Lang-Hong Wang

An in vitro investigation of the inhibitory mechanism of β-galactosidase by cinnamaldehyde alone and in combination with carvacrol and thymol

BACKGROUND Some antibacterial agents exert their antimicrobial action by targeting the cytoplasmic macromolecules, such as proteins or nucleic acids, to disturb the properties of macromolecules that may deeply influence their biological activities and functions. Cinnamaldehyde (CIN) is a natural antibacterial ingredient found in the bark and leaves of cinnamon trees. METHODS The inhibitory mechanism of a typical enzyme, β-galactosidase by CIN was investigated by UV-visible, fluorescence, 3-D spectroscopy, circular dichroism, atomic force microscopy and molecular modeling studies. RESULTS CIN decreased the activity of β-galactosidase by competitive inhibition through a multiphase kinetic process. 3-D spectroscopy and circular dichroism showed that the binding of CIN to β-galactosidase resulted in changes in micro-environment of tryptophan and tyrosine residues, and conformation of β-galactosidase. The molecular recognition was also analyzed through modeling which indicated that CIN was inserted into the active site pocket of β-galactosidase and interacted with amino acid residues, such as Met502, Trp568, Phe601 and Trp999. Atomic force microscopy showed that a serious destabilization of the native conformation of β-galactosidase occurred after binding with CIN, e.g., morphological changes and increased dimensions of the β-galactosidase molecule. Moreover, it was found that the combinations of CIN, carvacrol and thymol exposure displayed synergistic effects on the inhibition of β-galactosidase. GENERAL SIGNIFICANCE This study exhibits a comprehensively understanding about the action mechanism of CIN that affects the conformation and activity of β-galactosidase in biochemical processes and provides some new insights into the possible intracellular targeting behaviors of CIN at a molecular level.

Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol and Its Combined Effect with a Pulsed Electric Field

Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus.

Modification of membrane properties and fatty acids biosynthesis-related genes in Escherichia coli and Staphylococcus aureus : Implications for the antibacterial mechanism of naringenin

In this work, modifications of cell membrane fluidity, fatty acid composition and fatty acid biosynthesis-associated genes of Escherichia coli ATCC 25922 (E. coli) and Staphylococcus aureus ATCC 6538 (S. aureus), during growth in the presence of naringenin (NAR), one of the natural antibacterial components in citrus plants, was investigated. Compared to E. coli, the growth of S. aureus was significantly inhibited by NAR in low concentrations. Combination of gas chromatography-mass spectrometry with fluorescence polarization analysis revealed that E. coli and S. aureus cells increased membrane fluidity by altering the composition of membrane fatty acids after exposure to NAR. For example, E. coli cells produced more unsaturated fatty acids (from 18.5% to 43.3%) at the expense of both cyclopropane and saturated fatty acids after growth in the concentrations of NAR from 0 to 2.20mM. For S. aureus grown with NAR at 0 to 1.47mM, the relative proportions of anteiso-branched chain fatty acids increased from 37.2% to 54.4%, whereas iso-branched and straight chain fatty acids decreased from 30.0% and 33.1% to 21.6% and 23.7%, respectively. Real time q-PCR analysis showed that NAR at higher concentrations induced a significant down-regulation of fatty acid biosynthesis-associated genes in the bacteria, with the exception of an increased expression of fabA gene. The minimum inhibitory concentration (MIC) of NAR against these two bacteria was determined, and both of bacteria underwent morphological changes after exposure to 1.0 and 2.0 MIC.

Determination of membrane disruption and genomic DNA binding of cinnamaldehyde to Escherichia coli by use of microbiological and spectroscopic techniques

This work was aimed to investigate the antibacterial action of cinnamaldehyde (CIN) against Escherichia coli ATCC 8735 (E. coli) based on membrane fatty acid composition analysis, alterations of permeability and cell morphology as well as interaction with genomic DNA. Analysis of membrane fatty acids using gas chromatography-mass spectrometry (GC-MS) revealed that the proportion of unsaturated fatty acids (UFA) and saturated fatty acids (SFA) were the major fatty acids in plasmic membrane, and their levels were significantly changed after exposure of E. coli to CIN at low concentrations. For example, the proportion of UFA decreased from 39.97% to 20.98%, while the relative content of SFA increased from 50.14% to 67.80% as E. coli was grown in increasing concentrations of CIN (from 0 to 0.88mM). Scanning electron microscopy (SEM) showed that the morphology of E. coli cells to be wrinkled, distorted and even lysed after exposure to CIN, which therefore decreased the cell viability. The binding of CIN to genomic DNA was probed using fluorescence, UV-Visible absorption spectra, circular dichroism, molecular modeling and atomic force microscopy (AFM). Results indicated that CIN likely bound to the minor groove of genomic DNA, and changed the secondary structure and morphology of this biomacromolecule. Therefore, CIN can be deem as a kind of natural antimicrobial agents, which influence both cell membrane and genomic DNA.

Non-thermal technologies and its current and future application in the food industry: a review

In recent years, consumers have been demanding convenient and healthy foods which have ‘fresh-like’ characteristics while still being safe and a long shelf-life. These requirements are hard to achieve using existing traditional thermal food processing technologies and the innovative new food process and preservation technologies based on thermal processing systems are needed. However, non-thermal technologies in food processing do not generate high temperature and use short treatment times. This means that the nutritional components of foodstuffs are better retained, and the sensory properties of foods are less changed compared with traditional thermal processing. The aim of this review was to present non-thermal technologies applications and its mechanism in food industry in recently, and to explore the potential application prospects of combining non-thermal treatments applied in food industry because it not only could overcomes the drawback of single technology, but also can enhances the processing efficiency at lower treatment intensity.

Cinnamaldehyde inhibit Escherichia coli associated with membrane disruption and oxidative damage

In this study, the antimicrobial mechanism of cinnamaldehyde (CIN) against Gram-negative Escherichia coli ATCC 25922 (E. coli) based on membrane and gene regulation was investigated. Treatment with low concentration (0, 1/8, 1/4, 3/8 MIC) of CIN can effectively suppress the growth of E. coli by prolonging its lag phase and Raman spectroscopy showed obvious distinction of the E. coli after being treated with these concentration of CIN. The determination of relative conductivity indicated that CIN at relatively high concentration (0, 1, 2, 4 MIC) can increase the cell membrane permeability, causing the leakage of cellular content. Besides, the content of malondialdehyde (MDA) and the activity of total superoxide dismutase (SOD) of E. coli increased with increasing treatment concentration of CIN, implying that CIN can cause oxidative damage on E. coli cell membrane and induce the increase of total SOD activity to resist this oxidative harm. Moreover, quantitative real-time RT-PCR (qRT-PCR) analysis revealed the relationship between expression of antioxidant genes (SODa, SODb, SODc) and treatment CIN concentration, suggesting that SOD, especially SODc, played a significant role in resistance of E. coli to CIN. The underlying inactivation processing of CIN on E. coli was explored to support CIN as a potential and natural antimicrobial agent in food industry.