To delineate possible active metabolites produced by L. plantarum as
it transitions from exponential to stationary phase of growth, we
investigated the metabolomic changes using principal component
analysis (PCA). Apart from obtaining a graphical representation of
the similarities and differences between the bacterial strains, PCA
provides means of determining the variations among very similar
sample sets, as is the case for metabolomic changes through
transitioning phases of growth.24 The scores plot generated for midlog
and stationary-phase samples of L. plantarum resulted in a distinct
separation of the two groups with tight replicate clustering, indicating
the presence of previously anticipated metabolomic differences
between the two groups (Figure 1a). Detailed analysis of the loadings
plot (Figure 1b) and the metabolite features defining the separation
between the two groups identified a suite of metabolite features
pertaining to the stationary phase samples. Such analysis led to the
identification of a metabolite with a mass to charge ratio of 277.1
with a distinct retention time (16.4min, Figure 1c) and, as determined
from the t-test analysis and correlative with the growth-phase
transition, an 11-fold intensity increase. MS/MS analysis of
this metabolite produced a distinctive m/z 165.9 ion, corresponding
to a singly charged phenylalanine, and was correlative with a
loss of another proteinogenic amino acid—pyroglutamic acid
(Supplementary Figure S1). Following isolation and purification of
the compound using a series of normal- and reverse-phase chromatography
methods, conducting 1H, COSY (1H–1H) and HMBC (1H–
13C) NMR experiments provided sufficient information to elucidate
the structure (Supplementary Figures S2 and S6). From the 1H NMR
data, the first set of signals including a signal at 7.73 p.p.m. (s) that
corresponds to the secondary amine, 7.21 p.p.m. (t) and 7.15 p.p.m.