Extraction of rice bran protein concentrate

and its application in bread

Sudarat Jiamyangyuen
1
, Voranuch Srijesdaruk
2
,
and W. James Harper
3
Abstract
Jiamyangyuen, S.
1
, Srijesdaruk, V.
2
, and Harper, W.J.
3
Extraction of rice bran protein concentrate and its application in bread
Songklanakarin J. Sci. Technol., 2005, 27(1) : 55-64
In a study of rice bran protein concentrate (RBPC) preparation, the alkaline extraction was per-
formed. The objectives of this study were to determine the optimal extracting conditions of RBPC and apply
RBPC into a bread recipe in order to improve bread quality and make acceptable product to consumers. The
design used in extraction was a central composite design. The response surface methodology was chosen to
graphically express the relationship between pH and extraction time with the output variables of protein
content and percent yield of RBPC. It was found that optimal extracting conditions were pH 11 and 45 min.,
resulting in 69.16% protein content and 8.06% yield of RBPC. When incorporating 1-5% of RBPC in a
bread recipe, the weight loss and microbial counts of breads were decreased compared to those of control
bread. The higher protein content and fiber in bread was corresponding to the amount of RBPC added.
Therefore, adding RBPC can significantly increase protein and fiber content in bread. However, results from
sensory evaluation showed that adding more than 1% of RBPC decreased the liking scores of color, taste,
odor, texture, and overall liking. The results of this study could be used as a basic knowledge of RBPC
utilization in other food products.
Key words : rice bran protein concentrate, central composite design,
response surface methodology, bread
1
Ph.D.(Food Science and Technology), Asst. Prof., Department of Agro-Industry, Faculty of Agriculture,
Natural Resources and Environment, Naresuan University, Phitsanulok, 65000 Thailand.
2
M.Sc.(Food
Technology), Assoc. Prof., Department of Food Technology, Faculty of Technology, Khon Kaen University,
Khon Kaen, 40002 Thailand.
3
Ph.D.(Food Science and Technology), Prof., Department of Food Science and
Technology, The Ohio State University, 2015 Fyffe Road, Columbus, OH 43210, USA.
Corresponding e-mail: sudaratjiam@yahoo.com
Received, 20 April 2004 Accepted, 2 July 2004
ORIGINAL ARTICLE
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005 56
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al.
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Rice bran is an under-utilized milling by-
product of rough rice. At the present, a large
amount of rice bran has been discarded and used
as animal feed. Rice bran oil is produced from
rice bran and now commercially available. Several
studies reported the nutritional quality of rice bran,
which is composed of protein, fiber, vitamins, and
minerals. The protein found in rice bran is reported
approximately 12-15%. However, protein in rice
bran is still under-utilized. The interesting char-
acteristic of rice bran protein is that it is composed
of high amount of lysine, an essential amino acid.
Moreover, rice bran is considered a good source
of hypoallergenic proteins; therefore, rice bran
protein may serve as a suitable ingredient for
infant food formulations (Wang et al., 1999).
Several researches have been undertaken in an
attempt to increase utilization of rice bran as
human foods. For the protein aspect, most of the
literature available is focused on preparation and
functional property study of rice bran protein. The
commonly used preparation method of extracting
rice bran protein is solvent extraction. The solvent
extraction of protein from rice bran employs
alkaline condition and then precipitation at the
isoelectric point at pH 4.5 (Gnanasambandam and
Hettiarachchy, 1995).
Although the nutritional potential of rice bran
has been recognized, at present rice bran protein
concentrates and isolates are not commercially
available. A group of researchers (Wang et al., 1999;
Gnanasambandam and Hettiarachchy, 1995; Bera
and Mukherjee, 1989) was able to prepare rice bran
protein concentrate/isolate and propose potential
use as a food protein ingredient due to its
emulsifying and foaming properties. Even though
rice bran is a by-product produced in large quantity
in Thailand yearly, its application in foods is very
limited, as well as its application of such ingredient
to food products have not been widely studied and
published. Therefore, the purposes of this study
were to determine the optimal conditions for
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al. 57
preparing rice bran extract and utilize this extract
in bread. The specific purposes were to determine
the proper amount used to increase protein content
in bread, to study changes occurring in breads with
and without rice bran extract added, as well as to
determine consumers' acceptability of the product.
Materials and Methods
Defatting and extraction of rice bran protein
concentrate
Rice bran obtained from a local milling
factory was processed into two steps: defatting
and protein extraction. The defatting procedures
employed are according to Wang et al. (1999).
Briefly, rice bran is defatted twice using hexane in
bran to solvent ratio of 1:3 at a setting of 250 rpm
in a lab stirrer for 30 min and centrifuge at 5000 g
for 10 min. at room temperature (RT). The defatted
rice bran (DRB) is air-dried overnight, sieved
through a 100 mesh screen, packed in a bag and
stored at 5C. The extraction step is a modification
of Gnanasambandam and Hettiarachchy, 1995:
alkaline extraction followed by isoelectric pre-
cipitation are used to prepare rice bran extract. The
process is described as follows; Defatted rice bran
sample and distilled deionized water (1:4) was pH
adjusted to 9.5 and stirred 30 min at room temper-
ature (RT). The slurry was centrifuged (Sorvall

RC 28 S) at 5000 g for 30 min. (RT). The pH of

supernatant was adjusted to 4.5 and centrifuged
again at 5000 g for 30 min. (RT). Precipitate was
washed using water (pH 4.5). The residue was
suspended in distilled deionized water (pH 7.0)
and frozen overnight. The final product, which is
called rice bran protein concentrate (RBPC), was
then freeze dried (Freeze dryer, Flexi-Dry P FTS
system) and stored at -5 C.
Experimental Design
The experimental design was Central
Composite Design (CCD) with second order and
hexagon design points. There were two variables
including pH and time of extraction. According to
the hexagon design, the pH was varied to 5 differ-
ent levels; while the time of extraction was varied
to 3 different levels. Even though alkaline extract-
ion is commonly used for protein extraction, the
range of pH used in this study was set between
2-12 with the intention to cover a wide range for
comparing and determining the optimum condition.
Extraction time was set in the range 32-58 min.
For coded values, the pH was given coded 5 levels
as -1.000, -0.500, 0, 0.500, and 1.000. Coded values
for time of extraction and incubation were assigned
to 3 values as -0.8666, 0, and 0.8666.
For the whole experiment, there were a total
of 10 treatment combinations including 4 repeat-
ing combinations at center points in order to allow
curvature of the graph. Experiments were per-
formed in duplicate and in a completely random-
ized design.
Bread recipe and preparation
Ingredients for making 1 lb loaf of white
bread of control and RBPC-added bread are shown
in Table 1. In each formula, the total weight of
ingredients for making 1 loaf of white bread was
346 g. Rice bran protein concentrate is used to
substitute wheat flour and other ingredients were
kept constant for all formulae.
For bread preparation, the procedures are
as follows; 1) mix powder ingredients including
wheat flour, milk powder, and salt, then pass
through sieve 100 mesh before adding yeast and
RBPC. 2) In the mean time, mix sugar, egg, vanilla,
and water in another bowl and mix well. 3) Pour
ingredients from steps 1 and 2 together and mix
well. 4) Knead to form a dough (using a mixer for
bread making) until dough is smooth and elastic.
(5-8 min). 5) Leave the dough in ambient temper-
ature (approx. 1 hr), then transfer the dough to a
1 lb rectangular bowl and leave a dough for another
30 min 6) Spray some water on the surface of the
dough to prevent dryness occurring during baking.
7) Put in a bowl in an oven set at 180C. for 15-30
min. or until the surface turns yellow. 8) Take out
the product from an oven and a bowl, leave to cool
before further analysis.
Proximate analysis and percent yield
The proximate analysis of RBPC and bread
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005 58
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al.
samples was performed for protein, fat, moisture content, fiber, and ash, using method of AOAC (1995).
The percent yield was calculated as follows;
Percent yield =
Weight of RBPC
Weight of rice bran
100
Measurement of bread specific volume and weight loss
Breads with various RBPC added ranging from 1-5%, were determined for their specific volume
and weight loss and compared to the control formula (0% RBPC). Specific volume was performed
based on substitution of sesame seeds by bread in a certain volume container.
Specific volume =
vol. of sesame seeds in a container - vol. of sesame seeds in container with bread
Weight of bread
Weight loss =
Weight of dough before baking- Weight of dough after baking
Weight of dough before baking
100
Measurement of yeast and mold count
Sliced breads were kept at room temperature (27C, RH 50%) in a sealed plastic bag and micro-
bial counts were daily determined from day 1 to day 4 since colonies of yeast and mold started to appear
on day 5. Total plate count (TPA) was used to determine yeast and mold in bread samples.
Sensory evaluation
The sensory evaluation was conducted by 20 panelists, consisting of undergraduate and graduate
students from Department of Agro-Industry, Naresuan University. The test was carried out using a nine-
point hedonic scale for five attributes (color, odor, taste, texture, and overall liking), where 9 is like
extremely and 1 is dislike extremely. Samples were presented with 3-coded digit number. Water was
provided for rinsing between samples.
Table 1. Ingredients and recipe for bread.
% RBPC- added in bread (calculate from wheat flour weight)
Ingredients (g) Control 1% 2% 3% 4% 5%
RBPC 0 1.67 3.34 5.01 6.68 8.35
Wheat flour 167 165.33 163.66 161.99 160.32 158.65
Milk powder 8.3 8.3 8.3 8.3 8.3 8.3
Yeast 1.67 1.67 1.67 1.67 1.67 1.67
White sugar 41.69 41.69 41.69 41.69 41.69 41.69
Water 90 90 90 90 90 90
Salt 1.17 1.17 1.17 1.17 1.17 1.17
Egg 16.67 16.67 16.67 16.67 16.67 16.67
Vanilla 1.17 1.17 1.17 1.17 1.17 1.17
Butter 18.33 18.33 18.33 18.33 18.33 18.33
Total 346 346 346 346 346 346
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al. 59
Statistical analysis
Analysis of variance (ANOVA) technique
was used to compare mean differences of samples.
If the differences in mean existed, multiple com-
parisons were performed using the Duncan's
Multiple Range Test (DMRT). All analysis was
conducted using SPSS for Window Version 11.0.
Response Surface Methodology was per-
formed to determine optimal condition for RBPC
extraction by using software STATISTICA version
5.0.
Results and Discussion
Chemical composition of full fat rice bran
and defatted rice bran is shown in Table 2. Full fat
rice bran composed of 12.6 % protein, 21.13 % fat,
5.59 % fiber, 8.97 % ash, 8.5 % moisture, and
43.21 % carbohydrate. After a defatting process,
the fat content reduced from 21.13% to 1.92%,
(approximately 90% reduction). The color of
defatted rice bran was also observed as lighter
compared to the original rice bran.
Table 3 shows experimental conditions of
10 treatments originated from CCD as well as
percent protein and percent yield of RBPC. The
RBPC with the highest protein content (72.63%)
was extracted using pH 9.5 and 58 min. On the
other hand, using pH 4.5 and extraction time of
58 min. gave minimum protein content of RBPC
(6.75%). It is generally known that pH 4.5 is an
protein isoelectric point (pI) indicating that protein
has minimal solubility at this point. It was noted
that even at lower pH than pI, (pH 2.0), the protein
content of RBPC also increased (14.95%) com-
pared to that obtained at pH 4.5. It was reported
Table 2. Chemical compositions of full fat rice bran and defatted
rice bran.
Compositions (%) Full fat rice bran Defatted rice bran
Moisture 8.5 12.43
Protein 12.6 13.89
Fat 21.13 1.92
Crude fiber 5.59 6.03
Ash 8.97 10.13
Carbohydrate 43.12 55.6
Table 3. Percent protein, and percent yield of rice bran protein concentrates extracted
using different conditions
Code Values Experimental Values
pH Time pH Time (min.)
1 1.000 0 12 45 70.43 12.20
2 0.500 0.866 9.5 58 72.63 9.60
3 -0.500 0.866 4.5 58 6.75 1.36
4 -1.000 0 2 45 14.95 3.65
5 0.500 -0.866 9.5 32 71.59 9.58
6 0 -0.866 7 32 63.09 7.82
7 0 0 7 45 63.44 7.89
8 0 0 7 45 64.88 7.60
9 0 0 7 45 63.16 7.88
10 0 0 7 45 64.13 7.87
Treatments
Protein content
(%)
Yield
(%)
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005 60
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al.
that at low pH, phytate, a component in rice bran,
can interact with protein leading to decreased
solubility of protein (Bera and Mukherjee, 1989).
The protein content of RBPC extracted at pH 7.0
and 45 min. was about 63-65%. The results were
in agreement with the study of Chen and Houston
(1970), which reported that protein content of
product increased linearly from pH 7.5 to 11 and
decreased as pH reached to 12. At high pH, some
non-protein nitrogen could solubilize and contribute
to protein quality and purity.
The percent yield of different 10 treatments
varied from 1.36-12.20%. The percent yield was
highest when using pH 12 and extraction time of
45 min. The lowest percent yield was also obtained
when extracting at isoelectric point of protein. The
percent yield of RBPC extracted at pH 7.0 and 45
min. was approximately 8%.
In order to determine the optimal condition
for RBPC extraction when considering protein
content and percent yield simultaneously, the
analysis of result using RSM technique was per-
formed. The RSM is a means to find the optimal
operation conditions. The optimal conditions could
be the maximum or minimum point of response,
depending on the objective. In this study, the
Figure 1. Percent protein of rice bran protein concentrate extracted using different conditions
Figure 2. Percent yield of rice bran protein concentrate extracted using different conditions
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al. 61
maximum point of protein content as well as
percent yield was desirable. The graphical illus-
trations are shown in Figures 1 and 2. Both figures
show contour plots of percent protein content and
percent yield of RBPC, respectively. The X and Y
in both graphs represent pH and time, respectively;
whereas the Z in Figures 1 and 2 represents the
response value, which is protein content and yield,
respectively. From a statistical analysis, the model
that best fits the data in this study is quadratic with
adjusted R2 0.947 for equation in Figure 1 and
0.977 for equation in Figure 2 (data not shown).
The graphs of protein contents and percent yield
were then overlaid, as shown in Figure 3, and the
intersection (shaded) area was selected to determine
optimal extracting condition.
From Figures 1 and 2, it is shown that RBPC
contains higher protein content and percent yield
as the pH and time of extraction increase. The
results agreed with those reported by Chen and
Houston (1970), who found that protein content
increased linearly from pH 7.5 to 11 and decreased
at pH 12. At pH 12, the non-protein nitrogen
components were soluble resulting in a reduction
of protein purity of protein extracted at this pH.
In this study, it was also observed that using higher
alkaline condition gave a strong brown color of
RBPC. However, it was reported that using high
pH for protein extraction could bring some dis-
advantages; for example, (1) protein denaturation
and hydrolysis at high pH, resulting in undesirable
flavor and odor, (2) increase Maillard reaction,
leading to dark colored product, (3) decreased
nutritive value of protein, especially essential amino
acid such as lysine, and (4) increased extraction of
non-protein component, which also coprecipitated
with protein and lower protein purity (Wang et al.,
1999).
When overlaying in Figure 3, it was found
that there is an intersection area where both
protein content and percent yield were maximized.
Any point in this shaded area could be selected.
However, as discussed earlier, the high alkaline
condition should be avoided due to some negative
effects. In addition, at higher pH, some non-protein
nitrogen may be a component accounting for high
yield. Therefore, a condition chosen in this study
was to use the lowest pH as possible (in the shaded
Note: Intersection area ( ) is an overlap of maximum protein and percent yield,
dotted line (----------) is the selected optimum pH and time
Figure 3. Percent protein (bold line) and percent yield (thin line) of rice bran protein
concentrates.
1 = pH 12/45 min
2 = pH 9.5/58 min
3 = pH 4.5/58 min
4 = pH 2/45 min
5 = pH 9.5/32 min
6 = pH 7/32 min
7-10 = pH 7/45 min
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005 62
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al.
area), which is pH 11 and extraction time of 45
min. At this condition, the predicted protein and
yield were 76.34% and 12.2%, respectively.
The proximate analysis of control bread and
RBPC-added breads is shown in Table 4. The fat
and ash content of all samples were not statistically
different. On the other hand, each sample contained
statistically different amounts of moisture and
protein. The lowest and highest moisture contents
belonged to bread with 5% and 2%, respectively.
The protein content as well as fiber content of bread
increased as the amount of RBPC added increased.
However, there are two pairs, including 2% and
3% bread, and 4% and 5% bread, that are not
statistically different with respect to fiber content.
From this table, carbohydrate in samples ranged
from 49.03% to 53.86%. Due to the fact that rice
bran is a good source of fiber (Abdul-Hamid and
Luan, 2000) as well as these being a considerable
amount of protein in rice bran protein concentrate,
fiber and protein content were then affected most
when increasing amount of RBPC used in breads.
Breads were analyzed for their specific
volume and weight loss and data are shown in
Table 5. Adding more RBPC could decrease
specific volume of breads and reduce weight loss
as well. During bread making, it was observed that
when increasing amount of RBPC, dough weight
and size was reduced. This could be due to water
holding capacity of RBPC. As a result, breads with
RBPC- added were lower in specific volume (less
expanded) compared to the control bread.
Figure 4 shows microbial counts of bread
that were kept for 4 days at room temperature.
From this graph, when storage time increased, the
microbial count increased for all samples. How-
Table 4. Proximate analysis of breads containing different amounts of RBPC.
Composition (%)
Fat Ash Moisture Protein Fiber Carbohydrate
Control 7.53

0.20
a
0.69

0.01
a
28.53

0.27
c
9.01

0.05
a
0.49

0.02
a
53.86

0.11
c
1.0 7.67

0.22
a
0.71

0.01
a
29.74

0.04
e
9.84

0.16
b
1.68

0.15
b
50.25

0.00
b
2.0 7.71

0.02
a
0.70

0.02
a
30.55

0.17
f
10.16

0.15
c
2.00

0.01
c
49.03

0.46
a
3.0 7.63

0.56
a
0.69

0.02
a
28.82

0.01
d
10.52

0.15
d
2.19

0.12
c
50.07

0.30
b
4.0 7.95

0.32
a
0.67

0.02
a
27.81

0.05
b
11.59

0.12
e
2.50

0.01
d
49.65

0.17
ab
5.0 8.31

0.21
a
0.70

0.02
a
27.17

0.11
a
12.10

0.02
f
2.59

0.13
d
49.08

0.21
a
Mean SD
Means in column with same letters are not significantly different at = 0.05 (DMRT)
% RBPC-
added
Table 5. Specific volume and weight loss of breads contain-
ing different amounts of RBPC
% RBPC- added Specific volume (cm
3
/g) Weight loss (%)
Control 4.650
f
43.95
e
1.0 4.545
e
42.16
c
2.0 4.361
d
45.19
f
3.0 4.295
c
42.73
d
4.0 4.185
b
40.17
b
5.0 4.128
a
30.42
a
Means in column with same letters are not significantly different at
= 0.05 (DMRT)
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al. 63
Figure 4. Microbial count of breads during day 1-4 of storage
ever, when comparing the control and other RBPC-
added samples, it is noted that the control bread
showed the most rapid increase in microbial count
during storage time. Therefore, RBPC contributed
to reduced microbial count in breads.
Sensory evaluation results are shown in
Table 6. Hedonic scores of color and odor for all
RBPC- added breads were not different, but were
significantly lower when compared with control
sample. From this table, control bread received the
highest scores for taste, texture, and overall liking.
However, bread with 1% RBPC- added appeared
to be most similar to control in terms of liking for
all attributes evaluated. The darker color and un-
familiar taste to panelists of some treated samples
could be reasons for lower hedonic scores when
compared with the control bread. Since information
relating proper amount of RBPC that should be
used in bread formula is not available, it is difficult
to compare result of this study to others. From
the sensory result, it can be concluded that 1% is
the maximum use of RBPC in bread in order to
maintain consumers acceptability of the product.
Conclusions
Extraction of protein from rice bran using
pH 11 and 45 min was selected as the optimal
condition when considering protein content and
percent yield of RBPC simultaneously. RBPC
was analyzed for chemical composition and in-
corporated in bread formula ranging from 1% to
5%. It was found that increasing RBPC amount
resulted in bread with increased protein and fiber
content, but decreased specific volume and water
loss during baking. Mean values of microbial loads
Table 6. Hedonic mean scores of sensory evaluation of breads containing different amounts
of RBPC
% RBPC- added
0 1.0 2.0 3.0 4.0 5.0
Color 7.35

1.87
b
6.25

1.37
a
6.10

0.97
a
6.05

0.94
a
5.60

1.76
a
6.35

1.46
a
Odor 6.75

1.61
b
5.75

1.74
a
5.85

1.13
a
5.20

1.82
a
5.25

1.48
a
5.50

1.73
a
Taste 7.47

0.91
b
6.70

1.65
ab
6.07

1.54
a
6.20

1.05
a
5.90

1.91
a
6.42

1.38
a
Texture 6.95

1.87
b
6.85

1.26
ab
6.15

1.46
ab
5.85

1.81
ab
5.80

1.93
a
6.00

2.05
ab
Overall liking 7.55

1.39
c
6.80

1.28
ab
5.90

1.51
a
6.10

1.20
ab
5.80

1.60
a
6.25

1.33
ab
Mean SD. of 20 panelists
Means in row with same letters are not significantly different at = 0.05 (DMRT)
Attributes
Songklanakarin J. Sci. Technol.
Vol.27 No.1 Jan. - Feb. 2005 64
Extraction of rice bran protein concentrate
Jiamyangyuen, S., et al.
have shown that RBPC- added samples were lower
than that of control sample. However, sensory
results showed that using more than 1% or RBPC
could decrease consumers' acceptability of the
product. In conclusion, even though high use of
RBPC in bread is desirable in terms of chemical
and microbiological aspects, consumes' liking of
product has restricted its use up to 1%. More studies
should be conducted to investigate the possibility
of using RBPC as an ingredient in other food
products in order to increase applications of such
value-added food ingredient.
Acknowledgments
The authors greatfully acknowledge the
financial support from the Thailand Research Fund
(TRF) and the Commission on Higher Education,
Thailand.
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