Metabolic profiling of Lactococcus lactis under different culture conditions

Abstract

Gas chromatography mass spectrometry (GC-MS) and headspace gas chromatography mass spectrometry (HS/GC-MS) were used to study metabolites produced by Lactococcus lactis subsp. cremoris MG1363 grown at a temperature of 30 °C with and without agitation at 150 rpm, and at 37 °C without agitation. It was observed that L. lactis produced more organic acids under agitation. Primary alcohols, aldehydes, ketones and polyols were identified as the corresponding trimethylsilyl (TMS) derivatives, whereas amino acids and organic acids, including fatty acids, were detected through methyl chloroformate derivatization. HS analysis indicated that branched-chain methyl aldehydes, including 2-methylbutanal, 3-methylbutanal, and 2-methylpropanal are degdradation products of isoleucine, leucine or valine. Multivariate analysis (MVA) using partial least squares discriminant analysis (PLS-DA) revealed the major differences between treatments were due to changes of amino acids and fermentation products.

Figure 1

Metabolic profiles of L. lactis under growth conditions without agitation at 30 °C, and 37 °C, and 30 °C with agitation. Profiles were obtained using TMS derivatization and measured by GC-MS.

Figure 2

PLS-DA derived loading plot analysis of metabolites profiles. PLS-DA analysis with temperature as the y variable was used to identify the metabolites that distinguished L. lactis grown in non-agitated condition of 30 °C and 37 °C. Compounds marked red indicate metabolites with variable importance for projection (VIP) values exceeding 1.

Figure 3

Double hierarchical clustering analysis of detected amino acids from non-agitated conditions of 30 °C and 37 °C, and 30 °C with agitation. Red color indicates relatively high abundance; green represents a relatively low abundance.

Figure 4

Schematic representation of precursor relationship between amino acids and the central carbon metabolism. R5P represents ribose -5-phosphate, 3PG represents 3-phosphoglycerate and PEP represents phosphoenol pyruvate. OAA represents oxaloacetate and AKG represents 2-oxoglutarate while ADI represents the arginine deiminase.

Figure 5

Bar chart representing the relative abundances of detected branched-chain aldehydes using dynamic headspace (HS) coupled to GC-MS according to the three conditions. Condition with agitation showed less production of branched chain aldehydes. HS_30 represents condition of 30 °C, HS_37 represents condition of 37 °C and HS_30150 represents condition with agitation (150 rpm).

Figure 6

(a) PLS-DA score plots by the combination of PC1 and PC2 of TMS derivatised replicates. The ellipses represent confidences of 95% in the Hotelling T2 tests. 30 represents condition of 30 °C, 37 represents condition of 37 °C, 30150 represents condition with agitation (150 rpm). (b) PLS-DA score plots by the combination of PC1 and PC2 of MCF derivatised replicates. The ellipses represent confidences of 95% in the Hotelling T2 tests. 30 represents condition of 30 °C, 37 represents condition of 37 °C, 30150 represents condition with agitation (150 rpm). (c) Hierarchical clustering analysis of MCF derivatised metabolites according to conditions. Red represents condition of 30 °C, blue represents the condition of agitation and black represents the condition of 37 °C.