Starch from locally available corn was modified by hydrothermal method for evaluating its physiochemical, pasting, thermal properties and determining the rate of hydrolysis and resistant starch (RS) formation. The native corn starch was modified using six thermal cycles involving gelatinization at 121oC for 60 minutes and refrigeration for 24hour. Modified starch was studied by FT-IR (Fourier transform infrared spectroscopy), SEM (scanning electron microscopy), XRD (X-ray diffraction) and DSC (differential scanning calorimetry). Results shows that the smooth granular structure of starch changed into a continuous network with irregularities, the crystallinity of starch changed from A-type to B-type. Thermal studies showed a clear shifting of endothermic peaks towards lower temperatures. Modification of starch after two thermal cycles showed the highest (73.7 Â±0.00 %) RS content. Cycle 1, 2 and 3 samples however exhibited lower hydrolysis indicative of complex undigestible fractions. Cycle 2 samples exhibited lowest hydrolysis after 3 hours.
Keywords: Corn starch, XRD, SEM, FTIR, Hydrothermal method
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Starch is main source of energy in the human diet. It is the forms of storage polysaccharide present in plants which is most abundant and universally distributed, and occurs as granules in the chloroplast of green leaves and amyloplast of seeds, pulses and tubers (Tester, Karkalas, & Qi, 2004). The starch molecule is composed of linear amylose and branched amylopectin fractions responsible for its crystalline and amorphous properties, respectively. Starch is classified into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) according to the rate of glucose release and its absorption in the gastrointestinal tract (Englyst, Kingmann & Cummings, 1992). A fraction of the starch in starchy foods which escapes complete enzyme degradation depending on its granule size, amylose to- amylopectin ratio, starch-protein interactions, amylose-lipid complexes, percentage of retrograded starch, and degree of gelatinization is known as resistant starch (RS) (Englyst & Cummings, 1985; Leloup, Colona & Ring, 1993). In general, native starch presents low shear stress resistance and thermal decomposition, in addition to high retrogradation and syneresis (Whistler et al., 1984). Native corn starch can be modified to obtain pastes with specific attributes that can resist extreme food processing requirements. There are many health benefits of RS such as prevention of colon cancer, hypoglycemic effects, substrate for growth of the probiotic microorganisms, reduction of gall stone formation, hypocholesterolemic effects, inhibition of fat accumulation, and increased absorption of minerals (Sajilata et al., 2006). RS has been mainly divided into four categories which are type-1, -2, -3, and -4 for physically inaccessible starch, raw crystalline starch, retrograded starch, and chemically modified starch, respectively.
Autoclaving and cooling is a type of hydrothermal treatment that has been used to modify starch digestibility. When starch is gelatinized under high moisture content and allowed to cool, alignment of ordered amylose molecules with each other leads to the formation of a rigid gel. Resistant starch is produced as the insoluble crystallite formed by the process of controlled retrogradation (Englyst, kingmann & Cummings, 1992). Modification cause rupturing of some or all of the starch molecules, thus weakening them and decreasing their capacity to swell on pasting. Among the four types of RS, the type-3 is of great interest due to its thermal stability during food processing (Kim & Kwak, 2004). It can be obtained by hydrothermal treatments and retrogradation of starch derived from cereal grains, roots, tubers and legumes, such as wheat, corn, oat, rice, potato, tapioca and mung bean. Among these starches, high amylose corn starch (HACS) is most often used for preparation of RS3 (Dimantov, Kesselman, & Shimoni, 2004; Herman & Remon, 1989; Sievert & Pomeranz, 1989). Hydrothermal treatment of starch leads to B, C and V type X-ray diffraction pattern. Both B and C type starches appear to be more resistant to digestion with high-amylose maize producing RS which has been particularly useful in the preparation of foods (Brown, 2004). In order to produce the RS type-3, the granular structure of starch needs to be disrupted by heating in the presence of sufficient water, followed by the amylose re-association upon cooling.
Corn starch is a valuable ingredient to the food industry, being widely used as a thickener, gelling agent, bulking agent and water retention agent (Singh, Singh, Kaur, Sodhi, & Gill, 2003).
The objective of the present study was to investigate the effect of thermal treatment on the resistant starch formation, physicochemical, morphological and structural properties of starch from locally available corn varieties.
Materials and methods
Always on Time
Marked to Standard
Isolation of corn starch and yield
Corn kernels were cleaned to remove foreign materials before extraction. The clean kernels were steeped in 1% sodium metabisulfite at 40Â°C for 24 hours. The water was drained off and kernels were disintegrated with water in 1:1 ratio and ground in a laboratory blender (Philips juicer mixer grinder HL1632). The slurry was screened through a 75Âµm mesh and allowed to stand for 2-3 hour. The supernatant was drained and the settled starch layers were re-suspended in distilled water and centrifuged (Hettich Zentrifugen EBA 21) at 6000 rpm for 20 min at room temperature. The upper non-white layer was scrapped and the white layer was collected and dried at 40Â°C for 12 hour. The starch yield was calculated as:
% yield = (dry weight of starch recovered from extraction Ã- 100)/ dry weight of whole corn kernels.
Amylose content was determined by the method of Sowbhagya and Bhattacharya (1979).
Proximate analysis (Moisture, Crude fat, protein and ash determination)
Estimation of moisture, protein, fat and total starch in the isolated and native starch material was carried out following the AOAC (2005) protocols.
Hydro-thermal (Autoclaving-Cooling) Treatment
Autoclaved-cooled starches were prepared by the method of Sievert and Pomeranz (1989), with slight modification. Briefly, raw starch was suspended in distilled water at starch-to-water ratio of 1:3.5. The suspension was autoclaved (Lab Tech, Daihan labtech co ltd.) for 1 hour at 121Â°C, cooled to room temperature, and kept at 4Â°C for 24 hours which was termed as cycle 1. The autoclaving cooling cycle was repeated six times. The sample was dried at 40Â°C and grounded in a lab grain mill (Fritsch Pulverisette 14) using a 1mm sieve.
Moisture sorption capacity
The moisture sorption capacity of the native and modified starch was determined by following the method of Ohwoavworhua et al., (2004). It was calculated as:
Wa- Amount of water absorbed.
Wg- Weight gained.
W- Initial weight
Water absorption index (WAI) and water solubility index (WSI)
Water absorption and solubility index was determined by the method of Anderson, Conway, Pfeifer, and Griffin (1969). The water absorption and water solubility index were calculated by:
WAI = R/W
WSI (%) = (S/W) x 100
Where, R = weight of residue; W = dry weight of starch; S = weight of soluble starch .
The swelling capacity of the starch powders were determined by the method of Iwuagwu and Okoli (1992). The tapped volume occupied by 5g of the starch powder Vx was noted. The powder was then dispersed in distilled water and the volume was made up to 100ml. After 24hours of standing, the volume of the sediment, Vv was estimated and the swelling capacity was computed as;
Vv / Vx
Freeze thaw stability
The freeze-thaw stability of the gelatinized starch was measured by the method of Kaur, Singh, and Singh (2004) with some modifications. An aqueous suspension of starch (5%, w/w) was heated at 95 Â°C in a shaking water bath for 1 hour. The paste was weighed (exactly 20 g each) into previously weighed polypropylene centrifuge tubes and the tubes were closed tightly. The paste was centrifuged at 1000 rpm for 3 min to remove free water (supernatant). Alternate freezing and thawing was performed by freezing at 4Â°C for 24 hours and thawing for 4 hours at 30 Â°C, followed by centrifugation at 4500 rpm for 25 min. A total of eight freeze-thaw cycles were performed. The weight of water separated after each cycle was taken as the extent of syneresis and expressed as the percentage of water separated.
% Syneresis= Water separated / Total weight of sample Ã- 100
This was determined according to a method modified from Kornblum and Stoopak (1973). 1 gram samples (Y) was placed in centrifuge tubes and covered with 10ml of distilled water before keeping at RT (20Â±2oC), 8Â±2oC (refrigerator) and in the hot water bath at 500C (above RT) for 2 hours. The tubes were shaken intermittently during the time and then left to stand for 30minutes than centrifuged at 3000 rpm for 10 minutes. The supernatant was decanted and the weight of the powder after water uptake (X) was determined. Hydration capacity was calculated as:
Hydration capacity= X/Y
Light transmittance of starch suspensions was measured by a modified method of Perera and Hoover (1999). A 2% aqueous starch suspension was kept in a boiling water bath for 1 hour at 95Â°C with constant stirring. The suspension was cooled for 1 hour at 30 Â°C and then stored at 4 Â°C. Light transmittance was determined at 640 nm against a distilled water blank with a UV-VIS Spectrophotometer (Cecil Aquarius 7400) every 24 hours, for 5 days.
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Bulk and Tapped densities
Bulk and Tapped densities were determined by adopting the method of Olayemi, Oyi and Allagh (2008).
It was calculated as:
Where, Bd - Bulk density and TD - Tapped density
Wp- Initial weight, Vp- Volume occupied, VpT- Tapped volume
True Density and Porosity
The true density of the material was determined using xylene as the displacement liquid in a 100mL graduated measuring cylinder. 30g starch powder (W) was taken in a graduated 100ml measuring cylinder and 30ml xylene was poured. The volume of starch powder (V) occupied after adding xylene was noted. True density (Td) was computed as:
Td = W/V
The powder porosity (E) was calculated by the method of Ohwoavworhua and Adelakun (2005) as:
E = [1- (Bd/Td) Ã- 100]
Where Bb is bulk density.
The pasting profiles of starches were recorded using a Rapid Visco Analyser (RVA Starchmaster2, Newport Scientific Instruments). The viscosity profiles were recorded using starch suspensions (10% w/w; 28 g total weight). The Std1 profile of Newport Scientific was used, where the samples were held at 50 Â°C for 1 min, heated from 50 Â°C to 95Â°C at 12.16 Â°C/min, held at 95 Â°C for 2.30 min, cooled from 95 Â°C to 50 Â°C at 11.84 Â°C/min, and held at 50 Â°C for 2 min. The peak viscosity (PV), hot paste viscosity
(HPV), cold paste viscosity (CPV), breakdown (BD) and setback (SB) were recorded. HPV = minimum viscosity at 95 Â°C, CPV = final viscosity at 50 Â°C, BD = PV â€•HPV and SB = CPVâ€•HPV. (Dutta et al. 2011)
DSC Thermal Analysis
A differential scanning calorimeter (Shimadzu Model TGA50 DSC60), periodically calibrated with pure indium for heat flow temperature was used to determined changes in enthalpy (âˆ†H) of the starch, polysaccharide samples. About 10 mg, 1:2 flour to moisture ratio of each sample was taken, which was saturated for 12h at 4 0C in aluminum pans and sealed. The pans were heated from 200C to 2000C at the heating rate of 100C/minute. The pans were heated along with a reference empty aluminum pan. The heating of the pans were conducted in a nitrogen environment. The nitrogen flow rate of the instrument was 30ml/minute. The thermographs were obtained for each, onset (To), peak (Tp) and final conclusion temperature (Tf) using a TA-69 WS software. In case of polysaccharide the heating range was 200C to 3000C.
X-ray diffraction and FTIR spectroscopy
Wide angle X-ray diffractograms of starches were obtained with an X-ray diffractometer (Rigaku Miniflex) with a k value of 1.54040 operating at 30 kV acceleration potential and 15 mA current with a copper target. The scanning range was 2-40Â° of 2Î¸ values with a scan speed of 8Â° 2Î¸/min. The diffractogram patterns were evaluated according to Zobel, (1964). The percentage crystallinity was determined according to Singh, Ali, Somashekar and Mukherjee (2006)
% Crystallinity = Area under peaks/ Total areaÃ- 100
The infra-red spectra for starch and polysaccharide were obtained with a FTIR spectrometer (Nicolet Impact 410) equipped with KBr optics and a DTGS detector. The equipment was operated with a resolution of 2.0 cm-1 and scanning range of 4000-450 cm-1.
Starch granule size, structure and morphology
Native and modified starch granules were observed with a Scanning Electron Microscope (JEOL 6993V) operating at an acceleration voltage of 15 kV and 1200 Ã-magnification.
The colour of starch and cake was compared by analysing the samples in a Colour Measurement Spectrophotometer (Hunter Color- Lab Ultrascan Vis). The result was expressed as L, a, b values.
In vitro starch digestibility test and rate of hydrolysis
Starch samples (modified and native) were analyzed for the in vitro rate of starch digestibility by using the enzymatic, colorimetric method of Goni, Garcia and Saur (1997) with some modifications.
Briefly, 200ÂµL of KCl-HCl buffer (pH 2.0) containing 1g pepsin (sigma) was added to 25mg flour sample and kept at 40Â°C for 1 min in a shaking water bath for protein removal. The volume was made upto 6 ml with Tris-maleate buffer (pH 6.9) containing 2.6UI ï„ƒ-amylase (Hi-media) and incubated at 37Â°C. 200ÂµL aliquot samples were taken out from each tube after every 30 minute for 3 hours min and placed in a tube at 100Â°C with shaking for 5min for enyme inactivation to and stored in refrigerator. Than 60 Î¼l amyloglucosidase in 3mL sodium acetate buffer (0.4M, pH 4.75) was added to hydrolyze the digested starch into glucose at 60Â°C for 45 minute in a water bath. The mass was centrifuged (15 min, 4500 g) and the supernatant was analyzed for glucose. The digestible starch was calculated as mg of glucose Ã- 0.9.
The rate of digestion was expressed in terms of the digestible starch (%) formed in samples hydrolyzed at different times (0, 30, 60, 120, 160 and 180 min).
All the experimental analyses were done in triplicate. The results obtained were expressed as mean Â± standard deviation values and were subjected to statistical analysis using OrginPro 7.5 and GraphPad Prism 3 (version 3.02) software. The significance of differences between means was estimated with ANOVA-post hoc Turkey's multiple comparison tests at pÂ â‰¤Â 0.05.
Result and discussion
Compositional analysis of native corn starch and starch yield
The starch yield after extraction was around 62 Â± 0.02 %. The mean values of moisture, protein, fat and ash are 15.07 % Â± 0.02, 0.35025 Â± 0.02g/kg, 0.05 % Â± 0.00 and 0.24 Â± 0.02 respectively. The starch and amylose content was 99.1% Â± 0.04 (dry basis), 24.6 % Â± 0.3 respectively.
Moisture sorption capacity
The moisture sorption capacity is a measure of moisture sensitivity of a material. The result (Fig. 1.) shows that native starch absorbed the least moisture with time as compared to the modified starches. The modified starch from cycle 2 showed the highest sensitivity. This can be attributed to the retrogradation of starch molecules after heat treatment which enhances moisture absorption. This indicates that it is relatively more sensitive to atmospheric moisture, as supported by findings of Ohwoavworhua and Osinowo, (2010) in pregelatinized maize starch.
Water absorption capacity and water solubility index and
The water absorption capacity was observed lowest in the native starch and increased with increased treatment cycles (Fig. 2). This can be attributed to the presence of hydrophilic substituting groups that retains water (Betancur, Chel, & Canizares, 1987). The solubility of the native starch was comparatively higher than the modified starches. WSI: 3rd and 4th cycle solubility increased rest decrease.
Swelling: It is reasonable that reduction in swelling capacity is attributing to rearrangement of molecular chains during HMT, strengthening its maintained force, which restricted in absorbing water within starch matrices and therefore a more rigid starch structure were formed after
hydrothermal modifications (Eerlinger and Delcour, 1995; Franco et al., 1995). In addition to amylose, the bonds of amylopectin are weakened and gradually break with increasing temperature, resulting in increased solubility. The swelling capacity reflects the increase in volume of samples following water absorption, the damage of amylopectin by heat treatment can be attributed to the decrease in the swelling power of the granules. This observation concurred with the results of
Han and BeMiller (2007), Lim et al., (2002); and Sekine et al., (2000) for different starches.
Freeze thaw stability
The percent syneresis shown by the starches increased with increasing the treatment cycle (Fig. 3) (significantly differed at p<0.001). While the modified starches showed more syneresis than the native starch. Again with increase in freeze thaw cycles the syneresis increased on a gradual basis in all the starches except for cycle 3 and 4 in which slight decrease was noted on 6th and 5th day respectively. The increase in syneresi during the freeze thaw cycles is due to disruption of gel matrix (Lo & Ramsden, 2000) and on freezing starch rich regions are created in the matrix (Ferrero et al., 1994). The formation of such starch rich regions during the modification treatment may have occurred. This may be because on cooling, a three dimensional polymer network builds up in which double helices form junction points of the polymer chains and further cooling may leads to aggregation of these junction points building up a rigid gel structure. This tendency of intermolecular association might leads to the relatively high syneresis and low stability of starch gel as mentioned by Glicksman, (1982); Lee et al. (2002).
The parameter was found to increase with rise in temperature in most of the cases. The increased water absorption with increasing temperature was in agreement with the report of Hoover and Maunal (1996) in case of maize starch. The hydration capacity of the native starch (1.55Â± 0.01, 1.84Â± 0.01 and 2.39Â± 0.02 at 10, 25 and 50Â° C respectively) was lowest as compared to the modified starch samples, as also evident from the results of swelling capacity. However the values of hydration capacity did not show a regular trend with consecutive treatment cycle. The sample from Cycle 6 showed the highest hydration capacity (4.32Â±0.01, 5.25Â±0.03 and 7.3Â±0.00 at 10, 25 and 50Â°C respectively). The increase hydration and swelling of modified is attributed to increased numbers of small fragments of amylopectin or amylose leaching out through opened starch granules whose structures are changed during hydrothermal
treatment resulting in increased rate and extent of water penetration similar to findings of Lai,(2000) and Ohwoavworhua and Osinowo, (2010) in case of rice and maize starch respectively.
Turbidity values of all starch suspensions increased progressively during storage of starch gels at 4 Â°C. The absorbance at 640 nm of the native starch was the highest a shown in Fig. 4. However among the modified starches, samples from Cycle 2 exhibit the lowest % transmittance comparatively. This behaviour of modified starch samples may be attributed to the increased tendency towards retrogradation after modification. Turbidity development in starches during storage has been attributed to the interaction of several factors, such as granule swelling, granule remnants, leached amylose and amylopectin, chain length, intra or interbonding (Jacobson, Obanni, & BeMiller, 1997).The results revealed that the temperature treatment and the number of treatment cycles had different extent of changes in the amylose and amylopectin molecules that caused varied tendencies towards gelatinization and retrogradation.
Bulk, true, tapped density and porosity
The bulk density of the native starch was lowest (0.5Â±0.02) which means it has a fine structure as compared to the modified samples. The higher values of the modified starches (0.71Â±0.02, 0.62Â±0.00, 0.58Â±0.05, 0.62Â±0.01, 0.58Â±0.02 and 0.52Â±0.02 for Cycle 1 to 6 respectively) might be due to the presence of hydrophilic substituting groups that retained water (Betancur, Chel, & Canizares, 1997) after retrogradation by heat treatment of starch. Porosity of most of the modified starches (28.57Â± 0.04, 43Â± 0.01, 41.17Â± 0.02, 43.75Â± 0.02, 41.17Â± 0.01 and 60Â± 0.03 for Cycle 1 to 6 respectively) was lower than the native starch (55Â± 0.02). The lower porosity of the modified starches may be because the granular structure of native starch was totally broken down upon modification as confirmed by SEM images.
A severe decrease in peak viscosity was observed in all the modified starches as compared to the native starch. Extensive changes in the pasting patterns were observed in the processed samples and native starch are as shown in Table 1. The peak viscosity of native sample (4250 cp) dropped continuously with number of cycles run. Such decrease was also in accordance to the findings of Olu-Owolabi, Afolabi, and Adebowale (2010). Samples from cycle1 and 2 exhibited a higher setback (1669cp and 3525cp) respectively with a low breakdown (-291cp and -350cp) respectively indicating formation of longer chains that are more prone to retrograde on cooling with entrapment of higher number of water molecules. The extensive loss of peak viscosity and also the other viscosity parameters indicated tremendous breakdown of the amylose fine structures that failed to develop viscosity. Sample from cycle 6 showed to have lost all the pasting parameters indicating negligible development of viscosity (13.64cp). Native starch and modified starches form Cycle 1 & 2 gradual increase in viscosity with increase in temperature. The increase in viscosity with temperature may be attributed to the removal of water from the exuded amylose by the granules as they swell (Ghiasi, Varriano-Marston, & Hoseney, 1982). The negative breakdown values in cycles 1 to 5 (-291, -350, -57, -29 and -30 cp) respectively indicated complete loss of the starch granule structure as also indicated by SEM pictures.
3.9. Thermal properties of native and corn starch
The gelatinization transition temperatures onset [To], melting [Tm], and final [Tf]), gelatinization transition temperature range R, and gelatinization enthalpy (Î”H) of native, modified starches are presented in Table 2. The processed samples showed distinctly differently DSC patterns from the native starch. Sample from cycle 1 exhibited an additional sharp peak at 1100C indicative of amylose lipid complex and other peak at 73.40C may be attributed to ungelatinized starch fractions. The other processed samples did not show any formation of amylose-lipid complexes. If there was any such complex, the peak for it must have overlapped with the main starch melting peak. Clear shifting of the endothermic peak towards lower temperature with process severity was observed. This may be attributed to the melting of newly formed retrograded starch samples. The absence of peak for amylose lipid complexes better found to be present from the XRD studies may be explained as the complexes are thermally unstable when in suspension.
No clear inference should be driven from the thermal data obtained which may be because of differences in the sample sizes. However, among the modified starches, the sample from Cycle 2 showed the highest Î”Hgel value of 9.965 which may be attributed to the formation of more crystalline areas which favoured gelatinization at high temperature. After hydrothermal modification the starch especially from Cycle 2 shows slightly higher gelatinization enthalpy because more thermal energy is needed to initiate melting of the crystal-line structure in the absence of amylose-rich amorphous regions (Shih et al., 2007). The higher Tc-To for the native and modified starches reflects greater variation in crystalline stability.
3.10. Crystalline structure after hydrothermal modification
The X-ray diffraction pattern of native corn starch with major peaks at 2Î¸ values of 15.3, 18.1, and 13.1 was distinctive of A type crystalline pattern. (Zobel, 1964). Hydrothermal modification lead to formation of B type crystalline fractions, simultaneously with partial loss of A type pattern Fig 5. These may be because of entrapment of higher water molecules by the helices during retrogradation. This was indicated by major peak at 2Î¸ values 17.1, and minor peaks at 22.1, 24.0 for all the modified samples (Sievert & Pomeranz, 1989). In addition to these, there was distinct formation of amylose-lipid complexes as evident from the peaks near 2Î¸ values 20.0. This peak however was lost for the samples processed for 2nd and 3rd cycles, which however, reappeared as stronger peaks for the more severely processed samples. This may be explained as the complexes are heat labile and failed to reform them at lower extent of processing. However on sever treatments they develop thermal stability.
For the more severely processed samples, i.e, cycle 4, 5 and 6, the crystalline peaks become more prominent resulting in higher relative crystallinity as evident from diffractograms. This must be explained as development of more thermostable crystals on repeated gelatinization and retrogradation. The A type peaks at 2Î¸ near15.1 and 18.1 reappeared for these samples giving a pattern representative of both C (A+B) and V type crystallinity in the hydrothermally treated samples which arises from complexes formed by amylose with a variety of polar organic molecules (Zobel, 1988a,b; Zobel, et al., 1988). The appearance of V-type crystallites clearly indicates the molecular mobility of amylose chains during autoclaving (Song, Janaswamy, & Yao , 2010).A type 'C' pattern is believed to be a superposition of the 'A' and 'B' patterns (Buleon, Colonna, Planchot & Ball, 1998). This observation was further corroborated by Gidley, (1987) .
The % crystallinity of the native starch was found to be more then the modified starches (Table 2). The sample from cycle 2 showed the lowest % crystallinity but after that there was almost a gradual increase of crystallinity with severely treated samples indicating a loss of amorphous structure which may be due to amylose hydrolysis and the concentration of amylopectin in the samples similar to as in Dutta et al., 2011.
3.11. Morphological characteristics by SEM
The SEM images of modified corn starch sample with highest RS content was obtained and compared with that of native starch. Fig. 6 clearly illustrated that the modification of native starch by autoclaving cooling cycles altered the starch structure.
Native (unprocessed) maize starch showed smooth surfaces and irregular, polygonal shaped granules (Fig. 6. a). The size of corn starch granules ranged from 5 to 7 Âµm for small and 15 to 18 Âµm for larger granules. The granular structure disappeared and a continuous network with irregular shape was formed on hydrothermal treatment (Fig. 6. b-g). Owing to treatment starch gelatinization, swelling and rupturing of granules occured. There is an associated disintegration of the crystalline structure with the formation of a gel network (Doutch et al., 2012; Singh, Kaur, & McCarthy, 2007).
However, at temperatures exceeding 110â-¦ C, the formation of voids(hollow regions at the granule center) and the disappearance of birefringence at the granule center (indicates loss of radial orientation of helical structures) has been shown to occur in potato (Kawabata et al., 1994; Vermeylen et al., 2006) and maize(Kawabata et al., 1994) starches. However, in both starches, thegranule periphery was found to remain highly birefringent even after HMT (Kawabata et al., 1994; Vermeylen et al., 2006)â€¦critical revies
3.12. FTIR analysis
From the survey of literature for FTIR analysis it was deduced that, the peaks of at 1055 cm-1 is corresponded to O-C stretch (Fig. 7) and 1047 and 1022 cm-1 corresponded to C-O bond stretching and are linked to ordered/crystalline structures and amorphous regions in starch (Qin et al., 2012). The modified starches showed changes in the bond intensities of 1047 and1022 cm-1 peaks due to changes in the crystalline structure as inferred by X-Ray diffraction data. The absorption band in the range of 3426-3648 cm-1 was assigned to the stretching of -OH groups. The peak corresponding to 2978 cm-1 corresponds to C-H stretch which intensity has been found to slightly change in the modified starch after consecutive treatment cycles. However no overall distinct changes in the peak characteristic were observed compared to the native starch. This indicated that no change in the bonding pattern but with a little vibration occurred due to repeated gelatinization and retrogradation of the polymeric material (Soest, Tournois, Wit, & Vliegenthart, 1995).
3.13. Color measurements of starch samples
The colour values of all the samples shown in (Table 3). The L value for native starch is higher (86.73) than all the modified starches, which may be interpreted as more or less perfect white. Almost all the L values decreased with the treatment time. Yellowness, indicated by positive b value is higher than the native starch for all the modified starch. This decrease in whiteness and increase in yellowness of the starches with number of cycles run is may be due to sever treatment of the starch sample to high temperature causing Maillard reaction (Odjo et al., 2012).
2.14. Starch hydrolysis and RS content
The extent of hydrolysis increased with passage of time (Fig. 8a). The raw starch had a lower hydrolysis than cycle 4, 5 and 6 samples. This might be because high degradation of complex starch structure into simple digestible fragments. Cycle 1, 2, 3 samples however exhibited lower hydrolysis indicative of complex undigestible fractions. Once the highly organized structures within the swollen granules were completely destroyed as in case of samples from Cycle 1, 2 and 3 as the samples are treated above gelatinization temperature, the availability of starch was not increased further by swelling, rupture, and disintegration of the granular structure (Holm et al. 1987). Cycle 2 samples exhibited lowest hydrolysis after 3hours and also showed the highest RS content (71 Â±0.02%), at the end of 16 hour digestion, which reflects enhanced interactions between starch chains i.e. amylose-amylose chains and/or amylose-amylopectin chains on treatment (Fig. 8b). As seen from the analysis, sample from cycle 6 showed a sudden increase in hydrolysis after 2 hours which may be because with time, the starch molecules become more available to enzymes.
Starch susceptibility to enzyme digestion is substantially influenced by physical modification. Samples from Cycle 1, 2, 3 exhibited lower hydrolysis indicative of complex undigestible fractions. Cycle 2 samples exhibited lowest hydrolysis after 3 hours (180 min) and also showed the highest RS content, at the end of 16 hour digestion, which reflects enhanced interactions between starch chains. SEM studies revealed that after hydrothermal modification, the granular structure of the native starch was totally broken granular structure disappeared and a continuous network with irregular shape. RVA analysis showed that samples from cycle1 and 2 hydrothermal treatments exhibited a higher setback with a low breakdown indicating formation of longer chains that are more prone to retrograde on cooling with entrapment of higher number of water molecules. The negative breakdown values in cycles 1 to 5 (-291, -350, -57, -29 and -30 cp) respectively indicated complete loss of the starch granule structure as also indicated by SEM pictures. The x-ray diffraction pattern of native corn starch with major peaks at 2Î¸ values of 15.3, 18.1, and 13.1 was distinctive of A type crystalline pattern, which after modification changed to B type crystalline form. The study also revealed that the sever treatment caused greater changes in the bonding patterns, which resulted in lowering of the water holding capacities by the granules during continuous freezing and thawing, thus a higher syneresis. In the thermal analysis of starch samples, clear shifting of the endothermic peak towards lower temperature with process severity was observed. Sample from cycle 1 exhibited an additional sharp peak at 110 0C indicative of amylose lipid complex.
The study explored the optimal conditions of manufacturing resistant starch by hydrothermal (gelatinization and cooling) treatment from locally available corn which is a potent source of starch. Hydrothermally modified corn starch can be a valuable ingredient to the food industry, being used as a thickener, gelling agent, bulking agent and water retention agent etc.
We gratefully acknowledge the Vice Chancellor, Tezpur University (Central University, Tezpur, Assam, India) for providing facilities to carry out the study.