Study on Softening of Vinagrillo (Averrhoa bilimbi) Fruits during Ripening A. Guadarrama and E. González Universidad Central de Venezuela Facultad de Agronomía, Departamento de Botánica Agrícola Laboratorio de Fisiología Postcosecha Venezuela Keywords: Averrhoa bilimbi, postharvest, ripening, softening, pectinmethylesterase, polygalacturonase Abstract Vinagrillo is a climacteric fruit and ripening occurs in a few days after harvest. Ripening mainly involves a series of changes characterized by softening and changes of color by the combined action of ethylene and some hydrolases enzymes such as pectinmethylesterase (Pme), polygalacturonase (Pg) and cellulase (Ce). Some physical and chemical characteristics of the fruits during ripening were determined, the kinetic activity of enzymes previously mentioned and the activity of these enzymes with fruits softening were correlated. The fruits were harvested in different states from maturation: green, yellow green, ripened and overripened. The activity of Pme determined by the method of Hagerman and Austin (1986). The activity of enzymes Pg and Ce were determined according to the methodology of Durbin and Lewis (1988). Pme displayed greater activity at pH between 5 and 11 and performed a greater activity to 30°C. Pg showed greater activity in the green state and showed an optimal temperature of 30°C and the maximum activity to pH 7. Kinetic characterization of Pme and Pg was not made since they did not show a kinetic one of Michaelis-Menten. Detectable activity of CE in any of the ripening states was not demonstrated. Pectic substances in cellular wall undergo modifications due to the action of enzymes Pme and Pg. Statistical analyses were variance analysis with Tukey test and Kruskal and Wallis test with a significance level of 5%. INTRODUCTION Vinagrillo fruits is a climacterical fruit that ripens very fast reaching an overripe stage in approximately five days after harvest and tissues become extremely softened. Fruit ripening involves a sequence of physical chemical and biochemical changes conducting to an adequate color, flavor and texture for the consumer. Texture is related to softening that occurs by cell wall degradation including hydrolases enzymes such as cellulase (Ce), pectinmethylesterase (Pme) and polygalacturonase (Pg). Those hydrolases enzymes catalyze hydrolytic breakdown of esters and glucosydics links (Chin et al., 1999). Pme (E.C:3.1.1.11) catalyze methyl groups rupture from polygalacturonic acid chains increasing free carboxylic groups (Gordon et al., 2002). Pg enzyme breaks glucosydics α (1,4) links from pectin through an endo Pg (E.C:3.2.1.15) and exo Pg (E.C:3.2.1.67). Ce enzyme (E.C:3.2.1.4) is very specific (1-4-β endoglucosydase) and acts on cellulose to produce small fragments called cello-dextrin, cellobiose and glucose (Durbin and Lewis, 1988). MATERIALS AND METHODS Fruits growing under tropical conditions were harvested at four different stages of maturity. Texture analysis and pectins contents on Stage 1 (green color), stage 2 (greenyellow color ), stage 3 (full yellow color) stage 4 (deep yellow color) were performed. Texture determinations by using a penetrometer ELE-440 expressed in mm of penetration. Pectins analysis by AOAC methods (AOAC, 1980). Pme activity during ripening were determined by the methods of Hagerman and Austin (1986). Pg and Ce activity were monitored by the methods of Durbin and Lewis (1988). Kinectis assay was Proc. 1st IS on Biotechnol. of Fruit Species Eds.: M.-V. Hanke et al. Acta Hort. 839, ISHS 2009
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performed to determine Vmax and Km, optimal temperature and pH and heat stability of these enzymes. All analyses were performed on fruit pulp without seeds. RESULTS AND DISCUSSION Changes in Firmness Table 1 shows that during ripening of vinagrillo fruits the firmness (expressed in mm penetration) decreased from state green (1.8 mm penetration) to the overriped state (7.90 mm of penetration). Loss of firmness is produced mainly by changes in cell wall structure which have a thinning and reduced media laminilla, that is related to activity of hydrolytic enzymes activity acting on components of cell wall mainly pectic substance and cellulose (Hobson, 1996; Guadarrama, 2001). Pectin Changes The contents of pectins gradually decrease as vinagrillo fruits change from green state to overriped state. Figure 1 shows that as fruits have reached full maturity, pectins contents ns decreases by 50% aproximately in relation to green fruits. In mango fruits differences in pectin behaviors during ripening have been found. ‘Keitt’ mangoes pectin contents in physiological maturity were higher that ‘Tommy Atkins’ mangoes. In ‘Keitt’ mango it was observed that Pme activity remains high in early states of maturation in relation to Pg suggesting that it acts on pectins producing demetilation (El-Zoghbi, 1992). Pme Activity We evaluated the trend of PME activity in various states of maturation and that behavior is shown in Figure 2. Pme activity was similar in different stages of ripening, being in green state 0.11 ∆ Abs/h, then increased slightly to reach a value of 0.14 ∆ Abs/h in the green-yellow colour (turning) and then decreases when it reaches overriped state. El-Zoghbi (1994) and Andrews and Shulin (1995) have found that the behavior of Pme in fruits has variations. However, usually activity of this enzyme increases to the stage just before physiological maturity and then decreases in later stages of ripening. This behaviour was observed in guava, cherry, orange and banana fruits. The Pme activity in fruits of vinagrillo coincides with foregoing mentioned. Optimal pH and Temperature of Pme Figure 4 shows that Pme activity was higher at alkaline pHs in a range between 5 and 11. Usually activity curves in relation to pH are belt shaped but there are exceptions such as chollinesterase (Lehninger, 1995) whose activity vs pH is similar to vinagrillo fruits. Optimal temperature for vinagrillo fruits Pme was found to be 30°C showing a great difference with optimal temperature for ‘Bramley’ apples correponding to 60°C (King, 1990). PG Activity Figure 3 shows that Pg activity was higher at green stage of maturation then gradually decreasing until overripe state with no detectable activity of this enzyme. This behaviour is completely different to passionfruits (Passiflora edulis) according to Aponte and Guadarrama (2003) and Averrhoa carambola fruits according to Chin et al. (1999) where Pg activity was higher in overripe state. Optimal pH and Temperature of Pg Optimal pH for maximal activity of Pg was 7 showing that inferior or superior pH to these values drastically decrease enzyme activity as observed in Figure 5. In other fruits such as papaya and peach (Cham and Tam, 1982) the optimal pH values are similar. Pme activity optimal temperature for vinagrillo fruits Pg was found to be 30°C. 324
Celulase Activity Ce activity was not detectable in vinagrillo fruits during ripening. In avocado fruits according to Awad and Young (1979) Pg activity increases during ripening as wells in papaya fruits (Paull and Jung, 1983). CONCLUSIONS 1. Firmness and pectin contents were correlated with PME and Pg activities during fruit ripening. 2. Pme showed higher activity in green stage and PG activity was higher in green stage of ripening. 3. Pme activity was higher at alkaline pHs in a range between 5 and 11 and optimal temperature for Pme was found to be 30°C. 4. Optimal pH for maximal activity of Pg was 7 and optimal temperature for vinagrillo fruits Pg was found to be 30°C. 5. Cellulase activity was not detected during ripening of vinagrillo fruits. Literature Cited Andrews, P. and Shulin, L. 1995. Cell wall hydrolytic enzyme activity during development of non-climacteric sweet cherry (Prunus avium) fruits. J. Hort. Sci. 70(4):651-657. AOAC. 1980. Official Methods of Analysis of Association of Analytical Chemists. Washingthon, DC, USA. Awad, M. and Young, R. 1979. Postaharvest variations in cellulase, polygalacturonase and pectin methylesterase in avocado (Persea americana Mill) cv Fuerte. Plant Physiol. 64:306-308. Aponte, L. and Guadarrama, A. 2003.Actividad de las enzimas pectinmetilesterasa, poligalacturonasa y celulasa durante la maduración de frutos de parchita maracuyá (Passiflora edulis f. flavicarpa Degener). Revista de la Facultad de Agronomía(UCV): 29:145-160. Chan, H. and Tam, S. 1982. Partial separation and characterization of papaya endo and exo polygalacturonase. J. Food Sci. 47:1478-1483. Chin, I., Zainon, M. and Hamid, L. 1999. Cell wall modifications, degrading enzymes and softening of carambola fruit during ripening. J.Exp. Bot. 50:767-775. Durbin, M. and Lewis, L. 1988. Cellulase in Phaseolus vulgaris. Method in Enzymology 160:342-351. Gordon, A., Sekine, Y., Watanabe, N. and Barret, D. 2002. Thermal inactivation of pectinmethylesterase, polygalacturonase and peroxidase in tomato juice. J. Agric. Food Chem. 50:6153-6159. El-zoghbi, M. 1994. Biochemical changes in some tropical fruits during ripening. Food Chem. 49:33-37. Guadarrama, A. 2001. Fisiología postcosecha de frutos. Alcance 61. Revista de la Facultad de Agronomía. Universidad Central de Venezuela. 139p. Hagerman, A. and Austin. 1986. Continuous spectrophotometric assay for pectinmethylesterase. J. Agric. Food Chem. 34:440-444. Hobson, G. 1996. Maduración del fruto. p.463.478. En: J. Azcon-Bieto and M. Taylor (ed.), Fisiología y Bioquímica Vegetal. México. Interamericana McGraw-Hill. King, K. 1990. Partial characterization of the in situ pectinesterase in Bramley apple. Int. J. Food Sci. Tech. 25:188-197. Lehninger, A. 1995. Bioquímica. Ediciones Omega. Segunda Edición. Barcelona-España. 1117p. Paull, R. and Jung, E. 1983. Postharvest variations on cell wall degrading enzymes of papaya (Carica papaya L.) during fruits ripening. Plant Physiol. 72:382-385.
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Tables
Table 1. Variations of firmness of vinagrillo fruits during ripening. Stages of maturation Penetration in mm Green 1.80 Turning 2.70 Ripped 3.80 Overriped 7.90
g galacturonic acid /100 g pulp x10-2
Figures
16 14 12 10 8 6 4 2 0 Green
Turning
Ripped
Overriped
Maturity Stage
Fig. 1. Variations in pectin contents of vinagrillo fruits during ripening.
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14
Absorbance change per hour
12 10 8 6 4 2 0 Green
Turning
Ripped
Overriped
Maturity Stage
Viscosity Loss ( %) x 10-1
Fig. 2. Pectin methyl esterase (PME) activity of vinagrillo fruits during ripening.
45 40 35 30 25 20 15 10 5 0 Green
Turning
Ripped
Overriped
Maturity Stage
Fig. 3. Polygalacturonase (PG) activity of vinagrillo green fruits during ripening.
327
Absorbance change per hour x 10-2
160 140 120 100 80 60 40 20 0 2
3
4
5
6
7
8
9
10
11
pH
Fig. 4. Effect of pH on Pectin methylesterase (Pme) activity of vinagrillo green fruits.
Activity Loss (%) x 10-1
140 120 100 80 60 40 20 0 3
4
5
6
7
8
9
pH Fig. 5. Effect of pH on Polygalacturonase (Pg) activity of vinagrillo green fruits.
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