Comparison properties of rice bran protein hydrolyzate obtained by spray dry & freeze dry

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

Food Chemistry and Toxicology

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

ICE BRAN PROTEIN IS GAINING INTEREST IN THE FOOD INDUSTRY

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

Physicochemical properties and functionality of rice bran protein

protease, and to investigate their amino acid compositions, molecular sizes, thermal properties, and emulsifying and foaming properties. of nonreducing buffer and 0.025 mL 2-mercaptoethanol. The samples were heated at 95 C for 40 min and cooled to room temperature. Nonreduced/reduced protein solution (15 L) was loaded onto the gel wells. The gels were run in an electrode buffer (pH 8.3) containing 0.3% (w/v) Tris base, 1.44% (w/v) glycine, and 0.1% (w/ v) SDS with a Bio-Rad vertical slab gel electrophoresis unit model Mini-ProteanTM II and power supply model 3000 XI (Bio-Rad Laboratory, Richmond, Calif., U.S.A.) for 41 min. The gels were fixed and stained with 0.1% Coomassie Brilliant Blue R-250 in 10% acetic acid and 40% ethanol, then destained with 10% acetic acid and 40% ethanol, and dried in a Bio-Rad Model 543 Gel Dryer (Bio-Rad Laboratories). The molecular mass standards from Bio-Rad was composed of aprotinin (6.5 kDa), lysozyme (14.4 kDa), trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), serum albumin (66.2 kDa), phosphorylase B (97.4 kDa), -galactosidase (116.3 kDa), and myosin (200 kDa). One mL of this standard was diluted by 9 L of nonreducing buffer and loaded to the well.

Materials and Methods

SINGLE BATCH OF HEAT-STABILIZED DEFATTED RICE BRAN (HDRB)

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.

Amino acid composition

A modified AOAC method 982.30a (1990) was used for amino acid analysis of FD-RBP, SD-RBP, and SPI samples. For each 100 mg of sample, 5 mL 6 M HCl was added in a 50-mL stopper bottle and sealed. The air was removed by keeping the sample in the vacuum chamber. The sealed samples were taken to a drying oven to be hydrolyzed at 120 C for 16 h. After hydrolysis, 5 mL of 2 mM norleucine internal standard was added and the solution passed through a 0.2-L Gelman membrane filter. One milliliter of stock sample was pipetted into a 50-mL borosilicate glass serum bottle and dried in a freeze-drier. To the freeze-dried residue, 1 mL of sodium diluent buffer (pH 2.2) was added and transferred to a 1.5-mL micro-centrifuge tube for HPLC analysis. The prepared samples were injected as 2.5-L volumes and run on a Waters HPLC (Waters Corporation, Milford, Mass., U.S.A.) at a flow rate of 0.4 mL/min with a Pickering sodium ion-exchange column of 4 150 mm (Pickering Laboratories, Inc.) and sodium eluent (pH 3.15 and 7.40). TRIONE ninhydrin reagent was added with post column instrument (TRIONE Ninhydrin derivatization instrument, Pickering Laboratories, Inc.). The light absorbance of amino acids was detected with a UVVisible detector (Pickering Laboratories Inc., Mountain View, Calif., U.S.A.) at 570 nm wavelength and the amino acids were quantified by comparing with standard amino acid profiles.

Preparation of rice bran protein

Rice bran (50 g) was dispersed in 500 g deionized water. The pH was adjusted to 3.5, and the slurry was incubated at 45 C and shaken at 200 rpm in a Micro-Environ shaker (Melrose Park, Ill.) for 2.5 h with 8,750 units of pectinase. The residue of pectinase treatment was further treated with 60000 units of Protease P at pH 7.0, 45 C, and shaken at 200 rpm for 3.1 h. The treated product was heated at 95 C for 2 min to inactivate the enzymes, then cooled and centrifuged at 1100 g for 20 min in a centrifuge (model J2-21, Beckman, Fullerton, Calif., U.S.A.). The supernatants from pectinase and protease treatments were combined. Eighty percent of the total protein in rice bran was in combined supernatant. The combined supernatants were freeze-dried (FD-RBP) and spray-dried (SD-RBP). The protein contents in FD-RBP and SD-RBP were 32.8 and 32.5%, respectively. These proteins were used for investigating molecular size, amino acid composition, hydrophobicity, and functional properties. Soy protein isolate (SPI) was included for comparison. The rice bran protein extract and SPI varied in protein content were used at the same concentration for evaluation of functional properties.

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 separation of proteins

Electrophoresis of FD-RBP, SD-BRB, and SPI was conducted using a 12% separating gel with a 4% stacking gel (Laemmli 1970) under reducing and nonreducing conditions. For nonreducing conditions, 3 mg of protein was mixed with 0.5 mL nonreducing buffer, which consisted of 50.5 mL deionized water, 12.6 mL 0.5 M Tris-HCl (pH 6.8), 10.5 mL glycerol, 21.1 mL 10% (w/v) sodium dodecyl sulfate (SDS), and 5.3 mL 1% (w/v) bromophenol blue. For reduced conditions, the same amount of protein was added with 0.475 mL

Differential scanning calorimetry

Thermal properties of rice bran proteins were evaluated using differential scanning calorimetry (DSC). SPI was included for comparison. Seventy milligrams of FD-RBP, SD-RBP, or SPI was dis-

Food Chemistry and Toxicology

Physicochemical properties and functionality of rice bran protein

solved in 1 mL of 0.05 M phosphate buffer (pH 7.0) containing 0.1 M NaCl. This protein solution (45 L) was transferred and hermetically sealed in a stainless steel pan. The sample was heated by scanning from 25 to 135 C at a rate of 10 C per min against a reference containing 45 L buffer without protein in a differential scanning calorimeter (Perkin-Elmer Corp., Norwalk, Conn., U.S.A.) and denaturation peak temperature and enthalpy were calculated by a thermal analysis data software program (Pyris-I-DSC, Perkin-Elmer Corp., Norwalk, Conn., U.S.A.). of the treatments with JMP 4.02 (2000) software package (SAS, Cary, N.C., U.S.A.). The significance of differences between means was determined by the Tukey-Kramer HSD procedure at p < 0.05.

Results and Discussion

Protein contents of isoelectrically precipitated protein and FD-RBP and SD-RBP
The protein content of heat-stabilized defatted rice bran (HDRB) was 17.6%. Freeze-dried and spray-dried rice bran protein products from supernatants with 80% protein recovered contained 32.8 and 32.5% protein, respectively. The spray-dried and freezedried protein products were used for investigation of molecular size, amino acid composition, nitrogen solubility, thermal properties, hydrophobicity, and emulsifying and foaming properties.

Determination of surface hydrophobicity

Surface hydrophobicity of FD-RBP, SD-BRB, and SPI were determined by using the fluorescence 1-anilino-8-naphthalene sulfonate (ANS) binding method (Hayakawa and Nakai 1985). Rice bran protein solutions (0.0015, 0.003, 0.006, 0.012, 0.015%, w/v) were prepared in 0.01 M phosphate buffer (pH 7.0) and vortexed homogeneously. Ten L of 8 mM ANS in 0.01 M phosphate buffer (pH 7.0) was added into each of 4.0 mL of the protein solutions, then mixed well by vortexing (setting at 5) for 10 s. Fluorescence intensity of these solutions was measured at 390 nm of excitation and 484 nm emmision using a Kontron Spectrofluorometer (model SFM23/ B; Kontron Ltd., Zurich, Switzerland). The surface hydrophobicity, plotted as the slope of fluorescence intensity against protein concentration, was calculated by linear regression.

Food Chemistry and Toxicology

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.

Emulsifying activity and emulsion stability

Emulsifying activity (EA) and emulsion stability (ES) of rice bran proteins and SPI were determined by the turbidimetric methods of Pearce and Kinsella (1978). Six milliliters of 0.1% protein solution (w/v, pH 7.0) and 2 mL of soy oil were homogenized for 1 min using a Virtis homogenizer at 27000 g (Virtishear Tempest; The Virtis Co., Gardiner, N.Y., U.S.A.) to produce the emulsion. Portions of this emulsion (50 L) were pipetted into 5 mL of 0.1% SDS (w/v) at 0 and 10 min after homogenizing. The solution was mixed and the absorbance of the SDS solution was measured at 500 nm (Varian series 634 double-beam spectrophotometer). Absorbance measured soon after homogenizing was expressed as emulsifying activity of protein, and emulsion stability was determined as follows: ES = To t A where T is the decrease in turbidity (absorbance) of To in the time interval t and To is the absorbance of the emulsion after homogenization.

Amino acid composition

The amino acid compositions of FD-RBP, SD-RBP, and SPI are shown in Table 1. The FD-RBP and SD-RBP had very similar amino acid patterns, but FD-RBP had slightly higher amino acid contents than the SD-RBP. In comparison with SPI, both FD-RBP and SD-

Foam capacity and foam stability

Foam capacity (FC) and foam stability (FS) of rice bran protein was measured by the method of Kato and others (1983). Air (90 cm3/ min) was introduced into 5 mL of 0.2% protein in 0.05 M phosphate buffer (pH 7.4) in a glass tube (2.4 x 30 cm) for 15 s. The volume of foams was measured immediately for foam capacity. Foam stability (FS) was calculated from the following equation: FS=Vo t/V where V is the decrease in volume of foam during the time interval of t (15 min), and Vo is the volume of foam at 0 time (beginning after air introduction was stopped).

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.

Physicochemical properties and functionality of rice bran protein

Table 1Amino acid composition of freeze-dried (FD-RBP), spray-dried rice bran protein (SD-RBP), and soy protein isolate (SPI), g/100 g protein Amino acid Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine SPI** 13.8* 3.5 4.8 19.8 4.6 4.7 5.8 5.4 8.7 3.2 5.5 7.1 2.7 7.8 FD-RBP 11.2 3.7 4.5 18.1 6.2 7.3 7.0 4.5 8.0 3.7 5.1 5.4 3.3 10.2 SD-RBP 10.9 3.7 4.5 17.6 5.8 7.1 6.9 4.5 7.9 3.8 5.0 5.2 3.1 9.8 Table 2Thermal properties of freeze-dried/spray-dried rice bran protein (FD-RBP/SD-RBP) from heat-stabilized defatted rice bran and soy protein isolate (SPI) Protein source Onset FD-RBP** SD-RBP SPI 67.8* 73.9 75.4 Temperature, C Peak End Area, mJ 84.1 84.6 84.2 90.4 90.9 89.7 7.88 7.47 5.85

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.

Thermal properties of proteins

RBP contained higher amounts of the essential amino acids threonine, valine, tyrosine, histidine, and arginine. The lysine and threonine contents in FD-RPB and SD-RBP agreed with the data reported by Connor and others (1976), but arginine, phenylalanine, leucine, and valine contents contents in FD-RPB and SD-RBP were higher. Wang and others (1999) reported a much lower value of lysine content (3.99 g/100 g) in rice bran protein isolate. From the whole amino acid profile, amino acids in either FD-RBP or SD-RBP can meet the 2-y-old childrens requirement recommended by the FAO/WHO/UNU (Joint FAO/WHO/UNU 1985). Data from differential scanning calorimetry (DSC) measurements for FD-RBP, SD-RBP, and SPI are given in Table 2. FD-RBP, SD-RBP, and SPI had similar denaturation temperatures (84.1, 84.6, and 84.2 C, respectively). Biliaderis (1983) concluded that changes in protein enthalpy could be used to predict the extent of protein denaturation. 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 extracted with phytase and xylanase at pH 5.0. The enthalpies of FD-RBP and SD-RBP were 2.5 and 2.37 J/g, respectively. In this study, FD-RBP and SD-RBP may be less denatured than similar product reported by Wang and others (1999).

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-

Surface hydrophobicity of proteins

The surface hydrophobicities (So) of freeze-dried rice bran protein (FD-RBP), spray-dried rice bran protein (SD-RBP), and SPI were 206.77, 226.11, and 323.03, respectively, and the linear relationships between protein concentration and fluorescence intensity are shown in Figure 3. There was no significant difference in surface hydrophobicity between FD-RBP and SD-RBP (p > 0.05). However, the surface hydrophobicity value for SPI was significantly higher than either for FD-RBP or SD-RBP (p < 0.05). In the native proteins, the hydrophobic amino acids are buried in the central core of the protein molecule. This feature is lost when protein is denatured or hydrolyzed into shorter peptides. Wang and others (1999) measured the surface hydrophobicity of rice bran protein isolate and concentrate of full-fat nonstabilized rice bran, and reported hydrophobicity values of 14.0 and 20.4. Heat processing during oil extraction denatures the rice bran protein that could lead to a higher hydrophobicity values. Both FD-RBP and SD-RBP had higher hydrophobicities than protein isolate and concentrate of full-fat nonstabilized rice bran and this also could be due to hydrophobic amino acids exposed during heat-stabilization.

Emulsifying activity and emulsion stability of proteins

The emulsifying activities and emulsion stabilities of FD-RBP, SD-RBP, and SPI are shown in Table 3. SPI had significantly higher emulsifying activity and emulsion stability than both FD-RBP and SD-RBP (p < 0.05). However, no significant differences were observed between FD-RBP and SD-RBP in either emulsifying activity or emulsion stability (p > 0.05). Wang and others (1999) reported

Figure 2Effect of pH value on nitrogen solubility. *FDRBP and SD-RBP: freeze-dried and spray-dried rice bran protein; SPI: soy protein isolate.

Food Chemistry and Toxicology

Physicochemical properties and functionality of rice bran protein

Table 3Emulsifying activities and emulsion stabilities of rice bran proteins and soy protein isolate Protein FD-RBP** SD-RBP SPI Emulsifying activity 0.19b* 0.18b 0.24a Emulsion stability, min 16.5b 17.3b 27.5a Table 4Foam capacity and foam stability of freeze-dried/ spray-dried dried rice bran proteins and soy protein isolate Foaming capacity, mL FD-RBP** SD-RBP SPI 4.0b* 4.2b 47.6a Foaming stability,min 0 0 78.3

*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.

Food Chemistry and Toxicology

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

EAT-STABILIZED DEFATTED RICE BRAN WAS HYDROLYSED WITH

Foam capacity and stability of proteins

The foam capacities and stabilities of FD-RBP, SD-RBP, and SPI are given in Table 4. To have foam capacity, proteins should solubilize in the aqueous phase and rapidly unfold to form a cohesive layer of protein around gas/air droplets. This requires flexible protein molecules with few secondary and tertiary structures. To have foam stability, protein molecules should form continuous intermolecular polymers enveloping the air bubbles, since intermolecular cohesiveness and elasticity are important to produce stable foams. Wang and others (1999) reported that rice bran protein isolate had similar foaming properties (19.4 mL) in comparison with egg white (20.5 mL), but the foam stability (105 min) was slightly lower than that of egg white (120 min). Foam capacities were very low for FD-

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
American Association of Cereal Chemists [AACC]. 1990. Method 46-08. Approved methods of the American Association of Cereal Chemists, 8th Ed., Vol 2. St. Paul, Minn.: AACC. P 1-2. Association of Official Analytical Chemists [AOAC]. 1990. Method 982.30a. In: Official methods of analysis, 15th Ed. Arlington, Va.: Association of Official Analytical Chemists. P 1096-1097. Bae D, Jang S. 1999. Development of new food protein through chemical modification of rice bran proteins. Agric Chem Biotechnol 42(4):180-185. Bera MB, Mukherjee RK. 1989. Solubility, emulsifying, and foaming properties of rice bran protein concentrates. J Food Sci 54(1):142-145. Biliaderis CG. 1983. Differential scanning calorimetry in food research: A review. Food Chem 10:239-265. Connor MA, Saunders RM, Kohler GO. 1976. Rice bran protein concentrates obtained by wet alkaline extraction. Cereal Chem 53(4):488-496. Damodaran S. 1990. Interfaces, protein films, and foams. Adv Food Nutr Res 34:1-79. Gnanasambandam R, Hettiarachchy, NS. 1995. Protein concentrates from nonheat-stabilized rice bran: Preparation and properties. J Food Sci 60:1066-1069. Halling PJ. 1981. Protein-stabilized foams and emulsions. CRC Crit Rev Food Sci Nutri 21:155-203. Hamada J. 1997. Characterization of protein fractions of rice bran to devise effective methods of protein solubilization. Cereal Chem 74(5):662-668. Hamada J. 2000. Ultrafiltration of partially hydrolyzed rice bran protein to recover value-added products. JAOCS 77:779-784. Hayakawa S, Nakai S. 1985. Relationships of hydrophobicity and net charge to the solubility of milk and soy proteins. J Food Sci 50:486-491. Helm RM, Burks AW. 1996. Hypoallerginicity of rice protein. Cereal Foods World 41:839-842. Hermansson AM. 1978. Physicochemical aspects of soy proteins structure formation. J Text Stud 9:33-58. Joint FAO/WHO/UNU. 1985. Expert Consultation. Energy and protein requirements. WHO Tech. Rep. Ser. nr 724. WHO, Geneva, Switzerland. Juliano BO. 1994. Rice: Chemistry and technology. St. Paul, Minn.: American Association of Cereal Chemists. Kato A, Takahashi A, Matsudomi N, Kobayashi K. 1983. Determination of foam-

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.

Physicochemical properties and functionality of rice bran protein

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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).

Food Chemistry and Toxicology

Financial support provided by the Arkansas Rice Research and Promotion Board and TRQ are greatly appreciated.