Article pubs.acs.org/JAFC
Toward an Understanding of Mechanisms Involved in NonPolyphenol Oxidase (Non-PPO) Darkening in Yellow Alkaline Noodles (YAN) Robert E. Asenstorfer,*,† Marie J. Appelbee,‡,§ Christine A. Kusznir,† and Daryl J. Mares† †
School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA 5064, Australia South Australian Research and Development Institute, Grain Quality Research Laboratory, Waite Research Precinct, Glen Osmond, SA 5064, Australia
‡
ABSTRACT: Asian noodles prepared from bread wheat flour darken over time due to a combination of polyphenol oxidase (PPO) activity and non-PPO effects. Although the enzymatic mechanism associated with the PPO reaction is well established, the non-PPO component consists of both physical (e.g., changes in surface properties) and chemical reactions. Variations in pH and solvents were used to gain a quantitative estimate of the contribution of physical and chemical components to non-PPO darkening in yellow alkaline noodles (YAN). In a set of five common high-PPO Australian wheat cultivars it was estimated that on average non-PPO darkening accounted for 69% of total darkening, with approximately two-thirds of this due to physical darkening and one-third had a chemical origin. Data from the chemical portion of non-PPO darkening is consistent with the presence of a PPO-like enzyme that oxidizes tyrosine, has a pH maximum of 8.1, and is inhibited by 50% methanol or ethanol but in the noodle is insensitive to PPO inhibitors such as tropolone. Therefore, with low-PPO and PPO-free wheat varieties becoming available, it may be possible to further reduce darkening in YAN by breeding for wheat varieties with low or zero levels of this PPO-like enzyme. KEYWORDS: yellow alkaline noodle (YAN), polyphenol oxidase (PPO), non-PPO darkening
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INTRODUCTION Yellow alkaline noodles (YAN) are made from bread wheat (Triticum aestivum L.) flour treated with alkaline salts. A bright yellow color free from dark specks is highly desirable. YAN are usually sold as a fresh product, although they can be parboiled and refrigerated to extend storage time. Fresh YAN darken over time, which decreases their marketability. Polyphenol oxidase (PPO), which is inhibited by tropolone and salicylhydroxamic acid, causes about one-third to half of the darkening.1 It is, however, possible to reduce PPO darkening by the selection of wheat varieties with low or zero PPO activity.2,3 The remaining darkening is non-PPO darkening and results from both physical and chemical factors.1 The physical component of non-PPO darkening involves changes in the noodle matrix, which alter the reflectance properties of the noodle surface, whereas the chemical component of non-PPO darkening is thought to arise from oxidation of tyrosine to yield black pigments.4 In previous studies, non-PPO darkening as measured by a change in the CIE-Lab L* value was described using two time periods, 0−4 and 4−24 h.1,4 Non-PPO darkening during the first time period was correlated with protein content1 and was related to physical changes in the noodle.1 Non-PPO darkening in the second time period (4−24 h) was independent of total protein content and was thought to be predominantly due to chemical oxidation rather than physical changes in the noodle. Oxidation of tyrosine groups within the glutenin and gliadin proteins with subsequent polymerization provides a possible mechanism for darkening during the 4−24 h time period.1 Possible evidence for this was the addition of n-propanol extracts (containing predominantly gliadin and traces of unpolymerized glutenin), © 2014 American Chemical Society
tyrosine, or the peptide GQQGYYPTS (an analogue of the consensus motifs GYYPTS(L/G)QQ) to YAN increased darkening during the 4−24 h time period.1 Because a lack of specific inhibitors for non-PPO darkening,5 it has been difficult to determine the contribution of the chemical component to total non-PPO darkening in YAN. The aim of this paper is to gain an estimate of the proportion of physical and chemical components of non-PPO darkening and to examine the origin of the chemical portion of non-PPO darkening.
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MATERIALS AND METHODS
Materials. A hard white bread wheat with high-PPO activity (Triticum aestivum L. cv. Tasman) and a durum wheat with a near-zero PPO activity (Triticum durum Desf. cv. Kamilaroi) were selected. Grains of both cultivars were produced in field trials at the Waite Campus of the University of Adelaide.1,4 Four hard white high-PPO bread wheat cultivars (Lark, Sunvale, Janz, and Yitpi) and a hard white low-PPO bread wheat (Axe) produced in field trials at the Waite Campus of the University of Adelaide were also examined for chemical and physical components of non-PPO darkening. PPO activities for the cultivars as determined according to the method of Bernier and Howes2,6,7 were measured as an increase in absorbance of a tyrosine solution at 415 nm after 3 h: Tasman, 0.27 ± 0.05; Kamilaroi, 0.02 ± 0.01; Lark, 0.15 ± 0.02; Sunvale, 0.20 ± 0.01; Janz, 0.15 ± 0.09; Yitpi, 0.15 ± 0.02; Axe, 0.06 ± 0.01. Absorbance for Kamilaroi was not significantly different from control (solution with no grains), and indeed there was no visible change in color of the incubation solution or the grain coat. Received: Revised: Accepted: Published: 4725
January 13, 2014 April 4, 2014 May 1, 2014 May 1, 2014 dx.doi.org/10.1021/jf500206e | J. Agric. Food Chem. 2014, 62, 4725−4730
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Flour Milling and Noodle Sheet Preparation. Grain was conditioned overnight to 15% moisture and milled on a Bühler experimental mill (Bühler AG, Uzwil, Switzerland). The break and reduction flour streams were combined. The pollard stream was resieved on a shaker, and the recovered flour was added to the combined streams from the mill. Different batches of grain gave slightly different color responses, and therefore the same batch was used for each experiment and, when possible, for the different experiments. Flour (10 g) and 3.6 mL of 2% sodium carbonate solution, with or without the PPO inhibitor tropolone (0.01 mol L−1), were mixed into a dough in a cylindrical mixing bowl using a drill press with a modified mixing paddle. The dough was mixed for 105 s with intermittent pauses to clean dough adhering to the paddle. The dough was rolled into a ball and made into a noodle sheet using a domestic pasta maker (Atlas 150, Marcato S.P.A., Campodarego, Italy). The noodle sheet was then placed in a resealable plastic bag to prevent drying and to make noodle handling easier. Processing and storage were at room temperature (22 °C). Darkening Rates. CIE-Lab color measurements of raw noodles were made using a Dataflash 100 (Datacolor International, Lawrenceville, NJ, USA) reflectance spectrophotometer. Each data point represents the average of two noodle sheets, each of which was measured twice. The rates of darkening for the two time periods were determined from the L* value of the noodle sheets measured at 2, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, and 1440 min. Mixedorder rate equations (ΔL* = ktn + c, where ΔL* is the change in darkening between time 2 min and time t of darkening, k is the rate, t is time in h, and n and c are constants) were fitted to the noodle darkening and standard errors estimated using WinCurveFit.8 pH Analysis. Noodles from near-zero-PPO Kamilaroi flour were made in a series of buffers: 2% HCl, 0.2 mol L−1 sodium citrate (pH 4.0, 5.0, 6.0); 0.2 mol L−1 Tris (pH 7.0, 8.0, 9.0, 10.0); 0.2 mol L−1 NaCO3; 1% NaOH; 1.5% NaOH; 2% NaOH. The L* values were measured at 0, 2, 4, and 24 h. Noodles (1 g) were extracted in 2 mL of water and centrifuged, and the pH was measured. A curve9
ΔL* =
adding n-propanol (750 mL) to the precipitate and extracting for 30 min at 60 °C. This extract was centrifuged at 1500g for 10 min and the supernatant retained (if dried by rotary evaporation this yielded ≈10− 12 g). In preference to drying, different amounts of water or propanol were added, and the precipitates and supernatants were analyzed for noodle darkening activity. A fraction with the highest activity was obtained by diluting the supernatant to 9% propanol and allowed to stand for 72 h. The supernatant was decanted, and both supernatant and precipitate were dried by rotary evaporation (yields: precipitate, ≈1.5 g; supernatant, ≈9 g). The precipitate was washed with 200 mL of chloroform to give two fractions, chloroform soluble (≈0.7 g) and chloroform insoluble (≈0.7 g), which were dried by rotary evaporation. The chloroform-insoluble material was subsequently redissolved in warm 20% propanol (50 mL) and fractioned using a C18 column (90 mm × 20 mm dia., Bodesil-C18 40 μm, Varian, Palo Alto, CA, USA), collecting the elution with 100 mL of 20% propanol fraction (≈0.3 g) and then eluting with 50% propanol (100 mL) to obtain a 50% propanol fraction (≈0.4 g). Both fractions were dried by rotary evaporation. PPO Analysis. PPO analyses of protein extracts were based on the 96-well method of Bernier and Howes.6 A tyrosine solution (100 mL) was made with 0.01 mol L−1 tyrosine in 0.1 mol L−1 Tris base buffer (pH 9) with ≈40 μL of Tween 20 added. Protein extract (2 mg) was dissolved in 300 μL of tyrosine solution per microplate well and incubated for 3 h at 37 °C. Portions (100 μL) from each well were transferred into a new microplate, and the optical density was read by a microplate reader. Polyacrylamide Gel Electrophoresis (PAGE). SDS-PAGE separation was achieved byusing the method of Singh et al.14 with minor modifications.15 Acid-PAGE was performed according to the method of Clements.16 Gliadins were extracted from T. aestivum cv. Chinese Spring, Gabo, Cheyenne, and Tasman as reference standards.
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RESULTS AND DISCUSSION Effect of pH on YAN Non-PPO Darkening. It was necessary to establish the amount of physical and chemical components of non-PPO darkening. A higher proportion of chemical than physical darkening occurred during the 4−24 h time period.4 The pH data (Figure 1) indicated that there are
a + b10(pH − pK a1) + c10(2pH − pK a1− pK a1) 1 + 10(pH − pK a1) + 10(pH − pK a1− pK a2)
where a represents the ΔL* plateau at low pH, b is the theoretical maximum ΔL* value, and c represents the plateau at high pH, was fitted to the change in darkening (ΔL*) using WinCurveFit,8 which allowed estimation of pKa (acid dissociation constant) values and maximum and minimum ΔL* values. Incorporation of Solvents into Noodle Preparation. The 2% sodium carbonate solution used to prepare the noodles was made up with different nonaqueous solvents. These were glycerol (Merck Pty Ltd., Kilsyth, VIC, Australia), ethylene glycol (Ajax Chemicals Pty Ltd., Sydney, Australia), methanol (Chem-Supply, Gilman, SA, Australia), ethanol (Chem-Supply), n-propanol (Scharlab S.L., Sentimenat, Spain), and dimethyl sulfoxide (Merck Pty Ltd.). The rates of darkening were measured using the method described above. Mid-infrared (Mid-IR) Spectroscopy. Mid-IR frequencies used to examine changes in the protein quaternary structure were the amide I band (1580−1720 cm−1; 80% C−O stretch, 10% C−N bend, and 10% N−H bend) and amide II band (1480−1580 cm−1; 60% N−H bend and 40% C−N stretch).10,11 The spectrum of noodle sheets was obtained using a PerkinElmer (Waltham, MA, USA) Spectrum One FT-IR spectrometer equipped with a Pike Technologies (Madison, WI, USA) diffuse reflectance autosampler. The data were transferred to GRAMS/ AI12 software, and the areas of the amide bands were determined manually. n-Propanol Extract Fractionation. Initial separation was based on the Osborne method.13 Flour (200 g) was extracted with water (1.5 L) for 30 min at 60 °C and then centrifuged at 1500g for 10 min to give an albumin fraction. A globulin fraction was obtained by extracting the precipitate with a 0.2 mol L−1 NaCl solution (750 mL) for 30 min at 60 °C and then centrifuged at 1500g for 10 min. To the precipitate was added 30% ethanol (750 mL), and the mixture was extracted for 30 min at 60 ° and , then centrifuged at 1500g for 10 min to give gliadin fraction (A). The 50% propanol fraction (gliadin fraction B) was obtained by
Figure 1. Effect of pH on darkening during the 4−24 h time period in noodles made from near-zero-PPO Kamilaroi durum flour (gray area indicates the approximate pH of YAN).
both pH-dependent and pH-independent components to nonPPO darkening during the 4−24 h time period. The maximum darkening of noodles made from Kamilaroi flour occurs at pH 8.1, which is slightly lower than the pH of normal YAN (pH ≈9− 9.2) but similar to the pH of maximum PPO activity in noodles (pH ≈8.2).4 Plateaus associated with minima of non-PPO darkening were observed at pH values 11. These plateaus represent the pH-independent component of non-PPO darkening associated with physical darkening, whereas the pHdependent portion is associated with chemical darkening. In YAN, the chemical portion of non-PPO darkening was estimated 4726
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Table 1. Maximum and Minimum (Plateaus pH 11) Darkening (ΔL*) Values and pKa Values for Non-PPO Darkening in Noodles Made with Near-Zero-PPO Kamlaroi Durum with a Range of pH Values for Three Time Periods (0−4, 4−24, and 0−24 h) ΔL*min
pKa values
time period
ΔL*max (pH)
pH 11
pKa1
pKa2
0−4 h 4−24 h 0−24 h
7.07 (7.86) 5.02 (8.14) 12.09 (7.99)
4.35 ± 0.21 3.10 ± 0.17 7.49 ± 0.28
4.20 ± 0.30 2.16 ± 0.36 6.34 ± 0.48
6.07 ± 0.18 6.54 ± 0.22 6.27 ± 0.13
9.67 ± 0.21 9.91 ± 0.22 9.81 ± 0.16
by calculating the difference between maximum and minimum darkening, whereas the physical portion of non-PPO darkening was estimated by the difference between the minimum and zero darkening. Both of these were expressed as a percentage of maximum darkening. Without knowing the actual baseline darkening at alkaline noodle pH (9−9.2), two preliminary estimates for the chemical portion of non-PPO darkening were calculated by comparing maximum darkening at pH 8.1 with each of the plateaus. On the basis of the low-pH plateau, 38% of non-PPO darkening, or on the basis of the high-pH plateau, 57% of non-PPO darkening, was due to chemical processes during the 4−24 h time period. A similar analysis of the 0−4 and 0−24 h time periods suggested chemical darkening was responsible for approximately 38% (low-pH plateau) to 41% (high-pH plateau) and 38% (low-pH plateau) to 47% (high-pH plateau) of total non-PPO darkening, respectively (Table 1). pKa (acid dissociation constant) values were used to measure the midpoints between maximum and minimum darkening in YAN relative to pH (Figure 1; Table 1). The maximum for nonPPO darkening at pH 8.1 was flanked by a pKa measured for chemical darkening on the low-pH side of the peak (pKa1 = 6.54 ± 0.22) and one on the high-pH side of the peak (pKa2 = 9.91 ± 0.22), which are similar to the pKa values reported for PPO darkening in YAN made from Tasman flour (pKa1 = 7.0; pKa2 = 9.8).4 This suggests the chemical component of non-PPO darkening may be due to an enzyme. Effect of Solvents on YAN Non-PPO Darkening. In preliminary investigations, it was noted that different solvents have an effect on YAN darkening. The solvents investigated can be divided into two groups according to their effects on darkening in noodles made from Kamilaroi flour during the 4−24 h time period (Figure 2). Methanol, ethanol, and propanol show a linear inhibition of darkening with higher concentration until a critical concentration of solute was reached. This coincided with a concentration at which there is inadequate water to make a noodle. Dimethyl sulfoxide (DMSO) and ethylene glycol both showed a lower rate of inhibition until a critical concentration, when the inhibition of darkening increased rapidly (Figure 2). Glycerol at 10% showed a slight enhancement of darkening, and at lower concentrations a curve similar to DMSO and ethylene glycol could be fitted. In YAN made with near-zero-PPO Kamilaroi flour, the maximum inhibition of darkening by methanol or ethanol (50% solution) indicated that 61−64% of darkening during the 4−24 h time period (Figure 2A) and 33−39% of total darkening for the 0−24 h time period were due to non-PPO chemical reaction (Table 2). Whereas the estimate for the chemical portion of non-PPO darkening achieved by methanol/ethanol addition for the 4−24 h time period was slightly higher than the estimate derived from the pH data for the same period (38− 57%), the estimates obtained for the 0−24 h time period by 50% methanol/ethanol addition and by pH (38−47%) method were in reasonable agreement. For the 4−24 h time period, the level of
Figure 2. Effects of different solvents on YAN darkening in noodles made from near-zero-PPO Kamilaroi durum flour during the 4−24 h time period: (A) methanol (circles), ethanol (squares), propanol (diamonds); (B) ethylene glycol (diamonds) and dimethyl sulfoxide (triangles); (C) glycerol; panels B and C are fitted with modified logistic curves (bars represent standard error of the mean).
non-PPO darkening inhibited by 50% methanol or ethanol was similar to the high range for the pH data, indicating the plateau at high pH was a more appropriate baseline for physical non-PPO darkening than the low-pH plateau. Contrary to previous suggestions,1 a considerable amount of physical darkening occurred during the 4−24 h time period. Addition of nonaqueous solvents can displace water from the surface of the protein and cause changes in protein structure, which should be detected by IR spectroscopy.17−19 Whereas the amide II band is strongly influenced by hydration,19,20 no 4727
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Table 2. Comparison of Various Components of Total Darkening (ΔL*0−24) in YAN Made from a Near-Zero-PPO Durum Variety (Kamilaroi), Five High-PPO Bread Wheat Varieties (Tasman, Lark, Sunvale, Janz, and Yitpi), and a Low-PPO Bread Wheat Variety (Axe) av T. aestivum var. (high PPO)
Kamilaroi (near-zero PPO) darkening total PPO non-PPO total physical chemical
Axe (low PPO)
ΔL*0−24
%
ΔL*0−24
%
ΔL*0−24
%
10.68 ± 0.08 0.04 ± 0.15 10.64 ± 0.07 6.82 ± 0.19 3.82 ± 0.26
100 0 100 64 36
12.6 ± 0.32 3.93 ± 0.71 8.67 ± 0.39 5.93 ± 0.31 2.74 ± 0.70
100 31 69 47 22
7.78 ± 0.38 0.27 ± 0.58 7.51 ± 0.20 5.71 ± 0.51 1.80 ± 0.31
100 3 97 73 23
Figure 3. (A) Darkening in noodles made from zero-PPO Kamilaroi flour with 50% methanol (squares), and control (circles) with mixed-order rate equations fitted and their difference (diamonds) fitted with a first-order rate equation. (B) Darkening in noodles made from high-PPO Tasman flour with 0.1 mol L−1 tropolone (squares), 50% methanol (diamonds), 0.1 mol L−1 tropolone and 50% methanol (triangles), and control (circles) with mixed-order rate equations fitted. The difference between YAN made from Tasman flour with 0.1 mol L−1 tropolone and YAN made from Tasman flour with 0.1 mol L−1 tropolone and 50% methanol (cross) fitted a first-order rate equation.
Table 3. Rate (k1) and Order of the Rate Equations Fitted to the Difference in the Darkening (ΔL*) of the Control (0%) and Different Amounts of Alcoholic Solvent Added in YAN Noodles Made from Near-Zero-PPO Kamilaroi Flour methanol
ethanol −1
propanol −1
alcohol
order
k1 (h )
order
k1 (h )
order
k1 (h−1)
10% 20% 30% 40% 50% 60%
1st 1st 1st 1st 1st mixed
0.13 ± 0.06 0.12 ± 0.06 0.08 ± 0.03 0.11 ± 0.02 0.07 ± 0.02
1st 1st 1st 1st 1st
0.05 ± 0.04 0.11 ± 0.01 0.05 ± 0.02 0.08 ± 0.01 0.11 ± 0.02
0th 1st 1st 1st mixed
0.03 ± 0.03 0.02 ± 0.03 0.05 ± 0.03
indicates the effects of 50% methanol only. This curve fits a firstorder rate equation (ΔL* = a exp(−kt), where k is the rate, t is time in hours, and a is a constant), and from this it is possible to estimate the rate of darkening (0.07 ± 0.02 h−1). These difference rate data suggest that although there is sufficient water to make a noodle, methanol or ethanol inhibit first-order or pseudo-first-order reactions at all concentrations (Table 3). The rates of darkening for methanol and ethanol are similar. Additional darkening attributed to PPO, which was estimated by difference in the darkening of Tasman noodles made with and without 0.01 mol L−1 tropolone (a PPO inhibitor; Figure 3B), indicated that PPO darkening also follows a first- or pseudo-firstorder reaction, and the rate is similar to that for inhibition by methanol and ethanol (0.11 ± 0.01 h−1). A first-order or pseudofirst-order reaction is often indicative of an enzyme-catalyzed reaction.
differences were observed in the band areas of either the amide I or the amide II bands between 50% methanol or 50% ethanol and the control (data not shown), suggesting conformational changes in gluten structure were not involved in non-PPO darkening but rather a chemical reaction was involved. Noodles made with very high concentrations of DMSO, ethylene glycol, and glycerol showed little or no darkening, which clearly indicated that there is a requirement for water for both the physical and chemical components of non-PPO darkening to proceed. By calculating the difference in darkening between the control noodle (0% alcohol) and the noodle made with an inhibitor (or organic solvent), it is possible to estimate the rate of inhibition of darkening. For example, Figure 3A shows that the control noodle exhibits darkening at a faster rate and amount than the noodle made with 50% methanol. The difference between these curves 4728
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Table 4. Comparison of Fractions Obtained from T. aestivum cv. Tasman Flour on the Darkening (ΔL*0−24) of Yellow Alkaline Noodles (YAN) Made with Near-Zero-PPO Kamilaroi Flour (Adjusted to 0.1 g/10 g equiv) and the Oxidation of Tyrosine As Measured at 415 nm
Ethanol and methanol have the potential to alter enzyme conformation, leading to inhibition. Whereas the chemical portion of non-PPO darkening is inhibited by ethanol and methanol, PPO activity is only partially inhibited by both methanol and ethanol in noodles made from Tasman flour (Figure 3B). It is known that wheat PPO is resistant to inactivation by quite high levels of methanol and ethanol.21 By using a combination of 50% methanol and tropolone it is possible to estimate the contribution of the different processes to YAN darkening. For example, YAN made from Tasman flour with 0.01 mol L−1 tropolone compared (ΔL* = 9.26) with the control (ΔL* = 15.7) gave an estimate that 41% of darkening was due to PPO. Any darkening in YAN not inhibited by 50% methanol and 0.01 mol L−1 tropolone gives an estimate of the physical portion of non-PPO darkening (ΔL* = 6.36), which is about 41% in Tasman. The remaining darkening (18%) is due to the chemical portion of non-PPO darkening (Figure 3B). In this way, YAN made from five common high-PPO wheat varieties (including Tasman) and a low-PPO variety (Axe) were measured and compared with the near-zero-PPO Kamilaroi to give an estimate of the contribution of the PPO and the physical and chemical portions of non-PPO darkening in YAN (Table 2). There was no significant difference in noodle darkening in the presence or absence of the PPO inhibitor, tropolone. The relative contribution of the chemical portion of non-PPO darkening to total non-PPO darkening was similar in the high-PPO wheat varieties and Kamilaroi but not in the low-PPO wheat variety, Axe (Table 2). Other Possible Methods for Estimating the Chemical Portion of YAN Non-PPO Darkening. Asenstorfer et al.5 published the effects of a variety of enzyme inhibitors on noodle darkening, and although no specific non-PPO inhibitor was identified, the enzyme inhibitor phenylhydrazine (0.01 mol L−1) decreased darkening in Kamilaroi by an amount similar to the chemical portion of non-PPO darkening (ΔL0−24 = 4.16) and in Tasman by an amount similar to the combined contributions of PPO darkening and the chemical portion of non-PPO darkening (ΔL0−24 = 8.84). Phenylhydrazine is a PPO inhibitor;22 however, it appears to be inhibiting an enzyme in addition to PPO. This was further evidence that the chemical portion of non-PPO darkening is catalyzed by an enzyme, possibly an enzyme with PPO-like activity but which is insensitive to the PPO inhibitor, tropolone. Phenylhydrazine (or a less toxic alternative) may provide another method of estimating the contribution of the enzymatic (PPO and non-PPO) to YAN darkening. Extraction of YAN Darkening Components. Initial results suggested that gliadins and glutenins were involved in the chemical portion of non-PPO darkening.1 When the gliadin-rich extracts from Triticum durum cv. Kamilaroi were dried and added back to YAN, there was an increase in non-PPO darkening. The fraction with the highest YAN darkening activity was derived from a 50% propanol extract of Tasman flour. This 50% propanol extract was subsequently further fractionated by C18 chromatography with 20% propanol and 50% propanol. The fraction obtained from the 20% propanol was active with a ΔL*0−24 value of 3.10 ± 0.46, whereas the fraction obtained from the 50% propanol was inactive with a ΔL*0−24 value of −0.82 ± 0.52 (Table 4). These two propanol fractions were also analyzed for tyrosine darkening using the PPO assay. The 20% propanol fraction oxidized some tyrosine, whereas the 50% propanol fraction showed no darkening. Subsequent work confirmed the relationship between tyrosine darkening, as measured by the PPO assay, and the amount of noodle darkening (Figure 4) and
fraction supernatant precipitate CHCl3 soluble CHCl3 insoluble 50% propanol 20% propanol a
ΔL*0−24
ΔOD (mg−1 h−1 × 10−3)
0.14 ± 0.08 0.36 ± 0.06 −1.10 ± 0.02 0.90 ± 0.12 −0.82 ± 0.52 3.10 ± 0.46
nda nd nd nd −0.1 ± 0.8 14.0 ± 2.5
Not determined.
therefore indicated that the active fractions contained enzyme(s) rather than substrate.
Figure 4. Darkening of YAN (ΔL*0−24) made from zero-PPO Kamilaroi flour with a range of propanol extracts from Tasman flour added (adjusted to 0.1 g/10 g equiv) compared with the oxidation of tyrosine (ΔOD415) by the same extracts (bars represent standard error of the mean).
Analysis by SDS-PAGE and acid-PAGE showed that the 20% propanol fraction contained a higher proportion of α/β-gliadins than the 50% propanol fraction (data not shown). Mass spectrometry of the 20% propanol fraction revealed that the main constituents of this fraction were proteins with mass 30000−35000 (m/z), which was consistent with the presence of gliadins23 (data not shown). Typically wheat PPO enzymes are associated with the water-soluble (albumin/globulin) fraction24 and not the gliadin fraction. The active fractions appeared to contain an enzyme inhibited both by tropolone and by methanol (Table 5). In agreement with previous studies,21 PPO in YAN made from Tasman flour was only slightly inhibited by methanol (Figure 3B). However, partial purification of the unknown enzyme involved in non-PPO darkening may have increased its sensitivity to tropolone inhibition. It is therefore possible that this enzyme is a type of PPO. Anderson et al.25 reported a tropolone-insensitive PPO in immature wheat kernels. However, if this enzyme is indeed a PPO, it belongs to a different class of PPO enzymes than previously described in wheat bran.25,26 Data presented here are consistent with the suggestion that the chemical component of non-PPO darkening involves the presence of a PPO-like enzyme with a hydrophobicity similar to that of gliadin which is able to oxidize tyrosine, has a pH maximum of 8.1, and is sensitive to 50% methanol, ethanol, and 4729
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Table 5. Difference in Darkening ΔL* as a Result of Crude Enzyme Extract from Tasman Wheat Added to YAN Made from near-Zero-PPO Kamilaroi at a Concentration of 0.1 g/ 10 ga
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ΔL* extract (0.1 g/10 g) tropolone + extract methanol control methanol + extract expected methanol + extract
0.60 ± 0.37 0.04 ± 0.35 −2.41 ± 0.52 −2.54 ± 0.43 −1.81
a
Tropolone (PPO inhibitor) and 50% methanol were added to noodles containing the extract and compared with control noodles made with and without 50% methanol.
phenylhydrazine but is insensitive to tropolone in situ. With lowPPO and PPO-free wheat varieties becoming available, it may be possible to further reduce darkening in YAN by breeding for wheat varieties with low or zero levels of this PPO-like enzyme.
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AUTHOR INFORMATION
Corresponding Author
*(R.E.A.) Phone: +61 8 83137480. Fax: +61 8 83137109. E-mail:
[email protected]. Present Address §
(M.J.A.) LongReach Plant Breeders, Unit 1/18 Waddikee Road, Lonsdale, SA 5160, Australia. Funding
We thank The University of Adelaide (Adelaide, SA, Australia) and the Grains Research and Development Corporation (Barton, ACT, Australia) for research funding and support. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the use of a Bühler mill from the Australian Grain Technologies’ Wheat Grain Laboratory (Urrbrae, SA, Australia).
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REFERENCES
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dx.doi.org/10.1021/jf500206e | J. Agric. Food Chem. 2014, 62, 4725−4730
