J. Korean Soc. Appl. Biol. Chem. 53(1), 62-70 (2010) Article

Production and Physicochemical Properties of Rice Bran Protein Isolates Prepared with Autoclaving and Enzymatic Hydrolysis

Hye-Jung Yeom1, Eun-Hye Lee1, Mi-Sun Ha1, Sang-Do Ha2, and Dong-Ho Bae1*

1Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea2Department of Food Science & Technology, Chung-Ang University, Anseong 456-756, Republic of Korea

Received August 28, 2009; Accepted November 25, 2009

The objectives of this study were to increase the protein purity in the rice protein isolate, and to

enhance the functional properties of the extracted rice bran protein via enzymatic hydrolysis and/

or high temperature treatment. Washing the isolates with 30% ethanol solution achieved

significant improvements in the protein contents (77.62%) of the rice bran protein isolates. The

highest improvement in solubility was detected in the isolate autoclaved after enzymatic hydrolysis,

up to 97.4% at a pH of 10.0. A combined modification of the method involving autoclaving and

protease-hydrolysis improved the emulsion activity of the isolate at pH values above 6.0. The

autoclaving and enzymatic hydrolysis increased the foaming capacity of the isolate, but reduced

the foam stability at all pH ranges except 5.0. Enzymatically hydrolyzed isolates evidenced lower

minimum concentrations of gel formation proteins and higher gel strengths than were detected in

the other isolates.

Key words: enzymatic hydrolysis, functional properties, protease, protein isolate, rice bran

Rice bran is an inexpensive and underutilized by-

product of the rough rice milling process. In 2008,

approximately 4,843,000 tons of rice were produced in

Korea, ultimately yielding about 430,000 tons of bran

[National Agricultural Products Quality Management

Service, 2008]. Defatted rice bran is a by-product of rice

bran oil. Rice bran contains a not-insignificant amount of

protein (12-20%), with fairly high nutritional quality

[Saunders, 1990]. The lysine content of rice bran protein

is approximately 3-4%, which is higher than that of rice

endosperm protein or proteins from other cereal brans or

legumes. The PER of rice bran protein concentrate has

been measured at 2.0-2.5, which is not substantially lower

than that of casein (2.5). Rice bran protein is also very

digestible (more than 90%) [Wang et al., 1999] and may

be hypoallergenic [Helm and Burks, 1996]. Considering

its excellent protein compositions and abundance, rice

bran protein holds great promise as an alternative protein

resource such as a food ingredient. However, this

renewable protein resource has received only minimal

attention thus far, and this underutilized protein has thus

far only rarely been employed in the food industry. This is

due partly to the difficulty inherent to the extraction of a

large portion of rice bran protein, owing to extensive

disulfide bond cross-linkages and strong aggregation

[Hamada, 1997], and also partly because the protein has

classically exhibited poor functional properties.

Therefore, the current interest in rice bran protein for

use in food lies principally in the effort to improve its

recovery and functional properties, and to induce desirable

alterations in the structure, texture, and composition of the

final products, at an appropriate price. Protein hydrolysis is

an effective method for modifying functional properties

and improving extraction, in which the protein remains

highly soluble across a broad range of pHs and

temperatures [David et al., 2009]. Strong acids or bases

can also be applied to break the peptide bond, which is a

simple method, albeit harsh. Additionally, this either

destroys or modifies the essential amino acids, and

creates toxic by-products and undesirable side reactions

that reduce the quality of the protein [Humiski and Aluko,

2007]. However, enzymatic hydrolysis does not affect the

nutritional value of the proteins. Additionally, enzymatic

hydrolysis can improve the physicochemical, functional,

and sensory properties of native proteins [Kristinsson and

Rasco, 2000]. Considering the high costs associated with

*Corresponding authorPhone: +82-2-450-3756; Fax: +82-2-456-7011E-mail: donghoya@konkuk.ac.kr

doi:10.3839/jksabc.2010.011

Modified rice bran protein isolate 63

enzymatic hydrolysis or modification, high temperature

treatments may prove to effectively enhance the

extractability and functional properties of proteins. A

number of studies have demonstrated the utility of high

temperature treatment [Sereewatthanawut et al., 2008],

which was of relatively low cost and enhanced the

functional properties of protein, including its solubility,

emulsion properties, and foaming properties [Khuwijitjaru

et al., 2007].

The principal objectives of this study were to increase

protein purity in the rice protein isolate, and to increase

the functional properties of the extracted rice bran protein

via enzymatic hydrolysis and/or high temperature

treatment for the additional objective of utilizing a large

proportion of the underutilized rice bran protein,

hopefully allowing for its substitution for costly imported

soy protein isolates.

Materials and Methods

Optimization of conditions for rice bran protein

isolate production. Rice bran defatted with hexane was

dispersed in a 7-fold volume of distilled water, a volume

selected considering the extractability of protein and

recovery efficiency, and then mixed in a blender (IXM-

401, Shinil Co., Seoul, Korea) at high speed for 5 min.

The mixture was then adjusted to pH 9.0 with 1.0 N

NaOH, stirred for 2 h to extract the protein, and then

centrifuged for 20 min at 9,000×g at 4oC to remove the

insoluble materials. In order to facilitate the removal of

the non-protein materials and to improve the purity of the

resultant rice bran protein isolate, the effects of high

temperature treatment and/or carbohydrate-hydrolysis of

the supernatant on the final product, the rice bran protein

isolate, were determined. For the high temperature

treatment, the supernatant was autoclaved for 30 min at

121oC. For carbohydrate-hydrolysis, α-amylase (Liquozyme

Supra, Novo Nordisk, Bagsvaerd, Denmark) was applied

to the supernatant. The supernatant was heated at the

optimal temperature, 95oC, and then adjusted to the

optimum pH of 5.2-5.6 prior to the addition of enzymes.

The α-amylase (1% of supernatant, w/w) was added to

the supernatant and incubated for 1 h in a water bath.

After autoclaving and/or carbohydrate hydrolysis, the

protein in the supernatant was adjusted to a pH of 4.0

with 1.0 N HCl and centrifuged for 15 min at 8,000×g at

4oC in order to precipitate the protein. The precipitate was

then washed twice in 30% ethanol or distilled water for

30 min to remove the soluble materials. The precipitate

was then resuspended in distilled water (1:1, w/v),

neutralized by adjusting the pH to 6.5, and freeze-dried

(EYELA FD-1000, Rikakika Co., Tokyo, Japan). The

protein content of the isolate was determined by the

micro-Kjeldahl method (conversion factor of 5.95)

[AOAC, 2000; Method 979.09].

Enzymatic hydrolysis of rice bran protein. Enzymatic

hydrolysis of the protein isolate was conducted using a

mixture of Alcalase 2.4 L and Flavourzyme (Novo

Nordisk, Bagsvaerd, Denmark). The proper mixing ratios

of the proteases were determined in a preliminary study

as 9:1, considering efficiency and cost. Alcalase and

Flavourzyme are microbial and fungal proteases obtained

from Bacillus licheniformis and Aspergillus oryzae,

respectively. According to the instructions of the

manufacturer, Novo Nordisk, both proteases are GRAS.

The previously prepared protein isolate was then

dispersed in a 7-fold volume of distilled water, heated to

the optimum temperature of 50oC, and adjusted to the

optimum pH of 8.0. The protease mixture (1% of protein

isolate, w/w) was added to the dispersion and incubated

for 2 h in a water bath. After 2 h of enzymatic hydrolysis,

the protein solution was heated to 85oC for 10 min in a

water bath in order to deactivate the enzyme.

The DH was calculated in accordance with the method

developed by Yoon et al. [2009], in which the DH is

determined by measuring the soluble nitrogen content in

10% TCA. An aliquot (10 mL) of an aqueous dispersion

of RBPI (1%, w/v) was mixed with 10 mL of 20% TCA

and centrifuged for 20 min at 8,900×g at 4oC. The soluble

nitrogen in the supernatant was determined by the micro-

Kjeldahl method [AOAC, 2000; Method 979.09]. Total

nitrogen content was also determined using 10 mL of

protein dispersion prepared from the same method as was

used for enzymatic hydrolysis without the enzyme. The

DH was calculated as a percentage, as follows:

DH (%) =

Soluble nitrogen in 10% TCA solution (mg) ×100

Total nitrogen (mg)

Determination of nitrogen solubility. The nitrogen

solubility of rice bran protein was determined by the

method of Bera and Murkherjee [1989]. One gram of

sample was dispersed in 100 mL of distilled water and

adjusted to a pH of 2.0-10.0 using either 0.1 N NaOH or

HCl. The dispersion was stirred for 60 min at room

temperature, then centrifuged for 20 min at 4,500×g. The

nitrogen content in the supernatants was determined by

the micro-Kjeldahl method [AOAC, 2000; Method

979.09], and the percentage of soluble protein was

calculated as follows:

Solubility (%)=

Amount of nitrogen in supernatant ×100

Amount of nitrogen in sample

64 Hye-Jung Yeom et al.

Determination of emulsifying properties. Emulsifying

properties were assessed by the method of Pearce and

Kinsella [1979]. Twelve milliliters of soybean oil and 36

mL of 1% (w/v) rice bran protein solution, previously

adjusted to pH 5.0-8.0, were homogenized for 1 min at

8,000×g to prepare the emulsion. An aliquot (50 μL) of

the emulsion was taken from the bottom of the container

at 0 and 10 min after homogenization and mixed with 5

mL of 0.1% SDS solution. The absorbance of the emulsions

was measured at 500 nm using a spectrophotometer

(DU730 UV/Vis Spectrophotometer, Beckman Coulter,

Miami, FL, USA). The absorbance at 0 min was

expressed as the emulsion activity index. The emulsion

stability index (ESI) was determined as follows:

ESI=

Where A0 is the absorbance at 0 min and �A is the

change in absorbance, A0, occurring over the interval t

(10 min).

Foaming capacity and foam stability. The foaming

capacity and foam stability were evaluated by the

modified method of Kato et al. [1983]. One gram of

protein sample was dispersed in 100 mL of distilled water

and adjusted to pH 5.0-8.0 using either 0.1 N NaOH or

HCl. The foaming capacity of the dispersion was determined

and compared by measuring the volume of foams

immediately after 1 min of mixing with a homogenizer at

10,000×g. Foaming capacity was expressed by the

following equation:

Foaming capacity= Total volume−Drainage volume

Initial volume of 100 mL

Foaming stability was measured as the foam volume

remaining after 20 min and determined from the following

equation:

Foaming stability=

Where V0 is the initial foam volume at 0 min and �V is

the change in the volume of foam during the time interval

t (20 min).

Gel forming ability. The gel forming ability was

determined by the modified method of Coffmann and

Garcia [1977]. Sample dispersions of 5-15% (w/v) were

prepared in distilled water and adjusted to pH values of

4.0 and 7.0. The gelation properties of the samples

containing carrageenan (2%, w/v) were also determined at

protein concentrations of 0-10% (w/v) and pH values of

4.0 and 7.0. An aliquot (5 mL) of the prepared dispersion

was molded in a cap tube, which was heated to 95oC for

30 min in a water bath followed by rapid cooling in tap

water, followed by 2 h of maintenance in a cold room at

4oC. The final gel was carefully extracted from the cap

tube in which the protein dispersion was molded, and cut

in to a cube (15 mm×15 mm×15 mm). The rheological

properties of the gel were measured using a texture

analyzer (TA plus, Lloyd Instruments, Segensworth East,

Fareham, England) and subjected to a double-compression

test for hardness, adhesiveness, cohesiveness, and

springiness using a 10 N reversible load cell. The

diameter of the pin punch was 6 mm, and the moving

speed was 1 mm/s. The samples were compressed to

75% of their original height.

Results and Discussion

Rice bran protein isolate (RBPI) production. The

protein content of the rice bran defatted with n-hexane

was 19.30% (Table 1). A large portion of this protein

exhibits limited solubility in water, as it combines with

other components including starch and fiber. However,

many of these protein-involving bonds are pH-sensitive

[Shih et al, 1999], and thus might be cleaved under

alkaline pH conditions, as evidenced by the higher

protein purity of the protein isolates extracted at alkaline

pHs (Fig. 1(A)), whereas the isolates extracted at pH

values of 5.0 and 6.0 evidenced the lowest protein

contents. The extraction of rice protein at pH 9.0 also

resulted in the highest protein yield (42.6%) in the

resultant protein isolate, whereas the lowest protein yield

(37.6%) was noted at pH 5.0 (Fig. 1(B)). These results

were consistent with the data reported by Betschart et al.

[1977] and Bera and Mukherjee [1989], in which the

extraction of rice bran protein (RBP) exhibited the

minimum protein yield at pH 4.5 and the maximum at pH

9.0-10.5. Approximately 75% of the RBP is composed of

albumin and glutelin, which are soluble and insoluble,

respectively, at moderately alkaline or acidic pHs, which

rendered the extraction of glutelin at pH 9.0 and the

isolation of albumin at pH 4.0 difficult, thereby resulting

A0

t×

�A--------------

V0

t×

��--------------

Table 1. Compositions of defatted rice bran and ethanol-washed rice bran protein isolate produced in this study

Component

Composition (%)

Defattedrice bran

Ethanol-washedrice bran protein isolate

Crude protein 19.30±0.91 77.62±2.900

Carbohydrate 41.07±1.74 10.27±0.850

Crude fat 00.89±0.06 0.75±0.03

Moisture 10.31±0.34 3.82±0.20

Ash 12.36±0.13 4.98±0.21

Fiber 14.40±0.86 1.52±0.77

Modified rice bran protein isolate 65

in low yields (37.6-42.6%) of rice protein in RBPI.

Although extraction at pH 9.0 yielded the highest

protein contents (69.3%) in the resultant protein isolates,

these contents were still insufficient for commercial

application. Therefore, autoclaving, α-amylase treatment,

and ethanol-washing methods were performed in an

effort to improve the protein purity of the RBPI.

Autoclaving can cleave the hydrogen bonds of protein-

carbohydrate or protein-protein, and can also expose

some of the polar amino acid groups buried within the

folded protein structure, thereby inducing increases in

hydrophilicity, and thus extractability [Wu et al., 1998].

α-Amylase can hydrolyze the α-1,4 glucoside bond of

the starch bound to the protein, which facilitates the

liberation of starch-bound proteins and extraction by

increasing the solubility of unbound proteins [Shanhu et

al., 2002]. However, neither autoclaving nor α-amylase

significantly improved the protein purity of the resultant

isolates (Fig. 2). However, the washing of the isolates

with 30% ethanol solution achieved significant

improvements in the protein contents in the isolates,

regardless of whether or not they were autoclaved or

treated with α-amylase. Despite a previous report

showing that the protein content of isolate was improved

to up to 94% by alcohol treatment after acid precipitation

[Connor et al., 1976], a 30% ethanol solution was utilized

in this study, considering the flammability of the ethanol

solution with higher purity, and the implications of this

flammability in industrial applications. The proximate

compositions of the ethanol-washed RBPI are provided in

Table 1.

Enzymatic hydrolysis of RBPI. Humiski and Aluko

[2007] previously reported that Alcalase and Flavourzyme

hydrolyzed pea protein more effectively with a

significantly higher degree of hydrolysis than the other

proteases, such as papain, trypsin, and α-chymotrypsin.

Villanueva et al. [1999] also achieved the highest degree

of hydrolysis in sunflower protein with a combination of

Alcalase and Flavourzyme. On the basis of these previous

studies, RBPI was hydrolyzed by treatment with mixtures

of commercial food-grade Alcalase and Flavourzyme in

this study. Alcalase is a specific and economic endoprotease

[Doucet et al., 2003], with high tolerance for alkaline pHs

[Tardioli et al., 2003]. During protein hydrolysis,

undesirable bitter peptides can be generated, which limit

the use of protein hydrolysates as a food ingredient. The

bitterness is caused by exposure to a certain length of

hydrophobic residues at the end of the polypeptide chain.

Flavourzyme, which possesses both endoprotease and

exoprotease activities, is known to reduce the bitter taste

of protein hydrolysates [Pedersen, 1994]. Fig. 3 shows

the degree of RBP hydrolysis (DH) in the Alcalase and

Flavourzyme mixtures. The DH increased dramatically

during the initial 15 min, reaching 21.9%, and the rate of

Fig. 2. Protein contents in rice bran protein isolates(RBPI) pretreated by autoclaving (H-RBPI), amylase-hydrolysis (A-RBPI), and amylase-hydrolysis after

autoclaving (HA-RBPI). � and � are the rice branprotein isolates washed with distilled water and 30%alcohol after isolation.

Fig. 1. Protein contents (A) and yields (B) of rice branprotein isolates extracted at several pHs.

66 Hye-Jung Yeom et al.

change was reduced thereafter, ultimately reaching 27.7%.

This DH curve is consistent with the reports of Qi et al.

[1997] and Yoon et al. [2009]. Enzymatic hydrolysis

increases the number of exposed hydrophilic groups and

decreases the molecular weight of the protein, which

causes changes in the protein’s functional properties

[David et al., 2009].

Water solubility. Water solubility is one of the most

important characteristics of proteins, as it influences other

properties, including emulsion, foaming, and gel forming

ability. Water soluble proteins induce homogeneous

dispersibility of the molecules in colloidal systems, and

improve the interfacial properties [Villanueva et al.,

1999]. Fig. 4 shows the protein solubility profiles of

enzymatically and/or heat-induced modified RBPI. The

solubility curves of RBPI and modified RBPI evidenced

a typical ‘V’ pattern and the lowest solubilities at pH 4.0,

the isoelectric point of rice bran protein. The solubility

pattern was consistent with the reports of Gnanasambandam

and Hettiarachchy [1995] and Tang et al. [2003]. At

extremely acidic and alkaline pHs, proteins carry net

positive and negative charges, respectively, and thus,

electrostatic repulsion and ionic hydration promoted the

solubilization of the protein [El Nasri and El Tinay,

2007]. Autoclaving drastically increased the solubility of

RBPI at alkaline pHs [autoclaved rice bran protein isolate

(H-RBPI)]. Enzymatic hydrolysis profoundly increased

the solubility of RBPI at all pH ranges [protease-

hydrolyzed rice bran protein isolate (E-RBPI)]. The

highest improvements in RBPI solubility were detected in

the autoclaved rice bran protein isolate after protease-

hydrolysis (EH-RBPI), the RBPI autoclaved after

enzymatic hydrolysis, which reached a level of up to

97.4% at pH 10. The enzymatically modified RBPIs, E-

RBPI and EH-RBPI, evidenced significantly improved

solubility values, owing to the reduced molecular size of

the proteins. In particular, EH-RBPI evidenced solubility

in excess of 57% even at its isoelectric point, and thus,

might prove useful as a nutritional supplement in

beverages with acidic pH.

Emulsion activity and stability. The emulsion activity

and stability of RBPI, modified RBPIs, and BSA were

compared, as is shown in Fig. 5. The emulsion activities

of RBPIs increased with increases in pH, regardless of

modification (Fig. 5(A)). The emulsion activity of RBPI

was lower than that of bovine serum albumin at all pH

ranges because of the lower surface hydrophobicity of

RBP as compared with bovine serum albumin. In general,

the emulsion activity is affected principally by surface

hydrophobicity [Voutsinas et al., 1983]. The low surface

hydrophobicity of RBPI does not facilitate the interactions

between proteins and lipids [Wang et al., 1999].

However, a combined modified method involving heat

and protease treatments improved the emulsion activity

of RBPI to higher than that of bovine serum albumin at

pH values higher than 6. Although autoclaving did not, in

and of itself, increase the emulsion activity of RBPI to as

high a degree as the combined modification, it did

increase emulsion activity at all pH ranges tested. A

positive correlation between water solubility and

emulsion activity was noted, with the exception of the

low emulsion activity of the E-RBPI at pH values of 5

and 6, which may be attributable to the fact that the

increased water solubility facilitated the diffusion of RBP

and spread it rapidly at the oil/water interface [Wu et al.,

1998]. Although enzymatic hydrolysis increased the

water solubility of the RBPI at pH values of 5 and 6 (Fig.

Fig. 4. Nitrogen solubility profiles of rice bran protein

isolates (�) prepared by autoclaving (�), protease-hydrolysis (�), and autoclaving after protease-hydrolysis (�).

Fig. 3. Changes in the degree of hydrolysis of rice branprotein isolates resulting from reaction with protease.

Modified rice bran protein isolate 67

4), it did not enhance the structural flexibility of the RBP,

as did the autoclaving. The rigid structure of the E-RBPI

inhibited the rapid conformational changes at the oil/

water interface, although it diffused to the oil-water

interface. However, the rigid structure of the E-RBPI

induced a profound increase in emulsion stability at pH

values of 5 and 6 (Fig. 5(B)). With the exception of E-

RBPI at pH values of 5 and 6, neither enzymatic

hydrolysis nor autoclaving influenced the emulsion

stability as much as the emulsion activity. However, it

could also be concluded that the emulsion stability of rice

bran protein is inherently high, as the emulsion stabilities

of all of the RBPI samples were higher than that of

bovine serum albumin at all pH ranges. These results

were generally consistent with the data reported by Qi et

al. [1997] and David et al. [2009].

Foaming capacity and foam stability. The results of

foaming capacity and foam stability were described in

Fig. 6, which showed trends similar to those of the

emulsion properties. However, the effects of autoclaving

on foaming capacity were more profound than its effects

on emulsion activity (Fig. 6(A)), because the autoclaving

increased the surface hydrophobicity and flexibility of the

native proteins. Generally, the rapid protein adsorption at

the air-water interface during bubbling or whipping, the

ability to undergo rapid conformational change and

rearrangement at the air-water interface, and the resultant

rapid reduction in the surface tension are required for

high foaming capacity [Were, 1997]. The increased

surface hydrophobicity and flexibility of the RBP as the

result of autoclaving rendered possible both rapid adsorption

and conformational change. The soluble but rigid

structure of E-RBPI observed at acidic pH conditions

attenuated the foaming capacity of the RBP as it did the

emulsion activity; however, this reduction in foaming

capacity was moderate compared with the reduction in

emulsion activity, probably due to the higher surface

tension of the air-water interface.

Autoclaving and enzymatic hydrolysis reduced the

foam stability of RBPI at all pH ranges except 5 (Fig.

6(B)). These results are consistent with the report of

Fig. 6. Foaming capacity (A) and foam stability (B) of

rice bran protein isolates (�) prepared by autoclaving(�), protease-hydrolysis (�), and autoclaving afterprotease-hydrolysis (�).

Fig. 5. Emulsion activity (A) and emulsion stability (B)

of rice bran protein isolates (�) prepared byautoclaving (�), protease-hydrolysis (�), autoclavingafter protease-hydrolysis (�), and bovine serumalbumin (�).

68 Hye-Jung Yeom et al.

Bandyopadhyay et al. [2008] and Yoon et al. [2009].

Autoclaving and enzymatic hydrolysis resulted in an

unfolding of the protein structure and a reduction in the

molecular size, respectively, and thus, inhibited the

formation of thick and viscoelastic films around the air

bubbles, thus reducing foaming stability. The foaming

stability of RBPI increased between pH 5-7, but was

reduced at pH 8.0. Two macroscopic processes in foams

influence the kinetic stability of protein-stabilized foams:

the rate of liquid drainage from the lamellae, and film

rupture. The charge repulsion between the protein layers

tends to inhibit the thinning of the lamellae, the liquid

layer between bubbles. However, excessive electrostatic

repulsion between protein molecules within the layer was

shown to impair the integrity of the protein layer and thus

resulted in the breakage of the film [Yoon et al., 2009],

which reduced the foam stability of RBPI at pH 8.0.

Gel forming ability. A protein requires higher

concentration than a carbohydrate to form a gel, as

proteins harbor fewer hydroxyl groups than carbohydrates.

However, an excessively high concentration of protein

could result in its precipitation. Therefore, appropriate

protein concentration is critical to the process of protein

gelation. The minimum gelation concentrations of pea

and soybean proteins are generally approximately 10%

[Ee et al., 2009]. In this study, 5-15% protein solutions

adjusted to pH 4.0 and 7.0 were tested in order to

determine the minimum gelation concentration of the

RBPI and the modified RBPIs. However, the gelations of

all of the RBPIs failed under the tested conditions,

including the protein concentration and pH, regardless of

whether or not they were modified. Although protein

concentrations lower than 15% did not result in gel

formation, the application of higher concentrations would

be practically infeasible in industrial applications.

Therefore, an addition of 2% carrageenan to the protein

solution was attempted to enhance gel formation without

any further increases in the protein concentration of the

gel solution. The hardnesses of the gels formed with the

aid of the carrageenan are provided in Fig. 7. The

hardnesses of the gels formed at pH values of 4.0 and 7.0

were identical, thereby implying that the polymers,

carrageenan, and the protein were incompatible. If they

had been compatible, the gels formed at pH 4.0 should

have been harder than those formed at pH 7.0. As the

isoelectric points of the protein and the carrageenan were

pH 4.5 and 2.0, respectively, their net charges were

positive and negative at pH 4.0, thus resulting in the

formation of a harder gel than those formed at pH 7.0,

both of which carried net negative charges [Damodarran

and Paraf, 1997]. The gels were formed successfully with

all protein sample solutions, even at very low concentrations

(2-4%). Because the polymers, the protein, and the

carrageenan were incompatible, there were sizeable

differences in water content between the protein and

carrageenan phases. Usually, the highly concentrated

protein-rich phase achieves an equilibrium with a diluted

polysaccharide phase, leading to the concentration of a

rice bran protein phase, and thus, gelation at low protein

concentrations [Damodarran and Paraf, 1997].

The enzymatically hydrolyzed RBPIs--E-RBPI and

EH-RBPI--evidenced lower minimum concentrations of

proteins for gel formation and higher gel strengths than

the others--RBPI and H-RBPI; additionally, the

autoclaved RBPIs also exhibited higher gel strengths than

the unheated RBPIs. This might be because further

denaturations of the protein, induced by enzymatic

hydrolysis and autoclaving, promoted the hydrophobic

interactions between protein molecules, and the cleavages

of peptide bonds by enzymatic hydrolysis increased the

number of terminals carrying an electrical charge, and

thus, the opportunity to combine with water [Damodarran

and Paraf, 1997].

Fig. 7. Hardness of rice bran protein isolates (�)prepared by autoclaving (�), protease-hydrolysis (�),and autoclaving after protease-hydrolysis (�) at pH 4.0(A) and 7.0 (B).

Modified rice bran protein isolate 69

The rheological properties, including hardness,

adhesiveness, cohesiveness, and springiness, of the gels

prepared with 4 and 10% protein solutions at pH 7.0 are

provided in Table 2. Although there has been a study

reporting that gel properties are dependent on pH,

especially at neutral and alkaline pHs and protein

concentrations [El Nasri and El Tinay, 2007], the effects

of pH and the concentration of the protein solution on the

rheological properties were as profound as were observed

with the pretreatments (autoclaving and enzymatic

hydrolysis) utilized in this study. Autoclaving of RBPI

slightly increased the hardnesses of the gels, however, it

couldn’t increase the other rheological properties of the

gels. Hardness, adhesiveness, and cohesiveness of E-

RBPI and EH-RBPI were higher than those of RBPI.

Especially, the successive treatment with heat and

enzyme enhance most rheological properties of RBPI.

However, significant differences were not found in the

springinesses of RBPI, H-RBPI, E-RBPI, and EH-RBPI,

probably due to the fact that the effects of autoclaving and

enzymatic hydrolysis on the springiness of gel were

ignorable compared to the effect of carrageenan added for

gelation.

Protein concentrations in the rice protein isolates were

increased by amylase hydrolysis and ethanol-washing, by

up to 77.62%. Functional properties, such as water

solubility, emulsifying properties, foaming properties,

and gel forming ability, were also increased as the result

of autoclaving and protease-hydrolysis in this study.

However, the economic merits of these processes will

require closer examination, due to continued low protein

recovery (42.6%). In short, the economic processes

involved in the production of rice bran protein isolates to

substitute for soy protein isolates as a major product of

rice do not currently appear to be favorable. However,

were this product to be considered a byproduct (rice and

rice bran oil being the major products), the economics

become favorable and attractive.

Acknowledgments. This work was supported by the

GRRC program of Gyeonggi province [2009-0556,

Development of functional and safe materials from

defatted-bran protein in rice].

References

AOAC (2000) In Official Method of Analysis of AOAC Intl,

(17th ed.). Association of Official Analytical Communi-

ties, Gaithersburg, Maryland, U.S.A.

Bandyopadhyay K, Misra G, and Ghosh S (2008) Prepara-

tion and characterisation of protein hydrolysates from

indian defatted rice bran meal. J Oleo Sci 57, 47-52.

Bera MB and Mukherjee RK (1989) Solubility, emulsify-

ing, and foaming properties of rice bran protein concen-

trates. J Food Sci 54, 142-145.

Betschart AA, Fong RY, and Saunders RM (1977) Rice by-

products: Comparative extraction and precipitation of

nitrogen from U.S. and Spanish bran and germ. J Food

Sci 42, 1088-1093.

Coffmann CW and Garcia VV (1977) Functional properties

and amino acid content of a protein isolate from mung

bean flour. J Food Technol 12, 473-484.

Connor MA, Saunders RM, and Kohler GO (1976) Rice

bran protein concentrates obtained by wet alkaline

extraction. Cereal Chem 53, 488-496.

Damodarran S and Paraf A (1997) Food proteins and their

Table 2. Rheological properties of gel produced with 4 and 10% rice protein solutions via the addition of 2%carrageenan at pH 7.0

Rice branprotein isolate

Protein concentration in gel

Rheological properties of gels

Hardness (gf) Adhesiveness (g.m) Cohesiveness (g.m) Springiness (mm)

RBPI

4%

22.12±1.45 1.01±0.16 08.31±0.65 4.89±0.77

H-RBPI 24.77±1.60 0.96±0.19 08.72±1.23 6.08±0.15

E-RBPI 29.51±1.34** 1.98±0.32* 09.79±0.76* 5.96±0.36

EH-RBPI 33.97±2.02** 2.42±0.29** 16.66±1.08*** 5.30±0.74

RBPI

10%

24.61±1.25 1.12±0.20 10.60±1.19 5.78±0.66

H-RBPI 27.87±1.02* 1.74±0.18* 09.03±1.49 4.93±0.43

E-RBPI 36.19±2.31** 2.38±0.26** 12.83±1.10* 5.01±0.82

EH-RBPI 41.35±1.74*** 3.58±0.35*** 18.44±2.14** 5.45±0.51

RBPI: rice bran protein isolate

H-RBPI: autoclaved rice bran protein isolate

E-RBPI: protease-hydrolyzed rice bran protein isolate

EH-RBPI: autoclaved rice bran protein isolate after protease-hydrolysis

Reported values of each property are means of three replications±standard deviation.

*, **, and ***, significant differences at p<0.05, p<0.01, and p<0.001, respectively.

70 Hye-Jung Yeom et al.

applications. In Protein Gelation, pp.125-190. Marcel

Dekker Inc., New York, NY, U.S.A.

David BA, Rogelio MR, Alma CC, Arturo CR, Ma Euge-

nia JF, and Luis CG (2009) Functional properties of

hydrolysates from Phaseolus lunatus seeds. J Food Sci

Technol 44, 128-137.

Doucet D, Otter DE, Gauthier SF, and Foegeding EA

(2003) Enzyme-induced gelation of extensively hydro-

lyzed whey proteins by alcalase: peptide identification

and determination of enzyme specificity. J Agric Food

Chem 51, 6300-6308.

Ee KY, Rehman A, Agboola S, and Zhao J (2009) Influ-

ence of heat processing on functional properties of Aus-

tralian wattle seed (Acacia victoriae Bentham) extracts.

Food Hydrocolloids 23, 116-124.

El Nasri NA and El Tinay AH (2007) Functional proper-

ties of fenugreek (Trigonella foenum graecum) protein

concentrate. Food Chem 103, 582-589.

Gnanasambandam R and Hettiarachchy NS (1995) Protein

concentrates from nonheat-stabilized rice bran: Prepara-

tion and properties. J Food Sci 60, 1066-1069.

Hamada JS (1997) Characterization of protein fractions of

rice bran to devise effective methods of protein solubili-

zation. Cereal Chem 74, 662-668.

Helm RM and Burks AW (1996) Hypoallergenicity of rice

bran protein. Cereal Foods World 41, 839-843.

Humiski L and Aluko RE (2007) Physicochemical and bit-

terness properties of enzymatic pea protein hydrolysates.

J Food Sci 72, 605-611.

Kato A, Takahashi A, Matsudomi N, and Kobayashi K

(1983) Determination of foaming properties of proteins

by conductivity measurement. J Food Sci 48, 62-65.

Khuwijitjaru P, Nualchan P, and Adachi S (2007) Foaming

and emulsifying properties of rice bran extracts obtained

by subcritical water treatment. Food Sci Technol Res 13,

205-209.

Kristinsson HG and Rasco BA (2000) Biochemical and

functional properties of atlantic salmon (Salmo salar)

muscle proteins hydrolyzed with various alkaline pro-

teases. J Agric Food Chem 48, 657-666.

National Agricultural Products Quality Management Service

(2008) In Statistical Survey of Crops. Ministry of Food

Agriculture, Forestry and Fisheries, Seoul, Korea.

Pearce KN and Kinsella JE (1978) Emulsifying properties of

proteins: Evaluation of a turbidimetric technique. J Agric

Food Chem 26, 716-723.

Pedersen B (1994) Removing bitterness from protein

hydrolysates. Food Technol 48, 96-99.

Qi M, Hettiarachchy NS, and Kalapathy U (1997) Solubil-

ity and emulsifying properties of soy protein isolates

modified by pancreatin. J Food Sci 62, 1110-1115.

Saunders RM (1990) The properties of rice bran as a food

stuff. Cereal Foods World 35, 632-662.

Sereewatthanawut I, Prapintip S, Watchiraruji K, Goto M,

Sasaki M, and Shotipruk A (2008) Extraction of protein

and amino acids from deoiled rice bran by subcritical

water hydrolysis. Bioresource Technol 99, 555-561.

Shanhu T, Navam S, Hettiarachchy, Thomas H, and Shell-

hammer (2002) Protein extraction from heat-stabilized

defatted rice bran. 1. Physical processing and enzyme

treatments. J Agric Food Chem 50, 7444-7448.

Shih FF, Champagne ET, Daigle K, and Zarins Z (1999)

Use of enzymes in the processing of protein products

from rice bran and rice flour. Nahrung 43, 14-18.

Tang S, Hettiarachchy NS, Horax R, and Eswaranandam S

(2003) Physicochemical properties and functionality of

rice bran protein hydrolyzate prepared from heat-stabi-

lized defatted rice bran with the aid of enzymes. J Food

Sci 68, 152-157.

Tardioli PW, Pedroche J, Giordano RLC, Fernandez-Lafu-

ente R, and Guisan JM (2003) Hydrolysis of proteins by

immobilizedstabilized Alcalase–glyoxyl agarose. Biotech-

nol Progr 19, 352-360.

Villanueva A, Vioque J, Sanchez-Vioque R, Clemente A,

Pedroche J, Bautista J, and Millan F (1999) Peptide

characteristics of sunflower protein hydrolysates. J Am

Oil Chem Soc 76, 1455-1460.

Voutsinas LP, Voutsina P, Cheung E, and Nakai S (1983)

Relationship of hydrophobicity to emulsifying properties

of heat denatured proteins. J Food Sci 48, 26-32.

Wang M, Hettiarachchy NS, Qi M, Burks W, and Sieben-

morgen T (1999) Preparation and functional properties

of rice bran protein isolate. J Agric Food Chem 47, 411-

416.

Were L, Hettiarachchy NS, and Kalapathy U (1997) Modi-

fied soy proteins with improved foaming and water

hydration properties. J Food Sci 62, 821-823.

Wu WU, Hettiarachchy NS, and Qi M (1998) Hydrophobic-

ity, solubility, and emulsifying properties of soy protein

peptides prepared by papain modification and ultrafiltra-

tion. J Am Oil Chem Soc 75, 845-850.

Yoon JH, Jung DC, Lee EH, Kang YS, Lee SY, Park SR,

Yeom HJ, Ha MS, Park SK, Lee YS, Ha SD, Kim GH,

and Bae DH (2009) Characteristics of a black soybean

(Glycine max L. Merrill) protein isolate partially hydro-

lyzed by alcalase. Korean J Food Sci Technol 18, 488-

493.