Article pubs.acs.org/JAFC
Allium Discoloration: Color Compounds Formed during Pinking of Onion and Leek Roman Kubec,*,† Petra Urajová,† Ondřej Lacina,§ Jana Hajšlová,§ Marek Kuzma,# and Jakub Zápal# Department of Applied Chemistry, University of South Bohemia, Branišovská 31, 370 05 Č eské Budějovice, Czech Republic Department of Food Analysis and Nutrition, Institute of Chemical Technology, Technická 5, 166 28 Prague 6, Czech Republic # Laboratory of Molecular Structure Characterization, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic † §
S Supporting Information *
ABSTRACT: Structures and formation pathways of compounds responsible for pink discoloration of onion and leek were studied. A procedure was developed for the isolation and purification of the color compounds from various model systems and their identification by HPLC-DAD-MS/MS. In total, structures of 15 major color compounds were tentatively determined. It was found that the pigment is a complex mixture of highly conjugated species composed of two N-substituted 3,4-dimethylpyrrolederived rings linked by either a methine or a propenylidine bridge. These two-ring units are further modified by various C1- and C3-side chains. Experiments with isotope-labeled thiosulfinates revealed that the methine bridge and C1-side chains originate from the methyl group of methiin, whereas the C3 units are derived from the propenyl group of isoalliin. KEYWORDS: discoloration, plant pigment, pinking, onion, leek, Allium, thiosulfinate, isoalliin, dipyrromethane, pyrrole
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INTRODUCTION
The pinking of onion was once believed to be caused by the formation of a betanine type pigment2 or by the presence of some microorganisms.3 Surprisingly, only a single study has thus far been specifically dedicated to the pinking of leek.5 The authors (erroneously) concluded that this discoloration was a result of enzymatic oxidation of phenolic substances to quinones, which in turn reacted with amino acids. Despite the great visual difference in the colors formed, the discoloration of both garlic and onion/leek are of a closely related nature. In both cases, it is a very complex multistep process, consisting of enzymatic and nonenzymatic stages. (E)S-(1-Propenyl)cysteine sulfoxide (isoalliin, 1), together with other S-alk(en)ylcysteine sulfoxides (e.g., methiin, 2), is enzymatically cleaved upon disruption of the tissue, yielding a great number of various sulfur compounds. Among them, 1propenyl-containing thiosulfinates and cepathiolanes (often referred to as “color developers”) have been shown to subsequently react with amino acids to give N-substituted 3,4-dimethylpyrroles (called “color precursors”).8,10−12,22 These colorless pyrroles further react with various (thio)carbonyl species to produce the color compounds. It was reported that this process can also proceed without the catalytic action of alliinase on isoalliin. In that case, however, the formation of the pink color is considerably slower and takes several weeks.23 Although the primary precursor (isoalliin, 1) and general mechanism of the discoloration of onion and leek have already been identified, structures of the color compounds and exact pathways of their formation are still poorly understood. A
The formation of intensely colored compounds is often observed during the processing of many genus Allium species, including garlic (Allium sativum L.), onion (Allium cepa L.), leek (Allium porrum L.), or giant onion (Allium giganteum Regel).1−6 In the case of garlic, a vividly blue-green pigment is generated, whereas onion and leek homogenates may turn pink or red (Figure 1). The formation of these pigments significantly lowers the organoleptic quality of processed alliaceous vegetables and causes great economic losses to producers. Whereas the discoloration of garlic has already been studied quite thoroughly,1,7−17 the pinking of onion and leek has received considerably less attention.2−4,18−22 It is mainly due to much slower formation and paler intensity of the pink color compared with the vividly blue-green discoloration of garlic.
Received: September 21, 2015 Revised: November 2, 2015 Accepted: November 7, 2015
Figure 1. Discoloration of homogenized onion and leek flakes stored at 23 °C for 5 days. © XXXX American Chemical Society
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DOI: 10.1021/acs.jafc.5b04564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 2. 2D-HPLC chromatograms of color compounds formed after mixing extracts of homogenized onion (above) and leek (below) with alanine. operated using an information-dependent acquisition (IDA) method to collect full scan MS and MS/MS information simultaneously. The method consisted of a survey scan (m/z 100−800, 100 ms accumulation time) and a product ion scan (m/z 50−800, 40 ms accumulation time). Product ion spectra were collected automatically for the 10 most intense ions in the survey scan throughout the chromatographic run. Spectra were collected using a collision energy of 45 V with a collision energy spread of ±15 V. The mass resolving power over the acquired mass range was always R > 30000 fwhm. Collected data were processed with PeakView software 1.1.1.2 (AB SCIEX). The isolation of compound CC-371 was achieved by using a Dynamax SD-210 binary pump HPLC system (Varian, Palo Alto, CA, USA), employing a diode array detector (Varian 335) and a preparative Ascentis C-8 column (250 × 21.2 mm, 5 μm; Supelco, St. Louis, MO, USA). GC analyses were conducted on a Varian 3800 chromatograph (Varian), equipped with a Varian 4000 MS detector and an HP-5MS fused silica capillary column (30 m × 0.25 mm i.d.; film thickness = 0.25 μm; Agilent Technologies, Santa Clara, CA, USA). Mass spectra were obtained by EI ionization at 70 eV over the range of 30−350 mass units. NMR spectra were recorded on a Bruker AVANCE III 700 MHz spectrometer equipped with a cryoprobe (Bruker, Billerica, MA, USA). 1H and 13C chemical shifts were referenced to the signals of residual MeOH (δ 3.31 and 49.0, respectively). Chemicals and Plant Material. 1-Propenyl bromide, 1,2dibromoethane, magnesium, (13C)methyl iodide (99% 13C), propane-
better knowledge of the structure and formation of these compounds may help in the prevention of this undesirable process. Thus, this work was aimed to identify color compounds formed during pinking of onion/leek homogenates. Due to the overwhelming complexity of reactions taking place in real systems, structures and formation pathways of the color compounds were studied in model mixtures including the utilization of several isotopically labeled precursors.
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MATERIALS AND METHODS
General Methods. UHPLC analyses were performed using an UltiMate 3000 RS UHPLC system (Thermo Scientific, Waltham, MA, USA) equipped with an Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm, Waters, Milford, MA, USA) maintained at 45 °C. The mobile phase consisted of 10 mM formic acid in Milli-Q water (solvent A) and methanol (solvent B). The following gradient was used: A/B 80:20 (0 min) (flow rate = 0.4 mL min−1) and 0:100 (15 min) (flow rate = 0.6 mL min−1). UV−vis data were collected by using a diode array detector (UltiMate 3000, Thermo Scientific). ESI HRMS data were obtained by an AB SCIEX TripleTOF 5600 (AB SCIEX, Framingham, MA, USA), provided with a DuoSpray ion source. The needle voltage in (+)ESI was +5.0 kV and that in (−)ESI, −4.5 kV. Other ion source parameters were identical for both polarities: curtain gas, 35 psi; nebulizer and drying gas, 60 psi; turbo gas temperature, 600 °C; declustering potential, 60 V. The mass spectrometer was B
DOI: 10.1021/acs.jafc.5b04564 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry thiol, D,L-(3-D3)alanine (99% D), D,L-(15N)alanine (99% 15N), D,L-(2D)alanine (98% D), D,L-(1-13C)alanine (99% 13C), formic acid, MilliQ water, and methanol for LC-MS were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-Chloroperoxybenzoic acid, dansyl chloride, methyl thiocyanate, potassium thiocyanate, sodium, lithium, lithium aluminum deuteride (98% D), propargyl bromide, and tetrahydrofuran were obtained from Acros Organics (Geel, Belgium). Other chemicals were supplied by Lach-Ner (Neratovice, Czech Republic). Onion and leek were purchased in a local store. Synthesis of Thiosulfinates. Alk(en)yl 1-propenyl disulfides [alk(en)yl = CH3−, 13CH3−, or CH3CHCH−] were synthesized by following the methods reported in our previous study.8 13CH3SCN was obtained by the reaction of potassium thiocyanate with 13CH3I. (1,2-D2)1-Propenyl propyl sulfide was prepared according to the method of Block et al.24 by using LiAlD4 and used for the preparation of CH3CDCDSSCH3. Thiosulfinates [3a/b, (D2)3a/b, and (13CH3)3a/b] were obtained by oxidation of the corresponding disulfides with 3-chloroperoxybenzoic acid and used without further purification.8 Representative MS data of the synthesized disulfides and detailed description of experimental procedures can be found in the Supporting Information. Extraction of Thiosulfinates and Related Compounds from Plant Material. Peeled onions (500 g) were homogenized with 500 mL of H2O by a kitchen blender and allowed to stand for 30 min at room temperature. The slurry was filtered through a layer of cotton wool, and the filtrate was extracted by diethyl ether (3 × 300 mL). The combined ether portions were dried over MgSO4, and the solvent was removed at reduced pressure (
