Antimicrobial Activity of Eugenol Against Bacillus cereus and Its Application in Skim Milk.

Bacillus cereus is a foodborne pathogen widely distributed in the large-scale catering industry and produces spores. The study explored the antibacterial activity, potential mechanism of eugenol against B. cereus, and spores with germination rate. The minimum inhibitory concentration (MIC; 0.6 mg/mL) of eugenol to six B. cereus strains was compared with the control; B. cereus treated with eugenol had a longer lag phase. Eugenol at a concentration of more than 1/2MIC decreased viable B. cereus (∼5.7 log colony-forming unit [CFU]/mL) counts below detectable limits within 2 h, and eugenol of 3MIC reduced B. cereus (∼5.9 log CFU/mL) in skim milk below detectable limits within 30 min. The pH values of skim milk were unaffected by the addition of eugenol. The ΔE values below 2 show that the color variations of skim milk were not visible to the human eye. For sensory evaluation, eugenol did not significantly affect the color or structural integrity of the skim milk. It had a negative impact on the flavor and general sensory acceptance of the treated milk. Eugenol hyperpolarized B. cereus cell membrane, decreased intracellular ATP concentration, and increased intracellular reactive oxygen species contents and extracellular malondialdehyde contents, resulting in the cell membrane of B. cereus being damaged and permeabilized, and cell morphology being changed. In addition, according to the viable count, confocal laser scanning microscopy, and spore morphology changes, eugenol reduced the germination rate of B. cereus spores. These findings suggest that eugenol can be used as a new natural antibacterial agent to control B. cereus and spores in the food production chain.

The biochemical characteristics of viable but nonculturable state Yersinia enterocolitica induced by lactic acid stress and its presence in food systems.

The viable but nonculturable (VBNC) state is adopted by many foodborne pathogenic bacteria to survive in adverse conditions. This study found that lactic acid, a widely used food preservative, can induce Yersinia enterocolitica to enter a VBNC state. Y. enterocolitica treated with 2 mg/mL lactic acid completely lost culturability within 20 min, and 10.137 ± 1.693 % of the cells entered a VBNC state. VBNC state cells could be recovered (resuscitated) in tryptic soy broth (TSB), 5 % (v/v) Tween80-TSB, and 2 mg/mL sodium pyruvate-TSB. In the VBNC state of Y. enterocolitica induced by lactic acid, the intracellular adenosine triphosphate (ATP) concentration and various enzyme activities were decreased, and the reactive oxygen species (ROS) level was elevated, compared with uninduced cells. The VBNC state cells were significantly more resistant to heat and simulated gastric fluid than uninduced cells, but their ability to survive in a high-osmotic-pressure environment was significantly less than that of uninduced cells. The VBNC state cells induced by lactic acid changed from long rod-like to short rod-like, with small vacuoles at the cell edges; the genetic material was loosened and the density of cytoplasm was increased. The VBNC state cells had decreased ability to adhere to and invade Caco-2 (human colorectal adenocarcinoma) cells. The transcription levels of genes related to adhesion, invasion, motility, and resistance to adverse environmental stress were downregulated in VBNC state cells relative to uninduced cells. In meat-based broth, all nine tested strains of Y. enterocolitica entered the VBNC state after lactic acid treatment; among these strains, only VBNC state cells of Y. enterocolitica CMCC 52207 and Isolate 36 could not be recovered. Therefore, this study is a wake-up call for food safety problems caused by VBNC state pathogens induced by lactic acid.

Synergistic antibacterial and anti-biofilm mechanisms of ultrasound combined with citral nanoemulsion against Staphylococcus aureus 29213.

This study investigated the antibacterial and antibiofilm mechanism of ultrasound (US) combined with citral nanoemulsion (CLNE) against Staphylococcus aureus and mature biofilm. Combined treatments resulted in greater reductions in bacterial numbers compared to ultrasound or CLNE treatments alone. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein nucleic acid leakage, and N-phenyl-l-naphthylamine (NPN) uptake analysis showed that the combined treatment disrupted cell membrane integrity and permeability. Reactive oxygen species (ROS) and malondialdehyde (MDA) assays indicated that US+CLNE exacerbated cellular oxidative stress and membrane lipid peroxidation. Field emission scanning electron microscopy (FESEM) revealed that the synergistic processing of ultrasound and CLNE resulted in cell rupture and collapse. In addition, US+CLNE showed a more pronounced removal effect than both alone in the biofilm on the stainless steel sheet. US+CLNE reduced biomass, the number of viable cells in the biofilm, cell viability and EPS polysaccharide contents. The results of CLSM also showed that US+CLNE disrupted the structure of the biofilm. This research elucidates the synergistic antibacterial and anti-biofilm mechanism of ultrasound combined citral nanoemulsion, which provides a safe and efficient sterilization method for the food industry.

Citral and trans-cinnamaldehyde, two plant-derived antimicrobial agents can induce Staphylococcus aureus into VBNC state with different characteristics.

Viable but nonculturable (VBNC) state bacteria are difficult to detect in the food industry due to their nonculturable nature and their recovery characteristics pose a potential threat to human health. The results of this study indicated that S. aureus was found to enter the VBNC state completely after induced by citral (1 and 2 mg/mL) for 2 h, and after induced by trans-cinnamaldehyde (0.5 and 1 mg/mL) for 1 h and 3 h, respectively. Except for VBNC state cells induced by 2 mg/mL citral, the VBNC state cells induced by the other three conditions (1 mg/mL citral, 0.5 and 1 mg/mL trans-cinnamaldehyde) were able to be resuscitated in TSB media. In the VBNC state cells induced by citral and trans-cinnamaldehyde, the ATP concentration was reduced, the hemolysin-producing ability was significantly decreased, but the intracellular ROS level was elevated. The results of heat and simulated gastric fluid experiments showed different environment resistance on VBNC state cells induced by citral and trans-cinnamaldehyde. In addition, by observing the VBNC state cells showed that irregular folds on the surface, increased electron density inside and vacuoles in the nuclear region. What's more, S. aureus was found to enter the VBNC state completely after induced by meat-based broth containing citral (1 and 2 mg/mL) for 7 h and 5 h, after induced by meat-based broth containing trans-cinnamaldehyde (0.5 and 1 mg/mL) for 8 h and 7 h. In summary, citral and trans-cinnamaldehyde can induce S. aureus into VBNC state and food industry needs to comprehensively evaluate the antibacterial capacity of these two plant-derived antimicrobial agents.

Synergistic bactericidal effect of ultrasound combined with citral nanoemulsion on Salmonella and its application in the preservation of purple kale.

In this study, a novel citral nanoemulsion (CLNE) was prepared by ultrasonic emulsification. The synergistic antibacterial mechanism of ultrasound combined with CLNE against Salmonella Typhimurium and the effect on the physicochemical properties of purple kale were investigated. The results showed that the combined treatment showed obviously inactivate effect of S. Typhimurium. Treatment with 0.3 mg/mL CLNE combined with US (20 kHz, 253 W/cm2) for 8 min reduced S. Typhimurium populations in phosphate-buffered saline (PBS) by 9.05 log CFU/mL. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein and nucleic acid release assays showed that the US combination CLNE disrupt the integrity of S. Typhimurium membranes. Reactive oxygen species (ROS) and malondialdehyde (MDA) detection indicated that US+CLNE exacerbated oxidative stress and lipid peroxidation in cell membranes. The morphological changes of cells after different treatments by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) illustrated that the synergistic effect of US+CLNE treatment changed the morphology and internal microstructure of the bacteriophage cells. Application of US+CLNE on purple kale leaves for 6 min significantly (P < 0.05) reduced the number of S. Typhimurium, but no changes in the physicochemical properties of the leaves were detected. This study elucidates the synergistic antibacterial mechanism of ultrasound combined with CLNE and provides a theoretical basis for its application in food sterilization.

Inhibition of Cronobacter sakazakii by Litsea cubeba Essential Oil and the Antibacterial Mechanism.

Litsea cubeba essential oil (LC-EO) has anti-insecticidal, antioxidant, and anticancer proper-ties; however, its antimicrobial activity toward Cronobacter sakazakii has not yet been researched extensively. The objective of this study was to investigate the antimicrobial and antibiofilm effects of LC-EO toward C. sakazakii, along with the underlying mechanisms. The minimum inhibitory concentrations of LC-EO toward eight different C. sakazakii strains ranged from 1.5 to 4.0 µL/mL, and LC-EO exposure showed a longer lag phase and lower specific growth compared to untreated bacteria. LC-EO increased reactive oxygen species production, decreased the integrity of the cell membrane, caused cell membrane depolarization, and decreased the ATP concentration in the cell, showing that LC-EO caused cellular damage associated with membrane permeability. LC-EO induced morphological changes in the cells. LC-EO inhibited C. sakazakii in reconstituted infant milk formula at 50 °C, and showed effective inactivation of C. sakazakii biofilms on stainless steel surfaces. Confocal laser scanning and attenuated total reflection-Fourier-transform infrared spectrometry indicated that the biofilms were disrupted by LC-EO. These findings suggest a potential for applying LC-EO in the prevention and control of C. sakazakii in the dairy industry as a natural antimicrobial and antibiofilm agent.

Antibacterial Activity of Thymoquinone Against Shigella flexneri and Its Effect on Biofilm Formation.

Thymoquinone (TQ) has been demonstrated to have anti-cancer, anti-inflammatory, antioxidant, and anti-diabetic activities. Shigella flexneri is the main pathogen causing shigellosis in developing countries. In this study, the antibacterial activity of TQ against S. flexneri and its possible antibacterial mechanism were studied. In addition, the inhibitory effect of TQ on the formation of S. flexneri biofilm was also investigated. The results showed that both the minimum inhibitory concentration and the minimum bactericidal concentration of TQ against S. flexneri ATCC 12022 were 0.2 mg/mL. After treatment with TQ at 0.4 mg/mL in Luria-Bertani broth for 3 h, or treatment with 0.2 mg/mL TQ in phosphate-buffered saline for 60 min, the number of S. flexneri (initial number is 6.5 log colony-forming units/mL) dropped below the detection limit. TQ also displayed good antibacterial activity in contaminated lettuce juice. TQ caused an increase in intracellular reactive oxygen species level, a decrease in intracellular adenosine triphosphate (ATP) concentration, a change in the intracellular protein, damage to cell membrane integrity and changes in cell morphology. In addition, TQ showed the ability to inhibit the formation of S. flexneri biofilm; treatment resulted in a decrease in the amount of biofilm and extracellular polysaccharides, and the destruction of biofilm structure. These findings indicated that TQ had strong antimicrobial and antibiofilm activities and a potential to be applied in the fruit and vegetable processing industry or other food industries to control S. flexneri.

Antibacterial Mechanism of Eugenol Against Shigella sonnei and Its Antibacterial Application in Lettuce Juice.

Shigella sonnei is a species of Shigella, and the infection rate of S. sonnei is increasing year by year. Eugenol is an active ingredient in clove essential oil and is a generally recognized as safe (GRAS)-certified food ingredient. The mechanism of inhibition of S. sonnei by eugenol has been investigated in this study. The minimum inhibitory concentration of eugenol against both S. sonnei ATCC 25931 and S. sonnei CMCC 51592 was 0.5 mg/mL and minimum bactericidal concentration (MBC) for both strains was 0.8 mg/mL. The inhibition effect of eugenol against S. sonnei was due to increased levels of reactive oxygen species in cells, changed cell membrane permeability, and induced cell membrane dysfunction, for instance, cell membrane hyperpolarization and intracellular ATP concentration drops. The results of confocal laser scanning microscope and field emission scanning electron microscopy showed that eugenol leads to decreased cell membrane integrity, resulting in changed cell morphology. Moreover, eugenol inactivated S. sonnei in Luria-Bertani (LB) broth and lettuce juice. These results indicated that eugenol could inactivate S. sonnei and has the potential to control S. sonnei in the food industry.

Inhibitory effect of protocatechualdehyde on Yersinia enterocolitica and its critical virulence factors.

Yersinia enterocolitica (Y. enterocolitica) is a gastrointestinal pathogen that is distributed worldwide, involved in systemic, extraintestinal and invasive infections in immunocompromised patients. Establishment of antibiotic resistance in the pathogen has produced a need for new antibacterial agents. The purpose of this study was to elucidate antibacterial mechanism of protocatechualdehyde (PCA) extracted from the roots of Salvia miltiorrhiza towards Y. enterocolitica, and to investigate effects of PCA on key virulence factors associated with human infection. Present results indicated that PCA exerted its antibacterial activity against Y. enterocolitica mainly by the rapid rise of intracellular reactive oxygen species, leading to change in permeability and integrity of cell membrane, and ultimately decline of membrane potential and intracellular ATP. Furthermore, scanning electron microscopic analysis revealed that Y. enterocolitica presented gradually shrinkage in length and partial wrinkles upon PCA treatment. PCA also effectively decreased motility, biofilm formation, quorum sensing in a dose-dependent manner without affecting bacterial growth. Further, at SICs, PCA substantially suppressed the adhesion and invasion of Y. enterocolitica to HT-29 cells and the downregulation of essential virulence factor-encoding genes unveiled impaired virulence. Overall, the findings revealed the potential of PCA as an alternative antibacterial agent to combat Y. enterocolitica contamination and infections.

The Antimicrobial and Antibiofilm Activity of Oregano Essential Oil against Enterococcus faecalis and Its Application in Chicken Breast.

Oregano essential oil (OEO) possesses anti-inflammatory, antioxidant, and cancer-suppressive properties. Enterococcus faecalis is a foodborne opportunistic pathogen that can be found in nature and the food processing industry. The goal of this investigation was to explore the antimicrobial action and mechanism of OEO against E. faecalis, inactivation action of OEO on E. faecalis in mature biofilms, and its application in chicken breast. The minimum inhibitory concentration (MIC) of OEO against E. faecalis strains (ATCC 29212 and nine isolates) ranged from 0.25 to 0.50 µL/mL. OEO therapy reduced intracellular adenosine triphosphate (ATP) levels, caused cell membrane hyperpolarization, increased the intracellular reactive oxygen species (ROS), and elevated extracellular malondialdehyde (MDA) concentrations. Furthermore, OEO treatment diminished cell membrane integrity and caused morphological alterations in the cells. In biofilms on stainless-steel, OEO showed effective inactivation activity against E. faecalis. OEO reduced the number of viable cells, cell viability and exopolysaccharides in the biofilm, as well as destroying its structure. Application of OEO on chicken breast results in a considerable reduction in E. faecalis counts and pH values, in comparison to control samples. These findings suggest that OEO could be utilized as a natural antibacterial preservative and could effectively control E. faecalis in food manufacturing.

Effects of cinnamaldehyde against planktonic bacteria and biofilm formation of Shigella flexneri.

Cinnamaldehyde (CA) has demonstrated anti-inflammatory, anti-tumor and anti-cancer activities; Its antimicrobial and antibiofilm actions against Shigella flexneri, on the other hand, have not been investigated. Sh. flexneri is a gram-negative foodborne pathogen that can be widely found in nature and some industrial production environments. In this current research, our aim was to examine the influences of CA on planktonic bacteria and biofilm formation. The minimum inhibitory concentration (MIC) of CA against Sh. flexneri strain was 100 µg/mL, while bacteria treated with CA showed a longer lag phase compared with the untreated control. CA effectively inactivated the Sh. flexneri in LB broth and fresh lettuce juice. CA treatment resulted in cell membrane permeability changes and dysfunction, as proven by cell membrane depolarization, decreased intracellular ATP concentration. In addition, CA was also discovered to increase the level of reactive oxygen species (ROS) in cells, and induce morphological changes in cells. Crystal violet staining showed that the biomass of biofilm was decreased significantly with CA in 24 h. Light microscopy and field emission scanning electron microscopy (FESEM) observations demonstrated decreased biofilm adhesion and destruction of biofilm architecture after treatment with CA. These findings indicated that CA acts as a natural bacteriostatic agent to control Sh. flexneri in food processing and production.

Efficacy of 405 nm Light-Emitting Diode Illumination and Citral Used Alone and in Combination for Inactivation of Vibrio parahaemolyticus on Shrimp.

Vibrio parahaemolyticus is a widely distributed pathogen, which is frequently the lead cause of infections related to seafood consumption. The objective of the present study was to investigate the antimicrobial effect of the combination of 405 nm light-emitting diode (LED) and citral on V. parahaemolyticus. The antimicrobial effect of LED illumination and citral was evaluated on V. parahaemolyticus not only in phosphate-buffered saline (PBS) but also on shrimp. Quality changes of shrimp were determined by sensory evaluation. Changes in bacteria cell membrane morphology, cell membrane permeability, cell lipid oxidation level, and DNA degradation were examined to provide insights into the antimicrobial mechanism. The combination of LED treatments and citral had better antimicrobial effects than either treatment alone. LED combined with 0.1 mg/mL of citral effectively reduced V. parahaemolyticus from 6.5 log CFU/mL to below the detection limit in PBS. Combined treatment caused a 3.5 log reduction of the pathogen on shrimp within 20 min and a 6 log reduction within 2 h without significant changes in the sensory score. Furthermore, combined LED and citral treatment affected V. parahaemolyticus cellular morphology and outer membrane integrity. The profile of the comet assay and DNA fragmentation analysis revealed that combination treatment did not cause a breakdown of bacterial genomic DNA. In conclusion, LED may act synergistically with citral. They have the potential to be developed as novel microbial intervention strategies.

Citral mitigates inflammation of Caco-2 cells induced by Cronobacter sakazakii.

The aim of this study was to explore the anti-inflammatory effect and mechanism of citral in Cronobacter sakazakii-stimulated Caco-2 cells. Safe doses of citral were first determined in Caco-2 cells. Then, the effect of citral on the adhesion and invasion of C. sakazakii into Caco-2 cells and the translocation of C. sakazakii through Caco-2 monolayers were investigated. The release of nitric oxide (NO), interleukin (IL)-1ß, IL-6, and TNF-α, transcription of inflammatory genes, and expression of proteins associated with inflammatory signaling pathways were determined. Subsequently, activation of caspase-3, -8, and -9 and apoptosis induced by C. sakazakii were assessed. The results showed that up to 10 µg mL-1 citral had no cytotoxicity in Caco-2 cells. Citral protected Caco-2 cells by affecting the adhesion and invasion of C. sakazakii into Caco-2 cells and the translocation of C. sakazakii across Caco-2 monolayers. Additionally, inflammation induced by C. sakazakii was effectively inhibited by citral via suppression of inflammatory factors that included NO, IL-1ß, IL-6, and TNF-α, transcription of related genes, and expression of proteins associated with inflammatory signaling pathways. Moreover, the activation of caspase-3, -8, and -9, and apoptosis caused by C. sakazakii were suppressed by pretreatment with citral. These findings suggest that citral mitigates the inflammatory response of Caco-2 cells. Citral has the potential to prevent the inflammation of Caco-2 associated with C. sakazakii.