Aims

The aim for this experiment is to investigate the gel formation (tofu) from soya protein
isolate, determine the nitrogen content of red snapper fish and apply to the protein analysis
of red snapper fish and converts the derived nitrogen content into the protein content.

Introduction
Denaturation can be induced by chemical denaturants, such as urea and guanidium
hydrochloride. These denaturants stabilize denatured states relative to the native structure
because the amino acid side chains and peptide backbones of proteins are more stable in
guanidinium chloride or urea solutions, and the denatured state has more of those
structural features exposed to solvent than does the compact native state. Denaturation is
also induced by heating because the process of denaturation is endothermic at higher
temperature. In addition, because the denatured state of a protein has a much higher
specific heat than the native structure, proteins also denature on cooling; however,
the melting temperature for cold denaturation is generally below freezing. Most proteins
have maximal stability around physiological temperature. Denaturation may be reversible or
irreversible. Frequently, small, denatured proteins will spontaneously revert to their native
structure after denaturant has been removed or it has cooled down below its melting
temperature. At high enough concentrations, nearly all proteins denature irreversibly
because their denatured state aggregates and precipitates. Denaturation can also be caused
by chemical changes in a protein, such as oxidation of cysteine or methionine residues, or
by deamidation of glutamine or asparagine (Fersht, 2013). Coagulants such as calcium salt
are also added to the denatured protein to coagulate them into clumps, reducing the pH of
protein to its isoelectric point.

Kjeldahl method is a method used in analytical chemistry for quantitative determination of

nitrogen contained in organic substances plus the nitrogen contained in the inorganic
compounds such as ammonia and ammonium. This method was discovered by Johan
Kjeldahl in 1883 (Kjeldahl, 1883). In the Kjeldahl method, after digestion in
concentrated sulfuric acid, the total organic nitrogen is converted to ammonium sulfate.
Ammonia is formed and distilled into boric acid solution under alkaline conditions.
The borate anions formed are titrated with standardized hydrochloric acid, by which is
calculated the content of nitrogen representing the amount of crude protein in the sample.
Most proteins contain 16% of nitrogen, thus the conversion factor is 6.25. However, the
nitrogen from nonprotein additives or contaminants in the food, such as melamine in milk,
is also measured.

The aim of this experiment is to investigate the gel formation (tofu) from soya protein isolate,
determine the nitrogen content of red snapper fish and apply to the protein analysis of red
snapper fish and converts the derived nitrogen content into the protein content.
Methods
Part A: Gel formation
1. A 1:15 soy flour water mixture was prepared by adding 10g soy flour to 150mL of
distilled water and was stirred on a magnetic stir plate to form a uniform suspension.
2. The pH was adjusted to 8 with 0.1M NaOH and was stirred at room temperature for
20 minutes.
3. The suspension was transferred into two 50mL centrifuge tubes (30mL each) and
were centrifuged at 3500 rpm for 30 minutes.
4. 25mL of the supernatant was placed in a 100mL beaker, stirred and heated to
boiling.
5. The solution was removed from the heat as soon as it boiled and one drop of 5M
CaCl2 was added. The observation was recorded.
6. A few more drops of the CaCl2 was added and stirred with a glass stirring rod. The
observation was recorded.
7. A clump of tofu was picked out. The texture was felt and the observation was
recorded.

10g of soya flour was added to 150 ml of distilled water.

The mixture was stirred using a magnetic stir plate

The pH was adjusted to 8 with 0.1 M NaOH.

The suspension was stirred for 20 minutes and transferred

to two 50ml centrifuge tubes.

The tubes were centrifuged and 25ml of supernatant was

poured into a 100ml beaker, stirred and heated to boil.

One drop of 5M CaCl2 was added to the solution.

One drop of 5M CaCl2 was added to the solution.

A few drops of CaCl2 were added and stirred.

The texture of clump of tofu formed was felt with hand.

Part B: Kjeldahl Method
Sample Preparation
1. A sample of ca 0.5g of red snapper fish was taken and the fish was pat dry by using a
filter paper.
2. The Kjeldahl flask was weighed accurately, the fish was transferred into Kjeldahl flask
and reweighed to determine the mass of fish.
3. 4g of K2SO4 and 10mL of concentrated H2SO4 was added to the sample.

A sample of ca 0.5g of red snapper fish was taken

and the fish was pat dry by using a filter paper.

The Kjeldahl flask was weighed accurately, the

fish was transferred into Kjeldahl flask and
reweighed to determine the mass of fish.

4g of K2SO4 and 10mL of concentrated H2SO4 was

added to the sample.

Digestion
1. The flask was clamped in a slanted position, placed in a 500mL capacity heating
mantle. The mantle was turned to the highest setting.
2. The sample was digested until the colour became about colourless or a pale clear
yellow or orange.

The flask was clamped in a slanted position, placed

in a 500mL capacity heating mantle. The mantle was
turned to the highest setting.

The sample was digested until the colour became

about colourless or a pale clear yellow or orange.

Cooling
1. The flask was initially cooled in air for a few minutes and then in warm water before
immersing in an ice and water mix (without allowing the sample to solidify) for ca 10
minutes and then 100mL water was carefully added to the flask.
2. The outside of the Kjedahl flask and stopper were dried, labelled and stored securely
on a wooden triangle stand until week 2.

The flask was initially cooled in air for a few minutes

and then in warm water before immersing in an ice
and water mix for ca 10 minutes.

100mL of water was carefully added to the flask.

The outside of the Kjedahl flask and stopper were

dried, labelled and stored securely on a wooden
triangle stand until week 2.
Distillation
1. 25mL of 0.1M HCl was pipetted into the 250mL removing flask and the flask was
placed under the condenser, the tip of the delivery adapter was ensured to be
immersed in the acid solution.
2. With the Kjedahl flask held at an angle, 24mL of 50% NaOH was added carefully so
that the solution was not agitated.
3. The instructions given by the demonstrator were followed.
4. The Kjeldahl flask was checked with litmus paper.

25mL of 0.1M HCl was pipetted into the 250mL removing

flask and the flask was placed under the condenser, the
tip of the delivery adapter was ensured to be immersed
in the acid solution.
With the Kjedahl flask held at an angle, 24mL of 50%
NaOH was added carefully so that the solution was not
agitated.
The instructions given by the demonstrator were
followed.

The Kjeldahl flask was checked with litmus paper.

Titration
1. 2 drops of bromoscresol green was added to the receiver flask and the residual HCl
was titrated with 0.1M NaOH.
2. The number of moles of N and the mass of N in the original mass od red snapper fish
(atomic weight = 14.006) were calculated. The answer was expressed as a
percentage of the original mass and then converted to the percentage protein in the
red snapper fish.
3. The result was obtained was compared with percentage protein expected for fresh
red snapper fish.

2 drops of bromoscresol green was added to the receiver

flask and the residual HCl was titrated with 0.1M NaOH.

The number of moles of N and the mass of N in the

original mass of red snapper fish (atomic weight =
14.006) were calculated. The answer was expressed as a
percentage of the original mass and then converted to
the percentage protein in the red snapper fish.

The result was obtained was compared with percentage

protein expected for fresh red snapper fish.
Results
Part A: Gel formation
Table 1: Observations on the soya protein upon addition of 5M CaCl2.
Methods Observation
Upon addition 1 drop of 5M CaCl2 The solution turned white.
Upon addition of a few drops of White precipitates were formed at the bottom of the
5M CaCl2 beaker.
Texture of the final product Smooth and soft gel were formed.

Part B: Kjeldahl method

Table 2: Mass of red snapper fish, volume of NaOH used in the titration and the changes in
colour of litmus paper.
Mass of Kjeldahl flask (g) 147.3205
Mass of Kjeldahl flask + red snapper fish (g) 147.8291
Mass of red snapper fish (g) 0.5086
Initial burette reading (mL) 0.00
Final burette reading (mL) 14.60
Volume of NaOH added (mL) 14.60
Changes in colour of litmus paper Blue litmus paper: Blue litmus paper turned
red
Red litmus paper: No changes on red litmus
paper

Calculation
Volume of 0.1M HCl in receiving flask = 25.00 mL
(0.1)(25.00)
Number of moles of HCl =
1000
= 0.0025 mol
Volume of NaOH titrated = 14.60 mL
(0.1)(14.60)
Number of moles of NaOH =
1000
= 0.00146 mol
Mole of ammonia produced = no. moles of HCl – no. of moles of NaOH
= 0.0025 – 0.00146
= 0.00104 mol

Mass of N = no. of moles of ammonia x molar mass of nitrogen

= 0.00104 x 14.006
= 0.01457g
 mass of N
% of N content in red snapper fish = x 100%
mass of red snapper fish

0.01457
= x 100%
0.5086
= 0.02865 x 100%
= 2.86%
Conversion factor of protein-nitrogen in red snapper fish = 6.25

% protein content in fish = % of N content in red snapper fish x conversion factor of protein-
nitrogen in red snapper fish
= 2.86% x 6.25
= 17.875%

Expected % protein content in red snapper fish = 87%

Discussion
In part A, the clear brown solution turned white upon addition of one drop of 5M CaCl2 and
white precipitates formed when more drops of 5M CaCl2 were added. This was because
denaturation of soy protein took place when the solution was boiled to heating and the
structure of the protein molecules was disrupted. The hydrophobic part of the proteins was
exposed and formed precipitate when calcium salt was added to the heated solution.
Calcium ions in calcium chloride formed ionic bond with the negatively charged hydrophobic
part of the protein molecules to form calcium precipitates. The precipitates formed
suspended at the bottom of the beaker and clumped together in the form of tofu as the
final products.

In part B, the literature value of protein contained in the red snapper fish is found to be
87%. Meanwhile, the calculated % protein content in red snapper fish in this experiment is
calculated to be 17.875%. The huge difference between the calculated % protein content in
red snapper fish and the literature value most probably due to some errors involved in
performing this experiment. Incomplete titration of NaOH might be one of the errors
encountered in this experiment. The colour indicator used in this experiment was
bromoscresol green. Bromoscresol green gave a very pale colour when it was added to the
solution in the receiving flask. As for this, the changes in colour of the bromoscresol green
could hardly being judged by human naked eyes. This might cause lower amount of NaOH was
titrated to the receiving flask to completely neutralized the excess HCl in the receiving flask which
lead to the lower calculated % protein content in the red snapper fish. To overcome this error,
colour indicator with more distinctive colour change should be used in order to better determine the
end point of titration. The lower calculated % protein content in red snapper fish might also due to
the occurrence of protein degradation in the fish. The red snapper fish used in this experiment might
not be fresh and protein degradation might had taken place. The literature value of 87% protein
content in red snapper fish was analysed using fresh red snapper fish. Thus, the protein content in
red snapper fish analysed in this experiment was lower than the literature value. Despite that,
different part of the red snapper fish might also contain different amount of protein in it.
Conclusion
As to conclude, a soft and smooth solid gel like tofu was formed upon addition of calcium
salt to soy protein. Coagulation of protein in soy occurred. The nitrogen content in the red
snapper fish was determined to be 2.86% and the protein content in the red snapper fish
was calculated to be 17.875%. The difference in protein content between the literature
value and the calculated value might due to some experimental errors when performing the
experiment.
Questions
Part A: Gel formation
1. The solubilized soya protein denatures when it is heated to boiling. Denaturation of
solubilized soya protein will alter the protein structure by breaking the
intermolecular bonds such as ionic bonds, hydrogen bond, hydrophobic interactions
and disulphide bond between the protein molecules. By breaking the bonds
between protein molecules, protein molecules lose its secondary, tertiary and
quaternary structure and therefore these protein molecules fail to perform their
biological functions (Tanford, 1968). Exposure of hydrophobic part of the protein
lead to the precipitation of protein in the solution.

2. The function of calcium salt in the formation of tofu is to precipitate soy protein (Lu
et. al., 1980). Hydrophilic part of the protein molecules will be neutralized when
calcium salt is added. The neutralized protein will then be clumped together and
formed tofu as the product. This process is known as coagulation of protein.

Part B: Kjeldahl Method

1. A back titration is a titration method where the concentration of an analyte is
determined by reacting it with a known amount of excess reagent. The remaining
excess reagent is then titrated with another, second reagent. The second titration's
result shows how much of the excess reagent was used in the first titration, thus
allowing the original analyte's concentration to be calculated. A back titration may
also be called an indirect titration.

2. (NH4)2SO4 (aq) + 2NaOH(aq) 2NH3(g) + Na2SO4(aq) + 2H2O(l)

3. Amino acids that contain nitrogen other than that present in amino acid group are
histidine and arginine.

4. The conversion factor for cereal is much lesser than for other protein containing
food as cereal contain lesser amount of protein nitrogen than non-protein nitrogen.
Conversion factors depend on the ratio of protein nitrogen to non-protein nitrogen
contained in the food. The higher the ratio of protein nitrogen to non-protein
nitrogen contained in the food, the higher the conversion factor of that particular
food. Cereal has low amount of protein nitrogen, thus the conversion factor of
cereals is much lesser than other protein containing food.

5. Nitrogen sources other than protein that may be found in the fish are nucleic acid
including RNA and DNA molecules. These are accounted for in the determination of
protein as RNA and DNA sequences are responsible for coding of different types of
amino acids. Thus, the protein contained in the fish is determined by the
composition of nucleic acids present in the fish.
 6. Granulated zinc acts as an anti-bumping agent by providing nucleation sites as to
ensure the liquid boils smoothly without becoming superheated for bumping.

7. The tip of the condenser must be placed in the acid solution for distillation step to
prevent ammonia gas escaping to the air so that all the ammonia can fully react with
HCl in the conical flask.

References

1. Fersht A. R., 2013. Denaturation (Proteins). In Brenner’s Encyclopedia of Genetics 2nd

edition, pp. 302 – 303.

2. Kjeldahl, J., 1883. Neue Methode zur Bestimmung des Stickstoffs in organischen
Körpern (New method for the determination of nitrogen in organic substances). In
Zeitschrift für analytische Chemie, vol. 2, no.1, pp. 366 – 383.

3. Lu, JY, Carter, E & Chung, RA, 1980. Use of calcium salts for soybean curd
preparation. In Journal of Food Science, vol. 45, no. 1, pp. 32 – 34.

4. Tanford, C., 1968. Protein denaturation. In Advances in protein chemistry, vol. 23, pp.
121 – 282. Academic press.