Changes in the Chemical Composition and Bioactive Compounds of Quinoa Seeds by Germination ^†

Abstract

This research aimed to evaluate the changes that occur in the composition of macronutrients and soluble compounds of quinoa grains at different germination times. The seeds were soaked in water, drained, and then germinated in monolayers inside closed containers for 12, 24, 48, and 72 h and the germination was stopped by drying. Proteins, amino acids, fatty acids and antioxidant activity in flours were measured. A gradual reduction of carbohydrates is verified during the germination time with a concomitant increase in protein and lipid contents, while total minerals did not show modifications. The concentration effect due to metabolized carbohydrates seems responsible for the 33% rise in protein content 72 h after sprouting, but it is not enough to explain the almost 100% lipid increase for the same period. In general, amino acids and unsaturated fatty acids increase during germination, constituting a good resource for food and food ingredients intended for the general public, celiac patients, children, athletes, and elderly people.

1. Introduction

Quinoa (Chenopodium quinoa Willd.) is a native plant of the Andean region of South America, which has similar chemical characteristics and uses to cereals but does not belong to the same botanical family, so it is commonly referred to as a pseudo-cereal. It was part of the basic food of the pre-Hispanic people of South America, though, after the Spanish conquest, the crop was marginalized in isolated areas and was mostly replaced by wheat and barley. It has an outstanding nutritional quality, mainly represented by the content and biological value of its proteins, the starch structure, the fat content (rich in unsaturated fatty acids), and the significant number of bioactive compounds such as fiber, minerals, vitamins, phytosterols, polyphenols, among others [1]. People with celiac disease can consume both the grain and its derived products. During germination an increase in soluble compounds such as amino acids, fatty acids, sugars, vitamins, and minerals takes place, favoring the bioavailability of these nutrients [2]. This process is stopped by heat or drying, ending the so-called malting. In recent years, several investigations have been carried out into gluten-free malted cereals and pseudo-cereals especially aimed at the celiac population. In this work the changes in the macronutrients during the germination of quinoa grains are evaluated, improving the bioavailability of nutrients for celiac patients, but also for children, athletes, elderly people, and the general public.

2. Materials and Methods

2.1. Plant Material

The quinoa seeds (Chenopodium quinoa Willd.) were purchased from a quinoa producer from northwest Argentina. The seeds were cleaned and stored at 4 °C in sealed polyethylene bags until use.

2.2. Malted Quinoa Flours

Quinoa seeds were washed and moisturized in distilled water (1:10 ratio) for 2 h at 20 °C and then drained. Some of these seeds were dried in a fluidized bed dryer at 50 °C until moisture content was 11 ± 1% and then ground in a 0.25 mm hammer mill to obtain whole quinoa flour (QF0). The remaining seeds were arranged in a single layer on a plastic tray inside a closed container, away from direct sunlight. Germination was carried out at 25 °C and 50% relative humidity. The different stages of the process were monitored (Figure 1), stopping it at 12, 24, 48, and 72 h by drying and grinding following the same methodology described for QF0. Germination tests were carried out in triplicate, with the selection criteria being more than 90% sprouted grains [3].

2.3. Physicochemical Characterization of the Flours

2.3.1. Proximate Analysis

Proximate analysis was performed in triplicate using AOAC techniques [4]. Carbohydrates (C) were calculated by difference. All determinations were expressed on a dry basis.

2.3.2. Total Amino Acids Profile

Flours were subjected to acid hydrolysis with 6M HCl under reflux for 24 h, following AOAC 994.12 methodology [4]. Amino acids identification and quantification were carried out using a high-performance liquid chromatography (HPLC) equipment with UV-detector, while data acquisition and processing was done using a Total Chrom Workstation software (version 6.3), following the methodology described in Mufari et al. [5].

2.3.3. In Vitro Protein Digestibility

In vitro protein digestibility (PD) of samples was determined according to the technique of Dierick et al. [6]. PD results were expressed as a percentage on a dry basis.

2.3.4. Total Fatty Acids Profile

Quinoa flours were soaked in n-hexane at a 1:10 ratio for 24 h at 3 °C in darkness to extract lipids. Following extraction, the mixture was filtered, the solvent was evaporated, and the fatty acids were trans-methylated. Identification and quantification of the fatty acids in the extracted oils were performed using gas chromatography with a mass spectrometry detector (GC-MS) on a Clarus 600 Perkin Elmer instrument, following the procedure outlined by Mufari et al. [5].

2.3.5. Evaluation of Antioxidant Properties

The extraction of antioxidant compounds was carried out according to [7]. Total phenolic content (TPC) was determined using the Folin-Ciocalteu method [8], adapted to microplate determinations. The absorbance of samples was measured in a spectrophotometer at 760 nm (BMG Labtech GmbH, Germany), and results were expressed as mg gallic acid equivalents (GAE)/100 g flour. Total flavonoid content (TFC) was determined according to the AlCl₃ method [8] adapted to a microplate reading absorbance at 367 nm, and the results were expressed in mg Quercetin equivalents (QE)/100 g flour. The antioxidant activity of the extracts was determined using 2,2-dyphenil-1-picryl hydrazyl (DPPH•) [8], absorbance was measured at 517 nm, and radical scavenging capacity (RSC) was calculated by means of the following equation: %RSC= [1 − (Absorbance (DPPH•) − Absorbance sample))/Absorbance (DPPH•)] ×100.

2.4. Statistical Analysis

Results were expressed as mean ± standard deviation of the replicates. Data analysis was carried out using InfoStat^® professional (version 2020I). Significant differences between measurements were estimated by variance analysis (ANOVA). When statistically significant differences were observed (α = 0.05) a DGC multiple comparisons test was subsequently used.

3. Results and Discussion

Physico-Chemical Characterization of the Flours

Table 1. Proximal composition and digestibility of the different quinoa flours.

Components	Germination Time
	QF0	QF12	QF24	QF48	QF72
Moisture	9.59 a ± 0.23	16.49 d ± 0.01	13.24 b ± 0.01	14.33 c ± 0.01	16.86 e ± 0.14
Ash	1.97 a ± 0.02	1.68 a ± 0.10	2.29 a ± 0.13	2.05 a ± 0.11	1.89 a ± 0.26
Lipids	7.87 a ± 0.22	7.65 a ± 0.52	9.56 b ± 0.03	11.07 c ± 0.57	15.63 d ± 0.26
Proteins	13.56 a ± 0.30	17.19 b ± 0.10	17.38 b ± 0.25	18.34 c ± 0.10	18.03 c ± 0.16
Carbohydrates	76.60	73.48	70.77	68.54	64.45
% PD	71 b ± 2	72 b ± 1	81 c ± 1	82 c ± 1	62 a ± 2

The results are expressed in g/100 g of sample on a dry basis (except wet moisture content), means with standard deviations are reported (n = 3). Different letters in the same row denote statistically significant differences (p < 0.05). QF: quinoa flour, the number indicates the germination hours of the grains (0, 12, 24, 48, and 72 h); PD: protein digestibility.

shows the results of the proximal analysis of the flours. Although moisture was adjusted to 11% prior to milling, the flours showed different degrees of hydration; hence, wet content was between 9.59 and 16.86%.

The effect of sprouting on the nutritional composition of quinoa flour can be observed by the significant decrease of the total carbohydrate content (4 to 16% compared to QF0) with germination, a consequence of the metabolic activity of the seeds that takes place during germination. These results resemble the values reported for different varieties of sprouted rice, sorghum, millet, buckwheat, and amaranth, with decreases in carbohydrate content from 5 to 51% [9].

On the other hand, quinoa flours germinated for 48 and 72 h (QF48 and QF72) showed the highest protein values (Table 1). The synthesis of enzymes and non-protein nitrogenous substances (such as nucleic acids) can contribute to this increase. Diverse variations of protein contents are reported in sprouted cereals and pseudocereals, from reductions between 2 and 21% to increases between 5 and 100%. These differences depend on the plant material and on soaking, sprouting, and drying conditions [9]. In this investigation, an increase of 35% in protein content, between 0 and 72 h of germination, was observed, which implies an improvement in the nutritional profile. The same tendencies were found by Maldonado-Alvarado et al. [10] under similar conditions. A protein increase with carbohydrate reduction is good news for celiac patients commonly facing gluten-free foods that are highly energetic but poor in proteins.

Lipid contents were also increased with germination time, being almost double in QF72 compared to QF0 (Table 1).

In quinoa, lipids and protein are found mainly in the embryo while starch is in the perisperm. Amylases free glucose and maltose from perisperm starch, being the main energy source for germination; on the other hand, all the available nitrogen is endogenous as malting excludes any external source. So, the total nitrogen (as proteins) per 100 g existing at the beginning in QF0 should remain in the final stage 72 h later, then:

$${\displaystyle \frac{13.56}{\left(76.6-x\right)}}={\displaystyle \frac{18.03}{64.45}} x =28.2 \mathsf{g}$$

(1)

where x is the fraction of carbohydrates consumed by seed metabolism after 72 h of germination, nearly 37% of initial starch, showing the importance of carbohydrates for sprouting through providing the needed energy. The increase in lipids is greater than expected due to the concentration effect. Although some works show an opposite tendency for quinoa, Pachari Vera et al. [11] found the same behavior in four Peruvian quinoa varieties, in some cases with increases near 50%. The total mineral content did not show significant differences between germination stages (Table 1).

In vitro digestibility for proteins is presented in Table 1. The first 12 h of sprouting shows no significant changes, staying at 71–72%, but in the next 24–48 h it rises up to 81–82% and then drops to 62% after 72 h of germination (Table 1). A similar behavior was found by Prasad & Sahu [12] with values going from 72% to 87.5% after 48 h of sprouting, but these authors did not continue germination beyond.

Table 2. Amino acid profile (g/100 g of flour) of integral quinoa flour (control) and germinated flours at different times.

Amino Acids	QF0	QF12	QF24	QF48	QF72
Aspartic ac.	0.442 a ± 0.028	0.973 b ± 0.094	0.954 b ± 0.076	1.067 b ± 0.008	1.090 c ± 0.013
Glutamic ac.	1.487 a ± 0.013	1.601 c ± 0.022	1.580 b ± 0.061	1.500 a ± 0.055	1.553 b ± 0.037
Serine	0.025 a ± 0.003	0.354 b ± 0.004	0.348 b ± 0.025	0.379 c ± 0.004	0.502 d ± 0.105
Histidine	0.033 a ± 0.004	0.761 d ± 0.022	0.596 b ± 0.000	0.660 c ± 0.003	0.769 d ± 0.171
Glycine	0.438 a ± 0.004	0.638 c ± 0.003	0.570 b ± 0.009	0.550 b ± 0.002	0.559 b ± 0.071
Threonine	0.071 a ± 0.003	0.508 c ± 0.018	0.430 b ± 0.010	0.485 c ± 0.008	0.560 d ± 0.009
Arginine	0.758 a ± 0.005	1.281 d ± 0.029	1.075 c ± 0.032	1.128 c ± 0.020	0.877 b ± 0.056
Alanine	0.654 d ± 0.001	0.550 c ± 0.010	0.512 b ± 0.003	0.498 a ± 0.003	0.554 c ± 0.017
Proline	0.048 a ± 0.015	0.188 b ± 0.046	nd	nd	nd
Tyrosine	0.130 a ± 0.015	0.601 d ± 0.014	0.463 b ± 0.042	0.513 c ± 0.099	0.580 c ± 0.076
Valine	0.571 e ± 0.020	0.144 c ± 0.005	0.107 b ± 0.008	0.086 a ± 0.061	0.553 d ± 0.004
Methionine ± Cysteine	5.360 a ± 0.019	5.994 b ± 0.106	6.936 c ± 0.171	6.816 c ± 0.227	6.091 b ± 1.026
Isoleucine	0.404 a ± 0.002	0.616 d ± 0.006	0.583 c ± 0.016	0.518 b ± 0.007	0.687 d ± 0.103
Leucine	0.561 a ± 0.002	0.872 c ± 0.016	0.796 b ± 0.023	0.776 b ± 0.002	1.056 d ± 0.146
Phenylalanine	0.253 a ± 0.002	0.800 e ± 0.006	0.640 c ± 0.011	0.573 b ± 0.034	0.733 d ± 0.110
Lysine	0.480 a ± 0.004	0.471 a ± 0.003	0.530 b ± 0.010	0.602 c ± 0.005	0.774 d ± 0.126
% of Recovery	92.44	95.10	92.72	86.74	93.82

QF: quinoa flour, the number indicates the germination hours of the grains (0, 12, 24, 48, and 72 h). Means with standard deviations are reported (SD, n = 6), expressed in g/100 g of sample on a dry basis. nd: below the limit of quantification (0.012). Different letters in the same row denote statistically significant differences (p < 0.05).

summarizes the results of the total amino acid composition of the different flours. As can be seen in Table 2, lysine contents in quinoa flour (QF0) are almost double those of wheat, corn, and rice while sulfur amino acids are in a similar proportion to those in the above cereals, but with quinoa protein content being higher its total contribution of amino acids is clearly superior. Some amino acids may diminish during germination as pointed out by Moongngarm and Saetung [13].

Table 3. Fatty acid profile (g/100 g of flour) of integral quinoa flour (control) and germinated flours at different times.

Fatty Acids		QF0	QF12	QF24	QF48	QF72
Miristic	14:0	nd	0.20 a ± 0.01	0.23 a ± 0.03	0.24 a ± 0.01	0.22 a ± 0.03
Pentadecylic	15:0	nd	0.14 a ± 0.01	0.12 a ± 0.02	0.14 a ± 0.02	0.13 a ± 0.01
Palmitic	16:0	9.2 a ± 0.3	12.1 c ± 0.2	12.5 c ± 0.4	12.5 c ± 0.2	11.3 b ± 0.4
Palmitoleic	16:1	nd	0.41 a ± 0.02	0.42 a ± 0.03	0.38 a ± 0.03	0.42 a ± 0.02
Margaric	17:0	nd	nd	0.10 a ± 0.03	0.11 a ± 0.03	0.10 a ± 0.03
	17:1	nd	nd	0.12 a ± 0.03	0.12 a ± 0.03	0.13 a ± 0.03
Stearic	18:0	1.03 c ± 0.03	0.90 b ± 0.02	0.81 a ± 0.02	0.79 a ± 0.01	0.91 b ± 0.03
Oleic (ω9)	18:1	27.6 c ± 0.4	27.4 c ± 0.5	24.6 b ± 0.2	23.2 a ± 0.5	26.8 c ± 0.2
Linoleic (ω6)	18:2	54.9 c ± 0.2	44.9 a ± 0.4	44.3 a ± 0.2	45.4 b ± 0.3	45.8 b ± 0.2
Linolenic (ω3)	18:3	5.7 a ± 0.4	8.2 b ± 0.4	9.8 c ± 0.4	10.2 c ± 0.4	8.9 b ± 0.4
Arachidonic	20:0	0.33 a ± 0.02	0.60 b ± 0.03	0.78 c ± 0.02	0.81 c ± 0.02	0.58 b ± 0.04
Gondolic	20:1	1.19 a ± 0.01	2.49 c ± 0.02	2.50 c ± 0.02	1.72 b ± 0.03	2.68 d ± 0.03
	20:2	nd	0.18 a ± 0.02	0.21 a ± 0.01	0.20 a ± 0.01	0.22 a ± 0.03
Behenic	22:0	nd	0.98 b ± 0.02	1.10 b ± 0.03	1.11 b ± 0.03	0.89 a ± 0.02
Erucic	22:1	nd	1.91 b ± 0.05	2.45 c ± 0.02	2.71 d ± 0.02	1.58 a ± 0.03
Tricosylic	23:0	nd	0.10 a ± 0.02	0.1 a ± 0.01	0.11 a ± 0.02	nd
Lignoceric	24:0	nd	0.42 a ± 0.02	0.58 b ± 0.02	0.60 b ± 0.03	0.41 a ± 0.01
	24:1	nd	0.18 a ± 0.04	0.20 a ± 0.03	0.21 a ± 0.04	0.19 a ± 0.02
Saturated (S)		10.56	15.44	16.33	16.41	14.54
Monounsaturated (MI)		28.79	32.39	30.30	28.34	31.80
Poliunsaturated (PI)		60.6	53.28	54.31	55.80	54.92
ω6/ω3		9.63	5.47	4.52	4.45	5.15
PU/MU		2.10	1.64	1.79	1.97	1.73

QF: quinoa flour, the number indicates the germination hours of the grains (0, 12, 24, 48, and 72 h). Means with standard deviations are reported (SD, n = 6), expressed in g/100 g of sample on a dry basis. nd = below the limit of quantification (0.012). Different letters in the same row denote statistically significant differences (p < 0.05).

shows the changes in the fatty acid profile of quinoa flour depending on the germination time, with palmitic (16:0), oleic (18:1ω9), and linoleic (18:2ω6) acids being the majority. Guardianelli et al. [14] found similar trends in germinated amaranth seeds, with increases in palmitic, linoleic, and linolenic acids and a decrease in stearic and oleic acids. The ω6/ω3 ratio, which is 9.6 for QF0, is reduced to 5.3 in QF72. This result is close to the recommended 5:1 [14].

Antioxidant compounds significantly increased with germination time, until 24–48 h of germination, and then at 72 h a decrease occurs, a similar trend to several of the parameters previously determined in this work. The TPC of quinoa flour was 54 ± 2 mg GAE 100 g⁻¹ flour, a value that is within the range reported by other authors (25.0–71.7 mg GAE 100 g⁻¹ flour) [7,15]. Total phenols increased by 63% after a 24-h germination.

On the other hand, the TFC of quinoa flour was higher than the reported by Carciochi & Dimitrov [7] (24 ± 1 mg QC 100 g⁻¹ flour and 11 mg QC 100 g⁻¹ flour, respectively). These differences can be partially explained by the differences in the extraction methods, the variety of the grains and the growing conditions. Total flavonoids increased by 125% after germination for 24 h, in comparison to raw quinoa. These increases in the compounds with antioxidant capacity, are related to enzymes activated during germination that lead to the release of compounds bound to cell structure of the matrix or by de novo synthesis [7]. Although other authors reported a constant increase with the germination time, in this study, antioxidant compounds decrease after 48 h of germination, probably due to the fixation in the new matrix of the growing seedling that makes their extraction difficult.

Antioxidant activity of germinated quinoa seeds extracts assessed by DPPH• method, indicated that the germination process significantly increased the antioxidant activity, compared to the control sample. Maximum radical scavenging activities were obtained in the lipid (89%) and ethanolic fraction (77%). Similar results were reported for different cereals and pseudo cereals, but with different optimal times of germination to obtain a maximum amount of soluble or bioavailable nutrients [9]. In the case of quinoa under the established malting conditions, germination time should be between 24 and 48 h.

4. Conclusions

It has been shown that it is feasible to increase the nutritional quality of quinoa grains by germinating them for a period between 24 and 48 h. Flours obtained from sprouted grains demonstrated a greater bioavailability of nutrients. The preliminary results obtained in this work confirm that germination is an adequate process for improving the nutritional profile of quinoa flours, and for further production of healthy foods intended for vulnerable age groups such as children or the elderly.