Novel melon plants
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example 1
Preparation of Melon Extracts
[0193] A wedge of ca. 400 g was taken from a melon fruit, the seeds and the skin (1 cm thickness) were removed. The flesh was cut in small parts, which were blended for 30 seconds in a Warring blender until a smooth slurry was obtained. The slurry was filtrated over a Whatman paper filter, the juice centrifugated in an Eppendorf centrifuge at 10.000 g and stored at −20 degrees Celcius.
example 2
Determination of Citric Acid Content
[0194] Samples as prepared in Example 1 were incubated with citrate lyase (CL) to convert citric acid to oxaloactate and acetate. In the presence of the enzymes malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), oxaloacetate and its decarboxylated derivative pyruvate were reduced with NADH to respectively L-malate and L-lactate. The decrease of NADH is proportional with the quantity of citric acid in the sample and can be determined at 340 nm. The assay was conducted in microplates. 20 μl of diluted sample was added to a microtiterplate. 200 μl of assay mixture containing NADH, MDH and LDH was added and mixed to the plate. The reaction was started with 15 μl of start-solution containing CL. The plate was mixed and let the reaction proceed for 1 hour. Absorbance values were measured at 340 nm with a microtiterplate reader (Biotek EL808 reader with KCJunior software and computer). A calibration curve was used to calculate the concentratio...
example 3
Determination of Malic Acid Content
[0195] Samples as prepared in Example 1 were incubated with L-malate dehydrogenase (MDH) and NAD+ to convert L-malate to oxaloacetate. The equilibrium of the reaction is on the side of malate but was forced to the side of oxaloacetate with hydrazine by means of derivatization. NADH formed during the assay is proportional with the malic acid content in the samples. NADH can be determined at 340 nm as a measure for malic acid. The assay was conducted in microplates. 20 μl of diluted sample was added to a microtiterplate. The reaction was started by adding 200 μl of assay mixture containing hydrazine, MDH and NAD+ at pH 10. The plate was mixed and the reaction was allowed to proceed for 1 hour. Absorbance values were measured at 340 nm with a microtiterplate reader (Biotek EL808 reader with KCJunior software and computer). A calibration curve was used to calculate the concentration malic acid in the samples. MDH was purchased from Roche Diagnostics....
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