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Journal of Oleo ScienceCopyright ©2014 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess14030J. Oleo Sci. 63, (12) 1231-1241 (2014)

Effects of Rice Bran Protein Hydrolysates on the Physicochemical Stability of Oil-in-Water EmulsionsNopparat Cheetangdee＊

Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

1 INTRODUCTIONMany food products exist in a form of oil-in-water（O/W）

emulsions, e.g. milk, salad dressing, infant formula, etc. Im-portant factors determining the quality and shelf-life of the products include stability against phase separation and lipid oxidation. To maintain this physicochemical stability, an emulsifier plays important roles in facilitating emulsion formation and in forming interfacial films to act as a barrier against drop aggregation1） and pro-oxidant mobilization to non-polar phase2, 3）. Using effective emulsifiers may ensure the stability and quality of emulsion products. Recently there has been growing interest in employing natural ingre-dients in food products due to concerns about the health hazards of synthetic agents4）. Protein hydrolysates are promising candidate to be used as natural emulsifiers because of their safety, biodegradability, and producibility from renewable sources. Several recent studies have re-ported on the potent emulsifying activities of various protein hydrolysates, such as soy5）, and potato6） protein hydrolysates. Considering on the effects of protein hydro-lysates on oxidative stability of emulsion model, however, the data are still limited.

Rice bran is a by-product of rice milling, which is one of the most important agricultural industries in Thailand. It is generally used for low-value purposes, such as fertilizer

＊Correspondence to: Nopparat Cheetangdee, Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, ThailandE-mail: nopparat.ch@psu.ac.thAccepted August 11, 2014 (received for review February 17, 2014)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

and fuel. Rice bran contains sufficient protein amount（more than 10％）, with a good profile of essential amino acids7）. Some bioactivities of rice bran proteins, e.g. anti-carcinogenic capability, antiallergic property, and blood cholesterol reducibility, have been reported8）. Because of their functional and bioactivities, rice bran proteins may be useful for food application. Rice bran utilization, moreover, is part of a strategy of gaining value from agricultural waste.

To isolate proteins from rice bran, alkaline extraction（ca. ≥ pH 10）is conventionally conducted. Extremely basic con-dition, however, might affect protein structure and increase the liberation of other components, resulting in impaired functional properties of the derived proteins, e.g. solubility, foamability, and emulsifying ability9）. Alkaline extraction, moreover, can induce the formation of lysinoalanine, which poses a risk to human health10）. To overcome these issues, ex-traction with the aid of carbohydrases has been studied9－11）. Proteins are naturally present in the plant cellular matrix along with other components, especially cell wall structural carbohydrates10, 12）. Carbohydrases, therefore, could facili-tate protein recovery from plant cells by destroying the chemical bonds between carbohydrate residues10－12）. However, differences in plant sources and carbohydrases employed can affect extractability and properties of the

Abstract: Isolation of proteins from rice bran was studied, comparing alkaline- and carbohydrase-aided extraction. It was found that protein extractability could be effectively improved using carbohydrases (Viscozyme L and a-amylase), especially when mechanical force was incorporated. Then, rice bran protein hydrolysates (RBPH) were prepared at various degrees of hydrolysis (DH), and employed to stabilize soybean O/W emulsion. Improved colloidal stability of the emulsions could be achieved using RBPH, especially at higher DH level, as indicated by an increase in the emulsifying activity index and better long-term dispersibility. The present work novelty suggested the efficiency of RBPH to improve oxidative stability of the emulsions. The most potent antioxidant activity was exhibited by RBPH with DH of 6.4 and 7.6%. With their efficiency to promote physicochemical stability of the emulsions, RBPH might be potently employed as a natural additive in emulsified food products, which is significant to value addition of rice bran for further industrial application.

Key words: antioxidant activity, emulsifying activity, oil-in-water (O/W) emulsion, rice bran protein hydolysates (RBPH)

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derived proteins10－12）.In the present work, carbohydrase-assisted extraction

was studied compared with conventional means in order to improve protein extractability from rice bran. To accom-plish this goal, the effect of mechanical force on protein extractability was also reported. Then, improvement of the functional properties of the isolated proteins was investi-gated by preparing rice bran protein hydrolysates（RBPH）. It has been reported that protease aided hydrolysis could successfully improve solubility, emulsifying ability13）, and antioxidant activity14） of RBPH. However, the efficiency of RBPH to improve chemical stability of emulsion model has not been elucidated. Emulsion is a heterogeneous oil con-taining system which is prone to lipid oxidation since a presence of large interfacial areas15）. In this work, RBPH with different degrees of hydrolysis（DH）were prepared, and their abilities to retard both of phase separation and lipid oxidation in soybean O/W emulsions were observed. RBPH with rather low DH（less than 10％）were studied in the present work because of the previously reported disad-vantages of extensively hydrolyzed peptides, including bitter taste as well as inferior emulsifying, foaming, and an-tioxidant activities5, 16）.

2 EXPERIMENTAL PROCEDURES2.1 Materials

Defatted bran of rice（Oryza sativa L.）was supplied by Thai Edible Oil Co., Bangkok. The bran was kept in a poly-ethylene bag at 4℃ for less than 3 months before use. Refined soybean oil without exogenous antioxidants was provided by Lamsoon（Thailand）Public Company Limited. The enzymes, i .e. Viscozyme L from Aspergillus sp.（density of 1.2 g/mL）; a-amylase from A. oryzae with an activity of ca. 1.5 U/mg（1 U corresponds to the amount of enzyme that liberates 1 μmol maltose per min at pH 6.0 and 25℃, using starch as a substrate）; and protease from Bacillus licheniformis with an activity of ≥ 5 U/g（1 U cor-responds to the amount of enzyme that sets free 1 μmol Folin-positive amino acids and peptide（as tyrosine）per min at pH 7.0 and 37℃, using casein as a substrate）were purchased from Sigma-Aldrich（St. Louis, MO, USA）. Coo-massie Brilliant Blue G（Sigma-Aldrich）was used to deter-mine protein solubility. 2,4,6-trinitrobenzene sulfonic acid（TNBS）, 1-anilino-8-naphthalene sulfonate（ANS）, and thiobarbituric acid（TBA）were brought from Sigma-Aldrich（St. Louis, MO, USA）. Trichloroacetic acid was purchased

from Carlo Erba Reagenti（Rodano, Italy）. NaHPO4.2H2O, NaH2PO4.2H2O, and NaOH were procured from QRëC（Auckland, New Zealand）. HCl, isooctane, 2-propanol, methanol, and 1-butanol were products of J.T. Baker（De-venter, Netherlands）. All chemicals were analytical grade.

2.2 Preparation of rice bran protein isolates（RBPI）Firstly, the chemical composition of the defatted rice

bran was quantified by the AOAC standard method17）. To isolate protein from the bran, two methods, i.e. alkaline- and enzymatic-assisted extraction, were conducted with the presence/absence of sonication.

Alkaline-assisted extraction: Alkaline-assisted extrac-tion was conducted following the method of Paraman et al.9） with a slight modification. Rice bran and deionized（DI）water（pH 11）were mixed at room temperature for 1 h in a ratio of 1:7.5（w/w, rice bran:DI water）. A small aliquot of NaOH or HCl（0.1N）was used to re-adjust pH of the mixture to 11, before heating at 45±1℃ for 0–3 h with（sonicator: 40 Hz, Transsonic 460/H; Elma, Singen, Germany）or without sonication at 40 Hz（water bath: Memmert, Schwabach, Germany）.

Enzyme-assisted extraction: For enzyme-assisted ex-traction, rice bran and DI water were mixed at the above-mentioned ratio, and the pH of the mixture was adjusted to optimum conditions（pH 4.1 and 6.25 for Viscozyme L and a-amylase, respectively）18）. Enzymes were then introduced at a concentration of 0.5％ by weight of the bran, followed by heating at 45±1℃ for 0–3 h with or without sonication. After that, pH of the mixtures was elevated to pH 11 to terminate the enzyme reaction.

After completing the extraction, the mixtures were cen-trifuged at 3500 g for 10 min at 25℃（CR 22GIII high-speed refrigerated centrifuge; Hitachi, Tokyo, Japan）. The super-natant was collected and adjusted to pH 4, which is the isoelectric point of RBPI11）, using 1 N HCl. The precipitated proteins were washed with DI water, neutralized to pH 7 by 1 N NaOH, and then freeze-dried. Concentrations of isolat-ed proteins were determined by Bradford assay19）, follow-ing the method of Xie et al.20） Aqueous RBPI solutions（0.1％ w/v）were prepared using 10 mM glycine buffer, pH 11（basic pH provides the best solubility of RBPI）11）, then stirred at room temperature for 1 h and centrifuged at 4350 g for 20 min. The supernatant（100 μL）was separated to react with Coomassie Blue reagent（5 mL）. The mixtures were incubated at room temperature for 30 min before ob-serving the absorbance at 595 nm（Libra S22; Biochrom, Cambridge, UK）. The nitrogen content of the samples was calculated using the standard curve of bovine serum albumin（0–1 g/L in 10 mM phosphate buffer, pH 7）, and the protein contents of RBPI were calculated according to the following equation:

Extraction yield（％）＝

weight（g）of RBPI・protein content（％）of RBPI

weight of rice bran（g, dry basis）・ protein content（％）of rice bran

2.3 Preparation of rice bran protein hydrolysates（RBPH）RBPI and DI water（pH 9）were mixed in a ratio of 1:10

（w/w, RBPI:DI water）at room temperature for 1 h, then the

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protease was applied at a concentration of 0.5％ by weight of the RBPI. The reaction proceeded at 60℃ for 0, 15, 30, 45, and 60 min. The mixtures were heated at 90℃ for 5 min to terminate enzyme activity, and immediately cooled in an ice bath. The suspensions were neutralized, freeze-dried, and stored at 4℃. Degree of hydrolysis（DH）was de-termined using TNBS21）, following the modified method of Benjakul and Morrissey22）, and the DH was calculated by the following equation:

DH（％）＝［（Lt－Lo）/（Lmax－Lo）］・100

where Lt is the amount of a-amino acids released at time t, Lo the amount of a-amino acids in the original sample, and Lmax the total a-amino acids in the sample after complete hydrolysis with 6 N HCl at 100℃ for 24 h.

2.4 Solubility of rice bran proteinSolubility of RBPI and RBPH was estimated by preparing

aqueous protein solutions（0.5％, w/v）at the designated pH using 10 mM buffer solutions. The pH of the sample solu-tions was re-adjusted to the required values with a small aliquot of 0.1 N HCl or NaOH and mixed at room tempera-ture for 1 h. Samples were then centrifuged at 8000 g for 20 min. The protein concentration in the supernatant was determined by Bradford assay19）. Protein solubility was ex-pressed as the percentage of protein in the supernatant to the total protein content of the corresponding sample.

2.5 Electrophoresis of rice bran proteinSodium dodecyl sulfate polyacrylamide gel electrophore-

sis（SDS-PAGE）was used to examine the molecular size of rice bran proteins, according to the procedure of Laemmli23）. SDS-PAGE was carried out using a slab gel（4％ stacking gel, 12％ separating gel）in Tris/glycine/SDS dis-continuous buffer system. The protein samples（5 μg protein/μL）were prepared in both reducing and non-re-ducing buffer solutions（0.0625 M Tris-HCl, pH 6.8; 2％ SDS; 10％ glycerol; and 0.05％ bromophenol blue with/without 2-mercaptoethanol）. Five μL of the each protein solution was loaded onto the gel. Electrophoresis was per-formed at a constant current of 15 mA for approximately 1.5 h. The gel was stained with 0.1％ Coomassie Brilliant Blue in acetic acid/ethanol/water solution（10/40/50, v/v/v）and destained in the same solvent without the dye. The ap-proximate molecular weights of rice bran proteins were de-termined using molecular weight standard markers（Sigma-Aldrich）; the molecular weights ranged from 6.5 to 66 kDa.

2.6 Surface hydrophobicity（S0）determinationHydrophobicity of RBPH was determined through hydro-

phobic fluorescence measurement, following the method of Hayakawa and Nakai24）. A series of protein solutions was prepared at concentrations ranging from 0 to 0.02％（w/v）in 10 mM phosphate buffer, pH 7. The protein solution（4

mL）was reacted with 8 mM ANS solution（10 μL）, and the fluorescence intensities were read using a fluorescence spectrophotometer（RF-1501 spectrofluorophotometer; Shimadzu, Kyoto, Japan）at 390 and 484 nm excitation and emission wavelengths, respectively. S0 was determined as the slope of a linear regression between fluorescence in-tensity and protein concentration.

2.7 Emulsifying activities of RBPHTo observe the emulsifying activities of RBPH, soybean

O/W emulsion was used as a model. Soybean oil and RBPH solutions（1％, w/v in 10 mM phosphate buffer pH 7 with 0.02％, w/v NaN3）were homogenized（T 25 Ultra-Turrax®; Ika, Staufen, Germany）at 19,000 rpm for 2 min. The oil volume fraction was 0.2. Efficiency of the peptides to facili-tate emulsion formation was examined by measuring emul-sifying activity index（EAI）25）. For light scattering by dis-persed spherical particle, there is a relationship between interfacial areas and turbidity of emulsion prepared under a circumstance emulsification process, and turbidity（T）is provided by: 26）

T＝ 2.303・A500

φwhere A500 is the absorbance at 500 nm, φ is the optical path length（m）. For a spherical particle dispersing system, Mie theory mentioned that:

Total interfacial areas＝2T＝ 2・2.303・A500

φEAI is the factor indicating interfacial areas that can be covered by a weight of proteins, so it can be calculated as a following:

EAI（m2/g）＝ 2・2.303・A500

c・φ・（1－θ）where c the concentration of RBPH（g/m3）and θ the frac-tion of oil in the initial emulsion.

To determine EAI, the freshly prepared emulsion was diluted with SDS solution（0.1％, w/v）, and A500 was mea-sured following the procedure described by Pearce and Kinsella25）.

Oil droplet diameter was measured by an optical micro-scope（Labophot; Nikon, Tokyo, Japan）, and Sauter mean diameter（D32）was estimated from 12 views of each sample. Samples were independently diluted to optimum oil content. D32 was calculated using ImageJ software accord-ing to the equation:

D32＝Σnidi

3

Σnidi2

where ni is the number of oil droplets with diameter di

（μm）.To investigate emulsion dispersibility, turbidity loss rate

was measured following the method of Buffo et al.27） with a slight modification. The emulsions were diluted with SDS solution（0.1％, w/v）to an oil volume fraction of 0.0025, and kept at room temperature. The transmittance was observed

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at 500 nm periodically over 2 weeks. For each sample, the transmittance at different storage times was plotted by fitting a first-order model. The slope of the correlation in absolute value represented the turbidity loss rate（∆T）. Higher ∆T corresponds to a faster change in the degree of drop dispersion, indicating an unstable emulsion, and vice versa.

2.8 Oxidative stability of RBPH-added emulsionsSoybean emulsion was prepared using Tween 20 as an

emulsifier to ensure colloidal stability during storage. Soybean oil and Tween 20 solution（0.5％, w/v dissolved in 10 mM phosphate buffer pH 7 with 0.02％, w/v NaN3）were homogenized at 19,000 rpm for 2 min. The oil volume frac-tion of the initial emulsion was 0.2. The emulsion was diluted with RBPH solution（1％, w/v dissolved in 10 mM phosphate buffer pH 7 with 0.02％, w/v NaN3）at each DH level, and then homogenized for further 1 min. The final emulsions contained an oil volume fraction of 0.1 and RBPH 0.5％, w/v. The emulsions were stored in screw-capped bottles at 37℃ in the dark. The progression of lipid oxidation was monitored by measuring peroxide value（PV）and thiobarbituric acid reactive substances（TBARs）content over a period of 2 weeks.

PV was quantified according to the procedure of Hu et al.28）. Each emulsion sample（0.3 mL）was added with isooctane:2-propanol（3:1, 1.5 mL）before thoroughly mixing and centrifuging at 4000 g for 2 min. Two hundred μL of the solvent phase was taken out to react with 2.8 mL of methanol:1-butanol（2:1）, 15 μL of 3.97 M ammonium thiocyanate, and 15 μL of ferrous iron solution（composed of 0.132 M BaCl2 and 0.144 M FeSO4.7H2O）. After incubat-ing at room temperature for 20 min, the absorbance at 510 nm was observed. PV was estimated using cumene hydro-peroxide as a standard, and reported as mg Hydroperoxide equivalent/L of sample.

TBARs content was estimated following the method of Aewsiri et al.29） with a slight modification. Thiobarbituric solution was prepared by mixing 0.375％ TBA, 15％ tri-chloroacetic acid, and 0.25 N HCl. The emulsion（1 mL）was reacted with the thiobarbituric solution（5 mL）and heated at 100℃ for 10 min. After cooling to room temperature, the mixture was centrifuged at 8000 g for 5 min and the supernatant was then removed to observe the absorbance at 532 nm. TBARs content was estimated using malonalde-hyde（MDA）as a standard and reported as mg MDA equiva-lent/L of sample.

2.9 Statistical analysisAll experiments were carried out in triplicate, and the

mean values with standard deviations were calculated. Sta-tistical analysis of the data was performed by analysis of variance（ANOVA）using Duncan’s multiple range test（SPSS for Windows, SPSS Inc., Chicago, IL, USA）at a 95％

confidence level.

3 RESULTS AND DISCUSSION3.1 Preparation and characterization of rice bran protein

isolates（RBPI）Chemical composition of the defatted rice bran was

firstly quantified. Protein and fat contents of the bran were ca. 15 and 1％, respectively. The protein content in defat-ted rice bran was reported to be 12–20％7） and 15.4％13）. These data suggest that defatted rice bran could be a po-tential source of protein.

To isolate proteins from the bran, two methods – alkaline（pH 11）and enzymatic（Viscozyme L or a-amylase）extrac-tion, with the presence/absence of mechanical force – were carried out at different extraction times. Figure 1 shows

Fig. 1　�The protein extraction yield of RBPI prepared by (a) alkaline method, or with the aid of (b) Viscozyme L, and (c) a-amylase, with (closed symbols) or without (open symbols) sonication. The same letter indicates an insignificant differ-ence between the means (n=3, p<0.05) for each extraction condition.

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the extracted protein yields of RBPI derived under differ-ent conditions. Generally, protein content tended to in-crease with extraction time. The maximum yields obtained by alkaline（180 min）, Viscozyme L（60 min）, and a-amylase（60 min）extraction were ca. 25, 30, and 35％, respectively. This result suggested the superior ability of carbohydrases to recover protein from plant cells, compared with the al-kaline method. This was expected, given the ability of car-bohydrases to hydrolyze polysaccharide composites in plant cell walls10）. For the defatted rice bran used in the present work, it was treated by heating to inactivate lipoxy-genase during oil extraction process. This affected to stiff protein extractability since heating affected to enhance de-naturation and interaction of protein with other compo-nents18）. In plant cells, proteins were always bound with starch granules7） and other polysaccharide residues which were mainly cellulose, hemicelluloses, and xylose for rice bran12）. By using a-amylase, protein extraction could be promoted by hydrolyzing a-1,4-glycosidic linkage to release starch bound proteins7）. Tang et al.18）, also reported a potent ability of a-amylase to recover proteins from plant cellular matrix13）. Considering Viscozyme-L, it is a cocktail enzyme consisting of arabinase, cellulase, β-glucosidase, hemicellulase, and xylanase, so it might effectively enhance protein liberation from the matrix7, 18）.

Regarding the influence of mechanical force, sonication could facilitate protein extractability. After extraction for 1 h, the protein yield was raised from ca. 25 to 30％ with Vis-cozyme L, and from ca. 30 to 35％ with a-amylase, when sonication was incorporated. This result is in accordance with previous findings of improved protein extraction from plant cells with the application of other mechanical devices, e.g. high-pressure homogenizer7） and colloid mill30）. Mechanical force could facilitate cell breakage30） and cleaving of chemical bonds between molecular species in plant cellular matrix7）, thus enhancing protein extractabili-ty. As shown in Fig. 1, extraction using either Viscozyme L or a-amylase for 1 h with the presence of sonication was more effective to isolate protein from rice bran, as suggest-ed by the higher yields, compared with the conventional method（p＜0.05）. Next, protein extractability was im-proved by combining these conditions（extraction with Vis-cozyme L for 1 h, and subsequently with a-amylase for another 1 h, with sonication）. It was found that protein yield could be significantly increased（p＜0.05）, by up to ca. 50％（data not shown）. Therefore, this dual-enzyme ex-traction method was employed to prepare RBPI in the fol-lowing experiments.

Next, alkaline and enzymatic extractions providing the highest yield（i.e. extraction for 180 min with sonication for the former and dual-enzyme extraction with sonication for the latter）were conducted to prepare RBPI, and their properties were then characterized. Figure 2 illustrates the SDS-PAGE pattern of RBPI. There was no difference in the

molecular weight（MW）distribution of RBPI extracted by alkaline and enzymatic processes. The MW of the RBPI was within a range of 6.5–66 kDa, with distinct bands at ca. 6.5 and 33 kDa. The MW of RBPI prepared by alkaline extrac-tion was previously reported to be 6–97.4 kDa, with a pre-dominant band at 33 kDa representing a major rice bran protein, glutelin13, 30）. Tang et al.7） observed rice glutelin, globulin, and albumin with a MW range of 6.5–66.2 kDa in rice bran protein concentrate. In comparing reduced and non–reduced conditions, bands at MW of 24–29 kDa were observed only under reduced conditions, suggesting that some rice protein subunits interact through disulfide bonds13）.

Solubility of RBPI was measured at various pH, as shown in Fig. 3. The minimum solubility was found at pH 4, sug-gesting that the isoelectric point（IEP）of RBPI is around pH 4, which is in good agreement with the report of Wang et al.11）. U–shaped characteristics, and lowest solubility at around the IEP, have been confirmed in proteins isolated from various plants, e.g. soy5）, bitter melon seed31）, ungbean, black bean, and bambara groundnut32）. The IEP of most plant proteins is reportedly in the range of pH

Fig. 2　�SDS-PAGE pattern of RBPI: Lane 1, molecular weight marker; Lanes 2 and 3, RBPI prepared by alkaline extraction in non-reducing and re-ducing conditions, respectively; Lanes 4 and 5, RBPI prepared by enzymatic extraction in non-reducing and reducing conditions, respectively.

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4–531, 32）. In this work, there was no significant difference in solubility between alkaline– and enzymatic–extracted RBPI. It was suggested that carbohydrases only facilitated the degradation of polysaccharide composites in plant cell walls, but had no effect on the solubility of isolated pro-teins11）. However, Paraman et al.9） reported lower solubility of rice endosperm proteins liberated by the use of carbohy-drases than did the alkaline extraction; they postulated that this was due to a higher level of protein denaturation and greater loss of water–soluble protein fractions（albumin）upon enzymatic treatment.

In the present work, the difference on the properties of RBPI derived by alkaline and enzyme-aided extraction was not significant. Because of its higher isolated protein yield, enzymatic extraction was used to prepare RBPI for next study.

3.2 Preparation and characterization of rice bran protein hydrolysates（RBPH）

RBPH were prepared by hydrolyzing RBPI with the pro-tease at 60℃ for various times（0–60 min）. Table 1 shows the characteristics of RBPH prepared at various hydrolytic times, including DH, S0, and solubility.

As shown in Table 1, DH increased with hydrolytic time（p＜0.05）, a finding that is in accordance with prior studies5, 12, 20, 29）. In this work, RBPH with the highest DH of ca. 10％ could be prepared by hydrolyzing for 60 min. Re-garding the hydrophobicity, an increase in S0 with hydro-lytic time was observed; the highest value of ca. 155 was obtained after 45 min. Increased S0 with prolonged hydro-lytic time was due to the exposure of hydrophobic amino acids, originally buried in the core of the native state, as a result of partial denaturation5, 9）. An increase in S0 with higher DH was also observed for preparations of soy5） and rice bran7） protein hydrolysates. The hydrolysis process for at least 30 min could significantly elevate the solubility of RBPH（p＜0.05）. After hydrolyzing for 60 min, the solubili-ty was raised from ca. 20 to 52％. Noted that slightly lower solubility of the RBPH prepared by the aid of pectinase and protease P was reported with the value less than 50％16）. Increased solubility with DH levels could be expected by decreased molecular size of peptides as a result of molecu-lar bond breaking during hydrolysis treatment7）. In the present work, a diminishing in MW of the peptides at higher hydrolysis level was confirmed by SDS-PAGE pat-terns. One of the major bands at ca. 33 kDa of RBPI gradu-ally faded away, whereas the band at ca. 15 kDa, became more distinct when hydrolysis time, or in turn DH, was in-creased（data not shown）.

3.3 Emulsifying activities of RBPH To observe the emulsifying activities of RBPH, some col-

loidal stability parameters, i.e. EAI, ∆T, and D32 of the emulsions stabilized by RBPH at different DHs（0–10％）

Fig. 3　�Solubility of RBPI prepared by alkaline (●) and enzymatic (□) extraction at various pH.

Table 1　Characteristics of RBPH prepared at various hydrolytic times.

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were measured. Figure 4 depicts the EAI and ∆T of the emulsions stabilized by RBPH at various concentrations（0.5, 0.75, and 1％, w/v）. Generally, EAI tended to increase with increasing DH suggesting improved emulsion form-ability of the more hydrolyzed peptides. This behavior could relate to developed S0 and solubility at higher DH levels5, 18）. Increased S0 enhanced protein adsorption on oil drop surfaces, resulting in improved EAI5）. Increased solu-bility, moreover, effected to facilitate diffusion and spread-ing of the proteins at interfaces, thus achieving evolved EAI8）. By adding RBPH at a level of 0.5％, the EAI for 10％ DH-RBPH emulsions was significantly lower than the EAI for emulsions stabilized by 7.6％ DH-RBPH（p＜0.05）. At other RBPH concentrations（0.75 and 1％）, however, this behavior was not observed. Qi et al.5） also reported the reduced EAI of soy protein hydrolysate-based emulsions when DH was higher than 15％. For casein hydrolysates,

the effective DH for increasing the EAI was less than 8％16）. In these cases, the inferior EAI was explained by impaired interfacial adsorption of the peptides, because of imbalance in the hydrophilic–lipophilic ratio of the excessively hydro-lyzed peptides5）. By increasing concentration, however, added amount of the peptides might compensate for the adsorption, so higher EAI was observed. From Fig. 4a, moreover, a reduction of EAI with increased RBPH concen-tration was evident; the EAI for 7.6％ DH-RBPH based emulsions was ca. 0.7, 0.6, and 0.5 m2/g when RBPH con-centrations were 0.5, 0.75, and 1％, respectively. EAI implies how fast proteins are adsorbed to interfacial areas33）. A lower amount of emulsifier might allow more configuration changes of the peptides and lead to faster adsorption to interfacial areas under fixed emulsification circumstances（i.e. the same oil fraction and homogenizing conditions as in this work）. More pronounced protein ad-sorption was found for less-structural proteins（e.g. flexible coil of β-casein）than for rigid ones（e.g. compact globules of β-lactoglobulin and bovine serum albumin）, because of easier configuration changes during an emulsification process34）. Next, the transmission change over a period of 10 d was observed, in order to elucidate long-term dispers-ibility of the emulsions（Fig. 4b）. Lower ∆T indicated a slower change in turbidity, implying better colloidal dis-persibility of emulsion. A decline in ∆T was observed as DH increased, implying that the emulsifying activities of RBPH were related to DH level. Considering the effect of concen-tration, ∆T tended to decrease with increased RBPH con-centration: ∆T of the emulsions stabilized by RBPH at con-centrations of 0.5％ and 1％ at a DH of 6.4％ were ca. 0.08 and 0.06, respectively, and ca. 0.07 and 0.05 at a DH of 10％. Improved colloidal stability of emulsions with in-creased emulsifier concentration has been confirmed by previous studies1）.

The effect of DH on emulsion droplet size was deter-mined for various storage times（0–10 d）, as illustrated in Fig. 5. For the emulsion containing non-hydrolyzed（0％ DH）peptides, droplet sizes were larger with longer storage time: D32 increased from ca. 18 to 26 μm after 10 d. For the hydrolyzed peptides, on the other hands, D32 changed only slightly（less than 3 μm）through 10 d of storage, except for the 5.6％ DH-RBPH emulsion whose D32 increased from ca. 18 to 23 μm. This result indicated that RBPH with optimal DH could successfully improve the physical stability of the emulsions.

Referring from these results, improvement of colloidal stability of the emulsions could be accomplished by em-ploying RBPH as an emulsifier, and DH played a crucial role for this purpose. Generally, better abilities of RBPH were evident at higher DH levels, suggesting the impor-tance of improved solubility and S0 on the emulsifying ac-tivity of the peptides. Noted that RBPH prepared in the present work exhibited higher S0 than other hydrolysates:

Fig. 4　�(a) Emulsifying activity index (EAI) and (b) Transmission loss rate (∆T) of soybean O/W emulsions stabilized by RBPH at various de-grees of hydrolysis (DH). The emulsions were stabilized by RBPH at different concentrations: 0.5, 0.75, and 1%, w/v. The different letter indi-cates a significant difference between the means (n=3, p<0.05), by the capital and small letters denote the effects of DH and RBPH concentra-tion, respectively.

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S0 of soy protein hydrolysates prepared by papain for 60 min was less than 3035）, S0 of alcalase treated peanut pro-teins with DH of 5.4％ was ca. 10036）, and S0 of grass carp（Ctenopharyngodon idellus）protein hydrolysates pre-pared by papain（alcalase）with DH of 10％ was ca. 120（132）37）. To confirm the importance of S0 on emulsifying properties of RBPH, the relationships between S0 and EAI, ∆T, and D32 of the RBPH stabilized emulsions were plotted as shown in Fig. 6. It was suggested that improved emulsi-fying activity of the hydrolyzed peptides was related to the increased S0, as indicated by a fit linear relationship between a reduction of D32 and ∆T as well as increment of EAI with increased S0 level. To maintain physical stability of emulsions, emulsifiers must: i）be adsorbed to interfacial areas, where they lower interfacial tension and facilitate

drop formation, and ii）form barrier films around oil drop-lets to retard drop aggregation via electrostatic and steric mechanisms1, 38） Increased solubility and S0 of proteins fa-cilitated peptide partitioning at the interfaces, and thereby enhanced interaction between the peptides and oil phase5）. Thus, emulsion formation could be promoted, as suggested by the improvement in EAI with higher DH value（Fig. 4a）. Exposing hydrophobic amino acids, moreover, might promote interaction between adsorbed protein molecules, leading to effective formation of interfacial films. To form strong interfacial films, bond formation between adsorbed protein residues is necessary38）. This could explain the better long-term stability implied by lower ∆T（Fig. 4b）and less drop size increasing with storage time（Fig. 5）of the emulsions stabilized by RBPH with higher DH.

3.4 Antioxidant activity of RBPH in O/W emulsionsThe activity of RBPH in prolonging chemical stability of

O/W emulsions was further studied. Soybean O/W emul-sions stabilized by Tween 20 with the presence of RBPH（0.5％, w/v）were prepared. Formation of primary and sec-ondary products of lipid oxidation was quantified by moni-toring PV and TBARs content along 2 weeks. Figure 7a and b depict the relationship of storage time to PV and TBARs of RBPH-added emulsions.

Considering PV, a lag phase was observed for the first twice days for all samples. For the control（emulsion without RBPH）, PV rapidly increased to the maximum amount（ca. 7 mg Hydroperoxide equivalent/L）after 8 d, and then abruptly declined. A similar trend was found for the emulsions containing RBPH with DH of 0 and 5.6％, but a lower maximum value（ca. 4 mg Hydroperoxide equivalent/L）and longer storage time（10 d）were observed. A reduction in PV could be contributed, since hydroperox-ides were unstable products that tended to degrade further and/or interact with other species through progressive oxi-dation2）. Interestingly, PV of the emulsions containing 6.4 and 7.6％ DH-RBPH continually increased until the end of 2 weeks. This indicated better ability of the peptides with greater DH to retard the initial stage of oxidation. Higher TBARs formation was observed for the control compared with the RBPH added emulsions, especially when the RBPH with DH of 6.4 and 7.6％ were added. In this study, the effect of RBPH concentration on oxidative stability of emulsions was also investigated, by applying RBPH at dif-ferent concentrations（0.5, 0.75, and 1％, w/v）. However, there were no significant differences in PV and TBARs for the emulsions containing different concentrations of RBPH at all DH levels（data not shown）.

Efficiency of RBPH to prohibit lipid oxidation in emul-sion model was novelty elucidated in the present work. Due to a presence of large interfacial areas, emulsion is an oxidative susceptible model15） and possesses a different lipid oxidation mechanism compared with a homogeneous

Fig. 5　�Diameter of oil droplets (D32) dispersed in emul-sions stabilized by RBPH at various DH. The emulsions contained an oil fraction of 0.2 and were stabilized by RBPH (0.5%, w/v). D32 was measured at different storage times: 0, 4, 6, and 10 d after emulsification.

Fig. 6　�Correlation between surface hydrophobicity (S0) and emulsifying activity index (EAI), transmis-sion loss rate (∆T), and diameter of oil droplets (D32) of the emulsions stabilized by 0.5 %, w/v RBPH.

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counterpart, such bulk oil; in emulsion, oxidation is mainly initiated at the oil-water interfaces where dispersed oil drops are exposed to water-soluble pro-oxidants15）. Cheng et al.2） reported the ability of potato protein hydrolysates to inhibit oxidation in Tween 20-based emulsions and sup-posed due to improved S0 of the peptides. With greater S0, peptides could easily interact with emulsifiers, so larger amount of the peptides were accumulated at the interfaces resulting in enhanced interface films strength2, 3）. By an-choring at the interfaces, moreover, peptides could exhibit antioxidant activities effectively to protect oil drops from oxidative stress2）. It has been suggested that hydrolysis process could provoke antioxidant activity of proteins, by exposing some hydrophobic amino acids, e.g., Tyr, Phe, Trp, Leu, Val, Cys, Met, and His2, 20）. Potent antioxidant ac-tivities including free radical scavenging activity, reducing power, and ferrous iron chelating ability, of various protein hydrolysates were reported2）. The predominant amino acids in rice bran proteins were suggested to be sulfur-con-taining amino acids, i.e. Cys and Met13）. Met had a strong

radical scavenging activity, since its preference to donate proton to stable the structure via resonance mechanism16）. To more elucidate antioxidant mechanism of RBPH, com-posite amino acids of the peptides will be characterized in a future study.

4 CONCLUSIONSImproved protein extraction from rice bran could be ac-

complished by the application of carbohydrases and me-chanical force. Comparing the conventional alkaline method with enzymatic extraction, rice bran protein iso-lates with consistent characteristics could be prepared with different extracted yields（ca. 30％ for the conven-tional means using alkaline solution（pH 11）, and 50％ with the aid of Viscozyme L and a-amylase）. To improve func-tional properties of the isolated proteins, protease-assisted hydrolysis was conducted to prepare rice bran protein hy-drolysates（RBPH）at various degrees of hydrolysis（DH）, and their influence on physicochemical stability of O/W emulsions was observed. Emulsion formability and long-term dispersibility could be successfully improved by em-ploying RBPH as an emulsifier, especially at higher DH. Higher RBPH concentration effected to enhance colloidal stability of the emulsions. Interestingly, it was shown that RBPH could improve oxidative stability of emulsion, as in-dicated by lower PV and TBARs of the RBPH added emul-sions than control sample during a storage period of 2 weeks. The ability of RBPH to improve physicochemical stability of O/W emulsions was expected due to increased S0 and solubility of the peptides. With greater DH, RBPH might be better accumulated at the interfaces, and thereby effected to enhance interfacial film strength to prevent drop aggregation and pro-oxidants approaching to oil phase. By incorporating RBPH to the emulsion, the physi-cochemical stability could be successfully improved. The present results are significant to value addition of rice bran for further industrial application.

ACKNOWLEDGEMENTSThis research was supported by research funding of

Faculty of Agro-Industry, Prince of Songkla University（Seed Money）. The author wishes to thank: Prof. Dr. Soot-tawat Benjakul and Asst. Prof. Saowakon Wattanachant（Department of Food Technology, Faculty of Agro-Indus-try, Prince of Songkla University）for their kind guidance and use of laboratory facilities; Thai Edible Oil Co., Ltd., for providing rice bran; and Lamsoon（Thailand）Public Company Limited for supplying soybean oil.

Fig. 7　�Effect of storage time on (a) PV and (b) TBARs contents of emulsions containing RBPH (0.5%, w/v) at various DH: (○) control emulsion (with-out adding RBPH), (■) 0% DH, (□) 5.6% DH, (▲) 6.4% DH, (△) 7.6% DH, and (●) 10% DH.

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