Physico-chemical Characterisation of Ultrasound Processed Finger Millet Malt 

    
Literature has reported positive results on sonication of seeds and grains by increasing hydration and germination performance. Through acoustically induced cavitation activity, ultrasound treatment stands as a promising avenue for reducing processing time and diminishing the presence of anti-nutrients in finger millet. It also augments water binding capacity, starch solubility, starch particle size reduction and uniform dispersion (Yadav et al., 2021). This study aims to evaluate the impact of malting conditions on the physical, chemical and functional properties of ultrasonicated malted flour. The primary objective is to determine optimal malting conditions that preserve the nutritional content of finger millet, which is rich in essential minerals like calcium and iron. Additionally, the research assesses the bioaccessibility of minerals in control and optimized sonicated samples using solubility and dialysis methods. This comprehensive approach aims to deepen the understanding of finger millet’s nutritional significance and its potential to provide essential nutrients.

The present research was conducted at Dr. NTR College of Agricultural Engineering, Bapatla, Andhra Pradesh, India, over a period of six months in 2023. Finger millet grains of Tirumala variety were pre-treated with ultrasonic waves for 8, 13 and 18 minutes at varying different amplitudes (40, 70, 100%) and temperatures (30, 45, 60°C) using an ultrasonic probe Sonicator (Model: DP 120). The grains were steeped, sprouted for 48 hours, dried at 50±2°C for 12 hours and then milled into flour using a Wiley mill. A grain-to-water ratio of 1:3 was maintained throughout the hydration process. Angle of repose (Zaalouk and Zabady, 2009), water activity (Make: Rotronic, HydroLab), water holding capacity (Ramashia et al., 2018), colour (Magdic et al., 2009), moisture (AOAC 925.10) (George, 2019), phenol (Malick and Singh, 1980) and tannin content (Schanderi, 1970) of finger millet malt flour were performed in triplicate for analysis. Calcium and iron content were determined with the help of Atomic Absorption Spectrophotometer (Model: AA8000). The study assessed the bioaccessibility of micronutrients in finger millet malt samples using solubility and dialysis method. 16 g of pepsin was suspended in 100 mL of 0.1 M HCl to prepare pepsin digestion mixture. Pancreatin-bile extract mixture was prepared by dissolving 4 g pancreatin and 25 g of bile extract in 1 litre of 0.1 M NaHCO₃. The gastric stage involved homogenizing 1 g of flour sample with 80 mL of distilled water for 5 minutes, adjusting the pH to 2.0 by adding 6 M HCl and then adding 3 mL of fresh pepsin solution. The samples were then incubated at 37°C for 2 hours, then modified to 7.5 pH with 0.5 M NaOH. The assays were placed in an ice bath for 60 minutes to halt the gastric digestion process. The intestinal stage involved placing pepsin digest aliquots into the dialysis tube to which 5 g of pancreatin mixture was added and placed in a shaker incubator for 2 h at 37°C. The samples were then analysed for calcium and iron content using Atomic Absorption Spectrophotometer (Luten et al., 1996).
 
Process optimization and statistical analysis
 
Sonicated finger millet malt flour was optimized by using Design Expert Software (Version 12.0.6.0) through response surface methodology. Model was validated by comparing experimental values with predicted ones using a central composite design (Table 1). ANOVA in Design Expert was used to assess the impact of independent variables on quality characteristics.

Angle of repose for sonicated finger millet malt flour ranged from 27.45±0.95 to 35.52±0.82 (Fig 1). It was observed that increase in time duration, temperature and amplitude of sonication leads to an increased angle of repose due to the moisture content. As ultrasound waves pass through the grains they enhance liquid penetration into grains, facilitating deeper hydration. This causes the grains to swell and undergo changes in their physical properties, resulting in enhanced cohesion and reduced flowability, which indicates a more stable pile formation (Macho et al., 2020). Previous studies have reported that increase in moisture content of paddy from 8 to 16% has increased the angle of repose from 27.5 to 31.2° (Bhople et al., 2017). Response surface plots of angle of repose in relation to sonication time duration, temperature and amplitude are presented (Fig 1). A statistically significant difference was found (P<0.05) with a quadratic model (F-value-8.19). The angle of repose model regression equation (R²-0.8806 and adjacent R²-0.7731) is presented after excluding non-significant terms and affecting process parameters.
   


Ultrasound treatment affects water activity by enhancing the hydration process, creating microchannels and cavities within the food matrix. This accelerates water absorption and increases the equilibrium moisture content. The pressure differences caused by acoustic waves induce inertial flow and the “sponge effect,” where the food matrix alternately squeezes and relaxes, facilitating more efficient water uptake (Miano and Augusto, 2018). The water activity (aw) of finger millet malt flour ranged from 0.544±0.00 to 0.596±0.00 (Fig 2), indicating high microbial stability as the activity of most microorganisms would be inhibited at a water activity level below 0.6. These results closely align with kodo millet flour (Srilekha et al., 2019). Response surface plots of water activity relation to sonication time duration, temperature and amplitude are presented (Fig 2). Statistically, a significant difference was found (P<0.05) with a quadratic model being significant (F-value-6.40). Water activity model regression equation (R2-0.8521 and adjacent R2-0.7189) is presented below after excluding non-significant terms.
   
        

Water holding capacity of finger millet malt flour ranged from 201.36±0.95 to 218.36±1.96% (Fig 3), it was increased with time duration, temperature and amplitude. High shearing forces generated by ultrasound, increases water-binding capacity, leading to the formation of micro-jets that breaks down the starch granules. This process enhances water penetration and binding within the pores (Yadav et al., 2021). Mechanical energy generated by ultrasound leads to the physical modification (Vela et al., 2021) and it also dissociate proteins and disrupt their secondary structure, which affects their water-binding sites (Harasym et al., 2020). Similar findings were reported in finger millet (Yadav et al., 2021). Response surface plots of water holding capacity in relation to sonication time duration, temperature and amplitude are presented (Fig 3). Statistically significant difference was found with a quadratic model (F-value-33.37). WHC model regression equation (R2-0.9678 and adjusted R2-0.9388) is presented after excluding non-significant terms and impacting process parameters.
   
        

Colour plays a significant role in perception of food and it is greatly influenced by overall acceptability of consumers whether to accept it or reject (Imram, 1999). An increase in lightness with respect to control was observed among all treatments, contrary to the trend reported in sonicated rice by Ding et al., (2018). Ultrasound treatment may have removed the surface layer of grains, resulting in a brighter surface (Oladejo et al., 2017). Extent of colour change induced by ultrasound treatment appeared to be different among treatments in finger millet malt flour, as in sweet potato and wheat flour (Cui and Zhu, 2020). Largest DE was observed at sonication treatment of 18min, 60°C and 100%. Decrease in the red green value (a*) and blue-yellow value (b*) was observed with increase in temperature. The color changes were primarily due to the migration of colored components into the aqueous environment during incubation. Components mainly, the phenolic acids from the bran layer and carotenoids, are responsible for the dark and yellow/red hues, respectively (Xia et al., 2020). Additionally, Maillard reactions might occur during ultrasound treatments, further contributing to color changes in the flour (Cui and Zhu, 2020). Response surface plots of colour difference (DE) in relation to sonication time duration, temperature and amplitude are presented (Fig 4). Statistically, significant differences (P<0.05) were found with a quadratic model (F-value-11.15). The colour difference (DE) model regression equation (R2-0.9094 and adjacent R2-0.8278) is presented after deleting non-significant terms and highlighting the impact of process parameters.
   
        

Ultrasound treatment generates acoustic cavitation, which collapses bubbles and loosens the grain matrix in a grain-water system. This process creates micropores, increases porosity, and disrupts the cell wall, enhancing water mass transfer into the grain due to osmatic gradient difference (Bonto et al., 2021). As a result, moisture content in finger millet grains increases as water from the surrounding medium is absorbed (Oladejo et al., 2017) and it ranges from 7.27±0.56 to 7.88±0.48% (Fig 5) and it was observed that increase in sonication time duration, temperature and amplitude has increased the moisture content and these results agreed with finger millet flour (Ramashia et al., 2018). Response surface plots of moisture content in relation to sonication time duration, temperature and amplitude are presented (Fig 5). Statistically significant difference (P<0.05), was found with a quadratic model (F-value-13.17). Moisture content model regression equation (R2-0.9222 and adjacent R2-0.8522) is presented below after excluding non-significant terms, along with the impact of process parameters.

                                                                  Moisture content (%) = 7.80+0.1272A+0.1093B-0.0756A2-0.0756B2       

Total Phenol Content (TPC) of sonicated finger millet malt flour were found to be 50.11-126.22 mg/100 g (Fig 6). An extended sonication time duration increases total phenols, likely due to enhanced cell wall disruption caused by cavitation, which releases bound polyphenolic compounds. Increasing sonication amplitude and temperature has reduced phenols due to a lower grain-to-water ratio, which enhances cavitation and solvent penetration within the grain structure. This increased energy decreases the presence of galloyl moieties, leading to  reduction in phenols. Yadav et al., (2021) has reported that increase in sonication time duration and amplitude has decreased the phenolic content in finger millet. Response surface plots of phenolic content in relation to sonication time duration, temperature and amplitude are presented (Fig 6). A statistically significant difference was found with quadratic model (F-value-10.87). Phenolic content model regression equation (R2-0.9241 and adjacent R2-0.8558) is presented below after deleting non-significant terms and impacting process parameters.  
     

Tannin content of sonicated finger millet malt flour ranged from 50.64 to 107.91 mg/100 g (Fig 7), against the control sample 125.45±0.89 mg/100 g on dry weight basis and these results agreed with Yadav et al., (2021). Increased water content in the sample significantly reduces the tannin content. This reduction can be attributed to the disruptive effects of sonication on tannin molecules present in the grains which impacts the hydrolysis of ester linkages in the tannins, driven by the dissociation of water into OH- and H+ ions (Bhangu et al., 2018). Increasing the sonication time duration and amplitude creates disruptive effects on the grains, leading to a greater reduction of tannin content. Response surface plots of tannin content in relation to sonication time duration, temperature and amplitude are presented (Fig 7). A statistically significant difference was found with quadratic model being significant (F-value-8.15). Tannin content model regression equation (R2-0.8800 and adjacent R2-0.7721) is presented below after excluding non-significant terms and impacting process parameters. 
    

Analysis of minerals and trace elements is essential for assessing food nutritional quality. Microelements like copper, chromium, iron and zinc are vital for human metabolism (Ertas, 2013), but anti-nutritional factors like phytic acid in cereals can hinder their bioavailability. The present study found that ultrasound treatment significantly increased the soluble calcium and iron content (P<0.05), confirming a direct correlation between soluble minerals and bioaccessibility. Sonicated finger millet malt flour contained calcium ranging from 323±0.13 to 341±0.45 mg/100 g and iron from 3.73±0.54 to 7.12±0.22 mg/100 g (dry weight), consistent with findings by Singh and Raghuvanshi (2012). Ultrasound treatment enhances mineral composition in grains by improving germination rates, breaking seed dormancy and altering the nutritional profile and morphological structure (Gunathunga et al., 2024). Cavitation plays a crucial role in altering mineral levels by mobilizing endosperm nutrients through cell wall disruption (Miano and Augusto, 2018). This process increases calcium content by breaking down divalent metal ions (calcium, iron, zinc) that bind with proteins and form complexes with tannins, thus increasing the concentration of unbound calcium (Kassegn et al., 2018). Studies have demonstrated that ultrasound treatment has reduced calcium levels in PWR variety of rice (Xia et al., 2020). Additionally, sonicated finger millet malt exhibits increased iron content due to enhanced permeability of seed cell walls, promoting interaction between iron cations and polyphenols. This interaction leads to stable iron-polyphenol complexes, improving iron bioavailability and stability. Germination process further increases iron solubility by converting insoluble polyphenols into soluble forms (Xia et al., 2020). This phenomenon was in accordance with the findings of PWR variety of rice who reported that ultrasound treatment has increased the iron content (Xia et al., 2020). Response surface plots of calcium (Fig 8) and iron content (Fig 9) of sonicated finger millet malt flour in relation to sonication time duration, temperature and amplitude are presented. Calcium (R2-0.9549 and adjacent R2-0.9144) and Iron content model regression equation (R2-0.8812 and adjacent R2-0.7742) is presented below after excluding non-significant terms and impacting process parameters.
                                                                                                                                                   


 
  
Bio-accessibility of calcium and iron in sonication-treated finger millet malt significantly improved compared to the control, with calcium bioaccessibility increasing from 0.51% to 36.54% and iron from 1.92% to 58.26%. Ultrasound treatment enhances bioaccessibility by activating the phytase enzyme during germination, which reduces phytic acid, an antinutrient that binds essential minerals, making them less available for absorption (Nkhata et al., 2018). By hydrolyzing phytic acid into phosphoric acid and myoinositol, phytase releases these bound minerals, thereby increasing their bioaccessibility. The activation of hydrolytic enzymes during germination also facilitates the breakdown of carbohydrates, proteins, lipids and dietary fiber, further enhancing nutrient bioavailability (Gunathunga et al., 2024). Overall, mineral bioaccessibility in plant-based foods depends on interactions between food components and the food matrix structure (Xia et al., 2020). While germination and ultrasound treatment can enhance mineral availability, their effects can vary; for example, ultrasound-treated PWR variety rice showed increased calcium bioaccessibility but reduced iron bioaccessibility (Xia et al., 2020). Statistically, no significant difference was found in calcium and iron bioaccessibility percentages (P>0.05), suggesting that variations in sonication time, temperature and amplitude notably influenced bioaccessibility without a specific trend.
 
Process optimization and validation
 
Ultrasound-assisted hydration was optimized for tannin removal and retention of phenolic and mineral content using the desirability function. Optimal conditions for finger millet malt were 100% ultrasound amplitude, 18 minutes soaking time and 30°C temperature. Predicted optimized values were validated through laboratory experiments, showing close alignment with actual results and confirming the accuracy of the regression model (Table 2).

This study demonstrates that sonication is an effective processing technique to enhance the nutritional profile, functional properties and bioaccessibility of micronutrients in finger millet malt. Sonication significantly improved calcium and iron content while drastically reducing antinutritional factors, such as phenols and tannins, which are known to inhibit nutrient absorption. The resultant increase in calcium and iron bioaccessibility highlights the potential of sonicated finger millet malt to address micronutrient deficiencies. Moreover, optimized conditions of 18 minutes, 30°C and 100% amplitude yielded superior results, making the malt more palatable and functional as an ingredient in food formulations. This process offers a promising solution for tackling malnutrition and improving dietary diversity, particularly in regions where finger millet is a staple crop.

The authors declare no conflict of interest.