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Keywords:

  • amino acid;
  • blue pigment;
  • garlic;
  • greening;
  • thiosulfinate

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

ABSTRACT:  The color-forming ability of amino acids with thiosulfinate in crushed garlic was investigated. We developed reaction systems for generating pure blue pigments using extracted thiosulfinate from crushed garlic and onion and all 22 amino acids. Each amino acid was reacted with thiosulfinate solution and was then incubated at 60 °C for 3 h to generate pigments. Unknown blue pigments, responsible for discoloration in crushed garlic cloves (Allium sativum L.), were separated and tentatively characterized using high-performance liquid chromatography (HPLC) and a diode array detector ranging between 200 and 700 nm. Blue pigment solutions exhibited 2 maximal absorbance peaks at 440 nm and 580 nm, corresponding to yellow and blue, respectively, with different retention times. Our findings indicated that green discoloration is created by the combination of yellow and blue pigments. Eight naturally occurring blue pigments were separated from discolored garlic extracts using HPLC at 580 nm. This suggests that garlic discoloration is not caused by only 1 blue pigment, as reported earlier, but by as many as 8 pigments. Overall, free amino acids that formed blue pigment when reacted with thiosulfinate were glycine, arginine, lysine, serine, alanine, aspartic acid, asparagine, glutamic acid, and tyrosine. Arginine, asparagine, and glutamine had spectra that were more similar to naturally greened garlic extract.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

Garlic has traditionally been stored, transported, and sold in the bulb state and must be broken into cloves to remove the hull skin before consumption. Recently, steady growth in retail sales of fresh, ready-to-use vegetables, occurring in direct response to demands of the marketplace, has led to minimally processed prepeeled and crushed garlic products (Hong and Kim 2001; Lee and others 2007). However, during the processing of garlic, intensely colored pigments often form. In garlic homogenates, green, blue–green, or blue compounds are generated within several hours. Green discoloration is a major problem in crushed garlic and causes great economic losses. However, the discoloration phenomenon known as “greening” is poorly understood. It has been proposed that greening is similar to “pinking” in onion puree and is a multi-step process (Shannon and others 1967a,b; Imai and others 2006a,b; Lee and others 2006a,b; Kubec and Velíšek 2007). The 1st step for pigment development is formation of thiosulfinates, which is called “color developers,” resulting from hydrolysis of 1-propenyl-L-cysteine sulfoxide (1-PeCSO) and 2-propenyl-L-cysteine sulfoxide (2-PeCSO) by enzyme alliinase. Recently, Kubec and Velíšek (2007) reported that the color developers for blue pigments might contain di-1-propenyl or mono-1-propenyl group. The 2nd step corresponds to formation of blue pigments by reactions between thiosulfinates and amino acids (Figure 1). Lukes (1986) confirmed that storage of garlic at or below 12 °C increases the amount of 1-PeCSO. 1-PeCSO is the key compound in discoloration and a positive correlation exists between the degree of greening and concentration of 1-PeCSO. Kubec and others (2004) suggested discoloration occurs upon tissue disruption of Allium species that contain at least traces of 1-PeCSO. Although previously reported discoloration reactions are generally in agreement, the detailed reactions and the specific amino acids involved in pigment formation are still unknown. Glycine has usually been used in model reaction systems to generate green pigment. During our previous investigation on blue–green discoloration in mixed garlic and onion, we found that not only glycine but also other amino acids have the potential to form green pigments. Therefore, we attempted to find the optimal ratio of 1-PeCSO and 2-PeCSO for blue–green pigment formation and to develop a reaction system to identify the major candidate amino acids involved in blue–green pigment formation in crushed garlic cloves.

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Figure 1—. Reaction for blue pigment formation in crushed garlic.

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Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

Chemical reagents and standards

All chemicals were purchased from Sigma-Aldrich, Inc. (St. Louis, Mo., U.S.A.) or Fisher Scientific (Pittsburg, Pa., U.S.A.). The amino acid kits used for this study were purchased from Sigma-Aldrich, Inc. (St. Louis) and these are in free form. 1-PeCSO and 2-PeCSO were isolated from onion and garlic. (+)-S-(1-propenyl)-L-cysteine sulfoxide and (+)-S-(2-propenyl)-L-cysteine sulfoxide, the natural forms of 1-PeCSO and 2-PeCSO in onion and garlic, respectively, were obtained by the modified method of Shen and Parkin (2000). Onion and garlic juices prepared from heat-treated onion and garlic bulbs were adjusted to pH 6.5 with acetic acid. Onion and garlic juices were passed through a column of Dowex Marathon cation-exchange resin (Midland, Mich., U.S.A.) saturated with H+. Adsorbed amino acids and peptides were eluted with 0.1 N NaOH adjusted to pH 9 with acetic acid and then eluted with 0.25 N ammonium hydroxide. The eluate was tested with 0.8% ninhydrin in ethanol to confirm the presence of amino acids. Fractions containing 1-PeCSO or 2-PeCSO were separated using a Perkin Elmer (Shelton, Conn., U.S.A.) HPLC equipped with a Series 200 pump, Series 200 autoinjector, and Series 200 UV-vis detector and a preparative Econosil C-18 column (Alltech, 22 × 250 mm, 10 μm). The column was eluted at 8 mL/min with an isocratic mode of 100% H2O containing 0.5% acetic acid. Eluate was monitored at 220 nm.

Plant materials

Garlic bulbs (China) were purchased in a local store in College Station, Tex., U.S.A., in March 2007 and white onion bulbs were harvested in Weslaco, Tex., U.S.A., in March 2007.

Purification of alliinase in garlic

Purification of alliinase in garlic was carried out according to Imai and others (2006b). Unheated garlic juice chilled at 5 °C was adjusted to pH 4.0 with HCl. The precipitate was collected by centrifugation and redissolved in 0.05 M potassium phosphate buffer (pH 6.5) containing 10% glycerol and 20 μM pyridoxal-5′-phosphate.

Determination of optimal ratio of 1-PeCSO and 2-PeCSO for pigments formation

To investigate the optimal ratio of 1-PeCSO and 2-PeCSO for blue pigment formation, 1 mL each of 1-PeCSO solution (10 mg/mL) and 2-PeCSO (10 mg/mL) were mixed to obtain ratios of 0-100% (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) and were reacted with 20 μL of alliinase solution and 0.5 mL of glycine solution (10 mg/mL) in distilled H2O. After incubation at 60 °C for 3 h, the dark blue–green solutions were filtered, and their UV-vis spectra were recorded at 520 and 580 nm.

Preparation of greened garlic-onion juice

Garlic (200 g) and onion (100 g) were peeled and microwaved in a Magic Chef microwave oven for 3 min at 1000 W to inactive alliinase then fresh garlic (10 g) was added. Samples were homogenized with 300 mL of distilled H2O using a blender and the slurry was filtered through Miracloth. The filtrate obtained (400 mL) was centrifuged at 13000 ×g for 20 min. The aqueous upper layer was incubated at 60 °C for 3 h to generate blue–green pigments, then centrifuged at 13000 ×g for 20 min.

Reaction of thiosulfinate with amino acids

Garlic bulbs (100 g) and onion (50 g) were homogenized with 50 mL of distilled H2O using a blender. Thiosulfinates from samples were extracted with 100 mL of ethyl acetate and added to 100 mL of acetone to remove amino acids, and then added to 500 mL of distilled H2O to separate the ethyl acetate layer. The ethyl acetate layer was collected and evaporated using a rotary evaporator and 50 mL of distilled H2O was added to redissolve thiosulfinate in distilled H2O. Each amino acid was dissolved in distilled H2O (10 mg/mL) and 1 mL of each amino acid solution was reacted with 1 mL of thiosulfinate solution. Vials were shaken and incubated at 60 °C for 3 h to generate pink to blue pigments. Amino acids used in reactions with thiosulfinate solution are listed in Table 1.

Table 1—.  List of amino acids used in the reaction with thiosulfinate solution and abbreviations.
OrderAmino acidAbbreviationOrderAmino acidAbbreviation
1cysteineCys13alanineAla
2phenylalaninePhe14aspartic acidAsp
3glycineGly15histidineHis
4methionineMet16threonineThr
5arginineArg17leucineLeu
6valineVal18asparagineAsn
7isoleucineIle19glutamineGln
8prolinePro20cystineCyt
9lysineLys21glutamic acidGlu
10serineSer22tyrosineTyr
11tryptophanTrp   
12hydroxyl prolineh-Pro   

Analysis of blue–green pigment spectra

Blue–green pigments generated from a mixture of thiosulfinate and 22 kinds of amino acids solution were analyzed using a Perkin Elmer (Shelton) HPLC equipped with a Series 200 pump, Series 200 autoinjector, and Series 200 diode array detector ranging between 200 and 700 nm and an analytical Nova-Pak C-18 column (Waters, 4.6 × 150 mm, 4 μm). The column was eluted at 1 mL/min with a gradient of MeOH containing 0.5% phosphoric acid (40 min linear gradient 20% to 60% methanol, 3 min 100% methanol).

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

In the 1st stage of the study, an optimal ratio of 1-PeCSO and 2-PeCSO for blue–green pigment formation was investigated to develop a model reaction system that consisted of 1-PeCSO, 2-PeCSO, alliinase, and glycine. Different ratios of 1-PeCSO, 2-PeCSO, alliinase, and glycine were mixed and evaluated using a spectrophotometer. Pink to blue pigments developed in the model solutions (Table 2). Pigment was formed in solutions containing only 1-PeCSO, but no absorbance maximum was detected in a 100% 2-PeCSO solution. Blue pigments developed only in combinations containing both 1-PeCSO and 2-PeCSO, and the absorbance at 580 nm increased with increasing 1-PeCSO until reaching a maximum value. The highest value obtained (0.53) was for a 30% 1-PeCSO and 70% 2-PeCSO solution. However, there was no absorbance at 580 nm in solutions of 60% to 100% 1-PeCSO and resulting colors were violet to pink with no significant difference. Kubec and others (2004) reported 1-propenyl derivatives form pink, pink-red, and magenta compounds, whereas those containing allyl groups give rise to blue products after reacting with glycine. Imai and others (2006b) confirmed that a vivid-blue color could be produced using a highly defined model reaction system comprised only of isolated 1-PeCSO, 2-PeCSO, pure glycine, and purified garlic alliinase. Our results are consistent with these investigations.

Table 2—.  Absorbance of solutions consisting of 1-PeCSO and 2-PeCSO mixed in 0-100% ratios. Alliinase and glycine were present in all solutions.
1-PeCSO:2-PeCSOAbsorbance
520 nm580 nm
0: 1000  0  
10: 900.150.22
20: 800.260.45
30: 700.330.53
40: 600.360.48
50: 500.340.37
60: 400.270  
70: 300.260  
80: 200.280  
90: 100.270  
100: 00.290  

On the basis of absorbance at 580 nm, we conducted the next investigation with a 30% 1-PeCSO and 70% 2-PeCSO solution. All 22 amino acids were reacted with the thiosulfinate solution which comprised a 30% 1-PeCSO, 70% 2-PeCSO solution and alliinase. HPLC spectra were compared with naturally greened extracts from garlic-onion juice. Because blue–green pigments are easily generated by mixing juice from heated white onions (Imai and others 2006b), a good source of 1-PeCSO, we added white onions to the garlic juice. Peaks in HPLC spectra were detected at 440 nm and 580 nm in the visible range with different retention times: 2.7 to 5.6 min and 16.6 to 28.3 min, respectively (Figure 2). These spectra indicated that the green color was a mixture of yellow pigment (440 nm) and blue pigment (580 nm). It was previously assumed that at least 2 species of pigment existed in “Laba” garlic pickling solution using spectrophotometer with 2 maxima absorptions at 440 and 590 nm: one species with a yellow color and a second with a blue color. The combination of both species would therefore result in the observed green color of the pickling solution (Bai and others 2005; Wang and others 2008). In this study, we separated yellow and blue pigments using HPLC and diode array detector and we confirmed this discoloration was blueing, not greening. Figure 2 showed 8 blue peaks detected at 580 nm in natural garlic-onion juice. Their retention times were 16.6 (1), 19.7 (2), 20.8 (3), 21.7 (4), 23.5 (5), 25.1 (6), 26.9 (7), and 28.3 (8). Each peak was matched with an amino acid-thiosulfinate reaction solution. As shown in Figure 3, different kinds of blue pigments developed when different amino acids were reacted with thiosulfinate solution. In contrast to Kubec and others (2004), who reported that pink pigment is developed with only 1-PeCSO and alliinase, pink pigment did not develop in the absence of amino acid. Most of the amino acids formed blue pigments by reaction with thiosulfinate except Cys, Pro, Met, and h-Pro. However, peaks at 580 nm were detected only when mixed with Gly, Arg, Lys, Ser, Ala, Arp, His, Asn, Gln, and Tyr (Figure 4) and the greatest color-generating ability was exhibited by Gly, followed by Lys and Asn (data not shown). Compared with the natural greened extract, Arg matched with (1) and (2), Lys matched with (3), (4), and (7), Asn matched with (5), (7), and (8) and Gln matched with (5), (6), and (7), Gly and Ser matched with (7), and Asp matched with (8) (Table 3). Although glycine showed the greatest color-generating ability and most of previous researcher concluded glycine was the major amino acid involved in discoloration reaction (Shannon and others 1967a,b; Kubec and others 2004; Imai and others 2006b; Kubec and Velíšek 2007), the spectrum of glycine did not match well with the spectrum of greened garlic juice. Major peaks of glycine were detected at 24.9, 27.2, and 30.8 min, matching only 7, which is a minor peak in the natural greened extract. On the other hand, Arg, Asn, and Gln had spectra that were the most similar to the spectra of natural green garlic-onion juice. According to reports on the amino acid composition of garlic, Arg, Gln, Asn, and Glu are typically the most abundant free amino acids (Montaño and others 2004; Lee and others 2005) and these amino acids also have the ability to form blue pigments. Moreover, Gly was one of the minor amino acids and some varieties were not even detected glycine (Lee and Harnly 2005), although Gly showed the strongest blue color when reacted with thiosulfinate solution. Based on these findings, candidate amino acids for forming blue pigments in green garlic juice were Arg, Asn, and Gln rather than Gly.

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Figure 2—. Spectrum of blue pigments in natural garlic juice. Garlic bulbs were homogenated and filtered then the filtrate was incubated at 60 °C for 3 h to generate blue pigments.

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Figure 3—. Color variation by reaction with thiosulfinate and different amino acids. Each number corresponds to a different amino acid in Table 1.

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Figure 4—. Spectra of generated blue pigments by amino acids. Each number corresponds with a different amino acid in Table 1.

aSpectrum of natural green garlic juice.

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Table 3—.  Comparison of peaks at 580 nm in natural green garlic extract, and mixtures of amino acids and thiosulfinate.
Amino acidPeak no. of green garlic extract at 580 nm
12345678
3. Gly------O-
5. ArgOO------
9. Lys--OO--O-
10. Ser------O-
13. Ala--------
14. Asp-------O
15. His--------
18. Asn----O-OO
19. Gln----OOO-
22. Tyr--------

In the last step of this study, we compared spectra and chromatograms at 580 nm of blue pigments formed by the 3 major candidate amino acids: Arg, Asn, and Gln, with natural green juice to confirm retention time and spectra of each peak. Figure 5 shows that for addition of the candidate amino acids to both natural green juice and thiosulfinate solution each peak had the same retention time as one of the peaks in the natural green juice and no extra peaks developed due to treatment of the amino acid.

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Figure 5—. Spectra and chromatogram at 580 nm of blue pigments generated by major candidate amino acids; (A) addition of arginine to natural garlic juice; (B) addition of asparagine to natural garlic juice; (C) addition of glutamine to natural garlic juice; (D) addition of arginine to thiosulfinate solution; (E) addition of asparagine to thiosulfinate solution; (F) addition of glutamine to thiosulfinate solution.

aSpectrum of natural green garlic juice.

bChromatogram of each sample; pink line, control; brown line, arginine; blue line asparagine; green line, glutamine.

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

“Green discoloration” in crushed garlic represents a mixture of yellow and blue pigments, and the blue color results from at least 8 pigments, depending on amino acid composition, rather than a single blue pigment. Resulting color varied from pink to blue depending on the ratio of 1-PeCSO and 2-PeCSO. The optimal ratio for generating blue pigment was 30% 1-PeCSO and 70% 2-PeCSO. Apparently, greening in crushed garlic occurs when it contains 1-PeCSO, 2-PeCSO, and amino acids. Results indicated that amino acids other than glycine have the potential to form blue pigments. However, previous studies have only reported a role for glycine in greening (Shannon and others 1967a,b; Kubec and others 2004; Imai and others 2006a,b) and most of model reactions were conducted with glycine (Kubec and others 2004; Kubec and Velíšek 2007; Wang and others 2008). In this study, the mixture of glycine and thiosulfinate did not have a similar spectrum to the natural greened garlic extract despite the strong ability of glycine to generate blue pigments. The spectra of thiosulfinate reacted with arginine, glutamine, or asparagine, which are the most abundant amino acids in garlic, matched well with the spectrum of natural green garlic extract.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

This material was based upon work supported by the Cooperative State Research, Education, and Extension Service, U.S. Dept. of Agricultural under Agreement No. 2006-34402-17121, “Designing Foods for Health” through Vegetable & Fruit Improvement Center, Texas AgriLife Research.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References