Chapter 3: Materials and Methods
3.10 Growth Parameters
Four plants were taken from each treatment randomly as a replicate sample, the samples were used for biometric and biochemical measurements, which were plant height, number of leaves which was measured once every 10 days while other indices were measured at harvest (100 days). the minerals contained in fruits and all plant parts, the fresh weight (FW) for plants and fruits, also the contents of total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid, TSS, fruit size, sugars content (total sugars, reduced sugars), and firmness which used a penetrometer, all determined were used fresh samples, minerals in fruit and plants have been analyzed using dry samples.
The dry weight (DW) was determined using the oven-dried fruit samples (70°C for 3 days).
3.10.1 Determination of Chlorophyll and Carotenoid
The steps of chlorophyll determination were obtained from Lichtenthaler and Wellburn (1983). At first, half a gram of green plant tissue was weighed and grounded using mortar, 80% acetone was added to the crushed green tissue of the plant, as the acetone works to push all the cell contents out, including chlorophyll, this process was repeated until the plant tissue lost the green color and became white. The filtrate was collected and diluted with distilled water to reach a total volume of 50 mL. The absorbance of the extraction at 440 nm, 645 nm, and 663 nm was respectively measured by a model Helios Alpha 100-240 v spectrophotometer. The chlorophyll and carotenoid content were determined using the following equations:
Chl a (mg/g) = (12.7×OD663−2.59×OD645)V/W Chl b (mg/g) = (22.9×OD645−4.67×OD663)V/W
Total Chl (mg/g) = (20.2× (OD645) + 8.02(OD663)) ×Chl.a−104×Chl.b)V/W Car (mg/g) = ((OD440−3.27×Chl.a−104×Chl.b)/229)V/W
Where;
OD: is the optical density at a certain wavelength (645 nm or 663 nm or 440 nm).
V: is the total volume of acetone extract (50 mL).
W: is the fresh weight (500 g) of the sample.
3.10.2 Determination of Total Soluble Solids (TSS)
Fruit contains elements and nutrients such as sugars, acids, vitamins, pigments, proteins, minerals, phenols, and fructans. This large group of substances is called soluble solid. Total soluble solids (TSS) are one of the most important quality criteria that determine the sweetness of the product. Total soluble solids (TSS) are measured using the Brix scale and reported as degrees Brix which is equivalent to a percentage (%), but the Brix unit does not reflect the amount of net sugar in the samples, because watermelon and watermelon fruit juice does not only contain sugar (Magwaza &
Opara, 2015).
Brix used to determine the measure of maturity, flavor, and accumulation level of sugars and others solid in the fruits, it was determined by the index of refraction and is referred to it as the degrees Brix at 20°C. The TSS determination steps have collected the refractometer, cleaned the refractometer prism surface, placed a small amount of sample fresh juice (watermelon and sweet melon) onto the prism of the refractometer, and then taken the reading. The TSS content was determined using the following equations:
Water Soluble Content (g / 100 mL) % = ((BxV)/S) x100 Where,
B: is the degree of brix detected in the diluted sample.
V: is the volume in which the sample was diluted (ml).
S: is the sample amount (g).
3.10.3 Determination of Reducing Sugar
Reducing sugar consists of glucose and fructose that are obtained from sucrose hydrolysis. The procedure of reduced sugar determination was obtained from (Smri, 1997). There were a lot of tools that was used in this test, which were light-duty balance readable to 0.01 g, pipette: 5 cm3, volumetric flasks: 100, 200 and 1 000 cm3, burette: 50 cm3, Erlenmeyer flask: 500 cm3, hot plate, gauze or screen: 0.15 mm pore opening, stopwatch, glass beads, filter papers and reagents (pumice powder to prevent over-boiling, liquid paraffin, methylene blue solution (1%), ethylene diamine tetra acetic acid, disodium salt dihydrate (4%), copper sulfate pentahydrate, sodium potassium tartrate tetrahydrate (NaKC4H4O6· 4H2O), sodium hydroxide pellets, sodium hydroxide (NaOH, also called caustic, and fehlings solutions).
The procedure included several steps, at first standardization of the Fehlings solutions: 50 ml of standard invert solution was added in a volumetric flask and diluted it with 200 ml distilled water, filled the burette with the dilute standard invert solution, pipetted 5 cm3 Fehlings A solution and 5 cm3 Fehlings B solution into a 500 cm3 Erlenmeyer flask, added a little pumice powder, three glass beads, and four drops of liquid paraffin, this solution titrated against the dilute standard invert solution in the burette.
The sample preparation was poured to the sample of juice through a screen to rid of any solid particles, weighed 50.00 g of the screened sample, and placed in a volumetric flask, 10 cm3 EDTA (4%) was added, rinsed, and filled a 50 cm3 burette with the diluted juice and adjusted the level to zero, 5 cm3 of Fehlings A and 5 cm3 of Fehlings B solutions were placed into the Erlenmeyer flask, then a little pumice powder, 3 glass beads and four drops of liquid paraffin were added, this solution was titrated against the sample solution in the burette.
The final step was titration, where 15 cm3 of the solution from the burette added, placed the flask on a hot plate, and until the mixture boiled, 5 cm3 added each time until the original color of the reagent fades to bright orange.
Couple drops of the methylene blue indicator were added until the color changed from blue to orange, the solution was diluted by water when the amount used of the burette volume was less than 10 cm3, the reading from the burette as an approximate titration was taken, the second titration is done by added all but 1 cm3 of the approximate titer of the sample solution at once, the liquid was heated until the boiling point reached, three or four drops of the methylene blue indicator were added, the titration dropwise completed until the indicator became colorless. The amount of reducing sugars in the sample solution was determined using the following equations:
Reducing sugars (%) = (MRS/1000) x (1/MJ) x 100 Where;
MRS: is the mass reducing sugars (mg) in 100 cm3. MJ: is the mass of juice (g) in 100 cm3.
3.10.4 Determination of Total Sugar
The procedure of total sugars determination included several steps, which were weighed 0.5 out of fruit pulp and placed them in heat-tolerant test tubes, added 20 ml of 80% ethanol, samples were placed on the water bath for 10-15 minutes until the volatilized ethanol, the samples were transported in Persulin mortar, then 15 ml of ethanol was added during the grinding process to ensure that the sample was descended and washed in the mortar, samples were filtered with filter papers, the filtrate was placed in test tubes and raised on the water bath again until the added ethanol was volatilized after the filtration process.
Samples were removed from the water bath, then distilled water was added until the sample volume reached 15 ml, 5 ml of the sample was taken and placed in test tubes, then 1 ml lead acetate + 1.5 ml of NaH2PO4 was added to it, distilled water was added to the sample to reach a volume of 50 ml, after dilution, 2 ml of the sample was taken, then 0.05 phenol + 5 ml of sulfuric acid H2SO4 were added to it, the sample was mixed well and left at room temperature with cooled and then reading at 490 nm on a spectrophotometer.