AFIA - Laboratory Methods Manual - Australian Fodder Industry ...

4. Preheat extraction heating (reflux) unit to a temperature that permits boiling of neutral
detergent solution within 5 minutes.
5. Prepare an ice bath for cooling the beakers after they have been removed from the hotplates.
6. Add 50ml of neutral detergent solution and swirl beaker.
Note: Do not add 0.5 g of sodium sulphite as in Method 1.8R (NFTA method 5.1).
7. Put beakers onto preheated refluxing apparatus at 1 minute intervals.
8. Samples should come to a boil in 4 to 5 minutes. Timing is critical to the evaluation of the
enzymes because reaction rates are temperature and time dependent. It is also important that
enzymes be used in ascending order of concentration.
9. After the samples begin to boil (approximately 5 minutes after placing on hot plates), add one
of the selected volumes of Amylase Test Solution (from lowest to highest) to each of the
respective beakers.
10. Reflux for 10 minutes. One hour refluxing is not needed to evaluate amylases.
11. At 1 minute intervals remove each beaker from the refluxing apparatus.
12. Add the repeat volume of Amylase Test Solution to the respective beakers, swirl to mix and
rinse down the sides of the beaker using a minimum amount of room temperature neutral
detergent solution.
13. Let the amylase react with the beaker on the benchtop for 60 seconds (until after the next
sample is removed from the hotplate and enzyme is added to it).
14. Place the beaker in the ice bath to cool for 5 minutes.
15. Remove each beaker and place it on the benchtop until all beakers have been cooled.
16. Arrange beakers in order of increasing enzyme concentration, preferably on a white sheet of
paper or other light background colour surface.
17. Quickly add 0.5ml of Burke's iodine solution to each beaker, then swirl each beaker in turn.
18. Set timer to alarm in 90 seconds.
19. After 90 seconds evaluate the efficacy of raw corn starch (grits) hydrolysis using the following
scale:
a) Purple = not adequate enzyme
b) Amber = not adequate enzyme
c) Yellow = adequate enzyme.
20. Calculate the amount of enzyme needed in 2ml of standardised amylase solution to remove
interfering starch.
AFIA Laboratory Methods Manual – v7 September 2011 Page 42 of 103