**Changes in the Antioxidant Activity of Peptides Released during the Hydrolysis of Quinoa (***Chenopodium quinoa* **willd) Protein Concentrate †**

#### **Julio Rueda, Manuel Oscar Lobo and Nornma Cristina Sammán \***

Centro de Investigaciones Interdisciplinarias en Tecnologías y Desarrollo Social para el NOA (CIITED), CONICET-Facultad de Ingeniería, Universidad Nacional de Jujuy, Ítalo Palanca 10.4600,

San Salvador de Jujuy, Argentina; julioruedafca@gmail.com (J.R.); mlobo958@gmail.com(M.O.L.)


Published: 26 August 2020

**Abstract:** There is an increased interest in Andean crops as sources of nutritious compounds. This study evaluated changes in the antioxidant activity of quinoa protein hydrolysate with commercial enzymes. Aliquots at 0, 30, 60, 120 and 180 min were tested for DPPH (2,2ȝ-diphenyl-1 picrylhydrazyl) and ABTS ((2,2ȝ-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), antiradical activity. Initial DPPH inhibition rose from 9.2% ± 2.7% to 20.0% ± 4.0% (30 min) when employing alcalase and initial ABTS inhibition increased from 20.9 ± 0.2 to 105.1 ± 3.7 with ascorbic acid μg/mL (30 min). Protamex improved this to 75.7 ± 0.6 μg/mL (180 min). Alcalase and protamex are suitable enzymes for the production of rich peptides and hydrolysates as novel ingredients with antioxidant activity.

**Keywords:** enzymes; hydrolysate; peptides; protein; quinoa

#### **1. Introduction**

Several plant-derived foods exhibit health benefiting attributes and are suitable for healthy food production. Many of these properties are attributed to proteins and peptides. Peptides can be found in foods as individual parts of proteins or encrypted inside parent proteins.

Peptides and hydrolysates are produced from diverse protein sources. Although animal sources such as milk, eggs and meat proteins are the largest type of products employed, they are not cheap or easily accessible. This has led to an increased interest in vegetable proteins for the manufacturing of such products.

Quinoa has been largely consumed by early Latin American inhabitants and has a long tradition of well-known nutritive properties, now appreciated by different regions around the world [1]. There are few studies showing the potentiality of quinoa protein as substrate for the release of bioactive peptides or hydrolysate as novel ingredients [2]. The scope of this study was to evaluate the potentiality of quinoa for the production of protein concentrate and to select widely available enzymes for the production of protein hydrolysates as functional ingredients with antioxidant activity.

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

#### *2.1. Chemicals and Reagents*

Chemical and reagents employed were of analytical grade. The following were purchased from Sigma-Aldrich: 2,4,6-trinitrobenzenesulfonic (TNBS) acid solution (5%), L-leucine (ǃ98% HPLC), alcalase from *Bacillus lincheniformis* (activity ǃ 2.4 AU/g), flavourzyme from *Aspergillus oryzae* (activity ǃ 500 LAPU/g) and protamex from *Bacillus* sp. (activity ǃ 1.5 AU-NH/g). AU is defined as Anson units; LAPU is defined as leucine aminopeptidase units[3]. Reagents 2,2ȝ-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) were obtained from Merck Bio and Sigma-Aldrich, respectively. Quinoa seeds (*Chenopodium quinoa* willd. var INTA Hornillos) were provided by the National Institute of Agricultural and Livestock Technology (INTA-IPAF-NOA-Argentina). The pH was adjusted using NaOH or HCl 0.3 M. Assays were performed in duplicate (n = 2). A two-way ANOVA and the Tukey test (Graph Pad Prism 6.0.) were performed. Significant differences were detected at *p* < 0.05.

#### *2.2. Quinoa Protein Solubility Study*

#### 2.2.1. Quinoa Protein Solubility

Quinoa grains were ground in a mill and the flour was agitated for 2 h with petroleum ether (1:10 *w/v*). Protein solubility at different pH levels (212) was determined. Aqueous quinoa flour suspensions (1/10 *w*/*v*) were mixed for 20 min, at 30°C and 100 rpm. Proteins were measured at pH 2–12 using bovine serum albumin (BSA) as standard and Bradfordȝs reagent. Results are expressed as mg BSA equivalents/mL.

#### 2.2.2. SDS-PAGE

Electrophoresis in denaturing conditions was performed using acrylamide stacking (4%) and running (12%) gels. Samples were boiled for 3 min in separating buffer containing 2% SDS, 10% glycerol, 0.01% bromophenol blue, 0.0625 M tris-HCl pH 6.8 and 5% Ά-mercaptoethanol, loaded (5 μL) onto gels and run at a constant voltage (60 V first 10 min and 120 V) using Laemmli buffer. Gel staining was performed with Coomassie Blue R-250 and destaining was performed in methanol/acetic acid solution (50/20).

#### *2.3. Quinoa Protein Concentrate (QPC)*

A proportion of 1:10 (*w*/*v*) of defatted flour was agitated at 150 rpm, for 2 h, at 30 °C, at the desired pH, then centrifuged (10,000× *g*, 10 min), and proteins were precipitated at acid pH. The slurry was refrigerated for 30 min, at 4 °C, and centrifuged (10,000× *g*, 10 min, 4 °C). The pellet was air-dried in a flux oven (30 °C, 12 h). The protein concentrate was powdered and the protein content (N × 5.7) was determined by the Kjeldahl method.

#### *2.4. Proteolysis*

#### 2.4.1. Hydrolysis Conditions

Hydrolysis conditions of quinoa protein (10 mg/mL) at pH 7–10 and temperatures of 40–60 °C were determined. Proteases were added (1/10 w or *v*/*w*) and after 10 min, the reaction was stopped (1.0 mL TCA 10%). A blank for each assay was prepared without the enzyme. The slurry was refrigerated (4 °C), for 30 min and centrifuged (15,000× *g*, 4 °C and 10 min). TCA soluble peptides were determined.

#### 2.4.2. Protease Activity

Alpha amino groups were measured according [4]. Aliquots of 0.1 mL of peptides were mixed with 3.4 mL phosphate buffer pH 8.2 (0.2 M) and 0.5 mL of TNBS 0.05% and incubated in the dark at 50 °C, for 60 min at 200 rpm. The absorbance was measured at 420 nm in a UV-visible spectrophotometer, using leucine as standard. Proteolytic units were expressed as leucine equivalents mM/min (PU/min) [3].

#### 2.4.3. Quinoa Protein Hydrolysates (QPH)

Enzyme and substrate ratio was 1:10. Optimal conditions were adjusted. Aliquots were taken at 0, 30, 60, 120 and 180 min. Enzyme inactivation was performed at 85 °C for 10 min.

#### 2.4.4. Hydrolysis Degree Calculation

The hydrolysis degree percentage (HD%) was determined as follows:

$$HD\% = B \times N\_b \times \frac{1}{a} \times \frac{1}{PM} \times \frac{1}{h\_{tot}} \times 1000$$

*htot* is the total number of peptide bonds (7.21 miliequivalents/g protein), calculated on the basis of the amino acid occurrence in a local variety of quinoa [5], *B* is the volume (mL) of base necessary to keep the pH constant, *Nb* is the normality of the base, ΅ƺ1 is the calibration factor calculated as the reciprocal of the average degree of dissociation of ΅-NH amino groups, and *PM* is the mass of protein (g) in the total reaction.

#### *2.5. Antiradical Activity*

#### 2.5.1. Inhibition of Radical DPPH

QPH antiradical properties were tested following the methodology of Chakka et al. [6]. Briefly, 0.1 mL of sample or blank (distillate water) was mixed with 1.4 mL of DPPH at 0.1 mM in anhydrous methanol and incubated 30 min in the dark; absorbance readings were taken at 515 nm in a UV-visible spectrophotometer. The antiradical activity as percentage (ARA) was calculated according the following equation.

$$ARA\ (\%) = \left[\frac{1 - (A\_s - A\_b)}{A\_c}\right] \times 100\ $$

*Ac*, *As* and *Ab* are absorbance of control, sample and blank, respectively.

#### 2.5.2. Inhibition of Radical ABTS

The radical formed with 2,2ȝ-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt and potassium persulfate was employed. The radical solution was mixed with 50 μL of sample or standard and incubated for 6 min before absorbance reading. A linear calibration curve was prepared using ascorbic acid. Results are expressed as equivalent ascorbic acid μg/mL.

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

#### *3.1. Quinoa Protein Solubility*

Figure 1 shows the protein solubility in a wide range of pH levels; an increase in solubility profile at extreme acid and alkali conditions was observed. The maximal concentrations were measured at pH 9 (13.33 ± 0.71) and 10 (13.41 ± 0.62). Similar results were reported by Elsohaimy et al. [7]. In other Andean crops such as amaranth, pH values of 4 or 5 precipitated most proteins [8], which agrees with this study. The protein content of the concentrate was 61.61% ± 0.43%. This value is higher than the one (40.7% ± 0.9%) found by Nongonierma et al. [2], who employed mashed grains, an extraction time of 60 min compared to 120 min and quinoa defatted flour as starting material, as described in this study. The higher value may be due to the much smaller and homogeneous material employed, which allowed more protein extractability. Higher levels of protein content (65.5 ± 0.1 and 77.2 ± 0.1% *w*/*w*) were found in protein concentrates prepared by Aluko and Monu [9] and Abugoch et al. [10].

#### *3.2. Electrophoresis*

Figure 2 shows the electrophoretic run of proteins solubilized at pH 9 (lane 2) and 4 (lane3). While lane 3 shows no protein bands, indicating that the solubilized protein meets its isoelectric point, the pattern of proteins solubilized at pH 9 (lane 2) exhibits polypeptides of high molecular weight, ranging from 100.0 to 24.5 kDa. Major bands of polypeptides are estimated in molecular weights of 45,700 (A), 32,000–28,700 (B) and 25,900–24,480 (C) kDa. These sets of polypeptides have been initially described by Brinegar and Goudan [11] and recently by Vilcacundo et al. [1].

**Figure 1.** Quinoa protein solubility at different pH values. Obtained from aqueous suspensions of defatted flour (1:10 *w*/*v*) maintained at a constant pH (NaOH or HCl) for 10 min at 30 °C and 100 rpm. n = 2. Values (mean ± SD) are expressed as bovine serum album equivalents mg/mL.

The polypeptides belong to the main constituent protein in quinoa, chenopodin. Each group (B and C) is composed of subunits bonded by disulfide bonds. In Figure 2, bands B and C correspond respectively to the acidic and basic polypeptides found in 11S-type globulin family proteins. The 45,000 kDa protein appearing in Figure 2 may be a chenopodin A-B protein with a strong disulfide bond still remaining after reductive SDS-PAGE conditions; bands appearing under 20 kDa have been described as 2S type proteins present in many seeds, albumins mainly. Concerning amino acid composition, it has been reported that chenopodin exceeds the Food and Agriculture Organization(FAO) requirements and has a high chemical score [11].

#### *3.3. Hydrolysis Conditions*

Figure 3 shows the leucine equivalents at different conditions of pH and temperature, using quinoa protein as substrate. Figure 3B (flavourzyme) and Figure 3C (protamex) show an increase in the protease activity towards neutral conditions for all temperatures and pH levels. Flavourzyme showed no activity at pH 9–10, at 60 °C. On the other hand, alcalasa (Figure 3A) was very active at all pH levels and temperatures. Its activity increased noticeably as the medium turned alkaline. This increase was gradual at 40 °C but high and constant at 50 or 60 °C. Optimal conditions and proteolytic units (PU) are summarized in Table 1, solubilized from defatted flour 1:10 *w*/*v* in aqueous suspensions. Alkali and acid media were regulated with NaOH or HCl.

The optimal values of temperature found (flavourzyme and protamex) differ from those employed by Jung et al. [12]. This change could be attributed to the substrate type, enzyme: substrate ratio and conditions of reaction employed for the hydrolysis.




PU/min: Proteolytic units expressed as leucine equivalents mM/min.

**Figure 2**. SDS-PAGE characterization of quinoa proteins soluble at pH 9 (lane 2) and pH 4 (lane 3). MW: molecular weight marker; A: 7S globulin 45 kDa; B: globulin acid subunit; C: globulin basic subunit.

**Figure 3.** Leucine equivalents produced at different pH levels and temperatures by three enzymes (substrate enzyme ratio 1:10). (**A**) alcalase; (**B**) flavourzyme; (**C**) protamex. Temperatures: 40 °C (ź); 50 °C (×); 60 °C (Ŷ). Results (mean ± SD) are expressed as mM/mL of supernatant after 10 min of hydrolysis (n = 2).

#### *3.4. Quinoa Protein Hydrolysis*

As shown in Figure 4, the hydrolysis of quinoa protein follows an enzyme dependent hydrolysis pattern. Curves show a high rate of hydrolysis in the first 30 min with alcalsa (27.9%) and protamex (20.7%) and evolves at low velocity for flavourzyme (4.3%). Thamnarathip et al. [13] achieved 13–14 HD% by employing alcalasa, flavourzyme and rice bran protein after 6 h.

**Figure 4.** Hydrolysis degree progression of quinoa protein concentrate in aqueous suspension (10 mg/mL) using different enzymes (substrate enzyme ratio 1:10). Data are expressed as percentage (mean ± SD) of peptide bonds cleaved at defined time (min).

#### *3.5. Antiradical Activity*

Table 2 compares the antiradical activity measured by ABTS and DPPH methods as the hydrolysis progresses. DPPH antiradical activity with alcalasa was higher than that obtained with flavourzyme and protamex. From initial values of 9.3% ± 0.2%, alcalase increased activity to 20.0% ± 4.0% after 30-min hydrolysis. DPPH inhibition with flavourzyme and protamex decreased or remained fairly constant as the hydrolysis degree increased, with 14% being the average highest value after 180 min.

ABTS measurements were remarkably different among the enzymes employed. From initial values of 20.9 ± 0.2 ascorbic acid μg equivalents/mL of the non-hydrolyzed protein, the antioxidant activity increased to 105.1 ± 0.1 (30 min) and to 75.7 ± 0.6 μg/mL (180 min) using alcalasa and protamex, respectively. Flavourzyme reduced the antioxidant activity. Differences in the activities measured by the mentioned methods may be related to the solubility of the antioxidant compounds in the organic (methanol) and aqueous phases employed. This could be related to lipophilic and hydrophilic peptides and also to the low molecular weight.

**Table 2.** Antiradical activity of quinoa protein hydrolyzates at different times of hydrolysis.


ABTS expressed as ascorbic acid μg equivalents/mL of hydrolyzate; DPPH expressed as percentage of inhibition of 0.1 mL of hydrolyzate. Values followed by the same letter within each column are not significantly different, *p* > 0.05.

#### **4. Conclusions**

Results reported here show quinoa as a promising source for the production of protein concentrates and protein hydrolysates with potential antioxidant activity and the formulation of novel food ingredients with bioactive properties. Moreover, commercial enzymes were assayed from the hydrolysis of quinoa protein and the release of peptides, alcalasa and protamex being preferable for the hydrolysis of chenopodin at high rates in practically short times of reaction. Additional work needs to be performed in order to characterize the peptides responsible for the potential bioactivity.

**Acknowledgments:** This work was supported by grant Ia ValSe-Food-CYTED (119RT0567), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Secretaría de Ciencia y Técnica y Estudios Regionales (SECTER), Universidad Nacional de Jujuy, Argentina.

#### **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/).

### **Technological and Sensory Properties of Baby Purees Formulated with Andean Grains and Dried with Different Methods †**

#### **María Dolores Jiménez, Manuel Oscar Lobo and Norma Cristina Sammán \***

Faculty of Engineering- CIITED CONICET, National University of Jujuy, San Salvador de Jujuy 4600, Argentina; doloresjimenez4@gmail.com (M.D.J.); mlobo958@gmail.com@gmail.com (M.O.L.)


Published: 26 August 2020

**Abstract:** The aim of this work was to compare different cooking–drying methods to obtain dehydrated baby purees. Flours of quinoa and amaranth (native and germinated) were used to formulate them. Dry powders (DPs) were obtained by lyophilization (LD), convection (CD), and extrusion (ED). Proximal composition, particle size and morphology, water absorption capacity, and solubility were evaluated in DPs. Color, texture profile (TP), and sensory characteristics were determined in fresh pure and rehydrated powders (RPs). The LD particles were smaller and homogeneous; CD showed collapsed particles, and ED presented agglomerated particles. Different drying methods influenced the rehydration properties of DPs, as well as the color, TP, and sensory evaluation of RPs. The best method to obtain dehydrated baby purees was extrusion.

**Keywords:** amaranth; Andean potato; dehydrated powder; germination; puree; quinoa

#### **1. Introduction**

Complementary feeding begins after the first six months of life for babies. The feeding in this stage is fundamental for their physical and mental development. The incorporated foods must have semi-solid consistency and be very digestible [1].

The quinoa and amaranth Andean grains are free of gluten, have proteins of high biological value, and are rich in minerals, vitamins, fiber, and antioxidant compounds. During germination of grains, proteins, lipids, and starches are hydrolyzed, and the content of antioxidant compounds improves. The germinated grains are suitable for the formulation of baby foods due to their greater digestibility with respect to the native grains; however, the modifications that occur during germination influence the thermal, rheological, textural, and sensory characteristics of the final product [2].

The equilibrium humidity of dehydrated foods ensures their microbiological, chemical, and enzymatic stability over a prolonged period. It is important to determine the appropriate drying method for each type of food, because the variables of the process influence the nutritional, technological, and sensory characteristics of the dehydrated and rehydrated food [3,4].

The aim of this work was to formulate a dehydrated puree to reconstitute for babies that is made with quinoa and amaranth flours (germinated and non-germinated).

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

#### *2.1. Raw Material*

Quinoa (*Cica*), amaranth (*Mantegazzianus*), and Andean potato (*Collareja*) were obtained from Centro de Investigación y Desarrollo Tecnológico para la Agricultura Familiar, Hornillos, Jujuy-Argentina.

#### 2.1.1. Mashed Potato

Potatoes were washed, cooked in boiling water, peeled, and mashed.

#### 2.1.2. Non-Germinated and Germinated Grain Flours

The grains were washed, and the saponin of quinoa was removed. A part of the washed grains was soaked in tap water (6 h, at room temperature) and sprouted (22–24 °C, 80–90% RH, in darkness) 24 h quinoa and 48 h amaranth. Then, they were dried in a forced circulation oven (50 °C) and milled.

#### *2.2. Fresh Puree and Dry Powders*

#### 2.2.1. Fresh Puree (FP)

Mixture of mashed Andean potato (8.0 g), flours of quinoa (5.0 g), germinated quinoa (2.0 g), amaranth (3.0 g), and germinated amaranth (1.0 g) with dry milk (3.0 g), sugar (3.0 g), and tap water (81 mL) were cooked (30 min). Banana essence (400 μL) and citric and ascorbic acids (0.025 and 0.015 mL, respectively) were added. The cooked puree was packed in glass flasks with screwed metal caps and autoclaved (119 °C, 15 min).

#### 2.2.2. Freeze-Drying (LD)

The cooked puree was frozen at ƺ70 °C, and then freeze-dried at ƺ70 °C and pressure of 0.15 mbar during 24 h, in Heto FD4 (Heto-Holten, Denmark).

#### 2.2.3. Drying by Forced Air Circulation (CD)

It was carried out by using a convective dryer Memmert Radiant Warmer Model A52200-35\_Vac 230 (Memmert, Schwabach, Germany). The cooked puree was distributed with a thickness of 1.9 mm on a tray and dried (40 °C, 12 h). An air flow of 55 ft3/h was used.

#### 2.2.4. Extrusion (ED)

The mixture of the flours with the other ingredients were extruded, using a twin-screw extruder DT65 (Incalfer, Buenos Aires, Argentina), with a feed rate of 12 kg/h, water flow of 100 cc/min, and screw speed of 1500 rpm (die diameter 3.0 mm). Extrusion was carried out with three temperature sectors (45, 175, and 180 °C).

#### 2.2.5. Dry Powders (DPs)

They were obtained from fresh puree by freeze-drying, forced air circulation, and extrusion. The samples were milled in a centrifugal mill CHINCAN model FW 100 (CHINCAN, Hangzhou, China), vacuum-packed in polyethylene bags, and stored at room temperature.

#### *2.3. Proximal Composition*

It was determined by official techniques AOAC [5] moisture (method 925.10), ash (method 923.03), lipid (Soxhlet method), and total nitrogen (method 920.87). Nitrogen–protein conversion factors 6.25 was used. Carbohydrate was determined by difference.

#### *2.4. Particle Morphology*

Particle morphology was studied by scanning electron microscope (SEM), using SEM Zeiss Supra model 55 VP (Zeiss, Oberkochen, Germany). The powders were placed on a specimen holder, with the help of double-sided Scotch Tape and sputter-coated with gold (2 min, 2 mbar), and finally each sample was transferred to a microscope, where it was observed (15 kV, 9.75 × 10ƺ5 Torr).

#### *2.5. Rehydration Properties*

#### 2.5.1. Solubility and Water Absorption Capacity

The solubility and water absorption capacity (WAC) were studied according to Wani et al. [6]. DP was weight in a tared centrifuge tube, and water was added. It was mixed and then centrifuged. The supernatant was recovered and dried in a convection oven. The dry solid was weight, and the solubility calculated as g soluble solids/100 g product. The precipitate was weighed, and WAC was calculated as g water absorbed/g product.

#### 2.5.2. Water Adsorption

It was determined according to Tonon et al. [7]. DP was weight (2.2 g) in capsules and placed in a chamber (25 °C, 76% RH achieved with 6 molal NaCl solution). Water activity and moisture were registered. The experimental data were modeled with the BET equation (Equation (1)):

$$\text{BET (Brunauer, Emmertt, and Teller):} \quad \text{X = } \frac{\text{Xm } \text{Box}}{(1 + \text{new})[1 + (\text{C} - 1)\text{aw}]} \tag{1}$$

where Xm is the moisture of the product corresponding to a monomolecular layer of absorbed water, and C is the energy constant of the product related to the heat released in the process.

#### *2.6. Physical Characteristics*

#### 2.6.1. Color

The color of the rehydration powders (RPs) was evaluated with Color Quest XE colorimeter (Hunter Associates Laboratory, Virginia, USA) and was expressed with the L\*, a\*, and b\* parameters.

#### 2.6.2. Texture Profile Analysis (TPA)

The TPA of FP and RP was conducted by using a TA-XT Plus Texture Analysis (Stable Micro Systems, Godalming, UK). The factors determined were hardness (H), adhesiveness (A), cohesiveness (C), gumminess (G), and chewiness (C). The purees (50 g) were subjected to compressive force by probe, up to the distance of 5 mm, with a 1/4 "P/0-25 stainless-steel cylindrical probe. The conditions set were as follows: two penetration cycles, pre-test speed 0.5 mm/s; post-test speed 1.0 mm/s; depth of 16 mm; test time of 3 s; trigger force 5 g.

#### *2.7. Sensory Evaluation and Acceptability*

The sensory evaluation and acceptability of FP and RP were evaluated with 50 adults. The purees (10 g each) were presented in plastic cups. The acceptability was evaluated according to a hedonic scale of 9 points, with ends (1) "I do not like" and (9) "I like very much". Acceptability was calculated as the average score of the hedonic scale.

The sensory evaluation was studied by CATA (check-all-that-apply) questions. A list of 20 terms was presented to the consumers: pleasant, unpleasant, clear, dark, hard, soft, sandy, creamy, consistence, fluid, and flavor (smooth, intense, sweet, salty, cereal-like, acid, bitter, strange, artificial, rancid, and fruity). Consumers were instructed to tick the terms that most accurately describe the products. Frequency of mention for each term was determined by counting the number of consumers that used that term to describe each sample.

#### *2.8. Statistics Analysis*

All analyses were carried out in triplicate. Statistical analysis was done by using one-way analysis of variance (ANOVA). Tukey test was used to assess any differences between group means. Differences were considered significant at Ε < 0.05. Statistic for Windows version 9.0 (USA) was used.

#### **3. Results**

#### *3.1. Proximal Composition*

Table 1 shows the proximal composition of the mixture of ingredients to elaborate the puree and DP obtained by different drying methods. The protein and ash content did not show significant variations after drying; however, lipid content significantly decreased after cooking and dehydration.


**Table 1.** Proximal composition of fresh puree and dry powders (g/100 g db).

Values are means ± standard deviations from triplicate analysis. Different superscript letters in the same column indicated significant difference (*p* < 0.05). MP: mixture of ingredients to elaborate the purees; LD: lyophilized; CD: dehydrated by convection; ED: extruded.

#### *3.2. Particle Morphology*

Figure 1 shows the scanning electron micrographs of the DP. The LD had particles that were more homogeneous and smaller in size, while ED had larger particles with agglomerated appearance. The CD had particles with a collapsed appearance.

(**b**)

**Figure 1.** Scanning electron micrographs of dehydrated powders (1500X): (**a**) drying by forced circulation, (**b**) extruded, and (**c**) lyophilized.

#### *3.3. Rehydration Properties*

ED was the least soluble, probably due to the agglomeration of its particles [8], but it had the greatest WAC. The LD was the most soluble and had the least WAC (Table 2). The BET model had high fit with the experimental data (R2 > 0.90 and %E < 3.00) (Table 2). Xm represents the optimum moisture content in which the dehydrated product will have the maximum shelf life during storage. The ED had the lowest Xm value.


**Table 2.** Rehydration properties of the powders obtained by different drying.

Values are means ± standard deviations from triplicate analysis. Different superscript letters in the same file indicated significant difference (*p* < 0.05). LD: lyophilized; CD: dehydrated by convection; ED: extruded; Xm: moisture of the product corresponding to a saturated monomolecular layer of water; C: energy constant of the product; R2: linear correlation coefficient; %E: relative average error percentage.

#### *3.4. Physical Characteristics*

The color and texture profile of the obtained powders and rehydrated was influenced by different drying methods (Table 3). The LD was the clearest sample (highest L\*), while the powders obtained by heating (CD or ED) were darker (less L\*). Hardness of FP was similar to the RP obtained by convection, because the drying was carried out at a low temperature (50 °C), avoiding the formation of hard and dry rind on the surface [4]. The rehydrated LD had less studied textural parameters compared to the other RP and FP; while the rehydrated ED had more determined textural parameters with respect to the FP and the other RP. These results agreed with those informed by Xiao et al. [4] and Wang et al. [3], respectively.


**Table 3.** Color and texture profile of the rehydrated powders.

Values are means ± standard deviations from triplicate analysis. Different superscript letters in the same file indicated significant difference (*p* < 0.05). FP: fresh puree; LD: lyophilized; CD: dehydrated by convection; ED: extruded.

#### *3.5. Sensory Evaluation and Acceptability*

The FP was described as soft, consistent, clear, mild, pleasant, sweet, fruity, and slightly artificial taste (average acceptability of 7.4 on the hedonic scale). The PD by the different drying methods and reconstituted with water were described as follows: (i) lyophilized—soft, clear, intense flavor, bitter, strange, and unpleasant (3.7 on the hedonic scale); (ii) convection drying—soft, consistent, sandy, clear, consistent, and intense and strange taste (4.4 on the hedonic scale); and (iii) extruded—pleasant, consistent, sandy, dark, mild, fruity, and sweet (6.1 on the hedonic scale).

#### **4. Discussion**

The apparent decrease in the amount of lipids in the obtained powders was possibly due to the formation of amylose–lipid complexes during the cooking–dehydration processes [3] especially during the extrusion.

The particle size and morphology were different for each drying process, and this influenced the rehydration properties. All the DPs had pores that would facilitate hydration; however, the particles of LD showed homogenous size, which increases the exposure to water and improves the solubility. The particles of CD had contracted and collapsed appearance, which is characteristic when the water diffusion is slow and there is more time for the structures to deform. The ED presented the lowest solubility, probably due to the larger particle size and the agglomeration between them [8].

WAC was higher in this sample, possibly due to the formation of amylose–lipid complexes with water retention capacity and for the breakage of hydrogen bridges which favors hydration capacity [3]. A high WAC is a desirable feature for the preparation of soups, baby food, and instant puddings.

The RP from LP was the clearest, because during the drying by sublimation, non-enzymatic browning reactions are avoided [9]. Non-enzymatic browning reactions and sugar caramelization are promoted during high-temperature drying processes (mainly ED) [10].

The rehydrated of LD had the lowest values of hardness, adhesiveness, gumminess, and chewiness with respect to the other RD and the FP. This result coincides with that reported by [9], who explained that the ice crystals formed during freezing could break the cell structure of the sample and produce softer textures. The rehydrated ED was harder, with more adhesiveness, gumminess, and chewiness than FP and the other RP. This result could be due to the formation of amylose–lipid complexes. Moreover this result could be due to the formation of a hard crust on the particle surface by high temperatures, the rapid evaporation of water, and the high pressure generated between the particles during the process.

The rehydrated purees from LD and CD were described with negative sensory attributes (intense, bitter, and strange taste). In addition, the puree reconstituted from LD was described as being more fluid. Consumers differentiated the extruded sample by considering it darker, sweeter, and fruity, with more consistency and harder than the other samples. Therefore, the results of the evaluation of the sensory attributes by CATA method reflected the results obtained through instrumental measurements. Valentina et al. (2016) observed that, after lyophilization of some foods, some negative sensory attributes are highlighted with reduction in the acceptability of these products after lyophilization. Jafari et al. [10] observed improvement in the acceptability of bread composed of extruded sorghum-wheat, compared to that made with native sorghum-wheat. These authors explained that extrusion promotes Maillard reactions and caramelization, and therefore a darker and sweeter product is obtained.

#### **5. Conclusions**

The different drying methods influenced the technological and sensory features of the dehydrated and reconstituted powders.

Consumers were able to differentiate the samples obtained by different drying methods and attributed sensory terms to them that agreed with the instrumental determinations (such as color and texture) made in the samples.

The extruded powder had a better water retention capacity, with appropriate texture and sensory description. Therefore, the dehydrated powder that showed the best characteristics to produce a baby instant puree to reconstitute was obtained by extrusion.

**Acknowledgments:** This work was supported by grant IaValSe-Food-CYTED (Ref. 119RT0567), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Secretaría de Ciencia y Técnica y Estudios Regionales (SECTER), Universidad Nacional de Jujuy (Argentina).

#### **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/).

### **Design and Acceptability of a Multi-Ingredients Snack Bar Employing Regional PRODUCTS with High Nutritional Value †**

#### **Francisco Teodoro Rios, Argentina Angelica Amaya, Manuel Oscar Lobo and Norma Cristina Samman \***

Centro de Investigaciones Interdisciplinarias en Tecnologías y Desarrollo Social para el NOA-(CIITED), CONICET-Facultad de Ingeniería, Universidad Nacional de Jujuy, Ítalo Palanca 10.4600, San Salvador de Jujuy, Jujuy, Argentina; frios8078@gmail.com (F.T.R.); argentinaamaya12@gmail.com (A.A.A.); mlobo958email2@gmail.com (M.O.L.)


Published: 26 August 2020

**Abstract:** The aim was to develop a snack bar using regional food products. The formulation included traditional cereals and amaranth, quinoa, sunflower, flax, chia, sesame and poppy seeds subjected to different treatments. Two sensory evaluations were carried out to evaluate acceptability. Snack bars containing toasted seeds presented high acceptability by the consumer. Amaranth, quinoa, chia and sunflower significantly increased the acceptability. The sensory methods applied allowed for the selection of ingredients and processing technologies that increase the preference of consumers.

**Keywords:** amaranth; chia; quinoa; snack bar; sensory analysis

#### **1. Introduction**

The need to have a greater number of nutritious and healthy foods leads to the development of new products, with the incorporation of ingredients or active compounds that have beneficial effects for health [1]. A balanced and healthy diet depends on many factors, among which the quality and composition of raw materials that are used in the formulation of foods stand out. Snack bars are products that allow for the incorporation of multiple ingredients that, when properly selected, can increase the nutritional and functional value of the product. Andean grains such as quinoa (*Chenopodium quinoa*) and amaranth (*Amaranthus caudatus*), recognized for the nutritional quality of their proteins and their contribution in essential fatty acids, minerals and dietary fiber, are being reintroduced in the Andean region of the Argentine Northwest [2]. On the other hand, seeds such as chia (*Salvia hispanica* L*.*) and sesame (*Sesamum indicum*) are ingredients that can be used in the development of snack bars. Nutritionally, the seeds stand out in general due to the high content of lipids, proteins and fibers. The lipids of chia seeds have a high content of polyunsaturated fatty acids (PUFAs), particularly omega-3 (linolenic acid) and omega-6 (linoleic acid) [3]. Therefore, they could be adequate to supplement cereals nutritionally in the formulation of snack bars. In addition to their nutritional properties, seeds provide functional components such as polyphenols and flavonoids [4]. Therefore, the seeds have significant advantages over other ingredients used in the manufacturing of bars, such as oats and rice. However, it is necessary to prepare these raw materials prior to their use, which can induce negative effects in their nutritional and functional compounds and, consequently, in the final product. An important aspect to take into account in the design and development of products is to consider the preference and acceptance of the type and quantity of any ingredient by consumers.

This work proposes to develop a snack bar with high consumer acceptability by employing nutritious regional food products rich in functional compounds.

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

#### *2.1. Materials*

Quinoa (*Chenopodium quinoa* Willd. var. Inta Hornillos) and amaranth (*Amaranthus caudatus* var. rosado) were provided by the Instituto de Investigacion y Desarrollo Tecnologico para la Agricultura Familiar (IPAF) (Jujuy-Argentina). The seeds of chia (*Salvia hispanica* L.), sesame (*Sesamum indicum*), flax (*Linum usitatissimum*), sunflower (*Helianthus annuus*) and poppy (*Papaver somniferum*) were purchased from Melar S.A. (Argentina). Puffed rice and oats (rolled) were purchased at the local market (Jujuy). In the preparation of the binder, honey obtained from producers of the region of the Yungas (Jujuy) and commercial sucrose were used. The amounts and ratio of honey/sucrose remained constant in the preparation of the bars.

#### *2.2. Methods*

The nutritional compositions of quinoa and amaranth (proteins, moisture, lipids, fiber and ash) were determined by official AOAC methods [5].

#### *2.3. Design and Elaboration of the Snack Bars*

For the elaboration of the bar, the seeds and cereals were added to the union syrup and continuously mixed until obtaining a homogeneous composition. Then, they were placed in stainless steel molds, pressed for 10 min and left to stand (1 h) at room temperature. The product was packaged and stored at room temperature up to sensory analysis [6].

The design and development of the product was carried out through a sensory analysis with 160 consumers in two stages. Consumers were recruited among students, teachers and administrative staff of the Faculty of Engineering, with an age range between 18 and 50 years, of both genders. The evaluation was carried out in the sensory analysis laboratory of the Engineering Faculty—UNJu. Each consumer was given the total samples of each experimental design in 20 g portions of each, coded by three random digits. Next to the trays, a form was provided describing the objective of the study and the instructions to carry out the analysis as well as drinking water for mouth wash between samples.

#### *2.4. Sensory Analysis*

#### 2.4.1. Sensory Analysis 1

In the first place, the acceptability of the samples (seeds to be used as ingredients in the snack bar) was studied, to which different processes were applied depending on the temperature (T) and time (t). The applied processes were toasting (190 °C × 3 min), boiling (boiling: t1 = 15 min and t2 = 25 min), dry heating (T = 80 °C, t1 = 45 and t2 = 60 min) and baking (T = 130 °C, t = 30 and 45 min). In the first trial, a total of seven samples were analyzed (3 processes two times = 6 + 1 processes: roasting). The evaluation of acceptability was made with 75 consumers using a hedonic scale of 9 points with the following extremes: I dislike it a lot = 1 and I like it a lot = 9. In addition to the hedonic test, a survey was carried out with open questions to generate the descriptors of the samples. The attributes most frequently used (>20%) were selected as descriptive attributes of the samples.

#### 2.4.2. Sensory Analysis 2

Once the processes of adaptation of the raw materials were defined, a second sensory analysis was carried out with 85 consumers. The acceptability of the samples was evaluated in addition to a check-all-that-apply test that included 16 attributes grouped into appearance, taste, aroma and texture. The second sensory analysis was carried out with the objective of selecting the ingredients that produce the highest preference of the samples following a Taguchi L8 (27) design. According to the design, eight trials were carried out at two levels: absence and presence of each seed, which corresponds to the seven variables: quinoa, amaranth, chia, sesame, sunflower, flax and poppy.

#### *2.5. Statistical Analysis*

Samples from both trials were evaluated using the ANOVA, considering the processes and ingredients as variation factors. A principal component analysis (PCA) was applied to the descriptive data of each sensory analysis for its correlation with the acceptability of the samples.

#### **3. Results**

#### *3.1. Sensory Analysis. Process Selection*

Table 1 shows the multi-ingredient snack bar acceptability for a total of 75 consumers. Seeds and Andean grains were cooked by different processing conditions (n = 7) to study their incorporation into the bar. The ANOVA applied to the acceptability data determined significant differences (*p* < 0.05). The samples To and Ct2 presented the highest acceptability in opposition to Dt2. Figure 1 shows the PCA of the attributes and the relationship with the acceptability of the samples, which was used as a supplementary variable. The first two factors (F) explain 61.09% of the total variability of the data. The positive factor F1 indicates that the attributes that characterize the samples Ct1 and Dt1 were related to "good appearance*",* "bright" and "caramel color". However, the increase in acceptability was correlated to a negative F1, which is associated with samples To and Ct1, highlighting the attributes of "toasted", "crumbly in the mouth" and "very sweet". On the other hand, along the negative axis of F2, opposite to To, the samples of lower acceptance were observed: Bt1, Bt2 and Dt2. The B samples were strongly associated with the attributes "rancid taste" and "rancid smell"; for this reason, they would be rejected. In the case of D samples, although they were associated with "bright" and "good appearance", they would be rejected for "hard" and "sticky" attributes. Therefore, the processes that produced greater acceptance corresponded to the roasting of raw materials, followed by cooking.

**Figure 1.** Representation of F1–F2 factors of the principal component analysis applied to the data of the first sensorial analysis—process selection. (**a**) Variables: sensory attributes (NJ), supplementary variable: acceptability (ȣ). (**b**) Samples (Ƞ), supplementary variable: acceptability (ȣ).


**Table 1.** Conditions and response of acceptability of the different preparation processes.

Different letters in the same column indicate statistically significant differences, *p* < 0.05.

#### *3.2. Sensorial Analysis. Ingredient Selection*

Table 2 shows the ANOVA applied to the samples that presented a combination of the relative content of the different ingredients for their selection and incorporation into the final product. The samples with higher proportions of amaranth, quinoa, sunflower and chia presented the highest preference. In contrast, the samples in which flax and poppy predominated (b6 and b7) were significantly (*p* < 0.05) the least accepted. In Figure 2, the PCA shows the relationship between the attributes and the acceptability of the different formulations. The first two factors explained 68.94% of the total variability. The positive F1 factor indicated a weak correlation with the increase in the acceptability of the samples. According to the F1 axis, a separation of the samples into two groups was observed.

The samples placed in the F1 positive sector were those of greater acceptance and were associated mainly with the attributes "good appearance" and "sweet taste". In the case of the negative F2 axis, there was a negative correlation (r > 0.70) between the acceptability and the taste and smell attributes due to rancidity. The attribute "too rancid" was perceived mainly in seeds with high lipid content, such as flax, poppy, chia and sunflower. In addition, the "heterogeneous aspect" provided by flax and poppy, related to their color and, negatively influenced the acceptability of the samples. Therefore, due to the attributes provided by the flax and poppy seeds, their incorporation into the formulation of the bars was discarded.

**Figure 2.** Representation of F1–F2 factors of the principal component analysis applied to the data of the second sensorial analysis—ingredient selection. (**a**) Variables: sensory attributes (NJ), supplementary variable: acceptability (ȣ). (**b**) Samples (Ƞ), supplementary variable: acceptability (ȣ).


**Table 2.** Variables and experimental design response to the selection of ingredients.

\* Different letters in the same column indicate statistically significant differences, *p* < 0.05.

#### *3.3. Nutritional Analysis*

Table 3 shows the nutritional composition of the raw materials and selected snack bar. The chemical composition of the seeds showed a high content of lipids, followed by proteins. On the other hand, rice and oats have a high content of carbohydrates (CH). Therefore, the bar could be correctly complemented with seeds, cereals and Andean crops in order to elaborate a product of high nutritional value and greater acceptability. In the nutritional composition of the final product, lipids and proteins were in the range of FAO's nutrient contribution recommendations. Additionally, the use of seeds in an integral way was responsible for the high content of dietary fiber.

**Table 3.** Proximal composition of the selected ingredients and the final product.


\* Calculated by difference.

#### **4. Discussion**

According to the sensory study, the most important attributes for the preferences of snack bars were the appearance, "bright" and "good appearance", followed by the taste, principally "sweet" and "toasted". The "sweet taste" attribute is one of the most important and representative of snack bars. "Sweet taste" and its intensity can mask other ingredients, like antioxidants, which usually are rejected by consumers despite the fact that their consumption may be beneficial for health [6]. However, it should be mentioned that the increase in "sweet taste" implies the addition of simple sugars, which is negative from the point of view of health recommendations.

The "rancid taste" and "rancid smell" attributes were identified as rejected attributes for different products. The attribute "rancid" is characteristic of the oxidation of the lipids of the seeds due to processing at high temperatures for long times [7]. In this sense, roasting was the better process to use due to the short time involved [8]. Despite the fact that the seed content improves the nutritional composition of the product, both the processes and the consumers did not accept a high content of the seeds in the final product. Furthermore [9] determined that the health information did not determine the selection of a snack bar. Therefore, the design of a product with high nutritional and functional value does not only depend on the health benefits but also on aspects that condition the preference of the consumers.

#### **5. Conclusions**

The sensorial methods applied allowed for the selection of ingredients and processing technologies that increase the preference of consumers, identifying attributes of acceptability and rejection.

In general, the acceptance of snack bars depended mainly on attributes such as "sweetness" and "good appearance", and rejection occurred in formulations containing ingredients with high lipid content that were treated at high temperatures for a long time.

**Acknowledgments:** This work was supported by grant Ia ValSe-Food-CYTED (Ref. 119RT0567) and Consejo Nacional de Investigaciones Científicas y Técnicas CONICET and Secretaria de Ciencia y Técnica y Estudios Regionales SECTER, Universidad Nacional de Jujuy (Argentina).

#### **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*

### **Development of Gluten-Free Breads Using Andean Native Grains Quinoa, Kañiwa, Kiwicha and Tarwi †**

#### **Ritva Repo-Carrasco-Valencia 1,\*, Julio Vidaurre-Ruiz 1 and Genny Isabel Luna-Mercado 2**


Published: 26 August 2020

**Abstract:** The aim of this study was to develop gluten-free breads using the flours of Andean native grains. The following native grains were used: quinoa (*Chenopodium quinoa*) Pasankalla variety, kiwicha (*Amaranthus caudatus*) Centenario variety, kañiwa (*Cheopodium pallidicaule*) Illpa Inia variety and tarwi (*Lupinus mutabilis*) Blanco de Yunguyo variety. The formulations of the breads with Andean grains flours were optimized using the Mixture Design and the Central Composite Rotational Design, analyzing the dough's textural properties (firmness, consistency, cohesiveness and viscosity index), specific volume and crumb texture. Potato starch and xanthan gum were used in the preparation of the breads. The optimized formulations of the gluten-free breads with Andean grain flours were composed of quinoa (46.3%), kiwicha (40.6%), kañiwa (100%) and tarwi (12%) flours. The gluten-free breads developed showed acceptable specific volume and low crumb firmness and could help to improve the nutrition of celiac patients.

**Keywords:** quinoa; kiwicha; kañiwa; tarwi; gluten-free bread

#### **1. Introduction**

The gluten-free products market has increased throughout Latin America, especially in Peru, where there has been an increasing demand for gluten-free bread products in supermarkets, due to the increase in patients diagnosed with celiac disease and consumers seeking "healthier" alternatives. However, these are not necessary healthier, because the gluten-free bakery products on the market are made from starches or white rice flour, which are lacking in high quality proteins and important micronutrients.

Andean grains such as quinoa, kiwicha, kañiwa and tarwi do not contain peptides similar to wheat gluten; therefore, they are raw materials appropriate for consumption by celiacs. Likewise, these grains are sources of starches (more than 70% of their composition) [1–3], which are necessary to create bread structure [4]. On the other hand, tarwi is a legume also known as the Andean soybean because of its high protein and oil content (almost 50% and around 20%, respectively). The oil of tarwi could function as a natural emulsifier to retain the gas produced during the fermentation of glutenfree breads [5]. Quinoa is appreciated because of its high protein quality, having a balanced essential amino acid composition, and it is also considered a source of fiber and minerals [6,7]. Kiwicha is a very good source of iron, calcium and zinc. It contains more zinc and iron than conventional maize and beans [8]. Kañiwa, the least studied Andean grain, grows mainly in Peru and Bolivia, between 3500 and 4200 m above sea level where the climatic conditions are extreme [9]. Its small grains contain more protein than the common cereals; it has a good content of essential amino acids; it is rich in lysine, the first limiting amino acid in all cereals; it is rich in unsaturated fatty acids; and it is an excellent source of dietary fiber and an important source of minerals, especially iron, calcium, phosphorus and vitamins such as riboflavin [10].

The inclusion of these grains in the formulations of gluten-free breads is promising. Therefore, the aim of the research was to develop gluten-free breads with quinoa, kiwicha and kañiwa flours, and a bread with the mixture of quinoa and tarwi, using the response surface methodology, in order to find the proportions of the ingredients that can produce bread with acceptable quality.

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

#### *2.1. Conditioning and Characterization of Raw Materials*

Quinoa (*Chenopodium quinoa*) Pasankalla variety and kiwicha (*Amaranthus caudatus*) Centenario variety were supplied by the Cereals and Native Grains Program at the National Agrarian University La Molina, kañiwa (*Cheopodium pallidicaule*) Illpa Inia variety was supplied by ILLPA Puno Peru Agricultural Experimental Station, and tarwi (*Lupinus mutabilis*) Blanco de Yunguyo variety was bought from the local market of Cajamarca-Peru. Tarwi grains were conditioned, in order to eliminate the alkaloids, according to the procedure described by Jacobsen & Mujica [11]. All the grains were milled using a hammer mill (Retsch SR 300, Haan, Germany), and the proximal composition of the flours was determined following the procedures of the AOAC (2000) [12].

#### *2.2. Experimental Design*

The mixture design was used to optimize the textural properties (firmness, consistency, cohesiveness and viscosity index) of the gluten-free doughs with quinoa and kiwicha flours. For the inclusion of tarwi in the gluten-free bread formulation, the Central Composite Rotational Design (CCRD) was used; under the same principle of optimizing the dough textural properties and for the case of kañiwa bread, the CCRD was used to optimize the bread volume and backing loss.

The variables for the mixture design were the proportions of water (70–110%), xanthan gum (0.5–2%) and quinoa or kiwicha flour (10–50%). In the case of CCRD, the variables were the proportions of water (75–160%) and tarwi flour (10–30%).

In the case of the gluten-free breads with quinoa, kiwicha and tarwi, the target was to find the optimal levels of the variables that could imitate the textural properties of a dough control (3.69 ± 0.2 N of firmness; 56.5 ± 3.7 N.s of consistency; 2.7 ± 0.2 N of cohesiveness and 36.2 ± 2.1 N.s of viscosity index), which was previously developed and showed good quality properties [13].

In the case of the gluten-free bread with kañiwa, the variables were the proportions of water (75– 125%), xanthan gum (0.35–0.65%) and kañiwa flour (40%). The optimization criteria were the maximization of the specific volume of the bread and the minimization of the baking loss.

#### *2.3. Dough and Bread Preparation*

For the mixture designs (doughs with quinoa and kiwicha flours), 16 formulations were used for each experiment with different proportions of water, xanthan gum, and quinoa or kiwicha flour; the other ingredients for the dough preparation were potato starch, sugar (3%), salt (2%), soybean oil (6%) and yeast (3%). For the CCRD (doughs with tarwi flour), 13 formulations with different proportions of water and tarwi flour were used; these were mixed with potato starch, quinoa flour (46%), sugar (3%), xanthan gum (0.5%), salt (2%), soybean oil (6%) and yeast (3%). For bread with kañiwa flour, the same levels of sugar, salt, soybean oil and yeast were used.

All ingredients were mixed at two different speeds for 3 min in total and used to fill aluminum molds (300 g); then, they were fermented for 30 min at 30 °C and 85–90% RH, and finally, they were baked at 200 °C for 60 min.

#### *2.4. Dough Textural Properties*

The texture analysis of the doughs (without yeast) was carried out using the Back Extrusion accessory of the INSTRON universal texturometer (Model 3365, Canton, MA, USA), where a portion of dough was deposited in the Back Extrusion cylinder (diameter, 50 mm; height, 70 mm) and penetrated up to 50% with a plunger (diameter, 42 mm) at a speed of 1 mm/s and with a trigger force of 10 gf; finally, the plunger returned to its original position at the same speed. The textural properties determined were the firmness (N), consistency (N.s), cohesiveness (N) and viscosity index (N.s).

#### *2.5. Specific Volume*

The bread volume (mL) was measured by laser topography (BVM-6610, Perten Instruments, Hägersten, Sweden), and the specific bread volume (mL/g) was calculated by dividing the volume by the bread weight.

#### *2.6. Textural Properties of Bread*

The texture profile analysis (TPA) was carried out on breads 24 h after baking using an Instron Universal Testing Machine (Model 3365, Instron Co., Canton, MA, USA), according to the procedure described by Vidaurre-Ruiz et al. (2019).

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

The proximal composition of the quinoa, kiwicha, kañiwa and tarwi flours was 8.0–11.0% of moisture, 14.0–53.0% of protein, 6.0–22.0% of fat, 2.0–3.0% of ash, 2.5–9.0% of fiber and 12.8–69.5% of carbohydrates, respectively.

The gluten-free dough optimized with quinoa flour that was able to imitate the textural properties of the dough control was composed of quinoa flour (46.3%), potato starch (53.7%), xanthan gum (0.5%) and water (75.2%). In the case of the dough with kiwicha flour, the optimized formulation consisted of kiwicha flour (40.6%), potato starch (59.4%), xanthan gum (0.5%) and water (80.9%). The dough optimized with tarwi flour was composed of tarwi flour (12%), quinoa flour (46), potato starch (42%) and water (102%). The optimized formulation of the gluten-free bread with kañiwa flour was kañiwa flour (100%), xanthana gum (0.9%) and water (140%). The rest of the ingredients remained constant, according to the type of dough, as explained above.

The quality properties of the optimized breads, such as the specific volume and crumb texture, are shown in Table 1, where it can be seen that the gluten-free breads with Andean grains had acceptable specific volumes, as well as soft crumbs (Figure 1). According to Alvarez-Jubete et al. [14], the components of Andean grains such as the fats and starches, which include low levels of amylose, significantly help to improve the quality of gluten-free breads, producing breads with soft crumbs and with a lesser tendency to retrograde, therefore increasing the shelf life of the products.

The formulation optimized with 100% kañiwa flour demonstrates that the starches of this grain are propitious for baking and that when they are mixed with the appropriate levels of water and xanthan gum, they can produce breads of good physical and nutritional quality. Likewise, tarwi flour can be included in a smaller proportion (12%) due to its high content of proteins, which have a great capacity to absorb water [5].

The inclusion of xanthan gum was minimal (0.5–0.9%) in doughs with Andean grains; this shows that the ingredients of the grains can function as natural emulsifiers; however, the use of gums is still necessary to achieve the stability of the emulsion during baking.


**Table 1.** Characteristics of gluten-free breads (GFB) made with Andean grains.

**Figure 1.** Representative images of gluten-free breads (GFB) with quinoa, kiwicha, kañiwa and tarwi.

#### **4. Conclusions**

It was possible to develop gluten-free breads with quinoa (46.3%), kiwicha (40.6%), kañiwa (100%) and tarwi (12%) flours. The components of the Andean grains such as the lipids and starches help to improve the quality properties of gluten-free breads, producing soft crumbs and breads with acceptable specific volumes. The inclusion of xanthan gum in the gluten-free doughs with Andean grain flours was minimal, but its use is still necessary to achieve the stability of the emulsion during the baking process. The gluten-free breads developed contained a good amount of Andean grain flours and could help improve the nutrition of celiac patients.

**Funding:** This work was supported by the grant Ia ValSe-Food-CYTED (Ref. 119RT0567) and PROTEIN2FOOD Project (European Union's Horizon 2020, N° 635727).

#### **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* 

### **Nutritional Composition and Uses of Chia (***Salvia hispanica***) in Guatemala †**

#### **Elsa Julieta Salazar de Ariza \*, Ana Ruth Belloso Archila, Ingrid Odete Sanabria Solchaga and Sandra Beatriz Morales Pérez**

Department of Chemical and Pharmaceutical, School of Nutrition, Universidad de San Carlos de Guatemala, Cdad. de Guatemala 01012, Guatemala; anabelloso@gmail.com (A.R.B.A.); odette\_2865@hotmail.com (I.O.S.S.); sanbmor@gmail.com (S.B.M.P.)


Published: 10 September 2020

**Abstract:** *Salvia hispanica* L. (chia) is a seed native to Mexico and Central America; in Guatemala it is known as "chan". It is cultivated in small quantities and sold in neighborhood markets in different areas of the country. Little information exists on the nutritional composition of this seed, so chia samples were obtained in five regions of the country and studied for their macronutrients, minerals, and fatty acids, the form of consumption, and the adequate quantity to mix with water. We found an average of 22% for protein, 18.6% for fat, 67% for alpha-linoleic acid, 19% for raw fiber, and 9 mg/100 g of sodium, among other nutrients. The most frequent form of consumption is mixed with lemonade; the primary known benefits are to lose weight, improve digestion, and as a source of fiber; the adequate amount to mix with water is 0.8% of gel equivalent to 0.4% of seeds.

**Keywords:** alfa-linoleic acid; chia; consumers; *Salvia hispanica* L.

#### **1. Introduction**

*Salvia hispanica* L. is a seed native to Mexico and Central America; it has been known and cultivated in Guatemala since pre-Hispanic time [1,2], its common name is "chia", and traditionally, it has been sold in bulk in neighborhood markets, even though recently it has also been sold packaged and branded in supermarkets and gourmet stores. It is also found as an ingredient in processed food, which allows it to be advertised as a functional food. The chia seed has a high content of ΅-linoleic acid, protein, and dietary fiber, propertied which make it recommended for consumption as flour, as oil, or as a hydrolyzed protein [3].

Alvarado [4] quantified the macronutrients and fatty acids of the chia seed cultivated in the northern region of Guatemala, finding amounts of these nutrients similar to those reported by Bushway et al. [5], Ayerza and Coates [6] and Ixtaina [7].

The objective of the present study was to quantify the macronutrients, minerals, and fatty acids of *S. hispanica* sold in bulk in Guatemala, as well as to determine the form of consumption, the consumer benefits known by consumers, and the concentration of the gel that they consider suitable when mixed with water.

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

#### *2.1. Sample Collection*

The chia seeds were obtained in bulk in the markets of Cobán, Alta Verapaz (northern region), Atiquizaya, El Salvador (southern region), Jocotán, Chiquimula (eastern region), San Juan Ostuncalco (western region), Chimaltenango and San Agustín Acasaguastlán, El Progreso (central region).

#### *2.2. Macronutrient, Minerals and Fatty Acids Quantification*

Proximal analysis was used to determine the macronutrients. The humidity was determined in a convection oven at 60 °C, until it was a constant weight; the raw protein by the Kjeldhal method using Kjeltec Auto 1030 Analyzer equipment; the raw fiber by acid and alkaline digestion using Fibertec System I equipment; the fat by petroleum benzene extraction using Goldfish equipment, carbohydrates by difference, and energy by the Atwater factor. The minerals were determined by atomic absorption spectroscopy, using Perkin Elmer AAnalyst 100 equipment, except phosphorus, which was determined using a UV/VIS Lambda 11 colorimeter. The fatty acids were quantified using a GCMS-QP2020 gas chromatograph, with a 30-m-long DB-5MS column, an internal diameter of 0.25 mm, and a stationary phase thickness of 0.2 micrometers. The column temperature in the oven was 100.0 °C.

#### *2.3. Preparation of Chia Gel Solutions*

The gel was prepared by hydrating the chia seeds in water, at a 5:95 proportion, letting them stand for 24 h, and then separating the water and the gel by simple filtration. With the gel obtained, solutions were prepared at 0.4, 0.6, 0.8, and 1.6% in water, which were identified by three-digit codes, obtained from a random number table.

#### *2.4. Sensory Study*

We chose 50 young adults, students, and workers from the Universidad de San Carlos de Guatemala, who self-identified as chia consumers. In an appropriate room, they were presented with an ounce of each solution of gel in water, they were invited to taste each one and to qualify them as "lacking", "sufficient", or "a lot", which were respectively recorded as 1, 2, and 3; afterwards, they were asked two open questions: in what form have they consumed chia and what health benefits does it have. They were chia consumers.

#### *2.5. Statistical Analysis*

Macronutrient, mineral, and fatty acid content data were analyzed with descriptive statistics; the information about forms of consumption was analyzed by means of frequencies, and variance analysis was applied to the rating given to the chia gel solutions, through the Microsoft Excel 2010 statistical analysis.

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

#### *3.1. Macronutrients, Minerals, and Fatty Acidsin S. hispanica L.*

Table 1 shows the average content and standard deviation of macronutrients, minerals, and fatty acids of the chia samples collected from neighborhood markets from five regions of the country.

The chia is confirmed as a seed with high protein content, similar to the protein content of the bean (*Phaseolus vulgaris*), 20 to 22%, but with a digestibility of 29%, which is considerably lower than that of the bean, which is 79% [3,8]. In this study, 3% more protein was found than that reported by Alvarado [4], which is an acceptable variation, since values have been reported in the 18 to 24 g/100 g range [3,9].

The high content of alpha-linoleic acid in chia was also confirmed, representing 66% of total fatty acid of that oil seed, a similar concentration to that reported by Alvarado [4] and by Ixtaina [7].

#### *Proceedings* **2020**, *53*, 16

As for fat, it is notable that a much lower value was found than the range reported by the aforementioned authors, which is between 29 and 34 g/100 g.

The mineral content is similar to the values found in the United States Department of Agriculture database [10], which highlights its low sodium content and its high content of calcium, potassium, and magnesium.


**Table 1.** Macronutrient, energy, mineral, and fatty acid content in *S. hispanica* L.

#### *3.2. Forms of Consumption and Benefits of Consumption of S. hispanica*

Table 2 shows the forms of chia consumption in lemonade and water. It is important to observe that all the forms of consumption refer to the raw, whole seed, which has a digestibility of 29%, which means that only 6.38% of the protein and 19% of the alpha-linoleic acid are used. This indicates the need to promote the production of chia flour and its use in different preparations, through which up to 80% digestibility can be reached [3]. Despite the aforementioned, consumption of raw chia seeds is still important due to their raw fiber and dietary fiber content, made of xylose, glucose, and glucoronic acid, which increases the feeling of satiety and thereby decreases energy consumption, and decreases obesity, cardiovascular diseases, and type 2 diabetes risk factors [9,11].

The following benefits were mentioned regarding the consumption of chia seeds: it helps weight loss, improves digestion as a fiber source, prevents constipation as a source of omega-3 fatty acids, controls blood lipids and glycaemia. This indicates that consumers have correct information on the benefits of whole raw chia seed consumption, with the exception of the benefit of being an omega-3 fatty acid source. On the other hand, although it was requested that they mention only the benefits of consumption, three of the 50 consumers spontaneously mentioned that there is a risk of appendicitis, due the possibility of seeds accumulation on it.

#### *3.3. Adequate Quantity to Add to Water*

Table 3 shows the rating given by the consumers about the solutions of water and chia seed gel. The consumers' opinion did not show significant differences (*p* ǃ 0.05) for concentrations between 0.4 and 0.6%, indicating that they are lacking; the opinions were significantly different (*p* ǂ 0.05) when rating solution with 0.8 and 1.6%, indicating that they were sufficient and a lot, respectively.

**Table 2.** Form of consumption and benefits mentioned by consumers of *S. hispanica* L. in Guatemala.


**Table 3.** Percentage of *S. hispanica* gel adequate for a drink according to consumers' tastes.


Young Guatemalan adults, as residents of a tropical country, are in the habit of consuming water frequently throughout the day, so they carry a container of about a liter of water with them, to which they could add chia seeds. Taking into account that the chia seed doubles its volume due to gel formation when put in contact with water and left to stand for 2 h, 4 g of chia seeds could be added to a liter of water and consumed over the course of the day, which would allow the ingestion of 0.76 g of raw fiber and 1.26 g of dietary fiber, as well as 0.25 g of usable protein and 0.15 g of usable alpha-linoleic acid. The quantities are relatively small in relation to the recommended daily intake of nutrients, hence the need to promote chia consumption in the form of flour and through other

#### **4. Conclusions**

preparations, in order to increase daily intake.

Chia (*Salvia hispanica* L.) has 22% protein, 18% fat, 31% carbohydrates, 19% raw fiber, and 67% alpha-linoleic acid, as well as 9 mg/100 g of sodium, 512 mg/100 g of calcium, 722 mg/100 g of potassium and 358 mg/100 g of magnesium. In Guatemala, it is consumed as a raw seed mixed with lemonade and the benefits it provides as a source of dietary fiber are recognized. The consumer considers it adequate to use 0.8% gel mixed with water, which is equivalent to 4 g of chia seeds.

**Funding:** This work was supported by grant IaValSe-Food-CYTED (119RT0567) and Universidad de San Carlos de Guatemala.

#### **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* **Chia (***Salvia hispanica***): Nutraceutical Properties and Therapeutic Applications †**

#### **Talía Hernández-Pérez 1, María Elena Valverde 1, Domancar Orona-Tamayo 2 and Octavio Paredes-Lopez 1,\***


Published: 1 September 2020

**Abstract:** Chia seeds (*Salvia hispanica* L.) have high amounts of nutraceutical compounds and a great commercial potential. The aim of this work was to identify proximate composition, fatty acids profile, total phenolics and antioxidant capacity of chia, as well the protein fractions and determine their antihypertensive potential. The seeds exhibited high content of protein, fiber, and lipids, mainly polyunsaturated fatty acids. Important amounts of phenols and a high antioxidant activity (DPPH and ABTS) were found. Globulins fraction showed the most abundant concentration followed by albumins. Peptides from albumins and globulins exhibited the strongest potential against the angiotensin-converting enzyme (ACE) activity. In brief, this study demonstrates that chia can be considered a seed with high nutritional content, antioxidant activity and as a novel antihypertensive agent; important factors for the frequent incorporation of chia in the human diet.

**Keywords:** antioxidants; antihipertensive; chia; phenolics; nutraceutical; *Salvia hispanica*

#### **1. Introduction**

Salvia genus has around 900 species and belongs to the Plantae Kingdom and Lamiaceae family. Chia (*Salvia hispanica* L.) is an annual herb that can grow up to 1 m tall and has oppositely arranged leaves with small white or purple flowers, and oval seeds showing black, gray, and black spotted to white color (Figure 1) [1,2].

Chia is native to central Mexico up to northern Guatemala and began to be used as food in 3500 BC. Chia was a main crop of pre-Columbian societies; Aztecs, Mayas, and Incas (1500–900 BC) used it as medicine, food, painting, and as energizer. Aztecs received it as annual tributes, and as offering to gods. The Spanish conquest suppressed its use due to religious beliefs. In 2009, chia was approved as novel food by the European Parliament and the European Council. Chia seeds are good source of macronutrients, B vitamins, calcium, phosphorus, potassium, magnesium, iron, zinc, and copper, as well as phenolic compounds. Additionally, it can be incorporated to celiac diets due to the absence of gluten [2,3]. Nowadays, chia has shown several health benefits, i.e., antioxidant potential and antihypertensive, between others [1,4]. Thus, the aim of this work was to determine the proximate composition, fatty acid profile, total phenolic content and antioxidant capacity of chia, as well as its protein fractions in terms of their antihypertensive potential.

**Figure 1.** Chia plants, flowers and seeds. (**a**) Purple and white flowers, (**b**) Seeds from different commercial lines cultivated in Mexico.

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

Seeds from four varieties of commercial chia (*Salvia hispanica*): Black from Puebla, White and Pinta Jalisco (PJ) from Jalisco, and Xonotli from Guanajuato, Mexico, were soaked in distilled water (1:10, *w*/*v*) for 1 h to allow mucilage production. They were frozen overnight (ƺ80 °C) and freezedried, the dry mucilage was removed mechanically. Mucilage-free seeds were milled into flour and passed through a 0.5 mm mesh to obtain a uniform particle size. The flour was defatted with hexane (1:10, *w*/*v*) in a Soxhlet unit at 65ƺ70 °C and dried overnight at room temperature to remove remaining hexane; a second grinding was performed to obtain a smaller particle size (0.18 mm), and it was stored at 4 °C until use. Proximate composition was evaluated based on Sandoval-Oliveros and Paredes-López [5] method. Fatty acid profile was made using Guzmán-Maldonado et al. [6] procedure. Total phenolic compounds and antioxidant capacity were performed according to Martínez-Cruz and Paredes-López [3]. Antihypertensive potential was determined using the method reported by Orona-Tamayo et al. [7].

#### **3. Results**

#### *3.1. Proximate Composition*

The different types of chia analyzed presented an average of 22.7, 32.5 and 33.6 g/100 g dry basis of protein, lipids and dietary fiber, respectively (Table 1). As can be observed, these results agree with those previously reported by Ayerza and Coates [1]. Soluble and insoluble fiber fractions found in chia samples may improve glucose and lipidic profile, and intestinal function, thus reducing the risk for obesity, coronary heart disease, type II diabetes mellitus, metabolic syndrome and several types of cancer. It also is associated to increase post-meal satiety, decreasing subsequent hunger [6]. Chia fiber content (35–40 g/100 g) is equivalent to 100% of the daily recommendations for adult population [8]. On the other hand, protein content in chia seeds was higher than most of the traditionally staple grains, i.e., wheat (14%), corn (14%), rice (8.5%), oats (15.3%), and barley (9.2%) [1].


**Table 1.** Proximate composition and dietary fiber of chia seed.

Values are the mean ± SD of three determinations.

#### *3.2. Fatty Acids Profile*

*S. hispanica* seeds are worldwide recognized for their high content of lipid, which comprised mainly polyunsaturated fatty acids that play an important role in health [5,9]. The average amount of fatty acids in the chia varieties evaluated was 11.9 and 87.6 g/100 g of saturated and polyunsaturated fatty acids (PUFAs), respectively (Table 2). As it can be seen, three healthy fatty acids were identified in elevated amounts in the chia samples: linolenic (Ν-3), linoleic (Ν-6), and oleic (Ν-9). Otherwise, the saturated fatty acids, palmitic (16:0) and stearic (18:0), were found in very low concentrations. These data are in accordance with previous studies by Ayerza and Coates [9] and Mohd Ali et al. [2]. They have established that *S. hispanica* is an outstanding source of PUFAs (25– 40%), comprising 55–60% linolenic and 18–20% linoleic acids. Chia oil has the highest percentage (of any plant source) of *΅*-linolenic acid, which is considered essential because the human body cannot produce it and is also a potent lipid antioxidant [9,10].


**Table 2.** Fatty acid profile of chia cultivated in Mexico.

Values are the mean ± SD of three determinations.

#### *3.3. Protein*

The mean content of protein (22.7 g/100 g dry basis) of the evaluated chia seeds was high and is similar to that reported by Ayerza and Coates [1], and it provides essential amino acids to the daily diet [5]. Several important storage protein fractions have been found within chia. Based on solubility criteria, storage protein fractions were extracted. Table 3 shows the concentration of protein fractions from the four chia varieties evaluated. The fraction of globulins was the most abundant in all the lines studied, it ranged from 11.6 to 15.5 μg/mL, followed by the fraction of albumins 9.5 to 13.1 μg/mL, and glutelins with a concentration of 7.4 to 8.7 μg/mL, and the fraction of prolamins was found in the lowest concentration 4.4 to 5.2 μg/mL. These data were comparable to results obtained by Sandoval-Oliveros and Paredes-López [5] and Orona-Tamayo et al. [7].


Values are the mean ± SD of three determinations.

**Table 3.** Concentration of proteic fractions from chia seeds.

*3.4. Phenolic Compounds and Antioxidant Capacity* 

The concentration of total phenolic compounds of the *S. hispanica* lines evaluated was in the range of 0.78 and 0.97 g/100 g dry basis (Table 4). It can be observed that chia comprises a high concentration of phenolic compounds, thus, the antioxidant capacity was evaluated using the radical scavenging assays, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS [2,2ȝ-azinobis (3 ethylbenzothiazoline-6-sulphonic acid)]. Values for DPPH were in the range between 1.0 and 1.2 μg/g, and for ABTS from 1.0 to 1.6 μg/g. The concentration of total phenols and the antioxidant capacity of the chia seeds cultivated in different States of Mexico are remarkable and consistent with results obtained by Martínez-Cruz and Paredes-López [3] from *S. hispanica* cultivated in the Central area of Mexico.

**Table 4.** Total phenolic compounds and antioxidant capacity in chia.


DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2ȝ-azinobis (3-ethylbenzothiazoline-6-sulphonic acid). Values are the mean ± SD of three determinations.

#### *3.5. Antihypertensive Effect*

Chia contains high concentration of hydrophobic amino acids (proline, leucine, phenylalanine, and isoleucine) that generate peptides with high angiotensin converting enzyme (ACE)-inhibitory activity [11]. ACE controls blood pressure by regulating the volume of fluids in the body. It converts the hormone angiotensin I to the active vasoconstrictor angiotensin II. Our results indicate that the peptides obtained from the fraction of globulins promoted the highest inhibitory effect against ACE with an IC50 of 203.61, 148.23 and 110.11 μg/mL for White, Black, and Pinta Jalisco chia lines, respectively. Albumins, glutelins and prolamins demonstrated lower capacity against ACE, which is in accordance to data reported by Orona-Tamayo et al. [7]. They found that peptides from albumin and globulin fractions exhibited the highest ACE-inhibitory activity (IC50 377 and 339 μg/mL, respectively), followed by chia seed flour (IC50 516 μg/mL). It is interesting to note that albumin and globulins from Adzuki or red beans (*Vigna angularis*) [12] required higher protein concentration to inhibit ACE enzyme activity than the equivalent proteins of chia seeds from our study.

#### **4. Conclusions**

*Salvia hispanica* seeds showed important contents of proteins, dietary fiber and healthy lipids. In addition, they were a good source of total phenols and have high antioxidant capacity, which suggest that the scavenge ROS (reactive oxygen species) and may decrease or prevent chronic degenerative diseases, cancer and ageing. The chia protein profile showed that globulins are the major protein fraction followed by albumins, glutelins and prolamins. Chia peptides from the protein fractions exhibited capacity as ACE inhibitors, particularly, peptides from globulin and albumin fractions showed the strongest potential against ACE. These results can suggest that chia peptides could be outstanding natural antioxidants and ACE inhibitors for human health. Further research is required to develop novel chia cultivars with better nutraceutical attributes.

**Acknowledgments:** This work was supported by grant Ia ValSe-Food-CYTED (119RT0567) and Consejo Nacional de Ciencia y Tecnología (Conacyt), Mexico.

#### **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*  **Effect of Chia Seed Oil (***Salvia hispanica* **L.) on Cell Viability in Breast Cancer Cell MCF-7 †**

#### **Armando M. Martín Ortega and Maira Rubí Segura Campos \***

Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Periférico Norte Km. 33.5, Tablaje Catastral 13615, Col. Chuburná de Hidalgo Inn, Mérida, Yucatán C.P. 97203, Mexico; armandomarorte@gmail.com


Published: 1 September 2020

**Abstract:** Worldwide, cancer represents one of the main causes of mortality and morbidity, with breast cancer being the most diagnosed and the main cause of mortality among women. The purpose of this study is to evaluate the effect of chia seed oil on cell viability in the breast cancer line MCF-7. Tumor cells were treated to various concentrations of chia seed oil (12.5–400 ΐg/mL), then cellular viability was evaluated by (3-(4,5-dimethyl thiazole-2yl)-2,5-diphenyl tetrazolium bromide) MTT assay. Cellular viability was increased in the highest concentration group. Chia seed oil in high concentrations could potentially increase the viability of breast cancer cells.

**Keywords:** alternative; cancer; chia; nutraceutical; nutrition; oil

#### **1. Introduction**

Cancer is one of the main public health problems worldwide and represents the third leading cause of death in Mexico. Moreover, its incidence is increasing, without discriminating against countries or regions [1,2]. At the same time, national and international efforts to find more effective and less harmful treatments have been increased, including the investigation of bioactive compounds derived from food [3,4].

In addition to being an energy and structural source for the human body, fatty acids are bioactive lipids that regulate a large number of cellular processes, including growth regulation, apoptosis and cell proliferation [5,6]. Several in vitro and in vivo studies with isolated fatty acids and food oils have shown to have both anti-cancer and carcinogenic effects [7–10]. Thus, demonstrating an important role in the prevention and treatment of cancer. For its part, chia seed is an important source of Ν-3 polyunsaturated fatty acids, and is considered a functional food because of its ability to exert antiinflammatory, lipid-lowering, anti-hyperglycemic and metabolic regulating effects that are important in the treatment of chronic diseases, mainly metabolic [11,12]. Moreover, at present, there are international collaborations (Chia-Link International Network) for continuous research on the potential health benefits of chia seed, and the development of the functional foods derived from it.

The study of the effect of chia seed oil on cancer cells, would allow us to know its anti-cancer or carcinogenic potential, which would provide a basis for further studies in in vivo and clinical models. Likewise, the results of this study would serve as a guide for conducting studies of other oils, increasing the knowledge of the relationship between food and cancer, ultimately for the improvement of nutritional interventions in this disease.

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

#### *2.1. Seed Oil Extraction*

Dried whole seeds without mucilage were pressed in a cold pressing system until the oil was extracted, using a pressure of 8 ton/m2. The extracted oil was stored in an amber glass container inside a refrigerator at 4 °C away from the light, to allow sedimentation of seed residues and subsequent removal by centrifugation.

#### *2.2. Chemical Oil Hydrolysis (Ethanolysis)*

To obtain the free fatty acids (FFA), *Salvia hispanica* L. oil was chemically hydrolyzed by an alkaline hydrolysis with KOH and ethanol, following the methodology proposed by Riss et al. (2013). Then, 25 g of oil was mixed with 150 mL of 1 M KOH (95% EtOH) and placed in a 65 °C water bath for 2 h. Subsequently, to stop the hydrolysis, 100 mL of distilled water was added to the mixture. The non-hydrolyzed portion was removed by extraction with 100 mL of hexane. The aqueous alcohol phase, which contained the FFA, was acidified to pH 1 with 6N HCl to remove K from the carboxyl groups of fatty acids (R-COOK + HCl Æ R-COOH + KCl). The resulting free fatty acids were recovered by extraction with hexane and distilled water was added until a neutral pH was obtained. The phases formed by the mixture of water and hexane were separated with a separating funnel. Finally, the upper portion, which contains the FFA, was dried with anhydrous magnesium sulfate and the solvent was evaporated with a broken steam under vacuum at 35 °C [13].

#### *2.3. Determination of the Fatty Acid Profile by Gas Chromatography*

The composition of fatty acids was determined using the methodology proposed by Us-Medina (2015) with some modifications. First, 50 mg of oil was taken in a 50 mL test tube, 10 mL of 10% *w*/*v*  KOH in a methanol solution was added and allowed to reflux for 45 min in a controlled temperature bath (60 °C). At the end of the saponification, the sample was washed three times with 3 mL of hexane. Next, 2 mL of concentrated HCl was added and the fatty acids were extracted with three 2 mL portions of hexane, followed by drying with a nitrogen flow. Subsequently, a transesterification of the sample was performed by adding 420 ΐL of 5% HCl in a methanol solution to the fatty acids, then refluxing at 85 °C for 150 min in a controlled bath. The result of the transesterification was methyl fatty acid esters (FAME), which were extracted with three 2 mL portions of hexane. Then, 80 ΐL of a 10,000-ppm solution of the C17 standard in hexane was added. The hexenic phase was dried by a stream with nitrogen. After drying, the FAME were reconstituted with 1 mL of hexane to be injected into the chromatograph in split 25:1 mode with Helium (analytical grade) as the mobile phase. The gas chromatograph that was used is an Agilent Technologies 6890N, with an SP-2560 column, 100 m long, 0.25 mm internal diameter and 0.20 ΐm thick. The conditions that were used are: injector temperature of 250 °C, column flow of 1.0, oven temperature of 140 °C for 5 min and increased to 240 °C in a gradient of 4 °C/min, with a mass detector.

#### *2.4. Evaluation of Cytotoxic and Antiproliferative Activity In Vitro*

The cytotoxic and antiproliferative activities of the hydrolyzed oil were carried out by culturing the cell line with different concentrations of chia seed oil leaving them with the treatment for 48 h. The negative control was the cell line cultured only with the culture medium, while for the positive control Taxol was used, a drug commonly used in cancer chemotherapy and in vitro studies. At the end of the treatment, the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) was performed to evaluate cell viability (explained below).

#### 2.4.1. Preparation of the Compounds

The hydrolyzed oil was prepared at different concentrations for evaluation. A stock of 4 mg/mL of each compound was prepared by diluting 20 mg of the compound in 5 mL of fresh medium and subsequently passed through a 0.2 ΐm pore size nylon membrane syringe filter (cat. 431224, Corning, Monterrey, NL, Mexico). Serial dilutions were being made from the stock with the culture medium until concentrations of 12.5, 25, 50, 100, 200 and 400 ΐg/mL. These dilutions were prepared immediately before use.

#### 2.4.2. Cell Culture

Cells were grown in Dulbecco's Modified Eagle Medium (DMEM/F-12) medium without phenol red (cat. D2906, Sigma Aldrich, St. Louis, MO, USA) supplemented with 1.2 g/L NaHCO3 (cat. S5761, Sigma Aldrich, St. Louis, MO, USA) and 10% phosphate buffered saline (PBS) (cat. 10437028, Invitrogen, Carlsbad, CA, USA). The cells were incubated at 37 °C with 5% CO2 and a humidified atmosphere, in a Lab-Line incubator (Barnstead, Melrose Park, IL, USA). Each time the cell culture reached about 70–80% confluence, subcultures were performed.

#### 2.4.3. Cell Count and Viability

The cell count was performed with the Neubauer chamber and the determination of cell viability by the trypan blue exclusion technique.

For this procedure, a 200 ΐL sample of the suspended cells was taken, 20 ΐL of trypan blue was added, placed in a Neubauer chamber and observed under a microscope. Dead cells have a blue color. A concentration of cells was satisfied per ΐL, counting the living cells.

#### 2.4.4. Evaluation of Cytoxicity and Antiproliferative Effect by the MTT Technique

The determination of cellular cytoxicity/antiproliferative was performed using the MTT technique. This technique allows the proliferation to be measured indirectly by the detection of the coloration caused by the metabolic reduction in the tetrazolium salt bromide of 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), yellow, due to the action of mitochondrial dehydrogenase enzymes, so only viable cells can reduce it. The resulting compound, formazan blue, can be solubilized and quantified spectrophotometrically [14].

#### 2.4.5. Procedure

The procedure performed is described below.


% P = (ODw/compound)/(OD control) × 100

#### *Proceedings* **2020**, *53*, 18

#### where:

ODw/compound: optical density of cells with compound. OD control: optical density of the cell control (cells without compounds).

#### *2.5. Statistical Analysis*

All results were processed using descriptive statistics using measures of central tendency (mean) and dispersion (standard deviation). The data obtained from the biological activities were evaluated by means of one-way analysis of variance and a comparison of means (Student's T method) to establish statistical differences between treatments with a 95% confidence level (*p* < 0.05).

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

#### *3.1. Gas Chromatography*

The results of the gas chromatography demonstrated the presence of palmitic acid (7.7 ± 0.19%), stearic (4.45 ± 0.15%), oleic (9.57 ± 0.27%), arachidonic (0.30 ± 0.00%), linoleic (20.56 ± 0.14%) and alpha-linolenic (57.29 ± 0.07%). The fatty acid present in a greater proportion was alpha-linoleic acid, which is considered an essential fatty acid, and omega 3. The content of the latter is lower compared to that reported from other crops of Mexican origin [15]. However, it is known that the chemical composition of the seed is variable and depends on the region where it grows, with elevation above sea level being a determining factor.

#### *3.2. Cell Viability Assay*

Cellular viability was 159.8 ± 4.5% (*p* = 0.01), 144.4 ± 3.6% (*p* = 0.01), 109.5 ± 2.9% (*p* = 0.06), 105.1 (*p* = 0.09), 97.4 ± 3.1% (*p* = 0.31) and 91.8 ± 5.1% (*p* = 0.04) for 400, 200, 100, 50, 25 and 12.5 ΐg/mL of chia oil, respectively, compared with cells without treatment (control group: culture medium) (Figure 1). Cellular viability was significantly increased in the two major concentrations of the oil (400 and 200 ΐg/mL) and reduced in the lower concentration of the oil (12.5 ΐg/mL).

\* Statistically significant

The lower concentration (12.5 ΐg/mL) significantly reduced cell viability compared to the control. However, concentrations greater than 50 ΐg/mL increased cell viability. The two highest concentrations significantly increased cell viability. These results are due to the fact that alpha linolenic acid (omega 3) has antitumor potential, and at low concentrations (ǂ25 ΐg/mL) of chia oil its effect was prevalent. However, at high concentrations (>25 ΐg/mL) of the oil, the concentrations of linoleic acid (omega 6) are increased, which has been shown to increase the cell proliferation of breast cancer cells. In turn, it is important to consider that different studies have found an antitumor effect of alpha linolenic acid in various cell lines, including breast, colon and prostate cancer [16–18], while linoleic acid has demonstrated inverse effects, increasing the proliferation of breast cancer cell lines [19–21].

It is important to consider that the effects demonstrated in the *in vitro* study of the oil are not extrapolated at the systemic level, this is due to the possible metabolism and absorption of fatty acids by other organs before reaching the breast tissue. On the other hand, Espada et al. (2007) evaluated the effect of *Salvia hispanica* and *Carthamus tinctorius* oil on eicosanoid production, growth and metastasis in a murine model of mammary gland adenocarcinoma. This study found that the diet with chia oil produced a reduction in the amount of arachidonic acid and eicosanoids in the neoplasic cells (*p* < 0.05), as well as in the weight and number of tumor metastases (*p* < 0.05), compared with the *Carthamus tinctorius* diet and control diet. In addition, animals fed with chia oil showed a greater infiltration of T lymphocytes and apoptosis of the tumor cells with respect to the other diets (*p* < 0.05). Thus, the study authors concluded that *Salvia hispanica* oil is a rich source of polyunsaturated fatty acids Ν-3 with the potential to inhibit tumor growth and metastasis, at least in the murine model [8].

Interestingly, other studies conducted with seed oils such as flaxseed, canola, walnut, squash and neem, have found growth-inhibiting effects of various cancer cell lines [22–25]. In addition to the potential regulatory effect of fatty acids in oils, it is possible that the presence of phytochemicals may be contributing to the antitumor effects. However, in the case of the present study, it is likely that the linoleic acid content of chia seed oil has been the main contributor to the increase in cell viability at higher concentrations.

#### **4. Conclusions**

This study suggests that chia seed oil in high concentrations could potentially increase the viability of breast cancer cells. However, at low concentrations it could reduce cell viability. Thus, future research is necessary, specifically as regards employing the isolates of omega 6 and omega 3 to extend beyond our concluded results.

**Funding:** This work was supported by grant Ia ValSe-Food-CYTED (119RT0567).

#### **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* 
