Animal casings as a source of microorganisms involved in meat fermentation: evidence from culture-based and high-throughput molecular methods

Natural casings are portions of animal intestines that have been used for centuries in the manufacturing of many typical dry-fermented sausages. Because of their intestinal origin, casings have a high microbial load, but their possible role as inoculants of microorganisms that can play a role in the ripening of dry-fermented sausages has not been investigated yet in detail. Here, we analyzed by means of culture-dependent methods and Illumina high- throughput sequencing of 16S rRNA amplicons the bacterial ecology of hog, cow and ovine casings at different stages of their preparation for sausages production. We also compared the bacterial ecology of casings, meat mixtures and sausages at different ripening stages in the production chains of Salame Mantovano, a typical Italian dry-fermented sausage. Culture-based methods relied on the isolation and characterization of strains on different typical media, while for the molecular methods multi-million reads were originated and analyzed after amplification and Illumina MiSeq sequencing of 16S rRNA amplicons. From the animal casings, several strains of Staphylococcus, Lactobacillus, Bifidobacterium, Vagococcus and Clostridium were counted in significant amounts, isolated and characterized at phylogenetic level. High-throughput sequencing analyses revealed a high bacterial diversity, which differed strongly between casings of different animal species. It was also found that the technological processes had a strong impact on the casings bacterial ecology, with a significant reduction of undesired microorganisms, and an increase in the proportion of lactobacilli and staphylococci. Finally, the analyses on the production chain of Salame Mantovano revealed that several strains found in the casings before stuffing were also detected in the final ripened products, thus confirming our main hypothesis. This work shows the importance of the use of natural casings in the manufacturing of typical dry-fermented products, and highlights the role of high-throughput sequencing technologies as powerful tools to gain a better comprehension of food fermentations.