Millets, known as ‘Nutri-cereals’, are gaining immense attention due to their active promotion worldwide. These minor cereals contain health-beneficial nutrients, dietary fibers, and complex sugars. The high content of dietary fibers is pivotal in promoting digestive health, regulating bowel movements, and fostering a balanced gut microbiota. Complex sugars further contribute to sustained energy release, aiding in maintaining stable blood sugar levels. Moreover, their role in preventing lifestyle diseases, such as heart conditions and diabetes, underscores their significance in promoting long-term health [1]. They are a means of sustenance for many African and Asian populations and are grown mainly in semi-arid zones with little input. The world production of sorghum (Sorghum bicolor L) and millets stands at 61.36 MT and 30.09 MT, respectively [2]. Millets have varied uses, from food to feed and fodder, from ethanol production to pharmaceutical industries. Of late, with the growing awareness of the health benefits of millets and ardent popularization, millets are gaining footholds as food in non-millet-consuming populations. However, the presence of anti-nutritional factors, such as tannins, not only causes malabsorption of protein but also imparts bitterness and an astringency taste to the foods [3], which is one of the plausible reasons for the unpopularity of these Nutri-cereals. Several thermal and non-thermal processing techniques are used to improve the nutritional value and bioaccessibility of macronutrients and micronutrients in millets, including fermentation, which is an age-old process. The millet-consuming populations of Asia and Africa traditionally follow natural or spontaneous fermentation, using the natural microflora in the grains. These processes can vary from a few hours to a few days to reduce the anti-nutritional factors and improve the flavor and taste [4]. Fermentation using a dedicated microflora/starter culture supplemented with a growth media provides better control of microbial dynamics to achieve requisite quality [5]. Submerged LAB fermentation has demonstrated improved antioxidant activity, vitamin B, fatty acid profile, flavor, bioaccessibility of amino acids, and mineral digestibility in different millets [6, 7, 8, 9].

The parameters under scrutiny, including proximate composition, minerals, antioxidant activity, vitamin B, amino acid profile, and estimated glycemic index (eGI), hold considerable significance for human nutrition. Proximate composition analysis provides insights into macronutrient levels, aiding dietary planning [10]. Minerals play a vital role in bone health and immune function. The antioxidant activity of the grains is associated with potential benefits in preventing chronic diseases [11]. Vitamin B content evaluation shows the grains’ contribution to energy metabolism and overall well-being. A comprehensive amino acid profile analysis helps assess protein quality, which is crucial for tissue repair and growth. Lastly, the estimated glycemic index offers insights into the potential impact on blood sugar levels, contributing to understanding the suitability of these grains for individuals at risk of type 2 diabetes [12]. A detailed analysis of the fermented millet can suffice to assess the nutritional superiority of these ‘Nutri-cereals’ to adapt these value-added products better.

The fermentation process is affected by the type of culture, growth medium, substrate, temperature, and pH [13]. Tofu whey, a good source of polysaccharides, proteins, and minerals, is a suitable and low-cost culture medium for the growth of yeasts and LAB [14]. The economical use of tofu whey can also solve the wastewater disposal problem associated with tofu industries. In this investigation, the effect of submerged fermentation using mixed LAB culture grown in tofu whey-based media on the proximate composition, minerals, antioxidant activity, vitamin B and amino acid profile, and estimated glycemic index (eGI) of whole grain sorghum, pearl millet, and dehusked Kodo millet were investigated.

Sorghum, the great millet, and other millet are traditionally consumed in India and other regions worldwide. Many of these local dishes are fermented using back-slopping or spontaneous fermentation processes ranging from a few hours to a few days.

The enhancement of protein content in sorghum grain during fermentation could be due to single-cell protein production by the LAB culture [31] during the 17 h fermentation period with 1% culture. In the case of pearl millet and Kodo millet, the increase in protein content was more pronounced, even if the fermentation period was only for 8 h and 4 h, respectively, albeit at a higher cell culture concentration (2% and 3%, respectively). The fermentation media used in this investigation was tofu whey, which is a rich source of polysaccharides, such as stachyose, raffinose, sucrose, fructose, glucose, and protein [32] and has been used previously as growth media for LAB cultures [33]. The nutrient-rich media might have played a crucial role in the growth of the LAB during the fermentation and contributed to the production of single-cell protein [34] in the peripheral region of the whole grains. Despite a 17 h fermentation period with a 1% LAB culture concentration, the non-significant change in protein content observed in sorghum suggests that factors other than culture concentration alone might contribute to this outcome. While it is common to associate protein synthesis with higher culture concentrations, the extended fermentation duration in sorghum might have allowed for sufficient time for the LAB to metabolize and influence protein content [35].

In contrast, the non-significant change in protein content in Kodo millet, fermented for only 4 hours, hints at the importance of fermentation duration. The limited time might have been insufficient for the LAB to exert its full proteolytic activity, potentially impacting protein synthesis [36]. This underscores that culture concentration and fermentation duration are integral factors influencing protein content modulation by LAB during fermentation.

LAB derive energy from the lipids and fatty acid components of the food materials to carry out fermentation and other biochemical processes, such as lipolysis, oxidation, hydrolysis, and enzyme production [6, 37], resulting in a decrease in the total lipid/fat content.

The decrease in mineral content in the fermented millets can be explained by the fact that the submerged fermentation process was adopted for preparing these products, and it involved draining the media once fermentation was over. Since most of the minerals are concentrated in the outer layer or aleuronic layer of the grains, during this process of draining, the aleuronic layer, which also has the polysaccharides, might have been acted upon by the LAB, thus breaking away the outer layer, which might have helped to leach out of minerals. Moreover, the water-soluble minerals got solubilized in the media and eventually leached out. Our earlier work witnessed similar phenomena [38]. However, in the present case, the extent of mineral loss during fermentation was greater than in our earlier work for sorghum, where only 8 h of fermentation was involved. In contrast, in the current case, a higher period (17 h) was involved for sorghum, which might be the reason for more mineral loss. It is postulated that there could be a structural breakdown in the cell wall of the whole grains during fermentation [39], contributing to higher leaching losses. Hence, the time of submerged fermentation, media, and the type and concentration of culture will be vital for the retention of minerals in the fermented foods. The increase in Fe and Mn in the fermented grains may be due to the mineral absorption from the fermentation media, as tofu whey is rich in minerals, which was also observed in our earlier work [38]. As discussed in the previous sections, the decreased ash content indicates a decrease in most minerals after submerged LAB fermentation. This result is significant, as traditionally, people consume millets in their fermented form. In most cases, submerged fermentation is practiced through black slopping, spontaneous, or fermentation using a specific microbial culture.

The decrease in crude fiber in the fermented millets is in line with reports for sorghum [40] and pearl millet [41]. The reduction in the crude fiber during fermentation could be attributed to the breakdown of the cellulosic and hemicellulosic materials by the enzymatic action accompanied by morphological and chemical changes [42].

A significant increase in the carbohydrate content, especially in the case of Kodo millet, could be the higher degradation of crude fiber and loss of minerals, which ultimately increased the total carbohydrate content in the absence of the hard pericarp. This phenomenon is supported by the work of Alvarez et al. [43], who witnessed a faster reaction rate in the decorticated sorghum, implying the role of pericarp.

The overall increase in the antioxidant activity of the fermented grains can be explained by the fact that when the grains undergo fermentation, the breakdown of cell walls and membranes may result in the liberation of previously encapsulated bioactive compounds, particularly antioxidant phytochemicals. Notably, anthocyanins, a class of flavonoids responsible for the vibrant colors in many grains, fruits, and vegetables, are known for their potent antioxidant properties. The fermentation-induced degradation of the grain matrix may facilitate the release of anthocyanins and other phytochemicals, enhancing their bioavailability. Consequently, the heightened presence of these antioxidants in the fermented grains contributes to an increased overall antioxidant activity [44].

The increase in the antioxidant activity was more pronounced in sorghum than pearl millet and dehusked Kodo millet, indicating that the longer the fermentation period, the higher the release of bioactive compounds from the cell matrix, thus influencing the antioxidant activity. Even though pearl millet had a higher antioxidant potential than that of sorghum grain, the release of bioactive compounds from the cellular matrix seems slower in pearl millet compared to the sorghum grains and dehusked Kodo millet. This may be due to the compact cellular matrix of pearl millet compared to sorghum [45] and the absence of the harder pericarp in the Kodo millet, where dehusking eased the process of releasing bioactive compounds. It indicates that the surface area, cell wall thickness, and cellular components are crucial in defining the fermentation process.

The decrease in tannin content in the fermented millets may be attributed to the activity of the tannase enzyme, which breaks down the complex tannins. The breaking down of tannins by the tannase enzyme is reported to be produced by the LAB culture [46]. Therefore, LAB fermentation can be one of the most effective ways to reduce anti-nutritional factors, such as tannins, in sorghum and millets.

The increase in the multivitamin B can be attributed to the action of Lactococcus lactis, which was part of the culture media used in this investigation. Sybesma et al. [47] have also observed multivitamin B synthesis by Lactococcus lactis. Ochanda et al. [48] could achieve a four- to seven-fold increase in vitamin B content during 8 days of fermentation, where natural LAB fermentation was practiced, whereas we could achieve the same within 4–17 hours using selected media and mixed LAB cultures for different millets.

A significant increase in the amino acid profile of the fermented millets (F) over untreated (UT) is attributed to the metabolism of the LAB. This could be the action of Lactococcus lactis, known for its proteolysis and peptidolysis behavior [36]. The enzymes produced by the LAB are instrumental in breaking down the peptide bonds, hydrolyzing the high molecular protein structure into smaller units, thus releasing the amino acids. This has resulted in the accessibility of essential and non-essential amino acids. This implies the better absorption of amino acids in the gut, as indicated by the improvement in the estimated protein efficiency ratio and biological value in the fermented millets. The decrease in the essential to non-essential amino acids ratio suggests producing more non-essential amino acids through peptidolysis. Additionally, the significant increase in aromatic amino acids such as THR, TYR, PHE, and TRP indicates a change in the flavor of the fermented millet products. The flavor production in stinky tofu is also reportedly associated with aromatic amino acids such as phenylalanine [49]. This indicates a better flavor of the fermented millet due to fermentation.

It is postulated that during LAB fermentation, the bacteria use glucose for their metabolism, further converting glucose into lactic acid [50], which may be a plausible explanation for the decrease in the total glucose content in the fermented millets; however, the increase in the free glucose in the fermented millets could be the result of starch hydrolysis and breakdown of complex sugars into simpler forms. The LAB also utilizes resistant starch, thus decreasing the slowly digestible sugars in fermented millets. Our results are in accordance with the work of Ihediohanma [51], who also observed an increase in the eGI values of cassava starch with fermentation. On the contrary, fermentation has increased the resistant starch and decreased the eGI in millets [52, 53].

A controlled submerged LAB fermentation process has been shown to enhance the nutrient content of various millets, including whole grain sorghum, pearl millet, and dehusked Kodo millet. The biological value of protein increased in the proteins derived from pearl millet and Kodo millet, whereas it decreased in the case of sorghum. Moreover, fermentation not only contributes to these improvements but also facilitates the release of bioactive compounds, enhancing the flavor and antioxidant potential of fermented millet-based foods. Fermentation using suitable media, such as tofu whey and LAB cultures, can be adapted to commercialize millet products with better nutrients. In the future, the use of probiotics or the best vitamin B-producing and tannin-reducing cultures can be explored to improve the quality of fermented millet products.

All the data has been presented, the raw data will be provided on request.

DM conceptualized and prepared the manuscript and the principal investigator of the project. CN, YS, SRH: biochemical evaluation of the products. MKT developed the fermentation process protocol and identification of LAB culture. SSD developed the fermented products. AK provided laboratory facility and carried out mineral analysis, edited and reviewed the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Freshly harvested sorghum (Sorghum bicolor L) (commercial variety: Dadar) and pearl millet (Pennisetum glaucum) grains (commercial variety: Raj) sourced from Maharashtra, India, harvested in November 2016 were procured from the local grain market of Bhopal. Kodo millet (Paspalum scrobiculatum) harvested in January 2017 and sourced from the Mandla district of Madhya Pradesh, India, was procured from the farmer.

Authors wish to acknowledge ICAR-CIAE for providing facilities to carry out the experiments.

This research received no external funding.

Author Yogesh Sharma is employed by Waters India Pvt. Ltd., New Delhi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.