Formation of homocitrulline during heating of milk

Homocitrulline arises from the reaction between cyanate and the epsilon-amino group of lysine residues; in milk, cyanate derives from heat-induced urea breakdown. Since homocitrulline levels were unknown in heated milk, its formation was studied in the temperature range 100-150 degrees C. Firstly, an analysis method based on ion-exchange chromatography using an amino acid analyser was developed. Homocitrulline, liberated from milk protein by enzymic hydrolysis, was eluted well separated from other amino acids and could be readily distinguished using this technique. Secondly, homocitrulline levels were determined for various time-temperature combinations in samples of milk, milk to which 10 mM-urea had been added, and milk that was made urea-free. No homocitrulline was formed in urea-free milk, while homocitrulline formation was stimulated by urea addition. Upon prolonged heating, we found extensive subsequent breakdown of homocitrulline. The kinetics of homocitrulline formation was quite complicated, but an approximation was possible as the initial formation reaction could be modelled using zero order reaction kinetics. The apparent activation energy for homocitrulline formation was estimated at approximately 90 kJ/mol. In general, the levels of homocitrulline to be expected in heated milk appeared to be quite low: approximately 0.3 mM in in-bottle sterilized milk, and < 0.01 mM in UHT sterilized milk.