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Sequestration of edible oil from emulsions using new single and double layeredmicrocapsules from plant spores

Alberto Diego-Taboada,ab Priscille Cousson,c Elodie Raynaud,c Youkui Huang,c Mark Lorch,c

Bernard P. Binks,c Yves Queneau,cd Andrew N. Boa,c Stephen L. Atkin,ab Stephen T. Beckettbc

and Grahame Mackenzie*bc

Received 5th January 2012, Accepted 19th March 2012

DOI: 10.1039/c2jm00103a

Microcapsules were obtained conveniently from Lycopodium clavatum spores possessing either a single

layered shell of sporopollenin (exine) or double layered shell of sporopollenin and cellulose with an

inner layer (intine). These microcapsules were further modified by converting their surface hydroxyl

groups (alcohols, phenols carboxylic acids) into salts (Na+ and K+), acetates and methyl ethers

accordingly. All of these new types of microcapsules were found to sequester efficiently edible oils from

oil-in-water emulsions with the acetylated forms being the most efficient to sequester oils in near

quantitative fashion. The latter could be recycled without losing efficiency to recover oil. Oils could also

be released from the microcapsules in a stepwise manner by repeated rubbing.

Introduction

The protective exoskeletal shells of pollen and mature plant

spore grains possess two major layers. The outer layer (exine)

consists of the extremely resilient organic polymer sporopollenin,

whilst the inner (intine) is mostly cellulosic.1 The role of these

layers is to protect the fragile cytoplasmic materials against ultra-

violet light2 and aerial oxidation; hence empty pollen and spore

shells have interesting potential for microencapsulation appli-

cations.3–5 There has been much interest in recent years in both

microencapsulation6 and nanoencapsulation7 which has included

encapsulation of oils by different means and using different

materials to form the microcapsules.8–10

A key distinguishing feature of microcapsules from plant spore

shells is that the sporopollenin, the exine, is very stable both

physically and chemically. For example it is resistant to acetol-

ysis11,12 or long refluxing in strong base followed by strong acid1,3

such that the intine along with all the cytoplasmic materials are

degraded by such treatments.

Subsequent washing with solvents readily removes degraded

materials to leave only the exines as round single layered

microcapsules (SEC: sporopollenin exine capsules; Scheme 1, B)

with a large empty void which can be filled with a variety of polar

and non-polar materials over a wide range of molecular masses.3

aHull York Medical School, University of Hull, Hull HU6 7RX, UKbSporomex Ltd., 75 Ferriby Road, Hessle, HU13 0HU, UKcDepartment of Chemistry, University of Hull, Hull HU6 7RX, UK.E-mail: g.mackenzie@hull.ac.uk; Fax: +44(0)1482 466410; Tel: +44(0)1482 465479dICBMS, UMR 5246 CNRS, Universit�e Lyon 1, INSA Lyon, 69621Villeurbanne Cedex, France

This journal is ª The Royal Society of Chemistry 2012

The SEC are extracted by refluxing in strong base and acid

sequentially.3,13 Refluxing in strong base alone gives double

layered microcapsules (SCEC: sporopollenin cellulose exine

capsules; Scheme 1, A) in which the exine and intine remain

intact which have never been explored as potential microcapsules

and have only been mentioned in previous studies3,13,14 as inter-

mediates in stepwise protocols to obtain empty sporopollenin

exines (SEC) as the final product. Several scanning electron

microscopy (SEM) images of L. clavatum spores and SEC

microcapsules have been published:2–4,15 the SCEC retain much

the same morphology and size as the SEC. The size distribution

of L. clavatum spores has been shown16 by laser diffraction to be

very narrow with particles having a median diameter of 30.6 mm.

To use spore microcapsules, either SCEC or SEC, for micro-

encapsulation, the cytoplasm must first be evacuated and then

replaced by a required active component. This is achieved by

transport through a myriad of multidirectional channels which

are of nanometer dimensions (e.g., 15–20 nm for Lycopodium

clavatum)17 and penetrate both the exine and intine layers. They

are necessary in the living spore to hydrate and feed the spor-

oplasm with nutrients.17,18

The microencapsulation work reported in the literature using

microcapsules obtained from pollen and spores appears to have

focused on exines extracted from the spores L. clavatum.3,4 This is

perhaps not too surprising since the spores can be purchased from

a variety of vendors worldwide selling them, for example, as

components of herbal remedies. Also, the sporopollenin exines

(SEC) from this plant species are commercially available (e.g.,

Polysciences, Inc) and can be obtained rapidy in a one-pot

extraction.11Apart from the aforementioned nano-sized channels

the L. clavatum spore exines have a continuous surface, which

J. Mater. Chem., 2012, 22, 9767–9773 | 9767

Scheme 1 Conversion of L. clavatum spores into two families of microcapsules A and B, with and without intine respectively: both possess the outer

exine layer.

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lends them well for microencapsulation applications. They are

also highly resistant to various solvents including strong acids and

bases and do not swell in different solvents. They are also physi-

cally very stable but at the same time possess elasticity charac-

teristics.19They are also uniform inmorphology and diameter (ca.

25 mm). Current applications include drug delivery,15,20 taste

masking,21 micro-reactors,4 ion-exchange chromatography22 and

cosmetics.23

We have shown previously3 that oils can be encapsulated effi-

ciently and easily into SEC extracted as microcapsules from

L. clavatum spores. These microcapsules filled with fish oil can

encapsulate oil at high loadings, between ca. 3 : 1 oil:SECs w/w.3,21

They have the capacity to taste mask fish oil21 and of particular

note, deliver oil-soluble components such as eicosapentaenoic acid

(EPA) with much improved bioavailability as demonstrated in

clinical trials with human volunteers.20 The SEC type, as a pre-

formed microcapsule, has also been shown to exhibit antioxidant

properties hence encapsulated oil could be preserved from

becoming rancid for longer periods.24 Another approach to

microencapsulate and protect oil against oxidation, using a natural

material, has been to use sugar beet pectin to form the microcap-

sules by spray-drying.Whilst relatively inexpensive the particles are

not as robust and not as consistent in size and morphology.6

In the present study we were interested to use sporopollenin as

a component in an alternative and more robust microcapsule.

Such new types of renewable microcapsules offer advantages

such as recovering oil from compressed seed extracts also con-

taining water and scavenging oil from emulsions in cases such as

9768 | J. Mater. Chem., 2012, 22, 9767–9773

accidental spillage of toxic materials. The possibility to encap-

sulate oil from oil–water emulsions is based on the unique

characteristics of sporopollenin of fine porosity, relative lip-

ophilicity and presence of ionisable groups. The latter two

properties have recently been shown to enable SECs to stabilise

emulsions and form liquid marbles.25However, the proportion of

microcapsules used for the present work was chosen to be

sufficient to encapsulate the amount of oil known to be present in

the emulsion. This amount was between 2 : 1 and 4 : 1 w/w

oil:microcapsule, which we have seen to be feasible in our earlier

studies3,20 The exploration involved the two families of micro-

capsules with and without intine as depicted in Scheme 1, 1A and

1B respectively. Both families were modified to (i) increase their

lipophilicity by acetylation and methylation of available

hydroxyl groups and (ii) decrease their lipophilicity by forming

salts of the acidic functions (carboxylic acid and phenolic groups)

which are known to exist, at least on the sporopollenin moiety.13

With a view to applying such microcapsules to topical delivery

applications (e.g. cosmetics and pharmaceuticals), making use of

their elasticity, we also investigated the release of oil from the

microcapsules using repeated compression.19

Results and discussion

Extraction of SEC and SCEC from L. clavatum spores

Several protocols have been reported to obtain empty exines

(SEC) from L. clavatum spores. Some involve mild enzymatic

This journal is ª The Royal Society of Chemistry 2012

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treatments,26–32 whilst others1,12,33–40 employ aggressive methods.

The method used in this study to prepare SEC involved that

which we have previously reported involving sequential treat-

ment with acetone, KOH and phosphoric acid at reflux. This

method also provided access to SCEC (i.e. possessing the

sporopollenin exine and cellulose intine intact) which is an

intermediate in the process. Acidification of the SCEC used for

derivatisation and oil recovery was achieved with hydrochloric

acid. SCEC were also obtained by a one-pot process by omitting

the acetone pre-treatment. It was found that the SCEC, whether

pre-treated with acetone or not, had similar FTIR spectra and

combustion elemental analyses which showed them to be almost

devoid of nitrogen. They were recovered from the raw spores

with a typical mass loss of between 55–60% due to removal of

lipid and protein. SCEC were refluxed with phosphoric acid to

give SEC3 with a typical overall mass loss of 70–75% from the

parent spores, resulting largely from the removal of the intine.

An alternative and rapid one step method, involving acetic

anhydride catalysed by sulphuric acid11,12 was used to prepare

a sample of SEC for comparison of performance in oil seques-

tration with SEC obtained by the aforementioned method. All of

the microcapsules prepared in this study appeared to have much

the same outer surface morphology and size (diameter of 27 � 2

mm) as depicted in previous published works,3 irrespective of the

method of extraction; however, the SCEC were of a lighter buff

colour than the SEC. Both SEC and SCEC were obtained as free

flowing powders and did not agglomerate upon drying. It is of

note that SEC appeared to behave as monodisperse particles in

previous studies.25

Table 2 Percentage of oil recovered from fish oil-in-water emulsionsusing SCEC, SEC and their derivatives: each entry is the average of atleast 4 determination

Microcapsuletype

% ofoil recovered

Derivatisation of SEC and SCEC

The availability of hydroxyl, carboxyl and phenolic groups on

sporopollenin is known,13,29,31,41 however, their relative accessi-

bility to form salts has never been reported. Such accessibility

may offer a way of modifying the polarity of the microcapsule’s

surface. The Boehm titrimetric method42,43 could be considered

as a means to make this assay but to minimise possible errors in

titrations a modification of the method was employed using

flame photometry to determine concentrations of metal ions

released into solution from the microcapsules by treatment with

acid. The proportions of acidic groups present on SEC and

SCEC, as estimated by this method, are given in Table 1. Inter-

estingly, the total number of acidic sites available for salt

formation for both types of microcapsules is quite similar. The

only salt derivatives used in this study were those in which the

acidic sites on microcapsules were fully saturated with sodium or

Table 1 Loading of acidic groups on SEC and SCEC types (analysed byflame photometry as part of a modified Boehm method: each entry is anaverage of 3 determinations)

Microcapsuletype

Loading of functional groups on the microcapsules(mmol g�1)

carboxylicacids lactones phenols

SEC 0.70 � 0.02 0.15 � 0.02 0.35 � 0.02SCEC 0.44 � 0.03 0.25 � 0.03 0.36 � 0.03

This journal is ª The Royal Society of Chemistry 2012

potassium ions accordingly. Thus preparation of SEC and SCEC

in their sodium and potassium salt forms (Table 1) involved

stirring the protonated forms of the microcapsules with a large

excess of sodium hydroxide and potassium hydroxide solutions

at room temperature over 12 h. The lipophilicity of the micro-

capsules was increased by converting free acidic and hydroxyl

groups into their acetylated (SEC-Ac and SCEC-Ac) or meth-

ylated (SEC-Me) forms (Scheme 1, Table 2). Thus SEC and

SCEC were each treated with an excess of acetyl chloride in

dichloromethane and triethylamine to give SEC-Ac and SCEC-

Ac. A labelled form SEC-Ac has been previously synthesised by

Fawcett et al. to determine the hydroxyl content of some

sporopollenins using acetic 1-C14 anhydride.44 The methylated

forms SEC-Me and SCEC-Me were obtained by reaction of SEC

and SCEC, respectively with anhydrous powdered potassium

carbonate and dimethyl sulfate. It is of note that the acetylated

and methylated microcapsules appeared very similar by SEM

indicating the resilience of the microcapsules when chemically

modified as was previously found.13,14,22,36 None of the new

derivatives of either SCEC or SEC was found to form agglom-

erated solids in our experiments.

Encapsulation and sequestration of edible oils from emulsions

using SEC, SCEC and their derivatives

Dry SEC, SCEC and their derivatives were investigated to

compare their ability to encapsulate oil from oil-in-water emul-

sions and as a result sequester oil from the medium. Therefore the

microcapsules were each shaken for 10–15 s by hand in freshly

prepared oil-in-water (5.7% w/w oil-in-water) emulsions formed

by vigorous shaking for 20 min using a mechanical shaker. Fig. 1

shows an optical microscopy image of the fish oil-in-water emul-

sion with average droplet diameter of 30 � 20 mm.

The solid was removed by filtration leaving the filtrate free of

oil visible to the naked eye. To assess the typical ratio of water to

oil co-encapsulated within the microcapsules the loaded SEC

from this procedure, as an example, were examined by magic

angle spinning (MAS) NMR spectroscopy, Fig. 2. The sample

used was taken immediately after filtration, without drying.

Based on the comparison of NMR signals for water protons and

SCECa 89 � 4SCEC-Na 87 � 5SCEC-K 85 � 8SCEC-Ac 98 � 2SECb 90 � 7SEC-Na 92 � 3SEC-K 86 � 1SEC-Ac 98 � 1SEC-Me 94 � 2

a Average of three repeats each for SCEC prepared with and withoutacetone pre-treatment. b Average of three repeats each for SECprepared by ‘one-step’ acetolysis11 and from sequential, acetone, KOHand H3PO4 treatments.3

J. Mater. Chem., 2012, 22, 9767–9773 | 9769

Fig. 1 Fish oil-in-water emulsion (no added surfactant) having average

droplet diameter of 30 � 20 mm.

Fig. 3 CLSM image (field of view: 150 � 150 mm) of SEC containing

oil–water mixture (1.6 : 1 oil:water w/w) at a loading of ca. 3–3.5 g of

oil:water mixture to 1 g of microcapsules as determined by the NMR

spectrum in Fig. 2.

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C-3 protons of the fatty acids present in the oil, the total loading

of the oil–water mixture (1.6:1 oil:water w/w) was between 3–

3.5 g of oil to 1g of microcapsules. Confocal laser scanning

microscopy (CLSM) indicated the presence of materials encap-

sulated within the SEC due to refraction effects of oils, shown in

Fig. 3. Earlier studies3 also used confocal microscopy to view the

presence of substances encapsulated within SEC. The possibility

to encapsulate water and oil at the same time may be a means to

deliver such payloads to topical surfaces such as in cosmetics and

topical applications of drugs. Such relatively high loadings of oils

were also achieved in previous studies.3,21 Immediately after the

oil-filled microcapsules were removed by filtration, they were

Fig. 2 1H MAS (8Khz) NMR spectrum of filled SEC recovered after

sequestration of oil from a fish oil–water mixture. The peak at 4.67 ppm

corresponds to water. The remaining peaks are derived from the fish oil

which is predominantly glycerides of docosahexaenoic acid and eicosa-

pentaenoic acid. The integrations show the relative magnitudes of the

water and protons attached to C-3 of the fatty acids which correspond to

a mole ratio of 10 : 1. This translates to a mass ratio of 1 : 1.6 (water:oil)

assuming an average molecular mass of 300 for the fish oil.

9770 | J. Mater. Chem., 2012, 22, 9767–9773

directly extracted with either dichloromethane or hexane using

the same apparatus. Both solvents appeared to be equally effi-

cient in extracting the oil from the microcapsules. It is of note

that hexane is normally used for the extraction of vegetable oils

but dichloromethane was used in the majority of extractions in

this study. The organic solvent was removed from the oil by

evaporation and remaining water by lyophilisation. A compar-

ison of the efficiency by which the microcapsules were found to

sequester oil from the emulsions is given in Table 2. It is inter-

esting to note from Table 2 that all of the microcapsules are able

to recover oil reasonably efficiently from emulsions. In partic-

ular, the intine form SCEC and SEC without intine, performed

almost with the same efficacy such that the cellulosic intine of

SCEC failed to impede oil sequestration. Cellulose sheets have

been used in the chromatography of oils indicating that it does

have the capacity to absorb oil.9 Whilst it is realised that

a significant error margin was observed perhaps in part due to

error between operators and general handling, there appeared to

be almost quantitative and consistent recovery of oil using the

highly lipophilic esters SCEC-Ac and SEC-Ac. Perhaps not too

surprisingly, the poorest sequestration of oils was with the

sodium and potassium salts imposing polarity to the sporopol-

lenin surface.

Sporopollenin exines have been shown to possess antioxidant

properties.24 As with our previous findings3 the encapsulation

and recovered fish oil from SEC and SCEC proved not to be

deleterious to the oil as observed by retention of approximately

the same peroxide value (PV)3,45,46 as the parent oil. Thus the PV

of the fish oil before emulsification in water and extraction using

SCEC and SEC gave 13.4 � 1.4 meq kg�1 as determined on three

different samples. The oil recovered from both experiments gave

an average of 13.8 � 2.1 meq kg�1.

The capacity of the microcapsules to be recycled and hence

provide a potential economic advantage in their role to separate

oil from an oil–water emulsion was demonstrated (Table 3).

This journal is ª The Royal Society of Chemistry 2012

Table 4 Percentage of oil recovered using different edible oil-in-wateremulsions using SEC: each entry is the average of 3 repeats

Oil% ofoil recovered

fish 93 � 7sunflower 89 � 5corn 90 � 2

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Acetylated SEC and SCEC recovered after organic solvent

extraction of the oil were dried and put to a second extraction

and in some cases a third extraction. The data presented in

Table 3 indicate that the efficiency of recovery appears not to be

reduced by recycling the microcapsules. It is of note that

sporopollenin exines are known to be physically and chemically

resilient; hence, the observation that little or no apparent damage

during repeated use in an application is not surprising.

We chose some of the microcapsules to illustrate particular

properties that might likely be followed by the rest of those

synthesised in this programme. For example we chose SEC to

compare the sequestration of different edible oils, namely, fish

oil, sunflower oil and corn oil. It was shown that there was very

little difference between the oils, suggesting that the efficiency of

loading of oils in the microcapsules might be independent of the

type of edible oil (Table 4). Table 5 shows that even the more

polar microcapsules, SCEC-Na, were still able to recover oil, but

less efficiently, as the stability of the emulsion was increased with

increasing proportions of lecithin emulsifier.

Release of oil from SEC and SCEC

We have shown in this and a previous study that oils can be

extracted from microcapsules obtained from spores using

organic solvents (e.g. dichloromethane or hexane) helped by the

nanometer-sized channels penetrating both the exine and intine

layers. Diffusion through channels in SEC and SCEC can permit

release of an oil but it was thought that the elasticity19 and robust

physical stability of the microcapsules could also be employed to

aid stepwise release of an oil. Therefore oil was loaded into SEC

at increasing levels (1 : 1, 2 : 1 or 3 : 1 oil:SEC w/w), whilst still

retaining some powdery consistency as reported previously.3

Samples of a known mass were rubbed by a fingertip over paper

for 30, 45 and 60 s respectively.

These data presented in Table 6 were taken from 10 repeats at

each time point. Whilst the experiment is subject to high levels of

error due at least to oil released on to the fingertip not being

measured, an interesting comparative trend emerged. The most

effective loading to indicate a staged release by friction over the

time course was at 1 : 1 fish oil:SEC w/w: above this loading,

the longer periods of friction showed only a marginal increase in

the amount of oil release. It is possible that at higher loadings

more oil is absorbed at the outer surface which may contribute to

the release in the first compression at 30 s being comparatively

higher. This experiment demonstrates the potential of oil-filled

microcapsules to be used for the release of active lipid substances

topically such as in cosmetics and pharmaceutical applications.

Table 3 Percentage of oil recovered from fish oil-in-water emulsionsusing recycled SCEC-Ac and SEC-Ac: each entry is the average of 2repeats

Microcapsuletype recycled

% of oil recovered from recycling of themicrocapsules

% of oil cycle of use

SCEC-Ac 99 � 1 secondSCEC-Ac 97 � 1 thirdSEC-Ac 98 � 1 secondSEC-Ac 98 � 1 third

This journal is ª The Royal Society of Chemistry 2012

Experimental

General experimental procedures

Optical micrographic observations were performed on an upright

LMDB Leica Microscope from Leica, Germany. Confocal images

were obtained using a Bio-Rad Radiance 2100 confocal laser

scanning microscope (CLSM) equipped with Ar (488 nm), green

HeNe (563 nm) and red diode (637 nm) laser lines and connected to

a Nikon TE-2000E inverted microscope. Images were collected

using Lasersharp 2000 under the following conditions; laser exci-

tation lines Ar (488 nm) 15%, red diode (637) 38%. Fluorescence

from samples passed through 560 and 650 nm dichroic filters and

was collected in photomultiplier tubes equipped with the following

emission filters; 515/30, 590/70 and 600 long pass. The laser scan

speed was set at 166 lines per second, and the viewable area was

between 20 and 200 mm2 when using a 60� oil objective. The iris

was auto-set as optimal for conditions used. Gain was adjusted for

optimal signal/noise ratio. A Corning 400 Flame Photometer was

used for the analysis of sodium and potassium ions in solution. The

oils used were Seven Seas ‘Pure Cod Liver Oil’, Tesco ‘Pure

Sunflower Oil’ and Tesco ‘Mazola Pure Corn Oil’. The lecithin was

purchased from the Sigma Aldrich Company Ltd.

Magic angle spinning NMR measurements

All NMR experiments were carried out on a Bruker Avance II

500 MHz spectrometer using a 4 mm MAS probe operating at

500.1013 MHz (1H). 1H experiments were carried out with an

MAS speed of 8 kHz. Spectra were externally referenced to tet-

ramethylsilane at 0 ppm. 1H experiments were conducted with

a typical p/2 pulse length of 7 ms and a relaxation delay of 4 s.

Experiments were conducted at 303 K.

Preparation of SCEC

Spores (Lycopodium clavatum L., 250 g) were suspended in

acetone (750 cm3) and stirred under reflux for 4 h. Particles were

recovered by filtration, and dried overnight in open air. The

defatted spores were suspended in aqueous potassium hydroxide

solution (6% wt., 750 mL) and stirred under reflux for 12 h, with

filtration and refreshing of the alkaline solution after 6 h. The

Table 5 Percentage of oil recovered from fish oil-in-water emulsionsstabilized with lecithin using SCEC-Na

% lecithin (w/w) relativeto total oil-in-water emulsion

% ofoil recovered

0 87 � 55 63 � 610 48 � 7

J. Mater. Chem., 2012, 22, 9767–9773 | 9771

Table 6 Percentage of fish oil released from SEC by rubbing with onefingertip over paper, as an absorbent surface, for different times

Loading oil:SEC(w/w)

% of oil released after:

30 s 45 s 60 s

1 : 1 54.5 (�4.6) 65.7 (�8.3) 87.7 (�17.8)2 : 1 70.1 (�2.8) 75.2 (�4.3) 83.8 (�5.1)3 : 1 79.0 (�3.5) 84.3 (�9.9) 88.3 (�6.7)

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base-hydrolysed microcapsules were recovered by filtration, and

washed with hot water until the filtrate was neutral. The micro-

capsules were acidified with 4 M HCl (900 cm�3) by stirring for

12 h, washed with water until neutral and then with hot ethanol

until the filtrate was colourless and dried overnight in the open

air to give the SCEC (115 g, i.e. ca. 55% removal of material from

spores) with a typical combustion elemental analysis of %C

62.1� 0.5, %H 7.3� 0.3, %N 0.1� 0.1. This product was used to

synthesise the derivatives described in this paper.

The aforementioned method was repeated without the acetone

treatment to give a form of SCEC with a combustion elemental

analysis that fell within the data presented and showed the same

level of performance in oil recovery, within experimental error as

SCEC prepared with an acetone pre-treatment (first entry Table 2).

Preparation of SEC

SEC used in this paper for oil recovery and derivatisation were

prepared as previously reported.3 SECs were also prepared by the

method reported by Erdtman,12 for comparison of efficiency to

recover oil as reported in Table 2 but were not used for further

derivatisation.

Preparation of Na+ and K+ salt forms (SCEC-Na, SCEC-K,

SCEC-Na and SEC-K)

SCEC or SEC (10 g) was suspended in aqueous sodium

hydroxide or potassium hydroxide (2 M, 100 cm3) and stirred at

ambient temperature for 12h. The solid was recovered by filtra-

tion (porosity grade 3), washed with water (3 � 50 cm3) until

neutral pH of filtrate, ethanol (2 � 50 cm3) and acetone (50 cm3)

and air dried to constant weight. (NB, whilst sodium ion loadings

were not determined directly their recovery by acid treatment

was determined by flame photometry as shown in Table 1).

Preparation of SCEC-Ac and SEC-Ac

SCEC or SEC (5.1 g) were suspended in DCM (50 cm3) and Et3N

(3 cm3) and acetyl chloride (3 cm3) were added at 0 �C. Themixture was stirred for 4 h at room temperature. After this time,

the solid was recovered by filtration (porosity grade 3), washed

with aqueous hydrochloric acid (2 M, 100 cm3), water (3 �50 cm3) until neutral pH of filtrate, ethanol (2 � 50 cm3) and

acetone (50 cm3) and air dried to constant weight. SCEC-Ac

analysed for %C 71.1� 0.4, %H 7.5� 0.6, %N 0.1� 0.1; SEC-Ac

analysed for %C 68.1� 0.5, %H 7.8� 0.3, %N 0.0 (NB, a form of

SEC-Ac labelled with 1-C14 acetate was synthesised by Fawcett

et al.44 which involved reacting SEC from L. clavatum with acetic

1-14C anhydride to give a loading of 6.6 acetates to 90 carbon

atoms of SEC).

9772 | J. Mater. Chem., 2012, 22, 9767–9773

Preparation of SEC-Me

Dimethyl sulphate (4.75 cm3) was added in portions over 10 min

at room temperature to a stirred suspension of SEC (5 g) in

acetone (100 cm3) and anhydrous powdered potassium carbonate

(13.8 g). The mixture was then heated to reflux temperature for

3 h. The mixture was cooled to room temperature, the product

removed by filtration and subsequently washed with water and

then methanol and dried over P2O5 to give SEC-Me which

analysed for %C 66.4, %H 7.2, %N <0.1.

Oil recovery using SCEC, SEC and their derivatives

Fish oil, sunflower oil or corn oil (2 g) was added to water (35

cm3) and shaken in a mechanical shaker (Stuart SF1 Flask

Shaker) for 20 min at 900–1000 rpm. Sporopollenin (1 g) was

added to the emulsion, shaken vigorously for 1 minute manually

and set aside at room temperature for 4 h. The mixture was

filtered (porosity 3) and washed with either hexane or dichloro-

methane (3 � 50 cm3). The organic solvent was removed by

rotary evaporation and the remaining water removed by

lyophilisation. The yields of oil recovered are presented in

Tables 2–5.

Determination of acidic functional groups on SCEC-Na and

SEC-Na

The SCEC or SEC (0.5 g) were stirred in an excess of the alkali,

NaHCO3, Na2CO3 and NaOH (15 cm3, 0.1 M) accordingly and

stirred for 12 h. The microcapsules were separated by filtration

(porosity grade 3) and dried. A known mass (0.4 g) was added to

2M-HCl (20 cm3) and stirred for 12 h. The microcapsules were

filtered (porosity grade 3) and washed with water (3 � 20 cm3).

The subsequent filtrate was diluted with water to 100 cm3 and

analysed by flame spectrometry.

Release of encapsulated oil by friction

Samples of fish oil-loaded exine shells were prepared, as

described, at ratios of 1 : 1, 2 : 1 and 3 : 1. Approximately 20 mg

of the prepared sample was gently rubbed over a pre-weighed

piece of standard printer paper (1/6 of A4; Future multitech

white paper; 80 gsm) using one finger tip. These samples were

rubbed for 30, 45 or 60 s. The excess exines were then carefully

removed from the paper surface, and the paper weighed again. In

order to account for any exines that become embedded in the

surface of the paper, control experiments were performed where

exines not mixed with oil were rubbed for the same periods of

time; the average of these experiments was then deducted from

the absorption studies. Each experiment was conducted in trip-

licate and the results averaged.

Conclusion

Two new families of microcapsules have been obtained from

L. clavatum spores; one is composed of a double layer of exine

and intine and the other a single layer of only exines. All forms

were able to accumulate different edible oils efficiently and in

high yield from mixtures of oil in water. Their surface polarity

was modified to form salts (Na+ and K+), esters and ethers of the

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acidic and alcoholic groups on the respective surfaces. It was

found that the acetylated forms were the most efficient and could

recover oil in almost quantitative yields. The microcapsules were

also able to recover oil from surfactant-stabilised emulsions but

less efficiently. The known robust nature of the materials

composing the microcapsules was further demonstrated by their

being able to work equally well when recycled. This physical

stability along with the elasticity and porosity of the microcap-

sules was illustrated by their being able to release oil in stages by

repeated gentle pressure of a finger. Such properties offer the

potential of the microcapsules having a role in topical drug

delivery or cosmetics. Also, the microcapsules may have a role in

extracting and encapsulating oil from various aqueous media

which could include recovery of hydrophobic compounds in

fermentation liquids and in cases of accidental spillage.

In view of the similar efficiency of the SCECs and SECs in this

study, it is anticipated that the former double layered micro-

capsules, which are obtained by a cheaper and simpler process,

may offer valuable alternatives for several other microencapsu-

lation applications.

Acknowledgements

We thank Anthony D. Sinclair, Carol Kennedy, Bob Knight and

Lesley Galbraith for their technical assistance. We are grateful to

Sporomex Ltd and the University of Hull for providing funding

for the work.

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