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.

Temperature-mediated variations in cellular membrane fatty acid composition of Staphylococcus aureus in resistance to pulsed electric fields.

Effects of growth temperature on cell membrane fatty acid composition, fluidity and lethal and sublethal injury by pulsed electric fields (PEF) in Staphylococcus aureus ATCC 43300 (S. aureus) in the stationary phase were investigated. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) revealed that branched chain fatty acids (iso C14:0, iso C15:0, anteiso C15:0 and anteiso C17:0) and straight chain fatty acids (C12:0, C14:0, C16:0, C17:0 and C18:0) were primary constituents in the membrane. The S. aureus changed its membrane fatty acid composition and its overall fluidity when exposed to different temperatures. The PEF lethal and sublethal effects were assessed, and results suggested that the degree of inactivation depended on the cell membrane structure, electric field strength and treatment time. The PEF inactivation kinetics including lethal and sublethal injury fractions were fitted with non-linear Weibull distribution, suggesting that inactivation of the first log cycle of S. aureus population was significantly affected by growth temperature, and the membrane of cells became more fluid, and easier to induce electroportion in low temperatures. Moreover, the morphology of S. aureus cells were investigated by electron microscopy, showing that various temperature-modified cells were distorted to differing extents and some even collapsed due to deep irreversible electroporation after PEF treatment.

An in vitro investigation of the inhibitory mechanism of ß-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, ß-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 ß-galactosidase by competitive inhibition through a multiphase kinetic process. 3-D spectroscopy and circular dichroism showed that the binding of CIN to ß-galactosidase resulted in changes in micro-environment of tryptophan and tyrosine residues, and conformation of ß-galactosidase. The molecular recognition was also analyzed through modeling which indicated that CIN was inserted into the active site pocket of ß-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 ß-galactosidase occurred after binding with CIN, e.g., morphological changes and increased dimensions of the ß-galactosidase molecule. Moreover, it was found that the combinations of CIN, carvacrol and thymol exposure displayed synergistic effects on the inhibition of ß-galactosidase. GENERAL SIGNIFICANCE: This study exhibits a comprehensively understanding about the action mechanism of CIN that affects the conformation and activity of ß-galactosidase in biochemical processes and provides some new insights into the possible intracellular targeting behaviors of CIN at a molecular level.

Combination of microbiological, spectroscopic and molecular docking techniques to study the antibacterial mechanism of thymol against Staphylococcus aureus: membrane damage and genomic DNA binding.

Thymol (2-isopropyl-5-methylphenol) is a natural ingredient used as flavor or preservative agent in food products. The antibacterial mechanism of thymol against Gram-positive, Staphylococcus aureus was investigated in this work. A total of 15 membrane fatty acids were identified in S. aureus cells by gas chromatography-mass spectrometry. Exposure to thymol at low concentrations induced obvious alterations in membrane fatty acid composition, such as decreasing the proportion of branched 12-methyltetradecanoic acid and 14-methylhexadecanoic acid (from 22.4 and 17.3% to 7.9 and 10.3%, respectively). Membrane permeability assay and morphological image showed that thymol at higher concentrations disrupted S. aureus cell membrane integrity, which may decrease cell viability. Moreover, the interaction of thymol with genomic DNA was also investigated using multi-spectroscopic techniques, docking and atomic force microscopy. The results indicated that thymol bound to the minor groove of DNA with binding constant (K a) value of (1.22 ± 0.14) × 104 M-1, and this binding interaction induced a mild destabilization in the DNA secondary structure, and made DNA molecules to be aggregated. Graphical Abstract Thymol exerts its antibacterial effect throught destruction of bacterial cell membrane and binding directly to genomic DNA.

Improvement of betalains stability extracted from red dragon fruit peel by ultrasound-assisted microencapsulation with maltodextrin.

Natural betalains can be potential food additives because of their antioxidant activities, but they have poor thermal stability. In this study, betalains were extracted from red dragon fruit peel, and then encapsulated with maltodextrin by ultrasound method to increase the physicochemical properties of betalains microcapsules. The encapsulation efficiency of the betalains was above 79%, and the particle size and Zeta potential values were 275.46 nm and -29.01 mV, respectively. Compared to the control sample, onset temperature and DPPH free radical scavenging of betalains microcapsules under the modest ultrasound treatment (200 W, 5 min) was increased by 1.6 °C and 12.24%, respectively. This increase could be due to the ability of ultrasonification to create interactions between maltodextrin and betalains (as evidenced by FT-IR). Therefore, modest ultrasound treatment can be used for microcapsulation to improve the stability of betalains, and then expand the application of betalains in heat processed food field.

Review of the application of pulsed electric fields (PEF) technology for food processing in China.

With the improvement of living standards, growing consumer demand for high-quality and natural foods has led to the development of new mild processes to enhance or replace conventional thermal and chemical methods for food processing. Pulsed electric fields (PEF) is an emerging and promising non-thermal food processing technology, which is ongoing from laboratory and pilot plant level to the industrial level. Chinese researchers have made tremendous advances in the potential applications of PEF for processing a wide range of food commodities over the last few years, which contributes to the current understanding and development of PEF technology. The objective of this paper is to conduct a systematic review on the achievements of PEF technology used for food processing in China and the corresponding processing principles. Research on the applicability of PEF in food processing suggests that PEF can be used alone or in combination with other methods, not only to inactivate microorganisms and extract active constituents, but also to modify biomacromolecules, enhance chemical reactions and accelerate the aging of fermented foods, which are mainly related to permeabilization of biomembranes, occurrence of electrochemical and electrolytic reactions, polarization and realignment of molecules, and reduction of activation energy of chemical reactions induced by PEF treatments. In addition, some of the most important challenges for the successful implementation of large-scale industrial applications of PEF technology in the food industry are discussed. The results bring out the benefits of both researchers and the industry.

Hydroxyl-related differences for three dietary flavonoids as inhibitors of human purine nucleoside phosphorylase.

In this work, the hydroxyl-related differences of binding properties and inhibitory activities of dietary flavonoids, namely chrysin, baicalein and apigenin against purine nucleoside phosphorylase (PNP) were investigated. It was found that the hydroxylation on position C4' of chrysin (→apigenin) mildly decreased the binding affinities for PNP, whereas on the position C6 of chrysin (→baicalein) significantly increased binding affinities. Comparatively, the hydroxylation on position C4' and C6 greatly improved their PNP inhibitory effects. The IC50 values of apigenin and baicalein were 6.09 × 10-5 M and 8.94 × 10-5 M, respectively, which is significantly lower than that of chrysin (2.13 × 10-4 M). Results from molecular modeling revealed that there are two binding sites, i.e. active site (major) and tryptophan site (minor) on PNP, and the binding of these flavonoids might induce a serious conformational destabilization of PNP as a result of altering the micro-environment and morphology by flavonoids.

The preparation of Fe-glycine complexes by a novel method (pulsed electric fields).

A novel method for synthesizing Fe-glycine complex by pulsed electric fields (PEFs) was developed, and the physiochemical properties of Fe-glycine complexes were evaluated in this study. Results showed that the highest yield (81.2%) of Fe-glycine complexes was obtained by PEF treatment with 4.0kV/cm for 15min at 25°C, which was higher than thermal treatment of 79.5% at 60°C for 30min. Moreover, the highest Fe-chelating capacity was obtained at 107.13mg/L after PEF treatment with 4.0kV/cm for 15min. Results from Fourier transform infrared (FT-IR), X-ray diffraction (XRD) and thermal gravimetric (TG) analysis indicated that the structure of the ferrous ion-glycine complexes prepared by PEF and thermal treatment was similar. It was implied that the PEF treatment can be used as a novel and efficient method for synthesis of metal ion-amino acid complexes under moderate condition such as 25°C and pH 6.0.

Membrane and genomic DNA dual-targeting of citrus flavonoid naringenin against Staphylococcus aureus.

The antimicrobial mechanism of naringenin, one of the citrus antibacterial flavonoids against food-borne Staphylococcus aureus ATCC 6538, was investigated in this study. Analysis of gas chromatography-mass spectrometry (GC-MS) and fluorescence showed that relatively low concentrations of naringenin caused perturbations in the membrane fatty acid composition and the conformation of membrane proteins through changing the microenvironment of phenylalanine, tyrosine, and tryptophan residues. Exposure of naringenin at higher levels significantly increased membrane permeability and changed the morphology of S. aureus cells. The genomic DNA-binding of naringenin was also quantitatively monitored using UV-vis spectra in combination with multivariate curve resolution-alternating least squares (MCR-ALS) analysis, and the concentration and pure spectra profiles for the three reaction species (DNA, naringenin, and DNA-naringenin) were obtained. Moreover, the thermal behavior of DNA and docking studies revealed that naringenin preferentially bound to the A-T base pair regions of genomic DNA via groove binding, and atomic force microscopy and circular dichroism showed that naringenin induced mild secondary structure and obvious morphological variations of this biomacromolecule. These results suggested that naringenin exerting its antibacterial effects might be connected with disruption of the cytoplasmic membrane and DNA targeting effects in Staphylococcus aureus.

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.

Self-assembly behaviors of thermal- and pH- sensitive magnetic nanocarriers for stimuli-triggered release.

In the present work, we prepare thermo- and pH-sensitive polymer-based nanoparticles incorporating with magnetic iron oxide as the remote-controlled, stimuli-response nanocarriers. Well-defined, dual functional tri-block copolymer poly[(acrylic acid)-block-(N-isopropylacrylamide)-block-(acrylic acid)], was synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization with S,S'-bis(α,α'-dimethyl-α″-acetic acid)trithiocarbonate (CMP) as a chain transfer agent (CTA). With the aid of using 3-aminopropyltriethoxysilane, the surface-modified iron oxides, Fe3O4-NH2, was then attached on the surface of self-assembled tri-block copolymer micelles via 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinamide (EDC/NHS) crosslinking method in order to furnish not only the magnetic resources for remote control but also the structure maintenance for spherical morphology of our nanocarriers. The nanocarrier was characterized by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR), and ultraviolet-visible (UV/Vis) spectral analysis. Rhodamine 6G (R6G), as the modeling drugs, was encapsulated into the magnetic nanocarriers by a simple swelling method for fluorescence-labeling and controlled release monitoring. Biocompatibility of the nanocarriers was studied via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed that neither the pristine nanocarrier nor the R6G-loaded nanocarriers were cytotoxic to the normal fibroblast cells (L-929 cells). The in vitro stimuli-triggered release measurement showed that the intelligent nanocarriers were highly sensitive to the change of pH value and temperature rising by the high-frequency magnetic field (HFMF) treatment, which provided the significant potential to apply this technology to biomedical therapy by stimuli-responsive controlled release.