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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 Bestimmung des Verzweigungsgrades in normaler und amylo-
branched compared with normal potato varieties through the use of pektinreicher Kartoffelstarke mittels 'H-NMR-Spektroskopie:
'H-NMR spectroscopy. The average chain length, blue-value, and the Verbesserte Aufllisung und zweidimensionale Spektroskopie.
wavelength at maximum absorptivity clearly show that the new po- Durch I-H-NMR-Spektroskopie wurde gefunden, da13 Starke aus ge-
tato varieties produce amylopectin starch. Correlation between the netisch modifizierten Kartoffeln hoch verzweigt war im Vergleich zu
degree of branching as determined by 'H-NMR and starch-iodine normalen Kartoffelvarietaten. Die durchschnittliche Kettenlange,
complexation, expressed as blue-value, was good and the NMR-me- der Blauwert und die Wellenlange bei maximaler Absorptivitat zei-
thod gives low standard deviation. gen klar, da8 die neuen Kartoffelvarietaten Amylopektinstarken pro-
For the first time, the anomeric proton, H-1, of a (1-4)-a-linked duzieren. Die Korrelation zwischen dem durch 'H-NMR bestimmte
D-glucose residue and the H-1 of the glucose residue of a non-reduc- und als Blauwert ausgedriickte Starke-Jod-Komplexierungwar gut,
ing end have been assigned separate chemical shifts in starch. Assign- und die NMR-Methode gibt niedrige Standard-Abweichung.
ments were made as determined from two-dimensional homonu- Zum ersten Ma1 wurden durch das anomere H-1 eines (1-4)-gebun-
clear and 'H-I3C heteronuclear spectroscopy (COSY, HMQC, and dene D-Glucosereste und das H-1 des Glucoserestes eines nicht-re-
HMBC). duzierenden Endes getrennten chemischen Schichten in Starke zuge-
The molecular weight in degraded starch and pullulan were deter- teilt. Die Zuordnungen wurden durchgefiihrt eine bestimmt mittels
mined by means of NMR-spectroscopy. These results were in accor- durch zweidimensionale homonukleare und 'H-I3C heteronukleare
dance with determinations by size exclusion chromatography and Spektroskopie (COSY, HMQC, und HMBC).
with the known molecular weights of pullulan standards. Das Molekulargewicht in abgebauter Starke und Pullulan wurden in
Ubereinstimmung mit Bestimmungen durch GroBenausschluR-
Chromatographie und mit den bekannten Molekulargewichten von
Pullulan-Standards.

1 Introduction lose-free starch have been known since the beginning of this
century, of which waxy maize is the best known [ S ] . However,
1.1 Amylopectin type potato starch starch from tubers such as amylopectin type potato is a new
Starches from various plant species and with different ratio source for amylose free starch.
of amylose to amylopectin vary in their chemical and physical The aim of this work was to determine the degree of
properties [ 1, 21. Amylopectin, the highly branched macro- branching, by using NMR, and to compare genetically trans-
molecule, consists of short (1-4)-a-D glucan chains which are formed potatoes with normal potato varieties. As a compli-
joined together through a-( l+6)-branch points, whereas amy- mentary method, starch-iodine complexation was measured
lose is the essentially unbranched fraction consisting almost spectrophotometrically as a "blue-value", BV, a common
exclusively of a-( 1-4) linkages [3]. Since the number average means for determining amylose content [8].
chain length (CLn, number of glucose units) of amylopectin
is usually 20-25 glucose residues, it follows that each macro- 1.2 'H-NMR spectroscopy
molecule contains several thousand individual chains [3]. Nor- For the determination of the molecular characteristics of
mal potato starch contains approximately 22% amylose [l]. Po- starch, different chemical and enzymic methods are used
tato starch dispersions with different amylose contents differ which have been reviewed elsewhere [3,9]. Through the use
in rheological behaviour [4] and stability towards molecular of 'H and 13C nuclear magnetic resonance (NMR) spectros-
association, which are important properties for industrial ap- copy it is possible to determine the degree of branching with-
plications [ 11. Genetically modified starch, with either high out chemical or enzymic destruction of the molecules [lo, 111.
amount or without amylose, could offer properties similar to The high sensitivity of 'H-NMR allows for the resolution of
those of post-harvest modified starch with retained the anomeric proton (H-1) resonances of starch, sufficiently
characteristics of natural starch that depend on the granules to distinguish between the a-( 1-4) and a-(l+6)-linkages [lo,
being intact [5]. Instead of chemical and/or physical treatment 111. Determination of the degree of branching by NMR [lo,
in a factory, the potato itself is the producer of the desired 111 has been reported to be comparable in accuracy to other
starch structure and function [5,6]. Amylopectin type potato methods [9, 111.
starch, granular amylose-free starch, is highly interesting for Different pretreatment methods and solvents were com-
future industrial applications such as degradable packaging pared in this study. As there is a general problem with the
material [6]. New potatoes created by genetic engineering to solubilisation of native starch, different solvents have been
produce only amylopectin starch have been developed in Swe- tried and generally D20 has been used for polysaccharide
den [6,7]. This potato is analogous to waxy maize in its biosyn- NMR samples [9-141, although DMSO-d6 has also been used
thesis of starch [6]. Natural mutants of cereals producing amy- [lo, 151 for native starch. The chemical shifts of several, but

352 StarchlStCrke 48 (1996) Nr. 10. S. 352-357 @ VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1996 0038-9056/96/1010-0352$10.00+.25/0
not all protons of starch and related polymers have previously growth. The columns were kept at room temperature and the
been assigned [13,14,16]. This investigation was performed in detector temperature was 35°C. Pullulan standards were dis-
order to assign additional chemical shifts using 2D-spectros- solved in water (O.Smg/mL). Amylopectin was dissolved in ei-
copy. 'H-NMR spectroscopy has the advantage of giving infor- ther the mobile phase (OSmg/mL) at 100°C or in 0.1M NaOH
mation about the degree of branching, and the mol. wt. of de- at room temperature. The samples were filtrated through
graded starch simultaneously, as shown by Gidtey [lo]. Here, 0.45pm filter prior to injection.
determinations of the mol. wt. of polysaccharides are per-
formed by comparing 'H-NMR spectroscopy and size exclu- 2.4 NMR spectroscopy
sion chromatography (SEC). 2.4.1 Sample preparation
Two different pretreatment methods for normal potato
2 Experimental starch were compared; where the granules were dissolved ei-
ther in DMSO-d6 or in Dz0. When using DMSO-d6, dry starch
2.1 Materials was dissolved (lOmg/mL) in a screw capped bottle at 70°C
Starch samples from different potato varieties were gifts over-night (approximately 17h) with continuous stirring at
from the two Swedish manufacturers (Lyckeby Starkelsen, low speed, followed by freeze-drying. Exchange of hydroxylic
Kristianstad and Svalof-Weibull AB, Svalov) involved in the protons was performed in order to reduce interference from
development of amylopectin type potato starch (APPS). The the residual solvent resonance; 1.0ml DzO was added to 20mg
total ash and phosphorus contents in all starches were deter- of the sample, heated for 15min at 100°C followed by a sec-
mined by the manufacturers to be < 0.4% (w/w) and < 0.06% ond freeze-drying. The dried, deuterated sample was dis-
(w/w), respectively. Amylose and amylopectin from potato solved in D20 and heated for 15min at 100°C. The NMR-sam-
were purchased from Sigma (Sigma Chemical Co, St. Louis, ples were always prepared immediately before measurements
MO, USA, amylose type 3, cat. no. A-0512, lot 42H3861; amy- and never cooled below 70°C, in order to prevent retrograda-
lopectin cat. no. A-8515, lot 111H3901). Another amylopectin tion prior to analysis. When not using DMSO-d6 as a granule
sample from potato was purchased from Merck (Darmstadt, disperser, but simply DzO, the same procedure as described
Germany, cat. no. 1469, lot 818K3218369). Amylopectin from above for the exchange of hydroxylic protons was followed
corn was purchased from Sigma (cat. no. A-7780, lot 25F- with the exception that native starch was boiled for 30min, be-
0608). Pullulan standards were purchased from Shodex fore and also after freeze-drying, to obtain a homogeneous
(Showa Denko K. K.,Tokyo, Japan, from Aureobasidiurn pullu- sample.
lam, kit P-82, cat. no. 722106, lot no. 20101) with eight differ-
Amylose was pretreated in D M s 0 - d ~as described above
ent molecular weight fractions ranging from 853 kDa to 5.8 and freeze-dried. This sample was easily redissolved in D20
kDa. DzO was purchased from Dr. Glaser AG (Basel, Switzer- for exchange of hydroxylic protons, followed by freeze-drying.
land, cat. no. 57922, isotopic purity > 99.95 atom O/o D, which The amylose NMR-sample was finally prepared by dissolution
was used for proton exchange and cat. no. 57928, isotopic pu- (10mg/mL) in 90% DMSO-d6 in D 2 0 , 100°C for 15 min. Amy-
rity > 99.98 atom Yo D, which was used as a solvent for NMR lopectin was dissolved in DzO (2Omg/mL) at 100°C for 15min
samples). Deuterated DMSO (DMSO-d6) was purchased after exchange of hydroxylic protons as described above. Pul-
lulan was dissolved in DzO at room temperature, in accor-
from Dr. Glaser (cat. no. 12681, isotopic purity > 99.8 atom Yo
dance with the manufacturer's recommendations.
D) .
2.2 Blue-values 2.4.2 N M R spectroscopy
BV were measured according to the method described by The NMR measurements were performed with a spec-
Peng and Perlin [15], with the addition of 0.5mL of pure water trometer (mod. ARXSOO, Bruker Fallanden, Switzerland) op-
(Millipore, Milli-R04, CFOF 01205 Bedford, MA, USA) to erating at 500.25MHz for IH, and 125.76MHz for I3C. Sodium
5mL of DMSO and 50 mg of starch. The wavelength at maxi- 3-trimethylsilylpropionate-2,2,3,3-d4(TSP) was used as inter-
mum absorptivity (An3ax) of the polysaccharideiodine complex nal reference for the 'H and 13Cchemical shifts scales. Spectra
and BV were measured in a spectrophotometer (mod. U2000, were accumulated at 8OOC. A 'H selective 5mm probe was em-
Hitachi,Tokyo, Japan), using a l.Ocm cell. The iodine solution ployed for quantitative measurements. The probe was tuned
contained iodine (2 mg/mL) and potassium iodide (20mg/ to each sample to ensure optimal signal-to-noise ratio (S/N)
mL) in water. BV was measured at the approximate A,, for and consistency throughout the experimental series. Longitu-
amylose-iodine (640nm) and expressed as 4 Alconc. (mg/dL). dinal relaxation (TI) for 'H was estimated by the inversion-re-
covery method [17]. For quantitative measurements a 70' exci-
2.3 Size exclusion chromatography (SEC) tation pulse was employed along with the repetition time of
Sample solution (lOOpl) was injected into a guard column lOs, which was larger than 5tTl for the slowest relaxing pro-
(mod. Micro Aquagel, Chrompack, The Netherlands, 7.7 X ton of interest (i.e., T I for H4(t) was found to be ~ 2 . 0 ~2D
).
50mm,) followed by a SEC column (mod. TSK G4000PW, Ul- correlation spectroscopy was performed using an inverse
tropac, LKB, Bromma, Sweden, 7.5 X 600mm, exclusion lim- 5mm probe equipped with a shielded gradient coil. Gradient
its: 0.1-700 kDa). An on-line eluent degasser (mod. ERC- selection was used when acquiring the COSY [18] (Correla-
3112, ERMA CR inc, Tokyo, Japan) was connected before the tion Spectroscopy), HMQC [I91 (Heteronuclear Multiple
pump (mod. LKB 2150, Pharmacia, Bromma, Sweden). The Quantum Coherence), and HMBC [20] (Heteronuclear Multi-
eluate was monitored with a differential refractometer (mod. ple-Bond Correlation) spectral data. Refocusing delays in
ERC-7512 ERMA CR inc) and the data was evaluated with HMQC and HMBC were optimized for 'JcH=145Hz and
the software JCL 6000 (Jones Chromatography, Littleton, "Jc~=10Hz. The raw 1D data from quantitative measure-
Colorado, USA). The eluent was 1OOmM NaCl and 0.010mM ments were Fourier transformed and the resulting spectra
NaOH, containing 50p1 Kathon C G / L (1.125% methylchloroi- were baseline corrected by substraction of a matched polyno-
sothiazolinone and 0.375% methylisothiazolinone, Rohm and mial using Bruker UXNMR software (ver. 930901). Integra-
Haas Ltd, Croydon, England, UK) to prevent microbial tion of the peak areas of a-(1-4) and of a-(1+6) was per-

Slarch/Starke 48 (1996) Nr. 10, S. 352-357 353


formed at fixed ppm values on the spectrum at 5.47-5.23 and The viscosity of DMSO is higher than that of water and it is
5.01-4.89, respectively, using Bruker UXNMR software. The commonly known that highly viscous NMR-samples lead to
S/N of the a-(1+6) proton resonance was also calculated by line broadening. Furthermore, the signals become broader as
using a fixed spectral region and the spectrometer software. the molecular weight of a species increases, due to decreasing
motional rate of larger molecules in solution [22].Thus, starch
2.4.3 Notations of the protons and carbons consisting of very large molecules in viscous solutions has
The glucose residues in starch are divided into four groups; limitations as far as sample concentration is concerned. For
those involved in linear a-(1+4) linkages in a chain, in bran- native potato starch, a concentration of 10-20mg/mL in D20
ched a-(1+6) linkages, in terminal non-reducing ends, and in was found to be convenient.
one terminal reducing end. With this notation, it is assumed The concentration of DMSO in the solution for BV and
that the terminal glucose units are involved in linear linkages, I,,, measurements was limited to 0.5%, as suggested by Peng
in agreement with proposed structural models of amylopectin and Perlin [15]. For granular starch, it was found that adding a
[3]. The protons in a glucose residue involved in a linear chain small portion of water prior to dissolution in DMSO made the
are followed by (1-4) (e.g., H-l(1-4) for the anomeric pro- sample more clear and the starch-iodine complex solution
ton), those in a branching point are followed by (1-6), those more stable without any noticeable precipitation.
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 3.2 Assignments of chemical shifts
carbon atoms are denoted according to the same notation as Figure 1 shows a 'H-NMR spectrum of potato starch (la)
the protons. and of degraded amylopectin (Ib). The assignments of the
The average CLn. is calculated according to Eqn. 1. The de- various shifts that are of importance when determining the
gree of branching is calculated according to Eqn. 2, which ex- degree of branching and the mol. wt. are labelled in Figure 1.
presses the number of branching points compared with the to- The signals of the other protons are more or less overlapping,
tal number of linkages. 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-
Average CLn = integral (H-1(1+4) + H-l(t) + H-l(1-6)) sign new separate chemical shifts. Zang et al. [16] demonstrat-
om. 1) ed well resolved 2-D spectra of glycogen (a large and highly
branched a-D-glucose polymer) allowing for assignments of
Degree (integral H-1(1+6)) * 100 chemical shifts. In this work potato amylopectin and native
of branching = potato starch were used and additional assignments were
,
integral (H-I(lb4) + +
H-l(t) H-l(lb6)) made as determined from 2-D homonuclear and 'H-I'C
( E m 2) 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).
3 Results and Discussion This interpretation of 2D correlation spectra is supported by
H-l(l-4)
3.1 Preferred pretreatment methods and solvents I
A homogeneously dissolved sample is essential to obtain -w
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 oom 5.0
,
4.5
' ' " I " "
4.0
" ' '
3 5
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 Fig. 1. 'H-NMR spectrum of a) normal potato starch, and of b) de-
stable sample, whereas retrograded amylose requires a high graded amylopectin (degree of branching = 4.3%). (2Omg/mL D 2 0 ,
(autoclave) temperature to affect dissolution at neutral pH [l]. 80°C.)

354 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 3.3.2 Highly branched genetically engineered potato
range couplings are identified. Some of the nuclei are labelled starch
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 All the APPS samples are significantly more branched than
identified at 102.1 and 102.4ppm, respectively. Separate as- is normal starch from potato varieties that are not genetically
signments of chemical shifts were made also for H-6a:b(1-4) engineered. The degree of branching reflects the CLn in the
and H-6a:b(t), and for C-6a:b(l-4) and C-6(t). starch sample, since each chain has one branching point,
while a high BV and A,, indicate a high amylose content [8].
3.3 Determination of branching frequency These results show, see Table 2 and Figure 4, that the BV and
the degree of branching are similar in APPS and in commer-
3.3.1 Repetability of method cial amylopectin from potato and the new APPS was found to
Provided that certain precautions are taken when the have a high degree of branching, short average Cln, A,,,, and
NMR spectrum is recorded, the peak area is proportional to BV of what could be expected for potato amylopectin [2, 10,
the number of protons. The integration procedure [lo], the 11, 15,241 consisting of mainly branched polymers. The aver-
comparably small peak area arising from the H-(1-6) signal age Cln of the APPS samples was found to be around 24,
[I I], incomplete relaxation of the signals 1141, and the use of which is in the same range as reported values for amylopectin
water supression [14] are possible sources of error limiting the fractionated from potato starch (average CLM of 19-24) [2,3,
accuracy in determining the degree of branching. These prob- 10, 11, 241. All the APPS samples and also the purified amy-
lems have been addressed in this investigation. Relaxation lopectin sample from potato had approximately the same
times for the anomeric protons have been determined to con- A,,,,,, at 550 nm. Potato amylose, and normal potato starch
firm that these protons were fully relaxed and the time of samples had Lmax at approximately 640, and 600 nm, respecti-
spectral acquisition was sufficient. Water suppression was not vely. The CLM of amylopectin varies with its origin, e.g. corn
necessary, since the relatively low H D O signal did not inter- amylopectin has shorter chains than has potato amylopectin
fere with the signals from the anomeric protons, see Figures [2,24]. The corn amylopectin used in this investigation was, as
l a and b at 4.15ppm. (Exchange of hydroxylic protons in D20 expected, more branched than the potato amylopectin
followed by freeze-drying prevent a high H D O signal.) (Sigma) [2,24] and A,, was lower, at 540nm. The correlation
The accuracy in the integration of the signals was found to between the two methods, 'H-NMR spectroscopy and spec-
be highly dependent upon the S I N ratio of the integrated trophotometric measurement of starch-iodine complex, used
resonance peak, where S / N will improve by a factor of the for comparing the content of branched material in starch, is
square root of the number of scans. From empirical results it good as illustrated in Figure 4.
was here found that the S / N of the H-1(1+6) resonance must Values from the literature of the degree of branching in
exceed a value of 50 in order to give a consistent integral. normal potato starch [12, 151, as determined using 'H-NMR
With a sample concentration of lOmg/mL potato starch, the ranges from 2.8% to 3.4%. The starch samples from the nor-
S / N of 50 was achieved with 64 scans, giving an experimental mal potato varieties that were investigated here, range in de-
time of less than 15min. With a S I N larger than 50 for the gree of branching from 3.1% to 3.3%. Longer term studies
H-l( 1-6) and a proper baseline correction, the relative stan- could provide information about actual genetic differences in
dard deviation of the mean value (rsd) of the degree of the starch structure in different varieties. Starch from differ-
branching was low (rsd = 1.4°/o, n = 6 of commercial potato ent varieties and ages has been reported to be different in mo-
starch, including sample pretreatment). The values of the de- lecular structure, and ripening of potato tubers leads t o a hi-
gree of branching presented in Table 2 differed typically only gher amylose content [8] and elongation of the chains of both
k 0.02 points and always < k 0.07 points between two repeti- amylose, and amylopectin [25].
tions. This equals typically k 0.2 and always 5 f 0.6 glucose When determining the degree of branching according to
units in the calculated CLn. The repeatability in determining Eqn. 2, the reducing end was neglected and the branched link-
the degree of branching by this method is good compared
with previous investigations using N M R [lo], enzymic, and 1.o &.
AM
chemical methods [2, 11, 231. **.

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- K. Helmersson, K. Svegmark and 0. Wikstrom, Lyckeby Starkelsen
other way of expressing the degree of branching is to deter- for starch samples and valuable discussions.
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. Bibliography
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glucose residues (also including the H-l(r)) of a low molecu- 22 (1993), 19-29.
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(Merck) shown in Figure l b gives almost the same value, [6] Hofvander, P.,P. 7:Persson, A . Tallberg, and 0. Wikstrom: Swedish
4.30% and 4.22%, respectively. For samples of high mol. wt, Pat. 9004096-5, Amylogene HB (1992).
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due per molecule, is insignificant. tin Potato Starch, Starch Convention 1995 Detmold, Germany.
[8] Banks, I.I.: and C.T. Greenwood: Starch and its Components.
3.3 Determination of molecular weight by University Press, Edinburgh, UK, (1975).
~H-NMRspectroscopy [9] Banks, W , and C. 7: Greenwood: Arch. Biochem. Biophys. 136
'H-NMR spectroscopy gives information about the degree (1970), 320-322.
of branching, and the mol. wt. of degraded starch simultane- [lo] Gidley, M . J.: Carbohydr. Res. 139 (1985), 85-93.
[ l l ] Blake. J. D..M . L. Clarke, and J. Littlemore: Carbohydr. Res. 138
ously [lo]. The mol. wt. is determined by comparing the areas (1985), 161-167.
from the a + &free anomeric resonances [lo] with the total [12] McIntyre, D. D.,C. Ho, and H. J. Vogel: Starch/Starke 42 (1990),
anomeric resonances. This can be done in low mol. wt, sam- 2 6 0-2 67.
ples, where the number of reducing rings is relatively high. Ac- [13] Mclntyre, D.D.,and H. J . Vogel: Starch/Starke 42 (1990), 287-
cording to Mclnwvre and Vogel[14], the free anomeric protons 293.
were observed in a spectrum of dextran with a mol. wt. of 9.4 [14] Mclntyre, D.D.,and H. J. Vogel: Starch/Starke 43 (1991), 69-76.
kDa, but not in a spectrum of dextran having a mol. wt. of70.8 [15] Peng, Q.J..and A . S . Perlin: Carbohydr. Res. 160 (1987), 57-82.
kDa. Amylopectin from potato (Merck) was here found to be [16] Zang, L.-H., A . M . Howseman, and R. G. Shulmann: Carbohydr.
Res. 220 (1991), 1-9.
highly degraded. The number average mol. wt. as determined
[17] Vold, R. L., J.S. Waugh, M . P. Klein, and D.E. Phelps: J. Chem.
by NMR (see Figure lb) was 8.06 kDa, DP = 49.6 (degree of Phys. 48 (1968), 3831-3832.
polymerization, no. of glucose residues) (rsd = 4%, n=3). Re- [18] Hurd, R. E.: J. Magn. Reson. 87 (1990), 22-428.
sults from SEC agree well with mol. wt. determinations using [19] Hurd, R. E., and B. K. John: J. Magn. Reson. 91 (1991), 648-653.
'H-NMR spectroscopy. The peak maximum in the SEC-chro- [20] Wilker, W , D.Leibfritz. R. Kerssebaum, and W Bermel: Magn. Re-
matogram was determined to be DP = 50.1, and the peak son. Chem. 31 (1993), 287-292.
width ranging between DP = 700 to DP < 6 (totally perme- [21] Jackson, D.S.: Starch/Starke 43 (1991), 422-427.
ated). The larger mol. wt. potato amylopectin (Sigma) was al- [22] Mora-Gutierrez, A . , and I. C. Baianu: J . Agric. Food Chem. 39
most totally excluded by this column. Dissolution of the amy- (199 l), 1057- 1062.
[23] Banks, W., C. I: Greenwood, and K. M. Khan: Starch/Starke 22
lopectin samples in either the mobile phase (0.OlmM NaOH)
(1970), 292-296.
at 100°C or in 0.1M NaOH at room temperature gave the same [24] Hizukuri, S.: Carbohydr. Res. 141 (1985), 295-306.
results. Pullulan standards with DP 33.5 and 71.0 (measured [25] Praznik, W ; G. Burdicek, and R. H. R Beck: Starch/Starke 38
by the manufacturer by the ultracentrifugal sedimentation (1986), 181-184.
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. B. Sc. G. S. Nilsson and Associate Professor Dr. L. Gorton, Depart-
ment of Analytical Chemistry, University of Lund, P.O. Box 124,
Acknowledgements S-22100 Lund, Sweden.
Dr. K.-E. Bergquist and Dr. U.Nilsson, Department of Organic Chem-
This work was supported by Lyckeby Starkelsen, Kristianstad, istry 2, University of Lund, P.O. Box 124, S-22100 Lund, Sweden.
Sweden, and the Swedish Research Council for the Engineering Sci-
ences (TFR). The authors wish to thank P. Hofvander, Svalof-Weibull
AB, (Received: March 13, 1996)

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