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CHARACTERIZATION OF HEAT CURED AND
TRANSGLUTAMINASE CROSS-LINKED WHEY PROTEIN-
BASED EDIBLE FILMS

presented by

SAMIR AMIN

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5/08 KzlProlecc8IPres/CIRC/DaIeDue.indd

CHARACTERIZATION OF HEAT CURED AND TRANSGLUTAMINASE
CROSS-LINKED WHEY PROTEIN-BASED EDIBLE FILMS
By

Samir Amin

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Food Science
2008

ABSTRACT
CHARACTERIZATION OF HEAT CURED AND TRANSGLUTAMINASE CROSS-
LINKED WHEY PROTEIN-BASED EDIBLE FILMS
By
Samir Amin
Edible whey protein ﬁlms were produced using whey protein isolate (WPI),
glycerol and candelilla wax. Films were also produced with the addition of
microbial transglutaminase (T G) to the ﬁlm forming solution alter heating. Both
sets of ﬁlms were vacuum heat cured for 0, 12 and 24h at 90°C. The effect of heat
curing and transglutaminase treatments on mechanical properties [tensile strength
(TS), elongation at break (%E) and toughness], the free suﬂhydryl, disulﬁde, and
lysine contents and degree of cross-linking of WPI ﬁlms were assessed. Heat
cun'ng increased the T8 of WPI and WPI/T G ﬁlms compared to uncured ﬁlms.
Heat curing ﬁlms for 24h had increased %E compared to other ﬁlms. Heat curing
increased the toughness of WPI ﬁlms. Heat curing decreased the free sulfhydryl
content of WPI and WPI/l" G ﬁlms. The disulﬁde bond content of WPI ﬁlms heat
cured for 12h was higher than for ﬁlms heat cured at 0 and 24h. The disulﬁde
content of WPI/T G ﬁlms heat cured for 12 and 24h was higher than for ﬁlms at 0
and 48h. Heat cured WPI ﬁlms had lower lysine content compared to uncured
ﬁlms. WPI/TG ﬁlms heat cured for 24h had lower lysine content compared to ﬁlms

heat cured at 0 and 12h. SDS-PAGE was used to conﬁrm heat curing and TG

treatment resulted in the formation of covalent cross-links, between whey protein
fractions.

The water vapor permeability (WVP) of WPI and WPI/T G ﬁlms heat cured
for 0 and 24h was determined at 37.8°C and 85% RH as well as the oxygen
permeability at 23°C and 0% RH. Moisture sorption isotherms (MSI) of ﬁlms were
determined at 23°C and 5°C using the Guggenheim-Anderson—de Boer equation
(GAB). Heat curing WPI/T G ﬁlms reduced the WVP compared to uncured WPI,
WPlfl' G and cured WPI ﬁlms. The addition of TG reduced the oxygen permeability
(02P) compared to ﬁlms without TG. Heat cun'ng reduced the OzP of ﬁlms
compared to uncured ﬁlms. Heat curing of WPI/T G ﬁlms produced the greatest
reduction in 02P of ﬁlms tested. The moisture sorption isotherm of WPI and
WPI/T G ﬁlms heat cured for 0 and 24h at 90°C were found to closely follow the
GAB model at 5 and 23°C. The ﬁlms MSI, at 5°C were higher than the MSI of ﬁlms
at 23°C.

The ultraviolet and visible light transmission and the color characteristics of
WPI and WPI/T G ﬁlms heat cured for 0 and 24h were assessed. Heat curing
increased the yellovmess of ﬁlms. All ﬁlms had low transmission of ultraviolet light

The effect of heat curing and transglutaminase treatments on the thermal
properties (melting onset temperature, peak melting temperature and enthalpy of
fusion) of whey protein isolate ﬁlms with and without transglutaminase, and heat
cured at 90°C for 0, 12 and 24h was assessed. The peak melting temperatures for

ﬁlms increased with heat curing and time.

Copyright by
Samir Amin
2008

This dissertation is dedicated to Theresa, Chandrakant and Marilyn

ACKNOWLEDGMENTS

“If I have seen further it is by standing on the shoulders of giants.”

Isaac Newton, Letter to Robert Hooke, February 5, 1675
English mathematician & physicist (1642 - 1727)

I wish to express my appreciation to my advisors, Dr. Bruce Harte and Dr.
Alden Booren. Their guidance, support and encouragement were invaluable in the
completion of my Ph.D. I would also like to thank my other committee members Dr.
Gale Strasburg and Dr. Perry Ng whose suggestions helped to shape and guide
my research.

I wish to thank the many people who were instrumental in helping me to
complete my PhD. First I wish to thank my ﬁancée Theresa Martin and my father
Chandrakant Amin M.D. without their love and support I would have never under
taken such a monumental task, let alone been able to complete it. I wish to thank
Dustin Martin for providing motivation support even when he didn’t realize it. I wish
to thank my friends Ron Bigge, Rick Machung, Frank Monaco and Nick Monaco
for being there to chat with when I need to. To Keith Vorst and Lindsey Keskinen
thanks for the stimulating intellectual conversation we had in the graduate ofﬁce.
To Rafael Auras and Kn'ttika Tanprasert for all your help with mechanical, barrier
and UVNIS testing and for allowing me to pick your brains, thank you.

For those I have not acknowledged by name, thank you for your support

both professional and personal.

vi

Table of Contents

List of Tables ........................................................................................................... x

List of Figures ....................................................................................................... xiii

Abbreviations used in this thesis ....................................................................... xiv

Introduction ............................................................................................................. 1

Literature Review .................................................................................................... 5

1.1 Edible Films ................................................................................................. 5

1.1.1 Deﬁnition and Historical Background ................................................... 5

1.1.2 Formation of Protein-Based Films ........................................................ 8

1.1.2.1 Manufacture of Edible Films .............................................................. 8

1.1.2.2 Components of Protein-Based Films ................................................ 9
1.1.2.3 Forces Involved In the Formation and Stability of Protein-Based

Films ............................................................................................................ 10

1.2 Composition and Properties of Whey Protein Isolate ............................... 14

1.2.1 Manufacture of Whey Protein Isolate ................................................. 15

1.2.2 Protein Fractions in Whey .................................................................. 16

1.3 Properties of Whey Protein-Based Films .................................................. 19

1.3.1 Mechanical Properties ........................................................................ 19

1.3.2 Barrier Properties ................................................................................ 25

1.3.3 Thermal Properties ............................................................................. 31

1.4 Methods to Improve the Properties of WPI Films ..................................... 32

1.4.1 Chemical ............................................................................................. 32

1.4.2 Enzymatic ........................................................................................... 35

1.4.3 Physical .............................................................................................. 36

Mechanical and Molecular Properties of Heat - Cured Whey Protein Isolate -
Based Edible Films and Microbial Transglutaminase Heat - Cured Whey

Protein - Isolate Based Films. ............................................................................. 39
2.1 Abstract ......................................................................................................... 39
2.2 Introduction ................................................................................................... 40
2.3 Materials and Methods .................................................................................. 42

2.3.1 Materials ............................................................................................. 42
2.3.2 Film preparation .................................................................................. 43
‘ 2.3.3 Transglutaminase cross-linked ﬁlms .................................................. 43
2.3.4 Heat curing ......................................................................................... 45
2.3.5 Mechanical Properties ........................................................................ 45
2.3.6 Sulfhydryl and disulﬁde bond content ................................................. 46
2.3.7 Lysine content ..................................................................................... 47
2.3.8 Electrophoresis ................................................................................... 48
2.3.9 Statistical analysis .............................................................................. 49
2.4 Results and discussion .............................................................................. 49
2.4.1 Mechanical Properties ........................................................................ 49

vii

2.4.2 Tensile Strength .................................................................................. 49

2.4.3 Elongation at Break. ........................................................................... 53
2.4.4 Toughness. ......................................................................................... 54
2.4.5 Electrophoresis ................................................................................... 54
2.4.6 Sulfhydryl group, disulﬁde bond and lysine content ........................... 58
2.5 Conclusions ............................................................................................... 61
Barrier Properties of Heat - Cured and Microbial T ransglutaminase Heat -
Cured Whey Protein - Isolate Based Edible Films. ........................................... 62
3.1 Abstract ..................................................................................................... 62
3.2 Introduction .............................................................................. 63
3.3 Method ....................................................................................................... 64
3.3.1 Materials ............................................................................................. 64
3.3.2 Film preparation .................................................................................. 65
3.3.3 Transglutaminase cross-linked ﬁlms .................................................. 65
3.3.4 Heat curing ......................................................................................... 66
3.3.5 Water vapor permeability (WVP) ........................................................ 66
3.3.6 Oxygen permeability ........................................................................... 67
3.3.7 Moisture sorption isotherm ................................................................. 67
3.3.8 Statistical analysis .................................... 69
3.4 Results and Discussion ............................................................................. 69
3.4.1 Water Vapor Permeability ................................................................... 69
3.4.2 Oxygen Permeability .......................................................................... 71
3.4.3 Moisture sorption isotherms (MSI) ..................................................... 71
3.5 Conclusions ............................................................................................... 76

Optical Properties of Heat - Cured Whey Protein - Isolate Based Edible Films
and Microbial T ransglutaminase Heat - Cured Whey Protein - Isolate based

Films. ...................................................................................................................... 78
4.1 Abstract ..................................................................................................... 78
4.2 Introduction ................................................................................................ 78
4.3 Materials and Methods .............................................................................. 80

4.3.1 Materials ............................................................................................. 80
4.3.2 Film preparation .................................................................................. 80
4.3.3 Transglutaminase cross-linked ﬁlms .................................................. 81
4.3.4 Heat curing ......................................................................................... 81
4.3.5 Optical Properties ............................................................................... 81
4.3.6 Statistical analysis .............................................................................. 82
4.4 Results and Discussion ............................................................................. 82
4.4.1 Color ................................................................................................... 82
4.4.2 Transmission of UV and Visible light .................................................. 88
4.5 Conclusions ............................................................................................... 90

Thermal Properties Of Heat - Cured Whey Protein Isolate - Based Edible
Films Compared To Microbial Transglutaminase Heat - Cured Whey Protein
Isolate Films. ......................................................................................................... 91

viii

5.1 Abstract ..................................................................................................... 91

5.2 Introduction ................................................................................................ 92

5.3 Materials and Methods .............................................................................. 93
5.3.1 Materials ............................................................................................. 93

5.3.2 Film preparation .................................................................................. 93

5.3.3 Transglutaminase cross-linked ﬁlms .................................................. 94

5.3.4 Heat curing ......................................................................................... 94

5.3.5 Differential scanning calorimetry (DSC) ............................................. 94

5.4 Results and Discussion ............................................................................. 95

5.5 Conclusions ............................................................................................... 99
Conclusions ........................................................................................................ 100
Recommended Future Research ...................................................................... 103
APPENDIX A ........................................................................................................ 104
APPENDIX B ........................................................................................................ 110
References ........................................................................................................... 117

Table
1 .1

1.2

1.3

1.4

1.5

2.1
2.2

2.3

2.4

2.5

3.1

3.2

LIST OF TABLES

Page
Bond energies of typical hydrogen bonds found in proteins ................... 12

Common bond found in proteins and their average bond energies ............. 12

Mechanical properties of whey protein and B—lactoglobulin based
edible ﬁlms as inﬂuenced by various treatments ......................................... 20

Banier properties of whey protein and B—lactoglobulin based edible
ﬁlms as inﬂuenced by various treatments .................................................... 26

Glass transition temperature of whey protein based edible ﬁlms as
influenced by various treatments ...................................................... 33

Protein, lactose, ash, and moisture content of whey protein isolate ............ 44

Signiﬁcance of time and/or transglutaminase treatment on the
tensile strength, elongation at break and toughness of heat-cured
WPI ﬁlms .................................................................................... 51

Comparison of the signiﬁcance of heat curing time and
transglutaminase treatment on the tensile strength,elongation
at break and toughness of heat-cured WPI ﬁlms ................................. 51

Mechanical properties of whey protein-isolate based ﬁlms with
and without microbial transglutaminase heat cured at 90°C for
0, 12 and 24h .............................................................................. 52

Free sulfhydryl, disulﬁde bonds and lysine content of whey
protein-isolate based ﬁlms with and without microbial
transglutaminase heat cured at 90°C for 0, 12 and 24h ......................... 59

Water vapor permeability at 37.8°C and 85%RH and oxygen

permeability at 23°C and 0%RH of whey protein-isolate based

ﬁlms with and without microbial transglutaminase and heat cured

at 90°C for 0 and 24h .................................................................... 70

Parameters used to ﬁt the Guggenheim-Anderson-de Boer

equation at 23°C and 5°C of whey protein-isolate based ﬁlms with

and without microbial transglutaminase and heat cured at 90°C for

0 and 24h .................................................................................. 75

X

4.1

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

4.10

The L*, a*, b* values of whey protein-isolate based ﬁlms with
and without microbial transglutaminase heat cured at 90°C for
0, 12 and 24h ............................................................................. 83

Color difference between whey protein-isolate based ﬁlms
heat cured at 90°C 12 and 24h compared to ﬁlms cured for 0h ............... 83

Color difference between whey protein-isolate based ﬁlms
with microbial transglutaminase heat cured at 90°C 12 and 24h
compared to ﬁlms cured for 0h ...................................................... 83

The L*, a*, b* values of cellophane, polystyrene, low-density
polyethylene and polyethylene terephthalate .................................... 84

Color difference between whey protein-isolate based ﬁlms without
microbial transglutaminase heat cured at 90°C for 0h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate .............................................................................. 84

Color difference between whey protein-isolate based ﬁlms without
microbial transglutaminase heat cured at 90°C for 12h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate .............................................................................. 84

Color difference between whey protein—isolate based ﬁlms without

microbial transglutaminase heat cured at 90°C for 24h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate ............................................................................... 85

Color difference between whey protein-isolate based ﬁlms with

microbial transglutaminase heat cured at 90°C for 0h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate ............................................................................... 85

Color difference between whey protein-isolate based ﬁlms with

microbial transglutaminase heat cured at 90°C for 12h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate ................................................................................ 86

Color difference between whey protein-isolate based ﬁlms with

microbial transglutaminase heat cured at 90°C 24h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate ............................................................................... 86

xi

5.1

A.1

A2

A.3

8.1

3.2

83

Selected thermal properties of whey protein-isolate based ﬁlms
with and without microbial transglutaminase heat cured at 90°C
for 0, 12 and 24h ......................................................................... 98

Tensile strength of whey protein-isolate based ﬁlms cross-linked
with microbial transglutaminase inﬂuenced by heat curing
temperature, time and glycerol content ........................................... 107

Elongation at break (%) of whey protein-isolate based ﬁlms
cross-linked with microbial transglutaminase influenced by
heat curing temperature, time and glycerol content ............................. 108

Toughness (kJ/m3) of whey protein-isolate based ﬁlms cross-linked with
microbial transglutaminase inﬂuenced by heat curing temperature,
time and glycerol content .............................................................. 109

Tensile strength of whey protein-isolate based ﬁlms as inﬂuenced
by heat curing temperature, time and glycerol content ......................... 113

Elongation at break (%) of whey protein-isolate based ﬁlms as
inﬂuenced by heat curing temperature, time and glycerol content .......... 114

Toughness (kJ/m3) of whey protein-isolate based ﬁlms as inﬂuenced
by heat curing temperature, time and glycerol content ......................... 115

xii

LIST OF FIGURES

Figure Page

2.1

2.2

3.1

3.2

4.1

5.1

5.2

SDS-PAGE analysis of whey protein isolate (WPI) and
whey protein isolate based ﬁlms heat cured at 90°C for 0, 12,
and 24h. Using a 4% stacking gel and a 12% resolving gel made
from 37.5:1 acrylamide:bisacrylamide solution with 20ug of protein
loaded in each lane ...................................................................... 56

SDS-PAGE analysis of whey protein isolate and whey

protein isolate based ﬁlms with transglutaminase heat cured

at 90°C for 0, 12, and 24. Using a 4% stacking gel and a 12%

resolving gel made from 37.5:1 acrylamide:bisacrylamide solution

with 20pg of protein loaded in each lane ............................................ 57

Moisture sorption isotherm at 23°C of whey protein-isolate
based ﬁlms with and without microbial transglutaminase and
heat cured at 90°C for 0 and 24h ...................................................... 72

Moisture sorption isotherm at 5°C of whey protein-isolate based
ﬁlms with and without microbial transglutaminase and heat cured
at 90°C for 0 and 24h .................................................................... 73

% transmission versus wavelength of whey protein-isolate
based ﬁlms without (A) and with (B) microbial transglutaminase .............. 89

Differential Scanning Calorimetry therrnogram of whey protein-isolate
based ﬁlms without microbial transglutaminase heat cured at 90°C
for 0, 12, 24 and 48h ..................................................................... 96

Differential Scanning Calorimetry therrnogram of whey protein-isolate

based ﬁlms with microbial transglutaminase heat cured at 90°C
for 0, 12, 24 and 48h ..................................................................... 97

xiii

ASTM
AOAC
cur
DTNB
csc
'%E
EMC
GAB
IMC
MSI
was2
me-2
02F
PAGE

805

ABBREVIATIONS USED IN THIS THESIS

American Society for Testing and Materials
Association of Analytical Communities
Candeiilla Wax

5, 5'- dithiobis (2-nitrobenzoic acid)
Differential scanning calorimetry
Percent elongation

Equilibrium moisture content
Guggenheim-Anderson-de Boer

Initial moisture content

Moisture sorption isotherm

disodium 2-nitro-5 thiosulfobenzoate
2—nitro-5-thiobenzoate anion

Oxygen permeability

Polyacrylamide gel electrophoresis
Relative humidity

Sodium dodecyl sulfate

Microbial transglutaminase

Glass transition temperature

Melting temperature

Onset transition temperature

xiv

TS
WPI

Tensile strength
Whey protein isolate

Water vapor permeability

XV

INTRODUCTION

During the past two decades there has been a renewed interest in the use
of edible ﬁlms as a packaging material, with a number of materials emerging as
potential candidates. Edible ﬁlms based on proteins possess good mechanical
and oxygen bam‘er properties, but are poor moisture baniers. Edible ﬁlms could
serve as a secondary packaging material to reduce the amount of packaging
waste produced. In 2000 the total plastic packaging waste generated in the US
was 11.2 million tons (EPA 2001 ). Recycling recovered only 8.9% of the plastic
packaging waste which is much lower than the 45.0% recovery of aluminum
containers. One of the main challenges to increase the amount of plastic
packaging waste recovered through recycling is to ﬁnd secondary markets for it.
Some of the barriers that must be overcome include the lack of economic
incentives for people and companies to recycle, and the hidden taxes associated
with the municipal collection and disposal of the material (EPA 2000). When used
in conjunction with a primary package material, edible packaging can reduce the
amount of waste generated. This could also allow packers to change and/or
reduce the type of primary packaging used.

Many cities have or are reducing their recycling programs as a way to help
reduce budget deﬁcits (O'Connell 2002). The costs associated with recycling are
signiﬁcant. In 2002, New York City paid up to $240 per ton for its glass, metal, and
plastics recycling program. On the other hand, it would only cost the city $65-$85

per ton to dispose of the waste in landﬁlls (O'Connell 2002). The disposal of all
1

waste in landﬁlls would save the city ~$57 million. By reducing the amount of
packaging waste generated, the amount of waste entering landﬁlls would be
reduced.

The main function of edible ﬁlms is to act as a barrier to mass transfer into
and out of the product. By limiting the migration of moisture, oxygen, aroma, and
oil, both the shelf life and quality of the food product will be improved (Krochta
2002). In this capacity edible ﬁlms are intended to work in conjunction with
traditional packaging materials to decrease the amount of traditional packaging
used or and/or allow a packaging material that is more environmentally friendly to
be used. In 2002, 1.05 billion pounds of dry whey was produced for human
consumption, an increase of 7.5% over the previous year. Of this, only 608.8
million pounds were sold in 2002, leaving a surplus of441.2 million pounds of dry
whey in the US (USDA 2002). This surplus and the functional properties of whey
proteins make them an attractive choice as a possible raw material for the
production of edible ﬁlms. '

Kim (2000) produced ﬁlms using either WPI or whey protein concentrate.
She found that WPI ﬁlms possessed superior mechanical and barrier properties
compared to whey protein concentrate ﬁlms. Kim (2000) investigated the addition
of lipids on the mechanical, barrier and thermal properties. She observed that the
addition of candelilla wax enhanced the barrier properties of ﬁlms. Mert and
Ustunol (2004) investigated the effect of chemical cross-linkers on WPI ﬁlms

properties. They observed increased tensile strength and a decrease in oxygen

permeability for cross-linked ﬁlms. The main concern in using chemical cross-
linkers is that they are not suitable for human consumption.

Both heat curing and transglutaminase have been used to cross-link protein
- based ﬁlms (Mahmoud and Savello 1990; Gennadios et al. 1996; Miller et al.
1997; Yildirim and Hettiarachchy 1998; Kim et al. 2002; Kim et al. 2002). The
limited availability and the high cost of transglutaminase has restricted its use in
the past. However with the availability of transglutaminase from a microbial source
this has become a viable option for cross-linking ﬁlms. Preliminary studies on heat
curing WPI ﬁlms have been shown to enhance their mechanical properties
(Appendix A). Treating WPI ﬁlms with transglutaminase and heat curing also
enhanced the mechanical properties of the ﬁlms (Appendix B).

l hypothesize that heat curing and enzymatic cross-linking with
transglutaminase can be used to modify whey proteins by enhancing disulﬁde
bond formation and inducing the formation of Lys-Gln cross-links. By enhancing
the formation of these bonds, whey protein-based edible ﬁlms can be produced
with improved mechanical, barrier, thermal, and optical properties.

The objectives of my research are:

1. To compare the mechanical properties (tensile strength,

elongation at break and toughness) of whey protein isolate ﬁlms
with and without transglutaminase, and heat cured at 90°C for 0,

12 and 24h.

To determine the sulfhydryl, lysine and disulﬁde content of WPI
ﬁlms with and without TG heat cured for 0, 12 and 24h.

To determine the water vapor permeability and oxygen
permeability of WPI ﬁlms with and without TG, and heat cured at
90°C for 0 and 24h.

To characterize the moisture sorption isotherms of the ﬁlms at 5
and 23°C.

To determine the color characteristics and light transmission of
WPI ﬁlms with and without TG, and heat cured at 90°C for 0, 12
and 24h.

To compare the color values for the ﬁlms to commercially
available plastic packaging materials.

To determine thermal properties (melting onset temperature, peak
melting temperature and enthalpy of fusion) of whey protein
isolate ﬁlms with and without transglutaminase, and heat cured at

90°C for 0, 12 and 24h.

Chapter 1

LITERATURE REVIEW

1.1 Edible Films
1.1.1 Deﬁnition and Historical Background

Edible ﬁlms are deﬁned as a thin layer of edible material formed separately
from a food product which can act as a barrier to mass transfer and/or as a carrier
for food ingredients or additives. The ﬁrst free standing edible ﬁlms, produced
from boiled soymilk, were used in 15th century Japan for preservation and to
improve the appearance of food products (Guilbert and Biquet 1996). Even
though the Japanese have used edible ﬁlms since the 15th century, there have not
been many commercially successful edible ﬁlms.

Conventional packaging is used to prevent the mass transfer of moisture,
oxygen, aroma, or oil between a food product and the environment and to protect
the product from mechanical forces encountered during storage and distribution.
Edible ﬁlms are used to prevent mass transfer between a food product and the
environment. This leads to an increase in the quality and shelf life of the product.
Thus, edible ﬁlms are not intended to replace conventional packaging (Krochta
2002). Edible ﬁlms are to be used in conjunction with conventional packaging to
allow a less complex package to be used. Edible ﬁlms can provide additional

protection to a product after the package is opened.

Haugaard and others (2001) found that there were few examples of bio-
based (non-synthetic) materials used commercially as packaging material.
Potential applications in dairy, home meal replacement, and dry food categories
were identiﬁed. These include antimicrobial edible casings made from whey
protein for hot dogs to inhibit Listeria monocytogenes (Cagri et al. 2001). The
casings were effective in inhibiting the growth of L. monocytogenes. Antimicrobial
edible whey protein ﬁlms as a wrap for sliced summer sausage and bologna were
also investigated by Cagri and others (2002). The ﬁlms were effective in reducing
the growth of L. monocytogenes, Escherichia coli O157:H7, and Salmonella
enterica subsp. Enterica serovar Typhimurium DT104 by 3.1 - 4.1 logs. Kim
(2000) was able to produce heat sealed pouches from whey protein-based edible
ﬁlms ﬁlled with various dry food products such as oatmeal, mashed potato ﬂakes,
and hot cocoa mix. She also investigated the possible use of edible whey protein-
based ﬁlms as a wrap for processed cheese slices. The ﬁlms, however, were
found to be ineffective as a cheese wrap. The cheese slices wrapped in whey
protein-based ﬁlms had a signiﬁcant decrease in moisture content compared to
commercially wrapped cheese.

Edible ﬁlms could serve as a secondary packaging material to reduce the
amount of packaging waste produced. In 2000 the total plastic packaging waste
generated in the US was 11.2 million tons (EPA 2001). Recycling recovered only
8.9% of the plastic packaging waste which is much lower than the 45.0% recovery

of aluminum containers. One of the main challenges to increase the amount of

plastic packaging waste recovered through recycling is to ﬁnd secondary markets
for it Some of the barriers that must be overcome include the lack of economic
incentives for people and companies to recycle, and the hidden taxes associated
with the municipal collection and disposal of the material (EPA 2000). When used
in conjunction with a primary package material, edible packaging can reduce the
amount of waste generated. This could also allow packers to change and/or
reduce the type of primary packaging used.

Many cities are reducing their recycling programs as a way to help reduce
budget deﬁcits (O'Connell 2002). The costs associated with recycling are
signiﬁcant. In 2002, New York City paid up to $240 per ton for its glass, metal, and
plastics recycling program. On the other hand, it would only cost the city only $65-
385 per ton to dispose of the waste in landﬁlls (O'Connell 2002). The disposal of
waste in landﬁlls would save the city ~$57 million. By reducing the amount of
packaging waste generated, the amount of waste entering landﬁlls would be
reduced.

The main function of edible ﬁlms is to act as a barrier to mass transfer into
and out of the product. By limiting the migration of moisture, oxygen, aroma, and
oil, both the shelf life and quality of the food product will be improved (Krochta
2002). In this capacity edible ﬁlms are intended to work in conjunction with
traditional packaging materials to decrease the amount of traditional packaging
used or and/or allow a packaging material that is more environmentally friendly to

be used. In 2002, 1.05 billion pounds of dry whey was produced for human

consumption, an increase of 7.5% over the previous year. Of this, only 608.8
million pounds were sold in 2002, leaving a surplus of441.2 million pounds of dry
whey in the us (USDA 2002). This surplus and the functional properties of whey
proteins make them an attractive choice as a possible raw material for the
production of edible ﬁlms.
1.1.2 Formation of Protein-Based Films

The formation of protein-based ﬁlms usually follows three basic steps. The
ﬁrst step is the dispersion of high molecular weight polymer and other ﬁlm forming
constituents in a solvent. The second step is to cast the solution on a smooth level
surface or to coat the solution on to a food item. The third step is the drying
process where the solvent is evaporated which allows the protein to develop a
matrix thereby forming a free standing ﬁlm or coating (Cuq et al. 1995).
1.1.2.1 Manufacture of Edible Films

Solvent casting is commonly used to produce edible ﬁlms. In this process,
the ﬁlm-forming material is dispersed in a solution (generally this solution is water
or ethanol). The ﬁlm forming solution is cast by spreading or pouring in a thin layer
on a surface coated with polyteraﬂuorethylene (Teﬂon®), and then allowing the
solvent to be removed through evaporation. The dried ﬁlm can be detached from
the surface to produce a free-standing ﬁlm.

Solvent casting is not widely used in the commercial production of self-
supporting ﬁlms. The most common process for production of ﬁlms is extrusion. In

this process the solid polymer resin is converted into a melt. The melt is then

shaped using heat, shear and pressure. During the extrusion process the melt is
conveyed through the extruder barrel using a screw. The screw is usually divided
into three sections: conveying, compression or melting, and a metering section. At
the end of the extruder barrel the material is forced through a die and formed into a
ﬁlm (cast or blown) (Hernandez et al. 2000). Extrusion has been used in the
production of sausage casing from collagen (Osbum 2002) and corn zein (Ha and
Padua 2001). Currently, these are the only protein-based ﬁlms to be produced
using extrusion.

1.1.2.2 Components of Protein-Based Films

The basic requirement for the formation of an edible ﬁlm is the presence of
a high molecular weight polymer. The long chain polymeric structure provides the
cohesive strength required for the formation of a structural matrix (Banker 1966).
The two types of high molecular weight polymers commonly used in the formation
of edible ﬁlms are hydrocolloids (polysaccharides and proteins). Both
polysaccharides and proteins have better mechanical strength than lipid-based
ﬁlms, while lipid ﬁlms have better moisture barrier properties.

Proteins used in edible ﬁlms include soy, wheat, com, whey and collagen
(Gennadios 2002). Of these, only collagen has become commercially successful.
It is used in the processed meat industry primarily as an edible alternative to
natural casings (Osbum 2002) and a wrap for meat products.

By themselves, proteins form brittle ﬁlms. The brittleness of the ﬁlms can be

reduced by the addition of a plasticizer. Plasticizers reduce the intermolecular

interactions between the polymer chains. Plasticizers commonly used in edible
ﬁlms include glycerol, sorbitol, mannitol, xylitol and polyethylene glycol (McHugh et
al. 1994; McHugh and Krochta 1994; Tanaka et al. 2001; Shaw et al. 2002; Shaw
et al. 2002).

Due to the hydrophilic nature of proteins, they are poor moisture baniers.
The addition of lipids to protein ﬁlms can improve their moisture barrier properties.
Waxes such as candelilla, camauba and beeswax are commonly incorporated in
ﬁlm-forming solutions (McHugh and Krochta 1994; Shellhammer and Krochta
1997; Debeaufort et al. 2000; Gallo et al. 2000; Kim 2000; Perez-Gago and
Krochta 2001; Chick and Hernandez 2002; Gallstedt and Hedenqvist 2002).
1.1.2.3 Forces Involved In the Fonnatlon and Stability of Protein-Based
Films

Protein structures are stabilized through two groups of interactions:
intramolecular forces inherent to the protein molecule and intermolecular
interactions arising from the surrounding solvent or other molecules. Peptide
bonds, steric strain, and van der Waals interactions are intramolecular forces
inherent to protein and hydrogen bonding; electrostatic and hydrophobic
interactions can be both intramolecular and intermolecular forces (Damodaran
1996). Interaction among protein chains inﬂuence the characteristics of the ﬁlm
produced. The ability of proteins to form ﬁlms is inﬂuenced by the primary
structure of the protein and the formation of ionic cross-links, hydrogen bonds, and

inter- and intramolecular disulﬁde bonds (Gennadios 2002). The application of

10

heat to protein-ﬁlm—fon'ning solutions enhances the ﬁlm-forming properties
(Gennadios et al. 1994; Roy et al. 1999; Perez-Gago and Krochta 2001). Heating
the protein solution increases the denaturation of the protein, which exposes
hydrophobic and suﬁhydryl groups that were previously buried. During drying,
these groups interact to form high - molecular — weight aggregates. These
aggregates enhance the mechanical properties of the ﬁlms.

Hydrogen bonds are formed between a hydrogen atom covalently bonded
to an electronegative atom (usually N or O) and another electronegative atom.
Hydrogen bonds are one of the most important forces affecting the stability of
proteins. Hydrogen bonds are classiﬁed as a strong permanent dipole-dipole
interaction (Damodaran 1996). The bond energies of commonly found hydrogen
bonds in proteins are shown in Table 1.1.

Fairiey and others (1996) investigated the role of suflhydryI/disulﬁde
interchanges in whey protein ﬁlms. By inhibiting the interchange of suflhydryl and
disulﬁde bonds they observed an increase in ﬁlm solubility. The increase in
solubility was unexpected and attributed to the protein adopting a conformation
that increased hydrogen bonding. Subirade and others (1998) investigated the
change in conformation in glycinin in both an aqueous solution and ﬁlms. They
observed a decrease in the percentage of j3-sheet and unordered structure found
in the ﬁlm-forming solution and an increase a-helix conformation. Another
conformational change occurred while drying the solutions and that resulted in the

forming of intermolecular hydrogen bonded B-sheets.

11

Table1.1 Bond energies of typical hydrogen bonds found in proteins

(Rodriguez 1996).

 

Hydrogen

Bonds

Typical
Bond
Energy
(Kcal/mol)

 

O-H—O
O-H—N
N-H—O
N-H—N

3to6
3t06
4

3t05

 

Table 1. 2: Common bonds found in proteins and their average bond

 

 

energies (Rodriguez 1996).

Ave. Bond
Energy

Bond (Kcal/mol)

00 83

C-0 84

C-H 99

C=O 1 71

C-N 70

N-H 93

N=C 147

S-S 51

C-8 62

S-H 81

00 33

OH 1 1 1

 

12

The conformational changes induced in wheat gliadins were studied by Mangavel
and others (2001). They found that there was an increase in B-sheet formation in
the ﬁlm-forming solution. Drying the solution induced major conformational
changes in the protein. The conformational changes were attributed to antiparallel
hydrogen-bonded B—sheets and protein aggregates. The increase in hydrogen-
bonded p-sheets led to an increase in tensile strength of the dried ﬁlms. Even
though hydrogen bonds are relatively weak, their abundance signiﬁcantly
contributes to the stability of proteins. Increasing the amount of hydrogen bonding
can increase the mechanical strength of a protein ﬁlm.

Disulﬁde bonds are formed when two cysteine residues are brought in close
proximity. The suﬂhydryl groups undergo oxidation in the presence of molecular
oxygen which results in the formation of a disulﬁde bond. Disuiﬁde bonds can be
formed both inter-moleculariy and intra-molecularly in proteins. Disulﬁde bonds
help to stabilize the folded protein and are important in the stabilization of protein
ﬁlms. Even though disulﬁde bonds are important in stabilizing the structure of a
protein; they are relatively weak compared to other covalent bonds found in
proteins (Table 1.2). It is thought that the proteins in ﬁlm-forming solutions
denature through the reduction of SS bonds and reveal buried SH groups. During
the drying process, covalent S-S bonds are formed by air oxidation from the
exposed SH and suﬂhydryl-disulﬁde interchange reactions (McHugh, Aujard et al.
1994; Roy, Weller et al. 1999).

13

Perez-Gago and others (1999) investigated the solubility of native and
denatured whey protein isolate (WPI) ﬁlms and found that denatured WPI ﬁlms
had lower solubility in water then native WPI ﬁlms. The decrease in solubility was
attributed to the denaturation of the protein, the exposure of SH groups, and the
formation of intermolecular disulﬁde bonds. Kim (2000) observed that the
solubility of WPI ﬁlms differed when incubated in different bond dissociating
agents. Films incubated in p-mercaptoethanol (BME) had higher solubilities than
those in SDS. These results suggest that disulﬁde bonds play a role in stabilizing
the ﬁlm network. The solubility of ﬁlms incubated in urea was higher than in SDS,
indicating that hydrogen bonds are also important in stabilizing WPI ﬁlm networks
(Kim 2000).

Kayserilioglu and others (2001) determined that wheat gluten ﬁlm solutions
at pH 11 produced ﬁlms that were cross-linked through the formation of
Iysinoalanine. The ﬁlms showed no change in protein solubility with the addition of
sodium dodecyl sulfate (SDS), indicated that hydrophobic interactions were not as
important to the stability of the ﬁlms.

1.2 Composition and Properties of Whey Protein Isolate

Bovine milk contains ~3.5% protein, and of this, whey proteins account for 18%
(w/v) of the total protein content. Whey protein can be deﬁned as the proteins that
remain soluble alter the pH of milk has been adjusted to 4.6 (Fox and McSweeney
1998). Liquid whey from cheese production contains lactose, lipids and minerals

along with the soluble proteins. Cheese whey is further processed to remove

14

lactose, lipids and minerals to increase the protein concentration. Whey proteins
are used commercially as ingredients in baked goods, fortiﬁed beverages, dairy
and frozen desserts, dressings, sauces, dietetic products and processed meats
(Kinsella and Whitehead 1989).

1.2.1 Manufacture of Whey Protein Isolate

Due to the high moisture and low solids content of cheese whey it tends to
spoil rapidly. Moisture is removed through high velocity and low temperature spray
drying. High temperatures (>75°C) need to be avoided to minimize the
denaturation of whey proteins which alters their functional properties (Morr and Ha
1993).

The production of whey protein isolate begins with the pasteurization and
clariﬁcation of liquid whey. The whey is clariﬁed using a large sale centrifuge
(clariﬁer) to remove casein and cheese particles. The removal of lactose and
minerals are accomplished using ultraﬁltration, which concentrates the protein and
fat. Ultraﬁltration is used to produce whey protein concentrate (WPC) having a
protein content of 30-50%. Diaﬁltration is used to further concentrate the WPC to
80% protein (Morr and Ha 1993).

Two additional steps are required to produce whey protein isolate. After
clariﬁcation, fat and lactose are removed using microﬁltration. These processes
are then followed by ultraﬁltration and diaﬁltration. Once these processes are

completed, spray drying is used to produce a ﬁne white powder with low-fat and

15

lactose content. The composition of WPI is <90% protein, >1 % fat, and 1-4%
lactose and ash (Huffman 1996).
1.2.2 Protein Fractions in Whey

Whey proteins account for 15-25% of the total protein content of milk. Whey
proteins are globular and composed of B-lactoglobulin, a-lactalbumin, bovine
semm albumin, proteose-peptones, Iysozyme and lactoferrin (Brunner 1981;
Kinsella and Whitehead 1989).

1.2.2.1 B-Lactoglobulin
B—Iactoglobulin (j3-Lg) is the major protein fraction found in whey. It accounts

for 50% of the whey protein and has a molecular mass of 18,600 Daltons. In its
native state and in solution p-Lg exists as a dimer due to electrostatic interactions
between Asp13° and Glu134 with the correspOnding lysyl residues of another
monomer (Cramer et al. 1983). Native p-Lg possesses 2 pairs of SS between
Cyss‘s-Cys160 and Cysw‘s-Cys119 and a free SH group on Cys‘z‘. The free SH is
buried and inaccessible to solvents at neutral pH (Papiz et al. 1986). Bovine B-Lg
exists in seven different variants; of these seven variants A and B are the most
common. Variant A and B differ in their M composition at residues 64 and 118.
Variant A has Asp and Val at these residues and variant B has Gly and Ala (Cayot
and Lorient 1997). Bovine j3-Lg shows a high degree of secondary and tertiary
structural organization. It contains approximately 51% B-sheet, 15% a—helix, 17%

reverse tums and 17% random coils (Cramer, Parry et al. 1983).

16

Bovine p-Lg exists as a dimer at pH 58 Between pH 3-5 B-Lg undergoes a
pH dependant association to form octamers. Below pH 2 and above pH 8 p—Lg
dissociates into monomers (Pessan et al. 1985). Heating of B-Lg above 55°C
under alkaline conditions induces denaturation and aggregation. Denaturation of )3-
Lg exposes the SH at Cys121 which undergoes S—S/SH exchange which leads to
ineversible aggregation of the protein (Boye et al. 1998).
1.2.2.2 a-Lactalbumin

a—lactalbumin (or-La) is the second most abundant protein in whey, and
accounts for ~19% of the whey protein (Cayot and Lorient 1997). The molecular
mass of a-La is ~14,000 Da, and the protein contains 4 disulﬁde groups (cystine)
and no free sulfhydryl (cysteine). a-La contains 123 amino acid residues and
shows a strong homology to lysozymes (Fox and McSweeney 1998).

a—La is a globular protein and exhibits little organized secondary structure.
The secondary structure of a—La consists of 26% a—helix, 14% p-sheet and 60%
unordered structure (Kinsella and Whitehead 1989). a-La has an ellipsoidal tertiary
structure with a deep cleft dividing the molecule into two lobes. One lobe is
composed of 4 helices and the other lobe has 2 [3- sheets and a loop chain
sﬁ'ucture (Wong et al. 1996).

a—La is the most heat stable whey protein. a—La will undergo renaturation
after heat denaturation. However, in the presence of B-Lg, heated a—La will

undergo irreversible denaturation and form aggregates. Aggregation occurs

17

through the formation of disulﬁde bonded copolymers of a-La and B-Lg (Gezimati
et al. 1997).
1.2.2.3 Bovine Serum Albumin

Bovine serum albumin (BSA) accounts for ~5% of the whey proteins (Cayot
and Lorient 1997). BSA is a single peptide with a molecular mass of ~66,000 Da
and is comprised of 582 amino acid residues (Kinsella and Whitehead 1989). BSA
contains 17 disulﬁdes and 1 sulﬂ'lydryl group. The BSA molecule is elliptical and is
divided into three domains each with a different net charge (-10, -8, 0) (Fox and
McSweeney 1998). The denaturation of BSA occurs similarly to B—Lg and the
protein can be denatured by heat, acid or base treatment (Cayot and Lorient
1997). BSA forms disulﬁde-bonded polymers and aggregates through hydrophobic
interactions upon heating (Gezimati, Creamer et al. 1997).
1.2.2.4 Immunoglobulins and proteose-peptone

Immunoglobulins are made up of high molecular weight proteins, such as

lgG1, Ing, IgA, IgM, and IgE, all of which have antibiotic properties. The molecular
mass of immunoglobulins range from 15,000-1,000,000 daltons (Kinsella and
Whitehead 1989). Immunoglobulins account for ~13% of the whey protein (Cayot
and Lorient 1997). The function of immunoglobulins is to impart various types of
immunity to the body. In bovine milk ~80% of the immunoglobulins are lgG (Fox
and McSweeney 1998). Immunoglobulins are comprised of two light polypeptide
chains and two heavy polypeptide chains. Both light and heavy polypeptide chains

are cross-linked by disulﬁde bonds (Brunner 1981 ).

18

Proteose-peptones account for ~10% of the whey proteins. The molecular
mass of proteose-peptones range from 4,000 — 40,000 daltons (Kinsella and
Whitehead 1989). Proteose-peptones remain soluble after the precipitation of
casein at pH 4.6 and heat coagulation of 8-Lg and a-La at 95°C. Proteose-
peptones are deﬁned as a mixture of acid-soluble and heat stable
phosphoglycoproteins (Girardet and Linden 1996).

1.3 Properties of Whey Protein-Based Films
1.3.1 Mechanical Properties

Mechanical property requirements have practical importance in selecting
applications for which edible ﬁlms are best suited. The two most studied
mechanical properties of whey protein ﬁlms are tensile strength (TS) and
elongation-at-break (%E). Tensile strength is a measure of the maximum stress
(force per unit area) that a material can sustain before breaking. Elongation-at-
break is a measure of the maximum amount of strain (dimensionless fractional
length increase) a material can sustain before fracture (Hernandez, Selke et al.
2000)

The mechanical properties of whey protein-based (WPI) ﬁlms have been
extensively studied (Table 1.3). Mechanical properties of WPI ﬁlms heat cured 80,
90 and 100°C for 0, 12, 24, 48 and 72h treated with and without transglutaminase
is shown in Appendix A and Appendix B. Various compounds and treatments such
as plasticizers, denaturation, hydrolysis, chemical modiﬁcation, drying conditions,

and

19

Table 1.3: Mechanical properties of whey protein and B-Iactoglobulin based
edible films as influenced by various treatments.

 

 

Film e le Elastic
. 2 Treatment ' 5 Reference
Fm" corrpositiona Strength‘ Ewan” modulus8
. McHugh and
WPI/Gly 10.1.5 Glycerol 29.10 4.10 -—-— Km chta 19941
P G 1 ' 0 l 90 8 McHugh and
W II ly 0.3. Glycero 13. 30. 0 --- Krochta 19941
. . l McHugh and
WPI/Sor 10.3.0 Sorbrto 14.00 1.60 --- Km chta 19941
. Sothomvit and
BLglGly 10.5.8 Glycerol 16.01 76.46 705.60 Krochta 20011
. . 3 Sothomvit and
BLg/Sor 10.11.65 Sorbitol 10.06 65.85 38 .80 Krochta 20011
. P0|yemy|ene SOUIOITIVII and
BLg/PEG 200 10.128 Glycol 6.46 77.09 255.20 Krochta 20011
. Sothomvit and
lng/Suc 10.219 Sucrose 9.71 89.41 340.80 Krochta 20011
_ polyethylene Sothomvit and
BLgIPEG 400 1025.6 Glycol 2.88 32.31 117.20 Krochta 20011
Shaw and others
WPI/Gly 10:5.0 Glycerol 3.25 35.00 100.00 2002b‘
. Shaw and others
WPI/Gly 10.8.0 Glycerol 2.00 32.00 25.00 2002b‘
_ Shaw and others
WPI/Gly 10.10 Glycerol 1 .50 47.00 12.00 2002b‘
Shaw and others
WPI/Sor 1025.0 Sorbitol 9.00 10.00 325.00 1
2002b
Shaw and others
WPI/Sci 10:8.0 Sorbitol 3.50 15.00 150.00 1
2002b
_ Shaw and others
WPI/Sor 10:10 Sorbitol 3.30 25.00 100.00 1
2002b
0.5 X . I 8 00 5 225 Shaw and others
WPI/Xyl l . .0 ylrto . 1 .00 .00 2002b‘
_ . Shaw and others
WPI/Xyl 10.8.0 Xylrtol 4.50 7.00 175.00 2002b1
1 . _ l Shaw and others
WPI/Xyl 0.10 Xylrto 1.00 2.00 100.00 2002b‘
. Heat Perez-Gaga and
WPI/Gly 5.0.3.5 denatured 6.90 41.00 199.00 others 19991
. _ Perez-Gaga and
WPI/Gly 5.0.3.5 Native WPI 3.10 7.00 88.00 others 19991

 

20

Table 1.3 cont.: Mechanical properties of whey protein and e-Iactoglobulin

based edible ﬁlms as inﬂuenced by various treatments.

 

 

Film Tensile Elastic
. Treatment - 5 Reference
””2 corrlposiiion3 Strength“ Ewan“ modulus6 j
WPI/Gly 10-2 5 55% “99'“ °f 1 00 35 00 100 00 so m and
' ' hydrolysis ' ' ' Krodita 20001
, 10% degree of Sothomvit and
WPI/Gly 10.2.5 hydrdysis 2.00 2.50 25.00 mm 20001
' 5.5% deg'ee of Sothomvit and
WP'IG'V "155 hydrolysis "" ‘“ "'”' Krochta 20001
_ 10% degree of 3‘0me and
WPI/Gly 10.5.5 hydrdysis 1.75 60.00 25.00 K I ,3 20001
WPI/GI 10-3 3 0 0 mmol NEM 6 50 20 00 325 00 Fairley and
y . . . . . . others 19961
_ 0.01 rrmol Fairley and
WPI/Gly 10.3.3 NEM 7.00 23.00 375.00 others 19961
_ 0.02 rrmol Fairley and
WPI/Gly 10.3.3 NEM 7.50 20.00 325.00 cum 19961
_ 0.03 11111101 Fairley and
WPI/Gly 10.3.3 NEM 7.00 15.00 370.00 others 19961
'crowave Ka and Ka a
WPI/G 100100 N“ . 2.23 35.10 27.03 ya 1 y
drying 2000
. Room Kaya and Kaya
WPI/G 10.0100 conditions 1.94 26.10 20.37 20001
, dried at 2103 Nmntara and
WPI/Gly 11511.5 50% RH 030 42.00 23.90 others 1993‘
. dried at 950 3 Neantara and
WPI/Gly 11511.5 30% RH 1.40 32.10 42.80 others 19981
, _ dried at 250 3 Perez—Gage and
WPI/Gly/BW 10.0.3.3.40 40%RH 3.75 2.50 225.00 mm 20001
, , dried at 400 3 Perez—Gage and
WPI/GlyIBW 10.0.3.3.40 40%” 3.50 1.75 275.00 Krochta 20001
_ , dried at 300 3 Perez-6600 and
WPI/Gly/BW 10.0.3.3.40 40%RH 5.00 10.50 270.00 mm 20001
_ , dried at 250 3 Perez-Gage and
WPI/GIy/AMFF 10.0.3.3.40 40%RH 6.00 3.50 300.00 ME 20001
, , dried at 406 3 Perez-Gage and
WPI/GlylAMFF 10.0.3.3.40 40%RH 5.00 9.50 225.00 Krochta 20001
. . dried at 80C & Perez-Gaga and
WPI/GlylAMFF 10.0.3.3.40 40%RH 4.50 9.00 250.00 Krochta 20001
_ , dried at 250 3 Perez-Gage and
WPI/GIy/CW 10.0.3.3.40 40%RH —- —- mm 20001

 

21

Table 1.3 cont.: Mechanical properties of whey protein and e-Iactoglobulin

based edible ﬁlms as influenced by various treatments.

 

Film

Tensile

Elastic

 

Film2 corrposiiiona Treatment Strength‘ Elongation5 modulus“ Reference
WPI/Gly 10:2.5 5'5'3313932“ 1.00 35.00 100.00 $22222?
WPI/Gly 10:25 10:33:; °f 2.00 2.50 25.00 $022222?
WPI/Gly 10:5.5 55:33:.9y'3? -- -—-- --- mgvgoggﬁ’
WPI/Gly 10:55 10:33:; °f 1.75 60.00 25.00 $22222?
WPI/Gly 10:33 0.0 mmol NEM 6.50 20.00 325.00 Fairley andr

others 1996

WPI/Gly 10:3.3 0.0;;qu 7.00 23.00 375.00 0:113:35“

WPI/Gly 10:33 0033101 7.50 20.00 325.00 0:33 3321

WPI/Gly 1033 ”SJ“ 7.00 15.00 370.00 3:); 22:1
WPI/G 10.0:100 ”'ng 2.23 36.10 27.08 Kayazzgngaya
WPI/G 10.0:100 comg‘ns 1.94 26.10 20.37 Kayazggngaya
WPI/Gly 11511.5 “65%;: :L“ 0.80 42.00 23.90 mfg?
WPI/Gly 11511.5 “:33: '95,“ 1.40 32.10 42.30 22:23:?
WPI/Gly/BW 10.0:3.3:40 “"6202:ij 8‘ 3.75 2.50 225.00 Pﬁzer-1:230?
WPI/GlyIBW 1003.340 “egg? 8‘ 3.50 1.75 275.00 [33:338qu
WPI/GlyIBW 10.0:3.3:40 “8:037:13? 8‘ 5.00 10.50 270.00 meaaggo?“
WPI/Gly/AMFF 10.0:3.3:40 “ﬁg? 8‘ 6.00 3.50 300.00 P323230?
WPI/GlylAMFF 10.0:3.3:40 “92032;? 8‘ 5.00 9.50 225.00 Fiﬁgfaaggozqd
WPI/GlyIAMFF 10.0:3.3:40 “egg? 8‘ 4.50 9.00 250.00 P23223380?
WPI/Gly/CW 10.0:3.3:40 “6:037:43? 8‘ -- Paziaggozqd

 

22

Table 1.3 cont.: Mechanical properties of whey protein and 84actoglobulin
based edible ﬁlms as inﬂuenced by various treatments.

 

 

Film Tensile Elastic
' 2 Treatment ' 5 R f
F'Im composition3 Strength4 Elongatlon modulus6 e erence
, , dried at 40C & Perez-Sago and
WPI/GlleW 10.0.3.3.40 40%RH --- «— --- I( l ta 20001
_ , dried at 300 3 PereZ-Gago and
WPI/GlleW 10.03340 40% RH —— ---- -—— Krochta 20001
. . Shaw and others
WPI/Glylsoya OII 10:5.0:4.0 Soya orl 4.06 44.05 105.29 20021
_ . ' Shaw and others
WPI/Glylsoya orl 10:5.0:3.0 Soya OII 4.28 85.42 118.54 20021
. . Shaw and others
WPIIGIy/soya orl 10:50:20 Soya orl 3.98 60.75 130.19 20021
WPI/Gly/soya oil 10:50:00 Soya oil 4.26 33.63 159.49 Shawzadzf’the's

 

1Films tested according to ASTM 882

2WPl=whey protein isolate; Gly=glyoerol; Sor=sorbitol; Xyl=xylitol; G=glycerine; BW=beeswax;
AMFF=anhydrous milkfat fraction; CW=mndelilla wax; BLg=B-Lactoglobulin

3 %protein: %plasticizer. %lipid (w/w/w); ‘ MPa; 5 %; 6 Mpa

23

the addition of lipids affect the mechanical properties of WPI ﬁlms. Plasticizers
enhance the mechanical properties of protein-based edible ﬁlms, which otherwise
tend to be brittle by themselves. The addition of plasticizers decreases the
intermolecular interactions, which leads to an increased pliability in the ﬁlms.
Common plasticizers used for edible protein-based ﬁlms include glycerol, sorbitol
and polyethylene glycol (Gennadios 2002). Glycerol is the most effective
plasticizer, due to its smaller size and ability to interact with the polymers.
Increasing the level of plasticizers in the ﬁlm solution has been shown to decrease
the TS and increase the %E of WPI ﬁlms (McHugh and Krochta 1994; Sothomvit
and Krochta 2001; Shaw, Monahan et al. 2002) by reducing the intermolecular
attraction between the polymer chains. Increasing the molecular weight of the
plasticizer will affect the mechanical properties of WPI by decreasing the
elongation at break and by increasing the tensile strength (Sothomvit and Krochta
2001; Shaw, Monahan et al. 2002). The higher molecular weight plasticizers are
less effective in disrupting the intermolecular attraction between the polymer
chains.

Heating WPI solutions causes the proteins to denature and exposes
suﬂhydryl groups (SH) in the protein interior. Exposing the SH increases the
number of intermolecular disulﬁde bonds that may be formed during the drying
process and leads to increased intermolecular cross-linking. The increased TS of
denatured WPI ﬁlms have been attributed to the formation of covalent cross-links

during the heating process. Enzymatic hydrolysis of whey protein resulted in ﬁlms

24

that had lower TS compared to unhydrolyzed WPI (Sothomvit and Krochta 2000).
The decrease in TS in hydrolyzed WPI was attributed to the weaker protein
network formed by the shorter chains. The blocking of disulﬁde bond formation by
free sulﬂtydryl using N-ethylmaleimide (NEM) had no effect on the mechanical
properties of WPI ﬁlms (Fairley, Monahan et al. 1996). Conformational changes
induced through the heating cf protein ﬁlm solutions during the drying process
enhanced the mechanical properties of the ﬁlm. Increasing the drying temperature
and decreasing the humidity increases the TS and decreases the %E of WPI ﬁlms
(Alcantara et al. 1998). Drying of WPI ﬁlms by microwave radiation was shown to
increase the TS of ﬁlms compared to ﬁlms dried at room temperature (Kaya and
Kaya 2000). The addition of lipids to the ﬁlm forming solutions decreased the TS
of WPI ﬁlms (Perez-Gago and Krochta 2000; Shaw, Monahan et al. 2002). Drying
WPI ﬁlms at different temperatures and a constant humidity had no effect on the
mechanical properties of WPI ﬁlms containing lipids (Perez-Gago and Krochta
2000).
1.3.2 Barrier Properties

Barrier properties measure the resistance of a material to diffusion or
sorption of a substance (Hernandez, Selke et al. 2000). Permeability of a material
combines the effects of diffusion and sorption into a single value. Two important
barrier properties of WPI ﬁlms are water vapor permeability (WVP) and oxygen
permeability (OZP) (Table 1.4). Another barrier property that is important, but less

studied, is sorption of various volatile compounds. Protein based ﬁlms in general

25

Table 1.4: Barrier properties of whey protein and B-Iactoglobulln based
edible ﬁlms as inﬂuenced by various treatments.

 

 

F'lm
Film‘ ' , , 5 Treatment 0296 wvrv7 Reference
composrtlon
McHugh and
:1. , --
WPI/Gly 10 5 Glycerol 18 50 Krochta 19941
McHugh and
Pl/Gl 1 2 . l .1 --
W y 0 3 0 Glycero 76 0 Krochta 1994,
McHugh and
I 2 . ' , --
WPI Sor 10 3 0 Sorbltol 4 30 Krochta 19941
WPI/Gly 10:5.0 Glycerol M 116.00 Shaw and 2mm
2002b
h n
WPI/Gly 10:8.0 Glycerol --— 116.00 S aw a d 2mm
2002b
Shaw and others
WPI/Gly 10.10 Glycerol --- 150.00 2002b3
, Shaw and others
WPI/Sor 10.5.0 Sorbrtol -— 85.00 2002b3
, Shaw and others
WPI/Sor 10.8.0 Sorbitol -- 85.00 2002b3
, Shaw and others
WPI/Sor 10.1 0 Sorbitol -- 1 10.00 2002b3
. Shaw and others
WPI/Xyl 10.5.0 Xylrtol --- 90.00 2002b3
Shaw and others
WPI/X 10:8.0 X it I -— 90.00
y‘ y' ° 200263
Shaw and others
P l 10:1 ' l --- .
W l/Xy 0 Xyllto 90 00 2002b3
_ Heat Perez-Gago and
WPI/Gly 5.0.3.5 denatured -— 119.04 others 19993
_ . Perez-Gaga and
WPI/Gly 5.0.3.5 Native WPI --— 121.44 others 19993
5.5% degree of Sothomvit and
WPI/GI 1022.5 . 5. 0 ---
y hydrolysns 2 0 Krochta 20001
10% degree of Sothomvit and
WPI/GI 1022.5 . 25.00 ---
y hydrolySIS Krochta 20001
5.5% degree of Sothomvit and
WPI/GI 1025.5 . 300.00 ---
y hydrolysis Krochta20001
10% degree of Sothomvit and
WPI/GI 1025.5 . 300.00 ---
y hydI’OIYSIS Krochta 20001
F 'rte d th rs
WPI/Gly 10:3.3 0.0 mmol NEM --— 90.00 a' ‘23; e

 

26

Table 1.4 cont: Barrier properties of whey protein and B-Iactoglobulin based

edible ﬁlms as inﬂuenced by various treatments.

 

Film

 

Film4 compositions Treatment 02133 mp? Reference
WPI/Gly 10:3.3 O'ollgﬂm' -- 64.00 Fame); 3333“”
WPI/Gly 10:3.3 00:on ..- 84.00 Famey13323°mers
WPI/Gly 10:3.3 00:2,?” —— 84.00 Fame); 3323”“

WPI/G 100100 ”was“ -— 45.60 Kayazzggfaya

WPI/G 10.0:100 00$;an --— 50.16 Kayazzggfaya
WPI/Gly/BW 10.0:3.3:40 ”5:01;? 8‘ -- 54.00 Pig-1:83:02?
WPI/GlyIBW 10.0:3.3:40 “9:023? 8‘ --- 54.00 P22313330?“
WPI/GlleW 10.0:3.3:40 “36:01:30 8‘ --— 36.00 P33; 1333330?“
WPI/GIy/AMFF 1003.340 “320%? 8‘ -... 60.00 P23333022"
WPI/Gly/AMFF 10.0:3.3:40 “9:02;? 8‘ --— 48.00 $333;ng
WPI/GlyIAMFF 10.0:3.3:40 arr-320:3? 8‘ M 36.00 Piggfaaggoigd
WPI/GlleW 10.0:3.3:40 “6:01:30 8‘ -— 78.00 P22233302?
WPI/Gly/CW 10.0:3.3:40 “8:023:10 8‘ --— 72.00 P2223330?"
WPI/GlyICW 10.0:3.3:40 “ex/:3? 8' —— 60.00 $533330?
WPI/Glylsoya oil 10:50:40 Soya oil -— 126.41 Shawzao'gdzfmers
WPI/Gly/soya oil 10:50:30 Soya oil --.- 111.62 Shawzzrgdzfthe’s
WPI/Gly/soya oil 10:50:20 Soya oil M 116.32 Shawzao'gdzfmers
WPI/Gly/soya oil 10:50:00 Soya oil —— 115.72 Shawzaogdzfmers
BLg/Gly 10:1.5 Glycerol 10.00 55.92 Mate 3336??”

 

27

Table 1.4 cont: Barrier properties of whey protein and Iii-lactoglobulln based

edible ﬁlms as inﬂuenced by various treatments.

 

 

Film‘ comgggﬁoﬁ Treatment 025:5 WVP7 Reference
BLg/Gly 10:30 Glycerol 25.00 93.60 Mate 33$?“
BLg/Gly 10:40 Glycerol 50.00 136.72 Mate Eggmta
WPI/GlleW 5:3.3:0 Beeswj" 8‘ pH 120.00 $33339?“
WPI/Gly/BW 5:33:20 Bees”? 3‘ P” --.. 9.6.00 P22328339?
WPI/Gly/BW 5:33:40 Bees”? 8‘ PH _,_ P2233392?
WPI/GIy/BW 5:3.3:0 3°99:ng & pH ____ 126.00 P2232313; :26
WPI/GlleW 5:33:20 3635ng 8‘ 9” ~— 6000 P:::;:a$;9:2d
WPI/Gly/BW 5:33:40 3893ng 8‘ 9” --- 60.00 Pzzizagggzgd
WPI/Gly/BW 5:3.3:0 Bees“? 8‘ pH -— 120.00 P33233392?
WPI/Gly/BW 5:53:20 36°8ng 8‘ 9” --— 46.00 '32:; 13:33:92“
WPI/GIy/BW 5:53:40 3383ng 8‘ 9” --- 48.00 P3333392?

.. ..

9°”

 

1Films tested using ASTM 3985 at 23°C and 50%RH
2Films tested using ASTM 3985 at 23°C and 40%RH
3Films tested using ASTM E96 modiﬁed according to McHugh and others 1993

‘WPI=whey protein isolate; Gly=glyoerol; Sor=sorbitol; Xyl=xylitol; G=glycerine; BW=beeswax;

AMFF=anhydrous milkfat fraction; CW=candelilla wax; BL =B-Iact
5 %protein: % plasticizer (WM); 6 Oxygen permeability (c

permeability (g mm/d rn kPa)

28

m mm/m

oglobulin

d‘KPa); 7 Water vapor

and WPI speciﬁcally are poor barriers to moisture but are good barriers to oxygen
(Krochta and De Mulder-Johnson 1997). Their inadequacy as moisture barriers
has been attributed to their hydrophilic nature. The OzP of protein ﬁlms is
dependant on the relative humidity (RH) during testing; as RH increases the OzP
of the ﬁlm increases (Miller and Krochta 1997). Decreasing the WVP and OzP of
WPI ﬁlms will increase the likelihood of commercialization.

The plasticizer used has an effect on the permeability of WPI ﬁlms by
increasing the intermolecular distance between the proteins. This increases the
diffusion of water or oxygen through the ﬁlm (Hernandez, Selke et al. 2000). Films
which included glycerol had higher WVP compared to sorbitol plasticized ﬁlms
(Shaw, Monahan et al. 2002). The hygroscopic nature of glycerol increases the
adsorption of water, with the water acting as an additional plasticizer (Anker et al.
2000; Hernandez, Selke et al. 2000). Increasing the temperature and reducing the
RH during drying of ﬁlms leads to a decrease in WVP (Alcantara, Rumsey et al.
1998; Perez-Sago and Krochta 2000). The addition of lipids to WPI ﬁlms has
been investigated as a means to improve the WVP of the ﬁlms. However, Shaw
and others (2002) observed no effect on the WVP of WPI ﬁlms to which soya oil
had been added. The incorporation of lipids and a redUction in the level of
plasticizer can be used to improve the WVP of protein-based ﬁlms. Perez-Gago
and Krochta (1999) investigated the effect of pH on the WVP of WPI beeswax

emulsion ﬁlms. As the pH decreased to the isoelectric point (z 5), the WVP of the

29

ﬁlms increased. The increase in WVP was attributed an increase in viscosity of
the ﬁlm solution from protein aggregation and decreased lipid mobility.

Mate and Krochta (1996) compared the OzP of WPI ﬁlms and B-lactoglobulin ﬁlms.

They observed that despite the differences in molecular structure, hydrogen
bonds, hydrophobic interaction and disulﬁde bonds, there was no difference in OzP
between ﬁlms. They did observe that the amount of glycerol was more important
in lowering OzP than the stnlcture of the protein.

Edible protein-based ﬁlms are sensitive to changes in humidity due to the
hydrophilic nature of the proteins. However, there is little literature relating to the
moisture sorption isotherms of edible ﬁlms (Coupland et al. 2000; Kim and Ustunol
2001). The type and level of plasticizer inﬂuences the moisture sorption
characteristics of protein based ﬁlms. Films plasticized with glycerol had higher
equilibrium moisture content compared to sorbitol ﬁlms. This is due to the more
hygroscopic nature of glycerol compared to sorbitol. An increase and/or decrease
in the relative humidity affect the mechanical and barrier properties of protein-
based edible ﬁlms. Cho and Rhee (2002) investigated the relationship between
the moisture sorption characteristics of soy protein ﬁlms and their mechanical
properties. They observed that the TS decreased as RH% increased, and
attributed this to the higher moisture content of the ﬁlms; this correlated to the

higher equilibrium moisture content of the ﬁlms at higher water activities.

30

1.3.3 Thermal Properties

Thermal properties such as melting temperature (T m) and glass transition
temperature (T9) are important in the determination of processing conditions of
polymer ﬁlms. The Tm of a polymer is the temperature at which the polymer
molecules are free to move past each other and the crystalline regions begin to
break up (Rodriguez 1996). The Tm of ﬁlms can also be used to determine an
optimal temperature range for ﬁlm extrusion. For crystalline plastics, processing
temperatures are about 50°C above their Tm (Hemandez, Selke et al. 2000). The
T,I of a polymer is the temperature at which the polymer begins to have segmental
mobility. Above the T9 polymers are soft and ﬂexible, while below To polymers are
stiff andrigid (Hernandez, Selke et al. 2000). The T9 of a polymer is inﬂuenced by
the intermolecular forces. As the intermolecular forces increase, the T9 and
crystallinity of the polymer increases.

The T9 of plastic ﬁlms are inﬂuenced by various parameters (Hernandez,
Selke et al. 2000). These include the primary structure, intermdlecular forces, and
the ﬁlm additives. The T9 of protein ﬁlms is also, inﬂuenced by these same
parameters. The most common additives used in edible ﬁlms are plasticizers,
usually polyols such as glycerol and sorbitol. The effect of different plasticizers on
the T,I of WPI ﬁlms was investigated by Shaw and others (2002). They observed
that Tg’s were lower for glycerol-plasticized ﬁlms than for xylitol- and sorbitol-
plasticized ﬁlms. This was attributed to the higher solubility of glycerol in water

compared to the other plasticizers. Shaw and others (2002) investigated the

31

effects of glycerol concentration and addition of soya oil on the T,I of composite
WPI ﬁlms. As the concentration of glycerol increased, the T9 of the ﬁlms
decreased. Increasing the soya oil concentration decreased the T9 of the ﬁlms
(Table 1.5).

Differential scanning calorimetry (DSC) can be used to determine the
optimum heat seal temperature for WPI ﬁlms. The optimum seal temperature for
the ﬁlms corresponds to the onset transition temperature To (Kim and Ustunol
2001). Heat sealing at temperatures above To resulted in distorted seals and
decreased seal strength. Kim and Ustunol (2001) proposed that heat seal strength
may be due to covalent bond formation between the 8-NH2 group of lysine and the
carboxyl side groups of asparagine or glutamine.

1.4 Methods to Improve the Properties of WPI Films

Protein based edible ﬁlms have good mechanical and oxygen barrier
properties, but are poor moisture barriers. The incorporation of lipids into the ﬁlm-
fonning solution produces a protein/lipid emulsion ﬁlm. Cross-linking the proteins
can enhance their mechanical and barrier properties. Proteins can be cross-linked
through enzymatic, chemical or physical treatments.

1.4.1 Chemical

Chemical agents that are commonly used as cross linking agents include
gluteraldehyde, glyceraldehydes, formaldehyde, glyoxal and dialdehyde starch
(Gennadios 2002). Wheat gluten ﬁlms treated with formaldehyde vapors led to an

increase in tensile strength but had no effect on water vapor permeability

32

Table 1.5: Glass transition temperature of whey protein based edible ﬁlms as

inﬂuenced by various treatments.

 

Film

 

Fllm composition Treatment Tg (K) Reference
WPI/Gly 10:5.0 Glycerol 193.00 Shaw and others 2002b1
WPI/Gly 1028.0 Glycerol 193.00 Shaw and others 2002b1
WPI/Gly 10:10 Glycerol 183.00 Shaw and others 2002b1
WPI/Sor 10:5.0 Sorbitol 233.00 Shaw and others 20025‘
WPI/Sor 10:8.0 Sorbitol 233.00 Shaw and others 20025‘
WPI/Sor 10:10 Sorbitol 233.00 Shaw and others 2002b1
WPI/Xyl 10:50 Xylitol 223.00 Shaw and others 20025‘
WPI/Xyl 10:8.0 Xylitol 223.00 Shaw and others 2002b1
WPI/Xyl 10:10 Xylitol 223.00 Shaw and others 2002b1

WPI/GIy/soya oil 10:50:40 Soya oil 193.69 Shaw and others 20021
WPI/Gly/soya oil 10:5.0:3.0 Soya oil 192.65 Shaw and others 20021
WPI/GIy/soya oil 10:50:20 Soya oil 191.05 Shaw and others 2002‘
WPI/GIy/soya oil 1025.0:0.0 Soya oil 191.01 Shaw and others 20021

 

rFiIms tested using DSC and analyzed according to ASTM D3418

33

(Micard et al. 2000). Galietta and others (1998) cross-linked whey proteins with
formaldehyde and reported an enhancement in mechanical properties and a
decrease in water solubility. Under alkaline conditions formaldehyde and
gluteraldehyde form short methylene cross-links between lysine residues. The
abundance of lysine in B- Iactoglobulin and or—lactoglobulin (12% and 15%
respectively) makes the formation of methylene cross-links as a potential source to
enhance the mechanical properties of WPI ﬁlms. Both formaldehyde and
gluteraldehyde are chemically reacted during cross-linking, though there are
concerns of residual toxicity which limits their acceptability in the production of
edible protein-based ﬁlms (Gennadios, McHugh et al. 1994). Dialdehyde starch
has been studied as an alternative to glyceraldehydes and formaldehyde.
Dialdehyde starch is obtained by reacting native starch with periodic acid to
produce a polymeric aldehyde. Soy protein ﬁlms with dialdehyde starch were
essentially insoluble in water, 0.01 N HCI, 0.01 N NaOH, 4 M urea, and 0.2 M 2-
mercaptoethanol (Rhim et al. 2000). The addition of dialdehyde starch was shown
to be an effective cross-linker for the production of egg white ﬁlms (Gennadios et
al. 1998). An increase in the band intensity of higher molecular weight aggregates
was found using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) with increasing amounts of dialdehyde starch; this was coupled with a
reduction in total soluble matter of the ﬁlms. Calcium chloride was used as a

cross-linker for caseinate-based edible ﬁlms (Avena-Bustillo and Krochta 1993).

34

Calcium chloride induced cross-linking in mlcium caseinate ﬁlms, which
resulted in decreased water vapor permeability. Calcium salts were added as a
way to induce cross-linking of soy proteins ﬁlms to enhance their mechanical
properties (Park et al. 2001). Films with calcium sulfate had higher TS than those
with calcium chloride. Due to the pH of the ﬁlm solution, the protein side chains
had a predominantly negative charge. The increase in TS was attributed to the
bonding of divalent calcium ions to carboxyl groups of the protein. In the same
paper, Park and others (2001) investigated the effects of glucono-8-lactone (GDL)
on the mechanical properties of soy protein ﬁlms. They observed that the addition
of GDL increased the TS and puncture strength, and decreased the %E of soy
protein ﬁlms. The formation of soy protein ﬁlms using GDL occurred because of
hydrogen bonding, while electrostatic interactions and disulﬁde bonds were
involved in the heat formation of soy protein ﬁlms.

1.4.2 Enzymatic

Transglutaminase (T G) forms covalent cross-links between lysine and
glutamine. Mahmoud and Savello (1993) investigated the effects of TG on the
solubility and hydrolyzability of WPI ﬁlms. Cross-linking using T6 produced a
polymerized network of whey proteins. The solubility of ﬁlms increased in the
presence of urea compared to 8 mercaptoethanol (BME). Yildirim and
Hettairachchy (1998) examined the effects of TG on the solubility of WPI ﬁlms with
and without soy protein in a bond dissociating agent. Mechanical properties and

water vapor permeability were also studied. Films were found to be soluble in

35

solutions of urea and sodium dodecyl suifate (SDS) than in BME. They concluded
that hydrogen bonding and hydrophobic interactions are more important than
disulﬁde bonds in maintaining integrity of TG cross-linked ﬁlms. This is to be
‘ expected since transglutaminase induces cross-links through an acyl transfer
reaction with the y-carboxyamide group of glutamine and s-amino group of lysine
(Folk and Cole 1966). WPI ﬁlms cross-linked with TG had greater tensile strength
then control ﬁlms, but TG had no effect in lowering WVP. The two major proteins
found in whey are 8-lactoglobulin and or-lactalbumin; both of these proteins are
composed of ~ 9.0% lysine residues and ~3.0% glutamine residues (Kinsella and
Whitehead 1989). In the past TG was only obtained through animal sources which
limited its availability and use. Currently, TG from a microbial source,
Streptoverticillium mobaraense, is marketed under the name ActivaT" TG through
Ajinomoto Company Inc. (Japan) and has been approved as a GRAS substance.
The production of TG from a microbial source has reduced its cost, which has
increased the feasibility of using TG in commercial production of edible protein
ﬁlms.
1.4.3 Physical

Physical treatments such as y—irradiation, ultraviolet (UV) radiation and heat
curing have been shown to improve mechanical and barrier properties of protein
ﬁlms. Rhim and others (1999) showed that UV radiation increased tensile strength
in corn zein, egg albumen and wheat gluten ﬁlms. Exposure to UV radiation has

been shown to decrease the solubility of sodium caseinate ﬁlms without apparent

36

improvement in its tensile strength (Rhim, Gennadios et al. 1999). UV radiation
causes free radical formation among the aromatic amino acids. These free
radicals are then involved in the formation of intermolecular covalent bonds.
Neither ot-lactalbumin or 8-lactoglobulin have high amounts of these amino acids
(Rhim, Gennadios et al. 1999). Cross-linking of these proteins using UV radiation
will likely not be as effective as other physical treatments.

Caseinate ﬁlms had increased resistance to puncture and moisture when
cross-linked by y-irradiation (Brault et al. 1997). Cross-linking due to irradiation
was also enhanced in the presence of CBCIz. The use of y - irradiation on cast
wheat gluten ﬁlms resulted in an increase in tensile strength (43%) and a decrease
in elongation (79%). This was attributed to the formation of ditryosine, which
resulted in cross-links within the polypeptide macromolecule (Micard, Belamri et al.

2000). Sabato and others (2001) examined the effect of y - irradiation on a whey
protein/soy isolate ﬁlm. They found that y-irradiation was effective in increasing
mechanical strength. Vachon and others (2000) treated milk proteins, casein, and
whey protein (isolate and concentrate) both individually and as a mixture with y-
inadiation. They observed that y-irradiation was more effective on casein than
whey proteins. The lack of effectiveness of y-irradiation on the whey proteins was
attributed to the low tyrosine content.

Heat curing is a common method used to induce cross-linking in synthetic
therrnoset polymers. Heat curing is the exposure of a ﬁlm to an increased

temperature that allows the molecules to relax and/or rearrange. This leads to a

37

decrease in internal stresses (Soroka 1995). Heat curing has been shown to
increase tensile strength and reduce elongation-at-break. Heat curing decreases
water vapor permeability of protein-based edible ﬁlms (Gennadios et al. 1996;
Miller, Chiang et al. 1997). By exposing whey protein based edible ﬁlms to a heat
curing process the tensile strength can be increased, but elongation-at-break is
reduced. Yannas and Tobolsky (1967) reported that non-plasticized gelatin ﬁlms
had lower solubility when heat cured under vacuum. They attributed this to the
formation of a three dimensional network developed from interchain cross-linking.
Kim and others (2002) studied the effects of heat curing soy protein ﬁlms at
atmospheric and sub-atmospheric conditions. Heat curing ﬁlms under vacuum
enhanced the barrier and mechanical properties more rapidly than heat curing at
atmospheric conditions.

By gaining a better understanding of the effects physical and enzymatic
treatments on the mechanical, barrier, and thermal properties of edible ﬁlms
material can be produced that will have wider commercial application. The effect
of physical treatment such as heat curing, or the combination of heat curing with
enzymatic cross-linkers, on the molecular interaction of WPI ﬁlms have not been
extensively studied

Much work has been done the TS, %E, WVP, and OzP of WPI ﬁlms. There
is little or no literature reporting on other properties of WPI ﬁlms that have been
heat cured or treated with TG. These other properties include: toughness,

moisture sorption, melting temperature and heat of fusion.

38

Chapter 2

MECHANICAL AND MOLECULAR PROPERTIES OF HEAT - CURED WHEY
PROTEIN ISOLATE - BASED EDIBLE FILMS AND MICROBIAL
TRANSGLUTAMINASE HEAT - CURED WHEY PROTEIN - ISOLATE BASED
FILMS.
2.1 Abstract

Edible whey protein ﬁlms were produced using whey protein isolate (WPI)
(5% w/v), glycerol (3.5% w/v) and candelilla wax (0.8% w/v). Films were also
produced as described above with the addition of microbial transglutaminase
(0.001g/g WPI) to the ﬁlm-forming solution after heating. Both sets of ﬁlms were
vacuum-heat—cured for 0, 12 and 24h at 90°C. The effects of heat curing and
transglutaminase treatment on mechanical properties (tensile strength (TS),
elongation at break (%E) and toughness) of WPI ﬁlms were assessed. The effects
of heat curing and transglutaminase treatment on the free suﬂhydryl, disulﬁde, and
lysine content and degree of cross-linking were also determined. Heat curing
increased the T8 of WPI and WPI/T G ﬁlms compared to uncured ﬁlms. Heat
curing ﬁlms for 12h decreased %E while ﬁlms at 24h had higher %E compared to
ﬁlms heat cured for 0 and12h. Heat curing increased the toughness of WPI ﬁlms
with 24h ﬁlms having the highest toughness of all ﬁlms tested. All WPI ﬁlms had

lower free sulfhydryl, disulﬁde and lysine content compared to whey protein isolate.

Heat curing decreased the free sulfhydryl content of WPI and WPI/T G ﬁlms. The

39

disulﬁde bond content of WPI ﬁlms heat cured for 24h was higher than for ﬁlms
heat cured at 0 and12h. The disulﬁde content of WPI/T G ﬁlms heat cured for 12
and 24h were higher than ﬁlms at 0h. Heat cured WPI ﬁlms had lower lysine
content compared to uncured ﬁlms. WPI/T G ﬁlms heat cured for 24h had lower
lysine content compared to ﬁlms heat cured at 0 and12h. SDS-PAGE was used to
conﬁrm heat curing ”and TG treatment resulted in covalent cross-links.

2.2 Introduction

Edible ﬁlms consist of a thin continuous layer of edible material which can
act as a barrier to mass transfer and/or as a carrier for food ingredients or
additives. During the last 20 years a renewed interest has developed in the
potential use of edible ﬁlms as a packaging material, with a number of materials
emerging as potential resources. Edible ﬁlms based on proteins possess good
mechanical and oxygen barrier properties, but are poor moisture barriers. Proteins
used in edible ﬁlms come from soy, wheat, corn, whey and collagen (Gennadios
2002). Of these, only collagen has become commercially successful. It is used in
the processed meat industry primarily as an edible alternative to natural casings
(Osbum 2002) and as a wrap on meat products.

Mechanical properties have practical importance in determining which
edible ﬁlms are best suited for a particular application. The two most studied
mechanical properties of whey protein ﬁlms are tensile strength (TS) and
elongation-at-break (%E). Tensile strength is a measure of the maximum stress

that a material can sustain. Elongation-at—break is a measure of the maximum

40

amount of strain a material can sustain without breaking (Hernandez, Selke et al.
2000). Toughness, another mechanical property, is the energy absorbed by a
material before it breaks. It is the area under a stress-strain curve and materials
range from soft and weak to hard and tough (Hernandez, Selke et al. 2000).
Toughness provides another measure of the mechanical properties which is
different from either TS or %E. The toughness of protein-based edible ﬁlms has
not been previously published.

Cross-linking proteins through enzymatic or physical treatment can
enhance mechanical properties. Transglutaminase has been investigated as a
meﬂwod to induce cross-linking in protein ﬁlms (Mahmoud and Savello 1992;
Yildirim and Hettiarachchy 1998; Lim et al. 1999; Larre et al. 2000). Treating ﬁlm
solutions with T6 increased the tensile strength compared to control ﬁlms. The
increase in tensile strength was attributed to cross-linking of proteins. The enzyme

transglutaminase induces cross—links through an acyl transfer reaction with the y-

carboxyamide group of glutamine and e-amino group of lysine (Folk and Cole
1966). In the past TG, was only obtained from animal sources limiting its
availability and use. Currently, TG is available from a microbial source,
Streptoverticillium mobaraense, and is marketed under the name ActivaTM TG by
Ajinomoto Company Inc. (Japan). It has been approved as a GRAS substance.
The production of TG from a microbial source has reduced its cost. The reduced
cost increases the feasibility of using T6 in commercial production of edible protein

ﬁlms.

41

Heat curing is a common method used to induce cross-linking in synthetic
polymers. Heat curing is the exposure of a ﬁlm to an increased temperature that
allows the molecules to relax and/or rearrange which leads to a decrease in
internal stresses (Soroka 1995). Heat curing has been shown to increase tensile
strength and to reduce elongation-at-break. Water vapor permeability of protein-
based edible ﬁlms has also been shown to decrease due to heat curing
(Gennadios, Ghorpade et al. 1996; Miller, Chiang et al. 1997). Heat curing may
induce cross-linking in protein ﬁlms. Yannas and Tobolsky (1967) reported that
non-plasticized gelatin ﬁlms had lower solubility when heat cured under a vacuum.
They attributed this to the formation of a three dimensional interchain cross-link
network. Kim and others (2002) studied the effects of heat curing soy protein ﬁlms
at atmospheric and sub-atrnospheric conditions. Heat curing under vacuum
enhanced the mechanical properties more rapidly than heat curing at atmospheric
conditions.

The objective of this study is to compare the mechanical properties (TS, %E
and toughness) of whey protein isolate ﬁlms with and without transglutaminase,
and heat cured at 90°C for 0, 12 and 24h.

2.3 Materials and Methods
2.3.1 Materials

Whey protein isolate (WPI; Provan 190) was obtained from Glanbia

Ingredients Inc. (Monroe, WI). The whey proteins were extracted in a highly

puriﬁed, undenatured form using membrane technology. Composition of the WPI

42

was determined using AOAC methods (AOAC 2000) and compared to values
provided by Glanbia Ingredients (T able 2.1). Glycerol was purchased from J.T.
Baker Co. (Phillipsburg, NJ), and Candelilla wax was purchased from Stroh and
Pitsch Inc. (West Babylon NY.) Microbial transglutaminase (ActivaTM FP) was
obtained from Ajinomoto Company Inc. (Japan).
2.3.2 Film preparation

Whey protein isolate (WPI) (5% WM and glycerol (3.5% w/v) were mixed in
distilled water and the pH adjusted to 8.0 with 2N NaOH. These solutions were
then heated at 90°C for 15 min while being stirred continuously. Candelilla wax
(0.8% w/v) was added during the last 5 min of heating and allowed to melt into the
WPI solution. The WPI solutions were homogenized for 2 min, using a Polytron
PT 10/35 homogenizer with a PTA 20 TS homogenizing head (Tekmar Co.,
Cincinnati, OH). Each homogenized solution was then strained through a single
layer of cheesecloth to ensure complete incorporation of the lipid and allowed to
equilibrate at ambient temperature for 2 hours. Each solution was then degassed
for 30 min and cast on to a Teﬂon-coated metal pan. Each solution was dried at
2312°C and 35:1: 2%RH for 24h. Films were stored at 2312°C and 50: 2%RH for
24h prior to heat curing.
2.3.3 Transglutaminase cross-linked ﬁlms

Film forming solutions were prepared as above. Solutions were allowed to
cool to ~ 50°C and T6 was added (0.001g/ g of WPI) and allowed to incubate for

3h at 50°

43

Table 2.1: Protein, lactose, ash, and moisture content of whey protein

isolate.

 

Composition Experimental Published values
Values1 (%)

 

Protein 92.1:065 920-95090“
Lactose 001210.004 <1.0%3
Ash 2,620.01 <30%3
Moisture 4.13033 <50%3

 

1n=3 for all components tested.
2 Protein is on a dry basis and a nitrogen conversion factor of 6.38 was used.
3 Values obtained from Glanbia

Each TG containing solution was then degassed under vacuum for 30 min
and cast onto a Teﬂon-coated metal pan. The amount of TG used was based
upon the technical speciﬁcation received from Ajinomoto Company Inc. Each
solution was dried at 23¢2°C and 35: 5%RH for 24h. Films were stored at 23:2°C
and 503: 5%RH for 24h prior to heat curing.

2.3.4 Heat curing

Films were wrapped in aluminum foil and heated in a vacuum oven at 90°C
for 12 and 24h. After heat curing, ﬁlms were allowed to equilibrate at 45: 5% RH
for 24h prior to removal from foil. Films were conditioned at 2312°C and 50:
5%RH for 24h prior to testing.

2.3.5 Mechanical Properties

The mechanical properties of uncured WPI, uncured WPI/T G, heat-cured
WPI and heat-cured WPI/T G ﬁlms were compared. Samples were cut into 2.54
cm-wide strips using a Precision Sample Cutter (T hawing Albert Instrument
Company Philadelphia, PA). Film thickness was determined using a TMI model
549 M micrometer (Testing Machines, Inc. Amityville, NY). Measurements were
taken at 5 locations along the sample and averaged. Tensile strength (T S) and
percent elongation-at-break (%E) and tensile energy at break (toughness) were
determined according to ASTM D 882(ASTM 1997). Samples were tested using

an lnstron Universal Testing Machine Model 5500 (lnstron Corp., Canton MA) with

45

MerlinTM software to determine TS, %E and toughness. Initial grip separation was
50 mm and the crosshead speed was 500 mrn/min.
2.3.6 Sulfhydryl and disulﬁde bond content

The free sulﬂtydryl group and disulﬁde bond content were determined
according to Rangavajhyala and others (1997) based on the procedure developed
by Thannhauser and others (1987). Ellman’s reagent 5, 5'- dithiobis (2-
nitrobenzoic acid) (DTNB) and disodium 2-nitro-5 thiosulfobenzoate (NTBS'Z) were
used to determine the free sulﬂlydryl groups and disulﬁde bond content. Reaction
of sulfhydryl groups and disulﬁde bonds with DTNB and NTSB'2 (both colored
reagents) will yield soluble 2-nitro-5-thiobenzoate anion (NTB'Z).

Since NTSB‘2 is not commercially available it was synthesized from DTNB
according to Thannhauser and others (1987). DTNB was dissolved in 1M sodium
sulﬁte at 38°C and pH was adjusted to 7.5. Oxygen was bubbled through the
solution at 38°C until it changed from bright red to pale yellow.

To determine the free suﬂhydryl content, 10mg of a sample was added to 1
mL of reaction buffer containing 8M urea, 10mM DTNB, 3mM EDTA, 1% SDS, and
0.2M Tris-HCI, pH 8.0. The mixture was allowed to react for 15 min at room
temperature. The sample was centrifuged at 13,600 x g for 10 min to remove any
particulate matter. The absorbance was measured at 412 nm, and the sulﬁ'lydryl
content was calculated using the molar extinction coefﬁcient of NTB (13,600 M'

1cm").

46

To determine the disulﬁde bond content a 10 mg sample of WPI is added to
1 mL of reaction buffer containing 8M urea, 10mM NTSB'Z, 0.1M sodium sulﬁte,
'3mM EDTA, 1% SDS, and 0.2M Tris-HCI, pH 9.5. The mixture is allowed to react
for 25 min in a dark chamber at ambient temperature. The sample is centrifuged
at 13,600 x g for 10 min. The absorbance is measured at 412nm which represents
the total sulfhydryl content. The disulﬁde bond content is calculated as follows:

Qtotal sulfhydryl content - free sulﬂiydryl contentl)

Eiluation 2:1 Disulﬁde bonds = 2

 

2.3.7 Lysine content

Available lysine content was determined using o-phthalaldehyde (OPA)
and N-acetyI-L-cysteine (NAC)(Medina Hernandez and Garcia AIvarez-Coque
1992). Reagent buffer (OPA-NAC) was prepared by combining 25mL of 0.05M
OPA (EtOH), 25mL of 0.05M NAC (aqueous) and 200mL of 0.1M Boric acid-
Borate buffer (pH 9.5). Sample product (~35mg) was heated in 1.5mL of 0.0625M
Tris-HCI and 2% SDS (pH 6.8) at 90°C for 5 min. The material was then
centrifuged at 13,600 x g for 10 min and 0.75mL of supernatant was transferred
into a 10mL volumetric ﬂask. To the ﬂask 4.0mL of OPA-NAC solution was added
to the ﬂask and the volume was adjusted to 10mL with ddHZO. The reaction was
allowed to proceed for 10 min. The absorbance was determined at 335nm vs. a
reagent blank. The concentration (moles of NH2/ 9 of sample) was determined

using a molar absorptivity of 6830 M'1cm".

47

2.3.8 Electrophoresis

The protein content was determined by using the bicinchoninic acid (BCA)
protein assay (Smith et al. 1985) . Film samples were homogenized in 10 mL of
0.0625 M Tris, 3mM EDTA (pH 8.3) buffer for 20 sec. A 0.5 mL sample was then
transferred in to a microcentrifuge tube with 0.5 mL of 10% SDS and 6.6M urea
solution. The samples were heated at 50°C for 20 min, vortexed and heated for an
additional 5 min. After heating samples were centrifuged at 16,000 x g for 20 min.
Samples were diluted with equal volume of water and mixed with BCA reagent.
Samples were then heated at 37°C for 30 min. After heating samples were cooled
to room temperature, absorbance was read at 562nm. Protein concentrations were
calculated using a standard curve obtained using bovine serum albumen.

Molecular weight changes of the whey protein isolate, WPI and WPI/T G
ﬁlms (cured and uncured) were compared using electrophoresis. Sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according
to Laemmli (1970), in a Mini-Protean Il unit ( Bio-Rad Laboratories, Hercules, CA)
using a 4% stacking gel and a 12% resolving gel made from 37.5:1 acrylamide:
bisacrylamide solution. Samples (20mg) were incubated in 5 mL of 10% SDS, 10%
BME and 6.6M urea solution for 24h at room temperature. Samples were then
centrifuged at 13,600 x g for 15min. Film samples (2 mg protein/mL) were mixed
with denaturing sample buffer (0.0625 M Tris, pH 6.8, 2% SDS, 10% glycerol, 5%
mercaptoethanol, and 0.2% bromophenol blue) and heated at 90°C for 3min. Each

lane was loaded with 20119 of protein. The gel (7cm x 8 cm) was run at 200 volts

48

for 45min, stained with 0.1% w/v Coomassie blue (R250) dye in 50% v/v methanol
and 10% v/v acetic acid, and de—stained with 50% v/v methanol and 10% acetic
acid solution.
2.3.9 Statistical analysis
All experiments were replicated 3 times. Results were analyzed using the
mixed model procedure in SAS (SAS Institute, Cary, NC). Treatment means were

compared with signiﬁcance deﬁned as ps 0.05.

2.4 Results and discussion

2.4.1 Mechanical Properties

The mechanical properties of polymer ﬁlms can be improved by increasing
cross-linking between polymer strands which creates a three dimensional network.
Both heat curing and T6 are able to induce intermolecular cross-linking between
protein strands. The effect of heat curing time, TG treatment and the interaction of
time and TG had a signiﬁcant impact on the TS and %E. Heat curing time
signiﬁcantly impacted ﬁlm toughness (Table 2.2). Treating ﬁlms with TG produced
ﬁlms with signiﬁcantly different TS and %E compared to non-TG ﬁlms. However, it
had no effect on toughness. Films heat cured at 12 and 24h had a signiﬁcant effect
on the TS compared to Oh. Films heat cured for 24h had signiﬁcantly different %E
and toughness compared to the other ﬁlms (Table 2.3).
2.4.2 Tensile Strength.

The mechanical properties of WPI ﬁlms are shown in Table 2.4. Heat -

curing WPl/T G ﬁlms increased the TS compared to uncured WPI/T G ﬁlms
49

(p<0.05). Heat-curing WPI/T G ﬁlms for 24h produced the highest TS (p<0.05) of all
WPI/T G ﬁlms. The TS of WPI/T G ﬁlms decreased after 24h of heat curing. WPI
ﬁlms heat-cured for 24h were not signiﬁcantly different from uncured ﬁlms. WPl
ﬁlms heat-cured for 12h had the highest TS of all ﬁlms tested (p<0.05). WPI/T G
ﬁlms heat cured for 12h were not signiﬁcantly different compared to WPI ﬁlms heat
cured for 0 and 24h. WPI/T G ﬁlms heat cured for 24h were not signiﬁcantly
different compared to WPI ﬁlms heat cured for 0 and 24h. The treatment of protein
ﬁlm forming solutions with TG has been shown to increase their TS (Yildirim and
Hettiarachchy 1998; Larre, Desserrne et al. 2000). This is contrary to the results
obtained. The previous studies used transglutaminase obtained from mammalian
sources while transglutaminase from a microbial source was used in this study.
Transglutaminase from microbial sources cross-linked only one lysine residue
while mammalian transglutaminase cross-linked three lysine residues in 0.-
Iactalbumin (Lee et al. 2002). This difference in lysine cross-links may be
responsible for the lower TS observed with these ﬁlms. The initial heat treatment of
the aqueous solution may have produced intermolecular disulﬁde bonds which
decreased the accessibility of lysine. Miller and others (1997) reported heat curing
increased the TS of whey protein ﬁlms. They observed a linear relationship
between T8 and heat curing time as time increased. The results of this study show
an initial increase in TS with increasing time then a decrease in TS. The difference
in results from those reported by Miller and others can be attributed to a difference

in heat curing conditions.

50

Table 2.2: Signiﬁcance‘ of time andlor transglutaminase treatment on the
tensile strength, elomation at break and toughness of heat-cured WPI ﬁlms.

 

 

Source of Variation DFz TS3 %E‘ T5

Tune 3 <0.0001 0.0002 <0.0001
TG 1 <0.0001 0.0002 0.4560
Tlme x T6 3 <0.0001 0.0024 0.1082

 

‘ Probability values

2 OF = Degrees of freedom
3 TS = Tensile strength

4 %E = Elongation at break
5 T = Toughness

Table 2.3: Comparison of the significance‘ of heat curing time and
transglutaminase treatment on the tensile strength, elongation at break and
toughness of heat-cured WPI ﬁlms.

 

 

 

Effects Source of BF2 T81 %E‘ T'5
Variation
TG NOTG VS. TG 40 <0.0001 0.4560 0.4560
0h VS. 12h 40 <0.0001 0.6976 0.6976
Time 0h VS. 24h 40 <0.0001 <0.0001 <0.0001

12h VS. 24h 40 0.2503 <0.0001 <0.0001
‘ Probability values
2 DF = Degrees of freedom
3 TS = Tensile strength
4 %E = Elongation at break
T = Toughness

 

51

Table 2.4: Mechanical properties of whey protein-isolate based ﬁlms with
and without microbial transglutaminase heat cured at 90°C for 0, 12, and 24h.

 

 

 

Curing Elongation Tensile
Treatment Time Thickness at Break Toughness Strength
(h) (mil) (%) lkJIm°l (Mpa)
0 7.78:0.81‘ 7.781081“ 22516.72a 1.110.12a
WPIITG 12 5.161026“ 5.161026" 37.717.62a 3.81014”
24 72710.76“ 72710.76" 756117.07” 4610.10“
0 7.6610518 15118.15“ 406112.92a 4.2107299“
WPI 12 57511.57a 15013.06b 30.216.41a 5511.60
24 66611.14a 23.212.46°-d 67.41 6.66b 4.1101499"

 

°‘" means with different superscript are signiﬁcantly different (p<0.05),
Comparisons are made within each column, n=3 for all treatments

52

The previous study heat cured ﬁlms under atmospheric conditions at temperatures
below 90°C and at relative humidities of 40, 60 and 80%.

These lower temperatures and increased RH allowed the ﬁlms to retain
more moisture. Kim and others (2002) heat cured soy protein ﬁlms under sub-
atmospheric conditions and observed an initial increase in TS after 6 h of heat
curing then remained relatively unchanged for the remainder on the time. The heat
cured ﬁlms had lower moisture content compared to uncured ﬁlms. The difference
in moisture content of heat-cured ﬁlms inﬂuenced their mechanical properties. This
was attributed to cross-linking which results in a structure that restricts moisture

uptake.

2.4.3 Elongation at Break.

WPI ﬁlms heat cured for 24h had the highest %E of all ﬁlms tested
(p<0.05). The %E of all WPI/T G ﬁlms were signiﬁwntly lower compared to WPI
ﬁlms. WPI/T G ﬁlms heat cured for 24h and WPI ﬁlms heat cured for 24h were not
signiﬁcantly different. The %E of ﬁlms increased from 12 to 24h for both WPlfl' G
and WPI ﬁlms. Kim and others (2002) and Miller and others (1997) observed a
decrease in %E with an increase in heat curing time. In this research an initial
decrease in %E was observed in WPI/T G followed by an increase in %E with
increasing heat-curing time. This was not observed in WPI ﬁlms. The difference

between results and those reported previously may be attributed to the difference
53

in heat curing conditions. In this study the ﬁlms were completely wrapped in
aluminum foil, while the previous studies did not report wrapping the ﬁlms during
heat curing. Wrapping the ﬁlms would allow more moisture to be retained which
acts as a plasticizer and would increase %E.

2.4.4 Toughness.

Toughness is deﬁned as the energy absorbed by the sample at failure
(Rodriguez 1996). It is measured as the area under a stress-strain curve, and
takes into account both the TS and %E. There was no signiﬁcant difference
between WPI/T G and WPI ﬁlms heat cured at 0 and12h. Both ﬁlms with and
without TG and heat cured for 24h had the highest toughness and were not
signiﬁcantly different from each other. The toughness of edible ﬁlm has not been
previously reported. Films with moderate TS and high %E produced ﬁlms with the
highest toughness. Higher toughness ﬁlms may be better able to withstand ﬁll and
seal conditions, and product distribution.

2.4.5 Electrophoresis

Cross-linking of heat cured WPI and WPI/T G ﬁlms was veriﬁed using SDS-
PAGE under reducing conditions (Figure 2.1 and 2.2). The three most abundant
proteins found in whey are B-lactoglobulin, a-lactalbumin and bovine serum
albumin (BSA) with molecular weights of 18,363 daltons, 14,174 daltons and
66,000 daltons respectively(Kinsella and Whitehead 1989).

The effect of heat curing on WPI ﬁlms at 90°C for 0, 12 and 24h are shown in

Figure 2.1. In the heat cured WPI ﬁlms a decrease in the intensity of [3-

54

lactoglobulin, tat-lactalbumin and BSA was observed compared to WPI powder and
uncured WPI ﬁlms. This was accompanied by an increase in higher molecular
weight polymers. In the case of WPI ﬁlms heat cured for 12 and 24h high
molecular weight aggregates were observed at the top of the stacking gel. These
aggregates were too large to enter the stacking gel. Both uncured WPI ﬁlms and
WPI had similar electrophoretic patterns. The increase in higher molecular weight
polymer (25-41kDa) and aggregates (200+ kDa) in WPI ﬁlms heat cured for 12 and
24h suggests the formation of covalent protein cross-links other than disulﬁde
bonds(Rangavajhyala, Ghorpade et al. 1997; Yildirim and Hettiarachchy 1998).
The effects of heat curing WPI/T G ﬁlms at 90°C for 0, 12 and 24h are
shown in Figure 2.2. In all of the WPI/T G ﬁlms the formation of aggregates was
observed at the top of the stacking gel. These aggregates were unable to migrate
in to the stacking and resolving gels. The presence of protein aggregates in the
uncured WPI/T G ﬁlms indicates the formation of transglutaminase wtalyzed cross-
Iinks. The formation of aggregates in the uncured WPI/T G was similar to those
observed by Yildirim and Hettiarachchy (1998). The WPlfl' G ﬁlms heat cured at
90°C for 12 and 24h showed decreasing intensity in the B-lactoglobulin,ot-
lactalbumin and BSA bands with a corresponding increase in higher molecular
weight polymers. The increase in the higher molecular weight polymers in the heat

cured WPI/T G ﬁlms indicates the formation of protein cross-links.

55

200 kDa
116 kDa
97 kDa
66 kDa

45 kDa

31 kDa

21 kDa

14 .4kDa

 

Figure 2.1: SDS-PAGE analysis of whey protein isolate (WPI) and whey
protein isolate based ﬁlms heat cured at 90°C for 0, 12, and 24h. Using a 4%
stacking gelm and a 12% resolving gel made from 37. 5. 1

cu , --,' ' ' solution with 20119 of protein loaded in each lane.

 

 

200 kDa
‘39 EB:

66 kDa

45 kDa
31 kDa

21 kDa

  
 
  

14.4 kDa . _ .

6.5 kDa 1

Figure 2. 2: SOS-PAGE analysis of whey protein isolate (WPI) and whey
protein Isolate based ﬁlms with hansglutaminase heat cured at 90°C for 0,
12, and 24h. Using a 4% stacking gel and a 12% resolving gel made from
37.5:1 cu,‘ ” hisan,‘ ” solution with 201119 of protein loaded in each
lane.

 

 

57

2.4.6 Sulﬂ'lydryl group, disulﬁde bond and lysine content

The free sulfhydryls, disulﬁde bonds and lysine content of WPI ﬁlms with
and without TG heat cured at 90°C for 0, 12and 24h are shown in Table 2.5.
Uncured WPI ﬁlms (0h) had higher SH content than heat cured WPI and WPI/T G
ﬁlms (p<0.05). Uncured WPI/T G (0h) ﬁlms had higher SH content than heat cured
ﬁlms (p<0.05). Heat curing reduced the SH content of both WPlfl' G and WPI ﬁlms.
There was no difference in SH content between heat cured WPI/T G and heat
cured WPI ﬁlms.

Uncured WPI ﬁlms had higher S-S content than uncured WPI/T G ﬁlms
(p<0.05). There was no statistical difference between WPI ﬁlms heat cured for 0
and 24h. WPI ﬁlms heat cured for 12h were not different from WPlfl G ﬁlms heat
cured for 12 and 24h. Heat curing ﬁlms with WPI for 12h increased the SS content
compared to uncured WPI ﬁlms.

Films without TG had more available lysine compared to ﬁlms treated with
TG. Uncured WPI ﬁlms had the highest available lysine content of all ﬁlms tested.
Heat curing WPI ﬁlms reduced the available lysine content compared to the
uncured WPI ﬁlms. Increasing the heat curing time did not signiﬁcantly reduce the
available lysine content. Treating the ﬁlm solutions with TG prior to casting
decreased the available lysine content compared to the untreated ﬁlms. This was
expected since TG catalyzes the formation of cross-links between lysine and
glutamine. Heat cured ﬁlms (24h) had the lowest available lysine content

compared to the uncured ﬁlms with and without TG.

58

Table 2.5: Free sulﬂtydryl, disulﬁde bonds and lysine content of whey
protein-isolate based ﬁlms with and without microbial transglutaminase heat

cured at 90°C for 0, 12 and 24h.

 

 

 

Heat SH ( $617 ( (II-thslel
' . 0

$1.13) ("mug pmte'") Purge": ”groteln) g

0 13.0 1 1.671‘ 22.2 1 112° 15.5 1 060°
WPI 12 3.0 1 052° 25.9 1 041° 2.1 1 008°

24 3.0 1 030° 21.4 1 029° 1.6 1 0.11°-°

0 6.9 1 034° 19.7 1 0.31b 1.4 1 007°
WPI/T G 12 3.2 1 0.66° 24.6 1 0.27c 1.0 1 003°

24 4.5 + 077° 24.4 + 057° 0.3 1 0.04f

‘4 means with different s-uperscript are signiﬁcantly different (p<0.05),
Comparisons are made within each column, n=3 for all treatments.

59

The alkaline condition of ﬁlm formation in conjunction with heat curing may
induce racemization of amino acid residues which can lead to the formation of
covalent cross — links. An existing disulﬁde bond can form dehydroalanine through
a base catalyzed elimination of persulﬁde. In cysteine the [3 — elimination of the SH
will yield a dehydroprotein. The dehydroprotein is highly reactive and may form a
lysinoalanine cross - link with the 6- amino group of lysine (Friedman 1999;
Friedman 1999). For both WPI and WPI/T G ﬁlms, heat curing may result in the
formation of isopeptide cross-links. The formation of isopeptide cross-links can
also occur between the 13- amino group of lysine and the amide group of
asparagine or glutamine residues via a condensation reaction (Singh 1991 ). The
formation of isopeptide cross-links in both WPI and WPlfl‘ G ﬁlms maybe the
reason that the change in disulﬁde content was not proportional to the change in
sulfhydryl content (Table 2.5). The formation of isopeptide cross-links during heat
curing can be attributed to the decrease in lysine content observed in both WPI
and WPI/T G ﬁlms.

The electrophoresis patterns of the ﬁlms heat cured for 24h showed higher
molecular weight polymers compared to the other ﬁlms (Figure2.1 and 2.2). The
ﬁlms with the lowest lysine content also coincide with the ﬁlms that exhibit the
highest toughness (Table 2.4). This suggests that covalent cross - links involving
the e - amino group of lysine are important in enhancing the mechanical properties

0f WPI and WPI/T G ﬁlms.

60

2.5 Conclusions

The heat curing of WPI and WPI/T G ﬁlms generally enhanced the
mechanical properties of these ﬁlms. Heat curing WPI and WPI/T G ﬁlms for 24h
produced ﬁlms with the highest toughness of all ﬁlms tested. Toughness has not
been measured in previous studies. Toughness may relate to how a ﬁlm
withstands ﬁll and seal conditions. SDS-PAGE indicated that heat curing the WPI
ﬁlms induced the formation of covalent cross-links, which were also observed in all

WPI/T G ﬁlm samples. Heat curing may promote the formation of isopeptide bonds

involving the 6- amino groups of lysine.

61

 

Chapter 3

BARRIER PROPERTIES OF HEAT - CURED AND MICROBIAL
TRANSGLUTAMINASE HEAT - CURED WHEY PROTEIN - ISOLATE BASED
EDIBLE FILMS.

3.1 Abstract
Edible whey protein ﬁlms were produced using whey protein isolate WPI

(5% w/v), glycerol (3.5% WM and candelilla wax (0.8% w/v). Additional ﬁlms were
produced containing the above ingredients with the addition of microbial
transglutaminase (T G) (0.001g/g WPI) to the ﬁlm forming solution after heating.
Both sets of ﬁlms were vacuum — heat — cured for 0 and 24h at 90°C. The water
vapor permeability (WVP) of these ﬁlms was determined at 37.8°C and 85% RH,
while the oxygen permeability (OzP) was determined at 23°C and 0% RH. Moisture
sorption isotherms (MSI) of the ﬁlms were plotted at 5°C and 23°C using the
Guggenheim-Anderson-de Boer equation (GAB). Heat curing WPlrl’ G ﬁlms
reduced the WVP compared to uncured WPI, WPI/T G and cured WPI ﬁlms. The
addition of TG reduced the oxygen permeability compared to ﬁlms without TG.
Heat curing reduced the OzP of ﬁlms compared to uncured ﬁlms. Heat curing
WPI/T G ﬁlms resulted in the greatest reduction in OzP of ﬁlms tested. The
moisture sorption of ﬁlms at 5°C decreased as follows WPl> WPI/TG> WPI, 24h>
WPI/T G, 24h. At 23°C, the MS of ﬁlms decreased as follows WPI> WPI, 24h>
WPIfTG> WPI/T G, 24h. The ﬁlms MS determined at 5°C were higher than the MS

of ﬁlms determined at 23°C.

62

3.2 Introduction

Conventional packaging is used to prevent the mass transfer of moisture,
oxygen, aroma, and oil between a food product and the environment and protect
the product from mechanical forces encountered during storage and distribution.
Edible ﬁlms can be used to prevent mass ﬁansfer between a food product and the
environment, which can lead to an increase in the quality and shelf life of the
product. Thus, edible ﬁlms are not intended to replace conventional packaging
(Krochta 2002) in all functions. Edible ﬁlms can be used in conjunction with
conventional packaging which can result in a more biodegradable and sustainable
package. Edible ﬁlms add a second layer of protection and thus can help maintain
product quality when the package is opened.

Physical and enzymatic treatments such as heat curing and
transglutaminase - catalyzed cross linking have been investigated as methods to
enhance physical properties of protein based ﬁlms (Mahmoud and Savello 1993;
Miller, Chiang et al. 1997; Sabato, Ouattara et al. 2001; Kim, Weller et al. 2002;
Kim, Weller et al. 2002). Protein - based ﬁlms are good barriers to oxygen but are
poor moisture barriers. Their poor performance as moisture barriers is due to the
hydrophilicity of proteins and the use of hydrophilic plasticizers(Kester and
Fennema 1986) in their composition. Heat curing has been shown to improve the
moisture resistance of cast protein ﬁlms from soy (Rangavajhyala, Ghorpade et al.
1997; Rhim, Gennadios et al. 2000; Kim, Weller et al. 2002; Kim, Weller et al.

2002) and whey protein (Miller, Chiang et al. 1997). Transglutaminase catalyzed

63

cross-linking in whey protein and soy was investigated by Yildirim and
Hettiarachchy (1998). Other barrier properties such as oxygen and aroma
perrneabilities have not been as extensively studied. The effects of heat curing and
transglutaminase treatment on the oxygen permeability and moisture sorption
properties of protein-based ﬁlms have not been extensively reported. Lim and
others (1999) investigated the oxygen permeability of egg white protein ﬁlms
cross-linked with transglutaminase. They found that oxygen permeability increased
with increasing relative humidity.

The objectives of this study were: 1) to determine the water vapor
permeability (WVP) and oxygen permeability (OZP) of whey protein isolate based
edible ﬁlms treated with and without transglutaminase, and heat cured at 90°C for
0 and 24h; 2) to characterize the moisture sorption isotherms of these ﬁlms at 5
and 23°C.

3.3 Method
3.3.1 Materials

Whey protein isolate (WPI; Provan 190) was obtained from Glanbia
Ingredients Inc. (Monroe WI). The whey proteins were extracted in a highly
puriﬁed, undenatured form through membrane technology. Composition of WPI
was determined using AOAC methods (AOAC 2000) and compared to values
provided by Glanbia Ingredients. Glycerol was purchased from J.T. Baker Co.
(Phillipsburg, N.J.). Candelilla wax was purchased from Stroh and Pitsch Inc.

(West Babylon NY). Microbial transglutaminase was obtained from Ajinomoto
Company Inc. (Japan).
3.3.2 Film preparation

Whey protein isolate (WPI) (5% WM and glycerol (3.5% w/v) were mixed in
distilled water and the pH adjusted to 8 with 2N NaOH. Each solution was heated
at 90°C for 15 min while being stirred continuously. Candelilla wax (0.8% w/v) was
added during the last 5 min of heating and allowed to melt into the WPI solution.
The WPI solutions were then homogenized for 2 min, using a Polytron PT 10/35
homogenizer with a PTA 20 T8 homogenizing head (T ekmar Co., Cincinnati, OH).
Each solution was strained through a single layer of cheesecloth to ensure
complete mixing of the lipid, and than allowed to equilibrate at ambient
temperature for 2 hours. The ﬁlm solutions were degassed for 30 min and cast on
a Teﬂon-coated surface, and allowed to dry at 2312°C and 3515%RH for 24h.
Films were stored at 2312°C and 5015%RH for 24h prior to heat curing.
3.3.3 Transglutaminase cross-linked ﬁlms

Film forming solutions were prepared as described. Each solution was
cooled to ~ 50°C and TG was added (0.0019! g of WPI) and allowed to incubate
for 3h at 50°C. Each solution was then degassed under vacuum for 30 min and
cast onto a Teﬂon-coated surface. The amount of TG used was determined from
the technical speciﬁcation received from Ajinomoto Company Inc. The ﬁlms were
allowed to dry at 23i2°C and 3515%RH for 24h and then stored at 2312°C and

501 5%RH for 24h prior to heat curing.

65

3.3.4 Heat curing

Films were wrapped in aluminum foil and heated in a vacuum oven at 90°C
ﬁlms for 24h. After heat curing, ﬁlms were allowed to equilibrate at 4515% RH for
24h prior to removal from the foil. Films were then conditioned at 2312°C and
50:5%RH for 48h prior to testing.
3.3.5 Water vapor permeability (WVP)

The water vapor transmission rate (WVTR) of the ﬁlms was determined at

37.8°C and 85%RH using ASTM E 96-00 “Standard test method for water
vapor transmission of materials” (ASTM 2000). Specimens were cut using
a standard cutter and adhered with wax to standard size dishes containing
dessicant. Samples were placed into an environmental chamber maintained
at 37.8°C and 85% RH. The weight gain was recorded at 0, 0.5, 2.0, 3.0,
6.0 and 9.0h. The WVTR was calculated by linear regression of weight gain

and time per area. To determine WVP from WVTR the following equation

 

was used:
P _ WVT R x d
Equation 3:1 — Ap
P = permeability

WVTR = water vapor transmission rate

d = thickness of ﬁlm

Ap = difference of pressure between the internal
and external sides of the ﬁlm

66

3.3.6 Oxygen permeability

Oxygen permeability (OzP) was determined according to ASTM D 3985
“Standard test method for oxygen gas transmission rate through plastic ﬁlm and
sheeting using a coulometric sensor” (ASTM 2000) using a Oxtran 2/21 (Mocon
Minneapolis, MN). The testing surface area was reduced from 50 cm2 to 5 cm2
using aluminum foil tape. Film OzP was determined at 23°C and 0%RH. All ﬁlm
samples were conditioned at test conditions for 10h prior to testing. The test gas
was air (21% oxygen) and nitrogen was used as the carrier gas.
3.3.7 Moisture sorption isotherm

The hygroscopicity of a product relates to its ability to absorb moisture from
its surrounding environment. To determine if heat curing affects the hygroscopicity
of WPI ﬁlms, moisture sorption isotherms (MSI) were experimentally derived for
WPI and WPI/TG ﬁlms at 5 and 23°C. Initial moisture content (IMC) was
determined by weighing and then drying 3 0.3g sample in a convection oven at
100°C for 24h. Samples were allowed to cool in a desiccator for 30 min and then
weighed to determine loss of moisture. The IMC (dry basis) was determined using

the following equation:

AW x100

 

Equation 3:2 IMC (%) =
f

AW = change in weight
Wf = weight of sample before drying

67

To derive the MSI, samples were placed into containers with different humidity
conditions using saturated salt solutions. Saturated salt solutions were prepared
according to ASTM standard E-104 (ASTM 2000). Five humidity conditions were
prepared, including LiCI-Hzo (12%), MgCl2 (33%), Mg (N03)2-6H20 (50%), NaNOz
(60%), and NaCl (75%). Samples were placed into each relative humidity
condition (90 mil HDPE bucket) and allowed to equilibrate. Samples were
weighed every 2 days until they reach a constant mass. The moisture sorption
data was modeled using the Guggenheim-Anderson-de Boer equation as follows:

(W... CKa.)

(1 — Kerw + CKaw)

GAB = equilibrium moisture content

Wm = BET monolayer moisture content

C = Guggenheim constant

K = factor correcting properties of the multilayer
molecules corresponding to the bulk liquid

aw = water activity

 

Equation 3:3 GAB =

To determine the Wm, C, and K parameters the equation was transformed as

 

follows:
aw 2
Equation 3:4 EM = “(03) + I6(aw)+ 7
or = K/Wm(1/C-1)
3 = 1M... (1-2/C)

The values for or, [land y were determined by performing quadratic regression

analysis on the experimental equilibrium moisture content values.

68

3.3.8 Statistical analysis

All experiments were replicated 3 times. Results were analyzed using the
mixed model procedure in SAS (SAS Institute, Cary, NC). Pair - wise
comparisons were made between treatment means using Student-Newmans-Kuhl

with a signiﬁcance of difference deﬁned as ps 0.05.

3.4 Results and Discussion
3.4.1 Water Vapor Permeability

The VWP of WPI and WPlfI' G ﬁlms heat cured for 0 and 24h are shown in
Table 3.1. Heat curing signiﬁcantly (p<0.05) lowered the WVP of WPI/TG ﬁlms.
Heat curing lowered the WVP of WPI ﬁlms but was not signiﬁcantly different from
the control. Only WPI/T G heat cured for 24h was found to have a signiﬁcantly
lower WVP than the other samples. The values obtained for the WVP of the WPI
and WPI/T G ﬁlms are higher than those reported by other researchers (Miller,
Chiang et al. 1997; Yildirim and Hettiarachchy 1998; Kim 2000; Kim, Weller et al.
2002). The higher WVP values observed in this study may be due to the
differences in testing conditions. The previous studies were conducted at lower
temperatures (22-25°C) and relative humidity (SO-80%RH) while in this study the
testing conditions were 37.8°C and 85% RH. The higher temperature was chosen
to study the WVP of the ﬁlms under tropical conditions. An increase of ~10°C in
temperature will increase the permeability by a factor of 2 (Hernandez, Selke et al.

2000). The increase in relative humidity allows for an increase in absorbed water.

69

Table 3.1: Water vapor penneabllity at 37.8°C and 85%RH and oxygen
permeability at 23°C and 0%RH of whey protein-isolate based ﬁlms with
and without microbial transglutaminase and heat cured at 90°C for 0 and
24h

 

 

 

Treatment Heat WVP 02P(x 10-2
curing g*mmlm2*d*kPa cc*mmlm2*d*kPa
time (h)
0 33.712.9‘ 8.410.4‘
“MTG 24 20313.3” 5210.4"
WPI 0 36.5:I:3.2a 11.1:0.1°
24 30.714.6‘ 7.710.3‘

 

“ means with the same superscript are not signiﬁcantly different (p<0.05).
Comparisons are made within each column. n=3 for all treatments.

70

This absorbed water acts as a plasticizer and increases the free volume of the ﬁlm
(Hernandez, Selke et al. 2000).
3.4.2 Oxygen Permeability

The OP of WPI and WPI/T G ﬁlms heat cured at 90°C for 0 and 24h are
shown in Table 3.1. Heat curing both WPI/T G and WPI ﬁlms signiﬁcantly
decreased the 02P (p<0.05). WPl/T G ﬁlms heat cured for 24h had the lowest 02P
(p<0.05) of all ﬁlms tested. No signiﬁcant difference between uncured WPI/T G
ﬁlms and WPI ﬁlms heat cured for 24h were observed. Both TG and heat curing
induce cross-linking in WPI ﬁlms; thus the increase in cross-linking may reduce the
OzP of the ﬁlms. The polar nature of cross-linked protein based ﬁlms limits the
motion of the polymer chains(Chick and Ustunol 1998). Increased cross-linking
decreases the segmental mobility of the polymer, thus, decreasing the amorphous
regions in the ﬁlms. Permeability only occurs in the amorphous regions of a ﬁlm
(Hernandez, Selke et al. 2000).
3.4.3 Molsture sorption isotherms (MSI)

The GAB moisture sorption isotherm model has been shown to ﬁt well the
moisture sorption data of many food products and is the most accepted model for
foods (Bizot 1983). The MSl for WPI and WPlfT G ﬁlms heat cured at 90°C for 0
and 24h at 23°C (Figure 3.1) and 5°C (Figure 3.2) were determined. The
equilibrium moisture content (EMC) of WPI and WPlfl' G ﬁlms heat cured at 90°C
for O and 24h at 23°C increased slowly from 0.1 to 0.6 aW followed by an ‘

exponential increase in the EMC of the ﬁlms at higher aw. The EMC of ﬁlms at 5°C

71

 

 

 

.wm
05° - OWPI,24h
Awprrrc

oWPurc, 24h II;
0.50 + 'l o.

 

 

o
b
9

\~
\

0.30 -

0.20 —

Equilibrium Moisture content (919 solids)

0.10 4

 

 

 

0.00 i r r
0 0.2 0.4 0.6 0.8

Water Activity (aw)

 

 

 

 

Figure 3.1: Moisture sorption isotherm at 23°C of whey protein-isolate based
ﬁlms with and without microbial transglutaminase and heat cured at 90°C for
0 and 24h.

72

 

 

0.70 .

 

 

 

 

 

 

.WPI
o WPIITG
0'60 A WPI,24 ’—
0 WPI/TG,24 ’7:
0.50 - : .—

0.40

 

 

0.30

 

 

0.20

 

 

Equilibrium moisture content (gig solids)

0.10

 

 

 

 

0.00

 

T

0 0.2 0.4 0.6 0.8
Water activity (aw)

 

 

 

Figure 3.2: Moisture sorption isotherm at 5°C of whey protein-isolate based
ﬁlms with and without microbial transglutaminase and heat cured at 90°C for
0 and 24h.

73

 

showed a slow increase form 0 — 0.5 aW followed by an exponential increase in
EMC of the ﬁlms. At both 5 and 23°C the MSI of WPI and WPI/T G ﬁlms heat
cured for 24h were less pronounced than their corresponding uncured ﬁlms. The
difference in EMC of WPl and WPI/T G ﬁlms heat cured at 90°C for 0 and 24h
were not noticeable for aW ﬁom 005 at either 23 or 5°C. As aW increased above
0.5 the EMC of samples at 5°C were higher than those at 23°C. The EMC of TG
cross-linked egg white ﬁlms was seen to increase with decreasing temperature
(Lim, Mine et al. 1999). At 5°C and 23°C WPI and heat cured WPI ﬁlms had
sorption isotherms that showed higher moisture content at the same relative
humidity (aw) compared to WPI/T G and heat cured WPI/T G ﬁlms at the same
temperatures. The ﬁlms with lower isotherms (WPI, 24h and WPI/T G, 24h)
corresponded with ﬁlms with lower lysine content (Table 2.5).By increasing the
amount of cross-linking by heat curing produced ﬁlms with lower sorption
isotherms. The increased cross-linking in the heat — cured ﬁlms will decrease the
amorphous regions in the ﬁlms. As with permeability, sorption occurs in the
amorphous regions of the ﬁlm.

The GAB constants were determined by ﬁtting the GAB equation to the
experimental isotherm data for the ﬁlms at 23°C and 5°C (Table 3.2). The
monolayer moisture content (Wm) of heat cured WPI and WPI/T G ﬁlms were lower
than uncured WPI and WPlfl' G ﬁlms at both 23 and 5°C. The Wm of WPI/T G ﬁlms
were lower than WPI ﬁlms at both 23 and 5°C. At 23°C the Guggenheim constant

(C), a correction factor for the enthalpy difference between the monolayer and

74

Table 3.2: Parameters used to ﬁt the Guggenheim-Anderson-de Boer

equation at 23°C and 5°C of whey protein-isolate based ﬁlms with and

without microbial transglutaminase and heat cured at 90°C for 0 and 24h.

 

 

 

 

 

 

 

 

 

Treatment Heat
curing 1 2 3
time W... C k RMS‘

(0)
23°C 5°C 23°C 5°C 23°C 5°C 23°C 5°C
WPI/T6 0 0.1 46 0.148 96.90 91.44 0.980 1.01 6 5.13 2.72
24 0.1 36 0.134 99.89 59.89 0.985 1.01 0 4.34 4.44
WPI 0 0.1 49 0.152 202.77 91 .87 1 .009 1 .035 1.99 2.72
24 0.143 0.146 138.87 60.63 1.012 1.013 3.34 4.38

 

 

 

 

 

 

W... = monolayer moisture content
2C = factor correcting enthalpy difference between monolayer and multilayer of

water

3k = factor correcting enthalpy difference between free water and multilayer water
‘RMS = root mean square values

75

 

multilayer of water, for WPI and heat cured WPI ﬁlms was higher than for WPlfr G
and heat cured WPI/TG ﬁlms. At 5°C the C for uncured WPI and WPI/T G was
higher than heat cured WPI and WPI/TG ﬁlms. The k value, a correction factor for
the enthalpy difference between free and multilayer water, was higher for uncured
and heat cured WPI ﬁlms than uncured and heat cured WPlfr G at both 23 and
5°C. The root mean square (RMS) was used to evaluate the quality of the ﬁt
between the experimental and calculated values for ﬁlms at 23 and 5°C (Table
3.2).A lower RMS value indicates a good ﬁt. The RMS values determined for the
experimental and calculated values at both 23 and 5°C were in the low range. The
MSI can be used to predict barrier and mechanical properties of ﬁlms at various
RH (Chick 1998). From the MSI of WPI and WPI/T G at 5°C ﬁlms it could be
predicted that the ﬁlms would have increased water vapor permeability and
elongation, and decrease in tensile strength compared to ﬁlms at 23°C (Aydinli and
Tutas 2000; Cho and Rhee 2002).
3.5 Conclusions

At higher humidity and temperature the effectiveness of WPI ﬁlms as a
moisture barrier is drastically reduced. Cross-linking WPI ﬁlms with TG and heat
curing can improve the water vapor and oxygen permeability of the ﬁlms. The
cross-linking produced using heat curing and TG inhibits the diffusion of both water
vapor and oxygen through the ﬁlms. Cross-linking decreases the segmental
mobility of the polymer chains leading to a decrease in permeability. Heat cured

WPI/T G ﬁlms had the lowest WVP and OZP of all ﬁlms tested. Heat curing and TG

76

 

decreased the MSI of WPI ﬁlms at both 23 and 5°C. The EMC of the ﬁlms at 5°C
were slightly higher than those at 23°C. The higher MS of ﬁlms at 5°C could be a
problem for products that require refrigeration. At refrigerated temperatures the

ﬁlms would have higher WVP permeability and lower mechanical properties.

77

 

Chapter 4 .

OPTICAL PROPERTIES OF HEAT - CURED WHEY PROTEIN - ISOLATE
BASED EDIBLE FILMS AND MICROBIAL TRANSGLUTAMINASE HEAT -
CURED WHEY PROTEIN - ISOLATE BASED FILMS.

4.1 Abstract

The transmission of both ultraviolet and visible light are important factors in
considering possible applications for edible ﬁlms. The color characteristics of the
ﬁlm can impact consumer acceptability of the packaging. Edible whey protein ﬁlms
were produced using whey protein isolate WPI (5% w/v), glycerol (3.5% WM and
candelilla wax (0.8% w/v) with and without the addition of microbial
transglutaminase (0.001g/g WPI) to the ﬁlm forming solution after heating. Both
sets of ﬁlms were vacuum heat cured for 0, 12, and 24h at 90°C. The effect of heat
curing and transglutaminase treatment on the optical properties (ultraviolet and
visible light transmission and the color characteristics) of WPI ﬁlms were assessed
using spectrophotometer and a chromameter. Heat curing increased the
yellowness of ﬁlms. The yellowness of WPI ﬁlms increased from 11.81075 at Oh to
33.2:489 at 24h of heat curing. The yellowness of WPI/T G ﬁlms increased from
9.2:t0.15 at Oh to 33813.11 at 24h of heat curing. All ﬁlms showed low
transmission of ultraviolet light, from 0-60% transmission between 190 - 400nm.
WPI and WPI/T G ﬁlms are good barriers to UV radiation.
4.2 Introduction

One of the main factors related to food quality is the absorbance of

ultraviolet and visible light by the product. Light has been shown to affect the

78

ﬂavor and nutritional content of milk (Bradley 1980). Color characteristics and light
transmission are important properties in determining the acceptability of packaging
materials. The color (light and transmission) characteristics can be critical in
selecting the right ﬁlm for a food application. By reducing the exposure of food
products to ultraviolet and visible light the quality and shelf life of food products can
be enhanced (Hernandez, Selke et al. 2000).

Even though ultraviolet radiation accounts for only 3% of the total radiation
that reaches the earth it is able to cause chemical reactions in both polymers and
food products. These chemical reactions can have an undesirable effect on the
quality and shelf life of the food products. Ultraviolet radiation can be subdivided
into three distinctive wavelength regions. These three regions are UV-A (400-
315nm), UV-B (315-280nm), and UV-C (280-100nm). Visible light spans the
wavelengths from 400-700nm and produce colors from violet, blue, green, yellow,
and orange to red. There has been limited research on the color values of heat
cured protein based ﬁlms (Gennadios, Ghorpade et al. 1996; Rhim, Gennadios et
al. 2000; Kim, Weller et al. 2002). The research was conducted soy protein based
ﬁlms not on whey protein based ﬁlms.

The objectives of this study were to determine the color characteristics and
light transmission of whey protein isolate based edible ﬁlms with and without
transglutaminase, and heat cured at 90°C for 0,12 and 24h, and secondly, to
compare the color values for WPI ﬁlms to commercially available plastic packaging

materials.

79

 

4.3 Materials and Methods
4.3.1 Materials

Whey protein isolate (WPI; Provan 190) was obtained from Glanbia
Ingredients Inc. (Monroe WI). The whey proteins were extracted in a highly
puriﬁed, undenatured form using membrane technology. Glycerol was purchased
from J.T. Baker Co. (Phillipsburg, N.J.). and Candelilla wax was purchased from
Stroh and Pitsch Inc. (West Babylon NY). Microbial transglutaminase was
obtained from Ajinomoto Company Inc. (Japan).
4.3.2 Film preparation

Whey protein isolate (WPI) (5% w/v) and glycerol (3.5% w/v) were mixed in
distilled water and the pH adjusted to 8.0 with 2N NaOH. These solutions were
then heated at 90°C for 15 min while being stirred continuously. Candelilla wax
(0.8% w/v) was added during the last 5 min of heating and allowed to melt into the
WPI solution. The WPI solutions were homogenized for 2 min, using a Polytron
PT 10/35 homogenizer with a PTA 20 T8 homogenizing head (Tekmar Co.,
Cincinnati, OH). Each homogenized solution was then strained through a single
layer of cheesecloth to ensure complete incorporation of the lipid and allowed to
equilibrate at ambient temperature for 2 hours. Each solution was then degassed
for 30 min and mst on to a Teﬂon-coated metal pan, where it was dried at 2312°C
and 35: 5%RH for 24h. Films were stored at 2312°C and 50: 5%RH for 24h prior

to heat curing.

80

4.3.3 Transglutaminase cross-linked ﬁlms

Film forming solutions were prepared as described above. Solutions were
allowed to cool to ~ 50°C and TG was added (0.0019/ g of WPI) and allowed to
incubate for 3h at 50°C. Each solution containing TG was then degassed under
vacuum for 30 min and cast onto a Teﬂon-coated metal pan. The amount of TG
used was determined from the technical speciﬁcation received from Ajinomoto
Company Inc. Each solution was dried at 23t2°C and 35: 5%RH for 24h. Films
were stored at 2312°C and 501 5%RH for 24h prior to heat curing.
4.3.4 Heat curing

Films were wrapped in aluminum foil and heated in a vacuum oven at 90°C
ﬁlms for 12 and 24h. After heat curing, ﬁlms were allowed to equilibrate at 451 5%
RH for 24h prior to removal from foil. Films were conditioned at 2312°C and 50:
5%RH for 48h prior to testing.
4.3.5 Optical Properties

The color values (L*, a*, and b*) for WPI and WPI/T G heat cured ﬁlms were
measured with a Minolta Chromameter CR-310 (Commission D’Edairerage (ClE)
L*a*b*, Ramsey, NJ). The L* values measure the lightness and ranges from 0
(black) to 100 (white). The a* values measure the change from green (-) to red (+)
and b* measures the change from blue (-) to yellow (+). Total color diﬁerence (AE)

was calculated using equation 4:1:

81

Equation 4:1 AE = [(AL*) 2 + (Aa*) 2 + (Ab*) 2]"2
AE = Total color difference
AL* = .W _ UM
Aa* = an...” - ah...
Ab" = b*mpi. - b*m
The transmission of visible and UV light was determined for WPI and
WPI/T G heat cured ﬁlms. Transmission was measured using a Perkin Elmer
Lambda 25 UVNIS spectrometer equipped with a RSA-PE-20 Integrating Sphere
for Transmittance and Reﬂectance. Samples were scanned at wavelengths
between 190.0 to 900nm at 480 nm/min.
4.3.6 Statistical analysis
Color measurements were replicated 3 times. Results were analyzed using
the mixed model procedure in SAS (SAS Institute, Cary, NC). Treatment means
were compared with signiﬁcance deﬁned as ps 0.05.
4.4 Results and Discussion
4.4.1 Color
The color values for heat cured WPI and WPI/T G heat cured ﬁlms are
shown in Table 4.1. Both WPI and WPI/T G ﬁlms heat cured for 0 and 12h did not
have signiﬁcantly different L* values. Heat curing ﬁlms for 24h signiﬁwntIy
decreased (p<0.05) the L* values compared to uncured ﬁlms and ﬁlms heat cured
for 12h. There was no signiﬁcant difference in L* values between WPI ﬁlms treated

with and without TG. No signiﬁcant difference in a* values were observed

82

Table 4.1: The L*, a*, b* values of whey protein-isolate based ﬁlms with and
without microbial transglutaminase heat cured at 90°C for 0, 12 and 24h.

 

 

 

Time L" 3* b*
0 85.8102? 8.31809 11.810.75'
WPI 12 82.811 .021c 4610.27“ 24.713.33“
24 77.912402e -1 .811.29° 33.214896
0 87210.02a 8110.05a 9.210.15'
WPIITG 12 83.51016" 4.81:0.07‘ 22510.88b

24 77.611 .88“ -1 £11.02" 33313.1 1°

“3 means with different superscript are signiﬁcantly different (p<0.05),
Comparisons are made within each column, n=3 for all treatments values.

 

Table 4.2: Color difference between whey protein-isolate based ﬁlms heat
cured at 90°C 12 and 24h compared to ﬁlms cured for 0h.

 

 

Time AL" Aa” Ab" AE
o - - - -
.12 -2.92 0.71 12.89 13.24
24 -7.87 3.47 21.48 23.14

 

Table 4.3: Color difference between whey protein-isolate based ﬁlms with
microbial transglutaminase heat cured at 90°C 12 and 24h compared to
ﬁlms cured for 0h.

 

Time AL* Aa* Ab* AE
o - - .. -
12 -3.72 0.25 13.28 13.79
24 -9.58 3.31 24.61 26.61

 

83

Table 4.4: The L*, a*, b* values of cellophane, polystyrene, low-density
polyethylene and polyethylene terephthalate.

 

 

L* a* 0*
Cellophane 88.981010 08210.15 14710.04
PS‘ 89.821070 -0.76:l:0.05 05110.05
LDPE‘ 89.751010 09510.03 07010.04
PET‘ 88.151020 -1.0510.06 0.1010.05

 

‘ PS = polystyrene, LDPE = low density polyethylene, PET = polyethylene
terephthalate

Table 4.5: Color difference between whey protein-isolate based ﬁlms
without microbial transglutaminase heat cured at 90°C for 0h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate.

 

 

AL* Aa“ 110* AE
Cellophane 3.18 -4.43 10.31 11.87
PS -4.04 4.54 11.27 12.81
LDPE‘ 3.97 4.34 12.44 13.78
PET‘ .234 -4.19 11.88 12.83

 

‘ P8 = polystyrene, LDPE = low density polyethylene, PET = polyethylene
terephthalate

Table 4.6: Color difference between whey protein-isolate based ﬁlms
without microbial transglutaminase heat cured at 90°C for 12h compared to

cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate.

 

 

AL* Aa* Ab" AE
Cellophane 010 3.73 23.21 24.28
PS -6.96 3.84 24.17 25.44
LDPE‘ -6.89 3.84 25.34 28.51
PET‘ -5.26 3.49 24.58 25.37

 

‘ PS = polystyrene, LDPE = low density polyethylene, PET = polyethylene

terephthalate

Table 4.7: Color difference between whey protein-Isolate based ﬁlms
without microbial transglutaminase heat cured at 90°C for 24h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate.

 

 

AL" Aa* Ab* AE
Cellophane -1 1.05 -0.96 31 .80 33.68
PS 41.91 -1.07 32.78 34.87
LDPE‘ -11.84 087 33.93 35.94
PET‘ -1021 072 33.17 34.71

1 P8 = polystyrene, LDPE = low density polyethylene, PET = polyethylene
terephthalate

Table 4.8: Color difference between whey protein-isolate based ﬁlms with
microbial transglutaminase heat cured at 90°C for 0h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate.

 

 

AL" Aa" Ab” AE

Cellophane .175 .423 7.77 9.02
PS -2.61 .434 8.73 10.09
LDPE‘ .254 -4.14 9.90 11.02
PET‘ 091 3.99 9.14 10.01

 

‘ PS = polystyrene, LDPE = low density polyethylene, PET = polyethylene

terephthalate

85

Table 4.9: Color difference between whey protein-isolate based ﬁlms with
microbial transglutaminase heat cured at 90°C for 12h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene

terephthalate.

 

 

AL“ Aa‘ 10* AE
Cellophane -5.47 -3.98 21.05 22.11
PS 3.33 4.09 22.01 23.28
LDPE‘ -6.26 3.89 23.18 24.32
PET‘ 4.83 3.74 22.42 23.19

 

‘ PS = polystyrene, LDPE = low density polyethylene, PET = polyethylene
terephthalate

Table 4.10: Color difference between whey protein-isolate based ﬁlms with
microbial transglutaminase heat cured at 90°C 24h compared to
cellophane, polystyrene, low-density polyethylene and polyethylene
terephthalate.

 

 

AL* Aa* Ab* AE
Cellophane -11.33 -0.92 32.38 34.31
PS -12.19 -1.03 33.34 35.51
LDPE1 -12.12 -0.83 34.51 36.58
PET 1 -10.49 -0.68 33.75 35.35

 

‘ PS = polystyrene, LDPE = low density polyethylene, PET = polyethylene
terephthalate

86

between WPI and WPI/1' G ﬁlms heat cured for 0 and 12h. Films heat cured for 24h
had signiﬁcantly lower (p<0.05) a* values compared to ﬁlms heat cured at 0 and
12h. Both(Kinsella and Whitehead 1989) uncured WPI and uncured WPI/T G ﬁlms
had signiﬁcantly lower (p<0.05) b* values compared to heat cured WPI and

WPI/T G ﬁlms. Films heat cured for 12h had signiﬁcantly lower (p<0.05)
b*compared to ﬁlms heat cured for 24h. The increase in yellowness of heat cured
ﬁlms was also observed in soy protein isolate ﬁlms (Gennadios, Ghorpade et al.
1996; Rhim, Gennadios et al. 2000; Kim, Weller et al. 2002).

Tables 4.2 and 4.3 show the difference between heat cured WPI ﬁlms
treated with transglutaminase and uncured ﬁlms treated with transglutaminase. In
both cases the difference in the color values of the ﬁlms increased as heat curing
time increased. The increased AL* observed in the heat cured indiwted an
increased tightness in the ﬁlms compared to uncured ﬁlms. The increased Aa* in
the heat cured ﬁlms indicated an increased redness in the ﬁlms compared to
uncured ﬁlms. The increased Ab* observed in the heat cured ﬁlms indicated and
increased yellowness in the ﬁlms compared to uncured ﬁlms. The AL* and Ab“
observed in the heat cured ﬁlms resulted in the large AE obtained for the heat
cured ﬁlms. The increased yellowness observed in the heat-cured ﬁlms may be
from Maillard reactions occurring between whey proteins and residual lactose in
the ﬁlms.

The color differences (AE) between WPI ﬁlms and commercial ﬁlms are

shown in Table 4.5 through 4.10. All ﬁlm samples showed large degrees of color

87

difference compared to commercial ﬁlms. As heat-curing time increased the AE
values also increased. The greatest increase was observed between uncured ﬁlms
and those heat cured for 12h. As heat-curing time increased the increase in AE
was smaller. The large AE values observed can be attributed to the high b* values
of the WPI ﬁlms. This was observed as an increase in yellowness of the heat
cured ﬁlms. The large difference between commercial plastics and WPI ﬁlms may
have a beneﬁcial or a detrimental affect on consumer acceptance of products
packaged in WPI ﬁlms. More research needs to be conducted in this area to
determine the acceptability of heat cured WPI and WPI/T G ﬁlms.
4.4.2 Transmission of UV and Visible light

. The transmission of UV and visible light are critical parameters in choosing
the proper packaging applications for a ﬁlm. The transmission of UV and visible
light for WPI ﬁlms with and without TG heat cured at 90°C for 0, 12, 24h are shown
in Figure 4.1. All ﬁlms showed low transmission in the UV-C and UV-B range. At
~300nm the transmission of UV light increased in uncured WPI and WPIfl' G ﬁlms.
The increase in transmission continued through the visible spectrum from 400nm
to ~550nm (Figure 4.1). Both WPI and WPI/T G ﬁlms heat cured at 90°C for 12h
did not show any increase in UV transmission up to~350nm. Increased light
transmission then continued to ~550nm. The increase in UV and visible light
observed in the WPI and WPI/T G ﬁlms heat cured at 90°C for 24h was similar to
ﬁlms heat cured for 12h. The low UV transmission would allow WPI and WPI/T G

ﬁlms to be used to protect light sensitive products without the addition of UV

88

100. 1

%T

 

 

1.0

 

190 250300350400450500550800850700750800
"m

Figure 4.1: % transmission versus wavelength of whey protein-isolate
based ﬁlms with and without microbial transglutaminase heat cured at 90°C
for 0, 12 and 24h.

89

absorbers. The low transmission of UV light observed by the ﬁlms is to be
expected since proteins absorb UV radiation in the 190 to 280 nm range.
4.5 Conclusions

Heat curing increased the yellowness of WPI and WPI/T G ﬁlms. It also
increased the redness of WPI and WPI/T G ﬁlms. All WPI and WPI/T G ﬁlms have
low transmission of UV light which may impact oxidative rancidity of the food
packaged therein. The increased yellowness of heat cured WPI and WPI/T G ﬁlms
will probably decrease their acceptability in certain packaging applications. The
increased yellowness may lead consumers to view the package as old and that the
product is past its prime. The increased yellowness may serve to enhance other
food colors such as cured or smoked meat characteristic. The low transmission of
UV light indicates that WPI and WPI/T G ﬁlms could be used to protect light
sensitive products. Based on the low transmission of UV radiation WPI and
WPI/T G ﬁlms may be used as a packaging material for products such as dairy

foods and pharmaceutical products.

90

 

Chapter 5

THERMAL PROPERTIES OF HEAT - CURED WHEY PROTEIN ISOLATE -

BASED EDIBLE FILMS COMPARED TO MICROBIAL TRANSGLUTAMINASE
HEAT - CURED WHEY PROTEIN ISOLATE FILMS.
5.1 Abstract
Edible whey protein ﬁlms were produced using whey protein isolate WPI

(5% w/v), glycerol (3.5% w/v) and candelilla wax (0.8% w/v). Films were also
produced as described above with the addition of microbial transglutaminase
(0.0019/g WPI) to the ﬁlm forming solution alter heating. Both sets of ﬁlms were
vacuum heat cured for 0, 12 and 24h at 90°C. The effects of heat curing and
transglutaminase treatments on the thermal properties (melting onset temperature,
peak melting temperature and enthalpy of fusion) of the whey protein isolate ﬁlms
were assessed. The peak melting temperatures for ﬁlms increased with heat
curing and time. The peak melting temperatures of WPI ﬁlms increased from
140.57°C at 0 h to 158.1°C at 24h of heat curing. The peak melting temperatures
of WPI/T G ﬁlms increased from 137.36°C at Oh to 157.2°C at 24h of heat curing.
The increase in peak melting temperatures corresponded to an increase in cross-
linking in the ﬁlms. The higher peak melting temperatures observed for heat—cured
ﬁlms would indicate that higher processing temperatures would be needed to

produce ﬁlms and that the ﬁlms have greater thermal stability.

91

 

5.2 Introduction

Thermal properties such as melting temperature (T m) and glass transition
temperature (T9) are important in determining the processing conditions of polymer
ﬁlms. The T... of a polymer is the temperature at which the polymer molecules are
free to move past each other and the crystalline regions begin to break up
(Rodriguez 1996). The Tm of ﬁlms can also be used to determine an optimal
temperature range for ﬁlm extrusion. For crystalline plastics, ﬁlm processing
temperatures are about 50°C above their Tm (Hernandez, Selke et al. 2000).

Heat curing has been shown to enhance the mechanical and barrier
properties of protein based ﬁlms (Gennadios, Ghorpade et al. 1996; Miller, Chiang
et al. 1997; Kim, Weller et al. 2002; Kim, Weller et al. 2002). Cross-linking proteins
using transglutaminase has been shown to enhance the mechanical and barrier
properties of protein based ﬁlms (Mahmoud and Savello 1992; Mahmoud and
Savello 1993; Yildirim and Hettiarachchy 1998; Lim, Mine et al. 1999; Larre,
Desserme et al. 2000). There has been little research conducted on the thermal
properties of heat-cured ﬁlms and transglutaminase cross-linked ﬁlms.

The objectives of this study were to determine thermal properties (melting
onset temperature, peak melting temperature and enthalpy of fusion) of whey
protein isolate ﬁlms with and without transglutaminase, and heat cured at 90°C for

0, 12 and 24h.

92

5.3 Materials and Methods
5.3.1 Materials

Whey protein isolate (WPI; Provan 190) was obtained from Glanbia
Ingredients Inc. (Monroe WI). The whey proteins were extracted in a highly
puriﬁed, undenatured form using membrane technology. Glycerol was purchased
from J.T. Baker Co. (Phillipsburg, N.J.). and Candelilla wax was purchased from

Stroh and Pitsch lnc. (West Babylon NY.) Microbial transglutaminase was

 

obtained from Ajinomoto Company Inc. (Japan).
5.3.2 Film preparation -

Whey protein isolate (WPI) (5% wlv) and glycerol (3.5% wlv) were mixed in
distilled water and the pH adjusted to 8.0 with 2N NaOH. These solutions were
then heated at 90°C for 15 min while being stirred continuously. Candelilla wax
(0.8% wlv) was added during the last 5 min of heating and allowed to melt into the
WPl solution. The WPI solutions were homogenized for 2 min, using a Polytron
PT 10/35 homogenizer with a PTA 20 TS homogenizing head (T ekmar Co.,
Cincinnati, OH). Each homogenized solution was then strained through a single
layer of cheesecloth to ensure complete incorporation of the lipid and allowed to
equilibrate at ambient temperature for 2 hours. Each solution was then degassed
for 30 min and cast on to a Teﬂon-coated metal pan where it was allowed to dry at
2312°C and 351 5%RH for 24h. Films were stored at 2312°C and 501 5%RH for

24h prior to heat curing.

93

 

5.3.3 Transglutaminase cross-linked ﬁlms

Film forming solutions were prepared as above. Solutions were allowed to
cool to ~ 50°C and TG was added (0.001g/ g of WPI) and allowed to incubate for
3h at 50°C. Each solution containing T6 was then degassed under vacuum for 30
min and cast onto a Teﬂon-coated metal pan. The amount of TG used was
determined from the technical speciﬁcation received from Ajinomoto Company Inc.
Each solution was dried at 2312°C and 351 5%RH for 24h. Films were stored at
2312°C and 501 5%RH for 24h prior to heat curing.
5.3.4 Heat curing

Films were wrapped in aluminum foil and heated in a vacuum oven at 90°C
for 12 and 24h. After heat curing, ﬁlms were allowed to equilibrate at 451 5% RH
for 24h prior to removal from the foil wrap. Films were conditioned at 2312°C and
501 5%RH for 48h prior to testing.
5.3.5 Differential scanning calorimetry (DSC)
The melting temperatures Tm of uncured and heat-cured ﬁlms were determined
using a TA instruments Q 100 DSC, (TA instruments, Delaware) unit. Samples
(~10 mg) were weighed and sealed in an aluminum pan with an encapsulating
press. Samples were heated from room temperature (~22°C) to 250°C at a
temperature increase of 20°Clmin. An empty sample pan was used as a
reference. The DSC cell was ﬂushed with nitrogen at 20ml/min to maintain an inert

atmosphere. The differential temperature and enthalpy change were calculated

using TA Universal Analysis 2000 software. Transition temperatures were
determined according to ASTM standard D-3418.
5.4 Results and Discussion

The then'nograms for WPI and WPI/TG ﬁlms heat cured at 90°C for 0, 12
and 24h are shown in Figures 5.1 and 5.2. The onset temperature (To), peak
melting temperature (T m) and the enthalpy of fusion (AH) are shown in Table 5.1.
In all samples two peaks were observed at around 60°C, these two peaks
correspond to the melting temperatures of candelilla wax. The melting temperature
of candelilla wax has been reported as 640°C (Donhowe and Fennema 1993).

The T0 of WPI and WPI ﬁlms ranged from 98.6 to 153.1°C and the T...
ranged from 137.4 to 158.1°C. The WPI/T G ﬁlms had lower To and Tm compared
to WPI ﬁlms at corresponding heat curing times (Table 5.1). As heat curing time
increased a corresponding increase in To and T... was observed in both WPI ﬁlms
with and without transglutaminase. The increase in transition temperatures can be
attributed to an increase in cross-linking in the ﬁlms (Galietta, Di Gioia et al. 1998).
The cross-linking produced during heat curing may have increased the crystallinity
of the ﬁlms. The increase in crystalline regions in the heat-cured ﬁlms required
increased energy to break up. This was seen as an increase in Tm of heat-cured
ﬁlms as time increased.

Uncured WPI and WPI/T G ﬁlms showed a broad endothermic peak over

their melting range. As heat-curing time increased the endothermic peak narrowed

in both the WPI and WPI/T G ﬁlms (Figure 5.1 and 5.2). As heat curing time

95

Heat Flow (VII/g)

 

05
3.5,0h

 

---- 3,5,12h
—— 3.524h

 

 

 

 

 

 

 

-15‘
-2.0 I I I l I I I r I I I r I I 2 T I I I I I I I l T f I r j I I
40 60 80 100 120 140 160 180 200
Em Up TOMPOMUTO (0C) Universal V3.9ATA Instruments

Figure 5.1: Differential Scanning Calorimetry thennogram of whey protein-
isolate based ﬁlms without microbial transglutaminase heat cured at 90°C

for 0, 12, 24 and 48h.

 

Heat Flow (\ng)

 

 

 

 

 

 

 

 

0.5
—— WPITG
1 - - - - WPWG,12
— — WPWG24
0.0 ‘
~05 ‘
-1.0 ..
-1 5 T T ' l 1 I 1 I T I I I 1 I I T 1 1 ﬁi 1 17 I I T I I l T T f T
40 60 80 100 120 140 160 180 200
EXO Up Temperature (0C) Universal V3.9ATA Instruments

Figure 5.2: Differential Scanning Calorimetry thennogram of whey protein-
isolate based ﬁlms with microbial transglutaminase heat cured at 90°C for 0,

12, 24 and 48h.

97

Table 5.1: Selected thermal properties of whey protein-isolate based ﬁlms
with and without microbial transglutaminase heat cured at 90°C for 0, 12, 24

 

 

 

and 48h.
Heat Curing To 1 T...2 AH3
mm” Time (h) 1°C) 1°C) (Jig)
0 98.6 1 37.4 66.7
WPIITG 12 1 35.0 140.6 48.1
24 1 50.3 157.2 19.3
0 1 35.0 140.6 48.2
WPI 12 151.1 156.8 12.5
24 1 53.1 1 58.1 24.9

 

1T0: onset transition temperature
2Tm: peak melting temperature

3Ah = Enthalpy of fusion

98

 

increased the T0 of the ﬁlms became more pronounced. WPI ﬁlms exhibited a
sharper To compared to WPI/T G ﬁlms. The sharper To observed in heat cured ﬁlms
can be attributed to an increase in crystallinity in the ﬁlms. In amorphous polymers
the transition from a solid to a liquid is not well deﬁned; white crystalline polymers
exhibit a well deﬁned transition from a solid to a liquid (Hernandez, Selke et al.
2000).

Determination of proper seal temperature is important for the production of
pouches. Kim and Ustunol (2001) determined that the optimum seal strength was
obtained at a temperature that corresponded to the T0 of the ﬁlms. WPI ﬁlms
would need a higher temperature to obtain an optimal seal compared to WPI/T G
ﬁlms. Heat curing of ﬁlms would also, increase the temperature required to heat
seal the ﬁlms. The higher temperature need to seal the ﬁlms can be attributed to
the increase in crystallinity in the ﬁlms as seen in the higher To.

5.5 Conclusions

Heat curing ﬁlms produces ﬁlms of higher transition temperatures.
Increasing heat-curing time increased the transition temperatures of WPI ﬁlms with
and without transglutaminase. Increasing the cross-linking in WPI ﬁlms increased
the crystallinity of WPI ﬁlms. The higher T0 of heat cured ﬁlms would require higher

temperatures to obtain proper heat seals.

99

CHAPTER 6
CONCLUSIONS

To investigate the effects of heat curing and transglutaminase treatments

on the mechanical, molecular, barrier, optical and thermal properties of whey

protein ﬁlms a series of studies were conducted. The focus of these studies was to

determine that heat curing induced covalent cross-linking in WPI ﬁlms and to

characterize the effect cross-linking on the properties of WPI ﬁlms with and without

transglutaminase.

1.

The mechanical properties of whey protein ﬁlms (WPI) treated with
and without transglutaminase (T G) were inﬂuenced by the length
of heat curing. WPI ﬁlms had better tensile strength than WPI/T G,
while WPI/T G ﬁlms had higher elongation at break than WPI ﬁlms.
Heat curing both WPI and WPI/T G ﬁlms for 24h produced ﬁlms
with the highest toughness. These ﬁlms exhibited a balance
between high tensile strength and elongation at break.

Heat curing WPI and WPI/I“ G ﬁlms produced covalent cross-links
as seen in an increase in higher molecular weight polymers in
SDS-PAGE gels. The increase in higher molecular weight
polymers corresponded to ﬁlms with enhanced mechanical
properties.

The sulfhydryl content of WPI ﬁlms with and without TG were
inﬂuenced by heat curing. Heat curing both WPI and WPI/T G ﬁlms
decreased the sulﬂiydryl content compared to uncured ﬁlms.
Treatment with transglutaminase did not affect the sulﬂiydryl
content of heat-cured ﬁlms.

100

 

The lysine content of WPI ﬁlms with and without TG was
inﬂuenced by heat curing. Heat curing both WPI and WPI/1' G ﬁlms
decreased the lysine content compared to uncured ﬁlms.
Treatment with transglutaminase lowered the lysine content of
heat-cured ﬁlms compared to ﬁlms only heat cured. Films with
lower lysine content corresponded to ﬁlms with the highest
toughness. The formation of covalent cross-links by lysine plays a
signiﬁcant role in enhancing the mechanical properties of heat
cured WPI and WPI/T G ﬁlms.

Heat curing decreases the OzP of WPI and WPI/T G ﬁlms.
Treatment with TG and heat curing produces ﬁlms with improved
0213 compared to heat curing alone. Treatment with TG and heat
curing improved the WVP of WPI ﬁlms compared to heat curing or
TG treatments alone. Increasing the amount of covalent cross-
linking in WPI ﬁlms can be used to improve both the OzP and
WVP.

The moisture sorption isotherm of WPI and WPI/T G ﬁlms heat
cured at 90°C for 0 and 24h determined at 23 and 5°C ﬁt the
Guggenheim-Anderson-de Boer model. Sorption isotherms for
ﬁlms at 5°C had higher equilibrium moisture contents at all relative
humidity compared to ﬁlms at 23°C.

The color characteristics of WPI ﬁlms with and without TG, heat
cured at 90°C for 0, 12 and 24h. Treatment of ﬁlms with TG had
no impact on the color values. Heat curing increased the
yellowness of the ﬁlms. Both WPI and WPI/T G ﬁlms heat cured at

90°C for 0, 12 and 24h had a large AE (total color difference)
101

 

 

compared to polystyrene, low density polyethylene, polyethylene
terephthalate.

WPI ﬁlms with and without TG, heat cured at 90°C for 0, 12 and
24h had low transmission of ultraviolet radiation. Treating ﬁlms
with transglutaminase did not impact the VlS/UV transmission
properties of the ﬁlms. Heat curing ﬁlms decreased the
transmission of UV radiation.

Heat curing ﬁlms produces ﬁlms of higher transition temperatures.
Increasing heat-curing time increased the transition temperatures
of WPI ﬁlms with and without transglutaminase. Increasing the
cross-linking in WPI ﬁlms increased the crystallinity of WPI ﬁlms.

102

CHAPTER 7
Recommended Future Research

Currently WPI ﬁlms are produced in small test batches by casting.
Research needs to be conducted on methods to produce WPI
ﬁlms in a commercially viable manner.

Physical and molecular properties of commercially viable ﬁlms
need to be determined and compared to the research presented
here.

Barrier properties of commercially viable WPI ﬁlms need to be
determined over a broader range of temperature and relative
humidity.

The moisture sorption isotherms of commercially viable WPI ﬁlms
need to be determined and compared to the results obtained here.

The thermal properties of commercially viable WPI ﬁlms need to
be determined and used to determine correct conditions to
produce sealed pouches.

Determine the sensory attributes along with the acceptability of
commercially viable WPI ﬁlms

103

APPENDIX A

104

Mechanical Properties of Microbial Transglutaminase Heat Cured
Whey Protein Isolate Films

The objective of this study is to compare the tensile strength, elongation at
break and the toughness of TG treated WPI ﬁlms with varying levels of plasticizer
and heat cured at 80, 90 and 100°C for 0, 12, 24, 48, and 72h.

Film preparation: WPI (5% WM and Gly (2.7, 3.3, or 3.5% w/v) was mixed
in distilled water and the pH adjusted to 8 with 2N NaOH. Solutions were heated
at 90°C for 15 min while being stirred continuously. CW (0.8% wlv) was added
during the last 5 min of heating and allowed to melt into the WPI solution. The
WPI/CW solutions were homogenized for 2 min, using a Polytron PT 10/35
homogenizer with a PTA 20 TS homogenizing head (T ekmar Co., Cincinnati, OH).
Solutions were strained through a single layer of cheesecloth to ensure complete
mixing of the lipid. Solutions were allowed to cool to ~ 50°C and TG was added
(0.001g/ g of protein) and allowed to incubate for 3h at 50°C. Solutions were
equilibrated to room temperature. Solutions were degassed for 30 min and cast on
a Teﬂon-coated surface. Solutions were dried at 2312°C and 351 5%RH for 24h.
Films were stored at 2312°C and 501 5%RH for 24h prior to heat curing.

Heat curing: Films were wrapped in aluminum foil and heated in a vacuum
oven at 80, 90, or 100°C ﬁlms were removed after 12, 24,48 and 72h. After heat
curing, ﬁlms were allowed to equilibrate at 451 5% RH for 24h prior to removal

from foil. Films were conditioned at 2312°C and 501 5%RH for 48h prior to testing.

105

 

 

 

 

Mechanical Properties: Uncured WPI and heat-cured WPI ﬁlms were
compared. Samples were cut into 2.54cm-wide strips using a Precision Sample
Cutter (Thawing Albert Instrument Company Philadelphia, PA). Film thickness
was determined using a TMI model 549 M micrometer (Testing Machines, lnC.
Amityville, NY). Measurements were taken at 5 locations along the sample. An
average thickness value was used in the calculations. Tensile strength (TS) and
percent elongation-at-break (%E) and toughness were determined according to
ASTM D 882 (ASTM 1997). Samples were tested using an lnstron Universal
Testing Machine Model 5500 (lnstron Corp, Canton MA) with MeriinTM software to
determine TS, %E, and toughness. Initial grip separation was 50 mm and the
crosshead speed was 12.5 mein.

Statistical analysis: All treatments were replicated 3 times. Results were
analyzed using the mixed model procedure in SAS (SAS Institute, Cary, NC).

Treatment means were compared with signiﬁcance deﬁned as ps 0.05.

106

Results

Table A.1: Tensile strength of whey protein-isolate based ﬁlms cross-linked
with microbial transglutaminase inﬂuenced by heat curing temperature, time

and glycerol content.

 

 

 

 

Plasticizer Time 80°C 90°C 100°C
0 1.1510.05a 11510.05a 11510.05"
12 4.901075" 3.921018" 3.771015"
3.5% Gly 24 3.831051" 4.391085" 3.281074"
48 3.831082" 6.3510.92° 3.881084"
72 5.081234" 140812.15" 17.001448c
0 09510.143 09510.141 09510.14"
12 2.9310.63° 3.281035" 3.351040"
3.3% Gly 24 3.721053" 3.521018" 3.731043"
48 4.331029" 5.8511.83° 5.151041"
72 4.481108" 8.2511.77° 12.9815776
0 13710.14" 13710.14a 13710.14“
12 1.9110.15° 55310.98" 3.581088"
2.7% Gly 24 2.801126" 8.521048" 3.951079"
48 2.501051' 9.431338" 104714.51c
72 2.381027" 12.301295d 17.131248“

 

“ means with the same superscript are not signiﬁcantly different (p<0.05), n=3 for
all treatments. Comparisons are within each glycerol level and temperature

107

 

Table A2: Elongation at break (%) of whey protein-isolate based ﬁlms
cross-linked with microbial transglutaminase inﬂuenced by heat curing

temperature, time and glycerol content.

 

 

 

 

Plasticizer Time 80°C 90°C 100°C
0 11.501117a 11.501117a 11501117‘1
12 11.671108" 17.401110" 10.9211088
3.5% Gly 24 12.121505“ 140313.67“ 8.231082"
- 48 10.531255“ 2.9511236 4.881085"
72 8.801255" 5.131187" 1.151018"
0 5.4810.82“ 5.481082" 5.481082"
12 13.321269" 12.0812.88° 7.051172a
3.3% Gly 24 144314.04" 15.871179c 5.431059"
48 18.081480" 115013.77" 5.401111"
72 11.7716.24° 62011.84" 12510.13"
0 202513.81" 20.251381‘ 202513.81"
12 104313.42" 5.281139" 7.581245"
2.7% Gly 24 9.2813.39" 8.2013.28" 8.771212"
48 111812.25" 4.171228" 1.381013"
72 9.781321" 2.151091" 14010.37c

 

"° means with the same superscript are not signiﬁcantly different (p<0.05), n=3 for
all treatments. Comparisons are within each glycerol level and temperature

108

Table A.3: Toughness (lems) of whey protein-isolate based ﬁlms cross-
Iinked with microbial transglutaminase inﬂuenced by heat curing

temperature, time and glycerol content.

 

 

 

 

Plasticizer Time 80°C 90°C 100°C
0 8.71: 0.44 8.71 : 0.44 8.71 : 0.44
12 37.97: 6.96 45.92 : 1.65 26.82 : 3.85
3.5% Gly 24 28.00: 14.78 47.08 : 9.82 12.54 : 4.11
48 28.75: 6.16 20.39 : 11.24 10.41 : 0.22
72 16.73: 1.28 16.46 : 9.21 7.43 : 2.95
0 3.10: 0.48 3.10 : 0.48 3.10 : 0.48
12 25.05: 8.09 18.21 : 16.13 14.46 : 5.31
3.3% Gly 24 35.03: 8.91 35.61 : 3.74 11.76 : 1.15
48 46.59: 16.16 41.93 : 3.36 15.20 : 1.12
72 35.08: 12.47 21.47 : 23.27 2.70 : 2.34
0 17.73: 3.35 17.73 : 3.35 17.73 : 3.35
12 12.20: 4.82 16.10 : 2.28 16.24 : 4.96
2.7% Gly 24 11.36: 4.58 33.54 : 16.08 16.98 :I: 6.61
48 18.33: 1.80 27.14 : 16.44 4.60 : 2.15
72 15.08: 7.18 17.37 : 13.28 6.60 :l: 3.15

 

3'” means with the same superscript are not signiﬁcantly different (p<0.05),
n=3 for all treatments. Comparisons are within each glycerol level and temperature

109

APPENDIX B

110

Mechanical Properties of Heat Cured Whey Protein isolate Based
Edible Films compared to collagen and natural casings.

The objective of this study is to compare the tensile strength, elongation at
break and the toughness of WPI ﬁlms with varying levels of plasticizer and heat
cured at 80, 90 and 100°C for 0, 12, 24, 48, and 72h to collagen and natural
casings.

Film preparation: WPI (5% wlv) and Gly (2.7, 3.3, or 3.5% wlv) was mixed
in distilled water and the pH adjusted to 8 with 2N NaOH. Solutions were heated
at 90°C for 15 min while being stirred continuously. CW (0.8% wlv) was added
during the last 5 min of heating and allowed to melt into the WPI solution. The
WPI/CW solutions were homogenized for 2 min, using a Polytron PT 10/35
homogenizer with a PTA 20 T8 homogenizing head (T ekmar Co., Cincinnati, OH).
Solutions were strained through a single layer of cheesecloth to ensure complete
mixing of the lipid. Solutions were degassed for 30 min and cast on a Teﬂon-
coated surface. Solutions were dried at 2312°C and 35: 5%RH for 24h. Films
were stored at 2312°C and 50: 5%RH for 24h prior to heat curing.

Heat curing: Films were wrapped in aluminum foil and heated in a vacuum
oven at 80, 90 or 100°C ﬁlms were removed after12, 24, 48 and 72h. Alter heat
curing, ﬁlms were allowed to equilibrate at 45: 5% RH for 24h prior to removal
from foil. Films were conditioned at 2312°C and 501 5%RH for 48h prior to testing.

Mechanical Properties: Uncured WPI and heat-cured WPI ﬁlms were

compared. Samples were cut into 2.54cm-wide strips using a Precision Sample

111

Cutter (Thawing Albert Instrument Company Philadelphia, PA). Film thickness
was determined using a TMI model 549 M micrometer (Testing Machines, Inc.
Amityville, NY). Measurements were taken at 5 locations along the sample. An
average thickness value was used in the calculations. Tensile strength (TS) and
percent elongation-at-break (%E) and toughness were determined according to
ASTM D 882 (ASTM 1997). Samples were tested using an Instron Universal
Testing Machine Model 5500 (lnstron Corp., Canton MA) with MerlinTM software to
determine TS, %E, and toughness. Initial grip separation was 50 mm and the
crosshead speed was 12.5 mein.

Statistical analysis: All treatments were replicated 3 times. Results were
analyzed using the mixed model procedure in SAS (SAS institute, Cary, NC).

Treatment means were compared with signiﬁcance deﬁned as ps 0.05.

112

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References

116

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