EFFECT OF CHEMICAL AND THERMAL TREATMENTS ON INHIBITION OF PEROXIDASE ACTIVITIE OF PURPLE SKIN EGGPLANT (SOLANUMMELONGENA L.)

1. Department of Biochemistry and Microbiology, Agroforestry unit, University LorougnonGuédé, BP 150 Daloa (Côte d’Ivoire). 2. Department of Biochemistry-Genetics, University Peleforo Gon Coulibaly, Korhogo, (Côte d’Ivoire), BP 1328 Korhogo, Côte d’Ivoire. 3. Laboratory of Biochemistry and Food Technology, University NanguiAbrogoua, 02 BP 801 Abidjan 02 (Côte d’Ivoire). ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 05 February 2021 Final Accepted: 10 March 2021 Published: April 2021


ISSN: 2320-5407
Int. J. Adv. Res. 9(04), 304-312 305 importance, eggplant is unfortunately hampered by a phenomenon of enzymatic browning when the tissues are cut, peeled or crushed during processing for food or for storage (AnosikeandAyeebene, 1981). Enzymatic browning of fruits and vegetables during postharvest handling and processing degrades the sensory properties and nutritional value and discourages consumer purchase of fresh-cut products. Consequently, enzymatic browning results in significant economic losses for the fresh produce industry. Browning reactions which occur after infliction of a mechanical injury to some live fruitsand vegetable are initiated by the enzymes, polyphenol oxidase, peroxidase and to the production of polyphenols and derived products (Osagie and Opoku, 1984). Peroxidase (POD) (hydrogenperoxide oxidoreductase E.C. 1.11.1.7), is one of the major enzymatic compounds which catalyze redox reactions of a wide range of phenolic and non-phenolic substrates, in the presence of hydrogen peroxide as an electron acceptor, being found in bacteria, fungi, algae, plants and animals (Chakraborty et al., 2015). POD is one of the main quality deterioration indicators, such as flavor loss and different biodegradation reactions, being also relevant as a browning enzyme that contributes to the darkening of fruit and vegetable products during processing and storage (Jang and Moon, 2010). The inactivation of POD is essential in order to minimize losses that are caused by enzymatic browning, in such a manner that several methods and technologies have been studied. Currently, one of the most applied methods for the inactivation of oxidative enzymes is thermal treatment and using chemical agents, which are also being used to ensure product quality in the food industry. This technology provides the possibility to minimize the effect on flavor, color, and nutritional properties in order to obtain food products of high-quality. The data obtained in this studiemay be considered as a reference for the determination of treatment techniques effective thus ensuring the quality of purple skin eggplant (Solanum melongena L.).

Plant material and chemicals
Fresh purple skin eggplant (Solanum melongena L.) was purchased from tall market, of Lobia (Daloa, Côte d'Ivoire), during September and October 2020.All chemicals and reagents were analytical grade and purchased from the Merck A.G. (Darmstadt, Germany) and from the Sigma Chemical Company (St. Louis, USA).

Extraction of peroxidase (POD) of the purple skin eggplant
A sample of eggplant (150 g) was crushed in a blender (Moulinex, France) and homogenized for 10 min in 300 ml of NaCl 0.9% (w/v). The resulting homogenate was centrifuged at 8000 g for 10 min at 4°C (Refrigerated centrifuge TGL-16M, China). The collected supernatant was the crude enzymatic extract used for POD activity assays (Gnaguiet al., 2009).

Enzyme activity assay
The reaction mixture to determine the POD activity adjusted to 2 mL, consisted of 1.6 mL citrate 100 mM buffer, pH 6.0, 0.2 mL substrate solution (guaiacol 10 mM and dissolved in citrate buffer, pH 6.0), 0.1 mL hydrogen peroxide 3% and 0.1 mL enzyme extract. This reaction mixture was incubated at 25°C for 10 min. After incubation, the activity was determined by measuring the absorbance of the reaction mixture at 480nm. Experiments were performed in triplicate, and the results expressed as units of enzymatic activity per mg of protein. One unit of enzymatic activity (U) was defined as an increase in absorbance of 0.001 per min (Cong et al., 2005) Optimal pH and stability The optimum pH of POD extracted from purple skin eggplantwas determined by measuring the oxidation of substrate guaiacol in different buffers at various pH values ranging from pH 2.6 to 8.0. The buffers (100 mM concentration) used were acetate from pH 3.6 to 5.6, phosphate from 5.6 to 8.0 and citrate from pH 2.6 to 7.0. The pH stability of peroxidase was studied at a pH range of 2.6-8.0 with 100mM buffers. Buffers used were the same as in pH study. After 2 hours preincubation at 25 °C (room temperature), residual peroxidase activitie was measured at 25 °C for 10 min by adding substrate guaiacol. Experiments were performed in triplicate, and the results expressed as percentage activity of zero-time control of untreated enzyme.

Temperature optimal and Thermostability
The effect of temperature on PODactivitie was performed in 100 mM citrate buffer pH 6.0, after 10 min incubation at temperatures ranging from 10 to 80°C using a water bath under standard test conditions. The thermal inactivation was determined at 37 °C and at enzyme optimum temperature (35°C). Enzyme in appropriate buffer was exposed to 306 each temperature for 120 min. Then, aliquots were withdrawn at intervals (15 min) and immediately cooled. In the thermal denaturation tests, aliquots of each enzyme solution were preheated at different temperatures at a range of 10-90 °C for 15min. Residual activities, determined at 25 °C under the enzyme assay conditions were expressed as percentage of activity of zero-time control of untreated enzymes (Gnanguiet al., 2009).
The effect of temperature and the rate constant in a activation process was related according to the Arrhenius equation (Arrhenus, 1889): k = Ae(-Ea/RT) (1) Where; k is the reaction rate constant value, A is the Arrhenius constant, Ea is the activation energy (energy required for the activation to occur), R is the gas constant (8.31 Jmol -1 K -1 ), T is the absolute temperature in Kelvin. The Q 10 temperature coefficient is a measure of the reaction rate of temperature increase of 10 °Cthe Q 10 is calculated as: Q 10 = (X 2 / X 1 ) (2) where: X 1 represents the lower absorbance (D.O at 10 °C); X 2 represents the higher absorbance (D.O at 20 °C)

Effect of metal ions and chemical agents on enzyme activity
To determine the effect of various compounds as possible activators or inhibitors of the activitie peroxidase each enzyme solution was preincubated at 25 °C for 20 min with the compounds and the activity was assayed under the enzyme assay conditions. Residual activities were expressed as percentage referred to control without chemical agents.

Inactivation kinetics of POD
The thermal inactivation of POD activitie was determined at temperatures ranging from 40 to 80 °C. The crude enzymatic extract in citratebuffer 100 mMpH 6.0 was preincubated at different temperatures. Aliquots were withdrawn at intervals and cooled at room temperature for 10 min. The enzymatic activitie of the aliquots was measured under standard conditions. The Kinetic data analysis of thermal inactivation of the PODactivitie was done Where At is the residual enzymatic activity at time t, A 0 is the initial enzymatic activity, and k is the reaction rate constant (min -1 ) at the temperature studied.
The inactivation rate constant k was estimated by linear regression analysis of the logarithm of residual activity versus treatment time.

Statistical Analysis
The Statistical Analysis System (SAS) for the personal computer program (SAS Inst., 1988) was used for the ANOVA; LSD means separation, single, Pearson and stepwise regression analyses.

Results And Discussion:-
Effect of pH and pH stability pH is a very important parameter that affects the ionization of the exposed amino acids of an enzyme in such a manner that the low stability at very acidic pH values can be attributed to the instability of the binding site of the enzyme (Terefeet al., 2014). The peroxidase stability assessed under extreme conditions is a conclusive factor for its 307 industrial applications (Sonkaret al., 2015). The effect of pH on POD from purple skin eggplant was studied in the pH range of 2.6 to 8.0, at 25°C. Regarding the pH, the enzymatic activity of POD, the highest enzyme activity was found at pH 6.0, whereas at pH 5.6, the enzyme retained 88.01 % of the initial activity (Fig. 1). In the acidic pH range of 2.6 to 5.6, the enzymatic activity of POD displayed a progressive increase, while in the alkaline pH range from 7.6 to 8.0, a significant decrease of the enzymatic activity was observed. Since POD activitie from purple skin eggplant was optimal at pH 6.0, his activity could be inhibited when exposed in acidic environment (pH  5.6). The lowest values of the relative enzymatic activity were recorded at pH values of 2.

Optimum temperatureandactivation energy
To determine the optimum temperature of POD from purple skin eggplant, the activity was assessed at different temperatures ranging from 10 °C to 80°C. POD presented the highest activity at the temperature of 35° C (Fig. 3).

Thermal denaturation and thermal stability
The thermal denaturation shows that the POD from purple skin eggplant was fairly stable theoptimal temperature 35 °C. At higher temperatures, thermostability decreased progressively and the enzyme were completely inactivated at 80 °C (Fig. 4).
The peroxidase was fully active for more than 2 hours at his optimal temperature (35 °C) in citrate buffer pH 6.0 indicating a thermal stability at his optimal temperature (Fig. 5). However, his catalytic activitie was abruptly affected after 15 min incubation at 37 °C (Fig. 5). This result is similar to those obtained by Terefeet al. (2010) who have determined the temperature stability of POD extracted from strawberry puree in the temperature range 25-100°C and of Al-Senaidy and Ismael (2011) whodetermined the thermal stability of POD extracted from persimmon by incubating the enzyme at different temperatures for 60 min. In this context, running biotechnological processes at moderate temperatures would be advantageous for application of this enzyme.

Effect of Metal Ions and Chemical Agents on Enzyme Activity
From an industrial point of view, the possibility to identify the most potent inhibitory compound that can prevent enzymatic browning is very important in improving the sensorial properties not only for fresh fruit and vegetables, but also for their derived products. Based on the studies available in the literature, the process of enzymatic browning catalyzed by POD may be prevented by inhibition of the enzyme. In this context, the effect of some chemicals on the peroxidase activity was examined (Table 1). From Table 1, it can be seen that ions K + , Cu 2+ , Na + , Pb 2+ and Ba 2+ had almost no effect on the POD from purple skin eggplant, while Fe 2+ and Mg 2+ showed an inhibitory effect on the peroxidase activity at concentration of 1mM and 5 mM.These cations should not be included in the

Thermal inactivation
As concerned the effect of temperature on POD from purple skin eggplant, the optimal temperature, half-life (t 1/2value) and D-value which are important parameters for enzyme thermal stability evaluation were determined as shown in Table 2. The increase in temperature from 40 to 80 °C resulted in a decrease in PODactivitie, hence the decrease of t 1/2 -values and D-values. At 60 °C, the t 1/2 -value of the studied enzymatic activitiedecreased to value 16.34 min. This value was lower than those obtained by Muhammad et al. (2013) who reported a t 1/2 -value of 514.0 min at 60 °C for the POD of grape juice. Thus, the low t 1/2 -value obtained suggested that POD from purple skin eggplant is strongly inactivated at temperature from 60 °C. With respect to D-values, a 72 % reduction in activity of POD from purple skin eggplant was observed at time from54.24 min at 60 °C. The results for POD extracted from purple skin eggplant are consistent with those obtained by Suhaet al. (2013) who studied the POD extracted from potatoes, carrots, eggplants, and tomatoes and concluded that the rate of inactivation of the enzyme increased with the increasing temperature and time of treatment. POD inactivation extracted from purple skin eggplant was performed at 80°C for 4 to10 minutes.