A Milk Foodomics Investigation into the Effect of Pseudomonas fluorescens Growth under Cold Chain Conditions

Abstract

Pseudomonas fluorescens is a psychrotrophic species associated with milk spoilage because of its lipolytic and proteolytic activities. Consequently, monitoring P. fluorescens or its antecedent activity in milk is critical to preventing quality defects of the product and minimizing food waste. Therefore, in this study, untargeted metabolomics and peptidomics were used to identify the changes in milk related to P. fluorescens activity by simulating the low-temperature conditions usually found in milk during the cold chain. Both unsupervised and supervised multivariate statistical approaches showed a clear effect caused by the P. fluorescens inoculation on milk samples. Our results showed that the levels of phosphatidylglycerophosphates and glycerophospholipids were directly related to the level of contamination. In addition, our metabolomic approach allowed us to detect lipid and protein degradation products that were directly correlated with the degradative metabolism of P. fluorescens. Peptidomics corroborated the proteolytic propensity of P. fluorescens-contaminated milk, but with lower sensitivity. The results obtained from this study provide insights into the alterations related to P. fluorescens 39 contamination, both pre and post heat treatment. This approach could represent a potential tool to retrospectively understand the actual quality of milk under cold chain storage conditions, either before or after heat treatments.

Keywords: foodomics; milk contamination; milk quality; molecular marker; spoilage; untargeted profiling.

Figure 1

Heatmap-based unsupervised hierarchical cluster analysis (HCA—similarity index: Euclidean; dendrogram linkage method: Ward) of all compounds in the dataset of the non-heat-treated (A) and heat-treated (B) milk samples. Abbreviations: T1CN = non-inoculated sample incubated for 24 h; T1PF = inoculated sample incubated for 24 h; T6CN = non-inoculated sample incubated for 144 h; T6PF = inoculated sample incubated for 144 h.

Figure 2

OPLS-DA score plots were generated by considering the metabolomic profile of the non-heat-treated (A) and heat-treated (B) milk samples. Abbreviations: T1CN = non-inoculated sample incubated for 24 h; T1PF = inoculated sample incubated for 24 h; T6CN = non-inoculated sample incubated for 144 h; T6PF = inoculated sample incubated for 144 h.

Figure 3

Non-heat-treated sample group; (A) Venn analysis built by intersecting compounds of inoculated samples incubated at 24 (blue) and 144 (yellow), using up-regulated metabolites (FC >2) compared to non-inoculated samples incubated 24 and 144 h. (B) Volcano plot analysis (p-value < 0.05 and FC > 2) on 570 common VIP marker compounds identified at both 24 h and 144 h time points. (C) Chemical enrichment analysis generated from the significant compounds: T1 = significant chemical class for inoculated samples at 24 h; T6 = significant chemical class for inoculated samples at 144 h.