Physicochemical properties and antibacterial mechanism of theabrownins prepared from different catechins catalyzed by polyphenol oxidase and peroxidase

Theabrownins (TBs) are the characteristic functional and quality components of dark teas such as Pu-erh tea and Chin-brick tea. TBs are a class of water-soluble brown polymers with multi-molecular weight distribution produced by the oxidative polymerisation of tea polyphenols during the fermentation process of dark tea, both enzymatically and non-enzymatically. TBs have been extracted and purified from dark tea all the time, but the obtained TBs contain heterogeneous components such as polysaccharides and caffeine in the bound state, which are difficult to remove. The isolation and purification process was tedious and uses a lot of harmful organic solvents. This has made it difficult to industrialise TBs. In this study, epigallocatechin (EGC), epigallocatechin gallate (EGCG), epigallocatechin gallate (ECG), EGC/EGCG (mass ratio 1:1), EGCG/ECG (mass ratio 1:1), EGC/ECG (mass ratio 1:1), EGC/ECG (mass ratio 1:1), EGC/ECG (mass ratio 1:1) and EGC/EGCG/ ECG (mass ratio 1:1:1) as substrates and catalyzed by polyphenol oxidase (PPO) and peroxidase (POD) in turn to produce TBs, named TBs-dE-1, TBs-dE-2, TBs-dE-3, TBs-dE-4, TBs-dE-5, TBs-dE-6 and TBs-dE-7. The physicochemical properties and the antibacterial activity and mechanism of TBs-dE (1-7) were investigated. Sensory and colour difference measurements showed that all seven tea browning samples showed varying degrees of brownish hue. Zeta potential in aqueous solutions at pH 3.0-9.0 indicated that TBs-dE (1-7) was negatively charged and the potential increased with increasing pH. The characteristic absorption peaks of TBs-dE (1-7) were observed at 208 nm and 274 nm by UV-visible(UV-vis) scanning spectroscopy. Fourier infrared spectroscopy (FT-IR) indicated that they were phenolic compounds. TBs-dE (1-7) showed significant inhibition of Escherichia coli DH5α (E. coli DH5α). TBs-dE-3 showed the strongest inhibitory effect with MIC of 1.25 mg/mL and MBC of 10 mg/mL, followed by TBs-dE-5 and TBs-dE-6. These three TBs-dEs were selected to further investigate their inhibition mechanism. The results showed that TBs-dE inhibited bacteria by disrupting the extracellular membrane of E. coli DH5α, causing the leakage of contents and increasing the intracellular reactive oxygen content, resulting in abnormal cell metabolism due to oxidative stress. The TBs-dE was found to damage the extracellular membrane of E. coli DH5α, causing leakage of contents, and increase intracellular reactive oxygen content, resulting in abnormal cell metabolism due to oxidative stress. The results of the study provide a theoretical basis for the industrial preparation and product development of TBs.