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RESEARCH ARTICLE

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Chromatographic Study of High Amylose Corn Starch

Genotypes – Investigation of Molecular Properties after

Speciﬁc Enzymatic Digestion

Marco Ulbrich,* Fanni Scholz, and Eckhard Flöter

Two high amylose corn starches (HACS; HYLON V and HYLON VII) are

dissolved completely and subjected to speciﬁc enzymatic degradation by

means of diﬀerent amylases (AMY). The starches are digested using 𝜶-, 𝜷-,

and 𝜸-AMY (single) as well as in combination with the debranching enzyme

pullulanase (PUL; 𝜶-AMY-PUL and 𝜷-AMY-PUL), and the products are

characterized by means of size exclusion chromatography (SEC)-techniques

including multi angle laser light scattering-diﬀerential refractive index

detection (SEC-MALS-DRI) and conventional calibration-diﬀerential refractive

index detection (SEC-cal-DRI). Enzymolysis is resulted in largely maltose (Mal;

𝜶-and𝜷-AMY and respective combinations with PUL) or glucose (Glc; 𝜸-AMY;

almost complete digestion) as the major fraction, but also other residual

fractions of higher molar mass (MM), i.a. 𝜶-and𝜷-limit dextrins (𝜶-/𝜷-LDs).

The quantity (relative portion) and quality (speciﬁc molecular properties) of

the reaction products are found to be strongly dependent on both the

enzymatic treatment by itself (kind of enzyme/-combination and associated

speciﬁcity of action) and the molecular composition of the initial starch

(portion and speciﬁc molecular properties of the amylose [AM], intermediate

[IM], and amylopectin [AP] fraction), which is further investigated in detail.

1. Introduction

The polysaccharide starch is a mixture of diﬀerent structure

fractions. It consists of the mostly linear amylose (AM) with

anhydroglucose units (AGU) linked via 𝛼-1,4-glucosidic bonds

(weight average MM [Mw] between 105and 106gmol

−1), and

the amylopectin (AP), which is highly branched (𝛼-1,4- and 𝛼-

1,6-glucosidic linkages, Mwup to several 107gmol

−1). Normal

starches (isolated from, e.g., potato, wheat, or corn) feature an

M. Ulbrich, F. Scholz, E. Flöter

Department of Food Technology and Food Chemistry

Technische Universität Berlin

Chair of Food Process Engineering

Oﬃce ACK3, Ackerstraße 76, Berlin 13355, Germany

E-mail: [email protected]

The ORCID identiﬁcation number(s) for the author(s) of this article

can be found under https://doi.org/10.1002/star.202100303

This is an open access article under the terms of the Creative Commons

Attribution License, which permits use, distribution and reproduction in

any medium, provided the original work is properly cited.

DOI: 10.1002/star.202100303

AM content of about 20–30% w/w.[1] How-

ever, e.g. most pea starches possess a higher

AM content (e.g., 33% w/w [smooth pea],

71% w/w [wrinkled-pea]).[2,3] In contrast

to normal or regular starches, respectively,

starch varieties vary strongly with respect

to the AM/AP-ratio. On the one hand,

waxy varieties consist of AP (marginal AM

content),[4,5] on the other hand, the so-called

high AM starches have AM contents higher

than, e.g., 50% w/w,[6,7] in either case sig-

niﬁcantly higher compared to the regular

ones. The existence of an intermediate (IM)

fraction is commonly accepted).[8–10] The

starch type (source, variety) deﬁnes not only

the AM/AP-ratio, but rather the molecular

features of the polymer fractions speciﬁed

by parameters like MM and the correspon-

dent MM distribution (MMD), degree of

branching, AP branch chain (BC) length,

complexing with iodine and supramolec-

ular properties like type (crystal pattern)

and degree of crystallinity (granule charac-

teristics) (Cheetham and Tao[11]). Moreover,

minor components like fat/lipids, protein, and water vary

strongly related to their relative portion depending on the type

of starch. For example, the fat/lipids content of corn starches

is positively correlated with the AM content,[12–14] and potato

AP is characterized by a signiﬁcant portion of phosphate es-

ter groups bound covalently.[15–19] The determination of the AM

content and impacts of the diﬀerent methods (iodine complex-

ing/colorimetric, concanavalin A precipitation, 1D size exclusion

chromatography (SEC) fully branched, 1D SEC debranched, and

2D structural distributions from multidimensional SEC ×SEC)

on the values obtained were comprehensively discussed by Vila-

plana et al.[20]

These molecular starch properties determine inevitably the

behavior of a solution, physicochemical, and techno-/functional

properties like hot paste viscosity or the ability to form ﬁrm gels.

Since the AM fraction is mainly responsible for the development

of a 3-D gel network, high AM starches are basically suitable for

this purpose.[21,22] An appropriate solution state of such starches

is an essential requirement (Vesterinen et al.[23]). The inherent

starch properties, in particular on the molecular level, govern

the enzymatic digestion, which is of great importance for both

starch analytical problems and industrial processing. The char-

acteristics of molecules and molecule fractions (speciﬁc amount,

molecular properties), respectively, originated from the speciﬁc

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enzymatic digestion of a dissolved starch, are strongly dependent

on the composition of the initial starch material and, of course,

the speciﬁcity of the respective amylolytic enzyme.

Four groups of starch converting enzymes are diﬀerentiated,

the endo-AMY (1), the exo-AMY (2), debranching enzymes (3),

and transferases (4) (van der Maarel et al.[24]). The 𝛼-AMY (EC

3.2.1.1) is an endo-AMY, which is able to cleave 𝛼-1,4-glycosidic

linkages present in the inner part (endo-) of the molecule chain

producing oligosaccharides with varying chain length and 𝛼-limit

dextrins (𝛼-LDs), which constitute branched oligosaccharides.

Hydrolysis of granular starches (HACS, waxy CS [WxCS], nor-

mal CS, wheat starch, wrinkled pea starch) using 𝛼-AMY (As-

pergillus fumigatus) results in minor amounts of maltose (Mal)

and maltotriose, and the major product is glucose (Glc) (Plan-

chot et al.[25]). The release of Mal, maltotriose, and a resis-

tant amylodextrin (porcine-pancreatic 𝛼-AMY, human-salivary 𝛼-

AMY) and maltotriose, maltopentaose, maltohexaose, and a resis-

tant amylodextrin (Bacillus subtilis 𝛼-AMY) was reported by Jane

and Robyt.[26] The formation of 𝛼-LDs by hydrolysis of AP was

reported elsewhere.[27,28] The 𝛽-AMY (EC 3.2.1.2) is an exo-AMY

and cleaves exclusively 𝛼-1,4-glycosidic linkages (every 2nd) from

the non-reducing end of the chain. Since the digestion is termi-

nated on the polymer branches (𝛼-1,6-linkages in AP, IM fraction,

and AM), Mal as well as 𝛽-LDs are released,[29] which should have

a degree of polymerization (DP) of approximately 50% compared

to the AP fraction.[30] Landerito and Wang[31] reported contents of

𝛽-LDs between about 39% and 57% after hydrolysis for diﬀerent

CS (WxCS, common CS, HYLON V, and HYLON VII). The ﬁne

structure of 𝛽-LDs is based on chain length fractions of average

DP 50, 25, 15, 10, 7, 3, and 2 (WxCS) (Derde et al.[32]). The 𝛾-

AMY (glucoamylase, amyloglucosidase; EC 3.2.1.3) catalyzes the

hydrolysis of 𝛼-1,4- and 𝛼-1,6-glucosidic linkages from the non-

reducing end of the polymer chain (exo-AMY) to release predom-

inantly 𝛽-D-Glc and related poly- and oligosaccharides.[33] The

debranching enzyme PUL hydrolyzes starch by cleavage of the

𝛼-1,6-linkages (PUL type I; release of AP BC and basically BC

from other branched polymer fractions [IM and AM]; Li et al.,[34]

Hii et al.[35]). In particular, the combined application of PUL (de-

branching) and, e.g., 𝛼-AMY and 𝛽-AMY, respectively, enables the

degradation of the starch polymer structure for the most part with

the release of predominantly mono- and/or disaccharides.

A prerequisite for the (theoretically) complete enzymatic diges-

tion according to the particular speciﬁcity of the enzyme is the

accessibility to the respective sites, which is ensured by an opti-

mal solution state. Retrograded regions, but also chemical modi-

ﬁcation, can decrease the extent of the total hydrolysis or impede

the hydrolysis at least partially. This resistance of starch polymer

areas to molecular degradation is based on double helical struc-

tures and associated formation of crystalline regions (retrograda-

tion), which would be expected in particular for the AM fraction

but also for smaller unbranched residues originating, e.g., from

longer AP BC (e.g., after starch is debranched). The expected par-

tial resistance of starch polymers to enzymatic digestion due to

being protected by the higher organization level (retrogradation),

which is evidentially the case in starch gels, can be potentially

utilized for analytical purposes. Speciﬁc enzymatic degradation

of starch polysaccharides in a gel, which are not protected by

the structure (double helical, crystalline) and accessible to enzy-

matic cleavage, respectively, and subsequent special characteriza-

tion of the resistant residues by means of SEC-techniques (size

exclusion chromatography-multi angle laser light scattering-

diﬀerential refractive index detection, SEC-MALS-DRI, size ex-

clusion chromatography-conventional calibration-diﬀerential re-

fractive index detection, SEC-cal-DRI) could contribute to bet-

ter understanding of the gel structure. Hence, comprehensive

knowledge regarding the molecular properties of two HACS (HY-

LON V and HYLON VII), and their speciﬁc enzymatic degrada-

tion (single: 𝛼-AMY, 𝛽-AMY, 𝛾-AMY, combination: 𝛼-AMY-PUL

and 𝛽-AMY-PUL) in the solution state (complete dissolved) was

derived by SEC-techniques in the present study, which is a nec-

essary requirement for the successful and reliable analysis of the

gel ﬁne structure in a second step. This is the ﬁrst of a series of

publications.

2. Experimental Section

2.1. Starch Genotypes

Two commercial native HACS genotypes (HYLON V [HACS-50]

and HYLON VII [HACS-70], Ingredion Germany GmbH, Ham-

burg, Germany) were provided for the examination. The speciﬁ-

cations indicated are supplier information (HACS-50: 55% w/w

AM [nominal], 1.0% w/w total fat, 0.5% w/w protein, ≤0.1% w/w

ash; HACS-70: 73.8% w/w AM [colorimetric method], 0.58% w/w

protein). The dry matter contents (DM; HACS-50: 88.46% w/w,

HACS-70: 88.36% w/w) were determined using a moisture ana-

lyzer (MA 30, Sartorius, Göttingen, Germany).

2.2. Starch Degrading Enzymes

Diﬀerent starch degrading enzymes were used, an 𝛼-AMY (pow-

der, Optizym A 16126 [21 000 U g−1], SternEnzym GmbH &

Co. KG, Ahrensburg, Germany), a 𝛽-AMY (solution, Secura

[5000 BAMU g−1], Novozymes A/S, Bagsvaerd, Denmark), a 𝛾-

AMY (solution, AMIGASE MEGA L [≥36,000 AGI g−1], DSM

Food Specialties B.V., Delft, Netherlands) and a PUL (solution,

Promozyme D2 [200 U mL−1], Novozymes A/S, Bagsvaerd, Den-

mark). A solution of the 𝛼-AMY was prepared by dispersion of

the powder in water (addition of 9 g water to1genzyme;freshly

prepared before each experiment).

2.3. Molecular Characterization Using SEC-Techniques

Methodical steps of the comprehensive molecular characteriza-

tion of the starch genotypes as well as the products after enzy-

matic digestion are summarized schematically in Figure 1.

2.3.1. Preparing Starch Solutions

Starch solutions were prepared by heating aqueous dispersions

of 2.5% w/w in an autoclave (Model I, Carl Roth GmbH & Co.

KG,Karlsruhe,Germany)to145°C under continuous stirring

(300 min−1) for 30 min and subsequent high-shear-treatment us-

ing an Ultra-Turrax T25 (IKA-Werke GmbH & Co. KG, Staufen,

Germany) at 24,000 min−1for 2 min at about 80 ±5°C. An

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Figure 1. Schematic representation of the diﬀerent preparation steps for the SEC based molecular characterization.

aliquot of the dispersion was diluted 1:10 (v/v) in preheated

DMSO (2.5 mg mL−1), and the stabilized solution was passed

through a 5 μm PTFE ﬁlter (Carl Roth GmbH & Co. KG, Karl-

sruhe, Germany) before analysis (SEC-MALS-DRI, no enzymatic

treatment).

2.3.2. Speciﬁc Enzymatic Degradation

A volume of 10 mL of the freshly prepared starch solution (2.5%

w/w) was tempered to 40 °C and the respective volume of the en-

zyme solution was added (𝛼-AMY: 150 μL, 𝛽-AMY: 100 μL, 𝛾-AMY:

150 μL, PUL: 187 μL). For the degradation using enzyme combi-

nations (𝛼-AMY-PUL and 𝛽-AMY-PUL) the respective volumes

of both enzymes were added. The dispersion was gently stirred

at 40 °C for 20 min. After enzymatic digestion, the solution was

heated to 95 °C and tempered for 20 min for termination of the

enzyme. The solution was diluted 1:10 (w/v) in preheated DMSO

and passed through a 5 μm PTFE syringe ﬁlter (Carl Roth GmbH

& Co. KG, Karlsruhe, Germany) before analysis.

2.3.3. Separation Technique

The molecular characterization of the polydisperse solutions

was carried out by means of SEC-MALS-DRI. The separation

was executed with an SEC-3010 module (WGE Dr. Bures GmbH

& Co. KG, Dallgow-Döberitz, Germany) including degasser,

pump, and auto sampler connected to a MALS detector and a

diﬀerential refractive index detector (DRI). The MALS detector

was a Bi-MwA (Brookhaven Instruments Corporation, Holtsville,

NY, USA) ﬁtted with a diode laser operating at 𝜆=635 nm and

equipped with seven detectors at angles ranging from 35°to

145°. The DRI was a SEC-3010 RI detector operating at 𝜆=

620 nm. Three columns in a row were used: AppliChrom ABOA

DMSO-Phil-P-100 (100–2500 Da), P-350 (5–1500 kDa), and P-600

(20 to >20 000 kDa) (Applichrom, Oranienburg, Germany). The

samples were eluted with degassed DMSO (Carl Roth GmbH &

Co. KG, Karlsruhe, Germany) containing 0.1 M NaNO3at a ﬂow

rate of 0.5 mL min−1and a temperature of 70 °C. During the

sample run on the SEC-MALS-DRI system (single determina-

tion), the data from the MALS and DRI detectors were collected

and processed using ParSEC Enhanced V5.61 chromatography

software to give the concentration of the eluted solution and MM

at each retention volume (Mi). The basis for the molecular char-

acterization by means of SEC-MALS-DRI has been described

elsewhere.[36,37]

The separation system was additionally calibrated (SEC-cal-

DRI) using a set of 10 pullulan standards (800k, 400k, 200k, 110k,

50k, 22k, 10k, 6k, 1.3k, and 342) as well as Glc with a MM range

between 180 and 805 000 g mol−1(PSS Polymer Standards Ser-

vice GmbH, Mainz, Germany). The calibration limit (800k) is

indicated in Figure 2A. The standards were dissolved in DMSO

(2.5 mg mL−1w/v) and gently stirred 24 h at 80 °C. The standard

solutions were measured and the elution volume at the position

of the peak maximum was used as the reference for the particu-

lar Miand the calculation of the calibration curve. The calibration

related to the DP was calculated from the Midivided by 162. The

weight average DP (DPw) was calculated from the Mwdivided by

162.

2.3.4. Mathematical Peak Separation

The SEC-chromatograms (starch without enzymatic treatment

[Section 3.1.1], enzymatically debranched by means of PUL

[Section 3.1.1], digested with 𝛼-/𝛽-/𝛾-AMY and 𝛼-/𝛽-AMY-PUL

[Section 3.2.2]) were advanced analyzed using peak separa-

tion and analysis software PeakFit Version 4.12 as described

elsewhere.[38] Based on the ﬁtted SEC-chromatograms, single

peaks (chromatograms) representing diﬀerent fractions were

identiﬁed and calculated. The chromatograms originating from

the enzyme solution (Section 3.2.1) were subtracted, and the rel-

ative chromatogram area of each separated fraction was taken as

the relative amount. The values of Mwand DPwwere calculated

by means of the correspondent separated chromatogram and

the MM curve (ﬁt) from the MALS-detector (SEC-MALS-DRI)

or the standard calibration curve (SEC-cal-DRI), respectively,

according to the description elsewhere,[39,40] Speciﬁc band

broadening, induced by the injected volume or the injected

polymer concentration, respectively, was excluded based on

preliminary experiments. SEC-cal-DRI was used in Section 3.1.1

(characterization of the AP BC fraction [Figure 3]) and 3.2.2

(characterization of the obtained fractions [𝛼-AMY: F2 and F4,

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Figure 2. SEC-chromatograms of (A) Mal and Glc and (B) of the enzyme formulations (𝛼-, 𝛽-, and 𝛾-AMY as well as PUL) taking as reference or blank,

respectively. The calibration limit (SEC-cal-DRI) is indicated.

𝛼-AMY-PUL: F3, 𝛽-AMY: F3, 𝛽-AMY-PUL: F3; Figures 4 and 5]).

The applied method was indicated properly.

2.4. Determination of the AM Content

The AM content of the starch genotypes was determined by

means of diﬀerent methods, which are summarized in Table 1.

2.4.1. BV Method and Calibration Curve

Methods 1–4: The blue value (BV) was determined according to

the description elsewhere,[1,41] at 20±2°C after defatting with

EtOH (except method 4). The spectrophotometer used was a Jen-

way 6505 UV/visible (absorbance at 𝜆=635 nm). The calibration

curve for the determination of the AM content of the starch sam-

ples was prepared over a range of 2–64% w/w AM using mixtures

of a commercial WxCS (estimated AM content: 2% w/w, DM:

88.40% w/w; C*Gel 04201, Cargill B.V., Sas van Gent, Nether-

lands) and a HACS (nominal AM content: 64% w/w, DM: 88.40%

w/w; Megazyme International Ireland, Wicklow, Ireland). Mix-

tures were 100:0 (2% w/w AM), 80:20, 60:40, 40:60, 20:80, and

0:100 (64 % w/w AM). There was a linear correlation (R2=

0.9953, n=18) between the BV20 and the AM content [% w/w]

(BV20 =0.0399∙AM [%] +0.1763).

2.4.2. Defatting Using Organic Solvents

Methods 2–4: Partially defatted samples were prepared by treat-

ment using two diﬀerent solvent systems (TCM-MeOH [mixing

ration 1:2 v/v] and BuOH). An aqueous starch suspension

(50% w/w, 100 g) was prepared and a volume of 400 mL of the

respective organic solvent system added. The system was stirred

at 300 min−1for 24 h at 50 °C. After an initial centrifugation step

(10 min, 3000 min−1), the obtained sediment fraction was again

suspended in 200 mL of the solvent system and subsequently

centrifuged (10 min, 3000 min−1). After suspending the starch

again in 200 mL of the solvent system, the starch was achieved by

suction ﬁltration (ﬁlter paper: DP 1574 125, ALBETLabScience,

Hahnemuehle FineArt GmbH, Dassel, Germany). The samples

were dried in a climate cabinet at 40 °C and about 35% RH for

24 h, and subsequently ground (pulverisette 14, Fritsch, Idar-

Oberstein, Germany; 200 μm mesh), bottled in closed containers

and stored at 20 ±2°C.

Method 4: The starch samples partially defatted using

TCM-MeOH were disintegrated (Section 2.3.1). After high-shear-

treatment (Ultra-Turrax), an aliquot of the starch dispersion was

diluted appropriately and the BV determined (Section 2.4.1).

2.4.3. Peak Areas of SEC-Chromatograms

Methods 5 and 6: The peak areas of the separated chromatograms

(Sections 2.3.4 and 3.1.1) were evaluated. On the basis of the non-

debranched starch, three peaks were separated (chromatogram

1: AP fraction [F1], chromatogram 2: IM fraction [F2], and chro-

matogram 3: AM fraction [F3]), and F3/(F1+F2) ×100% w/w was

taken as the AM content (method 5, Figure 6C,D). On the basis

of the debranched starch, two peaks were separated and investi-

gated (chromatogram 1: AM fraction [F1] and chromatogram 2:

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Figure 3. Molecular characterization of HACS-50 and HACS-70 after complete debranching. A) SEC-chromatograms after complete enzymatic debranch-

ing and respective ﬁts (PeakFit), B) separated chromatograms for the AM fraction and the AP BC fraction based on A (PeakFit), C) calculated MMD

curves of the AM fraction (derived from the Figure 6C,D without debranching and Figure 3B), D) calculated MMD of the IM fraction (without debranch-

ing, derived from the Figure 6C,D), E) MMD curves of the AP fraction (without debranching, derived from the Figure 6C,D), and F) DDP curve of the AP

BC fraction (derived from the Figure 3B).

AP BC fraction [F2]), and F1/(F1+F2) ×100% w/w was taken as

the AM content (method 6, Figure 3B).

3. Results and Discussion

3.1. Molecular Characteristics of the Starch Genotypes

3.1.1. Chromatographic Investigation

Molecularly dispersed solutions of both HACS-50 and HACS-70

were investigated by means of SEC-MALS-DRI. Figure 6A shows

the chromatograms and the related MM ﬁts of the genotypes.

The ﬁrst chromatogram peak between about 15 and 17.5 mL cor-

responds presumably for the most part to the AP fraction, and the

diﬀerent speciﬁc chromatogram areas and height, respectively,

are ascribed to the diﬀerent AP contents of the corn starches.

However, the elution of AP of lower MM is strongly assumed

to be concomitant with the AM of higher MM. The slightly

diﬀerent positions of the MM ﬁts indicate diﬀerences regarding

molecule structure as well as solution state, i.e., the possible

existence of small particles. The MM ﬁt of the HACS-70 is lower,

most probably due to the signiﬁcantly higher AM content of the

sample eluting concomitantly with the highly branched polymer

molecules. The high degree of branching and the compact struc-

ture, respectively, result in a higher MM of the polymers eluting

at a speciﬁc elution volume. The huge diﬀerences between the

corn starch varieties are clearly obvious in the calculated MMD

curves in Figure 6B. The MMD of HACS-70 is broader and

shifted to lower MM (PDI: 9.27 HACS-50 and 12.60 HACS-70),

indicating lower MM in particular of the AM fraction. Since the

AM content of the HACS-70 (colorimetric method, supplier:

73.8% w/w) is supposed to be signiﬁcantly higher compared to

the HACS-50 (nominal, supplier: 55% w/w), the correspond-

ing Mwof the samples were strongly diﬀerent (HACS-50:

7.54 ×106gmol

−1, HACS-70: 3.63 ×106gmol

−1;Figure 7).

Based on the SEC-chromatograms (Figure 6A), separated

peaks were identiﬁed by means of deconvolution (Figure 6C:

HACS-50, and D: HACS-70). Two diﬀerent approaches resulted

in two and three chromatograms, respectively, where F1 repre-

sents the AP fraction, and F2 an IM fraction (branched). Frac-

tion F3 represents an AM containing polymer fraction (in the

case of two separated chromatograms) and the AM fraction (in

the case of three separated chromatograms), respectively. The

chromatograms calculated for F1 using the diﬀerent methods

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Figure 4. Molecular characterization of HACS-50 (A, C, and E) and HACS-70 (B, D, and F) after degradation using diﬀerent enzymes/combinations (A

and B: 𝛼-AMY and 𝛼-AMY-PUL, C and D: 𝛽-AMY and 𝛽-AMY-PUL, E and F: 𝛾-AMY). SEC-chromatograms after treatment of the starch dispersion with

enzyme/combination and subtraction of the respective blank (original), the chromatogram ﬁts (PeakFit), and the separated chromatograms of the frac-

tions F1–F5 calculated by means of mathematical deconvolution. Fractions F1–F5 correspond to elution volume at peak maxima of the chromatograms

of about 17–18 mL (F1), 20–21 mL (F2), 22–23 mL (F3), 24 mL (F4), and 25–26 mL (F5; Mal/Glc).

were similar, and used for further investigation. Moreover, the

existence of an IM branched fraction (F2) was obvious for both

starches, and likewise, the remarkable AM fraction (F3). The AM

contents determined using diﬀerent methods is presented in Sec-

tion 3.1.2.

For the purpose of an advanced and comprehensive molec-

ular examination, the completely enzymatically debranched

starch samples were investigated, too. Figure 3A shows the

SEC-chromatograms of debranched HACS-50/-70 (original) and

the respective chromatogram ﬁts (PeakFit). Based on the ﬁts,

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Figure 5. Relative portion and corresponding Mwof the diﬀerent fractions generated by degradation of HACS-50 and HACS-70 using diﬀerent en-

zymes/combinations (A and B: 𝛼-AMY and 𝛼-AMY-PUL, C and D: 𝛽-AMY and 𝛽-AMY-PUL, E and F: 𝛾-AMY). SEC-technique (SEC-MALS-DRI, SEC-

cal-DRI) used for calculation of Mwwas indicated (F1–F4), and attribution based on the respective peak maximum elution volume of Mal and Glc,

respectively (F5).

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Figure 6. Molecular characterization of HACS-50 and HACS-70. (A) SEC-chromatograms of the starch samples and corresponding MM ﬁts, (B) MMD

curves of the starch samples, (C) separated SEC-chromatograms of HACS-50 (PeakFit), (D) separated SEC-chromatograms of HACS-70 (PeakFit).

Table 1. Methods (1–6) for the determination of the AM content with varia-

tion of the basic principle (colorimetric and SEC-technique), the defatting

with EtOH, the starch basis (native and partially defatted), and starch state

(granular and dispersed).

Method 123456

BV ••••

Including defatting/EtOH •••

Native starch •••

Partially defatted starch TCM-MeOH ••

BuOH •

Granular •••

Dispersion/Autoclave •••

SEC technique (deconvolution) Non-debranched •

Debranched •

chromatograms of the fractions ascribed to the AM, the AP BC as

well as the enzyme solution (sucrose [Suc], Glc) were obtained by

deconvolution (Figure 3B; separated chromatogram arose from

enzyme solution is not shown).

By means of the MM ﬁts (Figures 6A and 3B), speciﬁc molecu-

lar data like MMD curves (AM fraction in Figure 3C [SEC-MALS-

DRI], IM fraction in Figure 3D [SEC-MALS-DRI], AP fraction in

Figure 3E [SEC-MALS-DRI]) and diﬀerential DP (DDP) curves

(AP BC fraction in Figure 3F [SEC-cal-DRI]), and corresponding

Mwand DPw(Figure 7, molecular properties), respectively, were

calculated.

AM: The molecular properties of the AM fraction strongly de-

pended on both method used and starch genotype in itself (Fig-

ure 7). The MwAM of HACS-70 was found to be signiﬁcantly

lower (non-debranched: 5.84 ×105gmol

-1, after debranching:

2.21 ×105gmol

-1) compared to HACS-50 (non-debranched: 1.44

×106gmol

-1, after debranching: 3.05 ×105gmol

−1), which

is supported by the respective shifts of the MMD curves (Fig-

ure 3C). The existence of a very small amount of comparatively

high MM polymers up to 4 ×106gmol

−1within the AM frac-

tion even after debranching is remarkable (Figure 3C). Since

there is a signiﬁcant diﬀerence between the MwAM derived

from the non-debranched and the debranched starch sample, the

data suggest a (slightly) branched structure of the AM fraction.

However, the respective portions calculated on the basis of the

chromatogram areas were basically comparable (HACS-50: about

48% w/w, HACS-70: about 57 % w/w; Figure 7, relative portion).

IM: An IM structure fraction was identiﬁed as a single peak

by means of deconvolution of the chromatogram of the non-

debranched starch resulting in three fractions (F1–F3), and it is

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Figure 7. Molecular data of the HACS-50/70 (Mwstarch [Figure 6A; SEC-MALS-DRI], MwAM fraction: based on the respective chromatogram cal-

culated by mathematical peak separation of the chromatogram derived from the non-debranched [Figure 6C,D; F3 with deconvolution to three chro-

matograms]/debranched starch [Figure 3B; SEC-MALS-DRI], MwIM fraction, and MwAP fraction: based on the respective chromatogram calculated by

mathematical peak separation of the chromatogram derived from the non-debranched starch [Figure 6C,D; SEC-MALS-DRI], DPwAP BC: based on the re-

spective chromatogram calculated by mathematical peak separation of the chromatogram derived from the debranched starch [Figure 3B; SEC-cal-DRI])

from SEC experiments.

consequently a constituent of F3 (besides AM) resulting in two

fractions using this method (Figure 6C,D). The IM fraction is of

branched nature, and it diﬀers in terms of molecular properties

and relative amount dependent on the genotype (Figure 7).

HACS-50, which has a lower AM content compared to HACS-

70, has a slightly higher portion of the IM polymer fraction

(about 35% w/w) compared to HACS-70 (30% w/w) exhibiting

concurrently a remarkably higher MM (HACS-50: about 7.5 ×

106gmol

-1, HACS-70: about 4.3 ×106gmol

-1). A diﬀerentia-

tion between AP and the IM fraction is permissible owing to

signiﬁcantly diﬀerent values of Mw. However, after complete

debranching, the separated BC fraction was assigned to AP

(Figure 3F).

AP: Since the AM content of the HACS-70 was higher com-

pared to HACS-50 (nominal and determined), a respective lower

AP content was expected, regardless of the existence of the

IM fraction. Depending on the method used (Figure 6C,D),

the AP content was estimated to be about 18 or 14% w/w

(HACS-70) and 22 or 17% w/w (HACS-50) (Figure 7, relative

portion). MwAP of HACS-50 was found to be signiﬁcantly

higher (25…27 ×106gmol

-1) compared to HACS-70 (15…17 ×

106gmol

-1). Moreover, diﬀerences between both genotypes were

also found with respect to the AP ﬁne structure. The DPwof the

AP BC fraction (Figure 7) of HACS-70 was higher (38.7) com-

pared to HACS-50 (33.3) and the DDP curves diﬀered (Figure 3F).

Surprisingly, a small amount of BC was calculated to have a DP

of up to 200–300 (Figure 3F), which is likely accountable to very

long B- and C-chains. Since this is not conﬁrmed by data pub-

lished elsewhere,[42–44] the contribution of small molecules from

the AM fraction is also conceivable.

3.1.2. AM Content Determined via Diﬀerent Methods

Diﬀerences with respect to molecular properties of the geno-

types (HACS-50/-70) are not only limited simply to the AM/AP

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Figure 8. AM contents of HACS-50/-70 determined using diﬀerent approaches: BV method including a defatting step using EtOH based on native/non

defatted starches[1] and partially defatted starches[2,3], BV method based on partially defatted starches after disintegration by means of pressure

cooking[4] without additional defatting using EtOH, and determination based on examining (including deconvolution) SEC-chromatograms of the na-

tive/non defatted starches non-debranched[5] and completely debranched[6].

ratio, but rather to all molecular parameters analyzed (Section

3.1.1). However, the AM content of the starch varieties was

hereinafter comprehensively examined, since it is the most im-

portant attribute. The AM contents were determined using the

BV method,[1,41] including a defatting step by means of EtOH

(method 1–3, Table 1). Because such genotypes normally contain

comparatively high amounts of lipids (Chen and Jane[12]), two dif-

ferently defatted samples (TCM-MeOH/EtOH and BuOH/EtOH)

of each genotype were included in addition to the respective na-

tive samples (method 2–3, Table 1). In addition, the determina-

tion was performed via BV after disintegration (method 4, Ta-

ble 1) as well as based on examination of the SEC-chromatograms

before (method 5, Table 1 [non-debranched]; Figure 6C,D) and af-

ter enzymatic degradation (method 6, Table 1 [debranched], Fig-

ure 3B).

The values calculated are summarized in Figure 8.AllAM

contents determined were lower compared to the expectation

based on the supplier information (HACS-50: 55% w/w, HACS-

70: 73.8 % w/w; nominal values) with a maximum of about 50%

w/w for the HACS-50 and about 62% w/w for the HACS-70, re-

spectively. Reasons are versatile and could be i.a. an incomplete

dispersion of the starch polysaccharides by processing according

to the method applied (method 1–3), despite the dissolving step

using DMSO. In an aqueous system (HYLON VII, 1% w/w, heat-

ingfrom50to95°C, heating rate of 5 K min−1), the maximum

values for solubility as well as degree of gelatinization were found

tobeabout15gg

−1and about 35%, respectively,[45] which is com-

paratively low. In particular, for HYLON VII (aqueous dispersion)

it was suggested, that even temperatures of 120 as well as 140 °C

are not high enough to gelatinize and disrupt the starch gran-

ules completely, even with continuous mechanical stirring (Wu

et al.[46]). The high AM content and accordingly low AP content

are surely responsible for the restricted swelling and prevention

of complete disintegration owing to the preparation steps (Ta-

ble 1, methods 1–3). Moreover, existing lipid remnants, despite

the diﬀerent defatting procedures applied, probably impact the

formation of the AM-iodine complexes reducing the BV. In addi-

tion, the tendency of high AM starches to comparatively fast and

comprehensive retrogradation could also inﬂuence the analysis

of the AM content.[47,48] Consequently, the calculated AM con-

tents would be quasi underestimated. Shi et al.[6] determined AM

contents of 56.8±0.4 (HYLON V) and 71.0±1.6% w/w (HYLON

VII) by means of potentiometric iodine titration method. They

concluded possibly i) overestimation in the presence of branched

molecules with long side chains which bind iodine, or ii) under-

estimation in the presence of linear low MM molecules which

bind less iodine than normal AM. The AM contents reported by

Duan et al.[49] for the same starches were slightly lower (HYLON

V: 55.1 ±0.6% w/w [potentiometric titration], 41.3 ±5.6% w/w

[colorimetric method]; HYLON VII: 66.5 ±0.6% w/w [potentio-

metric titration], 54.3 ±0.6% w/w [colorimetric method]), but ac-

cord reasonably with the values of the present study. Wu et al.[46]

reported an AM content of 51.8% w/w for HYLON V and 66.2%

w/w for HYLON VII (based on Con A). The impact of the analy-

sis method on the calculated AM content is huge. Schwall et al.[7]

investigated several high AM potato starches very systematically

and found strongly diﬀerent AM contents, which were up to 19%

w/w higher with the potentiometric compared to the colorimet-

ric method. The disintegration step using pressure cooking did

not result in higher calculated AM contents (method 4, Figure 8),

which supports the assumption of a signiﬁcant impact of struc-

tural features. Shi et al.[6] proved a remarkably higher portion of

branched structures (high MM and IM MM fraction) and corre-

sponding lower portion of linear polymers in both starch vari-

eties than the expected AP content would suggest. The portions

of the linear fraction (HYLON V: 41.8%, HYLON VII: 54.4%)

were in the same magnitude as the AM contents calculated in

the present study as well as when reported by Duan et al.[49]

based on the colorimetric method. Despite the fact of certain de-

viation dependent on the method used for examination of the

AM contents (methods 1–4, Figure 8), the estimation of the AM

content based on the analysis of SEC-chromatograms of the non-

debranched (method 5, Figure 8) and the completely debranched

starch (method 6, Figure 8) seems to provide reasonably reliable

information. The branched nature of the AM fraction of the in-

vestigated corn starch varieties was evidenced.

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Based on the results of the molecular characterization (3.1.1),

both genotypes were evaluated as very diﬀerent starches, which

don’t diﬀer simply with regard to the AM/AP-ratio (Jane et al.[50]).

In contrast, remarkable diﬀerences of the respective structure

fractions were pointed out such as AM, which was found to be

branched to a certain extent having a higher MM and degree

of branching with lower content in the starch (Cheetham and

Tao[51]). The existence of a branched IM fraction (Shi et al.[6]),

which is not clearly associated with both either AM or AP, could

be assumed. Moreover, the AP structure diﬀered in terms of

MM and ﬁne structure (Jane et al.[50]). Higher AM content of

the starch was found to be associated with lower Mwof the AP

fraction and with presumably lower degree of branching. It is hy-

pothesized, that the highly diﬀerent starch polysaccharide com-

positions and structures have enormous impact on the enzymatic

degradation and resulting speciﬁc products, respectively, as well

as functional properties.

3.2. Enzymatic Degradation of the Dissolved Starch Samples

3.2.1. Blank Enzymes

Aiming for the detailed analysis of the degradation products af-

ter treating the starch genotypes (molecularly dispersed solution)

with diﬀerent types of commercial enzymes (𝛼-, 𝛽-, 𝛾-AMY, PUL),

the speciﬁc contribution of each enzyme formulation to the SEC-

chromatogram was investigated, since the solutions contain dif-

ferent low MM substances (e.g., Glc) for stabilizing eﬀects. The

speciﬁc contribution (chromatogram area) due to the additives

of the enzyme solutions was considered as blank (𝛼-, 𝛽-, and 𝛾-

AMY) in the following experiments, which was deducted directly

from the chromatogram of the respective enzyme treated sample

or separated by means of mathematical deconvolution after com-

plete debranching (PUL; Section 3.1.1, Figure 3A,B). The latter

is an appropriate methodical approach when debranching (Ul-

brich et al.[52]), since the cleavage of the 𝛼-1,6-glycosidic bonds in

the starch (basis, e.g., native starch, not depolymerized by mod-

iﬁcation) results (polymer) molecules (AM fraction, AP BC frac-

tion [DPwabout 35]) signiﬁcantly diﬀer from mono- and disac-

charides.

Figure 2 shows the SEC-chromatograms of the disaccharide

Mal and the monosaccharide Glc A) as well as the enzyme formu-

lations B). The chromatograms in Figure 2B having a peak max-

imum between about 25.8 and 26.0 mL elution volume are very

probably assigned to Glc (𝛼-AMY), sorbitol/glycerin (𝛽-AMY),

Suc (𝛾-AMY) as well as Suc/Glc (PUL). However, the broad and

distinctive chromatogram region between 17.5 and 22.5 mL elu-

tion volume in the case of the 𝛾-AMY is presumably related to a

high MM polymer fraction (Mw>106gmol

−1) also contained in

the solution (enzyme formulation).

3.2.2. Molecular Characterization after Speciﬁc Enzymatic

Degradation

The starch genotypes were digested by means of the diﬀerent

enzymes (𝛼-, 𝛽-, and 𝛾-AMY) and enzyme combinations (𝛼-

AMY-PUL and 𝛽-AMY-PUL), and the resulting solutions were

Table 2. Residual polymer/molecule fractions (F1–F5) owing to digestion

using diﬀerent enzymes/combinations (𝛼-AMY, 𝛼-AMY-PUL, 𝛽-AMY, 𝛽-

AMY-PUL, and 𝛾-AMY) assigned to distinctive elution volume at peak max-

imum.

Range of elution volume at

peakmax [mL]

17–18 20–21 22–23 24 25–26

Enzyme/combination Fraction F1 F2 F3 F4 F5

𝛼-AMY •••

𝛼-AMY-PUL ••

𝛽-AMY •• •

𝛽-AMY-PUL ••

𝛾-AMY ••

characterized using SEC. The respective blanks were subtracted

from the chromatograms (Figure 4, SEC-chromatograms orig-

inal) and based on the chromatogram ﬁts (Figure 4, PeakFit),

diﬀerent molecule fractions generated due to speciﬁc enzymatic

cleavage of the glyosidic bonds were obtained by deconvolution

(Figure 4, fractions). Amount and molecular composition of

the fractions generated were strongly dependent on both the

starch genotype (HACS-50 and -70) and particularly the type of

enzyme (𝛼-AMY, 𝛼-AMY-PUL, 𝛽-AMY, 𝛽-AMY-PUL, and 𝛾-AMY)

digested with it. The classiﬁcation of the fractions is presented in

Table 2.

Degradation by means of 𝛼-AMY resulted at least three frac-

tions (Figure 4A,B), at which F5 is attributed predominantly to

generated Mal (relative portion about 83%, Figure 5A,B). Depen-

dent on the genotype, slight diﬀerences were obvious for F2 and

F4 (chromatograms Figure 4A,B), which is supported by the data

with respect to relative portion and Mw(Figure 5A,B). In partic-

ular Mwor DPw, respectively, of both fractions was slightly lower

with lower AP content of the initial starch sample (F2: DPw555–

405, F4: DPw11.4–8.6). Reasons for this are assumed to be the

remarkably diﬀerent molecular properties of both varieties. Par-

ticularly the AP and the IM fraction (branched structures) can

cause small branched residuals (𝛼-LDs) after degradation using

𝛼-AMY. Since both the MwAP and MwIM of HACS-50 were sig-

niﬁcantly higher compared to HACS-70, the MM of the respec-

tive 𝛼-LDs was accordingly. Fraction F4 could probably also be

released from (slightly) branched structures, possibly from the

AM. However, the existence of a high MM fraction (F3) after di-

gestion using the enzyme combination (𝛼-AMY-PUL) was not

actually expected (Figures 4A,B and Figure 5A,B). Characteriz-

ing 𝛼-LDs derived from a WxCS, Lee and Hamaker[53] identiﬁed

three sub-fractions (I–III) of branched 𝛼-LDs besides the linear

fraction consisting of maltooligosaccharides.

Degradation using 𝛽-AMY resulted in at least three fractions

(Figure 4C,D), similar to the treatment with 𝛼-AMY. Fraction F5

corresponds to the Mal, which is typically released due to spe-

ciﬁc cleavage of every 2nd 𝛼-1,4-glycosidic linkage starting on the

non-reducing end of the polymer molecules and ending near the

𝛼-1,6-glycosidic linkages, the branches within the molecule struc-

ture (relative portion of Mal of about 66% for both HACS-50 and

-70, Figure 5C,D). Fraction F2, having a relative portion of about

13% for both HACS-50 and -70 (Figure 5C,D), corresponds most

likely to the 𝛽-LDs, the “backbone” of the AP. This is conﬁrmed

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by the high Mwcalculated to be about 8–9 ×105gmol

−1, although

the level is low compared to literature data (Tester and Qi[30]). An

accordingly lower portion of the 𝛽-LDs fraction was initially ex-

pected for HACS-70, since the AP content and the overall amount

of branched molecules (AP and IM fraction) of HACS-70 is lower

compared to HACS-50 (Figure 7). In particular, the ratio (con-

tents) of 𝛽-amylolyse limits (F2 and F3, Figures 4C,D and 5C,D)

and initial branched fractions (F1 and F2, Figure 6C,D) was in ac-

cordance with data published elsewhere.[54,55] Fraction F3 could

originate from the IM fraction (branched), which means that it

is another 𝛽-LDs fraction. A higher relative portion of F3 (about

21%, Figure 5) compared to F2 supports this assumption, since

the ratio of IM and AP fraction is similar. Particularly F3 can also

contain residues of partly retrograded AM, residues of small AM

chains as well as residues of comparatively small branched dex-

trins (Zhao et al.,[56] and Manelius et al.[57]). Moreover, the contri-

bution of AM chains degraded up to the ﬁrst branch point consid-

ered from the non-reducing end is also possible or even likely in

F3. The digestion using the enzyme combination (𝛽-AMY-PUL)

resulted in about 6% (HACS-50) and 10% (HACS-70), respec-

tively, of a low MM fraction (F3), and Mal (F5; 94% and 90%),

which is obvious from Figure 5C,D. Analogous to 𝛼-AMY-PUL,

the existence of such fragments and also residues having 𝛼-1,6-

glycosidic linkages are supposable.

Since 𝛾-AMY, acting as an endo-enzyme (non-reducing end),

hydrolyses both 𝛼-1,4-glycosidic as well as 𝛼-1,6-glycosidic link-

ages of each AGU, the most important product on a quantity

basis was expected to be Glc, which is represented by F5 in

Figure 4E,F with a percentage of about 96% for both starches

(Figure 5E,F). However, a very small fraction (F1, relative portion

<4%, Figures 4E,F and 5E,F) having a comparatively high MM

was identiﬁed, too. The origin of F1 from the starch seems

absolutely implausible, since residues after digestion having

such high MM contradicts completely the mechanism and the

speciﬁcity of the enzymatic hydrolysis. It is rather supposed, that

the chromatogram area is caused by the enzyme formulation

itself (indication in Figure 2B), since marginal variation in terms

of the enzyme concentration or volume added, respectively, for

both digestion experiment and preparation of the corresponding

blank could not excluded.

The treatment with the speciﬁc enzymes resulted in an exten-

sive molecular degradation of the starch polymers. Compared to

the digestion with pure 𝛼-/𝛽-AMY, which results in appreciable

portions of 𝛼-/𝛽-LD in addition to the release of Mal, the com-

bined degradation with a speciﬁc debranching step included (𝛼-

/𝛽-AMY-PUL) minimized residual polymers in the solutions in-

vestigated. Comparing the “pattern” after enzymatic digestion

(resistant fractions), e.g., in terms of portion and molecular prop-

erties, can give an indication for the molecular structure of the de-

graded polymers on the one hand, and deliver possibly valuable

information on the residual molecules protected from degrada-

tion by its structural features on the other hand.

4. Conclusions

A speciﬁc method including pressure cooking and subsequent

high shear treatment was appropriate for preparing molecularly

disperse solutions of the HACS, which is an essential prerequi-

site for the analysis via SEC. The comprehensive molecular in-

vestigation of the genotypes in the ﬁrst part of the study, mainly

by means of SEC-techniques and special processing of the chro-

matograms, provided new insights regarding the molecular com-

position. The diﬀerences are not limited simply to the AM/AP-

ratio of the genotypes, but refer to basically all quantitatively (rel-

ative amount of diﬀerent structure fractions) and qualitatively

(e.g., MM, branched/not branched) analyzed molecular charac-

teristics. The molecular properties of the initial starch seriously

deﬁne the enzymatic digestibility of the starch polymers using

diﬀerent speciﬁc starch degrading enzymes and the properties

of the resulting reaction products, respectively. This is important

and should be considered, particularly when using the enzymatic

digestion as part of a special and complex analysis method based

on SEC experiments. The ﬁndings of the present study can con-

ceivably support the investigation of microstructural features of

aqueous starch gels, particularly the challenging interpretation

of SEC-chromatograms of enzymatically degraded gel matrices,

which is presented in a separate study.

Acknowledgements

This research was ﬁnancially supported by the German Federal Ministry

for Economic Aﬀairs and Energy (BMWi) within the scope of the Indus-

trial Collective Research (IGF) of the German Federation of Industrial Re-

search Associations (AiF; research project 20248 N). The authors would

like to thank Ingredion Germany GmbH for providing the starches, and

SternEnzym GmbH & Co. KG, Novozymes A/S as well as DSM Food Spe-

cialties B.V. for providing the enzymes. They also thank Ewgenia Kuhl and

Tim Alexander Terstegen for assistance in performing some experiments

and Donna Hastings for checking the manuscript linguistically.

Conﬂict of Interest

The authors declare no conﬂict of interest.

Data Availability Statement

Research data are not shared.

Keywords

amylose content, enzymatic degradation, high amylose corn starch,

molecular properties, size exclusion chromatography

Received: December 13, 2021

Revised: January 24, 2022

Published online: April 4, 2022

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