3.2 in 100mL of 0.01M CaCl2. The clotting assay

3.2       Methods

            3.2.1    Extraction of Proteolytic Enzyme

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This method is obtained from the research done by Al-hadban & Al-zubaidy (2016) and modified where 25g of pineapple core is crush with 50mL of
distilled water for 1 minute. Amount of pineapple fruit to volumes (mL) at
ratio 1: 0.5 of  0.1M sodium phosphate
buffer (pH 7) will be added into the crushed pineapple for 2 minutes and make
into a paste. The extract is separated by using filter paper and centrifuged at
6000rpm for 10 minutes. The supernatant will be collected and ready to use.   Papain

This method is obtained from Adachukwu et al., (2013) where leaves of Carica papaya is collected from farm at
Kuala Pilah. The sample need to wash immediately with clean water to remove
dirts, sand particles and other solid contaminants. The leaves will be dried
down under the sun for one week, pulverise into powder and stored in glass
bottle that has been autoclaved. The pulverised leaf will be measured at 10g
and adding 20mL of ethanol and distilled water into a beaker. After stirring,
the samples will be leave for 48 hours before filtering using Whattman filter
paper. The residues is thrown away and let the samples evaporate. The sample
will be stored in clean glass bottles until usage.


3.2.2    Milk Clotting
Activity (MCA)

This activity is obtained from Bruno et al. (2010) and Mazorra-Manzano et al.  (2013) are modified according to International Dairy Federation (IDF).
For the preparation of substrate, 12.5g of skim milk powder will be dissolve in
100mL of 0.01M CaCl2. The clotting assay will carried out by mixing
1mL of substrate with 0.1mL of pure enzyme that pre-equilibrate at
appropriate temperature depending on the activity of each enzyme where 40oC
for papain and 50oC for bromelain then adjust the pH at 6.5 and incubate
for 10 minutes. The assay will be performed at least in triplicate. Furthermore,
the activity will be repeat using different concentration of CaCl2 solution
at 0.03M, 0.05M, 0.07M and 0.1M and different dilution of enzyme at 1:2, 1:4,
1:12, 1:16, 1:20 to correlate with proteolytic activity. at The positive
control is replacing plant enzyme with rennet and the negative control is by
not adding any enzyme to the sample. Analysis of MCA

The MCA will be defined in terms of Soxhlet units (SU) , a standard
from International Dairy Federation (IDF,1992) 
as the quantity of protein needed to coagulate 1mL of milk in 40 minutes
(2400s) at the temperature evaluated: (SU) = 2400/t x S/E where t
is the clotting time (seconds), S is the volume of milk (mL) and E
is the volume of        extract (mL).


            3.2.3    Proteolytic Activity (PA) Preparation of
Calibration ( Tyrosine Standard ).

This method is the protocol obtained from Sigma-Aldrich (2017) and
use casein as a substrate. Tyrosine standard dilutions using six test tubes
including blank. To the tubes, 1.1 mM tyrosine standard stock solutions will be
add at following volumes in mL: 0.05, 0.10, 0.20, 0.40, and 0.50 except blank.
Once the tyrosine standard solution has been added, add an appropriate volume
of distilled water to each of the six standards to bring the volume to 2mL.

To all of the tubes containing the standards and standard blank, 5mL
of 0.5M sodium carbonate will be add. 1 ml of Folin’s reagent will be add
immediately afterwards and measure its absorbance at 660nm. Analysis of Calibration of Tyrosine Standard

Generate standard curve using a graphing program by plotting the
change in absorbance of standards on the Y axis, versus the amount in
micromoles for each of 6 standards of tyrosine on the X axis and generate best
fit line.

               Preparation of Samples for Proteolytic

Prepare 7 test tubes consist of 1 blank sample and 6 enzyme sample.
On each 7 tubes, 5mL of  0.65% casein solution
will be added and let them equilibrate in a water bath at 37°C for about 5
minutes. Then, add 0.5mL of enzyme solution at varying  dilution (pure enzyme, 1:2, 1:4 , 1:12, 1:16,
1:20) except the blank. Mix them by swirling and incubate at 40°C for papain
and 50°C for bromelain at exactly 10 minutes.

After incubate, 5 ml of the trichloroacetic acid (TCA) reagent to
each 5 tubes to stop the reaction. 0.5mL enzyme solution until it reach 1 mL of
enzyme solution and add 1mL of enzyme solution 
into the blank then incubate the solutions at 40°C for papain and 50°C
for bromelain at 30 minutes.

After the incubation, the test solutions will be filter using 0.45?m
polyethersulfone syringe filter and a syringe into new test tubes.

5mL of 0.5M sodium carbonate, Na2CO3 and 1mL
of Folin’s reagent add to test samples and test blank. The tubes are then mixed
by swirling and incubate at 40°C for papain and 50°C for bromelain exactly 30
minutes then absorbance is measure at 660nm.

               Analysis of Samples for Proteolytic Enzyme

Using the tyrosine standard curve calibration, the concentration of
tyrosine equivalent released will be determine from the measured absorbance
value. The enzyme activity will be calculate by the formula where a is total
volume of assay, b is time of assay and c is volume used in Calometric Determination:

Units/ml Enzyme =   

(umole tyrosine equivalents released)
x (a)

(volume of enzyme used) x (b) x (c)



3.2.4    Milk clotting Index (MCI)

                        This test obtained from Mazorra-Manzano et al.  (2013) where                                milk
clotting index can be obtained by calculate the ratio between                                  milk clotting
activity (MCA) and proteolytic activity (PA) based                             on correlation on the dilution of enzyme
where MCI = MCA/PA.