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Physicochemical, rheological, morphological and in vitro digestibility properties of pearl millet starch modified at varying levels of acetylation
International Journal of Biological Macromolecules 131 (2019) 1077–1083
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Physicochemical, rheological, morphological and in vitro digestibility properties of pearl millet starch modified at varying levels of acetylation Anil Kumar Siroha a, Kawaljit Singh Sandhu a,b, Maninder Kaur c,⁎, Varinder Kaur d a
Department of Food Science and Technology, Chaudhary Devi Lal University, Sirsa, India Department of Food Science and Technology, Maharaja Ranjit Singh Punjab Technical University, Bathinda, India Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India d Department of Chemistry, Guru Nanak Dev University, Amritsar, India b c
a r t i c l e
i n f o
Article history: Received 30 January 2019 Received in revised form 27 February 2019 Accepted 25 March 2019 Available online 26 March 2019 Keywords: Pearl millet starch Physicochemical Morphological Rheological properties
a b s t r a c t Pearl millet (PM) starch was reacted with acetic anhydride at different concentrations (1.25, 2.5, 3.75 & 5.0%) and its physicochemical, rheological, morphological and in vitro digestibility properties were compared with native starch. The acetyl (%) and degree of substitution (DS) of acetylated starches ranged between 1.07 and 2.15% and 0.040–0.081, respectively. Swelling power and solubility of acetylated PM starch increased progressively upto 3.75% level of acetylation, however, further increase in acetylation levels resulted in a decrease. Peak, setback and final viscosities of acetylated PM starches were higher than their native counterpart starch. Both native and acetylated PM starches showed similar A-type X-ray diffraction patterns. During heating, storage modulus (G ′) and loss modulus (G″) of acetylated starches ranged between 753 and 1177 Pa and 96–129 Pa, respectively. G′ was much higher than G′′ at all the values of angular frequency studied. Both native and acetylated PM starch pastes showed flow behaviour index value of b1. For acetylated starches, both, yield stress and consistency index values were higher than native starch. Readily digestible and slowly digestible starch contents of acetylated PM starches varied between 44.3 and 45.5% and 27.3–32.5%, respectively, the highest values observed for starch acetylated at 3.75% and 1.25% acetic anhydride concentrations. © 2019 Elsevier B.V. All rights reserved.
1. Introduction The industrial food applications of native starches are limited due to their low shear resistance, thermal decomposition and high tendency towards retrogradation [1]. These can be improved by modifications using physical, chemical and enzymatic methods. The free hydroxyl groups present at 2, 3 and 6 carbons of the glucose molecule enable native starch to be modified by different chemical treatments [2]. The chemical modification of starch can be done by employing acid hydrolysis, oxidation, esterification, etherification and cross-linking methods [3]. The physico-chemical and functional properties of starch can be improved by acetylation [4]. The number of acetyl groups incorporated into the starch molecule during acetylation depends on many factors, such as reactant concentration, starch source [5], reaction time, and the presence of a catalyst [6]. Starch acetylation results in an increased solubility and a decrease in bond strength, which improves clarity and freeze-thaw stability [7]. Food and Drug Administration recommends not N2.5% acetyl groups in starch acetate [8,9]. Pearl millet (Pennisetum glaucum) belongs to family Poaceae and is widely grown around the world for feed and fodder. Being drought ⁎ Corresponding author. E-mail address: [email protected] (M. Kaur).
https://doi.org/10.1016/j.ijbiomac.2019.03.179 0141-8130/© 2019 Elsevier B.V. All rights reserved.
tolerant cereal crop, it is grown primarily in India and Africa. India ranks first in annual production of millets (11,420,000 t in 2014), followed by Niger which produced 3,321,753 t [10]. Pearl millet (PM) has a starch content of 60–70% which is comparable with corn and this starch can be isolated easily from it. In our earlier study on Indian PM cultivars, we characterized starches isolated from these cultivars for their physicochemical, morphological, thermal, rheological and in vitro digestion properties [11]. Further, these PM starches were cross-linked by epichlorohydrin (0.5%) and studied for their various functional properties [12]. To the best of our knowledge no work has been reported on the acetylation of PM starch. In order to explore utilization of PM starch, the present investigation was aimed at acetylation of PM starch at varying levels of acetylation and analyzed for their physicochemical, rheological, morphological and in vitro digestibility properties. 2. Material and methods 2.1. Materials and starch isolation Pearl millet cultivar (HC-10) was procured from Chaudhary Charan Singh Haryana Agriculture University, Hissar, India. All chemicals used were of analytical grade. Starch was isolated from pearl millet grains
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by following the centrifugation method described by Sandhu and Singh [13]. Pearl millet grains (500 g) were added to 1.25 l of distilled water containing 0.1% sodium meta bisulphite. The mixture was maintained at 50 °C for 18–20 h with intermittent circulation of liquid. The steep water was then drained off and grains were ground in laboratory grinder (Maxie Plus, New Delhi, India). About 250 g of steeped grains were ground with 250 ml of distilled water. The ground slurry was passed through 250, 150, 100, 75, 45 mm mesh sieve. The starchprotein slurry was then allowed to stand for 4–5 h. The supernatant was removed by suction and the settled starch layer was resuspended in distilled water and centrifuged in wide mouthed cup centrifuge (Remi, New Delhi, India) at 3000 rpm for 10 min and the upper non-white layer was scrapped off. The white layer was re-suspended in distilled water and re-centrifuged 3–4 times. The starch was then collected and dried in an oven (NSW-143, New Delhi, India) at 45 °C for 12 h. 2.2. Preparation of acetylated starch, acetyl (%) and degree of substitution (DS) PM starch was acetylated by following the method described by Phillips et al. [14] using acetic anhydride at varying concentrations (1.25, 2.5, 3.75, 5.0 g). The percent acetylation (acetyl%) and degree of substitution (DS) were determined by following the method described by Wurzburg [15]. The acetyl (%) was calculated as: Acetyl ð%Þ ¼
ðBlank–SampleÞ Molarity of HCl 0:043 100 Sample weight
2.3. Amylose content, swelling power and solubility Amylose content of PM starches was determined by following the method described by Williams et al. [16]. Starch (20 mg) was thoroughly mixed with 10 ml of 0.5 N KOH and the dispersed sample were diluted to 100 ml with distilled water. An aliquot of test starch solution (10 ml) was pipetted into 50 ml volumetric flask and 5 ml of 0.1 N HCl and 0.5 ml of iodine reagent were added. The volume was diluted to 50 ml and the absorbance was measured at 625 nm in a spectrophotometer (Systronics, Ahmadabad, India). The measurement of the amylose was determined in triplicate from a standard curve developed using amylose and amylopectin blends. The method described by Leach et al. [17] was followed for swelling power and solubility measurements. Starch (1 g) was added to 99 ml of distilled water and heated to 90 °C for 1 h. The heated samples were rapidly cooled to 25 °C and centrifuged at 3000 rpm for 30 min. For measuring solubility, the supernatants were drained into pre-weighed moisture dishes, evaporated to dryness in a hot air oven at 100 °C and the dishes were reweighed. 2.4. Pasting properties The pasting properties of PM starches were determined using an inbuild starch cell of Modular Compact Rheometer (Model-52, Anton Paar, Austria). Starch slurries (1.2 g starch in 13.8 g distilled water)
were held at 50 °C for 1 min and then heated from 50 to 95 °C at a heating rate of 6 °C/min, held for 2.7 min, cooled to 50 °C at the same rate and again held at 50 °C for 2 min. Each sample was analyzed in triplicate. Peak viscosity, breakdown, setback, final viscosity and pasting temperature were obtained from the pasting graph. 2.5. Rheological properties 2.5.1. Dynamic properties A small amplitude oscillatory rheological measurement was made for starches with a Modular Compact Rheometer (Model-52, Anton Paar, Austria) equipped with parallel plate system (0.04 m diameter). The gap size, strain and frequency were set at 1000 μm, 2% and 10 rad/s, respectively. The linear viscoelastic range (LVR) was selected at a strain of 0.02%, and all experiments were carried out at the selected strain. The dynamic rheological properties, such as storage modulus (G′), loss modulus (G″) and loss factor (tanδ) were determined for the starches. Starch suspension of 10% concentration was loaded onto the ram of rheometer and covered with a thin layer of lowdensity silicon oil (to minimize evaporation losses). The starch samples were subjected to temperature sweep testing by heating them from 45 to 95 °C at the rate of 2 °C/min. For frequency sweep measurement, starch slurry (10%) was prepared, stirred and then heated at 90 °C in a water bath (Brookefield, TC-202, USA). The slurry was again stirred for 3 min and then cooled to room temperature. It was then loaded on the ram of rheometer and frequency sweep tests from 0.1 to 100 rad/s were performed at 25 °C. Parameters recorded were G′, G′′, and tan δ. 2.5.2. Steady shear measurement Steady shear properties of native and acetylated PM starches were determined by following the method described by Park et al. [18] with slight modification. The sample preparation method has been described in frequency sweep measurement method. The sample (10%) was sheared continuously from 1 to 100 s−1. The data was fitted to Herschel-Bulkley model to describe the variation in the rheological properties:
σ ¼ σ o þ Kð γ̇Þn
where σ is the shear stress (Pa), σo is the yield stress, γ̇ is the shear rate (s−1), K is the consistency index (Pa.sn), n is the flow behaviour index (dimensionless). 2.6. X-ray diffraction X-ray diffraction patterns of PM starches were recorded with a wavelength of 0.154 nm using X-ray diffractometer (Rigaku Miniflex, Japan). The diffractometer was operated at 45 kV and 40 mA. Diffractograms were acquired at 25 °C over a 2θ range of 4–40 with a step size of 0.02 and sampling interval of 10 s.
Table 1 Acetyl (%), degree of substitution (DS), amylose content, swelling power and solubility of native and acetylated PM starches. Acetic anhydride (%)
Acetyl (%)
DS
Amylose content (%)
Swelling power (g/g)
Solubility (%)
0 1.25 2.50 3.75 5.0
0.00a 1.07 ± 0.1b 1.50 ± 0.2c 1.72 ± 0.0d 2.15 ± 0.1e
0.00a 0.040 ± 0.00b 0.057 ± 0.01c 0.065 ± 0.01d 0.081 ± 0.00e
14.1 ± 0.5d 10.5 ± 0.3a 12.3 ± 0.2b 13.5 ± 0.6c 15.4 ± 0.2e
14.0 ± 0.6a 15.0 ± 0.7c 16.0 ± 0.5d 17.6 ± 0.6e 14.8 ± 0.8b
10.4 ± 0.4a 15.4 ± 0.3c 17.2 ± 0.1d 19.2 ± 0.2e 11.5 ± 0.1b
Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
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hydrolysing within 20 min of incubation was termed as readily digestible starch (RDS), fraction digested during the period between 20 and 120 min was slowly digestible starch (SDS), whereas resistant starch (RS) was that fraction which was not hydrolyzed with 120 min of incubation. 2.9. Statistical analysis The data reported in the tables were analyzed in triplicate and subjected to one-way analysis of variance (ANOVA) using Minitab Statistical Software version 15 (Minitab, Inc., State College, USA). 3. Results and discussion 3.1. Acetyl (%) and degree of substitution
Fig. 1. Pasting profiles of native and acetylated starches (A: Native starch; B: 1.25% acetic anhydride; C: 2.5% acetic anhydride; D: 3.25% acetic anhydride; E: 5.0% acetic anhydride).
2.7. Morphological properties The morphological properties of starches were studied by scanning electron microscope (Model EVOLS10 ZEISS, Oberkochen, Germany) using 1% starch suspension made by dissolving starch in ethanol. One drop of the suspension was applied on an aluminium stub, followed by coating with gold‑palladium (60:40). During micrography, an accelerating potential of 5 kV was used. 2.8. in vitro starch digestibility in vitro starch digestibility was analyzed by following the method described by Englyst et al. [19] and as modified by Chung et al. [20]. Porcine pancreatic alpha-amylase (No. 7545, Sigma-Aldrich, St. Louis, MO), and amyloglucosidase (No. 9913, Sigma-Aldrich) (3.89 g) were used for the analysis. Glucose content was measured using glucose oxidase and peroxidase assay kits (No. GAGO-20, Sigma-Aldrich). The starch fraction
Both acetyl (%) and DS of the acetylated PM starches increased with an increase in acetic anhydride concentration; their values ranged between 1.07 and 2.15% and 0.040–0.081, respectively (Table 1). Garg and Jana [21] also reported an increase in both DS and acetyl (%) for acetylated corn starch with an increase in concentration of acetic anhydride. The observed values of DS in the present study are in agreement with those reported by other workers such as 0.04–0.10 for acetylated Indian horse chestnut [4], 0.025–0.104 for acetylated rice [22], 0.115–0.154 for acetylated potato [5] and 0.03–0.08 for acetylated kidney bean starches [23]. 3.2. Amylose content, swelling power and solubility Amylose content of acetylated PM starches ranged between 10.5 and 15.4%, the highest and the lowest values were observed for starches modified at 5.0% and 1.25% acetic anhydride concentration, respectively (Table 1). Amylose content of acetylated starches were lower as compared to native PM starch at low acetic anhydride concentrations (1.25, 2.5 & 3.75%). However, at higher acetic anhydride concentration (5.0%) amylose content was observed to be higher (15.4%) than the native counterpart PM starch. Lawal et al. [24] reported a decrease in amylose content for acetylated corn, cassava and sweet potato starches in comparison to their counterpart native starches and attributed this to the leaching out of amylose during acetylation. Contrarily, an increase in the amylose content of acetylated starches from sorghum was observed by Singh et al. [25]. The decrease/increase in an amylose content
Table 2 Pasting properties of native and acetylated PM starches. Acetic anhydride (%)
Peak viscosity (mPa.s)
Breakdown viscosity (mPa.s)
Trough viscosity (mPa.s)
Setback viscosity (mPa.s)
Final viscosity (mPa.s)
Pasting temperature (°C)
0 1.25 2.5 3.75 5.0
1069 ± 15a 1301 ± 17b 1336 ± 12c 1379 ± 14d 1772 ± 19e
508 ± 7b 503 ± 5b 473 ± 8a 526 ± 5c 503 ± 7b
561 ± 5a 798 ± 5b 863 ± 6c 853 ± 5c 1269 ± 8d
352 ± 4a 606 ± 5c 556 ± 4b 632 ± 6c 851 ± 8d
913 ± 10a 1404 ± 12b 1419 ± 17b 1485 ± 11c 2120 ± 18d
81.2 ± 0.1c 80.6 ± 0.3b 80.6 ± 0.2b 79.4 ± 0.2a 84.2 ± 0.1d
Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
Table 3 Rheological properties of native and acetylated PM starches during heating. Acetic anhydride (%)
G′ (Pa)
G′′ (Pa)
Breakdown in G′ (Pa)
tan δ (G′′/G′)
TG′ (°C)
0 1.25 2.5 3.75 5.0
737 ± 9a 753 ± 11b 761 ± 13b 1147 ± 7c 1177 ± 9d
108 ± 4b 96 ± 2a 96 ± 1a 98 ± 2a 129 ± 3c
242 ± 5d 150 ± 6a 175 ± 4b 233 ± 6d 198 ± 3c
0.14b 0.12a 0.12a 0.08a 0.10a
75.0 ± 0.3a 85.0 ± 0.1b 85.0 ± 0.3b 85.1 ± 0.2b 85.0 ± 0.2b
Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
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after acetylation may be attributed to the interference of acetyl group with the functioning of amylose and amylopectin fractions of starch thereby affecting absorption of iodine during determination of amylose [26,27]. The swelling power (SP) and solubility of acetylated starches ranged between 14.8 and 17.6 g/g and 11.5–19.2%, respectively, the highest values were observed when acetylation was carried out at 3.75% acetic anhydride concentration. Starch modified at lower concentrations (1.25 to 3.75%) of acetic anhydride showed higher swelling power and solubility while at higher concentration (5.0%), a decrease was observed. On the other hand, the native PM starch showed lower SP (14 g/g) and solubility (10.4%) in comparison to acetylated starches. The increase in swelling and solubility of acetylated starches may be due to the introduction of acetyl groups to starch which could facilitate an access of water to amorphous areas [23]. 3.3. Pasting properties The pasting profiles of native and acetylated PM starches are shown in Fig. 1. Peak viscosity (PV) of acetylated starches ranged between 1301 and 1772 mPa.s, the highest and the lowest values were observed when starch was modified at 5.0 and 1.25% acetic anhydride concentration levels, respectively (Table 2). Breakdown viscosity (BV) of acetylated PM starches varied between 473 and 526 mPa.s, the highest value was observed when acetylation was carried out at 3.75% acetic anhydride concentration. Setback viscosity (SV) of acetylated starches ranged between 556 and 851 mPa.s. Final viscosity (FV) of acetylated PM starches was in the range between 1404 and 2120 mPa.s, the highest at 5.0% acetic anhydride concentration while the lowest was observed at 1.25% concentration. PV, TV, SV and FV of acetylated PM starches were higher than their counterpart native PM starch. Starch-starch interactions reduced and viscosity of the starch granules increased due to introduction of the bulky acetyl group in the starch molecules after acetylation [28]. 3.4. Rheological properties 3.4.1. Dynamic shear properties Rheological properties of starch suspensions from native and acetylated PM starches differed significantly (p b 0.05) during heating (Table 3). For native as well as acetylated starches, G′ and G′′ increased progressively during heating. The values of G′ and G′′ of acetylated PM starches ranged between 753 and 1177 Pa and 96–129 Pa, respectively, the highest values for both moduli were observed when acetylation was conducted at 5.0% acetic anhydride concentration. tan δ (G′′/G′) for both native as well as acetylated PM starches was b1 indicating their elastic behaviour. The increase in peak G′ of acetylated PM starches might be due better capture and retention of water molecules by acetyl groups thereby leading to the formation of more arranged structures, which were resistant to deformation [27]. Breakdown in G′ varied between 150 and 233 Pa, acetylated starches exhibited lower values in comparison to native counterpart (242 Pa) PM starch. The temperature at which G′ was maximum (TG′) showed a non-significant (p b 0.05) variation among acetylated starches; however, their values were higher than TG ′ of native starch. Singh et al. [5] also reported an increase in G′ and G′ ′ values after acetylation for corn and potato starches during heating. Fig. 2(A-C) show the change in the G′, G′′ and tan δ for native and acetylated PM starch pastes as a function of the frequency. Table 4 lists G′, G′′, tan δ and complex viscosity (η*) values for starch pastes at 6.28 rad/s and 25 °C. G′ for all the PM starch pastes showed an increase upon increase in angular frequency (ω); values of G′ (1216 to 1557 Pa) were found much higher than G′′ (64.7 to 76.0 Pa) at all values of ω, reflecting the visco-elastic behaviour of starch pastes. The behavior
Fig. 2. Angular frequency dependence at 25 °C for native and acetylated starches (A: G'; B: G"; C: tanδ) (Representations for curves are: A, Native; B, 1.25% acetic anhydride; C, 2.5% acetic anhydride; D, 3.25% acetic anhydride; E, 5.0% acetic anhydride).
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Table 4 Storage modulus (G′), loss modulus (G′′), loss factor (tan δ) and complex viscosity (η *) at 6.28 rad/s (at 25 °C) for native and acetylated PM starches. Acetic anhydride (%)
G′ (Pa)
G′′ (Pa)
tan δ
η * (Pa.s)
0 1.25 2.5 3.75 5.0
1216 ± 16a 1227 ± 15a 1296 ± 11b 1519 ± 18c 1557 ± 14d
73.8 ± 5c 64.7 ± 4a 65.1 ± 5b 76.0 ± 2d 65.5 ± 3b
0.03a 0.05a 0.05a 0.05a 0.04a
193 ± 5a 195 ± 6a 206 ± 4b 242 ± 4c 248 ± 2c
Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
observed for the starch pastes were similar to those of typical weak gels. No crossover between the two moduli was observed throughout the studied frequency range (0.1–100 rad/s), which reflects the stability of native and acetylated starches at this frequency range as well as weak dependence of G′ and G′′ values on the frequency. The highest and the lowest G′ values for acetylated starch pastes were observed when acetylation was carried out at 5.0% and 1.25% levels of acetic anhydride, respectively. tan δ (G′′/G′) values of acetylated starches ranged from 0.04 to 0.05. Acetylated PM starches showed higher G′ values as compared to the native starch, which might be attributed to their higher swelling power. Lee and Yoo [29] also reported an increase in G′ and G′′ values of sweet potato starch upon acetylation.
3.4.2. Steady shear properties Steady shear properties of native and acetylated PM starches are reported in Table 5. The flow behaviour index (n) of acetylated starches, which indicates the extent of shear-thinning behavior, ranged between 0.275 and 0.310, the highest value for starch acetylated at 2.5% acetic anhydride level was observed. Shear thinning behaviour might be due to cumulative orientation of soluble starch molecules in the direction of the flow as well as the breaking of hydrogen bonds formed between amylose molecules during shearing [30]. The K (consistency index) for acetylated PM starches varied between 43.5 and 52.8 Pa.s, which were higher than their native counterpart starch. Among acetylated starches, the highest K value was observed for starch acetylated at 1.25% acetic anhydride concentration. The higher K values of acetylated starches might be due to higher viscosity of acetylated starches than native starch. Yield stress value (σο ) of acetylated PM starches varied between 26.6 and 50.7 Pa, the highest value was observed when starch was acetylated at 5.0% acetic anhydride concentration. The value of σ ο increased upon an increase in the acetic anhydride concentration. Liu et al. [31] reported that the acetylation reduces interactions between starch chains due to the presence of hydrophilic substituents and increases the ability of granules to swell by loosening granule structure, resulting in an increase in both swelling power and solubility.
Table 5 Herschel-Bulkey model fitted to native and acetylated PM starch pastes during steady shear rate. Acetic anhydride (%)
σο (Pa)
K (Pa.sn)
n
R2
0 1.25 2.5 3.75 5.0
25.5 ± 2.12a 26.6 ± 3.31b 35.8 ± 2.42c 37.1 ± 3.76d 50.7 ± 5.45e
31.3 ± 1.21a 52.8 ± 2.48c 43.5 ± 3.61b 44.4 ± 2.33c 49.0 ± 4.51d
0.320 ± 0.008d 0.275 ± 0.003a 0.310 ± 0.005c 0.305 ± 0.005b 0.308 ± 0.002c
0.999 0.999 0.999 0.999 0.999
σo = yield stress; K = consistency index; n = flow behaviour index. Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
Fig. 3. X-ray diffraction pattern of native and acetylated starches (A: Native; B: 1.25% acetic anhydride; C: 2.5% acetic anhydride; D: 3.25% acetic anhydride; E: 5.0% acetic anhydride).
3.5. X-ray diffraction (XRD) The X-ray diffraction patterns of native and acetylated PM starches are shown in Fig. 3. All the starches showed A-type diffraction patterns with peaks at 15o, 17o, 18o and 23o (2θ), which is a characteristic of cereal starches. Suma and Urooj [32] reported A-type diffraction patterns for Indian pearl millet varieties with sharp peaks at 15o and 23o and diffused peaks at 17o and 18o (2θ). Acetylated PM starches also showed XRD pattern similar to that of counterpart native PM starch. 3.6. Morphological properties Scanning electron micrographs (SEM) of native and acetylated PM starches are shown in Fig. 4. Native starch showed starch granules varying in size and shape from small to large, spherical and polygonal. At lower concentrations of acetic anhydride, no detectable changes in the starch morphology were noticed. However, starch acetylated at 3.75 and 5.0% acetic anhydride concentrations, showed cavities on the surface of few starch granules. Acetylation changed external morphology of starch granules which might result in surface roughness, development of cavities, and binding of few starch granules [25]. 3.7. in vitro starch digestibility RDS, SDS and RS content of native and acetylated PM starches are shown in Table 6. RDS and SDS contents of acetylated starches varied between 44.3 and 45.5% and 27.3–32.5%, respectively, which were significantly lower than those observed for native counterpart PM starch. Among acetylated starches, the highest RDS and SDS contents were observed when starch was acetylated at 3.75% and 1.25% acetic anhydride concentrations, respectively. RS content of acetylated PM starches were significantly higher than native PM starch, values ranging between 22.5 and 27.3%, the highest value was observed for starch acetylated at 5.0%
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(A)
(B)
(C)
(D)
(E) Fig. 4. Morphological properties of native and acetylated starches (A: Native; B: 1.25% acetic anhydride; C: 2.5% acetic anhydride; D: 3.25% acetic anhydride; E: 5.0% acetic anhydride).
acetic anhydride concentration. Han and BeMiller [33] reported that acetylation, hydroxypropylation, and cross linking, reduced the extent of enzyme-catalyzed starch hydrolysis. Chung et al. [34] reported a decrease in SDS and RDS contents and an increase in RS content for corn starch acetylated at 6% acetic anhydride concentration. Ali and Hasnain
[35] reported non-significant correlation between RS content and degree of substitution; and suggested that some of the acetyl groups inserted as a result of modification could change the spatial structure of starch. Alpha-amylase may fail to act on this chemically altered starch structure resulting in reduced digestibility and higher RS content. Kaur
A.K. Siroha et al. / International Journal of Biological Macromolecules 131 (2019) 1077–1083 Table 6 in vitro digestibility properties of native and acetylated PM starches. Acetic anhydride (%)
RDS (%)
SDS (%)
RS (%)
0 1.25 2.5 3.75 5.0
49.9 ± 0.4d 45.0 ± 0.1b 44.3 ± 0.2a 45.5 ± 0.4c 45.4 ± 0.5c
38.1 ± 0.2e 32.5 ± 0.3d 31.6 ± 0.4c 29.5 ± 0.3b 27.3 ± 0.2a
12.0 ± 0.3a 22.5 ± 0.3b 24.1 ± 0.6c 25.2 ± 0.5d 27.3 ± 0.7e
RDS = readily digestible starch; SDS = slowly digestible starch; RS = resistant starch. Values within the same column followed by the same superscript are not significantly different (p b 0.05). Mean (±standard deviation) of triplicate analysis.
et al. [36] reported potential physiological benefits and unique functional properties of RS, similar to that of dietary fibers. 4. Conclusions Acetylation done using acetic anhydride at varying levels caused a significant change in physicochemical, pasting, rheological and in vitro digestibility properties of PM starch. Acetyl content and DS increased upon an increase in acetic anhydride concentration. Both swelling power and solubility increased with DS, however, the reverse trend was observed at higher DS. Pasting properties after acetylation also showed an increase as compared to native PM starch. G′ and G′′ of native and acetylated PM starches showed no crossover throughout the observed frequencies range, reflecting starch stability. Morphology of acetylated starches differed slightly from native PM starch at higher concentrations with a few starch granules having surface pores. Acetylation resulted in decrease in RDS and SDS contents while increase in RS content. Increase in degree of acetylation also resulted in a significant increase in RS content. Properties of pearl millet starch should be explored further by modifying it with different chemical agents so as to increase its utilization. Compliance with ethical standards The article does not contain any studies with human and animal subjects. Conflict of interest The authors declare no conflict of interest for this article. Acknowledgements The financial support provided by Chaudhary Devi Lal University, Sirsa, India (R&S/A-1/13/66-11/1263-1277) in the form of scholarship to Anil Kumar Siroha is deeply acknowledged. References [1] R. Hui, C. Qi-He, F. Ming-liang, X. Qiong, H. Guo-qing, Preparation and properties of octenyl succinic anhydride modified potato starch, Food Chem. 114 (2009) 81–86. [2] J. Bao, J. Xing, D.L. Phillips, H. Corke, Physical properties of octenyl succinic anhydride modified rice, wheat, and potato starches, J. Agric. Food Chem. 51 (2003) 2283–2287. [3] L. Jayakody, R. Hoover, The effect of lintnerization on cereal starch granules, Food Res. Int. 35 (2002) 665–680. [4] I.A. Wani Shubeena, A. Gani, P. Sharma, T.A. Wani, F.A. Masoodi, A. Hamdani, S. Muzafar, Effect of acetylation on the physico-chemical properties of Indian Horse Chestnut (Aesculus indica L.) starch, Starch/Stärke 67 (2015) 311–318.
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[5] J. Singh, L. Kaur, N. Singh, Effect of acetylation on some properties of corn and potato starches, Starch/Stärke 56 (2004) 586–601. [6] S.O. Agboola, J.O. Akingbala, G.B. Oguntimein, Production of low substituted cassava starch acetates and citrates, Starch/Stärke 43 (1991) 13–15. [7] H. Chi, K. Xu, X. Wu, Q. Chen, D. Xue, C. Song, P. Wang, Effect of acetylation on the properties of corn starch, Food Chem. 106 (2008) 923–928. [8] JECFA.Starches, Summary of Evaluations Performed by the Joint FAO/WHO Expert Committee on Food Additives, 2001. [9] Codex, F.C. Committee on Food Chemicals Codex. Food and Nutrition Board, Institute of Medicine, National Academy of Sciences. National Academy Press, Washington, DC, 1996. [10] FAO, http://faostat.fao.org/beta/en/#data/QC 2018, Accessed date: 27 December 2018. [11] K.S. Sandhu, A.K. Siroha, Relationships between physicochemical, thermal, rheological and in vitro digestibility properties of starches from pearl millet cultivars, LWTFood Sci. Technol. 83 (2017) 213–224. [12] A.K. Siroha, K.S. Sandhu, Physicochemical, rheological, morphological, and in vitro digestibility properties of cross-linked starch from pearl millet cultivars, Int. J. Food Prop. 21 (2018) 1371–1385. [13] K.S. Sandhu, N. Singh, Relationships between selected properties of starches from different corn lines, Int. J. Food Prop. 8 (2005) 481–491. [14] D.L. Phillips, H. Liu, D. Pan, H. Corke, General application of raman spectroscopy for the determination of level of acetylation in modified starches 1, Cereal Chem. 76 (1999) 439–443. [15] O.B. Wurzburg, Starch, Modified Starch and Dextrin. In: Products of the Corn Refining Industry; Seminar Proceedings, Corn Refiners Association, Inc, Washington, DC, 1978 22–32. [16] P.C. Williams, F.D. Kuzina, L. Hlynka, A rapid calorimetric procedure for estimating the amylose content of starches and flour, Cereal Chem. 47 (1970) 411–421. [17] H.W. Leach, L.D. McCowen, T.J. Schoch, Structure of the starch granule I. Swelling and solubility patterns of various starches, Cereal Chem. 36 (1959) 534–544. [18] S. Park, M.G. Chung, B. Yoo, Effect of octenyl succinylation on rheological properties of corn starch pastes, Starch/Stärke 56 (2004) 399–406. [19] H.N. Englyst, S.M. Kingman, J.H. Cummings, Classification and measurement of nutritionally important starch fractions, Eur. J. Clin. Nutr. 46 (1992) S33–S50. [20] H.J. Chung, H.S. Lim, S.T. Lim, Effect of partial gelatinization and retrogradation on the enzymatic digestion of waxy rice starch, J. Cereal Sci. 43 (2006) 353–359. [21] S. Garg, A.K. Jana, Characterization and evaluation of acylated starch with different acyl groups and degrees of substitution, Carbohydr. Polym. 83 (2011) 1623–1630. [22] K. Shon, B. Yoo, Effect of acetylation on rheological properties of rice starch, Starch/ Stärke 58 (2006) 177–185. [23] I.A. Wani, D.S. Sogi, B.S. Gill, Physicochemical properties of acetylated starches from some Indian kidney bean (Phaseolus vulgaris L.) cultivars, Int. J. Food Sci. Technol. 47 (2012) 1993–1999. [24] M.V. Lawal, M.A. Odeniyi, O.A. Itiola, Material and rheological properties of native, acetylated, and pregelatinized forms of corn, cassava, and sweet potato starches, Starch/Stärke 67 (2015) 964–975. [25] H. Singh, N.S. Sodhi, N. Singh, Structure and functional properties of acetylated sorghum starch, Int. J. Food Prop. 15 (2012) 312–325. [26] R.L. Whistler, J.R. Daniel, Carbohydrates, in: O.R. Fennema (Ed.), Food Chemistry, Marcel Dekker, New York 1995, pp. 69–137. [27] A.D. Betancur, G.L. Chel, H.E. Canizares, Acetylation and characterization of Canavalia ensiformis starch, J. Agric. Food Chem. 45 (1997) 378–382. [28] K.O. Adebowale, T.A. Afolabi, B.I. Olu-Owolabi, Functional, physicochemical and retrogradation properties of sword bean (Canavalia gladiata) acetylated and oxidized starches, Carbohydr. Polym. 65 (2006) 93–101. [29] H.L. Lee, B. Yoo, Dynamic rheological and thermal properties of acetylated sweet potato starch, Starch/Stärke 61 (2009) 407–413. [30] S.J. McGrane, D.E. Mainwaring, H.J. Cornell, C.J. Rix, The role of hydrogen bonding in amylose gelation, Starch/Stärke 56 (2004) 122–131. [31] H. Liu, L. Ramsden, H. Corke, Physical properties of cross-linked and acetylated normal and waxy rice starch, Starch/Stärke 51 (1999) 249–252. [32] P.F. Suma, A. Urooj, Isolation and characterization of starch from pearl millet (Pennisetum typhoidium) flours, Int. J. Food Prop. 18 (2015) 2675–2687. [33] J.A. Han, J.N. BeMiller, Preparation and physical characteristics of slowly digesting modified food starches, Carbohydr. Polym. 67 (2007) 366–374. [34] H.J. Chung, D.H. Shin, S.T. Lim, In vitro starch digestibility and estimated glycemic index of chemically modified corn starches, Food Res. Int. 41 (2008) 579–585. [35] T.M. Ali, A. Hasnain, Physicochemical, morphological, thermal, pasting, and textural properties of starch acetates, Food Rev. Int. 32 (2016) 161–180. [36] M. Kaur, K.S. Sandhu, S.T. Lim, Microstructure, physicochemical properties and in vitro digestibility of starches from different Indian lentil (Lens culinaris) cultivars, Carbohydr. Polym. 79 (2010) 349–355.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Physicochemical, rheological, morphological and in vitro digestibility properties of pearl millet starch modified at varying levels of acetylation Anil Kumar Siroha a, Kawaljit Singh Sandhu a,b, Maninder Kaur c,⁎, Varinder Kaur d a
Department of Food Science and Technology, Chaudhary Devi Lal University, Sirsa, India Department of Food Science and Technology, Maharaja Ranjit Singh Punjab Technical University, Bathinda, India Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India d Department of Chemistry, Guru Nanak Dev University, Amritsar, India b c
a r t i c l e
i n f o
Article history: Received 30 January 2019 Received in revised form 27 February 2019 Accepted 25 March 2019 Available online 26 March 2019 Keywords: Pearl millet starch Physicochemical Morphological Rheological properties
a b s t r a c t Pearl millet (PM) starch was reacted with acetic anhydride at different concentrations (1.25, 2.5, 3.75 & 5.0%) and its physicochemical, rheological, morphological and in vitro digestibility properties were compared with native starch. The acetyl (%) and degree of substitution (DS) of acetylated starches ranged between 1.07 and 2.15% and 0.040–0.081, respectively. Swelling power and solubility of acetylated PM starch increased progressively upto 3.75% level of acetylation, however, further increase in acetylation levels resulted in a decrease. Peak, setback and final viscosities of acetylated PM starches were higher than their native counterpart starch. Both native and acetylated PM starches showed similar A-type X-ray diffraction patterns. During heating, storage modulus (G ′) and loss modulus (G″) of acetylated starches ranged between 753 and 1177 Pa and 96–129 Pa, respectively. G′ was much higher than G′′ at all the values of angular frequency studied. Both native and acetylated PM starch pastes showed flow behaviour index value of b1. For acetylated starches, both, yield stress and consistency index values were higher than native starch. Readily digestible and slowly digestible starch contents of acetylated PM starches varied between 44.3 and 45.5% and 27.3–32.5%, respectively, the highest values observed for starch acetylated at 3.75% and 1.25% acetic anhydride concentrations. © 2019 Elsevier B.V. All rights reserved.
1. Introduction The industrial food applications of native starches are limited due to their low shear resistance, thermal decomposition and high tendency towards retrogradation [1]. These can be improved by modifications using physical, chemical and enzymatic methods. The free hydroxyl groups present at 2, 3 and 6 carbons of the glucose molecule enable native starch to be modified by different chemical treatments [2]. The chemical modification of starch can be done by employing acid hydrolysis, oxidation, esterification, etherification and cross-linking methods [3]. The physico-chemical and functional properties of starch can be improved by acetylation [4]. The number of acetyl groups incorporated into the starch molecule during acetylation depends on many factors, such as reactant concentration, starch source [5], reaction time, and the presence of a catalyst [6]. Starch acetylation results in an increased solubility and a decrease in bond strength, which improves clarity and freeze-thaw stability [7]. Food and Drug Administration recommends not N2.5% acetyl groups in starch acetate [8,9]. Pearl millet (Pennisetum glaucum) belongs to family Poaceae and is widely grown around the world for feed and fodder. Being drought ⁎ Corresponding author. E-mail address: [email protected] (M. Kaur).
https://doi.org/10.1016/j.ijbiomac.2019.03.179 0141-8130/© 2019 Elsevier B.V. All rights reserved.
tolerant cereal crop, it is grown primarily in India and Africa. India ranks first in annual production of millets (11,420,000 t in 2014), followed by Niger which produced 3,321,753 t [10]. Pearl millet (PM) has a starch content of 60–70% which is comparable with corn and this starch can be isolated easily from it. In our earlier study on Indian PM cultivars, we characterized starches isolated from these cultivars for their physicochemical, morphological, thermal, rheological and in vitro digestion properties [11]. Further, these PM starches were cross-linked by epichlorohydrin (0.5%) and studied for their various functional properties [12]. To the best of our knowledge no work has been reported on the acetylation of PM starch. In order to explore utilization of PM starch, the present investigation was aimed at acetylation of PM starch at varying levels of acetylation and analyzed for their physicochemical, rheological, morphological and in vitro digestibility properties. 2. Material and methods 2.1. Materials and starch isolation Pearl millet cultivar (HC-10) was procured from Chaudhary Charan Singh Haryana Agriculture University, Hissar, India. All chemicals used were of analytical grade. Starch was isolated from pearl millet grains
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by following the centrifugation method described by Sandhu and Singh [13]. Pearl millet grains (500 g) were added to 1.25 l of distilled water containing 0.1% sodium meta bisulphite. The mixture was maintained at 50 °C for 18–20 h with intermittent circulation of liquid. The steep water was then drained off and grains were ground in laboratory grinder (Maxie Plus, New Delhi, India). About 250 g of steeped grains were ground with 250 ml of distilled water. The ground slurry was passed through 250, 150, 100, 75, 45 mm mesh sieve. The starchprotein slurry was then allowed to stand for 4–5 h. The supernatant was removed by suction and the settled starch layer was resuspended in distilled water and centrifuged in wide mouthed cup centrifuge (Remi, New Delhi, India) at 3000 rpm for 10 min and the upper non-white layer was scrapped off. The white layer was re-suspended in distilled water and re-centrifuged 3–4 times. The starch was then collected and dried in an oven (NSW-143, New Delhi, India) at 45 °C for 12 h. 2.2. Preparation of acetylated starch, acetyl (%) and degree of substitution (DS) PM starch was acetylated by following the method described by Phillips et al. [14] using acetic anhydride at varying concentrations (1.25, 2.5, 3.75, 5.0 g). The percent acetylation (acetyl%) and degree of substitution (DS) were determined by following the method described by Wurzburg [15]. The acetyl (%) was calculated as: Acetyl ð%Þ ¼
ðBlank–SampleÞ Molarity of HCl 0:043 100 Sample weight
2.3. Amylose content, swelling power and solubility Amylose content of PM starches was determined by following the method described by Williams et al. [16]. Starch (20 mg) was thoroughly mixed with 10 ml of 0.5 N KOH and the dispersed sample were diluted to 100 ml with distilled water. An aliquot of test starch solution (10 ml) was pipetted into 50 ml volumetric flask and 5 ml of 0.1 N HCl and 0.5 ml of iodine reagent were added. The volume was diluted to 50 ml and the absorbance was measured at 625 nm in a spectrophotometer (Systronics, Ahmadabad, India). The measurement of the amylose was determined in triplicate from a standard curve developed using amylose and amylopectin blends. The method described by Leach et al. [17] was followed for swelling power and solubility measurements. Starch (1 g) was added to 99 ml of distilled water and heated to 90 °C for 1 h. The heated samples were rapidly cooled to 25 °C and centrifuged at 3000 rpm for 30 min. For measuring solubility, the supernatants were drained into pre-weighed moisture dishes, evaporated to dryness in a hot air oven at 100 °C and the dishes were reweighed. 2.4. Pasting properties The pasting properties of PM starches were determined using an inbuild starch cell of Modular Compact Rheometer (Model-52, Anton Paar, Austria). Starch slurries (1.2 g starch in 13.8 g distilled water)
were held at 50 °C for 1 min and then heated from 50 to 95 °C at a heating rate of 6 °C/min, held for 2.7 min, cooled to 50 °C at the same rate and again held at 50 °C for 2 min. Each sample was analyzed in triplicate. Peak viscosity, breakdown, setback, final viscosity and pasting temperature were obtained from the pasting graph. 2.5. Rheological properties 2.5.1. Dynamic properties A small amplitude oscillatory rheological measurement was made for starches with a Modular Compact Rheometer (Model-52, Anton Paar, Austria) equipped with parallel plate system (0.04 m diameter). The gap size, strain and frequency were set at 1000 μm, 2% and 10 rad/s, respectively. The linear viscoelastic range (LVR) was selected at a strain of 0.02%, and all experiments were carried out at the selected strain. The dynamic rheological properties, such as storage modulus (G′), loss modulus (G″) and loss factor (tanδ) were determined for the starches. Starch suspension of 10% concentration was loaded onto the ram of rheometer and covered with a thin layer of lowdensity silicon oil (to minimize evaporation losses). The starch samples were subjected to temperature sweep testing by heating them from 45 to 95 °C at the rate of 2 °C/min. For frequency sweep measurement, starch slurry (10%) was prepared, stirred and then heated at 90 °C in a water bath (Brookefield, TC-202, USA). The slurry was again stirred for 3 min and then cooled to room temperature. It was then loaded on the ram of rheometer and frequency sweep tests from 0.1 to 100 rad/s were performed at 25 °C. Parameters recorded were G′, G′′, and tan δ. 2.5.2. Steady shear measurement Steady shear properties of native and acetylated PM starches were determined by following the method described by Park et al. [18] with slight modification. The sample preparation method has been described in frequency sweep measurement method. The sample (10%) was sheared continuously from 1 to 100 s−1. The data was fitted to Herschel-Bulkley model to describe the variation in the rheological properties:
σ ¼ σ o þ Kð γ̇Þn
where σ is the shear stress (Pa), σo is the yield stress, γ̇ is the shear rate (s−1), K is the consistency index (Pa.sn), n is the flow behaviour index (dimensionless). 2.6. X-ray diffraction X-ray diffraction patterns of PM starches were recorded with a wavelength of 0.154 nm using X-ray diffractometer (Rigaku Miniflex, Japan). The diffractometer was operated at 45 kV and 40 mA. Diffractograms were acquired at 25 °C over a 2θ range of 4–40 with a step size of 0.02 and sampling interval of 10 s.
Table 1 Acetyl (%), degree of substitution (DS), amylose content, swelling power and solubility of native and acetylated PM starches. Acetic anhydride (%)
Acetyl (%)
DS
Amylose content (%)
Swelling power (g/g)
Solubility (%)
0 1.25 2.50 3.75 5.0
0.00a 1.07 ± 0.1b 1.50 ± 0.2c 1.72 ± 0.0d 2.15 ± 0.1e
0.00a 0.040 ± 0.00b 0.057 ± 0.01c 0.065 ± 0.01d 0.081 ± 0.00e
14.1 ± 0.5d 10.5 ± 0.3a 12.3 ± 0.2b 13.5 ± 0.6c 15.4 ± 0.2e
14.0 ± 0.6a 15.0 ± 0.7c 16.0 ± 0.5d 17.6 ± 0.6e 14.8 ± 0.8b
10.4 ± 0.4a 15.4 ± 0.3c 17.2 ± 0.1d 19.2 ± 0.2e 11.5 ± 0.1b
Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
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hydrolysing within 20 min of incubation was termed as readily digestible starch (RDS), fraction digested during the period between 20 and 120 min was slowly digestible starch (SDS), whereas resistant starch (RS) was that fraction which was not hydrolyzed with 120 min of incubation. 2.9. Statistical analysis The data reported in the tables were analyzed in triplicate and subjected to one-way analysis of variance (ANOVA) using Minitab Statistical Software version 15 (Minitab, Inc., State College, USA). 3. Results and discussion 3.1. Acetyl (%) and degree of substitution
Fig. 1. Pasting profiles of native and acetylated starches (A: Native starch; B: 1.25% acetic anhydride; C: 2.5% acetic anhydride; D: 3.25% acetic anhydride; E: 5.0% acetic anhydride).
2.7. Morphological properties The morphological properties of starches were studied by scanning electron microscope (Model EVOLS10 ZEISS, Oberkochen, Germany) using 1% starch suspension made by dissolving starch in ethanol. One drop of the suspension was applied on an aluminium stub, followed by coating with gold‑palladium (60:40). During micrography, an accelerating potential of 5 kV was used. 2.8. in vitro starch digestibility in vitro starch digestibility was analyzed by following the method described by Englyst et al. [19] and as modified by Chung et al. [20]. Porcine pancreatic alpha-amylase (No. 7545, Sigma-Aldrich, St. Louis, MO), and amyloglucosidase (No. 9913, Sigma-Aldrich) (3.89 g) were used for the analysis. Glucose content was measured using glucose oxidase and peroxidase assay kits (No. GAGO-20, Sigma-Aldrich). The starch fraction
Both acetyl (%) and DS of the acetylated PM starches increased with an increase in acetic anhydride concentration; their values ranged between 1.07 and 2.15% and 0.040–0.081, respectively (Table 1). Garg and Jana [21] also reported an increase in both DS and acetyl (%) for acetylated corn starch with an increase in concentration of acetic anhydride. The observed values of DS in the present study are in agreement with those reported by other workers such as 0.04–0.10 for acetylated Indian horse chestnut [4], 0.025–0.104 for acetylated rice [22], 0.115–0.154 for acetylated potato [5] and 0.03–0.08 for acetylated kidney bean starches [23]. 3.2. Amylose content, swelling power and solubility Amylose content of acetylated PM starches ranged between 10.5 and 15.4%, the highest and the lowest values were observed for starches modified at 5.0% and 1.25% acetic anhydride concentration, respectively (Table 1). Amylose content of acetylated starches were lower as compared to native PM starch at low acetic anhydride concentrations (1.25, 2.5 & 3.75%). However, at higher acetic anhydride concentration (5.0%) amylose content was observed to be higher (15.4%) than the native counterpart PM starch. Lawal et al. [24] reported a decrease in amylose content for acetylated corn, cassava and sweet potato starches in comparison to their counterpart native starches and attributed this to the leaching out of amylose during acetylation. Contrarily, an increase in the amylose content of acetylated starches from sorghum was observed by Singh et al. [25]. The decrease/increase in an amylose content
Table 2 Pasting properties of native and acetylated PM starches. Acetic anhydride (%)
Peak viscosity (mPa.s)
Breakdown viscosity (mPa.s)
Trough viscosity (mPa.s)
Setback viscosity (mPa.s)
Final viscosity (mPa.s)
Pasting temperature (°C)
0 1.25 2.5 3.75 5.0
1069 ± 15a 1301 ± 17b 1336 ± 12c 1379 ± 14d 1772 ± 19e
508 ± 7b 503 ± 5b 473 ± 8a 526 ± 5c 503 ± 7b
561 ± 5a 798 ± 5b 863 ± 6c 853 ± 5c 1269 ± 8d
352 ± 4a 606 ± 5c 556 ± 4b 632 ± 6c 851 ± 8d
913 ± 10a 1404 ± 12b 1419 ± 17b 1485 ± 11c 2120 ± 18d
81.2 ± 0.1c 80.6 ± 0.3b 80.6 ± 0.2b 79.4 ± 0.2a 84.2 ± 0.1d
Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
Table 3 Rheological properties of native and acetylated PM starches during heating. Acetic anhydride (%)
G′ (Pa)
G′′ (Pa)
Breakdown in G′ (Pa)
tan δ (G′′/G′)
TG′ (°C)
0 1.25 2.5 3.75 5.0
737 ± 9a 753 ± 11b 761 ± 13b 1147 ± 7c 1177 ± 9d
108 ± 4b 96 ± 2a 96 ± 1a 98 ± 2a 129 ± 3c
242 ± 5d 150 ± 6a 175 ± 4b 233 ± 6d 198 ± 3c
0.14b 0.12a 0.12a 0.08a 0.10a
75.0 ± 0.3a 85.0 ± 0.1b 85.0 ± 0.3b 85.1 ± 0.2b 85.0 ± 0.2b
Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
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after acetylation may be attributed to the interference of acetyl group with the functioning of amylose and amylopectin fractions of starch thereby affecting absorption of iodine during determination of amylose [26,27]. The swelling power (SP) and solubility of acetylated starches ranged between 14.8 and 17.6 g/g and 11.5–19.2%, respectively, the highest values were observed when acetylation was carried out at 3.75% acetic anhydride concentration. Starch modified at lower concentrations (1.25 to 3.75%) of acetic anhydride showed higher swelling power and solubility while at higher concentration (5.0%), a decrease was observed. On the other hand, the native PM starch showed lower SP (14 g/g) and solubility (10.4%) in comparison to acetylated starches. The increase in swelling and solubility of acetylated starches may be due to the introduction of acetyl groups to starch which could facilitate an access of water to amorphous areas [23]. 3.3. Pasting properties The pasting profiles of native and acetylated PM starches are shown in Fig. 1. Peak viscosity (PV) of acetylated starches ranged between 1301 and 1772 mPa.s, the highest and the lowest values were observed when starch was modified at 5.0 and 1.25% acetic anhydride concentration levels, respectively (Table 2). Breakdown viscosity (BV) of acetylated PM starches varied between 473 and 526 mPa.s, the highest value was observed when acetylation was carried out at 3.75% acetic anhydride concentration. Setback viscosity (SV) of acetylated starches ranged between 556 and 851 mPa.s. Final viscosity (FV) of acetylated PM starches was in the range between 1404 and 2120 mPa.s, the highest at 5.0% acetic anhydride concentration while the lowest was observed at 1.25% concentration. PV, TV, SV and FV of acetylated PM starches were higher than their counterpart native PM starch. Starch-starch interactions reduced and viscosity of the starch granules increased due to introduction of the bulky acetyl group in the starch molecules after acetylation [28]. 3.4. Rheological properties 3.4.1. Dynamic shear properties Rheological properties of starch suspensions from native and acetylated PM starches differed significantly (p b 0.05) during heating (Table 3). For native as well as acetylated starches, G′ and G′′ increased progressively during heating. The values of G′ and G′′ of acetylated PM starches ranged between 753 and 1177 Pa and 96–129 Pa, respectively, the highest values for both moduli were observed when acetylation was conducted at 5.0% acetic anhydride concentration. tan δ (G′′/G′) for both native as well as acetylated PM starches was b1 indicating their elastic behaviour. The increase in peak G′ of acetylated PM starches might be due better capture and retention of water molecules by acetyl groups thereby leading to the formation of more arranged structures, which were resistant to deformation [27]. Breakdown in G′ varied between 150 and 233 Pa, acetylated starches exhibited lower values in comparison to native counterpart (242 Pa) PM starch. The temperature at which G′ was maximum (TG′) showed a non-significant (p b 0.05) variation among acetylated starches; however, their values were higher than TG ′ of native starch. Singh et al. [5] also reported an increase in G′ and G′ ′ values after acetylation for corn and potato starches during heating. Fig. 2(A-C) show the change in the G′, G′′ and tan δ for native and acetylated PM starch pastes as a function of the frequency. Table 4 lists G′, G′′, tan δ and complex viscosity (η*) values for starch pastes at 6.28 rad/s and 25 °C. G′ for all the PM starch pastes showed an increase upon increase in angular frequency (ω); values of G′ (1216 to 1557 Pa) were found much higher than G′′ (64.7 to 76.0 Pa) at all values of ω, reflecting the visco-elastic behaviour of starch pastes. The behavior
Fig. 2. Angular frequency dependence at 25 °C for native and acetylated starches (A: G'; B: G"; C: tanδ) (Representations for curves are: A, Native; B, 1.25% acetic anhydride; C, 2.5% acetic anhydride; D, 3.25% acetic anhydride; E, 5.0% acetic anhydride).
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Table 4 Storage modulus (G′), loss modulus (G′′), loss factor (tan δ) and complex viscosity (η *) at 6.28 rad/s (at 25 °C) for native and acetylated PM starches. Acetic anhydride (%)
G′ (Pa)
G′′ (Pa)
tan δ
η * (Pa.s)
0 1.25 2.5 3.75 5.0
1216 ± 16a 1227 ± 15a 1296 ± 11b 1519 ± 18c 1557 ± 14d
73.8 ± 5c 64.7 ± 4a 65.1 ± 5b 76.0 ± 2d 65.5 ± 3b
0.03a 0.05a 0.05a 0.05a 0.04a
193 ± 5a 195 ± 6a 206 ± 4b 242 ± 4c 248 ± 2c
Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
observed for the starch pastes were similar to those of typical weak gels. No crossover between the two moduli was observed throughout the studied frequency range (0.1–100 rad/s), which reflects the stability of native and acetylated starches at this frequency range as well as weak dependence of G′ and G′′ values on the frequency. The highest and the lowest G′ values for acetylated starch pastes were observed when acetylation was carried out at 5.0% and 1.25% levels of acetic anhydride, respectively. tan δ (G′′/G′) values of acetylated starches ranged from 0.04 to 0.05. Acetylated PM starches showed higher G′ values as compared to the native starch, which might be attributed to their higher swelling power. Lee and Yoo [29] also reported an increase in G′ and G′′ values of sweet potato starch upon acetylation.
3.4.2. Steady shear properties Steady shear properties of native and acetylated PM starches are reported in Table 5. The flow behaviour index (n) of acetylated starches, which indicates the extent of shear-thinning behavior, ranged between 0.275 and 0.310, the highest value for starch acetylated at 2.5% acetic anhydride level was observed. Shear thinning behaviour might be due to cumulative orientation of soluble starch molecules in the direction of the flow as well as the breaking of hydrogen bonds formed between amylose molecules during shearing [30]. The K (consistency index) for acetylated PM starches varied between 43.5 and 52.8 Pa.s, which were higher than their native counterpart starch. Among acetylated starches, the highest K value was observed for starch acetylated at 1.25% acetic anhydride concentration. The higher K values of acetylated starches might be due to higher viscosity of acetylated starches than native starch. Yield stress value (σο ) of acetylated PM starches varied between 26.6 and 50.7 Pa, the highest value was observed when starch was acetylated at 5.0% acetic anhydride concentration. The value of σ ο increased upon an increase in the acetic anhydride concentration. Liu et al. [31] reported that the acetylation reduces interactions between starch chains due to the presence of hydrophilic substituents and increases the ability of granules to swell by loosening granule structure, resulting in an increase in both swelling power and solubility.
Table 5 Herschel-Bulkey model fitted to native and acetylated PM starch pastes during steady shear rate. Acetic anhydride (%)
σο (Pa)
K (Pa.sn)
n
R2
0 1.25 2.5 3.75 5.0
25.5 ± 2.12a 26.6 ± 3.31b 35.8 ± 2.42c 37.1 ± 3.76d 50.7 ± 5.45e
31.3 ± 1.21a 52.8 ± 2.48c 43.5 ± 3.61b 44.4 ± 2.33c 49.0 ± 4.51d
0.320 ± 0.008d 0.275 ± 0.003a 0.310 ± 0.005c 0.305 ± 0.005b 0.308 ± 0.002c
0.999 0.999 0.999 0.999 0.999
σo = yield stress; K = consistency index; n = flow behaviour index. Means followed by same superscript within a column do not differ significantly (p b 0.05). Mean (±standard deviation) of triplicate analysis.
Fig. 3. X-ray diffraction pattern of native and acetylated starches (A: Native; B: 1.25% acetic anhydride; C: 2.5% acetic anhydride; D: 3.25% acetic anhydride; E: 5.0% acetic anhydride).
3.5. X-ray diffraction (XRD) The X-ray diffraction patterns of native and acetylated PM starches are shown in Fig. 3. All the starches showed A-type diffraction patterns with peaks at 15o, 17o, 18o and 23o (2θ), which is a characteristic of cereal starches. Suma and Urooj [32] reported A-type diffraction patterns for Indian pearl millet varieties with sharp peaks at 15o and 23o and diffused peaks at 17o and 18o (2θ). Acetylated PM starches also showed XRD pattern similar to that of counterpart native PM starch. 3.6. Morphological properties Scanning electron micrographs (SEM) of native and acetylated PM starches are shown in Fig. 4. Native starch showed starch granules varying in size and shape from small to large, spherical and polygonal. At lower concentrations of acetic anhydride, no detectable changes in the starch morphology were noticed. However, starch acetylated at 3.75 and 5.0% acetic anhydride concentrations, showed cavities on the surface of few starch granules. Acetylation changed external morphology of starch granules which might result in surface roughness, development of cavities, and binding of few starch granules [25]. 3.7. in vitro starch digestibility RDS, SDS and RS content of native and acetylated PM starches are shown in Table 6. RDS and SDS contents of acetylated starches varied between 44.3 and 45.5% and 27.3–32.5%, respectively, which were significantly lower than those observed for native counterpart PM starch. Among acetylated starches, the highest RDS and SDS contents were observed when starch was acetylated at 3.75% and 1.25% acetic anhydride concentrations, respectively. RS content of acetylated PM starches were significantly higher than native PM starch, values ranging between 22.5 and 27.3%, the highest value was observed for starch acetylated at 5.0%
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(A)
(B)
(C)
(D)
(E) Fig. 4. Morphological properties of native and acetylated starches (A: Native; B: 1.25% acetic anhydride; C: 2.5% acetic anhydride; D: 3.25% acetic anhydride; E: 5.0% acetic anhydride).
acetic anhydride concentration. Han and BeMiller [33] reported that acetylation, hydroxypropylation, and cross linking, reduced the extent of enzyme-catalyzed starch hydrolysis. Chung et al. [34] reported a decrease in SDS and RDS contents and an increase in RS content for corn starch acetylated at 6% acetic anhydride concentration. Ali and Hasnain
[35] reported non-significant correlation between RS content and degree of substitution; and suggested that some of the acetyl groups inserted as a result of modification could change the spatial structure of starch. Alpha-amylase may fail to act on this chemically altered starch structure resulting in reduced digestibility and higher RS content. Kaur
A.K. Siroha et al. / International Journal of Biological Macromolecules 131 (2019) 1077–1083 Table 6 in vitro digestibility properties of native and acetylated PM starches. Acetic anhydride (%)
RDS (%)
SDS (%)
RS (%)
0 1.25 2.5 3.75 5.0
49.9 ± 0.4d 45.0 ± 0.1b 44.3 ± 0.2a 45.5 ± 0.4c 45.4 ± 0.5c
38.1 ± 0.2e 32.5 ± 0.3d 31.6 ± 0.4c 29.5 ± 0.3b 27.3 ± 0.2a
12.0 ± 0.3a 22.5 ± 0.3b 24.1 ± 0.6c 25.2 ± 0.5d 27.3 ± 0.7e
RDS = readily digestible starch; SDS = slowly digestible starch; RS = resistant starch. Values within the same column followed by the same superscript are not significantly different (p b 0.05). Mean (±standard deviation) of triplicate analysis.
et al. [36] reported potential physiological benefits and unique functional properties of RS, similar to that of dietary fibers. 4. Conclusions Acetylation done using acetic anhydride at varying levels caused a significant change in physicochemical, pasting, rheological and in vitro digestibility properties of PM starch. Acetyl content and DS increased upon an increase in acetic anhydride concentration. Both swelling power and solubility increased with DS, however, the reverse trend was observed at higher DS. Pasting properties after acetylation also showed an increase as compared to native PM starch. G′ and G′′ of native and acetylated PM starches showed no crossover throughout the observed frequencies range, reflecting starch stability. Morphology of acetylated starches differed slightly from native PM starch at higher concentrations with a few starch granules having surface pores. Acetylation resulted in decrease in RDS and SDS contents while increase in RS content. Increase in degree of acetylation also resulted in a significant increase in RS content. Properties of pearl millet starch should be explored further by modifying it with different chemical agents so as to increase its utilization. Compliance with ethical standards The article does not contain any studies with human and animal subjects. Conflict of interest The authors declare no conflict of interest for this article. Acknowledgements The financial support provided by Chaudhary Devi Lal University, Sirsa, India (R&S/A-1/13/66-11/1263-1277) in the form of scholarship to Anil Kumar Siroha is deeply acknowledged. References [1] R. Hui, C. Qi-He, F. Ming-liang, X. Qiong, H. Guo-qing, Preparation and properties of octenyl succinic anhydride modified potato starch, Food Chem. 114 (2009) 81–86. [2] J. Bao, J. Xing, D.L. Phillips, H. Corke, Physical properties of octenyl succinic anhydride modified rice, wheat, and potato starches, J. Agric. Food Chem. 51 (2003) 2283–2287. [3] L. Jayakody, R. Hoover, The effect of lintnerization on cereal starch granules, Food Res. Int. 35 (2002) 665–680. [4] I.A. Wani Shubeena, A. Gani, P. Sharma, T.A. Wani, F.A. Masoodi, A. Hamdani, S. Muzafar, Effect of acetylation on the physico-chemical properties of Indian Horse Chestnut (Aesculus indica L.) starch, Starch/Stärke 67 (2015) 311–318.
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