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1 EFFECTS OF DEGREE OF CROSS-LINKING ON PHYSICAL PROPERTIES, PASTING AND FREEZE-THAW STABILITY OF CASSAVA STARCH MODIFIED BY REACTIVE EXTRUSION PROCESS (REX) 1 HEEBTHONG, 2 KHANARAK, 3 RUTTARATTANAMONGKOL, 4 KHANITTA 1 Department of Agro-Industry, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok, Thailand 2 Center of Excellence in Biomaterial, Naresuan University, Phitsanulok, Thailand 1 khanakwangtung@gmail.com, khanittar@nu.ac.th Abstract- This study aimed to investigate a modification of cassava starch through phosphorylation and cross-linking reaction using a reactive extrusion process (REX). It combined chemical and physical alteration of starch in a single process. Cross-linking reaction of cassava starch was achieved using a pilot scale twin screw extruder under controlled temperature (40 C) and screw speed (200 rpm) conditions. A range of sodium trimetaphosphate (STMP) ( % dry weight starch), NaOH concentration ( M) and moisture content (50 85% dry weight starch) was preliminarily studied to produce cross-linked starch with three different degree of cross-linking; 1) low degree of cross-linking (50-69%); 2) medium degree of cross-linking (70-89%); and 3) high degree of cross-linking (>90%). Physical properties (water solubility, WSI and swelling power), pasting properties, gel properties and freeze-thaw stability of cross-linked cassava starch samples were studied in comparison with those of extruded starch control (non-cross-linked) and native cassava starch samples. The results indicated that the clarity and peak viscosity of starch decreased with an increase in degree of cross-linking. Cross-linking reduced the water solubility index and swelling power of starch. Introduction of phosphate cross-links into the starch restricted the mobility of the molecular structure, leading to a reduction in WSI and viscosities of starch. Extruded starch controls and all cross-linked cassava starch samples showed higher swelling power at 30 C than the native cassava starch. At 80 C, the swelling power of all cross-linked cassava starch samples was similar to the native starch but higher than the starch control equivalents. The maximum peak and final viscosities were shown in lowest degree of cross-linking. Gels made from cross-linked starch samples showed greater strength and stability against freeze-thaw process compared to the native starch. This study suggested that the low and medium degree of cross-linked starch could be use as thickener and stabilizer in a wide variety of food products. Keywords- Cassava starch, Cross-linking, Reactive Extrusion, Stabilities, Physical Properties. I. INTRODUCTION Cassava starch is one of the most important agricultural products in Thailand. Thailand is the world s largest exporter of cassava derived products that can be utilized in several food and non-food applications [1] due to its distinct characteristics including colorless, odorless, tasteless, high paste viscosity, and clear gel [2]. However, the use of cassava starch is limited due to its poor properties and instability under freeze-thaw, high acid, shear force, and heat conditions. To improve functionalities of native cassava starch, physical and chemical modification means have been used to alter its molecular structure and functional properties for specific applications such as increased water-holding capacity, high heat and shear resistance, minimized syneresis, and rheological behavior improvement [3], [4]. Phosphorylation is one of the most method used to modify starch and produce monostarch and distarch phosphate. The phosphate is bounded to starch molecules that causes changes in functional properties of starch [5]. Usually, STMP, STPP, POCl 3 and EPI are common chemical agents used for phosphorylation and cross-linking. For conventional starch modification, the reaction time is approximately more than 3 hour and generates a large amount of wastes [6], [7]. Reactive extrusion (REX) is versatile, continuous, commercial viability, easy to adapt to industry scale and cost-effective process. It offers short reaction time and produces a low level waste [8] [10]. In this process, in-barrel moisture content, reaction temperature, and time as well as the mechanical energy input can be manipulated by adjusting the operating conditions. This novel technique could replace the conventional starch modification process for industrial production. Therefore, the main objective of this research is to study the effect of different degree of cross-linking (low, medium, and high cross-linking) of cassava starch modified through phosphorylation using a pilot-scale twin screw extruder on physical properties such as water solubility and swelling power, pasting properties, and stabilities against freeze-thaw process of starch. II. MATERIALS AND METHODS 2.1 Materials and chemicals Cassava starch used to prepare phosphorylated crosslinked starches was obtained from Polymate Co., Ltd. (Bangkok, TH) with moisture of 10.98±0.57%; amylose content of 16.62±0.41%, starch content of 49
2 97.18±0.21% and phosphorus contents of 0.006%, respectively. Sodiumtrimetaphosphate (STMP) was used as the cross-linking agent (Sigma-Aldrich Inc., St. Louis, MO). Other chemical agents were supplied by RCI Labscan (Bangkok, TH). 2.2 Preparation of phosphorylated crosslinked starches The co-rotating twin screw extruder used in this studies was a pilot-scale (CTE D25L32, Chareon tut Co., Ltd, TH) with 7 zones of barrel, the screw diameter of mm and length to diameter ratio (L/D) of 32. Different STMP (%), NaOH concentration and water content levels were preliminarily designed to produce cross-linked cassava starch at low, medium and high degree of cross-linking, respectively as shown in Table 1. Feed rate of solid mixture was fixed at 5.3 kg/h. Water or NaOH solution was injected at zone 1 to adjust ph and in-barrel moisture. Extrusion process was performed at 40ºC and screw speed of 200 rpm using a 4-mm circular die diameter. The control starch sample was prepared without STMP and NaOH solution treatment. All starch extrudates were cut to 1 2 cm size, air dried at 60ºC for 8 hour, grounded, and rinsed with distilled water (starch : water ratio of 1 : 4) for 2 times to remove free STMP, then centrifuged at 9000 rpm for 10 minute. The ph of starch slurry was adjusted to 6.5 with 1 M HCl and washed with distilled water. The starch samples were dried at 60ºC for 8 hour, grounded, sieved through 100 mesh screening size, and stored at 4ºC until analysis. Table 1: Conditions for phosphorylated cross-linked starch production. cooled to 45ºC at the cooling rate of 10ºC/min. The peak viscosity values of native and cross-linked cassava starch were calculated degree of cross-linking according to [6] using (1), Degree of cross - linking(%) = ((A - B)/A) x 100 (1) Where A is peak viscosity of the native starch and B is peak viscosity of the modified starches. 2.4 Determination of water solubility index (WSI) and swelling power Water solubility index (WSI) and swelling power were determined according to Lee et al. [12]. The 2.5 g of starch was weighed (W 1 ) in 50 ml of preweighted centrifuge tube, then 30 ml of distilled water was added, capped the tube and shaking in water bath at 30ºC and 80ºC for 30 minute. The solution was centrifuged at 3000 rpm for 10 minute, the supernatant was poured to pre-weighted moisture can, dried at 105ºC until the weight was constant and record the dry dissolved starch (W 3 ). The sediment was weighted (W 2 ). The WSI and swelling power of starch samples were calculated as follows: weight of dry dissolved starch (W ) WSI = 3 x 100 (2) dry sample weight (W ) 1 sediment of starch after centrifuge(w ) Swelling factor = 2 (3) dry sampleweight (W ) Determination of freeze thaw stability The freeze-thaw stability of starch was measured according to the procedure of Wang et al. [13]. The 20 ml of 12% w/w starch suspension was prepared in 50 ml centrifuge tube, heated in water bath at 95ºC for 30 minute, cooled to room temperature and stored at -18ºC for 18 hour. After frozen, the starch gel was thawed for 6 hour at room temperature and centrifuged for 20 minute at 4000 x g, water was removed and weighted. The freeze-thaw stability was measured as % syneresis (percentage of released water weight to starch gel weight) for 5 cycles. 2.3 Determination of pasting properties and degree of cross-linking Pasting properties of a native starch and cross-linked starches were determined using a Rapid Visco Analyzer (RVA Model Super 3, Newport Scientific Pty. Ltd., AU) according to AOAC [11]. The 25 ml distilled water was added to the 3.0 g starch sample and held at 45ºC for 2 min, heated up to 95ºC at the heating rate of 10ºC/min, held for 3 minute, then 2.6 Determination of gel properties The paste clarity of starch suspensions was determined according to Wongsagonsup et al. [14]. The 1% w/v of starch suspension was heated in water bath at 95ºC with shaken every 5 minute for 30 minute total time and cooled down to room temperature. The transmittance (%T) of the starch suspension at 650 nm was measured using UV/VIS spectrophotometer and water was used as a blank sample. Gel properties were measured in terms of hardness and cohesiveness according to method described by Wattanachant et al. [15] with modification. The starch gel was prepared by heating 12% w/w starch 50
3 suspension at 95ºC for 30 minute in a 50 ml beaker with 2 cm height and kept for 24 hour at room temperature before texture analysis. The starch gel was compressed with 10 mm diameter cylindrical probe at 50% distance with speed of 2.0 mm/s using the Texture Analyzer (model QTS25, Brookfield Engineering Laboratories Inc., USA). 2.7 Statistical Analysis Statistical analysis was performed using One-way ANOVA and Duncan s Multiple Range Test (DMRT) by SPSS 17.0 software. III. RESULTS AND DISCUSSION 3.1 Pasting properties and degree of crosslinking The pasting properties and degree of cross-linking of phosphorylated cross-linked starches in comparison with native and control starch samples were presented in Table 2. Degree of cross-linking of phosphorylated cross-linked starch was determined and found in arrange of %. In this study, starch samples were categorized based on degree of crosslinking level as; high degree of cross-linking (>90%), medium degree of cross-linking (70-90%) and low degree of cross-linking (50-70%). There was a significant difference in pasting properties by degree of cross-linking. Lower peak viscosity with increasing degree of cross-linking was observed. The lower pasting temperature of starches compared to that of native starch indicated their lower resistance towards swelling. Substantial reduction in peak viscosity in cross-linked starch compared with that of native starch was due to phosphate intermolecular linkage in starch molecules. The pasting temperature among cross-linked starches was not significantly different, however, the peak viscosity and breakdown decreased with degree of cross-linking due to the intermolecular bridge in starch molecule. Crosslinking decreased amorphous chain mobility and strengthened the starch molecular structure. Higher degree of cross-linking suppressed swelling power and lowered the peak viscosity. The more hydrated granule had less space between starch granules resulting in a resistance to breakdown [14], [16]. According to Choi and Kerr [17], the cross-linking increased the temperature resistances of starch granules since cross-linked bonding strengthened the starch chains. Other values of pasting properties, breakdown viscosity, final viscosity, and set back all decreased with increase in degree of cross-linking. The breakdown viscosity is a measure of the fragility of the granules indicating the stability of starch pastes and their tendency to resist shear force during the heating process [18] and [19]. Final viscosity of the starch paste which represents an increase in viscosity of starch paste during cooling decreased significantly with degree of cross-linking. Highest final viscosity was shown in low degree of cross-linked starch sample that was non-significantly different (P>0.05) from native starches. The decrease in final viscosity was degree of cross-linking dependent. The final viscosity is largely due to re-ordering or polymerization of leached amylose and long linear amylopectin [18] and [20]. 3.2 Water solubility index (WSI) and swelling power The WSI and swelling power of starch samples at 30ºC and 80ºC were shown in Table 3. At 30ºC, extruded control and all cross-linked starches exhibited higher solubility than the native starch. This is due to the extrusion process promoting dextrinization and increasing solubility of starch [21]. However, at 80ºC, WSI of native starch and extruded starch control significantly increased due to its gelatinization, while all cross-linked starch samples showed slightly increase in solubility. Higher degree of cross-linking lowered WSI since phosphatelinkage prevented the breakage and leaching solid of starch granule that resulted in lower solubility. This result suggested that cross-linking strengthened the Table 2: Pasting profiles and degree of cross-linking of native cassava starch, control starch and phosphorylated cross-linked starch. The same lower case letters superscript (a) in the same column each sample group are not significantly different (P 0.05). molecular structure of starch and showed higher temperature resistance. Swelling power of starches was presented in Table 3. Swelling power decreased with an increase in crosslinking density. All starch samples possessed greater swelling power at 80ºC and showed similar value to the native starch. This is because the starch molecules 51
4 were gelatinized sufficiently and the amylopectin chains were hydrolyzed at a temperature of 80 C. Our observed results were similar to the studies reported by Wongsagonsup et al. [22]. It was shown that the swelling power increased with temperature but declined by cross-linking. At low level of crosslinking, the repulsion of negative charge of phosphate in starch chain could enhance hydration. On the other hand, at high degree of cross-linking, the bonding between adjacent molecules was denser and restricted the swelling and hydration of starch granules. 3.3 Freeze thaw stability The freeze-thaw stability of starch was presented as the percentage of syneresis which indicated the released water from starch gel during freeze-thaw cycle process. When starch paste was rapidly cooled down after heating process, the water could be released especially in starches contained high amylose. Syneresis is an important parameter critical to the tendency of retrogradation and the stability of a gel system [23]. No syneresis indicates good water holding capacity of the gel [24]. The syneresis of all starch samples as a function of 1-5 freeze-thaw cycling is shown in Table 4. In general, the native cassava starch contains low amylose content and low percentage of syneresis. However, the freeze-thaw stability of native starch was poorer compared to cross-linked starch samples. For starch gel, the starch granules absorbed more water during heating process which the gelatinization of starch occurred and formed gel after cooling. Low and medium degree of cross-linked starch gels had the lowest syneresis of % after the first cycle and it showed the highest stability compared with the native starch and extruded starch control, while the high degree of cross-linked starch showed the worst stability. However, the syneresis of all starch samples increased with increasing number of freeze-thaw cycles. When starch gels are frozen, phase separation occurs upon the formation of ice crystals. Upon thawing, syneresis occurs because the water can be easily expressed from the dense network [23]. The formation of dense network was mainly related to amylose amylose interaction which enhanced starch retrogradation [36]. At high degree of cross-linking, starch granules were highly dense and compacted with bridge of cross-link in the molecules that made it difficult to complete gelatinization and cannot form gel resulting the gel have high content of exceed water at the same concentration. The results also suggested that appropriate degree of cross-linking minimized freeze-thaw damage by reducing the available water to form ice crystals. As shown in Table 4, starches at low and medium degree of crosslinking displayed the best performance of total syneresis of % after five cycles. 3.4 Gel properties The clarity of starch solution was indicated by the percentage of transmittance (%T). The clarity of starch solution decreased with an increase in degree of cross-linking indicating lower solubility. As shown in Table 5, the native cassava starch and Table 3: Water solubility index and swelling power of native cassava starch, control starch and phosphorylated cross-linked starch at 30ºC and 80ºC The same lower case letters superscript (a) in the same column each sample group are not significantly different (P 0.05). Table 4: Syneresis values (%) of starch gels (12%, total solid) during freeze-thaw cycles 52
5 The same lower case letters superscript (a) in the same column each sample group are not significantly different (P 0.05). The same upper case letters superscripts (A) in the same row are not significantly different (P 0.05). starch control showed highest clarity with the transmittance of 58.10±0.26% and 58.57±0.49%, respectively. According to results of Wongsagonsup et al. [14] and [15], the cross-linking restricted the gelatinization of starch and showed turbid solution observed from the low percentage of light transmittance. Textural properties of starch gels are affected by different factors including interactions between dispersed and continuous phases, properties of the amylose matrix and volume fraction and amylose content [27]. Determination of the textural properties as hardness and cohesiveness of samples (Table 5) showed that with increasing the degree of crosslinking to medium level, hardness of both samples increased significantly and noticeably at highest level of cross-linking. Comparing the samples, medium degree of cross-linked starch gel was the hardest. Cohesiveness was slightly different among samples. The hardness of starch gel would indicate the gel strength since it showed the resistance against compression force and breakage. Wongsagonsup et al. [14] reported that at low level of cross-linking, the starch gel had a high space in molecules which was easily deformed. High degree of cross-linked starch showed a weak structure and lowest hardness value since the starch could not form gel. A similar finding was reported by Chatakanonda et al. [28] who indicated that cross-linked waxy rice starch paste was not gel after cooling because the cross-linking inhibited the swelling capacity of starch granules. Table 5: Gel properties of native, control and phosphorylated cross-linked starches. The same letters superscript (a) in the same column are not significantly different (P 0.05) CONCLUSIONS Different degree of cross-linked cassava starch was successfully produced by a pilot scale co-rotating twin screw extrusion. Water solubility, peak and final viscosities of the cross-linked cassava starch decreased with an increasing degree of cross-linking. Cross-linked cassava starch absorbed more water and 53 showed higher swelling power than native starch and extruded control samples at room temperature. The freeze-thaw stability and gel hardness of starch were significantly enhanced by cross-linking at optimal level. The modified starch produced by cross-linking in combined with reactive extrusion could be used as a thickener and stabilizer in a variety of food products that required stability against heat and freeze-thaw process. ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support of the National Science and Technology Development Agency (NSTDA) and Thailand Research Fund (TRF), Thailand. The authors have declared no conflicts of interest. REFERENCES [1] Wikipedia contributors, Native starch industry of Thailand, Wikipedia, The Free Encyclopedia, land, (accessed January 12, 2016). [2] K. Sriroth, and K. Piyachomkwan, Starch technology, 3 rd ed., Kasetsart University Press, Bangkok, [3] C. Gamonpilas, W. Pongjaruvat, P. Methacanon, N. Seetapan, A. Fuongfuchat, and A. Klaikherd, Effects of cross-linked tapioca starches on batter viscosity and oil absorption in deep-fried breaded chicken strips, Journal of Food Engineering, vol. 114, no. 2, pp , [4] C. Lobato-Calleros, C. 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