464 Chiang Mai J. Sci. 2010; 37(3) Chiang Mai J. Sci. 2010; 37(3) : Contributed Paper

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1 464 Chiang Mai J. Sci. 2010; 37(3) Chiang Mai J. Sci. 2010; 37(3) : Contributed Paper Partial Characterization of a New Peptide from Ivorian Red Onion that Inhibits Polyphenol Oxidase and Enzymatic Browning of Edible Yam (Dioscorea cayenensis-rotundata cv Longb ) Sophie N. Gnangui [a], Sebastien L. Niamke [b] and Lucien P. Kouame* [a] [a] Laboratoire de Biochimie et Technologie des Aliments de l Universit d Abobo-Adjam 22 BP 1297 Abidjan 02, C te d Ivoire. [b] Laboratoire de Biotechnologie de l Universit de Cocody (Abidjan, C te d Ivoire), 22 BP 582 Abidjan 22, C te d Ivoire. *Author for correspondence; kouame_patrice@yahoo.fr Received: 26 October 2009 Accepted: 17 August 2010 ABSTRACT The enzymatic browning of the yam was retarded by addition of fresh onion extract. The maximal activity of polyphenol oxidase inhibitor was increased with higher fresh onion extract concentrations. The inhibitor was a peptide and inhibited the polyphenol oxidase non-competitively. It was stable at ph 6.6 in phosphate buffer for 120 min at 35 o C. Rapid inactivation occurred from 50 to 60 o C and it was completely inactivated at 65 o C after 1h30 min incubation. From 40 to 60 o C, the activation energy value for thermal inactivation of the peptide was calculated to be kJ/mol. The D-values showed that the thermal treatment had more influence on the inactivation of the peptide. Keywords: enzymatic browning, inhibitory effect, polyphenol oxidase, onion, peptide, yam. 1. INTRODUCTION Polyphenol oxidase (PPO) is a major enzyme responsible for the browning reaction in damaged plant tissues and fruits [1]. Although browning reactions, in some food products, result in good appearance in terms of colour, these kinds of reactions, in general, lead to undesirable results with respect to texture, sweetness, and overall flavour. Therefore, inhibition studies have gained more importance for these types of reactions in food and vegetable processing technology [2]. PPO has been given more attention in food technology in this regard. Enzymatic browning of plants may be delayed or eliminated by removing the reactants, such as oxygen and phenolic compounds, or by using PPO inhibitors. Complete elimination of oxygen from plants is difficult, because oxygen is ubiquitous [3]. There are a number of inhibitors used by researchers to prevent enzymatic browning [4]. Sulfite-containing additives have been extensively used as anti-browning agents to keep vegetables and

2 Chiang Mai J. Sci. 2010; 37(3) 465 fruits fresh looking [5]. However, consumer awareness of the risks associated with sulfites and increased regulatory scrutiny have created the need for substitutes [6]. Inhibition by thiol compounds is attributed to either the stable colourless products formed trough an additional reaction with o-quinones or binding to the active centre of PPO, like metabisulfite. Ascorbate acts more as an antioxidant than as an enzyme inhibitor because it reduces the initial quinine formed by the enzyme to the original diphenol before it undergoes secondary reactions which lead to browning. Ascorbic acid has also been reported to cause irreversible inhibition [7]. There is increasing consumer demand to substitute synthetic compounds with natural substances as food ingredients [8]. Garlic (Allium sativum) and onion (Allium cepa), both from the Allium family, are two of the most commonly utilized ingredients as flavor enhancers for several foods [9]. They have been shown to possess antimicrobial [10], antioxidant [11,12], anticancer [13,14] and anti-browning [15-19] effects in many previous studies. Both fresh and heated onion extracts decreased the enzymatic browning and the heated onion extract was more effective in prevention of enzymatic browning. The inhibition of enzymatic browning by onion extract seemed to be due to the polyphenol oxidase inhibitor present in onion extract [15,16]. The authors assumed that thiol compounds in onion might be the active components responsible for this inhibitory effect [15,16]. To our knowledge, no investigation has been carried out on studying the onion peptide on polyphenol oxidase activity. The objective of this study was to characterize a peptide from ivorian red onion (Allium cepa) that inhibits polyphenol oxidase and enzymatic browning of edible yam (Dioscorea cayenensis-rotundata cv Longb ). This was done in order to find a new peptide as a natural antibrowning agent for use in food industry. 2. MATERIALS AND METHODS 2.1 Materials Yam and red onion were grown respectively at a Biological Garden of Universit d Abobo-Adjam (Abidjan, C te d Ivoire) and a plantation in Ferkessedougou (C te d Ivoire). Tubers and bulbs were randomly harvested at physiological maturity. These were immediately transported to the Laboratoire de Biochimie et Technologie des Aliments de l Universit d Abobo-Adjam, (Abidjan, C te d Ivoire) for subsequent storage (-20 o C) and biochemical analyses. Dopamine was purchased from Sigma Chemical Co. (St. Louis USA). All other chemicals used were of analytical grade. 2.2 Methods Polyphenol Oxidase Preparation Freshly peeled tubers (150 g) were homogenized for 10 min in 300 ml of phosphate buffer (100 mm, ph 6.6). The resulting homogenate was centrifuged at 20,000 g for 20 min at 4 o C. The supernatant collected was used as an edible yam polyphenol oxidase (PPO y ) solution throughout this experiment Onion Extract Preparation Onion (100 g) was homogenized with 100 ml of a 100 mm phosphate buffer at ph 6.6 for 10 min and the homogenate was centrifuged at 20,000 g for 20 min at 4 o C. The supernatant represented the fresh onion extract Assay of Polyphenol Oxidase Activity Under the standard test conditions, the activity of polyphenol oxidase was

3 466 Chiang Mai J. Sci. 2010; 37(3) determined with dopamine as substrate according to a spectrophotometric procedure [17]. An essay mixture (2 ml) consisting of a 100 mm phosphate buffer at ph 6.6, 5 mm dopamine and enzyme solution was incubated at 25 o C for 10 min. After incubation, the activity was determined by measuring the absorbance of the reaction mixture at 480 nm. The results were expressed as units of enzymatic activity per mg of protein. One unit of enzymatic activity was defined as an increase in absorbance of per min [17] Determination of Protein Protein was determined by the method of Lowry et al. [18] using bovine serum albumin as a standard Effect of Fresh Onion Extract on Browning of Edible Yam Tuber The effect of fresh onion extract on browning of edible yam tuber was study according to the method of Lee et al. [19]. Tubers of edible yam were cut into slices. Each slice placed in an individual Petri dish and was immersed for 2 min in 100 mm phosphate buffer at ph 6.6 or fresh onion extract. Then, 1.0 ml of 5 mm dopamine was spread over the whole surface of each slice. A lid was placed on each Petri dish to minimize evaporation. All the slices were then incubated at 25 o C for 10 min Effect of Fresh Onion Extract Concentration and Time on Polyphenol Oxidase Activity Under the standard test conditions, 0.3 ml of fresh onion extract (0.27, 0.54, 0.81, 1.20, 1.35 or 1.62, 1.90 mg/ml) was added to 0.6 ml of a 100 mm phosphate buffer at ph 6.6. Following the addition and mixing of 0.3 ml of edible yam polyphenol oxidase, the assay mixture was pre-incubated for a period from 5 to 120 min at 25 o C. Then, 0.8 ml of dopamine (5 mm) was added to the assay mixture and incubated at 25 o C for 10 min. The activity was determined by measuring the absorbance of the reaction mixture at 480 nm [20]. The degree of inhibition on the reaction was expressed as percent inhibition, calculated using the formula (100[A-B]/A), where A and B were enzyme activities in the control and test systems, respectively. Specific activity of inhibition was expressed as unit (U) inhibition per mg protein, where 1 U of inhibitor activity was defined as 10% inhibition of edible yam polyphenol oxidase [21] Thermal Inactivation of Onion Extract Inactivation reaction The thermal inactivation of the onion extract inhibitor was determined at constant temperature between 30 and 65 o C after exposure to each temperature for a period from 5 to 120 min. The onion extract was incubated in 100 mm phosphate buffer ph 6.6 Aliquots were drawn at intervals and immediately cooled in ice-cold water. The residual enzymatic activity was determined in both cases at 25 o C under the standard test conditions. The inhibitor activity on the polyphenol oxidase was expressed as unit (U) inhibition per mg protein Data Analysis The integral effect of an inactivation process at constant temperature (40-60 o C), where the inactivation rate constant is independent of time, is given in equation 1: Ln [A t /A o ] = -kt (1) Where A t is onion extract inhibitor activity at time t, A 0 is the initial onion extract inhibitor activity and k is the reaction rate constant (min -1 ) at a given condition. k values were obtained from the regression line of Ln [A t /A 0 ] versus time as -slope.

4 Chiang Mai J. Sci. 2010; 37(3) 467 The D-value represents the time required to reduce the onion extract inhibitor activity under examination to 90% of its initial value. The relationship between the decimal reduction time (D) and the inactivation rate constant is given by equation 2 [22]: D = 2.303/k (2) The temperature of treatment and the rate constant in a denaturation process was related according to the Arrhenius equation [23]: k = Ae (-Ea/RT) (3) Eq. 3 can be transformed to: Ln k = LnA - Ea/R x T (4) where k is the reaction rate constant value, A is the Arrhenius constant, Ea is the activation energy (energy required for the inactivation to occur), R is the gas constant (8.31 Jmol -1 K -1 ) and T is the absolute temperature in Kelvin. When the Ln of k was plotted against the reciprocal of the absolute temperature, a linear relationship should be observed in the temperature range studied. The slope of the line obtained permitted to calculate the activation energy and the ordinate intercept corresponds to Ln A Inhibition Kinetics The assay mixture contained 0.3 ml of edible yam polyphenol oxidase, 0.6 ml of a 100 mm phosphate buffer at ph 6.6, 0.3 ml of the fresh onion extract (0.97 or 2.28 mg/ml) and was pre-incubated for 2 h at 25 o C. Then, 0.8 ml of dopamine (5 mm) was added to the assay mixture and incubated at 25 o C for 10 min. The activity was determined by measuring the absorbance of the reaction mixture at 480 nm [20]. Values 1/V and 1/[S] were employed to draw Lineweaver-Burk graphs Molecular Weight Estimation and Research of Inhibitor Nature Molecular weight of fresh onion extract inhibitor was estimated by dialysis according to the method of Ates et al. [24]. Cellulose dialysis tubing, with protein retention of > 12 kda, was used to dialyze onion extract against 100 mm phosphate buffer ph 6.6 overnight in a cold room. Compounds in the dialyzate and cellulose dialysis tubing were analyzed for inhibition of polyphenol oxidase activity at 25 o C under the standard test conditions. To determine the inhibitor nature, native polyacrylamide gel electrophoresis (n-page) on a 14% separating gel and a 4% stacking gel (8.0 x 7.0 cm) was performed, according to the procedure of Laemmli [25] at 10 o C and constant current 20 ma in order to separate onion extract proteins. The sample was not boiled. Electrophoretic buffers did not contain sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. After running, the gel was washed with 100 ml of a 100 mm phosphate buffer at ph 6.6. It was divided in two parts and one was cut in eight fractions in order to detect inhibitory activity. Each fraction was homogenized with 0.5 ml of a 100 mm phosphate buffer at ph 6.6 for 10 min. The homogenate was centrifuged at 20,000g for 20 min at 4 o C. Peptides of the other part were stained with silver nitrate according to Blum et al. [26] 3. RESULTS AND DISCUSSION The inhibitory effect of ivorian red onion extract on enzymatic browning of edible yam is shown in Figure 1. The edible yam slice immersed in dopamine solution (Figure 1A) showed a rapid change to a black colour, whereas, that immersed in dopamine solution after dipping in fresh onion extract (Figure 1B) showed reduced intensity of the black colour formation. The inhibition of browning in edible yam by onion extract seemed to be

5 468 Chiang Mai J. Sci. 2010; 37(3) due to the polyphenol oxidase inhibitor present in onion extract. The result reported here was in good agreement with the result of a previous study of pear [15], potato [20] and taro [19] enzymatic browning. Figure 2 shows the effect of fresh onion extract concentrations on edible yam polyphenol oxidase (PPO Y ) activity at various durations. The maximal activity of polyphenol oxidase inhibitor was increased with higher fresh onion extract concentrations. This inhibitor was stable at ph 6.6 in phosphate buffer for 120 min at 25 o C. After this time, it had a dramatic effect on the polyphenol oxidase inhibitor. The finding in our investigation was contradictory to that reported by Tsukamoto et al. [27] who observed that the polyphenol oxidase inhibitor(s) in housefly pupae was quite stable over a wide range of ph (4.0 to 10.0). A B Figure 1. Effect of fresh onion extract on enzymatic browning of edible yam (Dioscorea cayenensis-rotundata cv Longb ). All slices were observed after incubating at 25 o C for 10 min. (A) Addition of substrate solution (5 mm dopamine). (B) Addition of substrate solution (1 ml of 5 mm dopamine) after dipping in fresh onion extract (0.27 mg/mg). Figure 2. Effect of fresh onion extract inhibitor concentration on edible yam (Dioscorea cayenensis-rotundata cv Longb ) polyphenoloxidase activity at various durations. 0.27mg/ml, 0.54 mg/ml, 0.81 mg/mg, 1.20 mg/ml, 1.35 mg/mg, 1.62 mg/ml, 1.90 mg/mg

6 Chiang Mai J. Sci. 2010; 37(3) 469 As shown in figure 3, Lineweaver-Burk plots of PPO Y in the presence of the fresh onion extract when stored at room temperature for 2 h was investigated. Increase of inhibitor concentration resulted in a decrease of the slope of the line, indicating that the inhibition of fresh onion extract on the enzyme was reversible. The K M value did not change with increasing ivorian red onion inhibitor concentration, while Vmax decreased (Table 1). The fresh onion extract inhibitor inhibited the PPO Y non-competitively with dopamine as the substrate (Table 1). This result suggested that the non-competitive inhibitor did not compete with substrate and the substrate concentration had no influence on the degree of inhibition of the enzyme s catalytic rate. In this case, where the noncompetitive inhibitor reacted with the enzyme at a site other than the active site, both the free enzyme (E) and the enzyme-substrate complex (E-S) reacted with inhibitor [28]. This finding was contradictory to that reported by McEvily et al. [29] who observed that proteins exerted an inhibitory effect on PPO activity by chelating the essential copper at the active site of PPO, and the substrate-similar compounds could work through competitive inhibition [29]. The copper maintains an equilibrium between enzyme-cu 2+ and enzyme-cu + during enzymatic browning [29,30]. However, this pattern seems to reflect the type of inhibition of polyphenol oxidase with onion [15] and honey [24] extracts. In order to estimate the molecular weight of the inhibitor of onion extract, we dialyzed this extract against 100 mm phosphate buffer ph 6.6 overnight in a cold room. Only Figure 3. Lineweaver Burk plot of on edible yam (Dioscorea cayenensis-rotundata cv Longb ) polyphenoloxidase activity in the presence of fresh onion extract mg of protein, 0.97 mg of protein, 0 mg of protein.

7 470 Chiang Mai J. Sci. 2010; 37(3) Table 1. Type of inhibition and kinetic parameters of dopamine oxidation by edible yam (Dioscorea cayenensis-rotundata cv Longb ) polyphenol oxidase in the presence of the onion extract inhibitor.. Protein content of onion extract V max K M V max / K M in the reaction (U/mg protein) (mm) (U/mg protein x mm) Type of inhibition mixture (mg) Non competitive moleculars in the dialyzate were found to cause inhibition of PPOy. This result suggested that the molecular weight of the ivorian red onion inhibitor was d 12 kda. To identify the inhibitor nature, native polyacrylamide gel electrophoresis on a 14% separating gel and a 4% stacking gel (8.0 x 7.0 cm) was performed. As can be seen in figure 4, eight fractions were obtained and designated as F 1, F 2, F 3, F 4, F 5, F 6, F 7 and F 8. Only the fraction F 4 was inhibitory, causing a 38.9 % inhibition of PPO Y. It seemed that the inhibitor was a peptide. This result was similar to that for the inhibitor from honey that inhibited mushroom polyphenol oxidase [24]. The inhibitory effect of onion extract, after heating at various temperatures on edible yam polyphenol oxidase was investigated (Figure 5). The onion extract inhibitor was stable at 30 and 35 o C after 2 h incubation. Rapid inactivation occurred from 50 to 60 o C and it was completely inactivated at 65 o C after 1h30 min incubation. We concluded that fresh onion extract was a stronger inhibitor than heated onion. The inhibitory effect of the onion extract was decreased with increasing heating temperature and time. The high temperature had a negative effect on the inhibitor native conformation. According to this criterion, onion extract inhibitor was concluded by as a heat-labile molecular. This result is contradictory to the findings of Kim et al. [15], Lee et al. [17,18] and Lee [1] Figure 4. Native polyacrylamide gel electrophoresis on a 14% separating gel and a 4% stacking gel. F 1, Fraction 1; F 2, fraction 2; F 3, fraction 3; F 4, fraction 4 (onion peptide); F 5, fraction 5; F 6, fraction 6; F 7, fraction 7; F 8, fraction 8.

8 Chiang Mai J. Sci. 2010; 37(3) 471 Specific inhibitor activity (U inhibition/mg protein) Pre-incubation time (min) Figure 5. Effect of heated onion extract inhibitor on edible yam (Dioscorea cayenensis-rotundata cv Longb ) polyphenoloxidase activity at different temperatures. One unit (U) of inhibitor activity was defined as 10% inhibition of edible yam PPO. 30 o C, 35 o C, 40 o C, 45 o C, 50 o C, 55 o C, 60 o C, 65 o C. who observed that the inhibitory effect of onion extract toward pear, taro, potato and banana polyphenol oxidases was increased with the heated extract. However, the polyphenol oxidase inhibitor isolated from tobacco horn worm was a heat-labile glycoprotein, totally inactivated by heating at 100 o C for 10 min [31]. The logarithmic linear relationship between onion extract inhibitor activity and treatment time for the temperature range of o C followed first-order kinetics (Figure 6) and was consistent with the relationships found in earlier studies on fruits and vegetables [24,32-34]. The first order inactivation constants (k values) are presented in table 2. From the table, it was clear that the peptide inhibitor was less thermostable at higher temperatures (55 and 60 o C) since a higher rate constant means that the molecular was less thermostable [35]. Rate of onion extract inhibitor inactivation, after logarithmic transformation, decreased linearly with the inverse of temperature (Figure 7). This relationship was described by the equation: Lnk = -13,229 (1/T) (R 2 = 0.988) where T represents absolute temperature (K). From 40 to 60 o C, the activation energy (Ea) value for thermal inactivation of onion extract inhibitor was calculated to be 10,993 kj/mol (Table 2). This activation energy was much higher than that reported for honey extract inhibitor (27.71 kj/mol, [24]). In order to establish the link between treatment time and onion extract inhibitor activity, the D-values were calculated. Kinetic data from the onion extract inhibitor inactivation studies measured as a function of preheating temperature are summarised in

9 472 Chiang Mai J. Sci. 2010; 37(3) Table 2. Rate constants (k), decimal reduction time (D), and activation energy (Ea)-values for thermal inactivation of onion extract inhibitor at temperature range o C. Pre-incubation temperature ( o C) k(min -1 ) D(min -1 ) Ea(kJ/mol) , Pre-incubation time (min) Ln At/A0 Figure 6. Thermal inactivation curves of onion extract inhibitor in sodium phosphate buffer (ph 6.6) in the temperature range o C. A 0 is the initial enzymatic activity and A t the activity at each holding time. 40 o C, 45 o C, 50 o C, 55 o C, 60 o C. 1/T ( o K -1 ) Lnk Figure 7. Temperature dependence of inactivation rate constant for thermal inactivation of onion extract inhibitor. 1/T represents the reciprocal of the absolute temperature.

10 Chiang Mai J. Sci. 2010; 37(3) 473 Table 2. At a pre-treatment temperature of 40 o C, the D-values obtained using the rate constant data (k) and Eq. (2) decreased in a linear manner (R 2 = 0.99) from to min -1, when the pre-treatment temperature was increased from 40 to 60 o C for onion extract inhibitor (Figure 8). D-values of onion extract inhibitor were relatively high (Table 2) compared with the D-values of peroxidase (POD), which is considered the most heat stable protein of vegetables [38]. In general, the larger an protein and the more complex its structure, the more susceptible it is to high temperature [39]. LogD Pre-incubation temperature ( o C) Figure 8. Effect of temperature on D-values for inactivation of onion extract inhibitor activity. 4. CONCLUSIONS Ivorian red onion contains a peptide inhibitor that inhibits polyphenol oxidase activity and enzymatic browning of edible yam. This peptide is thermostable at 35 o C and may be very promising for practical applications in the food industry. Additional research may reveal other potential new uses. ACKNOWLEDGEMENTS We are grateful to Laboratoire de Biochimie et Technologie des Aliments de l Universit d Abobo-Adjam, Abidjan (C te d Ivoire) for the support of this work. REFERENCES [1] Ricard-Forget C. and Gauillard A., Oxidation of chlorogenic asid, catechins, and 4-methylcatechol in modal solutions by combinations of pear (Pyrus communis Cv. Williams) polyphenol oxidase and peroxidase: a possible involvement of peroxidase in enzymatic browning, J. Agric. Food Chem., 1997; 45: [2] Vamos-Vigyazo L., Polyphenol oxidase and peroxidase in fruits and vegetables, CRC Crit. Rev. Food Nutr., 1981; 15:

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