Elsevier

Food Chemistry

Volume 131, Issue 3, 1 April 2012, Pages 852-861
Food Chemistry

Elucidation of chemical structures of pink-red pigments responsible for ‘pinking’ in macerated onion (Allium cepa L.) using HPLC–DAD and tandem mass spectrometry

https://doi.org/10.1016/j.foodchem.2011.09.059Get rights and content

Abstract

The chemical structures of pink-red pigments responsible for ‘pinking’ in macerated onion were tentatively elucidated using HPLC with a diode array detector and tandem mass spectrometry. All pigments were produced in conditions that approximated the natural process as closely as possible, using mixtures of onion thiosulphinates, the enzyme alliinase, and free amino acids. The isotopic distribution of protonated molecules of pink-red pigments produced from individual amino acids indicated the absence of sulphur, with the exception of pigment produced from cystine. The pigments had a basic polymethine framework containing two pyrrole rings (3,4-dimethyl-1H-pyrrole and 3,4-dimethyl-2,5-dihydro-1H-pyrrole) bridged by methines. The side chains attached to the nitrogen of the pyrrole rings were derived from the reacting amino acid. The simplest pink-red pigment, produced from glycine, was identified as 2-(2-(3-(1-(carboxymethyl)-3,4-dimethyl-1H-pyrrol-2(5H)-ylidene)prop-1-en-1-yl)-3,4-dimethyl-1H-pyrrol-1-yl)acetic acid. The pigment from alanine was identified as 2-(2-(3-(1-(carboxymethyl)-3,4-dimethyl-1H-pyrrol-2-yl)allylidene)-3,4-dimethyl-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid. The chemical structures of pink-red pigments from leucine, asparagine, glutamine, tyrosine, and cystine also were determined.

Highlights

► Pink pigments responsible for pinking in macerated onion were synthesised. ► Chemical structures of pink pigments were analysed using tandem mass spectrometry. ► The pigments share the same basic structure, two pyrrole rings bridged by methines. ► The pigments show considerably complex nature and will undergo changes with time.

Introduction

Allium discoloration mechanism following maceration, known as ‘greening’ in garlic or ‘pinking’ in onion, has been an interesting research area in food science since the 1950s (Bai et al., 2005, Bandyopadhyay and Tewari, 1973, Cho et al., 2009, Imai et al., 2006a, Imai et al., 2006b, Joslyn and Peterson, 1958, Kubec et al., 2004, Kubec and Velíšek, 2007, Kucerová et al., 2011, Lee et al., 2010, Lukes, 1959, Lukes, 1986, Shannon et al., 1967a, Shannon et al., 1967b). Recently, a new method to control greening by freeze-drying onion powder was suggested by Cho, Lee, Yoo, and Lee (2011). Greening is a well-known phenomenon because garlic products are widely used. Pinking of onion has received less attention due to the slower formation of the pink pigments, the low intensity of the colour, and the relatively limited use of minced onion products (Lee et al., 2010).

Since the proposal of a multi-step process of pinking in onion by Shannon et al., 1967a, Shannon et al., 1967b, a very similar mechanism was reported for pigment formation in both greening and pinking. The overall Allium discoloration mechanism was explained by the reaction of flavour compounds with the enzyme alliinase and amino acids (Bai et al., 2005, Cho et al., 2009, Imai et al., 2006a, Imai et al., 2006b, Kubec and Velíšek, 2007, Kubec et al., 2004, Lee et al., 2010, Li et al., 2008, Lukes, 1986). The first step in this process is the formation of thiosulphinates, resulting from the hydrolysis of S-1-propenyl-l-cysteine sulphoxide (1-PeCSO, isoalliin) and S-2-propenyl-l-cysteine sulphoxide (2-PeCSO, alliin) by the enzyme alliinase. The second step corresponds to the formation of multiple blue or pink pigments by non-enzymatic reactions between thiosulphinates and free amino acids. In our recent work, we confirmed that Allium discoloration is not caused by a single pigment, as reported by Lee, Cho, Kim, and Lee (2007), but by many pigments (Cho et al., 2009, Lee et al., 2010).

Recently, a new pink discoloration pathway was suggested by Dong, Wang, Li, Hu, and Zhao (2010). They confirmed that the key compound 1-PeCSO alone in a phosphate buffer at pH 5.0 could produce two red pigments with mass-to-charge (m/z) of 459 and 549, in the absence of enzymes or amino acids. This discoloration pathway has 1-PeCSO in common with the previously elucidated pathway (Bai et al., 2005, Cho et al., 2009, Imai et al., 2006a, Imai et al., 2006b, Kubec and Velíšek, 2007, Kubec et al., 2004, Li et al., 2008, Lukes, 1986), but differs in the absence of alliinase and amino acids.

Although the Allium discoloration mechanism has been extensively studied, little is known about pigment chemical structures, as attempts to isolate, purify, and identify pigments from naturally prepared samples have not been successful. Lee et al. (2007) first tried to purify a naturally-occurring green pigment (m/z 411) observed in macerated garlic, but they could not successfully elucidate its chemical structure. Recently, novel yellow pigments related to garlic greening were isolated from a model reaction system containing 2-(1H-pyrrolyl) carboxylic acids and pyruvic acids, and were characterised as water-soluble pyrrole derivative pigments (Hu et al., 2010, Wang et al., 2010).

In studies of onion pinking, Joslyn and Peterson (1958) first suggested that the pink pigment observed in macerated onion might be a new nitrogenous water-soluble pigment. Imai et al. (2006a) suggested that pink pigments are various N-substituted 3,4-dimethylpyrrole oligomers. However, the chemical structures of the naturally-occurring pigments responsible for Allium discoloration remain partially or wholly unknown. We assumed that these structures are considerably complex, as have been previously reported (Cho et al., 2009, Lee et al., 2010). In these studies, several pink-red pigments were synthesised using a single amino acid. Knowledge of the chemical structures of these pigments will be useful in understanding the mechanism of Allium discoloration, and in developing food quality control methods.

In this study, we synthesised several pink-red pigments in conditions that approximated to the natural process as closely as possible, using mixtures of onion thiosulphinates, the enzyme alliinase, and free amino acids. The pink-red pigments were purified and analysed using HPLC and tandem mass spectrometry. The chemical formulae and structures of these pigments were determined based on accurate mass, isotopic distribution, fragmentation pattern of the parent protonated molecule, and previously proposed chemical structures.

Section snippets

Chemical reagents and plant materials

All chemicals and amino acid kits were purchased from Sigma–Aldrich, Inc. (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA). The solid-phase extraction cartridge (SPE, C18, 500 mg) was purchased from Alltech, Inc. (Deerfield, IL). White onions and garlic bulbs were purchased from a local produce store. HPLC-grade acetonitrile and methanol were purchased from EMD chemicals (Darmstadt, Germany).

Preparation of crude garlic alliinase

Crude garlic alliinase was prepared as described by Imai et al. (2006b). Fresh garlic juice was

Analysis and isolation by HPLC–DAD

Separation and detection of pink-red pigments produced by the reaction of onion thiosulphinates with individual amino acids by HPLC–DAD at 515 nm are shown in Fig. 1 (left). An increase in retention time was observed as the hydrophobicity of the reacted amino acids increased from asparagine to leucine. A similar order of retention times has been reported previously (Lee et al., 2010). The UV–visible absorbance spectrum of each pigment at the specified retention time of Fig. 1 (left) is shown in

Conclusion

The chemical structure of Gly-p was the simplest of all pink-red pigments tested and its chemical structure was confidently identified. It was used as a base for a generalised chemical structure for the other pigments, where the only difference was in the identity of the two groups, R1 and R2. All pink-red pigments tested share the same basic structure, containing two pyrrole rings (3,4-dimethyl-1H-pyrrole and 3,4-dimethyl-2,5-dihydro-1H-pyrrole) bridged by methines. These pink-red compounds

Acknowledgements

This work was supported by the Cooperative State Research, Education, and Extension Service, US Department of Agriculture under Agreement No. 2009-34402-19831 and by “Designing Foods for Health” through the Vegetable & Fruit Improvement Center, Texas AgriLife Research.

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These authors contributed equally and should be considered joint first authors.

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