Rice Starch Isolation by Neutral Protease and High-Intensity Ultrasound 1

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1 RICE QUALITY AND PROCESSING Rice Starch Isolation by Neutral Protease and High-Intensity Ultrasound 1 L. Wang and Y.-J. Wang ABSTRACT The efficacy of neutral protease and combinations of neutral protease and highintensity ultrasound on rice starch isolation was investigated. Rice flour slurry (33%) was treated with neutral protease at 0.01, 0.03, or 0.05% (on flour basis) or combinations of 0.03% neutral protease and high-intensity ultrasound at 25, 50, or 75% amplitude for 15, 30, or 60 min concurrently or sequentially. The starch yield greatly improved to 79.8%-86.7% with % residual protein content and % damaged starch content when neutral protease treatment was combined with highintensity ultrasound. The preferred combinations were neutral protease digestion for 2 hr followed by sonication at 75 or 50% amplitude for 15 or 30 min. The starch structure analyzed by a high performance size-exclusion chromatography and a scanning electron microscope revealed no damaged on the molecule structure or on the surface of starch granules. The combination of neutral protease and high-intensity ultrasound was an effective technique for rice starch isolation without generating salt wastes. INTRODUCTION Rice starch is conventionally isolated by an alkaline steeping method because the main rice protein fraction, glutelin, is soluble in alkali. However, this method generates a large quantity of alkaline and salt waste, and the wastewater treatment is costly. Many efforts have been directed toward developing a process that would ef- 1 This is a completed study. 413

2 AAES Research Series 517 fectively isolate rice starch while preserving the environment (Guraya and James, 2002; Kun et al., 1987). High-pressure homogenization was recently studied for recovering rice starch and protein fractions (Guraya and James, 2002). The flour particle size was reduced to less than 10-µm after the rice flour slurry (32%) passed through the homogenizer two times. The starch yield was 72% with 2.7% residual protein. One neutral protease was found effective in rice starch isolation without using chemicals; however, one major drawback associated with the protease treatment was the long digestion time (18 hr) used in the study (Wang and Wang, 2001). This study continued the evaluation of protease treatment as an alternative method for rice starch isolation, but the digestion duration was shortened to avoid microbial spoilage. In addition, high-intensity ultrasound was employed before, during, or after the protease treatment for further improvement of starch isolation efficiency. The properties of the isolated starch were characterized to determine the effectiveness of the treatments. PROCEDURES Long-grain rice flour (RL-100) was purchased from Rivland Partnership, Houston, Texas. Neutral protease (N Amano with protease activity 150,000~190,000 units/g, optimum ph 7.5 and temperature 55 C) was in dry powder form and a gift from Amano Pharmaceutical Co., Ltd. (Nogoya, Japan). A high-intensity ultrasonic processor (750 Watt Model, 20 khz) with a 0.75-in. high gain probe was purchased from Sonics & Materials, Inc. (Newtown, Conn.). Starch isolation by alkaline steeping method served as the control isolation method. One-hundred-gram rice flour (as is) was soaked in 200 ml 0.1% sodium hydroxide (NaOH) for 18 hr. The slurry was blended with a Waring blender at a high speed for 2 min, passed through a 63-µm screen, and centrifuged at 1400 g for 10 min. The bottom starch layer was reslurried and washed with 0.1% NaOH and water, and then neutralized with 1.0 N hydrochloric acid to ph 6.5 and centrifuged. The starch was then washed with deionized water three more times and, dried at 45 C in an oven for 48 hr. Starch isolation by neutral protease used rice flour (100 g, as is) mixed with deionized water (200 ml) in a 500-mL reaction beaker. The temperature was maintained at 50 C with a circulator, and the slurry ph was adjusted to 7.0 with 1.0 N NaOH. Different levels of neutral protease (0.01, 0.03, or 0.05% on rice flour basis) were added to the slurry and reacted for 1, 3, or 5 hr with constant stirring using a magnetic stirrer. The flour slurry was then blended with a Waring blender at a high speed for 2 min after the protease digestion. The slurry was passed through a 63-µm screen and centrifuged at 1400 g for 10 min. The starch layer was reslurried and washed with deionized water 3 times. The isolated starch was dried at 45 C for 48 hr. Starch isolation by combinations of neutral protease and high-intensity ultrasound was as following. An ultrasonic treatment was added for pre-, during-, or post- 414

3 B.R. Wells Rice Research Studies 2003 protease treatment for starch isolation. Rice flour (100 g, as is) mixed with deionized water (200 ml) in a 500-mL reaction beaker. Neutral protease, 0.03% (on flour basis), was added, and the temperature of the slurry was maintained at 50 C by a circulator with constant stirring. For high-intensity ultrasonic treatment, the temperature was maintained at 40 C (Wang and Wang, 2001). The sonication amplitude was 25%, 50%, or 75% with 5-sec on and 5-sec off for 15, 30, or 60 min. The sonication time did not include the off-time. The starch isolation followed the same procedure as described in the neutral protease treatment. Moisture content, protein content, total starch, and damaged starch were determined according to AACC Approved Method 44-15A, 46-13, 76-13, and 76-30, respectively. The amylose content of the starch was determined using high-performance size-exclusion chromatography (Wang and Wang, 2000). Starch was dissolved in 90% dimethylsulfoxide and boiled for one hr, stirred overnight, and filtered through a 5-µm filter. The amount of amylose was calculated as the relative percentage of the amylose peak area over the total peak area. The scanning electron micrographs of isolated starches with or without applying high intensity ultrasound were taken with a Hitachi S-2300 scanning electron microscope (Tokyo, Japan) at an accelerating voltage of 25 kv. Experimental data were analyzed by using the General Linear Models Procedure (SAS Software Institute, Inc. Cary, N.C., 1999), and least significance differences were computed at p<0.05. RESULTS AND DISCUSSION The yield, residual protein, and damaged starch content of the starches isolated by neutral protease alone and by combinations of neutral protease with high-intensity ultrasound are listed in Tables 1 and 2, respectively. The rice flour used in this study had 87.6% (db) total starch, 7.7% (db) protein and 5.4% (db) damaged starch. When neutral protease was used alone, the starch yield increased and the residual protein decreased with increasing protease level and digestion time. The starch yield ranged from 62.5% to 71.8% with a residual protein and damaged starch of % and %, respectively. The control sample by the alkaline steeping method had a starch yield of 71.6%, a residual protein of 0.12%, and a damaged starch content of 1.59%. The results showed that neutral protease at 0.01% was not effective in separating protein and starch even after 5 hr of digestion. When neutral protease was increased to 0.03% and 0.05%, the resultant starch yields were comparable to that by the alkaline steeping. Both starch yield and residual protein were comparable for samples treated with 0.03% and 0.05% neutral protease for 3 and 5 hr. Thus 0.03% neutral protease and 2 hr digestion time were chosen for the following study by combining neutral protease and high-intensity ultrasound. Protease would hydrolyze the protein on the starch granule surface, thus possibly resulting in partial breakdown of the protein matrix and an increased susceptibility of rice flour to ultrasonic cavitation. The cavitation effect from high-intensity ultrasound in a water medium was assumed to be able to break the bonding between 415

4 AAES Research Series 517 starch granules and the protein matrix. The starch yield was significantly improved when high-intensity ultrasound was combined with the neutral protease treatment, ranging from 80.8 to 86.7%, compared with those with neutral protease only and the alkaline steeping method (Table 2). The residual protein of % (db) was higher but the damaged starch of % (db) was similar to the alkaline steeping control sample. Nevertheless, the residual protein of the starches treated with protease and high-intensity ultrasound was lower than one commercial rice starch (StarchPlus SPR rice starch from California Natural Products, Lathrop, Calif.) of 3.1% (as is). The starch yield increased as the sonication time increased at low amplitudes (25 and 50%), however the starch yield was similar about 84% at 75% amplitude of sonication regardless of treatment sequence. It was noted that starch was easier to separate from protein when sonication was applied after the protease digestion, suggesting that protease was more effective in loosening the protein matrix around the starch granules than was ultrasound. Therefore, the preferred combinations of neutral protease and high-intensity ultrasound were first protease digestion for 2 hr and then sonication at 50% or 75% sonication amplitude for 15 or 30 min (Table 2) because of a higher starch yield, a lower residual protein content, and ease of separation. The amylose content of the isolated rice starches as measured by HPSEC was similar for all treatments, suggesting that the sonication treatment below the gelatinization temperature did not depolymerize starch molecules. The SEM results did not show any damage on the surface of rice starch granules (Fig. 1). The combinations of protease and high-intensity ultrasound likely only affected non-covalent bonding between starch and protein without damaging the starch molecular and granular structures. SIGNIFICANT FINDINGS By combining neutral protease with high-intensity ultrasound, the efficacy of starch isolation was greatly improved, and sonication followed the protease digestion facilitated the separation process. A combination of protease and high-intensity ultrasound presented a great potential to achieve a high starch yield with similar physicochemical properties to the conventional method in a short period of time. ACKNOWLEDGMENTS The authors thank the Arkansas Rice Research and Promotion Board for financial support. 416

5 B.R. Wells Rice Research Studies 2003 LITERATURE CITED American Association of Cereal Chemists, Approved Methods of the AACC, 10 th Ed. The Association: St. Paul, Minn. Guraya, H.S. and C. James Deagglomeration of rice starch-protein aggregates by high-pressure homogenization. Starch/Starke 54: Kun, L.-L., H.-J. Chen, and H.-Y. Sung A new method for separation of rice protein and starch. J. Chinese Agri. Chem. Soc. 25: Wang, L. and Y.-J. Wang Comparison of protease digestion at neutral ph with alkaline steeping method for rice starch isolation. Cereal Chemistry 78: Wang, Y.-J. and L. Wang Effects of modification sequence on structures and properties of hydroxyproylated and crosslinked waxy maize starch. Starch/Starke 52: Table 1. Starch yield, protein content, and damaged starch content of rice starches isolated by neutral protease at different levels and durations. z Treatment Starch yield Protein content Damaged starch (% Starch db) (% db) (% db) Alkaline steeping 71.6 a 0.12 c % Protease 1 hr y 62.5 b 0.87 a 1.52 b 0.03% Protease 1 hr 63.5 b 0.78 a 1.02 d 0.05% Protease 1 hr 68.8 a 0.67 ab 1.81 a 0.01% Protease 3 hr 64.7 b 0.88 a 1.19 c 0.03% Protease 3 hr 69.5 a 0.67 ab 1.32 c 0.05% Protease 3 hr 71.3 a 0.77 a 1.34 c 0.01% Protease 5 hr 64.1 b 0.60 b 1.49 b 0.03% Protease 5 hr 70.8 a 0.53 b 0.99 d 0.05% Protease 5 hr 71.8 a 0.55 b 1.34 c z y Mean values in the same column followed by different letters are significantly different (P<0.05). Protease at 0.01% level and digestion for 1 hr. 417

6 AAES Research Series 517 Table 2. Starch yield, protein content, and damaged starch content of rice starches isolated by the combination of 0.03% neutral protease and high-intensity ultrasound. z Treatment Starch yield Protein content Damaged starch (% Starch db) (% db) (% db) Alkaline steeping 71.6d 0.12d 1.59b U15m50%amp-P2hr y 83.0b 0.76b 1.37c U30m50%amp-P2hr 85.7a 0.71bc 1.31c U15m75%amp-P2hr 84.6b 0.77b 1.45bc U30m75%amp-P2hr 83.5b 0.72bc 1.87a PU30m25%amp x 80.8c 0.96a 0.98e PU60m25%amp 86.7a 0.82b 1.11d PU15m50%amp 79.8c 0.84b 1.02e PU30m50%amp 83.9b 0.83b 0.99e PU60m50%amp 86.1a 0.67bc 1.38c PU30m75%amp 84.3b 0.68bc 1.57b P2hr-U15m50%amp w 83.4b 0.76b 1.08de P2hr-U30m50%amp 86.0a 0.50c 1.18d P2hr-U15m75%amp 85.7a 0.59c 1.18d P2hr-U30m75%amp 84.4b 0.68bc 1.82a z Mean values in the same column with different superscript letters are significantly different (P<0.05). y Sonication 15 min at 50% amplitude and then 2 hr protease digestion. x Protease digestion and sonication 30 min at 25% amplitude concurrently. w Protease digestion 2 hr and then sonication 15 min at 50% amplitude. 418

7 B.R. Wells Rice Research Studies 2003 A B Fig. 1. Scanning electron micrograph of rice starches isolated by (A) stirring at room temperature without sonication, and by (B) high-intensity ultrasound 30 min at 75% amplitude after 2 hr neutral protease digestion. 419