Practice 1: DETERMINE STARCH CONTENT

1. OBJECTIVE:
- Determination starch content from determine the content of reducing sugar by
Ferricyanide reduction method.

2. MATERIALS AND EQUIPMENT

- Sample: Turnip
- Erlenmeyer flask, volumetric flask, burette, pipette, beaker, stove, timer.

3. CHEMICALS
- 35% concentrated HCl
- 2% HCl (100 mL distilled water + 6ml 35% HCl)
- 10% NaOH solution
- 1% Methyl orange solution
- 1% Potassium ferricyanide solution
- 2.5N KOH solution
- 0.5% Methylene blue

4. PROCEDRURE
4.1. Starch hydrolysis by acid
- Weigh approx 5g of sample and transfer to a 250 mL Erlenmeyer flask or round
bottom flask.
- To remove dissolved sugar, add 100 mL of distilled water, shake well, and let
the sample stand for 10 minutes.
- Filter and continue to wash the residue with distilled water 2-3 times.
- Then, transfer the sample into a round bottom flask with 100 mL of 2% HCl and
connect the flask to a condenser (water -circulating condenser or air condenser
tube)
- Place the flask into a water bath, heat the bath and allow the boiling to occur for
60 minutes. The water level of bath must be always higher than that of the flask,
hot water must be always ready to refill the water bath.
- After hydrolysis, all of the starch is transformed into glucose (which can be
checked by iodine)
- Remove the flask and cool the contents to room temperature.
- Add 3-4 drops of methyl orange and use 10% NaOH to neutralize the acid until
the color changes.
- After the neutralization, filter and transfer the contents into a 100mL volumetric
flask, and carefully rinse the reaction flask with distilled water. Obtained sugar
sample is used to determine the content of reducing sugar by Ferricyanide
reduction method.

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 ATTENTION: The neutralization is conducted ONLY when the solution is cooled to
room temperature because high temperature and high alkaline conditions lead to
complete degradation of glucose and cause an error in the result.
4.2. Determine the content of reducing sugar by Ferricyanide reduction
method
a. Principle
Based on the chemical reaction:
K₃Fe(CN)₆ + 2KOH → K₄Fe(CN)₆ + H₂O + O
Next, sugar is oxidized into gluconic acid:
CH₂OH(CHOH)₄CHO + O → COOH(CHOH)₄COOH
Overall reaction is:
6K₃Fe(CN)₆ + 6KOH + CH₂OH(CHOH)₄CHO = 6K₄Fe(CN)₆ + COOH(CHOH)₄COOH + 4H₂O

b. Procedure
- Pipette 20mL Potassium ferricyanide solution into a 250 mL Erlenmeyer flask,
then add 5 mL KOH and 3-4 drops of methylene blue (if the concentration of
sugar solution < 0.25 then add only 10mL ferricyanide and 2.5 mL KOH)
- Shake and mix well.
- Place he flask on the stove for boiling in 1-2 minutes.
- Titrate the mixture with prepared sugar solution (dilute if needed) until there are
changes in the color of methylene blue: from blue to purple pink and finally to
orange yellow. If the solution is cooled, the purple – pink color will reappear.
- Prepare the standard sugar solution: weigh 0.5g pure glucose or fructose and
make sugar solution 0.5% in a 100mL volumetric flask. Conduct the titration as
mentioned above.

5. CALCULATION
 Content of reducing sugar in diluted solution:
a
RS (g/100ml) = 100
m
With:
a: grams of glucose in 0.5% glucose solution used to completely react to 20mL
ferricyanide solution.
m: mL of sample solution used to completely react to 20 mL ferricyanide
solution

 Content of starch in sample:

Content of starch (g/ml) = RS × 0.9
With RS is the content of reducing sugar in diluted solution
 Result:
1mL of 0.5% sugar solution contain 5mg glucose.

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To reduce 20mL ferricyanide solution, we need 1.6 mL of reducing sugar
solution 0.5% -> Amount of glucose in that solution is:
1.6 × 5 = 8 mg = 0.008g
=> a= 0.008 g
To completely react to 20 ML ferricyanide solution, we needed 11.5 ml of
sample solution => m = 11.5 mL
 Content of reducing sugar in diluted solution is:
a 0.008
RS (g/100ml) = 100 = ×100 = 0.07 (g/100ml)
m 11.5
The total volume of sample solution after neutralization is 250mL
Total reducing sugar = 0.07 × 2.5 = 0.175 / 250mL
The weight of turnip sample = 50g
 The amount of reducing sugar in 50g turnip sample is: 0.175g/50g=
0.35%
 Content of starch in sample = 0.35% × 0.9 =0.315%

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 Practice 2: EVALUATE OF STARCH HYDROLYSIS BY α -AMYLASE

1. OBJECTIVE
Evaluated the outcomes of starch hydrolysis catalyzed by α -Amylase under
different test conditions
2. CHEMICALS AND EQUIPMENT
- Cassava starch
- Enzyme product Termamyl 120L with diluted 1/20
- H2SO4 Solution
- 0,1M CaCl2 Solution
- Beaker 500-1000 mL
- Glass Stirring rod
- Pipette 1mL; 5mL
- Viscometer
- Balance, Hot Plate
3. PROCEDURE
- Prepare starch solution: Put 200g of cassava starch into a 2L pot. Gently mix
the starch with approx. 1,5L of water until it is completely dissolve. Then
divide equally the solution to 3 prepare beaker (cup 1, cup 2, cup 3)
- Using H2SO4 solution to adjust the pH and check by pH meter as follows
o Cup 1: Adjust to pH = 6,0
o Cup 2: Adjust to pH = 4,0
o Cup 3: Adjust to pH = 4,0
- Add 3mL of 0,1M CaCl2 solution in cup 3
- Add to all 3 cups: 0,5mL of diluted Enzyme solution each cup
- Starch the enzymatic hydrolysis on the heating sand bath: Place each cup on
and heat up to 80oC. Maintain this temperature for 60 minutes (with constant
mixing)
- Take the cups put and cool them down to room temperature
- Visually examine the difference in color, consistency, and stickiness between
these cups

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 - Measure the viscosity of the starch solution by the viscometer at 50 or 100 rp,
(revolution per minute)
4. CONCLUSION

Cup pH pH VH2SO4 (mL) Viscosity (cP)

before adjusting after adjusting used
Cup 1 6,92 6,04 0,47 4,80
Cup 2 6,9 4,08 1 -

Cup 3 6,9 4,06 1,24 -

 Comment: For Cup 2 and Cup 3 with pH~4, we cannot measure the
Viscosity of this starch solution because this solution is too viscous, this
viscometer cannot show the value of viscosity. In Cup 1, we have the
value of Viscosity is 4,80 cP.

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 Practice 3: EVALUATE QUALITY OF VEGETABLE OIL
1. OBJECTIVE:
Evaluate the quality of vegetable oil based on several values representing the
transformation of lipids.
2. SAMPLES
- Vegetable oil samples 1, 2, 3. These oil samples were heated to temperatures of
60oC; 100 oC; 140 oC and kept at this temperature for 30 minutes.
- For this experiment, our group used vegetable oil samples heated to temperature
of 60oC
3. DETERMINE ACID VALUE (AV):
a. Principle:
The acid value of oils and fats is defined as the number of milligrams of potassium
hydroxide necessary to neutralize the free fatty acids in one gram of sample. It is a
relative measure of rancidity as free fatty acids are normally formed during the
decomposition of triglycerides upon storage and processing. In this procedure, the
acid value is determined by directly titrating the oil/fat sample in an alcoholic
medium with a standard potassium hydroxide solution
Reaction:
RCOOH + KOH  RCOOK + H2O

Based on the amount of KOH used to neutralize the acids, calculate the acid value.
b. Chemicals:
- Ethyl ether
- 96% Ethanol
- 1% phenolphthalein in ethanol
- 0.1N KOH in ethanol
c. Procedure:
Put vegetable oil sample (5-7g) into a clean dry round bottom flask or conical
flask. Add 30 - 50ml of 96% ether-alcohol mixture (ratio 1:1) to dissolve the
sample. Titrate the sample solution with 0.1N KOH in ethanol in the presence of
phenolphthalein indicator until a pink color appears.
d. Calculation:
AV is calculated based on the formula:
5,611.b . f
A x=
m

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With:

b: ml of 0.1N KOH used for titration

m: amount of test sample (g)

f: concentration correction factor for used 0.1N KOH solution

5.611: mg of KOH in 1ml of 0.1N KOH

Titration result:

b1 = 0.35mL; b2 = 0.3mL => baverage = 0.325mL

m = 5 gram

f=1

5.611: mg of KOH in 1ml of 0.1N KOH

Hence, the acid value is:

5,611.b . f 5,611 ×0.325 ×1

A x= = =0.365
m 5

4. DETERMINE IODINE VALUE (IV)

a. Principle:
The iodine value or iodine index (commonly abbreviated as IV) is defined as
the amount of iodine absorbed by 100 g of oils and fats. This index is a measure
of the degree of unsaturation (number of double bonds) of a fatty sample. This
parameter is extremely important in food industry because it indicates the
oxidative stability and could be used to detect adulteration of fatty products
IV is determined by treating the sample with an excess of solutions of iodine
monochloride (ICl). Unreacted iodine monochloride is then converted to iodine
by potassium iodide. Subsequently, the liberated iodine is titrated with sodium
thiosulfate standard solution in the presence of starch indicator. The general
reaction scheme is as follows:
 Iodine chloride is formed in the reaction:
2 KI + KIO3 + 6 HCl → 3ICl + 3 KCl + 3 H2O
 Then, iodine chloride targets the double bonds of fatty acids in the oil:
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  The exceeded ICl is quantified by thiosulfate solution after adding KI
solution and distilled water to the mixture:

ICl + KI → KCl + I2

I2 + 2 Na2S2O3 → 2 NaI + Na2S4O6

The decrease in IV can be attributed to the destruction of double bonds by oxidation,

scission, and polymerization.

Note: When left for a long time, ICl can not only target double bonds but can also
replace the hydrogen atom in the saturated groups, so the operating conditions must be
strictly followed.

b. Chemicals

- Solution of ICl in 0.2 N HCl: put in a flask with ground joint 11.1g KI 7g
potassium iodate (KIO3), 50 mL distilled water and 50 mL concentrated HCl.
Shake well for desolation, the add 20mL chloroform. While shaking, add drops of
1% KIO3 one by one until the purple color of chloroform disappears. Transfer the
solution to the separation funnel and let it stand. Remove the lower layer and
transfer the upper layer to a 1L volumetric flask and add distilled water until
reaches the mark. Preserve the reagent in a brown flask and place it in the dark

- 10% KI solution

- 1% soluble starch solution

- 0.1N Na2S2O3 solution

- Ethyl ether

c. Procedure
- Take exactly 250mL vegetable oil (0.3 – 0.5g) into a flask with ground joint.
- Add 5 mL ethyl ether to dissolve the sample, then add 25mL ICl in 0.2N HCl.
Shake well, put the stopper on and let it stand for 15-20 minutes

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 - Add 10mL 10% KI solution, and 50mL distilled water
- Quantify the released iodine by 0.1N sodium thiosulfate solution until the
yellow color appears
- Add 1mL 1% soluble starch solution, and 3mL chloroform to release the iodine
absorbed by the oil

Notes:

- Remember to shake strongly during the titration

- Control sample is tested simultaneously
d. Calculation

IV is calculated based on the formula:

( b−a ) . f .0.01269 .100

I=
m

Where:

a: mL of 0.1N Na2S2O3 used to titrate the analyzed sample

b: mL of 0.1N Na2S2O3 used to titrate the control sample

m: Weight of sample (g)

f: Concentration correction factor for used 0.1N KOH solution

0.01269: g of iodine corresponds to 1mL 0.1N Na2S2O3 solution

Titration result:

Volume of 0.1 N Na2S2O3 used to titrate the analyzed sample: a = 45.8 mL

Volume of 0.1 N Na2S2O3 used to titrate the control sample: b = 65.0 mL

Mass of sample: m = 0.3 g

Concentration correction factor for used 0.1 N KOH solution: f = 1

Hence, the Iodine value (IV) is:

( 65.0−45.8 ) × 1× 0.01269 ×100

I= =81.216
0.3

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5. DETERMINE PEROXIDE VALUE

a. Principle:

Peroxide value is defined as the amount of iodine formed when potassium iodine
solution reacts with peroxides in 100g oil, is a useful indicator of the extent of
oxidation of fats and oils.

Hydroperoxide in acidic conditions can react with KI and form iodine following
reaction:

ROOH + 2KI + 2CH3COOH  ROH + 2CH3COOK + I2 + H2O

Formed iodine is quantified by the titration with sodium thiosulfate solution:

I2 + 2Na2S2O3  2NaI + Na2S4O6

The peroxide value is calculated based on the used amount of thiosulfate.

b. Chemicals:

- Glacial acetic acid

- Newly prepared saturated solution of KI

- 1% soluble starch solution (prepare right before use from 0,1N Na2S2O3 solution
using distilled water not containing CO2)

c. Procedure:

Take exactly 250ml of oil sample into a flask with ground joint. Add 5-10 ml of
chloroform to dissolve the sample, then add 10-20 ml of CH3COOH solution, and
1ml saturated solution of KI. Carefully shake in 1 minute. Add in the mixture
25mL distilled water and quantify the formed iodine by titration with 0,01N
sodium thiosulfate solution and 0,5mL 1% soluble starch solution as indicator.
Control sample is tested simultaneously (replace oil sample with 3-5mL distilled
water)

d. Calculation:

Peroxide value is calculated based on the formula:

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 ( a−b ) . f .0,01269.100
I=
m
Where:

a: ml of 0.01N Na2S2O3 solution used to titrate the analyzed sample

b: ml of 0.01N Na2S2O3 solution used to titrate the control sample

m: amount of sample (g)

f: concentration correction factor for used 0.01N Na2S2O3 solution

0.001269: g of iodine corresponds to 1 ml 0.01N Na2S2O3 solution

100: Conversion factor for vegetable oil

Titration result:

a = 2.4 mL

b = 0.3 mL

m = 5gram

f=1

0.001269: g of iodine corresponds to 1 ml 0.01N Na2S2O3 solution

100: Conversion factor for vegetable oil

Hence, the peroxide value (PV) is:

( a−b ) . f .0 .001269.100 ( 2.4−0.3 ) ×1 ×0.001269 ×100

P= = =0.053
m 5

6. CONCLUSION
Based on 3 values (Ax= 0.365, IV= 81.216, PV= 0.053) calculated for the 60 oC
vegetable oil sample, we could conclude that:
- The amount of free fatty acids increased over time when heating vegetable oil
sample as well as increasing the heating temperature
- Due to frying, the iodine value decreased along with the peroxide value
increased which indicated that oil had been oxidized resulting in reducing its

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quality.

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Practice 4: DETERMINE PROTEIN CONTENT IN MEAT AND FISH SAUCE
BY TOTAL NITROGEN ANALYSIS

1. OBJECTIVE
Evaluate the nutrition quality of milk based on protein content
2. CHEMICALS AND EQUIPMENT
- Meat, fish sauce
- Concentrated H2SO4
- 30 - 40% NaOH solution
- 3% H3BO3 solution
- 0.1N H2SO4 solution
- Catalyst mixture K2SO4: CuSO4 (2:1)
- Tashiro’s indicator: mixture of 0.1% methylene red and 0.1% methylene blue in
ethanol (or methanol) in ratio 2:1
- Digestion apparatus
- Kjeldahl apparatus
- Funnel ∅ 10
- Wash bottles

3. PROCEDURE
a. Sample preparation and digestion
- Weigh an amount of sample: 0.5g in case of meat; 1mL in case of fish sauce.
Transfer it to Kjeldahl’s flask and add 15-20mL concentrated H 2SO4 and 0,3g
catalyst mixture. Then, cover the flask with a glass funnel and start the
digestion process (using the pre-set program of the equipment). The digestion
process is finished when the sample solution changes color from blue to
completely transparent. Let it cool down. Rinse the funnel and the flask neck
with 10-15mL distilled water.
b. Distillation
- Place the Kjeldahl flask into the distiller.

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 - Pipette 10 mL 3% H3BO3 solution into a 250mL Erlenmeyer flask. Then add a
few drops of Tashiro's indicator and place the flask under the output of the
distiller.
- Carefully check the supply line of distilled water, 40% NaOH, and cooling
water.
- Start the distillation process using following the pre-set program. Use pH test
strips to monitor the process (check pH at the output of the distiller). The process
finishes when pH = 7
c. Titration
- Conduct direct titration by using 0.1 N H 2SO4 solution to quantify the produced
ammonium tetraborate until the light pink color appears.

4. CONCLUSION

a. Calculation
−3
a ×1,4. 10
X= ×(100∨10 00)
m
Where: X: Total nitrogen content (% or g/L)

a: Amount of 0,1N H2SO4 used in the titration of the sample (mL)

m: Amount of digested sample (g or mL)

1.4.10-3: Grams of nitrogen correspond to 1 mL 0,1N H2SO4

b. Result

Sample
Fish sauce Meat
m (g or mL) 1 mL 0.5 g
a1 (mL) 3.75 18.9
a2 (mL) 4.0 19.0
a (mL) 3.88 18.95

XN (%) 0.543 % 5.306 %

XP = 6.25XN: Total protein content (%) 3.395 % 33.163%

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Conclusion:

 Protein content in fish sauce is 3.395%

 Protein content in meat is 33.163%

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