Preparation of cross-linked maize (Zea mays L.) starch in different reaction media

Carbohydrate Polymers 124 (2015) 302–310

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Preparation of cross-linked maize (Zea mays L.) starch in different reaction media Jung Sun Hong ∗ , Sara V. Gomand, Jan A. Delcour Laboratory of Food Chemistry and Biochemistry, and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 7 November 2014 Received in revised form 4 February 2015 Accepted 11 February 2015 Available online 23 February 2015 Keywords: Chemical modification STMP Aqueous ethanol Aqueous acetone Degree of cross-linking RVA

a b s t r a c t Granular normal maize starch was reacted with sodium trimetaphosphate in deionized water (MSH2 O ), aqueous sodium sulfate solution (MSNa2 SO4 ), aqueous ethanol (MSethanol ) or aqueous acetone (MSacetone ) under otherwise identical reaction conditions. Analysis of the resultant starches by Rapid Visco Analysis (RVA) showed that the starch was cross-linked to a higher degree in aqueous ethanol or aqueous acetone than in water or sodium sulfate solution, and with minimal starch leaching. While MSacetone and MSethanol had incorporated similar levels of phosphorous, RVA analysis and microscopic analysis showed that MSacetone granules were more effectively stabilized by cross-linking than MSethanol granules. Cross-linking in aqueous acetone is believed to either contain the greater numbers of distarch monophosphate (versus monostarch monophosphate), or occur more intensively at the granule outer layers than that in aqueous ethanol and, at the same time, to account for the greater granular strength of MSethanol than that of MSacetone . © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Starch is a major functional biopolymer, which can be utilized as a texturant in food and non-food applications. It is often chemically modified to obtain desired functional properties. One way to modify starches is by cross-linking. Cross-linking introduces intra- and intermolecular bonds in starch molecules (Tattiyakul & Rao, 2000). It strengthens the granules and reduces their tendency to solubilize and rupture (Woo & Seib, 1997). Cross-linked (CL) starches are used as thickening agents in food applications and as fillers in non-food applications which require stability towards swelling, high temperature, shear, and acidic conditions (Singh, Kaur, & McCarthy, 2007). One of the reasons for the interest in CL starches is that crosslinking can render them enzyme resistant. Enzyme resistant starch (RS) is a collective name for starch fractions that are not digested in the small intestine, but are fermented in the colon. RS is subdivided into different groups (Eerlingen, Crombez, & Delcour, 1993; Englyst, Kingman, & Cummings, 1992). Several studies have reported on CL starch as resistant starch type 4 (RS4 ) (Juansang, Puttanlek, Rungsardthong, Puncha-arnon, & Uttapap, 2012; Koo, Lee, & Lee,

∗ Corresponding author. Tel.: +32 16 376824; fax: +32 16 321997. E-mail addresses: [email protected], [email protected] (J.S. Hong). http://dx.doi.org/10.1016/j.carbpol.2015.02.022 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

2010; Sang, Prakash, & Seib, 2007; Sang & Seib, 2006; Sang, Seib, Herrera, Prakash, & Shi, 2010; Shin, Song, & Seib, 2004; Shin, Woo, & Seib, 2003; Woo & Seib, 2002). In wheat starch, the RS4 content increases with the level of distarch monophosphate cross-linking (Sang et al., 2007, 2010). Here, further research efforts aiming at better understanding the cross-linking process are welcome. In general, CL starches are prepared by reacting granular starch with bi- or multifunctional reagents in an alkaline medium at temperatures ranging from 30 to 50 ◦ C (Kasemsuwan & Jane, 1994; Woo & Seib, 2002; Wu & Seib, 1990; Wurzburg, 1986). For reaction with sodium trimetaphosphate (STMP) and/or sodium tripolyphosphate (STTP), sodium sulfate has been often used in the starch crosslinking as a granule stabilizing salts (Lim & Seib, 1993; Woo & Seib, 1997). Woo and Seib (1997) reported that the addition of sodium sulfate enhances extent of cross-linking. The relative effects of various reaction parameters in determining the extent of starch modification in terms of total level of functional groups incorporated (i.e., degree of substitution, DS) and/or difference in swelling/gelatinization/pasting properties between the CL and native form of starches (i.e., degree of cross-linking) have been extensively investigated. The degree of cross-linking increases with pH, levels of cross-linking agent, reaction time, and the presence of a stabilizing salt or N,Ndimethylformamide (Chung, Woo, & Lim, 2004; Cooreman, van Rensburg, & Delcour, 1995; Hirsch & Kokini, 2002; Jyothi, Moorthy, & Rajasekharan, 2006; Kasemsuwan & Jane, 1994; Kaur, Singh,

J.S. Hong et al. / Carbohydrate Polymers 124 (2015) 302–310

& Singh, 2006; Lim & Seib, 1993; Liu, Ramsden, & Corke, 1999; Muhammad, Hussin, Man, Ghazali, & Kennedy, 2000; Wang, Thompson, & Liu, 2011; Wattanachant, Muhammad, Mat Hashim, & Rahman, 2003; Wongsagonsup, Shobsngob, Oonkhanond, & Varavinit, 2005; Woo & Seib, 1997; Wu & Seib, 1990; Yeh & Yeh, 1993). As an alternative to adding salts for inhibiting gelatinization, Han and Sosulski (1998) and Kweon, Bhirud, and Sosulski (1996) used a solution of ethanol in water for preparing cationized starches. This medium eliminates the need for extensive washing to remove salts after the reaction. At 50 ◦ C, a higher degree of substitution (DS), rate and efficiency of cationization were obtained in aqueous ethanol than in water. Although different reaction parameters and their impact on the degree of cross-linking have been studied, only a few studies (Jyothi et al., 2006; Kurakake, Akiyama, Hagiwara, & Komaki, 2009) have used mixtures of organic solvents–water mixtures. They confirmed that aqueous organic solvent is a valid medium to crosslink starches. Again this background, the objective of this study was to investigate the effect of reaction media containing ethanol or acetone on the properties of the resultant CL starches. To that end, normal maize starches were CL with sodium trimetaphosphate (STMP) in four different reaction media (deionized water, an aqueous sodium sulfate solution, aqueous ethanol and aqueous acetone) under otherwise constant reaction conditions in terms of starch concentration, reaction time, pH, temperature, and the level of reagent. The resultant CL starches were characterized and their properties were determined.

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recovered and suspended in 40.0 ml 70% (v/v) aqueous ethanol [or 70% (v/v) aqueous acetone for MSacetone ]. Next, the suspensions were centrifuged as described above. This washing step was executed three times to remove non-reacted STMP and/or residual salts. Finally, the starch samples were Büchner-filtered and airdried at room temperature for 24 h. Control samples underwent the same procedure but without adding STMP. The level of leached starch in the supernatant from each control was determined (cf. Section 2.3). Cross-linking was also performed using acetone concentrations ranging from 45 to 85%, v/v to result in MSacetone45 , MSacetone55 , MSacetone65 , MSacetone75 and MSacetone85 by following the above described procedure. Table 1 lists the reaction conditions. 2.3. Determination of leached starch Supernatants from the above control samples were concentrated to ≤5 ml by rotary evaporation at 50 ◦ C and freeze-dried. An aliquot (10.0 ml) of 90% (v/v) dimethyl sulfoxide (DMSO) was added to the freeze-dried samples which were then incubated for 60 min at 100 ◦ C and then for 24 h at room temperature under continuous stirring (150 rpm) (Han & Lim, 2004). An aliquot of this solution was used to determine leached carbohydrate (CHL) content by the phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) using a glucose standard. CHL levels were quantified as glucose × 0.9 and expressed as the percentage of the initial starch content. Measurements were conducted in duplicate. 2.4. Degree of substitution and reaction efficiency

2. Materials and methods 2.1. Starch sources and chemicals Normal maize starch was obtained from Cargill (Vilvoorde, Belgium). It contained 26.4% amylose, as assessed by the Megazyme amylose/amylopectin assay kit (Bray, Ireland), which itself is based on the method by Yun and Matheson (1990). All reagents, solvents, chemicals and enzymes used were of at least analytical grade and obtained from Sigma-Aldrich (Bornem, Belgium) unless indicated otherwise. 2.2. Starch cross-linking with sodium trimetaphosphate STMP CL starches were prepared in two fully aqueous reaction media as outlined by Woo and Seib (1997) (i.e. MSH2 O , CL starch reacted in deionized water and MSNa2 SO4 , CL starch reacted in an aqueous solution of sodium sulfate) and in two aqueous organic media (MSethanol , CL starch reacted in aqueous ethanol and MSacetone , CL starch reacted in aqueous acetone) under reaction conditions similar to those by Kweon et al. (1996). Table 1 summarizes the levels of the reagents used for each treatment. Starch (15.0 g, dry basis) was accurately weighed and suspended in 55.7 ml of the three media and 54.7 ml for MSNa2 SO4 . The media for producing MSH2 O , MSNa2 SO4 , MSethanol and MSacetone were H2 O, 1.8% (w/v) sodium sulfate [6.7% sodium sulfate (w/w, starch weight basis)], 87.6% (v/v) aqueous ethanol and 87.6% (v/v) aqueous acetone, respectively. An aliquot (0.3 ml) of 10.0% STMP solution (w/v, in deionized water) was added to the starch suspension. The reaction pH was then adjusted to 11.5 by adding 0.5 ml 2.0 M NaOH for producing MSH2 O , MSethanol and MSacetone and 1.5 ml 2.0 M NaOH for MSNa2 SO4 . The samples were incubated for 180 min at 45 ◦ C with continuous stirring. The reaction pH was maintained at 11.5 by adding a few additional drops of 2.0 M NaOH. The reaction was stopped by adjusting the pH to 6.0 using 1.0 M HCl. The mixture was then centrifuged (3000 × g, 10 min, 20 ◦ C). The starch pellets were

Phosphorus contents of control and CL starches were quantified by inductively coupled plasma-optical emission spectrometry (ICPOES) (Optima 3300 DV, Perkin Elmer, Waltham, MA, USA). Starch samples were prepared according to Anderson (1996) with slight modification. Starch samples (0.05 g, dry basis) were weighed into glass tubes and 1.0 ml 69% HNO3 (v/v) was added. The tubes were sealed and incubated for 180 min at 140 ◦ C. They were then cooled and diluted to a final volume (10.0 ml) with MilliQ water. All measurements were conducted in duplicate. The degree of substitution (DS) was calculated based on the amount (in g) of incorporated phosphorus in 100 g of starch, with the incorporated phosphorus (in g) being the difference between its levels in CL and control sample (P). To do so, the following equation was used (Paschall, 1964):

DS =

162 × np 162 × P = 100 − 102 × np 3100 − 102 × P

where nP , mole of incorporated phosphorus in 100 g starch (np = P/31); 162, the molar mass of anhydroglucose; 100, dry starch sample weight (in g); 102, the molar mass of NaPO3 2− . The reaction efficiency (RE) (Kweon et al., 1996) for each reaction medium is the ratio of the actual DS to the theoretical DS (i.e. assuming that all STMP reagents added would have reacted with starch). RE% =

DS × 162 mole of phosporus in STMP added/dry starch weight (g) × 100

2.5. Close packing concentration and swelling power The close packing concentration (C* ) of starch is the concentration (C) at which the swollen granules just fully fill up the available space in the starch suspension at a given temperature

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Table 1 Reaction medium composition and level of reagents for cross-linked (CL) maize starch (MS)1 with STMP. Samples

MSH2 O MSNa2 SO4 MSethanol MSacetone MSacetone 45 MSacetone 55 MSacetone 65 MSacetone 75 MSacetone 85

Used solvent

H2 O 6.7% Na2 SO4 3 Ethanol Acetone Acetone Acetone Acetone Acetone Acetone

Reaction medium composition (pH 11.5, 45 ◦ C) Starch (g, db2 )

10% STMP (ml)

Deionized water (ml)

Solvent (ml)

2.0 M NaOH (ml)

15 15 15 15 5 5 5 5 5

0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.2

55.7 54.7 6.9 6.9 16.1 13.1 10.1 7.1 4.1

0 0 48.8 48.8 13.5 16.5 19.5 22.5 25.5

0.5 1.5 0.5 0.5 0.2 0.2 0.2 0.2 0.2

1 MSH2 O , CL in deionized water; MSNa2 SO4 , CL in aqueous sodium sulfate solution; MSethanol , CL in aqueous ethanol; MSacetone , CL in aqueous acetone; MSacetoneX , CL in X% (v/v) aqueous acetone. 2 Dry weight basis. 3 Aqueous solution containing 6.7% sodium sulfate (w/w, starch weight basis).

(Eerlingen, Jacobs, Block, & Delcour, 1997). C* and swelling power (SP) for native and CL starches were determined as in Eerlingen et al. (1997) with slight modifications. Starch suspensions (100 mg in 9.0 ml of deionized water) were prepared in tubes (98 × 12 mm) which were sealed and heated for 30 min at 50, 70, or 90 ◦ C, with intermittent shaking every 5 min. After cooling for 5 min, starch suspensions were centrifuged (1000 × g, 30 min, 20 ◦ C). Sediments were weighed and the supernatants were analyzed for carbohydrate leaching (CHL) in duplicate. The C* and SP were determined according to the following equations: C∗ =

dry matter starch weight (g) × 100 sediment weight (g)

SP =

sediment weight (g) × 100 [dry matter starch weight (g)] × (100 − %CHL)

2.6. Differential scanning calorimetry (DSC) Starch gelatinization properties were analyzed using a Q2000 DSC (TA Instruments, New castle, DE, USA) as described in Gomand et al. (2010). Starch (3.00–4.00 mg, dry basis) was accurately weighed in an aluminum pan (Perkin Elmer, Waltham, MA, USA) and deionized water was added [1:3 w/w starch (dry basis): water]. The pan was hermetically sealed, equilibrated for at least 20 min and then heated from 20 ◦ C to 120 ◦ C at 4 ◦ C/min. An empty aluminum pan was used as reference, and calibration was done with indium. The onset (To ), peak (Tp ), and conclusion (Tc ) temperatures and enthalpy (H, J/g) were determined using TA Universal Analysis software. Measurements were conducted in triplicate. 2.7. Pasting properties in DMSO-water and deionized water Starch pastes in 92.5% DMSO (w/w) were studied by a Rapid Visco Analyzer 4 (RVA) (Perten Instruments, Hägersten, Sweden), using the method described by Cooreman et al. (1995). To starch (1.75 g, dry basis) 92.5% (w/w) DMSO was added to obtain a final net total weight of 25.0 g [starch concentration: 7.0% (w/w)]. The starch suspension was kept at 30 ◦ C for 1 min, heated from 30 ◦ C to 75 ◦ C in 3 min, and then kept at 75 ◦ C for 20 min. Throughout this temperature profile, it was constantly stirred at 160 rpm. The final viscosity was determined for each generated curve. Measurements were conducted in duplicate. The traditional RVA pasting properties in deionized water was also investigated. The starch suspension in deionized water [15% (w/w, dry basis), total weight 25.0 g] was prepared as described above, and was subjected to the following temperature-time profile: 1 min at 50 ◦ C, heating from 50 to 95 ◦ C in 9 min, and kept at

95 ◦ C for 15 min, cooling from 95 to 50 ◦ C in 15 min, and additional 10 min at 50 ◦ C, while being stirred at 160 rpm. A single measurement was conducted. 2.8. Light microscopy Samples withdrawn at the end of the RVA cycle were viewed using a Nikon (Melville, New York, NY, USA) Eclipse 80i microscope equipped with a Nikon digital Sight DS-U2 camera to visualize the granule (remnants). A small starch paste sample (∼50 ␮l) was diluted with 0.2 ml of deionized water and 0.1 ml of a solution containing 0.2% I2 and 2.0% KI. A few droplets of the diluted starch paste were placed on a microscope slide, and covered with a cover glass. Images were analyzed with Nikon NIS-Elements BR software. 2.9. Statistical design and statistical analysis Significant statistical differences (p < 0.05) for several variables were determined using the one-way ANOVA procedure and least significant difference (LSD) test with the Statistical Analysis System 9.3 (SAS, SAS Institute, Cary, NC, USA). 3. Results and discussion 3.1. Effects of reaction medium on starch leaching, degree of substitution (DS), and reaction efficiency (RE) Table 2 shows CHL levels and the DS and RE values of the CL starches. Very low levels of CHL (<0.06%) were observed for the CL starches. Cross-linking in aqueous organic media led to significantly less CHL than that in water or the salt solution, indicating that the former more efficiently inhibited starch swelling during cross-linking. Han and Sosulski (1998) for starch cationization also observed less CHL when using an ethanol solution instead of water. In the present case, no differences in CHL were observed between MSH2 O and MSNa2 SO4 on the one hand or between (MSethanol and MSacetone ) on the other hand. The total phosphorus content of native maize starch was 0.018%, which is in agreement with literature (0.019%) (Chung et al., 2004). The different reaction media resulted in a broad range of DS values (Table 2), even if equal levels of STMP reagent and identical reaction parameters had been used (Table 1). The DS values of MSH2 O and MSNa2 SO4 (0.0004 and 0.0002, respectively) were lower than those of MSethanol and MSacetone (0.0023 and 0.0025, respectively). This demonstrates that the RE highly depends on the reaction medium. The DS values in this study [DS 0.0002–0.0025, equivalent to 0.003–0.048% phosphorus (P, w/w)] are lower than those reported in literature [DS 0.0015–0.021, equivalent to 0.028–0.400% P w/w)]

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Table 2 Leached starch, degree of substitution (DS) and reaction efficiency (RE) for cross-linked (CL) maize starch (MS)1 . Values within a column sharing a common letter are not significantly different (p < 0.05). Means and standard deviations of at least two analytical replicates are given. Samples

Leached starch2 (%)

DS

Incorporated phosphorus (%)

RE (%)

MSH2 O MSNa2 SO4 MSethanol MSacetone MSacetone 45 MSacetone 55 MSacetone 65 MSacetone 75 MSacetone 85

5.7E − 02 ± 4.7E − 03a 5.9E − 02 ± 8.5E − 04a 4.2E − 03 ± 1.7E − 04b 3.1E − 03 ± 1.0E − 04b nd3 nd nd nd nd

4.4E − 04 ± 1.4E − 05b 1.8E − 04 ± 0.0E + 00c 2.3E − 03 ± 0.0E + 00a 2.5E − 03 ± 1.8E − 04a 1.7E − 03 ± 1.4E − 05d 2.2E − 03 ± 7.1E − 06c 2.7E − 03 ± 0.0E + 00b 3.0E − 03 ± 2.8E − 05a 3.0E − 03 ± 1.4E − 05a

8.4E − 03 ± 2.8E − 04b 3.4E − 03 ± 2.8E − 05c 4.5E − 02 ± 4.2E − 05a 4.8E − 02 ± 3.4E − 03a 3.3E − 02 ± 2.8E − 04d 4.2E − 02 ± 7.1E − 05c 5.1E − 02 ± 7.1E − 05b 5.6E − 02 ± 5.7E − 04a 5.7E − 02 ± 2.1E − 04a

13.9 ± 0.5b 5.5 ± 0.0c 73.6 ± 0.1a 79.2 ± 5.6a 53.8 ± 0.4d 68.7 ± 0.1c 83.7 ± 0.1b 92.9 ± 0.9a 93.5 ± 0.4a

1 MSH2 O , CL in a deionized water; MSNa2 SO4 , CL in aqueous sodium sulfate solution; MSethanol , CL in aqueous ethanol; MSacetone , CL in aqueous acetone; MSacetoneX , CL in X% (v/v) aqueous acetone. 2 Amount of starch leached from each reaction control (without addition of STMP). 3 Not determined.

(Chung et al., 2004; Lim & Seib, 1993; Mao, Wang, Meng, Zhang & Zheng, 2006; Sang et al., 2007; Woo & Seib, 1997). Jyothi et al. (2006) observed that the DS values of CL cassava starches produced with low levels of epichlorohydrin [0.25–0.50% (w/w), based on starch dry weight] were within the detectable range when performing the reaction in N,N-dimethylformamide (non-aqueous condition) but below the detection limit when performing the reaction in water. The DS values in their study were determined by RVA measurements as percentages of the difference in peak viscosity between CL and native starch to the peak viscosity of the native starch. For starch carboxymethylation, cyclohexane, N,N-dimethylformamide, acetone (Khalil, Hashem, & Hebeish, 1990), mixtures of organic solvents (e.g., ethanol–acetone, ethanol–isopropanol, and ethanol–benzene) (Kamel & Jahangir, 2007) or aqueous solutions of isopropanol, ethanol, methanol (Kweon et al., 1996) result in higher DS values than does water. In conclusion, the use of aqueous organic solutions yields CL starches with higher DS and RE values than does water (MSH2 O and MSNa2 SO4 ). The literature which reported similar effects with the organic solvent medium did not fully explain the reasons. The most probable hypothesis would be that the presence of organic solvent facilitates absorption of alkali by starch granules while limiting granule swelling (absorption of water) (Warren, Reid, & Hamalainen, 1952). For both aqueous organic solutions, no significant differences in DS and RE values of CL starches were observed. The DS and RE values of MSNa2 SO4 and MSH2 O differed only slightly (Table 2). The low DS value for MSNa2 SO4 is probably due to the loss of phosphorus from starch granules in the alkaline (pH 11.5) salt solution. More than 95% of P is removed from wheat starch when suspended in an aqueous sodium sulfate solution (16.0%) at pH 11.7 (42 ◦ C for 3 h) (Matsunaga & Seib, 1997). A lower phosphorus level was also found in the control sample of MSNa2 SO4 (without adding STMP) than in the other control samples (data not shown). Optimal concentrations for starch cross-linking have not been reported for aqueous acetone, while the optimal ethanol concentration was found to be 65% for starch cationization (Kweon et al., 1996). Table 2 lists DS and RE for acetone concentrations ranging from 45 to 85% (v/v). DS values increased with acetone concentrations up to 75%, but this trend tapered off above 75% acetone (Fig. 1). For a 16.7% (w/v) starch slurry, the highest DS values were obtained when using acetone concentrations of 75 and 85%. 3.2. Effects of reaction medium on swelling behavior Fig. 2A and B shows the SP and C* at 50, 70, and 90 ◦ C of native and CL starches. For all starch samples, SP gradually increased (and C* decreased) with temperature but to an extent depending on the reaction medium used.

Fig. 1. Degree of substitution (DS) of maize starches (MS) cross-linked in aqueous acetone as a function of the used acetone concentrations for the preparation [45, 55, 65, 75, and 85% (v/v) suspensions]. Standard deviations are indicated.

SP at 50 ◦ C was low for all starch samples, although slightly larger SP and lower C* were observed for MSH2 O than MSethanol or MSacetone , followed by native starch or MSNa2 SO4 . At 70 ◦ C, SP varied between 6.6 and 9.0 g/g. MSacetone had a lower SP and a higher C* than the other CL starches. At 90 ◦ C, SP values decreased and C* values increased with higher DS levels, except for MSH2 O (Table 2). The lower SP of MSNa2 SO4 , MSethanol and MSacetone can be attributed to a higher granular integrity at 90 ◦ C when cross-linking. Choi and Kerr (2004) suggested that cross-linking allows maintaining granular integrity during heating. The higher or unchanged SP (i.e., MSH2 O ) of starches CL only to a very low degree is in agreement with the outcome of different studies (Kartha & Srivastava, 1985; Kaur et al., 2006; Liu et al., 1999; Wurzburg, 1986). 3.3. Effects of reaction medium on thermal properties Table 3 lists the gelatinization characteristics of the native and CL starches. Only small differences in DSC gelatinization temperatures were observed. MSNa2 SO4 , MSethanol , and MSacetone had slightly higher To , Tp , and Tc than native starch, while those of MSH2 O and the native starch did not differ. In most cases, either unchanged (Chatakanonda, Varavinit, & Chinachoti, 2000; Gunaratne & Corke, 2007; Liu et al., 1999; Yeh & Yeh, 1993) or slightly increased (Choi & Kerr, 2004) starch gelatinization temperatures have been reported for cross-linked starches. A small increase in gelatinization temperature may be attributed to the reduced mobility of amorphous regions when introducing cross-links (Singh et al., 2007). Here,

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Table 3 Differential scanning calorimetry (DSC) gelatinization and Rapid visco analysis (RVA) pasting (in 92.5% DMSO) characteristics of native and cross-linked (CL) maize starch (MS)1 . Standard deviations of triplicate DSC measurements did not exceed 0.3 ◦ C for T and 0.5 J/g for H readings, while for RVA measurements they did not exceed 200 mPa s. Means of at least three analytical replicates are given. Values within a column sharing a common letter are not significantly different (p < 0.05). Sample

DSC gelatinization characteristics To (◦ C)

Native starch MSH2 O MSNa2 SO4 MSethanol MSacetone 1 2

c

63.7 63.3d 64.7a 64.0b 64.4a

Tp (◦ C) d

69.8 69.4e 71.1a 70.2c 70.6b

RVA pasting characteristics in 92.5% (w/w) DMSO Tc (◦ C) c

79.2 79.1c 81.0a 79.8b 79.9b

H (J/g) ab

14.2 14.1ab 14.4a 14.7a 13.5b

Peak viscosity (mPa s)2 c

3357 3129c 4957b 6720a 837d

Final viscosity (mPa s) 782c 882c 2488b 5025a 829c

MSH2 O , CL in a deionized water; MSNa2 SO4 , CL in aqueous sodium sulfate solution; MSethanol , CL in aqueous ethanol; MSacetone , CL in aqueous acetone. Peak viscosity was determined by the viscosity at 6 min.

CL and native starches had similar H values. This indicates that cross-linking primarily occurred in amorphous regions of the granules and implies that complete melting of crystallites can occur despite the presence of cross-links (Chatakanonda et al., 2000). Moreover, this shows that little if any starch gelatinization took place during the cross-linking reaction. 3.4. Effects of reaction medium on pasting in 92.5% DMSO

Fig. 2. Swelling power (SP, g/g of starch) (A), and close packing concentration (C* , %) (B) of native and cross-linked (CL) maize starches (MS) at 50, 70, and 90 ◦ C. MSH2 O (CL in a deionized water); MSNa2 SO4 (CL in aqueous sodium sulfate solution); MSethanol (CL in aqueous ethanol); MSacetone (CL in aqueous acetone). Standard deviations are indicated.

an increased gelatinization temperature was also observed for the reaction control of MSNa2 SO4 (data not shown), indicating that this shift was unlikely due to a greater degree of cross-linking in the MSNa2 SO4 sample. Its small increase in gelatinization temperatures may be due to the presence of residual sodium sulfate rather than to cross-linking. DSC measurements have shown that starch in excess water containing increasing levels of sodium sulfate has increased gelatinization temperatures (Evans & Haisman, 1982).

RVA analyses of CL starches in 92.5% DMSO are useful for characterizing the degree of cross-linking (Cooreman et al., 1995). In this method, a 7.0% starch suspension is heated to 75 ◦ C and held at 75 ◦ C in 92.5% DMSO while stirring. The viscosity in 92.5% DMSO is mainly determined by the solubility and the resistance to shear in the DMSO medium and provides information which differs from that obtained in aqueous environment (swelling/gelatinization/retrogradation). Indeed, in the DMSO solution, the viscosity of CL starch increased with degree of cross-linking even at a low concentration of the starch suspension (7%, w/w) (Cooreman et al., 1995). This approach is especially useful to assess the quality of CL starches when investigating CL starches which often fail to build up a detectable range of viscosity in aqueous suspension. Fig. 3A compares the relative degrees of cross-linking while Table 3 compares the peak and final viscosities for each starch sample. Peak viscosity was reached at the start of the isothermal phase at 75 ◦ C (4–6 min), after which the viscosity decreased, except for what was the case with MSacetone (Fig. 3A). The viscosity decreased over the pasting profile as the insoluble fraction of starch granules gradually diminished by the prolonged heating and shear (Cooreman et al., 1995). In addition, starch is susceptible to ‘shearthinning’ behavior in DMSO solution at a steady shear rate regime, as noted by Kapoor and Bhattacharya (2001). The highest peak viscosity was observed for MSethanol , followed by MSNa2 SO4 , MSH2 O or native starch, and MSacetone (Fig. 3A and Table 3). MSH2 O and native starch showed similar behavior, which is probably due to the very low DS value of MSH2 O . These two starches had similar C* and SP readings (cf. infra). To understand the impact of the DMSO solution in the RVA treatment, samples were stained using I2 /KI solution after RVA analysis and visualized by light microscopy (Fig. 4). Complete solubilization was seen for native starch and MSH2 O (Fig. 4A and B). MSNa2 SO4 retained its structural integrity slightly in the DMSO solution more than native starch since some granule remnants were detected (Fig. 4C). For MSethanol and especially MSacetone , the granular structure was largely retained with almost no leached molecules around the starch granules (Fig. 4D and E). These results suggest high degrees of cross-linking and great granular resistance for the MSethanol and MSacetone samples. The low viscosity reading for MSacetone is believed to result from the fact that, under the present experimental conditions, the MSacetone suspension does not reach the threshold concentration necessary for measurable

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Fig. 3. RVA pasting profiles of 7% (w/v) suspensions of native and cross-linked (CL) maize starches (MS) in 92.5% (w/w) DMSO solution. (A), MSH2 O (CL in deionized water); MSNa2 SO4 (CL in aqueous sodium sulfate); MSethanol (CL in aqueous ethanol); MSacetone (CL in aqueous acetone), and (B), MSacetoneX [CL in X% (v/v) aqueous acetone].

elasticity (near the close-packing point) (Evans & Lips, 1992) (Fig. 3A). Below such threshold concentration, the viscosity depends on the soluble fractions of the granules which were very low for MSacetone (Fig. 4E) (Cooreman et al., 1995). MSacetone showed even greater effect of cross-linking than MSethanol , although it had the same DS value as did MSethanol . These observations are in agreement with the differences in swelling parameters (cf. infra) which themselves are probably due to differences in the cross-linking reaction patterns. More cross-linking at the granule surface in MSacetone than in MSethanol most likely inhibits granule swelling to a greater extent at the beginning of the swelling process as the CL layer on the surface probably acts as a physical barrier against granule swelling and solubilization. Given

the similar DS values between the two media, another possibility accounting for the lower stability of MSethanol than of MSaceone granules is a higher number of monostarch monophosphate which destabilize starch. Additional work will be needed to understand the relationship between reaction patterns and physical properties at similar DS values. Fig. 3B shows the influence of acetone concentration on the cross-linking degree. The paste viscosity increased with the concentration of acetone in the reaction medium. This is in line with the increase in DS levels at higher acetone concentrations (Table 2). However, the most effective cross-linking was obtained for MSacetone75 , which showed lower pasting viscosity than the other starches. Iodine staining of the starch pastes showed that

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Fig. 4. Light microscopy images of native and cross-linked (CL) maize starches (MS) granules stained with 0.2% I2 /2.0% KI after pasting in 92.5% (w/w) DMSO solution: (A) native starch; (B) MSH2 O (CL in deionized water); (C) MSNa2 SO4 (CL in aqueous sodium sulfate); (D) MSethanol (CL in aqueous ethanol); (E) MSacetone (CL in aqueous acetone).

Fig. 5. Light microscopy images of native and cross-linked (CL) maize starches (MS) granules stained with 0.2% I2 /2.0% KI after pasting in 92.5% (w/w) DMSO solution: (A) MSacetone45 ; (B) MSacetone55 ; (C) MSacetone65 ; (D) MSacetone75 ; (E) MSacetone85 , where MSacetoneX is CL in X% (v/v) aqueous acetone.

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Cross-linking in aqueous ethanol or aqueous acetone resulted in CL starch with higher DS and RE levels than did reacting the starch in water. The DS levels were quite predictive for the extent of swelling and CHL. When using aqueous acetone as reaction medium, a concentration of 75% was most optimal in producing a high degree of cross-linking for a 16.7% (w/v) starch slurry based on the pasting properties. We conclude that aqueous solutions of organic solvents can be suitable for cross-linking which requires a great RE and a high DS levels. Furthermore, no extensive washing steps are needed as is the case when granule stabilizing salts are used. In addition to the present work, a study on reaction patterns within starch granules among various reaction medium compositions is needed to fully understand the differences in swelling behavior when cross-linking in aqueous ethanol and aqueous acetone.

Fig. 6. RVA pasting profiles of 15% (w/v) suspensions of native and cross-linked (CL) maize starches (MS) in deionized water. MSH2 O (CL in deionized water); MSNa2 SO4 (CL in aqueous sodium sulfate); MSethanol (CL in aqueous ethanol); MSacetone (CL in aqueous acetone).

the granular integrity of MSacetone75 was retained better than for the other starches (Fig. 5). This phenomenon mirrored what we observed in Fig. 4E for MSacetone , where a great degree of granule integrity with little evidence of starch leaching was noted. In addition, Fig. 3A showed little viscosity development for MSacetone . 3.5. Effects of reaction medium on pasting in deionized water Fig. 6 shows the pasting profiles of native and CL starches in water. The concentration of starch suspension was increased to 15% (w/w) to obtain a detectable range of viscosity development for the CL starches. The peak viscosities trended downward after cross-linking irrespective of the cross-linking medium. The greatest impact on the viscosity was observed for MSacetone over the entire profile, in agreement with its low viscosity in the DMSO solution (Fig. 3A). The breakdown (the difference between the peak and hot paste viscosity) appears to decrease in the order native MS > MSH2 O > MSNa2 SO4 > MSethanol > MSacetone . This indicates that the granular strength increased to a degree corresponding to that of cross-linking such as observed in the pasting profiles in the DMSO medium. This validates the DMSO RVA profile method for monitoring degrees of cross-linking. The final viscosities of CL starches (except for MSacetone ) increased as the degrees of cross-linking increased such as deduced from the pasting viscosities in the DMSO solution. The higher final viscosity than for the native counterpart has also been observed when cross-linking waxy maize starch with a low level of sodium trimetaphosphate (STMP) [2.0% (w/w, starch weight basis)] (Wongsagonsup et al., 2005), and ␥-irradiation followed by STMP/STPP (sodium tripolyphosphate) cross-linking with waxy maize starch (Chung et al., 2010). Chung et al. (2010) explained the increased final viscosity of the CL starches with increasing ␥-irradiation dose by that some fragmented structures would rather promote the starch aggregation to increase viscosity during cooling. Thus, it is plausible that the less degree of CL starches than MSacetone form partially broken structures during the heating and shearing process, which may facilitate the aggregation of starch molecules, thereby contribute to the rise of final viscosity. 4. Conclusion This study provides understanding of how starch cross-linking with STMP reagent is influenced by the reaction medium composition. The DS and RE values, thermal, swelling and pasting properties were compared for starches CL in four different media.

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