Food Chemistry 117 (2009) 296–301

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Food Chemistry

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Isolation and identification of one kind of yellow pigmentsfrom model reaction systems related to garlic greening

Dan Wang, Xiuli Yang, Zhengfu Wang, Xiaosong Hu, Guanghua Zhao *

College of Food Science and Nutritional Engineering, China Agricultural University, Engineering Research Centre for Fruits and Vegetables Processing,Ministry of Education, Key Laboratory of Fruits and Vegetables Processing, Ministry of Agriculture, Qinghuadonglu 17, Haidian District, Beijing 100083, China

a r t i c l e i n f o

Article history:Received 8 January 2009Received in revised form 24 February 2009Accepted 1 April 2009

Keywords:2-(1H-Pyrrolyl) acetic acidGarlic greeningPyruvic acidYellow pigmentStructure

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.04.021

* Corresponding author. Tel.: +86 10 62737465; faxE-mail address: gzhao318@yahoo.com.cn (G. Zhao

a b s t r a c t

It was established that green pigment(s) responsible for garlic greening is composed of yellow and bluespecies, and pyruvic acid (a product from 1-PeCSO or 2-PeCSO under the action of alliinase) reacted withpigment precursor (PP) model compounds, 2-(1H-pyrrolyl) carboxylic acids to produce yellow pigments.However, the structure of the yellow pigments is unknown. In present paper, we identified three yellowpigments (Y1, Y2 and Y3) from three reaction systems containing pyruvic acid and 2-(1H-pyrrolyl) aceticacid (P-Gly) or 1-(20-methyl-10-carboxy-propyl) pyrrole (P-Val) or 1-(20-methyl-10-carboxy-butyl) pyrrole(P-Ile), respectively, by LC-ESI MS/MS and IT-TOF mass spectrometry. The three pigments have a UV/vis-ible maximum absorbance between 400 and 434 nm and might be formed by dimerisation of the threecorresponding PP under participation of pyruvic acid, molecular formula of which are C16H16N2O4 (Y1),C22H28N2O4 (Y2) and C24H32N2O4 (Y3), respectively.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction di(1-propenyl) thiosulfinate and protein amino acids. Further sup-

Garlic is processed as powders, purees and juices, granules andoleoresin. During processing, garlic greening occurs, representinga major concern because of loss of the economic value of theseproducts (Adams & Brown, 2007; Kim, Cho, & Kim, 1999). In con-trast, the formation of green colour is desirable and required inthe traditional home-made Chinese garlic product, ‘‘Laba” garlic(Bai, Chen, Liao, Zhao, & Hu, 2005; Bai, Li, Hu, Wang, & Zhao,2006). Based on previous studies, it was established that the mech-anism of garlic greening is similar to that of onion redding, and thatthe discolouration is a multistep process consisting of enzymaticand nonenzymatic stages (Imai, Akita, Tomotake, & Sawada,2006b; Joslyn & Peterson, 1960; Lee & Parkin, 1998; Shannon,Yamaguchi, & Howard, 1967a, 1967b). The first step correspondsto the conversion of S-(1-propenyl)-L-cysteine sulfoxide (1-PeCSO)into di(1-propenyl) thiosulfinate (Imai, Akita, Tomotake, & Sawada,2006a) or 1-propenyl containing thiosulfinate (Kubec, Hrbácová,Musah, & Velíšek, 2004; Kubec & Velíšek, 2007) under the actionof alliinase. Indeed, Lukes found that 1-PeCSO is a key compound in-volved in the discolouration with a positive correlation with the de-gree of garlic greening (Lukes, 1986). Consistent with aboveobservation, Kubec et al. suggested that discolouration occurs upontissue disruption of any Allium species that contain a high enoughcontent of 1-PeCSO (Kubec et al., 2004). Step 2 corresponds to theformation of the pigment precursors (PP) from a reaction of the

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: +86 10 62737645 18.).

port for this conclusion came from previous results showing thatall of protein amino acids except for cysteine and proline were ableto form coloured products when mixed with 1-propenyl-containingthiosulfinates (Kubec & Velíšek, 2007), and that thiosulfinate con-sumption in the pickling solution of ‘‘Laba” garlic is proportionalto the formation of the pigments (Bai et al., 2005). Imai et al. iso-lated and characterised 2-(3,4-dimethyl-1H-pyrrolyl)-3-methylbu-tanoic acid (PP-Val) and 2-(3,4-dimethyl-1H-pyrrolyl) propanoicacid (PP-Ala) from the reaction of di(1-propenyl) thiosulfinate withL-valine or L-alanine. These two compounds were proposed to be pig-ment precursors due to findings that pigments were produced fromthe reaction of PP-Val and di(2-propenyl) thiosulfinate under heat-ing (Imai et al., 2006a). Recent studies showed that 2-(1H-pyrrolyl)carboxylic acids (model compounds of PP-Val) can turn garlic puree(which was prepared from freshly harvested garlic bulbs) green(Wang, Nanding, Han, Chen, & Zhao, 2008), confirming that PP-Valis a pigment precursor for garlic greening. The catalytic conversionof 2-PeCSO by alliinase into di(2-propenyl) thiosulfinate representsthe third step. Finally, formed thiosulfinate(s) reacts with PP toproduce green pigment(s), corresponding to the fourth step.

However, it was found that the green pigment(s) consists of yel-low and blue species which exhibited two maximal UV/visibleabsorbances at �440 and �590 nm, respectively (Bai et al., 2005;Kubec & Velíšek, 2007; Wang et al., 2008). Thus, above step 4 actu-ally corresponds to the formation of the blue pigment(s). In parallel,a reaction between the pigment precursor (PP) and pyruvic acid re-sulted in the formation of the yellow species, representing a path-way of the formation of the yellow species. The combination of

D. Wang et al. / Food Chemistry 117 (2009) 296–301 297

both yellow and blue pigments finally creates the green colouration(Wang et al., 2008). However, so far, there is no information on thestructure of the yellow species produced from pyruvic acid and PP.In the present study, three yellow pigments were isolated fromthree model systems by using column chromatography, and theircorresponding structures were identified with mass spectrometry.

(A)

(B)

Fig. 1. (A) Structures of 2-(1H-pyrrolyl) acetic acid (P-Gly), 1-(20-methyl-10-carboxy-propyl) pyrrole (P-Val), 1-(20-methyl-10-carboxy-butyl) pyrrole (P-Ile)2-(3,4-dimethyl-1H-pyrrolyl)-3-methylbutanoic acid (PP-Val) and 2-(3,4-dimethyl-1H-pyrrolyl) propanoic acid (PP-Ala) and pyruvic acid. (B) The proposed structure ofthe yellow pigments.

2. Materials and methods

2.1. Chemicals

All solvents/chemicals above were of analytical grade or purer.L-Glycine, L-valine, L-isoleucine, hydrochloric acid, acetic acid, so-dium acetate anhydrous, ethyl acetate, sodium sulphate anhydrousand potassium hydroxide were obtained from Beijing ChemistryCo. (Beijing, China). 2,5-Dimethoxytetrahydrofuran was purchasedfrom Fluka (Beijing, China). Pyruvic acid was obtained from Sinop-harm Chemical Reagent Co. (Beijing, China). Formic acid (P98%)was supplied by Sigma–Aldrich (Chemie GmbH, Germany), andmethanol and acetone were purchased from Honeywell Burdickand Jackson (SK Chemicals, Korea), and they were chromatographygrade. The solid phase extraction (SPE) cartridges cleanert ODS C18

(12 mL, 2000 mg) were purchased from Agela Technologies (Bei-jing, China). Syringe filter units 0.45 and 0.22 lm were suppliedby Hercules (Beijing, China).

2.2. Sample preparation

P-Gly, P-Val and P-Ile were synthesised as previously described(Wang et al., 2008). 1H NMR spectra were performed with a dpx-300 MHZ NMR spectrometer (Brucker Co., Germany). Deuteriumdimethyl sulfoxide (DMSO-d6) was used as a solvent with tetra-methylsilane (TMS) as an internal standard. Mass spectra were ob-tained by using LC–MS/MS (Alliance2695/Quattro Micro API,Waters Co., USA), and detection was performed in the positivemode. Their 1H NMR spectra and MS data were identical to thosein literature (Wang et al., 2008), confirming their purity andstructures.

To prepare the yellow pigment(s), both P-Gly (20 mM) andpyruvic acid (20 mM) in water were mixed thoroughly with a vol-umetric ratio of 1:1, and then stood at room temperature (23–25 �C) for 48 h. Resultant solution was filtered through a 0.45 lmsyringe filter for clean-up. In contrast, the reaction of P-Val or P-Ile with pyruvic acid was carried out by dissolving them in meth-anol instead of water followed by standing at 45 �C for 72 h. Otherprocedures are the same as those used as above for P-Gly and pyru-vic acid.

2.3. Fractionation by ODS C18 SPE cartridge

ODS C18 SPE cartridge was used for further clean-up. SPE car-tridge was conditioned consecutively with 8 mL of methanol and8 mL of water. One millilitre of resultant solution mentioned abovewas loaded on the SPE cartridge and effluent was collected. Subse-quently, a wash step was followed to elute yellow pigment re-tained on the SPE column. Water, acetone and methanolconsecutively were subjected to the column for eluting. Onlymethanol eluent was collected and concentrated to a small volumewith a rotary evaporator. Finally, resulting solution was filteredthrough a 0.22 lm syringe filter for LC–MS analysis.

2.4. LC–MS analysis

The LC–MS analysis of these yellow pigments was performed byan Alliance 2695 Separations Module containing an autosampler

(Waters, Milford, MA, USA) coupled to a Micromass Quattro Microtriple-quadrupole mass spectrometer (Micromass, Manchester,UK) with MassLynx software. Twenty microlitre of the reactionsolution containing P-Gly was injected onto an analytical scale re-versed ASB-C18 column (250 � 4.6 mm, 5 lm, Venusil, Agela, USA)maintained at 29 �C. The elution mode was isocratic with a mixtureof 40% methanol and 60% water containing 0.2% formic acid as mo-bile phase at a flow rate of 0.4 mL/min for LC–MS analysis.

The reaction solution containing P-Val or P-Ile (20 lL) wasloaded to an reversed MP-C18 column (250 � 4.6 mm, 5 lm, Venu-sil, Agela, USA), which was maintained at 29 �C. The elution modewas also isocratic using a mixture of 45% methanol and 55% watercontaining 0.2% formic acid as mobile phase at a flow rate of0.4 mL/min for LC–MS analysis. PDA detection ranging between200 and 800 nm was performed. Yellow pigments were detectedusing electrospray ionisation in the positive ion mode. Used MSparameters were as follows: capillary voltage, 2.5 kV; cone voltage,10 V; scan ranger, m/z 100–1000; source temperature, 110 �C;desolvation temperature, 400 �C; desolvation gas flow, 700 L/hnitrogen; cone gas flow, 50 L/h. High-resolution electrospray ioni-sation mass spectra were acquired on an FT-MS Bruker APEX IV(7.0 T) (Bruker Co., Germany) equipped with an ESI source in posi-tive ion mode.

0.0

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300 400 500 600 700 800

300 400 500 600 700 800

300 400 500 600 700 800

0.0

0.3

0.6

0.9

1.2

Wavelength (nm)

433.9c

400.9

a

Abs

432.9b

Fig. 3. UV/vis spectra of the yellow pigments. (a) The pigment (Y1) isolated fromthe reaction system containing pyruvic acid and P-Gly. (b) The pigment (Y2)isolated from the reaction system containing pyruvic acid and P-Val. (c) Thepigment (Y3) isolated from the reaction system containing pyruvic acid and P-Ile.

298 D. Wang et al. / Food Chemistry 117 (2009) 296–301

3. Results and discussion

Three model compounds P-Gly, P-Val and P-Ile (Fig. 1) wereprepared mainly based on the method recently described (Wanget al., 2008). All of them have very similar structures to those ofPP-Val and PP-Ala (pigment precursors, Fig. 1) (Imai et al.,2006a). Therefore, all three compounds were chosen to react withpyruvic acid, respectively, to produce the yellow species. Resultantreaction mixture of P-Gly or P-Val or P-Ile and pyruvic acid re-solved only available with polar solvents, so water, acetone andmethanol were used to consecutively elute the ODS C18 SPE car-tridge which had retained the analyte above through absorption.Three kinds of solvent separated the sample into three differentfractions. Since a visible yellow fraction can only be obtainedthrough methanol eluting, this fraction was further purified byhigh-performance liquid chromatography (HPLC).

To achieve efficient HPLC resolution of the yellow fraction, theanalytical condition first was optimised. The best separation condi-tion was obtained on an analytical ASB-C18 column for Y1, and MP-C18 column for Y2 and Y3. A mixture of 40% methanol and 60% watercontaining 0.2% formic acid as mobile phase at a flow rate of 0.4 mL/min was used for Y1 while a mixture of 45% methanol and 55% watercontaining 0.2% formic acid was used as mobile phase for both Y2and Y3. HPLC chromatograph of the yellow fraction containing Y1with 400 nm as a detection wavelength was shown in Fig. 2a whileHPLC spectra of the yellow fraction containing Y2 and Y3 at440 nm as a detection wavelength were given in Fig. 2b and c. Itwas observed that all three yellow fractions are complex mixtureswhich consist of several components, a result reflecting the com-plexity of garlic greening. This observation is in accordance with thatreported by Kubec et al. (2004), Kubec and Velíšek (2007). Since peakat 9.13 min (Fig. 2a), peak at 12.67 min (Fig. 2b) and peak at24.63 min (Fig. 2c) were completely separated from other peaks inHPLC, they further were analysed by LC–MS/MS in conjunction witha diode array detector. UV/visible spectra of these three peaks per-formed by a diode array detector were displayed in Fig. 3, whichwere characterised by only one absorbance at 400.9 nm (Y1),

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0 5 10 15 20 25 30

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Abs

(400 nm)

7.62

9.13

12.38

8.88

12.67

15.07

6.85

(440 nm)

Time (min)

12.50

24.63

31.70

(440 nm)

a

b

c

Fig. 2. HPLC chromatogram of the yellow fraction upon SOD C18 SPE cartridgeclean-up. (a) The fraction from reaction containing pyruvic acid and P-Gly, detectedat 400 nm. (b) The fraction from reaction containing pyruvic acid and P-Val,detected at 440 nm. (c) The fraction from reaction containing pyruvic acid and P-Val, detected at 440 nm.

432.9 nm (Y2) and 433.9 nm (Y3) in visible region, respectively.Their absorptions were in good agreement with that of yellow pig-ment (440 nm) obtained from either a pickling solution of ‘‘Laba”garlic or an extraction solution of garlic puree (Bai et al., 2005; Kubecet al., 2004, Kubec & Velíšek, 2007). According to the polarity of mo-bile phase, it could observe that a methanol/acidic water ratio usedfor Y1 was less than that for Y2 and Y3; and Y1 had shorter retentiontime than Y2 or Y3 did, indicating that Y1 has the strongest polarityamong the three compounds while Y3 exhibits the weakest. Thisfinding is in good agreement with the polarity order of reactants,namely, P-Gly > P-Val > P-Ile.

Furthermore, both MSn and high-resolution mass spectrometrywere used to detect relative purity and molecular mass of the com-pounds. Firstly, Y1 was detected in the positive ion mode withexperimental conditions as follows: capillary voltage, 2.5 kV; conevoltage, 10 V; scan ranger, m/z 100–1000, and result was given inFig. 4a. The method used above was performed in a wider massrange scan with a lower voltage. Under present conditions, all com-ponents could be detected as their protonated adducts (Pasch, Piz-zi, & Rode, 2001). There is a single prominent protonated molecularion peak at m/z 301 [M+H]+ in the MS spectrum of Y1. The accuratemass of this ion was determined to be 301.11917 by high-resolu-tion MS (Table 1), which gives a possible molecular formula asC16H16N2O4. The difference in mass between the measured massand a value calculated from their assigned formula has the leastexperimental error which was within 0.89 mDa unit, indicatingthat this formula could be the most reliable. Consistent with thisformula, the number of carbon atoms was calculated to be �16by the observed intensity of the M+1 peak over contribution percarbon atom (1.08%) (Harris, 2003). In addition, the molecularweight of Y1 (�300) is even, suggesting that this yellow speciescontains an even number of N atoms in this molecule. All thesedata are consistent with the above proposed formula. The sameMS conditions were used to analyse Y2, MS spectrum of whichexhibited a single prominent protonated molecular ion peak atm/z 385 [M+H]+, confirming its purity (Fig. 4b). Since Y2 was de-tected in the positive ion mode, its molecular weight was 384.

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(%

)

M/Z

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302.8

a

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(%

)

M/Z

384.9

385.9

386.9

b

Fig. 4. MS spectra of the yellow pigments at cone voltage 10. (a) MS spectrum of Y1.(b) MS spectrum of Y2.

D. Wang et al. / Food Chemistry 117 (2009) 296–301 299

Molecular formula of Y2 was determined through the high-resolu-tion MS analysis to be C22H28N2O4 (Table 1). Likewise, the high-res-olution MS analysis for Y3 gave its molecular formula asC24H32N2O4 (Table 1). Based on their formula, we calculated thenumber of rings + double bonds (R + DB) in the molecule accordingto rings + double bonds formula (Eq. (1)) (Harris, 2003). The R + DBof all Y1, Y2 and Y3 were 10.

R þ DB ¼ c � h=2þ n=2þ 1 ð1Þ

From the structure of P-Gly and P-Val, it was observed that thedifference in molecular weight between these two model com-pounds is around 42, corresponding to the mass difference be-tween –CH(CH3)2 and –H groups, C3H6. Interestingly, thedifference in M.W. between Y1 and Y2 is �84, exactly the sameas the mass of two C3H6, a resulting indicating that these two yel-low species Y1 and Y2 was formed likely through dimerisation ofP-Gly and P-Val, respectively. However, either Y1 (300) or Y2(384) is not the simple adduct of two P-Gly (125) or P-Val (167)molecules according to their M.W. In addition, Y1 and Y2 have yel-low colour but not contain metals (data not shown). These resultssuggested that both Y1 and Y2 are dimmers of their correspondingcompounds, P-Gly and P-Val bridged by two conjugated doublebonds as shown in Fig. 1B. Agreeing with this conclusion, the samefeatures were found between Y2 and Y3, and between Y1 and Y3.

Table 1Formula of the yellow pigments observed from high-resolution electrospray ionisation ma

Pigment Formula Measured (m/z)

Y1 C16H16N2O4 301.11917Y2 C22H28N2O4 385.21289Y3 C24H32N2O4 413.24354

The three new yellow species Y1, Y2 and Y3 were named as{2-[4-(1-carboxymethyl-1H-pyrrol-2-yl)-1-buta-1,3-dienyl]-pyr-rol-1-yl}-acetic acid, 2-(2-{4-[1-(1-carboxy-2-methyl-propyl)-1H-pyrrol-2-yl]-buta-1,3-dienyl}-pyrrol-1-yl)-3-methyl-butyric acidand 2-(2-{4-[1-(1-carboxy-2-methyl-propyl)-1H-pyrrol-2-yl]-buta-1,3-dienyl}-pyrrol-1-yl)-3-methyl-pentanoic acid, respectively.

To prove above conclusion, compound Y3 was chosen to analyseits structure with MS/MS in positive mode. Increasing cone voltagecould accelerate the collision of positive ions with N2 molecules tobreak into a few fragments (Harris, 2003). Small cone voltage fa-vours molecular ions whereas large voltage creates more frag-ments that aid in identification of analyte. Therefore, the degreeof fragmentation can be controlled by adjusting the cone voltage.MS spectra with changing cone voltages in a wide range wereshown in Fig. 5A. At low cone voltage (10 V), only a single peakat m/z 413 appeared, suggesting that peak 24.63 min containsone compound, Y3 (Fig. 2c). The difference in M.W. is �1 unit be-tween Y3 and recently reported compound purified from garlicgreen puree, M.W. of which is about �411 and might contain sul-phur (Lee, Cho, Kim, & Lee, 2007), a result indicating that Y3 is dis-tinct from the reported species.

If the proposed structure of Y3 were correct, we would expectthat the C–N bond between pyrrole ring moiety and a-carbon atomin Y3 be broken first due to its most instability. Indeed, withincreasing the cone voltage to 70 V, this mother ion 413 [M+H]+

produced its first daughter ion at m/z 298 through losing one grouphaving mass as 413 � 298 = 115, indicating that lost fragment wasthe side chain of Y3, –CH(COOH)CH(CH3)CH2CH3. The second posi-tive ion was produced at m/z 232, a result indicating that the sec-ond group eliminated from this mother ion is pyrrole ring moietywith mass as 298 � 232 = 66. These results are in agreement withabove proposed structure of Y3 (Fig. 1B). When the cone voltage in-creased to 80 V, three new peaks at m/z 283, 183 and 117 appearedexcept for the two above peaks. The peak at m/z 283 corresponds toloss of –CH3 from ion with m/z 298. Cation at m/z 183 was pro-duced through losing another side chain from the ion at m/z 298.Cation at m/z 117 was produced by several pathways depicted inFig. 5B. Thus, all MS/MS spectra are in accordance with the pro-posed structures. Although Y1, Y2 and Y3 were yellow pigments,it is not known whether they were predominant yellow pigmentsrelating to garlic greening. Previous studies showed that unidenti-fied yellow pigments were also generated from degradation of bluespecies (Bai et al., 2005). However, the elucidation of the molecularstructures of Y1, Y2 and Y3 would obtain insights into the mecha-nism of garlic greening. According to the structure of three yellowspecies, a possible formation pathway was proposed, which likelycontains two main steps as depicted in Fig. 6. Step 1 corresponds toan nucleophilic addition reaction occurring between carbon atomin carbonyl of pyruvic acid and C-2 in pyrrole moiety of 2-(1H-pyrrolyl) carboxylic acid, resulting in the formation of intermediateI, which loses its –COOH group to produce intermediate II. Thisproposal is consistent with the fact that the reactivity of C-2 isgreater than that of C-3 of 2-(1H-pyrrolyl) carboxylic acid due toits higher acidic property, and carbon atom of carbonyl group ofpyruvic acid takes some positive charge. Step 2 corresponds tothe formation of final yellow species through a dimerisation reac-tion in which 2 mol of intermediate II were polymerised followedby other reactions into 1 mol of corresponding yellow pigments.

ss spectra.

Calculated (m/z) Error (mDa) Error (ppm)

301.11828 0.89 2.94385.21218 1.82 0.7413.24348 0.15 0.06

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10V412.9

413.9

414.9

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N

CHHC COOH

CH CH CH CHN

CHHOOC CH

CH3

CH2 CH3

CH3

H2CH3C

+H

[M+H]+=C24H33N2O4

(m/z 413)

NCH CH CH CH

N

CHHOOC CH

CH3

CH2 CH3

+H

(m/z 298)

CH CH CH CHN

CHHOOC CH

CH3(m/z 232)

+

CH2 CH3

CH C CH CHN

CH CH

CH3

CH2 CH3

+

CH C CH CHN+

(m/z 117)

N

CHCH COOH

CH CH CH CHN

CH3

H2CH3C

+H

(m/z 183)N

CH CH CH CHN

+H

HOOC(m/z 117)

CH CH CH CHN

+

CH C CH CHN+

A

B

Fig. 5. (A) MS/MS spectra of the Y3 at different cone voltages 10, 70 and 80 V. (B) The proposed guideline for mass spectral data (m/z) of Y3.

300 D. Wang et al. / Food Chemistry 117 (2009) 296–301

N

CHHOOC

+ H3C C

O

COOH

N

CHR

C CH3

N

CHR

CH CH

N

CH R

CH

Addition reaction

Dimerization

CH

Step 1

R

COOH

COOH HOOC

O

Intermediate II

N

CHR

C

CH3

OH

COOH

COOH

-CO2

Step 2

Intermediate I

Fig. 6. Proposed two-step pathway of the formation of yellow pigments related to garlic greening.

D. Wang et al. / Food Chemistry 117 (2009) 296–301 301

Although the above two-step pathway was proposed, other path-ways can not be excluded. More detailed mechanism about the for-mation of the yellow species is under investigation.

4. Conclusion

In this study, three yellow pigments from model systems wereisolated and identified by HPLC, LC–MS/MS and high-resolutionMS. The molecular formula of Y1, Y2 and Y3 was C16H16N2O4,C22H28N2O4 and C24H32N2O4, respectively. All of them containtwo pyrrole ring moieties bridged by two conjugated doublebonds. The elucidation of the three yellow species would benefitto understand the mechanism of garlic greening.

Acknowledgements

This work was supported by China High-Tech (863) Project(2007AA10Z333), the National Natural Science Foundation of Chi-na (30570181) and the Ministry of Education of the People’sRepublic of China, Specialised Research Fund for the Doctoral Pro-gram of Higher Education (20070019004).

References

Adams, J. B., & Brown, H. M. (2007). Discoloration in raw and processed fruits andvegetables. Food Science and Nutrition, 47, 319–333.

Bai, B., Chen, F., Liao, X., Zhao, G., & Hu, X. (2005). Mechanism of the greening colorformation of ‘‘Laba” garlic, a homemade Chinese food product. Journal ofAgricultural and Food Chemistry, 53, 7103–7107.

Bai, B., Li, L., Hu, X., Wang, Z., & Zhao, G. (2006). Increase in the permeability oftonoplast of garlic (Allium sativum) by monocarboxylic acids. Journal ofAgricultural and Food Chemistry, 54, 8103–8107.

Harris, D. C. (2003). Quantitative chemical analysis (6th ed., pp. 522–526). W. H.Freeman and company.

Imai, S., Akita, K., Tomotake, M., & Sawada, H. (2006a). Identification of two novelpigment precursors and a reddish-purple pigment involved in the blue-greendiscoloration of onion and garlic. Journal of Agricultural and Food Chemistry, 54,843–847.

Imai, S., Akita, K., Tomotake, M., & Sawada, H. (2006b). Model studies on precursorsystem generating blue pigment in onion and garlic. Journal of Agricultural andFood Chemistry, 54, 848–852.

Joslyn, M. A., & Peterson, R. G. (1960). Reddening of white onion tissue. Journal ofAgricultural and Food Chemistry, 8, 72–76.

Kim, W. J., Cho, J. S., & Kim, K. H. (1999). Stabilization of ground garlic color bycysteine, ascorbic acid, trisodium phosphate and sodium metabisulfite. Journalof Food Quality, 22, 681–691.

Kubec, R., Hrbácová, M., Musah, R. A., & Velíšek, J. (2004). Allium discoloration:Precursors involved in onion pinking and garlic greening. Journal of Agriculturaland Food Chemistry, 52, 5089–5094.

Kubec, R., & Velíšek, J. (2007). Allium discoloration: The color-forming potential ofindividual thiosulfinates and amino acids: Structural requirements for thecolor-developing precursors. Journal of Agricultural and Food Chemistry, 55,3491–3497.

Lee, C. H., & Parkin, K. L. (1998). Relationship between thiosulfinates and pinkdiscoloration in onion extracts, as influenced by pH. Food Chemistry, 61,345–350.

Lee, E. J., Cho, J. E., Kim, J. H., & Lee, S. K. (2007). Green pigment in crushed garlic(Allium sativum L.) cloves: Purification and partial characterization. FoodChemistry, 101, 1677–1686.

Lukes, T. M. (1986). Factors governing the greening of garlic puree. Journal of FoodScience, 51(1577), 1581–1582.

Pasch, H., Pizzi, A., & Rode, K. (2001). MALDI-TOF mass spectrometry ofpolyflavonoid tannins. Polymer, 42, 7531–7539.

Shannon, S., Yamaguchi, M., & Howard, F. D. (1967a). Reactions involved information of a pink pigment in onion purees. Journal of Agricultural and FoodChemistry, 15, 417–422.

Shannon, S., Yamaguchi, M., & Howard, F. D. (1967b). Precursors involved information of a pink pigment in onion purees. Journal of Agricultural and FoodChemistry, 15, 423–426.

Wang, D., Nanding, H., Han, N., Chen, F., & Zhao, G. (2008). 2-(1H-Pyrrolyl)carboxylic acids as pigment precursors in garlic greening. Journal of Agriculturaland Food Chemistry, 56, 1495–1500.