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 Electronic supplementary material The online version of this article (10.1007/s11356-019-05688-9) contains supplementary material, which is available to authorized users.

This study investigated the application of water-oil-water (W/O/W) 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 W and external W 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.

W/O/W 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 W/O/W 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 W/O/W 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 W/O/W emulsion. The viability of L. paracasei encapsulated in W/O/W emulsion was enhanced during storage and after exposure to simulated gastrointestinal conditions. L. paracasei remained within the inner W phase till the end of the storage period (28 days at 4 °C). Moreover, W/O/W emulsion altered physicochemical and textural properties; however, these were within acceptable range. These results demonstrate the capability of W/O/W emulsion to be utilised for probiotic fortification of yogurt to increase functionality without interfering with starter culture and fermentation.

The results suggest that release of bacteria from W1/O/W2 emulsion can be controlled by varying the formulation. Release occurs due to oil globule bursting independent to diffusion.

Highlights A multi-frequency study of ultrasound (US) and nisin for microbial inactivation. US impacts E. coli at 500 kHz only; L. innocua resists all frequencies studied. Nisin applied before US enhances inactivation of E. coli but not when applied after. Attributed to outer membrane destabilisation by US allowing nisin penetration. System structure (viscosity) reduces US inactivation efficacy.