Gas chromatographic detection of d-amino acids in natural and thermally treated bee honeys and studies on the mechanism of their formation as result of the Maillard reaction

Relative quantities of d-amino acids, (%D) calculated from the sum of d- and l-amino acids were determined in bee honeys (n=6) by GC-SIM-MS. Amino acids were isolated by treatment with Dowex 50 W X8 cation exchanger and converted into N(O)-perfluoroacyl amino acid propyl esters. In all honeys d-Ala, ranging from 2.2–6.2% d-Ala, was detected. Other d-amino acids were also found, albeit not in all honeys and approached 5.9% d-Glx, 5.4% d-Lys, 3.0% d-Phe, 2.1% d-Orn, 1.7% d-Asx, 1.5% d-Ser, 0.1% d-Pro, and 0.4% d-Val in certain honeys. Quantities of d-amino acids increased very much on experimental heating of honeys in an oven and on a microwave treatment. Conventional heating of a forest honey (no. 1) at 65 °C for 450 h leads to an increase of d-Ala (2.2–12.5%), d-Pro (0.0–5.0%), d-Ser (1.5–9.1%), d-Asx (1.7–9.8%), d-Phe (0.4–5.0%) and d-Glx (1.5–5.8%); first numbers in parentheses refer to unheated honeys. Relative quantities of other d-amino acids also increased. Experimental heating of another forest honey (no. 2) in a microwave oven for 3 min at 180 W leads to an increase of d-Ala (3.7–11.0%), d-Glx (1.5–13.7%), d-Asx (0.7–10.2%), d-Phe (0.3–4.8%), d-Val (0–4.2%), and d-Pro (0.1–2.3%). Microwave treatment at 700 W for 1 min of a blossom honey (no. 3) leads to an increase of d-Ala (6.2–26.7%) and of d-Phe (3.0–10.9%). Microwave treatments were accompanied by intensive destruction of amino acids. Heating of a model mixture mimicking the major components of honey (d-glucose, d-fructose, and l-amino acids at 20% water content) at pH 2.6–9.0 and at 180 W for 1–3 min leads to the generation of d-amino acids and was also accompanied by intensive decay of amino acids. From the data it is concluded that d-amino acids are formed in honeys in the course of the Maillard reaction. A mechanism is presented based on amino acid racemization of reversibly formed Heyns and Amadori compounds (fructose-amino acids).Parts of the results have been presented at 9th International Congress on Amino Acids and Proteins, August 8–12, 2005, Vienna, Austria, and Euro Food Chem XIII, September 21–23, 2005, Hamburg, Germany.