1.0 PURPOSE
The purpose of the document is to establish a general and uniform method for the determination of protein in food and feeds.
2.0 SCOPE
This standard testing procedure is suitable for the determination of total nitrogen content and specifies the factors used in converting nitrogen to protein for foods and feeds.
3.0 RESPONSIBILITY
The expert and trained chemist are responsible for following this procedure. The technical manager is responsible for implementing this procedure.
4.0 REQUIREMENTS
4.1 Apparatus
4.1.1 For Digestion - Conduct digestion over a heating device. Adjusted to bring 250 ml water at 25°C to a rolling boil approximately for 30 minutes if the electric heater is used. To test heaters, preheat for 10 minutes, to prevent superheating. Add 3 to 4 boiling chips or glass beads.
4.1.2 For Distillation: - Use distillation apparatus and assembly for distillation.
§ Kjeldohl flask 500ml
§ Erlenmeyer flask 500ml
4.2 Glassware
4.2.1 Burette: Graduated in 0,l ml and with an accuracy of 0.05 ml
4.2.2 Pipettes: capacity 2 ml
4.2.3 Measuring cylinders: of capacity 50 ml
4.2.4 Conical flasks, of capacity 100 or 150 ml
4.3 Reagent
4.3.1 Concentrated Sulphuric Acid - 93 to 98 percent by mass
4.3.2 Mercuric Oxide or Metallic Mercury.
4.3.3 Potassium Sulphate or Anhydrous Sodium Sulphate.
4.3.4 Zinc (Granulated)
4.3.5 Thiosulphate Solution - Dissolve 80 g of hydrated sodium thiosulphate in 1 liter of distilled water.
4.3.6 Sodium Hydroxide-solution- For solution, dissolve about 450 g solid sodium hydroxide in
Distilled water, cool, and dilute to 1 liter.
4.3.7 Sulphuric Acid, Standard Solution - 0.1N Standardize against a primary standard
4.3.8 Sodium Hydroxide Standard Solution - 0.1 N. Standardize against a primary standard.
4.3.9 Methyl Red Indicator - Dissolve 1 g methyl red in 200 ml alcohol.
5.0 PROCEDURE
5.1 Digestion - Accurately weigh 0.7 to 2.2 g of the sample into the digestion flask. Add 0.7g mercury
oxide or 0.65 g mercury and 15g powdered potassium sulfate or anhydrous sodium sulfate, and 25 ml sulphuric acid. The ratio of salt to acid ( m/v ) should be approximately 1: 1 at the end of digestion for proper temperature control. Digestion may be incomplete at a lower ratio and nitrogen may be lost at a higher ratio.
5.2 Each gram of fat consumes 10 ml and each gram of carbohydrate 4 ml of sulphuric acid during digestion. Place the flask in an inclined position on a heater and heat gently until foaming ceases. A small amount of paraffin or silicon antifoam may be added to reduce foaming. Boil vigorously until the solution becomes clear and then continue boiling it for 1 to 2 hours.
5.3 Distillation - Cool, add about 200 ml distilled water, and in order to avoid complex formation, add 25 ml of the thiosulphate solution. Mix to precipitate the mercury. Add a few zinc granules to prevent bumping, and set the flask on distillation assembly. Drop sodium hydroxide solution with a dropping funnel. A change of color from black to red indicates a solution is strongly alkaline. ( distillation bulb on the condenser, and, with the tip of the condenser immersed in a measured quantity of standard acid (usually 50 ml, the quantity of 0.1 N ) in the receiver, the heat immediately until all ammonia has distilled over ( at least 150 ml distillate ) Lower the receiver before stopping distillation and wash the tip of the condenser with distilled water. Back-titrate excess acid with standard 0.1 N sodium hydroxide, using methyl red as an indicator. Correct for blank determination in reagents.
6.0 CALCULATION
Nitrogen content (N) in g= (A-B) - (C-D) x 0.0014
Where,
A= Volume in ml 0.1N acid measured for main distillation
B= Volume in ml 0.1N alkali used for back titration A
C= Volume in ml 0.1N acid measured for blank distillation
D= Volume in ml 0.1N alkali measured for back titration C
Protein % by mass = (N x 100 x Conversion factor)/W
Where,
N= Mass of nitrogen content in mg of the original sample
W=Mass of sample in g
Milk Protein in milk SNF % by mass=
Protein% by mass/(100 -MIV + Fat) x 100
MIV= Moisture in Volatile content
7.0 REVISION/CHANGE HISTORY: NA
8.0 ATTACHMENTS
· Appendix-1:FLOW CHART PROTEIN IN FOOD AND FEE
9.0 REFERENCES
· IS 7219:1973; RA 2010; Amendment 2016
END OF THE DOCUMENT
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