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Journal of Cereal Science 26 (1997) 211221

Simultaneous Spectrophotometric Determination of


Amylose and Amylopectin in Starch from Maize
Kernel by Multi-wavelength Analysis
M. Se ne, C. The venot and J. L. Prioul
Universite Paris XI et Universite Paris VI, Institut de Biotechnologie des Plantes (associe au C.N.R.S.
U.R.A. 1128), Laboratoire Structure et Me tabolisme des Plantes, Batiment 630, 91405 Orsay Cedex,
France
Received 31 July 1996
ABSTRACT
A six-wavelength spectrophotometric measurement of amylose- and amylopectin-iodine complexes
has been improved and adapted for the lipid-rich maize kernel. Use of a spreadsheet program allowed
graphical and mathematical resolution of a six-wavelength multi-component analysis. Lipid removal
by aqueous acetone was found to minimise interference in the amylose determination. Solubilisation
of maize starch in 4x NaOH at ambient temperature was found to be rapid and ecient, without
aecting the stability of amylose or amylopectin. The method was accurate, simple and applicable to
routine analysis of the genotypic variability of starch composition in normal and mutant maize grains.
1997 Academic Press Limited
Keywords: amylopectin, amylose, spectrophotometry, kernel, maize, starch.
are branched at one or several positions. The single
INTRODUCTION
chain amylopectin molecule with one reducing end
Starch, the major storage component in mature
is called the C chain. The branches formregularly
maize (Zea mays) kernel is composed of amylose
spaced clusters along the main axis
1
. In addition,
(AM), and amylopectin (AP). Amylose is essentially
starch frequently contains lipid, especially in
a linear polymer of glucose residues, linked by
cereals
2
.
(14)-glucosidic bonds plus occasional (16)-
In normal maize lines the AM is organised in
glucosidic linkages. Amylopectin is a highly
an helical conformation, tightly associated with
branched polymer. Approximately every 20 gluc-
lipids. The complex between amylose and lipids
ose units along a linear chain in which the glucose
have been established using techniques based on
units are linked by (14) bonds, a (16)- 13
C-cross polarisation-magic angle spinning nuc-
glucosidic bond produces a branch, which in turn
lear magnetic resonance
2
. Acomplex is also formed
may itself be branched. Three chain typesA, B
with tri-iodide ion giving a blue colour with
max
and Care formed in an asymmetric process. A
at 630 nm. The iodine anity of AM varies be-
chains are linked only by their reducing ends, they
tween 17 to 22 g I
2
per 100 g AM in maize,
are not substituted at carbon six. The B chains
according to the genotype, whilst the anity of
AP for iodine is only 105125 g I
2
per 100 g AP
and the iodine complex has a purple colour with

max
at 530 nm.
\nnnr\i\+ioxs tsrn: AM=amylose; AP=amylopectin;
The ne structure of AP is the subject of much
DMSO=dimethylsulphoxide; FPLC=fast Phar-
current research aimed at elucidation of the av-
macia liquid chromatography; HP-SEC=high per-
erage chain length, A/B chain ratio and genetic
formance size exclusion chromatography; d.b= dry
control of their distribution. The relative pro-
basis; c.v.=coecient of variation; SE=standard error.
Corresponding author. E-mail: prioul@ibp.u-psud.fr portion of AM and AP varies considerably within
07335210/97/050211+11 $25.00/0/jc970124 1997 Academic Press Limited
M. Sene et al. 212
the plant species, plant organs, and as a function of simpler procedure was proposed for maize amy-
lose, which took advantage of the spontaneous organ development and growth conditions (Sene,
unpubl. data). Biochemical and genetic data have formation, in DMSO, of the tri-iodide ions neces-
sary for the initiation of AM-I
3
complex and of lead to a route for starch biosynthesis in the cereal
endosperm, but the precise understanding of the the good solubility of starch in this solvent
5
. The
simultaneous determination of AM and AP was synthesis of the two components in relation to
starch structure (AM/AP ratio and branching of rst described for potato starch
11
; it was based on
a two-wavelength measurement of AM- and AP- AP) are still a matter of debate
3
.
Our group is currently involved in the study of iodine complexes. Accuracy was improved by
using a six-wavelength multi-component analysis the genetic diversity of AM/AP ratio in maize
kernels in relation to the molecular bases of its which allowed the simultaneous quantitative de-
termination of AM, AP and starch in low lipid regulation. For this purpose, a simple, rapid and
accurate method suitable for simultaneous de- material like potato
4
. This procedure was not suit-
able for maize kernel starch determination due to termination of amylose and amylopectin in hun-
dreds of samples was needed. Acommon limitation interference from lipids and structural dierences
of AM between various genotypes. for several methods of starch determination in
maize is the quantitative solubilisation of starch. The technique of high-performance size-ex-
clusion chromatography (HP-SEC) is now used For starches resistant to gelatinisation, even in
boiling water (e.g. high amylose maize), solvents widely to estimate the relative amounts, and the
apparent molecular weights, of AM and AP in such as potassium hydroxide
4
or di-
methylsulphoxide (DMSO)
5
are commonly used. native starch
12
. This method is useful for the precise
identication of all starch components including Various additional pre-treatment steps may be
required to ensure rapid and eective solu- the so-called intermediate fraction with a mo-
lecular mass between that of AM and AP. Fur- bilisation. For example, the addition of 3x CaCl
2
and sonication at 70 C, have been shown to thermore, the use of a debranching enzyme
allowed the analysis of the chain length distribution reduce the solubilisation time
6
.
The enzymic method
7
for starch quantication in starch samples
13,14
. Unfortunately, this technique
is time consuming and not suitable for routine is highly specic, but may underestimate starch in
materials containing starch resistant to enzymatic analysis.
In this report, we describe a simple and rapid, hydrolysis or to gelatinisation. Therefore, a pro-
cedure combining simultaneous solubilisation in six-wavelength, multi-component analysis method,
for the simultaneous quantication of AM and AP dimethylsulphoxide (DMSO) and the use of ther-
mostable alpha-amylase at 100 C, followed by in maize kernel starch, combining the advantage
of NaOH solubilisation, and reaction with iodine pullulanase/beta-amylase treatment at 50 C was
developed. The maltose and maltotriose generated solubilised in DMSO. Evaluation of the eciency
of this procedure was rst checked on reconstituted were hydrolysed further by amyloglucosidase, and
the resultant glucose measured by a glucose-ox- starch solutions containing various amounts of AM
and AP, then with starches from a normal dent idase/peroxidase reagent
8
. Such a procedure is
both expensive and time consuming and thus not (F-2) and a dent int (Io) maize kernel lines, and
from mutants aected in starch composition and suitable for routine analysis of numerous samples.
In the past, amyloglucosidase alone has been used content. The results were compared with those
obtained by enzymic determination of car- for gelatinised starch.
The iodine reaction has been proposed to quant- bohydrate from the butanol fractionation of the
same starch samples. ify amylose content in maize starches using
measurement made at one wavelength (600 nm)
9
.
However, the accuracy of a colorimetric procedure
MATERIALS AND METHODS
using one wavelength for the quantitative de-
termination of AM in cereals is limited by in-
Maize genotypes
terference from AP and lipids. Thus, the
measurement was not an absolute indicator of AM Starch was extracted from dent (Io), dent-int (F-
2) genotypes and from the mutant lines: waxy(wx), and gave only apparent amylose content. The
problem has been partly addressed by a method shrunken 2 (sh2), amylose-extender (ae), shrunken 1 (sh1),
dull (du), sugary 1 (su1) and sugary 2 (su2). The Io for total AM estimation after lipid removal
10
. A
Assay of amylose/amylopectin from maize kernels 213
and F-2 mature kernels were provided by la Ferme sampled for reaction with iodine reagent (05 mL).
Another aliquot (015 mL) was used for measure- du Moulon (INRA-Universite Paris-Sud-CNRS,
Gif-sur-Yvette, France). The mutant genotypes ment with amyloglucosidase.
were a gift from M. Pollacsek (INRA, Clermont-
Ferrand, France).
Preparation of samples
Eight maize kernels per genotype were air dried
Preparation of standards
overnight at 50 Cand ball-milled to a ne powder.
The AM standard was obtained from Sigma
A sample (007 g) was twice shaken in 70% (v/v)
Chemical Co, France, but, since this AMcontained
aqueous acetone (2mL) to eliminate lipids and
approximately 30% AP, it was repuried by bu-
then centrifuged at 1600 g for 15 min. The starch
tanol fractionation according to the Schoch
in the dry pellet was suspended in H
2
O (10 mL),
method
15
as modied by Wang et al.
16
. This pro-
solubilised in 5x NaOH (4 mL) with constant stir-
cedure involved defatting commercial AM (1g) by
ring for 1 h at room temperature, then neutralised
shaking in 70% aqueous acetone (v/v) for 1 h at
with HCl.
room temperature. Defatted AM was dispersed in
85% (v/v) DMSO (100 mL), under constant stir-
ring in a boiling water bath for 15 min. After
Enzymic determination of AM, AP and starch
cooling at room temperature, absolute methanol
The total carbohydrate in AM, AP standards or
(500 mL) was added for precipitation for more
butanol fractionated starch was determined by
than 2 h at 0 C. After centrifugation at 2500 g
enzyme-coupled assay: NaOH-solubilised samples
for 15 min, the pellet was air-dried and gelatinised
(01 mL) were diluted with 03 x citrate buer
in water (250 mL) at 100 C, for 1 h. After cooling,
(04 mL), pH 46 and hydrolysed with 50 L amy-
the pH was adjusted to 63 with 005 x NaOH.
loglucosidase (Boehringer; 150 unit/mL) for 1 h
Aqueous butanol (20%, v/v) was used to complex
at 50 C. The resultant glucose was quantied
AM. After centrifugation at 2500 g for 15 min,
in an aliquot (005 mL); it was introduced in a
three layers were obtained: an upper aqueous
spectrophotometric cell containing 075 x tri-
supernatant containing AP, a precipitate of AM-
ethanolamine buer, pH 76 (025 mL) deionised
butanol complex, and a middle layer. The AM-
water (04 mL), 40 mx ATP (005 mL), (75 mx)
butanol complex was puried further by three
NAD (005 mL), and absorbance was measured at
repetitions of the same protocol. Finally, the pellet
340 nm(A
1
). Then, 2 unit/mL (nal concentration)
was dissolved in either 85% DMSO (5 mL) or 4 x
glucose 6-phosphate dehydrogenase (Boehringer)
NaOH (5 mL) depending on further use. The
and 4 unit/mL (nal concentration) hexokinase
purity of each fraction was checked by size ex-
(Boehringer) were added. After 1 h incubation at
clusion chromatography and AM content was
25 C, the absorbance was measured at 340 m
measured by the enzymic method.
(A
2
). The dierence (A
2
A
1
) allowed calculation
The AP standard was obtained from Sigma
of AM, AP and starch.
Chemical Co and was derived from a waxy geno-
type known to be amylose free.
Spectrophotometric determination of AM, AP
and starch Size exlusion chromatography
The fractionation procedure was derived from Starch solubilised in 4 x NaOH and neutralised
with Hcl (025 mL) was diluted with water the original method of Jane and Chen
17
. FPLC
(Pharmacia) was used, with a Superose 6 column (075 mL). An aliquot (003 mL) of this diluted
solution was added to a cuvette containing 005 x (1.530 cm), operating at a ow rate of 05 mL/
min. The puried sample (>15 mg) was dissolved sodium phosphate buer, pH 7 (092 mL); then,
iodine reagent (02 g iodine in 100 mL 85%, v/v, in 85% DMSO (2 mL) and an aliquot (05 mL)
was injected onto the column under 1 MPa pres- DMSO, (005 mL) added. The mixture (nal pH
72) was mixed and the colour allowed to develop sure, and run in ascending mode with water
adjusted to pH 9 by 01 x NaOH. Fractions for 10 min. This procedure was repeated for stand-
ard solutions containing AM (15 g/mL), AP (025 mL) were collected and aliquots (003 mL)
M. Sene et al. 214
800
Wavelength (nm)
A
b
s
o
r
b
a
n
c
e
0.25
500 600 700
0.00
400
a
b
d
c
0.50
(b)
A
b
s
o
r
b
a
n
c
e
1.00
0.00
AM + AP
AP
AM
0.50
(a)
530
(nm)
AM AP
16.41 3.09
545 18.65 3.13
560 21.12 3.08
600 25.24 2.42
630 25.00 1.93
680 22.88 1.27
Figure 1 (a) Absorbance spectra of iodine-reacted AM (15 g/mL), AP (75 g/mL) and reconstituted starch containing
15 g AM+75 g AP per mL. (b) Absorbance of iodine-reacted defatted starch extracted from Io (genotype) kernels after
dierent solubilisation procedures: a4 x NaOH (40 min, room temperature); b1 x NaOH (6 h, room temperature); c85%
(v/v) DMSO (6 h, room temperature); dwater (24 h, 100C).
(75 g/mL) and an AM+AP mixture (15+75 g/ six wavelengths, against a reagent blank from
which starch was omitted: mL) [Fig. 1(a)].
Absorbances were read, using a Perkin Elmer 530 nm: greatest dierence between AM and AP
spectra when AP absorbance is higher than AM, (Lambda 5) spectrophotometer, at the following
Assay of amylose/amylopectin from maize kernels 215
545 nm: maximum AP absorbance, and was computed using Excel 4 (Microsoft)
560 nm: maximal absorbance for an AM+AP spreadsheet. The graphical solution was given by
mixture (15+85 g/mL), the coordinates of the converging points of the six
600 nm: maximal absorbance for an AM+AP straight lines derived from Equation (2) and is
mixture (30+70 g/mL), helpful for visualising the alteration of the results
630 nm: maximal AM absorbance, caused by interfering materials such as lipids.
680 nm: greatest dierence between AM and AP When such a problem occurred the crossing point
spectra when AM absorbance was greater than of the dierent straight lines was no longer unique
AP. The dierence further increased for higher (Fig. 2).
wavelengths, but the coecient of correlation be-
tween AM concentration and absorbance cor-
RESULTS AND DISCUSSION relatively decreased (r
2
=0972 at 700 nm
compared to 0990 at 630 nm).
Starch solubilisation
Those selected wavelengths for maize are
slightly dierent fromthe ones established by Jarvis
Solubilisation is a limiting factor for accurate quan-
and Walker
4
for potato which may be explained
titative determination of starch components (AM
by the dierence in the molecular structure for
and AP). In contrast to puried starch, which
AM and AP in the two species.
gelatinises spontaneously in alkaline solutions or
The basic relation for absorbances:
in water at 100 C, the solubilisation of unpuried
maize kernel starch generally leads to an under-
A
AM+AP
=A
AM
+A
AP
(1)
estimated value, particularly in genotypes having
a high amylose content
4,5
. Another problem, spe-
was experimentally veried at each wavelength
cic to the spectrophotometric determination of
(see results).
AM and AP, concerns solubilisation procedures
The following equation was dened at any se-
that do not modify their molecular structure and
lected wavelength, assuming that a linear re-
hence their iodine anity.
lationship was observed between AM or AP
From methods described for improving or ac-
concentration and absorbance:
celerating solubilisation of starch, we have ex-
amined the eciency of the following procedures:
A
(i)
=
AM(i)
C
AM
L+
AP(i)
C
AP
L (2)
starch gelatinisation in boiling water, solubilisation
in 85% DMSO at ambient temperature or at
A
(i)
=sample absorbance at wavelength i
100 C, or solubilisation in concentrated NaOH

AM(i)
=AM molar absorption factor at wavelength
solution at room temperature. Solubilisation in
i
the dierent solvents was evaluated with defatted
C
AM
=AM concentration
starch from the Io genotype and the yield from

AP(i)
=AP molar absorption factor at wavelength
the dierent procedures were compared from the
i
absorbance spectra obtained after reaction with
C
AP
=AP concentration
iodine. Solubilisations of maize starch in boiling
L=pathlength (1 cm).
water for 24 h lead to an under-estimated value
In this equation A
(i)
was measured and
AM(i)
,
which could have resulted from starch degradation

AP(i)
were calculated from standard solutions.
by heating or more likely from AP retrogradation
Thus, a system of six equations with two unknown
during cooling [Fig. 1(b), line d]. Eciency of
variables (C
AM
and C
AP
) corresponding to the six
solubilisation in 85% (v/v) DMSO at room tem-
selected wavelengths was obtained. It was solved
perature was better than that in boiling water and
both graphically and analytically for the amylose
could be improved by heating or prolonging the
and amylopectin contents. The mathematical so-
solubilisation time [Fig. 1(b), line c]. In NaOH at
lution was demonstrated to be:
roomtemperature, the time required for maximum
solubilisation of the starch from ground maize
C
AM
=[(
AM

AP
)
i
(A
AP
)
i
][(
AM
A)
i
kernels was dependent on concentration of NaOH:
(
AP
)
2
i
] / [(
AM

AP
)]
2
[(
AP
)
2
(
AM
)
2
]
40 min for 4 x NaOH, 6 h for 1 x and 15 h for 05 x
were observed [Fig. 1(b), lines a, b]. Therefore, in
C
AP
=[(
AM

AP
)
i
(A
AM
)
i
][(
AP
A)
i further experiments, gelatinisation of starch was
performed in 4 x NaOH at ambient temperature. (
AM
)
2
i
] / [(
AM

AP
)]
2
[(
AP
)
2
(
AM
)
2
]
M. Sene et al. 216
40
250
AM mg/mL
A
P

m
g
/
m
L
200
150
100
50
10 20 30 0
40
200
A
P

m
g
/
m
L
150
100
50
10 20 30 0
(a)
(b)
Figure 2 Graphical determination of amylose (AM) and amylopectin (AP) in starch from Io genotype, after iodine-complex
measurements at six wavelengths (see Materials and Methods): (a)70% (v/v) acetone defatted sample; (b)undefatted sample.
Assay of amylose/amylopectin from maize kernels 217
This procedure did not depolymerise starch since
Precision of the method
the FPLC elution prole for NaOH solubilised The graphical and mathematical resolutions of the
starch was not dierent from that obtained with equation were rst tested with reconstituted starch
starch dissolved in 85% (v/v) DMSO solution and obtained by mixing puried AM and AP standards
which is known to preserve polymerisation (result in dierent proportions.
not shown). As a preliminary test for Equation (1) the dier-
ence between absorbance of the reconstituted
starch A
AM+AP
and the sum A
AM
+A
AP
was measured
at six wavelengths and a systematic positive de-
viation ranging from 0 to 005 at 530 nm, and
Simultaneous determination of AM and AP
from 0 to 01 at 680 nm, was observed increasing
Formation and stability of the iodine complexes
with wavelength. Such a deviation introduced a
Tri-iodide formation in DMSO has been studied
bias in the calculated values which ought to be
by Knutson
5
. In our experience, the advantages
corrected. The best correction leading to the ex-
of dissolving the I
2
reagent in 85% (v/v) DMSO,
pected AM/AP ratio in the reconstituted starch,
without KI were:
appeared to be by the addition of the observed
(i) reduction of the dierences observed between
deviation to the AP absorbance. This deviation
the sum A
AM
+A
AP
of standard solutions at the given
may result from interactions of AM and AP
wavelengths, compared to A
AM+AP
(Equation 1),
molecules when they are mixed in solution, thus
(ii) increased stability of the complexes (for ex-
modifying the I
3
complexation.
ample, 25 x KI in the cuvette caused aggregation
The results obtained with the spec-
of the iodine complexes in buered medium after
trophotometric procedure and the multi-com-
only 5 min compared with DMSOsolution without
ponent analysis are given in Table I. The mean
KI which remained stable for 1 h).
value of AMand AP, calculated for 10 independent
Despite these advantages, the iodine colour
determinations of the three mixtures of AM and
stability in DMSO is still dependent on pH and
AP (mg/5 mL): M1 (115/45), M2 (175/31.5),
this was observed from pH 49, using 005 x
M3 (22/20), were close to the expected values. The
phosphate buer and measuring absorbance at six
precision evaluated by the coecient of variation
wavelengths as a function of time. The absorbance
(c.v.) and the mean standard error (SE) showed a
of the AP-I
3
complex decreased regularly with
good precision for AM; but the c.v. and SE for
time in acidic solution or when pH>75. Full
AP increased linearly with AM/AP ratio. To over-
colour development was reached after 10 min and
come this problem, the concentration of AM and
was stable for 10 min at pH 72. The AM-I
3 AP in standard solution should be chosen in order
complex colour developed immediately and was
to give AM/AP ratio close to that of the sample
stable for 1 h independent of pH. Therefore, fur-
to be assayed.
ther measurements were done in 005 x potassium
phosphate buer, nal pH 72. Interferences from other kernel components
In order to evaluate interference by lipids and
possibly proteins present in maize kernels on the
AM and AP molar absorption factor
precision of the method, samples from the Io
The validity of the BeerLambert law was veried
genotype were, either defatted with aqueous 70%
at each wavelength. A linear relationship between
(v/v) acetone treatment, or highly puried by
absorbance and nal concentration was observed
sequential NaOH solubilisation and ethanol pre-
in the 580 g/mL range for AM and 20150 g/ cipitation; the latter treatment is also known to
mL for AP (r
2
>0998 for both AM and AP). Using remove proteins. The maximum absorbance (
max
)
an iodine solution blank the molar absorption after iodine reaction and the proles of the graph-
factor () for AM and AP, as determined for ical resolution of the defatted starch were com-
solutions containing respectively 15 g/mL AM pared to that of un-defatted samples. The
and 75 g/mL AP (corresponding to the average maximum absorbance (
max
) of the lipid containing
amount of AM or AP in the kernels of non-mutant starch from genotype Io was at 545 nm (Fig. 3).
genotypes), was dramatically dierent for AM(16 The defatting process caused a shift of this
max
to 26) and AP(1 to 4) depending on the wavelength from 545 to 600 nm. The interference of lipids
with the starch-iodine complex also altered the [Fig. 1(a)].
M. Sene et al. 218
Table I Simultaneous spectrophotometric determination of amylose and amy-
lopectin in reconstituted starches containing three dierent ratios of AM/AP (mg/
5 mL)
M1 M2 M3
AM AP AM AP AM AP
Original ratio 115 450 175 315 220 200
Mean 116 418 173 309 218 202
SE 026 031 019 07 026 09
c.v. 22% 07% 11% 02% 12% 40%
The iodine starch complexes were measured at six wavelengths and the obtained
concentrations expressed as the meanstandard error (SE) and coecient of
variation (c.v.) from 10 independent measurements.
800
0.80
Wavelength (nm)
A
b
s
o
r
b
a
n
c
e
0.40
500 600 700
0.00
400
a
b
d
c
Figure 3 Absorbance spectra of iodine-reacted starch from Io (genotype) kernels after dierent purication processes: ano
purication; b01 x HCl treatment; c70% (v/v) acetone defatted; ddeproteinisation and defatting by sequential NaOH
solubilisation and ethanol precipitation.
graphic resolution, as illustrated by the imperfect
Application to AM, AP and starch determination
convergence of the straight-lines for undefatted
in various maize genotypes
samples compared to the defatted ones (Fig. 2).
Table II presents the AM and AP content (%, w/w,
The changes in the absorbance spectra may be
d.b.) of two normal (Io, F-2) and seven mutant
explained in part by the decreased availability of
genotypes, using either butanol fractionation fol-
AM for iodine complexation because of com-
lowed by the enzymic determination of AM and
petition with lipids. This is supported by the under-
AP fractions (method E) or spectrophotometric estimation of AM values in the lipid containing
assay (method C). The AM+AP assay does not starch: 193% instead of 224% (w/w, d.b.) in
defatted starch. take into account the water soluble starch fraction
Assay of amylose/amylopectin from maize kernels 219
Table II Determination of amylose and amylopectin content in kernels from dierent maize genotypes
(expressed as %, w/w, d.b.). Two methods are compared: (i) Enzymic measurement from butanol fractionated
starch (E) (2 measurements from the same extract), (ii) six wavelength spectrophotometric determination of
the iodine complexes (C) (10 independent measurements)
Genotypes AM AP AM+AP AM/AP
E CSE E CSE E C E C
Io 163 157014 573 581035 736 738 028 027
F-2 133 125013 584 552095 717 677 023 023
wx 08 0 479 461038 487 461 002 0
sh2 47 50023 321 296055 368 346 015 017
ae 297 288023 326 316036 623 604 091 091
sh1 122 142036 521 53204 643 677 023 026
du 157 87014 468 58103 625 668 033 015
su1 91 54018 453 35306 544 406 020 015
su2 116 65013 389 58207 505 647 030 011
SE=standard error of the mean, wx=waxy, sh2=shrunken 2, ae=amylose extender, sh1=shrunken 1, du=dull,
su1=sugary 1, su2=sugary 2.
which may represent up to 10% of the total starch tions the chain length ranged from 1619 glucose
units for the short chains, to 4749 for the long (results not shown). The agreement between the
two independent methods was good for the normal chains, whilst in the normal dent genotype the
lengths were 20 and 45 units, respectively. These genotypes and for wx, sh2, ae and sh1 mutants.
However the spectrophotometric method tended structural dierences may introduce variations in
AP molar absorption factor between the presently to underestimate both AM and AP content by 27
to 6%, except in the Io and sh1 genotypes. By used AP-waxy standard and AP standard puried
from the normal genotypes. Similarly, amylose- contrast, a large discrepancy between both
methods was observed for amylose content in du, extender mutant (ae) has longer inner and outer
chains than those of normal AP
20
. However, our su1 and su2 mutants.
The dierence between the two methods may results (Table II) suggest that the reported struc-
tural dierences were unlikely to aect the AM and originate from the quality of the fractionation by
method E and/or from the adequacy of the AM AP molar absorption factor, at least for normal, ae,
sh1 and sh2 genotypes. This is in agreement with and AP standards for the starch samples to be
measured. The selectivity of fractionation may be Yun and Matheson
21
who observed that the aver-
aged
AM
and
AP
from wx and ae were less than questioned for waxy since a small, so called amylose
fraction, is obtained whereas this genotype is 10% dierent from normal genotype. This small
dierence could produce the observed un- known to be amylose free. Another explanation
for the lower values derived from the spec- derestimation.
The failure of the present spectrophotometric trophotometric method could be that the molar
absorption factor of AM and AP standards may method to evaluate amylose in starch from dull,
su1 and su2 mutants, compared with available data be slightly dierent from that of AM and AP in
the measured genotypes. Indeed, the molecular of the literature
22
, may also be explained by larger
molecular structural dierences than in the mut- structure of each component i.e. molecular weight,
chain length distribution and branching are known ants examined previously. Gel ltration chromato-
graphy applied to dull and sugary mutants showed to inuence iodine anity and thus absorbance
of the starchiodine complex since the chain length an elution prole for native starch with no obvious
sharp separation between AM and AP
22
. An inter- and iodine anity of AM and AP are corre-
lated
18,19
. mediate component consisting of a branched
molecule with molecular weight lower than that The commercial AP used as a standard in this
experiment was prepared from a waxy genotype. of AP but more branched than AM was present
in amount ranging from 45 to 15% of total starch Pullulanase treatment, followed by liquid column
chromatography of the resultant linear malto oli- in dull mutant
19
. This intermediate material is likely
to be a source of error since its properties are gosaccharides
14
have shown that in waxy AP frac-
M. Sene et al. 220
between those of AM and AP. Moreover, dull and between the two methods except for some mutants
known to contain intermediate material. The spec- su1 starches had higher amounts of A and B
chains in their AP molecules which could introduce trophotometric method appeared adequate for
comparing genotypic variability among normal further dierence in iodine complex molar ab-
sorption factor. genotypes, even when using a commercial AP
standard derived from the waxy mutant. The comparison of composition between the
two normal genotypes (Table II) shows that the
dent genotype (Io) had a signicantly higher AM
content compared to the dent-int genotype (F-
Acknowledgements
2). The results from waxy kernel, conrmed the
near absence of AM but also showed a slight We thank very much J. P. Rocher for sharing his large
experience in carbohydrate biochemistry and Dr A. deciency in AP synthesis compared to normal
Reyss for her ecient help in preparing the revised
(46%, w/w, d.b. vs 5558%). The sh2 mutation
version of the manuscript.
aected ADP-glucose pyrophosphorylase activity
which provides the substrate for starch synthesis.
Accordingly, a substantial decrease in starch con-
tent was observed in the sh2 mutant, but the AM/
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