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Esterification of potato starch by a biocatalysed reaction in an ionic liquid
Carbohydrate Polymers 137 (2016) 657–663
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Esterification of potato starch by a biocatalysed reaction in an ionic liquid Arkadiusz Zarski a , Sylwia Ptak a,b , Przemyslaw Siemion a , Janusz Kapusniak a,∗ a Institute of Chemistry, Environmental Protection and Biotechnology, Faculty of Mathematics and Natural Sciences, Jan Dlugosz University in Czestochowa, 42-200 Czestochowa, Poland b Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 90-924 Lodz, Poland
a r t i c l e
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Article history: Received 13 July 2015 Received in revised form 6 November 2015 Accepted 7 November 2015 Available online 14 November 2015 Keywords: Potato starch Oleic acid Esterification Immobilised lipase 1-Butyl-3-methylimidazolium chloride
a b s t r a c t In this study, potato starch was esterified with oleic acid, using 1-butyl-3-methylimidazolium chloride as a reaction medium and an immobilised lipase from Thermomyces lanuginosus as a catalyst. The degree of substitution (DS) of the products was determined by the volumetric method; and the best esterified product (with the highest DS) was determined by an elemental analysis. The effect of the reaction parameters on the DS, such as the time and the temperature, were also studied. The product with the highest DS (0.22) was found in the reaction carried out at 60 ◦ C for 4 h. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) analyses confirmed the esterification of the potato starch. Furthermore, the results of X-ray diffraction (XRD) and a scanning electron microscopy (SEM) revealed that the crystallinity and the morphology of the native potato starch was slightly changed during its partial gelatinisation in the ionic liquid, and was completely destroyed as a result of the formation of the esters. The thermal stability of the starch oleate decreased, when compared to the unmodified starch, as was indicated by a thermal gravimetric analysis (TGA). © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Natural polymers which are non-toxic, biodegradable and inexpensive, such as starch, are of great interest regarding to their possible use as materials in various industries, such as the food, paper, pharmaceutical and packaging industries (Fang & Fowler, 2003). Starch granules consist of two types of ␣-glucan: amylose and amylopectin (representing approximately 98–99% of the dry weight). These form linear polymer chains and hydrogen-bonded supramolecular structures (Jie, Wen-ren, Manurung, Ganzeveld, & Heeres, 2004). Native starch is characterised by high fragility and an incompatibility with hydrophobic polymers. It also has a low moisture resistance, meaning it has low processing qualities (due to high viscosity) and is highly hydrophilic. These properties can significantly limit the use of a starch to obtain a new type of material. Therefore, different types of modifications have been implemented to improve the mechanical properties of starch and its hydrophobisation (Fang, Fowler, Tomkinson, & Hill, 2002; Tomasik & Schilling, 2004).
∗ Corresponding author. E-mail address: [email protected] (J. Kapusniak). http://dx.doi.org/10.1016/j.carbpol.2015.11.029 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
Esterification is one of the oldest methods used for the modification of carbohydrate polymers (Mullen & Pacsu, 1942). The most important starch derivative – fatty acid starch esters – can be obtained through an acylation reaction of the free hydroxyl groups in the anhydroglucose unit (AGU). Usually, in order to enable an esterification reaction with the acids, a starch is first dissolved in dimethyl sulfoxide (DMSO) (Junistia et al., 2009), pyridine (Aburto et al., 1999) or another commonly used solvent, such as dimethylformamide (DMF) or tert-butanol (Boruczkowska et al., 2013; Lukasiewicz & Kowalski, 2012). However, the use of solvents also has some limitations and disadvantages, such as volatility, flammability and high levels of toxicity. They not only represent a significant hazard during the separation process, but can also be a source of environmental pollution. Therefore, it has become important to look for a “green”, environmentally friendly solvent, which can be used for the esterification of polymers. A few years ago, researchers discovered the some ionic liquids (ILs) have the ability to dissolve carbohydrate polymers (Wilpiszewska & Spychaj, 2011). One of the first of these ionic liquids was 1-butyl-3-methylimidazolium chloride, which was used during the chemically catalysed synthesis of a starch acetate using acetic anhydride (Biswas, Shogren, Stevenson, Willett, & Bhowmik, 2006). In 2007, a study was conducted on the influence of the same ionic liquid on the dissolution of starch from different botanical
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origins (corn, rice, wheat and potato) (Stevenson, Biswas, Jane, & Inglett, 2007). From this, it is known that the solubility of starch in an ionic liquid not only depends on its structure, but also on the temperature of the reaction. In order to reduce the time for starch depolymerisation, while maintaining a highly efficient reaction, the depolymerisation can be carried out in an oil bath, or even by heating using a microwave reactor in the presence of a chemical catalyst (Rajan, Prasad, & Abraham, 2006). Different esterifying factors have been used for the non-catalysed synthesis of starch esters in an ionic liquid, including: acetic and succinic anhydride (Luo & Zhou, 2012; Xie, Shao, & Liu, 2010); fatty acids such as stearic acid, lauric acid, and palmitic acid; or esters of these acids such as vinyl stearate (Biswas, Shogren, & Willett, 2009; Gao, Luo, & Luo, 2012). The chemically catalysed (using pyridine) esterification of starch in an ionic liquid has also been carried out, by reactions with acetic anhydride (Biswas et al., 2006); hexanoic, phthalic, and propionic anhydride (Lehmann & Volkert, 2009); and with fatty acid esters such as methyl laurate or stearate (Xie & Wang, 2011). It is commonly known that the esterification of starch with fatty acids can be performed in the presence of biocatalysts (like lipases). The main sources of these enzymes are fungi, including: Candida antarctica (Xu et al., 2012); Thermomyces lanuginosus (Alissandratos et al., 2011); Burkholderia cepacia (Rajan & Abraham, 2006); and Candida rugosa (Rajan, Sudha, & Abraham, 2008). Sometimes, enzymes from bacteria are also used, such as: Staphylococcus aureus (Horchani, Chaabouni, Gargouri, & Sayari, 2010), and Pseudomonas sp. (Qiao, Gu, & Cheng, 2006). Studies have confirmed that some ionic liquids improved the stability of the enzymes, and in addition could be their activators (Van Rantwijk, Madeira Lau, & Sheldon, 2003). In many cases, the enzymatic esterification in an ionic liquid has been the selective and efficient method (Ganske & Bornscheuer, 2005; Sheldon, Madeira Lau, Sorgedrager, van Rantwijk, & Seddon, 2002), such as a successful attempt at the lipase-catalysed esterification of high-amylose corn starch in a mixture of imidazolium-based ionic liquids (Lu, Luo, Yu, & Fu, 2012). Fatty acid starch esters have also been prepared via a twostep method using two different types of imidazolium-based ionic liquids, where the pregelatinisation of the starch was performed using chloride, but the lipase catalysed synthesis was carried out in tetrafluoroborate (Lu, Luo, Fu, & Xiao, 2013). The aim of present study is to optimise the conditions for the preparation of new functional materials based on starch, by a biocatalysed esterification with unsaturated fatty acids in an ionic liquid. Thus, potato starch was esterified with oleic acid, in the presence of an immobilised fungal lipase from T. lanuginosus as the catalyst, and with 1-butyl-3-methylimidazolium chloride as the reaction medium. The esterification of the starch with an unsaturated fatty acid may provide opportunities to further modify the obtained potato starch fatty acid esters, by an addition reaction to unsaturated double bond. Important and novel aspects of the study are the application of lipase immobilised on a polymer carrier, which can be used several times; and a simplification of the process by using the same ionic liquid for both the pregelatinisation of the starch and the preparation of the starch oleates. It should be emphasised that the proposed model esterification reaction may be applied to the esterification of potato starch with oils rich in oleic acid, such as high oleic sunflower oil and high oleic canola oil.
2. Materials and methods 2.1. Materials The materials used in the study include: potato starch purchased from Roth (Karlsruhe, Germany); pure oleic acid purchased from Chempur (Piekary Slaskie, Poland); 99.8% pure anhydrous
ethanol purchased from POCH (Gliwice, Poland); 95% ionic liquid 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) purchased from Sigma–Aldrich (Germany); and lipase from T. lanuginosus (Immozyme TLL-T1-350) adsorbed on a polymer carrier (with an activity of 10,000 TBU/g), purchased from Chiral Vision (The Netherlands).
2.2. Determination of lipase activity The lipase activity was measured by the liberation of butyric acid during the hydrolysis of tributyrin (glycerol tributyrate) according to the assay method described by Chiral Vision for their Immozyme TLL-T1-350 lipase. Hydrolysis of tributyrin was monitored titrimetrically in a pH-stat titration system (Metrohm titrator). The butyric acid, which is formed, was titrated with 0.1 mol/l sodium hydroxide and the consumption of the latter recorded as a function of time. Used method is based on the speed at which the enzyme hydrolyses tributyrin at pH 7.5 and 40 ◦ C. The activity of enzyme is expressed as tributyrin units per gram enzyme (TBU/g). 1 TBU (lipase unit) is the amount of enzyme which releases 1 mol titratable butyric acid per minute under the given standard conditions.
2.3. Partial gelatinisation of the starch and preparation of the starch oleates Initially, the potato starch was dried at 105 ◦ C, to a water content below 5%. The dried starch was then added to the ionic liquid ([BMIm]Cl) at a concentration of 10% (w/w) in a two-necked round flask flushed with an inert gas (Ar). The suspension was heated in an oil bath at approx. 90 ◦ C for 1 h. After the ionic liquid melted (and the suspension became viscous), the whole starch paste was stirred vigorously with a magnetic stirrer at 500 rpm. Then, the pregelatinised starch was cooled to the desired temperature for synthesis, and the oleic acid and the biocatalyst were added to initiate the esterification (Lu et al., 2013). A constant molar ratio of the starch (anhydroglucose unit, AGU) to the oleic acid (1:3) was maintained, as well as a constant weight ratio of the immobilised lipase (1.176 g per 1 g of the native starch). The synthesis of esters was carried out in an oil bath at different temperatures (60, 70 or 80 ◦ C) for 4, 6 or 8 h, and then the mixture was again stirred using a magnetic stirrer at 500 rpm. After the completion of the reaction, the mixture was cooled to room temperature. In a further step, the products were precipitated using anhydrous ethanol. Then, the precipitates were centrifuged and washed with anhydrous alcohol to separate out the main product of the esterification – the starch oleates. The solid residues were rewashed (three times) to eliminate the byproducts, the ionic liquid and the untreated reagents. Finally, the residues from the filtration were dried at 50 ◦ C for 36 h. The dried products were ground and then subjected to a physicochemical analysis.
2.4. Determination of the degree of substitution (DS) The DS of the fatty acid starch esters was determined according to the titration method, with a slight modification (Varavinit, Chaokasem, & Shobsngob, 2001). A half gram of powdered starch ester was weighed accurately, and was then dispersed in 25 ml of deionised water containing 5 ml of a 0.5 mol/l NaOH solution. The mixture was stirred vigorously at room temperature for 1 h. After this time, a few drops of phenolphthalein were added as an indicator, and the mixture was titrated using a 0.5 mol/l HCl solution. A blank sample was simultaneously titrated using native potato starch instead of the starch ester. Each sample was measured in
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triplicate, and the DS value of the ester was calculated using the following equation: DS =
162 × (V0 − V ) × CHCl W − [281 × (V0 − V ) × CHCl ]
where 162 – is the molecular weight of the anhydroglucose unit (AGU); 281 – is the molecular weight of the oleic acid residue; V0 – is the volume of HCl consumed for titrating the blank; V – is the volume of HCl consumed for titrating the sample; C(HCl) – is the exact molarity of the HCl solution used; and W – is the exact weight of the dry sample. The highest degree of substitution for the starch ester, which was first determined using the volumetric analysis presented above, was also verified by an elemental analysis. In the second method, the DS was calculated by comparing the theoretical values (the percentages of the elements: C, H, O) with the experimental values, ranging from 0 to 3, for the actual DS. 2.5. Elemental analysis The elemental analyses (to determine the percentages of carbon and hydrogen) were carried out using a micro-analyser, with a simultaneous CHNS determination using a VARIO Micro Cube (Elementar Analysensysteme GmbH, Hanau/Frankfurt, Germany). The measurements were carried out at the Centre of Molecular and Macromolecular Studies (Polish Academy of Sciences, Lodz, Poland).
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Table 1 Degree of substitution (DS) of starch oleates at different temperatures and times. Temperature [◦ C]
60 70 80
Time [h] 4
6
8
0.22 0.21 0.14
0.19 0.18 0.11
0.15 0.14 0.09
2.9. Scanning electron microscopy (SEM) Morphological changes of the native starch, the pregelatinised starch and the fatty acid starch ester were observed using a Tescan VEGA 3SBU scanning electron microscope (Tescan, Brno, Czech Republic). The accelerating voltage was 3 kV, and the samples were not coated with any conductive material.
2.10. Thermal stability analyses The thermal analyses were performed using an STA 409C simultaneous thermal analyser (Netzsch, Selb, Germany). The thermogravimetric (TG) and the differential thermogravimetric (DTG) methods were used, and the measurements were conducted under an argon flow. The samples were heated to temperatures from 25 to 500 ◦ C, with a temperature rate increase of 10 ◦ C/min (Kapusniak & Siemion, 2007).
2.6. Fourier transform infrared spectroscopy (FTIR) The FTIR spectra of the native potato starch, the partly gelatinised starch and the fatty acid starch ester (with the highest value of DS) were recorded using a Nexus Nicolet spectrophotometer (Madison, WI, USA). The powdered samples were mixed separately with an analytical grade KBr and then pressed into discs. Their spectra were recorded in the region of 4000–400 cm−1 , with 32 scans in total. Before the recording, the baseline was adjusted against a KBr background. 2.7. Proton nuclear magnetic resonance spectroscopy (1 H NMR) The 1 H NMR spectra of the samples were recorded using a Bruker Avance III spectrometer (Billerica, MA, US) at 500 MHz. The samples were dissolved in DMSO-d6 (99.8% D, Armar Chemicals, Döttingen, Switzerland) and the solution was then heated at 343 K in a water bath for 12 h. Next, 0.70 ml of the solution was placed in a 5 mm NMR tube. All of the NMR measurements were carried out at 295 K with a 5 mm BBI probehead and a z-gradient coil. Prior to Fourier transformation of the FIDs, the Lorenzian apodisation function (0.3 Hz) was applied and the spectra were zero-filled twice. These measurements were also carried out at the Centre of Molecular and Macromolecular Studies (Polish Academy of Sciences, Lodz, Poland). 2.8. X-ray diffraction analyses (XRD) X-ray powder diffraction patterns were obtained with a Bruker D8 Advance powder diffractometer (Billerica, MA, US), in a theta2-theta configuration, using a Johansson monochromator and a ˚ Data was collected in LynxEye strip detector (Cu K␣1 = 1.5406 A). the 2 theta range of 4–45◦ , with a 1 s step of 0.02◦ . For one sample (Sample A) additional data was collected in the 2 theta range of 4–8◦ , with a 3 s step of 0.01◦ . The measurements were performed at the Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University (AMU) in Poznan (Poland).
3. Results and discussion 3.1. Effect of temperature and the reaction time on the degree of substitution The degree of substitution of a starch ester can be defined as the number of hydroxyl groups that are substituted by acyl groups in each anhydroglucose unit. The theoretical maximum value of the DS is 3, because each glucose unit contains three reactive hydroxyl groups. Table 1 presents the results of the calculated DS for the prepared esters (determined by the volumetric method). The potato starch that produced the esterification with the highest degree of substitution was found in those reactions that were carried out at 60 ◦ C for 4 h. This value amounted to 0.22 and was additionally confirmed by the results obtained from the elemental analysis (DS 0.20). Table 1 also illustrates the relationship between the DS and the conditions at the time of the reaction. With an increased temperature and a prolonged reaction time, the value for the degree of substitution decreased. The esterification of the starch at 80 ◦ C for 8 h was the least effective of the samples tested. This phenomenon may be due to direct or indirect influences of the reaction environment on the enzyme. For example, a longer reaction time tends to generate a greater amount of water, as a byproduct of the esterification. An increase in the water content can then cause changes in the pH of the reaction system, and stimulate hydrolytic reactions. In addition, an increase in the water activity also contributes to a decrease in the catalytic activity of lipase (Eckstein, Wasserscheid, & Kragl, 2002). The inhibition of the catalytic activity can also be caused by small changes in the temperature of the synthesis, especially when the temperature is far from its optimal value. However, based on the results of the DS, it can be seen that it is possible to increase the thermal stability of the enzyme with the use of an ionic liquid as the reaction medium. Because the melting point of the ionic liquid used as solvent in the reaction is about 70 ◦ C, esterification at a temperature lower than 60 ◦ C is not performed.
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Fig. 3. XRD patterns of: (a) native potato starch; (b) partly gelatinised starch; and (c) starch oleate (with the DS 0.22).
Fig. 1. FTIR spectra of: (a) native potato starch; (b) partly gelatinised starch in [BMIm]Cl; and (c) starch oleate with the DS 0.22.
Fig. 2.
1
H NMR spectra of: (a) native potato starch; and (b) starch oleate (with the DS 0.22).
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Fig. 4. SEM images of: (a) native potato starch; (b) partly gelatinised starch; and (c) starch oleate (with the DS 0.22).
1H
3.2. FTIR measurements
3.3.
The FTIR spectra of the native potato starch, the partly gelatinised starch and the esterified starch are presented in Fig. 1. In the spectrum for native potato starch, a strong broad band appeared with three peaks between 980 and 1200 cm−1 (the finger print region). These can be attributed to C O stretching vibrations, which are the most characteristic ones for polysaccharides. A second characteristic band between 3000 and 3500 cm−1 can be assigned to stretching vibrations from hydrogen-bonded hydroxyl groups. In comparing the spectrum of the native starch and the partly gelatinised starch, no significant changes in these regions were observed, which is in agreement with previously reported results (Lu et al., 2013). However, in the starch oleate (with the DS 0.22) the broad peak at about 3400 cm−1 was reduced in intensity. Such a phenomenon may be due to the reduction in the number of hydroxyl groups, which have been converted into ester groups. In addition, in the FTIR spectrum of the starch oleate a new band appeared at 1730 cm−1 . This band was not present in the spectra of either the native starch or the partly gelatinised starch, and its occurrence is attributed to the carbonyl group in the ester. Therefore, this could confirm the esterification of the starch with the oleic acid (Fang et al., 2002; Mathew & Abraham, 2007). For comparison, the carbonyl signal in the FTIR spectrum for oleic acid appears at about 1711 cm−1 (Spectral Database for Organic Compounds, 2015).
The 1 H NMR spectra for native potato starch and for the starch oleate with the DS 0.22 are presented in Fig. 2. Chemical shifts are reported in parts per million, and spectral signals between 3.50 and 5.50 ppm correspond to the protons of the anhydroglucose units (of the 1,6-anhydro--D-glucose). Chemical signals of the protons at 3.30 ppm for H-2, 3.65 ppm for H-3, and 3.55 for H-5 appeared in the spectra of the native potato starch, and the shifts of H-1 and OH-2, 3, 6 were assigned to peaks between 4.58 and 5.50 ppm (Namazi, Fathi, & Dadkhah, 2011). The changes in the appearance of the NMR spectra of the ester when compared with the spectra of the native starch may indicate the occurrence of a modification in the structure of the polymer. Namely, during the esterification, the hydrogen atom of the hydroxyl groups in the unmodified starch has been substituted by an acyl group (from the oleic acid), leading to a change in the proton resonances of the AGU units. Such a substitution is visible in the spectrum of the starch oleate, as weaker signals are assigned to the hydroxyl groups of the anhydroglucose. Furthermore, the presence of characteristic peaks for the ester of an unsaturated fatty acid was confirmed. The NMR spectra for the starch oleate showed signals from three protons (a triplet peak) at around 0.8–0.9 ppm, which can be linked to the protons in the terminal methyl group of the acyl chain. The proton signal at 2.2 ppm may be related to the methylene group, which occurs before the carbonyl group, and another proton peak at around 1.5 ppm is associated with the methylene
NMR measurements
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group directly before it. In addition, a signal at 5.3 ppm refers to the protons associated with carbon atoms which are connected by a double bond. In the spectrum of the starch oleate, signals of the methylene groups can also be observed close to this double bond (at around 2.0 ppm) and all the other groups included in the acyl chain (at around 1.25 ppm). This result is in agreement with previously reported results (Kapusniak & Siemion, 2007; Lu et al., 2012) 3.4. XRD analysis X-ray diffraction was performed to investigate the difference in the crystallinity between the unmodified and the modified starch (Fig. 3). The XRD pattern for the native potato starch displayed strong reflections at about 5.6◦ and 17◦ (2), with the addition of a few smaller reflections at about 15◦ , 20◦ , 22◦ , 24◦ (2), which indicated a typical B-type crystalline pattern (Mathew & Abraham, 2007). The XRD pattern changed slightly after the initial pregelatinisation of the starch in the ionic liquid (i.e. the main signals became less intense or partly disappeared). The crystal region of the starch was also slightly destroyed, which might confirm that the ionic liquid partially disrupted the intra- and intermolecular hydrogen bonds in the starch molecules, which are responsible for a highly ordered crystalline structure (Xu, Miladinov, & Hanna, 2004). As is shown in Fig. 3, the diffractogram of the starch oleate (DS 0.22) did not exhibit any diffraction peaks, which could confirm that the esterification did not lead to the formation of new crystalline regions in the ester (Gao et al., 2012). The overall appearance of the diffraction pattern might indicate only the presence of amorphous regions, suggesting that after the modification with oleic acid, the bonds were completely disrupted. This is because during the esterification, the acidic residues of the oleic acid replaced some of the hydroxyl groups in the potato starch, resulting in a loss of crystallinity from the original structure caused by a reduction of the intermolecular hydrogen bonds (Desalegn et al., 2014).
Fig. 5. TG thermograms of: (a) native potato starch; (b) partly gelatinised starch; and (c) starch oleate (with the DS 0.22).
Fig. 6. DTG curves of: (a) native potato starch; (b) partly gelatinised starch; and (c) starch oleate (with the DS 0.22).
3.5. SEM analysis Fig. 4 illustrates the morphologies of the native starch, the partly gelatinised starch and the starch oleate (DS 0.22), investigated using a scanning electron microscopy technique. According to the SEM images, most of the potato starch granules ranged in size from 20 to 60 m and were oval to ellipsoid in shape. After the initial pregelatinisation process in 1-butyl-3methylimidazolium chloride, the starch partially lost its granular structure, and some of the granules became more irregular, with an increased surface area. However, in comparison with the native and pregelatinised starches, the starch oleate exhibited a very different morphology, where the starch granules were completely destroyed, and lost their individuality and smoothness. The SEM images demonstrate that the products were agglutinated. The morphological changes in the ester can be attributed to the effect of the gelatinisation process, when the ionic liquid as a reaction medium disrupts the hydrogen bonds and destroys the crystalline structure of the starch granules (Lu et al., 2013). In addition, the changes can be attributed to the effect of the substitution of the hydroxyl groups during the esterification (Xie & Wang, 2011). 3.6. Thermal stability analysis The thermal properties of native potato starch, the partly gelatinised starch and the starch oleate were studied by a TGA. The resulting TG and DTG curves are presented in Figs. 5 and 6, respectively. These were used to determine the weight loss of the material after heating, and the changes in thermal stability caused by the esterification. The thermograms and curves of the potato starch,
Table 2 Thermal characteristics of the native potato starch, partly gelatinised starch, and starch oleate (DS 0.22). Sample
TG ◦
DTG
ti [ C]
Weight loss [%]
to [◦ C]
Native potato starch
38.8 251.8
7.82 53.75
98.8 300.1
Partly gelatinised starch
40.3 229.3
4.94 49.89
94.8 286.3
Starch oleate (DS 0.22)
41.5 198.4
2.84 41.27
58.0 250.7
partly gelatinised starch and starch oleate (DS 0.22) show the main stages of this weight loss. The first step is related to the evaporation of water below 100 ◦ C; whereas the other step corresponds to the decomposition processes, which are mainly relating to the degradation of the starch chain. The maximum temperature for decomposition was found at about 300 ◦ C for the native starch, 286 ◦ C for the partly gelatinised starch and 250 ◦ C for the starch oleate. The temperatures for the initial degradation of were reached at about 251.8 ◦ C, 229.3 ◦ C and 198.4 ◦ C, respectively (Table 2). The extra weight loss of the starch oleate at higher temperatures was probably due to the decomposition of the ester moiety that was formed during the synthesis of the oleic acid starch ester (Desalegn et al., 2014). Comparing the results of the unmodified starch and the starch oleate, it can be concluded that the biocatalysed esterification in the ionic liquid
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reduced the initial temperature for decomposition, as well as the thermal stability of the final product (Lu et al., 2012). 4. Conclusion An ester of potato starch and oleic acid was successfully synthesised by a lipase catalysed esterification in only one ionic liquid (1-butyl-3-methylimidazolium chloride), as a reaction medium for both the gelatinisation and the esterification of the starch. The proposed method suggests the possibility of producing esters with varying degrees of substitution, in the range of 0.1–0.2. The highest DS was found in the product from the reaction carried out at 60 ◦ C for 4 h. Based on these results, we can conclude that the chloride did not provide a complete dissolution of the potato starch, but only resulted in its partial gelatinisation and the relaxation of the macrostructure. However, this was enough for the synthesis of new potato starch esters. Multiple uses of a lipase from T. lanuginosus were achieved in this newly developed method, due to the fact that it was immobilised. The application of an unsaturated fatty acid, such as oleic acid, as an esterifying agent may provide opportunities for the further functionality of the resulting ester. Moreover, there is a chance that this method could be successfully applied for the transesterification of starch with high oleic acid sunflower and canola oils. Materials based on high fatty acid starch esters, with improved processing properties, could be used to produce new biodegradable packaging or as carriers for bioactive agents. References Aburto, J., Alric, I., Thiebaud, S., Borredon, E., Bikiaris, D., Prinos, J., et al. (1999). Synthesis, characterization, and biodegradability of fatty acid esters of amylose and starch. Journal of Applied Polymer Science, 74, 1440–1451. Alissandratos, A., Baudendistel, N., Hauer, B., Baldenius, K., Flitsch, S., & Halling, P. (2011). Biocompatible functionalisation of starch. Chemical Communication, 47, 683–685. Biswas, A., Shogren, R. L., Stevenson, D. G., Willett, J. L., & Bhowmik, P. K. (2006). Ionic liquids as solvents for biopolymers: Acylation of starch and zein protein. Carbohydrate Polymers, 66, 546–550. Biswas, A., Shogren, R. L., & Willett, J. L. (2009). Ionic liquid as a solvent and catalyst for acylation of maltodextrin. Industrial Crops and Products, 30, 172–175. ˙ z, ˙ W., Boruczkowska, H., Boruczkowski, T., Tomaszewska-Ciosk, E., Drózd ˙ A., et al. (2013). Enzymatic esterification of starch Bienkiewicz, M., Zołnierczyk, phosphate with oleic acid. Chemical Review, 92, 1078–1082. Desalegn, T., Garcia, I. J. V., Titman, J., Licence, P., Diaz, I., & Chebude, Y. (2014). Enzymatic synthesis of epoxy fatty acid starch ester in ionic liquid–organic solvent mixture from vernonia oil. Starch, 66, 385–392. Eckstein, M., Wasserscheid, P., & Kragl, U. (2002). Enhanced enantioselectivity of lipase from Pseudomonas sp. at high temperatures and fixed water activity in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide. Biotechnology Letters, 24, 763–767. Fang, J., & Fowler, P. (2003). The use of starch and its derivatives as biopolymer sources of packaging materials. Food, Agriculture & Environment, 1(3&4), 82–84. Fang, J. M., Fowler, P. A., Tomkinson, J., & Hill, C. A. S. (2002). The preparation and characterization of a series of chemically modified potato starches. Carbohydrate Polymers, 47, 245–252. Ganske, F., & Bornscheuer, U. T. (2005). Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH. Journal of Molecular Catalysis B: Enzymatic, 36, 40–42. Gao, J., Luo, Z., & Luo, F. (2012). Ionic liquids as solvents for dissolution of corn starch and homogeneous synthesis of fatty-acid starch esters without catalysts. Carbohydrate Polymers, 89, 1215–1221.
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Horchani, H., Chaabouni, M., Gargouri, Y., & Sayari, A. (2010). Solvent-free lipase-catalyzed synthesis of long-chain starch esters using microwave heating: Optimization by response surface methodology. Carbohydrate Polymers, 79, 466–474. Jie, Y., Wen-ren, C., Manurung, R. M., Ganzeveld, K. J., & Heeres, H. J. (2004). Exploratory studies on the carboxymethylation of cassava starch in water-miscible organic media. Starch, 56, 100–107. Junistia, L., Sugih, A. K., Manurung, R., Picchioni, F., Janssen, L. P. B. M., & Heeres, H. J. (2009). Experimental and modeling studies on the synthesis and properties of higher fatty esters of corn starch. Starch, 61, 69–80. Kapusniak, J., & Siemion, P. (2007). Thermal reactions of starch with long-chain unsaturated fatty acids. Part 2. Linoleic acid. Journal of Food Engineering, 78, 323–332. Lehmann, A., & Volkert, B. (2009). Investigations on esterification reactions of starches in 1-N-butyl-3-methylimidazolium chloride and resulting substituent distribution. Journal of Applied Polymer Science, 114, 369–376. Lu, X., Luo, Z., Fu, X., & Xiao, Z. (2013). Two-step method of enzymatic synthesis of starch laurate in ionic liquids. Journal of Agricultural and Food Chemistry, 61, 9882–9891. Lu, X., Luo, Z., Yu, S., & Fu, X. (2012). Lipase-catalyzed synthesis of starch palmitate in mixed ionic liquids. Journal of Agricultural and Food Chemistry, 60, 9273–9279. Lukasiewicz, M., & Kowalski, S. (2012). Low power microwave-assisted enzymatic esterification of starch. Starch, 64, 188–197. Luo, Z., & Zhou, Z. (2012). Homogeneous synthesis and characterization of starch acetates in ionic liquid without catalysts. Starch, 64, 37–44. Mathew, S., & Abraham, T. E. (2007). Physico-chemical characterization of starch ferulates of different degrees of substitution. Food Chemistry, 105, 579–589. Mullen, J. W., & Pacsu, E. (1942). Starch studies. Preparation and properties of starch triester. Industrial & Engineering Chemistry, 34, 1209–1217. Namazi, H., Fathi, F., & Dadkhah, A. (2011). Hydrophobically modified starch using long-chain fatty acids for preparation of nanosized starch particles. Scientia Iranica, 18, 439–445. Qiao, L., Gu, Q. M., & Cheng, H. N. (2006). Enzyme-catalyzed synthesis of hydrophobically modified starch. Carbohydrate Polymers, 66, 135–140. Rajan, A., & Abraham, T. E. (2006). Enzymatic modification of cassava starch by bacterial lipase. Bioprocess and Biosystems Engineering, 29, 65–71. Rajan, A., Prasad, V. S., & Abraham, T. E. (2006). Enzymatic esterification of starch using recovered coconut oil. International Journal of Biological Macromolecules, 39, 265–272. Rajan, A., Sudha, J. D., & Abraham, T. E. (2008). Enzymatic modification of cassava starch by fungal lipase. Industrial Crops and Products, 27, 50–59. Sheldon, R. A., Madeira Lau, R., Sorgedrager, M. J., van Rantwijk, F., & Seddon, K. R. (2002). Biocatalysis in ionic liquids. Green Chemistry, 4, 147–151. Spectral Database for Organic Compounds, SDBS. (2015). National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan. SDBS No.: 1035. http://sdbs.db.aist.go.jp/sdbs/cgi-bin/direct frame disp.cgi?sdbsno=1035. Stevenson, D. G., Biswas, A., Jane, J., & Inglett, G. E. (2007). Changes in structure and properties of starch of four botanical sources dispersed in the ionic liquid, 1-butyl-3-methylimidazolium chloride. Carbohydrate Polymers, 67, 21–31. Tomasik, P., & Schilling, C. H. (2004). Chemical modification of starch. Advances in Carbohydrate Chemistry and Biochemistry, 59, 175–403. Van Rantwijk, F., Madeira Lau, R., & Sheldon, R. A. (2003). Biocatalytic transformations in ionic liquids. Trends in Biotechnology, 21, 131–138. Varavinit, S., Chaokasem, N., & Shobsngob, S. (2001). Studies of flavor encapsulation by agents produced from modified sago and tapioca starches. Starch, 53, 281–287. Wilpiszewska, K., & Spychaj, T. (2011). Ionic liquids: Media for starch dissolution, plasticization and modification. Carbohydrate Polymers, 86, 424–428. Xie, W., Shao, L., & Liu, Y. (2010). Synthesis of starch esters in ionic liquids. Journal of Applied Polymer Science, 116, 218–224. Xie, W., & Wang, Y. (2011). Synthesis of high fatty acid starch esters with 1-butyl-3-methylimidazolium chloride as a reaction medium. Starch, 63, 190–197. Xu, Y., Miladinov, Y., & Hanna, M. A. (2004). Synthesis and characterization of starch acetates with high substitution. Cereal Chemistry, 81, 735–740. Xu, J., Zhou, C. W., Wang, R. Z., Yang, L., Du, S. S., Wang, F. P., et al. (2012). Lipase coupling esterification of starch with octenyl succinic anhydride. Carbohydrate Polymers, 87, 2137–2144.
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Esterification of potato starch by a biocatalysed reaction in an ionic liquid Arkadiusz Zarski a , Sylwia Ptak a,b , Przemyslaw Siemion a , Janusz Kapusniak a,∗ a Institute of Chemistry, Environmental Protection and Biotechnology, Faculty of Mathematics and Natural Sciences, Jan Dlugosz University in Czestochowa, 42-200 Czestochowa, Poland b Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 90-924 Lodz, Poland
a r t i c l e
i n f o
Article history: Received 13 July 2015 Received in revised form 6 November 2015 Accepted 7 November 2015 Available online 14 November 2015 Keywords: Potato starch Oleic acid Esterification Immobilised lipase 1-Butyl-3-methylimidazolium chloride
a b s t r a c t In this study, potato starch was esterified with oleic acid, using 1-butyl-3-methylimidazolium chloride as a reaction medium and an immobilised lipase from Thermomyces lanuginosus as a catalyst. The degree of substitution (DS) of the products was determined by the volumetric method; and the best esterified product (with the highest DS) was determined by an elemental analysis. The effect of the reaction parameters on the DS, such as the time and the temperature, were also studied. The product with the highest DS (0.22) was found in the reaction carried out at 60 ◦ C for 4 h. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) analyses confirmed the esterification of the potato starch. Furthermore, the results of X-ray diffraction (XRD) and a scanning electron microscopy (SEM) revealed that the crystallinity and the morphology of the native potato starch was slightly changed during its partial gelatinisation in the ionic liquid, and was completely destroyed as a result of the formation of the esters. The thermal stability of the starch oleate decreased, when compared to the unmodified starch, as was indicated by a thermal gravimetric analysis (TGA). © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Natural polymers which are non-toxic, biodegradable and inexpensive, such as starch, are of great interest regarding to their possible use as materials in various industries, such as the food, paper, pharmaceutical and packaging industries (Fang & Fowler, 2003). Starch granules consist of two types of ␣-glucan: amylose and amylopectin (representing approximately 98–99% of the dry weight). These form linear polymer chains and hydrogen-bonded supramolecular structures (Jie, Wen-ren, Manurung, Ganzeveld, & Heeres, 2004). Native starch is characterised by high fragility and an incompatibility with hydrophobic polymers. It also has a low moisture resistance, meaning it has low processing qualities (due to high viscosity) and is highly hydrophilic. These properties can significantly limit the use of a starch to obtain a new type of material. Therefore, different types of modifications have been implemented to improve the mechanical properties of starch and its hydrophobisation (Fang, Fowler, Tomkinson, & Hill, 2002; Tomasik & Schilling, 2004).
∗ Corresponding author. E-mail address: [email protected] (J. Kapusniak). http://dx.doi.org/10.1016/j.carbpol.2015.11.029 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
Esterification is one of the oldest methods used for the modification of carbohydrate polymers (Mullen & Pacsu, 1942). The most important starch derivative – fatty acid starch esters – can be obtained through an acylation reaction of the free hydroxyl groups in the anhydroglucose unit (AGU). Usually, in order to enable an esterification reaction with the acids, a starch is first dissolved in dimethyl sulfoxide (DMSO) (Junistia et al., 2009), pyridine (Aburto et al., 1999) or another commonly used solvent, such as dimethylformamide (DMF) or tert-butanol (Boruczkowska et al., 2013; Lukasiewicz & Kowalski, 2012). However, the use of solvents also has some limitations and disadvantages, such as volatility, flammability and high levels of toxicity. They not only represent a significant hazard during the separation process, but can also be a source of environmental pollution. Therefore, it has become important to look for a “green”, environmentally friendly solvent, which can be used for the esterification of polymers. A few years ago, researchers discovered the some ionic liquids (ILs) have the ability to dissolve carbohydrate polymers (Wilpiszewska & Spychaj, 2011). One of the first of these ionic liquids was 1-butyl-3-methylimidazolium chloride, which was used during the chemically catalysed synthesis of a starch acetate using acetic anhydride (Biswas, Shogren, Stevenson, Willett, & Bhowmik, 2006). In 2007, a study was conducted on the influence of the same ionic liquid on the dissolution of starch from different botanical
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origins (corn, rice, wheat and potato) (Stevenson, Biswas, Jane, & Inglett, 2007). From this, it is known that the solubility of starch in an ionic liquid not only depends on its structure, but also on the temperature of the reaction. In order to reduce the time for starch depolymerisation, while maintaining a highly efficient reaction, the depolymerisation can be carried out in an oil bath, or even by heating using a microwave reactor in the presence of a chemical catalyst (Rajan, Prasad, & Abraham, 2006). Different esterifying factors have been used for the non-catalysed synthesis of starch esters in an ionic liquid, including: acetic and succinic anhydride (Luo & Zhou, 2012; Xie, Shao, & Liu, 2010); fatty acids such as stearic acid, lauric acid, and palmitic acid; or esters of these acids such as vinyl stearate (Biswas, Shogren, & Willett, 2009; Gao, Luo, & Luo, 2012). The chemically catalysed (using pyridine) esterification of starch in an ionic liquid has also been carried out, by reactions with acetic anhydride (Biswas et al., 2006); hexanoic, phthalic, and propionic anhydride (Lehmann & Volkert, 2009); and with fatty acid esters such as methyl laurate or stearate (Xie & Wang, 2011). It is commonly known that the esterification of starch with fatty acids can be performed in the presence of biocatalysts (like lipases). The main sources of these enzymes are fungi, including: Candida antarctica (Xu et al., 2012); Thermomyces lanuginosus (Alissandratos et al., 2011); Burkholderia cepacia (Rajan & Abraham, 2006); and Candida rugosa (Rajan, Sudha, & Abraham, 2008). Sometimes, enzymes from bacteria are also used, such as: Staphylococcus aureus (Horchani, Chaabouni, Gargouri, & Sayari, 2010), and Pseudomonas sp. (Qiao, Gu, & Cheng, 2006). Studies have confirmed that some ionic liquids improved the stability of the enzymes, and in addition could be their activators (Van Rantwijk, Madeira Lau, & Sheldon, 2003). In many cases, the enzymatic esterification in an ionic liquid has been the selective and efficient method (Ganske & Bornscheuer, 2005; Sheldon, Madeira Lau, Sorgedrager, van Rantwijk, & Seddon, 2002), such as a successful attempt at the lipase-catalysed esterification of high-amylose corn starch in a mixture of imidazolium-based ionic liquids (Lu, Luo, Yu, & Fu, 2012). Fatty acid starch esters have also been prepared via a twostep method using two different types of imidazolium-based ionic liquids, where the pregelatinisation of the starch was performed using chloride, but the lipase catalysed synthesis was carried out in tetrafluoroborate (Lu, Luo, Fu, & Xiao, 2013). The aim of present study is to optimise the conditions for the preparation of new functional materials based on starch, by a biocatalysed esterification with unsaturated fatty acids in an ionic liquid. Thus, potato starch was esterified with oleic acid, in the presence of an immobilised fungal lipase from T. lanuginosus as the catalyst, and with 1-butyl-3-methylimidazolium chloride as the reaction medium. The esterification of the starch with an unsaturated fatty acid may provide opportunities to further modify the obtained potato starch fatty acid esters, by an addition reaction to unsaturated double bond. Important and novel aspects of the study are the application of lipase immobilised on a polymer carrier, which can be used several times; and a simplification of the process by using the same ionic liquid for both the pregelatinisation of the starch and the preparation of the starch oleates. It should be emphasised that the proposed model esterification reaction may be applied to the esterification of potato starch with oils rich in oleic acid, such as high oleic sunflower oil and high oleic canola oil.
2. Materials and methods 2.1. Materials The materials used in the study include: potato starch purchased from Roth (Karlsruhe, Germany); pure oleic acid purchased from Chempur (Piekary Slaskie, Poland); 99.8% pure anhydrous
ethanol purchased from POCH (Gliwice, Poland); 95% ionic liquid 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) purchased from Sigma–Aldrich (Germany); and lipase from T. lanuginosus (Immozyme TLL-T1-350) adsorbed on a polymer carrier (with an activity of 10,000 TBU/g), purchased from Chiral Vision (The Netherlands).
2.2. Determination of lipase activity The lipase activity was measured by the liberation of butyric acid during the hydrolysis of tributyrin (glycerol tributyrate) according to the assay method described by Chiral Vision for their Immozyme TLL-T1-350 lipase. Hydrolysis of tributyrin was monitored titrimetrically in a pH-stat titration system (Metrohm titrator). The butyric acid, which is formed, was titrated with 0.1 mol/l sodium hydroxide and the consumption of the latter recorded as a function of time. Used method is based on the speed at which the enzyme hydrolyses tributyrin at pH 7.5 and 40 ◦ C. The activity of enzyme is expressed as tributyrin units per gram enzyme (TBU/g). 1 TBU (lipase unit) is the amount of enzyme which releases 1 mol titratable butyric acid per minute under the given standard conditions.
2.3. Partial gelatinisation of the starch and preparation of the starch oleates Initially, the potato starch was dried at 105 ◦ C, to a water content below 5%. The dried starch was then added to the ionic liquid ([BMIm]Cl) at a concentration of 10% (w/w) in a two-necked round flask flushed with an inert gas (Ar). The suspension was heated in an oil bath at approx. 90 ◦ C for 1 h. After the ionic liquid melted (and the suspension became viscous), the whole starch paste was stirred vigorously with a magnetic stirrer at 500 rpm. Then, the pregelatinised starch was cooled to the desired temperature for synthesis, and the oleic acid and the biocatalyst were added to initiate the esterification (Lu et al., 2013). A constant molar ratio of the starch (anhydroglucose unit, AGU) to the oleic acid (1:3) was maintained, as well as a constant weight ratio of the immobilised lipase (1.176 g per 1 g of the native starch). The synthesis of esters was carried out in an oil bath at different temperatures (60, 70 or 80 ◦ C) for 4, 6 or 8 h, and then the mixture was again stirred using a magnetic stirrer at 500 rpm. After the completion of the reaction, the mixture was cooled to room temperature. In a further step, the products were precipitated using anhydrous ethanol. Then, the precipitates were centrifuged and washed with anhydrous alcohol to separate out the main product of the esterification – the starch oleates. The solid residues were rewashed (three times) to eliminate the byproducts, the ionic liquid and the untreated reagents. Finally, the residues from the filtration were dried at 50 ◦ C for 36 h. The dried products were ground and then subjected to a physicochemical analysis.
2.4. Determination of the degree of substitution (DS) The DS of the fatty acid starch esters was determined according to the titration method, with a slight modification (Varavinit, Chaokasem, & Shobsngob, 2001). A half gram of powdered starch ester was weighed accurately, and was then dispersed in 25 ml of deionised water containing 5 ml of a 0.5 mol/l NaOH solution. The mixture was stirred vigorously at room temperature for 1 h. After this time, a few drops of phenolphthalein were added as an indicator, and the mixture was titrated using a 0.5 mol/l HCl solution. A blank sample was simultaneously titrated using native potato starch instead of the starch ester. Each sample was measured in
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triplicate, and the DS value of the ester was calculated using the following equation: DS =
162 × (V0 − V ) × CHCl W − [281 × (V0 − V ) × CHCl ]
where 162 – is the molecular weight of the anhydroglucose unit (AGU); 281 – is the molecular weight of the oleic acid residue; V0 – is the volume of HCl consumed for titrating the blank; V – is the volume of HCl consumed for titrating the sample; C(HCl) – is the exact molarity of the HCl solution used; and W – is the exact weight of the dry sample. The highest degree of substitution for the starch ester, which was first determined using the volumetric analysis presented above, was also verified by an elemental analysis. In the second method, the DS was calculated by comparing the theoretical values (the percentages of the elements: C, H, O) with the experimental values, ranging from 0 to 3, for the actual DS. 2.5. Elemental analysis The elemental analyses (to determine the percentages of carbon and hydrogen) were carried out using a micro-analyser, with a simultaneous CHNS determination using a VARIO Micro Cube (Elementar Analysensysteme GmbH, Hanau/Frankfurt, Germany). The measurements were carried out at the Centre of Molecular and Macromolecular Studies (Polish Academy of Sciences, Lodz, Poland).
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Table 1 Degree of substitution (DS) of starch oleates at different temperatures and times. Temperature [◦ C]
60 70 80
Time [h] 4
6
8
0.22 0.21 0.14
0.19 0.18 0.11
0.15 0.14 0.09
2.9. Scanning electron microscopy (SEM) Morphological changes of the native starch, the pregelatinised starch and the fatty acid starch ester were observed using a Tescan VEGA 3SBU scanning electron microscope (Tescan, Brno, Czech Republic). The accelerating voltage was 3 kV, and the samples were not coated with any conductive material.
2.10. Thermal stability analyses The thermal analyses were performed using an STA 409C simultaneous thermal analyser (Netzsch, Selb, Germany). The thermogravimetric (TG) and the differential thermogravimetric (DTG) methods were used, and the measurements were conducted under an argon flow. The samples were heated to temperatures from 25 to 500 ◦ C, with a temperature rate increase of 10 ◦ C/min (Kapusniak & Siemion, 2007).
2.6. Fourier transform infrared spectroscopy (FTIR) The FTIR spectra of the native potato starch, the partly gelatinised starch and the fatty acid starch ester (with the highest value of DS) were recorded using a Nexus Nicolet spectrophotometer (Madison, WI, USA). The powdered samples were mixed separately with an analytical grade KBr and then pressed into discs. Their spectra were recorded in the region of 4000–400 cm−1 , with 32 scans in total. Before the recording, the baseline was adjusted against a KBr background. 2.7. Proton nuclear magnetic resonance spectroscopy (1 H NMR) The 1 H NMR spectra of the samples were recorded using a Bruker Avance III spectrometer (Billerica, MA, US) at 500 MHz. The samples were dissolved in DMSO-d6 (99.8% D, Armar Chemicals, Döttingen, Switzerland) and the solution was then heated at 343 K in a water bath for 12 h. Next, 0.70 ml of the solution was placed in a 5 mm NMR tube. All of the NMR measurements were carried out at 295 K with a 5 mm BBI probehead and a z-gradient coil. Prior to Fourier transformation of the FIDs, the Lorenzian apodisation function (0.3 Hz) was applied and the spectra were zero-filled twice. These measurements were also carried out at the Centre of Molecular and Macromolecular Studies (Polish Academy of Sciences, Lodz, Poland). 2.8. X-ray diffraction analyses (XRD) X-ray powder diffraction patterns were obtained with a Bruker D8 Advance powder diffractometer (Billerica, MA, US), in a theta2-theta configuration, using a Johansson monochromator and a ˚ Data was collected in LynxEye strip detector (Cu K␣1 = 1.5406 A). the 2 theta range of 4–45◦ , with a 1 s step of 0.02◦ . For one sample (Sample A) additional data was collected in the 2 theta range of 4–8◦ , with a 3 s step of 0.01◦ . The measurements were performed at the Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University (AMU) in Poznan (Poland).
3. Results and discussion 3.1. Effect of temperature and the reaction time on the degree of substitution The degree of substitution of a starch ester can be defined as the number of hydroxyl groups that are substituted by acyl groups in each anhydroglucose unit. The theoretical maximum value of the DS is 3, because each glucose unit contains three reactive hydroxyl groups. Table 1 presents the results of the calculated DS for the prepared esters (determined by the volumetric method). The potato starch that produced the esterification with the highest degree of substitution was found in those reactions that were carried out at 60 ◦ C for 4 h. This value amounted to 0.22 and was additionally confirmed by the results obtained from the elemental analysis (DS 0.20). Table 1 also illustrates the relationship between the DS and the conditions at the time of the reaction. With an increased temperature and a prolonged reaction time, the value for the degree of substitution decreased. The esterification of the starch at 80 ◦ C for 8 h was the least effective of the samples tested. This phenomenon may be due to direct or indirect influences of the reaction environment on the enzyme. For example, a longer reaction time tends to generate a greater amount of water, as a byproduct of the esterification. An increase in the water content can then cause changes in the pH of the reaction system, and stimulate hydrolytic reactions. In addition, an increase in the water activity also contributes to a decrease in the catalytic activity of lipase (Eckstein, Wasserscheid, & Kragl, 2002). The inhibition of the catalytic activity can also be caused by small changes in the temperature of the synthesis, especially when the temperature is far from its optimal value. However, based on the results of the DS, it can be seen that it is possible to increase the thermal stability of the enzyme with the use of an ionic liquid as the reaction medium. Because the melting point of the ionic liquid used as solvent in the reaction is about 70 ◦ C, esterification at a temperature lower than 60 ◦ C is not performed.
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Fig. 3. XRD patterns of: (a) native potato starch; (b) partly gelatinised starch; and (c) starch oleate (with the DS 0.22).
Fig. 1. FTIR spectra of: (a) native potato starch; (b) partly gelatinised starch in [BMIm]Cl; and (c) starch oleate with the DS 0.22.
Fig. 2.
1
H NMR spectra of: (a) native potato starch; and (b) starch oleate (with the DS 0.22).
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Fig. 4. SEM images of: (a) native potato starch; (b) partly gelatinised starch; and (c) starch oleate (with the DS 0.22).
1H
3.2. FTIR measurements
3.3.
The FTIR spectra of the native potato starch, the partly gelatinised starch and the esterified starch are presented in Fig. 1. In the spectrum for native potato starch, a strong broad band appeared with three peaks between 980 and 1200 cm−1 (the finger print region). These can be attributed to C O stretching vibrations, which are the most characteristic ones for polysaccharides. A second characteristic band between 3000 and 3500 cm−1 can be assigned to stretching vibrations from hydrogen-bonded hydroxyl groups. In comparing the spectrum of the native starch and the partly gelatinised starch, no significant changes in these regions were observed, which is in agreement with previously reported results (Lu et al., 2013). However, in the starch oleate (with the DS 0.22) the broad peak at about 3400 cm−1 was reduced in intensity. Such a phenomenon may be due to the reduction in the number of hydroxyl groups, which have been converted into ester groups. In addition, in the FTIR spectrum of the starch oleate a new band appeared at 1730 cm−1 . This band was not present in the spectra of either the native starch or the partly gelatinised starch, and its occurrence is attributed to the carbonyl group in the ester. Therefore, this could confirm the esterification of the starch with the oleic acid (Fang et al., 2002; Mathew & Abraham, 2007). For comparison, the carbonyl signal in the FTIR spectrum for oleic acid appears at about 1711 cm−1 (Spectral Database for Organic Compounds, 2015).
The 1 H NMR spectra for native potato starch and for the starch oleate with the DS 0.22 are presented in Fig. 2. Chemical shifts are reported in parts per million, and spectral signals between 3.50 and 5.50 ppm correspond to the protons of the anhydroglucose units (of the 1,6-anhydro--D-glucose). Chemical signals of the protons at 3.30 ppm for H-2, 3.65 ppm for H-3, and 3.55 for H-5 appeared in the spectra of the native potato starch, and the shifts of H-1 and OH-2, 3, 6 were assigned to peaks between 4.58 and 5.50 ppm (Namazi, Fathi, & Dadkhah, 2011). The changes in the appearance of the NMR spectra of the ester when compared with the spectra of the native starch may indicate the occurrence of a modification in the structure of the polymer. Namely, during the esterification, the hydrogen atom of the hydroxyl groups in the unmodified starch has been substituted by an acyl group (from the oleic acid), leading to a change in the proton resonances of the AGU units. Such a substitution is visible in the spectrum of the starch oleate, as weaker signals are assigned to the hydroxyl groups of the anhydroglucose. Furthermore, the presence of characteristic peaks for the ester of an unsaturated fatty acid was confirmed. The NMR spectra for the starch oleate showed signals from three protons (a triplet peak) at around 0.8–0.9 ppm, which can be linked to the protons in the terminal methyl group of the acyl chain. The proton signal at 2.2 ppm may be related to the methylene group, which occurs before the carbonyl group, and another proton peak at around 1.5 ppm is associated with the methylene
NMR measurements
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group directly before it. In addition, a signal at 5.3 ppm refers to the protons associated with carbon atoms which are connected by a double bond. In the spectrum of the starch oleate, signals of the methylene groups can also be observed close to this double bond (at around 2.0 ppm) and all the other groups included in the acyl chain (at around 1.25 ppm). This result is in agreement with previously reported results (Kapusniak & Siemion, 2007; Lu et al., 2012) 3.4. XRD analysis X-ray diffraction was performed to investigate the difference in the crystallinity between the unmodified and the modified starch (Fig. 3). The XRD pattern for the native potato starch displayed strong reflections at about 5.6◦ and 17◦ (2), with the addition of a few smaller reflections at about 15◦ , 20◦ , 22◦ , 24◦ (2), which indicated a typical B-type crystalline pattern (Mathew & Abraham, 2007). The XRD pattern changed slightly after the initial pregelatinisation of the starch in the ionic liquid (i.e. the main signals became less intense or partly disappeared). The crystal region of the starch was also slightly destroyed, which might confirm that the ionic liquid partially disrupted the intra- and intermolecular hydrogen bonds in the starch molecules, which are responsible for a highly ordered crystalline structure (Xu, Miladinov, & Hanna, 2004). As is shown in Fig. 3, the diffractogram of the starch oleate (DS 0.22) did not exhibit any diffraction peaks, which could confirm that the esterification did not lead to the formation of new crystalline regions in the ester (Gao et al., 2012). The overall appearance of the diffraction pattern might indicate only the presence of amorphous regions, suggesting that after the modification with oleic acid, the bonds were completely disrupted. This is because during the esterification, the acidic residues of the oleic acid replaced some of the hydroxyl groups in the potato starch, resulting in a loss of crystallinity from the original structure caused by a reduction of the intermolecular hydrogen bonds (Desalegn et al., 2014).
Fig. 5. TG thermograms of: (a) native potato starch; (b) partly gelatinised starch; and (c) starch oleate (with the DS 0.22).
Fig. 6. DTG curves of: (a) native potato starch; (b) partly gelatinised starch; and (c) starch oleate (with the DS 0.22).
3.5. SEM analysis Fig. 4 illustrates the morphologies of the native starch, the partly gelatinised starch and the starch oleate (DS 0.22), investigated using a scanning electron microscopy technique. According to the SEM images, most of the potato starch granules ranged in size from 20 to 60 m and were oval to ellipsoid in shape. After the initial pregelatinisation process in 1-butyl-3methylimidazolium chloride, the starch partially lost its granular structure, and some of the granules became more irregular, with an increased surface area. However, in comparison with the native and pregelatinised starches, the starch oleate exhibited a very different morphology, where the starch granules were completely destroyed, and lost their individuality and smoothness. The SEM images demonstrate that the products were agglutinated. The morphological changes in the ester can be attributed to the effect of the gelatinisation process, when the ionic liquid as a reaction medium disrupts the hydrogen bonds and destroys the crystalline structure of the starch granules (Lu et al., 2013). In addition, the changes can be attributed to the effect of the substitution of the hydroxyl groups during the esterification (Xie & Wang, 2011). 3.6. Thermal stability analysis The thermal properties of native potato starch, the partly gelatinised starch and the starch oleate were studied by a TGA. The resulting TG and DTG curves are presented in Figs. 5 and 6, respectively. These were used to determine the weight loss of the material after heating, and the changes in thermal stability caused by the esterification. The thermograms and curves of the potato starch,
Table 2 Thermal characteristics of the native potato starch, partly gelatinised starch, and starch oleate (DS 0.22). Sample
TG ◦
DTG
ti [ C]
Weight loss [%]
to [◦ C]
Native potato starch
38.8 251.8
7.82 53.75
98.8 300.1
Partly gelatinised starch
40.3 229.3
4.94 49.89
94.8 286.3
Starch oleate (DS 0.22)
41.5 198.4
2.84 41.27
58.0 250.7
partly gelatinised starch and starch oleate (DS 0.22) show the main stages of this weight loss. The first step is related to the evaporation of water below 100 ◦ C; whereas the other step corresponds to the decomposition processes, which are mainly relating to the degradation of the starch chain. The maximum temperature for decomposition was found at about 300 ◦ C for the native starch, 286 ◦ C for the partly gelatinised starch and 250 ◦ C for the starch oleate. The temperatures for the initial degradation of were reached at about 251.8 ◦ C, 229.3 ◦ C and 198.4 ◦ C, respectively (Table 2). The extra weight loss of the starch oleate at higher temperatures was probably due to the decomposition of the ester moiety that was formed during the synthesis of the oleic acid starch ester (Desalegn et al., 2014). Comparing the results of the unmodified starch and the starch oleate, it can be concluded that the biocatalysed esterification in the ionic liquid
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reduced the initial temperature for decomposition, as well as the thermal stability of the final product (Lu et al., 2012). 4. Conclusion An ester of potato starch and oleic acid was successfully synthesised by a lipase catalysed esterification in only one ionic liquid (1-butyl-3-methylimidazolium chloride), as a reaction medium for both the gelatinisation and the esterification of the starch. The proposed method suggests the possibility of producing esters with varying degrees of substitution, in the range of 0.1–0.2. The highest DS was found in the product from the reaction carried out at 60 ◦ C for 4 h. Based on these results, we can conclude that the chloride did not provide a complete dissolution of the potato starch, but only resulted in its partial gelatinisation and the relaxation of the macrostructure. However, this was enough for the synthesis of new potato starch esters. Multiple uses of a lipase from T. lanuginosus were achieved in this newly developed method, due to the fact that it was immobilised. The application of an unsaturated fatty acid, such as oleic acid, as an esterifying agent may provide opportunities for the further functionality of the resulting ester. Moreover, there is a chance that this method could be successfully applied for the transesterification of starch with high oleic acid sunflower and canola oils. Materials based on high fatty acid starch esters, with improved processing properties, could be used to produce new biodegradable packaging or as carriers for bioactive agents. References Aburto, J., Alric, I., Thiebaud, S., Borredon, E., Bikiaris, D., Prinos, J., et al. (1999). Synthesis, characterization, and biodegradability of fatty acid esters of amylose and starch. Journal of Applied Polymer Science, 74, 1440–1451. Alissandratos, A., Baudendistel, N., Hauer, B., Baldenius, K., Flitsch, S., & Halling, P. (2011). Biocompatible functionalisation of starch. Chemical Communication, 47, 683–685. Biswas, A., Shogren, R. L., Stevenson, D. G., Willett, J. L., & Bhowmik, P. K. (2006). Ionic liquids as solvents for biopolymers: Acylation of starch and zein protein. Carbohydrate Polymers, 66, 546–550. Biswas, A., Shogren, R. L., & Willett, J. L. (2009). Ionic liquid as a solvent and catalyst for acylation of maltodextrin. Industrial Crops and Products, 30, 172–175. ˙ z, ˙ W., Boruczkowska, H., Boruczkowski, T., Tomaszewska-Ciosk, E., Drózd ˙ A., et al. (2013). Enzymatic esterification of starch Bienkiewicz, M., Zołnierczyk, phosphate with oleic acid. Chemical Review, 92, 1078–1082. Desalegn, T., Garcia, I. J. V., Titman, J., Licence, P., Diaz, I., & Chebude, Y. (2014). Enzymatic synthesis of epoxy fatty acid starch ester in ionic liquid–organic solvent mixture from vernonia oil. Starch, 66, 385–392. Eckstein, M., Wasserscheid, P., & Kragl, U. (2002). Enhanced enantioselectivity of lipase from Pseudomonas sp. at high temperatures and fixed water activity in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide. Biotechnology Letters, 24, 763–767. Fang, J., & Fowler, P. (2003). The use of starch and its derivatives as biopolymer sources of packaging materials. Food, Agriculture & Environment, 1(3&4), 82–84. Fang, J. M., Fowler, P. A., Tomkinson, J., & Hill, C. A. S. (2002). The preparation and characterization of a series of chemically modified potato starches. Carbohydrate Polymers, 47, 245–252. Ganske, F., & Bornscheuer, U. T. (2005). Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH. Journal of Molecular Catalysis B: Enzymatic, 36, 40–42. Gao, J., Luo, Z., & Luo, F. (2012). Ionic liquids as solvents for dissolution of corn starch and homogeneous synthesis of fatty-acid starch esters without catalysts. Carbohydrate Polymers, 89, 1215–1221.
663
Horchani, H., Chaabouni, M., Gargouri, Y., & Sayari, A. (2010). Solvent-free lipase-catalyzed synthesis of long-chain starch esters using microwave heating: Optimization by response surface methodology. Carbohydrate Polymers, 79, 466–474. Jie, Y., Wen-ren, C., Manurung, R. M., Ganzeveld, K. J., & Heeres, H. J. (2004). Exploratory studies on the carboxymethylation of cassava starch in water-miscible organic media. Starch, 56, 100–107. Junistia, L., Sugih, A. K., Manurung, R., Picchioni, F., Janssen, L. P. B. M., & Heeres, H. J. (2009). Experimental and modeling studies on the synthesis and properties of higher fatty esters of corn starch. Starch, 61, 69–80. Kapusniak, J., & Siemion, P. (2007). Thermal reactions of starch with long-chain unsaturated fatty acids. Part 2. Linoleic acid. Journal of Food Engineering, 78, 323–332. Lehmann, A., & Volkert, B. (2009). Investigations on esterification reactions of starches in 1-N-butyl-3-methylimidazolium chloride and resulting substituent distribution. Journal of Applied Polymer Science, 114, 369–376. Lu, X., Luo, Z., Fu, X., & Xiao, Z. (2013). Two-step method of enzymatic synthesis of starch laurate in ionic liquids. Journal of Agricultural and Food Chemistry, 61, 9882–9891. Lu, X., Luo, Z., Yu, S., & Fu, X. (2012). Lipase-catalyzed synthesis of starch palmitate in mixed ionic liquids. Journal of Agricultural and Food Chemistry, 60, 9273–9279. Lukasiewicz, M., & Kowalski, S. (2012). Low power microwave-assisted enzymatic esterification of starch. Starch, 64, 188–197. Luo, Z., & Zhou, Z. (2012). Homogeneous synthesis and characterization of starch acetates in ionic liquid without catalysts. Starch, 64, 37–44. Mathew, S., & Abraham, T. E. (2007). Physico-chemical characterization of starch ferulates of different degrees of substitution. Food Chemistry, 105, 579–589. Mullen, J. W., & Pacsu, E. (1942). Starch studies. Preparation and properties of starch triester. Industrial & Engineering Chemistry, 34, 1209–1217. Namazi, H., Fathi, F., & Dadkhah, A. (2011). Hydrophobically modified starch using long-chain fatty acids for preparation of nanosized starch particles. Scientia Iranica, 18, 439–445. Qiao, L., Gu, Q. M., & Cheng, H. N. (2006). Enzyme-catalyzed synthesis of hydrophobically modified starch. Carbohydrate Polymers, 66, 135–140. Rajan, A., & Abraham, T. E. (2006). Enzymatic modification of cassava starch by bacterial lipase. Bioprocess and Biosystems Engineering, 29, 65–71. Rajan, A., Prasad, V. S., & Abraham, T. E. (2006). Enzymatic esterification of starch using recovered coconut oil. International Journal of Biological Macromolecules, 39, 265–272. Rajan, A., Sudha, J. D., & Abraham, T. E. (2008). Enzymatic modification of cassava starch by fungal lipase. Industrial Crops and Products, 27, 50–59. Sheldon, R. A., Madeira Lau, R., Sorgedrager, M. J., van Rantwijk, F., & Seddon, K. R. (2002). Biocatalysis in ionic liquids. Green Chemistry, 4, 147–151. Spectral Database for Organic Compounds, SDBS. (2015). National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan. SDBS No.: 1035. http://sdbs.db.aist.go.jp/sdbs/cgi-bin/direct frame disp.cgi?sdbsno=1035. Stevenson, D. G., Biswas, A., Jane, J., & Inglett, G. E. (2007). Changes in structure and properties of starch of four botanical sources dispersed in the ionic liquid, 1-butyl-3-methylimidazolium chloride. Carbohydrate Polymers, 67, 21–31. Tomasik, P., & Schilling, C. H. (2004). Chemical modification of starch. Advances in Carbohydrate Chemistry and Biochemistry, 59, 175–403. Van Rantwijk, F., Madeira Lau, R., & Sheldon, R. A. (2003). Biocatalytic transformations in ionic liquids. Trends in Biotechnology, 21, 131–138. Varavinit, S., Chaokasem, N., & Shobsngob, S. (2001). Studies of flavor encapsulation by agents produced from modified sago and tapioca starches. Starch, 53, 281–287. Wilpiszewska, K., & Spychaj, T. (2011). Ionic liquids: Media for starch dissolution, plasticization and modification. Carbohydrate Polymers, 86, 424–428. Xie, W., Shao, L., & Liu, Y. (2010). Synthesis of starch esters in ionic liquids. Journal of Applied Polymer Science, 116, 218–224. Xie, W., & Wang, Y. (2011). Synthesis of high fatty acid starch esters with 1-butyl-3-methylimidazolium chloride as a reaction medium. Starch, 63, 190–197. Xu, Y., Miladinov, Y., & Hanna, M. A. (2004). Synthesis and characterization of starch acetates with high substitution. Cereal Chemistry, 81, 735–740. Xu, J., Zhou, C. W., Wang, R. Z., Yang, L., Du, S. S., Wang, F. P., et al. (2012). Lipase coupling esterification of starch with octenyl succinic anhydride. Carbohydrate Polymers, 87, 2137–2144.