Journal of Agricultural Science and Technology B 2 (2012) 1148-1157 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
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Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening Ă ngel Guadarrama and Scarlett Andrade Department of Agricultural Botany, College of Agriculture, Central University of Venezuela, Maracay, Aragua 2021,Venezuela Received: July 11, 2012 / Published: November 20, 2012. Abstract: This study aimed at the physical, chemical and biochemical changes during ripening of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) fruits during ripening as important features to better understand their postharvest handling. It was carried out physical analysis such as firmness and chemical analysis such as total chlorophyll, total carotenoids, soluble solids, pectins and titrable acidity and biochemical analysis such as pectin methyl esterase, polygalacturonase, cellulase, and peroxidase and polyphenoloxidase activities in crude extract. Fruits were harvested at different stages of ripening. Experimental design was completely randomized and was carried out analysis of variance and Tukey tests. Total chlorophyll was decreasing in later stages of ripening, total soluble solid contents increased as the fruits ripen, while the acidity expressed percentage of citric acid decreased during fruits ripening. The loss of firmness and soluble solids content increased as the fruit get ripped stage, while the content of pectin decreased. Activity was observed for pectin methyl esterase and polygalacturonase enzymes during all stages of maturation, presenting the highest activity for both enzymes in the mature state. No cellulase activity detected at any stage during the ripening of these fruits. Activity of the enzyme polyphenoloxidase and peroxidase, associated with pulp browning was higher in the last stages of ripening of these fruits. Physical, chemical and biochemical patterns during ripening were different according to fruit species suggesting differential postharvest handling requirements. Key words: Firmness, pectin, pectin methyl esterase, polygalacturonase, peroxidase.
1. Introductionď€ Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) are underutilized fruits in Venezuela despite the ecological conditions are favorable to become commercial fruits due to their attractive acid-sweet taste for consumers and potential for juices and jams processing. Fruit ripening is related to a series of processes that produce organic compounds, increase respiratory activity and ethylene production leading to certain physical and chemical changes associated with softening of the tissue, pigmentation as chlorophyll
Corresponding author: Angel Guadarrama, Ph.D., research field: postharvest physiology of tropical fruits. E-mail: angelguadarrama6@gmail.com.
and carotenoids, solid solubles, titrable acidity and enzyme activities related to softening and browning. Firmness is one of the most important factors in assessing the degree of ripening in different kinds of fruits. Fruit softening in peach is a continuous process and correlated closely with the depolimerization of matrix glycans, which proceeded throughout development, including ripening [1]. Pectic degradation causes weakening of the cell wall with consequent changes in fruit tissues which lead to softening. The loss of pectins in persimmon fruits was also accompanied by a depolimerization of the polysaccharides extracted in the pectic fractions [2]. The color changes are due to the synthesis of pigments that will be desirable depending on the type
Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening
of fruit. Ripening involves a series of changes in pigmentation mainly chlorophyll degradation and synthesis of carotenoids. Carotenoids biosynthesis and its regulation during tomato fruit development and ripening is a complex process that occurs alongside the differentiation of chloroplasts into chromoplasts and changes to the organoleptic properties of the fruit [3]. Chlorophyll degradation is affected by many factors such as chlorophyllase enzyme activity, changes in pH and oxidative changes in fruit tissues in skin and mesocarp [4]. Total soluble solids primarily reflecting the sugar content in fruits are used as an indicator of quality and physiological maturity of fruits. Most fruits are rich in organic acids such as citric, malic and tartaric acids. The titratable acidity increases during fruit development to reach a maximum value approximately at physiological maturity and then begin to decrease with ripening fruit where they are used in part as a substrate for respiration [5]. During fruit softening, the protopectins and hemicelluloses suffer solubilization and depolimerization which contribute to laxity and disintegration of the cell wall [6]. The involvement of enzymes of cell wall hydrolases constitute key elements in fruit softening, mainly pectin methyl esterase, polygalacturonase and cellulase. Pectin methyl esterase (EC 3.1.1.11) is an enzyme that catalyzes pectin demetilation from polygalacturonic acid [7] which results in generation of free carboxyl groups in pectins affecting the pH and ionic equilibrium within the cell wall and activity of hydrolases enzymes associated and interactions between cell wall structural components. Polygalacturonase (EC 3.2.1.15) is an enzyme that catalyzes the hydrolysis of glycosidic bonds α-1,4 polygalacturonic acid. This enzyme is classified into endo and exo-polygalacturonase, according to the inner side or terminal, respectively of polygalacturonic acid [8]. Cellulase (EC 3.2.1.4) is very specific and acts on the β-1,4 glycosidic bonds of cellulose cell wall and
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converts it into smaller fragments called dextrins and then heat-in cellobiose. Cellulolytic enzymes are a complex of enzymes consisting of exo-β-1,4-glucanase (EC 3.2.1.91), endo-β-1,4-glucanase (EC 3.2.1.4) and β-glucosidases (EC 3.2.1.21), which act synergistically to degrade cellulose to glucose and other oligosaccharides [9, 10]. During the postharvest of many fruits, undesirable reactions occur that degrade fruit quality named enzymic browning. In this process participates enzymes such as polyphenoloxidase and peroxidase. Polyphenoloxidase is a copper-containing enzyme catalyzes the oxidation of phenolic compounds and makes melanoid pigments which give undesirable traits to the harvested fruits and their processed products. Peroxidase is a cupper-containing enzyme that is used as a catalyst for the oxidation of hydrogen peroxide by any of several substrates [11]. The purpose of this study was to determine some physical, chemical and biochemical changes in Annona squamosa L. and Spondias citherea Sonner fruits during ripening in order to stimulate further studies to improve postharvest handling in these underutilized fruits with potentials in Venezuela.
2. Materials and Methods 2.1 Plant Material Sweetsop fruits (Annona squamosa L.) were harvested in San Francisco de Asis, Aragua, Venezuela. Golden apple fruits (Spondias citherea Sonner) were harvested in Cumana, Sucre State, Venezuela. Both fruit species were selected for each ripening stage: green at physiological maturity (stage 1), half-ripe (stage 2), ripe (stage 3) and overriped (stage 4) based on subjective criteria of peel color and firmness to the touch. 2.2 Physical Analysis Firmness: we made determination of firmness in fruit equatorial zones by a penetrometer ELE-400.
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Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening
2.3 Chemical Analysis Soluble solids content: using a refractometer Atago N1 and results expressed in Brix degrees as indicators of soluble solids (%). Pectins: by AOAC method [12] and using a standard curve of galacturonic acid in a range between 0 and 60 mg/mL of galacturonic acid. Total carotenoids: by McCollum method [13] using hexane as a solvent and an average specific absorption coefficient of 224 obtained from α-carotene (248), β-carotene (228) and criptoxantol (216) and readings were performed on a Spectronic 20 at 470 nm. Results were expressed as mg of total carotenoids/100 g of peel. Total chlorophyll: by Comar and Zscheille method [14] with 80% aqueous acetone as a solvent and specific absorption coefficients of chlorophyll a and b indicated by Arnon [15]. 2.4 Biochemical Analysis Extraction of hydrolases enzymes: Twenty five gram of fruit pulp (in different stages of maturity) in 50 mL of a buffer solution of 10% NaCl, homogenized for 1 min in cold (-5 °C). The extract was centrifuged for 30 min 15,000 rpm at 5 °C in a Sorvall RC.2B. The supernatant obtained was filtered through glass wool and stored frozen at -10 °C for analysis of the corresponding enzyme activities. Extraction of Oxidase Enzymes: Twenty five gram of fruit pulp (in different stages of maturity) were added in 50 mL of a buffer solution of buffer phosphate 0.1 M, pH 6.2 and blended for 1 min at 5 °C. The extract was centrifuged for 30 min 15,000 rpm at 5 °C in a Sorvall RC.2B. The supernatant or crude extract was used for enzymatic assays. Pectin methyl esterase activity was evaluated by the spectrophotometric method of Hagerman and Austin [16]. The reaction mixture consisted of 2.5 mL of a solution of 1% citrus pectin (w/v) of distilled water and 0.75 mL of crude enzyme extract and two drops of blue bromophenol. The mixture reaction was incubated
in a thermal bath for 1 h at 30 °C. Pectin methyl esterase activity was measured by the change in absorbance at 620 nm in a Milton Roy Spectronic 401. The activity was expressed as the change in absorbance at 620 nm per hour (Δabs 620/h). Polygalacturonase activity: using the method of Durbin and Lewis [17]. We used a reaction mixture of 2.5 mL 0.2% (w/v) polygalacturonic acid plus 0.75 mL of crude enzymatic extract crude. The mixture reaction was incubated at 30 °C in a thermal bath for 1 hour. PG activity was expressed as the percentage loss in viscosity of the reaction mixture in an Oswald viscometer and the activity was expressed as the percentage loss of viscosity when the reaction mixed. Cellulase activity: by Durbin and Lewis method [17], in conditions similar to the activity of polygalacturonase but using 0.2% carboxymethyl cellulose as substrate. The activity was expressed as percentage of loss of viscosity. Polyphenoloxidase activity: enzyme activity was determined by the method of Ponting and Joslyn [12-18] using a reaction mixture comprising 2 mL of substrate (Chatecol 0.01 M), 0.9 mL of 0.1 M phosphate buffer, pH 6.2 and 0.1 mL of crude enzyme extract. Peroxidase activity: by the method of Ponting and Joslyn [18] using a reaction mixture comprising 2 mL of substrate (Guaiacol 0.01 M + Hydrogen peroxide), 0.9 mL of 0.1 M phosphate buffer, pH 6.2 and 0.1 mL of crude enzyme extract. Statistical analysis: samples of fruits were taken randomly using three replicates and three fruits for each physical and chemical analysis and enzyme activities. An analysis of variance and Tukey tests were performed.
3. Results and Discussion 3.1 Establishment of Different Stages of Ripening Based on subjective criteria of pigmentation of the peel and firmness to the touch, we designed a hedonic scale to the ripening stages for sweetsop and golden apple fruits (Tables 1 and 2, Figs. 1 and 2).
Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening Table 1 Peel pigmentation and firmness to the touch of sweetsop fruits. Ripening Peel color stages Light green with hints of violet on the Stage 1 scales and yellow pigmentations including Scales greenish-yellow with yellow Stage 2 colors including Scales with features yellow purple and Stage 3 yellow colors including Purple scales less yellowing Stage 4 including
Firmness to the touch Higher firmness Intermediate firmness Intermediate firmness Lower firmness
Table 2 Peel pigmentation and firmness to touch of golden apple fruits. Ripening stages Stage 1 Stage 2 Stage 3 Stage 4
Peel color Full green Green with light yellow Full bright yellow Pale yellow
Firmness to the touch Higher firmness Medium firmness Regular firmness lower firmness
3.2 Physical Changes Fruits with green color at physiological maturity showed the higher firmness on the first day of evaluation and therefore more resistant to penetration of the conical tip of the instrument and less degree of penetration on the surface of the fruit. Subsequently,
Stage 1 Fig. 1
Fig. 2
the resistance tended to decrease as time of ripening goes on, as seen in Fig. 3, mainly caused by changes in the structure and composition of the cell walls by enzymatic degradation or hydrolysis of cellulosic and pectic substances. Softening and changes in firmness during fruit ripening occurs to the extent that the cell wall is modified and partially degraded by hydrolase enzymes [1]. Fast softening of climacteric fruit such as sweetsop is the main limiting factor in postharvest life during storage. This physiological process of ripening is associated with changes in texture as a product of cell wall degradation. During the softening of the fruit, pectins and hemicellulose suffer solubilization and depolimerization. As part of the physiological process of ripening, fruit softening is one of the factors limiting the useful life of the fruit postharvest and is associated with firmness changes that occur as a result of hydrolysis of the modified cell wall structure and composition of its constituents, mainly transformations of insoluble protopectins soluble pectins.
Stage 2
Stage 3
Stage 4
Stages of ripening in sweetsop (Annona squamosa L.) fruits.
Stage 1
Stage 2
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Stage 3
Stages of ripening in golden apple (Spondias citherea Sonner) fruits.
Stage 4
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Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening
Fig. 3 Variations in firmness loss in sweetsop and golden apple fruits during ripening.
Fig. 4 Variations in pectins in sweetsop and golden apple fruits during ripening.
3.3 Chemical Changes Pectin content decreases as the fruits passed from a condition more firmly in the green fruit at physiological maturity (stage 1) to a less firmness in overripe fruit (stage 4), as illustrated in Fig. 4. Pectin content in soursop fruits was always higher than golden apple in every state of maturation suggesting its greater potential for preparation of jams. As mentioned above during the softening of the fruits, pectins and hemicellulose suffer solubilization and depolimerization probably following different patterns for each studied fruit. Persimmon fruits (Diospyros kaki L.) have a large proportion of pectins, 46% by dry weight that decreased to 20% with ripening. This decrease
occurred mainly in fractions composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The loss of pectins in persimmon fruits was also accompanied by a depolimerization of the polysaccharides extracted in the three pectic fractions [2]. Fruit softening in peach is a continuous process and correlated closely with the depolimerization of matrix glycans, which proceeded throughout development. However, numerous other cell wall changes also took place, such as the deglycosylation of particular polymers and the solubilization and depolimerization of chelator-soluble polyuronides, but these were transient and occurred only at specific phases of the softening process. Fruit softening and other textural changes in peach appear to have a number of stages, each involving a different set of cell wall modifications [1]. Pectin methyl esterase demethoxylates pectins and is believed to be involved in degradation of pectic cell wall components by polygalacturonase in ripening tomato fruit. Antisense and sense chimeric pectin methyl esterase genes have introduced into tomato to elucidate the role of pectin methyl esterase in fruit development and ripening. Fruits from transgenic plants expressing high levels of antisense pectin methyl esterase showed <10% of wild-type enzyme activity and undetectable levels of pectin methyl esterase protein and mRNA. Lower pectin methyl esterase enzyme activity in fruits from transgenic plants was associated with an increased molecular weight and methylesterification of pectins and decreased levels of total and chelator soluble polyuronides in cell walls. The fruits of transgenic plants also contained higher levels of soluble solids than wild-type fruits. This trait was maintained in subsequent generations and segregated in normal Mendelian fashion with the antisense pectinmethylesterase gene. These results indicate that reduction in pectin methyl esterase enzyme activity in ripening tomato fruits had a marked influence on fruit pectin metabolism and increased the soluble solids
Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening
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content of fruits, but did not interfere with the ripening process [19]. 3.4 Soluble Solids Content As fruits ripe the soluble solids content increased as shown in Fig. 5, which is attributed to degradation of starch probably due to an increase in the activity of starch hydrolases enzymes. Soluble solids content was higher in sweetsop fruits in all stages of ripening comparing to those of golden apple. generally the solids content (primarily sugars) increases in ripening fruit conferring to a more pleasant flavor as it improves the balance sugar-acid. Compositional changes of fruit pulp and peel during ripening of white- and pink-fleshed guava fruits were studied [20]. Fruit tissue firmness decreased progressively, in a similar manner, in both guava fruit types. Total soluble solids (TSS) and total sugars increased in pulp and peel of both guava types with decrease in flesh firmness. More increase in total sugars was observed after the climacteric peak of respiration. Reducing sugars and titratable acidity increased up to the full-ripe stage and then decreased. The peel showed higher values of ascorbic acid, total protein and phenolic compounds than the pulp. The white-fleshed guavas had higher levels of TSS than the pink-fleshed fruits. 3.5 Titrable Acidity Fig. 6 shows a decrease in titrable acidity as ripening progresses. This decline is the most evident when fruits change from stage 1 to stage 2 in which almost there are reductions of about 50% for both fruit species. Organic acids generally fall during ripening because are used as respiratory substrates or are converted into sugars. Titrable acidity was higher in golden apple fruits at all stages of ripening. Organic acids are important in relation to the fruits flavor and that influence perception of sweetness and the acidity reduction plays an important role in acid-sugar balance. The organic acids present in the pulp of Keitt mangoes at various stages of ripeness
Fig. 5 Variations in soluble solids in sweetsop and golden apple fruits during ripening.
Fig. 6 Variations in titrable acidity in sweetsop and golden apple fruits during ripening.
were analyzed by HPLC [21]. Acidity loss was shown by decreasing titratable acidity and increasing pH values. Citric and malic acids were found to be the major organic acids. A large decrease in citric acid and a small reduction in malic acid were responsible for the loss of acidity. Tartaric, ascorbic, oxalic and Îą-ketoglutaric acids were also shown to be present at low concentrations. 3.6 Total Carotenoids The production of carotenoids in golden apple fruits has a dramatic increasing trend in ripe stage as observed in Fig. 7. Total carotenoids were higher in golden apple fruits than sweetsop fruits at all stages of ripening. Carotenoids biosynthesis and its regulation during tomato fruit development and ripening is a complex process that occurs alongside the differentiation of chloroplasts into chromoplasts and changes to the organoleptic properties of the fruit. Unusually for plants,
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Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening
Fig. 7 Variations in total carotenoids in sweetsop and golden apple fruits during ripening.
Fig. 8 Variations in total chlorophyll in sweetsop and golden apple fruits during ripening.
the ripe tomato fruit accumulates large amounts of lycopene, as the pattern of gene expression found in green fruit changes during fruit ripening. Although the control of gene expression is thought to be the main regulatory mechanism for these alterations in carotenoids, postâ&#x20AC;?transcriptional regulation has also been reported, including feedback inhibition. The use of genetic manipulation of carotenogenesis in tomato has been used primarily for biotechnological reasons, but it has also facilitated investigations into these regulatory mechanisms, as well as into the effects of such perturbations on other isoprenoids such as gibberellins, tocopherols and sterols [3].
levels of transcripts of various genes coding for proteins of the photosystem I, photosystem II and the stroma decrease when plastids differentiate from chloroplasts to chromoplasts. The amount of plastid ribosomal RNA also decreases. Transcripts of the genes for the P700 reaction center protein, for the photosystem II-associated proteins and for the large subunit of ribulose-1,5-bisphosphate carboxylase cannot be detected in chromoplasts [22].
3.7 Total Chlorophyll The increased synthesis of carotenoids in sweetsop and golden apple fruits coincides with chlorophyll degradation. Loss of Chlorophyll from the peel was almost similar at latest stage of ripening for both fruit species as shown in Fig. 8. Indicator clear of fruit ripening is the degradation of chlorophyll in the peel, so the fruit appearance for consumption will depend on the relative amounts of individual pigments existing in the peel. Chlorophyll degradation is affected by many factors such as chlorophyllase enzyme activity, changes in pH and oxidative changes in fruit tissues in skin and mesocarp as mentioned in the introduction. At molecular level, the expression of plastid genes during tomato fruit ripening has been studied. The
3.8 Biochemical Changes Trends of pectin methyl esterase and polygalcturonase enzyme activities follow the same pattern in both fruit species for all stages of ripening, but is always higher activities in sweetsop fruits as shown in Figs. 9 and 10, respectively As the fruit advances in ripening reduces the strength of them due to structural and compositional changes in the cell walls by enzymatic hydrolysis of cellulosic substances, pectic acid and polygalacturonic. So in fruits of papaya [23], it was determined the apparent molecular mass range of different extractable fractions of pectin and hemicellulose, finding that the molecular mass of pectin decreased and solubility increased during fruit ripening , while the water-soluble uronic acid increased during ripening. The loss of high molecular weight pectins decreases during maturation, while the rate of demethylation was greater at the beginning of ripening. The pectin fractions are mainly composed of rhamnose, glucose,
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Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening
is responsible exclusively of this physiological process, but has a synergistic joint participation of various hydrolytic enzymes of the cell wall. Cellulase activity there was no detectable activity of the cellulase enzyme in any of the stages of ripening with the methodology used in this study, suggesting that sweetsop and golden apple fruits its participation is not as crucial to the softening of these fruits such as pectinmethylesterase and polygalacturonase are. Fig. 9 Variations in pectin methyl esterase in sweetsop and golden apple fruits during ripening.
3.9 Polyphenoloxidase and Peroxidase Activities Polyphenoloxidase
and
peroxidase
activities
increased in pulp tissues up to the stage 2 of ripening and then decreasing to late stages of ripening in both fruits species but showing different paterns as showed in Figs. 11 and 12, respectively Postharvest browning of fruit pericarp is a major problem, resulting in accelerated shelf life and reduced commercial value of the fruits. The browning was generally thought to be a rapid degradation of Fig. 10 Variations in polygalcturonase activity in sweetsop and golden apple fruits during ripening.
phenolic compouds caused by polyphenol oxidase and
xylose, galactose, mannose and arabinose, in descending order of concentration. These results suggest that the hydrolysis of pectin and modification of the hemicellulose were involved in the softening of papaya fruit during ripening and hydrolysis of the pectin appears to be most important during the final phase of the softening. All changes in the cell wall on the fruits are related to the activity of hydrolytic enzymes. The principal enzymes that have been identified as involved in the metabolism of the cell wall during softening of the fruits are polygalacturonase, pectin methyl esterase, cellulase, xylanase and β-galactosidase, which can vary the importance of each of these hydrolases depending on the fruit species considered. For example, the cellulase activity correlates well with the avocado fruit softening, whereas polygalacturonase correlates well with the tomato fruit softening. It is also likely that none of the enzymes mentioned above,
are among the most studied enzymes in fruits and
peroxidase [24]. Polyphenoloxidases and peroxidases vegetables. Owing to the deleterious effects of discoloration and off-flavor formation induced by their actions, these enzymes have not ceased to be a matter of concern to food technologists, while their versatility as catalyst and their diversity as protein present a challenge to the biochemist [11].
4. Conclusions (1) Variations were detected in firmness, soluble solids, chlorophylls, carotenoids and pectins during ripening of the sweetsop and golden apple fruits following different patterns according to the fruit species suggesting differences at genetic level. (2) There were pectin methyl esterase and polygalacturonase activities in all stages of ripening, presenting maximum values for both enzymes in half-ripe sweetsop and overriped golden apple fruits, respectively.
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Physical, Chemical and Biochemical Changes of Sweetsop (Annona squamosa L.) and Golden Apple (Spondias citherea Sonner) Fruits during Ripening [2]
[3]
[4]
Fig. 11 Variations in polyphenoloxidase activity in sweetsop and golden apple fruits during ripening. [5]
[6] [7]
[8]
Fig. 12 Variations in peroxidase activity in sweetsop and golden apple fruits during ripening.
(3) In both fruit species there was no detectable activity of the enzyme cellulase for any of stages of ripening evaluated. We can infer a negligible or no activity of this enzyme. (4) Hydrolysis of pectic substances during the softening is probably a consequence of the action of pectin methyl esterase and polygalacturonase enzymes in sweetsop and golden apple fruits. (5) This study can lead to stimulate further studies in fruit physiology area in order to improve postharvest handling in these underutilized fruits with potentials for fresh consumption and processing in Venezuela and why not in other tropical countries.
[9]
[10]
[11]
[12]
[13]
[14]
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