Role of Pectic Enzymes in Fruit Ripening Process

Role of Pectic Enzymes in Fruit Ripening Process ABSTRACT Fruits are important natural sources of human nutrition for regulation of several metabolic functions and maintenance of good health. They are one of the significant commercial food commodities and their economic value depends on several limiting factors. They easily undergo ripening in a shorter period of time and have a limited post-harvest existence. Fruit ripening is a common phenomenon occurring in fruits based on which they are harvested, consumed directly or processed for various purposes. It is an intricate phenomenon involving extremely synchronized, genetically encoded and an irreversible process that results in a sequence of biochemical, physical and organoleptic alterations in a fruit. This finally develops into soft, palatable and a ripe fruit which possess necessary quality characteristics. On the other hand, excessive ripening results in spoilage of fruits. Ripening is related to the modifications of cell wall polysaccharides like starch, cellulose, hemicellulose and p ectins. Despite of several changes occurring in fruits such as loss of turgor, starch degradation and loss of contents during ripening, enzyme-catalysed variations to cell wall composition and structure are considered as main reason for softening of fruits. Pectic enzymes are a group of enzymes responsible for pectin degradation in fruits. They are termed as pectinase that includes pectic lyase, polygalacturonase, pectic methyl esterase and rhamnogalacturonase. Pectic enzymes degrade pectin resulting in softening and senescence in fruits. Pectins are the important polysaccharides occurring in middle lamella and primary cell wall. It contributes to the texture, consistency and quality of fruits. Degradation of pectin during ripening process was known to cause fruit tissue softening. Recent developments in molecular biology have helped in better understanding of fruit ripening mechanism due to enzymic activity. A review about the role of pectic enzymes on the plant cell walls to bring about softening during ripening process is discussed. Keywords: Fruits, cell wall polysaccharides, pectin, pectic enzymes, ripening INTRODUCTION Fruits are important sources of human nutrition and it is a commercially essential food commodity. Fruits are greatly specialized structures developed from a matured and pollinated ovary which have aesthetic characteristics with delicate aroma, striking colours, flavour, taste and juiciness from the edible seed vessels or receptacles (Prasanna et al., 2007). Prasanna et al., (2007) have indicated that fruits possess a wide variety of nutritional substances like vitamins (B-complex, C and K), phenolics and carotene providing higher energy value. They are classified as temperate, tropical and sub-tropical fruits based on their distribution in nature (Prasanna et al., 2007). When completely matured, harvesting of fruits is done. Maturity and ripening of fruits depends on their catalytic mechanism and based on the respiration pattern and biosynthesis of ethylene during ripening, harvested fruits are grouped as climacteric (capable of ripening after detachment from parent plant) and non-c limacteric (unable to continue ripening when detached from parent plant) type (Prasanna et al., 2007). Fruit ripening is an intricate irreversible phenomenon which is highly synchronized and genetically encoded causing biochemical, physiological and organoleptic alterations resulting in a ripened edible fruit (Prasanna et al., 2007). Several factors involved in ripening of fruits are shown in Figure 1. Figure 1: Factors involved in fruit ripening (Koning, 1994) Plant cell walls are complex structures which contain polysaccharides, proteins and enzymes. Pectin which contributes about one-third of the plant cell wall in dicots plays a crucial role in tissue development, adherence of cells, apoplastic porosity and so on (Almeida and Huber, 2007). Pectin is in control of maintenance of integrity of cell wall. It forms a cross-link between cellulose microfibrils and xyloglucans as shown in Figure 2. During ripening, pectin is greatly modified by the enzyme actions resulting in relaxing of cellular interconnections. This leads to loosening and hydrolysis of cell wall polymers which is the main reason for softening of fruit tissues (Gwanpua et al., 2014). Figure 1: Plant cell wall structure (Sticklen, 2008) Texture, an important quality attribute in fruits is altered leading to softening of fruits during ripening (Barret and Gonzalez, 1994). The mechanisms involved in fruit ripening were not understood clearly. It may be due to several factors like turgor loss, starch deprivation, loss of nutritional contents and so on (Ali et al., 2004). The major changes in texture causing softness where due to enzyme-catalysed changes in the cell wall assembly and composition, cell wall polysaccharides (pectin and cellulose) solubilization partially or completely (Waldron and Faulds, 2007). It has been studied by Rodriguez and Restrepo (2011) that, Pectic constituents is an important determinant of the firmness and consistency of the fruits (Van Buren, 1991). Pectinesterase (PE), polygalacturonase (PG) and Pectateliase (PL) are major pectic enzymes responsible for softening of fruit tissues during ripening (Rodriguez and Restrepo, 2011). Rodriguez and Restrepo (2011) have also indicated that, combine d action of these enzymes add to the final texture of fruit and when these enzymes undergo excessive reactions with pectin, it results in notorious softening and provides perfect conditions for microbial attack. Gwanpua et al., (2014) studied that softening is one of the significant changes occurring in climacteric fruit ripening. They were more prone to microbial infections and physical damages and may adverse during prolonged storage. Fruit firmness determines its economic value and considerable effects were taken to control softness of fruits due to enzymic action, metabolic changes and changes in storage conditions (Gwanpua et al., 2014). Softening associated with the ripening mediated by the pectic enzyme action on plant cell walls of different fruits is discussed in this study. 2. Pectic enzymes Pectinases are commonly referred to as pectic enzymes. They act upon pectin constituents of cell wall matrix resulting in softening of fruits. Polygalacturonase (PG), Pectin methyl esterase (PME), Pectin lyase (PL) are some of the major pectic enzymes classified based on their type of action involved in degradation of pectins. The modes of action of these enzymes are as shown in figure 2. Figure 3: Mode of action of pectolytic enzymes (Van Rensburg and Pretorius, 2000). PME causes de-esterification by catalysing the pectin methyl group hydrolysis (Prasanna et al., 2007). They were known to usually attack on the methyl ester linkages of a galacturonate entity which lies next to non-esterified galacturonate component. This process acts as a precondition for polygalacturonase activity (Prasanna et al., 2007). Rodriguez and Restrepo (2011) implicated that pectin methyl esterases (PME) were found to alter pectin in case of low methoxy pectin and in polygalacturonic acid by hydrolysis of methyl ester bonds in esterified carboxyls. It was observed that the softening process could be controlled by the control of this enzyme action in fruits like pear, grape, citrus, tomato, strawberry, arracacha, potato, papaya, kiwi and guava (Rodriguez and Restrepo, 2011; Prasanna et al., 2007). PG, referred as Pectolytic glycanase is an essential hydrolytic enzyme playing an important role in pectin termination (Prasanna et al., 2007). They act on pectic acids and ÃŽ ±-1, 4- glycosidic bonds are hydrolysed between the residues of galacturonic acid in galacturonans. They are categorized as endo-PG and exo-PG (Prasanna et al., 2007). Prasanna et al., (2007) studied that Endo-PG act randomly in pectin de-polymerization and exo-PG releases galacturonic acid by catalysing the glycosidic bonds hydrolysis from the non-reducing end of the pectic substances. It is recognized that middle lamella dissolution in the course of fruit ripening were done by PG. PL undergoes mechanism of ÃŽ ²-elimination of the glycosidic bonds (Prasanna et al., 2007). They also indicated that PL acts an endo-enzyme which acts randomly during cleavage of esterified galacturonate units. PL was known to act at methyl-ester bonds leading to beta-elimination of glycosidic bonds by formation of double bonds in galacturonic acids (Rodriguez and Restrepo, 2011). Rodriguez and Restrepo (2011) specified that pectin lyase (PL) enzyme were responsible for cell wall degradation resulting in softening of banana and strawberries. The changes in pectin due to these enzymes action were as shown in Figure 4. Figure 4: Action of Pectolytic enzymes on pectin (Prasanna et al., 2007) 2.1. Role of pectic enzymes in softening during ripening in different fruit varieties Pectic enzymes play an important role in ripening of fruit by degradation of pectic substances found in fruits. Several researches were conducted in different fruit varieties and in their respective juices to understand the role of pectic enzymes in softening of fruit tissues and their intended use in preparation of fruit juices to enhance mashing of fruits, for clarification of fruit juices and also to increase the release of flavours from fruits (Whitaker, 1984). Invitro-studies of some selected fruits and their pectic enzymic activity is discussed. Besford and Hobson (1972) studied the pectic enzyme activity in tomato fruit and concluded that action of PG/ PME or transelimination by pectic transeliminase were not responsible for tomato ripening. Almedia and Huber (2008) have indicated that softening of tomato fruit during ripening were not associated with the PG activity eventhough they remained active in ripened fruit. Enzymic activities of other fruits are studied comparing with tomato as model fruit (Ali et al., 2004). Pires and Finardi-Filho (2005) have extracted PE and PG from Peruvian carrots to understand the pectic enzyme activities in carrot and found that pectinases along with amylases would act together to bring about spoilage in tubers. Owino et al., (2004) have studied about the alterations in fig fruits during ripening due to changes in cell-wall polysaccharides. They have concluded that qualitative differences in both receptacle and drupelets were observed in pectic polymers compared to the hemi-cellulosic substances. This indicates that ripening of fig is associated with modifications in pectin. Several studies were done to understand ripening of strawberries genetically as it softens rapidly (Pombo et al., 2009; Bustamante et al., 2006). It was observed that cell wall degradation were due to pectin dissoloution by pectic enzyme activities in strawberries which enters ripening phase quicker overlapping with the senescence. Manrique and Lajolo (2004) discussed about the cell-wall modifications in papaya fruit during postharvest ripening. They found that several enzymic actions are involved in papaya fruit during the entire period. There were notable changes in pectic polymers during papaya ripening which were mainly due to PG activity and probably due to PME as there were charge density modifications in polymers (Manrique and Lajolo, 2004). Avocado fruit undergoes pectin depolymerisation in contrast to other fruits and it was observed that it was the only fruit in which huge amounts of pectin were readily solubilizing under milder conditions (Pesis et al., 1978; Huber at al., 2001). In case of apples, exo-PG, PME and PL activity were observed to cause changes in cell wall polysaccharides (Goulao et al., 2007; Oritz et al., 2011). Studies related to cell wall modification during ripening of apples reveals that exo-PG, PME and PL activity influences softening of apples (Goulao et al., 2007). Studies by Gw anpua et al., (2014) in jonagold apples have shown that increment of PG activity occurred late during ripening and PME activity did not influence the ripening process. Cheng et al., (2011) observed that banana fruit which enters softening during ripening were associated with pectic hydrolytic activity and it was mainly due to the combined effects of PG and PME activity. Abu-Goukh and Bashir (2003) have observed that softening of guava fruit and mango were mainly due to PG and cellulose activity during ripening which resulted in drop in firmness of the fruits. Similarly in case of peaches, softening is due to depolymerisation of pectin by endo-PG activity resulting in solubilization of cell-wall polymers (Yoshioka et al., 2011). 3. CONCLUSION Pectin polymers form a gel-like matrix which provides cell-wall integrity contributing to the firmness of fruits. It stabilizes the texture and colour of the fruit based on which freshness of fruits is determined. Commercial value of fruits is based on these quality attributes. Several researches conducted on different types of fruits indicate that pectic enzymes have an important role in softening of fruit tissues during ripening. They were known to cause pectin solubilization in cell-wall matrices by different modes of action like lysis, de-esterification and glycanase activity. Research studies conducted on selected types of fruits under in-vitro conditions indicated that Polygalacturonase activity is predominant in almost all-types of fruits compare to pectin methyl-esterase and Pectic-lyase activities. Pectic enzymes were not the only factor for softening of fruits during ripening as it is influenced by several other factors like changes in pH, temperature, harvesting period, st orage conditions, physical damage, microbial attack, presence of sugars, other hydrolytic, kinetic and amylase enzymes and so on. Better understanding of mechanism of pectic enzymes on primary cell-walls of fruits would enhance the control of softening and ripening process. This in turn, will improve the quality of fruits extending its shelf-life. Recent advancements favoured the isolation of pectic enzymes commercially which is used intentionally for accelerating the maturation of fruits, added in production of fruit juices to easily crush the fruits, to solubilize the complex cell-wall matrices, to release all nutrients, to control haze formation in juices, to enhance flavour and colour characteristics of fruit juices, and so on. Recent developments in molecular biology have paved a way for genetically understanding the pectic enzyme action on primary cell-walls in fruits. Henceforth, pectic enzymes play a determining role in softening associated with ripening of fruits.