Influence of postharvest ethylene treatment dehydration and

14th Workshop on the Developments in the Italian PhD Research on Food Science Technology and Biotechnology - University of Sassari Oristano, September 16 – 18, 2009 Influence of postharvest ethylene treatment dehydration and maceration of Aleatico grapes Luigi Lodola (l.lodola@politicheagricole.gov.it) Dept. Food Science and Technology, Postharvest Laboratory (LAPO) University of TUSCIA Viterbo, Italy Tutor: Prof. Fabio Mencarelli The objective of this experimental work has been to evaluate the effect of ethylene on weight loss of Aleatico grape during dehydration and during grape maceration. Pectinmethylesterase, polygalacturonase, α-galactosidase increased significantly their activities soon after the ethylene treatment. In parallel, the increase of weight loss was measured and at the end of dehydration period, ethylene-treated berries lost approximately 5-6% more water than untreated samples. VOCs increased at the end of dehydration period in both samples above all in untreated one. Ethylene had negative effect on enzyme activities during maceration. Influenza del trattamento postraccolta con etilene sulla disidratazione e macerazione su uve di Aleatico L'obiettivo di questo lavoro sperimentale è stato quello di valutare l'effetto dell’etilene sulla perdita di peso di uva di Aleatico durante la disidratazione e la macerazione delle uve. L’attività della pectinmetilesterasi, poligalatturonasi e α-galattosidasi (altre glicosidasi sono state misurate ma non vengono riportate) era significativamente aumentata dal trattamento con etilene fatto prima del trattamento di disidratazione. Parallelamente si assisteva ad una più elevata perdita di acqua (5-6%) nei campioni trattati precedentemente con etilene. L’etilene ha un effetto negativo sulla attività enzimatica durante la macerazione. Keywords: postharvest treatments, ethylene, dehydration, cell wall enzymes. 1. Introduction Grape is considered non climacteric fruit meaning that ethylene production does not increase during ripening and not all the ripening features (colour, softening, sugar increase, acidity decrease) respond to exogenous ethylene treatment. Postharvest ethylene treatments are used in non climacteric fruit i.e. for the degreening of citrus and pineapple and, in peppers, 100 ppm ethylene treatment after harvest accelerated the appearance of full red colour (Fox et al., 2005). Grape berry cell walls form a barrier to the diffusion of components such as aromas and polyphenols, which are key compounds for determining the quality of both wine and table grapes. PG has been seen to increase with ripening (Cabanne et al, 2001), and just recently PME specific activity of grape skin has been seen to rise with ripening in parallel with the strong expression of VvPME1(Deytieux-Belleau et al. 2008). If ethylene hastens the cell wall degradation through the stimulation of cell wall hydrolases, then the hormone can accelerate water release from berry during the postharvest grape drying, affecting the release of cell compounds, i.e. polyphenols, or volatile compounds. Here we report the results of an experimental work to study the effect of exogenous ethylene treatment after harvest on cell wall enzymes of Aleatico grape and consequently on the grape drying in environmental controlled conditions. Terpenols are very important for the aroma profile of Aleatico grape thus the objective of this experimental work has been the study of cell wall enzymes in relation to ethylene treatment and dehydration, and their role on aroma profile. 2. Experimental Procedure Grapes (var. Aleatico) were treated with 1000 ppm ethylene gas for 48 h a 20°C and high RH; CO2 was absorbed by calcium carbonate. After the treatment, grape bunches were removed and placed in ventilated room at 20°C and 45% RH for dehydration. Samplings were carried out when the bunches lost 15%, 30%, and 40% of their weight. Control grapes were untreated with ethylene and subjected to the same dehydration treatment. During samplings, berry were removed and immediately frozen at -20°C for the further biochemical and chemical analyses. The third year has been lead a test of maceration of the grapes in containers of glass with a champion treated with ethylene (1000 ppm 48 h to 20°C at intervals of 12 hours with bubbling and then closed in saturation) and another one without bubbling. At the beginning of the experiment samplings were carried at 24 and 48 hours and at the final time (after 7 days of maceration). The champions have been frozen and then subordinates to analysis 14th Workshop on the Developments in the Italian PhD Research on Food Science Technology and Biotechnology - University of Sassari Oristano, September 16 – 18, 2009 of the glycosidases, polyphenols totals and anthocyanins, to estimate the effect of ethylene on the champions. T0 C6 geraniol acetaldehyde ethyl acetate acetic acid ethanol isoamyl acetate isoamyl alcohol hexyl acetate ethyl hexanoate ethyl eptanoate 34850c 233ab 16792d 328e 2907d 8410e 55073b 17151c 356c 248b 6239b 15% CK 47552b 214b 42600c 620d 2817d 15923b 50559b 17256c 241d 140d 5436c 15% ET 46847b 255a 47182b 520d 2752d 9317d 38530d 15646d 272d 174c 8393a 30% CK 47906b 210b 47236b 1085c 3062d 16226b 46500c 16562c 249d 135d 5132c 30% ET 57518a 249a 45000b c 1582b 4342c 10747c 43200c 16439c 324c 197c 6033b 40% CK 47553b 237a 64930a 2195a 5464b 27781a 66022a 21254a 430b 237b 3785d 40% ET 48170b 212b 62497a 2126a 6641a 16814b 37057d 19608b 580a 336a 8636a Table 1 Selected volatile compounds of Aleatico grape berries treated with ethylene (ET) or untreated (CK) and then dried; sampling at 15%, 30, 40% of weight loss. T0 refers to the initial time before ethylene treatment. Data are expressed as peak area, divided by 1000. 3 GC runs of the headspace of the vials containing berries juice of 3 different bunches were performed . Anova was performed and mean separation by the LSD test. Different letters on each row indicate significant difference per P=0.05. 3. Materials and Methods Glycosidases extraction has been conducted by using 5 g of berries powder obtained as described for PME,were dissolved in 4 mL of 50 mM Na-acetate buffer (pH 5.2) adding of 1.4 mM NaCl, 0.2% (w/v) cystein and 1% (w/v) PEG 3350, homogenized with UltraTurrax for 2 minutes in ice, then centrifuged at 17000 rpm for 30 min at 4°C (FILS et al. 1991). 2.5 ml of surnatant was filtered through PD10 Sephadex preequilibrated at 4°C with Na-acetate buffer and enzyme was recovered by using Na-acetate buffer. Assay mixture consisted of 100µl of enzyme extract in 0.9 ml of corresponding p-nitrophenil derivatives of specific substrate. Final concentrations in 50 mM sodium acetate buffer (pH 5) were 10 mM α-D-mannopyranoside, 15 mM α-D-glucopyranoside , 3 mM β-D-glucopyranoside, 3 mM α-D-galactopyranoside, and 6 mM β-D-galactopyranoside. Reaction mixture was incubated at 37°C for 60 min. After stopping the reaction, the formed p-nitrophenol was determined at 405 nm absorbance. Enzyme acitivity is espressed as µmoles of p-nitrophenol formed per second per g of f.w. Pectinmethylesterase (PME) was extracted with 0,2 M phosphate buffer (pH 7,5) plus 1mM EDTA, 5% of PVPP and 2M NaCl. Reading at 620 nm. Polygalacturonase (PG) extraction was similar but with 0,5 M NaCl + 2% PeG. Incubation at 37°C and addition of 20 µl DNS (3,5 dinitrosalicylate). Reading at 540 nm. For glycosidases, extraction with sodium acetate buffer, NaCl, cystein, and PEG. The singol glycosidase (α-β galactosidase, α-β glucosidase et α mannosidase) was reading by using as substrate the correspondent pyranosidic sugar in 50 mM sodium acetate. 405 nm was used for enzyme reading. VOCs analyse was performed by SPME technique (Bellincontro et al., 2004). Total polyphenols: They were defined with the application of the Folin-Ciocalteu method, anthocyanins were determined according to maximum absorbance in visible range (536 - 540 nm) against a blank (solution: ethanol/ water/HCl 70:30:1) (Di Stefano et al., 1989). 4. Results and discussion Ethylene increased weight loss of berries around 5 % (untreated = 43 % and ethylene treated= 48 %). During dehydration, enzymes activity (FIG. 1) was higher in sample pretreated with ethylene, thus, it appears a close relationship among ethylene, cell walls enzymes, and water loss from berries. PG was higher in ethylene-treated sample than untreated one in all the dehydration period. PME was higher in ethylene treated sample until 30% of weight loss, then declined. α-galactosidase was significantly higher in ethylene-treated sample in all the period. Same behaviour was observed for the other enzymes. Total VOCs content (without ethanol) initially slight decrease but not significantly in both samples then increased significantly above all in untreated sample due to a concentration effect (Table1). Ethylene treated sample showed a significant higher content and increase of acetic acid + ethylacetate and less ethanol content to indicate an advanced anaerobic process. C6 compounds increased in both samples at the same extent while 14th Workshop on the Developments in the Italian PhD Research on Food Science Technology and Biotechnology - University of Sassari Oristano, September 16 – 18, 2009 terpenols (mainly linalool and geraniol) greatly decreased until 30% of weight loss in both samples to increase at the end due to a concentration effect. No relation was found between glycosidases and terpenols but the increase in anaerobic metabolites let us to suppose an accelerated degradation process which involve the oxidation of terpenols which will explain the increase in C6 compounds and the increase of isoamylic alcohol. Ethanol was lower in ethylene treated sample while acetic acid and ethylacetate was higher. Terpenols declined until 30% of weight loss then increased at 40% greatly in both samples. A strong anaerobic catabolism due to cell wall degradation is induced thus a strong oxidation occurs. 0,9 0,8 100 0,7 0,6 0,5 60 0,4 0,1 0,3 0,2 0,1 0 0 0,15 0,3 0,4 weight loss 40 80 0,15 0,2 A 120 B 0,25 C Ethylene CK 20 Ethylene CK 0,05 Ethylene CK 0 0 0,15 0,3 0,4 weight loss 0 0 0,15 0,3 0,4 weight loss Figure 1 Activity of PG (a), α galactosidase (b) and PME (c) versus weight loss in the experiment conditions reported in the text. 40 35 30 25 20 15 10 5 0 to t24 t48 finale α ma nno e ti α ma nno 10 β gluc o e ti β gluc o 5 0 Glyc osida se s 25 Glyc osida se s 45 40 G lyc osida ses 20 35 30 15 25 20 15 5 10 α ga la tto e ti α ga la tto 0 to t24 t48 finale to t24 t48 finale Figure 2 Glycosidases activity (α mannosidase, β glucosidase et α galactosidase) in the must with the experiment conditions reported in the text. Antociani 450 400 350 300 250 200 150 100 50 0 to t24 t48 Final 1400 catechina mg*l-1 1200 1000 800 600 400 200 0 to Polifenoli m g*l-1 eti normale eti normale t24 t48 Final Figure 3 Total anthocyanins and total polyphenols of must with the experiment conditions reported in the text. 5. Conclusions and future perspectives Following the results of the first step of the experimental work studying the influence of ethylene on grape berry during dehydration procedure, we have studied the response of cell wall enzymes in the must after bubbling with ethylene. Cell wall enzymes are very sensitive to ethylene and our objective was to observe the possibility to use ethylene in the must to favour the phenols and anthocyanins extraction. In contrast with our expectations, ethylene bubbling reduced the extraction of these compounds and the reason was the reduction of the activity of cell wal enzymes. α-mannosidase, β-glucosidase and α-galactosidase activities was significantly lower during maceration than the one of the samples macerated with air bubbling. It is difficult to rise hypothesis of this behaviour because there are not publications on this type of treatment. Ethylene is known to stimulate glycosidases activity as we mentioned before. Green cell, following a microorganism attak, can produce inhibitor protein for cell wall enzymes such as polygalacturonase produced by 14th Workshop on the Developments in the Italian PhD Research on Food Science Technology and Biotechnology - University of Sassari Oristano, September 16 – 18, 2009 fungi. No knowledge about inhibitors for glycosidases. Other possibility is a mass effect which means that an excess of ethylene directly of free cells in the must works as toxic effect and this would explain why above all galactosiase is inhibited. Indeed, galactose is reported stimulated ethylene production (Gross, 1985). Galactosidase cleaves the galactose from cell wall. An excess of ethylene could have an opposite effect, inhibiting galactosidase and thus the release of galactose. Grape squeezing induces a strong wound response and this could have stimulated the synthesis of inhibitors protein reducing the cell wall enzymes activity. This is a new subject of research. 6. References Bellincontro A., DeSantis D., Botondi R., Villa I., Mencarelli F. (2004) Different Postharvest Dehydration Rate Affects Quality Characteristics and Volatile Compounds of Malvasia, Trebbiano, and Sangiovese Grapes for wine production. J. Science and Food Agric. 84:1791-1800. Cabanne C. and Doneche B., (2001) Changes in polygalactorunase activity and calcium content during ripening of grape berries. Amer. J. of Enol. and Vitic., 52, 331-335. Cardarelli M., Botondi R., Vizovitis K., Mencarelli F. (2002) Effects of exogenous propylene on softening, glycosidase, and pectinmethylesterase activity during postharvest ripening of apricots. J. Agric. Food Chem.50(6):1441-1446). Deytieux-Belleau C., Vaillet A., Doneche B. and Geny L., (2008) Pectin methylesterase and polygalactorunase in the developing grape skin. Plant Physiol.Biochem. 46, 638-646. Di Stefano, R., Guidoni, S., (1989) La determinazione dei polifenoli totali nei mosti e nei vini. Vignevini, N. 1/2. Fils-Lycaon B. and Buret M., (1991) Changes in glycosidase activities during development and ripening of melon. Postharvest Biol. Technol. 1, 143-151. Fox A J., Pozo-Insfran D., LEE J.H., Sargent S.A. and Talcott S.T., (2005) Ripening-induced chemical and antioxidant changes in bell peppers as affected by harvest maturity and postharvest ethylene exposure. HortScience, 40, 732-736. Gross K. C., (1985) Promotion of Ethylene Evolution and Ripening of Tomato Fruit by Galactose. Plant Physiol. 79(1):306– 307. Pérez-Magariño and González-San José, (2006) Polyphenols and colour variability of red wines made from grapes harvested at different ripeness grade, Food Chemistry 96 (2006), pp. 197–208.