Three Successive Infusions: Scientific Insights Into a Traditional Green Tea Drinking Custom

This study examines the scientific basis of a traditional multi-infusion tea-brewing practice common in Asia by evaluating compositional changes, antioxidant capacity, and sensory attributes across successive green tea infusions. Using UPLC-MS, GC-IMS, and elemental profiling, we characterized how repeated extractions alter key tea constituents. Although flavonoids, polyphenols, polysaccharides, and amino acids remained detectable across all infusions, the concentrations of major bioactive compounds including gallic acid, its esters, and L-theanine declined progressively from the first to the third infusion (GT1-GT3), corresponding with reduced in vivo radical-scavenging activity. Changes in volatile profiles and sensory evaluation further indicated noticeable shifts in flavor and taste with each infusion. Pearson correlation analysis revealed that most quantified compounds were positively associated with puckery, bitter, and acidic attributes, and negatively associated with sweetness and freshness. Variable Importance in Projection (VIP) analysis identified L-lysine, L-tyrosine, epicatechin, myricetin, theophylline, quercetin, and epigallocatechin as key contributors to sensory perception. These findings demonstrate that multiple infusions diversify flavor characteristics while modulating the bioactive profile of green tea, offering insights relevant to product development and consumer brewing practices.