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• Melanoidins were mostly produced during distillation. • Melanoidins were primarily composed of carbohydrates. • Melanoidins formed during fermentation and distillation had structural differences. • Carbohydrates, proteins and phenols are involved in melanoidin formation diversely. Chinese liquor (baijiu) is a typical fermented food. In which production, melanoidins are formed in fermenting grains during low-temperature fermentation with long-term, and in distilled grains with high-temperature distillation for short-term. Here, the formation and structural characterization of melanoidins in these two distinct stages were first investigated through chemical composition analysis and spectroscopic identification. Our research showed that proteins and phenols continuously participate in melanoidin formation during fermentation and distillation processes. Distillation could produce melanoidins with larger amounts, darker colors, higher molecular weights, and more stable states than fermentation. The chemical composition including 10 carbohydrates, 17 amino acids, 5 free phenolic acids, and 7 bound phenolic acids was successfully identified in melanoidins. Ion chromatography was proposed to be an efficient method to investigate carbohydrates in melanoidins. Moreover, the potential impact of microorganisms on melanoidins was first revealed to be the possible utilization of glucose in melanoidins. [ABSTRACT FROM AUTHOR]