Effect of different alcoholic-alkaline treatments on physical and mucoadhesive properties of tapioca starch

Journal Pre-proofs Effect of different alcoholic-alkaline treatments on physical and mucoadhesive properties of tapioca starch Zahra Kaveh, Sodeif Azadmard-Damirchi, Gholamhossein Yousefi, Seyed Mohammad Hashem Hosseini PII: DOI: Reference:

S0141-8130(19)35283-3 https://doi.org/10.1016/j.ijbiomac.2019.10.230 BIOMAC 13731

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

9 July 2019 22 September 2019 24 October 2019

Please cite this article as: Z. Kaveh, S. Azadmard-Damirchi, G. Yousefi, S.M.H. Hosseini, Effect of different alcoholic-alkaline treatments on physical and mucoadhesive properties of tapioca starch, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.230

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Effect of different alcoholic-alkaline treatments on physical and mucoadhesive properties of tapioca starch Zahra Kaveha, Sodeif Azadmard-Damirchia,b *, Gholamhossein Yousefic,d, Seyed Mohammad Hashem Hosseinie a

Department of Food Science and Technology, School of Agriculture, University of Tabriz, P.O.

Box 51666-16471, Tabriz, Iran b

Food and Drug Safety Research Center, Health Management and Safety Promotion Research

Institute, Tabriz University of Medical Sciences, Tabriz, Iran c

Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, P.O.

Box 71345-1583, Shiraz, Iran d

Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz,

Iran e

Department of Food Science and Technology, School of Agriculture, Shiraz University, P.O. Box

7144165186, Shiraz, Iran * Corresponding author: Sodeif Azadmard-Damirchi, Email address: [email protected], Tel: +984133392032, Fax: +984133345332, Postal address: Faculty of Agriculture, University of Tabriz, P.O. Box 5166616471, Tabriz, Iran.

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Abstract Alcoholic-alkaline treatment (AAT) is a physical modification method for the production of granular cold water swelling starch and should be optimized for each starch source. The main objective of this research was to investigate physical and mucoadhesive properties of tapioca starch (TS) modified by different AATs. Improvement of cold-water absorption, solubility, rheological properties at low temperatures, clarity, freeze-thaw stability, and mucoadhesion was positively correlated with the alkali amount and reaction temperature and negatively correlated with ethanol content. Morphological studies demonstrated different degrees of swelling, birefringence loss, and surface wrinkling of granules depending on modification degree. Starch pastes, modified in a higher degree, showed a change from rheopexy to thixotropy and from translucency to turbidity over time. The highest quality along with maintaining granular integrity was obtained by treating starch (10 g) with 30 g alkaline solution (2.5 M) and 110 g aqueous ethanol (40%) at 25 ºC. The characteristics of this sample were higher than those of corn counterpart except for viscosity, consistency, and freeze-thaw stability and were almost similar to those of thermally gelatinized TS (TGTS). Therefore, this AA-modified TS can be an alternative for TGTS in instant and heatsensitive foods and delivery of bioactives as a mucoadhesive polymer. Keywords: Tapioca starch; Alcoholic-alkaline treatment; cold water swelling; Physical property; Mucoadhesion; Corn starch

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1.

Introduction Native starch requires heat treatment to exhibit its functional properties. It cannot be used

in different types of products such as instant foods, formulations with heat sensitive ingredients, and those with no or insufficient heat treatment in their production process [1, 2]. Therefore, efforts have been made to modify starch in order to eliminate this drawback. Among chemical, physical, enzymatic and genetic starch modification methods, physical modifications including pregelatinization and production of granular cold water swelling (GCWS) starch are preferred due to the lack of chemical changes in starch and the absence of chemical residues, which make it riskless for humans and environmentally friendly [3]. In pregelatinization, equipment such as drum dryer, extruder and spray dryer is used to destroy the crystalline structure of starch granules. The starch can consequently form a paste with high viscosity at room temperature [4]. Nevertheless, extensive damage or destruction of starch granules, due to the severity of the process, and retrogradation of the wet starch during drying step lead to starch pastes with reduced viscosity, insufficient consistency, and weak gels compared to their heat treated native counterparts [5]. In addition, previous studies have shown that the physical properties of pregelatinized starch were more affected by other components of the system such as acid and salt than GCWS starch [2, 6]. Therefore, a greater tendency has been toward the application of GCWS starch which is gelatinized while maintaining the integrity of granules and provides paste with more similar properties to cook-up starches. There are different methods to produce GCWS starches. Heating in an aqueous ethanol and spray drying are two of the common methods for their production [7, 8]. Starch granules can also be gelatinized by high hydrostatic pressure at room temperature. However, it is a time consuming process with high operating costs and is not economical for industrial applications [9]. 3

An alternative non-thermal process for the production of GCWS starch is alcoholicalkaline treatment (AAT) that involves using sodium hydroxide for starch gelatinization and alcohol for maintenance of granular integrity. Despite using chemicals, it has been demonstrated that no chemical changes occurs in the starch structure [10, 11]. This technique does not require any special equipment and consumes significantly lower amounts of energy. Unlike the other three methods, it is applicable for all types of starch especially waxy starches [1]. Despite the costs associated with the required chemicals and waste treatment, the total cost of AAT seems to be less than that of thermal treatment due to lower capital costs and significant contribution of energy costs in the final price of products in many countries. GCWS corn [10, 11], banana [12], potato [13], sago [14], buckwheat [15], and very recently waxy rice [16] starches have been prepared and characterized by common features like high viscosity, cold water solubility and improved freeze-thaw stability. In other studies, alcoholicalkaline freeze drying and sonication-assisted treatments were optimized to obtain the maximum solubility in cold water [17, 18]. A lower degree of changes in the properties of GCWS corn starch (CS) than in those of pregelatinized starch after treatment with different concentrations of acetic acid and salt has been reported by Hedayati et al. [6] and Majzoobi et al. [2]. The properties of alcoholic alkaline treated starches depend not only on the starch type, but also on the amount and concentration of ethanol and sodium hydroxide and also the applied temperature [10]. Therefore, this treatment should be optimized for the starches obtained from different botanical sources. Tapioca starch (TS), extracted from cassava root, has attractive characteristics for the application in food formulations especially baby foods. It is not allergic and has high molecular weight amylose, very low non-starch residues and has no interference with the perception of 4

original food taste due to its bland flavor. It has less amylose content (17%-20%) than normal CS (25%-31%), and therefore, develops gel with less firmness, which is favorable for special applications such as cream filling, smooth pudding, and pourable salad dressing [19]. On the other hand, it has a high swelling power due to its high amylopectin content which makes it an appropriate mucoadhesive polymer for the delivery of different bioactive compounds. Mucoadhesion is the result of physical entanglements and secondary intermolecular interactions, especially hydrogen bonding, between polymer and mucin molecules. It leads to increased retention time in gastrointestinal tract or other sites of action, resulting in improved bioavailability and absorption and reduced required dosage of nutraceutical or drug [20]. Mucoadhesion can lead to a steady state release of flavor constituents and hence prolongs the flavor perception [21]. The functional properties of different cold water soluble TS, prepared by various methods such as drum drying [22], aqueous ethanol treatment [8, 23], spray drying [24] and extrusion [25] have been studied previously. To the best of our knowledge, there is no scientific published report studying the physical and mucoadhesive properties of modified TS prepared by different AATs. Therefore, the main purpose of this study was to characterize TS treated under different amounts of sodium hydroxide and ethanol at different temperatures. The properties of AAT-modified TSs were compared with those of various control samples including thermally gelatinized (TG) TS, GCWS CS, and TG CS.

2.

Materials and methods

2.1. Materials

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Native tapioca and corn starches (NTS and NCS, respectively) were purchased from Pars Khoosheh Pardaz Company (Shiraz, Iran), which contained 9.77% and 6.43% moisture, 0.28% and 0.49% protein, 0.091% and 0.15% ash, 0.12% and 0.68% lipid (quantified according to the Approved Methods of the AACC [26]), and 17.57% and 27.81% total amylose (measured using the method of Morrison and Laignelet [27]), respectively. Food grade ethanol (96%, v/v) was supplied by Parsian Company (Shiraz, Iran). Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Merck Co. (Darmstadt, Germany). 2.2. Alcoholic-alkaline treatments for the modification of tapioca and corn starches The AATs were carried out according to the procedure described by Chen and Jane [10]. 10 g (dry basis) of NTS was dispersed in either 70 or 110 g aqueous ethanol (40%). 20, 25, or 30 g of sodium hydroxide solution (2.5 M) was gradually added to the dispersion at two different temperatures (25 or 35 ºC). After gentle stirring with a magnet stirrer (Heidolph, MR Hei-Tec, Germany) for 15 min, aqueous ethanol (40%, w/w) was added to the suspension and stirring was continued for 10 min. Then, the reaction vessel was removed from the stirrer, allowing the starch to settle at ambient temperature. After 30 min, the supernatant was discarded, and the starch was washed with ethanol solution. The substrate was then suspended in aqueous ethanol (40%, w/w) and neutralized using hydrochloric acid solution (2.5 M). The neutralized starch was washed with 60%, followed by 95% ethanol, filtered under vacuum, and dehydrated with absolute ethanol. Finally, the resultant starch was dried at 40 ºC for 12 h, ground with mortar and pestle and screened. The starch with the particle size of < 125 µm was stored in an airtight container at ambient temperature for further experiments.

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GCWS CS was prepared with 110 g aqueous ethanol (40%, w/w) and 30 g sodium hydroxide solution (2.5 M) at 25 ºC to investigate the effect of a same set of conditions on the properties of modified starches obtained from different sources (tapioca and corn). 2.3. Production of thermally gelatinized tapioca and corn starches TG TS and CS dispersions (1%, w/v) and pastes (5% and 10%, w/v) were prepared by heating aqueous native starch suspensions to 95 ºC in a water bath followed by stirring at 50 rpm for 30 min. They were subsequently cooled to room temperature for further studies. Dispersions were utilized for cold water solubility, water absorption, and morphological tests. Pastes were prepared for rheological, mucoadhesive, turbidity, and freeze-thaw stability studies. 2.4. Determination of cold water solubility and water absorption Alcoholic alkaline treated starch dispersions were prepared by gradually adding starch (0.15 g) to 15 mL distilled water, while stirring with an overhead stirrer (Type BS, VELP Scientifica, Italy) at 50 rpm. Stirring was continued for 30 min at 25 ºC. TG tapioca and CSs were prepared as described in section 2.3. Then each dispersion was centrifuged at 1200 g for 10 min and the supernatant was transferred into pre-weighed glass plate and dried to a constant weight at 110 ºC. Solubility was determined by Eq. (1): Solubility (%) = (WDSN / WS)

´

(1)

100

where, WDSN is the weight of dried supernatant (g) and WS is the dry weight of starch (g). The pellet was also weighed and dried at 110 ºC in order to calculate water absorption by Eq. (2) [28]: Cold water absorption (g/g) = (WWP – WDP) / WS

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(2)

where, WWP is the weight of wet pellet (g), WDP is the weight of dried pellet (g), and WS is the dry weight of primary starch (g). 2.5. Morphological studies An Optical microscope (Olympus, BX41TF, Olympus Optical Co. Ltd, Japan) in bright field and polarization modes was used to observe the morphology of aqueous starch dispersions (1%, w/v). Further information about surface morphological properties were obtained by using scanning electron microscope (Tescan-Vega 3, Tescan, Czech Republic). The dried samples were fixed on the stub using double-sided adhesive tape and coated with gold. The micrographs were acquired at an accelerating voltage of 5 kV and the magnification of 3000x. 2.6. Rheological properties Steady shear flow properties of starch samples were determined using a rotational rheometer (Brookfield, R/S Plus, Brookfield Viscometers Ltd, England) with a cone and plate geometry (50 mm diameter, 1º cone angle, and 0.5 mm gap). Starch samples were prepared by adding alcoholic alkaline treated starches (1 g) to distilled water (10 mL), while stirring with an overhead stirrer at 50 rpm for 30 min at room temperature. TGTS and CS pastes were produced as described in section 2.3. The resultant pastes were stored individually at 4, 25 and 40 ºC for 1 h. Other samples were refrigerated at 4 ºC for 24 h and 1 week to determine the changes in the rheological properties over time. Then, 2 mL of each sample was placed onto the rheometer plate and the test was carried out by increasing the shear rate from 0 to 600 s-1 for 1 min (upward stage), followed by decreasing from 600 to 0 s-1 (downward stage). The rheological properties were described using Power Law equation (Eq. (3) and (4)): τ=K

n

(3) 8

ηapp = K

n-1

(4)

where, τ is the shear stress (Pa); is the shear rate (s-1); K is the consistency coefficient (Pa.sn); n is the flow behavior index (dimensionless); and ηapp is the apparent viscosity (Pa.s) [29, 30]. Thixotropic indices of the starch samples were also calculated using the equation reported by Wei et al. [31]: (5)

α = (At / Aup) ´ 100

where, α is the thixotropic index (dimensionless), At (also known as thixotropic area or hysteresis) is the area between the upward and downward curves (Pa.s-1) and Aup is the area under the upward flow curve. 2.7. Mucoadhesive properties Tensile study was performed to investigate the mucoadhesion of starch samples using a texture analyzer (TA) (Brookfield, CT3, Brookfield engineering, USA) according to a modified method of Thirawong, Nunthanid, Puttipipatkhachorn, and Sriamornsak [32]. 2 g of each 10% (w/v) starch sample was weighed into 10 mL glass beaker and left for 1 h at room temperature. The small intestine from freshly slaughtered sheep, used as a model for the intestinal mucosa, was equilibrated at 37 °C for 30 min in phosphate buffer (pH 6.8, Fig. 1SA (supplementary data)). For each test, 4-cm long pieces of the intestine were cut (Fig. 1SB) and opened longitudinally (Fig. 1SC). Then, it was fixed on the instrument probe (15 mm diameter) with the help of a small rubber band in such a way that the inner surface of the intestine was exposed to the environment (Fig. 1SD). 30 μL of buffer solution was poured onto the mucosa, and the TA probe was lowered until the contact of mucosal tissue to the gel surface at the preload of 20 mN (Fig. 1SE1 & 1SE2). After a contact for 1 min, a vertical force was applied in the opposite direction by raising the TA probe 9

at the test speed of 1 mm/s (Fig. 1SF) until the complete failure of the adhesive joint (the interfacial bonds (layer) between the surface of mucosal tissue and the starch gel surface molecules). From the resulting curve, fracture strength (mN/cm2) was defined as the maximum adhesive force (mN) per unit contact area (cm2): (6)

Fracture strength = Fmax/A

Work of adhesion (mN.mm) was determined from the area under the force versus distance curve. 2.8. Turbidimetric analysis The turbidity of different starch dispersions was measured during storage at 4 ºC for 72 h. Starch pastes (5%, w/v) were prepared and diluted to 1% just before the test. Following the method of Singh and Singh [13], the absorbance was measured using a UV/Visible spectrophotometer (SHIMADZU, UV- 1650 PC, SHIMADZU Corporation, Japan) at 640 nm against a water blank after 0, 24, 48 and 72 h. 2.9. Freeze-thaw stability The syneresis (%) of TG and alcoholic alkaline treated starch samples (10%) was determined for evaluation of their stability after consecutive freeze-thaw cycles. 10 g of each sample, prepared as described in section 2.3 and 2.6, was weighed in 15 mL centrifuge tubes and subjected to 1-5 repeated freeze-thaw cycles (24 h freezing at -18 ºC and 4 h melting at room temperature). After each cycle, a set of three samples was selected for each starch type, centrifuged at 1100 g for 25 min and the other samples were returned to the freezer. Syneresis (%) was reported as the percent of released liquid from the initial sample [2].

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2.10. Statistical analysis All experiments were carried out at least in triplicate and the resultant data were exported to Excel 2013 (64-Bit Edition, Microsoft Corporation, USA) and SAS software V9.0 (SAS Institute, Cary, NC). Significant differences (P < 0.05) between the mean values were evaluated using oneway Analysis of variances (one-way ANOVA) and Duncan’s multiple range post-hoc tests.

3.

Results and discussion Preliminary studies revealed that four treatments (Table 1, treatments No. 6, 9, 11, and 12) led

to the gelatinization of starch along with complete disintegration of granules. Because of losing granular integrity, these four treatment conditions were not suitable for our purpose and therefore, they were excluded from further experiments. Taking into account the control samples (TG TS, GCWS CS, and TG CS), a total of 11 treatments were further studied (Table 1). 3.1. Cold water absorption and solubility Increasing the reaction temperature (from 25 to 35 ºC) and the amount of added alkaline solution had a positive effect on the cold water absorption and solubility. In contrast, increasing ethanol content showed an inhibitory effect on these properties (Table 2). Treatments with 25 and 30 g of sodium hydroxide solution (2.5 M) and 110 g of aqueous ethanol (40%) at 35 and 25 ºC could dramatically improve the water absorption and solubility from 3.64 (g/g) and 2.36% in ESTS110(40),20,25 to 18.81 (g/g) and 20.89% in ESTS110(40),25,35 and 18.48 (g/g) and 20.20% in ESTS110(40),30,25, respectively, that were close to those of TGTS (21.29 g/g and 21.65%). Similar effects have been obtained for the alcoholic alkaline treated CS [10, 18]. Strong semi-crystalline structure of native starch prevents sufficient penetration of cold water into the starch granules [33]. In contrast, sodium hydroxide can disrupt hydrogen bonds in the starch structure [14], leading to 11

uncoiling of amylopectin double helices and destruction of the crystalline structure. As a result, starch would be able to absorb significant amounts of water at room temperature [10]. Under identical conditions, GCWS and TG CSs had significantly (p < 0.05) less water absorption (11.16 and 14.92 g/g vs. 18.48 and 21.29 g/g) and solubility (7.34% and 9.06% vs. 20.20% and 21.65%) than GCWS and TG TSs; which can be the result of lower amylopectin and higher lipid content of CS [33]. Although both TS and CS have the A-type crystalline pattern with a closed-packed arrangement [23, 34], the amount of crystallinity in TS is lower than that of the CS due to the higher polymerization degree of tapioca amylose [23]. The lower crystallinity of TS leads to its lower resistance to alkali or heat treatment, and therefore, a higher degree of modification, greater solubility, and water absorption at a same modification condition. 3.2. Microscopic observations Microscopic observations revealed that after AATs, the smooth surface with round to polygonal shape of NCS and spherical and truncated shape of NTS granules (Figs. 1A1 and 1K1) changed into irregular wrinkled shapes that were most evident in ESTS110(40),30,25 and ESTS110(40),25,35 (Figs. 1C1-1I1). However, under identical conditions, granule deformation was less pronounced in GCWS CS compared to tapioca counterpart (Fig. 1L1 vs 1H1). Deformation of potato, sago, buckwheat, and corn starch granules after AAT has also been reported previously [2, 11, 13, 14, 15]. AAT intensification increased the number of swollen granules with enlarged diameter up to a critical point (Figs. 1B-H and 1L). Then, the disruption of granules was initiated (Fig. 1I) and finally the granules were completely disintegrated (micrographs not shown). The swelling of granules was positively correlated with the amount of added sodium hydroxide and had a negative relationship with ethanol content. Furthermore, granules with larger size were obtained from the 12

treatment at 35 ºC compared to 25 ºC (Figs. 1C2 vs 1B2, 1G2 vs 1D2, and 1I2 vs 1E2). Swelling and deformation of granules have been attributed to the electrostatic repulsion between negatively charged dissociated hydroxyl groups as a function of alkali and also the resulting tension on adjoining remaining crystalline regions, respectively. Ethanol decreases the available water for starch and hence, it acts as a restrainer [11]. There was not any noticeable difference between the granular structure of ESTS110(40),20,25 (Fig 1B) and NTS. Despite the maintenance of granules integrity, a complete lack of birefringence was observed for the ESTS70(40),25,25, ESTS70(40),20,35, and ESTS110(40),30,25 (Figs. 1F3, 1G3, and 1H3). A mixture of native and modified (non-birefringent) granules was observed in ESTS110(40),20,35, ESTS70(40),20,25, and ESTS110(40),25,25 (Figs. 1C3, 1D3, and 1E3). This behavior suggested that the disappearance of the Maltese cross pattern that is indicative of destruction of the initial crystalline structure depends on the severity of the treatment and resistance of individual granules toward the reaction conditions. ESTS110(40),25,35 exhibited the agglomeration of non-birefringent granules and disintegrated remnants (Fig. 1I), which is indicative of exceeding the required amounts of variables (critical point). Similar behaviors were also found for the starch treated with aqueous ethanol at elevated temperatures varied in starch:water:ethanol ratios [8]. A honeycomb-like microstructure with complete loss of birefringence and destruction of granules was observed for the freeze-dried TG corn and TSs (Fig. 1J and 1M), similar to that observed earlier for CS [35]. 3.3. Rheological properties Among four common rheological models, namely Power Law, Herschel-Bulkley, Bingham, and Casson, the shear stress vs. shear rate data was best fitted to the Power Law model with the highest values of R2 (Table 1S & 2S, supplementary data).

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Unlike ESTS110(40),20,25, starches treated under more intense conditions were able to develop viscosity at low temperatures (4, 25 and 40 ºC). The consistency coefficient increased from 6103.3 mPa.sn in ESTS110(40),20,35 to a maximum of 16616.5 and 20089.7 mPa.sn in ESTS110(40),30,25 and ESTS110(40),25,35 at 4 ºC (downward stage), respectively (Table 3). However, the consistency coefficient of all modified TSs was significantly (p < 0.05) lower than that of GCWS CS (80411.5 in upward and 36686.5 mPa.sn in downward stage at 4 ºC). Amylose is essential for gel formation and strength [33]; therefore, the TS with lower amylose content can only form a weak gel; whereas, higher amylose content of CS can lead to the formation of a strong interconnected three dimensional network and hence greater consistency and viscosity. Increasing the storage temperature from 4 to 40 ºC, reduced the apparent viscosity and consistency coefficient of all samples except for the ESTS110(40),20,35. Higher viscosity of starch pastes at lower temperature (especially at 4 ºC), is the result of the general effect of temperature reduction on increasing the intermolecular forces and its specific effect on cross-linking the starch granules by amylose molecules (early stage of retrogradation) [36]. The results of microscopic observations and water absorption (sections 3.1 and 3.2) of ESTS110(40),20,35 confirmed the inadequacy of its treatment condition to reach the maximum swelling; therefore, the expected decrease in viscosity was not observed due to the continued swelling at 40 ºC. The flow behavior indices (n) of all samples were less than one, which indicated the nonNewtonian characteristic with shear thinning behavior of starch samples. During the first day of experiment, applying shear led to further swelling of starch granules mainly due to the dissociation and uncoiling of amylopectin molecules that had remained native after AAT. Higher swelling increased the friction between swollen granules, resulted in enhanced viscosity and consistency in the downward stage (Table 3), and therefore, negative values of thixotropic areas and thixotropic 14

indices (Table 1S, supplementary data) (time-dependent rheopectic behavior). A similar behavior was observed in TGTS samples but to a lesser extent (lower thixotropic area and thixotropic index); which indicated the amount of native amylopectin molecules after heat treatment was lower than that after AAT. Starch pastes had enough time to swell and to build structure during storage for 24 h as well as after one week; however, simultaneous retrogradation of amylopectin molecules led to the weakening of starch pastes structure and thixotropy (time-dependent thinning) was observed for this reason. ESTS110(40),20,35 and TGTS exhibited the lowest and the highest degree of thixotropy, respectively (Table 4); which confirmed the positive correlation between the thixotropic behavior and the destruction of amylopectin and amylose leaching. Rheopexy followed by thixotropy was explained earlier for the TGTS paste kept at 5 ºC [37]. At the first day, ndownward was lower than nupward for each sample. In contrast, lower n values were measured in upward than downward stage after storage for one day and one week. These differences can be related to the different amounts of intermolecular interactions. The stronger the total interaction, the higher the structure breakdown, resulting in greater differences between the maximum and minimum viscosities (i.e., lower n values). GCWS and TGCSs displayed thixotropic behavior in every three days of the experiment but to a lesser extent after one week compared to the TS counterparts which was attributed to the lower content of amylopectin in CS compared to the TS. The opposite behavior (thixotropy) of CS on the first day, shown with positive values of the thixotropic area and thixotropic index (Table 1S), arises from breaking the strong network of amylose after increasing the shear rate and insufficient time for the CS paste to recover its structure. A similar result was observed for drum dried pregelatinized CS [38].

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The rheological properties of GCWS CS were less similar to those of TG CS as compared to the similarities of GCWS TS and TG TS. This result confirms the possibility of applying more severe conditions for the production of modified CS by alcoholic-alkaline method. 3.4. Mucoadhesive properties Fig. 2 demonstrates greater fracture strengths and works of adhesion for the starches treated with higher amounts of alkaline solution and at 35 ºC compared to 25 ºC. In addition, the lower the amount and concentration of alcohol, the greater the mucoadhesion. As discussed in previous sections, above mentioned conditions resulted in higher water absorption and swelling. The positive correlation between swelling and adhesiveness has been already confirmed for rice and waxy corn starch pastes [39, 40]. Water absorption and swelling allow polymer molecules to interpenetrate more efficiently into the mucosal glycoproteins and increase interlocking and interactions between them which is considered as a higher mucoadhesion [20]. Therefore, amongst the TSs, the least and highest bioadhesion were observed for ESTS110(40),20,25 and TGTS, respectively. In spite of higher fracture strength of GCWS and TG CSs, the required energy for detachment was significantly (p < 0.05) less than those of TSs. A negative correlation between the amylose content and adhesiveness was also reported by Kong, Zhu, Sui and Bao [41]. Although the amylose chain associations build a strong network which can resist greater force before the rupture of adhesive joint, they reduce penetrability. In addition, less water absorption of CS may have led to a lesser entanglement which resulted in faster detachment and less work. It has been reported that the work of adhesion is the best indicator of bioadhesion [42]. 3.5. Turbidity Unlike ESTS110(40),20,25, the turbidity of other starch samples increased during storage at 4 ºC similar to those reported for GCWS banana and corn starches [2, 12] mainly due to the re16

association of starch molecules and formation of microcrystals [36]. The turbidity development of TS dispersions followed this order: TGTS> ESTS110(40),25,35> ESTS110(40),30,25> ESTS70(40),20,35> ESTS70(40),25,25> ESTS110(40),25,25> ESTS70(40),20,25> ESTS110(40),20,35 (Table 5); which suggests that the rate of retrogradation was dependent on the amounts of leached amylose and dissociated amylopectin that increase the incidence of new hydrogen bonding. However, the initial transmittance was also effective on the final turbidity and hence, ESTS110(40),30,25 had the third highest value of clarity after 72 h (after ESTS70(40),20,35 and ESTS70(40),25,25). Amylose is more responsible for the early stages of retrogradation; whereas, amylopectin is associated with slower re-association [36]. Therefore, the major retrogradation of GCWS and TG CSs, occurred during the first 24 h of storage, unlike the tapioca counterparts. 3.6. Freeze-thaw stability The water that remains unfrozen during freezing results in the formation of concentrated starch pastes with a higher degree of interactions between starch molecules followed by retrogradation. Afterwards, the water molecules are ejected from the gel network and crystallize and hence can readily release from the network during thawing. This phenomenon, also known as syneresis, is increased by repeating freeze-thaw cycles [30]. ESTS110(40),20,25 lost much of the trapped water after the first cycle (69.67%) (Fig. 3) due to the low water absorption and holding capacity of their semi-crystalline structures. After starch modification under more intense conditions, the water holding capacity and deformation resistance toward centrifugal force were enhanced. So that the syneresis after 5 cycles was reduced from 54.69% in ESTS110(40),20,35 to 12.77% in ESTS110(40),30,25. On the other hand, the rate of water loss induced by repeated freeze-thaw cycles was more pronounced in the samples subjected to higher degree of modification. For instance, the final water separation was 1.41 times the initial value in ESTS110(40),20,35 compared to the ESTS110(40),30,25 with

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that of 2.46 times. The maximum rate of change was observed in TGTS (4.96 times). The greater freeze-thaw stability of GCWS corn and banana starches compared to TG counterparts have been already reported [2, 6, 11, and 12]. The sigmoid manner of water separation in GCWS TS was also observed in corn counterpart but with a greater syneresis at 3rd cycle and a lower final water loss.

4. Conclusions The results of this study showed that the physical and mucoadhesive properties of modified TSs were wholly dependent on the AAT conditions. Increasing the temperature and alkali amount and decreasing the ethanol content led to higher cold water absorption, solubility, viscosity, consistency coefficient, clarity, freeze-thaw stability and interestingly mucoadhesion of starch samples. The modification of starch was also evident in morphological observations. Increasing the severity of the process gradually changed the starch microstructure into non-birefringent swollen granules with surface wrinkling. However, cold storage and repeated freeze-thaw cycles were more destructive to the rheological properties, clarity and paste structure of starches subjected to a higher degree of modification. Therefore, ESTS70(40),25,25, ESTS70(40),20,35, and especially ESTS110(40),30,25 are suitable for frozen products that are not exposed to more than two freeze-thaw cycles. In addition, these starches are the best candidates when more translucency and viscosity are required or when they are utilized as a delivery system for heat sensitive bioactives, although, a proper strategy must be devised to improve the product stability over time. ESTS 110(40),20,35 and ESTS70(40),20,25 are the preferred options in opaque products with desired lower consistency. Furthermore, these starches are suggested in formulations that need a mixture of native and pregelatinized starches such as puffable snacks that are prepared in a microwave oven. In spite of lower degrees of water absorption, clarity and mucoadhesion of GCWS CS than tapioca 18

counterpart, it is most suitable in the products with high viscosity and strong structure requirement that might be exposed to consecutive freeze-thaw cycles. ESTS110(40),30,25 had close properties to TGTS while maintaining the integrity of granules. Therefore, it can be an appropriate alternative of TGTS in instant foods and formulations with heat sensitive ingredients. Acknowledgements The authors would like to thank University of Tabriz for the financial support and Shiraz University and That of Medical Sciences for equipment and technical supports. References [1] J.N. BeMiller, K.C. Huber, Physical modification of food starch functionalities, Annu Rev Food Sci Technol. 6(1) (2015) 19-69. https://doi.org/10.1146/annurev-food-022814-015552. [2] M. Majzoobi, Z. Kaveh, C.L. Blanchard, A. Farahnaky, Physical properties of pregelatinized and granular cold water swelling maize starches in presence of acetic acid, Food Hydrocoll. 51 (2015) 375-382. https://doi.org/10.1016/j.foodhyd.2015.06.002. [3] B. Kaur, F. Ariffin, R. Bhat, A.A. Karim, Progress in starch modification in the last decade, Food Hydrocoll. 26(2) (2012) 398-404. https://doi.org/10.1016/j.foodhyd.2011.02.016. [4] A.O. Ashogbon, E.T. Akintayo, Recent trend in the physical and chemical modification of starches from different botanical sources: A review, Starch-Stärke 66(1-2) (2014) 41-57. https://doi.org/10.1002/star.201300106. [5] S. Rajagopalan, P.A. Seib, Granular cold-water-soluble starches prepared at atmospheric pressure, J Cereal Sci. 16(1) (1992) 13-28. https://doi.org/10.1016/S0733-5210(09)80076-3. [6] S. Hedayati, M. Majzoobi, F. Shahidi, A. Koocheki, A. Farahnaky, Effects of NaCl and CaCl2 on physicochemical properties of pregelatinized and granular cold-water swelling corn starches, Food Chem. 213 (2016) 602-608. https://doi.org/10.1016/j.foodchem.2016.07.027. 19

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24

Table 1 Different treatment conditions for the modification of starch samples. Treatment number

Starch sample symbol

Starch type (10 g)

Treatment

Weight (g) of sodium hydroxide solution (2.5 M)

Weight (g) of ethanol solution (40%, w/w)

Reaction temperature (ºC)

1

ESTS110(40),20,25 a

tapioca

Alcoholicalkaline treatment

20

110 (40)

25

2

ESTS110(40),25,25

tapioca

Alcoholicalkaline treatment

25

110 (40)

25

3

ESTS110(40),30,25

tapioca

Alcoholicalkaline treatment

30

110 (40)

25

4

ESTS110(40),20,35

tapioca

Alcoholicalkaline treatment

20

110 (40)

35

5

ESTS110(40),25,35

tapioca

Alcoholicalkaline treatment

25

110 (40)

35

6b

ESTS110(40),30,35

tapioca

Alcoholicalkaline treatment

30

110 (40)

35

7

ESTS70(40),20,25

tapioca

Alcoholicalkaline treatment

20

70 (40)

25

8

ESTS70(40),25,25

tapioca

Alcoholicalkaline treatment

25

70 (40)

25

9b

ESTS70(40),30,25

tapioca

Alcoholicalkaline treatment

30

70 (40)

25

10

ESTS70(40),20,35

tapioca

Alcoholicalkaline treatment

20

70 (40)

35

11b

ESTS70(40),25,35

tapioca

Alcoholicalkaline treatment

25

70 (40)

35

12 b

ESTS70(40),30,35

tapioca

Alcoholicalkaline treatment

30

70 (40)

35

13

TGTS c

tapioca

Thermal gelatinization

0

0

90

14

ESTS(corn)110(40),30,25d

corn

Alcoholicalkaline treatment

30

110 (40)

25

15

TGCS e

corn

Thermal gelatinization

0

0

90

a

ESTSa(b),c,d indicates ethanol-sodium hydroxide treated tapioca starch treated with “a” gram aqueous ethanol (b%) and “c” gram sodium hydroxide solution (2.5 M) at d ºC; b treatment conditions that led to complete disintegration of starch granules; therefore, they were excluded from further experiments; c Thermally gelatinized tapioca starch; d ESTS(corn)110(40),30,25 indicates ethanol-sodium hydroxide treated corn starch treated with 110 g aqueous ethanol (40%) and 30 g sodium hydroxide (2.5 M) at 25 ºC; e Thermally gelatinized corn starch.

25

Table 2 Cold water absorption (g/g) and solubility (%) of thermally gelatinized and modified corn and tapioca starches, produced with different amounts of ethanol and sodium hydroxide solutions at 25 and 35 ºC, measured at ambient temperature.a Starch sample Cold water absorption (g/g) Cold water solubility (%) 3.64i 2.36h ESTS110(40),20,25 b 14.18e 10.70d ESTS110(40),25,25 18.48b 20.20b ESTS110(40),30,25 h 9.02 5.03g ESTS110(40),20,35 c 17.68 16.96c ESTS70(40),20,35 f 13.28 7.44f ESTS70(40),20,25 b 18.81 20.89b ESTS110(40),25,35 c 17.15 16.32c ESTS70(40),25,25 21.29a 21.65a TGTS g 11.16 7.34f ESTS(corn)110(40),30,25 d 14.92 9.06e TGCS a For each property, means followed by different letters are significantly different (P < 0.05). All coefficient of variations (%) were less than 5%. b Abbreviations are defined in Table 1.

26

Table 3 Rheological properties of thermally gelatinized and modified corn and tapioca starches, produced with different amounts of ethanol and sodium hydroxide solutions at 25 and 35 ºC, measured after 1 h storage at 4, 25, and 40 ºC. a Starch sample

Stage

K (mPa.sn) of the samples measured

n of the samples measured

ηapp at 31.56 s-1 (mPa.s) of the

ηapp at 600 s-1 (min, mPa.s)

after 1-hour storage at

after 1-hour storage at

samples measured after 1-hour storage at

of the samples measured after 1hour storage at

4 ºC

25 ºC

40 ºC

4 ºC

25 ºC

40 ºC

4 ºC

25 ºC

40 ºC

4 ºC

25 ºC

40 ºC

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

Dd

N.D. e N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

ESTS110(40),25,25

U D

5868.2NOa 9265.2Ka

4469.8Ob 8844.7Kb

3928.2NMc 6845.9Kc

0.735Cb 0.667Da

0.767Ca 0.662Ea

0.703Dc 0.622Fb

2779.6La 3019.8Ka

2230.8Nb 2711.7KLb

1493.5Oc 1953.2Mc

1145.4Fa 1142.7Fa

1035.0Eb 1017.2Eb

612.8Ec 617.0Ec

ESTS110(40),30,25

U

7097.4Ma

6241.3Mb

4424.4Lc

0.754Bc

0.769Cb

0.804ABa

3752.5Ia

2903.1Ib

2289.3Jc

1606.5Ca

1481.2Cb

1318.4Bc

Fb

Ga

Eb

Fc

Ca

Cb

ESTS110(40),20,25 b

ESTS110(40),20,35

Uc

Ha

D

16616.5

U

4103.0Pa

D

6103.3

NOb Na

16022.0

Hb

2966.6Pc 4972.5

Nb

4009.0NLMb 7546.8

Nc

0.627

0.679

4956.8

EFa

4670.5

3445.5

1604.6

1486.3

1340.1Bc

435.12Fc

569.0Ea

0.666Db

0.694Da

0.694Da

1288.1Ob

1150.4Qc

1517.2Oa

497.4Gb

Ga

0.615

Ha

FGa

Nb

Pc

1824.7

Na

Hc

0.796

Aa

2208.4

Kc

0.609

0.601

1400.9

1274.4

401.7

435.1

570.9Ea

Cb

1440.0

1095.3Cc

ESTS70(40),20,25

9950.4Ic 4178.0LMc

0.626Fb 0.748Bc

0.630Gb 0.772BCb

0.654Ea 0.785CBa

4603.4Ga 2518.9Ma

4328.3Fb 2148.6Ob

2921.6Gc 2110.9Lb

1543.1Da 1101.7Fa

1495.8Cb 1031.1Eb

1097.2Cc 1076.2Ca

D

9184.0Ka

7893.7Lb

7467.6Jb

0.671Db

0.684Da

0.696Da

2823.8La

2656.8Lb

2533.6Ic

1101.1Fa

1049.6Eb

1077.5Cab

U

7873.9La

6022.8Mb

4095.7NLMc

0.750Bc

0.783Bb

0.820Aa

3617.1Ia

2830.2Jb

2157.2KLc

1618.0Ca

1493.8Cb

1318.1Bc

Aa

TGTS

D U

20089.7 6298.2Na

17241.7 5019.4Nb

15672.0 3638.1Nc

0.606 0.642Ea

0.620 0.777BCa

D

12708.0Ja

12039.3Jb

9598.2Ic

0.627Fa

U

21621.3Fa

18406.7Fb

17650.0Fc

D

Ea

Eb

Ec

22533.7

Ba

ESTS(corn)

U

80411.5

110(40),30,25

D

36686.5Da

TGCS

U D

Aa

113390.5 38465.5Ca

20599.0 58880.0

Bb

19356.5 42842.0

Bc

0.607 0.772Ca

Ea

5106.9 3062.8Ka

0.643Fa

0.624Fa

0.573Hb

0.612Ha

Ib

Ia

0.553 0.290

Lc

GHa

0.586 0.323

Lb

FGb

4735.0 2518.4Mb

Dc

4074.1 1977.6Mc

Ca

1616.1 1248.0Ea

1732.4 1205.2Da

1357.4Bb 870.9Db

3925.8Ha

3499.9Hb

2730.8Hc

1251.0Ea

1206.1Da

869.9Db

0.604FGa

4560.1Ga

4239.2Gb

4135.9Db

1327.3Eb

1452.5Ca

1322.7Bb

Ga

Fa

Db

Cc

1317.1

Eb

Ca

1313.6Bb

1860.8

Ba

Bb

1345.0Bc

0.593

0.344

Ia

4922.3 7046.3

Ba

DEb

1501.6

Da

15429.7Ib 4421.4Ob

Gb

2750.3

Kb

15940.0Ia 5510.8Oa

Gb

3425.7

Ja

D U

Ga

0.805

ABa

6430.7

Gc

0.768

Ab

Fb

U

ESTS70(40),25,25

3652.0

Ja

0.630

Ea

ESTS70(40),20,35

ESTS110(40),25,35

5239.5

Nc

10455.3

Hc

4758.2

6014.7

Bb

4295.4 4688.7

Bc

1440.0 1631.2

35372.0Ca

27006.0Db

0.412Ja

0.400Ja

0.410Ha

5454.4Da

5061.0Cb

3955.2Ec

1854.8Ba

1625.5Bb

1348.9Bc

Ab

Ac

Mb

Mb

Ia

Aa

Ab

Ac

Aa

Ab

1673.9Ab 1672.2Ab

96195.5 34722.0Db

59423.0 33379.5Cb

0.232 0.401Ka

0.229 0.387Kb

a

0.324 0.406Ha

9400.4 5770.6Ca

7613.6 5018.2Cb

6172.3 4681.3Bc

1993.2 1982.9Aa

1727.5 1704.5Ab

For each parameter, means values followed by different lowercase letters in the same line are significantly different (P < 0.05), which is the indicator of the difference between the samples stored at different temperatures. Means values followed by different uppercase letters in the same column are significantly different (P < 0.05). All coefficient of variation (%) were less than 5%. b Abbreviations are defined in Table 1. c Upward; d Downward; e Not detectable;

27

Table 4 Rheological properties of thermally gelatinized and modified corn and tapioca starches, produced with different amounts of ethanol and sodium hydroxide solutions at 25 and 35 ºC, measured after refrigerated storage for 1 h, 24 h and 1 week. a Starch sample

Stage

K (mPa.sn) of the samples measured

n of the samples measured after

ηapp at 31.56 s-1 (mPa.s) of the

ηapp at 600 s-1 (min, mPa.s) of

after storage for

storage for

samples measured after storage for

the samples measured after storage for

1h ESTS110(40),20,25

b

ESTS110(40),25,25 ESTS110(40),30,25 ESTS110(40),20,35 ESTS70(40),20,35 ESTS70(40),20,25 ESTS110(40),25,35 ESTS70(40),25,25 TGTS ESTS(corn) 110(40),30,25

TGCS

Uc Dd U D U D U D U D U D U D U D U D U D U D

24 h e

N.D. N.D. 5868.2NOb 9265.2Ka 7097.4Mb 16616.5Ha 4103.0Pb 6013.3NOa 6430.7Nb 15940.0Ia 5510.8Ob 9184.0Ka 7873.9Lb 20089.7Ga 6298.2Nb 12708.0Ja 21621.3Fa 22533.7Ea 80411.5Bb 36686.5Da 113390.5Ab 38465.5Ca

1h

24 h

1 week

1h

24 h

1 week

1h

24 h

1 week

N.D.

1 week N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D. 6429.0Ka 5974.7Lb 11166.0Ea 10731.0Fb 5168.3Ma 3882.9Nb 8727.3GHa 8107.9Ib 6922.8Ja 6306.5KLb 9105.2Ga 7922.1Ib 8653.5Ha 7835.1Ib 7328.7Jb 5155.4Mb 102649.0Ba 33712.5Db 144092.0Aa 39989.0Ca

N.D. 545.0Lc 411.0Lc 1398.2JKIc 419.2Lc 3084.0Fc 2599.1Gc 1910.5Hc 1164.2JKc 1802.6HIc 1010.9Kc 1407.3JKIc 433.5Lc 3488.1Ec 2765.9FGc 1500.7JHIc 439.9Lc 42507.0Bc 16932.0Dc 57293.5Ac 23136.5Cb

N.D. 0.735Cb 0.667Db 0.754Ba 0.630Fb 0.666Da 0.609Ga 0.768Aa 0.626Fb 0.748Ba 0.671Db 0.750Ba 0.606Gb 0.642Ea 0.627Fa 0.573Hc 0.553Ic 0.290La 0.412Ja 0.232Ma 0.401Ka

N.D. 0.605FEGDc 0.616EDc 0.604FEGDb 0.609FEDc 0.570Hb 0.612EDa 0.662Bb 0.669Ba 0.586GHc 0.598FEGc 0.568Hb 0.589FHGc 0.626CDb 0.640Ca 0.641Ca 0.694Ab 0.213Kb 0.393Ib 0.160Lb 0.364Jb

N.D. 2779.6La 3019.8Ka 3752.5Ia 4956.8EFa 1288.1Oa 1400.9Na 3425.7Ja 4603.4Ga 2518.9Ma 2823.8La 3617.1Ia 5106.9Ea 3062.8Ka 3925.8Ha 4560.1Ga 4922.3Fa 7046.3Ba 5454.4Da 9400.4Aa 5770.6Ca

N.D. 1720.0NOb 1668.2Ob 3016.8Eb 2940.3Fb 1223.4Pb 1092.2Qb 2666.2Gb 2589.6Hb 1943.2Lb 1868.4Mb 2114.5Kb 1971.9Lb 2505.4Ib 2233.8Jb 2189.0JKb 1760.7Nb 7713.6Ba 4618.2Db 10105.8Aa 5158.5Cb

N.D. 223.2Oc 211.1QOPc 486.1Kc 199.8QPc 726.0Gc 676.4Hc 536.8Ic 412.0Mc 513.5Jc 376.6Nc 435.4Lc 192.4Qc 936.9Ec 831.2Fc 441.8Lc 216.2OPc 4728.2Bb 2673.9Dc 5133.63Ab 3188.5Cc

N.D. 1145.4Fa 1142.7Fa 1606.5Ca 1604.6Ca 497.4Ga 401.7Ha 1501.6Da 1495.8Da 1101.7Fa 1101.1Fa 1618.0Ca 1616.1Ca 1248.0Ea 1251.0Ea 1327.3Ea 1317.1Ea 1860.8Ba 1854.8Ba 1993.2Aa 1982.9Aa

N.D. 559.2GHb 557.5IHb 876.6Db 876.3Db 347.6Jb 347.0Jb 984.5Cb 979.7Cb 530.8Ib 513.1Ib 606.5Gb 600.6GHb 807.3Eb 794.1Eb 748.3Fb 739.3Fb 1527.5Bb 1519.5Bb 1737.2Ab 1734.7Ab

N.D. 109.2IJHc 108.9IJHc 100.6Jc 101.7Jc 209.7Ec 209.1Ec 114.1IGHc 106.0IJc 150.5Fc 147.8Fc 100.5Jc 91.9Kc 296.4Dc 294.0Dc 120.3Gc 117.5GHc 913.3Bc 902.8Cc 936.4Ac 908.3BCc

N.D. 0.751Ca 0.790Ba 0.565GHc 0.755Ca 0.577Gb 0.602Fa 0.561Hc 0.613Fc 0.603Fb 0.698Da 0.573GHb 0.758Ca 0.611Fb 0.640Ea 0.613Fb 0.803Aa 0.275Ka 0.421Ia 0.236La 0.376Jb

a For each parameter, means values followed by different lowercase letters in the same line are significantly different (P < 0.05), which is the indicator of the difference between the samples stored for different times. Means values followed by different uppercase letters in the same column are significantly different (P < 0.05). All coefficient of variation (%) were less than 5%. b Abbreviations are defined in Table 1. c Upward; d Downward; e Not detectable;

28

Table 5 Turbidity of thermally gelatinized and modified corn and tapioca starches, produced with different amounts of ethanol and sodium hydroxide solutions at 25 and 35 ºC, during storage for 0, 24, 48 and 72 h at 4 ºC.a Starch sample

Duration of refrigerated storage (h) 0 24 48 72 2.660Aa 2.660Ca 2.659Ca 2.655Ba ESTS110(40),20,25 b 1.920Ed 2.225Dc 2.234Db 2.243Ca ESTS110(40),25,25 1.207Id 1.630Ic 1.695Hb 1.729Ga ESTS110(40),30,25 2.156Bc 2.171Eb 2.186Ea 2.190Da ESTS110(40),20,35 1.260Hd 1.574Jc 1.585Ib 1.603Ia ESTS70(40),20,35 1.960Cd 2.224Dc 2.239Db 2.245Ca ESTS70(40),20,25 1.180Jd 1.900Fc 2.014Fb 2.093Ea ESTS110(40),25,35 1.356Gd 1.682Hc 1.691Hb 1.701Ha ESTS70(40),25,25 1.150Kd 1.872Gc 1.987Gb 2.062Fa TGTS ESTS(corn) 110(40),30,25 1.946Dd 2.744Ac 2.769Ab 2.779Aa 1.591Fd 2.720Bc 2.754Bb 2.772Aa TGCS a Means followed by different lowercase letters in the same line are significantly different (P < 0.05). Values followed by different uppercase letters in the same column are significantly different (P < 0.05). All coefficient of variation (%) were less than 3%. b Abbreviations are defined in Table 1.

29

Figure Captions Fig. 1. Optical and electron micrographs of native, thermally gelatinized and modified corn and tapioca starches, produced with different amounts of ethanol and sodium hydroxide solutions at 25 and 35 ºC. NTS (A), ESTS110(40),20,25 (B), ESTS110(40),20,35 (C), ESTS70(40),20,25 (D), ESTS110(40),25,25 (E), ESTS70(40),25,25 (F), ESTS70(40),20,35 (G), ESTS110(40),30,25 (H), ESTS110(40),25,35 (I), TGTS (J), NCS (K), ESTS(corn)110(40),30,25 (L), TGCS (M). 1, 2, and 3 following (A)-(M) refer to electron, bright field, and polarized light micrographs, respectively. Bars on the electron micrographs = 10 µm (magnification = 3000x) and those on the optical micrographs = 100 µm. Fig. 2. Fracture strength (mN.cm-2) ( ) and work of adhesion (mN.mm) (

) of thermally

gelatinized and modified corn and tapioca starches, produced with different amounts of ethanol and sodium hydroxide solutions at 25 and 35 ºC, as the indicators of mucoadhesion. Data are presented as mean values ± standard deviation. For each property, different letters represent significant differences between mean values (p < 0.05). Fig. 3. Syneresis (%) of thermally gelatinized, and modified corn and tapioca starches, produced with different amounts of ethanol and sodium hydroxide solutions at 25 and 35 ºC, during 5 freezethaw cycles (24 h freezing at -18 ºC and 4 h thawing at 25 ºC). All coefficient of variation (%) were less than 10%.

30

A

A

A

B

B

B

C

C

C

D

D

D

E

E

E

31

F

F

F

G

G

G

H

H

H

I

I

I

J

J

J

32

K

K

K

L

L

L

M

M

M

International Journal of Biological Macromolecules - Kaveh et al. - Fig. 1

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International Journal of Biological Macromolecules - Kaveh et al. - Fig. 2

34

ESTS110(40),20,25

80.00

ESTS110(40),30,25

70.00

ESTS110(40),20,35

60.00

Syneresis (%)

ESTS110(40),25,35 ESTS70(40),25,25 ESTS110(40),25,25 ESTS70(40),20,25

50.00 40.00 30.00 20.00

ESTS70(40),20,35

10.00

TGTS

0.00

ESTS(corn) 110(40),30,25

0

1

2

3

4

5

6

TGCS

Syneresis (%)

Freeze-thaw cycles

25

ESTS110(40),30,25

20

ESTS110(40),25,35 ESTS70(40),25,25

15

ESTS70(40),20,35

10

TGTS

5 0

0

1

2

3

4

5

Freeze-thaw cycles

6

ESTS(corn)110(40) ,30,25 ESTS110(40),25,25 TGCS

International Journal of Biological Macromolecules - Kaveh et al. - Fig. 3

35

Highlights ·

Tapioca starch (TS) was modified by different alcoholic-alkaline treatments.

·

Granular cold water swelling (GCWS) TS paste showed a notable mucoadhesion.

·

Intense modification conditions led to translucent cold-set starch gels.

·

Less modified granules were better able to keep the quality attributes over time.

·

GCWS TS developed a weaker gel as compared to its corn counterpart.

36

Graphical abstract

37