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

Partial Characterization of a New Peptide from IvorianRed Onion that Inhibits Polyphenol Oxidase andEnzymatic Browning of Edible Yam (Dioscoreacayenensis-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; e-mail: kouame_patrice@yahoo.fr

Received: 26 October 2009Accepted: 17 August 2010

ABSTRACTThe 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 onionextract concentrations. The inhibitor was a peptide and inhibited the polyphenol oxidasenon-competitively. It was stable at pH 6.6 in phosphate buffer for 120 min at 35oC. Rapidinactivation occurred from 50 to 60oC and it was completely inactivated at 65oC after 1h30min incubation. From 40 to 60oC, the activation energy value for thermal inactivation of thepeptide was calculated to be 109.93kJ/mol. The D-values showed that the thermal treatmenthad more influence on the inactivation of the peptide.

Keywords: enzymatic browning, inhibitory effect, polyphenol oxidase, onion, peptide, yam.

1. INTRODUCTIONPolyphenol oxidase (PPO) is a major

enzyme responsible for the browningreaction in damaged plant tissues and fruits[1]. Although browning reactions, in somefood products, result in good appearancein terms of colour, these kinds of reactions,in general, lead to undesirable results withrespect to texture, sweetness, and overallflavour. Therefore, inhibition studies havegained more importance for these types ofreactions in food and vegetable processingtechnology [2].

PPO has been given more attention infood technology in this regard. Enzymaticbrowning of plants may be delayed oreliminated by removing the reactants, such asoxygen and phenolic compounds, or by usingPPO inhibitors. Complete elimination ofoxygen from plants is difficult, because oxygenis ubiquitous [3]. There are a number ofinhibitors used by researchers to preventenzymatic browning [4]. Sulfite-containingadditives have been extensively used asanti-browning agents to keep vegetables and

Chiang Mai J. Sci. 2010; 37(3) : 464-475www.science.cmu.ac.th/journal-science/josci.htmlContributed Paper

Chiang Mai J. Sci. 2010; 37(3) 465

fruits fresh looking [5]. However, consumerawareness of the risks associated with sulfitesand increased regulatory scrutiny have createdthe need for substitutes [6]. Inhibition by thiolcompounds is attributed to either the stablecolourless products formed trough anadditional reaction with o-quinones or bindingto the active centre of PPO, like metabisulfite.Ascorbate acts more as an antioxidant thanas an enzyme inhibitor because it reducesthe initial quinine formed by the enzyme tothe original diphenol before it undergoessecondary reactions which lead to browning.Ascorbic acid has also been reported to causeirreversible inhibition [7]. There is increasingconsumer demand to substitute syntheticcompounds with natural substances as foodingredients [8].

Garlic (Allium sativum) and onion (Alliumcepa), both from the Allium family, are two ofthe most commonly utilized ingredients asflavor enhancers for several foods [9]. Theyhave been shown to possess antimicrobial [10],antioxidant [11,12], anticancer [13,14] andanti-browning [15-19] effects in manyprevious studies. Both fresh and heated onionextracts decreased the enzymatic browningand the heated onion extract was moreeffective in prevention of enzymaticbrowning. The inhibition of enzymaticbrowning by onion extract seemed to be dueto the polyphenol oxidase inhibitor present inonion extract [15,16]. The authors assumedthat thiol compounds in onion might be theactive components responsible for thisinhibitory effect [15,16]. To our knowledge,no investigation has been carried out onstudying the onion peptide on polyphenoloxidase activity.

The objective of this study was tocharacterize a peptide from ivorian red onion(Allium cepa) that inhibits polyphenol oxidaseand enzymatic browning of edible yam(Dioscorea cayenensis-rotundata cv Longb ).

This was done in order to find a new peptideas a natural antibrowning agent for use infood industry.

2. MATERIALS AND METHODS2.1 Materials

Yam and red onion were grownrespectively at a Biological Garden ofUniversit d’Abobo-Adjam (Abidjan, C ted’Ivoire) and a plantation in Ferkessedougou(C te d’Ivoire). Tubers and bulbs wererandomly harvested at physiological maturity.These were immediately transported to theLaboratoire de Biochimie et Technologie desAliments de l’Universit d’Abobo-Adjam ,(Abidjan, C te d’Ivoire) for subsequentstorage (-20oC) and biochemical analyses.Dopamine was purchased from SigmaChemical Co. (St. Louis USA). All otherchemicals used were of analytical grade.

2.2 Methods2.2.1 Polyphenol Oxidase Preparation

Freshly peeled tubers (150 g) werehomogenized for 10 min in 300 ml ofphosphate buffer (100 mM, pH 6.6). Theresulting homogenate was centrifuged at20,000 g for 20 min at 4oC. The supernatantcollected was used as an edible yam poly-phenol oxidase (PPOy) solution throughoutthis experiment.

2.2.2 Onion Extract PreparationOnion (100 g) was homogenized with

100 ml of a 100 mM phosphate buffer atpH 6.6 for 10 min and the homogenate wascentrifuged at 20,000 g for 20 min at 4oC.The supernatant represented the fresh onionextract.

2.2.3 Assay of Polyphenol OxidaseActivity

Under the standard test conditions, theactivity of polyphenol oxidase was

466 Chiang Mai J. Sci. 2010; 37(3)

determined with dopamine as substrateaccording to a spectrophotometric procedure[17]. An essay mixture (2 ml) consisting of a100 mM phosphate buffer at pH 6.6, 5 mMdopamine and enzyme solution was incubatedat 25oC for 10 min. After incubation, theactivity was determined by measuring theabsorbance of the reaction mixture at 480 nm.The results were expressed as units of enzy-matic activity per mg of protein. One unit ofenzymatic activity was defined as an increasein absorbance of 0.001 per min [17].

2.2.4 Determination of ProteinProtein was determined by the method

of Lowry et al. [18] using bovine serumalbumin as a standard.

2.2.5 Effect of Fresh Onion Extract onBrowning of Edible Yam Tuber

The effect of fresh onion extract onbrowning of edible yam tuber was studyaccording to the method of Lee et al. [19].Tubers of edible yam were cut into slices. Eachslice placed in an individual Petri dish andwas immersed for 2 min in 100 mMphosphate buffer at pH 6.6 or fresh onionextract. Then, 1.0 ml of 5 mM dopamine wasspread over the whole surface of each slice.A lid was placed on each Petri dish to minimizeevaporation. All the slices were then incubatedat 25oC for 10 min.

2.2.6 Effect of Fresh Onion ExtractConcentration and Time on PolyphenolOxidase Activity

Under the standard test conditions, 0.3ml of fresh onion extract (0.27, 0.54, 0.81,1.20, 1.35 or 1.62, 1.90 mg/ml) was addedto 0.6 ml of a 100 mM phosphate buffer atpH 6.6. Following the addition and mixingof 0.3 ml of edible yam polyphenol oxidase,the assay mixture was pre-incubated for aperiod from 5 to 120 min at 25oC. Then, 0.8

ml of dopamine (5 mM) was added to theassay mixture and incubated at 25oC for 10min. The activity was determined bymeasuring the absorbance of the reactionmixture at 480 nm [20].

The degree of inhibition on the reactionwas expressed as percent inhibition, calculatedusing the formula (100[A-B]/A), where A andB were enzyme activities in the control andtest systems, respectively. Specific activity ofinhibition was expressed as unit (U) inhibitionper mg protein, where 1 U of inhibitor activitywas defined as 10% inhibition of edible yampolyphenol oxidase [21].

2.2.7 Thermal Inactivation of Onion Extract2.2.7.1 Inactivation reaction

The thermal inactivation of the onionextract inhibitor was determined at constanttemperature between 30 and 65oC afterexposure to each temperature for a periodfrom 5 to 120 min. The onion extract wasincubated in 100 mM phosphate buffer pH6.6 Aliquots were drawn at intervals andimmediately cooled in ice-cold water. Theresidual enzymatic activity was determined inboth cases at 25oC under the standard testconditions. The inhibitor activity on thepolyphenol oxidase was expressed as unit (U)inhibition per mg protein.

2.2.7.2 Data AnalysisThe integral effect of an inactivation

process at constant temperature (40-60oC),where the inactivation rate constant isindependent of time, is given in equation 1:

Ln [At/Ao] = -kt (1)

Where At is onion extract inhibitor activity attime t, A0 is the initial onion extract inhibitoractivity and k is the reaction rate constant(min-1) at a given condition. k values wereobtained from the regression line of Ln[At /A0] versus time as -slope.

Chiang Mai J. Sci. 2010; 37(3) 467

The D-value represents the time requiredto reduce the onion extract inhibitor activityunder examination to 90% of its initial value.The relationship between the decimal reductiontime (D) and the inactivation rate constant isgiven by equation 2 [22]:

D = 2.303/k (2)

The temperature of treatment and the rateconstant in a denaturation process was relatedaccording 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 activationenergy (energy required for the inactivation tooccur), R is the gas constant (8.31 Jmol-1K-1)and T is the absolute temperature in Kelvin.When the Ln of k was plotted against thereciprocal of the absolute temperature, a linearrelationship should be observed in thetemperature range studied. The slope of theline obtained permitted to calculate theactivation energy and the ordinate interceptcorresponds to Ln A.

2.2.8 Inhibition KineticsThe assay mixture contained 0.3 ml of

edible yam polyphenol oxidase, 0.6 ml of a100 mM phosphate buffer at pH 6.6, 0.3 mlof the fresh onion extract (0.97 or 2.28mg/ml) and was pre-incubated for 2 h at25oC. Then, 0.8 ml of dopamine (5 mM) wasadded to the assay mixture and incubated at25oC for 10 min. The activity was determinedby measuring the absorbance of the reactionmixture at 480 nm [20]. Values 1/V and 1/[S]were employed to draw Lineweaver-Burkgraphs.

2.2.9 Molecular Weight Estimation and

Research of Inhibitor NatureMolecular weight of fresh onion extract

inhibitor was estimated by dialysis accordingto the method of Ates et al. [24]. Cellulosedialysis tubing, with protein retention of> 12 kDa, was used to dialyze onion extractagainst 100 mM phosphate buffer pH 6.6overnight in a cold room. Compounds in thedialyzate and cellulose dialysis tubing wereanalyzed for inhibition of polyphenol oxidaseactivity at 25oC under the standard testconditions.

To determine the inhibitor nature, nativepolyacrylamide gel electrophoresis (n-PAGE)on a 14% separating gel and a 4% stackinggel (8.0 x 7.0 cm) was performed, accordingto the procedure of Laemmli [25] at 10oCand constant current 20 mA in order toseparate onion extract proteins. The samplewas not boiled. Electrophoretic buffers didnot contain sodium dodecyl sulfate (SDS) andbeta-mercaptoethanol. After running, the gelwas washed with 100 ml of a 100 mMphosphate buffer at pH 6.6. It was divided intwo parts and one was cut in eight fractionsin order to detect inhibitory activity. Eachfraction was homogenized with 0.5 ml of a100 mM phosphate buffer at pH 6.6 for10 min. The homogenate was centrifuged at20,000g for 20 min at 4oC. Peptides of theother part were stained with silver nitrateaccording to Blum et al. [26]

3. RESULTS AND DISCUSSIONThe inhibitory effect of ivorian red onion

extract on enzymatic browning of edible yamis shown in Figure 1. The edible yam sliceimmersed in dopamine solution (Figure 1A)showed a rapid change to a black colour,whereas, that immersed in dopamine solutionafter dipping in fresh onion extract (Figure1B) showed reduced intensity of the blackcolour formation. The inhibition of browningin edible yam by onion extract seemed to be

468 Chiang Mai J. Sci. 2010; 37(3)

due to the polyphenol oxidase inhibitorpresent in onion extract. The result reportedhere was in good agreement with the resultof a previous study of pear [15], potato [20]and taro [19] enzymatic browning.

Figure 2 shows the effect of fresh onionextract concentrations on edible yam poly-phenol oxidase (PPOY) activity at variousdurations. The maximal activity of polyphenoloxidase inhibitor was increased with higherfresh onion extract concentrations. Thisinhibitor was stable at pH 6.6 in phosphatebuffer for 120 min at 25oC. After this time,it had a dramatic effect on the polyphenoloxidase inhibitor. The finding in our investi-gation was contradictory to that reported byTsukamoto et al. [27] who observed that thepolyphenol oxidase inhibitor(s) in houseflypupae was quite stable over a wide range ofpH (4.0 to 10.0).

Figure 1. Effect of fresh onion extract onenzymatic browning of edible yam (Dioscoreacayenensis-rotundata cv Longb ). All slices wereobserved after incubating at 25oC for 10 min.(A) Addition of substrate solution (5 mMdopamine). (B) Addition of substrate solution(1 ml of 5 mM dopamine) after dipping infresh onion extract (0.27 mg/mg).

Figure 2. Effect of fresh onion extract inhibitor concentration on edible yam (Dioscoreacayenensis-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

A B

Chiang Mai J. Sci. 2010; 37(3) 469

As shown in figure 3, Lineweaver-Burkplots of PPOY in the presence of the freshonion extract when stored at room tempera-ture for 2 h was investigated. Increase ofinhibitor concentration resulted in a decreaseof the slope of the line, indicating that theinhibition of fresh onion extract on theenzyme was reversible. The KM value did notchange with increasing ivorian red onioninhibitor concentration, while Vmax decreased(Table 1). The fresh onion extract inhibitorinhibited the PPOY non-competitively withdopamine as the substrate (Table 1). This resultsuggested that the non-competitive inhibitordid not compete with substrate and thesubstrate concentration had no influence onthe degree of inhibition of the enzyme’scatalytic rate. In this case, where the non-competitive inhibitor reacted with the enzyme

Figure 3. Lineweaver–Burk plot of on edible yam (Dioscorea cayenensis-rotundata cv Longb )polyphenoloxidase activity in the presence of fresh onion extract. 2.28 mg of protein,

0.97 mg of protein, 0 mg of protein.

at a site other than the active site, both thefree enzyme (E) and the enzyme-substratecomplex (E-S) reacted with inhibitor [28]. Thisfinding was contradictory to that reported byMcEvily et al. [29] who observed that proteinsexerted an inhibitory effect on PPO activityby chelating the essential copper at the activesite of PPO, and the substrate-similarcompounds could work through competitiveinhibition [29]. The copper maintains anequilibrium between enzyme-Cu2+ andenzyme-Cu+ during enzymatic browning[29,30]. However, this pattern seems to reflectthe type of inhibition of polyphenol oxidasewith onion [15] and honey [24] extracts.

In order to estimate the molecular weightof the inhibitor of onion extract, we dialyzedthis extract against 100 mM phosphatebuffer pH 6.6 overnight in a cold room. Only

470 Chiang Mai J. Sci. 2010; 37(3)

moleculars in the dialyzate were foundto cause inhibition of PPOy. This resultsuggested that the molecular weight of theivorian red onion inhibitor was d” 12 kDa.

To identify the inhibitor nature, nativepolyacrylamide gel electrophoresis on a 14%separating gel and a 4% stacking gel (8.0 x7.0 cm) was performed. As can be seen infigure 4, eight fractions were obtained anddesignated as F1, F2, F3, F4, F5, F6, F7 and F8.Only the fraction F4 was inhibitory, causinga 38.9 % inhibition of PPOY. It seemed thatthe inhibitor was a peptide. This resultwas similar to that for the inhibitor fromhoney that inhibited mushroom polyphenoloxidase [24].

The inhibitory effect of onion extract,after heating at various temperatures on edibleyam polyphenol oxidase was investigated(Figure 5). The onion extract inhibitor wasstable at 30 and 35oC after 2 h incubation.Rapid inactivation occurred from 50 to 60oCand it was completely inactivated at 65oC after1h30 min incubation. We concluded thatfresh onion extract was a stronger inhibitorthan heated onion. The inhibitory effect ofthe onion extract was decreased with increasingheating temperature and time. The hightemperature had a negative effect on theinhibitor native conformation. According tothis criterion, onion extract inhibitor was

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 extractinhibitor..

Protein contentof onion extract V

maxK

MV

max /

K

M

in the reaction (U/mg protein) (mM) (U/mg protein x mM) Type of inhibitionmixture (mg)

0 100.02 80.01

0.27 41.66 1.23 33.32 Non competitive

0.97 24.39 19.51

Figure 4. Native polyacrylamide gel electro-phoresis on a 14% separating gel and a 4%stacking gel. F1, Fraction 1; F2, fraction 2; F3,fraction 3; F4, fraction 4 (onion peptide); F5,fraction 5; F6, fraction 6; F7, fraction 7; F8,fraction 8.

concluded by as a heat-labile molecular.This result is contradictory to the findings ofKim et al. [15], Lee et al. [17,18] and Lee [1]

Chiang Mai J. Sci. 2010; 37(3) 471

who observed that the inhibitory effect ofonion extract toward pear, taro, potato andbanana polyphenol oxidases was increasedwith the heated extract. However, thepolyphenol oxidase inhibitor isolated fromtobacco horn worm was a heat-labileglycoprotein, totally inactivated by heating at100oC for 10 min [31].

The logarithmic linear relationship betweenonion extract inhibitor activity and treatmenttime for the temperature range of 40-60oCfollowed first-order kinetics (Figure 6) andwas consistent with the relationships foundin earlier studies on fruits and vegetables[24,32-34]. The first order inactivationconstants (k values) are presented in table 2.From the table, it was clear that the peptideinhibitor was less thermostable at highertemperatures (55 and 60oC) since a higher rateconstant means that the molecular was less

thermostable [35].Rate of onion extract inhibitor inactiva-

tion, after logarithmic transformation, decreasedlinearly with the inverse of temperature(Figure 7). This relationship was describedby the equation: Lnk = -13,229 (1/T) + 35.91(R2 = 0.988) where T represents absolutetemperature (K). From 40 to 60oC, theactivation energy (Ea) value for thermalinactivation of onion extract inhibitorwas calculated to be 10,993 kJ/mol (Table2). This activation energy was much higherthan that reported for honey extract inhibitor(27.71 kJ/mol, [24]).

In order to establish the link betweentreatment time and onion extract inhibitoractivity, the D-values were calculated. Kineticdata from the onion extract inhibitorinactivation studies measured as a function ofpreheating temperature are summarised in

Figure 5. Effect of heated onion extract inhibitor on edible yam (Dioscorea cayenensis-rotundatacv Longb ) polyphenoloxidase activity at different temperatures. One unit (U) of inhibitoractivity was defined as 10% inhibition of edible yam PPO. 30oC, 35oC, 40oC, 45oC,

50oC, 55oC, 60oC, 65oC.

Pre-incubation time (min)

Spec

ific i

nhib

itor a

ctiv

ity(U

inhi

bitio

n/m

g pro

tein

)

472 Chiang Mai J. Sci. 2010; 37(3)

Figure 6. Thermal inactivation curves of onion extract inhibitor in sodium phosphate buffer(pH 6.6) in the temperature range 40-65oC. A

0 is the initial enzymatic activity and At the activity

at each holding time. 40oC, 45oC, 50oC, 55oC, 60oC.

Table 2. Rate constants (k), decimal reduction time (D), and activation energy (Ea)-values forthermal inactivation of onion extract inhibitor at temperature range 40–60oC.

Pre-incubation temperature (oC) k(min-1) D(min-1) Ea(kJ/mol)40 0.002 1,151.5045 0.003 767.6750 0.006 383.83 109.9355 0.012 191.9260 0.024 95.96

Pre-incubation time (min)

Ln

At/

A0

0 50 100 150

Figure 7. Temperature dependence of inactivation rate constant for thermal inactivation ofonion extract inhibitor. 1/T represents the reciprocal of the absolute temperature.

1/T (oK-1)0.00295 0.003 0.00305 0.0031 0.00315 0.0032 0.00325

Lnk

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Figure 8. Effect of temperature on D-values for inactivation of onion extract inhibitor activity.

Table 2. At a pre-treatment temperature of40oC, the D-values obtained using the rateconstant data (k) and Eq. (2) decreased in alinear manner (R2 = 0.99) from 1151.50 to95.96 min-1, when the pre-treatmenttemperature was increased from 40 to 60oCfor onion extract inhibitor (Figure 8). D-values

of onion extract inhibitor were relatively high(Table 2) compared with the D-values ofperoxidase (POD), which is considered themost heat stable protein of vegetables [38].In general, the larger an protein and the morecomplex its structure, the more susceptible itis to high temperature [39].

Log

D

Pre-incubation temperature (oC)

4. CONCLUSIONSIvorian red onion contains a peptide

inhibitor that inhibits polyphenol oxidaseactivity and enzymatic browning of edibleyam. This peptide is thermostable at 35oCand may be very promising for practicalapplications in the food industry. Additionalresearch may reveal other potential new uses.

ACKNOWLEDGEMENTSWe are grateful to Laboratoire de

Biochimie et Technologie des Aliments del’Universit d’Abobo-Adjam , Abidjan (C ted’Ivoire) for the support of this work.

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