Protein is an essential component of all cell and tissue structures. It is the most important material basis for human life activities. In human cells, proteins account for about 1/3 of the total protein, and adults average about 16.3% of protein. The main functions of proteins in the human body are: (1) Constitute enzymes, hormones, antibodies, and body tissues. (2) Promote human growth and development. (3) Maintain capillary osmotic pressure. (4) Supply part of human body energy. Proteins are ubiquitous in life activities and have a variety of important functions. Once the organism loses its protein, all life activities will stop and the end of life. So, protein is a substance of life, and it is also an important nutritional indicator in food.
Proteins in foods are complex nitrogen-containing organic compounds with large molecular weights. They are composed of 20 amino acids that are bound together in certain ways through amide bonds and have a certain spatial structure. The main chemical elements contained are C, H, and O. N, N, nitrogen is the main sign of protein differentiation from other organic compounds, Kjeldahl determination of protein in food by measuring the nitrogen content, and then multiplied by the protein conversion factor to get the protein content.
1 Principle of Kjeldahl method
Kjeldahl determination of protein in food is based on the principle that the sample is heated and digested with concentrated sulfuric acid, potassium sulfate, and copper sulfate to decompose the protein, in which carbon and hydrogen are oxidized into carbon dioxide and water escapes, and the organic nitrogen in the sample is converted. For ammonia and sulfuric acid combined ammonium sulfate, and then add alkali distillation, so that ammonia vaporized, absorbed with boric acid and then titrated with standard hydrochloric acid solution, according to the standard acid consumption can be calculated protein content.
2 Reaction Equations for Each Step in Protein Determination
(1) Digestion
2NH2 (CH2) 2COOH + 13H2SO4 = (NH4) 2SO4 + CO2↑ + SO2↑ + 16H2O
(2) Distillation
In the digested sample solution is added concentrated sodium hydroxide is alkaline, the heating distillation can release ammonia, the reaction equation is as follows: NaOH + (NH3) 2SO4 = 2NH3↑ + Na2SO4 + H2O
(3) Absorption
Ammonia released by heat distillation can be absorbed by boric acid solution. The reaction equation is as follows:
2NH34H2BO3 = (NH4) 2B4O7 + 5H2O
(4) Titration
(NH4) 2B4O7 + 5H2O + 2HCl = 2NH4Cl + 4H3BO3
3 Kjeldahl test should pay attention to matters
(1) The sample should try to select a representative, large solid sample to be finely and evenly applied by the crushing equipment, and the liquid sample should be uniformly mixed.
(2) When weighing in Kjeldahl flasks, care should be taken not to allow the sample to stick to the neck of the flask. This may cause the nitrogen-containing compound adhered to the neck to be undigested in the absence of sulfuric acid. Nitrogen loss.
(3) Do not use strong fire during digestion. Especially when sample fat or sugar content is high, a lot of foam is easily generated during digestion. Strong fire can spill the sample out of the bottle or splash and adhere to the Kjeldahl flask wall. Digestion is complete and causes nitrogen loss. Therefore, at the beginning of digestion, it should be heated with low heat to maintain gentle boiling, so that the fire focus on the bottom of the Kjeldahl flask.
(4) The Kjeldahl flask must be rotated from time to time during the digestion process in order to use condensing acid to wash the solid residue attached to the bottle wall and promote its digestion.
(5) When the fat content in the sample is too high, the amount of sulfuric acid must be increased because of the large amount of sulfuric acid consumed during digestion, which results in the loss of nitrogen due to the lack of ammonium sulfate.
(6) After digesting until the liquid is clear and transparent, it only needs to continue heating for 0.5h. After heating for too long, the sulfuric acid is continuously decomposed, the water continuously escaping and the concentration of potassium sulfate increases, the boiling point rises, and the generated ammonium salt is easily formed. The occurrence of heat breaks down the loss of ammonia and results in loss, especially for foods with a low protein content.
(7) Before distillation, check the sealing of the distillation apparatus. Use a water seal on the funnel containing sodium hydroxide to prevent the ammonia from escaping due to leakage of the distillation apparatus and affect the measurement results.
(8) The standard acid for titration must be formulated and calibrated according to the standard. The accuracy of the standard acid concentration will affect the accuracy of the test data.
(9) In order to improve the accuracy of test data and reduce random errors, a sample must be tested for parallel testing. Kjeldahl determination of protein content in food more steps, in the inspection process should pay attention to the above matters, reduce the experimental error in each step in order to improve the accuracy of the test results.