Thinned-Young Apple Polyphenols Inhibit Halitosis-Related Bacteria Through Damage to the Cell Membrane

Abstract

The thinned young apple is a by-product and is generally discarded in the orchard during fruit thinning. The polyphenol content of thinned young apples is about 10 times more than that of ripe apples. In our study, the antibacterial effect of thinned young apple polyphenols (YAP) on the halitosis-related bacteria including Porphyromonas gingivalis, Prevotella intermedius, and Fusobacterium nucleatum was investigated. The minimum inhibitory concentrations of YAP against P. gingivalis, P. intermedia, and F. nucleatum were 8.0, 8.0, and 12.0 mg/ml, while the minimum bactericidal concentrations were 10.0, 10.0, and 14.0 mg/ml, respectively. The scanning electron microscopy and transmission electron microscopy analyses showed that after YAP treatment, the membrane surface of halitosis-related bacterial cells was coarse and the cell wall and membrane were separated and eventually ruptured. The integrity of the cell membrane was determined by flow cytometry, indicating that the cells with the integrity membrane significantly reduced as the YAP concentration treatment increased. The release of proteins and nucleic acids into the cell suspension significantly increased, and the membrane potential reduced after the YAP treatment. This research illustrated the antibacterial mechanism of YAP against halitosis-related bacteria and provided a scientific basis of utilizing the polyphenols from the discarded thinned young apples.

Keywords: Thinned-young apple polyphenols; antibacterial mechanism; cell membrane; halitosis; membrane potential.

FIGURE 1

HPLC chromatograms of YAP. YAP was 70% ethanol-eluted fractions from thinned young apples.

FIGURE 2

The outer wall structure changes of bacterial cells treated with different concentrations of YAP. (A1) P. intermedia with untreated, as control; (A2) P. intermedia treated with MIC; (A3) P. intermedia treated with MBC; (B1) P. gingivalis with untreated, as control; (B2) P. gingivalis treated with MIC; (B3) P. gingivalis treated with MBC; (C1) F. nucleatum with untreated, as control; (C2) F. nucleatum treated with MIC; (C3) F. nucleatum treated with MBC. MIC, minimum inhibition concentration; MBC, minimum bactericide concentration.

FIGURE 3

The intracellular microstructure changes of bacterial cells treated with different concentrations of YAP. (A1) P. intermedia with untreated, as control; (A2) P. intermedia treated with MIC; (A3) P. intermedia treated with MBC; (B1) P. gingivalis with untreated, as control; (B2) P. gingivalis treated with MIC; (B3) P. gingivalis treated with MBC; (C1) F. nucleatum with untreated, as control; (C2) F. nucleatum treated with MIC; (C3) F. nucleatum treated with MBC. MIC, minimum inhibition concentration; MBC, minimum bactericide concentration.

FIGURE 4

Flow cytometric analysis. Data acquisition was set to 30 μl for each experiment. (A) F. nucleatum; (B) P. gingivalis; (C) P. intermedia. MIC, minimum inhibition concentration; MBC, minimum bactericide concentration.

FIGURE 5

Release of 260 nm absorbing material from (A) F. nucleatum; (B) P. gingivalis; and (C) P. intermedia treated with YAP for 32 h. MIC, minimum inhibition concentration; MBC, minimum bactericide concentration.

FIGURE 6

Release of protein-absorbing material from (A) F. nucleatum; (B) P. gingivalis; and (C) P. intermedia treated with YAP for 32 h. MIC, minimum inhibition concentration; MBC, minimum bactericide concentration.

FIGURE 7

Membrane potentials of P. intermedia, P. gingivalis, and F. nucleatum treated with different concentrations of YAP. MIC, minimum inhibition concentration; MBC, minimum bactericide concentration.

FIGURE 8

Loadings of P. intermedia, P. gingivalis, and F. nucleatum treated with different concentrations of YAP on the first and second principal components. F.n, F. nucleatum; P.g, P. gingivalis; P.i, P. intermedia; MIC, minimum inhibition concentration; MBC, minimum bactericide concentration.