12th IFDC 2017 Special Issue – Influence of germination of quinoa (Chenopodium quinoa) and amaranth (Amaranthus) grains on nutritional and techno-functional properties of their flours

Journal of Food Composition and Analysis 84 (2019) 103290

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Original Research Article

12th IFDC 2017 Special Issue – Influence of germination of quinoa (Chenopodium quinoa) and amaranth (Amaranthus) grains on nutritional and techno-functional properties of their flours⋆ María Dolores Jimenez, Manuel Lobo, Norma Sammán

T

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Faculty of Engineering-CIITED CONICET, National University of Jujuy, Jujuy, Argentina

A R T I C LE I N FO

A B S T R A C T

Keywords: Food analysis Effect of germination on food composition Sprouted grains Andean grains Nutrition improvement Starch hydrolysis Protein degradation Thermal behavior

The germination process produces changes in grains that may affect the nutritional properties and technological characteristics of their flours. The aim of this work was to determine nutritional changes in quinoa (Chenopodium quinoa) and amaranth (Amaranthus) flours, induced by germination under controlled conditions. Proximate composition, protein digestibility, starch and fiber were determined by AOAC methods. Total and reducing sugars (dinitrosalicylic acid method) and protein fractions (SDS–PAGE) were determined in flours. Amylose content (spectrophotometric method) was determined in starch. Thermal behavior of flour was studied by DSC. Protein content and digestibility, and reducing and total sugars were increased by germination. Protein degradation was observed in fractions with molecular weights higher than 24 kDa in quinoa, while in amaranths the degradation was in all the molecular weight range 14–66 kDa. Apparent amylose content increased, possibly due to the formation of dextrins and linear chains of glucans from amylopectin. Gelatinization temperatures were similar between samples before and after germination. Gelatinization enthalpies of flours were significantly lower in sprouted than unsprouted grains; also a greater tendency to retrogradation was determined. Germination improved the nutritional contributions of quinoa and amaranth flours, but the starch content decreased and the gel became more unstable, important features if they are to be used as ingredients in food formulations.

1. Introduction Quinoa (Chenopodium quinoa) and amaranth (Amaranthus) grains grow throughout the Andean region, and have been used by the inhabitants of South America for centuries. Known as indigenous pseudocereals or Andean grains, they can be adapted to different environmental conditions. Pseudocereals are dicotyledonous species which are not closely related to each other or to the true monocotyledonous cereals ; the name pseudocereals derives from their production of small grain-like seeds that resemble in function and composition those of the true cereals (Valcárcel-Yamani and da Silva Lannes, 2012). There is very wide genetic variability among the Andean grains, which is reflected in the differing nutritional contents and phenotypic characteristics. The Andean grains are a good source of nutrients with high protein content (12–16% db) and good lipid profiles (lipid content between 5% and 10% db). The fraction of dietary fiber varies from 12 to

14% db in quinoa varieties, and from 11 to 21% db in amaranth varieties. Moreover, these crops have important amounts of vitamins, minerals and antioxidant compounds (Repo-Carrasco-Valencia and Serna, 2011; Valcárcel-Yamani and da Silva Lannes, 2012; Carciochi et al., 2014; Nascimento et al., 2014). Germination is a biological process that can be applied in an easy and economical way in order to obtain biotechnologically processed new food products. The consumption of sprouted products is increasing because numerous studies have documented their advantages and health benefits (Moongngarm and Saetung, 2010; Murugkar, 2015). During the germination process, hydrolytic enzymes are activated and they are also the newest enzymes synthesized which, along with the reserve substances in the seed, are mobilized to be used in the initial growth of the seedling (Bedón Gómez et al., 2013). This process causes changes in the content and composition of proteins, carbohydrates and lipids. The proteins are hydrolyzed and consequently their digestibility

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This paper was originally submitted as a poster presentation at the 12th International Food Data Conference held from 11 to 13 October 2017 in Buenos Aires, Argentina. ⁎ Corresponding author. E-mail addresses: [email protected] (M.D. Jimenez), [email protected] (M. Lobo), nsamman@fi.unju.edu.ar (N. Sammán). https://doi.org/10.1016/j.jfca.2019.103290 Received 31 January 2018; Received in revised form 15 September 2018; Accepted 8 August 2019 Available online 13 August 2019 0889-1575/ © 2019 Published by Elsevier Inc.

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is improved (Chaparro et al., 2010; Omary et al., 2012; Świeca et al., 2013; Hager et al., 2014; Carciochi et al., 2014; Devi et al., 2015). The protein profile of grains and legumes has been studied by analysis in polyacrylamide gel electrophoresis (PAGE) in faba beans (Goyoaga et al., 2011), lupin (Rumiyati et al., 2012), and quinoa (Valenzuela et al., 2013), to determine modifications caused by different processes such as germination, thermal treatments, storage, etc. Some authors showed that germination of grains and legumes (such as quinoa, soybean, chickpea, beans, peas, millet, rice and corn) may decrease the content of antinutrients such as phytates, tannins and inhibitory proteases (Omary et al., 2012; Kanensi et al., 2011; Wang et al., 2015; Jan et al., 2017). Besides, other researchers observed improvements of the polyphenol contents and the antioxidant capacity in wheat, buckwheat, chickpea, quinoa and amaranth during germination under certain conditions (Khalil et al., 2007; Paśko et al., 2009). Simultaneously, germination causes starch degradation by the action of amylases producing mainly dextrins, glucose and sucrose that will be oxidized to produce the energy necessary for the development of the embryo. Sucrose is the main source of energy storage in the plant (Bedón Gómez et al., 2013). These modifications can also affect the functional, rheological, textural and thermal properties of sprouted grain flours, which are important characteristics if they will be used as food ingredients. There are previous records about using cereals, legumes and grains in food formulations with particular nutritional or technological characteristics (Murugkar, 2015; Yang et al., 2012; Fu et al., 2014; Marengo et al., 2015; Hallen et al., 2004; Jideani and Onwubali, 2009; among others), demonstrating that sprouted Andean grains may be used for this purpose. The aim of this work was to study changes caused by germination in nutritional and thermal properties of flours of three varieties of quinoa (Cica, Kamiri and Inga Pirca) and two varieties of amaranth (Mantegazzianus and Rosado).

in polyethylene bags and stored at room temperature. 2.2. Proximate composition The proximate composition of flours was determined by official techniques AOAC (2017): moisture (method 925.10), ash (method 923.03), fat (method 963.15), total nitrogen (method 920.87); and N × 6.25 factor was used to calculate total protein content (Chaparro et al., 2010; Kanensi et al., 2011; Nascimento et al., 2014). Total carbohydrates were calculated by the sum of total fiber, total starch and total sugar. All analyses were carried out in triplicate. 2.2.1. Soluble, insoluble and total dietary fiber Dietary fiber, both soluble and insoluble, were determined according to AOAC Method 991.43 (1995), using the Megazyme assay kit (Wicklow, Ireland). Samples (1.000 ± 0.005 g) were suspended in 40 mL MES-TRIS buffer (pH 8.2) (MES: 2-(N-morpholino)ethanesulfonic acid; TRIS: tris-(hydroxymethyl) aminomethane) and digested sequentially with 50 μL heat-stable α-amylase (98–100 °C, 30 min), 100 μL protease (60 °C, 30 min), and 200 μL amyloglucosidase (60 °C, 30 min). Digested samples were filtered through fritted glass crucibles; these insoluble dietary fibers were rinsed with 95% ethanol followed by pure acetone, and oven dried overnight at 105 °C. Filtrates were mixed with 95% ethanol (4× volume) to precipitate soluble fibers; after 1 h they were filtered through tared fritted glass crucibles. Both soluble and insoluble dietary fiber residues were corrected for protein and ash content calculated according to the following equations (Eq.1 and Eq.2):

Fiber (Soluble or Insoluble) =

R1 + R2 + R3 − p− 3 m1 + m2 + m3 3

B= (BR1 + BR2 + BR3)/3 − BP− BA

A−B

100 (1) (2)

where mi = sample weight, i = 1, 2 and 3, Ri = residue weight from mi, i = 1,2, and 3), p = protein weight, A = ash weight, B = blank, BRi = blank residue, i = 1, 2 and 3; BP = blank protein, and BP = blank ash. Total dietary fiber was calculated as the sum of soluble and insoluble dietary fiber. Analyses were run in triplicate.

2. Materials and methods 2.1. Andean crops Quinoa (Cica, Kamiri and Inga Pirca varieties) and amaranth (Mantegazzianus and Rosado varieties) were obtained from Centro de Investigación y Desarrollo Tecnológico para la Agricultura Familiar (CIPAF, Research and Technological Development Center for Family Agriculture Hornillos, Jujuy, Argentina).

2.2.2. Resistant, digestible and total starch The resistant and digestible starches were determined using the Megazyme assay kit (Wicklow, Ireland) following the AOAC method (2003). Flour samples (100 ± 0.5 mg) were incubated with 4 mL pancreatic α-amylase (10 mg/mL) solution containing amyloglucosidase (AMG) for 16 h at 37 °C with constant shaking (200 strokes/min); in order to terminate the reaction, ethanol (99% v/v) was added. The pellet was separated by centrifugation (1500 g, 10 min) and re-suspended in ethanol (50% v/v); then it was digested with 2 mL KOH (2 M) in an ice bath with vigorous magnetic stirring. Digested pellet and supernatant were separately incubated with AMG. The D-glucose released was measured using a glucose oxidase-peroxidase (GOPOD) reagent kit, and the absorbance was read at 510 nm against the reagent blank (UV6300PC, MAPADA, Shanghai). The total starch was calculated as the sum of resistant and digestible starch. Analyses were run in triplicate.

2.1.1. Unsprouted grain flours The saponin of the quinoa was removed with successive washes using tap water at room temperature with manual friction, until bitter taste was eliminated. Amaranth was also washed to eliminate dust. Both grains were dried in a forced circulation oven at 50 °C until constant weight and then the whole grains were milled in a centrifugal mill (CHINCAN model FW 100, China). Flours were vacuum-packed in polyethylene bags and stored at room temperature. 2.1.2. Sprouted grain flours The washed and saponin-free grains were soaked in tap water (1:5 w/v) for 6 h at room temperature. Water was drained and wet grains were spread in a thin layer in plastic trays covered with wet filter papers and incubated under controlled conditions: 22–24 °C and 80–90% relative humidity in darkness, 24 h for quinoa and 48 h for amaranth until both germinated grains reached the same length of radical (1.0–1.5 cm). The germinative capacity was determined according to Hager et al. (2014), counting germinated grains and expressing it as a percentage of the total number of grains. The germinated grains were dried in a forced circulation oven at 50 °C until constant weight. Dried grains were milled in a centrifugal mill (CHINCAN model FW 100, China). The flours were vacuum-packed

2.2.3. Reducing and total sugar 2.2.3.1. Reducing sugar. The reducing sugar was determined by the spectrophotometric method (Miller, 1959) with minor modifications mentioned by Başkan et al. (2016), based on cupric reduction. DNS (3,5-dinitrosalicylic acid) reagent was prepared with 1:1:1:0.5 (v/v/v/v) ratio of: DNS solution (1% w/v), phenol solution (0.2% w/v), NaOH solution (1.5% w/v) and sodium biftalate (0.15% w/v). Flour samples (1.000 ± 0.005 g) were suspended with 10 mL distilled water and the mixture was shaken. Solution was centrifuged (10,000 rpm, 10 min, 2

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20 °C). An aliquot (25 μL) was solubilized in 75 μL water and 1 mL DNS solution was added. The mixture was shaken and incubated in a boiling water bath for 5 min, then 1 mL sodium potassium tartrate (Rochelle salt) (40% w/v) was added and it was immediately cooled in ice bath during 15 min. The absorbance of sample was recorded at 540 nm against a reagent blank (UV-6300PC, MAPADA, Shanghai). A linear regression equation was obtained using a D(+)-glucose standard solution (Sigma-Aldrich, Steinheim, Germany). Analyses were run in triplicate.

blended slurry was filtered through 400 (37 μm) mesh. The filtrate was centrifuged (9000 g, 5 °C, 20 min), and the residue was re-suspended in water and neutralized with 1 M HCl. The resultant suspension was then filtered through a filter paper. The retained material (starch) was washed five times for purification with sufficient water and 200 mL of ethanol (96% v/v) and then dried in air oven at 40 °C 12 h. The dried starch was milled in a centrifugal mill (CHINCAN FW 100, China). The starch was vacuum-packed in polyethylene bags and stored at room temperature.

2.2.3.2. Total sugars. Total sugar content was determined by the method described by Başkan et al. (2016) with slight modifications. Hydrolysis of sucrose was performed in the supernatant (3 mL), obtained in point 2.2.3.1, with 0.5 mL of concentrated HCl, 15 min in a boiling water bath; the mixture was neutralized with 2 M NaOH. An aliquot (25 μL) was solubilized in 75 μL water and then analysed as indicated for reducing sugars. A linear regression equation was obtained using a hydrolyzed sucrose standard solution (Sigma, Steinheim-Germany). Analyses were run in triplicate.

2.5.2. Amylose Amylose content was measured by the method described by Juliano et al. (1981) and mentioned by Lu et al. (2013) with minor modifications, based on the measurement of the iodine–amylose complex. Starch samples (100 mg) plus 1 mL of 96% (v/v) ethanol and 9 mL of 1 M NaOH were kept at room temperature for 24 h, and then distilled water was added to make 100 mL solution. An aliquot of 0.5 mL was transferred to a 10-mL volumetric flask containing 5 mL of distilled water, and 0.1 mL 0.4 M Na2HPO4 buffer (pH 7.2) was added. Then 2 mL of 0.2 g/L iodine solution (I2: 2 g/KI: 20 g/L) and distilled water were added to make up exactly 10 mL. Spectrophotometer measurements were made at 620 nm (UV-6300PC, MAPADA, Shanghai) after the above starch–iodine solution was incubated for 20 min at room temperature. A standard curve was generated using potato amylose–amylopectin (A0512, Sigma Chem. Co., St. Louis, MO). Analyses were run in triplicate.

2.3. Protein fractions The protein fractions of samples, before and after germination, were separated by SDS–PAGE. Alkaline proteins extraction of the flours was performed with NaOH 0.2% w/v (ratio 1:10) and vortexed for 30 min (Rumiyati et al., 2012). The mixture was centrifuged (8000 g, 25 °C, 5 min). The supernatant was dissolved with sample buffer pH 6.8 (ratio 1:1) containing 10% (v/v) glycerol, 1% (w/v) SDS and 0.05% (w/v) bromophenol blue. To determine the disulfide bonds, the samples were run in conditions similar to those above but with the addition of 5% (v/ v) β-mercaptoethanol and heating in a boiling water bath for 5 min (denaturing and reducing conditions). Molecular weight (MW) markers from Sigma (6.5–205 kDa) were used. The stacking gel had 5%T acrylamide:bisacrylamide (0.049 M Tris−HCl, pH 6.8) and the separating gel was 10%T and 12%T for runs without and with β-mercaptoethanol respectively (0.375 M Tris−HCl, pH 8.8); both contained 1% (w/v) SDS. Running buffer (pH 8.3) was prepared with 0.025 M Tris−HCl, 0.186 M glycine and 1% (w/v) SDS. The electrophoresis runs were conducted at a constant voltage of 100 V. Gels were washed with washing solution of acetic acid (10%) for 10 min and stained with 0.1% (w/v) Coomassie Brilliant Blue R 250 (Sigma, St. Louis, MO) in 10% (v/v) acetic acid for 15 min. Gels were scanned, and the molecular mass of the bands assessed using the software GelAnalyzer2010 (J&B Lab. S.A.C, Lima, Peru).

2.5.3. Thermal behavior Thermal properties of flours were determined by differential scanning calorimeter (DSC) using a TA Instrument model Q2000 (TA Instruments, New Castle, DE). Flour (2.0 mg) was weighed into an aluminum pan and 10 μL distilled water were added. The pan was hermetically sealed and equilibrated at room temperature for 1 h, then scanned at a heating rate of 10 °C/min from 25 to 120 °C with an empty sealed pan as a reference. Onset (To), peak (Tp), final (Tf) temperatures, and enthalpy of gelatinization (ΔH) were determined by Universal Analysis 2000 Program (TA Instruments). After cooling, the scanned sample pans were placed in a refrigerator at 4 °C for 21 days. Retrogradation properties were measured by rescanning the sample pans under the same conditions. The percentage of retrogradation (R%) was calculated as (ΔHr/ΔH) × 100. The experiments were carried out in triplicate. 2.6. Statistical analysis Results were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to compare the means. Differences were considered significant at p < 0.05. Statistical analyses were performed with Statistix for Windows version 9.0 (Analytical Software, Tallahassee, FL).

2.4. Protein digestibility In vitro protein digestibility was determined by AOAC 971.09 method modified by Miller (2002). Defatted flours (1 g) were digested with 150 mL of pepsin solution in 0.075 M HCl (0.0002% v/v) and incubated in a shaking bath (45 °C, 16 h) (Viking Shaker, Argentina). The digested samples were vacuum-filtered and washed three times with water and acetone. In the residue, the nitrogen content was determined by Kjeldahl method (AOAC 920.87). The value was corrected by a nitrogen determination in an HCl solution free of pepsin; then the protein was calculated by using 6.25 factor. Analyses were run in triplicate.

3. Results Quinoa and amaranth grains possessed high germinative capacity. Quinoa reached a length of radicle of 1.0–1.5 cm in 24 h while the amaranth reached the same length after 48 h. 3.1. Proximate composition

2.5. Starch Table 1 shows the proximate composition of unsprouted and sprouted grains. The germination increased the protein content significantly in all varieties of quinoa and amaranth (p < 0.05), except in the Inga Pirca variety; in which the protein content did not undergo significant change. On the other hand, the lipid content was not modified significantly with the germination. The ash content presented a slight increase with the germination, only significant (p < 0.05) in the

2.5.1. Isolation Starch was isolated from the flours of the quinoa and amaranth varieties by alkaline steeping method (Jan et al., 2017) with slight modifications in the procedure. Defatted flour was dispersed in water (1:10 w/v) and the pH was adjusted to 9.0 with 2 M NaOH and placed at room temperature for 24 h. The samples were mixed for 30 min and the 3

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Table 1 Proximate composition of grain flours. Grain variety Quinoa Cica Sprouted Quinoa Cica Quinoa Kamiri Sprouted Quinoa Kamiri Quinoa Inga Pirca Sprouted Quinoa Inga Pirca Amaranth Rosado Sprouted Amaranth Rosado Amaranth Mantegazzianus Sprouted Amaranth Mantegazzianus

Table 2 Protein digestibility.

Ash 2.16 2.26 2.36 2.58 2.25 2.37 2.88 3.03 3.05 3.53

Protein ± ± ± ± ± ± ± ± ± ±

e

0.10 0.01de 0.02d 0.14c 0.02de 0.02d 0.05b 0.05b 0.05b 0.06a

12.74 14.31 12.97 15.78 14.55 14.00 12.51 13.94 13.74 14.69

± ± ± ± ± ± ± ± ± ±

Fat f

0.31 0.14bcd 0.22ef 0.12a 0.24bc 0.31cd 0.12f 0.21cd 0.16de 0.19bc

7.48 5.61 6.53 5.99 6.75 6.79 7.78 8.81 6.66 7.00

± ± ± ± ± ± ± ± ± ±

ab

0.14 1.94b 0.18ab 0.08b 0.27ab 0.05ab 0.02ab 0.33a 0.14ab 0.25ab

Grain variety

Protein digestibility (%)

Quinoa Cica Sprouted Quinoa Cica Quinoa Kamiri Sprouted Quinoa Kamiri Quinoa Inga Pirca Sprouted Quinoa Inga Pirca Amaranth Rosado Sprouted Amaranth Rosado Amaranth Mantegazzianus Sprouted Amaranth Mantegazzianus

61.27 87.11 63.24 72.89 63.23 72.89 72.33 86.73 71.57 85.34

± ± ± ± ± ± ± ± ± ±

0.38b 0.41a 3.58b 5.30b 1.21b 0.24b 3.69b 2.19a 4.35b 2.56a

Values are means ± standard deviations of data from triplicate analysis. Values followed by different superscript letters in the same column are significantly different (p < 0.05).

Values are means ± standard deviations (g/100 g db) from triplicate analysis. Values followed by different superscript letters in the same column are significantly different (p < 0.05).

other authors (Bedón Gómez et al., 2013; Janssen et al., 2017). They also differentiated the protein fractions of quinoa into albumins (14–66 kDa), globulins (14–36 kDa), prolamins (24, 29, 36 and 55 kDa) and glutelins (14–55 kDa); and albumins1 (20–36, 45, 66 kDa), albumins2 (29, 36, 45, 66 and > 66 kDa) and globulins (29 a > 66 kDa) for amaranth. Some protein bands are smaller in the profiles of germinated grain samples, indicating enzymatic hydrolysis of proteins during

Kamiri varieties for quinoa and Mantegazzianus for amaranth. 3.2. Protein fractions The polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the protein extracts of unsprouted and sprouted quinoa and amaranth is shown in Fig. 1a and b, respectively. Proteins of molecular weights between 14 and 66 kDa were identified (Fig. 1a and b); this agrees with

Fig. 1. Representative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (a and b) without and (c and d) with β-mercaptoethanol. P: MW standard; 1: Quinoa Cica; 2: Quinoa Kamiri; 3: Quinoa Inga Pirca; 4: Sprouted Quinoa Cica; 5: Sprouted Quinoa Kamiri; 6: Sprouted Quinoa Inga Pirca; 7: Sprouted Amaranth Mantegazzianus; 8: Sprouted Amaranth Rosado; 9: Sprouted Amaranth Mantegazzianus; 10: Sprouted Amaranth Rosado. 4

Journal of Food Composition and Analysis 84 (2019) 103290

73.80 74.24 74.74 71.75 72.81 72.88 72.77 71.44 73.79 71.22

germination. The electrophoretic profile with β-mercaptoethanol showed new bands (Fig. 1c and d) with respect to electrophoretic profile without this reducing agent (Fig. 1a and b), which means that the mercaptoethanol broke the disulfide bonds, evidencing their presence in the proteins of quinoa and amaranth grains, before and after germination.

0.56a 0.36a 0.45a 0.85a 0.79a 1.01a 0.52a 0.47a 0.50a 1.01a

Total carbohydrate

M.D. Jimenez, et al.

Soluble fiber

3.85 3.55 3.71 3.75 3.28 2.55 3.54 4.17 2.99 3.44

Insoluble fiber

7.99 ± 0.46d 9.37 ± 0.28bcd 7.91 ± 0.25d 8.86 ± 0.94cd 9.02 ± 0.29cd 11.85 ± 1.32ab 12.00 ± 1.93ab 11.29 ± 1.44abc 12.19 ± 0.27a 11.85 ± 0.14ab

± ± ± ± ± ± ± ± ± ±

3.3. Protein digestibility in vitro Table 2 presents the protein digestibility in vitro of sprouted and unsprouted grain flours. The protein digestibility was improved significantly with germination in all varieties of both grains. This increase was also observed by Chaparro et al. (2010) for quinoa and amaranth grains. Omary et al. (2012) observed increments in protein digestibility with the germination of millet, corn and sorghum, among others.

0.51bc 0.32abc 0.35c 0.89abc 0.54abc 1.17abc 1.23a 0.96a 0.39ab 0.57a

3.4. Carbohydrates

11.83 12.92 11.62 12.61 12.29 14.40 15.54 15.46 15.18 15.28 5.41 ± 0.47e 29.73 ± 1.18c 4.42 ± 0.05e 34.02 ± 0.87b 4.75 ± 0.17e 42.75 ± 1.52a 5.48 ± 0.45e 18.97 ± 1.03d 5.02 ± 0.05e 29.41 ± 1.41c

Values are means ± standard deviations (g/100 g db) from triplicate analysis. Values followed by different superscript letters in the same column are significantly different (p < 0.05).

0.25e 0.77a 0.09ef 0.75c 0.14ef 0.60b 0.33ef 0.68d 0.30f 0.66c 17.58 52.28 14.05 35.57 14.73 42.98 14.62 29.63 13.23 39.23 43.69 ± 0.28ab 8.56 ± 0.55f 48.51 ± 1.64a 22.97 ± 0.55d 45.10 ± 1.55ab 15.00 ± 1.61e 37.57 ± 0.57c 21.67 ± 1.28d 41.27 ± 1.76bc 12.86 ± 1.95ef Quinoa Cica Sprouted Quinoa Cica Quinoa Kamiri Sprouted Quinoa Kamiri Quinoa Inga Pirca Sprouted Quinoa Inga Pirca Amaranth Rosado Sprouted Amaranth Rosado Amaranth Mantegazzianus Sprouted Amaranth Mantegazzianus

44.39 ± 0.30ab 9.04 ± 0.54e 49.07 ± 1.63a 23.57 ± 0.68c 45.79 ± 1.58ab 15.50 ± 1.63d 42.61 ± 0.55b 26.35 ± 0.93c 45.38 ± 1.67ab 16.71 ± 1.59d

0.70 0.47 0.56 0.59 0.69 0.50 5.05 4.68 4.12 3.86

± ± ± ± ± ± ± ± ± ±

0.02d 0.01d 0.01d 0.13d 0.03d 0.02d 0.02a 0.35ab 0.09bc 0.35c

Total sugar

Table 4 shows the amylose content in the starches of all studied grain samples. It can be observed that the apparent amylose increased significantly (p < 0.05) in all grains with germination. The increase was between 13 and 65% in quinoa while in amaranth it was higher than 100% for both studied varieties.

Digestible Starch Resistant Starch Total starch Grain variety

Table 3 Carbohydrates composition of grain flours.

3.5. Amylose

± ± ± ± ± ± ± ± ± ±

Total Fiber Reducing sugar

± ± ± ± ± ± ± ± ± ±

Table 3 shows the carbohydrates composition of the different flours. The resistant starch did not show significant variations by germination, while the digestible starch decreased significantly in all the varieties studied. This was observed by other authors (Omary et al., 2012; Wu et al., 2013; Hager et al., 2014) in legumes, millet, rice, amaranth and quinoa. The total, soluble and insoluble fiber contents did not show significant variations during germination, except in the variety of quinoa Inga Pirca where insoluble fiber increased (p < 0.05). Insoluble dietary fiber was the main fraction of total dietary fiber in all samples, since it represented from 67 to 80%.

3.6. Thermal behaviors Table 5 shows thermal behavior of quinoa and amaranth flours before and after germination. The gelatinization temperatures (initial, peak and final) did not show significant variations between unsprouted and sprouted grain flours, which indicated that the cooking temperature of the grains before and after germination would be similar. However, the gelatinization energy (ΔH) was significantly less in the sprouted grain flours. The reduction of the enthalpy (ΔH) with germination (Table 5) could be Table 4 Apparent amylose content of starch. Grain variety

Amylose

Quinoa Cica Sprouted Quinoa Cica Quinoa Kamiri Sprouted Quinoa Kamiri Quinoa Inga Pirca Sprouted Quinoa Inga Pirca Amaranth Rosado Sprouted Amaranth Rosado Amaranth Mantegazzianus Sprouted Amaranth Mantegazzianus

9.45 ± 0.04d 14.24 ± 0.11a 7.45 ± 0.26e 12.32 ± 0.15b 7.76 ± 0.04e 8.74 ± 0.15d 1.45 ± 0.26f 8.76 ± 0.41d 1.74 ± 0.15f 11.47 ± 0.22c

Values are means ± standard deviations (g/100 g db) from triplicate analysis. Values followed by different superscript letters in the same column are significantly different (p < 0.05). 5

Journal of Food Composition and Analysis 84 (2019) 103290

0.90 2.53a 2.29a 1.95a 1.31a 0.98a 2.42a 1.95a 0.80a 2.22a ± ± ± ± ± ± ± ± ± ± 81.53 81.58 81.74 81.32 81.69 81.37 86.26 81.10 88.38 82.33 0.46 1.70a 1.89a 2.37a 0.88a 0.69a 2.12a 1.97a 0.87a 1.41a ± ± ± ± ± ± ± ± ± ± 65.24 67.93 67.57 67.66 67.52 68.82 68.32 70.37 72.19 70.68 1.22 1.83a 1.62a 1.50a 1.31a 1.18a 1.41a 1.30a 2.02a 1.67a ± ± ± ± ± ± ± ± ± ± 60.83 61.09 62.20 62.62 62.18 62.04 64.05 65.03 66.14 66.80

To (ºC)

Values are means ± standard deviations from triplicate analysis. Values followed by different superscript letters in the same column are significantly different (p < 0.05). Q.: Quinoa; A.: Amaranth; Sp.: Sprouted; To: initial gelatinization temperature; Tp: peak gelatinization temperature; Tf: final gelatinization temperature; ΔH: gelatinization enthalpy; To(r): initial retrogradation temperature; Tp(r): peak retrogradation temperature; Tf(r): final retrogradation temperature; ΔHr: retrogradation enthalpy; R%: percentage of retrogradation.

0.04a 0.02b 0.02bc 0.02bcd 0.02cde 0.02de 0.02cd 0.02ef 0.01bc 0.02bc ± ± ± ± ± ± ± ± ± ± 1.21 0.89 0.76 0.69 0.57 0.52 0.63 0.40 0.27 0.75 2.14 0.97a 1.67a 1.53a 1.04a 1.02a 1.22a 1.59a 2.06a 0.91a ± ± ± ± ± ± ± ± ± ± 64.88 64.84 66.84 63.75 60.89 60.18 64.26 63.55 64.35 60.71 1.34 1.67a 0.80a 1.37a 1.61a 0.69a 1.23a 0.57a 1.63a 1.82a ± ± ± ± ± ± ± ± ± ± 51.57 52.13 50.14 50.70 50.44 49.45 49.27 47.52 53.31 50.69 0.32 1.39a 1.01a 1.11a 0.80a 0.95a 1.21a 1.10a 1.24a 0.60a ± ± ± ± ± ± ± ± ± ± 39.58 40.79 38.96 39.53 37.96 37.86 38.98 39.15 41.45 40.15 8.62 ± 0.28 4.87 ± 0.13cd 5.46 ± 0.11c 3.28 ± 0.03ef 5.71 ± 0.11c 3.66 ± 0.05def 10.35 ± 0.25a 2.59 ± 0.08f 3.98 ± 0.03de 2.92 ± 0.07ef

To(r) (ºC)

a

Q. Cica Q. Cica Sp. Q. Kamiri Q. Kamiri Sp. Q. Inga Pirca Q. Inga Pirca Sp. A. Rosado A. Rosado Sp. A. Mantegazzianus A. Mantegazzianus Sp.

Grain variety

Table 5 Thermal behavior of flours.

Gelatinization

a

Tp (ºC)

a

Tf (ºC)

a

ΔH (J/g)

b

Retrogradation

Tp(r) (ºC)

a

Tf(r) (ºC)

a

ΔHr (J/g)

R%

14.04 ± 0.03d 18.28 ± 0.09c 13.91 ± 0.23d 21.02 ± 1.35b 9.97 ± 0.45e 14.19 ± 0.81d 6.09 ± 0.06f 15.43 ± 0.33d 6.78 ± 0.36f 25.67 ± 0.49a

M.D. Jimenez, et al.

related to the reduction in the starch content (Table 3). The retrogradation enthalpies (ΔHr) in germinated grain flours were less than in non-germinated only in the Cica quinoa variety and in the Rosado amaranth variety, while in other varieties ΔHr did not suffer significant variations with germination. 4. Discussion The ash content increased slightly with the germination of Kamiri quinoa and Mategazzianus amaranth. The mineral content of tap water used for germination (wash and soaked) may have contributed to the ash content (Suma and Urooj, 2014) or it could be explained by a loss of dry matter which produced an apparent increase of ash content (Khalil et al., 2007). In other varieties, the ash content did not present significant variation. These results were in agreement with other authors (Omary et al., 2012). The lipid content did not show variations during the germination and the obtained values agreed with the ones observed by Omary et al. (2012). However, other research showed a reduction of the lipid content with germination, as lipids would have been used as an energy source (Rumiyati et al., 2012; Devi et al., 2015). Protein content increased during germination, from 12 to 22% db in quinoa and between 7 and 11% db in amaranth. Apparent increases in protein content (and other nutrients) could be explained by a loss of dry matter, particularly through carbohydrates oxidation and the release of CO2, H2O and small amounts of ethanol. These results agree with those shown by Khalil et al. (2007) and Omary et al. (2012). On the other hand, Kanensi et al. (2011) did not report significant changes in protein content in the germination of amaranth grains, with 5 h of soaking and 24 h of germination; but by varying the germination time to 48 h, these authors found that the protein content increased significantly. In addition, electrophoretic profiles showed that proteins, especially those of high molecular weight, were hydrolyzed and mobilized during germination. High molecular weight proteins were enzymatically degraded with germination; in quinoa the proteins greater than 24 KDa were mainly degraded, while in amaranth the degradations were produced in all fractions between 14 and 66 KDa. Degradation is observed by the reduction of band width, which would involve less protein of those molecular weights in the samples of sprouted grains. The decrease in the molecular size of proteins could be related to the increased in protein digestibility, which was 15–42% in quinoa and 19–20% in amaranth (Chaparro et al., 2010; Omary et al., 2012). Quinoa and amaranth proteins have disulfide bonds (ValcárcelYamani and da Silva Lannes, 2012); germination did not break these unions completely as shown in Fig. 1, comparing the protein profile in the SDS–PAGE without and with β-mercaptoethanol. If the flours are used in a food formulation where the viscosifying and gelling properties will stabilize the food matrix (Liu et al., 2016), the presence of disulfide bonds is important. In addition, the existence of disulfide bonds indicates the presence of essential sulfur amino acids (methionine and cysteine) which would nutritionally improve the formulated products (Valcárcel-Yamani and da Silva Lannes, 2012). Total, soluble and insoluble fiber contents did not change by germination (except in quinoa Inga Pirca variety), a result which coincided with those of some authors (Repo-Carrasco-Valencia and Serna, 2011; Quinto et al. 2015). Nevertheless, other authors have reported that the fiber content increased with germination (Moongngarm and Saetung, 2010) while other researchers have reported that the fiber decreased in germination of buckwheat, rice, sorghum and amaranth (Omary et al., 2012). Dueñas et al. (2016) reported a notable increase during germination in the level of insoluble and total dietary fiber in beans and, on the contrary, germinated lentils showed a decrease in the total dietary fiber as a consequence of the marked decrease in the insoluble dietary fiber. These results are in concordance with Gong et al. (2018) who indicated an increase in the soluble fiber and a decrease in the insoluble fiber upon germination of corn. Dueñas et al. (2016) indicated that an 6

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M.D. Jimenez, et al.

sensory properties of formulated food with germinated grain flours. Thus, as this study shows, germination is a low-cost technology that improves the nutritional contributions of quinoa and amaranth grains. However, if these flours are used as ingredients in food formulations, modifications of the thermal behavior and rheological properties must be considered.

increase in soluble fiber may be a consequence of the rise of cellulosic glucose due to the metabolic reactions undergone by seeds during germination. Thus the effect of germination in total, soluble and insoluble dietary fiber content depends on the type of grain. These grains exhibited different behaviors during the process, due to the different structures and compositions of the cell wall as well as the conditions of germination process (Omary et al., 2012; Dueñas et al., 2016; Gong et al., 2018). Digestible starch decreased during germination by action of amylolytic enzymes. The partial hydrolysis of starch produced a significant increase (p < 0.05) in reducing and total sugars higher than 100%; these results are similar to those reported by Hager et al. (2014) for quinoa and amaranth. The starch hydrolysis would increase its digestibility, which could produce an increase in the glycemic and insulinemic response (Bedón Gómez et al., 2013; Świeca et al., 2013). On the other hand, the resistant starch did not suffer significant variations because it cannot be attacked by enzymes during germination. The amylolytic enzymes, α-amylase and β-amylase, hydrolyzed the starch, producing mainly dextrin, glucans, sucrose, fructose, glucose, maltose, oligomaltose and maltotriose. The increment of apparent amylose content (between 13–65% in quinoa grains and over 100% in amaranth grains) could originate from the hydrolysis of primary amylopectin that releases linear branches of glucan chains and dextrins which are reactive to the iodine just like the amylose (Juliano et al., 1981). This assumption is consistent with the results obtained by Wu et al. (2013), who determined the amylose/amylopectin ratio in flours of different cultivars of brown rice after different germination times, by enzymatic method amylose–amylopectin assay kit. These authors reported that amylose and amylopectin gradually decreased with germination. Gelatinization enthalpy decreased probably by starch hydrolysis with increased amylose/amylopectin ratio due to greater degradation of the amylopectin during germination. These results are similar to the ones obtained by Wu et al. (2013), who studied the changes in rice starch after germination; they noted a slight decrease in gelatinization enthalpy, probably due to an increase in the amylose/amylopectin ratio during germination. Enthalpy of retrogradation did not change in the same way for all varieties of grain flours before and after germination. This measure depends on several variables, such as the molecular ratio of amylose/ amylopectin, structures of the amylose and amylopectin molecules (which are determined by the botanical source of the starch), starch content, the presence and concentration of other components (Fennema, 2017). On the other hand, germination increased the retrogradation (R%) in quinoa and amaranth caused by the higher amylose/amylopectin ratio (Table 4). Although R% increased, it is important to emphasize that the total content of starch in sprouted grains was lower (Table 3). Therefore, the amount or crystalline structures capable of retaining water was also lower. Yu et al. (2009) reached the same conclusions in the study of the impact of amylose content on retrogradation of rice starch.

Declaration of Competing Interest The authors declare do not have conflict of interest of any kind. Acknowledgements The authors would like to thank Silvia Rebeca Chañi for her technical assistance in the determinations in differential scanning calorimeter (DSC), in National University of Jujuy (Jujuy, Argentina). This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ministerio de Ciencia y Tecnología, Argentina. References AOAC, 2017. Association of Official Analytical Chemists. Methods of Analysis. AOAC. http://www.aoac.org/. Başkan, K.S., Tütem, E., Akyüz, E., Özen, S., Apak, R., 2016. Spectrophotometric total reducing sugars assay based on cupric reduction. Talanta 147, 162–168. Bedón Gómez, M., Nolasco Cárdenas, O., Santa Cruz Carpio, C., Gutiérrez Román, A., 2013. Partial Purification and Characterization of Alpha Amylase from germinated grains from Chenopopdium quinoa (Quinua). J. Int. Sci. Meet. 10 (1), 51–57. Carciochi, R.A., Manrique, G.D., Dimitrov, K., 2014. Changes in phenolic composition and antioxidant activity during germination of quinoa seeds (Chenopodium quinoa Willd). Int. Food Res. J. 21 (2), 767–773. Chaparro, D.C., Pismag, P., Correa, E., Quila, V., Caicedo, C.A., 2010. Effect of the germination on the protein content and digestibility in amaranth, quinoa, soy bean and grandul seeds. Biotecnología en el Sector Agropecuario y Agroindustrial 8 (1), 35–42. Devi, C., Kushwaha, A., Kumar, A., 2015. Sprouting characteristics and associated changes in nutritional composition of cowpea (Vigna unguiculata). J. Food Sci. Technol. 52 (10), 6821–6827. Dueñas, M., Sarmento, T., Aguilera, Y., Benitez, V., Mollá, E., Esteban, R.M., MartínCabrejas, M.A., 2016. Impact of cooking and germination on phenolic composition and dietary fibre fractions in dark beans (Phaseolus vulgaris L.) and lentils (Lens culinaris L.). Lwt - Food Sci. Technol. 66, 72–78. Fennema, O.R., 2017. Food Chemistry, 5th edition. ed. Acribia, Zaragoza (España). Fu, B.X., Hatcher, D.W., Schlichting, L., 2014. Effects of sprout damage on durum wheat milling and pasta processing quality. Can. J. Plant Sci. 94, 545–553. Gong, K., Chen, L., Li, X., Sun, L., Liu, K., 2018. Effects of germination combined with extrusion on the nutritional composition, functional properties and polyphenol profile and related in vitro hypoglycemic effect of whole grain corn. J. Cereal Sci. 83, 1–8. Goyoaga, C., Burbano, C., Cuadrado, C., Romero, C., Guillamón, E., Varela, A., Pedrosa, M.M., Muzquiz, M., 2011. Content and distribution of protein, sugars and inositol phosphates during the germination and seedling growth of two cultivars of Vicia faba. J. Food Compos. Anal. 24, 391–397. Hager, A.S., Mäkinen, O.E., Arendt, E.K., 2014. Amylolytic activities and starch reserve mobilization during the germination of quinoa. Eur. Food Res. Technol. 239 (4), 621–627. Hallen, E., IIbanoglu, S., Ainsworth, P., 2004. Effect of fermented/germinated cowpea flour addition on the rheological and baking properties of wheat flour. J. Food Eng. 63, 177–184. Jan, K.N., Panesar, P.S., Rana, J.C., Singh, S., 2017. Structural, thermal and rheological properties of starches isolated from Indian quinoa varieties. Int. J. Biol. Macromol. 102, 315–322. Janssen, F., Pauly, A., Rombouts, I., Jansens, K.J.A., Deleu, L.J., Delcour, J.A., 2017. Proteins of Amaranth (Amaranthus spp.), Buckwheat (Fagopyrum spp.), and Quinoa (Chenopodium spp.). A Food Science and Technology Perspective. Comprehens. Rev. Food Sci. Food Safety 16, 39–58. Jideani, V.A., Onwubali, F.C., 2009. Optimization of wheat-sprouted soybean flour bread using response surface methodology. Afr. J. Biotechnol. 8 (22), 6364–6373. Juliano, B.O., Perez, C.M., Blakeney, A.S., Castillot, D., Kongseree, N., Laingnelet, B., Lapis, E.T., Murty, V.S., Paule, C.M., Webb, B.D., 1981. International Cooperative testing on the amylose content of milled rice. Starch 33, 157–162. Kanensi, O.J., Ochola, S., Gikonyo, N.K., Makokha, A., 2011. Optimization of the period of steeping and germination for amaranth grain. J. Agric. Food Technol. 1 (6), 101–105. Khalil, A.W., Zeb, A., Mahmood, F., Tariq, S., Khattak, A.B., Shah, H., 2007. Comparison of sprout quality characteristics of desi and kabuli type chickpea cultivars (Cicer arietinum L.). Lwt - Food Sci. Technol. 40, 937–945. Liu, G., Jaeger, T.C., Lund, M.N., Nielsen, S.B., Ray, C.A., 2016. Effects of disulphide bonds between added whey protein aggregates and other milk components on the

5. Conclusions Germination improved the nutritional contributions of the quinoa and amaranth flours, as the proteins and carbohydrates were hydrolyzed during germination, and the protein hydrolysis improved their digestibility. The decrease in the starch content and the changes in its quality, produced by germination, generated alterations in the gelatinization enthalpy, but the cooking temperatures were not modified. However, germination increased the retrogradation of the flours, due to the structural modifications that carbohydrates and proteins undergo. In this way, the changes in the starch and proteins during germination could surely produce changes in the rheological and textural behavior of the products elaborated with these flours. and probably in the 7

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