Acid stable amino acids
A portion of sample is mixed with hydrochloric acid solution in a modified Kjeldahl flask. To prevent oxidation of the amino acids, as much oxygen as possible is removed from the flask by repeated freezing and thawing under vacuum. The neck of the flask is heat-sealed and the flask is heated in a 110°C oven for 20 hours. Protein in the sample is hydrolyzed to amino acids by the hot hydrochloric acid solution.
Acid Stable Amino Acids, Free
Samples are extracted at ambient temperature in dilute hydrochloric acid and neutralized. Extracts are separated and quantitated by ion exchange chromatography with post-column ninhydrin detection using a dedicated amino acid analyzer.
The sample is suspended in water, and made alkaline with an excess of magnesium oxide. Any free ammonia present is volatilized and distilled into a known amount of standard acid. The amount of acid reacting with the ammonia is quantitated by titration of the excess acid with standard alkali. Ammonia is calculated from the known amount of reacting acid.
Cysteine and methionine by oxidation
Cystine acid and methionine sulfone are separated on an ion-exchange column and reacted with ninhydrin, forming color-complex solutions that are measured spectrophotometrically. The concentration of cysteine and methionine are quantitated against a combined standard of cysteic acid, methionine sulfone, and an internal reference standard, which is taken through the method and injected onto the amino acid analyzer. (Any cystine present in the original sample will be quantitated as cysteine.)
Hydroxyproline can be used to estimate the amount of connective tissue in a sample of meat. A portion of sample is mixed with hydrochloric acid and heated in a sealed flask at 110°C for 24 hours. Protein in the sample is hydrolyzed to hydroxyproline by the hot hydrochloric acid solution. Hydrolysates are neutralized, diluted in buffer and analyzed using an amino acid analyzer with ninhdrin detection. The concentration of hydroxyproline in the sample is quantitated against a standard solution of known concentration.
Nitrogen solubility index
A portion of sample is suspended in water with stirring at 30°C for two hours. It is then diluted to a known volume with water. A portion of sample extract is centrifuged and an aliquot analyzed for protein. A separate portion of sample is analyzed for total protein by the same method.
Non-protein nitrogen, protein equivalent
The sample is digested for 1 hour in a urease enzyme solution (pH approximately 7.0). Any urea present is separated into carbon dioxide and ammonia. The resultant solution is made alkaline with magnesium oxide. Any ammonia present is liberated and distilled into a known amount of standard acid. The distillate is titrated with standard base. Percentage urea is calculated from the amount of acid consumed in the reaction. For comparison, this non-protein nitrogen may be calculated to a protein equivalent (PE) using the appropriate nitrogen to protein factor.
The sample is weighed, and defatted in refluxing petroleum ether. It is then incubated at 45oC, for 16 hours, with continuous agitation in acidic pepsin solution. The sample is filtered, and the residue analyzed for Kjeldahl protein. This value, subtracted from the total protein value, divided by the total protein value, times 100, is equal to the percent pepsin digestible protein.
Protein is determined by either the traditional kjeldahl method or combustion analysis.
Protein dispersibility index
The sample is placed in suspension and blended at 8500 rpm for 10 minutes. A portion of sample slurry is centrifuged and an aliquot of the supernatant is analyzed for Kjeldahl protein. The supernatant protein value is divided by the sample protein value and multiplied by 100 to give the percent protein dispersibility index.
Protein solubility in 0.2% KOH
The sample is suspended in 0.2% KOH solution for 20 minutes. This solution is filtered, and an aliquot of the filtrate is analyzed for Kjeldahl protein. This value, divided by the total Kjeldahl protein value, times 100, is equal to the percentage protein solubility in 0.2% KOH.
A portion of sample is treated with hydrochloric acid in a modified Kjeldahl flask. To prevent oxidation of the amino acid, oxygen is removed from the flask by freezing while under vacuum. The neck of the flask is heat sealed, and the flask heated at 110°C for 16 hours. The hydrolysate is diluted with buffer and analyzed by ion exchange chromatography with ninhydrin detection.
Nitrogen containing compounds in the sample are reduced using boiling sulfuric acid and a catalyst consisting of a mixture of potassium sulfate/titanium dioxide/cupric sulfate to form ammonium sulfate. The ammonium sulfate solution is cooled, diluted, and made alkaline with a sodium hydroxide solution. Ammonia is liberated and distilled into a known amount of standard acid. The distillate is titrated and nitrogen is calculated from the known amount of standard acid solution consumed in the reaction.
This procedure measures tryptophan in foods, feeds, ingredients, and physiological samples. Samples are hydrolyzed with sodium hydroxide in an evacuated sealed glass vessel. Hydrolysates are analyzed on a high pressure liquid chromatograph (HPLC) using UV detection and quantitated from standards of known concentration.