Laboratory Exercise: Enzyme Inactivation via Blanching

Objective

1. To practice testing for blanching adequacy.

Equipment

• Visible spectrophotometer and cuvettes
• Pipettes, 1 and 2 mL
• Test tubes
• Parafilm
• Small blender
• Whatman No. 1 filter paper
• Knife
• Ice bucket
• Erlenmeyer flask, 125 mL
• Beaker, 600 mL
• Graduated cylinders, 50 mL
• Forceps
• Hot plate
• Water baths at 30, 60, and 80°C

Reagents and Materials

• Small potatoes (held in refrigerator overnight)
• Ice (in the ice bucket)
• Buffer A: sodium phosphate buffer, 0.1 M, pH 6.8, containing 0.1 M NaF
• Buffer B: sodium phosphate buffer, 0.1 M, pH 6.8
• Dopa: 4 mg/mL in buffer B
• Guaiacol solution (1% v/v in 95% ethanol)
• Hydrogen peroxide (0.5% v/v)

Procedure

Blanching of the Raw Potatoes

1. Blanch a potato at the assigned temperature (in the proper water bath or in the beaker with boiling water) for the following times 0, 2.5, 5, 10, 20, 30 minutes
2. Immediately put the potato on ice to cool it down

Preparation of Crude Enzyme Extract

1. Peel each blanched potato and cut into small pieces.
2. Rapidly weigh about 10 g potato and mix with 50 mL ice cold buffer A.
3. Grind the mixture in a blender for about 1 min.
4. Filter the mixture with Whatman No. 1 paper into an iced 125 mL Erlenmeyer flask and hold on ice until needed.

Enzyme Assay

For Polyphenol Oxidase

1. Transfer 2.0 mL of buffer B and 0.5 mL of dopa solution to cuvettes.
2. Set the wavelength on the spectrophotometer to 475 nm and zero the instrument against distilled water. Re-zero after each assay.
3. When everything is set, initiate the reaction by adding 0.5 mL of the enzyme extract to the prepared cuvette. Invert to mix and begin recording absorbance readings immediately (to do this, one person should make and record the readings while the other watches the clock and indicates when readings should be taken).
4. Take readings at 15 s intervals for 2 min for each tube. Record all readings in the table below for each blanching temperature.

For Peroxidase

1. Transfer 0.6 mL of buffer B and 0.6 mL of the enzyme extract to a test tube.
2. Incubate for 5 minutes at 30°C.
3. Transfer the content of the test tube to a cuvette.
4. Set the wavelength on the spectrophotometer to 410 nm and zero the instrument against distilled water. Re-zero after each assay.
5. When everything is set, initiate the reaction by adding 1.2 mL of the hydrogen peroxide solution and 0.6 mL of the guaiacol solution to the prepared cuvet. Invert to mix and begin recording absorbance readings immediately.
6. Take readings at 15 s intervals for 2 min for each tube. Record all readings in the table below for each blanching temperature.