The study aimed to evaluate the synergistic interaction of organic acids (OAAs) or lemon extract (LE) plus mild heat (MH; 55 °C) against Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes inoculated in beetroot and watermelon juices. A mixed culture cocktail of E. coli O157:H7, S. Typhimurium or L. monocytogenes was inoculated in beetroot or watermelon juice, followed by treatments with MH, citric acid + MH, malic acid + MH, tartaric acid + MH, and LE + MH. Approximately < 2.0-log reductions in the number of E. coli O157:H7, S. Typhimurium, and L. monocytogenes were observed when these bacteria were heated in juices at 55 °C for 5 min. A combination of 1.0% OAAs or 20% LE and MH (55 °C) for 5 min resulted in an additional log-reduction in the count of E. coli O157:H7, S. Typhimurium, and L. monocytogenes by 2.2-5.0, 4.5-5.0, and 1.5-5.0, respectively.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.

Cancer therapeutic vaccine can induce antigen-specific immune response, which has shown great potential in cancer immunotherapy. As the key factor of vaccine, antigen plays a central role in eliciting antitumor immunity. However, the insufficient antigen delivery and low efficiency of antigen presentation by dendritic cells (DCs) have greatly restricted the therapeutic efficiency of vaccine. Here we developed a kind of DC hybrid zinc phosphate nanoparticles to co-deliver antigenic peptide and photosensitive melanin. Owing to the chelating ability of Zn2+, the nanoparticles can co-encapsulate antigenic peptide and melanin with high efficiency. The nanovaccine showed good physiological stability with the hydration particle size was approximately 30 nm, and zeta potential was around - 10 mV. The nanovaccine showed homologous targeting effect to DCs in vivo and in vitro, efficiently delivering antigen to DCs. Meanwhile, the nanovaccine could effectively reflux to the tumor-draining lymph nodes. When combined with near-infrared irradiation, the nanovaccine induced effective mild heat in vitro and in vivo to promote antigen presentation. After administrating to MC38 tumor-bearing mice, the hybrid nanovaccine effectively promoted the maturation of DCs, the expansion of cytotoxic T lymphocytes and helper T cells, and the secretion of immunostimulatory cytokines, thereby significantly inhibiting tumor growth.

Salmonella spp. is one of the leading causes of foodborne outbreaks worldwide. Salmonella spp. has been associated with a variety of food sources, particularly egg products. They can enter a viable but nonculturable (VBNC) state in response to harsh stress. VBNC cells still retain membrane integrity and metabolic activity, which may pose health risks. However, the formation mechanism and resuscitation ability of VBNC cells are not well understood. In this work, Salmonella spp. cocktails, including Salmonella enterica serovar Newport and Salmonella enterica serovar Enteritidis, in liquid egg products was induced into a VBNC state by mild heat treatment, a commonly used method to inhibit the growth of pathogenic in liquid egg industry. Mild heat induced VBNC cells were found to resuscitate in liquid egg yolk (LEY) and liquid whole egg (LWE), but they failed to recover in liquid egg white (LEW). In addition, a certain number of cells remained as VBNC state after in vitro digestion. The membrane vesicle (MV) protein encoding gene pagC, two-component system encoding genes phoP/Q and sigma factor encoding gene rpoS were highly expressed in VBNC cells compared with the culturable counterparts. The results of this study can contribute to a better understanding of the health risks associated with Salmonella spp. in VBNC state and provide a theoretical basis for formation mechanism of VBNC state.

Antibiotics have been widely used to treat several infectious diseases. However, the overuse of antibiotics has promoted the emergence and spread of antibiotic resistant bacteria (ARB) in various fields, including the food industry. In this study, the antimicrobial efficacies of two conventional sterilization methods, mild heat, and sonication, were evaluated and optimized to develop a new strategy against ARB. Simultaneous mild heat and sonication (HS) treatment led to a significant reduction in viable cell counts, achieving a 5.58-log reduction in 4 min. However, no remarkable decrease in viable cell counts was observed in individually treated groups. Interestingly, the release of antibiotic resistance genes (ARGs) increased in a time-dependent manner in the heat-treated and HS-treated groups. The inactivation levels of ARGs increased as the HS treatment time increased from 2 to 8 min, and most ARGs were degraded after 8 min. In contrast, no significant inactivation of ARGs was observed in the heat-treated and sonication-treated groups after 8 min. These results reveal the synergistic effect of the combination treatment in controlling not only ARB but also ARGs. Finally, on applying this newly developed combination treatment to fresh food (cherry tomato and carrot juice), 3.97- and 4.28-log microbial inactivation was achieved, respectively. In addition, combination treatment did not affect food quality during storage for 5 days. Moreover, HS treatment effectively inactivated ARGs in fresh food systems.

Ultrasound-assisted probiotic fermentation suspension treatment under mild heat was applied to freshly cut lotus root. Combined treatment significantly (p < 0.05) improved the bactericidal effect: S. aureus, E. coli O157:H7, A. flavus spores, and yeast were completely killed at 55℃ for 6 min, morphological and structural changes of cells were observed. Combined treatment inhibited enzymatic browning via decreasing the activities of PPO, POD, PAL, and decreasing TPC, soluble quinone content. The decrease of external resistance, capacitance and the increase of internal resistance reflected the loss of structural integrity of cells after combined treatment. For combined treatment samples, no obvious color change was observed during 12 days of storage but weight loss rate, total acid increased significantly (p < 0.05) and hardness decreased, the shelf life was extended to 12 d. Ultrasound-assisted probiotic fermentation suspension treatment under mild heat had the dual effects of sterilization and inhibition of browning.

Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persist in leafy green vegetables and subsequently produce biofilms. Biofilm formation is an ongoing concern in the food industry as biofilms are relatively resistant to a variety of antimicrobial treatments. In the present study, we evaluated the combined effects of phage FP43 and mild-heated slightly acidic hypochlorous water (SAHW) in reducing established biofilms on lettuce. Prior to the sequential treatments involving phage-SAHW and SAHW-phage for long-term storage, equal inoculum densities of E. coli O157:H7 and E. coli O91:H- were added on iceberg lettuce surfaces and the lettuce samples were stored at 10 °C for 48 h to allow biofilm formation. The sequential treatment with phage FP43 and SAHW significantly decreased the number of adhered cells, especially the combination of phage FP43 at 25 °C for 2 h and mild-heated SAHW, which considerably eliminated E. coli viable biofilm cells to undetectable levels (>3 log CFU/piece). However, the biofilms were not completely removed, as evidenced via SEM observation. Additionally, sequential treatment with SAHW and phage caused continuous reductions in viable counts, decreasing the viability of E. coli O157:H7 and total E. coli to the lower limit of detection after incubation for 5 d. Meanwhile, bacterial regrowth was observed after treatment with SAHW alone. These results indicated that the combination of phage and SAHW could be considered as a promising strategy to control the formation of E. coli O157:H7 biofilms on lettuce.

Mild heat treatment of fruit juices in combination with natural aroma compounds has been reported as an alternative to conventional pasteurization to better preserve their nutritional value. However, its antimicrobial efficiency varies from one juice to another. This study aims at developing a secondary predictive model of microbial inactivation scale during such combined process. Carvacrol was used as aroma compound and acid-adapted L. monocytogenes as target microorganism. The inactivation kinetics of this bacteria were followed in simulated fruit juices using a Central Composite Design with pH (2-6), °Brix (0-24), temperature (55-65 °C), and carvacrol concentration (0-60 μL/L) as independent variables. Curves were fitted to the Weibull inactivation model, and data collected used to generate two predictive models of the inactivation scale parameter through multiple regression analysis following an empirical and a mechanistic (based on Gamma concept) approach. The best of the two models was then validated using real fruit (orange, pineapple, and watermelon) juices. The empirical model where only the four variables tested were considered showed a lower statistical performance compared to the mechanistic model where octanol-water partition coefficient (Ko/w) and vapour pressure (Vp) of carvacrol at the treatment temperature were integrated (R2 0.6 and 0.9; Accuracy factor 1.5 and 1.3; Sum of Squared Error 3.6 and 1.1, respectively). No significant difference was observed between inactivation scale values obtained with real juices and the predicted values calculated using this mechanistic model. The Ko/w and Vp of the aroma compound used are key parameters that determine the efficiency of the above-described combined treatment.

Klebsiella pneumoniae is an important foodborne pathogen with high biofilm-forming ability, which is continuously detected in food products in recent years. The antibacterial and antibiofilm activities and mechanism of ultrasonication in combination with heat treatment against K. pneumoniae were studied. K. pneumoniae planktonic and biofilm cells were treated with ultrasound (US), mild heat treatment (HT50, HT60, and HT70), and combinations of US and mild heat treatment (UH50, UH60, and UH70) for 5, 10, 20, 30, and 60 min. Results showed that the combination of US and mild heat treatment was more effective in inactivating K. pneumoniae planktonic and biofilm cells than the single treatment by counting viable bacteria. In addition, confocal laser scanning microscopy, scanning electron microscopy, and analysis of leakage of intracellular substances have revealed that the combination treatment effectively damaged the integrity of bacterial cell membrane and increased cell permeability, which led to the quick release of adenosine triphosphate (ATP) and macromolecular substances of nucleic acids and proteins. Moreover, the activities of respiratory chain dehydrogenase in planktonic and biofilm cells significantly decreased after UH treatment. The results indicated that ultrasonication and mild heat treatment had a synergistic effect on the inactivation of K. pneumoniae planktonic and biofilm cells by damaging the cell membrane and inhibiting intercellular cell respiration.

The aim of this work was to evaluate the potential of ultrasound (US), alone or in combination with mild heating and/or EDTA towards reduction of As, Cd, I, and Hg content of Laminaria hyperborea. Concentrations of As, Cd, I, and Hg of 56.29, 0.596, 7340, and <0.01 mg kg-1 of dry weight, respectively, were found in L. hyperborea blades. Treatment with US at 50 °C increased approx. 2-fold the amount of As released, although did not affect significantly the content of Cd or I, as compared to control (no US) samples. Reducing the temperature to 8 °C significantly decreased the effect of US, but heating at 80 °C did not cause a significant effect as compared to treatments at 50 °C. On the other hand, treatment with 0.1 N EDTA at 50 °C enhanced the percentage of Cd released by approximately 7-fold, regardless of sonication. In the present work, the combination of US and EDTA at 50 °C for 5 min led to a significant reduction of the As (32%), Cd (52%) and I (31%) content in L. hyperborea, thus improving the product\'s safety for consumers.