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This is to certify that the

dissertation entitled

DEVELOPMENT AND CHARACTERIZATION OF EDIBLE
AND/OR DEGRAOABLE FILMS FROM WHEAT PROTEINS

presented by

Luis Martin Rayas

has been accepted towards fulﬁllment
of the requirements for

PhoDo degreein FOOd SCTence

Department of Food Science
and Human Nutrition

 

Dr. James F. Steffe, P.E.

Major professor
jg“, f 054242;.

[hue December 27, 1995

MSU i: an Afﬁrmative Action/Equal Opportunity Institution 0- 12771

 

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University

 

 

 

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TO AVOID FINES return on or baton date duo.

DATE DUE DA'KﬁaéJaE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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MSU It An Affirmative Action/Equal Opportunity Institution
Walla-M

 

DEVELOPMENT AND CHARACTERIZATION OF EDIBLE
AND/OR DEGRADABLE FILMS FROM WHEAT PROTEINS

BY

Luis Martin Rayas

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1995

ABSTRACT

DEVELOPMENT AND TESTING OF EDIBLE AND/OR
DEGRADABLE FILMS FROM WHEAT PROTEINS

By
Luis Martin Rayas

 

A new procedure for making degradable ﬁlms was developed which
resulted in the issue of US. patent No. 5,472,511 (issued December 5, 1995).
This procedure consisted of bringing the proteins into solution using wheat ﬂour
as raw material. A centrifugation step yielded a supernatant, containing the
solubilized wheat proteins, called the ﬁlm-fanning solution (FFS). Several ﬁlms
were produced by casting the FPS on a ﬂat inert surface. Depending on the
FFS characteristics (e.g., pH and cross-linker), ﬁlms with different properties
were fabricated.

Analyses of the ﬁlms were performed with different techniques
involving mass transfer, rheological, chemical, and physical studies. Mass
transfer was studied by oxygen permeability, showing low values, similar to
those of nylons, and inﬂuenced mainly by temperature and type of ﬂour used to
make the ﬁlms. Rheological methods included tensile stress and tensile creep:
Tensile stress demonstrated the effect of a cross-linker on the mechanical
properties and tensile creep showed the importance of the environmental
conditions on long-term behavior. Chemical studies involved the analysis of the

ﬁlms by Fourier-Transformed Infrared Spectrometry (FTIR) and swelling

behavior. FTlR showed important chemical groups or bonds present in the films.
Swelling behavior was used to determine a solubility parameter for the protein
ﬁlms. Physical studies included the color analysis by the Commission
lntemationale de I’Eclairage Laboratory (ClE-Lab) technique. An increase in the
yellow and red values upon cross-linking with glutaraldehyde was found while a
reduction in the yellowness was observed when the ﬁlms were cross-linked with

cysteine or formaldehyde.

Copyright by
Luis Martin Rayas
1995

DEDICATION

To my wife, Ana Teresa, with all my love

To my sons and daughter, Luis Alberto, Anna Teresa,
and Diego Alejandro

To my parents, Luis and lrrna, for their inﬁnite love and support

ACKNOWLEDGMENTS

The author wants to deeply thank Dr. James F. Steffe for the patience,
guidance, friendship, and support acquired during the time in which the author
was fortunate to have him as his major professor. Sincere appreciation to Dr.
Bruce R. Harte, Dr. Ruben J. Hernandez, and Dr. Gale M. Strasburg, members
of the committee, is acknowledged for their instruction, time, and help received
throughout this research. Special thank to Mike Rich, mainly from whom the
author learned to operate the MDSC, TMA, and FTIR equipment and to Chris
Daubert and Danny Campos, for teaching the author to use the RheoStress RS-
100. Also, very appreciated is the aid provided by Dr. Jack Giacin for the
interpretation of the FT IR data and Dr. Guoquan Hou for the interpretation of the
SDS-PAGE bands.

Special mention is made for the support and affection that the author
gained from fellow friends Dr. Nirmal Sinha, Dr. Christine Bergman, Chris
Daubert, Danny Campos, Jenni Briggs, Emily Schluentz, Dr. Guoquan Hou,
Scott Worthington, Arti Arora and Afclabi Abinusawa.

The author also wishes to thank the King Milling Company for the
supply of the hard red winter and soft white ﬂours. Assistance and the use of
facilities and equipment during this study from the Food Engineering and
Rheology, the Cereal Science, and the Baking and Rheology Laboratories,
Department of Food Science and Human Nutrition; the Permeation & Material
Testing and Food and Pharmaceutical Laboratories, School of Packaging; and
the Composite Materials and Structures Center Laboratory, Engineering

Research Complex, at Michigan State University, is greatly appreciated.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................. x
LIST OF FIGURES ............................................................................................. xii
NOMENCLATURE ............................................................................................. xv
1. INTRODUCTION .............................................................................................. 1
2. LITERATURE REVIEW .................................................................................... 4
2.1 CHARACTERIZATION OF FLOUR ............................................................ 4
2.1.1 Moisture Content ................................................................................. 4
2.1.2 Protein Content ................................................................................... 5
2.1.3 Falling Number .................................................................................... 6
2.1.4 Farinography ....................................................................................... 7
2.2 CHARACTERISTICS OF WHEAT PROTEINS ........................................... 8
2.3 SEPARATION OF GLUTEN FROM FLOUR. ........................................... 13
2.3.1 Industry vs. Laboratory ...................................................................... 13
2.3.2 Wheat Proteins for Film Formation ................................................... 14
2.4 GENERAL PROCEDURES FOR MAKING EDIBLE/BIODEGRADABLE

FILMS ........................................................................................................ 15

2.5 CURRENT METHODOLOGY FOR PREPARATION OF GLUTEN-BASED
FILMS ........................................................................................................ 27
2.5.1 Overview ........................................................................................... 27
2.5.2 Cross-linking of Proteins ................................................................... 29
2.6 MASS TRANSFER IN POLYMERIC FILMS FILMS .................................. 34
2.6.1 Permeability of Gases ....................................................................... 36
2.6.2 Permeability of Water Vapor ............................................................. 40
2.7 RHEOLOGICAL METHODS ..................................................................... 42
2.7.1 Tensile Strength ................................................................................ 43
2.7.2 Tensile Creep .................................................................................... 44
2.7.3 Thermal Mechanical Analysis ........................................................... 50
2.7.4 Differential Scanning Calorimetry ..................................................... 51
2.8 CHEMICAL AND PHYSICAL CHARACTERIZATION OF POLYMERS 57
2.8.1 Swelling of Polymer Networks ........................................................... 59

vii

2.8.2 Fourier-Transform Infrared Spectroscopy ......................................... 61

2.8.3 Plastic/Film Color Measurements ...................................................... 66

3. MATERIALS AND METHODS ........................................................................ 69
3.1 FLOUR SAMPLES .................................................................................... 69
3.2 FLOUR CHARACTERIZATION ................................................................ 70
3.2.1 Protein Content ................................................................................. 70
3.2.2 Flour Moisture ................................................................................... 71
3.2.3 Falling Number .................................................................................. 72
3.2.4 Farinography ..................................................................................... 72
3.2.5 SDS-PAGE ........................................................................................ 73
3.3 FILM PREPARATION ............................................................................... 74
3.3.1 Protein Solubilization ........................................................................ 74
3.3.2 Protein Cross-linking ......................................................................... 76
3.3.3 Film Formation .................................................................................. 77
3.4 ANALYTICAL TESTS OF FILMS ........................... 79
3.4.1 Barrier Properties .............................................................................. 79
3.4.1.1 Oxygen Permeability .................................................................. 79
3.4.2 Rheological Methods ......................................................................... 81
3.4.2.1 Mechanical Properties ............................................................... 81
3.4.2.1.1 Tensile Strength ................................................................. 81
3.4.2.1.2 Tensile Creep ..................................................................... 82

3.4.2.2 Film Characterization ................................................................. 86
3.4.2.2.1 Thermal Mechanical Analysis ............................................. 86
3.4.2.2.2 Modulated Differential Scanning Calorimetry ..................... 86

3.4.3 Swelling Study ................................................................................... 89
3.4.4 Fourier-Transform Infrared Spectroscopy ......................................... 91
3.4.5 Color Study ....................................................................................... 92
3.5 STATISTICAL ANALYSIS ........................................................................ 92
4. RESULTS AND DISCUSSION ....................................................................... 93
4.1 FLOUR CHARACTERIZATION ................................................................ 93
4.1.1 Moisture Content ............................................................................... 93
4.1.2 Protein Content ................................................................................. 93
4.1.3 Falling Number .................................................................................. 96
4.1.4 Farinography ..................................................................................... 98
4.1.5 SDS-PAGE ...................................................................................... 102
4.2 FILM FORMATION PROCESS ............................................................... 105
. 4.3 BARRIER PROPERTIES ........................................................................ 105

viii

4.3.1 Oxygen Permeability ....................................................................... 105

4.4 RHEOLOGICAL MEASUREMENTS OF FILMS ..................................... 112

4.4.1 Mechanical Properties ..................................................................... 112

4.4.1.1 Tensile Strength ....................................................................... 112

4.4.1.2 Tensile Creep .......................................................................... 117

4.4.2 Therrnomechanical Properties ........................................................ 138

4.4.2.1 Thermal Mechanical Analysis .................................................. 138

4.4.2.2 Modulated Differential Scanning Calorimetry .......................... 138

4.5 CHEMICAL CHARACTERIZATION ........................................................ 140

4.5.1 Fourier-Transform Infrared Spectroscopy ....................................... 140

4.5.2 Swelling Study ................................................................................. 144

4.5.3 Color Study ..................................................................................... 153

5. SUMMARY AND CONCLUSIONS ................................................................ 159

6. SUGGESTIONS FOR FUTURE RESEARCH ............................................... 162
APPENDIX

APPENDIX A ............................................................................................... 164

APPENDIX B ............................................... 165

APPENDIX c ................ ' .................. - .................................... V .......................... 1 as

APPENDIX D ................................................................................................ 167

APPENDIX E ................................................................................................ 168

APPENDIX F .............................. ’ .................................................................. 169

7. BIBLIOGRAPHY ........................................................................................... 177

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.
Table 9.

Table 10.
Table 11.
Table 12.

Table 13.
Table 14.

Table 15.

LIST OF TABLES

Solubility fractionation of the endosperm proteins of hard
red spring wheat .................................................................... 10

Typical glass transition temperatures (T,) values of some
synthetic polymers ................................................................. 55

Some typical group stretching vibrational absorption
frequencies for polymer analysis ........................................... 65

Films produced and tested for oxygen permeability and
ultimate tensile properties in this study ................................. 80

Film variable and conditions used to perform creep tests in
this study ................................................................................ 87

Films produced and examined by thermal mechanical
analysis (TMA) and modulated differential scanning
calorimetry (MDSC) techniques .............................. 88

Films produced for determination of swelling coefﬁcient (Q),
analysis by Fourier-Transformed Infrared Spectroscopy

(FTIR), and measurements of color by ClE-Lab system ........ 90
Moisture content of the ﬂours used in this study ...... 94
Protein content of the ﬂours used in this study ...................... 95
Falling Number of the flours used in this study ..................... 97
Farinograph results for the ﬂours used in this study ............. 101
High-molecular-weight glutenin subunits (HMW-GS) present

in the ﬂours used in this study ............................................... 104
Oxygen permeability values for selected ﬁlms at different
temperatures ............... _ .......................................................... 1 06
Arrhenius constant (P°) and activation energy (expressed as

E. R") for selected ﬁlms ........................................................ 110
Ultimate tensile properties for selected ﬁlms ......................... 113

Table 16.

Table 17.
Table 18.

Table 19.
Table A1 .

Table F1 .

Four-element Burgers Model constants for wheat protein

ﬁlms ....................................................................................... 127
Peleg Model constants for wheat protein films ...................... 137
Swelling coefﬁcients of selected protein ﬁlms in several

solvents ................................................................. _ ................ 145
Color analysis of selected wheat protein ﬁlms ...................... 155
Correction of sample weight to 14% moisture basis (AACC
56-81A) .................................................................................. 164
Raw data for calculation of creep curves .............................. 169

xi

Figure 1.

Figure 2.

Figure 3.

Figure 4.
Figure 5.
Figure 6.

Figure 7.
Figure 8.

Figure 9.

Figure 10.
Figure 11.

Figure 12.
Figure 13.

Figure 14.
Figure 15.
Figure 16.
Figure 17.

LIST OF FIGURES

Scheme of reaction between the e-amino group of lysine

residues of two protein chains and glutaraldehyde ............... 33
Experimental setup for oxygen permeability studies on

ﬁlms ....................................................................................... 37
Example of dumbbell-shaped polymer sample specimen to
evaluate tensile properties .................................................... 45
Schematic representation of a creep curve ........................... 47
Burgers Model diagram of creep behavior of a polymer ........ 48

Schematic of a typical differential scanning calorimetry
(DSC) heating curve for a polymeric ﬁlm ............................... 52

Schematic of ﬁlm-making process developed in this study 75

Transfer of ﬁlm-forming solution (FFS) to TLC spreader with

thickness regulator ................................................................ 78
Diagramof tensile creep machine made for this study .......... 84
Type M II tension test specimen dimensions (mm) ................ 85
Farinograms of the ﬂours used to make the ﬁlms in this

study ...................................................................................... 99
One-dimensional sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS—PAGE) of C, H, and S ﬂours ............... 103
Oxygen permeability of selected ﬁlms as a function of
temperature ........................................................................... 108
Arrhenius plot of selected ﬁlms ...... 109
Engineering strain (elongation) at break of selected ﬁlms 114
Tensile strength of selected ﬁlms .......................................... 115
Creep compliance of ﬁlms at 5°C and 85% RH ..................... 118

xii

Figure 18.
Figure 19.
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.

Figure 33.

Figure 34.
Figure 35.

Figure 36.

Figure 37.

Creep compliance of ﬁlms at 10°C and 40% RH ................... 119

Creep compliance of ﬁlms at 20°C and 35% RH ................... 120
Creep compliance of ﬁlms at 20°C and 55% RH ................... 121
Creep compliance of ﬁlms at 25°C and 25% RH ................... 122
Creep compliance of ﬁlms at 25°C and 30% RH ................... 123
Creep compliance of ﬁlms at 40°C and 25% RH ................... 124
Linearized creep curves of ﬁlms at 5°C and 85% RH ............ 128
Linearized creep curves of ﬁlms at 10°C and 40% RH .......... 129
Linearized creep curves of ﬁlms at 20°C and 35% RH ........... 130
Linearized creep curves of ﬁlms at 20°C and 55% RH .......... 131
Linearized creep curves of ﬁlms at 25°C and 25% RH .......... 132
Linearized creep curves of ﬁlms at 25°C and 30% RH .......... 133
Linearized creep curves of ﬁlms at 40°C and 25% RH .......... 134
FT IR spectra for selected wheat protein ﬁlms ....................... 141
Typical transmission spectra in the infrared region for some

commercial polymers ............................................................. 142
Swellingcoefﬁcient (Q) of different protein ﬁlms, calculated

using dry weight of ﬁlm before immersion ............................. 146
Weight loss of the ﬁlms during time of solvent immersion ..... 148

Swelling coefﬁcient (Q) of different protein ﬁlms, calculated
using dry weight of ﬁlm after immersion ................................ 150

Swelling coefficient, Q, as a function of solubility parameter,
6, of various solvents ............................................................. 152

Three-dimensional color evaluation using the CIE-Lab
system ................................................................................... 154

xiii

Figure 38. Transmittance and reﬂectance signiﬁcance for color
measurement ......................................................................... 1 56

Figure B1. Cross-sectional area of films as a function of grip
displacement for correction of tensile creep data .................. 165

Figure C1. TMA curve for glutaraldehyde-cross-linked protein ﬁlm ........ 166

Figure D1. MDSC curve for a glutaraldehyde-cross-Iinked wheat _
protein ﬁlm. Shows total heat ﬂow, along with reversible
and non-reversible heat ﬂow ................................................. 167

Figure D2. MDSC curve for a gIutaraldehyde-cross-linked wheat
protein ﬁlm. Shows total heat ﬂow with a thermal change
around 0°C attributed to phase transition change of water 157

Figure E1. Thermal change of wheat proteins observed in the FFS ....... 168

xiv

NOMENCLATURE

ﬁlm area, m2

green-to-red [CIE-Lab system], - to +
blue-to-yellow [CIE-Lab system], - to +
velocity of light in vacuum, 2.9979 x 10" m s'1
diffusion constant, m2 5'1

energy of photon, ergs or J

, molecular quantized energy. ergs or J

activation energy, Kcal mol'1
constant in Burgers model, Pa
constant in Burgers model, Pa
complex modulus, Pa

Planck’s constant, 6.6262 x 10'“ J s
creep compliance (s/o), Pa'1

oxygen transmission rate (STP = standard temperature and pressure
= 273.15 K; 1.013 x 105 Pa), m° (STP) m'2 s'1

asymptotic creep compliance, Pa'1
instantaneous compliance, Pa'1

retarded compliance, Pa'1

constant in Peleg model, Pa 5

constant in Peleg model, Pa

lightness, %

length, m

initial length or length of undeformed sample, m
ﬁlm thickness (1 mil = 0.001 in), mil or m
desired moisture basis (wet basis), %

moisture loss, 9

weight of the swollen sample, g

weight of the dry sample, 9

rotor speed, rpm

moles of polymer chains per unit volume, mole m“3

P2

00

AG"
AHM
AP

AS"

8A
8h

permeability (STP = standard temperature and pressure = 273.15 K;
1.013 x 105 Pa), m3 (STP) m m'2 s" Pa“

Arrhenius constant, m3 m rn‘2 3'1 Pa'1

partial water vapor pressure inside face (product) of ﬁlm, mmHg or
Pa

partial water vapor pressure outside face (environment) of ﬁlm,
mmHg or Pa

amount of penetrant passing through a ﬁlm, m3
swelling coefficient, ml 9‘1

radius from centerline of rotor to the point in the tube where relative
centrifugal force (RCF) is required, cm

gas constant per mole, 1.987 cal mol'1 K’1

solubility coefﬁcient (SSTP = standard temperature and pressure =
273.15 K; 1.013 x 10 Pa), cm° (STP) cm° Pa'1

original weight of sample, 9

time, s

time for weight change, day

absolute temperature, K

glass transition temperature, °C or K

melting temperature, °C or K

water vapor transmission rate, 9 m'2 day'1
weight of water absorbed in the cup (at t=tw), 9
weight fraction of component 1, dimensionless
weight fraction of component 2, dimensionless
extension ratio, dimensionless

solubility parameter, MPa"

change in Gibb’s free energy, J

enthalpy of mixing, J

partial gas or vapor pressure differential across the ﬁlm, Pa
entropy of mixing, J K ‘

strain/elongation (or-1), dimensionless
asymptotic tensile strain, dimensionless
Hencky strain, dimensionless

shear strain, dimensionless

wavelength, cm

retardation time, s

constant in Burgers model, Pa 5
constant in Burgers model, Pa s
wavelength, um

density of swelling agent, 9 ml"
shear stress, Pa

retractive force per unit area (f Au ), where A..' 13 original cross-
sectional area, kg cm“2

constant stress, Pa
wavenumber, cm"
frequency (cycles s"), Hz
angular velocity, rad s"

xvii

1. INTRODUCTION

Most industries package their products to protect and contain them,
thus preventing contamination, physical damage, chemical changes, etc. This
practice is done so commodities reaching the consumer will have the expected
quality and shelf life (especially in foodstuffs). As a result, capital loses due to
the product-environment interaction is minimized. In this study, discussions will
be focused on the possibilities of ﬁlm usage in food markets, although other
applications can be identiﬁed for these ﬁlms.

In the last ten years, several new types of ﬁlms have been developed
that are degradable and/or edible. Knowledge of these materials, in terms of
mechanical and barrier properties, among others, is increasingly being
demanded by the packaging industry, government and the general public.

Several potential advantages for using degradable/edible ﬁlms include:
an improvement in the mechanical-handling properties or structural integrity of
the packaged food; if disposed, quick degradation in the landﬁlls; if consumed
along with the food it may increase the food value (especially in protein-based
ﬁlms) of the product; different nutrients and additives can be incorporated thus
enhancing organoleptic properties and preventing deterioration of the packaged
foods; control of gases and moisture migration from and into the product can be
achieved; and, if used as coatings (ﬁlms that are formed directly on the surface

of the foods) these ﬁlms can be used to package small foods or to prevent

2
deterioration of larger food items (Donhowe and Fennema, 1994 and Gennadios

and Weller, 1990).

Although degradable/edible ﬁlms can be successfully formed using
carbohydrates, lipids, and proteins, today there is limited information available
on degradable packaging materials produced from these goods. Development
of ﬁlms utilizing wheat proteins present a good opportunity because in the
United States, approximately 57 million metric tons of wheat are produced per
annum. At the farm gate, this ﬁgure contributes more than $ 7 billion to the US
Gross Domestic Product. Given that edible/degradable ﬁlms, if commercialized,
could replaceisome synthetic packaging materials, their future seems assured.
It is estimated that growth of 4% in the global consumption of plastic resins from
about 101 million metric tons in 1993 to about 123 million metric tons in 1998 will
be observed, with the US consumption estimated to rise from about 30.5 to
about 36 million metric tons in these same period (Modern Plastics
Encyclopedia, 1995). From these ﬁgures, packaging is the single largest market
for plastics, and most processed foods are packaged. Therefore, the possibility
of ﬁnding a market for edible/degradable ﬁlms seems promising.

Growth in packaging will be stimulated by rebounding markets as the
global economy emerges from the economic slowdown of the early 1990’s.
While the stronger global economy over the next few years will foster improved
end-use markets for plastics, growth will also stem from the quality, versatility,
durability, and light weight of plastics, as well as technological and resin

improvements (Modern Plastics Encyclopedia, 1995).

3
Wheat proteins present unique characteristics for use as

edible/degradable ﬁlms. First, they account for 8-15% of the dry weight of wheat
kernels and about 70% of the total protein is contained in the wheat endosperm,
the major source of wheat flour (Gennadios et al., 1994). Second, the presence
of high molecular weight groups of proteins is highly advantageous in order to
form a ﬁlm with desirable rheological properties. Third, the barrier
characteristics of ﬁlms obtained from proteins in general has been determined to
be low for gases (Donhowe and Fennema, 1994); therefore, good control of
permeability to gases such as oxygen and carbon dioxide can be achieved using
these ﬁlms.

In this research, the main objective was to develop a new process to
make ﬁlms from wheat ﬂour proteins. This resulted in the issue of US. patent
No. 5,472,511 (issued December 5, 1995). The method consisted of separating
proteins from wheat ﬂour by solubilizing them, followed by separating the
insoluble material, and casting the protein solution on an inert ﬂat surface.
Other objectives of the proposed study were to gather rheological, oxygen
barrier, chemical, and physical information on ﬁlms made using the above
procedure and to compare some of these values with current synthetic ﬁlms
used in several food applications. Results should establish the potential for

commercialization and utilization of these edible! degradable ﬁlms.

2. LITERATURE REVIEW

2.1 CHARACTERIZATION OF FLQQR

Different types of flours can be characterized on a compositional basis
according to their moisture and protein content, and on a quality basis by
checking the amylase content and the protein behavior under mixing. All of
these characteristics are important to help identify a ﬂour and recommend
appropriate end uses for the ﬂour. It" is important to mention that even variation
in factors (eg. proteins) within cultivars for a single variety could affect the end-

use property of the ﬂours (McLendon et al., 1993).

2.1.1 MOISTURE CONTENT

According to Kent (1983), the optimum ﬂour moisture level is between
12 and 13 percent. At a moisture below 12%, there is a risk of increasing fat
oxidation and rancidity, while at moisture above 13% mold growth may develop.

When ﬂour is to be stored for long periods of time, it is generally
recommended storage be in a closed atmosphere for the following reasons
(Kent, 1983): a) ﬂour acidity increases due to the lipolysls of linoleic and
Iinolenic acids (which are slowly oxidized); b) slow reduction of disulﬁde groups
(S-S) and therefore little increase in sulfhyde groups (-SH); c) decrease in the
solubility of gluten protein; and d) changes in processing strength, although this

is minor.

2.1.2 PROTEIN CONTENT

The protein present in the wheat flour plays a very important role in the
formation of a strong, cohesive dough capable of retaining gas. This unique
characteristic is attributed to protein, and particularly to the gluten protein
fraction (Hoseney, 1994).

Albumins and globulins in ﬂour are commonly referred to as the soluble
proteins (Kent, 1983). Albumin proteins of wheat, according to Kent (1983),
have molecular weights of 17,000 to 28,000. They are responsible in part for the
differences in baking characteristics among ﬂours. This author also indicated
that globulins are essential for proper baking performance. Both, albumin and
globulins, have metabolic and structural functions.

The insoluble proteins (gliadin and glutenin) are regarded as the
storage proteins of wheat and are contained primarily in the endosperm. The
gluten fraction is composed of two main groups of proteins: a) gliadin, soluble in
70% ethanol, and b) glutenin, soluble in dilute acetic acid. According to
Hoseney, (1994), the gliadins are a heterogeneous group of proteins which have
an average molecular weight of about 40,000 and are single-chained. Kent
(1983) mentioned reported values of 42,000 to 47,000 for the molecular weight
of the gliadins. These-proteins are extremely sticky when hydrated with little or
no resistance to extension. Gliadins are thought to be responsible for dough
cohesiveness.

The molecular weight of the peptide chains of the glutenin fraction is

on the order of 20,000 (Kent, 1983). Hoseney (1994) indicated that the range of

6
the molecular weights of the glutenin subunits is from 16,000 to about 133,000.

They are a heterogeneous group of proteins bonded together by disulfide bonds
into macrounits with molecular weight from about 100,000 to several million
(Hoseney, 1994 and Kent, 1983). Their average complex molecular weight is
about 3 million. A physical characteristic of this group of proteins is that they are
resilient but not cohesive, therefore these proteins are known to give dough its
property of resistance to extension (Hoseney, 1994).

The determination of ﬂour protein is important because it is recognized
(Baik et al., 1994; Hay, 1993; and Roels et al., 1993) that two factors, protein
quantity and protein quality, may produce signiﬁcant differences in the end-
product quality. The quantity of protein in a ﬂour can be resolved with great
accuracy by several techniques, however, the quality cannot be determined so

easily (Hoseney, 1994).

2.1 .3 FALLING NUMBER

The Falling Number method is an internationally standardized method
for the determination of a—amylase in grain, ﬂour and other starch containing
products, particularly wheat and rye. It determines a-amylase activity using the
starch in the sample as the substrate. The method is based upon the rapid
gelatinization of a suspension of ﬂour or meal in a boiling water bath and the
subsequent measurement of the liquefaction of the starch slurry by a-amylase.
Falling Number values bear a complex inverse relationship with the quantity of
a—amylase in the sample. This relationship is known as the Perten Liquefaction

equaﬁon.

7
The activity of a-amylase indicates the extent to which the viscosity of

a ﬂour/water paste is reduced by amylolytic hydrolysis of the starch. A
ﬂour/water suspension in a tube immersed in a boiling water bath is stirred for
exactly 60 sec. A plunger is then allowed to fall freely through the suspension
under standardized conditions (Kent, 1983). The Falling Number is deﬁned as
the total time in seconds from the immersion of the viscometer tube into the
water bath until the viscometer stirrer has fallen the prescribed distance through

the gelatinized suspension. Thus, the stirring time is included (Perten, 1988).

2.1.4 FARINOGRAPHY

Farinography is used by the cereal industry as a relative measure of
the dough strength, that is, a farinograph records the power that is needed to
mix dough at a constant speed (Pomeranz, 1987). A farinograph chart is
composed of two parts: one in which an amount of water is added (start of
mixing) and the point of maximum resistance (or minimum mobility), is identiﬁed;
the second part consists of a moderate decrease in consistency and resistance
to mixing. These parts were identiﬁed by Bloksma (1984) as the stages in which
the farinograph mixes ﬂour and water into a dough, develops it, and ﬁnally
overrnixes it.

The general practice has been to determine the standard absorption by
“titration curve” with water added from a buret to the ﬂour as it is mixed. This
technique has been most useful in determining water absorption, although
information on mixing time, mixing tolerance index and dough stability, among

other values, can be also obtained from the same chart. The standard water

8
absorption is one of the most commonly used and widely accepted farinograph

measurements. It is defined as “the amount of water required to center the
farinograph curve on the 500-BU [Brabender Units of torque] line for a ﬂour-
water dough” (Shuey, 1984a).

The dough development time in a farinogram is deﬁned as “the time, to
the nearest half minute, between the ﬁrst addition of the water and the
' development of the dough’s maximum consistency, or minimum mobility, i.e., the
point immediately before the ﬁrst indication of weakening” (Shuey, 1984b).
Stability is deﬁned as “the difference in time, to the nearest half minute, between
the point at which the top of the curve ﬁrst intercepts the 500-BU line (arrival
time) and the point at which the top of the curve leaves the 500-BU line
(departure time). The mixing tolerance index (MTI) is deﬁned as “the difference,
in Brabender units, between the top of the curve at the peak and the top of the

curve measured 5 min after the peak is reached” (Shuey, 1984b).

2. 2 QHARACTERISTIC§ OF WHEA T PR0 TEIN§ .

Until the eighteenth century, there was very little knowledge regarding
the role of the proteins in the dough formation. It was ﬁrst observed that the
properties of the dough and wet gluten did not vary signiﬁcantly, therefore, the
water absorbing power was attributed mainly to the proteins of the system
(Meredith, 1969). Various authors have reported the importance of the protein in
the development of good pasta, bread, cake, cookies, and other ﬂour-related
products depending upon the percent protein present in the ﬂour as well as the

quality of it.

9
Wheat flour proteins are very unique and therefore could be studied

apart from other cereal proteins. Krull and Wall (1969), reported the
composition of wheat proteins based on solubility and identified the following
types: albumin (water soluble), globulin (salt solution soluble), gliadin (70%
ethanol soluble), and glutenin (dilute acid soluble) proteins. Similarly, Chen and
Bushuk (1970) reported the components of hard red spring wheat using the
classical protein fractionation procedure of Osborne (see Table 1), in which
components were analyzed for protein content and the fraction that each one
represented as compared to the total sample weight.

Each of these fractions have a different amino acid composition which
in turn gives them their solubility and functional characteristics. Krull and Wall
(1969) reported that concentrations of ionizable amino acids (acidic: glutamic,
aspartic; and basic: lysine, histidine and arginine) are low for gliadin and
glutenin but high for albumin and globulin. Glutamine, accounts for more than
35% of the total amino acids present in wheat gluten (Hoseney, 1994),
corresponding to similar values reported by Wall and Beckwith (1969), who
indicated that about 37% of the total amino acid content in wheat gluten proteins
was glutamine and Krull and lnglett (1971) reported that about 37% of glutamine
was present in gluten. This is in contrast with a more recent publication
(Gontard et al., 1993) which reported that about 45% of the amino acid present
in wheat gluten proteins is glutamine. It is possible that this last value is higher
than the actual glutamine value since most of the literature reports values similar

to the ﬁrst three citations. Another important amino acid in gluten is proline

10

Table 1. Solubility fractionation of the endosperm
proteins of hard red spring wheat.1

 

 

 

 

 

Component Protein content Fraction of total protein
(%) (%)
Water-soluble fraction 53.3 11.9
Salt-soluble fraction 76.2 5.2
Alcohol-soluble fraction 89.7 28.5
Acetic-acid-soluble fraction 70.2 16.6
Residue 6.4 34.0

 

1Adapted from Chen and Bushuk (1970)

 

11
because of its influence in the structural conformation of the proteins. This

amino acid comprises 12.5% and 13.9% in glutenin and gliadin proteins,
respectively. When a proline residue is present in an a-helix, it imparts a twist to
the polypeptide chains. In addition, it is known that proline residues are
frequently found in ﬂexible regions of proteins (Branden and Tooze, 1991),
which suggests that the gluten proteins be elastic.

According to Krull and Wall (1969), physical bonds (i.e. hydrogen
bonding, electrostatic interaction, and hydrophobic interaction) can be broken by
the use of speciﬁc solvents, changing the pH of the solution, increasing the
temperature or altering the salt content. Also, using an acetic acid solution (pH
3.8) gliadin had approximately 23% helix as compared to 14% for glutenin. They
also indicated that gliadin has a more stable structure as compared to glutenin
due to internal disulﬁde bonds, and found that glutamine residues and non polar
amino acids are responsible for the aggregation of gluten proteins in aqueous
media. In this study, they also reported that amide groups are the primary sites
of association of wheat ﬂour proteins through hydrogen bond interaction, which
in part give the dough cohesive and elastic characteristics.

Vital wheat gluten is the name used to indicate wheat gluten that, when
hydrated, has the ability to form an elastic dough-like material, i.e., has not lost
its ability to form a cohesive mass during its production. It is readily available
today in the form of powder. It is, in general, a creamy-tan to light tan powder,
produced from wheat ﬂour by drying freshly washed gluten at a controlled

temperature. Several companies (e.g., ADM-Arkady’ and Tenstar Aquitaine)

12
produce starch and gluten from wheat flour. Gluten is obtained as a co-product

in this process. The approved method to obtain vital wheat gluten is AACC 38-
20 (AACC, 1990a). This method is the basis for producing dried vital wheat
gluten utilized by the refining industry.

Several authors (Gontard et al., 1993; Gennadios et al., 1993; Gontard
et al., 1992; Gennadios and Weller, 1990;_and Krull and lnglett, 1971), among
others have prepared ﬁlms using vital wheat gluten obtained either from the
industry (i.e. Pro-80TM and F 33000) or prepared in the laboratory according to
AACC Method 38-10 (AACC,1990b). Different properties and characteristics of
ﬁlms have been obtained depending of the process, but most gluten protein ﬁlm
producers agree that the commercial vital wheat gluten yields more opaque and
granular ﬁlms (Gennadios and Weller, 1990 and Krull and lnglett, 1971).
Furthermore, the industrially prepared vital wheat gluten is made from hard
wheat ﬂours, and its application is typically in the bakery industry for ﬂour
fortiﬁcation of bakery products (Magnuson, 1985). ,. According to Magnuson
(1985), other applications include meat, ﬁsh, and poultry products; pet foods;
breakfast cereals, nutritional snacks; breading and batter mixes, coatings; pasta
products; cheese analogues, pizza; aquaculture; and some nonfood appli-
cations.

One important characteristic that must be considered when a process
requires the extraction of starch and gluten is the parameters that must be
maintained for the operation to be successful. These requirements are usually

based on the grain quality (i.e. wheat), and according to Jones (1987) the most

13
important process parameters to consider when extracting wheat starch and

gluten from ﬂour are: a) process economics, including raw material costs, yields,
losses and waste treatment costs, b) final product quality, and c) trouble-free
plant operation. In choosing which wheat to purchase, it is necessary to
consider: a) wheat class in relation to price, b) wheat seed variety, c) growing
conditions and agricultural treatment, and d) climatic variations. He concluded
that some of these factors are beyond processor control, therefore ﬂexibility in
the operating conditions must exist to control minor changes due to wheat qual-
ity or varietal mix. This is important to consider ‘Since there are various reports
(Jones, 1987; Miller, 1974) indicating that in the production of wheat starch,
gluten is a commercial co-product.

Gontard et al. (1992) reported that proteins have been studied less
extensively than lipids or polysaccharides in terms of their ﬁlm-forming ability,
especially those Of wheat gluten. According to Gennadios et al. (1993) and
Gennadios and Weller (1990), commercial ﬁlm-forming preparations are limited

and include mainly those prepared from collagen, gelatin, and zein.

2.3 §EPARA TION OF GLUTEN FRQM FLQQR.
2.3.1 INDUSTRY vs. LABORATORY

The aim of gluten production is, until today, that of improving the
gluten quality as related to a higher bread making quality of the ﬂour. One
important difference between the commercial and the laboratory preparation of
gluten powder is that laboratory-prepared gluten generally has less starch and

impurities as compared to an industrially-prepared gluten powder (Godon et al.,

14
1983). These authors also reported that the Martin process (in which a dough is

prepared before the extraction of gluten) is the most frequently used process in
the world. They developed a laboratory-size machine to separate the gluten
from the starch using flour as raw material. This produced material of compa-
rable quality and with similar yields to those found in industry. Also, no
relationship between particle size and performance of the gluten was found in
this study. I

McDermott (1985) reported that the simplest test for gluten quality (in
terms of baking performance) is probably the lactic acid sedimentation test. This
procedure requires that 1 ml of ethyl alcohol and 25 ml of lactic acid (0.03M) are
combined with 100 mg of gluten and mixed every 2 minutes for 10 minutes. A
volume is transferred to a colorimeter tube and the turbidity of the gluten
dispersion is measured at 2 times (0 and 3 minutes). The sedimentation value is
calculated as the percent reduction in turbidity after 3 minutes. Best glutens

have a lower sedimentation value.

2.3.2 WHEAT PROTEINS FOR FILM FORMATION

The above information does not necessarily indicate the production of
better gluten quality for ﬁlm formation is needed. Furthermore, details of
manufacturing methods are not obtained easily when companies are asked
whether they used the "dough" or "batter" method (McDermott, 1985), and what
wheat varieties they use for the process.

There is evidence that different uses for gluten (i.e. wheat gluten) exist

and could increase beyond the current use as bread ﬂour improvers. It is

15
important to cite the production of gluten by companies that produce starch

utilizing ﬂours as the raw material (eg. ADM Arkady, Olathe, KS among others).
This type of gluten is the one that most researchers use (Krull and lnglett, 1971;
Anker et al., 1972; Kester and Fennema, 1986; Gennadios and Weller, 1990;
and Gontard et al., 1992) as raw material for the manufacture of protein-based

ﬁlms derived from cereal grains.

. 2.4 GENERAL PRQCEDQRES FOR MAKINQ EDIBLE/BIODEGRADABLE
FILM

There are three terms which could create confusion because only
recently (1989) was a formal deﬁnition proposed for them. The terms are
degradable, bio-degradable, and photo-degradable plastics. The deﬁnition of
degradable plastics, according to Narayan (1989), is "plastic materials that
undergo bond scission in the backbone of a polymer through chemical,
biological and/or physical forces in the environment at a rate which is reasonably
accelerated, as compared to a control, and which leads to fragmentation or
disintegration of the plastic.” Bio-degradable plastics are "those degradable
plastics where the primary mechanism of degradation is through the action of
microorganisms such as bacteria, fungi, algae, and yeast.” Photo-degradable
plastics are "those degradable plastics where the primary mechanism of
degradation is through the action of sunlight.” From these deﬁnitions, and
because degradable plastics is a broader deﬁnition, protein ﬁlms are going to be
considered and referred to as degradable packaging materials hereafter. In

addition, Gennadios and Weller (1990) deﬁned the terms edible ﬁlm and

16
coatings to mean "thin layers of edible material applied on (or even within) foods

by wrapping, immersing, brushing, or spraying in order to offer a selective barrier
against the transmission of gases, vapor, and solutes while also offering
mechanical protection.”

Because of the properties of a protein are generally related to its amino
acid composition and sequence, it is recognized that the large percentage of
glutamine in the gluten result in extensive intermolecular interactions (Gontard et
al., 1993), which in turn are in great part responsible for the formation of the
protein ﬁlm. Gontard et al. (1993) indicated that the disulfide bonds found in
gliadin are only intramolecular, while glutenin contain both types (intramolecular
and intermolecular) of disulﬁde bonds that link protein chains into high-
molecular-weight polymers. This is very important when considering the overall
properties of each of these fractions. In protein ﬁlm formation, gluten can be
used as the raw material. It is necessary, however, to add a plasticizer to
prevent the ﬁlm from becoming brittle (Krull and lnglett, 1971; Wall and
Beckwith, 1969; Gennadios and Weller, 1990). The function of plasticizers is to
reduce intermolecular forces, softening the ﬁlm structure, increasing the mobility
of the biopolymer chains, and thereby improving the mechanical properties of
the ﬁlms (Gennadios and Weller, 1990). According to Davies et al. (1991),
plasticisation is responsible for the change of brittle to ﬂexible behavior in a
material. This is due to increased segmental mobility of chains in the
amorphous regions of glassy and partially crystalline polymers. Since

plasticizers loosen the protein structure, diffusion of various gases and vapors

17 '
through the ﬁlm is faster; therefore, the type and quantity of plasticizer are very

important aspects of ﬁlm fabrication.

Examples of plasticizers, according to Gennadios and Weller (1990)
are glycerol, diglycerol, polypropylene glycol, and polyethylene glycols.
According to Kester and Fennema (1986), all of the preceding examples are
food grade plasticizers. Gontard et al. (1993) reported that polyols and lactic
acid were the only substances tested that plasticized gluten film (increased
ﬂexibility), and they determined that glycerol was the most effective plasticizer.
They also indicated that the amphipolar substances tested (glycol monostearate,
acetic ester of monoglyceride, sucrose ester of stearic acid and diacetyl tartaric
ester of monoglyceride) had no substantial plasticizing effect while hydrophobic
substances (i.e. beeswax and fatty acids such as lauric, stearic, and oleic acids)
had an anti-plasticizing effect on gluten ﬁlm, that reduced ﬂexibility. Kester and
Fennema (1986) stated that food-grade pasticizers are in general polyols,
including also sorbitol, and mannitol. Magnuson (1985) reported that aqueous-
alcoholic dispersions of wheat gluten have been prepared to form strippable,
edible coatings, such as sausage casings. He also reported that vital wheat
gluten, when hydrated, can be cast into ﬁlms.

There are certain conditions that must be met. for a protein ﬁlm to form.
Okamoto (1978) studied some of the main factors that affect the formation of a
protein ﬁlm. They used a protein ﬁlm from soy milk called "yuba,” a popular
foodstuff in Japan, as well as ﬁlms from wheat ﬂour proteins, and ﬁlms from

gliadin/keratin mixture. The yuba ﬁlm had approximately 55% protein and 25%

18
lipid. They found that protein contributes to the structure while lipids and

carbohydrates contribute significantly to the flavor and physical properties of the
ﬁlm. Also, they reported that a yuba-like ﬁlm can be formed from a solution
containing soybean protein and no lipids or carbohydrates. Okamoto (1978),
reported that the following conditions are required for a yuba ﬁlm to form: a) a
speciﬁc range of pH for the ﬁlm-forming solution (for complete protein
dissolution); b) possibly heating to a certain temperature and time interval (to
produce a partial heat denaturalization of the proteins); c) free evaporating
surface (no saturation with water vapor) and d) possibly a selective solvent (for
complete protein dissolution). They determined that a very important condition
to meet for ﬁlm formation is the dissolution of the proteins in the solution, and
this can be attained by a) or d) (adjusting pH or by selecting suitable solvents).
Kamper and Fennema (1986) deﬁned the process of ﬁlm formation by this
manner as "simple coacervation,” where a single hydrocolloid is driven from
aqueous suspension or caused to undergo a phase change by evaporation of
the solvent, addition of a water-miscible non electrolyte in which the hydrocolloid
is not soluble (e.g., alcohol), addition of an electrolyte to cause salting out or
cross-linking, or alteration of pH.

The way in which the ﬁlm was prepared in the Kamper and Fennema
(1986) study was similar to the yuba ﬁlm procedure. This process included the
complete dissolution of gluten in water by increasing the pH beyond 10 and then
heating the solution. Gennadios and Weller (1990), explained that the heating

process facilitates the sulfhydryl-disulﬁde interchange through unfolding of the

19
polypeptide chains. One important difference with respect to pH that Okamoto

(1978) found is that a wheat gluten film formed at pH 10, had rubber elasticity
while at pH 11 the-film was similar to yuba films (full of creases and translucent).
This author attributed this fact to disulfide bonds, which contribute to the
viscoelasticity of gluten may remain at pH 10 but are hydrolyzed at pH 11, as

follows:

2 RS-SR + 4 OH- :> 3 RS- + RSOa- + 2 H20 (1)

One important fact that Okamoto (1978) reported was that the addition of
mercaptoethanol (ME) resulted in a signiﬁcant increase in strength of the ﬁlm
formed.

Gennadios and Weller, (1990), explained how the cleavage of S-S
bonds by reducing them into SH groups results in polypeptide chains with lower
molecular weights, destroying elasticity and cohesiveness of gluten. Then, the A
dispersed gluten is reoxidized in the air and the‘reformation of S-S bonds yields
the ﬁlm structure. Okamoto (1978) also studied the development of ﬁlms from a
gliadin/keratin mixture and found that both proteins spread at random in the ﬁlm
and few available linkages that are built function property. Also, there is a
limitation in making ﬁlms using two or more proteins since a solvent suitable for
dissolving them both in the same solution is necessary. An interesting
observation by Anker et al. (1972) is that up to 20% and up to 50% of the gluten
in a ﬁlm-forming mixture can be successfully substituted with corn zein and soy

protein isolate, respectively.

20
Gennadios and Weller (1990) reported various advantages of using

edible ﬁlms from wheat and corn proteins such as: 1) the film and product can be
consumed simultaneously (environmentally ideal package); 2) since the ﬁlms are
made of a base of organic material (in the case a ﬁlm is not consumed), it can
degrade more easily compared to current synthetic materials; 3) additives can
be incorporated into the ﬁlm enhancing its general properties (i.e. flavor, color,
antimicrobial and antioxidant agents, etc); 4) nutritional value of the food is
supplemented since the ﬁlm contain proteins; 5) when ﬁlms are used between
two different structures in a food, they can reduce moisture and solute migration;
6) if an edible film is used along with non edible ﬁlms (i.e., synthetic polymer) in
a multilayer structure, the edible ﬁlms could be the internal layers (in direct
contact with the food). Similarly, some of the properties of these ﬁlms as
suggested by Kester and Fennema _ (1986) include retardation in moisture
migration, gas diffusion (oxygen, carbon dioxide), oil and fat migration, solute
migration, improvement in mechanical-handling properties of foods, added
structural integrity to foods, retention of volatile ﬂavor compounds, and incorpo-
ration Of food additives.

Golding (1959) reported the use of some proteins as industrial plastics.
Some of these include polyamides from milk, speciﬁcally casein (used in the
fabrication of buttons, buckles, ornamental jewelry, and wool-like ﬁbers); soy
bean polyamides (used as extenders in phenolic molding powders and as ﬁber
for staple-ﬁber form in automobile upholstery); polyamides from corn, particularly

zein (used in coating of paper which provides scuff-resistant surface without high

21
gloss, plastics for manufacture of buttons, buckles, and other novelties); and

polyamides from peanuts (used -currently discontinued- by combining equal
mixtures of Ardil -peanut protein fiber- and wool, cotton or rayon to produce
textile fabric products). In addition, there have been several reports on the
preparation of ﬁlms made from corn and soybean (Sian and lshak, 1990;
Gennadios and Weller, 1990; and Andres, 1984). It is important to realize that
the properties Of these ﬁlms are less desirable compared to those obtained from
wheat proteins. Gennadios et al. (1993), reported that ﬁlm formation can be
attained using wheat gluten, collagen, gelatin, casein, whey proteins, corn zein,
soy protein, and peanut protein. A ﬁlm based on soybean protein was discussed
before (the yuba ﬁlm). Sian and lshak (1990) studied various factors inﬂuencing
yuba ﬁlm. They took a solution of soybean milk and adjusted the solution with
NaOH or HCI to pH values of 2.0, 6.7, 7.5, 9.0, and 11.0. Samples of soy milk
were preheated to 84-86 °C and poured into a tray ﬁxed in a boiling water bath.
The ﬁlm which formed on the surface of the solution was picked up with an L-
shaped wire every 20 minutes and numbered according to that sequence. From
these ﬁlms, they determined protein, fat, moisture, and ash contents as well as
ﬁlm yield (weight of the ﬁlm after drying at 25 °C for 48 hours). They found that
protein and carbohydrate composition increased as the sequence number of the
ﬁlm increased, while the lipid content decreased." Percent moisture and ash also
increased with the ﬁlm number. They attributed the lower carbohydrate content
with ﬁlm number to the presence of more fat (in the ﬁrst ﬁlms) which tends to

bind less carbohydrate and minerals (ash). Also, they indicated that at higher

22
pH, the protein solubility was high, and thus more protein was incorporated into

the ﬁlm. This higher incorporation of negatively charged proteins at higher pH
probably increased its capacity to hold more polar groups such as minerals and
carbohydrates but reduced the tendency to hold the non polar lipids. An
important consideration is that at elevated pH an increase in emulsifying
capacity of the proteins was achieved. Kester and Fennema (1986) indicated
' that emulsifying agents may be incorporated when a stable Oil-in-water, ﬁlm-
forming emulsion or micro emulsion, is desired.

In general, zein ﬁlms are prepared and currently used as coating
materials rather than film sheets (Gennadios and Weller, 1990 and Andres,
1984). Andres (1984) indicates that an increase in shelf life of food products
can be Obtained by using an edible coating composed of corn protein (zein) and
vegetable oils. Torres et al. (1985) developed a zein ﬁlm in which incorporation
of preservatives was performed in order to control microbial growth at the
surface Of intermediate moisture-coated foods when exposed to temperature
changes (emulating environmental changes during production, distribution and
storage). Andres (1984) suggested that some applications could include:
coating of nuts to prevent rancidity and maintain desirable texture; in
confectionery products coating could be useful for maintaining desirable levels
of moisture and texture; coating of fruit pieces or raisins to retain moisture when
these food particles are incorporated as .dry ingredients in breakfast cereals or

mixes.

23
Andres (1984) depicted a commercial edible coating composed of

alcohol, zein, special vegetable oils, glycerin, citric acid, BHA, and BHT. This is
a good example of the incorporation of an antioxidant into a protein-based
coating film. The mode of application of the coating includes dipping, misting,
and spraying. In this type of process, when the coating is still in liquid form, zein
interacts primarily with the alcohol. Once the alcohol evaporates, the zein
hydrophobically interacts with the vegetable oil, and produces a non-greasy,
transparent, shiny ﬁlm (Andres, 1984).

Andres (1984) also studied storage tests with pecans. The pecans
were held at room temperature (70 °F), 50% relative humidity, and in normal
indoor light. Non-coated pecans developed abnormal ﬂavor and chewiness after
one month. On the other hand, coating with 1% zein prevented off ﬂavors and
chewiness to appear providing pecans that were fresh and felt crisp after three
months. It was also found that uncoated pecans darken more rapidly than
coated ones, and Andres (1984) attributed this to absorption of ultraviolet light
by zein in the coating.

A different approach is given by Gennadios and Weller (1990) in which
they report the use of zein coatings for pharmaceutical tablets along with
foodstuffs. They also reported the use of colorants in the coatings. Other
possible uses of zein coating ﬁlms include application on candy products (sugar-
burnt peanuts, candy corn and butter creams, chocolate-panned confections,
hard gum candles, and sugar coated pan candles), on fortiﬁed rice, on egg shell

(to improve quality), and micro encapsulation of food products (Gennadios and

24
Weller, 1990). These authors also explain that zein films can be formed in a

similar way as that for wheat gluten films, that is, aqueous-alcoholic solutions of
zein can be cast on flat, non reactive surfaces and then peeled off after drying.
One problem with corn protein films formed in this way is that the resulting films
are brittle so, the presence of a plasticizer is required to give some ﬂexibility
(Gennadios and Weller, 1990). These authors indicated that zein films prepared
with and without plasticizers resulted in that: a) the unplasticized films were
yellowish and transparent to translucent, and b) the plasticized ﬁlms were
yellow, opaque and had higher permeability to water vapors and oxygen. Also
the mechanical properties of both type of ﬁlms were inferior as compared to
commercial synthetic polymer materials in use today.

Kester and Fennema (1986) indicated that edible ﬁlms and coatings
could never replace non edible, synthetic packaging materials when used in
storage for prolonged periods of time. They suggested that the utility of these
ﬁlms is based primarily on their capacity of improving overall food quality and
extending shelf life as well as improving the economic efﬁciency of packaging
materials. One important point to consider is that synthetic polymer chains are
very well controlled during polymerization (Sperling, 1992 and Birlet et al.,
1992). On the other hand, edible polymers are already formed in the material
used for ﬁlm preparation so the size cannot be controlled, except by cross-
linklng existing chains. The size of protein molecules could be increased by
chemical and/or physical speciﬁc conditions (addition Of reducing and oxidant

agents to the ﬁlm-forming solution, pH control, etc.) This is important since, as

25
polymer chain length and polarity increase, structural cohesion is enhanced, and

this increase in cohesion generally reduces film flexibility, porosity, and
permeability to gases, vapors, and solutes (Gennadios and Weller, 1990 and
Rodriguez, 1989). Gennadios and Weller (1990) proposed some ways to
increase cohesion: a) produce a uniform distribution of polar groups along the
polymer chain which increases the likelihood of interchain hydrogen bonding
and ionic interactions; b) produce maximum solvation and extension of the
polymer molecules by using ethanol and water-ethanol combination as solvents;
c) maintain good environmentalicontrol during film formation (i.e., application of
warm ﬁlm-forming solutions to a warm receiving surface yields) to avoid
excessive temperatures since an excessive rate of evaporation during ﬁlm drying
could immobilize polymer molecules before they have a chance to interact to
form a continuous, coherent ﬁlm (is. defects such as pinholes or non uniform
thickness). Pinholes have been reported as important permeability-increasing
factors (Labuza and Contreras-Medellin, 1981, Kamper and Fennema, 1984b, .
and Gennadios and Weller, 1.990).

Disulﬁde bonds play a very important role in the protein conformation
of the gluten proteins. Beckwith et al. (1965) studied the effects of a reduction
and reoxidation process when applied to gliadin. They found that gliadin can be
reduced with B-mercaptoethanol at pH 7.4 in solution of 6M urea, and can also
be reoxidized in acid solvents at 0.1% protein concentration. An important
ﬁnding by these authors is that gliadin proteins can be reduced and then

reoxidized to yield a product closely resembling the native protein. Similar

26
reports of the reduction-reoxidation capability of the gliadin molecules was

reported by Schofield et al. (1983). Beckwith et al. (1965) assessed the
restoration of native structure by immunological studies in which antibodies to
gliadin present in the sera of individuals who have celiac disease were used.
They found that gliadin proteins can be reducedand then reoxidized to yield a
product closely resembling the native protein. It was suggested, however, that
the conditions for reoxidation of the proteins must be controlled (mainly protein
concentration in the solution) to get a high restoration of the native structure.
For instance, Beckwith et al. (1965) indicated that previous studies involving
microimmunodiffusion resulted with immunochemical properties of the original
gliadin was restored at 0.1% protein concentration. On the other hand, the
properties were not recovered when the reoxidation took place at a 5% protein
concentration. This information could be useful in case disruption of the gluten
protein is necessary to introduce functiOnal groups to improve the general
properties Of the wheat protein ﬁlms. A

Stewart and Mauritzen (1966) studied the feasibility of adding cysteine
to proteins Of wheat dough. Their ﬁndings were that the incorporation of
cysteine into dough was possible and increased in the absence of air. This sug-
gested that a S-S exchange reaction was the formation mode rather than oxida-
tion. They mixed a dough with cysteine in a Brabender farinograph in which ni-
trogen was flushed to maintain anaerobic conditions during the mixing process.
In the case of gluten ﬁlms, this information could be useful in an attempt to

increase the molecular weight of the proteins so that tensile strength may be

27
increased. Rodriguez (1989), and Brown (1981) have reported that an increase

in molecular weight of the polymer system will yield an increase in tensile
strength. There have been few papers published related to direct measurement
of the disulfide bond and its characterization as related to protein ﬁlm per-

forrnance.

.5 RRENT METH DOLO Y F R PREPARATI N F LUTEN-BA ED
FILM

2.5.1 OVERVIEW
Krull and lnglett (1971) developed ﬁlms from whole gluten obtained

both in the laboratory and from the industry. These films were cast from a 20%
solution of gluten in a solvent comprised 60% ethanol, 20% lactic acid, and 20%
water. Lactic acid is a non-volatile acid (Gontard et al. 1992), which double
functions as glutenin dissolving agent and plasticizer. All of these ﬁlms (Krull
and lnglett, 1971) were extremely brittle, not water resistant, nor did they
compare favorably in tensile strength to some synthetic ﬁlms.

Gennadios and Weller (1990) prepared ﬁlms using commercial Pro-
80TM vital wheat gluten. They put 15 g of the gluten in 72 ml of 95% ethanol.
Then added 6 g of glycerol as plasticizer (instead of lactic acid used in the
previous method) and boiled the solution to disperse the wheat gluten. They
added 48 ml distilled water and increased the pH with 14 ml of 6N ammonium
hydroxide to dissolve the glutenin fraction. The resulting ﬁlms, after casting and

drying, were strong, ﬂexible, and translucent, but not transparent. Good barrier

28
properties to oxygen and carbon dioxide were obtained for the films, but they

had a very high water permeability.

More recently, Gontard et al. (1992) developed films using commercial
F 33000 vital gluten. These ﬁlms were prepared from a solution of gluten in
absolute ethanol, acetic acid, and water with glycerol added as plasticizer. The
concentrations of gluten (gl100 ml solution), ethanol (ml/100 ml solution, and the
pH of the solutiOn (adjusted with acetic acid) were varied to test for the influence
of these variables on ﬁlm properties. They did not ﬁnd an optimum condition for
the formation of a ﬁlm with "ideal" characteristics. The signiﬁcant ﬁndings were
that pH and ethanol concentration of the ﬁlm-fornﬁng solution were the two most
important factors inﬂuencing ﬁlm opacity, water solubility and water vapor
permeability. Also, mechanical properties appeared to be strongly inﬂuenced by
the concentration of gluten and the pH. They suggested that depending on the
ﬁlm use, application technique or any other consideration, a particular ﬁlm-
formation combination may be chosen which could cover the basic properties to
optimize.

In a more recent study, Gontard et al. (1993) investigated the inﬂuence
of plasticizers and how they affect mechanical and water vapor barrier
properties of gluten ﬁlms. They prepared the ﬁlms using commercial F 33000
vital wheat gluten in a concentration of 7.5I100 ml solution, 45 ml ethanol I 100
ml solution. The pH of the solution was adjusted to 4 with acetic acid. Glycerol
was added as the variable to test in concentrations from 0 to 33.3 91100 g dry

ﬁlm matter. Their ﬁndings were that polyols (glycerol, sorbitol, propanediol) and

29
lactic acid were the only substances that especially plasticized gluten film.

Amphipolar substances (glycol monostearate, acetic ester of monoglyceride,
sucrose ester of stearic acid and diacetyl tartaric ester of monoglyceride) had no
real plasticizing effect. The hydrophobic substances (beeswax and fatty acids
such as lauric, stearic, and oleic acids) had an anti-plasticizing effect on gluten
ﬁlm. The most effective plasticizer according to Gontard et al. (1993) was
‘ glycerol. Another signiﬁcant ﬁnding was that water activity (a...) and temperature
were crucial parameters for establishing glutenﬁlms properties. Gennadios et
al. (1993) fabricated gluten ﬁlms by following the technique of Gennadios et al.
(1990) in which 15 g wheat gluten, 72 ml 95% ethanol, and 6 9 glycerol were
mixed. This particular study showed, the temperature effect on oxygen
permeability of the gluten ﬁlm. The result was that oxygen permeability "values
increased with an increase of the testing temperature. This was due to
enhanced motion Of the polymer segments and to increased energy levels of the
permeating oxygen molecules. The oxygen permeability vs. temperature plot
ﬁtted the Arrhenius model. They proposed that probably both intermolecular and
intramolecular disulﬁde bonds are signiﬁcant in the complex formation.
Furthermore, Gennadios et al. (1993) suggested that the extended random-
coiled glutenin polypeptides provide the frame Of the structure, and the smaller

globular gliadin polypeptides are packed into the network.

2.5.2 CROSS-LINKING OF PROTEINs

The term ”cross-linking” denotes stable association of generally large

elements at speciﬁc places to create a new entity that has distinct properties as

30
a result of the juncture (Friedman, 1977). In the case of proteins, cross-linking

promotes changes in the chemical, functional, nutritional, and biochemical
properties, in addition to physical properties as related to the new molecular size
and shape.

In synthetic polymerization < reactions, ordered step-by-step
progressions do not occur precisely. That is, each macromolecule will react at
different rates, thus, the final product is represented by a mixture of
macromolecules with different molecular weights. Usually a bimodal distribution
for the molecular weight is obtained. In some cases, controlled rheology resins
with a narrow molecular weight distribution are preferred (Gruenwald, 1993).
More importantly, it is generally recognized (Gruenwald, 1993; Nicholson, 1991;
Sperling, 1992; Mandelkem, 1993) that the molecular weight of a polymer
greatly inﬂuences different properties, including the viscosity of the polymer melt
and the mechanical properties of the ﬁlms Obtained.

Cross-linking Of proteins has been reported to be successful by various
authors (Lotan and Sharon, 1977; Uy and Wold, 1977; Harland and Feairheller,
1977; Jane et al., 1993; Mahmoud and Savello, 1993). Protein polymers, in
contrast to synthetic polymers, are already formed in the source material used to
make the ﬁlms. This is easily understood since the synthesis of the former occur
inside an organism upon the expression Of genetic information which starts the .
mechanism of protein synthesis. There are two points for comparing the
complexity of protein and synthetic polymers. The ﬁrst is that proteins are more

complex than most linear polymers in that they can incorporate 20 different

31
monomers (amino acids) in their manufacture instead of one or two for synthetic

polymers. On the other hand, proteins are structurally less complex since most
chemical polymers are synthesized by polymerizing a mixture of monomers,
thereby producing a distribution Of chain lengths and (if more than one type of
monomer is present) an approximately random sequence of monomers.
Proteins are linear and unbranched and have precise lengths and exact
sequences of amino acids. In fact, it is only the differences in length and
sequence that distinguish one protein frOm any other, and make possible a
diversity of structures and functions. The linear polymeric chain of almost every
natural protein has the property of being able to assume a speciﬁc three-
dimensional folded conformation (Creighton, 1993). In addition, these
conﬁgurations, ranging from random coils to highly complex helices, are
essential to the behavior and role of proteins in the life processes. This would
make the chemical complexity of protein macromolecules essentially limitless
(Battista, 1958).

Mahmoud and Savello (1993) used transglutaminase to cross-link
covalently concentrated proteins solutions of a 1:1 (wt/wt) mixture of a-
lactalbumin and B—lactoglobulin to form gels. Thesengels were dehydrated to
produce transparent ﬁlms. An important ﬁnding by these authors was that
although the protein ﬁlms were insoluble in aqueous buffers at various pH and
heat treatments, these ﬁlms were protease-digestible. Golding (1959) reported
that when casein is immersed in a 4 to 5 percent formaldehyde solution, the 8-

amino group (of lysine), as well as the unsubstituted a—amino group, can bind

32
one or two moles of formaldehyde, depending on the concentration of the latter.

This author also reported that zein and casein plastics can be cured by
immersing the plastics in formaldehyde solution. This reaction can be catalyzed
by acids, being HCI the most effective.

Lim and Jane (1993) reported that mixtures of starch and zein with
cross-linking agents including formaldehyde and glutaraldehyde yielded plastic-
like materials when a compression-molding technique was used. They found
that the effect of the cross-linking in the material was that of the enhanced
physical strength and water-resistance. They indicated that their starch-zein
plastics were economically feasible replacements for petroleum-based plastics.

Cross-linking of proteins with glutaraldehyde was reported successful
by Richards and Knowles (1968), and indicated that the protein cross-linking is
irreversible, surviving treatments with urea, semicarbazide, and wide ranges of
pH, ionic strength and temperature. Richards and Knowles (1968) and Chatterji
(1989) reported the formation of a Shiff base by the interaction between the
aldehyde and the amino groups Of the amino acids. Chatterji (1989) attributed
the change in color from pale yellow to deep orange to the formation of the Schiff
(aldimine) linkage between the free amino groups of the proteins and
glutaraldehyde. This author also reported that when native gelatin is treated with "
glutaraldehyde, the reaction exclusively involves the lysines, to almost 100%.
The schematic of the reaction is represented in Figure 1. Satyanarayana and
Chatterji (1991) demonstrated that the cross-linking of gelatin granules with

glutaraldehyde involves the s-amino groups of the lysine residues of the protein

33

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34
and the aldehyde functionality of the glutaraldehyde yielded a versatile cross-

linked matrix.

2.6 MASS TRANSFER IN PQL YMERIC FILMS FILMS

Gas and vapor transport in polymeric materials is of great importance
to the packaging industry because this is one of the functions of a package in
protecting contents. No polymer ﬁlm is known to provide a complete barrier to
the transport of a gas or vapor molecule (Brown, 1981). In the absence of
cracks, pinholes, or other defects the mechanism for gas flow through a polymer
ﬁlm is by activated diffusion, driven by a concentration gradient. The penetrant
dissolves in the film matrix at the high concentration side, and then diffuses
through the ﬁlm towards the low concentration side. The overall phenomenon is,
inﬂuenced primarily by the chemical composition, size, shape, and polarity of the
penetrating molecule and chemical c0mpOsition andpolymer-chain segmental
motion within the film matrix. When the concentration of the perrneant at both
sides of the ﬁlm are kept constant, the system eventually reaches a steady state
after an initial period of transient ﬂow. The determination of the permeability of
edible ﬁlms is carried out in a similar fashion as for non-edible ﬁlms using i

continuous ﬂow and quasi-isostatic methods (Donhowe and Fennema, 1994).

The solution to the second Fick’s law for a continuous flow permeation
experiment for oxygen (Hernandez et al., 1986) is given by the following

equaﬁon:

35

_n)2€2 (2)

.EL

11'"sz 40:),ésex‘" 4Dt)

 

where F, is the ﬂow rate of oxygen permeating the ﬁlms at time t, F... is the
oxygen ﬂow rate under steady state conditions, 2 is the ﬁlm thickness, and D is
the oxygen diffusion coefﬁcient. By taking only the ﬁrst term of the series, the
permeation experiment up to a value of the flow ratio of 0.95 can be simplified to

the following equation:

..fr_.._4_ vz _
°‘F,",/;’X epr X) . (3)

where X = (21(4Dt), and <1) is the ratio between the ﬂow rates at time t and at

steady state. From a continuous ﬂow permeability experiment, F. values can be
Obtained as a function of time from t=0 to the steady state. The Newton-
Raphson method can be used to evaluate X from the Eq. (3) as a function of
time. The diffusion coefﬁcient D is determined from the slope of the straight line

of the plot X‘ vs. time for values within the' range of 0.05<4><0.95 (Hernandez et

al., 1986). The oxygen permeability constant, P, can be determined directly from

the steady state value of each permeability experiment:

Fm!
AP

P = (4)

where AP is the driving force given by the oxygen or permeant pressure gradient

across the ﬁlm.

36
Finally, according with Hernandez (1994), Gavara and Hernandez

(1994), and Kester and Fennema (1986), the solubility (S) of the permeant into
the film can be calculated by applying F ick’s and Henry’s laws with the following

equaﬁon:
P
S=— , (5)
D

It is assumed, in Eq. 5, that D is independent of the concentration of
the penetrant. Figure 2 presents a diagram of a setup for oxygen permeability
studies using a diffusion cell, with carrier gas (nitrogen) and test gas (oxygen)
ﬂushed on either side Of the cell. The terminology of the units is also explained
in this figure. Although this study focused only on measurement of permeability
of oxygen (gas) through the ﬁlms produced,“ both, gas and water vapor
permeability on other ﬁlms are discussed. It must remain understood that
permeability is not a universal property of the ﬁlm, but rather a characteristic of
both ﬁlm and penetrant under speciﬁc environmental conditions. Permeability of
water vapor through a polar ﬁlm matrix, for example, increases significantly as

vapor pressure is increased (Kester and Fennema, 1986).

2.6.1 PERMEABILITY OF GAsEs

Brown (1981) explained the various methods which can be employed
for gas permeability measurements: a) manometric method in which the quantity
of gas that has permeated through a test piece in a given time is measured as a
change in pressure and volume b) constant volume method in which the steady

state portion of the plot of pressure versus time may be near linear, hence the

37

r/ difussion cell

 

 

ﬁlm specimen
coulox cell

(detector)

 

 

 

 

 

 

or [m302(stp)](m)
P = DS = = 2
At(AP) . (m 4. )(s)(Pa)
where: P = Permeability
D = Diffusion constant
8 = Solubility coefﬁcient
Q Amount of oxygen passing through the ﬁlm at standard

temperature and pressure (stp)
g = Film thickness
A = Film area
= Time
AP = Oxygen gas partial differential pressure

Figure 2. Experimental setup for oxygen permeability studies on ﬁlms.

38
calculation of the gas transmission rate can be carried out either at a single point

or over an extended time interval, c) the constant pressure method in which
pressure is kept constant in the low chamber of a cell and the volumetric change
in permeated gas is measured, and d) carrier gas method in which the test gas
ﬂows at a constant rate through one chamber and a second gas, the carrier,
flows through the other chamber at a constant rate. The test gas'which
permeates through the polymer is swept away to a detector which may be of the
absorptiometric or thermal conductivity type.

Another important consideration in permeability of protein-based ﬁlms
(wheat gluten, corn zein, and wheat gluten/soy protein isolate) was discussed by
Gennadios et al. (1993). According to Gennadios et al. (1993) not much infor-
mation is available on the mechanical properties and barrier characteristics for
protein ﬁlms because much of the information on the preparation of these edible
ﬁlms derives from patents. These authors found that the differences amOng
oxygen permeability in the three ﬁlms at each temperature were signiﬁcant.
They also suggested that, as a general rule, permeability values follow a- direct
proportion effect meaning there is an increase in peMeability with increase of ’
temperature. This is attributed to enhanced motion of the polymer chains and to
the greater energy level of the permeating molecules due to a more energetic
system. Gennadios ef al. (1993) reported that wheat gluten ﬁlms had a lower
permeability compared to corn zein ﬁlms underithe same conditions. This was
attributed to the more complex structure and closer polymer segments in the

gluten ﬁlms. They concluded that oxygen molecules can permeate more readily

39
through a zein helical conformation (about 50%) than through the highly cross

linked gluten structure, and the nature of interaction involves disulﬁde bonding.
One of the major contributions of this study was that it gives practical
comparisons between the three protein films with respect to other films. An
important characteristic of these ﬁlms was that the oxygen permeability values
were lower compared to other polysaccharide and composite
polysaccharide/lipid edible ﬁlms. These values were also lower than some of
the common plastic ﬁlms used, such as low and high density polyethylene,
polypropylene, polystyrene, and unplasticized polyvinyl chloride. They mention
that the protein ﬁlms were even less permeable to oxygen than polyamide-6
(Nylon-6) which is known as a good oxygen barrier. It is important to note,
however, that the oxygen permeability values were obtained at 0% relative
humidity. Oxygen permeability may be very different if tested at higher relative
humidities because of the hydrophilic nature of the protein molecules
(Gennadios et al., 1993). Gontard et al. (1993), demonstrated that during
hydration of a gluten ﬁlm (increase of relative humidity), mechanical properties
such as puncture strength and elasticity decrease while water vapor
transmission rate and extensibility decreased. According to Gennadios et al.
(1993) the response of protein ﬁlms showing increased oxygen permeability with
an increase in relative humidity is comparable to synthetic moisture-sensitive
ﬁlms such as polyvinyl alcohol, polyvinyl acetate, cellophane, cellulose acetate,
polyamide, and ethylene-vinyl alcohol. A very interesting suggestion made by

Gennadios et al. (1993) is that of the possible application of these types of ﬁlms

40
in multilayer laminates. In this case, the good oxygen barrier properties of the

protein ﬁlms could be used in one of the layers of the laminates, while the other
layers could protect the moisture sensitive characteristic of the protein films.
This type of laminate is used in industry, i.e. ethylene-vinyl alcohol (EVOH). It
has very good oxygen barrier properties but is moisture sensitive (water vapor
reduces its oxygen barrier properties). Thus, EVOH is placed in a multilayer

laminate structure in which the other materials prOtect it from moisture.

2.6.2 PERMEABILITY OF WATER VAPOR

Brown (1981) indicated that water vapor is generally measured by the
gravimetric method. This method consist of introducing a desiccant or water into
the bottom of a dish and the test piece is sealed onto the lip of the dish using
wax. The assembly is then placed in an atmosphere of controlled temperature
and humidity. The weight gain or loss is measured at regular intervals of time
and, when this becomes approximately constant, the rate of weight change is
used to calculate the quantity of water vapor which has permeated through a unit
area of the test piece per unit time.

Kamper and Fennema (1984a) explained that water permeability
through ﬁlms varies signiﬁcantly with ﬁlm composition and with orientation of
molecules in the ﬁlm. They suggested that lipids appear to be the most effective
barrier to the movement of water through an edible ﬁlm.

Water vapor permeability of edible ﬁlms was studied for a bilayer ﬁlm
by Kamper and Fennema (1984a). In this-study, the ﬁlm matrix was composed

of hydroxypropyl methyl cellulose, a carbohydrate. This ﬁlm was considered a

41
layer and additional lipid layers were tested for water vapor permeability using

the test mentioned above (gravimetric method). They determined water vapor

transmission rate (WVTR) and permeability (P) by using the following equations:

WVTR = ML (6)
Afw

and

we

= (7)
Atw(P2 ‘ P1)

 

where: P2 - P1 = vapor pressure differential across the ﬁlm (AP).

They found that water vapor transmission through the emulsion ﬁlm was highly
dependent on chain length and degree of saturation of the fatty acids. The
introduction Of one double bond to the fatty acid hydrocarbon chain increased
the WVTR. Decreasing the chain length of the saturated fatty acid also
increased the permeability of the emulsion ﬁlm to water vapor.

In a similar work, Kamper and Fennema (1984b) evaluated the water,
vapor permeability of a bilayer ﬁlm composed Of stearic and palmitic acids as
one layer and hydroxypropyl methyl cellulose as the other layer. They found
that the amount of lipid added to the ﬁlm forming solution inﬂuenced the WVTR,
and this amount resulted in a signiﬁcant reduction in permeability for additions of
0.46 mg/cm2 or more. They also noted that when the hydrophilic side of the ﬁlm
was exposed to relative humidities other than 0%, the ﬁlm would absorb water
from the atmosphere, apparently resulting in an alteration Of the lipid layer and

discontinued resistance to the water vapor through the ﬁlm. Kamper and

42
Fennema (1984b) also noted that the amount of water vapor transmission

through the films increased as the temperature decreased. lt_is important to
note, however, that this effect may vary significantly depending of the film, since
Labuza and Contreras-Medellin (1981) showed that polyethylene had a greater
permeability to water at -30 °C than at 35 °C. Also, a small disruption in ﬁlm
integrity could signiﬁcantly diminish the ability of a ﬁlm to delay the permeability
of water vapor (Kamper and Fennema, 1984b).

A different approach was taken by Kamper- and Fennema (1985) in
which the water vapor gradient was retained for longer periods of time when an
edible ﬁlm composed of a layer of stearic-palmitic acid and a layer of hy-
droxypropyl methyl cellulose was placed between two food components. One
had a high water activity (tomato paste) while the other had low water activity
(ground crackers). The ﬁlm signiﬁcantly retarded the transfer of water from the
tomato paste to the crackers in two of the three conditions studied: 14 days at 25
°C, and 21 days at 5°C. They also found that the ﬁlm basically halted the
transfer from tomato paste to the crackers during 70 days at -20 °C. All ﬁlms,
after storage for 5 or more weeks, were more elastic, due to hydration of the ﬁlm.
In this case, an increase on water permeability could be predicted (Kamper and

Fennema, 1985).

2.7 RHE L I AL METH D

Rheology is ”the science of the deformation and ﬂow of matter; the
study of the manner in which materials respond to applied stress or strain”

(Steffe, 1992). A polymeric material possesses a wide range of material

43
properties and among these the most significant are hardness, deformability,

toughness, and ultimate strength (Cowie, 1991 ). Alfrey (1948) raised a question
that any polymer-material design engineer would like to answer: “If an object of
some known shape is constructed from a polymer of known properties and is
subjected to a known sequence of surface forces or constraints, what will be the
detailed response of this object?.” The answer has to be studied by different
' techniques involving different behavioral characteristics. This usually involves
the knowledge of the properties of the polymer by solving differential equations
of practical interest which can be formulated in terms of stress and strain. \
According to Cowie (1991), most of the time one would expect certain
desired characteristics for a material but, unfortunately, many times these would
correspond to conflicting properties. A polymer, for example, with a high
modulus and low creep response does not absorb energy by deforming easily,
hence it has poor impact strength. This indicates that a compromise must be
sought depending on the ﬁnal use of the polymer, and this requires a detailed
knowledge of the mechanical properties. There exist, in the broader" sense, two
main types of mechanical properties that can be analyzed based on the type Of
study performed: at small deformations, in which creep tests are included, and at
large deformation, including the ultimate properties, in which tensile strength is

determined.

2.7.1 TENSILE STRENGTH

The terms “ultimate strength” or “tensile strength” of a material is the

stress at or near failure and is usually measured in tension. It is amongst the

44
most significant properties studied in polymers (Cowie, 1991). Tensile strength

correlates with toughness, tear resistance, and fatigue, hence it has real merit.

Toughness can be deﬁned as the energy absorbed at failure. The area
under the stress-strain curve has the units of energy per unit volume, or joules
per cubic meter (when stress has units of pascals and strain of meters per
meter). The energy may be stored elastically or may be dissipated as heat
causing a permanent deformation.

A common tensile test involves elongating a dumbbell-shaped sample
held in jaws that separate at a constant rate (Figure 3). The stress is measured
as a function of time. Despite the difﬁculty of measuring strain accurately, the
dumbbell samples have the great advantage that ultimate failure will take place
in the center of the sample and will not be affected by stress c0ncentration at the
jaws.

2.7.2 TENSILE CREEP

In order for an object made from a polymeric material be of any
practical use, it must be able to retain its shape when subjected to tenSion or
compression over long periods of time. This particulardimensional stability is an
important consideration when choosing a polymer to use in the manufacture of
an item.

Cowie (1991) deﬁned creep as “a progressive increase in strain,
observed over an extended time period, in a polymer subjected to a constant

stress.” Steffe (1992) indicated that in a creep test, an “instantaneous stress” is

45

 

    

 

r1 r-I r----------'
I I I I I
I I _\ I I I
I I " I I I
I I I I I
I I I I I
I- .r I- .r I
Metal plates 55;,
Specimen
rjfrﬁ I
I I I I I
I I I I I
I I I I I
I I I I I
I I I I I
I_ _, I__ _, I

 

(a) (b)

Figure 3. Example of dumbbell-shaped polymer sample specimen to evaluate
tensile properties.
(a) side view, (D) front view.

46
applied to the sample and the strain (creep) is observed over time. Data are

usually presented in terms of creep compliance:
J = f(t) = —°— (8)
CO

which is deﬁned as the ratio of the relative elongation at time t to the stress.
Ferry (1970) indicated that for the cross-linked rubbers, J(t) at long times
approaches a limiting value J., the equilibrium compliance, which according to
the theory of rubber—like elasticity is proportional to the number-average
molecular weight between cross-links in the network.

This idealized picture of creep behavior in a polymer has a simple
mechanical equivalent constructed from springs and dashpots. Figure 4
represents an schematic representation of a creep curve, and in Figure 5 gives a
series of diagrams (four-element Burgers model) which explain the simple,
idealized creep behavior of a polymer. In diagram (i) the system is at rest. In
diagram (ii), an instantaneous stress (00) has been applied and the free spring
extends by an amount O'o/Eo. This is followed by a decreasing rate of creep with
a progressively increasing amount of stress being carried by E. Given sufﬁcient

time, both dashpots are fully extended (iii). ,Such behavior is described by

8 = f(t) = 241+— exp[-iit-):l (9)

where the retardation time (A... = LII/E1) is the time taken for the delayed strain

(E and I11) to reach 1-1/e or approximately 0.632 of the total deformation. A

47

Stress removed

 

 

 

C m h ’
' a
D
b
b,
a c’

 

 

OI e t
Stress applied

Figure 4. Schematic representation of a creep curve.

48

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49
considerably longer time may be required for complete deformation to occur.

When spring E1 is fully extended the creep attains a constant rate corresponding
to movement in the dashpot restricted by #0 Viscous flow continues and the

dashpot (Do) is deformed until the stress is removed.

When the stress is removed, the deformation of the free spring is
recovered quickly, followed by a slow recovery in the parallel spring-dashpot
system. At that time, E0 retracts quickly (Figure 5) along section a’ and a period
of recovery results (b’). During this time spring E1 forces the dashpot plunger in

(H1) back to its original position (iv).

As no force acts on No it remains in the extended state, and
corresponds to the non-recoverable viscous ﬂow (v), deﬁned by c’ in Figure 4.

The ﬂow of the dashpot (lie) is retained as a permanent “set.”

The complete four-element Burgers model is represented by

_ t]-E_I__ Lot
s=f(t)- ET: +E1[1- exp[ I11 ]+ I40 (10)

in which the ﬁrst part of the sum corresponds to the elastic behavior of the free
spring (E0), the second to the behavior of the combination of the spring and
dashpot in parallel (E1 and I11), and the third to the Newtonian behavior of the
free dashpot (00). Writing Eq. (10) in terms of creep compliance yields

J=f(t)=Jo+JI[1—ex i)]+_t_ (11)
ret I10

50
where Jo = oolEo and J1 = co/EI.

Creep compliance data can also be modeled using an empirical

equation proposed by Peleg (1980):
t
T] = k1 + k2 t (12)

Eq. (12) can be very useful in modeling complexcbiological systems.

' 2.7.3 THERMAL MECHANICAL ANALYSIS

Thermal mechanical analysis (TMA) deforms a sample under a static
load as its temperature is changed. At very low‘loads, it measures the volume
change of the sample with temperature (dilatometry). The applied load can be in
tension, compression or in ﬂexure. Oscillating load studies are performed by
dynamic mechanical analyses (DMA).

Tension loads are used to study ﬁlms and ﬁbers (Grulke, 1994).
According to this author, the equipment measures the linear expansion of the
polymer and works with solid samples only. The volume change of a sample is
an integral change, while the change in heat capacity is a differential change.
The sensitivity of TMA, coupled with accurate temperature control, enables such
factors as volume changes, melting properties and various rheological
parameters to be measured (Ma et al., 1990). According to these authors,
volumetric changes in the substance measured can be Obtained as a function of
temperature or time.

Another advantage of using TMA, is that it can measure the amount of

orientation of polymers, which can be used in turn to optimize the spinning or

51
drawing process (Grulke, 1994). Ma et al. (1990) indicated that in the tension

mode configuration, samples are held in jaws that are attached to the probe,
allowing the examination of ﬁlms and fibers under tension load. According to
these authors, this could be used to measure the tensile strength of films and

other materials commonly used in food packaging.

2.7.4 DIFFERENTIAL SCANNING CALORIMETRY

Differential scanning calorimetry (DSC) is a technique in which the
difference in heat ﬂow between a sample and an inert reference can be
measured as a function of time and temperature as both are subjected to a
controlled environment of temperature, atmosphere, and pressure. This
technique has become an essential tool in measuring and understanding thermal
transitions in polymeric materials (Progelhof andThrone, 1993). A typical DSC
heating curve is seen in Figure 6. The differential amount of energy needed to
maintain the rate is plotted as the abscissa. Either time or temperature is plotted
as the ordinate. These two parameters are related since the hating/cooling rate
is predetermined. Points (2) and (3) represent phase changes or ﬁrst-order
thermodynamic phase transitions (Progelhof and Throne, 1993 and Gruenwald,
1993). These transitions are identiﬁed by discontinuities in the curve. The
polymer at point O has enough molecular mobility that it continues to crystallize.
At point 0), the formed crystallites now begin to melt. Point CD represents a
second-order thermodynamic phase transition known as the glass transition (T,).

This transition is identified by a discontinuity in the slope of the curve.

52

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53
The most important factor that determines the glass transition

temperature of a polymer is backbone flexibility. The phenomenon is observed
mainly in linear amorphous polymers (Nicholson, 1991). According to
Gruenwald (1993), a viscosity of 10‘° to 1012 Pa-s is generally regarded as a
reasonable region to separate liquid from solid behavior, which represents the
characteristic attribute for the glass transition temperature. It is important to
realize that To is neither a thermodynamic transition such as the melting
transition nor a sharp temperature point.

Polymers with ﬂexible backbones have low glass transition temperature
values and the opposite is true for polymers with rigid backbones (Eisenberg,
1993). Steric conformation, that is, hindrance caused by location or size of a
bulky group, can also increase TII. Important for proteins is the fact that an
increase in factors such as molecular asymmetry and polarity of the polymer
structure, are known to cause an increase in T, values (Progelhof and Throne,
1993 and Painter and Coleman, 1994). Also, these authors indicated that
additives and low molecular weight polymers can severely reduce the glass
transition temperature of a polymer. This, based on free volume arguments, is
explained by the increase of the free volume of the system due to these low
molecular weight species, and is assumed that the relationship between the free
volume of the mixture and the components are simply additive. The equation
that relates the TII of the mixture to that of its components, is

1 -mgvz

__ 13
T9 TQ1 T92 ( )

54
where T91 and T92 are the Tg’s of the pure components and W1 and W; are the

respective weight fractions present in the mixture.

Painter and Coleman (1994) discussed the effect of cross-linking
polymeric structures on the T9. They indicated that the effect of cross-linking
can be explained on the basis of simple free volume reasoning. Since parts of
the chain are tied more closely together because cross-linking decreases free
volume, TII increases. Since cross-linking is usually accomplished by the
addition of a speciﬁc cross-linking agent (which can be considered a comonomer
in addition to being a cross-linker), two different effects must be considered: a
copolymer effect, resulting from the incorporation of a second unit, and a cross-
linking effect (Eisenberg, 1993).

T, is marked by a change in the polymer character from a brittle solid to
a rubbery state. Typically the modulus Or stiffness of the polymer drops by a"
factor of a thousand or more (Progelhof 'and Throne, 1993). Table 2 shows
typical values of TI, for several polymers. Rosen (1982) categorizes polymer _
molecular motions as: O vibrations of atoms about equilibrium positions (similar
to crystal lattice vibration except that there is no precise location of the atoms in
amorphous polymers and only an imperfect one in crystalline polymers); O
motion of a few (5 to 10) atoms along the main Chain or rotation and vibration of
side chains and pendant groups; O cooperative wriggling and jumping of
segments of molecules approximately 40 to 50 carbon atoms in length which
permits chain ﬂexing and uncoiling (high temperature creep and second stage

creep are also thought to be examples of this type of motion); and O

55

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56
translational motion (sliding, reptation) of entire molecules, also called flow or

permanent deformation. Below the glass transition temperature, only polymer
motions of type O and O are found. Type O is important between To and
melting temperature (Tm), and type O dominates in the liquid state.

Progelhof and Throne (1993) suggested that because of the nature of
polymer macromolecules, “transitions are not asinstantaneous as they are with
simple molecules.” Therefore, to obtain useful thermodynamic data, the DSC
heating/cooling rate must be sufficiently slow to allow for near-isothermal
transitions. Sperling (1992) suggests that both the sample and the reference
material be heated at a uniform rate between 10 to 20 °C per minute. According
to Progelhof and Throne (1993), the imperfect nature of the polymer structure
allows some void (free volume) around the polymer chains. As the polymer is
heated, the free volume increases and the increased space allows for more
molecular motion. This is important because if an external stress is applied to
the polymer in this state, the molecular motion will be such as to relieve the
stress. Also, T. determination in polymers is important because, as indicated by ‘
Nicholson (1991), the coefﬁcient of thermal expansion, heat capacity, refractive
index, mechanical damping, and electrical properties are among the many
physical properties that change profoundly at the glass transition.

A new technique that can be used to determine the parameters
discussed was recently developed and consists of a variation in the Standard
DSC technique. It is referred as modulated differential scanning calorimetry

(MDSC). This technique provides the same information as conventional DSC

57
plus additional benefits which significantly increase the understanding of

material properties. A heater control and data analysis capability can make two
simultaneous analyses on the same sample. In addition, (a) MDSC furnishes
separation of complex transitions into more easily interpreted components; (b)
has the ability to determine the initial (i.e., prior to heating) crystallinity of a
material; (c) has increased sensitivity for detecting weak T, and T... values; (d)
increases resolution without loss of sensitivity; (e) measures heat capacity and

heat ﬂow in a single experiment and; (f) can measure thermal conductivity.

2.8 QHEMIQAL AND PH Y§IQAL QHARAQTERIZA TION QF POL YMER§

Chemically, two components with similar chemical groups or similar
polarities will generally be compatible and soluble in each other. The free
energy of mixing (Eq. 14) is used to quantitatively predict the solubility between
two components,

AG" = AH" - TASM (14)
where AG" is the change in Gibb’s free energy, AH” is the enthalpy of mixing, T
is the absolute temperature, and AS” is the entropy of mixing.

During mixing, there is always an increase in the entropy of mixing, so
the term TASM is always positive. The Sign of the term 116., indicates whether
or not the solution process will occur spohtaneOusly.” For instance, a negative
value of AG... indicates that the solution process will occur spontaneously. An
ideal solution has a zero heat of mixing (AHM = 0), therefore since the entropy of

mixing is always positive, mixing in all proportions will occur spontaneously.

58
However, in a regular solution (practical solution), the term AHM is larger than

zero, and if the value of AHM is greater than T A8,, then the process will yield a
phase separation. In practice, since the value of AHM Controls the sign of AGM, a
solvent may or may not dissolve a polymer in this type of solution. Rodriguez
(1989) indicated that dissolving a polymer in a low-molecular-weight liquid
causes the random coil to expand and occupy a greater volume than it would in
the dry, amorphous state. If the polymer is composed of single molecules,
viscous ﬂow can occur, and the viscosity will be increased as the polymer
expands. It is expected that when the polymer and solvent have the same
solubility parameter (5), the maximum expansion will occur ”and therefore the
highest viscosity (for a given concentration) will be obtained. If the polymer
comprises a cross-linked network, a solution cannot occur, but individual parts of
the polymer Chains (polymer segments) can solvate to give a swollen gel.
Nicholson (1991) indicated the importance of the swelling experiments, since
this behavior depends on the nature and extent of interchain covalent bonds.
For instance, an non-cross-linked polymer will usually dissolve in an apprOpriate
solvent, given appropriate polymer-solvent compatibility and sufﬁcient time. By
contrast, Nicholson (1991) also indicated that cross-linked polymers will not
dissolve. However, these materials will swell and become softer by admitting
solvent. Birley et al. (1992) suggested that, for uncross-linked polymers, the
swelling effect is seen as a breakdown of intermolecular bond attraction due to

the presence of the migrating species. However, these authors also indicated

59
for the cross-linked polymers this effect is reduced, so swelling experiments are

a useful empirical tool for determining the degree of cross-linking. Such swelling
is generally reversible Nicholson (1991) and, given appropriate conditions,
solvent that has entered a cross-linked structure can be removed and the

polymer will return to its original size.

2.8.1 SWELUNG OF POLYMER NETWORKS

When a piece of a cross-linked polymer is put in contact with a
compatible solvent, a swelling process occurs. Mark (1993) indicated that this
process is a three-dimensional dilation in which the polymer absorbs solvent and
reaches an equilibrium degree of swelling. At equilibrium, the free energy
decrease due to the mixing of the solvent with the network chains is balanced by
the free energy increase due to the stretching of the chains. The theoretical
extent of swelling is predicted by the F lOry-Rehner theory on the basis of the
cross-link density and the attractive forces between the solvent and the polymer
(Sperling, 1992). Forces considered arise from three sources: (a) the entropy
change caused by mixing polymer and solvent (the entropy change from this
source is positive and favors swelling); (b) the entropy change caused by
reduction in numbers of possible chain conformations on swelling (the entropy
change from this source is negative and opposes swelling); and (c) the heat of
mixing of polymer and solvent, which may be positive, negative, or zero (usually,
it is slightly positive, opposing mixing).

In a cross-linked polymer, the best solvent is deﬁned for the purposes

of the experiment as the one with the closest solubility parameter. This solvent

60
also swells the polymer the most. The swelling coefficient, Q (ml 9"), is defined

by

 

$0110)
mo 95

where m is the weight of the swollen sample, mo is the dry weight, and p. is the

density of the swelling agent.

According to Grulke (1994), mixed solvents can be treated as a single
solvent by determining the solubility parameter of the solvent mixture. However,
if both the solvents and the polymer interact, then the description becomes more
complicated. Rodriguez (1989) and Sperling (1992), have indicated that liquids
with like solubility parameters are apt to dissolve the same solutes and to be
mutually compatible. ' .

Solubility data are useful for determining chemical resistance of plastic
materials, due to the fact that a solvent (i.e. organic media, polyelectrolytes, etc.)
can penetrate the amorphous phase of. the polymer investigated. Materials
manufacturer’s data sheets often deﬁne chemical resistance in an arbitrary
manner (Birley et al., 1992), which can be useful ifuthe precise experimental
constraints (reagent concentration, temperature, etc.) are deﬁned. Yeo (1986)
peforrned swelling experiments of perfluorosulfonic acid membranes using
various solvents, with a range of solubility parameters of 7.4 (triethyl-amine
solvent) to 23.4 [cal cm"]" (water solvent). He found that both the sample

pretreatment and the functional groups present in the membranes had a

61
significant effect on their swelling properties. He also observed an increase in

the swelling of the membranes with an increase in the solvent temperature.
Chatterji (1989) studied the effect of swelling on gelatin networks. He
also pointed out that swelling behavior is “entirely determined by the chemical
nature of the chains.” The gelatin macromolecules were formed by reacting
gelatin chains with glutaraldehyde, which yielded an insoluble random protein
network. This author indicated that swelling behavior can be used to
differentiate cross-linked copolymers from one another. Among the gelatin
samples, he reported that when gelatin was not cross-linked, it dissolved in
water but did not Observe any uptake when methanol or benzene/T HF
(tetrahydrofuran) were used. This same behavior was Observed by

Satyanarayana and Chatterji (1991).

2.8.2 FOURIER-TRANSFORM INFRARED SPECTROSCOPY

Fourier-Transform Infrared SpectroscOpy (FTIR) has been recognized
as an important tool for studying spatial Chain conformation and structure of
proteins (Wang and Smith, 1994 and Creighton, 1993). Garton (1992) published
a comprehensive review on FTIR as applied to polymeric materials. This. author
explained the important operational parameters that a spectrometer must have
to produce quality data. Among them, .apodization (function introduced to
truncate the data when spectra are to be compared); spectral range resolution;

reproducibility; mirror velocity; phase correction; and Fourier deconvolution.

62
According to Garton (1992), the frequency of an absorption band in the

infrared spectra is usually expressed in terms of wavenumbers (0), whose unit is
the reciprocal centimeter (cm"). The wavenumber is the reciprocal of the
wavelength (7).) in centimeters, or when the wavelength is in microns, the
wavenumber is expressed by Eq. 16

1x10“
0:
I.

 

(16)
u

where wavenumber (v) is in cm" and A, is wavelength in microns.
According to quantum theory, the frequency (v’) in cycles/second of a

photon is proportional to its energy (E) in so that
E = h u’ (17)

and the frequency (0’) is therefore related to the wavelength (7).) in cm and to the

wavenumber (u) in cm" as shown in Eq. 18:
u = — = no (18)

The useful area in the infrared spectrum for analyzing polymeric
materials using FT IR is between 4000 cm" and 600 cm" (2.5 - 16.6 It). In
addition, identiﬁcation of polymer samples can be made by making use of the
“ﬁnger-print” region, where it is least likely for one polymer to exhibit exactly the
same spectrum as another (Cowie, 1991). This region lies within the range 1500
to 800 (6.6 to 12.5 p). Garton (1992) indicatedthat the energy involved in an

infrared (IR) transition is insufﬁcient to break most chemical bonds, but it is

63
enough to impart bond deformation associated with a vibrational transition.

Giacin (1995) indicated that in molecular absorption of radiant energy, several
components must be considered, which sum for the total quantized energy levels

within the molecules:

Etotnl = Emu 1' Evtbmttomr 1' Emotional 1' Etnnslational (19)

Translational energies can be disregarded since radiation energy is not
imparted through spectroscopy. Also, the energy difference between electronic
energy levels is greater than that for vibrational energy, which are greater than
those for rotational energy (Giacin, 1995). In addition, the energies associated
with vibrational and rotational changes are comparable to those for infrared
radiation, and it is the vibrational and rotational energy changes which are
measured in the infrared region. Gruenwald (1993) indicated that, depending on
their chemical makeup, each speciﬁc chemical group possesses characteristic
absorption bands making it possible to readily identify the chemical composition
of any plastic. It should be emphasized that motion is being described for the
entire molecule, and not simply bending or stretching of one set of bonds.
Therefore, what is observed on the atomic level (although quantum restrictions
prevent from doing so) would be superposition of the many normal modes of

vibration (Garton, 1992).

Giacin (1995) points out some criteria for obtaining an accurate
interpretation of an infrared spectrum:

1. The spectrum must be adequately resolved and of adequate intensity;

64
2. The spectrum should be of a reasonably pure compound;

3. The spectrophotometer should be calibrated so that bands are
observed at their proper frequencies or wavelengths;
4. The method of sample handling must be specified.

There are two important areas for examination of an infrared spectrum,
the region above 1300 cm", and the 909-650 cm" region. The higher frequency
' portion of the infrared spectrum is called the functional group region. The
characteristic stretching frequencies for important functional groups such as OH,
NH, CN and C=O occur in this region. The other region (909-650 cm") provides
with information about aromatic or heteroaromatic compounds. These low
frequency bands are due to strong out-of-plane C-H bending and ring bending
modes (Giacin, .1995).

The intermediate portion of the spectrum, 1300-909 cm", on the other
hand, is usually referred to as the “ﬁngerprint” region. Absorption in this
intermediate region is unique for every molecular species (Giacin, 1995). Some
typical functional groups with their absorption frequency ranges are presented in
Table 3. .

Satyanarayana and Chatterji (1991) observed that the IR spectra for
three different polymeric materials: cross-linked gelatin (Gel-x), polymethyl
acrylate (PMA), and cross-linked gelatin with polymethyl acrylate grafts (Gel-
xIPMA). They Observed an absorption band at 1640 cm" which they attributed
to the amide and aldimine linkages. They also observed an absorption peak.

between 3200-3500 cm" which was attributed to N-H stretching and another for

65

Table 3. Some typical group stretching vibrational absorption frequencies for
polymer analysis.“

 

Functional Group

Structure Found

Absorption Frequency (cm")

 

Hydroxyl (O-H)

Amino (N-H)

33037113365977"
bond (C-H)

-—---—---------—‘b

Nitrile (CEN)
2899191819. ﬂag-19)..

Carbonyl (C=O)

Halide

-------—----------—-

A

. Alcohols, ROH

2. Phenols, ROH
(where R = aromatic)

Acids, R-COOH
Amine, R-NH-

Amide, R-CONH;
Amide, R-CONH-

(where R = aromatic)
R-CHz'H . I
(where R= aliphatic)
10. R-C= N

11.5:82929 ..........
12. General Region

13. Aldehyde, R-COH

14. Ketone, R-CO-R
aliphatic and saturated

15. Carboxylic acid,
R-CO-OH
Monomer
Dimer

16. Ester, R-CO-OR
saturated

17.Amide, R-CO-NH;

 

 

3650-3584
3546-31 94

3300-2500

3500-3300 (two bands for
primary, one band for
secondary)

3521-2941
N-H @ 3450; amide I @

3000-2840

2962-2872

1 870-1 540
1 740-1 720
1 720

1760
1720-1706
1 751 -1 736

1 650-1 689

C-H @ 3030; C=C @ 1640
(intensity varies with

 

f' Modiﬁed from Giacin (1995) and Garton (1992)

66
N-H deformation at 1540 cm". They also reported that the peak observed at

about 2920 and 1440 cm" was due to the C-H stretching and deformation,
respectively. They attributed the appearance of an absorption band at 1740 cm'1
to the grafting of the polymer during its formation. Chatterji (1989) analyzed
these same polymeric materials and indicated that the characteristic absorption
of the backbone peptide bond occurring at 1540 and 1650 cm" were the only
distinguishing features of the gelatin. However, this author attributed the extra
absorption peak observed for the cross-linked gelatin at 1450 cm" to the
aldimine linkages, as opposed to 1640 cm" by Satyanarayana and Chatterji
(1991).

Wang and Smith (1994) studied the effect of isothermal heating on
spectra for chicken breast myosin. The IR spectra was useful to investigate
unfolding of the proteins. They found that. irregular structures were formed and
were accounted for by irreversible unfolding of helices and B—structures which
occur when the protein was heated at 55 °C. It is important to note that these
authors worked with a very pure protein system, myosin from chicken breast,
thus making the IR analysis very suitable according to the guidelines of Giacin

(1995) explained above.

2.8.3 PLASTIC/FILM COLOR MEASUREMENTS

Color was deﬁned by Francis (1985) as a term to denote the human
eye’s perception of colored materials. Another definition by Progelhof and
Throne (1993), is “color is the property of radiant energy that permits a living

organism to distinguish by eye between two uniform, structure-free patches of

67
identical size and shape. For instance, color of a protein-based ﬁlm would have

a significant effect on the acceptability of such film as “edible” by the population,
due to several factors, including cultural, geographical, and sociological.
According to Francis (1985), “certain food groups are acceptable only if they fall
within a certain color gamut.” This may be applicable to edible films because
they could be used in the preparation of foods, especially when used as coatings
or wrapping materias. I

Gruenwald (1993) indicated that accurate measurement of color is
important in plastic materials so the manufacturer can offer uniform colored
products. For this purpose, he pointed out that there are several methods to
physically describe any colored surface. In these systems, as Gruenwald (1993)
indicated, three numbers must be designated: the hue (the dominant color
relating to the whole visible spectrum); the chroma (indicating the intensity or
purity Of the color); and the value (representing the lightness or superimposed
gray scale of the color, with pure black and white at Opposite poles on the color
sphere). Color matching involves establishing the X," Y, Z coordinates for two
specimens, then determining the length of the vector between the two coordinate
points. Progelhof and Throne (1993) emphasized that nowadays computers are
used to determine the vector length in a chromaticity diagram. They indicated
that the units are always listed as CIELab units [“ClELab” refers to Commission
lntemationale de I’Eclairage Laboratory, an international laboratory that

established the unit system in 1931].

68
The importance of these values is of greatest interest when differences

among plastics can be observed. Examples include those of GPPS and PMMA
which are water-white transparent. Others such as polyethylene are translucent.
Some are milky white and nearly opaque, like polyacetal and nylon. Others are
off-white and opaque, like plasticized PVC and ABS. Some are hay- or straw-
colored, such as polyimides and polyphenylene sulﬁde. Polysulfones are
amber. And some, such as polyamide-imide, phenolic and resorcinol are cherry
red when ultra-pure. Progelhof and Throne (1993) indicated that polymers are
quite easily colored and so provide products that have the color “all the way
through.” Color measurements are very important for assuring a consistent color
of the plastic products. In addition, Gruenwald (1993) also indicated the
importance of the product lighting condition on acceptability. Lighting may
simulate daylight, ﬂuorescent, or incandescent light, and pigments can be
applied for products to match a speciﬁc color. -

More speciﬁcally, an example of the importance of color related to
proteins include those changes observed by Chatterji (1989) in the color of
gelatin granules from pale yellow to deep orange within minutes upon cross-
Iinking treatment of the gelatin with glutaraldehyde. This author attributed the
color change to the establishment of aldimine (Schiff) linkage between the free
amino groups of the protein and the glutaraldehyde (Figure 1). The
measurement of color in edible ﬁlms is important since that will be the ﬁrst

contact of the consumer with the new ﬁlm/coating material.

3. MATERIALS AND METHODS

QLELQIJBﬁAMELES

The ﬂours used in this study included three types:

a) commercial bread ﬂour (C), bought from a commercial store in
Lansing. Label stated it was “Bakers and Chefs” brand, an
“enriched, bleached bread ﬂour, blend of hard spring and winter
wheat class ﬂours," distributed by North Arkansas Wholesale Co.
Inc. (Bentonville, Arkansas);

b) hard red winter ﬂour (H) class (King Milling Co., Lowell, Michigan);

c) soft white ﬂour (S) class (King Milling Co., Lowell, Michigan).

The basis for the use of these three ﬂours was the difference that a
commercial (usually blend of different classes of wheat ﬂours and added vital
wheat gluten to it) versus pure-class wheat ﬂours may have on the ﬁlm
performance. Also, hard versus the soft classes represent unique groups since
generally these classes are used in different food applications (Hoseney; 1994)
due to their distinct physical and chemical characteristics. In addition, the soft
wheat class is of particular interest since over 70 percent of Michigan’s wheat is
white grained. White wheat bran is in far greater demand than red wheat bran
due to ﬂavor an color problems associated with the red bran pigments after
processing. White ﬂour can be used in the production of edible and/or

degradable ﬁlms, among other industrial uses.

69

70
The flours used in this study were first characterized for protein

content, moisture content, falling number, farinography, and by Sodium Dodecyl
Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), under reduced and
unreduced conditions. SDS-PAGE was done to confirm that there were
differences between each ﬂour so that the films produced from them may show

differences in the properties to be measured.

3.2 FLQQR QHARA C TERIZA TIQN

The ﬂours were characterized on the basis of protein content, percent

moisture, falling number, farinography, and SDS-PAGE.

3.2.1 PROTEIN CONTENT

The protein of the ﬂours was determined according to an American
Association of Cereal Chemists approved micro-Kjeldahl standard method AACC
46-13. A Tecator 1008 Control Unit with a Digestion System 40 1006 Heating
Unit (Silver Spring, Maryland) was used to digest the ﬂour samples following
distillation using a Kjeltec System 1002 Distilling Unit (Silver Spring, Maryland).
Finally, a titration of the extracted samples was performed using hydrochloric

acid. The % nitrogen was then calculated according to the following equation:

(ml HCI sample - ml blank) x N HCI x equiv wt N X 100

(20)
wt of sample (mg)

% Nitrogen =

 

where: ml HCI sample = milliliters of HCI used in titration of sample; ml
blank = milliliters of HCI used in titration of a blank (no ﬂour sample); N HCI =

normality of HCI used for titration; and equiv wt N = number of equivalent

71
weights of nitrogen. From this, the crude protein (%) was calculated by the

following expression:

% Crude Protein = (% Nitrogen) X (5.7) (21)

where: 5.7 represents the factor obtained by dividing 100 mg protein 1 17.54 mg
nitrogen (for wheat).

The perCent crude prOtein was then adjusted to 14 % moisture basis
' (m.b.) for all the flour samples, since this basis is the most commonly used in the
United States (Pomeranz, 1987). The adjustment was done according to the

equation in AACC method 46-14A (Crude protein _- Udy Dye Method):

 

100-M
% rote'n °/ m.b. =‘V rot. measured (22)
p | (o ) Op [100%-%moisture measured]

where M represents the desired moisture basis (wet basis).

3.2.2 FLOUR MOISTURE

The water content in the ﬂour was deterwned according to AACC
Method 44-15A. This method required a two tothreegram portion with a tared
moisture dish. Five tared moisture dishes (replications) for each ﬂour were used
in this determination. Once the ﬂour samples were weighed, they were heated
for exactly 60 minutes in an oven held at 130°C (01°). The dishes were removed
from the oven and transferred to a desiccator as quickly as possible. Weight of
dishes was determined after they reached room temperature.

The % moisture was calculated by use of the following equation:

% moisture = (ijﬁ 00) (23)
3w

72
where M, represents moisture loss and SW is the original weight of the sample.

The % moisture values were used as a basis for the adjustments in the
quantities of flour used in the preparation of the films, to assume consistent dry

weight values.

3.2.3 FALLING NUMBER

The Falling Number was determined using a Falling Number Apparatus
Type 1400 (Perten Instruments, Huddinge, Sweden), which conforms with AACC
Method 56-81-B. In general, Falling number determination on flour falls from 0-
30 units higher than that for the corresponding grain. This methodology required
that the weight of the ﬂour samples be corrected to a 14% moisture content

according to Table A1 in the Appendix A.

3.2.4 FARINOGRAPHY

A Farinograph (C.W. Brabender Instruments, Inc., South Hackensack,
USA.) composed of a dynamometer type PL-2H and a measuring head type S-
300 was used to evaluate the ﬂow behavior of the ﬂour.

A sample of 300 g for each type Of ﬂour used in this study wasiplaced
inside the chamber of the measuring head and water was added continuously so
that mixing followed, and gave, a maximum peak value. Titration followed until a
curve with a peak value of 500 120 BU was obtained. A horizontal line crossing
half way between the lowest and the highest value at the peak was drawn and
calculations of the percent water absorption, dough development time (mixing
time), mixing tolerance index (MTI), and stability were calculated for each

farinogram obtained.

73
3.2.5 SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) was done according to the technique described by Ng et al. (1986) to
determine differences in the chemistry of the proteins present in the three
experimental flours. This technique consisted of extracting the protein from the
ﬂour by adding extracting buffer solution to the ﬂour sample. The extracting
buffer solution contained mercaptoethanol (ME), which is known to reduce the
disulﬁde bonds Of wheat proteins. For the unreduced samples, no ME was
added to the solution. These mixtures were heated in a boiling water bath for
about 2.5 minutes. Polymerization of both, separating-gel and stacking-gel,
were accomplished separately. Once the gels solidiﬁed and were ready for the
sample application, eight ul of the clear extract from the ﬂour mixture were
deposited into each slot of the stacking-gel. Also ﬂour from Neepawa (pure
variety), which is a Canadian cultivar, was prepared and included as a standard
for the electrophoretic analysis. A d.c. electric current was passed through the
gel and separation Of the protein subunits was achieved after about 24 hours.
After the current was stopped, and the electrophoregram was developed by
rinsing the gel with a rinsing solution. Staining the gel followed by covering the
gel with a staining solution which contained commasie brilliant blue and gently
shaking it overnight by sitting the gel on a variable speed shaker. Destaining
the gel was accomplished by rinsing the staining solution out and putting in a
new rinsing solution for a few hours, after which a ﬁnal rinse with water was

done. A photograph of the gel was taken for later analysis.

74
3.3 FILM PREPARATION

A new procedure for film formation was developed based on utilizing
the flour as the raw material and directly extracting the protein fraction into a
solution. A schematic representation of the general process is depicted in

Figure 7.

3.3.1 PROTEIN SOLUBIIJZATION

The general procedure is described below, with variables that will be
discussed for each particular ﬁlm in the next section.

To approximately 150 g of wheat ﬂour (actual quantity adjusted based
on protein present with a 14% moisture content for the particular ﬂour) 360 ml of
a 70% ethanol solution was added and mixed using a magnetic stirrer (Fisher
Scientiﬁc Co., Pittsburgh, USA.) to form a slurry. Agitation sufﬁcient to form a
vortex in the mixture continued under magnetic stirring for ‘16 hour at room
temperature. Two different basic procedures, low and high pH, were used. For
the low pH procedure, an adjustment-of the pH to a value of 4.0 was done using
acetic acid (glacial), and for the high pH procedure, an adjustment to a value of
11.0 was done using a GM solution of sodium hydroxide (NaOH). The pH was
measured using a Fisher Accumet pH Meter model 810 (Fisher Scientiﬁc,
Pittsburgh, USA.) Mixing continued for one more hour at 40°C. Centrifugation
of the slurry followed at 10,500 x gravity (9) using a Sorvall Refrigerated

Superspeed Centrifuge model RC-5B (Du Pont Co., Newton, CT, USA.)

75

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76
The Relative Centrifugal Force (RCF), “g,” was determined according

to the following equation' :

 

1/2
n = [(RCF)(10002 )] (24)
(1 117)(r)

where n is the rotor ‘speed (rpm) and r is the radius from the centerline of the
rotor to the point in the tube where RCF value is required. The rotor used with
the centrifuge was a Sorvall GSA superspeed ﬁxed-angle rotor. For this rotor,
the maximum radius has a value of 14.57 cm, and the calculated rotor speed
was 7011 rpm (734 rad/s).

After the centrifugation step, the supernatant contained solubilized the
proteins that were responsible for ﬁlm formation. This supernatant was deﬁned
as the “ﬁlm forming solution,” FFS, since by just letting it evaporate on a ﬂat
surface it was capable of forming a ﬁlm. The FFS was then decanted from the
precipitate and poured into a 170 x 90 crystallizing dish (Fisher Scientiﬁc 00.,

Pittsburgh, USA).

3.3.2 PROTEIN CROSS-LINKING

Films in which cross-linking of the proteins was required were
produced by adding one of the following reagents to the FFS: a) 15 ml 0.1 M
cysteine (Sigma Chemical Co., St. Louis, MO, USA); b) 0.75 ml of formaldehyde

solution (Mallinckrodt Specialty Chemicals Co., Paris, KY, USA); c) 0.75 ml of

 

' Modiﬁed from Du Pont (1980).

77
glutaraldehyde solution (Aldrich Chemical Co., Milwaukee, WI, USA). The

addition of these reagents was done slowly after boiling the FFS for about 5
minutes, except for the glutaraldehyde solution which was added just prior to
casting the ﬁlm. This was done to avoid an excessive change in color from the
typical light-yellow-brownish (obtained for all ﬁlms produced without cross-linker
and those produced using cysteine or formaldehyde) to brown-reddish of the

ﬁlms produced in using glutaraldehyde.

3.3.3 FILM FORMATION

Glycerol, the plasticizer, was added to each FFS. This polyalcohol was
added in a concentration of 3.6 g/150 g (14% me.) ﬂour. Evaporation of the
solvent was permitted to continue. After about 75% of the original volume was
evaporated, the solution was poured into a heated PyrexTM glass funnel. This
step was done to avoid bubbles from entering a thin-layer chromatography (TLC)
spreader with layer thickness regulator model # 611 (Desaga Co., Heidelgerg,
Germany) when the solution was transferred to it via a Tygon’ hose connected
to the funnel (see Figure 8). The thickness in the TLC spreader was set at 0.75
mm. Spreading of the solution was done by moving the TLC spreader across
the glass surface.

Films were dried for 48 hours in a Forced Air lsotemp Oven model 737-
F (Fisher Scientiﬁc 00., Pittsburgh, USA.) set at 45°C. Films were then placed
in a room with controlled temperature (25 11°C) and relative humidity (12 11%)
for 24 hours. The relative humidity was measured using a HygrocheckT'“

hygrometer (Hanna Instruments, Woon Socket, USA.) Films were then peeled

78

020.300. 0005.25 53> 0000050 04... 2 02560 mc_F:o._-EE ho 000000.... .0 059“.

 

000.0

 

000200 mc_E._oc_-E__n_ \

79
off the glass surface and put, depending on the specifications of the film, into

closed containers with controlled relative humidity for 72 hours so that the film
equilibrated with this environment. The analytical tests were then performed on
the ﬁlms. Films based on the above procedure with different variables were
produced and studied. The conditions and ﬁlm fabrication processes for each
test were selected based on the variable combinations considered most

important for obtaining and influencing results.

3.4 ANALYTIQAL TE§T§ QF FILMS

3.4.1 BARRIER PROPERTIES

3.4. 1.1 Omen Permeability
Oxygen permeability was determined for selected ﬁlms (Table 4)

prepared according to the general procedure elucidated above. Two 11 x 11 cm
samples of the ﬁlm to be tested were cut and each masked with an aluminum foil
mask (Mocon, Inc, Minneapolis, MN., USA.) so that a circular ﬁlm area of 5 cm2
was tested. The masked ﬁlms were placed in a 50 cm’ cell of an Oxtran 200
permeability tester apparatus (Modern Controls, Inc, Minneapolis, MN... USA.)
equipped with an Endocal temperature control bath model RTE 100 (Neslab
Instruments, Inc., Newington, USA.), which conforms with standard method
ASTM D-3985.

The tests were run at 0% relative humidity at temperatures of 10, 25,
and 40°C using nitrogen (containing 2% hydrogen) as carrier gas and air (21%

oxygen) as test gas. The oxygen permeability (P) is reported in m3 m rn'2 5'1 Pa‘1

80

Table 4. Films produced and tested for oxygen permeability
and ultimate tensile properties in this study.

 

 

 

 

 

 

 

 

 

 

 

Film Flour‘ pH Cross-linker
1 C 4.0 none
2 C 1 1.0 none
3 C 1 1.0 cysteine
4 H 4.0 none
5 H 4.0 cysteine
6 H 1 1.0 none
7 S 4.0 none
8 S 4.0 cysteine
9 S 1 1.0 none

 

 

 

 

* C = commercial bread ﬂour; H = hard red winter flour;
8 = soft white flour

 

81
for all films by converting the oxygen transmission rate values (J) obtained from

the instrument (co m2 day") by the use of the appropriate conversion units and

the following equation:

F - if. (25)
AP

3.4.2 RHEOLOGICAL METHODS
3.4.2. 1 Mechanical Properties

Two techniques to determine mechanical properties of the ﬁlms were
used: ultimate properties (in tension mode) and tensile creep. Ultimate
properties include tensile strength, or ultimate strength, which is the tensile
stress at or near failure (Rodriguez, 1989) and elongation or engineering strain,

also known as Cauchy strain (Steffe, 1992).

3.4.2.1.1 Tensile Strength
The ﬁlms (Table 4) were analyzed using an Instron Model 4201

equipped with a 1 kN static load cell (Canton, USA.) This test was done in
conformance to test method A (static weighing, constant-rate-of-grip separation
test) of the standard ASTM method 0882-91 (ASTM, 1991a). This method
employs a constant rate of separation of the grips holding the ends of the test
specimen. The extension of the specimens may be measured in theSe test
methods by grip separation, extension indicators, or displacement of gage
marks. The values stated in SI units are to be regarded as the standard.
Tensile properties measured by this method may vary with specimen thickness,

method of preparation, speed of testing, type of grips used, and manner of

82
measuring extension. Consequently, these factors were carefully controlled.

Results may be utilized to provide data for research and development and
engineering design as well as quality control and specification. The tensile
modulus of elasticity (E) is an index of the stiffness of thin plastic sheeting. The
method specifies that when different materials (i.e., ﬁlm processes) are tested
and compared, specimens of identical dimensions must be employed.

The width of the ﬁlm samples were cut to 25.4 mm (1 in) using a JDC
Precision Sample Cutter model 25 (Thwing Albert Instrument Co., Philadelphia,
PA, USA) and the edges were cut with scissors to about 200 mm of length. The
separation between the grips of the Instron was set to 50.8 mm (2 in). The cross
head speed for the test was set to 508 mmlmin (20 inlmin). The method
indicates that a width-thickness ratio of at least eight shall be used. This is
because narrow specimens magnify effects of edge strains or ﬂaws, or both. It is
recommended that the utmost care be exercised in cutting specimens to prevent
nicks and tears which are likely to cause premature failure. Also, the edges
shall be parallel to within 5% of the width over the length of the specimen
between the grips. All ﬁlms were conditioned at 2:1 °C with a 5012% relative
humidity for 48 hours, and testing was performed at these environmental,

conditions.

3.4.2.1.2 Tensile Creep
The ﬁlms were analyzed using an apparatus fabricated in our

laboratory in which ﬁlms were gripped between two sets of aluminum plates so

that the upper plates were immobilized and the IOwer plates had a known weight

83
attached to them. The measure of strain was determined over real time by

mounting a video camera to the apparatus while having a constant stress
applied on the film (see Figure 9).

The geometry of the ﬁlm samples for the creep tests conformed to type
M-II tension test specimen specifications (Figure 10), as described in the
standard method ASTM D-638M-91a, “Standard Test Method for Tensile
Properties of Plastics (Metric)“ (ASTM, 1991b). The grip separation was set as
indicated by the specimen speciﬁcation standard at 80 mm. Creep tests were
performed according to the standard method ASTM D-2990-91 (ASTM, 1991c).
This method indicates that for measurements of creep-rupture, tension is the
preferred stress mode, and the values stated in SI units are to be regarded as
standard. This method consisted of measuring the extension as a function of
time and time-to-rupture, or failure of the specimen subjected to constant tensile
load under the speciﬁed environmental conditions. The method explains that
data from creep and creep-rupture tests are necessary to predict the creep
modulus and strength of materials under long-term loads and to predict
dimensional changes that may occur as a result of such loads. These data can
be used for many purposes: (1) to compare materials; (2) in the design of
fabricated parts; (3) to characterize ﬁlms for long-term performance under
constant load; and (4) under certain conditions, for speciﬁcation purposes.

Film specimens were put into a chamber maintained at the appropriate
temperature and relative humidity for at least 40 hours so that the ﬁlms

equilibrated with that environment. Analytical tests were then performed on the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

upper
lates
./ p
‘_______..—-—i
_ ‘ ﬁlm
g _ ____,./ sample
é _ . lower
1 ____..L/ plates
_ * wei ht
5 if <————-—-‘// 9
E millimetric
g // ruler
: __,__/ cushion

 

 

 

 

 

 

 

 

 

 

 

 

T T

Figure 9. Diagram of tensile creep machine made for this study.

85

4— 25+

 

 

 

 

 

 

 

v,
115 ->6<— :3
/\

 

 

 

Figure 10. Type M II tension test specimen dimensions (mm).
[shown to actual size]

86
films using a creep apparatus located inside the environmental chamber.

Different ﬁlms, produced with their respective variables used for measuring
creep, are presented in Table 5. The reduction in cross-sectional area during
the creep experiment for all films was adjusted to obtain more accurate stress
values. This adjustment was calculated on the basis of a linear relationship of
the cross-sectional area as a function of the differential displacement (6L)
observed. Figure 81 (Appendix) shows the linear relationship and the equation

obtained for performing this adjustment.

3.4.2.2 Film Characterization

3.4.2.2.1 Thermal Mechanical Analysis
Thermal mechanical analysis (TMA) was used with the intention of

determining the coefﬁcient of expansion and T, of selected ﬁlms in Table 6. A
then'no-mechanical analyzer model 943 (Dupont Instruments, Wilmington, DE.,
USA) located in the Composite Materials and Structures Center Laboratory at
the Engineering Research Complex, Michigan State University, was used. The
method used was as follows: (1) equilibrate at -80 °C; (2) ramp 10.00 °Clmin to

100 °C.

3.4.2.2.2 Modulated Differential Scanning Calorimetry
Modulated differential scanning calorimetry (MDSC) technique was

used to examine ﬁlms. The apparatus was a Modulated DSC instrument model
DSC 2920 (TA Instruments, Inc, New. Castle, DE., USA) located in the

Composite Materials and Structures Center Laboratory at the Engineering

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88

Table 6. Films produced and examined by thermal mechanical
analysis (TMA) and modulated differential scanning
calorimetry (MDSC) techniques.*

 

 

 

 

 

 

 

 

 

 

 

 

 

Film # pH Cross-linker
1 4.0 None
2 4.0 Cysteine
3 4.0 Formaldehyde
4 4.0 Glutaraldehyde
5 1 1.0 None
6 1 1.0 _ Cysteine
7 1 1.0 Formaldehyde
8 1 1.0 Glutaraldehyde

 

* Commercial bread ﬂour was used for their fabrication

89
Research Complex, Michigan State University. This apparatus was equipped

with a refrigerated cooling system and a heater control. Data analysis capability
made two simultaneous analyses on the same sample in an attempt to determine
glass transition temperature (T,) and melting temperature (Tm). Various film
samples (Table 6) were analyzed.

Films were fabricated and the samples kept in a desiccator containing
Drierite (W.A. Hammond Drierite Co., Xenia, OH. USA) until tested. The method
used was as follows: (1) equilibrate at -55 °C; (2) modulate +l- 1.000 °C for 5.00

min; (3) isothermal for 5.00 min; (4) ramp 5.00 °Clmin to 184.70 °C.

3.4.3 SWELLING STUDY

Determination of the swelling coefﬁcient, Q, was performed for ﬁlms
shown on Table 7. Pieces of ﬁlm weighing between 0.2 and 0.4 g were cut from
the ﬁlms studied. Three replications were made for each ﬁlm. Five different
solvents were used: (1) n-butyl alcohol (Mallinckrodt, lnc., Paris, KY); (2) hexane
(EM Industries, Inc, Gibbstown, NJ); (3) distilled water (obtained from the
laboratory); (4) 70% ethanol (prepared from 95% ethanol bulk-obtained from
general stores, MSU), with a pH adjusted to 4 using glacial acetic acid; and (5)
methanol (J.T. Baker, lnc., Pillipsburg, NJ).

The ﬁlms were kept at 55 °C for 24 hours in a forced-air lsotemp oven
model 737-F (Fisher Scientiﬁc, Pittsburgh, \PA) prior to immersion in the
solvents. Weight of the ﬁlms (before immersion) were recorded. 250 ml of each

solvent was placed in a 400 ml beaker and three pieces of ﬁlm (replications)

90

Table 7. Films produced" for determination of swelling
coefﬁcient (Q), analysis by Fourier-Transformed
Infrared Spectroscopy (FTIR), and measurements of
color by ClE-Lab system.

 

 

 

 

 

 

Film # Cross-linker
1 None
2 Cysteine
3 Formaldehyde
4 Glutaraldehyde

 

 

 

* Commercial bread ﬂour was used for their fabrication; low pH
process (4.0) was used

91
were added. The polymer-solvent system was gently-stirred continuously for 24

hours at 23 °C using a magnetic stirrer.

After this time, the ﬁlms were removed from the solvent and placed in
between two circles of Whatman paper #42 (ashless) and a book (about 600 g)
was placed on top. This was done for 1 minute to remove the excess solvent
from the surface of the samples. The weight of the ﬁlm was recorded by placing
the swollen ﬁlms on previously tared aluminum plates. Films were then dried for
24 hours at 55 °C. The weight of the ﬁlms were then recorded again. The

swelling coefﬁcient (Q) was calculated according to Eq. 15.

3.4.4 FOURIER-TRANSFORM INFRARED SPECTROSCOPY

The Fourier-Transform Infrared Spectra were obtained by using a
Perkin-Elmer FT -lR Spectrophotometer model Paragon 1000 (Perkin-Elmer
Limited, Beaconsﬁeld Bucks, England) located in the Engineering Research
Complex, MSU. The spectra were obtained for the wavenumber range between
4000 cm‘1 to 600 cm". Each spectrum represents the average of 16 runs and
the data were collected at room temperature using a very thin ﬁlm deposited on
the surface of potassium bromide (KBr) tablets. The tablets were fabricated from
approximately 300 mg of Kbr by placing the powder into a metallic cylinder and
holding it at nine tons of pressure for 3 minutes using a Carver Lab. Press model
SP-F 6030 (Menomonee Falls, WI, USA). The tablets were prepared within 24
hours of using them in the FT-IR spectrophotometer. The dimensions of the
tablets were approximately 12.8 mm diameter and 1 mm thickness. Two drops

of FFS were deposited on the surface of a tablet for each ﬁlm studied and

92
allowed to dry for 12 hr at room temperature. The films studied are presented in

Table 7.

3.4.5 COLOR STUDY

The physical measurement of color for the ﬁlms listed on Table 7 was
done using a Color Hunter Lab instrument model 45/0 ColorQUEST (Hunter
Associates Laboratory, lnc., Reston, VA. USA) located in the Permeation
Laboratory in the School of Packaging, MSU. ClE-Lab [CIE are the French
initials for “Commision lntemationale de ‘Eclairage”] values for “L”, “a”, and “b”
were obtained. The ﬁlms were conditioned overnight to 50% relative humidity.
prior to testing. Five replications were performed and both, values in the
reﬂectance (with black background) and transmittance (with white background)

modes, are reported.

3.5 STA TISTIQAL ANALY§IS

The numerical results were analyzed by either one-way or two-way
analysis of variance, and the comparisons among different factors were
investigated according to either of one of the Tukey’s or Student-Neuman-Keul’s
multiple comparison tests. MSTAT-C statistical program version 2.10 (Michigan
State University, East Lansing, MI., USA) and Minitab statistical software release
10.0 (Minitab lnc., State College, PA., USA) were used to perform all statistical

calculations.

4. RESULTS AND DISCUSSION

4. 1 FLOUR CHARACTER/2A TION

4.1.1 MOISTURE CONTENT

Moisture content of the ﬂours (wet basis) is reported in Table 8.
Results indicate that these ﬂours were in the lower recommended moisture level
of 12 to 13% so that minimum oxidation or mold growth would occur. From this,
it was expected and assumed that no signiﬁcant detrimental changes that could
vary the results would be found in the ﬂours. As a preventive action, all three

ﬂours were stored at 4 °C in sealed containers while not in use.

4.1.2 PROTEIN CONTENT

The results for the protein tests of the three ﬂours used in this study
are presented in Table 9. From these results, it is possible to observe that a
signiﬁcant difference in the protein content “existed among all three ﬂour
samples.

The end purpose of the C was to serve as a bread ﬂour, and it was
labeled as a blend of hard spring and winter wheat, therefore, it was expected
that the C would have the highest protein content. This was assumed because
ﬂour companies usually add vital wheat gluten to the blend of wheat ﬂours sold
to adjust or replace the original protein content of the ﬂours, depending on ﬁnal
use (McDermott, 1985 and Magnuson, 1985). As for the H, it is a blend of ﬂours
from a single class (hard red winter wheat), with the characteristic, when

compared to soft wheat classes, of having a higher water absorption and tighter

93

94

Table 8. Moisture content of the ﬂours used in this study.*

 

 

 

 

 

Flour Mozatrre
c 12.11 1 0.08
H 11.80 1 0.07
8 11.96 1 0.07

 

 

 

* Average :1: standard value of 5 replications

95

Table 9. Protein content of the ﬂours used in this study.1

 

 

 

 

Flour Fragin’ Prgtsin’
C 13.86 :I: 0.09 13.56 :I: 0.09
H 12.93 :i: 0.06 12.61 :I: 0.06
S 9.83 :l: 0.20 9.60 :I: 0.20

 

 

 

 

 

‘ Average :I: standard deviation values of 5 replications
2 Samples dried for 48 hours at 120 i 1 °C
3 Adjusted values to a 14% moisture content

96
adherence (stronger interaction) of the protein and starch. The protein content

of this flour falls in between the C and S. S is also a blend of flours within a
class, but with the characteristic of being from all soft white wheat. This flour
sample had the lowest protein content.

According to D’Appolonia (1994), H with a protein range of 10.0-11.0
percent would be used as an “all purpose” ﬁour. On the other hand, S with a
protein range of 80-100 percent could have typical applications in ﬂat bread,
noodles, and pastries. Finally, since C is a “mixture of hard spring and winter
wheat class ﬂours,” it may be expected to be applicable in making white pan

bread, which typically requires a protein range of 11.0-13.0 percent.

4.1 .3 FALUNO NUMBER

Results of the Falling Number determination are presented in Table 10.
The values for the C and S fall within the range indicated by Perten (1988) for
unsprouted wheat. The value corresponding to the H indicated low amylase
activity with the value in the lower limit for a sound wheat. From these values, it
was anticipated that possible problems associated with the a-amylase activity,
such as presence of free sugars in the FFS (non-enzymatic browning, etc.),
would have be minimum, or absent from this study. In addition, since ﬂours
milled from sprouted wheat may present not only a-amylase but protease activity
(since both are enzymatic changes involved in the development of the new
plant), it was assured that the ﬂours used in this study contained the proteins
with minimum, or no degradation. Overall, the determination of a-amylase as a

variable is expected to play a minor role in the process since one of the

Table 10. Falling Number of the ﬂours used in this study.

 

Falling Number '

 

 

 

 

 

Flour (s)
C 241 :I: 11.20
H 351 :t 8.40
S 271 :t 2.70

 

* Average :I: standard deviation of 5 replications

 

98
objectives of the preparation of the film is to separate the starch by

centrifugation.

4. 1 .4 FARINOGRAPHY

Figure 11 show the farinograms obtained from the studies performed
for the C, H, and S ﬂours, respectively. Dough resistance to mixing during the
successive stages of its development is measured with the farinograph. It was
observed from. these ﬁgures that a different shape among the three different
ﬂours was obtained (Figure 11). This indicated that there was a difference
among individual ﬂour constituents of the ﬂours analyzed. A strengthening of
the farinograph curve decreased from Figures 11-C, 11-H, and 11-S,
respectively. This behavior agrees with a report by D’Appolonia (1984) in which
the author indicated that, with an increase in prOtein content, a deﬁnite
strengthening of the farinograph curve occurs. It was also observed that the
gluten-ﬂour bread of the C in Figure 11-C as compared to the H ﬂour (no gluten
added) showed a strengthened (or improved) ﬂour curve. A similar report was
made by Lorenz (1984). Also, by comparing the shapes from a study by Preston
and Kilbom (1984), it was established that C and H could be considered strong
ﬂours. This was further validated since the values for MTI of 50 and 40 (Table
11), respectively, fell between the 30 (strong) to 80 (medium strength) according
to the classiﬁcation developed by these authors. On the other hand, 8 was
considered a weak ﬂour in agreement with Preston and Kilbom (1984) since the
MTI value of 170 (Table 11) for the S was similar to the 180 value determined by

, these authors for a weak ﬂour.

99

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100
The calculated values obtained from the curves in Figure 11 for water

absorption, dough development time (mixing time), and stability are also
reported in Table 11. The water absorption value of H fell within the typical
absorption levels for bread flours of 58 to 66 percent (D’Appolonia, 1994). Since
absorption is correlated positively by the protein content and the damaged
starch (D’Appolonia, 1994), and C contained added gluten, it was expected that
the higher protein level in C yielded a slightly higher value of the maximum
typical value expected. The absorption values for the three types of flours
studied indicated a resemblance with the observations made by Shuey (1984a)
in which absorption generally increases, when all other variables are kept
constant, with an increase in the protein content of the ﬂour. This indicated that
C, H, and S required, respectively, greater amounts of water to give the same
consistency. The mixing time values showed that the weak ﬂour (8), required
about half the time to reach the maximum consistency ”as compared to the strong
ﬂours (C and H). The stability values showed a decrease from C, H, and S,
respectively, which corresponded to an indication for a greater tolerance to
mixing from values of 7.25 minutes to 1.50 minutes.

The above results indicated that differences in physical behavior
existed among the C, H, and S ﬁours. It is important to mention, however, that
these differences were produced predominantly by the protein and starch

interaction of the ﬂours used (D’Appolonia, 1984).

101

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4.1.5 SDS-PAGE

The one-dimensional sodium dodecyl sulfate-polyacrylamide gel
electrophoretic (SDS-PAGE) patterns of the C, H, and S flours, both under
reduced and unreduced conditions are presented in Figure 12. Both C and H
ﬂours are mixtures of two or more varieties, evidenced by more than ﬁve high
molecular weight glutenin subunits (HMW-GS) present in each ﬂour on the SDS-
PAGE gel. From the analysis of the different bands obtained for the high
molecular weight (HMW) glutenin subunits region for each ﬂour, different
subunits were identified and are presented in Table 12. It was observed that
both, C and H ﬂours, contained the HMW-GS pair “5+10,” whose presence has
been suggested as an indicator of strong gluten (Gupta and MacRitchie, 1994). '
On the other hand, soft white ﬂour contained the HMW glutenin subunit pair
“2+12,’ whose presence has been suggested to be an indicator of weak gluten,
by producing a smaller percentage of larger sized polymers as compared to
those of ﬂours containing the subunit pair “5+10" (Gupta and MacRitchie, 1994).

These observations on the differences in the HMW-GS composition
found in the ﬂours may in part explain the ﬁlm behavior discussed later on. In
addition, it was also noted that there were some differences in the regions
corresponding to the low molecular weight glutenin subunits (LMW-GS). These
variations in the types of LMW-GS, which have been studied less extensively
than HMW-GS, may also be responsible for the differences in the ﬁlm properties
observed. Similarly, differences in the unreduced electrophoregram patterns are

evident: Both the intensity and the number of bands present increased from S,

 

unreduced reduced
r"———"—"l l

1
SHC CtHSNP-

HMW
Glutenin

subunits
_l

Gliadins

LMW
Glutenin
subunns

Nonstorage
proteins

 

 

Figure 12. One-dimensional sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) of C, H, and S flours
C = Commercial flour
H = Hard red winter flour
S = Soft white flour
NP = Neepawa flour (Canadian hard wheat -pure variety-)

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105
H, and C flours. The films were formed using the unreduced flours except when

a reducing agent (cross-linker) was used (i.e., tensile strength, tensile creep,
FTIR and color studies). This is important because, as observed in Figure 12, a
great number of polymeric protein molecules could not enter the separating gel,
an indication of the great size that these proteins have before a reducing
treatment is applied. Nonetheless, these results are preliminary and further
conﬁrmation of these observations is needed to present more conclusive

statements.

4.2 FILM FQRMA TIQN PRQQE§§

Several advantages were found by using the methodology deﬁned in
this study as compared to previously published reports for making ﬁlms using
wheat proteins. These include: (a) the ﬂour disperses more easily and does not
form clumps while mixing it with solvents at room temperature; (b) after
centrifugation, only soluble materials (proteins, etc.) are present, because the
starch and other insoluble materials that may be found in the commercial wheat
gluten have been removed; (c) the ﬁlms are homogeneous with a smooth (not
granular) texture, and; (d) a wider variety of raw materials can be used to

prepare the ﬁlms including ﬂour and protein powder.

4. 3 BARRIER PRQPERTIE§

4.3.1 OXYGEN PERMEABILITY

The oxygen permeability, determined for films 1, 2, 4, 5, 7, and 8

(Table 4) with the data at 10, 25, and 40 °C, are presented in Table 13. A plot of

106

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107
permeability vs. temperature is shown in Figure 13. It was observed from these

values that the temperature was found to be the most significant, factor (p<0.05)
affecting oxygen permeability. The values showed that an increase in
temperature corresponded an increase in the permeability values for films. This
behavior is in agreement with that observed previously by Gennadios et al.
(1993). The second important factor that inﬂuenced the oxygen permeability
was the type of flour used to prepare the ﬁlms: ﬂour type was found highly
signiﬁcant (p<0.01) with the effect of oxygen permeability. Finally, it was found
that the pH of the solution did not have a signiﬁcant effect (p>0.05) on the
oxygen permeability for the ﬁlms prepared and analyzed in this study.

The oxygen permeability values were ﬁtted to an Arrhenius model,

obeying the following equation:
P = P ° 55"" (26)

An Arrhenius plot was then generated by plotting the logarithmic form of the

above equation:

In P= In P°-E.IRT (27)
and is presented in Figure 14. From this plot, the Arrhenius factor (P°) and the
Energy of Activation (E.) were calculated and reported in Table 14. Note that E.
is eXpressed as E. R", thus the values of Table 14 can be multiplied by the gas
constant, R (0.001987 Kcal mol'1 K‘ 1) to obtain E. in Kcal mol". There was an

agreement between the oxygen permeability values at the different temperatures

and the values of E. found for these ﬁlms. At higher temperatures, the term

108

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111
(E.R"T') becomes smaller so that the exponential e"E"‘Rm becomes larger.

Therefore, as the temperature increases, the oxygen permeability (Eq. 26)
increases meaning that oxygen molecules permeate faster through the films.
Also the term P°, which in this case is the probability of an oxygen molecule
colliding with a polymer (protein) molecule, was found to be larger in the ﬁlms
made from commercial ﬂour and hard red 'winter flour using the low pH method
(Table 14). This may suggest that a more compact structure is found in these
ﬁlms as compared to the other films studied. A!

The activation energy values of the oxygen permeability process
between 9.1-14.5 kcal mol‘1 fell within the range of those found for synthetic
polymers. It appeared that, at pH 4, C and H ﬁlms showed higher values than 8
ﬁlms. The activation energy of the permeation process is the addition of the '
activation energy of the diffusion process and the enthalpy of sorption of the
permeant in the ﬁlm. Unfortunately, there are no data regarding the enthalpy of
sorption of oxygen in these wheat ﬁlms. But in general it is less than the
activation energy of the ﬁlms. Nevertheless, assuming that the activation energy
of the diffusion process is the dominating term, it can be suggested that a higher
value of the activation energy of the permeation process implies a higher energy
value for the oxygen molecule to diffuse through the polymeric matrix. This also
implies that C and H ﬁlms are produced with the characteristic of having a more
tortuous path than 8 ﬁlms.

No signiﬁcant correlation between the protein content in the flour and

oxygen permeability of the resulting ﬁlms was found (p>0.05) when measured at

112
dry conditions (moisture content < 2.5 % w/v for all films). Although this is not

completely understood, it may indicate that the total protein content present in
the ﬂour is less important than the type of protein in the determination of the
morphology of the polymers which ultimately determine the gas barrier
characteristics. The oxygen permeability values of the wheat film measured at
25°C ranged from 5.9 to 18.5 x 102° m3 02 (STP) m rn'2 5'1 Pa". These values
were similar to those for the oxygen permeability of nylon-6, reported to have a
value of 13.7 x 10’20 m3 02 (STP) m rn'2 s'1 _Pa'1 at 23°C and 0% RH (Gavara and
Hernandez, 1994). The similarity values may be explained by the similar
chemical structure of these materials based on the polyamide functionality.

The non-proportional protein content/oxygen permeability ratio for the
different temperatures studied may indicate that it is not only the total protein
content present in the ﬁlm which is responsible for the total oxygen permeability,
but also the type of protein (referred as “quality” in baking) present in the wheat

ﬂour

4.4 RHEQLOGICAL MEA§QREM§NT§ QF FILM§

4.4.1 MECHANICAL PROPERTIES

4.4. 1. 1 Tensile than th
The ultimate properties were studied on ﬁlms 1, 3, 4, 6, 7, and 9 (Table

4). These properties include elongation, tensile strength, and modulus of
elasticity. Results are presented in Table 15. Measurements were made with

Films conditioned at 50% RH. Figures 15 and 16 represent the elongation and

113

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116
tensile strength values, respectively for the films studied. From these results it

was observed that differences in elongation for the films were signiﬁcant
(p<0.05) for the ﬁlms prepared using the commercial bread flour and the others.
The two classes of flour, hard red winter and soft white, did not present
signiﬁcant (p>0.05) differences in the values obtained for them. On the other
hand, differences observed using the reducing agent (cysteine) were not
signiﬁcant for ﬁlms made of the same ﬂour. Gennadios et al (1993) reported
elongation at break values for a wheat gluten ﬁlm ranging 216-260%, measured
at 50% RH. These values were lower in magnitude than ﬁlms made in this study
having a range of 490 to 640%. Elongation at break values of ﬁlms produced
with reducing agent were not signiﬁcantly different from ﬁlms produced without it.
This may be attributed to the fact that the measurements were made at 50% RH
and because of the hydrophilic nature of the ﬁlms, water served as a plasticizer.
This may have softened the structure resulting in elongation values that were
higher than if the tests were performed at a lower relative humidity. According to
Modern Plastics Encyclopedia (1995), typical values of elongation at break for
selected synthetic polymers are: low density polyethylene (LDPE), 300-1000%;
high density polyethylene (HDPE), 100-1000%; ethylene-vinyl acetate (EVA),
GOO-900%; and polypropylene (PP), 20-800% (ProgelhOf and Throne 1993).
These numbers indicate that elongation values for wheat ﬁlms (485-640%) are
similar to those found for synthetic polymers.

Tensile strength was observed to be signiﬁcantly different (p<0.05)

among the ﬁlms in which the reducing agent (cysteine) was added. The values

117
of the tensile strength among the films that did not have cysteine, although made

with different flour, were not significantly different (p>0.05). This may support the
suggestion by Guthrie and Gruen (1990), that the use of cysteine promotes
inter-molecular cross-links. The cross-linking process involves the exchange of
disulﬁde groups present in the molecules, acting as a catalyst for the “disulﬁde
interchange” reaction. The result of this would be an increase in the ﬁlm tensile
strength. Values of the tensile stress at break of the wheat protein ﬁlm ranged
from 0.2-0.4 x 10‘ Pa and were more than one order of magnitude lower in than
the commercial synthetic plastics such as LDPE, HDPE, EVA, and PP that
showed a tensile stress at break from 8-37 x 10‘ Pa (Modern Plastics
Encyclopedia 1995).

Values for modulus of elasticity (E) for the protein ﬁlms were in the
range of 3.7-7.8 x 105 Pa (Table 15). Literature reports values of 2.0 x 10" for
soft rubber, 2.2 x 10" for LDPE, 1.08 x 10’ ror HDPE, 1.19 x 10’ for Nylon-6, and
2.52 x 10° for Nylon-66 (Sperling 1992 and Modern Plastics Encyclopedia 1995).
This indicates that the wheat protein ﬁlms were from less than one to almost four
orders of magnitude, less resistant to stretching compared to these commercial

polymeric materials.

4.4. 1.2 Tensile Qreeg

All the creep curves (Figures 17 to 23) were obtained for time values
up to two hours, which resulted in reaching the point of failure (rupture) of the
protein ﬁlms for most of the samples. It was observed that, as the temperature

and/or relative humidity increased, the ‘time “to reach failure was reduced

118

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considerably. It was also observed that temperature had a greater effect on the

creep behavior of the ﬁlms than the relative humidity.

By comparing the experimental curves of Figures 17 to 23, it was
observed that creep compliance of films made with the cross-linking agents were
smaller than those for ﬁlms made without a cross-linking agent. Also, by
comparing the experimental curves of ﬁlms made with the same cross-linking
agent (formaldehyde or glutaraldehyde) in Figures 21 and 23, it was found that
an increase in temperature produced an increase in the creep compliance. This
was expected since the creep compliance is proportional to strain and proteins,
as well as many other polymers, increase their molecular mobility (i.e. become
more rubbery) as temperature increases. This process may, have promoted
successive changes in the proteins so that the biopolymers changed from hard
(brittle) to viscoelastic materials.

Similarly, an increase in the relative humidity yielded higher values of
the creep compliance for both pair of conditions investigated (Figures 19 & 20
and 21 & 22). This was attributed to the plasticizing effect of the water in the ﬁlm
which is known to decrease the modulus of elasticity of the protein biopolymers.
This behavior is explained by the action of water that reduces the intermolecular
forces (dipole and dispersion forces and hydrogen bonding), loosening the
bonding of polymer molecules with each other. Also at the same temperature, at
low relative humidity the ﬁlms were stiffer to the touch than the ones at higher
relative humidity. This was in accordance with the thermodynamic theory of

Iplasticization. This theory explains that in a stiff, brittle polymer the

126
intermolecular separations are small, compared with an elastomer, and every

deformation causes internal stresses which the molecules cannot accommodate;
consequently, the elasticity of the protein ﬁlms was lower producing a hard
rubber type consistency.

The plots in Figures 17 to 23 include curves for the four-element
Burgers model obtained by calculating element values from experimental data.
Table 16 shows the numerical values obtained for the Burgers Model as deﬁned
by Eq. (10). At lower temperatures, better curve ﬁtting was achieved. This
result may be interpreted on the basis of molecular theory. The increased
temperature permitted assemblies of protein chains to move in a coordinated
manner, being governed by the principles of reptation and diffusion. Since the
protein molecules were cross-linked, this motion was thought to involve several
chain segments which are bound together. This behavior may be predicted
using more complicated mathematical models (e.g., introduce one or more
additional Kelvin elements) which accounts for the new thermodynamic
molecular behavior exhibited by the biopolymeric ﬁlm with the increase of the

temperature.

Analysis of the raw data using the model proposed by Peleg (1980)
yielded a much better ﬁtting. It was observed (Figures 24 to 30) that the greater

the value of the intercept (k1), the lower the creep compliance differential over
time. This inverse of k1 was indicative of an ability to exhibit a lower tensile

defamation over longer periods of time. The Value of the inverse of the slope

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135
(Mg), on the other hand, represents a value of constant creep, that is, when the

hypothetical equilibrium (if failure of the specimen did not occur) was reached.

Such behavior is observed in the followingrelationship:
JA - —— = — (28)

in which JA = asymptotic creep compliance and a. = asymptotic tensile strain.

From this, the asymptotic tensile strain can be calculated:
3,. = ﬂ = JAao (29)
k2

which may be a more useful parameter for determining the overall deformation
rate of the protein ﬁlm. It was observed that the lower the a. number, the lower
the deformation rate for the curves studied. This value appears to accOunt for
the initial instantaneous deformation (Jo in the Burgers Model) since the
magnitude of the a. value was related to the free spring behavior of the creep
curves. The limitation appears to be that it does not account for the total
experimental time required for strain measurements for each ﬁlm. These values

of k; are also sensitive to the environmental conditions, which in biological

materials would account particularly for the influence of temperature and relative
humidity.

Therefore, for practical purposes, it is recommended that comparison
for these values for different ﬁlms: (1) are' made on different ﬁlms tested at the
same environmental conditions (eg., temperature and relative humidity) to

account for ﬁlm differences; or (2) are made of the same ﬁlms at the different

136
environmental conditions to account for the influence of these variables on the

creep behavior.

Parameters for Peleg Model, given by Eq. 12, are summarized in Table
17. Unlike the Burgers equation, all curves could be plotted, with an r2 2 0.98 in
all cases except one using the Peleg relationship. The exception was for the
glutaraldehyde-cross-linked ﬁlm tested at 20°C and 35% RH, with an r2 of 0.87,
° which was attributed to a very small strain presented by the sample (see Hencky
strain in Table 17). Thus, a Hencky strain of about 0.30 (the next value up
observed for the fonnaldehyde-cross-linked ﬁlm in Table 17) was determined to
be the limit for obtaining creep curves with good accuracy using the creep tester. f

Results indicated that le- is related to a large initial change in strain.
Values for k- were obtained for all ﬁlms studied. By comparing the different ﬁlms

tested under the same environmental conditions, it was generally observed that
the cross-linked ﬁlms had higher values as compared to the uncross-linked ﬁlms.
This indicated that a long time passed without ~ major change in creep
compliance. By comparing groups of ﬁlms tested at different conditions, it was
observed that the ﬁlms at lower temperatures and lower relative humidity,
exhibited a smaller change in creep compliance, as noted in Eq. 28.

It is important to mention that there is possibility for a better ﬁtting of
the curves using a 6- or more-element Burgers model. However, this model
becomes more complicated when compared to the Peleg model. Consequently,
it was observed that in a comparison of both 4-element Burgers and Peleg

models, the last was superior for examining the creep behavior of the ﬁlms.

137

Table 17. Peleg Model constants (le- and leg) for wheat protein films.

 

 

 

 

 

 

 

 

 

T-RH k1 k2 (2 2.3
”W (°C-%) MPa'1-s MPa" s 5"
24 l N 05-85 -‘ -‘ 1 1.42
24 / G 05—85 9.3 1.3 420 1.82
24 I F 05-85 3.9 1.5 85 1.55
25 I N 1040 109.9 1.9 42 0.72
25 I G 10-40 1532.5 1.9 5400 1.12
25 I F 10.40 4840.1 2.4 5400 0.83
26 I N 20-35 55.0 2.2 10 0.35
25 I G 20-35 53351.0 9.5 05 0.14
25 I F 2085 19755.0 5.7 0 0.30
27 I N 2055 1.5 2.4 2 1.43
27 I G 2055 755.7 2.3 05 1.14
27 I F 20-55 720.2 2.7 0 1.15
28 I N 25.25 5.5 2.1 12 1.39
28 I G 2525 148.2 4.9 220 ‘ 1.21
28 I F 25.25 302.9 3.4 900 1.11
29 I N 2530 19.9 3.7 4 0.47
29 I G 2530 25.2 4.4 70 1.29
29 I F 25-30 83.1 3.9 350 1.15
30 I N 40-25 11.3 1.7 4 0.87
30IG . 40.25 124.9 2.4 50 0.77
30 I F 40.25 1.0 2.0 70 1.53

 

 

 

 

 

 

‘ Figure # / Letter (N=non-;
cross-linked)

2 Value at failure

3 Hencky strain

‘ Could not be determined due to insufﬁcient data points

5 Did not break in the 2 hr. observation period

=glutaraldehyde-; and F=formaldehyde-

t
—=le kt
J 1+ 2

 

1 38
4.4.2 THERMOMECHANICAL PROPERTIES

4.4.2.1 Thermal Mechanical Analysis
TMA was peformed for the films but no useful results were obtained for

these tests. The main limiting factor was the thickness of the ﬁlms. It was not
possible to produce a ﬁlm thicker than 1.25 mm (approx. 0.05 in). The particular
TMA instrument considered required samples at least 125 mm (approx. 0.5 in) in
thickness. The intended thermal expansion coefﬁcient was not obtained using
this technique. A sample output of a glutaraldehyde-cross-Iinked ﬁlm measured

using this instrument is presented in the Appendix (Figure C1).

4.4.2.2 Modulated Differential cannin lorimet
A couple of samples of MDSC curves obtained for the glutaraldehyde-

cross-linked ﬁlm are presented in Figures D1 and DZ in the Appendix. This
technique although very useful for obtaining Ta and T... values in polymeric
materials, was not very useful for the protein ﬁlms studied. It is suspected that
no data were obtained primarily because of the process used for fabricating the
ﬁlm. The protein solution was heated to boiling temperature (measured to be
between 82-83 °C) thus making the proteins denature.

Hoseney (1994) reported that a non-reversible thermal effect on the
gluten-water system occurs between around 40 and 70 °C, which represents
values below the boiling temperature of the FFS during ﬁlm fabrication. The
non-reversible thermal effect of the proteins in the FFS was demonstrated by the
use of RheoStress” Measuring System model RS-100 (Haake Mess-Technik

GmbH u. 00., Karlsruhe, Germany). FFS was taken and placed between two 60

139
mm diameter parallel plates using a solvent trap. The distance between the

plates was set to 0.25 mm and an oscillatory test was performed. Results for
this test can be seen in Figure E1 in Appendix E. In this ﬁgure, the values for G*
were observed to signiﬁcantly increase when the temperature reached about
62°C from a value of about 0.007 Pa to a ﬁnal value (in the ramping-up process)
of just under 600 Pa which was attributed to the unfolding of the protein
molecules. Once it reached the upper end temperature, the FFS was cooled
down immediately and, as expected, a decrease of the 6* value was not
observed. In the cooling process (ramping-down), the 6* value increased from
around 600 to about 1000 Pa in the interval from about 80 to around 65°C, and
remained constant at lower temperatures. Based on this experiment, for the
actual process it was assumed that the structure opened and, when a cross-
linker was added to the FFS, a macromolecule formed as the ﬁlm formed.
Similarly, in the FFS with no cross-linker added, the structure remained open, in
a disordered structure (Hoseney, 1994) with no formation of a macromolecule.
Since MDSC measures the denaturation as T, being a change in the baseline of
the heat ﬂow, and no change in the baseline was observed, it was assumed that
the biopolymeric structure was opened. In addition, the protein ﬁlms degraded
before the T... temperature was reached (especially for the non-cross-linked
ﬁlms, since the cross-linked ﬁlm would not show a true melting point). This
degradation temperature was evidenced by the sudden increase of the heat ﬂow

around 150 °C observed for all the ﬁlms (see Figure D1 in Appendix).

140
4.5 CHEMICAL CHARACTERIZATION

4.5.1 FOURIER-TRANSFORM INFRARED SPECTROSCOPY

Spectra obtained experimentally for the wheat protein ﬁlms (Table 7)
by Fourier-Transform Infrared Spectroscopy (FTIR) are presented in Figure 31.
The FTIR spectra obtained from the literature for somecommercial polymers are
shown in Figure 32. It is not coincidental that the. overall shape and a number of
the characteristic absorption bands observed in the nylon-6 spectrum (Figure
32), are similar to those obtained for the protein ﬁlms (Figure 31), since all of
these ﬁlms have the polyamide functionality. For instance, proteins are
sometimes referred as nylon-2 (Sperling, 1992), and have the following general

structure:

_. T T.
—N—cl;4— . (30)
R n

—l—

0
II
C

 

 

b

The “R” group will depend on which of the 20 common types of amino
acids are present in the protein structure. These amino acids follow very
speciﬁc sequences, but, according to Sperling (1992), they are copolymers in
the broad sense of the term.

The main features observed for the bands in Figure 31 are: a broad
band around 3300 cm" attributed primarily to the hydroxyl (-OH) groups from the
plasticizer (glycerol) and to presence of water molecules (Colthup et al., 1964).

According to Colthup et al. (1964) and Satyanarayana and Chatterji (1991), the

141

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l r
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4.000 3.000 2.000 1.500 1.000 800 700 550

Wovenumber, cm"

Figure 32. Typical transmission spectra in the infrared region for some
commercial polymers. The ordinate in each diagram is %
transmission (T).

[from Rodriguez, 1989]

143
close narrower bands observed at around 2950 and 2920 cm'1 are typical of the

methyl group (-CH3) and C-H bond stretching, respectively, which are typical
groups found in the protein structure.

There was a small band present at around 2350 cm", which is not
found for many of the synthetic polymers. This is probably due to rotational
vibration of free water molecules (Colthyp et al., 1964) which were present in the
' protein ﬁlms. These ﬁlms were very hydrophilic (see results of swelling studies),
which made it easy for them to pick up water from the environment.

There were two very typical absorption bands found for the ﬁlms, which
are typical of polyamides. The ﬁrst at around 1650-1640 cm", which
corresponded to the amide-l band (Giacin, 1995). This band was found due to
the carbonyl (C=C) stretching in the peptidic bond (see Eq. 30). The second
one, known as amide-ll, due to the N-H bending, was observed at around 1540
cm“. This helped to conﬁrm that polyamide functionality in the ﬁlms was
preserved after the casting process. In addition, it can also be concluded that
the cross-linking process did not affect the peptide bond. Finally, a (band found
at 1450 cm'1 attributed to the aldimine absorption (Satyanarayana and Chatterji,
1991) was also observed, particularly for the glutaraldehyde-cross-linked ﬁlm.
The peaks for the disulﬁde (-S-S-) bonds are typically present between 525-509
cm'1 (Colthup et al., 1964). Because data were collected only up to 600 cm",

this research could not account for the FTIR spectra.

144
4.5.2 SWELLING STUDY

The swelling coefficient (Q) values obtained for the different film
samples calculated after immersion in different solvents are presented in Table
18. Comparisons for Q values for all the ﬁlms in the solvents used did not show
signiﬁcant differences for hexane and n-butanol, with mean values of 0.063 and
0.069 ml 9", respectively. However, Q was found to be signiﬁcantly different for
methanol, 70% ethanol with a pH of 4.0 (to be referred as the “ethanol” solvent
this point fonIvard, which was intended to simulate the evaporated solvent when
the ﬁlms formed), and water (p<0.05), with mean values of 0.803, 3.192, and
2.294, respectively. When the comparisons for all treatments were analyzed for
the Q values on the ﬁlms, it was found that signiﬁcant differences were present
for the uncross-linked ﬁlms with the cysteine-cross-linked and glutaraldehyde-
cross-linked but not with the formaldehyde cross-linked. Their mean Qwvalues
were 0.303, 1.109, 1.531, and 1.252, respectively. It is important here to note
that the mean value for water is much smaller than what would theoretically be
due to the dissolution of the ﬁlm in the ethanol and water solvents. Figure 33
represents the mean values obtained for the ﬁlms as calculated using Eq. 14 by
using the dry weight values of the ﬁlms before immersion in the solvents (mo in
Eq. 14). In this ﬁgure, it is observed that very little uptake of hexane and butanol
was performed by all of the ﬁlms. As for methanol, the smallest molecule of the
alcohol series (CHs-OH) with a relatively bulky polar group, little uptake was

observed for cross-linked ﬁlms but not for water. This may be attributed to the

145

Table 18. Swelling coefﬁcients* of selected protein ﬁlms in several solvents.

 

 

 

 

Crosslinker Solvent Swelling coefﬁcient (ml 9")
none hexane 0.0652 :l: 0.0072
cysteine hexane 0.0347 1 0.0023
formaldehyde hexane 0.0848 1 0.0352
glutaraldehyde hexane 0.0682 3: 0.0167

75.13 """""""""" n’- 6613361 """"""" 0. 6902’i070095 """"
cysteine n-butanol 0.0542 :1: 0.0154
formaldehyde n-butanol 0.0626 :l: 0.0127
glutaraldehyde n-butanol . ' 0.0589 1 0.0139 "

‘rTJn'e """""""""""" 1?: 51715661 """"""""" 0'. 5829’; 0700713 """"
cysteine methanol ' H " 0.9441 :1: 0.0535
formaldehyde methanol 0.7552 :l: 0.1022
glutaraldehyde methanol 0.9296 :1: 0.0405

'aan'; """"""" “"7035 51713361751311 """""" r ﬁrﬁ'd'iéBTv'eE """"
cysteine 70% ethanol, pH 4 3.0131 :l: 0.4222
formaldehyde 70% ethanol, pH 4 3.7796 :l: 0.3088
glutaraldehyde 70% ethanol, pH 4 2.7830 1 0.1296

'HJn'e """""""""" Rafe-r """"""""" f ﬁrﬁ'd'isTsBTv‘eE """"
cysteine water 1.4998 :1: 0.0433
formaldehyde water 2.9729 :I: 0.5326
glutaraldehyde water 2.4086 :1: 0.1819

 

* Average 1 standard deviation of three replications

146

 

 

   
      
 

m\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\

//

 

 

Cl un-cross-linked

I cysteine-cross-linked

lformaldehyde-cross-linked -

I glutaraldehyde-orass-linked

 

 

\\\\\\\\\\\\\\\\\\\\\\\\

////

water

paAlossm

ethanol*

paAlossm

methanol

butanol

 

hexane

 

 

2.0

1.6 _...... ,4...

1.4 l

0 6
0 2
0 0

Figure 33. Swelling coefﬁcient (Q) of different protein ﬁlms, calculated using dry weight of

ﬁlm before immersion.
* 70% (vlv), pH 4.0

147
fact that some glycerol escaped to the solvent and, in the case of the cross-

linked biopolymers, the stress created by the glycerol voids in the
macromolecules provided the driving force for the introduction of some methanol
molecules. A much greater uptake was observed by the use of both the ethanol
and water solvents, with uptakes of more than 250% in weight for the solvents.
The uncross-linked ﬁlms dissolved in both the ethanol and water solvents, which
was expected, as similar results were reported for uncross-linked gelatin
proteins in a swelling study by Chatterji (1989). For this case, the dissolving of
the biopolymer in the solvent causes the random coil to expand and occupy a
greater volume than it would in the dry, amorphous state. In this case, viscous
ﬂow occurred, and the viscosity, although not measured, would increase as the
polymer expands (Rodriguez, 1989). According to Rodriguez (1989), it is
expected that when the polymer and solvent have the same solubility
parameters, 6, the maximum expansion will occur and therefore the highest
viscosity (for a given concentration) will be obtained.

Figure 34 represents the weigh loss observed on the ﬁlms during the
24 hours of solvent immersion. These values were obtained by difference of the
dry weight before after immersion in the solvents. The order of the solvents, in
ascending order with respect to their solubility parameter, 6, (from hexane to
water) have values of 14.9, 28.7, 29.7, 32.6, and 48.0 (Mpa)". This is important
because an increase in the weight loss is observed as polarity is increased for
the solvent. It is suspected that glycerol was extracted during the immersion

time. This was partially conﬁrmed by the greater ﬂexibility retained by the ﬁlms

148

 

 

 

 

 

El un-cross-linked

I cysteine-cross-linked

I fonnaldehyde-cross-linked

.glutamldehydgaogg'inked ..

 

 

    

water

         

     
   
   

3 .

peAIossm

ethanol‘

peAl9ssm

i

memanol

 

 

butanol

hexane

 

0.40

0.35

 

0 10

ID
or m
o

% ‘ssoi tuﬁgeM

Figure 34. Weight loss of the ﬁlms during time of solvent immersion.

* 70% (vlv), pH 4.0

149
immersed in the hexane and butanol solvents as compared to the ones

immersed in the ethanol and water solvents (after they were dried in the oven).
Glycerol is a very polar molecule, withtthree hydroxyl groups present in the
molecule, thus very compatible to water solvent. There is the possibility that
some loose proteins came into solution as well, especially with the ethanol and
water solvents, although this was not conﬁrmed in this study.

Figure 35 presents the swelling coefﬁcient values of the protein ﬁlms
calculated from the dry weight of the ﬁlm after immersion. This may be more
representative of the actual solubility parameters (as compared to the values of
Figure 33) because in the calculation of these values it was used the
biopolymer's dry weight, which does not account for the weight loss (which is
included in the dry weight before immersion). Thus, it is assumed that only the
evaporated solvent occupied the rest of the volume in the swollen state. Very
similar behavior was observed when compared to Figure 33 except that the
values are greater (takes in account the weight lost during the immersion time).
Comparison among the Q values for ﬁlms immersed in hexane does not show a
signiﬁcant difference among them (p>0.05). Similar results were found for the
ﬁlms immersed in butanol, in which no signiﬁcant differences on the Q values
were observed (p>0.05). However, for the ﬁlms immersed in methanol, the non-
cross-linked value was found to have a smaller swelling coefﬁcient as compared
to the rest, followed by the formaldehyde-cross-Iinked ﬁlm, with mean values of
0.5829 and 0.7552 ml 9“, respectively. The cysteine- and glutaraldehyde-cross-

linked ﬁlms were found to have greater solubility values, different than the other

150

 

 

  
 
   

water

 
 

,PGAIQSSW

ethanol"

    
  

 

  

   

 

 

 

 

 

4.0

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; g E C
‘1 3 l/ (U
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8 ,= i ?
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D . .Il |
.2. c; .2. ., c3 «3 e.
('0 00 N > ‘- 1- O O

o
E 2.0~
d

P
|

ﬁlm after immersion.

Figure 35. Swelling coefﬁcient (Q) of different protein ﬁlms, calculated using dry weight of
* 70% (vlv), pH 4.0

151
two described above but not different among them. Swelling coefficients for

these ﬁlms were determined to be 0.9441 and 0.9296 ml 9", respectively.
Comparison of the swelling coefﬁcients among the cysteine- and glutaraldehyde-
cross-linked ﬁlms show no significant difference among these two but were
found to have lower values as compared to the formaldehyde-cross-linked ﬁlms
(p<0.05), as shown in Figure 35. When water was used as the solvent, Q values
were signiﬁcantly smaller for the cysteineecrosselinked ﬁlm as compared to the
other two, formaldehyde- and glutaraldehyde-cross-linked ﬁlms (p<0.05).
Solubility parameters for the solvents used were obtained from the
literature (Rodriguez, 1989). The swelling coefﬁcients calculated during this
study for eachﬁlm and solvent are presented, as a function of the solubility
parameter, in Figure 36. This representation is useful to determining the
solubility parameter for any ﬁlm since the swelling coefﬁcient reaches a
maximum when the solubility parameter of the solvent nearly matches that of the
polymer (Rodriguez, 1989). Rodriguez (1989) indicated that the solubility
parameter is very useful because when the hydrogen-bonding index for various
solvents are plotted in a two-dimensional plot, along with the solubility
parameters, a swell map is obtained. Such a map has great value in selecting
solvents for a coating system or in judging probable resistance to swelling for a
particular application (Rodriguez, 1989). in the case of these ﬁlms, the curves
for the non-cross-linked ﬁlms follow only up to the third point [methanol, with a 5
value of 29.7 (Mpa)"] because they dissolved in the other solvents. Similar

shaped-curves for polyurethane, polystyrene, and polyurethane-polystyrene

152

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153
interpenetrating polymer networks are presented in a study by Sperling (1992)

using different solvents for each polymer system. Since most synthetic polymers
are formed by chemical reactions, and the polymer formed has an “unknown”
chemical property, a common way to determine their solubility parameters is to
do swelling studies. The curves as presented were expected because in the
case of these particular protein ﬁlms, they were cast directly from a solvent
composed of 70% ethanol having a pH of 4.0 (referred to as the “ethanol”
solvent throughout this discussion). There is an imminent advantage that the
solvent (in which the proteins came into solution, thus being the “optimum”
solvent) is known. Therefore, this makes it possible that the actual (if not very

close) solubility parameter, 6, for the protein ﬁlms is 32.6 (MPa)".

4.5.3 COLOR S'ruov

A diagram showing the ClE-Lab three-dimensional color evaluation
parameters and signiﬁcance is shown in Figure 37. Determination of color ClE-
Lab parameters for the protein ﬁlms are shown in Table 19. Results for the
luminous transmittance mode (see schematic of Figure 38 for luminous mode
explanation) showed the cysteine- and formaldehyde-cross-linked ﬁlms have the
lightest color (L values around 90%) among the ﬁlms studied, with no signiﬁcant
difference among these two (p>0.05). They are closely followed by the uncross-
linked ﬁlm with a value in the lower eighties. The glutaraldehyde-cross-linked
ﬁlm, was signiﬁcantly less light in color (L value of about 56%). The color
change, due to the formation of the aldimine linkage between the free amino

. groups of the protein and the glutaraldehyde during the cross-linking reaction,

154

L = 100 white

£27

yellow

ﬂ

+8

 

 

I red

 
 

 

 

L = '0 black

Figure 37. Three-dimensional color evaluation using the ClE-Lab system.
[Adapted from Birley et al., 1992]

155

Table 19. Color analysis of selected wheat protein ﬁlms.

 

 

 

 

 

 

 

 

 

 

 

Crosslinker CIELab Value“ 1 std. dev.
parameter
Luminous Transmittance Mode
none L 83.33 1 0.24
cysteine L 89.71 1 0.54
formaldehyde L 89.28 1 1.15
glutaraldehyde _ __l._______ _____5_6._5_9_13._5_5 ______
‘55.; """""""""""" a V 2.17 1 0.08
cysteine a -1.82 1 0.13
formaldehyde a -1.40 1 0.23
_g_l1_1taraldehyde a _ _ _______2_7.29 13.05 ______
non-e """"""""""""""" b" "" ' 3972—93: 0.39
cysteine b 29.79 1 1.38
formaldehyde b 29.50 1 2.18
glutaraldehyde b 61.79 1 1.63
Luminous Reﬂectance Mode
none L 32.66 1 0.42
cysteine L 28.89 1 0.37
formaldehyde L 31.27 1 0.23
-9'2919192'1Y93 ________________ L_ ______ __-_-_1§-_9_9_£9§§ ______
none a -0.53 1 0.12
cysteine a -2.47 1 0.05
formaldehyde a. -2.00 1 0.06
2091399899 _________ _ ______ 9. ------ -----.11183 19:5 _____
none b - 17.55 £0.35"
cysteine b 10.32 1 0.92
formaldehyde b 11.19 1 0.99
glutaraldehyde b 25.83 1 0.41

 

* Average of 5 replications. L is expressed as %, a & b as positive or negative

values with color corresponding to scheme presented in Figure 37.

156

 
   

/////////////////////////////////////////////////////%

7

->
(a)
/
\

I E
\\ 7%/////////////////////////////////////////%//////////,/%

Figure 38. Transmittance (a) and reﬂectance (b) signiﬁcance for color

 

measurement.

157
was observed for the films. Chatterji (1989) reported this color change when the

color of gelatin granules turned from pale yellow to deep orange within minutes
upon cross-linking of the gelatin with glutaraldehyde. There is a significant
increase in the “a” (red) and “b” (yellow). color components for these ﬁlms. As
observed in Table 19, the color of the ﬁlms changed from light yellow, with
values of about 40 for the non-cross-linked to over 60 for the glutaraldehyde-
‘ cross-linked ﬁlms. The red-green component, “a”, remained very close to 0,
which in practice meant that the main color observed for all ﬁlms (except for the
glutaraldehyde cross-linked) had a yellow tone.

The same observations can be made for the ﬁlms studied in the
luminous reﬂectance mode. The values'were lower since most of the incident
light was absorbed by the black background and only the color reﬂected by the
ﬁlm was picked up by the machine. The main effect observed was related to
lightness: It was reduced about three-fold, while approximately a two-fold
reduction was observed for the yellowness. It was also observed that the
yellowness in the ﬁlms was reduced for the cysteine- and -formaldehyde-cross-
linked ﬁlms by a proportion of about 75% and 60% for the transmittance and
reﬂectance mode, respectively.

In practice, the luminous reﬂectance mode color values may be more
signiﬁcant for the protein ﬁlms studied. This is because if commercialized as
ﬁlms (with function of containing and protecting the food product), and since they
are not transparent, the primary contact that the consumer would have prior to

. opening and consuming the items contained would be the ﬁlms themselves.

158
On the other hand, if the FFS is cast directly on the food items (as a

coating) then the luminous transmittance mode would be possibly of more value.
This because the food product would be reflecting the transmitted light back to
the eyes of the consumer, and the color of the products inside the coatings will

look different than without them.

 

5. SUMMARY AND CONCLUSIONS

A new procedure for making ﬁlms from wheat proteins was developed.
Barrier, rheological, chemical, and physical properties were studied for different
films prepared using this methodology. Three types of ﬂours, two different pH
ﬁlm-forming solutions, three reducing (cross-linking) agents, and different test
temperatures and relative humidities were considered for the preparation of the
different wheat protein-based ﬁlms. From the tests performed to the ﬂours used
in this study, both farinography and SOS-PAGE results were the best methods to
aid in the selection of good ﬂours in ﬁlm making.

Oxygen permeability was affected primarily by the temperature and the
type of ﬂour used to form the ﬁlm. The pH of the solutions tested did not
signiﬁcantly inﬂuence this factor. The non-proportional change in permeability
for a speciﬁc temperature, with respect to the percent protein present in the ﬂour
sample, suggests that the permeability is affected not only by the quantity but
also by the type of protein (referred to astquality’ in baking technology) present
in the material used to make the ﬁlms. The elongation deformation was found
not to be affected by the use of cysteine, and the only signiﬁcant difference
found was the use of commercial bread flour as compared to the other two.

Tensile strength was found to be inﬂuenced by the cysteine and the
type of ﬂour (protein) used. Signiﬁcant differences were found for the elongation
values of the ﬁlms based on the type of ﬂour alone. Creep behavior of wheat

protein ﬁlms is very sensitive to differences in temperature and relative humidity.

159

160
Temperature was found to be the most Significant factor affecting the tensile

creep behavior, followed by relative humidity. Cross-linking of the biopolymers
produced a stronger ﬁlm capable of supporting higher loads, with formaldehyde-
cross—linked films being able to stand smaller deformations than glutaraldehyde-
cross-linked ﬁlms. Similarly, cross-linked ﬁlms were able to resist deformation at
high stresses as compared to the non-cross-linked ﬁlms.

Fourier-Transformed Infrared Spectrometry (FT lR) showed a pattern
similar to that of the nylons. Appearance of an absorption peak for the cross-
linked ﬁlms compared to the- uncross-li'nked "ones indicated that chemical
changes occurred during the cross-linking process.

Swelling Studies of ﬁlms helped to ﬁnd the value of the solubility
parameter, 6, for the ﬁlms studied. The ﬁlms were observed to have a maximum
swelling coefﬁcient when immersed in a solvent whose composition was similar
to that of the original FFS. This conﬁrmed that the main chemical groups that
aid in the interaction solvent-polymer molecule are kept and probably unaltered
while only speciﬁc groups react forming the network. Further, this may indicate
that the chemical properties of the cross-linked molecules are similar to that of
the uncross-linked molecules. No swelling in solvents with low values of 6 was
observed on the protein ﬁlms studied.

The color analysis of the ﬁlms indicated that a reduction in the
yellowness occur with cross-linking using cysteine or formaldehyde, as

compared to the uncross-linked ﬁlms. On the other hand, a signiﬁcant increase

161
in yellowness and redness (whose combination yields orange) is observed for

the ﬁlms cross-linked with glutaraldehyde.

The development and analysis of edible/degradable films is relatively
new and thus the area of study is broad as new techniques for the analysis of
these ﬁlms are introduced. In general, techniques used with synthetic polymeric
materials can be applied towards the study of these new materials, because they
are formed from biopolymers. Both areas, Food and Polymer Science converge
when studies of these materials are required.

In general, there is interest by the food industry and general public
towards the development of a successful, useful package that will serve as a
nutrient if consumed, or will be environmentally-friendly if disposed. This
possibility is offered by the development of ﬁlms and coatings made from
polymers that nature provides. However, pilot-plant studies and new techniques

to produce these materials on a large scale are still required.

6. SUGGESTIONS FOR FUTURE RESEARCH

There is a need to looking for more quantitative methods to
characterize ﬂours for their film-making-potential. This could lead to define good
specific methods for ﬂour selection in ﬁlm making. Molecular weight
determination for the uncrosslinked ﬁlms. by SDS-PAGE seems possible and
different densities of the cross-linked SDS-PAGE gels may be experimented to
obtain good separation and resolution. As for the cross-linked ﬁlms,
determination of the Flory-Huggins polymer-solvent dimensionless interaction
term, X1. and application of the Flory-Rehner theory principle’s could be used to f
determine the molecular weight between cross-links. This could be tried with
different cross-linkers and at different concentration levels.

Further studies are needed to investigate the possible differences
between the type of protein present in the material as how it inﬂuences the
oxygen permeability of ﬁlms made from wheat ﬂour. There is also need to test
for differences in color by using different variables (e.g., pH, solvents, prea
treatment of ﬂour, etc.) on the initial ﬂour or the solutions prepared since the
pigments present in the FF 8 may be affected by this factor. This is supported by
simple visual comparison of ﬁlms that were made using the same procedure
except for the pH of the solution. In addition, in order to estimate the shelf-life
for speciﬁc applications, ﬁlm aging studies could be considered given that, as
suggested in the literature, association of protein chains via dissulﬁde bonds

slowly increases during aging.

152

163
Over the last ten years scientists have been linking the theory and

analysis of polymer rheology, processing, engineering, and science as it is
related to synthetic polymers and starting to apply this information to the study of
the edible/degradable ﬁlms. There is, however, need for more work on these
converging areas so that less general and more conclusive statements can be
drawn with respect to the relationship between film chemistry and ﬁlm properties.
Particularly important in this respect is the application of polymer rheology and
processing to commercialization of wheat protein films.

Finally, this study opens the possibility of using different techniques to
modify or test variables that could improve the overall performance of the ﬁlms
formed by using this new methodology. These could include improvements
and/or studies on both the engineering of the ﬁlm forming solutions (rheological

methods) and the properties of the resulting ﬁlms.

APPENDIX

1‘ ‘11:

APPENDIX A

-. or 0 WW: ._

- u

-up‘re‘ null-.01

The ofﬁcial method AACC 56-81A recommends correction of the

sample weight used in the determination of the Falling Number. Table A1 shows

the required sample weight, at different moisture contents, corresponding to 7

grams at 14% moisture (no change is made in the quantity of water used).

Table A1. Correction of sample weight to 14% moisture basis (AACC 56-81A).*

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

% ”ism” .0 .2 .4 .5 .8
in sample
8 6.54 6.56 6.57 6.59 6.60
9 6.62 6.63 6.64 6.66 6.67
10 6.69 6.70 6.72 6.73 6.75
11 6.76 6.78 6.80 6.81 6.83
12 6.84 6.86 6.87 6.89 6.90
13 6.92 6.94 6.95 6.97 6.98
14 7.00 7.02 7.03 7.04 7.07
15 7.08 7.10 7.12 7.13 7.15
16 7.17 7.18 7.20 7.22 7.24
17 7.25 7.27 7.297 7.31 7.32
grams needed for test

 

 

* From Perten (1988)

154

 

 

APPENDIX B

111-010 ‘0, 2."- '0. o 011‘ r 10 -‘,o -. -.‘-. '1']

An adjustment in the cross-sectional area obtained experimentally was

necessary to get more accurate values of the stress measured at a given time,

since stress, by deﬁnition, was calculated by dividing force over the cross-

sectional area. Figure B1 shows the relationship of the cross-sectional area as a

function of the displacement observed.

Cross-sectional area, CSA (m 2)

2.15E-06 .-- 1-11am-

 

 
   

2.105-05 .-

; CSA = 00000055393 (5L) + 0.0000021
2.055-05 J.
2.005-05 4..
1.955-05 .-
1905-05 -
1.855-05 ._

1.80E-06 ._ --.. -
0 0.01 0.02 0.03 0.04 0.05

5L (m)

 

 

Figure B1. Cross-sectional area of ﬁlms as a function of grip displacement for

correction of tensile creep data.

165

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166

APPENDIX D

Samples of MDSC curves for ﬁlms. Curves for the other ﬁlm types were similar.

.4 .o L. ancestor L“:
vez':: *2: -55°: '3 :84 ": Ben Cate 6-A8;-35 22 SA

::*~e~t '; 5 TM dete'nznatzon

 

 

 

 

 

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”-0 20
-0 3 3 5° 0 50 100 so 00 go'0 25
-10 - ‘
Temperatures 1°c1 “058 V! II '4 son: 2200
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LJ E5 Lu Joerggar (an
“on Date ?-Aue-95 00 27
0 2

 

Ned! I In- III/0|

-0 2'1 \

1100 -50 0 so 16 50

 

 

 

Figure D. MDSC curves for a glutaraldehyde-cross-linked wheat protein ﬁlm.
(1) shows total heat ﬂow, along with reversible (lower line) and non-
reversible (upper line) heat ﬂow.

(2) shows total heat ﬂow with a thermal change around 0°C
attributed to phase transition change of water.

167

APPENDIX E

Rh I i Im rem nt fth FF inh tin Iin ces iml in.
Figure E1 represents the output of the complex modulus (6") over a
temperature ramp (heating curve by the triangular marker; cooling curve by
rounded marker). Test settings for RheoStress” RS-100 were: o=0.5 Pa;

w=0.100 Hz; heating temperature ramp=2°Clmin from 25 to about 80°C

(simulating heating of the FFS during the actual process). The solution was
immediately cooled down at 2°C/min. This showed an irreversible thermal
change of the wheat proteins in the F FS, since 6* relates to the increase of the

more elastic behavior provided by the unfolding of the protein molecules.

 

 

 

 

 

 

 

[Pa]

6.

"TI”: 3; .......... :l :“H;-UDI as:
_‘— 3'

, none-0111.. 050

 

_.- J!

 

 

 

T 2°21
lDSZJO-OSC 1.3.‘ Sensor 52 Ster:33§C<0.250m-.): SgstemQSICO:
laceratorzeuis ". al.,IS: -omoangztcoc Rheology Laboratcru: Hismrqﬁ'rs (9" 4)
Lose-rm. as: 2.5: 9a .12: a: 25.20 - 80.0: of

 

 

Figure E1. Thermal change of wheat proteins observed in the FFS.

168

APPENDIX F

The data points of Figures 17-30 were obtained from the raw experimental
values of Table F1. "
Note: N = non-cross-linked.

G = glutaraldehyde-cross-linked.

F = formaldehyde-cross-linked.

Table F1. Raw data for calculation of creep curves.

 

I ’ 5°C; 85 %RH; N I

 

Time Time Distance 6L area 0 LILo 8,, J
(min) (s1 (cm) (m1 (m2) Ma) Ma")

 

 

0.00 0 8.10 0.00E+00 1.83E-06 2.78689 1.00000 0.00000 0.00000

 

 

 

 

 

 

 

 

 

0.02 1 18.50 1.04E-01 1.15E-06 4.43684 4.15152 1.42347 0.32083

 

 

j 5°c; 85 %RH; G |
Time Time Distance 6L area 0 ULo 8,. J
(min) (s) (cm) (m) (m2) tMPa) (MPa‘)

 

0 00 0 8.20 0.00E+00 2.29E-06 2.22951 1.00000 0.00000 0.00000

 

0:50 30 20.70 1.25E-01 2.20E-06 2.31219 4.78788 1.56609 0.67732

 

1.00 60 22.00 1.38E-01 2.20E-06 2.32114 5.18182 1.64516 0.70877

 

1.50 90 22.80 1.46E-01 2.19E-06 2.32668 5.42424 1.69088 0.72673

 

2.00 120 23.30 1.51E-01 2.19506 2.33016 5.57576 1.71843 0.73747

 

2.50 150 23.70 1.55E-01 2.18E-06 2.33295 5.69697 1.73993 0.74581

 

3.00 180 23.90 1.57E-01 2.18E-06 2.33435 5.75758 1.75052 0.74990

 

3.50 210 24.20 1.60E-01 2.18E-06 2.33645 5.84848 1.76618 0.75593

 

4.00 240 24.40 1.62E-01 2.18E-06 2.33785 5.90909 1.77649 0.75988

 

4.50 270 24.50 1.63E-01 2.18E-06 2.33855 593939178161 0.76184

 

5.00 300 24.70 1.65E-01 2.18E-06 2.33995 6.00000 1.79176 0.76572

 

5.50 330 24.80 1.66E-01 2.18E-06 2.34066 6.03030 1.79680 0.76765

 

6.00 360 25.00 1.68E-01 2.18E—06 2.34206 6.09091 1.80680 0.77146

 

6.50 390 25.10 1.69E-01 2.18E-06 2.34277 6.12121 1.81176 0.77334

 

 

 

 

 

 

 

 

 

 

7.00 420 25.20 1.70E-01 2.17E-06 2.34347 6.15152 1.81670 0.77522

 

 

J 5°C; 85 %RH; F J
Time Time Distance 6L area a L/Lo 8.. J
(min) ls) (cm) (m) (m’) (We) (MPa“>

 

0.00 0 8.10 0.00E+00 2.06E—06 2.47723 1.00000 0.00000 0.00000

 

0.08 5 16.20 8.10E-02 2.00E-06 2.54270 3.45455 1.23969 0.48755

 

0.17 10 18.00 9.90E—02 1.99E-06 2.55772 4.00000 1.38629 0.54200

 

0.25 15 18.70 1.06E-01 1.99E-06 2.56360 4.21212 1.43797 0.56092

 

 

0.33 20 19.30 1,12E-01 1.98E-06 2.56867 4.39394 1.48023 0.57626

 

 

 

 

 

 

 

 

 

169'

 

 

 

 

170

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.42 25 19.60 1.15E—01 1.98E-06 2.57122 4.48485 1.50070 0.58366
0.50 30 20.00 1.19E-01 1.98E-06 2.57461 4.60606 1.52737 0.59324
0.58 35 20.30 1.22E-01 1.98E-06 2.57717 4.69697 1.54692 0.60024
0.67 40 20.60 1.25E-01 1.98E-06 2.57973 4.78788 1.56609 0.60707
0.75 45 20.80 1.27E-01 1.97E-06 2.58144 4.84848 1.57867 0.61155
0.83 50 20.90 1.28E-01 1.97E-06 2.58229 4.87879 1.58490 0.61376
0.92 55 21.10 1.30E-01 1.97E-06 2.58400 4.93939 1.59724 0.61813
1.00 60 21.30 1.32E-01 1.97E-06 2.58572 5.00000 1.60944 0.62243
1.08 65 21.50 1.34E-01 1.97E-06 2.58743 5.06061 1.62149 0.62668
1.17 70 21.60 1.35E-01 1.97E-06 2.58829 5.09091 1.62746 0.62878
1.25 75 21.80 1.37E-01 1.97E-06 2.59001 5.15152 1.63929 0.63293
1.33 80 21.90 1.38E-01 1.97E-06 2.59087 5.18182 1.64516 0.63498
1.42 85 21.90 1.38E-01 1.97E-06 2.59087 5.18182 1.64516 0.63498
I 10°C; 40 %RH; N I

Time Time Distance 6L area a ULo 8.. J

(min) (s) (cm) (m1 (m2) (MPa) LMPa")
0.00 0 8.10 0.00E+00 1.83E-06 2.78692 1.00000 0.00000 0.00000
0.10 6 8.60 5.00E-03 1.80E-06 2.83765 1.15152 0.14108 0.04972
0.20 12 9.10 1.00E-02 1.76E-06 2.89026 1.30303 0.26469 0.09158
0.30 18 9.60 1.50E-02 1.73E-06 2.94487 1.45455 0.37469 0.12724
0.40 24 10.00 1.90E-02 1.70E-06 2.99006 1.57576 0.45474 0.15208
0.50 30 10.50 2.40E-02 1.67E-06 3.04853 1.72727 0.54654 0.17928
0.60 36 11.00 2.90E-02 1.64E-06 3.10934 1.87879 0.63063 0.20282
0.70 42 11.60 3.50E-02 1.60E-06 3.18559 2.06061 0.72300 0.22696

[ 10°C; 40 %RH; G |

Time Time Distance 6L area 0 L/Lo 8,. J

(min) (s) (cm) (m) (nﬂ (MPa) <MPa“)
0.00 0 7.90 0.00E+00 2.51 E-06 2.02685 1.00000 0.00000 0.00000
5.00 300 8.90 1.00E-02 2.45E-06 2.08096 1.30303 0.26469 0.12720
10.00 600 9.80 1.90E-02 2.39E-06 2.13220 1.57576 0.45474 0.21327
15.00 900 10.50 2.60E-02 2.34E-06 2.17383 1.78788 0.58103 0.26728
20.00 1200 11.20 3.30E-02 2.30E-06 2.21711 2.00000 0.69315 0.31263
25.00 1500 11.70 3.80E-02 2.27E-06 2.24910 2.15152 0.76617 0.34066
30.00 1800 12.10 4.20E-02 2.24E-06 2.27537 2.27273 0.82098 0.36081
35.00 2100 12.50 4.60E-02 2.21E-06 2.30225 2.39394 0.87294 0.37917
40.00 2400 12.80 4.90E-02 2.19E-06 2.32284 2.48485 0.91021 0.39185
45.00 2700 13.10 5.20E-02 2.17E-06 2.34379 2.57576 0.94614 0.40368
50.00 3000 13.40 5.50E-02 2.15E-06 2.36513 2.66667 0.98083 0.41470
55.00 3300 13.60 5.70E-02 2.14E-06 2.37957 2.72727 1.00330 0.42163
60.00 3600 13.80 5.90E-02 2.13E-06 2.39419 2.78788 1.02528 0.42824
65.00 3900 13.90 6.00E-02 2.12E-06 2.40157 2.81818 1.03609 0.43142
70.00 4200 14.10 6.20E-02 2.11E-06 2.41646 2.87879 1.05737 0.43757

 

 

 

 

 

 

 

 

 

 

 

 

171

 

75.00

4500

14.30

6.40E-02

2.10E-06

2.43154

2.93939

1 .07820

0.44342

 

80.00

4800

14.50

6.60E-02

2.08E-06

2.44680

3.00000

1.09861

0.44900

 

85.00

5100

14.60

6.70E-02

2.08E-06

2.45451

3.03030

1.10866

0.45168

 

 

90.00

 

5400

 

14.70

 

6.80E-02

 

2 .07E-06

 

2.46226

 

3.06061

 

1.11861

 

 

0.45430

 

10 °C; 40 %RH; F

 

Time
(min)

Time

(5)

Distance

(cm)

5L
1m)

area
(m2)

0'
(MPa)

ULo

8h

J
(MPa")

 

0.00

7.90

0.00E+00

2.13E-06

2.38879

1 .00000

0.00000

0.00000

 

5.00

300

8.30

4.00E-03

2.11E-06

2.41843

1.12121

0.11441

0.04731

 

10.00

600

8.70

8.00E-03

2.08E-06

2.44883

1 .24242

0.21706

0.08864

 

15.00

900

9.20

1 .30E-02

2.05E-06

2.48791

1 .39394

0.33213

0.13350

 

20.00

1200

9.50

1 .60E-02

2.03E-06

2.51197

1 .48485

0.39531

0.15737

 

25.00

1 500

9.90

2005-02

2005-06

2.54478

1 .60606

0.47378

0.18618

 

30.00

1 800

10.20

2.30E-02

1.98E-06

2. 56995

1 .69697

0.52884

0.20578

 

35.00

2100

10.40

2.50E-02

1 .97E-06

2.58701

1.75758

0.56394

0.21799

 

40.00

2400

10.70

2.80E-02

1 .95E-06

2.61303

1.84848

0.61437

0.23512

 

45.00

2700

10.90

3.00E-02

1 .94E-06

2.63067

1 .90909

0.64663

0.24580

 

50.00

3000

11.10

3.20E-02

1 .925-06

2.64855

1 .96970

0.67788

0.25594

 

55.00

3300

11.30

3.40E-02

1.91 E-06

2.66667

2.03030

0.70819

0.26557

 

60.00

3600

11.40

3.50E-02

1 .90E-06

2.67583

2.06061

0.72300

0.27020

 

65.00

3900

11.60

3.70E-02

1 895-06

2.69433

2.12121

0.75199

0.27910

 

70.00

4200

11.70

3.80E-02

1 .89E-06

2.70367

2.15152

0.76617

0.28338

 

75.00

4500

11.80

3.90E-02

1 .88E-06

2.71308

2.18182

0.78016

0.28755

 

80.00

4800

11.90

4.00E-02

1.87E-06

2.72256

2.21212

0.79395

0.29162

 

85.00

5100

12.10

4.20E—02

1 .86E-06

2.74172

2.27273

0.82098

0.29944

 

 

90.00

 

5400

 

12.20

 

4.30E-02

 

1 .85E-06

 

2.75139

 

2.30303

 

0.83423

 

 

0.30320

 

20°C; 35 %RH; N

 

Time
(min)

Time

(5)

Distance

(cm)

6L
(m)

area
(m2)

0’
(MPa)

L/Lo

8h

J
(MPa")

 

000

8.20

0.00E+00

1 835-06

2.78689

1 .00000

0.00000

0.00000

 

0.03

8.50

3.00E-03

1.81 E-06

2.81711

1 .09091

0.08701

0.03089

 

0.07

8.70

5.00E-03

1 .80E-06

2.83762

1.15152

0.14108

0.04972

 

0.10

9.00

8.00E-03

1 .78E-06

2.86896

1 .24242

0.21706

0.07566

 

0.13

9.20

1 .00E—02

1 .76E-06

2.89023

1 .30303

0.26469

0.09158

 

 

0.17

 

..L
000015160

 

9.60

 

1.40E-02

 

1.74E-06

 

2.93375

 

1 .42424

 

0.35364

 

0.12054

 

20°C; 35 %RH; G

 

Time
(min)

Time

(5)

Distance

(cm)

5L
(m)

area
(m2)

0'
(MPa)

L/Lo

8h

J
(MPa‘)

 

0.00

8.00

0.00E+00

2.29E-06

2.22951

1 .00000

0.00000

0.00000

 

10.00

600

8.10

1 .00E-03

2.28E-06

2.23591

1 .03030

0.02985

0.01335

 

20.00

1 200

8.15

1 .50E-03

2.28506

2.23912

1.04545

0.04445

0.01985

 

 

30.00

1 800

8.20

2.00E-03

2.27E-06

2.24234

1.06061

0.05884

0.02624

 

 

 

 

 

 

 

 

 

 

 

172

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

40.00 2400 8.20 2.00E-03 2.27E-06 2.24234 1.06061 0.05884 0.02624
50.00 3000 8.25 2.50E-03 2.27E-06 2.24557 1.07576 0.07303 0.03252
60.00 3600 8.30 3.00E-03 2.27E-06 2.24881 1.09091 0.08701 0.03869
70.00 4200 8.35 3.50E-03 2.26E-06 2.25206 1.10606 0.10080 0.04476
80.00 4800 8.40 4.00E-03 2.26E-06 2.25532 1.12121 0.11441 0.05073
90.00 5400 8.40 4.00E-03 2.26E-06 2.25532 1.12121 0.11441 0.05073
100.00 6000 8.45 4.50E-03 2.26E-06 2.25858 1.13636 0.12783 0.05660
110.00 6600 8.45 4.50E-03 2.26E-06 2.25858 1.13636 0.12783 0.05660
120.00 7200 8.50 5.00E-03 2.25E-06 2.26186 1.15152 0.14108 0.06237
I 20°c; 35 %RH; F I
Time Time Distance 6L area 0 ULo 8,. J
(min) (8) (cm) (m) ("12) (MPa) (MPa'U
0.00 0 8.10 0.00E+00 2.06E-06 2.47723 1.00000 0.00000 0.00000
10.00 600 8.30 2.00E-03 2.04E-06 2.49308 1.06061 0.05884 0.02360
20.00 1200 8.50 4.00E-03 2.03E-06 2.50913 1.12121 0.11441 0.04560
30.00 1800 8.65 5.50E-03 2.02E-06 2.52131 1.16667 0.15415 0.06114
40.00 2400 8.75 6.50E-03 2.01E-06 2.52949 1.19697 0.17979 0.07108
50.00 3000 8.90 8.00E-03 2.01 E-06 2.54187 1.24242 0.21706 0.08540
60.00 3600 8.95 8.50E-03 2.00E-06 2.54602 1.25758 0.22919 0.09002
70.00 4200 9.00 9.00E-03 2.00E-06 2.55018 1.27273 0.24116 0.09457
80.00 4800 9.10 1.00E-02 1.99E-06 2.55856 1.30303 0.26469 0.10345
90.00 5400 9.15 1.05E-02 1.99E-06 2.56276 1.31818 0.27625 0.10780
100.00 6000 9.20 1.10E-02 1.99E-06 2.56698 1.33333 0.28768 0.11207
110.00 6600 9.20 1.10E-02 1.99E-06 2.56698 1.33333 0.28768 0.11207
120.00 7200 9.25 1.155-02 1.98E-06 2.57122 1.34848 0.29898 0.11628
I 20°C; 55 %RH; N I
Time Time Distance 6L area a LILo 8.. J
(min) (s) (cm) (m1 (m2) (MPa) (MPa")
0.00 0 8.00 0.00E+00 1.83E-06 2.78689 1.00000 0.00000 0.00000
0.02 1 12.50 4.50E-02 1.53E-06 3.32131 2.36364 0.86020 0.25899
0.03 2 18.50 1.05E-01 1.14E-06 4.46224 4.18182 1.43075 0.32063
I 20°C; 55 %RH; G I
Time Time Distance 6L area 0 LILo 8,, J
(min) (s) (cm) (m1 (m2) (MP5 (MPa“)
0.00 0 8.20 0.00E+00 2.29E—06 2.22951 1.00000 0.00000 0.00000
5.00 300 10.50 2.30E-02 2.14E-06 2.38653 1.69697 0.52884 0.22160
10.00 600 11.50 3.30E-02 2.07E-06 2.46191 2.00000 0.69315 0.28155
15.00 900 12.20 4.00E-02 2.02E-06 2.51758 2.21212 0.79395 0.31536
20.00 1200 12.60 4.40E-02 2.00E-06 2.55053 2.33333 0.84730 0.33220
25.00 1500 13.00 4.80E-02. 1.97E-06 2.58436 2.45455 0.89794 0.34745
30.00 1800 13.40 5.20E-02 1.95E-06 2.61910 2.57576 0.94614 0.36125
35.00 2100 13.60 5.40E-02 1.93E-06 2.63682 2.63636 0.96940 0.36764

 

 

 

 

 

 

 

 

 

 

 

 

 

173

 

40.00

2400

13.80

5.60E-02

1.92E-06

2.65479

2.69697

0.99213

0.37371

 

45.00

2700

13.90

5.70E-02

1.91E-06

2.66386

2.72727

1 .00330

0.37663

 

50.00

3000

14.20

6.00E-02

1 .89E-06

2.69146

2.81818

1 .03609

0.38496

 

55.00

3300

14.40

6.20E-02

1 .88E-06

2.71018

2.87879

1 .05737

0.39015

 

60.00

3600

14.50

6.30E-02

1 .87E-06

2.71963

2.90909

1 .06784

0.39264

 

65.00

3900

14.60

6.40E-02

1.87E-06

2.72916

2.93939

1.07820

0.39507

 

70.00

4200

14.70

6.50E-02

1 .86E-06

2.73875

2.96970

1 .08846

0.39743

 

75.00

4500

14.80

6.60E-02

1 .85E-06

2.74840

3.00000

1 .09861

0.39973

 

80.00

4800

14.80

6.60E-02

1 .85E-06

2.74840

3.00000

1 .09861

0.39973

 

85.00

5100

14.90

6.70E-02

1 .85E-06

2.75813

3.03030

1.10866

0.40196

 

90.00

5400

15.00

6.80E-02

1 .84E-06

2.76792

3.06061

1.11861

0.40413

 

95.00

5700

15.00

6.80E-02

1.84E-06

2.76792

3.06061

1.11861

0.40413

 

100.00

6000

15.05

6.85E-02

1.84E-06

2.77285

3.07576

1.12355

0.40520

 

105.00

6300

15.10

6.90E-02

1 .835-06

2.77779

3.09091

1.12847

0.40625

 

110.00

6600

15.15

6.95E-02

1 .83E-06

2.78275

3.1 0606

1.13336

0.40728

 

115.00

6900

15.15

6.95E-02

1 .83E-06

2.78275

3.10606

1.13336

0.40728

 

 

120.00

 

7200

 

15.20

 

7.00E-02

 

1 .83E-06

 

2.78773

 

3.12121

 

1.13822

 

0.40830

 

20°C; 55 %RH; F

I

 

Time
(min)

Time

18)

Distance

AWL

6L
(m)

area
(m2)

0’
(MPa)

ULo

8h

J
(MPa")

 

0.00

8.20

0.00E+00

2.06E-06

2.47723

1 .00000

0.00000

0.00000

 

5.00

300

10.70

2.50E-02

1 .89E-06

2.69107

1 .75758

0.56394

0.20956

 

10.00

600

11.70

3.50E-02

1 .83E-06

2.78731

2.06061

0.72300

0.25939

 

15.00

900

12.40

4.20E-02

1.78E-06

2.85887

2.27273

0.82098

0.28717

 

20.00

1200

12.90

4.70E-02

1 .755-06

2.91229

2.42424

0.88552

0.30406

 

25.00

1 500

13.30

5.10E-02

1.72E-06

2.95648

2.54545

0.93431

0.31602

 

30.00

1 800

13.60

5.40E-02

1 .70E—06

2.99051

2.63636

0.96940

0.32416

 

35.00

2100

13.80

5.60E-02

1 .69E-06

3.01363

2.69697

0.99213

0.32921

 

40.00

2400

14.00

5.80E-02

1 .68E-06

3.03712

2.75758

1.01435

0.33398

 

45.00

2700

14.30

6.10E-02

1.66E-06

3.07305

2.84848

1 .04679

0.34063

 

50.00

3000

14.50

6.30E—02

1 .65E-06

3.09747

2.90909

1.06784

0.34475

 

55.00

3300

14.60

6.40E-02

1.64E-06

3.1 0983

2.93939

1 .07820

0.34671

 

60.00

3600

14.70

6.50E-02

1 .63E-06

3.12229

2.96970

1 .08846

0.34861

 

65.00

3900

14.80

6.60E-02

1.63E-06

3.13485

3.00000

1.09861

0.35045

 

70.00

4200

14.90

6.70E-02

1 .62E—06

3.14751

3.03030

1.10866

0.35224

 

75.00

4500

15.00

6.80E-02

1.61 E—06

3.16027

3.06061

1.11861

0.35396

 

80.00

4800

15.10

6.90E-02

1.61E-06

3.17314

3.09091

1.12847

0.35563

 

85.00

5100

15.15

6.95E-02

1 .60E-06

3.17961

3.10606

1.13336

0.35644

 

90.00

5400

15.20

7.00E-02

1.60E-06

3.18611

3.12121

1.13822

0.35725

 

95.00

5700

15.25

7.05E-02

1 .60E-06

3. 1 9263

3.13636

1.14306

0.35803

 

100.00

6000

15.35

7.15E-02

1 .59Ev06

3.20577

3.16667

1.15268

0.35956

 

105.00

6300

15.35

7.15E—02

1 .59E-06

3.20577

3. 16667

1.15268

0.35956

 

 

11 0.00

 

6600

 

15.35

 

7.15E-02

 

1 .59E-06

 

3.20577

 

3.16667

 

1.15268

 

0.35956

 

 

 

 

174

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

@1500 6900 15.40 7.20E-02 1.59E-06 3.21237 3.18182 1.15745 0.36031]
ﬁ20.00 7200 15.40 7.20E-02 1.59E-06 3.21237 3.18182 1.15745 0.36031I
I 25°C; 25 %RH; N |

Time Time Distance 6L area a LlLo 8.. J
(min) (5) (cm) (m) (m2) (MPa) (MPa’1)
0.00 0 8.20 0.00E+00 1.98E-06 2.57251 1.00000 0.00000 0.00000
0.03 2 10.20 2.00E-02 1.85E-06 2.75433 1.60606 0.47378 0.17201
0.07 4 12.20 4.00E-02 1.72E-06 2.96381 2.21212 0.79395 0.26788
0.10 6 14.20 6.00E-02 1595-06 3.20778 2.81818 1.03609 0.32299
0.13 8 16.10 7.90E-02 1.46E-06 3.47991 3.39394 1.22199 0.35116
0.17 10 17.30 9.10E-02 1.39E-06 3.67691 3.75758 1.32377 0.36002
0.20 12 18.20 1.00E-01 1.33E-06 3.83995 4.03030 1.39384 0.36298
| 25°C; 25 %RH; G |
Time Time Distance 6L area 0 ULo 8., J
(min) 1s) (cm) (m) ("ﬂ (MPa) (MPa">
0.00 0 8.20 0.00E+00 1.37E-06 3.71585 1.00000 0.00000 0.00000
0.17 10 8.80 6.00E-03 1.33E-06 3.82528 1.18182 0.16705 0.04367
0.33 20 9.40 1.20E-02 1.29E-06 3.94134 1.36364 0.31015 0.07869
0.50 30 9.80 1.60E-02 1.27E-06 4.02271 1.48485 0.39531 0.09827
0.67 40 10.50 2.30E-02 1.22E-06 4.17350 1.69697 0.52884 0.12671
0.83 50 10.90 2.70802 1.20E-06 4.26485 1.81818 0.59784 0.14018
1.00 60 11.50 3.30E-02 1165-06 4.40962 2.00000 0.69315 0.15719
1.17 70 12.00 3.80E-02 1.12E-06 4.53800 2.15152 0.76617 0.16883
1.33 80 12.50 4.30E—02 1.09E-06 4.67407 2.30303 0.83423 0.17848
1.50 90 12.90 4.70E-02 1.06E-06 4.78895 2.42424 0.88552 0.18491
1.67 100 13.30 5.10E-02 1.04E-06 4.90962 2.54545 0.93431 0.19030
1.83 110 13.60 5.40E-02 1.02E-06 5.00419 2.63636 0.96940 0.19372
2.00 120 13.90 5.70E-02 9.99E—07 5.10247 2.72727 1.00330 0.19663
2.17 130 14.20 6.00E—02 9.79E-07 5.20469 2.81818 1.03609 0.19907
2.33 140 14.50 6.30E-02 9.60E-07 5.31109 2.90909 1.06784 0.20106
2.50 150 14.70 6.50E-02 9.47E-07 5.38448 2.96970 1.08846 0.20215
2.67 160 14.90 6.70E-02 9.33E-07 5.45992 3.03030 1.10866 0.20305
2.83 170 15.10 6.90E-02 9.20E-07 5.53750 3.09091 1.12847 0.20379
3.00 180 15.30 7.10E-02 9.07E-07 5.61732 3.15152 1.14788 0.20435
3.17 190 15.50 7.30E-02 8.94E-07 5.69948 3.21212 1.16693 0.20474
3.33 200 15.70 7.50E-02 8.81 E-07 5.78407 3.27273 1.18562 0.20498
3.50 210 15.90 7.70E-02 8.68E-07 5.87122 3.33333 1.20397 0.20506
3.67 220 16.00 7.80E-02 8.62E-07 5.91578 3.36364 1.21302 0.20505
I 25°C; 25 %RH; F I
ITime Time Distance 6L area a L/Lo 8., J I
(min) (s) (cm) (m1 (m2) 1MPa) (MPa‘D
I 0.00 0 8.20 0.00E+00 1.68E-06 3.04027 1.00000 0.00000 0.00000]

 

 

 

 

 

 

 

 

 

 

175

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.00 60 9.70 1.50E-02 1.58E-06 3.22922 1.45455 0.37469 0.11603
2.00 120 10.70 2.50E-02 1.51 E-06 3.36880 1.75758 0.56394 0.16740
3.00 180 11.50 3.30E-02 1.46E-06 3.48946 2.00000 0.69315 0.19864
4.00 240 12.10 3.90E-02 1.42E-06 3.58578 2.18182 0.78016 0.21757
5.00 300 12.50 4.30E-02 1.40E-06 3.65300 2.30303 0.83423 0.22837
6.00 360 12.90 4.70E-02 1.37E-06 3.72280 2.42424 0.88552 0.23786
7.00 420 13.20 5.00E-02 1.35E-06 3.77692 2.51515 0.92233 0.24420
8.00 480 13.50 5.30E-02 1.33E-06 3.83264 2.60606 0.95784 0.24992
9.00 540 13.80 5.60E-02 1.31 E-06 3.89002 2.69697 0.99213 0.25504
10.00 600 14.00 5.80E-02 1.30E-06 3.92925 2.75758 1.01435 0.25815
11.00 660 14.20 6.00E-02 1.28E-06 3.96927 2.81818 1.03609 0.26103
12.00 720 14.40 6.20E-02 1.27E-06 4.01011 2.87879 1.05737 0.26368
13.00 780 14.50 6.30E-02 1.26E-06 4.03085 2.90909 1.06784 0.26492
14.00 840 14.70 6.50E—02 1.25E-06 4.07298 2.96970 1.08846 0.26724
15.00 900 14.90 6.70E—02 1.24E—06 4.11600 3.03030 1.10866 0.26935
I 25°C; 30 %RH; N I
Time Time Distance 6L area 5 ULo 8., J
(min) (sec) (cm) (m) (m2) (MPa) (MPa")
0.00 0 8.20 0.00E+00 1.37E-06 3.71585 1.00000 0.00000 0.00000
0.03 2 9.30 1.10E-02 1.30E-06 3.92151 1.33333 0.28768 0.07336
0.07 4 10.20 2.00E-02 1.24E-06 4.10751 1.60606 0.47378 0.11535
I 25°C; 30 %RH; G I
Time Time Distance 6L area 6 ULo 8,. J
(min) 1s) (cm) (m1 (m2) (MPa) Me")
0.00 0 8.50 0.00E+00 1.37E-06 3.71585 1.00000 0.00000 0.00000
0.17 10 10.50 2.00E-02 1.24E-06 4.10751 1.60606 0.47378 0.11535
0.33 20 13.50 5.00E-02 1.04E-06 4.87889 2.51515 0.92233 0.18905
0.50 30 14.80 6.30E-02 9.60E-07 5.31109 2.90909 1.06784 0.20106
0.67 40 15.70 7.20E-02 9.01 E-07 5.65810 3.18182 1.15745 0.20457
0.83 50 16.20 7.70E-02 8.68E-07 5.87122 3.33333 1.20397 0.20506
1.00 60 16.70 8.20E-02 8.35E-07 6.10101 3.48485 1.24842 0.20464
1.17 70 17.20 8.70E-02 8.03E-07 6.34953 3.63636 1.29098 0.20334
I 25°C; 30 %RH; F I
Time Time Distance 6L area 0 LlLo 8.. J
(min) (S) (cm) (m) (m2) (MPa) (MPa'1)
0.00 0 8.50 0.00E+00 1.52E-06 3.34427 1.00000 0.00000 0.00000
0.33 20 10.30 1.80E-02 1.41E-06 3.62418 1.54545 0.43532 0.12011
0.67 40 11.40 2.90E-02 1.33E-06 3.81955 1.87879 0.63063 0.16510
1.00 60 12.10 3.60E—02 1.29E-06 3.95523 2.09091 0.73760 0.18649
1.33 80 12.70 4.20E~02 1.25E-06 4.07945 2.27273 0.82098 0.20125
1.67 100 13.10 4.60E-02 1.22E-06 4.16668 2.39394 0.87294 0.20950
22.00 120 13.50 5.00E-02 1.20E-06 4.25773 2.51515 0.92233 0.21663

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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2.33 140 13.80 5.30E-02 1.18E-06 4.32867 2.60606 0.95784 0.22128
2.67 160 14.10 5.60E-02 1.16E-06 4.40202 2.69697 0.99213 0.22538
3.00 180 14.30 5.80E-02 1.14E-06 4.45231 2.75758 1-01435 0.22783
3.33 200 14.50 6.00E-02 1.13E-06 4.50377 2.81818 1.03609 0.23005
3.67 220 14.70 6.20E-02 1.12E-06 4.55643 2.87879 1.05737 0.23206
4.00 240 14.90 6.40E-02 1.11E-06 4.61033 2.93939 1.07820 0.23387
4.33 260 15.00 6.50E-02 1.10E-06 4.63777 2.96970 1.08846 0.23469
4.67 280 15.20 6.70E-02 1.09E-06 4.69363 3.03030 1.10866 0.23621
5.00 300 15.30 6.80E-02 1.08E-06 4.72206 3.06061 1.11861 0.23689
5.33 320 15.50 7.00E-02 1.07E-06 4.77998 3.12121 1.13822 0.23812
5.67 340 15.60 7.10E-02 1.06E-06 4.80948 3.15152 1.14788 0.23867
6.00 360 ' 15.70 7.20E-02 1.05E-06 4.83934 3.18182 1.15745 0.23918
I 40°C; 25 %RH; N I
Time Time Distance 6L area 6 LILo 8., J
(min) (sec) (cm) (111) (m2) (MPa) (MPa'1)
0.00 0 8.20 0.00E+00 1.60E-06 3.18501 1.00000 0.00000 0.00000
0.03 2 10.20 2.00E-02 1.47E-06 3.46850 1.60606 0.47378 0.13660
0.07 4 12.80 4.60E-02 1.30E-06 3.92234 2.39394 0.87294 0.22256
I 40°c; 25 %RH; G |
Time Time Distance 6L area a ULo 8., J
min) (8) (cm) (m) ("12) (MPa) (MPa")
0.00 0 8.20 0.00E+00 1.60E-06 3.18501 1.00000 0.00000 0.00000
0.17 10 9.00 8.00E-03 1.55E—06 3.29266 1.24242 0.21706 0.06592
0.33 20 9.80 1.60E-02 1.50E-06 3.40783 1.48485 0.39531 0.11600
0.50 30 10.70 2.50E-02 1.44E-06 3.54743 1.75758 0.56394 0.15897
0.67 40 11.30 3.10E-02 1.40E-06 3.64703 1.93939 0.66238 0.18162
0.83 50 12.00 3.80E-02 1.35E-06 3.77054 2.15152 0.76617 0.20320
I 40°C; 25 %RH; F I
Time Time Distance 6L area a L/Lo 8., J
(min) (8) (CIT!) (m) ("12) (MP8) @1138")
0.00 0 8.20 0.00E+00 2.44E-06 2.09017 1.00000 0.00000 0.00000
0.17 10 16.50 8.30E-02 1.90E-06 2.68862 3.51515 1.25708 0.46756
0.33 20 18.50 1.03E-01 1.76E-06 2.88786 4.12121 1.41615 0.49038
0.50 30 19.50 1.13E-01 1.70E-06 2.99899 4.42424 1.48710 0.49587
0.67 40 20.20 1.20E—01 1.65E-06 3.08200 4.63636 1.53393 0.49771
0.83 50 20.90 1.27E-01 1.61E-06 3.16974 4.84848 1.57867 0.49804
1.00 60 21.30 1.31E-01 1.58E-06 3.22216 4.96970 1.60336 0.49760
1.17 70 21.70 1.355-01 1.56E-06 3.27634 5.09091 1.62746 0.49673

 

 

 

 

 

 

 

 

 

 

 

 

 

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