N2 gas flushing alleviates the loss of bacterial diversity and inhibits psychrotrophic pseudomonas during the cold storage of bovine raw milk

Abstract

The quality and safety of rawmilk still remains a worldwide challenge. Culture-dependent methods indicated that the continuous N2 gas-flushing of raw milk reduced the bacterial growth during cold storage by up to four orders of magnitude, compared to cold storage alone. This study investigated the influence of N2 gas-flushing on bacterial diversity in bovine raw-milk samples, that were either cold stored at 6C or additionally flushed with pure N2 for up to one week. Next-generation sequencing (NGS) of the V1-V2 hypervariable regions of 16S rRNA genes, derived fromamplified cDNA, which was obtained from RNA directly isolated from rawmilk samples, was performed. The reads, which were clustered into 2448 operational taxonomic units (OTUs), were phylogenetically classified. Our data revealed a drastic reduction in the diversity of OTUs in raw milk during cold storage at 6C at 97%similarity level; but, the N2-flushing treatment alleviated this reduction and substantially limited the loss of bacterial diversity during the same cold-storage period. Compared to cold-storedmilk, the initial raw-milk samples contained less Proteobacteria (mainly Pseudomonadaceae, Moraxellaceae and Enterobacteriaceae) but more Firmicutes (mainly Ruminococcaceaea, Lachnospiraceae and Oscillospiraceaea) and Bacteroidetes (mainly Bacteroidales). Significant differences between cold-stored and additionally N2-flushed milk were mainly related to higher levels of Pseudomononadaceae (including the genera Pseudomonas and Acinetobacter) in cold-stored milk samples; furthermore, rare taxa were better preserved by the N2 gas flushing compared to the cold storage alone. No major changes in bacterial composition with time were found regarding the distribution of themajor 9 OTUs, that dominated the Pseudomonas genus in N2-flushed or non-flushedmilk samples, other than an intriguing predominance of bacteria related to P. veronii. Overall, this study established that neither bacteria causingmilk spoilage nor any wellknown human pathogen or anaerobe benefited from the N2 gas flushing even though the N2-flushed and non-flushed cold-stored milk differed in bacterial counts by up to 104-fold.