INHIBITION OF POLYPHENOL OXIDASE IN BANANA, APPLE AND MUSHROOM BY USING DIFFERENT ANTI- BROWNING AGENTS UNDER DIFFERENT CONDITIONS

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1 Int. J. Chem. Sci.: 8(5), 2010, S550-S558 INHIBITION OF POLYPHENOL OXIDASE IN BANANA, APPLE AND MUSHROOM BY USING DIFFERENT ANTI- BROWNING AGENTS UNDER DIFFERENT CONDITIONS SAMANTA ARPITA, DAS SUBROTO, BHATTACHARYYA PINAKI and BANDYOPADHYAY BIDYUT * Post Graduate Department of Biotechnology, Oriental Institute of Science and Technology, Dewandighi, Mirjapur, BURDWAN 02 (W.B.) INDIA ABSTRACT Fruit are highly perishable and maintain an active metabolism during the postharvest phase. Polyphenol oxidase or PPO (EC ) is considered the enzyme responsible for quality deterioration and browning in different fruits during postharvest period. Since enzymatic browning causes deterioration of sensory and nutritional quality and affects appearance and organoleptic properties, inactivation of PPO is desirable for preservation of foods. The objective of this study was to evaluate the antibrowning (inhibition of polyphenol oxidase activity) effect of Cysteine (Cys), Ascorbic acid (AA), citric acid (CA), sodium metabisulphite(smb) alone or in combination, at three different ph (3.5,4 and 4.5) in banana (Musa paradisiaca L. var. Kanthali), apple (Malus pumila Mill. var. Ambri kashmiri), and mushroom (Agaricus bisporus). All the samples were mixed with Cys (100, 200 and 300 mg/kg.), AA(250,500 and 1000 mg/kg.), CA (250, 500 and 1000 mg/kg.) and SMB (100,200 and 300 mg/kg.) to assess their effect on PPO. PPO activity was analyzed spectrophotometrically at 420 nm (30 0 C). The most effective PPO inhibitors were AA and SMB and in combination with CA and Cys in all the samples tested. No significant differences were observed for PPO activity among concentrations of Cys and CA when both anti-browning agents were used alone or in combination and mixed with the samples. Key words: Inhibition, Anti-browning agents, Banana, Apple and Mushroom Polyphenol oxidase, Chemical treatment. INTRODUCTION Enzymatic browning is one of the most studied reactions in fruits and vegetables during handling, processing and storage. The main enzyme involve in browning reaction is polyphenol oxidase (PPO, Ec ). The activity of polyphenol oxidase has been extensively studied and reported as the key factor in browning reaction 1. PPO, also called tyrosinase, phenol oxidase, cresolase are copper containing enzyme, synthesized in plants and stored in chloroplast 2. Polyphenol oxidase catalyzes the O-hydroxylation of monophenols to O- diphenols, which are further catalyzed to produce O-quinones. Once formed, these quinones undergoes * Author for correspondence; bidyut2006@gmail.com

2 Int. J. Chem. Sci.: 8(5), 2010 S551 polymerization reactions leading to the production of black, brown or red pigments (Polyphenols) which is the cause of enzymatic browning in fruits and vegetables. So, it is desirable to inactivate the PPO activity during preservation of foods as the enzymatic browning change the physical appearance, sensory and nutritional quality of fruits and vegetables 3. Now a days, several methods have been used to inhibit the PPO activity, such as the use of Antibrowning agents, removal of its one of the necessary component: O 2, Cu 2+ etc or by thermal processing to inactivate PPO activity is limited due to loss of sensory and nutritional quality of fruits and vegetables 4. Removal of oxygen from PPO can also check enzymatic browning but browning may restart when oxygen is available 5. So, the best way to prevent enzymatic browning is the use of anti-browning agents. These agents act on the enzyme or react with the substrate and/or products of enzymatic catalysis and thereby formation of browning pigment is inhibited 6. Several Anti-browning agents such as sulphites (Sulphur dioxide, sodium or potassium metabisulfite, sodium or potassium bisulfite); acidulants such as citric, malic or phosphoric acid; chelators (EDTA); reducing agents (ascorbic acid alone or in combination, Cysteine); 4-hydroxyrecercinol etc have been extensively used for preventing browning of foods 7,8. Ascorbic acid reduces o-quinones to diphenols and prevents the formation of browning pigments 9. Sometimes ascorbic acid is oxidized completely and browning may occur due to formation of melanins 10. Acidifiers or acidulants can inhibit PPO activity by reducing ph and or chelating copper in fruits and vegetables 11. Cysteine reacts with O- quinones and forms a colourless compound and thereby reduces PPO activity by delaying discolorations 12. Another anti-browning agent, 4- hydroxyresorcinol binds with PPO to form an inactive complex and thereby reduces the browning reaction 13. The objective of this study was to evaluate the antibrowning (inhibition of PPO activity) effect of Cysteine, ascorbic acid, citric acid, sodium metabisulfite alone or in combination, at three different ph (3.5, 4 and 4.5) in banana, apple and mushroom. Materials and methods Materials EXPERIMENTAL Healthy, disease free Banana (Musa paradisiaca L. var. Kanthali), apple (Malus pumila Mill. var. Ambri kashmiri), and mushroom (Agaricus bisporus) were purchased from the local market. Preparation of Crush The materials were washed, held in ice water (1 2 0 C). Apple and banana were peeled, de-seeded and cut into pieces. Mushrooms were sliced. Materials were blended in a domestic blender to obtain a crush and kept in beakers (around min) surrounded with ice water to slow down discoloration. The crush was adjusted to desired ph (3.5, 4.0, or 4.5) with phosphoric acid immediately after preparation. Crush (50 g) was weighed and mixed with sodium metabisulphite (E. Merck), L-ascorbic acid (E. Merck), or L-cysteine hydrochloride hydrate (E. Merck) and Citric acid (E. Merck) alone, or in combination as presented in Table 1. Each crush

3 S552 A. Samanta et al.: Inhibition of Polyphenol Oxidase. was weighed (35 g) and placed into a sterile sample bag with remaining air evacuated and tabs folded 8 times to seal the opening. Bags were then held in ice water until analysis for PPO activity. The study was made in duplicate. Table 1: Combination of different anti-browning agents used in this experiment Treatments - Anti-browning agents SMB Cys (mg/kg) AA (mg/kg) CA(mg/kg) (mg/kg) PPO assay PPO activity was assessed spectrophotometrically at 420 nm (30 0 C) in a UV-Vis spectrophotometer (Elico, India) 14. The PPO extract was obtained by mixing 10 g of apple, banana and mushroom crush with 10 ml of sodium phosphate buffer (ph 6.6) at 5 o C and centrifuging at 4000 rpm for 40 min at 4 o C. The extract was filtered with Whatman paper No. 1. The supernatant was denominated as enzyme extract. The reaction mixture consisted of 2.2 ml of buffer, 0.5 ml of catechol (0.175 mol/l), and 0.3 ml of enzyme extract. After mixing the mixture the absorbance was recorded every 15 s for 3 min. The linear portion obtained in plotting absorbance as a function of time was used to compute PPO activity. One unit of PPO activity was defined as A420/min/mL. All extracts were analysed in triplicate. Statistical analysis Data analysis was carried out using a Microsoft Excel 2003 and analysis of variance (ANOVA) was performed with the SPSS 12.0 software. *P < 0.05 was selected as the decision for significant differences. RESULTS AND DISCUSSION Banana, apple and mushroom crush containing SMB, Cys, AA and CA PPO activity PPO activity in banana, apple and mushroom crush at different ph (3.5,4.0 and 4.5) and different concentrations of anti-browning agents were presented in Table 2,3 and 4 respectively. From the tables, it can be concluded that the PPO activity decreased as the ph of the crush materials decreased in case of all the three species studied. It was also observed that the PPO activity was drastically decreased at ph 4 and ph 3.5 in all the three species, as compared to ph 4.5 (Fig. 1, 2 and 3). As the PPO activity is maximally observed between ph 5 and 7 13, low ph

4 Int. J. Chem. Sci.: 8(5), 2010 S553 may inhibit, or minimize PPO activity due to acidification 11. According to McEvily et al. 9, PPO was completely inhibited at a ph below 3. But fruit products generally do not possess this much low ph and in this low ph the taste of such products become changed and it may be extremely acidic for regular consumption. The anti-browning effect of SMB, AA, CA and Cys (used alone) in banana, apple and mushroom crush were presented in Table 2, 3 and 4 respectively and a comparative study was also carried out to determine the potentiality of different selected antibrowning agents and were presented in Fig. 1 (for Banana), Fig. 2 (for Apple) and Fig. 3 (for Mushroom). In general, maximum inhibition activity of PPO were observed at a concentration of 200 mg/kg SMB at ph-3.5 and 1000 mg/kg AA at ph-3.5 in case of banana crush; 300 mg/kg SMB at ph-3.5 and 1000 mg/kg AA at ph-3.5 in case of apple crush; 200 mg/kg SMB at ph-4 and 1000 mg/kg AA at ph- 4 and 300 mg/kg cysteine at ph-4 in case of mushroom crush. McEvily et al. 9 suggested that ascorbic acid inhibits PPO activity by reducing o-quinones to diphenols and cysteine reacts with o-quinones to form a colourless compound and thereby reduces PPO activity. AA contributes both weak acidic and strong reducing properties 15 that may reduce the ph of crush. However, from the two-way ANOVA study, we can conclude that the residual PPO activity at different ph and at different concentration of selected anti-browning agents were significantly different from control (*P < 0.05). Table 2: Residual PPO activity in banana crush containing different anti-browning agents at various ph Anti-browning agents Enzyme activity units Species Concentration (mg/kg) Control ± ± ±2.4 Sodium metabisulfite Ascorbic acid Citric acid Cystein PH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.5

5 S554 A. Samanta et al.: Inhibition of Polyphenol Oxidase. Table 3: Residual PPO activity in apple crush containing different anti-browning agents at various ph Anti-browning agents Enzyme activity Units Species Concentration (mg/kg) Control ± ± ±1.5 Sodium metabisulfite Ascorbic acid Citric acid Cysteine PH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.4 Table 4: Residual PPO activity in mushroom crush containing different anti-browning agents at various ph Anti-browning agents Enzyme activity Units Species Concentration (mg/kg) Control ± ± ±1.4 Sodium metabisulfite Ascorbic acid Citric acid PH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.6 Cont

6 Int. J. Chem. Sci.: 8(5), 2010 S555 Anti-browning agents Enzyme activity Units PH Species Concentration (mg/kg) ± ± ±1.6 Cysteine ± ± ± ± ± ±2.8 Enzyme Activity Units ph 3.5 ph 4 ph 4.5 C P1 P2 P3 Q1 Q2 Q3 R1 R2 R3 S1 S2 S3 Different antibrowning agent at different concentration Fig.1: Residual PPO activity in banana treated with different anti-browning agents C = Control; P1, P2 and P3 = SMB (100, 200 and 300 mg/kg ); Q1,Q2 and Q3 = AA (100,200 and 300 mg/kg ); R1, R2 and R3 = CA (100, 200 and 300 mg/kg ); S1,S2 and S3 = Cys (100,200 and 300 mg/kg ) Enzyme activity units ph 3.5 ph 4 ph C P1 P2 P3 Q1 Q2 Q3 R1 R2 R3 S1 S2 S3 Different antibrowning agent at different concentration Fig. 2: Residual PPO activity in apple treated with different anti-browning agents. C = Control; P1, P2 and P3 = SMB (100, 200 and 300 mg/kg ); Q1,Q2 and Q3 = AA(100, 200 and 300 mg/kg ); R1,R2 and R3 = CA(100,200 and 300 mg/kg ); S1,S2 and S3 = Cys (100,200 and 300 mg/kg )

7 S556 A. Samanta et al.: Inhibition of Polyphenol Oxidase. Enzyme activity units ph 3.5 ph 4 ph 4.5 C P1 P2 P3 Q1 Q2 Q3 R1 R2 R3 S1 S2 S3 Different antibrowning agent at different concentration Fig. 3: Residual PPO activity in mushroom treated with different anti-browning agents C = Control; P1,P2 and P3 = SMB(100, 200 and 300 mg/kg ); Q1,Q2 and Q3 = AA(100, 200 and 300 mg/kg ); R1,R2 and R3 = CA(100, 200 and 300 mg/kg ); S1,S2 and S3 = Cys (100, 200 and 300 mg/kg ). Banana, apple and mushroom with SMB/Cys, SMB/AA and SMB/CA PPO activity Table 5, 6 and 7 represents the residual PPO activity in banana, apple and mushroom crush respectively (ph 3.5) containing different combinations of anti-browning agents. The lowest PPO activity in banana crush was obtained after adding combinations of SMB/Cys (200/300 mg/kg) and SMB/AA (100/500 mg/kg) (Table 5). The combinations of SMB/Cys (100/200 mg/kg) and SMB/AA (100/250 mg/kg) were most suitable in reducing PPO activity in apple (Table 6). From Table 7, it was observed that SMB/AA (100/500 mg/kg) and SMB/CA (100/250 mg/kg) combinations in mushroom crush showed reduced PPO activity. Table 5: Residual PPO activity in banana crush containing different anti-browning agents at various ph SMB Enzyme activity units Cys (mg/kg) AA (mg/kg) CA (mg/kg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.5

8 Int. J. Chem. Sci.: 8(5), 2010 S557 Table: 6. Residual PPO activity in apple crush containing different anti-browning agents at various ph SMB Enzyme activity units Cys (mg/kg) AA (mg/kg) CA (mg/kg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.1 Table 7: Residual PPO activity in mushroom crush containing different anti-browning agents at various ph SMB Enzyme activity units Cys (mg/kg) AA (mg/kg) CA (mg/kg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.7 REFERENCES 1. J. R. L. Walker and P. H. Ferrar, Diphenol Oxidases, Enzyme catalysed Browning and Plant Disease Resistance, Biotechnol. Genetic Engg. Rev., 15, (1998). 2. V. Pilizota and D. Subaric, Control of Enzymatic Browning of Foods, Food Technol. Biotechnol., 36, 219 (1998). 3. M. Hendrickx, L. Ludikhuyze, I. Vandenbroeck, and C. Weemaes, Effects of High Pressure on Enzymes Related to Food Quality, Trends Food Sci. Technol., 9, (1998). 4. N. Sun, S. Lee, and K. B. Song, Effect of High-Pressure Treatment on the Molecular Properties of Mushroom Polyphenoloxidase, Lebensmittel- Wissenschaft Und-Technol., 35, (2002). 5. T. T. Langdon, Preventing of Browning in Fresh Prepared Potatoes without the use of Sulfiting Agents, Food Technol., 41(64), (1987). 6. O. Arslan and S. Dogan, Inhibition of Polyphenol Oxidase obtained from Various Sources by 2,3-Diaminopropionic Acid, J. Sci. Food Agri., 85, (2005).

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