Abstract

ABSTRACT: Commercially obtained defatted (DF), full-fat stabilized (FFS), and full-fat unstabilized (FFU) rice bran were processed by colloid milling and homogenization to affect bran breakdown and extraction of rice protein. Relative to unprocessed samples, there were moderate to slight increases in the amount of protein extracted from the various fractions of processed bran. Colloid milling and homogenizing slightly influenced the distribution of proteins in the various fractions obtained, with the FFU showing the greatest effect compared to DF and FFS protein fractions. The protein content of the supernatant fraction of FFU bran increased from 21.8 to 33.0% after colloid milling with a further increase to 38.2% after homogenizing, representing an overall increase of 75.2% in protein content. The supernatant fractions of DF bran increased from 13.9 to 14.7% after colloid milling, and to 16.5% after colloid milling and homogenizing, for an overall increase of 18.7%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a molecular weight distribution ranging from 6.0 to 97.4 kDa. Few detectable differences between protein bands of unprocessed and processed DF and FFU bran were observed. However, FFS bran showed breakdown in size distribution of protein after colloid milling and homogenizing, because certain high molecular weight proteins shifted to lower molecular weight units. Paper no. J9912 in JAOCS 78, 969-972 (September 2001). KEY WORDS: Colloid milling, homogenizing, physical processing, protein distribution, rice bran, rice protein. Industrial processing of rice bran into edible products is attractive due to the abundance of rice bran as a by-product in the rice milling industry and the recognition of its commercial potential. Hammond (1) described a method of processing rice bran into products, such as milk replacers, a slow-release carbohydrate product, fiber in health foods, and ingredients in cosmetics and pharmaceuticals. Rice bran contains 14-16% crude protein, of which 3-4% is lysine (2), and is therefore of high nutritional value. Rice bran protein can have significant usage as hypoallergenic milk replacers in infant formulas (3). However, procedures for extracting protein from rice bran must be carefully selected to produce protein concentrates and isolates with desirable functional properties (4), because the extensive network of disulfide bonding and aggregation renders much of the rice bran protein insoluble in ordinary aqueous solvents such as salt, alcohol, and acids (5). Various approaches have been used to enhance the protein value of rice bran. These include dry milling of bran followed by air classification, isolation of protein by precipitation at the isoelectric point, and separation of protein by enzyme treatment (6). Alkaline extraction procedures are normally used to prepare protein concentrates from rice bran. However, exposure of protein to extreme alkaline conditions may change its nutritional characteristics, such as the conversion of cysteine and serine residues of protein to toxic lysinoalanine In spite of its ready availability and nutritional quality, rice bran continues to be underutilized and used mainly as an ingredient in animal feed production (2). Data on processing of rice bran to extract proteins through mechanical operations, such as size reduction and/or mechanical shearing, have not been reported. The increase in consumer demand for highfiber, high-protein food products, coupled with the necessity to reduce processing costs, requires a more efficient and environmentally friendly way to process rice bran. This study was conducted to understand the efficiency of using the physical processes of colloid milling and homogenizing on breakdown of rice bran and extraction of protein and protein electrophoretic properties. EXPERIMENTAL PROCEDURES Extraction of protein from rice bran. Full-fat stabilized (FFS) and defatted (DF) rice bran were obtained from Riceland Foods (Stuttgart, AR). Full-fat unstabilized (FFU) rice bran was obtained from Sage V Foods (Los Angeles, CA). Based on preliminary investigations, a 10% (wt/vol) slurry of each sample was prepared by stirring 200 g of each bran sample in 1800 mL of deionized water for 1 h at room temperature. Two approaches were adopted to subject rice bran to physical processing as follows: (i) Colloid milling. Slurries were first subjected to continuous flow with high speed, high shear in a Bematek Model 2-V colloid mill (Bematek Systems, Inc., Beverly, MA) for 30 min with the rotor speed set at 7500 rpm. The colloid mill subjected the rice bran slurry to very high levels of mechanical shear forces. As a result, the slurry's internal phase solid particles and liquid droplets were reduced in size and distributed in the fluid dispersion. The precise degree of particle size reduction was controlled by adjusting the gap between the rotor and stator. The temperature of the colloid-milled (CM) product was 38-39°C. (ii) Homogenization. After colloid milling, the slurries were transferred to a homogenizer (Manton Gaulin, Inc., Everett, MA). Homogenization was done for about 10 min by forcing the slurry through a narrow orifice at a homogenization pressure of ~1.7 × 10 4 kPa. After homogenization, slurries were centrifuged for 20 min at 20,000 × g to obtain a supernatant product (SP), a residue product (RP), and a layer of insoluble fiber product (FP) between the supernatant and residue fractions. The supernatant fraction was decanted, and the insoluble fiber fraction was carefully scraped with a spatula from the surface of the residue. In this manner, three different products were processed for each sample as follows: (i) Product 1, unmilled (UM). The 10% slurry was fractionated by centrifuging and lyophilized. (ii) Product 2, CM. The 10% slurry was CM. The product was fractionated by centrifuging and was lyophilized. (iii) Product 3, CM and homogenized (CMH). The 10% slurry was CM followed by homogenization. The slurry was fractionated and lyophilized. Scheme 1 is an outline of the processing procedure. The products after each processing step were lyophilized. Freeze-dried samples were ground using a mortar and pestle, and the fine rice bran flour passing through an 80-mesh screen was collected. The moisture contents of freeze-dried samples were 4-5%. All the dried and sieved samples were stored in glass jars at 4°C until further analysis. Analysis of protein content. After each stage of processing, samples of slurries were taken and analyzed for soluble protein content using bicinchoninic acid, following the method of Chan and Wasserman (9). For determining protein content in the lyophilized products, a LECO FP-428 nitrogen analyzer (LECO Corp., St. Joseph, MI) was used to determine the nitrogen content, which was then converted to percentage protein by using the factor of 5.95. Protein yields of various processed fractional products were calculated as (weight of fraction × % protein content)/(weight of bran × % protein content) × 100. Electrophoresis. Samples of freeze-dried supernatant after each processing step were dissolved in Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, pH 8.45, and subjected to SDS-PAGE to determine if there were any changes in molecular weight (M r ) patterns of samples during processing. Electrophoresis was conducted using a 10-20% precast mini Tricine gel (8 × 8 × 0.1 cm) (Novex, San Diego, CA). A Profile TM Mini Electrophoresis System (Schleicher & Schuell, Inc., Keene, NH) and power supply model PS 500XT (Hoefer Scientific, San Francisco, CA) were used. Protein extracts (15-25 µg) were loaded into slots, and gels were run at a constant voltage of 100 V with starting and ending currents at 152 and 61 mA, respectively. A Mark 12 wide-range protein standard (Novex) was used as standard to determine approximate M r of protein bands. Statistical analysis. Data means were analyzed and compared at the 5% level by the one-way analysis of variance and means matrix using the StatPlus Add-In software in MS Excel 2000. RESULTS AND DISCUSSION Extracted protein fractions and yields. Soluble protein contents in the supernatant fractions of the various processed products are shown in Freeze-dried products from the 10% unprocessed bran slurries, colloid milling, and homogenizing gave supernatant fractions from the FFU bran with higher protein contents than did DF and FFS samples, indicating higher protein extractability in FFU bran. Similar increases in protein content of rice bran as a result of milling have previously been reported (6). Moreover, a significant reduction in percentage protein extracted was recently observed in stabilized rice bran compared to unstabilized rice bran (4). Before processing, protein was concentrated in the residue fractions of all bran samples, with the lowest protein yield in the middle fractions SDS-PAGE. There were no differences in protein bands in DF samples ( The FFS samples showed some distinctive differences between protein bands of unprocessed bran and those from CM and CM/homogenized bran The physical processing of rice bran influenced the distribution of protein in the recovered products, and colloid milling followed by homogenizing was more effective in protein redistribution in FFU rice bran than in DF and FFS bran under the conditions studied. Physical processing, on the other hand, did not significantly improve protein recovery compared to reported chemical and enzymatic extractions REFERENCES