Reference:
(D. R. Mertens, 1992, USDA Dairy Forage Research Center, Personal
communication)
Scope:
This method is applicable for the determination of alpha-amylase
activity of solutions to be used in neutral detergent fiber
procedure (5.1).
Basic Principle:
To insure that the amylase activity is sufficient to remove the
majority of starch in samples and reduce filtering difficulties,
it is critical to determine the amount of any specific enzyme
source that is needed for the aNDF method. Activity of
alpha-amylase is determined under conditions existing during aNDF
procedure.
Equipment:
Refluxing apparatus
Berzelius beakers (600 mL)
Analytical electronic balance, accurate to 0.1 mg
Ice bath
Reagents:
Dried hominy corn (sold in most grocery stores as corn grits,
raw, not instant) that has been ground to pass a 1-mm screen
(Wiley mill) Burke's iodine solution (2 g KI, 1 g I2 and 100 mL
H2O) or iodine solution from Sigma (Cat. No. 700-2) Amylase test
solution or extraction The ratio of powder or stock solution to
water can vary greatly depending on the enzyme activity. After
some experience with an amylase source is gained, it may be
possible to select a dilution or extraction rate that is most
appropriate for the specific amylase source. If a liquid enzyme
source is used, dilute a small volume to 100 mL final volume with
distilled water (e.g. approximately 1.25 mL Sigma Cat. No. A5426
or 5 mL of NOVO Termamyl diluted to 100 mL). Powdered enzymes
must be extracted with water. It is recommended to start with 5 g
of powder extracted for 20 min with 100 mL of distilled water.
Concentration of amylase test solution (C) is expressed as g
(powder) or mL (liquid) of enzyme/100 mL test solution.
Safety Precautions:
- Sodium lauryl sulfate is irritating to mucous membranes.
Wear dust mask and gloves while handling.
Procedure:
A. Determine appropriate amount of amylase needed.
Use a range of enzyme concentrations to detect differences in
the activity of an enzyme and determine the amount of the enzyme
that is needed for aNDF. It is suggested that six doses of most
enzyme dilutions is all that need to be evaluated (0, 1.0, 1.5,
2.0, 2.5, & 3.0 mL). However, if an unknown source is used,
it may be wise to use a geometric progression of doses (0, 0.5,
1.0, 2.0, 4.0 & 8.0 mL) the first time to establish the range
to be used in a second trial that will determine the final amount
of an unknown amylase to use.
- Weigh 0.5 g (± 0.005 g) of ground, dried hominy corn
into each of six Berzelius beakers.
- Preheat extraction heating (reflux) unit to a temperature
that permits boiling of neutral detergent solution within
5 min. Prepare an icebath for cooling the beakers after
they have been removed from the hotplates.
- Add 50 mL of neutral detergent solution and swirl beaker.
Do not add 0.5 g of sodium sulfite as in method 5.1.
- Put beakers onto preheated refluxing apparatus at 1
minute intervals. Samples should come to a boil in 4 to 5
min. 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.
- After the samples begin to boil (approximately 5 min
after placing on hot plates), add one of the doses of
enzyme solution to each of the respective beakers.
- Reflux for 10 min. One hr refluxing is not needed to
evaluate amylases.
- At 1 minute intervals remove each beaker from the
refluxing apparatus. Add the second dose of amylase,
swirl to mix and rinse down the sides of the beaker using
a minimum amount of room temperature neutral detergent
solution. Let the amylase react with the beaker on the
benchtop for 60 sec (until after the next sample is
removed from the hotplate and enzyme is added to it).
- Place the beaker in the ice bath to cool for 5 min.
- Remove each beaker and place it on the benchtop until all
beakers have been cooled.
- Arrange beakers in order of increasing enzyme
concentration, preferably on a white sheet of paper or
other light background color surface.
- Quickly add 0.5 mL of Burke's iodine solution to each
beaker, then swirl each beaker in turn. Set timer to
alarm in 90 sec.
- After 90 sec evaluate the efficacy of raw corn starch
(grits) hydrolysis using the following scale:
- Purple = not adequate enzyme
- Amber = not adequate enzyme
- Yellow = adequate enzyme.
The volume of the amylase test solution or extraction
(Vs) that indicates no starch (yellow color) is used to
calculate the standardized amylase solution.
It is best not to look at the beakers while waiting for
the 90 sec to elapse, instead look away from the beakers
and after the alarm sounds make a quick decision (before
120 sec has elapsed) about the lowest amount of enzyme
that gives a yellow starch reaction with iodine. Caution:
Excess enzyme is not beneficial and can be detrimental.
Too much amylase is expensive, can cause retrograde
starch synthesis and may increase the amount of
contaminating enzymes in the amylase preparation that can
cause problems.
- Calculate the amount of enzyme needed in 2 mL of
standardized amylase solution to remove interfering
starch.
B. Verify the absence of undesirable fiber-degrading
enzyme activity in any unknown amylase source
Use Novo Termamyl or the AOAC Dietary Fiber method amylase
(Sigma Cat. No. A5426) which have minimal fiber-degrading
activity under the conditions of aNDF analysis. However, if other
sources are used, use beta-glucan (barley - Sigma No. G-6513),
arabinogalactan (larchwood - Sigma No. A-2012 or Dietary Fiber
control - Sigma No. A9788) and pectin (citrus- ICN No. 102587 or
Sigma No. P9135) to determine unacceptable fiber-degrading
activities.
- Weigh each of the following into separate Berzelius
beakers in duplicate: 0.1 g of beta-glucan, 0.5 g
arabinogalactan, or 0.5 g pectin.
- Follow the usual aNDF procedure using the standardized
amylase solution determined previously, EXCEPT:
Add the amylase to one of the duplicate beakers, but not
the other. To aid filtering of these difficult materials,
add 0.25 g of glass wool or glass microfiber filters
(Whatman GF/D, 4.25 cm) to the crucibles prior to initial
drying of the crucible (W1) before obtaining the tared
weight. Make sure wash water is boiling.
- After calculating the percentage aNDF for each compound,
divide the %aNDF with amylase by the %aNDF without
amylase. If this ratio is less than 0.9 for any of the
compounds, reject the source of enzyme and choose another
source to use because the unknown amylase source has
significant fiber degrading activity.
Comments:
- Each new source or lot of enzyme should be standardized.
- If a single lot is being used over a period of time it
should be checked every month for activity.
Calculation: Amount of enzyme preparation needed for
the standardized amylase solution.
Rather than vary the amount of enzyme solution to use with
each source or lot, it is desirable to calculate concentration of
a specific enzyme that will be added in the aNDF method as 2 mL
of solution. Then an automatic pipet or syringe, precalibrated to
2 mL, can be used to dispense the enzyme regardless of its
initial activity. Two mL of dilute enzyme is used to minimize the
error associated with a drop of solution that may be retained on
the syringe or pipet, because one drop of a concentrated enzyme
solution contains significant activity. The calculation that
follows is for dilution of an enzyme stock solution or extraction
to allow 2 mL of standardized amylase solution to be added during
the aNDF procedure.
E = 100 mL X (Vs X C) / 2 mL
- E = Enzymepowder (g) or liquid (mL) needed to make 100 mL
of standardized amylase solution
- Vs = Minimum volume of test solution or extract resulting
in yellow color with iodine (no starch)
- C =Concentration of solution (g powder/100 mL or mL
liquid/100 mL)
- 2 mL = Volume to be added in aNDF procedure
- 100 mL = Volume of standardized amylase solution to be
prepared
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