Crude protein (CP) determination
CP is the total amount of protein in a sample, calculated by multiplying the total nitrogen content by 6,25. Nitrogen was determined using the Kjeldahl technique. After adding a catalyst tablet, weighed samples were digested in concentrated H2SO4. By adding H2O2 the burning process was accelerated. The samples were heated for one hour in a preheated block digester. The digestion results in conversion of nitrogen to ammonia. After digestion the samples were cooled down to room temperature and demineralised water and boiling chips were added. NaOH was added tot neutralize. Titration flasks were prepared by adding boric acid solution (1%) and methyl red indicator to an amount of water. The samples were distilled with a distillation apparatus by cooking it for 25 minutes, in which distillation of the ammonia into the boric acid solution occurred. The distillate containing the ammonium was titrated with a 0,1N HCl solution to red endpoint, to quantify the amount of ammonia.
To calculate the percentage of CP on a DM base in a sample, the following formula was used;
% Crude protein (DM) = (6,25 x 14 x v x N) / (p x DM of AD)
Where 6,25 is the conversion factor 100/16; 14 is the atomic weight of N; v is the quantity HCl added after titration (ml); N is the normality of HCl (=0,1N); p is the weight in mg of the sample and DM of AD is the percentage of dry matter in the air-dried sample.