Impact of Environmental Stresses on the Antibacterial Activity of Graphene Oxide (GO) Nanoparticles against P. putida Biofilms.

As the production of graphene-based nanomaterials such as GO is increasing, it is expected that a large amount of GO waste will be generated. The environment (i.e., soil and aquatic systems) will be amongst the final repositories of these wastes which means important natural microbial communities in such environments will be at risk of GO exposure. However, little is known about how these communities respond to environmental stresses in synergy with the presence of GO. In this study, the effect of three different stress conditions: temperature (5, 25 and 40 °C); pH (5 to 9) and osmotic stress (51, 219 and 320 mM NaCl) in addition to GO treatment was investigated on the viability and physiology of biofilms and planktonic cells of soil bacterium P. putida. It was found that planktonic cells were more resistant to GO alone compared to biofilms. However, the cells were sensitive to GO when exposed to pH or osmotic stresses. Temperature was not found to influence the survival of biofilm with or without exposure to GO. However, low pH caused a reduction in colony-forming units (CFU) at pHs 5 and 6 for the pre-treated samples, while biofilms at pH 7-9 did not show any decrease. Interestingly, the post-treatment of planktonic cells or biofilms with GO showed a significant reduction in CFU at all pH ranges. The effect of higher osmotic stress in combination with GO resulted in a significant reduction in biofilms. These results show that the effect of stresses naturally occurring in the environment can be affected and changed when in combination with GO and can potentially affect the balance of natural biofilms.

The Effect of Bacteria on the Stability of Microfluidic-Generated Water-in-Oil Droplet.

Microencapsulation in emulsion droplets has great potential for various applications such as food which require formation of highly stable emulsions. Bacterial-emulsion interactions affect the physiological status of bacteria while bacterial cell characteristics such as surface-active properties and metabolic activity can affect emulsion stability. In this study, the viability and growth of two different bacterial species, Gram-negative Escherichia coli and Gram-positive Lactobacillus paracasei, encapsulated in water-in-oil (W/O) droplets or as planktonic cells, were monitored and their effect on droplet stability was determined. Microencapsulation of bacteria in W/O droplets with growth media or water was achieved by using a flow-focusing microfluidic device to ensure the production of highly monodispersed droplets. Stability of W/O droplets was monitored during 5 days of storage. Fluorescence microscopy was used to observe bacterial growth behaviour. Encapsulated cells showed different growth to planktonic cells. Encapsulated E. coli grew faster initially followed by a decline in viability while encapsulated L. paracasei showed a slow gradual growth throughout storage. The presence of bacteria increased droplet stability and a higher number of dead cells was found to provide better stability due to high affinity towards the interface. The stability of the droplets is also species dependent, with E. coli providing better stability as compared to Lactobacillus paracasei.

Effect of Wheat Replacement by Pulse Flours on the Texture, Color, and Sensorial Characteristics of Crackers: Flash Profile Analysis.

Pulse flours are growing in popularity as alternatives to wheat in bakery products due to their high protein and nutritional value. However, the effect of different pulse species and substitution on sensory perception is unclear. The sensory perception of crackers made by partially replacing wheat with chickpea (40-80%) and lupin flour (10-30%) was evaluated using Flash profile analysis in association with instrumental analysis of texture and color. Flash profile analysis was conducted in Greece and Indonesia in order to allow culture comparison of the profiling of the samples and language by the subjects of the panel. Lightness (L∗) and hardness of crackers were decreased by the addition of pulses. Flash profile analysis indicated an association among color, texture, and sensory perception by judges. Derived attributes were associated with the physicochemical characteristics and raw materials of crackers for both panels. GPA analysis of Greek panel indicated that increasing the replacement of wheat led to the generation of more attributes regardless of pulse species, while the Indonesian panel was able to detect differences among pulse species.

Mechanism of antimicrobial activity of honeybee (Apis mellifera) venom on Gram-negative bacteria: Escherichia coli and Pseudomonas spp.

Honeybee venom (Apitoxin, BV), a secretion substance expelled from the venom gland of bees, has being reported as antimicrobial against various bacterial species; however, the mechanism of action remains uncharacterized. In this study, the antibacterial activity of BV was investigated on hygiene indicator Escherichia coli and the environmental pathogen and spoilage bacterial species, Pseudomonas putida and Pseudomonas fluorescens. An array of methods was combined to elucidate the mode of action of BV. Viability by culture on media was combined with assessing cell injury with flow cytometry analysis. ATP depletion was monitored as an indicator to metabolic activity of cells, by varying BV concentration (75, 225and 500 µg/mL), temperature (25 [Formula: see text] and 37 [Formula: see text]), and time of exposure (0 to 24 h). Venom presented moderate inhibitory effect on E. coli by viability assay, caused high membrane permeability and significant ATP loss where the effect was increased by increased concentration. The viability of P. putida was reduced to a greater extent than other tested bacteria at comparable venom concentrations and was dictated by exposure time. On the contrary, P. fluorescens appeared less affected by venom based on viability; however, flow cytometry and ATP analysis highlighted concentration- and time-dependent effect of venom. According to Transmission Electron Microscopy results, the deformation of the cell wall was evident for all species. This implies a common mechanism of action of the BV which is as follows: the cell wall destruction, change of membrane permeability, leakage of cell contents, inactivation of metabolic activity and finally cell death.

Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12.

Antimicrobial resistance is an ever-growing health concern worldwide that has created renewed interest in the use of traditional anti-microbial treatments, including honey. However, understanding the underlying mechanism of the anti-microbial action of honey has been hampered due to the complexity of its composition. High throughput genetic tools could assist in understanding this mechanism. In this study, the anti-bacterial mechanism of a model honey, made of sugars, hydrogen peroxide, and gluconic acid, was investigated using genome-wide transposon mutagenesis combined with high-throughput sequencing (TraDIS), with the strain Escherichia coli K-12 MG1655 as the target organism. We identified a number of genes which when mutated caused a severe loss of fitness when cells were exposed to the model honey. These genes encode membrane proteins including those involved in uptake of essential molecules, and components of the electron transport chain. They are enriched for pathways involved in intracellular homeostasis and redox activity. Genes involved in assembly and activity of formate dehydrogenase O (FDH-O) were of particular note. The phenotypes of mutants in a subset of the genes identified were confirmed by phenotypic screening of deletion strains. We also found some genes which when mutated led to enhanced resistance to treatment with the model honey. This study identifies potential synergies between the main honey stressors and provides insights into the global antibacterial mechanism of this natural product.

Propolis particles incorporated in aqueous formulations with enhanced antibacterial performance.

In recent years, the use of natural bioactives in food, pharmaceutical and cosmetic industries has emerged as a global formulation development trend. Although natural bioactives exhibit promising properties, they are also associated with chemical instability or poor aqueous solubility. One such bioactive with beneficial functionalities but limited industrial applicability within industry is propolis. The purpose of this study was to investigate means to enable enhancement to the antibacterial activity of propolis-based aqueous formulations. Dry propolis was firstly extracted from crude material and the effect of common carrier phases used for dissolution of propolis for antibacterial assays was investigated. Consequently, the extract was formulated into propolis sub-micron aqueous dispersions via direct ultrasonication. Processing time was varied, and all formed particles were characterised immediately after production in terms of size, polydispersity and zeta potential, and then again after a month-long storage period. When tested on E. coli cells, 15% propolis dispersions caused a bactericidal effect, which was sonication time and time of exposure dependent. Particles formed at the shortest sonication period (4 min) resulted in higher cell injury while those processed the longest (10 min) caused greater cell death and with AFM imaging, cell membrane alterations were confirmed. Chemically, for whole dispersions and carrier phases alone, free radical scavenging activity and total phenol content were slightly enhanced at longer sonication times. Overall, the present work suggests that formulating propolis extract sub-micron aqueous dispersions via sonication enhances their antibacterial performance via a synergistic effect involving both their carrier and dispersed phases.

Optimisation of bacterial release from a stable microfluidic-generated water-in-oil-in-water emulsion.

Application of droplet microfluidics for the encapsulation of bacteria in water-in-oil-in-water (W/O/W) emulsion allows for production of monodisperse droplets with controllable size. In this study the release of bacteria from W/O/W emulsion, the effect of the double emulsion structure on bacterial growth and metabolic activity, and the stability and mechanism of bacterial release were investigated. W/O/W emulsions were formed using a double flow-focusing junction microfluidic device under controlled pressure to produce droplets of approximately 100 µm in diameter containing an inner aqueous phase (W1) of about 40-50 µm in diameter. GFP-labelled Escherichia coli (E. coli-GFP) bacteria were encapsulated within the W1 droplets and the stability of emulsions was studied by monitoring droplet size and creaming behaviour. The double emulsions were stabilised using a hydrophilic (Tween 80) and a lipophilic surfactant (polyglycerol polyricinoleate) and were destabilised by altering the osmotic balance, adding NaCl either in the inner W1 phase (hypo-osmotic) or outer W2 phase (hyper-osmotic). The release of E. coli-GFP was monitored by plating on agar whereby the colony form unit (CFU) of the released bacteria was determined while fluorescent microscopy was employed to observe the mechanism of release from the droplets. The release of E. coli-GFP was significantly increased with higher concentrations of NaCl and lower amounts of Tween 80. Microscopic observation revealed a two-step mechanism for the release of bacteria: double W/O/W emulsion droplet splitting to release W1 droplets forming a secondary double emulsion followed by the collapse of W1 droplets to release E. coli-GFP into the continuous aqueous phase.

Use of a model to understand the synergies underlying the antibacterial mechanism of H2O2-producing honeys.

Honey has been valued as a powerful antimicrobial since ancient times. However, the understanding of the underlying antibacterial mechanism is incomplete. The complexity and variability of honey composition represent a challenge to this scope. In this study, a simple model system was used to investigate the antibacterial effect of, and possible synergies between, the three main stressors present in honey: sugars, gluconic acid, and hydrogen peroxide (H2O2), which result from the enzymatic conversion of glucose on honey dilution. Our results demonstrated that the synergy of H2O2 and gluconic acid is essential for the antibacterial activity of honey. This synergy caused membrane depolarization, destruction of the cell wall, and eventually growth inhibition of E. coli K-12. The presence of H2O2 stimulated the generation of other long-lived ROS in a dose-dependent manner. Sugars caused osmosis-related morphological changes, however, decreased the toxicity of the H2O2/gluconic acid. The susceptibility of catalase and general stress response sigma factor mutants confirmed the synergy of the three stressors, which is enhanced at higher H2O2 concentrations. By monitoring cellular phenotypic changes caused by model honey, we explained how this can be bactericidal even though the antimicrobial compounds which it contains are at non-inhibitory concentrations.

Diversity of Lactobacillus Species of Stilton Cheese Relates to Site of Isolation.

This study has characterized the dominant non-starter Lactobacillus species isolated from different sites in a Stilton cheese to establish its diversity, stress-tolerance, anti-microbial activity and potential contribution to quality of cheese. Fifty-nine Lactobacillus isolates were cultured from the outer crust, blue veins and white core of the cheese and were speciated phenotypically and by 16S rDNA sequence analysis. Lactobacillus plantarum was the dominant species detected with only two isolates identified as Lactobacillus brevis. Strains were typed by pulse-field gel electrophoresis (PFGE) using the enzyme NotI to examine their genomic diversity. Cluster analysis of PFGE patterns produced five major clusters which associated isolates with their sites of isolation within the cheese. One L. plantarum isolate from each cheese site was selected and evaluated for salt, acid, relative humidity, and heat tolerance to determine whether stress conditions within the isolation site selected their phenotype. D 72°C values were 6, 13, and 17 s for strains from the crust, veins and core, respectively, suggesting strains on the crust may not have been able to survive pasteurization and therefore had been added post-pasteurization. All strains recovered from heat injury within 24-48 h at 4°C. pH values of 3, 3.5, and 4 suppressed growth but strains showed a varying ability to grow at pH 4.5 and 5; isolates from the core (which has the lowest pH) were the most acid-tolerant. All strains grew at 3.5 and 5% salt but were suppressed at 10%; those from the crust (which has a lower water activity) were the most halo-tolerant, growing at 8% salt whereas strains from the core were sensitive to this salt concentration. All 57 L. plantarum isolates were examined for antimicrobial activity and variable activity against Lactobacillus pentosus and other genera was demonstrated; plantaricin EF genes were present in 65% of strains. It was concluded that there are varied phenotypes and genotypes of Lactobacillus in a Stilton cheese according to site of isolation. Occurrence of different L. plantarum genotypes could contribute to variation in the cheese quality from batch to batch and provides criteria for selecting isolates as potential adjunct cultures.

The synergistic effect of enzymatic detergents on biofilm cleaning from different surfaces.

Biofilm growth is a significant source of contamination in the food industry. Enzymes are considered green countermeasures against biofilm formation in the food industry owing to their biodegradability and low toxicity. In this study, the synergistic effect of enzymes was studied against biofilm cleaning from hard surfaces. A mixed-microbial sample was sourced from a meat packaging line and biofilms were grown under high shear conditions on stainless steel and polyethylene surfaces. A model cleaning-in-place (CIP) parallel-plate flow chamber was used for firstly, the enzymatic cleaning and secondly, a disinfection step. The cleaning effectiveness was evaluated in response to different formulations containing non-foaming commercial surfactants among with amylase, protease and lipase at neutral pH. The formulation combining all three enzymes was the most effective, showing a synergy essential for the deformation of biofilm structure and consequently better disinfection of both material surfaces.

Antibacterial effect of graphene oxide (GO) nano-particles against Pseudomonas putida biofilm of variable age.

Graphene oxide (GO) has been reported to possess antibacterial activity; therefore, its accumulation in the environment could affect microbial communities such as biofilms. The susceptibility of biofilms to antimicrobials is known to depend on the stage of biofilm maturity. The aim of this study was to investigate the effect of GO nano-particles on Pseudomonas putida KT2440 biofilm of variable age. FT-IR, UV-vis, and Raman spectroscopy confirmed the oxidation of graphene while XPS confirmed the high purity of the synthesised GO over 6 months. Biofilms varying in maturity (24, 48, and 72 h) were formed using a CDC reactor and were treated with GO (85 µg/mL or 8.5 µg/mL). The viability of P. putida was monitored by culture on media and the bacterial membrane integrity was assessed using flow cytometry. P. putida cells were observed using confocal microscopy and SEM. The results showed that GO significantly reduced the viability of 48-h biofilm and detached biofilm cells associated with membrane damage while the viability was not affected in 24- and 72-h biofilms and detached biofilm cells. The results showed that susceptibility of P. putida biofilm to GO varied according to age which may be due to changes in the physiological state of cells during maturation. Graphical abstract.

Soy sauce fermentation: Microorganisms, aroma formation, and process modification.

Soy sauce is an increasingly popular oriental fermented condiment produced through a two-step fermentation process called koji (solid-state fermentation) and moromi (brine fermentation). Complex microbial interactions play an essential role in its flavor development during the fermentation. Tetragenococcus halophilus and Zygosaccharomyces rouxii are predominant among the microbes involved in the moromi stage. Despite their importance for producing a wide range of volatile compounds, antagonism can occur due to different growth condition requirements. Furthermore, microbial interactions in moromi fermentation are affected by current efforts to reduce salt in soy sauce, in order to tackle slow fermentation due to low metabolic activity of microbes and increased health risk related to high sodium intake. Attempts to enhance and accelerate flavor formation in the presence of high salt concentration include the inoculation with mixed starter cultures, genetic modification, cell, and enzyme immobilization. Although salt reduction can accelerate the microbial growth, the flavor quality of soy sauce is compromised. Several approaches have been applied to compensate such loss in quality, including the use of salt substitutes, combination of indigenous cultures, pretreatment of raw material and starter cultures encapsulation. This review discusses the role of microorganisms in soy sauce production in relation to flavor formation and changes in production practices.

Acid production, growth kinetics and aroma profiles of Lactobacillus flora from Stilton cheese.

The effect of Lactobacillus plantarum isolates from Stilton cheese on aroma profiles of milk fermentation was examined. Representative Lb. plantarum isolates were cultured alone and in combination with acid-producing and non-acid producing Lactococcus lactis NCIMB 9918 in UHT milk at 30 & 18 °C for 48 h & 12 weeks, respectively in presence and absence of salt, simulating cheese production and ripening. During long-term ripening, Lb. plantarum grew faster when co-cultured with non-acid producing Lc. lactis in the presence of salt. One isolate of Lb. plantarum produced the highest concentration of alcohols, organic acids and acetoin. Co-culture of Lb. plantarum with acid-producing Lc. lactis enhanced acid and alcohol production, whereas co-inoculation with non-acid producing Lc. lactis increased acetoin synthesis. Lb. plantarum is an incidental organism in cheese and its presence is unpredictable. Occurrence of different genotypes of Lb. plantarum could contribute to batch to batch variation in the cheese aroma characteristics.

Water-in-oil-in-water double emulsion for the delivery of starter cultures in reduced-salt moromi fermentation of soy sauce.

This study investigated the application of water-oil-water (W1/O/W2) double emulsions (DE) for yeast encapsulation and sequential inoculation of Zygosaccharomyces rouxii and Tetragenococcus halophilus in moromi stage of soy sauce fermentation with reduced NaCl and/or substitution with KCl. Z. rouxii and T. halophilus were incorporated in the internal W1 and external W2 phase of DE, respectively. NaCl reduction and substitution promoted T. halophilus growth to 8.88 log CFU/mL, accompanied with faster sugar depletion and enhanced lactic acid production. Reducing NaCl without substitution increased the final pH (5.49) and decreased alcohols, acids, esters, furan and phenol content. However, the application of DE resulted in moromi with similar microbiological and physicochemical characteristics to that of high-salt. Principal component analysis of GC-MS data demonstrated that the reduced-salt moromi had identical aroma profile to that obtained in the standard one, indicating the feasibility of producing low-salt soy sauce without compromising its quality.

Utilisation of water-in-oil-water (W1/O/W2) double emulsion in a set-type yogurt model for the delivery of probiotic Lactobacillus paracasei.

W1/O/W2 emulsion in set-type yogurt has the potential to segregate probiotics in order to avoid interference with the starter culture as well as protection against harsh processing and digestion conditions. Lactobacillus paracasei subsp. paracasei DC 412 probiotic cells in milk-based W1/O/W2 emulsions were incorporated in yogurt, in addition to starter cultures Lactobacillus bulgaricus and Streptococcus thermophilus, and the effect on the fermentation, bacterial growth kinetics, physicochemical properties, and structural characteristics was investigated. Stability of W1/O/W2 was monitored with optical microscopy and cryo-SEM and localisation of encapsulated L. paracasei in yogurt was monitored using fluorescent microscopy. During fermentation, starter culture was not affected by introduction of L. paracasei and/or W1/O/W2 emulsion. The viability of L. paracasei encapsulated in W1/O/W2 emulsion was enhanced during storage and after exposure to simulated gastrointestinal conditions. L. paracasei remained within the inner W1 phase till the end of the storage period (28 days at 4 °C). Moreover, W1/O/W2 emulsion altered physicochemical and textural properties; however, these were within acceptable range. These results demonstrate the capability of W1/O/W2 emulsion to be utilised for probiotic fortification of yogurt to increase functionality without interfering with starter culture and fermentation.

Segregation of Tetragenococcus halophilus and Zygosaccharomyces rouxii using W1/O/W2 double emulsion for use in mixed culture fermentation.

Antagonism in mixed culture fermentation can result in undesirable metabolic activity and negatively affect the fermentation process. Water-oil-water (W1/O/W2) double emulsions (DE) could be utilized in fermentation for segregating multiple species and controlling their release and activity. Zygosaccharomyces rouxii and Tetragenococcus halophilus, two predominant microbial species in soy sauce fermentation, were incorporated in the internal W1 and external W2 phase of a W1/O/W2, respectively. The suitability of DE for controlling T. halophilus and Z. rouxii in soy sauce fermentation was studied in relation to emulsion stability and microbial release profile. The effects of varying concentrations of Z. rouxii cells (5 and 7logCFU/mL) and glucose (0%, 6%, 12%, 30% w/v) in the W2 phase were investigated. DE stability was determined by monitoring encapsulation stability (%), oil globule size, and microstructure with fluorescence and optical microscopy. Furthermore, the effect of DE on the interaction between T. halophilus and Z. rouxii was studied in Tryptic Soy Broth containing 10% w/v NaCl and 12% w/v glucose and physicochemical changes (glucose, ethanol, lactic acid, and acetic acid) were monitored. DE destabilization resulted in cell release which was proportional to the glucose concentration in W2. Encapsulated Z. rouxii presented higher survival during storage (~3 log). The application of DE affected microbial cells growth and physiology, which led to the elimination of antagonism. These results demonstrate the potential use of DE as a delivery system of mixed starter cultures in food fermentation, where multiple species are required to act sequentially in a controlled manner.

Effects of co-inoculation and sequential inoculation of Tetragenococcus halophilus and Zygosaccharomyces rouxii on soy sauce fermentation.

The use of Tetragenococcus halophilus and Zygosaccharomyces rouxii as starter cultures is essential for desirable volatiles production during moromi stage of soy sauce fermentation. In this study, the effect of simultaneous and sequential inoculation of cultures in moromi fermentation models, with respect to viability, physicochemical changes, and volatiles formation (using SPME-GC/MS) was investigated. Interestingly, an antagonism was observed as T. halophilus only proliferated (3 log increase) in the presence of Z. rouxii, while Z. rouxii growth was suppressed by 4 log in concurrence with pH increase to 7.31. Final content of reducing sugars, ethanol, acetic acid, and amino nitrogen did not differ significantly (p<0.05) between co-inoculation and sequential inoculation. However, Z. rouxii promoted alcohols formation and produced a more complex aroma profile under suppression. According to Principal Component Analysis (PCA), the inoculation sequence (co-inoculation and sequential) has impacts on volatile compound profiles during moromi fermentation.

Do oil-in-water (O/W) nano-emulsions have an effect on survival and growth of bacteria?

Nano-emulsions (typically droplet diameter<1µm) are common in foods, and have been extensively reported to present antimicrobial activity, however, the mechanism is not well defined, and some studies reported no effect. A review of the literature was conducted and revealed strongly contradictory reports regarding the antimicrobial effect of nano-emulsions even in reference to similar microbial species and formulations. Following up, this study aimed to investigate the effect of nano-emulsions on four bacterial species (Staphylococcus epidermidis, Bacillus cereus, Lactobacillus acidophilus and five Escherichia coli strains) possessing different surface charge and hydrophobicity. Model oil-in-water (O/W) emulsions with different size of oil droplets were prepared with sunflower oil stabilised by polysorbate 80 (Tween80) emulsifier (hydrophilic), using high shear mixing followed by ultrasonication. The viability of bacteria was monitored by culture, membrane integrity was assessed with flow cytometric analysis with propidium iodide (PI) staining and fluorescence microscopy monitored the spatial distribution of cells within the O/W emulsions. The stability of the nano-O/W emulsions in the presence of bacteria was assessed by monitoring the droplet size [D (4, 3)] and creaming height. In contrast to other reports the survival and growth of bacteria was not affected by the size of the oil droplets, no damage to the bacterial membrane was evident with flow cytometry and emulsion stability was not affected by the presence of bacteria during 7days of storage. Furthermore, the antimicrobial activity of caprylic acid (CA) was compared between O/W coarse and nano-emulsions while varying the concentration of the hydrophilic surfactant Tween80. The activity of CA was similar in nano-emulsion and coarse emulsion; however, it was higher than in bulk oil and was reduced with increasing Tween80 concentration, suggesting that its efficacy is dictated by formulation rather than oil droplet size. The results demonstrated no enhanced antimicrobial activity due to nano-sized oil droplets and that conclusions on nano-emulsions should be taken with caution.

Incorporation of water-in-oil-in-water (W1/O/W2) double emulsion in a set-type yogurt model.

The effect of W1/O/W2 emulsion incorporation in set-type yogurt on the acidification process, physicochemical properties, bacterial growth kinetics and structural characteristics was investigated. The W1/O/W2 emulsion was formed by using a two-step homogenisation process and milk as the W1 and W2 phases, and stability was monitored with optical microscopy and cryo-SEM. Adding the W1/O/W2 emulsions reduced the acidification rate, viscosity and water retention capacity. Texture (adhesiveness, cohesiveness, hardness, and gumminess) differed in yogurts containing W1/O/W2 emulsion compared to controls during the acidification process, however, trends became stable during storage. The growth of S. thermophilus during the acidification process of yogurt was reduced in the presence of W1/O/W2 emulsion while L. bulgaricus trended higher during storage. This study shows that yogurts containing W1/O/W2 emulsion are feasible subject to processing modification.