Maltogenic α-amylase modification of pulse and maize ...

Maltogenic α-amylase modification of pulse and maize starches to enhance the

functional properties and resistant starch content

A Thesis Submitted to the

College of Graduate and Postdoctoral Studies

In Partial Fulfillment of the Requirements

For the Degree of Master of Science

In the Department of Food and Bioproduct Sciences

University of Saskatchewan

Saskatoon, Saskatchewan, Canada

By

Jiayi Li

2020

© Copyright Jiayi Li, December 2020. All rights reserved

i

PERMISSION TO USE

In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from

the University of Saskatchewan, I agree that the Libraries of this University may make it freely available

for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part,

for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in

their absence, by the Head of the Department or the Dean of the College in which my thesis work was done.

It is understood that any copying, publication, or use of this thesis or parts thereof for financial gain shall

not be allowed without my written permission. It is also understood that due recognition shall be given to

me and to the University of Saskatchewan in any scholarly use which may be made of any material in my

thesis.

Requests for permission to copy or to make other use of material in this thesis in whole or part

should be addressed to:

Head of the Department of Food and Bioproduct Sciences

Room 3E08, Agriculture Building, 51 Campus Drive


Saskatoon, Saskatchewan, S7N 5A8, Canada

OR

Dean

College of Graduate and Postdoctoral Studies


116 Thorvaldson Building, 110 Science Place

Saskatoon, Saskatchewan S7N 5C9 Canada

ii

ABSTRACT

The overarching objective of this thesis research was to modify granular lentil (LS), faba

bean (FBS), pea (PS), waxy maize (WMS), normal maize (NMS) and high-amylose maize (HAMS)

starches with maltogenic α-amylase (MGA) from Bacillus stearothermophilus and to characterize

the functional properties and in vitro digestibility of modified starches. Pulse starches have higher

amylose contents and longer amylopectin branch chains than most of the cereal starches. The first

study aimed to modify LS, FBS and PS with MGA in a native granular form and to characterize

and compare the structural features, functional properties, and in vitro digestibility of the modified

pulse starches with that produced from NMS. Three pulse starches showed comparable

characteristics before and after MGA treatment. Upon MGA hydrolysis, the granules of pulse

starches were broken into small pieces. In contrast, numerous pores were observed in MGA-

modified NMS granules. MGA treatment did not change the wide-angle X-ray diffraction (WAXD)

patterns for all the starches, but slightly reduced the relative crystallinity of the three pulse starches.

The chain lengths of amylose and long branch chain of amylopectin of all the starches were

shortened by MGA hydrolysis. The shortening of amylopectin chain length reduced starch

retrogradation rates. The degradation of molecular and granular structures led to the extremely low

pasting viscosities of all MGA-modified starches. The 24-h MGA modification increased the

resistant starch (RS) contents of cooked LS, FBS, PS and NMS by 5.9%, 6.5%, 4.2% and 4.7%,

respectively, which could be attributed to the formation of retrograded amylose. The research

demonstrated that MGA modification was an effective method to alter the functional properties

and increase the RS contents of normal maize and pulse starches.

In the second study, the effects of MGA modification on the starches from the same crop

type but with different amylose contents were investigated. WMS, NMS and HAMS were selected

for the modification with MGA. The structural features, functional properties, and in vitro

digestibility of the modified maize starches were examined and compared with those of their

respective controls. MGA performed limited enzymatic hydrolysis on HAMS because of its high

amylose content and the B-type polymorphic structure. The MGA-modified WMS and NMS

exhibited numerous pores in their granules. The WAXD patterns of the three maize starches were

not changed by MGA treatment. MGA treatment significantly reduced the percentages of long

branch chains and increased the percentages of short branch chains of WMS and NMS by

iii

hydrolyzing the α-1,4 linkages. The percentages retrogradation of WMS and NMS were

substantially decreased due to the shortening of the branch chains. MGA hydrolysis degraded both

granular and molecular structure of WMS and NMS, which resulted in extremely low pasting

viscosities. Among the three modified maize starches, only the cooked NMS showed an increase

of RS from 2.6% (control) to 7.3% due to the formation of retrograded amylose. The new findings

from the research advanced our understanding of the hydrolysis patterns and resultant effects of

MGA on starches with different amylose contents.

The thesis research illustrated the new method of using MGA to modify starches in a native

granular form and provided new insights into the impacts of MGA modification on the structures,

functionality and in vitro digestibility of different pulse and maize starches. The most attractive

features of the MGA-modified starches for food applications included substantially low

retrogradation rate (except for HAMS) and increased RS contents (except for WMS). The new

information as presented in this thesis will be meaningful for utilizing MGA treatment as a clean-

label method to diversify the functional attributes and improve the nutritional value of starches

from different botanical sources for wider industrial applications.

iv

ACKNOWLEDGEMENTS

I would like to express my sincere thanks and gratitude to my supervisor, Dr. Yongfeng Ai,

for his kind guidance and support. He inspired me to begin this new chapter in my life. I would

not be able to complete my thesis research without his continuous assistance and encouragement.

His positivity and encouragement brightened my days during the three years of M.Sc. study. I

would also like to thank my Advisory Committee members, Drs. Michael T. Nickerson, Robert

Tyler, and Takuji Tanaka, for their time and valuable advice on the completion of this research

project and the writing of my M.Sc. thesis.

I am very thankful to my previous and current lab mates in Dr. Yongfeng Ai’s group (Dr.

Claire Chigwedere, Fan Cheng, Xinjing Kong, Dr. Dongxing Li, Liying Li, Dr. Siyuan Liu,

Yingying Lu, Yikai Ren, Rashim Setia, Xinya Wang, Hanyue Yin, and Tommy Yuan). I also

greatly appreciate the help provided by Ms. Ann Harley and Ms. Donna Selby in the Department

of Food and Bioproduct Sciences.

I am grateful to Dr. Jianfeng Zhu at the Saskatchewan Structural Sciences Centre for his

help with wide-angle X-ray diffraction and proton nuclear magnetic resonance analyses, Dr. Eiko

Kawamura and Mr. Osai J. R. Clarke at the Western College of Veterinary Medicine Imaging

Centre for their aid on scanning electron microscopy, and Dr. Peiqiang Yu in the Department of

Animal and Poultry Science his help with statistical analysis.

I greatly appreciate the financial support from the Saskatchewan Ministry of Agriculture –

Agriculture Development Fund for my graduate study at the University of Saskatchewan. I am

also thankful to AGT Food and Ingredients, Cargill Inc., and Ingredion Inc. for the donation of

samples for the research project.

Finally, I would like to express my deepest gratefulness to my beloved parents and

girlfriend, Jun Li, Yaping He, and Shiyi Li, for their unconditional help and support in my life.

Their love gives me the strength I need to overcome all the difficulties and achieve important goals

in the different stages of my life.

v

TABLE OF CONTENTS

PERMISSION TO USE ................................................................................................................. i

ABSTRACT ................................................................................................................................... ii

ACKNOWLEDGEMENTS ........................................................................................................ iv

TABLE OF CONTENTS ............................................................................................................. v

LIST OF TABLES ..................................................................................................................... viii

LIST OF FIGURES ..................................................................................................................... ix

LIST OF SYMBOLS AND ABBREVIATIONS ....................................................................... xi

1. INTRODUCTION .................................................................................................................... 1

1.1 Overview .............................................................................................................................. 1

1.2 Objectives ............................................................................................................................. 2

1.3 Hypotheses ........................................................................................................................... 3

1.4 Organization of the Thesis ................................................................................................... 3

2. LITERATURE REVIEW ........................................................................................................ 5

2.1 Utilization of pulses in human foods .................................................................................... 5

2.2 Fractionation of pulses using dry and wet milling methods ................................................. 6

2.3 Structure of starch granules .................................................................................................. 7

2.3.1 Starch granular morphology and structures of amylose and amylopectin ...................... 7

2.3.2 Organization of starch molecules within granules ....................................................... 11

2.4 Maltogenic α-amylase (MGA) modification of starch for enhancing functional properties

and resistant starch (RS) content ........................................................................................ 12

2.4.1 Overview of starch use and modification in the industry ............................................. 12

2.4.2 Effects of MGA hydrolysis on physicochemical properties of starch .......................... 13

2.4.2.1 Gelatinization properties ........................................................................................ 14

2.4.2.2 Retrogradation........................................................................................................ 15

2.4.2.3 Pasting properties ................................................................................................... 16

2.4.3 Digestibility of MGA-modified starch ......................................................................... 18

3. UTILIZATION OF MALTOGENIC α-AMYLASE TREATMENT TO ENHANCE THE

FUNCTIONAL PROPERTIES AND REDUCE THE DIGESTIBILITY OF PULSE

STARCHES ............................................................................................................................ 21

3.1 Abstract .............................................................................................................................. 21

3.2 Introduction ........................................................................................................................ 22

3.3 Materials and methods ........................................................................................................ 24

vi

3.3.1 Materials ....................................................................................................................... 24

3.3.2 Modification of starch using MGA .............................................................................. 24

3.3.3 Amylose content of starch ............................................................................................ 25

3.3.4 Granular morphology of starch ..................................................................................... 25

3.3.5 WAXD of starch ........................................................................................................... 25

3.3.6 Molecular-weight distribution of debranched starch .................................................... 26

3.3.7 Percentage of α-1,6 branch linkages of starch .............................................................. 27

3.3.8 Thermal properties of starch ......................................................................................... 27

3.3.9 Pasting properties of starch ........................................................................................... 28

3.3.10 In vitro digestibility of cooked starch ......................................................................... 28

3.3.11 Thermal properties of RS residue ............................................................................... 28

3.3.12 Statistical analysis....................................................................................................... 29

3.4 Results and discussion ........................................................................................................ 29

3.4.1 Percentage of starch hydrolysis by MGA ..................................................................... 29

3.4.2 Apparent Amylose content of starch ............................................................................ 31


3.4.4 WAXD and relative crystallinity of starch ................................................................... 32





3.4.9 In vitro digestibility of cooked starch ........................................................................... 46

3.5 Conclusions ........................................................................................................................ 51

3.6 Connection to the Next Study ............................................................................................ 51

4. MODIFICATION OF GRANULAR WAXY, NORMAL AND HIGH-AMYLOSE

MAIZE STARCHES USING MALTOGENIC α-AMYLASE TO IMPROVE THE

FUNCTIONALITY ................................................................................................................ 53

4.1 Abstract .............................................................................................................................. 53

4.2 Introduction ........................................................................................................................ 54

4.3 Materials and methods ........................................................................................................ 56

4.3.1 Materials ....................................................................................................................... 56

4.3.2 Modification of starch using MGA .............................................................................. 56

4.3.3 Amylose content of starch ............................................................................................ 57

vii


4.3.5 WAXD of starch ........................................................................................................... 57





4.3.10 In vitro digestibility of cooked starch ......................................................................... 60

4.3.11 Thermal properties of RS residue ............................................................................... 60

4.3.12 Statistical analysis....................................................................................................... 61

4.4 Results and discussion ........................................................................................................ 61

4.4.1 Percentage of starch hydrolysis by MGA ..................................................................... 61

4.4.2 Apparent amylose content of starch ............................................................................. 63


4.4.4 WAXD and relative crystallinity of starch ................................................................... 64





4.4.9 In vitro digestibility of cooked starch ........................................................................... 78

4.5 Conclusions ........................................................................................................................ 83

5. GENERAL DISCUSSION ..................................................................................................... 84

6. GENERAL CONCLUSIONS AND FUTURE STUDIES ................................................... 87

7. REFERENCES ....................................................................................................................... 89

8. APPENDICES ....................................................................................................................... 101

8.1 List of Tables .................................................................................................................... 101

8.2 Copyright approval ........................................................................................................... 104

viii

LIST OF TABLES

Table 2.1 Apparent amylose contents of representative pulse starches and cereal starches ...........7

Table 2.2 Amylopectin branch-chain-length (BCL) distributions of lentil, faba bean, pea and

normal maize starches. ....................................................................................................9

Table 2.3 Enthalpy change of retrograded amylopectin of bread crumb without and with MGA

treatment before and after reheating ..............................................................................14

Table 2.4 In vitro digestibility of raw and cooked pulse and maize starches ...............................18

Table 3.1 Apparent amylose contents of control and MGA-modified starches ............................32

Table 3.2 Molecular-weight distributions of debranched control and MGA-24 h-modified

starches ..........................................................................................................................39

Table 3.3 Percentages of α-1,6 branch linkages of control and MGA-24 h-modified maize

starches ..........................................................................................................................40

Table 3.4 Gelatinization and retrogradation properties of control and MGA-24 h-modified

starches ..........................................................................................................................42

Table 3.5 In vitro digestibility of cooked control and MGA-24 h-modified starches ..................46

Table 3.6 Thermal properties of RS residue samples recovered from control and MGA-24 h-

modified starches after cooking and amylolysis ..........................................................47

Table 4.1 Apparent amylose contents of control and MGA-modified maize starches ................64

Table 4.2 Molecular-weight distributions of debranched control and MGA-24 h-modified maize

starches .........................................................................................................................71

Table 4.3 Percentages of α-1,6 branch linkages of control and MGA-24 h-modified maize

starches .........................................................................................................................72

Table 4.4 Gelatinization and retrogradation properties of control and MGA-24 h-modified maize

starches .........................................................................................................................74

Table 4.5 In vitro digestibility of cooked control and MGA-24 h-modified maize starches .......79

Table 4.6 Thermal properties of RS residue of control and MGA-24 h-modified starches .........80

ix

LIST OF FIGURES

Fig. 2.1 Scanning electron microscopy images of native lentil, faba bean and pea starches ......6

Fig. 2.2 Proposed structure of the branched amylose molecule incorporating immature clusters

.......................................................................................................................................................7

Fig. 2.3 Proposed cluster model of amylopectin molecule comprising A, B1, B2, and B3 chains

.......................................................................................................................................................8

Fig. 2.4 Schematic of the organization of a starch granule .......................................................10

Fig. 2.5 Packing patterns of double-helical crystallites in A- and B-type polymorphs .............10

Fig. 2.6 Schematic presentation of the action of MGA on amylopectin ...................................12

Fig. 2.7 A typical differential scanning calorimeter (DSC) thermogram of a wheat starch-water

system ...........................................................................................................................13

Fig. 2.8 A typical starch pasting curve measured using Rapid Visco-Analyzer (RVA) ...........16

Fig. 2.9 Effect of MGA addition on RVA profiles of wheat starch slurries ..............................17

Fig. 2.10 RDS, SDS and RS contents of raw normal maize starch after MGA hydrolysis .......19

Fig. 3.1 Degrees of starch hydrolysis after incubating LS, FBS, PS and NMS with maltogenic α-

amylase (MGA) for 4, 8 and 24 h ................................................................................29

Fig. 3.2 Scanning electron microscopy (SEM) images of control and MGA-24 h-modified maize

starches .........................................................................................................................33

Fig. 3.3 X-ray diffraction patterns of control and MGA-24 h-modified starches .....................35

Fig. 3.4 Normalized high-performance size-exclusion chromatograms (HPSEC) of debranched

control and MGA-24 h-modified starches ....................................................................38

Fig. 3.5 Pasting properties of control and MGA-modified starches analyzed using a RVA with

28.0 g starch suspension containing 8% (w/w, db) starch ............................................44

Fig. 3.6 DSC thermograms of RS residue samples recovered from control and MGA-24 h-


Fig. 4.1 Degrees of starch hydrolysis after incubating WMS, NMS and HAMS with maltogenic

α-amylase (MGA) for 4, 8 and 24 h .............................................................................61


starches .........................................................................................................................65

Fig. 4.3 X-ray diffraction patterns of control and MGA-24 h-modified maize starches ...........67

x


control (A) and MGA-24 h-modified (B) maize starches ............................................70

Fig. 4.5 Pasting properties of control and MGA-modified maize starches analyzed using a RVA

with 28.0 g starch suspension containing 8% (w/w, db) starch ...................................76

Fig. 4.6 DSC thermograms of RS residue samples recovered from control and MGA-24 h-


xi

LIST OF SYMBOLS AND ABBREVIATIONS

AM Amylose

ANOVA Analysis of variance

BCL Branch-chain-length

DMSO Dimethyl sulfoxide

DMSO-d6 Deuterated dimethyl sulfoxide

DP Degree of polymerization

DSC Differential scanning calorimetry

FACE Fluorophore-assisted capillary electrophoresis

FBS Faba bean starch

HAMS High-amylose maize starch

HPSEC High-performance size-exclusion chromatography

IBC Intermediate branch chain

LBC Long branch chain

LS Lentil starch

MGA Maltogenic α-amylase

NMR Nuclear magnetic resonance

NMS Normal maize starch

PS Pea starch

RDS Rapidly digestible starch

RI Refractive index

RS Resistant starch

RVA Rapid Visco Analyzer

SBC Short branch chain

SDS Slowly digestible starch

SEM Scanning electron microscopy

Tc Conclusion gelatinization temperature

To Onset gelatinization temperature

Tp Peak gelatinization temperature

WAXD Wide-angle X-ray diffraction

xii

WMS Waxy maize starch

1H NMR Proton nuclear magnetic resonance

ΔH Enthalpy change

1

1.INTRODUCTION

1.1 Overview

Pulses refer to dried edible seeds of plants that belong to the Leguminosae family,

excluding those used for oil production, such as soybean and peanut. Common pulse crops include

pea (Pisum sativum L.), lentil (Lens culinaris L.), faba bean (Vicia faba L.), dry bean (Phaseolus

vulgaris L.), and chickpea (Cicer arietinum L.) (Asif et al., 2013; McDermott & Wyatt, 2017). In

recent years, pulses have gained growing popularity for food use due to the high nutritional value

and affordable prices (Dilis & Trichopoulou, 2009; Kumar & Masood, 2001). Pulses are processed

into various functional ingredients to prepare high-quality and nutritious food products for

consumers. Pulse starch is one of the major co-products from such value-added processing. Like

starches from many other botanical sources, however, certain undesirable inherent properties of

pulse starch limit the industrial applications (Li et al., 2019). Therefore, in the starch industry, it is

a common practice to apply different modifications, including chemical, physical, enzymatic and

genetic approaches, to improve the functionalities and nutritional quality of pulse and other

starches for increased industrial uses (Kaur et al., 2012).

Among the different modification approaches, the enzymatic method has drawn increasing

attention from the food industry primarily because the derived starch ingredients not only meet the

“clean-label” trend but also have special functional properties (Maniglia et al., 2020; Saltmarsh,

2015). Various enzymes have been commonly applied for starch modification, such as α-amylases,

β-amylases, amyloglucosidases, isoamylases and debranching enzymes (Ao, Simsek, et al., 2007;

Cai & Shi, 2010; Dura et al., 2014; Mendez-Montealvo et al., 2011). More recently, a new type of

hydrolytic enzyme known as maltogenic α-amylase (MGA) has been used to increase the resistant

starch (RS) content and enhance the cold-storage stability of starch (Zhang et al., 2008). MGA can

effectively cleave the α-1,4 glycosidic bonds of starch, which thus shortens the length of amylose

and amylopectin branch chains and increases the percentage of α-1,6 branch linkages

(Christophersen et al., 1998; Miao et al., 2014). Therefore, MGA has been widely used as an anti-

2

staling agent in bakery products (Hug‐Iten et al., 2003; Purhagen et al., 2011). Nevertheless, MGA

has not been used for modifying pulse starch in the current literature. In addition, enzymatic

modifications by MGA as reported in previous studies were carried out on starch in a cooked and

dispersed/solubilized state, rather than the native granular form typically used for modifying starch

at a commercial scale.

1.2 Objectives

(1) To modify lentil, faba bean, and pea starches with MGA and to characterize the structural

features, functional properties, and in vitro digestibility of native and enzyme-treated pulse

starches in comparison with those of native and MGA-modified normal maize starch;

(2) To modify waxy, normal, and high-amylose maize starches with MGA and to compare their

structural features, functionalities, and in vitro digestibility after MGA modification;

(3) To elucidate the interrelationships among the granular and molecular structures, functional

attributes, and in vitro digestibility of the pulse and maize starches of various amylose contents

before and after the MGA treatment.

The first study of this thesis project aimed to modify three different pulse starches – lentil, faba

bean and pea – in a granular form using MGA. We hypothesized that the hydrolysis by MGA could

inhibit the retrogradation and enhance the resistant starch contents of the pulse starches. The

structures, functional properties, and in vitro digestibility of MGA-modified pulse starches were

characterized and compared with their native counterparts, with commercial normal maize starch

being included for comparison. Our research objective of Study 2 was to examine the effects of

MGA modification on starches from the same type of crop but with different amylose contents.

Waxy, normal and high-amylose maize starches were selected and then modified using MGA in a

native granular form in the second study. The granular and molecular structures, functional

properties, and in vitro digestibility of the maize starches were comprehensive characterized and

compared before and after MGA modification.

This thesis research will greatly advance our understanding of how MGA treatment altered

the granular and molecular structures of pulse and maize starches of various amylose contents and

how the changes in the structures influenced the functional attributes and digestibility of the

3

starches. The new knowledge as presented in this thesis will be meaningful for the starch industry

to utilize MGA hydrolysis as an effective enzymatic modification method to improve the techno-

functional properties and enhance the enzymatic resistance of starch for promoted industrial

applications. This will be particularly important for the pulse industry to find new markets for the

underutilized pulse starches.

1.3 Hypotheses

(1) Compared to their respective native counterparts, the MGA-modified lentil, faba bean, pea,

waxy maize, normal maize, and high-amylose maize starches will have: 1) lower amylose contents,

decreased molecular weights, and shorter amylopectin branch-chain lengths; 2) lower pasting

viscosities, lower gelatinization temperatures, and reduced retrogradation; and 3) higher enzymatic

resistance.

(2) Like the similarity among their native counterparts, the MGA-treated lentil, faba bean and pea

starches will show comparable functionalities and in vitro digestibility because of the similar

granular and molecular structures of these pulse starches before and after the enzymatic

modification.

(3) As the amylose content increased, the degree of hydrolysis by MGA will show a decreasing

trend from waxy to normal and to high-amylose maize starches and thus the effects of the

enzymatic modification on the functional attributes and in vitro digestibility of the starches will

progressively become less obvious.

1.4 Organization of the Thesis

This thesis research shows the impacts of MGA modification on the structures,

functionality and in vitro digestibility of different pulse and maize starches. The organization of

this thesis is as follows.

Section 2 introduces the background information: utilization and fractionation of pulses,

structures, functionalities, and in vitro digestibility of native and MGA-modified starch. Section 3

is the study 1 of this thesis research and describes the effects of MGA hydrolysis on functional

4

properties and digestibility of pulse starches. Section 4 is the second study of this thesis research

and illustrates the effects of MGA hydrolysis on functional properties and digestibility of maize

starches. Section 5 discusses the results of both studies in detail. Section 6 provides the general

conclusions and offers recommendations for improvements to this thesis research and future

studies.

5

2.LITERATURE REVIEW

2.1 Utilization of pulses in human foods

According to the definition given by the Food and Agriculture Organization (FAO), pulses

are dried, edible seeds of certain plants in the Leguminosae family, and the most widely grown

pulse crops include pea, lentil, faba bean, dry beans, and chickpeas (Ratnayake et al., 2001). As of

2015, India was the largest producer of pulses in the world, followed by Canada, Myanmar and

China (Pulses, 2020). Canada was the largest producer of pea and the second largest producer of

lentil globally. More specifically, Saskatchewan is the leading producer of pea, lentil and chickpea

in the country (Hoover et al., 2010).

Pulses have been consumed as an important component of human diets in different regions

of the world. Pulses are a good source of complex carbohydrates (e.g., oligosaccharides, resistant

starch and other dietary fibers). Because of this, pulses show lower glycemic impact compared to

cereal grains and most other starchy foods, and thus they are recognized as a good choice for

providing potential health benefits to humans and are also recommended for improving the

glycemic control of individuals with diabetes (Rizkalla et al., 2002). Pulses are also rich in proteins

and vitamins, which makes them a good protein alternative for vegetarians (Tiwari et al., 2011;

Tosh & Yada, 2010). In addition, pulses are gluten-free, which renders them suitable to prepare

gluten-free foods for people who are adversely affected by celiac disease (Han et al., 2010).

Conventionally, pulses are consumed as whole seeds. However, directly consuming or

using whole seeds in human foods may also bring the challenges of undesirable beany flavors and

odors, hard texture, and long cooking time (Ma et al., 2016). Therefore, there is growing interest

from the industry in processing pulses into various functional ingredients that can be used for the

preparation of high-quality and nutritious food products. Pulse starch, protein concentrate/isolate,

and hull fiber are the major products from the value-added processing of pulses. These ingredients

6

are widely used in the food industry to deliver attractive functional and nutritional attributes in

different food products (Byars & Singh, 2016).

2.2 Fractionation of pulses using dry and wet milling methods

Pulses can be processed using different methods to prepare ingredients with enhanced

functional properties for wider food uses. Two fractionation methods are commonly employed:

dry and wet milling methods (Hoover et al., 2010).

Dry milling process typically includes sequential steps of dehulling, milling, and air

classification. Dehulling is the first and key step of dry milling because it can remove 97-98% of

the fiber component of pulse seeds (Tulbek, 2010). Hammer mill, pin mill, or impact mill are

commonly applied to reduce the particle size of pulse flours. Air classification is a fractionation

method that separates starch (in a form of “starch-rich flour”) and protein (in a form of “protein-

rich flour”) components based on the differences in their sizes and densities (Rumpf, 1990). After

air classification, the heavy, coarse fraction is starch-rich flour and the light, fine fraction is

protein-rich flour. This air classification step can be repeated for several times to separate the

starch from protein more effectively (Boye et al., 2010). However, the dry milling process can

only produce starch of a low purity because the air classification step cannot separate protein and

fiber from starch completely (Meuser et al., 1995).

Wet milling process includes a series of steps of dehulling, soaking, milling, extraction of

protein, washing and recovering of starch. The pulse seeds are firstly dehulled to remove the seed

coat, followed by soaking in water and milling into fine particles. Protein in the resultant

suspension is extracted by different approaches, such as alkaline extraction/isoelectric

precipitation, acid extraction, water extraction, salt extraction, and ultrafiltration (Boye et al.,

2010). After further washing, the starch can be recovered by filtration or centrifugation and then

dried. Compared to the described dry milling process, wet method can lead to a higher purity of

the recovered starch. However, because of the higher capital and processing costs and larger use

of water, wet method is less commonly utilized in the pulse industry in Canada (Sosulski &

Sosulski, 1986).

7

The obtained protein fraction can be used to provide suitable functional properties for the

development of bakery goods, meat analogues, snacks, beverages and other food products (Toews

& Wang, 2013). In contrast, the pulse starch has undesirable inherent properties, and thus it has

very limited applications in the food industry. Therefore, the pulse industry in Canada is exploring

new methods that can improve the functional and nutritional properties of pulse starch for broader

industrial uses.

2.3 Structure of starch granules

2.3.1 Starch granular morphology and structures of amylose and amylopectin

Previous studies have been undertaken to advance our understanding of the structure,

functionality and digestibility of pulse and maize starches (Hoover et al., 2010; Li et al., 2019).

Most of pulse starches have granules of an oval shape with smooth surface, although spherical,

round or irregular shapes also exist (Fig 2.1) (Tiwari et al., 2011). Pulse starches have comparable

granule sizes: lentil starch has granules of 6 to 37 μm; faba bean starch of 9 to 48 μm; and pea

starch of 14 to 37 μm (Hoover et al., 2010; Hoover & Ratnayake, 2002). Normal maize starch and

waxy maize starch have granules of polygonal, spherical and irregular shapes with some

indentations on the surface (Fig 2.1). The granule sizes of waxy maize starch and normal maize

starch ranged between 5 to 30 μm, which were smaller than those of pulse starches. High-amylose

maize starch granules exhibits similar shapes to waxy maize and normal maize starches. The starch

granules of high-amylose maize starch exhibit heterogeneous shapes consisting of polygonal

granules, spherical granules, irregular granules, and elongated granules (Fig 2.1) (Jiang, Horner,

et al., 2010).

Lentil Faba bean Pea

8

Fig. 2.1 Scanning electron microscopy (SEM) images of native lentil, faba bean, pea, waxy maize,

normal maize and high-amylose maize starches. The images were taken at 1,500× magnification

for lentil, faba bean, pea, waxy maize and normal maize starches. For high-amylose maize starch,

the scale bar = 10 μm [with permission from Li et al. (2019) and Cai et al. (2014)].

Amylose and amylopectin are the two major components of starch (Hoover et al., 2010).

Amylose is an essentially linear α-D-glucose polymer linked by α-1,4 glycosidic linkages and a

few branches of α-1,6 linkages (Fig 2.2) (Takeda et al., 1984). Amylose can greatly affect the

granular structure, functional properties, and digestibility of starch. Many studies have reported

that the apparent amylose contents of the pulse starches are higher than those of most of the cereal

starches (e.g., waxy maize, normal maize and tapioca), but lower than that of high-amylose maize

starch (Hoover et al., 2010; Li & Yeh, 2001; Li et al., 2019; Wang et al., 2014). The apparent

amylose contents of some representative pulse starches and cereal starches are presented in Table

2.1.

Fig. 2.2 Proposed structure of the branched amylose molecule incorporating immature clusters.

EL, extremely long; L, long; and S, short; Ø, reducing end [with permission from Takeda et al.

(1990)].

Waxy maize Normal maize High-amylose maize

9

Table 2.1 Apparent amylose contents of representative pulse starches and cereal starches.a

Starch Apparent amylose content (%)

Lentilb 38.0

Faba beanb 39.9

Peab 41.1

Waxy maizeb 1.9

Normal maizeb 31.2

High-amylose maizec 85.0

Tapiocab 30.6

a Determined using iodine colorimetry (Chrastil, 1987).

b Li et al. (2019)

c Wang et al. (2014)

Amylopectin, with approximately 5% α-1,6 branch linkages, is a highly branched molecule

(Hizukuri et al., 1981). Amylopectin branch-chain-length (BCL) is one important factor in starch

that determines the structure, functionality and digestibility (Jane et al., 1999). Amylopectin

branch chains have been classified into three different categories, A, B and C chains, by Hizukuri

(1986). A chains have a degree of polymerization (DP) of 6 to 12 with no other A or B chains

attached onto them. B chains can be further categorized to B1 (DP 13-24, spanning one cluster),

B2 (DP 25-36, spanning two clusters), B3 (DP > 37, spanning three clusters) and B4 (DP > 37,

spanning four or more clusters) based on their lengths and the number of clusters they extend

through (Hoover et al., 2010). C chain carries the only reducing end of amylopectin molecule

(Ratnavathi & Komala, 2016). Branch chains of amylopectins form double helices and contribute

to the crystalline structure of native starch granules (Hoover et al., 2010). The amylopectin BCL

distributions of lentil, faba bean, pea, waxy maize, normal maize and high-amylose maize starches

are shown in Table 2.2. Li et al. (2009) have reported that the average BCL of pulse starches are

higher than that of waxy maize and normal maize starches.

10

Fig. 2.3 Proposed cluster model of amylopectin molecule comprising A, B1, B2, and B3 chains.

Ø represents the reducing end. The chain with Ø is C chain. C. L., chain length [with permission

from Hizukuri (1986)].

Table 2.2 Amylopectin branch-chain-length (BCL) distributions of lentil, faba bean, pea, waxy

maize, normal maize and high-amylose maize starches.a

Starch Amylopectin

average chain length

Amylopectin chain length distribution (%)

DP 6-12 DP 13-24 DP 25-36

Lentilb 20.4 22.1 55.0 13.6

Faba beanb 20.4 21.5 56.0 12.8

Peab 20.1 22.0 56.1 13.1

Waxy maizeb 18.4 30.6 51.9 10.4

Normal maizeb 19.7 27.0 51.5 12.1

High-amylose maizec 26.9 18.5 45.3 14.4

a Determined by fluorophore-assisted capillary electrophoresis (FACE).

b Li et al. (2019).

c Lin et al. (2016).

11

2.3.2 Organization of starch molecules within granules

According to Jane et al. (1992), amylose and amylopectin are synthesized simultaneously

from the hilum to the periphery of starch granules and amylose is interspersed with amylopectin

molecules (Fig. 2.4). Amylose is present in the amorphous phase of starch granules, and branch

chains of amylopectin develop double helices and lead to the formation of crystalline structure.

Therefore, native starch granules have a semi-crystalline structure. X-ray diffraction patterns of

starch granules reveal that there are three different types of polymorphs: A-, B-, and C-type (Zeng

et al., 2011). Generally, A-type starches exist in common cereal crops (e.g., waxy and normal

maize, waxy and normal wheat, waxy and normal rice), which consist of a large proportion of

short branch chains (DP 6-12) in the amylopectin and have double-helical crystallites packed in

monoclinic unit cells (Fig. 2.5 A). Compared to A-type starches, B-type starches are mostly found

in tuber and root crops and high-amylose varieties (e.g., potato starch, high-amylose maize). They

are composed of a smaller proportion of short chains (DP 6-12) in the amylopectin and have

double-helical crystallites packed in hexagonal unit cells (Fig. 2.5 B) (Buleon et al., 1998; Gallant

et al., 1997; Jane et al., 1999; Robyt, 2012). C-type starch has a combination of A- and B-type

polymorphs, rather than a new organization pattern of double helices (Man et al., 2013). Most of

the pulse starches, including lentil, faba bean, pea, chickpea and bean, have been revealed to have

the C-type X-ray diffraction pattern (Chung et al., 2008; Robyt, 2012).

Fig. 2.4 Schematic of the organization of a starch granule [with permission from Jane (2009)].

12

Fig. 2.5 Packing patterns of double-helical crystallites in A- and B-type polymorphs. (A)

Monoclinic unit cell in A-type starch. (B) Hexagonal unit cell in B-type starch [with permission

from Pérez et al. (2009)].

2.4 Maltogenic α-amylase (MGA) modification of starch for enhancing functional

properties and resistant starch (RS) content

2.4.1 Overview of starch use and modification in the industry

In the food industry, starch plays important roles in the quality and nutritional value of

many food products. Starch can provide functions as a thickener, gelling agent, binder, stabilizer,

encapsulant and/or emulsifier (Ai & Jane, 2015). Nevertheless, the undesirable inherent properties

of starch, such as rapid retrogradation and associated poor cold-storage stability, and poor

resistance to acid, shear, and high temperature, limit the application value of starch (Singh et al.,

2017). Meanwhile, as one of the most important glycemic carbohydrate components in human

diets, starch is a good source of glucose for energy supply. However, long-term consumption of

starch or starchy foods with a high level of rapidly digestible starch can also increase the risk of

chronic diseases (e.g., type-2 diabetes, cardiovascular diseases) (Zhang & Hamaker, 2009).

Therefore, there is growing interest in developing food ingredients rich in resistant starch (RS).

RS refers to a portion of starch that cannot be digested in the small intestine of humans after

ingestion (Englyst & Macfarlane, 1986). RS can provide various health benefits to humans after

intake, but most of the RS ingredients in the market possess poor functional properties, which

13

impede their food uses (Reddy et al., 2014). Therefore, many researchers and industries are

exploring new methods to modify starch and overcome these undesirable properties.

Starch modification methods include physical, chemical, enzymatic and genetic

approaches. Recently, enzymatic modification method has drawn much academic and private

sector attention because this method can lead to desired functional properties and the resultant

modified starch can meet the requirements of “clean-label” ingredients (Saltmarsh, 2015). In the

past decade, studies have used different enzymes, such as α-amylase, β-amylase, amyloglucosidase,

isoamylase and debranching enzymes, to modify starches (Ao, Simsek, et al., 2007; Kim et al.,

2014; Mendez-Montealvo et al., 2011). Among the different starch-related enzymes, maltogenic

α-amylase (MGA) has received considerable attention due to its unique action pattern.

MGA from Bacillus stearothermophilus is a thermostable α-amylase that can catalyze the

hydrolysis of α-1,4 linkages in starch to release maltose as the main product from the non-reducing

ends of the chain (Christophersen et al., 1998). This enzyme has a high degree of multiple attack

action and has been reported to exhibit exo-action at a relatively low temperature (35 °C). However,

when the temperature is increased (e.g., 70 °C), MGA mainly performs endo-hydrolysis (Fig. 2.6)

(Grewal et al., 2015).

Fig. 2.6 Schematic presentation of the action of MGA on amylopectin: (—), α-1,4 linkages; (→),

α-1.6 linkages; G-G, maltose; G1-6, saccharides with 1-6 glucose units [with permission from

Grewal et al. (2015)].

2.4.2 Effects of MGA hydrolysis on physicochemical properties of starch

MGA can effectively shorten amylose chains and branch chains of amylopectin of starch

(Leman et al., 2006). Consequently, the proportion of the α-1,6 linkages is increased by the

enzymatic attack. The MGA-treated starch exhibits greater cold-storage stability, reduced

14

retrogradation, and decreased viscosities due to the shortening of starch branch chains (Leman et

al., 2006; Miao et al., 2014). Therefore, MGA has been widely used as an anti-staling agent to

extend the shelf life of bread and other bakery products (Goesaert, Leman, et al., 2009).

2.4.2.1 Gelatinization properties

Gelatinization is a process where the double-helical crystallites in starch granules are

melted into an amorphous state after the starch is heated in the presence of a sufficient amount of

water (Hoover et al., 2010). Differential scanning calorimetry (DSC) is widely used to determine

the gelatinization properties of starch. An endothermic peak in the DSC curve reflects the process

of gelatinization of the starch (Fig. 2.7). Onset temperature (To) indicates the beginning of

gelatinization, and conclusion temperature (Tc) records the end of the process. Enthalpy change

(ΔH), which is calculated based on the area under the DSC curve, indicates the energy required to

melt double helices during gelatinization (Cooke & Gidley, 1992). Starches from various botanical

sources have different gelatinization temperatures and ΔH, which could be attributed to the

differences in their amylopectin BCL distributions, ratio of amylose to amylopectin, and ratio of

amorphous region to crystalline region (Ai & Jane, 2015; Gunaratne & Hoover, 2002). It has been

reported that starches with shorter branch chains of amylopectin and fewer double-helical

crystallites will have lower gelatinization temperatures and ΔH (Jane et al., 1999). During MGA

treatment, the double-helical structure of the MGA-treated starch will be broken down and

removed, thus decreasing the ΔH of the starch. Meanwhile, MGA can shorten the chain length of

the starch, which also limits the formation of double helices. The degree of heterogeneity of

crystallites can also be decreased by MGA treatment because MGA-modified starch shows a

narrowed range of gelatinization temperatures (Qin et al., 2011).

15

Fig. 2.7 A typical DSC thermogram of a wheat starch-water system (water : starch = 4 : 1) with a

heating rate of 10 °C/min. To, onset temperature; Tp, peak temperature; Tc, conclusion temperature

[with permission from Wang et al. (2016)].

2.4.2.2 Retrogradation

When gelatinized starch is cooled and stored at a low temperature (e.g., 4 °C), it will return

from a solvated, dispersed and amorphous state to an insoluble and crystalline state (Bello-Perez

et al., 2005). This process is defined as “retrogradation”. Retrogradation of starch leads to an

increased degree of crystallinity, an increased gel firmness, and enhanced syneresis (Hoover, 1995;

Miles et al., 1985). The differences in retrogradation rates among starches can be attributed to

various factors: water content, starch source, storage conditions and additives (Wang et al., 2015).

Generally, starch with a higher amylose content and longer amylopectin branch chains tends to

have a higher retrogradation rate and extent (Jane et al., 1999; Liu et al., 2019). MGA modification

has been proven to be a useful method to retard the retrogradation of starch (Goesaert, Leman, et

al., 2009; Grewal et al., 2015; Hug‐Iten et al., 2003). The enthalpy changes of retrograded

amylopectin of bread crumb with and without MGA modification are summarized in Table 2.3.

16

MGA can shorten the amylopectin branch chains, which will therefore prevent the formation of

double helices and hinder water immobilization, thus suppressing the retrogradation of starch

(Goesaert, Leman, et al., 2009; Goesaert, Slade, et al., 2009).

Table 2.3 Enthalpy change of retrograded amylopectin of bread crumb without and with MGA

treatment before and after reheating.a,b

Aging time (day) Enthalpy change of retrograded amylopectin (J/g)

Without MGA addition With MGA addition

7 (before reheating) 2.60 0.29

0 (after reheating) ndc nd

1 (after reheating) 0.50 nd




a Measured using differential scanning calorimetry.

b Hug‐Iten et al. (2003).

c Not detectable.

2.4.2.3 Pasting properties

Pasting of starch occurs when it is further heated and stirred after gelatinization. The

increase in the viscosity of starch paste is primarily attributed to the swelling of starch granules

and the leaching out of some amylose molecules and amylopectins of small molecular weights

from the swollen granules (Atwell, 1988). A typical pasting profile of starch as determined using

a Rapid Visco-Analyzer (RVA) is illustrated in Fig. 2.8. RVA has been widely used to study the

pasting characteristics of different starches. During the procedure of continuous heating and

stirring, gelatinized starch will hydrate and develop viscosity. Pasting temperature is defined as

the temperature at which starch begins to develop detectable viscosity. After reaching the

maximum viscosity (peak viscosity), the swollen starch granules collapse. The collapse of the

swollen starch granules results in a “breakdown” in the viscosity. During the breakdown process,

17

amylose molecules diffuse out of the disrupted starch granules. When the starch paste is cooled,

viscosity increased because of the re-association between starch molecules, especially amylose.

The difference between the final viscosity and the holding strength is defined as the setback

viscosity (Yildiz et al., 2013). It has been demonstrated that the pasting properties of starches are

affected by amylose content, amylose molecular weight, and amylopectin BCL (Jane et al., 1999;

Leman et al., 2006).

Fig. 2.8 A typical starch pasting curve measured using Rapid Visco-Analyzer (RVA) [with

permission from Wani et al. (2012)].

Many studies have been carried out to use MGA to modify the pasting properties of starch

(Leman et al., 2006; Leman et al., 2005; Van Steertegem et al., 2013). MGA modification can

significantly reduce the pasting viscosities by degrading starch molecules (Fig. 2.9). However, the

rate of viscosity development is not affected by MGA because this enzyme is mainly active at

higher temperature (Leman et al., 2006). MGA-modified starches can be used in beverages and

18

confectioneries to increase the solids content without considerably affecting the texture of the

products.

Fig. 2.9 Effect of MGA addition on RVA profiles of wheat starch slurries. 1, 4.52 EU/g; 2, 9.03

EU/g; 3, 18.1 EU/g; 4, 36.1 EU/g [with permission from Leman et al. (2005)].

2.4.3 Digestibility of MGA-modified starch

Starch is one of the most important carbohydrates in human diets. Starch can be hydrolyzed

by different amylolytic enzymes, including salivary α-amylase, pancreatic α-amylase,

glucoamylase, maltase, sucrase and isomaltase, into glucose for energy supply (Ao, Quezada-

Calvillo, et al., 2007; Chen et al., 2011). According to Rooney and Pflugfelder (1986) and Bird et

al. (2009), variations in the digestibility of starch can be attributed to many factors, including

composition of starch, physical form of starch, granular architecture, granular size, and interaction

between starch and other components (e.g., lipids, fiber and protein).

According to the study of Englyst et al. (1982), starch can be classified into three different

fractions based on the digestibility: rapidly digestible starch (RDS), slowly digestible starch (SDS)

and resistant starch (RS). RDS is hydrolyzed into glucose by amylolytic enzymes within the first

20 min; SDS is the starch fraction that can be completely hydrolyzed between 20 and 120 min;

and RS remains resistant after 120 min of hydrolysis. The Englyst method is one of the reliable

19

and common methods that can be used to determine the digestibility of starch in vitro. Studies have

been carried out to compare the digestibility of pulse starches with other starches (Ambigaipalan

et al., 2011; H.-J. Chung et al., 2009; Li et al., 2019). As shown in Table 2.4, raw native pulse

starches have higher RS contents than those of waxy and normal maize starches due to their higher

amylose contents and longer amylopectin BCL (Li et al., 2019). Higher amylose contents and

longer branch chains can form more stable double-helical crystallites, which results in higher

enzymatic resistance (Zhu et al., 2011). However, it is also important to note that the differences

in the digestibility of pulse starches and cereal starches disappear after the starches are cooked (Li

et al., 2019). With relatively greater amylose contents and longer amylopectin BCL, pulse starches

are considered as a promising raw material to prepare RS in food applications.

Table 2.4 In vitro digestibility of raw and cooked pulse and maize starches.a

Starch Raw Cooked

RDS (%) SDS (%) RS (%) RDS (%) SDS (%) RS (%)

Peab 21.0 38.3 35.5 86.0 2.9 5.9

Lentilb 19.0 37.6 41.3 89.3 1.8 6.9

Faba beanb 15.3 34.5 46.7 88.1 2.5 5.9

Waxy maizeb 34.7 51.9 10.9 90.3 0.6 6.6

Normal maizeb 20.8 56.7 18.3 88.1 2.3 5.4

High-amylose maizec 14.9 13.3 71.7 71.7 4.2 24.1

a Determined using Englyst method (Englyst et al., 1992).

b Li et al. (2019)

c Ai et al. (2013)

Starch modified by MGA has been shown to have reduced digestion rate and extent, which

has been attributed to the decrease in the chain lengths of starch molecules and the increase in α-

1,6 branch linkages (Le et al., 2009; Miao et al., 2014). As presented in Fig. 2.10, the SDS and RS

20

contents of normal maize starch gradually increased and the RDS decreased upon MGA

modification (Miao et al., 2014).

Fig. 2.10 RDS, SDS and RS contents of raw normal maize starch after MGA hydrolysis. Control,

native starch; MS1, 1-h hydrolysis; MS2, 2-h hydrolysis; MS3, 3-h hydrolysis; MS4, 6-h

hydrolysis; MS5, 12-h hydrolysis; MS5, 24-h hydrolysis [with permission from Miao et al. (2014)].

Despite the noticeable changes in the structure, functionalities and digestibility of starch as

induced by MGA modification, this α-amylase has been mainly used to modify starch in a

gelatinized and dispersed form (Ao, Simsek, et al., 2007; Grewal et al., 2015; Miao et al., 2014).

In the starch industry, however, it is a more common practice to perform starch modification in a

raw and granular form. In addition, to the best of our knowledge, MGA has not employed to modify

pulse starch in the current literature. The aforementioned impacts of MGA modification on the

techno-functional characteristics of starch, such as reduced retrogradation rate and extent,

controlled pasting viscosity, and decreased digestibility, can be beneficial to modify pulse starch

into a more versatile and nutritious food ingredient.

21

3. UTILIZATION OF MALTOGENIC α-AMYLASE TREATMENT TO ENHANCE

THE FUNCTIONAL PROPERTIES AND REDUCE THE DIGESTIBILITY OF

PULSE STARCHES

3.1 Abstract

This study aimed to modify lentil (LS), faba bean (FBS) and pea (PS) starches with

maltogenic α-amylase (MGA) from Bacillus stearothermophilus in a native granular form, and the

MGA-modified starches were then characterized and compared with that produced from native

normal maize starch (NMS). Upon a 24-h MGA treatment, the granules of pulse starches were

broken into small pieces. Numerous pores were observed in MGA-modified NMS granules. MGA

treatment did not change the WAXD patterns for all starches, but slightly reduce the relative

crystallinity of three pulse starches. The chain lengths of amylose and long branch chain of

amylopectin of all starches were shortened by MGA hydrolysis. The shortening of amylopectin

chain length resulted in the retardation of retrogradation rates. The degradation of molecular and

granular structures was attributed to the extremely low pasting viscosities of all MGA-modified

starches. The 24-h MGA modification increased the resistant starch (RS) contents of cooked LS,

FBS, PS and NMS by 5.9%, 6.5%, 4.2% and 4.7%, respectively, which could be attributed to the

retrograded amylose formation. The information presented in this study will be particularly

important for the pulse industry to improve functional properties and find new markets for the

underutilized pulse starches.

Keywords: Maltogenic α-amylase modification; Granular pulse starch; Granular normal maize

starch; Starch structure; Functional properties of starch; In vitro digestibility of starch

22

3.2 Introduction

Pulses are plants that belong to the Leguminosae family, which include a variety of dry,

edible seeds, such as peas, lentils, chickpeas and faba beans (McDermott & Wyatt, 2017). Pulses

are a good source of complex carbohydrates (e.g., resistant starch and other dietary fibers), proteins,

vitamins, and minerals in the diets of populations in different regions of the world (Tosh & Yada,

2010). Because of the balanced nutritional profile, continuous consumption of pulses has been well

documented to enhance the health of humans (Hall et al., 2017; Mudryj et al., 2014). More recently,

there is growing interest in processing pulses into various functional ingredients that can be used

for the preparation of nutritious and high-quality food products.

Like many other plants, pulses reserve starch as the most abundant carbohydrate polymers

in the seeds. Starch consists of two major components: amylose and amylopectin (Hoover et al.,

2010). Amylose is an essentially linear α-D-glucopyranose polymer linked by α-1,4 glycosidic

bonds and a few α-1,6 branch linkages (Takeda et al., 1984). In contrast, amylopectin, with

approximately 5% α-1,6 branch linkages, is a highly branched molecule (Hizukuri, 1986). During

the fractionation of pulses to develop new food ingredients in the industry, starch is generated as

a major product that is underutilized. Compared with important commercial starches such as waxy

maize, normal maize and tapioca, common pulse starches (e.g., pea, lentil and faba bean) have

higher amylose contents (38.0-41.1%) and longer amylopectin branch chains (Li et al., 2019).

Primarily due to these structural features, pulse starches tend to display higher retrogradation rates,

develop lower peak and breakdown viscosities but higher final viscosities, and form more opaque

pastes upon cooking and cooling, which may limit the industrial applications of pulse starches (Li

et al., 2019; Sangokunle et al., 2020). On the other hand, the aforementioned structural traits render

pulse starches more suitable for the development of resistant starch (RS) (Chung et al., 2010; Li et

al., 2020), a nutritionally important food constituent attracting growing attention from food

companies. Therefore, modifications that improve the functional properties and enhance the RS

contents of pulse starches can potentially promote the industrial utilization of this main product

from the pulse processing industry.

Among the chemical, physical and enzymatic methods commonly applied in the industry

to modify starch to achieve the abovementioned goals, enzymatic method has received increasing

interest because it not only confers unique functional properties to starch but also enables the

resultant modified starch to meet the growing demands for “clean-label” ingredients (Maniglia et

23

al., 2020; Sorndech et al., 2018). In the enzymatic approach, various types of starch-related

enzymes, such as α-amylases, β-amylases, transglucosidases, pullulanases, isoamylases, and

amylosucrases, have been widely employed to retard retrogradation and increase enzymatic

resistance of starch (Ao, Quezada-Calvillo, et al., 2007; Cai & Shi, 2010; Demirkesen-Bicak et al.,

2018; Kim et al., 2014; Mendez-Montealvo et al., 2011). Among the starch-related enzymes,

maltogenic α-amylase (MGA) has attracted tremendous research attention due to its unique action

pattern on starch. This enzyme can catalyze the hydrolysis of α-1,4 glycosidic bonds in starch to

liberate maltose, resulting in a higher proportion of α-1,6 branch linkages (Christophersen et al.,

1998). MGA exhibits both endo- and exo- hydrolysis patterns: it mainly shows exo-action at a

relatively low temperature (such as 35 °C) but performs more endo-hydrolysis with a rise in the

incubation temperature (Bijttebier et al., 2007). Consequently, MGA has been used to shorten the

length of starch chains to slow down retrogradation (Goesaert, Leman et al., 2009). Additionally,

the increase in α-1,6 branch linkages by MGA hydrolysis has been proposed to enhance the slowly

digestibly starch (SDS) and RS contents of normal maize starch (Ao, Simsek, et al., 2007; Miao et

al., 2014).

To the best of our knowledge, despite the promising ability of MGA to improve the inherent

properties and elevate the RS content of starch, this enzyme has not been utilized to modify pulse

starches — particularly in a granular form — to enhance their potential for industrial applications.

It is a common industrial practice to modify starch in a native granular state to better control the

process and facilitate the recovery of final product. In the present study, granular lentil (LS), faba

bean (FBS), and pea starches (PS) were modified using a commercial Bacillus stearothermophilus

MGA enzyme. The effects of MGA modification on the structures, functional properties, and in

vitro digestibility of the three most representative pulse starches were comprehensively

characterized and compared. A commercial normal maize starch (NMS) was included in the starch

modification and subsequent characterization as a check for comparison. The new knowledge will

be meaningful for the use of MGA to modify pulse starches to develop clean-label starch

ingredients with improved functional attributes and enhanced nutritional value, which will help

the pulse processing industry explore new markets for this predominant co-product.

24

3.3 Materials and methods

3.3.1 Materials

Lentil (LS) and faba bean starches (FBS) were isolated from starch-rich flours of lentil

(Homecraft® 2103) and faba bean (Homecraft® 3103) air classified by AGT Food and Ingredients

(Saskatoon, SK, Canada). The wet isolation was carried out following the method reported by Li

et al. (2019), and the isolated LS and FBS had a purity comparable to that of commercial starches

(~96% starch, dry basis, db; determined using Megazyme Total Starch Assay Kit). Pea starch (PS)

was kindly donated by Roquette Canada Ltd. (Winnipeg, MB, Canada). Normal maize starch

(NMS; Cargill GelTM 03420) was generously provided by Cargill Inc. (Minneapolis, MN, U.S.A.).

Total Starch Assay Kit, D-Glucose Assay Kit, potato amylose standard, and isoamylase were

procured from Megazyme International Ltd. (Co. Wicklow, Ireland). Deuterated dimethyl

sulfoxide was purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, U.S.A.).

Maltogenic α-amylase (MGA) from Bacillus stearothermophilus, maize amylopectin standard,

pancreatin from porcine pancreas, amyloglucosidase and deuterated trifluoroacetic acid were

obtained from Sigma-Aldrich Canada Co. (Oakville, ON, Canada). All the other chemicals used

in this study were purchased from Fisher Scientific Company (Ottawa, ON, Canada) or Sigma-

Aldrich Canada Co. (Oakville, ON, Canada), which were of reagent grade or higher purity.

3.3.2 Modification of starch using MGA

The native granular LS, FBS, PS and NMS (20 g each, db) were mixed with 50.0 mM

sodium acetate buffer (pH 5.5, containing 0.02% sodium azide) to reach 100.0 g total weight. After

adding MGA to the starch slurry at a level of 1% (w/w, db; equivalent to 110 MANU per g of dry

starch), the sample bottle was placed horizontally in a 50 °C water bath with 150 rpm shaking to

initiate the enzymatic modification. The enzymatic hydrolysis was continued for 4, 8 and 24 h. At

the end of the modification, four volumes of anhydrous ethanol were added to the mixture,

followed by vigorous manual shaking to terminate the enzymatic reaction. The modified starch

was further washed with 200 mL anhydrous ethanol twice, recovered by centrifugation at 5,000 g

for 10 min, and then dried at 40 °C overnight.

To determine the percentages of starch hydrolysis by MGA at 4, 8 and 24 h, the total mass

of released soluble carbohydrates was quantitated (Mendez-Montealvo et al., 2011). After

vigorously mixing with four volumes of anhydrous ethanol to stop the enzymatic hydrolysis, an

25

aliquot (1.0 mL) of the starch suspension was collected into a 1.5-mL microcentrifuge tube. Upon

centrifugation at 3,000 g for 10 min, the concentration of soluble carbohydrates in the supernatant

was measured using a phenol-sulfuric acid method (Dubois et al., 1956). The total mass of released

soluble carbohydrates was applied to calculate the degree of starch hydrolysis: %Degree of starch

hydrolysis = [Total mass of released soluble carbohydrates × (162 / 180)] / (Initial dry mass of

starch) ×100.

The native starch that was subjected to the same treatment for 24 h without the use of MGA

was designated as the control for the subsequent characterization experiments.

3.3.3 Amylose content of starch

Amylose contents of the control and MGA-modified starches were measured using iodine

colorimetry (Chrastil, 1987). A standard curve was drawn using the blends of commercial potato

amylose and maize amylopectin standards at different ratios: 0.0%, 25.0%, 50.0%, 75.0% and

100.0% amylose.

3.3.4 Granular morphology of starch

Granular morphology of the control and MGA-24 h-modified starches was observed using

a field-emission scanning electron microscope (SEM, SU810, Hitachi High Technologies Canada

Inc., Rexdale, ON, Canada) (Li et al., 2019). After being gold coated using a sputtering technique,

each starch sample was viewed under the conditions of 3.0 kV and 10 μA. Representative images

were captured at magnifications of 500×, 1,500× and 3,000×.

3.3.5 WAXD of starch

WAXD patterns of the control and MGA-24 h-modified starches were detected using a

Rigaku X-ray diffractometer (Model Ultima IV, Rigaku Corp., The Woodlands, TX, U.S.A.). For

each treatment, 1.0 g starch was collected from each of the three independent batches of

modification and homogeneously mixed to obtain a composite sample. After the equilibration at

25 °C with 100% relative humidity for 24 h, the composite starch sample was scanned from

diffraction angle (2θ) of 3° to 40° at a rate of 1.3°/min under the conditions of 40 kV and 44 mA

with Cu Kα radiation (λ = 1.5406 Å) at room temperature. The relative crystallinity of the starch

26

was calculated using Origin Software (Origin, OriginLab Corp., Northampton, MA, U.S.A.)

following the method described by Hayakawa et al. (1997).

3.3.6 Molecular-weight distribution of debranched starch

Molecular-weight distributions of the debranched control and MGA-24 h-modified

starches were analyzed to assess the effects of MGA modification on the molecular structures of

amylose and amylopectin. The starch sample (20.0 mg) was dispersed in 2.0 mL of 90% dimethyl

sulfoxide (DMSO) by boiling in a water bath for 20 min with gentle magnetic stirring. After

cooling to room temperature, the starch molecules were precipitated by adding five volumes of

anhydrous ethanol. Upon centrifugation at 3,000 g for 15 min, the supernatant was carefully

decanted. The centrifuge tube was inverted and placed on a paper towel for 10 min to remove the

liquid residue. An aliquot of deionized water (1.75 mL) was added to disperse the starch pellet in

a boiling water bath with mild stirring for 10 min. Upon cooling to room temperature, 250 μL of

sodium acetate buffer (160 mM, pH 4.75, containing 0.16 % sodium azide) and 2 μL of isoamylase

(500 U/mL) were added to the sample. The sample tube was placed in a water bath of 37 °C for

24 h to completely debranch the starch (Li et al., 2008). After the debranching step, the sample

was boiled for 10 min and then cooled to ambient temperature, followed by the addition of 20

volumes of anhydrous ethanol to precipitate the fully debranched starch molecules. The mixture

was centrifugated at 5,000 g for 15 min, the supernatant was carefully removed, and the pellet was

re-dispersed in 4.0 mL of DMSO containing 0.5% lithium bromide by boiling in a water bath with

gentle stirring for 10 min. After cooling to ambient temperature, the starch dispersion was filtered

through a 5-µm disc filter. A 20-μL aliquot of the prepared sample was automatically injected into

a high-performance size exclusion chromatography (HPSEC) system (1260 Infinity II, Agilent

Technologies Canada, Mississauga, ON, Canada) that was composed of an isocratic pump, a

vialsampler, and a refractive index (RI) detector (Peng & Yao, 2018). Two Zorbax gel PSM 60-S

columns (Agilent Technologies Canada, Mississauga, ON, Canada) were connected to separate

amylose and amylopectin branch chains of different molecular weights for subsequent

quantification by the equipped RI detector. The analytical columns were stored at 30 °C in the

equipped Multicolumn Thermostat component, and the RI detector was maintained at 35 °C. The

mobile phase was DMSO with 0.5% (w/w) lithium bromide at a flow rate of 0.5 mL/min.

Calibration of the HPSEC system was performed using glucose, maltotriose, maltohexaose and

27

three pullulan standards (6.2 × 103, 48.8 × 103 and 348 × 103 g/mol). The collected raw data were

normalized based on the total peak area and then used for the drawing of HPSEC profiles.

3.3.7 Percentage of α-1,6 branch linkages of starch

Percentages of α-1,6 branch linkages of the control and MGA-24 h-modified starches were

measured using proton nuclear magnetic resonance (1H NMR) on a Bruker AVIII HD 600 NMR

Spectrometer following the method described by Tizzotti et al. (2011) with slight modifications.

Starch sample (~10 mg) was weighed into a 1.5-mL microcentrifuge tube, followed by the addition

of 1.0 mL of deuterated dimethyl sulfoxide (DMSO-d6) to reach a concentration of 1% (w/v). The

sample was incubated in an 80 °C water bath with 300 rpm shaking for 1 h. After the incubation,

the sample was cooled to room temperature and an aliquot of 0.5 mL was transferred into a 5-mm

NMR tube. A small amount of deuterated trifluoroacetic acid (TFA-d1) (5.66 μL) was immediately

added into the tube prior to the NMR scanning. The 1H NMR spectrum was collected at 70 °C and

Larmor frequency of 600.17 MHz with 300 scans: 6.25 μs 30° pulse; a repetition time of 6.6 s (an

acquisition time of 5.45 s and a relaxation delay of 1.15 s). The percentage of α-1,6 branch linkages

of the starch was calculated according to the following equation:

DB% = I(4.80)

I(5.10)+I(4.80)

Where DB% is the degree of branching; I(4.80) is the integrated intensity of peak at 4.80 ppm;

I(5.10) is the integrated intensity of peak at 5.10 ppm. Peaks at 5.10 ppm and 4.80 ppm

corresponded to α-1,4 and α-1,6 glycosidic bonds, respectively (Gidley, 1985).

3.3.8 Thermal properties of starch

Thermal properties of the control and MGA-24 h-modified starches were analyzed using a

differential scanning calorimeter (DSC 8000, Perkin Elmer, Waltham, MA, U.S.A.) in accordance

with the method of Song and Jane (2000). The starch sample (~10 mg) was accurately weighed

and then wet with three volumes of distilled water in a stainless-steel DSC pan. After being

hermetically sealed, the sample was equilibrated at ambient temperature for more than 2 h. The

hydrated starch was scanned from 10 to 160 °C at a rate of 10 °C/min. After the first scan, the

gelatinized sample was stored at 4 °C for 7 days and then scanned for the second time using the

same procedure to examine the retrogradation behavior. The endothermic peaks of starch

gelatinization (in the first scan) and melting of retrograded starch (in the second scan after the 7-

28

day cold storage) in the DSC thermograms were analyzed using Pyris Manager Software (Perkin

Elmer, Waltham, MA, U.S.A.). Onset temperature (To), peak temperature (Tp), conclusion

temperature (Tc), and enthalpy change (ΔH) were reported. Percentage of starch retrogradation

was calculated as: % retrogradation (%R) = (ΔH of melting of retrograded starch) / (ΔH of starch

gelatinization) × 100%.

3.3.9 Pasting properties of starch

A Rapid Visco-Analyzer (RVA Super 3, Newport Scientific, Sydney, Australia) was

deployed to measure the pasting properties of the control and MGA-modified starches following

a previous method (Li et al., 2019). The starch sample was mixed with distilled water to prepare a

slurry of 28.0 g total weight containing 8% (db) starch. The starch suspension was loaded onto the

RVA instrument and run using the Standard Method 2 in the Thermocline Software.

3.3.10 In vitro digestibility of cooked starch

In vitro digestibility of the control and MGA-24 h-modified starches after cooking was

evaluated using the method of Englyst et al. (1992) with some modifications (Ai et al., 2013). The

starch sample (containing 600 mg starch, dry starch basis, dsb) was suspended in 15.0 mL distilled

water. The starch was cooked in a boiling water bath for 10 min with vigorous magnetic stirring.

After the tube was cooled to ambient temperature, 5.0 mL of sodium acetate buffer (0.4 M, pH 5.2,

containing 0.08% sodium azide) was added to the cooked sample. Equilibration in a 37 °C water

bath with 160 rpm shaking was carried out before the addition of fresh enzyme cocktail (5.0 mL)

that contained porcine pancreatin extract and amyloglucosidase. The amounts of glucose released

from the amylolysis in the same water bath at 20 and 120 min were quantitated using Megazyme

D-glucose Assay Kit. Rapidly digestible starch (RDS), slowly digestible starch (SDS), and

resistant starch (RS) contents were calculated following the method reported by Englyst et al.

(1992).

3.3.11 Thermal properties of RS residue

To better explain the increases in the RS contents of cooked pulse and maize starches after

the 24-h MGA modification, RS residue of the samples immediately after the 120-min digestion

as described in Section 3.3.10 was collected and further characterized for thermal properties. At

29

the end of the 120-min amylolysis, five volumes (125 mL) of anhydrous ethanol were added to the

starch hydrolyzate to stop the enzymatic reaction. After vigorous manual shaking, the mixture was

centrifuged at 4,000 g for 15 min, followed by careful decanting of the supernatant to recover the

RS sediment. This anhydrous ethanol washing step was carried out for two additional times. After

drying at 40 °C in a convection oven overnight, the collected RS residue was ground into a fine

powder using a mortar and pestle, which was then scanned and analyzed following the same

procedure as described in Section 3.3.8.

3.3.12 Statistical analysis

The enzymatic modification of LS, FBS, PS and NMS by MGA at 4, 8 and 24 h was carried

out in three independent batches. Except for WAXD, all the described characterization

experiments were performed in one replicate on each independent batch of the control and MGA-

treated samples (n = 3). WAXD was conducted in one replicate for each of the composite starch

samples (n = 1) (Li et al. 2020). Statistical significance was evaluated using one-way ANOVA,

Tukey’s multiple comparison test at a significance level of 0.05 on IBM SPSS Statistics Analysis

(Version 24.0; IBM Corp., Armonk, NY, U.S.A.).

3.4 Results and discussion

3.4.1 Percentage of starch hydrolysis by MGA

The degrees of hydrolysis of granular LS, FBS, PS and NMS by MGA for 4, 8 and 24 h

are presented in Fig. 3.1. During the 24-h hydrolysis, PS showed the highest hydrolysis rate, while

LS, FBS and NMS had similar hydrolysis rates. Upon 4-h MGA treatment, the degrees of

hydrolysis of PS, LS, FBS and NMS were 23.6%, 19.7%, 20.7% and 19.8%, respectively; values

gradually increased to 38.4%, 34.7%, 34.4% and 35.9% at 24 h, respectively. The reason for the

difference in hydrolysis profile between PS and other pulse starches could be that the PS was

purchased from the company, and the isolation method was different to that used on LS and FBS.

However, pulse starch is C-type starch and it includes both characteristics of A-type starch and B-

type starch (Buléon et al., 1998). C-type starch is more resistant than A-type starch under

enzymatic attack because it has larger population of long chains (Srichuwong et al., 2005). At the

first 4 h of MGA treatment, pulse starches exhibited similar or higher hydrolysis rate compared

30

with NMS. With the modification going on, the degrees of hydrolysis of all starches reached

similar value upon 24-h MGA treatment.

Fig. 3.1 Degrees of starch hydrolysis after incubating LS, FBS, PS and NMS with maltogenic α-

amylase (MGA) for 4, 8 and 24 h. At the same modification time, the numbers with different letters

are significantly different at p < 0.05

31

3.4.2 Apparent Amylose content of starch

The apparent amylose contents of control LS, FBS, PS and NMS were 40.1%, 40.2%, 40.3%

and 29.9%, respectively (Table 3.1). After 24-h hydrolysis by MGA, the apparent amylose

contents of all the three pulse starches were gradually decreased to 33.7%, 35.6% and 37.5%,

respectively, which were significantly lower than those of their corresponding control starches. In

contrast, 24-h treatment only slightly diminished that of NMS from 29.9% to 27.9%, and the

difference was statistically insignificant. It has been reported that the relative ratio of amylose and

amylopectin will not change as hydrolysis progressed (Zhang et al., 2006). In this study, the larger

difference in the amylose contents of MGA-modified pulse starches could be attributed to the

higher amylose contents of their controls.


Under SEM observation, all the control pulse and maize starches displayed morphology

resembling that reported in previous research (Fig. 3.2) (Hoover et al., 2010; Li et al., 2019).

Interestingly, the morphology of MGA-24 h-modified starches revealed that the enzyme

hydrolyzed pulse and maize starches following distinctly different patterns. It is known that pea,

lentil and faba bean starch granules possessed a solid and homogenous internal structure (Chung

et al., 2008; Li et al., 2019). Consequently, amylolytic enzymes, including amylases and

amyloglucosidases, hydrolyze pulse starch granules through “surface pitting” (Gallant et al., 1992;

Sujka & Jamroz, 2007), which was supported by the rougher surface of the MGA-modified pulse

starches. With the high percentages of starch hydrolysis at 24 h (34.4% to 38.4%), MGA was able

to split the pulse starch granules into small pieces (marked by up arrows). For some broken

granules, prominent growth rings were observed (marked by chevrons) in the internal regions. For

some granules resistant to MGA hydrolysis, fissures (marked by triangles in FBS) were found in

them, which supported the aforementioned “surface pitting” hydrolysis pattern. By contrast, NMS

were not split into small pieces by MGA. Instead, a hydrolysis degree of 35.9% generated

numerous holes into the interior of NMS granules. It is a well-accepted fact that native NMS has

endogenous pores and channels (Huber & BeMiller, 1997; Li et al., 2019), which could probably

be further enlarged at 50 °C (i.e., the incubation temperature used for MGA modification in the

present study). MGA was likely to initiate the hydrolysis from these “weak points”, created tunnels

into the granule interior, and then hydrolyzed the granules using the “inside-out” pattern to

32

generate those large holes in NMS granules (MacGregor & Ballance, 1980; Zhang et al., 2006).

The morphology of MGA-treated NMS resembled that of NMS hydrolyzed by other α-amylases,

such as α-amylase from porcine pancreas (Zhang et al., 2006) and α-amylase from Aspergillus

fumigatus (Planchot et al., 1995).

3.4.4 WAXD and relative crystallinity of starch

As shown in Fig 3.3, the control LS, FBS and PS exhibited the typical C-type WAXD

pattern, whereas the control NMS had the typical A-type WAXD pattern (Chung et al., 2010; Li

et al., 2019). Overall, the 24-h MGA modification did not change the WAXD patterns of the

starches. The enzymatic modification marginally reduced the percentages of crystallinity of the

three pulse starches, suggesting that the “surface pitting” hydrolysis pattern caused the loss of

some crystalline structure in the pulse starch granules. The enzymatic modification, however, did

not obviously alter the relative crystallinity of NMS. The observation could be explained by that

the “inside-out” action pattern of MGA simultaneously degraded and removed amylose and

amylopectin from NMS granules (MacGregor & Ballance, 1980; Zhang et al., 2006), which is

consistent with the result that the MGA treatment did not significantly change the amylose content

of NMS as reported in Table 3.1.

Table 3.1 Apparent amylose contents of control and MGA-modified starches.1, 2


LS3 FBS PS NMS

Control 40.1 ± 0.6b 40.2 ± 1.7b 40.3 ± 0.9b 29.9 ± 1.4a

4 h 35.4 ± 1.0a 37.8 ± 1.5ab 39.3 ± 1.7ab 30.4 ± 0.7a

8 h 35.3 ± 1.3a 36.8 ± 0.8ab 38.0 ± 0.8ab 29.1 ± 1.6a

24 h 33.7 ± 1.6a 35.6 ± 1.2a 37.5 ± 0.4a 27.9 ± 0.5a

1 Values are presented as average ± standard deviation (n = 3); in the same column, the numbers with the same letter are not significantly

different at p < 0.05.

2 Determined using iodine colorimetry (Chrastil, 1987).

3 LS: lentil starch; FBS: faba bean starch; PS: pea starch; NMS: normal maize starch; 4 h, 8 h and 24 h: 4-, 8- and 24-h modified starch.

33

34

LS-C (500×) LS-C (1500×) LS-C (3000×)

LS-24 h (500×) LS-24 h (1500×) LS-24 h (3000×)

FBC-S (500×) FBC-S (1500×) FBC-S (3000×)

FBS-24 h (500×) FBS-24 h (1500×)

PS-C (500×) PS-C (1500×) PS-C (3000×)

FBS-24 h (3000×)

35


starches. LS-C: lentil control; LS-24 h: lentil 24-h modified; FBS-C: faba bean control; FBS-24 h:

faba bean 24-h modified; PS-C: pea control; PS-24 h: pea 24-h modified; NMS-C: normal maize

control; NMS-24 h: normal maize 24-h modified. The magnification at which the image was taken

is given in parentheses. Up arrows mark broken starch granules caused by MGA hydrolysis;

chevrons mark grow rings observed in the internal regions of broken starch granules; triangles

mark fissures found in FB starch granules caused by MGA hydrolysis through “surface pitting”

pattern; and right arrows mark large holes in NMS caused by MGA hydrolysis through “inside-

out” pattern.

PS-24 h (500×) PS-24 h (1500×) PS-24 h (3000×)

NMS-C (500×) NMS-C (1500×) NMS-C (3000×)

NMS-24 h (500×) NMS-24 h (1500×) NMS-24 h (3000×)

36

Fig. 3.3 WAXD patterns of control and MGA-24 h-modified starches. LS-C: lentil control; LS-24

h: lentil 24-h modified; FBS-C: faba bean control; FBS-24 h: faba bean 24-h modified; PS-C: pea

control; PS-24 h: pea 24-h modified; NMS-C: normal maize control; NMS-24 h: normal maize 24-

h modified. Relative crystallinity is given in parentheses.

3 6 9 12 15 18 21 24 27 30

Inte

nsity

2 (°)

LS-C (27.1%)

LS-24 h (24.0%)

FBS-C (25.3%)

FBS-24 h (24.6%)

PS-C (27.6%)

PS-24 h (26.2%)

NMS-C (28.4%)

NMS-24 h (28.8%)

37


According to Fig 3.4 A, the normalized HPSEC profiles of debranched control starches

were divided into four areas: Area 1 corresponding to amylose [peak value = degree of

polymerization (DP) 1,074 for LS; DP 1,081 for FBS; DP 1,096 for PS; and DP 1,051 for NMS];

and Area 2 (DP 23-236), Area 3 (DP 11-23), and Area 4 (DP 3-11) corresponding to long,

intermediate and short branch chains (LBC, IBC and SBC) of amylopectin, respectively. As

summarized in Table 3.2, the percentages of amylose of the control LS, FBS, PS and NMS

determined by the HPSEC method were 31.5%, 33.1%, 33.9% and 26.2%, respectively, which

were consistently lower than the apparent amylose contents measured using the iodine colorimetry

approach (40.1%, 40.2%, 40.3% and 29.9% as reported in Table 3.1). The differences in the data

between these two methods could be attributed to the fact that a portion of the LBC of the

amylopectin (DP 23-236) could also bind with iodine in the colorimetric method to inflate the

apparent amylose contents (Jane et al., 1999). All the three control pulse starches had similar

branch-chain-length (BCL) distributions for their amylopectin molecules: ~ 27.6% for LBC, ~34.2%

for IBC, and ~38.3% for SBC. Compared with the pulse starches, NMS showed the lowest

percentage of LBC (26.7%) and the highest percentage of SBC (39.8%), which could contribute

to the smallest inflation in the apparent amylose content of NMS (3.7% versus 6.4%-8.6% of pulse

starches).

After 24-h MGA modification, the amylose and LBC peaks of all the four starches were

shifted toward smaller DP (Fig. 3.4 B): the amylose peak was changed from DP 1,074 to 835 for

LS, from DP 1,081 to 781 for FBS, from DP 1,096 to 615 for PS, and from DP 1,051 to 603 for

NMS, respectively; and the LBC peak range was altered from DP 23-236 to DP 21-121 for all the

four starches. The shift of the amylose and LBC peaks toward smaller DP was a result of molecular

breakdown by MGA.

The 24-h treatment with MGA did not considerably alter the percentages of amylose of all

the starch samples (Table 3.2), which is inconsistent with the changes in their apparent amylose

contents as reported in Table 3.1. The obvious reduction in the apparent amylose contents of the

starches, particularly in the pulse starches, could be attributed to the shortening of amylose and

LBC by MGA hydrolysis as discussed above. The modified amylose and LBC with markedly

smaller DP possessed lower iodine affinity in comparison with their counterparts in the control

starches. With respect to the BCL distributions of amylopectins, the modification with MGA

38

largely decreased the proportions of LBC or IBC and increased that of SBC, with the most

significant change being found with NMS. The results are in good agreement with previous studies

showing that MGA hydrolysis resulted in a higher proportion of SBC of amylopectin molecules

(Ao, Simsek, et al., 2007; Grewal et al., 2015).


All the three control pulse starches exhibited comparable percentages of α-1,6 branch

linkages (2.5-2.6%), while control NMS contained 3.0% α-1,6 glycosidic bonds (Table 3.3). The

differences are generally in good accordance with their different amylose contents as demonstrated

in Tables 3.1 and 3.2 because amylose is primarily linear with several branch linkages only and

amylopectin is highly branched with around 5% branch linkages (Hizukuri, 1986; Takeda et al.,

1984). After 24-h MGA treatment, the percentages of α-1,6 glycosidic bonds of LS, FBS, PS and

NMS were increased by 0.5%, 0.6%, 0.6% and 0.7% respectively, resulting from that MGA can

only hydrolyze α-1,4 glycosidic bonds under the applied conditions (Christophersen et al., 1998).

39


control (A) and MGA-24 h-modified (B) starches. LS-C: lentil control; LS-24 h: lentil 24-h

modified; FBS-C: faba bean control; FBS-24 h: faba bean 24-h modified; PS-C: pea control; PS-

24 h: pea 24-h modified; NMS-C: normal maize control; NMS-24 h: normal maize 24-h modified.

The solid line separates the amylose (AM) and amylopectin branch chains; and the dash lines

separate the long (LBC; corresponding to DP 23-236 for A; DP 21-121 for B), intermediate (IBC;

corresponding to DP 11-23 for A; DP 11-21 for B), and short (SBC; corresponding to DP 6-11in

both A and B) branch chains of amylopectin.

9 10 11 12 13 14 15 16 17 18 19 9 10 11 12 13 14 15 16 17 18 19

RI

sig

nal

Elution time (min)

AM LBC IBC SBC

10

00

10

05

0

20

10

8 3

LS-C

FBS-C

PS-C

NMS-C

A

Elution time (min)

B AM LBC IBC SBC

10

00

10

05

0

20

10

8 3

Degree of polymerization (DP)

LS-24 h

FBS-24 h

PS-24 h

NMS-24 h

Table 3.2 Molecular-weight distributions of debranched control and MGA-24 h-modified starches.1

Starch2 Amylose (%) Amylopectin branch-chain-length distribution (%)3

Long branch chain Intermediate branch chain Short branch chain

LS, control 31.5 ± 0.1c 27.8 ± 0.1bc 34.4 ± 0.1d 37.8 ± 0.2a

LS, 24 h 32.8 ± 0.0cd 27.3 ± 0.1b 34.3 ± 0.3d 38.4 ± 0.4abc

FBS, control 33.1 ± 0.3d 28.0 ± 0.2bc 33.7 ± 0.3cd 38.3 ± 0.5ab

FBS, 24 h 33.5 ± 0.6d 27.9 ± 0.7bc 33.0 ± 0.9bc 39.1 ± 0.3bcd

PS, control 33.9 ± 0.3d 26.9 ± 0.1b 34.4 ± 0.1d 38.7 ± 0.1abcd

PS, 24 h 32.4 ± 0.7cd 29.0 ± 0.6c 31.5 ± 0.7a 39.5 ± 0.2cd

NMS, control 26.2 ± 0.1a 26.7 ± 0.0b 33.5 ± 0.1cd 39.8 ± 0.1d

NMS, 24 h 27.7 ± 1.0b 24.3 ± 1.3a 32.0 ± 0.4ab 43.7 ± 0.9e



2 LS: lentil starch; FBS: faba bean starch; PS: pea starch; NMS: normal maize starch.

3 Percentages of long, intermediate and short branch chains were calculated as the respective fractions divided by the total area of

amylopectin branch chains as shown in Fig. 3.4.

40

Table 3.3 Percentages of α-1,6 branch linkages of control and MGA-24 h-modified maize starches.1

Starch2 α-1,6 linkage (%)3

LS, control 2.6 ± 0.1a

LS, 24 h 3.1 ± 0.0b

FBS, control 2.5 ± 0.1a

FBS, 24 h 3.1 ± 0.0b

PS, control 2.5 ± 0.0a

PS, 24 h 3.1 ± 0.0b

NMS. control 3.0 ± 0.1b

NMS, 24 h 3.7 ± 0.0c

1 Values are presented as average ± standard deviation (n = 3); in the same column, the numbers with different letters are significantly



3 %Degree of branching = (Integrated intensity of peak at 4.80 ppm) / (Integrated intensity of peak at 5.10 ppm + Integrated intensity of

peak at 4.80 ppm) × 100.

41

42


The control pulse starches had significantly lower gelatinization temperatures (To of 59.8-

64.9 °C and Tc of 75.6-79.6 °C; Table 3.4) than control NMS (To of 70.9 °C and Tc of 81.9 °C),

consistent with the findings from previous studies (Hoover et al., 2010; Li et al., 2019). The

gelatinization ΔH of control LS and FBS (16.0-16.2 J/g) were similar to that of control NMS (16.6

J/g, whereas that of control PS (13.4 J/g) was the lowest among all the four starches. The lowest

gelatinization temperatures and ΔH of PS from a commercial source might be related to the

destabilization of double-helical crystallites in the high-temperature drying step during the wet

milling process to isolate the starch (Haros et al., 2003). Upon 24-h MGA treatment, the

gelatinization temperatures of all the four starches were considerably increased and their

gelatinization ΔH in general showed a decrease trend. The data suggested that MGA tended to

break down and remove double-helical crystallites with lower thermal stability.

The percentages of retrogradation of control pulse starches (46.2-56.9%) were remarkably

higher than that of control NMS (34.8%), which were related to the longer branch chains of

amylopectins and the greater contents of amylose of pulse starches (Tables 3.1 and 3.2) (Li et al.,

2019). The 24-h MGA treatment markedly lowered the percentages of retrogradation of all the

four starches, which could be partly attributed to the shortening of amylopectin branch chains by

MGA (Fig. 3.4 and Table 3.2). Therefore, the modified amylopectin molecules were unable to

reform double-helical crystallites as stable as those of control samples during retrogradation: Tp

and Tc of the retrograded MGA-modified starches were apparently lower than those of their

respective controls. The observation is in good accordance with the data illustrated in the study of

Ao, Simsek, et al. (2007). This research explicitly demonstrated that MGA treatment could be an

effective approach to inhibiting the retrogradation pulse starches to reduce their syneresis.

Table 3.4 Gelatinization and retrogradation properties of control and MGA-24 h-modified starches.1

Starch2 Gelatinization of starch3 Melting of retrograded starch3 % Retrogradation

(%R)4

To (°C) Tp (°C) Tc (°C) ∆H (J/g) To (°C) Tp (°C) Tc (°C) ∆H (J/g)

LS, control 64.9 ± 0.2c 70.3 ± 0.4b 79.6 ± 0.7bc 16.0 ± 1.7c 42.5 ± 0.6a 59.3 ± 0.8b 72.4 ± 0.8d 9.0 ± 0.3e 56.9 ± 7.5e

LS, 24 h 72.3 ± 0.1e 75.8 ± 0.2d 81.5 ± 0.3cd 15.2 ± 0.5abc 49.5 ± 3.6bc 56.8 ± 0.3a 67.6 ± 0.9bc 2.1 ± 0.2b 13.7 ± 1.4b

FBS, control 63.8 ± 0.4b 69.7 ± 0.3b 78.3 ± 1.3b 16.2 ± 1.3c 47.4 ± 3.2ab 60.4 ± 1.1b 70.3 ± 3.4cd 7.4 ± 0.6d 46.2 ± 5.5d

FBS, 24 h 72.5 ± 0.2e 75.7 ± 0.2d 81.7 ± 0.3cd 15.6 ± 0.1bc 43.5 ± 0.3ab 56.5 ± 0.5a 64.9 ± 2.9ab 2.1 ± 0.3b 13.6 ± 1.6b

PS, control 59.8 ± 0.0a 66.8 ± 0.0a 75.6 ± 2.2a 13.4 ± 0.3ab 46.2 ± 0.5ab 59.6 ± 0.0b 71.4 ± 0.2cd 7.5 ± 0.2d 56.2 ± 2.6de

PS, 24 h 71.4 ± 0.1d 74.3 ± 0.1c 79.6 ± 0.1bc 13.0 ± 0.1a 47.6 ± 5.2ab 57.2 ± 0.3a 67.7 ± 1.7bcd 2.2 ± 0.0b 16.5 ± 0.7b

NMS, control 70.9 ± 0.2d 75.8 ± 0.3d 81.9 ± 0.3cd 16.6 ± 0.3c 42.1 ± 0.1a 56.4 ± 0.0a 66.7 ± 0.4bc 5.8 ± 0.2c 34.8 ± 2.0c

NMS, 24 h 72.5 ± 0.3e 77.4 ± 0.1e 83.1 ± 0.2d 14.4 ± 0.5abc 53.9 ± 1.1c 56.6 ± 0.3a 61.3 ± 0.0a 0.2 ± 0.0a 1.5 ± 0.0a

1 Values are presented as average ± standard deviation (n = 3); in the same column, the numbers with different letters are significantly different at p < 0.05.

2 LS: lentil starch; FBS: faba bean starch; PS: pea starch; NMS: normal maize starch; 24 h: 24-h modified starch.

3 To: onset temperature; Tp: peak temperature; Tc: conclusion temperature; ∆H: enthalpy change.

4 % Retrogradation (%R) = (ΔH of melting of retrograded starch / ΔH of starch gelatinization) × 100%.

43

44


The MGA treatment diminished the pasting properties for all the starches substantially (Fig.

3.5). Simply after 4-h MGA hydrolysis, the peak viscosity was decreased from 1,647 to 35 cP,

from 1,346 to 8 cP, from 1,348 to 49 cP, and from 1,666 to 168 cP for LS, FBS, PS and NMS,

respectively. A longer enzymatic modification period further reduced the peak viscosities of the

starches. Additionally, all the modified starches showed very low viscosity after the pasting peak.

The marked decreases in the pasting viscosities of the starches upon MGA modification resulted

from the degradation of both granular (Fig. 3.2) and molecular (Fig. 3.4 and Table 3.2) structures

of the starches. Furthermore, the extremely low viscosities of the MGA-modified starches allowed

them to disperse easily during pasting. This behavior effectively reduced the molecular proximity

of these samples, which could be another factor contributing to their lower percentages of

retrogradation as demonstrated in Table 3.4 (Jane et al., 1999).

Fig. 3.5 Pasting properties of control and MGA-modified starches analyzed using a Rapid Visco-Analyzer with 28.0 g starch suspension

containing 8% (w/w, db) starch. A: lentil; B: faba bean; C: pea; D: normal maize; 4 h, 8 h and 24 h: 4-, 8- and 24-h modified.

45

46


The RDS (85.9%-90.5%), SDS (2.4%-6.5%), and RS (2.6%-5.8%) contents of the cooked

LS, FBS, PS and NMS controls did not show significant differences (Table 3.5), suggesting their

similar in vitro digestibility (Li et al., 2019). The 24-h MGA modification significantly increased

the RS contents of cooked LS, FBS, PS and NMS by 5.9%, 6.5%, 4.2% and 4.7%, respectively.

The improvement in the RS contents of the MGA-24 h-treated pulse and normal maize starches

could not be attributed to the increases in the percentages of α-1,6 branch linkages of the modified

starches: the MGA-treatment for 24 h noticeably increased the percentages of α-1,6 branch

linkages of waxy maize starch as well but did not elevate its RS content in a cooked form according

to the results reported in the subsequent Study 2 of this thesis.

To elucidate the factor(s) contributing to the increased RS contents of the enzymatically

modified starches, the RS residue was collected after the Englyst Assay, dried, and subjected to

DSC scanning. As presented in Fig. 3.6, two endothermic peaks were observed in the RS residue

samples of all the starches: the first peak (Tp ranging from 78.4 to 82.4 °C of all the residue samples)

represented the melting of remaining double-helical crystallites of amylopectin, while the second

peak (Tp ranging from 105.4 to 129.0 °C of the RS residue of the controls and Tp ranging from

123.6 to 129.6 °C of the RS residue samples of MGA-treated counterparts) represented the melting

of retrograded amylose (Sievert & Wuesch, 1993). In comparison with those of the corresponding

controls, the RS residue samples of the MGA-modified starches showed a higher melting

temperature and a greater enthalpy change of amylose double-helical crystallites (Table 3.6),

which could contribute to the larger RS contents of the cooked modified starches (Li et al., 2020;

Sievert & Pomeranz, 1989). The formation of such amylose double-helical crystallites probably

occurred during the incubation of cooked starch samples with the amylolytic enzyme cocktail in

the Englyst Method (Ring et al., 1988). Because the MGA treatment shortened amylose chains in

all the four starches (Section 3.4.5), the resulting amylose molecules in the cooked modified

starches could have better molecular mobility to re-associate with each other to develop stable

double-helical crystallites during the incubation at 37 °C in the in vitro starch digestibility analysis

(Buléon et al., 2007; Li & Hu, 2020).

Table 3.5 In vitro digestibility of cooked control and MGA-24 h-modified starches.1, 2

Starch3 RDS (%)4 SDS (%) RS (%)

LS, control 85.9 ± 2.3abc 6.5 ± 2.3a 3.2 ± 0.9a

LS, 24 h 81.6 ± 1.1a 4.7 ± 0.3a 9.1 ± 0.6bc

FBS, control 86.0 ± 0.9abc 6.3 ± 0.3a 3.1 ± 1.1a

FBS, 24 h 83.8 ± 2.5ab 2.3 ± 0.9a 9.6 ± 1.2c

PS, control 87.0 ± 2.5bc 3.1 ± 1.9a 5.8 ± 1.4ab

PS, 24 h 84.4 ± 2.2ab 2.0 ± 1.2a 10.0 ± 0.6c

NMS, control 90.5 ± 1.3c 2.4 ± 2.2a 2.6 ± 2.0a

NMS, 24 h 85.6 ± 1.6abc 2.5 ± 2.1a 7.3 ± 0.8bc



2 Starch samples were cooked in a boiling water bath for 10 min.


4 RDS: rapidly digestible starch; SDS: slowly digestible starch; RS: resistant starch; values were calculated on a dry starch basis.

47

Table 3.6 Thermal properties of RS residue samples recovered from control and MGA-24 h-modified starches after cooking and

amylolysis in the Englyst Method.1

RS residue2 Melting of amylopectin double-helical crystallites3 Melting of amylose double-helical crystallites3

To (°C) Tp (°C) Tc (°C) ΔH (J/g) To (°C) Tp (°C) Tc (°C) ΔH (J/g)

LS, control 71.5 ± 2.2a 80.1 ± 0.7ab 85.2 ± 0.9a 1.1 ± 0.2a 99.3 ± 0.6a 106.5 ± 0.3a 111.2 ± 0.8a 0.7 ± 0.4ab

LS, 24 h 72.4 ± 4.4a 79.7 ± 0.4ab 85.7 ± 0.2a 1.1 ± 0.3a 118.0 ± 4.8b 126.5 ± 1.1b 131.8 ± 5.5b 2.8 ± 0.6c

FBS, control 74.1 ± 2.0a 79.8 ± 0.6ab 84.6 ± 1.3a 1.6 ± 0.7ab 99.9 ± 1.0a 106.1 ± 1.6a 110.8 ± 1.6a 0.7 ± 0.2ab

FBS, 24 h 71.8 ± 3.5a 79.9 ± 0.7ab 85.3 ± 0.7a 0.9 ± 0.2a 103.7 ± 4.9a 123.6 ± 2.8b 136.4 ± 5.1b 3.1 ± 0.0c

PS, control 72.1 ± 2.3a 80.1 ± 0.4ab 86.4 ± 0.3a 1.7 ± 0.3ab 100.7 ± 1.2a 105.4 ± 1.1a 110.3 ± 1.8a 0.2 ± 0.1a

PS, 24 h 68.9 ± 3.1a 78.4 ± 0.8a 85.2 ± 1.6a 1.6 ± 1.0ab 113.9 ± 0.7b 125.4 ± 5.4b 139.3 ± 2.7b 2.7 ± 0.9c

NMS, control 74.6 ± 1.1a 82.4 ± 1.1c 89.3 ± 2.2a 3.6 ± 0.8c 122.1 ± 1.1b 129.0 ± 2.4b 136.6 ± 2.1b 0.8 ± 0.1ab

NMS, 24 h 74.1 ± 0.5a 80.8 ± 0.3bc 90.4 ± 5.8a 2.9 ± 0.5bc 116.2 ± 5.0b 129.6 ± 2.4b 138.3 ± 2.1b 2.4 ± 0.8c

1 Values are presented as average ± standard deviation (n = 3); in the same column, the numbers with different letters are significantly different at p

< 0.05. DSC thermograms of the RS residue samples are shown in Figure 3.6.



48

50

Fig. 3.6 DSC thermograms of RS residue samples recovered from control and MGA-24 h-modified

starches after cooking and amylolysis in the Englyst Method. A: lentil; B: faba bean; C pea; D:

normal maize. The black solid line with two end points is the baseline for the thermogram. The

first endothermic peak of lower temperatures corresponded to the melting of remaining

amylopectin double-helical crystallites, and the second endothermal peak of higher temperatures

corresponded to the melting of amylose double-helical crystallites.

51

3.5 Conclusions

Using MGA to modify granular pulse starches is a novel method to improve the functional

properties and RS contents. In this study, LS, FBS and PS were modified with 1% (w/w, db) MGA

for 4, 8 and 24 h. The MGA-modified LS, FBS and PS exhibited comparable structures, functional

properties and in vitro digestibility, but different from that of MGA-modified NMS. MGA

treatment degraded the structures of pulse starch granules and split them into broken granules,

while it generated numerous pores into NMS granules by using the “inside-out” hydrolysis pattern.

MGA treatment did not alter the WAXD patterns of pulse and maize starches. DP of amylose and

LBC of amylopectin were reduced by 24-h MGA hydrolysis. The retardation of the retrogradation

rates was due to the shortening of the amylopectin chains. The higher retrogradation rates of MGA-

modified pulse starches than NMS could be attributed to the higher amylose contents and longer

branch chains of pulse starches. MGA caused degradation in both molecular and granular structure

to all starches and results in the extremely decrease of pasting viscosities. The in vitro digestibility

of cooked pulse starches was also decreased after 24-h MGA treatment, which could be explained

by the recrystallization of the shortened amylose molecules. The results obtained in this study

would help the pulse industry find new approaches to develop “clean-label” starch ingredients with

improved functional properties and higher nutritional value.

3.6 Connection to the Next Study

In the present study, MGA-modified pulse starches were compared with MGA-modified

NMS. Based on the reported results, the variations in the structures, functional properties, and in

vitro digestibility among the pulse and normal maize starches after MGA treatment were mainly

attributed to their differences in botanical origins, amylose contents, and polymorphic patterns. In

the current literature, however, there is a lack of understanding regarding the hydrolysis patterns

and resulting effects of MGA modification on granular starches from the same crop type but with

various amylose contents. Therefore, waxy (3.3% apparent amylose), normal (29.9% apparent

amylose) and high-amylose maize (58.6% apparent amylose) starches were selected for the

subsequent study to modify with MGA treatment. Interrelationships between the structures,

functional attributes, and in vitro digestibility of the MGA-modified maize starch samples were

examined to investigate the influence of MGA hydrolysis on the starches. The second study greatly

advanced our understanding of MGA modification on starches with a broad range of amylose

52

contents (3.3-58.6%) and clearly illustrated and highlighted the special functionality and

nutritional quality of the enzymatically modified maize starches. The new insights will be

meaningful for the starch industry to apply MGA modification to prepare “clean-label” maize

starches with more diverse techno-functional characteristics and enhanced nutritional value to

promote their industrial utilization.

53

4. MODIFICATION OF GRANULAR WAXY, NORMAL AND HIGH-AMYLOSE

MAIZE STARCHES USING MALTOGENIC α-AMYLASE TO IMPROVE THE

FUNCTIONALITY

4.1 Abstract

Maltogenic α-amylase (MGA) was used to modify granular waxy (WMS), normal (NMS),

and high-amylose maize (HAMS) starches for improved functional attributes. MGA treatment for

24 h significantly shortened the amylopectin branch chains of WMS and NMS, which retarded

their retrogradation during cold storage. Due to the effective degradation at both granular and

molecular levels, MGA modification markedly diminished the pasting viscosities of WMS and

NMS. The MGA treatment increased the resistant starch (RS) content of cooked NMS from 2.6%,

to 7.3%, resulting from the formation of retrograded amylose in MGA-modified NMS during

incubation at 37 °C in the Englyst Assay. Different from the effective hydrolysis on WMS and

NMS using an “inside-out” pattern, MGA hydrolyzed HAMS to a very low degree through

“surface pitting”, thus showing limited influence on its functionality. The reported novel findings

will be meaningful for utilizing MGA to develop “clean-label” starch ingredients with enhanced

functional properties.

Keywords: Maltogenic α-amylase modification; Granular maize starch; Amylose content; Starch

structure; Starch functionality; In vitro digestibility of starch

54

4.2 Introduction

Starch is one of the most abundant polysaccharides produced by green plants. In nature,

starch exists as water-insoluble, semi-crystalline granules that have a particle density of around

1.5 g/cm3 (Marousis & Saravacos, 1990). The insolubility and larger density of starch granules

than that of water allow easy isolation and recovery in producing and modifying starch. Most

starch granules are composed of two types of α-glucans: amylose and amylopectin. Amylose is

essentially a linear glucan of α-1,4-linked D-glucopyranose with several α-1,6 branch linkages

(Takeda et al., 1990), whereas amylopectin is a highly branched glucan with α-1,4-linked linear

chains connected by approximately 5% α-1,6 branch linkages (Hizukuri, 1986). Based on the

amylose content, starch can be broadly classified into three categories: waxy type with 0-8%

amylose, normal type with 20-40% amylose, and high-amylose type with 50-90% amylose (Ai &

Jane, 2015). Amylose content and branch-chain length of amylopectin are known to be two crucial

structural features that govern the physicochemical properties and digestibility of starch (Ai et al.,

2016).

In the food industry, starch is commonly utilized as a thickener, binder, stabilizer, gelling

agent, emulsifier and/or encapsulant in various products. Nevertheless, native starch has certain

undesirable inherent properties that limit its industrial use, such as high tendency to retrograde,

fast digestion rate upon gelatinization, and poor resistance to high temperature, acid and shear

(BeMiller, 1997). Therefore, native starch is usually modified to improve the functional properties

and enhance the enzymatic resistance for wider industrial applications. In the past few decades,

chemical methods have been primarily employed for this purpose (Chen et al., 2015). Recent

research efforts, however, have concentrated on developing new enzymatic and physical methods

that not only impart target physicochemical properties to native starch but also meet the

requirements of clean label (Saltmarsh, 2015).

In the enzymatic approach, different types of starch-related enzymes have been utilized for

modifications, such as α-amylases, β-amylases, amyloglucosidases, α-glucanotransferases,

debranching enzymes and amylosucrases (Ao, Quezada-Calvillo, et al., 2007; Cai & Shi, 2010;

Dura et al., 2014; Kim et al., 2014; Mendez-Montealvo et al., 2011; van der Maarel & Leemhuis,

2013). Among these enzymes, maltogenic α-amylase (MGA) has attracted tremendous research

interest because of the unique action pattern. For example, MGA from Bacillus stearothermophilus,

as the most researched commercial enzyme of the type, has been revealed to have the following

55

important features: (1) This enzyme is a thermostable α-amylase with a high degree of multiple

attack action on starch (Christophersen et al., 1998); (2) This enzyme displays both exo- and endo-

hydrolysis patterns ‒ at a relatively low temperature (e.g., 35 °C), it primarily exhibits exo-action,

releasing α-maltose as the main product from starch; as the temperature rises (e.g., at 70 °C), it

tends to show increased endo-action (Bijttebier et al., 2007). Consequently, MGA has been used

to shorten the branch chains of amylopectin and amylose chains to effectively retard the

retrogradation of starch (Grewal et al., 2015; Leman et al., 2009). In the baking industry, MGA is

commonly added in bread formulations to deliver the anti-staling effect, which has been related to

the inhibition of both amylopectin recrystallization and amylose rearrangement during bread

storage (Goesaert, Leman et al., 2009; Hug‐Iten et al., 2001). Moreover, extensive hydrolysis by

MGA has been demonstrated to enhance the enzymatic resistance of starch as determined in vitro,

which was attributed to the increase in the proportion of α-1,6 glycosidic bonds as the presence of

more branch linkages was proposed to decrease the hydrolysis rate of starch by important

amylolytic enzymes (e.g., porcine pancreatic α-amylase and amyloglucosidase) (Ao, Simsek, et

al., 2007).

In the current literature, however, the modification of starch by MGA has only been carried

out on a gelatinized/solubilized form (Ao, Simsek, et al., 2007; Grewal et al., 2015), rather than

the common insoluble granular form of starch used in industrial modification processes. Modifying

starch in a gelatinized/solubilized state may not be easily controlled and complicates the recovery

and drying steps, which is thus an undesirable practice in the industry (Richardson et al., 2000).

To further explore the application value of MGA for starch modification, a commercial Bacillus

stearothermophilus MGA enzyme was applied to modify granular waxy (WMS), normal (NMS)

and high-amylose maize starch (HAMS) of a broad range of amylose contents in the current study.

The structural features, functional properties, and in vitro digestibility of the modified maize

starches were comprehensively characterized and compared with those of their respective controls.

The fundamental understanding as presented in this work is of great value for the utilization of

MGA to prepare “clean-label” starch ingredients with diverse functional attributes and increased

resistant starch (RS) content.

56

4.3 Materials and methods

4.3.1 Materials

Waxy maize starch (WMS; AMIOCA TF) and high-amylose maize starch (HAMS;

HYLON®VII) were obtained from Ingredion Inc. (Bridgewater, NJ, U.S.A.). Normal maize starch

(NMS; Cargill GelTM 03420) was provided by Cargill Inc. (Minneapolis, MN, U.S.A.). D-Glucose

Assay Kit, potato amylose standard, and isoamylase were purchased from Megazyme International

Ltd. (Co. Wicklow, Ireland). Maltogenic α-amylase (MGA) from Bacillus stearothermophilus.,

maize amylopectin standard, pancreatin from porcine pancreas, α-amylase, amyloglucosidase and

deuterated trifluoroacetic acid were procured from Sigma-Aldrich Canada Co. (Oakville, ON,

Canada). Deuterated dimethyl sulfoxide was purchased from Cambridge Isotope Laboratories Inc.

(Tewksbury, MA, U.S.A.). All the other chemicals used in this study were of reagent grade or

higher purity, and they were purchased from Fisher Scientific Company (Ottawa, ON, Canada) or

Sigma-Aldrich Canada Co. (Oakville, ON, Canada).

4.3.2 Modification of starch using MGA

Each of the WMS, NMS and HAMS (20.0 g, dry basis, db) was slurried in 50.0 mM sodium

acetate buffer (pH 5.5, containing 0.02% sodium azide) to reach 100.0 g total weight. MGA was

added to the starch slurry at a level of 1% (w/w, db; equivalent to 110 MANU per g of dry starch),

and the sample bottle was placed horizontally in a 50 °C water bath with shaking of 150 rpm for

the enzymatic hydrolysis to occur. The enzymatic modification was carried out for 4, 8 and 24 h.

At the end of the modification step, four volumes of anhydrous ethanol were added to the mixture,

followed by vigorous manual shaking to terminate the enzymatic reaction. The modified starch

was recovered by centrifugation at 5,000 g for 10 min, which was then washed with 200 mL

anhydrous ethanol twice. The recovered starch was dried at 40 °C overnight.

To determine the percentages of starch hydrolysis by MGA at 4, 8 and 24 h, the total mass

of released soluble sugars was quantitated (Mendez-Montealvo et al., 2011). After the addition of

four volumes of anhydrous ethanol to stop the enzymatic hydrolysis, a 1.0-mL aliquot of the starch

suspension was transferred into a 1.5-mL microcentrifuge tube, which was centrifuged at 3,000 g

for 10 min. The concentration of soluble sugars in the supernatant was measured using a phenol-

sulfuric acid method (Dubois et al., 1956). The total mass of released soluble sugars was used to

57

calculate the degree of starch hydrolysis: %Degree of starch hydrolysis = [Total mass of released

soluble sugars × (162 / 180)] / (Initial dry mass of starch) ×100.

The corresponding native starches that underwent the same treatment for 24 h without the

addition of MGA were used as the controls for the subsequent characterization tests.

4.3.3 Amylose content of starch

Amylose contents of the control and MGA-modified starches were determined using an

iodine colorimetric method (Chrastil, 1987). Five different ratios of commercial potato amylose

and maize amylopectin (0.0%, 25.0%, 50.0%, 75.0%, 100.0%) were used as the standards to draw

a standard curve for the quantification.


Granular morphology of the control and MGA-24 h-modified starches was viewed using a

field-emission scanning electron microscope (SEM, SU810, Hitachi High Technologies Canada

Inc., Rexdale, ON, Canada) (Li et al., 2019). The starch sample was coated with gold using a

sputtering method and then observed under the conditions of 3.0 kV and 10 μA. Representative

images were taken at 500×, 1,500× and 3,000× magnifications.

4.3.5 WAXD of starch

WAXD patterns of composite control and MGA-24 h-modified starches were determined

using a Rigaku X-ray diffractometer (Model Ultima IV, Rigaku, The Woodlands, TX, U.S.A.).

For each treatment, 1.0 g starch was taken from each of the three independent batches of

modification and then homogeneously blended to prepare the composite sample. The composite

starch sample was equilibrated at 25 °C with 100% relative humidity for 24 h before the

measurement. The diffractometer was operated at 40 kV and 44 mA with Cu Kα radiation

(λ = 1.5406 Å) and the scanning diffraction angle (2θ) ranged from 3° to 40° at a rate of 1.3°/min

at ambient temperature. The relative crystallinity of the starch was calculated using Origin

Software (OriginLab Corporation, Northampton, MA, U.S.A.) according to a previous method

(Hayakawa et al., 1997).

58


Molecular-weight distributions of the control and MGA-24 h-modified starches were

analyzed after debranching to examine the effects of MGA hydrolysis on the molecular structures

of amylose and amylopectin. The starch sample (20.0 mg) was dispersed in 2.0 mL of 90%

dimethyl sulfoxide (DMSO) and boiled in a water bath for 20 min with mild magnetic stirring.

After cooling to room temperature, five volumes of anhydrous ethanol were added to the sample

to precipitate the starch. Upon centrifugation at 3,000 g for 15 min, the supernatant was poured

out. The tube was inverted and dried on a clean paper towel for 10 min to remove almost all the

ethanol. Deionized water of 1.75 mL was then added to disperse the pellet in a boiling water bath

for 10 min with gentle stirring. After cooling to room temperature, 250 μL of sodium acetate buffer

(160 mM, pH 4.75, containing 0.16 % sodium azide) and 2 μL of isoamylase (500 U/mL) were

added to the sample. The mixture was incubated at 37 °C for 24 h to completely debranch the

starch (Li et al., 2008). After the debranching reaction, the mixture was boiled for 10 min and then

cooled to room temperature, which was followed by the addition of 20 volumes of anhydrous

ethanol to precipitate the fully debranched starch chains. Upon centrifugation at 5,000 g for 15

min, the supernatant was carefully decanted, and the pellet was re-dispersed in 4.0 mL of DMSO

containing 0.5% lithium bromide and then boiled in a water bath for 10 min with mild magnetic

stirring. After cooling to room temperature, the starch dispersion was passed through a disc filter

with a 5 μm filtration level. A 20-μL aliquot of the prepared sample was injected into a high-

performance size exclusion chromatography (HPSEC) system (1260 Infinity II, Agilent

Technologies Canada, Mississauga, ON, Canada) consisting of an isocratic pump, a vialsampler,

and a refractive index (RI) detector (Peng & Yao, 2018). Two Zorbax gel PSM 60-S columns

(Agilent Technologies Canada, Mississauga, ON, Canada) were used jointly to separate amylose

and amylopectin branch chains of different molecular weights for subsequent quantification by the

RI detector. The analytical columns were kept at 30 °C by using the equipped Multicolumn

Thermostat component, and the RI detector was set at 35 °C. The mobile phase was DMSO with

0.5% (w/w) lithium bromide at a flow rate of 0.5 mL/min. Calibration of the system was completed

using glucose, maltotriose, maltohexaose and three pullulan standards (6.2 × 103, 48.8 × 103 and

348 × 103 g/mol). The collected raw data were normalized on the basis of the total area under the

curve and then used for the construction of HPSEC profiles.

59


Percentages of α-1,6 branch linkages of the control and MGA-24 h-modified starches were

determined using proton nuclear magnetic resonance (1H NMR) on a Bruker AVIII HD 600 NMR

Spectrometer following the method of Tizzotti et al. (2011) with modifications. A quantity of

starch sample (about 10 mg) was weighed into a 1.5-mL microcentrifuge tube, followed by the

addition of 1.0 mL of deuterated dimethyl sulfoxide (DMSO-d6). The sample was incubated in an

80 °C water bath with shaking of 300 rpm for 1 h. After the incubation, the sample was cooled to

room temperature and 5.66 μL of deuterated trifluoroacetic acid (TFA-d1) was added immediately

before the NMR measurement. Approximately 0.5 mL of the prepared sample was transferred into

a 5-mm NMR tube. The 1H NMR spectrum was obtained at 70 °C and Larmor frequency of 600.17

MHz with 300 scans: 6.25 μs 30° pulse; and a repetition time of 6.6 s composed of an acquisition

time of 5.45 s and a relaxation delay of 1.15 s. The percentage of α-1,6 branch linkages of the

starch sample was calculated using the following equation:

DB% = I(4.80)

I(5.10)+I(4.80)

Where DB% is the degree of branching; I(4.80) is the integrated intensity of peak at 4.80

ppm; I(5.10) is the integrated intensity of peak at 5.10 ppm. Peaks at 5.10 ppm and 4.80 ppm were

assigned to α-1,4 and α-1,6 glycosidic bonds, respectively (Gidley, 1985).


A differential scanning calorimeter (DSC 8000, Perkin Elmer, Waltham, MA, U.S.A.) was

used to determine the thermal properties of the control and MGA-24 h-modified starches according

to the method of Song and Jane (2000). The starch sample (approximately 10 mg) was accurately

weighed and thoroughly mixed with distilled water (three volumes) in a stainless-steel sample pan.

After being hermetically sealed, the sample was equilibrated at room temperature for at least 2 h.

The scanning of the hydrated starch was performed from 10 to 160 °C at a rate of 10 °C/min. After

the gelatinization step, the starch was stored at 4 °C for 7 days and scanned again following the

same procedure. The thermal transition peaks of starch gelatinization (in the initial scan) and

melting of retrograded starch (in the second scan after the 7-day storage) in the DSC thermograms

were analyzed using Pyris Manager Software (Perkin Elmer, Waltham, MA, U.S.A.). Onset

temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy change (ΔH)

60

were reported. Percentage of starch retrogradation was calculated as: % retrogradation (%R) =

(ΔH of melting of retrograded starch / ΔH of starch gelatinization) × 100%.


Pasting properties of the control and MGA-modified starches were measured using Rapid

Visco-Analyzer (RVA Super 3, Newport Scientific, Sydney, Australia) following the method of

Li et al. (2019). The starch sample was suspended in distilled water at a concentration of 8% (db)

to prepare a starch slurry of 28.0 g total weight, which was loaded to the RVA instrument and run

using the Standard Method 2 in the Thermocline Software.


In vitro digestibility of the control and MGA-24 h-modified starches after cooking was

determined using the Englyst Method (Englyst et al., 1992) with minor modifications (Li et al.,

2019). The starch sample (containing 600 mg starch, dry starch basis, dsb) was suspended in 15.0

mL distilled water. The starch suspension was cooked in a boiling water bath for 10 min with

vigorous magnetic stirring. After cooling to ambient temperature, 5.0 mL sodium acetate buffer

(0.4 M, pH 5.2, containing 0.08% sodium azide) was added into the cooked sample. Upon the

equilibration at 37 °C with shaking (160 rpm) for at least 15 min, a freshly prepared enzyme

solution (5.0 mL) containing porcine pancreatin extract and amyloglucosidase was added into the

mixture to initiate the amylolysis. D-glucose Assay Kit was used to quantitate the amounts of

glucose released from the enzymatic hydrolysis of starch at time intervals of 20 and 120 min.

Rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) contents

were calculated in accordance with the method reported by Englyst et al. (1992).

4.3.11 Thermal properties of RS residue

To better elucidate the mechanisms responsible for the different effects of MGA treatment

on the RS contents of the maize starches after cooking (data reported in Section 4.4.9), RS residue

of the samples immediately after digestion for 120 min as described in Section 4.3.10 was collected

and further analyzed for thermal properties. At the end of the 120-min amylolysis, the RS residue

was precipitated by adding five volumes (125 mL) of anhydrous ethanol, followed by vigorous

manual shaking to stop the enzymatic reaction. The mixture was then centrifuged at 4,000 g for 15

61

min, and the supernatant was carefully decanted. This anhydrous ethanol washing step was

repeated two more times. After drying at 40 °C overnight in a convection oven, the recovered RS

residue was ground into a fine powder using a mortar and pestle, which was then subjected to the

same DSC scanning as indicated in Section 4.3.8.

4.3.12 Statistical analysis

The enzymatic modification of WMS, NMS and HAMS by MGA was conducted in three

independent batches to prepare control, 4-, 8- and 24-h modified samples. The characterization

experiments, except for WAXD (using composite samples only as described above; n = 1), were

carried out in one replicate for each of the control and MGA-modified starches (n = 3). Statistical

differences among the control and modified maize starches were evaluated using one-way

ANOVA test with Tukey’s adjustment at a significance level of 0.05 on IBM SPSS Statistics

Analysis (Version 24.0), IBM Corp., Armonk, NY, U.S.A.).

4.4 Results and discussion

4.4.1 Percentage of starch hydrolysis by MGA

Fig. 4.1 shows the hydrolysis profiles of granular WMS, NMS and HAMS by MGA for a

total period of 24 h. Overall, the three starches displayed hydrolysis rates in a descending order of

WMS > NMS > HAMS. Upon 4-h hydrolysis, the degrees of hydrolysis of WMS, NMS and

HAMS reached 22.5%, 19.8% and 4.8%, respectively; the values progressively increased to 39.7%,

35.9% and 11.2% at 24 h. The observed different hydrolysis rates of the starches are in good

accordance with the data reported for the hydrolysis of granular starches by other α-amylases (Ai,

Nelson, et al., 2013; Li et al., 2019), which could be primarily attributed to the difference in their

amylose contents (data presented in Section 4.4.2): enzymatic resistance of granular starch tends

to increase with a larger amylose content (Jane et al., 2003). In addition, the substantially greater

enzymatic resistance of HAMS could be ascribed to the B-type WAXD pattern (versus A-type of

the other two maize starches; confirmed in Section 4.4.4) as well as the presence of more amylose-

lipid complexes in this high-amylose starch (Ai, Hasjim, et al., 2013; Jane et al., 1997).

62

Fig. 4.1 Degrees of starch hydrolysis after incubating WMS, NMS and HAMS with maltogenic

α-amylase (MGA) for 4, 8 and 24 h. At the same modification time, the numbers with different

letters are significantly different at p < 0.05.

c

c

c

b

b

b

a

a

a

0

10

20

30

40

0 4 8 12 16 20 24

Deg

ree

of

hyd

roly

sis

(%)

Hydrolysis time (h)

WMS NMS HAMS

63

4.4.2 Apparent amylose content of starch

The apparent amylose contents of control and MGA-modified starches are presented in

Table 4.1. The waxy, normal and high-amylose nature of the starches was confirmed by the

apparent amylose contents of the controls: 3.3%, 29.9% and 58.6%, respectively. Interestingly, the

extensive hydrolysis by MGA did not appear to significantly alter the apparent amylose contents

of the starches, consistent with the hydrolysis of granular starches by porcine pancreatic α-amylase

(Tawil et al., 2012; Zhang et al., 2006). The data suggested that MGA, like porcine pancreatic α-

amylase, simultaneously hydrolyzed the amylose and amylopectin in the starch granules (Zhang

et al., 2006).


As shown in Fig. 4.2, the control WMS and NMS had granules of polygonal, spherical and

irregular shapes with some indentations on the surface. The majority of control HAMS granules,

despite having relatively smaller sizes, exhibited morphology similar to that of the other two maize

starches. A notable feature of the control HAMS was the existence of elongated starch granules

(indicated by chevrons), which resulted from the fusion of multiple small starch granules into one

granule in the amyloplast during starch biosynthesis (Jiang, Horner, et al., 2010). The described

granular morphologies of the WMS, NMS and HAMS controls are in good agreement with

previous observations (Jiang, Horner, et al., 2010; Li et al., 2019).

The modification by MGA for 24 h led to the formation of numerous holes into the interior

of WMS and NMS granules (indicated by up arrows), which resembled the morphological features

of granular WMS and NMS hydrolyzed by other α-amylases (Karim et al., 2008; Zhang et al.,

2006). It is a known fact that the native WMS and NMS granules possess endogenous pores and

channels (Huber & BeMiller, 1997; Li et al., 2019), and the openings were likely further enlarged

under 50 °C incubation (i.e., the temperature used for MGA modification in the present study).

MGA probably initiated the hydrolysis from these discrete structures, formed tunnels into the

granule interior, and then hydrolyzed the granules following the “inside-out” pattern to generate

those large holes in WMS and NMS granules (MacGregor & Ballance, 1980; Zhang et al., 2006).

In contrast, HAMS granules, with a remarkably larger amylose content (58.6%; Table 4.1) and the

B-type polymorphic structure (confirmed in Section 4.4.4), do not have the same endogenous pores

and channels as the other two maize starches (Glaring et al., 2006; Jiang, Horner, et al., 2010).

64

Thus, MGA was only able to hydrolyze HAMS through “surface pitting”, leading to the formation

of small holes (indicated by triangles) and fissures (indicated by right arrows) in some granules

(Gallant et al., 1992). The extents of hydrolysis by MGA as illustrated in the SEM images

correspond well with the different degrees of hydrolysis of the three starches at 24 h (Fig. 4.1).

4.4.4 WAXD and relative crystallinity of starch

The control WMS and NMS displayed the typical A-type WAXD pattern, whereas the

control HAMS had the typical B-type pattern (Fig. 4.3), consistent with previous literature (Jiang,

Campbell, et al., 2010; Li et al., 2019). MGA treatment for 24 h did not alter the WAXD patterns

of the starches. A slight decrease in the relative crystallinity was observed for WMS hydrolyzed

by MGA for 24 h, suggesting that the extensive hydrolysis (39.7% in Fig. 4.1) resulted in the loss

of some crystalline structure. The effect of 24-h MGA treatment on the relative crystallinity of

NMS was negligible. By contrast, a considerable increase in that of HAMS was observed after the

24-h treatment, suggesting that the limited hydrolysis through surface erosion by MGA (11.2% in

Fig. 4.1) preferably removed the molecules present in the amorphous phase of the granules

(Gallant et al., 1992).

Table 4.1 Apparent amylose contents of control and MGA-modified maize starches.1, 2


WMS3 NMS HAMS

Control 3.3 ± 0.1a 29.9 ± 1.4a 58.6 ± 0.8a

4 h 3.6 ± 0.1a 30.4 ± 0.7a 60.7 ± 1.8a

8 h 3.5 ± 0.2a 29.1 ± 1.6a 60.6 ± 0.7a

24 h 3.3 ± 0.2a 27.9 ± 0.5a 58.5 ± 0.3a



2 Determined using an iodine colorimetric method (Chrastil, 1987).

3 WMS: waxy maize starch; NMS: normal maize starch; HAMS: high-amylose maize starch; 4 h, 8 h and 24 h: 4-, 8- and 24-h modified

starch.

65

66

WMS-C (500×) WMS-C (1,500×) WMS-C (3,000×)

WMS-24 h (1,500×) WMS-24 h (3,000×) WMS-24 h (500×)

NMS-C (500×) NMS-C (1,500×) NMS-C (3,000×)

NMS-24 h (3,000×) NMS-24 h (1,500×) NMS-24 h (500×)

HAMS-C (500×) HAMS-C (3,000×) HAMS-C (1,500×)

67


starches. WMS-C: waxy maize control; WMS-24 h: waxy maize 24-h modified; NMS-C: normal

maize control; NMS-24 h: normal maize 24-h modified; HAMS-C: high-amylose maize control;

HAMS-24 h: high-amylose maize 24-h modified. The magnification at which the image was taken

is given in parentheses. Up arrows mark large holes in waxy and normal maize starches caused by

MGA hydrolysis through “inside-out” pattern; chevrons mark elongated starch granules in high-

amylose maize starch; and triangles mark small holes and right arrows mark fissures caused by

MGA hydrolysis of high-amylose maize starch through “surface pitting”.

HAMS-24 h (500×) HAMS-24 h (3,000×) HAMS-24 h (3,000×)

68

Fig. 4.3 X-ray diffraction patterns of control and MGA-24 h-modified maize starches. WMS-C:

waxy maize control; WMS-24 h: waxy maize 24-h modified; NMS-C: normal maize control;

NMS-24 h: normal maize 24-h modified; HAMS-C: high-amylose maize control; HAMS-24 h:

high-amylose maize 24-h modified. Relative crystallinity is given in parentheses.

3 6 9 12 15 18 21 24 27 30

Inte

nsity

2 (°)

WMS-C (38.3%)

WMS-24 h (36.1%)

NMS-C (28.4%)

NMS-24 h (28.8%)

HAMS-C (18.0%)

HAMS-24 h (21.1%)

69


Based on the study of Peng and Yao (2018), the normalized HPSEC profiles of debranched

control starches were divided into four areas (from short to long elution time): Area 1 [peak value

= degree of polymerization (DP) 1,051 for NMS and DP 988 for HAMS) corresponded to amylose,

and Area 2 (DP 22-251), 3 (DP 11-22) and 4 (DP 6-11) corresponded to long, intermediate and

short branch chains (LBC, IBC and SBC) of amylopectin, respectively (Fig. 4.4 A). The

percentages of amylose in starch and the proportions of LBC, IBC and SBC in amylopectin of the

debranched WMS, NMS and HAMS are summarized in Table 4.2. The percentages of amylose of

the control WMS, NMS and HAMS as determined using the HPSEC method were 1.8%, 26.2%

and 51.9%, respectively, consistently lower than the values measured by the iodine colorimetric

method (3.3%, 29.9% and 58.6%, respectively; Table 4.1). The differences between these two

methods could be attributed to that some of the LBC of amylopectin were able to bind with

polyiodide ions to inflate the amylose contents in the iodine colorimetry (Jane et al., 1999). The

amylopectin molecules of control WMS and NMS had comparable branch-chain-length (BCL)

distributions: ~26.3% LBC, ~33.7% IBC, and ~40.0% SBC, whereas those of the control HAMS

showed a significantly larger proportion of LBC (56.0%) and smaller proportions of IBC and SBC

(26.1% and 17.9%, respectively). The observed differences in the amylopectin BCL distributions

among the three control starches are in good agreement with previous research (Jane et al., 1999).

Among the three starches, WMS had the highest percentage of SBC and the lowest percentage of

LBC in amylopectin, which might explain the smallest inflation in the apparent amylose content

of WMS (1.7%) when compared with those of NMS and HAMS (3.7% and 6.7%, respectively).

In contrast, because of the largest proportion of LBC (56.0%), the most significant difference in

the amylose contents as measured using the two abovementioned methods was found with HAMS

(6.7%; Tables 4.1 and 4.2).

After the 24-h treatment with MGA, the amylose and amylopectin LBC peaks of the maize

starches were shifted toward longer elution times (i.e., smaller DP): amylose peak was diminished

from DP 1,051 to DP 603 for NMS and from DP 988 to DP 482 for HAMS, respectively; and LBC

peak area was shifted from DP 22-251 to DP 22-101 for all the three starches (Fig. 4.4 and Table

4.2). Meanwhile, the DP ranges of the IBC and SBC largely remained unchanged after the 24-h

enzyme treatment. The comparison between the HPSEC curves of the control and MGA-modified

starches clearly illustrated that the enzymatic hydrolysis primarily degraded amylose and LBC of

70

amylopectin. Based on the new lines applied to divide the four areas in Fig. 4.4 B, the percentage

of amylose of 24-h modified WMS slightly increased (Table 4.2), which was an artifact caused by

the shifting of the dividing line between amylose and amylopectin branch chains toward a longer

elution time. The 24-h treatment with MGA did not substantially change the percentage of amylose

of NMS (Table 4.2), corresponding well with the results presented in Table 4.1. The modification

noticeably reduced the percentage of amylose of HAMS (Table 4.2), which is in good accordance

with the increase in the relative crystallinity of this starch as demonstrated in Fig. 4.3. The surface

pitting by MGA in HAMS (Fig. 4.2) preferably broke down amylose existing in the amorphous

phase of its granules as explained above.

Furthermore, the modification by MGA significantly reduced the percentages of LBC and

elevated the percentage of SBC of WMS and NMS and marginally changed the proportions of IBC

of both starches. The data are in good agreement with previous findings that MGA hydrolysis

resulted in a higher proportion of SBC of amylopectin molecules (Ao, Simsek, et al., 2007; Grewal

et al., 2015). The enzymatic modification also significantly reduced the percentage of LBC of

HAMS but only showed a slight increasing effect on the percentage of SBC.


WMS, NMS and HAMS controls consisted of 4.4%, 3.0% and 1.0% α-1,6 branch linkages

(Table 4.3), respectively, which are in good accordance with the results reported by Tizzotti et al.

(2011) and correspond well with the differences in their amylose contents (Tables 4.1 and 4.2) as

α-1,6 linkages are essentially found in highly branched amylopectin molecules. After 24-h MGA

treatment, the percentages of α-1,6 linkages of WMS, NMS and HAMS increased by 0.8%, 0.7%

and 0.1%, respectively, corresponding well with their degrees of hydrolysis as reported in Fig. 4.1.

As one type of α-amylase, MGA can only cleave α-1,4 glycosidic bonds and release maltose as

the primary product (Christophersen et al., 1998), thus proportionally increasing the percentage of

α-1,6 linkages.

71


control (A) and MGA-24 h-modified (B) maize starches. WMS-C: waxy maize control; WMS-24

h: waxy maize 24-h modified; NMS-C: normal maize control; NMS-24 h: normal maize 24-h

modified; HAMS-C: high-amylose maize control; HAMS-24 h: high-amylose maize 24-h

modified. The solid line separates amylose (AM) and amylopectin branch chains; and the dash

lines separate the long (LBC; corresponding to DP 22-251 in A and to DP 22-101 in B),

intermediate (IBC; corresponding to DP 11-22 in both A and B), and short (SBC; corresponding

to DP 6-11 in both A and B) branch chains of amylopectin.

9 10 11 12 13 14 15 16 17 18 19 9 10 11 12 13 14 15 16 17 18 19

RI

sig

na

l

Elution time (min)

AM LBC IBC SBC

10

00

10

05

0

20

10

8 3

Degree of polymerization (DP)

WMS-C

NMS-C

HAMS-C

WMS-24 h

NMS-24 h

HAMS-24 h

A

Elution time (min)

B IBCAM LBC SBC

10

00

10

05

0

20

10

8 3

Table 4.2 Molecular-weight distributions of debranched control and MGA-24 h-modified maize starches.1

Starch2 Amylose (%) Amylopectin branch-chain-length distribution (%)3

Long branch chain Intermediate branch chain Short branch chain

WMS, control 1.8 ± 0.3a 25.8 ± 0.0bc 33.9 ± 0.3de 40.3 ± 0.2b

WMS, 24 h 4.7 ± 0.2a 21.5 ± 0.1a 34.4 ± 0.2e 44.1 ± 0.2c

NMS, control 26.2 ± 0.1b 26.7 ± 0.0c 33.5 ± 0.1d 39.8 ± 0.1b

NMS, 24 h 27.7 ± 1.0b 24.3 ± 1.3b 32.0 ± 0.4c 43.7 ± 0.9c

HAMS, control 51.9 ± 0.4d 56.0 ± 0.2e 26.1 ± 0.1a 17.9 ± 0.2a

HAMS, 24 h 44.1 ± 4.2c 53.6 ± 0.1d 27.5 ± 0.6b 18.9 ± 0.5a



2 WMS: waxy maize starch; NMS: normal maize starch; HAMS: high-amylose maize starch.

3 Percentages of long, intermediate and short branch chains were calculated as the respective fractions divided by the total area of

amylopectin as shown in Fig. 4.4.

72

Table 4.3 Percentages of α-1,6 branch linkages of control and MGA-24 h-modified maize starches.1

Starch2 α-1,6 linkage (%)3

WMS, control 4.4 ± 0.1d

WMS, 24 h 5.2 ± 0.0e

NMS, control 3.0 ± 0.1b

NMS, 24 h 3.7 ± 0.0c

HAMS, control 1.0 ± 0.1a

HAMS, 24 h 1.1 ± 0.1a

1 Values are presented as average ± standard deviation (n = 3); in the same column, the numbers with different letters are significantly



3 %Degree of branching = (Integrated intensity of peak at 4.80 ppm) / (Integrated intensity of peak at 5.10 ppm + Integrated intensity of

peak at 4.80 ppm) × 100.

73

74


Control WMS and NMS exhibited similar starch gelatinization temperatures (ranging from

To of ~70.2 °C to Tc of ~83.0 °C; Table 4.4), which were noticeably lower than those of HAMS

(ranging from To of 73.3 °C to Tc of 104.6 °C). The gelatinization ΔH of WMS, NMS and HAMS

were 17.4, 16.6 and 14.0 J/g, generally consistent with their different amylose contents (Table 4.1

and 4.2) as amylopectin is the main constituent contributing to the formation of double helices in

starch (Ai & Jane, 2015). The noted differences in the gelatinization properties of the three control

starches are in good consistency with the findings reported in previous studies (Ai & Jane, 2015;

Kibar et al., 2010).

The 24-h treatment with MGA elevated the gelatinization temperatures of WMS and NMS

and lowered their gelatinization ΔH, suggesting that the extensive enzymatic hydrolysis removed

double helices with poorer thermal stability. By contrast, the MGA modification increased

gelatinization To, lowered Tc, and marginally increased gelatinization ΔH of HAMS. The opposite

trend of change in the gelatinization ΔH of HAMS could be attributed to the considerably lower

percentage of starch hydrolysis (Fig. 4.1) and the occurrence of such enzymatic reaction primarily

to amylose in HAMS (Sections 4.4.4 and 4.4.5).

Percentages of starch retrogradation were in an ascending order of WMS (7.5%) < NMS

(34.8%) < HAMS (70.6%) (Table 4.4), which agrees well with previous observation of different

retrogradation rates of waxy, normal and high-amylose maize and barley starches (Singh et al.,

2006; Song & Jane, 2000). The 24-h modification by MGA substantially lowered the percentages

of retrogradation of WMS and NMS, which could be explained by that the enzymatic modification

shortened the branch chains of amylopectins (Fig. 4.4 and Table 4.2) and increased the percentages

of α-1,6 linkages of WMS and NMS (Table 4.3). The results are in good accordance with the data

reported before (Ao, Simsek, et al., 2007). On the contrary, the 24-h enzyme treatment noticeably

enhanced the percentage of retrogradation of HAMS from 70.6% to 96.9%. The opposite trend on

HAMS retrogradation could be explained by that the limited enzymatic hydrolysis shortened the

amylose molecules (Fig. 4.4 and Section 4.4.5) to improve the mobility, which thus allowed the

amylose molecules to partly recrystallize with each other or to recrystallize with long branch chains

of amylopectin during the 7-day storage of fully gelatinized HAMS at 4 °C. This explanation was

supported by the observation that the retrograded modified HAMS showed considerably higher

75

melting Tp (96.3 versus 90.4 °C) and Tc (111.9 versus 106.5 °C) when compared with the

retrograded control.


MGA treatment was very effective to diminish the pasting properties of WMS and NMS

(Fig. 4.5). Upon 4-h MGA hydrolysis, the peak viscosities of WMS and NMS decreased from

2,835 to 229 cP and from 1,666 to 168 cP, respectively. Extended hydrolysis for up to 24 h further

reduced the peak viscosities of both starches. In addition, modified starches from both types

displayed little viscosity after the pasting peak. The marked decreases in the pasting viscosities of

enzyme-treated WMS and NMS could be related to the breakdown of the two starches at both

granular (Fig. 4.2) and molecular (Fig. 4.4 and Table 4.2) levels. Moreover, because of the

substantially low pasting viscosities, the MGA-modified WMS and NMS could be easily dispersed

after cooking. Therefore, the enzyme-treated WMS and NMS had considerably poorer molecular

proximity than their respective controls upon gelatinization, which could be another factor

responsible for the considerably slower retrogradation of 24-h modified WMS and NMS as shown

in Table 4.4 (Jane et al., 1999). All the control and modified HAMS did not exhibit any detectable

viscosity by RVA, resulting from their high Tc (~103.5 °C) as shown in Table 4.4. The HAMS

samples were only partially gelatinized and swelled to a very low extent under the applied pasting

conditions (heating temperature = 95 °C) (Ai, Hasjim, et al., 2013; Liu et al., 2019).

Table 4.4 Gelatinization and retrogradation properties of control and MGA-24 h-modified maize starches.1

Starch2 Gelatinization of starch3 Melting of retrograded starch3 % Retrogradation

(%R)4

To (°C) Tp (°C) Tc (°C) ∆H (J/g) To (°C) Tp (°C) Tc (°C) ∆H (J/g)

WMS, control 69.5 ± 0.3a 76.5 ± 0.1a 84.0 ± 0.2c 17.4 ± 0.4b 42.0 ± 1.1a 56.3 ± 0.3b 67.2 ± 0.3c 1.3 ± 0.3b 7.5 ± 1.8a

WMS, 24 h 73.2 ± 0.0d 78.4 ± 0.2a 85.7 ± 0.2d 13.5 ± 0.4a 43.5 ± 0.6a 45.9 ± 0.3a 49.2 ± 0.7a 0.2 ± 0.0a 1.4 ± 0.2a

NMS, control 70.9 ± 0.2b 75.8 ± 0.3a 81.9 ± 0.3a 16.6 ± 0.3b 42.1 ± 0.1a 56.4 ± 0.0b 66.7 ± 0.4c 5.8 ± 0.2c 34.8 ± 2.0b

NMS, 24 h 72.5 ± 0.3c 77.4 ± 0.1a 83.1 ± 0.2b 14.4 ± 0.5a 53.9 ± 1.1b 56.6 ± 0.3b 61.3 ± 0.0b 0.2 ± 0.0a 1.5 ± 0.0a

HAMS, control 73.3 ± 0.2d 88.2 ± 2.5b 104.6 ± 1.2f 14.0 ± 1.2a 54.0 ± 0.4b 90.4 ± 0.6c 106.5 ± 3.4d 9.8 ± 0.2d 70.6 ± 7.2c

HAMS, 24 h 75.7 ± 0.2e 89.3 ± 0.6b 102.3 ± 0.7e 14.6 ± 0.4a 54.1 ± 2.0b 96.3 ± 0.4d 111.9 ± 2.3e 14.2 ± 0.4e 96.9 ± 4.8d

1 Values are presented as average ± standard deviation (n = 3); in the same column, the numbers with different letters are significantly different at p < 0.05.



4 % Retrogradation (%R) = (∆H of melting of retrograded starch / ∆H of starch gelatinization) ×100%.

76

Fig. 4.5 Pasting properties of control and MGA-modified maize starches analyzed using a Rapid Visco-Analyzer with 28.0 g starch suspension

containing 8% (w/w, db) starch. A: waxy maize; B: normal maize; C: high-amylose maize; 4 h, 8 h and 24 h; 4-, 8- and 24-h modified starch.

77

78


The cooked control WMS and NMS displayed comparable digestibility: 91.7% and 90.5%

RDS, 0.6% and 2.4% SDS, and 4.5% and 2.6% RS, respectively (Table 4.5). Compared with these

two starches, the digestibility of cooked control HAMS was noticeably lower, which possessed

58.2% RDS, 4.7% SDS and 32.4% RS. The markedly greater enzymatic resistance of control

HAMS after cooking was a result of its high gelatinization Tc (Table 4.4). Upon boiling in water,

the starch was only partially gelatinized and swelled to a limited level (Fig. 4.5), thus displaying

the lowest digestibility among all the three control starches (Ai, Hasjim, et al., 2013; Liu et al.,

2020). The 24-h modification by MGA did not obviously alter the digestibility of WMS; however,

the modification significantly decreased the RDS content of NMS from 90.5% to 85.6% and

elevated the RS content from 2.6% to 7.3%, indicating that the enzyme treatment improved the

enzymatic resistance of cooked NMS.

Previous studies attributed the enhancement in the enzymatic resistance of soluble normal

maize starch by MGA treatment to the increase in the percentage of α-1,6 linkages (Ao, Simsek,

et al., 2007). However, in the present study, the increase in the percentage of α-1,6 linkages did

not appear to play such an important role to increase the enzymatic resistance of the cooked MGA-

treated starches because the enzymatic treatment markedly increased the percentage of α-1,6

linkages in WMS from 4.4% to 5.2% (Table 4.3) but did not significantly change the RDS, SDS

and RS contents of the cooked WMS (Table 4.5). Thus, to reveal the mechanism(s) responsible

for the enhanced enzymatic resistance of MGA-treated NMS, RS residue samples of the control

and MGA-24 h-modified maize starches were collected and scanned by DSC as indicated in Study

1 of the thesis. The RS residue from MGA-24 h-treated NMS after cooking and amylolysis in the

Englyst Assay showed a considerably larger melting peak (ΔH = 2.4 J/g) of retrograded amylose

of 116.2-138.3°C when compared with the counterpart from NMS control (ΔH = 0.8 J/g and

meting temperatures of 122.1-136.6°C) (Table 4.6). The structure of retrograded amylose is known

to be enzymatic resistant (Sievert & Pomeranz, 1989). The formation of more amylose double-

helical crystallites in the MGA-24 h-modified NMS could be explained by that the 24-h MGA

treatment shortened the amylose chains (Fig. 4.4) for enhanced molecular mobility, which was

favorable for the development of such amylose double-helical crystallites during the incubation of

cooked starch at 37 °C in the amylolysis step in the Englyst Method (Buléon et al., 2007; Li & Hu,

2020; Ring et al., 1988). However, only remaining amylopectin double-helical crystallites were

79

found in the control and MGA-24 h-modified WMS, with no amylose double-helical crystallites

due to the absence of this linear component, which explained why the MGA treatment did not

improve the enzymatic resistance of WMS after cooking.

The 24-h MGA treatment showed negligible effect on the in vitro digestibility of cooked

HAMS, which could be related to the following two factors: (1) The MGA treatment was only

able to modify the granular HAMS to a very low extent (11.2% hydrolysis at 24 h; Fig. 4.1); (2)

The MGA-modified HAMS was only partially gelatinized and swelled to a limited extent upon

cooking (Fig. 4.5), and thus the amylose did not have good mobility to retrograde to further

increase the enzymatic resistance of cooked HAMS as described above for cooked NMS.

Table 4.5 In vitro digestibility of cooked control and MGA-24 h-modified maize starches.1,2

Starch3 RDS (%)4 SDS (%) RS (%)

WMS, control 91.7 ± 1.7c 0.6 ± 0.7a 4.5 ± 1.3ab

WMS, 24 h 89.6 ± 1.8c 2.4 ± 0.2ab 2.8 ± 1.6a

NMS, control 90.5 ± 1.3c 2.4 ± 2.2ab 2.6 ± 2.0a

NMS, 24 h 85.6 ± 1.6b 2.5 ± 2.1ab 7.3 ± 0.8b

HAMS, control 58.2 ± 1.0a 4.7 ± 1.4b 32.4 ± 1.4c

HAMS, 24 h 59.2 ± 0.8a 2.8 ± 0.6ab 33.4 ± 1.1c



2 Starch samples were cooked in a boiling water bath for 10 min.


4 RDS: rapidly digestible starch; SDS: slowly digestible starch; RS: resistant starch; values were calculated on a dry starch basis

80

Table 4.6 Thermal properties of RS residue of control and MGA-24 h-modified starches after cooking and amylolysis in the Englyst

Method.1

RS residue2 Melting of amylopectin double-helical crystallites3 Melting of amylose double-helical crystallites3

To (°C) Tp (°C) Tc (°C) ΔH (J/g) To (°C) Tp (°C) Tc (°C) ΔH (J/g)

WMS, control 75.6 ± 1.7a 81.4 ± 0.6ab 86.3 ± 2.0a 2.1 ± 0.3ab N.D.4 N.D. N.D. N.D.

WMS, 24 h 75.5 ± 1.9a 79.9 ± 0.6a 84.6 ±1.2a 1.2 ± 0.3a N.D. N.D. N.D. N.D.

NMS, control 74.6 ± 1.1a 82.4 ± 1.1b 89.3 ± 2.2a 3.6 ± 0.8c 122.1 ± 1.1a 129.0 ± 2.4a 136.6 ± 2.1a 0.8 ± 0.1a

NMS, 24 h 74.1 ± 0.5a 80.8 ± 0.3ab 90.4 ± 5.8a 2.9 ± 0.5bc 116.2 ± 5.0a 129.6 ± 2.4a 138.3 ± 2.1ab 2.4 ± 0.8b

1 Values are presented as average ± standard deviation (n = 3); in the same column, the numbers with different letters are significantly different at p

< 0.05. DSC thermograms of the RS residue samples are shown in Fig. 4.6.

2 WMS: waxy maize starch; NMS: normal maize starch.


4 N.D. = Not detectable.

81

82

Fig. 4.6 DSC thermograms of RS residue samples recovered from control and MGA-24 h-modified

starches after cooking and amylolysis in the Englyst Method. A: waxy maize; B: normal maize.

The black solid line with two end points is the baseline for the thermogram. The first endothermic

peak of lower temperatures corresponded to the melting of remaining amylopectin double-helical

crystallites, and the second endothermal peak of higher temperatures corresponded to the melting

of amylose double-helical crystallites.

83

4.5 Conclusions

MGA was used as a clean-label modification approach to diversifying the functionality of

granular maize starches having a wide range of amylose contents for the first time. After up to 24-

h incubation, MGA hydrolyzed the granular maize starches in a descending order of WMS > NMS >

HAMS. SEM observation revealed the “inside-out” hydrolysis pattern on WMS and NMS and the

“surface erosion” pattern on HAMS by MGA, corresponding well with their different degrees of

hydrolysis. The 24-h MGA modification did not alter the WAXD patterns of the three maize

starches, only slightly decreasing the relative crystallinity of WMS but increasing that of HAMS.

The 24-h enzyme treatment effectively reduced the retrogradation rates of WMS and NMS, which

was mainly attributed to the shortening of their amylopectin branch chains by MGA. In contrast,

the same modification enhanced the retrogradation of HAMS, which might be explained by that

the limited hydrolysis by MGA (11.2% at 24 h) shortened amylose chains for improved molecular

mobility to facilitate retrogradation during cold storage. MGA-treated WMS and NMS exhibited

extremely low pasting viscosities, which was ascribed to the breakdown of WMS and NMS by

MGA at both granular and molecular levels. Among the three maize starches, 24-h MGA treatment

was only able to elevate the RS content of cooked NMS from 2.6% to 7.6%, resulting from the

formation of more retrograded amylose in cooked, MGA-modified NMS during incubation at

37 °C in the Englyst Method. The unique technological characteristics of the MGA-treated

granular maize starches offered new insights for employing this commercial α-amylase to develop

novel starch ingredients for broader food applications.

84

5. GENERAL DISCUSSION

The overarching goal of this thesis project was to investigate the impacts of MGA

modification on the structural features, functional properties, and in vitro digestibility of different

starches. The results obtained from this thesis project will be important for utilizing MGA to

modify starches in a native granular form for diversified functionality and enhanced RS contents

in the starch industry. In the first study, three representative pulse starches (LS, FBS and PS) were

subjected to MGA treatment and then compared with their corresponding native counterparts.

NMS was included as the control and treated under the same modification conditions as pulse

starches. The four native starches were modified with 1% (w/w, db) MGA at 50 °C for 4, 8 and 24

h. The degrees of hydrolysis of LS, FBS, PS and NMS were 19.7%, 20.7%, 23.6% and 19.8% at

4 h, respectively; and the values increased to 34.7%, 34.4%, 38.4% and 35.9% at 24 h, respectively.

Upon 24-h MGA treatment, the apparent amylose contents of LS, FBS, PS and NMS decreased

6.4%, 4.6%, 2.8% and 2.0%, respectively. The SEM images revealed the different hydrolysis

patterns of MGA on pulse and maize starches. After 24-h MGA hydrolysis, the granules of the

three MGA-modified pulse starches were disrupted into small pieces by the enzyme through

“surface pitting” pattern due to the homogenous and solid internal structure of native starch

granules. In contrast, MGA hydrolysis generated numerous pores in NMS granules via “inside-

out” pattern, which was related to the endogenous pores and channels in native NMS granules.

The findings are consistent with the results reported in previous studies (Planchot et al., 1995;

Zhang et al., 2006). The WAXD patterns of pulse starches and NMS remained the same after MGA

treatment. However, the percentages of crystallinity of the three pulse starches were reduced,

which could be explained by that the “surface pitting” hydrolysis pattern of MGA disrupted some

crystalline structure during the enzymatic modification. After 24-h MGA treatment, the amylose

and LBC of amylopectin peaks were shifted to smaller DP, indicating the shortening of starch

chains due to molecular breakdown by MGA. Based on the NMR results, the percentages of α-1,6

linkages of LS, FBS, PS and NMS were increased 0.5%, 0.6%, 0.6% and 0.7% after 24-h MGA

treatment, respectively. During the MGA-modification, the double helices with poor stability in

the starches could be removed and thus the modified starches displayed considerably higher

gelatinization temperatures. The percentages of retrogradation of LS, FBS, PS and NMS were

56.9%, 46.2%, 56.2% and 34.8%, respectively. With the shortening of amylopectin branch chains

85

by 24-h MGA treatment, the retrogradation rates of the pulse and normal maize starches were

significantly decreased to 13.7%, 13.6%, 16.5% and 1.5%, respectively. All MGA-modified

starches displayed extremely low pasting viscosities, resulting from enzymatic breakdown of

starch at both granular and molecular levels. The MGA-24 h-modified starches showed an increase

in RS contents of cooked LS, FBS, PS and NMS by 5.9%, 6.5%, 4.2% and 4.7%, respectively. A

more obvious endothermic peak of retrograded amylose was observed in the RS residue samples

recovered from MGA-modified starches after cooking and amylolysis in the Englyst Method than

those recovered from their corresponding controls, suggesting that the increased RS contents could

be attributed to the formation of retrograded amylose with great enzymatic resistance.

According to Study 1, MGA modification was a useful method to improve the functional

properties and in vitro digestibility of pulse and normal starches. To gain a greater understanding

of the effects of MGA modification on the techno-functional attributes of starches with a broader

range of amylose content will be valuable for using this α-amylase to modify starch in the industry.

Thus, in Study 2, native granular WMS, NMS and HAMS with apparent amylose contents ranging

from 3.3 to 58.6% were hydrolyzed by MGA under the same conditions as described in Study 1.

Upon 24-h MGA treatment, the degrees of hydrolysis of WMS, NMS and HAMS were 39.7%,

35.9% and 11.2%, respectively, which were inversely associated with their apparent amylose

contents. The apparent amylose contents of maize starches were not significantly changed by

MGA treatment. MGA hydrolysis created numerous pores into the interior of WMS and NMS

granules, resulting from the “inside-out” hydrolysis pattern on these two maize starches. By

contrast, only small holes and fissures were found in HAMS granules, which was due to the fact

that MGA could only hydrolyze this B-type starch with exceptionally high enzymatic resistance

through surface pitting. MGA treatment for 24 h did not alter the WAXD patterns of the three

maize starches. The relative crystallinity of WMS was slightly decreased, suggesting the loss of

some crystalline structure during the extensive 24-h MGA hydrolysis. In contrast, HAMS

exhibited a noticeable increase in the relative crystallinity, which might be because of the removal

of molecules in the amorphous regions of granules by MGA (Gallant et al., 1992). MGA hydrolysis

for 24 h led to the shift of amylose and LBC peaks toward smaller DP, suggesting that MGA

modification degraded the starch molecules and shorten the amylose and LBC of amylopectin.

Consequently, the proportion of amylopectin SBC was increased for three MGA-modified maize

starches. The NMR data revealed that 24-h MGA treatment elevated the percentages of α-1,6

86

linkages of WMS, NMS and HAMS by 0.8%, 0.7%, 0.1%, respectively. After 24-h MGA

modification, the gelatinization temperatures of WMS and NMS were increased, while their ΔH

values were decreased. In contrast, ΔH of HAMS was elevated, which is in good agreement with

the increase in the relative crystallinity. Moreover, percentages of retrogradation of WMS and

NMS were markedly diminished to 1.4% and 1.5% after MGA treatment for 24 h, which was

attributed to the shortening of amylopectin branch chains. However, MGA treatment increased the

retrogradation rate of HAMS from 70.6% to 96.9%. One possible explanation for this opposite

trend on HAMS could be that the shortening of amylose chains fostered the recrystallization of

amylose with each other or with amylopectin LBC. Both WMS and NMS displayed extremely low

pasting viscosities after MGA treatment due to the degradation by the enzyme. For HAMS, it did

not exhibit any detectable viscosity before and after MGA hydrolysis, which could be explained

by the fact that control and modified HAMS was only partially gelatinized during pasting. Among

the three maize starches, only MGA-24 h-modified NMS exhibited a significant increase in RS

content from 2.6% to 7.3% as analyzed in a cooked form in the Englyst Assay, which could be due

to the development of retrograded amylose with great enzymatic resistance in NMS as shown in

Study 1.

This thesis research clearly demonstrated that MGA treatment can be employed as a clean-

label method to markedly modify the granular and molecular structures, functionality, and in vitro

digestibility of starches having amylose contents of 3.3-58.6%. The most desirable features of the

MGA-modified starches included considerably low retrogradation rate (except for HAMS) and

enhanced RS contents (except for WMS). The enzymatically modified starches can be utilized as

a novel ingredient to prepare food products with good cold-storage stability and enhanced

carbohydrate nutritional value.

87

6. GENERAL CONCLUSIONS AND FUTURE STUDIES

The main goal of this thesis project was to modify granular starches with MGA and to

develop “clean-label” starch ingredients with improved functional properties and nutritional value

for promoted industrial applications. Three pulse starches (LS, FBS and PS) and three maize

starches (WMS, NMS and HAMS) were chosen for this thesis research and modified with MGA

in a native granular form. Structural features, functional properties and in vitro digestibility of

MGA-modified starches were characterized and compared with their corresponding controls.

According to the presented results, MGA hydrolyzed granular pulse and maize starches

with different hydrolysis patterns. The C-type pulse starches and B-type HAMS were hydrolyzed

by a “surface-pitting” pattern mainly due to their relatively high amylose contents (38.4-58.6%)

and homogeneous, solid internal granular structure, while the A-type WMS and NMS were

hydrolyzed via a “inside-out” pattern because of the endogenous pores and channels in their

granules. MGA modification did not change the WAXD patterns of the studied starches. The chain

lengths of amylose and LBC of amylopectin were reduced by MGA attack and thus the proportions

of amylopectin SBC were increased for the starch samples. Consequently, the retrogradation rates

of the starches (except for HAMS) were significantly reduced by 24-h MGA treatment, which have

the potential to be used in food products to improve the cold-storage stability. After 24-h MGA

hydrolysis, all the starches displayed extremely low pasting viscosities, which renders them

suitable for the applications in beverage and confectionery products to increase the solids content

without noticeably changing the viscosity. Upon 24-h MGA treatment, cooked pulse starches and

NMS exhibited significant increases in the RS contents when compared with their respective

control starches.

Future studies can be conducted to evaluate the application value of MGA-modified

starches in a broad range of food products, such as beverages, confectioneries, energy bars, meat

products, and sauces. In addition, the control and MGA-modified HAMS exhibited little viscosity

after being pasted as 95 °C because of limited starch gelatinization. The pasting behaviors of the

control and MGA-modified HAMS can be determined at 95-140 °C using Rapid Visco Analyzer

4800 (Liu et al., 2019), which will not only advance our understanding of the influence of MGA

treatment on the pasting properties of HAMS but also generate new information useful for the

88

applications of native and modified HAMS in high-temperature food processing, such as canning,

retorting, and extrusion. Additional research can also be carried out to combine MGA treatment

with other clean-label modification methods, such as annealing and heat-moisture treatment, to

further improve the RS contents of starch.

89

7. REFERENCES

Ai, Y., Hasjim, J., & Jane, J.-l. (2013). Effects of lipids on enzymatic hydrolysis and physical

properties of starch. Carbohydrate Polymers, 92(1), 120-127.

Ai, Y., Jane, J., Gunaratne, A., & Corke, H. (2016). Starch: Structure, property, and determination.

Encyclopedia of Food and Health, 165-174.

Ai, Y., & Jane, J. l. (2015). Gelatinization and rheological properties of starch. Starch‐Stärke,

67(3-4), 213-224.

Ai, Y., Nelson, B., Birt, D. F., & Jane, J.-l. (2013). In vitro and in vivo digestion of octenyl succinic

starch. Carbohydrate Polymers, 98(2), 1266-1271.

Ambigaipalan, P., Hoover, R., Donner, E., Liu, Q., Jaiswal, S., Chibbar, R., . . . Seetharaman, K.

(2011). Structure of faba bean, black bean and pinto bean starches at different levels of

granule organization and their physicochemical properties. Food Research International,

44(9), 2962-2974.

Ao, Z., Quezada-Calvillo, R., Sim, L., Nichols, B. L., Rose, D. R., Sterchi, E. E., & Hamaker, B.

R. (2007). Evidence of native starch degradation with human small intestinal maltase-

glucoamylase (recombinant). FEBS Letters, 581(13), 2381-2388.

Ao, Z., Simsek, S., Zhang, G., Venkatachalam, M., Reuhs, B. L., & Hamaker, B. R. (2007). Starch

with a slow digestion property produced by altering its chain length, branch density, and

crystalline structure. Journal of Agricultural and Food Chemistry, 55(11), 4540-4547.

Asif, M., Rooney, L. W., Ali, R., & Riaz, M. N. (2013). Application and opportunities of pulses

in food system: A review. Critical Reviews in Food Science and Nutrition, 53(11), 1168-

1179.

Atwell, W. (1988). The terminology and methodology associated with basic starch phenomena.

Cereal Foods World, 33, 306-311.

Bello-Perez, L. A., Ottenhof, M.-A., Agama-Acevedo, E., & Farhat, I. (2005). Effect of storage

time on the retrogradation of banana starch extrudate. Journal of Agricultural and Food

90

Chemistry, 53(4), 1081-1086.Bemiller, J. N. (1997). Starch modification: Challenges and

prospects. Starch‐Stärke, 49(4), 127-131.

Bijttebier, A., Goesaert, H., & Delcour, J. A. (2007). Temperature impacts the multiple attack

action of amylases. Biomacromolecules, 8(3), 765-772.

Bird, A. R., Lopez-Rubio, A., Shrestha, A. K., & Gidley, M. J. (2009). Resistant starch in vitro

and in vivo: Factors determining yield, structure, and physiological relevance Modern

Biopolymer science (pp. 449-510): Elsevier.

Boye, J., Zare, F., & Pletch, A. (2010). Pulse proteins: Processing, characterization, functional

properties and applications in food and feed. Food Research International, 43(2), 414-431.

Buléon, A., Colonna, P., Planchot, V., & Ball, S. (1998). Starch granules: Structure and

biosynthesis. International Journal of Biological Macromolecules, 23(2), 85-112.

Buléon, A., Véronèse, G., & Putaux, J.-L. (2007). Self-association and crystallization of

amylose. Australian Journal of Chemistry, 60(10), 706-718.

Byars, J. A., & Singh, M. (2016). Rheological and textural properties of pulse starch gels. Starch‐

Stärke, 68(7-8), 778-784.

Cai, L., & Shi, Y.-C. (2010). Structure and digestibility of crystalline short-chain amylose from

debranched waxy wheat, waxy maize, and waxy potato starches. Carbohydrate Polymers,

79(4), 1117-1123.

Chen, Q., Yu, H., Wang, L., ul Abdin, Z., Chen, Y., Wang, J., . . . Zhang, H. (2015). Recent

progress in chemical modification of starch and its applications. Rsc Advances, 5(83),

67459-67474.

Chen, Y., Huang, S., Tang, Z., Chen, X., & Zhang, Z. (2011). Structural changes of cassava starch

granules hydrolyzed by a mixture of α-amylase and glucoamylase. Carbohydrate Polymers,

85(1), 272-275.

Chrastil, J. (1987). Improved colorimetric determination of amylose in starches or flours.

Carbohydrate Research, 159(1), 154-158.

Christophersen, C., Otzen, D. E., Noman, B. E., Christensen, S., & Schäfer, T. (1998). Enzymatic

characterisation of novamyl®, a thermostable α‐amylase. Starch‐Stärke, 50(1), 39-45.

Chung, H.-J., Liu, Q., & Hoover, R. (2009). Impact of annealing and heat-moisture treatment on

rapidly digestible, slowly digestible and resistant starch levels in native and gelatinized

corn, pea and lentil starches. Carbohydrate Polymers, 75(3), 436-447.

91

Chung, H.-J., Liu, Q., & Hoover, R. (2010). Effect of single and dual hydrothermal treatments on

the crystalline structure, thermal properties, and nutritional fractions of pea, lentil, and navy

bean starches. Food Research International, 43(2), 501-508.

Chung, H. J., Liu, Q., Donner, E., Hoover, R., Warkentin, T. D., & Vandenberg, B. (2008).

Composition, molecular structure, properties, and in vitro digestibility of starches from

newly released canadian pulse cultivars. Cereal Chemistry, 85(4), 471-479.

Cooke, D., & Gidley, M. J. (1992). Loss of crystalline and molecular order during starch

gelatinisation: Origin of the enthalpic transition. Carbohydrate Research, 227, 103-112.

Demirkesen-Bicak, H., Tacer-Caba, Z., & Nilufer-Erdil, D. (2018). Pullulanase treatments to

increase resistant starch content of black chickpea (cicer arietinum l.) starch and the effects

on starch properties. International Journal of Biological Macromolecules, 111, 505-513.

Dilis, V., & Trichopoulou, A. (2009). Nutritional and health properties of pulses. Mediterranean

Journal of Nutrition and Metabolism, 1(3), 149-157.

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. t., & Smith, F. (1956). Colorimetric method

for determination of sugars and related substances. Analytical Chemistry, 28(3), 350-356.

Dura, A., Błaszczak, W., & Rosell, C. M. (2014). Functionality of porous starch obtained by

amylase or amyloglucosidase treatments. Carbohydrate Polymers, 101, 837-845.

Eerlingen, R., & Delcour, J. (1995). Formation, analysis, structure and properties of type iii

enzyme resistant starch. Journal of Cereal Science, 22(2), 129-138.

Eerlingen, R., Jacobs, H., & Delcour, J. (1994). Enzyme-resistant starch. 5. Effect of retrogradation

of waxy maize starch on enzyme susceptibility. Cereal Chemistry, 71(4), 351-355.

Englyst, H., Wiggins, H., & Cummings, J. (1982). Determination of the non-starch

polysaccharides in plant foods by gas-liquid chromatography of constituent sugars as

alditol acetates. Analyst, 107(1272), 307-318.

Englyst, H. N., Kingman, S., & Cummings, J. (1992). Classification and measurement of

nutritionally important starch fractions. European Journal of Clinical Nutrition, 46, S33.

Englyst, H. N., & Macfarlane, G. T. (1986). Breakdown of resistant and readily digestible starch

by human gut bacteria. Journal of the Science of Food and Agriculture, 37(7), 699-706.

Gallant, D., Bouchet, B., Buleon, A., & Perez, S. (1992). Physical characteristics of starch granules

and susceptibility to enzymatic degradation. European Journal of Clinical Nutrition, 46(2),

3-16.

92

Gallant, D. J., Bouchet, B., & Baldwin, P. M. (1997). Microscopy of starch: Evidence of a new

level of granule organization. Carbohydrate Polymers, 32(3-4), 177-191.

Gidley, M. J. (1985). Quantification of the structural features of starch polysaccharides by nmr

spectroscopy. Carbohydrate Research, 139, 85-93.

Glaring, M. A., Koch, C. B., & Blennow, A. (2006). Genotype-specific spatial distribution of

starch molecules in the starch granule: A combined clsm and sem approach.

Biomacromolecules, 7(8), 2310-2320.

Goesaert, H., Leman, P., Bijttebier, A., & Delcour, J. A. (2009). Antifirming effects of starch

degrading enzymes in bread crumb. Journal of Agricultural and Food Chemistry, 57(6),

2346-2355.

Goesaert, H., Slade, L., Levine, H., & Delcour, J. A. (2009). Amylases and bread firming–an

integrated view. Journal of Cereal Science, 50(3), 345-352.

Grewal, N., Faubion, J., Feng, G., Kaufman, R. C., Wilson, J. D., & Shi, Y.-C. (2015). Structure

of waxy maize starch hydrolyzed by maltogenic α-amylase in relation to its retrogradation.

Journal of Agricultural and Food Chemistry, 63(16), 4196-4201.

Gunaratne, A., & Hoover, R. (2002). Effect of heat–moisture treatment on the structure and

physicochemical properties of tuber and root starches. Carbohydrate Polymers, 49(4), 425-

437.

Hall, C., Hillen, C., & Garden Robinson, J. (2017). Composition, nutritional value, and health

benefits of pulses. Cereal Chemistry, 94(1), 11-31.

Han, J. J., Janz, J. A., & Gerlat, M. (2010). Development of gluten-free cracker snacks using pulse

flours and fractions. Food Research International, 43(2), 627-633.

Haros, M., Tolaba, M. P., & Suárez, C. (2003). Influence of corn drying on its quality for the wet-

milling process. Journal of Food Engineering, 60(2), 177-184.

Hayakawa, K., Tanaka, K., Nakamura, T., Endo, S., & Hoshino, T. (1997). Quality characteristics

of waxy hexaploid wheat (triticum aestivum l.): Properties of starch gelatinization and

retrogradation. Cereal Chemistry, 74(5), 576-580.

Hizukuri, S. (1986). Polymodal distribution of the chain lengths of amylopectins, and its

significance. Carbohydr. Res., 147, 342-347.

Hizukuri, S., Takeda, Y., Yasuda, M., & Suzuki, A. (1981). Multi-branched nature of amylose and

the action of debranching enzymes. Carbohydrate Research, 94(2), 205-213.

93

Hoover, R. (1995). Starch retrogradation. Food Reviews International, 11(2), 331-346.

Hoover, R., Hughes, T., Chung, H., & Liu, Q. (2010). Composition, molecular structure, properties,

and modification of pulse starches: A review. Food Research International, 43(2), 399-

413.

Hoover, R., & Ratnayake, W. (2002). Starch characteristics of black bean, chick pea, lentil, navy

bean and pinto bean cultivars grown in canada. Food Chemistry, 78(4), 489-498.

Huber, K. C., & BeMiller, J. N. (1997). Visualization of channels and cavities of corn and sorghum

starch granules. Cereal Chemistry, 74(5), 537-541.

Hug‐Iten, S., Escher, F., & Conde‐Petit, B. (2001). Structural properties of starch in bread and

bread model systems: Influence of an antistaling α‐amylase. Cereal Chemistry, 78(4), 421-

428.

Hug‐Iten, S., Escher, F., & Conde‐Petit, B. (2003). Staling of bread: Role of amylose and

amylopectin and influence of starch‐degrading enzymes. Cereal Chemistry, 80(6), 654-

661.

Jane, J.-l. (2009). Structural features of starch granules ii Starch (pp. 193-236): Elsevier.

Jane, J.-l., Ao, Z., Duvick, S. A., Wiklund, M., Yoo, S.-H., Wong, K.-S., & Gardner, C. (2003).

Structures of amylopectin and starch granules: How are they synthesized? Journal of

Applied Glycoscience, 50(2), 167-172.

Jane, J.-l., Wong, K.-s., & McPherson, A. E. (1997). Branch-structure difference in starches of a-

and b-type x-ray patterns revealed by their naegeli dextrins. Carbohydrate Research,

300(3), 219-227.

Jane, J., Chen, Y., Lee, L., McPherson, A., Wong, K., Radosavljevic, M., & Kasemsuwan, T.

(1999). Effects of amylopectin branch chain length and amylose content on the

gelatinization and pasting properties of starch. Cereal Chemistry, 76(5), 629-637.

Jane, J., Xu, A., Radosavljevic, M., & Seib, P. (1992). Location of amylose in normal starch

granules. I. Susceptibility of Amylose and Amylopectin to Cross-Linking Reagents, 69, 405-

409.

Jane, J. l., Chen, Y., Lee, L., McPherson, A., Wong, K., Radosavljevic, M., & Kasemsuwan, T.

(1999). Effects of amylopectin branch chain length and amylose content on the

gelatinization and pasting properties of starch. Cereal Chemistry, 76(5), 629-637.

94

Jiang, H., Campbell, M., Blanco, M., & Jane, J.-L. (2010). Characterization of maize amylose-

extender (ae) mutant starches: Part ii. Structures and properties of starch residues remaining

after enzymatic hydrolysis at boiling-water temperature. Carbohydrate Polymers, 80(1), 1-

12.

Jiang, H., Horner, H. T., Pepper, T. M., Blanco, M., Campbell, M., & Jane, J.-l. (2010). Formation

of elongated starch granules in high-amylose maize. Carbohydrate Polymers, 80(2), 533-

538.

Karim, A., Sufha, E., & Zaidul, I. (2008). Dual modification of starch via partial enzymatic

hydrolysis in the granular state and subsequent hydroxypropylation. Journal of

Agricultural and Food Chemistry, 56(22), 10901-10907.

Kaur, B., Ariffin, F., Bhat, R., & Karim, A. A. (2012). Progress in starch modification in the last

decade. Food Hydrocolloids, 26(2), 398-404.

Kibar, E. A. A., Gönenç, İ., & Us, F. (2010). Gelatinization of waxy, normal and high amylose

corn starches. The Journal of Food, 35(4), 237-244.

Kim, B. K., Kim, H. I., Moon, T. W., & Choi, S. J. (2014). Branch chain elongation by

amylosucrase: Production of waxy corn starch with a slow digestion property. Food

Chemistry, 152, 113-120.

Kumar, S., & Masood, A. (2001). Pulses an essential ingredient of food basket. Indian Farming

(India).

Kwon, K., Auh, J., Choi, S., Kang, G., Kim, J., & Park, K. (1999). Characterization of branched

oligosaccharides produced by bacillus licheniformis maltogenic amylase. Journal of Food

Science, 64(2), 258-261.

Le, Q.-T., Lee, C.-K., Kim, Y.-W., Lee, S.-J., Zhang, R., Withers, S. G., . . . Park, K.-H. (2009).

Amylolytically-resistant tapioca starch modified by combined treatment of branching

enzyme and maltogenic amylase. Carbohydrate Polymers, 75(1), 9-14.

Leman, P., Bijttebier, A., Goesaert, H., Vandeputte, G. E., & Delcour, J. A. (2006). Influence of

amylases on the rheological and molecular properties of partially damaged wheat starch.

Journal of the Science of Food and Agriculture, 86(11), 1662-1669.

Leman, P., Goesaert, H., & Delcour, J. A. (2009). Residual amylopectin structures of amylase-

treated wheat starch slurries reflect amylase mode of action. Food Hydrocolloids, 23(1),

153-164.

95

Leman, P., Goesaert, H., Vandeputte, G. E., Lagrain, B., & Delcour, J. A. (2005). Maltogenic

amylase has a non-typical impact on the molecular and rheological properties of starch.

Carbohydrate Polymers, 62(3), 205-213.

Li, C., & Hu, Y. (2020). In vitro digestibility of short-term retrograded rice starches is driven by

the amylose molecular size and amount of amylose short to medium chains. Food

Hydrocolloids, 106071.

Li, J.-Y., & Yeh, A.-I. (2001). Relationships between thermal, rheological characteristics and

swelling power for various starches. Journal of Food Engineering, 50(3), 141-148.

Li, L., Jiang, H., Campbell, M., Blanco, M., & Jane, J.-l. (2008). Characterization of maize

amylose-extender (ae) mutant starches. Part i: Relationship between resistant starch

contents and molecular structures. Carbohydrate Polymers, 74(3), 396-404.

Li, L., Yuan, T. Z., & Ai, Y. (2020). Development, structure and in vitro digestibility of type 3

resistant starch from acid-thinned and debranched pea and normal maize starches. Food

Chemistry, 126485.

Li, L., Yuan, T. Z., Setia, R., Raja, R. B., Zhang, B., & Ai, Y. (2019). Characteristics of pea, lentil

and faba bean starches isolated from air-classified flours in comparison with commercial

starches. Food Chemistry, 276, 599-607.

Lin, L., Cai, C., Gilbert, R. G., Li, E., Wang, J., & Wei, C. (2016). Relationships between

amylopectin molecular structures and functional properties of different-sized fractions of

normal and high-amylose maize starches. Food Hydrocolloids, 52, 359-368.

Liu, S., Reimer, M., & Ai, Y. (2020). In vitro digestibility of different types of resistant starches

under high-temperature cooking conditions. Food Hydrocolloids, 105927.

Liu, S., Yuan, T. Z., Wang, X., Reimer, M., Isaak, C., & Ai, Y. (2019). Behaviors of starches

evaluated at high heating temperatures using a new model of rapid visco analyzer‒rva 4800.

Food Hydrocolloids, 94, 217-228.

Ma, Z., Boye, J. I., Azarnia, S., & Simpson, B. K. (2016). Volatile flavor profile of saskatchewan

grown pulses as affected by different thermal processing treatments. International Journal

of Food Properties, 19(10), 2251-2271.

96

MacGregor, A., & Ballance, D. (1980). Hydrolysis of large and small starch granules from normal

and waxy barley cultivars by alpha-amylases from barley malt. Cereal Chemistry, 57(6),

397-402.

Man, J., Yang, Y., Huang, J., Zhang, C., Chen, Y., Wang, Y., . . . Wei, C. (2013). Effect of

simultaneous inhibition of starch branching enzymes i and iib on the crystalline structure

of rice starches with different amylose contents. Journal of Agricultural and Food

Chemistry, 61(41), 9930-9937.

Maniglia, B. C., Castanha, N., Le-Bail, P., Le-Bail, A., & Augusto, P. E. (2020). Starch

modification through environmentally friendly alternatives: A review. Critical Reviews in

Food Science and Nutrition, 1-24.

Marousis, S., & Saravacos, G. (1990). Density and porosity in drying starch materials. Journal of

Food Science, 55(5), 1367-1372.

McDermott, J., & Wyatt, A. J. (2017). The role of pulses in sustainable and healthy food systems.

Annals of the New York Academy of Sciences, 1392(1), 30-42.

Mendez-Montealvo, G., Wang, Y.-J., & Campbell, M. (2011). Thermal and rheological properties

of granular waxy maize mutant starches after β-amylase modification. Carbohydrate

Polymers, 83(3), 1106-1111.

Meuser, F., Pahne, N., & Möller, M. (1995). Extraction of high amylose starch from wrinkled peas.

Starch‐Stärke, 47(2), 56-61.

Miao, M., Xiong, S., Ye, F., Jiang, B., Cui, S. W., & Zhang, T. (2014). Development of maize

starch with a slow digestion property using maltogenic α-amylase. Carbohydrate Polymers,

103, 164-169.

Miles, M. J., Morris, V. J., Orford, P. D., & Ring, S. G. (1985). The roles of amylose and

amylopectin in the gelation and retrogradation of starch. Carbohydrate Research, 135(2),

271-281.

Mudryj, A. N., Yu, N., & Aukema, H. M. (2014). Nutritional and health benefits of pulses.

Applied Physiology, Nutrition, and Metabolism, 39(11), 1197-1204.

Ochubiojo, E. M., & Rodrigues, A. (2012). Starch: From food to medicine. Scientific, Health and

Social Aspects of the Food Industry, 355-380.

97

Peng, X., & Yao, Y. (2018). Small-granule starches from sweet corn and cow cockle: Physical

properties and amylopectin branching pattern. Food Hydrocolloids, 74, 349-357.

Pérez, S., Baldwin, P. M., & Gallant, D. J. (2009). Structural features of starch granules i Starch

(pp. 149-192): Elsevier.

Planchot, V., Colonna, P., Gallant, D., & Bouchet, B. (1995). Extensive degradation of native

starch granules by alpha-amylase from aspergillus fumigatus. Journal of Cereal Science,

21(2), 163-171.

Pulses. (2020). Where do pulses grow? Retrieved from https://pulses.org/what-are-pulses/where-

do-pulses-

grow#:~:text=As%20of%202015%2C%20the%20world's,France%2C%20Ethiopia%2C

%20and%20Turkey.

Purhagen, J. K., Sjöö, M. E., & Eliasson, A.-C. (2011). Starch affecting anti-staling agents and

their function in freestanding and pan-baked bread. Food Hydrocolloids, 25(7), 1656-1666.

Qin, F., Man, J., Xu, B., Hu, M., Gu, M., Liu, Q., & Wei, C. (2011). Structural properties of

hydrolyzed high-amylose rice starch by α-amylase from bacillus licheniformis. Journal of


Ratnavathi, C., & Komala, V. (2016). Sorghum grain quality Sorghum Biochemistry (pp. 1-61):

Elsevier.

Ratnayake, W., Hoover, R., Shahidi, F., Perera, C., & Jane, J. (2001). Composition, molecular

structure, and physicochemical properties of starches from four field pea (pisum sativum

l.) cultivars. Food Chemistry, 74(2), 189-202.

Reddy, C. K., Haripriya, S., Mohamed, A. N., & Suriya, M. (2014). Preparation and

characterization of resistant starch iii from elephant foot yam (amorphophallus

paeonifolius) starch. Food Chemistry, 155, 38-44.

Richardson, S., Nilsson, G. S., Bergquist, K.-E., Gorton, L., & Mischnick, P. (2000).

Characterisation of the substituent distribution in hydroxypropylated potato amylopectin

starch. Carbohydrate Research, 328(3), 365-373.

Ring, S. G., Gee, J. M., Whittam, M., Orford, P., & Johnson, I. T. (1988). Resistant starch: Its

chemical form in foodstuffs and effect on digestibility in vitro. Food Chemistry, 28(2),

97-109.

98

Rizkalla, S. W., Bellisle, F., & Slama, G. (2002). Health benefits of low glycaemic index foods,

such as pulses, in diabetic patients and healthy individuals. British Journal of Nutrition,

88(S3), 255-262.

Robyt, J. F. (2012). Essentials of Carbohydrate Chemistry: Springer Science & Business Media.

Rooney, L., & Pflugfelder, R. (1986). Factors affecting starch digestibility with special emphasis

on sorghum and corn. Journal of Animal Science, 63(5), 1607-1623.

Rumpf, H. (1990). Particle technology (english edition). Powder Technology Series. London:

Chapman and Hall.

Saltmarsh, M. (2015). Recent trends in the use of food additives in the united kingdom. Journal of

the Science of Food and Agriculture, 95(4), 649-652.

Sangokunle, O. O., Sathe, S. K., & Singh, P. Purified starches from 18 pulses have markedly

different morphology, oil absorption and water absorption capacities, swelling power, and

turbidity. Starch‐Stärke, 2000022.

Shamai, K., Bianco-Peled, H., & Shimoni, E. (2003). Polymorphism of resistant starch type iii.

Carbohydrate Polymers, 54(3), 363-369.

Sievert, D., & Pomeranz, Y. (1989). By enzymatic, thermoanalytical, and microscopic methods'.

Cereal Chem, 66(4), 342-347.

Sievert, D., & Wuesch, P. (1993). Amylose chain association based on differential scanning

calorimetry. Journal of Food Science, 58(6), 1332-1335.

Singh, A., Geveke, D. J., & Yadav, M. P. (2017). Improvement of rheological, thermal and

functional properties of tapioca starch by using gum arabic. Lwt, 80, 155-162.

Singh, N., Inouchi, N., & Nishinari, K. (2006). Structural, thermal and viscoelastic characteristics

of starches separated from normal, sugary and waxy maize. Food Hydrocolloids, 20(6),

923-935.

Song, Y., & Jane, J.-L. (2000). Characterization of barley starches of waxy, normal, and high

amylose varieties. Carbohydrate Polymers, 41(4), 365-377.

Sorndech, W., Tongta, S., & Blennow, A. (2018). Slowly digestible‐and non‐digestible α‐glucans:

An enzymatic approach to starch modification and nutritional effects. Starch‐Stärke, 70(9-

10), 1700145.

99

Sosulski, F., & Sosulski, K. (1986). Composition and functionality of protein, starch, and fiber

from wet and dry processing of grain legumes: ACS Publications.

Srichuwong, S., Isono, N., Mishima, T., & Hisamatsu, M. (2005). Structure of lintnerized starch

is related to x-ray diffraction pattern and susceptibility to acid and enzyme hydrolysis of

starch granules. International Journal of Biological Macromolecules, 37(3), 115-121.

Sujka, M., & Jamroz, J. (2007). Starch granule porosity and its changes by means of amylolysis.

International Agrophysics, 21(1).

Takeda, Y., Shirasaka, K., & Hizukuri, S. (1984). Examination of the purity and structure of

amylose by gel-permeation chromatography. Carbohydrate Research, 132(1), 83-92.

Takeda, Y., Shitaozono, T., & Hizukuri, S. (1990). Structures of sub-fractions of corn amylose.

Carbohydrate Research, 199(2), 207-214.

Tawil, G., Viksø-Nielsen, A., Rolland-Sabaté, A., Colonna, P., & Buléon, A. (2012). Hydrolysis

of concentrated raw starch: A new very efficient α-amylase from anoxybacillus

flavothermus. Carbohydrate Polymers, 87(1), 46-52.

Tian, Y., Li, Y., Jin, Z., & Xu, X. (2009). A novel molecular simulation method for evaluating the

endothermic transition of amylose recrystallite. European Food Research and Technology,

229(6), 853-858.

Tiwari, B. K., Gowen, A., & McKenna, B. (2011). Pulse Foods: Processing, Quality and

Nutraceutical Applications: Academic Press.

Tizzotti, M. J., Sweedman, M. C., Tang, D., Schaefer, C., & Gilbert, R. G. (2011). New 1h nmr

procedure for the characterization of native and modified food-grade starches. Journal of


Toews, R., & Wang, N. (2013). Physicochemical and functional properties of protein concentrates

from pulses. Food Research International, 52(2), 445-451.

Tosh, S. M., & Yada, S. (2010). Dietary fibres in pulse seeds and fractions: Characterization,

functional attributes, and applications. Food Research International, 43(2), 450-460.

Tulbek, M. C. (2010). Pulse-milling: Wet and dry fractionation applications of peas, lentils and

chickpeas in gluten-free foods. Paper presented at the Institute of food technologists 2010

Annual Meeting. Fargo, ND, US.

van der Maarel, M. J., & Leemhuis, H. (2013). Starch modification with microbial alpha-

glucanotransferase enzymes. Carbohydrate Polymers, 93(1), 116-121.

100

Van Steertegem, B., Pareyt, B., Brijs, K., & Delcour, J. A. (2013). Combined impact of bacillus

stearothermophilus maltogenic alpha-amylase and surfactants on starch pasting and

gelation properties. Food Chemistry, 139(1-4), 1113-1120.

Wang, S., Li, C., Copeland, L., Niu, Q., & Wang, S. (2015). Starch retrogradation: A

comprehensive review. Comprehensive Reviews in Food Science and Food Safety, 14(5),

568-585.

Wang, S., Wang, J., Yu, J., & Wang, S. (2014). A comparative study of annealing of waxy,

normal and high-amylose maize starches: The role of amylose molecules. Food

Chemistry, 164, 332-338.

Wang, S., Zhang, X., Wang, S., & Copeland, L. (2016). Changes of multi-scale structure during

mimicked dsc heating reveal the nature of starch gelatinization. Scientific Reports, 6, 28271.

Wani, A. A., Singh, P., Shah, M. A., Schweiggert‐Weisz, U., Gul, K., & Wani, I. A. (2012). Rice

starch diversity: Effects on structural, morphological, thermal, and physicochemical

properties—a review. Comprehensive Reviews in Food Science and Food Safety, 11(5),

417-436.

Yildiz, Ö., Yurt, B., Baştürk, A., Toker, Ö. S., Yilmaz, M. T., Karaman, S., & Dağlıoğlu, O. (2013).

Pasting properties, texture profile and stress–relaxation behavior of wheat starch/dietary

fiber systems. Food Research International, 53(1), 278-290.

Zeng, J., Li, G., Gao, H., & Ru, Z. (2011). Comparison of a and b starch granules from three wheat

varieties. Molecules, 16(12), 10570-10591.

Zhang, G., Ao, Z., & Hamaker, B. R. (2006). Slow digestion property of native cereal starches.

Biomacromolecules, 7(11), 3252-3258.

Zhang, G., Ao, Z., & Hamaker, B. R. (2008). Nutritional property of endosperm starches from

maize mutants: A parabolic relationship between slowly digestible starch and amylopectin

fine structure. Journal of Agricultural and Food Chemistry, 56(12), 4686-4694.

Zhang, G., & Hamaker, B. R. (2009). Slowly digestible starch: Concept, mechanism, and proposed

extended glycemic index. Critical Reviews in Food Science and Nutrition, 49(10), 852-

867.

Zhu, L.-J., Liu, Q.-Q., Wilson, J. D., Gu, M.-H., & Shi, Y.-C. (2011). Digestibility and

physicochemical properties of rice (oryza sativa l.) flours and starches differing in amylose

content. Carbohydrate Polymers, 86(4), 1751-1759.

101

8. APPENDICES

8.1 List of Tables

Table 1 DP for AM peak of debranched control and MGA-24 h-modified starches.

Starch1 DP for AM peak

LS, control 1074

LS, 24 h 835

FBS, control 1081

FBS, 24 h 781

PS, control 1096

PS, 24 h 615

NMS, control 1051

NMS, 24 h 603 1 LS: lentil starch; FBS: faba bean starch; PS: pea starch; NMS: normal maize starch.

102

Table 2 DP for AM peak of debranched control and MGA-24 h-modified maize starches.

Starch1 DP for AM peak

NMS, control 1051

NMS, 24 h 603

HAMS, control 988

HAMS, 24 h 482 1 NMS: normal maize starch; HAMS: high-amylose maize starch.

103

Table 3 DP range for AM fraction of debranched control and MGA-24 h-modified maize

starches.

Sample DP range for Amylose fraction

Control 251-84557

MGA-24 h modified 101-3011

104

8.2 Copyright approval

ELSEVIER LICENSE

TERMS AND CONDITIONS

Oct 16, 2020

This Agreement between Jiayi Li ("You") and Elsevier ("Elsevier") consists of your license details and

the terms and conditions provided by Elsevier and Copyright Clearance Center.

License Number 4930671378798

License date Oct 16, 2020

Licensed Content Publisher Elsevier

Licensed Content Publication Food Chemistry

Licensed Content Title

Characteristics of pea, lentil and faba bean starches

isolated from air-classified flours in comparison with

commercial starches

Licensed Content Author Liying Li,Tommy Z. Yuan,Rashim Setia,Ramadoss

Bharathi Raja,Bin Zhang,Yongfeng Ai

Licensed Content Date Mar 15, 2019

Licensed Content Volume 276

Licensed Content Issue n/a

Licensed Content Pages 9

Start Page 599

End Page 607

Type of Use reuse in a thesis/dissertation

Portion figures/tables/illustrations

Number of figures/tables/illustrations 1

Format electronic

Are you the author of this Elsevier article? No

Will you be translating? No

Title

Maltogenic α-amylase modification of pulse and

maize starches to enhance the functional properties

and resistant starch content

Institution name University of Saskatchewan

Expected presentation date Jan 2021

Portions Fig. 2.1

Requestor Location

Jiayi Li

123 Wesolowski Cove

Saskatoon, SK S7W 0E9

Canada

Attn: JIAYI LI

Publisher Tax ID GB 494 6272 12

Total 0.00 USD

ELSEVIER LICENSE


Oct 16, 2020

105




License date Dec 28, 2020


Licensed Content Publication Carbohydrate Polymers


Morphology, structure and gelatinization properties

of heterogeneous starch granules from high-amylose

maize

Licensed Content Author Canhui Cai,Lingxiao Zhao,Jun Huang.Yifang

Chen,Cunxu Wei

Licensed Content Date Feb 15, 2014




Start Page 606

End Page 614




Format electronic



Title






Portions Fig. 2.1

Requestor Location

Jiayi Li

123 Wesolowski Cove


Canada

Attn: JIAYI LI


Total 0.00 USD

ELSEVIER LICENSE


Oct 16, 2020

106






Licensed Content Publication Carbohydrate Research

Licensed Content Title Structures of sub-fractions of corn amylose

Licensed Content Author Yasuhito Takeda,Tetsuya Shitaozono,Susumu

Hizukuri

Licensed Content Date Jun 1, 1990


Licensed Content Issue 2


Start Page 207

End Page 214




Format electronic



Title






Portions Fig. 2.2

Requestor Location

Jiayi Li

123 Wesolowski Cove


Canada

Attn: JIAYI LI


Total 0.00 USD

ELSEVIER LICENSE


Oct 15, 2020



107




Licensed Content Publication Carbohydrate Research

Licensed Content Title Polymodal distribution of the chain lengths of

amylopectins, and its significance

Licensed Content Author Susumu Hizukuri





Start Page 342

End Page 347




Format electronic



Title

Maltogenic α-amylase modification of pulse and maize

starches to enhance the functional properties and

resistant starch content



Portions Fig. 2.3

Requestor Location

Jiayi Li

123 Wesolowski Cove


Canada

Attn: JIAYI LI


Total 0.00 USD

ELSEVIER LICENSE


Oct 16, 2020






108

Licensed Content Publication Elsevier Books

Licensed Content Title Starch

Licensed Content Author Jay-lin Jane

Licensed Content Date Jan 1, 2009


Start Page 193

End Page 236




Format electronic



Title






Portions Fig. 2.4

Requestor Location

Jiayi Li

123 Wesolowski Cove


Canada

Attn: JIAYI LI


Total 0.00 USD

ELSEVIER LICENSE


Oct 16, 2020






Licensed Content Publication Elsevier Books

Licensed Content Title Starch

Licensed Content Author Serge Pérez,Paul M. Baldwin,Daniel J. Gallant

109

Licensed Content Date Jan 1, 2009


Start Page 149

End Page 192




Format electronic



Title






Portions Fig. 2.5

Requestor Location

Jiayi Li

123 Wesolowski Cove


Canada

Attn: JIAYI LI


Total 0.00 USD

112

ELSEVIER LICENSE


Oct 16, 2020





Licensed Content Publisher John Wiley and Sons

Licensed Content Publication Comprehensive Reviews in Food Science and Food

Safety


Rice Starch Diversity: Effects on Structural,

Morphological, Thermal, and Physicochemical

Properties—A Review

Licensed Content Author Idrees Ahmed Wani, Khalid Gul, Ute Schweiggert‐Weisz,

et al

Licensed Content Date Aug 24, 2012




Type of Use Dissertation/Thesis

Requestor type University/Academic

Format Electronic

Portion Figure/table

Number of figures/tables 5


Title


starches to enhance the functional properties and resistant

starch content



Portions Fig. 2.8

Requestor Location

Jiayi Li

123 Wesolowski Cove


Canada

Attn: JIAYI LI


Total 0.00 USD

ELSEVIER LICENSE


113

Oct 16, 2020







Licensed Content Title Maltogenic amylase has a non-typical impact on the

molecular and rheological properties of starch

Licensed Content Author Pedro Leman,Hans Goesaert,Greet E. Vandeputte,Bert

Lagrain,Jan A. Delcour

Licensed Content Date Dec 1, 2005




Start Page 205

End Page 213




Format electronic



Title






Portions Fig. 2.9

Requestor Location

Jiayi Li

123 Wesolowski Cove


Canada

Attn: JIAYI LI


Total 0.00 USD

ELSEVIER LICENSE


Oct 16, 2020



114





Licensed Content Title Development of maize starch with a slow digestion

property using maltogenic α-amylase

Licensed Content Author Ming Miao,Shanshan Xiong,Fan Ye,Bo Jiang,Steve

W. Cui,Tao Zhang





Start Page 164

End Page 169




Format electronic



Title






Portions Fig. 2.10

Requestor Location

Jiayi Li

123 Wesolowski Cove


Canada

Attn: JIAYI LI


Total 0.00 USD

Maltogenic α-amylase modification of pulse and maize ...

Documents

Transcript of Maltogenic α-amylase modification of pulse and maize ...