Antifungal activity of citral,octanal and α-terpineol against Geotrichum citri-aurantii

This study investigated the antifungal activity and potential antifungal mechanisms of three volatile compounds (i.e., citral, octanal, and α-terpineol) against Geotrichum citri-aurantii, one of the main postharvest pathogens in citrus. Results showed that the volatile compounds exhibited strong antifungal activity against the targeted pathogens, with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 0.50 μL/mL and 1.00 μL/mL for citral, 0.50 μL/mL and 2.00 μL/mL for octanal, and 2.00 μL/mL and 4.00 μL/mL for α-terpineol. The volatile compounds alter the morphology of G. citri-aurantii hyphae by causing loss of cytoplasm content and distortion of the mycelia. The membrane permeability of the G. citri-aurantii increased with increasing concentrations of the three volatile compounds, as evidenced by cell constituent release, extracellular conductivity, and pH. Moreover, the volatile compounds induced a decrease in the total lipid content of G. citri-aurantii cells, indicating the destruction of cell membrane structures. These results suggest that the antifungal activity of citral, octanal, and α-terpineol against G. citri-aurantii could be attributed to the disruption of cell membrane integrity and leakage of cell components.     

Chemical composition,antibacterial activity of Cyperus rotundus rhizomes essential oil against Staphylococcus aureus via membrane disruption and apoptosis pathway

The chemical composition, antibacterial activity and mechanism of essential oil from Cyperus rotundus rhizomes against Staphylococcus aureus were investigated in this study. Results showed that α-cyperone, cyperene and α-selinene were the major components of the essential oil. The essential oil exhibited strong antibacterial activity against S. aureus with the minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) were 10 and 20 mg/mL respectively, and the antibacterial effects increased with increasing essential oil concentrations and treatment time. The electric conductivity, cell membrane integrity, NPN uptake, and membrane potential assays demonstrated that essential oil disrupted the membrane integrity of S. aureus. Electron microscope observations further confirmed that essential oil destroyed cell membrane. Moreover, we found that essential oil could induce cells death of S. aureus through apoptosis pathway based on apoptosis analysis. These findings suggested that essential oil mainly exerted antibacterial activity by damaging cell membrane and membrane-mediated apoptosis pathway.     

An insight into the inhibitory activity of dihydromyricetin against Vibrio parahaemolyticus

The objective of this study is to investigate the antibacterial activity of dihydromyricetin (DMY) against Vibrio parahaemolyticus. The dilution method indicated that the minimum inhibitory concentration (MIC) of DMY against V. parahaemolyticus was at 0.625 mg/mL. The inhibitory effects of DMY against V. parahaemolyticus was further studied by analyzing cell morphology, cell injury, cell permeability, cell surface hydrophobicity (CSH) and antibacterial rate. The results showed bacterium cells are completely inactivated in a higher concentration (10 MIC). DMY treatment also lead to an increase in cell membrane permeability, cell injury as well as CSH. A good correlation between antibacterial rate and CSH was also observed. These findings indicated DMY could be used as a new alternative natural antibacterial agent for control pathogen growth in aquatic food.     

Inhibitory effect of nerol against Aspergillus niger on grapes through a membrane lesion mechanism

The antifungal activity of Nerol (NEL) was evaluated against Aspergillus niger, a known cause of grape spoilage, and possible modes of action were explored. The antifungal efficacy of NEL against A. niger has been proven in a dose-dependent manner through in vitro (mycelial growth and spore germination) and in vivo (grapes) tests. An obvious increase in the membrane permeability was detected through the determination of extracellular pH and conductivity. The damage in the cell membrane was measured by monitoring the influx of Propidium iodide (PI) using flow cytometry and was further verified through the inhibition of ergosterol synthesis in the cell membrane. NEL induced morphological changes, and the integrity of the A. niger fungal cells decreased with increasing NEL concentrations. A dose-dependent decrease in ergosterol production was also seen in A. niger cell membranes upon exposure to NEL. The mechanism-of-action study revealed that NEL activated a membrane-active mechanism that inhibits ergosterol synthesis and consequently compromised the membrane integrity of A. niger. As a result, the membrane permeability changes and causes cell death. The membrane lesion mechanism makes NEL a natural alternative to commercial fungicide for control of stored grape spoilage.     

Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes

Bifidocin A, produced by Bifidobacterium animalis BB04, is a novel bacteriocin with antimicrobial activity against a wide range of gram-positive and gram-negative foodborne bacteria. The objective of this study was to investigate the antibacterial activity and mechanism of action of bifidocin A against Listeria monocytogenes, one of the most susceptible bacteria to this bacteriocin. The minimum inhibitory concentration (MIC) of bifidocin A for L. monocytogenes 35152 was 0.029 mg/mL. Time-kill assays showed that bifidocin A effectively inhibited the growth of L. monocytogenes in a time-and concentration-dependent manner. The mechanism of action of bifidocin A was studied by analyzing its effects at a MIC on the cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of L. monocytogenes. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of L. monocytogenes cells. Confocal laser scanning microscopy images showed that L. monocytogenes cells treated with bifidocin A took up propidium iodide. Bifidocin A treatment also induced the leakage of K⁺ and inorganic phosphate, the hydrolysis and release of ATP, and a collapse of the transmembrane electrical potential and pH gradient in L. monocytogenes cells. These results suggested that bifidocin A exerted its anti-Listeria monocytogenes effect through the dissipation of the cytoplasmic membrane PMF, increased membrane permeability, cell membrane pore formation, destruction of membrane integrity, and ultimately complete disintegration of the cells.     

Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus

Cinnamon essential oil (EO) exhibited effective antibacterial activity against foodborne spoilage and pathogenic bacteria in model systems using Escherichia coli and Staphylococcus. The minimum inhibition concentration (MIC) of cinnamon EO was similar for both bacteria (1.0 mg/ml) while the minimum bactericide concentration (MBC) were 4.0 mg/ml and 2.0 mg/ml for E. coli and Staphylococcus aureus. GC–MS analysis confirmed that cinnamaldehyde was the major constituent in cinnamon EO (92.40%). Much effort was focused on elucidating the mechanism of antibacterial action of cinnamon EO against E. coli and S. aureus by observing the changes of cell microstructure using scanning electron microscope, determination of cell permeability, membrane integrity and membrane potential. After adding cinnamon EO at MIC level, there were obvious changes in the morphology of bacteria cells indicating cell damage. When cinnamon EO were added at MBC levels, the cells were destroyed. Cinnamon EO led to leakage of small electrolytes, causing rapid increase in the electric conductivity of samples at the first few hours. The values for E. coli and S. aureus reached 60% and 79.4% respectively at 7 h. Moreover, the concentration of proteins and nucleic acids in cell suspension also rose with increased cinnamon EO. Bacterial metabolic activity was decreased 3–5 folds as reflected by the results of membrane potential. Overall, S. aureus was more susceptible to cinnamon EO than E. coli.     

Antibacterial mechanism of bifidocin A,a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04

Bifidocin A, a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04, was isolated from the feces of a healthy centenarian. To understand the mechanism of the antibacterial action of bifidocin A against gram-negative bacteria, its effects at a minimum inhibitory concentration on cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of Escherichia coli 1.90 were investigated. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of E. coli cells. The intracellular organization was more susceptible to changes induced by bifidocin A than the morphology. Bifidocin A treatment caused the leakage of K⁺ and inorganic phosphate, the release of ATP and UV-absorbing materials, and a collapse of the transmembrane electrical potential and pH gradient in E. coli cells. Confocal laser scanning microscopy images showed that E. coli cells treated with bifidocin A took up propidium iodide. These results suggested that the mechanism of action bifidocin A against E. coli involved dissipation of the PMF of the cytoplasmic membrane, an increase in membrane permeability, pore formation in the cell membrane, a change in membrane integrity, and complete cell disintegration.     

Combination effects of ultrasound and citral nanoemulsion against Shigella flexneri and the preservation effect on fresh-cut carrots

This present study evaluated the effect of ultrasound (US), citral nanoemulsion (CLON) and combined treatments on the inactivate effectiveness of Shigella flexneri in phosphate-buffered saline (PBS) and on the surface of fresh-cut carrots. In this study, CLON was produced by ultrasonic nanoemulsification. Results demonstrated that US + CLON treatment significantly (P < 0.05) improved the bactericidal effect against Sh. flexneri, especially 0.15 mg/mL of CLON combined with US (20 kHz, 345 W/cm²) treatment for 9 min decreased the number of Sh. flexneri by 8.55 log CFU/mL. The assay of reactive oxygen species (ROS) and malondialdehyde (MDA) levels indicated that US + CLON aggravated oxidative stress and lipid peroxidation in cell membranes. The leakage of cell constituents (proteins and nucleotide) demonstrated that the permeability and integrity of the cell membrane was significantly (P < 0.05) impaired. Field-emission scanning electron microscopy further showed that US + CLON exacerbates the disruption of the bacteria morphology. Confocal laser scanning microscopic and flow cytometry observed US + CLON treatment enhanced the changes of bacterial cell membrane integrity. Meanwhile, US + CLON was effective in reducing the presence of bacteria on the surface of fresh-cut carrots, and had no negative effects on the quality of fresh-cut carrots in terms of colour, hardness and weight during storage. Overall, this present study revealed ultrasound combined with CLON is a promising and viable cleaning method for improving the microbiological safety of fresh produce.     

Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane

Gram-negative Escherichia coli (ATCC 8735) and Gram-positive Staphylococcus aureus (ATCC 3101) were selected as model bacteria to determine the antimicrobial mechanism of cinnamaldehyde. Several techniques were utilized to investigate the effects of cinnamaldehyde on food-borne bacterial membranes. The ultraviolet (UV) absorption and electrical conductivity of the culture supernatant were used to determine membrane integrity. β-Galactosidase activity was determined to detect inner membrane permeability. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to observe bacterial morphology. Samples from both strains exposed to cinnamaldehyde showed higher UV absorptions, conductivity values, and β-Galactosidase activities compared with the control group and displayed a rapid rise trend. Thereafter, the values stabilized at a relatively steady state. SEM shows that treated E. coli and S. aureus cell samples exhibited rough cell membranes with particulate matter, and some of the S. aureus cells split due to deep wrinkle formation and distortion, unlike the control group. TEM shows that the bacteria treated with cinnamaldehyde exhibited numerous abnormalities, including cytoplasmic membrane separation from the cell wall, cell wall and cell membrane lysis, cytoplasmic content leakage, cytoplasmic content polarization, cell distortion, and cytoplasmic content condensation. These results indicate that bacterial cell morphology, membrane integrity, and permeability are damaged when the E. coli and S. aureus cells are exposed to the minimum inhibitory concentrations of cinnamaldehyde (0.31 mg/mL). In addition, the higher the cinnamaldehyde concentration, the more serious the bacterial membrane damage is.     

Growth inhibition and morphological alterations of Fusarium verticillioides by cinnamon oil and cinnamaldehyde

Fusarium verticillioides is a filamentous fungus and a widely distributed pathogen having the ability to infect and cause destruction in economically important crops and grains by producing fumonisin mycotoxins. In the present study, the inhibitory effect of cinnamon, citral, Litsea cubeba oil, clove, eucalyptus, anise, spearmint and camphor oils on F. verticillioides was investigated, and cinnamon oil proves to be the most effective in inhibition. The antifungal effect of cinnamon oil was studied with special reference to its mechanism of inhibition of F. verticillioides growth at the morphological and ultrastructural levels. For F. verticillioides, the minimal inhibitory concentrations (MICs) of cinnamon oil (85% cinnamaldehyde), natural cinnamaldehyde (95%), and synthetic cinnamaldehyde (99%) were 60, 50, and 45 μL/L, respectively. The antifungal activity of cinnamon oil was proportional to its cinnamaldehyde concentration. Scanning electron microscopy and transmission electron microscopy of F. verticillioides exposed to MIC of cinnamaldehyde showed irreversible deleterious morphological and ultrastructural alterations, such as lack of cytoplasmic contents, loss of integrity and rigidity of the cell wall, plasma membrane disruption, mitochondrial destruction, folding of the cell. These modifications induced by cinnamaldehyde may be due to its interference with enzymatic reactions of cell wall synthesis, thus affecting the morphogenesis and growth of the fungus. These results further emphasized the toxicity of cinnamon oil against F. verticillioides attacking grains, and that cinnamon oil could be safely used as an alternative to chemical fungicides during grain storage and in the field.     

Application of ultraviolet-C light on oranges for the inactivation of postharvest wound pathogens

Germicidal effects of ultraviolet-C (UV-C) light on the postharvest wound pathogens of citrus fruits namely Penicillium digitatum and Penicillium italicum were investigated. P. digitatum and P. italicum spores were inoculated (4.00–4.50 log cfu/orange) onto Washington navel oranges (Citrus sinensis L. Osbeck cv Washington navel) by using wound and spot inoculation methods and subjected to eight different UV-C doses in the range of 0.26–15.84 kJ/m². Maximum reductions of 2.75 and 3.33 log cfu/orange of P. digitatum were obtained at the UV-C dose of 3.17 kJ/m² for spot and wound inoculation methods, respectively. P. italicum was more resistant than P. digitatum to UV-C treatments. The results suggest that UV-C treatments designed to reduce P. italicum spores will provide an adequate degree of protection against P. digitatum spores. UV-C light could be an alternative technique for the use of synthetic chemicals to reduce the development of postharvest pathogens of oranges.     

Inhibitory effects of a novel antimicrobial peptide from kefir against Escherichia coli

Antimicrobial peptide F1, a novel antimicrobial peptide from Tibetan kefir, have shown strong antimicrobial activity against several bacteria and fungi. We identified the amino acid sequence and studied the antimicrobial mechanism of peptide F1 against Escherichia coli. Our results showed that antimicrobial peptide F1 contained 18 amino acids (Thr-DAP-Asn-Thr-PEA-His-Pro-Asn-Thr-His-Leu-Ile- PEA-CySH-Val-Asn-PEA-Tau), which increased the outer and inner membrane permeability of E. coli, and the leakage of the cytoplasmic β-galactosidase and potassium ions was detected in the process. Morphologies of E. coli were observed by confocal laser scanning microscopy and transmission electron microscopy, which visually showed that antimicrobial peptide F1 could penetrate and accumulate into cell causing disruption of cell membrane functions. Furthermore, we elucidated the DNA binding ability of antimicrobial peptide F1 by agarose gel retardation and atomic force microscopy. Our findings indicated that antimicrobial peptide F1 has multiple targets in the killing of E. coli.     

Combined effect of carvacrol and citral on the growth of Listeria monocytogenes and Listeria innocua and on the occurrence of damaged cells

The aim of this study was to evaluate the growth kinetics of Listeria innocua Serovar 6a (CECT 910) and Listeria monocytogenes Serovar 4b (CECT 4032) exposed to combinations of carvacrol and citral (0.0 μL/mL (control), 0.050 μL/mL of carvacrol and 0.075 μL/mL of citral, 0.050 μL/mL of carvacrol and 0.125 μL/mL of citral, 0.085 μL/mL of carvacrol and 0.075 μL/mL of citral, and 0.085 μL/mL of carvacrol and 0.125 μL/mL of citral), with two initial inoculum concentrations, and also the occurrence of sublethal damage in these cell populations. The terpene combinations exhibited antibacterial activity against L. innocua and L. monocytogenes and the effects were dependent on the concentration of terpenes present in the culture medium (p ≤ 0.05). When terpene-treated L. innocua and L. monocytogenes were incubated in TSB, significant differences in lag phase and growth rate were observed between low and high inoculum concentrations (p ≤ 0.05), indicating that the inoculum level should be taken into account in modeling studies. When bacterial cells were exposed to terpenes the proportion of sublethally injured cells increased with the increase in the terpene dose (p ≤ 0.05). In conclusion, all of these results show that carvacrol and citral can be used in combination at 25% of the MIC in order to control Listeria growth.     

Chemical composition,antibacterial activity and mechanism of action of essential oil from seeds of fennel (Foeniculum vulgare Mill.)

Fennel (Foeniculum vulgare Mill.) is widely cultivated and used as a culinary spice. In this work, the chemical composition of the essential oil obtained by hydrodistillation of fennel seeds was analyzed by gas chromatography-mass spectrometry (GC–MS), and 28 components were identified. Trans-anethole (68.53%) and estragole (10.42%) were found to be the major components. The antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericide concentration (MBC) of essential oil against several food-borne pathogens were evaluated. The results showed that the gram positive and gram negative strains of bacteria had different sensitivities to essential oil of fennel seeds, the essential oil exhibited antibacterial activity against Staphylococcus albus, Bacillus subtilis, Salmonella typhimurium, Shigella dysenteriae and Escherichia coli according to the results of MIC and MBC. Among these bacteria, S. dysenteriae was the most sensitive to essential oil, showing the lowest MIC and MBC values of 0.125 and 0.25 mg/mL respectively. In addition, kill-time assay also showed that the essential oil had a significant effect on the growth rate of surviving S. dysenteriae. We concluded that the mechanism of action of the essential oil against S. dysenteriae might be described as essential oil acting on membrane integrity according to the results of the leakage of electrolytes, the losses of contents (proteins, reducing sugars and 260 nm absorbing materials) assays and electron microscopy observation.     

Inactivation by 405 ± 5 nm light emitting diode on Escherichia coli O157:H7, Salmonella Typhimurium,and Shigella sonnei under refrigerated condition might be due to the loss of membrane integrity

The objective of this study was to evaluate the antibacterial effect of 405 ± 5 nm light emitting diode (LED) on Escherichia coli O157:H7, Salmonella Typhimurium and Shigella sonnei. Its antibacterial mechanism was also investigated by determining the permeability of bacterial membrane and DNA degradation. Bacterial strains in phosphate-buffered saline were exposed to 405 ± 5 nm LED to a final dose of 486 J/cm² (7.5 h) at 4 °C. The inactivation curves were fitted by Weibull model to compare the sensitivities of pathogens to the LED illumination by calculating the decimal reduction times (t_R). The bacterial sensitivity to bile salts and NaCl by LED illumination was also determined. LIVE/DEAD^® BacLight™ staining as well as comet assay and DNA ladder analysis were carried out to determine the bacterial membrane integrity and DNA degradation, respectively. Results showed that LED illumination inactivated 1.0, 2.0, and 0.8 log CFU/ml for E. coli O157:H7, S. Typhimurium, and S. sonnei for 7.5 h, respectively. The comparison of t_R values demonstrated that S. Typhimurium was found to be the most (P < 0.05) susceptible strain to LED illumination. Regardless of the bacterial strain, the sensitivity of illuminated bacterial cells to bile salts and NaCl considerably increased compared to non-illuminated controls. Furthermore, LIVE/DEAD^® assay clearly showed that LED illumination resulted in loss of bacterial membrane permeability. On the other hand, no DNA degradation was observed by both comet assay and DNA ladder analysis. Therefore, these results suggest that the antibacterial effect of 405 ± 5 nm LED might be partly attributed to the physical damage to bacterial cell membrane. This study proposes that 405 ± 5 nm LED under refrigerated conditions may be effective to control the pathogens on foods.     

Combination treatment of ohmic heating with various essential oil components for inactivation of food-borne pathogens in buffered peptone water and salsa

Consumer preference for minimally processed foods has steadily increased for several years, while foodborne outbreaks from under-processed foods continue to be reported worldwide. We investigated the combination effect of ohmic heating with various essential oil components for inactivation of foodborne pathogens in buffered peptone water and salsa. We choose carvone, eugenol, thymol, and citral to combine with ohmic heating, which are registered for use as flavorings in foodstuffs. Combination treatment of ohmic heating with citral showed the most synergistic bactericidal effect against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in buffered peptone water followed by thymol, eugenol, and carvone. When enumerated on selective media, the reductions were 4.8, 5.7, and 4.3 log CFU/ml for E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively. Cell membrane destruction by combination treatment and the loss of cell membrane potential by essential oil components were proposed as the bactericidal mechanism. When applied in salsa, inactivation of bacterial pathogens was the greatest with the ohmic and thymol combination treatment followed by citral, eugenol, and carvone. A synergistic virucidal effect was observed for MS -2 bacteriophage, which was used as a norovirus surrogate. Color (b* values) of salsa were improved by combination treatment of ohmic heating and thymol compared to ohmic treated samples. Therefore, the combination treatment of ohmic heating and thymol could be used effectively to pasteurize salsa.     

Evaluation of monolaurin from camphor tree seeds for controlling food spoilage fungi

The preservative ability between monolaurin and potassium sorbate or sodium benzoate against Saccharomyces cerevisiae, Aspergillus niger and Penicillium glaucum was compared to determine the potential use of monolaurin as a novel food preservative. The mode of action for monolaurin has also been investigated. The minimum inhibitory concentrations (MIC) of monolaurin against S. cerevisiae, A. niger and P. glaucum were determined to be 0.32, 0.32 and 0.16 mg/mL, respectively, and the minimum bactericidal concentrations (MBC) were 1.25, 2.50 and 0.63 mg/mL, respectively, and monolaurin began to degrade in the cultures at 100 h, 3rd day and 2nd day, respectively. The results showed that monolaurin had better inhibition abilities against these fungi than potassium sorbate and sodium benzoate. Furthermore, unlike potassium sorbate or sodium benzoate, monolaurin was independent of pH. The exposure of S. cerevisiae cells, A. niger and P. glaucum spores to monolaurin at MIC resulted in the release of intercellular ions, nucleic acids and proteins with molecular weights of 15–100, 15–70 and 35–70 kDa, respectively. Monolaurin was found to be incorporated into the membrane of bacteria, leading to the changes in its permeability and fluidity. In addition, the mode of monolaurin's action may involve inhibition of cell respiration.     

Carvacrol as potential quorum sensing inhibitor of Pseudomonas aeruginosa and biofilm production on stainless steel surfaces

Pseudomonas aeruginosa biofilm confers resistance to antibiotics and biocides; therefore, it represents a problem to clinical and industrial settings. This bacterial organization is controlled by quorum sensing (QS), which depends of autoinducer molecules, e.g. acyl-homoserine-lactones that regulate production on virulence factors as pyocyanin. As a solution to this problem, carvacrol, present in most of the antibacterial essential oils could be a potential agent to inhibit QS for its ability to interact with cell membrane and protein receptors involved in biofilm formation. Therefore, this work evaluated the effect of carvacrol on pyocyanin production and biofilm formation of P. aeruginosa. Minimal inhibitory concentration (MIC) of carvacrol against planktonic P. aeruginosa was 7.9 mM; in addition, carvacrol was tested against Chromobacterium violaceum as model for anti-QS agents, showing a MIC of 0.7 mM. Lower concentrations of carvacrol to observed MICs were applied to observe changes in QS activity and biofilm production to avoid effect of cell death on mentioned parameters. Carvacrol inhibited P. aeruginosa biofilms (1.5–3 Log CFU/cm²) at 0.9–7.9 mM, compared to non-treated bacteria on stainless steel surface. Pyocyanin production by P. aeruginosa was reduced up to 60% at 3.9 mM of carvacrol. Higher doses of carvacrol affected P. aeruginosa viability. Similar results were obtained for violacein production that is related to QS of C. violaceum, where carvacrol reduced up to 50% at 0.7 mM without affecting cell viability. These results showed that the inhibition of QS could be related with reduction of bacterial virulence and biofilm formation on stainless steel surfaces exposed to carvacrol.     

Antifungal action and inhibitory mechanism of polymethoxylated flavones from Citrus reticulata Blanco peel against Aspergillus niger

Polymethoxylated flavones (PMFs), isolated from the peels of Citrus reticulata Blanco, were identified and quantitated as tangeretin (TAN) (33.87%), nobiletin (NOB) (20.98%), 5-demethylnobiletin (3.52%), tetramethyl-o-scutellarein (1.61%), tetramethyl-o-isoscutellarein (1.23%), pentamethoxyflavone (1.08%) and sinensetin (0.35%). PMFs are promising natural antimicrobial compounds with potential applications in the food industry. The antifungal effects of PMFs on Aspergillus niger (A. niger) were evaluated by microbroth dilution assay and growth curve determination. The minimum inhibition concentration (MIC) of PMFs extract and of TAN against A. niger was determined to be 0.12 mg/mL and 1.5 mg/mL respectively. PMFs affected the permeability of cytomembrane, resulting in instant increased flux of K⁺ and increased relative electrical conductivity. PMFs also dose-dependently reduced the chitin production. These results suggest that the antifungal effects of PMFs could be explained by the permeability change of cytomembrane and the fragility of cell walls caused by chitin inhibition.