Carbohydrate Polymers 136 (2016) 930–935
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Deep eutectic solvents as efficient solvent system for the extraction of -carrageenan from Kappaphycus alvarezii Arun Kumar Das a , Mukesh Sharma b,c , Dibyendu Mondal b,c , Kamalesh Prasad b,c,∗ a Analytical Division and Centralized Instrument Facility, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar 364 002, India b Marine Biotechnology & Ecology Division, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar 364 002, India c Academy of Scientific and Innovative Research (AcSIR), Central Salt and Marine Chemicals Research Institute, G. B. Marg, Bhavnagar 364 002, India
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Article history: Received 16 June 2015 Received in revised form 29 September 2015 Accepted 30 September 2015 Available online 9 October 2015 Keywords: Deep eutectic solvents Hydrated deep eutectic solvent Extraction -Carrageenan
a b s t r a c t Three different deep eutectic solvents (DESs) prepared by the complexation of choline chloride with urea, ethylene glycol and glycerol along with their hydrated counterparts were used for the selective extraction of -carrageenan from Kappaphycus alvarezii. Upon comparison of the quality of the polysaccharide with the one obtained using water as extraction media as well as the one extracted using widely practiced conventional method, it was found that, the physicochemical as well as rheological properties of -carrageenan obtained using DESs as solvents was at par to the one obtained using conventional method and was superior in quality when compared to -carrageenan obtained using water as solvent. Considering the tedious nature of the extraction method employed in conventional extraction process, the DESs can be considered as suitable alternative solvents for the facile extraction of the polysaccharide directly from the seaweed. However, among the hydrated and non-hydrated DESs, the hydrated ones were found to be more effective in comparison to their non-hydrated counterparts. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Carrageenan is a commercially important phycocolloid extracted from red seaweeds and is used mostly as stabilizers and texture providers in food and ice-cream industries (Van de Velde & Ruiter, 2005). Chemically, it consists of alternating 1,3-linked ␣-d-galactopyranose (Gal) and 1,4-linked -(3,6anhydro-) d-galactopyranose [(3,6-A)Gal] (Fig. 1). Carrageenan is mostly present in three varieties known as -, -, and - and they differ from each other in the number and position of sulphate groups on the repeating galactopyranose units. -Carrageenan has one negative charge per the disaccharide units, while - and -carrageenans have 2 and 3 negative charges per the disaccharide units, respectively. Carrageenan, alone find application in food and beverage industries and their functional hybrids find applications as matrices for the slow release of drug and volatile flavoured compounds (Bylaite, Ilgunaite, Meyer, & Adler-Nissen, 2004; Siepmann et al., 2007; Tapia, Corbalan, Costa, Gai, & Yazdani-Pedram, 2005).
∗ Corresponding author at: Marine Biotechnology & Ecology Division, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar 364 002, India. E-mail addresses: [email protected], [email protected] (K. Prasad). http://dx.doi.org/10.1016/j.carbpol.2015.09.114 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
Due to unique properties such as negligible vapour pressure, high boiling point, large electrochemical window, recyclability, etc., ionic liquids (ILs) are much preferred choice for various applications such as in materials design (Lodge, 2008), to dissolve and extract various biopolymers and as extraction media for polysaccharides (Swatloski, Spear, Holbrey, & Rogers, 2002). The unique characteristics of ILs made them useful in many processes in carbohydrate chemistry (Murugesan & Linhardt, 2005). ILs having the imidazolium structure, such as 1-butyl-3-methylimidazolium chloride (BMIMCl), 1-allyl-3-methylimidazolium chloride (AMIMCl) are used to dissolve cellulose and chitin, respectively (Prasad et al., 2009b; Swatloski et al., 2002; Wu et al., 2004; Zhang, Wu, Zhang, & He, 2005). Although, there were no attempts made so far to extract carrageenan directly from seaweeds using ILs but formation of strong ion gels of different carrageenans and their composites with cellulose were observed in BMIMCl (Prasad, Kaneko, & Kadokawa, 2009), which envisaged the possibility of extraction of the polysaccharide in ILs. Moreover, recently we have reported selective precipitation of agarose from seaweed (Gracilaria dura) extractives using bio-based ionic liquids (Sharma, Chaudhary, Mondal, Meena, & Prasad, 2015; Sharma, Mondal, Singh, & Prasad, 2015). It should be noted that, -carrageenan is conventionally extracted using alkali following the method reported by Craigie and Leigh (1978).
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Handa, & Stoddart, 2005). In a typical reaction, choline chloride (hydrogen bond acceptor) and ethylene glycol, glycerol or urea (hydrogen bond donors) were mixed separately in the optimized molar ratio of 1:2 and were heated at 80 ◦ C until a transparent solution was obtained. 2.3. Extraction of -carrageenan from seaweed using DES Fig. 1. Repeating units in -carrageenan.
After the discovery of deep eutectic solvents (DESs) by Abbott, Capper, Davies, Rasheed, and Tambyrajah (2003) as an alternative to conventional ILs, the application of the DESs is increasing exponentially in various applications. Further the properties of DESs such as non-toxicity, cheaper cost, ease of syntheses, etc., make them lucrative for number of applications e.g., as reaction media, reactants, and catalysts (Zhang, Vigier, Royer, & Jérôme, 2012) and even preferable over ILs in number of applications. In general, DESs can be obtained by the complexation of the halide salts of quaternary ammonium or phosphonium cations (as the hydrogen bond acceptor, HBA) with hydrogen bond donor (HBD) moieties such as urea, glycerol, ethylene glycol, mannitol, recorcinol, etc., (Sirviö, Visanko, & Liimatainen, 2015). There are reports on the applications of DESs for the dissolution of biopolymers (Mondal, Sharma, Mukesh, Gupta, & Prasad, 2013; Sharma, Mukesh, Mondal, & Prasad, 2013), extraction of phenolic compounds from plants (Dai, Spronsen, Witkamp, Verpoorte, & Choi, 2013), extraction of proteins (Zeng et al., 2014), extraction of glaucarubinone from Simaruba glauca (Kholiya, Bhatt, Rathod, Meena, & Prasad, 2015) and many more applications of the novel liquid are continuously being explored. We have shown the excellent capability of certain bio-based DESs for the dissolution and morphological transformation of DNA as well as material preparation using this bio-macromolecule as building blocks employing DESs as dissolution media (Bhatt, Mondal, Bhojani, Chatterjee, & Prasad, 2015; Mondal, Bhatt, Sharma, Chatterjee, & Prasad, 2014; Mukesh & Prasad, 2015). Although the DESs are turned to be good alternate solvents for biopolymers but extraction of biopolymers especially any seaweed based are not attempted so far. Herein we have employed DESs obtained by the complexation of choline chloride with urea, ethylene glycol and glycerol as well as their hydrated forms for the efficient extraction of -carrageenan from Kappaphycus alvarezii. The seaweed belongs to the family Rhodophycea and commercially exploited for -carrageenan.
All the three DESs were used separately to extract -carrageenan from K. alvarezii. Experiment I: In a typical experiment, 500 mg of powdered seaweed was taken separately in 10 g of DES in a beaker. The mixture was heated at 85/95 ◦ C for 1 h. Experiment II: 500 mg of powdered seaweed was heated with 10 g of DES having 10% water for 1 h. The mixtures thus obtained from both the experiments were centrifuged separately and the mass obtained at the bottom of centrifuge tube was separated, washed several times using IPA and dried under vacuum. A control experiment was carried out by mixing 500 mg of seaweed powder and 10 ml of water keeping the temperature and time same as that of the previous set of reactions. The supernatant was precipitated in IPA (1:3, v/v). The precipitated -carrageenan was vacuum dried. 2.4. Extraction of -carrageenan employing conventional method -Carrageenan was extracted from K. alvarezii following the method reported by Craigie and Leigh with minor modifications (Craigie & Leigh, 1978). In a typical experiment, 10 g of dried K. alvarezii was soaked in 200 ml of 0.5% calcium hydroxide solution for 2 h. The soaked seaweed was autoclaved at 107 ◦ C for 1.5 h after the addition of 200 ml water. After autoclaving, the hot mixture was ground in a kitchen blender and centrifuged. The supernatant was slowly added in to IPA under constant stirring (1:3, v/v). The precipitated -carrageenan was vacuum dried. 2.5. Preparation of -carrageenan gel For the preparation of gel, 10 mg sample of each extracted carrageenan was dissolved in 1% 1 ml KCl solution by heating at 90 ◦ C for 5 min in beakers. The gel obtained at room temperature was further kept in refrigerator at 5 ◦ C for 12 h for curing. 2.6. Characterization
Choline chloride (AR grade) and Urea was purchased from SD Fine chemicals, Mumbai, India. Ethylene Glycol and glycerol was procured from Merck Chemicals, Mumbai. HPLC grade isopropyl alcohol (IPA) was purchased from Loba Chemie, Mumbai. All chemicals were used as received. Fresh K. alvarezii was collected from the cultivation sites of west coast of India, Simar (20◦ 42 N; 71◦ 01 E) during February, 2015. The seaweed was sun dried and was washed several times with water to remove epiphytes and kept under shadow for drying. After drying, the seaweed was crushed into very fine powder using mortar and pestle in the presence of appropriate amount of liquid nitrogen.
FTIR analyses of the -carrageenan samples were carried out on a Perkin-Elmer FTIR spectrometer instrument (Spectrum GX, USA) using KBr disc in the range 400–4000 cm−1 . The 1 H NMR was recorded on a Bruker Avance-II, 500 MHz spectrometer. Rheological measurements were performed on an Anton Paar, Physica MCR 301 rheometer USA, using parallel plate PP50/P-PTD200 geometry (49.971 mm diameter; 0.75 mm gap). Temperature was maintained by Anton Paar, Viscotherm VT2 circulating water bath. The dynamic viscosities were determined varying the shear rate at 25 ◦ C e.g., 0.1–50 s−1 followed by measurement of steady shear viscosity in the range of 10–50 s−1 . Frequency sweep was performed to calculate storage modulus, G and the loss (or viscous) modulus, G where angular frequency was set from 0.05 to 1. Time dependant G and G measured at amplitude 0.1% and frequency 0.01 Hz by setting 20 measuring points of 2 s duration each in the time frame of 0–200 s. The time and frequency dependence of the storage and loss moduli (G and G ) for the gels were measured at 25 ◦ C.
2.2. Synthesis of deep eutectic solvents
3. Results and discussion
ChoCl. Urea 1:2, ChoCl-EG 1:2 and ChoCl-Gly 1:2 were synthesized and characterized as described previously (Abbott, Bell,
Well ground K. alvarezii powder was soaked separately in ChoClUrea 1:2; ChoCl-EG 1:2 and ChoCl-Gly 1:2 for 10 min at 1:20 (w/w)
2. Experimental 2.1. Materials
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Fig. 2. Photographic demonstration for the extraction of -carrageenan from Kappaphycus alvarezii using deep eutectic solvents.
Table 1 Yield of -carrageenan obtained from Kappaphycus alvarezii using different solvent systems. S. no.
Solvent system/method
Yield % (±S.D)
Viscosity (cP) of solvent at 25 ◦ C
Sample code
1
Choline-ChlorideUrea 1:2 Choline-ChlorideEthylene glycol 1:2 Choline-ChlorideGlycerol 1:2 10% Hydrated cholineChloride-Urea 1:2 10% Hydrated choline-ChlorideEthylene glycol 1:2 10% Hydrated cholineChloride-Glycerol 1:2 Water Conventional methoda
37.60 ± 1.20
289.10
A
50.66 ± 1.78
35.49
B
30.93 ± 0.90
266.60
C
53.64 ± 1.25
34.49
D
46.02 ± 2.30
16.50
E
60.25 ± 1.10
52.64
F
46.87 ± 2.00 36.58 ± 1.90
0.90 NA
G H
2 3 4 5
6 7 8
NA: not applicable. a Craigie and Leigh (1978).
ratio followed by heating at 85 ◦ C for 1 h. The mixture thus obtained was centrifuged, which showed formation of two layers as shown in Fig. 2. The isolated bottom layer after several washings with IPA and vacuum dry yielded -carrageenan. -Carrageenan was also extracted using 10% hydrated DESs, pure water and by conventional method for comparison of the physicochemical properties. The upper layer upon addition in IPA yielded a white precipitate (0.5–1% with respect to the DESs), which turned hard upon drying and the FT-IR of the precipitate showed the presence of traces of the polysaccharide (supporting Fig. S1). The result confirms the presence of majority of the polysaccharide in the bottom layer. The yields of -carrageenan (with respect to dry seaweed) obtained using all the above solvent systems are depicted in Table 1. It can be observed from Table 1 that, the yield of -carrageenan has increased substantially when hydrated DESs were used for extraction in comparison to the one extracted using pure DESs, water and conventional method. The yield was highest when 10% hydrated ChoCl-Gly 1:2 was used as solvent for the extraction and lowest for the -carrageenan extracted using ChoCl-Gly 1:2. Almost two fold increases in the yield was observed in the case of hydrated DESs mediated extractions in comparison to the extractions carried out using the pure ones. Extraction of -carrageenan from K. alvarezii involve breaking of the seaweed cell walls first followed by interaction of the polysaccharide with the solvents. As can be
seen from Fig. 1, -carrageenan is sulphated at C-4 position and normally potassium cation present as counter ion. During the interaction process, the choline cations replace the potassium cations simply by ion exchange. Such ion exchange was also observed during reaction of -carrageenan with a polymerizable ionic liquid (Prasad & Kadokawa, 2010). Since choline has very high solubility in water and perhaps the reason behind the higher extraction ability of the hydrated DESs. Although in the case of hydrated ChoCl-EG, the hydration has reduced the viscosity by 92% over the original but the extraction of the polysaccharide did not improve even got marginally lower. So, lower viscosity is not only the case of higher extraction efficiency. Hence, the interaction of the polysaccharide with the DES must be playing crucial role in the extraction process. The marginally higher yield in the case of the hydrated DESs in comparison to water may be due to the higher affinity of the charges (due to choline) towards -carrageenan present in the seaweed, which is absent in water. The electrostatic interactions between the solvent and carrageenan in the hydrated DESs as well as their lower viscosity are perhaps responsible for the observed higher yield of -carrageenan. Such interaction was found to be responsible for selective agarose extraction from G. dura, an agarophyte (Sharma, Chaudhary, et al., 2015; Sharma, Mondal, et al., 2015). The indication of the interaction between the sulphate group of -carrageenan and the DESs are also evident from the elemental analyses data (supporting Table S1). In Table S1, the % S for the -carrageenan extracted using hydrated ChoCl-Urea 1:2 was the lowest in comparison to the rest of the samples, which showed the removal of some portion of the anion due to the strong interaction with the DES. However the % N was more in the samples extracted using DESs or their hydrated counterparts in comparison to the sample extracted using water as solvent. After detailed evaluation of the elemental analyses data, a plausible mechanism for the extraction of -carrageenan using different DESs is proposed as shown in Scheme 1. The calculated %N and %S in the proposed structure of A is 4.10 and 4.60%, respectively, while the values were 4.77 and 3.36 in B, which nearly matches with the measured values. Similarly in the case of extraction using hydrated ChoCl-Urea 1:2 (pH 9.76), the alkaline hydrolysis has removed the sulphate group of -carrageenan and the elemental analysis suggested the structure D. The elemental analyses also suggested the structures of E and F for the -carrageenan extracted using hydrated ChoCl-EG 1:2 and ChoCl-Gly 1:2, respectively. In order to find out quality as well as preservation of chemical structures of -carrageenan extracted using various solvents, the FT-IR spectra was recorded for all the samples and is shown in Fig. 3 and Fig. S2. In all the -carrageenan samples extracted using various solvents systems, the characteristic bands between
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Scheme 1. Plausible mechanism showing structure of -carrageenan extracted using different DESs and their hydrated counterparts.
Fig. 3. FT-IR spectra of -carrageenan extracted using different solvent systems.
845 and 848 cm−1 due to d-galactose-4-sulphate was visible. However the intensity of the band was relatively better in the case of the samples extracted using hydrated ChoCl-EG 1:2 (E) and pure ChoClgly 1:2 (C). The characteristic bands due to 3,6 anhydrogalactose was also observed between 924 and 927 cm−1 in all the samples extracted using various solvents. However the intensity of the band was very low in the case of -carrageenan extracted using both pure and hydrated ChoCl-urea 1:2 (A and D), which may be due to the strong interaction of sulphate of carrageenan with the DESs eventually resulting modification in the structure of the polysaccharide (Scheme 1). The presence of characteristic bands of the polysaccharide indicates that the solvent systems employed are suitable to extract the polysaccharide directly from the seaweed. However, among all the solvent systems, 10% hydrated ChoClEG 1:2 and 10% hydrated ChoCl-Gly 1:2 were emerged as the superior solvent systems among all the solvents used in this study for the extraction of the polysaccharide. 1 H NMR was recorded for the -carrageenan extracted using 10% hydrated ChoCl-Gly 1:2 (which yield the highest amount of -carrageenan) and was compared with the one extracted using
Fig. 4. Steady shear viscosity of -carrageenan gel prepared from the sample extracted using various solvent systems.
conventional method (supporting Figs. S3 and S4). The spectra were same for both the samples with the chemical shifts (␦) of the protons comparable to the reported values for -carrageenan (Van de Velde, Knutsen, Usov, Rollema, & Cerezo, 2002). Since -carrageenan is used mostly in the gel form and hence detail rheological investigation was carried out to study the nature of the -carrageenan gels prepared from the samples obtained using different solvent systems and to compare their rheological properties with the one extracted using water as solvent and the sample extracted using conventional method. All the C gels shown in Fig. 4 have revealed different flow behaviour with pseudoplastic nature in all the cases. The gel prepared from C by conventional method (H) had showed lower degree of flow in comparison to the rest of the samples with highest flowable property for the sample obtained with hydrated ChoCl-Gly 1:2. -Carrageenan is known to form strong gels with KCl. The stiffer behaviour of the C gel prepared using conventional method and water as solvent in comparison to the C gel extracted using the DES or their hydrated counterparts indicated some sort
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Fig. 6. Frequency dependence of G and G for -carrageenan gel prepared from -carrageenan extracted using different solvents.
the hydrated DESs showed liquid viscous behaviour in the angular frequency range 0.05–1000 s−1 , however the gels obtained by conventional methods as well as using pure DESs as solvents showed viscous liquid behaviour in the angular frequency range above 100 s−1 . After the crossover of the moduli, the storage modulus started to become predominant over the loss modulus indicating elastic nature of the gels at higher frequency range (Fig. 6). 4. Conclusions
Fig. 5. (a): Time dependent viscoelasticity profile of -carrageenan gel prepared from the samples extracted using various solvent systems. (b) Time dependence of G and G for -carrageenan gel prepared from -carrageenan extracted using different solvents.
of hindrance in the gel formation in the C samples extracted using the DESs. Furthermore the C extracted using hydrated DESs found to form weaker gels in comparison to those extracted using their pure counterparts. Perhaps this is due to the presence of little amount of the DESs in the C (indicated in the elemental analyses) responsible for the disturbance in the formation of three dimensional helices during the gel formation. Time dependent viscoelasticity measurements showed gel like behaviour with G > > G for all the C gels irrespective of the solvents used for extraction in the entire time period of investigation. However, the differences in the magnitude of G and G was maximum in the case of the C gels extracted using ChoCl-gly 1:2 and hydrated ChoCl-EG 1:2 in comparison to the rest of the samples. It should be noted that, the difference between G and G was minimum for the C samples extracted using conventional method or by water (Fig. 5a and b). From the flow behaviour and viscoelasticity profile it can be inferred that, although the C gels obtained in the presence of DES are less stiff in nature but possess better viscoelasticity in comparison to their counter parts extracted using conventional method or water as solvent. In all the C gels, the crossover of G and G was observed with increasing frequency indicating frequency dependence signature of weak gels. However the value of frequency at which they crossover was different for different gels. It was interesting to note that in all the cases the loss modulus predominant over the storage modulus in a wide frequency range indicating the presence of more flow components in the gels making the gels behave like viscous liquid at lower frequencies. The C gels obtained using water and all
Three different deep eutectic solvents prepared by the complexation of choline chloride with urea, ethylene glycol and glycerol as well as their hydrated counterparts were used for the selective extraction of -carrageenan from the carrageenophyte K. alvarezii. The quality of thus obtained -carrageenan was compared with the quality obtained using water as extraction media as well -carrageenan extracted using well practiced conventional method. However, traces of the DESs were found in the polysaccharide samples. Considering the non toxic nature of choCl-EG and ChoCl-Gly, the impurities may not pose any difficulty in the application of -carrageenan. However the impurity of DES consisting of Choline chloride and urea may pose some problem. A plausible mechanism for the modification of chemical structure of the polysaccharide during the extraction process is proposed. The detail rheological investigation of the carrageenan gels was studied. It was inferred from the studies that, the physicochemical as well as rheological properties of the polysaccharide obtained using DESs as solvents was at par to the one obtained using conventional method and was superior in comparison to the carrageenan obtained using water as solvent. Considering the tedious nature of the carrageenan extraction method employed in conventional extraction method, the DESs can be considered as suitable alternative solvents for the facile extraction of the polysaccharide directly from seaweed. However, among the hydrated and non-hydrated DESs, the hydrated ones were found to extract -carrageenan more efficiently in comparison to their non-hydrated counterparts. Acknowledgments KP thanks Council of Scientific and Industrial Research, New Delhi for granting CSIR-Young Scientist Awardees Project (CSIRYSP/2011-12), CSIR Network project CSC10130 and OLP0080 for overall financial support. MS and DM are grateful to UGC and CSIR for senior research fellowships and to AcSIR for PhD registration. Analytical Division & Centralized Instrument Facility of the Institute
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is gratefully acknowledged for providing all necessary analytical facilities for the work. This is CSIR-CSMCRI communication No. 090/2015. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.carbpol.2015.09.114. References Abbott, A. P., Bell, T. J., Handa, S., & Stoddart, B. (2005). O-Acetylation of cellulose and monosaccharides using a zinc based ionic liquid. Green Chemistry, 7, 705–707. Abbott, A. P., Capper, G., Davies, D. L., Rasheed, R. K., & Tambyrajah, V. (2003). Novel solvent properties of choline chloride/urea mixtures. Chemical Communications, 70–71. Bhatt, J., Mondal, D., Bhojani, G., Chatterjee, S., & Prasad, K. (2015). Preparation of bio-deep eutectic solvent triggered antibacterial silver chloride-DNA hybrid material having cephalopod like architecture. Material Science & Engineering C, 56, 125–131. Bylaite, E., Ilgunaite, Z., Meyer, A. S., & Adler-Nissen, J. (2004). Influence of -carrageenan on the release of systematic series of volatile flavor compounds from viscous food model systems. Journal of Agricultural and Food Chemistry, 52, 3542–3549. Craigie, J. S., & Leigh, C. (1978). In J. A. Hellebust, & J. S. Craigie (Eds.), Hand book of phycological methods (pp. 109–131). Cambridge: Cambridge Univ. Press. Dai, Y., Spronsen, J., Witkamp, G.-J., Verpoorte, R., & Choi, Y. H. (2013). Ionic liquids and deep eutectic solvents in natural products research: Mixtures of solids as extraction solvents. Journal of Natural Products, 76, 2162–2173. Kholiya, F., Bhatt, N., Rathod, M. R., Meena, R., & Prasad, K. (2015). Fundamental studies on the feasibility of deep eutectic solvents for the selective partition of glaucarubinone present in the roots of Simarouba glauca. Journal of Separation Science, http://dx.doi.org/10.1002/jssc.201500470 Lodge, T. P. (2008). A unique platform for materials design. Science, 321, 50–51. Mondal, D., Bhatt, J., Sharma, M., Chatterjee, S., & Prasad, K. (2014). A facile approach to prepare dual functionalized DNA based material in a bio-deep eutectic solvent. Chemical Communications, 50, 3989–3992. Mondal, D., Sharma, M., Mukesh, C., Gupta, V., & Prasad, K. (2013). Improved solubility of DNA in recyclable and reusable bio-based deep eutectic solvents with long-term structural and chemical stability. Chemical Communications, 49, 9606–9608. Mukesh, C., & Prasad, K. (2015). Formation of multiple structural formats of DNA in a bio-deep eutectic solvent. Macromolecular Chemistry & Physics, 216, 1061–1066.
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Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Deep eutectic solvents as efficient solvent system for the extraction of -carrageenan from Kappaphycus alvarezii Arun Kumar Das a , Mukesh Sharma b,c , Dibyendu Mondal b,c , Kamalesh Prasad b,c,∗ a Analytical Division and Centralized Instrument Facility, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar 364 002, India b Marine Biotechnology & Ecology Division, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar 364 002, India c Academy of Scientific and Innovative Research (AcSIR), Central Salt and Marine Chemicals Research Institute, G. B. Marg, Bhavnagar 364 002, India
a r t i c l e
i n f o
Article history: Received 16 June 2015 Received in revised form 29 September 2015 Accepted 30 September 2015 Available online 9 October 2015 Keywords: Deep eutectic solvents Hydrated deep eutectic solvent Extraction -Carrageenan
a b s t r a c t Three different deep eutectic solvents (DESs) prepared by the complexation of choline chloride with urea, ethylene glycol and glycerol along with their hydrated counterparts were used for the selective extraction of -carrageenan from Kappaphycus alvarezii. Upon comparison of the quality of the polysaccharide with the one obtained using water as extraction media as well as the one extracted using widely practiced conventional method, it was found that, the physicochemical as well as rheological properties of -carrageenan obtained using DESs as solvents was at par to the one obtained using conventional method and was superior in quality when compared to -carrageenan obtained using water as solvent. Considering the tedious nature of the extraction method employed in conventional extraction process, the DESs can be considered as suitable alternative solvents for the facile extraction of the polysaccharide directly from the seaweed. However, among the hydrated and non-hydrated DESs, the hydrated ones were found to be more effective in comparison to their non-hydrated counterparts. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Carrageenan is a commercially important phycocolloid extracted from red seaweeds and is used mostly as stabilizers and texture providers in food and ice-cream industries (Van de Velde & Ruiter, 2005). Chemically, it consists of alternating 1,3-linked ␣-d-galactopyranose (Gal) and 1,4-linked -(3,6anhydro-) d-galactopyranose [(3,6-A)Gal] (Fig. 1). Carrageenan is mostly present in three varieties known as -, -, and - and they differ from each other in the number and position of sulphate groups on the repeating galactopyranose units. -Carrageenan has one negative charge per the disaccharide units, while - and -carrageenans have 2 and 3 negative charges per the disaccharide units, respectively. Carrageenan, alone find application in food and beverage industries and their functional hybrids find applications as matrices for the slow release of drug and volatile flavoured compounds (Bylaite, Ilgunaite, Meyer, & Adler-Nissen, 2004; Siepmann et al., 2007; Tapia, Corbalan, Costa, Gai, & Yazdani-Pedram, 2005).
∗ Corresponding author at: Marine Biotechnology & Ecology Division, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar 364 002, India. E-mail addresses: [email protected], [email protected] (K. Prasad). http://dx.doi.org/10.1016/j.carbpol.2015.09.114 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
Due to unique properties such as negligible vapour pressure, high boiling point, large electrochemical window, recyclability, etc., ionic liquids (ILs) are much preferred choice for various applications such as in materials design (Lodge, 2008), to dissolve and extract various biopolymers and as extraction media for polysaccharides (Swatloski, Spear, Holbrey, & Rogers, 2002). The unique characteristics of ILs made them useful in many processes in carbohydrate chemistry (Murugesan & Linhardt, 2005). ILs having the imidazolium structure, such as 1-butyl-3-methylimidazolium chloride (BMIMCl), 1-allyl-3-methylimidazolium chloride (AMIMCl) are used to dissolve cellulose and chitin, respectively (Prasad et al., 2009b; Swatloski et al., 2002; Wu et al., 2004; Zhang, Wu, Zhang, & He, 2005). Although, there were no attempts made so far to extract carrageenan directly from seaweeds using ILs but formation of strong ion gels of different carrageenans and their composites with cellulose were observed in BMIMCl (Prasad, Kaneko, & Kadokawa, 2009), which envisaged the possibility of extraction of the polysaccharide in ILs. Moreover, recently we have reported selective precipitation of agarose from seaweed (Gracilaria dura) extractives using bio-based ionic liquids (Sharma, Chaudhary, Mondal, Meena, & Prasad, 2015; Sharma, Mondal, Singh, & Prasad, 2015). It should be noted that, -carrageenan is conventionally extracted using alkali following the method reported by Craigie and Leigh (1978).
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Handa, & Stoddart, 2005). In a typical reaction, choline chloride (hydrogen bond acceptor) and ethylene glycol, glycerol or urea (hydrogen bond donors) were mixed separately in the optimized molar ratio of 1:2 and were heated at 80 ◦ C until a transparent solution was obtained. 2.3. Extraction of -carrageenan from seaweed using DES Fig. 1. Repeating units in -carrageenan.
After the discovery of deep eutectic solvents (DESs) by Abbott, Capper, Davies, Rasheed, and Tambyrajah (2003) as an alternative to conventional ILs, the application of the DESs is increasing exponentially in various applications. Further the properties of DESs such as non-toxicity, cheaper cost, ease of syntheses, etc., make them lucrative for number of applications e.g., as reaction media, reactants, and catalysts (Zhang, Vigier, Royer, & Jérôme, 2012) and even preferable over ILs in number of applications. In general, DESs can be obtained by the complexation of the halide salts of quaternary ammonium or phosphonium cations (as the hydrogen bond acceptor, HBA) with hydrogen bond donor (HBD) moieties such as urea, glycerol, ethylene glycol, mannitol, recorcinol, etc., (Sirviö, Visanko, & Liimatainen, 2015). There are reports on the applications of DESs for the dissolution of biopolymers (Mondal, Sharma, Mukesh, Gupta, & Prasad, 2013; Sharma, Mukesh, Mondal, & Prasad, 2013), extraction of phenolic compounds from plants (Dai, Spronsen, Witkamp, Verpoorte, & Choi, 2013), extraction of proteins (Zeng et al., 2014), extraction of glaucarubinone from Simaruba glauca (Kholiya, Bhatt, Rathod, Meena, & Prasad, 2015) and many more applications of the novel liquid are continuously being explored. We have shown the excellent capability of certain bio-based DESs for the dissolution and morphological transformation of DNA as well as material preparation using this bio-macromolecule as building blocks employing DESs as dissolution media (Bhatt, Mondal, Bhojani, Chatterjee, & Prasad, 2015; Mondal, Bhatt, Sharma, Chatterjee, & Prasad, 2014; Mukesh & Prasad, 2015). Although the DESs are turned to be good alternate solvents for biopolymers but extraction of biopolymers especially any seaweed based are not attempted so far. Herein we have employed DESs obtained by the complexation of choline chloride with urea, ethylene glycol and glycerol as well as their hydrated forms for the efficient extraction of -carrageenan from Kappaphycus alvarezii. The seaweed belongs to the family Rhodophycea and commercially exploited for -carrageenan.
All the three DESs were used separately to extract -carrageenan from K. alvarezii. Experiment I: In a typical experiment, 500 mg of powdered seaweed was taken separately in 10 g of DES in a beaker. The mixture was heated at 85/95 ◦ C for 1 h. Experiment II: 500 mg of powdered seaweed was heated with 10 g of DES having 10% water for 1 h. The mixtures thus obtained from both the experiments were centrifuged separately and the mass obtained at the bottom of centrifuge tube was separated, washed several times using IPA and dried under vacuum. A control experiment was carried out by mixing 500 mg of seaweed powder and 10 ml of water keeping the temperature and time same as that of the previous set of reactions. The supernatant was precipitated in IPA (1:3, v/v). The precipitated -carrageenan was vacuum dried. 2.4. Extraction of -carrageenan employing conventional method -Carrageenan was extracted from K. alvarezii following the method reported by Craigie and Leigh with minor modifications (Craigie & Leigh, 1978). In a typical experiment, 10 g of dried K. alvarezii was soaked in 200 ml of 0.5% calcium hydroxide solution for 2 h. The soaked seaweed was autoclaved at 107 ◦ C for 1.5 h after the addition of 200 ml water. After autoclaving, the hot mixture was ground in a kitchen blender and centrifuged. The supernatant was slowly added in to IPA under constant stirring (1:3, v/v). The precipitated -carrageenan was vacuum dried. 2.5. Preparation of -carrageenan gel For the preparation of gel, 10 mg sample of each extracted carrageenan was dissolved in 1% 1 ml KCl solution by heating at 90 ◦ C for 5 min in beakers. The gel obtained at room temperature was further kept in refrigerator at 5 ◦ C for 12 h for curing. 2.6. Characterization
Choline chloride (AR grade) and Urea was purchased from SD Fine chemicals, Mumbai, India. Ethylene Glycol and glycerol was procured from Merck Chemicals, Mumbai. HPLC grade isopropyl alcohol (IPA) was purchased from Loba Chemie, Mumbai. All chemicals were used as received. Fresh K. alvarezii was collected from the cultivation sites of west coast of India, Simar (20◦ 42 N; 71◦ 01 E) during February, 2015. The seaweed was sun dried and was washed several times with water to remove epiphytes and kept under shadow for drying. After drying, the seaweed was crushed into very fine powder using mortar and pestle in the presence of appropriate amount of liquid nitrogen.
FTIR analyses of the -carrageenan samples were carried out on a Perkin-Elmer FTIR spectrometer instrument (Spectrum GX, USA) using KBr disc in the range 400–4000 cm−1 . The 1 H NMR was recorded on a Bruker Avance-II, 500 MHz spectrometer. Rheological measurements were performed on an Anton Paar, Physica MCR 301 rheometer USA, using parallel plate PP50/P-PTD200 geometry (49.971 mm diameter; 0.75 mm gap). Temperature was maintained by Anton Paar, Viscotherm VT2 circulating water bath. The dynamic viscosities were determined varying the shear rate at 25 ◦ C e.g., 0.1–50 s−1 followed by measurement of steady shear viscosity in the range of 10–50 s−1 . Frequency sweep was performed to calculate storage modulus, G and the loss (or viscous) modulus, G where angular frequency was set from 0.05 to 1. Time dependant G and G measured at amplitude 0.1% and frequency 0.01 Hz by setting 20 measuring points of 2 s duration each in the time frame of 0–200 s. The time and frequency dependence of the storage and loss moduli (G and G ) for the gels were measured at 25 ◦ C.
2.2. Synthesis of deep eutectic solvents
3. Results and discussion
ChoCl. Urea 1:2, ChoCl-EG 1:2 and ChoCl-Gly 1:2 were synthesized and characterized as described previously (Abbott, Bell,
Well ground K. alvarezii powder was soaked separately in ChoClUrea 1:2; ChoCl-EG 1:2 and ChoCl-Gly 1:2 for 10 min at 1:20 (w/w)
2. Experimental 2.1. Materials
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Fig. 2. Photographic demonstration for the extraction of -carrageenan from Kappaphycus alvarezii using deep eutectic solvents.
Table 1 Yield of -carrageenan obtained from Kappaphycus alvarezii using different solvent systems. S. no.
Solvent system/method
Yield % (±S.D)
Viscosity (cP) of solvent at 25 ◦ C
Sample code
1
Choline-ChlorideUrea 1:2 Choline-ChlorideEthylene glycol 1:2 Choline-ChlorideGlycerol 1:2 10% Hydrated cholineChloride-Urea 1:2 10% Hydrated choline-ChlorideEthylene glycol 1:2 10% Hydrated cholineChloride-Glycerol 1:2 Water Conventional methoda
37.60 ± 1.20
289.10
A
50.66 ± 1.78
35.49
B
30.93 ± 0.90
266.60
C
53.64 ± 1.25
34.49
D
46.02 ± 2.30
16.50
E
60.25 ± 1.10
52.64
F
46.87 ± 2.00 36.58 ± 1.90
0.90 NA
G H
2 3 4 5
6 7 8
NA: not applicable. a Craigie and Leigh (1978).
ratio followed by heating at 85 ◦ C for 1 h. The mixture thus obtained was centrifuged, which showed formation of two layers as shown in Fig. 2. The isolated bottom layer after several washings with IPA and vacuum dry yielded -carrageenan. -Carrageenan was also extracted using 10% hydrated DESs, pure water and by conventional method for comparison of the physicochemical properties. The upper layer upon addition in IPA yielded a white precipitate (0.5–1% with respect to the DESs), which turned hard upon drying and the FT-IR of the precipitate showed the presence of traces of the polysaccharide (supporting Fig. S1). The result confirms the presence of majority of the polysaccharide in the bottom layer. The yields of -carrageenan (with respect to dry seaweed) obtained using all the above solvent systems are depicted in Table 1. It can be observed from Table 1 that, the yield of -carrageenan has increased substantially when hydrated DESs were used for extraction in comparison to the one extracted using pure DESs, water and conventional method. The yield was highest when 10% hydrated ChoCl-Gly 1:2 was used as solvent for the extraction and lowest for the -carrageenan extracted using ChoCl-Gly 1:2. Almost two fold increases in the yield was observed in the case of hydrated DESs mediated extractions in comparison to the extractions carried out using the pure ones. Extraction of -carrageenan from K. alvarezii involve breaking of the seaweed cell walls first followed by interaction of the polysaccharide with the solvents. As can be
seen from Fig. 1, -carrageenan is sulphated at C-4 position and normally potassium cation present as counter ion. During the interaction process, the choline cations replace the potassium cations simply by ion exchange. Such ion exchange was also observed during reaction of -carrageenan with a polymerizable ionic liquid (Prasad & Kadokawa, 2010). Since choline has very high solubility in water and perhaps the reason behind the higher extraction ability of the hydrated DESs. Although in the case of hydrated ChoCl-EG, the hydration has reduced the viscosity by 92% over the original but the extraction of the polysaccharide did not improve even got marginally lower. So, lower viscosity is not only the case of higher extraction efficiency. Hence, the interaction of the polysaccharide with the DES must be playing crucial role in the extraction process. The marginally higher yield in the case of the hydrated DESs in comparison to water may be due to the higher affinity of the charges (due to choline) towards -carrageenan present in the seaweed, which is absent in water. The electrostatic interactions between the solvent and carrageenan in the hydrated DESs as well as their lower viscosity are perhaps responsible for the observed higher yield of -carrageenan. Such interaction was found to be responsible for selective agarose extraction from G. dura, an agarophyte (Sharma, Chaudhary, et al., 2015; Sharma, Mondal, et al., 2015). The indication of the interaction between the sulphate group of -carrageenan and the DESs are also evident from the elemental analyses data (supporting Table S1). In Table S1, the % S for the -carrageenan extracted using hydrated ChoCl-Urea 1:2 was the lowest in comparison to the rest of the samples, which showed the removal of some portion of the anion due to the strong interaction with the DES. However the % N was more in the samples extracted using DESs or their hydrated counterparts in comparison to the sample extracted using water as solvent. After detailed evaluation of the elemental analyses data, a plausible mechanism for the extraction of -carrageenan using different DESs is proposed as shown in Scheme 1. The calculated %N and %S in the proposed structure of A is 4.10 and 4.60%, respectively, while the values were 4.77 and 3.36 in B, which nearly matches with the measured values. Similarly in the case of extraction using hydrated ChoCl-Urea 1:2 (pH 9.76), the alkaline hydrolysis has removed the sulphate group of -carrageenan and the elemental analysis suggested the structure D. The elemental analyses also suggested the structures of E and F for the -carrageenan extracted using hydrated ChoCl-EG 1:2 and ChoCl-Gly 1:2, respectively. In order to find out quality as well as preservation of chemical structures of -carrageenan extracted using various solvents, the FT-IR spectra was recorded for all the samples and is shown in Fig. 3 and Fig. S2. In all the -carrageenan samples extracted using various solvents systems, the characteristic bands between
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Scheme 1. Plausible mechanism showing structure of -carrageenan extracted using different DESs and their hydrated counterparts.
Fig. 3. FT-IR spectra of -carrageenan extracted using different solvent systems.
845 and 848 cm−1 due to d-galactose-4-sulphate was visible. However the intensity of the band was relatively better in the case of the samples extracted using hydrated ChoCl-EG 1:2 (E) and pure ChoClgly 1:2 (C). The characteristic bands due to 3,6 anhydrogalactose was also observed between 924 and 927 cm−1 in all the samples extracted using various solvents. However the intensity of the band was very low in the case of -carrageenan extracted using both pure and hydrated ChoCl-urea 1:2 (A and D), which may be due to the strong interaction of sulphate of carrageenan with the DESs eventually resulting modification in the structure of the polysaccharide (Scheme 1). The presence of characteristic bands of the polysaccharide indicates that the solvent systems employed are suitable to extract the polysaccharide directly from the seaweed. However, among all the solvent systems, 10% hydrated ChoClEG 1:2 and 10% hydrated ChoCl-Gly 1:2 were emerged as the superior solvent systems among all the solvents used in this study for the extraction of the polysaccharide. 1 H NMR was recorded for the -carrageenan extracted using 10% hydrated ChoCl-Gly 1:2 (which yield the highest amount of -carrageenan) and was compared with the one extracted using
Fig. 4. Steady shear viscosity of -carrageenan gel prepared from the sample extracted using various solvent systems.
conventional method (supporting Figs. S3 and S4). The spectra were same for both the samples with the chemical shifts (␦) of the protons comparable to the reported values for -carrageenan (Van de Velde, Knutsen, Usov, Rollema, & Cerezo, 2002). Since -carrageenan is used mostly in the gel form and hence detail rheological investigation was carried out to study the nature of the -carrageenan gels prepared from the samples obtained using different solvent systems and to compare their rheological properties with the one extracted using water as solvent and the sample extracted using conventional method. All the C gels shown in Fig. 4 have revealed different flow behaviour with pseudoplastic nature in all the cases. The gel prepared from C by conventional method (H) had showed lower degree of flow in comparison to the rest of the samples with highest flowable property for the sample obtained with hydrated ChoCl-Gly 1:2. -Carrageenan is known to form strong gels with KCl. The stiffer behaviour of the C gel prepared using conventional method and water as solvent in comparison to the C gel extracted using the DES or their hydrated counterparts indicated some sort
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Fig. 6. Frequency dependence of G and G for -carrageenan gel prepared from -carrageenan extracted using different solvents.
the hydrated DESs showed liquid viscous behaviour in the angular frequency range 0.05–1000 s−1 , however the gels obtained by conventional methods as well as using pure DESs as solvents showed viscous liquid behaviour in the angular frequency range above 100 s−1 . After the crossover of the moduli, the storage modulus started to become predominant over the loss modulus indicating elastic nature of the gels at higher frequency range (Fig. 6). 4. Conclusions
Fig. 5. (a): Time dependent viscoelasticity profile of -carrageenan gel prepared from the samples extracted using various solvent systems. (b) Time dependence of G and G for -carrageenan gel prepared from -carrageenan extracted using different solvents.
of hindrance in the gel formation in the C samples extracted using the DESs. Furthermore the C extracted using hydrated DESs found to form weaker gels in comparison to those extracted using their pure counterparts. Perhaps this is due to the presence of little amount of the DESs in the C (indicated in the elemental analyses) responsible for the disturbance in the formation of three dimensional helices during the gel formation. Time dependent viscoelasticity measurements showed gel like behaviour with G > > G for all the C gels irrespective of the solvents used for extraction in the entire time period of investigation. However, the differences in the magnitude of G and G was maximum in the case of the C gels extracted using ChoCl-gly 1:2 and hydrated ChoCl-EG 1:2 in comparison to the rest of the samples. It should be noted that, the difference between G and G was minimum for the C samples extracted using conventional method or by water (Fig. 5a and b). From the flow behaviour and viscoelasticity profile it can be inferred that, although the C gels obtained in the presence of DES are less stiff in nature but possess better viscoelasticity in comparison to their counter parts extracted using conventional method or water as solvent. In all the C gels, the crossover of G and G was observed with increasing frequency indicating frequency dependence signature of weak gels. However the value of frequency at which they crossover was different for different gels. It was interesting to note that in all the cases the loss modulus predominant over the storage modulus in a wide frequency range indicating the presence of more flow components in the gels making the gels behave like viscous liquid at lower frequencies. The C gels obtained using water and all
Three different deep eutectic solvents prepared by the complexation of choline chloride with urea, ethylene glycol and glycerol as well as their hydrated counterparts were used for the selective extraction of -carrageenan from the carrageenophyte K. alvarezii. The quality of thus obtained -carrageenan was compared with the quality obtained using water as extraction media as well -carrageenan extracted using well practiced conventional method. However, traces of the DESs were found in the polysaccharide samples. Considering the non toxic nature of choCl-EG and ChoCl-Gly, the impurities may not pose any difficulty in the application of -carrageenan. However the impurity of DES consisting of Choline chloride and urea may pose some problem. A plausible mechanism for the modification of chemical structure of the polysaccharide during the extraction process is proposed. The detail rheological investigation of the carrageenan gels was studied. It was inferred from the studies that, the physicochemical as well as rheological properties of the polysaccharide obtained using DESs as solvents was at par to the one obtained using conventional method and was superior in comparison to the carrageenan obtained using water as solvent. Considering the tedious nature of the carrageenan extraction method employed in conventional extraction method, the DESs can be considered as suitable alternative solvents for the facile extraction of the polysaccharide directly from seaweed. However, among the hydrated and non-hydrated DESs, the hydrated ones were found to extract -carrageenan more efficiently in comparison to their non-hydrated counterparts. Acknowledgments KP thanks Council of Scientific and Industrial Research, New Delhi for granting CSIR-Young Scientist Awardees Project (CSIRYSP/2011-12), CSIR Network project CSC10130 and OLP0080 for overall financial support. MS and DM are grateful to UGC and CSIR for senior research fellowships and to AcSIR for PhD registration. Analytical Division & Centralized Instrument Facility of the Institute
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