Colorimetric determination of dehydroalanine in wheat gluten
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Transcript of Colorimetric determination of dehydroalanine in wheat gluten
1
This is the final draft post-refereeing. 1
The publisher’s version can be found at http://dx.doi.org/ 10.1016/j.jcs.2011.04.007 2
Please cite this article as: Rombouts, I.; Lagrain, B.; Brijs, K.; Delcour, J. A. Colorimetric determination of 3
dehydroalanine in wheat gluten. Journal of Cereal Science. 2011, 54, 148-150. 4
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Colorimetric determination of dehydroalanine in wheat gluten 6
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Ine ROMBOUTS*, Bert LAGRAIN, Kristof BRIJS, and Jan A. DELCOUR 8
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Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre 11
(LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium 12
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*Corresponding author 15
Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre 16
(LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium. 17
Tel.: (+32)-16-321634. Fax: (+32)-16-321997. E-mail: [email protected] 18
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Keywords 21
Dehydroalanine; Gluten; Lysinoalanine; Lanthionine 22
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Alphabetical list of abbreviations 25
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Abstract 29
The present paper describes a method for quantifying dehydroalanine (DHA) in wheat gluten. 30
The procedure consists of hydrolysis, transforming peptide-bound DHA into free pyruvic acid, 31
followed by colorimetric determination of the latter. Optimal conditions for the hydrolysis reaction 32
and the colorimetrical assay of pyruvic acid have been determined for the specific case of gluten 33
proteins. 34
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Introduction 36
Dehydroalanine (DHA) residues can be formed in proteins by heating and/or under alkaline 37
conditions by a -elimination reaction of e.g. cystine. Subsequent reaction of DHA with cysteine or 38
lysine leads to lanthionine or lysinoalanine, respectively. The formation of such non-reducible cross-39
links affects protein structure (Friedman, 1999). Furthermore, lysinoalanine formation reduces the 40
bio-availability of lysine, and forms a potential risk for food safety (Abe et al., 1984). Thus, it is 41
important to quantify DHA for structural, toxicological and nutritional reasons. Alkaline processing 42
also decreases the nutritional value of food proteins, e.g. by the formation of D-amino acids (Gilani 43
et al., 2005; Sanderson et al., 1978), so DHA can be an indicator for the impact of heat/alkaline 44
treatment. 45
This paper deals with DHA determination in gluten proteins, the storage proteins of wheat. 46
Gluten proteins are a co-product of the industrial purification of wheat starch. Previous research 47
showed that heating at alkaline pH of gliadin, the monomeric gluten fraction, results in formation of 48
DHA, lanthionine, and lysinoalanine (Rombouts et al., 2010). These reactions can e.g. be relevant for 49
pretzels, which are dipped in a hot alkaline solution prior to baking, and for yellow alkaline noodles, 50
which are prepared with addition of alkaline reagents, usually sodium or potassium carbonates (Fu, 51
2008). Also, alkali is used for extrusion cooking of wheat flour (Bredie et al., 2002), to improve gluten 52
solubility and functionality (Van Der Borght et al., 2005), and to produce gluten based biofilms 53
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(Kayserilioglu et al., 2001). In spite of the numerous food and non-food purposes of DHA 54
determination in gluten, no convenient general method has yet been reported. 55
Patchornik and Sokolovsky (1964) and Steenken and Zahn (1984) converted DHA in non-gluten 56
proteins and peptides to pyruvic acid and ammonia by acid hydrolysis and subsequently quantified 57
pyruvic acid colorimetrically. While this method is accurate and user-friendly, it requires some 58
optimization for gluten proteins. Indeed, for each specific protein, hydrolysis for several time 59
intervals are necessary to correct for hydrolytic decomposition of pyruvic acid (Steenken and Zahn, 60
1984). Rombouts and co-workers (Rombouts et al., 2010) optimized this method for determining 61
DHA contents in gliadin, but the optimized method was not reproducible when applied to the 62
complete gluten fraction, probably due to formation of side products during hydrolysis which 63
interfere with the colorimetric assay. 64
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Materials and Methods 66
Materials. Wheat gluten from Syral (Aalst, Belgium) contained 76.6 % protein (N x 5.7) on dry 67
basis. Heat/alkali treated gluten was obtained by heating gluten (1.00 g) for different times (0-120 68
min) at 130 °C in closed test tubes in 5.0 ml 50 mM sodium phosphate buffer (pH 8.0). After 69
treatment, samples were freeze-dried, ground in a laboratory mill (IKA, Staufen, Germany) and 70
sieved (250 µm). Chemicals and reagents were from VWR International (Leuven, Belgium) unless 71
specified otherwise, and of at least analytical grade. 72
Acid hydrolysis. To liberate DHA in gluten proteins as pyruvic acid, gluten proteins (100 mg) were 73
heated in sealed reaction tubes in 1.5 N HCl (0.50 ml) at 110 °C for different times (30, 90, 120, 180, 74
240, 300, 360, 1200 and 1440 min). 75
Clarification and colorimetric determination of pyruvic acid. The mixture obtained after acid 76
hydrolysis, further referred to as the hydrolyzate, was clarified to avoid interference during the 77
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colorimetric assay. Carrez I solution [0.50 ml, 85 mM K4[Fe(CN)6], Sigma-Aldrich, Steinheim, 78
Germany], 0.5 ml Carrez II solution [250 mM ZnSO4 (Sigma-Aldrich)] and 1.00 ml 0.10 N NaOH 79
(Mallinckrodt Baker, Deventer, The Netherlands) were subsequently added to the hydrolyzate. The 80
mixture was shaken vigorously after addition of each reagent, diluted to 10.0 ml, and filtered 81
through a filter paper (MN615, diameter 90 mm, Macherey-Nagel, Düren, Germany). An aliquot 82
(1.30 ml) of the clear mixture was pipetted into a cuvet, and 0.13 ml 1.0 N NaOH (Mallinckrodt 83
Baker), 1.17 ml Tris/HCl buffer (pH 7.4, 0.50 M), and 200 µl 2.82 mM NADH (Megazyme, Bray, 84
Ireland) were added. The extinction at 340 nm was measured before and exactly 3 min after addition 85
of 20 µl D-lactate dehydrogenase (D-LDH) solution (2000 units/ml, units as defined by the supplier, 86
Megazyme). The pyruvic acid concentration was calculated from the extinction drop caused by the 87
consumption of NADH, the extinction coefficient of which at 340 nm is 6.3 . 10³ mol-1.l.cm-1 (O'Neill 88
et al., 2001). 89
Statistical analysis. Standard addition plots were made by adding different levels of standard 90
pyruvic acid (0.0-3.0-6.0-9.0-12.0 µM) to the hydrolyzates of non-treated and heat/alkali treated 91
gluten sample. After clarification and dilution, the extinctions were measured at 340 nm, and 92
plotted as a function of the level of added pyruvic acid in the cuvet. Pearson correlation coefficient 93
(r), slope, and intercept were calculated using the Analyse-It software from Microsoft Excel 2010. 94
The extinction coefficient was calculated from the slope value. 95
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Results and discussion 97
The first goal of this study was to determine the hydrolysis time that maximizes the release of 98
DHA in gluten proteins as pyruvic acid, and minimizes the possible degradation of the latter. 99
Steenken and Zahn (1984) hydrolyzed wool protein in 6.0 N HCl at 105 °C, but the transformation of 100
peptide bound DHA to free pyruvic acid can be achieved under less drastic pH conditions (Patchornik 101
and Sokolovsky, 1964). We hydrolyzed gluten proteins in 1.5 N HCl at 110 °C. The formation of 102
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pyruvic acid during acid hydrolysis, as reported by Damodaran and Ramachandran (1940), was 103
evaluated by hydrolyzing gluten proteins for different times (Figure 1). No pyruvic acid was found in 104
non-treated gluten samples after hydrolysis for 120 min or less, indicating that short hydrolysis times 105
do not result in spontaneous formation of pyruvic acid. However, longer hydrolysis times led to 106
overestimation of DHA. To evaluate degradation of pyruvic acid during acid hydrolysis, heat/alkali 107
treated (pH 8.0, 130 °C) gluten proteins, containing DHA, were hydrolyzed for 30, 90 and 180 min 108
(Figure 1). The pyruvic acid level increased with hydrolysis time. Furthermore, heat treatment of 109
pyruvic acid in 1.5 N HCl at 110 °C for 120 min did not decrease the pyruvic acid level. Thus, it is 110
suggested to hydrolyze gluten proteins in 1.5 N HCl at 110 °C for 120 min, because the above results 111
indicate that such conditions minimize spontaneous formation and degradation of pyruvic acid. 112
Following conversion of the DHA residues to pyruvic acid, levels of the latter were determined 113
colorimetrically. For some protein samples, like wool and gliadin, pyruvic acid can be determined 114
without hydrolyzate pretreatment. For gluten, clarification of the hydrolyzate is required to avoid 115
interference during the colorimetric assay. Because the clarified neutralized mixture may still contain 116
impurities interfering with the absorbance reading, different levels of pyruvic acid standard were 117
added to hydrolyzed non-treated and heat/alkali treated gluten sample. Based on the standard 118
addition plots (r > 0.95), an extinction coefficient of 6.2 . 10³ mol-1.l.cm-1 was obtained for gluten 119
proteins. This value is in close agreement with the extinction coefficient of NADH at 340 nm (6.3 . 10³ 120
mol-1.l.cm-1). 121
The determination of DHA has, to the best of our knowledge, not been optimized for gluten 122
proteins. Literature reports that alkaline pH favors DHA formation during heating, and this was 123
confirmed for gluten (Figure 2). Standard deviations ranged from 0.0 to 0.9 µmol/g protein. Lagrain 124
and co-workers (2010) showed that the sum of cystine, DHA and lanthionine levels in gliadin remains 125
constant during hydrothermal treatment at alkaline pH. In such reactions, cystine is converted into 126
cysteine, sulfur and DHA, and some of the formed DHA reacts with cysteine to form lanthionine. 127
Table 1 shows that the molar loss of cystine during hydrothermal treatment of gluten at pH 8.0 and 128
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130 °C corresponds to the sum of the lanthionine and DHA levels formed. Thus, the measured DHA 129
levels are in line with the theoretical values based on the stoichiometry of the reaction, which 130
supports the accuracy of the present method. The limit of detection of the present method is 0.3 131
µmol/g protein. 132
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Conclusions 134
An accurate method was developed for quantifying DHA in wheat gluten. The procedure consists 135
of the transformation of DHA into pyruvic acid in 1.5 N HCl (120 min, 110 °C) and subsequent 136
colorimetric determination of the formed pyruvic acid. Hydrolyzate mixtures are clarified by the 137
Carrez reagents prior to the colorimetric assay, and an extinction coefficient of 6.3 . 10³ mol-1.l.cm-1 is 138
used to calculate the pyruvic acid concentration from the extinction values. 139
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Acknowledgments 141
This work is a part of the Methusalem programme “Food for the Future” at the K.U.Leuven. B. 142
Lagrain wishes to acknowledge the Research Foundation – Flanders (FWO, Brussels, Belgium) for a 143
function as postdoctoral researcher. K. Brijs wishes to acknowledge the Industrial Research Fund 144
(K.U.Leuven, Leuven, Belgium) for a position as Industrial Research Fund fellow. 145
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Literature cited 147
Abe, K., Suehara, S., Homma, S., Fujimaki, M., Arai, S., 1984. Metabolic transit of lysinoalanine in the rat-148
body - Observation of feeding tests with alkali-treated casein and gluten. Agricultural and 149
Biological Chemistry 48, 579-584. 150
Bredie, W.L.P., Mottram, D.S., Guy, R.C.E., 2002. Effect of temperature and pH on the generation of 151
flavor volatiles in extrusion cooking of wheat flour. Journal of Agricultural and Food Chemistry 152
50, 1118-1125. 153
Damodaran, M., Ramachandran, B.V., 1940. Amino-acids of casein phosphopeptone. Nature 145, 857. 154
Friedman, M., 1999. Chemistry, biochemistry, nutrition, and microbiology of lysinoalanine, lanthionine, 155
and histidinoalanine in food and other proteins. Journal of Agricultural and Food Chemistry 47, 156
1295-1319. 157
Fu, B.X., 2008. Asian noodles: History, classification, raw materials, and processing. Food Research 158
International 41, 888-902. 159
Gilani, G.S., Cockell, K.A., Sepehr, E., 2005. Effects of antinutritional factors on protein digestibility and 160
amino acid availability in foods. Journal of AOAC International 88, 967-987. 161
Kayserilioglu, B.S., Stevels, W.M., Mulder, W.J., Akkas, N., 2001. Mechanical and biochemical 162
characterisation of wheat gluten films as a function of pH and co-solvent. Starch-Stärke 53, 381-163
386. 164
Lagrain, B., De Vleeschouwer, K., Rombouts, I., Brijs, K., Hendrickx, M.E., Delcour, J.A., 2010. The kinetics 165
of beta-elimination of cystine and the formation of lanthionine in gliadin. Journal of Agricultural 166
and Food Chemistry 58, 10761-10767. 167
O'Neill, M.J., Smith, A., Heckelman, P.E., 2001. The Merck Index: an encyclopedia of chemicals, drugs, 168
and biologicals, 13th ed. Merck & Co. Inc., Whitehouse Station, NJ. 169
Patchornik, A., Sokolovsky, M., 1964. Nonenzymatic cleavages of peptide chains at cysteine + serine 170
residues through their conversion into dehydroalanine .1. Hydrolytic + oxidative cleavage of 171
dehydroalanine residues. Journal of the American Chemical Society 86, 1206-1212. 172
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Rombouts, I., Lagrain, B., Brijs, K., Delcour, J.A., 2010. [beta]-Elimination reactions and formation of 173
covalent cross-links in gliadin during heating at alkaline pH. Journal of Cereal Science 52, 362-174
367. 175
Sanderson, J., Wall, J.S., Donaldson, G.L., Cavins, J.F., 1978. Effect of alkaline processing of corn on its 176
amino-acids. Cereal Chemistry 55, 204-213. 177
Steenken, I., Zahn, H., 1984. Determining the dehydroalanine and amide contents of wool as a means of 178
estimating damage caused by alkaline treatments. Textile Research Journal 54, 429-433. 179
Van Der Borght, A., Goesaert, H., Veraverbeke, W.S., Delcour, J.A., 2005. Fractionation of wheat and 180
wheat flour into starch and gluten: overview of the main processes and the factors involved. 181
Journal of Cereal Science 41, 221-237. 182
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Figure captions 186
Figure 1: Levels of pyruvic acid in untreated (▲) and heat/alkali treated (●) gluten after hydrolysis in 187
1.5 N HCl solution at 110 °C for different times. 188
Figure 2: Dehydroalanine levels in gluten after heating at 130 °C in 50 mM sodium phosphate buffer 189
(pH 7.0, 8.0, or 13.0) as a function of heating time. Samples were hydrolyzed in 1.5 N HCl solution at 190
110 °C for 120 min. 191
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Table 1: Levels (µmol/g protein) of dehydroalanine, cystine, and lanthionine, and their sum, in gluten 200
after hydrothermal treatment at 130 °C in sodium phosphate buffer (50 mM, pH 8.0). Standard 201
deviations are reported between brackets. 202
Heating time (min)
Cystine Dehydroalanine Lanthionine Sum
0 78 (1) 0,0 (0,0) 0 (0) 78
30 48 (2) 5,6 (0,7) 24 (1) 78
45 42 (1) 5,4 (0,1) 30 (0) 77
60 40 (2) 6,4 (0,0) 32 (2) 78
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