Antioxidant Activities of Selective Gluten Free Ancient Grains

Ancient grains were known for special nutritional values along with gluten free qualities. Amaranth, quinoa, teff, and buckwheat flours were evaluated for pasting properties, water holding capacities, phenolic contents, and antioxidant activities (free and bound). They all had higher water holding capacities than wheat flour. Amaranth, quinoa, and teff showed higher pasting viscosities than wheat flour. Buckwheat flour had the highest free, bound and total phenolic contents among the flours in all aqueous extracts. The bound phenolic contents were higher than the free phenolic contents regardless of the solvents with the exception of water extraction of quinoa and buckwheat. The free phenolic compounds for all four flours were highest in water extract, and least in 100% ethanol. Bound antioxidant activities were much higher than the free antioxidant activities regardless of solvents for all products. The free antioxidant activities from water and 50% ethanol were higher than 100% ethanol. In contrast, more bound phenolics were extracted with 100% ethanol than water and 50% ethanol. Our study suggested that the total phenolic contents and antioxidant activities of grains could be underestimated in the literature without considering the bound phenolic compounds. These ancient grains have nutrition, antioxidants, and textural qualities suitable for functional foods.

amaranth flour certified by international certification services Inc. was purchased from Dakota Prairie Organic Flour Co. Harvey, ND, USA. Gluten free quinoa and teff flour was purchased from Bob's Red Mill, Milwaukie, OR, USA.

Water-Holding Capacity
The water-holding capacity (WHC) of the samples was determined according to a previous procedure with minor modifications [22]. Each sample (2 g, dry weight) was mixed with 25 g of distilled water and vigorously mixed for 1 min to a homogenous suspension using a Vortex stirrer, held for 2 h, and centrifuged at 1590 g for 10 min. Each treatment was replicated twice. Water-holding capacity was calculated on dry basis by the difference between the weight of water added and decanted.

Pasting Property Measurement
The pasting properties of samples were evaluated using a Rapid Visco Analyzer (RVA-4, Perten Scientific, Springfield, IL). Samples (2.24 g, dry basis) were made up to a total weight of 28 g with distilled water in a RVA canister (8% solids, w/w). The viscosity of the suspensions was monitored during the following heating and cooling stages. Suspensions were equilibrated at 50˚C for 1 min, heated to 95˚C at a rate of 6.0˚C/min, maintained at 95˚C for 5 min, and cooled to 50˚C at rate of 6.0˚C/min, and held at 50˚C for 2 min. For all test measurements, a constant paddle rotating speed (160 rpm) was used throughout the entire analysis except for 920 rpm in the first 10 s to disperse sample. Each sample was analyzed in duplicate. The results were expressed in Rapid Visco Analyser units (RVU, 1 RVU = 12 centipoises).

Sequential Alkaline Extraction
Step 1: double extraction for free compounds. The extraction conditions were modified based on the research by Serpen (2008) [21]. Samples (1 g) were extracted with 10 ml of 50% ethanol twice under N 2 by shaking in a water bath for 20 min. Each extraction step was followed by centrifugation at 3000 rpm (1462 g) for 10 min. Combined supernatants were used for free phenolic and antioxidant activity measurements.
Step 2: alkaline extraction for bound compounds. The solid residue from double extraction was hydrolyzed with 20 mL of 2 N sodium hydroxide for 2 h under N 2 by shaking in the dark at room temperature [23]. The alkaline extracts were acidified using about 20 mL of 2 N HCl and centrifuged at 3000 rpm (1462 g) for 10 min. The supernatants were used for bound phenolic and antioxidant activity measurements.

Total Phenolic Content
Phenolic content was determined by the Folin-Ciocalteau colourimetric method with minor modifications [24] [25]. To 100 µL of extract, 7.9 mL of deionized water and 0.5 mL of Folin-Ciocalteau reagent (F9252, Sigma Aldrich, St Louis, MO) were added, mixed on a vortex mixer, and 1.5 mL of 1.85 M Na 2 CO 3 was added after 15 min. Absorbance of samples was measured at 765 nm after 2 h. Gallic acid was used as a standard and results were expressed as mg of gallic acid equivalents per g (d.m.).

Statistical Analysis
Data from replicated samples were analyzed using SAS software using analysis of variance with Duncan's multiple comparison adjustment to determine significant differences (p < 0.05) between treatments [27].

Nutrition Composition
The ancient gain flours in Table 1 contain more proteins and minerals including calcium, iron, magnesium, phosphorus, potassium, and zinc than wheat flour. Amaranth contains the highest magnesium and phosphorus;  quinoa has the highest potassium; and teff has the highest calcium, iron, and zinc of all products. Also they are all rich in vitamins. Amaranth has the highest vitamin C, and vitamin B-6, quinoa has highest folate, vitamin E and D; buckwheat has highest riboflavin and niacin contents. Quinoa and teff contain higher thiamin (B1) than the other two ancient grain flours. Vitamin B1 helps the body metabolizes fats and protein, and is required for healthy skin, hair, eyes, and liver. It also helps the nervous system function properly and good brain functions [28]. Particularly, teff is the only one containing Vitamin K among the products in the Table 1. Vitamin K is a fat-soluble vitamin that the body stores it in fat tissue and the liver. It is best known for its role in helping blood clot and bone health [28]. Both quinoa and teff have high quality lipids including monounsaturated and polyunsaturated fatty acid contents. These ancient grains have endured the ages from early civilizations as an important food source to its current resurgence as a highly nutritious gluten-free source. Therefore, the nutritional value of gluten free baked products could be improved by adding ancient grain products to recipes.

Water-Holding Capacity
The WHC of the ancient grains and wheat flours are shown in Figure 1. Among the four gluten free flours, buckwheat had the highest WHC (164%), the teff ranked in second (144%), and WHC of amaranth was the least (125%), that was slightly lower than WHC of quinoa (130%). Overall, all ancient grain flours in this study had higher WHC than that of wheat flour (92%). These gluten free ancient gain flours could be widely used in different food applications because of better WHC for improved texture along with nutrition compared to wheat flour.

RVA Pasting Properties
The pasting curves of all products were obtained by RVA expressed as RVU (1 RVU = 12 centipoises). As shown in Figure 2, the pasting curves for all samples had dissimilar patterns. The Amaranth pasting viscosity exhibited a significantly high (~52 RVU) and sharp viscosity peak (~5 RVU/min) during the initial 11 min  heating period at ~90˚C followed by a gradually decrease in viscosity (1 RVU/min) to ~41 RVU during continued heating and cooling at 18 min, and increase to the final viscosity (53 RVU) during cooling down showing a break down (peak viscosity minus the lowest point of viscosity after peak). The viscosity of quinoa showed the initial peak (~33 RVU) at 95˚C, and then the viscosity showed a shallow break down and then decreased to a final viscosity (~28 RVU) that was lower than that of amaranth (~53 RVU). The viscosity of teff increased to the initial peak (~18 RVU) after temperature reaching 95˚C at 12 min, keeping a near constant viscosity during heating, and then increased sharply (~2 RVU/min) during cooling resulting in a final viscosity (37 RVU) that was higher than the final viscosity of quinoa (~28 RVU). Perhaps, it was due to highest calcium content that reacted with protein in teff. It could be a similar mechanism to make tofu by coagulating proteins in soymilk with calcium or magnesium sulfate. The proteins coagulate when bonding occurs between the positively charged calcium ions and negatively charged anionic groups of the protein molecules. The calcium and magnesium in teff could be reacted with protein causing the increased viscosity during cooling. Buckwheat flour had the lowest initial viscosity peak (~6 RVU) than other samples at 95˚C, and then kept nearly constant value to the final viscosity (~7 RVU) that was lowest among all samples. The initial peak (~17 RVU) for wheat was similar to teff but it decreased sharply on 12 min at 95˚C. It is known that the viscosity of a completely gelatinized starch slurry decreases during heating [29].
The RVA data could provide useful information for food processing and product development. The product with low viscosity may be suitable for products such as cookies, muffin, or nutritional bars. The product with high viscosities could be used for products such as breads for improved the texture quality and health benefits.

Free, Bound and Total Phenolic Contents
Buckwheat flour had the highest free, bound and total phenolic contents among the four flours when extracted with water, 50% ethanol and 100% ethanol ( Table 2). Buckwheat (Farinetta) flour contains a considerable amount of aleurone layer along with embryo. Cereals are rich in substituted cinnamic acids such as ferulic acid that are esterified to cell walls, to arabinoxylan and arabinogalactan in the aleurone layer, and pericarp [30]. The free phenolic contents were ~20% -60% of total phenolic contents while the bound phenolic contents were ~22% -79% of total phenolic contents. Overall, the bound phenolic contents were higher than free phenolic contents regardless of the ethanol concentrations with exception of water extraction of quinoa and buckwheat. Studies have reported that the phenolic compounds are primarily bound to cell walls for most cereal grains [23]. The bound phenolic compounds probably are released under alkaline extraction conditions. Amaranth, quinoa, and teff flour were all significantly lower in the free, bound, and total phenolic contents than that of buckwheat regardless of the ethanol concentrations. Interestingly, the free phenolic compounds for all four products were significant highest in water extract, secondly in 50% ethanol, and were least in (the) 100% ethanol. Cinnamic and benzoic acid derivatives are generally water soluble and universally present in plant foods [31]. It may be the explanation for the more free phenolic compounds when water or 50% ethanol was used for all four flours, respectively ( Table 2). In contrast to the free phenolic contents, more bound phenolics were extracted with 100% ethanol than water and 50% ethanol. It implied that bound phenolics are more soluble in 100% ethanol.
The different letters indicated the significances between treatments in column (p < 0.05).

Free, Bound and Total Antioxidant Activities
The similar trends were observed for antioxidant activities as phenolic contents ( Table 3). In general, the bound antioxidant activities are all much higher statistically than the free antioxidant activities for all products as phenolic content in spite of solvents. However, the differences of antioxidant activities bound among products seem not as large as bound phenolic contents. The free antioxidant activities ranged from 1.3% to 33.55% of total antioxidant activities whereas the bound antioxidant activities were ranged from 67.40% to 98.7% of total antioxidant activities. Our study suggests that the total phenolic contents and antioxidant activities of grains could be underestimated in the literature if not including the bound phenolic compounds. The free antioxidant activities from water and 50% ethanol were higher than those from absolute alcohol. The free antioxidant activities from water were higher than those from 50% ethanol for amaranth and quinoa. In contrast, the free antioxidant activities from water were lower than those from and 50% ethanol for teff and buckwheat. Therefore, the conclusion of solvent selection for free antioxidant activities regarding water and 50% ethanol are inclusive.

Conclusion
Amaranth, quinoa, teff and buckwheat all have special nutrients. Higher water holding capacities were found for the ancient grain flours than wheat flour. Buckwheat flour had the highest free, bound and total phenolic contents among the four products. In general, the bound phenolic contents and antioxidant properties were higher than the free phenolic contents and antioxidant activates regardless of the ethanol concentrations. The free phenolic compounds and antioxidant activities for all four products were significantly higher in extracts using water and 50% ethanol than 100% ethanol. In contrast, more bound phenolics were in 100% ethanol extracts than water and 50% ethanol extracts. Our study suggested that the total phenolic contents and antioxidant activities of grains could be underestimated in the literature without considering the bound phenolic compounds. These ancient grains have nutritional value, antioxidants, and texture qualities suitable for functional food applications.