Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy

Abstract

BackgroundQuenching 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.ResultsOur 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 10 % 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.ConclusionThe 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.

Bacterial Metabolomics: Sample Preparation Methods

“…Villas-Boas and Bruheim [ 85 ] reported that cold glycerol-saline (3 : 2) showed much higher intracellular metabolite recovery in Pseudomonas fluorescens compared to cold methanol. A similar finding showed that cold glycerol-saline only caused minimal cell damage towards Lactobacillus paracasei [ 84 ]. Chen et al [ 91 ] demonstrated that the use of methanol/glycerol (−20°C) successfully recovered and identified a high concentration of intracellular metabolites in Lactobacillus bulgaricus .…”

“…Studies have reported that quenching process can be performed before, during, or after harvesting of sample [ 36 , 49 , 58 , 80 – 82 ]. The step is essential to “quench” or stop or at least slow down the turnover rate and in vivo metabolic reactions [ 30 , 67 , 83 , 84 ]. Quenching is done by exposing the sample to solvent at either extreme temperatures (cold and hot) [ 24 , 52 , 75 ] or extreme pH conditions (highly acidic or alkaline) [ 13 , 30 , 68 ].…”

“…The choice of extraction solvent depends on a number of factors including the total sample volume, the extraction time, and the coverage of metabolites [ 96 ]. The mechanical methods include the use of ultrasonic bath [ 83 ], glass bead beating [ 49 , 52 , 72 , 82 ], bead mill [ 53 , 87 ], freeze-thaw [ 55 , 56 , 59 , 71 , 84 , 97 ], and the least favourable, supercritical fluid extraction [ 98 ]. Mechanical extraction is commonly applied for animal and plant samples but it is least preferred for microbial intracellular metabolites as the method releases both small and large metabolites [ 30 ].…”

Erratum to: Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy

“…Unfortunately, the original version of this article [ 1 ] contained an error within the abstract. The sentence beginning with “Membrane integrity assayed by propidium iodide” reads “approximately 10 % of the L. paracasei cell membranes were damaged” but should read “approximately 100 % of the L. paracasei cell membranes were damaged”.…”

Improved Sample Preparation for Untargeted Metabolomics Profiling of Escherichia coli

“…As an alternative, a fast filtration system combined with quenching/extraction has been proposed as an effective strategy to reduce the loss of intracellular metabolites ( 20 ). However, performing manual fast filtration and clogging of filters are inevitable drawbacks of this approach ( 21 , 22 ). Alternative quenching strategies that reduce the leakage of intracellular metabolites and stabilize bacterial cells have been tested with limited success, including using different methanol concentrations ( 14 ), addition of buffering solutions ( 13 , 23 , 24 ), and changing the ratios of bacterial culture to quenching solvents.…”