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Physicochemical, rheological and structural characterization of acetylated oat starches
LWT - Food Science and Technology 80 (2017) 19e26
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Physicochemical, rheological and structural characterization of acetylated oat starches Asima Shah, F.A. Masoodi*, Adil Gani, Bilal Ahmad Ashwar Department of Food Science and Technology, University of Kashmir, Srinagar, 190006, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2016 Received in revised form 26 January 2017 Accepted 28 January 2017 Available online 30 January 2017
Starches isolated from three different varieties of oat were modified with acetic anhydride to make starches with high contents of type 4 resistant starch (RS4). The acetyl group percentage (Ac) and degree of substitution values (DS) of oat starches ranged from 0.64 to 1.50 and 0.02e0.05, respectively. The absorption peaks at 1737 cm1, 1337 cm1 and 1243 cm1in the FTIR spectrum confirms the presence of acetyl groups in the starch molecule. SEM micrographs revealed significant changes in external morphology of acetylated starches due to the formation of small pores. XRD analysis reflects the formation of amyloseelipid complex with decreased crystallinity in acetylated starches. The viscosity of starches decreased on acetylation, exhibited shear-thinning behavior as reflected from the convex shaped graph with viscosities of acetylated starches following the order; SKO90 > SKO20 > Sabzaar. © 2017 Published by Elsevier Ltd.
Keywords: RS4 Acetylation Oat starch
1. Introduction Oat is receiving increased scientific and public interest because of nutritional and neutraceutical health benefits associated with its consumption (Chu et al., 2013; Butt, Nadeem, Khan, Shabir, & Butt, 2008). Mostly oat is consumed as whole grain products like breakfast cereals, infant foods, porridge, and specialty breads (Tester & Karkalas, 1996). Besides it oat can be an important starch source which is also the major component of grain and could be usually found at levels between 60 and 65 g/100 g. But native starch has very narrow range of food applications due to their less stability to retrogradation, high gelatinization temperatures and high gel turbidity. Also to achieve the target of food as a neutraceutical there is need to modify the structural architecture of starch in order to develop appreciable quantity of resistant starch (RS). Different modifications of starch have been employed for improving the functionality and increase the resistance to enzymatic hydrolysis (Ashwar, Gani, Shah, Wani, & Masoodi, 2015). Acetylation is one such chemical modification in which some of the hydroxyl groups of starch chain are replaced by acetyl group, thereby altering its molecular structure and hence its properties. The maximum limit of acetyl content for food starches permitted by the FDA is 2.5 g/ 100 g. In India the demand of modified starch is growing day by day
* Corresponding author. E-mail address: [email protected] (F.A. Masoodi). http://dx.doi.org/10.1016/j.lwt.2017.01.072 0023-6438/© 2017 Published by Elsevier Ltd.
and is approximately 27,500 tons per year with 8e9 percent of growth rate per annum (Anon, 2006, pp. 1e17). Therefore modified oat starch could be a better alternative to meet the demands of food industries which has been otherwise ignored. So far very little work reporting properties of acetylated oat starch are available (Mirmoghtadaie, Kadivar, & Shahedi, 2009; Galdeano, Mali, Grossmann, Yamashita, & Garcia, 2009; Berski et al., 2011). The present work was undertaken to investigate the effect of acetylation on the RS4 (resistant starch type 4) content inclusive of physicochemical, morphological, structural and rheological properties of oat starch from three varieties. This comprehensive information on the properties of acetylated oat starch can be helpful to expand its use as an ingredient in food industry which otherwise utilizes primarily potato, corn, wheat, and rice starches as raw material. 2. Materials and methods 2.1. Materials Three oat varieties were procured form Sher-e-Kashmir University of Agricultural Sciences and Technology (SKAUST), Shalimar, Srinagar, J&K, India. Amyloglucosidase, pancreatin, and sodium acetate buffer (3 mol/L) were obtained from Sigma- Aldrich, St. Louis, USA. GOPOD Assay Kit was procured from Megazyme International Ldt, Bray, Ireland. All the other chemicals used were of analytical grade and purchased from HIMEDIA, Mumbai- India.
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2.2. Starch extraction Starch was isolated according to the method described by Shah, Masoodi, Gani, and Ashwar (2016). Briefly aqueous oat flour slurry (flour: water, 1:10) was maintained at pH 9, mixed intermittently for 1 h and filtered through muslin cloth. This was followed by centrifugation of filtrate at 3000 g for 15 min and then scraping off the upper layer containing impurities while as lower starch layer was washed three times with distilled water and then dried at 40 C. 2.3. Acetylation of oat starchwith acetic acid Oat starches were acetylated according to the method described by Otto, Baik, and Czuchajowska (1997) with some modifications. Starch dispersion (100 g/500 mL of H2O) was stirred for 20 min on magnetic stirrer. The pH of starch slurry was adjusted to pH 8.0 (1 mol/L NaOH) followed by addition of 7.65 g of acetic anhydride drop wise with continuous stirring while maintaining the pH between 8.0 and 8.5. The reaction was allowed to proceed for 5 min and then stopped by adjusting the pH to 4.5 with HCl (0.5 mol/L). The final suspension was centrifuged for 3 min at 1000 g and the recovered starch was washed three times with distilled water, then dried at 30 C. 2.4. Determination of acetyl percentage and degree of substitution Percent of acetyl groups (Ac) and degree of substitution (DS) were determined by the method of Wurburg (1964). Starch (1.0 g) was dispersed in a 250 mL flask containing 50 mL of ethanol (75 mL/L). The flask was covered properly with aluminum foil before heating at 50 C for 30 min in a water bath. After cooling the suspension, potassium hydroxide (KOH) (40 mL, 0.5 mol/L) was added and titrated out with HCl (0.5 mol equi/L). The solution was allowed to stand for 2 h, and again titrated to remove any additional alkali, which may have leached from the sample. Native starch sample was used as a blank.
Relative percent crystallinityðRCÞ ¼ ½Ac =ðAc þ Aa Þ 100; where Ac is the crystalline area; Aa is the amorphous area on the Xray diffractograms. 2.7. Scanning electron microscopy The fine dried powder of starch samples were placed on an adhesive tape attached to an aluminum stub and coated vertically with gold-palladium. The samples were examined by scanning electron microscope (Hitachi S-300H-Tokyo, Japan) to study its morphological characteristics. 2.8. Resistant starch content Resistant starch (RS) content was determined using glucose oxidase/peroxidase (GOPOD) assay Kit (Megazyme International Ldt, Bray, Ireland), by AACC method (2000). 2.9. Rheological measurement The flow properties of starch suspension were measured using rotational rheometer (MCR 102, Anton Paar) according to the method mentioned in Shah, Masoodi, Gani, and Ashwar (2016). The flow behavior index, yield stress and consistency coefficient were determined from the flow curve of shear rate (r) versus shear stress (t) using Herschel-Bulkley model
t ¼ t0 þ Kgn t (Pa) is the shear stress, t0 (Pa) is the yield stress, K is the consistency index (Pa sn), and n is the flow behavior index. To determine the zone of linear viscoelasticity, amplitude sweep test at constant frequency of 1 Hz and shear stress ranging between 0.01e100 Pa was carried for each starch gel separately. After choosing the appropriate shear stress (1 Pa) the frequency sweep test using a frequency range of 0.1e100 Hz.
Ac ðg=100gÞ ¼ ½ðVB VS Þ Molarity of HCl 0:043 100 =Weight of sample VB and VS represent titration volume of blank and sample expressed in mL, and sample weight was expressed in grams. Degree of substitution (DS) is defined as the average number of sites per glucose unit that possesses asubstituent group (Whistler & Daniel, 1995).
DS ¼ 162 Ac=½4300 ð42 AcÞ: Where Ac is the acetyl group percent.
2.10. Water and oil absorption capacity 2.5 g starch on dry weight basis (db) was mixed with 20 mL distilled water or mustard oil and then stirred for 30 min at 25 C. The slurry was then centrifuged at 3000 g for 10 min and the supernatant was decanted. The gain in weight was expressed as water/oil absorption capacity (Gani et al., 2014). 2.11. Freeze thaw stability
The infrared spectra of starch samples were obtained using ATRFTIR spectrophotometer (CARY 630, Agilent Technologies, USA) within the range of 600e4000 cm1 at a resolution of 4 cm1.
Aqueous starch suspension (6 g/100 g) was heated in a water bath at a temperature of 90 C, for 30 min. To measure freeze thaw stability, the gels were frozen at 16 C for 24 h, thawed at 25 C for 6 h and then refrozen at 16 C. Five cycles of freeze thaw were performed. The tubes were centrifuged at 1000 g for 20 min at 10 C and the released water was measured as freeze thaw stability (Shah et al., 2016).
2.6. XRD analysis
2.12. Statistical analysis
XRD patterns of starch samples were recorded with a wavelength of 0.154 nm using X-ray diffractometer (X'Pert PRO, Panalytical, Netherlands). The relative percent crystallinity (RC) of starch was calculated as:
Mean values, standard deviation, two-way analysis of variance (ANOVA) were computed using a commercial statistical package SPSS (IBM statistics 22). These data were then compared using Duncan's multiple range tests at 5% significance level.
2.5. ATR-fourier transforms infrared (FTIR) spectroscopy
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3. Results and discussion 3.1. Acetyl group percent (Ac) and degree of substitution (DS) Oat starches from three different varieties had 10.03e10.99 g/ 100 g moisture, 0.32e0.34 g/100 g protein, 0.25e0.44 g/100 g lipid and 0.42e0.57 g/100 g ash content (data already reported Shah et al., 2016). Acetyl group percent and DS values of oat starches ranged from 0.645 to 1.505 and 0.024e0.057, respectively as shown in Table 1. The values of Ac and DS were significantly (p < 0.05) found higher in Sabzaar than SKO20 and SKO90. However, Berski et al., 2011 reported higher acetyl percentage and degree of substitution in acetylated oat starches using 10 g of acetic anhydride. This difference in percent acetylation and degree of substitution arise due to variation in the factors like amylose content, difference in size, packing and fragility of starch, and reaction conditions
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(Sodhi & Singh, 2005). Also oat starch granules are smaller in size and compactly arranged which restricts access of acetic anhydride therefore low DS (Mirmoghtadaie et al., 2009). Starches with DS between 0.01 and 0.2 are classified as low DS and are used in food industry as thickener, binder, film former, gelling and texturing agent, water binder and encapsulating agent and has numerous other applications both in food and non-food areas (Zhang, Wang, Wang, & Dong, 2014). Acetylation of starch takes place by an additioneelimination mechanism. Hydroxyl groups particularly C2, C3, and C6 of starch shows different reactivity. Primary hydroxyl group (eOH) on C6 is more reactive and is acetylated more readily than eOH groups on C2 and C3 due to steric hindrance. However, of two secondary OH groups, C2 is more reactive than C3 mainly because the former is closer to hemi-acetal and more acidic than the latter (Xu, Miladinov, & Hanna, 2004). 3.2. ATR-fourier transform infrared (FT-IR) spectra
Table 1 Acetyl percentage, and degree of substitution of oat starches (n ¼ 3).
Acetyl percentage (g/100 g) Degree of substitution (DS)
Sabzaar
SKO20
SKO90
1.50 ± 0.01b 0.05 ± 0.001c
1.50 ± 0.01b 0.05 ± 0.000b
0.64 ± 0.05a 0.02 ± 0.000a
Results are mean ± S.D. Means in the row within a particular parameter with different superscript are significantly different at (P < 0.05).
The FTIR spectra of oat starches displayed absorptions peaks in the range of 3400e3465 cm1, which is assigned to stretchingabsorption bands of hydrophilic hydroxyl groups (eOH). An intense peak at 2929 cm1 is related to stretching-vibrations of eCH2 functional group. Absorption peak at 1500 and 1600 cm1 corresponds to CeOeC and eCOO functional groups (Gani et al., 2017). The appearance of strong absorption peaks in the region of
Fig. 1. ATR-FTIR spectra of representative native (A), acetylated Sabzaar (B), acetylated SKO20(C), and acetylated SKO90 (D) of oat starches. R 1047/1022 cm1; spectral ratio representing the crystalline to amorphous phase.
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1737 cm1 (carbonyl C]O stretching of acetyl group), 1337 cm1 (CeH symmetry deformation vibration) and 1243 cm1 (CeO stretching of acetyl group) confirms the presence and extent of acetyl groups in the starch molecule. Similar observation was reported by Colussi et al. (2015). FTIR spectra of native and acetylated oat starches are represented in Fig. 1. Moreover, band at 1047 cm1 reflects the crystalline regions of starch while as absorption peak at 1022 cm1 is characteristic to an amorphous domain (Gani et al., 2016). So their ratio of intensity, 1047 cm1/1022 cm1 can be used to express the molecular order in starches. The ratio of intensity of native oat starches showed higher values then acetylated counterparts. This decrease can be ascribed due to acetyl group substitution which altered packing and generated more amorphous regions. 3.3. XRD analysis In native and acetylated oat starches peaks were observed at 15 and 23 , doublet peak at 17 and 18 , and small peak at 20 of 2q (Fig. 2). These peaks are characteristics of A-type X-ray pattern, which are more densely packed, stable monoclinic structure characteristics of cereal starches. Acetylation did not alter the crystalline pattern however reduction in crystallinity and peak intensities at 15 , 17, 18 , and 23 , except 20 was observed. Peak at 20 reflects formation of amyloseelipid complex in starches, and its intensity was found higher in acetylated oat starches. Acetylated SKO20 oat starch displayed higher peak intensity (1486) at 20 indicating higher amount of amyloseelipid complex in this starch followed by SKO90 (1399) and sabzaar (1249). Generally, a typical amyloseelipid complex increases the resistance of starches against the digestive enzyme attack (Dupuis, Liu, & Yada, 2014). Also decrease in crystallinity on acetylation is due to substitutions of hydroxyl groups by bulky acetyl moieties in starch chains that reduce the formation of junction zones between starch molecules due to steric hinderances and results in limited destruction of the ordered crystalline structure (Zhang, Xie, Zhao, Liu, & Gao, 2009). 3.4. Scanning electron microscopyand light microscopy Scanning electron microscopy revealed significant differences in external morphology of native and acetylated starches (Fig. 3). Acetylated oat starches still showed the presence of smaller, intact, oval or irregular shaped starch granules with varying dimensions,
Fig. 2. XRD pattern of representative native (A), acetylated Sabzaar (B), acetylated SKO20 (C), and acetylated SKO90 (D) oat starches. RC ¼ Percent relative crystallinity.
but their smooth surfaces turned rough with prominent presence of small pores. Length range, width range and mean granule width of native and acetylated starch were 5.85e9.13 mm, 3.0e7.5 mm, and 1.5e9.7 mm, respectively (Table 2). Ashwar et al. (2016) and Bartz et al. (2015) also observed similar change in the morphology of rice and barnyardgrass starch at low DS. 3.5. Resistant starch content Resistant starch (RS4) content of acetylated oat starches varied significantly in the range of 35.81e48.88 g/100 g with highest in Sabzaar and lowest in SKO90 (Table 2). Acetylated oat starches showed significantly (p < 0.05) higher RS contents as compared to their native starches. RS4 is a type of starch that is resistant to digestive enzymes because of chemical modification. Higher RS4 content in acetylated starches can be due to following reasons: (A) Free hydroxyl groups (eOH) particularly on C2, C3, and C6 are substituted by acetyl groups. These acetate ester groups produce steric hindrances, which restricts digestive enzymes to bind properly to starch, thereby restricting their hydrolytic action, (B) Acetylation results in higher amount of amyloseelipid complex which is resistant to digestive enzyme breakdown (Czuchajowska, Sievert, & Pomeranz, 1991). Results were in consistent with XRD analysis that revealed higher amyloseelipid complex (higher peak intensity at 20 ) in acetylated starches compared to their native counterparts. Similar increase in RS content by acetylation were reported by Simsek, Ovando-Martinez, Whitney, and Bello-Perez (2012) on bean starch and Chung, Shin, and Lim (2008) on normal corn starch. 3.6. Rheological measurement The flow behaviors of native and acetylated oat starch suspensions at a concentration of 6 g/100 g, displaying changes in viscosity with increase in applied shear stress compared to native, are shown in Fig. 4 (a, b, & c). In acetylated starches the shear stress appeared to be proportional to the shear rate for Sabzaar whereas a non linear curve was observed for SKO20, SKO90 and all native ones. This implies that all the samples exhibited shear-thinning behavior as reflected from the convex shaped graph with viscosities of acetylated starches following the order; SKO90 > SKO20 > Sabzaar. Native starches showed higher shear stress than that of acetylated starches indicating their higher resistance to structure breaking at same shear rate. Acetylation resulted in a weak gel structure with higher ability to flow. Effect was seen higher in sabzaar starch, followed by SKO20 and least in SKO90. This implies that starch with highest DS has lowest values in shear stress. Similar effect of acetylation on viscosity was earlier seen in banana and corn starches (Rivera et al., 2013). Moreover, shear thinning behavior can be fitted in a Herschel-Bulkely model and results of these parameters are displayed in Table 2. Flow behavior index (n) of acetylated starches was found higher (0.24e0.74) than native ones (0.42e0.50) but lesser than unity further confirming that oat starch solutions exhibited shear-thinning behaviors. Shear-thinning behavior is ascribed due to the slower rate of polymer gel reformation as the shear rate is increased (Salamone, 1996). Acetylation reduces interchain association due to acetyl group substitution and disorganizes the structure by weakening of intra- and intermolecular hydrogen bonding in the starch network resulting in highly shear thinning fluids (Liu, Ramsden, & Corke, 1999). Dynamic stress sweep test at constant frequency was carried to measure the linear viscoelastic limit (LVE) of oat starch gels (Fig. 5A). The G0 reflects the elastic behavior of gels resulting from the formation of three dimensional gel network via junction zones and G00 represents the viscous nature. At low shear rate, inall oat starch gels G0 was observed higher than G00 upto a certain place of
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Fig. 3. SEM micrographs of representative native (A) acetylated Sabzaar (B), acetylated SKO20 (C), and acetylated SKO90 (D) oat starches.
Table 2 Morphological properties, resistant starch content, rheological parameters of oat starches (n ¼ 3). Sabzaar Native Morphological properties 6.26 ± 0.20b Mean granule length (mm) Mean granule width (mm) 3.2 ± 0.07ab Width range (mm) 2e3.8ab Resistant starch (g/100g) 23.9 ± 1.27b Herschel-Bulkley model fitted parameters Yield stress [to] [Pa]a 12.02 Flow behavior index [ na] 0.42 a Consistency index, K 5.65
SKO20
SKO90
Modified
Native
Modified
Native
Modified
7.2 ± 0.21d 6.5 ± 0.07d 1.5e3.8ab 48.88 ± 0.29e
5.85 ± 0.07 a 3.0 ± 0.20a 2e3 a 17.39 ± 0.57a
9.13 ± 0.17 e 3.6 ± 0.21c 5.2e7.8 d 39.14 ± 0.61d
6.9 ± 0.07 c 7.5 ± 0.11e 5e9.7 e 17.14 ± 0.06a
7.5 ± 0.17 d 6.5 ± 0.03 d 3e8.7 c 35.81 ± 1.04c
0.24 0.63 0.24
0.001 0.502 0.758
0.457 0.741 0.06
1.615 0.43 3.36
7.42 0.47 2.20
Results are mean ± S.D. Means in the rows within a particular parameter with different superscript are significantly different at (P < 0.05). a Parameters obtained by fitting the curve by the Herschel-Bulkley equation. to ¼ yield stress; Ka ¼ consistency index; na ¼ flow behavior index.
intersection called flow point. Above this point and with further increase in shear rate G00 becomes higher than G0 , representing a typical gel behavior. The acetylated oat gels showed lower values for G0 and G00 compared to their native ones. Tan (d) or loss factor (reflecting the ratio of G00 /G0 ) values of acetylated and native pastes were within the range of 0.09e0.12 and 0.07e0.11, respectively.
Lower the values of Tan (d), more is the gel like behavior (Lertphanich et al., 2013). Frequency dependence of G00 and G0 was measured in the LVR within the frequency range of 0.1e100 Hz (Fig. 5B). In both native and acetylated starches, G0 was greater than G00 throughout the frequency range which implies that under small deformation they behave as strong gels but as strain is increased
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Fig. 4. Flow curves of native ( ) and acetylated ( ) starch dispersions fitted with Herschel-Bulkley model (dashed lines); (a) sabzaar, (b) SKO20 and (c) SKO90.
their network structure breakdowns. However native oat starches produce gels that were stiffer than their acetylated samples. This may be due to a weaker granular integrity caused by increased uptake of water in the swollen granules of acetylated pastes (Lee & Yoo, 2009). 3.7. Water and oil absorption capacity Water and oil absorption capacity of native and acetylated oat starches are shown in Table 3. Water absorption capacity was in the range of 0.77e0.86 g g1 for native oat starches that varied significantly (P < 0.05). Water absorption capacity relies upon the molecular structure, crystalline and amorphous regions within the starch and distribution of granular size (Gani, Masoodi, Wani, & Salim, 2013). The oil absorption capacity involves physical entrapment of oil by capillary action (Kinsella, 1976). Oil absorption
capacity of native oat starches was in the range of 0.29e0.34 g g1. Water absorption capacity of acetylated oat starches was found in the range of 1.33e1.54 g g1 and oil absorption capacity in the range of 0.57e0.87 g g1, significantly (p 0.05) higher than their respective native starches. The introduction of acetyl groups can elevate interactions between water and the starch molecules thereby decreasing the intermolecular hydrogen bonding in starch which facilitates access of more water/oil to the amorphous regions (Ayucitra, 2012; Betancur, Chel, & Canizares, 1997). Similar studies, using rice starch (Das, Singh, Singh, & Riar, 2010), and horse chestnut starch (Shubeena et al., 2015), the authors also affirmed an increase in water and oil absorption of starch by acetylation. 3.8. Freeze thaw stability Freezeethaw stability is the ability of starch gels to resist
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Fig. 5. Oscillatory (d) and mechanical spectra (e) measured as function of storage modulus (G0 ) and loss modulus (G00 ); native sabzaar ( 1G0 4G00 ); native SKO20 ( 2G0 2G00 ) & acetylated SKO20 ( 5G0 5G00 ); native SKO90 ( 3G0 3G00 ) & acetylated SKO90 ( 6G0 6G00 ).
1G00 ) & acetylated sabzaar ( 4G0
Table 3 Physicochemical properties of oat starches (n ¼ 3). Sabzaar
Water absorption capacity (g/g) Oil absorption capacity (g/g) Freeze thaw stability (g/100g) 0 thaw 1 thaw 2 thaw 3 thaw 4 thaw 5 thaw
SKO20
SKO90
Native
Modified
Native
Modified
Native
Modified
0.86 ± 0.0b 0.34 ± 0.02b
1.54 ± 0.03e 0.87 ± 0.01e
0.78 ± 0.01a 0.32 ± 0.01ab
1.40 ± 0.07d 0.64 ± 0.02d
0.77 ± 0.01a 0.29 ± 0.02a
1.33 ± 0.01c 0.57 ± 0.01c
22.80 23.93 27.15 30.93 34.56 36.88
± ± ± ± ± ±
0.63cbp 1.5bp 0.72bq 0.86br 0.72bs 0.78bs
19.41 21.74 25.26 26.82 30.59 31.45
± ± ± ± ± ±
0.50ap 0.35aq 0.11ar 0.33as 0.33at 0.13au
25.41 28.92 32.41 34.96 39.21 45.41
± ± ± ± ± ±
0.50dp 1.04cq 0.50cr 0.22cs 0.66ct 1.12cv
20.80 22.08 26.08 32.58 32.96 41.55
± ± ± ± ± ±
0.69bp 0.31aq 0.42abr 0.60cs 1.51bs 0.67ct
25.24 27.64 30.91 35.02 38.89 45.85
± ± ± ± ± ±
0.63ep 1.16cq 0.72cr 0.62cs 0.99ct 0.94cu
22.37 24.18 28.92 32.51 34.29 42.21
± ± ± ± ± ±
0.41cp 0.27bq 0.95cr 1.17cs 0.44bt 0.66cu
Results are mean ± S.D. Means in the rows within a particular parameter with different superscript are significantly different at (P < 0.05).
repeated freezing and thawing cycles without any undesirable physical changes. It is directly related to the tendency of starch to retrogradation and is an important parameter to be taken into consideration in the formulation of starch rich food products. Freeze thaw stability of native and acetylated oat starch gels is presented in Table 3. There was a significant (p < 0.05) decrease in freeze thaw stability from 0 h (22.80e25.41 g/100 g) to 120 h (36.88e45.85 g/100 g) of storage in native starches. Increase in syneresis during the frozen storage would be the result of increased aggregation of the starch chains via hydrogen bonds, causing increase in exudation of water from gels. Furthermore, syneresis of acetylated starch gels during the frozen storage was significantly (p 0.05) lower than the native starch. This may be attributed to the fact that steric hindrances produced by the acetyl groups restricts the re-association of amylose, resulting in enhanced water retention capacity of the starch molecules during storage. This property of the acetylated starch has also been reported by many other authors (Mbougueng, Tenin, Scher, & Tchiegang, 2012; Saartrat, Puttanlek, Rungsardthong, & Uttapap, 2005; Simsek et al., 2012). 4. Conclusion Introduction of acetyl groups in starch chains significantly
improved its physicochemical properties like water/oil absorption capacity and freeze thaw stability. Rheological behaviors of oat starches were found to vary with type of variety and degree of substitution. Shear-thinning depicts that it can be operated at lower energy andcan find applications in many ready to eat food products. Also by varying acetyl content it is possible to produce starches with specific flow properties. Furthermore, the resistance of acetylated starches to digestion suggests its function as prebiotic, thus improving the gut health. Acknowledgements Authors are thankful to the Department of Biotechnology (Grant number: D.O.BT/PR6701/FNS/20/674/2012), Government of India for their financial support. References AACC. (2000). Approved methods of the AACC International, methods (10th ed., pp. 32e40). St. Paul, MN: The Association AACC. Anon. (2006). Starch production in India. Modified starch.com. Availablefrom www. tidco.com. Ashwar, B. A., Gani, A., Shah, A., Wani, I. A., & Masoodi, F. A. (2015). Preparation, €rke, 67, health benefits, and applications of resistant starch- a review. Starch/Sta 1e15. Ashwar, B. A., Gani, A., Shah, A., & Masoodi, F. A. (2016). Production of RS4 from rice
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by acetylation: Physicochemical, thermal, and structural characterization. Starch/Starke, 68, 1e10. Ayucitra, A. (2012). Preparation and characterisation of acetylated corn starches. International Journal of Chemical Engineering and Applications, 3, 156e159. Bartz, J., Goebel, J. T., Giovanaz, M. A., Zavareze, E. R., Schirmer, M. A., & Dias, A. R. G. (2015). Acetylation of barnyardgrass starch with acetic anhydride under iodine catalysis. Food Chemistry, 178, 236e242. Berski, W., Ptaszek, A., Ptaszek, P., Ziobro, R., Kowalski, G., Grzesik, M., et al. (2011). .Pasting and rheological properties of oat starch and its derivatives. Carbohydrate Polymers, 83, 665e671. Betancur, A. D., Chel, G. L., & Canizares, H. E. (1997). Acetylation and characterization of Canavalia ensiformis starch. Journal of Agricultural and FoodChemistry, 45, 378e382. Butt, M. S., Nadeem, M. T., Khan, M. K. I., Shabir, R., & Butt, M. S. (2008). Oat: Unique among the cereals. European Journal of Nutrition, 47, 68e79. Chu, Y. F., Wise, M. L., Gulvady, A. A., Chang, T., Kendra, D. F., Klinken, B. J. W. V., et al. (2013). In vitro antioxidant capacity and anti-inflammatory activity of seven common oats. Food Chemistry, 139, 426e431. Chung, H., Shin, D., & Lim, S. (2008). In vitro starch digestibility and estimated glycemic index of chemically modified corn starches. Food Research International, 41, 579e585. Colussi, R., Halal, S. L. M. El, Pinto, V. Z., Bartz, J., Gutkoski, L. C., Zavareze, E. D. R., et al. (2015). Acetylation of rice starch in an aqueous medium for use in food. LWT - Food Science and Technology, 62, 1076e1082. Czuchajowska, Z., Sievert, D., & Pomeranz, Y. (1991). Enzyme-resistant starch. IV. Effects of complexing lipids. Cereal Chemistry, 68, 537e542. Das, A. B., Singh, G., Singh, S., & Riar, C. S. (2010). Effect of acetylation and dual modification on physico-chemical, rheological and morphological characteristics of sweet potato (Ipomoea batatas) starch. Carbohydrate Polymers, 80, 725e732. Dupuis, J. H., Liu, Q., & Yada, R. Y. (2014). Methodologies for increasing the resistant starch content of food starches: A review. Comprehensive Reviews in Food Science and Food Safety, 13, 1219e1234. Galdeano, M. C., Mali, S., Grossmann, M. V. E., Yamashita, F., & Garcia, M. A. (2009). Effects of plasticizers on the properties of oat starch films. Materials Science and Engineering C, 29, 532e538. Gani, A., Masoodi, F. A., Wani, S. M., & Salim, R. (2013). Characterization of rice starches extracted from Indian cultivars. Food Science and Technology International, 19, 143e152. Gani, A., Nazia, S., Rather, S. A., Wani, S. M., Shah, A., Bashir, M., et al. (2014). Effect of g-irradiation on granule structure and physicochemical properties of starch extracted from two types of potatoes grown in Jammu & Kashmir, India. LWT Food Science and Technology, 58, 239e246. Gani, A., Jan, A., Shah, A., Masoodi, F. A., Ahmad, M., Ashwar, B. A., et al. (2016). Physico-chemical, functional and structural properties of RS3/RS4 from kidney bean (Phaseolus vulgaris) cultivars. International Journal Biological Macromolecule, 87, 514e521. Gani, A., Ashwar, B. A., Akhter, G., Shah, A., Wani, I. A., & Masoodi, F. A. (2017). Physico-chemical, structural, pasting and thermal properties of starches of fourteen Himalayan rice cultivars. International Journal of Biological Macromolecules, 95, 1101e1107. Kinsella, J. E. (1976). Functional properties of proteins in foods: A survey. Critical Reviews in Food Science and Nutrition, 7, 219e280. Lertphanich, S., Wansuksri, R., Thierry, T., Da, G., Nga, L. H., Dufour, D., Piyachomkwan, K., & Sriroth, K. (2013). Comparative study on physicochemical
properties of ensete and water caltrop with other root, tuber, and legume €rke, 65, 1e13. starches. Starch/Sta Lee, H. L., & Yoo, B. (2009). Dynamic rheological and thermal properties of acetylated sweet potato starch. Starch/Starke, 61, 407e413. Liu, H., Ramsden, L., & Corke, H. (1999). Physical properties of crosslinked and acetylated normal and waxy rice starch. Starch/Starke, 51, 249e252. Mbougueng, P. D., Tenin, D., Scher, J., & Tchiegang, C. (2012). Influence of acetylation on physicochemical, functional and thermal properties of potato and cassava starches. Journal of Food Engineering, 108, 320e326. Mirmoghtadaie, L., Kadivar, M., & Shahedi, M. (2009). Effects of cross-linking and acetylation on oat starch properties. Food Chemistry, 116, 709e713. Otto, T., Baik, B. K., & Czuchajowska, Z. (1997). Wet fractionation of garbanzo bean and pea flours. Cereal Chemistry, 74, 141e146. Rivera, M. M. S., Benitez, S. A., Perez, L. A. B., Montealvo, G. M., Santiago, M. C. N., Ambriz, S. L. R., et al. (2013). Acetylation of banana (Musa paradisiacaL.) and corn (Zea mays L.) starches using a microwave heating procedure and iodine as catalyst: II. Rheological and structural studies. Carbohydrate Polymers, 92, 1256e1261. Saartrat, S., Puttanlek, C., Rungsardthong, V., & Uttapap, D. (2005). Paste and gelproperties of low-substituted acetylated canna starches. Carbohydrate Polymers, 61, 211e221. Salamone, J. C. (1996). Polymeric materials encyclopedia. Boca Raton, FL: CRC Press. Shah, A., Masoodi, F. A., Gani, A., & Ashwar, B. A. (2016a). In-vitro digestibility, rheology, structure, and functionality of RS3 from oat starch. Food Chemistry, 212, 749e758. Shah, A., Masoodi, F. A., Gani, A., & Ashwar, B. A. (2016b). Newly released oat varieties of himalayan region-Techno-functional, rheological, and nutraceutical properties of flour. LWT - Food Science and Technology, 70, 111e118. Shah, U., Gani, A., Ashwar, B. A., Shah, A., Wani, I. A., & Masoodi, F. A. (2016c). Effect of infrared and microwave radiations on properties of Indian Horse Chestnut starch. International Journal of Biological Macromolecules, 84, 166e173. Shubeena, Wani, I. D., Gani, A., Sharma, P., Wani, T. A., Masoodi, F. A., et al. (2015). Effect of acetylation on the physico-chemical properties of indian horse chestnut (Aesculus indica L. Starch/Starke, 67, 311e318. Simsek, S., Ovando-Martinez, M., Whitney, K., & Bello-Perez, L. A. (2012). Effect of acetylation, oxidation and annealing on physicochemical properties of bean starch. Food Chemistry, 134, 1796e1803. Sodhi, N. S., & Singh, N. (2005). Characteristics of acetylated starches prepared using starches separated from different rice cultivars. Journal of Food Engineering, 70, 117e127. Tester, R. F., & Karkalas, J. (1996). Swelling and gelatinization of oat starches. Cereal Chemistry, 73, 271e277. Whistler, R. L., & Daniel, J. R. (1995). Carbohydrates. In O. R. Fennema (Ed.), Food chemistry (pp. 69e137). New York: Marcel Decker. Wurburg, O. B. (1964). Acetylation. In R. L. Whistler, R. J. Smith, & M. L. Wolfram (Eds.), Methods in carbohydrate chemistry (pp. 240e241). New York, IV: Academic press. Xu, Y., Miladinov, V., & Hanna, M. A. (2004). Synthesis and characterization of starch acetates with high substitution. Cereal Chemistry, 81, 735e740. Zhang, L., Xie, W., Zhao, X., Liu, Y., & Gao, W. (2009). Study on the morphology, crystalline structure and thermal properties of yellow ginger starch acetates with different degrees of substitution. Thermochimica Acta, 495, 57e62. Zhang, H., Wang, R., Wang, J., & Dong, Y. (2014). Microwave-assisted synthesis and characterization of acetylated corn starch. Starch/Starke, 66, 515e523.
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Physicochemical, rheological and structural characterization of acetylated oat starches Asima Shah, F.A. Masoodi*, Adil Gani, Bilal Ahmad Ashwar Department of Food Science and Technology, University of Kashmir, Srinagar, 190006, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2016 Received in revised form 26 January 2017 Accepted 28 January 2017 Available online 30 January 2017
Starches isolated from three different varieties of oat were modified with acetic anhydride to make starches with high contents of type 4 resistant starch (RS4). The acetyl group percentage (Ac) and degree of substitution values (DS) of oat starches ranged from 0.64 to 1.50 and 0.02e0.05, respectively. The absorption peaks at 1737 cm1, 1337 cm1 and 1243 cm1in the FTIR spectrum confirms the presence of acetyl groups in the starch molecule. SEM micrographs revealed significant changes in external morphology of acetylated starches due to the formation of small pores. XRD analysis reflects the formation of amyloseelipid complex with decreased crystallinity in acetylated starches. The viscosity of starches decreased on acetylation, exhibited shear-thinning behavior as reflected from the convex shaped graph with viscosities of acetylated starches following the order; SKO90 > SKO20 > Sabzaar. © 2017 Published by Elsevier Ltd.
Keywords: RS4 Acetylation Oat starch
1. Introduction Oat is receiving increased scientific and public interest because of nutritional and neutraceutical health benefits associated with its consumption (Chu et al., 2013; Butt, Nadeem, Khan, Shabir, & Butt, 2008). Mostly oat is consumed as whole grain products like breakfast cereals, infant foods, porridge, and specialty breads (Tester & Karkalas, 1996). Besides it oat can be an important starch source which is also the major component of grain and could be usually found at levels between 60 and 65 g/100 g. But native starch has very narrow range of food applications due to their less stability to retrogradation, high gelatinization temperatures and high gel turbidity. Also to achieve the target of food as a neutraceutical there is need to modify the structural architecture of starch in order to develop appreciable quantity of resistant starch (RS). Different modifications of starch have been employed for improving the functionality and increase the resistance to enzymatic hydrolysis (Ashwar, Gani, Shah, Wani, & Masoodi, 2015). Acetylation is one such chemical modification in which some of the hydroxyl groups of starch chain are replaced by acetyl group, thereby altering its molecular structure and hence its properties. The maximum limit of acetyl content for food starches permitted by the FDA is 2.5 g/ 100 g. In India the demand of modified starch is growing day by day
* Corresponding author. E-mail address: [email protected] (F.A. Masoodi). http://dx.doi.org/10.1016/j.lwt.2017.01.072 0023-6438/© 2017 Published by Elsevier Ltd.
and is approximately 27,500 tons per year with 8e9 percent of growth rate per annum (Anon, 2006, pp. 1e17). Therefore modified oat starch could be a better alternative to meet the demands of food industries which has been otherwise ignored. So far very little work reporting properties of acetylated oat starch are available (Mirmoghtadaie, Kadivar, & Shahedi, 2009; Galdeano, Mali, Grossmann, Yamashita, & Garcia, 2009; Berski et al., 2011). The present work was undertaken to investigate the effect of acetylation on the RS4 (resistant starch type 4) content inclusive of physicochemical, morphological, structural and rheological properties of oat starch from three varieties. This comprehensive information on the properties of acetylated oat starch can be helpful to expand its use as an ingredient in food industry which otherwise utilizes primarily potato, corn, wheat, and rice starches as raw material. 2. Materials and methods 2.1. Materials Three oat varieties were procured form Sher-e-Kashmir University of Agricultural Sciences and Technology (SKAUST), Shalimar, Srinagar, J&K, India. Amyloglucosidase, pancreatin, and sodium acetate buffer (3 mol/L) were obtained from Sigma- Aldrich, St. Louis, USA. GOPOD Assay Kit was procured from Megazyme International Ldt, Bray, Ireland. All the other chemicals used were of analytical grade and purchased from HIMEDIA, Mumbai- India.
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2.2. Starch extraction Starch was isolated according to the method described by Shah, Masoodi, Gani, and Ashwar (2016). Briefly aqueous oat flour slurry (flour: water, 1:10) was maintained at pH 9, mixed intermittently for 1 h and filtered through muslin cloth. This was followed by centrifugation of filtrate at 3000 g for 15 min and then scraping off the upper layer containing impurities while as lower starch layer was washed three times with distilled water and then dried at 40 C. 2.3. Acetylation of oat starchwith acetic acid Oat starches were acetylated according to the method described by Otto, Baik, and Czuchajowska (1997) with some modifications. Starch dispersion (100 g/500 mL of H2O) was stirred for 20 min on magnetic stirrer. The pH of starch slurry was adjusted to pH 8.0 (1 mol/L NaOH) followed by addition of 7.65 g of acetic anhydride drop wise with continuous stirring while maintaining the pH between 8.0 and 8.5. The reaction was allowed to proceed for 5 min and then stopped by adjusting the pH to 4.5 with HCl (0.5 mol/L). The final suspension was centrifuged for 3 min at 1000 g and the recovered starch was washed three times with distilled water, then dried at 30 C. 2.4. Determination of acetyl percentage and degree of substitution Percent of acetyl groups (Ac) and degree of substitution (DS) were determined by the method of Wurburg (1964). Starch (1.0 g) was dispersed in a 250 mL flask containing 50 mL of ethanol (75 mL/L). The flask was covered properly with aluminum foil before heating at 50 C for 30 min in a water bath. After cooling the suspension, potassium hydroxide (KOH) (40 mL, 0.5 mol/L) was added and titrated out with HCl (0.5 mol equi/L). The solution was allowed to stand for 2 h, and again titrated to remove any additional alkali, which may have leached from the sample. Native starch sample was used as a blank.
Relative percent crystallinityðRCÞ ¼ ½Ac =ðAc þ Aa Þ 100; where Ac is the crystalline area; Aa is the amorphous area on the Xray diffractograms. 2.7. Scanning electron microscopy The fine dried powder of starch samples were placed on an adhesive tape attached to an aluminum stub and coated vertically with gold-palladium. The samples were examined by scanning electron microscope (Hitachi S-300H-Tokyo, Japan) to study its morphological characteristics. 2.8. Resistant starch content Resistant starch (RS) content was determined using glucose oxidase/peroxidase (GOPOD) assay Kit (Megazyme International Ldt, Bray, Ireland), by AACC method (2000). 2.9. Rheological measurement The flow properties of starch suspension were measured using rotational rheometer (MCR 102, Anton Paar) according to the method mentioned in Shah, Masoodi, Gani, and Ashwar (2016). The flow behavior index, yield stress and consistency coefficient were determined from the flow curve of shear rate (r) versus shear stress (t) using Herschel-Bulkley model
t ¼ t0 þ Kgn t (Pa) is the shear stress, t0 (Pa) is the yield stress, K is the consistency index (Pa sn), and n is the flow behavior index. To determine the zone of linear viscoelasticity, amplitude sweep test at constant frequency of 1 Hz and shear stress ranging between 0.01e100 Pa was carried for each starch gel separately. After choosing the appropriate shear stress (1 Pa) the frequency sweep test using a frequency range of 0.1e100 Hz.
Ac ðg=100gÞ ¼ ½ðVB VS Þ Molarity of HCl 0:043 100 =Weight of sample VB and VS represent titration volume of blank and sample expressed in mL, and sample weight was expressed in grams. Degree of substitution (DS) is defined as the average number of sites per glucose unit that possesses asubstituent group (Whistler & Daniel, 1995).
DS ¼ 162 Ac=½4300 ð42 AcÞ: Where Ac is the acetyl group percent.
2.10. Water and oil absorption capacity 2.5 g starch on dry weight basis (db) was mixed with 20 mL distilled water or mustard oil and then stirred for 30 min at 25 C. The slurry was then centrifuged at 3000 g for 10 min and the supernatant was decanted. The gain in weight was expressed as water/oil absorption capacity (Gani et al., 2014). 2.11. Freeze thaw stability
The infrared spectra of starch samples were obtained using ATRFTIR spectrophotometer (CARY 630, Agilent Technologies, USA) within the range of 600e4000 cm1 at a resolution of 4 cm1.
Aqueous starch suspension (6 g/100 g) was heated in a water bath at a temperature of 90 C, for 30 min. To measure freeze thaw stability, the gels were frozen at 16 C for 24 h, thawed at 25 C for 6 h and then refrozen at 16 C. Five cycles of freeze thaw were performed. The tubes were centrifuged at 1000 g for 20 min at 10 C and the released water was measured as freeze thaw stability (Shah et al., 2016).
2.6. XRD analysis
2.12. Statistical analysis
XRD patterns of starch samples were recorded with a wavelength of 0.154 nm using X-ray diffractometer (X'Pert PRO, Panalytical, Netherlands). The relative percent crystallinity (RC) of starch was calculated as:
Mean values, standard deviation, two-way analysis of variance (ANOVA) were computed using a commercial statistical package SPSS (IBM statistics 22). These data were then compared using Duncan's multiple range tests at 5% significance level.
2.5. ATR-fourier transforms infrared (FTIR) spectroscopy
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3. Results and discussion 3.1. Acetyl group percent (Ac) and degree of substitution (DS) Oat starches from three different varieties had 10.03e10.99 g/ 100 g moisture, 0.32e0.34 g/100 g protein, 0.25e0.44 g/100 g lipid and 0.42e0.57 g/100 g ash content (data already reported Shah et al., 2016). Acetyl group percent and DS values of oat starches ranged from 0.645 to 1.505 and 0.024e0.057, respectively as shown in Table 1. The values of Ac and DS were significantly (p < 0.05) found higher in Sabzaar than SKO20 and SKO90. However, Berski et al., 2011 reported higher acetyl percentage and degree of substitution in acetylated oat starches using 10 g of acetic anhydride. This difference in percent acetylation and degree of substitution arise due to variation in the factors like amylose content, difference in size, packing and fragility of starch, and reaction conditions
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(Sodhi & Singh, 2005). Also oat starch granules are smaller in size and compactly arranged which restricts access of acetic anhydride therefore low DS (Mirmoghtadaie et al., 2009). Starches with DS between 0.01 and 0.2 are classified as low DS and are used in food industry as thickener, binder, film former, gelling and texturing agent, water binder and encapsulating agent and has numerous other applications both in food and non-food areas (Zhang, Wang, Wang, & Dong, 2014). Acetylation of starch takes place by an additioneelimination mechanism. Hydroxyl groups particularly C2, C3, and C6 of starch shows different reactivity. Primary hydroxyl group (eOH) on C6 is more reactive and is acetylated more readily than eOH groups on C2 and C3 due to steric hindrance. However, of two secondary OH groups, C2 is more reactive than C3 mainly because the former is closer to hemi-acetal and more acidic than the latter (Xu, Miladinov, & Hanna, 2004). 3.2. ATR-fourier transform infrared (FT-IR) spectra
Table 1 Acetyl percentage, and degree of substitution of oat starches (n ¼ 3).
Acetyl percentage (g/100 g) Degree of substitution (DS)
Sabzaar
SKO20
SKO90
1.50 ± 0.01b 0.05 ± 0.001c
1.50 ± 0.01b 0.05 ± 0.000b
0.64 ± 0.05a 0.02 ± 0.000a
Results are mean ± S.D. Means in the row within a particular parameter with different superscript are significantly different at (P < 0.05).
The FTIR spectra of oat starches displayed absorptions peaks in the range of 3400e3465 cm1, which is assigned to stretchingabsorption bands of hydrophilic hydroxyl groups (eOH). An intense peak at 2929 cm1 is related to stretching-vibrations of eCH2 functional group. Absorption peak at 1500 and 1600 cm1 corresponds to CeOeC and eCOO functional groups (Gani et al., 2017). The appearance of strong absorption peaks in the region of
Fig. 1. ATR-FTIR spectra of representative native (A), acetylated Sabzaar (B), acetylated SKO20(C), and acetylated SKO90 (D) of oat starches. R 1047/1022 cm1; spectral ratio representing the crystalline to amorphous phase.
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1737 cm1 (carbonyl C]O stretching of acetyl group), 1337 cm1 (CeH symmetry deformation vibration) and 1243 cm1 (CeO stretching of acetyl group) confirms the presence and extent of acetyl groups in the starch molecule. Similar observation was reported by Colussi et al. (2015). FTIR spectra of native and acetylated oat starches are represented in Fig. 1. Moreover, band at 1047 cm1 reflects the crystalline regions of starch while as absorption peak at 1022 cm1 is characteristic to an amorphous domain (Gani et al., 2016). So their ratio of intensity, 1047 cm1/1022 cm1 can be used to express the molecular order in starches. The ratio of intensity of native oat starches showed higher values then acetylated counterparts. This decrease can be ascribed due to acetyl group substitution which altered packing and generated more amorphous regions. 3.3. XRD analysis In native and acetylated oat starches peaks were observed at 15 and 23 , doublet peak at 17 and 18 , and small peak at 20 of 2q (Fig. 2). These peaks are characteristics of A-type X-ray pattern, which are more densely packed, stable monoclinic structure characteristics of cereal starches. Acetylation did not alter the crystalline pattern however reduction in crystallinity and peak intensities at 15 , 17, 18 , and 23 , except 20 was observed. Peak at 20 reflects formation of amyloseelipid complex in starches, and its intensity was found higher in acetylated oat starches. Acetylated SKO20 oat starch displayed higher peak intensity (1486) at 20 indicating higher amount of amyloseelipid complex in this starch followed by SKO90 (1399) and sabzaar (1249). Generally, a typical amyloseelipid complex increases the resistance of starches against the digestive enzyme attack (Dupuis, Liu, & Yada, 2014). Also decrease in crystallinity on acetylation is due to substitutions of hydroxyl groups by bulky acetyl moieties in starch chains that reduce the formation of junction zones between starch molecules due to steric hinderances and results in limited destruction of the ordered crystalline structure (Zhang, Xie, Zhao, Liu, & Gao, 2009). 3.4. Scanning electron microscopyand light microscopy Scanning electron microscopy revealed significant differences in external morphology of native and acetylated starches (Fig. 3). Acetylated oat starches still showed the presence of smaller, intact, oval or irregular shaped starch granules with varying dimensions,
Fig. 2. XRD pattern of representative native (A), acetylated Sabzaar (B), acetylated SKO20 (C), and acetylated SKO90 (D) oat starches. RC ¼ Percent relative crystallinity.
but their smooth surfaces turned rough with prominent presence of small pores. Length range, width range and mean granule width of native and acetylated starch were 5.85e9.13 mm, 3.0e7.5 mm, and 1.5e9.7 mm, respectively (Table 2). Ashwar et al. (2016) and Bartz et al. (2015) also observed similar change in the morphology of rice and barnyardgrass starch at low DS. 3.5. Resistant starch content Resistant starch (RS4) content of acetylated oat starches varied significantly in the range of 35.81e48.88 g/100 g with highest in Sabzaar and lowest in SKO90 (Table 2). Acetylated oat starches showed significantly (p < 0.05) higher RS contents as compared to their native starches. RS4 is a type of starch that is resistant to digestive enzymes because of chemical modification. Higher RS4 content in acetylated starches can be due to following reasons: (A) Free hydroxyl groups (eOH) particularly on C2, C3, and C6 are substituted by acetyl groups. These acetate ester groups produce steric hindrances, which restricts digestive enzymes to bind properly to starch, thereby restricting their hydrolytic action, (B) Acetylation results in higher amount of amyloseelipid complex which is resistant to digestive enzyme breakdown (Czuchajowska, Sievert, & Pomeranz, 1991). Results were in consistent with XRD analysis that revealed higher amyloseelipid complex (higher peak intensity at 20 ) in acetylated starches compared to their native counterparts. Similar increase in RS content by acetylation were reported by Simsek, Ovando-Martinez, Whitney, and Bello-Perez (2012) on bean starch and Chung, Shin, and Lim (2008) on normal corn starch. 3.6. Rheological measurement The flow behaviors of native and acetylated oat starch suspensions at a concentration of 6 g/100 g, displaying changes in viscosity with increase in applied shear stress compared to native, are shown in Fig. 4 (a, b, & c). In acetylated starches the shear stress appeared to be proportional to the shear rate for Sabzaar whereas a non linear curve was observed for SKO20, SKO90 and all native ones. This implies that all the samples exhibited shear-thinning behavior as reflected from the convex shaped graph with viscosities of acetylated starches following the order; SKO90 > SKO20 > Sabzaar. Native starches showed higher shear stress than that of acetylated starches indicating their higher resistance to structure breaking at same shear rate. Acetylation resulted in a weak gel structure with higher ability to flow. Effect was seen higher in sabzaar starch, followed by SKO20 and least in SKO90. This implies that starch with highest DS has lowest values in shear stress. Similar effect of acetylation on viscosity was earlier seen in banana and corn starches (Rivera et al., 2013). Moreover, shear thinning behavior can be fitted in a Herschel-Bulkely model and results of these parameters are displayed in Table 2. Flow behavior index (n) of acetylated starches was found higher (0.24e0.74) than native ones (0.42e0.50) but lesser than unity further confirming that oat starch solutions exhibited shear-thinning behaviors. Shear-thinning behavior is ascribed due to the slower rate of polymer gel reformation as the shear rate is increased (Salamone, 1996). Acetylation reduces interchain association due to acetyl group substitution and disorganizes the structure by weakening of intra- and intermolecular hydrogen bonding in the starch network resulting in highly shear thinning fluids (Liu, Ramsden, & Corke, 1999). Dynamic stress sweep test at constant frequency was carried to measure the linear viscoelastic limit (LVE) of oat starch gels (Fig. 5A). The G0 reflects the elastic behavior of gels resulting from the formation of three dimensional gel network via junction zones and G00 represents the viscous nature. At low shear rate, inall oat starch gels G0 was observed higher than G00 upto a certain place of
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Fig. 3. SEM micrographs of representative native (A) acetylated Sabzaar (B), acetylated SKO20 (C), and acetylated SKO90 (D) oat starches.
Table 2 Morphological properties, resistant starch content, rheological parameters of oat starches (n ¼ 3). Sabzaar Native Morphological properties 6.26 ± 0.20b Mean granule length (mm) Mean granule width (mm) 3.2 ± 0.07ab Width range (mm) 2e3.8ab Resistant starch (g/100g) 23.9 ± 1.27b Herschel-Bulkley model fitted parameters Yield stress [to] [Pa]a 12.02 Flow behavior index [ na] 0.42 a Consistency index, K 5.65
SKO20
SKO90
Modified
Native
Modified
Native
Modified
7.2 ± 0.21d 6.5 ± 0.07d 1.5e3.8ab 48.88 ± 0.29e
5.85 ± 0.07 a 3.0 ± 0.20a 2e3 a 17.39 ± 0.57a
9.13 ± 0.17 e 3.6 ± 0.21c 5.2e7.8 d 39.14 ± 0.61d
6.9 ± 0.07 c 7.5 ± 0.11e 5e9.7 e 17.14 ± 0.06a
7.5 ± 0.17 d 6.5 ± 0.03 d 3e8.7 c 35.81 ± 1.04c
0.24 0.63 0.24
0.001 0.502 0.758
0.457 0.741 0.06
1.615 0.43 3.36
7.42 0.47 2.20
Results are mean ± S.D. Means in the rows within a particular parameter with different superscript are significantly different at (P < 0.05). a Parameters obtained by fitting the curve by the Herschel-Bulkley equation. to ¼ yield stress; Ka ¼ consistency index; na ¼ flow behavior index.
intersection called flow point. Above this point and with further increase in shear rate G00 becomes higher than G0 , representing a typical gel behavior. The acetylated oat gels showed lower values for G0 and G00 compared to their native ones. Tan (d) or loss factor (reflecting the ratio of G00 /G0 ) values of acetylated and native pastes were within the range of 0.09e0.12 and 0.07e0.11, respectively.
Lower the values of Tan (d), more is the gel like behavior (Lertphanich et al., 2013). Frequency dependence of G00 and G0 was measured in the LVR within the frequency range of 0.1e100 Hz (Fig. 5B). In both native and acetylated starches, G0 was greater than G00 throughout the frequency range which implies that under small deformation they behave as strong gels but as strain is increased
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Fig. 4. Flow curves of native ( ) and acetylated ( ) starch dispersions fitted with Herschel-Bulkley model (dashed lines); (a) sabzaar, (b) SKO20 and (c) SKO90.
their network structure breakdowns. However native oat starches produce gels that were stiffer than their acetylated samples. This may be due to a weaker granular integrity caused by increased uptake of water in the swollen granules of acetylated pastes (Lee & Yoo, 2009). 3.7. Water and oil absorption capacity Water and oil absorption capacity of native and acetylated oat starches are shown in Table 3. Water absorption capacity was in the range of 0.77e0.86 g g1 for native oat starches that varied significantly (P < 0.05). Water absorption capacity relies upon the molecular structure, crystalline and amorphous regions within the starch and distribution of granular size (Gani, Masoodi, Wani, & Salim, 2013). The oil absorption capacity involves physical entrapment of oil by capillary action (Kinsella, 1976). Oil absorption
capacity of native oat starches was in the range of 0.29e0.34 g g1. Water absorption capacity of acetylated oat starches was found in the range of 1.33e1.54 g g1 and oil absorption capacity in the range of 0.57e0.87 g g1, significantly (p 0.05) higher than their respective native starches. The introduction of acetyl groups can elevate interactions between water and the starch molecules thereby decreasing the intermolecular hydrogen bonding in starch which facilitates access of more water/oil to the amorphous regions (Ayucitra, 2012; Betancur, Chel, & Canizares, 1997). Similar studies, using rice starch (Das, Singh, Singh, & Riar, 2010), and horse chestnut starch (Shubeena et al., 2015), the authors also affirmed an increase in water and oil absorption of starch by acetylation. 3.8. Freeze thaw stability Freezeethaw stability is the ability of starch gels to resist
A. Shah et al. / LWT - Food Science and Technology 80 (2017) 19e26
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Fig. 5. Oscillatory (d) and mechanical spectra (e) measured as function of storage modulus (G0 ) and loss modulus (G00 ); native sabzaar ( 1G0 4G00 ); native SKO20 ( 2G0 2G00 ) & acetylated SKO20 ( 5G0 5G00 ); native SKO90 ( 3G0 3G00 ) & acetylated SKO90 ( 6G0 6G00 ).
1G00 ) & acetylated sabzaar ( 4G0
Table 3 Physicochemical properties of oat starches (n ¼ 3). Sabzaar
Water absorption capacity (g/g) Oil absorption capacity (g/g) Freeze thaw stability (g/100g) 0 thaw 1 thaw 2 thaw 3 thaw 4 thaw 5 thaw
SKO20
SKO90
Native
Modified
Native
Modified
Native
Modified
0.86 ± 0.0b 0.34 ± 0.02b
1.54 ± 0.03e 0.87 ± 0.01e
0.78 ± 0.01a 0.32 ± 0.01ab
1.40 ± 0.07d 0.64 ± 0.02d
0.77 ± 0.01a 0.29 ± 0.02a
1.33 ± 0.01c 0.57 ± 0.01c
22.80 23.93 27.15 30.93 34.56 36.88
± ± ± ± ± ±
0.63cbp 1.5bp 0.72bq 0.86br 0.72bs 0.78bs
19.41 21.74 25.26 26.82 30.59 31.45
± ± ± ± ± ±
0.50ap 0.35aq 0.11ar 0.33as 0.33at 0.13au
25.41 28.92 32.41 34.96 39.21 45.41
± ± ± ± ± ±
0.50dp 1.04cq 0.50cr 0.22cs 0.66ct 1.12cv
20.80 22.08 26.08 32.58 32.96 41.55
± ± ± ± ± ±
0.69bp 0.31aq 0.42abr 0.60cs 1.51bs 0.67ct
25.24 27.64 30.91 35.02 38.89 45.85
± ± ± ± ± ±
0.63ep 1.16cq 0.72cr 0.62cs 0.99ct 0.94cu
22.37 24.18 28.92 32.51 34.29 42.21
± ± ± ± ± ±
0.41cp 0.27bq 0.95cr 1.17cs 0.44bt 0.66cu
Results are mean ± S.D. Means in the rows within a particular parameter with different superscript are significantly different at (P < 0.05).
repeated freezing and thawing cycles without any undesirable physical changes. It is directly related to the tendency of starch to retrogradation and is an important parameter to be taken into consideration in the formulation of starch rich food products. Freeze thaw stability of native and acetylated oat starch gels is presented in Table 3. There was a significant (p < 0.05) decrease in freeze thaw stability from 0 h (22.80e25.41 g/100 g) to 120 h (36.88e45.85 g/100 g) of storage in native starches. Increase in syneresis during the frozen storage would be the result of increased aggregation of the starch chains via hydrogen bonds, causing increase in exudation of water from gels. Furthermore, syneresis of acetylated starch gels during the frozen storage was significantly (p 0.05) lower than the native starch. This may be attributed to the fact that steric hindrances produced by the acetyl groups restricts the re-association of amylose, resulting in enhanced water retention capacity of the starch molecules during storage. This property of the acetylated starch has also been reported by many other authors (Mbougueng, Tenin, Scher, & Tchiegang, 2012; Saartrat, Puttanlek, Rungsardthong, & Uttapap, 2005; Simsek et al., 2012). 4. Conclusion Introduction of acetyl groups in starch chains significantly
improved its physicochemical properties like water/oil absorption capacity and freeze thaw stability. Rheological behaviors of oat starches were found to vary with type of variety and degree of substitution. Shear-thinning depicts that it can be operated at lower energy andcan find applications in many ready to eat food products. Also by varying acetyl content it is possible to produce starches with specific flow properties. Furthermore, the resistance of acetylated starches to digestion suggests its function as prebiotic, thus improving the gut health. Acknowledgements Authors are thankful to the Department of Biotechnology (Grant number: D.O.BT/PR6701/FNS/20/674/2012), Government of India for their financial support. References AACC. (2000). Approved methods of the AACC International, methods (10th ed., pp. 32e40). St. Paul, MN: The Association AACC. Anon. (2006). Starch production in India. Modified starch.com. Availablefrom www. tidco.com. Ashwar, B. A., Gani, A., Shah, A., Wani, I. A., & Masoodi, F. A. (2015). Preparation, €rke, 67, health benefits, and applications of resistant starch- a review. Starch/Sta 1e15. Ashwar, B. A., Gani, A., Shah, A., & Masoodi, F. A. (2016). Production of RS4 from rice
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