Module 2: CARBOHYDRATE EXTRACTION AND QUANTIFICATION BY DNS

Reducing sugars are carbohydrates that can act as reducing agents due to the presence of free
aldehyde groups or free ketone groups. All monosaccharides are reducing sugars, along with some
disaccharides, oligosaccharide.
Principle: When 3,5-dinitrosalicylic acid (DNS) reacts with reducing sugars in an alkaline medium,
the DNS (yellow) is reduced to 3-amino-5-nitrosalicylic acid (orange to redbrown) which can be
quantified by spectrophotometry at 540 nm.

Table 1: The calibration curve of DNS method

Glucose
Test Vol of glucose Vol of deionized Corrected OD540nm
concentration ( μ OD540nm
tube 500 μg/mL (mL) water (mL) (OD540nm)
g/mL)
Blank 0 5
1 1 4
2 2 3
3 3 2
4 4 1
5 5 0
OD = ODstandard/sample – OD0

Table 2: Determination of the reducing sugar concentration in the sugar extracts

Reducing
Actual
sugar
concentration
concentration
Sample OD540nm OD540nm OD540nm OD540nm Dillution of reducing
OD540nm calculated
s (1) (2) (3) (Average) factor sugars in the
based on the
extract ( μ
standard curve
g/mL)
( μg/mL)
Sugar
extract
Blank
C=axn

Table 3: Determination of the reducing sugar amount in 100 grams of samples

Volume Actual concentration The amount of The amount of The amount of
Sampl (V) of the of reducing sugars in reducing sugars in sample used for reducing sugars
e name sugar the sugar extract ( μ V mL of the sugar making V mL of the in 100 grams of
extract g/mL) extract ( μg) sugar extract (g) samples

m=VxC
m ×100
The amount of reducing sugars in 100 grams of samples (g) = 6
msample ×10
m: The amount of reducing sugars in V mL of the sugar extract ( μg)
V: Volume of the sugar extract (mL)
C: Actual concentration of reducing sugars in the extract ( μg/mL)
msample: The amount of sample used for making V mL of the sugar extract (g)

Module 3: BROMELAIN EXTRACTION AND CHARACTERIZATION

A proteolytic enzyme called Bromelain, which breaks down protein, is found naturally in the stems
of pineapples.
 Bradford Protein Assay:
Principle: The assay is based on the observation that the absorbance maximum for an acidic
solution of Coomassie Brilliant Blue shifts from 465 nm to 595 nm when binding to protein occurs.
This method actually measures the presence of the basic amino acid residues, arginine, lysine and
histidine, which contributes to formation of the protein-dye complex. The amount of the complex
present in solution is a measure for the protein concentration, and can be eatimated by use of an
absorbance reading.
The Bradford protein assay can measure protein quantities as little as 1 to 20 μg.

Table 1: Bradford protein assay standard curve

Test Vol of 0.1 mg/mL Vol of deionized Protein concentration Corrected OD595nm
OD595nm
tubes BSA (mL) water (mL) ( μg/mL ¿ (OD595nm)
Blank
1
2
3
4
5

Table 2: Determination of protein concentration in samples (I-II) using the standard curve
Protein concentration
Actual protein
OD595 OD595n OD595 OD595nm OD595 calculated based on Dilution
Samples concentration
nm (1) m (2) nm (3) (Average) nm the standard curve ( factor
( μg/mL ¿
μg/mL ¿
Solution
(I)
Solution
(II)

Bromelain Assay:
Principle: In this assay, casein acts as a substrate. When the protease we are testing (bromelain)
digests substrate (such as casein, hemoglobin), the amino acid tyrosine is liberated along with other
amino acids and peptide fragments. The Folin-Ciocalteus reagent, or Folin’s phenol reagent
primarily reacts with free tyrosine to produce a blue colored compound, which is quantifiable and
measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from
casein, the more the blue colored compounds are generated and the stronger the activity of the
protease.

Table 3: Determination of bromelain activity in samples (I-II)

Solution (I) Solution (II)
Samples
Blank Tyrosine Sample Blank Tyrosine Sample

OD620nm

OD620nm (Average)
OD620nm
Dilution factor
Amount of
tyrosine ( μg ¿ in
0.5 mL solution
used in
"tyrosine" batch
Amount of
tyrosine ( μg ¿
produced in
“sample” batch
Bromelain
activity (UI/mL)
Tyrosine: 45 mg Tyrosine in 100 mL HCl 0.2N
? mg Tyrosine in 0.5 mL HCl 0.2N
3
45 x V x 10
mT = ( μg)
100
mT x ΔO D S 45 x V x 10 3 x ΔO DS
mS = =
Δ OD T 100 x Δ OD T
3
mS 45 x V x 10 x ΔO DS
Bromelain activity = =
C N x t 100 x Δ ODT x C N x t

Table 4: Bromelain purification table

Total Total Specific Protein Bromelain
Volume Purificatio
Step protein activity activity recovery activity
(mL) n fold
(mg) (UI) (UI/mg) (%) recovery (%)
Homogenate /
(crude extract) (I)
Solvent
precipitation (II)
Total protein (mg) = C x Volume
Total activity (UI) = Bromelain activity x Volume
Total activity
Specific activity (UI/mg) =
Total protein
Purification fold(I) = 1; Protein recovery(I) = 100%; Bromelain activity(I) = 100%
Purification fold (II) ×1
Purification fold(II) (%) =
Purification fold(I )
Protein recovery (II) ×100
Protein recovery(II) (%) =
Protein recovery (I )
Bromelain activity recovery (II) ×100
Bromelain activity recovery(II) (%) =
Bromelain activity recovery( I)
SDSPAGE is a technique used to separate proteins according to their molecular size through the
gel.
Proteins are unfolded and migrate from cathode to anode terminal at different rates.
Stacking gel (2-4% acrylamide) allows to concentrate all the proteins in one band, so that they will
start migrating in running gel at the same time.
Separating gel (7 –15% acrylamide) or running gel allows to separate the proteins based on their
molecular weight.

Module 4: VITAMIN C EXTRACTION AND QUANTIFICATION BY IODINE

Principle: The vitamin C concentration in a solution is determined by a redox titrtion using iodine.
As the iodine is added during the titrtion, the vitamin C (more properly called ascordic acid) is
oxidized to dehydroascorbic acid, while the iodine is reduced to iodine ions.
Ascorbic acid + I3- -> 3I- + dehydroascorbic acid
2 requirements when quantifying vitamin C by iodine: Iodine must have a very precise
concentration and that concentration must be unchanged (Iodine concentration tends to decrease
(due to sublimation) => must determine the calibration ratio to bring the Iodine concentration to the
correct concentration be available at the beginning to avoid incorrect value during Iode
sublimation).
The endpoint of the titration: Once all the ascorbic acid has been oxidized, the excess iodine is free
to react the starch indicator, forming the blue-black starch-iodine complex.

Table 1: Determination of the calibration ratio

1st trial 2nd trial 3rd trial
Vol of 0.01N Na2S2O3 (mL)
Vol of iodine titrant (mL)
Average vol of iodine titrant (mL)
x ratio
V
x=
V
Table 2: Determination of the Vitamin C content in the sample
1st trial 2nd trial 3rd trial
Vol of the filtrate of sample extract (mL)
Vol of Iodine titrant (mL)
Average vol of Iodine titrant (mL)
Dilution factor
nI2 needed for titrating v mL the filtrate (mol)
nascorbic acid reacting (equivalent to nascorbic acid in v mL of the filtrate)
(mol)
mascorbic acid reacting ( equivalent to mascorbic acid in v mL of the
filtrate) (g)
mascorbic acid in V mL of the filtrate (eqivalent to mascorbic acid in P or m
(g) sample) (g)
Vit C content in the sample (g per 100 g)
CM = CN / δ = CN / 2
nI2 = CM x a x x x 10-3
Ascorbic acid + I2  2I- + dehydroascorbic acid
 nI2 = nascorbic acid = CM x a x x x 10-3
mascorbic acid = Mascorbic acid x nascorbic acid = 176 x CM x a x x x 10-3
−3
mascorbic acid x V 176 ×C M ×a × x ×10 x n x V
m’ascorbic acid = =
v v
m' x 100 176 ×C M ×a × x ×10−3 x n x V ×100
mVit C/100g = ascorbic acid =
m v×m
m xm
mVit C/sample = Vit C/ 100 g
100