Abstract
Sporopollenin exine capsules (SECs) extracted from Lycopodium clavatum spores are an attractive biomaterial possessing a highly robust structure suitable for microencapsulation strategies. Despite several decades of research into SEC extraction methods, the protocols commonly used for L. clavatum still entail processing with both alkaline and acidolysis steps at temperatures up to 180 °C and lasting up to 7 days. Herein, we demonstrate a significantly streamlined processing regimen, which indicates that much lower temperatures and processing durations can be used without alkaline lysis. By employing CHN elemental analysis, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and dynamic image particle analysis (DIPA), the optimum conditions for L. clavatum SEC processing were determined to include 30 hours acidolysis at 70 °C without alkaline lysis. Extending these findings to proof-of-concept encapsulation studies, we further demonstrate that our SECs are able to achieve a loading of 0.170 ± 0.01 g BSA per 1 g SECs by vacuum-assisted loading. Taken together, our streamlined processing method and corresponding characterization of SECs provides important insights for the development of applications including drug delivery, cosmetics, personal care products, and foods.
Highlights
Natural capsules obtained from plant spores and pollens are drawing significant attention for diverse microencapsulation applications[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]
The scanning electron microscopy (SEM) cross-section showed a significant reduction in the sporoplasmic debris
The spore intine layer is reported to be undamaged by alkaline lysis and requires acidolysis to remove[2,6,7]
Summary
Several L. clavatum SEC extraction processes have been developed involving acetolysis, mild enzymatic chemical treatments, or aggressive non-oxidative reagents[4,5,6,7,8,9,10,11,35,36,37]. Our DIPA data indicates that the average diameter of intact natural untreated L. clavatum spores is 31.0 ± 2.2 μ m (Fig. 6A) This is in agreement with reported results obtained by laser diffraction, which identified. The circularity and aspect ratio distributions of the acid treatment data (Fig. 6B) both show clear shifts towards lower values when acidolysis treatment is prolonged beyond 30 h These shifts indicate decreases in the overall uniformity of particles. These results suggest that SEC structural integrity is progressively reduced with increasing acidolysis treatment times. Upon examination of our SEC protein content data (Fig. 5), acid processing demonstrated a much greater effect on total protein removal than the alkali treatment steps. The 30 h SECs had a loading of 0.170 ± 0.010 g of BSA per 1 g of SECs, whereas, the natural untreated spores had a loading of 0.131 ± 0.012 g
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