Determination of PROTEIN content 

What are 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 bond.
• They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine & valine, which are essential to human health but which the body cannot synthesize. 

Industrial applications of determination of protein content :

• In order to formulate/design the diet of a particular individual
• In Quality assurance laboratory
• In order to check the optimum quality of the food products
• To determine the final nutritional & textural qualities of food

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Principle of protein determination & method

The protein content is determined from the organic Nitrogen content by Kjeldahl method. The various nitrogenous compounds are converted into ammonium sulphate by boiling with concentrated sulphuric acid (HCl). The ammonium sulphate formed is decomposed with an alkali (NaOH) and the ammonia liberated is absorbed in excess of standard solution of acid and then back titrated with standard alkali.

Apparatus

1. Kjeldahl digestion flask – 500 or 800 ml
2. Kjeldahl distillation apparatus
3. Conical flask, 250 ml
4. Burette 50 ml

Reagents used for Protein analysis

• Concentrated Sulphuric acid (H₂SO₄₎ – Specific gravity 1.84
• 45% Sodium Hydroxide (NaOH)- Dissolve 450 gm of Sodium hydroxide pellets in 1000 ml water
• Standard Sulphuric acid (H₂SO₄) solution – 0.1 N (Normal)
• Standard Sodium Hydroxide (NaOH) solution – 0.1 N
• Methyl Red Indicator solution – Dissolve 0.5 gm methyl red in 100 ml of alcohol

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Three main steps involved in determination of PROTEIN content : Digestion, Distillation & Titration

Digestion : 

1. Take a 2 gm of powder sample
2. Mixed 2 catalyst (copper sulphate & potassium sulphate) in the ratio of 1:5.( Mix thoroughly)
3. Take a 2 gm of sample & 2 gm of catalyst mixture in heating tube
4. Add a 10 ml 98% concentrated H₂SO₄ & then add 20 ml of 40 % NaOH in the heating tube
5. Adjust the temperature at 420°C & put the tube in a heating mental
6. Observed color changes i.e (final end point is light green) 

Distillation : 

1. Add 40 ml NaOH to reservoir
2. Automatically 40 ml NaOH is received in heating tube through coil. (continuous heating) 
3. Because of NaOH, amino acids are breakdown into N₂
4. Then liberated N₂ is passed through Nebulizer
5. Add 4 % boric acid to the Nebulizer. Then acid N₂ is dissolved & form Sodium borate
6. We will get N2 is as a borate salt.

We have to find how much N2 is dissolved in boric acid ? 

Titration :

1. borate salt (alkali) is titrated with 1 N HCL 2)
2. Add 1 ml phenophthelin indicator.3)
3. titrate the sample with 1 N HCL until the solution changes to greenish to pink in color.

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Calculation formula

Calculate protein as = N (% nitrogen) x 6.25

Protein on dry wt. basis = Protein content x 100 (100 – Moisture content)

Ideally the protein content of food stuff is calculated by multiplying its total nitrogen content by a factor 6.25

This factor is used whenever the nature of the protein is unknown or when the product to be analyzed is a mixture of different proteins with different factors.  However use of different Nitrogen conversion factors for different matrices may lead to better accuracy of results.

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