Lyophilized chloroplasts as molecular capsules: A scalable platform for stable and functional human α-lactalbumin...

The growing world population and the increasing demand for proteins present significant challenges to food security. Advances in synthetic biology have enabled the development of transplastomic plants engineered to produce milk proteins, offering a promising solution for the large-scale, economical production of alternative protein sources. This study demonstrated the extended stability of human α-lactalbumin (hLA) in freeze-dried transplastomic tobacco chloroplasts. hLA was expressed at high levels, constituting 39.1 % of the total soluble protein in chloroplasts (66.5 mg/g isolated chloroplasts). Western blot analysis of lyophilized tobacco chloroplasts stored at room temperature for up to six months indicated that the hLA protein remained stable, soluble, and properly folded over extended periods. The freeze-drying process of isolated chloroplasts resulted in a 34-fold increase in hLA protein concentrations. Importantly, the biological functionality of purified hLA from lyophilized chloroplasts was confirmed through an in vitro lactose synthesis assay, where recombinant hLA successfully activated galactosyltransferase, leading to efficient lactose production at level comparable to commercial standard. Lyophilized chloroplasts, free from nicotine and bacterial contamination, are suitable candidates for the oral administration of the dietary protein alpha-lactalbumin. These findings suggest that recombinant protein production in transplastomic plants could serve as a viable food source and alternative to conventional sources. Lyophilization further reduces expenses and streamlines downstream processing, purification, and storage. These methods facilitate relevant formulation practices for these compounds to meet the demand-oriented requirements. Future research should prioritize translation of this technology to edible plant hosts to enable direct human consumption and undertake comprehensive in vivo and clinical studies to validate safety, bioavailability, and efficacy.