j ourna l ho me page: www.elsev ier .com/ locate /carbpol
green approach for starch modification: Esterification by lipase andovel imidazolium surfactant
unita Adak, Rintu Banerjee ∗
epartment of Agricultural & Food Engineering, Indian Institute of Technology, Kharagpur 721302, India
r t i c l e i n f o
rticle history:eceived 16 December 2015eceived in revised form 9 May 2016ccepted 12 May 2016vailable online 16 May 2016
a b s t r a c t
Starch being one of the most abundant polysaccharides in nature has been subjected to modification toenhance its applicability. Modification by esterification involves acylation of hydroxyl groups of glucoseunits to form starch esters. Lipases, as catalysts have emerged as a promising alternative to chemicalprocesses. Although ionic liquids and microwave assisted heating are emerging as green technology yettheir use along with lipases for starch modification has not been probed. In the present study esterification
of corn starch employing Rhizopus oryzae lipase, microwave irradiation and novel imidazolium surfactantshas been attempted. At 80% irradiation, 1:3 starch/oleic acid molar ratio, 150 IU enzyme, and 50 �mol of[C16-3-C16im]Br2 maximum degree of substitution (DS = 2.75) was attained. The modified starch showedbetter hydrophobicity and thermoplasticity with corresponding structural changes depicted by FTIR, XRDand SEM. These properties advocate the usefulness of the modified starch in food and biopolymer sectors.
Starch being one of the most abundant polysaccharides in natureas been used for multifarious applications. It comprises of twoolymeric units made up of glucose; amylose and amylopectin,hich are present in specific proportions depending on the source.ased on composition the physicochemical properties of starchary and accordingly it is applied in food and non-food sectorsKaur, Ariffin, Bhat, & Karim, 2012; Kavlani, Sharma, & Singh, 2012).et native starch has many shortcomings for industrial applica-ions (Abbas, Khalil, & Hussin, 2010; Xie, Liu, & Cui, 2005) whichnspired researchers to modify starch to overcome these hurdles.
odification of starch by esterification has been predominant andnvolves acylation of hydroxyl groups of anhydrous glucose unitsn starch to form starch esters. Different types of starch esters haveeen developed with varying degree of substitution (DS) (molecyl/mole anhydroglucose), which are of commercial importanceAlissandratos & Halling, 2012). Among these, long chain fatty acid
sters have been extensively studied and show promising proper-ies of hydrophobicity, thermoplasticity and biodegradability. Theyave potential applications as dip coatings in food and non-food
∗ Corresponding author at: Microbial Biotechnology and Downstream Processingaboratory, Department of Agricultural and Food Engineering, Indian Institute ofechnology, Kharagpur 721302, India.
industries, binders, coating material, adhesives, and for preparationof drug delivery system and biomaterials (Alissandratos & Halling,2012; Grote & Heinze, 2005; Lu, Luo, Yu, & Fu, 2012).
Esterification by chemical means employs harsh chemicalslike solvents, alkali, pyridine, catalysts at high temperatures andalso require sophisticated instrumentation (Rajan & Abraham,2006; Xu et al., 2012). These conditions restrict their use inindustries like food and pharmaceutical as they involve environ-mental, economical and safety risks. Alternative environmentallyfriendly approaches have been looked for process developmentviz. enzymes, ionic liquids and microwave irradiation. Biocatalysts,mainly extracellular lipases have been successfully applied in laband large scale for esterification of starch with long chain fatty acids,oils or other acyl donors under different system conditions (Kumar,Yadav, Jahan, & Saxena, 2014; Namazi, Fathi, & Dadkhah, 2011; Xuet al., 2012). Similarly ionic liquids and microwave irradiation dueto their unique properties have been used for starch esterificationas a green alternative to organic solvents and conventional heatingtechniques respectively. Ionic liquids made of methylimidazoliumcations are non-flammable, have low melting point and low orinsignificant vapour pressure. These features make them betterthan organic solvents which bear many disadvantages like flamma-bility, toxicity and volatility (Horchani, Chaabouni, Gargouri, &Sayari, 2010; Lu, Luo, Fu, & Xiao, 2013; Rajan, Sudha, & Abraham,
2008). Presence of imidazolium head group imparts amphiphiliccharacter to these ionic liquids as a result they show aggregationproperties like surfactants at different concentrations. Hence long
hain imidazolium based ionic liquids both monomer ([C10-16-im]+)nd gemini ([C10-16-n-C10-16im]+) are emerging as novel surfac-ants having special qualities compared to conventional cationicurfactants like stronger self aggregation, lower critical micelle con-entration (CMC) and novel surface properties (Zhao, Gao, Wang,
Tang, 2008). Pal, Datta, Aswal, and Bhattacharya (2012) andatta, Biswas, and Bhattacharya (2014) reported ionic liquid-typeemini cationic imidazolium surfactants with shorter spacer ([C16--C16im]Br2) have lower critical micelle concentration (CMC) valuehan conventional ammonium surfactants depicting better surfacective property. Thus they find potential application in the fieldf separation science, electrochemistry, material science, petro-hemistry, organic synthesis and biocatalysis. Apart from reactionedium, improvisation in the heating systems has been also tar-
eted for various synthesis processes. Compared to conventionaleating systems, microwave heating gives fast and uniform heat-
ng with less startup time and is energy efficient. Apart from this,t is also found to accelerate a wide range of organic reactionsPalav & Seetharaman, 2007; Perreux & Loupy, 2001; Shogren &iswas, 2006). Chemical and enzymatic modification of starch haslso been successfully carried out employing microwave irradia-ion (Horchani, Chaabouni, Gargouri, & Sayari, 2010; Rajan, Prasad,
Being a progressive research area, there is plenty of scope for fur-her investigation in the field of starch esterification. Simultaneousse of lipase, ionic liquid and microwave irradiation for starch ester-
fication is yet to be probed. In the present study an attempt wasade to improve upon the hydrophobic and thermoplastic prop-
rty of maize starch by esterifying with oleic acid using Rhizopusryzae lipase. The esterification process was further modified bysing microwave irradiation and novel imidazolium based cationic
onic liquid-type surfactants (monomeric and gemini forms). Theodified starch so obtained was finally characterized using dif-
erent techniques FTIR, XRD, SEM, TGA and DSC etc. to verify theesired changes.
. Materials and methods
.1. Materials
p-Nitrophenyl acetate (pNPA) and maize starch (amylose 30%nd amylopectin 70%) were procured from Hi-Media, Mumbai,ndia (CAS No: 9005-25-8). Three novel ionic liquid-type cationicmidazolium surfactants were given by Prof. S. Bhattacharya,rganic Chemistry Department, Indian Institute of Science, Banga-
ore. Their synthesis and characterization has been already reportedy Pal, Datta, Aswal, and Bhattacharya (2012) and Datta, Biswas,nd Bhattacharya (2014). One was momomeric – (C16mim)Br andwo were gemini – [C16-3-C16im]Br2 and [C16-12-C16im]Br2 (Fig.1). All other reagents were of analytical grade.
.2. Lipase
Lipase of Rhizopus oryzae NRRL 3562 was produced and fur-her purified by following the procedure of Kumari, Mahapatra,arlapati, and Banerjee (2008) and Adak and Banerjee (2013)
espectively.
.3. Determination of microwave treatment stability of lipase
The stability of lipase on subjecting to microwave heating at00% power for different time interval (10–150 s) was checked. Theesidual activity was measured considering the untreated enzyme
olymers 150 (2016) 359–368
at t0 as control using p-nitrophenyl palmitate (pNPP) as substrate(Pencreach & Baratti, 2001).
2.4. Lipase mediated synthesis of starch esters of oleic acid
To 1 g dried maize starch 5 mL phosphate buffer (10 mM, pH 7)was added and subjected to heating for 2 min over boiling waterbath. Oleic acid was added to it at required molar ratio of oleicacid/anhydrous glucose unit (AGU) in starch (1:1, 2:1, 3:1 and4:1) and mixed thoroughly using magnetic stirrer. 1 mL lipase ofrequired activity (100 IU, 150 IU, 200 IU and 300 IU) was added fol-lowed by microwave irradiation at desired power (50%, 80% and100%) for 1 min given intermittently for 10 s using a domesticmicrowave oven (Bajaj 2004ETB, India; power input 1250 W andoutput 800 W with operation frequency of 2450 MHz). The reac-tion was further allowed to proceed for required time (2 h, 4 h, 6 hand 8 h) at 30 ◦C under shaking condition at 100 rpm. Reactionsunder only shaking condition and microwave treatment were alsocarried out to check the efficiency of the process. Further reactionswere also performed in presence of novel ionic liquid-type imi-dazolium cationic surfactants ((C16mim)Br, [C16-3-C16im]Br2 and[C16-12-C16im]Br2) at different amounts with the aim to enhancethe process efficiency. Surfactants were added to the phosphatebuffer (pH 7, 5 mL) and dissolved prior to the addition of maizestarch which was followed by other mentioned steps carried outfor esterification. For each set of experiments the control experi-ments were carried out keeping all the reaction conditions sameexcept for the addition of enzyme. The starch sample derived fromthe control sets were termed as control starch. After completion ofthe reaction, the starch esters were precipitated by adding an equalamount of ethanol. The precipitated starch esters were separatedby centrifugation and further washed with ethanol and distilledwater repeatedly (4–5 times) and filtered. The filtered modifiedstarch was dried at 40 ◦C for 2 days, powdered and used for furtheranalysis.
2.5. Determination of degree of substitution (DS)
Degree of substitution was determined following Lin, Li, Long,Su, and Huang (2014) method with modification. To 0.2 g of sample10 mL of DMSO, 4 mL of 0.2 M NaOH was added and the mixturewas stirred for 4 h at 50 ◦C. Excess NaOH was titrated with 0.1 MHCl using phenolphthalein as indicator. DS value of starch esterwas calculated as follows:
DS = MAGU × C (V0 − V)
W −[(
MFA − MH2O)
× C (V0 − V)]
where, MAGU = Molecular weight of AGU = 162,MFA = Molecular weight of oleic acid = 282.47,MH2O = Molecular weight of water = 18,V0 = Titration volume of HCl consumed for control starch (mL),V = Titration volume of HCl consumed for starch ester (mL),C = Molar concentration of HCl used for titration (M),W = Weight of the sample (g).
2.6. Characterization of modified starch
2.6.1. Water and oil binding capacityWater and oil binding capacity of starch samples were deter-
mined by following the method of Yousif, Gadallah, and Sorour(2012). The samples were ground and passed through (0.420 mm)
80 mesh size and dispersed in 10 mL of water/olive oil in pre-weighed centrifuge tubes. The resulting suspension was gentlystirred in shaking water bath for 30 min at 30 ◦C, followedby centrifugation at 5000 rpm for 15 min. The supernatant was
rate Polymers 150 (2016) 359–368 361
dws
2
satws
2
bmss
2
DtAam
2
1caAm
p1(
3
3
s8iaebhPd&A
3
s
3
ois
S. Adak, R. Banerjee / Carbohyd
ecanted and centrifuge tube was reweighed. Water/oil bindingas expressed as water/oil bound in terms of weight (g/g) of starch
ample.
.6.2. Fourier transform infrared spectrometric (FTIR) analysisTo analyze the changes in the chemical structure of the starch
amples, FTIR was carried out. The starch samples were mixed withnhydrous potassium bromide in 1:10 (w/w) ratio and compressedo form disk shaped pellet, which was analyzed. Sample spectraere obtained over the range of 400 cm−1 and 4000 cm−1 with a
pectral resolution of 1 cm−1.
.6.3. X-ray diffraction (XRD) studyChanges in the structural arrangement of the starch samples
efore and after esterification was determined by XRD1710 equip-ent using Cu K� radiation (� = 1.79 Å) set at 40 kV and 20 mA. All
amples were scanned at diffraction angle (2�) from 5 to 50◦ withcanning speed of 3◦ min−1.
.6.4. Scanning electron microscopy (SEM)The morphology of the starch samples was studied by SEM.
ried samples were mounted on a metal stub with double stickyape and coated with gold to make them conductive in nature.fter gold coating the samples were observed and photographedt 20 kV accelerating voltage. The pictures were presented in theagnifications 1000× and 2000×.
0 mg of sample was weighed in aluminium pans and hermeti-ally sealed. The samples were then heated from 30 to 250 ◦C with
heating rate of 10 ◦C.min−1 in nitrogen atmosphere (20 mL/min).n empty pan was used as reference for all measurements. Theelting temperature was determined from the thermogram.The thermogravimeteric analysis (TGA) measurements were
erformed by heating 4 mg of starch samples at the rate of0 ◦C min−1 from 30 to 600 ◦C in continuous flushing of nitrogen100 mL/min).
. Results and discussion
.1. Microwave irradiation stability of Rhizopus oryzae lipase
The enzyme irradiated at 100% power for several secondshowed gradual reduction in the activity (Fig. 1). Till 30 s exposure5% of initial activity was retained. Enzyme showed half of its orig-
nal activity after 60 s of irradiation. Further exposure lowered thectivity to 10% which remained constant even on increasing thexposure time. Similar effect was observed in different lipases ofoth bacterial and fungal origin. Denaturation of enzyme structureas been attributed to be the probable cause for the loss in activity.roduction of heat by in situ vibration of water molecules leads toenaturation of enzyme structure (Horchani, Chaabouni, Gargouri,
.2. Enzymatic esterification of starch with oleic acid
Under this section effect of different reaction conditions ontarch esterification with oleic acid have been discussed.
.2.1. Mode of heating for esterification
In the present study the effect of mode of heating was observed
n starch esterification with oleic acid (Fig. S2). Conventional heat-ng was performed by carrying out the reaction in water bathhaker set at 30 ◦C; the optimum temperature of the lipase for 6 h.
Fig 1. Microwave irradiation stability of Rhizopus oryzae lipase.
Microwave treatment was given at 100% power for 1 min (temper-ature of the medium- 70 ◦C) as lipase maintained half of its activitytill 1 min. Another set was placed combining both the processes toanalyze the synergistic effect. Only microwave treatment resultedin a DS of 0.91, whereas DS of 0.62 was obtained under shakingcondition. The result obtained in this study in terms of DS wasfound to be higher than other works (Alissandratos, Baudendistel,Ritsch, Hauer, & Halling, 2010; Alissandratos, Baudendistel, Haueret al., 2010; Gao et al., 2014; Xin et al., 2012). Under liquid stateshaking condition using Rhizopus oryzae lipase Kumar et al. (2014)reported production of starch palmitate esters with high DS (1.68)in organic solvent compared to water. The result obtained in thepresent study show similar DS value as obtained for water byKumar, Yadav, Jahan, and Saxena (2014) but in lesser reaction time.Rajan, Prasad, and Abraham (2006) and Rajan, Sudha, and Abraham(2008) reported higher modification in terms of DS of starch sam-ples in lesser time using hydrolyzed coconut oil when subjectedto microwave heating compared to shaking under liquid state withorganic solvents as medium. Compared to conventional heating theobserved high DS in microwave treatment has been proposed to bethe result of higher collision efficiency, faster diffusion rates due tomolecular vibrations along with dielectric heating effect (Perreux &Loupy, 2001; Singh & Tiwari, 2008). Moreover, being non-ionizingin nature microwave treatment gives uniform heating without acti-vation of any specific bond at 2450 MHz. As a result it does not giverise to any kinetic difference as found in case of other heating tech-niques (Lewandowicz et al., 2000; Rajan, Sudha, & Abraham, 2008).A higher DS (1.22) was obtained when microwave treatment wasfollowed by shaking in water bath. This proves that the combinedprocess gives better result compared to their individual effect. Sim-ilar effect has been observed by Horchani, Chaabouni, Gargouri,and Sayari (2010) where individual microwave and shaking yieldedas DS of 1.6 and 1.8 respectively, while when combined togetherresulted in 2.86 DS under optimized condition. To enhance the DSin the present system study on different parameters were furtherundertaken.
3.2.2. Effect of microwave irradiation power on esterificationMicrowave irradiation of different power (50, 80 and 100%) was
applied during the esterification reaction, to observe its effect on DS
of the reaction and thus optimize the reaction conditions. Fig. 2(A)shows maximum DS (1.42) was obtained at 80% power. DS at 50%(0.78) was low compared to 80 and 100%. With increasing power,value of DS rises initially till 80%, beyond which it decreases. This
362 S. Adak, R. Banerjee / Carbohydrate Polymers 150 (2016) 359–368
F ation
n
cmtsoop
3c
rda&fiosAtTL
3
oto
ig. 2. Effect of different process parameters on esterification. (A) microwave irradiovel imidazolium surfactants (F) amount of [C16-3-C16]Br2.
ould be attributed to the greater loss in enzyme activity whichight have occurred due to denaturation of enzyme on exposure
o high power irradiation compared to that of 80%. The rise in DShows correlation between power and reaction. Similar effect wasbserved by Liao, Raghavan, and Yaylayan (2002), where the yieldf butylparaben ester was found to raise with increasing irradiationower.
.2.3. Effect of reaction time on esterification during shakingondition
As coupled method of microwave treatment and shaking at 30 ◦Cesulted in better DS an insight into the progression of reactionuring shaking condition is of importance to optimize the processnd in turn enhance the cost-effectiveness of the process (Garlapati
Banerjee, 2013). The effect of time on the DS value of esteri-ed starch with oleic acid has been given in Fig. 2(B). The valuef DS increases from 0.76 to 1.48 after 2–4 h of reaction progres-ion. Thereafter (>4 h) no further increase in DS value was observed.nalogous trend has been observed in lipase mediated esterifica-
ion of starch under varying reaction systems (Chakraborty, Sahoo,eraoka, Miller, & Gross, 2005; Lin, Li, Long, Su, & Huang, 2014; Lu,uo, Yu, & Fu, 2012; Lu, Luo, Fu, & Xiao, 2013; Xu et al., 2012).
.2.4. Effect of molar ratio of acyl donor/starch on esterification
Molar ratio of oleic acid to starch (AGU) had immense influence
n the value of DS of starch. In Fig. 2(C) this effect has been illus-rated. The value of DS increased from 0.62 to 1.48 as the ratio ofleic acid/AGU rose from 1 to 2. With further increase the value
power (B) shaking time (C) molar ratio of acyl donor/starch (D) enzyme amount (E)
of DS attained maximum value at molar ratio 3 (1.63) and subse-quently remained almost constant. Lu, Luo, Yu, and Fu (2012) andLu, Luo, Fu, and Xiao (2013) also obtained maximum DS value at3:1 molar ratio of acyl donor/starch. In other starch esterificationsystems researchers had come across similar pattern of observa-tion (Lin, Li, Long, Su, & Huang, 2014; Xin et al., 2012; Xu et al.,2012), but their optimum ratio showed wide variation. The opti-mum molar ratio reported by Lin, Li, Long, Su, and Huang (2014)and Xin et al. (2012) for their esterification system was 2:1 and6:1 respectively. The probable reason behind such variation couldbe the difference in the enzymes, substrates used and the reac-tion conditions employed during the esterifcation process. Steadyincrease in DS value initially with the rise in the oleic acid/AGUmolar ratio could be the result of more availability of acyl donor(Xu et al., 2012). The maximum ratio of 3:1 was fixed for furtherreactions in the present study.
3.2.5. Effect of amount of enzyme on esterificationFor enzyme catalyzed reactions amount of enzyme plays an
important role in reaction rates which also affects the product yieldwithin a fixed reaction time (Raghavendra, Panchal, Divecha, Shah,& Madamwar, 2014). From the obtained result (Fig. 2(D)) it canbe observed that increasing the enzyme amount from 100 to 150IU raised the value of DS from 1.63 to 1.88. Beyond this amount
there was no significant increase in the DS value. This shows therate of esterification enhanced upon increasing the enzyme amountwithin the same reaction time. Such result was also obtained dur-ing other esterification processes by lipases (Garlapati & Banerjee,
rate Polymers 150 (2016) 359–368 363
2S
3
tiTatihmLtacimtabogstrsitlsBft
3
tavSvaabasIa[yae
3d
tretMKeo
Table 1Water and oil binding capacity of the starch samples.
Sample Water binding capacity (g/g) Oil binding capacity (g/g)
.2.6. Effect of novel imidazolium surfactants on esterificationBoth surfactants and ionic liquids have been applied for the syn-
hesis of starch esters to promote substrate dispersion and increasenterfacial area, which enhances the substrate enzyme interaction.o improve the contact between starch and fatty acid Rajan, Prasad,nd Abraham (2006) added Triton-X-100 in gelatinized starch sys-em, which resulted in starch ester with DS value of 0.43. Similarly,midazolium based ionic liquids have been used as medium foromogenous esterification of starch both chemically and enzy-atically (Lehmann & Volkert, 2011; Lu, Luo, Yu, & Fu, 2012; Lu,
uo, Fu, & Xiao, 2013; Xie, Shao, & Liu, 2009). To further enhancehe esterification in terms of DS value in the present study anttempt has been made by adding novel ionic liquid-type longhain cationic imidazolium surfactants (100 �mol). From Fig. 2(E)t can be concluded that among the surfactants, [C16-3-C16]Br2 have
aximum influence. The DS was increased to 2.59, which is twicehe value compared to the system without surfactant. Xie, Shao,nd Liu (2009) reported similar DS value for starch esters using 1-utyl-3-methylimidazolium chloride (BMIMCl) as medium. Theybserved better dissolution of starch granules due to strong hydro-en bonding between starch and ionic liquid. Out of the two geminiurfactants, one with short spacer length showed better effect thanhe monomer, while the other did not had much influence on theeaction. This difference could have arisen due to variation in theurfactant structure. Gemini surfactants show better surface activ-ty compared to monomer (Ao, Huang, Xu, Yang, & Wang, 2009),hus promote better interaction between the starch, oleic acid andipase causing higher esterification. Apart from this in the priortudy (Adak, Datta, Bhattacharya, & Banerjee, 2015a, Adak, Datta,hattacharya, & Banerjee, 2015b) influence of [C16-3-C16]Br2 was
ound to be greater on enhancing the activity of lipase compared tohe other two.
.2.7. Effect of amount of [C16-3-C16]Br2 on esterificationSurfactants bring about changes in lipase activity in a concentra-
ion dependent manner. Most cases show gradual enhancement inctivity with rise in concentration of surfactant attaining maximumalue followed by deactivation (Mogensen, Sehgal, & Otzen, 2005;alameh & Wiegel, 2010). As [C16-3-C16]Br2 yielded in best DSalue for esterified starch, it was used in the subsequent study. Themount of surfactant was varied to monitor its effect on the reactionnd also establish the minimum amount required for getting theest conversion. In Fig. 2(F) DS values obtained for [C16-3-C16]Br2t various quantities (10–100 �mol) have been given. The geminiurfactant was found to be effective from 25 �mol and beyond.ncrease in DS value (2.63) has been seen from 10 to 25 �mol,ttaining maximum value (2.75) at 50 �mol. Further increase inC16-3-C16]Br2 lowered the DS value. Though the variation is minoret this could be attributed to reduction in lipase activity at highmount of the gemini surfactant as observed in earlier study (Adakt al., 2015b) due to greater loss in the secondary structure.
.2.8. Effect of [C16-3-C16]Br2 on reaction time for esterificationuring shaking condition
Presence of surfactants and ionic liquids at specific concen-ration range enhances the activity, stability of lipases as well aseduces the reaction time. Many reactions including hydrolysis,sterification etc. have been found to take place in faster pace inurn causing reaction completion in lesser duration (Goto, Kamiya,
iyata, & Nakashio, 1994; Harjani, Naik, Nara, & Salunke, 2007;o, Wang, Hwang, & Hsieh, 2006). To monitor this effect, starchsterification reaction with oleic acid was carried out in presencef [C16-3-C16]Br2 (50 �mol). Reaction time of 2 h was sufficient to
achieve the required DS value of 2.75 (Fig. S3). On extending thereaction time beyond this did not show any further improvement.Similar effect has been monitored in different surfactant modifiedlipase reactions, where rate of reaction was found to be faster andbetter conversion was observed (Mogensen, Sehgal, & Otzen, 2005;Song, Ding, & Wang, 2007).
3.3. Characterization of starch ester
The starch esters obtained after drying showed variation in theirappearance and were light yellow in colour compared to maize andcontrol starch sample. This difference in colour has also been seenin case of other starch esters substituted with fatty acids (Kapusniak& Siemion, 2007). To further probe into the changes in the physical,chemical, structural, thermal properties characterization study wasperformed.
3.3.1. Water and oil binding capacityAbsorption of water and oil by starch is useful for food prepa-
ration/processing to impart desired texture, flavor and even toextend the shelf life. The water and oil binding capacity mainlydepends on the size, shape, conformation and composition of thestarch (Ikegwu, Okechokwu, & Ekumankana, 2010; Rajan, Sudha, &Abraham, 2008) and thus tends to vary. In Table 1 the water and oilbinding capacity of starch esters and their comparison with maizeand control starch has been given. Compared to maize starch (starchwithout any treatment), the control starch sample (starch subjectedto estrification devoid of only enzyme keeping all other parameterssame) shows better water binding property, though little increasein oil binding capacity have also been observed. This could be theresult of changes in the internal structure of starch granules causedby the heat treatment while carrying out the esterification process.Such observations have been reported for pregelatinized starches(Majzoobi et al., 2011; Wootton & Bamunuarachchi, 1978; Yousif,Gadallah, & Sorour, 2012). For the starch esters, with increasingDS value the water binding capacity lowered and almost becamehalf of the value obtained in case of control starch. On the con-trary the oil binding capacity of starch esters was doubled. Thuswith the increasing DS value, hydrophobicity of the starch estersincreased leading to lesser water absorption and higher oil bind-ing. For starch acetate esters Diop, Li, Xie, and Shi (2011) foundequivalent tendency towards water absorption. Compared to thepresent study the decrease in water absorption was found to belower, which could be due to the lesser hydrophobic character ofacetic acid used.
3.3.2. FTIR analysisIn the present study FTIR spectra of maize starch (Fig. 3(A))
shows the characteristic transmittance pattern. Compared to maizestarch, the spectra of control starch sample (Fig. 3(B)) shows differ-ence in transmittance pattern; inferring corresponding changes inthe structure of maize starch during the esterification. Starch estersFTIR spectra illustrated in Fig. 3(C) and (D), represent characteristic
changes along with basic structural features of starch molecules.Formation of starch oleate esters was confirmed by the appear-ance of a new peak at 1721 cm−1. This band represents stretchingvibrations of C O of ester group, which has been reported in case
364 S. Adak, R. Banerjee / Carbohydrate Polymers 150 (2016) 359–368
ontrol
oCRiAaosb(aT22sprC
3
h
Fig. 3. FTIR spectra of starch samples (A) maize starch, (B) c
f other starch esters in the range of 1754–1712 cm−1 (Horchani,haabouni, Gargouri, & Sayari, 2010; Kapusniak & Siemion, 2007;ajan, Sudha, & Abraham, 2008; Xie, Shao, & Liu, 2009). With the
ncrease in DS the intensity of this peak was found to increase.nother major change was observed in the region between 3000nd 3500 cm−1, where greater reduction in the peak intensityccurred compared to control and maize starches. This fall in inten-ity has been ascribed to the decrease in concentration of hydrogenonded O H groups due to formation of ester bonds. Aburto et al.1999) reported similar changes in the FTIR spectra of potato starchnd amylose on esterification with long chain fatty acid chlorides.hey also observed enhancement in the band intensity in the range800–2950 cm−1 with increasing DS value. Bands in this region at926 and 2855 cm−1 was also found to increase in intensity intarch oleate samples in this study in a similar manner. As thiseak arises due to aliphatic alkyl chain C H stretching, analogousesults have been reported for other starch esters as well (Horchani,haabouni, Gargouri, & Sayari, 2010; Lu, Luo, Fu, & Xiao, 2013).
.3.3. XRD analysisThe diffractogram of the starch samples of the present study
as been given in Fig. 4. Maize starch (Fig. 4(A)) gives character-
istics peaks at 2� = 15.1, 17.1 and 23◦, which have been found incase of A type starch. Smaller peaks were also observed at 20 and26◦ specific for A type starch (Gao et al., 2014). In control starchsample (Fig. 4(B)) no sharp peaks were observed, only a broad peakwas obtained which indicates loss of crystalline arrangement due tothe rupture of intra and intermolecular hydrogen bonding. Similarresult was reported by Lu, Luo, Fu, and Xiao (2013) for gelatinizedcorn starch used for synthesis of starch laurate esters. Both thestarch oleate samples of different DS values, had same diffractionpattern with prominent peaks at 2� = 13.1 and 19.7◦ (Fig. 4(C) and(D)). These peaks are characteristics of V type starch crystallinearrangement (Diop, Li, Xie, & Shi, 2011; Lu, Luo, Yu, & Fu, 2012).This type of arrangement is obtained by collapse of amylose heliceswith adjuncts like fatty acids trapped inside (Zobel, Young, & Rocca,1988). For starch laurate similar results were obtained and suchpattern was inferred to be formed by single helical structure madeup of starch molecules and esterified laurate (Lu, Luo, Fu, & Xiao,2013).
3.3.4. SEM analysisMaize starch SEM images (Fig. 5(A)) showed granular struc-
tures having irregular and mostly polygonal shape and smooth
S. Adak, R. Banerjee / Carbohydrate Polymers 150 (2016) 359–368 365
contro
scsimebPacZf(cb
3
t63LowcrrpT
Fig. 4. Diffractogram of starch samples (A) maize starch, (B)
urface. Gao et al. (2014) also observed similar microstructure inorn starch under SEM. Compared to maize starch, control starchample microstructure (Fig. 5(B)) had no granular appearance. Thentact granules were disrupted and were fused together forming a
olecular network. Temperature treated starch granules in pres-nce of water form similar collapsed sheet like structure as depictedy SEM analysis (Majzoobi et al., 2011; Ratnayake & Jackson, 2006).regelatinization of starch and dissolution with ionic liquids hasnalogous effect on the starch structure which favoured esterifi-ation (Lu, Luo, Yu, & Fu, 2012; Lu, Luo, Fu, & Xiao, 2013; Luo &hou, 2012; Xie, Shao, & Liu, 2009). Loss of granular structure andormation of fussed masses was also found in case of starch estersFig. 5(C) and (D)). The fused masses were bigger for starch estersompared to control starch sample and surface was also found toe altered to a greater extent.
.3.5. TGAThe TGA curves have been given in Fig. 6. The maize and con-
rol starch samples both show three stage weight losses below00 ◦C. Evaporation of water leads to the first loss in the range0–120 ◦C that is generally reported near 100 ◦C (Zhang, Xie, Zhao,iu, & Gao, 2009). The weight loss is found to be greater in casef maize compared to that of control starch sample. The secondeight loss was found to be the major one with maximum per-
ent loss (60–65%) for both the starch samples in the temperature
ange 280–370 ◦C. Remaining 20% weight loss was observed in theange 370–540 ◦C. These two weight losses occur due to decom-osition of starch amylose and amylopectin (Beninca et al., 2008).he starch decomposition was found to occur at 310 ◦C. For starch
ester (DS 2.75) the initial weight loss (%) was found to be far lesscompared to both maize and control starch samples. Further heat-ing showed reduction in the thermal stability of starch ester withan early decomposition at 230 ◦C and little shift in starch decom-position temperature (308 ◦C). The weight loss (16%) in the region180–270 ◦C could be due to the evaporation of incorporated oleicacid. Yunos and Rahman (2011) reported analogous effect of lower-ing of decomposition temperature in thermoplastic starches due tothe evaporation of plasticizer, glycerol. The lowering of thermal sta-bility was also observed in case of starch palmitate as a result of lossof crystalline structure (Lu et al., 2012) corroborated by XRD andSEM studies. Hermawan, Rosyanti, Megasari, Sugih, and Muljana(2015) reported disintegration of intramolecular interactions instarch matrices by esterification with long chain fatty acids, whichin turn reduced the thermal decomposition temperature. Differ-ent cellulose esters also depicted similar property on esterification(Jandura, Riedl, & Kokta, 2000).
ring in the starch samples over a range of temperature. Differentendothermic and exothermic changes take place with increase inthe temperature which is found to differ with sample. Wang et al.(2008) reported that both glass transition temperature (Tg) andmelting temperature (Tm) changed after acetylation of native yam
starch. In the present study, DSC curve of maize starch showedmultiple endothermic peaks (Fig. S4). Occurrence of such multiplemelting transition peaks have been reported in case of low moisturecontent potato starch and also observed in case of thermoplastic
366 S. Adak, R. Banerjee / Carbohydrate Polymers 150 (2016) 359–368
Fig. 5. SEM images of starch samples (A) maize starch, (B) control starch, (C) starch ester (1.88 DS), (D) starch ester (2.75 DS).
S. Adak, R. Banerjee / Carbohydrate Polymers 150 (2016) 359–368 367
ve of s
cSa2tr2RpbeLpihiwHPaws
4
lfwttrrsfCiaft
Fig. 6. TGA cur
orn starch composite (Da Roz, Carvalho, Gandini, & Curvelo, 2006;teeneken & Woortman, 2009). The three transition peaks weret 139 ◦C, 147 ◦C and 155 ◦C which represent melting transition 1,
and high temperature transition respectively. Low temperatureransition peak was not observed in the present sample, which waseported to be at 78 ◦C for potato starch (Steeneken & Woortman,009). Absence of low transition peak was also observed by Daoz, Carvalho, Gandini, and Curvelo (2006). For control starch sam-le the two melting peaks were found to merge together into aroad peak, which shifted to lower temperature (135 ◦C). Such anffect has been observed after gelatinization of rice starch (Chung,ee, & Lim, 2002). For starch ester (DS 2.75) single endothermiceak was observed at 123 ◦C which is much lower than the melt-
ng transitions of maize and control starch sample. Replacement ofydroxyl groups of sugar molecules by oleic acid reduced the melt-
ng temperature. Similar effect has been reported in the literatureith starch ester of different fatty acids (Diop, Li, Xie, & Shi, 2011;ermawan, Rosyanti, Megasari, Sugih, & Muljana, 2015; Rajan,rasad, & Abraham, 2006; Wang et al., 2008). Thus the acyl groupsct as internal plasticizers. Incorporation of glycerin (plasticizer) inheat starch has been found to lower the melting temperature of
tarch making it thermoplastic in nature (Liu, 2001).
. Conclusion
The present study was conducted to synergize the use ofipase, novel imidazolium surfactants and microwave irradiationor starch modification. Esterification of corn starch with oleic acidas performed employing Rhizopus oryzae lipase. Different reac-
ion parameters affecting the process were monitored. An exposureo 80% irradiation for 1 min followed by 4 h shaking at 30 ◦C of theeaction mixture yielded in high DS with 1:3 starch/oleic acid molaratio and 150 IU enzyme. Further presence of novel imidazoliumurfactants enhanced the etherification process, where gemini sur-actant [C16-3-C16im]Br2 (50 �mol) yielded in maximum DS (2.75).haracterization of modified starch showed significant changes in
ts properties. The modified starch showed better water resistance,nd oil holding capacity. FTIR spectra data confirms ester bondormation and corresponding changes in the crystallinity and struc-ure was depicted by XRD and SEM analysis respectively. Thermal
tarch samples.
analysis shows the esterified starch to be thermoplastic in nature.These properties of modified starch advocate for its potential appli-cation in food and biopolymer sectors.
Acknowledgements
Ms Sunita Adak gratefully acknowledges the financial sup-port provided by CSIR, New Delhi, by granting her the SeniorResearch Fellowship. The authors are also thankful to Prof. SantanuBhattacharya, Organic Chemistry Department, Indian Institute ofScience, India for providing the novel imidazolium surfactants.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.carbpol.2016.05.038.
References
Abbas, K. A., Khalil, S. K., & Hussin, A. S. M. (2010). Modified starches and theirusages in selected food products: a review study. Journal of Agricultural Science,2(2), 90–100.
Aburto, J., Alric, I., Thiebaud, S., Borredon, E., Bikiaris, D., Prinos, J., et al. (1999).Synthesis, characterization: and biodegradability of fatty acid esters ofamylase and starch. Journal of Applied Polymer Science, 74, 1440–1451.
Adak, S., & Banerjee, R. (2013). Biochemical characterisation of a newly isolatedlow molecular weight lipase from Rhizopus oryzae NRRL 3562. EnzymeEngineering, 2, 118. http://dx.doi.org/10.4172/2329-6674.1000118
Adak, S., Datta, S., Bhattacharya, S., & Banerjee, R. (2015a). Imidazolium based ionicliquid type surfactant improves activity and thermal stability of lipase ofRhizopus oryzae. Journal of Molecular Catalysis B: Enzymatic, 119, 12–17.
Adak, S., Datta, S., Bhattacharya, S., & Banerjee, R. (2015b). Role of spacer length ininteraction between novel gemini imidazolium surfactatns and Rhizopusoryzae lipase. International Journal of Biological Macromolecules, 81, 560–567.
Alissandratos, A., & Halling, P. J. (2012). Enzymatic acylation of starch. BioresourceTechnology, 115, 41–47.
Alissandratos, A., Baudendistel, N., Hauer, B., Baldenius, K., Flitsch, S., & Halling, P.(2010). Biocompatible functionalisation of starch. Chemical Communications,47, 683–685.
Alissandratos, A., Baudendistel, N., Ritsch, S. L., Hauer, B., & Halling, P. J. (2010).
Lipase-catalysed acylation of starch and determination of the degree ofsubstitution by methanolysis and GC. BMC Biotechnology, 10, 82.
Ao, M., Huang, P., Xu, G., Yang, X., & Wang, Y. (2009). Aggregation andthermodynamic properties of ionic liquid-type gemini imidazolium surfactantswith different spacer length. Colloid and Polymer Science, 287, 395–402.
Zhao, Y., Gao, S., Wang, J., & Tang, J. (2008). Aggregation of ionic liquids [Cnmim]Br(n = 4, 6, 8, 10: 12) in D2O: a NMR study. The Journal of Physical Chemistry B, 112,2031–2039.
68 S. Adak, R. Banerjee / Carbohyd
eninca, C., Demiate, I. M., Lacerda, L. G., Filho, M. A. S. C., Ionashiro, M., &Schnitzler, E. (2008). Thermal behaviour of corn starch granules modified byacid treatment at 30 and 50 ◦C. Eclética Química, 33(3), 13–18.
hakraborty, S., Sahoo, B., Teraoka, I., Miller, L. M., & Gross, R. A. (2005).Enzyme-catalyzed regioselective modification of starch nanoparticles.Macromolecules, 38, 61–68.
hung, H. J., Lee, E. J., & Lim, S. T. (2002). Comparison in glass transition andenthalpy relaxation between native and gelatinized rice starches. CarbohydratePolymers, 48, 287–298.
a Roz, A. L., Carvalho, A. J. F., Gandini, A., & Curvelo, A. A. S. (2006). The effect ofplasticizers on thermoplastic starch compositions obtained by melt processing.Carbohydrate Polymers, 63, 417–424.
atta, S., Biswas, J., & Bhattacharya, S. (2014). How does spacer length ofimidazolium gemini surfactants control the fabrication of 2D-Langmuir filmsof silver-nanoparticles at the air-water interface? Journal of Colloid andInterface Science, 430, 85–92.
iop, C. I. K., Li, H. L., Xie, B. J., & Shi, J. (2011). Effects of acetic acid/acetic anhydrideratios on the properties of corn starch acetates. Food Chemistry, 126,1662–1669.
ao, Y., Wang, L., Yue, X., Xiong, G., Wu, W., Qiao, Y., et al. (2014). Physicochemicalproperties of lipase-catalysed laurylation of corn starch. Starch/Starke, 66,450–456.
arlapati, V. K., & Banerjee, R. (2013). Solvent-free synthesis of flavour estersthrough immobilized lipase mediated transesterification. Enzyme Research,http://dx.doi.org/10.1155/2013/367410 [Article ID 367410]
oto, M., Kamiya, N., Miyata, M., & Nakashio, F. (1994). Enzymatic esterification bysurfactant-coated lipase in organic media. Biotechnol Progress, 10, 263–268.
rote, C., & Heinze, T. (2005). Starch derivatives of high degree of functionalization11: studies on alternative acylation of starch with long-chain fatty acidshomogeneously in N,N-dimethyl acetamide/LiCl. Cellulose, 12, 435–444.
arjani, J. R., Naik, P. U., Nara, S. J., & Salunke, M. M. (2007). Enzyme mediatedreactions in ionic liquids. Current Organic Synthesis, 4, 354–369.
ermawan, E., Rosyanti, L., Megasari, L., Sugih, A. K., & Muljana, H. (2015).Transesterification of sago starch using various fatty acid methyl esters indensified CO2. International Journal of Chemical Engineering, 6(3), 152–155.
orchani, H., Chaabouni, M., Gargouri, Y., & Sayari, A. (2010). Solvent-freelipase-catalyzed synthesis of long-chain starch esters using microwaveheating: optimization by response surface methodology. CarbohydratePolymers, 79, 466–474.
kegwu, O. J., Okechokwu, P. E., & Ekumankana, E. O. (2010). Physico-chemical andpasting characteristics of flour and starch from Achi Brachystegia eurycomaseed. Journal of Food Technology, 8(2), 58–66.
andura, P., Riedl, B., & Kokta, V. (2000). Thermal degradation behaviour ofcellulose fibres partially esterified with some long chain organic acids. PolymerDegradation and Stability, 70, 387–394.
apusniak, J., & Siemion, P. (2007). Thermal reactions of starch with long-chainunsaturated fatty acids. Part 2. Linoleic acid. Journal of Food Engineering, 78,323–332.
aur, B., Ariffin, F., Bhat, R., & Karim, A. A. (2012). Progress in starch modification inthe last decade. Food Hydrocolloids, 26, 398–404.
avlani, N., Sharma, V., & Singh, L. (2012). Various techniques for the modificationof starch and the applications of its derivatives. International Research Journalof Pharmacy, 3(5), 25–31.
o, W. C., Wang, H. J., Hwang, J. S., & Hsieh, C. W. (2006). Efficient hydrolysis oftuna oil by a surfactant-coated lipase in a two-phase system. Journal ofAgricultural and Food Chemistry, 54, 1849–1853.
umar, V., Yadav, S., Jahan, F., & Saxena, R. K. (2014). Organic synthesis of maizestarch-based polymer using Rhizopus oryzae lipase, scale up: and itscharacterization. Preparative Biochemistry and Biotechnology, 44, 321–331.
umari, A., Mahapatra, P., Garlapati, V. K., & Banerjee, R. (2008). Comparative studyof thermostability and ester synthesis ability of free and immobilized lipaseson cross linked silica gel. Bioprocess and Biosystems Engineering, 31, 291–298.
ehmann, A., & Volkert, B. (2011). Preparing esters from high-amylose starch usingionic liquids as catalysts. Carbohydrate Polymers, 83, 1529–1533.
ewandowicz, G., Fornal, J., Walkowski, A., Maczynski, M., Urbaniak, G., &Szymanska, G. (2000). Starch esters obtained by microwave radiation-structure and functionality. Industrial Crops and Products, 11, 249–257.
iao, X., Raghavan, S. V., & Yaylayan, V. A. (2002). A novel way to preparen-butylparaben under microwave irradiation. Tetrahedron Letters, 43, 45–48.
in, R., Li, H., Long, H., Su, J., & Huang, W. (2014). Synthesis of rosin acid starchcatalyzed by lipase. BioMed Research International, http://dx.doi.org/10.1155/2014/647068 [Article ID 647068]
iu, Z. Q. (2001). Effects of glycerin and glycerol monostearate on performance ofthermoplastic starch. Journal of Materials Science, 36, 1809–1815.
u, X., Luo, Z., Yu, S., & Fu, X. (2012). Lipase-catalyzed synthesis of starch palmitatein mixed ionic liquids. Journal of Agricultural and Food Chemistry, 60,9273–9279.
u, X., Luo, Z., Fu, X., & Xiao, Z. (2013). Two-step method of enzymatic synthesis ofstarch laurate in ionic liquid. Journal of Agricultural and Food Chemistry, 61,9882–9891.
ajzoobi, M., Radi, M., Farahnaky, A., Jamalian, J., Tongdang, T., & Mesbahi, G.
(2011). Physicochemical properties of pre-gelatinized wheat starch producedby a twin drum drier. Journal of Agricultural Science and Technology, 13,193–202.
olymers 150 (2016) 359–368
Mogensen, J. E., Sehgal, P., & Otzen, D. E. (2005). Activation: inhibition anddestabilization of Thermomyces lanuginosus lipase by detergents. Biochemistry,44, 1719–1730.
Namazi, H., Fathi, F., & Dadkhah, A. (2011). Hydrophobically modified starch usinglong-chain fatty acids for preparation of nanosized starch particles. ScientiaIranica, 18(3), 439–445.
Pal, A., Datta, S., Aswal, V. K., & Bhattacharya, S. (2012). Small-angleneutron-scattering studies of mixed micellar structures made of dimericsurfactants having imidazolium and ammonium headgroups. The Journal ofPhysical Chemistry B, 116, 13239–13247.
Palav, T., & Seetharaman, K. (2007). Impact of microwave heating on thephysico-chemical properties of a starch-water model system. CarbohydratePolymers, 67(4), 596–604.
Pencreach, G., & Baratti, J. C. (2001). Comparison of hydrolytic activity in water andheptane for thirty-two commercial lipase preparations. Enzyme and MicrobialTechnology, 28, 473–479.
Perreux, L., & Loupy, A. (2001). A tentative rationalization of microwave effects inorganic synthesis according to the reaction medium, and mechanisticconsiderations. Tetrahedron, 57(45), 9199–9223.
Raghavendra, T., Panchal, N., Divecha, J., Shah, A., & Madamwar, D. (2014).Biocatalytic synthesis of flavour ester pentyl valerate using Candida rugosalipase immobilized in microemulsion based organogels: effect of parametersand resusability. BioMed Research International, http://dx.doi.org/10.1155/2014/353845 [Article ID 353845]
Rajan, A., & Abraham, T. E. (2006). Enzymatic modification of cassava starch bybacterial lipase. Bioprocess and Biosystems Engineering, 29, 65–71.
Rajan, A., Prasad, V. S., & Abraham, T. E. (2006). Enzymatic esterification of starchusing recovered coconut oil. International Journal of Biological Macromolecules,39, 265–272.
Rajan, A., Sudha, J. D., & Abraham, T. E. (2008). Enzymatic modification of cassavastarch by fungal lipase. Industrial Crops and Products, 27, 50–59.
Ratnayake, W. S., & Jackson, D. S. (2006). Gelatinization and solubility of cornstarch during heating in excess water: new insights. Journal of Agricultural andFood Chemistry, 54(10), 3712–3716.
Salameh, M. A., & Wiegel, J. (2010). Effects of detergents on activity:thermostability and aggregation of two alkalithermophilic lipases fromThermosyntropha lipolytica. The Open Biochemistry Journal, 4, 22–28.
Shankar, S., Agarwal, M., & Chaurasia, S. P. (2013). Study of reaction parameters andkinetics of esterification of lauric acid with butanol by immobilized Candidaantarctica lipase. Indian Journal of Biochemistry and Biophysics, 50, 570–576.
Shogren, R. L., & Biswas, A. (2006). Preparation of water-soluble andwater-swellable starch acetates using microwave heating. CarbohydratePolymers, 64(1), 16–21.
Singh, V., & Tiwari, A. (2008). Microwave-accelerated methylation of starch.Carbohydrate Research, 343(1), 15–154.
Song, B. D., Ding, H., & Wang, S. C. (2007). Hydrolysis of olive oil catalyzed bysurfactant-coated Candida rugosa lipase in a hollow fibre membrane reactor.Biotechnology and Bioprocess Engineering, 12, 121–124.
Staroszczyk, H., & Janas, P. (2010). Microwave-assisted synthesis of zincderivatives of potato starch. Carbohydrate Polymers, 80(3), 962–969.
Steeneken, P. A. M., & Woortman, A. J. J. (2009). Identification of the thermaltransitions in potato starch at low water content as studied by preparativeDSC. Carbohydrate Polymers, 77, 288–292.
Wang, X., Gao, W. Y., Zhang, L. M., Xiao, P. G., Yao, L. P., Liu, Y., et al. (2008). Studyon the morphology, crystalline structure and thermal properties of yam starchacetates with different degrees of substitution. Science in China Series B:Chemistry, 51(9), 859–865.
Wootton, M., & Bamunuarachchi, A. (1978). Water binding capacity of commercialproduced native and modified starches. Starch/Starke, 30, 306–309.
Xie, S. X., Liu, Q., & Cui, S. W. (2005). Starch modification and application. In S. W.Cui (Ed.), Food carbohydrates: chemistry, physical properties, and applications(pp. 357–406). Boca Raton, FL, USA: CRC Press, Taylor & Francis Group.
Xie, W., Shao, L., & Liu, Y. (2009). Synthesis of starch esters in ionic liquids. Journalof Applied Polymer Science, 116, 218–224.
Xin, J. Y., Wang, Y., Liu, T., Lin, K., Chang, L., & Xia, C. G. (2012). Biosynthesis of cornstarch palmitate by lipase novozyme 435. International Journal of MolecularSciences, 13, 7226–7236.
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.
Yousif, E. I., Gadallah, M. G. E., & Sorour, A. M. (2012). Physico-chemical andrheological properties of modified corn starches and its effect on noodlequality. Annals of Agricultural Science, 57(1), 19–27.
Yunos, M. Z. B., & Rahman, W. A. W. A. (2011). Effect of glycerol on performance ricestraw/starch based polymer. Journal of Applied Sciences, 11(13), 2456–2459.
Zhang, L., Xie, W., Zhao, X., Liu, Y., & Gao, W. (2009). Study on the morphology,crystalline structure and thermal properties of yellow ginger starch acetateswith different degrees of substitution. Thermochimica Acta, 495, 57–62.
Zobel, H. F., Young, S. N., & Rocca, L. A. (1988). Starch gelatinization: an X-raydiffraction study. Cereal Chemistry, 65(6), 443–446.
