Exploring the ecological functioning of an anticlostridial Mammaliicoccus sciuri strain in view of its application as a meat starter culture

Onderzoeksoutput: PhD Thesis

Samenvatting

The concept of meat fermentation can be traced back to ancient empirical practices that enabled the preservation of raw meat. Nowadays, fermented meat products are mostly produced on an industrial scale with the use of starter cultures but are nevertheless still marked by a large variety of different production practices and end-products. In most cases, curing salts are still an integral part of recipe formulations,as their use incurs important technological properties such as colour formation and microbiological stability. However, the use of these chemical preservatives has comeunder pressure as they are being linked with the formation of carcinogenic compounds and colorectal cancer. Therefore, a phasing-out urges itself within the industry, which is further accelerated by current clean-label trends. The present PhD study explored the potential of Gram-positive catalase-positive cocci (GCC) to replace the antibacte-rial activity that is attributed to nitrate and nitrite salts, thereby focusing on the food pathogen Clostridium botulinum. An assessment of their application potential as non-conventional starter cultures was performed in view of the production of fermented meat products without the addition of nitrate and/or nitrite.

To facilitate the selection of a bacterial strain with anticlostridial activity (i.e., inhibitoryactivity towards C. botulinum), a large collection of GCC strains, mainly isolated from meat, was build-up following authenticity approval with (GTG)5-PCR fingerprinting of genomic DNA and subsequent sequencing of the 16S rRNA, rpoB, and/or tuf genes. Screening of this strain collection via a deferred antagonism test revealed a low incidence of antibacterial activity among GCC. From the eighteen strains that showed antibacterial activity towards closely related species, only Mammaliicoccus sci-uriIMDO-S72 was able to inhibit group I (proteolytic) and group II (non-proteolytic) C. botulinum. Also, the occurrence of biogenic amine production was assessed among the GCC collection, through ultra-performance liquid chromatography coupled to tandem mass spectrometry. In general, tyramine andβ-phenylethylamine were the mostproduced biogenic amines. The production of these compounds was found to be a strain-dependent trait, except for the species Staphylococcus carnosus. While longer incubation times could allow for higher biogenic amine concentrations, no threat to food safety was found.

The anticlostridial M. sciuriIMDO-S72 strain was further characterized during in vitro single and co-culture fermentation processes, next to two other antibacterial strains namely Staphylococcus xylosus IMDO-S216 and Staphylococus equorumIMDO-S257. The antibacterial compound of M. sciuriIMDO-S72 followed secondary metabolite production kinetics and showed a bacteriostatic mode of action, while no antagonistic activity was found for S. xylosusIMDO-S216 and S. equorumIMDO-S257 during liquid culturing. The three strains did not express arginine deiminase activity, a trait which is considered to confer extra competitiveness within a fermented meat matrix. Absenceof biogenic amine production was again confirmed. Despite its strong antagonistic activity, M. sciuriIMDO-S72 failed to overrule S. xylosusIMDO-S216 in a co-culture set-up, unless given an initial competitive advantage.

The genome of M. sciuriIMDO-S72 was sequenced and de novo assembled using a combination of both short and long sequencing technologies. Annotation and genome mining revealed a plasmid-associated biosynthetic gene cluster (BGC) which was responsible for the antibacterial phenotype of M. sciuriIMDO-S72, the product of which was structurally identified as micrococcin P1. Using comparative (patho)genomics, a safety assessment of M. sciuriIMDO-S72 was performed. The strain was shown to have a low cytolytic potential, especially compared to pathogenic Staphylococus aureus strains, and mainly contained virulence factors that are involved in surface colonization. Similar genes are ubiquitously found among (non-pathogenic) staphylococcal species as well. While antibiotic resistance genes could be found within the genome of M. sciuriIMDO-S72, they were not located on mobile genetic elements (MGEs) and could not be linked with a resistant phenotype. Overall,M. sciuriIMDO-S72 was free of virulence and antibiotic resistance-associated MGEs, with the exception of a plasmid-encoded ica cluster, supporting its safety as a potential candidate starter.

To facilitate further insights into the metabolism of the selected anticlostridial strain and aiding to understand the ecological functioning of this strain/species, an RNA-seq workflow was successfully set up and optimized. This allowed a genome-wide differential gene expression analysis in combination with target metabolite analyses.An attempt to chart the respiratory chain of M. sciuriIMDO-S72 was undertaken, but many uncertainties remained based on inconsistent annotations and expression pro-files. Much clearer trends were observed for genes involved in nitrate-based respiration, being under the regulatory control of the oxygen-sensing two-component system NreBC. These genes were marked by a strong upregulation under anaerobic conditions. In the presence of oxygen, acetic acid and gluconic acid were the major metabolites produced by M. sciuriIMDO-S72 whereas a rapid switch to lactic acid production was found in response to decreasing oxygen levels. Amino acids represented important alternative carbon sources for M. sciuriIMDO-S72, the degradation products of which most likely fuelled the tricarboxylic acid cycle under aerobic conditions. While some gene expression profiles were in agreement with the metabolite profiles, this was not always the case, suggesting multiple levels of (post-transcriptional) regulation, putative misannotated genes, or the presence of yet unknown pathways. Map-ping of RNA-seq reads revealed erroneous gene annotations as well as unannotated genome regions that were marked by high read coverage. The generated RNA-seq datasets can therefore be further (re)analyzed and (re)used to fine-tune genome an-notations and detect unannotated features as well as to further explore the regulatory mechanisms behind the metabolism of M. sciuriIMDO-S72 in future research.

As a last part of this PhD study,M. sciuriIMDO-S72 was applied as starter culture in nitrate/nitrite-free fermented sausages to assess its in situ competitiveness, as well as its anticlostridial potential within a fermented meat matrix. Mammaliicoccus sciuriIMDO-S72 showed a limited proliferation potential within fermenting meat, especially under more pronounced acidification conditions and in the presence of Latilactobacillussakeiwithin the starter culture formulation. Using challenge tests with atoxigenic pro-teolytic C. botulinum strains, no additional anticlostridial effect by M. sciuriIMDO-S72was found. Despite the lack of such antagonistic activity,C. botulinum failed to pro-liferate within nitrate/nitrite-free fermented sausages even in a worst-case scenario in which no acidification occurred. These results question the anticlostridial role that is attributed to nitrate/nitrite. The fermented meat matrix might thus be restrictive enough to limit C. botulinum outgrowth, even in the absence of these preservatives.More research is however needed to substantiate these results, given the seriousness of botulism, and to exclude the in-situ production of toxins by this pathogen.

Overall, this PhD study provided insights into the applicability of GCC as bioprotective starter cultures for meat fermentation. It focused on the characterization of an anticlostridial M. sciuri strain, using omics-based analyses (i.e., genomics, transcriptomics, and metabolomics). It provided one of the first RNA-seq analyses of thisspecies and linked the limited competitiveness of the selected strain with its behaviour and metabolism under in vitro conditions.


Originele taal-2English
Toekennende instantie
  • Vrije Universiteit Brussel
Begeleider(s)/adviseur
  • Leroy, Frederic, Promotor
Datum van toekenning21 apr 2023
StatusPublished - 2023

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