Bacillus spore germination: mechanisms, identification, and antibacterial strategies.

Bacterial spores are metabolically inactive and highly resistant to harsh environmental conditions in nature and during decontamination processes in food and related industries. However, inducing germination using specific germinants in dormant spores can convert them into vegetative cells which are metabolically active and fragile. The potential utility of a "germinate to eradicate" strategy, also known as germination-inactivation, has been validated in foods. Meanwhile, the strategy has sparked much interest in triggering and maximizing spore germination. Although many details of the spore germination process have been identified over the past decades, there remain many uncertainties, including some signal transduction mechanisms involved in germination. In addition, the successful implementation of the germination-inactivation strategy relies on the sensitive detection of germinative biomarkers within minutes of germination initiation and the optimal timing for the subsequent inactivation step. Meanwhile, the emergence of biomarkers has renewed attention to the practical application of the spore germination process. Here, this review presents the current knowledge of the germination mechanisms of Bacillus spore, influencing factors, and germination biomarkers. It also covers a detailed discussion on the development of germination-inactivation as a spore eradication strategy.

Recent advances on the application of UV-LED technology for microbial inactivation: Progress and mechanism.

Conventional technologies for the inactivation of microorganisms in food products have their limitations, especially changes in quality attributes that have led to quality deterioration, low consumer acceptance, impact on the environment, and potential health hazards (carcinogens). Ultraviolet (UV) light is an emerging promising nonthermal technology employed for microbial inactivation in water, liquid, and solid food products to curtail the limitations above. This review provides an insight into UV light-emitting diodes (UV-LEDs)' potential as an alternative to the traditional UV lamps for microbial inactivation in liquid and solid media. Also, the mechanisms of inactivation of lone and combined UVA-, UVB-, and UVC-LEDs were discussed. The strategies utilized to improve the efficacy between the UV-LED treatments at various wavelengths were summarized. Combining different UV-LEDs treatments at different wavelengths have a synergistic effect and suppression of microbial cell reactivation. The UV-LED-based advanced oxidation processes (AOPs) also have high germicidal action against numerous microorganisms and are efficient for the degradation of micropollutants. Among the UV-LEDs discussed, UVC-LED has the most antimicrobial effect with the most efficient micropollutants decomposition with regards to UV-LED-based AOPs. This review has provided vital information for future application, development, and customization of UV-LED systems that can meet the food and water safety requirements and energy efficiency.

The comparison of ultrasound-assisted thawing, air thawing and water immersion thawing on the quality of slow/fast freezing bighead carp (Aristichthys nobilis) fillets.

Effects of ultrasound-assisted thawing (UAT), air thawing (AT) and water immersion thawing (WIT) on the quality of slow freezing (SF)/fast freezing (FF) bighead carp (Aristichthys nobilis) fillets were investigated. The thawing time of AT, WIT and UAT were 138, 30 and 12 min, respectively, indicating that UAT improved the thawing efficiency. UAT maintained the color and pH, and inhibited the lipid oxidation of SF/FF samples. For SF samples, thawing methods did not affect water retention and muscle structure. However, massive water loss and muscle destruction were observed in FF-WIT and FF-UAT samples. The AT did not produce any structural muscle damage; instead, it maintained the water retention in FF samples and prevented the FF fish fillets from massive water loss. No significant difference in the primary protein structure was observed among all samples. UAT can be an alternative strategy to the traditional thawing of SF fish fillets.

Hurdle enhancement of acidic electrolyzed water antimicrobial efficacy on Bacillus cereus spores using ultrasonication.

This study evaluated the inactivation effect of ultrasonic treatment combined with acidic electrolyzed water (AEW) on Bacillus cereus spores. AEW treatment reduced the spores by 1.05-1.37 log CFU/mL while the sporicidal effect of ultrasound was minor. More strikingly, simultaneous ultrasonic and AEW treatments for 30 min led to 2.29 log CFU/mL reduction and thus, considered a synergistic effect. Flow cytometry combined with SYTO/PI staining analysis revealed that ultrasound hydrolyzed the cortex while the AEW partially damaged the integrity of the inner membrane. Scanning and transmission electron microscopies were used to characterize the ultrastructural changes. The detachment of the exosporium induced by ultrasound was the most apparent difference compared with the control group, and the electron density of spores appeared to be heterogeneous after treatment with AEW. These results indicated that combining ultrasound with AEW is a promising decontamination technology with potential uses in the food industry and environmental remediation.

Ultrasound pretreatment enhances the inhibitory effects of nisin/carvacrol against germination, outgrowth and vegetative growth of spores of Bacillus subtilis ATCC6633 in laboratory medium and milk: Population and single-cell analysis.

This study evaluated the synergetic inhibitory effects of ultrasound and nisin/carvacrol on spore germination, outgrowth, and subsequent growth of vegetative cell of Bacillus subtilis in laboratory medium and milk. Ultrasound pretreatment (3.33 W/mL, 15 min) and nisin/carvacrol (0.01%, 0.02%) synergistically inhibited spore germination, outgrowth, and vegetative growth of spores in laboratory medium. Whereas no such inhibitory effect was observed in milk even with a 10-fold increase in the concentration (1%) of nisin. Flow cytometry analysis showed that the germination capacities of ultrasound pretreated spores combined with nisin/carvacrol (67.3% and 30.5%, respectively) was lower than that of the untreated spores (95.1%). These results quantitatively revealed the inhibitory effect of the combined treatments which were confirmed by phase-bright spore observations at single cell level. In general, the current work identified the combined ultrasound-carvacrol treatment as an effective strategy to control spores and vegetative cells of B. subtilis in the laboratory medium and milk during abusive storage.

Modeling the Inactivation of Bacillus cereus in Tiger Nut Milk Treated with Cold Atmospheric Pressure Plasma.

The impact of cold atmospheric pressure plasma treatment on the inactivation kinetics of Bacillus cereus ATCC 14579 and the resulting quality changes was investigated in tiger nut (Cyperus esculentus L.) milk (TNM). The effect of input power (39, 43, and 46 W) and treatment time (0 to 270 s) was fitted using the Weibull model to represent the microbial kinetic inactivation in the treated TNM. Inactivation efficacy increased with an increase in treatment time and input power. A 5.28-log reduction was achieved at 39 to 46 W without significant changes in titratable acidity, whereas no reduction in titratable acidity was observed in the pasteurized sample. The inactivation kinetics was adequately described by the Weibull model. Higher input power of 43 and 46 W and 120 s of treatment resulted in marked decreases in pH, flavonoid concentration, and antioxidant activity compared with those parameters in pasteurized TNM. Increases in total color difference and phenolic concentrations also were observed. The results indicate that these changes were caused by the immanent plasma reactive species. This study provides valuable inactivation kinetics information for food safety assessment studies of B. cereus vegetative cells in TNM.

Thermosonication damages the inner membrane of Bacillus subtilis spores and impels their inactivation.

The extreme resistance of bacterial spores to most killing agents makes them a major concern to the food industry and consumers. This gave rise an increasing interest in developing new strategies to inactivate spores and understand the mechanisms of inactivation by various agents. In this study, ultrasound combined with heat (thermosonication, TS) was used to inactivate the spores of Bacillus subtilis and the factors that influence the resistance to TS were analyzed. The spores of wild-type B. subtilis and isogenic mutants were subjected to heat at 80 °C and ultrasound at 6.67-20 W/mL and 23 °C for 0-40 min. TS treatment has synergistically resulted in spore inactivation, and spores of wild-type B. subtilis and isogenic mutants showed different resistance to TS treatment, which was in the following order: Strains 533 (wild-type) ≈ strains PS3518 (gfp) ≈ strains PS2318 (recA-) > strains PS578 (α-ß-), and spores of strains PS3328 (cotE-) were also more susceptible than those of wild-type strains. The inactivated spores lost some proteins in the spore core but initiated germination normally. The germinated inactivated spores did not swell and their plasma membrane permeability was equally altered. It was concluded that the damage to spores' inner membrane (IM) proteins or the IM itself has led to the leakage of intracellular substances and the death of a spore by TS treatment. Our results could support the development and optimization of TS treatment, which has great significance for its further utilization in food industry.

Fabrication of (-)-epigallocatechin-3-gallate carrier based on glycosylated whey protein isolate obtained by ultrasound Maillard reaction.

This study investigated the effect of glycation on the binding of whey protein isolate (WPI) with (-)-epigallocatechin-3-gallate (EGCG), and the physicochemical stability and bioaccessibility of the formed complex. The WPI-gum Acacia (GA) conjugate was prepared by ultrasound-assisted Maillard reaction. Results indicated that conjugated WPI showed stronger binding and entrapping ability to EGCG than that of WPI. The protein aggregation induced by EGCG was partly inhibited by glycosylation, presumably due to the steric hindrance of polysaccharide chains. The greatest protection of EGCG and its antioxidant activity were also obtained by complexing it with WPI-GA conjugate. The in vitro bioaccessibility analysis demonstrated that the bioaccessibility of EGCG cloud be significantly (p < 0.05) enhanced by complexing it with WPI, especially WPI-GA conjugate. These findings are important to design promising and effective EGCG carriers for its wide application in food industry.

Time effect on structural and functional properties of whey protein isolate-gum acacia conjugates prepared via Maillard reaction.

BACKGROUND: The functional properties of whey protein isolate (WPI) are sensitive to pH, ionic strength, and temperature. This prevents its application in various food systems and processing technologies. The conjugation of proteins with polysaccharides via the Maillard reaction is an efficient method to improve the functionality of proteins. The purpose of this work was to conjugate gum acacia (GA) with WPI via the dry-heating Maillard reaction and to investigate the effect of reaction time on the physicochemical and functional properties of WPI-GA conjugates. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance size exclusion chromatography confirmed the formation of higher molecular weight conjugates. The degrees of glycation for WPI-GA conjugates incubated for 1, 3, 5, and 7 days were 28.14%, 44.98%, 49.50%, and 51.20%, respectively. The glycation reaction reduced the surface hydrophobicity and fluorescence intensity of WPI significantly (P < 0.05). Functional properties of the conjugates, such as solubility, stability against heat-induced insolubility, and emulsion properties were all superior to the control WPI. However, a reaction time longer than a day resulted in a high degree of browning and decreased the functionality of the conjugate significantly (P < 0.05). CONCLUSION: The results indicated that conjugation of WPI with GA can be a promising way to enhance its functional properties. However, the reaction time suitable for producing conjugates with superior functional properties was not necessarily the highest glycation degree that could be reached. © 2019 Society of Chemical Industry.

Comparison of Biogenic Amines in Chinese Commercial Soy Sauces.

Soy sauce contains a series of biogenic amines (BAs) which is a kind of bioactive organics relating to food quality and safety. High concentration of BAs may lead to remarkable physiological and toxicological influences on human bodies, including hypotension, dizziness, and headaches. Here, we systematically compared the levels of ten main BAs among 53 Chinese commercial soy sauces using an improved high-performance liquid chromatography (HPLC) method. The results showed that the brands and production regions were both important factors accounting for the BAs' content. The contents of Cad, Spm, Try, Phe, His, and Tyr in dark soy sauces were higher than those in light soy sauces. His and Phe in dark soy sauces were 3.7 and 1.84 times higher than in light samples, respectively. Besides, it was surprising that the content of BAs in soy sauces hugely varied from place to place. This work comprehensively compared the content of BAs in soy sauces, showing the relation between soy sauce processes and BAs, offering abundant information for further research on BAs control.

Ultrasound-assisted thawing of mango pulp: Effect on thawing rate, sensory, and nutritional properties.

Sensory and nutritional properties of mango pulp thawed by different ultrasound intensities and temperatures were studied. Compared to water immersion thawing, a thawing time reduction of 16-64% (p < 0.05) and more phenolic acids contents (gallic, hydroxybenzoic, and caffeic acid) were observed after ultrasonic thawing. The sensory evaluation revealed texture and aroma deterioration at higher ultrasonic intensities (4 °C:0.074 W/mL; 25 °C:0.123 W/mL) due to increase in viscosity and some volatile compounds in the mango pulp. Ultrasonic thawing treatment at 25 °C reduced thawing time by 51-73% compared to that at 4 °C. Mango pulp processed at 25 °C exhibited better sensory quality and retained 26.5-58.5% more total phenol and 8.7-11.0% more total carotenoid contents. In short, higher ultrasonic intensities (0.074-0.123 W/mL) at a temperature of 25 °C contributed to better thawing efficiency and nutritional quality. The results demonstrated that ultrasound processing at optimized conditions could serve as a potential alternative to conventional thawing processing of mango pulp.

Synergistic inactivation and mechanism of thermal and ultrasound treatments against Bacillus subtilis spores.

Some Bacillus species are causative agents of food spoilage and a wide array of diseases. Due to their ability to form highly heat-resistant spores, it is of great interest to develop more effective inactivation strategies whereby these spores could be inactivated. Therefore, this work assessed inactivation of thermal and ultrasound treatments against Bacillus subtilis spores. The study further investigated the thermosonication (thermal and ultrasound, TS) -induced inactivation to the spores through a combination of morphology observation and internal factor analyses. The results of TS indicated that the TS combination synergistically inactivated spores by the maximum log reduction of 2.43 ±â€¯0.08 at 80 °C and 20 W/ml and caused severe cell damage. The visual images revealed that the destructive mode of action of TS had multitarget sites, including coat, cortex, and inner membrane. Three distinct sub-populations were detected by Flow cytometry (FCM), and an unknown step with some physical compromise of the spore's inner membrane and partially hydrolyzed cortex involving the three steps model of inactivation was suggested. The combination of DPA (pyridine-2,6 dicarboxylic acid) content and the relative viabilities of the fractions suggested that during the TS treatment DPA release took place largely after spore death. The dead spores that retained DPA germinated relatively normally, but outgrow poorly, indicating that some key enzymes of intermediary metabolism has been damaged by TS treatment. Such understanding of the lethal action of TS may lead to the development of novel strategies involving spore destruction.

Effect of ultrasonication and thermal and pressure treatments, individually and combined, on inactivation of Bacillus cereus spores.

Bacillus cereus spores are a concern to the food industry due to their high resistance to processing and their ability to germinate to vegetative cells under suitable conditions. This research aimed to elucidate the mechanisms of Bacillus cereus spore inactivation under ultrasonication (US) combined with thermal (thermosonication, TS) treatments, with pressure (manosonication, MS) treatments, and with thermal and pressure (manothermosonication, MTS) treatments. Electronic microscopy, dipicolinic acid (DPA) release, and flow cytometric assessments were used to investigate the inactivation effect and understand the inactivation mechanisms. The sporicidal effects of the US and thermal treatment were slight, and the MS and TS also showed little inactivation effect. However, ultrasonication promoted the detachment of the exosporium, thereby reducing the spore's ability to adhere to a surface, while the thermal treatment induced a decrease in the electron density in the nucleoid of bacterium, which retained a relatively intact exosporium and coat. MS caused 92.54% DPA release, which might be due to triggering of the germinant receptors or releasing of ions and Ca2+-DPA. In addition, the morphological changes such as core hydration and cortex degradation were significant after treatment with MS. The release of DPA and the morphological changes were responsible for the reduction in thermal resistance. The MTS showed a remarkable inactivation effect of 3.12 log CFU/mL reductions after 30 min of treatment. It was the most effective treatment and exhibited a large fraction of damage. In addition, the MTS had a significant impact on the intracellular structure of the spores, with the coat destroyed and the cortex damaged. These results indicated that ultrasonication combined with thermal and pressure treatments had a significant sporicidal effect on Bacillus cereus spores and could be a promising green sterilization technology.

Bacterial spore inactivation induced by cold plasma.

Cold plasma has emerged as a non-thermal technology for microbial inactivation in the food industry over the last decade. Spore-forming microorganisms pose challenges for microbiological safety and for the prevention of food spoilage. Inactivation of spores induced by cold plasma has been reported by several studies. However, the exact mechanism of spore deactivation by cold plasma is poorly understood; therefore, it is difficult to control this process and to optimize cold plasma processing for efficient spore inactivation. In this review, we summarize the factors that affect the resistance of spores to cold plasma, including processing parameters, environmental elements, and spore properties. We then describe possible inactivation targets in spore cells (e.g., outer structure, DNA, and metabolic proteins) that associated with inactivation by cold plasma according to previous studies. Kinetic models of the sporicidal activity of cold plasma have also been described here. A better understanding of the interaction between spores and cold plasma is essential for the development and optimization of cold plasma technology in food the industry.

Combating Staphylococcus aureus and its methicillin resistance gene (mecA) with cold plasma.

The increase in antibiotic resistance has become a global challenge to public health. In this study, an atmospheric cold plasma (ACP) system was applied for combating methicillin-resistant Staphylococcus aureus (MRSA) and its methicillin resistance gene (mecA) during food wastewater treatment. The plate count and flow cytometry methods were employed to estimate the damage in MRSA induced by plasma treatment. A quantitative real-time PCR (qPCR) method was used to assess the plasma-induced degradation of the mecA genes. The inactivation of MRSA and degradation of extracellular (e-) and intracellular (i-)mecA genes were investigated in phosphate buffered solution as a function of plasma exposure. A relatively low plasma influence of 0.12 kJ/cm2 accounted for 5-log MRSA and 1.4-log e-mecA genes reduction, while only around 0.19-log degradation for i-mecA genes. As the plasma intensity was accumulated to 0.35 kJ/cm2, the reduction of e- and i-mecA genes was increased to 2.6 and 0.8 logs, respectively. The degradation of i-mecA genes was much slower than that of e-mecA genes due to the protective effects of the outer envelopes or intracellular components against plasma. The matrix effect of wastewater effluents shielded both antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) from plasma disinfection, which led to a lower degradation efficacy. Our results could support the development and optimization of plasma-based wastewater treatment.

Application of a Dielectric Barrier Discharge Atmospheric Cold Plasma (Dbd-Acp) for Eshcerichia Coli Inactivation in Apple Juice.

Atmospheric cold plasma (ACP) is a promising non-thermal technology in food industry. In this study, a dielectric barrier discharge (DBD)-ACP exhibited strong bactericidal effect on Escherichia coli in apple juice. Under a 30 to 50 W input power, less than 40 s treatment time was required for DBD-ACP to result in 3.98 to 4.34 log CFU/mL reduction of E. coli in apple juice. The inactivation behavior of ACP on E. coli was well described by the Weibull model. During the treatment, the cell membrane of E. coli was damaged severely by active species produced by plasma, such as hydrogen peroxide, ozone and nitrate. In addition, the ACP exposure had slight effect on the °Brix, pH, titratable acidity (TA), color values, total phenolic content, and antioxidant capacity of apple juice. However, higher level of DBD-ACP treatment, 50 W for more than 10 s in this case, resulted in significant change of the pH, TA, color and total phenolic content of apple juice. The results in this study have provided insight in potential use of DBD-ACP as an alternative to thermal processing for fruit juices in food industry. PRACTICAL APPLICATION: Escherichia coli O157:H7 in apple juice is a potential risk for public health. This study demonstrated that 30 s cold plasma treatment resulted in more than 4 log CFU/mL reduction under 50 W, while the quality attributes of apple juice were not significantly affected. Therefore, cold plasma technology is a promising alternative substitute of traditional thermal processing for juice pasteurization.

Effects of Nonthermal Plasma Technology on Functional Food Components.

Understanding the impact of nonthermal plasma (NTP) technology on key nutritional and functional food components is of paramount importance for the successful adoption of the technology by industry. NTP technology (NTPT) has demonstrated marked antimicrobial efficacies with good retention of important physical, chemical, sensory, and nutritional parameters for an array of food products. This paper presents the influence of NTPT on selected functional food components with a focus on low-molecular-weight bioactive compounds and vitamins. We discuss the mechanisms of bioactive compound alteration by plasma-reactive species and classify their influence on vitamins and their antioxidant capacities. The impact of NTP on specific bioactive compounds depends both on plasma properties and the food matrix. Induced changes are mainly associated with oxidative degradation and cleavage of double bonds in organic compounds. The effects reported to date are mainly time-dependent increases in the concentrations of polyphenols, vitamin C, or increases in antioxidant activity. Also, improvement in the extraction efficiency of polyphenols is observed. The review highlights future research needs regarding the complex mechanisms of interaction with plasma species. NTP is a novel technology that can both negatively and positively affect the functional components in food.