Since the prices of Alternaria toxin standards are quite high, the toxins used to prepare contaminated apple juice were extracted from microbiological medium on which toxigenic A. alternata grew instead of spiking toxins directly to the juice. For this aim, an A. alternata strain (American Type Culture Collection-ATCC 34957, isolated from sorghum hybrid “Pioneer 8442”) which is known to produce TeA, AOH and AME toxins (40) was used. Before being used in the experiments, the strain was activated on potato dextrose agar (PDA) at 30 °C for 10 days. After activation, spores were harvested by adding 10 mL of sterile distilled water and scraping the surface of the medium with a Drigalski loop. The obtained suspension was filtered through a cheesecloth and the density of the spores in the suspension was measured with a hemocytometer. The required dilutions were made with sterile distilled water to obtain an inoculum density of 5.3 × 10⁶ spores mL⁻¹. A total of 40 petri dishes containing PDA were prepared and each dish center was inoculated with 10 μL of the spore suspension prepared. The inoculated dishes were incubated at 30 °C for 10 days. After this incubation period, the surface of the medium was completely covered with fungal mycelia and toxins were extracted from this medium.

For the extraction of Alternaria toxins, PDA media on which the fungus had grown for 10 days were transferred from 40 petri dishes to a Waring blender and blended for 1 min at high speed. Then, 200 mL of methanol was added to the blender and blended again for 1 min. The contents of the blender were transferred to an Erlenmeyer flask and this flask was shaken for 30 min on an orbital shaker (OS-20, Boeco, Hamburg, Germany). This mixture was filtered through a coarse filter paper and the resulting filtrate was refiltered through Whatman No. 1 (Cytiva, Little Chalfont, Buckinghamshire, United Kingdom) into a rotary evaporator flask. The filtrate in the flask was evaporated in a rotary evaporator (Scilogex RE 100-PRO, CT, United States) at 50 °C to a volume of ~30 mL. This methanolic extract served as the toxin stock solution. A defined volume of this extract was then mixed with 1,300 mL of apple juice (11.2° Brix) to obtain initial toxin concentrations of approximately 791.50 μg L⁻¹ for TeA, 71.37 μg L⁻¹ for AOH, and 34.39 μg L⁻¹ for AME in the contaminated juice. Before UV-C treatment, toxin concentrations in the contaminated apple juice were quantified by HPLC and toxin homogeneity was confirmed by analyzing samples taken from different depths. A portion of 300 mL of the contaminated apple juice that was not treated with UV-C was served as the positive control. The contaminated juice sample was kept at +4 °C until use.

UV-C treatment of juice samples was carried out in a system consisted of a stainless-steel cylindrical chamber with a wall thickness of 1.5 mm, a flow-speed adjustable peristaltic pump (G328A, Grothen, China) and a magnetic stirrer (MSH-20A, Daihan Scientific, South Korea) to ensure homogeneity of the sample. A cooling liquid at +4 °C obtained from a refrigerated circulator (BD 402, Nuve, Ankara, Türkiye) was circulated through the outer jacket of the chamber to remove the heat coming from the UV-C lamps. By this way, the temperature of the juice circulating through the system could be maintained at 20–22 °C throughout the treatment. Maintaining the juice at 20–22 °C prevents any thermal contribution to toxin degradation and avoids temperature-induced biochemical responses that could confound the effects of UV-C. As UV-C exposure and temperature shifts may stimulate oxidative stress pathways and influence phenolic metabolism, controlling the temperature ensures that the reductions observed are due solely to UV-C treatment (41). A photograph of experimental setup for UV-C treatment is given in Figure 1.

The juice was passed through a quartz tube (height: 37 cm; inside diameter: 5 mm; outside diameter: 6 mm) placed along the axis of the cylindrical chamber at a flow rate of 33 mL min⁻¹. Eight UV-C lamps (254 nm, 48 W, 436 mm; LMP-GPH436T5L/HO/4PSE, Atlantic Ultraviolet Co., Hauppauge, NY, United States) were symmetrically positioned around the quartz tube at an equal distance of 2 cm to ensure uniform irradiation along the entire flow path. The central placement of the quartz tube, together with the symmetrical lamp arrangement, provided homogeneous UV-C exposure, while the controlled flow rate and continuous mixing minimized shadowing effects and helped maintain uniform light distribution within the treated juice (Figure 2). This configuration ensured that all portions of the juice were repeatedly exposed to the same UV-C intensity distribution, providing a uniform and reproducible treatment. Juice samples were treated with UV-C light for 1, 2, 4 and 8 h and each treatment time was repeated in triplicates.

UV-C dosage in our system was calculated according to Equation 1 suggested by Keyser et al. (42).

The Retention time in Equation 1 was calculated using Equation 2.

Here, the flow rate was adjusted using the peristaltic pump and set to 33 mL min⁻¹ (0.00055 L s⁻¹). The Volume of the quartz tube was calculated using Equation 3.

The height of the tube was 37 cm and inner radius was 0.25 cm. So, the volume of the quartz tube was calculated as 7.26 cm³ (0.00726 L). Then, the retention time was calculated as 0.00726 L/0.00055 L s⁻¹ = 13.2 s. The intensity in Equation 1 was determined using a radiometer (HD 2302, Delta OHM, Italy), the probe of which was placed at the same position as that of quartz tube. Three replicate measurements were taken from three different positions using the radiometer. The intensity was measured as 37.61 W m⁻² for one single lamp and calculated as 300.84 W m⁻² for 8 lamps in total. Then, the dosage was calculated as 300.84 W m⁻² × 13.2 s = 3971.09 J m⁻².

Analysis of Alternaria toxins in treated and untreated apple juice samples was carried out according to the method of da Motta and Valente Soares (43) with a slight modification. Accordingly, 10 mL of apple juice sample and 40 mL of methanol were mixed and this mixture was stirred for 2 min in a magnetic stirrer. After adding 12 mL of ammonium sulfate solution (10%, w/v), the mixture was stirred for an additional 10 min and filtered through coarse filter paper. The filtrate was transferred into a separating funnel and 10 mL of distilled water at 8 °C was added. The mixture in the separating funnel was extracted with 4 mL of chloroform for 2 min. This extraction step was repeated one more time using 4 mL of fresh chloroform. The chloroform extracts-which contain AOH and AME-were combined in a beaker. The remaining phase in the separating funnel was transferred to an Erlenmeyer flask and its pH was adjusted to 2 with 6 N HCl. This solution was extracted two times with chloroform (2 × 4 mL) as explained above. The resulting extract—which contains TeA—was combined with the previously obtained extracts. The combined extracts were filtered over anhydrous sodium sulfate and the filtrate was evaporated to dryness in the rotary evaporator at 40 °C. The residue was dissolved in 4 mL of high performance liquid chromatography (HPLC) grade methanol and the resulting sample was filtered through a 0.22 μm filter.

HPLC analysis was carried out using a Thermo Scientific Dionex Ultimate 3000 chromatograph equipped with a pump (LPG-3400SD), a column compartment (TCC-3000SD) and a diode array detector (DAD-3000) set at 258 nm for AOH and AME, and at 280 nm for TeA determination. The analytical column was Inertsil ODS 3 (GL Sciences, 5 μm, 250 × 4.6 mm i.d., Tokyo, Japan), the mobile phase was a mixture of methanol:water (80:20, v/v) containing 300 mg L⁻¹ ZnSO₄·H₂O at a flow rate of 0.3 mL min⁻¹ and the injection volume was 20 μL.

Stock solutions of AOH, AME and TeA were prepared by dissolving analytical standards of these toxins in HPLC grade methanol (10,000 μg L⁻¹, 10,000 μg L⁻¹ and 50,000 μg L⁻¹, respectively). Standard solutions prepared by appropriate dilutions of these stock solutions were used to construct five-point calibration curves covering the range of 5–250 μg L⁻¹ for AOH, 2.5–50 μg L⁻¹ for AME and 100–2,500 μg L⁻¹ for TeA. Calibration curves with low concentrations of toxins (0.25–2.50, 0.5–2.0 and 1–20 μg L⁻¹ for AOH, AME and TeA, respectively) were also drawn to determine limit of detection (LOD) and limit of quantification (LOQ) values. LOD and LOQ values were calculated from these curves by multiplying the standard deviation of the response by 3.3 and 10, respectively, and dividing by the slope of the calibration curve. LOD values were determined as 0.32, 0.14 and 1.31 μg L⁻¹ and LOQ values were determined as 0.98, 0.41 and 3.98 μg L⁻¹ for AOH, AME and TeA, respectively. Recovery tests were conducted by spiking apple juice samples with the standard solutions of the toxins to obtain known final concentrations. After leaving the samples for 30 min at room temperature, the extraction and the injection procedures were done as explained above. Triple injections were performed and the mean recovery values were calculated as 96, 88 and 120% for AOH, AME and TeA, respectively. All results given in this study were corrected for average recovery values.

The soluble solids, pH, titratable acidity, turbidity and color values of the apple juice samples were determined before and after the UV-C treatments. Soluble solid content was determined using an Abbe refractometer (Abbe-Ref 2, PCE Instruments, Germany). A pH-meter (Isolab, Eschau, Germany) was used to measure the pH values of the samples. Titratable acidity was determined by titrating with 0.1 N sodium hydroxide solution and expressed as malic acid (g 100 mL⁻¹). Turbidity values were determined using a turbidimeter (2100Q, Hach, Loveland, CO, United States) and expressed as nephelometric turbidity unit (NTU). All parameters were evaluated based on the official methods provided by the International Fruit and Vegetable Juice Association (44).

The effects of the treatments on juice color were determined by measuring the transmittance of the samples at 440 nm against a blank (distilled water) using a spectrophotometer (UV-1201V, Shimadzu spectrophotometer, Kyoto, Japan). In addition, colorimetric values ( L 0 ∗ , a 0 ∗ and b 0 ∗ ) of the juice samples were determined using a colorimeter (CSM 1, PCE Instruments, Southampton, United Kingdom). The color difference (∆E) between treated and untreated samples was calculated using Equation 4.

In the equation, L 0 ∗ , a 0 ∗ , b 0 ∗ were the values of the untreated sample and L ∗ , a ∗ , b ∗ were the values of the treated samples. According to Yamauchi and Kanazawa (45), a classification based on ∆E values could be done as follows: larger than 12, another color group; 6.0–12.0, large difference in the same color group; 3.0–6.0, detectable by ordinary people; 1.5–3.0, detectable by trained people; 0.5–1.5, hard to detect with the human eye; 0–0.5, trace difference.

A gallic acid stock solution was prepared by dissolving 5 mg of gallic acid in 10 mL of distilled water. A series of standard solutions covering a concentration range of 5–100 mg L⁻¹ were prepared from this stock solution. A calibration curve (R² = 0.9974) was constructed using these standard solutions. The total phenolic contents of the samples were determined using this curve and expressed as mg gallic acid equivalent (GAE) per 100 mL. Total phenolic contents of control and UV-C treated apple juice samples were determined according to the method of Singleton et al. (46). Briefly, 1 mL of 2-fold diluted juice sample and 5 mL of 10-fold diluted Folin–Ciocalteu reagent were put in a test tube and mixed vigorously using a vortex mixer for 1 min. The tube was held in the dark for 1 min and 4 mL of sodium carbonate solution (20%, w/v) were added. After mixing for an additional 1 min and holding at 20 °C for 2 h, the absorbance measurements were taken at 760 nm by the spectrophotometer.

A trolox stock solution was prepared by dissolving 5 mg of trolox in 10 mL of distilled water. A series of standard solutions covering a concentration range of 10–50 μM were prepared from this stock solution. A calibration curve (R² = 0.9982) was constructed using these standard solutions. Antioxidant activities of the samples were determined using this curve and expressed as μmol trolox equivalent (TE) per 100 mL. Antioxidant activities of control and UV-C treated juice samples were determined according to the method of Brand-Williams et al. (47). A stock solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was prepared by dissolving 24 mg of DPPH in 100 mL of methanol. Then, DPPH working solution was prepared by diluting this stock solution to give an absorbance of about 1.1 at 515 nm. According to the method, 150 μL of 2-fold diluted juice sample and 2,850 μL of DPPH working solution were put in a test tube and mixed vigorously using a vortex mixer for a min. After holding at 20 °C for 1 h in the dark, the absorbance measurements were taken at 515 nm by the spectrophotometer.

Statistical analysis of the experimental data was obtained from Minitab 16 Statistical Software (Minitab LLC, State College, PA, United States). One-way analysis of variance (ANOVA) was performed to determine the significance of differences between group means. All experiments were performed in triplicate (n = 3) and statistical analyses were conducted using the same number of independent measurements. The differences among the means were tested using Tukey’s multiple comparison test at a significance level of p < 0.05.

Chromatograms showing peaks of Alternaria toxins (for TeA at 280 nm; for AOH and AME at 258 nm) in control and UV-C treated apple juice samples can be seen in Figure 3. Retention times of TeA, AOH and AME were 8.0, 17.4 and 31.6 min, respectively, under the experimental conditions tested. These retention times were in accordance with those of the analytical standards. Moreover, the UV-spectra obtained for the peaks of each analyte were in accordance with the published data of Ashour et al. (48). UV-spectra of the toxins are given as Figure 4.

The concentrations of Alternaria toxins in control and UV-C treated apple juice samples are given in Table 1. As can be seen from the table, TeA was the most abundant toxin in control samples, followed by AOH and AME. The concentrations of these toxins in apple juice were 791.50, 71.37 and 34.39 μg L⁻¹, respectively. These concentrations are quite rational when the indicative levels introduced by EFSA (12) for these toxins in various food products are taken into consideration (2–30 μg L⁻¹, 2–30 μg L⁻¹ and 100–10,000 μg L⁻¹ for AOH, AME and TeA, respectively).

Degradation of all Alternaria toxins tested in the present study increased significantly with increasing the UV-C treatment time (p < 0.05). It might be due to the enhanced formation of reactive oxygen species (ROS)—which are capable of degrading toxins—as the UV-C treatment time prolonged. A similar situation was also reported by Udovicki et al. (24) who studied the effect of UV-C light on aflatoxin B1. In addition to ROS accumulation, prolonged UV-C exposure provides more opportunities for hydroxyl radicals (•OH) to attack the aromatic rings and phenolic hydroxyl groups of Alternaria toxins, initiating oxidative cleavage and structural destabilization. According to photochemical principles and earlier research, UV-C photons possess sufficient energy to break O–H, C–C, and C–O bonds, and this cumulative bond-scission effect becomes more pronounced as the irradiation time increases (23). Therefore, both the increased radical density and the extended duration of molecular photolysis likely contribute to the higher degradation rates observed at longer treatment times.

The shortest treatment time tested in the present study (1 h) resulted in 19.12, 12.89 and 51.02% degradation in the levels of TeA, AOH and AME, respectively. It shows that after 1 h treatment, AME was the most affected one among the tested toxins. On the contrary, Han et al. (23) reported that AOH was more easily degraded than AME after a 90-min UV-C treatment in model system. According to the authors, there are three hydroxyl groups of AOH, which caused it to be more easily oxidized than AME that possesses only two hydroxyl groups on its benzene ring. The same authors reported that citric and malic acid, abundantly found in our samples (apple juice) (49), could increase the degradation of both AME and AOH (23).

Degradation of all Alternaria toxins continued throughout the treatment period tested. Towards the end of the treatment, degradation rate of TeA and AOH increased compared to the beginning. For example, degradation rates recorded for TeA were 19.2 and 42.85% at the end of 1 h- and 2 h-treatments, respectively. Also, AOH was degraded by 24.48, 48.25 and 77.01% with UV-C treatment for 2, 4 and 8 h, respectively. It is obvious that UV-C affects not only the toxins but also the other compounds like phenolics, vitamins in the apple juice (50). At the beginning of the treatment, these compounds (the ones other than toxins) might absorb a substantial amount of UV-C (51). Later on, their structure might change, and their UV-C absorption might be reduced. This can be a logical explanation for the enhanced degradation of the two most abundant toxins in our samples.

In the literature, there are very few studies investigating the effect of UV-C treatment on Alternaria toxins. In one of these studies, UV-C light was used to reduce the amount of toxins produced by A. alternata and to inhibit their penetration into tomato fruit. The authors found that both toxin production and penetration could be effectively reduced by UV-C treatment (35). In another study, the effect of UV-C light on degradation of Alternaria toxins was investigated. After UV-C treatment for 90 min, AOH in methanol, aqueous solution and solid state was degraded by 89.1, 72.9 and 53.2%, respectively, while the degradation percentages of AME were 86.6, 50.1 and 11.1%, respectively (24). It can be clearly seen from these results that the effectiveness of UV-C light strongly depends on the medium treated. The penetration of the UV-C light is higher in liquids than in solids. Our sample (juice) is similar to the aqueous solution among the media tested in the aforementioned study. The degradation rates of AOH and AME reported by the authors for this medium (50.1–72.9%) are quite close to those obtained in the present study.

It was shown that Alternaria toxins can transfer from the infected fruits into the juice if these fruits are used as raw material (52). Heat treatment is the most common technique used in the juice industry to inactivate microorganisms and degrade toxins in the juices. However, scientific studies revealed that pasteurization (95 °C) and even heating over 100 °C were not sufficient to reduce some Alternaria toxins in various juice samples such as tomato juice (53) and pomegranate juice (19). Therefore, nonthermal treatments were also tested for degrading Alternaria toxins in juice and juice-like media. For example, Qi et al. (54) evaluated the efficiency of pulsed light on the degradation of AOH and AME in apple juice. The maximum degradation rates obtained for AOH and AME were 79.2 and 68.7%, respectively. The authors also determined that the degradation rates of the toxins were significantly increased by decreasing the depth of the juice and the distance during the pulsed light treatment. Wang et al. (55) investigated the effectiveness of ozone treatment in degrading AOH and AME in aqueous solution and orange juice. The authors reported that the degradation percentages of AOH and AME in the juice after ozone treatment were 51.86 and 49.04%, respectively, which were comparatively lower than those observed in aqueous solution. In the present study, we tested another nonthermal treatment for degrading Alternaria toxins in apple juice. The maximum degradation rates were observed after the longest treatment time tested (8 h). These rates were recorded as 90.52, 77.01 and 75.92% for TeA, AOH and AME, respectively. These degradation rates are very close to (and even higher than) the ones obtained by the other techniques reported in the literature (54, 55). Due to these promising results, UV-C treatment can be a potential alternative to thermal and nonthermal treatments in order to effectively degrade Alternaria toxins in apple juice. In contrast to conventional thermal pasteurization, which has been reported to be largely ineffective for the degradation of Alternaria toxins (19, 53), UV-C irradiation offers a nonthermal, energy-efficient, and more effective approach capable of achieving substantially higher reduction rates. Although a direct experimental comparison with conventional thermal pasteurization was beyond the scope of the present study, a conceptual comparison can be made based on existing literature. Thermal treatments are well established for microbial safety in fruit juices; however, they are generally ineffective in degrading Alternaria toxins and are often associated with adverse effects on heat-sensitive quality attributes such as color, aroma, and nutritional compounds. In contrast, UV-C treatment offers a nonthermal alternative that has shown potential for toxin degradation while operating at lower energy inputs, although its industrial feasibility and cost-effectiveness depend strongly on juice optical properties and reactor design. Besides, before using UV-C treatment in practical applications, its potential effects on quality characteristics of apple juice should be revealed.

The effect of UV-C treatment on soluble solid content, pH, titratable acidity, turbidity and color values of apple juice is given in Table 2. Soluble solid content, pH and titratable acidity values did not change significantly after the UV-C treatments (p > 0.05). It shows that the UV-C light does not have a substantial impact on sugars and acids, which are the main contributors to the soluble solids of the apple juice. Similar results were reported for other kinds of juices (56–58). On the other hand, turbidity values of the samples increased as the UV-C treatment time increased. Turbidity value of the control sample was 0.76 NTU whereas the turbidity values of the samples treated with UV-C for 1, 2, 4 and 8 h were 0.88, 0.93, 1.27 and 1.46 NTU, respectively. The turbidity of apple juice is primarily attributed to pectin, proteins, free amino acids, phenolic compounds, and the presence of living or dead microorganisms (56). While thermal treatments are known to disrupt cell wall structures and release pectin, proteins, and other macromolecules into the juice—thereby increasing turbidity—previous studies have not reported a comparable effect for UV-C irradiation. In fact, UV-C has been described as capable of reducing turbidity and improving the visual clarity of apple juice (56). Nevertheless, turbidity responses to UV-C exposure may vary depending on juice composition and matrix complexity. In beverages that are initially low in turbidity, haze formation can occur through protein–polyphenol interactions, leading to the formation of colloidal aggregates (59). Considering these mechanisms, the increase in turbidity observed in the present study may be associated with UV-C-induced alterations in phenolic compounds, partial destabilization of pectin-protein structures, or the formation and dispersion of fine suspended particles within the apple juice matrix.

Color is one of the most important attributes that affect the consumers’ preference for the juices, as well as other food products. Table 2 shows the effect of UV-C treatment on various color parameters of the apple juice. The transmittance value of the apple juice sample significantly increased as the UV-C treatment time increased (p < 0.05). It means that the UV-C treated samples are lighter in color compared to control samples. However, this effect was not correlated with L^* values (an indicator of luminosity) which did not change significantly due to UV-C treatment. On the other hand, a^* and b^* values significantly decreased as the UV-C treatment time increased (p < 0.05). Decreases in a^* and b^* values mean decreases in redness and yellowness, respectively, which is probably due to photodestruction of the color pigments of the juice. These pigments can either be originally present in the juice or can be formed later by the rapid action of polyphenol oxidase (enzymatic browning reactions) as well as the Maillard reaction between sugars and amino acids (58). Our results are in accordance with the ones of Guerrero-Beltrán and Barbosa-Cánovas (60) who reported a simultaneous decrease in a^* and b^* values of UV-C treated apple juice. They observed that the transparent yellow-brown color of apple juice turned slightly to less red and to less yellow in UV-C treated samples. Decreases in a^* and b^* values in the juices of other fruits such as peach and lemon as a result of UV-C treatment were also reported (61).

We also calculated the ∆E values to evaluate the color differences between the control and UV-C treated samples. These values significantly increased by increasing the UV-C treatment time. For example, ∆E values were 4.11 and 4.72 after UV-C treatments for 1 h and 2 h, respectively, which means that the difference observed is “appreciable; detectable by ordinary people” according to the Handbook of Color Science (45). After treatments for 4 h and 8 h, ∆E values were 8.46 and 12.78, respectively. These values correspond to differences that can be classified as “large difference in the same color group” and “another color group,” respectively. Discoloration of apple juice due to UV-C treatment is clearly seen in Figure 5.

The concentration and composition of phenolics in apple juice are not constant and affected by the raw material and the technology used in production (62). But in general, apples and their products are rich in phenolic acids such as chlorogenic acid, gallic acid, caffeic acid, coumaric acid and flavonoids such as catechin, phloridzin, quercetin, quercetin glycosides (63). The effect of UV-C treatments for different time periods on the total phenolic content of apple juice is shown in Figure 6. As can be seen from the figure, the total phenolic content did not change during the first 4 h of UV-C treatment and was not significantly different from the control (p > 0.05). However, a clear decrease was observed only after 8 h of UV-C exposure (p < 0.05), with the 8-h treatment resulting in a 16% reduction in the total phenolic content of the juice. Our results are in parallel with those of Xiang et al. (63), who reported decreases of 3.03–10.45% in the total phenolic content of apple juice following exposure to UV-C LEDs at doses of 200–1,200 mJ cm⁻². Similar reductions were also noted by Noci et al. (64), who observed a statistically significant decrease in total phenolics after UV-C treatment. In contrast, Islam et al. (38) reported that the total phenolic content of apple juice remained unchanged regardless of UV-C exposure. Additionally, Barut Gök (37) found that UV-C treatment significantly increased the total phenolic content of apple juice, further highlighting the variability in phenolic responses to UV-C depending on the juice matrix and processing conditions.

The effect of UV-C treatments for different time periods on the antioxidant activity of apple juice is shown in Figure 7. It is seen from the figure that the antioxidant activity gradually decreased as the treatment time increased. The antioxidant activity of apple juice sample treated with UV-C for 8 h was about only 1.7% of that of the control. In the literature, it was indicated that the antioxidant activity of apple juice is mostly due to phenolic compounds and in particular polyphenolic acids, flavonoids and proanthocyanidins (65). However, our findings on the decrease in antioxidant activity cannot be explained by the decrease in phenolic content since total phenolic content values of our samples maintained for the first 4 h of the treatment. Antioxidant compounds other than phenolics such as chlorophylls and some antioxidant vitamins (vitamins A, C and E) found in apple juice might play a role in the decrease of antioxidant activity observed. Barut Gök (37) reported that fruit juices contain different antioxidant compounds that could react in different mechanisms when treated with UV-C. In accordance with our results, some authors reported decreases in antioxidant activity of UV-C treated apple juice (38, 39, 56). However, it was also reported that antioxidant activity of apple juice did not change (66) and even increased (37) as a result of UV-C treatment. According to Alothman et al. (67), the behavior characteristics of antioxidants in plants and plant-based products differ greatly and are not fully enlightened yet. The effects of ongoing chemical reactions such as enzymatic and non-enzymatic browning reactions on antioxidant activity of the product and the interactions of UV-C light with all these compounds make the situation more complicated to interpret.

Although UV-C treatment substantially reduced toxin levels, its potential impacts on sensory properties such as flavor and aroma should also be considered, especially given the observed changes in color, turbidity, and phenolic quality. While UV-C exposure yielded 70–90% degradation of Alternaria toxins, it remains essential to evaluate whether the residual concentrations fall within regulatory or health-based safety thresholds; however, no specific limits have yet been defined for apple juice. These findings highlight the intrinsic balance between food safety and food quality: higher UV-C doses or prolonged exposure may be necessary to achieve sufficient toxin degradation in opaque matrices such as apple juice, but such conditions can simultaneously intensify quality deterioration, as demonstrated in our experiments. From a technological perspective, strategies such as blending UV-C treated juice with untreated batches may help achieve an acceptable compromise by improving toxin safety while preserving sensory and nutritional attributes. In practical industrial applications, the optimization of UV-C dose and treatment time—aimed at achieving significant toxin reduction with minimal sensory and nutritional losses—should therefore be carefully considered. In order to address the quality deterioration that has been observed at prolonged UV-C treatment times, additional process optimisation approaches may be implemented. These include the treatment with higher UV intensities for shorter exposure times, multiple-pass short-time treatments, or improved reactor designs that enhance UV dose distribution and reduce overall treatment time. Furthermore, the combination of UV-C processing with other non-thermal technologies (e.g., ultrasound, pulsed light) may facilitate synergistic toxin degradation while minimising adverse effects on sensory and nutritional attributes. Preliminary steps, such as clarification or filtration, have been shown to enhance UV penetration in opaque juice matrices. In addition, post-treatment strategies, including antioxidant addition, have been reported to mitigate oxidative quality losses associated with intense UV exposure. Collectively, these integrated approaches underscore the potential of process integration and optimization to surmount the quality challenges associated with protracted UV-C exposure in industrial juice processing.

The findings of this study provide specific insights into the effectiveness of UV-C treatment for reducing Alternaria toxins in apple juice and its impact on juice quality, thereby allowing validation of the proposed hypotheses. The first hypothesis was confirmed as UV-C treatment resulted in a statistically significant degradation of the major Alternaria toxins (AOH, AME, and TeA), with degradation efficiencies up to 90% at prolonged treatment times in comparison with untreated controls. The reduction of toxins was found to increase in a consistent manner as a function of the duration of UV-C exposure, thereby demonstrating the efficacy of UV-C irradiation in mitigating Alternaria toxins in apple juice. With regard to the second hypothesis, which was partially confirmed, UV-C treatment caused limited changes in selected quality characteristics at shorter treatment periods; however, extended UV-C exposure led to pronounced reductions in antioxidant activity, exceeding the predefined 20% threshold.

The limitations of the present study are as follows: The experiments were conducted using artificially spiked apple juice samples containing three Alternaria toxins (AOH, AME, and TeA). It should be noted that these toxins may not fully reflect the complexity of naturally contaminated matrices where toxins coexist with fungal spores, secondary metabolites, and native microflora. The study was confined to a single juice matrix and did not include validation against naturally contaminated samples or pathogenic microorganisms. Furthermore, it did not assess potential synergistic or antagonistic interactions among multiple contaminants. Moreover, the shelf-life stability, microbiological safety, and sensory attributes of UV-C-treated juice during storage were not evaluated. Despite the substantial toxin degradation achieved, the relatively long treatment times applied under laboratory conditions give rise to questions regarding industrial feasibility and throughput constraints in commercial juice processing. Additionally, the absence of established regulatory maximum limits for Alternaria toxins in apple juice complicates direct compliance assessment. Finally, the chemical identity and toxicological relevance of degradation by-products formed during UV-C treatment were not investigated and remain a critical knowledge gap that must be addressed before industrial implementation.