Zein is a prolamine derived from corn [Zea mays Linné (Fam. Gramineae)].
Sample solution: Dissolve 0.1 g in 10 mL of 0.1 N sodium hydroxide.
Analysis: To the Sample solution add a few drops of cupric sulfate TS. Warm in a water bath.
Acceptance criteria: A purple color develops.
Sample solution: In a test tube add 1 mL of nitric acid to 25 mg of Zein.
Analysis: Agitate the Sample solution vigorously.
Acceptance criteria: The solution becomes light yellow. Further addition of about 10 mL of 6 N ammonium hydroxide produces an orange color.
Delete the following:• C. It is insoluble in water, but 100 mg/mL in a mixture of isopropyl alcohol and water (17:3), at a temperature of 37, yields a clear to cloudy solution.NF30
Add the following:• C. SDS-Polyacrylamide Gel Electrophoresis
Solvent: 55% isopropyl alcohol with 2% beta mercaptoethanol
Sample loading buffer:1 0.5 M Tris hydrochloride pH 6.8, 20% glycerin, 4% sodium dodecyl sulfate (SDS), and 0.005% bromophenol blue
Gel running buffer stock solution:2 0.25 M Tris base pH 8.6, 1.92 M glycine, and 1.0% SDS
Gel running buffer: Gel running buffer stock solution and water (1:9)
Gel staining solution: A suitable Coomassie blue-based solution3
Molecular weight marker: Use a suitable molecular weight marker4 containing protein bands at 10190 kDa. [NoteA molecular weight marker with protein bands at 10100 KDa can also be used. ]
Molecular weight standard solution: Dilute the Molecular weight marker (1:1) with the Sample loading buffer. Incubate the mixture in a closed microcentrifuge tube for 10 min at 95. After incubation, allow the tube to flash-cool on ice. Place the tube in a microcentrifuge, spin at a top speed for a few seconds, and allow to stop on its own to collect any condensation on the sides and top of the tube.
Sample stock solution: 10 mg/mL of Zein in Solvent. Mix on a vortex mixer until the sample is fully dissolved. Centrifuge at 10,00012,000 rpm in a microcentrifuge with a fixed rotor for 10 min to pellet any undissolved material.
Sample solution: Dilute the Sample stock solution (1:1) with the Sample loading buffer. Incubate the mixture in a closed microcentrifuge tube for 10 min at 95. After incubation, allow the tube to flash-cool on ice. Place the tube in a microcentrifuge, spin at a top speed for a few seconds, and allow to stop on its own to collect any condensation on the sides and top of the tube.
(See Electrophoresis 726.)
SDS-PAGE gel and apparatus setup: Following the manufacturer's instructions, assemble and fill a precast 16% Tris-Glycine Gel5 in an appropriate electrophoresis module.
Running buffer: Gel running buffer
Voltage: 100 V
Run time: 2.5 h or until the upper dye front is at the bottom of the gel. [NoteThe total run time may need to be altered, depending on the molecular weight standards as well on as laboratory equipment variability, because the dye front may co-migrate with or close to the lowest bands of the set. ]
Samples: Molecular weight standard solution and Sample solution
Gel loading: Load 25 µL of the Molecular weight standard solution. Load a volume of the Sample solution, equal to approximately 25 µg of calculated Zein. [NoteThe actual amount of Zein extracted from the starting material cannot be quantified. Therefore, an estimated amount is derived. ]
Gel staining: After electrophoresis, carefully remove the gel from the plates. Rinse the gel three times with water. Stain the gel by following the manufacturers directions for the stain used.
Destaining: After staining as directed by the manufacturer, destain as directed by the manufacturer.
Gel scan procedure: Set up a gel scanner according to the manufacturers instructions. Place the gel in the detector, and obtain a single image of all loaded lines of the gel.
Acceptance criteria: Zein has two major bands: the band is at 2125 kDa, and the band is at 1718 kDa.NF30
• Residue on Ignition 281: NMT 2.0%, using an ignition temperature of 800 ± 25
• Heavy Metals, Method II 231: NMT 20 ppm
Add the following:Organic Impurities
• Limit of Hexane-Soluble MatterNF30
Sample: 15 g of Zein
Solvent: Alcohol and water (17:3, w/w)
Analysis: Dissolve the Sample in 150 mL of Solvent. Stir the mixture, using a magnetic stirrer, and heat the solution to 30. Once the Sample is dissolved, transfer the solution to a 500-mL separatory funnel.
Add 60 mL of n-hexane. Shake the mixture, and allow the phases to separate. Discharge the bottom layer (alcohol) to a beaker, and transfer the top layer (hexane) to a first 500-mL flask. Weigh the first 500-mL flask, and record the weight. Pour the bottom layer of alcohol back into the separatory funnel. Repeat this step four more times.
After the five 60-mL hexane solutions have been added to the first 500-mL flask, attach it to a rotary evaporator to distill the hexane. Collect the hexane in a second 500-mL flask.
The first 500-mL flask contains a yellow to reddish oil. Record the weight of the flask containing this oil.
Calculate the percentage of hexane-soluble matter in the portion of Zein taken:
Result = [(WT WF)/W] × 100
For Zein from normal dent corn: NMT 12.5% for hexane-soluble matter
For Zein from waxy corn: NMT 16.0% for hexane-soluble matter
• Loss on Drying 731: Dry a sample at 105 for 2 h: it loses NMT 8.0% of its weight.
• Microbial Enumeration Tests 61 and Tests for Specified Microorganisms 62: The total bacterial count does not exceed 103 cfu/g, and the tests for Salmonella species and Escherichia coli are negative.
Change to read:• Protein Content
Analysis: Proceed as directed in Nitrogen Determination, Method I 461. Calculate the weight percentage of the protein content in Zein by multiplying the percentage of nitrogen found by 6.25.
Acceptance criteria: 81.9%100.0%NF30 on the dried basis
• Packaging and Storage: Preserve in well-closed containers, and store at room temperature.
Add the following:• Labeling: Label it to indicate the corn source from which it is derived.NF30
1 Available from Invitrogen as Tris-Glycine SDS Sample Buffer (2X), catalog number LC2676.
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USP35NF30 Page 2019Pharmacopeial Forum: Volume No. 36(6) Page 1641