Ascorbic Acid Content and Antioxidant Activities of White and Brown Teff [Eragrostic tef (Zucc.)Trotter] Grains and Injera

Teff [Eragrostis tef (Zuccagni) Trotter] is a cereal grain originating in Ethiopia as a staple food for millions of people. Its grain is a gluten-free superfood and got acceptance as a medicinal ingredient. Therefore, it is worthwhile to determine the antioxidative activities and L-ascorbic acid contents of teff grain and its baked food (injera). This study aimed to determine the ascorbic acid contents and antioxidant activities in the aqueous extract of the white and brown teff grains and their injera samples using iodimetric titration and UV-Vis spectrophotometric methods, respectively. The ascorbic acid contents in the white and brown teff ranged from 67.9–112.6 mg/100 g and 69.2–117.2 mg/100 g, respectively, and those in injera of the selected teff samples ranged from 30.5–32.9 mg/100 g and 37.3–43.0 mg/100 g, respectively. The antioxidant activities ranged from 1.26–7.04 μmol AAE/g for the white teff grains, 1.44–6.29 μmol AAE/g for the brown teff grains, 1.81–2.47 μmol AAE/g for white teff injera, and 3.89–4.86 μmol AAE/g for the brown teff injera samples. Findings of the present study have revealed that white teff and brown teff grains and their injera were found to have a higher content of ascorbic acid than commonly consumed grains and vegetables. No significant difference (α = 0.05) has been observed between the two varieties of teff grains with respect to the ascorbic acid content and antioxidant activities. However, there was a statistically significant difference (α = 0.05) in the ascorbic acid content and antioxidant activities between the teff grains and their injera samples. Therefore, this study indicated that teff grains and injera are rich in ascorbic acid content and antioxidant activities as compared to other cereal grains and are very crucial for human nutrition and health.


Introduction
Te need for food security and Ethiopia's diverse climatological and ecological conditions has driven most subsistence farmers to grow various crops [1]. Cereal crops are the staples and the most important source of nutrients for mankind [2]. Tef [Eragrostis tef (Zuccagni) Trotter] is a cereal grain originating in Ethiopia as a staple food to millions of people [3]. Only fve cereals (rice, wheat, maize, sorghum, and millet) account for more than half of global bread consumption. Excessive use of these selected cereals has the potential to result in genetic losses and difculty meeting future agricultural demands [4]. Tef ofers significantly higher nutritional values than most other cereal Furthermore, tef bread has a longer shelf life and a slower aging rate than rice, wheat, maize, sorghum, and barley [9,11]. Tef can also be used as a fat substitute in producing low-calorie foods due to its high viscosity and low gelling ability [4]. Due to this reason, tef is increasingly studied under the lens of local and international research to support its cultivation and commercialization [12].
Vitamins are essential micronutrients that can be fatsoluble (A, D, E, and K) or water-soluble (B and C) [13,14]. Tey are benefcial for the prevention and treatment of various diseases, including heart disease, high cholesterol levels, eye disorders, and skin disorders [7,11]. Furthermore, vitamins are essential for growth, metabolism, reproduction, and overall health. Dietary vitamin intake is critical except for vitamins D and B1, which the human body cannot synthesize [13].
Niacin, vitamin B6, thiamin, ribofavin, vitamin K (phylloquinone), vitamin A, α-tocopherol [7,15], and vitamin C [4,7] are all abundant in tef grains. Vitamin C (Lascorbic acid) has antioxidant properties and antiinfammatory and antiapoptotic properties. It can also improve the immune system in humans by lowering the body's susceptibility to viral infections. It is one of the most commonly used health supplements to boost immunity and alleviate symptoms caused by COVID-19 infections [16,17].
Most of plants and animals produce ascorbic acid from D-glucose or D-galactose [18]. L-ascorbic acid, as an antioxidant, lowers the risk of arteriosclerosis, cardiovascular disease, infectious diseases, asthma, cataract, diabetes mellitus, and some types of cancer [19]. It helps relieve common cold symptoms and plays an important role in wound healing. It also prevents free radical oxidation, preventing cell damage, and is commonly used as a food additive [20,21]. L-ascorbic acid is also necessary to prevent scurvy and maintain healthy skin, gums, and blood vessels. It aids in the formation of collagen, the absorption of inorganic iron, the reduction of plasma cholesterol levels, the inhibition of nitrosoamine formation [21,22], and the metabolism of tyrosine, folic acid, and tryptophan [21]. L-ascorbic acid less than 300 mg in the body results in scurvy and other disease symptoms. Te maximum ascorbic acid in the body is limited to about 2 g for normal health. With high doses of L-ascorbic acid (over 2 g), unabsorbed ascorbate is degraded in the intestine, causing diarrhea. Furthermore, excessive L-ascorbic acid consumption resulted in renal problems, nausea, and gastric irritation [23]. Despite the importance of tef grains for human food and nutritional security, there needs to be more qualitative and quantitative information on vitamins in general, and ascorbic acid in particular in the literature.
Te inclusion of antioxidant foods in daily diet is critical to deactivating free radicals [24]. Tef grains have shown better antioxidant potential than other cereals [9]. Tis makes injera (ready-to-eat food) to be healthy food. Among the two tef varieties, brown tef injera had shown superior antioxidant potentials compared to white tef injera [25].
Various analytical methods for determining ascorbic acid content in fruits and vegetables have been reported in the literature, including potentiometric and reductometric methods [18], high-performance liquid chromatography coupled with ultraviolet spectrophotometry (HPLC-UV) [20,26], and volumetric and spectrophotometric methods [27,28]. However, most of these methods are timeconsuming, involve multiple chromatographic steps, and require highly skilled technicians, making them problematic. To the best of the researcher's knowledge, no analytical methods for determining L-ascorbic acid contents in cereal grains, specifcally tef grain and injera samples, have been reported. As a result, this study aimed to (1) determine the Lascorbic acid contents in the aqueous extract of the white and brown tef grains and their injera using redox titration with a standardized solution of iodine, (2) evaluate the antioxidant activities of the white and brown tef grains and their injera using the DPPH assay, and (3) correlate the Lascorbic acid contents and antioxidant activities of the white and brown tef grains.

Apparatus and Instrument.
Te experiment was carried out using an electronic balance (model: PW254, China) with a precision of 0.0001 g, a grinder (high-speed multifunctional grinder, Shanghai, China), a centrifuge (model: 80-2, China), and a burette set up (10 mL). Te absorbance of the prepared standards and sample extracts was measured using a double beam UV-VIS-NIR spectrometer (Lambda 950, Perkin Elmer, UK) with a 1 cm path length quartz cuvette.

Sample Collection and Pretreatment.
Te sample collection and pretreatment were described elsewhere [3,9].
Injera was made to compare the ascorbic acid content of white and brown tef grains. Te traditional fermentation of tef four was used to prepare injera. Tef four was combined with the previous batch's water and ersho (without yeast additives). For primary fermentation, the mixture was fermented for 42 hours. A portion of the batter was mixed and boiled after primary fermentation to produce absit (gelatinization process). Te prepared absit mixes were added to the primary fermented batter and allowed to ferment for 4 hours.
Finally, the batter was made to injera using Mitad. Te prepared injera was dried for four days at room temperature before being ground to mesh size with an electronic grinder, and it was prepared for extraction.

Extraction of Ascorbic Acid in the White and Brown Tef
Grains and Injera Samples. Yisak et al. [9] described the adopted method to extract the ascorbic acid content of white and brown tef grains and injera samples. In brief, 0.25 g of ground white tef, brown tef, and injera samples was soaked for 10 min in 20 mL of the extraction solvent (deionized water). Handshaking extracted the ascorbic acid from the wetted samples for 20 minutes, and the mixture was centrifuged for 15 minute at 3000 rpm. Finally, the supernatant was fltered through Whatman flter paper and prepared for analysis.

Preparation of Reagents and
Standards. Te preparation of 3 M H 2 SO 4 , 0.5% starch indicators and preparation of iodine solution for the titration method were described elsewhere [29].

Preparation of the Ascorbic Acid Standard (100 mg/L) Solution for Standardization in the Titration Method.
A 100 mg/L-ascorbic acid standard solution was made by dissolving 0.005 g ascorbic acid (L-ascorbic acid) in a 50 mL volumetric fask with distilled water and then flling the fask to the mark with the solvent.

Standardizing Iodine Solutions for the Titration Method.
A 100 mL Erlenmeyer fask was flled with 20 mL of 100 mg/L-ascorbic acid standard solution, and 1 mL of 0.5% starch solution was added. Tis solution was titrated with a small volume of iodine solution (1.95 mL) until the endpoint was reached. Te endpoint was discovered when the frst sign of the purple-blue color appeared during the titration process. Te initial and fnal volumes of the iodine solution were then measured. Te average concentration of the iodine solution was calculated by repeating titration three times and averaging the three results.

Determination of Ascorbic Acid Content by Iodimetric
Titration. Many researchers reported titration as a preferable method for ascorbic acid determination due to its simplicity, low cost, and speed. Te ascorbic acid determination method was adopted from Belete et al. [29] and Satpathy et al. [30] with some modifcations. Te oxidationreduction reaction was carried out based on iodimetric titration of the sample. To determine the amount of ascorbic acid, 20 mL of the sample extract was taken and 1 mL of 0.5% starch solution was added to each extract.
Te solution was titrated against the prepared iodine solution while shaking continuously, and the endpoint for each sample was recorded. Te titration was performed in triplicate, and the results were presented as a mean ± SD on a dry basis from triplicate measurements.

Determination of Antioxidant Activities of the White and Brown Tef Grains and Teir Injera Samples.
Te antioxidant activity of the white and brown tef grains and their injera extracts was determined using the 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity method described by Yisak et al. [9] with some modifcations. In a 200 mL volumetric fask, 0.02 g of DPPH was dissolved with a small amount of methanol. After DPPH was completely dissolved, the fask was flled to the mark with methanol to achieve a solution concentration of 253.60 μmol/L. 3 mL of methanol and 2 mL of DPPH solution were used as controls. Furthermore, an ascorbic acid stock solution (1135.6 μmol/L) was prepared by dissolving 0.02 g of ascorbic acid in 100 mL of the volumetric fask with methanol. A calibration curve was established by preparing diferent concentrations of the stock solution (136.3, 102.2, 68.14, 17.74, and 6.036 μmol/L). A volume of 1 mL of each standard ascorbic acid solution was transferred into fve diferent 25 mL volumetric fasks, and each fask received 3 mL of methanol and 2 mL of DPPH solution before being incubated in the dark at room temperature for 60 min. Finally, the absorbance at 517 nm was measured.
A 1 mL portion of the extract was mixed with 3 mL of methanol and 2 mL of DPPH solution for the samples. For 60 min, the mixture was kept in the dark at room temperature. Using a calibration curve, the results were expressed as micromoles of ascorbic acid equivalent/g on a dry basis (μmol AAE/g). Each sample was scanned three times.

Statistical Analysis.
All analyses were performed in triplicate. On a dry matter basis, the results were reported as a mean ± SD. Te diferences in mean values between tef grain varieties and injera were determined using one-way ANOVA, followed by Tukey's honestly signifcant diference (HSD) multiple rank test (α � 0.05) [31]. Minitab 17 software was used for all statistical analyses. Tables 1 and 2 show the determined ascorbic acid contents of white tef grains and brown tef grains and their injera samples in milligrams per 100 g of four on a dry basis (mg/ 100 g dry basis). Te ascorbic acid contents of the white and brown tef aqueous extracts were 67.9-112.6 mg/100 g and 69.2-117.2 mg/100 g, respectively, and those in injera of the selected tef samples ranged from 30.5-32.9 mg/ 100 g and 37.3-43.0 mg/100 g, respectively. As shown in Table 1, the brown tef grain sample from Were Ilu district (South Wollo zone) contained the highest quantity of ascorbic acid. In contrast, the white tef grain sample from Minjar Shenkora district (North Shewa zone) contained the least quantity of all the tef grains analyzed. Te ascorbic acid content of tef grains is higher than that of the selected tef injera samples. Because ascorbic acid is susceptible to food-processing procedures and radiation due to its high solubility in water, cooking can afect its content. However, signifcant losses do not occur with the typical household cooking method [21]. Furthermore, Otemuyiwa et al. [32] described that a combination of leaching and chemical destruction causes vitamin loss during cooking.

Determination of Ascorbic Acid Content in Tef.
Gebremariam et al. [7] reported an ascorbic acid content in tef of 88 mg/100 g, which is within the range of the tef grains and higher than the content in the current study's injera samples. However, the authors did not provide any International Journal of Analytical Chemistry information about the methods used to determine ascorbic acid in the tef grain samples. In comparison to this study, the ascorbic acid content of raw rice (0.3-1.1 mg/100 g), cooked rice (0.04-0.56 mg/100 g) [32], and barley grains (0.35-0.38 mg/100 g is very low [33]. Furthermore, Satpathy et al. [30] reported ascorbic acid concentrations in some vegetables, including garlic (40.95 mg/100 g), onion (30.79 mg/ 100 g), potato (33.65 mg/100 g), tomato (16.47 mg/100 g), pea (50.84 mg/100 g), common bean (41.28 mg/100 g), pumpkin (36.30 mg/100 g), and cucumber (24.23 mg/100 g), which are in agreement with the content of ascorbic acid in the selected tef injera samples and lower than the tef grain samples of the present study. Terefore, it is evident from the present fndings that one can get enough ascorbic acid in a given tef injera food.
One-way ANOVA (α � 0.05) was used to test for the presence of signifcant diferences in the mean concentration of ascorbic acid in white and brown tef grains (Table 1) and their injera samples (Table 2). Te ANOVA test revealed no statistically signifcant diferences (α � 0.05) between the two tef grain varieties. Still, there was a signifcant diference (α � 0.05) in the mean concentration of ascorbic acid between the tef grains and their injera samples.

Precision and Recovery of the Method.
Te precision (% RSD) and accuracy (% recovery) of the titration method for determining ascorbic acid content were assessed. As a result, the method's repeatability was evaluated by calculating the relative standard deviation (RSD) of triplicate measurements, which yielded 1.8-7.6%, indicating that the method is precise. Te reproducibility (recovery) test was carried out by adding a known amount of ascorbic acid to injera, white, and brown tef extracts. Te spiked solution was analyzed three times to obtain the average recovery (R), which was calculated using the formula: [(C S − C)/C A ] × 100 � %R, where C S represents the concentration of the spiked sample extract, C represents the concentration of the unspiked sample extract, and C A represents the concentration of the spiked ascorbic acid. Te percent recovery results (98.6-104%) in Table 3 demonstrate that the method is reproducible for determining ascorbic acid in tef grain and injera extracts.

Determination of Antioxidant Activities of the White and Brown Tef Grains and Teir Injera Samples.
Te antioxidant capacity of cereal grains can be determined using a variety of assays. Te DPPH assay was used in this study to assess the antioxidant activities of the ascorbic acid in white and brown tef grains and their respective injera samples (Table 4). Figure 1 shows the calibration curve (y � −0.0051x + 0.94686) of ascorbic acid standard DPPH scavenging activities determined by its regression coefcient (R 2 � 0.9995). Figure 2 depicts the UV-Vis overlay spectra of ascorbic acid standard DPPH scavenging activities.
Te antioxidant activity levels in white and brown tef grains were 1.26-7.04 μmol AAE/g and 1.44-6.29 μmol AAE/g, respectively. Te lowest and highest values of DPPH radical scavenging activities in white tef grain samples were determined for Ada'a district (East Shewa zone) and Goncha Siso Enese district (East Gojjam zone), respectively, while  Note. Values (means ± SD, n � 3) within a row and column with diferent superscript letters are signifcantly diferent (α � 0.05).
those of brown tef grain samples were determined for Dessie Zuria district (South Wollo zone) and Gomibora district (Hadiya zone). Furthermore, the DPPH scavenging activities of white and brown tef injera were found to be 1.81-2.47 μmol AAE/g and 3.89-4.86 μmol AAE/g, respectively.
Tis study found that the antioxidant activities of white and brown tef grains are comparable to those of the literature reports of other cereal grains presented as μmol of trolox equivalent antioxidant capacities per g of the sample (μmol TE/g sample) like rice (1.39-10 μmol TE/g) [34], wheat (7-10 μmol TE/g) [35], white tef (4.32-6.36 μmol TE/g), and brown tef (6.54-7.16 μmol TE/g) [36], but they are higher than the antioxidant activities of the white tef (2.10-2.14 μmol TE/g) and brown tef grains (2.14-4.30 µmol TE/g) reported by Kotaskova et al. [37]. One-way ANOVA indicated that there was no signifcant diferences (α � 0.05) in the mean antioxidant activity content between the white and brown tef grains. Te low correlation (r � 0.14937) between ascorbic acid contents and in vitro antioxidant activities (Figure 3) in the tef grains may suggest that the major antioxidant compounds in the sample might be bioactive compounds other than ascorbic acid.
Te antioxidant activities of some selected white and brown tef injera prepared in 42 hours of fermentation were evaluated using the DPPH assay, as shown in Table 5. As a result, the antioxidant activity content of the injera samples was lower than that of the tef grains using the DPPH method. Tis is because bioactive compounds such as ascorbic acid, which acts as an antioxidant, are susceptible to cooking and radiation, resulting in a loss of DPPH    International Journal of Analytical Chemistry 5 scavenging activity in the injera samples. Te one-way ANOVA test revealed signifcant diferences (α � 0.05) between the white and brown tef grains and their injera samples (Table 5).

Conclusion
Te tef's ascorbic acid content is important for human health and is used as a quality indicator parameter. As a result, the study reported ascorbic acid contents and antioxidant activities in aqueous extracts of white tef grains and brown tef grains and their injera samples. Te mean ascorbic acid content and antioxidant activities determined in the white and brown tef grains have shown no signifcant diferences (α � 0.05). However, the mean concentration of ascorbic acid and antioxidant activities of the selected tef grains and their injera samples difered signifcantly (α � 0.05). According to this study, tef grains and their corresponding injera have high ascorbic acid content and antioxidant activity. Furthermore, the ascorbic acid content of tef grain and injera was higher than that of commonly consumed grains and vegetables. Were Ilu district of the South Wollo zone had the highest ascorbic acid content in white and brown tef grains. To the best of our knowledge, this is the frst study to determine the ascorbic acid content of tef grains and injera. As a result, this fnding can serve as a foundation for future research on using tef grains in functional foods.

Recommendations
It is fact that tef grain is typically used in fermented foods. Te endogenous and microbial enzymes derived from tef grain four initiate injera dough fermentation which could be an interesting area of future research. Besides, establishing standards for the quality and quantity of starter culture, fermentation conditions, and other ingredients can let researchers investigate biological activities and prospective applications.

Data Availability
All the data are included in the manuscript. Tere are no additional data with the authors.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
BSC, MR, and EEY were responsible for conceptualization. HY and AB were responsible for experimental data collection and analysis. HY was responsible for writing of the original draft. BSC were responsible for writing, reviewing,   International Journal of Analytical Chemistry and editing the manuscript. BSC, MR, and EEY were responsible for supervision. All authors have read and agreed to the published version of the manuscript.