Ensuring global food security in the future requires the identification and sustainable utilization of resilient plant-based resources. Grain amaranth (Amaranthus spp.) represents one such overlooked and underexploited crop with strong adaptability to drought and considerable nutritional potential that remains insufficiently studied (Akin-Idowu  et al., 2017). The seeds contain a high-quality protein content ranging from 13-19%, with a well-balanced amino acid profile and notably high levels of lysine an essential amino acid that is typically limited in most cereal grains (Amare et al., 2015). The protein composition of amaranth is reported to closely align with the amino acid recommen-dations set by FAO/WHO due to its desirable nutritional balance (Murya and Pratibha, 2018).
       
In terms of mineral composition, grain amaranth exhibits levels comparable to widely consumed cereals such as sorghum, millet, wheat, rice and maize (Mustafa et al., 2011). Additionally, it contains beneficial lipids, carbohydrates, antioxidants and various bioactive substances associated with positive health outcomes (Karamac et al., 2019). Unlike conventional cereal grains especially wheat, amaranth holds promise as a gluten-free ingredient for formulating foods suitable for individuals with celiac disease (Martinez-Villaluenga et al., 2020). Although antinutritional factors in amaranth can restrict its direct use, pretreatments such as soaking, roasting and germination help reduce these compounds, making the grain more suitable for consumption. Evaluating its functional properties is therefore important for its incorporation into diverse food systems and processing technologies (Teresa and Pag, 2021). Amaranth is a small-seeded grain rich in nutrients and phytochemicals, offering several health benefits and protection against chronic diseases such as hypertension, diabetes, cancer and cardiovascular disorders. Despite these advantages, it has declined in popularity due to its coarse nature and is underutilised in developed regions. Recent research efforts aim to reintroduce and valorise this grain for modern food applications (Bhat et al., 2018; Jan et al., 2023).
       
Hence, the present research was conducted to assess effect of different pretreatments namely soaking, roasting and germination on the proximate composition, mineral profile, antinutrient content, colour characteristics and functional properties of amaranth seed flour. The findings aim to support its potential application in the development of diverse value-added food products.

Materials
 
The present study took place during 2024-2025 at the “College of Food Technology, Vasantrao naik Marathwada Krishi Vidyapeeth (VNMKV), Parbhani.” Amaranth seed grains, purchased from local market of Parbhani and cleaned to remove unwanted materials. Four batches were made 250 g each portion for three pretreatments (soaking, roasting and germination) and one used as a raw sample.
 
Sample preparation and pretreatments
 
Untreated amaranth seed flour
 
Amaranth seeds were cleaned and then milled through lab scale hammer mill to get fine flour of 70 mesh size. The untreated amaranth seed flour was stored in polyethylene zipper plastic bag at room temperature.
 
Soaked amaranth seed flour
 
Soaking treatment for amaranth seeds was carried out with slight modifications to the procedure described by Thakur et al., (2021). The grains were washed and soaked in distilled water (1:5 ratio) for 12 h. After soaking, they were dried at 65^oC for 4 h, cooled to room temperature and ground into flour. The flour was then sieved through a 70-mesh screen, packed in polyethylene zipper bags and stored at room temperature for further analysis.
 
Roasted amaranth seed flour
 
Roasting was conducted with slight modifications to the method reported by Shinde (2023). Cleaned amaranth grains were roasted in an oven at 120^oC for 10 min, then cooled to room temperature. They were ground into flour and sieved through a 70-mesh screen. The flour was packed in polyethylene zipper bags and stored at room temperature for later use and analysis.

Germinated amaranth seed flour
 
The germination method was followed with slight changes based on Thakur et al., (2021). Cleaned grains (250 g) were soaked in 1000 mL of water for 8 h in a muslin-covered beaker under dark at room-temperature. After draining the water, the grains were wrapped in a moist muslin cloth and allowed to germinate for 72 h, with occasional sprinkling of water to keep them moist. Once germination was complete, the grains were dried in a cabinet dryer at 60^oC for 4 h, cooled, ground into flour, sieved through a 70-mesh screen and packed in polyethylene zipper bags for later analysis.
 
Proximate composition
 
The proximate composition of both untreated and pretreated amaranth seed flour samples was assessed to determine moisture, fat, protein, crude fiber, ash and carbohydrate content. All analyses were carried out following the standard procedures described by AOAC (2005).
 
Mineral composition
 
Mineral content of the samples was evaluated using the analytical methods described by AOAC (2005). The minerals quantified included calcium, phosphorus, iron, magnesium, potassium, zinc, manganese and copper.
 
Antinutritional factor
 
Tannin and phytate was determined as per the method suggested by (Mane et al., 2023). For tannin estimation, about 0.1 ml of sample extract was transferred to a 100 ml volumetric flask containing 75 ml of distilled water. Folin-Denis reagent (5 ml) and sodium carbonate solution (10 ml) were then added and the volume was adjusted to 100 ml with distilled water. The concentration of tannic acid used was 0.1 mg/ml. The resulting solution was incubated for 30 min and then the absorbance was recorded against an experimental blank at 760 nm. The data was expressed as milligram tannic acid equivalent/100 g dry basis of flour weight (mg TAE/100 g).
       
For the phytate test, the powder sample (0.15 g) was combined with 10 ml of HCl (2.4%) and the extraction was performed using a shaking incubator (1 h /25-27^oC). The resulting solution was centrifuged (3000 rpm/30 min) and filtered to obtain a pure phytate extract. Briefly, 1 ml of wade reagent (0.3% of sulfosalicylic and 0.03% FeCl₃.6H₂O acid in water) was mixed with sample extract (3 ml) and vortexed for 5 s. The concentration of phytic acid used was 1 mg/ml. The reading was immediately recorded at a wavelength of 500 nm and the results reported as mg/100 g dry basis of sample weight.
 
Colour analysis
 
The colour attributes of untreated and treated amaranth flour were measured using a Hunter Lab Colorimeter (Color Flex EZ). The device was calibrated with a white tile before use and L*, a* and b* values were recorded. Mean readings from three replicates were reported for each sample (Bachate et al., 2025).

Functional properties
 
Determination of water absorption capacity
 
The water absorption capacity of untreated and pretreated (soaked, roasted and germinated) amaranth flours was determined following Joshi et al., (2022). 1 g of flour was mixed with 10 mL of distilled water, equilibrated for 30 min at 30±2^oC, centrifuged at 3000 rpm for 30 min and expressed as the percentage of water retained per gram of flour.
 
Water solubility index (WSI)
 
The water solubility index of untreated and pretreated (soaked, roasted and germinated) amaranth flour was evaluated following the procedure of Joshi et al., (2022), with slight modifications. 1 g of flour was mixed with 10 mL of water, incubated at 37^oC for 30 min and centrifuged at 3000 rpm for 15 min. The supernatant was transferred to a pre-weighed dish and dried at 110^oC to constant weight and the residue was used to calculate the water solubility index.
 

                      

                     
 

  

 
                         
Bulk Density
 
Bulk density was measured by filling 100 g of flour into a 250 mL graduated cylinder, tapping gently until constant volume and calculating density from the final volume and weight (Joshi et al., 2022).
 

  

                                
Oil absorption capacity
 
The oil absorption capacity of flour was determined as per Singh and Punia (2020). One gram of flour was mixed with 10 mL hydrogenated fat, centrifuged at 2000 rpm for 30 min, excess fat removed and the sample weighed. Oil absorption was calculated as the percentage of fat retained relative to the flour’s initial weight.
 
Statistical analysis
 
Data were analysed using a completely randomized design (CRD) and treatment differences were tested through ANOVA as per Panse and Shukhatame (1985).

Proximate composition
 
The effect of different pretreatments like soaking, roasting and germination on proximate composition of amaranth seed flour was presented in Table 1. Moisture content decreased in all pretreated amaranth flours compared to raw flour. Soaked and germinated samples showed 8.7% and 8.1% moisture, respectively, due to drying after treatment, while roasting caused the largest reduction from 10.2% to 6.6%, likely from heat-induced removal of free and bound water (Komi et al., 2008).


       
A noticeable decline in fat content was observed in the treated samples compared with the untreated amaranth flour, which initially contained 4.95% fat. The soaked amaranth flour showed a similar fat content to the raw flour, measuring 4.91%. Roasting led to a reduction, with roasted flour containing 3.65% fat. The germinated flour demonstrated the most significant decrease, with the fat level dropping to 2.59%. During germination, the decline in fat content occurs because stored fats are used to supply energy for seed sprouting (Sade, 2009). The reduction is also linked to the enhanced action of lipolytic enzymes, which break down fats to support the metabolic activities essential for seed growth (Beniwal et al., 2019).
       
Protein content increased after soaking (16.63%) and germination (17.62%) but decreased with roasting (15.42%), likely due to amino acid formation during germination and heat-induced protein denaturation during roasting. However, roasting led to a reduction in protein content, possibly due to structural alterations in native proteins caused by heat treatment (Fasasi, 2009).
       
Crude fiber was highest in germinated flour (6.39%) and lowest in roasted flour (2.58%). Thakur et al., (2021) observed significant increases in protein and crude fiber following germination, confirming that sprouting enhances nutrient contents by activating endogenous enzymes. while ash remained relatively stable (2.25-2.45%). Carbohydrates increased in all treated samples, with the highest in roasted flour (69.50%) and germinated flour (63.04%), compared to raw flour (60.69%). Comparable increases in carbohydrate fractions following soaking and germination have also been documented by Thakur et al., (2021), who reported gains in digestible carbohydrates with reduced antinutrients during processing. These results align with previous reports on processed amaranth flour (Bachate et al., 2025).
 
Mineral composition
 
The effect of different pretreatments soaking, roasting and germination on mineral composition of amaranth seeds was assessed and the results are depicted in Table 2.


       
Germination resulted in the highest calcium level (79.64 mg/100 g), exceeding that of the untreated seeds (74.28 mg/100 g). A minor reduction in calcium was observed after soaking (73.98 mg/100 g), while roasted flour showed a similar value to the control (74.06 mg/100 g).
       
Phosphorus content in raw amaranth flour measured 528.6 mg/100 g and exhibited a slight increase after germination (557.3 mg/100 g). Roasting also enhanced phosphorus content to 556.2 mg/100 g, whereas soaking resulted in a marginal decrease to 527.4 mg/100 g. Magnesium levels decreased in soaked (253 mg/100 g) and roasted samples (240 mg/100 g). However, germination improved magnesium content to 299 mg/100 g compared to the untreated sample (261 mg/100 g).
       
The potassium concentration was highest in germinated amaranth flour (554.6 mg/100 g), followed by roasted flour (533.1 mg/100 g), while the untreated seeds contained 508.2 mg/100 g. A slight reduction was recorded in soaked flour, which measured 507.9 mg/100 g. Iron content also showed variation across treatments, with a small decrease observed in soaked samples (13.24 mg/100 g) compared to the control (13.95 mg/100 g). In contrast, roasting and germination enhanced iron levels to 16.01 mg/100 g and 16.87 mg/100 g, respectively.
       
Zinc content increased after roasting and germination, reaching 5.72 and 5.88 mg/100 g, while untreated and soaked samples had 4.12 and 4.03 mg/100 g, respectively. Manganese ranged from 2.90 mg/100 g in soaked flour to 3.27 mg/100 g in germinated flour, with untreated and roasted samples at 3.18 and 3.22 mg/100 g. Copper also improved with processing, measuring 0.93, 0.88 and 1.32 mg/100 g in soaked, roasted and germinated flours, compared to 0.82 mg/100 g in the untreated sample.
       
Soaking reduced minerals content as some minerals solubilized into the water (Chavan et al., 2013). Germination showed the highest mineral increase due to enzyme activity, nutrient release and breakdown of antinutrients like tannins and phytates (Khan et al., 2013). The results are consistent with previous studies showing increased mineral content in processed amaranth. Thakur et al., (2021) reported iron and zinc contents of 16.2 mg/100 g and 5.6 mg/100 g, respectively, in germinated amaranth, attributing the rise to phytate degradation. Similarly, Shinde (2023) observed higher calcium and magnesium levels after germination. In line with these findings, germinated amaranth flour in this study exhibited the highest mineral concentrations (Table 2), confirming that pretreatments effectively enhance mineral content and potential bioavailability.
 
Antinutritional factors
 
Anti-nutrients like tannins and phytic acid were analysed for amaranth seed flours (Table 3). Processing of amaranth seed flours i.e., soaking, roasting and germination caused decrease in phytic acid content 1.42% to 1.27%, 0.79% and 0.78% respectively and tannin 0.120% to 0.093%, 0.051% and 0.048% respectively.

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The decrease in tannin and phytic acid content during soaking may be attributed to their decline into the soaking water, while during germination, enzymatic activity plays a major role. In the case of phytic acid, soaking increases the activity of natural enzymes such as phytases, which break down phytic acid into lower inositol phosphates and free inorganic phosphate (Larsson and Sandberg, 1992). The present results are similar with those reported by Singh et al., (2017).
 
Colour analysis
 
The colour of amaranth flour was evaluated after soaking, roasting and germination by measuring L* (lightness), a* (red–green) and b* (yellow–blue) values, as presented in Table 4. L* ranges from 0 (black) to 100 (white), with positive a* indicating redness and negative a* greenness, while positive b* represents yellowness and negative b* blueness. The untreated seeds had L*, a* and b* values of 64.17, 7.80 and 31.56, whereas the untreated flour showed 77.71, 3.69 and 19.47.

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Soaking treatment reported in an increase in L* value of amaranth flour, recorded as 79.80. The a* value was 3.18, while the b* value was 18.53. The increase in L* value shows the flour became lighter, while small decreases in a* and b* values indicate less red and yellow color. The lighter color may result from pigments and phenolic compounds leaching into the soaking water (Rao et al., 2013).
       
During roasting process L* value of amaranth flour was indicated as 75.96, while a* value was 5.95 and the b* value was 25.10. The increase in a* and b* values recorded an enhancement in the redness and yellowness of the flour as the roasting temperature increased. The increase in a* and b* values and the decrease in L* value reflect the reddish-yellow darkening of flour due to browning reactions like Maillard and caramelization during roasting, which form brown pigments (Bello et al., 2017).
       
Germination treatment also affects the colour values of amaranth flour, with L* value 76.47, a* value 3.64 and b* value 22.17 after 72 h of germination. Germination slightly darkens flour (lower L*) due to enzyme activity and phenolic formation, while redness and yellowness (a* and b*) increase from carotenoids and flavonoids in germinated seeds (Sogut and Seydim, 2018). Roasting produces the highest redness and yellowness, showing a stronger colour effect (Beniwal et al., 2019).
 
Functional properties
 
Functional properties are the characteristics of a substances that influence its performance and interaction with other components during food processing. They are commonly assessed through parameters such as water absorption capacity (WAC), water solubility index (WSI), oil absorption capacity (OAC) and bulk density (BD). The effects of soaking, roasting and germination on amaranth flour’s functional properties are presented in Table 5.


       
WAC increased from 2.66 g/g in untreated flour to 2.83, 2.92 and 2.91 g/g in soaked, roasted and germinated samples, respectively. The higher WAC after roasting is likely due to heat-induced denaturation of proteins and structural changes in starch, exposing more water-binding sites (Singh and Singh, 2020). Germination enhances WAC through enzymatic breakdown of starch and proteins, producing more soluble molecules capable of retaining water (Maldonado-Cervantes  et al., 2019), while soaking leads to a slight increase by softening and hydrating the grains (Olagunju and Ifesan, 2021).
       
Similarly, WSI rose with pretreatments, from 6 g/100 g in untreated flour to 8, 10 and 14 g/100 g in soaked, roasted and germinated samples, respectively. The pronounced increase during germination results from enzymatic hydrolysis of starch into dextrins and simple sugars, which improves solubility (Gamel et al., 2016). Roasting also elevated WSI, likely due to partial starch gelatinization and formation of Maillard reaction products (Mbithi-Mwikya  et al., 2018), whereas soaking caused a noticeable increase, possibly due to leaching of soluble compounds.
       
Oil absorption capacity of amaranth flour increased with pretreatments, from 1.65 ml/g in untreated flour to 2.40 ml/g in roasted and 2.26 ml/g in germinated samples, while soaked flour showed a slight increase to 1.73 ml/g. The higher OAC after roasting is attributed to thermal protein denaturation, which exposes hydrophobic side chains that bind oil (Altan et al., 2009). Germination improves OAC by breaking down high molecular weight compounds and increasing porosity, enhancing oil retention. Structural changes, such as partial starch-protein breakdown and more porous microstructures, further facilitate oil absorption (Onwuka, 2005; Gao et al., 2014).
       
Bulk density decreased from 0.74 g/ml in untreated flour to 0.67, 0.59 and 0.60 g/ml in soaked, roasted and germinated samples, respectively. Roasting reduces density through puffing, lower particle compactness and increased porosity (Awolu et al., 2017), while germination softens polysaccharides, producing lighter, porous flour. Soaking caused only a slight decrease (Gamel et al., 2006). Overall, pretreatments enhanced functional properties by disrupting structure and increasing porosity, exposing hydrophilic and hydrophobic sites, which improve water and oil retention. These modifications make the flour suitable for bakery, protein-enriched snacks and beverage applications where hydration and oil binding are essential (Onwuka, 2005; Gao et al., 2014).

The findings of this study indicate that pretreatments such as soaking, roasting and germination enhanced the nutritional, mineral, colour and functional properties of amaranth flour, with germination proving the most effective. These treatments also reduced antinutritional factors, improving nutrient availability. This work demonstrates the strong potential for the commercial scale up, shelf-life enhancement and the use of combined pretreatments to further enhance flour quality.

The authors extend their gratitude to Dr. V. S. Pawar, Research Guide, for continuous guidance and support.
 
Disclaimer
 
The views presented in this manuscript are those of the author, who is solely responsible for its content and accuracy.

The authors declare no conflict of interest.