Comparison of Two Methods for the Extraction of Fractionated Rice Bran Protein. College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 1. China. 2College of Food Science, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 1. China. 3College of Food Science, Beijing Technology and Business University, 3. Fucheng Road, Beijing 1. China. Copyright . This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Two different methods for extracting fractionated rice bran protein (FRBP) from defatted rice bran were investigated according to the solubility of protein in different extraction solvents. The yields of the obtained proteins and their purity were first compared.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, differential scanning calorimetry, protein surface hydrophobicity, and protein secondary molecular structure analyses were subsequently applied to identify and compare the compositional, structural, and functional characteristics of the obtained proteins. The highest yield (1. FRBP products were obtained using 0. M Na. Cl, 8. 0% ethanol, and 0.
Determination of Protein Content in Biomass. Laboratory Analytical Procedure (LAP) Issue Date: 05/23/2008.
M Na. OH as extraction solvents to fractionate albumin, globulin, prolamin, and glutelin. Several good properties were exhibited, including good functionality, specific denaturation temperature, and enthalpy values, for FRBP products prepared by the above method. Introduction. Cereals are the major source of dietary protein for a large population. The intake of rice protein exceeds other cereal protein, such as wheat protein and maize protein. Moreover, rice protein is hypoallergenic and rich in lysine. Thus, rice protein is widely used in infant foods and the formulation of restricted recipes for children with food allergies . Compared with casein and soy isolate protein, the amino acid profile of rice protein satisfies the demands of 2- to 5- year- old children .
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms.
Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins (e.g., for detecting proteins, for isolating and. The electrophoretic separation of proteins can be achieved by various methods, matrices, and buffer systems. By choosing suitable separation matrices and. Solubility of Protein. The solubility of FRBP products prepared by the two extraction methods was determined at different pH levels and is shown in Figure 2.
Various proteins from vegetable sources have been studied for a long time . There is as much as 1. China. Rice bran protein, which is extracted from defatted rice bran, can be added in various foods as a nourishing ingredient .
Thus, rice bran is a type of high value- added raw material that shows great potential in food applications. Although the availability and nutrition values of rice bran have been widely accepted, it is difficult to separate rice bran protein due to its association with phytate acid and cellulose .
Moreover, the solubility of rice bran protein is undesirable due to the high number of disulphide groups. At present, rice bran, which is mainly used for producing animal feeds, is insufficiently utilized . The methods including alkaline extraction are followed by isoelectric precipitation . Despite the extensive efforts of researchers, the extraction approach for satisfactory fractionation of rice bran protein while maintaining its functionality is yet to be established.
In this study, two FRBP extraction methods were compared, and the optimum method was selected based on the functionality of the obtained FRBP products. Materials and Methods. Materials. Full- fat rice bran (Kongyu 1.
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Chahayang Farm, Nongkenzongju, Heilongjiang province, China. The defatted rice bran was prepared with the following procedures: full- fat rice bran was extracted with a 1. Extraction Methods. Method 1. The protein extraction was performed at room temperature. Defatted rice bran (1. L of distilled water for 1 h using a magnetic stirring apparatus followed by centrifugation at 4,5. The precipitate was extracted following the procedures as described above.
Each supernatant was collected and filtered to obtain the albumin extract. The residue was subsequently extracted with 1 M Na. Cl following the procedure as described above to obtain the globulin extract. After removing albumin and globulin, the residue was extracted with 8.
After removing albumin, globulin, and prolamin, the residue was extracted with 5. L of 0. 0. 1 M Na. OH according to the procedures described for the extraction of albumin to obtain the glutelin extract.
The albumin, globulin, and glutelin fractions were obtained by adjusting the p. H of the extracts to their isoelectric points of 4. The precipitates were allowed to rest for 1 h.
The precipitated proteins were centrifuged at 4,5. H was neutralized before freeze- drying. Method 2. The protein extraction was performed at room temperature. Defatted rice bran (1.
L of different extraction solvents for 1 h using a magnetic stirring apparatus. The sequence of the extraction solvents was as follows: 0. M Na. Cl, 8. 0% alcohol and 0. M Na. OH. The extract was kept separately, and the residue was reextracted. Each extraction step was followed by centrifugation at 4,5.
The sequential extraction step was repeated on the residue with 4. L of each extracting solution. Each extraction was combined and filtered. The 0. 4 M Na. Cl filtrate was dialyzed against water for 7. The albumin and globulin fractions were collected from the supernatant and precipitate, respectively. Each of the fractions was separately freeze- dried.
The solvent in the 8. The prolamin concentrate was extracted with hexane and then freeze- dried to obtain the prolamin fraction. The p. H of the 0. M Na. OH filtrate was adjusted to 4. The precipitate was allowed to rest for 1 h.
The precipitated protein was centrifuged at 4,5. H was neutralized before freeze- drying.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS- PAGE) Analysis. SDS- PAGE analysis was performed on the FRBP products obtained by the two extraction methods. The analysis was performed using a SDS- Tris- glycine buffer system with 5% (w/w) stacking gels and 1.
Laemmli . Electrophoresis was initially performed at a constant voltage of 8. V followed by an increase to 1. V. At the end of electrophoresis, the gel was separated. The peptide and protein bands were stained with Coomassie brilliant blue G- 2. The gel images were scanned using an Alphalmager HP scanner (Proteinsimple, USA) and then subjected to analysis. Protein Solubility.
The FRBP sample (0. L of distilled water using a magnetic stirring apparatus and then diluted to a final volume of 2. L. The dispersion (4 m. L) was added to a 1. L centrifuge tube and centrifuged at 4,0. The protein concentration of the supernatant was measured by Lowry’s method .
Absorbance was measured at 5. BSA; 9. 8% pure, Sigma, US).
The protein solubility (expressed by dissolved nitrogen index, NSI) was calculated with (1) as follows. Evaluation of Emulsifying Properties. The emulsifying properties of the FRBP products obtained by the two methods were evaluated by the turbidimetric method. To prepare the emulsion, 1. L of FRBP solution and 5.
L of soy oil were homogenized with an Ultraturrax device (T- 2. S2. 5N1. 0G, IKA Labortechnik, Karlsruhe, Germany) at 1. After shaking in a vortex mixer, the absorbance of the diluted emulsions was measured at 5. SDS solution. The emulsifying activity index (EAI) of FRBP was calculated using (2) as follows.
DF is the dilution factor (1. L); is the fraction of oil used to form the emulsion; and is the absorbance of diluted emulsions. Foaming Properties. A 1% (w/v) FRBP solution was prepared. The p. H of the protein solution was adjusted to 2, 3, 4, 4. M HCl or 0. 1 M Na.
OH. The solutions were stirred at 1. The blend was immediately transferred into a 1.
L graduated cylinder, and the volume was recorded before and after stirring. The foaming ability was calculated using (3) as follows.
Fat Absorption Capacity (FAC) and Water Absorption Capacity (WAC)The FAC and WAC of FRBP were measured as previously described . The FRBP sample (0. L preweighed centrifuge tube and mixed with 3 m. L of soybean oil. The emulsion was incubated at 3.
The supernatant was then carefully removed, and the tube was reweighed. WAC Determination. The FRBP sample (2.
L preweighed centrifuge tube. Distilled water was added in small increments to the tube under continuous stirring with a glass rod until the mixture became a syrup and no water separation occurred. The tube was centrifuged at 2,5. The supernatant was then removed, and the tube was weighed. FAC or WAC was calculated using (4) as follows. FRBP. 2. 2. 7. Circular Dichroism (CD) Spectrum Analysis. CD spectrum analysis was performed according to the method of Wu et al.
The measurement was conducted under the following conditions: scanning velocity of 1. Surface Hydrophobicity. The surface hydrophobicity was measured with the fluorescence probe, 1- aniline- 8- naphthalene sulfonate (), according to the method of Jiang and Zhao . The protein solution was excited at 3. FI) was scanned at wavelengths from 4. The slit width for both excitation and emission was 5 nm.
Intrinsic Fluorescence Spectroscopy. The intrinsic fluorescence spectroscopy was measured as previously described . The FRBP sample was weighed and dissolved in 0. M phosphate buffer at p.
H 7. 0 with magnetic stirring. The dispersion was then centrifuged at 4. The protein solution was excited at 2. Differential Scanning Calorimetry (DSC) Analysis. The denaturation of the FRBP products was evaluated based on DSC analysis according to the method of Haskard and Li- Chan .
The sample (2 mg) was accurately weighed into an aluminium liquid pan, and 1. The pan was hermetically sealed and held at 4. The pan was heated from 3. A sealed empty pan was used as a reference. The denaturation temperature of protein and enthalpy change of the endotherm (. Statistical Analysis.
All data were expressed as the means . Differences between the mean values of multiple groups were analyzed by one- way analysis of variance (ANOVA) with Duncan’s multiple range tests. MS Excel 2. 00. 3 was used to analyze and report the data. Results and Discussion. Effect of Extraction Method on the Yield of FRBPThe yields of FRBP products prepared by the two extraction methods were compared and are shown in Table 1.
The yields of the FRBP products were 1.