Apples are a significant crop globally, valued for their taste and nutritional content. Several studies have explored the use of non-thermal atmospheric plasmas (NTAPs) to enhance apple properties, either treating apple juice directly or the whole fruit. NTAPs generate reactive oxygen species (ROS) and reactive nitrogen species (RNS), potentially modifying the physicochemical, microbiological, and cytotoxic properties of the treated product. Previous research has examined the impact of NTAPs on various aspects of apple juice, including color, total polyphenol content, titratable acidity, °Brix value, microbial inactivation, and pH. However, most studies used stationary, non-flow-through systems with limited throughput and potential for uncontrolled plasma-liquid interactions. Additionally, optimization of NTAP treatment parameters was often done using a one-factor-at-a-time approach, limiting the understanding of parameter interactions. This research aimed to optimize a high-throughput, continuous-flow NTAP system for controlled AJ treatment, aiming to improve nutritional value, shelf life, and overall quality without altering desirable organoleptic properties. The research utilized a design of experiments (DOE) approach followed by response surface methodology (RSM) to achieve this optimization.

Existing literature demonstrates the potential of NTAP treatment to improve the quality and shelf life of apple juice. Studies have shown that NTAP treatment can inactivate various microorganisms, such as *Escherichia coli*, *Citrobacter freundii*, *Zygosaccharomyces rouxii*, and *Salmonella* species, leading to extended shelf life. The impact of NTAP treatment on physicochemical properties, like color and total polyphenol content, has also been investigated, with results varying depending on the type of NTAP system and treatment parameters. However, limitations in previous research included the use of small-scale, non-flow-through systems, and often a less rigorous optimization methodology than DOE and RSM. The lack of comprehensive studies considering multiple parameters simultaneously and their interactions on final features of apple juice was a major gap addressed in this study. There was also a lack of data about the influence of NTAP treatment on the element concentration in the apple juice.

This study employed a novel high-throughput, continuous-flow NTAP system based on direct current atmospheric pressure glow discharge (dc-APGD) generated in contact with a flowing liquid cathode (FLC). Commercially available apple juice was filtered and then continuously treated using the FLC-dc-APGD system. A Box-Behnken design was used to investigate the effects of three operating parameters: flow rate of AJ (F), discharge gap (d), and discharge current (I). The responses measured were the absorbance (A) at 284 nm (indicative of phenolic compounds) and the temperature (T) of the juice. These responses were modeled using full quadratic functions, and the models were statistically evaluated using ANOVA. Optimal operating conditions were determined based on maintaining the absorbance while maximizing temperature (up to 50 °C) to achieve prolonged shelf-life. After optimization, apple juice processed under optimal conditions was analyzed for its nutritional, physicochemical, microbiological, and cytotoxic properties. Elemental analysis was performed using ICP-OES. Physicochemical analysis included attenuated total reflectance Fourier transform-infrared spectroscopy (ATR FT-IR) to assess organic compound structure, °Brix measurement for saccharide content, colorimetry (CIE L*a*b*), Folin-Ciocalteu assay for total phenolic content, and FRAP assay for antioxidant power. Microbiological analysis involved plating on TSA agar to determine CFUs and assess shelf life. Cytotoxicity was evaluated using the MTT assay and apoptosis analysis (Annexin V/PI staining) on Caco-2 (human colorectal adenocarcinoma) and FHs 74 Int (normal human intestinal epithelial) cell lines.

The optimized NTAP treatment conditions were determined to be: flow rate = 6.0 mL min⁻¹, discharge gap = 4.0 mm, and discharge current = 50 mA. Under these conditions, the NTAP treatment resulted in a significant increase in the concentration of various elements. The concentration of Ca, Fe, K, Mg, Na, and Sr increased by 8-10%, while Al, B, Ba, Cu, Mn, and Zn increased by 11-15%. The total phenolic content increased by 23%, and the FRAP value increased by 12%, indicating enhanced antioxidant capacity. Notably, the NTAP treatment did not significantly affect the structure of organic compounds, °Brix value, or color of the apple juice. Microbiological analysis showed a significant extension of shelf life by 12 days, attributed to the antimicrobial effects of NTAP, with no observed microbial contamination. Cytotoxicity assays revealed no adverse effects on normal human intestinal cells (FHs 74 Int), while a significant cytotoxic effect was observed against human colorectal adenocarcinoma cells (Caco-2) after 72 hours of incubation, particularly at higher concentrations. This suggests that NTAP-treated AJ exhibits selective cytotoxicity towards cancer cells.

The results demonstrate the effectiveness of the optimized NTAP system in improving apple juice properties without compromising its nutritional value or sensory attributes. The increased mineral and phenolic content enhances the nutritional profile of the juice, while the extended shelf life improves its marketability. The observed selective cytotoxicity against cancer cells opens up the possibility of utilizing this treatment to develop functional foods with potential health benefits. The continuous-flow nature of the system addresses the limitations of previous studies by enabling high-throughput processing while maintaining controlled treatment conditions. The generation of reactive species within the plasma likely contributes to the observed effects, however further investigations are needed to identify exact mechanisms responsible for changes in the concentrations of specific elements and bioactive compounds.

This study successfully optimized a continuous-flow NTAP system for improving the quality and shelf life of apple juice. The resulting product exhibits enhanced nutritional value, extended shelf life, and selective cytotoxicity against cancer cells. Future research could focus on further optimization of the NTAP process, mechanistic studies to understand the precise biochemical modifications, and scaled-up production to assess the feasibility of industrial applications. Further investigation of the long-term effects and potential health benefits of consuming this NTAP treated apple juice are also warranted.

This study focused on a specific type of apple juice and a particular NTAP system. The findings may not be directly generalizable to other juice types or plasma technologies. The sample size for some analyses, while appropriate statistically, may be too small for more granular analyses or to generalize with high certainty across all possible apple juice types. Further research is needed to assess the stability of the improved properties over longer storage periods under various conditions. The mechanisms of action associated with observed increase in the concentration of minerals remain to be elucidated.