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Physicochemical Properties and Functionality of Rice Bran Protein Hydrolyzate Prepared from Heat-stabilized Defatted Rice Bran with the Aid of Enzymes
S. TANG , N.S. H ETTIARACHCHY , R. H ORAX, AND S. E SWARANANDAM
ABSTRACT: Molecular size, thermal properties, hydrophobicity, nitrogen solubility, and emulsifying and foaming properties were determined for protein products from heat-stabilized defatted rice bran. The freeze-dried and spray-dried proteins had molecular sizes between 6.5 to 66.2 kDa; denaturation temperatures of 84.1 and 84.6 C, enthalpies of 2.5 and 2.37 J/g, hydrophobicities of 20677 and 22611, maximum solubilities of 66.3% and 66.1% at pH 12.0, emulsifying capacities of 0.19 and 0.18, emulsion stabilities of 16.5 and 17.3 min, foam capacities of 4.0 mL and 4.2 mL, and negligible foam stabilities. These results demonstrated that the extracted rice bran protein has potential as a nutraceutical ingredient in food applications. Keywords: rice bran protein, hydrophobicity, DSC, functionality
Introduction
because of its unique nutritional value and nutraceutical properties (Saunders 1990). Its nutritional value is much higher than rice endosperm protein or protein from any other cereals or legumes (Juliano 1994). Rice bran protein also is highly digestible. In addition to the high nutritional value, rice protein is a hypoallergenic food ingredient and may serve in infant formulations (Helm and Burks 1996). Furthermore, rice bran protein has been reported to have anti-cancer properties (Kawamura and Muramoto 1993). Knowledge on its functional properties is needed for this ingredients use in a range of food applications. Protein functional properties are determined to a large extent by a proteins physicochemical and structural properties (Damodaran 1990). Protein solubility is an important prerequisite for food protein functional properties, and it is a good index of potential applications of proteins (Kinsella 1976). Researchers have reported that protein solubility has a close relationship with emulsifying (Yatsumatsu and others 1972a,b; McWatters and Holmes 1979) and foaming properties (Yatsumatsu and others 1972a). Amino acid composition and molecular size are the foundations of protein functionality (Kinsella 1981 ). Differential scanning calorimetry (DSC) has been used to study the thermal properties and the structural changes of proteins (Hermansson 1978; Patel and others 1990; Reaker and Johnson 1995). Surface hydrophobicity has also been reported to correlate to surface tension, interfacial tension, and emulsifying activity (Nakai and others 1980; Halling 1981; Petruccelli and Anon 1994; Phillips and others 1994). Bera and Mukherjee (1989) showed that rice bran protein concentrate from alkali extraction had higher nitrogen solubility at higher or lower pH values than its isoelectric point (pH 4.5). Wang and others (1999) evaluated the solubility of rice bran protein isolate in the pH range of 2.0 to 12.0 with varying concentrations of NaCl. These researchers found that the nitrogen solubility of rice bran protein isolate was between 70.0 and 90.0% in the broad pH range of 6.0 to 12.0. Rice bran protein
isolate had the lowest solubility (10%) at pH 4.0. Hamada (2000) reported that protein hydrolysate of protease treatment from defatted nonheat-stabilized rice bran had very good nitrogen solubility. Wang and others (1999) with SDS-PAGE and Hamada (1997, 2000) with size-exclusion HPLC reported the molecular sizes of rice bran protein from nonheat-stabilized rice bran. Wang and others (1999) reported that the denaturation temperature and enthalpy changes were 83.4 C and 0.96 J/g for rice bran protein isolate. These investigators also reported that the hydrophobicity values of rice bran protein isolate (obtained after treating with phytase and xylanase) and rice bran concentrate without enzyme treatment were 14.0 and 20.4, respectively. Bae and Jang (1999) reported that succinylated rice bran protein concentrate had good functional properties, including solubility, emulsion properties, and oil absorption capacity. Wang and others (1999) reported emulsifying capacity and emulsion stability were 0.3 to 0.37 and 3.9 to 4.2 min, and foam capacity and stability were 0.3 to 0.5 and 4.1 to 4.3 min, respectively, for rice bran protein isolate prepared from nonstabilized rice bran. In addition to the characteristics and functional properties, Connor and others (1976) and Wang and others (1999) reported on the amino acid composition of rice bran proteins. Heat processing during oil extraction denatures rice bran protein, which could lead to higher hydrophobicity and affect functional properties. Information about molecular size, hydophobicity, nitrogen solubility, and thermal and functional properties of proteins from heat-stabilized defatted rice bran are not available in the published literature. Commercially, the oil is extracted from rice bran. During the oil extraction process, rice bran goes through a heat stabilization step to inactivate the lipase enzyme to preserve the quality of oil. The rice bran after its oil removal with heat treatment is a co-product and is designated as heat-stabilized defatted rice bran. Protein extracted from heat-stabilized defatted rice bran could be utilized as a nutraceutical food ingredient. The objectives of this study were to characterize freeze-dried (FD-RBP) and spraydried rice bran protein (SD-RBP) hydrolyzate with pectinase and
was obtained from Riceland Foods, Inc (Stuttgart, Ark., U.S.A.). Commercial food-grade enzymes, pectinase II (3500 units/g) and Protease P (600000 units/g) were obtained from Amano Pharmaceutical Co. (Nagoya, Japan). Soy protein isolate (PRO FAM) was obtained from Archer Daniels Midland Co. (Decatur, Ill.. U.S.A.). Soy protein isolate contain 90% protein and pH is about 7.0 in 1:10 dispersion in water. Electrophoresis reagents were purchased from Bio-Rad Laboratories (Hercules, Calif., U.S.A.). Other analytical grade chemicals were purchased from Fisher Scientific (Pittsburgh, Pa., U.S.A.) and Sigma Chemical Co. (St. Louis, Mo., U.S.A.). Sodium ion exchange column was used to separate amino acids (Pickering Laboratories, Inc., Mountain View, Calif., U.S.A.). Reagents for amino acid analysis sodium eluant (pH 3.15, pH 7.40), sodium sample diluent (pH 2.20) and TRIONE ninhydrin reagent were purchased from Pickering Laboratories, Inc. All chemical used were of reagent grade.
Nitrogen solubility
Nitrogen solubility was determined according to the procedure of Bera and Mukherjee (1989). One hundred mg of FD-RBP, SDBRB, or SPI protein was dispersed in 10 mL of distilled deionized water. The suspensions were adjusted to pH 2.0/4.0/6.0/8.0/10.0/ 12.0 using either 0.1 M HCl or 0.1 M NaOH. These suspensions were shaken (Lab-Line Environ-Shaker; Lab-Line Instrument, Inc., Melrose Park, Ill., U.S.A.) for 30 min at room temperature (approximately 25 C) and centrifuged at 4000 g for 30 min. The nitrogen content of the supernatant was determined by the Kjeldahl method and percent nitrogen solubility was calculated as follows:
amount of nitrogen in supernatant 100 nitrogen solubility, % = total amount of nitrogen in 100 mg sample
Protein content
Protein contents in rice bran and extracted rice bran protein products were determined by the automated Kjel-Foss method 4608 (AACC 1990). Samples were digested with Kjeldahl Digestion System 6 for 1 h at 420 C, and protein contents were determined with KjelTech Analyzer 2000 (Tecator Co., Hoganas, Sweden ). Percent total protein content in rice bran and extracted products were read from the output of the instrument using a conversion factor of 5.95 (Juliano 1994).
Electrophoresis of proteins
The electrophoresis patterns of FD-RBP, SD-RBP, and SPI are shown in Figure 1. The nonreducing and reducing samples showed that the rice bran protein products had 4 components of protein. The molecular weights of all 4 components were between 6.5 and 66.2 kDa. The largest molecular size was between 45.0 and 66.2 kDa. Two higher-density protein bands were observed between 6.5 and 14.4 kDa. The SPI had more than 10 bands and their molecular sizes ranged from 14.4 to 97.5 kDa. Wang and others (1999) reported that rice bran protein isolate had more than 12 bands. It is possible that protease hydrolyzed some higher-molecular-weight protein fraction into smaller-molecular-weight peptides that ran off the gel.
Statistical analysis
Data were analyzed for variance and multiple mean comparison
Figure 1Electrophoretogram of rice bran protein from heat-stabilized defatted rice bran. ST: standard (kDa); FRP and FRPR: nonreduced and reduced freeze-dried rice bran protein; SRP and SRPR: nonreduced and reduced spraydried rice bran protein; SPI and SPIR: nonreduced and reduced soy protein isolate; STR: reduced standard.
DH J/g
2.50 2.37 1.86
*Values are means of duplicate analyses **FD-RBP and SD-RBP: freeze-dried and spray-dried rice bran protein SPI: soy protein isolate
*Values are means of duplicate analyses **FD-RBP and SD-RBP: freeze-dried and spray-dried rice bran protein SPI: soy protein isolate
tein concentrate by Bera and Mukherjee (1989), Gnanasambandam and Hettiarachchy (1995), and Wang and others (1999). At pH 12.0, the solubility of SPI reached 82%, while solubility for both rice bran protein products was only 66%. The solubilities of freeze-dried and spray-dried products were very similar from pH 2.0 to 12.0.
Protein solubility
The solubilities of FD-RBP, SD-RBP, and SPI at pH 2.0/4.0/6.0/ 8.0/10.0/12.0 are presented in Figure 2. At pH 2.0, soy protein isolate (SPI) and freeze-dried and spray-dried rice bran protein products had similar solubilities (below 30%). At pH 4.0, all 3 sources of protein had lower solubilities, 10.9% for FD-RBP, 9.8% for SD-RBP, and 1.7% for SPI. Above pH 6.0, the nitrogen solubilities increased rapidly with an increase in pH up to pH 12.0. These trends in solubilities are in agreement with the data reported for rice bran pro-
Figure 2Effect of pH value on nitrogen solubility. *FDRBP and SD-RBP: freeze-dried and spray-dried rice bran protein; SPI: soy protein isolate.
*Values are means of triplicate analyses and the values followed by different letters in the same column are significantly different (p<0.05). **FD-RBP and SD-RBP: freeze-dried and spray-dried rice bran protein SPI: soy protein isolate
*Values are the means of triplicate analyses and values followed by different letters in the same column are significantly different (P<0.05). nd is not determinable . **FD-RBP and SD-RBP: freeze-dried and spray-dried rice bran protein; SPI: soy protein isolate
that the emulsifying activity and emulsion stability with canola, corn, and soybean oils for rice bran protein isolate from full-fat rice bran were 0.30 to 0.37 and 3.90 to 4.20 min, respectively, and for rice bran protein concentrate were 0.30 to 0.5 and 4.10 to 4.30 min. Hamada (2000) reported 0.10 to 0.51 of emulsifying activity with a stability of approximately 11 min for freeze-dried protease extracted protein hydrolysates from nonstabilized rice bran at pH 5, 7, and 9. Petruccelli and Anon (1994) reported that emulsifying properties are closely associated with protein surface hydrophobicity. Our data showed that both freeze-dried and spray-dried products had relatively good emulsifying properties and SPI had highest emulsifying activity and stability. This could be due to the exposure of hydrophobic groups of denatured proteins.
RBP and SD-RBP; and the foam stabilities were almost negligible. This could be due to the lack of sufficient amounts of secondary and tertiary structures that are needed to form cohesive layers around air droplets. It is also possible that the hydrolyzed peptides in the FD-RBP and SD-RBP products could not form cohesive layers to stabilize the foam.
Conclusions
pectinase and protease to produce hydrolyzate and then freeze-dried and spray-dried to obtain rice bran protein. Electrophoresis of rice bran protein hydrolyzate showed only 4 bands for FD-RBP and SD-RBP and their molecular weights ranged from 6.5 to 66.2 kDa. In comparison with SPI, both FD-RBP and SD-RBP contained higher amounts of the essential amino acids threonine, valine, tyrosine, histidine, and arginine. Solubilities of both FD-RBP and SD-RBP at pH 4.0 were similar but higher than SPI. Both FDRBP and SD-RBP had higher hydrophobicities than protein isolate and concentrate of full-fat nonstabilized rice bran but lower than SPI. Emulsifying activities and emulsion stabilities and foam capacity of FD-RBP and SD-RBP were lower than SPI. FD-RBP and SDRBP products could find applications as ingredients in nutraceutical products.
References
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Figure 3Fluorescence intensity of rice bran protein and soy protein solutions. *FD-RBP and SD-RBP: freeze-dried and spray-dried rice bran protein; SPI: soy protein isolate.
Authors Tang, Hettiarachchy, Horax, and Eswaranandam are with the Dept. of Food Science, Univ. of Arkansas, 2650 North Young Ave., Fayetteville, AR 72704. Direct inquiries to author Hettiarachchy (E-mail: nhettiar@mail.uark.edu).
Financial support provided by the Arkansas Rice Research and Promotion Board and TRQ are greatly appreciated.