*Sicana odorifera* **"Kurugua" from Paraguay, Composition and Antioxidant Potential of Interest for the Food Industry †**

#### **Coronel Eva, Caballero Silvia, Baez Rocio, Villalba Rocio and Mereles Laura \***

Departamento de Bioquímica de Alimentos, Facultad de Ciencias Químicas, Universidad Nacional de Asunción, P.O. Box 1055, San Lorenzo, Paraguay; ecoronel@qui.una.com (C.E.); scaballero@qui.una.com (C.S.); rocio.baez15@gmail.com (B.R.); rvillalba@qui.una.com (V.R.)


Published: 6 August 2020

**Summary:** The aim of this study was to evaluate the physicochemical characteristics, centesimal composition and antioxidants of the *Sicana odorifera* pulp and the antioxidant potential of the seeds and fruit peel harvested in a culture of the city of San Lorenzo, Paraguay. These fruits harvested in Paraguay present an antioxidant potential, interesting for food industry, especially in a ripe and semi-ripe state, where the highest content of vitamin C and total phenols was observed, as well as the total antioxidant capacity (ABTS).

**Keywords:** *Sicana odorifera*; composition; antioxidants; Curcubitaceae; total phenols; vitamin C

#### **1. Introduction**

Worldwide, the search for food components as additives with functional properties represents a great demand, where biodiversity and the systematic study of species of interest is fundamental to generating knowledge about potential uses of native fruits, with known medicinal applications described in ethnobotany [1]. This specie is cultivated in several countries in South America and Central America, known as melao de croa, cassabanana, or red melon [2]. In Brazil, the composition of the fruit of the kurugua was recently described [2], however, the phytochemical composition of the fruit pulp has been little explored in Paraguay, despite being described as an autochthonous fruit, which limits its use at an industrial level, mainly due to the ignorance of its nutritional potential and consequently of its potential applications. The fruit of the kurugua is used in juices, jams and preserves, it is used as an infusion in popular wisdom for liver diseases [3], and some species of the same family Cucurbitaceae have known hepatoprotective properties [4]. It is known that antioxidants can contribute to liver protection, and they vary in their concentration and quality with the stage of maturity. The objective of the present work was to evaluate the physicochemical characteristics, centesimal composition and antioxidants of the *Sicana odorifera* pulp and to evaluate the antioxidant potential in the seeds and fruit peel harvested in a culture in San Lorenzo city, Paraguay.

#### **2. Materials and Methods**

#### *2.1. Sampling*

Samples of *Sicana odorifera* fruits "kurugua" were collected from healthy mature trees (12 years old) from an orchard placed at San Lorenzo, Central Department, Paraguay (GPS ƺ25.3266340 N,

#### *Proceedings* **2020**, *53*, 10

ƺ57.4832020 E). The yearly average sunlight in the cultivation place ranges between 6 and 14 h per day. The orchard is used as a seedbed of the "Kurugua Poty" foundation, which ensured the traceability of the variety analyzed. Samples were collected for herbarium material for botanical identification by the Botánica Department of the Facultad de Ciencias Químicas of the Universidad Nacional de Asunción. Random sampling from the crop of fruits in three stages of maturity—unripe, semi-ripe, and ripe—was carried out. Fourteen kilograms of fruits were obtained. After the morphological measurements, the pulp was separated from the peel and seeds. The centesimal composition was made in the edible fraction (pulp) of ripe fruit.

#### *2.2. Physicochemical Characters*

Morphological studies were performed on unripe, semi-ripe and ripe fruits without previous treatment, as described by Mereles and Ferro [5]. The pH, titratable acidity and soluble solids were measured according to AOAC Methods [6]. A potentiometer (Accurate pH 900, Horiba, Kyoto, Japan) at 25 °C and an analytical balance (AYD HR 120, Bradford, England) were used. All measurements were made in triplicate. All the reagents used were of analytical grade.

#### *2.3. Chemical Analysis*

Moisture, protein, total carbohydrates, dietary fiber, total mineral content or ash were determined, all according to official AOAC methodologies [6]. To estimate the antioxidant potential of the fruit, the content of total phenols, monomeric anthocyanins, vitamin C and the total antioxidant capacity were evaluated by the ABTS method. The determination was made using the spectrofluorometric method 967.22 of AOAC [6]. For the measurements, an L-ascorbic acid calibration curve (2.5–20 ΐg/mL) was used. The results were expressed in mg of vitamin C per 100 g of pulp fresh weight. The determination of anthocyanins was carried out by the spectrophotometric method of differential pH, based on the color loss of the monomeric anthocyanins at pH 4.5 and presence of color at pH 1, measuring at 510 and 700 nm [1]. The final concentration of anthocyanins (mg/100 g) was calculated based on the volume of extract and sample fresh weight. It is expressed in cyanidinidine 3-glucoside (MW: 449.2 and Ή: 26,900). An extraction of 2 g of lyophilized pulp with methanol:water (60:40) in an ultrasonic bath for 15 min was performed and subsequent centrifugation (15 min, 10,000× *g* rpm, 4 °C). After separating the supernatant, a second extraction was carried out with acetone:water (70:30) with the same treatment described. Extractions and measurements were carried out by triplicates. Total phenolic compounds (TPC) were determined by the Folin–Ciocalteau described by Singleton & Rossi [7], colorimetric method using a calibration curve obtained with gallic acid (0–120 ΐg/mL aqueous solution). The mixture was stirred and kept for 30 min at room temperature in the dark. The absorbance was measured spectrophotometrically at 765 nm against a blank reagent. For measurements, a gallic acid standard curve (concentration interval 0–120 ΐg/mL aqueous solution) was plotted at 765 nm in the UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The results were expressed as mg of gallic acid equivalents (GAE) per 100 g of pulp (mg of GAE/100 g). The antioxidant activity was determined by the TEAC assay using the radical cation ABTS+• [8] (Re et al. 1999). The ABTS+• stock solution (7 mM) was prepared using ammonium persulphate (NH4)2S2O8 as the oxidant agent. The working solution of ABTS+• was obtained by diluting the stock solution in ethanol to give an absorption of 0.70 ± 0.02 at Ώ = 734 nm. For measurements, a calibration curve with Trolox (0–500 ΐM aqueous solution) was plotted at 730 nm. The results were expressed as micromoles of Trolox equivalents (TEAC) per gram of pulp fresh weight.

#### *2.4. Statistical Analysis*

The results were expressed as means ± standard deviation (SD) from three independent replicates. Data were stored in a spreadsheet, when appropriate, to compare stages of maturity values (unripe, semiripe and ripe) ANOVA and Tukey's post-test were used. Values with *p* ǂ 0.05 were considered as statistically significant with the assistance of Graph Pad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA) for the calculations.

#### **3. Results and Discussion**

#### *3.1. Physicochemical Characters*

The physicochemical characteristics of the whole fruit of *S. odorifera* were determined in three stages of maturity (Table 1). Variations in weight, diameter and height were observed among unripe fruits compared to semi-ripe and ripe fruits. These results are consistent with those reported by Paula Filho et al. (2015) in *S. odorifera* harvested in Brazil, for the weight and average diameter of the ripe fruits (2510 g, 9.72 cm, respectively) [3]; however these authors reported that the fruits studied were longer (36.91 cm, approximately).

Regarding soluble solids (SS), significant differences were observed between maturity stages, increasing with it, observing 8.24 Brix in mature state, higher than the one reported (4.15 Brix) in Minas, Brazil [3]. In the composition of the ripe fruit (Table 2), it was observed that it has a low caloric intake with high water content (88.0 ± 0.1 g/100 g).

**Table 1.** Physicochemical characterization of the unripe, semi-ripe and ripe *Sicana odorifera* fruit pulp.


The values are means ± SD. Different letters indicate significant differences between means (ANOVA and Tukey's a posteriori test, *p* ǂ 0.05). \* Determinations made in fresh fruit pulp (n = 3).



The values are means ± DS (n = 3). Determinations made in fresh fruit pulp.

#### *3.2. Antioxidants Analysis*

Statistically significant differences were observed in the contents of total phenols between the three maturity stages evaluated (Figure 1), unripe, semi-ripe and ripe (13.8 ± 0.8, 22.8 ± 2.2 and 37.5 ± 4.2 mg of GAE/100 g, respectively). The increase in total phenols as the fruit matures is important from the alimentary point of view, since it is the state in which it is consumed directly, and the health benefits associated with these compounds are expected. These results are also superior to those reported by Contreras-Calderón et al. [9] of fruits of *S. odorifera* harvested in Colombia (15.7 ± 1.1 mg of GAE/100 g) in fresh pulp and Marquez et al. [10] in fruits of another Cucurbitaceae, *Citrullus lanatus* (8.8 mg of GAE/100 g sample).

**Figure 1.** Total phenols content in unripe, semi-ripe and ripe *Sicana odorifera* fruit pulp. The bars represent the mean ± SD (n = 3). Different letters on the bars indicate significant differences between the means (ANOVA, Tukey's test a posteriori, *p* ǂ 0.05).

The content of anthocyanins was higher (2.64 ± 0.10 mg/100 g) in the unripe fruit (Table 3), compared to the semi-ripe fruit (1.55 ± 0.70 mg/100 g) or ripe (not detectable); this behavior was contrary to what was observed in the content of total phenols, whose content increased with maturity (Figure 1). This could be due to the fact that the anthocyanins are stable at acid pH and the pH in the fruit of *Sicana odorifera* increased with maturity (Table 2). Anthocyanins are natural compounds present in a wide variety of plants, fruits and vegetables that are of great interest to the food industry, due to their qualities as colorants (given their range of colors from red to blue) and their antioxidant properties normally due to the presence of phenols in its structure. Although for *S. odorifera* no values of anthocyanins are reported, the content in other cucurbits such as watermelon is lower (0.4 mg/100 g) than observed in this work [10].

Regarding the vitamin C content, it was observed that it was higher in the ripe pulp (21.8 ± 4.7 mg/100 g) compared to the previous maturity stages. This result is higher than the value reported in pulp of *S. odorifera* harvested in Colombia (16.0 mg/100 g) reported by Contreras et al. [9] and Brazil (3.21 mg/100 g) reported by de Paula Filho et al. [3].


**Table 3.** Anthocyanins, vitamin C content and total antioxidant capacity (ABTS) in three stages of maturity in *Sicana odorifera* fruit pulp.

The values are means ± SD. Different letters indicate significant differences between means (ANOVA and Tukey's test a posteriori, *p* ǂ 0.05).

In the total antioxidant capacity determination by the ABTS method (Table 3), the behavior was different; the semi-ripe fruit (normally used as a vegetable in soups) presented a statistically higher value (6.75 ± 0.90 ΐM TEAC/g) in comparison with the values of unripe and ripe fruit (3.18 ± 0.39 and 4.54 ± 0.24 ΐM TEAC/g, respectively). The value observed in semi-ripe fruit is consistent with the value reported by Contreras et al. (2011) in ripe fruit (6.49 ± 0.47 ΐM TEAC/g) [9], although it is expected that there are differences between the chemical concentrations of fruits compared, due to the genetic variability in some criollo varieties of Cucurbitaceae. In the seeds and skin of the fruit, high contents of total phenols were observed (Table 4), and the total antioxidant capacity was higher (18–19 ΐmol of Trolox/100 g of ripe fruit peel).

**Table 4.** Total phenols content and total antioxidant capacity (ABTS) in *Sicana odorifera* fruits seeds and peel.


The values are means ± SD. Different letters indicate significant differences between means (ANOVA and Tukey's a posteriori test, *p* ǂ 0.05).

#### **4. Conclusions**

The *Sicana odorifera* fruits analyzed present an antioxidant potential of interest for the food industry, especially in its ripe and semi-ripe state, where the highest content of vitamin C and total phenols was observed, as well as the total antioxidant capacity (ABTS). This fruit can contribute to the supply of vitamin C in the diet in its fresh state and reduce the food insecurity of the population.

This work contributes to the scientific basis on the antioxidant potential of *S. odorifera* grown in Paraguay and opens a path towards the revaluation of the fruit. Continuing studies on its potential as a coloring and flavoring in the replacement of critical ingredients in foods, such as artificial additives, is a viable alternative based on its composition.

**Acknowledgments:** This work was supported by grant Ia ValSe-Food-CYTED (119RT0567). The authors are especially grateful to the "Kurugua poty" Foundation for the provision of the samples and the Facultad de Ciencias Químicas de la Universidad Nacional de Asunción for providing their facilities.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Proceedings*

### **Microencapsulation of Sacha Inchi (***Plukenetia huayllabambana***) Oil by Spray Drying with Camu Camu (***Myrciaria dubia* **(H.B.K.) Mc Vaugh) and Mango (***Mangifera indica***) Skins †**

#### **Rafael Alarcón 1, Billy Gonzales 1, Axel Sotelo 1, Gabriela Gallardo 2, María del Carmen Pérez-Camino 3 and Nancy Chasquibol 1,\***


Published: 26 August 2020

**Abstract:** Sacha inchi (*Plukenetia huayllabambana*) oil was microencapsulated by spray drying with gum arabic and with extracts of camu camu (*Myrciaria dubia* (HBK) Mc Vaugh) and mango (*Mangifera indica*) skins, obtained by assisted microwave. The physicochemical characteristics, such as moisture content, encapsulation efficiency, particle size, morphology, fatty acid composition and oxidative stability, were evaluated in order to select the best formulation for the development of functional foods. The most important results indicate that the microcapsules formulated with extracts of the fruit skins provide greater protection to sacha inchi oil (*P. huayllabambana*) against oxidation compared to commercial antioxidant BHT (Butylated Hydroxytoluene), resulting in a slight loss of Ν-3 fatty acids.

**Keywords:** antioxidants; microencapsulation; assisted microwave extraction; oxidative stability; *Plukenetia huayllabambana*

#### **1. Introduction**

The camu camu (*Myrciaria dubia* (H.B.K.) Mc Vaugh) and mango (*Mangifera indica*) skins shown high antioxidant activity. Camu camu is a low-growing shrub found throughout the Amazon rainforest of Peru, Colombia, Venezuela and Brazil. The camu camu fruit is mainly consumed after being processed into juices, concentrates, and for the production of vitamin C capsules. As a result, a great volume of residue of seeds, skins and pulp that represent around 40% of the fruit in weight, are generated [1]. The importance of antioxidants is crucial for health, due to its ability to neutralize free radicals, which contain one or more unpaired electrons, being responsible for many degenerative diseases. Sacha inchi *Plukenetia huayllabambana* grows in the province of Rodríguez de Mendoza, Department of Amazonas-Peru, its oil contains a higher percentage of Ν-3 (55.62 to 60.42% ΅-linolenic acid) [2]. However, an existing problem is that, due to its chemical structure, Ν-3 acids have a high susceptibility to oxidation. A technology that emerges as an alternative to delay or inhibit its deterioration is microencapsulation [3], which consists in the preparation of an oil-in-water emulsion,

#### *Proceedings* **2020**, *53*, 11

containing encapsulating agents (or carriers, such as gums, fibers, proteins or carbohydrates) and their subsequent drying. This process aims to protect the poly-unsaturated fatty acids (PUFAs) from environmental factors, such as light, air or humidity. The aim of this work was to compare physicochemical characteristics and the oxidative stability of sacha inchi oil microcapsules elaborated with gum arabic and different mixtures of camu camu and mango skin extracts, in order to select the best formulation, which will allow for obtaining functional foods once the production of the microcapsules has escalated.

#### **2. Materials and Methods**

#### *2.1. Raw Material*

Cold pressed sacha inchi oil from the ecotype *P. huayllabambana* was obtained in the laboratory of *Centro de Estudios e Innovación del Alimento Funcional* (CEIAF) from the Universidad de Lima (Peru) and kept at 4 °C. Additionally, the camu camu and mango skins were washed and dried by infrared dryer (IRC DI8, Spain) at 40 °C, then ground into the food shredder (Grindomix GM200/Restch) and kept at ƺ5 °C in polyethylene bags prior to phenolic extraction. The arabic gum (AG) was the agent encapsulant (wall material) due its versatility, good solubility, low viscosity at high concentrations and very good emulsifying properties.

#### *2.2. Microwave-Assisted Extraction (MAE) of Polyphenols*

The extraction of polyphenols from camu camu and mango dried skins were performed using a microwave oven (CW-2000, China) with ethanol:water. The optimal MAE conditions were previously determined. The resulting extracts were evaporated at 30 °C using a rotary evaporator (Büchi rotavapor R100 Labortechnic AG Switzerland).

#### *2.3. Samples Proposed*

A total amount of five samples to be analyzed was proposed, making mixtures of camu camu skin (CCS) extract, mango skin extract (MSE) with gum arabic (GA) and sacha inchi (*P. huayllabambana)* (SIPH) oil (Table 1).

**Table 1.** Samples of sacha inchi, *Plukenetia huayllabambana* oil microcapsules (SIPH) elaborated.


a Camu camu skin extract, b Mango skin extract, c Commercial antioxidant.

#### *2.4. Microencapsulation*

The solutions were prepared with GA and distilled water containing 180 g of camu camu and mango skins extracts. Sacha inchi oil was then added at a concentration of 18% with respect to total solids. Emulsions were formed using a Silverson homogenizer L5M-A-England, operating at 9000 rpm for 10 m. The solutions were dried by spray dryer (Büchi B-290-Switzerland). Inlet and outlet air temperature were 140 °C and 70 °C, respectively, and the feed flow rate was 55 mL/min. The dried powders collected were stored in opaque hermetic bags at ƺ5 °C for further analysis.

#### 2.4.1. Moisture Determination

The moisture of the encapsulated samples was determined gravimetrically by drying until constant weight using halogen moisture analyzers (Sartorius MA-30, Germany), operating at 103 ± 2 °C.

#### 2.4.2. Total Phenolic Content (TPC) and Surface Phenolic Content (SPC)

The TPC and SPC of the extract and powder was determined by the Folin–Ciocalteu method [4] with some modifications. The absorbance of the solution was measured at 760 nm using a spectrophotometer (1205 Vis Spectrophotometer UNICO). The results were expressed as μg of equivalent gallic acid (GAE) per gram of microcapsules (powder). All analyzes were done in triplicate. For the determination of the surface phenolic content (SPC), 24 mg of microcapsules were dissolved in 4.5 mL of methanol and stirred using a vortex for 1 min and then filtered through a Whatman filter paper number 2. The surface phenolic content was measured according to the same method described for TPC determination.

The percentage of efficiency of TPC microencapsulation was calculated using the following equation: Percentage Efficiency (%) = [(TPC) ƺ (SPC)/TPC] × 100.

#### 2.4.3. Determination of Antioxidant Activity on DPPH Radical

The determination of antioxidant activity was determined using DPPH as a free radical according the procedures described previously [5]. The absorptions of samples were then detected (Abs 517sample). The percentage inhibition (% I) of free radicals was calculated using the following equation: Percentage Inhibition (% I) = [(Abs517 control) ƺ (Abs517 sample)/(Abs517 control)] × 100.

#### 2.4.4. Fatty Acid Composition

The fatty acid methyl esters (FAMEs) were prepared according to the International Union of Pure and Applied Chemistry, IUPAC [6] and the FAMEs formed were analyzed using a 7890B Agilent gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a SP2380 polar capillary column and a flame ionization detector (FID). The injector and detector temperatures were maintained at 225 and 250 °C, respectively. Hydrogen was used as carrier gas at a flow rate of 1.0 mL/min. The oven temperature was set at 165 °C and increased to 230 °C at 3 °C/min maintaining this temperature for 2 minutes. The injection volume was 1 μL.

#### 2.4.5. Particle Size Distribution and Morphology

The particle size distribution was determined by laser diffraction spectroscopy on a Master Sizer Micro equipment (measuring range: 0.3 μm–300 μm). The average diameter of the equivalent volume or D [4,3] was informed. The photomicrographs were analyzed by a FEI scanning electron microscope, model QUANTA 250 FEG, (Hillsboro, OR, USA). Samples were previously gold sputtered with an Edwards Sputter Coater S150B (Crawley, England).

#### *2.5. Oxidative Stability*

The thermal analysis was carried out by differential scanning calorimetry (DSC), for the determination of oxidation onset temperature (OOT) according to the ASTM E2009-08 Standard Test Method for Oxidation Onset Temperature of Hydrocarbons by Differential Scanning Calorimetry.

#### **3. Results and Discussion**

According to Figure 1 the moisture content was between 3.74 and 4.32% being the highest value for the mixtures of antioxidants extracts, and the lowest values for the mango skin extract. The higher inlet (140 °C) and outlet temperature (80 °C) promoted the drying rate of droplets and resulted in low moisture content [7].

**Figure 1.** Moisture content (%) of sacha inchi, *P. huayllabambana* oil microencapsulated (SIPH). Different capital letters indicate significant differences (*p* ޒ 0.05(.

The total phenolic content (TPC) was found to be between 357.7 and 1677.9 μg GAE g-1 powder, and the highest amount of phenolic compound was obtained with camu camu and mango skin extract. There was a statistically significant difference in TPC between them. The encapsulation efficiency ranged from 90.25 to 98.28 %.

The microencapsulates showed the highest antioxidant activity (75.29 I% to 91.76 I%) and a composition very similar to the starting oil with a slight loss of omega-3 (57 vs. 58%) and a slight amount of *trans* fatty acid isomers (0.05–0.09%).

According to the particle size determination (Table 2), microcapsule diameters were between 1.6 and 20.9 μm. All samples showed a monodisperse distribution. The morphological analysis performed by SEM microscopy allowed us to distinguish rounded and concave microcapsules (Figure 2). This was an expected behavior for samples obtained by spray drying [8].


**Table 2.** D [4,3] values obtained from sacha inchi, *P. huayllabambana* oil microencapsulated (SIPH).

**Figure 2.** Morphology of microcapsule: SIPH + GA + CCSE (110 ppm) + MSE (110 ppm).

The results obtained for the oxidative stability of microcapsule with naturals antioxidants were to be between 188 to 198 °C and for the commercial antioxidant were 174 °C, thus showed that the antioxidant extracts from natural origin provided greater protection to sacha inchi oil (*P. huayllabambana*) against oxidation.

#### **4. Conclusions**

The microcapsules of sacha inchi (*P. huayllabambana*) oil showed a low percentage of moisture content (3.74 to 4.32%), a high amount of phenolic compound (357.7 to 1677.9 μg GAE g-1 power) and a high encapsulation efficiency (90.25 to 98.28 %). The microencapsulates showed high antioxidant activity (75.29 I% to 91.76 I%) with a composition very similar to the starting oil with a slight loss of omega-3 (57 vs. 58%) and a slight amount of *trans* fatty acid isomers (0.05 to 0.09%). The morphological analysis allowed us to distinguish rounded and concave microcapsules, and they vary in their shape due to their chemical composition. The results obtained from OOT showed that the antioxidant extracts from natural origin provide greater protection to sacha inchi oil (*P. huayllabambana*) against oxidation. For this reason, the microcapsules can be used as a natural antioxidant in functional foods or nutraceutical products, with possible health benefits.

**Acknowledgments:** This research is part of the project N° 093-INNOVATEPERU-IDIBIO "Development of functional drink, source of omega-3 and antioxidants microencapsulated from camu camu and mango skins, to promote the commercial development of Peruvian biodiversity" the INNOVATE PERU, belonging to the Ministry of Production—Peru, and Universidad de Lima—Peru. The authors want to thank Ia ValSe-Food-CYTED (119RT0567) for financing the participation in the CYTED meeting.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
