METHODOLOGY The purple product was immediately read at

620nm using a spectrophotometer. The

A. Preparation of human salivary α-
amylase
Before collection, the mouth of the donor
was rinsed with distilled water for 10 minutes to
avoid any contamination that can comprise the
assay. By a passive unstimulated drool, enough
volume (>1 mL) of saliva was collected in a
beaker. A milliliter of the sample was then
diluted with 99 mL of 100mM phosphate buffer
(pH 6.9) with 1mM NaCl to make a 1:100 enzyme
solution.
B. Preparation of 0.1% starch and Iodine
reagent
A 0.5 g soluble starch was dissolved in a 100
mL distilled water. The solution was heated until
it became homogenous. An aliquot of 4 mL was
then diluted with 16 mL of distilled water to
make a 0.1 % starch solution.
absorbance readings were graph against
concentration to generate the starch standard
curve.
D. Amylase activity assay
For the enzymatic activity assay, 1 mL of
saliva solution was added to 9 mL 0.1 % starch.
Afterwards, an aliquot of 1 mL was transferred
into a tube containing 2 mL phosphate buffer
and 50 µL iodine reagent. This was done
continuously for every 30 seconds starting from
the incubation time. Using the linear equation
generated from the standard curve, the
absorbance readings were converted into starch
concentrations. The average velocity of
enzymatic activity was calculated and was
graphed versus time.

To prepare the iodine solution, 6g of KI was

added to 100mL distilled water followed by the
addition of 2g iodine.

C. Preparation of standard curve

To prepare the standards, seven empty test

tubes were filled with components according to
the proportion found on table 1.

Table 1. Components of Starch Standards

pH 6.9
0.1 %
100mM KI/Iodine
Test starch
Phosphate reagent
tube No. soln.
buffer (µL)
(mL)
(mL)
1 0 3 50
2 0.1 2.9 50
3 0.3 2.7 50
4 0.6 2.4 50
5 0.9 2.1 50
6 1.2 1.8 50
7 1.5 1.5 50