Art_15981.indd Journal of Applied Botany and Food Quality 94, 169 - 175 (2021), DOI:10.5073/JABFQ.2021.094.020 1Ingeniería en Agroindustrias, Universidad de la Costa, Oaxaca, México 2Departamento de Fitotecnia, Universidad Autónoma Chapingo, Estado de Mexico 3Departamento de Ingeniería Agroindustrial, Universidad Autónoma Chapingo, Estado de Mexico Sweetened nopal flakes: a functional snack Leidy Laura Cruz-de la Cruz1, Rosario García-Mateos2*, Carmen Ybarra Moncada3, Joel Corrales-García3 (Submitted: May 7, 2021; Accepted: September 22, 2021) * Corresponding author Summary Nopal (Opuntia spp. and Nopalea spp. genera) is a crop, recognized for its nutritional and medicinal properties; however, there are some underused species, despite the great genetic diversity in Mexico. The genus Opuntia spp. is the most consumed nopal, whereas Nopalea spp. has low commercial demand, possibly because their nutraceutical attributes are unknown. Additionally, the nopal pads or cladodes are little accepted by many consumers, due to their texture and flavor. The study objectives were 1) evaluate the nutraceutical content and antioxidant activity of four nopal cultivars: Nopalea cochenillifera cv. Texas (NT) and Opuntia ficus-indica cv. Jade (OJ), Milpa Alta (OMA), and Atlixco (OA); 2) develop nopal flakes, sweetened with rebaudioside A, from the cultivar with the best nutraceutical quality and sensory acceptability. Ascorbic acid, total phenolics, and total flavonoids were determined by spectrophotometric methods, individual flavonoids (quercetin, kaempferol, and isorhamnetin) by HPLC, and antioxidant activity by the DPPH assay. OA was the cultivar with the best nutraceutical quality. The sweetened nopal flakes of OA, at a concentration of 1.1 mg g-1 rebaudioside A, had the highest sensory acceptability by the panelists in intensity and sweetness preference. The addition of rebaudioside A improved the product’s flavor and contributed to preserve the flavonoids and antioxidant activity. These results will contribute to the chemotaxonomy of O. ficus-indica and N. cochellinifera species, and to the utilization of nopals as functional foods, due to their nutraceutical quality. Keywords: antioxidant activity, chemotaxonomy, Nopalea, Opuntia, nutraceuticals, rebaudioside A. Introduction Nopal is an endemic plant of America that is distributed as wild or cultivated forms in desert and semi-desert areas of the continent. Nopal belongs to the Cactaceae family and includes the Opuntia and Nopalea genera. Opuntia spp. is the largest genus with 377 identified species; 104 of them are found in wild-type form in Mexico (ANAYA- PÉREZ, 2001; STINTZING and REINHOLD, 2005). Despite the great diversity of nopal cultivars, few studies have been done to charac- terize their nutraceutical components, which are metabolites that prevent diseases and maintain the good health of consumers. In the nutraceutical, medicinal, and chemotaxonomical context, the ‘Jade’ and ‘Texas’ cultivars from the Opuntia genus haven been little studied, whereas these properties are unknown in species from the Nopalea genus. Nopals have been consumed since ancient times for their diverse medicinal properties, such as antidiabetic (SHANE-MCWHORTER, 2009), anticholesterolemic (YANG et al., 2008), anticarcinogenic (ABOU-ELELLA and ALI, 2014), anti-inflammatory (PARK et al., 2001), antiulcerogenic, diuretic and antioxidant (ALIMI et al., 2010) effects. Moreover, nopals have been used for their cicatrizing properties in dermal wounds and gastric ulcers (ALIMI et al., 2010), which have been attributed to their content of dietary fiber (polysaccharides), mucilage (polysaccharide), amino acids, vitamins, minerals, and phenolic compounds (OSUNA-MARTÍNEZ et al., 2014). In recent years, steviol glycosides (chemical compounds identified in Stevia rebaudiana, a native plant of South America) have obtained relevance in the food, cosmetic, and pharmaceutical industries, since they are non-caloric sweeteners. Rebaudioside A is the most abun- dant steviol glycoside in S. rebaudiana, the most accepted by con- sumers, and has no bitter flavor. In vitro and in vivo studies have reported that rebaudioside A has no mutagenic, teratogenic, or fer- tility effects (ARANDA-GONZÁLEZ et al., 2014). The nopal pads or cladodes are little accepted by many consumers, due to its mucilaginous texture, acidity, astringency, and herbal fla- vor. As an alternative to increase their utilization and consumption, nopal cladodes were transformed into a functional snack by prepara- tion of dehydrated flakes and sweetened with rebaudioside A to im- prove their texture and flavor, as well as to maintain the nutritional, medicinal, and nutraceutical properties of nopal. In this context, the objectives were to evaluate the ascorbic acid, total phenolic, and fla- vonoid (quercetin, kaempferol, and isorhamnetin) contents, and the antioxidant activity of four nopal cultivars (Nopalea cochenillifera cv. Texas and Opuntia ficus-indica cv. Jade, Milpa Alta, and Atlixco), as well as to develop nopal flakes, sweetened with rebaudioside A, from the cultivar with best nutraceutical quality and sensory accept- ability. Materials and methods Plant material Small nopal cladodes (20 days of age) of O. ficus-indica cv. Jade (OJ), Atlixco (OA) and Milpa Alta (OMA), and N. cochellinifera cv. Texas (NT) were harvested free from diseases, physical damages, and mor- phological alterations in Campo de Experimental La Nopalera of Universidad Autónoma Chapingo. Samples were freeze-dried (model 7670520, Labconco, Kansas, USA) at -50 °C and 0.1 mbar for 18 h. Each experimental unit consisted of three different plants, and each analysis was done with three replicates. Quantification of ascorbic acid The quantification of ascorbic acid was done with the spectro- photometric method described by DÜRÜST et al. (1997), which is based on color reduction of the indicator dichloroindophenol. The results were expressed in mg of ascorbic acid per 100 g dry weight (mg 100 g-1 dw). Total phenolics Freeze-dried nopal (0.3 g) was pulverized and mixed with 10 mL of 80% (v/v) MeOH aqueous solution. The mixture was homogenized 170 L.L. Cruz-de la Cruz, R. García-Mateos, C. Ybarra Moncada, J. Corrales-García by using a vortex and sonicating for 20 min at room temperature (25 ± 2 °C). The extracts were left to rest for 12 h under refrigera- tion (4 °C) and darkness. Afterwards, the extracts were filtered with Whatman filter paper No. 1 for the quantification of total phenolics, flavonoids, and antioxidant activity. The content of total phenolics was obtained by the Folin-Ciocalteu method (GÜLÇIN et al., 2004). Total phenolics were expressed in mg of gallic acid equivalents per g dw (mg GAE g-1 dw). Total flavonoids Total flavonoids were determined, based on the spectrophotometric method described by GÜLÇIN et al. (2011), where 10% (w/v) AlCl3 was used as indicator. The results were expressed in mg of quercetin equivalents per g dw (mg QE g-1 dw). Antioxidant activity Antioxidant activity was measured by the DPPH assay (KUSKOSKI et al., 2005). DPPH (100 μM) in 80% (v/v) methanol was mixed with the sample, and the reaction was incubated for 60 min. The results were expressed in μmol Trolox equivalents per g dw (μmol TE g-1 dw). The percentage of inhibited DPPH was determined by using the formula %DPPH inhibited = (Absblank – Abssample) / (Absblank) *100 (Eq. 1) where Abs = absorbance and blank = DPPH solution at a concentra- tion of 100 μM. Identification and quantification of individual flavonoids Identification and quantification of individual flavonoids was per- formed, based on the methodology reported by GRAY et al. (2006). Freeze-dried nopal powder (1.5 g) was blended with 25 mL of etha- nol and 10 mL of distilled water. The mixture was sonicated for 30 min at room temperature. Thereafter, 4 mL of concentrated HCl were added and the solution was maintained at reflux for 3 h. The volume of the cold mixture was completed to 50 mL with ethanol and was filtered through a Millipore nylon membrane (0.22 μm). The ex- tracts were analyzed with a reverse phase HPLC system (Shimadzu, Kyoto, Japan), equipped with a diode array detector (DAD) and a C18 analytic column (250 × 4.6 mm i.d.: 5 μm) (Phenomenex® 110A, Germany). The injection volume was 10 μL. As mobile phase, a mix- ture (50:50, v/v) of methanol and an aqueous solution of phosphoric acid (0.5%) was used in isocratic conditions, with a flow rate of 1.2 mL min-1, and a total elution time of 17 min. Column temperature and pressure were 35 °C and 1900-1910 psi, respectively. Quercetin, kaempferol, and isorhamnetin were detected at 252 nm and identified by using their respective standards. Preparation of sweetened nopal flakes For the preparation of nopal flakes, homogenous nopal cladodes were used with a weight of 187 ± 5.26 g from the OA cultivar, which pre- sented the highest phenolic content and antioxidant activity, com- pared with the other cultivars. Fresh, disinfected nopals were cut transversely into flakes (3 mm thick). The flakes were submerged in different solutions of the sweetener rebaudioside A (2, 4, 6, and 8 g L-1) for one hour in a 1:1.5 proportion (nopal:solution). Un- sweetened flakes were used as control. Previously, the optimal freeze-drying time was determined by dry- ing kinetics and desorption isotherms with three different concen- trations of rebaudioside A (0, 5, and 10 g L-1) at a temperature of -50 °C and a pressure of 0.01 mbar. Equilibrium moisture content and water activity (aw) of the flakes were measured in 3-h intervals until concluding 21 h. Equilibrium moisture content was determined with the equation (GEANKOPLIS, 1998) Xt = (mi − mss) / mss (Eq. 2) where Xt = equilibrium moisture content (g of water g-1 dry weight); mi = flake weight (g) per time; mss = flake dry weight (g). Moisture content was determined according to AOAC method and aw with a aw meter (AquaLab, Decagon Devices, Washington, USA). Afterwards, both sweetened and unsweetened flakes were frozen in liquid nitrogen and then dehydrated by freeze-drying. The freeze- dried flakes were covered with aluminum foil and placed in a desic- cator to prevent the absorption of moisture. Quantification of rebaudioside A Sweetened and pulverized flakes (2.0 g) were blended with 10 mL of 70% (v/v) ethanol solution. The mixture was placed in a water bath at 70 °C for 30 min. Subsequently, the extracts were filtered with a Millipore nylon membrane (0.22 μm). The extracts were analyzed in a reverse phase HPLC system (Perkin Elmer, USA), equipped with a DAD and a C18 analytic column (150 × 4.6 mm i.d.: 5 um) (Thermo Scientific® Hipersil ODS). Injection volume was 10 μL. As mobile phase, acetonitrile and water (80:20) were used, acidified to pH 3 with phosphoric acid, with a flow rate of 1 mL min-1 and a total elu- tion time of 3.5 min. Column temperature was room temperature. Rebaudioside A was detected at 210 nm and identified by using its respective standard. Sensory evaluation Sensory evaluation of unsweetened flakes and flakes sweetened with different concentrations of rebaudioside A (2, 4, 6, and 8 g L-1) was done to identify the rebaudioside A concentration with most accept- ability by the panelists. The evaluation was done by applying two reference scales: intensity (just about right, JAR) and preference (he- donic). The panel was made of 100 non-trained judges (53 men and 47 women) of 15-26 years of age. Each panelist evaluated at random the flakes with the five different concentrations of rebaudioside A. Statistical analysis The data were analyzed using a randomized complete block design (RCBD), and statistical differences were determined by an analysis of variance (ANOVA), followed by Tukey’s mean difference test (p < 0.05). All data were analyzed by using SAS software. Results and discussion Nutraceutical content and antioxidant activity in four nopal cultivars The contents of ascorbic acid, total phenolics, total flavonoids, antioxidant activity, and inhibited DPPH after 60 min of reaction are presented in Tab. 1 for the cultivars OJ, OA, and OMA (O. ficus- indica) and NT (N. cochellinifera). The ascorbic acid content in the OMA and OA cultivars was superior (38.5 and 43.1 mg 100 g-1 dw, respectively) to the same cultivars (19.21 and 25.52 mg 100 g-1 dw, respectively) reported by RAMÍREZ-MORENO et al. (2013); but MEDINA-TORRES et al. (2011) found a greater concentration (205.62 mg 100 g-1) for the OMA cultivar recollected from different regions to that of the present study. On the other hand, the concentration of phenolic compounds in the studied cultivars (2.9-8.1 mg GAE g-1 dw) was within the interval published by GUEVARA-FIGUEROA et al. (2010) for other Mexican cultivars of the Opuntia genus: Tapón (I and II), Blanco, and Manso (2.0, 5.2, and 11.7 mg GAE g-1 dw, respectively). Total flavonoids are not affected by mechanical dam- ages or manipulation of the food, just as it has been reported in other Sweetened nopal fl akes 171 phenolic compounds that respond to stress applied to food, such as storage time, refrigeration temperature, and UV light, conditions which cause an increase in the phenolic content as a defense mecha- nism (DE ANCOS et al., 2009). Finally, the cultivars OJ, OA, and OMA presented an antioxidant activity that was superior to cultivar NT (N. cochellinifera). The OA cultivar excelled for its high content of phenolic compounds and antioxidant activity, reason for which it was selected to prepare the sweetened fl akes. In Fig. 1 are shown the chromatograms of the fl avonoid profi le of the four cultivars evaluated in this study. Quercetin, kaempferol, and iso- rhamnetin were identifi ed in all cultivars. These three fl avonoids have been found in several species of the Opuntia genus (O. fi cus-indica, O. humifusa, O. monacantha, O. lindheimeri, O. robusta, O. strepta- cantha, O. undulata, O. rastrera y O. leucotricha) (MEDINA-TORRES et al., 2011; JUN et al., 2013; VALENTE et al., 2007; SANTOS-ZEA et al., 2011). Furthermore, glycosylated fl avonoids have also been identifi ed in the Opuntia genus (quercetin-3-O-β-glucopyranoside, quercetin-3-O-rutinoside or rutin, kaempferol-3-O-rutinoside or nicotifl orin, isorhamnetin-3-O-rutinoside or narcissin and isorham- netin-3-O-glucoside) (GUEVARA-FIGUEROA et al., 2010). However, these metabolites have not been studied in the Nopalea genus. The results of the present study could contribute to the chemotaxonomic differences between the species O. fi cus-indica and N. cochellinifera. In addition, the identifi cation of these fl avonoids could explain some of the medicinal properties of nopal. Fig. 2 depicts the concentrations of quercetin, kaempferol, and iso- rhamnetin of the cultivars of this study. Isorhamnetin was the fl a- vonoid that was found in the highest concentration in OMA culti- var (3.902 mg g-1 dw), whereas the most prevalent fl avonoid in the OJ, OA, and NT cultivars was kaempferol with similar concentra- tions between them (1.611- 2.284 mg g-1 dw). The concentrations of the three fl avonoids of all cultivars were inferior to those found by MEDINA-TORRES et al. (2011) for the same nopal species (O. fi cus- indica) (1.995, 2.201, and 4.065 mg g-1 dw, respectively). However, the concentrations of kaempferol and isorhamnetin were superior Tab. 1: Nutraceutical content and antioxidant activity of four nopal cultivars. Cultivar Ascorbic acid Total phenolic compounds Total fl avonoids Antioxidant activity DPPH Inhibited (mg 100 g-1 dw) (mg GAE g-1 dw) (mg QE g-1 dw) (μmol TE g-1 dw) (%) NT 28.4 ± 1.51 d 4.0 ± 0.51 b 1.9 ± 0.01 bc 5.9 ± 0.58 b 42.6 ± 4.06 b OJ 48.9 ± 1.11 a 2.9 ± 0.27 c 2.0 ± 0.14 b 8.1 ± 0.16 a 57.2 ± 1.11 a OA 43.1 ± 0.42 b 8.1 ± 0.46 a 1.7 ± 0.06 c 8.3 ± 0.35 a 58.4 ± 1.18 a OMA 38.5 ± 1.51 c 4.6 ± 0.21 b 2.3 ± 0.04 a 8.2 ± 0.17 a 58.9 ± 2.40 a Values represent the mean ± standard deviation of 3 replicates. Different letters in the same column indicate statistically signifi cant difference by Tukey’s test (p < 0.05). Abbreviations: NT = Nopalea cochellinifera cv. Texas; OJ, OA, and OMA = O. fi cus-indica cv. Jade, Atlixco, and Milpa Alta, respectively; GAE = gallic acid equivalents; QE = quercetin equivalents; TE = Trolox equivalents; DPPH = free radical 2,2-diphenyl-1-picrylhydrazyl. Fig. 1: Chromatograms obtained by reverse phase HPLC of the fl avonoid profi le identifi ed in four nopal cultivars. a) O. fi cus-indica cv. Atlixco; b) O. fi cus- indica cv. Milpa Alta; c) O. fi cus-indica cv. Jade; d) N. cochellinifera cv. Texas. Abbreviations: Q = quercetin, K = kaempferol, I = isorhamnetin. Fig. 2: Flavonoid content in four nopal cultivars. Values represent the mean ± standard deviation of 3 replicates. Bars with different letters for the same fl avonoid aglycone indicate statistically signifi cant differ- ence by Tukey’s test (p < 0.05). Abbreviations: dw = dry weight; NT = N. cochellinifera cv. Texas; OJ, OA, and OMA = O. fi cus-indica cv. Jade, Atlixco, and Milpa Alta, respectively. QUERCETINE KAEMPFEROL ISORAMNETINE FLAVONOIDS AGLYCONE OA OMA OJ NT C O N C E N TR AT IO N (m g ·g -1 d .w .) 172 L.L. Cruz-de la Cruz, R. García-Mateos, C. Ybarra Moncada, J. Corrales-García to those reported for the cultivars Jalapa and Villanueva (O. fi cus- indica) (0.119 and 0.474 mg g-1 dw kaempferol, and 0.726 and 0.654 mg g-1 dw isorhamnetin, respectively) by SANTOS-ZEA et al. (2011). The signifi cant differences observed in the nutraceutical content and antioxidant activity between the cultivars of the present study and the differences found with other studies could be due, as in other foods, to diverse factors: genetic (RAMÍREZ-TOBÍAS et al., 2012), physiological response to manipulation (DE ANCOS et al., 2009), ripening stage (BAKHSHI and ARAKAWA, 2006), agronomic practi- ces, environmental conditions (HAGEN et al., 2007), and postharvest handling (TSAO, 2007). In the case of environmental conditions and postharvest handling, plants synthesize fl avonoids and other phenolic compounds as a defense mechanism against UV light, water stress, and predators. However, the chemical composition of a food can also affects the content of some metabolites. VILLAÑO et al. (2007) found that the content of phenolic compounds can vary, depending on the composition of the food matrix. In this context, nopal is character- ized by a high concentration of mucilage (polysaccharide consist- ing of galactose, rhamnose, arabinose, and xylose), and its binding to phenolics could explain the low phenolic concentration when only free phenolics are quantifi ed, which could also explain a possible de- crease in antioxidant activity (KUMAR et al., 2006). Nopal fl akes from O. fi cus-indica cv. Atlixco (OA) Drying kinetics and desorption isotherms For the preparation of nopal fl akes, cladodes from the OA cultivar were used, which presented the highest content of phenolic com- pounds and antioxidant activity compared with the remaining cul- tivars. The preliminary study of drying kinetics and desorption iso- therms, obtained with different rebaudioside A concentrations (0, 5, and 10 g L-1), allowed to determine the optimal drying time, where moisture content and aw of the nopal fl akes were maintained constant (Fig. 3). Nopal fl akes from OA cultivar, with an initial moisture content of 94.5 ± 0.56% (17.18 gH2O g-1 dw) and aw of 0.987 ± 0.059, were dehydrated until reaching the equilibrium moisture content of 3.36 ± 0.25 % (0.592 gH2O g -1 dw) and aw of 0.217 ± 0.003 at -50 °C and a pressure of 0.01 mbar (Fig. 3a and 3b). From 18 h of freeze-drying onwards, the moisture content and aw of the nopal fl akes were main- tained constant; therefore, 18 h was chosen as the optimal drying time for the preparation of nopal fl akes, sweetened with rebaudioside A at the concentrations of 0, 2, 4, 6, and 8 g L-1. The concentration of rebaudioside A (0, 5, and 10 g L-1) added to the nopal fl akes did not cause a signifi cant effect over the drying kinetics by freeze-drying. However, the fl akes sweetened with rebaudioside A had a lower aw than unsweetened fl akes (control) (Fig. 3b). This could have been due to an interaction between rebaudioside A and the fl akes’ surface, leading to a decrease of available water of the food (SHIVHARE et al., 2004). The desorption isotherms (Fig. 3b) correspond to Type III, according to Brunauer’s classifi cation (MATHLOUTHI and ROGE, 2003), characteristic of foods with high content of polysaccharides, such as mucilage of nopal. After 18 h of dehydration, the nopal fl akes had a crispy texture and were stable, and the low aw value is asso- ciated with a high level of preservation and shelf-life, since foods with an aw below 0.4 are resistant to microbial deterioration and are less susceptible to enzymatic reactions (IGATHINATHANE et al., 2007). Sensory evaluation Flakes sweetened with 6 g L-1 of rebaudioside A were the best evalu- ated sensorially (Tab. 2), since most panelists (64%) rated their sweet- Tab. 2: Sensory evaluation of nopal fl akes of O. fi cus-indica cv. Atlixco, sweetened with different concentrations of rebaudioside A. Rebaudioside A JAR scale Hedonic scale (g L-1) Response “little sweet” Response “adequate” Response “very sweet” Intensity score Preference score % % % 0 95 5 0 1.1 e 3.3 c 2 46 48 6 2.2 d 5.3 b 4 31 55 14 2.7 c 5.4 ab 6 12 64 24 3.2 b 5.9 a 8 5 52 43 3.8 a 5.7 ab Values represent the mean ± standard deviation of 100 panelists. Different letters in the same column indicate statistically signifi cant difference by Tukey’s test (p < 0.05). Abbreviations: JAR = just about right. Fig. 3: Drying kinetics (a) and desorption isotherms (b) of nopal fl akes of O. fi cus-indica cv. Atlixco, preliminarily sweetened with three concentrations of rebaudioside A (0, 5, and 10 g L-1). Values represent the mean ± standard deviation of 3 replicates. Abbreviations: Xt = equilibrium moisture content, aw = water activity, dw = dry weight. Sweetened nopal fl akes 173 ness intensity as “adequate” (JAR scale) and were the fl akes that obtained the highest score in preference (hedonic scale) (ROTHMAN and PARKER, 2009). Therefore, these fl akes were selected for further analyses. Incorporated rebaudioside A HPLC analysis allowed to identify and establish for all treatments the real concentration of rebaudioside A incorporated by immersion to the nopal fl akes. Fig. 4 shows the chromatograms of rebaudioside A as a standard (Fig. 4a) and in sweetened nopal fl akes (Fig. 4b). The fl akes that were best scored in the sensory evaluation were those at a concentration of 6 g L-1 of rebaudioside A and presented a concen- tration determined by HPLC of 1.1 ± 0.013mg g-1 of the sweetener (Tab. 3). fl akes. The signifi cant differences found between the nutraceutical content of sweetened and unsweetened fl akes could be attributed to leaching of soluble nutraceuticals during immersion in the rebaudio- side A solution (NAIDU, 2003). However, KUMAR et al. (2006) ex- plained that a decrease in phenolic compounds could be also due to the presence of hydrophilic interactions with components of the food matrix; in this case, an interaction with rebaudioside A. But, despite the loss of phenolic compounds, the antioxidant activity was less affected, probably caused by rebaudioside A (TAVARI et al., 2015). This phenomenon was observed by CARBONELL-CAPELLA et al. (2013) in papaya, mango, and orange juices, processed with high pressures, where the incorporation of glycosides from S. rebaudiana increased the antioxidant activity of these beverages. Fig. 5 depicts the concentrations of quercetin, kaempferol, and iso- rhamnetin in sweetened and unsweetened fl akes. No signifi cant differences were observed between the fl akes for none of the fl avo- noids, but the amounts were inferior to those published by MEDINA- TORRES et al. (2011) in nopals of O. fi cus-indica (1.99, 2.20, and 4.06 mg g-1, respectively). However, the authors reported that the content of these metabolites was reduced almost entirely after ex- posure of convective drying at a temperature of 65 °C. The consumption of quercetin (2 mg per kg body weight in humans) has shown signifi cant effects in the reduction of triglycerides, choles- Fig. 5: Concentration of quercetin, kaempferol, and isorhamnetin in un- sweetened fl akes and fl akes sweetened with rebaudioside A (6 g L-1) of nopal O. fi cus-indica cv. Atlixco. Values represent the mean ± standard deviation of 3 replicates. Bars with different letters for the same fl avonoid aglycone indicate statistically signifi cant difference by Tukey’s test (p < 0.05). Abbreviations: dw = dry weight. Tab. 3: Concentration of rebaudioside A incorporated to nopal fl akes of O. fi cus-indica cv. Atlixco after being submerged in different solu- tions of rebaudioside A. Concentration of Real concentration of rebaudioside A rebaudioside A in the immersion solution in the nopal fl akes (g L-1) (mg g-1) 0 ND 2 0.8 ± 0.04 d 4 1.0 ± 0.05 c 6 1.1 ± 0.01 b 8 1.3 ± 0.03 a Values represent the mean ± standard deviation of 3 replicates. Different letters in the same column indicate statistically signifi cant difference by Tukey’s test (p < 0.05). Abbreviations: ND = not detected. Fig. 4: Chromatograms of pure rebaudioside A (a) and nopal fl akes of O. fi cus-indica cv. Atlixco, sweetened by immersion in a rebaudioside A solution (6 g L-1) (b). Nutraceutical content The nutraceutical content of the most sensorially accepted sweetened fl akes of the OA cultivar (6 g L-1) was evaluated and compared with unsweetened fl akes. Tab. 4 shows the levels of ascorbic acid, total phenolics, total fl avonoids, and antioxidant activity of both types of Tab. 4: Nutraceutical content and antioxidant activity of unsweetened fl akes and fl akes sweetened with rebaudioside A (6 g L-1) of nopal O. fi cus- indica cv. Atlixco. Nutraceutical Unsweetened Sweetened Recovery property fl akes fl akes (%) AA (mg 100 g-1) 38.0 ± 2.21 a 33.9 ± 1.25 b 89.20 TP (mg GAE g-1) 5.9 ± 0.05 a 5.0 ± 0.19 b 84.87 TF (mg QE g-1) 2.9 ± 0.22 a 2.9 ± 0.11 a 99.74 AOX (μmol TE g-1) 9.8 ± 0.09 a 9.0 ± 0.07 b 91.35 (% DPPHinhibited) 55.8 ± 0.47 a 51.1 ± 0.39 b 91.71 Values represent the mean ± standard deviation of 3 replicates. Different let- ters in the same row indicate statistically signifi cant difference by Tukey’s test (p < 0.05). Abbreviations: AA = ascorbic acid, TP = total phenolics, TF = total fl avonoids, AOX = antioxidant activity, GAE = gallic acid equivalents, QE = quercetin equivalents, TE = Trolox equivalents, DPPH = free radical 2,2-diphenyl-1-picrylhydrazyl. QUERCETINE KAEMPFEROL ISORAMNETINE FLAVONOIDS AGLYCONE HSE HER C O N C E N TR AT IO N (m g ·g -1 d .w .) 174 L.L. Cruz-de la Cruz, R. García-Mateos, C. Ybarra Moncada, J. Corrales-García terol, and glucose in blood, as well as decrease in weight gain (RIVERA et al., 2008). This dose would be equivalent to eating approximately 130 g of sweetened flakes. Furthermore, it has been reported that kaempferol inhibits proliferation of cancer cells and preserves the viability of normal cells (CHEN and CHEN, 2013). Moreover, recent studies have revealed that isorhamnetin glucosides of O. ficus-indica prevent the development of metabolic abnormalities associated with obesity induced by a high-fat diet and cause anti-inflammatory ef- fects (ANTUNES-RICARDO et al., 2015) in mice. Finally, steviosides (rebaudioside A and stevioside) provide some benefits to health, such as reduction of blood pressure (HSIEH et al., 2003) and can be con- sumed by patients with diabetes type I and type II without presenting adverse effects (BARRIOCANAL et al., 2008). Hence, sweetened nopal flakes could be a functional snack and could possibly be consumed by patients with diabetes. Conclusions The nopal cultivars with greater commercial demand O. ficus-indica presented higher antioxidant activity than the underused NT culti- var from N. cochellinifera. The profile of quantified and identified flavonoids in this study could contribute to the chemotaxonomy of both nopal species. The nopal flakes of the OA cultivar (O. ficus- indica), sweetened with rebaudioside A (1.118 mg g-1), had a good sensory acceptability. The nutraceutical content of the flakes was de- creased during immersion in the rebaudioside A solution; however, the antioxidant activity was preserved, possibly due to the presence of rebaudioside A. Due to their nutraceutical content and antioxidant activity, the freeze-dried and sweetened nopal flakes could be used as a functional snack and could be possibly be consumed by patients with diabetes. The results of this study could contribute to the utiliza- tion and consumption of nopal. Conflict of interest No potential conflict of interest was reported by the authors. References ABOU-ELELLA, F.M., ALI, R.F.M., 2014: Antioxidant and anticancer activi- ties of different constituents extracted from Egyptian prickly pear cactus (Opuntia ficus-indica) peel. Biochem. Anal. Biochem. 3, 158. 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