Development of Bioactive Compounds Enriched Malted Milk Powder by Incorporating Selected Germinated Cereals and Pulses

At present, people are seeking ready-to-eat food products which have optimum nutritional value as well as health benefits due to urbanisation, loss of traditional food culture, concern about personal health led by the busy lifestyle, and increase the risk of non-communicable diseases. Hence, the development of functional foods has become a trend that can improve human well-being. Cereals and pulses are considered as functional food ingredients of providing proteins, carbohydrates, dietary fibres, vitamins, minerals, and phytochemicals such as polyphenols and flavonoids. Though cereal and pulses provide nutritional value, antinutritional factors such as oxalate, phytates and saponins and protease inhibitors limit their nutritional quality and bioavailability of nutrients. Therefore, researchers applied various processes to improve nutritional quality of cereal and pulses such as dehulling, cooking, germination, soaking and fermentation. Among all processes, gemination can be used as an economical, environmentally friendly and effective way to enhance the nutritional value and functional properties of cereal and pulses. Germination alters seeds’ biochemical and physical properties and enhances bioactive compounds due to enzymatic action during germination. Germinated seeds are considered beneficial and superior to non-germinated seeds due to their increased antioxidant activity and nutritional value. Germination also improves digestibility, leads to the formation of new bioactive compounds, and helps mask unpleasant flavours and odours. Hence, germinated cereal and pulses can be used as a potential functional food ingredient rather than a raw type. This study primarily aimed to develop a malted milk powder enriched with bioactive compounds from germinated cereals and pulses, intended as a grain- and dairy-based functional beverage. It also investigated the bioavailability and bioaccessibility of nutrients and bioactive compounds, with and without zinc fortification, using a simulated in vitro digestion model. In the first stage of this research, the investigation of the optimum germination condition which gives maximum bioactive compounds was carried out. The selected cereal and pulses (rice, maize, green gram and soy) were germinated for 2, 4, 6 and 8 days at 30 ºC after steeping 12 hrs in tap water. The ungerminated cereal and pulses were used as the control. Then the germinated cereal and pulses were ground into fine particles. The highest total polyphenol content, flavonoid content was obtained in 8 days germinated cereal and pulses compared to control type. The lowest IC50 concentrations for DPPH and ABTS radicals and high FRAP activity were obtained in 8 days germinated cereal and pulses indicating the highest antioxidant activity. Based on the results obtained from the first stage of the research, 8 days germinated cereal and pulses was selected to formulate the malted milk powders. To obtain the malt mix, 35 % of rice, 35 % of maize and 30 % of green gram which germinated for 8 days were mixed together and obtained malt mix were incorporated into fresh pasteurised liquid milk at three different malt mix to milk ratios of 40:60, 50:50, and 60:40. The malted milk slurry was dried by using the vacuum oven at 50 ºC under 50 mbar pressure for 24 hrs and crushed it into fine powder. The obtained malted milk powder formulations were subjected to characterisation of nutritional, physical and bioactive compound analysis. Increasing the proportion of germinated malt mix in malted milk powder significantly (p < 0.05) enhanced protein, polyphenol, flavonoid content, antioxidant activity, and mineral levels, while reducing fat content compared to powders made with ungerminated mixes. These improvements are attributed to enzymatic hydrolysis during germination, which boosted nutritional quality and bioactivity. Notable changes in microstructure and protein secondary structure also contributed to better dispersibility and solubility. However, formulations with germinated mixes exhibited a darker colour. In the next step of the research project, all six malted milk formulations were subjected to the in vitro gastrointestinal digestion. Polyphenol and Flavonoid content progressively increased across oral, gastric, and intestinal digestion phases, with MM 60:40 formulations exhibiting superior bioaccessibility compared to CMM samples. Germination-induced enzymatic activity was identified as a key factor in breaking down polyphenol bound complexes, promoting easy exposure to digestive enzymes than CMM samples. The antioxidant activity, measured through DPPH radical scavenging assays, showed a significant rise throughout digestion, aligning with the release of polyphenols and flavonoids. Amino acid bioavailability also improved, with the intestinal phase driving the highest release, supported by enzymatic hydrolysis and reduced starch-protein interactions. SDS-PAGE analysis confirmed extensive protein hydrolysis, particularly in germinated samples, resulting in smaller peptides and free amino acids. Furthermore, mineral bioavailability (Na, K, Mg, Ca, Zn, and Fe) significantly improved in MM formulations due to the reduction of antinutritional factors like phytic and oxalic acids. The intestinal phase displayed the highest mineral absorption, aided by pancreatic enzymes and casein phosphopeptides (CPP), which maintained mineral solubility. In the final stage of the project, the impact of zinc (Zn) fortification on the nutritional and functional properties of malted milk powder formulations was examined as a strategic response to the global challenge of micronutrient deficiencies. Zinc fortification in malted milk powders significantly enhanced the initial extractable total polyphenol content (TPC), flavonoid content (TFC), and antioxidant activity, particularly in formulations incorporating germinated malt mixes (Zn-MM). MM 60:40 consistently showed the highest bioactive compound levels and antioxidant potential across digestion phases. Zn fortification likely stimulated polyphenol biosynthesis through Reactive Oxygen Species (ROS)-mediated stress and improved solubility of bioactives by altering matrix interactions. However, despite these improvements, Zn fortification occasionally reduced bioactive compound bioavailability during digestion, likely due to complex formation which can reduce solubility. Nevertheless, antioxidant activity (DPPH) remained elevated in Zn-fortified samples, suggesting the presence of stable, functional antioxidant complexes. Amino acid release varied by phase and formulation, with MM 60:40 showing the highest intestinal bioavailability. Some amino acids strongly bound to Zn (e.g., Cys, His, Asp), limiting their release. Mineral bioavailability, particularly for Zn, Na, Ca, and K, improved during digestion, especially in Zn-MM 60:40. Dialyzable Zn content in this formulation reached 2.20 mg /20 g, covering ~18.6% of the adult RDI. While one serving fell short of delivering half the Recommended Dietary Intake (RDI), 3-4 servings could meet the target. Zn fortification also altered protein secondary structure by reducing β-sheets and increasing random coils, enhancing protein flexibility, solubility, and digestibility. Together, this study demonstrated that germination significantly enhances the nutritional, functional, and structural properties of cereal and pulses. Incorporating germinated blends into milk not only improved polyphenol and flavonoid content, antioxidant activity, amino acid and minerals but also their bioavailability along the enhanced protein digestibility. Zinc fortification further amplified the antioxidant potential and mineral content, particularly in germinated formulations like MM 60:40, despite some reductions in bioaccessibility of bioactive compounds due to complex formation.<p></p>