Microwave-assisted aqueous two-phase extraction of phenolic compounds from Ribes nigrum L. and its antibacterial effect on foodborne pathogens

Abstract

Microwave-assisted ammonium sulfate/ethanol aqueous two-phase system was used to extract the phenolic compounds from blackcurrant (Ribes nigrum L.) fruits. After purification with AB-8 macroporous resin, the phenolics (RMAP) was obtained with a purity of 80.50 ± 0.26%. The minimum inhibitory concentration (MIC) and inhibition zone tests showed that RMAP exhibited more potent antibacterial properties against Staphylococcus aureus than against Escherichia coli and Salmonella typhimurium. Additionally, high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis confirmed RMAP was mainly composed of four phenolic compounds including kaempferol-3-O-rutinoside, rutin, cyanidin-3-O-glucoside (C3G) and hyperoside. MIC and time-kill curve assays indicated that C3G displayed the highest inhibitory activity on S. aureus among four compounds. Moreover, crystal violet and MTT assays showed that C3G exhibited obviously inhibitory effect on fresh biofilm formation of S. aureus. Transmission electron microscopy observation, alkaline phosphatase, lactate dehydrogenase, protein and DNA leakage assays verified that C3G damaged cell wall and membrane of S. aureus. On the other hand, UV absorption spectroscopy and DNA cleavage assay revealed that C3G was able to interact with DNA in the form of chimera, but had no direct effect on DNA cleavage. Whereas, DNA topoisomerase IV (DNA Topo IV) inhibition assay and molecular docking model manifested that C3G could combine with DNA Topo IV to affect its activity and further restrained the synthesis of DNA. These results indicated that the antibacterial mechanism of C3G against S. aureus was mainly related to its inhibiting biofilm formation, disrupting cell structure and resisting DNA Topo IV activity.