Effect of jujube polysaccharide on CP degradation of AOH and AME
Polysaccharide, an essential constituent of jujube nutrients, was found to influence the degradation of AOH and AME by CP as depicted in Fig. 1. In aqueous solutions, CP treatment at 30 kV for 4 min resulted in complete degradation of both AOH and AME. However, their degradation rates decreased upon exposure to jujube polysaccharide. While AOH was completely degraded within 7 min, AME required only 5 min, indicating a relatively lower impact of jujube polysaccharide on AME degradation compared to AOH. Nevertheless, the inhibitory effect on the degradation of both toxins did not increase with increasing concentrations of jujube polysaccharide.
Based on these findings, it was evident that all tested jujube polysaccharides effectively inhibited the degradation of Alternaria mycotoxins by CP. This inhibition might be attributed to the enhanced barrier formed by jujube polysaccharide molecules, resulting in improved toxin protection. Fan et al. (2020) reported that CP treatment led to a more compact and orderly arrangement of polysaccharides, with a transformation of monosaccharide ring from β-pyran sugar to β-furan sugar. Benoit et al. (2011) demonstrated that cellulose could undergo oxidation due to reactive species generated by plasma, while •OH played a critical role in the plasma hydrolysis process of polysaccharides. From this perspective, carbohydrates might partially consume active particles produced by CP, thereby inhibiting plasma-mediated degradation of mycotoxins. The prolonged time required for complete toxin degradation could be attributed to the depletion of highly reactive particles generated by CP since jujube polysaccharide exhibits effective reducing power and various antioxidant activities such as free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating.
Effect of jujube protein on the degradation of AOH and AME by CP
The degradation percentages of AOH and AME by CP at different concentrations of jujube protein were illustrated in Fig. 2, demonstrating the significant impact of jujube protein on the plasma-mediated degradation of AOH and AME. As the concentration of jujube protein increased from 0 g/mL to 0.00750 g/mL, the degradation percentages of AOH after 5 minutes of plasma treatment decreased from 100–43.2%, while AME degradation reduced from 100–43.1%. After being treated with a voltage of 30 kV for 9 minutes, AOH was degraded by 98.2%, 93.4%, and 94.2% when exposed to jujube protein concentrations of 0.00350, 0.00550, and 0.00750 g/mL respectively; whereas AME was degraded by 96.9%, 92.3%, and 87.2%, respectively. When treatment time was extended to 10 min, both toxins could be completely degraded. Based on these findings, it is evident that all tested concentrations of jujube protein could inhibit the degradation of Alternaria mycotoxins by CP to some extent.
The inhibition of jujube protein on mycotoxin degradation by cold plasma may be attributed to the protection of proteins. During CP discharge, ROS including •OH, atomic oxygen (O), 1O2, •O2− and H2O2, accompany O3 was produced (Jiang et al., 2020; Misra et al., 2019). These ROS can disrupt the initial protein structure and enhance the mechanical properties of the interfacial film, thereby increasing their activity and expansion at interfaces (Jiang et al., 2020). Following CP treatment, the protein structure underwent changes leading to the formation of a robust network structure that acted as a stronger barrier between mycotoxin molecules and the surrounding environment. The interfacial film formed by CP is likely to be the primary factor inhibiting AOH and AME degradation. Moreover, •OH is presumed to attack C-H bonds during protein modification by CP, forming CH-OH H-bonded complexes followed by hydrogen motion. Additionally, H2O2 is also related to the dissociation of the C-O bond, but with less effectiveness (Yusupov et al., 2013). It is suggested that jujube protein could scavenge free radicals produced by CP. However, as treatment time prolongs, active particles generated by plasma become sufficient to overcome this blocking effect of proteins and completely degrade toxins.
Effect of Vc on the degradation of AOH and AME by CP
Chinese jujube has been found to possess a high content of Vc (Chen et al., 2019), which may influence the degradation of mycotoxins by CP. Therefore, it is imperative to investigate the impact of Vc on the degradation of Alternaria mycotoxins in jujube juice through CP treatment. As depicted in Fig. 3, in an aqueous solution, the degradation percentages of AOH were 73.8%, 93.3%, 96.7%, and 100% after plasma treatment for 1, 2, 3, and 4 min respectively; while the degradation rates of AME were recorded as 78.4%, 94.0%, 98.4%, and ultimately reaching complete degradation at a rate of 100%. Under identical treatment conditions with a Vc concentration of 0.350 mg/mL, the degradation percentage of AOH were observed as 0%, 72.3%, 84.3%, 91.7%, 97.6%, and 100% respectively, whereas for AME, the corresponding values were 0%, 67.5%, 80.0%, 84.5%, 96.2%, and ultimately reaching complete degradation respectively. When exposed to varying concentrations of Vc, the plasma degradation of AOH and AME increased with discharge time until complete degradation was achieved after 5 min, a slightly longer duration compared to that in aqueous solution.
The results indicated that the degradation of AOH by cold plasma was not significantly impacted by Vc, while AME degradation showed slight susceptibility. The potential inhibition of AME degradation by Vc might be attributed to the antioxidant capacity of VC, wherein the free radicals generated by CP could be scavenged, resulting in a partial reduction in their attack on both AOH and AME. The disparity between AOH and AME might be ascribed to the presence of three hydroxyl groups on the benzene ring of AOH, rendering it more susceptible to oxidation compared to AME which possesses only two hydroxyl groups along with one methoxy group. Similar findings have been reported by Surowsky (2016) who suggested that Vc exhibited protective effects against reactive oxygen species-induced damage in food matrices owing to its inherent antioxidant properties.
Effect of Ca2+ and K+ on the degradation of AOH and AME by CP
Jujube fruit has been demonstrated to possess a high content of essential micronutrients, including Ca2+ and K+, which play crucial roles in promoting health and preventing diseases(Lu et al., 2021). The degradation of AOH in aqueous solution, with or without the presence of Ca2+, was effectively facilitated by CP in a time-dependent manner, as demonstrated in Fig. 4. The degradation percentages of AOH when exposed to Ca + concentrations of Ca2+ of 0.0050, 0.00750, and 0.100 mg/mL were found to be 73.8%, 73.4%, 80.5% and 77.6%, respectively, whereas for AME, the corresponding degradation percentages were observed as 78.4%, 62.3%, 58.7%, and 60.7%, respectively. After being treated by CP at 30 kV for 4 min, the degradation percentages of AME in 0, 0.100, 0.250, and 0.400 mg/mL KCl solution were 100%, 96.4%, 97.8%, and 97.6%, respectively, suggesting that KCl slightly decreased AME degradation. For both Ca2+ and K+, no obvious effect was observed on AOH degradation, while slight inhibition on AME degradation was presented.
It has been reported that the degradation of mycotoxin by CP is attributed to the oxidation ability of ROS and RNS generated by CP. Neves et al. (2022) have suggested that Ca2+ may mitigate the toxic levels of NO2− in pacu juveniles, potentially through alleviating oxidative stress-induced ROS or RNS production. This implies that Ca2+ could reduce the levels of RON and ROS, leading to a slight decrease in AME degradation rate. Feng et al. (2006) have mentioned that KCl acts as a scavenger for •OH. However, it has been established that •OH produced by CP plays a major role in mycotoxin degradation (Attri et al., 2015). It was hypothesized that the partial consumption of •OH by KCl may be the reason for the reduction of Alternaria mycotoxin degradation. Moreover, the impact of K+ on AME degradation surpasses AOH, which could be attributed to the presence of three hydroxyl groups on AOH's benzene ring compared to two hydroxyl groups plus one methoxy group on AME.
Effect of oleic acid and linoleic acid on the degradation of AOH and AME by CP
The impact of oleic acid on the plasma degradation of AOH and AME was illustrated in Fig. 5. Even when exposed to varying concentrations of oleic acid, AOH could be completely degraded after a 3-min treatment with CP. For AME, the degradation percentage was 95.5%, 93.7%, and 96.9% at oleic acid concentrations of 0.0500, 0.100, and 0.150 mg/mL respectively; however, complete degradation occurred within 5 min regardless of concentration level. These results demonstrated that oleic acid had little effect on the degradation of AOH and AME by CP. Figure 5 also depicted the effect of linoleic acid on plasma degradation of AOH and AME; no significant impact was observed for AOH while slight effects were noted for AME at the highest concentration tested (0.15 mg/mL) under these experimental conditions.
Batista et al. (2021) demonstrated that the fatty acid profile remains unaffected by CP technology. Perez-Andres et al. (2020) investigated the impact of cold atmospheric plasma treatment on commercially packaged mackerel fillets and reported insignificant alterations in both fatty acid composition and nutritional quality indices post-treatment. The fact that the degradation of mycotoxin by CP was not significantly affected in the presence of oleic acid and linoleic acid might be because oleic acid and linoleic acid did not consume the active substances produced by CP.
Mechanism of the effect of jujube components on mycotoxin plasma degradation
The effect of jujube components on the electrochemical properties of the systems after cold plasma discharge
ORP is a measure of a solution's ability to oxidize or reduce another substance, reflecting the overall level of RONS present in the solution. A higher ORP value indicates a stronger oxidation capacity (Thirumdas et al., 2018). As shown in Fig. 6, plasma treatment resulted in varying degrees of increase in the ORP of the solution. The results of this experimental study demonstrated that the changes in △ORP for jujube protein solution, jujube polysaccharide, and simulated jujube juice were lower compared to an aqueous solution. However, solutions with different concentrations of Vc, K+, Ca2+, oleic acid, and linoleic acid exhibited similar changes in △ORP as observed for an aqueous solution. The variation trend of △ORP was highly consistent with the degradation pattern of toxins, suggesting that the effect of jujube juice components on AOH and AME degradation might be attributed to the oxidative capability of certain RONS generated by CP discharge.
H2O2, a major active substance generated during CP discharge, exhibits strong oxidation effects (Ma et al., 2021). As depicted in Fig. 7, the H2O2 content of solutions containing various jujube components such as jujube protein, jujube polysaccharide, Ca2+, K+, Vc, oleic acid, and linoleic acid was significantly lower than that of aqueous solution after CP treatment. Tripathi and Mishra (2009) suggested that H2O2 can degrade mycotoxins via oxidation. Additionally, Ma et al. (2021) highlighted the crucial role of H2O2 in chitosan degradation during CP discharge and its decomposition to produce •OH. The impact of different jujube juice components on AOH and AME degradation by CP might be attributed to their partial consumption of H2O2.
The effect of CP on the •OH scavenging rate of various jujube juice component model systems was shown in Fig. 7. The results showed that with the increase of jujube protein concentration from 0 g/mL to 0.00750 g/mL, the •OH clearance decreased significantly from 70.3–24.1%. Furthermore, an increase in jujube polysaccharide concentration to 0.0750 g/mL resulted in a reduction of •OH clearance decreased by 6.19%. Conversely, no notable change was observed in •OH clearance following CP treatment across different concentrations of linoleic acid, Ca2+, K+, and Vc environments. As for oleic acid, there was an initial significant decrease followed by an increase in •OH clearance with increasing concentration. Notably, compared to the aqueous solution, the multi-component model system of jujube juice exhibited a substantial decrease in •OH clearance with increasing concentration. It is worth mentioning that •OH is one of the primary short-lived reactive species generated during CP discharge (Kurake et al., 2017). Several scholars have argued that the oxidizing effectors produced by CP are primarily attributed to •OH radicals which can disrupt structurally important bonds within peptidoglycan (i.e., C-O, C-N, or C-C bonds) (Yusupov et al., 2013). Moreover, variations observed in •OH clearance were highly consistent with the degradation effects exerted by AOH and AME under different jujube component model systems; thus indicating a strong correlation between their removal from jujube juice through CP treatment and •OH generation during discharge.
In addition to ROS, CP discharge also generates extremely rich RNS (NO3− and NO2−, etc.) (Sardella et al., 2021; Yusupov et al., 2013; Lukes et al., 2014). Therefore, it is imperative to investigate the impact of CP on NO3− and NO2− in various model systems representing jujube juice components (Fig. 8). The results revealed that following CP treatment, the concentration of NO2− significantly decreased for different jujube juice components compared to the aqueous solution. This reduction could be attributed to two factors. Firstly, diverse jujube juice components might consume some NO2− through chemical reactions with them. Secondly, there might be a conversion of NO2− into NO3−. Luke et al. (2014) proposed that in aqueous solutions, unstable NO2− could undergo transformation into more stable form i.e., NO3−, leading to pH decrease and increased acidity.
The impact of CP on the NO3− content in various jujube juice component model systems was illustrated in Fig. 8. Following an equivalent duration of CP treatment, distinct jujube juice component solutions exhibited varying levels of NO3− content, with significantly lower concentrations observed when exposed to jujube polysaccharide and protein compared to aqueous solution. However, the presence of Ca2+, K+, oleic acid, linoleic acid, and Vc had minimal influence on the NO3− concentration generated by CP. Consequently, the degradation of AOH and AME by CP in jujube juice was found to be closely associated with the production of NO3− during CP discharge.
The correlation analysis between the changes of electrochemical properties of the system and the CP degradation of toxins
After CP treatment, the Pearson correlation analysis revealed in Fig. 9 and Fig. 10 demonstrated that the degradation of AOH and AME was significantly positively correlated with H2O2, •OH, NO3−, NO2−, and ORP while being negatively correlated with pH when exposed to jujube protein and polysaccharide. Furthermore, a significant correlation was observed between the physical and chemical properties as well as active substance content of jujube protein and polysaccharide solution after CP treatment. This suggested that the inhibition of toxin degradation by jujube protein and polysaccharide was closely related to changes in physicochemical properties and active substances produced by CP.
In the Pearson correlation analysis of AOH and AME degradation and the changes in Vc model system properties, the degradation trend of AOH exhibited significant positive correlations with H2O2, •OH, NO3−, and NO2− contents, while showing a negative correlation with pH value. However, no significant correlations were observed between △ORP and conductivity. Meanwhile, the degradation trend of AME showed positive correlations with H2O2 content, NO2− content, and △ORP; whereas it displayed a negative correlation with pH.
Regarding the Ca2+, K+, oleic acid, and linoleic acid model systems, the degradation trend of AOH showed only partial or insignificant correlation with certain properties or active substances. In summary, following CP treatment, there was a significant positive correlation between the change in △ORP, the contents of H2O2, •OH, NO3−, and NO2− of jujube polysaccharide, jujube protein, and multi-component model system and the degradation trend of toxins. Additionally, the degradation trend of toxins in Vc system exhibited a high correlation with H2O2, •OH, NO3−, and NO2−. However for oleic acid, linoleic acid, Ca2+, and K+, the degradation trend of toxins displayed less correlation with the properties of these systems. Furthermore, the results from Pearson correlation analysis further demonstrated that jujube components could influence the degradation of AOH and AME by CP through consumption of active particles including H2O2, •OH, NO3−, and NO2−. It can be inferred that RONS produced by CP is considered as a primary factor responsible for toxin degradation.
Overall, the present study demonstrated the effective degradation of AOH and AME in both aqueous solution and jujube juice by CP. However, the degradation was partially inhibited in jujube juice. Among these tested components of jujube juice, jujube protein exhibited the highest inhibitory effect on plasma degradation of these two toxins, followed by jujube polysaccharide. Other components including Vc, Ca2+, K+, oleic acid, or linoleic acid showed insignificant effects on AOH and AME degradation. Furthermore, the degradation of AOH and AME by CP was closely associated with the presence of H2O2, •OH, NO3−, and NO2− active particles in different discharge modes during CP treatment. After CP treatment, consumption of RONS such as H2O2, •OH, NO3−, and NO2− by jujube protein or polysaccharide may be a key factor contributing to inhibition of AOH and AME degradation. This study provides a theoretical basis for understanding the mechanism underlying Alternaria mycotoxin degradation in jujube juice system using CP technology and promotes its application for efficient targeted detoxification in food systems.