Several nutraceutical compounds, such as polyphenols and peptides, modulate the enzymatic antioxidant defence system, providing an avenue for the development of multifunctional functional foods.

To enhance the effects of kombucha’s beneficial compounds and their functional properties, studies on kombucha fermentation using alternatives ingredients are needed. The aim of this study was to formulate and optimize kombucha using hempseed as a high antioxidant, phenolic compound, and protein contributor. An experimental central composite design (CCD) with response surface methodology (RSM) was used for maximizing the antioxidant capacity (AC), total phenolic content (TPC), and protein concentrations (PC) of this product. The optimized infusion concentrations were observed at 0.017% of hempseeds and 0.00046% of black tea leaves. AC was determined by the DPPH⁺ assay in microplate. TPC was determined in microplate using the Folin–Ciocalteu method. PC was determined by Peterson’s modification of the micro-Lowry method. The optimized kombucha results were 0.134 ± 0.002 mg Trolox Eq/mL for AC, 0.473 ± 0.027 mg GA Eq/mL for TPC, and 6.535 ± 0.477 μg/mL of PC. RSM can be developed to optimize the formulation of kombuchas to increase the amounts of desirables compounds. This study demonstrated that hempseeds added to kombucha have a higher antioxidant capacity, total phenolic content, and protein concentration than traditional kombucha.