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Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 10 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.710.362 Effect of Ultrasound Treatment on Physicochemical and Functional Properties of Cassava Starch T. Krishnakumar* and M.S. Sajeev Division of Crop Utilization, ICAR-Central Tuber Crops Research Institute (CTCRI), Sreekariyam, Thiruvananthapuram, Kerala – 695 017, India *Corresponding author ABSTRACT Keywords Ultrasound, Cassava, Modification of starch, Functional properties, Pasting properties, Rheological properties, Textural properties Article Info Accepted: 24 September 2018 Available Online: 10 October 2018 Ultrasound is a non-thermal processing technique that offers the opportunity to modify the functionality of starch in terms of physicochemical and functional properties. The present work was aimed to study the effect of ultrasound treatment on physicochemical and functional properties of cassava tuber starch. The cassava starch extracted using conventional extraction technique was used in the present study. A 3 × 2 factorial design with three factors and two level, i.e., sonication temperature (40 and 50°C), sonication time (10 and 20 min) and solid-liquid ratio (1:1 and 1:2) was adopted in this study. A significant increase in solubility, swelling power, textural, pasting and rheological properties of the ultrasonicated starch was observed. A slight decrease in clarity of the sonicated cassava starch paste was observed compared to the control starch, but the differences were not much significant statistically. The whiteness of the sonicated cassava starch powder was lower compared to control native starch, but the differences were not statistically significant. Freeze-thaw stability of the treated cassava starches was found to be better compared to the control native cassava starch. Introduction Tropical tuber crops are rich sources of starch. Cassava (Manihot esculenta Crantz) is one of the major tuber crops locally called as Tapioca and cultivated in tropical and subtropical regions of the world. It is the third largest source of carbohydrates after rice and wheat for people all over the world and the starch content of cassava tubers varies according to varieties. In India, it is cultivated about 0.20 Million hectares with a total production of 8.13 Million Tonnes and a productivity of 22.3 Metric Tonnes per hectare (India Agristat, 2014). In India, cassava starch is a major industrial starch and a large portion of this used for sago production. The native and different modified forms of cassava starch is used as a base material for an array of processed products viz., sago, dextrin, glucose, core binder, stabilizer, adhesives, sizing yarns and as thickener for printing clothes (Sheriff et al., 2005). Processing of cassava starch is an easy process compared to that of cereal starches because of the presence of relatively small amount of non-starchy substances in the cassava tubers. 3122 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 Cassava starch is immiscible in cold water and some of its natural properties limit its usefulness in many commercial applications (Jyothi et al., 2011). The presence of amorphous and crystalline regions in the starch granules offers wide opportunity for modification of cassava starch (Moorthy, 2002). In order to make tailor-made starches suitable for specific end uses, modification is highly required. Modified starches are widely used in food, textile, paper and adhesive industries. Physical modification of starches are gaining more attention in recent years due to less amount of byproducts and chemical agents and makes this approach more sustainable and environment-friendly (Carmona et al., 2016; Krishnakumar and Sajeev, 2017). Ultrasound (US) is a sound waves having frequency above 20 kHz (beyond human hearing range) that passes in liquid medium creates cavitation. This mechanical action of cavitation with high velocity and shear force lead to high penetration in to cell membranes that causes cell disruption (Ahmad et al., 2015). Ultrasound treatment (UT) is a physical method for modification of starch that has many advantages such as less usage of chemicals and processing time and environment friendly processing (Krishnakumar and Sajeev, 2017; Monroy et al., 2018). Ultrasound treatment (UT) is useful to modify the functionality of starch in terms of physico-chemical and functional properties (Luo et al., 2008;Chan et al., 2010; Jambrak et al., 2010; Manchun et al., 2012; Zheng et al., 2013; Carmona et al., 2016). To the best of our knowledge, only very limited studies have been reported on the effect of ultrasound treatment on the modification of functional properties of native cassava starch. Thus, the objectives of the present work were to study the influence of ultrasound on the physicochemical and functional properties and correlation between the different properties of native cassava starches under different sonication temperature, solid-liquid ratio and time. Materials and Methods Raw material Matured cassava (Manihot esculenta Crantz) variety of Sree Pavithra collected from the CTCRI Research farm was used as raw material for the extraction of starch. Extraction of starch Cassava tuber starch was extracted from the freshly harvested cassava tubers using the standard procedure (Krishnakumar and Sajeev, 2017). The method used in the study was very similar to extraction of starch from sweet potato and arrowroot. The fresh tubers were thoroughly rinsed, manually peeled and were cut into small pieces using a motor operated chipping machine, then crushed in a mobile type starch extraction unit with supply of adequate water. The crushed starch milk was passed through a 150-mesh sieve and the resultant cassava starch milk was allowed to settle in a sedimentation tank for 12 hr. The settled cassava starch removed from sedimentation tank was washed with excess water for three times to obtain bright white colour. The starch was then dried in tray drier at 50ºC for 12 h and stored under airtight condition with moisture content of 12 % (dry basis) for further ultrasound treatment. Ultrasound treatment of cassava starch Ultrasound treatment (US) of cassava starch was conducted as per the method reported by ying et al., (2011), using a probe ultrasonicator (Sonic, Model: VCX750) operating frequency of 30 ± 3 kHz, input voltage of 230 V and heating strength of 750 W, attached with digital timer. The schematic 3123 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 diagram of the probe type ultrasound treatment system used in the study is shown in Figure 1. The aqueous cassava starch suspension obtained from the isolated cassava starch were treated with a constant ultrasound power of 750 W and 50 % amplitude during different sonication temperature (40, 50°C), sonication time (10, 20 min), solid-liquid ratio (1:1, 1:2 %). The temperature during the extraction was maintained by circulating water through the double-jacketed glass beaker. Ultrasonic probe of 19 mm diameter was directly placed in the suspension (cassava mash + distilled water) at a depth of 28 mm from the suspension surface and the desired amplitude (%) and extraction time (min) were maintained by means of digital amplitude and time controller. After the treatment, the pure starch was dried, powdered using pestle and mortar, sieved through standard BSS 100 mesh sieve and then stored in airtight container for further analysis. Solubility and swelling power Solubility index (%) of the cassava starch was determined using Ding et al., (2006). The 2.5 g of cassava starch was weighed into 50 ml centrifuge tube and heated in 30 ml distilled water in a water bath at 60°C for 30 min without mixing and then centrifuged at 3000 rpm for 10 min. The supernatant was dried at 105°C to constant weight and the weight of the dry solids was measured. All the experiments were made in triplicate. The following equation used to calculate the solubility index. 𝑆𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 𝑖𝑛𝑑𝑒𝑥 % = 𝑚𝑠 𝑚𝑑 × 100 (1) Swelling power (g/g) was determined by modified method of Betancur et al., (2001). 2.5 g of the ultrasonicated cassava starch sample was weighed into 50 ml centrifuge tube. Then 30 ml of distilled water was added and mixed gently. The sample was heated in a water bath at 60°C for 30 min and centrifuged at 3000 rpm for 10 min. The supernatant was decanted immediately after centrifuging. The weight of the sediment was taken and recorded. Swelling power g g = Weight of sedimented starch paste g Weight of starch sample on dry basis x (100−% solubility ) x100 (2) (2) Paste clarity Paste clarity of the ultrasonicated (US) cassava starch was measured according to the procedure described by Sandhu and Singh (2007). Aqueous starch suspension containing 1% starch was prepared by heating 0.2 g starch in 20 ml water in a shaking water bath at 90ᴼC for 1 h. The starch paste was cooled to room temperature, and the transmittance was measured at 640 nm in a spectrophotometer (Spectra scan uv-2600, Thermo fisher scientific, India). Colour of starch The colour of the US starch sample was analyzed using a colorimeter (Hunter Lab, Virginia). The primary colour parameters „L‟, ‟a‟, ‟b‟ were measured by placing samples in the sample holder. The „L‟ parameter represent light dark spectrum with a range from 0 (black) to 100 (white), „a‟ represents green red spectrum ranging from -60 (green) to +60 (red) and „b‟ represents blue yellow spectrum with a range from -60 (blue) (1) to +60 (yellow) dimensions respectively. Where, Freeze-Thaw stability ms - Weight of soluble starch (g) md – Weight of starch sample on dry basis (g) Freeze-thaw stability was determined according to the method of Singhal and 3124 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 kulkarni (1990). Ultrasonicated (US) starch at a concentration of 5% (w/v) was heated in distilled water at 95ᴼC for 30 min with constant stirring. Ten milliliters of paste was transferred into the weighed centrifuge tube. This was subjected to alternate freezing and thawing cycles (22h freezing at -20ºC followed by 2 h thawing at 30ºC) for 3 days and centrifuged at 5000×g for 10 min after each cycles. The percentage (%) of syneresis was then calculated as the ratio of the weight of the liquid decanted and the total weight of the gel before centrifugation multiplied by 100. Totally three freeze-thaw cycles were conducted for each sample. and recovered from it per cycle while the loss modulus (G") is a measure of the energy dissipated or lost per cycle of sinusoidal deformation (Ferry, 1980). The ratio of the energy lost to the energy stored for each cycle may be defined by tan δ, which is another parameter indicating the physical behaviour of a system. In dynamic oscillation measurements, the frequency sweep of the two moduli (G' and G"), may be used to distinguish between the elastic and viscous properties of a material over a spectrum of times. When the viscous properties dominate, G" exceeds G', and vice versa (G' > G") when the elastic properties prevail. Pasting properties of starch The dynamic rheological properties (storage modulus, loss modulus and phase angle) for the 10% (w/v) gel suspension of the cassava control and ultrasonicated starch samples obtained from RVA studies were determined using Rheoplus MCR 51 Rheometer (M/s Anton Paar GmbH. Germany) at 30ºC, using a parallel plate geometry system (PP20SN5912, 1mm diameter) at 1 mm gap. The following experimental conditions were selected: frequency 1 to 10 Hz; strain of 1 per cent (%). Fifteen measuring points were recorded for each experiment. For each sample, storage modulus (G’), loss modulus (G’’) and phase angle (δ) were recorded and the measurements were conducted at least in duplicates. The viscosity of control and ultrasound treated samples was determined using a rapid visco analyzer (RVA-4, Newport Scientific, and Warriewood, Australia). The powdered sago sample (2.5 g dry weight) was accurately weighed out into the aluminum canister and distilled water (25 g) was added and mixed well. The canister was placed in the RVA unit and the heating/cooling cycle was performed according to Standard I profile. The slurry was heated from 50 to 95ºC at 12ºC/min and held at 95ºC for 2 min. The paste was cooled to 50ºC at 12ºC/min and finally maintained at 50ºC for 2 min. The parameters such as peak viscosity, breakdown viscosity, setback viscosity in terms of centipoises (cP) and pasting temperature (ºC) were measured. The breakdown ratio was calculated as the ratio of breakdown to peak viscosity. Dynamic rheological properties of starch In dynamic oscillation measurements, the potential energy and the energy that is dissipated as heat may be separated into storage modulus and loss modulus, respectively. Storage dynamic modulus (G') is a measure of the energy stored in the material Textural analysis Textural properties (cohesiveness, consistency, firmness) of the cassava starch were determined by using Texture Analyser (TA.XT plus, Stable Micro Systems, UK) equipped with a 50 kg load cell. A probe adapter was used to connect the compression plate to the movable bar. Stainless steel cylindrical probe (P/35) attached in the movable bar was used. The following experimental conditions were adopted for the 3125 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 analysis, Option-return to start, Pre-test speed: 2mm/s, Test speed: 2mm/s, Post-test speed: 2mm/s, Distance: 8 mm, Force: 10 g, Time: 5s, Data acquisition rate: 200 pps. Starch paste sample was prepared at a concentration of 5% (w/v) for measuring the textural properties. The sample was compressed to 80 % of their original height, a cross head with speed of 2 mm/s twice in two cycles. The aqueous cassava starch suspension at a concentration of 10 % (w/v) was prepared. Experimental design and statistical analysis A three factor two level full factorial design was employed to study the effect of ultrasound treatment on the physico-chemical and functional properties of cassava starch. The factors selected were sonication temperature (40, 50°C), sonication time (10, 20 min) and solid-liquid ratio (1:1, 1:2). A total of nine experiments with three replications were conducted including control (Table 1). Data analysis was performed using R software. Factorial completely randomized design (FCRD) was applied for all the statistical analysis. Analysis of variance (ANOVA) and pair-wise mean comparison were performed to determine the significant effect of the independent variables on the response variables. The treatments and their interactions were compared at p<0.05 level using least significant difference (LSD) method. Results and Discussion Effect of sonication on solubility and swelling power of cassava starch The solubility and swelling power of the ultrasonicated cassava starch samples increased significantly compared to control sample (Table 2). The treatments were found to be statistically significant except for solubility at Sonication time for 10 min (T2) and swelling power at T2, T4, and T6, which might be due to lower sonication time and solid-liquid ratio, which resulted in a lesser degradation of starch molecules. This is due to the role of ultrasound makes starch molecules to increase solubility, particle content easy expansion, thereby weakening the reflection and refraction of light (Singh et al., 2003; Lida et al., 2008; Luo et al., 2008; Jambrak et al., 2010; Manchun et al., 2012). Similar increase in starch swelling and solubility due to ultrasonication has been reported by Zheng et al., (2013) for sweet potato starch and by Yu et al., (2013) for non-waxy rice starch. The solubility and swelling power increased when temperature and solid-liquid ratio of ultrasound treatment was less. This might be due to the fact that with increase in temperature, the degradation of starch was more, and some degraded soluble starch might have been lost during recovery. Similar trend was also observed with solid liquid ratio and evinced that some soluble starch might have been lost during treatment with high solid liquid ratio. Effect of sonication on paste clarity of cassava starch The major factors affecting the clarity of starch paste includes distribution of different starch size particles and the proportion of amylose and amylopectin (Sitohy et al., 2000). The clarity of the pastes prepared using ultrasonicated starch samples was observed to be lower compared to control but was not much statistically different (Table 2). The result showed that the clarity of the natural cassava starch was 21.42 %, but the clarity of ultrasonicated starch was decreased with increase in sonication temperature, time and solid-liquid ratio. The results were not in agreement with the results obtained by Jambrak et al., (2010) for corn starch, Sujka and Jamroz (2013) for potato starch and Zheng et al., (2013) for sweet potato starch. It can be seen that clarity decreased with 3126 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 increase in time and temperature as well as solid-liquid ratio. This might be due to highest disintegration of the starch. The starch granules with more extraction time, solidliquid ratio and higher temperature which allowed more water to be absorbed and thereby led to more viscous starch paste, thus decreasing the transmittance. It could also be due to disintegration of starch molecules by ultrasound made some bonds to be weaken which might allowed some impurities with the starch molecules, thus decreasing the clarity of starch paste. Effect of sonication on colour of the starch powder Colour is an important parameter to estimate starch quality. The variation of colour “L” value of ultrasonically treated cassava starch with respect to different sonication time, temperature and solid liquid ratio is presented in Table 3. The colour variation in L* value was found to be non-significant with control. The colour values increased with increase in the ultrasound treatment time and temperature. This might be due to the long exposure time leading to rapid hydration action hence leading to change in colour. This result was not in agreement with the study conducted by Nadir et al., (2015) that potato starch samples modified by ultrasonication methods had lower values of lightness (L) value when compared with native starch. The “L” value of ultrasonically extracted starch ranged from 92.57 to 95.82, while those of conventionally extracted starch were 94.36. The lightness value greater than 90 confirms the purity of starch (Perez Sira and Amaz, 2004). Ultrasonically extracted starches also exhibited more redness and yellowness compared to control starch. This might be attributed to the disintegration of starch molecules during ultrasonication which might have allowed some impurities to bond with the starch molecules, thereby reducing the whiteness and increasing the redness and yellowness. Effects of sonication on freeze-thaw stability of the cassava starch paste Syneresis (%) is used to find out the freezethaw stability of starches to withstand the undesirable physical changes during freezing (Adebowale et al., 2005). Starch paste after freeze and thawing occur syneresis phenomenon, which is due to the freezing of starch paste. The percent syneresis of the starch gels measured by ultrasound treatments was found to be substantially lower than that of the control starch gel during all the three freeze-thaw cycles (Table 4). Moreover, ultrasonically extracted starches were observed to be more stable under repeated freeze-thaw cycles as less difference was noticed in percent syneresis between the freeze-thaw cycles. Because of the ultrasonic treatment, gel structure is destroyed, resulting in precipitation of free water, breakage of starch chains in the amorphous region caused extensive reordering of the chain segments. Therefore, amount of water expelled during thawing was comparatively less in ultrasonicated starches compared to that in control sample as due to breakage and reordering, a greater number of hydrophilic bonds were exposed which could hold more water during thawing, thereby reducing syneresis. This result was in agreement with the study conducted by Luo et al., (2008) on ultrasonically treated maize starch. A similar observation on freeze-thaw behavior of ultrasonically treated maize starch was made by Hu et al., (2014). Native starch retrogradation is considered to be unacceptable for many food applications (Nwokocha et al., 2012; Collar and Rosell 2013; Wang et al., 2015). Better freeze-thaw stability of the sonicated cassava starch is more acceptable for food application, meant for refrigeration. 3127 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 Table.1 Treatment combinations at different ultrasound conditions S. No 1. 2. 3. 4. 5. 6. 7. 8. 9. Treatment Temperature, °C Time, min Solid-Liquid ratio, g/mL 0 40 50 40 50 40 50 40 50 0 10 10 20 20 10 10 20 20 0 1:1 1:1 1:1 1:1 1:2 1:2 1:2 1:2 Control T1 T2 T3 T4 T5 T6 T7 T8 Table.2 Effect of different ultrasonic treatments on solubility and Swelling power of the cassava starch Treatments Control T1 T2 T3 T4 T5 T6 T7 T8 Solubility (%) 28.20±0.18g 30.36±0.23b 28.45±0.31g 31.26±0.35a 28.96±0.28d 29.56±0.16c 28.98±0.14e 30.44±0.18b 28.66±0.20f Swelling power (g/g) 7.52±0.12f 8.67±0.15c 7.85±0.22f 9.62±0.05a 7.69±0.14f 7.42±0.12e 7.33±0.13f 7.75±0.12d 7.94±0.18b Clarity (% T) 21.42±1.10a 21.27±1.11a 21.14±0.95b 21.38±0.82a 19.56±0.15c 20.05±0.64d 19.45±0.23c 20.15±0.32d 19.35±0.56c Values are mean ± standard deviation of three replications. Means followed by same letters in the superscript were found not significantly different at p>0.05. Table.3 Effect of different ultrasonic treatments on colour of the cassava starch Treatments Control T1 T2 T3 T4 T5 T6 T7 T8 L value 94.36±1.24a 92.57±1.56a 93.39±0.98a 93.27±1.57a 95.82±2.12a 95.26±1.32a 95.32±2.03a 95.18±1.26a 94.97±1.05a a value 0.23±0.04f 0.89±0.03b 0.56±0.02d 1.24±0.05a 0.59±0.03d 0.81±0.04c 0.52±0.02e 0.84±0.01c 0.75±0.02d b value 5.94±0.05i 7.17±0.04b 6.46±0.03e 7.34±0.04a 6.89±0.01c 6.21±0.02g 5.91±0.04h 6.36±0.03f 6.85±0.02d Values are mean ± standard deviation of three replications. Means followed by same letters in the superscript were found not significantly different at p>0.05. 3128 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 Table.4 Freeze-thaw stability of cassava starch samples under different treatments Treatments Control T1 T2 T3 T4 T5 T6 T7 T8 First cycle 29.15±1.12l 17.24±1.32hj 19.36±1.57j 13.37±2.01bc 15.28±1.68eh 16.88±1.39hj 14.86±1.83ce 15.94±2.04eh 16.19±2.11fh Second cycle 27.21±1.37l 16.37±1.22gh 18.34±1.26ij 13.89±1.53bc 14.67±1.84ce 11.32±1.67ab 14.83±1.52ce 13.27±1.68ad 15.53±2.16eh Third cycle 24..36±1.55k 15.27±1.35df 15.32±1.62eh 12.45±1.95ac 14.83±1.54ce 10.57±2.16a 13.38±2.17bc 12.53±2.14ac 14.88±2.18ce Values are mean ± standard deviation of three replications. Means followed by same letters in the superscript were found not significantly different at p>0.05. Table.5 Textural Properties of sonicated cassava starch gel Treatments Control T1 T2 T3 T4 T5 T6 T7 T8 Cohesiveness (g) 8.92±0.03c 8.97±0.05b 8.80±0.15d 8.34±0.12f 9.06±0.09a 8.57±0.05e 8.91±0.13c 8.65±0.05e 8.96±0.02b Consistency (g/s) 145.36±1.12d 153.91±0.15c 156.86±1.32a 152.59±1.42c 153.79±0.83c 152.10±1.13c 153.03±0.92c 158.39±1.16a 154.32±1.45b Firmness (g) 10.54±0.06e 12.31±0.02c 12.53±0.15b 12.01±0.04d 12.64±0.08b 12.11±0.14d 12.52±0.06b 13.01±0.05a 12.52±0.11b Values are mean ± standard deviation of three replications. Means followed by same letters in the superscript were found not significantly different at p>0.05. Table.6 Pasting profile of starch pastes under different ultrasonic treatment conditions Treatment Peak viscosity Holding (cP) viscosity (cP) f 2938±45 1273±28f Control 3363±40c 1785±19b T1 e 3232±41 1731±18c T2 3729±38a 1865±17a T3 3584±24b 1743±11c T4 3339±21d 1736±31c T5 e 3276±24 1718±23c T6 2741±38h 1486±20d T7 2861±37g 1371±18e T8 Breakdown Final viscosity (cP) viscosity (cP) 1665±14b 2190±19g 1578±11d 2878±36c e 1501±16 2915±30b 1864±19a 3113±42a 1843±12a 2888±32b 1605±14c 2833±38c d 1560±17 2737±19d 1257±20f 2386±24e 1492±12e 2261±36f Setback viscosity (cP) 917±18f 1093±15d 1184±12b 1248±09a 1145±08c 1097±14d 1019±17e 900±11g 891±15g Pasting Temp (°C) 71.15±0.06f 72.35±0.03b 72.35±0.01b 71.85±0.04d 71.95±0.05c 72.75±0.02a 71.55±0.03e 70.75±0.01h 71.05±0.02g Values are mean ± standard deviation of three replications. Means followed by same letters in the superscript were found not significantly different at p>0.05. 3129 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 Fig.1 Schematic diagram of the probe ultrasound treatment system Fig.2 Pasting profiles of starch paste under different ultrasound treatments 3130 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 3122-3135 Fig.3 Dynamic moduli of the pastes (10%) of native and treated cassava starch 3131