Antibacterial effect of ultrasound combined with Litsea cubeba essential oil nanoemulsion on Salmonella Typhimurium in kiwifruit juice.

This study investigated the antibacterial effect of ultrasound (US) combined with Litsea cubeba essential oil nanoemulsion (LEON) on Salmonella Typhimurium in kiwifruit juice and effect on the quality and sensory properties of kiwifruit juice. In this study, LEON prepared by ultrasonic emulsification method had a good particle size distribution and high stability. The US+LEON treatment significantly (P < 0.05) improved antibacterial efficacy, compared to the control, and would not destroy the nutritional components containing ascorbic acid, flavonoids, total phenol and total soluble solids. Meanwhile, US+LEON treatment enhanced 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS) radical scavenging capacity and ferric ion reducing antioxidant power (FRAP). In terms of sensory properties, US and LEON had a significant (P < 0.05) effect on the odor and overall morphology of kiwifruit juice. The enhance of antibacterial efficacy and the retention of nutrients by combined treatments shows that US+LEON is a promising antibacterial method that will provide new ideas for the processing and safety of fruit juices, and the US parameters and LEON concentration should be adjusted to reduce the effect on food sensory properties in future studies.

Inhibition and Mechanism of Citral on Bacillus cereus Vegetative Cells, Spores, and Biofilms.

Bacillus cereus causes food poisoning by producing toxins that cause diarrhea and vomiting and, in severe cases, endocarditis, meningitis, and other diseases. It also tends to form biofilms and spores that lead to contamination of the food production environment. Citral is a potent natural antibacterial agent, but its antibacterial activity against B. cereus has not been extensively studied. In this study, we first determined the minimum inhibitory concentrations and minimum bactericidal concentrations, growth curves, killing effect in different media, membrane potential, intracellular adenosine triphosphate (ATP), reactive oxygen species levels, and morphology of vegetative cells, followed by germination rate, morphology, germination state of spores, and finally biofilm clearance effect. The results showed that the minimum inhibitory concentrations and minimum bactericidal concentrations of citral against bacteria ranged from 100 to 800 µg/mL. The lag phase of bacteria was effectively prolonged by citral, and the growth rate of bacteria was slowed down. Bacteria in Luria-Bertani broth were reduced to below the detection limit by citral at 800 µg/mL within 0.5 h. Bacteria in rice were reduced to 3 log CFU/g by citral at 4000 µg/mL within 0.5 h. After treatment with citral, intracellular ATP concentration was reduced, membrane potential was altered, intracellular reactive oxygen species concentration was increased, and normal cell morphology was altered. After treatment with citral at 400 µg/mL, spore germination rate was reduced to 16.71%, spore morphology was affected, and spore germination state was altered. It also had a good effect on biofilm removal. The present study showed that citral had good bacteriostatic activity against B. cereus vegetative cells and its spores and also had a good clearance effect on its biofilm. Citral has the potential to be used as a bacteriostatic substance for the control of B. cereus in food industry production.

Synergistic bactericidal effect and mechanism of ultrasound combined with Lauroyl Arginate Ethyl against Salmonella Typhimurium and its application in the preservation of onions.

In the present study, the synergistic bactericidal effect and mechanism of ultrasound (US) combined with Lauroyl Arginate Ethyl (LAE) against Salmonella Typhimurium were investigated. On this basis, the effect of US+LAE treatment on the washing of S. Typhimurium on the surface of onions and on the physical and chemical properties of onion during fresh-cutting and storage were studied. The results showed that treatment with US+LAE could significantly (P < 0.05) reduce the number of S. Typhimurium compared to US and LAE treatments alone, especially the treatment of US+LAE (230 W/cm2, 8 min, 71 µM) reduced S. Typhimurium by 8.82 log CFU/mL. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein and nucleic acid release and N-phenyl-l-naphthylamine (NPN) assays demonstrated that US+LAE disrupted the integrity and permeability of S. Typhimurium cell membranes. Reactive oxygen species (ROS) and malondialdehyde (MDA) assays indicated that US+LAE exacerbated oxidative stress and lipid peroxidation in cell membranes. Field emission scanning electron microscopy (FESEM) demonstrated that US+LAE treatment caused loss of cellular contents and led to cell crumpling and even lost the original cell morphology. US+LAE treatment caused a significant (P < 0.05) decrease in the number of S. Typhimurium on onions, but there was no significant (P > 0.05) effect on the color, hardness, weight and ascorbic acid content of onions. This study elucidated the synergistic antibacterial mechanism of US+LAE and verified the feasibility of bactericidal effect on the surface of onions, providing a theoretical basis for improving the safety of fresh produce in the food industry and to propose a new way to achieve the desired results.

Antibacterial effect of ultrasound and ß-citronellol against Listeria monocytogenes and its application in carrot preservation.

This study investigated the antibacterial effects of ultrasound (US), ß-citronellol (CT), and a combination of the two treatments on Listeria monocytogenes. Results showed that US or CT alone did not show apparent antibacterial effect (0.02-0.76 log CFU/mL reduction). The combined treatment showed obviously inactivate effect of L. monocytogenes, the populations of L. monocytogenes decreased by 8.93 log CFU/mL after US (253 W/cm2, 20 kHz) + 0.8 mg/mL CT treatment. US + CT treatment also had a significant (P < 0.05) antibacterial effect on isolates of L. monocytogenes from three different serotypes. In this study, the damage of US + CT on cell morphology had been observed using field emission scanning electron microscopy, while the damage to cell membranes by US + CT was observed by confocal laser scanning microscopy and flow cytometry. Meanwhile, the uptake of N-phenyl-l-naphthylamine and the absorbance at 260 and 280 nm also indicated that the combined treatment disrupted the permeability and integrity of L. monocytogenes membranes. Reactive oxygen species and malondialdehyde assays showed that US + CT exacerbated cellular oxidative stress and lipid peroxidation. In addition, the US + CT treatment reduced L. monocytogenes by 3.14-4.24 log CFU/g on the surface of carrots. Total phenolic and carotenoid contents in carrots were elevated after US + CT treatment. During storage, compared to control, US + CT did not significantly (P > 0.05) change the surface color of carrots but significantly (P < 0.05) decreased both hardness and weight, and has an impact on the sensory. This study showed that US + CT is a promising cleaning method that will provide new ideas for the preservation of fresh agricultural produce.

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.

Inactivation of Salmonella using ultrasound in combination with Litsea cubeba essential oil nanoemulsion and its bactericidal application on cherry tomatoes.

The presence of Salmonella in nature poses a significant and unacceptable threat to the human public health domain. In this study, the antibacterial effect and mechanism of ultrasound (US) combined with Litsea cubeba essential oil nanoemulsion (LEON) on Salmonella. LEON + US treatment has a significant bactericidal effect on Salmonella. Reactive oxygen species (ROS), malondialdehyde (MDA) detection, N-phenyl-l-naphthylamine (NPN) uptake and nucleic acid release assays showed that LEON + US exacerbated cell membrane lipid peroxidation and increased the permeability of the cell membrane. The results of field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) showed that LEON + US treatment was able to alter cell morphology. It can be observed by flow cytometry (FCM) that LEON + US treatment can cause cell apoptosis. In addition, bacterial counts of cherry tomatoes treated with LEON (0.08 µL/mL) + US (345 W/cm2) for 9 min were reduced by 6.50 ± 0.20 log CFU/mL. This study demonstrates that LEON + US treatment can be an effective way to improve the safety of fruits and vegetables in the food industry.

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.

Inhibitory Effects of Trans-Cinnamaldehyde Against Pseudomonas aeruginosa Biofilm Formation.

Pseudomonas aeruginosa biofilm formation has been considered to be an important determinant of its pathogenicity in most infections. The antibiofilm activity of trans-cinnamaldehyde (TC) against P. aeruginosa was investigated in this study. Results demonstrated that the minimum inhibitory concentration (MIC) of TC against P. aeruginosa was 0.8 mg/mL, and subinhibitory concentrations (SICs) was 0.2 mg/mL and below. Crystal violet staining showed that TC at 0.05-0.2 mg/mL reduced biofilm biomass in 48 h in a concentration-dependent mode. The formation area of TC-treated biofilms was significantly declined (p < 0.01) on the glass slides observed by light microscopy. Field-emission scanning electron microscopy further demonstrated that TC destroyed the biofilm morphology and structure. Confocal laser scanning microscopic observed the dispersion of biofilms and the reduction of exopolysaccharides after TC treatment stained with concanavalin A (Con-A)-fluorescein isothiocyanate conjugate and Hoechst 33258. Meanwhile, TC caused a significant decrease (p < 0.01) in the component of polysaccharides, proteins, and DNA in extracellular polymeric substance. The swimming and swarming motility and quorum sensing of P. aeruginosa was also found to be significantly inhibited (p < 0.01) by TC at SICs. Furthermore, SICs of TC repressed the several genes transcription associated with biofilm formation as determined by real-time quantitative polymerase chain reaction. Overall, our findings suggest that TC could be applied as natural and safe antibiofilm agent to inhibit the biofilm formation of P. aeruginosa.

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.

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.