Phenolic Compounds, Antioxidant Capacity, and Protein Content of Three Varieties of Germinated Quinoa (Chenopodium quinoa Willd)

Quinoa (Chenopodium quinoa Willd) is a pseudocereal with a high nutritional potential and a significant content of bioactive compounds, which is consumed mainly by the inhabitants of the South American Andes. The aim of this study was to evaluate the protein content, total phenols, and antioxidant activity of quinoa grains of the Salcedo INIA, Pasankalla, and Negra collana varieties, germinated for 24 and 48 hours at 35 ◦C. Organic quinoa grains were grown in the Andahuaylas province in Peru, at an altitude of 3582 m. The protein content was determined through the Kjeldahl method, total phenols, in turn, by spectrophotometry with the Folin-Ciocalteu reagent, and the antioxidant activity of the DPPH type were registered. The data were analyzed through an Analysis of Variance (ANOVA), a Tukey test, and Pearson’s correlation at 5% significance. The germinated quinoa grains showed a considerable increase (p < 0,05) in their protein content, total phenolic compounds, and antioxidant activity, as well as a strong positive correlation with the size of sprouts during the germination time. Therefore, germinated quinoa could be considered as a promising product for human nutrition and health.


Introduction
Quinoa (Chenopodium quinoa Wild) is an endemic South American plant typical, domesticated thousands of years ago by the Inca people of the Andes of Peru and Bolivia, as well as parts of Ecuador, Argentina, and Chile. It comes with a diversity of forms, genotypes, and wild progenitors (Navruz-Varli, and Sanlier, 2016), as well as a high resistance and How to cite: Choque-Quispe, D., Ligarda-Samanez, C. A., Ramos-Pacheco, adaptability to climatic and edaphic conditions (Jacobsen, Mujica, and Jensen, 2003;Filho et al., 2015).
Quinoa grain is considered to be a pseudocereal with a high nutritional value, mainly due to its high protein content and essential amino acids, which are higher than traditionally consumed cereals (Filho et al., 2015;Dakhili, Abdolalizadeh, Hosseini, Shojaee-Aliabadi, and Mirmoghtadaie, 2019), whose importance is increasingly recognized in food safety, since it considerably replaces and complements the diet, especially for people who rarely consume meat and dairy products (Elsohaimy, Refaay, and Zaytoun, 2015;Navruz-Varli, and Sanlier, 2016). It is one of the main sources of protein in developing countries (Repo-Carrasco-Valencia, and Serna, 2011).
The digestibility of quinoa protein is a limiting factor in their use in food (Elsohaimy et al., 2015), but it improves considerably when subjected to germination, fermentation, and thermal treatments, thus increasing the bioavailability of its amino acids (Graf et al., 2014;Navruz-Varli and Sanlier, 2016;Nickel, Spanier, Botelho, Gularte, and Helbig, 2016).
The aim of this research was to evaluate the effect of the germination of quinoa (Chenopodium quinoa Willd) of the Salcedo INIA, Pasankalla, and Negra collana varieties on phenolic compounds, antioxidant capacity, and protein content.

Vegetable material
Organic quinoa grains (grown without the addition of pesticides and synthetic fertilizers) of the Salcedo INIA, Pasankalla, and Negra collana varieties were provided by the Machu Picchu agrarian cooperative in the province of Andahuaylas, Perú. These grains were cultivated during the vegetative period of 2017-2018, in fields located at 13 • 39'26" S, 73 • 17'32" W, and 3 682 m of altitude.

Obtaining germinated quinoa
Quinoa grains were washed with abundant treated water (pH 7,5), generating friction between grains to eliminate saponin. The washing continued until an evident absence of foam was reached. Then, they were subjected to a humidity between 43% and 45%, and packed a in wet gauze at 35 • C, for periods of 24 and 48 h, in order to cause germination, and the size of the sprout was measured with a Vernier. The sprouts were taken to a horizontal dryer at 60 • C, until constant humidity was reached, and then they were ground to 250 microns in an Agate mortar.

Protein quantification
The nitrogen content was determined through AOAC method 984.13 (AOAC, 2016). The protein content was calculated by N × 6,25, and the results were expressed as percentage on dry basis (d.b).

Total phenol quantification
The spectrophotometric method used by Ah-Hen et al. (2012) was followed with some modifications. 1,0 g of sample was taken (germinated quinoa ground) and mixed with 10 ml of acidified methanol with 1% HCl. The homogenate was stored for 24 hours at 4 • C in darkness. Then, it was centrifuged for 15 min at 7000 rpm. An aliquot of the supernatant was taken in a test tube, thus obtaining a germinated quinoa extract, which was stored in a dark refrigerator. 100 µL of the extract were taken and caused to react under agitation with 750 µL of the reagent 1,0 N and 750 µL of sodium carbonate (60 g/L). The mixture was stored for 30 minutes in darkness at room temperature. In the same way, a blank was prepared using distilled water, which served to calibrate the spectrophotometer. Then, the absorbance was read at 755 nm in a spectrophotometer, model T80+ from PG Instruments.
The total phenolic compounds (TP) were estimated from a standard curve made with an aqueous gallic acid solution (R 2 = 0,96). The results were expressed as the mg gallic acid equivalent (GAE) per 100 g sample on dry basis (d.b).

Antioxidant capacity quantification
The method of Brand-Williams, Cuvelier, and Berset, (1995) was followed with some modifications. 5,0 g of sample (germinated quinoa ground) were weighed, and 20 ml of 80% methanol were added and mixed for 15 min at 800 rpm. The extract was stored for 24 hours at 4 • C in darkness. Then, it was centrifuged at 3000 rpm for 20 minutes, taking an aliquot of the supernatant (extract) and placed in the darkness at 4 • C lined with aluminum foil. 150 µL of the extract were taken and 2850 µL of diluted DPPH solution (24 mg/100 ml methanol) were added. Identically, a blank was prepared with 150 µL of 80% methanol to obtain a correction factor due to the dilution. The mixture was left to react in darkness for 30 min at 20 • C and then taken to thr spectrophotometer; the absorbance was read at 515 nm.
The antioxidant capacity was calculated using a standard Trolox curve (0,5 mM) and expressed as the µmol Trolox equivalent (TE)/g sample on dry basis (d.b.).

Statistic analysis
The data were collected in triplicate, and an analysis of variance (ANOVA) and Tukey test were applied. Correlations between variables were determined by the Pearson correlation coefficient (Rs) at 5% significance.
On the other hand, it was observed that the protein content in the germinated quinoa increased significantly (p < 0,05) for the Salcedo INIA and Pasankalla varieties, unlike the Negra  During the germination process, the enzymatic systems are activated, mobilizing reserve proteins located in the cotyledons of the quinoa grain. In the same way, changes in the amino acid composition occur due to enzymatic activity (Gan et al., 2016;Televičiutė et al., 2020), which allows the production of peptides of intermediate molecular weight due biological activity (Torres, Cova, and Valera et al., 2018;Banchuen et al., 2009). This also happens with the mobilization of nitrogen stored in the quinoa grain, which allows a significant increase in protein, as observed by El-Safy, Mukhtar, and Salem (2013), Graf et al. (2014), . However, this depends on the humidity and temperature of the environment.
The TP for the quinoa grains was between 159,69 and 198,23 mg GAE/100 g on dry basis (Table 2). These values are similar to those reported by Abderrahim et al. (2015), Nickel et al. (2016), and Saad-Allah and Youssef (2018). The differences in the total phenol content are due to growing conditions such as soil type (Nsimba, Kikuzaki, and Konishi, 2008;Liberal, Calhelha, Pereira, and Adega, 2016;Huang, Qin, Shi, and Wen, 2017), as well as the variety, since the colored varieties have a higher TP, as reported by Tang et al. (2015), Abderrahim et al. (2015), and Han et al. (2019). Pasankalla (red coloration) reported the highest content, followed by the Negra collana variety (shiny black coloration). Evaluated through a Tukey test at 5% significance.

Source: Authors
It was observed that, after 24 h of germination, the TP for the Salcedo INIA variety had an increase of 61,3% and, 79,0% after 48 h. The Pasankalla variety increased to 55,2% and 110,7% after 24 and 48 h, respectively (Table 2). It could also be observed that Negra collana reported the highest increase (152,6% after 48 h), which is due to its genotypic differences; since this variety is darker, it releases a greater amount of phenolic compounds due to the leaching effect during germination (Televičiutė et al., 2020;Piñuel et al., 2019).
The TP increase during the germination process of quinoa has been observed by Alvarez-Jubete, Wijngaard, Arendt, and Gallagher (2010), Filho et al. (2015), Gan et al. (2016), and Piñuel et al. (2019). It is due to the fact that quinoa grains suffer biotic and abiotic stress during germination, which induces the generation mainly of oxygen-reactive spices (ROS). These compounds are vital to protecting the grains during germination (Shulaev, Cortes, Miller, and Mittler, 2008;Televičiutė et al., 2020) and for the biochemical and physiological functions of the sprouts, releasing aglycones due to enzymatic activity, which translates into an increase in phenols (Shetty, 2004;Sani et al., 2012). Another critical aspect is the germination temperature, which was 35 • C for this research. The recommended standard is between 35 and 45 • C (Televičiutė et al., 2020).

Antioxidant capacity (AA)
AA is an aspect that evaluates the ability of a compound to reduce the impact of ROS, and quinoa grains are an excellent source of antioxidants, being superior to many cereals, pseudocereals, and legumes (Pasko et al., 2009;Tang and Tsao, 2017).
The increase in antioxidant activity is due to the response of the seed to the physiological and biochemical changes to which they are subjected at the beginning of germination, so germination is used as a strategy to increase this capacity (Banchuen et al., 2009;Sani et al., 2012;Filho et al., 2015;Torres et al., 2018).

Correlation between variables
It was observed that the size of shoots evinced a high positive correlation with proteins, total phenols, and antioxidant activity (Rs > 0,81) (Table 4), which indicates that germination time causes these variables to increase considerably. Likewise, it was observed that the germination process of the three varieties of quinoa showed a high correlation between the study variables.
The protein content reported high correlation with the total phenols, with values above 0,80, as well as with the antioxidant capacity. This indicates that quinoa grains, during the stress that occurs during germination, produce phenols and proteins in parallel to condition the sprouts of the grains Televičiutė et al., 2020).

Source: Authors
In the same way, the TP shows a high positive correlation with the AA for the three quinoa varieties (> 0,86), which suggests that the phenol content is a good indicator of the antioxidant activity (Pasko et al., 2009;Tang et al., 2014;Contreras-Jiménez et al., 2019), thus making germinated quinoa highly bioaccessible and bioavailable to human health (Tang et al., 2015;Navruz-Varli, and Nevin Sanlier, 2016;Vilcacundo and Hernández-Ledesma, 2017).

Conclusions
The study showed that unsaponified quinoa grains of the Salcedo INIA, Pasankalla, and Negra collana varieties, subjected to germinations of 24 and 48 hours at 35 • C, increased their content of proteins and total phenolic compounds, as well as their antioxidant activity, presenting a strong positive correlation between them.
The results allow us to confirm that germinated quinoa is a promising product for human nutrition and health.