Fresh-cut produce like lettuce may contain a very high microbial load, including human pathogens. Therefore, the need for antimicrobial agents at post-harvest stages to mitigate microbial cross-contamination and growth is evident. Sanitation based on non-thermal plasma (NTP) reveals innovative food processing possibilities by application at different points along the food chain, for production, modification, and preservation, as well as in packaging of plant- and animal-based food. The aim of the present study was to evaluate the applicability of plasma-treated water (PTW) as antimicrobial process water additives for washing in fresh-cut lettuce processing. Antibacterial activities of PTW the natural occurring microflora of lettuce were examined. Different process variants of PTW application inside the washing process were investigated. Fresh-cut lettuce were investigated regarding microbiological safety and food quality. Samples were analyzed for antimicrobial and metabolic activity as well as metabolic vitality to prove food safety. The investigations for food quality included color and texture analyses and nitrate concentration detection in fresh tissue as well as microscopic measurements by scanning electron microscopy (SEM) and atomic force microscopy (AFM) for tissue surface structure and transmission electron microscopy (TEM) for cell organelle investigations. The application of PTW allowed up to 5 log₁₀ cycle reduction, depending on the process variant and scale (lab and pilot scale). The increase of antimicrobial activity was accompanied by a reduction of metabolic activity, but not consequently by a decrease in metabolic vitality. Food quality was not affected by the use of PTW in the washing process of the fresh-cut lettuce. The promising results in color and texture were supported by the results of the microscopic assays. These promising results may lead to an industrial application of PTW as process water additive in fresh-cut produce processing to reduce the microbial load on the food surface and in addition in the process water or on food processing surfaces.

The synergistic antimicrobial effects of plasma-processed air (PPA) and plasma-treated water (PTW), which are indirectly generated by a microwave-induced non-atmospheric pressure plasma, were investigated with the aid of proliferation assays. For this purpose, microorganisms (Listeria monocytogenes, Escherichia coli, Pectobacterium carotovorum, sporulated Bacillus atrophaeus) were cultivated as monocultures on specimens with polymeric surface structures. Both the distinct and synergistic antimicrobial potential of PPA and PTW were governed by the plasma-on time (5–50 s) and the treatment time of the specimens with PPA/PTW (1–5 min). In single PTW treatment of the bacteria, an elevation of the reduction factor with increasing treatment time could be observed (e.g., reduction factor of 2.4 to 3.0 for P. carotovorum). In comparison, the combination of PTW and subsequent PPA treatment leads to synergistic effects that are clearly not induced by longer treatment times. These findings have been valid for all bacteria (L. monocytogenes > P. carotovorum = E. coli). Controversially, the effect is reversed for endospores of B. atrophaeus. With pure PPA treatment, a strong inactivation at 50 s plasma-on time is detectable, whereas single PTW treatment shows no effect even with increasing treatment parameters. The use of synergistic effects of PTW for cleaning and PPA for drying shows a clear alternative for currently used sanitation methods in production plants. Highlights: Non-thermal atmospheric pressure microwave plasma source used indirect in two different modes—gaseous and liquid; Measurement of short and long-living nitrite and nitrate in corrosive gas PPA (plasma-processed air) and complex liquid PTW (plasma-treated water); Application of PTW and PPA in single and combined use for biological decontamination of different microorganisms.

Microorganisms are predominantly organized in biofilms, where cells live in dense communities and are more resistant to external stresses compared to their planktonic counterparts. With in vitro experiments, the susceptibility of Candida albicans biofilms to a non-thermal plasma treatment (plasma source kINPen®09), in terms of growth, survival, and cell viability was investigated. Behind that background, the C. albicans strain SC5314 (ATCC® MYA-2876™) was plasma treated for different time periods (30 s, 60 s, 120 s, 180 s, 300 s). The results of the experiments embracing colony forming units, fluorescence LIVE/DEAD™ assays, and XTT assays and revealed a negative influence of the plasma treatment on the proliferation ability, vitality, and the metabolism of C. albicans biofilms, respectively. Morphological analysis of plasma treated biofilms using atomic force microscopy supported the indications for lethal plasma effects concomitant with membrane disruptions and the loss of intracellular fluid. Controversial to other publications, fluorescence- and confocal laser scanning-microscopic inspection of plasma treated biofilms indicated that an inactivation of cells mainly appeared on the bottom side of the biofilms. If this inactivation leads to a detachment of the biofilms from the overgrown surface, it might offer completely new approaches in the plasma treatment of biofilms. Because of its biochemical-mechanical mode of action, resistance of microbial cells against plasma are unknown at this state of research. Importance Microbial communities are an increasing problem in medicine but also in industry. Thus, an efficient and rapid removal of biofilms is becoming increasingly important. With the aid of the kINPen®09, a radiofrequency plasma jet (RFPJ), decisive new findings on the effects of plasma on C. albicans biofilms could be obtained. This work was able to show that the inactivation of biofilms mainly takes place on the bottom side, which in turn offers new possibilities for the removal of biofilms by other strategies, e.g. mechanical treatment.This results demonstrated that non-thermal atmospheric pressure plasma is well-suited for biofilm decontamination.

Summary: The susceptibility of Candida albicans biofilms to a non‐thermal plasma treatment has been investigated in terms of growth, survival and cell viability by a series of in vitro experiments. For different time periods, the C. albicans strain SC5314 was treated with a microwave‐induced plasma torch (MiniMIP). The MiniMIP treatment had a strong effect (reduction factor (RF) = 2.97 after 50 s treatment) at a distance of 3 cm between the nozzle and the superior regions of the biofilms. In addition, a viability reduction of 77% after a 20 s plasma treatment and a metabolism reduction of 90% after a 40 s plasma treatment time were observed for C. albicans . After such a treatment, the biofilms revealed an altered morphology of their cells by atomic force microscopy (AFM). Additionally, fluorescence microscopy and confocal laser scanning microscopy (CLSM) analyses of plasma‐treated biofilms showed that an inactivation of cells mainly appeared on the bottom side of the biofilms. Thus, the plasma inactivation of the overgrown surface reveals a new possibility to combat biofilms.

For the decontamination of surfaces in the food production industry, plasma-generated compounds such as plasma-treated water or plasma-processed air offer many promising possibilities for future applications. Therefore, the antimicrobial effect of water treated with microwave-induced plasma (MidiPLexc) on Pseudomonas fluorescens biofilms was investigated. A total of 10 mL deionized water was treated with the MidiPLexc plasma source for 100, 300 and 900 s (pretreatment time) and the bacterial biofilms were exposed to the plasma-treated water for 1, 3 and 5 min (post-treatment time). To investigate the influence of plasma-treated water on P. fluorescens biofilms, microbiological assays (colony-forming units, fluorescence and XTT assay) and imaging techniques (fluorescence microscopy, confocal laser scanning microscopy, and atomic force microscopy) were used. The colony-forming units showed a maximum reduction of 6 log₁₀ by using 300 s pretreated plasma water for 5 min. Additionally, a maximum reduction of 81% for the viability of the cells and a 92% reduction in the metabolic activity of the cells were achieved by using 900 s pretreated plasma water for 5 min. The microscopic images showed evident microbial inactivation within the biofilm even at the shortest pretreatment (100 s) and post-treatment (1 min) times. Moreover, reduction of the biofilm thickness and increased cluster formation within the biofilm was detected. Morphologically, the fusion of cell walls into a uniform dense cell mass was detectable. The findings correlated with a decrease in the pH value of the plasma-treated water, which forms the basis for the chemically active components of plasma-treated water and its antimicrobial effects. These results provide valuable insights into the mechanisms of inactivation of biofilms by plasma-generated compounds such as plasma-treated water and thus allow for further parameter adjustment for applications in food industry.

Plasma-generated compounds conceived by microwave-induced plasma (MidiPLexc) were physicochemically investigated in their molecular compositions. Therefore, plasma-treated water (PTW) was generated by treatment with the MidiPLexc within a 1 l glass bottle for a pre-treatment time of 100 s, 300 s and 900 s. The PTW was further investigated in its composition of reactive oxygen/nitrogen species. For this purpose, the hydrogen peroxide (H ₂ O ₂ ) concentration of the PTW was determined electrochemically using chronoamperometry, and the anionic components were determined using ion chromatography. As a result, H ₂ O ₂ concentrations of up to 720 mg l ⁻¹ , nitrite concentrations of 1600 mg l ⁻¹ and nitrate concentrations of 72.3 mg l ⁻¹ could be detected after 900 s pre-treatment time of the water. The compounds obtained by ion chromatography were fractionated and subsequently confirmed by mass spectrometry. Once an overview of the chemical composition of PTW had been obtained, its effects on pathogens like Pseudomonas fluorescens and L. monocytogenes , which are both of particular importance as pathogenic contaminants during food production, were tested. In that process, a maximum reduction in the colony forming units of 4 log ₁₀ steps for P. fluorescens and 3 log ₁₀ steps for L. monocytogenes could be observed. The LIVE/DEAD assay showed a maximum reduction in the ratio G/R of 67% for P. fluorescens and 38% for L. monocytogenes . In addition, the XTT assay results showed a maximum cell metabolism reduction of 96% for P. fluorescens and 91% for L. monocytogenes .