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| Utilisation of dates in bio technology III:
The Enzymology of Dates Ripening |
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Enzymes are the biological catalysts of all reactions in all forms of life. They are proteins with widely different specificities and carefully controlled by special gene for each enzyme (i.e. one gene one polypeptide chains). The ripening process of fruits is catalyzed by a group of hydrolytic enzymes where the optimal ultimate result is observed in superior fruit quality (texture, flavor, color and nutritive value). In dates, the activity levels of glycosidases (carbohydrate splitting enzymes) dictates dates classification (i.e. dry, semi-dry or soft cultivar). These enzymes are classified as hydrolases where they catalyze the breakage of the glycosidic bonds in carbohydrate molecules utilizing water molecules (one water molecule per each cleaved bond). These enzymes include invertase, pectinesterase, polygalacturonase, cellulase(s) and β-galactosidase. However, the discovery and study of these enzymes varied in the depth and date of reporting. Among these enzymes, invertase was the first and most studied (since the first decade of the past century) while β-galactosidase was the last enzyme discovered (in 1988).
The present article is an attempt to point out the significance of the role of these enzymes in dictating the ultimate quality of dates with a special emphasis on dates β-galactosidase. |
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| Invertase |
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This is the most extensively studied among date enzymes and it was reported for the first time in 1907. The enzyme hydrolyzes sucrose to glucose and fructose (invert sugars) from the very beginning of rutab stage. In terms of specificity, dates invertase is classified as β-fructofuranosidase which means that the enzyme binds the fructose moiety of the sucrose molecule. In the presence of suitable moisture levels (i.e. higher than 25%), invertase activity converts sucrose almost completely to invert sugars which enhance the remarkable sweet taste of soft date cultivars. At decreased moisture content in the rutab stage (e.g. in dry and semi-dry date cultivars) sucrose inversion reaction ceases since water represents the second substrate. Increasing the moisture level by re-hydration process restarts the reaction and causes remarkable change in the fruits quality (usually better color and flavor) as in Deglet Noor cultivar. "Sucrose cultivars" (dry and semi-dry) are characterized by their light color and moderately sweet taste mainly due to the low invertase activity during the natural ripening process. In fact, natural dates color depends greatly on invertase activity level because reducing sugars (i.e. glucose and fructose) react with amino acids and peptides through non-enzymatic browning and producing the characteristic brown color and contributing to the flavor of fully ripe soft date cultivars. However, such events do not occur in dry and semi-dry cultivars due to the non-reducing (i.e. not reactive) nature of sucrose. Furthermore, physical field treatments such as `bunch bagging causes increased invertase activity and faster ripening due to cumulative effects of higher temperature, moisture and increased ethylene level around bunch vicinity.
Invertase seems to be a cell wall bound enzyme and it is present in all dates maturity stages with high activity jump in the rutab stage. It is believed that most (if not all) date biosynthesis of ripening associated enzymes are triggered by fruit ethylene production which is one of the major indicators of cell and tissue senescence. It was shown that extracting dates invertase with low ionic strength buffer solutions causes the formation of strong ionic complex between invertase and a high molecular weight pectic substance fragment.
Thus, controlling invertase activity during ripening leads to the control of sucrose level and color intensity in dates.
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| Pectic Enzymes |
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| Two types of pectinases were reported to be present in dates: |
- Pectinesterases which hydrolyze the ester bonds between the methyl ester groups and galacturonic acid in the pectin molecules without affecting the polysaccharide chain (i.e. decreasing the degree of esterification).
- Endo-polygacturonase which hydrolyzes the polysaccharide chains of pectin randomly from the causing rapid decrease in molecular weight and producing soluble pectin fragments with variable molecular sizes. The enzyme was purified from Hallawi cultivar by affinity chromatography on cross-linked pectate and it was characterized as an endo-splitting polygalacturonase.
Since pectin is the "cement" of plant cell walls, it affects plant tissues firmness to a great extent. The rigid and crunchy texture of Bisr (Khalal) stage is due to intact cell walls and the presence of relatively high content of insoluble pectic substances (protopectin). Pectinesterase activity is present in all of dates maturity stages while polygalacturonase activity starts in the beginning of Bisr stage and increases remarkably in the rutab stage. Pectin degradation causes the loss of cell wall integrity which is reflected in fruit softening (as in rutab) in addition to the release of various cell wall enzymes through the exposure of protected cell wall components to specific enzymes. Polygalacturonase biosynthesis is also believed to be induced by ethylene and consequently its activity levels would be positively affected by pre-harvest treatments such as bunch bagging. Thus, dry cultivars contain higher levels of insoluble pectic substances than soft cultivars due to the limited polygalacturonase activity. The type of pectic substances (soluble or insoluble) has an essential role in the processing of date extracts (i.e. filtration) which represents the first stage in date transformational industries. |
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| Cellulases |
These are the least studied among date enzymes in spite of their important effect on dates texture during the ripening process of dates. Although it is well known in many fruits and vegetables that cellulose(s) activity include a mixture of two or three enzymes, there are no similar details available about date cellulases. Basically, the main types of higher plant cellulases hydrolyze cell wall cellulose in different mechanisms yielding small cellulose fragments, cellobiose (disaccharide) and glucose. The extent of these activities contributes to fruit texture softness during the ripening process. The available information about these activities in dates indicated the presence of increasing cellulase(s) activity in dates as they start ripening from early rutab to tamr stage. Bunch bagging caused a clear increased in Zahdi and Khastawi cultivar fruits and showed softer texture compared to the control bunches. Again this represent an indirect evidence on ethylene induction of date cellulases.
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| β-Galactosidase |
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This enzyme is probably the latest discovered among date ripening enzymes where its |
presence was reported for the first time in 1988 in Zahdi dates. The common name lactase was given to β-galactosidase due its hydrolytic activity on its natural substrate lactose (milk sugar). The enzyme is `widely spread in nature being present in microorganisms, plants and animals. However, the enzyme function in higher plants is entirely different from that in microorganisms and animals since lactose hydrolysis does not represent significant metabolic process. The presence of β-galactosidase activity was reported in apples, wheat leaves, wheat grains, sugar cane leaves, Petunia hybrida and lily pollen. The biochemical and physiological roles of the enzyme range from apple ripening to degradation of stylar arabinogalactans to provide the required carbon source for pollen tube nutrition.
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While β-galactosidase was present in all examined maturity stages of Zahdi cultivar fruits, the |
| specific activity of the enzyme increased 35 folds as the fruits reached the full rutab stage as compared to the early green stage (kimri). Also soluble and ionically bound enzyme forms were detected and the former was the most abundant. Although three different purification procedure were used, only partial purification was achieved. In all three procedures the large carbohydrate fraction which was associated with the enzyme was the major obstacle in the achievement of purified homogenous enzyme preparation. It was concluded that a high molecular weight enzyme-carbohydrate covalent complex existed and caused incomplete purification. In fact, the carbohydrate fraction represented 95.3%, 90.0% and 75.6% of the partially purified enzyme preparation of purification procedures I, II and II, respectively. Partially purified dates β-galactosidase by procedures I and II had invertase activity (bound) of 18.9% and 3.7%, respectively. The enzyme demonstrated clear hydrolytic activity on dates cell wall fractions yielding D-galactose among mixture of oligosacchrides. This confirms the ripening function of the enzyme in dates which makes it as one of the important markers of dates ripening process. The techniques which were used in the purification procedures include ethanol fractionation, gel filtration on various types of columns and ion exchange chromatography. |
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Histological studies showed that while most cell walls integrity was maintained in dry date |
| cultivar tissues, cell walls were massively degraded in soft cultivars. These experimentally obtained facts about ripening enzyme must be carefully considered in the design and timing of pre-harvest treatments, post-harvest treatments and date processing operations in order to achieve the targeted improvements and product developments. |
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“ Inquiries about references and further technical details should be forwarded to OASIS Ltd”
Email::info@oasisnakhoil.com |
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