Ultrasonic effect on the octenyl succinate starch synthesis and substitution patterns in starch granules

Food Hydrocolloids 35 (2014) 636e643

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Ultrasonic effect on the octenyl succinate starch synthesis and substitution patterns in starch granules Hai-ming Chen, Qiang Huang*, Xiong Fu*, Fa-xing Luo College of Food Sciences, South China University of Technology, 381 Wushan Road, Guangzhou 510640, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2013 Accepted 8 August 2013

Octenyl succinate starch (OS-starch) was prepared with ultrasonic-assisted treatment. The substitution patterns in starch granule was estimated by acid hydrolysis, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, confocal laser scanning microscopy and chemical surface gelatinization. The pasting properties of starch derivatives were studied by Brabender pasting analyzer. Results showed that ultrasound could reduce reaction time and improve the reaction efficiency over the control by 15e20%. Substitution pattern with the OS-groups located predominantly in the amorphous domains and on the granule surface with a surface effect that is more pronounced than in acetylation reactions. Ultrasonic treatment during octenyl-succinylation results in a somewhat less pronounced surface effect. Compared with OS-starch made by traditional method (OSS), OS-starch prepared under ultrasonic condition (UOSS) has slightly higher pasting temperature, break down and setback values, lower peak viscosity, hot paste viscosity and final viscosity. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Ultrasonic Octenyl succinate starch Synthesis Substitution patterns

1. Introduction OS-starch is an important chemically modified starch, which was first synthesized with water phase method by Caldwell and Wurzburg (1953). Because both hydrophilic and hydrophobic groups are present, OS-starch is widely used as emulsifier in food and industrial areas. During recent years, preparation methods and functional properties of OS-starch have been studied extensively (Angellier, Molina-Boisseau, Belgacem, & Dufresne, 2005; Bao, Xing, Phillips, & Corke, 2003; Huang, Fu, et al., 2010; Song, He, Ruan, & Chen, 2006). Normally, the reaction between octenyl succinic anhydride (OSA) and starch granules is retarded due to poor penetration of the big oily droplets of OSA into the starch granules in an aqueous suspension, and the site of reaction is limited to the surface of starch granules. As a consequence, the OS groups are not evenly distributed throughout the starch granule (Huang, Fu, et al., 2010; Shogren, Viswanathan, Felker, & Gross, 2000). Abbreviations: OSA, octenyl succinic anhydride; OSS, octenyl succinic anhydride modified starch; UOSS, OSS prepared under ultrasonic conditions; CLSM, confocal laser scanning microscopy; DS, degree of substitution; FTIR, Fourier transform Infrared spectroscopy; XRD, X-ray diffraction; Tp, pasting temperature; BD, back down value; SB, setback value; PV, peak viscosity; FV, final viscosity; HPV, hot paste viscosity; PMT, photomultiplier; MBþ, methylene blue; RE, reaction efficiency; RT, reaction time; BU, Brabender units. * Corresponding authors. Tel.: þ86 20 8711 3845; fax: þ86 20 8711 3848. E-mail addresses: [email protected] (Q. Huang), [email protected] (X. Fu). 0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2013.08.009

Ultrasonic treatments have been reported to accelerate chemical reaction, such as saccharification (Montalbo-Lomboy et al., 2010), acetylation (Zhang, Zuo, Wu, Wang, & Gao, 2012), oxidation (Orozco, Sousa, Domini, Araujo, & Band, 2013), transesterification (Gharat & Rathod, 2013) and degradation (Xu, Chu, Graham, & Nigel, 2013). When ultrasound passes through a liquid medium, the interaction among ultrasonic waves, liquid and dissolved gas leads to acoustic cavitation that may affect morphology and structure of the starch granules. Previous reports have studied the ultrasound effects on the supramolecular structure of potato and maize starches, suggesting that notch and groove were formed on the starch granule surface (Huang, Li, & Fu, 2007; Zhu, Li, Chen, & Li, 2012). As a result, the surface area of starch particles is increased, which may improve reaction efficiency (RE). Besides, starch degradation could happen, possibly due to the breakage of a-1,6 glycosidic bond (Iida, Tuziuti, Yasui, Towata, & Kozuka, 2008; Isono, Kumagal, & Watanabe, 1994). Furthermore, ultrasonic processor also can be used in the generation of nano-size dispersions of the reagent in water, because of the de-agglomeration effect and the reduction of droplet size. Kentish et al. (2008) reported that low frequency ultrasound (20 kHz) was employed to efficiently reduce the size of oil droplet in an aqueous suspension. However, shear forces generated at high frequency are relatively weaker than that generated at lower frequency, and not suitable for emulsification applications (Chemat, Zill-e-Huma, & Khan, 2011; Hielscher, 2005; Chandrapala, Oliyer, Kentish, & Ashokkumar, 2012).

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The patterns of substitution starches are distributed nonuniformly throughout the starch granules. It was found that amylose is higher substituted than amylopectin (Steeneken & Smith, 1991; Van der Burgt et al., 1998; Van der Burgt et al., 2000). The crystalline regions are less accessible for substitution than amorphous ones (Hood & Mercier, 1978; Steeneken & Smith, 1991; Van der Burgtetal et al., 2000). In the crystalline domains, branching zones are favored over linear regions (Van der Burgt et al., 1998; Van der Burgt et al., 1999). Although many studies on the synthesis and physicochemical characteristics of OS-starch have been conducted (Bhosale & Singhal, 2007; Franco, Cabral, & Tavares, 2002; Huang, Fu, et al., 2010; Jyothi, Raiasekharan, Moorthy, & Sreekumar, 2005; Park, Chung, & Yoo, 2004; Song et al., 2006), little work has been reported about ultrasonic effect on the OS-starch synthesis and OS group substitution in starch granules. The use of chemical gelatinization for revealing surface effects in starch modification has been described in rapid esterifications (Chen, Schols, & Voragen, 2004; Steeneken & Woortman, 2008), but not in slow etherification reactions. Steeneken and Woortman (2008) have obtained depth profiles of acetate groups in granular starch acetate by chemical gelatinization. In this study, OS-starch preparation was assisted by ultrasonic power (UOSS) and the products were compared to reference starches prepared without ultrasonic treatment (OSS). OS group substitution in amorphous/crystalline and surface/inner region of starch granules were further investigated by acid hydrolysis, chemical surface gelatinization, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and confocal laser scanning microscopy (CLSM) techniques. 2. Materials and methods 2.1. Materials Maize starch was purchased from Dacheng Company (Changchun, China). The original moisture content was 13.44%. High purity 2-Octen-1-ylsuccinic anhydride (OSA) was obtained from Sigmae Aldrich Chemical Co. (Milwaukee, WI) and all other chemicals were commercial products of analytical reagent grade. 2.2. Preparation of OS-starch The preparation of OS-starch was conducted in an ultrasonic dispersion instrument NP 8000 (Newpower Ultrasonic Equipment Co., Ltd, Guangzhou, China). Fig. 1 shows the schematic diagram of this apparatus. Pliers type ultrasonic transducers (single frequency factor at 20 kHz) are not contact with the samples, in order to avoid metal ion pollution. Transducers are powered by a generator which maximum output power 1000 W. The combined use of jacketed heat exchanger and a radiator fan ensure the temperature being controlled in an appropriate range. The probes of pH monitor and thermometer are installed in the storage tank (6 L). A pneumatic pump fitted with an air compressor was utilized in the samples circulation at a rate between 1 and 6 L/min. After the experiment is finished, the slurry was sucked out with the aid of vacuum pump. Maize starch (1400 g, dry basis) was suspended in 2600 mL distilled water (35%, w/w) with agitation in a 5000 mL beaker. After the slurry was poured into the storage container, the air compressor was started to activate circulating pump. The pH of the suspension was adjusted to 8.5 with 3% (w/v) NaOH solution, and the temperature was controlled at 35  3  C and then ultrasonic power (0e 1000 W) was switched on. Various doses of OSA (1e5%, based on the starch dry basis) was added slowly over 1 h. The reactions were carried out under ultrasonic condition. After completion of the reaction when pH in a constant state (all samples in Table 1) or

10

637

5 11 3

2

4

5 6

10 7

1

9 8 Fig. 1. Schematic diagram of the equipment for ultrasonic 1, Storage Tank; 2, pH Monitor; 3, Thermometer; 4, Jacketed Heat Exchanger; 5, Pliers Type Ultrasonic Transducers; 6, Radiator Fan; 7, Centrifugal Pump; 8, Air Compressor; 9, Pneumatic Pump; 10, Pipeline; 11, Pipe Clamp.

breaking reaction to obtain specific degree of substitution values (some samples in Table 2), the suspension was neutralized to pH 6.5 with 3% HCl and vacuum-filtered through filter paper, then the resulted modified starch was washed three times with distilled water and once with 95% alcohol to remove the residual reagents, and the solid was oven dried at 40  C for 24 h. After drying, the samples were passed through a 100-mesh standard sieve. 2.3. Determination of the degree of substitution The degree of substitution (DS) is the average number of hydroxyl groups substituted per glucose unit. The DS of OS-starch was determined by titration as previously reported (Song et al., 2006). OS-starch sample (5 g, dry weight) was accurately weighed and suspended by stirring for 30 min in 2.5 M HCl in isopropanol (25 mL). 100 mL 90% (v/v) aqueous isopropyl alcohol solution was added and the slurry stirred for an additional 10 min. The suspension was filtered through a glass filter and the residue was washed with 90% isopropyl alcohol solution until no Cl- could be detected any longer (using 0.1 M AgNO3 solution). The starch was re-dispersed in 300 mL distilled water, and then the dispersion was heated in a boiling water-bath for 20 min with stirring. The starch solution was titrated with 0.1 M standard NaOH solution, using phenolphthalein as an indicator. A blank was simultaneously

Table 1 a Effect of ultrasonic on the DSb and RE of OS-starch. No.

UP/W

OSA/%

RT/h

DS(  103)

1 2 3 4 5 6

0 200 400 600 800 1000

3 3 3 3 3 3

6.5 1.5 1.5 1.5 1.5 1.5

17.58 17.95 19.87 20.33 20.32 18.02

     

0.10a 0.06b 0.09c 0.05d 0.04d 0.08b

RE/% 75.78 77.37 85.65 87.63 87.59 77.67

     

0.43a 0.30b 0.23c 0.16d 0.16d 0.22b

a The data are averages of two measurements with standard deviation. Means in a column with different superscript letters (a-d) are significantly different (p < 0.05) by Duncan’s least significant test. b DS, degree of substitution, RE, reaction efficiency, UP, ultrasonic power, RT, reaction time. All the experimental conditions were controlled pH 8.5, temperature  35 C and starch concentration 35% (w/w).

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Table 2 Brabender viscosity parameters of native starch and OS-starcha. Tp/ C

DSb

Viscosity (BU) PV

c

0 0.00695d1 0.01908d2 0.01908e 0.02883d3

71.5 71.0 66.3 66.8 53.8

    

0.2d 0.6c 0.3b 0.4b 0.6a

180 285 373 335 499

HPV     

1.7a 6.6b 6.6d 5.5c 11.0e

160 160 269 212 207

BD     

1.7a 6.6a 2.6c 9.2b 5.3b

20 25 104 123 292

FV     

1.7a 1.7a 5.6b 3.6c 5.3d

294 312 382 362 344

SB     

5.3a 3.6b 4.2e 2.8d 5.0c

134 152 113 150 137

    

2.0b 4.4c 2.0a 5.6c 4.6b

a

Starch concentration: 6%. DS, degree of substitution, Tp, pasting temperature, PV, peak viscosity, HPV, hot paste viscosity, FV, final viscosity, SB, set back value (SB ¼ FV-HPV), BD, beak down value (BD ¼ PV-HPV), BU, Brabender units. The data are averages of three measurements with standard deviation. Means within a column with different online letters (aee) are significantly different (p < 0.05). c Normal maize starch. d OSS (d1:1% OSA, 6.5 h; d2: 5% OSA, 1.8 h; d3: 5% OSA, 6.5 h). e UOSS (3% OSA, 1 h, 600 W). b

titrated with native starch as a control. The DS was calculated by the following equation:

DS ¼

0:162  ðA  MÞ=W 1  ½0:210  ðA  MÞ=W

where: A is the titration volume of NaOH solution (mL), M is the molarity of NaOH solution, and W is the dry weight (g) of the OSstarch. The reaction efficiency was calculated as follows:

RE ¼

Actual DS  100% Theoretical DS

The theoretical DS was calculated by assuming that all of the added anhydride reacted with starch to form the ester derivative. 2.4. Acid hydrolysis OS-starch was hydrolyzed with HCl (5%, w/w) at 55  C (1.0 g starch/1.27 mL acid) for periods ranging from 0 to 12 h in the waterbathing constant temperature vibrator (Changzhou Aohua instrument co., LTD, Jiangsu, China). Aliquots taken at specific time intervals were neutralized and centrifuged at 2000 g for 10 min. The granular residues were washed with deionized water until the pH was 6, then the products were vacuum-filtered through filter paper, the solid was oven dried at 40  C for 24 h. After drying, the samples were passed through a 100-mesh standard sieve. And the total carbohydrate content in the supernatant was measured using the phenol-H2SO4 method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956; Huang, Fu, et al., 2010). The DS of the remaining cores were determined according to 2.3. The degree of hydrolysis (HD) was confirmed by the lost quantity accounts for the proportion of the total. 2.5. Fourier transform infrared spectroscopy During the esterification, hydroxyl groups of starch molecules were substituted by carbonyl groups of OSA that can be confirmed by FTIR spectroscopy. Changes in the chemical structure of starch were analyzed by FTIR spectroscopy (Vector 33, Bruker Optics, Ettlingen, Germany) according to the method of Song et al. (2006). Native and OS-starch were dried at 105  C for 12 h before analysis to avoid interference from water. Samples were prepared by finely grinding starch with KBr in a ratio of 1: 150 (w/w) and scanned over a wave number range from 400 to 4000 cm1. 2.6. X-ray diffraction The crystalline structure and relative crystallinity of OS starch was identified by an X-ray diffractometer (D/Max-200, Rigaku

Denki Co. Ltd., Tokyo, Japan). The instrument was operated at 30 mA and 30 kV with a wavelength of 0.1542 nm monochromatic Cu-Ka radiation and a theta compensating slit. The scanning of diffraction angle (2q) was from 5 to 35 at the speed of 5 /min. Prior to the analysis, the samples were equilibrated at 25  C and 100% relative humidity for 24 h, then lightly ground in an agate mortar prior to X-ray analysis. Percentages of crystallinity were calculated using the HermanseWeidinger method (Hermans & Weidinger, 1961). 2.7. Confocal laser scanning microscopy Native starch and OS-starch were dye-stained for CLSM as following (Zhang et al., 2011). Specimens (0.5 g) were suspended in 30 mL water. The pH of the suspensions was adjusted to 8.0, then 1% Methylene Blue (MBþ) solution was added into each sample. The mixture was incubated in a water-bathing constant temperature vibrator at room temperature for 3 h and the granules were washed with methanol to remove the residual dye. A TCS SP5 CLSM fitted with an Argon ion laser (Leica, Wetzlar, Germany) was used for the detection of the fluorescence signal from the dye-stained starch granules. The details of the Leica objective lens used were 40  /1.25 oil. The excitation wavelength was 514 nm with 56 capacity (Jayaraj, Umadevi, & Ramakrishnan, 2001). During the image acquisition, each line was scanned four times and averaged to reduce noise. 2.8. Chemical surface gelatinization Chemical surface gelatinization was conducted according to the method of Koch and Jane (2000) with some modifications. The OSS and UOSS with same DS (0.01908) were used for chemical gelatinization. Starch samples (10 g) were suspended in a CaCl2 (4 M) solution (75 mL) and stirred with a magnetic stirrer at room temperature for different periods of time to achieve different levels of surface gelatinization. The reaction was stopped by quickly adding 750 mL of cold (4  C) distilled water. Samples were centrifuged at 5, 000  g for 20 min, washed twice with H2O, washed once with ethanol, and dried at 40  C overnight. 2.9. Pasting properties of starches The pasting properties were determined using a Brabender viscoamylograph (Melchers Co., Germany). Samples were prepared by mixing starch (6.0 g) and 100 mL distilled water. The mixture was stirred manually for 1 min to facilitate dispersion before testing (Shih & Daigle, 2003). A 700 cm/g cartridge was fitted with the rotation speed set at 75 rpm. The heating and cooling cycles were programmed in the following manner: the samples were heated at

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All tests were performed in triplicate. Analysis of variance was performed and results were evaluated by Turkey-Kramer Multiple Comparison Test (p < 0.05) using the statistical software SPSS 18.0 (SPSS Inc., Chicago, Illinois, USA). 3. Results and discussion

30

0.022

A

0.020

25

0.018

B

0.016

20

0.014

15

0.012 0.010

10

B

0.008

A

0.006

5

0.004

Degree of Hydrolysis(%)

2.10. Statistical analysis

0.024

Degree of Substitution(DS)

7.5  C/min from 30 to 95  C, then held for 5 min at 95  C, cooled to 50  C at 7.5  C/min, and then held for 5 min at 50  C. The pasting temperature (Tp), peak viscosity (PV), hot paste viscosity (HPV), break down (BD) and setback (SB) values were recorded. The reference sample is normal maize starch. Two samples which have the same DS in Table 2 were synthesized under different conditions as stated in the footnotes in Table 2.

639

0 0

2

4

6

8

10

12

Acid Hydrolysis Time(h) Fig. 2. The effect of hydrolysis time on DS and DH of OS-starch (A(OSS) and B (UOSS) have the same DS (0.01908)).

3.1. Preparation of OS-starch with ultrasonic-assisted treatment OS-starch has been permitted by FDA to be used within food products at maximum 3.0% (Bhosale & Singhal, 2007). Therefore, 3% OSA content of the dry starch basis was chosen to investigate the ultrasonic effect on the esterification reaction. By varying the power of ultrasound, a series of products (Table 1) with different DS were prepared while holding the starch concentration (35%), pH (8.5) and addition of OSA (3%). It was marked the end of the reaction when the pH was unchanged. We also synthesized reference derivatives (Table 2) by changing the OSA dosage (1e5%) and reaction time. The degree of substitution (DS), reaction efficiency (RE) and reaction time (RT) of OS starch are shown in Table 1. With the gradually increase of ultrasonic power (0e1000 W), DS (RE) increased from 0.01758 (75.78%) to the peak value 0.2033 (87.63%) when the power was 600 W, and then dropped to 0.01802 (77.67%). The results indicated that ultrasonic can assist the esterification reaction moderately to achieve higher RE over the control by 15e 20% at most. However, continued increase of ultrasonic power could not further increase its DS and RE. Comparing with the control, the reaction time of OS-starch prepared by ultrasonic assistance was reduced drastically from 6.5 h to 1.5 h. The insufficient mixing between the water-insoluble OSA and starch granules by traditional reaction method was observed in previous studies (Ruan, Chen, Fu, Xu, & He, 2009; Zhang et al., 2011). Higher DS and RE may be caused by the ultrasonic cavitation effect, which increased the surface area by destroying the surface of the starch granules and created some pores and grooves (Huang et al., 2007; Zhu et al., 2012). In addition, strong shear forces generated by lower ultrasonic frequency (20 kHz) can reduce the oil droplet size and uniformize the droplets of OSA, which may reduce the reaction time effectively. However, when ultrasound spread through the liquid medium, a number of physical forces generates, such as shock waves, vibration, agitation and microjets (Chandrapala et al., 2012), the new generated compounds may be not stabilized or even easily hydrolyzed, result in the decrease of DS and RE with the continuous increasing of ultrasonic power.

The DS of the control decreased significantly in the first 4 h of hydrolysis, and little change was found for the rest of hydrolysis time. The DS of UOSS decreased drastically in the first 4 h of hydrolysis, and continued decreasing for 2 more hours. In general, the DS of UOSS was higher than that of OSS during the whole hydrolysis process. Normal cereal starch consists of three distinct components: (1) highly crystalline regions formed by double-helical amylopectin chains, (2) solid-like regions formed from lipid-complexed amylose, and (3) completely amorphous regions as associated with amylopectin branching regions and possibly the lipid-free amylose (Lauro, Forssell, Suortti, Hulleman, & Poutanen, 1999; Morgan, Furneaux, & Larsen, 1995). Reflections become stronger at increasing degree of hydrolysis as the degree of crystallinity increases (see Fig. 3). These results are due to the amorphous regions are more rapidly hydrolyzed by acid comparing with crystalline region. The residue after prolonged acid hydrolysis consisted of acid-resistant fractions with crystalline amylopectin (Robin, Mercier, Duprat, & Guilbot, 1975). Former studies have shown that amylose is higher substituted than amylopectin, and the substitute groups are mostly located in the amorphous region than crystalline ones (Cowie & Greenwood, 1957; Hood & Mercier, 1978; Huang, Fu, et al., 2010; Shogren et al., 2000; Steeneken & Smith, 1991; Van der Burgt et al., 2000). In the crystalline domains, branching zones are favored over linear regions (Van der Burgt et al., 1998; Van der Burgt et al., 1999). Therefore, DS of the OS-

3.2. OS group distribution in amorphous or crystalline regions In order to investigate the OS group distribution in amorphous or crystalline regions, OS-starch was subjected to acid hydrolysis to preferentially remove the amorphous region. The OS-starch (UOSS and the control) used in the hydrolytic reaction had the same DS (0.01908). The effect of hydrolysis time on DS and DH of the OSstarch is shown in Fig. 2. No pronounced difference was observed in DH between two samples, while there was a clear distinction between the two starches in the trend of DS with hydrolysis time.

Fig. 3. X-ray diffraction and relative crystallinity of OSS (DS ¼ 0.01908) with different DH (%).

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starch decreased rapidly in the beginning of the hydrolysis reaction (Fig. 2). After acid treatment for 4 h, the dropping trend of DS of OSstarch became slower. It demonstrated that there were still less numbers of OS groups bonded with amylose or relatively solid region. After 6 h hydrolysis, DS of OSS was basically stable, whereas the DS of UOSS still dropped at a low level. The results implied that OS groups could reach deeper layer of the granules or even got to the crystalline regions assisted by ultrasonic. In addition to investigate the presence of OS-groups in starch granules, FTIR also can be used to determine the DS of OS-starch (Biswas, Shogren, Kim, & Willett, 2006; Cizova, Srokova, Sasinkova, Malovikova, & Ebringerova, 2008). We tried to determine the impact of acid hydrolysis on OS-starch by comparing the corresponding FTIR peaks. Fig. 4 is a partially enlarged FTIR spectra of the samples. UOSS (C1) and OSS (C2) had the same DS (0.01908) and conspicuous peak at 1724 cm1, but not the normal maize starch (A). After acid hydrolyzed for 6 h, the residue of UOSS (B1) showed more conspicuous peaks comparing with OSS (B2), suggesting that UOSS has retained more OS groups comparing with control after hydrolysis. The results indicated that more OS groups can be induced by ultrasonic to crystalline regions of the starch granules, which was consistent with the acid hydrolysis results. 3.3. OS groups distribution on the periphery or inward part of starch granules Confocal laser scanning microscopy (CLSM) has been successful used for monitoring the visualization of starch granule morphologies (Chen et al., 2009; Van de Velde, Van Riel, & Tromp, 2002), pasting of teff and maize starches (D’Silva, Taylor, & Emmambux, 2011), microstructures of cornstarches (Chen et al., 2009), and distribution of amylose, amylopectin and OS groups within the starch granules (Glaring, Koch, & Blennow, 2006; Zhang et al., 2011). In this study, we investigated the distribution of OS groups in starch granules according to the method of Zhang et al. (2011).

Fig. 4. FT-IR spectra of acid hydrolyzed OS-starch. C1 (UOSS) and C2 (OSS) have the same DS (0.01908), after their hydrolysis to obtain B1 and B2 respectively, A is normal maize starch. Acid hydrolysis time is 6 h.

Samples were stained by Methylene Blue (MBþ) before detected by CLSM, and fluorescence intensity induced by OS groups was also determined. Fig. 5 shows the CLSM optical sections of OS-starch granules obtained at the same photomultiplier (PMT) after being stained with MBþ. The native starch granules (DS: 0; Fig. 5A) did not show fluorescence, while increasingly stronger fluorescence was observed with the DS of OS-starch increasing (DS: 0.01758; 0.01908; Fig. 5B, and 5 C respectively). A more uniform distribution of negatively charged groups (-COO- of OS groups) was available to stain with MBþ when samples have a higher DS (Zhang et al., 2011). CLSM of UOSS (DS: 0.01908) is showed in Fig. 5D. Compared with Fig. 5C, it showed almost the same fluorescence intensity. However, the distribution of the fluorescence spots between Fig. 5C and Fig. 5D were slightly different. Fluorescence spots were mainly located in the surface of the granules in Fig. 5C. A relatively uniform distribution of the fluorescence spots could be obtained from Fig. 5D, which implied that OS groups are slightly more evenly distributed in the granules of UOSS. In other words, a little more OS groups were induced to the inner region of the OS starches when the reaction was assisted by ultrasound. This is due to ultrasonic procedure can promote the de-agglomeration effect of the starch slurry and the reduction and uniformity of droplet size. The use of chemical gelatinization for revealing surface effects in starch modification has been described (Chen et al., 2004; Steeneken & Woortman, 2008). Steeneken and Woortman (2008) have obtained depth profiles of acetate groups in granular starch acetate by chemical gelatinization. In order to further study the distribution of OS groups in periphery and inward of the two starches (OSS and UOSS), treatment with CaCl2 (4 mol/L) was performed at 20  C. The degree of starch gelatinization depended on the exposure time in the CaCl2 solution, and various types of starch required different reaction time to achieve a similar degree of surface gelatinization (Huang, Zhang, Chen, & Li, 2010; Kuakpetoon & Wang, 2007). Two starch samples were treated with CaCl2 solution for various times to achieve different degree of gelatinization. As shown in Fig. 6, the changing of DS of the two starches (UOSS and OSS) with the degree of chemical gelatinization is different. During the early stage of the CaCl2 treatment (degree of gelatinization < 40%), a layer of gelatinized starch developed from the periphery of the granules uniformly and slowly, but the decline speed of the DS (OSS and UOSS) was very fast (Jane, 1993; Kuakpetoon & Wang, 2007). This can be explained by the OS groups mainly concentrated in the periphery of the starch granules. The gelatinization speed of the two types of ‘peeled’ remaining granules was gradually accelerated for the rest of reaction time, and the gelatinization process was stopped when the starch was completely gelatinized. By compared the DS of two types of starch granules in similar degree of gelatinization, the DS of UOSS was slightly higher than that of OSS in the process of gelatinization, which the effects of ultrasonic treatment on the substituent distribution in OS-starch is moderate and may promote OS group to enter inward of the starch granules. An explanation was that, as the ultrasound pass through the suspension liquid, the notch and groove were formed on the starch granule surface, due to the acoustic cavitation caused by the interaction of ultrasonic waves, liquid and dissolved gas (Huang et al., 2007; Zhu et al., 2012). The small size OSA droplets reduced by ultrasonic can reach the inward part of the starch granule via the notch and groove. The result was consistent with CLSM. When compared to a common acetylation with acetic anhydride (Steeneken & Woortman, 2008), the surface effect in the esterification with OSA appears to be much more pronounced which is probably due to the higher water solubility and diffusion rate of acetic anhydride as compared to OSA. In a word, Figs. 5 and 6 suggest that the effect of ultrasonic treatment on the substituent distribution in OS-starch is very

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641

Fig. 5. CLSM optical sections of starches with different DS. A, native starch; B, OSS (DS: 0.01758); C, OSS (DS: 0.01908); D: UOSS (DS: 0.01908).

moderate. The decrease in DS of OSS as a function of degree of gelatinization is slightly more pronounced than in UOSS. 3.4. Pasting properties The Brabender viscosity parameters reflect the pasting characteristics of starch during processing and use (Deffenbaugh & Walker, 1989). The pasting temperature (Tp) is the temperature at

0.020

Degree of substitution(DS)

0.018

UOSS OSS

0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 0

21

43

80

Degree of gelatinization(%) Fig. 6. The effect of degree of gelatinization on DS of remaining OS-starch granule (the initial two samples (UOSS and the control) have the same DS (0.01908)).

which the viscosity starts to rise. Lower pasting temperature means faster swelling. Peak viscosity (PV) reflects the extent of granule swelling. Hot paste viscosity (HPV) is the viscosity after holding for 30 min at 95  C. The breakdown (BD) of the starch paste, defined as the difference between the peak viscosity and hot paste viscosity (PV-HPV). BD reflects the stability of the paste during cooking, whereas the final viscosity (FV) at 50  C indicates the stability of the cooked paste. Setback value (SB) shows the viscosity increase on cooling to 50  C (FV-HPV), indicating the extent of retrogradation of the starch product. The pasting parameters of normal maize starch and OS-starches with different DS are shown in Table 2. All OS-starches exhibited lower pasting temperature, higher peak viscosity and higher break down values compare with the native starch, which was consistent with previous studies (Bao et al., 2003; Song et al., 2006). Compared with the control (DS: 0.01908), a slightly higher Tp, BD, SB and lower PV were observed in the UOSS (DS: 0.01908). Peak viscosity is a consequence of starch granule swelling, which is generally caused by weakening of the internal network structure of the granule by gelatinization. Electrostatic forces will also enhance swelling, especially in distilled water. Because hydrophobic interactions may suppress swelling, the results suggest that electrostatic effects dominate. The incorporation of a bulky OS group destroys the ordered structure and changes the pasting properties of native starch. With the increase of DS, the structure of starch becomes more inflexible and the negative charged OS group repels with each other, so the OS-starches tend to be gelatinized at lower temperature (Shih & Daigle, 2003; Song et al., 2006; Song, Zhu, Li, & Zhu, 2010). The

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PV but not the HPV was increased with increase of DS, which was due to the hydrophobic alkenyl groups were incorporated into a normally hydrophilic native starch granule, the modified starch attains surface active properties that lead to starch granule aggregation. The aggregated granules might be broken down quickly at high temperature, causing the viscosity at 95  C to decrease quickly, resulting in a lower HPV (Bao et al., 2003). And according to Flory (1953), chap. XIII, charged networks have a higher elastic modulus than uncharged ones at the same cross-link density which may also affect HPV. With assistance of ultrasound, small part of bonds between molecular chains, which have low energy, such as hydrogen bond, can easily be damaged by shear force. The remaining part (UOSS) is more difficult to be destroyed by heat compared with OSS. So the UOSS has slightly higher pasting temperature. OSS also showed higher peak viscosity than the both native counterpart and UOSS, which is due to the introduction of OS groups, and the reduction in starch molecular weight caused by ultrasonic. Molecular degradation will result in a strong decrease in swelling power because of disruption of the internal network structure of the granule. The higher Tp and lower PV and HPV of UOSS as compared to OSS (Table 2) could possibly be caused by a more even substituent distribution in UOSS. Hydrophobic interactions are operating between OS-groups within a single granule. Thereby the swelling of individual granules is restricted, causing an increase in Tp and a decrease of PV. The break down viscosity is the difference between the peak viscosity and the viscosity after holding for certain minutes at 95  C (Huang et al., 2007; Muhammad, Hussin, Man, Ghazali, & Kennedy, 2000). The higher break down viscosity indicates granule disruption or the less tendency of starch to resist shear force during heating (Huang, Fu, et al., 2010). The breakdown value of UOSS (123) was higher than that of OSS (104), indicating that gelatinized UOSS had a weaker resistance to shear force. The setback value of UOSS (150) was higher than the control (113), which may be due to the increase of amylose content caused by ultrasonic and key role of amylose in restructuring of starch network structure (Chan, Bhat, & Karim, 2010; Miles, Morris, Orford, & Ring, 1985). 4. Conclusions Ultrasound cannot change the crystalline type of the starch, while it could lead to dispersion, surface area expanding, and increase the reaction efficiency. Reaction of starch with OSA results in a heterogenous substitution pattern with the OS groups located predominantly in the amorphous domains and on the granule surface with a surface effect that is more pronounced than in acetylation reactions. Ultrasonic treatment during octenylsuccinylation results in a somewhat less pronounced surface effect and lower viscosity characteristics (PV, HPV and FV) of OSstarch. Acknowledgments The financial supports received from the National Natural Science Foundation of China (31101378), China Postdoctoral Science Foundation (2012M511813), Science and Technology Planning Project of Guangdong Province, China (2012A020602005) are acknowledged. References Angellier, H., Molina-Boisseau, S., Belgacem, M. N., & Dufresne, A. (2005). Surface chemical modification of waxy maize starch nanocrystals. Langmuir, 21(6), 2425e2433.

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