Food Science Experiment- Protein Analysis

Posted by Buddhi Katangodage
 

Introduction of Proteins

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize.

Determination of Protein Concentration by using Kjeldahl method

It is considered to be the standard method of determining protein concentration in food. It does not measure the protein content directly and needs a conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein and this is an average value, each protein has a different conversion factor depending on its amino-acid composition). This method has 3 steps:

• Digestion
• Neutralization 
• Titration

Apparatus 

• Digestion unit
• Scrubber Unit/Exhaust system
• Distillation unit
• Titration burette of 50 ml
• Digestion flasks
• Distillation flasks
• Measuring cylinders
• Pipettes
• Conical flasks- 250 ml
• Retort Stand & clamp
• Small funnel
• Necessary Glassware

Methodology

Digestion

1. The food sample to be analyzed is weighed into a digestion flask.(m g)
2. It is digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst( ex:copper, selenium, titanium, or mercury (to speed up the reaction)). 
3. Digestion converts any nitrogen in the food (other than that which is in the form of nitrates or nitrites) into ammonia, and other organic matter to C02 and H20. Ammonia gas is not released to the solution because the ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in solution as:  N(food) --> (NH4)2SO4

 Solutions before the Digestion

Solutions after the digestion

Neutralization

1. Digestion flask is connected to a recieving flask by a tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide(converts the ammonium sulfate into ammonia gas):

          (NH4)2SO4 + 2 NaOH --> 2NH3 + 2H2O + Na2SO4

Adding NaOH to the flask

          The ammonia gas that is formed is released from the solution and moves out of the digestion flask and   into the receiving flask - which contains an excess of boric acid. The low pH of the solution in the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts the boric acid to the borate ion:

                  NH3 + H3BO3 (boric acid)--> NH4+ + H2BO3- (borate ion)

Titration

1. Titration of the ammonium borate formed, with standard hydrochloric  acid using an indicator to determine the end-point of the reaction.(xM HCl)

H2BO3- + H+ --> H3BO3

The concentration of acid required to reach the end-point is equivalent to the concentration of nitrogen that was in the original food. A blank sample is usually ran at the same time.

%N=(x M/1000 cm3)x((vs-vb)/m g)x14x100%

vs =     titration volume

vb =     blank volume

m g =   sample weight

x M =  HCl solution's concentration

14 =    molecular weight of nitrogen

       2. Nitrogen content is converted to a protein content using the conversion factor: 

%Protein = F x %N

Advantages and Disadvantages of  Kjeldahl Method

Advantages  

• It is a standard method of determining nitrogen content on protein in the food.

Disadvantages 

• It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein.
• Different proteins need different correction factors because they have different amino acid sequences.
• The use of concentrated sulfuric acid at high temperatures make a some kind of hazard, as does the use of some of the possible catalysts 
• The is time consuming to method