| It is well known that microbial contamination can cause food spoilage and nutrient losses,which even endanger people's health.Synthetic preservatives are widely applied to prevent the growth of the pathogen and to extend the shelf life of food.However,some people have expressed strong concerns about potential risks to human health posed by these substances.In addition,an increasing number of consumers prefer natural or “green” preservation techniques that both protect against the growth of food-borne pathogens and retain nutritional quality.In recent years,the pulsed electric field(PEF),one of the most widely nonthermal techniques,has attracted much attention for its effective sterilization and maximal retaining food nutrition.The inactivation of microorganism induced by PEF was mainly via penetrating or destroying the cytomembrane.However,the specific sterilization mechanism is still unclear,especially in the relationship between the membrane fatty acid composition and PEF sterilization.In order to obtain a satisfactory sterilization effect,it is often necessary to increase the field strength and treatment time,but in this condition,it will increase the equipment load and energy consumption,and even lead to thermal effect in samples due to increasing temperature.Therefore,various synergistic treatments with PEF have been a research hotspot.Naringenin is a bioactive flavonoid compound found in citrus plants,exhibits potentially exploitable activities.However,few studies have been reported evaluating the antibacterial mechanism of naringenin,and its mode of action remains not been fully understood.Based on these considerations,the aim of this work was to study the antibacterial behavior of naringenin,and used it as a stress factor to explore the relationship between changes in physicochemical properties including fluidity and membrane fatty acid composition,etc.with the resistance of Escherichia coli and Staphylococcus aureus to PEF treatment.Moreover,the combined sterilization effect of naringenin with PEF was investigated to further explore their synergistic mechanism.This work is expected to provide a theoretical basis for the looking for a new PEF combined technology and a method for the modification of microbial cell membranes,which has important theoretical significance and application value.It is of great theoretical significance and application value.Specific research results are as follows:The growth of Escherichia coli and Staphylococcus aureus cells was inhibited by naringenin in low concentrations.Combination of gas chromatography–mass spectrometry with fluorescence polarization analysis revealed that Escherichia coli and Staphylococcus aureus cells increased membrane fluidity by altering the composition of membrane fatty acids after exposure to naringenin.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 presence of naringenin.For Staphylococcus aureus cells,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 naringenin 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.Moreover,naringenin can bind directly to cell membrane proteins of Escherichia coli and Staphylococcus aureus resulting in a significant change in membrane protein conformation,caused perturbations in the conformation of membrane proteins through changing the microenvironment of phenylalanine,tyrosine,and tryptophan residues.Additionally,it was found that naringenin can cause these two bacteria to depolarize,to reduce their membrane potential and increase the cell membrane permeability,which resulted in intracellular material leaked,and underwent morphological changes such as cell surface shrinkage,collapse,and even cell lysis.The genomic DNA-binding of naringenin were quantitatively monitored using Uv-vis spectra with the combination of multivariate curve resolution--alternating least(MCR-ALS)analysis,and the concentration and pure spectra profiles for the three-reaction species(DNA,naringenin and DNA-naringenin)were obtained.Moreover,thermal behavior of DNA and docking studies revealed that naringenin preferentially bound to the A-T base pairs region of genomic DNA via groove mode,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 exerts its antibacterial effects might be connected with disruption of the cytoplasmic membrane and DNA targeting effects of Staphylococcus aureus.This work was aimed to investigate the stress induced by naringenin,one of the natural antibacterial components in citrus plants on the resistance of Escherichia coli and Staphylococcus aureus cells to pulsed electric fields(PEF).Results revealed that Escherichia coli and Staphylococcus aureus subjecting to naringenin shock(2.0 h)did not significantly change the resistance to the subsequent PEF treatment.However,naringenin adaptation of Staphylococcus aureus and Escherichia coli cells decreased their resistance by exhibiting higher inactivation levels with more damage in cell membrane integrity and more morphological change after being exposed to PEF treatment.This difference in response to PEF treatment for Escherichia coli and Staphylococcus aureus might be due to the difference in the alterations of membrane fatty acids during preliminary naringenin exposure.Escherichia coli cells caused a significant increase of unsaturated fatty acids at the expense of saturated and cyclic fatty acids,whereas Staphylococcus aureus adapted to naringenin remarkably increased the content of anteiso-branched chain fatty acids at the same time decreased the levels of straight and iso-branched chain fatty acids.Comparatively,no significant changes were observed in the membrane fatty acid composition of cells after being subjected to naringenin shock.The combination of naringenin(0 ~ 0.2 g/L)and PEF on Escherichia coli and Staphylococcus aureus in orange juice,grape juice and lychee juice were studied.Results have shown that naringenin has a mild inhibitory effect on bacteria in these three kinds of juice at 0.05 to 0.2 g/L.The inactivation effect of PEF on Escherichia coli and Staphylococcus aureus has a certain correlation with its pH value(grape juice < lychee juice < orange juice),among them,grape juice has the highest degree of bacterial inactivation by PEF,following by lychee and orange juice.The combination of naringenin and PEF produced a certain synergistic effect on Escherichia coli and Staphylococcus aureus in orange juice,grape juice and lychee juice.Moreover,the synergistic effect was more obvious with the higher concentration of naringenin.Additionally,it was found that the bacterial growth in naringenin-PEF-treated juices were inhibited compared to the control,naringenin-treated,and PEF-treated juices. |
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