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Abstract
The present thesis dealt with the papaya fruit as a tropical one recently cultivated in Egypt and its cultivated area is expected to increase. So, to provide the market with high quality papaya products, it is necessary to study chemical and biochemical changes occurring in papaya fruit during three stages of maturity. Also, papaya jam was prepared and evaluated through six mouths of storage. The main points of this thesis can be summarized as follows:
1-Chemical composition:
The chemical composition of the fruit during mature green, semi ripe (25% yellow) and ripe (100% yellow) stages was carried out. The chemical determinations included moisture content, total soluble solids, titratable acidity, pH, protein, total cartenoids, ascorbic acid and alcohol insoluble solids (AIS).
2- Enzymatic studies:
Enzymatic studies were carried out on poly-phenoloxidase (PPO) and pectinesterase (PE).
A – Polyphenoloxidase:
Studies on PPO included:
Establishing a valid assay of the enzyme.
Establishing the optimum conditions to obtain highest extractability of the enzyme.
Characterization of the enzyme properties towards its synthetic substrate (catechol) ; Michaelis constant (Km) and maximum velocity . The pH and substrate concentration on the enzyme activity, and determination of Km and Vmax values.
The characterization of the pH optima and the temperature optima for PPO activity .
Identifying the enzyme stability at different pH values and at different temperatures.
Assaying for the papaya PPO activity during the different stages of ripening.
The effect of freezing storage on the enzyme activity.
B – Pectinesterase:
Studies on PE included:
Establishing a valid assay of the enzyme.
Establishing the optimum conditions for maximum extractability of the enzyme from papaya pulp.
Characterization of the enzyme properties towards its substrate (citrus pectin ) ; Michaelis constant (Km) and maximum velocity . The pH and substrate concentration on the enzyme activity, and determination of Km and Vmax values.
The characterization of the pH optima and the temperature optima for PE activity .
Identifying the enzyme stability at different pH values and at different temperatures.
Assaying for the papaya PE activity during the different stages of ripening.
C – Purification of pectinesterase:
Purification steps included
Extraction, precipitation and molecular sieving of the enzyme on CM–sephadex C–50 columns .
Determination of the molecular weight of the purified pectinesterase using SDS – PAGE.
3– Preparation of jam:
Preparing of papaya jam and studying the changes in its chemical composition during 6 months storage effect of storage at room temperature
Analysis of jam monthly by determining:
Total solids, total soluble solids, protein, titratable acidity, pH, total carotenoids and color index.
Sensory evaluation of jam.
The obtained results can be summarized as follows:
1 – Chemical composition
During the fruit ripening the obtained data indicated that: the fruit moisture, total soluble solids (TSS), pH, protein, ascorbic acid and total carotenoids were increased, moisture increased from 62% to 89.4%, TSS from 8.7 to 12.5%, pH from 5.5 to 6.5, protein from 0.195 to 0.8%, ascorbic acid from 47 to 96 mg / 100 gm pulp, total carotenoids from 0.9 to 2.19 mg / 100g . However, the acidity and alcohol insoluble solids were decreased, from 0.15 to 0.08% and from 3.26 to 1.98%, respectively.
2 – Enzymatic studies:
A – Polyphenoloxidase
from the valid assay study, it was found that 0.5 ml was the appropriate volume for the enzymatic assay in a standard assay mixture containing 0.2 M dsodium phosphate buffer containing 1.5 N NaCl at pH 7.0 for
The highest extractability of the enzyme was obtained by using 0.2M sodium phosphate buffer (pH7) containing 1.5M of sodium chlorid with incubation for 6 hours at 4ºC.
The optimum pH of the enzyme = 7.
The Km = 0.029 M and Vmax = 9.1 Units / mg protein The enzyme was most stable at pH range of 5.5 – 7.5.
The enzyme gave 96% of its maximum activity at 20 ºC, and more than 50% at 30- 50 ºC and completely inactivated at 85ºC.
During ripening the enzyme activity increased from 3.69 to 10.0 unit/ mg protein.
Freezing storage at -20ºC steadily increased the enzyme activity in the first 4 months from 10.9 to 24.5 unit / mg protein then the enzyme activity declined to 9.6 unit/mg protein at the end of
storage 6th month.
B – Pectinesterase:
The valid assay study revealed that 10 ml (of extract) was the suitable concentration.
The highest extractability of the enzyme was obtained by using 2M sodium chlorid (pH = 8) with incubation for 6 hours at 4ºC.
The optimum pH of the enzyme = 7.5.
The optimum temperature of the enzyme =65 ºC.
Km = 0.013mg/ ml and Vmax = 42.7 E U / mg protein The residual activity of the enzyme was maximum at pH= 8 and it decreased at acidic and basic values. Also, it gave 80% of its maximum activity at pH range of 4 – 10.
Regarding thermal stability, the enzyme showed high stability at 0-50ºC , then its activity was decreased at more than 50ºC till complete inactivation at 75 ºC.
During ripening enzyme activity was increased from 2.8 to 4.8 units/mg protein for mature-green and rip tissue, respectively.
C – Purification of pectinesterase:
The enzyme was purified and it gave a single band on SDS-PAGE with a molecular weight = 33000 Da.
3 –The chemical composition of papaya jam:
During storage the jam total solids, TSS, pH, color increased. Slightly. However, the acidity and the total carotenoids were slightly decreased.
Sensory evaluation:
It shows that high the prepared jam was acceptable regarding color, taste and flavor. The acceptability was maintained throughout the six months storage period.