Modelling of Inactivation Kinetics of Escherichia coli on Radish and Hemp seeds and Their Physicochemical Properties after Non-Thermal Plasma Treatment: A Comparative Study of Two Plasma Sources

Modelling of Inactivation Kinetics of Escherichia coli on Radish and Hemp seeds and Their Physicochemical Properties after Non-Thermal Plasma Treatment: A Comparative Study of Two Plasma Sources | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Modelling of Inactivation Kinetics of Escherichia coli on Radish and Hemp seeds and Their Physicochemical Properties after Non-Thermal Plasma Treatment: A Comparative Study of Two Plasma Sources Silvia Mošovská, Mohamed Khalaf Abdelmajeed Fawwaz, Petra Šrámková, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7392470/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract In recent years, seed sprout consumption has been associated with several infectious outbreaks. To ensure sprouts are safe to eat, any potential pathogens must be eliminated before the sprouting process begins. The objective of this study was to compare the efficacy of two different plasma sources, producing non-thermal plasma under ambient air conditions, on the inactivation kinetics of Escherichia coli on radish and hemp seeds intended for sprouting. Plasma treatments were conducted using Diffuse Coplanar Surface Barrier Discharge (DCSBD) and piezoelectric direct discharge plasma called the Piezobrush PZ3. Microbial inactivation data were modelled using the Bigelow log-linear, biphasic, and Weibull models. After 90 seconds of DCSBD plasma treatment, reductions of 4.79 log10 CFU/g in radish seeds and 5.27 log10 CFU/g in hemp seeds were achieved. The inactivation effect of Piezobrush PZ3 was less effective in comparison to DCSBD and more pronounced against E. coli on hemp seeds than on radish seeds. Plasma treatments enhanced the physicochemical properties of seeds without causing significant changes to their surface morphology. Treatment with both plasma sources led to enhanced wettability in both seed types and an increase in soluble protein content in radish seeds. In the treatment with Piezobrush PZ3, reducing sugar content in both seed types increased up to 180 seconds of plasma exposure; however, prolonged exposure resulted in a subsequent decrease. Non-thermal plasma is a promising technology for the decontamination of seeds intended for sprouting; however, selecting the appropriate plasma discharge is crucial. Inactivation kinetics Escherichia coli non-thermal plasma seeds physicochemical properties sprouts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Germination is recognized as an efficient bioprocess for enhancing the levels of bioactive compounds in seeds, such as γ-aminobutyric acid, polyphenols, and vitamins. These compounds can function as dietary antioxidants, playing a significant role in protecting the body from oxidative stress (Liu et al. 2022). Consequently, the consumption of sprouts may contribute to the prevention of multiple chronic disorders (Wuytack et al. 2003). On the other hand, seeds are germinated under warm, moist, and nutrient-rich conditions that promote the proliferation of microorganisms, including pathogens such as Escherichia coli or Salmonella spp. Because sprouts are typically consumed raw, they represent a potential high-risk factor for consumer safety (Budryn et al. 2019, Machado-Moreira et al. 2021). Over the past decade, the consumption of seed sprouts has been epidemiologically associated with numerous infectious disease outbreaks on a global scale. Notable cases include the outbreak in Germany and other European countries in 2011 which was associated with the consumption of fenugreek sprouts contaminated with E. coli O104:H4. The outbreak resulted in 4,000 cases of infection, including 50 deaths (EFSA 2011). Sprouts can become contaminated through many ways; however the seeds themselves are primarily source of contamination (Machado-Moreira et al. 2021). Due to their ability to rapidly proliferate during sprouting, even a small number of pathogenic cells on seeds can pose a significant health risk. Therefore, preventing seed contamination or removing pathogens before sprouting is essential to ensure product safety (Wuytack et al. 2003). Chemical disinfectants such as chlorine compounds, trisodium phosphate, and acids have been widely used to reduce pathogens on seeds, though their effectiveness is limited, and their use is not permitted in organic production in some countries, such as Germany. Hence, increasing concerns about health and environmental impact of chemical agents have driven interest in alternative techniques, such as low-temperature plasma (Puligundla et al. 2017). Low-temperature plasma (LTP) has attracted increasing attention in recent years for its antimicrobial potential. Plasma, a partially or fully ionized gas consisting of reactive oxygen and nitrogen species (RONS), UV photons, and charged particles (Mandal et al. 2018), has demonstrated significant antimicrobial activity against a broad spectrum of microorganisms inoculated on the surface of many food matrices (Hertwig et al. 2017; Wiktor et al. 2020; Casado et al. 2024). LTP treatment offers several advantages for food processing applications primarily due to its ability to be generated continuously under atmospheric and/or reduced pressure conditions (Hertwig et al. 2018). On the other hand, the effect of cold plasma has been found to be highly dependent on several parameters, including working gas, discharge characteristics (dielectric barrier discharge, microwave discharge, gliding arc, plasma jet, etc.), input parameters (frequency, voltage, power density, etc.), and gas flow (Guo et al. 2015). One of the most commonly used plasma systems is dielectric barrier discharge (DBD) system (Hertwig et al. 2018). In DBD, a plasma discharge is generated between two parallel electrodes, with one or both electrodes covered by a dielectric material. This plasma system offers several benefits, including adaptable geometric configurations and scalability, ease of operation, straightforward and compact design, cost-effectiveness, safety, and favourable power supply features (Feizollahi et al. 2021). On the other hand, although DBD plasma is widely used for microbial inactivation due to its ability to generate reactive species at atmospheric pressure and ambient temperature (Ziuzina et al. 2014; Laroque et al. 2022; Yang et al. 2025), it also has several limitations. As mentioned in the section above, the effectiveness of this system is highly influenced by process parameters such as gas pressure, type, flow rate, frequency and power of plasma excitation. Additionally, factors like reactor geometry and the nature of the treated material can play a crucial role in its overall efficacy (Feizollahi et al. 2021). The growing need for compact, cost-effective, and versatile plasma devices has driven the development of a new class of piezoelectric cold plasma generators to produce the piezoelectric direct discharge (PDD). PDD is characterized by the initiation of micro-discharges in proximity to the treated surface, which defines its distinctive physical behaviour and broad application potential. The relatively low temperature of the plasma gases (only a few kelvins above ambient) enables the safe treatment of heat-sensitive materials such as fruits, seeds, and biological tissues. Additionally, the generation of high ozone concentrations enhances the system’s effectiveness for disinfection and sterilization applications (Korzec et al. 2021). The objective of this study was to investigate and compare the effects of two different plasma sources on the kinetic behaviour of Escherichia coli inoculated on the surface of radish and hemp seeds. The generated plasmas were characterized using optical emission spectroscopy. Following plasma treatments, surface diagnostics (scanning electron microscopy and wettability) and physicochemical characteristics (soluble proteins and reducing sugars) of the seeds were also examined to assess their impact on seed quality. Material and Methods Tested samples Radish ( Raphanus sativus L.) and hemp ( Cannabis sativa ) seeds were purchased at a local retail store (Bratislava, Slovakia). Samples were stored at room temperature in the dark. Bacterial strain and cell suspension preparation In this study, vegetative cells of E. coli CCM 3988 (the Collection of Microorganisms, Masaryk University, Brno, Czech Republic) was used. For inoculum preparation, 20 ml of sterile Mueller Hinton broth was aseptically inoculated with a colony of the tested bacterial strain. The cell suspension was incubated for 16 h at 37°C under continuous shaking (250 rpm) to reach the stationary phase of E. coli (approximate cell density: 10 8 cells/ml). Plasma source and plasma characterization The present study compared two different sources of non-thermal plasma operating at atmospheric pressure in ambient air. First plasma device, Diffuse Coplanar Surface Barrier Discharge (DCSBD) (Roplass, s.r.o, Modřice, CZ), is an effective source of non-equilibrium plasma that is being widely explored for various applications such as material processing (Šrámková et al. 2021), seed treatment (Ďurčányová et al. 2023; Tomeková et al. 2024) and pathogen decontamination (Mošovská et al. 2019; Medvecká et al. 2020). Detailed specifications and properties of the DCSBD plasma source have already been published (Černák et al. 2009; Černák et al. 2011). For our experiments, the electrode system was operated by an alternating current high voltage of up to 20 kV (peak-to-peak) with a frequency of ~ 15 kHz (HV generator VF 700, Lifetech s.r.o., CZ). The plasma treatment was performed with an input power of 400 W at atmospheric pressure in ambient air. The second plasma device is the commercially available piezoelectric direct discharge Piezobrush® PZ3 (Relyon plasma GmbH, Regensburg, Germany) (Korzec et al. 2021). The core component of this device is the piezoelectric cold plasma generator type CeraPlas™ F (TDK Electronics GmbH, Austria). It represents a piezoelectric resonant transformer with a maximum input power of 8 W, which operates at a resonant frequency of 50 kHz and sinusoidal waveform with peak-to-peak ~ 40 kV (Korzec et al. 2020). During the plasma treatment, the Piezobrush PZ3 was placed in a vertically stable position in the special holder as is depicted in the Fig. 1a). Electrical and optical properties of plasma generated by DCSBD using different experimental conditions have already been published (Šerá et al. 2021; Tomeková et al. 2024). The optical emission spectra (OES) of the plasma generated by Piezobrush PZ3 in ambient air were measured at an input power of 8 W, while the perpendicular distance between the optical fibre and the piezoelectric generator was approximately 8 cm. The radiation from the plasma was collected with the optical fibre (Avantes FC-UV200-2-SR, F1000 UV–VIS SR) connected to the AvaSpec-2048 TEC (Thermo-Electric-Cooled) spectrometer with a range of ~ (300–400) nm and a resolution of 20 px/nm. Characteristic lines identified from the measured optical emission spectra were used to estimate the vibrational temperatures in the Spectrum Analyzer 1.8 program (Navratil et al. 2006), and the rotational temperatures were determined using the peaks of the simulated spectra in the Specair 3.0 program (Laux 2002). Sample preparation Before plasma treatment, seeds were sterilized in an autoclave at 121 ᵒ C and 120 kPa for 20 minutes. Subsequently, seeds were inoculated with 1000 µL (hempseeds) and 600 µL (radish seeds) cell suspension. Then, the seeds were shaken by hand for 5 minutes to obtain a homogenous coating of E. coli on the seeds surface. Afterwards, the inoculated seeds were allowed to dry for about 24 h under at ambient temperature. Plasma treatment and determination of microbial inactivation For the DCSBD treatment, inoculated seeds (1 g) were spread on the ceramic plate within the plasma region (see Fig. 1d), e)). The seeds were treated in ambient air at exposure times of 15, 30, 60 and 90 seconds. During the treatment, the samples were stirred manually with the silicone brush. After each treatment, the ceramic plate and the other used tools were cleaned with isopropanol. For the treatment with the PiezoBrush PZ3, inoculated seeds (0.5 g) were transferred into the plastic container measuring 2 x 3 cm 2 . During the treatment, the container was manually moved back and forth vertically to obtain a homogenous treatment (see Fig. 1c)). Plasma treatment was applied for 60, 120, 180, 240 and 300 seconds. Trials were performed at least in duplicate. Immediately after treatment, the viable cell count was determined using the standard plate count method in duplicate (Houghtby et al. 1992). The recovery of viable of E. coli cells was performed by shaking the seeds (1 g for each treatment condition) in 9 mL of 0.85% sterile saline solution containing 0.1% Tween 80 (Merck, Germany) for 1 hour at 140 rpm. The resulting bacterial suspensions were serially diluted in 0.85% saline solution, and each dilution was plated on a Mueller-Hinton agar plate. Colony-forming units (CFU) were counted after 24 hours of incubation at 37 ᵒ C. Modelling of inactivation kinetics The experimental data were fitted using a biphasic (Cerf 1977), a log-linear (Bigelow and Esty 1920) and a Weibull (Mafart et al. 2002) models. The mean values of the inactivation data were modelled with GInaFit software (version 1.7 for Microsoft Excel24). The mathematical equations describing inactivation kinetics are shown in Table 1. Where N 0 is the initial microbial population (log CFU/g); N t is the microbial population after plasma treatment at the time t (log CFU/g); k max1 , k max2 and k are the inactivation rate constant (s − 1 ); f is the fraction of the initial population corresponding to the subpopulation more sensitive to the plasma treatment; δ is the scale parameter; p represents the shape parameter Surface diagnostic Scanning electron microscope (SEM) Possible micromorphological changes on the surface of studied seeds were monitored by scanning electron microscopy (SEM). Tescan Lyra 3 SEM microscope (Tescan, CZ) with an accelerating voltage of 1 kV in regime of secondary electrons and with magnifications of 2.5-10kx was employed. The samples subjected to this analysis were treated with both plasma devices at the longer used exposure times: DCSBD – 90 s, Piezobrush PZ3–300 s, and compared to reference seeds without plasma treatment. Wettability The changes in wettability were monitored by the water uptake of the seeds immersed in the water. Dry seeds (hemp and radish) in the amount of 50 pieces were placed in dry beakers. Untreated seeds (labelled REF) and plasma-treated seeds were weighed on an analytical balance (KERN ABT220-4NM, KERN & SOHN GmbH, Germany) to determine their initial weight in the dry state (m i ). Each set of seeds contained 50 seeds. Then 50 ml of deionized water was poured into the beakers to let the seeds imbibe. After 120 minutes, the seeds were removed from the water, the excess water was collected with filter paper and the imbibed seeds were weighed (m t ). The percentage water uptake (%) was expressed as the mass gain of the sample after 120 minutes (average value determined from triplicates) according to the following equation (Eqs. (1)): $$\:Water\:uptake\:m\:\left[\%\right]=\:\frac{{m}_{t}-{m}_{i}}{{m}_{i}}*100\%\:$$ 1 Physicochemical characteristics Measurement of soluble protein content Analysis of soluble protein content was performed using the Lowry method (Lowry et al. 1951). A 0.1 g defatted sample was placed in a microcentrifuge tube and vortexed with 1 mL of 0.1 M sodium phosphate buffer (pH 7.4) for 10 seconds. The mixture was then allowed to stand for 15 minutes, followed by centrifugation at 13,000 rpm for 10 minutes. A 0.25 mL aliquot of the supernatant was mixed with 1.25 mL of alkaline copper reagent and incubated at room temperature for 10 minutes. Subsequently, 0.125 mL of Folin–Ciocalteu reagent was added, and the mixture was incubated for an additional 30 minutes. Absorbance was measured at 660 nm using a spectrophotometer. Soluble protein concentration was determined from a standard curve prepared with bovine serum albumin (BSA). Measurement of reducing sugars Reducing sugars were analysed using the 3,5-dinitrosalicylic acid (DNS) colorimetric method with slight modification (Miller 1959). A 2 g sample was mixed with 10 mL of distilled water preheated to 80°C. The mixture was left to stand for 30 minutes under constant agitation on an orbital shaker at 200 rpm. Subsequently, 0.5 mL of Carrez I solution was added under continuous stirring, followed by the addition of 0.5 mL of Carrez II solution. The mixture was then diluted with distilled water to a final volume of 25 mL, thoroughly mixed, and filtered through filter paper (Whatman 1). A 25 µL aliquot of the filtrate was pipetted into a test tube, followed by the addition of 200 µL of DNS reagent. The mixture was thoroughly mixed and incubated in a boiling water bath for 5 minutes. After incubation, the sample was cooled, 2 mL of distilled water was added, and the tube was mixed again. Absorbance was measured at 540 nm. The concentration of reducing sugars was determined from a standard curve prepared using glucose. Statistical analysis The values represent the mean ± standard deviation (SD). Significant differences between means were determined using one-way ANOVA followed by Bonferroni correction post hoc test. Differences at p < 0.05 were considered significant. Results and discussion Plasma diagnostics Optical emission spectroscopy (OES) was used to diagnose the plasma generated by the plasma sources studied. DCSBD has been extensively studied in previous experiments and OES spectra have been collected under different operating conditions. The typical emission spectrum of a DCSBD plasma generated in ambient air contains the second positive system of N 2 (C-B) as the most intense spectral band in the UV region (~ 300–400 nm). From this spectral system, the vibrational (T vib ) and rotational (T rot ) temperatures were determined in the previous study (Tomeková et al. 2024) as follows: T vib = 2610 ± 225 K, T rot = 385 ± 30 K. For comparison, we have measured the OES spectrum of Piezobrush PZ3 plasma. Since this plasma is similarly generated in ambient air at atmospheric pressure like in case of DCSBD, the emission spectrum is very similar to that of DCSBD (Fig. 2). The dominant spectral system is the second positive system of N 2 (C-B), which was detected in the UV spectral range. We calculated the vibrational and rotational temperatures from this spectral band: T vib = 3110 ± 170 K, T rot = 560 ± 30 K. The significant difference between T vib and T rot is clear evidence of the non-equilibrium nature of the plasma generated by both plasma sources. Moreover, the UV radiation can also contribute to decontamination. Plasma-induced microbial inactivation and kinetic modelling The surviving population of E. coli inoculated on the surface of radish and hemp seeds after plasma treatments is presented in Fig. 3. The obtained results indicated varying sensitivity of E. coli inoculated on the surface of tested seeds to plasma treatments, depending on both the plasma source and the seed type. It was evident that the bactericidal effect of DCSBD plasma treatment was significantly higher compared to that of the Piezobrush PZ3. As shown in Fig. 3 (A, B), a considerable reduction of E. coli inoculated on surface of seeds was observed with increasing exposure time. This finding is in agreement with our previous work (Mošovská et al. 2018; Mošovská et al. 2019; Medvecká et al. 2020), as well as with the research of Hertwig et al. (2017), which indicated the strong antimicrobial potential of DCSBD plasma. The current work used DCSBD plasma system generated in ambient air. Ambient air plasma produces high concentrations of NOx and OH radicals (Mošovská et al. 2019), all of which have a direct impact on microorganisms and may result in microbial inactivation (Laroussi and Leipold 2004; Hertwig et al. 2017). Table 2 Statistical analysis of different models for the inactivation of E. coli on radish and hemp seeds treated with DCSBD and Piezobrush PZ3 plasma treatments Plasma source Parameters Radish seeds Hemp seeds DCSBD R 2 0.9933 0.9974 Adj. R 2 0.9731 0.9965 MSSE 0.1203 0.0150 RMSE 0.3468 0.1225 Piezobrush PZ3 R 2 0.9674 0.9613 Adj. R 2 0.9457 0.9354 MSSE 0.0144 0.0589 RMSE 0.1198 0.2427 Mathematical modelling of inactivation kinetics is essential for quantitative exposure and risk assessment, and it also enables comparison of different processing technologies in terms of their effectiveness in reducing microbial populations (Esua et al. 2022). E. coli displayed different inactivation patterns depending on the seed type under DCSBD plasma treatment (Fig. 3). The biphasic model (Cerf 1977) successfully described the inactivation curve of plasma-treated cells on radish seeds (Fig. 3A). The high R 2 value (> 0.9) indicates a good fit of the model to the inactivation kinetics (Table 2). The model parameters k max1 and k max2 confirmed the presence of two subpopulations with different sensitivities to plasma treatment (Table 3). The k max1 -value was significantly higher than the k max2 -value, indicating that a substantial portion of the initial E. coli population was inactivated during the first phase. In contrast, E. coli on hemp seeds showed a linear inactivation behaviour (Fig. 3B). However, despite the absence of a resistant subpopulation, the inactivation rate of E. coli on hemp seeds was slower compared to that on radish seeds within the first 60 seconds. The decimal reduction time ( D -value) is one of the key parameters in inactivation kinetics, offering valuable insight into process efficiency (Samioti et al. 2024). D -value for E. coli on radish seeds was almost half that on hemp seeds (Table 3) which indicates a more intense effect of the plasma treatment. Subsequently, the rate of CFU reduction on radish seeds began to decline. Tailing in microbial inactivation curves has also been observed for E. coli , Salmonella Enteritidis, and endospores of Bacillus subtilis , B. atrophaneus , and Alicyclobacillus acidoterrestris treated with plasma treatment (Hertwig et al. 2015; Mošovská et al. 2023; Ding et al. 2024). The occurrence of tailing can be explained by heterogeneity in bacterial susceptibility to the lethal agent or by adaptive responses that enhance the resistance of residual cells (Sehrawat et al. 2021). Even in pure cultures like the one used in this study, biological variability among cells leads to subpopulations exhibiting distinct inactivation behaviours. While some cells can adapt to the stress applied, others are more susceptible and get inactivated early during the process. Consequently, the inactivation kinetics often deviate from a first-order model, resulting in nonlinear survival curves (Samioti et al. 2024). In this study, the observed tailing could be associated with the slight surface irregularities of radish seeds compared to the smoother surface of hemp seeds (Fig. 4). Table 3 Fitted parameters of inactivation kinetics for different models of E. coli on radish and hemp seeds treated with DCSBD and Piezobrush PZ3 plasma treatments Plasma source Parameters Radish seeds Hemp seeds DCSBD log N 0 (CFU/g) 6.46 ± 0.23 7.23 ± 0.34 Model Biphasic Log-linear k max1 (s − 1 ) 0.217 (0.038) 0.132 (0.004) k max2 (s − 1 ) 0.016 (0.041) NA f 1.0000 (0.0002) NA D -value (s) 10.61 17.51 Piezobrush PZ3 log N 0 (CFU/g) 6.42 ± 0.12 7.62 ± 0.57 Model Weibull Weibull δ (s) 146.10 56.93 p 0.46 (0.12) 0.57 (0.15) Note: NA – not applicable The inactivation profile of E. coli reflected variation in the sensitivity of the seed types to treatment with the Piezobrush PZ3 (Fig. 3C, D). The results suggested higher sensitivity of tested bacteria on hemp seeds compared that on radish seeds. Specifically, treatment with the Piezobrush PZ3 reduced the initial population of E. coli on hemp seeds by 1.29 log 10 CFU/g within the first 60 seconds of exposure. On the other hand, the reduction in the population of E. coli on radish seeds was limited. After treatments for 300 seconds, a 1.51 log 10 CFU/g reduction was achieved. For kinetic analysis, E. coli on both seed types showed non-linear inactivation behaviour (Fig. 3C, D). Experimental data were fitted using the Weibull model (Mafart et al. 2002); the strong fit of the model was supported by high R 2 and low RMSE values (Table 2). As shown in Table 3, the value of the shape parameter ( p ) was less than 1, indicating upward concavity in the survival curve of the tested bacteria. Although the Weibull model provides a statistical description of the survival curve, the shape parameter can offer insights into the physiological response of the cells to stress. Hence, the obtained values of the shape parameter ( p < 1) indicate the ability of the remaining cells to adapt to the applied stress and/or exhibit increased resistance to plasma treatment (Rojas et al. 2017). The scale parameter (δ; D -value) also confirmed that E. coli on hemp seeds required less time for population reduction compared to that on radish seeds (Table 3). The Weibull model has successfully described the inactivation curves of plasma-treated microorganisms in previous studies (Hertwig et al. 2015; Mošovská et al. 2023). However, the inactivation effect of piezoelectric direct discharge plasma generated by the Piezobrush PZ3 has not been investigated until now. The UV spectrum (Fig. 2) was dominated by molecular bands of the second positive system of N 2 (C-B), which is characteristic for air plasmas. Air plasmas generate a range of reactive species, including reactive nitrogen and oxygen species, which can interact directly with microorganisms and contribute to their inactivation (Laroussi and Leipold 2004). In addition, UV radiation may also have played a role in the decontamination process (Fig. 2). Moreover, the Piezobrush PZ3 plasma produces ozone (Korzec et al. 2020), which is widely recognized for its potent antimicrobial properties (Hertwig et al. 2017). On the other hand, the interaction between low-temperature plasma and microorganisms is quite complex and depends not only on plasma source, microorganism species, and working gas, but also on the nature of application. Furthermore, the penetration depth of UV light and reactive species is limited. As a result, they can only affect individual bacteria, and in the case of aggregates, primarily those located in the top layer (Hertwig et al. 2015). In the case of DCSBD plasma, the plasma treatment area was larger compared to Piezobrush, which allows for better contact between the sample and the plasma. Therefore, the antibacterial effect of DCSBD plasma may be more pronounced than that of the Piezobrush PZ3. Surface diagnostic Scanning electron microscope (SEM) To assess the possible morphological changes on the seed surface induced by plasma treatment we examined the seed surface with the scanning electron microscope (Fig. 4). When comparing the surface morphology of both seed types, the hemp seeds are smoother and do not have a distinct characteristic structure like the radish seeds. The smooth surface of the hemp seeds was only slightly roughened after DCSBD plasma treatment, as small pinholes formed on the surface (Fig. 4c)). In comparison, the Piezobrush treatment was gentler and did not cause any significant etching (Fig. 4d)). For radish seeds, plasma treatment with both plasma sources did not affect the natural morphology of this seed type (Fig. 4g), h)) compared to the reference seed (Fig. 4f)), even at the higher exposure times. Wettability Plasma treatment of the seed surface often leads to activation and oxidation of the hydrophobic waxy layer of different seed types, which results in improved wettability. Due to the small size of the analysed seeds, we were not able to measure the water contact angle at the surface, which normally provides information on wettability. Therefore, we measured the amount of water absorbed by seeds during 2 hours after plasma treatment compared to the reference seeds (Fig. 5). When comparing the seed types and their ability to absorb water, the hemp seeds absorbed about half as much water as the radish seeds. The plasma treatment clearly led to a higher water uptake of the seeds compared to the reference seeds, which is due to the higher wettability of the seed surface, which promotes better water uptake. Similar results have been reported for wheat, maize and pea seeds treated with plasma generated by DCSBD (Stolárik et al. 2015; Zahoranová et al. 2018; Ussenov et al. 2022). According to Švubová et al. (2020), plasma produced reactive oxygen and nitrogen species cause the oxidation of lipids and polysaccharides on the seed surface leading to the formation of functional groups that increase wettability. When comparing the plasma sources, both led to a comparable increase in water uptake in both seed types. The difference was in the amount of water taken up by the seeds after plasma treatment: the hemp seeds absorbed about 27–55% more water than the reference seeds, while the radish seeds took up only 12–19% more water compared to the reference. Physicochemical characteristics Based on the OES results (Fig. 2), the significant difference between T vib and T rot clearly confirms the non-equilibrium nature of the plasma generated by both plasma sources. This is also the sign of the low temperature of such plasma, which is capable of generating reactive species without causing excessive thermal damage to the substrate. However, these reactive species can interact with the surface of biological material and induce various biochemical processes (Šerá et al. 2021). In our study, the levels of soluble proteins in radish seeds increased significantly ( p < 0.05) after treatment with DCSBD ambient air and Piezobrush plasma (Table 4), compared to the untreated sample. Moreover, the increase in soluble protein levels was more pronounced with longer exposure times. Our results correlate with Ling et al. (2014), who showed that plasma treatment led to a noticeable increase in the soluble protein content of soybean seeds. Sadhu et al. (2017) also observed an enhancement in soluble protein content after plasma treatment of maize seeds. According to Ling et al. (2014), plasma treatment has the potential to promote the conversion of seed storage proteins from complex to simpler soluble protein forms, which are thought to support reserve utilization and promote to seedling development (Zahoranová et al. 2018). Moreover, plasma treatment could enhance the activity of protease and thus stimulate the accumulation of soluble proteins (Yin et al. 2016). Table 4 Effect of the DSCBD and Piezobrush PZ3 plasma treatments on the physicochemical properties of radish and hemp seeds Physicochemical properties DCSBD treatment time (s) Radish seeds Hemp seeds Soluble proteins (mg albumin/g) Reducing sugars (mg glucose/g) Soluble proteins (mg albumin/g) Reducing sugars (mg glucose/g) 0 513.41 ± 14.98 a 32.26 ± 1.54 a 133.54 ± 3.88 a 3.96 ± 0.15 a 15 706.37 ± 24.10 b 34.65 ± 0.54 a,b 130.88 ± 7.32 a 4.03 ± 019 a 30 718.83 ± 17.92 b 35.84 ± 1.49 b,c 130.06 ± 2.47 a 4.14 ± 0.20 a 90 925.66 ± 19.56 c 37.35 ± 0.83 c 130.44 ± 5.62 a 3.98 ± 0.10 a Piezobrush PZ3 treatment time (s) Radish seeds Hemp seeds Soluble proteins (mg albumin/g) Reducing sugars (mg glucose/g) Soluble proteins (mg albumin/g) Reducing sugars (mg glucose/g) 0 513.41 ± 14.98 a 32.26 ± 1.54 a 133.54 ± 3.88 a 3.96 ± 0.15 a 60 663.24 ± 29.27 b 36.20 ± 0.31 b 126.82 ± 3.06 a 4.65 ± 0.12 b 180 706.07 ± 31.21 c 39.09 ± 0.91 c 128.04 ± 6.05 a 4.07 ± 0.20 a,b,c 300 736.42 ± 34.78 c 30.38 ± 1.35 a 126.13 ± 1.05 a 3.48 ± 0.12 c Mean values marked with the same letters within the same column are not significantly different ( p > 0.05). Statistical analyses were performed separately for the DCSBD and Piezobrush PZ3 treatments. On the other hand, no significant differences were found in the content of soluble proteins between untreated and treated hemp seeds using DCSBD or Piezobrush plasma ( p > 0.05). A similar trend was reported by Švubová et al. (2020), who found that plasma treatment using DSCBD in ambient air did not affect the soluble protein content in pea seeds. The physiological response to the plasma treatment is quite complex, depending on the species, the variety, hybrid, and cultivar. Moreover, the selection of an appropriate plasma dosage has been identified as a critical factor determining treatment efficacy (Šerá et al. 2010; Meng et al. 2017). Proteins in hemp seeds are predominantly located in the inner layer of the seed (Farinon et al. 2020). Since hemp seeds possess a thicker outer layer compared to radish seeds, the plasma exposure may not be sufficient to release storage proteins from the inner structures. In addition to soluble protein levels, plasma can also affect the concentration of sugars. As shown in Table 4, the effect of plasma treatment on reducing sugar content in the tested seeds depended on the plasma source and the seed species. Regarding the DCSBD treatment of hemp seeds, the level of reducing sugars were not significantly different ( p > 0.05) between untreated and treated samples. Comparable results were observed in rapeseed sprouts grown from seeds treated with a corona discharge plasma jet (Puligundla et al. 2017). On the other hand, an increase in reducing sugar content in radish seeds was observed with increasing exposure time to DSCBD plasma treatment ( p < 0.05). This increase is likely due to plasma-induced depolymerization of polysaccharides and oligosaccharides, as well as the possible hydrolysis of sucrose (Fernandes and Rodrigues 2021), potentially associated with increased α-amylase activity (Ling et al. 2014). Treatment with Piezobrush PZ3 led to an increase in the content of reducing sugars up to 180 seconds of plasma exposure in both radish and hemp seeds ( p 0.05), whereas in hemp seeds, a further decline below the control level was recorded ( p < 0.05). These findings are consistent with the results of Ji et al. (2022), who observed a decrease in reducing sugar content in soybean sprouts treated with plasma activated water. The observed decrease in reducing sugar content may be explained by their consumption during defence responses triggered by stress-induced metabolic activity (Fernandes and Rodrigues 2021; Ji et al. 2022). Sugars are converted to phosphoenolpyruvate and erythrose-4-phosphate via glycolysis and the pentose phosphate pathway (Fernandes and Rodrigues 2021). Conclusion The present study provides an overview of the effects of two different plasma sources, which generate non-thermal plasma in ambient air, on the inactivation kinetics of E. coli on the surface of seeds as well as on the levels of soluble proteins and reducing sugars. DCSBD plasma effectively inactivated E. coli on both radish and hemp seeds. Piezobrush PZ3 treatment showed promising results in inactivating E. coli on hemp seeds; however, E. coli on radish seeds displayed slight resistance to Piezobrush PZ3 plasma exposure. SEM analysis revealed no significant changes on the surface structure of the seeds. Plasma treatment improved wettability in both seed types and increase soluble protein levels in radish seeds. Nevertheless, prolonged exposure to Piezobrush PZ3 led to a decrease in reducing sugar content in hemp seeds. The findings of this study demonstrated the antibacterial potential of the plasma treatments used and confirmed that both plasma sources are suitable for application on dry and heat-sensitive food products, such as seeds. However, further research is required to fully understand and optimize their use, particularly that of the Piezobrush PZ3, and its interactions with treated biological materials. Declarations Acknowledgments This work was supported by the Slovak Grant Agency VEGA, grant No. 1/0688/22, and funded by the EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under project No. 09I03-03-V04-00143. Author contributions S.M.: design the study; S.M., P.Š.: writing – original draft; M.K.A.F.: performed plasma treatment, wettability measurement; S.M.: performed microbial analysis, measurement of soluble proteins, reducing sugars; J.T.: performed OES measurements; L.S.: performed SEM measurements; P.Š.: evaluated OES, SEM, wettability data; Ľ.V.: performed and evaluated modelling of inactivation kinetics; A.M. evaluated soluble proteins and reducing sugars; V.M.: had oversight of the experiments; V.M., P.Š.: funding acquisition. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the Slovak Grant Agency VEGA, grant No. 1/0688/22, and funded by the EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under project No. 09I03-03-V04-00143. Data availability The original contributions presented in the current study are available from the corresponding author upon request. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7392470","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":505212506,"identity":"fa7e12a3-8322-4afb-a2b7-0489202915a7","order_by":0,"name":"Silvia Mošovská","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIie3PMUvEMBTA8VcCnR507XFivkJDIC6HX8SlRegtZlZQaqVQl8Nbdbqv0Mk5EohLwbVjxdXhJukgcukNikjuVof8h5BAfuQFwOf7jxFSQvp9UnBo13DcJm4S/Cbcgj0ELPlJQVbuI/Q2qOP+Ck6iSJvJ0B7PV6uF6AcouIsk2pLUgHy4z/MpdqeyMXjEFqCFk5CgStIQZNMh57AmsglRxAhq5hysGsmXJS8tZ8P6ek5rFJNPKJwEdHDTZ7Ul6oz12OkUDIopAnEPtiV38fiX7A3bZ9aY/JwfJNr5fbrU6mn4mMllNG7MJaWVfnx9vyhY6ZxsW/zn9d33fT6fz7e7DR4mVaUJsuJXAAAAAElFTkSuQmCC","orcid":"","institution":"Slovak University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Mošovská","suffix":""},{"id":505212507,"identity":"cfa8463d-e123-4357-b079-8afc913d0e2d","order_by":1,"name":"Mohamed Khalaf Abdelmajeed Fawwaz","email":"","orcid":"","institution":"Comenius University Bratislava","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Khalaf Abdelmajeed","lastName":"Fawwaz","suffix":""},{"id":505212508,"identity":"3210dfaa-b911-44de-93cb-e7db030c01b7","order_by":2,"name":"Petra Šrámková","email":"","orcid":"","institution":"Comenius University Bratislava","correspondingAuthor":false,"prefix":"","firstName":"Petra","middleName":"","lastName":"Šrámková","suffix":""},{"id":505212509,"identity":"7c2602a2-5743-46c7-abef-c77e35ea9134","order_by":3,"name":"Ľubomír Valík","email":"","orcid":"","institution":"Slovak University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ľubomír","middleName":"","lastName":"Valík","suffix":""},{"id":505212510,"identity":"6c36ac5f-0419-4bbf-9258-3d0b5032f6ac","order_by":4,"name":"Anna Mikulajová","email":"","orcid":"","institution":"Slovak University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Mikulajová","suffix":""},{"id":505212511,"identity":"b2f83bc2-3db4-4737-8171-0c4817734792","order_by":5,"name":"Juliána Tomeková","email":"","orcid":"","institution":"Comenius University Bratislava","correspondingAuthor":false,"prefix":"","firstName":"Juliána","middleName":"","lastName":"Tomeková","suffix":""},{"id":505212512,"identity":"2d8e41e8-de8e-457d-becc-91675b44af68","order_by":6,"name":"Leonid Satrapinskyy","email":"","orcid":"","institution":"Comenius University Bratislava","correspondingAuthor":false,"prefix":"","firstName":"Leonid","middleName":"","lastName":"Satrapinskyy","suffix":""},{"id":505212513,"identity":"fae435f1-03ff-4905-abcc-fe2dd76b09f4","order_by":7,"name":"Veronika Medvecká","email":"","orcid":"","institution":"Comenius University Bratislava","correspondingAuthor":false,"prefix":"","firstName":"Veronika","middleName":"","lastName":"Medvecká","suffix":""}],"badges":[],"createdAt":"2025-08-17 13:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7392470/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7392470/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90043760,"identity":"437efeee-6df4-4479-8c23-c165abaf04ea","added_by":"auto","created_at":"2025-08-27 17:38:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12252980,"visible":true,"origin":"","legend":"\u003cp\u003ea) Photo of the Piezobrush PZ3 device placed in the holder above the movable table with a plastic container loaded with radish seeds; b) view of the piezoelectric generator CeraPlas™ F generating cold plasma; c) detail of the radish seed treatment by Piezobrush PZ3; d) photo of the DCSBD electrode with the active plasma area and hemp seeds placed on it (a green silicone brush was used for seed movement); e) detailed of the hemp seeds placed on the DCSBD electrode\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7392470/v1/830cc0af3ab1ee10daf8e32c.png"},{"id":90042455,"identity":"0b885dd5-d84c-4264-b598-7c67d43203f5","added_by":"auto","created_at":"2025-08-27 17:22:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":622995,"visible":true,"origin":"","legend":"\u003cp\u003eOES spectrum of Piezobrush PZ3 plasma generated in ambient air at an input power of 8 W\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7392470/v1/7d1a7d080f09bf623a9adef4.png"},{"id":90042456,"identity":"da7e8b97-fb76-4303-8202-ba63047142e9","added_by":"auto","created_at":"2025-08-27 17:22:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187192,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DCSBD and Piezobrush PZ3 plasma treatments on \u003cem\u003eE. coli\u003c/em\u003e inactivation survival curves on radish (A, C) and hemp seeds (B, D), fitted with biphasic (A), log-linear (B), and Weibull models (C, D)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7392470/v1/d909b29d3f2cb5c88f53ac67.png"},{"id":90043176,"identity":"b2a7071a-63f2-4f1f-b933-87ead70eec7c","added_by":"auto","created_at":"2025-08-27 17:30:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16045896,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of hemp and radish seeds: a) reference hemp seed detail at 50x magnification; b) reference hemp seed, c) DCSBD-treated hemp seed (90 s), and d) PZ3-treated hemp seed (5 min), all at 10 kx magnification; e) reference radish seed detail at 80x magnification; f) reference radish seed, g) DCSBD-treated radish seed (90 s), and h) PZ3-treated radish seed (5 min), all at 500x magnification\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7392470/v1/2423d1a7b277cc9f8d0ae752.png"},{"id":90043171,"identity":"ccbd4efe-90e2-46e7-bd84-d1f365aec89d","added_by":"auto","created_at":"2025-08-27 17:30:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":455028,"visible":true,"origin":"","legend":"\u003cp\u003eGraphs of water uptake (%) of the seeds after plasma treatment by DCSBD and Piezobrush PZ3\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7392470/v1/90cfbbbb1a1f6ea2e554d37e.png"},{"id":90044197,"identity":"341deb00-722c-4b99-aeae-335defd02114","added_by":"auto","created_at":"2025-08-27 17:47:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28584107,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7392470/v1/a32cae5a-b0fc-4340-807b-3c947fff6df9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modelling of Inactivation Kinetics of Escherichia coli on Radish and Hemp seeds and Their Physicochemical Properties after Non-Thermal Plasma Treatment: A Comparative Study of Two Plasma Sources","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGermination is recognized as an efficient bioprocess for enhancing the levels of bioactive compounds in seeds, such as γ-aminobutyric acid, polyphenols, and vitamins. These compounds can function as dietary antioxidants, playing a significant role in protecting the body from oxidative stress (Liu et al. 2022). Consequently, the consumption of sprouts may contribute to the prevention of multiple chronic disorders (Wuytack et al. 2003).\u003c/p\u003e\u003cp\u003eOn the other hand, seeds are germinated under warm, moist, and nutrient-rich conditions that promote the proliferation of microorganisms, including pathogens such as \u003cem\u003eEscherichia coli\u003c/em\u003e or \u003cem\u003eSalmonella\u003c/em\u003e spp. Because sprouts are typically consumed raw, they represent a potential high-risk factor for consumer safety (Budryn et al. 2019, Machado-Moreira et al. 2021). Over the past decade, the consumption of seed sprouts has been epidemiologically associated with numerous infectious disease outbreaks on a global scale. Notable cases include the outbreak in Germany and other European countries in 2011 which was associated with the consumption of fenugreek sprouts contaminated with \u003cem\u003eE. coli\u003c/em\u003e O104:H4. The outbreak resulted in 4,000 cases of infection, including 50 deaths (EFSA 2011).\u003c/p\u003e\u003cp\u003eSprouts can become contaminated through many ways; however the seeds themselves are primarily source of contamination (Machado-Moreira et al. 2021). Due to their ability to rapidly proliferate during sprouting, even a small number of pathogenic cells on seeds can pose a significant health risk. Therefore, preventing seed contamination or removing pathogens before sprouting is essential to ensure product safety (Wuytack et al. 2003). Chemical disinfectants such as chlorine compounds, trisodium phosphate, and acids have been widely used to reduce pathogens on seeds, though their effectiveness is limited, and their use is not permitted in organic production in some countries, such as Germany. Hence, increasing concerns about health and environmental impact of chemical agents have driven interest in alternative techniques, such as low-temperature plasma (Puligundla et al. 2017).\u003c/p\u003e\u003cp\u003eLow-temperature plasma (LTP) has attracted increasing attention in recent years for its antimicrobial potential. Plasma, a partially or fully ionized gas consisting of reactive oxygen and nitrogen species (RONS), UV photons, and charged particles (Mandal et al. 2018), has demonstrated significant antimicrobial activity against a broad spectrum of microorganisms inoculated on the surface of many food matrices (Hertwig et al. 2017; Wiktor et al. 2020; Casado et al. 2024). LTP treatment offers several advantages for food processing applications primarily due to its ability to be generated continuously under atmospheric and/or reduced pressure conditions (Hertwig et al. 2018). On the other hand, the effect of cold plasma has been found to be highly dependent on several parameters, including working gas, discharge characteristics (dielectric barrier discharge, microwave discharge, gliding arc, plasma jet, etc.), input parameters (frequency, voltage, power density, etc.), and gas flow (Guo et al. 2015).\u003c/p\u003e\u003cp\u003eOne of the most commonly used plasma systems is dielectric barrier discharge (DBD) system (Hertwig et al. 2018). In DBD, a plasma discharge is generated between two parallel electrodes, with one or both electrodes covered by a dielectric material. This plasma system offers several benefits, including adaptable geometric configurations and scalability, ease of operation, straightforward and compact design, cost-effectiveness, safety, and favourable power supply features (Feizollahi et al. 2021). On the other hand, although DBD plasma is widely used for microbial inactivation due to its ability to generate reactive species at atmospheric pressure and ambient temperature (Ziuzina et al. 2014; Laroque et al. 2022; Yang et al. 2025), it also has several limitations. As mentioned in the section above, the effectiveness of this system is highly influenced by process parameters such as gas pressure, type, flow rate, frequency and power of plasma excitation. Additionally, factors like reactor geometry and the nature of the treated material can play a crucial role in its overall efficacy (Feizollahi et al. 2021).\u003c/p\u003e\u003cp\u003eThe growing need for compact, cost-effective, and versatile plasma devices has driven the development of a new class of piezoelectric cold plasma generators to produce the piezoelectric direct discharge (PDD). PDD is characterized by the initiation of micro-discharges in proximity to the treated surface, which defines its distinctive physical behaviour and broad application potential. The relatively low temperature of the plasma gases (only a few kelvins above ambient) enables the safe treatment of heat-sensitive materials such as fruits, seeds, and biological tissues. Additionally, the generation of high ozone concentrations enhances the system\u0026rsquo;s effectiveness for disinfection and sterilization applications (Korzec et al. 2021).\u003c/p\u003e\u003cp\u003eThe objective of this study was to investigate and compare the effects of two different plasma sources on the kinetic behaviour of \u003cem\u003eEscherichia coli\u003c/em\u003e inoculated on the surface of radish and hemp seeds. The generated plasmas were characterized using optical emission spectroscopy. Following plasma treatments, surface diagnostics (scanning electron microscopy and wettability) and physicochemical characteristics (soluble proteins and reducing sugars) of the seeds were also examined to assess their impact on seed quality.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eTested samples\u003c/h2\u003e\n \u003cp\u003eRadish (\u003cem\u003eRaphanus sativus\u003c/em\u003e L.) and hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e) seeds were purchased at a local retail store (Bratislava, Slovakia). Samples were stored at room temperature in the dark.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eBacterial strain and cell suspension preparation\u003c/h3\u003e\n\u003cp\u003eIn this study, vegetative cells of \u003cem\u003eE. coli\u003c/em\u003e CCM 3988 (the Collection of Microorganisms, Masaryk University, Brno, Czech Republic) was used. For inoculum preparation, 20 ml of sterile Mueller Hinton broth was aseptically inoculated with a colony of the tested bacterial strain. The cell suspension was incubated for 16 h at 37°C under continuous shaking (250 rpm) to reach the stationary phase of \u003cem\u003eE. coli\u003c/em\u003e (approximate cell density: 10\u003csup\u003e8\u003c/sup\u003e cells/ml).\u003c/p\u003e\n\u003ch3\u003ePlasma source and plasma characterization\u003c/h3\u003e\n\u003cp\u003eThe present study compared two different sources of non-thermal plasma operating at atmospheric pressure in ambient air. First plasma device, Diffuse Coplanar Surface Barrier Discharge (DCSBD) (Roplass, s.r.o, Modřice, CZ), is an effective source of non-equilibrium plasma that is being widely explored for various applications such as material processing (Šrámková et al. 2021), seed treatment (Ďurčányová et al. 2023; Tomeková et al. 2024) and pathogen decontamination (Mošovská et al. 2019; Medvecká et al. 2020). Detailed specifications and properties of the DCSBD plasma source have already been published (Černák et al. 2009; Černák et al. 2011). For our experiments, the electrode system was operated by an alternating current high voltage of up to 20 kV (peak-to-peak) with a frequency of ~ 15 kHz (HV generator VF 700, Lifetech s.r.o., CZ). The plasma treatment was performed with an input power of 400 W at atmospheric pressure in ambient air.\u003c/p\u003e\n\u003cp\u003eThe second plasma device is the commercially available piezoelectric direct discharge Piezobrush® PZ3 (Relyon plasma GmbH, Regensburg, Germany) (Korzec et al. 2021). The core component of this device is the piezoelectric cold plasma generator type CeraPlas™ F (TDK Electronics GmbH, Austria). It represents a piezoelectric resonant transformer with a maximum input power of 8 W, which operates at a resonant frequency of 50 kHz and sinusoidal waveform with peak-to-peak ~ 40 kV (Korzec et al. 2020). During the plasma treatment, the Piezobrush PZ3 was placed in a vertically stable position in the special holder as is depicted in the Fig.\u0026nbsp;1a).\u003c/p\u003e\n\u003cp\u003eElectrical and optical properties of plasma generated by DCSBD using different experimental conditions have already been published (Šerá et al. 2021; Tomeková et al. 2024). The optical emission spectra (OES) of the plasma generated by Piezobrush PZ3 in ambient air were measured at an input power of 8 W, while the perpendicular distance between the optical fibre and the piezoelectric generator was approximately 8 cm. The radiation from the plasma was collected with the optical fibre (Avantes FC-UV200-2-SR, F1000 UV–VIS SR) connected to the AvaSpec-2048 TEC (Thermo-Electric-Cooled) spectrometer with a range of ~ (300–400) nm and a resolution of 20 px/nm. Characteristic lines identified from the measured optical emission spectra were used to estimate the vibrational temperatures in the Spectrum Analyzer 1.8 program (Navratil et al. 2006), and the rotational temperatures were determined using the peaks of the simulated spectra in the Specair 3.0 program (Laux 2002).\u003c/p\u003e\n\u003ch3\u003eSample preparation\u003c/h3\u003e\n\u003cp\u003eBefore plasma treatment, seeds were sterilized in an autoclave at 121 \u003csup\u003eᵒ\u003c/sup\u003eC and 120 kPa for 20 minutes. Subsequently, seeds were inoculated with 1000 µL (hempseeds) and 600 µL (radish seeds) cell suspension. Then, the seeds were shaken by hand for 5 minutes to obtain a homogenous coating of \u003cem\u003eE. coli\u003c/em\u003e on the seeds surface. Afterwards, the inoculated seeds were allowed to dry for about 24 h under at ambient temperature.\u003c/p\u003e\n\u003ch3\u003ePlasma treatment and determination of microbial inactivation\u003c/h3\u003e\n\u003cp\u003eFor the DCSBD treatment, inoculated seeds (1 g) were spread on the ceramic plate within the plasma region (see Fig.\u0026nbsp;1d), e)). The seeds were treated in ambient air at exposure times of 15, 30, 60 and 90 seconds. During the treatment, the samples were stirred manually with the silicone brush. After each treatment, the ceramic plate and the other used tools were cleaned with isopropanol.\u003c/p\u003e\n\u003cp\u003eFor the treatment with the PiezoBrush PZ3, inoculated seeds (0.5 g) were transferred into the plastic container measuring 2 x 3 cm\u003csup\u003e2\u003c/sup\u003e. During the treatment, the container was manually moved back and forth vertically to obtain a homogenous treatment (see Fig.\u0026nbsp;1c)). Plasma treatment was applied for 60, 120, 180, 240 and 300 seconds.\u003c/p\u003e\n\u003cp\u003eTrials were performed at least in duplicate.\u003c/p\u003e\n\u003cp\u003eImmediately after treatment, the viable cell count was determined using the standard plate count method in duplicate (Houghtby et al. 1992). The recovery of viable of \u003cem\u003eE. coli\u003c/em\u003e cells was performed by shaking the seeds (1 g for each treatment condition) in 9 mL of 0.85% sterile saline solution containing 0.1% Tween 80 (Merck, Germany) for 1 hour at 140 rpm. The resulting bacterial suspensions were serially diluted in 0.85% saline solution, and each dilution was plated on a Mueller-Hinton agar plate. Colony-forming units (CFU) were counted after 24 hours of incubation at 37 \u003csup\u003eᵒ\u003c/sup\u003eC.\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eModelling of inactivation kinetics\u003c/h2\u003e\n \u003cp\u003eThe experimental data were fitted using a biphasic (Cerf 1977), a log-linear (Bigelow and Esty 1920) and a Weibull (Mafart et al. 2002) models. The mean values of the inactivation data were modelled with GInaFit software (version 1.7 for Microsoft Excel24). The mathematical equations describing inactivation kinetics are shown in Table 1.\u003cimg 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\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere N\u003csub\u003e0\u003c/sub\u003e is the initial microbial population (log CFU/g); N\u003csub\u003et\u003c/sub\u003e is the microbial population after plasma treatment at the time t (log CFU/g); \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003emax1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003emax2\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e are the inactivation rate constant (s\u003csup\u003e− 1\u003c/sup\u003e); f is the fraction of the initial population corresponding to the subpopulation more sensitive to the plasma treatment; δ is the scale parameter; \u003cem\u003ep\u003c/em\u003e represents the shape parameter\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSurface diagnostic\u003c/h3\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eScanning electron microscope (SEM)\u003c/h2\u003e\n \u003cp\u003ePossible micromorphological changes on the surface of studied seeds were monitored by scanning electron microscopy (SEM). Tescan Lyra 3 SEM microscope (Tescan, CZ) with an accelerating voltage of 1 kV in regime of secondary electrons and with magnifications of 2.5-10kx was employed. The samples subjected to this analysis were treated with both plasma devices at the longer used exposure times: DCSBD – 90 s, Piezobrush PZ3–300 s, and compared to reference seeds without plasma treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eWettability\u003c/h2\u003e\n \u003cp\u003eThe changes in wettability were monitored by the water uptake of the seeds immersed in the water. Dry seeds (hemp and radish) in the amount of 50 pieces were placed in dry beakers. Untreated seeds (labelled REF) and plasma-treated seeds were weighed on an analytical balance (KERN ABT220-4NM, KERN \u0026amp; SOHN GmbH, Germany) to determine their initial weight in the dry state (m\u003csub\u003ei\u003c/sub\u003e). Each set of seeds contained 50 seeds. Then 50 ml of deionized water was poured into the beakers to let the seeds imbibe. After 120 minutes, the seeds were removed from the water, the excess water was collected with filter paper and the imbibed seeds were weighed (m\u003csub\u003et\u003c/sub\u003e). The percentage water uptake (%) was expressed as the mass gain of the sample after 120 minutes (average value determined from triplicates) according to the following equation (Eqs.\u0026nbsp;(1)):\u003c/p\u003e\n \u003cdiv id=\"Equ1\"\u003e\n \u003cdiv id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\:Water\\:uptake\\:m\\:\\left[\\%\\right]=\\:\\frac{{m}_{t}-{m}_{i}}{{m}_{i}}*100\\%\\:$$\u003c/div\u003e\n \u003cdiv\u003e1\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003ePhysicochemical characteristics\u003c/h2\u003e\n \u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eMeasurement of soluble protein content\u003c/h2\u003e\n \u003cp\u003eAnalysis of soluble protein content was performed using the Lowry method (Lowry et al. 1951). A 0.1 g defatted sample was placed in a microcentrifuge tube and vortexed with 1 mL of 0.1 M sodium phosphate buffer (pH 7.4) for 10 seconds. The mixture was then allowed to stand for 15 minutes, followed by centrifugation at 13,000 rpm for 10 minutes. A 0.25 mL aliquot of the supernatant was mixed with 1.25 mL of alkaline copper reagent and incubated at room temperature for 10 minutes. Subsequently, 0.125 mL of Folin–Ciocalteu reagent was added, and the mixture was incubated for an additional 30 minutes. Absorbance was measured at 660 nm using a spectrophotometer. Soluble protein concentration was determined from a standard curve prepared with bovine serum albumin (BSA).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eMeasurement of reducing sugars\u003c/h2\u003e\n \u003cp\u003eReducing sugars were analysed using the 3,5-dinitrosalicylic acid (DNS) colorimetric method with slight modification (Miller 1959). A 2 g sample was mixed with 10 mL of distilled water preheated to 80°C. The mixture was left to stand for 30 minutes under constant agitation on an orbital shaker at 200 rpm. Subsequently, 0.5 mL of Carrez I solution was added under continuous stirring, followed by the addition of 0.5 mL of Carrez II solution. The mixture was then diluted with distilled water to a final volume of 25 mL, thoroughly mixed, and filtered through filter paper (Whatman 1). A 25 µL aliquot of the filtrate was pipetted into a test tube, followed by the addition of 200 µL of DNS reagent. The mixture was thoroughly mixed and incubated in a boiling water bath for 5 minutes. After incubation, the sample was cooled, 2 mL of distilled water was added, and the tube was mixed again. Absorbance was measured at 540 nm. The concentration of reducing sugars was determined from a standard curve prepared using glucose.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThe values represent the mean ± standard deviation (SD). Significant differences between means were determined using one-way ANOVA followed by Bonferroni correction post hoc test. Differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 were considered significant.\u003c/p\u003e\n \n\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003ch2\u003ePlasma diagnostics\u003c/h2\u003e\u003cp\u003eOptical emission spectroscopy (OES) was used to diagnose the plasma generated by the plasma sources studied. DCSBD has been extensively studied in previous experiments and OES spectra have been collected under different operating conditions. The typical emission spectrum of a DCSBD plasma generated in ambient air contains the second positive system of N\u003csub\u003e2\u003c/sub\u003e (C-B) as the most intense spectral band in the UV region (~ 300–400 nm). From this spectral system, the vibrational (T\u003csub\u003evib\u003c/sub\u003e) and rotational (T\u003csub\u003erot\u003c/sub\u003e) temperatures were determined in the previous study (Tomeková et al. 2024) as follows: T\u003csub\u003evib\u003c/sub\u003e = 2610 ± 225 K, T\u003csub\u003erot\u003c/sub\u003e = 385 ± 30 K. For comparison, we have measured the OES spectrum of Piezobrush PZ3 plasma. Since this plasma is similarly generated in ambient air at atmospheric pressure like in case of DCSBD, the emission spectrum is very similar to that of DCSBD (Fig.\u0026nbsp;2). The dominant spectral system is the second positive system of N\u003csub\u003e2\u003c/sub\u003e (C-B), which was detected in the UV spectral range. We calculated the vibrational and rotational temperatures from this spectral band: T\u003csub\u003evib\u003c/sub\u003e = 3110 ± 170 K, T\u003csub\u003erot\u003c/sub\u003e = 560 ± 30 K. The significant difference between T\u003csub\u003evib\u003c/sub\u003e and T\u003csub\u003erot\u003c/sub\u003e is clear evidence of the non-equilibrium nature of the plasma generated by both plasma sources. Moreover, the UV radiation can also contribute to decontamination.\u003c/p\u003e\u003ch2\u003ePlasma-induced microbial inactivation and kinetic modelling\u003c/h2\u003e\u003cp\u003eThe surviving population of \u003cem\u003eE. coli\u003c/em\u003e inoculated on the surface of radish and hemp seeds after plasma treatments is presented in Fig. 3.\u003c/p\u003e\u003cp\u003eThe obtained results indicated varying sensitivity of \u003cem\u003eE. coli\u003c/em\u003e inoculated on the surface of tested seeds to plasma treatments, depending on both the plasma source and the seed type. It was evident that the bactericidal effect of DCSBD plasma treatment was significantly higher compared to that of the Piezobrush PZ3. As shown in Fig. 3 (A, B), a considerable reduction of \u003cem\u003eE. coli\u003c/em\u003e inoculated on surface of seeds was observed with increasing exposure time. This finding is in agreement with our previous work (Mošovská et al. 2018; Mošovská et al. 2019; Medvecká et al. 2020), as well as with the research of Hertwig et al. (2017), which indicated the strong antimicrobial potential of DCSBD plasma. The current work used DCSBD plasma system generated in ambient air. Ambient air plasma produces high concentrations of NOx and OH radicals (Mošovská et al. 2019), all of which have a direct impact on microorganisms and may result in microbial inactivation (Laroussi and Leipold 2004; Hertwig et al. 2017).\u003c/p\u003e\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eStatistical analysis of different models for the inactivation of \u003cem\u003eE. coli\u003c/em\u003e on radish and hemp seeds treated with DCSBD and Piezobrush PZ3 plasma treatments\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003ePlasma source\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eRadish seeds\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eHemp seeds\u003c/p\u003e\n \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eDCSBD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9933\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9974\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAdj. R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9731\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9965\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eMSSE\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1203\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0150\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eRMSE\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3468\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1225\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003ePiezobrush PZ3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9674\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9613\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAdj. R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9457\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9354\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eMSSE\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0144\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0589\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eRMSE\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1198\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2427\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003cp\u003eMathematical modelling of inactivation kinetics is essential for quantitative exposure and risk assessment, and it also enables comparison of different processing technologies in terms of their effectiveness in reducing microbial populations (Esua et al. 2022). \u003cem\u003eE. coli\u003c/em\u003e displayed different inactivation patterns depending on the seed type under DCSBD plasma treatment (Fig. 3). The biphasic model (Cerf 1977) successfully described the inactivation curve of plasma-treated cells on radish seeds (Fig. 3A). The high R\u003csup\u003e2\u003c/sup\u003e value (\u0026gt; 0.9) indicates a good fit of the model to the inactivation kinetics (Table 2). The model parameters \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003emax1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003emax2\u003c/em\u003e\u003c/sub\u003e confirmed the presence of two subpopulations with different sensitivities to plasma treatment (Table 3). The \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003emax1\u003c/em\u003e\u003c/sub\u003e-value was significantly higher than the \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003emax2\u003c/em\u003e\u003c/sub\u003e-value, indicating that a substantial portion of the initial \u003cem\u003eE. coli\u003c/em\u003e population was inactivated during the first phase. In contrast, \u003cem\u003eE. coli\u003c/em\u003e on hemp seeds showed a linear inactivation behaviour (Fig. 3B). However, despite the absence of a resistant subpopulation, the inactivation rate of \u003cem\u003eE. coli\u003c/em\u003e on hemp seeds was slower compared to that on radish seeds within the first 60 seconds. The decimal reduction time (\u003cem\u003eD\u003c/em\u003e-value) is one of the key parameters in inactivation kinetics, offering valuable insight into process efficiency (Samioti et al. 2024). \u003cem\u003eD\u003c/em\u003e-value for \u003cem\u003eE. coli\u003c/em\u003e on radish seeds was almost half that on hemp seeds (Table 3) which indicates a more intense effect of the plasma treatment. Subsequently, the rate of CFU reduction on radish seeds began to decline. Tailing in microbial inactivation curves has also been observed for \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e Enteritidis, and endospores of \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eB. atrophaneus\u003c/em\u003e, and \u003cem\u003eAlicyclobacillus acidoterrestris\u003c/em\u003e treated with plasma treatment (Hertwig et al. 2015; Mošovská et al. 2023; Ding et al. 2024). The occurrence of tailing can be explained by heterogeneity in bacterial susceptibility to the lethal agent or by adaptive responses that enhance the resistance of residual cells (Sehrawat et al. 2021). Even in pure cultures like the one used in this study, biological variability among cells leads to subpopulations exhibiting distinct inactivation behaviours. While some cells can adapt to the stress applied, others are more susceptible and get inactivated early during the process. Consequently, the inactivation kinetics often deviate from a first-order model, resulting in nonlinear survival curves (Samioti et al. 2024). In this study, the observed tailing could be associated with the slight surface irregularities of radish seeds compared to the smoother surface of hemp seeds (Fig. 4).\u003c/p\u003e\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eFitted parameters of inactivation kinetics for different models of \u003cem\u003eE. coli\u003c/em\u003e on radish and hemp seeds treated with DCSBD and Piezobrush PZ3 plasma treatments\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003ePlasma source\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eRadish seeds\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eHemp seeds\u003c/p\u003e\n \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eDCSBD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003elog N\u003csub\u003e0\u003c/sub\u003e (CFU/g)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e6.46 ± 0.23\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e7.23 ± 0.34\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eBiphasic\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eLog-linear\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003emax1\u003c/em\u003e\u003c/sub\u003e (s\u003csup\u003e− 1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.217 (0.038)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.132 (0.004)\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003emax2\u003c/em\u003e\u003c/sub\u003e (s\u003csup\u003e− 1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.016 (0.041)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ef\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0000 (0.0002)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eD\u003c/em\u003e-value (s)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10.61\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e17.51\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003ePiezobrush PZ3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003elog N\u003csub\u003e0\u003c/sub\u003e (CFU/g)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e6.42 ± 0.12\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e7.62 ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eWeibull\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eWeibull\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eδ\u003c/em\u003e (s)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e146.10\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e56.93\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.46 (0.12)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.57 (0.15)\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: NA – not applicable\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003cp\u003eThe inactivation profile of \u003cem\u003eE. coli\u003c/em\u003e reflected variation in the sensitivity of the seed types to treatment with the Piezobrush PZ3 (Fig.\u0026nbsp;3C, D). The results suggested higher sensitivity of tested bacteria on hemp seeds compared that on radish seeds. Specifically, treatment with the Piezobrush PZ3 reduced the initial population of \u003cem\u003eE. coli\u003c/em\u003e on hemp seeds by 1.29 log\u003csub\u003e10\u003c/sub\u003e CFU/g within the first 60 seconds of exposure. On the other hand, the reduction in the population of \u003cem\u003eE. coli\u003c/em\u003e on radish seeds was limited. After treatments for 300 seconds, a 1.51 log\u003csub\u003e10\u003c/sub\u003e CFU/g reduction was achieved.\u003c/p\u003e\u003cp\u003eFor kinetic analysis, \u003cem\u003eE. coli\u003c/em\u003e on both seed types showed non-linear inactivation behaviour (Fig.\u0026nbsp;3C, D). Experimental data were fitted using the Weibull model (Mafart et al. 2002); the strong fit of the model was supported by high R\u003csup\u003e2\u003c/sup\u003e and low RMSE values (Table\u0026nbsp;2). As shown in Table\u0026nbsp;3, the value of the shape parameter (\u003cem\u003ep\u003c/em\u003e) was less than 1, indicating upward concavity in the survival curve of the tested bacteria. Although the Weibull model provides a statistical description of the survival curve, the shape parameter can offer insights into the physiological response of the cells to stress. Hence, the obtained values of the shape parameter (\u003cem\u003ep\u003c/em\u003e \u0026lt; 1) indicate the ability of the remaining cells to adapt to the applied stress and/or exhibit increased resistance to plasma treatment (Rojas et al. 2017). The scale parameter (δ; \u003cem\u003eD\u003c/em\u003e-value) also confirmed that \u003cem\u003eE. coli\u003c/em\u003e on hemp seeds required less time for population reduction compared to that on radish seeds (Table\u0026nbsp;3).\u003c/p\u003e\u003cp\u003eThe Weibull model has successfully described the inactivation curves of plasma-treated microorganisms in previous studies (Hertwig et al. 2015; Mošovská et al. 2023). However, the inactivation effect of piezoelectric direct discharge plasma generated by the Piezobrush PZ3 has not been investigated until now. The UV spectrum (Fig.\u0026nbsp;2) was dominated by molecular bands of the second positive system of N\u003csub\u003e2\u003c/sub\u003e (C-B), which is characteristic for air plasmas. Air plasmas generate a range of reactive species, including reactive nitrogen and oxygen species, which can interact directly with microorganisms and contribute to their inactivation (Laroussi and Leipold 2004). In addition, UV radiation may also have played a role in the decontamination process (Fig.\u0026nbsp;2). Moreover, the Piezobrush PZ3 plasma produces ozone (Korzec et al. 2020), which is widely recognized for its potent antimicrobial properties (Hertwig et al. 2017). On the other hand, the interaction between low-temperature plasma and microorganisms is quite complex and depends not only on plasma source, microorganism species, and working gas, but also on the nature of application. Furthermore, the penetration depth of UV light and reactive species is limited. As a result, they can only affect individual bacteria, and in the case of aggregates, primarily those located in the top layer (Hertwig et al. 2015). In the case of DCSBD plasma, the plasma treatment area was larger compared to Piezobrush, which allows for better contact between the sample and the plasma. Therefore, the antibacterial effect of DCSBD plasma may be more pronounced than that of the Piezobrush PZ3.\u003c/p\u003e\u003ch2\u003eSurface diagnostic\u003c/h2\u003e\u003ch2\u003eScanning electron microscope (SEM)\u003c/h2\u003e\u003cp\u003eTo assess the possible morphological changes on the seed surface induced by plasma treatment we examined the seed surface with the scanning electron microscope (Fig.\u0026nbsp;4). When comparing the surface morphology of both seed types, the hemp seeds are smoother and do not have a distinct characteristic structure like the radish seeds. The smooth surface of the hemp seeds was only slightly roughened after DCSBD plasma treatment, as small pinholes formed on the surface (Fig.\u0026nbsp;4c)). In comparison, the Piezobrush treatment was gentler and did not cause any significant etching (Fig.\u0026nbsp;4d)). For radish seeds, plasma treatment with both plasma sources did not affect the natural morphology of this seed type (Fig.\u0026nbsp;4g), h)) compared to the reference seed (Fig.\u0026nbsp;4f)), even at the higher exposure times.\u003c/p\u003e\u003ch2\u003eWettability\u003c/h2\u003e\u003cp\u003ePlasma treatment of the seed surface often leads to activation and oxidation of the hydrophobic waxy layer of different seed types, which results in improved wettability. Due to the small size of the analysed seeds, we were not able to measure the water contact angle at the surface, which normally provides information on wettability. Therefore, we measured the amount of water absorbed by seeds during 2 hours after plasma treatment compared to the reference seeds (Fig.\u0026nbsp;5). When comparing the seed types and their ability to absorb water, the hemp seeds absorbed about half as much water as the radish seeds. The plasma treatment clearly led to a higher water uptake of the seeds compared to the reference seeds, which is due to the higher wettability of the seed surface, which promotes better water uptake. Similar results have been reported for wheat, maize and pea seeds treated with plasma generated by DCSBD (Stolárik et al. 2015; Zahoranová et al. 2018; Ussenov et al. 2022). According to Švubová et al. (2020), plasma produced reactive oxygen and nitrogen species cause the oxidation of lipids and polysaccharides on the seed surface leading to the formation of functional groups that increase wettability. When comparing the plasma sources, both led to a comparable increase in water uptake in both seed types. The difference was in the amount of water taken up by the seeds after plasma treatment: the hemp seeds absorbed about 27–55% more water than the reference seeds, while the radish seeds took up only 12–19% more water compared to the reference.\u003c/p\u003e\u003ch2\u003ePhysicochemical characteristics\u003c/h2\u003e\u003cp\u003eBased on the OES results (Fig.\u0026nbsp;2), the significant difference between T\u003csub\u003evib\u003c/sub\u003e and T\u003csub\u003erot\u003c/sub\u003e clearly confirms the non-equilibrium nature of the plasma generated by both plasma sources. This is also the sign of the low temperature of such plasma, which is capable of generating reactive species without causing excessive thermal damage to the substrate. However, these reactive species can interact with the surface of biological material and induce various biochemical processes (Šerá et al. 2021).\u003c/p\u003e\u003cp\u003eIn our study, the levels of soluble proteins in radish seeds increased significantly (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) after treatment with DCSBD ambient air and Piezobrush plasma (Table\u0026nbsp;4), compared to the untreated sample. Moreover, the increase in soluble protein levels was more pronounced with longer exposure times. Our results correlate with Ling et al. (2014), who showed that plasma treatment led to a noticeable increase in the soluble protein content of soybean seeds. Sadhu et al. (2017) also observed an enhancement in soluble protein content after plasma treatment of maize seeds. According to Ling et al. (2014), plasma treatment has the potential to promote the conversion of seed storage proteins from complex to simpler soluble protein forms, which are thought to support reserve utilization and promote to seedling development (Zahoranová et al. 2018). Moreover, plasma treatment could enhance the activity of protease and thus stimulate the accumulation of soluble proteins (Yin et al. 2016).\u003c/p\u003e\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of the DSCBD and Piezobrush PZ3 plasma treatments on the physicochemical properties of radish and hemp seeds\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003ePhysicochemical properties\u003c/p\u003e\n \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDCSBD treatment time (s)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eRadish seeds\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHemp seeds\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eSoluble proteins\u003c/p\u003e\n \u003cp\u003e(mg albumin/g)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eReducing sugars\u003c/p\u003e\n \u003cp\u003e(mg glucose/g)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eSoluble proteins\u003c/p\u003e\n \u003cp\u003e(mg albumin/g)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eReducing sugars\u003c/p\u003e\n \u003cp\u003e(mg glucose/g)\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e513.41 ± 14.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e32.26 ± 1.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e133.54 ± 3.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e3.96 ± 0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e706.37 ± 24.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e34.65 ± 0.54\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e130.88 ± 7.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e4.03 ± 019\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e718.83 ± 17.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e35.84 ± 1.49\u003csup\u003eb,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e130.06 ± 2.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e4.14 ± 0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e925.66 ± 19.56\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e37.35 ± 0.83\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e130.44 ± 5.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e3.98 ± 0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePiezobrush PZ3 treatment time (s)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eRadish seeds\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHemp seeds\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eSoluble proteins\u003c/p\u003e\n \u003cp\u003e(mg albumin/g)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eReducing sugars\u003c/p\u003e\n \u003cp\u003e(mg glucose/g)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eSoluble proteins\u003c/p\u003e\n \u003cp\u003e(mg albumin/g)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eReducing sugars\u003c/p\u003e\n \u003cp\u003e(mg glucose/g)\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e513.41 ± 14.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e32.26 ± 1.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e133.54 ± 3.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e3.96 ± 0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e663.24 ± 29.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e36.20 ± 0.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e126.82 ± 3.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e4.65 ± 0.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e706.07 ± 31.21\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e39.09 ± 0.91\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e128.04 ± 6.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e4.07 ± 0.20\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e736.42 ± 34.78\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e30.38 ± 1.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e126.13 ± 1.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e3.48 ± 0.12\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003cp\u003eMean values marked with the same letters within the same column are not significantly different (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Statistical analyses were performed separately for the DCSBD and Piezobrush PZ3 treatments.\u003c/p\u003e\u003cp\u003eOn the other hand, no significant differences were found in the content of soluble proteins between untreated and treated hemp seeds using DCSBD or Piezobrush plasma (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). A similar trend was reported by Švubová et al. (2020), who found that plasma treatment using DSCBD in ambient air did not affect the soluble protein content in pea seeds. The physiological response to the plasma treatment is quite complex, depending on the species, the variety, hybrid, and cultivar. Moreover, the selection of an appropriate plasma dosage has been identified as a critical factor determining treatment efficacy (Šerá et al. 2010; Meng et al. 2017). Proteins in hemp seeds are predominantly located in the inner layer of the seed (Farinon et al. 2020). Since hemp seeds possess a thicker outer layer compared to radish seeds, the plasma exposure may not be sufficient to release storage proteins from the inner structures.\u003c/p\u003e\u003cp\u003eIn addition to soluble protein levels, plasma can also affect the concentration of sugars. As shown in Table\u0026nbsp;4, the effect of plasma treatment on reducing sugar content in the tested seeds depended on the plasma source and the seed species. Regarding the DCSBD treatment of hemp seeds, the level of reducing sugars were not significantly different (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) between untreated and treated samples. Comparable results were observed in rapeseed sprouts grown from seeds treated with a corona discharge plasma jet (Puligundla et al. 2017).\u003c/p\u003e\u003cp\u003eOn the other hand, an increase in reducing sugar content in radish seeds was observed with increasing exposure time to DSCBD plasma treatment (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). This increase is likely due to plasma-induced depolymerization of polysaccharides and oligosaccharides, as well as the possible hydrolysis of sucrose (Fernandes and Rodrigues 2021), potentially associated with increased α-amylase activity (Ling et al. 2014).\u003c/p\u003e\u003cp\u003eTreatment with Piezobrush PZ3 led to an increase in the content of reducing sugars up to 180 seconds of plasma exposure in both radish and hemp seeds (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Plasma exposure for 300 seconds resulted in a decrease in reducing sugar content. In radish seeds, the value returned to the baseline observed in untreated seeds (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05), whereas in hemp seeds, a further decline below the control level was recorded (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). These findings are consistent with the results of Ji et al. (2022), who observed a decrease in reducing sugar content in soybean sprouts treated with plasma activated water. The observed decrease in reducing sugar content may be explained by their consumption during defence responses triggered by stress-induced metabolic activity (Fernandes and Rodrigues 2021; Ji et al. 2022). Sugars are converted to phosphoenolpyruvate and erythrose-4-phosphate via glycolysis and the pentose phosphate pathway (Fernandes and Rodrigues 2021).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study provides an overview of the effects of two different plasma sources, which generate non-thermal plasma in ambient air, on the inactivation kinetics of \u003cem\u003eE. coli\u003c/em\u003e on the surface of seeds as well as on the levels of soluble proteins and reducing sugars.\u003c/p\u003e\u003cp\u003eDCSBD plasma effectively inactivated \u003cem\u003eE. coli\u003c/em\u003e on both radish and hemp seeds. Piezobrush PZ3 treatment showed promising results in inactivating \u003cem\u003eE. coli\u003c/em\u003e on hemp seeds; however, \u003cem\u003eE. coli\u003c/em\u003e on radish seeds displayed slight resistance to Piezobrush PZ3 plasma exposure.\u003c/p\u003e\u003cp\u003eSEM analysis revealed no significant changes on the surface structure of the seeds. Plasma treatment improved wettability in both seed types and increase soluble protein levels in radish seeds. Nevertheless, prolonged exposure to Piezobrush PZ3 led to a decrease in reducing sugar content in hemp seeds.\u003c/p\u003e\u003cp\u003eThe findings of this study demonstrated the antibacterial potential of the plasma treatments used and confirmed that both plasma sources are suitable for application on dry and heat-sensitive food products, such as seeds. However, further research is required to fully understand and optimize their use, particularly that of the Piezobrush PZ3, and its interactions with treated biological materials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Slovak Grant Agency VEGA, grant No. 1/0688/22, and funded by the EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under project No. 09I03-03-V04-00143.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.M.: design the study; S.M., P.\u0026Scaron;.: writing \u0026ndash; original draft; M.K.A.F.: performed plasma treatment, wettability measurement; S.M.: performed microbial analysis, measurement of soluble proteins, reducing sugars; J.T.: performed OES measurements; L.S.: performed SEM measurements; P.\u0026Scaron;.: evaluated OES, SEM, wettability data; Ľ.V.: performed and evaluated modelling of inactivation kinetics; A.M. evaluated soluble proteins and reducing sugars; V.M.: had oversight of the experiments; V.M., P.\u0026Scaron;.: funding acquisition. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Slovak Grant Agency VEGA, grant No. 1/0688/22, and funded by the EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under project No. 09I03-03-V04-00143.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the current study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBigelow, W. ., \u0026amp; Esty, J. R. (1920). 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The Response of Physiological Characteristics, Expression of OSC Genes, and Accumulation of Triterpenoids in Betula platyphylla Sukto MeJA and SA Treatment. \u003cem\u003ePlant Molecular Biology Reporter\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e(2), 427\u0026ndash;439. https://doi.org/10.1007/s11105-015-0931-5\u003c/li\u003e\n\u003cli\u003eZahoranov\u0026aacute;, A., Hoppanov\u0026aacute;, L., \u0026Scaron;imončicov\u0026aacute;, J., Tučekov\u0026aacute;, Z., Medveck\u0026aacute;, V., Hudecov\u0026aacute;, D., et al. (2018). Effect of Cold Atmospheric Pressure Plasma on Maize Seeds: Enhancement of Seedlings Growth and Surface Microorganisms Inactivation. \u003cem\u003ePlasma Chemistry and Plasma Processing\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e(5), 969\u0026ndash;988. https://doi.org/10.1007/s11090-018-9913-3\u003c/li\u003e\n\u003cli\u003eZiuzina, D., Sonal, P., Cullen, P., Keener, K., \u0026amp; Bourke, P. (2014). Atmospheric Cold Plasma Inactivation of Escherichia Coli , Salmonella Enterica Serovar Typhimurium and Listeria Monocytogenes Inoculated on Fresh Produce. \u003cem\u003eFood Microbiology\u003c/em\u003e, \u003cem\u003e42\u003c/em\u003e, 109\u0026ndash;116. https://doi.org/10.1016/j.fm.2014.02.007\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Inactivation kinetics, Escherichia coli, non-thermal plasma, seeds, physicochemical properties, sprouts","lastPublishedDoi":"10.21203/rs.3.rs-7392470/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7392470/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent years, seed sprout consumption has been associated with several infectious outbreaks. To ensure sprouts are safe to eat, any potential pathogens must be eliminated before the sprouting process begins. The objective of this study was to compare the efficacy of two different plasma sources, producing non-thermal plasma under ambient air conditions, on the inactivation kinetics of \u003cem\u003eEscherichia coli\u003c/em\u003e on radish and hemp seeds intended for sprouting. Plasma treatments were conducted using Diffuse Coplanar Surface Barrier Discharge (DCSBD) and piezoelectric direct discharge plasma called the Piezobrush PZ3. Microbial inactivation data were modelled using the Bigelow log-linear, biphasic, and Weibull models. After 90 seconds of DCSBD plasma treatment, reductions of 4.79 log10 CFU/g in radish seeds and 5.27 log10 CFU/g in hemp seeds were achieved. The inactivation effect of Piezobrush PZ3 was less effective in comparison to DCSBD and more pronounced against \u003cem\u003eE. coli\u003c/em\u003e on hemp seeds than on radish seeds. Plasma treatments enhanced the physicochemical properties of seeds without causing significant changes to their surface morphology. Treatment with both plasma sources led to enhanced wettability in both seed types and an increase in soluble protein content in radish seeds. In the treatment with Piezobrush PZ3, reducing sugar content in both seed types increased up to 180 seconds of plasma exposure; however, prolonged exposure resulted in a subsequent decrease. Non-thermal plasma is a promising technology for the decontamination of seeds intended for sprouting; however, selecting the appropriate plasma discharge is crucial.\u003c/p\u003e","manuscriptTitle":"Modelling of Inactivation Kinetics of Escherichia coli on Radish and Hemp seeds and Their Physicochemical Properties after Non-Thermal Plasma Treatment: A Comparative Study of Two Plasma Sources","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 17:22:47","doi":"10.21203/rs.3.rs-7392470/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-16T02:13:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T15:06:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-10T13:31:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-08T08:27:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45266643062340079998846564164721917595","date":"2025-08-25T02:29:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328251212856113780326541475081976976644","date":"2025-08-21T12:14:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32420330218482910929446315558648175716","date":"2025-08-20T19:37:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91207936573618678271010102512906074207","date":"2025-08-20T06:53:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278011714093435641986093153435344823204","date":"2025-08-20T06:00:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41271564659609358893967019106387888705","date":"2025-08-19T19:20:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-19T17:38:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-19T07:38:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-19T01:05:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2025-08-17T12:53:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"07ad971d-91bd-4096-971e-39e3459d8f9e","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-18T09:38:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 17:22:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7392470","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7392470","identity":"rs-7392470","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}