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 raw milk 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 6°C 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 from amplified cDNA, which was obtained from RNA directly isolated from raw-milk 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 6°C 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-stored milk, 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 the major 9 OTUs, that dominated the Pseudomonas genus in N2-flushed or non-flushed milk samples, other than an intriguing predominance of bacteria related to P. veronii. Overall, this study established that neither bacteria causing milk spoilage nor any well-known 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.

Fig 1. Time course analyses of the number of bacteria present in the three truckloads of bovine raw-milk samples (L1, L2, and L3), enumerated on PCA agar after 3 days incubation at 30°C under aerobic conditions: C (cold-stored milk at 6°C), N (milk flushed with N₂ gas while in cold storage at 6°C).

Error bars indicate standard deviations. The blue line corresponds to the 3x10⁵ (5.5 log-units) threshold value for raw milk acceptance for dairy processing.

Fig 2. Rarefaction curves of partial 16S rRNA transcript sequences after RNA extraction, cDNA synthesis and PCR amplification from three truckloads of milk samples divided into 15 subsamples as follows: L10, L20, L30 (initial samples), cold-stored at 6°C for 3 to 4 days (L1C4, L2C4, L3C3) for 6 to 7 days (L1C7, L2C7, L3C6); cold-stored at 6°C and N₂-flushed for 3 to 4 days (L1N4, L2N4, L3N3) for 6 to 7 days (L1N7, L2N7, L3N6).

Fig 3. Venn diagrams of Illumina sequence data comparing (A) initial and cold-stored raw-milk samples (B) initial and cold-stored N₂-flushed raw-milk samples.

OTUs shared between the conditions are indicated in boldface.

Fig 4. Composition of the bacterial communities based on 16S rRNA transcripts in initial raw milk, in cold-stored milk, and in cold-stored while N₂-flushed for either 3 or 4 and 6 or 7 days, based on partial 16S rRNA gene sequences after RNA extraction, cDNA synthesis and PCR amplification.

(a) Total phyla, (b) total classes, (c) families of Bacteroidetes, (d) genera of Bacteroidetes, (e) families of Bacilli, (f) genera of Bacilli, (g) families of Clostridia, (h) genera of Clostridia, (i) families of Gammaproteobacteria, (j) genera of Gammaproteobacteria.

Fig 5. Relative abundance of raw milk taxa that were significantly affected by cold storage at 6°C and by cold storage combined with N₂ gas flushing.

Error bars represent the standard deviation of mean (n = 3) values. Average values sharing a common letter are not significantly different with a level of significance of 0.05 over all comparisons. (u_: unclassified).