Comparative Activity of Peroxidase and Polyphenoloxidase during Fruit Ripening in golden apple (Spondias citherea Sonner), loquat (Eryobotria japonica Lindl) and rose apple (Syzygium malaccense L. Merr and Perry) Growing under Tropical Conditions ﾃ］gel Guadarrama Universidad Central de Venezuela Facultad de Agronomﾃｭa Instituto de Botﾃ｡nica Agrﾃｭcola Campus Maracay-Aragua Venezuela angelguadarrama6@gmail.com Abstract There are a series of biochemical changes during fruits ripening , including browning reactions and lignification of fruit tissues with the involvement of polyphenoloxidase and peroxidase enzymes, respectively. These biological process could affect the organoleptic properties desired by consumers. Fruits of Spondias citherea Sonner, Eryobotria japonica Lindl and Syzygium malaccense L. Merr and Perry were used at different stages of maturation (green, turning, ripe and overripe) utilizing 50 grams of pulp of each state of maturation. Polyphenoloxidase and peroxidase enzymes activitiies were determined in crude extracts by method of Ponting and Josling(1948). Protein content determination was performed by the method of Bradford(1976). Studied fruits showed variable activity of polyphenol oxidase and peroxidase enzymes In different stages of ripening, with exception of Syzygium malaccense fruits which there was no detectable activity of both enzymes. Peroxidase enzyme activity could be a biochemical indicator to determine the degree of lignification of these fruits underexploited in Venezuela. Polyphenoloxidase enzyme activity seems to be an indicator of enzymatic browning of studied fruits. Spondias citherea (qualitatively very lignified) and Syzygium malaccense (qualitatively not lignified) could serve as model systems for studying lignification processes in fruits. Keywords: enzymatic browning, lignification, golden apple ,loquat, rose apple INTRODUCTION During postharvest of fruits there are a series of changes in physical, chemical, physiological and biochemical changes that leading to the ripening of each item in particular with well defined characteristics. All these changes are catalyzed by enzymes, resulting in softening of exocarp and mesocarp, degradation and synthesis of pigments, starch degradation, synthesis and processing of sugars, changes in respiratory activity, browning reactions and tissues lignification of fruits. Polyphenoloxidase and peroxidase enzymes are involved in both physiological processes.
Polyphenoloxidase is a copper-containing enzyme that catalyzes the oxidation of mono-and diphenols to quinones then undergo polymerization as a visual expression of browning of fruits after harvest (Wesche-Ebeling and Montgomery 1990). Peroxidase is an enzyme that contains iron and is involved as a participant in mechanisms of plant tissue lignification (Vamos-Vigyazo ,1981). The aim of this study was to determine the comparative behavior of polyphenol oxidase and peroxidase enzymes in these fruits which show contrasting differences qualitatively in terms of browning and lignification enzyme which could lead to the establishment of biochemical indicators for these processes as well as model systems to further studies on the metabolic pathways responsible for browning and lignification of fruit tissues that could affect their organoleptic properties during postharvest. MATERIAL AND METHODS Fruits were used at different stages of maturation (green, turning, ripe and overripe). Tissues was extracted from the mesocarp which were stored under freezing at -10 C for a month to conduct further tests for enzyme activities.For the extraction we used 50 g of fruit mesocarp at each stage of maturation, which were homogenized for 1 minute in a blender with 100 ml of 0.1 M phosphate buffer, pH 6.2 and centrifuged at 15,000 rpm for 30 minutes. The precipitate was discarded and to the supernatant was applied a double volume of cold acetone (- 15 degrees Celsius) in order to precipitate soluble proteins. Then the new precipitate was dissolved in 20 ml of 0.1 M phosphate buffer, pH 6.2, using this extract for determinations of protein content and enzymatic activity. Peroxidase activity determination was performed by of Ponting and Joslyn method using the following reaction mixture: 2 ml of substrate containing 0.04 M guaiacol and hydrogen peroxide 0.020 M, 0.9 ml of 0.1 M phosphate buffer pH 6.2 and 0.1 ml of enzyme extract. Enzyme activity at 30 degrees Celsius and 420 nm was measured on a Spectronic 401 - Roy Milton with thermal cell equipped and attached to a Forma Scientific model 2067 bath and circulator. Results were expressed in ∆A/min.mg Proteín. Polyphenoloxidase enzyme activity was also performed by the method of Ponting and Joslyn utilizing the following reaction mixture: 2 ml of substrate containing 0.03 M catecol , 0.9 ml of 0.1 M phosphate buffer pH 6.2 and 0.1 ml of enzyme extract. Enzyme activity at 30 degrees Celsius and 470 nm was measured on a Spectronic 401 - Roy Milton with thermal cell equipped and attached to a Forma Scientific model 2067 bath and circulator Enzymatic activity was expressed in ∆A/min.mg Proteín. Determinations of protein were performed by Bradford method (1976) refined by Spector (1978).
RESULTS AND DISCUSION Table 1 shows comparative results of specific enzyme activity in the three fruits studied. Spondias citherea fruits exhibited the highest activity in all stages of maturation, reaching its peak in turning fruits to decrease in mature and over mature fruits. Eryobotria japonica fruits showed a peroxidase activity from 18 to 40 times lower in over mature and mature states, respectively, compared with
Spondias citherea fruits at the same stages of maturity.In Syzygium malaccense fruits were no detectable peroxidase activity during ripening. From a qualitative point of view is evident the high degree of lignification or fiber formation that occur in fruits of Spondias citherea, being fruits of Eryobotria japonica lightly lignified and fiber formation is almost nonexistent in Syzygium malaccense fruits. These results suggest a possible relationship between lignification and peroxidase activity on which the literature (Bohn et al,2006; M채der, M and F체ss,1982) suggest as one likely enzymes involved in lignification processes. Quantitative determinations of lignin content of the fruits during different stages of maturity could contribute more to establish the relationship of peroxidase and lignification, and would open the possibility that this enzyme can serve as a biochemical indicator of lignification process in fruits under study.Table 5 shows the comparative activity of the polyphenoloxidase enzyme from studied fruits. In relation to the behavior of polyphenoloxidase enzyme is shown that fruits of Eriobotrya japonica are those with higher activity of this enzyme, then a lesser extent the fruits of Spondias citherea fruits not being detectable activity in Syzygium malaccense fruits. CONCLUSIONS 1.- Different states of maturation of fruits under study shows variable activity of peroxidase and polyphenoloxidase enzymes except Syzygium malaccense fruits which there was no detectable activity of both enzymes. 2.-Peroxidase enzyme could be a biochemical indicator to determine the degree of lignification of these fruits at different stages of maturity. 3.-The activity of polyphenoloxidase enzyme seems to be an indicator of enzymatic browning of fruits under study 4.-Both fruits of Spondias citherea (qualitatively very lignified) and Syzygium malaccense (qualitatively not lignified) could serve as model systems to study lignification processes in fruit
Literature cited 1.-Bohn, P., Zanardo, F., L. Ferrarese and O. Ferrarese. (2006). Peroxidase activity and lignification in soybean root growth-inhibition by juglone. Biol. Plantarum 50: 315-317 2.-Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein dye binding. Anal. Bioch 72 : 248-254 3.-M채der, M and F체ss, Rl. (1982). Role of Peroxidase in Lignification of Tobacco Cells: II. Regulation by Phenolic Compounds. Plant Physiology. 70 : 1132-1134 4.-Ponting, J.D and Joslyn, M.A. (1948). Ascorbic acid and oxidation and browning in apple tissue extyracts. Arch. Bioch. 19: 47-63.
5.-Spector, T. (1978). Refinement of the coomasie blue method of protein quantitation. Anal. Biochem. 86: 142-146 6.-Vamos-Vigyazo L. (1981). Polyphenoloxydase and peroxidase in fruits and vegetables. CRC Critical Reviews in Food Science and Nutrition 1: 49–127 7.-Wesche-Ebeling P. and Montgomery M.W. (1990).Strawberry polyphenoloxidase: extraction and partial characterization. J. Food Sci. 55: 1320– 1324
Tables Table 1. Specific activity (∆A/ min. mg Protein) of partially purified peroxidase with acetone in Spondias citherea, Eriobotrya japonica and Syzygium malaccense fruits Fruit species Spondias citherea Eriobotrya japonica Syzygium malaccense
Green 6.20 0.20
Turning 8.75 0.23
Riped 5.05 0.52
Overriped 3.50 0.45
Non detectable
Non detectable
Non detectable
Non detectable
Table 2. Specific activity (∆A / min. mg Protein) of partially purified poliphenoloxidase with acetone in Spondias citherea, Eriobotrya japonica and Syzygium malaccense fruits Fruit species Spondias citherea Eriobotrya japonica Syzygium malaccense
Green 0.05 0.40
Turning 0.15 0.55
Riped 0.16 0.65
Overriped 0.10 0.25
Non detectable
Non detectable
Non detectable
Non detectable