Microbiome-based analytical approaches and biopreservation agents for improving meat quality and safety

Resumo

Meat and meat processing environments can host a wide range of microorganisms. They can be found all along the processing lines, in raw materials, final products, food contact and non-food contact environments. When added as starter cultures, they are beneficial as they carry out important functions, for example leading fermentation processes or competing against harmful microorganisms. On the contrary, others are spoilers or pathogens, being detrimental for the quality and safety of meat products. Generally, the study of microorganisms in the food chain has been approached through classical culture-dependent methodologies, although in the last years new developments and improvements have led to the increased implementation of more comprehensive methodologies providing information on whole food microbiomes. High-throughput sequencing (HTS) technologies offer several advantages over culture-dependent methodologies. However, they are still underutilised in general in the food industry, and particularly in meat production systems. The objective of the first three experimental chapters of this PhD Thesis was to develop HTS methods and protocols to study the composition and function of the microbiomes of meat and associated processing environments. Briefly, Chapter I describes a procedure to study through whole metagenome sequencing the resident microbiome of meat processing environments. It covers the sampling strategy in the industry and the approach for extraction of total metagenomic DNA, with an improved protocol to increase the yield considering the low microbial biomasses that can be found in processing plants after cleaning and disinfection. The protocol can yield DNA loads >10 ng in >98% of samples and allows the collection of, on average, 12.2 metagenome assembled genomes (MAG) per sample, which markedly improves other available alternatives. Chapter II presents and discusses the results obtained during the validation of the protocol described in chapter I for mapping the microbial communities prevailing specifically in meat, meat products, and associated processing environments of 19 meat processing facilities. It shows that bacterial species belonging to the genera Pseudomonas, Staphylococcus, Brochothrix, Acinetobacter and Psychrobacter dominated across sample types, with Pseudomonas fragi and different Psychrobacter species being found as the most abundant core species in processing environments. It also highlights the occurrence of a high diversity of putative new species in meat processing environments, demonstrating an important microbial diversity still to be unravelled. Chapter III is focused on the development of a novel approach for the study of class I integrons, based on their amplification and sequencing using long-read nanopore technology. Its use for the characterization of the resistome associated with these genetic elements in meat, meat products, and associated processing environments allowed the resolution in single reads of class I integrons containing up to 7 different antimicrobial resistance genes, mainly linked to resistance to aminoglycosides, betalactams and folate pathway antagonists. On the basis that meat can be the source of some foodborne pathogens associated with public health and food safety risks, there is an urging need to validate novel effective strategies of preservation and pathogen control in the meat industry. In addition, the current trend towards naturally produced foods is promoting the search for new alternatives to traditional preservative additives. Lactic acid bacteria (LAB) are good candidates as food biopreservative agents, due to their generally safety qualification and their promising antimicrobial effect against foodborne pathogens. The objective of the last three chapters of the PhD Thesis was to design and validate biopreservation strategies based on the use of LAB to improve the quality and safety of meat products. Chapter IV presents a study evaluating the combination of a commercial starter culture with High-Pressure Processing (HPP) to control the growth of Listeria monocytogenes and Salmonella Typhimurium in a dry fermented sausage (chorizo de León). The application of HPP in the early stages of ripening, alone or in combination with a starter culture containing Pediococcus acidilactici, Latilactobacillus sakei and Staphylococcus carnosus, achieved a successful control of Listeria monocytogenes and S. Typhimurium, with log reductions of up to 4.8 and 2.4, respectively, and no adverse effects regarding physico-chemical or sensory quality attributes. Chapter V describes the screening and selection of three LAB strains (Lactococcus lactis, Lacticaseibacillus paracasei and Lactiplantibacillus plantarum), based on their aptitude to be employed as antilisterial agents in different meat products, and their further assessment in cooked ham in combination with vacuum packaging, modified atmosphere packaging and HPP. Finally, Chapter VI is focused on the study of the performance of the previously selected LAB strains to control L. monocytogenes in a marinated pork product. The detailed challenge tests undertaken with the cocktail of the three LAB strains in ready-to-eat cooked meat products and marinated pork showed that the LAB cocktail was able to adapt to the food matrix and dominate over the background microbiota during storage, demonstrating control over L. monocytogenes and some spoilage-associated taxa such as Brochothrix, without noticeable adverse effects on the physico-chemical or sensorial characteristics of the products. Altogether, the six experimental chapters included in this thesis contributes to provide solutions to obtain safer meat products through combined mitigation strategies, using next generation sequencing methodologies as an innovative tool to study the microbiome of meat products and associated processing environments, and applying biopreservative agents capable of modulating the microbiome of selected meat products.