Detection and quantitation of 2,5-diketopiperazines in wheat sourdough and bread

Liquid chromatography mass spectrometry (LC-MS) was used to quantify the levels of the 2,5-diketopiperazines cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) in acidified dough and bread. Dough acidification led to a significant increase in the level of cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) over 48 h compared to a nonacidified dough. However, no differences were found between chemically (mix of lactic and acetic acid in the presence of antibiotics) and biologically acidified doughs. On examination of the levels of cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) in bread crumb and crust, it was found that temperature is the main causative agent of 2,5-diketopiperazine formation during the baking process. Bread crumb and crust contained almost 100 and 2000 times respectively the levels found in dough prior to baking. cis-Cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) were also found to be at sensorally active levels in bread crust, however both 2,5-diketopiperazines were found to be below the minimum inhibitory concentration for antifungal activity in bread.