Lars Rehmann, Hidefumi Yoshii, Takeshi Furuta
Department of Biotechnology, Tottori University, Tottori, Japan
Characteristics of Modified β -Cyclodextrin Bound to Cellulose Powder
Monochlorotriazinyl-β-cyclodextrin (MCT- β -CD) was covalently bonded to cellulose powder. The
amount of MCT- β -CD bonded to cellulose could be determined by infrared spectroscopy. The
coupling reaction was characterized as a physical adsorption of the MCT-β-CD on the cellulose
powder followed by an apparently zero order chemical reaction. The reaction rate constant was
found to be k = 6.43 10-4 + 0.11 10^ g g-1 s-1. The immobilized cyclodextrin was able to include and
release d-limonene as a model flavor compound. The maximum molar inclusion ratio was 0.85,
which is the same inclusion ratio as for cMimonene in native p-cyclodextrin. The release rates of dlimonene included in the fixed MCT- β -CD were monitored at various relative humidities and 50
°C. The release kinetics could be modeled using the Avrami equation. These results demonstrate
that flavors as well as other hydrophobic compounds can be included and released from MCT- β CD immobilized on cellulose.
Keywords: Microencapsulation; Immobilized β -cyclodextrin; Surface modification; Flavor
compound
1 Introduction
Cyclodextrins have been known for the last century [1]. Cyclodextrins are cyclic oligosaccharides
and natural byproducts of enzymatic starch degradation [2]. The most common and industrially
produced Cyclodextrins are a-, P- and y-cyclodextrin, containing 6, 7 and 8 o-(+)-glucopy-ranose
units, respectively. Cyclodextrins having more units in their ring structure are possible and have
been reported [3], but as the number of glucopyranose units increases, the structure relaxes, and
they tend to react like their linear homologues. In the crystalline state and in solution, Cyclodextrins
form a cone shaped molecule with the hydroxyl groups on the outside of the molecule, providing a
hydrophilic outside and a hydrophobic cavity [2, 4, 5]. This results in high solubility in water and
the ability to host hydrophobic non-water-soluble substances in the hydrophobic cavity. The
encapsulated molecules are protected from UV radiation and oxidative processes [6]. This makes
Cyclodextrins interesting substances for drug delivery purposes [7, 8] and the inclusion of volatile
flavor [9, 10] for long-term storage. Monochlorotriazinyl-(MCT)-p-cyclodextrin is a commercially
available (Cavasol W7 CT, Wacker Chemicals East Asia, LTD, Tokyo. Japan), reactive form of pcyclodextrin. Each β -cyclodextrin molecule contains on average two to three monochlorotriazinyl
Correspondence: Takeshi Furuta. Department of Biotechnology, Tottori University, Tottori 6808552, Japan. Phone: +81-857-31-5273, Fax: +81-857-31-0881, e-mail: takeshi@bio.tottori-u.ac.jp.
groups, able to form covalent bonds to nucleophilic groups, such as -OH in cellulose [11, 12].
Therefore, β -cyclodextrin offers the possibility to immobilize flavors or any hydrophobic
includable component at a cellulose surface such as paper or wallpaper, providing an interesting
way of surface modification. Tanabe et al. could successfully immobilize modified cyclodextrin on
a cellulose membrane and use it as a molecular detector [13]. Fluorescing cyclodextrin containing a
dansyl-(1-dimethy-laminonaphthalene-5-sulfonyl-) glycine group was used and the cellulose had to
be activated before the bonding. The fluorescence intensity decreased as a hydrophobic component
was included in the cavity, and the immobilized cyclodextrin could function as a chemosensor. The
aim of this study, however, was to easily bond a high amount of cyclodextrin to a cellulose surface
to include flavors or antibacterial components in its future applications. Therefore, the process has
to be kept simple and a commercially available product was preferred in this study.
2 Materials and Methods
2.1 Materials
MCT- β -CD with degrees of substitution (DS) between 0.2 and 0.46 was purchased from Wacker
Chemicals East Asia LTD. Unless further indicated, MCT- β -CD with DS of 0.46 was used.
Cellulose was supplied by Funakoshi Co. Ltd., Tokyo, Japan. Sodium carbonate, sodium chloride,
chloroform, potassium bromide, d-limonene, magnesium nitrate and potassium chloride were from
Wako Pure Chemical Company, Tokyo, Japan.
2.2 Chemical bonding of MTC-β-CD to cellulose
Two different approaches to bind MCT- β -CD to cellulose were investigated in this study. In the
first process [11] 2.5 g cellulose (microcrystalline), 0.85 g MCT- β -CD (Cavasol W7), 12 g H2O
and 0.05 g Na2CO3 were heated to 98 °C for 90 min in a programmable water bath. At 30 and 60
min 0.25 g NaCI and at 90 min 0.15 g Na2CO3 were added. The reaction temperature was held for 1
to 3 h. The product was washed and filtered on a 47 mm diameter and 0.2 mm pore size cellulose
filter (Toyo Roshi Kaisha, Ltd. Tokyo, Japan) and dried under vacuum at 70°C for 24h.
In the second process, the same initial composition with only 6 g water was used and no NaCI was
added. The resulting slurry was pre-dried under vacuum at 70 °C for 2 h to a water content of 10%
(w/w). The powder was exposed to 110 - 160 °C for 2-6 min, washed and dried as described before.
2.3 Determination of the MCT- β -CD content in the MCT- β -CD-cellulose powder
The final MCT- β -CD content in the powder was measured by CHNOS elemental analysis (Vario
EL III CHNOS Elemental Analyzer, Elementar Analysensysteme GmbH, Hanau, Germany) and
infrared spectroscopy (FTIR 8300, Shimadzu). CHNS-analysis provides the elemental concentration of carbon, hydrogen, nitrogen and sulfur in a given substance. Assuming water, MCT- β CD and cellulose to be the only substances in the sample allows to calculate the MCT- β -CD
content. A nitrogen content of MCT- β -CD = 0.073 g/g was obtained from NMR measurements
(270 MHz NMR and HMR). The degree of substitution (monochlorotriazinyl groups per
glucopyranose unit) was found to be 0.459. CHNS-analysis of pure MCT- β -CD yielded a nitrogen
content of 0.071 g/g; for the further calculation the more accurate value from the NMR measurements was used.
The infrared spectrum was recorded from 4000 cm-1 to 500 cm-1, and the MCT- β -CD peak was
detected at 1540 cm~1 (conjugated cyclic >C=N-systems show an absorption at wave numbers
between 1660 and 1480 cm-1). Measurement was performed in KBr pellets [14]. To this end 200
mg KBr and 2 mg of the sample were mixed and pressed to disks 10 mm in diameter and 1 mm
thick. The pressing was done under vacuum and with a force of 11-12 t for 5 min. The peak
intensity was used to correlate the MCT- β -CD content in the powder, utilizing the Lambert-Beer
equation.
2.4 Inclusion of d-limonene as a model flavor compound
Five grams of MCT- β -CD-cellulose powder, 12 g H2O and the required amount of d-limonene to
obtain a 0.3 to 5 fold molar ratio were stirred constantly for 24 h at room temperature. The powder
was freeze-dried for 24 h and additionally dried under vacuum at 70 °C for 24 h. During vacuum
drying all non-included, on the cellulose surface adsorbed d-limonene was removed as shown in
previous studies [15].
2.5 Release characteristic of cMimonene
d-Limonene included in MCT- β -CD-cellulose powder (0.5 g) was weighed into a 15 mL glass
bottle and placed in a desiccator containing the appropriate saturated salt solution to obtain relative
humidity of 50, 75 and 98% for 1 to 100 h. Magnesium nitrate, sodium chloride and potassium
chloride were used for 50, 75 and 98% relative humidity, respectively. The d-limonene
concentration was measured by gas chromatography after chloroform extraction as reported
previously [16]. About 500 mg powder was weighed in a capped glass tube, and 8 mL of water and
2 mL of chloroform was added in that order. The solution was heated in a water bath for 25 min at
80 °C to extract the d-limonene into the chloroform phase. During heating the tube was periodically
mixed with a vortex mixer. The concentration of d-limonene in chloroform was determined by FID
gas chromatography (GC-8A, Shimadzu, Kyoto, Japan). The sample was separated on a PEG 20M
column (Shimadzu) at 100 °C with nitrogen as carrier gas. The injection and detector temperature
was 150 °C.
3 Results and Discussion
3.1 Chemical bonding of MTC- β -CD to cellulose
The first process was taken from a Wacker Chemie patent [11]. The maximum achievable MCT- β CD content in the final product was only 3.3% (w/w). Thus, experiments with a variation in the
water and NaCI content, the reaction time and temperature, the type of cellulose, the pH value and
additional stirring during the reaction time, were carried out. The water content and the reaction
temperature seemed to have the most significant influence on the product yield. Decreased water
content and an increased temperature positively affected the yield, whereas additional stirring
decreased it. The optimal reaction time was 2 h. If the cellulose was exposed to 98 °C for more than
2 h a decrease in the product yield was observed. These results are due to a general decrease in the
amount of recovered cellulose because of hydrolysis. The other parameters were found to be of
minor significance.
Fig. 1. Decrease of the MCT-β-CD concentration during the reaction time. The reaction temperature
was 140 °C and the initial MCT- β -CD concentration 0.255 g/g. The solid line represents a linear
regression; the dotted lines show the upper and lower confidence limit.
In the second process, described before to modify cotton [17], a MCT- β -CD content of more than
20% (w/w) in the product could be achieved. The optimal reaction conditions were found to be 5
min at 140 °C. The exposure to higher temperatures for more than 5 min could increase the amount
of bonded MCT- β -CD, but it came along with a strong yellowing of the cellulose caused by the
heat. These results indicate a two-step reaction mechanism. First, the MCT- β -CD was adsorbed on
the cellulose surface, a process inhibited by high water content and stirring, followed by an
apparently zero-order coupling reaction. The stirring might cause sheer stress, interfering with
adsorption. As shown in Tab. 1, even if the initial MCT- β -CD contents were varied, appoximately
the same amount was bonded per gram of cellulose, verifying the zero order assumption. At an
initial MCT- β -CD content of 0.21 g/g the amount of bonded MCT- β -CD was slightly lower. This
can be explained by a complete reaction of the
Tab. 2. Effect of the reaction temperature during the coupling reaction of MCT- β -CD and
cellulose on the final MCT- β -CD content and the maximum inclusion ratio for d-limonene. The
initial MCT- β -CD amount was 25% (w/w).
Tab. 1. Effect of initial MCT- β -CD content on its binding efficiency at 140 °C with a 5-min
reaction time.
available MCT- β -CD, even though only about 91% of the initially available MCT- β -CD was
found in the product. Approximately 7% of the initially available cellulose was lost during the
washing steps. Assuming this cellulose to have the same MCT- β -CD content as the recovered
cellulose gives a MCT- β -CD utilization of 99%. To determine the reaction rate constant the
reaction time was varied, as shown in Fig. 1 . The reaction rate constant was found to
3.2 Effect of reaction temperature on inclusion ratio
The effect of temperature on the binding reaction can be seen in Tab. 2. These samples were
prepared using different reaction temperatures during the coupling reaction, using the same initial
amount of MCT- β -CD. MCT- β -CD with two different degrees of substitution was used. The
relative errors in determination of the MCT- β -CD content and the molar inclusion ratio were less
than 1.5% of the mean values for all reaction temperatures. The molar inclusion ratio for samples
prepared at a reaction temperature higher than 150 °C is significantly lower than for those prepared
at 130 and 140 °C. At 160 °C the molar inclusion ratio drops to about 50% of the value at 140 °C.
Two possible mechanisms could cause this effect. A heat degradation of the cyclodextrin, which
inactivates its inclusion ability, or a decrease in the availability of the cyclodextrin's cavity due to
sterical reasons, caused by mul-
tiple binding of the MCT-β-CD to the cellulose. The multiple binding could require a higher
reaction temperature and therefore would not occur at temperatures below 150 °C. The results
shown in Tab. 2 strongly indicate heat degradation of the MCT- β -CD at temperatures above 140
°C. The decrease in the inclusion ability, caused by high temperature, does not depend on the
degree of substitution of the utilized MCT- β -CD. At a higher degree of substitution multiple
cellulose bonds would more likely be formed, therefore a lower inclusion ability of the sample with
a higher DS would be expected. This is not the case, and the decrease of the inclusion ability at high
temperatures can be explained best by heat degradation of the cyclodextrin. The actual mechanism
could be a ring opening reaction caused by thermal stress. Thus, 140 °C was chosen as the reaction
temperature resulting in low heat degradation of the MCT- β -CD and a high binding efficiency.
These results suggest that it is necessary to investigate the detailed reaction mechanism including
the regioselectivity and structure of MCT- β -CD bound to cellulose with a particular interest in
their dependence on the reaction temperature.
3.3 Correlation of the MCT- β -CD content in the modified powder by Lambert-Beer equation
The intensity of the expected MCT- β -CD peak (1660-1480 cm-1) was used to correlate the MCTβ -CD content in the powder, utilizing the Lambert-Beer equation
Where / is the peak intensity (area), e is the extinction coefficient, d the path length of the light
through the sample and c the concentration of the component causing the peak. The constants /0 and
£ have to be determined from experimental values. To simplify the calculations, instead of e and d,
the constant b = E d was used. The constants could be determined as /0 = 115.0 ± 0.5 and b = 1.673
±0.021.
3.4 Inclusion of d-limonene as a model flavor
The inclusion of d-limonene was investigated by changing the initial amount of d-limonene during
the inclusion reaction. Fig. 2 shows the achieved molar inclusion ratio as a function of the initially
available amount of d-limonene. The molar inclusion ratio increases linearly with an increase in the
initial d-limonene content up to a maximum value. A further increase in the initial d-limonene
concentration does not affect the inclusion ratio anymore; also showing the efficient removal of
non-included d-limonene from the cellulose surface during the drying process. The maximum molar
inclusion ratio was found to be 0.85 mol d-limonene per mol cyclodextrin. This ratio could be
achieved when the initial d-limonene cyclodextrin ratio was higher than 1.2 mol/mol. The
maximum inclusion ratio is not significantly lower than the value for native b-cyclodextrin, which
can include 0.9 mol d-limonene per mole [9, 15]. The inclusion ratio shows that sterical effects,
caused by the bonding to cellulose do not influence the d-limonene inclusion. To be able to
compare the d-limonene inclusion ratio in the MCT- β -CD modified cellulose powders with
different MCT- β -CD concentrations, the initial amount of MCT- β -CD during the production was
varied. The conditions during the coupling reaction were the same for all samples, i. e., the reaction
mix was held for 5 min at 140 °C. The maximum inclusion ratio was not affected by the amount of
MCT- β -CD bonded to the cellulose, the maximum value of 0.85 mol/mol could be achieved with
various samples having MCT- β -CD contents between 11 and 18%.
3.5 Release characteristic of cMimonene
The release behavior of d-limonene from the MCT- β -CD-cellulose powder is shown in Fig. 3.
Over a period of 100 h, the d-limonene retention decreased to almost 0.1 at a relative humidity of
98% and to 0.2 at 75% RH, whereas it only decreased to 0.7 at a relative humidity of 50%. The
retention time curve could be described by the Avrami equation.
Fig. 3. Release behavior of d-limonene included in modified cyclodextrin bonded to cellulose
powder. The relative humidity was 50 (▲), 75 (■) and 98% (●); the temperature was 50 °C. The
solid lines show the results of the Avrami equation for both relative humidities.
Where R is the flavor retention (the amount of included flavor divided by the amount of initially
included flavor), k the release rate constant and n a parameter representing the release mechanism.
Theoretically, the parameter n= 1 corresponds to a first order release kinetics and n = 0.54 to
diffusion limitation. The values for k and n are shown in Tab. 3. In all cases the flavor release is
diffusion limited. The conditions used in this experiment are very harsh and might not be realistic
for future applications. The chosen conditions should just prove the general abilities of the MCT-βCD-cellulose powder to release its included compounds. As expected from previous studies [10,18]
the relative humidity has a strong influence on the release rate, and the release goes remarkably
slower at low relative humidities. This can be explained the release mechanism. Yoshii et al. have
shown that during the inclusion of guest molecules a minimum amount of water molecules is
required [16]. The inclusion only takes place if the flavor can substitute an included water molecule.
Tab. 3. Parameters of the Avrami equation (R = exp[- (kt/)ⁿ]) describing the release of d-limonene
from MCT- β -CD-cellulose at various relative humidities at 50 °C.
The release follows the same mechanism; water substitutes the flavor.
4 Conclusions
MCT- β -CD could be successfully bonded to cellulose powder with high efficiency. The evaluated
process can be easily transferred to non-powdery cellulose, like paper or wallpaper due to the
physical adsorption as the first reaction step. The inclusion and release of flavors could be shown
using d-limonene as an example. The produced MCT- β -CD-cellulose has the potential to include
various other substances, as shown for native cyclodextrins [19-21]. Possible applications can be
flavored or antibacterial surfaces.
Acknowledgement
This study was financially supported partly by a Grant-in-Aid for Scientific Research (C)
(14550737) by the Ministry of Education, Culture, Sports, Science and Technology.
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(Received: July 29, 2002) -(Revised: February 10, 2003) (Accepted: February 11, 2003)