Determination of the Degree of Branching in
Normal and Amylopectin Type Potato Starch
with H-NMR Spectroscopy
Improved resolution and two-dimensional spectroscopy
Gunilla S. Nilsson, Karl-Erik Bergquist,
Ulf Nilsson, and Lo Gorton,
Lund (Sweden)
Starch from genetically modified potatoes was found to be highly
branched compared with normal potato varieties through the use of
'H-NMR spectroscopy. The average chain length, blue-value, and the
wavelength at maximum absorptivity clearly show that the new po-
tato varieties produce amylopectin starch. Correlation between the
degree of branching as determined by 'H-NMR and starch-iodine
complexation, expressed as blue-value, was good and the NMR-me-
thod gives low standard deviation.
For the first time, the anomeric proton, H-1, of a (1-4)-a-linked
D-glucose residue and the H-1 of the glucose residue of a non-reduc-
ing end have been assigned separate chemical shifts in starch. Assign-
ments were made as determined from two-dimensional homonu-
clear and 'H-I3C heteronuclear spectroscopy (COSY, HMQC, and
HMBC).
The molecular weight in degraded starch and pullulan were deter-
mined by means of NMR-spectroscopy. These results were in accor-
dance with determinations by size exclusion chromatography and
with the known molecular weights of pullulan standards.
1 Introduction
1.1 Amylopectin type potato starch
Starches from various plant species and with different ratio
of amylose to amylopectin vary in their chemical and physical
properties [ 1, 21. Amylopectin, the highly branched macro-
molecule, consists of short (1-4)-a-D glucan chains which are
joined together through a-( l+6)-branch points, whereas amy-
lose is the essentially unbranched fraction consisting almost
exclusively of a-( 1-4) linkages [3]. Since the number average
chain length (CLn, number of glucose units) of amylopectin
is usually 20-25 glucose residues, it follows that each macro-
molecule contains several thousand individual chains [3]. Nor-
mal potato starch contains approximately 22% amylose [l]. Po-
tato starch dispersions with different amylose contents differ
in rheological behaviour [4] and stability towards molecular
association, which are important properties for industrial ap-
plications [ 11. Genetically modified starch, with either high
amount or without amylose, could offer properties similar to
those of post-harvest modified starch with retained
characteristics of natural starch that depend on the granules
being intact [5]. Instead of chemical and/or physical treatment
in a factory, the potato itself is the producer of the desired
starch structure and function [5,6]. Amylopectin type potato
starch, granular amylose-free starch, is highly interesting for
future industrial applications such as degradable packaging
material [6]. New potatoes created by genetic engineering to
produce only amylopectin starch have been developed in Swe-
den [6,7]. This potato is analogous to waxy maize in its biosyn-
thesis of starch [6]. Natural mutants of cereals producing amy-
352 StarchlStCrke 48 (1996) Nr. 10. S. 352-357 @ VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1996
Bestimmung des Verzweigungsgrades in normaler und amylo-
pektinreicher Kartoffelstarke mittels 'H-NMR-Spektroskopie:
Verbesserte Aufllisung und zweidimensionale Spektroskopie.
Durch I-H-NMR-Spektroskopie wurde gefunden, da13 Starke aus ge-
netisch modifizierten Kartoffeln hoch verzweigt war im Vergleich zu
normalen Kartoffelvarietaten. Die durchschnittliche Kettenlange,
der Blauwert und die Wellenlange bei maximaler Absorptivitat zei-
gen klar, da8 die neuen Kartoffelvarietaten Amylopektinstarken pro-
duzieren. Die Korrelation zwischen dem durch 'H-NMR bestimmte
und als Blauwert ausgedriickte Starke-Jod-Komplexierungwar gut,
und die NMR-Methode gibt niedrige Standard-Abweichung.
Zum ersten Ma1 wurden durch das anomere H-1 eines (1-4)-gebun-
dene D-Glucosereste und das H-1 des Glucoserestes eines nicht-re-
duzierenden Endes getrennten chemischen Schichten in Starke zuge-
teilt. Die Zuordnungen wurden durchgefiihrt eine bestimmt mittels
durch zweidimensionale homonukleare und 'H-I3C heteronukleare
Spektroskopie (COSY, HMQC, und HMBC).
Das Molekulargewicht in abgebauter Starke und Pullulan wurden in
Ubereinstimmung mit Bestimmungen durch GroBenausschluR-
Chromatographie und mit den bekannten Molekulargewichten von
Pullulan-Standards.
lose-free starch have been known since the beginning of this
century, of which waxy maize is the best known [ S ] . However,
starch from tubers such as amylopectin type potato is a new
source for amylose free starch.
The aim of this work was to determine the degree of
branching, by using NMR, and to compare genetically trans-
formed potatoes with normal potato varieties. As a compli-
mentary method, starch-iodine complexation was measured
spectrophotometrically as a "blue-value", BV, a common
means for determining amylose content [8].
1.2 'H-NMR spectroscopy
For the determination of the molecular characteristics of
starch, different chemical and enzymic methods are used
which have been reviewed elsewhere [3,9]. Through the use
of 'H and 13C nuclear magnetic resonance (NMR) spectros-
copy it is possible to determine the degree of branching with-
out chemical or enzymic destruction of the molecules [lo, 111.
The high sensitivity of 'H-NMR allows for the resolution of
the anomeric proton (H-1) resonances of starch, sufficiently
to distinguish between the a-( 1-4) and a-(l+6)-linkages [lo,
111. Determination of the degree of branching by NMR [lo,
111 has been reported to be comparable in accuracy to other
methods [9, 111.
Different pretreatment methods and solvents were com-
pared in this study. As there is a general problem with the
solubilisation of native starch, different solvents have been
tried and generally D20 has been used for polysaccharide
NMR samples [9-141, although DMSO-d6 has also been used
[lo, 151 for native starch. The chemical shifts of several, but
0038-9056/96/1010-0352$10.00+.25/0
not all protons of starch and related polymers have previously
been assigned [13,14,16]. This investigation was performed in
order to assign additional chemical shifts using 2D-spectros-
copy. 'H-NMR spectroscopy has the advantage of giving infor-
mation about the degree of branching, and the mol. wt. of de-
graded starch simultaneously, as shown by Gidtey [lo]. Here,
determinations of the mol. wt. of polysaccharides are per-
formed by comparing 'H-NMR spectroscopy and size exclu-
sion chromatography (SEC).
2 Experimental
2.1 Materials
Starch samples from different potato varieties were gifts
from the two Swedish manufacturers (Lyckeby Starkelsen,
Kristianstad and Svalof-Weibull AB, Svalov) involved in the
development of amylopectin type potato starch (APPS). The
total ash and phosphorus contents in all starches were deter-
mined by the manufacturers to be < 0.4% (w/w) and < 0.06%
(w/w), respectively. Amylose and amylopectin from potato
were purchased from Sigma (Sigma Chemical Co, St. Louis,
MO, USA, amylose type 3, cat. no. A-0512, lot 42H3861; amy-
lopectin cat. no. A-8515, lot 111H3901). Another amylopectin
sample from potato was purchased from Merck (Darmstadt,
Germany, cat. no. 1469, lot 818K3218369). Amylopectin from
corn was purchased from Sigma (cat. no. A-7780, lot 25F-
0608). Pullulan standards were purchased from Shodex
(Showa Denko K. K.,Tokyo, Japan, from Aureobasidiurn pullu-
lam, kit P-82, cat. no. 722106, lot no. 20101) with eight differ-
ent molecular weight fractions ranging from 853 kDa to 5.8
kDa. DzO was purchased from Dr. Glaser AG (Basel, Switzer-
land, cat. no. 57922, isotopic purity > 99.95 atom O/o D, which
was used for proton exchange and cat. no. 57928, isotopic pu-
rity > 99.98 atom Yo D, which was used as a solvent for NMR
samples). Deuterated DMSO (DMSO-d6) was purchased
from Dr. Glaser (cat. no. 12681, isotopic purity > 99.8 atom Yo
D) .
2.2 Blue-values
BV were measured according to the method described by
Peng and Perlin [15], with the addition of 0.5mL of pure water
(Millipore, Milli-R04, CFOF 01205 Bedford, MA, USA) to
5mL of DMSO and 50 mg of starch. The wavelength at maxi-
mum absorptivity (An3ax) of the polysaccharideiodine complex
and BV were measured in a spectrophotometer (mod. U2000,
Hitachi,Tokyo, Japan), using a l.Ocm cell. The iodine solution
contained iodine (2 mg/mL) and potassium iodide (20mg/
mL) in water. BV was measured at the approximate A,, for
amylose-iodine (640nm) and expressed as 4 Alconc. (mg/dL).
2.3 Size exclusion chromatography (SEC)
Sample solution (lOOpl) was injected into a guard column
(mod. Micro Aquagel, Chrompack, The Netherlands, 7.7 X
50mm,) followed by a SEC column (mod. TSK G4000PW, Ul-
tropac, LKB, Bromma, Sweden, 7.5 X 600mm, exclusion lim-
its: 0.1-700 kDa). An on-line eluent degasser (mod. ERC-
3112, ERMA CR inc, Tokyo, Japan) was connected before the
pump (mod. LKB 2150, Pharmacia, Bromma, Sweden). The
eluate was monitored with a differential refractometer (mod.
ERC-7512 ERMA CR inc) and the data was evaluated with
the software JCL 6000 (Jones Chromatography, Littleton,
Colorado, USA). The eluent was 1OOmM NaCl and 0.010mM
NaOH, containing 50p1 Kathon C G / L (1.125% methylchloroi-
sothiazolinone and 0.375% methylisothiazolinone, Rohm and
Haas Ltd, Croydon, England, UK) to prevent microbial
Slarch/Starke 48 (1996) Nr. 10, S. 352-357
growth. The columns were kept at room temperature and the
detector temperature was 35°C. Pullulan standards were dis-
solved in water (O.Smg/mL). Amylopectin was dissolved in ei-
ther the mobile phase (OSmg/mL) at 100°C or in 0.1M NaOH
at room temperature. The samples were filtrated through
0.45pm filter prior to injection.
2.4 NMR spectroscopy
2.4.1 Sample preparation
Two different pretreatment methods for normal potato
starch were compared; where the granules were dissolved ei-
ther in DMSO-d6 or in Dz0. When using DMSO-d6, dry starch
was dissolved (lOmg/mL) in a screw capped bottle at 70°C
over-night (approximately 17h) with continuous stirring at
low speed, followed by freeze-drying. Exchange of hydroxylic
protons was performed in order to reduce interference from
the residual solvent resonance; 1.0ml DzO was added to 20mg
of the sample, heated for 15min at 100°C followed by a sec-
ond freeze-drying. The dried, deuterated sample was dis-
solved in D20 and heated for 15min at 100°C. The NMR-sam-
ples were always prepared immediately before measurements
and never cooled below 70°C, in order to prevent retrograda-
tion prior to analysis. When not using DMSO-d6 as a granule
disperser, but simply DzO, the same procedure as described
above for the exchange of hydroxylic protons was followed
with the exception that native starch was boiled for 30min, be-
fore and also after freeze-drying, to obtain a homogeneous
sample.
Amylose was pretreated in D M s 0 - d ~as described above
and freeze-dried. This sample was easily redissolved in D20
for exchange of hydroxylic protons, followed by freeze-drying.
The amylose NMR-sample was finally prepared by dissolution
(10mg/mL) in 90% DMSO-d6 in D 2 0 , 100°C for 15 min. Amy-
lopectin was dissolved in DzO (2Omg/mL) at 100°C for 15min
after exchange of hydroxylic protons as described above. Pul-
lulan was dissolved in DzO at room temperature, in accor-
dance with the manufacturer's recommendations.
2.4.2 N M R spectroscopy
The NMR measurements were performed with a spec-
trometer (mod. ARXSOO, Bruker Fallanden, Switzerland) op-
erating at 500.25MHz for IH, and 125.76MHz for I3C. Sodium
3-trimethylsilylpropionate-2,2,3,3-d4(TSP) was used as inter-
nal reference for the 'H and 13Cchemical shifts scales. Spectra
were accumulated at 8OOC. A 'H selective 5mm probe was em-
ployed for quantitative measurements. The probe was tuned
to each sample to ensure optimal signal-to-noise ratio (S/N)
and consistency throughout the experimental series. Longitu-
dinal relaxation (TI) for 'H was estimated by the inversion-re-
covery method [17]. For quantitative measurements a 70' exci-
tation pulse was employed along with the repetition time of
lOs, which was larger than 5tTl for the slowest relaxing pro-
ton of interest (i.e., T I for H4(t) was found to be ~ 2 . 0 ~2D
).
correlation spectroscopy was performed using an inverse
5mm probe equipped with a shielded gradient coil. Gradient
selection was used when acquiring the COSY [18] (Correla-
tion Spectroscopy), HMQC [I91 (Heteronuclear Multiple
Quantum Coherence), and HMBC [20] (Heteronuclear Multi-
ple-Bond Correlation) spectral data. Refocusing delays in
HMQC and HMBC were optimized for 'JcH=145Hz and
"Jc~=10Hz. The raw 1D data from quantitative measure-
ments were Fourier transformed and the resulting spectra
were baseline corrected by substraction of a matched polyno-
mial using Bruker UXNMR software (ver. 930901). Integra-
tion of the peak areas of a-(1-4) and of a-(1+6) was per-
353
formed at fixed ppm values on the spectrum at 5.47-5.23 and
5.01-4.89, respectively, using Bruker UXNMR software. The
S/N of the a-(1+6) proton resonance was also calculated by
using a fixed spectral region and the spectrometer software.
2.4.3 Notations of the protons and carbons
The glucose residues in starch are divided into four groups;
those involved in linear a-(1+4) linkages in a chain, in bran-
ched a-(1+6) linkages, in terminal non-reducing ends, and in
one terminal reducing end. With this notation, it is assumed
that the terminal glucose units are involved in linear linkages,
in agreement with proposed structural models of amylopectin
[3]. The protons in a glucose residue involved in a linear chain
are followed by (1-4) (e.g., H-l(1-4) for the anomeric pro-
ton), those in a branching point are followed by (1-6), those
in a terminal non-reducing end are denoted by the addition of
(t), and those in the reducing end with (r) (e.g. H-l(a-r)). The
carbon atoms are denoted according to the same notation as
the protons.
The average CLn. is calculated according to Eqn. 1. The de-
gree of branching is calculated according to Eqn. 2, which ex-
presses the number of branching points compared with the to-
tal number of linkages.
Average CLn = integral (H-1(1+4) + H-l(t) + H-l(1-6))
om. 1)
Degree (integral H-1(1+6)) * 100
of branching =
,
integral (H-I(lb4) + +
H-l(t) H-l(lb6))
( E m 2)
3 Results and Discussion
3.1 Preferred pretreatment methods and solvents
A homogeneously dissolved sample is essential to obtain
well resolved N M R signals. According to Jackson [21], the dis-
persibility of starch granules in DMSO is most efficient with a
concentration of 90% DMSO in water, although rarely starch
is totally dissoved. In this work 100% DMSO-d6,90°/o DMSO-
d6 in D20, and 100% D20 were compared when dispersing
starch granules, followed by freeze-drying, and also as sol-
vents for NMR-samples. When DMSO was used to disperse
starch granules, deuterated solvent had to be used, since all
the solvent was not removed during the freeze-drying. It was
found to be easier to freeze-dry samples dissolved in 100%
DMSO-d6 than samples in 90% DMSO-d6 which was the rea-
son why pure DMSO-d6 was used in the pretreatment step.
The use of DMSO-d6 to disperse starch granules did not im-
prove the quality of the NMR-sample (results not shown).
For potato starch and amylopectin, simple dissolution by heat
treatment in D 2 0 was sufficient, followed by freeze-drying
prior to dissolution of the NMR-sample in D20.
D20 has generally been used [lo-14, 161 as a solvent for
NMR-samples of starch and starch related polysaccharides.
For granular starch, however, DMSO-d,j has been used [lo, 151
and it has been stated to furnish more homogeneous samples
[15]. For NMR measurements, 90% DMSO-d6 was used for
the amylose sample, but for potato starch and amylopectin
D20 was preferred in this investigation. Amylose is more eas-
ily dissolved in DMSO than is amylopectin [21]. In water, on
the other hand, amylopectin in low concentrations produces a
stable sample, whereas retrograded amylose requires a high
(autoclave) temperature to affect dissolution at neutral pH [l].
354
The viscosity of DMSO is higher than that of water and it is
commonly known that highly viscous NMR-samples lead to
line broadening. Furthermore, the signals become broader as
the molecular weight of a species increases, due to decreasing
motional rate of larger molecules in solution [22].Thus, starch
consisting of very large molecules in viscous solutions has
limitations as far as sample concentration is concerned. For
native potato starch, a concentration of 10-20mg/mL in D20
was found to be convenient.
The concentration of DMSO in the solution for BV and
I,,, measurements was limited to 0.5%, as suggested by Peng
and Perlin [15]. For granular starch, it was found that adding a
small portion of water prior to dissolution in DMSO made the
sample more clear and the starch-iodine complex solution
more stable without any noticeable precipitation.
3.2 Assignments of chemical shifts
Figure 1 shows a 'H-NMR spectrum of potato starch (la)
and of degraded amylopectin (Ib). The assignments of the
various shifts that are of importance when determining the
degree of branching and the mol. wt. are labelled in Figure 1.
The signals of the other protons are more or less overlapping,
except for H-4(t) and for H-2(TJ-r) (upfield of H-4(t) [13]). Im-
proved resolution, made it possible in this investigation to as-
sign new separate chemical shifts. Zang et al. [16] demonstrat-
ed well resolved 2-D spectra of glycogen (a large and highly
branched a-D-glucose polymer) allowing for assignments of
chemical shifts. In this work potato amylopectin and native
potato starch were used and additional assignments were
made as determined from 2-D homonuclear and 'H-I'C
heteronuclear spectroscopy (COSY, HMQC and HMBC). In
1D spectra, the small "hump" slightly upfield from the signal
of the H-l(1-4) was assigned to be the resonance of H-l(t).
This interpretation of 2D correlation spectra is supported by
H-l(l-4)
I
-w
oom 5.0
,
4.5
' ' " I " "
4.0
" ' '
3 5
Fig. 1. 'H-NMR spectrum of a) normal potato starch, and of b) de-
graded amylopectin (degree of branching = 4.3%). (2Omg/mL D 2 0 ,
80°C.)
StarchlStPrke 48 (1996) Nr. 10, S . 352-357
 1

t
' Hlft) - Ha(:
I " , 1I:;
3.5

H I (1-4) - Hi
4.0

4.5

IP
5.0

Fig. 2. COSY spectrum of potato starch. (Smg/mL D20, 8 0 T .

Presaturation of residual water and gradient selection were
5.0 4.5 4.0 3.5 ppm used.)

the observation that the size of this "hump" varied with the Table 1. Proton and Carbon Chemical Shifts of Starch at 80" in D 2 0 .
degree of branching of the sample (compare Figures l a and Scale relative TSP.
lb). The H-l(lP4) and H-l(t) protons in starch have for the
first time been assigned separate chemical shifts, at 5.35ppm Position Chemical shifts (p.p.m)
and 5.33ppm, respectively. This finding made it necessary to
'H(1-4) 'H(1-6) 'H(t) I3C(1+4) 13C(t)
include the integral of H-l(t) in Eqns. 1 and 2 when determin-
ing the average CLn and the degree of branching. 1 5.35 4.94 5.33 102.1 102.4
Further assignments of 'H-shifts and their correlations to 2 3.63 3.59 3.58 14.2 14.3
I3C chemical shifts, in chain- and terminal residues, were 3 3.94 3.69 75.8 15.1
4 3.62 3.41 80.1 72.3
made from the 2D correlation spectra. A COSY spectrum of
5 3.82 3.11 74.0 75.4
potato starch is shown in Figure 2, where the separate signals 6a; 6b 3.88; 3.80 3.15; 3.85 63.3 63.4
of H-l(t) and H - l ( l b 4 ) are circled. In Figure 3 a HMBC spec-

C4(1)-H3( t ) : 5 ( t )

Fig. 3. HMBC spectrum of amylopectin

(80mg/mL DzO, 80°C) showing long
range couplings. (Gradient selection was
used. Correlation between carbon and
proton chemical shifts are labelled for
some of the nuclei in glycosyl linked and
5.5 5.0 4.5 4.0 3.5 ppm terminal non-reducing residues.)

Starch/Starke 48 (1996) Nr. 10, S . 352-357 355

trum of amylopectin from potato (Sigma) is shown and long
range couplings are identified. Some of the nuclei are labelled
in Figure 3. A summation of the data is presented in Table 1.
New separate chemical shifts of C-l(1-4) and C-l(t) were
identified at 102.1 and 102.4ppm, respectively. Separate as-
signments of chemical shifts were made also for H-6a:b(1-4)
and H-6a:b(t), and for C-6a:b(l-4) and C-6(t).
3.3 Determination of branching frequency
3.3.1 Repetability of method
Provided that certain precautions are taken when the
NMR spectrum is recorded, the peak area is proportional to
the number of protons. The integration procedure [lo], the
comparably small peak area arising from the H-(1-6) signal
[I I], incomplete relaxation of the signals 1141, and the use of
water supression [14] are possible sources of error limiting the
accuracy in determining the degree of branching. These prob-
lems have been addressed in this investigation. Relaxation
times for the anomeric protons have been determined to con-
firm that these protons were fully relaxed and the time of
spectral acquisition was sufficient. Water suppression was not
necessary, since the relatively low H D O signal did not inter-
fere with the signals from the anomeric protons, see Figures
l a and b at 4.15ppm. (Exchange of hydroxylic protons in D20
followed by freeze-drying prevent a high H D O signal.)
The accuracy in the integration of the signals was found to
be highly dependent upon the S I N ratio of the integrated
resonance peak, where S / N will improve by a factor of the
square root of the number of scans. From empirical results it
was here found that the S / N of the H-1(1+6) resonance must
exceed a value of 50 in order to give a consistent integral.
With a sample concentration of lOmg/mL potato starch, the
S / N of 50 was achieved with 64 scans, giving an experimental
time of less than 15min. With a S I N larger than 50 for the
H-l( 1-6) and a proper baseline correction, the relative stan-
dard deviation of the mean value (rsd) of the degree of
branching was low (rsd = 1.4°/o, n = 6 of commercial potato
starch, including sample pretreatment). The values of the de-
gree of branching presented in Table 2 differed typically only
k 0.02 points and always < k 0.07 points between two repeti-
tions. This equals typically k 0.2 and always 5 f 0.6 glucose
units in the calculated CLn. The repeatability in determining
the degree of branching by this method is good compared
with previous investigations using N M R [lo], enzymic, and
chemical methods [2, 11, 231.
3.3.2 Highly branched genetically engineered potato
starch
All the APPS samples are significantly more branched than
is normal starch from potato varieties that are not genetically
engineered. The degree of branching reflects the CLn in the
starch sample, since each chain has one branching point,
while a high BV and A,, indicate a high amylose content [8].
These results show, see Table 2 and Figure 4, that the BV and
the degree of branching are similar in APPS and in commer-
cial amylopectin from potato and the new APPS was found to
have a high degree of branching, short average Cln, A,,,, and
BV of what could be expected for potato amylopectin [2, 10,
11, 15,241 consisting of mainly branched polymers. The aver-
age Cln of the APPS samples was found to be around 24,
which is in the same range as reported values for amylopectin
fractionated from potato starch (average CLM of 19-24) [2,3,
10, 11, 241. All the APPS samples and also the purified amy-
lopectin sample from potato had approximately the same
A,,,,,, at 550 nm. Potato amylose, and normal potato starch
samples had Lmax at approximately 640, and 600 nm, respecti-
vely. The CLM of amylopectin varies with its origin, e.g. corn
amylopectin has shorter chains than has potato amylopectin
[2,24]. The corn amylopectin used in this investigation was, as
expected, more branched than the potato amylopectin
(Sigma) [2,24] and A,, was lower, at 540nm. The correlation
between the two methods, 'H-NMR spectroscopy and spec-
trophotometric measurement of starch-iodine complex, used
for comparing the content of branched material in starch, is
good as illustrated in Figure 4.
Values from the literature of the degree of branching in
normal potato starch [12, 151, as determined using 'H-NMR
ranges from 2.8% to 3.4%. The starch samples from the nor-
mal potato varieties that were investigated here, range in de-
gree of branching from 3.1% to 3.3%. Longer term studies
could provide information about actual genetic differences in
the starch structure in different varieties. Starch from differ-
ent varieties and ages has been reported to be different in mo-
lecular structure, and ripening of potato tubers leads t o a hi-
gher amylose content [8] and elongation of the chains of both
amylose, and amylopectin [25].
When determining the degree of branching according to
Eqn. 2, the reducing end was neglected and the branched link-
1.o &.
AM
**.

Table 2. The Degree of Branching, Average CLn, and BV of Commer- I

cial Native Potato Starch, Native Starch from Different Normal Po-
tato Varieties (PS. l-PS 3), Amylopectin Type Starch from Different
Genetically Modified Potato Varieties (APPS.l -APPS.3), Amylose,
and Amylopectins. (For information about repeatability, see section
3.3.1).

i
Sample Degree of C Ln BV
PAP.
branching ( o h ) APPS o@..
0.2
Potato Amylose 1.oo 100 1.01
CAP 'O
Commercial Starch 3.05 32.8 0.52
PS.1 3.39 29.5 0.49 0.0
PS.2 3.09 32.4 0.51 0.0 I.o 2.0 3.0 4.0 5.0
PS.3 3.33 30.0 0.50
APPS.l 4.17 24.0 0.22 Degree of branching (%)
APPS.2 4.20 23.8 0.24
APPS.3 4.20 23.8 0.23 Fig. 4. Correlation between the degree of branching and BV of corn
Potato Amylopectin (Sigma) 4.07 24.6 0.24 amylopectin (CAP), potato amylopectin (PAP), amylopectin type
Corn Amylopectin 4.77 21.0 0.12 starch (APPS) and normal starch (PS) from different potato varieties,
and of potato amylose (AM).

356 StarchlStarke 48 (1996) Nr. 10, S. 352-357

ages were compared with the number of total linkages. An-
other way of expressing the degree of branching is to deter-
mine the number of glucose residues involved in a branched
linkage compared with the total number of residues. This can
be estimated from a 'H-NMR spectrum when a low mol. wt.
sample is used and the number of reducing ends is detectable
(see also Section 3.3). Previously [lo, 111, the degree of branch-
ing has been determined by NMR spectroscopy and the num-
ber of linkages estimated. However, calculating the degree of
branching according to Eqn. 2 or determining the number of
glucose residues (also including the H-l(r)) of a low molecu-
lar weight sample such as the degraded potato amylopectin
(Merck) shown in Figure l b gives almost the same value,
4.30% and 4.22%, respectively. For samples of high mol. wt,
such as native starch, the non-detectable, one reducing resi-
due per molecule, is insignificant.
3.3 Determination of molecular weight by
~H-NMRspectroscopy
'H-NMR spectroscopy gives information about the degree
of branching, and the mol. wt. of degraded starch simultane-
ously [lo]. The mol. wt. is determined by comparing the areas
from the a + &free anomeric resonances [lo] with the total
anomeric resonances. This can be done in low mol. wt, sam-
ples, where the number of reducing rings is relatively high. Ac-
cording to Mclnwvre and Vogel[14], the free anomeric protons
were observed in a spectrum of dextran with a mol. wt. of 9.4
kDa, but not in a spectrum of dextran having a mol. wt. of70.8
kDa. Amylopectin from potato (Merck) was here found to be
highly degraded. The number average mol. wt. as determined
by NMR (see Figure lb) was 8.06 kDa, DP = 49.6 (degree of
polymerization, no. of glucose residues) (rsd = 4%, n=3). Re-
sults from SEC agree well with mol. wt. determinations using
'H-NMR spectroscopy. The peak maximum in the SEC-chro-
matogram was determined to be DP = 50.1, and the peak
width ranging between DP = 700 to DP < 6 (totally perme-
ated). The larger mol. wt. potato amylopectin (Sigma) was al-
most totally excluded by this column. Dissolution of the amy-
lopectin samples in either the mobile phase (0.OlmM NaOH)
at 100°C or in 0.1M NaOH at room temperature gave the same
results. Pullulan standards with DP 33.5 and 71.0 (measured
by the manufacturer by the ultracentrifugal sedimentation
equilibrium method) were here determined by NMR to have
DP 34.7 and 71.8, respectively. This shows a good agreement
between mol. wt. determinations by SEC and by NMR for de-
graded starch.
Acknowledgements
This work was supported by Lyckeby Starkelsen, Kristianstad,
Sweden, and the Swedish Research Council for the Engineering Sci-
ences (TFR). The authors wish to thank P. Hofvander, Svalof-Weibull
AB,
Starch/Starke 48 (1996) Nr. 10, S. 352-357
K. Helmersson, K. Svegmark and 0. Wikstrom, Lyckeby Starkelsen
for starch samples and valuable discussions.
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B. Sc. G. S. Nilsson and Associate Professor Dr. L. Gorton, Depart-
ment of Analytical Chemistry, University of Lund, P.O. Box 124,
S-22100 Lund, Sweden.
Dr. K.-E. Bergquist and Dr. U.Nilsson, Department of Organic Chem-
istry 2, University of Lund, P.O. Box 124, S-22100 Lund, Sweden.
(Received: March 13, 1996)
357