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Diss. ETH No.

13779

Staling

of

Bread

and

Bread Model Systems

Role of Starch

and

Amylases

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY


ZURICH

for the

degree

of

Doctor of Technical Sciences

presented by
Susanna

Hug-lten

Dipl. Lm.-lng.
born 30

July,

citizen of

accepted

on

1970

Untergeri (ZG), Switzerland

the recommendation of
Prof. Dr. F.
Dr. B.

Escher, examiner

Conde-Petit,

Prof. Dr. K.

Zurich 2000

ETH

co-examiner

Poutanen, co-examiner

13/2/01

Acknowledgement
Prof

Dr

Felix Escher gave

of bread

fascinating phenomenon

and constructive discussions I

specially

guidance

never-ending
a

am

very

be

study

the

For his continuous

guidance, support

perfect

balance between scientific freedom

I thank her for the uncountable hours she invested


enthusiasm and her

co-examiner

creativity

in

my work, her

In many fruitful discussions I learned

of the field

view

of this thesis and for

I wish to thank Novo Nordisk

(VTT Finland)

Spendler

A/S, Copenhagen,

accepting

Denmark for
A

support

and Jack Bech Nielsen who gave

the world of enzyme business and

for

to

critically reading my manuscript

financial and technical

investigation possible by
extended to Tina

and

interesting

grateful

very thankful to Prof Dr Kaisa Poutanen

am

to

Batrice Conde-Petit for her excellent supervision and

thank Dr

lot from her broad

opportunity

staling

support She constantly provided


and

the

me

supported

me

this

making

special

me an

thank

insight

constantly during my

is

into

work with

many scientific discussions

My

sincere

Science for

always

thanks

providing

remember the

motivated

me

extended to all my

good

time I had

good

who introduced

suitable methods for

semester and

Crespo,

Matthias

Franoise
explicitly

diploma

carefully
into the

me

studying
into

support

bread

manuscript,

who

and with

field of microscopy and

under the

X-ray diffractometry

works Rita

I will

help

Christoph Zweifel,

read the

great

and

light microscope

and DSC

analysis

Blattmann, Chantal Bussmann, Pamela

Huber, Rena Rekeweg, Sarah Robbiani, Thomas Wren and

Zuber contributed to this work

mentioned for

interesting

me

their

the lab E24 1 with

humor and

Jeannette Nussh introduced

During

in

at the Institute of Food

colleagues

working environment,

a nice

with his

Stephan Handschin,
developed

are

making my

time

am

thankful to all friends who

enjoyable

and for

making

are

life

not

more

Trudi and Peter

My parents,
and have given

me

Iten, have accompanied

unconditional

parents, Evelyn, Ralph, Silvia,

support

in

everything

me

throughout my

I have done

life

I thank my

Alex and Dominik for their love and their belief

in

me

My

husband

encouragement,
marvellous

Florian
the

Hug

many

great experiences

Thank you for

being

Zurich, July 7 2000

was

constantly

inspiring
in

there

discussions,

the Swiss mountains

the very person you

for

his

me

His

love,

his

and

our

enthusiasm

were an

invaluable

Susanna

Hug-lten

are

support

Table of Contents

abbreviations

SUMMARY

VII

Zusammenfassung

ix

INTRODUCTION

LITERATURE REVIEW

2.1

Staling

of bread

2.2

Role of starch in bread

2.3

2.2.1 Transformations of starch

during baking

2.2.2Transformations of starch

during staling

13

Current

antistaling

understanding

2.3.1 General features of

of the

amylases

2.3.2 Antistaling mechanism of

in

effect of

amylases

baking technology

amylases

17
17
18

EXPERIMENTAL

23

3.1

Material

23

3.2

Preparation

of bread

3.3

Preparation

of

3.3.1 Starch

gels

gels

3.3.2Wheat flour

25

and starch

dispersions

26
26
27

gels

3.3.3 Low concentration starch

dispersions

29

11

3.4

Table of Contents

Mechanical measurements
3.4.1 Instrumental texture
3.4.2 Uniaxial

30

profile analysis

compression

test of

of bread

30
32

gels

3.5

Light microscopy

33

3.6

Physicochemical methods

34

3.6.1 Determination of water content

34

3.6.2 Determination of iodine

X-ray powder

3.6.3 Wide-angle
3.6.4 Differential

staining

of starch

34

dispersions

diffraction

35

Scanning Calorimetry

3.6.5 Determination of starch and starch

35
36

degradation products

RESULTS AND DISCUSSION

39

4.1

Mechanical

39

properties

4.1.1 Influence of

of bread and bread models

amylases

on

the texture of bread

amylases
gels

on

the mechanical

4.1.2 Influence of
and flour

40

properties

of starch
45

4.1.3 Conclusions
4.2

52

Microstructure of

dough,

4.2.1

of method for

Development

bread and bread models

aged

amylases

on

54

light microscopy

4.2.2 Localization of starch in the microstructure of


4.2.3 Influence of

dough

and bread

starch

.55

62

amylases

on

the microstructure of flour

gels

and
71

gels

4.2.5 Influence of

the microstructure of fresh and

bread

4.2.4 Influence of

amylases

on

iodine

staining

of starch

4.2.6 Conclusions
4.3

54

81
89

Physicochemical properties

of starch in bread and in bread models91

4.3.1 Influence of

amylases

on

the

crystallinity

4.3.2 Influence of

amylases

on

the

melting

retrograded

starch

of starch

91

transitions of
99

Ill

4.3.3 Influence of

amylases
degradation products

on

the starch content and the starch


106

4.3.4 Conclusions

111

A COMPREHENSIVE APPROACH TO STALING

5.1

Comparison
organization

of bread and bread models at different levels of

Relationship

between starch structure and

5.2

113

5.2.11nfluence of

staling

5.2.3 Influence of

117

Soy--amylase

119

5.2.4 Contribution of different starch fractions to


Model for the
of

staling

amylases

5.4

Outlook

REFERENCES

115
115

Novamyl

5.2.2 Influence of BAN

5.3

113

of bread and the

staling

antistaling

120

effect
122
128

131

IV

Abbreviations
[mm2]

area

AL

amylose

AP

amylopectin

AS

available starch

BANU

-amylase

CLSM

confocal laser

day

db

dry

DP

degree

DSC

differential

ED

modulus of

eH

true strain

ESEM

environmental

force

height [mm]

Ah

compression

AH

enthalpy [J/g sample db]

HT

high-temperature

ITS

intermediate thermal

wavelength [nm]

KNU

kilo Novo

LM

light microscopy

MANU

maltogenic amylase

MW

molecular

Mi,2

melting

transition native

M3,4

melting

transition

amylose-lipid complexes

NMR

nuclear

magnetic

resonance

true stress

rH

relative

humidity [%]

Ri

melting

transition

retrograded amylopectin

R2

melting

transition

retrograded amylose

RS

resistant starch

Novo unit

scanning microscopy

base
of

polymerization
scanning calorimetry
deformability [N/mm2]

Hencky

strain

scanning

[-]

electron

microscopy

[N]

distance

oc-amylase

[mm]

stability

unit

Novo unit

weight
crystalline amylopectin

[N/mm2]

VI

temperature [C]

Tg

glass

Tm

melting temperature [C]

volume

weight

wb

wet base

26

scattering angle [degrees]

WAXS

wide-angle X-ray

transition

diffraction

VII

Summary
Starch

the

is

component

main

known to contribute

significantly

antistaling agents

has been

tion deals with the

antistaling

(Novamyl),

bread and bread models

wheat starch
The

key

elements

as

Bread

investigated

and flour

dispersions (40 %)

and

staling

were

followed

wide-angle X-ray diffraction,

and

gels

were

and bread

was

dough

by

Addition of

and to reduced

firming

and flour

gels

firmness

Unexpectedly,

amylase,
ther

on

to the

increase

prepared by

without

during baking

as

Novamyl

nor

as

This

granules

they

maintained the

granules

part

of the

Only

in

baked bread and

systems

and did not

and

by enzy

degradation products
systems investigated
rate of bread

firming

gel

of bread crust

typical birefringence

amylose

gels Soy--

fraction accumulated

also observed

weak

in

birefringence

models which became

Novamyl strong birefringence

more

birfringent

structures of

rich regions within the starch

systems

granules

with

were

In control

and
in

gelati

and formed

amylopectin

the center of

flour and starch

of starch

and

granules

during aging

already

Novamyl

not

of native starch

intense

occurred

nei

gels

gelatinized

polymers amylose

was

revealed

were

change during staling By combining bright-field

microscopy the

amylose

with

gels during

light microscopy,

calonmetry (DSC)

of bread crumb

phase separation

gels Polanzed-light microscopy


freshly

bread and

but without increasing the initial

granules

continuous starch network The two starch

starch

concentrated

closed moulds

in

affect texture of bread and flour

showed that starch

and

in

on

conventional

hand, reduced only the firmness of starch gels but did

On the other hand, starch

phase separated

yeast

from soy

it did not influence the firmness of starch

initial firmness

Light microscopy
nized

extent

same

the other

-amylase

BAN reduced the

during aging

rate

serves as a

maltogenic oc-amylase

as

staling

led to enhanced initial firmness of all

Novamyl

turn,

in

mechanical measurements,

differential scanning

amylases

is

present investiga

prepared by heating

matic determination of starch content and starch

of

for

responsible

structural, physical and chemical changes of starch

baking

1950 The

amylases which,

oc-amylase (BAN)

of

importance

degrading enzymes, namely

was

baking procedure Starch

early

as

reorganization during aging

The

staling

mechanism of

classical bacterial

to bread

recognized

base for the identification of

The influence of starch

of bread and its

after

baking

polanzed-light

were

samples

identified
and

in

as

sys-

VIII

Summary

terns with BAN

or

after several

birfringent only
gence

developed
It

granules
not

Soy--amylase

and that both

changes

polymers

which

is

the

does not
was

the

three

an

increase

necessarily

of

was

less intense birefrin

rich outer

zones

bread

during

fraction but also of

induced

during aging

fraction

confirmed with

crystallinity

and of

of the

staling

does

amylose polymers

higher

In the

case

initial crys

of

Novamyl,

polanzed-light microscopy

enthalpy changes

an increase in

which almost blocked

firmness

of

Fur

amylopectin

during aging Novamyl


of

rtrogradation

hand, the antistaling effect of Novamyl

one

tially degrade amylopectin

and

this hinder its

by

hand, the slight degradation of amylose


of this

polymer Thereby

birefringence
starch

and

sticky

hindered

explanation
staling

responsible

bread and flour

in

texture

found

amylopectin

in

all

behavior of

of

Soy--amylase
which

and

A model for bread

serves as

staling

is

in

on

the

ability

promotes

to par

the other

the

gelation

was

BAN induced substantial

as

gels

reduction of firmness

large

prevented

since

starch

crystalline

of starch

low MW dexnetwork

gels

No

The anti

be ascribed to the presence

can

accessible substrate before enzyme


is

mostly

inactivated before

accessible substrate

proposed

of

aging

the enzyme

on

the base of the

containing amylases

formation of starch networks contributes

recrystallization

on

prevent firming

starch

gels,

terization of bread and bread models

the effects of

endo attack

continuous

serves

based

recrystallization On

gels causing

BAN did not

In bread and flour

gelatinizes

is

for the enhanced initial firmness and

Firming upon aging

why

pregelatinized starch,

starch

is

the formation

was

inactivation

it

via an

prevents rearrangements

degradation

but also

of

center became

investigated systems

On the

tnns

granule

firming during aging

rate

correlate with

only amylase

of starch

crystalline amylose

crystalline amylose fraction

thermore,

amylopectin

amylopectin

firming

by

the

in

rich starch

Additionally,

rtrogradation

contribute to

caused

amylose

of aging

systems

in

the

Amylases reducing
tallinity

days

concluded that

is

involve

only

these

in

the

significantly

amylopectin

to

experimental

It

is

firming

charac

concluded that the


and overshadows

IX

Zusammenfassung
Starke stellt den
Backen
den

wesentlich

tragen

als

Amylasen

Der Einfluss

starkeabbauenden

von

Soja

einer

durch

Erhitzung

Brotteig

von

konzentrierten

in

(DSC)

Bereits 1950

erkannt

Die

namhch

Enzymen,

einer

und

Formen

Die

hergestellt

Mechanismus

maltogenen
und

einer

oc-

Brot wurde mit


wurden

Mehlgele

und

von

strukturellen, phy

der Starke wahrend des Backens

Brot und Gelen wurden mit mechanischen

und mit

vorliegenden

oc-Amylase (BAN)

produziert Starke-

Vernderungen

wur

Brotalterung

kroskopie, Weitwinkel-Rontgendiffraktionsmessungen,
metne

nach dem

Weizenstarkesuspensionen (40 %)

geschlossenen

sikalischen und chemischen


von

Brot bei

auf Brot und Brotmodelle wurde untersucht

ohne Hefe

und Alterns

Umwandlungen

alterungsverzogernden

klassischen bakteriellen

konventionellen Backverfahren

einem

von

Mittel

altbackenverzogernde

Amylase (Novamyl),
Amylase

Altbackenwerden

und den Schlusselfaktoren der

Amylasen

aus

zum

Brot dar Ihre

von

befassen sich mit dem

Untersuchungen
von

Hauptbestandteil

Differential-Raster-Kalon-

des

enzymatischer Bestimmung

Lichtmi-

Messungen,

Starkegehaltes

und der

Starkeabbauprodukte verfolgt
Die

Zugabe

von

reduzierten

einer

Systeme

Novamyl

wie

Novamyl,

hen

nur

die

Die Textur

Die

Erhhung

erhhte

jedoch

Brot und

krume und bildeten

Starke
und

in

Brot und

Amylopektin,

akkumulierte

chung
der

bei Brot und

in

in

nicht

im

glei

Zudem

ohne die

jedoch

Anfangsfestigkeit

zu

erho

nicht beeinflusst

typische Doppelbrechung

verkleisterten die Starkekorner der Brot

den Gelen induzierte


ein

Mehlgelen

dass die Starkekorner der Brotkruste wahrend

Hingegen

wobei sich

zu

Starkegelen Soja--Amylase

kontinuierliches Starkenetzwerk

Polarisierte

der Starke

Alterung

ein

und

aller untersuchten

Alterung

des Backens nicht verkleisterten und daher noch die


nativer Starke aufwiesen

Anfangsfestigkeit

Anfangsfestigkeit

von

wurde

Mehlgelen

Lichtmikroskopie zeigte,

die

Festigkeit

Festigkeit von Starkegelen,

von

der

wahrend der

Festigkeitszunahme

hatte BAN keinen Einfluss auf die


reduzierte

zur

Festigkeitszunahme

BAN reduzierte die

chen Masse

fhrte

Teil der

eine

Amylosefraktion

Gelen,

Nur bei Brot und Gelen mit

Verkleisterung

Phasentrennung

Lichtmikroskopie zeigte

frischen Broten und

Die

eine

im

von

der

Amylose

Starkekornzentrum

schwache

Doppelbre

welche intensiver wurde wahrend

Novamyl lag

eine

starke

Doppelbre-

Zusammenfassung

bereits

chung

zugeordnet werden

-Amylase

wurden

In den

Starkekorns

als auch

von

der

eine

kristalline

Festigkeit

dass

gezeigt,

nicht

eine

und beide

wahrend der
der

war

Polymere

Zonen des
Brot sowohl

Festigkeits

zur

Abbau

der

verhindert und
stantiellen

Amylose,

so

die

Festigkeitsreduktion

Effekt

Amylase,

eines

BAN

Starkegelen
von

in

welche die

Novamyl

von

zurck

der

Brot

Alterung

erfolgt
Folge

und

zu einer

wird

wurde keine

Soja--Amylase

vorverkleisterten Starke

inaktivierung vorliegt

der

werden,

eine

weitere molekulare

ein

von

klebrigen

der

Textur fhrte

in

Die

Erklrung gefunden

Starkegelen

Mehlgelen

Model fur die

sub

einen

grossen

Festigkeits

verhindert, da niedermolekulare Dextrine die


Fur die
Der

Alterung

Starkenetzwerken tragt wesentlich

deckt die Effekte der Rekristallisation

zur

von

von

wird durch die Anwesenheit der

ist das

von

Hemmung

festigkeitsreduzie

verursacht, die als zugngliches Substrat

In Brot und

rasche

Umordnung

einer

zu

teil-

dessen

eine

reduziert BAN verursachte

Mehlgelen,

einen

der

durch den Endo-Abbau

vor

der

Enzym-

Enzym praktisch vollstndig

Enzymabbau zugnglich

experimentellen Charakterisierung

Amylasen wird

wahrend

Rtrogradation

kann einerseits auf

gefuhrt

inaktiviert bevor die Starke verkleistert und fur den

Aufgrund

Schmelzenthalpie

Festigkeitszunahme

kontinuierlichen Starkenetzwerkes stren

Bildung

rende Effekt

was in

aber auch

zunahme wahrend der

Novamyl,

von

polarisierter Lichtmikroskopie besttigt

Festigkeitszunahme

Starkeabbau

verursach

blockierte

Amylopektin

von

Im Falle

der Knstallinitat und der

die einzige

festigkeitsreduzierende

Aggregation

von

von

einigen

eine weni

Alterung reduzierten,

korreliert mit der

Rekristallisation hindert Andererseits

mit

nach

erst

Alterung

Amylosefraktion

mit

Erhhung

unbedingt

Amylopektin weitgehend

in

oderSoja-

mit BAN

amylopektinreichen

dass wahrend der

gefolgert,

Amylosefraktion

Alterung Novamyl

weisen

Systemen

entwickelte sich zudem

den usseren,

Anfangsknstallinitat

Amylopektin

Der

in

Amylose retrogradiert

welche die

erhhte

Es wurde

und

beitragen

Amylasen
wurde

Systemen

In diesen

Daraus wird

Amylopektin

polarisierter Lichtmikroskopie

amylosereichen Starkekornzen

den

Kontrollproben

Doppelbrechung

ger intensive

eine

Hellfeld und

von

amylosereichen Starkekornzentren

die

Tagen doppelbrechend

zunahme

und vernderte sich nicht wahrend der

vor

doppelbrechenden Strukturen

konnten die

ten

Systemen

frischen

Durch die Kombination

Lagerung
tren

in

Brot

von

Brot und Brotmodellen

vorgeschlagen

Festigkeitszunahme

Amylopektin

wird

Die

Bildung

bei und ber

1
Introduction

Bread

of the most

is one

important

known for several thousand years


around 4000 B C
civilization

of

human nutrition The

high acceptance

properties,

particular

in

cereals for bread


for

special type
In

and

principle,
baking

physical

large

carbohydrates, proteins,

dietary fiber for


with the

In

making

steps

are

flavor of bread

mastered

parallel

of the world it

provides

substantial

E, minerals and

nutritional value of bread

significant
food

with the Western

fundament of human

several B vitamins, vitamin

daily

breadmaking

paired

due to its attractive sensory

item

while other cereals

is

are

used to

present
a

the primary

lesser extent and

breads

consists of the three processes mixing, fermentation

involve

transformations

constituents

foods and has been

texture and flavor Wheat and rye

production,

bread

All

in

as

Egypts

indispensable

an

areas

staple

almost

preparation developed

many cultures

in

the old

Already

Therefore, bread has been

existence
source

Bread

cereal based

by

complex

chemical and biochemical reactions and

which cereal flour

nutritionally accessible,

are

formed Textural

converted into

is

and

by

which the

quality usually

combines

product

typical
a

whose

texture and

crisp crust with

soft crumb of fresh bread

Immediately
bread, all

after

baking,

whole array of

at different rates and intensities

changes

starts to take

Except for microbiological

place

in

deterioration

1. Introduction

these

changes

referred to

are

of the

primarily by firming
of

changes
affects

aroma

that stale

products

at

The

Freshness is

large.

bread is unsalable and

produced

aging process

generally rejected.

are

the freshness

aspect

crumb, loss of crispness of the crust, and loss

compounds.

bakery products

staling. Staling impairs

as

As

presents

is not restricted to

important

to most

bread, but

consumers so

result, around 3 % of the annually

sizable economic loss

or

(Zobel

and

Kulp1996).
The

phenomenon

research

the

controlling

in

the

changes

has been

staling
it

Today,

groups.

transformations

of

is

starch

well

established

fraction

Due

proposed

in the texture. Gluten

the

on

or

of selected

expand knowledge
transformation
mechanism of
on

on

and

undesired

of bread in

and

aging.

As

should be elucidated

have

been

methods

of the starch fraction.

antistaling enzymes.
as a

among

of view of

antistaling

deal with the role of starch in


as

are

point

approaches

changes

and

factors

important

pentosans

Most of the

by many

migration

water

most

many

the contribution of starch

amylases

two

aging

prevent staling.

amylases

during baking

that

proteins

perspectives,

counteracting against

present investigations

application

clues

economic

to retard and limit

concentrate

The

to

the

are

the further constituents which influence the


texture.

since decades

investigated

staling

The main

goal

major texturogen

second

and the
was

and

on

to

its

focus, the antistaling

which, in turn, would give further

the structure and function of starch in the different

stages

of bread

making.
The

experiments
On

bread.

the

complement

the

were

other

carried out

hand,

interpretation

on one

model

hand with standard

systems

of the behavior of bread

gels

in closed containers

formed

system
This

was

type

bread

so

reduced

introduced

were

material. For this purpose, small concentrated


that neither

even more

of concentrated starch

crust

was

by preparing
gels

was

as a

nor

gels

wheat flour

in

order

to

complex composite

of wheat flour

similar

used

type

pores

were

prepared

developed.

The

of pure wheat starch.

successfully

in earlier studies

on

staling (Krsi 1983, Conde-Petit 1992).

As it

was

gel systems
staling,

postulated

that information

is needed for

on

developing

characterization of bread and

all dimensional levels of bread and the

comprehensive

model

gel samples spanned

on

baking

and

the range from

macro-

to nanoscale

mechanical,

as

illustrated and out-lined

microscopic

interdisciplinary approach
whose structural

amylose

and

in

Fig

and

physicochemical

is

general requirement

organization

from the starch

amylopectin comprises

six

1 1

methods
when

granule

orders of

Accordingly,
were

magnitude

applied

in

The

with starch

dealing

to the two

different

biopolymers

scale

Fig.

1.1:

on

of

Phase

and

amylopectin

protein

networks

of starch

granules

Composite

Networks

10u

Meter

LEVEL

in bread and bread model

characteristics

measurements:

properties

Compression

of starch

separation

Starch and

Disintegration

Light microscopy:

Mechanical

10"J

Millimeter

MACROSCOPIC

investigated levels of structures of starch

amylose

the different

Degradation

Overview

domains

Microstructure

10"

Micrometer

SUPRAMOLECULAR

starch determination:

Crystalline

Enzymatic

amylose

amylopectin

and

of

Phase transitions

WAXS:

DSC:

Physicochemical properties

10a

Nanometer

MOLECULAR

systems

2
Literature Review

Staling

2.1

of bread

The textural deterioration of bread upon


which has

intrigued

apparently

made the first scientific

between fresh and

researchers for

aged

bread

molecular state, which forms

pioneering

studies of Katz

of

aimed

starch

Interestingly,
a

prohibition

affecting
the

of

are

of

of

and

the

initiated

on

by

is

one

of the

phenomena

century. Boussingault (1852)


He

recognized

that differences

water loss but

develops

on

basis

particular

of bread. The

aging

the chemical and

molecular

by

crystalline

of

bread

discussions in the Dutch

nature

staling.

parliament

on

work for bakers. Over the years, various factors

have been

reviews

by

staling.

(1913a,b, 1930)

was

than

not caused

cooling

on

unhealthy night
staling

study

understanding

his research

bread

number

at

more

storage

investigated extensively,

published

(Bechtel

D'Appolonia 1977a, Knightly 1977, Kulp

1955,

and Ponte

which is reflected

Maga

1975,

Kim

by

and

1981, Hebeda and Zobel

1996).
The term bread
other than

changes

staling

refers to all

changes

microbiological spoilage (Bechtel

which

et al.

occur

1953).

in both the crust and the crumb and leads to

acceptance.

The crust, which is

becomes soft and

leathery

on

relatively dry, crisp

staling

due to

in bread after

Bread

staling

decreasing

baking

includes

consumer

and brittle in the fresh state

migration

of water from crumb to

2. Literature Review

and Larsson

(Eliasson

crust

crumbliness is observed

as

of the

typical

aroma

of fresh

Reineccius

(Achermann 1976,

Since starch presents the


view starch

having

as

major

quantity, protein

role in

of

of its contribution to the

discussion.

Investigations

Senti

cross-linking

and

rate

content

(Axford

and

(Kim

causes

et al.

of the starch

but to

Mita

gluten

higher protein

loaf volume

1968). Overall,
component

it

can

found to correlate

was

staleness and
monitor
like

dynamic

mechanical

Weipert 1997)
Russell

and

et al.

applied

been used

1999).

A
a

proteins

1963, Axford

followed

widely applied

by

At the molecular

different methods.

to follow starch

1991).

(Zobel
a

and

lower

higher protein
softer crumb
act

diluent

as

mechanism is caused

by

the

Kulp 1996).

valid

degree

et al.

et al.

(Knorr

instrumental

of

staling.

method to

1968). Rheological
1990, Vodovotz

et al.

methods

et al.

1996,

1976, Wassermann 1979,

1990, He and Hoseney 1990, Giovanelli

frequently.

et al.

with sensory evaluation of bread

significantly

tests

et al.

(Martin

to follow the rate and

analysis (Persaud

compression

(Cluskey

1983, Hibberd and Parker 1985, Redlingeret al. 1985, Baker

Faridi and Faubion

was

and

(Zobel

therefore, its determination is

staling (Szczesniak

gluten undergoes

investigators proposed

be concluded that the

increase of order in the starch fraction

Firmness

of bread. The

in bread is under

which, in turn, results in

firming

and is

Kulp 1996).

content of bread often results in

and that the basic

Numerous methods have been

and

(Zobel

not been confirmed

et al.

bread,

rtrogradation

of bread

aging

on

to

changes including

firming

Other

1990).

D'Appolonia 1977b, Callejo

higher

of

of

aging

much lesser extent than starch

1960,

and

have shown that

cross-links, however, has

Kulp 1996). Interestingly,

loss

stale flavor

important component

development

gluten gels

between starch and

The formation of such

staling

on

Dimler

of

bread, it is reasonable

series of

of bread crumb

firming

significance

1959,

of wheat

staling. On cooling

is the second most

changes during storage,

accompanied by

This transformation is called

cause

1968).

1992).

major component

crystallization.

to be the

thought
In

and

are

et al.

(Axford

development

in the starch fraction lead to

rearrangements

gelation

bread and the

significant

in the crust

crispness

increase in firmness and

an

in the crumb

typical change

of the crumb and loss of

Firming

In contrast,

1993).

et al.

et al.

1988,

1997)

have

level, the ordering of the starch fraction

Differential

rtrogradation

scanning calorimetry (DSC)

in bread

(Fearn

and Russell

is

1982,

2.2 Role of starch in bread

Hoseney 1986, Czuchajowska

Zeleznak and
et al.

1995, Schiraldi

Crystallinity

1996, Leon

et al.

of starch in bread

was

(WAXS) (Katz 1930, Dragsdorf


al.

(Wilson

spectroscopy

spectroscopy (Kulik
water is

further

and

has

al.

1991, Chen

et al.

which

is

during

storage temperature

of

for

one

are

factors

exist

crumb

2.2

and Ponte

investigations

on

on

starch in bread.

and Ponte
in

1981).
and

mobility

1983, Kim-Shin

formulation

retard

to

firming. Mixing conditions,

in the field

component

and

of

prevention
Zobel and

(1981),

et

i.e.

As

etc.

Pentosans,

staling.

are

influencing staling
of

the

primary

fermentation time and


of bread. In the
the

staling prevention, namely

Antistaling enzymes mainly

act

the

on

bread, which makes it necessary

general.
of

staling

For

comprehensive

to

overviews

the reader is referred to

Maga

Kulp (1996).

present

the function of starch in bread is abundant. In

starch itself

were

During processing

different levels of

and

state of

Role of starch in bread

The literature

is

NMR

by many factors,

Monoglycerides

process variables

the bulk

as

softness.

will be discussed.

affecting staling

(1975), Kulp

et al.

is affected

parameters,

describe also the role of starch in bread in


on

dynamic

changes

staling (Leung

and

1995)

also used. The

study

staling

methods

possibility

amylases,

starch fraction

al.

et

staling process (Kulp

bread

bread

surfactants used to inhibit crumb

application

et

and sugar influence for instance the moisture level of bread

important

following, only

are

to

production

different

oligosaccharides

applied

protein,

temperature,

consequence,

1980, Varriano-Marston

1997).

Besides starch and

moisture,

Champenois

factor in the

micro-redistribution of water

diffraction

by wide-angle X-ray

1983, Zeleznak and Hoseney 1987a). Infrared

1991,

been

1989, Champenois

1997, Defloor and Delcour 1999).

determined

Haverkamp 1997)

important

NMR

Recently,

al.

et

et al.

and Varriano-Marston

et al.

1980, Pisesookbunterng

and Pomeranz

initiated in order to understand the role of

bread, starch undergoes

granule organization

in the form of native starch

retrogrades during storage

overview

of

on

the

native

fact, much basic

and structure. In the

severe

changes

dough stage,

at

starch

granules. Starch gelatinizes during baking

of bread. The

organization

present chapter

of starch

and

its

role

contains
in

dough.

short
The

2. Literature Review

transformations of starch
in

chapter

2.2.1 and 2.2.2.

Native

starch

is

in

organized

consist

of

glucose

105

between

amylose ranges

basic

as

contains

amylopectin additionally

exhibit

1987).

Three
A

amylopectin:
1984).

(outer),

chains
B

The

respectively.
1988). Mainly
amylopectin

are

responsible

starch

on

and

use

amorphous

an

overview

Angstrm

of different

proportion

amylose

molecular

and Bowler
branched

and Daniel

where B- and

DP of approx. 15 and

in

granules

Based

15 to 45%

45,

(Zobel

et al.

by

on

the

1997).

nm

in the

on

X-ray diffraction,

the

an

A-type pattern.

The

amylose

structures

are

and

amylopectin.

packed

organization

in different

of the starch

range and also review the starch

are

dependent

conditions. While the structure of

of

of

microscopic and spectroscopic techniques.

The characteristics of cereal starch

growing

degree

multiple

crystallinity (Gallant

consists of both

granules

(1997) give

the

has

of

double helices of the side chains of

characterized

are

from the micrometer to the

structure based

packed

C-chain,

molecules is the

granule (French 1984).

fraction of starch

shells. Gallant et al.

to

chain) (Whistler

linked

weight

molecule is described to extend from 120 to 400

Crystalline, partially crystalline

on

main

for the observed

of native cereal starches

amorphous

(the

the

of native starch varies from

ordered structures, i.e.

radial direction of

granule

and C

amylopectin

crystallinity

single amylopectin

crystals

in

connected. The A- and B-chains have

are

The molecular

points (Galliard

distinguished

are

(inner)

The backbone of the

A-chains

The

of

both of

whereas the branched

(Zobel 1988). Amylopectin

around 108 with approx. 4 to 5% branch

details

(1^4)

corresponds

weight

types

amylopectin,

block.

glucan amylose,

106 which

and

more

partially crystalline

and

building

oc(1^6) linkages.

of approx. 3000

polymerization (DP)

and

in

explained

are

biopolymers amylose

units form the linear

oc-D-glycopyranosyl

staling

granules

structure. Starch is built of the two

which

and

during baking

genotypic

on

and

amylose

and the amount of

lipid

variation and

amylopectin,

are

under

the

genetic

control, the size and number of starch granules, composition and gelatinization
behavior of starch

development

of the

wheat starch is
ones

(A-type)

granules

are

influenced

grain (Tester

composed

are

(B-type)

of

by

et al.

spherical

during

the

1995, Tester 1997). Like barley and rye,

bimodal distribution of starch

lenticular and have


are

the environmental conditions

with

diameter of 25
a

diameter

granules.

The

large

40 urn, and the small

ranging

from

5-10

urn

2.2 Role of starch in bread

(Seidemann 1966).

B-Starch constitutes 24 -28 % of the total starch volume but

about 90 % of the starch

proportion

of small

flour. A- and

(Soulaka
29

B-granules

than in

%)

free

more

acids

starch

with the

granules

1970).

granules
The

Eliasson

have

and Eliasson

described

as a

protein

with

of the

hydrolytic

surface coat of

Transformations of starch
starch

partially

(Keetels

et al.

et al.

and Larsson

in

the

polarized light

granules

has been

act

as

inert

studies showed that

a mere

surface

filler

(Hibberd
and

(Larsson

are

considered to be

protein (Eliasson

free"

water

are

was

and Larsson

associated

forming

protein phase.

during baking

same

which

as

but still maintain their


et al.

1984).

granular

The swollen

essential structural elements of

time, the gluten transforms from

means

that the

during baking

wb, which is close

occurs

gel

gel

looses its cohesiveness

crumb

does

not

at intermediate water

to the moisture content of

baking. Oven temperatures during baking range


temperature

spherical

1993).

Transformation of starch
i.e. around 46 % w/w

as

1985).

dough. Recently, dough

1980, Pomeranz

At the

have similar

degrading enzymes (Lindahl

the continuous

solubilized starch act

1996a).

grain

A-granules

and Morrison

granule

starch

granules gelatinize

coagel by polymerization,

(Eliasson

starch

wheat

properties

controversially. Dough

behavior of wheat

than

%)

same

function than that of

complex

The

A-granules (approx.

(Bloksma 1990). Other

matrix

of

and

when observed under

is discussed

interpenetrated by

identity (Varriano-Marston

to

(Soulaka

bicontinuous network of starch and

During baking,

bread

of the

1993).

hand, B-granules contain

material where the native starch

rheological

network which is

and

is found in

the other

B-granules

cross

dough

a more

1993). Starch granules

granules

composition

1992, Martfnez-Anaya and Jimenez 1997)

for the

2.2.1

Maltese

and the presence of

important

baking performance

in the native state and appears

specific properties

1997)

%). On

measured with DSC

present

composite

as a

amylose

27

A- and

Its role in

fillers in the continuous


starch

as

typical

(Seidemann 1966).
described

is

for the

lysophospholipids (approx.

%). Generally,

dough,

More

1985).

and Larsson

(Eliasson

of wheat starch vary in

and

gelatinization properties
In

important

B-granules (approx.

fatty

0.8

(approx.

is

granules

and Morrison

in number

granules

exceed

content,

dough prior

to

from 180 to 250 C whereas the


100 C

due

to

constant water

10

2. Literature Review

and condensation. These conditions

evaporation

starch in the crumb at the level of


molecular

of starch

morphology

The term

arrangements.

gelatinization

connection with starch and refers to irreversible

heating

upon

melting

of

(Atwell

et al.

improved

of starch in water

due to

glass-to-rubber

origin

and

(ii)

melting

The transition

of

Fig.

(melting

melting process. One


can

or

Both

complexes,

which

melting

complexes

are

of

occurs

as

melting

transition

the botanical

on

to follow

gelatinization

1979, Biliaderis

excess

M-,.

et al.

amylopectin crystallites

endogenous lipids

the

water,

Reduced water content

temperatures
at limited and

M2) (Biliaderis 1998). Therefore,

crystallites,

of

around 60 C

temperatures

transition which extends to

of

is

two-stage

such

(M3

as

and

cereal

amylose-lipid

(Biliaderis 1998). Contrary

dissociation
et al.

of

to

amylose-lipid

1980). During baking

of

on

cooling (Eberstein

are

melted, which allows swelling and solubilization of

of starch

of the starch

dependent

transition

between 100 and 150 C

and Larsson

1993).

amylose leaching

have shown that

into the center of the

layers

glass

(i)

temperature Tm (Roos 1995,

applied

is detected at

melting

amylopectin

dispersions

the

attributed to the order-disorder transition of

bread, starch crystallites

Upon heating

as

heating,

two additional endothermic first-order transitions

is reversible

(Eliasson

and

of cereal starch at intermediate water

be detected in starches which contain

starches.

starch

curve

melting

transition

intermediate water contents,

starch

are

known

leads

granules

2.1. When wheat starch is heated in

second

1988).

and Bamunuarachchi

(Wootton

also referred to

appearance of

around 99 C

in

and starch solubilization

hydration

at the

crystals

temperatures

amylopectin crystallites

(Eliasson 1980),
causes

in

presented

of

in the

as

(double-helical) order,

nature of starch

amorphous phase,

1980, Eliasson 1980). A typical DSC

the

upon

of starch. DSC measurements have been

melting

of

physical changes taking place

and Bock

transitions

phase

transition for the

1998).

content is

well

as

has become established

disruption

level, the partially crystalline

starch in presence of water

M4)

and

gelatinization (Ranhotra

temperature Tg,
Biliaderis

granules

changes

1988, Biliaderis 1998). Furthermore, the digestibility of starch is

characteristic

two

irreversible

loss of molecular

involving

crystallites, granular swelling

At the molecular
to

cause

granules,

amylose

whereas the

granules (Langton

occurs.

Microscopical

is leached out of the

amylopectin

and Hermansson

studies of

granules

and

remains in the outer

1989, Conde-Petit

et al.

11

2.2 Role of starch in bread

1998). Leaching
amylopectin
1987).

polymers
occurs

amylose

due to mutual

Phase

moderate

of

separation

is

result of

and

amylose

amylopectin,

to the solvent water

effective

(Hsu

and Prausnitz

concentration

of

both

microdomains is raised which may lead to the

(Biliaderis 1998).

essential

structuring

In

retains the

1974).

When

occurs

Ring

even

element

(Eliasson

and Larsson

Conde-Petit et al.

and

their

of

two

respective

composite gel

network which is

an

1993).

of the

granular

starch structure

complete disintegration

1998). Starch

granular identity. Light microscopy

at

phase separation

in

development

bread, leached amylose builds

place involving swelling, collapse

(Seidemann 1966,

are

which

polymers

During gelatinization, morphological changes


take

and

thermodynamic immiscibility (Kalichevsky

of

and

amylose

concentrations, is promoted by the asymmetry in the affinity of the

the

network

of

phase separation

of bread

of the

granule

crumb, however,

revealed that the starch

granules

swollen, deformed and aligned along the pore surfaces (Burhans and Clapp

1942, Sandstedt

granules

is

et al.

thought

1954, Varriano-Marston

et al.

1980).

The orientation of the

to arise from the extension of the lamellae due to the

gas cells in the initial

stage

of

baking.

growing

2. Literature Review

_|

40

Temperature (C)
2.1:

L_

140

90

Typical DSC curve of cereal starch at intermediate water content


(50 w/w wb). M-, and M2 indicate melting of amylopectin crystallites. M3
and

M4
1998).

refer to dissociation of

amylose-lipid complexes (Biliaderis

13

2.2 Role of starch in bread

2.2.2

Transformations of starch
in the starch

Rearrangements
bread, play
introduced

staling

important

an

is not

only

staling process.

of starch from

solubility

process which

defined

(1988)

of starch

bread crumb.

staling

may

develop into

conditions,
includes

more

gelation

and

synonymous, and
formation

regarded

of

rtrogradation

three-dimensional

crystalline

the roles

pure

have the

order

(Zobel

and

faster than that of

follows:

as

In its initial

definition, rtrogradation

to form

and

and

crystallinity

Juncture

order.

are

not

result in the immediate

interchain

points

be

can

associations, i.e.

of such double helices

can

lead to the

Kulp 1996).
and

were

the

gels, although

amylopectin. Gelation

is

In order to

amylopectin.
studies

amylopectin,

solutions

amylopectin

tendency

this

necessarily

result from

amylose

and

played by amylose

amylose

on

crystalline

Aggregation

involves both

Rtrogradation

Based

does not

rtrogradation

formation of double helices.


formation of

performed.

gelation

of

dependent

the

on

separate

rtrogradation

Both starch

much

amylose proceeds

on

length

amylose (DP

660)

Bulpin

<

1989).

of DP

tend to

Gelation

measurements of the

(DP

>

2500)

faster with

showed
shorter

explained by

of

polymer concentration,

1100 whereas short and middle molecular

storage
slow

solutions

modulus

development

in G' whereas
et

al.

the formation of cross-links between

mobility

Gelation of

amylose

and

thereby

is followed

slow down the

by

interchain associations with

a
a

followed

(G'). Amylose

(Clark

chains

was

slow

with

This

very

high

was

behavior

kinetics of

crystallization

DP of approx. 30

in the

long

DP

attained

long amylose chains,

gelation

and

by rheological

plateau G'

1989).

weight

yield partially crystalline precipitates (Gidley


amylose

amylose

retard chain

involving

>

of

polymers

DP, temperature and time (Biliaderis 1998). Gelation of amylose requires


minimum chain

simple juncture point which then

crystallization. Thus, rtrogradation

molecular order which

as

Nowadays,

extensively ordered regions. Ultimately, under favorable

crystalline order appears."

during

when the starch molecules of

occurs

starch chains may form

or more

was

but is related to

gelatinized starch begin to reassociate into ordered structures.


phases, two

of

aging

rtrogradation

change

used in connection with bread

basic starch transformation. Atwell et al.

Starch rtrogradation is

The term
a

and

cooling

occur on

Lindet and described

by

which reduces the

rtrogradation

fraction, which

role in the

in 1902

already

during staling

was

which

chains.

B-type

form

(Ring 1987). Thus, aged

14

2. Literature Review

amylose gels
junction

consist of double

zones, and

networks, which
al.

gel

can

be melted at

only

of

recrystallization

structure

development
amylopectin

and

temperatures

thermostable

are

around 150 C

of

in

starch

networks is related to

gel

(Eberstein

et

long-term development

of

are

primarily

systems (Miles

ordering

et al.

(DP 10-20) (Ring

amylopectin gels
et al.

determines the

amylopectin

crystallinity

chains

(Kalichevsky
a

crystallites (Biliaderis 1998). Amylose gels

1980, Ring 1987).


The

of

helices, small aggregates of double helices, i.e.

determined

1990). Unlike amylose,

melting temperature

1987).

around 60 C in

and

gelling

the

length

ordered

The

1985b).

crystallization

The

by

al.

et

of the short

time and the

rigidity

of the side chains

amylopectin

structures exhibit

water. In contrast to native wheat

excess

starch, where crystalline amylopectin is ordered in the A-type form, recrystallized


structures show

found between

B-type pattern (Miles

amylose

and

et al.

amylopectin regarding
is

rtrogradation. Amylopectin crystallization

solutes at the molecular level

reducing

chain

Sugars,

solutes.

small-molecular-weight

mobility

and

1985b). Thus,

instance,

by

act

the

are

after

presence of

antiplasticizing

as

action of water

plasticizing

of

crystallization

crystalline pattern

influenced

for

to the

(relative

the

differences

no

amylopectin (Slade

and

alone)
Levine

1991).
Due to its

bread. Swollen starch

granules

During baking, amylose forms


as

well

as

added

in the central

mainly

affects starch

event is obtained at

of

since

amylopectin

temperatures

(1947)

of bread is due to

can

The

continuous

with

leads to

single

reorganization

be followed

C,

is

hardly

by DSC.

matrix.

amylose

endogenous

amylopectin

around 60

amylose

Recently,

were

wheat

lipids

helix with the

of

amylopectin

leached

during

An endothermic

which is often termed the

the first researches who

rtrogradation

retrogrades during cooling.

on

complexes

staling

(Eliasson 1985).

and that the linear

based

inclusion

granule rigidity

Schoch and French

their work.

embedded in

cavity (V-type amylose).

baking. Rtrogradation

staling

are

lipids (Zobel 1988). Complexation

ligand

endotherm

is essential for the structure of fresh

rapid rtrogradation, amylose

of the branched molecule

fraction has

no

The current

understanding

staling

models of Schoch

model

(1965)

suggested

was

influence

on

presented by

and Lineback

staling

of

amylopectin

since it

staling

that

already

still builds

Zobel and

(1984) including

on

Kulp (1996)
the current

15

2.2 Role of starch in bread

molecular
describes

view

of

changes

starch

and

starch

Additionally,

indicated. In the

Fig. 2.2,

starch

and

indicating

bread

as

continuous

as

crystalline fraction

indicated

amylose

Fig.

2.2:

as a
on

refreshment of

granules

in

dough

are

aged

whereas

characteristic

the

granules.

amylose

is in the

component

In

dough stage
bread

model
to fresh

by heating

is

smaller than those in fresh

dough. Gluten

is shown

dough, amylopectin

is shown

their unswollen native state in

phase surrounding

The

(Fig. 2.2).

of starch at the molecular level from the

and stale bread crumb.

aged

rtrogradation

amorphous

state. Polar

of wheat starch which

can

lipids

are

interact with

gelatinization.

Model for bread crumb

(Zobel

and

Kulp 1996).

staling showing molecular starch structures

16

2. Literature Review

In fresh

bread, crystallites

shown in the
can

into the

intragranular space.

part

of the

therefore, amylopectin in Fig. 2.2 is


is located

Leached

amylose which
wheat

endogenous

is indicated

amylose
which is

retrograded

as

granules

forms inclusion

but

double helices.

complexes

with

measurements of fresh

lipids during baking. Thus, X-ray

V-type pattern (Zobel 1988).

in double helical structures

During aging, amylopectin reorganizes resulting


and

granules

for the initial loaf firmness. That

important

remains in the

in the

mainly

is shown in both the inter- and

intergranular space. Amylose

promotes gelation

bread show

melted and

amorphous stage. Amylopectin

protrude

This form

are

From the model

crystalline regions.
of

reorganization

(Fig. 2.2)

it

be concluded that the

can

to the swollen

amylopectin imparts rigidity

At the

granules.

same

time, cross-links develop between amylose and the remaining granules in the
matrix of the bread. The formation of
to

network structure is

increasing rigidity. Cross-linking promotes

can

be

(Senti

and Dimler

According
amylopectin

fraction since

are

pointed

characteristics

different

of
of

phases

dependent

the

amylose

gelatinized

are

100

as

Redistribution takes
and starch.
redistributed

water

place

However, it

properties

the

on

of

composite food

order

volume

be

of water

during aging (Willhoft 1971).

the

The

rigidity

of bread.

only

plasticization

during baking
Although

redistributed

estimated that

starch.

like

1993). Additionally,

the extent of network

firming

of

fraction,

and Larsson

between crumb and crust

was

temperatures

and the interactions between the

(Eliasson

absorption

may

the

hand, remains unchanged

molecular

granules

well

dependent

Increased

the

matrix,

the initial softness and decrease the

during staling,

is melted at

primarily

C).

on

starch

important

(Biliaderis 1998).

occurs

the other

on

out that the mechanical

the final firmness is also


water

crystalline regions

of bread affects

retrograded amylopectin

stability (>

only

not

(deformability)

(Fig. 2.2), reheating

Retrograded amylose,

because of its thermal

It should be

of this network which

continuity

than the extent of

firming

contributing

1960).

to the model

around 60 C.

bread

for bread

important

more

the

factor

one

no

can

well

as

increase

loss of water

during aging

as

with

of

between

bread.

gluten

about 3 % of the total water is

2.3 Current

of the

understanding

effect of

antistaling

Current

2.3

understanding
amylases

2.3.1

General features of

Amylases
influence
Mulvihill

are

the

on

sugars in

dough

product

at all

and

of sugars with

Little is known

staling

levels of

proteins.

the

production

of Maillard

have

an

and

aging (Fox

level of fermentable

products

due to the reaction

the influence of

regarding

and water retention in crumb.

are

amylases

Finally, amylases

in cereals.

endogenously present

4 d

over a

or

breadmaking by producing

-amylase

is found in

via disulfide bonds


used to

optimize
are

amylases

can

on

gas

retard the

the

specificity,

(Kruger
wheat

the

origin

of the

plants,

animals

and malt

degradation products

amylase

1987).

endogenous enzyme

from

and

are

oc-amylase

increases
This

1987).

wet, sticky bread crumb. Unlike oc-amylase,

and Lineback

fungal, bacterial,
the

intact cereals have low

and Lineback

period (Kruger

sound, intact grains and is attached

produced

from

5 d

Sound,

the level of

oc-amylase. However, upon germination

several hundred fold

quality,

higher

they

of bread.

Amylases

affects

and

as

therefore, improves the loaf volume. Additionally, flavor and

by

dough

industry

production

results in

amylases

effect of

baking technology

of bread

stages

crust color is intensified

retention in

in

antistaling

used for various purposes in the bread

Addition of

1982).

of the

amylases

17

amylases

levels and to

for

influence

microorganisms.
often used.

sources are

important

glutenin proteins

Technical enzymes, which

and

and the

to the

In

are

bread

baking,
like

Properties

thermostability are dependent

on

such

as

technological processes

breadmaking.
Amylases

-amylase

are

and

classified

glucoamylase,

and release maltose

Thus, -amylases

and

the

can

amylopectin

-limit

dextrins.

act

the

on

products

of

nonreducing

cannot attack the

fully degrade

end of

molecule to the

oc-(1-6)

On the other hand,

oc-amylase

action

on

oc-(1-6) linkages

the linear

randomly hydrolyzing oc-(1-4) linkages


final

endoenzymes. Typical exoenzymes,

like

starch molecule

by hydrolyzing oc-(1-4) linkages (Hoseney 1986). Unlike

glucoamylase, -amylase

strips

as exo- or

branch

to

are

amylopectin.

-maltose,

points resulting

endoenzymes

of starch

starch

amylose

of

like

but

only

in maltose

oc-amylases

act

(Hoseney 1986). Therefore,

the

low MW dextrins of

varying

size.

18

2. Literature Review

The

of

extent

starch

and

temperature

degradation

hydrolyze native, undamaged


starch

dough,
The

Hebeda

is

hydrolysis

al.

et

(1991)

starch at

dependent

low

65

70 C.

main

60

C)
of

part

C)

results in residual

sticky

example

are

The

On

from Bacillus

(barely

by

suggested

responsible

Hoseney 1991,

firming

can

for

1999).

have maximum
inactivated after

fungal

et al.

of

the

thermal

other

The

higher stability
for

gummy

degradation. ITS
and

sources

and Norman

hand,

stability (Toptimum

responsible

1991)

stability

inactivated before

originate

and from

for

selected

1984).

is known since decades.


and bacterial

of starch

effect.

oc-Amylases

sources

by amylases

mainly

leads to

bread

Martin and

as

changes

in

staling (Schultz
et al.

or

the

different mechanisms

particular

Hoseney 1994, Leon

serve

mechanism

antistaling

Consequently,

that dextrins of

from

Thus, it is conceivable that

extend bread freshness have been

retarding

Akers and

be

of low MW dextrins.

antistaling

high

amylases

malt), fungal,

production

and

thermal

due to excessive starch

amylases

oc-amylases

Defloor and Delcour


that

mechanism of

cause an

which

are

which

starch structure is involved in the

Several authors

amylase

product

baking. Degradation

changed

formed dextrins
of action

baking

higher

(Hammer 1995).

megaterium (Hebeda

wheat

starch structure and

and

after

effect of

or

additives in bread

either the

much

stearothermophilus (Outtrup

Antistaling

cereals

to ITS enzymes

activity

antifirming

lower

gelatinizes (Hammer 1995).


have

of

attack.

temperature optimum

have

isolated from both bacterial and

strain of Bacillus

2.3.2

have

enzymes

starch

texture of the final

enzymes

are

are

oc-amylases

to

(intermediate thermostability) enzymes

fungal oc-amylases

compared

as

ITS

starch, but

able

amylases.

intermediate

low,

than ITS enzymes and in consequence

conventional bacterial
70 -80

termed ITS

The

1991).

Conventional

50

(""optimum
the

al.

et

were

of the

retarding staling

of

gelatinization temperature

baking. Such enzymes


(Hebeda

for

is

amylolytic

to

thermostability

between

distinguished

pH,

on

During heating

rate.

susceptible

the

thermostability enzymes. Optimal amylases


activities above the

degradation

more
on

dependent

Only oc-amylase

substrate.

and becomes

gelatinizes

of

degree

of the

accessibility

is

by amylases

proposed.

size

produced by

et al.

1952, Martin

1997, Min

Hoseney (1991 ) presented

of bread crumb results from cross-links between the

et al.

model

gluten

1998,

stating

matrix and

2.3 Current

starch

of the

understanding

found

that

added

starch-protein

oc-amylase

branched-chain dextrins of low molecular


are

believed to hinder the

developed

wheat bread
concluded

in

firming

bread

is

dextrins

when

measured

maltotetraose
bread

involves

dough

DSC.

with

Min

produced by

is

amylopectin
(1999),
a

al.

et

different

method such

does not
the

necessarily
firmness

oc-amylases

opposition
firming

of

The authors
starch-starch

antistaling
a

effect of
decrease

amylopectin rtrogradation

as

the

primarily

due to dextrins which

molecules. Similar conclusions

who showed that

reduction of

addition of

amylopectin rtrogradation
that

(1998) suggested
oc-amylases

an

maltotriose

responsible

are

for

and

retarding

of

(Zobel

and

Senti

to note that

measured with DSC.

as

amylopectin rtrogradation
Kulp 1996). X-ray

higher

starch

1980). Only

firming

Brthen

based
causal

only

on

one

relationship

(1997) reported

rate of bread but had

firming

and

and

only

that

small effects

on

However, this stands in


is

mainly responsible

for

diffraction showed that bread with

crystallinity

softer than untreated bread

and Varriano-Marston

crystallinity

Sahlstrom

bread.

amylopectin

were

important

are

exist between the structure of starch at the molecular level

addition had

although they

and

DSC. It is

to the view that

of bread

oc-amylase

as

reduced the

rtrogradation

Zobel

in

regular

to

rtrogradation.

specific

of

into the role of

oc-amylase.

Some conclusions of the studies mentioned above

and

(1997)

by glucoamylase. Therefore,

oc-amylase

caused

products

et al.

similarly

changes

to

which could not be detected when

removed

were

Defloor and Delcour

maltodextrins to bread
as

insight

more

breads behaved

adding oc-amylases,

interfere with the reassociation of

by

gain

test and reduced

produced by oc-amylase

drawn

amylopectin. Morgan

also concluded that the

(1997)

authors concluded that the action of

were

some

amylopectin

These branched-chain

rate and the addition of

by compression

by DSC

detected

degraded

related to the starch fraction. The authors found

mainly

rate

of

firming essentially

Leon et al.

interactions.

oc-amylase

These starch

regarding firming

that

weight.

rtrogradation

interactions.

starch-gluten

of

positive impact

cross-links. Lin and Lineback

partially

starch bread in order to

gluten-free

19

amylases

the bread texture with the formation of maltodextrins of DP 3-9

on

which interfere with the formation of

(1990)

effect of

remnants. The authors correlated the

granule

oc-amylases

antistaling

than

(Zobel

unsupplemented

and Senti

bread

1959, Dragsdorf

in bread without enzyme addition

an

increase

could be correlated. On the basis of these

findings,

(1959) postulated

that

oc-amylases

attack the

long

starch

20

2. Literature Review

molecules of the

amorphous regions

Cleavage

long

of the

starch

freedom for

crystalline regions

the entire structure is less

crystal

chains, which

weaken the three-dimensional

regions,

chains

which link the

near

to

structure within those

action of

endoamylases.

The

and

network

laterally by

Kulp 1996). On

protrude

into the

amylopectin

interaction with

permitting greater

another. As

result,

time, greater freedom of


and

enlarge

the

perfect

model accounts for the

some

exoamylases

chains to the branch

model of Zobel and

remove

since

between

crystalline

could

points.

adjacent crystalline regions (Zobel

staling

amylases

different

branches to form double helices and

or no

intergranular space

cross-linking

consequence,

of

one

to

presented

antistaling properties

it is conceivable that

(Fig. 2.2),

same

segments

The

regions.

the basis of the

of

independently

enable such

Thus, amylopectin has fewer, shorter

through

of the network

strength

be attributed to the removal of the outer

to build

pass

i.e. softer. At the

rigid,

crystalline regions

move

can

crystalline regions (Fig. 2.3).

they

amylopectin

branches which

the most accessible. As

are

and

amylose

Kulp (1996)

is

amylopectin

hindered

(Bowles 1996).
Recent work concluded that dextrins
correlated with the

(Gerrard

et

al.

Duedahl-Olesen

al.

et

modification of the starch. Krsi and Neukom


effect of added dextrins is

gels

amounts of dextrins

in

gluco-oligomeres
is

responsible

postulated

that

the formation
starch.

to act

for

the

gels.

(>26 %)
he

positive way,

as

Based

Although

plasticizer

antistaling

that

seems

that

in contrast to added

to influence starch

starch structure,

of starch

properties
not

enough

and that rather the modified starch fraction

effect

of

amylases.

and

rtrogradation

Kweon

et

al.

(1994)

amylopectin, thereby promoting


which retard

understood.

are

responsible

of starch would be affected

the exact nature of

for the

of

rtrogradation

maltodextrins, in the right place and

rtrogradation. However,

which

high

the addition of

only

by

structure, it may well be that the maltodextrins in situ formed by amylase

present,

plasticizing

effect in

antifirming

oc-amylases produce

amylopectin-lipid complexes,
it

found that the

for their

be

can

reflect

1999). They merely

influenced the mechanical

proposed

loaf

staling process directly

the results that

on

amylase degrades amylose


of

amylase-treated

(1984b)

mainly responsible

concentration wheat starch

high

an

rate but do not inhibit the

staling

1997,

in

present

at the

right

changes

antistaling effect,

its

are

time

in the

is not

fully

2.3 Current

understanding

of the

antistaling

effect of

77/
'

//

'

''

<''

/y
F/g.

2.3:

i
W

--'

Network structure of starch in the


indicated with

xKv

<

//'.''<:

regions
by bars (Zobel and Senti 1959).
are

21

amylases

arrows

% /'"

gel phase of bread crumb. Crystalline


points of amylase action

and possible

22

3
EXPERIMENTAL

3.1

Material

Commercial low-extraction wheat flour, type 400,


Stadtmhle Zrich
13.4 to 14.7

(Zurich, Switzerland).

g/100 g wb, protein

content from 0.35 to 0.38

Pressed

yeast

was

g/100 g

purchased

Native wheat starch and

(Wdenswil, Switzerland).
was

potato

received from

g/100 g db,

The

as

measured

enzymes

by

listed

from

starch

local grocery store.

was

amylose

Klinglerfrom

size exclusion

in

Tab. 3.1

(Bagsvaerd, Denmark). They

and ash

db.

supplied by

without

Blattmann Cerestar AG

amylose-butanol complexes

TFH Berlin. This

extracted

weight

is around

chromatography.

were

had been

was

amylose

from aqueous solution of smooth pea starch and the molecular


300'000

Swissmill,

Moisture content of flour varied from

content from 11.8 to 12.1

Isolated

received from Prof. R.

was

received

purified by

from

Novo

Nordisk A/S

Novo Nordisk down to very

limited side activities.

Unless otherwise

(Buchs, Switzerland)
or

specified,
or

all chemicals

Merck Ltd.

were

purchased

(Darmstadt, Germany)

and

from Fluka Ltd.

were

of

analytical

puriss quality.

23

ADN

07154)

KNU2Vg

(Kilo

a-Amylase Unit)

is defined

Novo

Unit)

is

a measure

per min.

259 Merck

Amylum

solubile

Erg. B.6)

per h.

the amount of enzyme which, under standard conditions

(101

as

for

dextrinizes 5.26 g starch db

(-Amylase

M)

Novo

80 C

(37 C, pH 5.6,

7.0

Ca-concen-

concentra

5.0-7.0

5.0

4.8-6.0

pH optimum

(substrate

55-60C

60

45-75C

stability

under standard conditions

1585BANU3Vg

256

MANU1Vg

Thermal

Properties

maltotetraose to maltose. Thereafter, maltose is further

-amylase activity and is determined relatively with a known standard by hydrolyzing


degraded to gluconate-6-phosphate by means of maltose Phosphorylase (MP),
-phosphoglucomutase (-PGM) and -glucose-6-phosphate dehydrogenase (GPD). During the reaction sequence nicotinamide-ade
nine dinucleotide (NAD+) is reduced to NADH+H+. The speed of NADH+H+ production is proportional to -amylase activity.

3) BANU

bean

from Bacillus

-amylase extracted from soy


purified (Nagase, Japan)

and

oc-amylase
amyloliquefaciens

bacterial

1500

Specific activity

(Maltogenic Amylase Novo Unit) is defined as the amount of enzyme which,


mg/ml, 37 C, pH 5.0, incubation time 30 min) hydrolyzes 1 uJVI of maltotriose

tration 0.0003

2) 1 KNU

tion 10

1) 1 MANU

Soy--amylase
(batch F47)

(batch

BAN

maltogenic oc-amylase from Bacillus


stearothermophilus produced by
genetically modified Bacillus subtilis

antistaling agents.

Novamyl
(batch AB2 1330)

as

Description

Properties of amylases used

Enzyme

Tab. 3.1:

3.2

25

of bread

Preparation

Preparation of

3.2

White pan bread


the enzymes
flour.

The

were

was

bread

prepared

from low-extraction wheat flour. 42 g

stirred into different

optimal

of

amount

deionized

between 64 -67 g water/100 g flour

(Pitec AG, Oberriet, Switzerland)


20 g salt

600 g each
moulds.

Proofing

was

temperature

are

some

(3

of

100

dough preparation

No.

for 4 to 5

listed

in

dosages

PR

7 d.

Steam

After

were

was

The breads

cooled to

were

to

sealing.

concentration

at

The

three

the bread

was

to 7 d at

the

times

again

in

the

higher

sealed in

initial

method

dosages

as

of the

end

an

aging

baking phase

plastic bags

and

for further 4 d at 20 C.

Tab. 3.2:

Dosage of amylases.

Enzyme

Dosage

Novamyl

750

BAN

Addition

MANU/kg

KNU/kg

flour

flour

as

powder,

1.172

ml/kg

500

mg/kg

flour

flour of 1 %

(w/v)

solution of BAN

Soy--amylase

5283

BANU/kg

flour

as

powder,

3333

of

control.

reheated at 170 C for 20 min after

added

of

baked at 200 C

used. Bread without enzyme served

was

high

(Pitec AG, Oberriet,

rapeseed displacement

formerly 72-10) prior

bread

cooling,

4 min at

GmbH, Affalterbach, Germany).

Wiwa

the

mixer

100, Eiken, Switzerland)

chamber

proofing

by

speed. Dough pieces

by

h, sealed in plastic bags and stored for 0

Tab. 3.2.

was

kneaded in

was

at low

baking phase.

measured

was

and varied

followed

mixing

(Wiesheu

oven

115/1)

was

speed,

greased (Trennaktiv

added in the initial

experiments

ml).

dough

for 3 min at low

carried out in

Method 55-50

recommended

period

was

Bread volume

(AACC, 2000,

In

in

placed

ventilated

100 ml steam

enzymes

for

at 30 C and 80 % rH for 90 min. The loaves

for 40 min in

20 C.

water

The

(wb).

added after 2 min

was

were

Switzerland)

room

of the water and added to 1000 g

by recording farinograms (ICC-Standards,

determined

speed.

portions

and

yeast

mg/kg

flour

aged

26

3.

3.3

Preparation of gels

3.3.1

Starch

heating

starch

method

adapted

suspensions

pregelatinized
starch

In
and

starch

starch/100 g

gel)

were

first

the

step,

stirring (90 rpm)

followed

with and without enzymes in closed moulds


and Krsi and Neukom

(1983)

the main

stirring

(1984a)

in

starch

pregelatinized
450 g of

Brabender

by cooling

stirred for 5 min

cylindrical

suspension (4 g dry

rate of 3 C/min. In

prepared by heating

was

from 30 to 95 C with

Viscograph

were

dispersion

native starch

to 30 C at

added to the

second

greased (Trennaktiv

Germany)

30 mm,

(diameter:

step,

PR

100,

sealed and heated in

for 60 min at 96 C. The

immersion in cold water

(10 C)

ventilated

gels

were

for 10 min.

Biocides, Manchester, UK),

0.14g/100g paraffin).

trimethylene-4-isothiazolin-3-one
calculated

so

that the

served

control.

some

Gel

The

ratio is

enzyme

equal

experiments

aging

was

procedure

was

the moulds

were

removed

(Zeneca

to

paraffin (approx.
2-methyl 4,5-

concentrations

in bread and starch

gels

altered

as

were

gels

without enzyme

follows:

extended up to 182 d.

pregelatinized

dispersion

the standard

were

Promexal X50

starch content of 70 % w/w wb for flour. Starch

assuming
as

starch/enzyme

The

previously

prevent

5% aqueous solution of

(MTI).

into

oil to

paraffin

added

was

filled

moulds

opening

Thereafter, the gels

preservative,
a

The

cooled without

microbiological spoilage,

Promexal is

was

(WTB Binder, Tuttlingen,

oven

from the moulds and stored for up to 12 d at 20 C in


moisture loss. In order to avoid

and

dispersion

which had

mm),

Switzerland).

Eiken,

20

native starch

DZM, Janke & Kunkel,

RW20

height:

starch/100 g

rate of 1.5 C/min

starch

pregelatinized

(500 rpm, anchor-shaped stirrer,

brass moulds

hermetically

In

shown in

as

of the starch in

portion

Labortechnik, Stauten, Germany). The starch dispersion

been

using

in order to avoid the sedimentation of native

dispersion

and the enzyme

powder

IKA

prepared by

during gelatinization.

suspension)

by

from Krsi

gels (40 g dry

3.1. This method consists in

Fig.

dispersions

and starch

gels

concentration starch

High

Experimental

wheat starch

(0.4g/100g).

dispersion

Xanthan

powder

was
was

replaced by
carefully

xanthan

dispersed

in

3.3

Preparation

of gels and starch

deionized water and heated in


for 30 min and stirred with
the xanthan

steps

are

dispersion

which

The heat treatment


30 min followed

3.3.2

Wheat flour

Flour
moulds
in

gels
as

chapter

sealed.

were

at 96 C

cooling,

subsequent

and

deionized

water

used instead of deionized water.

were

as

Ca/I,

50 mg

approx.

prolonged by holding

prepared by heating
gels. Dough

3.2 and filled into the


and

cooling

Promexal to

experiments

stirrer at 300 rpm. After

the

temperature

at 60 C for

described in the standard

procedure.

gels

(chapter 3.3.1). Likewise,

some

held at 40 C

was

procedure (Fig. 3.1).

(180 mg Ca/I)

by heating

dispersion

mixed with starch and enzyme. The

was

was

used for starch

Heating

containing

anchor-shaped

contained

enriched with calcium

an

water bath. The

identical to the standard

Tap water,

27

dispersions

the

the flour

dough

without

greased

were

prevent

bread

yeast

brass

carried

gels

was

out

were

prepared

on

as

were

stored

at 20 C

in

same

described

hermetically

described for starch

as

gels

paraffin

microbiological spoilage.

conditions

elucidate the influence of enzyme inactivation

in the

yeast

moulds, which

moisture loss and

time/temperature

without

aging

were

varied

of flour

gels.

in

oil
In

order to

28

3.

Experimental

Heating / Pregelatinization
Brabender-Viscograph
20 C to 95 C, 1.5 C/min, 90 rpm
starch

Addition of native
wheat starch

Addition of
-

Novamyl

g/100 g

wb

Cooling
Brabender-Viscograph
C to 30 C, 3 C/min, 90 rpm

amylases:

Mixing
(40 g/100 g wb)

total starch

-BAN
-

95

dispersion:

and enzyme for 5 min

Soy--amylase

I
Filling
greased moulds
(cylindrical brass moulds)
the

I
Heating
(60

min / 96

/ Gelatinization

C,

ventilated

oven)

I
Cooling in water
(10 min/10 C)

I
in

Fig.

3.1: Standard

Storage
paraffin with Promexal
(20C/0to12days)

procedure for the production of starch gels.

3.3

Preparation

3.3.3

of gels and starch

Low concentration starch

The influence of enzymes


studied

in

low

dispersions

prepared

starch

ratios

acetate buffer of

pH

The tubes

heated for 5 min in

with

magnetic

being placed

in

The

After

for 30

required

were

according

(pyrex

were

and

amylopectin

to Tab. 3.3 in order to

The

then air-cooled for

Novamyl,

were

was

g/100 g, respectively.

dispersions

were

in 2 g 0.01 M

with teflon lined

screw

caps).

stirring

few minutes before

70 C for BAN and 60 C for


added to the substrate.

was

heated for another 5 min at 150 C in

order to inactivate the enzymes and then air cooled to

dispersions

amylopectin

amylopectin (28 mg)

amount of enzyme

min, the tubes

and

oil bath at 150 C under constant

an

water bath at 65 C for

Soy--amylase.
incubating

or

5.5 in 10 ml culture tubes

stirrer. The tubes


a

amylose

in Tab. 3.2.

as

prepared by suspending amylose (12 mg)

were

of

dispersions. Amylose

calculated

were

starch/enzyme

constant

staining

at concentrations of 0.6 and 1.4

The enzyme concentrations


obtain

dispersions

iodine

on

concentration

were

29

dispersions

room

temperature.

The

stored for 0 to 48 d at 20 C.

Tab. 3.3: Calculation

of enzyme

dosage used for low concentration starch

dispersions.
Dosage/kg
flour1)

Dosage/kg
starch
(70%

Novamyl

BAN

Soy-amylase

of

flour)

Dosage/g
amylopectin
(70%

of

starch)

Dosage/g
amylose
(30%

of

starch)

750 MANU

1071 MANU

1.5 MANU

3.5 MANU

(500 mg)

(714 mg)

(1.00 mg)

(5.25 mg)

3 KNU

4.3 KNU

0.0061 KNU

0.0143 KNU

(11.7mg)

(16.8 mg)

(0.024 mg)

(0.056 mg)

5283 BANU

7547 BANU

10.7 BANU

25.2 BANU

(3333 mg)

(4762 mg)

(6.75 mg)

(15.90 mg)

1) values from Tab. 3.2

30

3.

ZA

Mechanical measurements

3.4.1

Instrumental texture

Bread crumb
machine
loaves

was

profile analysis

subjected

to

of bread

compression

(Zwick 1445, Ulm, Germany) equipped

were

cut in 4 slices of 30

and

two

repeating cycles

texture

mm

profile analysis (TPA)

test with

with

universal

an

each. The crust of the slices


carried out

room

temperature,

which simulates the

slice. The bread slices

were

compressed

15

curve

crosshead

speed

of 50 mm/min.

of bread crumb which

converted

recovery

was

into deformation

(2)

were

evaluated

Firmness

[N]

Elastic recovery

of 1st

[%]=

biting
40

typical

of

bread

plunger

force-time

can

and

(1)

removed

mm

twice. The time axis

The crumb firmness

described

Fmax

with

3.2 illustrates

compressed

values.
as

Fig.

(50 %)

was

each slice with

by compressing

mm

testing

1000 N load cell. The

at

and

Experimental

also be

the elastic

by Szczesniak (1963).

cycle

(1 )

(2)

-100
a

F:

force

a:

compression

distance

b:

compression

distance of reversible deformation

Besides these two


from the

slope

parameters,

of the linear

compression cycle
carried out

[N]
[mm]

the modulus of

region

according

to

equation (3)

deformability ED

of the force-deformation

between 5 and 10 %
in

compression.

chapter

[mm]

3.4.2.

was

curve

calculated
of the first

The calculation of

ED

was

31

3.4 Mechanical measurements

Time

>-<

1st Cycle

Fig.

3.2:

Evaluation of

[s]

2nd Cycle

typical force-time

TPA. The time axis

can

curve

of bread crumb obtained

also be converted into deformation values.

by

32

3.

Uniaxial

3.4.2

Uniaxial

were

the universal

compressed

50 mm/min.

previously

The

been

plates (Bagley
the

slope

12

testing

greased

1985).

of the linear

to

92

paraffin

were

carried out at 20 C

plates

at

and

mm)

crosshead

the

lower

deformability ED

of the force-deformation

curve

was

The

gels

speed

plate

to reduce the friction between

The modulus of

region

compression according

between two

plate (diameter:
with

gels

(Zwick 1445, Ulm, Germany).

machine

(60 %)

mm

upper

et al.

gels

test of starch and flour

compression

using again

test of

compression

Experimental

gel

of

had
and

calculated from

between 5 and 10 %

equation (3).

AFh
F

(3)

ArM

modulus of

ED:
^'

'

deformability [N/mm2]
~

10 % compression

"5

% compression

h0:

initial

A0:

initial cross-sectional

Ah:

compression

For the calculation of the


transformed to true stress

1987).

of the

height

The true stress

actual cross-sectional

(a)

that the

gels

of the

A(t)

are

gel [mm2]

[mm]

true

the force-deformation

Hencky's

or

calculated

by dividing

of the deformed

CT

Assuming

area

breaking stress,

was

area

gel [mm]

distance

versus

L'^J

strain

curves were

relationship (Peleg

the actual force

F(t) by

the

gel:

F{t)

incompressible, A(t)

A(t)=A0

is

given by

hn
'0

h0-Ah

(5)

3.5

33

Light microscopy

The true strain

(eH)

was

calculated

H=\n

by

(h \
^

Ih J

true stress

F(t):

actual

A(t):

actual cross-sectional

A0:

initial cross-sectional

h0:

initial

Ah:

compression

eH:

true strain

breaking

maximum

compression

height

stress

stress and

f ^h

IK-

[N/mm2]

a:

The

=ln

of the

force

[N]

area
area

of the

of the

gel [mm2]

gel [mm2]

gel [mm]

distance

[mm]

[-]

(aBreak)

and

strain

breaking

(eHBreak)

defined

were

as

the

strain, respectively, that the gels could sustain before

failure.

3.5

Light microscopy

Small

pieces

of

dough,

bread

were

cut from the center of

were

immediately

were

soaked for 15 min under

crumb, bread

dough,

crumb and

frozen with carbon dioxide


vacuum

without

prior

fixation.

samples

thickness.

microscope slides,
improve
coating
for

The

samples

were

microscope

slides

polarized-light microscopy.

The bread crumb

sucrose

then

was

mounted
a

used for

cut

in

in

the

were

C)

for sections of

frozen

state

glycerol/gelatin
No

which
either

to -150

Reichert-Jung

displayed

solution of

samples

The thin sections

samples

compound (Miles,

isopentan (-160

cryosections during staining.


was

mm)

solution, and then frozen

were

Cryostat

which had been covered with

the adhesiveness of the


of the

(-78 C).

frozen with

at -20 C.

7x7x7

gels, respectively. Dough samples

Thereafter, the samples

cryostat (Leica, Vienna, Austria)

10|im

were

gels (approx.

in Tissue-Tek O.C.T.

Kankakee, USA) diluted 1:4 with 20 % (w/v)


with carbon dioxide. Gel

crust and

to
to

glycerol/gelatin

were

determined

directly

observed

34

under

polarized light

in order to

to

localize the

amylopectin,
Light Green

well

as

14

mM, Kl

were

by

droplet

1:10

of

to

described above. All

specimens

were

examined in

in the

3.6.1

Determination of water content

were

then

total water content

were

ground
was

was

was

determined

3.6.2

staining

photometric

and dried for

Kontron

(,max)

for

to

2 min.

solution

as

and

polarization

mode.

at 130 C for 90 min in

The water content of bread

predried

second time at 130 C for 90 min. The

drying steps.

gravimetrically using

an

The water content

infrared

at 120 C for 10 min. All

dryer (LP 16,

analyses

were

duplicate.

staining

of low concentration

determination of

with

before

Axioplan photomicroscope

by drying

calculated from the two

determined

added to 1.5 ml starch

performed

The

s.

stained, the thin

cooled for 2.5 h at ambient conditions. The

Determination of iodine

Iodine

bread crumb at 103 C for at least 12 h. Before

Mettler-Toledo, Greifensee, Switzerland)


carried out at least in

l2

methods

by predrying

samples

of native starch

bright-field

min,

covered

glass

glycerol/water

an

(WTB Binder, Tuttlingen, Germany).

oven

determined

samples

with

solution

to 20

were

cover

starch had to be

the

Physicochemical

the

only

for 30

solution

(stock

samples

and

covered

3.6

weighing,

The

Lugol's

(Zeiss Ltd., Oberkochen, Germany)

was

stained with aqueous

was

iodine vapor of

were

The water content of flour

(1:1 v/v)

When

microscope.

Thereafter, the sections

ventilated

first stained

fractions, amylose and

solution

staining step.

solution

glycerol/water

exposed

were

or

AG, Buchs, Switzerland)

(v/v) Lugol's

rinsed in water after each

examination under the


sections

contrast. Protein

Fluka Chemie

diluted

birefringence

Experimental

mM, Fluka Chemie AG, Buchs, Switzerland) for 10

44

slides

improve

(1 g/l,

starch with iodine in

of starch

degree

and the two starch

proteins
to

as

solution

the

assess

3.

light

dispersion.

scanning speed

of starch

starch

absorbance.
A

wavelength

dispersions

dispersions
Lugol's
scan

of 100 nm/min in

was

solutions

analyzed by

(10|il)

from 500 to 750

was

nm was

photometer (Uvikon 940,

Instruments, Milan, Italy). The wavelength of the maximum absorbance

was

evaluated.

3.6

Physicochemical

3.6.3

equilibrated

over

least 12 h before
13

using CuKoc

radiation

3.6.4

slit of 1

Differential

gels

after various

DSC

Kristalloflex

selected.

were

Samples

Elmer

(Perkin

pans

carried out

empty pan

heating

was

analysis

was

of 5-30

on

used

was

melting enthalpy (AH)

Thermal

as

slit of 2

mm

recorded with

degrees

Analyst

was

software program. The exact

with

crumb, flour

bread loaf

or

gel

directly weighed

USA).

CT,

DSC

(DSC 2910,

calibrated before

analysis

samples were

TA
with

heated from

nitrogen flushing (20 cm3/min).

until 200 C. The

matter content

individual pan after the DSC

scan

by puncturing

16 h. The

were

expressed

in

2000

and of

amylose-lipid

the TA Instruments
determined for each

was

the pan and

J/g sample

In

melting temperature

thermograms using

dry

bread

were

recrystallized amylopectin

evaluated from the

enthalpy changes

on

Norwalk,

reference. The

performed

of

carried out

Ltd.,

rate of 10 C/min with

experiments, heating

were

temperature

divergence

of approx. 30 to 40 mg

an

complexes

The

D500, Karlsruhe, Germany)

taken from the center of

were

indium, and

and

diameter

holder.

sample

X-ray intensity

was

Ltd., Newcastle, UK). The system

(Tm)

on

for at

degree/min.

Instruments

some

thickness and

scattering angle range (26)

times.

were

mm

C)

was

Scanning Calorimetry

pressure

4 to 160 C at

% rH at 20

(91.2

with 35 mA and 40 kV. A

degree

gels. Samples

aging

measurements

The freeze-dried material

mounted

Scanning Calorimetry (DSC)

and starch

into

of 0.05

Differential

was

(Siemens

(1.54 )

scintillation counter in

scanning speed

powder.

(WAXS), samples

carried out in the transmission mode at ambient

were

receiving

it to thin disks of 1-2

pressed sample

diffractometer

powder

to

saturated barium chloride solution

compressing

The

mm.

measurements

and

diffraction measurements

wide-angle X-ray powder

frozen, freeze-dried and ground

were

on

diffraction

Wide-angle X-ray powder

For the

of

35

methods

drying

db.

at 105 C for

36

3.

3.6.5

Determination of starch and starch

Total starch content of freeze dried flour

gels (0.4 g)
starch

gels

were

incubated with 0.5 ml thermostable

and cooled to

with 5 ml buffer

(buffer:

amyloglucosidase

by

using

ml)

and

acetic acid.

the

60 C water bath. The

determined

and maltose

as

method 61B/1.1

12

h).

gels

and

was

L, Novo

4.6-4.8

to 4.6 with

of 1 ml

Diagnostic

samples

cooled to

were

determined

were
room

enzymatically

(Swiss

Absorbance

).

Official

measured in

was

and the amount of starch

was

used for the determination of starch

was

the

ethanol, samples

kit number 1 113 950

incubated

with

analyzed

were

for

Diagnostic AG,

Roche

and

amyloglucosidase

glucose content
Maltose and

of bread

glucose

were

glucose

were

determined from dried crumb

extracted

10 g crumb to 100 ml deionized water and


After

temperature.

of flour

glucose.

evaporating
(test

remove

described above.

Maltose and

adding

After

Germany)

Mannheim,

(103 C,

water),

samples

glucose

pH

After addition

dissolved in 1 ml nanopure

the basis of the released

glucose

to

Roche

Analysis (1994),

degradation products.

120

(10 mg amyloglucosidase (208469

The ethanol extract of flour

free

adjusted

was

hexokinase/glucose-6-phosphate-dehydrogenase

on

step

sodium-hydroxide, pH adjusted

photometer (4010 Clinicon, Mannheim, Germany)

calculated

Freeze dried flour

oc-amylase (Termamyl

The mixture

and filtered. The amount of

Manual of Food
a

1000

to

AG, Mannheim, Germany)

temperature

Analysis (1994).

the

following

water bath for 15 min under constant

boiling

70 ml 0.5 M

solution

stirred for 30 min in

temperature.

room

acid, and diluted

acetic

determined

The residue of the ethanol extraction

Nordisk, Bagsvaerd, Denmark) in

stirring,

was

two times extracted with 50 ml 40 % ethanol in order to

degradation products.

was

degradation products

gels

method of the Swiss Official Manual of Food

Experimental

filtering,

maltose and

glucose

the

by grounding

stirring

content

crumb,

for 20 min at

were

room

determined

as

described above.

Resistant and available starch

(5 g)

was

bread

The

determined

added to 10 ml deionized water and

suspension,

weighed

were

and

which

dispersed

samples

were

correspond

to

dry

with

0.2 ml

homogenized.

Bread crumb

Around 550 mg

substance of 100 mg,

in 10 ml KCI-HCI buffer

incubated

simultaneously.

pH

1.5

(50

mM

KCl,

was

exactly

30 mM

porcine pepsin (Merck

No.

HCl).
7190,

3.6

Physicochemical

2000

FIT-U/g)

for

protein degradation

9 ml Tris-maleate buffer
0.04 %

(w/v)

added and

degradation

6.9

pH

sodium azide

(0.1

(NaN3)

at 40 C for 60 min. For the

samples kept

Tris-maleate, 4 mM CaCI2) containing

added and the

was

remaining

the resistant starch fraction. The latter fraction

kept

at

room

was

in the residue

was

for

washed twice

corresponds

to

in 3 ml deionized

dispersed

temperature

shaking

under constant

stirring

for

Then, 3 ml sodium-acetate buffer of pH 4.75 (0.4 M sodium acetate,

CaCI2)

supernatant,

was

added and

supernatant

(Fluka 10115)

pH

was

analysis

4.75 and

and the residue

adjusted

were

of starch.

The

with 2 M HCl.

of available

pH adjusted
then

for 30 min at 60 C. Glucose

for determination

summing up

pH

which is used for the

8 ml sodium-acetate buffer
of the

to 6.9 with

supernatant containing

collected and the sediment

was

water and 3 ml 4 M KOH and

the

centrifugation

with 10 ml of water. The starch fraction

20 mM

pH adjusted

for 16 h at 37 C under continuous

of available starch. After

the available starch fraction

30 min.

following step,

pancreatic oc-amylase (Sigma A-3176, Buchs, Switzerland)

NaOH. Solution of
was

37

methods

amount

hydrolyzed

was

with

determined

of total

starch

the fractions of resistant and available starch.

the

was

mixed with

(2 M).

The starch

starch,

with HCl

Similarly,

amyloglucosidase

as

described above

was

calculated

by

38

4
Results

4.1

Mechanical

During baking

and

macroscopic level,
a

system

(Eliasson
by

in

of bread

storage

and Larsson

bread and bread models

complex

an

1993)

(Axford

open pore

system,

Firmness

readings

et al

and flour

gels,

which

baking,

was

were

not

only

considered

bread crust had

that

freshly

of

around 46 to 47

was

content to around 44

types

studied
as

in

of

gluten (Kokini

reveal that
is

et al

an increase

accompanied by

1994)

wb

g/100 g

was

change

foam to

are

bread

from

sponge

influenced

strongly

et al

of porous structures

1996b)

Therefore,

crumb, but also

starch

in

found

was

slight

(Zeleznak
g/100 g

and

leathery

During

in

diagrams

in

bread crust

components

In bread

water

Hoseney 1987b)

found

as

to rubber transition of the two

consequence, the crust becomes soft and

decrease

The state

during storage

and wheat starch

wb

similar to the water content

Only

wb

g/100 g

On the other hand,

wb

g/100 g

of moisture from 10 to 30

glass

from

At the

occur

bread models without pores

baked bread crumb

g/100 g

is

water content of around 10

the water content of


which

and

dough

of bread

aging, the water content increased to around 30

dough

changes

1968, Wassermann 1979, Keetels

turn reflects different densities and

amylases

structural

induces the solidification of

with gas cells to

the influence of

After

properties of

baking

the bread volume

which

Discussion

and

As

crumb, the rather

39

40

4. Results and Discussion

high

moisture

promotes rearrangements

of the

mobility

g/100 g

wb for flour

moisture loss

4.1.1

changes

of

amylases

gels

negligible

was

quality

of bread. The

crumb since it is the bulk

wb for starch

and

The

gels.

therefore, rheological

be excluded.

in

aging,

on

following

phase

can

of crust,

crispness

g/100 g

between 46 and

was

the texture of bread

on

in texture of bread

crumb and the loss of


textural

bread models

and around 60

gels

during storage

Influence of

The

based

gel

due to variations in water content

changes

high

polymers.

The water content of the


47

in the starch fraction because of the

are

particular

the

essential factors in

discussion is limited to

which is most affected

of bread

firming

the

reducing

changes

by changes

in bread

in the starch

fraction.

The bread loafs


varied

depending
had

Novamyl

-amylase
and

(crumb
on

the

specific

of 3.6 0.1

Soy--amylase

and

crust)
of

type

had

weight

amylase

added. Control bread and bread with

cm3/g

volume of 3.3 0.1


and 3.2 0.1

cm3/g

did not

or

of approx. 500 g. The volume

and bread with BAN and

Soy-

cm3/g, respectively. Hence, Novamyl

only slightly

affect the volume whereas BAN

increased the volume.

The

changes

of bread

instrumental Texture Profile


on

crumb

at the

macroscopic

Analysis (TPA).

the firmness of bread crumb is shown in

showed

higher

initial firmness and

did not affect the initial firmness


rate

over

influence

dough

an
on

and

aging period
firmness and

sticky

lower

compared

of 7 d.

firming

Fig.

Novamyl

studied

firming

by

amylases

4.1. Bread baked with

Novamyl

rate than control bread. BAN

to the control but reduced the

Soy--amylase

had

was

firming

only

Bread baked with BAN gave

bread crumb and the porous structure

bread. In contrast,

were

The influence of different

The added
rate.

level

little

sticky

different from control

did not induce stickiness and the crumb

quality

was

similar to control bread.

The influence of
in

Fig.

amylases

on

the elastic recovery of bread crumb

4.2. The elastic recovery is

deformation, which is equivalent

a measure

to the

for the

reversibility

springiness.

are

shown

of bread crumb

The elastic recovery of

4.1 Mechanical

properties

control bread

41

of bread and bread models

reduced from 96.3 % to 75.9 %

was

On the other hand, the elastic recovery of fresh bread with


reduced

to control bread but did not

compared

to the

control, but in

elastic recovery still decreased

on

on

the

containing Novamyl

from 94.0 % to 83.8 %. BAN

bread showed reduced elastic recovery after

slightly

was

the elastic recovery

contrast to bread

aging

Novamyl

aging (elastic recovery

on

%). Soy--amylase improved

between 88.8 % and 91.1

aging compared

change

of 7 d.

storage period

over a

containing

and further decreased upon

baking

aging.
The modulus of

deformability ED

compression cycle

and is

shows

bread

trends

of

ED
as

aging

already

of the first

measure

containing

of

compression cycle

reduced the increase of


BAN reduced the

of bread

different

with the firmness

seen

rigidity

firming

calculated from the

was

amylases. ED

TPA). Novamyl

aging compared

on

rate and

enhanced the initial

Soy--amylase

had

same

(maximum
and

rigidity

an

enzyme-free

only

little effect

to

4.3

Fig.

showed the

deformation

high

at

of the first

at small strain.

rigidity

readings

slope

control.
bread

on

rigidity.
Keetels

ascribed the increase of

(1995)

increase in stiffness of swollen starch

amylopectin. However,

granules

bread crumb is

due to

much

aging

on

of starch

reordering

more

of

gels

to

amylose

an

and

which

complex system

aging (Cluskey

et

1959, Senti and Dimler 1960, Mita 1990) it is assumed that the properties

at

contains also
al.

ED

the

But since

protein.

macroscopic

level

are

protein changes only

mainly

determined

little

by changes

on

in the starch fraction.

The elastic recovery and the firmness reveal mechanical characteristics of


bread crumb at

large

deformation which

during chewing (Szczesniak


recovery and firmness

are

et al.

are

1963).

dependent

on

comparable
The

the

type

of

leads to low firmness values and

which is

a-amylase,

to

great

classical

extent. In terms of

elastic recovery.

amylase

amylase

high

added. An ideal

BAN,

reduced the firmness but also the elastic recovery

by

an

excessive

degradation

It is assumed that due to the low thermal


was

between elastic

elastic recovery.

of starch via endo

hand, Soy--amylase had only minor effects

the enzyme

perception

texture, low elastic recovery correlates with sticky

bread crumb which is caused


attack. On the other

consumer

relationships

antistaling enzyme
a

to

inactivated before most starch

on

firmness and

stability

gelatinized

of

Soy--

and could be

42

4. Results and Discussion

which

degraded. Novamyl,

(Outtrup

and Norman

is

1984),

actually

turned out to be

showed clear

antifirming effects,

retained. This

can

other

partly

amylase

(Dauter

with

degradation

on

et al.

unusual

and at the

as

an

same

ideal

maltogenic a-amylase
antistaling enzyme.

time the elastic recovery

the characteristics

1999)

of

properties

mechanism. The

Novamyl
in

present

terms

(Christophersen

et al.

1998)

showed that this enzyme is


of enzyme

structure

results corroborate these

and

findings.

It

was

stability. On

be ascribed to its intermediate thermal

hand, investigations

the structure

classified

the
and

an a-

starch

4.1 Mechanical

properties

43

of bread and bread models

35

Control

Soy--amylase
CA
CA
d>
C

Novamyl

E
BAN

Aging

Fig.

4.1:

Influence of

Novamyl,
n
12).

aging time

BAN and

time

10

[d]

the firmness of bread crumb

on

Soy-$-amylase. (Mean

containing

with standard deviation,

100

Novamyl

90-

Soy--amylase
80a>
>

Control

o
o

70<

BAN

CA
re

UJ

60-

50
0

Aging

Fig.

4.2:

Influence

of

6
time

10

[d]

aging time on the elastic recovery of bread crumb


containing Novamyl, BAN and Soy-$-amylase. (Mean with standard
deviation, n
12).
=

4. Results and Discussion

0.15-

Control

it

0.10-

Soy--amylase

0.05-

10

Aging
Influence of
crumb

aging time

on

[d]

the modulus of

containing Novamyl,
n
12).

standard deviation,

time

BAN and

deformability ED of bread

Soy-$-amylase. (Mean

with

4.1 Mechanical

4.1.2

properties

Influence of
flour

on

the mechanical

of starch and flour

properties

compression

of starch and

properties

gels

as

of

measure

gel rigidity

characterized with

were

ED

measurements. The modulus

determined

were

Gbreak

amylases

gels

The mechanical
uniaxial

45

of bread and bread models

and the

breaking

at small and

stress

large strain,

respectively.
stress-strain

Typical
times

are

defined

the

as

and starch

strain

as

of flour

of

compared

apart

together

and

deformability

Fig.

of flour

on

the other

fractures

to

the

of the starch

Keetels

strain

breaking

behavior of flour

lower stress and lower

observed

(1995)

network

of the

granules

were

is

on

compression

hand, showed large fractures and tended

compression.

stiffening

the

breaking

occurred at

gels

gels. Small

to starch

before 60 %

4.5 and

gels

and starch

firming

Fig.

swollen

results in

4.6 the influence of

and starch

gels

with

gels containing

Novamyl

rate than control

breaking

lowest
the

Failure of flour

whereas

aging

which

breaking stress,

found in the

were

at various

gels

attributed the increase

holding

granules

the starch
whereas

decrease of the

granules
reduced

strain of

breaking

gels.

In

the

increased

stress,

stress to the formation of

breaking

the

gels.

the

During aging,

Differences

gels.

gels. Starch gels,

to break

4.4.

Fig.

maximum

decreased for all

gels

in

presented

of control flour and starch

curves

stress.

breaking

stress and also

starch

compression experiments

stress but

to starch
a

In flour

gels.

firming

as

higher

they

the

initial

breaking

stress abreak

Both flour

presented.

breaking

stress and

gels, Soy--amylase only slightly


gels

reduced

gels.

on

different enzymes is

In contrast, starch

Novamyl containing

opposition

showed

time

aging

Flour

were

with

Soy--amylase

aging rate,

gels

was

gels.

reduced

comparable
during

to

the

deformed. This stands in

which showed

rate similar to control starch

lower

showed the

with BAN did not break

only plastically

gels containing BAN,

which

reduced

breaking

46

4. Results and Discussion

0.25

0.0

0.2

0.4

Hencky

Fig.

4.4:

0.6

0.8

strain eH

[-]

1.0

0.0

0.2

0.4

Hencky

0.6

0.8

strain eH

1.0

[-]

Representative stress-strain curves of control flour gels and control


starch gels after an aging time of2h, 3d, 7 d and 10 d at 20 C.

4.1 Mechanical

properties

47

of bread and bread models

0.25

0.20-

Control

Soy--amylase

0.15-

Novamyl
CA
CA

0.10-

CA

O)
C

!2

0.05-

re

0.00
4

Aging

Fig.

4.5:

Influence of

aging time

time

the

on

and

containing Novamyl
deviation, n
6-12).

10

12

14

[d]

breaking stress

Soy-$-amylase.

<5break of flour

with

(Mean

gels

standard

0.25

ir

Control

BAN

Novamyl

0.05

re

Soy--amylase

0.00

Aging

Fig.

4.6:

Influence of

aging time

containing Novamyl,
deviation, n
6-12).
=

on

time

the

BAN and

10

12

14

[d]

breaking stress

g break

Soy-$-amylase. (Mean

of starch

gels

with standard

48

4. Results and Discussion

The modulus

shown

are

enzymes

scales should be
reduced the

for flour and starch

ED

in

trends

bread crumb
on

enzymes

rigidity

were

well

as

as

the

observed after
whereas

aging period

an

Soy--amylase

rate of starch

without

gels

increasing

the effect

was more

bread. It is assumed that the


the

low

comparatively

of starch

protein

dry

matrix

rigidity by Novamyl

has to

antifirming

an

the initial

by Novamyl
in starch

pronounced

particular

and Zobel

be taken

into

rigidity

and

rigidity

whereas

In

general,

gels.

gels

and

The influence of

was

magnified

effect

rigidity

gels

only

was

of starch

reducing

the

in

gels

firming

rigidity.

effect of

was

found for all

systems,

gels

than in flour

gels

in starch

Novamyl

matter content of starch

system (Maxwell

ordinate

is different from flour

gels

of 7 d. BAN did not affect the

An enhanced initial firmness induced

although

amylases.

the most effective enzyme in

was

time for various

deformation of flour

large

of starch

properties

the

on

rate in flour

firming

with the consequence that

gels,

impact

the influence of

(Fig. 4.3) regarding

aging

enhanced the initial

had little

found at small and

the mechanical

function of

gels, Novamyl

and bread. The enhancement of the initial

pure starch

as

Fig. 4.8, respectively (different

Soy--amylase

rate.

BAN reduced the initial

comparable

In flour

noted).

firming

4.7 and

Fig.

gels

gels,

gels

is due to

which retards the

1978). Furthermore,

and

firming

the absence of the

account, which facilitates starch-starch

interactions, for example the build up of intergranular amylose networks.


In order to further
for 175 d.

aged

were

The

increase of

ED

Fig.

4.9 illustrates the modulus

means

containing Novamyl hardly


deformation

(data

not

of
no

shown).

Novamyl
protein

play

can

be

matrix is

increased

compression

clearly

ED

of

in starch

Novamyl,

starch

gels

long aged

starch

gels.

showed

over

rate did not decrease between

rigidity

of control

gels

is not attained

the

long aging period. Similarly,

reduced the firmness and the

measurements show that

gels,

it

pronounced

hand, the modulus ED of starch gels

observed in starch

present

minor role in the

firming

the other

(abreak), Novamyl

The

the

that the maximal

long aging period. On

large

effect of

antifirming

aging. Interestingly,

on

10 and 175 d which


after this

the

plotted logarithmically. Control gels

time is

aging

investigate

gels

seems

staling phenomenon.

after
that

an

firming

antifirming

at

rate

effect

long aging period. Since

protein-starch

interactions

4.1 Mechanical

properties

49

of bread and bread models

E
E

Control

Soy--amylase

UJ
>

!5
re

CA
3

4. BAN

Novamyl

c
O

Aging

Fig.

4.7:

Influence of aging time

containing Novamyl,
deviation, n
6-12).

on

8
time

10

12

[d]

the modulus of deformability

BAN and

14

ED of flour gels

Soy-$-amylase. (Mean

with standard

1.0

E
E

i.

BAN

Al

Control

0.8-i

UJ
>
=

0.6

1 Novamyl

re

E
i_

0.4-I

CA
3

0.2-

Soy--amylase

c
O

0.0

1i1i111111111

Aging

Fig.

4.8:

Influence of

aging time

on

8
time

gels containing Novamyl,


standard deviation, n
6-12).

12

14

[d]

the modulus of
BAN

10

and

deformability ED of starch

Soy-$-amylase.

(Mean with

4. Results and Discussion

2.0-

1.5-

10"'

10"

10u

Aging
Influence of aging time
starch

on

10'
time

10'

10J

[d]

the modulus of deformability

gels containing Novamyl. (Mean

ED of long aged

with standard deviation,

4).

4.1 Mechanical

Soy--amylase

The effect of
model
flour

systems

not

was

Soy--amylase

that

hypothesize

substrate is accessible
case

of starch

sedimentation of native
order to test this

the

First,

reduced the
that

the

around 60 C

was

was

Soy--amylase
flour

gels (data

degrade

starch

not

was

to

tested. BAN

viscosity

that BAN

are

xanthan

prolonged

starch

heat treatment at

gelatinization

with

gels

procedure

an

ED

of

capable

to

inactivated. In

xanthan, standard flour

gelatinized

Therefore, it could only degrade

was

being

Soy-

before

to this

Soy--amylase

at 60 C before

could have

became

stress and the modulus

breaking

was

and

gelatinized

and could

serve

as

small amount of starch.

antistaling

effect in bread if it

stability.
BAN did not show the

influenced the

suspension,

and the

BAN

rigidity
was

which

breaking

was

and

firming

active to

serves as

gels

reduced. It is

rate of starch

i.e.

since deionized water

was

that BAN

was

4.6

heating

had

very

reduced. It is conceivable

pregelatinized
granules

not

used and therefore calcium

systems

gels (Fig.

greased moulds,

was

network into which the starch

hypothesized

effect in all

certain extent before

filled into the

stress of the

same

consequence the fracture stress of this matrix and the

gels

In

This indicates

shown).

gels produced according

only degraded intergranular starch,

starch, which

not

starch

inactivated in starch

Fig. 4.8). Nevertheless,

since the starch

replaced by

ED (data

promote

holding period

Soy--amylase,

hardly

Soy--amylase.

carried out.

was

It is assumed that

Soy--amylase

thermal

higher

were

before

and bread before most of the starch

Similarly

low

the

during

It is concluded that

and

inactivated

shown).

if the

xanthan, the added Soy--amylase only slightly

able to reduce the

accessible substrate.

had

substrate for

as

dispersion

inactivated. In flour

contrast, the enzyme

gels

serve

introduced in order to

was

only

can

which is added in order to avoid

enzymatic degradation. Second,

accessible for

gels

before the enzyme is inactivated. In

stress and the modulus

was

rate of

in starch

effects

good antistaling

experiments

starch

with

gels

breaking

amylase

-amylase

two

pregelatinized

In starch

dispersion.

shows

starch, may

firming

Fig. 4.8). One

4.6 and

procedure (Fig.

pregelatinized starch,

hypothesis

of bread and bread

antifirming properties

(gelatinized starch)
the

gels

properties

The enzyme did not reduce the

congruent.

to the standard

produced according

the

the mechanical

on

and bread but had remarkable

gels

51

of bread and bread models

properties

embedded. As

breaking

fully
as

are

and leached

stress of the

active in starch

gels

activator and stabilizer

52

4. Results and Discussion

was

To

missing.

maximum stabilization the calcium concentration should

ensure

be approx. 100-180

calcium concentrations
tested.

stress which

was

observed

was

It

shown).
in

high

medium

On the other hand,


in

seems

and 180

(50

Unexpectedly,

breaking

et al.

mg/l (Madsen

observed

breaking

that calcium does have

of starch and

inhomogeneous

on

ED.

modulus

(180 mg/l) (data

ED
not

remains unclear. It is conceivable that

water distribution

seen

are

of the

responsible

for the
starch

pregelatinized

starch sedimentation

possible

in standard starch

mechanical
remarkable

firming

on

antistaling

gels.

also

in

et al.

systems during

firmness of

firming

Interestingly,
gels

Novamyl

rate whereas

the
were

led to

prevented firming

different.

effect

slight
on

increase
BAN

had

amylases

Soy--amylase

the

on

aging.

Soy--amylase

influence of the

of

the

but

initial

was

firmness

most

due

to

Novamyl

in starch

pronounced

other authors

1997).

(Qi

only
the

on

showed
nor

the

gels.

addition
The

was

antifirming

Si 1996, Gerrard et al. 1997, Leon et al.

Little attention has been

the first hours after

starch is accelerated

by

to the initial firmness of

that the

the action of the enzyme. A similar

emulsifiers, which
are

paid

sample preparation.

Novamyl containing systems suggests

amylose fraction,

comparable

bread, porous starch gels and low concentration starch gels

reported by

1997, Morgan

the

effects whereas BAN did not influence the firmness

systems

Novamyl

as

and

had

gels.

enhancement

effect of

and of bread.

gels

of starch

properties

detectable in all

well

as

texture.

rate of starch

The

of flour

amylases

freshly prepared systems

reduced the firmness


small effects

be said that

can

properties

in firmness of the

when

effect

no

influence, but the mechanism of BAN

xanthan solution in order to avoid

conclusion, it

mechanical

the

was

Conclusions

4.1.3

was

gels

induced

(50 mgl/l)

concentration

an

gels

did not show any different effects than

in starch

activity

stress but

phenomenon. However, replacement

dispersion by

In

BAN

concentration

calcium

high

concentration wheat starch

demixing

on

to the control and enhanced modulus

comparable

with

gels

mg/l)

calcium

reduction in the

The influence of two different

1973).

are

gelation process

phenomenon

able to form helical inclusion

added to starch

The enhanced initial

systems (Conde-Petit

of

is found

complexes

with the

and Escher

1995).

4.1 Mechanical

properties

53

of bread and bread models

In summary, the main conclusions from the characterization of the mechanical

properties

of bread and

Influence of

amylases

Thus, the porous

gels present

on

the

is

protein

present

probably

of bread and flour

gels

is

comparable.
and flour

firming

on

aging,

caused

by

various

suitable bread model.

matrix

(which

in starch

Degradation

firming

are:

structure is not determinant for the

Differences between starch


i.e.

gels during aging

gels)

of starch

caused

by

is

gels

and flour

lacking

and

dry

in starch

are

gels), pregelatinized

factors,

starch

(which

matter content.

by Novamyl

accelerated

gels

induces

gelation

higher

initial firmness which is most

of starch.

54

4. Results and Discussion

4.2

Microstructure of

4.2.1

Development

Light

microscopy

microstructure of
and
al.

of method for

presents

dough

Clapp 1942,

bread and bread models

dough,

light microscopy

valuable

and bread

experiments,

(Barthel

method with

and
as

et al.

Raymond 1990,

few

dough

and

gels

crumb

was

soaked in diluted O.C.T.

was

consisted of

the

freezing

samples

compound

during cryosectioning.

A rather short

artefacts

by

described

times

hydration

Moss

soaking

(1975)

are

in

(18-24h). However,

of

shrinkage

protein

and starch due to

1994)

as

the

1915, Burhans

of

time

et al.

1999).

on

preliminary

was

developed
from

specimens

prior

fixation. Bread

for 15 min before

freezing.

The

distortion of the pores

selected, since the

was

probably promoted by long

most

of the short

spite

and

possible

without

prevent

hydration time,

completely. On

of bread crumb could not be inhibited

swelling

Flint

preparation

to stabilize the porous structure and to

as

of

study

1996, Hug-lten

preparatory steps

in order to minimize the risk of artefacts. The

aim

the

Sandstedt et al. 1954, Bechtel et al. 1978, Varriano-Marston et

literature

on

for

and Van Teutem

(Verschaffelt

1980, Pomeranz and Meyer 1984, Autio

Based

method

dehydration

the other

avoided

was

the

hand,

by using

the

cryosectioning technique.
Carbon dioxide

(-78 C)

within the porous

samples

stress
were

was

Iodine

to differentiate between

in

selected

amylose

potential

aqueous solutions,

samples
solutions

changes

which had
were

source

i.e.

only

found to

caused

be

to
as

and

of

temperature gradient

dough

and bread from

uniform structure without pores,


to -150

negligible

since

C).

The risk of ice

O.C.T.

acts

as

granules. Staining

Samples

and

exposed

by hydration during

is

with aqueous solution

which had been stained with

Lugol's solution,

were

compared

to iodine vapor. The aqueous

increase the

for starch because it allows

amylopectin. However, staining intensity

Light Green

slightly

an

piece

staining agent

of artefacts.

been

the

isopentan (-160

known to vary between the starch

presents

cryogen to limit the

prevented

considered

was

as

which have

by rapid freezing

formation

cryoprotectant.

used

which

cracking. Gel samples,

stabilized

crystal

was

swelling

stabilization and

of starch.

staining

to

staining

Overall,

the

did not induce

4.2 Microstructure of

in the starch fraction. The starch

major changes

Neither the localization of starch in the


two starch

4.2.2

polymers

protein

matrix

4.10

presents

light micrograph
Light Green.

of

cryosection

Green. The starch

show the characteristic

recognizable.
regions

granules

Protein and starch

et al.

more

1978)

on

surface coat of

not

evenly

granules

microscopy,

recent structural

1993).

model of

the microstructure of
it remains to be

dough. However,

(mixing, proofing

and

microstructure of

dough. According

segregation

occurs

reshaping)

during

partial phase separation


resistance in uniaxial

granules

thought

dough

are

reason

by

bicontinuous

granules

phase.

The

have

gluten

The results of the

consistent with the latter

how the

dough

et al.

is surrounded

as

granules.

protein quality

to be the

and

in bread

(Sandstedt

The starch

investigated

of the

dough,

protein

granule

processing steps

of wheat influence the

to Kieffer and Stein

reshaping

is also

(1997), starch-protein

after

for the

period.

rest

large

The

increase of

extension, called strain hardening. This stands in opposition

to the view that starch

determines the

is

the

and the

dough

Light

of native wheat starch

continuous

fills the space between the water-fused starch


on

slightly

accumulated.

are

stain

distributed in the

model described

and Larsson

dough

colored with

as

several authors

water and tend to fuse into

free"

present investigation

shape

concluded that each starch

starch-protein system (Eliasson

gel

are

wheat

granules

the distribution of starch and

regarding

is controversial. Based

protein.

and bread

proofed

bimodal size distribution of wheat starch

typical

The literature data

1954, Bechtel

of

fraction appears green

found where several starch

are

dough

The native starch

protein

The

the distribution of the

nor

dough

violet with iodine while the

granules.

shape.

affected.

were

stained with iodine and

dough

maintained their

granules

Localization of starch in the microstructure of

Microstructure of

Fig.

55

bread and bread models

dough,

simply

acts

rheological properties

as

of

filler in bread

dough,

dough (Bloksma 1990).

while

gluten

56

4. Results and Discussion

Microstructure of bread

Fig.

4.11a-b show

different

magnifications. Again,

the two fractions. At low


swollen and

elongated,

aligned parallel
areas

at

sections of pore walls of fresh bread crumb at two

cross

starch and

but still retain their

fused with

homogeneously
The

neighboring granules.
stain

fraction of starch

granules, only

to

bread, but

separation
and

interface. In

amylose

Fig. 4.11b,

resolution level

as a

amylose

of starch is

in the starch

explained by
and

granule

be observed

concentric

granules

granule

the starch

as

dark lines
as one

visible for

are

not

granules.

growth rings

granules

and

surrounding

Clapp 1942,

inhomogeneous

by

one

starch, mainly amylose. The

center is also documented for rye

the

thermodynamic immiscibility

Ring 1987).

(Autio

The

was

et al.

elongated

reported

in

1996).
of

amylose

previous

Sandstedt et al. 1954, Varriano-Marston et al.

of

baking.

The fact that

in bread crumb proves that in

spite

growth rings

of the

partial amylose-amylopectin phase separation

Phase

form of starch

to arise from the extension of the pore walls due to the

early stage

starch and

and consists of

the authors

and

recognizable

are

starch

by

(Burhans

and the

respectively,

within the starch

can

and leached

and their orientation in bread crumb

in the

amylose

studied, the pore walls of bread crumb

The starch fraction itself is

amylopectin (Kalichevsky

cells

oriented and

bicontinuous structure, which is built up

not further commented

thought

are

reveals that

large

green

recognizable

whereas the brown/violet

granules

It is

as

are

are

phase separation. Outside

rich in

phase separated" granules

accumulation of

granules

granule.

microscopical

protein, respectively.

granules

of the

zone

of the internal structure of starch

may be described

swollen

Most

rich structures. In contrast, the small

does not show this

swollen starch

At the

inner

granules appear

distinguishable

brown/violet,

amylose

zones

staining

phase separated

the

amylopectin

protein

particular aspect
highly

in

areas

few and small

the starch

is

the starch

Iodine

and

are

to accumulations of

phase corresponds

along

blue

distributed and

blue, elongated

correspond

granular identity.

the bread crumb. Details of the starch fraction

which

amylopectin,

the starch

protein phase

higher magnification (Fig. 4.11b). Clearly,

partly

stained in order to localize

are

magnification (Fig. 4.11a)

to the pore surface. The

throughout

protein

studies

1980).

growing gas

of starch

are

morphological changes

the native

organization

of

4.2 Microstructure of

starch

which is

granules,

orientation of

57

bread and bread models

dough,

mainly

determined

amylopectin (Gallant

et al.

by

the

packing density

is

largely preserved.

1997),

It is concluded that the transformation of starch


continuous

starch

network.

macroscopic properties
Sandstedt et al.
starch is

(1954),

replaced by

are
a

an

it

Therefore,
determined

typical

by

is
the

not

suitable

to

starch

systems

with

inert filler such

as

glass

1997).

and

are

The

resulting

determined

by

the other

sponge structure

(e.g. xanthan, pregelatinized starch)


al.

mechanical
the

swelling

of the thickness of the pore walls

surprising

to

leads to

find

that

the

starch. As shown

by

bread crumb structure cannot be obtained when

consistency. On
a

during baking

gelatinized

beads. The

authors led to the conclusion that starch does not

gluten

and the radial

to

simply

act

experiment by
as a

filler that dilutes

hand, it is possible

by combining

these

to

generate

starch with other

polymers

replace gluten (Keetels 1995, Morgan

properties

are

state of starch

(Keetels 1995).

et

similar to those of bread crumb

granules

and

by

the distribution

58

Fig.

4. Results and Discussion

4.10: Light

micrograph of proofed control dough stained with Light Green and

iodine,

(scale bar

Fig.

25

pm)

micrographs of control bread crumb at two different


magnifications. Samples were stained with Light Green and iodine,
a) scale bar 50 pm
b) scale bar =10 pm

4.11: Light

4.2 Microstructure of

Besides the soft

59

bread and bread models

dough,

crumb, the crisp

crust is the second

of fresh bread. Characterization of the microstructure of bread

complemented by
the

observations

bright-field

and

steam. Starch

was

stained with iodine vapor and


observed

(Fig. 4.12b-d). Contrary

to bread

in the bread crust. Starch is


characteristic
In

with

starch

present investigations,

the crust which is detected


zone

of crust still shows


whether

Regardless

interesting
surrounding

observation
the pores

around the lower

The

investigations

gelatinized.
the crust

This is

is

probably

not

stained

which exhibit the

granules

was

cross

only

the

are

are

not swollen.

similar to

injected during baking,

over

as

generally applied

of starch in the outmost

in the form of Maltese


without

or

upper

part

strong

of bread. A further

of the

while this

birfringent,

of

crosses.

the

steam,

zones

layer

The inner

birefringence (Fig. 4.12d).

with

all

the whole bread crust

baking phase,

towards the inner

on

starch
was

granules

not the

case

crust revealed that most of the starch fraction is not


to be the consequence of

the initial

baking phase,

When steam is

rapid

water

evaporation

which reduces the

added, starch of the

availability

in
of

outmost crust

due to condensation and release of latent heat of steam at the


The

observations

present

which showed with different

McDonough

granules

et al.

and

in

are

agreement

microscopy techniques
in the crust

1980, Pomeranz

are

et al.

not

or

with other

such

as

SEM,

only partially

1984, Freeman and

Rooney 1999).

for the

around the pores


water

were

omitted

of the pores.

possible explanation

granules

baked

the crust

gelatinized (Varriano-Marston

the loss of

that

ESEM and LM that the starch

Shelton 1991,

steam

Maltese

disappears

thought

surface.

investigations

no

gelatinization

was

gelatinization.

layer gelatinized
dough

led to

by

in

was

strongly birfringent

are

strong birefringence

near

during

zones

water for starch

cold

part

When

typical

bread

of the crust

birefringence

crust

of steam in the initial

(Fig. 4.12b,c). Injection


in the

polarized-light

of native wheat starch and which

(Fig. 4.12b-d).

showed the

granules

protein staining

in the form of

addition, starch granules of bread

native wheat starch

micrographs

crumb, the starch granules appear almost intact

present

spherical shape

4.12 shows

Fig.

therefore

was

mode of bread crust baked with and without

polarized-light

Samples

(Fig. 4.12a).

bread crust.

on

characteristic

important

inhomogeneous gelatinization

is also related to the

evaporates very rapidly

at the

availability

warmer

of the starch

of water.

Most

side of the pore, which is

60

4. Results and Discussion

always

the side

near

the crust. Then water

migrates through

condenses at the colder side of the pores, which is


releases its latent heat and starch
and condensation
and

Larsson

zones.

are

very

rapid

steam

in

the

caused

positions

by gravity

in the pores

outer

part

gelatinization during cooling


partly

as

compared
is

found between crust

bread,

of the pores, where

and would result in


on

samples

loaf it is concluded that the

condensation rather than

of

of the bread. This would

depending

which

is caused

in crust

condenses

by

and

it is available for starch

mean

top,

(Eliasson

is caused

that the

phenomenon

birefringence occurring

taken from the

There, it

observed

the location of the crust. Since

phenomenon

gravity.

only

and

evaporation

to the crumb

inhomogeneous gelatinization
zones

phase

the crumb.

the processes of

1993), inhomogeneous birefringence

accumulates in the lower

were

in the crust

It is also conceivable that the

superheated

is

gelatinizes. Since

near

the gas

no

at different

differences

the bottom and the side of

by rapid evaporation

and

4.2 Microstructure of

Fig.

dough,

61

bread and bread models

micrographs of control bread crust


bright-field mode (scale bar 25 pm)
25 pm)
baked without steam, polarized-light mode (scale bar
baked without steam, polarized-light mode (scale bar =100 pm)
(arrow indicates inhomogeneous gelatinization around pores of the
crust)
100 pm)
baked with steam, polarized-light mode (scale bar
indicates
outer
of
(arrow
gelatinized
layer
crust)

4.12: Light

a)
b)
c)

d)

baked without steam,

62

4. Results and Discussion

4.2.3

Influence of

amylases

the microstructure of fresh and

on

aged

bread
The microstructures of fresh and 7 d
BAN

or

Soy -amylase

microstructure

characterized with

of fresh

crumb

bright-field

different from control

Fig.

also

illumination.

blue-violet and

Color

Soy--amylase

of starch

impression

to control

is altered

(Banks
of

development process

in

on

the

bread

type

reasons

Aging

of bread had

granules

in bread with

lost their

of

and Greenwood

photo micrographs,

was

still

samples.

an

inactivated

active

disintegration

influence

Novamyl

structure

on

when

Novamyl

is not

during aging

of starch.

swollen

more

on

and

are

Soy--amylase

is

are

1975). On
which

was

conceivable for the


a

reduced

degree

consequence,

the other
not

impression

in

iodine

hand, the film

standardized, could
of the

micrographs.

the microstructure of starch. The starch

BAN,

during baking

with

In

amylase.

Soy--amylase

or

In bread with

typical granule

completely

found in

amylases

shows

of starch leads to

result in color shifts. Both facts influence the color

in the fresh

are

bread, whereas starch of Novamyl and BAN

Enzymatic degradation

polymerization depending
staining

different

were

to the control. Differences in the colors of starch

comparable

color differences.

with

Bread crumb with

crumb stains violet to brown. Two

containing

Little differences

protein

bread, apart from color differences of iodine stained starch.

granules compared

observed.

containing Novamyl,

4.13 to 4.16. Starch and

produced

On the other hand, bread with BAN and


starch

bread crumb

Light Green, respectively.

stained with iodine and


the

shown in

are

aged

on

aging.

were

the other

smaller in the

aged

hand, the starch granules

It is conceivable that BAN

due to its

high

degraded

thermal

starch

than

was

not

stability. Therefore,

which

led

to

it

further

4 2 Microstructure of

Fig.

dough,

bread and bread models

micrographs of fresh and aged control bread crumb


(scale bar 25 pm)
a) fresh (0 d)
b) aged (7 d)

4.13: Light

Fig.

micrographs of fresh and aged bread crumb with Novamyl


(scale bar 25 pm)
a) fresh (0 d)
b) aged (7 d)

4.14: Light

63

64

Fig.

4. Results and Discussion

micrographs of fresh and aged bread crumb with


(scale bar 25 pm)
a) fresh (0 d)
b) aged (7 d)

4.15: Light

BAN.

Fig.

micrographs of fresh and aged bread crumb with Soy-$-amylase.


(scale bar 25 pm)
a) fresh (0 d)
b) aged (7 d)

4.16: Light

4.2 Microstructure of

The

dough,

with

investigations

regular

control bread and bread with


not stained. As

were

birefringence (Fig. 4.17a)

bright-field

4.17 and

polarized-light microscopy. Fig.

samples

65

bread and bread models

Fig.

of

no

the

assessed with

as

bread

shown). During aging,

bread, which

means an

hand, developed
bread

Fig.

not

in

magnification
was

and

iodine vapor. This

technique

and the

of the starch

an

intensity

inside the

stained

was

adopted

was

rich

revealed that

granule

large

starch

to

visible

as a

on

the other

between control and

Novamyl

for

bright longish

prevent

BAN,

zone

zone can

example

zone

the

the

in the

and

of starch
the

out of

washing

amylose

of

in

bread

granules.

Fig.

to

amylose
The

amylose

rich

phase

corresponding

phase separated

is observed at the outer


crumb

be detected in the

seen

the

within the

high

protein

solutions.

amylopectin

granules. On

birefringence

only
be

an

at

cryosection

reveals accumulations of

High magnification

birfringent
can

Bread with

by exposing

granules

A less intense

zones.

center. This

was

behaved like control

granules by aqueous staining

at the outer

birfringent.

amylopectin

aging

Soy--amylase

The

polarized-light micrograph (Fig. 4.19b)


is

on

polarized-light mode, respectively.

bright-field micrograph (Fig. 4.19a) clearly

(violet/brown colored)

change

(7 d)

omitted, and starch

(blue colored)

contrast, Novamyl

of stored control bread crumb

same area

bright-field

swelling

they may

polarized-light microscopy (micrographs

birefringence.

with

as

of

shown).

4.19a-b show the

granules

out of focus. In

pattern

cross

disregarded

containing Soy--amylase

increase of

birefringence

(micrographs

staining

planes

birefringence

bright irregular

as

Maltese

The microstructure of fresh bread with BAN and

similar to control bread


not

is visible

should be

spots

in

laying

marked increase of

typical

is

(Seidemann 1966).

in the fresh bread crumb which did not

birefringence

(Fig. 4.18).

of the

recurrence

dust

molecules

birefringence

native starch is observed. The circular

induced

The

starch

order

magnification,

birfringent

magnification.

of

control bread crumb shows

be the result of

at low

granule swelling upon gelatinization

loss

and

the microstructure of

since starch

Interestingly, aged

longish structures,

by

fresh crumb of control bread exhibits little

expected,

At low

present

Novamyl, respectively,

accompanied by

(Fig. 4.17b).

4.18

complemented

were

with

amylose

4.20d-f. The

Novamyl
rich starch

birefringence

is

66

Fig.

4. Results and Discussion

4.17: Polarized-light

micrographs
Samples were not stained,
(scale bar 50 pm)
a) fresh (0 d)

of fresh and

aged control bread crumb.

b) aged (7 d)

m/*m

IM
f:/-#^4#S*

Fig.

micrographs of fresh and aged bread crumb with


Novamyl. Samples were not stained,
(scale bar 50 pm)
a) fresh (0 d)
b) aged (7 d)

4.18: Polarized-light

4.2 Microstructure of

Fig.

4.19:

dough,

67

bread and bread models

Corresponding light micrographs of aged control bread


Samples are stained with iodine vapor,
(scale bar= 10 pm)
a) bright-field mode
b) polarized-light mode

crumb

(7 d).

68

4. Results and Discussion

The

influence of

reheating

investigated by heating
aged

After 7 d of
the

aging,

outer

starch

rich starch

amylose

of

zones

the

the microstructure of starch

granules
granule

polarized-light

of control bread crumb show

center and

After

granules (Fig. 4.20a).

birefringence

in the

birefringence

remained in the center of starch

aging period

of 4

Polarized-light
Fig.

of the

starch

are

of

granule

zones

of

birfringent

birfringent

granules.

birefringence

outer

Novamyl

after

an

center remained

with

Novamyl

mainly

the

of 7 d

and the

not

birefringence

disappeared

aging period

was

4.20.

whereas
the

in

in the

reheating (Fig. 4.20b),

detectable in the

bread crumb

Fig.

the

some

following

granules regained birefringence.

crumb

reheating step

in

birefringence

granules. During

bread crumb

zones are

Due to the

zones

of the starch

bread

Novamyl containing

centers

and very weak

d, the

micrographs

4.20d-f. In

granule

amylopectin

rich outer

less intense

was

of bread crumb with

presented

are

was

bread

aging. Thereafter,

High magnification micrographs

observed with

Novamyl

on

bread at 60 C after 7 d

for further 4 d at 20 C.

and without

of bread

shown

are

amylose

rich starch

(Fig. 4.20d). Only

amylopectin

rich

birfringent (Fig. 4.20e,f).

and the

few

regions

subsequent aging,

changed,

in

amylose

the
rich

4.2 Microstructure of

Fig.

dough,

69

bread and bread models

micrographs of bread crumb without and with Novamyl.


Samples were not stained, (scale bar 10 pm)
Bread before reheating (7 d aged):
a) Control
d) Novamyl
Bread directly after reheating:
b) Control
e) Novamyl
Control
Bread 4 d after reheating:
c)
f) Novamyl

4.20: Polarized-light

70

4. Results and Discussion

The combination of

light microscopy

in the

allowed localization and identification of the


zones

detected with

as

structures but not

in

zone

the

of

C,

60

starch

>

Czuchajowska

and Pomeranz

1989),

during baking,

regions
aging

become

of control bread
of bread

cooling

(Fig. 4.17),

(Fig. 4.18).

al.

1985a,b, Clark

partial crystallization

reorganization

of

et al.

of

amylose

is

or

Based

1989)

birfringent

by

on

of

reorganized amylose.
et al.

(Eberstein

necessary to melt ordered


followed

either due to

birfringent

It is concluded that the

has not been

birfringent amylose structures,

Birfringent

structures.

anisotropic

around 150 C

are

mode

to

consists

granules

probably

most

of starch

separation

crystallinity.

to

Temperatures

Phase

birfringent

polarized-light

polarized-light microscopy correspond

necessarily

center

and

bright-field

amylose.

the formation of ordered

by

reported

1980,

to date. The

amylose

rich

spontaneous reorganization during

the action of

studies

on

Novamyl during baking

pure

and

amylose systems (Miles

et

it is conceivable that lateral chain association favors


The

amylose during aging.


artefact introduced

an

by

that

possibility

iodine

complexation

excluded, since the samples of Fig. 4.17, 4.18 and 4.20

the

can

be

not stained.

were

However, the ordering of the amylose fraction may have been promoted by

complexation
The

with

wheat starch

endogenous

slight birefringence

in the outer

amylopectin. During

the

bread crumb due to

could

never

when

Novamyl

completely

of

which

be attributed to

recurrence

added. This

of

was

birefringence disappeared
i.e.

melting

only

retrograded

of

which

retrograded

subsequent aging birefringence reappeared

in control

amylopectin rtrogradation. Birefringence

amylopectin
suggests

birefringence

rich outer

that

in the outer

with the results of Jacobson et al.

near

granules,

zones

reorganization

of starch
of

granules

amylopectin

was

blocked.

The observed

granule

can

anisotropic structures,

be detected in the
was

aging,

at 60 C this

amylopectin. During reheating


destruction

of starch

zones

observed in control bread crumb after 7 d

reflects the

lipids.

zones

(1997). They

remnants of low-concentrated starch

the outer

edges

concluded that the


crumb involves

of intact swollen

development

changes

in both

of starch

is consistent

described that upon

storage

pastes regained slight birefringence

granules.

of ordered

granules

Based

zones

on

microscopy,

in starch

granules

fractions, amylose and amylopectin.

it

can

be

of bread

4.2 Microstructure of

4.2.4

dough,

Influence of

71

bread and bread models

amylases

on

the microstructure of flour

gels

and starch

gels
Flour

gels

Stained flour

gels

were

polarized-light

mode.

the

mode. The

bright-field

assessed

by light microscopy

4.21 illustrates flour

Fig.

granular identity.
and

neighboring granules,
the

interface. Starch

protein

those in

flour

aged

is distributed

control flour

Flour

gels

the

gels

with added

granule

4.21

is

Novamyl (Fig.
as

Novamyl

swollen and

is also visible

are

as

are

in

well

more

4.21
as

granules

gels

are

show

within

along

larger

which stains

the

than

blue,

of fresh bread whereas in

accumulated in the center of

c-d)

in

fused with

dark blue lines

in fresh control flour

in the

partly

elongated,

phase separated

a-b). Furthermore, amylose,

amylose

center in fresh

are

granules

amylopectin

granules

homogeneously

more

aged

the

gels (Fig.

with and without

granules

The starch

and

amylose

granules. Intergranular amylose

starch

and

bright-field

matrix is colored green whereas starch appears

protein

blue to violet. Like in bread crumb the starch


but still retain their

gels

in the

enrichment of

an

aged gels.

As

granules.

amylose

in

in bread crumb

seen

shifts the color of iodine stained starch from blue towards

(Fig. 4.14), Novamyl


violet.

The

tendencies
with

as

is

more

mainly

occurs

enriched, and does

of control flour

gels

intense

not

compared

birefringence

to

control

in the center of starch

change

increased

on

aging. On

aging

on

showed the

gels (Fig. 4.22)

observed in bread. The

already

Novamyl

birefringence
is

of flour

polarized-light micrographs

the other

of fresh flour

model

granules,

same

systems.

where the

gels
The

amylose

hand, the birefringence

and is distributed

over

the whole starch

granule.
In

conclusion, it

be said that the microstructure of flour

can

to that of bread crumb.

Novamyl

induces the

same

color shift form blue to violet and increased initial


measurements

(see chapter

4.1

also showed that

bread and flour

gels.

It is assumed that BAN and

microstructure

in

similar

microstructure of flour

gels

way

as

seen

with BAN and

in

effects

gels

is

as seen

comparable
in

bread, i.e.

birefringence. Compression

Novamyl

had similar effects in

Soy--amylase
bread

Soy--amylase

crumb.
was

not

also affect the

Therefore,

the

investigated.

72

Fig.

4. Results and Discussion

micrographs of fresh and aged flour gels without and with


Novamyl. Stained with iodine and light green,
(scale bar 25 pm)
a) Control fresh (0 d)
b) Control aged (7 d)
c) Novamyl fresh (0 d)
d) Novamyl aged (7 d)

4.21: Light

4.2 Microstructure of

Fig.

dough,

bread and bread models

73

micrographs of fresh and aged flour gels without and


Novamyl. Samples were not stained,
(scale bar 50 pm)
a) Control fresh (0 d)
b) Control aged (7 d)
c) Novamyl fresh (0 d)
d) Novamyl aged (7 d)

4.22: Polarized-light

with

74

4. Results and Discussion

Starch

gels

In contrast to bread and flour


solution.
starch

The

accumulations

space

clearly

are

microstructure of starch

Fig.

4.24.

granule

on

the

distinguished
with

partially

to starch

In

gels

In

starch

gels

with

some

with

Novamyl

are

amylose

intergranular

are

granules

more

violet

found in the

when observed in the

an

in starch

the

within the

the starch

amylose

the blue coloration


of

aged gels

cryosection
was

for

observed in starch

gels

since the

not rotated

no

are

visible

indicating amylose
blue

areas are

is

were

of the

not

are

In other areas,

(Fig. 4.24c).

In

can

of starch

accumulation is

visible

(Fig. 4.24e),

possible (Fig. 4.24f).

Soy--amylase,
detected.

caused either

viscosity

evidence

gels were

centers

is not stained and the starch

gels (Fig. 4.24b).

staining

two

differently

stainable

It is conceivable that the

by demixing

gels. Mainly

of starch

in starch

gels

pregelatinized suspension

which could facilitate starch sedimentation.

macroscopic

granule

are

starch

elongated

BAN, inter- and intragranular amylose enrichment

with BAN and

same

observed that the

BAN show

accumulations

intense blue iodine

gels

Soy--amylase

BAN, iodine staining is inhomogeneous

uniform distribution of the enzyme in the

no

changes

no

with BAN and

gels

Novamyl,

some areas

regions

inhomogeneous staining

was

and in the

Soy--amylase gels (Fig. 4.24d). During aging

in fresh

Soy--amylase,

lost. In

Only

blue

gels,

the center of starch

gels containing

with

aged gels

region.

whereas in others

regions

aged

intragranular amylose

contrast to starch

gels

granules

almost

center remained uncolored like in fresh

blue inter- and

be

control

Novamyl,

with and without

gels

Fresh starch

(Fig. 4.24a).

depending

with

gels. On aging,

of fresh and

granules. Similarly
stained

In

gels.

granules

mode.

Micrographs
shown in

gels

the microstructure of

and therefore starch

missing
flour

on

stained with iodine

Furthermore, the starch granules stain generally

of control

granules

in

only

were

Novamyl

visible in the center of

In starch

remained uncolored.

bright-field

than

together

(Fig. 4.23a-b).

than the

matrix is

protein

closer

spatially

gels

4.23 illustrates the influence of

Fig.

gels.

starch

gels,

or

by

with

was

a non

BAN, it

very low,

However, baked starch gels showed

sedimentation.

with BAN. This cannot be

during baking. One

hard

core

explained by
reason

for the

was

sometimes

starch

demixing

inhomogeneous

4.2 Microstructure of

staining
would

and the hard

lead to

consequence,
of starch

dough,

core

could be

inhomogeneous
staining

is altered

rtrogradation

75

bread and bread models

can

starch

only

also be

in

non-uniform enzyme distribution. This

degradation throughout
some areas

expected.

the

gel.

As

and differences in the extent

76

Fig.

4 Results and Discussion

micrographs of fresh and aged starch gels without and with


Novamyl Stained with iodine solution (10 %) for 16 s
(scale bar 25 pm)
a) Control fresh (0 d)
b) Control aged (7 d)
c) Novamyl fresh (0 d)
d) Novamyl aged (7 d)

4.23: Light

4 2 Microstructure of

Fig.

dough,

bread and bread models

77

micrographs of fresh and aged starch gels with BAN and


Soy-$-amylase Stained with iodine solution (10 %) for 16 s Aged gels
(7 d) represent two different regions of the same gel
(scale bar 25 pm)
a) BAN fresh (0 d)
b) and c) BAN aged (7 d)
fresh
d) Soy-$-amylase
(0 d)
e) and f) Soy-$-amylase aged (7 d)

4.24: Light

78

4. Results and Discussion

Like

in

bread

and

flour

complemented by observing
were

taken in the

always

found. The

(Fig. 4.25)
fresh

show the

control

the

same

starch

control

gels

but

gels

but less

in the

birefringence
are

is

observed in the

In summary, it
but could not be
eliminate

described in

Only

starch

reducing

control with

matrix in starch

be said that

iodine

gels

staining

of

high

starch

gels.

BAN

BAN

and
show

compared

gels.

the

Birefringence

and in the
show

gels.

to

amylopectin

The

very weak
increase of

containing gels.

Differences

of starch

amylases

clarification

in starch

occurs
can

change

of this

or even

point,

and low concentration starch

gels

further

systems

are

4.2.5.

firming

showed similarities with bread and flour

birefringence. Soy--amylase was

rate of starch

gels

birefringence compared

observed in bread with

gels

increases

gels.

BAN which

compression

tests.

with BAN showed

to fresh control starch

is not

and bread with BAN

effective

but could not be differentiated from

polarized-light microscopy. Starch gels

between starch

gels

bread, flour gels and starch gels

since

For

amylose.

with

The

gels.

Novamyl. On aging,

starch

phase separation

detected in all
of

in

Novamyl

starch

intense

Soy--amylase

birefringence

the microstructure and

the

with

granules

control

to

gels containing Novamyl

enhanced initial

level with

of

protein

staining

chapter

gels regarding
in

on

gels

was

fresh

with

gels

gels

more

control

with

while

birefringence

Starch

4.26.

to

the

starch

aged

than in starch

comparable

clearly

iodine

investigations

can

and

gels

with and without

Novamyl containing

rich centers of starch

intensity

due to the absence of

the

Fig.

birefringence

birefringence,

is similar to control and BAN

aging

on

in

Fresh starch

which

birefringence,

in

comparably

amylose

regions.

no

were

Pictures

polarized light.

observed in bread and flour

as

of fresh

pronounced

increases

birefringence

gels

in the fresh state, which is

birefringence already

rich outer

of starch

almost

hardly

presented

are

with

gels

microstructure

where the most intense

tendencies
exhibit

the

on

clearly birfringent. On aging,

are

systems

Soy--amylase

develops

unstained starch

regions

Polarized-light micrographs

control

investigations

polarized-light micrographs

containing Novamyl
in

gels,

surprising

were

since

gels.

This

large

also detected at the

slightly
was

not

differences

macroscopic

4.2 Microstructure of

Fig.

dough, bread and bread models

79

micrographs of fresh and aged starch gels without and


Novamyl. Samples were not stained,
(scale bar 25 pm)
a) Control fresh (0 d)
b) Control aged (7 d)
c) Novamyl fresh (0 d)
d) Novamyl aged (7 d)

4.25: Polarized-light

with

80

Fig.

4. Results and Discussion

micrographs of fresh and aged starch gels with BAN and


Soy-$-amylase. Samples were not stained,
(scale bar 25 pm)
a) BAN fresh (0 d)
b) BAN aged (7 d)
c) Soy-$-amylase fresh (0 d)
d) Soy-$-amylase aged (7 d)

4.26: Polarized-light

4.2 Microstructure of

4.2.5

dough,

Influence of

In the
with

amylases

bright-field

and the

amylases

of starch

gels

mode

are

bright-field

granules

with

center of the starch

same area

polarized-light
and

illustrated

mode.

gels

gels
with

the effect of

study

of starch

gels

Additionally,

were

iodine

incubated

amylopectin dispersions

gels

in

Fig.

4.27.

show blue

concentrations observed
Phase

amylose

separated

(7.5 MANU/kg flour)

granules.

is visible

At low concentrations of

the blue coloration is weakened and

starch

enrichment in the

granules (Fig. 4.27a). Intergranular amylose

between the swollen starch

spots

of the

increasing Novamyl

visible. Control starch

are

of starch

in order to

performed

amylose

centers of starch

analyzed.

was

Micrographs

granule

Light microscopy

staining. Micrographs

of low concentration

staining

was

of starch

staining

shown that starch

was

concentration

iodine

on

recorded in the

in the

it

iodine

on

do not stain with iodine.

increasing Novamyl

with

amylases

previous chapter

Novamyl

81

bread and bread models

more

violet

as

blue

Novamyl

regions

are

visible, but inter- and intragranular amylose is still detectable (Fig. 4.27b). Starch

gels

with

standard

with iodine

staining

be observed in starch

gels

than in bread and in flour

4.28 shows the

Novamyl

The

respectively.
as

well

the

as

in the control

gels

are

Based

of stored

same area

in

the

rich outer

amylopectin

gels (Fig. 4.28a-b). On

these observations

parts

The

by

it

of starch

can

starch
and

of the starch

gels

amylopectin,
in starch

with and without

polarized-light mode,
amylose

granules

are

rich center

birfringent

hand, Novamyl containing starch

granule

be concluded that

of starch

gels

differences in iodine

microscopy (data
weak

blue

gels

reveals

center

(Fig. 4.28c-d).

amylose

as

well

as

without enzyme. In contrast, in


an

increase in molecular order

by microscopy.

inhomogeneity

detected

i.e.

pronounced

reveal that the

the other

Novamyl containing systems only amylose


detected

more

bright-field

in the uncolored starch

amylopectin reorganize upon aging

as

(182 d)

corresponding micrographs

only birfringent

on

staining structures,

no

gels.

high magnification

at

violet

This effect is much

gels.

show

Novamyl (750 MANU/kg flour)

(Fig. 4.27c). Only

can

Fig.

of

dosage

not

birefringence,

staining intensity,

shown). Intensively

which

was

Soy--amylase,

with BAN and

mainly

stained

is confirmed

cryosections

detectable in the starch

which

was

by polarized

showed rather

granule

center.

82

4. Results and Discussion

Vice versa, starch

gels

strong birefringence
of the

granules.

structures. As
a

less intense

in which

in the starch

mainly amylose
granule

high intensity

consequence,

staining (Banks

of

centers

could not be stained exhibited


as

well

as

birefringence points

complexation

in the outer
to

highly

regions
ordered

with iodine is reduced and results in

and Greenwood

1975).

4.2 Microstructure of

dough,

bread and bread models

Fig. 4.27-.Light micrographs of aged starch gels (7d) containing different


Novamyl concentrations. Samples were stained with iodine solution
(10 %) for 16 s. (scale bar =10 pm)
a) Control
b) Novamyl (low dosage: 7.5 MANU/kg flour)
c) Novamyl (standard dosage: 750 MANU/kg flour)

84

Fig.

4. Results and Discussion

4.28:

Corresponding light micrographs of long aged starch gels (182d)


Novamyl. Samples were stained with iodine solution
(10 %) for 16 s. (scale bar 10 pm)
a) Control, bright-field mode
b) Control, polarized-light mode
c) Novamyl, bright-field mode
d) Novamyl, polarized-light mode
without and with

4.2 Microstructure of

Iodine

of low concentration

staining
In

investigated.
interest since

iodine

particular,

shows the colors of iodine stained

enzyme.

The

prepared

control

incubated with

did

amylose

dispersions

with iodine. The control

temperature.
fresh

and

with

(,max)

are

control

shorter

the

Fig.

nm

with

visible in

that the

wavelength

lower values. After


BAN and

still blue whereas

colorless.

Additionally,

with

Novamyl.

amylopectin dispersions

at the maximum absorbance

609

and

598

Incubation

nm.

with

showed

Novamyl

during subsequent aging.

A shift towards lower

shift in color towards violet.

Soy--amylase.

aging period

Visually,

no

amylose dispersions

differences in

and

dispersions

The absorbance measurements showed

of 14

was

only slightly

shifted towards

d, color differences between control and


both visible and measurable. BAN and

stained violet and the maximum absorbance

lowered to 566 and 569 nm,

respectively.

dispersions
Fig.

at

maximum

incubated with

the

of 7 d it decreased to

During

Soy--amylase dispersions were

wavelengths

of

storage period

nm.

Soy--amylase dispersions

The

room

4.31. The maximum absorbance of

of the maximum absorbance

an

aged

shifted the maximum absorbance towards

color were detectable between fresh control


incubated with BAN and

and

amylose dispersions

and remained constant


indicates

wavelengths
Fig.

were

in

for 17 h at

aged dispersions

amylose

between

Novamyl

Fresh

wavelengths.

wavelengths

were

was

became

Novamyl

4.30 and

maximum absorbance at 590


563

with

shifted

The violet color of

kept

were

staining

slightly

was

pronounced

more

dispersions

after

directly

dispersions.

slightly

the control

amylose dispersions

the color

amylose dispersions

period,

amylose dispersions

amylose dispersions

although

was

recorded. The

in

with and without

Amylose dispersions

color differences

in the visible range of

were

presented

same

still blue

amylose aggregates

stained with iodine

4.29

Fig.

blue. In contrast,

deep

Novamyl

After this time

of

stained

to the fresh control

staining,

Absorption spectra

not stainable.

was

was

amylose-butanol complexes. Freshly

aged dispersions

sedimented

Novamyl

was

not contain

was

compared

After

dispersions.

degraded by Novamyl

stained violet instead of blue.

Novamyl

amylose dispersions

amylose dispersions

amylose dispersion (0.6 %)

stored for 7 d at 20 C showed the

towards violet

of starch

with

gels

and

amylopectin

staining

of starch

amylose

85

bread and bread models

dough,

absorbance of iodine

Novamyl,

BAN and

4.31. Differences between control and

stained

Soy--amylase

was

amylopectin

are

shown in

amylopectin dispersions

incubated

86

with

4. Results and Discussion

amylases

maxima and also


with

dispersions

phenomenon,
occurred in

small. Almost

were

visually only

was

amylose dispersions,
incubated with
violet.

observed in

were

aggregation

no

and became violet

after

only

*C.IJ

(0 d)

mixing

after

standing

\ I'N

directly

after

"

staining

was

After 17

at

observed in

aged (7 d)
4.29: Photographs of fresh

without and with

Control

Novamyl

'

also

In contrast to

amylopectin dispersion

iMICIHJ
/

Novamyl,

amylopectin dispersions

temperature

aged (7 d)

bleaching

h, control dispersions

blue color similar to


room

with

colorless.

were

was

amylose dispersions
for several hours.

i WW

directly

JlClfl

Fig.

recognizable. Amylopectin

amylose dispersions

addition of iodine the


it had

found in the absorbance

less intense coloration. The

Novamyl dispersions

Novamyl. During

Interestingly,

fresh

showed

were

amylopectin dispersions with Novamyl.

violet whereas the

were

differences

minor differences

Soy--amylase

which

no

after

^ N'

/17 h after

staining

staining

(0 d) and aged (7 d) amylose dispersions (0.6 %)


Novamyl. Dispersions were stained with iodine.

4.2 Microstructure of

dough,

Aging

Fig.

87

bread and bread models

time

[d]

Aging

time

[d]

4.30: Influence of

aging on iodine staining of 0.6 % amylose dispersions


incubated with Novamyl, BAN and Soy-$-amylase. The wavelength of
the maximum absorbance

(kmax)

was

evaluated.

630

o
C
re

610

CA

2
re

ll

IJ
c
a>

590

570

550

a>
>
re

530
0

Aging

Fig.

4.31 .-Influence of

time

48

[d]

Aging

14
time

[d]

aging on iodine staining of 1.4 % amylopectin dispersions


Novamyl, BAN and Soy-$-amylase. The wavelength of

incubated with

the maximum absorbance

(kmax)

was

evaluated.

88

4. Results and Discussion

In summary, it

can

be said that the color of

induced

changed by enzyme activity. Novamyl


violet. This color

aging.

is

Soy--amylase

BAN and

It is concluded that

dispersions.
or

not stable and the

was

not stainable. In

even

stained with iodine

amylose

color shift from blue towards

dispersions

induced

and

degraded

became colorless

color shift

be

can

after

only

during
of the

aging

stains violet

aggregated amylose

contrast, the color of amylopectin dispersions

was

not

changed by amylases.
On the microstructure level of bread and bread model systems,
induce structures which
starch

with

gels

uncolored and
of starch
center

Novamyl

in

granules

was

polar light

not

showed

with

gels

empty although

would

1998).
to

of starch

endo attack within

rapidly

of starch with

decreases

is

(Outtrup

was

polysaccharide

observed

with 1 %

that

no

longer

mentioned that

ascribed this

complexed"

in

performed

and Norman

Novamyl

chains

was

means

that the

ability

cross

that the enzyme


to form inclusion

of

1984, Christophersen
was

(Dauter

1 %

amylose

stained with iodine.

originates

Starch would have to be

30 % of the starch

a-amylase

et al.

to

more

similar

whereas the

ability

to carry out

1999). Regarding

typical endo-amylase.

maltose
or

the

from

degraded

(Banks

extensive
to

reaction

is unable to take up iodine.

double helix model where

an

the

to

can

degradation

amylose

(total amylose content)

of

1975).

They

would be

be excluded that the

of the

amylose

DP smaller than 10 to lose the

and Greenwood

Liebl et

starch led to the formation of

with itself and thus unable to bind iodine. It

to stain with iodine

found to be

et al.

Furthermore, Banks and Greenwood

retrograded amylose

phenomenon

to stain

endo-manner and results

an

than to

with

by degrading amylopectin

glucanotransferase (GTase)

inability

center

iodine, Taniguchi (1991 ) reported that the blue iodine value

addition

(1975)

suggests

found to be consistent with the

that

(1992)

products

birefringence

way that it lost the

cyclodextrin glycosytransferase (CGTase)

staining

al.

exhibited

The three-dimensional structure of

structure of the active site


an

the

it remained uncolored. Holes observed with

by Novamyl

amounts of maltose

high

where

Primarily

detectable. The fact that the center

black. The observation rather

stay

with iodine.

can

with iodine.

Degradation
in

staining

granules

was

Novamyl

modified the starch fraction in such

complexes

starch

intergranular amylose

no

from

prevent amylose

amylases

This would

capability

that around

mean

would have had to be

fraction.

degraded

to

4.2 Microstructure of

impede
of

with iodine.

complexation

starch, that

means

shown).

The

amylose

of iodine

exact

3.5 %

the

to

and

et al.

and

gels

Novamyl

were

shifted towards violet

starch

gels

the

consequence,

local

amylopectin may

be different in

and

crystallization

aggregation, gelation

concentration and chain

investigated

would decrease the

length (Ring

whether

swelling

et

reduced

of starch and

as a

staining.

and

their

Phase

of

starch

amylopectin

granules,

amylose

tends to be

amylose

and

during

aging

two

polymers

can

be

visualized

its

by Novamyl.

birefringence

As

birefringence already

their

promoted by

iodine

staining

in the center of the

Due

to

the

and

granules,

by
with

where

directly

degradation

long range

compared

an

endo
are

effect, bread and gels with Novamyl exhibit

Novamyl

rapid aggregation
contribute to the

to control. It is conceivable that

also reduces the

via

ordered structures

in the fresh state. It is assumed that the

enhanced initial firmness

visible in low

endoactivity, amylose fragments

and induce

result of this

was

starch

in fresh bread and bread models with

amylose aggregation

observed

which accumulates in the

by

aggregation. Aggregation

readily aggregate

urn).

is

the

already freshly prepared systems

amylose dispersions which suggest

15-25

induces

of

accumulated, suggests that Novamyl partially degrades

promotes

which

amylose

phase separate upon heating

amylose fraction,

The fact that

showed enhanced

produced

The

leads to the

tend to

of the

separation

Novamyl

mechanism

polarized-light microscopy

reorganization

polarized-light microscopy.

concentration

and

bright-field

thermodynamic immiscibility.

of

As

and

It remains to be

iodine

amylose

that

birefringence.

(approx.

with

Interestingly,

Conclusions

conclusion that

center

amylose

by polymer

of water in starch

improve

i.e.

The kinetics of

1989).

The combination of

starch

slightly

between

phase separation.

polymer,

known to be influenced

4.2.6

gels

prevent

granule

are

differences

understood.

not

could be due to differences in the extent of starch

investigated systems.

consequence

fully

(data

and

by Novamyl

portion

protein

the

availability

The

the extent of

1987, Clark

not

are

less intense and

was

rather small

and

concentration of each

al.

induced

are

40 % ethanol extraction

by

of bread and flour

granules

control.

containing systems
swelling

which

complexation, however,

although staining

compared

Novamyl degrades only

measured

as

structures

the centers of the starch


stained

89

bread and bread models

dough,

firming

rate of bread and

an

enhanced

gels by producing

90

4. Results and Discussion

less

perfect amylose network,

which rearranges less

on

aging (Conde-Petit

1992).
In

the

summary,

microstructure of

and

conclusions

from

the

bread, flour gels and of starch gels

The two starch

baking

main

polymers amylose

part

of the

amylose

and

characterization

of

the

are:

amylopectin phase separate during

fraction accumulates in the center of starch

granules.

Amylose

rich

reorganization (takes

Most
via

an

endo-mechanism and
and firmness

It is conceivable that
rate of bread and

several

Based

on

bread

and

an

it

thereby

compared

enhanced

can

model

amylopectin fraction,

or

the

gels by producing

microscopy,
bread

birfringent

days)

probably Novamyl promotes

birefringence

become

regions

by

either

the action of

aggregation
is

of

responsible

to control

less

but also in the

does

amylose by degrading

it

for the enhanced initial

reduces the

perfect amylose

not

spontaneous

Novamyl.

amylose aggregation
a

to

systems.

be concluded that the

systems

due

only

network.

rtrogradation
involve

amylose polymers.

firming

of starch in

changes

in

the

4.3

Physicochemical properties

of starch in bread and in

Physicochemical properties

4.3

91

of starch in bread and in bread models

bread models
The influence of

investigated

was

assessed
followed
was

prior

by X-ray

diffraction is
et al.

on

the

crystallinity

degradation

and Leiievre
wb. The

determined

largely

1982). Therefore,

The dried

1978).

of starch

the water content

freeze-dried

samples

were

amylases

were

(Cleven

by X-ray

1978, Bulon

et al.

conditioned

order of

water content

measured

as

was

freeze-dried

polymer

had

samples

of starch

crystallinity

by

samples

is known to maintain the

Freeze-drying

g/100 g

was

of starch

diffraction. For this purpose, the

(Ahmed

of approx. 2-3

and

of starch

was

methods.

amylases

to measurements.

wheat starch

(WAXS). Rtrogradation

diffraction

structure of bread and bread models with added

crystalline

determined

Crystallinity

methods.

physicochemical

scanning calorimetry (DSC),

by enzymatic

Influence of

The

different

by

differential

by

the starch fraction of bread and bread models

on

by wide-angle X-ray

determined

4.3.1

amylases

saturated

over

barium chloride solution for at least 12 h before measurements. This resulted in


water

of

contents

8-11

approx.

wb,

g/100 g

which

led

satisfactory

to

diffractograms.
Diffractograms

Soy--amylase
pronounced
first

are

presented

reflection

indication

Additionally,

of

for

reflections of the

the

characteristic

1993).

In

of V- and

which

gelatinized,

and

were

but not

caused

B-type

structures

13

degrees,
in

al.

et

which

bread.

as a

1993).

are

Beside

also
the

by endogenous amylose-lipid

yet recrystallized.

In

aged

control

bread,

5.6,15 (weak) and 17, and between 22 and 24 (broad

corresponds essentially

retrograded

Kksel

observed. This indicates that starch of

to

scattering angles (26) 5.6, 15, 17,

of

of fresh control bread

1988,

detected

were
are

case

BAN and

Novamyl,

This reflection often appears

(Zobel

7.5

at

V-type pattern,

addition, the reflection

organization

degree.

formation

V-type pattern,

This

4.32. In the

at 20

reflections

detected at

were

bread without and with

Fig.

further reflections

no

peak) degrees.

in

V-complex

fresh control bread is


reflections

aged

appeared

weak

two

characteristic

complexes,

of fresh and

at 20

degrees

22 and 24

can

which has the

degrees (Le

is still visible.

starch of control bread

(Dragsdorf and

B-type pattern,

Thus, the crystalline

be described

Varriano-Marston

Bail et al.

1980).

as a

The

mixture

origin

of

92

4. Results and Discussion

the

crystalline structure,

i.e.

crystalline amylose

and/or

amylopectin,

determined in combination with other methods since both


as

B-type

cases

detected

at 20

of bread crumb with

Novamyl

and

degrees. Novamyl

breads

containing

was

V- and

BAN

recrystallization
gels

amylases

on

degrees
on

containing

crumb exhibited

degrees already

in the fresh

whereas

BAN

In

supplementation

resulted

crystallinity, Soy--amylase containing

to that of control bread. Fresh crumb exhibited

and

essentially

in

B-type pattern

single

resulted due to starch

aging.

behaved
the

the main reflection

breads, the crystallinity only slightly increased in

B-type patterns.

comparable

reflection at 20

Flour

of these two

aging

superimposed
crumb

be

polymers recrystallize

amylase addition,

additional reflections at 17 and between 22 and 24


state. On

only

structures.

In all three
was

can

similarly

crystalline

to

bread

fraction of starch

crumb

regarding

(Fig. 4.33).

the

influence

of

4.3

Physicochemical properties

93

of starch in bread and in bread models

10

15

20

25

30

Scattering angle 20 [degrees]

Fig. 4.32:X-ray diffractograms of fresh (0 d) and aged (7 d) bread crumb without


and with Novamyl, BAN and Soy-$-amylase.

94

4. Results and Discussion

10

15

20

25

30

Scattering angle 20 [degrees]

Fig.

diffractograms of fresh (0 d) and aged (7 d) flour gels without and


Novamyl, BAN and Soy-$-amylase.

4.33: X-ray

with

4.3

of wheat starch

X-ray diffractograms

to bread crumb and flour

in fresh control

angles

of

mainly

to

period

of 7 d and 10
at

17

starch

aged

gels

with

intermediate

an

after 7 d of
narrower

aging.

peaks

Novamyl

From

the

gels

Novamyl.

as

A-or

crystalline

where

Intermediate

crystallinity
gels

refers to

but less

starch

recrystallization

and to differ from


in control

the other

gels

with

than in

gels

was

presented

The

narrow

in

Fig.

exhibited

On

were
a

one

gels

slightly

indicates

possible

not

to

be described

can

as

Soy--amylase

with

was

recognizable

pronounced

and

reflections than in

gels

more

and

of

more

gels

led

starch

to

gels

control and

expected

firmer after 10 d

are

the

amylase

second

were

for

aged

Novamyl

of the starch

gels

with

in

highest
a

long

starch

gels

B-type pattern superimposed


Novamyl

with

were

still

to the control which indicates

the two reflections at


in

addition. A

could have been

the most effective

but

pronounced compared

clearly distinguishable

broad

was

long aged

pattern

Soy--amylase

and

Novamyl gels

reduction,

4.35 and show

higher crystallinity. Additionally,


degrees

gels. Only,

two

found to be rather low in control

investigations,

X-ray pattern

The reflections of the

V-pattern.
more

firmness

of starch. For further

period (182 d).


are

aging

an

detected in fresh

was

structure

developed

Novamyl

hand, Soy--amylase

gels regarding

crystallinity

be attributed

During

is

the

degrees,

which

B-type pattern

scattering

since the reflection at 17

B-type

highest crystallinity

The

crystallinity

it

pattern

since firmness measurements showed that control

starch

can

pronounced,

more

with V-structures. Starch

than in the control

higher crystallinity

aging. On

which

observed

was

at 17

of wheat starch.

observed

crystallizes

compared

addition.

Interestingly,
starch

crystalline pattern

V-type pattern

became

degrees

B-pattern superimposed

with

to

is characteristic for both. The

showed

differences

d, the crystallinity hardly increased in these

whether starch

distinguish

some

with weak reflections at

containing gels

correspond

of starch.

recrystallization

and

A weak

endogenous amylose-lipid complexes

reflection

degree

showed

7.5, 13, 17 and 20 degrees. Apart from the peak

observed reflections

the

gels

gels (Fig. 4.34).

and in BAN

gels

95

of starch in bread and in bread models

Physicochemical properties

scattering angles

Novamyl gels

22 and 24

whereas the control

gels

peak.

hand, the finding that amylases induce higher crystallinity compared

the control is consistent with the view of

Dragsdorf

On the other hand, contrary to the these authors

and Varriano-Marston

no

A-type structures,

to

(1980).

but

more

96

4. Results and Discussion

less

or

pronounced B-type patterns

which is in

agreement

and firmness it

with Zobel and Senti

found that

was

were

observed in

(1959).

do not

they

When

necessarily

with

systems

amylases

comparing crystallinity

correlate. In the

case

of

BAN, the highest starch crystallinity but the softest bread crumb and flour gels
were

observed. On the other

crystallinity
impact

no

of starch

on

and

crystallinity

control

and

crystallinity

crystallinity

was

enhanced initial firmness


Based

during aging.
crystallinity

of

systems

presented results,
not
to

necessarily

produce

on

in

it

as

and

systems

as

initial

light microscopy

with

can

well

Novamyl

systems

induced

by

crystallinity

crystallinity

with

on

aging.

rate

which both

Fresh

amylose-lipid

increased.

Novamyl

hardly changed

it is assumed that the enhanced initial

is caused

by crystallized amylose.

and that increased

X-ray

crystallinity

From the

measurements

are

alone is insufficient

rearrangements

order, i.e. starch granules and starch networks,

increase in firmness

firming

Novamyl.

internal

firmer crumb. It is conceivable that structural

lower level of

had

correlation between firmness

be concluded that firmness and

agreement

rate and the

Conversely, Soy--amylase

gels. High

both firmness and

aging,

on

firming

of bread crumb but reduced the

V-type pattern

not alter the

to control.

of starch

found in control

showed

systems

complexes

gels compared

texture and

and enhanced the

hand, BAN did

at

account for the

4.3

Physicochemical properties

10

97

of starch in bread and in bread models

15

20

25

30

Scattering angle 20 [degrees]

Fig. 4.34:X-ray diffractograms of fresh (0 d) and aged (7 d,


without and with Novamyl, BAN and Soy-$-amylase.

10

d) starch gels

98

4. Results and Discussion

10

15

20

25

30

Scattering angle 20 [degrees]

Fig. 4.35:X-ray diffractograms of long aged starch gels (182 d) without and with
Novamyl.

4.3

Physicochemical properties

4.3.2

Influence of

99

of starch in bread and in bread models

amylases

the

on

melting

transitions of

retrograded

starch
The

rtrogradation

staling. DSC
Novamyl,

of

measurements

BAN and

is

amylopectin

carried out in order to

were

Soy--amylase

on

the

amylose. Thermograms

of bread and

retrograded amylopectin

and

fresh and

crumb shows

only

one

rtrogradation

amylopectin

allowed to discriminate

gels

of bread

the effect of

study

rate of

phase

are

in

presented

between

C,

retrograded amylopectin

and

second endothermic

In the

starch

systems upon rtrogradation

peaks
dry

were

matter content of the

Fig.
of

4.37

presents

enthalpy (AH)

the influence of

of

melting

aging

in

control

the

breads.

retrograded amylopectin
storage period

oven

showed

of 7

enthalpy (AH)

hardly changed by

of

the basis of the

the

on

rtrogradation

but not

visible

was

reduced

the

(0.083 d)

temperature

yet retrograded.

(thermograms

retrograded amylopectin

to

bread.

not

increased

melting enthalpy

containing

melting enthalpy

did not at all

The

melting

the action of the enzymes

reduction of the dissociation of


of bread

R-,

The

staling.

transitions in the

Soy--amylase

control

complexes

was

no

d, bread with Novamyl presented

compared

aging

on

in all

in bread whereas BAN showed minor effects. After

rtrogradation

seen on

amylases

gelatinized

was

Novamyl clearly

six times lower than the control.

slight enthalpy

and of

amylose-lipid complexes

shown). During aging,


most

calculated

was

occurs

sample.

the loaf from the

removing

the

since it

and is not limited to bread

range of 60 C which indicates that starch

Only

R-,

as

in bread crumb. The measurements of bread crumb 2 h

amylopectin

after

this transition is referred to

and the

integrated

melting

staling endotherm (Eliasson

is often termed

1985).

present study,

of

which reflects the

event becomes visible at around 60 C. This transition is attributed to the

of

and

4.36. Fresh bread

Fig.

transition at around 114-117

amylose-lipid complexes (M3). On aging,

dissociation of

cause

amylose-lipid complexes. Typical thermograms

control bread crumb

aged

to be the main

thought

BAN.

of

(data

that

of
a

was

influence the

amylose-lipid
not

shown).

amylose-lipid complexes

was

only

100

4. Results and Discussion

fresh

(0 d)

re

1'
o
c

115.8C

1 mW

UJ

40

60

80

100

120

140

160

Temperature [C]

Fig.

4.36:

Typical DSC thermograms of fresh (0 d) and aged (7 d) control bread


crumb. Water content of fresh crumb sample: 46 g/100 g wb. Water
content of aged crumb sample: 44 g/100 g wb.

c
o
0)

Q.
o

Control

'^

Soy--amylase

>

E 5"
re

"c

re

<i

BAN

Novamyl

Aging

Fig.

4.37:

time

10

[d]

Melting enthalpy of amylopectin (R^ for bread crumb

as

function of

aging time for different amylases. (Mean with standard deviation,


n
4). Control (a), Novamyl (m), BAN (+), Soy-$-amylase ()
=

4.3

Physicochemical properties

The thermal behavior of flour


similar.

4.38

Fig.

rtrogradation

of

and

and

Novamyl

amylopectin

and starch

show the

in flour

reduced the

melting enthalpy

starch and flour


effect of BAN

was

similar to control

of

which

found in flour

gels.

that starch

The

gels

gels

have

seen

of

It

can

gels

than for flour

enthalpy

always

amylopectin

as

antifirming

in

consistent with the

were

increase in the

an

which does have

effect

the

bread,

of
no

gels

enthalpy

calculated

on

the basis of the

enthalpies
that

are

on

dry

starch basis

only slightly higher

gels.

correlate with

assessed

rtrogradation

pronounced amylopectin rtrogradation.

a more

an

antifirming
not

and
of

findings

hinders the

effect in bread and

significantly

by DSC, although
bread

melting enthalpy

firming. Novamyl

hand, BAN and Soy--amylase did


starch,

bread,

values of flour and starch

retrograded amylopectin

be concluded that

does not

Like in

baking.

was

as

The calculation of the

for starch

gels, respectively (different

increase

gels

matter content of the

for

the

in bread. In contrast to

enthalpies

gives enthalpy changes

on

completely gelatinized during

This is due to the fact that the

sample.

was

retrograded amylopectin during aging

and starch

comparison

addition

enzymes

did not influence starch

already

was

amylase

of the

two hours after

gels. Soy--amylase

gels,

with

influence

starch

noted). Again,

yet retrograded

gels

and starch

gels

not

was

of flour and starch

suggests

gels

4.39

Fig.

ordinate scale should be

heating

101

of starch in bread and in bread models

starch

Longton

BAN and

and

gels. On

have

These

of

the other

rtrogradation

Soy--amylase

LeGrys (1981)

amylopectin

rtrogradation

reduce the

gels, respectively.

of

of

strong

results

are

and Sahlstrom and

Brthen (1997). They reported that firmness and starch rtrogradation during

aging

of bread and starch

gels

fact that enzymes may have


of starch is not hindered
a

lower level of

did not

necessarily

strong antifirming

points

organization.

out

again

the

show

effects

causal

although

importance

relationship.

the

The

rtrogradation

of structural elements at

102

4. Results and Discussion

c
o
0)

Q.
o

Control

Soy--amylase

>

E 5"
re

"c

re

^i

BAN

Novamyl

c
0)

Aging

Fig.

4.38:

time

10

[d]

Melting enthalpy of amylopectin (RJ for flour gels as function of aging


time for different amylases. (Mean with standard deviation, n
4).
Control (a), Novamyl (m), BAN (+), Soy-$-amylase ()
=

-^BAN

Control

o
0)

Soy--amylase

Q.
o

>

E
re

II
re

<

CD
c

Aging

Fig.

4.39:

time

[d]

Melting enthalpy of amylopectin (R-,) for starch gels as function of aging


amylases. (Mean with standard deviation, n 4).
Control (a), Novamyl (m), BAN (+), Soy-$-amylase ()

time for different

4.3

Physicochemical properties

The

transitions of selected

phase

samples
a

for

182d

heating

transition

in

and

(R-,)

occurred

4.40.

Fig.

amylose-lipid

around

150-170 C.

to the

starch

of

melting

R2

evaluated

was

reproducibility.
found to be
in

plotted

The

enthalpy (AH)

4.41

as

inversely dependent

on

the

the

on

containing gels, high enthalpy


small.

was

comparison,
Fig.

4.41.

the

As

independent

expected,

the

of

it

enthalpy

DSC

difficult to evaluate the

signal

was

parameter

In

The

gels

of the

same

At

of

are

of

and

is true for the

of the transition

reduced the

(R2)

for

of the

aged

gels, respectively.

sample weight
transition.

independent

on

the

was

sample age (data

R2

melting enthalpy

transition

gels.

flour

shown).

130

is
of

C)

because the

was

by high
the

only

Tab. 4.1 indicates the

gels

noticed that the


not

For

also shown in

accurately

melting temperature by

It

was

Novamyl

high temperatures (above

melting temperature

R2

amylose-lipid complexes

the control which resulted in transitions 162 and 154 C for

was

R2

was

transition is

enthalpy

detected when the

enthalpy

good

transitions

R2 melting

control

which could be evaluated in the

Novamyl

flour and starch

155 C.

difficult to obtain

AH of the

only

shown).

of the HT transition

melting enthalpy
both cases,

was

Novamyl

very often disturbed due to tensions in the pans induced

pressure of the water. The


reliable

smaller in

was

amylose-lipid complexes

not

R2. Control gels

high-temperature (HT)

were

retrograded amylopectin (data


was

since it

sample weight.

The

endothermic

temperatures approx.

melting enthalpy

sample weight.

third

therefore termed

was

reduced the

melting enthalpy

of the

(M3),

retrograded

et al.

sample weight.

Novamyl slightly

of

melting

gels aged

retrograded amylose (Eberstein

sample weight.

values

of starch

most

of these

function of the

g wb and measured

latter endothermic event

mainly qualitatively

dependent

Fig.

This

and occurred at lower

gels

the

at approx. 170 C. This transition

large R2 peak

containing

Besides

complexes

1980, Sievert and Wrsch 1993) and


a

H2O/100

Representative thermograms

presented

to 80 g

measured up to 200 C. The

rate of 10 C/min.

probably corresponds

showed

were

rehydrated

are

amylopectin

samples

freeze dried and

were

with

103

of starch in bread and in bread models

and starch
-

8 C

gels.

In

compared

to

Novamyl containing
melting temperature

104

4. Results and Discussion

Amylopectin (R.,)
Amylose-lipid complex (M3)
o

Amylose (R2)

re
0)

0)

Novamyl

O
a
c

UJ

1 mW
-i

30

1i

50

1i

70

1i

90

1i|

110

1|

130

150

1|i

170

190

Temperature [C]

Fig.

4.40:

Typical DSC thermograms of aged (182 d) and freeze dried starch gels
up to high temperatures. Samples were rehydrated to

recorded
80 g

H2O/100

g wb.

24

High-temperature melting

transition

(R2)

Sample weight [mg]


Fig. 4.41-.Influence of sample weight
AH of

on

AH of HT

melting transition (R2) and

on

amylose-lipid complex (M3) of freeze dried and rehydrated

(80 g H2O/100 g wb) starch gels.


R2: Control (a), Novamyl (n)

M3: Control (+), Novamyl (o).

4.3

Physicochemical properties

Tab. 4.1:

105

of starch in bread and in bread models

Melting temperature (TmR2) of HT melting transition (R2) of starch gels


and flour gels without and with

Novamyl.

Melting temperature TmR2 [C]


Starch

Flour

gels
(-46 g H20/100 g wb)

gels
(-80 g H20/100 g wb)

Control

169

162

Novamyl

162

154

Based

these results it is assumed that

on

starch since it

can

therefore

protein-starch

no

temperature

flour

interactions

differently. Starch gels

which is

gels

high

transition

water contents

changes.
the

reflects

(R2)

(40

80

Differences in the

melting temperature

with lower

found for control


more

gels

where the
more

X-ray

than the

(Fig.

as

measurements. Control starch

Novamyl containing gels, although

4.34

and

Fig. 4.35).

No

R2

of the

in

less

Fig.

showed

the latter

of

amylopectin

supramolecular

amylose-lipid

perfect amylose
contrary

is

and

crystals

are

built.

4.41

are

inconsistent

clearly

for this

complexes

to

more

AH for

R2

crystalline

incongruence.

It

polymer fraction,

the

formation

of

structures.

In summary, it
increased

and

that

amylose requires

reinforces how difficult it is to estimate the contribution of each

amylose,

suggest

slightly higher

were

is found

explanation

the HT

detected with DSC. On the other

presented
gels

No

and volume

than the control. The

as

than

gels.

Probably,

transition

perfect amylose aggregates

higher melting temperature

degraded

of starch in combination with

spontaneous aggregation

hand, the enthalpy values of starch gels


with

evaluation.

by Novamyl give

melting temperatures

time, and therefore,

The result is

induced

melting

temperatures

g/100 g wb), high pressure (5-15 bar)

rapid amylose aggregation

aggregates

enthalpy

physical change

the

water content in starch

higher

and

present,

Additionally,

occur.

showed lower transition

the

explained by

is

protein

no

transition of

by melting

addition which indicates that

conclusive results could be obtained from

melting

where

gels

changed by Novamyl

was

starch behaves

be found in starch

is caused

R2

can

be said that the

during aging

and that

melting enthalpy

Novamyl

was

the

of

retrograded amylopectin

only amylase

which almost

106

4. Results and Discussion

blocked

rtrogradation

difficult and not

4.3.3

amylopectin. DSC

of

necessarily

in

agreement

Influence of

amylases
degradation products

on

with

measurements of

fraction, the starch

determined in fresh
of bread
a

(0 d)

the starch content and the starch

classified

was

as

amylases, mainly

content and the starch

and

resistant starch

(RS)

of starch

individuals

method
starch

et al.

(Asp

and available starch

quantitative

1996).

In the

at 37 C for 16 h.

termed RS. The

as

the

determination is

Starch,

bread

which

crumb

was

not

were

(AS).

RS is

of starch and

sum

dependent

present investigation,

on

The starch content

gels.

It is difficult to compare RS contents

analyzed by incubating

was

a-amylase

its

as

Novamyl,

not absorbed in the small intestine of

degradation

(Euresta 1992).

different studies

of

degradation products

bread and flour

aged (7 d)

nutritional classification of starch and is defined

products

were

diffraction.

X-ray

In order to understand the mode of action of


the starch

amylose

analyzed

the

on

healthy
in

analytical

resistant and available


with

slurry

degraded by

this

pancreatic
step,

was

of RS and AS results in the total starch content of the

sum

sample.
Starch,

maltose and

presented

are

in Tab. 4.2. The

without enzymes gave

the amount of RS

nor

with
the

changed

of RS

the RS fraction of

was

comparable

around 2.6 times

Novamyl containing

g/100 g

higher

to control bread.

portion

Interestingly,

than in control bread. On

bread did not

change

apparent

56.2

Novamyl during storage

db in bread with

db. A small

bread

Neither the total starch content

starch content decreased. The

g/100 g

freshly prepared

The total starch content of fresh bread

aging.

was

amylase supplementation

of AS and RS in

(1.7 g/100 g db).


on

Novamyl (69.0 g/100 g db)

proportion

sum

total starch content of 69.4

found to be resistant starch

was

content of bread with

glucose

aging,

whereas the total

reduction of starch content from 69.0 to


can

be

unsuitable starch determination method

or

may have contributed to further starch

degradation during aging

by

residual enzyme

explained by

activity.

an

The latter

and/or

during

extraction of starch.

The literature data

(Eerlingen
aging,

et al.

which

regarding RS

in bread is inconsistent.

1994, Sahlstrom and Brthen 1997) found

they

attributed to

rtrogradation

of

an

Several authors

increase of RS

on

amylopectin. Others (Berry 1986,

4.3

1988, Siljestrom

Hansen et al.
the

of

course

also the

aging

et al.

1988)

observed

present investigation.

enhanced the RS content of bread. This


of starch

degradation

starch fraction.

X-ray

No

and

significant

as

glucose

activity

stability.

source

Soy--amylase

no

changes

BAN, it is

no

thermostability.

for

was

not

an

points

to

and

Novamyl

expected

highest

maltose

gels

further starch

Soy--amylase

produced.

high

can

presented

were

standard deviations

of bread

during aging

were

because its

produced by

yeast
starch

by BAN, mainly oligomers


BAN inactivation it would

complemented by investigations

gels

are

be reached.

were

of starch

of 7 d.

Fresh bread

change during aging.

heated in closed

with DP

>

In order to achieve

first baked at 96 C for 1 h

and

starch

overlapped. Generally,

the

flour

gels.

temperature

(standard conditions)

were

on

complete enzyme

degradation products

Differences in starch content

on

moulds, temperatures

in Tab. 4.3. The starch content remained constant

aging period

low thermal

glucose.

heated to 120 C for further 1.5 h.

analysis

degradation products

in

step

due

in bread. A residual

during baking

on

gels

The

degradation

suitable bread models to examine the influence of

than 100 C

second

not

level

which

inactivation flour
a

formed

maltose contents derive from

To draw conclusions

of starch content

are

and

d, the level of

investigations.

glucose

weight products,

enzyme inactivation. Since flour

higher

that BAN is inactivated

expected

rather than maltose and

Flour

in the

produced sugars

of 7

aging period

level, which did

therefore be necessary to determine starch

Analysis

mainly

since the enzyme has

occurred in maltose and

It is assumed that the

are

occur

found in fresh bread due

was

with BAN. Due to the endo attack of starch

degradation

bread is

addition. It is assumed that the

yeast. During

fermentation of low molecular

with DP

rearrangements

This result needs to be confirmed with further

Although

Novamyl

evidence that due to the

Novamyl

and maltose

glucose

inactivation of

with BAN contained the

with

structural

was

crystalline amylose.

and maltose increased which

of

to note that

This

compression measurements, light microscopy

increase of

energy

incomplete

an

on

Soy--amylase

and

Novamyl

served

to

Based

during baking.

important

provides

measurements it is concluded that RS of

by aggregated

to

by Novamyl major

It is

in RS content in

changes

no

and concluded that total RS is formed

in the

case

107

of starch in bread and in bread models

Physicochemical properties

in

over

not

flour

gels

is

the observed

significant

supplementation

and

of

since

Novamyl

108

4. Results and Discussion

slightly

decreased the

products

starch

content

and the maltose and

glucose

products,

maltose and

according

to the standard

in flour

with

gels

procedure. On

of flour

can

gels

showed that

inactivated with the


concluded that

effect, but it

by heating

Novamyl

second

Novamyl

has

hand,

Novamyl

mainly

caused

by

also conceivable that


due to the

the

would indicate

amylases

same

higher

is

times.

starch

inactivation

systems Novamyl

respectively. By
complex system
they

are

and

Maltose and

during aging

for

even

if it is
it

is

antistaling

an

during baking.
gels

and bread crumb

of

cleavage

>

baking,

Novamyl
display

and
the

of starch

2. The action

the added

starch

in

is

not

bread, however, the amylases

are

matrix which exerts

gels.

are

were

This

leads

of BAN and

and

exo-way,

completely

not

and maltose. The

surprising.

In

aqueous

at 65 C and 75

activity
not

gels

the results

by Novamyl

endo-

optimal activity
the

an

amylases

temperature

embedded in

in flour

pattern

producing glucose
BAN

which

gels. Nevertheless,

degradation

to

degrade

BAN

in flour

the

higher

were

in flour

degradation

glucose contents,

systems,

with DP

further increase of
like

Its

measurements

shown). Therefore,

not

time for starch

degradation

due

standard

inactivated and continue to

incomplete

found

during storage.

good antistaling properties

more

during baking

different

respectively. During

as

had

degradation products mainly

Soy--amylase

was

inactivated in flour

Compression

to 120 C.

sufficient starch

method in both

lead to the conclusion that


to

increase

two different methods used for starch determination. It is

longer baking

analyzed by

degradation

Novamyl produced

fully

It is assumed that differences in starch content of flour


are

starch

starch

degraded

heating step (data

degrade

no

not

was

does not have to be active

must be able to

with

degradation

heating steps.

at 96 C and therefore still

be achieved

starch

aging,

gels

the other

with two

The results lead to the conclusion that

inactivation

contents. On

increased in flour

glucose

Novamyl produced

gels during heating

but enhanced the

is reduced.

completely

protective

effect.

C,

In

inactivated

4.3

Physicochemical properties

Tab. 4.2:

109

of starch in bread and in bread models

Starch, maltose and glucose content of bread crumb with Novamyl,


BAN and Soy-$-amylase. (Mean with standard deviation, n
1-8).
=

Starch

AS1)

RS2)

Maltose

Glucose

Total

[g/100 g db] [g/100 g db] [g/100 g db] [g/100 g db] [g/100 g db]
Control

Od

67.67 2.08

1.69 0.02

69.36

3.31

0.06

7d

67.20 2.63

1.76 0.01

68.96

3.50

0.08

64.48 3.20

4.48 0.12

68.96

3.12

0.14

7d

51.82 4.79

4.37 0.07

56.19

4.51

0.16

Novamyl 0

BAN

Soy-amylase

Od

nd3)

nd

4.36

0.10

7d

nd

nd

4.63

0.10

0 d

nd

nd

3.77

0.07

? rf

nd

nd

6.32

0.09

1)

available starch

2)

resistant starch

3)

not determined

110

4. Results and Discussion

Tab. 4.3: Influence

products

of
in

Novamyl on starch content and starch degradation


flour gels. (Mean with standard deviation, n
2-4).
=

Starch

[g/100gfd1)
flour

C,

96

1h

Control

Novamyl

C,

96

1h

Control

Novamyl

gels]

Starch

degraproducts
[g/100g fd flour
gels]

dation

Maltose

Glucose

[g/100gfd
gels]

flour

flour

[g/100gfd
gels]

(standard conditions)
Od

59.79 1.61

7.05 0.56

3.98 0.12

0.09 0.03

7d

62.75 0.90

3.85 0.95

3.19 1.55

0.09 0.00

Od

56.32 1.94

11.68 1.31

5.16 0.63

0.20 0.01

7d

54.41 1.16

13.00 1.27

6.70 0.07

0.25 0.03

120

C,

1.5h

Od

55.55 2.81

4.53 0.93

4.55 0.37

0.12 0.03

7d

55.251.45

4.18 0.41

4.08 0.50

0.13 0.03

Od

50.97 4.19

9.51 0.73

8.45 0.22

0.24 0.03

7d

48.61 2.36

9.17 0.68

8.14 0.38

0.19 0.00

freeze dried

4.3

Physicochemical properties

4.3.4

Conclusions

Based

following

the

on

physicochemical

main conclusions

Increase of
firmness

crystallinity

compared

to control

The increased

crystallinity

mainly

Determination

of

with

was

The added

Novamyl
have

correlate with

an

increase in

as

of fresh

systems

higher

is most

initial

probably

of

increase of resistant starch

an

aggregated

and

amylose by DSC

crystallinity

caused

by

already

in fresh bread

crystalline amylose.

was

difficult

and

necessarily

in

rtrogradation

of

not

firmness, light microscopy and X-ray.


the

only amylase

which

almost

blocked

measured with DSC.

amylases

are

completely

not

has not to be active

enough

rate induce

firming

fraction.

which consists

Novamyl

necessarily

systems.

addition leads to

amylopectin

does not

which reduce the

agreement

be drawn:

can

Amylases

Novamyl

data of starch in bread and bread models the

during aging.

crystalline amylose

111

of starch in bread and in bread models

time for starch

inactivated

during aging

for

an

during heating

antistaling effect,

degradation during baking.

at 96 C.

but it must

112

5
A COMPREHENSIVE APPROACH TO STALING

Comparison of
of organization

5.1

One

main

bread and bread models at different levels

difference between bread

macroscopic appearance of these


i e

sponge, which

pore

size

starch

pores

in

turn, exhibit

amylases

volumes and pore

compared

retard the

on

homogeneous

is an

open pore

dense and crisp crust The

the structural

sizes as

pointed out, however,


lower

Bread crumb

properties

of bread

that the

to flour

of starch

firming

found

dry

gels

in

without

strength

bread when

being

adding amylases

matter content of starch

and bread

54

(approx

systems (Maxwell

flour and starch

fused

together,

Differences exist
a

bicontinuous

starch

only

gels
and

in

In all three

systems,

and Zobel

starch

amylose/amylopectin

protein-starch

gels

Flour and
without

measure

the

different

by

It should be

gels (40 g/100 g wb)

g/100 g wb)

which

can

1978)

granules

phase

the matrix In bread and flour

to

the

and the

gel

biased

As for the microstructure, many characteristics of bread crumb


in

the

throughout

texture

is

system,

porosity

Thus, flour and starch gels provide the possibility

crust

influence of

is

by

systems

distribution determine the mechanical

gels,
nor

coated

is

crumb, flour gels and starch gels

are

were

also found

elongated, partly

separation

was

the microstructure

matrix whereas the matrix of starch

which facilitates starch-starch interactions The

gels

found

presents

consists of

comparatively

low

dry

113

114

5. A

matter content of starch

starch. The

gels

look

granules

has

an

influence

swollen and

more

that the extent of

slightly

At the molecular
three

and

systems

of starch

content calculated

approx. 40

content is related to the

folded and

more

amylose

are more

it is assumed
is visible in the

gels.

level, starch
was

more

staling

behavior of

gelatinization

gels. Additionally,

is different since

phase separation

intergranular space

the

on

hooked into each other than in bread and flour

to

comprehensive approach

starch content is then approx. 75

g/100 g

was

similar in all

wb. Differences exist when the starch

g/100 g

matter of each

dry

wet basis

on

In bread and flour

system.

db and in starch

100

gels

gels

the

g/100 g

db.

Furthermore, with exception of the damaged starch through the milling process,
the entire starch fraction is in the native state
In

gels.

starch

gels,

the

on

pregelatinized (4 g/100 g wb)


The

The

investigate staling

properties

macroscopic

and the

gels

can

screening

tool

for

temperatures higher
can

be

important

consequences

for

on

gels

are

produced,

potential

starch

testing

gels

are

dough handling

different at all

Consequently, amylases

bread.

Therefore, starch gels

enzymes. On the other hand,

gels

are

enzymes.

In

similar from the

proofing

investigated

on

levels

using
flour

flour

high

not suitable

system.

This

and its

not allow to

aging.

screening

information and may contribute to understand the

as

baking

complex systems

compared

concentration starch

gels

gels,

to flour

may show different effects in starch

are

smaller

production,

hand, the closed system does


and

of

antistaling enzymes.

be reached due to the closed

the other

consists

suitable bread models in

to bread

inactivation of enzymes in

staling. On

fraction

which is essential when

can

of bread and flour

(36 g/100 g wb).

Compared

antistaling

than 100 C

of bread.

heating

behavior of bread and flour

bread.

during staling

to

of bread and the mechanism of

aging

be

follow the influence of

Starch

and native starch

to the molecular level.

batches of

the

hand,

shows that flour

present investigation

order to

other

prior

tools for

gels

gels

and

and in

antistaling

gels give complementary

changes

in the starch fraction

5.2

Relationship

between starch structure and

Relationship

5.2

Based

on

if

even

an

the

not

was

on

in bread and model

the main influences of

surprising
the

same

changes

Soy--

BAN and
is

at the

at different

systems

systems

same.

in terms of

systems

Novamyl,

behavior of bread and model

aging

it is not

necessarily

the detailed examination of the

justifies

staling

systems,

that the enzyme induces the

mean

scales. An overview
on

systems

amylase performs similarly

molecular level. This

amylase

in different

amylase

an

texture, this does not

length

between starch structure and

the dissimilarities of bread and model

that the effect of


But

115

staling

presented

in

Tab. 5.1.

5.2.1

Influence of

Novamyl
it

showed similar effects in

that the

seems

by Outtrup

degrading

and Norman

of low molecular

is formed in the

concluded that

Both DSC and

degradation product
X-ray

aging. However, DSC

Rtrogradation

structures.

by DSC

whereas

method.

X-ray diffraction,

and

aging

Based

on

therefore,

DSC and

crystallinity

of

of

on

no

X-ray

systems

of

the other

both

with

Novamyl

amylopectin

and that this is

Novamyl.

maltose

amylose by Novamyl
The authors

amylose.
a

non-reducing

end

in the starch fraction

not

easily

equally

during

sensitive to starch

be followed and

quantified

by

the latter

amylopectin yield

possible

B-type pattern

between the two

polymers.

measurements it is concluded that the enhanced initial

degradation

effect of

Primarily

is difficult to be followed

and

discrimination is

that the

reorganization

leads to the

hand, allows the detection of low levels of

amylose

amylopectin rtrogradation
of

are

can

amylose

and that

its

of wheat

changes

diffraction

amylopectin

rtrogradation

crystallinity. Unfortunately,
on

X-ray

in all

is maltose.

diffraction reveal

and

of DP 1-9.

and does not need

endo-activity

same

has been characterized

by Novamyl

distribution of

weight

the

et al. 1998 and Dauteret al. 1999.

weight oligosaccharides

has

basically

Novamyl

of starch

hydrolysis

molecular

Novamyl

the main

although

mechanism of

oc-configuration. Degradation

reduces the average

is

Novamyl

1984, Christophersen

The authors showed that the

production

bread, flour gels and starch gels. Therefore,

mechanism of

antistaling

The starch

systems.

Novamyl

one

is induced

by

is almost blocked

crystalline amylose
during aging.

side chains to the


factor which is

It is assumed

branching points

responsible

fraction

for the

hinders

antifirming

1.39
4.35

BAN

Soy--amylase

4)

amylopectin
amylose

2) not visible
3)

lower than control

than control

1.65

Soy--amylase

higher

4.38

BAN

ED(7d)/ED(0d)

AM+

1.36

Novamyl

nv

nv

nv

4.65

nv

Control

gels

AM+

1.95

Novamyl
nv

nv

4.83

nv

Control

Starch

1>

6.75

Soy--amylase

Flour gels

1.67

BAN
nv

AM+

2.04

Novamyl

Od

AM4)

AP+AM

4.894

4.035

0.997

AM"
AP+AM

3.995

2.111

AP+AM

AP+AM

2.064

0.847

AM"
AP+AM

2.208

2.645

1.437

0.249

2.835

7d

[J/g db]

AH of AP

AP+AM

AP+AM

AP+AM

AM"

AP3)
+

7d

Birefringence

gels during aging induced by amylases.

nv2)

7d

Firmness1) ["]

Relative

of bread, flour and starch

11.99

Changes

Control

Bread

Tab. 5.1:

B+

B+

B+

B+

B+

B+

Od

Crystalline

B+

B+

B+

B~

B+

B~

7d

structure

5.2

between starch structure and

Relationship

The

that

hypothesis

supported by

the observation that the

birfringent already
starch

produces long range


Novamyl promotes
via

amylose

birfringent

the

aggregation
Thereby

of

that

amylose

amylose

|im).

in fresh

amylose

in fresh bread and

the reduced
network is

firming

of

amylopectin

firming.

built-up,

but also the

Due to the

which

rate is reduced in

aggregated amylose

probably rearranges
with

also achieved because the enhanced


formation between different

of

rapid aggregation

systems

less

on

the blocked

less

for

perfect

therefore, the

antifirming

an

systems,

of
an

responsible

and

aging,

inhibits

effect is

cross-link

i.e. starch

granules,

amylose aggregation
of the

is

amylose

Novamyl. Possibly,

components

aggregation

only

it

of the

enhanced initial firmness and to

increased resistant starch content. It is conceivable that not

rtrogradation

probably,

mobility

chains which facilitates association of the molecules. The


an

and

systems by degrading

the enzyme increases the

leads to

are

rich center of

Most

amylose

gels

granules

amylose aggregates

15-25

(approx.

is

by Novamyl

rich center of starch

means

ordered structures

endo mechanism.

an

is induced

amylose

in the fresh state. The fact that the

becomes

granules

of

crystallization

117

staling

matrix, amylose and amylopectin rich phases within the granules. This and other

investigations (Conde-Petit 1992)


starch

systems during aging

is

show that the

influenced

remarkably

behavior of wheat

rheological

by changes

in the

amylose

fraction.

5.2.2

Influence of BAN

BAN, which is

by

an

conventional bacterial

a-amylase, degrades

endo-mechanism. This leads to the reduction of the molecular

starch and the formation of dextrins. The

viscosity
effects

starch

loss. Influence of BAN

seen

in

starch

on

different

gels. Therefore,

gels

weight

results in

endo-degradation

bread and flour

randomly

was

of

rapid

different from the

mechanisms

appear to

be

involved.

The firmness of bread and flour

gels

with BAN is

aging although amylopectin rtrogradation


is

enhanced.

Based

enhanced initial

systems

regard

with

to the

on

DSC

crystallinity

Novamyl,
antifirming

and

is caused

these

drastically

is not altered and the initial

X-ray diffraction,
by

crystals

reduced

it

is

during

crystallinity

assumed

that the

crystalline amylose fraction. Unlike

did not enhance initial firmness.

effect of BAN in bread and flour

gels

the

With

several factors

118

5. A

comprehensive approach

appear to be involved. It is conceivable that the substantial starch


the endo-mechanism results in
not interconnected

are

as

the

produced

reduces the overall firmness of the

and

to

continuous

of the

intergranular

gels

correlate with the mechanical

BAN

only slightly

the firmness and the

properties

reduced the

gels,

it is reasonable to

presence

of

concentrations
the

vicinity

1997).

assume

inhibitor

or

induce

an

can

of the

supposed

i.e. starch

of the bulk.

stress and

breaking

some or

the

absence

matter

the other way inhibited

hydrolysis products, by

of

activator.

an

play

that BAN

Since the

role.

only

of starch

breaking

weakened the

and

gels

stress

proposed

that

linking

an

firming

antifirming

between starch and

due to the absence of

and

gels

Novamyl

in pure starch

(Hill

the
was

in

et al.

starch

slightly

portion

of

starch,

embedded in the matrix.

is caused

by cross-linking

and that low MW dextrins would interfere with it.


would have

activity

starch matrix

intergranular

but did not affect the main

the

High sugar

and thus inhibit the enzyme

content

no

the mechanism of BAN in starch

in

by

had

of starch in starch

The effect of BAN would corroborate the model of Martin and


who

The effects

osmotic effect which reduces the water

granules together,

granules

on

was

enzyme-starch complex

concentration could also

which holds the

the

by

Furthermore, the dry

reduced it is

that BAN

Inhibition could be caused

an

properties.

physicochemical properties

While it is difficult to draw conclusions

gels.

the

and bread demonstrate that the molecular level does not

gels.

in starch

during

as

starch network and of the starch

of BAN in flour

on

low MW dextrins act

BAN is not inactivated

Finally,

in turn, influence the overall mechanical

impact

and

therefore, further degrades starch during storage. This leads

weakening

Unexpectedly,

which

rearrangements during aging

granules which,

necessarily

degradation by

low MW dextrins interfere with network

system. Additionally,

and thus reduce firmness.

baking process

staling

independent (dispersed) crystalline regions

formation. This structure hinders molecular

plasticizers

to

to starch

systems.

Hoseney (1991 ),

between starch and

According

to this

model, BAN

effect because low MW dextrins hinder the

protein

protein

whereas

matrix.

showed that

no

effect is

expected

gluten

cross-

in starch

gels

However, the addition of Soy--amylase


an

This indicates that

antifirming

effect

can

amylases primarily

also be obtained

influence the starch

5.2

Relationship

between starch structure and

fraction rather than the starch


action of BAN

in starch

interface. To obtain

protein
starch

gels,

119

staling

clearer view

on

the

would have to be

degradation products

determined.

5.2.3

Soy--amylase

Influence of

Soy--amylase

is

of maltose from the

non-reducing

configuration,

hence the

has

influence

no

major

Soy--amylase

bread and flour


It showed

on

the molecular

an

nor

did it have

bread crumb.

It is assumed that

during baking

of bread and flour

amylase (55

60

As

C)

and

degradation
In starch

BAN, Soy--amylase

of starch. Starch

degradation by

the

the

only

gels.

no

on

of starch occurred

Due to the low thermal

stability

inactivated before starch


no

influence

accessible

on

systems.

of the elastic recovery of

slight degradation

had

of starch in

the firmness of these

preservation

mostly

amylase

therefore, had

physicochemical properties

influence

an

the enzyme is

consequence,

accessible substrate exists.

only potential regarding

the successive removal

and

Novamyl

weight

neither influenced the

gels

catalyzes

ends of starch. Maltose is released in the

In contrast to

name.

is fast if

Soy--amylase

and

typical exo-amylase

staling

substrate

of flour

gels

Soy--

of

gelatinizes.
for

starch

and bread.

on

the other

hand, the pregelatinized starch fraction, which is

specific component

of starch

gels,

main

gels,

of starch is

portion

the enzyme
inhibited
as

birefringence

pregelatinized

interfering

the

starch

reorganization

mobility

might

be

starch.

can

and

of the
act

as

portion

of

It is assumed that

gelatinizes

amylopectin

from

serves

nor

on

the

to the

crystalline amylose
amylopectin

of the

branching points,

which

polymer. Furthermore,
antiplasticizer

it has to be taken

at the molecular level thus

amylopectin which,

in turn, reduces

that maltose is formed in situ at the

with network formation. It is

and

of starch is not affected. The

probably

of

before the

extent but is then

large

The side chains of

crystallization

important

of starch

rtrogradation

possibly degraded

are

heating step.

starch to

portion

derives most

pregelatinized

the

chain

It

on

crystallinity

into account that maltose

firming.

second

is observed since the main

fraction of the

reducing

pregelatinized

No influence

enhanced initial

complicates

in

before the main

during baking

substrate.

gelatinized

the

degrades

Soy--amylase

is accessible for

hypothesized

that the

right place

intergranular

network, which is mainly formed by the pregelatinized starch, does

for

starch

not rearrange

120

5. A

and that

cross-links between the matrix and the starch

no

the

amylase although
granules,

Like in
the

with

Novamyl,

effect of

antifirming

was

supramolecular
on

enhanced.

during baking

Due

structures of

the mechanical

amylose

fraction

in starch

the

to

it

is

different

amylose

are

due to its

higher

formed which have

thermal

Novamyl systems.

temperature

behavior of the

rheological

affected

are

stabilities.

role

during staling.

numerous

is

early stages

dominant factor in the


this

by

amylose

becomes

aged systems. Amylases


systems

systems. Thus,
accelerates the
network

starch,
induced

and

degrade

the

starch

intragranular

aggregates

of

micro-domains, i.e.

Soy--amylase

due

on

the

aging

is

in

the presence of

shows that

amylose

of bread and of bread

models,

antifirming

properties

of

effect in bread and model

of starch. The combination of DSC and X-

responsible

for the

partial cleavage

of

crystallinity

rearrangements

of the

intragranular amylose

have shown that the


inclusion

aggregation

complexes

of fresh

amylose by amylase

with

of

amylose

networks with

aged systems. Preliminary experiments

amylose

an

fraction has received less

amylose

of starch and hinders

lipid-free starch,

by

of

The result is inter- and

strength

of

staling

present investigation

an

crystallinity

it is concluded that

gelation

molecular

determinant for the mechanical

mainly amylose

during aging.

low structural

The

which have

enhance the initial

ray revealed that

the initial

publications, amylopectin plays

In contrast, the

staling.

and

with

to the initial firmness of

and

by Novamyl

gels

This, in turn, has different impacts

attention in connection with


a

additionally

role in

system.

As has been documented in

important

key

mechanism

that

assume

Contribution of different starch fractions to

5.2.4

Soy--

different influence

is able to

This leads to the conclusion that different

intragranular starch,

to the different

stability. Therefore,

contribute

granules

although

dissimilar

properties. Additionally, Novamyl

within the starch

inter- and

with

In contrast to

degradation

that

supposed

play

to

seems

gels.

starch fraction is also affected. It is reasonable to

amylose

gels

starch, i.e. starch in

did not enhance the initial firmness

Soy--amylase

and

the

Soy--amylase

Novamyl, Soy--amylase

Novamyl

of

portion

staling

is not inhibited.

systems

crystallinity

of the main

rtrogradation

to

granules develop

Both weaken the overall firmness of the starch

during storage.

the

comprehensive approach

with

amylose

potato
is not

endogenous lipids.

5.2

Relationship

The
on

between starch structure and

hydrolysis products

staling

plasticizer

as

well.

formed

Dextrins

at the textural

the action of

by

amylases

have

influence

an

interfere with network formation and act

can

level.

121

staling

as

Thus, the antistaling effect of enzymes is

combination of the modified starch structure and the

hydrolysis products

formed

in situ.

Finally,

it should be

pointed

critical in connection with


different

structural

deformation.

possible

out that the

firming.

elements

By testing

In

different

give

properties

granules

interfaces. For further elucidation of the


more
a

investigations

are

required

comprehensive understanding

of different structural levels

by

are

structure of starch is

materials such

composite

mechanical

to reveal if the starch

supramolecular

mechanical
of the

deformed

relationships

or

as

bread the

responses

bulk material

if failure

occurs

it

upon
is not

along

the

between starch and texture

on

the network level of starch. It is evident that

on

antistaling enzyme requires

the assessment

combination of different methods.

122

5. A

Model for the

5.3

staling

to

comprehensive approach

of bread and the

staling

effect of

antistaling

amylases
Based
model

points
Fig.

and

baking

on

of attack for

antistaling enzymes

5.1 illustrates the

system

components.

changes

be described

The

properties

as

reflect the real dimensions.


dimension.
10

nm

Amylopectin

in

reality,

smaller in relation to the

Dough
granules

is

depicted
partly

are

and

nm

granules

The

whereas

starch contains

lipids

fraction

in the native

which

which results in

biopolymers.
introduce

The

i.e.

system.

are

of nanoscale
are

about

The estimated

length

Fig.

5.1a

nm

(French

to

granular

5.1.

spaces

forms the

is

part

of

amylose
of

and

zones

zones

in the

portion

of the

Wheat

amylose

1993).

of

starch

amylopectin

(Fig. 5.1b).

in

rich in either

granular

Light
starch

bakery products

one

of these two

phase separation

of starch

with different interfaces in the structure of

built between swollen starch


between

are

starch

framework of the

confirmed the maintenance of the

clearly distinguishable

intergranular amylose,

granules

the

amorphous regions.

with

structure of starch and the

are

starch

et al.

gelatinization

between

crystalline

of the

partly complexed

phase separation

bread crumb. Interfaces

in

1997).

(B granules)

granules (Morrison

immiscibility

Fig.

fills the

protein

amylose

are

leads

microscopical investigations
structure and the

of

starch-protein system (Fig. 5.1a). Starch

bicontinuous

Part of

dough

et al.

than drawn in

amylopectin

of

of different

is in the order of 200-400

native state in

Baking

and

presentation.

consisting

molecules

depicted

as

long (Gallant

and small

already

schematic

amylopectin

amylopectin

fused.

granules

during baking

and starch molecules does not

granules

granules. Large (A granules)

native starch

potential

the extension of the molecules is almost 1000 times

as a

dough.

in

macroscopic properties

side chain clusters

in diameter and about 6

1984). Thus,

and the

of each element and the interactions between the

Amylose

for the macromolecules of

comprehensive

structures

material

composite

the size ratio of starch

Fig. 5.1,

far,

developed,

resulting

amylase supplementation

can

so

have been defined.

and the

different constituents determine the

In

presented

of bread has been

staling

of bread without

aging
The

the results and conclusions

on

amylose

and

granules

and

amylopectin

leached,

within

the

5.3 Model for the

of bread and the

staling

of

nature

side chains

amylopectin

rich starch

amylose

is

amylopectin

granule

center.

It

is

is

to be

and Larsson

Anisotropic

essential

structures

Besides free

are

the

another

amylose,

contribute

The

reorganization
the

to

formed since

in each

of

of

in

Cross-linking

reorganizes

as

well and is

structures in the starch

Probably,

present

in

Networks

amorphous

through crystalline

and

intergranular amylose
level.

From

amylopectin

the
form

by hydrogen

have

an

impact

on

amylopectin

bread.

centers

networks induces
work

zones

but do not

interwoven

and

by

beside

presents

of bread crumb.

level

regions

during aging

the

mainly

rigidity

starch

of the

granules.

contributes

crystalline regions
The observation

can

on

contribute to the

crystallites

are

it

can

zones.

granule rigidity.
of

amylose

by

the formation of intra- and

concluded

that

both

macroscopic
amylose

It is conceivable that the formation of

entanglements
strength

between

chains

sequentially

pass

firmness increase at the

be

amylose

anisotropic

cross-linked

molecules

to

the formation of

partial crystallization

Single amylose

the mechanical

rich

is

gelatinized

amorphous regions. Possibly,

bonds and

certain extent.

for firmness. The formation of

built where

areas.

crystalline

structures leads to the conclusion that

granule

present

links

and

the

rigidity.

important

are

to

present,

macroscopic

influences

lateral chain association favors

during aging.

fraction

during baking

the

at

into

(Eliasson

and formation of interconnected

zone

between

birfringent amylose

occurs

results in double helices of the outer branches and

network formation which increases


ordered

still

are

into the

cannot be detected.

birefringence

starch

amylopectin

amylopectin

as

some

intergranular amylose

already gelled

is located inside the

polymer

Reorganization
crystallites.

continuous

firming perceived

since the

the

important structuring component

(Fig. 5.1c). Rtrogradation


granules

yet

matrix. Protein denatures

protein

Molecular

of fresh bread is

not

well

as

element of fresh bread crumb

amylose, amylose-lipid complexes

change during staling.


continuous

structuring

1993). Amylose

that

During baking, amylose leaching

intergranular regions (grey colored). Mainly


an

crumb, the

conceivable

intergranular space

intra- and

thought

In fresh bread

protein.

melted.

into the

protrude

123

effect of amylases

and between starch and

gelatinized granules
crystalline

antistaling

amylose

and

and

cross

amylopectin

of the interfaces between the

amylose

which, in turn, influence the mechanical properties of

124

Fig.

5. A

5.1:

to

staling

Schematic
based

comprehensive approach

on

presentation of a model for baking and staling of bread


changes in the starch fraction.

Amorphous amylose

HM

Retrograded amylose

&SW--

Amylose-lipid complex

HB

Amylose

rich

regions

Crystalline amylopectin
(native and recrystallized)
Gelatinized amylopectin

Free

polar lipid

as

5.3 Model for the

staling

of bread and the

Starch in

Dough

tarch in
resh Bread

Starch in
Stale Bread

antistaling

effect of amylases

125

126

5. A

To

protein

at the molecular

speculative. On

remain

bread consists of

Specific bindings

staling

level,

the basis of the


of

reorganization

Martin and

proposed by

as

present

work it is

amylopectin

as

Hoseney (1991),
that

proposed

well

polymers

enhance

granule rigidity.

The intra- and

enhances the overall firmness and

during aging

amylases

bread models results from the


to the

crystallinity

systems

aggregates reorganize

less

consequence the different

leads to
This

expect

starch

rtrogradation.

network formation
The

increase in firmness

an

of bread and

properties

several constituents. With

presented staling model,

to

play

firming

evidence

degradation

role.

key

Increased

during aging

that

rapidly

of

amylose

behavior has shown to be

on

the nature of chain

amylose

can

be

chain

imperfect

the

rate

length

of

induced
and

as a

important.
it is

organization,

involves different

prevented,

or even

is

degradation

which

likely

firming

prevents

crystalline regions

are

it is conceivable that the reduced chain

crystallites.

during cooling
intergranular

to be

but

critical

step.

does

not

It is assumed that the small branched molecules

but that the

In contrast to

of bread. On

networks.

together

thermal

intragranular

both

in firmness decrease.

hydrolyzes

firming

The

reorganization

that the reduction of

The extent of starch

effects

or

reduced

during aging.

network formation. Network formation

of small

seems

provides

While it is difficult to draw conclusions


reasonable to

one

results and the above

experimental

of fresh

of

weakening

systems containing amylases.

resulting

for

the mechanical

on

of starch network formation

prevention

and the

the interfacial adhesion.

responsible

of

of bread.

The influence of the

regard

intergranular

strengthens

combination of the different processes is

staling

amylose

as

are

between starch

formation of starch networks. The increase of molecular order of the

aging

to

date, interactions between starch and protein polymers in bread crumb

less understood than starch-starch interactions.


and

comprehensive approach

induce

aging,

sticky

length

of

the

amylose

crystalline amylose

maltose is

and the

formed

are

does not form intra- and

the formed dextrins act

endo-mechanism of

Similarly,

leads to the formation

plasticizers.

as

and gummy crumb. In contrast,

particular

starch

crystallize during

and not continuous.

without deterioration of the crumb structure. As

stability

extensively

impede

amylopectin, amylose crystallites

Additionally,
a

dispersed

BAN

Novamyl

All

reduces

result of the lower

Novamyl,

formed, it is supposed that Novamyl degrades starch

to

where
a

mainly

lesser extent

5.3 Model for the

than

BAN.

of bread and the

staling

Most

the

probably,

antistaling

chains

outer

127

effect of amylases

of

molecules

amylopectin

removed, which prevents formation of double helices. Amylose,


hand, is less degraded than by BAN, but it is degraded
more

mobile than the uncleaved

ranged

ordered

imparts rigidity
in the

amylose
no

anisotropic

The

systems.

to the

are

and

granules

structures

extent that it is

and build

network structure

which

is

did not show

birefringence.

matter of concentration. In

it is assumed that

intergranular amylose

exist in the fresh state and that the structure is settled very

amylose networks, resulting

initial firmness

assumption

in

changes

reduced firmness

low

of the

of the

regardless

and flour

since

blockage

approach

of

during staling

the starch
and

amylose aggregates
within

the

be

can

reorganization
completely

of

starch

rearrangements

of the

reorganization only

would

intergranular

responsible

for

within the starch

component

should attack

an

networks in the
not

the

be

promising

a-amylase

with

only amylose

which is

This would

lead to

intergranular space only.

affected.

granules

to the

to test whether

would be

For this purpose,

Novamyl

within

This leads to the

bread, it might be interesting

imperfect

the

transferable to bread

granules during gelatinization.

granules

reorganization

only.

increasing

missing.

firming.

mechanism close to

released from

of

starch

intergranular

for the reduction of

degradation

Starch

matrix is

protein

without

starch

starch

not

are

gels

it is still difficult to estimate the contribution of each

increase of firmness
a

gels

intragranular

during aging.

pregelatinized

of the molecular

results of starch

Although

rate

intergranular

granules. However,
gels

firming

reduced the firmness of starch

by degradation

that

aggregated,

This indicates that

well. This hinders the increase of molecular order of inter- and

Soy--amylase

long

thus, the initial firmness is enhanced. Leached

intragranular amylose

aggregates already

birfringent

built, which could be

were

an

the other

This results in the formation of

intragranular amylose,

intergranular space

to the

analogy

which

aggregates

in fresh

already

amylose.

to such

on

are

is

As
not

starch network formation

consequence,

altered
are

whereas

complicated.

As

result, the overall firmness is reduced by weakening the cohesion between the

granules although granule rigidity

increased.

128

5. A

staling

Outlook

5.4

In the

the

present investigation,

properties

based

mainly

are

on

the

and networks formed due to starch


would therefore be to

challenge
obtain

information

more

freeze-fracture

techniques present
the mechanical

like bread.

Confocal

laser

information

starch

granule

in the

center

as

X-ray

measurements

as

investigations

to the

investigations

be

protein

used

would

on

manipulation

interfaces and
materials

composite

in situ.

performed

ideally

to

obtain

more

networks.

crystallinity

of starch. It

the contribution of each micro-domain of the


starch

crystallinity. Especially

birfringent

are

investigated by

where the relative

microscopy

would have to be

could

of starch and

amylopectin

Difficulties

investigations.

role. The main

could be identified. Micro-mechanical

quantify

control bread crumb where


well

interactions could be

measurements revealed the overall


to

aggregates

structures in order to

investigate supramolecular

protein-starch

continuity

interesting

gelatinized

important

an

of the different constituents of

strength

angle X-ray

would be

degradation play

scanning microscopy

the

on

that the nature of the

hypothesis

further suitable methods for

the

in the mechanical

changes

the network structure and interfaces. The nature and

zones

Preferably,

of

explanation

combined with electron

techniques

weakness of interfacial

Wide

on

of starch-starch and

strength

on

to

comprehensive approach

zones were

rich outer

expected

detected in the

regions

probably present

granules

are

in the extraction of

Synchrotron

method

single

aged

amylose-rich

of interest.

suitable

of

for

starch

these

granules

from the crumb matrix.

Quantification of the phase transition of amylose aggregates in situ would be


of

high

interest. So

attempted

to

far,

measure

no

conclusive results

in

the

120

C,

(1993)

presence of

DSC method

lysophosphatidylcholine (LPC)

quantified by following

during

heated to

the

cooling phase.

temperatures

In consequence,

by DSC

where it

the
In

melting

second

around 170 C

LPC-amylose complex

quantifiable during reheating.

procedure

high

proposed by Sievert

and

so

step

that

systems

to

the

of the
the

non

could be heated

temperature

The combination

around

aggregated amylose

amylose-LPC complexes
system

with LPC would be

amylose aggregates

formation is

was

to

information could be obtained. The

cooled and reheated. Due to this

could be
formed

more

obtained

directly amylose aggregates by heating samples

temperatures (200 C). By adapting


Wrsch

were

are

melted.

possible during cooling

of the two

and

heating steps

to

129

5 4 Outlook

different

would

temperatures

allow

to

quantitatively

estimate

amylose

aggregates

High performance liquid chromatography


information

more

the

on

different

degradation products by
starch

degradation by amylases

It would

be

interesting

granules

and

degrade

that

they

act

the

on

suitable

test the

hypothesis

space would reduce


cannot

penetrate

starch

depending

is

inner

surface

firming,

to the starch it has

of the

it would be

detailed

storage

knowledge
is

required

to

is

the

complement

only

staling

focused

is a minor

proposed

that redistribution of water

Based

The

on

on

nutritional

would

properties
It

of

the small intestine

intergranular
that

in

on

the

wheat

changes

compared

of bread

properties

protein during processing

is

of

and

staling

described whereas

available and many

the

protein

resonance

on

the

speculations
and starch

studies it

amorphous phase

is

was

and exerts

1991)

Energy dispersive X-ray

probably present

another suitable method for

is

nutritionally important

Addition

be

influenced

characterized
At

present,

of

little

SA, Denmark,

amylases

the

by

Novamyl

structural

leads to the

by resisting amylolytic

starch fractions

Novo Nordisk

flavor of bread

can

that the addition of

was seen

(Euresta 1992)

analysis performed by
impacts

al

et

food

formation of resistant starch which

structure of

In order to

water micro-redistribution

characteristics of starch

in

are

occurs in

(Kim-Shin

effect

microanalysis (Davies 1991)


investigations

likely

huge enzyme

primarily

component

170 nuclear magnetic

for

antiplasticizing

present understanding

few data

important

an

is more

into the

to create

interesting

assumed that redistribution of water between

staling

if it

or

starch

of the enzymes would

amylose leaching

the transformations of

micro-distribution of water
It

of the enzyme

the microstructure

in

Diffusion of water between crumb and crust

exist

granules

extent of

penetrate

can

major contribution to the structural

on

factors, the

formed

granules

Although protein

of the

the

on

acting place

Immunolabelling

So far, the characterization of bread


of the starch fraction

part

to localize enzymes

degradation

into the

the

on

other

order to obtain

in

and

degradation

amylases Among

the

in

granule

that

starch

performed

know whether the enzymes

to

technique

present

of

extent

could be

is

known

Besides,
it

which

in

induce

regarding
some

was seen

attack

that

the

sensory

Novamyl

specific

starch

130

5. A

structures could

to further understand the

help

comprehensive approach

relationship

to

staling

between sensory and

starch structure.

In the

present investigation, only

the

tendency

in

particular

of

consequences

rtrogradation

the
on

presence
the

physiological aspect

efficacy

of

is
of

of

low extraction wheat flour

high. Specific properties


pentosans

amylases

and

and
on

was

of other

hemicellulose,

staling

nutrition, high extraction flour

or

of bread.

used because

types

of

might

flour,
have

Regarding

the

wholemeal is favored.

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Curriculum Vitae

Susanna
born

Hug-lten

July 30,

citizen of

1970

Untergeri ZG

Switzerland

1996-2000

Ph.D.

student and

Research Assistant of

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the

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Swiss

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Federal

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Food

Institute

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Science,

Technology

Zrich

Diploma

in

Degree

Science

Food

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Technology (Dipl. Lm.-lng. ETH)


1990-1996

Studies in Food Science and


the Swiss Federal

(ETH),

Technology

Institute of

at

Technology

Zrich

9 Months

practical experience

at

Mineralquelle Eglisau AG, Eglisau


Landw. Institut des Kantons
Milchwirtschaftliches

Freiburg,

Bildungszentrum,

Grangeneuve/Posieux
Selectchemie AG, Zrich
Carma-Pfister AG, Dbendorf
1992-1995

Technical

Assistant

at

Selectchemie

AG,

Zrich

1989-1990

Kantonsschule

completed
examination

1985-1989

with

type

Kantonsschule

completed

Enge,

Swiss

Federal

Maturity

Enge,

with

Zrich

Zrich

Federal

Diploma

Commerce
1977-1985

Primary

and

Secondary School, Horgen

for

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