Date Started: 2023/11/17

Date Finished:

Formulation of Microencapsulated Okra (Abelmoschus esculentus (L.) Moench)

Extract into a ReadyTo-Drink Juice: Towards Improved Taste Preference,
Manageable Shelf-Life, and Validated Anti-hyperglycemic Property

I. Objectives
 To prepare a microencapsulated okra mucilage as part of a ready-to-drink
okra juice product that will improve the taste preference, prolonged shelf-
life and preserved anti-hyperglycemic activity.
 To optimize okra extract microencapsulation materials and parameters
based on anti-hyperglycemic property (in-vitro)

II. Chemicals, Materials, Equipment

 Okra  Beakers
 α-amylase  Pipettes
 3,5-dinitrosalicylic acid (DNS)  Reagent bottle (transparent
 0.1% w/v starch solution and amber)
(cornstarch)  Volumetric flasks
 16 mmol sodium acetate buffer  Graduated cylinders
 Distilled water  Stir plate
 Sodium potassium tartrate,  Magnetic stirring bar
tetrahydrate  Micropipettes
 Sodium hydroxide (for pH
adjustment)
 Hydrochloric acid (for pH
adjustment)
 III. Laboratory Safety Measures

Chemical GHS Hazard Statement Storage

 Tightly closed. Dry.
 H302 – Harmful if
Keep locked up or in
swallowed
an area accessible
 H334 – May cause
Health hazard, only to qualified or
α-amylase allergy or asthma
Irritant authorized person
symptoms or
 Recommended
breathing difficulties
storage
if inhaled
temperature: 20 °C
 Tightly closed and
 H302 – Harmful if
away from sources
Corrosive, swallowed
3,5-DNS of ignition and heat.
Irritant  H318 – Causes
Observe national
serious eye damage
regulations
 Store in cool
location
 H302 – Harmful if
Sodium acetate Irritant  Avoid heat sources
swallowed
 Avoid oxidizing
agents
 H226 – Flammable  Store locked up
liquid and vapor  Store in a well-
Flammable,
Glacial acetic acid  H314 – Causes ventilated place.
Corrosive
severe skin burns  Keep container
and eye damage tightly closed
 Keep container tight
Cornstarch N/A  N/A  Store in cool, dry,
and ventilated are
Sodium N/A  N/A  Storage conditions:
potassium tartrate Keep only in the
 original container in
a cool, well-
ventilated place
away from:
incompatible
materials. Keep
container closed
when not in use.
 Incompatible
products: Strong
bases. Strong acids.
 Incompatible
materials: Sources
of ignition. Direct
sunlight.
 H290 – May be
corrosive to metals  Storage conditions:
Sodium hydroxide Corrosive  H314 – Causes No metal containers.
severe skin burns Tightly closed. Dry.
and eye damage
Hydrochloric acid Corrosive  H314 – Causes  Keep only in the
severe skin burns original container in
and eye damage a cool, well
 H318 – Causes ventilated place
serious eye damage away from:
incompatible
materials. Keep
container closed
when not in use.
 Incompatible
products: metals.
 cyanides. Strong
bases.
 Incompatible
materials: Direct
sunlight.
 Packaging
materials: Do not
store in corrodable
metal.

IV. Waste Disposal

Waste Identity/Composition Waste Container Code

α-amylase G704
3,5-DNS G704
Sodium acetate G704
Glacial acetic acid G704
Cornstarch G704
Sodium potassium tartrate G704
Sodium hydroxide D499
Hydrochloric acid D499
*D499 – Inorganic waste

*G704 – Non-halogenated organic waste

*G703 – Halogenated organic waste

*B206 – Sulfuric and Hydrochloric acid waste

*B203 – Nitric acid waste

*C301 – Caustic soda waste

V. Methodology
A. Preparation of Reagents

80.00 mL dist. H2O

 + 88.38 mg sodium acetate

 + 31.38 mg acetic acid
 Adjust using NaOH or HCl
 + dist. H2O until volume is 100 mL

100 mL 16 mmol sodium acetate buffer (pH 5) solution

0.1 g cornstarch

 + 100 mL buffer solution

 Cover beaker
 Heat below boiling for 15 min w
constant mixing
 Cool to RT
 Bring to final volume using dist. H2O

100 mL 0.1% w/v starch solution

27.5 mg α-amylase

 + 100 mL dist. H2O

100 mL enzyme solution

0.8 g NaOH pellets

 + 10 mL dist. H2O
 Heat at 50-70 °C

10 mL 2 M NaOH solution
 12.0 g potassium sodium tartrate

 + 8.0 mL warm 2.0 M NaOH

 Dissolve while maintaining temp and
constant stirring (DO NOT BOIL)

8 mL 5.3 M potassium sodium tartrate solution

438 mg 3,5-DNS

 + 20.00 mL dist. H2O

 Heat at 50-70 °C
 Dissolve while maintaining temp and
constant stirring (DO NOT BOIL)

20 mL 96 mM 3,5-DNS solution

12 mL dist. H2O

 Heat at 60 °C
 + 8 mL potassium sodium tartrate
 + 20 mL 3,5-DNS
 Stir
 Store in amber flask at RT

Coloring reagent solution (stopper)

2 g maltose

 Dissolve in 10 mL dist. H2O

10 mL 0.2% w/v maltose solution

 B. Analysis of in vitro anti-diabetic activity of α-amylase enzyme assay (DNSA
method)
a. The control tube contains an only reagent and the test sample in the
range of 100–500 µg/ml was prepared. From this, 500 µl of sample
was mixed with 500 µl of starch solution and 500 µl of alpha-amylase
solution which is incubated at 37 ° C for 10 min.
b. The reaction was stopped by the addition of 1 ml of 3, 5 Di-nitro
salicylic acids and incubated in boiling water bath for 5 min, cooled at
room temperature.
c. The reaction mixture was then diluted by adding 10 ml of distilled
water.
d. The absorbance was measured at 540 nm.
e. Control was tested by replacing test sample with DMSO. A similar
procedure was also followed for the standard drug Acarbose. The
percentage of inhibition was calculated using the formula

% inhibition=
[ ( O Dcontrol −O Dtest sample )
O Dcontrol ] ×100 (Equation 1)

Alternatively,

1. Determine ∆A540 of standard and blank

Δ A 540 (std)= A 540 (std) – A540 (blk) (Equation 2)

2. Prepare a standard curve by plotting the ΔA 540 of the standards vs. mg of

maltose using linear regression.
3. Determine ∆A540 of standard and blank

Δ A 540 ( sample)=A 540 (sample ) – A 540 (blk) (Equation 3)

4. Determine the mg of cornstarch released using the standard curve

units (m cornstarch released (¿ mg))(df )

= (Equation 4)
mL enzyme V enzyme (¿ mL)
 units
units mL enzyme
= (Equation 5)
mg solid mg solid
mL enzyme
VI. Data and Calculation