Name: extraction of plant-based capsules for microencapsulation applications
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Description: Watch this Scientific Journal Video about Extraction of Plant-based Capsules for Microencapsulation Applications at JoVE.com

This work was supported by the National Research Foundation (NRF-NRFF2011-01) and the National Medical Research Council (NMRC/CBRG/0005/2012).

1. Extraction of Sporopollenin Exine Capsules (SECs) from L. clavatum Spores
Note: The SEC extraction process involves a flammable powder (L. clavatum), hot corrosive acids, and flammable solvents, hence proper personal protective equipment (goggles, face mask, gloves, lab coat), approved risk assessment on usage, and disposal of chemicals by authorized laboratory personnel is essential.
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	<li>Spore Defatting
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		<li>Prepare a reflux set-up in a fume hood by using a gla...

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	<li>Paunov, V. N., Mackenzie, G., Stoyanov, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporopollenin+micro-reactors+for+in-situ+preparation,+encapsulation+and+targeted+delivery+of+active+components.">Sporopollenin micro-reactors for in-situ preparation, encapsulation and targeted delivery of active components.</a> J. Mater. Chem. 17 (7), 609-612 (2007).</li><li>Barrier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physical and chemical properties of sporopollenin exine particles. Doctoral dissertation thesis. , (2008).</li><li>Beckett, S. T., Atkin, S. L., Mackenzie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosage+Form.">Dosage Form.</a> US Patent. , (2009).</li><li>Lorch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+contrast+agent+delivery+using+spore+capsules:+controlled+release+in+blood+plasma.">MRI contrast agent delivery using spore capsules: controlled release in blood plasma.</a> Chem. Comm. (42), 6442-6444 (2009).</li><li>Wakil, A., Mackenzie, G., Diego-Taboada, A., Bell, J. G., Atkin, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+bioavailability+of+eicosapentaenoic+acid+from+fish+oil+after+encapsulation+within+plant+spore+exines+as+microcapsules.">Enhanced bioavailability of eicosapentaenoic acid from fish oil after encapsulation within plant spore exines as microcapsules.</a> Lipids. 45 (7), 645-649 (2010).</li><li>Barrier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporopollenin+exines:+A+novel+natural+taste+masking+material.">Sporopollenin exines: A novel natural taste masking material.</a> LWT-Food Sci. Technol. 43 (1), 73-76 (2010).</li><li>Barrier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+of+plant+spore+exine+capsules+for+microencapsulation.">Viability of plant spore exine capsules for microencapsulation.</a> J. Mater. Chem. 21 (4), 975-981 (2011).</li><li>Hamad, S. A., Dyab, A. F., Stoyanov, S. D., Paunov, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+living+cells+into+sporopollenin+microcapsules.">Encapsulation of living cells into sporopollenin microcapsules.</a> J. Mater. Chem. 21 (44), 18018-18023 (2011).</li><li>Diego-Taboada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequestration+of+edible+oil+from+emulsions+using+new+single+and+double+layered+microcapsules+from+plant+spores.">Sequestration of edible oil from emulsions using new single and double layered microcapsules from plant spores.</a> J. Mater. Chem. 22 (19), 9767-9773 (2012).</li><li>Diego-Taboada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+free+microcapsules+obtained+from+plant+spores+as+a+model+for+drug+delivery:+Ibuprofen+encapsulation,+release+and+taste.">Protein free microcapsules obtained from plant spores as a model for drug delivery: Ibuprofen encapsulation, release and taste.</a> Mater. Chem. B. 1 (5), 707-713 (2013).</li><li>Atwe, S. U., Ma, Y., Gill, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollen+grains+for+oral+vaccination.">Pollen grains for oral vaccination.</a> J. Control. Release. 194, 45-52 (2014).</li><li>Mackenzie, G., Beckett, S., Atkin, S., Diego-Taboada, A., Gaonkar, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+24.">Ch 24.</a> Microencapsulation in the Food Industry: A Practical Implementation Guide. , 283-297 (2014).</li><li>Diego-Taboada, A., Beckett, S. T., Atkin, S. L., Mackenzie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hollow+Pollen+Shells+to+Enhance+Drug+Delivery.">Hollow Pollen Shells to Enhance Drug Delivery.</a> Pharmaceutics. 6 (1), 80-96 (2014).</li><li>Ma, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+a+novel+rape+pollen+shell+microencapsulation+and+its+use+for+protein+adsorption+and+pH-controlled+release.">Preparation of a novel rape pollen shell microencapsulation and its use for protein adsorption and pH-controlled release.</a> J. Microencapsul. 31 (7), 667-673 (2014).</li><li>Archibald, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+iron+interact+with+sporopollenin+exine+capsules?+An+X-ray+absorption+study+including+microfocus+XANES+and+XRF+imaging.">How does iron interact with sporopollenin exine capsules? An X-ray absorption study including microfocus XANES and XRF imaging.</a> J. Mater. Chem. B. 2 (8), 945-959 (2014).</li><li>Southworth, D., et al., Blackmore, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+10.">Ch 10.</a> Microspores Evolution and Ontogeny: Evolution and Ontogeny. , 193-212 (2013).</li><li>Stanley, R. G., Linskens, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pollen: Biology, Biochemistry, Management. , 307 (1974).</li><li>Ariizumi, T., Toriyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+sporopollenin+synthesis+and+pollen+exine+development.">Genetic regulation of sporopollenin synthesis and pollen exine development.</a> Annu. Rev. Plant Biol. 62, 437-460 (2011).</li><li>Mundargi, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+Sunflower+Pollen+as+a+Drug+Delivery+Vehicle.">Natural Sunflower Pollen as a Drug Delivery Vehicle.</a> Small. 12 (9), 1167-1173 (2015).</li><li>Mundargi, R. 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Chem. 461 (1), 89-109 (1928).</li><li>Zetschke, F., Kaelin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+&#252;ber+die+membran+der+sporen+und+pollen+v.+4.+Zur+autoxydation+der+sporopollenine.">Untersuchungen &#252;ber die membran der sporen und pollen v. 4. Zur autoxydation der sporopollenine.</a> Helv. Chim. Acta. 14 (1), 517-519 (1931).</li><li>Zetzsche, F., Vicari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+&#252;ber+die+Membran+der+Sporen+und+Pollen+III.+2.+Picea+orientalis+Pinus+silvestris+L.,+Corylus+Avellana+L.">Untersuchungen &#252;ber die Membran der Sporen und Pollen III. 2. Picea orientalis Pinus silvestris L., Corylus Avellana L.</a> Helv. Chim. 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Mitteilung &#252;ber die Membran der Sporen und Pollen.</a> Journal f&#252;r Praktische Chemie. 148 (9-10), 267-286 (1937).</li><li>Shaw, G., Yeadon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+studies+on+the+constitution+of+some+pollen+and+spore+membranes.">Chemical studies on the constitution of some pollen and spore membranes.</a> Grana. 5 (2), 247-252 (1964).</li><li>Shaw, G., Yeadon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+studies+on+the+constitution+of+some+pollen+and+spore+membranes.">Chemical studies on the constitution of some pollen and spore membranes.</a> J. Chem. Soc. C Org. , 16-22 (1966).</li><li>Dom&#236;nguez, E., Mercado, J. A., Quesada, M. A., Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollen+sporopollenin:+degradation+and+structural+elucidation.">Pollen sporopollenin: degradation and structural elucidation.</a> Sex. Plant Reprod. 12 (3), 171-178 (1999).</li><li>Mundargi, R. C., Tan, E. L., Seo, J., Cho, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+and+controlled+release+formulations+of+5-fluorouracil+from+natural+Lycopodium+clavatum+spores.">Encapsulation and controlled release formulations of 5-fluorouracil from natural Lycopodium clavatum spores.</a> J. Ind. Eng. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lycopodine+triggers+apoptosis+by+modulating+5-lipoxygenase,+and+depolarizing+mitochondrial+membrane+potential+in+androgen+sensitive+and+refractory+prostate+cancer+cells+without+modulating+p53+activity:+signaling+cascade+and+drug-DNA+interaction.">Lycopodine triggers apoptosis by modulating 5-lipoxygenase, and depolarizing mitochondrial membrane potential in androgen sensitive and refractory prostate cancer cells without modulating p53 activity: signaling cascade and drug-DNA interaction.</a> Eur. J. 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In this work, a systematic analysis of SEC extraction from L. clavatum spores is presented and this report shows that it is possible to produce higher quality capsules while also achieving a significant streamlining of the pre-existing commonly used protocol.11 In contrast to the existing protocol requiring a high process temperature (180 &#176;C) and a long process duration (7 days),11 the current SEC extraction processing optimization was primarily focused on reducing the temperature and ...

There is significant interest in natural plant-based capsules obtained from plant spores and pollens for use in microencapsulation applications.1-15 In nature, spores and pollen provide protection for sensitive genetic materials against harsh environmental conditions. The basic structure of plant spores and pollen typically comprises an outer shell layer (exine), an inner shell layer (intine), and the internal cytoplasmic material. The exine is comprised of a chemically robust biopolymer1,9,10,13,16 referred to as sporopollenin and the intine is comprised primarily of cellulosic materials.16-18 Empty capsules can be isolated by various processes7,9 for removing cytoplasmic material, proteins, and the intine layer.2,12,16 These sporopollenin exine capsules (SECs) provide a compelling alternative to synthetic encapsulants due to their narrow size distribution and uniform morphology.7,9,13,19,20 The development of standardized processes to obtain SECs from various plant species, such as Lycopodium clavatum, opens the potential for a wide range of microencapsulation applications in the fields of drug delivery, foods, and cosmetics.6,10-13,21
In order to obtain SECs, researchers first treated spores and pollen with organic solvents and refluxed in alkaline solutions to remove cytoplasmic contents.22-25 However, the remaining capsule structure was determined to still contain the cellulosic intine layer. In order to remove this, researchers explored the use of prolonged acidic hydrolysis processing using hydrochloric acid, hot sulfuric acid, or hot phosphoric acid over several days,22-25 with phosphoric acid becoming the preferred method of SEC intine removal.2 However, ongoing research over the years has shown that various spores and pollens have differing degrees of resilience to the harsh processing methods commonly used.26,27 Some spores and pollens are completely degraded and lose all structural integrity in strong alkaline solutions, or become heavily damaged in strong acidic solutions.16 The variability in SEC response to treatment conditions is due to subtle variations in the chemical structure and exine morphology of the sporopollenin exine material between species.28 Due to the variability in the robustness of sporopollenin exine capsules (SECs), it is necessary to optimize the processing conditions for each species of spore and pollen.
Plant spores from the species L. clavatum have become the most widely studied single source of SECs. It is proposed that this is primarily due to its widespread availability, low cost, monodispersity, and chemical robustness.9,29 The spores can be easily harvested and contain sporoplasmic contents in the form of groupings of 1 - 2 &#181;m cellular organelles and biomolecules.11 L. clavatum spores have been used as a natural powder lubricant,30,31 a base for cosmetics,30 and in herbal medicine32-36 for a wide range of therapeutic applications. The SECs obtained from L. clavatum have been shown to be more resilient to processing than SECs from other species of spores and pollen.2 After processing, the resulting SECs have been shown to retain their intricate microridge structures and high morphological uniformity while providing a large internal cavity for encapsulation.7 Studies indicate that L. clavatum SECs can be used for the encapsulation of drugs,10,13 vaccines,11 proteins,7,14 cells,8 oils,5-7,9 and food supplements.5,15 Observed SEC loading efficiencies are relatively high in comparison to conventional encapsulation materials.7 There are also a number of reported benefits to SEC encapsulation such as the ability to mask tastes,6,10 and to provide some degree of natural protection against oxidation.12 In the existing studies, the most commonly used SEC extraction method for L. clavatum is based on four main steps. Step one is solvent refluxing in acetone for up to 12 hr at 50 &#176;C to defat the spores.11 Step two is alkaline refluxing in 6% potassium hydroxide for up to 12 hr at 120 &#176;C to remove cytoplasmic and proteinaceous materials.11 Step three is acid refluxing in 85% phosphoric acid for up to 7 days at 180 &#176;C to remove the cellulosic intine material.11 Step four is a comprehensive washing process using water, solvents, acids, and bases to remove all remaining non-exine material and chemical residues.
The main goals of SEC extraction in relation to encapsulation applications are to produce capsules which are empty of cytoplasmic material, free from potentially allergenic proteins, and morphologically intact.2,37 However, from an industrial manufacturing perspective, it is also important to consider additional economic and environmental factors, such as, energy efficiency, production duration, safety, and resulting waste. With regards to energy efficiency, both high temperatures and long processing times affect production costs as well as environmental footprint. Production duration and turnaround time are key factors influencing processing profitability. Of particular concern is that high temperature phosphoric acid processing increases safety issues and is known to result in corrosive scaling which leads to significant increases in infrastructure maintenance and delays in batch turnaround times.38-40 Where possible, minimizing the number of steps required may lead to significantly reducing the waste produced. However, the commonly used four-step process of L. clavatum SEC extraction has simply evolved from decades of research and has had little actual process optimization. Recently, Mundargi et al.,41 made a significant contribution to the ongoing work in this field by systematically evaluating and optimizing one of the most commonly reported SEC extraction techniques.
In the first section of this study: spore defatting is demonstrated utilizing acetone processing at 50 &#176;C for 6 hr; sporoplasm and intine removal procedures are demonstrated utilizing 85% phosphoric acid processing at 70 &#176;C for 30 hr; extensive washing with water, solvents, acid, and base is used to demonstrate the removal of residual sporoplasmic contents; and SEC drying is demonstrated utilizing convection drying and vacuum oven drying. In the third section, SEC vacuum loading is demonstrated utilizing vacuum loading of a model protein, bovine serum albumin (BSA), followed by BSA-loaded-SEC washing and lyophilization. In the fourth section, the determination of the BSA encapsulation efficiency is demonstrated utilizing centrifugation, probe sonication, and UV/Vis spectrometry.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Lycopodium clavatum spores (s-type)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">19108-500G-F</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin</td>
			<td style="width:235px;">Sigma</td>
			<td>A2153-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">FITC-conjugated BSA</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">A9771-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Phosphoric acid (85 % w/v)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">438081-2.5L&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Hydrochloric acid</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">V800202&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Sodium hydroxide</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">S5881-1KG&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Acetone</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">V800022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ethanol</td>
			<td style="width:235px;">AcME&#160;</td>
			<td style="width:171px;">C000356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Deionized water</td>
			<td style="width:235px;">Millipore purified water&#160;</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">Qualitative filter paper (grade No. 1, cotton cellulose)</td>
			<td style="width:235px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Polystyrene microspheres (50 &#177; 1 &#181;m)&#160;</td>
			<td style="width:235px;">Thermoscientific (CA, USA)</td>
			<td style="width:171px;">4250A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Vectashield&#160;</td>
			<td style="width:235px;">Vector labs (CA, USA)</td>
			<td style="width:171px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:319px;">Sticky-slides, D 263 M Schott glass, No.1.5H (170 &#956;m, 25 mm x 75 mm) unsterile glass slide</td>
			<td style="width:235px;">Ibidi GmbH (Munich, Germany)</td>
			<td style="width:171px;">10812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Commercial Lycopodium SECs (L-type)</td>
			<td style="width:235px;">Polysciences, Inc. (PA, USA)</td>
			<td style="width:171px;">16867-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Heating plates</td>
			<td style="width:235px;">IKA, Germany</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Scanning electron microscope</td>
			<td style="width:235px;">Jeol, Japan</td>
			<td style="width:171px;">&#160;JFC-1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Elemental analyzer&#160;</td>
			<td style="width:235px;">Elementar, Germany</td>
			<td style="width:171px;">VarioEL III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">FlowCam: The benchtop system</td>
			<td style="width:235px;">Fluid Imaging Technologies, USA</td>
			<td>FlowCamVS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Confocal laser scanning microscope</td>
			<td style="width:235px;">Carl Zeiss, Germany</td>
			<td style="width:171px;">LSM710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Freeze dryer</td>
			<td>Labconco, USA&#160;</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">UV Spectrometer</td>
			<td>Boeco, Germany</td>
			<td style="width:171px;">S220</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction of plant-based capsules for microencapsulation applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

Streamlined Extraction Process for Sporopollenin Exine Capsules 
The L. clavatum SEC extraction was achieved by three main steps: (1) Defatting using acetone; (2) Acidolysis using phosphoric acid 85% (v/v); and (3) Extensive SEC washing using solvents. The flow of the streamlined SEC extraction process is presented in Figure 1 A - I. Briefly, the process involves a defatting step with a...

Watch this Scientific Journal Video about Extraction of Plant-based Capsules for Microencapsulation Applications at JoVE.com

Extraction of Plant-based Capsules for Microencapsulation Applications

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

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