Study of Two Main Approaches-Electrophoresis and Chromatography as Varietal Identification Methods in Rice
Rice is a major worldwide crop that cultivated in the most areas of the
north of Iran (Mazandaran and Gillan Province). An increase in the assortment
of rice varieties is making it progressively more difficult to distinguish
between the many cultivars by traditional visual identification methods.
The more advanced identification techniques of electrophoresis and chromatography
offer an effective solution to this emerging identification dilemma. This
paper reviews the application of these two evaluation techniques. An Electrophoresis
analysis includes gel electrophoresis and capillary electrophoresis and
compares them with a popular chromatography technique, namely reversed-phase,
size exclusion anion-exchange high performance liquid chromatography (HPLC).
This paper will also include an interpretation of the results.
Rice, in terms of total production and value, is the most important cereal
in the world. As a crop, it is especially important to developing countries
(Juliano, 1995). There are hundreds of rice varieties. The quality of
these different varieties varies considerably and as a result, proper
identification of each variety has become a crucial matter (Juliano, 1995;
Lookhart and Wrigley, 1995). When countries export rice, a declaration
of variety is often used as the basis for defining quality type. Specialty
varieties of rice include rice varieties such as Italian Arborio, Thai
Jasmine, Indian Basmati, Japanese Short Grain, Japanese Mochi, Wild Rice,
Red Rice, Black Rice and others. Some of these varieties are now grown
in the United States and some are still imported. The founder and president
of the company was the Senior VP of Marketing for the largest rice milling
and exporting company in the U.S. for 6 years in the late 1980s. During
this time period, he spent most of his time traveling the world buying
and selling rice and studying the varieties grown in different countries.
Sage V Foods takes advantage of this knowledge to help customers source
specialty varieties. As the number of rice varieties continues to increase,
visual methods for identification become increasingly unreliable (Juliano,
1995). An arising practice of developing rice with unusual quality characteristics
has further heightened the necessity of establishing a set of requirements
for identification (Juliano, 1995; Cooke, 1995). Many studies have reported
various methods of identifying rice based on protein composition. Polyacrylamide
gel electrophoresis (PAGE) and high-performance liquid chromatography
(HPLC) are among todays laboratory techniques of choice. Gel electrophoresis
methods, acid (A-PAGE) or sodium dodeeyl sultfate (SDS-PAGE), are established
techniques for separation of proteins. However, they have several drawbacks
that include the use of toxic reagents, long analysis times and data that
are difficult to quantify or interpret (Bietz and Schmaizreid, 1992).
Recently Bean and Lookhart (2000) reported that rice cultivars were consistently
differentiated in less than 15 minutes by capillary electrophoresis (CE).
Capillary electrophoresis (CE) is a comparatively modem method of identification
with the potential to distinguish between cereal varieties. It has been
used to characterize cereal proteins, demonstrating excellent resolution
and reproducibility. Additionally, CE is a very fast method and does not
necessarily require extensive skilled manpower (Lookhart and Bean, 1995).
Separation of rice proteins: Provided efficient methods of protein
analysis can be developed, a determination of the grain-protein composition
using this analysis shows promise as a reliable identification procedure.
Various other methods of analysis have been evaluated; most of these have
involved either chromatographic or electrophoretic techniques (Wrigley
et al., 1982; Bietz and Simpson, 1992).
The protein of rice varies from 7 to 15% in brown rice and from 6 to
13% in rough and milled rice (dry basis). Milled rice protein is 15% salt
soluble (albumin and globulin), 20% ethanol soluble (prolamin) and 65%
alkali soluble (glutelin) (Ogawa et al., 1987) Separation
of these rice protein can be performed by either traditional (Osborne
solubility) methods or modern methods.
Traditional method (Osborne solubility): According to the Osborne
solubility method (Osborne, 1907), cereal proteins are classified into
five groups on the basis of their solubility in a series of solvents.
However, the Osborne system does not provide accurate quantitative data
and many researchers have criticized its weak points (Shewry and Mifflin,
1985). These detractors emphasize that the extractability of the proteins
is strongly dependent on many factors. These include flour particle size,
the composition of the extraction solvents, the conditions of extraction
(such as the fineness of milling or the vigor of shaking and stirring),
the number and sequence of extraction steps, the temperature and also
the type of mixing. Additionally, they point out that cross-contamination
of fractions can occur, as can be seen from the wide ranging results of
many different workers. However, the classic Osborne procedure is still
widely used and is reserved as the first step of the purification process.
Modern techniques of protein separation: As discussed earlier,
the separation of protein by the Osborne system is suitable only as the
first step for a purification process. To have more precise preparative
procedures for the preparation and identification of protein fractions,
other techniques such as electrophoresis or chromatography should be employed.
Chromatographic and electrophoretic techniques have long been used for
protein identification. Chromatography is an analytical tool which selectively
partitions components between two phases, while in electrophoresis, charged
species are separated, based on their electrophoretic mobilities.
Chromatographic techniques: Chromatographic techniques have long
been used for cereal protein isolation and characterization. These include
size-exclusion high performance liquid chromatography (SE-HPLC), ion exchange
chromatography (IE-HPLC) and reversed phase chromatography (RP-HPLC) (Beitz,
1986). SE-HPLC separates proteins primarily by size IE-HPLC fractionates
them by charge differences (Huebner and Wall, 1966) and RP-HPLC separates
them by differences in hydrophobicity.
Size-exclusion chromatography (SE-HPLC): Size-exclusion chromatography,
also called gel permeation or gel filtration, achieves fractionation with
a gel column. It separates molecules according to their size (Autran,
1994). Size-exclusion high performance chromatography (SE-HPLC) has been
applied to cereal proteins to relate the quantity of protein fractions
to breadmaking characteristics (Huebner and Bietz, 1985; Dachkevitch and
Autran, 1989; Huebner and Bietz, 1993). It has commonly been used to provide
information about protein aggregates and for further studies into their
structures and interactions between the protein components (Autran, 1994).
Ion-Exchange chromatography (IEC): Ion-exchange fractionates proteins
by differences in charge since, a charged compound in solution is attracted
to and binds electrostatically to a support bearing a group with the opposite
charge. However, it has not become a common technique in cereal protein
separation due to the limited applications of various methods of ion-exchange
HPLC to cereal proteins. In contrast, it has been widely used for biological
and medical purposes (Batey, 1994; Bietz, 1986).
Reversed phase chromatography: Reversed-phase high performance
liquid chromatography (RP-HPLC) separates proteins on the basis of differences
in surface hydrophobicity (Unger et al., 1991). It has been used
to identify rice proteins by several researchers (Huebner et al.,
1991; Hussain et al., 1989; Lookhart et al., 1987) who have
shown that rice varieties could be identified by RP-HPLC. The method demonstrated
that eleven brown rice varieties were successfully differentiated, but
the method was less effective on sister-line IR rice, excepting IR 36
and IR 42. However, various bonded phases from different manufacturers
differ significantly in selectivity, which causes elution patterns to
vary. Solvent gradient modification can also significantly affect resolution
(Huebner et al., 1991).
Electrophoresis techniques: Although chromatographic techniques
have been gaining acceptance for protein separation, electrophoresis has
remained the most popular procedure for analysis of cereal storage proteins
(Lookhart and Wrigley, 1995). Various electrophoretic techniques have
been reported such as polyacrylamide gel electrophoresis (PAGE) at pH
3 and pH 7.6-8.9 gradient, sodium dodecyi sulfate (SDS) and isofocusing
(IEF) (Huebner et al., 1991; Guo et al., 1986). Starch gel
electrophoresis was reported to separate rice proteins from 14 rice varieties
(Li and Worland, 1993) Capillary electrophoresis has been shown to offer
excellent capabilities for rice identification in recent years (Lookhart
and Bean, 1995; Bean and Lookhart, 2000).
Gel electrophoresis: Gel electrophoresis has long been used for
protein separations. Eiton and Ewart (1960) were first to describe the
use of starch ge1 electrophoresis to separate cereal proteins. Other studies
have reported the improvement of this procedure for varietal identification
of wheat. However, starch gel electrophoresis may have poor reproducibility
due to variable starch quality. Polyacrylamide gel electrophoresis (PAGE)
using a synthetic polymer of acrylamide monomer, usually as acid-PAGE
or with sodium dodecyi sulphate (SDS-PAGE), provides reproducible results.
On the other hand, the main drawbacks of this method are that the preparation
of gel usually requires a long time, the toxicity of acrylamide and data
are difficult to quantify and interpret (Loohkart and Bean, 1995; Hames
and Rickwood, 1981).
The ability to correctly differentiate and identify cultivars of cereal
grains is an important aspect of cereal science. Polyacrylamide gel electrophoresis
(PAGE) and high-performance liquid chromatography (HPLC) are the laboratory
methods of choice. Gel electrophoresis methods, usually acid (A)-PAGE
or sodium dodecyl sulfate (SDS)-PAGE, are the established techniques for
separation of proteins. However, they have several drawbacks that include
the use of toxic reagents, long analysis times and data that are difficult
to quantify and interpret. The electrophoretic methods and extraction
conditions used to differentiate cultivars of all major cereal crops were
recently reviewed by Lookhart (1990). Lookhart and Wrigley (1995) reviewed
electrophoretic methods for varietal identification. The methods varied
ideally in extraction procedures, in proteins analyzed and in the type
of electrophoresis. Analysis times ranged from 1 to 12 h (Lookhart, 1990;
Lookhart and Wrigley, 1995).
Jahani et al. (2002) studied the Variation of glutelin seed storage
protein in Bangladesh rice cultivars. based on their work Glutelin, a
major storage protein in rice, is synthesized as 57 kD proglutelin on
the rER, transported to the vacuole via the Golgi apparatus and formed
by proteolysis of the proglutelin through post-translational cleavage
into acidic (alpha) and basic (beta) subunits in the vacuole (Yamagata
et al., 1982; Takaiwa et al., 1986; Masumura et al.,
1989). Uemura et al. (1996) reported that sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) gel electrophoresis
are useful to detect the variation of glutelin seed storage protein. Kagawa
et al. (1988) found the variation in glutelins of local rice cultivars
by SDS-PAGE analysis. Satoh et al. (1990) reported that a large
variation exists in glutelin polypeptides in rice collected in Tanzania.
This report deals with the variation of glutelin polypeptides in rice
cultivars of Bangladesh, an area considered to be one of the centers of
origin of cultivated rice (Chatterjee, 1951).
Five hundred and seventy six Bangladesh rice cultivars preserved at the
Laboratory of Plant Genetic Resources, Kyushu University, Japan, were
used in this study. Extracted proteins were separated by SDS-PAGE using
the discontinuous buffer system of Laemmli (1970) on a slab gel containing
a linear of 15 to 25% acrylamide, 0.05 to 0.67% BIS concentration gradient.
Rice glutelin was composed of alpha and beta subunits, which were separated
into alpha-1 (39 kD), alpha-2 (38 kD), alpha-3 (37.5 and 37 kD) and alpha-4
(34 and 33 kD) for alpha subunit and beta-1 (23 kD), beta-2 (22.5 kD)
and beta-3 (22 kD) bands for beta subunit, respectively. Uemura et
al. (1996) reported that the alpha-3 band of Japanese rice cultivar
Kinmaze is smaller in molecular size than that of rice cultivar IR36 developed
at IRRI, while the alpha-4 (34 kD) band of Kinmaze is larger than that
of IR36 (33 kD). Bangladesh rice cultivars varied significantly in SDS-PAGE
profiles of glutelin storage protein. In addition to Kinmaze (type 1)
and IR36 (type 5) types, mutant types with decreased alpha-2 band and
with two alpha-3 bands were observed. One mutant was characterized by
decreased intensity of alpha-2 band, as alpha-2 deficient mutants induced
by N-methyl-N-nitrosourea (MNU) treatments (Satoh et al., 1997).
The other mutant possessed two alpha-3 bands with different molecular
mass (type 2). The decreased intensity of alpha-2 band was accompanied
by an increased intensity of alpha-1 band, suggesting that the total glutelin
content remained unchanged. On the other hand, the cultivars having decreased
intensity of alpha-2 bands (types 3 and 4) differed in SDS-PAGE pattern
of alpha-3 band, indicating that alpha-2 and alpha-3 bands were controlled
by different genes. This is the first report on spontaneous glutelin mutants
detected by SDS-PAGE from Bangladesh rice cultivars, suggesting that rice
genetic resources of Bangladesh provide a rich source of genetic diversity.
In Bangladesh, rice is grown in three seasons, aus in summer, aman in
autumn and boro in winter. They showed that ecotypic distribution for
glutelin variation. In all of the ecotypes, type 5 was most frequent,
indicating that selection preference was higher for this type. Types 2,
3 and 4 were confined to T. Aman ecotype, even if their collection places
were different. SDS-PAGE separates proteins based on molecular masses,
while IEF separates them depending on electric charges. SDS-PAGE analysis
does not elucidate the genetic traits of the polypeptides because each
of the SDS-PAGE bands consists of several polypeptides by IEF (Wen and
Luthe, 1985). After extraction by 1% lactic acid, the glutelins of 74
cultivars were analyzed by the horizontal slab gel IEF system. At least
13 types were detected among the cultivars studied for IEF. Types 1 and
5 of SDS-PAGE were separated to types 4 to 10 and types 14 to 16 of IEF
respectively, suggesting that both SDS-PAGE and IEF analyses were important
for detecting glutelin variation in rice. The alpha-2 band deficient mutants
lacked in pI 6.80 band for IEF, while all the cultivars having alpha-2
band, such as IR36, possessed pI 6.80 band, suggesting that pI 6.80 band
was the major polypeptide component of alpha-2 subunit. Similarly, increased
intensity of pI 6.59 band and the presence of pI 6.30 band in types 12
and 13 in common suggested that they were the polypeptide components of
alpha-1 subunit. These results suggest that the mutated subunits were
controlled by structural genes. Meanwhile, IEF profile of cultivars having
two alpha-3 bands showed increased intensity of pI 7.52 band and reduced
intensity of pI 7.19 band, a pattern similar to IR24 by IEF. Rice seed
stores most of the proteins as dilute acid/alkali soluble glutelin (about
75% of total protein) which is superior in quality due to its easy digestibility
and the presence of high amount of first limited amino acid, lysine (Huebner
et al., 1990). The glutelin variation observed in Bangladesh rice
cultivars may serve as useful materials to improve rice grain quality.
Capillary electrophoresis: Capillary electrophoresis (CE) is a
modem analytical technique. It has proved to be rapid, sensitive and can
be automated, providing high resolution, separation and reproducibility
(Lookhart and Bean, 1995). Furthermore, the availability of advanced commercial
instrumentation of CE has led to its use for the analysis of various compounds
such as proteins, sugars, oligosaccharides, anions and soluble vitamins
(Cancalon, 1995). Capillary zone electrophoresis (CZE) of endosperm storage
proteins was used to differentiate cultivars of both oats and rice in
less than 12 min (Lookhart and Bean, 1995). This is the first study that
proteins of these two cereals have been separated by CZE. Cultivars were
chosen for the difficulty of different-were no differences tiating them
by other means, electrophoretic or chromatographic. Ethanol (70%) extracts
of the oat samples were separated on a 20-yrm i.d. untreated fused-silica
capillary, whereas rice samples were extracted with 60% 1-propanol and
the solubilized proteins were separated on a 50-,um i.d. untreated fused-silica
capillary. The CZE separation buffer was 0.1M phosphate, pH 2.5, containing
0.05% hydroxypropylmethylcellulose (HPMC). Most cultivars were differentiated
quickly and easily. Only the patterns of two rice cultivars, IR36 and
IR50, were nearly identical. There were no differences between IR36 and
IR50 extracts by high-performance liquid chromatography (HPLC) or acid
(A)-polyacrylamide gel electrophoresis (PAGE). CZE is a faster method
of separating endosperm storage proteins than A-PAGE and separates as
least as well and better in most cases than either A-PAGE or reversed
The use of CE for rice varietal identification has been reported over
the past decade (Lookhart and Bean, 1995; Bean and Lookhart, 2000). At
present, the use of CE for variety identification has so far involved
the use of sophisticated equipment that has been of considerable size
and cost, warranting a place in a central laboratory. In the future, the
approach of CE would offer the prospect of small, portable equipment that
would offer the ideal combination of convenience, speed of analysis and
good resolution, together with portability and consequently on-site use
(Wrigley and Bekes, 2002).
Capillary Zone Electrophoresis was successfully used for the first time to
differentiate rice and oat cultivars (Although RP-HPLC was able to distinguish
oat cultivars with identical A-PAGE patterns, the different separation modes
enabled more differences to be detected with CZE than with RP-HPLC. In addition,
CZE analysis required considerably less time (24 min) to analyze the storage
proteins than A-PAGE (2-4 h) and were consistently shorter than RP-HPLC analysis
times (30-60 min), even when equilibration was considered. CZE is complementary
to RPHPLC, where it has the advantage of speed and resolution with the similarity
of ease of automation and differs in mode of separation, charge versus hydrophobicity.
Oat avenins were separated in less than 6 min by free-zone capillary electrophoresis
using a low pH phosphate buffer. Rice cultivars, both from the U.S. and from
the IRRI, were consistently differentiated by CZE in less than 12 min. U.S.
long-grain cultivars L202 and Newbonnet, which had identical HPLC patterns,
were also readily differentiated by CZE. Cultivars with close genetic relationships
may exhibit similar or identical prolamin patterns by CZE, as they do by A-PAGE
or RP-HPLC. The fast speed, high resolution and complementary nature of CZE
make it well suited to quickly differentiate rice cultivars, even those with
similar or identical A-PAGE and RPHPLC patterns (Lookhart and Wrigley, 1995).
Variety identification of rice: Identification of rice varieties
is very important because of the differences in quality associated with
different varieties (Juliano, 1995). The declaration of variety for rice
deliveries is used in many rice producing countries as a basis for defining
quality type (Lookhart and Wrigley, 1995). As the number of rice varieties
increase over the years, visual methods for identification of these varieties
have become insufficient. Because there is a need to keep pace with the
growing number of quality characteristics, the requirements for varietal
identification are under continuous development (Lookhart and Wrigley,
In summary, the routine methods can be compared as in Table
1 (Wrigley and Bekes, 2002). CE and RP-HPLC have advantages with respect
to speed of analysis, instant data interpretation and low labor cost.
In contrast, gel electrophoresis has potential for greater through-put
of samples with relatively low capital costs for the extra equipment needed.
Health risks are lower in CE and pre-cast gel than in RP-HPLC and conventional
Use of storage protein for variety identification: Storage proteins
(tertiary sementides) especially prolamin have long been used for varietal
identification because of the consistency of prolamin (gliadin) composition.
This despite a wide range of variations in growth and treatment conditions.
This aspect of phenotype for the genetic material is extremely stable
under variable environmental conditions, such as growth location, growth
season and soil conditions. Lookhart et al. (1987). Huebner and
Bietz (1985) also studied the effects of growth locations and climactic
conditions on gliadin composition, using RP-HPLC. He found that total
amounts of specific gliadins vary with both location and conditions, but
the qualitative aspects of the chromatograms of gliadin were not altered.
Recently, Anjum et al. (2000) reinforced previous reports that
gliadin composition obtained from A-PAGE bands was not affected by growth
locations and crop years, with only minor differences in band densities
being observed (Bietz, 1986; Hames and Rickwood, 1981).
Summary: The difficulties in identifying different rice varieties
are compounded by the use of insufficient, ineffective and subjective
visual identification methods.
The relative effectiveness of routine methods of variety identification
based on protein analysis (Wrigley and Bekes, 2002
Two main techniques have shown improved abilities to better separate
rice proteins and hence provide more effective and reliable variety identification.
However, it has been found that the two techniques share a frequently
encountered problem, being that a particular variety can be polymorphic
for protein composition depending on the electrophoretic or chromatographic
method used; that is, analysis of individual grains of the same variety
gives more than one electrophoretic or chromatographic pattern due to
the presence of multiplebiotypes within the variety. A solution to this
problem may lie in analysis of whole meal samples, which account for the
main biotypes to define the variety. Alternatively, the use of a less
discriminating method may reduce the risk of distinguishing between biotypes.
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