3.2. Effects of UV-C Irradiation and Thermal Treatment on the Natural Microbiota of Red Prickly Pear Juice (PPJP)
The UV-C radiation doses (9.87, 15.13, and 31.78 mJ/cm2) for both juices at pH 3.6 and 7 effectively reduced the aerobic plate count, total coliforms, yeasts, and mold counts. The thermal treatments (80 °C for 30 s, and 130 °C for 3 s) eliminated the microbial load after treatment at any pH (pH 3.6, 7) or day (0, 5, 10, and 20).
Table 2 shows the microbial load of the untreated red prickly pear juice (control), juices treated with UV-C (three doses), and thermal treatment (HTST and UHT), respectively. Total coliforms, yeasts and molds, and aerobics microorganisms grew in untreated juice at pH 7, but from day five of storage, a decrease was observed. This is related to the pH of the juice (
Table 3); although the juice was at a pH of 7, storage for more than five days caused an increase in the microorganism’s growth, reducing the pH, increasing the acidity of the juice, and inhibiting the growth of the microbiota. Total coliforms are indicators of contamination and can be used to evaluate food safety. UV-C radiation was effective in both pHs (3.6 and 7) as no total coliform growth was observed after treatment. However, the total coliforms were better controlled in acidified juice. This was observed with the control storage time, where these microorganisms were not detected at a pH of 3.6 after five days. An acidic pH helps generate an environment that is not suitable for most microorganisms that harm consumer health and food quality [
19].
Yeasts and molds are considered some of the most difficult microorganisms to eradicate because of their size and production of spores. Ultra-pasteurization and pasteurization were effective for the inactivation of yeasts and molds during the 20 days of storage time. Similarly, the UV-C doses used effectively eliminated the initial 3.26 and 2.32 log CFU/mL loads present in juices at pH 3.6 and 7 on day 0, respectively.
Consistent with these reports, all juices with a pH of 7.0 presented no mold or yeast growth. However, juices treated with a low UV-C radiation dose (9.87 mJ/cm
2) and a pH of 3.6 did contain yeasts and molds from day five of storage time. Yeast and mold growth were also reported in Aloe vera gel acidified with UV-C radiation [
30]. The low efficiency of UV-C treatment in eliminating molds and yeast is attributed to the larger size and thickness of the cell wall [
32]. A higher irradiation dose (31.78 mJ/cm
2) and lower pH (pH = 3.6) prevented the growth of yeasts and molds during total storage time (20 days).
Aerobic microorganisms, such as mesophiles and psychrophiles, showed inactivation of the initial load at all UV-C doses and pH 3.6. A microorganism inactivation level of 5 log CFU/mL is recommended by the FDA to consider UV-C radiation as a pasteurization method [
9]. Mesophilic aerobics had an inactivation of initial load (6.17 and 6.41 log mesophilic CFU/mL) for juice at pH 3.6 and 7.0, respectively. Psychrophiles were diminished by 5.55 log CFU/mL for the juice at pH 3.6 and 5.57 log CFU/mL for juice at pH 7.0. An effective decline in total aerobic microorganisms is not always achieved. Pala and Toklucu [
33] processed orange juice with an initial total aerobe load of 3.25 log CFU/mL using UV-C radiation and reduced it to 2.96 log CFU/mL using a dose of 48.12 KJ/L. This study demonstrated that the UV-C processing of acidified red prickly pear juice (pH 3.6) could be used to provide a safe product for public consumption.
3.3. Effect of UV-C Irradiation and Thermal Treatment on the Physical Properties of Red Prickly Pear Juice (PPJP)
The total soluble solids did not differ significantly (
p > 0.05) between the control juice (4.0 Brix) and the juice processed using both treatments (UV-C and thermal) and were maintained during storage. Shamsudin et al. [
34] also reported that UV-C radiation did not affect the total soluble solids of pineapple juice.
The pH and acidity differed significantly (
p < 0.05) during storage time (
Table 3), specifically with the pH-neutral (pH 7) untreated juice, which showed a decrease in pH and an increase in acidity. These changes are related to microbial growth in the control juice (untreated and not-acidified), specifically the total coliform, mold, and yeast loads, as well as mesophilic microorganisms (
Table 2). However, treated juices (UV-C and thermal) did not show changes in pH during storage time. Stable pH values achieved through UV-C radiation or thermal treatment were also reported for pomegranate [
15].
Color measurement is an important evaluation of juice quality. Color changes in red prickly pear juice are mainly due to the betalain pigments (betacyanins or betaxanthins).
L* is a parameter that measures the luminosity (0 = black, 100 = white) of the juice. It is observed that
L* values ranged from 51.96 to 59.34 (
Table 4). A difference was noted in
L* values between UV-C doses, thermal treatment, and pH. The juice has less turbidity at a pH of 3.6 (
Table 1), which will consequently result in a high
L* value. Juices at both pH levels (3.6 and 7) treated with the highest UV-C radiation dose and HTST showed the same tendency in luminosity due to the loss of compounds such as betalains and polyphenols.
In this study, there was a significant change (
p < 0.05) in
L* during storage of processed juices, which increased with the number of days compared to the control (untreated juice). Juice treated with UV-C radiation showed a significantly lower
L* value (
p < 0.05) during storage compared to UHT treatment, indicating the degradation of red pigments with this thermal treatment. Kathiravan et al. [
6] evaluated a beet-based drink that was pasteurized at 96 °C at different times (540, 720, and 900 s) and stored at 27–30 ± 2 °C for 180 days, evaluating the juice every 15 days, reporting a significant difference (
p < 0.05) in the color parameters due to betalain degradation.
The color parameter
a*, which represents the trend from green (−) to red (+), presented values ranging from 31.38 to 63.98 (
Table 4), indicating a trend towards red (
Figure 1). Color degradation was minimal for UV-C treatments, preserving the red prickly pear juice color, but differed significantly (
p < 0.05) during storage. Juices with a pH of 3.6 presented stable
a* values during storage, indicating betalain stability at an acidic pH. The acidification of juice using citric acid is advantageous due to its chelating property, which improves the stability of betalains against factors such as temperature, light, and the presence of oxygen [
35]. Regarding the juice at pH 7,
a* values remained stable in untreated juice due to the pH decrease during storage resulting in greater stability (
Table 3), while
a* values decreased slightly during storage for UV-C treatments. Conversely, thermal treatment (UHT 130 °C/3 s) significantly increase (
p < 0.05)
a* values during storage in juice at pH 7. Temperature is the main factor affecting betalain stability, changing its hue value from a characteristic red to brownish-red tonality [
36]. Kathiravan et al. [
6] reported that betacyanin changes to yellowish-brown from a deep-violet-red in beet-based drinks pasteurized at 96 °C at different times (540, 720, and 900 s).
The color parameter,
b*, represents the color trend from blue (−) to yellow (+), which ranged from 9.98 to 33.92 (
Table 4), with a tendency to yellow color. This parameter differed significantly (
p < 0.05), with the highest values recorded in the juice at pH 7.0. A significant difference (
p < 0.05) between UV-C radiation and thermal treatments (HTST and UHT) was observed. Increased color degradation was observed at higher temperatures due to the thermolability of the color compounds. The juices at pH 3.6 treated with UV-C radiation presented higher values of the parameter
b* compared to the thermal treatment. However, at pH 7, an increase in the
b* value is observed in UV-C and thermal treatments, so we can observe that the UV-C treatments show an increase during storage, while the thermal one presents an opposite behavior.
Other color parameters,
chroma* and
hue angle represent the saturation and purity of the color, respectively. These differed significantly (
p < 0.05) during the storage of juices processed at pH 7 for both treatments (UV-C and thermal treatment) (
Table 5).
UHT treatment decreased both parameters due to the processing temperature (130 °C), while the juice at pH 3.6 treated with both UV-C and HTST maintained saturation and color purity.
Chroma* values ranged between 45 and 64, with maximum reductions of 23.26% observed in juices treated by UHT. Decreases in
chroma* are related to a decrease in
a* values. The
a* parameter was reduced by thermal treatment (UHT) due to betalain degradation caused by the high temperature (
Table 4).
Hue angle values ranged from 0.75 to 1.40, with maximal reductions of 42.3% in the juice processed by UHT. However, all
hue angle values remained in a magenta color (0°). The
hue angle presents the same tendency as the
a* parameter. Herbach et al. [
37] identified a drastic change in the
hue angle in beet juice heat-treated at 85 °C for 8 h, with a value of 358° for the juice without treatment, thus, reducing this value to 62°, altering the product characterization to a yellowish color.
Color changes (ΔE) were calculated with respect to untreated juice daily, and additionally, color difference value was calculated with respect to untreated juice on day zero (
Table 6). Both values increased significantly (
p < 0.05) during storage. No significant difference was found between UV-C for both pHs (3.6 and 7) on day 0. However, a difference was noted with the thermal treatments, which was related to the
a* parameter and betalain degradation. Most of the color change values (from 1 to 6) were within the range of “perceptible” to “well perceptible” [
38]. However, in juice at pH 7 to 10 and 20 days of storage and in juices treated by UHT (ΔE > 27), the color difference was “very perceptible” [
38] (ΔE > 8.0). In juices processed by HTST and UHT, the color differences decreased during storage. This could be due to the fact that betalains tended to partially regenerate when exposed to optimal conditions [
39], including temperature (4 °C), pH (3.6), and darkness, the storage conditions applied in this research. In contrast, Riganakos et al. [
32] used carrot juice stored for 16 days at 4 °C to investigate UV-C radiation (227.5 mJ/cm
2) and thermal treatment (65 °C for 30 min) and found a significant difference in ΔE, which increased during storage for both treatments.
3.4. Effect of UV-C Irradiation and Thermal Treatment on the Chemical Properties of Red Prickly Pear Juice (PPJP)
Chemical analyses showed that the total polyphenol content in untreated juice at pH 3.6 (
Figure 2a) and 7 (
Figure 2b) decreased significantly (
p < 0.05) during storage, with contents of 159.32 and 153.18 mg GAE/L at day 0, respectively. The juice treated at day 0 by UV-C irradiation showed contents of 170.88 mg GAE/L (31.87 mJ/cm
2) and 175.32 mg GAE/L (9.81 mJ/cm
2), and HTST contents of 156.12 mg GAE/L for juice at pH 3.6. UV-C treatment showed values of 140.81 (31.87 mJ/cm
2) to 152.38 mg GAE/L (9.81 mJ/cm
2), and UHT treatment showed values of 156.98 mg GAE/L for juice at pH 7. Treated juices (UV-C, HTST, and UHT) did not show changes in polyphenols contents from day 5, observing differences only in pH. Moldovan et al. [
40] indicated the importance of pH with regard to polyphenols, and oxidative degradation was more stable at pH levels below 5. UV-C irradiation did not significantly affect (
p > 0.05) the total polyphenol content of the evaluated juices. However, it has been reported that while the content of some polyphenols is reduced, the content of others increases, both changes due to photooxidation or photoinduced molecular rearrangement [
41], so the change will not be observed in the total quantification. Additionally, the UV-C system used is derived in a short time of exposure that minimizes the changes in bioactive compounds, as has been reported by Rodríguez-Rodríguez [
18]. Caminiti et al. [
42] report similar behavior in apple juice treated with doses in the range of 2.66 to 53.10 J/cm
2. However, a decrease of 21.3% of polyphenol content was observed on day 20 in the thermally treated (HTST 80 °C) prickly pear juice at pH 3.6, this due to the polyphenols being considered thermolabile compounds. Santhirasegaram et al. [
43] investigated mango juice treated at 90 °C for 60 s and reported a 38% loss in total polyphenols. This was re-evaluated after five weeks of storage at 4 ± 1 °C, with no significant change in polyphenol content.
The antioxidant activity values in untreated juices were 1004.5 and 610.49 mmol TE/L at pH 3.6 (
Figure 3a) and 7 (
Figure 3b), respectively. UV-C treatment at day zero presented values of 877.1 mmol TE/L (31.87 mJ/cm
2) to 900.6 mmol TE/L (9.81 mJ/cm
2). HTST treatment at day zero presented a value of 434.6 mmol TE/L in juice at pH 3.6. UV-C treatment at day zero presented values of 524.6 mmol TE/L (31.87 mJ/cm
2) to 604.65 mmol TE/L (9.81 mJ/cm
2) and UHT treatment of 255.6 mmol TE/L in juice at pH 7. The antioxidant activity was significantly (
p < 0.05) affected by storage time and pH (
Figure 3). Changes in antioxidant activity are mainly due to bioactive compound alterations of the fruit such as polyphenols, ascorbic acid, carotenoids, and pigments, among others, that can be affected by the processing. Although, in this study, the concentrations of polyphenols did not show noticeable changes due to the effects of the study variables, trends or changes in antioxidant activity can be attributed, in addition to the content, to the type of polyphenol and therefore on its structure [
44] and the radical scavenging capacity of betalains, which could be reduced/increased depending on their structural features [
2]. After processing, there was a reduction in the content of betacyanins and betaxanthins, and it has been reported that betalain degradation products may show enhanced antioxidant properties [
4]. After storage under suitable conditions, betalains can be regenerated or have a change in structure [
39], impacting the antioxidant activity. The changes observed in antioxidant activity at storage day five can be explained by the degradation and regeneration of betalains. There were no changes at storage times greater than five days. The UV-C doses applied did not show a significant difference (
p > 0.05) in the juices treated, presenting a decrease on day zero of 12.68 and 14.06% in juices at pH 3.6 (
Figure 3a) and 7 (
Figure 3b), respectively. Juices irradiated with the higher dose (31.78 mJ/cm
2) in both pHs, and with storage day 20, they decreased antioxidant activities by 40% (pH 3.6) and 36.22% (pH 7). Santhirasegaram et al. [
43] reported no significant difference (
p > 0.05) in antioxidant activity by UV-C dose (3.52 J/m
2) at different irradiation times (15, 30, and 60 min). The thermal treatments significantly affected (
p < 0.05) the antioxidant activity, decreased by 56.73 and 58.13% for HTST and UHT on day 0, respectively. Although phenolic compounds and betalains are considered thermolabile, they are components that present antioxidant activity. A decrease of 37.9 and 40.79% was obtained for HTST and UHT after 20 days of storage, respectively. This could be due to the partial regeneration of betalains when exposed to optimal storage conditions [
39]. Due to storage under stable conditions, antioxidant activity stabilized after five days.
Whit respect to pigments, betacyanin content in untreated juices was 16.65 and 15.66 mg/L at pH 3.6 (
Figure 4a) and 7 (
Figure 4b), respectively. UV-C treatment at day zero presented values of 13.87 mg/L (31.87 mJ/cm
2) to 15.22 mg/L (9.81 mJ/cm
2). HTST treatment at day zero presented a value of 14.18 mg/L in juice at pH 3.6. UV-C treatment at day zero presented values of 12.4 mg/L (31.87 mJ/cm
2) to 15.01 mg/L (9.81 mJ/cm
2) and UHT treatment of 5.87 mg/L in juice at pH 7. UV-C irradiation significantly affected (
p < 0.05) the pigment content at pH 7. The betalain transformation in the presence of light (UV and visible range) leads to electronic excitation, characteristic of the betalain chromophore [
2]. Additionally, in these juices, it was observed that both pigments, betacyanins (red-purple pigments) and betaxanthins (orange-yellow pigments), were significantly affected (
p < 0.05) by storage time. The treatments at pH 7 (
Figure 4b) showed that betacyanin contents decreased as storage time for UV-C treatments. The betalains may be altered/transformed/degraded during storage since they are sensitive to several factors such as pH, and the optimal pH for betalains stability ranges from 5.5 to 5.8 [
2]. All UV-C treatments in juices at pH 3.6 showed betacyanin (
Figure 4a) stability. Citric acid, which was used to acidify the samples, is considered a stabilizer of these pigments [
35]. Changes were noted at doses of 15.13 and 31.78 mJ/cm
2, and immediately after the process, the reductions were 11.11 and 16.69% for betacyanins in juice at pH 3.6 (
Figure 4a) and 19.66 and 20.81% for juice at pH 7 (
Figure 4b), respectively. Values similar to the 22% reduction were reported in the Aloe vera and pitaya blend processed by UV-C light [
30]. Contents observed at lower doses were similar to those of non-treated juice. At a dose of 9.87 mJ/cm
2, betacyanin reductions of 8.58 and 4.15% were observed in juice at pH 3.6 and 7 (
Figure 4a,b), respectively.
Betaxanthin content in untreated juices was 10.07 and 10.36 mg/L at pH 3.6 (
Figure 4c) and 7 (
Figure 4d), respectively. UV-C treatment at day zero presented values of 9.04 mg/L (31.87 mJ/cm
2) to 9.83 mg/L (9.81 mJ/cm
2). HTST treatment at day zero presented a value of 8.62 mg/L in juice at pH 3.6. UV-C treatment at day zero presented values of 8.69 mg/L (31.87 mJ/cm
2) to 10.75 mg/L (9.81 mJ/cm
2) and UHT treatment of 5.63 mg/L in juice at pH 7. The betaxanthin content in juice at pH 3.6 (
Figure 4c) was not affected by UV-C irradiation and showed the lowest reductions, ranging from 2.38 to 10.22%. However, the betaxanthin content of juice at pH 7 (
Figure 4d) reacted similarly to the betacyanin content, showing a significant difference (
p < 0.05) according to UV-C doses. Reductions of 11 and 16.11% were observed at doses of 15.13 and 31.78 mJ/cm
2, respectively. However, despite reductions in pigment content, degradation from UV-C treatments was minimal compared to thermal treatments, which showed a high reduction in the ultra-high pasteurization process. Ochoa-Velasco and Guerrero [
16] reported significant reductions in the pigments of a pitaya drink irradiated with a UV-C light system (57 μW/cm
2) at different flow rates and exposure times, reporting losses ranging from 3.89 to 20.21%, which was reported as total betalains.
Thermal treatments significantly affected (
p < 0.05) both betalains. Both HTST and UHT treatments resulted in diminished pigment content. HTST treatment (80 °C/30 s) reduced (
p < 0.05) the 14.83% of betacyanins and 14.39% of betaxanthins compared to the untreated juice. UHT also significantly decreased (
p < 0.05) betacyanin and betaxanthin contents by 63.09 and 45%, respectively, in comparison with untreated juice. Processing at high temperatures causes hydrolysis, generating betalamic acid and cyclo-dopa-5-O-glucoside [
5]. However, betalains increased from day 5, caused by betalain regeneration due to the hydrolysis occurring due to the high temperature, which could be partial in the bond of betalamic acid, and therefore this bond can be conjugated with precursor amino acids, such as proline [
45]. Additionally, after storage, the content of pigments at pH 3.6 reaches the UV-C treatments by the partial regeneration of betalains when exposed to optimal storage conditions [
39]. Jiménez-Aguilar et al. [
7] investigated a drink based on crystalline prickly pear and red prickly pear, treated with an unconventional method of high hydrostatic pressure (550 MPa/tC 2 min), in comparison with an ultra-pasteurization (UHT) treatment at 138 °C for 2s. UHT treatment resulted in considerable losses of 7 to 45% for betaxanthins and 18 to 26% for betacyanins. However, the unconventional treatment significantly increased the betaxanthin (6–8%) and betacyanin (4–7%) contents, emphasizing the need for alternative non-thermal processing.
The main betalains present in red prickly pear are betanin and indicaxanthin, from the betacyanin and betaxanthin groups, respectively [
46]. Betanin, was identified at 551 m/z ([M+H]
+) and indicaxanthin was identified at 309 m/z ([M+H]
+). These specific pigments were used to monitor the changes for different treatments immediately after processing and at the end of the 20-day storage period. Betanin retention is shown in
Figure 5a. The retention percentage was significantly affected (
p < 0.05) by treatment and pH.
In the juice undergoing pH 3.6 treatments, retention ranged from 53.62 to 87.24%, with retention decreasing as the UV-C dose increased, thereby identifying the lowest retention percentage for the thermal treatment (HTST). Retentions were lower in juice at pH 7 (
Figure 5a), with values ranging from 11.93 to 30.32%, showing no significant difference (
p > 0.05) between the different treatments. It has been reported that betacyanins have more thermal stability at pH 4 [
47], indicating greater stability at a low pH. The retention percentages were significantly reduced (
p < 0.05) for untreated juice and UV-C treatments after storage for 20 days (
Figure 5b), except for the thermal treatment, where an increase in the retention of this pigment was observed. This may be due to partial betalain regeneration when exposed to optimal conditions during storage [
39]. The retention percentage of indicaxanthin was greater than betanin, with values ranging from 41.73 to 92.27% (
Figure 5c). Betaxanthins have shown more stability under heating at pH 6 than betacyanins [
47] and after UV-C treatment [
30]. However, the low retention of betanin and high retention of indicaxanthin could be related because betaxanthin formation from betacyanins has been observed in food systems [
5]. Retention of betaxanthins was significantly affected (
p < 0.05) by pH, indicating that the juice at pH 7 processed with the highest UV-C dose had lower retention than the other UV-C treatments, including the thermal treatments (HTST and UHT), which did not significantly differ. Reduced retention was observed in the untreated juice and in the UV-C treatments after storage for 20 days of storage (
Figure 5d), except in the juice at pH 7 treated with the highest UV-C dose. Similarly, retention was maintained in thermally treated juices. The high reduction in the retention of betanin and indicaxanthin that occurred during storage (
Figure 5) is contrary to the results observed in the total content of betacyanins and betaxanthins (
Figure 4), where it is observed that the content of both pigment groups remains stable (at pH 3.6). This is due to the fact that when quantifying the total betalains per group, reductions in some of the specific pigments attributed to such changes as isomerization or deglycosylation cannot be determined, which has been reported in the processing of betalains [
46]. Another difference identified is, under thermal treatment at pH 7, the results of total betacyanins and betaxanthins showed the lowest contents. However, the quantification of betanin and indicaxanthin shows statistically equal contents.