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Metabolic footprinting for investigation of antifungal properties of Lactobacillus paracasei

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Abstract

Lactic acid bacteria with antifungal properties are applied for biopreservation of food. In order to further our understanding of their antifungal mechanism, there is an ongoing search for bioactive molecules. With a focus on the metabolites formed, bioassay-guided fractionation and comprehensive screening have identified compounds as antifungal. Although these are active, the compounds have been found in concentrations that are too low to account for the observed antifungal effect. It has been hypothesized that the formation of metabolites and consumption of nutrients during bacterial fermentations form the basis for the antifungal effect, i.e., the composition of the exometabolome. To build a more comprehensive view of the chemical changes induced by bacterial fermentation and the effects on mold growth, a strategy for correlating the exometabolomic profiles with mold growth was applied. The antifungal properties were assessed by measuring mold growth of two Penicillium strains on cell-free ferments of three strains of Lactobacillus paracasei pre-fermented in a chemically defined medium. Exometabolomic profiling was performed by reversed-phase liquid chromatography in combination with mass spectrometry in electrospray positive and negative modes. By multivariate data analysis, the three strains of Lb. paracasei were readily distinguished by the relative difference of their exometabolomes. The relative differences correlated with the relative growth of the two Penicillium strains. Metabolic footprinting proved to be a supplement to bioassay-guided fractionation for investigation of antifungal properties of bacterial ferments. Additionally, three previously identified and three novel antifungal metabolites from Lb. paracasei and their potential precursors were detected and assigned using the strategy.

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Abbreviations

ArAT:

Aromatic aminotransferase

BCAA:

Branched-chain amino acid

BcAT:

Branched-chain aminotransferase

BPC:

Base peak chromatogram

CDIM:

Chemically defined interaction medium

CF:

Cell-free ferment

Da:

Dalton

ESI:

Electrospray ionization

FID:

Flame ionization detector

GC:

Gas chromatography

id:

Internal diameter

ID:

Inhibition degree

IS:

Internal standard

LC:

Liquid chromatography

m/z :

Mass-to-charge ratio

MIC:

Minimal inhibitory concentration

MS:

Mass spectrometry

Neg:

Negative, as for negative electrospray mode

OD:

Optical density

PC:

Principal component

PCA:

Principal component analysis

PLSR:

Partial least squares regression

Pos:

Positive, as for positive electrospray mode

ppm:

Parts per million

REF:

Reference, un-inoculated substrate

TIC:

Total ion chromatogram

UPLC:

Ultra-performance liquid chromatography

VIP:

Variable importance in projection

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Acknowledgments

This work was partially financed by the Danish Ministry of Science, Innovation and Higher Education and by the University of Copenhagen as a scholarship for Stina Dissing Aunsbjerg. The analytical support for GC work by Research Associate Marianne Termansen and Technician Lasse Hørup, DuPont Nutritional BioSciences ApS was highly appreciated.

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Authors and Affiliations

  1. DuPont Nutrition Biosciences ApS, Edwin Rahrs Vej 38, 8220, Brabrand, Denmark

    Anders H. Honoré, Michael Thorsen & Connie Benfeldt

  2. Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958, Frederiksberg C, Denmark

    Anders H. Honoré, Stina D. Aunsbjerg, Parvaneh Ebrahimi, Susanne Knøchel & Thomas Skov

Authors
  1. Anders H. Honoré
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  2. Stina D. Aunsbjerg
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  3. Parvaneh Ebrahimi
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  4. Michael Thorsen
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  5. Connie Benfeldt
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  6. Susanne Knøchel
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  7. Thomas Skov
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Correspondence to Anders H. Honoré.

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The authors declare that they have no conflict of interest.

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Anders H. Honoré and Stina D. Aunsbjerg contributed equally to the manuscript.

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Honoré, A.H., Aunsbjerg, S.D., Ebrahimi, P. et al. Metabolic footprinting for investigation of antifungal properties of Lactobacillus paracasei . Anal Bioanal Chem 408, 83–96 (2016). https://doi.org/10.1007/s00216-015-9103-6

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  • DOI: https://doi.org/10.1007/s00216-015-9103-6

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