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International Journal of Polymer Analysis and Characterization

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Functional and Morphological Characterization of Low-Substituted Acetylated White Sorghum (Sorghum bicolor) Starch
Tahira Mohsin Ali & Abid Hasnain
a a a

Department of Food Science and Technology, University of Karachi, Karachi, Pakistan Version of record first published: 25 Apr 2011.

To cite this article: Tahira Mohsin Ali & Abid Hasnain (2011): Functional and Morphological Characterization of Low-Substituted Acetylated White Sorghum (Sorghum bicolor) Starch, International Journal of Polymer Analysis and Characterization, 16:3, 187-198 To link to this article: http://dx.doi.org/10.1080/1023666X.2011.562690

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International Journal of Polymer Anal. Charact., 16: 187198, 2011 Copyright # Taylor & Francis Group, LLC ISSN: 1023-666X print=1563-5341 online DOI: 10.1080/1023666X.2011.562690

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FUNCTIONAL AND MORPHOLOGICAL CHARACTERIZATION OF LOW-SUBSTITUTED ACETYLATED WHITE SORGHUM (Sorghum bicolor) STARCH Tahira Mohsin Ali and Abid Hasnain
Department of Food Science and Technology, University of Karachi, Karachi, Pakistan
Starch isolated from white sorghum (Sorghum bicolor) was acetylated by addition of acetic anhydride at concentrations of 3%, 6%, and 9% w/w of starch under alkaline conditions to produce starch acetates of three different degrees of substitutions. Morphological characterization through scanning electron microscopy revealed roughness on the surface of acetylated starch granules. The modied biopolymer exhibited improved swelling power, solubility, water binding capacity, and paste clarity compared to its native counterparts. Decrease in percent syneresis and gel hardness and extent of reduction in percent transmittance indicate reduced retrogradation tendency of white sorghum starch acetates. Keywords: Acetylated starches; Degree of substitution (DS); Functional properties; Morphological characteristics; White sorghum starch

INTRODUCTION White sorghum (Sorghum bicolor (L.) Moench), a non-celiac crop,[1] is an underutilized source of starch. The starch isolated from sorghum has properties very similar to cornstarch[2] and thus could be a good alternative to cornstarch in the future. Sorghum is a highly drought-tolerant crop and was found to be superior to both maize and pearl millet under moderate irrigation conditions, while maize performed poorly under drought conditions.[3] Sorghum crops have the ability to extract water from deep soil layers. In addition to high drought tolerance, sorghum can tolerate salinity better than maize and uses the C4 malate cycle, which is the most efcient form of photosynthesis.[4] The physical and chemical makeup of the grain also makes it resistant to diseases and pests.[5] These physiologically advantageous properties of sorghum have increased its importance as an alternative starch source. Chemical modications are known to improve the functional properties of polymers.[6,7] A lot of work has been done to improve sorghum starch yields by employing different wet milling procedures.[8,9] However, considerably less work
Submitted 2 January 2011; accepted 18 January 2011. Correspondence: Tahira Mohsin Ali, Department of Food Science and Technology, University of Karachi, 75270 Karachi, Pakistan. E-mail: tahiramohsin@gmail.com 187

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has been done to improve the functional properties of sorghum starch via modications. The aim of present study is to evaluate the effect of different levels of acetic anhydride on the morphological and functional characteristics of modied white sorghum starch. MATERIALS AND METHODS Materials

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Cleaned and sound white sorghum grains from a single cultivar, free from molds and insects, were used for isolation of starch. All chemicals used were reagent grade. Isolation of Starch Starch was isolated by combining wet-milling procedures reported by Wang et al.[9] and Olayinka et al.[10] Starch isolation from white sorghum grains was carried out in two steps: (a) steeping and (b) wet milling. Cleaned grains were steeped in 0.5% lactic acid and 0.2% SO2 solution for 24 h at 50 C. The steeped grains were then washed twice for the complete removal of the steeping solution. Grains were then coarsely ground and blended in a Waring blender for 5 min at 8000 rpm. The slurry thus obtained was adjusted to pH 8.5 using 0.5 M NaOH solution and was allowed to stand for 30 min. It was then sieved through a stack of 80, 170, and 270 wire mesh sieves from top to bottom. The overs from the sieves were reblended in a Waring blender for 2 min, followed by sieving. This step was repeated twice. The starch slurry thus collected was homogenized using a homogenizer (Polytron PT 2100, Kinematica Inc., Switzerland) for 60 s, and starch was allowed to sediment at 4 C. The protein layer was scraped off using a spatula. The sedimented starch was washed with water and homogenized again to remove the residual protein. The washed starch was then dried at 45 C in a forced air oven. Modication Procedure The method described by Philips et al.[11] was employed for preparing acetylated white sorghum starch by addition of acetic anhydride at three different concentrations (3%, 6%, and 9%) w=w of starch. Determination of Acetyl (%) and Degree of Substitution The method of Wurzburg[12] was used to determine percent acetylation and degree of substitution in acetylated white sorghum starch. Swelling Power and Solubility Swelling power and solubility of starch was determined using the method of Bello-Perez et al.[13]

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Water Retention Capacity (WRC) Water retention capacity was determined by using the method of Bryant and Hamaker.[14]

Microviscoamylography Pasting properties of native and acetylated white sorghum starches were studied using a Brabender Microviscoamylograph (Model 803201, Brabender, Germany) equipped with 300 cmg sensitivity cartridge. A slurry of 8% starch in 100 mL water was heated from 40 to 95 C at a heating rate of 3 C=min. The slurry was kept at this temperature for 10 min and subsequently cooled back to 50 C at a cooling rate of 3 C=min. The slurry was held at 50 C for 10 min.

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Syneresis The method of Singh et al.[15] was used to estimate percent syneresis.

Paste Clarity The method of Perera and Hoover[16] was used for determining paste clarity.

Gel Hardness The starch gels prepared through microviscoamylography were poured into plastic bowls with 3.5 cm internal diameter. The gels were covered with paralm and stored at 4 C. Gel hardness was determined using a Universal Testing Machine (D-89079, Zwick=Roell, GmbH & Co., Ulm, Germany). The gel was compressed at a speed of 1 mm=s to a distance of 5 mm using a 5 mm cylindrical probe. The gel hardness was measured after 1, 3, and 7 days of refrigerated storage.

Morphological Properties Granular morphology of native and acetylated white sorghum starches was studied through scanning electron microscopy (SEM) by sprinkling a thin layer of starch on an aluminum specimen holder by a double-sided tape. The starch was then coated with gold and examined under 1600 and 8000 magnications.

Statistical Analysis Analysis of variance was used to calculate signicant differences between the means, and Duncans test at p 0.05 was used to separate means. The Pearson coefcient of correlation was also calculated using SPSS software (SPSS version 17, USA).

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RESULTS AND DISCUSSION Percent Acetyl Content and Degree of Substitution The percent acetyl content and degree of substitution of white sorghum starch acetates prepared by addition of acetic anhydride at three different levels (3%, 6%, and 9%) w=w of starch is presented in Table I. Increase in level of acetic anhydride resulted in progressive increments of percent acetyl content and degree of substitution of sorghum starch with Pearson correlation coefcients of 0.983 and 1.000, respectively. According to the U.S. Food and Drug Administration (FDA), modied starches prepared via acetylation must not have percent acetyl content greater than 2.5%.[17] The percent acetyl groups as determined in sorghum starch acetates are thus within the range proposed by the FDA. Swelling Power and Solubility Swelling power and solubility of native and acetylated white sorghum starch are presented in Table II. Swelling power is an important functional property of starch. Acetylated white sorghum starches had signicantly higher swelling power than native white sorghum starch. Swelling power at 90 C was found to be positively correlated with the degree of substitution with a Pearson correlation coefcient of
Table I. Effect of acetic anhydride levels on degree of substitution (DS) and % acetyl content of white sorghum starch Acetic anhydride g=100 g of starch 3 6 9 % Acetyl 0.88a 1.06b 1.41c DS 0.03a 0.04b 0.05c

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All values are mean of duplicate determinations. Means within a column with different superscript letters are signicantly different at p 0.05.

Table II. Effect of degree of substitution on the swelling power and solubility of white sorghum starch DS of starch Swelling power (g=g) 0 0.03 0.04 0.05 Solubility (g=100 gm) 0 0.03 0.04 0.05 60 C 2.27a 2.46b 3.03c 4.67d 1.26a 2.39b 2.91c 3.83d 70 C 5.82a 7.72b 8.13c 8.57d 1.89a 2.89b 5.58c 6.30d 80 C 8.68a 9.08b 9.67c 11.02d 4.80a 5.14a 7.60b 8.30c 90 C 10.30a 13.75b 16.70c 18.04d 7.12a 12.24b 13.73c 14.36d

All values are mean of triplicate determinations. Means within a column with different superscript letters are signicantly different at p 0.05.

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r 0.983, p 0.05. Starch acetate prepared by addition of acetic anhydride at 9% level exhibited the highest swelling power at all temperatures. The marked improvement in swelling power of starches following acetylation is due to the substitution of hydroxyl groups by bulky acetyl moieties. These hydrophilic acetyl groups disrupt the hydrogen bonding present between the glucan chains, thus allowing higher percolation of water into the starch granules. Higher percentage of acetyl groups results in increased disruption of intragranular binding forces within the starch granule and subsequently improves swelling power of starch acetates. Swelling power of both native and modied white sorghum starch increased linearly with rise in temperature. Increase in temperature results in further weakening of intragranular binding forces and thus improves the ability of starch granules to hold water. Solubility of starch is yet another important functional property of starch that is affected by modication. Solubility of sorghum starch acetates increased signicantly after acetylation (Table II). The starch acetate with DS-0.05 showed highest solubility, followed by starch acetates with DS-0.04 and 0.03, respectively within the entire temperature range of 60 90 C. Solubility progressively improved with the increase in degree of acetylation at all temperatures with r 0.991, p 0.05 for solubility estimated at 90 C. This could be attributed to the vitiation of the intra-granular structure of starch due to introduction of acetyl groups, thus facilitating less restricted amylose leaching. Solubility, like swelling power, was found to be temperature dependent, as increasing the temperature from 60 to 90 C continuously improved solubility of both native and modied white sorghum starch.

Water Retention Capacity (WRC) The water retention capacity of native and acetylated starches with three different degrees of substitutions is presented in Table III. WRC is the measure of the hydrophilic tendency of starch granules. Acetylated starches showed signicantly higher WRC than the native starch within the temperature range of 70 90 C. Second, water retention capacity increased with the increase in acetyl content. The improvement in WRC of starch reects the fact that acetylation increases the number of available water binding sites, which subsequently alters the ability of starch granules to bind water. The substitution of hydroxyl groups by acetyl groups creates steric hindrances and affects the interaction between the starch chains. The loosely associated starch chains in acetylated starches demonstrate higher water retention

Table III. Effect of degree of substitution on water retention capacity (g=g) of white sorghum starch DS of starch 0 0.03 0.04 0.05 60 C 1.06a 1.13a 1.35a 2.38b 70 C 1.66a 4.67b 5.70c 6.36d 80 C 6.22a 6.60b 6.87c 7.71d 90 C 7.87a 8.92b 11.50c 14.56d

All values are mean of duplicate determinations. Means within a column with different superscript letters are signicantly different at p 0.05.

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capacity.[18] Moreover, WRC was found to be a function of temperature, as WRC increased linearly with the rise in temperature for both native and acetylated starches. As temperature increases, hydrogen bonds present between the polymer chains are disrupted, thus improving the availability of water-binding sites. WRC of native and acetylated starches was highest at 90 C, implying that the hydrophilicity of starch is maximum at this temperature owing to gelatinization of starch, which results in completely disordered granular structure. Pasting Properties The pasting properties of native and acetylated white sorghum starches are presented in Table IV. Pasting is an important phenomenon following gelatinization that relates to many functional properties of starches. The commonly studied parameters of pasting are pasting temperature, peak viscosity, time to reach peak viscosity, hot paste, cold paste, breakdown, and setback viscosities. Pasting temperature decreases with increase in degree of substitution, indicating ease of pasting due to the introduction of acetyl groups (r 0.963, p 0.05). Acetylated sorghum starch with DS-0.05 showed a 8.70 C reduction in pasting temperature compared to native starch. Ease of gelatinization is further evidenced by the reduction in time to reach peak viscosity. Presence of acetyl groups causes less restricted swelling of starch granules, thus resulting in quick attainment of peak viscosity. Acetylated starches with DS-0.03 and 0.88% acetyl content showed a signicant improvement in peak viscosity. However, further increase in acetyl content in starches with DS-0.04 and DS-0.05 resulted in higher but statistically insignicant peak viscosity than that of native white sorghum starch. The interplay between reduction of hydrogen bonds due to steric hindrance created by acetyl groups and participation of acetyl groups in improved hydrogen bonding with other starch chains could have resulted in insignicant changes in the peak viscosity of aforementioned acetylated starches. Hot paste viscosities of acetylated starches were signicantly higher than that of native white sorghum starch. The higher water retention capacity of acetylated starches could be the reason for improvement in hot paste viscosity of starch acetates. When starch slurry is held at 95 C, the swollen starch granules begin to break down due to solubilization of linear amylose chains, which

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Table IV. Effect of degree of substitution on pasting properties of white sorghum starch DS of starch 0 0.03 0.04 0.05 PT 73.15a 69.3b 68.2c 64.45d PV 245.5a 258b 252a 255.5a TTP 17.2a 15.65b 15.03b,c 14.18b HPV 163.5a 173b 171b 183.5c CPV 401.5a,b 386.5a 411b 480.5c BD 82a 84.5a 81a 59.5b SB 271a 274.5a 266a 295b

All values are mean of duplicate determinations. Means within a column with different superscript letters are signicantly different at p 0.05. PT pasting temperature ( C), PV peak viscosity (BU), TTP time to reach peak viscosity (min), HPV hot paste viscosity (BU), CPV cold paste viscosity (BU), BD breakdown viscosity (BU), SB setback viscosity (BU).

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subsequently results in loss of structural integrity. Therefore, breakdown viscosity is said to be the measure of fragility of starch granules. The results revealed that breakdown of acetylated starch with DS-0.05 was signicantly reduced, indicating improvement in stability of starch granules. The improvement in breakdown viscosity post acetylation is also reported for low substituted barley starches.[19] However, acetylated starches with DS < 0.05 showed insignicant changes in breakdown viscosity, implying that very low degree of substitution was not enough to bring any changes in fragility of starch granules. As starch slurry is cooled, a second rise in viscosity is recorded that is due to the re-association of starch chains. Cold paste and setback viscosities of acetylated starches prepared with 3% and 6% acetic anhydride were statistically insignicant from those of native starches. But cold paste and setback viscosities of sorghum starch acetate with DS-0.05 were signicantly higher than those of native starch, which is contrary to studies conducted by many other researchers.[20,21] However, Hoover and Soluski[22] in their study on acetylated Phaseolus vulgaris starches suggested that higher setback viscosity was due to increased exudation of amylose chains following acetylation and nullication of any steric effects of acetyl groups due to shearing action of blades. Syneresis The results of percent syneresis are presented in Table V. Acetylation reduced percent syneresis in white sorghum starch. Precent syneresis after seven days of refrigerated storage was found to be negatively correlated with degree of substitution (r 0.988, p 0.05). Native starch and DS-0.03 starch acetate showed timedependent increment in percent syneresis (exudation of water from starch gel) on refrigerated storage, whereas acetylated starches with DS > 0.03 did not show much alteration in percent syneresis on storage, which lends credence to the notion that acetylation improves low-temperature stability of starches. Improvement in percent syneresis on acetylation could be attributed to improved water retention capacity of starches (Table III). Moreover, the presence of acetyl groups on leached amylose chains hinders the formation of junction zones or re-association of amylose chains, which subsequently improves the water-holding capacity of starch gels.

Table V. Effect of degree of substitution on % syneresis of white sorghum starch Storage (days) DS of starch 0 0.03 0.04 0.05 1 75.07 64.52b 68.23c 65.33b
a

3 76.21 69.88b 68.50c 65.26d

a

7 78.47a 70.35b 68.56b 65.43c

Values are mean of duplicate determinations. Means within a column with different superscript letters are signicantly different at p 0.05.

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Paste Clarity The paste clarity (%T) of native and acetylated starches is presented in Table VI. Paste clarity of all starch acetates was signicantly higher than that of native white sorghum starch. The higher swelling power of sorghum starch acetates could be one of the reasons for the improvement in percent transmittance. The insertion of bulky acetyl groups in the amorphous region of starch granules causes repulsion and facilitates improved water percolation into the starch granules. These highly swollen starch granules improve transmittance of light.[23] Paste clarity reduces when starch solutions are stored at low temperature after gelatinization. The reduction in %T is due to re-association of leached out amylose chains and is therefore a measure of retrogradation tendency. Our results clearly show decrease in paste clarity with the increase in number of storage days. However, the extent of reduction in paste clarity varied signicantly between the starches. Percent reduction in transmittance after 72 h of cold storage for native, DS-0.03, DS-0.04, and DS-0.05 starches was found to be 69.5%, 61.94%, 56.78%, and 48.55%, respectively. Extent of reduction in %T after three days of refrigerated storage negatively correlated with degree of substitution (r 0.957, p 0.05) and percent acetyl content (r 0.955, p 0.05). These results clearly demonstrate the fact that increase in degree of acetylation decreased the retrogradation or re-association tendency of sorghum starch. Gel Hardness Gel hardness values of native and acetylated white sorghum starch is presented in Table VII. The gel strength of all three acetylated starches with different degrees of substitutions was signicantly lower than that of native white sorghum starch gel. According to Lawal[20] gel strength depends upon intra-granular binding forces. The hampering of these intra-granular binding forces due to introduction of acetyl substituting groups results in the formation of weaker gels. The re-association of amylose chains (retrogradation) affects the gel strength. The gel strength of native white sorghum starch was more than doubled after seven days of refrigerated storage. However, acetylated starches with DS-0.04 and DS-0.05 did not show much change in gel hardness, indicating restricted re-association of amylose chains, which subsequently results in better water retention capacity and reduced percent syneresis.

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Table VI. Effect of degree of substitution on paste clarity (%T) of white sorghum starch Storage (hours) DS of starch 0 0.03 0.04 0.05 0 4.89 7.54b 7.96b 6.55d
a

24 2.14 3.17b 3.45b 3.29b

a

48 1.88 3.09b 3.27c 3.26c

a

72 1.49a 2.87b 3.44c 3.37c

Values are mean of triplicate determinations. Means within a column with different superscript letters are signicantly different at p 0.05.

CHARACTERISTICS OF WHITE SORGHUM STARCHES Table VII. Effect of degree of substitution on gel hardness (N) of white sorghum starch Storage (days) DS of starch 0 0.03 0.04 0.05 1 0.170a 0.046b 0.030b 0.030b 3 0.187a 0.083b 0.033c 0.040c

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7 0.43a 0.17b 0.037c 0.033c

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Values are mean of triplicate determinations. Means within a column with different superscript letters are signicantly different at p 0.05.

Morphological Characteristics Scanning electron micrographs of native and acetylated white sorghum starches at 1600 and 8000 magnications are presented in Figures 1 and 2, respectively. Native white sorghum starches appear to be spherical and polygonal in shape (Figure 1(a)). The size of the starch granules ranged between 6 and 20 mm. There were fewer small-sized granules than large-sized granules. A low degree of acetylation did not signicantly alter the shape and size of the starch granules (Figures 1(b), (c), and (d)). However, DS-0.05 acetylated starches showed clustering or fusion of starch granules (Figure 1(d)). Both native and acetylated starches showed random distribution of pores under 8000 magnication (Figures 2(a), (b), (c), and (d)). The diameter of pores ranged between 178 and 350 nm for both native and acetylated starches. The presence of pores on starches is also reported

Figure 1. Scanning electron micrographs of native and acetylated white sorghum starches under 1600 magnication: (a) native, (b) DS-0.03 acetylated starch, (c) DS-0.04 acetylated starch, and (d) DS-0.05 acetylated starch.

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Figure 2. Scanning electron micrographs of native and acetylated white sorghum starches under 8000 magnication: (a) native, (b) DS-0.03 acetylated starch, (c) DS-0.04 acetylated starch, and (d) DS-0.05 acetylated starch.

by Huber and BeMiller.[24] These external pores are connected to the internal cavity at the hilum via radial tube-like channels.[25] Scanning electron micrographs under 8000 magnication revealed damage and roughness on the surface of white sorghum starch acetates with three different degrees of substitutions. CONCLUSION The study on morphological and functional characteristics of low substituted acetylated white sorghum starches has shown that swelling power, solubility, water retention capacity, and paste clarity improved via acetylation. Degree of substitution was found to be positively correlated with swelling power, WRC, and solubility. Acetylation decreased the pasting temperature of starches, which indicates weakening of starch granules due to disruption of intra-granular binding forces. Gel hardness and percent syneresis reduced after acetylation, which lends credence to the notion that acetylation improves low-temperature stability of starches. Also, the extent of reduction in percent transmittance was curtailed after acetylation compared to native starch, which indicates that the re-associative tendency of starch granules is reduced due to acetylation. Therefore, acetylated starches could nd applications in foods intended for refrigeration. REFERENCES
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