Improved Antioxidant Capacity by Block Cryoconcentration of Opuntia ficus-indica L. Mill (Green and Red) Juice

Abstract

The presence of bioactives in prickly pear has been documented, including flavonoids and betalains, which are compounds highly unstable to thermal processing. An alternative to the thermal processing of foods is the use of cryoconcentration. The objective of this work was to use cryoconcentration assisted by centrifugation to obtain prickly pear (Opuntia ficus-indica L. Mill) concentrate from two ecotypes (green and red) and evaluate their impact on the polyphenol profile and betalains. Prickly pear juice was obtained and cryoconcentrated. The process parameters of cryoconcentration were obtained. The highest solute yield (Y) was observed for red prickly pear juice (0.42 ± 0.03 kg solute × kg initial solute⁻¹), but the efficiency (η) did not show differences between ecotypes (green 51.0 ± 7.0 vs. red 55.0 ± 7.0%), physicochemical parameters (pH, titratable acididty, °Bx), reducing sugars, or color. The highest total phenolic content (TPC) (1843 ± 153), total flavonoid content (TFC) (759 ± 17), betanin (801.6 ± 19), and indicaxanthin (453.7 ± 19) were observed in cryoconcentrated red prickly pear juice, while the antioxidant activity (ABTS, FRAP, and ORAC) was higher in cryoconcentrated green prickly pear juice (except ABTS). Betalains showed a high correlation with the ABTS antioxidant results, and the TPC showed a high correlation with the ORAC results. Cryoconcentration technology has a high potential to process prickly pear juice, preserving its bioactives.

1. Introduction

Mexico is the main producer of opuntia in the world, and among the main products obtained are the nopal vegetable and prickly pear [1]. Opuntia ficus-indica (L.) Mill is the most important species from an agro-economic point of view among the different species of opuntia [2]. The fruit (prickly pear) harvest season is from April to August in the northern hemisphere. It has an elongated oval shape, like an oval apple or a pear, with a weight range of 60–200 g [3] and a wide range of peel colors.

Unfortunately, the shelf life of prickly pear is short, as it has been reported from 2 to 4 weeks [2], which complicates its storage, transfer, and distribution, as its main sales channel is in the form of fresh fruit, even though there are some reports on the industrialization of prickly pear [4]. Most studies have focused on obtaining juice from prickly pears, including the development of various food products such as concentrated juices, beverages, or even pasteurization of the juice. The presence of bioactives in prickly pear juice has been documented [2]: flavonoids are present in white and green prickly pear [3], betalains in the juice from red and purple prickly pear [4], and phenolic acids in prickly pears of any color [3]. However, thermal processes affect the stability of these bioactives [3,4]. Thus, non-thermal technologies that allow both the juice to be processed and the bioactive compounds present in the juice and their antioxidant activity to be preserved are needed. One such process is cryconcentration. Because this process does not use thermal treatment, thermal degradation is minimized. Cryoconcentration can be assisted by vacuum, centrifugation, microwaves, etc.

The use of block cryoconcentration assisted by centrifugation has been reported to produce concentrated blueberry and pineapple juices [5]. While it has been reported for processing concentrated Chilean prickly pear juice [6], this process has not been reported for processing Mexican Opuntia ficus-indica L. Mill of different colors (green and red). Thus, the objective of the present work was to use cryoconcentration assisted by centrifugation to obtain prickly pear (Opuntia ficus-indica L. Mill) concentrate from two ecotypes (green and red) and evaluate its impact on the polyphenol profile and betalains.

2. Materials and Methods

2.1. Materials

Catechin, gallic acid, dimethyl sulfoxide (DMSO), 2,2-diphenyl-picrylhydrazyl (DPPH), Folin–Ciocalteu reactive (2N), sodium carbonate, sulfuric acid, 2,2-azinobis (ethyl-2,3-dihydrobenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), potassium persulfate, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), and fluorescein (sodium salt) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Iron (III) chloride hexahydrate (FeCl₃·6H₂O), 2,4,6-tripyridyl-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and sodium acetate trihydrate were supplied by Sigma-Aldrich (Toluca, Edo de Mex, Mexico).

The prickly pear (Opuntia ficus-indica L. Mill, two ecotypes: green and red) was acquired at a wholesale market in Durango, Dgo., Mexico, from the municipality of Pinos, Zacatecas, Mexico.

2.2. Juice Obtention

The fruit samples were selected following the same ripening stage, according to Kataria et al. [7], which included ripe color for each ecotype (green and red), soft texture, sweet taste, and low acidity. The fruits had no presence of external damage, and fruits of a similar size were selected for each group (10 kg) (Figure 1). The fruits were washed, and the pericarp of the selected prickly pears was manually removed. The juice was obtained using a mechanical pulper (Polinox, Mexico City, Mexico) and then frozen and stored at −20 °C until use (no more than one week).

2.3. Cryoconcentration

Falcon centrifuge tubes of 25 mL were used, isolated by a polypropylene layer (8 mm thickness, thermal conductivity K = 0.035 W × m⁻¹ × K⁻¹), allowing heat flow only in one direction (axial). Each tube was added with 15 mL of previously thawed juice (in refrigeration, for 12 h) and frozen for 24 h. Thawing was subsequently carried out (2 h, at room temperature), which was followed by centrifugation (5000 rpm, 20 min). Two freezing cycles were carried out, while the concentrate was recovered, and the ice was again concentrated, repeating the process.

2.4. Determination of Process Parameters of Cryoconcentration by Block Assisted by Centrifugation

The parameters process efficiency (η) and percentage of solute recovery (Y) were evaluated according to Orellana-Palma et al. [8], following the next expressions:

$$\eta \left(\%\right)=\left[{\displaystyle \frac{\left(C_0-C_i\right)}{C_0}}\right]\times 100$$

(1)

where η is the efficiency (%), and $C_0$ and $C_i$ are the solutes (°Brix) in cryoconcentrated and ice fractions, respectively.

$$Y\left(\mathrm{k}\mathrm{g}/\mathrm{k}\mathrm{g}\right)={\displaystyle \frac{m_s}{m_0}}$$

(2)

where Y is the solute yield (kg solute per 1 kg initial), m_s is the solute mass in concentrated solution and $m_0$ is the initial solute mass.

2.5. Physicochemical Parameters

2.5.1. Titratable Acidity

The titratable acid content was evaluated by the titration method with sodium hydroxide (NaOH) in the presence of phenolphthalein.

2.5.2. pH

The pH value was determined using a potentiometer (pH meter SenIon TM 1 Hach, Loveland, CO, USA), according to the described method (potentiometer) [9] (NOM-F-317-S-1978, 1978). The pH meter was calibrated at pH 4, pH 7, according to the product type.

2.5.3. °Bx

The °Bx content was determined by the method described in [10] (NMX-F-103-1982, 1982), using an Atago digital handheld refractometer (Atago, Tokyo, Japan). The refractometer was calibrated with water at 20 °C, and then a sample was put in the instrument.

2.5.4. Color

The color was determined in a Minolta colorimeter (Konica-Minolta, Tokyo, Japan), recording the data of L, a, and b, to subsequently evaluate the color change (ΔE) using the following formula:

ΔE = √ (L² + a² + b²)

(3)

2.6. Reducing Sugars

The total reducing sugar content was determined by the phenol–sulfuric method [11]. Briefly, 0.5 mL of the experimental sample was added with 0.5 mL of 5% phenol solution and vortexed. Then, 2.5 mL of concentrated H₂SO₄ was added directly into the solution within 1–2 s. The mixture was vortexed and allowed to stand for 30 min at room temperature (20 °C), reading the samples at 490 nm in a UV-Vis spectrophotometer.

2.7. Total Phenolic Content (TPC)

The total phenolic content was determined by the Folin–Ciocalteu method, described by Agbor et al. [12] in a microplate reader (96-well plates). Briefly, 25 µL of experimental sample and 5 µL of Folin–Ciocalteu reagent were placed in each well, allowing to react for 6 min in dark conditions, stopping the reaction by adding 80 µL of 7% sodium bicarbonate. The solution was allowed to stand for 30 min in the dark, and then the absorbance at 765 nm was measured in a Daigger microplate reader (Vernon Hills, IL, USA). A gallic acid standard curve was used, and the results were reported as mg of gallic acid equivalents ×L⁻¹ (mg GAE × L⁻¹).

2.8. Total Flavonoids Content (TFC)

The determination of total flavonoids content (TFC) was performed according to the methodology proposed by Agbor et al. [12]. Briefly, 20 µL of sample, 7.5 µL NaNO₂ at 5%, 15 µL AlCl₃ at 10% and 50 µL of NaOH were placed in a flask. It was shaken for one minute and left to stand in the dark for 5 min. Absorbance readings were performed at 570 nm in the Daigger microplate reader (Vernon Hills, IL, USA). A catechin curve was used, and the results were expressed as mg of catechin equivalents ×L⁻¹ (mg CE × L⁻¹).

2.9. Betalains

The betalain content (BC) was evaluated according to the method described by Stintzing et al. [13]. Briefly, the experimental samples were diluted with McIlvaine buffer (pH 6.5, citrate-phosphate) to obtain absorption values between 0.9 and 1.0 at their respective absorption maxima. The BC was calculated as follows: BC [mg/L]) = [(A × DF × MW × 1000/ǫ × l)] where A is the absorption value at the absorption maximum corrected by the absorption at 600 nm, DF the dilution factor, l is the path length (1 cm) of the cuvette, and MW is the molecular weight of betalains (MW of betanin, 550,000 mg/mol or indicaxanthin, 308,000 mg/mol).

2.10. 2,2′-Azinobis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Assay

The determination was based on the methodology proposed by Re et al. [14]. Briefly, the ABTS stock solution was prepared by an initiative reaction of 7 mM ABTS stock solution with 140 mM potassium persulfate and incubating overnight in the dark for 12–16 h. The ABTS•+ solution was diluted with absolute ethanol to adjust the absorbance at 734 nm to 0.70 ± 0.02. Then, 1 mL of experimental sample was mixed with 3 mL of the diluted ABTS•+ solution. After 5 min of the mixing, the decrease in absorbance was recorded. Trolox was used as the standard compound.

2.11. Ferric Reducing Antioxidant Power (FRAP) Assay

It was performed according to the methodology used by Benzie and Strain [15]. Briefly, the FRAP reagent was made up of 300 mM acetate buffer (3.1 g C₂H₃NaO₂·3H₂O and 16 mL C₂H₄O₂), 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution dissolved in 40 mM HCl and 20 mM FeCl₃·6H₂O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer at pH 3.6, 2.5 mL TPTZ solution and 2.5 mL FeCl₃·6H₂O solution. The mixture was warmed at 37 °C prior to analysis. Then, 2 mL of warmed distilled water at 37 °C was added to 50 μL of experimental sample and 2 mL of reagent in a test tube. The mixture was incubated at 37 °C for 4 min in the dark condition and monitored until 8 min. Absorbance was taken against methanol as blank at 593 nm. The standard curve was obtained using Trolox.

2.12. Oxygen Radical Absorbance Capacity (ORAC)

It was evaluated according to the methodology proposed by Swada et al. [16]. Briefly, AAPH (2,2-azobis [2-amidinopropane] dihydrochloride) was used as a peroxyl radical generator and fluorescein as a fluorescent probe. Filters were used to select an excitation wavelength of 485 nm and an emission wavelength of 535 nm [17]. A total of 175 μL mixture containing fluorescein (3 μM) and AAPH (221 mM) was injected into each well of the microplate. All solutions were prepared in phosphate buffer 1 M pH 7.4. Then, 25 μL of experimental sample at an appropriate dilution, blank, or Trolox calibration solution (50–200 μM) were added. The fluorescence at 37 °C was recorded every 2 min for 1.5 h in a multiplate reader (Synergy™ HT Multi-Detection Microplate Reader, Bio-Tek, Winooski, VT, USA). The final ORAC value was calculated from the net area under the fluorescence decay curve. All assays were performed in triplicate.

2.13. Rheology

Simple shear tests were carried out on fresh prickly pear juice, using an HR-2 rheometer (TA Instruments, Wilmington, DE, USA), using a cone (2°) and plate (40 mm) geometry with a gap of 500 microns, temperature control by means of element peltier, and using a shear speed range of 0.01–1000 s⁻¹ [17]. The data were analyzed using the Trios software 5.1 (TA Instruments, Wilmington, DE, USA).

2.14. Data Analysis

Data were analyzed using analysis of variance (ANOVA) and a means comparison test (Tukey) (p < 0.05) and correlation analysis using the software Statistica v12.5 (StatSoft, Tulsa, OK, USA). All experiments were performed in triplicate.

3. Results

The results of the yield of solutes (Y) and efficiency (η) are shown in

Table 1. Process parameters of cryoconcentration of prickly pear juice.

Sample	Solute Yield (Y), (Kg Solute × kg Solute Initial−1)	Efficiency (η), (%)
Cryoconcentrated green prickly pear juice	0.18 ± 0.02 a	51.0 ± 7.0 a
Cryoconcentrated red prickly pear juice	0.42 ± 0.03 b	55.0 ± 7.0 a

Data mean ± standard deviation, different superscript letter in the same column indicates statistical differences (p < 0.05, Tukey test, n = 3).

. No statistical differences were observed in efficiency (51–55%). These values are on the average level described for the conventional cryoconcentration described by Swada et al. [16]. One of the reasons that could have influenced such a high value may be associated with the physical characteristics of the ice. Since the efficiency of any freeze concentration technology is dependent on the ability to separate ice crystals from viscous concentrate, the ice crystal size distribution, general crystal morphology, and surface characteristics are critical to obtain an efficient design [18].

The titratable acidity results are shown in

Table 2. Physicochemical parameters and chemical characterization.

	Green Uncryoconcentrated	Green Cryoconcentrated	Red Uncryoconcentrated	Red Cryoconcentrated
Titritable acidity	0.05 ± 0.01 b	0.07 ± 0.01 c	0.03 ± 0.01 a	0.04 ± 0.03 b
pH	5.40 ± 0.01 b	5.1 ± 0.04 a	5.00 ± 0.04 a	4.90 ± 0.04 a
°Bx	14.9 ± 0.20 a	23.4 ± 0.50 c	13.9 ± 0.10 b	23.0 ± 1.10 c
Reducing sugars (mg glucose × L−1)	4166 ± 183 a	18,195 ± 353 c	10,135 ± 147 b	21,217 ± 350 d
Total phenolic content (TPC) mg GAE × L−1	764 ± 46 a	1426 ± 136 b	737 ± 26 a	1843 ± 153 b
Total flavonoid content mg CE × L−1 (TFC)	230 ± 27 a	437 ± 16 b	383 ± 34 b	759 ± 17 c
Betanin mg BE × L−1	3.63 ± 0.30 a	3.43 ± 0.10 a	53 ± 1 b	802 ± 19 c
Indicaxanthin mg IE × L−1	3.40 ± 0.30 a	3.30 ± 0.10 a	36 ± 1 b	454 ± 19 c

Data showed mean ± standard deviation. Different superscript letter in the same row indicates statistical differences (p < 0.05, Tukey test, n = 3). GAE = gallic acid equivalent; CE = catechin equivalent; BE = betanin equivalent, IE = indicaxanthin equivalent.

, where the fresh juice of green prickly pear (0.048 ± 0.003) showed a higher acidity value than the juice of the red one (0.027 ± 0.003). The pH results of green and red prickly pear juices are shown in Table 2, where a pH value of 5.43 ± 0.06 is observed for green prickly pear, while for red prickly pear, the pH was 5.50 ± 0.03. The results of total reducing sugars are shown in Table 2. As expected, for two different opuntia prickly pears (green and red), values of different reducing sugars are observed, with red prickly pear juice showing the highest value of 10,162 mg glucose ×L⁻¹ of nectar, while the green prickly pear presented 4176 mg × L⁻¹. The total phenolic content (TPC) is shown in Table 2; only changes related with the concentration process were observed. The results obtained for total flavonoid content (TFC) are shown in the Table 2. A higher TFC value was observed for red prickly pear juice (383 mg CE × L⁻¹). The betalains content is shown in Table 2. Betalain concentration was found for green prickly pear as 3.63 ± 0.26 mg BE × kg⁻¹ and 3389 ± 0.28 mg IE × kg⁻¹; while for red prickly pear, the concentration was 53.17 ± 1.45 mg BE × kg⁻¹ and 36.22 ± 1.07 mg IE × kg⁻¹.

The antioxidant activity results using the ABTS, FRAP and ORAC methods are shown in

Table 3. Antioxidant capacity of prickly pear juice (cryoconcentrated and uncryoconcentrated).

Sample Juice (Opuntia ficus-indica)	ABTS (µM Trolox × L−1)	FRAP (µM Trolox × L−1)	ORAC (µM Trolox × L−1)
Green uncryoconcentrated	5587 ± 348 a	5963 ± 588 a	18,437 ± 345 a
Red uncryoconcentrated	6657 ± 415 a	7963 ± 696 b	19,818 ± 21 b
Green cryoconcentrated	4817 ± 433 a	7544 ± 768 b	43,265 ± 4299 c
Red cryoconcentrated	11,357 ± 860 b	13,655 ± 1074 c	49,099 ± 1147 c

Data show mean ± standard deviation. Different superscript letter in the same column indicates statistical differences (p < 0.05, Tukey test, n = 3), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC).

.

A correlation analysis (

Table 4. Pearson correlation of TPC (total phenolic content), TFC (total flavonoid content), betalains, antioxidant test (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, ferric reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) from prickly pear juice.

Variable	TPC	TFC	Betanin	Indicaxanthin	ABTS	FRAP	ORAC
TPC	1.000
TFC	−0.643	1.000
Betanin	0.405	0.357	1.000
Indicaxanthin	0.476	0.262	0.976 *	1.000
ABTS	0.405	0.357	0.637	0.976 *	1.000
FRAP	0.666	−0.071	0.833 *	0.881 *	0.833 *	1.000
ORAC	0.881 *	−0.524	0.548	0.643	0.548	0.881 *	1.000

* Indicates significant correlation at p < 0.05.

) clearly shows that the antioxidant activity measured by FRAP is associated with the content of betanin (r = 0.83), while the content of indicaxanthin is related to FRAP (r = 0.88) and ABTS (r = 0.98), while TPC was associated with ORAC (r = 0.88).

4. Discussion

The values observed of solute yield and efficiency are in the average range described for the conventional cryoconcentration by Aider and Halleux [18]. However, when comparing green vs. red cryoconcentrated prickly pear juice, a low solute yield was obtained in the cryoconcentrated green prickly pear juice (0.18 ± 0.02). An explanation of this behavior could be related with the formation of ramifications in the ice structure, which cause the formation of a network arrangement, which occlude the diffusion of solutes, causing a decrease in separation and lowering the solute yield [19].

The previous phenomenon was less important in the cryoconcentrated red prickly pear juice (0.42 ± 0.03). According to Petzold and Aguilera [19], if the ice block behaves as a highly compressible porous medium, it is possible to obtain a maximum separation speed, but a further increase in the centrifugal speed will enhance the mass centrifugal force together with small deformations in the pores, which in turn will produce important variations in the shear speed in the pores. This effect is magnified in fluids with pseudoplastics characteristics, which is reflected in a lower efficiency and recovery of solutes, as a similar behavior was reported by Rezzadori et al. [20].

About titratable acidity, Meraz-Maldonado et al. [21] evaluated the titratable acidity of prickly pear juice, obtaining for red prickly pear values of 0.030 ± 0.006% of citric acid, which were like those found in the present work. Regarding the effect of cryoconcentration, a higher acidity value is observed in cryoconcentrated juices (green and red prickly pears). This behavior has been documented by Orellana-Palma et al. [22], since when juice is processed by cryoconcentration, the concentration of organic acids increases due to the decrease in water content.

The registered pH value was more acidic than that reported by Aparicio-Fernández et al. [23] for prickly pear (6.9 ± 0.3), but it was less acidic than that reported by Mendez et al. [24] for green Opuntia ficus-indica (4.71 ± 0.07). The pH value is associated with the maturity of the fruit as well as the cultivation and harvest conditions, which may explain the observed differences. Regarding the effect of cryoconcentration on the pH values, only an effect was observed on green prickly pear juice, going from 5.43 to 5.12, a phenomenon that may be associated with an increase in the content of organic acids, which promotes a decrease in pH. However, it is noteworthy that no changes are observed in the pH value of red prickly pear (4.99 and 4.88, respectively).

Regarding °Bx, Aparicio-Fernández et al. [23] reported 12.3 ± 0.5 °Bx, which is a value slightly below that found in this work. However, Monroy-Gutiérrez et al. [25] reported 13.25 °Bx for red prickly pear, which is a value that is in accordance with our work. Regarding the effect of cryoconcentration, it can be seen that in both green and red prickly pears, there is an increase in the content of soluble solids. The °Bx values obtained (around 23) were lower than those reported for pineapple juice and blueberry juices (30 °Bx) [5]. However, such a value was reached after three concentration cycles, and in this work, only one cycle was carried out. Thus, when comparing the results by cycles in the juices, they only reached 18–19 °Bx, which is lower than in our present report. In any case, our °B value is higher than the documented averages for centrifugation-assisted block cryoconcentration [16].

As expected, for two different opuntia prickly pears (green and red), different reducing sugars contents were observed, with red prickly pear juice showing the highest content of 10,162 mg glucose equivalents for each liter of nectar, while the green prickly pear presented 4176 mg × L⁻¹. These amounts are like those reported by Feugang et al. [2] (10,000 mg × L⁻¹) for prickly pears of different origins. Regarding the effect of cryoconcentration, the effect was more marked, especially in green prickly pear juice, with a 4.36-fold increase in sugar concentration, while in red prickly pear juice, the increase was 2.09-fold.

Cruz-Cansino et al. [26] evaluated the concentration of TPC in purple prickly pear (Opuntia ficus-indica L. Mill), reporting values close to 1600 mg GAE × L⁻¹, which is higher than the 736 ± 20.71 mg GAE × L⁻¹ reported in this research. This difference may be attributable to the different variety evaluated, as well as to the climate conditions, soil type and crop origin. Regarding the effect of cryoconcentration, a higher increase in TPC was observed in red prickly pear (2.5-fold with respect to the fresh juice), while in green prickly pear, the increase was 1.8-fold.

A higher TFC value was observed for red prickly pear juice (383 mg CE × L⁻¹). Chavez-Santoscoy et al. [27] reported flavonoids from nine cultivars of opuntia, ranging between 96 and 376 mg CE × L⁻¹, which are similar values to those found in the present work. Meanwhile, the output obtained after cryoconcentration shows that the concentration process is highly efficient for phenolics and flavonoids.

Regarding betanin content, Chavez-Moreno et al. [1] found 270.02 ± 0.44 mg BE × kg⁻¹ in prickly pear, while Stintzing et al. [13] reported a concentration of betanin of 78.4 ± 0.2 mg × L⁻¹, while that of betaxanthin was 48.6 ± 1 mg × L⁻¹. In all cases, the values were higher than those reported in the present investigation.

We confirmed the presence of small amounts of betalains in green prickly pear juice, while a higher amount of betalains in red prickly pear was observed. Regarding the effect of cryoconcentration, no variation was detected between the betalain values in fresh and concentrated green prickly pear juices, which implies a net loss, since its value did not increase, even as a function of the observed concentration. On the other hand, a strong increase in the content of betanin in red prickly pear juice was observed, going from 53 to 801 mg × Kg⁻¹ equivalents of betanin: a 15-fold increase.

This increase cannot be explained by the concentration factor of around 1.9 times. In this sense, Khan [28] found that betanin, in acid solution at room temperature for 36 h, can be isomerized to isobetanin, which cannot be evaluated by the described method. This could cause an initial underestimation in the betanin content, while it may have been degraded in the fresh juice, increasing the content of betalamic acid in solution.

In the case of indicaxanthin, the phenomenon is similar although less evident than for betanin (12 times more concentrated). Moßhammer et al. [3] reported the presence of an indicaxanthin isomer, called isoindicaxanthin, which can be evaluated by HPLC. Although such an isomer is less likely in acidic conditions, there are no reports of a possible regeneration of indicaxanthin in prickly pear Opuntia ficus-indica L. Mill.

The obtained results on antioxidant activity for uncryoconcentrated juices are similar to those reported by Zenteno-Ramírez et al. [29] for red prickly pear juice from Opuntia ficus-indica. Once subjected to cryoconcentration, in the case of green prickly pear juice, no increase in antioxidant activity was observed. This was contrary to red prickly pear juice, which showed an increase in antioxidant activity, which was directly related to the relative increase in TPC produced during concentration. Meanwhile, the FRAP assay showed (Table 3) a similar behavior to that observed for ABTS. In the case of ORAC (Table 3), its value in uncryoconcentrated prickly pear juice did not show a difference in terms of the type of prickly pear (white or red), but it did show the influence of the process.

A correlation analysis (Table 4) clearly shows that the antioxidant activity measured by FRAP is associated with the content of betanin (r = 0.83), while the content of indicaxanthin is related to FRAP (r = 0.88) and ABTS (r = 0.98), and that of TPC was associated with ORAC (r = 0.88). Such betanin behavior has been discussed by Gliszczyńska-Świgło [30]. He showed that there is a significant increase in the antioxidant activity of betanin as the pH increases, finding a maximum in pH > 4, which coincides with those found in this work. This effect is observed in the ABTS method due to the formation of different mono, di and triprotonated forms that increase their electron donation capacity in the ABTS assay. Regardless of whether both ABTS and FRAP are methods that share the same basic principle of electron transfer, the pH at which the evaluation is carried out is not the same, which can lead to different conclusions [31].

It is important to note that the amount of bioactive compounds, including betalains, flavonoids, and phenolic acids, among others, is influenced by several environmental conditions [32,33] that were not controlled in the present experiment. Therefore, it is necessary to develop experiments with controlled harvesting and growing conditions to evaluate their effect on the quality and quantity of bioactive compounds in the prickly pear cultivars.

There are several limitations of the study, including a lack of knowledge on the growing and harvesting conditions as well as the storage and handling of the fruits that could impact the observed behaviors. Also, the lack of information and identification of flavonoids and phenolic acids present in the juice limits the discussion about the nutraceutical influence of the cryoconcentration process.

5. Conclusions

An important increase in betanin content was observed for cryoconcentrated red prickly pear juice and also for indicaxanthin. The increase in betalains was not a related concentration factor. High values of total phenolic content (TPC), total flavonoid content (TFC) and high antioxidant activity were observed for cryoconcentrated prickly pear juices (red and green). As expected, higher amounts of betalains were observed in red prickly pear juices, although green prickly pear juice showed also their presence. Betalains showed high correlation with ABTS antioxidant results and TPC, exhibiting a high correlation with ORAC results. The use of cryoconcentration technology on prickly pear juice has a high potential for processing this functional fruit, preserving their important bioactives (flavonoids, betalains).