Solution to blue garlic challenge

Abstract

The article presents a solution to blue garlic challenge. The pigment precursor was a 3,4-dimethylpyrrole derivative. It is thought to be formed by condensation of the amine group of Allium amino acids with the thial/thial S-oxide formed by - sigmatropic rearrangement of bis-1-propenyl thiosulfinate, in turn formed by the action of alliinase on 1-PeCSO (isoalliin). The thial/thial S-oxide is an intermediate in the formation of zwiebelanes, and is closely related in structure to (Z,Z)-d,1-2,3-dimethyl-1,4-butanedithial S,S'-dioxide, a compound isolated from onion preparations, which could play a role in forming the pigment precursor. The plant chemistry, however, is always wonderfully complex, so that other studies are not useless. The active compound that yielded blue color when combined with unheated onion juice was isolated from unheated garlic juice and was confirmed to be allicin, which derived from alliin by the action of alliinase.

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ANALYTICAL CHALLENGE
Solution to blue garlic challenge
Hervé This
#Springer-Verlag Berlin Heidelberg 2014
The winner of the blue garlic challenge (published in volume
406 issue 1) is:
Wai-Yin Lau, Department of Chemistry, University of Hong
Kong, Hong Kong, China
The award entitles the winner to select a Springer book of
her choice up to a value of €100.
Our Congratulations!
Solution
The explanation of the “greening”or “blueing”of garlic
(Allium sativum L.) and “pinking”of onion (Allium cepa L.)
given here will conclude with a new challenge of its own.
Color changes of Allium tissues have been studied by
several investigators since Joslyn, in 1958 [1]. In the 1960s,
the reactions involved in the formation of pink pigment in
onion purée were investigated [2] and a three-step reaction
scheme was proposed: (1) the formation of a colorless, ether-
soluble substance [the color developer (CD)] by the catalytic
action of alliinase (enzyme EC 4.4.1.4) upon then-unknown
precursors in the fraction of neutral and basic amino acids; (2)
the formation of a colorless, ether-insoluble pigment precursor
(PP) from the color developer and an amino acid such as
glycine; (3) the formation of a pink pigment from the pigment
precursor PP and a naturally occurring carbonyl (NOC) sub-
stance such as formaldehyde. It was then assumed [3]thata
blue pigment-forming “carbonyl”was present in garlic, and
when reacted with the pigment precursor PP in place of NOC
from onion, a blue color would form.
Subsequently, it was found [4] that the blue pigment-forming
“carbonyl”was derived from isoalliin (a substrate for alliinase, a
minor precursor in garlic flavor, but the major precursor of onion
flavor), and the same reactions were responsible for “greening“
of garlic and of the mixture of garlic and onion.
But our understanding of the Allium chemistry advanced
significantly when Eric Block and his colleagues [5–8]intro-
duced methods for the study of Allium chemistry and pro-
duced a wealth of results based on such methods. Based on
their work, it was established (see Fig. 1) that the pigment
precursor was a 3,4-dimethylpyrrole derivative. It is thought
to be formed by condensation of the amine group of Allium
amino acids with the thial/thial S-oxide formed by [3]-
sigmatropic rearrangement of bis-1-propenyl thiosulfinate, in
turn formed by the action of alliinase on 1-PeCSO (isoalliin).
The thial/thial S-oxide is an intermediate in the formation of
zwiebelanes, and is closely related in structure to (Z,Z)-d,l-
2,3-dimethyl-1,4-butanedithial S,S'-dioxide, a compound iso-
lated from onion preparations, which could play a role in
forming the pigment precursor. A second key aspect to the
formation of colors in Allium preparation requires the inter-
mediacy of thioacrolein.
Using such information, a positive correlation between
thiosulfinate concentration and pink pigment formation was
first observed [9], and 1-propenyl-containing thiosulfinates
were confirmed to be the major color-developing compounds
[10]. Then in 2005, Bai et al. [11] studied the mechanisms of
the green color formation in “Laba”garlic, a preserve of garlic
including vinegar and sometimes sugar: both alliinase and
acetic acid are required for the color formation, and the de-
crease in the total thiosulfinates in garlic cloves is associated
with the pigment formation.
The plant chemistry, however, is always wonderfully com-
plex, so that other studies are not useless. Imai et al. [12]
established a model reaction system that comprised only well-
defined constituents and reported identifications of new
This article is the solution to the Analytical Challenge to be found at
http://dx.doi.org/10.1007/s00216-013-7464-2
H. This (*)
INRA/AgroParisTech, UMR 1145, Group of Molecular Gastronomy,
16 rue Claude Bernard, 75005 Paris, France
e-mail: herve.this@paris.inra.fr
Anal Bioanal Chem (2014) 406:2743–2745
DOI 10.1007/s00216-014-7699-6

substances, which were involved in the pigment formation
along with various conditions that affected color development.
Addition of glycine suggested that proteins in the garlic juice
participated presumably in the pigment formation and that
they were less reactive than 75 % MeOH-soluble free amino
acids, such as glycine. The active compound that yielded blue
color when combined with unheated onion juice was isolated
from unheated garlic juice and was confirmed to be allicin,
which derived from alliin by the action of alliinase. It was also
confirmed that a vivid-blue color could be produced by using
a highly defined model reaction system comprising only iso-
lated alliin, pure glycine, and purified garlic alliinase. Later,
Imai et al. [13,14] isolated two pigment precursors and a
reddish-purple pigment (PUR-1) and determined their chem-
ical structures.
In 2007, Lee et al. [15] purified the green pigment, respon-
sible for greening in crushed garlic cloves, and they analyzed
it by liquid chromatography–electrospray ionization mass
spectrometry (LC-ESI-MS), fast atom bombardment mass
spectrometry (FAB-MS), matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF-
MS), and nuclear magnetic resonance (NMR) spectroscopy.
The purified green pigment was highly polar and slightly
viscous, with a garlic odor, and easily turned to a yellow or
brown color with exposure to room temperature. The absorp-
tion spectrum in methanol showed a crude methanolic green
pigment-like profile with two absorbance maxima at 440 and
590 nm. Although complete isolation of the 411 Da com-
pound for proper structure elucidation was not achieved in
their experiments, the MS and NMR spectra of the 411 Da
green pigment suggested the ambiguous structural assignment
of one sulfur atom and odd number of nitrogen atoms, with
25–30 carbon atoms, including aromatic ring. Therefore, it
was envisioned that the green pigment observed in crushed
garlic cloves was a new sulfur-containing nitrogenous water-
soluble compound differing significantly from all previously
reported green pigments in plants.
Obviously, the story is not fully over, but the worst is
when one tries to get this color, as I did. When I simply
put peeled garlic cloves in vinegar, the color did not
appear, whether I boiled the system or not, and even after
2 wk of maceration. According to Block [15], garlic heads
should be aged before immersion in vinegar in order for
the color to appear. Let’s meet again in 4 months from
now!
References
1. Joslyn MA, Peterson RG (1958) Food discoloration, reddening of
white onion bulb purees. J Agric Food Chem 6:754–765
2. Shannon S, Yamaguchi M, Howard FD (1967) Reactions involved in
formation of a pink pigment in onion purees. J Agric Food Chem 15:
417–422
isoalliin, 1-PeCSO
alliin, 2-PeCSO
alliinase SS+
O
S
OH
CO2H
S+
O
H2N
SS+
O
HO2C
NH2
R
N
HO2CR
alliinase SS+
O
S
OH
CO2H
S+
O
H2N
N
HO2CR
N
CO2H
R
+
pigment
precursor
max 230
allicin
dipyrrole pigment max 570
N
HO2CR
N
CO2H
R
+
tripyrrole pigment (proposed; blue-green)
N
R
CO2H
+
steps N
HO2CR
+
N
HO2CR
+
SH
H
N
HO2CR
H+
N
HO2CR
S
S
Fig. 1 Formation of various
visible light absorbing
compounds from Allium bulbs
(from [15])
2744 H. This

3. Yamaguchi M, Shannon S, Howard FD, Joslyn MA (1965) Factors
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Solution to blue garlic challenge 2745