Physical, Chemical and Microbiological Properties during Storage of Red Prickly Pear Juice Processed by a Continuous Flow UV-C System

Abstract

The effects of pH (3.6 and 7.0) and irradiation UV-C dose irradiation (0, 9.81, 15.13, and 31.87 mJ/cm²) on the physicochemical properties and natural microbiota of red prickly pear juice were evaluated during processing and storage. Thermal treatments were used as the control applying high temperatures for a short time (HTST 80 °C/30 s) or ultra-high temperature (UHT 130 °C/3 s). UV-C treatments applied to juices with both pHs inactivated coliforms and mesophiles with the same efficacy as thermal treatments. Yeasts and molds were inactivated at a dose of >15.13 mJ/cm² at both pHs. The UV-C doses showed no differences in betalains, polyphenols, or antioxidant activity. However, a decrease in these compounds was observed during storage. The lowest reductions in betacyanins (11.1–16.7%) and betaxanthins (2.38–10.22%) were obtained by UV-C treatment at pH 3.6. Thermal treatments (HTST and UHT) caused a reduction greater than UV-C irradiation in betacyanins, betaxanthins, polyphenols, and antioxidant activity after treatment. However, after storage at pH 3.6, the contents of these compounds reached those of the UV-C treatments, except for polyphenols. In specific pigments, betanin retention was highest at pH 3.6 (62.26–87.24%), and its retention decreases with UV-C dose increase and storage. The indicaxanthin retentions were higher (75.85–92.27%) than those of betanin, and the reduction was mainly due to storage. The physical properties (pH, acidity, and °Brix) were not affected by treatments, except for the color. The results suggest that a dose of 15.13 mJ/cm² of a continuous UV-C system is a non-thermal alternative for the processing of red prickly pear juice at pH 3.6, preserving its properties.

1. Introduction

The consumer demand for foods produced with natural ingredients is increasing. Commercial juices may contain synthetic colorants or color enhancers since they are stable when subjected to different preservation processes. Despite this advantage, the addition of colorants has been related to such health problems as indigestion, anemia, allergic reactions (asthma and urticaria), pathological brain lesions, tumors, cancer, paralysis, intellectual disability, abnormal offspring, growth inhibition, and ocular defects resulting in blindness [1]. An enormous variety of exotic fruit and vegetables are used to produce beverages as these contain various vitamins, minerals, and some bioactive compounds. A Mexican fruit with high nutritional value and good organoleptic properties associated with health benefits (functional foods) is the red prickly pear (Opuntia ficus indica). In addition to its nutritional value, the fruit provides other properties such as antiradical or antioxidant, anti-inflammatory, and other properties, which are attributed to the presence of betalains [2] and polyphenols [3]. Betalains are pigments that can be divided into two types, betaxanthins and betacyanins, and provide yellow-orange and purple-red colorations, respectively [4]. Betacyanins are most abundant in the red prickly pear, resulting in its characteristic color. Red prickly pear is mostly consumed fresh and near to production areas because of postharvest losses as it is highly perishable. These disadvantages can be addressed by processing beverages with functional and nutritional benefits. However, betalains are affected by changes in pH and water activity, the presence of light and oxygen, and high temperatures associated with processing [5], rendering thermal treatment inadequate. Several studies have reported betalains content reductions associated with thermal treatment. The pasteurization of beetroot juice caused pigment losses of 39.9 and 42.28% for betacyanins and betaxanthins, respectively. However, an increase in these pigments (67.42 to 64.13%) and a reduction in antioxidant activity (63.87%) were observed during storage [6]. The ultra-pasteurization of prickly pear beverages caused betaxanthin and betacyanin losses of 45 and 26%, respectively, and a 52 and 45% reduction in total polyphenols and antioxidant activity, respectively [7]. A non-thermal method can be used to reduce the loss of compounds of interest, such as the application of short-wavelength ultraviolet (UV-C) radiation. UV-C is approved by the Food and Drug Administration and can preserve heat-sensitive compounds, among other advantages [8,9]. UV-C technology has been used in a variety of juices, including apple and grape [10], onion [11], orange [12], guava, pineapple, apple cider, and mango nectar [13]. The main objective of these studies was to prove the effectiveness of UV-C in the inactivation of bacteria, yeasts, and molds and to evaluate the behavior of these microorganisms during storage. However, the immediate and shelf-life changes in bioactive compounds, which contribute favorably to the nutritional and functional quality, or acceptability of the product, have not yet been evaluated in depth. Some studies have been reported on the study of changes in pigments such as carotenoids [14] and anthocyanins [15] related to acceptability attributes such as color. Recent studies have reported the effects of UV-C light on bioactive compounds and pigments. Pigments and phenolic compounds showed a reduction in pitaya (Stenocereus griseus) juice treated with UV-C, attributed to the action of photons produced by UV-C light. The photons could be absorbed by organic molecules such as betalains that have conjugated bonds and aromatic rings, provoking changes in the structure. However, the microbial reduction was not sufficient to consider it a safe product [16]. A cranberry-flavored beverage processed using UV-C treatment did not show changes in the content of anthocyanins [17]. In pineapple (Ananas comosus) juice, UV-C and heat treatments slightly reduced phenolic acids and antioxidant activity immediately after treatment, with greater reductions after storage [14]. Although the efficacy of UV-C light has been demonstrated for the inactivation of microorganisms, this is dependent on the physical, chemical, and optical properties of the liquid medium [8,18]. Additionally, other factors such as pH help to generate an unsuitable environment for most of the bacteria involved in food spoilage [19] and are related to the stability of bioactive compounds in the food matrix. These variables, in combination with the operating conditions such as irradiation dose, could impact, in addition to the microbiological quality, the changes that occur in the bioactive compounds in irradiated beverages, mixtures, or juices. Therefore, the composition of the medium, the pH, and the operating conditions during UV-C irradiation should be evaluated, considering that the information on the processing with UV-C irradiation in pigmented matrices is scarce and its impact on the physical and chemical characteristics and microbiological behavior during storage is not clear. This study aimed to evaluate the effect of thermal treatments (HSTS and UHT) and UV-C radiation at different doses (0, 9.81, 15.13, and 31.87 mJ/cm²) on the physicochemical properties and natural microbiota of prickly pear juice at different pH (3.6 and 7) levels during processing and storage.

2. Materials and Methods

2.1. Solvents and Reagents

Analytical grade methanol, hydrochloric acid, sodium hydroxide, and standard buffers with a pH of 7.0 (±0.01) and 4.0 (±0.01) were obtained from J. T. Baker (Mexico City, Mexico). DPPH (2,2-diphenyl-1-picrylhydrazyl), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Folin–Ciocalteu reactive, sodium carbonate, and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Juice Preparation

Red prickly pears (Opuntia ficus indica) [60 kg] harvested from central Mexico were classified, washed, disinfected (NaCIO, 150 ppm), cut, peeled, and homogenized using Pulper equipment (Bertuzzi Brugherio, Milano, Italy) and a 0.1 mm sieve. The juice was centrifuged (IEC Centra CL3R centrifuge, Thermo Electron Corporation, Waltham, MA, USA) at 3600× g, at 4 °C for 10 min and filtered (No. 1; Whatman, Kent, United Kingdom). Then, a 1:3 dilution with purified water (agUach^®) was prepared. A portion of the diluted juice was acidified (pH 3.6) using citric acid (20%). The remaining juice was kept at its natural pH of 7. The pH, soluble solids (Brix), color (L*, a*, and b*), antioxidant activity, and total polyphenol and betalain (betacyanins and betaxanthins) contents were analyzed. The juice was frozen and stored at −20 °C in darkness until processing.

2.3. UV-C Treatment

UV-C irradiation was performed using a commercial CiderSure 3500 UV continuous flow reactor (FPE Inc., Macedon, NY, USA). This unit consists of an outer casing of stainless steel, which includes eight low-pressure mercury lamps located concentrically inside a quartz cylinder. The fluid to be processed is exposed to ultraviolet radiation. The UV-C unit operates at an irradiation wavelength of 254 nm and has two UVX-25 sensors (UVP, Inc., Upland, CA, USA) placed in the upper and lower parts of the cylinder to detect passing radiation. The UV-C dose administered was previously determined [18]. The exposure time was determined by dividing the average flow (GPH) of each experimental operation by the volume of the equipment:

$$UV-C dose \left(\frac{\mathsf{\mu }\mathrm{J}}{{\mathrm{cm}}^2}\right)=\mathrm{Irradiance}\mathrm{intensity}\left(\frac{mW}{c{m}^2}\right)Exposure time\left(s\right)$$

(1)

The doses calculated were 9.87 ± 0.62, 15.13 ± 0.35, and 31.78 ± 0.17 mJ/cm². The physical, chemical, and microbiological properties of unirradiated (control) and UV-C irradiated juices were analyzed. Treatments were performed in duplicate. The processed juices were aseptically poured into sterile glass bottles with screw caps (250 mL). Bottles were stored at 4 °C in the dark, and physicochemical and microbiological analyses were performed on days 1, 5, 10, and 20.

2.4. Thermal Treatment

Thermal treatments were performed using an ultra-high-temperature–high-temperature short-time (UHT-HTST) LAB-25-DH MicroThermics system (Sigma Equipment, EV, IN, USA). HTST treatment was used for acidified juice (pH = 3.6). It was processed at 80 ± 1 °C for 30 s. Juice with a pH = 7.0 was processed using UHT treatment at 130 ± 1 °C for 3 s. Physical, chemical, and microbiological properties of juices with and without thermal treatment (control) were analyzed. Treatments were performed in duplicate. The processed juices were aseptically poured into sterile glass bottles with screw caps (250 mL). Bottles were stored at 4 °C in the dark, and physicochemical and microbiological analyses were performed on days 1, 5, 10, and 20.

2.5. Optical Properties

Turbidity (T), absorption coefficient (α), and water depth penetration (λ) were evaluated as optical properties. Turbidity, expressed in nephelometric turbidity units (NTU), was determined using a Micro 100 HF Scientific, Inc. turbidimeter (Fort Myers, FL, USA). The absorption coefficient, expressed in 1/cm or 1/mm, was measured according to Gayán et al. [20], using FireflySci detachable quartz cells of different depths (0.1, 0.2, 0.5, and 1 mm) at 254 nm and a UV/Vis Lambda 25 spectrophotometer (Perkin-Elmer, Waltham, MA, USA). The coefficient was calculated by determining the slope of the absorbance graph. The penetration depth parameter (λ) was measured using Equation (2) [20].

$$\lambda =\frac{1}{\alpha }$$

(2)

2.6. Microbiological Analysis

A total of 1 mL of decimal dilution of untreated and treated red prickly pear juice samples were pipetted into Petri dishes using the pour plate method. Total aerobic plate counts and psychrophiles were enumerated using standard count agar. Incubation was performed at 35 °C for 48 h and 4 °C for 7 d. Total coliforms were enumerated on red-violet-bile-lactose agar. Incubation was performed at 35 °C for 24 h. Total yeast and mold counts were enumerated on potato dextrose agar. Total yeast and mold counts were incubated at 25 °C for 5 d. Each test was performed in duplicate, and the results were expressed as colony-forming units (CFU) per mL. The shelf life of fresh red prickly pear juice was tested by determining the aerobic plate count, psychrophiles, total coliforms, and total yeast and mold counts of treated and untreated samples stored at 4 °C in darkness for 1, 5, 10, and 20 days.

2.7. Soluble Solids and pH

Soluble solids (°Brix) and pH were measured at 25 ± 1 °C using a manual refractometer (ATAGO N-1α. CO. Ltd., Tokyo, Japan), and a pH meter (HANNA Instruments, model EDGE HI2020, Woonsocket, RI, USA) calibrated at 7.0 and 4.0 pH with standard buffers, respectively. The analysis was performed in triplicate.

2.8. Titratable Acidity

The official AOAC 942.15 [21] method was used. The juice (10 mL) was titrated with 0.01 N sodium hydroxide (NaOH) using a pH meter to measure the end point (pH 8.2 ± 0.1). Titratable acidity was calculated using the following equation and was reported as citric acid (%):

$$TA (\%)=\frac{V\times 0.01 NaOH\times 0.067\times 100}{M}$$

(3)

where V is the volume of NaOH and M is the sample volume.

2.9. Color Parameters

The color was determined [16] using a Konica Minolta CR-400/410 colorimeter (Minolta Co., Osaka, Japan), calibrated using a standard tile (X = 94.9, y = 0.3185, and x = 0.3124). Color was expressed as L* (lightness), a* (greenness-redness), and b* (blueness-yellowness) values. The determination was carried out in triplicate. These values were then used to calculate the chroma* and hue angle. The chroma* value, which indicates color intensity, was determined using the following formula:

$$Chrom{a}^{*}={\left(a^{*2}+b^{*2}\right)}^{1/2}$$

(4)

Hue angles can vary between 0° (pure red color), 90° (pure yellow color), 180° (pure green color), and 270° (pure blue color). The angle was calculated using the following formula:

$$hue angle=arctan\left(\frac{b^{*}}{a^{*}}\right)$$

(5)

The color differences between the treatment control and processed juice, and color differences by storage time, were calculated using the following formula:

$$∆E^{*}={\left[\left(L_c^{*}-L^{*}\right)+\left(a_c^{*}-a^{*}\right)+\left(b_c^{*}-b^{*}\right)\right]}^{1/2}$$

(6)

where L*_c, a*_c, and b*_c correspond to the control or treated juice on day 0.

2.10. Extracts for Chemical Determination

The juice samples were centrifuged at 3600× g at 4 °C for 10 min (IEC Centra CL3R centrifuge, Thermo Electron Corporation, USA). Supernatants were filtered through a 0.45 μm nylon filter (Millipore Corp., Bedford, MA, USA) for the determination of betalains, and one 0.22 μm polyethylene (Millipore Corp., Bedford, MA, USA), for total polyphenols and antioxidant activity.

2.11. Total Phenolics

Total polyphenol content was determined using the Folin–Ciocalteu spectrophotometric method [22], with some modifications. We reacted 30 μL extract, 3 mL distilled water, and 200 μL Folin–Ciocalteu reagent for 10 min at room temperature (25 °C). Then, 600 μL of a 20% sodium carbonate solution was added and incubated for 20 min at 40 °C in a temperature-controlled bath (Fisher Scientific, model 210, Waltham, MA, USA). It was cooled on ice and measured at 760 nm using a UV/Vis Lambda 25 spectrophotometer (Perkin-Elmer, USA). A gallic acid standard was used to obtain the calibration curve. The analysis was performed in triplicate. The results were expressed as the mg equivalents of gallic acid (mg GAE)/L.

2.12. Antioxidant Activity

Antioxidant activity was determined using the DPPH˙ spectrophotometric method [23]. First, 0.1 mL of sample was added to a DPPH˙ solution in methanol (3.9 mL, 80 μM), shaken, and stored in the dark for 3 h. Absorbance was measured at 517 nm using a UV/Vis Lambda 25 spectrophotometer (Perkin-Elmer, USA). A Trolox standard calibration (0.2 to 1.4 mM) was used to determine the antioxidant activity. Measurements were taken in triplicate, and the results were expressed as mmol Trolox equivalent (TE)/L.

2.13. Betalain Quantification

The betalain content was determined photometrically [24] using a UV-Vis Lambda 25 spectrophotometer (Perkin Elmer, USA). Red prickly pear juice, with and without treatment, was diluted in a 1:1 ratio with buffer Mcllvain (pH 6.5, citrate-phosphate) to obtain absorption values in the range of 0.9–1.0. The betacyanin and betaxanthin contents were measured at 537 and 492 nm, respectively, and calculated according to the following equation:

$$BC=\frac{A\ast DF\ast M_w\ast 1000}{\epsilon \ast l}$$

(7)

where BC is the betalain content (mg/L), A is the absorbance, DF is the dilution factor, l is the trajectory length of the quartz cells (1 cm), ε is the molar excitation coefficient (60,000 L/(mol cm) for ε-betacyanins, and 48,000 L/(mol cm) for betaxanthins), and M_w is the molecular weight (550 and 308 g/mol for betacyanin and betaxanthin, respectively). Measurements were performed in triplicate.

2.14. Retention of Betanin and Indicaxanthin

Juice samples processed on day 0 and day 20 of storage were filtered through 0.45 μm nylon filters (Millipore Corp., Bedford, MA, USA). A total of 10 μL of an internal standard (Umbelliferone, 1000 ppm, Sigma-Aldrich, St. Louis, MO, USA) was also added. Samples were analyzed using triple quadrupole liquid chromatography equipment with a PDA detector (WatersTM 996) and an Ultra AQ C18 column (3 μm × 100 × 2.2 mm) coupled to a WatersTM 2695 mass spectrum. Samples were analyzed by electrospray in mode positive (ESI+) under the following conditions: nitrogen as carrier gas at 450 °C, m/z range of 100–1500, with a flow of 350 L/h and a capillary voltage of 3000 V. Mobile phase A of 2% (v/v) formic acid in methanol and phase B of 0.1% formic acid in methanol at a flow of 0.25 mL/min [25]. Monitoring was performed at 537 and 492 nm for betacyanins and betaxanthins, respectively.

2.15. Experimental Design and Statistical Analysis

A completely randomized factorial design was used (4 × 2). Independent variables were the UV-C irradiation doses (0, 9.81, 15.13, and 31.87 mJ/cm²) and the pH (3.6 and 7.0). Analyses of variance (ANOVA) were performed to evaluate the effect of UV-C and thermal treatments. Differences between samples were determined using Tukey’s test at a 95% significance level. Experimental design and data analyses were performed using Minitab version 17 software [26] and SPSS version 22 software [27].

3. Results

3.1. Optical Properties of Red Prickly Pear Juice

Optical properties are the most important characteristics to consider when exposing a beverage to UV radiation as a preservation method. Juices were analyzed before treatment to verify their optical properties and to determine the effectiveness of UV-C radiation treatment.

Table 1. Optical properties of red prickly pear juice.

	T (NTU)	α (1/cm)	λ (cm)
Juice no-diluted	274.8 ± 0.56 a	24.36 ± 0.31 a	0.04 ± 0.00 b
PPJP at pH 3.6	21.35 ± 0.14 c	5.97 ± 0.03 b	0.17 ± 0.00 a
PPJP at pH 7.0	68.70 ± 0.30 b	6.09 ± 0.07 b	0.16 ± 0.00 a

Mean ± standard deviation. T = Turbidity, α = absorption coefficient, λ = depth penetration, PPJP = prickly pear juice diluted (1:3). Different superscript by column represent significantly different mean (p < 0.05), by Tukey’s test.

shows the properties of turbidity, absorption coefficient, and penetration length of the diluted and non-diluted red prickly pear juice. The three optical properties of the diluted juice samples differed significantly (p < 0.05). Non-diluted juice presented different optical property values as it was made from fruit and had an opaque appearance, mainly due to the soluble solids present, as well as the color of the pigments of the fruit [28]. The absorption coefficient determined in diluted samples, both acidified and non-acidified, was approximately 6/cm. This was similar to the result communicated by Kaya and Unluturk [29], who reported coefficient values of absorption of 5.63 ± 0.01/cm for clarified grape juice, higher than those reported in Aloe vera and pitaya blends [30]. An absorption coefficient of less than 15/cm is desirable to ensure a 5 log of Escherichia coli K12 reduction in fruit juices [31].

The penetration depth was 75% less in the undiluted juice than that in both diluted juices (acidified and non-acidified), favoring UV-C radiation efficiency. The penetration depth (0.16 and 0.17 cm) was lower compared to the Aloe vera and pitaya blend [30]. This is likely because the juice they studied only contained 5% pitaya juice. The turbidity of non-diluted juice was 274.8 ± 0.56 NTU, and the opacity is caused by the intense redness of red prickly pear juice. The turbidity, absorption coefficient, and depth penetration were reduced when the juice was diluted (1:3), making the red prickly pear juice prepared (PPJP) suitable for efficient UV-C processing (Table 1).

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. Effect of UV-C irradiation and thermal treatment on the microbiota of red prickly pear juice.

Treatment	Total Coliforms (Log CFU/mL)
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	4.94 ± 0.05	ND	ND	ND	3.95 ± 0.03	4.8 ± 0.01	3.29 ± 0.03	ND
UV-C (9.87 mJ/cm2)	ND	ND	ND	ND	ND	ND	ND	ND
UV-C (15.13 mJ/cm2)	ND	ND	ND	ND	ND	ND	ND	ND
UV-C (31.78 mJ/cm2)	ND	ND	ND	ND	ND	ND	ND	ND
HTST (80 °C)	ND	ND	ND	ND	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	ND	ND	ND	ND
Treatment	Yeast and Molds (Log CFU/mL)
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	2.32 ± 0.00	3.46 ± 0.26	4.3 ± 0.00	1.44 ± 0.04	3.26 ± 0.01	1.88 ± 0.05	1.81 ± 0.16	1.66 ± 0.09
UV-C (9.87 mJ/cm2)	ND	0.57 ± 0.18	1.13 ± 0.03	1.34 ± 0.06	ND	ND	ND	ND
UV-C (15.13 mJ/cm2)	ND	ND	ND	0.74 ± 0.29	ND	ND	ND	ND
UV-C (31.78 mJ/cm2)	ND	ND	ND	ND	ND	ND	ND	ND
HTST (80 °C)	ND	ND	ND	ND	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	ND	ND	ND	ND
Treatment	Mesophilic (Log CFU/mL)
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	6.17 ± 0.08	6.28 ± 0.13	5.22 ± 0.15	ND	6.41 ± 0.01	8.23 ± 0.01	8.21 ± 0.05	ND
UV-C (9.87 mJ/cm2)	ND	ND	ND	ND	ND	ND	ND	ND
UV-C (15.13 mJ/cm2)	ND	ND	ND	ND	ND	ND	ND	ND
UV-C (31.78 mJ/cm2)	ND	ND	ND	ND	ND	ND	ND	ND
HTST (80 °C)	ND	ND	ND	ND	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	ND	ND	ND	ND
Treatment	Psycrophilic (Log CFU/mL)
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	5.57 ± 0.02	4.94 ± 0.03	4.25 ± 0.06	ND	5.55 ± 0.05	4.47 ± 0.00	ND	ND
UV-C (9.87 mJ/cm2)	ND	ND	ND	ND	ND	1.06 ± 0.00	3.47 ± 0.00	ND
UV-C (15.13 mJ/cm2)	ND	ND	ND	ND	ND	1.5 ± 0.11	3.47 ± 0.01	ND
UV-C (31.78 mJ/cm2)	ND	ND	ND	ND	ND	ND	3.47 ± 0.02	ND
HTST (80 °C)	ND	ND	ND	ND	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	ND	ND	ND	ND

Mean ± standard deviation. ND = Not Detectable N/A = Treatment not performed.

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. Effect of UV-C irradiation and thermal treatment on pH and acidity of red prickly pear juice.

Treatment	pH
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	3.7 ± 0.0 d	3.7 ± 0.1 b	3.7 ± 0.1 c	3.6 ± 0.0 b	7.0 ± 0.0 bc	3.8 ± 0.0 b	3.8 ± 0.0 c	3.8 ± 0.0 b
UV-C (9.87 mJ/cm2)	3.7 ± 0.0 d	3.7 ± 0.0 b	3.7 ± 0.0 c	3.7 ± 0.0 b	7.1 ± 0.0 a	7.0 ± 0.0 a	6.9 ± 0.0 ab	6.8 ± 0.0 a
UV-C (15.13 mJ/cm2)	3.7 ± 0.0 d	3.7 ± 0.0 b	3.7 ± 0.0 c	3.7 ± 0.0 b	7.1 ± 0.0 ab	7.0 ± 0.0 a	6.8 ± 0.0 b	6.9 ± 0.1 a
UV-C (31.78 mJ/cm2)	3.7 ± 0.0 d	3.7 ± 0.0 b	3.8 ± 0.0 c	3.7 ± 0.0 b	7.00 ± 0.0 c	7.0 ± 0.0 a	6.9 ± 0.0 ab	6.9 ± 0.1 a
HTST (80 °C)	3.8 ± 0.0 d	3.7 ± 0.0 b	3.6 ± 0.0 c	3.6 ± 0.0 b	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	7.0 ± 0.0 abc	7.1 ± 0.0 a	7.0 ± 0.0 a	7.1 ± 0.0 a
Treatment	Titratable Acidity (%)
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	0.02 ± 0.0 a	0.02 ± 0.0 a	0.02 ± 0.0 ab	0.02 ± 0.0 a	0.001 ± 0.00 c	0.02 ± 0.00 a	0.020 ± 0.00 b	0.020 ± 0.00 a
UV-C (9.87 mJ/cm2)	0.02 ± 0.0 a	0.02 ± 0.0 a	0.02 ± 0.0 a	0.01 ± 0.0 b	0.001 ± 0.00 c	0.001 ± 0.00 b	0.002 ± 0.00 c	0.001 ± 0.00 c
UV-C (15.13 mJ/cm2)	0.02 ± 0.0 a	0.02 ± 0.0 a	0.02 ± 0.0 a	0.01 ± 0.0 b	0.001 ± 0.00 c	0.001 ± 0.00 b	0.002 ± 0.00 c	0.001 ± 0.00 c
UV-C (31.78 mJ/cm2)	0.02 ± 0.0 a	0.02 ± 0.0 a	0.02 ± 0.0 a	0.01 ± 0.0 b	0.001 ± 0.00 c	0.001 ± 0.00 b	0.002 ± 0.00 c	0.001 ± 0.00 c
HTST (80 °C)	0.02 ± 0.0 b	0.02 ± 0.0 a	0.02 ± 0.0 ab	0.02 ± 0.0 a	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	0.001 ± 0.00 c	0.001 ± 0.00 b	0.001 ± 0.00 c	0.001 ± 0.00 c

Mean ± standard deviation. Different superscript in each column and same day by parameter and pH indicate significantly different mean (p < 0.05), by Tukey’s test. N/A = Treatment not performed.

); 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/cm²) 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/cm²) 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. Effect of UV-C irradiation and thermal treatment on color parameters (L*, a* and b*) of red prickly pear juice.

Treatment	L*
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	54.5 ± 0.2 cd	53.9 ± 0.2 b	53.1 ± 0.1 cd	52.8 ± 1.0 d	52.8 ± 0.3 e	52.6 ± 0.1 b	52.0 ± 0.0 d	52.0 ± 0.2 d
UV-C (9.87 mJ/cm2)	54.7 ± 0.3 cd	55.2 ± 0.1 b	54.6 ± 0.2 bc	54.4 ± 0.1 c	52.9 ± 0.2 e	54.4 ± 0.1 b	55.2 ± 0.1 b	59.3 ± 0.0 b
UV-C (15.13 mJ/cm2)	54.8 ± 0.3 cd	55.6 ± 0.1 b	54.5 ± 0.1 bc	54.5 ± 0.2 c	52.9 ± 0.2 e	54.4 ± 0.1 b	55.3 ± 0.2 b	58.3 ± 0.1 b
UV-C (31.78 mJ/cm2)	55.1 ± 0.1 bc	55.7 ± 0.7 b	55.1 ± 0.2 b	54.8 ± 0.1 c	53.3 ± 0.1 de	54.5 ± 0.1 b	55.2 ± 0.2 b	59.1 ± 0.3 b
HTST (80 °C)	56.5 ± 0.4 b	54.0 ± 0.0 b	54.4 ± 0.1 bc	55.0 ± 0.4 c	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	68.6 ± 1.0 a	63.86 ± 6.4 a	64.2 ± 1.3 a	64.4 ± 0.4 a
Treatment	a*
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	64.0 ± 0.1 a	63.5 ± 0.5 a	63.5 ± 0.0 a	63.3 ± 0.1 a	63.7 ± 0.2 ab	63.6 ± 0.1 a	62.5 ± 0.1 a	62.1 ± 0.1 ab
UV-C (9.87 mJ/cm2)	62.0 ± 0.4 abc	61.9 ± 0.0 a	63.4 ± 0.2 a	63.2 ± 0.1 a	60.1 ± 0.2 c	58.3 ± 0.2 bc	56.8 ± 0.3 b	47.4 ± 0.8 d
UV-C (15.13 mJ/cm2)	61.3 ± 0.3 bc	61.9 ± 0.2 a	63.0 ± 0.1 a	62.8 ± 0.4 a	60.2 ± 0.1 c	58.3 ± 0.1 bc	56.5 ± 0.0 b	50.1 ± 0.0 c
UV-C (31.78 mJ/cm2)	61.2 ± 0.1 bc	61.0 ± 0.2 ab	62.2 ± 0.0 a	62.4 ± 0.0 ab	59.7 ± 0.3 c	57.5 ± 0.0 c	56.8 ± 0.1 b	48.7 ± 0.0 cd
HTST (80 °C)	59.8 ± 0.5 c	62.6 ± 0.2 a	62.3 ± 0.2 a	60.9 ± 0.1 b	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	32.8 ± 1.8 d	31.4 ± 2.2 d	39.5 ± 1.3 c	38.4 ± 0.8 e
Treatment	b*
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	14.5 ± 0.0 c	13.2 ± 0.4 de	13.2 ± 0.0 d	13.1 ± 0.1 c	17.6 ± 0.3 b	13.9 ± 0.1 c	14.0 ± 0.1 d	13.3 ± 0.1 c
UV-C (9.87 mJ/cm2)	15.8 ± 0.7 c	14.6 ± 0.1 d	11.6 ± 0.2 e	10.2 ± 0.0 d	20.3 ± 0.2 b	20.4 ± 0.2 b	23.8 ± 0.2 b	29.5 ± 0.0 a
UV-C (15.13 mJ/cm2)	15.4 ± 0.4 c	14.4 ± 0.2 d	11.6 ± 0.1 e	10.4 ± 0.0 d	20.2 ± 0.1 b	20.2 ± 0.1 b	24.2 ± 0.1 b	27.9 ± 0.2 ab
UV-C (31.78 mJ/cm2)	15.2 ± 0.1 c	14.0 ± 0.1 d	11.7 ± 0.4 e	10.6 ± 0.1 d	20.1 ± 0.2 b	20.1 ± 0.2 b	22.4 ± 0.1 c	28.8 ± 0.6 a
HTST (80 °C)	10.0 ± 0.3 d	11.9 ± 0.5 e	12.3 ± 0.1 e	12.3 ± 0.2 cd	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	31.7 ± 0.8 a	33.9 ± 1.4 a	28.0 ± 0.3 a	26.3 ± 1.7 b

Mean ± standard deviation. Different superscript in each column and same day by color parameter and pH indicate significantly different mean (p < 0.05), by Tukey’s test. N/A = Treatment not performed.

). 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. Effect of UV-C irradiation and thermal treatment on color parameters (hue angle and chroma*) of red prickly pear juice.

Treatment	Hue angle
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	1.3 ± 0.0 b	1.3 ± 0.00 a	1.4 ± 0.0 bc	1.4 ± 0.0 ab	1.3 ± 0.0 c	1.3 ± 0.0 ab	1.4 ± 0.0 c	1.3 ± 0.0 b
UV-C (9.87 mJ/cm2)	1.3 ± 0.0 bc	1.3 ± 0.00 a	1.4 ± 0.0 a	1.4 ± 0.0 a	1.3 ± 0.0 bc	1.2 ± 0.0 b	1.2 ± 0.0 e	1.0 ± 0.0 cd
UV-C (15.13 mJ/cm2)	1.3 ± 0.0 bc	1.3 ± 0.00 a	1.4 ± 0.0 a	1.4 ± 0.0 a	1.3 ± 0.0 c	1.2 ± 0.0 b	1.2 ± 0.0 e	1.1 ± 0.0 c
UV-C (31.78 mJ/cm2)	1.3 ± 0.0 bc	1.3 ± 0.00 a	1.4 ± 0.0 a	1.4 ± 0.0 a	1.3 ± 0.0 c	1.2 ± 0.0 b	1.2 ± 0.0 d	1.0 ± 0.0 c
HTST (80 °C)	1.4 ± 0.0 a	1.4 ± 0.00 a	1.4 ± 0.0 ab	1.4 ± 0.0 ab	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	0.8 ± 0.0 d	0.8 ± 0.1 c	1.00 ± 0.1 f	1.0 ± 0.0 d
Treatment	Chroma*
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	64.8 ± 1.2 a	65.2 ± 0.6 a	65.0 ± 0.3 a	64.8 ± 0.0 a	64.4 ± 1.0 a	64.3 ± 0.1 ab	65.7 ± 0.0 a	60.5 ± 0.4 d
UV-C (9.87 mJ/cm2)	64.1 ± 0.1 a	63.6 ± 0.0 abc	64.4 ± 0.2 ab	64.0 ± 0.1 ab	60.0 ± 3.8 a	61.7 ± 0.1 cd	61.6 ± 0.4 c	55.9 ± 0.7 e
UV-C (15.13 mJ/cm2)	63.2 ± 0.4 a	63.6 ± 0.2 abc	64.1 ± 0.1 ab	62.6 ± 0.4 abc	62.0 ± 1.1 a	60.9 ± 0.1 cd	61.5 ± 0.1 c	57.3 ± 0.1 e
UV-C (31.78 mJ/cm2)	62.8 ± 0.4 a	62.6 ± 0.0 bcd	63.3 ± 0.0 b	63.3 ± 0.1 bc	60.9 ± 2.1 a	61.0 ± 0.2 d	61.1 ± 0.1 c	56.6 ± 0.3 e
HTST (80 °C)	60.7 ± 0.3 a	63.8 ± 0.2 ab	63.6 ± 0.2 b	62.5 ± 0.6 c	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	45.7 ± 2.0 b	47.0 ± 1.5 e	48.4 ± 0.8 d	46.4 ± 0.1 f

Mean ± standard deviation. Different superscript in each column and same day by color parameter and pH indicate significantly different mean (p < 0.05), by Tukey’s test. N/A = Treatment not performed.

).

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. Effect of UV-C irradiation and thermal treatment on color parameters of color difference (ΔE) of red prickly pear juice.

Treatment	ΔE: Compared to Untreated Juice
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
UV-C (9.87 mJ/cm2)	2.0 ± 0.5 c	2.4 ± 0.0 e	2.04 ± 0.3 c	3.19 ± 0.1 d	3.6 ± 0.2 c	6.6 ± 0.1 c	11.8 ± 0.0 b	23.1 ± 0.6 b
UV-C (15.13 mJ/cm2)	2.8 ± 0.0 c	2.6 ± 0.1 e	2.03 ± 0.0 c	3.09 ± 0.2 d	3.6 ± 0.1 c	6.6 ± 0.1 c	12.3 ± 0.0 b	20.0 ± 0.2 c
UV-C (31.78 mJ/cm2)	3.4 ± 0.6 c	3.4 ± 0.1 d	2.79 ± 0.0 c	3.19 ± 0.1 d	4.0 ± 0.2 bc	7.3 ± 0.1 b	10.7 ± 0.0 b	21.7 ± 0.3 bc
HTST (80 °C)	6.4 ± 0.1 b	1.7 ± 0.2 f	1.80 ± 0.1 c	3.19 ± 0.2 d	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	37.4 ± 1.7 a	35.6 ± 0.0 a	29.5 ± 1.7 a	29.9 ± 1.3 a
Treatment	ΔE: Compared to Untreated Juice, day 0
	pH 3.6				pH 7
	Day 0	Day 5	Day 10	Day 20	Day 0	Day 5	Day 10	Day 20
Untreated juice (Control)	N/A	1.4 ± 0.5 e	1.5 ± 0.1 d	1.5 ± 0.2 f	N/A	2.0 ± 0.0 de	3.8 ± 0.2 c	4.4 ± 0.0 d
UV-C (9.87 mJ/cm2)	2.0 ± 0.5 c	1.8 ± 0.1 de	3.0 ± 0.3 cd	4.4 ± 0.0 d	3.6 ± 0.2 c	4.6 ± 0.1 b	8.4 ± 0.1 b	19.8 ± 0.6 b
UV-C (15.13 mJ/cm2)	2.8 ± 0.0 c	2.2 ± 0.2 cde	3.0 ± 0.1 cd	4.2 ± 0.1 de	3.6 ± 0.1 c	4.7 ± 0.1 b	8.8 ± 0.0 b	16.5 ± 0.0 c
UV-C (31.78 mJ/cm2)	3.4 ± 0.6 c	3.1 ± 0.3 c	3.4 ± 0.3 cd	4.1 ± 0.0 de	4.0 ± 0.2 bc	5.3 ± 0.1 b	7.4 ± 0.0 b	18.4 ± 0.2 b
HTST (80 °C)	6.4 ± 0.1 b	2.4 ± 0.2 cd	2.4 ± 0.2 cd	2.8 ± 0.1 ef	N/A	N/A	N/A	N/A
UHT (130 °C)	N/A	N/A	N/A	N/A	37.4 ± 1.7 a	33.7 ± 0.0 a	27.2 ± 1.7 a	27.7 ± 1.0 a

Mean ± standard deviation. Different superscript in each column and same day by color parameter and pH indicate significantly different mean (p < 0.05), by Tukey’s test. N/A = Treatment not performed.

). 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/cm²) 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/cm²) and 175.32 mg GAE/L (9.81 mJ/cm²), 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/cm²) to 152.38 mg GAE/L (9.81 mJ/cm²), 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/cm². 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/cm²) to 900.6 mmol TE/L (9.81 mJ/cm²). 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/cm²) to 604.65 mmol TE/L (9.81 mJ/cm²) 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/cm²) 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/m²) 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/cm²) to 15.22 mg/L (9.81 mJ/cm²). 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/cm²) to 15.01 mg/L (9.81 mJ/cm²) 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/cm², 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/cm², 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/cm²) to 9.83 mg/L (9.81 mJ/cm²). 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/cm²) to 10.75 mg/L (9.81 mJ/cm²) 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/cm², 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/cm²) 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.

4. Conclusions

Effective microorganism inactivation was observed with the various irradiation doses for juices at both pHs. Microorganism growth was controlled by applying UV-C irradiation at doses >15.13 mJ/cm² as efficient thermal processes. Properties, such as pH, Brix, and acidity, were not affected by the UV-C dose irradiation. However, changes were detected during storage time. The chemical compounds such as betalains and total polyphenols, and antioxidant activity, were diminished by UV-C radiation during storage. However, the juice at pH 3.6 was stable from day 5, while the juice at pH 7 showed a gradual decrease over time. Thermal treatments (HTST and UHT) produced a considerable decrease in chemical properties. The greatest reduction was observed with the UHT treatment; however, a partial regeneration of compounds was observed after five days of storage. Betanin and indicaxanthin were reduced with UV-C and pH treatments, but pigment regeneration occurred in some treatments. The improved control observed at an acidic pH contributes to microbiological stability and physical and chemical properties. The application of a 15.13 mJ/cm² irradiation treatment in juice at pH 3.6 effectively inactivates microorganisms and preserves the physicochemical properties. Thus, this method presents a processing alternative that avoids the loss of sensory properties, such as color and functional physicochemical properties.