Sensitive determination of lysinoalanine for distinguishing natural from imitation mozzarella cheese

A new method of reverse-phase HPLC was used to determine the crosslinked amino acid lysinoalanine in natural Mozzarella cheese, dairy-based substitutes, and related ingredients. Commercial samples manufactured under known conditions or collected at the market were analyzed. The acid-hydrolyzed sample derivatized by 9-fluorenyl-methylchloro-formate was submitted to solid-phase extraction on an amino cartridge to extract selectively the lysinoalanine derivatives that were chromatographed under fluorescence detection. Lysinoalanine was not found (<0.4 ppm in protein) in pasteurized milk, but 0.4 to 4 ppm ((X) over bar = 1.7; n = 30) were present in natural Mozzarella cheese. Because of the ingredient characteristics and the more severe thermal processing conditions, the different types of processed cheese and imitation Mozzarella cheese exhibited much higher lysinoalanine contents, ranging from 15 to 421 ppm ((X) over bar = 54; n = 29). Hence, a highly significant distinction between natural Mozzarella cheese and imitations, even those that did not contain added milk protein, could be achieved by the lysinoalanine index. Conversely, the furosine index distinguished the imitation products only when the quantity of reducing sugars allowed the early Maillard reaction to be extensive.