ࡱ> JLIG )bjbjَ Q#]T}~~~~~      $3'D9"DM ~~y~M M M f~~M *M wh~b@e;[v  Potassium salt of 2,5-dimercapto-1,3,4-thiadiazole as potential inhibitor of enzymatic browning D. `ubari,1 R. Vukovi,2 A. Erceg,2 V. Pili~ota,1 T. Lovri,3 N. Nedi1 1Faculty of Food Technology, F. Kuha a 18, HR-31 000 Osijek, Croatia 2INA, Research and Development Institute, HR-10 000 Zagreb, Croatia 3Faculty of Food Technology and Biotechnology, Pierottijeva 6, HR-10 000 Zagreb, Croatia Abstract The purpose of this study was to investigate the possibility of monopotassium salt of 2,5-dimercapto-1,3,4-thiadiazole (DMTD) as potential inhibitor of enzymatic browning of fresh-cut fruits and vegetables. The synthesis of monopotassium salt of 2,5-dimercapto-1,3,4-thiadiazole is presented. The oxidation of phenolic compounds, chlorogenic acid (CA) and 3,4-dihydroxy phenilalanine (l-DOPA), catalized by mushroom polyphenol oxidase (PPO), was investigated in the presence of DMTD. Also DMTD was evaluated on the cut surface of apples (Jonagold variety), as browning inhibitor. Inhibition of PPO activity in model solutions of phenolic compounds was measured by spectrophotometer, and the enzymatic discoloration on the cut surface of apples was measured by the tristimulus colorimeter. DMTD was effective as an inhibitor of oxidation of phenolic compounds used in assay as well as inhibitor of browning on cut surfaces of apples. For the comparison, as inhibitors of enzymatic browning, ascorbic acid (AA), and benzoic acid (BA) were used too. Introduction Browning of raw fruits and vegetables, after tissues damage, during postharvest handling and processing, is one of the main causes of quality loss. Enzymatic browning represents a serious problem for the food processing industry, especially with recent restrictions in the use of sulfites in raw fruits and vegetables and other products [1, 2]. Enzymatic browning has a deleterious effect on fresh-cut fruits and vegetables and limit product quality and shelf-life [3, 4, 5]. In order to prevent browning of raw fruits and vegetables, many investigations has been done to develop the method for eliminating or retarding the process [6], as well as to find alternatives to sulfites [7, 8]. The objective in the present study was to evaluate the performance of DMTD as potential PPO inhibitor. Experimental Material Mushroom polyphenol oxidase (PPO; EC 1.14.18.1) product T-7755, with activity of 3000 units/mg of solid (Sigma Chemical Co., St. Louis, MO, U.S.A.) as a dry powder was dissolved in phosphate buffer (pH 6.5, 47 mM). PPO substrates, chlorogenic acid (Sigma) and l-DOPA (Sigma) were prepared as 2.5 mM solutions by disolving in phosphate buffer (pH 6.5). Synthesis of monopotassium salt of 2,5-dimercapto-1,3,4-thiadiazole (DMTD) The compound was prepared by dissolving of 0.6 g dimercapto-thiadiazole in 4.6 mL of n-KOH, and than evaporated in vacuum to dryness. Crystalline product (0.8 g) was dissolved in 12 mL of ethanol and recrystallized at room temperature. Yild of 0.31 g of yellow product in the form of the long fine needles was get. The product has m. p. above 270 C. Analysis: Calculated for C2HN2S3K (188); Theoreticaly: C, 12.77; H, 0.56; N, 14.89; S, 51.06; Found: C, 12.03; H, 0.60; N, 13.45; S, 49.31 (Fig. 1).  Figure 1 Structure of monopotassium salt of 2,5-dimercapto-1,3,4-thiadiazole (DMTD) Activity assay The enzyme activity was assayed spectrophotometrically using CECIL 2000 (CECIL INSTRUMENTS , England) spectrophotometer. Reaction mixture, in a total volume of 3 mL, included l-DOPA (2.5 mM), mushroom PPO (16 (g/mL) dissolved in phosphate buffer (pH 6.5, 47 mM) and certain concentration of DMTD solutions. Before measurements, reaction solution (PPO substrate and DMTD in phosphate buffer) were thermostated for 20 min at 25 C. The enzyme solution was added in treatment solution before measurement. The appearance of the brown colour, in the case of the l-DOPA was measured at 475 nm as a function of time (for 10 min), and in the case of the chlorogenic acid at 420 nm. The enzyme activity was calculated from the initial part of the curve of the absorption vs reaction time ((A/min) (4). Measured values were transformed into % inhibition. Apple preparation for browning measurements Apples (Jonagold variety) were purchased from the local growers and stored at 4 C until needed. Before samples preparation apples were held at least one hour at room temperature. To prepare samples, apples were washed with water, peeled and cut into dices (1 cm x 1 cm x 1 cm) with a sharp knife. 100 g samples were immediately immersed for 120 sec in treatment solution (DMTD, 0.01, 0.05 and 0.1%). The excess solution was than blotted with adsorbent tissue and samples were packed in plastic boxes, covered with plastic film to prevent evaporation, and stored at 4 C. The degree of browning of apple samples was monitored by reflectance measurements immediately after cutting, treatment with DMTD solutions (0 time), and during storage of apple samples (untreated and treated) on day 1, 4, 8 and 12. L*, a*, and b* values were measured by the tristimulus colorimeter Minolta CR-300 (Minolta Camera Co., Osaka, Japan) using the averaging mode with fifteen replications. Based on the measured data the calculation of effectiveness of each inhibitor was performed by equation: (Eab = (((L*)2+((a*)2+((b*)2)1/2 Results and Discussion To determine whether or not monopotassium salt of 2,5-dimercapto-1,3,4-thiadiazole (DMTD) can be used as potential PPO inhibitor, model solutions with phenolic compounds CA and l-DOPA with mushroom PPO were used. Apple dices as a cut surface system were used to find out whether DMDT has anti-browning activity. Results of DMDT efficiency to prevent an oxidation of phenolic compounds are presented in Tab. 1 and Tab. 2. It is evident that DMTD was more effective in prevention of oxidation than ascorbic acid (AA) and benzoic acid (BA). For the 50% inhibition of oxidation of l-DOPA, 13 (M of DMTD was used. At the same conditions, for the 50% inhibition of l-DOPA, 232 (M of AA, and 875 (M of BA were used. For the 96.54% of inhibition of oxidation of l-DOPA, 0.05 mM of DMTD was needed. Table 1 The effectiveness of different inhibitors on PPO activity in solutions of l-DOPA SubstratesInhibitorsI50 (mM)*l-DOPADMTD Benzoic acid Ascorbic acid0.013 0.875 0.232* I50= the concentration of the inhibitors that causes 50% inhibition of PPO activity In the case of CA, as PPO substrate, the most effective as inhibitor of PPO activity was again DMTD. Table 2 The effectiveness of different inhibitors on PPO activity in solutions of chlorogenic acid SubstratesInhibitorsI50 (mM)*Chlorogenic acidDMTD Benzoic acid Ascorbic acid0.051 0.658 0.414* I50= the concentration of the inhibitors that causes 50% inhibition of PPO activity Monopotassium salt of 2,5-dimercapto-1,3,4-thiadiazole (DMTD) showed considerable effectiveness as an inhibitor of oxidation of phenolic compounds CA and l-DOPA. Table 3 summarizes some results on colour parameters L*, a* and "E, of enzymatic browning of apple dices by treatment by DMTD. From the data it can be seen that DMTD was very effective as an inhibitor of discoloration of cut surface of apple dices. The most effective was 0.1% DMTD. Fresh-cut apples had good colour after 8 days, and some even after 12 days. Table 3 Effect of monopotassium salt of 2,5-dimercapto-1,3,4-thiadiazole, as potential browning inhibitor, applied to apple dices Colour parametersTreatmentDayL* value 1a* value 1(E2Control 0 1 4 8 1280.01 (0.84) 76.22 (1.16) 76.32 (1.10) (1.44) 75.28 (1.00)-3.18 (0.46) -1.66 (0.66) -0.81 (0.66) -0.43 (0.61) -0.27 (0.51) 5.13 5.57 6.62 6.180.01% DMTD0 1 4 8 1279.61 (0.88) 77.66 (0.73) 76.60 (1.77) (2.61) 71.06 (2,63)-3.59 (0.60) -2.79 (0.86) -1.36 (0.92) -0.47 (1.37) 1.00 (1,28) 2.46 4.03 6.91 10.990.05% DMTD0 1 4 8 1280.54 (0.93) 78.74 (1.20) 77.62 (1.91) (1.02) 75.05 (2.01)-3.74 (0.39) -3.49 (0.32) -2.73 (0.43) -2.51 (0.59) -1.64 (0.59) 2.04 3.39 3.95 5.950.10% DMTD0 1 4 8 1280.96 (1.75) 80.23 (2.20) 80.07 (1.79) (2.19) 79.56 (1.29)-3.77 (0.65) -2.68 (1.58) -2.05 (1.28) -1,21 (0.92) -1.07 (0.98) 1.71 2.56 3.33 3.84 1L and a values (means of 15 replicates) Value in parentheses are standard deviation (SD) 2Total colour change References Anon.: Fed. Reg. 51 (131): 25021, 1986. Anon.: Fed. Reg. 52 (237): 46968, 1987. Rolle R.S., Chism G.W.: J. Food Qual. 10, 157-177, 1987. King A. D., Bolin H. R.: Food Technol. 43, 132-135, 1989. Brecht J. K.: Hort Sci. 30, 18-22, 1995. Hsu A. F., Shieh J. J., Bills D. D., White K.: J. Food. Sci. 53, 765-771, 1988. McEvily A. J., Iyengar R., Otwell W. S.: Crit. Rev. Food Sci. & Nutr. 32, 253-273, 1992. Sapers G. M.: Food Technol. 47, 75-84, 1993. Keywords Polyphenol oxidase, enzymatic browning, inhibition, potassium salt of 2,5-dimercapto-1,3,4-thiadiazole.  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