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Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

Jäpelt KB, Christensen JH, Villas-Bôas SG - Microb. Cell Fact. (2015)

Bottom Line: However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging.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.

View Article: PubMed Central - PubMed

Affiliation: Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. dkkba@chr-hansen.com.

ABSTRACT

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 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

L. paracasei cells assayed with PI to assess cell membrane integrity during quenching. a Cells quenched with cold buffered methanol at −40 °C. b Cells quenched with glycerol saline solution at −30 °C
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Fig1: L. paracasei cells assayed with PI to assess cell membrane integrity during quenching. a Cells quenched with cold buffered methanol at −40 °C. b Cells quenched with glycerol saline solution at −30 °C

Mentions: While it is essential to maintain an unbiased metabolic fingerprint by halting metabolic activity, a second requirement for successful quenching deals with the maintenance of cell membrane integrity. The PI membrane integrity assay showed that the L. paracasei cell membrane was significantly damaged by contact with buffered methanol (Fig. 1a). The damage was limited to a few cells when performing glycerol saline quenching (Fig. 1b). The variation in quenching conditions led to around 10 % PI-labelled cells for glycerol saline while nearly 100 % labelling was demonstrated for cold buffered methanol quenching.Fig. 1


Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

Jäpelt KB, Christensen JH, Villas-Bôas SG - Microb. Cell Fact. (2015)

L. paracasei cells assayed with PI to assess cell membrane integrity during quenching. a Cells quenched with cold buffered methanol at −40 °C. b Cells quenched with glycerol saline solution at −30 °C
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4559878&req=5

Fig1: L. paracasei cells assayed with PI to assess cell membrane integrity during quenching. a Cells quenched with cold buffered methanol at −40 °C. b Cells quenched with glycerol saline solution at −30 °C
Mentions: While it is essential to maintain an unbiased metabolic fingerprint by halting metabolic activity, a second requirement for successful quenching deals with the maintenance of cell membrane integrity. The PI membrane integrity assay showed that the L. paracasei cell membrane was significantly damaged by contact with buffered methanol (Fig. 1a). The damage was limited to a few cells when performing glycerol saline quenching (Fig. 1b). The variation in quenching conditions led to around 10 % PI-labelled cells for glycerol saline while nearly 100 % labelling was demonstrated for cold buffered methanol quenching.Fig. 1

Bottom Line: However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging.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.

View Article: PubMed Central - PubMed

Affiliation: Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. dkkba@chr-hansen.com.

ABSTRACT

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 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus