FOOD BIOCHEMISTRY

(FD-204)
OPEN ENDED LAB
GROUP MEMBERS:
SARA SOHAIL FD-21031
ALINA FATIMA FD-21010
SAMRA NAZ FD-21024
AYESHA KHAN FD-21011

Topic: Estimate the activity of any specific enzyme, CHO, Reducing sugar
and protein for selected food products.
 BIURET TEST
Objective:
Detection of amino acids and proteins in a given food sample.

Apparatus:
Beakers, stirrer, spatula, weighing machine, test tubes, pipette, test tube holder.

Reagents:
1. Copper sulfate solution (CuSO4) 1%
2. Sodium hydroxide solution (NaOH) 1%

Food samples:
1. Butter
2. Milk

Theory:
Proteins are nitrogen containing compounds made up of amino acids joined together by peptide linkages.
They play crucial roles in biological processes, including structure, catalyzing reactions, transport, gene
expression regulation, and cell signaling. Proteins' primary structure is the sequence of amino acids, and their
folding into secondary, tertiary, and quaternary structures determines their function. These structures result
from interactions like hydrogen bonds and disulfide bonds. Proteins exhibit specificity and functionality based
on their three-dimensional structure, essential for proper functioning. They are involved in enzymatic
reactions, signaling, immune response, muscle contraction, and more. Overall, proteins are fundamental for
life, contributing to diverse biological functions.
Biuret test is done for peptide linkages. This method is based on the reaction which occurs between cupric
ions in the biuret reagent and peptide bonds of the protein molecule present in an alkaline solution. The
purple colored complex is the result of products forms peptide nitrogen with copper the amount of product
forms depend upon the concentration of protein.
Protein + Copper ions  protein-copper complex (purple)

Preparation of reagents:
Preparation of sodium hydroxide solution: We weighted 5gm sodium hydroxide in powder form then added it
in 100 ml distilled water and stirred until a homogenous mixture was formed.
Preparation of copper sulfate solution: We weighted 1gm copper sulfate in powder form then add this in 100
ml distilled water lastly stirred for a homogenous mixture.

Procedure:
1. Take out 5ml sample in a test tube using a pipette for melted butter and milk.
2. Then add 1 ml sodium hydroxide (NaOH) solution in the test tube containing the sample.
3. After that add 1-2 drops of copper sulfate (CuSO4) solution in the same test tube.
4. Shake the test tube gently for proper mixing.
5. Violet or purple color will appear which indicates presence of proteins.

Results:
The solution turned purple indicating the presence of protein in the sample of butter and milk.

Milk Butter
 MOLISCH TEST
Objective:
To detect carbohydrates (CHO) in the given sample by molisch test.

Apparatus:
Beakers, stirrer, spatula, test tubes, pipette, test tube holder.

Reagents:
1. Molisch reagent ( 5g of alpha napthol n 100ml of ethanol)
2. Sulphuric acid (H2SO4)

Food sample:
1. Corn
2. Butter

Theory:
Carbohydrates are a diverse group of organic compounds composed of carbon, hydrogen, and oxygen. They
serve as a vital source of energy and play essential roles in living organisms. Carbohydrates include simple
sugars, such as glucose and fructose, as well as complex polysaccharides like starch and cellulose. They are
found in various foods like grains, fruits, and vegetables. In the body, carbohydrates are broken down during
digestion into glucose, which is then used by cells as an energy source. Carbohydrates also contribute to
structural components in organisms, such as cell walls. Overall, carbohydrates are crucial for energy
metabolism and provide important structural and functional roles in biological systems.
When disaccharides and polysaccharides are treated with strong inorganic acids that are Sulphuric acid, these
hydrolyzed into monosaccharaides, undergoing dehydration by these acids. These dehydrated
monosaccharaides which are now referred to as furfural are then condensed with ethanolic alpha-napthol to
form purple colored complex.

Procedure:
1. Firstly take 2ml sample in a test tube.
2. After that add 2ml molisch reagent using a pipette.
3. Lastly add 2-8 drops of Sulphuric acid (H2SO4) in the test tube containing the sample.
4. Shake gently to observe a purple ring.

Results:
A purple ring was observed on the top of the mixture of both samples indicating that carbohydrates are
present in the given samples.

Corn Butter
 BENEDICT’S TEST
Objective:
To detect the presence of reducing sugar in the sample by benedict’s test.

Apparatus:
Beakers, stirrer, spatula, test tubes, pipette, test tube holder.

Reagent:
1. Benedict’s reagent:
By mixing copper sulfate pentahydrate (CuSO4. 5H2O), sodium citrate (Na3C6H5O7), and sodium
carbonate (Na2CO3) in distilled water.

Food sample:
1. Watermelon juice
2. Peach juice

Theory:
Reducing sugars are a type of carbohydrate that can undergo oxidation reactions. They have a free aldehyde
or ketone functional group that is capable of reducing other compounds. Common examples of reducing
sugars include glucose, fructose, and lactose. These sugars can react with specific reagents, such as Benedict's
solution or Fehling's solution, producing a color change from blue to green, yellow, or brick-red, indicating the
presence of reducing sugars. The ability of these sugars to act as reducing agents is important in various
physiological processes and biochemical reactions.
Sugar under alkaline conditions tautomerize and form endiols which are powerful reducing agents. They can
reduce cupric ions to cuprous from which is the basis of benedicts reaction. The cupric hydroxide from is not
easily soluble. An order to keep the hydroxide in solution a metal chelator like citrate is included in the
solution. The cupric hydroxide during the process of heating is converted into red cuprous oxide. Benedict test
is a qualitative test for reducing sugars. It is more specific than Fehling’s test because in this test uric acid,
creatinine, and other compounds do not interfere.

 Endiols + cupric-sodium citrate complex  copper ion + sugar acid

 Reducing sugar  endiol form of reducing sugar
 Copper ions + OH  Cu(OH)2
 CuSO4  Cu + Na-citrate  cupric-sodium-citrate complex

Procedure:
1. Add 7-10 drops of sample in a test tube.
2. After that add 5ml Benedict’s reagent in the sample’s test tube.
3. Mix the solution in the test tube thoroughly.
4. Lastly heat the test tube in a double boiler heating system or low flame for 8-10 minutes.
5. Observe a light blue ring at the top of the mixture inside the test tube.

Result:
A light blue ring was observed at the top of the solution in the test tube indicating the presence of reducing
sugar in the samples of watermelon and peach juice.

Peach juice Watermelon juice

 DEACTIVATION OF CATALASE
Objective:
To observe the deactivation of catalase through blanching.

Reagents:
1. Hydrogen peroxide (H2O2)

Food samples:
1. Papaya
2. Peach

Theory:
Enzymes play diverse roles in food. Naturally occurring or added during processing, they act as catalysts,
accelerating chemical reactions. Digestive enzymes like amylase, lipase, and protease aid in nutrient
breakdown during digestion. In food processing, enzymes are used to enhance characteristics; rennet
coagulates milk for cheese-making, and alpha-amylase improves dough texture. Fermentation relies on
microbial enzymes for producing yogurt, bread, wine, and beer. Enzymes like papain and bromelain tenderize
meat, while others contribute to flavor development in coffee and fruit ripening. Enzymes are highly specific,
and their activity is influenced by factors like temperature and pH. Utilizing enzymes improves food quality
and offers various benefits in the culinary industry.
Blanching is a step used in processing facilities in order to prepare the product for additional food steps. This is
accomplished by stopping enzymatic activity in the product. Blanching is usually used prior to other processing
steps that would not cause product to reach conditions in which the enzymes would be the denatured. These
processing steps include freezing and dehydration.
Blanching is used in the industry for several reasons. The first is in the inactivation of enzymes. By deactivating
the enzymes the product is protected from sensory and nutritional degradation. Second use of blanching is to
remove air from the intercellular spaces with an product. This can make the color of the product more
appealing and can also aid in headspace formation if the product can be canned. In addition blanching can
make canning easier by softening the tissues of the product making it easier to cram into a can. Finally
blanching conditions can remove surface microorganisms from the product in an attempt to make it easier for
the processor to remove all of the microorganisms in later processing steps.

Procedure:
1. Slice the food sample into two uniform pieces.
2. Boil one of the slices in the hot water bath for 3-5 minutes.
3. After blanching the slice place the unblanched and blanched slices of food sample side by side.
4. Pour few drops of hydrogen peroxide in each slice.
5. Bubbles will form on the unblanched slice whereas blanched slice shows no bubbles.

Results:
No bubble formation in the blanched food sample determines reduced to no enzyme activity whereas the
bubble formation in raw food sample determines that the catalase present in the sample breaks down the
hydrogen peroxide.

Peach Papaya
CONCLUSION:
All of the above mentioned test were performed on different food samples and
these food samples contained carbohydrate, protein, reducing sugar and enzyme
according to their specifications. Some of the test were performed again due to
handling mistakes but finally were achieved. There are many other methods for
detection of proteins for instance ninhydrin test or lead acetate test. For the
detection of carbohydrates one can also perform barfoed’s test or iodine test.
Then for reducing sugars Fehling’s test or tollen’s test may be performed as well.
Lastly for enzymes different types of test may be performed for different enzymes.