Effect of modification with 1,4-α-glucan branching enzyme on the rheological properties of cassava starch

Effect of modification with 1,4-α-glucan branching enzyme on the rheological properties of cassava starch

Accepted Manuscript Title: Effect of modification with 1,4-␣-glucan branching enzyme on the rheological properties of cassava starch Authors: Yadi Li,...

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Accepted Manuscript Title: Effect of modification with 1,4-␣-glucan branching enzyme on the rheological properties of cassava starch Authors: Yadi Li, Caiming Li, Zhengbiao Gu, Yan Hong, Li Cheng, Zhaofeng Li PII: DOI: Reference:

S0141-8130(16)31941-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.05.045 BIOMAC 7534

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

10-10-2016 28-4-2017 11-5-2017

Please cite this article as: Yadi Li, Caiming Li, Zhengbiao Gu, Yan Hong, Li Cheng, Zhaofeng Li, Effect of modification with 1,4-␣-glucan branching enzyme on the rheological properties of cassava starch, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.05.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of modification with 1,4-α-glucan branching enzyme on

the rheological properties of cassava starch Yadi Li b, Caiming Li a,b,c, Zhengbiao Gu a,b,c,*, Yan Hong a,b,c, Li Cheng a,b, Zhaofeng Li a,b,c,* a

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122,

China b

c

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi,

Jiangsu 214122, China *Correspondence:

Zhaofeng Li and Zhengbiao Gu, School of Food Science and Technology,

Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China. Tel/fax: +86-510-85329237 E-mail address: [email protected] (Z. Li) ; [email protected] (Z. Gu).

Highlights 

GBE modification improved the fluidity and the shear resistance of cassava starch paste.



GBE modification produced cassava starch paste with a more structured glucan network.



GBE modification made cassava starch paste more elastic with higher gel rigidity.



GBE modification reduced the frequency and temperature dependence of cassava starch paste.

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Abstract Steady and dynamic shear measurements were used to investigate the rheological properties of cassava starches modified using the 1,4-α-glucan branching enzyme (GBE) from Geobacillus thermoglucosidans STB02. GBE treatment lowered the hysteresis loop areas, the activation energy (Ea) values and the parameters in rheological models of cassava starch pastes. Moreover, GBE treatment increased its storage (G') and loss (G") moduli, and decreased their tan δ (ratio of G"/G') values and frequency-dependencies. Scanning electron microscopic studies showed the selective and particular attack of GBE on starch granules, and X-ray diffraction analyses showed that GBE treatment produces significant structural changes in amylose and amylopectin. These changes demonstrate that GBE modification produces cassava starch with a more structured network and improved stability towards mechanical processing. Differential scanning calorimetric analysis and temperature sweeps indicated greater resistance to granule rupture, higher gel rigidity, and a large decrease in the rate of initial conformational ordering with increasing GBE treatment time. Pronounced changes in rheological parameters revealed that GBE modification enhances the stability of cassava starch and its applicability in the food processing industry. Keywords: cassava starch; 1,4-α-glucan branching enzyme; steady shear rheology; dynamic shear rheology; temperature dependence 1. Introduction Starch is an important food additive because of its thickening and gelling properties. It helps provide proper texture and controls moisture mobility, improving the quality and stability of processed foods [1, 2]. The rheological properties of starch products are the 2

major functional properties that govern their use in food processing applications. Rheological properties are closely related to the quality of starch-based foods (e.g., hardness, stickiness, and chewiness), as well as being crucial in processing-related parameters such as transportation, agitation, mixing, and energy consumption [3]. The key rheological characteristics of starch pastes include their flow behavior, their viscoelastic properties, and the effect of temperature on the behavior of the starch paste. Starches with different molecular conformations and structures exhibit different flow behaviors and deformation characteristics in response to applied stress [4, 5]. The rheological properties of starch are very sensitive to several factors, including the type of starch, temperature, starch concentration, pH, and the presence and concentration of other components (gums, proteins, salts and acids). In particular, the amylose/amylopectin ratio, molecular weight, and the chain length distribution of starch have tremendous effects on its rheological behavior [6, 7]. Native starch is often extensively modified to achieve the rheological properties necessary for specific industrial applications [8, 9]. In previous studies, 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) has been shown to catalyze the intramolecular or intermolecular transglycosylation of starch molecules through the cleavage of α-1,4-glycosidic bonds and subsequent transfer of the cleaved oligosaccharide to create α-1,6 branches [10-12]. Thus, GBE decreases the amylose content of starch and produces shorter branches within a more highly branched structure [13, 14]. A higher proportion of short-chain amylopectin results in a greater decrease in the viscosity of starch, and a lower amylose content means a better structured network [4, 15]. In particular, the rheological properties of starch modified by GBE are much less studied. Systematic investigation of the rheological properties of cassava starch may 3

comprehensively elucidate its functionality and physicochemical properties, enhancing its use in food industries. The objective of this study was to investigate the effect of GBE treatment on the rheological properties of cassava starch, thereby clarifying the relationships between its rheological parameters and molecular structure. The results should provide insights that allow us to understand and optimize the utilization of GBE-modified cassava starch. 2. Materials and methods 2.1 Materials Commercial cassava starch was purchased from Cargill, Inc. (Shanghai, China). The moisture content of the starch (12.63 wt%, wet basis) was determined by drying a sample to constant weight in an oven at 105 °C. The GBE from Geobacillus thermoglucosidans STB02 was expressed as a recombinant enzyme in Escherichia coli BL21 and purified using the established procedure [13]. 2.2 Preparation of GBE-treated cassava starch pastes Cassava starch suspensions (30%, w/v) were prepared in deionized water and adjusted to pH 7.5 with NaOH solution (1 M). The suspensions were incubated with GBE (300 U/g starch) in a water bath at 50 °C for 4, 6, 8, or 10 h. After the incubation, the starch samples were washed with deionized water and dried in a vacuum desiccator at 45 °C and approximately 0.01 MPa for 12 h. The final products, GBE-treated cassava starches, were obtained by passing the dried starches through a 100-mesh sieve [13].

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Modified and native cassava starch dispersions (5%, w/w) were moderately stirred for 10 min at ambient temperature, and then heated at 95 °C in a water bath for 30 min with mild agitation provided by a magnetic stirrer in order to obtain fully gelatinized pastes. At the end of the heating period, the hot starch pastes were immediately transferred to the rheometer plate for rheological measurements. 2.3 Scanning electron microscopy Starch samples were sprinkled onto circular aluminum stubs with double sticky tape. The excess powder was brushed to get a uniform layer of sparsely scattered powder particles. The stubs were coated with gold in a Hitachi 1B-3 ion coater. The ion current was maintained at 6 mA with a fine vacuum of 0.07 Torr for 4 min. Scanning electron microscopy (SEM) was performed using a Hitachi S-405 Electron Microscope at 2400× and 5000× magnification and an accelerating potential of 25 kV. The images of selected areas were recorded on black and white high-speed (200 ASA) photographic film with the help of an attached camera assembly [16]. 2.4 X-ray diffraction analysis The X-ray diffraction patterns of amylose (Cargill Amylogel 03003, 70% purity, USA), amylopectin (Sigma Aldrich 10120, 75% purity, USA) and cassava starches were determined using an X-ray diffractometer (Bruker AXS Inc., Germany) according to a published method [17]. The samples were exposed to the X-ray beam generated at 40 kV and 30 mA and containing Cu-Kα radiation. The scanning region of the diffraction angle (2θ) was from 3° to 40° with a rate of 2°/min and a step size of 0.05°. All samples were stored in a desiccator, where a saturated solution of NaCl maintained a constant-humidity atmosphere with a relative 5

humidity of 75%, at 25 °C for 1 week before measurements. The relative crystallinity of each starch sample was calculated from the ratio of the area of the sharp crystalline peak to the total area of the diffractogram using Jade 5.0 software (Materials Data Inc., Livermore, CA, USA) [13]. 2.5 Differential scanning calorimetric analysis Thermal analyses of starch samples were performed using a differential scanning calorimeter (DSC8000, Perkin–Elmer Co., USA). Each sample of 2.0 ± 0.1 mg was placed in an aluminum pan, and then deionized water was added to each with a microsyringe to achieve a solid-water ratio of 1:2 (w/w). The sample pans were hermetically sealed, equilibrated at room temperature for 24 h, and then heated from 40 to 95 °C at a heating rate of 5 °C/min. An empty aluminum pan was used as a reference. During the experiment, the space surrounding the sample chamber was filled with dry nitrogen. The onset temperature (To), peak temperature (Tp) and conclusion temperature (Tc) were determined from each thermogram. The gelatinization enthalpy (ΔHg) was calculated from the total peak area of the endotherm using differential scanning calorimetry software (Universal Analysis 2000 TA Instruments, USA). 2.6 Steady and dynamic shear rheological measurements Rheological measurements were carried out using a stress-controlled rheometer (DHR3; TA Instruments Ltd., Crawley, UK). Steady shear properties and small amplitude oscillatory rheological properties were obtained at 25 °C using a rheometer equipped with a parallel plate system (4 cm diameter) at a gap of 1000 μm. Before each experiment, the samples on the rheometer plate were equilibrated for 5 min. For steady shear 6

measurements, the samples were sheared continuously from 0.1 to 200 s–1 and their timedependent flow behaviors were determined using ascending and descending shear cycles. Dynamic shear properties were obtained from frequency sweeps over the range of 0.63–62.8 rad·s-1 at 2% strain. The 2% strain was in the linear viscoelastic region. Mechanical spectra were obtained by recording the storage modulus (G'), loss modulus (G") and loss tangent (tan δ = G"/G') as a function of frequency (ω). To determine the effect of temperature on rheological properties, the apparent viscosities at constant shear rates of 0.34 or 100 s-1, in the temperature range of 25–90 °C were measured [18]. 2.7 Temperature sweeps of cassava starch Cassava starch dispersions (5%, w/w) were subjected to temperature sweeps during heating and cooling. Before measurements were taken, the parallel plate was preheated to 40 °C and the sample was placed on it for 5 min to allow stress relaxation and temperature equilibration. The sample perimeter was covered with a thin layer of silicone oil to prevent sample dehydration. Strain and frequency were set at 2% and 6.28 rad·s-1, respectively. The temperature was increased from 40 to 95 °C and then decreased to 25 °C with holding for 5 min. The heating rate was 5 °C·min-1 and the cooling rate was 5 °C·min-1 [19]. 2.8 Statistical analysis All measurements were performed in triplicate. Results reported were obtained using TA rheometer Data Analysis software (version VI. 1.76). The mean and standard deviations of the data collected were calculated using SPSS 17.0 software (SPSS Incorporated, Chicago, Illinois, USA). 3. Results and discussion 7

3.1 Effect of GBE treatment on the morphology of cassava starch SEM was used to observe the submicroscopic shape and surface characteristics of cassava starch granules (Fig. 1). Native starch granules were generally round in shape, although a few possessed an irregular truncated structure. GBE-modified starch granules exhibited significant variations in shape, compared with native starch granules. Surface erosion and micro-holes as well as sparse cracks were observed in starch granules exposed to GBE for 4 h, indicating that the starch granules were attacked by GBE. With increasing GBE modification time (6 h to 8 h), the attack was selective and some starch granules exhibited an increased number of cracks and even large openings. After 10 h, the starch granules behaved in the same manner and the particular attack was evident; starch granules were broken, with larger openings. The GBE modification of starch granules is a selective process that can be quantified by determining the intensity and manner in which the erosion and corrosion take place [20]. Cassava starch granules were first hydrolyzed superficially. Larger openings were presented as the cracks became larger, allowing GBE access to the interior of the granules. This confirms that the selective attack and different susceptibilities of starch granules to GBE modification produce the particular characteristics of cassava starch. 3.2 Effect of GBE treatment on the X-ray diffraction patterns of cassava starch The crystalline properties of starch granules have been widely examined using X-ray diffraction studies. Native starch granules are semi-crystalline and can be found in two polymorphic forms (A- and B-types) that differ in conformation and structural water content [21]. Starches can be classified as A-type (A-type polymorph), B-type (B-type polymorph) or

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C-type (a mixture of A- and B-type polymorphs) depending upon the types of crystalline polymorphs present in their granules. In Fig.2 (A), the X-ray diffraction patterns of cassava starches are compared with the known diffraction patterns of A- and B-type polymorphs. The cassava starches showed strong diffraction peaks at 2θ values of approximately 15° and 23°, along with an unresolved doublet at 17° and 18°. These features are very similar to the typical A-type X-ray diffraction pattern seen in starches containing amylopectin with larger proportions of short chains (degrees of polymerization between 6 and 12) [22]. The type of starch is related to the presence of amylose or amylopectin. The crystalline lamellae contain double helices formed by clusters of side chains, which branch off the radially arranged amylopectin molecules. The amorphous regions are composed of other parts of amylopectin and long chains of linear amylose [21]. In order to identify the crystalline structure of cassava starch, the well-established powder diffraction files (PDF) database was directly inspected. As is well known, only the structure of amylose has been reported (α-amylose 431858) (JCPDS-International Centre for Diffraction Data 1997) [21], while the structure of amylopectin is still under study. From a physics point of view, the intensity of each of the peaks that form a pattern composed of a mixture of different phases is proportional to the concentration of each of the crystalline components [23]. Fig. 2(B) shows the X-ray diffraction patterns of amylose, amylopectin, and native cassava starch, as well as a cassava starch sample that had been modified using GBE for 10 h. The continuous lines in this figure represent the diffraction planes of amylose. According to this figure, cassava starch does not exhibit the characteristic peaks of amylose located at 19.70° (0 0 4), 22.25° (2 2 0), and 24.03° (1 3 0). The peak located at 23.033° is present only in cassava starch and amylopectin. This is an indication 9

that cassava starch is rich in amylopectin. The arrow in this figure located at 16.10° (0 2 1) points out the peak due to amylose recrystallization, because the peak does not appear in the patterns of cassava starch, amylopectin, or amylose [20]. Fig. 2(C) shows the X-ray diffraction patterns of native cassava starch and cassava starches that had been modified using GBE for 4, 6, 8 and 10 h. GBE modification increased the relative crystallinities of cassava starches from 34.8% to 39.5%, and produced amylose recrystallization. The intermolecular transglycosylation activity of GBE might also prevent the introduction of branch points in the double helices and produce high amounts of short chains within the amylopectin, helping to conserve the crystalline structure. These results indicate that modified cassava starches adopt a more close-packed arrangement with water molecules between each double-helical structure. This arrangement produces particular characteristics and likely results in increased granular stability and an increasingly structured network in aqueous systems. 3.3 Effect of GBE treatment on the flow behavior of cassava starch paste The flow behaviors of cassava starch pastes were characterized using the experimental data displayed in Fig. 3. At the same shear rate, the shear stresses of the starch pastes increased with increasing GBE treatment time. The shear stress (σ) versus shear rate (γ) data were fitted to a power law model: σ = K × γn (1)

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where σ is the shear stress (Pa), γ is the shear rate (s-1), K is the consistency index (Pa·sn), and n is the flow behavior index (dimensionless). The data were well fit, with determination coefficients (R2) from 0.998 to 0.999, as shown in Table 1. All of the starch pastes were pseudoplastic, with values of the flow behavior index (n) well below one, and exhibited shear-thinning behavior. Shear-thinning behavior, in which the material behaves as a gel or very viscous liquid at rest or when subjected to mild shear but flows freely when subjected to a larger shear, is a very desirable and crucial rheological property in typical food processing procedures. For example, shear-thinning behavior is shown by some jams and ketchups that become temporarily fluid when shaken or stirred. This behavior is beneficial because it prevents in-can settling and sagging and provides sufficient film build-up and suitable brush or roller drag without brush marks. The shear-thinning behavior of starch pastes can be ascribed to the disentangling, stretching, and ultimately breaking of long polysaccharide chains in the starch network by increasing shear rates [24]. The consistency coefficient (K), which can be taken as a viscosity criterion, decreased with GBE treatment time, indicating that GBE treatment resulted in a decrease in the apparent viscosity of cassava starch paste. This might be attributed to the decrease of integer starch grains and starch amylose content, and the inhibition of starch granule swelling by extensive cross-linking [4]. The values of n and K obtained using the power law model (Table 1) were subjected to regression analysis to investigate the effect of GBE treatment time on n and K. An exponential function and a polynomial function were found suitable for describing the relationship between these two parameters and GBE treatment time (Table 2). The η values of cassava starch pastes increased as γ approached zero, which suggested the existence of σ0 [25]. Therefore, the Casson model: 11

σ0.5 = σ00.5 + (ηcγ)0.5 (2) where σ0 is the yield stress (Pa), ηc is the Casson plastic viscosity (Pa·s), σ is the shear stress (Pa), and γ is the shear rate (s-1), could be used to describe the flow behavior of the tested cassava starch pastes with high determination coefficients (R2 = 0.927–0.965; Table 1). The relationship between σ0 and GBE treatment time was also shown in Table 2. The σ0 values of the cassava starch pastes, which represent the finite stress required to initiate flow, decreased linearly with the GBE treatment time. GBE modification decreased the percentage of long starch chains, weakening the entanglements of ordered chain segments and decreasing the interaction between different starch molecules, which restricts molecular motion [26]. Hence, σ0 decreased. 3.4 Effect of GBE treatment on the thixotropic behavior of cassava starch Thixotropic behaviors were observed in all the shear stress-shear rate curves obtained by first increasing and then decreasing the shear rate in the range of 0.1–200 s-1 (Fig. 4). For shear-sensitive samples, the two flow curves did not coincide. This caused a hysteresis loop, which indicates flow-time dependence. This behavior can be interpreted as structural breakdown by the shear field, which causes structural alteration or forms a new structure that maintains its shear-thinning characteristic in following shear sweeps [27]. The hysteresis loop areas of cassava starch pastes decreased with GBE treatment time, in accordance with changes of the plastic viscosity [28]. Generally, a larger hysteresis loop area suggests that shearing causes greater destruction of the gel structure and that more energy per unit time and unit volume is needed to eliminate the influence of time on flow behavior [3]. These led to the conclusion that GBE treatment could reduce the loss of 12

cassava starch paste structure and its time dependence, indicating that the shear resistance of cassava starch paste had been enhanced. 3.5 Effect of GBE treatment on the dynamic shear properties of cassava starch The storage shear moduli (G') and loss shear moduli (G"), which were obtained from the dynamic measurements, are measures of the elastic and the viscous components of measured samples, respectively. Mechanical spectra of the storage moduli (G') and the loss moduli (G") as a function of the frequency (ω) were displayed in Fig. 5. These rheograms showed that, in all cases, the storage modulus (G') was significantly greater (2-fold) than the loss modulus (G"). The G' and G" values of the modified starch pastes were larger than those of the native starch pastes. Both moduli increased with increasing ω, indicating a frequency dependence and a typical weak gel structure with a viscoelastic nature after GBE treatment [6, 29, 30]. The storage modulus (G') is directly related to the cross-link density of the network in a gel [31]. The rheological measurements indicated that the rigidity, strength and viscoelasticity of the gels significantly increased as the GBE treatment time increased, with a lower degree of deformation in response to stress. This behavior was also indicated by the relatively low loss tangent values (tan δ = G"/G'), which were lower than one within the experimental frequency range (Fig. 5). The tan δ values of the modified starches were lower than those of the control and decreased with the GBE treatment time, indicating that the modified starches were more structured and more elastic gel-like with an increase in the relative elastic contribution to their viscoelastic properties [18, 30]. A larger G' and a smaller tan δ suggests improved gelling ability of the starch; whereas, in high-amylose starches, the lower degree of swelling retards gelatinization, thereby inhibiting the formation of a gel 13

network [32]. These observations indicate that GBE modification may decrease the amylose content of cassava starch, stabilize and reinforce the network structure of cassava starch pastes, resulting in more solid-like behavior. They also demonstrate that the elastic properties of cassava starches were affected by GBE modification, and that the magnitude of the effect was dependent on the GBE treatment time. The frequency dependence of G' and G" can provide valuable information about the structure of a gel. To investigate the viscoelasticity of the starch pastes, the dependence of G' and G" on frequency was subjected to linear regression analysis. Table 3 provides the magnitudes of the slopes (n’ and n”), the intercepts (K’ and K”) and R2 values obtained from these analyses. The intercepts correspond to the magnitudes of G' and G", and the slopes provide information about the frequency dependence of the sample [33]. As indicated by the decreased slope and increased intercept, GBE treatment decreased the frequency dependence of G' and G", which indicated a more ordered structure that is consistent with greater alignment of the starch chains [34]. The magnitudes of K' and K" increased with the GBE treatment time and the values of K' were much larger than those of K". There was a substantial difference between the K' values of the pastes and the control as compared with their K" values. The result indicates that modification of the pastes using GBE may increase the elastic properties of the pastes [18]. From a structural point of view, a true gel shows frequency independence with zero slopes for G' and G". Low slope values indicate an intermediate network structure containing highly crosslinked material mixed with some uncrosslinked material [35]. They also indicate that the starch pastes have improved stability towards thermal and mechanical processing [4]. The lower 14

slope of the G' versus frequency observed with the gels might be attributed to an improved network structure, with an increased number of junction zones among the biopolymers [34]. In [34]. In general, starch pastes can be viewed as composite systems consisting of swollen amylopectin particles embedded in a three-dimensional network of aggregated amylose polymers [36]. Thus, the overall rheological properties of the starch system could be determined by the volume fraction and viscoelastic properties of the dispersed phase (swollen particles), the rheological properties of the continuous phase (amylose chains) and the interactions between the dispersed and continuous phases [37]. The results of this study suggest that those GBE-mediated cassava starch modifications that enhance the interactions and/or associations between the swollen particles and amylose chains of starch lead to a more structured network system. 3.6 Effect of temperature on the apparent viscosity of cassava starch The effect of temperature (25–90 °C) on the apparent viscosity of cassava starch pastes at 0.34 or 100 s–1 can be determined using an Arrhenius model: ηa = A × exp (Ea/RT) (3) where ηa is the apparent viscosity (Pa·s) at 0.34 or 100 s–1, A is a constant (Pa·s), T is the absolute temperature (K), R is the gas constant (8.3144 J/mol·K), and Ea is the activation energy (kJ·mol-1). In this model, the apparent viscosity (ηa) decreases exponentially with temperature. Investigating the effect of temperature on the rheological properties of food dispersions is important because food dispersions are, in general, processed and stored at a wide range of temperatures [6]. The stability of food during storage is affected by very low shear rates (0.34 s-1); however, industrial production subjects food to high shear rates (100 s-1). Thus, the values 15

of Ea calculated at both low and high shear rates could reflect the effect of temperature on cassava starch during processing and storage. The magnitudes of Ea and the constant A at γ = 0.34 s-1 and 100 s-1 were determined from regression analysis of the 1/T versus ln ηa (Table 1), with high determination coefficients (R2 = 0.981–0.998 and 0.976–0.989, respectively). In some cases, prolonged GBE treatment resulted in a decrease in Ea, compared with that for the native starch paste, and a viscosity less sensitive to temperature [18, 38]. Thus, the decrease in viscosity with increasing temperature was always more pronounced in the native starch pastes than in the modified starch pastes. This indicates that the viscosities of modified starch pastes are less responsive to temperature changes during processing and storage than those of native pastes. 3.7 Temperature sweeps Changes in G' observed during temperature sweeps of starch systems are presented in Fig. 6. The increase in G' was relatively slower during the early stage of heating; thus, amylose molecules dissolved from the starch granules and the suspension became a sol. The G' increased more steeply from the pasting onset temperature, reached a maximum value (G'max), and then decreased as the temperature increased further, indicating that the gel structure had broken down. The removal of water that leached from the amylose as the granules swelled and the formation of a three-dimensional gel network are two of the factors responsible for the initial increase of G'. The breakdown of the gel structure can be attributed to the disentanglement of the amylopectin molecules present in the swollen granule, and the loss of interaction between the granules and the network matrix (composed mainly of amylose) [39]. It must be noted that heating induces starch granule 16

swelling, which leads to a volume exclusion effect, so that particles have to rearrange in a lower available volume. In these cases, minimal water loss due to evaporation during the heating of small suspension volumes contributed to the instability of the suspension, which further increased the value of the elastic modulus [19]. The maximum values of G' obtained by heating, together with the loss tangent values calculated at the maximum values of G', reveal the ability of the granules to swell freely before their physical breakdown, the rigidity of the gel structure, and the contribution of elastic behavior [40]. As seen in Table 4, modified cassava starches exhibited higher G'max values and lower tan δ values than did native starch. These data indicate that increased GBE treatment time leads to greater resistance to granule rupture and higher gel rigidity. When heated in the presence of excess water, starch undergoes an order-to-disorder phase transition known as “gelatinization”. The structural transitions associated with phase changes in these starch systems during the heating stage were investigated using differential scanning calorimetry. The results, summarized in Table 4, showed that GBE modification slightly increased the onset and peak gelatinization temperatures, compared with those of native cassava starch. A high gelatinization onset temperature indicates that starch granules have a high resistance to swelling. Gelatinization temperatures have been related to the molecular structure of amylopectin and amylose, starch composition, and the architecture of the granules [20]. In this case, it is clear that the increase in onset temperature is due to morphological changes of the starch granules, as well as the recrystallization process of amylose, which were shown by SEM images and X-ray diffraction analysis. Moreover, the increase of G' up to its maximum value occurred in a temperature range (Tp–To) that decreased with GBE treatment time. This 17

result indicates that, after reaching the gelatinization onset temperature, the kinetics of gel formation are faster for GBE-treated cassava starches [19]. The gelatinization enthalpy of cassava starch was 11.42 J/g, and GBE modification increased the gelatinization enthalpy of modified cassava starches. The energy required for gelatinization had been related to the degree of starch granule crystallinity. The results of this research were consistent with the X-ray diffraction analysis, reflecting a more stable starch granule structure. During cooling from 95 to 25 °C, the G' values of the starch pastes increased continuously with decreasing temperature (Fig. 6). The values of G' in the initial stages of cooling did not change significantly until G' increased rapidly at around 60 °C. The observed sharp increase in G' can be attributed to the onset of intermolecular associations among amylose chains released from swollen granules [41]. The increases in G' values observed in the final stages of cooling reflect a predominantly solid-like structure. This seems to be due to the association of flexible strands into stiff helices, making it more difficult for individual molecules to move through the surrounding matrix of neighboring chains [42]. The extent of the increase in the rheological parameters during cooling was observed to be influenced by the tendency of these starch pastes to retrograde, which is, in turn, due to their amylose content [43]. To describe the differences in the extent of amylose association that occurred during cooling, the change in G' values (ΔG') between 25 and 90 °C can be calculated (Table 4). These values are related to the rate of amylose association. The magnitudes of ΔG' decreased with GBE treatment time, indicating GBE modification caused a large decrease in the rate of initial conformational ordering (double helix formation) during cooling. 18

4. Conclusion Enzymatic modification of cassava starch with GBE resulted in significant changes in its molecular structure and rheological properties. SEM studies showed the selective and particular attack of GBE on starch granules, some of which were broken and had larger openings. X-ray diffraction analyses showed that GBE treatment produces amylopectin with higher proportions of short chains and an amylose recrystallization process. The enzymatic modification with GBE increased the shear resistance, fluidity, and elastic characteristic of cassava starch paste, and regression analysis of steady and dynamic rheological data demonstrated cassava starch with improved stability towards mechanical processing. Moreover, the viscosities of modified starch pastes were less responsive to temperature changes during processing and storage than those of native pastes. Differential scanning calorimetric analysis and temperature sweeps indicated greater resistance to granule rupture, faster kinetics of gel formation, higher gel rigidity, and a large decrease in the rate of initial conformational ordering upon treatment with GBE. These results will provide a new method of enzymatic modification to design and produce cassava starch-based products with desirable rheological properties. Acknowledgements This work was financially supported by Science and Technology Support (Agriculture) program of Jiangsu Province (No. BE2014305) , China Postdoctoral Science Foundation (No. 2014M560394, 2016T90420), the Doctoral Fund of Ministry of Education of China (No. 20130093130001), the Jiangsu Planned Projects for Postdoctoral Research Funds (No. 1401100C), and the program of "Collaborative innovation center of food safety and quality control in Jiangsu Province". 19

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24

Figure captions Fig. 1. SEM images of native cassava starch and cassava starches modified using GBE for 4, 6, 8 and 10 h: (A) – (E) 2400×, (F) – (J) 5000×. Fig. 2. (A) X-ray diffraction patterns typical of A- and B-type polymorphs, and the X-ray diffraction pattern of native cassava starch. (B) X-ray diffraction patterns typical of amylose and amylopectin, as well as those of native cassava starch and CS–10. (C) X-ray diffraction patterns of cassava starches. CS, cassava starch; CS–4, cassava starch treated with GBE for 4 h; CS–6, cassava starch treated with GBE for 6 h; CS–8, cassava starch treated with GBE for 8 h; CS–10, cassava starch treated with GBE for 10 h. Fig. 3. Flow behaviors of cassava starch pastes at 25 °C. ■, Native cassava starch; □, Cassava starch treated with GBE for 4 h; ○, Cassava starch treated with GBE for 6 h; △, Cassava starch treated with GBE for 8 h; ▽, Cassava starch treated with GBE for 10 h. Fig. 4. The hysteresis loops of cassava starch pastes. A, Native cassava starch; B, Cassava starch treated with GBE for 4 h; C, Cassava starch treated with GBE for 6 h; D, Cassava starch treated with GBE for 8 h; E, Cassava starch treated with GBE for 10 h. Fig. 5. Plots of G', G" and tan δ vs. log ω for cassava starch pastes. ■, Native cassava starch; □, Cassava starch treated with GBE for 4 h; ○, Cassava starch treated with GBE for 6 h; △, Cassava starch treated with GBE for 8 h; ▽, Cassava starch treated with GBE for 10 h. Fig. 6. Temperature sweeps of cassava starch dispersions (heating from 40 to 95 °C, cooling from 95 °C to 25 °C and then holding at 25 °C for 5 min). ■, Native cassava starch; □, Cassava starch treated with GBE for 4 h; ○, Cassava starch treated with GBE for 6 h; △, Cassava starch treated with GBE for 8 h; ▽, Cassava starch treated with GBE for 10 h. 25

26

Fig. 1

27

A Intensity (arb. units)

B-type

A-type

Cassava Starch

20 24 2Degrees

28

32

36

(130)

16

(220)

12

(004)

8

(020) (021) (103)

4

B 23.033

Intensity (arb. units)

Amylose

Amylopectin

Cassava Starch

CS-10

4 4

8 8

12 12

16 16

20 2Degrees 20

24 24

28 28

32 32

36 36

Intensity (arb. units)

C 39.5%

CS-10

38.5%

CS-8

36.9%

CS-6

35.7%

CS-4

34.8%

CS

4

8

12

16

20 24 2Degrees

28

32

36

Fig. 2 28

180 160

Shear stress (Pa)

140 120 100 80 60 40 20 0 0

50

100

150

200

-1

Shear rate (s )

Fig. 3

29

180

A

160

160

140

140

Shear stress (Pa)

Shear stress (Pa)

180

120 100 80

B

120 100 80

60

60

40

40

20

20 0

0 0

50

100

150

0

200

50

180

C

160

160

140

140

Shear stress (Pa)

Shear stress (Pa)

180

120 100 80

150

200

150

200

D

120 100 80

60

60

40

40

20

20 0

0 0

50

100

150

0

200

180

E

160 140 120 100 80 60 40 20 0 0

50

100

50

100

-1 Shear rate (s )

-1 Shear rate (s )

Shear stress (Pa)

100

-1 Shear rate (s )

-1 Shear rate (s )

150

200

-1 Shear rate (s )

Fig. 4

30

1000

G' (Pa)

100

10

1 0.1

1

10

100

Angular frequency (rad/s) 1000

G" (Pa)

100

10

1 0.1

1

10

100

Angular frequency (rad/s)

0.7

Tan 

0.6

0.5

0.4

0.3 0.1

1

10

100

Angular frequency (rad/s)

Fig. 5

31

120

100

G' (Pa)

80

60

40

20

0 0

10

20

30

40

50

60

70

Temperature (C)

Fig. 6

32

80

90

100

Table Table 1 Effect of 1,4-α-glucan branching enzyme (GBE) treatment on the flow rheological properties and slopes (n' and n") and intercepts (K' and K") of ln (G', G") vs ln ω (rad/s) data of cassava starch pastes. Arrhenius model Power law

Casson model

K

σ0

ηc

(Pa)

(Pa·s)

Sam ple

n

(Pa·s )

CS

n (-)

R2

R2

γ = 0.34 s-1

γ = 100 s-1

A

Ea

A

Ea

(mPa·s

(kJ/mo

(mPa·s

(kJ/mo

)

l)

)

l)

R2

R2

19.61± 0.42±0 0.9

10.37± 0.69±

0.9

1.27±

27.55

0.9

0.15±

22.60

0.9

0.23

0.23

27

0.01

±0.60

90

0.01

±0.13

89

.003

99

0.04

CS–

11.07± 0.44±0 0.9

7.74±0 0.43±

0.9

7.24±

20.12

0.9

0.44±

17.94

0.9

4

0.08

.17

39

0.24

±0.48

91

0.03

±0.88

86

CS–

9.78±0 0.44±0 0.9

7.48±0 0.40±

0.9

9.85±

18.52

0.9

3.21±

14.85

0.9

6

.19

.14

47

0.19

±0.88

98

0.11

±0.68

87

CS–

7.45±0 0.45±0 0.9

4.78±0 0.31±

0.9

55.96

13.06

0.9

5.01±

11.46

0.9

8

.11

.15

30

±0.69

±0.30

81

0.17

±0.22

82

CS–

6.16±0 0.47±0 0.9

4.74±0 0.26±

0.9

61.08

12.75

0.9

9.64±

10.39

0.9

10

.11

.10

65

±0.35

±0.15

85

0.23

±0.07

76

.002

.004

.003

.004

99

98

99

99

0.03

0.02

0.03

0.02

CS, cassava starch; CS–4, cassava starch treated with GBE for 4 h; CS–6, cassava starch treated with GBE for 6 h; CS–8, cassava starch treated with GBE for 8 h; CS–10, cassava starch treated with GBE for 10 h. Values are mean ± standard deviation for triplicate measurements. 33

Table 2 Relationships between the parameters of rheological models and 1,4-α-glucan branching enzyme treatment time for the cassava starch pastes.

a

Parametera

Equation

R2

K

K = 16.026 – 1.289t + 0.030t2

0.9905

n

n = 0.454 + 0.014t – 0.006t2 + 4.351 × 10-4t3

0.9098

σ0

σ0 = 10.346 – 0.594t

0.9209

ηc

η = 0.685 – 0.067t + 0.002t2

0.9782

K and n are from the power law model, ηc and σ0 are from the Casson model.

Table 3 Slopes (n' and n") and intercepts (K' and K") of ln (G', G") vs ln ω (rad/s) data of cassava starch pastes. G'

G"

Sample K' (Pa·s)

n'

R2

K" (Pa·s)

n"

R2

CS

26.603±0.275

0.175±0.004

0.9940

14.897±0.054

0.152±0.002

0.9978

CS–4

35.285±0.225

0.164±0.002

0.9973

19.052±0.075

0.145±0.001

0.9986

CS–6

42.905±0.322

0.159±0.003

0.9960

21.066±0.066

0.140±0.001

0.9990

CS–8

47.376±0.299

0.155±0.004

0.9971

24.058±0.120

0.134±0.001

0.9974

CS–10

58.470±0.290

0.141±0.002

0.9976

27.209±0.204

0.127±0.003

0.9931

CS, cassava starch; CS–4, cassava starch treated with GBE for 4 h; CS–6, cassava starch treated with GBE for 6 h; CS–8, cassava starch treated with GBE for 8 h; CS–10, cassava starch treated with GBE for 10 h. Values are mean ± standard deviation for triplicate measurements.

34

Table 4 Gelatinization temperatures, gelatinization enthalpies, and rheological properties of cassava starch pastes during temperature sweeps.

b

Sample

To (°C)

Tp (°C)

Tc (°C)

ΔHg (J/g)

G'max (Pa)

tan δb

ΔG' (Pa)

CS

62.72

66.05

81.05

11.42

90.8

0.298

27.5

CS–4

63.72

66.99

83.19

12.68

92.7

0.202

11.2

CS–6

64.04

67.30

85.32

13.47

94.6

0.185

12.7

CS–8

64.37

67.58

85.03

14.41

100.0

0.176

7.5

CS–10

64.56

67.55

84.96

14.80

108.0

0.170

2.2

The values of tan δ are calculated at G’max.

TG'0, the onset temperature of gelatinization; G'max, the maximum value of G'; TG'max, the temperature for G'max; ΔG', the change in G' values between 25 and 90 °C; CS, cassava starch; CS–4, cassava starch treated with GBE for 4 h; CS–6, cassava starch treated with GBE for 6 h; CS–8, cassava starch treated with GBE for 8 h; CS–10, cassava starch treated with GBE for 10 h.

35