cheese quality and product authenticity.Cheese microbiota play a pivotal role in the development of cheese flavor and the product quality and safety of cheese. These attributes can be achieved by tightly controlled manufacturing conditions, excellent hygiene and use of commercial starter cultures (usually strains of Lactococcus lactis). Following manufacture, when starter bacteria cease to dominate, cheese must be ripened for full flavour development. This is a highly complex process, with successions within microbial communities, and their associated enzymes and biochemical reactions occurring over time to release numerous flavoursome organic compounds 1 . Ripening can be controlled to some extent by the addition of known adjunct cultures, usually strains of lactobacilli, but adventitious bacteria from the factory environment also contribute to the ripening process and to the composition of the cheese microbiota 2 .Advances in DNA sequencing using DNA extracted directly from samples have enabled improved understanding of the microbial communities found in cheese, and especially of less-abundant microorganisms which can nevertheless have significant impacts on cheese flavour and safety 3,4 . Although microbial communities vary depending on the cheese type, in hard cheeses such as cheddar that undergo ripening, after the starter bacteria die off lactobacilli are dominant, and may be found in combination with a number of other genera not belongin...

Microbiota and Metabolite Profiling Combined With Integrative Analysis for Differentiating Cheeses of Varying Ripening Ages

“…has demonstrated that strong and significant associations exist between cheese microbiota and metabolites. To our knowledge, some of these associations have not been previously described, such as the positive association between the levels of phenylalanine and the presence of Thermus, while others were consistent with the known biochemical characteristics of bacterial species, such as the association between urease-positive S. thermophilus and decreased levels of urea (Afshari et al, 2020).…”

“…Total DNA was extracted from 200 mg of each cheese sample using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, United States) following the manufacturer's instructions. DNA purity and concentration were determined and PCR amplification and DNA sequencing of bacterial 16S rRNA genes were performed as described previously (Afshari et al, 2020). Briefly, the V4 region of DNA was amplified using primers 515F and 806R.…”

“…Raw Illumina fastq files were demultiplexed, quality filtered, and analyzed using GHAP v2.1 (Greenfield Hybrid Amplicon Pipeline, developed by Paul Greenfield at CSIRO, Canberra, ACT, Australia) as described previously (Afshari et al, 2020). The relative abundance of each taxon in each sample was determined using the vegan package Rv.3.4.3.…”

“…Freeze-dried cheese (60 mg) was extracted as described by Afshari et al (2020). Briefly, 60 mg of cheese was extracted in 500 µL of MeOH/H 2 O/CHCl 3 (2.5:1:1, v:v:v).…”

“…Following extraction, 40 µL aliquots were transferred into glass vial inserts and dried in vacuo for subsequent trimethylsilyl (TMS) polar metabolite derivatization. Online, chemical derivatization and acquisition were performed as previously described by Afshari et al (2020). This method specifically enables the extraction of non-volatile compounds.…”