Physicochemical stability and antibacterial mechanism of theabrownins prepared from tea polyphenols catalyzed by polyphenol oxidase and peroxidase

Abstract

Tea polyphenols were used as substrates and oxidized successively by polyphenol oxidase and peroxidase to prepare theabrownins (TBs-dE). The conversion rate of catechins to TBs-dE was 90.91%. The ultraviolet and infrared spectroscopic properties and zeta potential of TBs-dE were characterized. TBs-dE is more stable at pH 5.0-7.0, about 25 °C or in dark environment. Ultraviolet light and sunlight can deepen its color due to the further oxidative polymerization. Mg²⁺, Cu²⁺, and Al³⁺ had a significant effect on the stability of TBs-dE. The inhibitory rates of TBs-dE (1 mg/mL) against Staphylococcus aureus and Escherichia coli DH5α were 51.45% and 45.05%, respectively. After TBs-dE treatment, the cell morphology of both bacteria changed, some cell walls were blurred, and the cytoplasmic content leaked. The research results can provide theoretical support for the industrialization of theabrownins.

Keywords: Antibacterial mechanism; Enzymatic oxidation; Physicochemical stability; Theabrownins.

Fig. 1

A HPLC analysis of tea polyphenols. B HPLC analysis of TBs-dE. C SDS-PAGE for detecting PPO and POD in TBs-dE. (The band 1 and 8 referred to marker protein, the band 2 referred to tea polyphenols, the band 3 referred to PPO, the band 4 referred to POD, the band 5 referred to TBs-dE before protein removal, and the band 6 and 7 referred to TBs-dE samples). D Ultraviolet–visible spectra of TBs-dE. E Fourier-transform infrared spectra of TBs-dE. F Zeta potential curve of TBs-dE aqueous solution in pH range 3.0–9.0. All data were expressed as the mean values ± standard deviation (n = 3)

Fig. 2

Effects of different conditions on the stability of TBs-dE. A The color of newly prepared TBs-dE in different-pH and ultra-pure aqueous solutions. B, C Absorbance change curves and total chromatic difference curve of TBs-dE in pH 3.0–9.0 solutions and purified aqueous solution (CK) during 5-day storage. D, E Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution stored at 25 °C, 50 °C, and 90 °C for 10 h. F, G Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution stored for 5 h under different lighting conditions. H, I Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution under different metal ions treatments. J, K Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution under different oxidants and reductants. L, M Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution treated with different food additives. All data were expressed as the mean values ± standard deviation (n = 3). Different lowercase letters indicate statistically significant differences (p < 0.05)

Fig. 2

Effects of different conditions on the stability of TBs-dE. A The color of newly prepared TBs-dE in different-pH and ultra-pure aqueous solutions. B, C Absorbance change curves and total chromatic difference curve of TBs-dE in pH 3.0–9.0 solutions and purified aqueous solution (CK) during 5-day storage. D, E Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution stored at 25 °C, 50 °C, and 90 °C for 10 h. F, G Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution stored for 5 h under different lighting conditions. H, I Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution under different metal ions treatments. J, K Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution under different oxidants and reductants. L, M Absorbance change curves and total chromatic difference curve of TBs-dE aqueous solution treated with different food additives. All data were expressed as the mean values ± standard deviation (n = 3). Different lowercase letters indicate statistically significant differences (p < 0.05)

Fig. 3

Inhibitory effect of tea polyphenols and TBs-dE on S. aureus and E. coli DH5α. A The growth curve of S. aureus. B The growth curve of E. coli DH5α

Fig. 4

A SEM micrographs of S. aureus and E. coli DH5α treated with tea polyphenols and TBs-dE. B TEM micrographs of S. aureus and E. coli DH5α treated with tea polyphenols and TBs-dE

Fig. 5

Flow cytometric analysis. A PI fluorescence intensity of untreated S. aureus cells. B PI fluorescence intensity of S. aureus cells treated with tea polyphenols. C PI fluorescence intensity of S. aureus cells treated with TBs-dE. D PI fluorescence intensity of untreated E. coli DH5α cells. E PI fluorescence intensity of E. coli DH5α cells treated with tea polyphenols. F PI fluorescence intensity of E. coli DH5α cells treated with TBs-dE

Fig. 6

A Changes in the membrane permeability of S. aureus. B Changes in the membrane permeability of E. coli DH5α. C Intracellular β-galactosidase leakage of S. aureus. D Intracellular β-galactosidase leakage of E. coli DH5α. All data were expressed as the mean values ± standard deviation (n = 3). E Effect of TBs-dE on the production of intracellular ROS in S. aureus and E. coli DH5α. All data were expressed as the mean values ± standard deviation (n = 3). Different lowercase letters indicate statistically significant differences (p < 0.05)