Rice Starch Isolation by Alkaline Protease Digestion

of Wet-milled Rice Flour

N. Lumdubwong and P. A. Seib

Kansas State University, Department of Grain Science and Industry, Manhattan, KS 66506, U.S.A.

ABSTRACT
Alkaline protease digestion with a food-grade enzyme was used to produce rice starch from wet-
milled rice flour (WMRF). In a 33 factorial modelling experiment, recoveries of starch and levels
of protein contamination were determined at pH 85100, protease levels of 0515% (based on
WMRF), and digestion times of 50300 h. The following digestion conditions were kept constant;
55 C with mild agitation, 3437% (w/v) flour solids, and alkalinity to within 02 pH units.
Regression equations with the three variables explained 92% and 98%, respectively, of the variances
in starch recovery and protein contamination. Upon digestion with 11% protease at pH 100 and
180 h, starch recovery was 95% and protein contamination was 05%. Most hydrolysis of rice protein
occurred in the first 34 h of digestion as determined by the consumption of sodium hydroxide
(NaOH). Rice starch also was isolated by extraction of WMRF with c. 25 parts of 005 NaOH at
c. pH 12. The recovery of starch was c. 10% higher with the protease method than with the NaOH
method, and the effluents contained mostly amino acid salts as opposed to protein mixed with alkali.
The rice starch isolated by protease digestion was lighter in appearance, contained more lipid, and
gave a somewhat lower consistency after pasting. The raw materials used to isolate rice starch by
the protease method were approximately twice as costly in 1996 as those in the NaOH method,
principally because of the cost of the protease (55% of total).
2000 Academic Press

Keywords: starch, rice, isolation, protease digestion.

INTRODUCTION that removes the bran and germ. Broken rice, a

Milled rice, essentially the endosperm of the rice
kernel, is the major form of rice consumed by
humans. However, rice flour and starch are being
used increasingly in breakfast cereals, hypo-
allergenic foods, infant formulas, seasoned multi-
grain products, reduced calorie items, and bakery
foods13.
Milled rice is derived from rough (paddy) rice
collected by a threshing machine and dehulled
with rubber rolls to give brown rice. Brown rice
is converted to milled rice by an abrading machine
: DAPU=detergent alkaline pro-
tease units; DSC=differential scanning calorimetry;
WMRF=wet milled rice flour.
Corresponding author: P. A. Seib.
by-product of rice milling, can be ground directly
into flour, or it can be tempered4 to c. 30% moisture
content on a wet basis, then ground and dried to
obtain wet-milled rice flour (WMRF). The small
particle size and low starch damage of WMRF
make it attractive for starch isolation.
Rice starch is manufactured around the world,
but its tonnage is low because of its price. Rice
starch contains tiny granules (<5 lm) with a nar-
row size distribution, which makes it ideally suited
as a cosmetic dusting powder, a textile stiffening
agent, and a fat mimetic in foods. In addition, rice
starch causes minimal allergic reactions and its
flavour is bland5,6. High purity rice starch with
low surface protein-lipid contamination is desired
to minimise rancidity during storage and for use
as a starting material for chemical modification,
fermentation, and industrial applications.

07335210/00/010063+12 $35.00/0 2000 Academic Press

64 N. Lumdubwong and P. A. Seib
Compared with maize and wheat starches, isol-
ation of rice starch is difficult and therefore costly.
The starch in rice endosperm is associated with
proteins occurring as protein bodies I and II (PB
I and PB II)7. PB I is a prolamin accounting for
20% of rice protein, whereas PB II is a glutelin
accounting for 60% of total protein7. Both proteins
are hydrophobic and resist swelling in water at
neutral pH. Moreover, PB II is cross-linked with
disulphide bonds8. Uncharacterised proteinaceous
material is also localised in small pockets sur-
rounding both the compound granules and the
individual rice starch granules9. Besides problems
associated with rice protein, the tiny granules of
rice starch are slow to sediment in water, resulting
in losses during separation and purification.
Rice starch has been purified from endosperm
by way of alkali, detergent, and protease di-
gestions1015. Alkali gave high purity starch, but its
use commercially would generate an alkaline or
salty effluent. Anionic detergent was satisfactory
in removing protein and fibre from rice endo-
sperm, but detergent also would cause an effluent
problem and probably would reduce starch paste
consistency. Protease digestion of rice endosperm
near neutral pH required 24 h at 38 C to release
the starch. Such a long digestion period would
increase capital costs in a commercial process, and
would intensify microbial problems. Increasing the
pH of the protease digest of rice endosperm should
increase the hydration of rice proteins and ac-
celerate the release of starch. Also, if the protease
were a food-grade enzyme, the by-products of
digestion could be added directly as nutrients to
animal feeds, fermentation broths, or cell culture
media. Enzyme-assisted wet-processing of barley16
and amaranth17 has been reported recently.
The objectives of this research were: to isolate
rice starch from WMRF by alkaline protease di-
gestion with a food-grade enzyme; to optimise
the recovery of starch containing less than 05%
protein with respect to digestion time, pH, and
level of protease; and to compare properties of
rice starches isolated by protease digestion of flour
and by NaOH extraction, with both having <05%
protein contamination.
EXPERIMENTAL
Materials
A protease, Optimase APL-440, and casein sub-
strate were obtained from Solvay Enzymes, Inc.
(Elkhart, IN). The enzyme was provided as a
solution (density at 25 C was 110 g/mL) and
contained 88 mg protein/mL with an activity of
440 detergent alkaline protease units (DAPU)/g
of solution or c. 50 DAPU/mg of protein (see
protease assay). Commercial WMRF flour with
127% moisture content and 80% protein (dry
basis) was purchased from Erawan Co. Ltd., Bang-
kok, Thailand. Polysorbate 80 was from Sigma
Chemical Co (St Louis, MO), and other chemicals
were reagent-grade.
General methods
Unless otherwise stated, assays were performed in
duplicate. The particle-size distribution of WMRF
(50 g) was determined with an air jet sieve (Model
A200LS, Alpine Co., Augsburg, Germany) fitted
with sieves having openings of 38, 53, 75, 106 and
125 lm. Moisture and ash contents were meas-
ured, respectively, by oven-drying18 for 1 h at
130 C and by incinerating18 at 550 C for 2 h.
Nitrogen was measured by the Kjeldahl pro-
cedure18, and protein contents of rice flour, en-
zyme, and starch were calculated by multiplying
nitrogen contents by 595. Protein content of Op-
timase was measured by the biuret procedure19.
Total starch20 and damaged starch21 were de-
termined by enzyme digestion using kits from
Megazyme Ltd. (Wicklow, Ireland). Fatty acids in
rice starch were quantified as methyl esters by gas
chromatography22, and the colour of rice starch
was determined by a Minolta Chroma Meter
(Model CR 210, Minolta Corporation, Ramsey,
NJ).
Differential scanning calorimetry (DSC) meas-
urements were made with a Perkin-Elmer DSC-2
(Norwalk, CT) equipped with an FTS system
cooler and temperature controller (FTS Systems,
Inc., Stone Ridge, NY). The instrument was cal-
ibrated with indium. Starch samples (c. 27 mg)
were weighed into a DSC pan, and water
(c. 68 mg) was added. The sealed pan was heated
at 10 K/min from 280 to 400 K, and an empty
pan was used as reference.
Pasting properties of starch were determined by
a Rapid Visco-Analyzer (Newport Scientific Pty.
Ltd., Narrabeen, Australia). Rice starch (27 g, ds)
and water (25 mL) at 50 C were mixed; the slurry
was held at that temperature for 2 min and then
heated from 50 C to 95 C at a rate of 75 C/
min. The hot paste was held at 95 C for 2 min,
 Isolation of rice starch
cooled to 50 C at 75 C/min, and then held at
that temperature for 4 min.
Swelling powers of starch were determined by
modifying a previously published method.23 Rice
starch (05 g dry basis) was weighed into a 50 mL
polycarbonate centrifuge tube, water (30 mL) was
added, and the contents were mixed on a vortex
mixer. The tube was placed in a water bath at
925 C and inverted twice every 20 s for 3 min,
every 30 s for 2 min, every 1 min for 5 min, and
every 5 min for 20 min. The tube was centrifuged
at 7700 g at 25 C for 20 min, and the supernatant
was removed carefully and dried at 105 C for
5 h. The sedimented starch gel was weighed, and
swelling power was calculated as the water ab-
sorbed by the starch corrected for soluble starch.
Alpha-Amylase Assay
Alpha-Amylase activity was determined by di-
gestion of soluble starch and measuring the in-
crease in reducing power by the Nelson copper
reagents.24 Optimase was diluted 20 000-fold by
volume with 2% aqueous pentasodium tri-
polyphosphate, which had been adjusted to pH 85
by adding 1 hydrochloric acid (c. 01 mL). An
aliquot (1 mL) of the diluted enzyme solution was
mixed with 1% starch solution in 06 borate
buffer at pH 85 (10 mL), and the mixture in-
cubated at 25 C for 10 min. For blank samples,
2% aqueous tripolyphosphate solution was used
instead of diluted enzyme solution. An aliquot
(08 mL) of the digest was mixed with alkaline
copper reagent (10 mL) and water (02 mL) and
the mixture was heated for 20 min in a boiling
water bath. After the reaction mixture cooled to
25 C, an aliquot (1 mL) of molybdate/arsenate
reagent was added, and the solution was held
10 min at room temperature. The reaction mixture
was made to volume (25 mL) with water, and
absorbance was read at 520 nm was 0001 com-
pared with zero for the blank. A standard curve
was prepared with maltose solutions (10 mL) at
concentrations between 532 lg/mL.
Protease assay
Protease activity of Optimase APL-440 was as-
sayed by the method supplied by the manufacturer.
Aqueous pentasodium tripolyphosphate (2% w/v)
(pH 85) was used to dilute the commercial pro-
tease 20 000-fold by volume. Casein (133 g, db)
65
was dissolved in water (700 mL) and 06 borate
buffer (pH 87) (50 mL) was added. The mixture
was warmed to 40 C, adjusted to pH 85 by adding
1 NaOH, and then made to volume (1 L). A
portion of the protease solution was warmed to
40 C, and an aliquot (1 mL) was mixed with an
aliquot (5 mL) of the casein solution at 40 C. The
mixture was incubated for 40 min at 40 C, and
proteolysis was stopped by adding 50 mL of aque-
ous acid, which was a mixture of 18% tri-
chloroacetic acid, 18% sodium acetate, 3% acetic
acid, and 0024% Polysorbate 80. After cooling
in an ice bath, the acidified digest was agitated on
a vortex mixer and centrifuged for 15 min at
1800 g and 1015 C. The absorbance of the clear
supernatant at 275 nm in a 1 cm cuvette was 0732.
Blanks were done by the same procedure, except
incubation time was 10 min, and the blank A275 nm
was 0270. An extinction of 132103 L/mol-cm
was used to convert absorbance to lmol of tyros-
ine, and one detergent alkaline protease unit
(DAPU)/g was equal to the release of 4 lmol/
min/g of enzyme solution or 0050 lmol/min/mg
of protein in the enzyme solution.
Rice starch isolation by the NaOH method
The NaOH method of Yamamoto et al.11 was used,
except the ratio of rice flour to dilute NaOH
solution was increased, and the starch was neut-
ralised with acid prior to washing. The WMRF
(20 g) was mixed for 3 h at 25 C with 02% (005 )
aq. NaOH (50 mL) and the slurry filtered through
a No. 200 wire mesh sieve (75 lm opening). The
filtrate was centrifuged at 3000 g for 20 min, the
supernatant was discarded, and the sediment was
washed twice with water (50 mL) and centrifuged.
The residue was suspended in water and adjusted
to pH 7 by adding 1 hydrochloric acid, and
the slurry was centrifuged. The supernatant was
discarded, and the dark tailings layer atop the
starch was carefully scraped away and discarded.
The starch was washed three times with water
(50 mL) until the tailings fraction became neg-
ligible after centrifuging. The starch was dried in
a convection oven at 40 C for 48 h. Yield was
65% based on flour, and the starch contained
04% protein (db).
Rice starch isolation by protease digestion
In the protease method of isolating rice starch
from WMRF, the following variables were fixed
66 N. Lumdubwong and P. A. Seib

in all digestions; the level of flour solids in a digest Table I Commercial wet-milled rice flour
was 3437% (w/v), the temperature was 55 C, (WMRF) from Thailand
and the agitation by a magnetic stir bar was mild. Composition Granulation
The levels of protease, pH, and digestion time
were the independent variables, and the recovery Component Contenta Opening Through
of rice starch and the protein content were the % (lm) (%)
dependent variables. All experiments were con- Moisture 127 38 31
ducted with a Box-Bechken design with 12 treat- Protein 80 53 53
ment combinations and three replications at the Starch 761 75 72
centre point, which gave a total of 15 experiments Ash 04 106 93
per set. All data were analysed by the Statistical Damaged 30 125 98
starch
Analysis System (SAS 1985). Alpha-amylase none (?) 150 100

Modelling experiment I
The WMRF (20 g, dry solids) was placed in a glass
jar (c. 120 cm3) with 0001 sodium hydroxide
(50 mL) and Optimase protease (010, 020, and
03 g of solution or 05, 11, and 15% based on
WMRF) was added. The jar was covered with
Parafilm (American National Can, Neenah, WI)
that contained a single hole for insertion of a pH
electrode and a 3 mm (o.d.) delivery tube. The jar
was placed in a water bath at 55 C and stirred
with a magnetic stir bar. The mixture was adjusted
to pH 85, 90, and 95, which were maintained
within 02 units by a pH meter/controller
(Model No. H-05652-00) with an attached peri-
staltic pump (Model No. H-77120-50), both from
Cole-Parmer (Chicago IL). The acidity developed
during digestion was countered by intermittent
additions of 1 NaOH, which amounted at least
to 35 mL and at most to c. 9 mL. Reaction mix-
tures were stirred for 50, 100, and 150 h and
then filtered through a No. 200 wire screen (75 lm
opening). The sediment was centrifuged at 3000 g
for 20 min, and the dark yellow supernatant dis-
carded. The sediment was washed with water
(50 mL) and the mixture centrifuged for 15 min.
After the supernatant was discarded, the washing
and the centrifugation steps were repeated. The
sediment was suspended in water (50 mL), and
adjusted to pH 7 with 1 hydrochloric acid, and
the slurry was centrifuged at 10 000 g for 20 min.
The supernatant was discarded, and the upper
dark layer scraped from the starch. The dark layer
was dried under ambient conditions and assayed
for nitrogen by the Kjeldahl procedure. After being
washed three more times with water, the starch
was dried in a convection oven at 40 C for 48 h.
Modelling experiments II and III
Modelling experiment II was similar to modelling
experiment I, except pHs were 90, 95, and 100
a
Content on dry basis, except moisture on a wet
basis.
and digestion times were 120, 160, and 180 h.
Modelling experiment III was carried out with
protease added at 014, 022, and 030 g (07
15%); pHs 90, 95, and 100; and digestion times
of 180, 240, and 300 h.
RESULTS AND DISCUSSION
WMRF and alkaline protease
The properties of a sample of WMRF from Thai-
land are given in Table I. The product was finely
granulated with a median particle size of ap-
proximately 50 lm, yet contained only 30% dam-
aged starch. Heating the flour with 25 parts
of water in the DSC showed an endotherm at
To 678 C, Tp 735 C, and Tc 793 C with
DH 102 J/g, which was assigned to starch gel-
atinisation. The relatively high gelatinisation tem-
perature, which indicated that the flour was from
long grain rice25, permitted the use of an elevated
temperature in the protease digestion step during
starch isolation.
At least three food-grade alkaline proteases are
available commercially in the U.S.A. All are bac-
terial enzymes from Bacillus species, and they pos-
sess a broad range of optimum pH and broad
specificity toward substrates. We decided on Op-
timase APL-440, a commercial, foodgrade pro-
tease from B. licheniformis that is active at pH 610
(Table II). The supplier reported it to be free of
alpha-amylase activity at pH 95 and 55 C, which
was verified at pH 85 and 25 C. The protease
shows optimum activity at pH 910 and an opti-
mum temperature of 60 C. Its proteolytic activity
was found to be 453 DAPU/g of enzyme solution,
 Isolation of rice starch
Table II Characteristics of optimase APL-440
from B. licheniformis
Property Reported
or
found
Level of protein (mg/ml) 88a
Density of solution at 25 C (g/mL) 110a
Stable pH range 610b
Optimum pH range 910b
Optimum temperature (C) 60b
Temperature stability (C) <70b
Activity at pH 85, 40 C, DAPU/g 453a
Alpha-amylase activity at pH 85 and nonea
25 C
a
Determined in this work.
b
Properties reported by manufacturer were: level
of protein, 63 mg/ml; activity, 440 DAPU/g of
solution at pH 85 and 40 C; a-amylase at
pH 95 and 55 C, none.
which agreed with the labelled activity of
440 DAPU/g.
Protease digestion of WMRF; general
considerations
The digestion temperature was set at 55 C, which
was near the optimum of the protease yet below
the gelatinisation temperature of WMRF. The
operating pH was selected between pH 85 and 10,
which is within the activity range of the Optimase
protease (Table II). The glutelin proteins of rice
are soluble8 above pH 10, and at pH 85100 they
would be expected to swell. The levels of protease
were chosen by preliminary experiments and were
set in the range of 0515% protease solution
added based on flour weight. A low level of enzyme
was insufficient to digest the rice proteins in 30 h,
whereas a high level resulted in increased protein
contamination of the rice starch. The combination
of elevated pH and temperature eliminated the
need for use of an antimicrobial agent during the
digestion period.
Protein digestion of rice flour by protease at
alkaline pH decreased the pH of the medium, so
that during digestion with 11% protease, pH 10,
and 18 h, up to 045 eq of alkali per kg of flour
(wb) had to be added to maintain alkalinity. The
maximum volume of 1 sodium hydroxide added
for pH control was approximately 450 mL/kg
flour, which corresponded to an 18% increase in
the initial volume (25 L/kg flour) of the liquid
67
phase. After digestion, all the digests were treated
by conventional methods to isolate fine screenings
(overs), supernatant, tailings, and starch. Figure 1
gives a partial mass balance for the optimum
digestion conditions derived in the modelling ex-
periments (see discussion below). The screenings
and tailings were mostly endosperm cell wall ma-
terial as indicated by their low protein levels.
The supernatant in the preferred process gave no
precipitate upon addition of trichloroacetic acid,
which indicated that the proteins had been con-
verted to peptides and/or amino acids. Under the
mild alkaline conditions (at most pH 10 for 30 h
at 55 C) used for digestion, it is presumed that
the amino acids in the protein underwent minimal
structural changes. Some lysinoalanine may have
formed during digestion, but racemisation should
have been slight26.
After a digest was neutralized a high grav-
itational force (10 000 g) in the centrifugation step
was necessary to sediment all the starch, as evid-
enced by the clarity of the supernatant and by the
appearance of the dark tailings layer atop the
starch. The tailings layer contained no protein
(<01%), indicating that layer was mostly fine
fibrous material must likely discoloured by phen-
olic compounds. In contrast, the tailings fraction
from barley starch isolated by protease digestion
contained 23% protein27.
Modelling studies of protease digestion of
WMRF; recovery of rice starch and protein
contamination
In the first modelling study (pH 8595, protease
0515% and time 50150 h), starch recovery
was 7395%, and protein contamination was 10
16%. Theoretically, the lowest protein con-
tamination in isolated rice starch is c. 012%, which
is the level of granule-bound starch synthases in
the granule28. In the second modelling experiment,
at the same protease level but with increased
alkalinity (pH 90100) and increased digestion
time (120180 h), starch recovery increased to
8695%, yet the starch still contained 0610%
protein.
In the final modelling experiment, protease
levels were narrowed to 0715%, digestion times
were increased to 180300 h, but alkalinity was
kept the same (pH 90100). The final regression
models are given in the following equations.
Starch recovery (%)=16721103x12838 x2
68
Wet-milled rice flour (17.5 g, d.s.)
Screenings, or overs (solids 0.3 g)
Supernatant
Tailing
Solids 1.2 g
Protein <0.1%
Starch recovered 12.7 g (d.s.) (95.1%)
Protein level 0.52%
Figure 1 Isolation of rice starch from wet-milled rice flour (WMRF) after alkaline protease digestion under optimum
conditions.
1519 x12+0057 x22+1524 x1x30654 x32.
Protein contamination (%)=2745+1620 x1
0205 x25120 x3+0216 x12+0007 x1x2
+0001 x220225 x1x3+0014 x2x3+0255 x32.
In the equations x1 is protease level in % based
on WMRF; x2 is time in h and x3 is pH units. The
models showed that the three variables of pH,
protease level, and digestion time explained the
variability of starch recovery with an R2 092 and
the variability of protein contamination of starch
with an R2 098. The response surfaces in Figure
2(b, c) show that at the medium and high levels,
respectively, of protease, a 513% rise in starch
recovery occurred as alkalinity increased from
pH 90 to 100 both at 180 and 300 h of digestion,
which agrees with increased swelling of rice protein
and increased digestion as pH approached 10. At
the low level of protease shown in Figure 2(a)
starch recovery at pH 100 was overall 46% lower
than at the two higher levels of protease [Figure
2(b, c)]. Those results indicate that the protease
N. Lumdubwong and P. A. Seib
Starch 13.3 g
Protein 1.4 g
Add 0.001 M NaOH (50 mL) and
protease (0.22 mL).
Adjust and maintain pH 10.0 with 1 M NaOH
using pH controller.
Stir at 18 h and filter through wire mesh (75 m).
Starch slurry
Centrifuge at 3,000 g for 20 min,
discard supernatant.
Wash sediment with water (2 50 mL)
and centrifuge at 3,000 g for 15 min.
Adjust the slurry to pH 7 with 1 M HCl.
Centrifuge at 10,000 g for 20 min,
discard supernatant, and scrape the dark tailings layer off
and discard.
Starch
Wash the sediment (3 50 mL) with water
and centrifuge at 3,000 g for 15 min.
Starch cake
Dry at 40 C for 48 h.
Starch
probably was undergoing some denaturation at
pH 10. Remaining unexplained is the reduced
recovery of starch at the intermediate digestion
time of c. 24 h observed in Figure. 2(ac).
The response surfaces for protein contamination of
the starch are shown in Figure 3. At all three levels
of protease, protein contamination of rice starch was
reduced as digestion time increased from 18 to 30 h.
However, as pH varied from 90 to 100, protein
contamination reached a minimum value at
pH 9497. At a given level of protease and without
denaturation of the protease, prolonged digestion with
agitation would be expected to erode more and more
protein from the surface of starch granules. However,
the minimum protein contamination observed at
pH 9497 indicates again that some of the protease
may have been denatured on the surface of starch
granules at c. pH 10. Protein-coated granules might
repel each other during centrifugation, and that could
explain the reduced recovery of starch at prolonged
digestion times (Figure 2).
 Isolation of rice starch 69

(a)
(a)

100 1.0

0.8
95

(%)
ry (%)

t
Protein conten
0.6
Starch recove

90
10.0
0.4
9.8
85 30 9.6

pH
0.2
27 9.4

80 24 h) 0.0 9.2
21
e(

9.8 24 27 9.9
21
m

9.6 30
Time (h)
Ti

pH 9.4 9.2
9.0 18

(b)
(b)

100 1.0

0.8
95
t (%)
ry (%)

Protein conten

0.6
Starch recove

90
10.0
0.4
9.8
85 30 9.6

pH
0.2
27 9.4
80 24 0.0 9.2
h)

21
e(

9.8 24 27 9.9
21 30
m

9.6 Time (h)

Ti

9.4
pH 9.2
9.0 18

(c)
(c)

100 1.0

95 0.8
t (%)
ry (%)

Protein conten

0.6
Starch recove

90
10.0
0.4
9.8
85 30 9.6
pH

0.2
27 9.4

80 24 0.0 9.2
h)

21
e(

9.8 24 27 9.9
21 30
m

9.6 Time (h)

Ti

9.4
pH 9.2
9.0 18
Figure 3 Response surfaces of protein contents of rice
Figure 2 Response surfaces of starch recoveries after di- starch after digestion of WMRF at pH 90100; digestion
gestion of WMRF at pH 90100, 180300 h and protease times of 180300 h; and protease levels of (a) 07%, (b)
levels of (a) 07%, (b) 11%, and (c) 15% (WMRF basis). 11%, and (c) 15% (WMRF basis).
70 N. Lumdubwong and P. A. Seib
Figure 4 Boundary lines on contour plots giving 94%
recovery of starch ( ) with 05% protein (- - -) after
digesting WMRF (c. 34% solids) with 11% protease at 55 C.
Shaded area indicates pH and digestion times satisfying both
requirements.
At the lowest level (07%) of protease [Fig.
3(a)], the shortest digestion time to ensure 05%
protein contamination of the starch was 24 h. To
reduce digestion time, a protease level of 11%
was chosen. Setting the requirement of [94%
recovery of rice starch with 05% protein (db)
at 11% protease for digestion of WMRF gave the
solution area shown in Figure 4. Finally, we chose
the digestion conditions of pH 100, 11% protease,
and 18 h digestion, which experimentally gave
95% recovery of starch with 05% protein. The
isobar of 05% protein contamination shown in
Figure 4 predicts that a minimum digestion time
of 21 h is needed at pH 10, but experimentally we
found that 18 h digestion was sufficient to obtain
starch with 05% protein. Under the conditions
preferred for rice starch isolation, uptake of alkali
was most rapid in the first 3 h of digestion (data not
shown), and at that digestion time, the regression
equation predicts the protein contamination of
rice starch to be 12%. Use of shear forces might
reduce the protein contamination from 12% to
below 05%, and thereby reduce digestion time
from 18 h to 3 h. However, we did not investigate
that possibility.
Rice starch isolated using the NaOH method
The initial NaOH concentration was 50 times
greater in the NaOH procedure (Figure 5) than
in the protease digestion at pH 10. However, a
total of c. 9 g (022 eq) of NaOH was consumed to
produce 1 kg of rice starch at 10% m.c. with
<05% protein for the NaOH procedure vs c. 25 g
(062 eq) for the protease procedure at pH 10,
11% protease, and 18 h digestion. The con-
sumption of alkali was due to neutralisation of the
carboxyl groups that were released by the action
of the protease.
If the average formula weight of the amino acids
in rice protein is assumed to be 110 g per mole,
then each kilogram of the flour (wb) containing
70 g of protein is estimated to contain 064 eq. of
amino acids. The consumption of alkali (062 eq)
observed during protease digestion indicated al-
most complete hydrolysis of the rice protein to
amino acids.
Starch recovery with the NaOH method (Figure
5) was c. 10% below (85% vs 95%) that with the
protease method. The slurry of WMRF at pH 12
in the NaOH method had a higher viscosity than
the slurry after protein digestion, and the high
viscosity reduced the recovery of starch during
sieving and centrifugation. The increased viscosity
of the pH 12 slurry of WMRF may be attributed
to dissolved proteins and non-cellulosic poly-
saccharides. Total solids recoveries were almost
the same (81 vs 78%) for the two methods (Figures
1 and 4).
The protein contamination of the rice starch in
the NaOH method (04%) was less than that (05%)
found for the preferred protease method. Rice
starch purified by either method contained the
same levels of starch, damaged starch, and ash
(Table III). The 1996 prices of reagents used in
starch isolation are given in Table IV. The cost
of the reagents to isolate 1 kg of rice starch (w.b.)
by the NaOH procedure was estimated to be
$076, which was one-half that for the protease
method ($175). About 55% of those costs are
attributable to the protease (Table IV).
Physical properties of rice starch isolated by the
protease and NaOH methods
Rice starch isolated by the NaOH method ge-
latinized at approximately 2 C lower than the
starch isolated by protease digestion (Table V). In
addition, loss of the enthalpy of gelatinisation was
 Isolation of rice starch 71

Wet-milled rice flour (17.5 g, d.s.)

Starch 13.3 g
Protein 1.4 g
Add 0.05 M NaOH (50 mL) and
stir 3 h at room temperature.
Screenings, or overs (solids 0.5 g)

Filter through wire mesh (75 m).

Starch slurry
Centrifuge at 3,000 g for 20 min,
Supernatant discard supernatant.
Wash sediment with water (50 mL)
and centrifuge at 3,000 g for 15 min; repeat.
Tailing
Solids 1.8 g Add water (50 mL) and adjust the slurry to pH 7 by 1 M HCl.
Protein <0.1%
Centrifuge at 3,000 g for 20 min,
discard supernatant, and scrape the dark tailings layer off
and discard.
Starch
Wash with water (3 50 mL)
and centrifuge each time at 3,000 g for 15 min.
Discard supernatant and tailings.
Starch cake
Dry at 40 C for 48 h.
Starch
Starch recovered 85.4%
Protein level 0.42%

Figure 5 Isolation of rice starch from WMRF by the sodium hydroxide (NaOH) method11.

Table III Recoveries and compositions of rice starches isolated by two methods

Protein
Recovery content
(%) (%) Starch Damaged Fatty acid
content Ash starch methyl estersc
Methoda Theoryb Exper. Theoryb Exper. (%) (%) (%) (mg/100 g)

Protease
Digestion 971 951 060 052 990 016 21 544
NaOH
Extraction ND 854 ND 042 989 019 26 390
a
The protease method was digestion of WMRF at 3437% flour solids, 55 C, pH 100 with
I.1% protease (flour basis) for 18 h. The sodium hydroxide (NaOH) method was extraction of
WMRF with 005 M NaOH at 25 C.
b
Predicted recovery and protein content were calculated from the regression equation in
Table III.
c
Fatty acid methyl esters were determined by gas-liquid chromatography after acid-catalyzed
methanolysis of starch followed by lipid extraction and methylation of the extract.
72 N. Lumdubwong and P. A. Seib

Table IV Material costs to produce 1 kg of rice starch (db) than that from the alkali method (Table V); also,
by the NaOHa and Proteaseb Methods it was lower in yellow hue (b=+11 vs +21) but
Amount (kg) Cost ($) almost the same in green hue (a=14).
The pasting peak of the NaOH-isolated rice
Material Pricesc, NaOHd Proteaseb NaOH Protease starch at 93% solids in water was higher than
$/kg that of the protease-isolated rice starch, as was its
WMRFd 048 154 138 074 066 setback (Figure 6). The reduced lipid in the alkali-
NaOH 250 0009 0028 002 007 isolated rice starch (Table III) probably accounted
HCl (36%) 006 006 006 0004 0004 for the difference in paste consistency. It is well
Protease 59 0 0019 0 102 known that the monoacyl lipids in cereal starches
Total Cost 076 175 reduce hot-paste swelling and solubility.30,31 Set-
back is increased by amylose leached from swollen
a
NaOH method is extraction of WMRF with 005
sodium hydroxide at 25 C. Yield of starch 65%.
starch during pasting. The swelling power of the
b
The protease method was digestion or WMRF at 3437% NaOH-isolated rice starch measured at 925 C
flour solids, 55 C, pH 100, and 11% protease for 18 h. was the same as that of the protease-isolated starch
Yield of starch=73%.
c
(Table V), but its solubility was elevated. The
Chemical Marketing Reporter, Apr. 24, 1995. increased solubility of the NaOH-isolated starch
d
Wet-milled rice flour (WMRF) purchased in local market,
Bangkok, Thailand, 1996.
was in accord with its increased setback in the
pasting curve (Figure 6). To measure swelling
power and solubility of rice starch pastes, the
gravitational force in the measurement was in-
c. 13%. Lugay and Juliano29 previously found that creased to 7700 g from the 1000 g used previously
rice starch isolated in 005 NaOH had reduced for wheat and corn starches.23 The increased grav-
x-ray crystallinity compared to detergent-isolated itational force was required to sediment the swol-
rice starch. The reduction of rice starch lipids in len, but relatively small, rice starch granules.
005 NaOH (Table III) might have allowed extra
swelling of the starch and reduced the gel-
atinisation temperature. The similarities of the
CONCLUSIONS
magnitude and the range of gelatinisation tem-
peratures of the rice starch isolated using protease- A food-grade, alkaline protease can be used to
digestion compared to those of rice flour indicate recover 95% of the rice starch from WMRF after
no annealing of the starch during digestion at it is digested at 55 C and pH 10 for 18 h. Aqueous
55 C at pH 100. Rice starch isolated by protease NaOH must be added during the digestion to
digestion gave a slightly higher lightness (L) value maintain pH, especially during the first 34 h. The

Table V Characteristics of rice flour and rice starches isolated by the NaOH and the protease methodsa

Materialb Gelatinisationc Starch or Flour Swelling Solubilitye

Colourd Powera

To(C) Tp(C) 7c(C) DH( J/g) L a b (g/g) (%)

Rice starch by protease 676 a 739 a 793 b 131 a 948 b 14 a 11 c 136 a 97 b

digestion
Rice starch by NaOH 656 b 728 c 799 a 112 b 934 c 14 a 21 b 136 a 123 a
extraction
Rice flour 678 a 735 b 793 b 102 b 932 d 15 b 29 a
a
Data in columns followed by the same letter are not statistically significant at p<005.
b
The protease method was digestion of WMRF at 3437% flour solids, 55 C, pH 100, and 11% protease for 18 h. The
sodium hydroxide (NaOH) method was extraction of WMRF with 005 M NaOH at 25 C.
c
Measured by DSC at a starch/water ratio of 10/25 (w/w).
d
Commercial rice starch (Sigma) and WMRF gave L-values of 932 and 953, respectively with LSD005=0014.
e
Measured at 925 C and 167% starch solids.
 Isolation of rice starch 73

Temperature ( C)
50 95 95 50 50
350 100

300
80

250
Consistency (RVU)

Temperature ( C)
60
200

150
40

100

20
50

0 0
0 5 10 15 20
Time (min)

Figure 6 Rapid viscograms of rice starches isolated either by extraction of WMRF with 005 sodium hydroxide (
) at 25 C or with protease digestion at pH 100 and 55 C ( ). Solid line ( ) is the temperature profile. The
viscograms were measured at a dry starch solids level of 93% (w/w) in water and at a heating and cooling rate of 75 C/
min.

rice starch isolated by protease digestion has
c. 05% protein and is bright white in appearance.
Compared with rice starch isolated by slurrying
and washing with 005 NaOH (pH c. 12) at
25 C, the protease-isolated starch contains more
lipid and its hot paste peak and setback con-
sistencies are lower. The recovery of starch was
10% higher in the protease method, but twice as
much NaOH was required compared to the NaOH
method. The protease method generated a dilute
solution of amino acid salts as a co-product,
whereas the NaOH method gave protein. Our
results suggest that a two-step starch isolation
procedure could be developed. In the first step,
fluid shear would be used on rice flour to produce
rice starch with 24% protein, and in the second
step, alkaline protease digestion could reduce pro-
tein contamination of the rice starch to <05%.
Acknowledgements
The authors thank Dr Thomas M. Loughin for his
assistance with the statistical methods. This is con-
tribution no. 99-325-J from the Kansas Agricultural
Experiment Station, Manhattan, KS.
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