Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.
BACKGROUND
Quenching in cold buffered methanol at -40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline.


RESULTS
Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in Lactobacillus paracasei subsp. paracasei (L. paracasei). Membrane integrity assayed by propidium iodide revealed that approximately 100 % [Corrected] of the L. paracasei cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the L. paracasei culture, fast filtration took several minutes, which is far from ideal for metabolites with high intracellular turnover rates.


CONCLUSIONS
The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.
Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. dkkba@chr-hansen.com.|Chr. Hansen A/S, Hoersholm, Denmark. dkkba@chr-hansen.com.
Kristina B
KB
Jäpelt
Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. jch@plen.ku.dk.
Jan H
JH
Christensen
The Metabolomics Lab, School of Biological Sciences, University of Auckland, Auckland, New Zealand. s.villas-boas@auckland.ac.nz.
Silas G
SG
Villas-Bôas
D005990:Glycerol / Q000494:pharmacology; D007778:Lactobacillus / Q000378:metabolism*; D055432:Metabolomics / Q000379:methods*; D000432:Methanol / Q000494:pharmacology; D015203:Reproducibility of Results
D005990:Glycerol; D000432:Methanol