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Properties of whey protein/lipid
emulsion edible films

presented by

Seong-joo Kim

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and Human Nutrition

 

 

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moo mummy)“

PROPERTIES OF WHEY PROTEIN/ LIPID
EMULSION EDIBLE FILMS

By

SEONG-JOO KIM

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

2000

ABSTRACT

PROPERTIES OF WHEY PROTEIN/ LIPID EMULSION EDIBLE FILMS
By

SEONG-JOO KIM

Methodologies were developed to produce edible ﬁlms ﬁom whey protein isolate
(WPI) and concentrate (WPC), and ﬁlm-forming procedure was optimized. Lipids, butter
fat (BF) and candelilla wax (CW), were added into ﬁlm-forming solutions to produce
whey protein/lipid emulsion edible ﬁlms. Signiﬁcant reduction in water vapor and oxygen
permeabilities of the ﬁlms could be achieved upon addition of BF and CW. Mechanical
properties were also inﬂuenced by the lipid type. Microstructures of the ﬁlms accounted
for the diﬂ‘erences in their barrier and mechanical properties. Studies with bond-
dissociating agents indicated that disulﬁde and hydrogen bonds, cooperatively, were the
primary forces involved in the formation and stability of whey protein/lipid emulsion ﬁlms.
Contribution of hydrophobic interactions was secondary.

Thermal properties of the ﬁlms were studied using differential scanning
calorimetry, and the results were used to optimize heat-sealing conditions for the ﬁlms.
Electron spectroscopy for chemical analysis (ESCA) was used to study the nature of the
interfacial interaction of scaled ﬁhns. All ﬁlms were heat scalable and showed good seal
strengths while the plasticizer type inﬂuenced optimum heat-sealing temperatures of the
ﬁlms, 130°C for sorbitol-plasticized WPI ﬁlms and 110°C for glycerol-plasticized WPI

ﬁlms. ESCA spectra showed that the main interactions responsible for the heat-sealed

joint of whey protein-based edible ﬁlms were hydrogen bonds and covalent bonds
involving C-O—H and N-C components.

Finally, solubility in water, moisture contents, moisture sorption isotherms and
sensory attributes (using a trained sensory panel) of the ﬁlms were determined. Suitability
of WPI-based pouches in packaging of powder cocoa mix was investigated. Solubility
was inﬂuenced primarily by the plasticizer in the ﬁlms, and the higher the plasticizer
content, the greater was the solubility of the ﬁlms in water. Moisture contents of the ﬁlms
showed a strong relationship with moisture sorption isotherm properties of the ﬁlms.
Lower moisture content of the ﬁlms resulted in lower equihbrium moisture contents at all
aw levels. Sensory evaluation of the ﬁlms revealed that no distinctive odor existed in WPI
ﬁlms. All ﬁlms tested showed slight sweetness and adhesiveness. Films with lipids were
scored as being opaque while ﬁlms without lipids were scored to be clear. Whey
protein/lipid emulsion edible ﬁlms may be suitable for packaging of powder cocoa mix and

should be suitable for packaging of non-hygroscopic foods.

To my parents

iv

ACKNOWLEDGMENTS

This dissertation has been made by the support and help from many people.
Foremost among them is my academic advisor Dr. Zeynep Ustunol. I would like to
express my sincerest and most heartfelt thanks to her. Since she took me under her wings
years ago, her thoughtful guidance and advice helped me grow educationally and
professionally. I cannot thank her enough for the all the support she has provided me for
the successful completion of this research project.

I would like to express my sincere appreciation to all my committee members for
their guidance and support. I am grateful to Dr. Bruce Harte for all his support and advice
throughout my graduate program. Special thanks goes to Dr. Jack Giacin for his helpful
suggestion and enthusiasm for this research project. I would also like to appreciate Dr.
Gale M. Strasburg for use of the equipment in his laboratory, for help with data analysis
and for all the helpful meetings and advice.

I would like to thank Center for Food and Pharmaceutical Packaging Research,
and Crop and Food Bio-Processing for their ﬁnancial support of this research. New
Zealand Milk Product, Inc., Strahl and Pitch, Inc., and Lonza, Inc. are also acknowledged
for providing me with the whey proteins, candelilla wax, and food grade plasticizers,
respectively.

I sincerely appreciate sensory panel members for serving on my sensory panel, and
Dr. Janice Harte for her advice on setting up the sensory tests. I want to thank Dr. Denise
M. Smith in Dept. of Food Science and Human Nutrition for her help with protein

analysis, and Dr. Michael Rich and Dr. Per Askeland at Composite Materials and

Structures Center for their help and advice with DSC and ESCA analysis. I would like to

acknowledge all the past and present colleagues in my laboratory for their continuous

encouragement and ﬁ'iendship. My deepest appreciation goes to my family and ﬁiends

who supported me while accomplishing this work.

vi

TABLE OF CONTENTS

LIST OF TABLES ......................................................................... xi
LIST OF FIGURES ........................................................................ xiii
ABBREVIATIONS ........................................................................ xvi
INTRODUCTION ........................................................................... 1
CHAPTER 1
LITERATURE REVIEW ................................................................... 3
1.1 Edible ﬁlms ..................................................................... 3
1.1.1 Deﬁnition and historical background ............................ 3
1.1.2 Formation of edible ﬁlms ......................................... 4
1.1.2.1 Components of edible ﬁlms ............................ 4
1.1.2.2 Manufacture of edible ﬁlms ............................ 5
1.1.2.3 Forces involved in the formation and stability of
protein-based edible ﬁlms .............................. 6
1.2 Properties of protein/lipid emulsion edible ﬁlms ........................... 8
1.2.1 Water vapor permeability ......................................... 8
1.2.2 Oxygen permeability ............................................... 10
1.2.3 Mechanical properties ............................................. l l
1.3 Composition and properties of components used in edible ﬁlms ........ 12
1.3.1 Whey proteins ...................................................... 12
1.3.1.1 Manufacture of whey proteins ........................ 13
1.3.1.2 Protein ﬁ‘action of wheys .............................. l4
1.3.1.2.1 B-Lactoglobulin .............................. l4
1.3.1.2.2 a-Lactoalbumin .............................. 16
1.3.1.2.3 Bovine serum albumin ...................... 17
1.3.1.2.4 Immunoglobulin and proteose-peptone.. 18
1.3.2 Lipids ............................................................... 18
1.3.3 Plasticizers ......................................................... 20
1.4 Properties of ﬁlms important for food applications ....................... 21
1.4.1 Barrier properties ................................................. 21
1.4.2 Mechanical properties ........................................... 26
1.4.3 Thermal properties ............................................... 28
1.4.4 Sealing properties ................................................ 32
1.4.5 Moisture sorption properties ................................... 35

vii

CHAPTER 2
DEVELOPMENT OF WHEY PROTIEN/ LIPID EMULSION EDIBLE FILMS
AND DETERMINATION OF THEIR MICROSTRUCTURE, BARRIER AND

MECHANICAL PROEPRT'IES ............................................................ 40
2.1 Abstract ........................................................................ 40
2.2 Introduction .................................................................... 41
2.3 Materials and methods ....................................................... 43
2.3.1 Materials ............................................................ 43
2.3.2 Film preparation ................................................... 43
2.3.3 Determination of thickness ....................................... 46
2.3.4 Water vapor permeability ........................................ 48
2.3.5 Oxygen permeability .............................................. 49
2.3.6 Mechanical properties ............................................ 49
2.3.7 Scanning Electron Microscopy .................................. 50
2.3.8 Statistical analysis ................................................. 51
2.4 Results and discussion ....................................................... 51
2.4.1 Water vapor permeability ........................................ 53
2.4.2 Oxygen permeability .............................................. 53
2.4.3 Tensile strength and percent, elongation ........................ 63
2.4.4 Microstructure ..................................................... 69
CHAPTER 3
FORCES INVOLVED IN THE FORMATION AND STABILITY OF WHEY
PROTEIN/ LIPID EMULSION EDIBLE FILMS ...................................... 75
3.1 Abstract ......................................................................... 75
3.2 Introduction .................................................................... 76
3.3 Materials and methods ........................................................ 78
3.3.1 Materials ............................................................ 78
3.3.2 Film preparation ................................................... 78
3.3.3 Solubility in bond-dissociating agents ........................... 80
3.3.4 Determination of free sulﬂlydryl group and disulﬁde
bond contents ...................................................... 81
3.3.5 Determination of hydrophobicity ................................ 82
3.3.6 Statistical analysis ................................................. 83
3.4 Results and discussion ........................................................ 84
3.4.1 Solubility of the ﬁlms in bond-dissociating agents ............ 84
3.4.2 Free sulfhydryl group and disulﬁde bond contents of the
ﬁlms ................................................................. 88
3.4.3 Hydrophobicity of the ﬁlms ....................................... 90

viii

CHAPTER 4

THERMAL PROPERTIES, HEAT SEALABILITY AND SEALING
PROPERTIES OF WHEY PROTEIN/ LIPID EMULSION EDIBLE FILMS. . 94

4.1 Abstract ......................................................................... 94
4.2 Introduction ................................................................... 95
4.3 Materials and methods ....................................................... 97
4.3.1 Materials ........................................................... 97
4.3.2 Film preparation ................................................... 97
4.3.3 Thermal analysis of the ﬁlms .................................... 99
4.3.4 Determination of seal strength ................................... 101
4.3.5 Surface analysis of sealed ﬁlms using Electron
Spectroscopy for Chemical Analysis (ESCA) ................. 102
4.3.6 Statistical analysis ................................................. 103
4.4 Results and discussion ........................................................ 103
4.4.1 Thermal properties of the ﬁlms .................................. 103
4.4.2 Heat scalability and seal strength of the ﬁlms .................. 111
4.4.3 Surface elemental compositions of the ﬁlms determined
by Electron Spectroscopy for Chemical Analysis
survey spectra ...................................................... 116
4.4.4 Surface compositional changes of the ﬁlms upon
heat-sealing ........................................................ 120
CHAPTER 5
SUITABILITY OF WHEY PROTEIN/ LIPID EMULSION EDIBLE FILMS
AS FOOD PACKAGING MATERIALS ................................................. 131
5.1 Abstract .......................................................................... 131
5.2 Introduction ..................................................................... 132
5.3 Materials and methods ......................................................... 133
5.3.] Materials ............................................................. 133
5.3.2 Films preparation ................................................... 134
5.3.3 Moisture content of the ﬁlms ..................................... 134
5.3.4 Solubility of the ﬁlms in water .................................... 135
5.3.5 Moisture sorption isotherm of the ﬁlms ......................... 135
5.3.6 Sensory evaluation of the ﬁlms ................................... 137
5.3.7 Whey protein—based pouch for powder cocoa mix ............. 138
5.3.8 Statistical analysis ................................................... 139
5.4 Results and discussion ........................................................ 140
5.4.1 Moisture content of the ﬁlms .................................... 140
5.4.2 Solubility of the ﬁlms in water ................................... 142
5.4.3 Moisture sorption isotherm of the ﬁlms ........................ 144
5.4.4 Sensory evaluation of the ﬁlms .................................. 148
5.4.5 Whey protein-based pouch for powder cocoa mix ............ 151

ix

CONCLUSIONS ............................................................................ 157

APPENDIX I Amino acid composition of B-Lactoglobulin and

a-Lactalbumin ........................................................... 161
APPENDIX 11 Structures ofpolyol plasticizers ....................................... 163
APPENDIX [D Schematic diagram of Scanning Electron Microscope ............. 165
APPENDIX IV Written consent form ................................................... 167
APPENDIX V Whey protein-based edible ﬁlms as cheese wraps .................. 179
REFERENCES .............................................................................. 188

CONCLUSIONS ............................................................................ 157

APPENDIX I Amino acid composition of B-Lactoglobulin and

ct-Lactalbumin ........................................................... 161
APPENDIX II Structures of polyol plasticizers ....................................... 163
APPENDIX IH Schematic diagram of Scanning Electron Microscope ............. 165
APPENDIX IV Written consent form ................................................... 167
APPENDIX V Whey protein-based edible ﬁlms as cheese wraps .................. 179
REFERENCES .............................................................................. 188

Table 2.1

Table 2.2

Table 2.3

Table 2.4

Table 2.5

Table 2.6

Table 2.7

Table 2.8

Table 3.1

Table 3.2

Table 3.3

Table 4.1

Table 4.2

Table 4.3

Table 4.4

LIST OF TABLES

Composition of whey proteins used .......................................... 44

Composition of whey protein/lipid emulsion edible ﬁlms produced. . . 47

Water vapor permeability (WVP) of whey protein/lipid emulsion
edible ﬁlms (at 25°C) ........................................................... 56
Water vapor permeability (WVP) of various protein-based edible

ﬁlms and synthetic polymers .................................................. 57
Oxygen permeability (OP) of whey protein/lipid emulsion edible

ﬁlms (23°C, 0% RH) .......................................................... 61
Oxygen permeability (OP) of various protein-based edible ﬁlms

and synthetic polymers ........................................................ 62
Mechanical properties of whey protein/lipid emulsion edible ﬁlms.

(23 °C, 50% RH) ............................................................... 66

Mechanical properties of various protein-based edible ﬁlms and
synthetic polymers .............................................................. 67

Composition of whey protein/lipid emulsion edible ﬁlms produced. . 79

Free sulﬂiydyrl group and disulﬁde bond contents of whey

protein/lipid emulsion edible ﬁlms .......................................... 89
Hydrophobicity (8,) values of whey protein/lipid emulsion

edlble ﬁhns ..................................................................... 92
Composition of whey proteins used ......................................... 98

Composition of whey protein/lipid emulsion edible ﬁlms produced. . . .. 100

Thermal properties of ﬁlm-forming components as determined by
Diﬂ‘erential Scanning Calorimetry ........................................... 107

Therm] properties of whey protein/lipid emulsion edible ﬁlms as
determined by Differential Scanning Calorimetry ......................... 108

xi

Table 4.5

Table 4.6

Table 4.7

Table 4.8

Table 4.9

Seal strength (N/m) of whey protein/lipid emulsion edible ﬁlms
plasticized with sorbitol ....................................................... 112

Seal strength (N /m) of whey protein/lipid emulsion edible ﬁlms
plasticized with glycerol ....................................................... 113

Seal strength (N/m) of various edible ﬁlms and synthetic polymers ...... 114

Surface elemental compositions of whey protein/lipid emulsion
edible ﬁlms before and aﬁer heat-sealing determined with
survey Spectra of Electron Spectroscopy for Chemical Analysis ......... 119

Binding energies of peaks in the Electron Spectroscopy for Chemical
Analysis spectra of whey protein/lipid emulsion edible ﬁlms .............. 122

Table 4.10 Surface compositions of whey protein/lipid emulsion edible ﬁlms

Table 4.11

Table 4.12

Table 5.1

Table 5.2

Table 5.3

Table 5.4

Table 5.5

Table 5.6

before and alter heat-sealing as determined by the Electron

spectroscopy for Chemical Analysis spectra Cls ........................... 123
Surface compositions of whey protein/lipid emulsion edible ﬁlms

before and after heat-sealing as determined by the Electron

spectroscopy for Chemical Analysis spectra Ols .......................... 124
Surface compositions of whey protein/lipid emulsion edible ﬁlms

before and aﬁer heat-sealing as determined by the Electron

spectroscopy for Chemical Analysis spectra N1 8 .......................... 125
Moisture content of whey protein/lipid emulsion edible ﬁlms ............ 141

Water solubility and elapsed time to solubilize whey protein/lipid
emulsion edible ﬁlms (at 20°C for 24 h) .................................... 143

Parameters of ﬁtted Guggenheim-Anderson-de Boer equation
for whey protein/lipid emulsion edible ﬁlms ................................ 147

Sensory characteristics of whey protein/lipid emulsion edible
ﬁlms .............................................................................. 149

Moisture content of cocoa mix packaged in whey protein-based
pouches stored for 40 days ................................................... 152

Moisture content of whey protein-based pouches used to store
cocoa mix for 40 days ........................................................ 153

xii

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Figure 1.8

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

LIST OF FIGURES

Manufacturing process of whey proteins ................................... 15
Permeability model for gas or vapor transfer through a barrier

polymer ......................................................................... 22
Diagram of the water vapor permeability measurement using the

inﬁ'ared sensor technique ..................................................... 24
Diagram of the oxygen permeability measurement using the

coulometric sensor technique ................................................ 27
Diagram of mechanical properties measurement ........................... 29
Schematic diagram of the Differential Scanning Calorimetry

instrument ...................................................................... 31
Schematic diagram of the photo emission process ........................ 36

Schematic diagram of the Electron Spectroscopy for Chemical
Analysis instrument ........................................................... 37

Schematic diagram of ﬁlm-forming process ............................... 45

A representative whey protein isolate-based ﬁlm;
whey protein isolate-based ﬁlm containing candelilla wax .............. 52

Water vapor permeability (WVP) of whey protein/lipid emulsion
edible ﬁlms (tested at 25°C and 50% RH) ................................ 54

Water vapor permeability (WVP) of whey protein/lipid emulsion
edible ﬁlms (tested at 25°C and 80% RH) ................................ 55

Oxygen permeability (OP) of whey protein/lipid emulsion
edible ﬁlms (tested at 23°C and 0% RH) .................................. 60

Tensile strength (TS) of whey protein/lipid emulsion edible ﬁlms
(tested at 23°C and 50% RH) .............................................. 64

Percent elongation (%E) of whey protein/lipid emulsion edible ﬁlms
(tested at 23°C and 50% RH) .............................................. 65

xiii

Figure 2.8 Scanning Electron Micrographs of cross-sections of whey
protein/lipid emulsion ﬁlms. Magniﬁcation 400x. (a) IS;
(b) IS-BF4%; (c) IS-CW4%; (d) CS; (e) CS-BF4%;
(t) CS-CW4% ................................................................. 70

Figure 2.9 Scanning Electron Micrographs of top surfaces of whey protein
isolate/lipid emulsion ﬁlms. Magniﬁcation 1,500X. (a) IS;
(b) IS-BF2%; (c) IS-BF4%; (d) IS-CW4%; (e) IS-CW16% ............ 71

Figure 2.10 Scanning Electron Micrographs of top surfaces of whey protein
concentrate/lipid emulsion ﬁlms. Magniﬁcation 1,500X. (a) CS;
(b) CS-BF2%; (c) CS-BF4%; (d) CS-CW4%; (e) CS-CW16% ........ 72

Figure 3.1 Solubility of whey protein/lipid emulsion edible ﬁlms in protein
bond-dissociating agents ..................................................... 85

Figure 3.2 Relative ﬂuorescence intensity (RFI) of ANS at 486nm with
concentration of whey protein isolate/lipid emulsion edible ﬁlms
in the range of 0.05 to 0.5 mg/ml in aqueous phosphate buffer ......... 91

Figure 4.1 Diﬁ‘erential Scanning Calorimetry thermogram of ﬁlm components
(20°C/min) ...................................................................... 104

Figure 4.2 Differential Scanning Salorimetry thermogram of ﬁlms plasticized
with sorbitol (20°C/min) ..................................................... 105

Figure 4.3 Differential Scanning Salorimetry thermogram of ﬁlms plasticized
with glycerol (20°C/min) ...................................................... 106

Figure 4.4 Thermogravimetric Analysis thermogram of whey protein isolate
powder (20°C/min) ............................................................ 109

Figure 4.5 Electron Spectroscopy for Chemical Analysis survey spectra of
whey protein/lipid emulsion edible ﬁlms plasticized with sorbitol.
A. (a) IS ﬁlm unsealed, (b) IS ﬁlm sealed;
B. (a) IS—BF4% ﬁlm unsealed, (b) IS-BF4% ﬁlm sealed;
C. (a) IS-CW16% ﬁlm unsealed, (b) IS-CW16% ﬁlm sealed ............ 117

Figure 4.6 Electron Spectroscopy for Chemical Analysis survey spectra of
whey protein/lipid emulsion edible ﬁlms plasticized with glycerol.
A. (a) 16 ﬁlm unsealed, (b) 16 ﬁlm sealed;
B. (a) IG-BF4% ﬁlm unsealed, (b) IG-BF4% ﬁlm sealed;
C. (a) IG-CW16% ﬁlm unsealed, (b) IG-CW16% ﬁlm sealed ........... 118

xiv

Figure 4.7 Representative Electron Spectroscopy for Chemical Analysis
spectra of unsealed and sealed sorbitol-plasticized whey protein
isolate ﬁlms. unsealed (a) Cls; (b) 013; (c) le,
sealed (a) Cls; (b) 018; (c)N1s ............................................. 121

Figure 4.8 Proposed model for hydrogen bonding between (a) plasticizer-
plasticizer, (b) plasticizer-protein, (c) protein-protein upon
heat-sealing of glycerol- or sorbitol-plasticized whey
protein-based edible ﬁlms ..................................................... 128

Figure 4.9 Proposed model for covalent bonding between (a) lysine-asparagine,
(b) lysine-glutamine upon heat-sealing of whey protein-based
edible ﬁlms ...................................................................... 129

Figure 5.1 Moisture sorption isotherm of whey protein/lipid emulsion edible
ﬁlms plasticized with sorbitol ................................................ 145

Figure 5.2 Moisture sorption isotherm of whey protein/lipid emulsion edible
ﬁlms plasticized with glycerol ................................................ 146

Figure 5.3 Various food products in pouches manufactured using whey
protein-based edible ﬁlms ..................................................... 155

XV

ANS
ASTM
BF
CFR
CW
DTNB
DSC
%E
EMC
ESCA

GAB
GRAS
HPMC
IMC
2-ME
MSI
NTSB"
NTB”
or

m

SDS
SEM

ABBREVIATIONS

1-anilino-8-naphthalene sulfonic acid
American Society for Testing and Materials
Butter fat

Code of Federal Regulation
Candelilla wax

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

Equilibrium moisture content
Electron spectroscopy for chemical analysis
Food and Drug Administration
Glycerol

Guggenheim-Anderson-de Boer
Generally-recognized-as—safe
Hydroxypropyl methylcellulose

Initial moisture content
2-mercaptoethanol

Moisture sorption isotherm
Disodium 2-nitro-5-thiosulfobenzoate
2-nitro-5-thiobenzoate anion

Oxygen permeability

Relative ﬂuorescence intensity
Relative humidity

Sorbitol

Hydrophobicity value

Sodium dodecyl sulfate

Scanning electron microscopy

xvi

TGA
TS
WPC
WPI
WVP

Degradation temperature
Glass transition temperature

Melting temperature

Onset transition temperature
Peak transition temperature
Thermogravimetric analysis
Tensile strength

Whey protein concentrate
Whey protein isolate

Water vapor permeability

xvii

INTRODUCTION

Edible ﬁlms such as wax coatings, sugar and chocolate covers, and sausage
casings, have been used in food applications for years (Guilbert, 1986). However, interest
in edible ﬁlms and biodegradable polymers has been renewed due to concerns about the
environment, a need to reduce the quantity of disposable packaging, and demand by the
consumer for higher quality food products.

Edible ﬁlms can function as secondary packaging materials to enhance food quality
and reduce the amount of traditional packaging needed. For example, edible ﬁlms can
serve to enhance food quality by acting as moisture and gas barriers, thus, providing
protection to a food product after the primary packaging is opened. Edible ﬁlms are not
meant to replace synthetic packaging materials; instead, they provide the potential as food
packagings where traditional synthetic or biodegradable plastics cannot ﬁmction. For
instance, edible ﬁlms can be used as convenient soluble pouches containing single-servings
for products such as instant noodles and soup/seasoning combination. In the food
industry, they can be used as ingredient delivery systems for delivering pre-measured
ingredients during processing. Edible ﬁlms also can provide the food processors with a
variety of new opportunities for product development and processing.

Research on milk protein-based edible ﬁlms has been reviewed by McHugh and
Krochta (1994a). Film formation from nonfat dry milk (NFDM) was interfered due to
lactose crystallization, and use of total milk proteins as ﬁlm-forming materials was only
possible after removal of lactose from NFDM (Maynes and Krochta, 1994). Earlier

studies with whey protein- and casein-based edible ﬁlms involved enzymatic crosslinking

1

of proteins using transglutaminase (Motoki et al., 1987; Mahmoud and Savello, 1992,
1993). Ho (1992) reported on water vapor permeability (WVP) of caseinate ﬁlms
produced without crosslinking enzymes. He also reported on the eﬂ‘cct of incorporating
lipids on the WVP of these ﬁlms. McHugh et a1. (1994) produced ﬁlms ﬁom whey
protein isolate by applying heat and monitoring pH and protein concentration of the ﬁlm-
forming solutions without the use of crosslinking enzymes. Typically whey protein-based
ﬁlms are not good moisture barriers due to the hydrophilic nature of proteins. McHugh
and Krochta (1994b) suggested incorporation of lipids into whey protein ﬁlm-forming
solutions since lipids are the most effective edible barriers to moisture transfer. Until now
much of the research on milk protein-based edible ﬁlms have focused on their
development and testing for their barrier and mechanical properties. There is very little or
no other information available on other properties of these ﬁlms.

I hypothesize that it is possible to make whey protein-based edible ﬁlms with
improved moisture barrier properties without signiﬁcantly altering other properties by
producing whey protein/lipid emulsion ﬁlms and these ﬁlms will be suitable for food
applications. The following are the speciﬁc objectives of this research:

1. Develop whey protein/lipid emulsion edible ﬁlms and determine their microstructures,
barrier (moisture and oxygen) and mechanical (tensile strength and elongation)
properties.

2. Study the nature of interactions involved in the formation and Stability of the ﬁlms.

3. Investigate thermal properties, heat scalability, and scaling properties of the ﬁlms.

4. Demonstrate suitability of their application in foods as packaging materials.

CHAPTER 1

LITERATURE REVIEW

1.1 EDIBLE FILM

1.1.1 Definition and historical background

Edible ﬁlm can be deﬁned as a thin and continuous layer of edible materials that
can provide a barrier to mass transfer, like moisture, oxygen, lipids and solutes, and/or act
as a carrier for food ingredients and additives. Edible ﬁlms diﬁ‘er from edible coatings in
that they are prc-formed and ﬁecstanding sheets (Krochta, 1992; Chen, 1995). In ancient
China, wax has been used on citrus fruit to delay dehydration and fat coatings have been
applied to meats to prevent shrinkage since the 12th century. Yuba, the ﬁrst ﬁ'eestanding
edible ﬁlm appeared in the 15‘h century Japan, was obtained from boiled soymilk and was
used on food products for preservation and improvement of appearance (Guilbert and
Biquct, 1996).

It was in the last few decades that edible ﬁlms received scientiﬁc attention and
validation for their potential as food packaging materials. Over the last 40 years, a great
number of scientiﬁc articles and patents have been published on the characterization and
application of edible ﬁlms, and myriad resources were investigated as edible ﬁlm-forming

materials. Some of these edible ﬁlms are now available in the commercial market.

1.1.2 Formation of edible ﬁlms
1.1.2.1 Components of edible ﬁlms

Formation of ﬁlms requires use of at least one component capable of forming a
structural matrix with enough cohesive strength (Banker, 1966). Hydrocolloids,
polysaccharides and proteins, meet this requirement and offer good mechanical strength
compared to that of lipids. However, hydrocolloid ﬁlms are poor moisture barriers and
lipids are often combined with them to improve moisture Met properties by increasing
hydrophobicity of the ﬁlms (Krochta, 1997a; Debeaufort ct al., 1998).

The polysaccharides used to form edible ﬁlms are alginate, dextrin, starch, pectin,
carrageenan, chitosan, gum arabic and cellulose derivatives. Proteins used in edible ﬁlms
include wheat gluten, collagen, gelatin, corn, soy, peanut, and milk proteins (Kester and
Fennema, 1986). Lipids used in edible ﬁhns are typically waxes (beeswax, candelilla wax,
carnauba wax), sm'ﬁdctants (glycerol monostcaratc, acetate glycerol monostcaratc, citrate
glycerol monostcaratc and sorbitol monostcaratc), and fatty acids (lauric acid, palmitic
acid and stearic acid) (Donhowe and Fennema, 1993; Park et al., 1994b; Debeaufort and
Voillcy, 1995).

Like synthetic ﬁlms, edible ﬁlms also oﬁen require use of plasticizers to enhance
ﬁlm pliability. Protein-based ﬁlms in particular by themselves form very brittle ﬁlms,
however the brittleness can be decreased with the aid of a plasticizer. Plasticizers reduce
the level of intermolecular interactions in polymer chains and enhance pliability of ﬁlms.
Common food-grade plasticizers include sorbitol, marmitol, sucrose, glycerol, propylene
glycerol, polyethylene glycol, triethylene glycol, fatty acid, and monoglyccrides (Reiners,

1973; Krochta, 1997b).

A solvent system is required to produce a hydrocolloid or an emulsion ﬁlm. The
solvent allows solubilization and uniform spreading of high molecular weight polymer to
form a thin layer ﬁlm. Water and ethanol are the two typical solvents used for edible ﬁlm
formation (Kester and Fennema, 1986). There are also a number of additives that can be
used in the ﬁlm-forming solutions to inﬂuence properties of edible ﬁlms. These include
crosslinkers, various nutrients, ﬂavoring and coloring agents, antioxidants, and

antimicrobials (Donhowe and F cnnema, 1994; Krochta, 1997b).

1.1.2.2 Manufacture of edible ﬁlms

Solvent casting is the common process to form hydrocolloid edlble ﬁlms. A ﬁlm-
forming material is dispersed in an aqueous solution, generally water, ethanol, or a
combination of both The ﬁlm-forming solution is distributed by spreading or pouring it in
a thin layer, then allowing it to dry for solvent removal. Dried ﬁlm is detached ﬁ'om the
support and the freestanding ﬁlm is complete. In the industry, solvent casting has been
adopted commercially to manufacture hydroxypropyl methylcellulose (HPMC) edible ﬁlms
(Donhowe and Fennema, 1994; Krochta, 1997b). There are several types of surfaces that
can be used for edible ﬁlm casting, such as glass, teﬂon (polyetraﬂuorethylene),
polystyrene, plcxiglass (polymethacrylate), polyethylene (PE), and polyvinyl chloride
(PVC) (Gennadios et al., 1993b; McHugh et al., 1994; Maynes and Krochta, 1994).

Extrusion is another technique to obtain self-supporting ﬁlms. In the extrusion
process, a material is compacted and melted in the heated machine barrel and forced
through a die to be shaped as ﬁnished products (Koyich, 1992). Extrusion has been
applied to produce sausage casings from collagen. Several patents can be found referring

5

to the production of collagen casing ﬁlms by extrusion (Lieberman, 1964, 1965 & 1967;

Fagan, 1970; Miller, 1972).

I. I. 2. 3 Forces involved in the formation and stability of protein-based edible ﬁlms

In general, the protein network formation is resultant of protein-protein and
protein-solvent interactions, and a balance between attractive and repulsive forces between
polypeptide chains. Hydrophobic interaction (enhanced at high temperature), hydrogen
bonding (enhanced by cooling), and disulﬁde cross-links are known to be the attractive
forces in the protein network formation (Cheﬁel et al., 1985). According to F arnum ct a1.
(1976), the ﬁlm structure is a protein matrix formed by heat-catalyzed protein-protein
interactions with disulﬁdc, hydrogen, and hydrophobic bonds.

Disulﬁde bonds are oxidized forms of sulﬂIydryls, formed ﬁ'om free sulﬂiydryl
groups of two cysteine molecules in proteins. These bonds are linked together to form
polypeptide chains which contribute to a protein’s tertiary structure and produce a ﬁlm. A
heating process is important in the formation of protein ﬁlm network since it alters the
three-dimensional structure of proteins, and reveals ﬁee sulﬂiydryl groups and
hydrophobic side chains. For example, heating will expose ﬁee sulﬂiydryl groups in B-
lactoglobulin, then sulﬂtydryl/ disulﬁdc interchange reaction may occur (Gennadios et al.,
1994). Hydrolysis with acid or alkali is another factor for denaturation of proteins. An
alkaline condition aids ﬁlm formation because disulﬁde bonds are cleaved and reduced to
free sulﬂiydryl when dispersed in alkaline condition Disulﬁde bonds are reformed upon

drying of ﬁlm solutions.

Hydrogen bond is the electrostatic interaction force between polar molecules. The
H atom is shared between a proton donor group (acid) and a proton acceptor group
(base), for instance -OH or -NH, and : C=O, respectively (Howell, 1991). These
intermolecular association results in brittle ﬁlms. Thus, addition of plasticizers is
necessary to disrupt some of these associations and decrease rigidity of the ﬁlm structure.

Hydrophobic interactions can be deﬁned as the attractive force between nonpolar
molecules or nonpolar groups of molecules which induce association of these molecules in
an aqueous environment (Stenesh, 1989). The majority of the hydrophobic groups of the
native protein exist as buried inside of the molecule, therefore only a small amount of
hydrophobic interactions occur when protein is not heated. When heated, the hydrophobic
side chains may be exposed, and the hydrophobic interaction can occur. Upon drying,
exposed hydrophobic residues come closer due to the evaporation of the solvent and form
the intermolecular hydrophobic interactions, which contribute to the formation of protein
network (Chcﬁel et al., 1985).

Forces involved in the formation and stability of protein-based edible ﬁlms will
depend on the protein used and its amino acid composition. According to Gennadios et al.
(1994), the ﬁlm-forming ability of corn zein is primarily through hydrophobic interactions
and hydrogen bonding in the protein network. Contribution of disulﬁde bonds is
secondary due to low content of cystine in zein. Rangavajhyala et a1. (1997) studied
solubilities of soy protein-based ﬁlms in 2-mercaptocthanol (2-ME; a disulﬁde bond-
dissociating agent) and urea (a hydrogen bond-dissociating agent and also a weak
hydrophobic bond-dissociating agent). They concluded that disulﬁde and hydrogen bonds

play an important role in the formation of soy protein-based ﬁlm matrix. Roy et a1. (1999)

investigated molecular properties of wheat gluten-based ﬁlms using sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Reportedly, cross-linking
through disulﬁde bonds was responsible for the polymerization of ﬁlm-forming solutions.
They also suggested that similar ﬁlm-forming mechanism could be involved for other

sulﬂrydryl groups containing proteins such as soy and whey proteins.
1.2 PROPERTIES OF PROTEIN/ LIPID EMULSION EDIBLE FILMS

Although protein-based edible ﬁlms Show good oxygen barrier properties
compared to synthetic ﬁlms and posses suﬂicient mechanical strength to be utilized as
packaging materials, they are poor water vapor barriers due to proteins’ hydrophilic
characteristic (Krochta, 1992). Thus, lipids have been combined into protein-based ﬁlms
to produce ﬁlms with improved moisture banier properties (Gennadios et al., 1994). The
following reviews the inﬂuence of lipids on barrier and mechanical properties of

protein/lipid emulsion edible ﬁlms.

1.2.1 Water vapor permeability

Gontard et a1. (1994) reported water vapor permeability (WVP) of wheat gluten!
lipid emulsion ﬁlms with various lipid materials, such as soya lecithin, acetylated
monoacylglycerol, beeswax, carnauba wax and paraﬂin wax. A lipid material was added
into the ﬁlm forming solution and mixed together, then warmed until it melted. Addition
of soya lecithin and acetylated monoacylglycerol (both polar lipids) increased WVP of the

wheat gluten/lipid emulsion ﬁlms. On the contrary, beeswax, carnauba wax and parafﬁn

wax (all posses strong hydrophobic characteristics) contributed to improve resistance of
the ﬁlms to water vapor transport. These results were also related to melting point
diﬁ‘erences among lipids. Melting points of soya lecithin and acetylated monoacylglycerol
are 20°C, while beeswax, carnauba wax, and reﬁned paramn wax have higher melting
points, 61°C, 70°C, and 75°C, respectively. They concluded that use of lipids with high
melting point resulted in decreased WVP of wheat gluten/lipid emulsion ﬁlms.

Avena-Bustillos and Krochta (1993) determined WVP of acetylated
monoacylglycerol- and beeswax-added sodium caseinate-based edible ﬁlms. Beeswax was
more effective than acetylated monoacylglycerol in reduction of WVP. Acctylated
monoacylglycerol-added sodium caseinate ﬁlms had almost twice the permeability of
beeswax-added sodium caseinate ﬁlms. Chick (1998) studied the effect of carnauba and
candelilla wax on WVP of lactic acid casein-based ﬁlms. WVP was signiﬁcantly
decreased as wax concentration increased. Candelilla wax was more effective in reducing
WVP of the ﬁlms than carnauba wax.

McHugh and Krochta (1994c) investigated effects of incorporating fatty acids into
whey protein-based edible ﬁlms to reduce their WVP. Incorporation of fatty acids
signiﬁcantly decreased WVP of whey protein/lipid emulsion ﬁlms. Increased chain length
of fatty acids lowered WVP of the ﬁlms. WVP of palmitic acid (C16) and myristic acid
(C14) incorporated whey protein-based ﬁlms were 19.2 and 23.8 g. nun/m2 - day~ kPa,
respectively. Shellhammer and Krochta (1997) investigated WVP of whey protein/lipid
emulsion ﬁlms containing beeswax, candelilla wax, carnauba wax, and a high-melting
fraction of anhydrous milk fat. Each lipid was added at same level, 40% of the ﬁlm dry

weight, beeswax and milk fat fraction distinctively decreased WVP of the whey

9

protein/lipid emulsion ﬁlms from 45.4 to 10.8 and 21.9 g - mm/m2 - day - kPa, respectively.
With all lipid types, WVP of the ﬁlms were signiﬁcantly reduced as lipid concentration
increased. They concluded that the lipid type and concentration were important in

controlling WVP of the whey protein/lipid emulsion ﬁhns.

1.2.2 Oxygen permeability

Unlike the results observed in WVP of protein/lipid emulsion ﬁlms, no speciﬁc
trends were observed in oxygen permeability (OP). Gennadios et al. (1993c) investigated
the effect of acetylated monoacylglycerol on OP of wheat gluten/lipid emulsion ﬁlms.
They reported that acetylated monoacylglycerols signiﬁcantly lowered OP of the ﬁlms by
about 30% than that of the control ﬁlm. McHugh (1996) reported that OP of whey
protein/beeswax emulsion ﬁlms at two relative humidity testing conditions of 46 and 70%
were 8.6 and 101.1 cm3 - rim/m2 - day - kPa, respectively. OP of whey protein ﬁlms with no
lipid at the same relative humidity conditions were 1.5 and 42.3 crn3 - tun/tn2 - day- kPa,
respectively. Although no statistical comparisons were made, it is apparent that addition
of beeswax increased OP of whey protein-based ﬁhns at both relative humidity conditions.
Chick (1998) studied effect of carnauba and candelilla waxes on OP of lactic acid casein-
bascd ﬁlms. Reportedly, concentration of carnauba and candelilla waxes had no

signiﬁcant effect on OP of lactic acid casein/lipid emulsion ﬁlms and neither did the wax

type.

10

1.2.3 Mechanical properties

Lai et al. (1997) prepared zein/lipid emulsion ﬁlms with palmitic acid (0, 0.25, 0.5,
0.75 and 1g of palmitic acid per gram of zein), and reported on their tensile strength (TS).
Films with no pahnitic acid showed the lowest TS. Addition of palmitic acid increased
TS, however when it was added at more than 0.75g/g of zein TS decreased continuously
as the concentration of palmitic acid increased. Santosa and Padua (1999) investigated
mechanical properties of zein/lipid emulsion ﬁlms with oleic acid. Oleic acid was added
into zein ﬁlm-forming solutions at various ratios. TS of the ﬁlms decreased ﬁ‘om 9.4 to
2.2 MPa as oleic acid levels increased from 0.5 to 1.0 g/g of zein. On the other hand,
addition of oleic acid increased the ﬁlm’s percent elongation (%E), which is a measure of
ﬁlm’s ability to stretch However, excess oleic acid decreased %E, probably due to
weakened structure of the ﬁlms. Rhim et al. (1999a) investigated the eﬁ‘ects of lauric and
stearic acids on TS of soy protein/lipid emulsion ﬁlms. They reported decreased TS and
%E upon incorporation of these fatty acids.

Banerjee and Chen (1995) investigated TS and %E of whey protein/lipid emulsion
ﬁlms. Acctylated monoacylglycerol was added into whey protein concentrate and isolate
ﬁlm-forming solutions at 1:2 ratio (w/w, lipidzprotein). TS and %E of acetylated
monoacylglycerol-added whey protein-based ﬁlms were signiﬁcantly lowered in both whey
protein types compared to ﬁlms without lipids. Whey protein concentrate-based ﬁlms had
3.4 MPa and 20.8 % of TS and %E, respectively. These values were decreased to 1.1
MPa and 13.6 % alter addition of acetylated monoacylglycerol A similar trend was
observed in the values of whey protein isolate-based ﬁlms. Shellhammer and Krochta

(1997) studied effects of beeswax, candelilla wax, carnauba wax, and a high-melting

11

ﬁaction of anhydrous milk fat on TS and %E of whey protein-based ﬁlms. Lipid type and
concentration aﬁ‘ected TS of the ﬁlms. Increasing lipid levels linearly decreased TS of the
ﬁlms for all lipid types. However, carnauba wax-added ﬁlms were the strongest at all
concentrations. No signiﬁcants eﬂ‘ect of the lipid type and concentration on %E of the
ﬁlms were observed except for the milk fat-added ﬁlms. As the concentration of milk fat
increased, %E of the ﬁlms increased signiﬁcantly, probably resulting ﬁ'om plasticizing
elfects of unsaturated and low molecular weight triacylglycerols in the milk fat. Overall,
incorporation of lipids into protein-based ﬁlms altered ﬁlms mechanical properties. Type
of lipid and concentration seemed to be the most important factors ali‘ecting mechanical

properties of protein/lipid emulsion ﬁlms.

1.3 COMPOSITION AND PROPERTIES OF COMPONENTS USED IN EDIBLE
FILMS.

1.3.1 Whey proteins

Cheese whey is the liquid remaining alter the precipitation and removal of casein
during cheese manufacturing. About 9 kg of whey is generated to produce 1 kg of cheese
(Kosikowski, 1979). Liquid whey represents 85-95% of the milk volume, and contains
lactose, soluble proteins, lipids and mineral salts. Among them soluble proteins make up
06-08% (w/v) of liquid whey (Siso, 1996). Cheese whey is typically processed to
concentrate the whey proteins. Whey proteins are sold commercially in various forms as
ingredients to be used in baked goods, processed meats etc. due to their desirable
functional properties. Following are the manufacturing processes for whey protein
products.

12

1.3.1.1 Manufacture of whey proteins

Cheese whey tends to spoil easily, due to its low solids and high moisture content
and presence of cheese starter organisms. Thus, moisture is removed to prolong its shelf
life. This is achieved by high velocity and low temperature spray drying. High
temperature (>75 °C) is avoided to prevent denaturation of whey proteins, which may alter
their ﬁmctional properties (Renyard and Whitehead, 1992; Morr and Ha, 1993).
Composition of whey powders varies with the manufacturing process. Whey powder can
be obtained with reverse osmosis by removing water. This has 13% protein, 1% ﬁt, 76%
lactose and 10% ash. The 35% whey protein concentrate (WPC) contains 35% protein,
4% ﬁt, 53% lactose and 8% ash. The 50% WPC has 53% protein, 5% fat, 35% lactose
and 7% ash. In 80% protein WPC, lactose is reduced to 7%, and the ﬁt and ash range
from 4 to 7%. Whey protein isolate (WPI) has more than 90% protein with less than 1%
ﬁt, and lactose and ash vary between 1 to 4% (Early, 1992; Huﬂinan, 1996).

To make a WPC, whey is pasteurized and clariﬁed. The clariﬁer (a large scale
centrifuge) puriﬁes whey by removing small particles of cheese and casein. Then,
ultraﬁltration (UF) physically removes lactose and minerals, and concentrates whey
protein and ﬁt. The membrane molecular weight (MW) cut-oﬁ‘ is typically 20,000, thus
smaller particles, such as water, salts and lactose are readily removed. Following spray
drying a ﬁne white powder is produced. Diaﬁltration (DF) can further concentrate the
whey protein up to 80%. This membrane process involves applying a water stream to
wash out lactose and minerals (Mon and Ha, 1993; Huffman, 1996).

To produce a WPI, two additional processing steps are required. Microﬁltration

eliminates fat, and lactose hydrolysis removes the lactose. Both processes are followed by

13

UF and DF to make a low-ﬁt, low-lactose WPI (Huﬂinan, 1996). Ion-exchange is
another way to obtain a WPI. This process is a pretreatment prior to UP to produce WPI
(Early, 1992; Morr and Ha, 1993). An outline of the whey protein manufacturing process

isgiveninFigure 1.1.

1.3.1.2 Protein ﬁaction of whey

Whey proteins represent 15-25% of the total milk protein. Whey proteins consist
of [l-lactoglobulin, a-lactalbumin, bovine serum albumin, immunoglobulins, and proteose
peptone (Brunner, 1981). Whey proteins also include a number of enzymes such as
alkaline phosphatase, lactoperoxidase, sulﬂrydryl oxidase and catalase, and

metalloproteins, like lactoferrin (Cayot and Lorient, 1997).

1.3.1.2.] QLactogIobulin ——- B-lactoglobulin (Ii-Lg) makes up approximately 50% of
the whey proteins (Cayot and Lorient, 1997), and 7-12% of the total milk protein
(Brunner, 1981). B-Lg is a globular protein with a molecular mass of approximately
18,300 daltons (Eigcl et al., 1984). It has 162 amino acid residues and contains one ﬁ'ee
sulﬂrydryl group and two disulﬁde bonds. The amino acid composition of B-Lg is
provided in Appendix 1. Seven genetic variants of B-Lg have been characterized, among
themAandBarethemainvariants. TheAvarianthasAspandVal, andBvarianthas
Gly and Ala at residue 64 and 118, respectively (Cayot and Lorient, 1997). Bovine B-Lg
shows a high degree of organization in the secondary and tertiary structures with 50% [3
sheets, 15% or helix, and 15-20% [3 turns. Its compact structure is a result of two
disulﬁde bonds and stacked nine [3 sheets (Creamer et al., 1983). The free Cys 121

14

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residue is located at the sheet-helix interﬁce, and two disulﬁde bonds are formed Cys 66
to Cys 160 and Cys 106 to Cys 119 (Wong et al., 1996). Most of non-polar residues in B-
Lg are buried in the protein’s interior, forming a hydrophobic pocket, and a majority of the
polar groups are exposed at the surﬁce (Cayot and Lorient, 1997).

Bovine B-Lg exhibits pH-dependent association-dissociation behavior. B-Lg
generally exits as a dimer at pH 5-8. At pH 3-5 the dimers tend to form octamers. Below
pH 2 and above pH 8, B-Lg dissociates into monomers (Passen et al., 1985; Monaco et
al., 1987). Thermal denaturation and aggregation of B-Lg occur above 65°C.
Denaturation of B-Lg initiates with exposure of the reactive Cys 121. This reversible
conformational change leads sulﬂrydryl/disulﬁde exchange to form disulﬁde bonds then
association of unfolded species through hydrophobically bonded aggregates. This
aggregation is irreversible and displays time and temperature dependency (Gough and

Jenness, 1962; Sawyer et al., 1971; Gezimati et al., 1996).

1.3.1.2.2 q-Lactalbumin — cr-lactalbumin (or-La) represents approximately 19% of the
whey proteins (Cayot and Lorient, 1997), and 2-5% of the total milk protein (Brunner,
1981). It has four disulﬁde groups with a molecular mass of 14,147 daltons for genetic
variant A and 14,175 daltons for B. ct-La contains 123 anrino acid residues, and the
variants A and B differ at residue position 10; Gln for A and Arg for B (Brew et al., 1970;
Eigel et al., 1984). The amino acid composition of ol-La is provided in Appendix I.

or-La exhibits a very low tendency of organized secondary structure. It consists of

30% or helix and 9% [3 sheets (Alexandrescu et al., 1993). The a—La molecule shows

16

ellipsoidal shape, and a deep cleft divides the molecule into two lobes. One side of the
lobe comprises of four helices, and the other lobe contains two [3 strands and loop-like
chain (Wong et al., 1996). Heat treated (at 80°C) or-La recovers its structure aﬁer heat
treatment; this reversrhility makes it more heat resistant than B-Lg. The apparent
diﬁ'erence in thermal aggregation behavior between or-La (reversible) and B-Lg
(irreversible) is likely to be based on the fact that B-Lg has self initiation ability of
sulﬂiydryl/disulﬁde exchange reaction, but not or-La. However, the aggregation rate of or-
La is increased when ct-La is heated in the presence of B-Lg. This aggregation occurs
through disulﬁde bonded copolymer formation between a-La and B-Lg (Hines and

Foegcding, 1993; Gezimati et al., 1997).

1.3 1.2.3 Bovine serum albumin —— Bovine serum albumin (BSA) makes up about 5%

 

of whey proteins (Cayot and Lorient, 1997), and 0.7-1.3% of the total milk proteins
(Brunner, 1981). BSA is a carrier protein, functioning to transport nonpolar molecules in
biological ﬂuids (Whitney et al., 1976). The BSA molecule is a single peptide chain with
582 amino acid residues with a molecular mass of approximately 66,000 daltons. The N-
terminal amino acid residue is Asp and Ala is the C-terminal. It appears to be that the
BSA molecule has an ellipsoidal shape and the N-terminal region is more compact than the
C-terminal region. It has 17 intramolecular disulﬁde bonds with one free sulﬂrydryl group
at residue 34 (Eigel et al., 1984). BSA can be denatured by heating or by acid or base
treatment (Cayot and Lorient, 1997). BSA shows very similar denaturation behavior to
that of B-Lg. Upon heating, BSA forms disulﬁde bonded-polymers and aggregates

through hydrophobic interactions (Gezimati et al., 1996).

17

1.3. 1.2.4 Immunoglobulins and protease-peptone — Immunoglobulins form family of
high molecular weight proteins, such as IgGr, Ing, IgA, IgM and IgE, with antibody
properties. Immunoglobulins represent 1.4 -3. 1% of total milk proteins (Brunner, 1981),
and make up around 13% of whey proteins (Cayot and Lorient, 1997). Immunoglobulins
have molecular mass of 15,000 to 1,000,000 daltons (Whitney et al., 1976; Eigel et al.,
1984). The irmnunoglobulin molecule consists of two light polypeptide chains (~20,000
daltons), and two heavy polypeptide chains (~50,000 to 70,000 daltons). The light and
heavy polypeptide chains are cross-linked by disulﬁde bonds (Whitney et al., 197 6;
Brunner, 1981). About 80% of irnmunoglobulins in milk are IgGs (Eigel et al., 1984).
Proteose-peptoncs (PP) account for 2-4% of total milk proteins, and 10% of whey
proteins. Their molecular masses range ﬁom 4,000 to 40,000 daltons (Brunner, 1981).
The PP components remain soluble after precipitation of casein at pH 4.6, and heat
coagulation of the B-Lg and or-La at 95°C for 30 min. Thus, the PP fraction is deﬁned as
a mixture of acid-soluble and heat-stable phosphoglycoproteins (Whitney et al., 197 6;

Girardet and Linden, 1996).

1.3.2 Lipids

Candelilla wax (CW) is a vegetable wax ﬁ'om a reed-like plant (E uphorbia
Antisiphilitica, Euphorbia Cerifera, and Pedilanthus Pavanois), which grows in
northwestern Mexico and southern Texas. When the plant reaches a height of one to
three feet, stalks are uprooted and utilized. The plant is immersed into boiling water
containing sulfuric acid and wax is extracted, strained, and cooked to remove excess water

(Bennett, 1975). The wax consists of 50-51% hydrocarbons and 29% wax esters, with

18

the remainder consisting mainly of alcohols and free acids. The melting point of CW
ranges between 66 and 71°C. CW exists as hard, brittle, and lustrous granules and has a
honey-like aromatic odor (Bennett, 1975). CW belongs to the non-polar lipid class; it is
insoluble in water and has high hydrophobicity like other waxes (Callegrain et al., 1997).
CW is GRAS (generally-recognized-as-safe) and is approved by the Food & Drug
Administration (FDA) for use in ﬁ'uit and vegetable coatings, confections and beverages.
There are no limitations in usage of CW other than good manuﬁcturing practice (CFR,
184.1976; Hernandez and Baker, 1991; Hernandez, 1994; Baldwin et al., 1997)

Butter ﬁt (BF) or milk ﬁt accounts for 3.5-3.8% of bovine milk. The milk ﬁt
exists in milk as small ﬁt globules emulsion dispersed in the aqueous phase. The ﬁt
globules range from 2 to 10m in diameter. Total milk lipids contain 97-98%
triacylglycerols. The remaining lipids are di- and monoacylglycerols, phospholipids, ﬁ'ee
ﬁtty acids, and cholesterol and its esters. Triacylglycerol consists primarily of oleic, stearic
and palmitic acids with smaller amount of triacylglycerols of butyric, caproic, caprylic and
capric acids (Swaisgood, 1985; Sax and Lewis, 1987; Igoc, 1989; Muir, 1992). Milk ﬁt
triacylglycerols contain approximately 10-15% high melting, 30—45% middle melting and
35-55% low melting acylglycerols, and their melting points are around 20-40°C, 0-20°C
and 0°C, respectively. Since the main components of BF are triacylglycerols, BF is
classiﬁed as a polar lipid (Lane, 1992; Callegrain et al., 1997). Commercial butter is
required to contain at least 80% ﬁt by weight and maximum 16% in moisture (Lane,

1992).

19

1.3.3 Plasticizers

Sorbitol (D-glucitol) was discovered ﬁom the ripe berries of mountain ash (Sorbus
aucuparia L.) in 1872. It can be produced by high-pressure hydrogenation or electrolytic
reduction of D-glucose, or by catalytic hydrogenation of dextrose. The molecular weight
of sorbitol is 182.17 daltons, and it consists of a 6 carbon chain with 6 hydroxyl groups
(C5H1406). Its structure is shown in Appendix II. Sorbitol is stable and chemically
unreactive (Budavari et al., 1989). Sorbitol is a polyol (polyhydric alcohol) with good
solubility in water and poor solubility in oil. It is slightly soluble in methanol, ethanol,
acetic acid, phenol and acetamide, and almost insoluble in most other organic solvents.
The relative sweetness of sorbitol is approximately 60 % that of sucrose. Sorbitol is a
white, odorless and hygroscopic crystalline powder. Hydrated sorbitol crystals melt below
100°C, while anhydrous sorbitol melts at 110-112°C. Sorbitol is approved by FDA for
food use as a sweetener, humectant, emulsiﬁer, thickener, anticaking agent and dietary
supplement (Sax and Lewis, 1987; Budavari et al., 1989; Igoe, 1989).

Glycerol is a three-carbon molecule with one hydroxyl group (Cd-1303), and its
structure is shown in Appendix II. It has a molecular weight of 92.09 daltons. It is a
colorless, odorless clear syrupy liquid with high solubility of 71 g per 100g of water at
25°C. It is insoluble in ether, benzene, and chloroform. Glycerol has medium to high
hygroscopicity. Melting temperature of glycerol is 17.8°C. It is about 60% as sweet as
sucrose (Budavari et al., 1989; Igoc, 1989). Glycerol is a byproduct of soap manuﬁcture.
It can be produced by catalytic hydrogenation of carbohydrates or isomerization of

propylene oxide to allyl alcohol, which is then hydrolyzed to glycerol. Glycerol is used as

20

a humectant in pharmaceuticals, a sweetener in confectionerics, fermentation nutrient in

antibiotic production, and as a plasticizer for regenerated cellulose (Sax and Lewis, 1987).

1.4 PROPERTIES OF FILMS IMPORTANT FOR FOOD APPLICATIONS

1.4.1 Barrier properties

Food product quality depends on loss of or exposure to vapor and gases, such as
water, oxygen, carbon dioxide, or volatile compounds. Functions of edible ﬁlms include
providing barriers to the transfer of water, gas, or solute (salts, pigment, or lipids).
Therefore, the measurements of permeability values of edible ﬁlms are important in
determining their performances as packaging materials (Debeaufort et al., 1998). Water
vapor pemreability (WVP) and oxygen permeability (OP) are the most commonly studied
barrier properties of edible ﬁlms.

Permeability is deﬁned as transmission of vapor or gases through polymer
materials. This permeation involves adsorption of the vapor or gas into the polymer
surﬁce, its diffusion through the polymer, and its desorption through the opposite surface
by evaporation (Sperling, 1992). A diagram of the permeability model is shown in Figure
1.2. This is expressed by the following equation:

P = D x S (1)
P is the permeability coeﬁcient and commonly called permeability, D is the diﬁ‘usion
coefﬁcient, and S is the solubility coefﬁcient (Giacin and Hernandez, 1997). The diffusion
coefﬁcient refers to the speed that the molecules move into the polymer. Diﬁ'usion

movement occurs ﬁ'om the side of the polymer that is in contact with high concentration

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or partial pressure of permeant to the side that is in contact with a low concentration of
permeant. The dilfusion process ﬁrrther depends on the size, shape and polarity of the
diﬁ‘using molecules, and the structure and characteristics of the ﬁlms. The solubility
coeﬁcient is the number of permeant molecules that are diffusing. Solubility coeﬂicient
refers to the solubility parameters of vapors or gases to the particular polymer. The
solubility inﬂuences dissolution and evaporation of the permeating substance at the
interface of the ﬁhn. A low permeability is the result of a low diﬁ‘usion coeﬂicient or a
low solubility coefﬁcient or both. The permeation test for vapor or gas is run until steady
state is reached (Guilbert and Biquct, 1996; Delassus, 1997).

Water vapor transmission rate (WVTR) is the rate of water vapor ﬂow to the
surﬁce, under steady-state conditions, per unit area. Water vapor permeance is the ratio
of a barrier’s WVTR to the vapor pressure difference between the two surﬁccs. Water
vapor permeability (WVP) is the product of the permeance and the thickness of the ﬁlm
(ASTM, 1997). WVTR can be tested using an inﬂated or coulometric sensors;
spectrophotometric, gas chromatograhic or gravimetric techniques.

Only an inﬁared sensor method, ASTM F -1249 “Standard test method for water
vapor transmission rate through plastic ﬁlm and sheeting using a modulated inﬁared
sensor” (ASTM, 1997), is reviewed here since this method was the one adopted to
measure WVP of the ﬁlms developed in this present research. The operation diagram of
an infrared sensor method is shown in Figure 1.3. First, desired temperature and humidity
conditions are set. Test ﬁlm is placed in a diffusion cell to separate a dry chamber and a
wet chamber. Then, the dry chamber side is swept with dry air, which carries diffused

water vapor through the test ﬁlm into an infrared sensor. The sensor detects infrared

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energy absorbed by the water vapor and yields an electrical signal, which is proportional to
water vapor concentration. WVTR is obtained by the equation:
WVTR = C x (ES - E0) (2)
Where, C = a calibration ﬁctor
ES = eqlrihbrium voltage obtained with the test ﬁlm
E0 = steady-state voltage produced by dry air
This value is used to calculate water vapor permeance:
Pcnneance = WVTR / Ap (3)
Where, WVTR = water vapor transmission rate of the test ﬁlm
Ap = water vapor partial pressure gradient across the test ﬁlm
Then, permeability is calculate as following:
WVP = permeance x d (4)
Where, WVP = water vapor permeability of the test ﬁlm
d = thickness of the test ﬁlm
WVP can be expressed as the g - mm/m2 - day - kPa and since the permeance is the function
of relative humidity (RH) and temperature, the test condition must be stated.

Oxygen transmission rate (OTR) is the quantity of oxygen that passes through a
unit area of a test ﬁlm per unit time. Oxygen permeance is the ratio of the OTR to the
difference between the partial pressure of oxygen across the test ﬁlm. Oxygen
permeability (OP) is the product of permeance and the thickness. Coulometric sensor
technique is the most commonly used method to determine OP of ﬁlms, and this method

was also adopted in this present study.

25

ASTM D-3 985 “Standard test method for oxygen gas transmission rate through
plastic ﬁlm and sheeting using a coulometric sensor” (ASTM, 1997) can be used to
obtain OTR. A test ﬁlm is placed between two chambers at ambient atmospheric
pressure. OTR is determined after the test ﬁlm is equilibrated in a dry condition, which is
less than 1% RH. One side of the chamber is purged with nitrogen and the other side by
air containing 21% of oxygen. Nitrogen gas carries permeated oxygen through the test
ﬁlm into the coulometric sensor, which produces an electrical current. This electrical
current is proportional to the amount of oxygen ﬂowing into the sensor per unit time.
Operation diagram of the measurement of OTR is given in Figure 1.4. To obtain the OP,
thickness of the ﬁlm and the partial pressure are ’taken into account shown by the
equation:

OP = (OTR x d) / Ap (5)
where d is thickness, and Ap is partial pressure of oxygen. OP can be expressed as the

cm3 - urn/m2 - day - kPa and the test condition must be stated.

1.4.2 Mechanical properties

Edible ﬁlms need to have a certain degree of mechanical strength with suﬁcient
ﬂexibility to resist breakage during distribution and handling of packaged products. Thus
determination of mechanical properties is necessary to provide information to set
appropriate storage and handling requirement of edible ﬁlms (Gontard and Guilbert,
1994). Various mechanical properties of edible ﬁhns have been evaluated, such as tensile
strength, puncture strength, extensibility to puncture, torsion resistance, elasticity,

elongation at break, elongation at yield, etc. (Guilbert and Biquct, 1996). Among them,

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tensile strength (TS) and percent elongation (%E) are the most common mechanical
properties to evaluate edible ﬁlms and to predict their performance as packaging materials
(Gennadios et al., 1994).

TS refers to the maximum tensile stress that a material can sustain before the onset
of pemianent deformation or failure. This is determined by measuring the force per unit
area (F/A) while the material is pulled apart, and is usually expressed in pounds per square
inch (lbs/inz) or megapascals (MPa). TS is an indicator of how strong a material is. %E
is the deformation caused by stretching and fractional increase in length while a material is
stressed in tension. %E is determined by measuring the length of displacement per
original length (AL / Li, reported as percentage) while pulling apart the material. %E is an
indicator of the material’s toughness and ﬂexibility (Symonds, 1989; Koyich, 1992). The
established method to determine tensile strength and elongation at break is ASTM D-882,
“Standard test method for tensile properties of thin plastic sheeting” (ASTM, 1997).

Diagram of mechanical properties measurement is shown in Figure 1.5.

1.4.3 Thermal properties

In considering thermoplastic processing, for instance heat-sealing or extrusion, for
edible ﬁlms, one of the most important limitations is the lack of information on their
thermal properties. Thus, exploiting thermal properties of biopolymer-based ﬁlms and
their components is crucial in the development of edible packaging rmterials. Several
different thermal analysis techniques are available that measure temperature of transition,
heat capacity change, and energies of transition or enthalpic change (AH) of processing

materials, such as differential scanning calorimetry (DSC), differential thermal armlysis

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(DTA), thermogravimetric analysis (TGA), thermomechanical analysis (TMA) and
dynamic mechanical thermal analysis (DTMA). Among them, DSC is the most common
thermal analysis technique. DSC measures the diﬂ‘erential temperature or heat ﬂow to or
from a sample versus a reference material (Figure 1.6). This can be plotted as a function
of temperature or time (Davis, 1994).

Thermoplastic processing is possible when the raw materials have a wide plastic
range between room temperature and its degradation temperature (Td). In general, ﬁlm
production is carried out at a temperature moderately above glass transition temperature
(T g), or at melting temperature (Tm) of the polymer (Hernandez, 1997; Metzger, 1997).

T8 is the temperature where the reversible change occurs in amorphous polymer or
in amorphous regions of partially crystalline polymer ﬁom (or to) a viscous and rubbery
condition to (or from) a relatively brittle one. The T3 of polymers range from -25°C to
365°C (ASTM, 1997; Hernandez, 1997). Heating amorphous thermoplastic biopolymers
above their T,3 results in soﬁ and rubbery materials, which allows them to be shaped as
packaging materials. Cooling at room temperature reconverts the rubbery materials into a
glassy product with desired shape (Cuq, 1997a; Krochta and De Mulder-Johnston, 1997).
Tm is the temperature of molten polymers. Most semicrystalline polymers have a Tm range
instead of having a sharp melting peak. Amorphous polymers don’t have a Tm. The TIn of
materials could range &0m 2°C to 455°C depending on the polymer. Ta is the point where
thermal decomposition of polymers occurs. Thermal degradation of polymers is the
breaking of bonds by heat in the absence of oxygen. As the temperature increases,

chemical bonds with low energy values will be broken ﬁrst, and thermally stable bonds will

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resist thermal degradation and require higher energy to dissociate them. Thus Td can
occur as multiple peaks (Throne, 1986; ASTM, 1997; Hernandez, 1997).

ASTM D-3418 “Standard test method for transition temperatures of polymers by
thermal analysis” (ASTM, 1997) covers determination of transition temperatures of
polymers by DSC. This standard method also shows determination of onset (To), peak
(T9), and end (Te) temperatures of ﬁrst-order transition. Tm is a ﬁrst-order transition
which exhibits a discontinuity with temperature. On the other hand, T8 is a second-order
transition which exhibits a temperature continuity (Rosen, 1982).

Cuq et al. (1997a) studied thermal properties of myoﬁbrillar proteins and
myoﬁbrillar protein-based edible ﬁlms. They reported that T8 of the myoﬁbrillar proteins
were drastically decreased when it was formed as dried ﬁlms, from 215-250°C to 130-
185°C, respectively. They explained that this larger decrease (> 75°C) in T8 is due to
addition of glycerol. Similar trends were observed with zein, corn gluten, and gelatin-
based edible ﬁlms upon incorporation of plasticizers (O’Donnell et al., 1997; Di Gioia and
Guilbert, 1999; Menegalli et al., 1999). Plasticizers separate interactions between
polymers causing reduction of their resistance to applied stress, which results in reduced
T3 (Throne, 1986). The presence of hydrophilic plasticizers in ﬁlms also decreases the Tm
by enhancing molecular mobility of the ﬁlm structure (Gutiérrez-Rocca and McGinity,

1994; Aravanitoyannis et al., 1998).

1.4.4 Sealing properties
Scalability of the edible ﬁlms is an appealing attnbute, and important in the

development of pouches or sachets. It is desirable to have materials that are not only

32

edible but also heat-scalable from the viewpoint of edible-packaging operation. There are
diﬁ‘erent heat-sealing techniques such as bar sealing, band sealing, impulse sealing, hot
wire or knife sealing, gas sealing, contact sealing, hot-melt sealing, and pneumatic sealing,
etc. (Young, 1997). Although there are many different methods for heat-sealing, the basic
principles remain the same. Surface regions of polymer melt when heat is applied. The
bonding of two polymer surfaces is initiated by bringing them together while they are in
partially molten state. With the application of pressure, polymer-chain segments ﬁ'om
opposite sides of the interface diﬂ‘use across the interface, and molecular entanglements
are induced between the melted surface. After cooling, a heat sealed joint of the two
polymer surfaces is completed (Stehling and Mcka, 1994).

The effect of heat-sealing depends on sealing temperature, pressure, and dwell
time. To achieve sealing with suﬁcient strength, the surface must be pressed together an
adequate length of time and pressure so the polymer chains can diﬁuse across the interface
and form bridged bonds (Meka and Stehling, 1994; Mueller et al., 1998). However, the
important seal properties of the ﬁlms such as seal strength, toughness, and appearance are
much affected by the sealing temperature. Appropriate sealing temperature or sea] range
is determined according to data obtained ﬁom thernm] analysis of the polymer. Seal range
is the range of temperature in which eﬁ‘ective seal can be obtained at constant dwell time
and pressure, and the highest sealing temperature should be determined at which a seal can
be obtained without deterioration of the seal or the polymer structure (Martin, 1986).

ASTM F -88 “Standard test method for seal strength of ﬂexible barrier materials”
(ASTM, 1997) is designed to provide a standard seal strength test for packaging materials
and adopted in this present study. Studies have demonstrated heat scalability of

33

biopolymer-based ﬁlms from casein, carrageenan and soy protein (Georgevits, 1967;
Ninomiya et al., 1990; Rhim et al., 1999a). However, there are no reports on the seal
properties of whey protein-based edible ﬁlms.

The sealed joints studied by seal strength test provides macroscopic information on
the bond strength, however it doesn’t give information on the nature of the sealing
mechanism or on the chemical characteristics of the sealed surfaces. Thus, a surface
analytical technique that provides information about the interfacial chemistry of polymers
may be necessary to gain further understanding. Electron spectroscopy for chemical
analysis (ESCA) has been the principal technique that deﬁnes interfacial chernistries
associated with adhesive behavior of materials. ESCA allows identiﬁcation and
quantitation of all elements with the exception of hydrogen (Ruse and Smith, 1990; Ratner
and Castner, 1997). ESCA is an ideal technique to study surfaces of sealed ﬁlms because
of its surface sensitivity (l-lOnm; Gerenser, 1993).

Lee (1994) studied surfaces of polyimide ﬁlms using ESCA. Polar functional
groups, such as hydroxy (OH), aldehyde (CH0), and carboxylic acid (COOH), have been
identiﬁed on the surfaces of polyimide ﬁlms. Reportedly, modiﬁcations of these functional
groups were responsrble for the adhesion strength differences of the ﬁlms. Kawabe et al.
(1999) employed ESCA to investigate surface modiﬁcation of adhesive tapes on their
adhesion behavior. Nitrogen plasma-treated tapes showed enhanced adhesion. They
conclude that adhesion strength increased due to cross-linking reactions across the
surfaces as determined by ESCA. As with many other biopolymer-based ﬁlms, the sealing
mechanism of whey protein-based ﬁlms are not known, and no study has ever employed
ESCA to investigate seal properties of biopolymer-based ﬁlms.

34

The principle of the ESCA technique involves detecting the electrons emitted ﬁ'om
specimen’s atoms by absorption of photons. Another acronym simultaneously used is X-
ray photoelectron spectroscopy (XPS), since X-ray is the source of the photons. X-ray
excitation is used to yield emission of electrons form the core levels. This involves
ionization of core electrons with bonding energies smaller than 1000 eV (Cayless, 1991).
A schenmtic illustration of photoelectron emission is shown in Figure 1.7. A sample is
irradiated with X-ray of known energy, hv, and electrons of binding energy, Eb, are
ejected, where E, < hv. These electrons have a kinetic energy, E, which can be measured
in the spectrometer.

Ek=hv -Eb —<l>Sp (6)
where (1),, is the work ﬁtnction of the spectrometer and a constant. Thus, by measuring
the kinetic energy of the photoelectrons, binding energy of the electrons can be obtained.
An ESCA spectrum can be generated by plotting the measured photoelectron intensity as
a function of binding energy. The binding energy is characteristic for each element.

Eb = 17" - Bk (7)

A schematic diagram of the main components of an ESCA instrument is shown in Figure
1.8. The main components of the system, the X-ray source, sample stage, lens, analyzer,

and detector are enclosed in an ultra high vacuum chamber (Kibel, 1991; Oleﬁord, 1997).

1.4.5 Moisture sorption properties
A moisture sorption isotherm (M81) is a plot of correspondent equilibrium
moisture content (EMC) of a material with its water activity (aw) at constant temperature.

Adsorption isotherms are obtained by addition of water to previously dried samples.

35

 

 

 

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hv = energy of X-ray; E, = binding energy of electrons; E. = kinetic energy of electrons;
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Desorption isotherms are prepared by removal of water from samples. Although these
two sorption isotherms will not necessarily be superimposed, an adsorption isotherm is of
particular interest when considering MSI of edible ﬁlms. Moisture adsorption signiﬁcantly
affects stability, quality attributes, acceptability, and packaging and storage requirements
of food products (Kinsella, 1984; Fennema, 1985).

Examining moisture sorption properties of edible ﬁhns is useﬁil in predicting water
vapor transfer through ﬁlms under various relative humidity (RH) conditions. The MSI
also can be used to estimate properties of ﬁlms at different environmental conditions for
their appropriate application and to predict potential problems like reduced storage
stability (McHugh and Krochta, 1994a; Jangchud and Chinnan, 1999). One of the
simplest methods for obtaining a sorption isotherm is suggested by Labuza (1984). Films
are placed in hermetically sealed containers at controlled RH and temperature. To obtain
speciﬁc RH inside of containers, saturated salt solutions must be prepared by ASTM E-
104 “Standard practice for maintaining constant relative humidity” (ASTM, 1997).
After equilibrium is reached, moisture content of ﬁlm samples are measured. This method
has been employed in several studies to examine moisture sorption properties of protein-
based edible ﬁlms. Gontard et al. (1993) showed the difference between sorption and
desorption isotherms of wheat gluten-based edible ﬁlms. Lim et al. (1998) investigated
MSI of egg-white protein ﬁlms plasticized with varying plasticizer levels. Reportedly, the
higher plasticizer levels induced higher EMC in the ﬁlms resulted in ﬁlms with increased
hydrophilicity. Lai and Padua (1998) studied MSI of zein-based ﬁlms. They reported that

the MSI of zein-ﬁlms were not typical sigmoidal curves as in the case of foods. Instead, it

38

resembled the MSI of crystalline sugar: very little moisture gain at aw < 0.7, and
exponential moisture sorption increase at aw > 0.7.

There are number of food isotherm equations available in the literature. The four
most commonly used isotherm equations are, Guggenheim-Anderson-de Boer (GAB),
Braunauer—Ennnett-Teller (BET), Smith, and Henderson equation (Jangchud and Chinnan,
1999). Among them, GAB equation is considered the most appropriate equation for
modeling the moisture sorption characteristics of the ﬁlms (Lim et al., 1999; Coupland et
al., 2000). GAB equation is given as:

MC=kaCaw/ [(1 -kaw) (l-kaw+ckaw)] (8)
where, MC = moisture content of the samples on a dry basis (g / 100 g dry solids)
Wm = monolayer moisture content
k, C = factors correcting differences in enthalpy of free and monolayer water
compared to that of multilayer water, respectively.
aw = water activity

In order to estimate parameters, the equation was transformed into a quadratic form as :

a../EMC=aaw2+Baw+y (9)
where, a=k/Wm[1/C—1] (10)
B=l/Wm[1—2/C] (11)
y=l/Wm[ka] (12)

39

CHAPTER 2

DEVELOPMENT OF WHEY PROTEIN/ LIPID EMULSION EDIBLE FILMS
AND DETERMINATION OF THEIR MICROSTRUCTURE,

BARRIER AND MECHANICAL PROPERTIES.

2.1 ABSTRACT

Methodologies were developed to produce edible ﬁlms ﬁ‘om whey protein isolate
(WPI) and concentrate (WPC), and a ﬁlm-forming procedure was optimized. Lipids,
butter fat (BF) and candelilla wax (CW), were added into ﬁlm-forming solutions to
produce whey protein/lipid emulsion edible ﬁlms. Effects of protein and lipid types and
concentrations on moisture and oxygen barrier properties of whey protein/lipid emulsion
ﬁlms were investigated. Cross-sections and top surfaces of the ﬁlms were examined using
scanning electron microscopy. Signiﬁcant reductions in water vapor and oxygen
permeabilities of the ﬁlms could be achieved upon addition of BF and CW. Tensile
strength and percent elongation of whey protein/lipid emulsion ﬁlms were affected
primarily by protein type with some inﬂuenced by the lipid. Differences in the

microstructures of the ﬁlms accounted for the differences in their barrier and mechanical

properties.

40

2.2 INTRODUCTION

Poor performance of whey protein-based ﬁlms as moisture barriers is one of the
main limitations for their application as food packaging materials. This is due to the
hydrophilic nature of the proteins. Lipid ﬁlms, on the other hand, act as good moisture
barriers but have poor oxygen barrier properties and mechanical strength. The advantages
of each can be achieved by combining whey proteins with lipids to produce composite
ﬁlms with improved properties (McHugh, 1996).

Protein/lipid composite ﬁlms can be obtained using emulsions or layering
technique. Generally, two or more layered ﬁlms show better mechanical and barrier
properties than emulsion ﬁlms. However, the layering technique requires a complex and
time-consuming ﬁlm-manuﬁicturing process, and the ﬁlms tend to delaminate due to high
surface energy on the interﬁace (Shellammer and Krochta, 1997). Emulsion ﬁlms,
however, require only one-step operation, and the emulsifying property of whey proteins
makes them good candidates to be used in emulsion ﬁlms.

Various lipids have been examined for their effectiveness on properties of whey
protein/lipid emulsion ﬁlms. Fatty acids, waxes and butter fat (or milk fat) have been used
in whey protein/lipid emulsion ﬁlms and their properties have been reported (McHugh and
Krochta, 1994c; l994e; Shellhammer and Krochta, 1997). McHugh and Krochta (1994c;
1994c) investigated effects of fatty acids and beeswax on water vapor permeability (WVP)
of whey protein/lipid emulsion ﬁlms. They reported that increasing fatty acids or beeswax
concentrations reduced the WVP of whey protein/lipid emulsion ﬁlms. Shellhammer and
Krochta (1997) studied mechanical properties of whey protein/lipid emulsion ﬁlms when
high-melting milk fat fraction, beeswax, carnauba wax and candelilla wax were

41

incorporated into ﬁlm-forming solutions. The high-melting milk fat ﬁ'action and beeswax-
added ﬁlms showed relatively lower WVP compared to those of candelilla wax- or
carnauba wax-added ﬁlms (40% of the ﬁlm dry wt.). Lipid type and concentration
aﬁ’ected mechanical properties of the ﬁlms. Overall, increasing the lipid concentration
reduced the tensile strength (TS) of the ﬁlms. Wax-added ﬁlms showed no diﬁ‘erence in
percent elongation (%E) upon increasing lipid levels. However, there was a signiﬁcant
increase in %E of the ﬁlms as the high-melting milk fat ﬁaction level was increased.
Previous studies on whey protein/lipid emulsion ﬁlms have been directed at their
moisture barrier or mechanical properties, however, they have been limited. Fewer studies
have been conducted on oxygen permeability (OP) of whey protein/lipid emulsion ﬁlms.
The main objective of this study was to evaluate the eﬂ‘ect of lipids on moisture and
oxygen barrier, and mechanical properties of whey protein/lipid emulsion ﬁlms. Two
types of whey proteins, whey protein isolate and whey protein concentrate, were selected
to determine their feasibility and effectiveness as ﬁlm-forming agents. Lipids (butter fat
and candelilla wax) were added into ﬁlm-forming solutions in various concentrations.
Film formation procedure and formula ratio were optimized for whey protein/lipid
emulsion ﬁlms. The effect of protein and lipid type and concentration on barrier (WVP
and OP) and mechanical properties (TS and %E) was investigated. Microstructures of the
ﬁlms were determined to illustrate the relationship between microstructures, and barrier

and mechanical properties of the ﬁlms.

42

2.3 MATERIALS AND METHODS

2.3.1 Materials

Whey protein isolate (ALACEN 891) and whey protein concentrate (ALACEN
879) were obtained ﬁom New Zealand Milk Products (North America) Inc., (Santa Rosa,
CA). Table 2.1 shows the composition of the whey proteins used in the production of
edible ﬁlms in this study. Their protein contents were conﬁrmed using AOAC 930.29
Kjeldahl method (AOAC, 1990). Candelilla wax was purchased ﬁom Strahl and Pitsch
Inc. (West Babylon, NY), and unsalted butter ﬁt was obtained ﬁom Land O’Lakes Inc.
(Arden Hills, MN). D-Sorbitol was purchased ﬁom Sigma Chemical Co.(St. Louis, MO),

and NaOH was purchased from Mallinckrodt Specialty Chemical Co. (Paris, KY).

2.3.2 Film preparation

Figure 2.1 shows the schematic diagram of the ﬁlm-forming process. Edible ﬁlm-
forming solutions were prepared by mixing whey protein isolate (WPI) or whey protein
concentrate (WPC; 5%, w/v) with sorbitol, 2N NaOH (to adjust pH), and distilled H20 to
a ﬁnal volume of 100ml. The pH of the solutions were adjusted to 8. These solutions
were heated to a ﬁnal temperature of 90 i 2 °C for 15 minutes while being stirred
continuously (Model 4820-4 “Magna ” nmgnetic stirrer and hot plate, Cole-Parmer,
Chicago, IL). Butter ﬁt (BF; 0.1 or 0.2%, w/v) or candelilla wax (CW; 0.2, 0.4, or 0.8%,
w/v) was added during the heat treatment and allowed to melt into the protein solutions.
BF used in this study contained 15.8% of moisture (w/w), determined in drying oven

(Precision Scientiﬁc model 524, Chicago, IL) at 100 i 2°C for 3 h (AOAC, 1990). BF

43

Table 2.1. Composition of whey proteins used‘.

 

 

Components Whey protein isolate Whey protein concentrate
ALACEN 891 ALACEN 879
Protein (N x 6.38) % 90.0 80.0
Ash % 2.0 4.3
Moisture % 3.8 4.2
Fat % 0.2 5.2
Lactose % 4.5 5.2
sz 6.5 7.0

 

iValues based on speciﬁcations provided by New Zealand Milk Products (N. America) Inc.

2 5% at 20°C

44

Mix ﬁlm forming components

WPI or WPC, sorbitol and distilled H20
U

Adjust solution pH to 8 with 2N-NaOH
U

Heat treatment ( 90°C, 15 min, stirring )

Add lipids, BF or CW, during heat treatment
U
Homogenize (2 min)
U
Equilibrate (2 hours at 23 i 2°C)
U
Filter through cheese cloth
U
Vacuum for de-gassing
U
Cast solution onto teﬂon coated pans using pipette
U
Dry (23 :t 2°C, 30 :1: 5% RH, 18 :l: 3hr)
U
Peel ﬁlms from casting surface

U

Store samples at ambient condition before testing (23 i 2°C, 50 i 5 % RH)

Figure 2.1. Schematic diagram of ﬁlm-forming process.

45

was chosen due to its compatibility with whey powders; also it has been reported to be an
effective material in reducing WVP of protein-based ﬁlms (Shellhammer and Krochta,
1997). CW was selected after the preliminary test. It showed no waxy texture like
beeswax and provided better WVP than that ﬁlms containing carnauba wax.

Next the solutions were homogenized for 2 minutes using the Polytron PT 10/35
homogenizer with a PTA 20 TS generator (homogenizing head) and a PCU 11 power
control unit at setting 5 (Tekmar Co., Cincinnati, OH). The solutions were allowed to
equihhrate at 23 i 2°C for 2 h The mixtures were ﬁltered through two layers of
cheesecloth, and were de-gassed using a hydrometric vacuum system. Casting was
performed by pipetting the solutions on to teﬂon coated pans, 18.5 cm in diameter and
placed on a leveled surﬁce. Drying was carried out at 23 i 2°C and 30 :1: 5% RH for 18 i
3 h. Films were peeled from the casting surﬁce and stored at 23 i 2°C and 50 i 5% RH

until tested. Table 2.2 shows the composition of the ﬁlms produced.

2.3.3 Determination of thickness

A TMI model 549 M micrometer (Testing Machines, Inc. Amityville, NY) was
used to measure ﬁlm thickness. For determining barrier and mechanical properties,
measurements were taken at ﬁve different location and the mean value was used for
calculations. Barrier and mechanical testing were done on ﬁlm samples of 0.140 t 0.019

mm (5.5 mils).

46

Table 2.2. Composition of whey protein/lipid emulsion edrhle ﬁlms produced.

 

 

 

 

% w/v of aqueous solution % w/w of protein
Treatments ‘ Protein 2 Sorbitol Lipid Sorbitol Lipid
IS 5 5.0 0 100 0
IS-BF2% 5 4.9 BF = 0.1 98 BF = 2
IS-BF4% 5 4.8 BF = 0.2 96 BF = 4
IS-CW4% 5 4.8 CW = 0.2 96 CW = 4
IS-CW8% 5 4.6 CW = 0.4 92 CW = 8
IS-CW16% 5 4.2 CW = 0.8 84 CW = 16
CS 5 5.0 0 100 0
CS-BF2% 5 4.9 BF = 0.1 98 BF = 2
CS-BF4% 5 4.8 BF = 0.2 96 BF = 4
CS-CW4% 5 4.8 CW = 0.2 96 CW = 4
CS-CW8% 5 4.6 CW = 0.4 92 CW = 8
CS-CW16% 5 4.2 CW = 0.8 84 CW = 16

 

 

 

I I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; C = whey protein
concentrate.
2 whey protein isolate or whey protein concentrate

47

2.3.4 Water vapor permeability

Water vapor permeability (WVP) was tested using the Permatran-W3/3l (MoCon,
Inc., Minneapolis, MN), according to ASTM F-1249, “Standard test method for water
vapor transmission rate through plastic ﬁlm and sheeting using a modulated infrared
sensor” (ASTM, 1997). The testing surface area of the whey protein-based ﬁlm samples
were reduced ﬁom 50cm2 to 5cm2 with the use of a aluminum foil backing to stay within
sensor range.

Two different relative humidities (50 i 3 and 80 i- 3% RH) were used while
temperature was kept constant at 25 i 0.5 °C. Samples were conditioned for 5 h at the
RH condition which testing was conducted. Permeability was determined once a steady
state was reached. Steady state occurs when permeation of the gas or vapor is constant.
Calibration of the system was executed with 0.025mm (1mil) thick standard polyester
(PET) ﬁlm. The WVP wasreported ing-mm/mz-day-kPa, forallﬁhnsby converting the
water vapor transmission rate values (WVTR) obtained ﬁom the instrument (g/rn2 - day),
with the use of the appropriate conversion units. WVP was calculated by the following
equation:

WVP=WVTRxd/Ap (13)
WVTR = water vapor transmission rate of the test ﬁlm
d = thickness of ﬁhn

Ap = pressure differential acting on ﬁlm

48

2.3.5 Oxygen permeability

Oxygen permeability (OP) test was conducted according to ASTM D-3985,
“Standard test method for oxygen gas transmission rate through plastic ﬁlm and sheeting
using a coulometric sensor” (ASTM, 1997), using the Oxtran 200 (MoCon, Inc.,
Minneapolis, MN). Films were cut into 11 x 11cm square samples and masked with an
aluminum foil mask, making the area to be tested 5cm2, and placed in the 50cm2 cell of the
tester. The tester was equipped with an Endocal Temperature Control Bath model RTE
100 (Neslab Instrument Inc., Newington, USA.), and conformed with standard ASTM D-
3985 method.

The test was run at 23 i 0.5°C and 0% RH using nitrogen (containing 2%
hydrogen) as the carrier gas, and air (21% oxygen) as the test gas. All samples were
conditioned for 5 h under the same conditions prior to testing. The ﬂow was measured
until steady state was reached. Calibration of the system was executed with 0.025mm
(1mil) standard PET ﬁlm. The or was reported in em3 - um/mz-day-kPa for all ﬁlms by
converting the oxygen transmission rate values (OTR) obtained from the instrument
(cm3/m2 - day) with the use of the appropriate conversion units and the following equation:

P= (O'I'Rxd)/Ap (14)
d = thickness of ﬁlm

Ap = pressure differential acting on the ﬁlm

2.3.6 Mechanical properties
Films samples were cut into strips of 2.54cm wide using a Precision Sample Cutter
(Thawing Albert Instrument Co., Philadelphia, PA). All ﬁlms were conditioned for 48 h at

49

the same test conditions prior to testing. Tensile strength (TS) and percent elongation
(%E) was determined according to ASTM D882, “Standard test method for tensile
properties of thin plastic sheeting” (ASTM, 1997). Test were run using the Instron
Universal Testing Machine Model 2401 (Instron Corp., Canton, MA), at 23 i 2°C and 50
i 5% RH. A 1 kN static load cell and cross head speed of 20 in/min was used. Testing
sample size was 2.54cm x 8cm. The MPa value of TS was calculated from the following
equation:
TS = load / area (15)
load = peak force
area = sample width x sample thickness
%E was determined by the equation:
%E = AL / Li x 100 (expressed as a percentage) (16)
AL = change in length
L,- = original length of sample

2.3.7 Scanning Electron Microscopy

Microstructure of the ﬁhns were determined using the Scanning Electron
Microscope (SEM) JSM-6400V (Japan Electron Optics Laboratory, Tokyo, Japan;
Appendix III). Cross-sections and surﬁces of the ﬁlms were examined. A ﬁlm cross-
section was obtained by cross cutting using a razor blade. Both samples were mounted on
aluminum stubs with double-sided cellophane tape and carbon conductive paint. Each
sample was ion sputtered with gold-palladium alloy (gold deposit: 10 nm) for 3 minutes
with 20 mA current. Samples were observed with accelerating voltage of 15 kV. Two

50

diﬂ‘erent magniﬁcations of 400x and 1,500x were used to examine cross-sections and

surﬁces of the ﬁlms, respectively.

2.3.8 Statistical analysis

All experiments were replicated three times in a randomized block experiment. A
new ﬁlm forming solution and new set of ﬁlms were prepared for each replicate.
Statistical analysis were made using Sigma Stat 2.0 (Jandel Corp., San Raﬁel, CA) and

appropriate comparisons were done using the Student-Newman-Keuls method for multiple

comparisons.

2.4 RESULTS & DISCUSSION

Analysis of the whey protein isolate (WPI) and concentrate (WPC) powders
showed them to contain 90.8 and 81.8% protein, respectively. Films produced from WPI
and WPC with sorbitol as the plasticizer were clear, smooth, and ﬂexible. CW added ﬁlms
showed slight opaqueness in both WPI and WPC ﬁlms. Figure 2.2 shows representative
ﬁlms. CW could not be incorporated above 16% (dry basis, w/w of protein), because
higher wax contents produced ﬁlms that could not be peeled oﬁ‘ ﬁom the casting surface.
Above 2% (dry basis, w/w of protein) BF incorporated ﬁlms had a greasy surface.
Overall, increased lipid levels induced more translucent ﬁlms. The thickness of the ﬁlms
averaged of 140 :1: l9um. Whey protein type and incorporation of lipids did not affect ﬁlm

thickness.

51

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-833 £895 hon? 0335858 < .N.N wing

”82 no 9325 SE 339

 

52

2.4.1 Water vapor permeability

Figures 2.3 and 2.4 show WVP of BF- and CW—added whey protein-based ﬁlms
tested at 50 and 80% RH, respectively. Table 2.3 shows the statistical comparison of
WVP among treatments and protein types, WPI and WPC. Addition of lipids signiﬁcantly
(p < 0.05) lowered WVP of whey protein-based ﬁlms at both 50 and 80% RH (Figures
2.3, 2.4). At 50% RH, no signiﬁcant difference in WVP was observed between the ﬁlms
containing BF 2 and 4% in both WPI and WPC ﬁlms (Figures 2.3a, b). Overall, BF levels
did not affect WVP of whey protein-based ﬁlm. CW was much more effective in lowering
WVP of the ﬁlms. As the concentrations of CW were increased ﬁom 4 to 16% (w/w of
protein), there was a signiﬁcant decrease (p < 0.05) in WVP of both WPI and WPC ﬁlms
(Figures 2.4a, b). CW16%-added WPI ﬁlms had the lowest WVP at both RHs (Table
2.3). Overall, similar trends were observed with the WVP of the ﬁlms at 80% RH (Figure
2.4).

Reduction of WVP, by incorporation of CW into whey protein-based ﬁlms, was
expected since waxes (non-polar) are signiﬁcantly more resistant to moisture vapor
migration than most polar-lipids, such as fatty acids and acylglycerol type lipids (Cuq et
al., 1995a). Initially, it may seem unclear whether the reduction of WVP occurred due to
increased hydrophobicity of the ﬁlms or decreased plasticizer levels, since plasticizer
concentrations were subtracted from the total ratio of the ﬁlms to compensate for added
lipid amounts. In general, barrier properties of ﬁhns are negatively affected by plasticizers.
Since plasticizers reduce the rigidity of ﬁlm structure, diffusion of moisture vapor
molecules through ﬁlms become easier at higher plasticizer concentrations (Gennadios et

al., 1993c). However, Chick and Ustunol (1998) reported that the protein/plasticizer ratio

53

a)

b)

9
a:

 

A

 

9’
m on

 

 

N
on

7‘
UI

wwmmm/m‘daykra)
M

.3

.0
an

 

 

 

0

IS lS-BF2% lS-BF4’/o IS-CW4% lS-CW8% IS-CWI6%
Treatment

 

P
UIUI

 

&

 

.0
on

 

(A)

 

N
on

.3
din

WVP(gmm/m2daykPa)
N

.0
UI

 

 

 

0

cs CS-BF2% CS-BF4% CS-CW4% CS-CW8% CS-CW16%
Treatment

Figure 2.3. Water vapor permeability (WVP) of whey protein/lipid
emulsion edible ﬁlms (tested at 25°C and 50% RH).

(a) whey protein isolate ﬁlms; (b) whey protein concentrate ﬁlms

I = whey protein isolate; C = whey protein concentrate;

S = sorbitol; BF = butter fat; CW = candelilla wax

54

a)

b)

 

 

gas

 

 

WVP(gmm/m2daykPa)
3

 

 

 

IS IS-BF2% ls-BF4% IS-CW4% lS-CW8% lS-CW16%
Treatment

 

 

 

 

 

WVP(gmm/mzdaykPa)

 

 

 

cs CS-BF2% CS-BF4% cs-CW4% CS-CW8% CS-CW16%
Treatment

Figure 2.4. Water vapor permeability (WVP) of whey protein/lipid
emulsion edible ﬁlms (tested at 25°C and 80% RH).

(a) whey protein isolate ﬁlms; (b) whey protein concentrate ﬁlms

I = whey protein isolate; C = whey protein concentrate;

S = sorbitol; BF = butter fat; CW = candelilla wax

55

Table 2.3. Water vapor permeability (WVP) of whey protein/lipid emulsion edible ﬁlms

 

 

 

(at 25°C).
WVP (g. mm/ m2. d. kPa)

Treatment ' 50% RH 80% RH

IS 3.23 i 0.37“ 28.5 i 1.2“”
IS-BF2% 2.85 i 0.36” 25.7 :1; 1.6"b
IS-BF4% 2.64 :1; 0.23 “b 25.6 i 1.1 “’
IS-CW4% 2.39 i 0.42 b 24.1 :1: 2.5 "’
IS-CW8% 2.36 i 0.21 b 23.8 :t 2.1 "’
IS-CW16% 1.98 i 0.27 b 20.7 3; 1.4 "
cs 3.75:0.58' 31.1 i4.4'
CS-BF2% 2.69 a: 0.74 'b 28.4 i 0.7 "’
CS-BF4% 2.63 i 0.68 “b 25.4 i 1.1'”
CS-CW4% 2.50 :t 0.59“” 23.5 :1: 0.8 "’
CS-CW8% 2.19 :i: 0.35 b 24.4 :l: 0.3 “b
CS-CW16% 2.05 :l: 0.43 b 22.9 :1: 0.7“

 

 

 

"b Different letters columnwise denote signiﬁcant difference (p < 0.05), n=3 for all treatments.
1 l = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; C = whey protein

concentrate.

56

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Table 2.4. Water vapor permeability (WVP) of various protein-based edible ﬁlms and

 

 

 

 

 

 

synthetic polymers.
Fillns ' WVP Test conditions References
(g.mm/ (temp., RH)
m2.d.kPa)

WG : G (5:1) 8.3 30°C, 0/ 100% Gontard et al (1994)

WG : AM : G (5:1.5:1) 10.5 30°C, 0/100% Gontard et a1 (1994)

WG : SL : G (5:1.5:l) 9.0 30°C, 0/ 100% Gontard et al (1994)

WG : PW : G (5:1.5:l) 6.4 30°C, 0/100% Gontard et al (1994)

WG : CBW : G (5:1.5:1) 6.0 30°C, 0/100% Gontard et al (1994)

WG : BW : G (5:1.5:l) 3.0 30°C, 0/ 100% Gontard et al (1994)

SC (no plasticizer) 36.7 25°C, 0/31% Avena-Bustillos and Krochta
(1993)

SC : AM (4:1) 25.4 25°C, 0/88% Avena-Bustillos and Krochta
(1993)

SC : BW (4:1) 13,2 25°C, 0/88% Avena-Bustillos and Krochta
(1993)

LAC : G (1:1) 59.3 38°C, 0/90% Chick and Ustunol (1998)

LAC : G (0.6:1) 54.9 38°C, 0/90% Chick and Ustunol (1998)

LAC : 8 (1:1) 2.8 38°C, 0/50% Chick (1993)

LAC:CBW : S (1.1:0.l:1) 2.9 38°C, 0/50% Chick (1998)

LAC :CBW : S (l.4:0.4:l) 1.3 38°C, 0/50% Chick (1998)

LAC:CW : S (l.l:0.l :1) 1.7 38°C, 0/50% Chick (1998)

LAC:CW : S (l.4:0.4:l) 0.8 38°C, 0/50% Chick (1998)

LAC:CW : S (l.l:0.1 :1) 8.1 38°C, 0/70% Chick (1998)

LAC:CW : S (l .4:0.4:l) 6.2 38°C, 0/70% Chick (1998)

WPI : S (1.3:1) 52.0 25°C, 0/70% McHugh and Krochta (l994c)

WPI : MA : s (1 .3:1.8:1) 23.8 25°C, 0/70% McHugh and Krochta (1994c)

WPI : PA : s (1 .3:1.8:1) 19.2 25°C, 0/70% McHugh and Krochta (1994c)

WPI : G (15:1) 45.4 25°C, 0/88% Shellhammer and Krochta
(1997)

WPI : CW : G (15:1 1:1) 30.9 25°C, 0/88% Shellhammer and Krochta
(1997)

WPI : MF : G (15:11:1) 21.9 25°C, 0/88% Shellhammer and Krochta
(1997)

WPI : BW : G (15:11:1) 10.8 25°C, 0/88% Shellhammer and Krochta
(1997)

Low density polyethylene 0.08 38°C, 90/O% Smith (1986)

High density polyethylene 0.02 38°C, 90/0% Smith (1936)

Cellophane 7.27 38°C, 90/0% Taylor (1936)

 

' WG = wheat gluten; SC = sodium caseinate; LAC = lactic acid casein; WPI = whey protein
isolate; G = glycerol; S = sorbitol; AM = acetylated monoacylglycerol; BW = beeswax; SL = soya
lecithin; PW = paraﬁn wax; CBW = carnauba wax; CW = candelilla wax; MA = myristic acid;
PA = palmitic acid; MF = milkfat.

57

had no effect (p > 0.05) on WVP of lactic acid casein-based ﬁlms (Table 2.4).
Apparently, a protein:plasticizer ratio change ﬁ'om 1:1 to 0.6:] was not signiﬁcant enough
to aﬁ‘ect WVP of the ﬁlms. In the present study, the change in the proteinzplasticizer ratio
was less than that reported by Chick and Ustunol (1998), and also there were diﬁ‘erences
in WVP of BF4%- and CW4%-added ﬁlms with the same amount of plasticizer (Tables
2.2, 2.3). This leads me to believe that the decrease in WVP is due to the lipids
incorporated rather than due to the reduction in the amount of plasticizer in the ﬁlms.

The protein type, WPI and WPC, did not affect WVP of whey protein-based ﬁlms
(Table 2.3). With an increase in RH from 50 to 80%, the WVP of all treatments increased
~10 times. Similar results were observed by Chick (1998). Reportedly, RH aﬁ‘ected
WVP of CW-added lactic casein acid-based ﬁlms exponentially. According to Gennadios
et al. (1993c), protein-based ﬁlms are prone to swell in high RH environments because of
high water sorption rates. This leads to a diffusivity increase of water molecules and
results in increased WVP of protein-based ﬁlms at high RH.

The WVP results of this study were comparable to those of most protein/lipid
emulsion edible ﬁlms (Table 2.4). In particular, when comparing WVP data in this present
study to the results ﬁom the study by Shellhammer and Krochta (1997), the WVP data
from this study showed a greater effectiveness of lipid addition on lowering WVP of whey
protein-based ﬁlms. They reported WVP of 30.9 and 21.9 g.mm/m2.day.kPa for CW and
milk fat-added WPI ﬁlms, respectively (Table 2.4). Approximately 70% (w/w of protein)
lipid were incorporated in the ﬁlms to obtain their results, while only 4% BF and 16% CW
(w/w of protein) were required to achieve similar WVP values in our study, 24.1 and 20.7

g.mm/m2.day.kPa, respectively (Table 2.3). However, caution needs to be taken in

58

comparing data obtained ﬁom different studies by different investigators, since the
material resources, ﬁlm preparation, procedures, equipment and testing conditions were

slightly different in each of the studies.

2.4.2 Oxygen permeability

Figure 2.5 shows OP of whey protein/lipid emulsion ﬁlms. A statistical
comparison of OP between WPI and WPC ﬁlms is shown in Table 2.5. A signiﬁcant
decrease (p < 0.05) was observed in OP with the addition of lipids, BF and CW, to WPI
and WPC ﬁlms (Figures 2.5a, b). All treatments with lipids showed lower OP compared
to those with no lipid added. Similar results were observed by Gennadios et al. (19930).
Incorporation of a lipid (acetylated monoacylglycerol) decreased OP of wheat gluten-
based ﬁlms ﬁ'om 3.9 to 2.7 cm3 - urn/m2 - d - kPa (Table 2.6).

In comparing effect of lipid types (BF and CW) on OP of WPI ﬁlms, CW was
more eﬂ'ective (p < 0.05) than BF in reducing OP of the ﬁlms (Figure 2.5a). This
comparison was made within the same lipid level of 4% (w/w of protein). A similar trend
was observed in WPC ﬁlms (Figure 2.5b). In general, waxes show good barrier properties
since waxes present a tight orthorhombic crystalline arrangement that is perpendicular to
the direction of the gas ﬂow (Donhowe and Fennema, 1993). According to Callegarin et
al. (1997), in addition to the nature of ﬁlm-forming substances, surface morphology and
homogeneity of the ﬁlm matrix are also important factors that aﬂ‘ect barrier properties of
ﬁlms. Detailed discussion with regards to surface morphology and microstructure will be

provided in section 2.4.5 including Scanning Electron Micrographs.

59

a)

b)

0P (cm3 “In / m2 day kPa)

 

IS lS-BF2% IS-BF4% IS-CW4% IS-CW8% IS-CW16%
Treatment

 

 

 

 

 

 

 

OP (cm3 pm / rn2 day kPa)

 

 

 

CS CS-BF2% CS-BF4% CS-CW4% cscws% CS-CW16%
Treatment

Figure 2.5. Oxygen permeability (OP) of whey protein/lipid
emulsion edible ﬁlms (tested at 23°C and 0% RH).

(a) whey protein isolate ﬁlms; (b) whey protein concentrate ﬁlms
I = whey protein isolate; C = whey protein concentrate;

S = sorbitol; BF = butter ﬁat; CW = candelilla wax

60

 

 

Table 2.5. Oxygen permeability (OP) of whey protein/lipid emulsion edible ﬁlms

 

 

(23°C, 0% RH).
Treatment1 OP (cm’. um/ m2. d. kPa)
IS 3.74 1 0.45 °
IS-BF2% 1.59 :1: 0.12 “
IS-BF4% 1.95 :1: 0.25 "
IS-CW4% 1.02 i 0.42 °
IS-CW8% 1.23 3. 0.28 °
IS-CW16% 1.55 :1: 0.12 “
cs 3.67 i 0.29:
CS-BF2% 1.55 :l: 0.20be
CS-BF4% 1.93 :1: 0.21 "
CS-CW4% 1.22 i 0.22 “
CS-CW8% 1.52 i 0.10be
CS-CW16% 1.49 i 0.15 b°

 

 

"Different letters columnwise denote signiﬁcant difference (p < 0.05), n=3 for all treatments.
l I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; C = whey protein
concentrate.

61

Table 2.6. Oxygen permeability (OP) of various protein-based edible ﬁlms and synthetic

 

 

 

polymers.
FilmsT OP Test References
(cm’pm/ conditions
m2.d.kPa) (temp, RH)
WG : G (2.5:1) 3.9 23°C, 0% Gennadios et al. (1993c)
WG : AM : G (2.5:0.2:l) 2.7 23°C, 0% Gennadios et al. (1993c)
LAC : s (1 :1) 0.4 23°C, 0% Chick (1998)
LAC: CBW : S (l.l:0.l:l) 0.3 23°C, 0% Chick (1998)
LAC: CBW : S (l.4:0.4:l) 0.5 23°C, 0% Chick (1998)
LAC: CW : S (l.l:0.l:l) 0.3 23°C, 0% Chick (1998)
LAC: CW : S (l.4:0.4:l) 0.6 23°C, 0% Chick (1998)
WPI : S (3.5:1) 1,5 23°C, 46% McHugh (1996)
WPI : BW : S (3.5:1.8:l) 8.6 23°C, 46% McHugh (1996)
WPI : s (3.5:1) 42.3 23°C, 70% McHugh (I996)
WPI : 13w : s 05:18:” 101.1 23°C, 70% McHugh (1996)
Low density polyethylene 1865 23°C, 50% Salame (1986)
High density polyethylene 427 23°C, 50% 531311160936)
Cellophane 0.7 23°C, 0% Salame (1986)

 

 

 

 

' WG = wheat gluten; LAC = lactic acid casein; WPI = whey protein isolate; G = glycerol; 8 =
sorbitol; AM = acetylated monoacylglycerol; CBW = carnauba wax; CW = candelilla wax; BW =

beeswax.

62

Whey protein type, WPI and WPC, did not aﬂ‘ect OP of the ﬁlms (Table 2.5). The
CW4%-added WPI ﬁlm had the lowest 01> of 1.02 cm3 - urn/m2 - d - kPa. This was
approximately a 70% reduction compared to OP of the WPI ﬁlm with no lipid added, 3.74
cm3 - urn/m2 -d- kPa. Further addition of CW (8 and 16%, w/w of protein), however,
increased OP. The oxygen barrier properties of whey protein/lipid emulsion ﬁlms in this
study were comparable to those of acetylated monoacylglycerol-added wheat gluten-based
ﬁlms, carnauba wax-added lactic acid casein-based ﬁlms, and cellophane (Table 2.6).
However, it is important that the OP of ﬁlms are compared between ﬁlms tested under the
same testing conditions. RH conditions in particular have an exponential eﬂ‘ect on OP of
the protein-based ﬁlms (Mujica-Paz and Gontard, 1997).

Whey protein-based ﬁlms make excellent oxygen barriers because of their highly
polar nature (Chen, 1995). Highly polar polymers like proteins exhibit high degrees of
hydrogen bonding, which creates limited polymer chain motion, resulting in low gas
permeability (McHugh and Krochta, 1994a). Thus, OP of protein-based ﬁlms is often
lower than that of common synthetic ﬁlms such as LDPE and HDPE. For example, OP of
WPI-based ﬁlm was 500 times lower than that of LDPE, 3.74 compared to 1865

cm3 - tun/m2 - d - kPa (Tables 2.5, 2.6).

2.4.3 Tensile strength and percent elongation

Figures 2.6 and 2.7 show TS and %E of whey protein/lipid emulsion ﬁlms. Table
2.7 shows the statistical comparison of TS and %E among protein types. With WPI ﬁlms,
addition of BF had no signiﬁcant eﬂ‘ect on TS and %E of the ﬁlms. On the other hand,
increasing CW levels increased TS of WPI ﬁlms while a decline in %E was observed (p <

63

a)

b)

 

 

 

 

 

TS (MPa)

O-tNU-hUIONOO

 

 

 

IS lS-BF2% lS-BF4% IS-CW4% Is-CW8% lS-CW16%
Treatment

 

 

 

 

 

TS (MPa)

 

 

 

CS CS-BF2% CS-BF4% CS-CW4% CS-CW8% CS-CW16%
Treatment

Figure 2.6. Tensile strength (TS) of whey protein/lipid
emulsion edible ﬁlms (tested at 23°C and 50% RH).

(a) whey protein isolate ﬁlms; (b) whey protein concentrate ﬁlms
I = whey protein isolate; C = whey protein concentrate;

S = sorbitol; BF = butter fat; CW = candelilla wax

64

a) 40

 

 

 

 

%E

 

 

 

IS IS-BF2% IS-BF4% ISCW4% IS-CW8% IS—CW16%
reatmeut

b)

 

 

 

 

%E

 

 

 

CS CS-BF2% CS-BF4% cs-CW4% CS-CW8% CS-CW16%
Treatment

Figure 2.7. Percent elongation (%E) of whey protein/lipid
emulsion edible ﬁhns (tested at 23°C and 50% RH).

(a) whey protein isolate ﬁlms; (b) whey protein concentrate ﬁlms
I = whey protein isolate; C = whey protein concentrate;

S = sorbitol; BF = butter fat; CW = candelilla wax

65

Table 2.7. Mechanical properties of whey protein/lipid emulsion edible ﬁlms

 

 

(23°C, 50% RH).

Treatment r Tensile strength (MPa) Percent elongation (%)
IS 3.92 3 0.45 d‘ 24.2 3 2.8 "
IS-BF2% 4.01 3 0.64dc 24.6 3 3.1 ”
IS-BF4% 3.69 3 0.66“ 24.9 3 6.1 "
IS-CW4% 5.51 3 1.11be 23.3 3 3.2b
IS-CW8% 6.46 3 1.10 b 15.6 3 34°
IS-CW16% 7.89 3 1.01 ‘ 13.7 3 2.6c
cs 3.88 3 0.43 d‘ 33.1 3 3.1'
CS-BF2% 3.96 3 0.39 d: 35.3 3 2.3 '
CS-BF4% 2.80 3: 0.88 f 35.6 3 3.2 '
CS-CW4% 4.56 3 0.59““ 26.5 3 3.2 "
CS-CW8% 4.79 3 0.60cd 25.6 3 1.3 "
CS-CW16% 5.09 3 0.20c 26.3 3 2.0 "

 

 

 

a{Different letters columnwise denote signiﬁcant difference (p < 0.05), n=3 for all treatments.
I I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; C = whey protein
concentrate.

66

 

Table 2.8. Mechanical properties of various protein-based edible ﬁlms and synthetic

 

 

 

 

 

 

polymers.
Films ‘ Tensile Percent References

strength elongation

(MP8) (70)

CZ : PA (1:0.5) 5.2 0.5 Lai et al. (1997)
CZ : PA (1:0.75) 14.6 1.0 Lai et al. (1997)
CZ : PA(1:1) 11.6 0.8 Laietal. (1997)
CZ : OA (1:0.5) 9.4 5.9 Santosa and Padua (1999)
CZ : OA (1:0.75) 4.2 46.9 Santosa and Padua (1999)
CZ : OA (1:1) 2.2 7.5 Santosa and Padua (1999)
SP1 : G (2:1) 6.3 112 Rhim et al. (1999a)
SP1 : LA : G (220.6:1) 4.2 35.0 Rhim et al. (1999a)
SPI : PA : G (220.6:1) 4.8 20.0 Rhim et al. (1999a)
LAC : S (1:1) 6.2 156.0 Chick (1998)
LAC : CW : S (l.4:0.4:l) 8.3 37 Chick (1998)
WP] : G (2:1) 5.9 22.7 Banerjee and Chen (1995)
WPC : G (2:1) 3.4 20.8 Banerjee and Chen (1995)
WPI : AM : G (2:1 :1) 3.2 10.8 Banerjee and Chen (1995)
WPC :AM:G(2:1:1) 1.1 13.6 Banerjeeand Chen (1995)
Low density polyethylene 8.6 - 17 500 Briston (1988)
High density polyethylene l7 - 35 300 Briston (1988)
Cellophane 48 - 110 15 - 25 Briston (1988)

 

lCZ = corn zein; SPI = soy protein isolate; LAC = lactic acid casein; WPI = whey protein isolate;
WPC = whey protein concentrate; G = glycerol; S = sorbitol; PA = palmitic acid; OA = oleic acid;
LA = lauric aicd; CW = candelilla wax; AM = acetylated monoacylglycerol.

67

0.05; Figures 2.6a, 2.7a). Similar trends were observed with TS and %E of WPC ﬁlms,
except that the addition of BF decreased TS of WPC ﬁlms (Figures 2.6b, 2.7b). The
effectiveness of CW on TS and %E of lactic acid casein-based ﬁlms was studied by Chick
(1998). Similar to the results from my study, addition of CW signiﬁcantly increased TS
and decreased %E of lactic acid casein-based ﬁlms. Reportedly, CW induced rigidity of
whey protein/lipid emulsion ﬁlms, which resulted in increased TS and decreased %E upon
raised CW levels.

Unlike CW-added ﬁlm, BF -added ﬁlms exhibited somewhat different
characteristics in terms of mhanical properties. Increased BF level did not increase TS
values, while %E values remained the same compared to ﬁlms without lipids
incorporated. According to Callegarin et al. (1997), acylglycerol type lipids may increase
ﬂexibility of the ﬁlms by weakening the intermolecular forces between adjacent polymer
chains. BF contains 97-98% triacylglycerols (Sax and Lewis, 1987). Banerjee and Chen
(1995) reported addition of acetylated monoacylglycerols reduced TS of whey protein-
based ﬁlms (Table 2.8).

In Table 2.7, WPI ﬁlms showed higher (p < 0.05) TS values while WPC ﬁlms had
higher (p < 0.05) %E at all treatments. The highest TS of 7.89 MPa was observed with
CW16%-added WPI ﬁlms, and BF4%-added WPC ﬁhns had the highest %E of 35.6%.
Banerjee and Chen (1995) also reported WPI ﬁlms to have higher TS than WPC ﬁlms,
5.94 and 3.36 MPa, respectively. They suggested a lower protein concentration was
responsible for the weaker ﬁlms with WPC. The TS and %E values of my ﬁlms were

comparable to those of most protein/lipid emulsion edible ﬁlms. The TS and %E values

68

were also comparable to those of low density polyethylene ﬁlm and cellophane,

respectively (Table 2.8).

2.4.5 Microstructure

SEM micrographs of whey protein/lipid emulsion ﬁlms are shown in Figures 2.8,
2.9 and 2.10. Magniﬁcation of 400x was used for cross-sections of the ﬁlms (Figure 2.8).
The top surface (open to the environment while drying) of the ﬁlms were magniﬁed to
1,500X (Figures 2.9, 2.10), and these SEM micrographs were taken at a tilted angle (45°)
to show a cross-sectional top surface of the ﬁlms. SEM micrographs of the bottom
surfaces, in contact with the casting surface while drying, of ﬁlms were not shown here
since no differences were observed among treatments.

In Figure 2.8, cross-sections of both WPI and WPC ﬁlms are shown with no lipids
(Figures 2.8a, d), with 4% BF (Figures 2.8b, e), and with 4% CW (Figures 2.8c, t). In
overall comparison, WPC ﬁlms showed a coarse protein matrix while WPI ﬁlms appeared
to be more compact and less coarse. This cross-sectional analysis showed that WPI ﬁlms
were more continuous and homogeneous (Figures 2.8a-c) than WPC ﬁlms (Figures 2.8d-
f). Diﬁ‘erences in composition between WPC compared to that of WPI probably account
for the loosely packed open ﬁlm matrix as apparent in the microstructure, thus the
previously reported lower TS of these ﬁlms (Tables 2.1, 2.7).

Figures 2.9 and 2.10 shows top surfaces, lipid-oriented sides, of WPI and WPC
ﬁlms, respectively. In Figures 2.9a and 2.10a, top surfaces of WPI and WPC ﬁlms
without lipids (BF and CW) appeared to be smooth. Addition of lipids to both WPI and

WPC ﬁlms induced irregular morphology on the top surfaces (Figures 2.9b-e, 2.10b-e).

69

 

Figure 2.8. Scanning electron micrographs of cross-sections of whey protein/lipid
emulsion ﬁlms. Magniﬁcation 400x.

(a) IS; (b) IS-BF4%; (c) IS-CW4%; (d) CS; (e) CS-BF4%; (t) CS-CW4%.

I = whey protein isolate; C = whey protein concentrate; S = sorbitol;

BF = butter fat; CW = candelilla wax

70

;-_1.'l.. F 7‘

. :3 ."1 131 3 ‘3 m m

 

Figure 2.9. Scanning electron micrographs of top surﬁices of whey protein isolate/lipid
emulsion ﬁlms. Magniﬁcation 1,500X.

(a) IS; (b) IS-BF2%; (c) IS-BF4%; (d) IS-CW4%; (e) IS-CW16%.

I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax

71

 

Figure 2.10. Scanning electron micrographs of top surfaces of whey protein
concentrate/lipid emulsion ﬁlms. Magniﬁcation 1,500X.

(a) CS; (b) CS-BF2%; (c) CS-BF4%; (d) CS-CW%4; (e) CS-CW16%.

C = whey protein concentrate; S = sorbitol; BF = butter ﬁat; CW = candelilla wax

72

Top surface micrographs of ﬁlms containing CW showed the appearance of wax droplets
dispersed throughout the ﬁlm, giving the ﬁlms a hilly appearance, however, the structure
appeared fairly compact (Figures 2.9d-e, 2.10d-e). No signiﬁcant morphological
diﬁ'erences could be seen among the top surface of ﬁlms containing diﬁ'erent levels of CW.
On the other hand, increased BF contents can be observed by a BF layer distinctive ﬁ'om
the top surfaces of the ﬁhns (Figures 2.9b-c, 2.10b-c). As BF concentration increased in
both WPI and WPC ﬁlms, a thicker layer of BF and bigger void spaces were introduced
on the top surfaces of the ﬁlms (Figures 2.9c, 2.10c).

In particular, BF4%-added WPC ﬁlm was coarse looking in the protein matrix and
had open structures in lipid layer (Figures 2.8c, 2.10c). Earlier I reported this ﬁlm to have
the lowest TS (Table 2.7). This porous structure may also explain the high OP value
obtained with these ﬁlms compared to CW containing ﬁlms (Figure 2.5). Apparently,
oxygen molecules can more readily permeate through the ﬁlm matrix when some holes and
pores are formed on the surface (Figures 2.9c, 2.10c).

This present study showed that the lipids, CW and BF, oriented to the top side of
dried ﬁlms, which were exposed to the environment during drying. Greener and F ennema
(1989a) found a similar morphology in methylcellulose/beeswax ﬁlms. Beeswax was
formed on the top surface of the ﬁhns. Avena-Bustillos and Krochta (1993) observed the
same tendency in the casein/lipid emulsion ﬁlms. This phase separation probably occurred
in whey protein/lipid emulsion ﬁlms due to density differences between lipids and whey
proteins, and responsible for the appearance of the dull and shiny sides of lipid added ﬁlms
(Figure 2.2). Moderate emulsifying ability of whey proteins may be responsible for this

emulsion instability (McHugh and Krochta, 1994c; 1994c).

73

In summarizing the results of this chapter, addition of lipids, BF and CW,
enhanced moisture barrier properties of the whey protein-based edible ﬁlms. The most
desirable WVP was observed with CW16%-added WPI ﬁlms. BF and CW addition also
improved oxygen barrier properties of the ﬁlms. Whey protein type (WPI and WPC) did
not affect barrier properties (WVP or OP) of the ﬁlms. Protein, and lipid type and
concentration inﬂuenced mechanical properties of the ﬁlms. Overall, higher TS was
observed with CW-added WPI ﬁlms while BF -added WPC ﬁlms showed higher %E.
SEM micrographs illustrated relationship between the ﬁlm’s microstructure, and barrier

and mechanical properties.

74

CHAPTER 3

FORCES INVOLVED IN THE FORMATION AND STABILITY OF

WHEY PROTEIN/ LIPID EMULSION EDIBLE FILMS.

3.1 ABSTRACT

Whey protein isolate (WPI; 5%, w/v) or concentrate (WPC; 5%, w/v) ﬁlms were
plasticized with sorbitol (4.2-5%, w/v), and butter fat (BF; 0.2%, w/v) or candelilla wax
(CW; 0.8%, w/v) was added into the ﬁlm-forming solutions. Forces involved in formation
and stability of the ﬁlms were studied by determining their solubilities in 2-
mercaptoethanol (2-ME; a disulﬁde bond-dissociating agent), urea (a hydrogen bond-
dissociating agent and also a weak hydrophobic bond-dissociating agent) and sodium
dodecyl sulfate (SDS; a hydrophobic bond-dissociating agent). Free sulfhydryl groups and
disulﬁde bonds concentrations of the ﬁlms were determined using Ellman’s reagent 5,5'-
dithiobis (2-nitrobenzoic acid) and disodium 2-nitro-5-thiosulfobenzoate, respectively.
Additionally, hydrophobicity of the ﬁlms was investigated using an extrinsic ﬂuorescence
probe 1-anilino-8-naphthalene sulfonic acid. Disulﬁde and hydrogen bonds, cooperatively,
were the primary forces involved in the formation of whey protein/lipid emulsion ﬁlms.
Contribution of hydrophobic interactions was secondary. Sulﬂiydryl contents of the ﬁlms
ranged ﬁ'om 2.5 to 3.0, and 1.39 to 1.44 1.1ng of ﬁlm for WPI and WPC ﬁlms,
respectively. Disulﬁde contents were not signiﬁcantly different among treatments and

ranged from 1.8 to 3.6 umol/g of ﬁlm. Hydrophobicity of the ﬁlms increased with

75

incorporation of lipids. Although CW containing ﬁlms were more hydrophobic than BF

containing ﬁlms, these differences were not statistically signiﬁcant.

3.2 INTRODUCTION

Proteins such as wheat gluten, soy, corn zein, and whey proteins have been studied
as ﬁlm-forming materials in the production of protein-based edible ﬁlms (Gennadios et al.,
1994). For effective formation of ﬁlms, extensive protein-protein interactions are
necessary to form a continuous ﬁlm network with suﬁicient mechanical strength. Protein-
based ﬁlm formation is thought to occur through protein polymerization and solvent
evaporation at the surface of the aqueous dispersion (Gennadios and Weller, 1991). For
instance, wheat proteins are capable of forming wheat gluten colloidal complex upon
hydration in alkaline solutions. Intramolecular and intermolecular disulﬁde bonds in the
gluten complex are cleaved and reduced to free sulﬁrydryl groups. Reformation of
disulﬁde bonds occurs upon casting and drying to produce the ﬁhn structure. Hydrogen
bonds and hydrophobic interactions are also thought to contribute to ﬁlm matrix
(Gennadios et al., 1994). Similar to wheat protein-based ﬁlms, soy protein-based ﬁlms
result due to the polymerization of heat denatured proteins through disulﬁde bonds.
Hydrogen bonds and hydrophobic interactions are also important in maintaining the soy
protein-based ﬁlm’s stability (Farnum et al., 1976). Zein, on the other hand, produces ﬁlm
structure primarily through hydrogen bonding and hydrophobic interactions. The role of
disulﬁde bonds in the formation of the zein-based ﬁlms is minimal due to its low cysteine

content (Reiners et al., 1973).

76

Two primary proteins that make up whey proteins are B-lactoglobulin and 01-
lactoalbumin. Secondary and tertiary structures of these proteins are facilitated by
hydrogen bonds. Intramolecular disulﬁde bonds also contribute to their stability (Brunner,
1981; Shimada and Cheﬁel, 1989). As the temperature of the solution is increased as in
the case of ﬁlm-forming process, polymerization of whey protein molecules occurs after
the conformational change of their tertiary structures. The heat denaturation of whey
proteins induces exposure of internal ﬁee sulﬂrydryl groups, promoting intermolecular
disulﬁde bond formation. Free sulﬂrydryl groups also can be involved in interchange
reactions with existing disulﬁde bonds upon heating and regenerate free sulﬁlydryl groups.
These regenerated ﬁ'ee sulﬂrydryl then react again in free sulfhydryl/disulﬁde bond
interchange reactions. The heat treatment also results in exposed hydrophobic groups at
the molecular surface (Shimada and Cheﬁel, 1989; Damodaran, 1996). This process may
result in the formation of a protein network, which possesses suﬁcient mechanical
strength to produce ﬁ'eestanding ﬁlms upon drying (Cheﬁel et al., 1985; Gennadios et al.,
1994)

Mahmoud and Savello (1993) studied forces involved in formation and stability of
transglutaminase cross-linked whey protein-based ﬁlms. Transglutaminase catalyzes the
covalent binding of lysine with a glutamic acid residue, and generates intramolecular and
intermolecular s-(y—glutaminyl)lysine cross-links. The transglutaminase cross-linked ﬁlms
produced by Mahmoud and Savello (1993) were insoluble in 2-mercaptoethanol (a
disulﬁde bond-dissociating agent) and sodium dodecyl sulfate (a hydrophobic bond-
dissociating agent), but were soluble in urea and guanidine hydrochloride (a hydrogen

bond-dissociating agent and also a weak hydrophobic bond-dissociating agent). This led

77

them to believe that in addition to the covalent bonds formed by cross-linking, hydrogen
bonding also contributed to the stability of these ﬁhns. Limited information is available on
forces inﬂuencing the structure of heat catalyzed whey protein-based ﬁlms. Thus, the
intent of this research was to gain an understanding of the molecular forces involved in the
formation and stability of heat catalyzed whey protein-based and lipid emulsion ﬁlms so

that their properties can be improved or altered by enhancing or altering these interactions.

3.3 MATERIALS AND METHODS

3.3.1 Materials

Ellman’s reagent [5,5'-dithiobis (2-nitrobenzoic acid) (DTNB)], l-anilino-8-
naphthalene sulfonic acid (ANS), 2-mercaptoethanol (2-ME), ethylenediaminetetraacetic
acid (EDTA), and Tris-HCl were purchased ﬁ'om Sigma Chemical Co. (St. Louis, MO).
Urea, trichloroacetic acid (TCA), and sodium sulﬁte were purchased from J .T. Baker Co.
(Phillipsburg, NJ). Sodium dodecyl sulfate (SDS) was obtained from Pierce Co.

(Rockford, IL).

3.3.2 Film preparation

Composition of the ﬁlms prepared to study the effect of bond-dissociating agents,
free sulﬂlydryl groups and disulﬁde contents, and hydrophobicity are shown in Table 3.1.
The ﬁlm preparation was carried out as described in section 2.3.2 of chapter 2 (Figure

2.1).

78

Table 3.1. Composition of whey protein/lipid emulsion edible ﬁlms produced.

 

 

 

 

% w/v of aqueous solution % w/w of protein
Treatments ' Protein 2 Sorbitol Lipid Sorbitol Lipid
IS 5 5.0 0 100 0
IS-BF4% 5 4.8 BF = 0.2 96 BF = 4
IS-CW16% 5 4.2 CW = 0.8 84 CW = 16
CS 5 5.0 O 100 0
CS-BF4% 5 4.8 BF = 0.2 96 BF = 4
CS-CW16% 5 4.2 CW = 0.8 84 CW = 16

 

 

 

I I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; C = whey protein
concentrate.
2 whey protein isolate or whey protein concentrate

79

3.3.3 Solubility in bond-dissociating agents

The effect of bond-dissociating agents on each ﬁlms solubility and stability was
determined according to the method of Mahmoud and Savello (1993) with slight
modiﬁcations. 2-ME has a ﬁ'ee sulﬂrydryl group which can dissociate disulﬁde bonds
(Stenesh, 1989). Urea breaks hydrogen bonds of proteins and interacts with unfolded
proteins through hydrogen bonding altering the physical properties of water. Urea also
can weaken hydrophobic bonds by altering the structures of water (Creighton, 1993;
Lefebvre-cases et al., 1998). SDS is a hydrophobic bond-dissociating agent. SDS, an
ionic detergent, has both hydrophobic and hydrophilic portions in its molecular structure,
and can break hydrophobic interaction (Lapanje, 1978; Lefebvre-cases et al., 1998).

Film (30mg) was placed in 6ml of protein bond-dissociating agents: 0.1M 2-ME,
0.5M SDS and 6.6M urea. The samples were incubated at room temperature for 24h, then
centrifuged at 9,000 x g for 20min (Biofuge 22R, Heraeus Instruments, Hanau, Germany).
The total absorbance of the supernatant (2ml) was measured at 280nm (Spectronic 1001+,
Milton Roy Co., Rochester, NY). Proteins exhlbit strong absorption at UV 280 nm due to
tryptophan and tyrosine residues (Chang, 1994). The supernatant (2ml) was then mixed
with 6% TCA (2ml), allowed to react for 30 min and centrifuged at 9,000 x g for 20min.
The A230 of the supernatant represented non-protein absorbance. Soluble protein
absorbance was calculated as following:

Soluble protein absorbance = total absorbance - non protein absorbance (17)

80

3.3.4 Determination of free sulﬂlydryl group and disulﬁde bond contents

Free sulﬂrydryl groups and disulﬁde bonds in whey protein/lipid emulsion ﬁlms
were determined according to Rangavajhyala et al. (1997) with slight modiﬁcations. This
procedure was developed based upon methods of Thannhauser et al. (1987) and Chan and
Wasserman (1993). Ellman’s reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and
disodium 2-nitro-5-thiosulfobenzoate (NT 8B2) were used for the determination of free
sulﬂrydryl group and disulﬁde bond contents, respectively. Reaction with these color
reagents yield soluble 2-nitro-5-thiobenzoate anion (N TBZ').

Since NTSBZ' was not commercially available, DTNB was used to synthesize
NTSBZ' according to Thannhauser et al. (1987). DTNB (0.2g) was dissolved in 1M
sodium sulﬁte (20ml) at 38°C, and pH was adjusted to 7.5. While keeping this solution at
38°C, oxygen was bubbled into the solution until the bright red solution turned to a pale
yellow (about 45 min). This stock solution was then used to prepare NTSB assay
solution.

To assay free sulﬂ'lydryl group content in the ﬁlms, a 10 mg of ﬁlm was added into
1 ml of reaction buffer containing 8 M urea, 10 mM DTNB, 3 mM EDTA, 1% SDS, and
0.2 M Tris-HCl, pH 8.0. The reaction mixture was held at room temperature (~ 23°C) for
15 min. To remove particulate matter, the mixture was centrifuged at 13,600 x g for
10min. A 02an aliquot of supernatant was removed and diluted with 1.8 n11 of 8M urea,
3mM EDTA, 1% SDS, and 0.2M Tris-HCl, pH 8.0. Then its absorbance was measured at
412 nm (Spectronic 1001+, Milton Roy Co.). Sulﬂrydryl content was calculated by using

the extinction coefﬁcient ofNTB (13,600 M‘om") at 412nm.

81

To assay disulﬁde bond content in the ﬁlms, a 10 mg of ﬁlm was added into 1 ml
of reaction buffer containing 8 M urea, 10 mM NTSBZ', 0.1 M sodium sulﬁte, 3 mM
EDTA, 1 % SDS, and 0.2 M Tris-HCl, pH 9.5. The reaction mixture was held in the dark
chamber for 25 min at room temperature (~ 23°C), then centrifuged at 13,600 x g. A
0.2ml aliquot of supernatant was removed and diluted with 1.8m] of 8M urea, 0.1 M
sodium sulﬁte, 3mM EDTA, 1% SDS, and 0.2 M Tris-HCI, pH 9.5. Its absorbance was
measured at 412nm, and referred to as total sulihydryl group content. Disulﬁde bond
content was determined as following:

Disulﬁde bond =

1/2 x ( I total sulﬂlydryl group content - free sulﬂlydryl group content I) (18)

3.3.5 Determination of hydrophobicity

Hydrophobicity of the ﬁlms was determined using an extrinsic ﬂuorescence probe
l-anilino-8-naphthalene sulfonic acid (ANS) according to Lakkis and Villota (1992).
Standard plot of hydrophobicity was established by measuring hydrophobicity of ten
different concentrations (0.05mg/ml to 0.5mg/ml of ﬁlms in phosphate buﬁ‘er, pH 7). To
obtain ﬁlm-dispersed solutions, 500mg of ﬁlm pieces were dissolved into 10ml of ultra
puriﬁed H20 with sodium azide (0.02%, to prevent microbial growth) for 24 hr at room
temperature (~ 23°C).

To assay intensity of the ﬁlm hydrophobicity, different concentrations of ﬁlm
dispersions and ANS (101.11, 8mM in 0.1 M phosphate buffer) were added to a cuvette.
Phosphate buffer, pH 7.0, was added to bring the total volume to 2m]. Relative

ﬂuorescence intensity (RFI) of samples was measured using a Model 4800

82

spectroﬂuorometer (SLM Instruments, Urbana-Champaign, IL) connected to a data
acquisition and operating system (On-Line Instrument Systems, Bogart, GA).
Fluorescence measurement was done with semi-micro quartz ﬂuorescence cuvettes (4 x
10 mm) held in a thermostable block (22°C). The excitation wavelength was 390nm
(excitation wavelength of ANS) and emission was scanned (400-600nm) with slit width of
4am. The ﬂuorescence intensity based on emission maximum wavelength at 486nm was
measured. RFI of ﬁlms in phosphate buﬂ‘er (pH 7.0) was also measured without ANS.
The net RFI for each sample was obtained by subtracting the RFI without ANS.

The initial slope (So) of the RFI versus ﬁlm concentrations plot was determined
and referred to as a hydrophobicity index of the sample. Under conditions with excessive
probe, the So value has shown a close correlation to the hydrophobicity of proteins (Kato

and Nakai, 1980; Yildirim et al., 1996; Haskard and Li-Chan, 1998).

3.3.6 Statistical Analysis

All experiments were replicated three times in a randomized block experiment. A
new ﬁlm forming solution and new set of ﬁlms were prepared for each replicate.
Statistical analysis were made using Sigma Stat 2.0 (Jandel Corp., San Rafael, CA) and
the appropriate comparisons were done using the Student-Newman-Keuls method for

multiple comparisons.

83

3.4 RESULTS & DISCUSSION

3.4.1 Solubility of the films in bond-dissociating agents

Solubilities of whey protein/lipid emulsion ﬁlms in bond-dissociating agents, 2-ME,
urea, and SDS, are shown in Figure 3.1. Relatively higher protein solubilities were
observed when 2-ME (a disulﬁde bond-dissociating agent) and urea (a hydrogen bond-
dissociating agent and also a weak hydrophobic bond-dissociating agent) were the
solvents rather than SDS (a hydrophobic bond dissociating-agent).

The strong effect of 2-ME on protein dissociation in WPI and WPC ﬁlms with and
without lipids reﬂected the important contribution of disulﬁde bonds to the formation and
stability of these ﬁlms. Disulﬁde bond formation is promoted during the heat treatment of
the ﬁlm-forming solutions and then during drying of the ﬁlms. These results are consistent
with those of Pérez-Gago et al. (1999). They prepared WPI-based ﬁlms with or without
heat treatment of the ﬁlm-forming solutions, and compared the mechanical properties,
tensile strength (TS) and elongation at break (%E), of the ﬁlms. Reportedly, TS and %E
of the WPI-based ﬁlms were much lower (p < 0.05) when the ﬁlms were prepared without
heat treatment compared to those of the heat denatured WPI-based ﬁlms. They concluded
that the disulﬁde bond formation through heat-denaturing process was important for the
formation of the WPI-based ﬁlm network and the ﬁlm’s structural integrity.

A similar action of urea as with 2-ME on protein dissociation in both whey protein
type ﬁlms suggested the presence of hydrogen bonds in the stabilization of the ﬁlm
network (Figure 3.1). Urea is believed to alter the physical properties of water; at high
concentrations, it hydrogen bonds to water in an aqueous system, thus, disrupting the

84

9°

N
DUI-IGNUIOOUI

Absorbance (280nm)

O

 

S S-BF4% S-CW16%

9°
tea:

19

.°

 

Absorbance(280nm)
0 0| a a N 0|

S S-BF4% S-CW16%

9’
teen

1"

 

 

.0

 

Absorbance(280nm)
0 0| -| :1: N 0|

S S-BF4% S-CW16%

Treatments

Figure 3.1. Solubility of whey protein/lipid emulsion edible ﬁlms in
protein bond-dissociating agents. I = whey protein isolate;

C = whey protein concentrate; S = sorbitol; BF = butter fat;

CW = candelilla wax.

85

usual hydrogen bond network of the aqueous system Urea also interacts with polar and
nonpolar surfaces more favorably than water, although the physical interactions with
nonpolar groups are not understood. Creighton (1993) reported that demturants such as
urea are similar to water in their degree of hydrogen bonding but different geometries.
The importance of hydrogen bonds for the formation and stability of whey protein-based
ﬁlms was expected since plasticizer (sorbitol) must be added to produce ﬂexible ﬁlms.
Sorbitol reduces protein-protein interactions by forming hydrogen bonds with
polypeptides, thus increasing ﬂexibility of the protein-based ﬁlms. Without the aid of
plasticizer (sorbitol in this case), protein-based ﬁlms become too brittle to handle
(McHugh and Krochta, l994d). When Fairley et al. (1996) added SDS as a plasticizer to
WPI-based ﬁlms, SDS was unable to plasticize the ﬁlms. This led them to conclude that
hydrophobic interactions probably were not the main attractive forces in WPI-based ﬁlms
since a hydrophobic bond-dissociating agent SDS was not an effective plasticizer for the
ﬁlms. The lower protein solubility with SDS (Figure 3.1) may indicate the contribution of
hydrophobic interactions to ﬁlm formation and stability were not as signiﬁcant as disulﬁde
or hydrogen bonds.

Solubilities of whey protein-based ﬁlms in protein bond-dissociating agents were
only determined on transglutaminase cross-linked ﬁhns by Mahmoud and Savello (1993)
and Yildirim and Hettiarachchy (1998). Their studies showed that whey protein-based
ﬁlms were stable in 2-ME while the ﬁlms were highly soluble in urea. They concluded that
the result suggested the insigniﬁcance of disulﬁde bonds and the signiﬁcance of hydrogen
bonds for the formation and stability of the ﬁlms. However, contribution of hydrophobic

bonds for the formation and stability of the ﬁlms were diﬂ‘erent. Mahmoud and Savello

86

(1993) reported that SDS was unable to solubilize transglutaminase cross-linked whey
protein-based ﬁlms and concluded that hydrophobic bonds' contrrhution was minimal. On
the other hand, solubility of transglutaminase cross-linked whey protein-based ﬁlms in
SDS was higher than with urea in the study by Yildirim and Hettiarachchy (1998), which
suggested the importance of hydrophobic bonds for the formation and stability of the
ﬁlms. Unfortunately, no studies investigated solubility of heat-catalyzed whey protein-
based ﬁlms in protein bond-dissociating agents other than this present study.

In this present study, the ﬁlm’s solubilities in bond-dissociating agents, 2-ME, urea
and SDS, showed no relevancy to their mechanical properties (TS and %E; Figure 3.1,
Table 2.7). Although the TS and %E values of the ﬁlms were signiﬁcantly (p < 0.05)
changed upon addition of BF (4%, w/w of protein) and CW (16%, w/w of protein), no
diﬁ‘erences were observed in the ﬁlms’ solubilities in the different bond-dissociating
agents. Protein type (WPI and WPC) also did not affect the ﬁlms’ solubilities in bond-
dissociating agents. A protein content difference (~10%) between WPI and WPC, 90 and
80%, respectively, perhaps was not signiﬁcant enough to affect chemical bond formation
in the ﬁlms. Neither there was a relationship between chemical bonds in the ﬁlms, and
their moisture and oxygen barrier properties. In Figure 3.1, solubilities of the ﬁlms in
bond-dissociating agents (2-ME, urea and SDS) were not different among treatments
while water vapor and oxygen permeabilities of the ﬁlms were signiﬁcantly changed (p <
0.05) depending on the protein and lipid type and concentration (Table 2.3, 2.5).
Structural and chemical natures of polymers are thought to play signiﬁcant roles in the
permeability of the ﬁlms (McHugh and Krochta; 1994a). Results ﬁ'om this study showed

that chemical bond formation among treatments was not statistically different (Figure 3.1).

87

Perhaps barrier properties of whey protein/lipid emulsion ﬁlms are more inﬂuenced by

structural differences, and surface morphology or matrix density of the ﬁlms.

3.4.2 Free sulfhydryl group and disulﬁde bond contents of the films

The ﬁee sulﬂlydryl group and disulﬁde bond contents of whey protein/lipid
emulsion ﬁlms are shown in Tables 3.2. Free sulﬂrydryl group content of WPI ﬁlm was
higher (p < 0.05) than those of WPC ﬁlms, 2.50-3.24 and 1.39-1.44 mng of ﬁlm,
respectively. This is probably due to the differences in the total protein amounts between
WPI and WPC, 90 and 80%, respectively (Table 2.1). Lipid type (BF or CW) had no
effect on ﬁ'ee sulﬂrydryl group contents of the WPI and WPC ﬁlms. This was expected
since protein amounts were kept the same (50% w/w, dry basis) throughout the treatments
(Table 3.1).

Disulﬁde contents of the WPI and WPC ﬁlms ranged from 2.75 to 3.58, and 1.84
to 2.35umol/g of ﬁlm, respectively (Table 3.2). However, no statistical diﬁerences were
observed among treatments. Lipid type (BF and CW) also did not affect disulﬁde contents
of the ﬁlms. Although there were no statistical differences in disulﬁde contents of the
ﬁlms, a relationship was observed between disulﬁde contents of the ﬁlms and TS of the
ﬁlms. Lower disulﬁde contents of WPC ﬁlms (1.84-2.35pmol/g of ﬁlm) coincided with
lower TS values (2.8-5.1 MPa) of the ﬁlms compared to those of WPI ﬁlms, while higher
disulﬁde contents of WPI ﬁlms (2.75-3.58umol/g of ﬁlm) agreed with higher TS values
(3.7-7.9 MPa) of the ﬁlms (Tables 2.7, 3.2). Similar results were reported by Were et al.
(1999) when they investigated disulﬁde contents of soy-wheat gluten composite ﬁhns.

Reportedly, the highest TS was observed with the ﬁlm that exhibited the highest disulﬁde

88

Table 3.2. Free sulfhydryl group and disulﬁde bond contents of whey protein/lipid
emulsion edible ﬁlms.

 

 

Treatments 1 SH (umol/g of ﬁlm) S-S (11ng of ﬁlm)
IS 3.043017“ 2.75 30.41 '
IS-BF4% 2.50 3 0.39 ' 3.01 3 0.45 '
IS-CW16% 2.89 3 0.31 “ 3.58 3 0.48 ‘
cs 1.39 3 0.37 " 2.33 3 0.69 "
CS-BF4% 1.44 3 0.26 b 1.84 3 1.02 '
CS-CW16% 1.39 3 0.31 b 2.35 3 0.90 "

 

 

 

*5 Different letters columnwise denote signiﬁcant difference (p < 0.05), =3 for all treatments.
1 I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; C = whey protein
concentrate

89

content. They also suggested that additional introduction of disulﬁde bonds to ﬁlms may
increase TS of the ﬁlms, and suggested use of a free sulﬂrydryl group containing
compound like cysteine in ﬁlm-forming solutions to induce ﬁ'ee sulﬂrydryl/disulﬁde

interchange reaction and rearrangement of disulﬁde bonds.

3.4.3 Hydrophobicity of the ﬁlms

Relative ﬂuorescence intensities (RFI) of ANS at 486nm with varying
concentrations of whey protein/lipid emulsion ﬁlms, 0.05 to 0.5 mg/rnl in phosphate
buffer, are shown in Figure 3.2. These RFI versus ﬁlm concentrations plots were used to
obtain initial slope (So), hydrophobicity values, of the ﬁlms. The S0 values of whey
protein/lipid emulsion ﬁlms ranged ﬁom 8.40 to 9.38 (Table 3.3). Addition of lipids, BF
and CW, resulted in increased hydrophobicity of WPI ﬁlms ﬁom 8.40 to 8.61 and 9.05,
respectively. A similar trend was observed for the hydrophobicity of WPC ﬁlms. Higher
S, values for BF or CW added WPC ﬁlms were obtained compared to that of the ﬁlm
without lipids, 8.44, 8.52 and 9.38, respectively. However, these differences were not
statistically signiﬁcant.

Although no statistical differences were observed among treatments, overall,
CW16%-added WPI and WPC ﬁlms exhibited higher hydrophobicity values compared to
those of the ﬁlms without lipids and with BF4% (Table 3.3). This was expected since CW
belongs to the non-polar lipid group (Callegarin et al., 1997). Incorporation of BF4% to
WPI and WPC ﬁlms was not as effective as CW to increase hydrophobicity of the ﬁlms.
This is probably due to the nature of BF. About 98% of BF is composed of
triacylglycerols, which make BF a polar lipid (Sax and Lewis, 1987).

90

(a)

0))

Relative ﬂuorescence Intensity

Relative fluorescence Intensity

3.5

 

2.5

.. 1+1s-CW16%j

   
 

 

+1TT‘T
--o--IS-BF4% '

 

 

3.5

  

 

 

I J
f

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Film concentration (mglml)

 

2.5 ..

 

 

 

- - 3 - -cs I
—o—CS—BF4%
+CS-CW16%

 
  
    

 

 

 

 

0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Film concentration (mglml)

0.05 0.1

Figure 3.2. Relative ﬂuorescence intensity (RF I) of ANS at 486nm with
concentration of whey protein/lipid emulsion edible ﬁlms in the range of
0.05 to 0.5 mg/ml in aqueous phosphate buffer (Data shown here are

the average of three replicates).

(a) whey protein isolate ﬁlms; (b) whey protein concentrate ﬁlms;

I = whey protein isolate; C = whey protein concentrate;

S = sorbitol; BF = butter fat; CW = candelilla wax.

91

Table 3.3. Hydrophobicity (So) values of whey protein/lipid emulsion edible ﬁlms.

 

 

Treatments ' S0

IS 8.40 :l: 0.47 ‘
IS-BF4% 8.61 i 0.35 ‘
IS-CW16% 9.05 i 0.72 '
CS 8.44 i 0.32 '
CS-BF4% 8.52 i 0.46 '
CS-CW16% 9.38 :1: 0.60 '

 

 

*b Different letters columnwise denote signiﬁcant difference (p < 0.05), n=3 for all treatments.
I I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax;
C = whey protein concentrate.

92

This trend in hydrophobicity of the ﬁlms coincided with reduced (p < 0.05) water
vapor permeability (WVP) of the ﬁlms upon addition of lipids (Tables 2.3, 3.3). For
instance, the highest WVP of WPI ﬁlms was observed with WPI ﬁlms without lipids added
which exhibited the lowest hydrophobicity value, 28.5 g.mm/m2.day.kPa at 80% RH and
8.40, respectively. On the other hand, CW16%-added WPI ﬁlms showed the lowest WVP
20.7 g.mm/m2.day.kPa had the highest hydrophobicity value 9.05. A similar trend was
observed with WPC ﬁlms’ WVP and hydrophobicity values (Tables 2.3, 3.3).
Unfortunately, no other studies are found reporting hydrophobicity of protein/lipid
emulsion ﬁlms.

In summary, the results of this study demonstrated that disulﬁde and hydrogen
bonds were the main forces involved in the formation and stabilization of whey
protein/lipid emulsion edible ﬁlms. The contrrhution of hydrophobic interactions to ﬁlm
formation and stability were secondary. Protein type (WPI and WPC) affected the free
sulﬂlydryl group contents of the ﬁlms: 2.50-3.04 mng of ﬁlm for WPI ﬁlms, and 1.39-
1.44 11ng of ﬁlm for WPC ﬁlms. Although as a general trend WPI ﬁlms had higher
disulﬁde contents than WPC ﬁlms, no statistical differences were observed among
treatments. Hydrophobicity of the ﬁlms increased with addition of lipids. CW-added ﬁlms

were more hydrophobic than ﬁlms containing BF, however, these differences were not
statistically signiﬁcant.

93

CHAPTER 4

THERMAL PROPERTIES, HEAT SEALABILITY AND SEALING PROPERTIES

OF WHEY PROTEIN/ LIPID EMULSION EDIBLE FILMS.

4.1 ABSTRACT

Whey protein isolate (WPI; 5%, w/v) ﬁlms were plasticized with sorbitol (4.2-5%,
w/v) and glycerol (2.7-3.5%, w/v), and butter fat (0.2%, w/v) or candelilla wax (0.8%,
w/v) was added to produce whey protein/lipid emulsion edible ﬁlms. Thermal properties of
the ﬁlm-forming components and of the ﬁlms were studied using Differential Scanning
Calorimetry (DSC). DSC analysis results were used to optimize heat-sealing conditions of
the ﬁlms. The effect of heat-sealing temperature, pressure, and dwell time on sea] strength
were investigated. Electron spectroscopy for chemical analysis (ESCA) was used to study
the nature of interfacial interactions in heat sealed whey protein/lipid emulsion edible ﬁlms.
All ﬁlms were heat sealable. The seal strengths of the ﬁlms ranged ﬁ'om 110 to 323 N/m
Pressure and dwell time variation did not affect the seal strength of the ﬁlms. However,
the plasticizer type inﬂuenced heat-sealing temperature of the ﬁlms, 130°C for sorbitol-
plasticized ﬁhns and 110°C for glycerol-plasticized ﬁlms. ESCA spectra showed main
components on the surfaces of unsealed and heat sealed ﬁlms and gave evidence that
hydrogen and covalent bonds involving C-O-H and N-C components, respectively, may be
the main forces responsible for the sealed joint of whey protein-based ﬁlms. Model

structures for the interfacial interactions of the ﬁlms upon heat-sealing were proposed.

94

4.2 INTRODUCTION

Edible packaging materials have received much interest in recent years since they
provide unique and new opportunities for food processing and product development. In
chapter 2, development, barrier and mechanical properties of whey protein/lipid emulsion
edlhle ﬁlms were reported. Some proposed applications of these protein-based edible
ﬁlms include pouches or sachets to package individual portions of dry ingredients, such as
beverage mixes or soups for convenience. Other possible applications include ‘ingredient
delivery systems’ that deliver pre-measured ingredients during processing operations, thus,
offering convenience and preventing human error in weighing and handling (Debeaufort et
al., 1998).

Sealability and sealing properties of materials are important in development of
pouches sachets or ‘ingredient delivery systems’. Application of heat is widely used in the
packaging industry to seal polymer ﬁlms. Seal properties of a ﬁlm are typically inﬂuenced
by sealing temperature, pressure, and dwell time. However, sealing temperature of the
ﬁlms has been reported to be most important in inﬂuencing seal strength (Meka and
Stehling, 1994). Upon application of heat, surface regions of crystalline polymer melt and
application of pressure leads to difﬁlsion and entanglements of the melted surface. The
intermolecular diffusion across the joint surfaces is a necessary step to give sufﬁcient seal
strength to the sealed ﬁlms. The diffusion step requires time. After cooling,
recrystallization occurs producing a heat-sealed joint (Yeh and Benatar, 1997 ; Mueller et
al., 1998). This joint formation occurs through interfacial interactions on the polymer

surface, which is dependent on the surface chemistry of the polymer (Allen, 1987).

95

 

Few studies have demonstrated heat scalability of edible ﬁlms. Ninomiya et aL
(1990) reported on heat scalability and seal strength of carageenan-based edible ﬁlms.
Glycerol- and sorbitol-plasticized carageenan-based ﬁlms were heat scalable, and had seal
strength of 137 and 130 N/m, respectively. Chick (1998) investigated heat scalability of
lactic acid casein-based ﬁlms. The ﬁlms were heat scalable at around 107-120°C. Seal
strength of the ﬁlms ranged ﬁ'om 153 - 247 N/m. No studies have been reported on the
mechanism involved in heat-sealing of protein-based edible ﬁlms.

Electron spectroscopy for chemical analysis (ESCA) is a useﬁll tool that provides
qualitative and quantitative characterization of the near surface regions of materials
(Cayless, 1991). Wu et al. (1995) investigated effects of ammonia plasma treatment on
LDPE and HDPE surfaces to enhance seal strength of the two polymers. Surface analyses
by ESCA revealed interactions occurred between nitrogen and oxygen containing
ﬁinctional groups on the ammonia plasma treated polymer surfaces. Possart and
Dieckhoﬁ' (1999) also employed ESCA to study the surface of polycyanurates to
determine which chemical structures are capable of interfacial interactions with various
substrate surﬁtces. Reportedly, hydrogen bonds involving OH-groups were responsible
for the interactions at the interfacial region.

Much of the research on protein-based edible ﬁlms until now has focused on their
development, determining their barrier and mechanical properties. Very little information
is available on their thermal properties. There is no information available on heat
scalability and seal properties of whey protein-based cdrhle ﬁlms. In this study, whey
protein isolate ﬁlms were plasticized with sorbitol and glycerol, and butter fat and

candelilla wax were incorporated to produce whey protein/lipid emulsion ﬁlms. The intent

96

was to determine thermal properties of the ﬁlms using differential scanning calorimetry to
optimize heat-sealing temperatures. Heat scalability and seal strength of the ﬁlms were
determined at various temperatures, pressures and dwell times to obtain optimum sealing
conditions. Surface chemical properties of the unsealed and sealed ﬁlm were investigated
using ESCA to gain an understanding for the interﬁcial interactions in the formation of the
scaled joint with whey protein/lipid emulsion ﬁlms. In addition, model structures for
interﬁcial interactions of the ﬁlms were proposed to illustrate bonding formation on the

surfaces of the ﬁlms upon heat—scaling.

4.3 MATERIALS AND METHODS

4.3.1 Materials

Whey protein isolate (ALACEN 895) was obtained ﬁom New Zealand Milk
Products (North America) Inc., (Santa Rosa, CA). Table 4.1 shows the composition of
the whey proteins used in the production of edible ﬁlms in this study. Its protein content,
93.2%, was conﬁrmed using AOAC 930.29 Kjeldahl method (AOAC, 1990). Glycerol
was purchased from J.T. Baker Co. (Phillipsburg, NJ). D-Sorbitol, candelilla wax, butter
fat and NaOH were purchased ﬁom various sources as mentioned in chapter 2 section

2.3.2.

4.3.2 Film preparation
Edible ﬁlm forming solutions were prepared by mixing whey protein isolate (WPI)

(5%, w/v) with sorbitol or glycerol, 2N NaOH (to adjust pH), and distilled H20 to a ﬁnal

97

Table 4.1. Composition of whey proteins used'.

 

 

Components Whey protein isolate
ALACEN 895
Protein (N x 6.38) % 93.5
Ash % 1.6
Moisture % 4.0
Fat % <1.0
Lactose % <1 .0
pH2 6.8

 

1Values based on speciﬁcation provided by New Zealand Milk Products (N. America) Inc.
2 5% at 20°C

98

volume of 100ml. Rests of the procedures were the same as described in section 2.3.3 of
chapter 2 (Figure 2.1). Table 4.2 shows the composition of the ﬁlms. Thicknesses of the
ﬁlms were measured using a TMI model 549 M micrometer (Testing Machines, Inc.,
Amityville, NY). Thicknesses of sorbitol- and glycerol-plasticized WPI ﬁlms were 140 :1:

l9 and 120 :l: 15 pm, respectively.

4.3.3 Thermal analysis of the ﬁlms

Differential scanning calorimetry (DSC) was employed to determine thermal
properties and processing conditions of whey protein/lipid emulsion ﬁlm. Thermal
transition temperatures of the ﬁlm forming components and ﬁlms were evaluated. A Du
Pont 2920 DSC unit (Wilmington, DE) was used to measure the differential temperature
and enthalpy change (AH). Approximately 10mg of sample was weighed and sealed in an
aluminum sample pan (TA Instruments, Newcastle, DE) by an encapsulating press.
Samples were heated from 0°C to 250°C with increase in temperature of 20°C/min. An
empty sample pan was used as a reference. The DSC cell was ﬂushed with nitrogen at 20
ml/min to maintain an inert environment during the measurement. The transition
temperatures of the ﬁlm forming components and ﬁlms were determined by the General V
4.1 C software program (Wilmington, DE), which controlled the DSC unit. Onset (To)
and peak (Tp) temperatures were assigned according to ASTM D-3418 “Standard test
method for transition temperature of polymers by thermal analysis” (ASTM, 1997).

Thermogravimetric Analysis (TGA) was used to determine weight loss of whey
protein powder during heating under conditions close to that of the DSC experiment.
TGA is a technique in which the mass of a substance is measured as a function of

99

Table 4.2. Composition of whey protein/lipid emulsion edlhle ﬁlms produced.

 

 

 

 

% w/v of aqueous solution % w/w of protein
Treatments 1 Protein 2 Plasticizer Lipid Plasticizer Lipid
IS 5 5.0 0 100 0
IS-BF4% 5 4.8 BF = 0.2 96 BF = 4
IS-CW16% 5 4.2 CW = 0.8 84 CW = 16
IG 5 3.5 0 70 0
IG-BF4% 5 3.3 BF = 0.2 66 BF = 4
IG-CW16% 5 2.7 CW = 0.8 54 CW = 16

 

 

 

' I = whey protein isolate; S = sorbitol ; BF = butter fat ; CW = candelilla wax ; G = glycerol

2 whey protein isolate

100

temperature or time (ASTM, 1997). A Du Pont 2200 TGA unit (Wilmington, DE) was
employed and approximately 10mg of WPI powder was prepared as described in DSC
procedure. A sample was heated ﬁom 20 to 150°C with increase in temperature of 20°C/

min under a nitrogen atmosphere. The mass of the sample was measured concurrently.

4.3.4 Determination of seal strength

Film samples were cut into strips of 7.62cm x 2.54cm using a Precision sample
cutter (Thawing Albert Instrument Co., Philadelphia, PA). Two layers of ﬁlm strips were
sealed together using a thermal heat sealer Model-12ASL (Sencorp System Inc., Hyannis,
MA). The seal area was 2.54cm x 1.5cm. Three different seal temperatures 110, 120 and
130°C were investigated. These temperatures were selected based on DSC results. Dwell
times were 1 or 3 seconds, with a seal pressure of 40 or 60psi. All scaled specimens were
conditioned (23 3: 2°C, 50 :t 5 % RH) for 48 hours prior to testing for strength of the seal.

Seal strength of the ﬁlms were determined using a ASTM F-88 “Standard test
method for seal strength of ﬂexible barrier materials” (ASTM, 1997). Tests were
conducted using the Instron Universal Testing Machine Model 2401 (Instron Corp.,
Canton, MA) at 23 i 2°C and 50 .+_ 5% RH. Each leg of the specimen was clamped into
the testing machine. The distance between clamps was 5.08 cm with a loading rate of
25.4 cm/min. During the test, each end of the sealed ﬁlm was held perpendicularly to the
direction of pull as the specimen was stressed. The maximum force required to cause seal
failure were reported in newtons/meter (N/m). Seal strength was calculated ﬁom the

following equation:

101

Seal strength = load/w (19)
load = peak force

w=ﬁlmwidth

4.3.5 Surface analysis of unsealed and sealed films using Electron Spectroscopy for
Chemical Analysis (ESCA)

Whey protein/lipid emulsion ﬁlms were sealed at 110°C for 1 sec at pressure of
40psi. The surface elemental compositions and bonding distributions of unsealed, and
sealed ﬁlms were determined using ESCA with a PHI 5400 ESCA lab workstation
(Physical Electronics, Eden Prairie, MN). A 15 mm diameter of circular sealed or
unsealed ﬁlm was placed in sample holder and monochromatic X-rays were used as the
radiation source. All spectra were collected using a Mg anode operated at a power of 300
W with an analyzer pass energy of 33 eV. An electron kinetic energy analyzer plotted the
intensity of the emitted photoelectrons according to their binding energies. The optimum
spot size for the conditions used in this experiments was 1 mm diameter aperture.

The shape of the spectra indicated that no compensation for diﬂ‘erential surﬁce
charging was needed. The bonding scale was calibrated to 284.6 eV for the main Cls (C-
H) feature. Spectra were run in both low resolution (survey scan) and high resolution
modes for the Cls, Ols and N18 regions. Elemental compositions were calculated from
the survey scan spectra. Chemical information indicating changes in the surface of
polymers was elucidated by curve-ﬁtting the carbon 1s (Cls), nitrogen ls (N Is), and
oxygen 1s (Ols) spectra. Curve-ﬁtting deﬁned and interpreted the carbon chemistry as

detected at the sample surface by allowing the user to distinguish overlapping features

102

within the spectral envelope. The spectra were ﬁt with a Lorentzian-Gaussian mix Voigt
proﬁle function using a nonlinear least-square curve-ﬁtting program PHI PC Explorer
Software multipack (Physical Electronics, Eden Prairie, MN). The resulting curve ﬁts

have levels of experimental error of approximately 5%.

4.3.6 Statistical analysis

The seal strength experiments were replicated three times in a randomized block
experiment. A new ﬁlm forming solution and new set of ﬁlms were prepared for each
replicate. Statistical analysis were made using Sigma Stat 2.0 (Jandel Corp., San Rafael,
CA) and the appropriate comparisons were made using the Student-Newman-Kculs
method for multiple conrparisons. DSC and ESCA were conducted on one set of each

replicate. Representative data were selected for presentation in this chapter.

4.4 RESULTS & DISCUSSION

4.4.1 Thermal properties of the films

DSC results of the ﬁlm forming components, and sorbitol- and glycerol-plasticized
WPI ﬁlms are shown in Figures 4.1, 4.2 and 4.3, respectively. Onset (To) and peak (Tp)
temperatures were assigned according to ASTM D-3418 (Tables 4.3, 4.4). WPI powder
exhibited a broad endothermic peak of ﬁrst-order transition between 125 and 173°C,
similar to the distinctive melting transition characteristic of semicrystalline polymers,
suggesting that WPI may be a partially amorphous semicrystalline polymer. It is thought

that all polymers have at least some amorphous material (Rosen, 1982). WPI powder had

103

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Table 4.3. Thermal properties of ﬁlm forming components as determined by Diﬂerential

 

 

 

 

 

 

 

Scanning Calorimetry.
Film components 1 Transition temperatures (°C) 2 Heat ﬂows

T. Tp 3 (J/g)
WPI 125 156 193.2

215 241 65.0

Sorbitol 96 101 192.2
Glycerol 165 178 66.7

BF 2 10 30.9

29 30 10.2

99 101 181.1

CW 57 68 211.2

 

 

 

1 WPI = whey protein isolate; BF = butter fat; CW = candelilla wax.
2 To = onset transition temperature; Tp = peak transition temperature.

3 Only the ﬁrst peak temperature was reported if more than two peaks were observed.

107

Table 4.4. Thermal properties of whey protein/lipid emulsion edible ﬁlms as determined
by Differential Scanning Calorimetry '.

 

 

 

 

 

 

 

 

Treatmentsl Transition temperatures (°C) 2 Heat ﬂows
To Tp 3 (J/g)
IS 126 143 15.6
IS-BF4% 127 160 202.9
IS-CW16% 60 66 5.4
127 135 84.0
16 108 145 169.9
IG-BF4% 122 132 31.8
IG-CW16% 6O 66 7.0
116 142 208.0

 

 

 

' WPI = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; G = glycerol
2 To = onset transition temperature; Tp = peak transition temperature.
3 Only the ﬁrst peak temperature was reported if more than two peaks were observed.

108

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T0 at 125°C and TI) at 156°C with heat ﬂow change of 193.2 J/g (Table 4.3). WPI
powder had one more peak at 241°C, suggesting degradation of the protein.

In Figure 4.1 and Table 4.3, sorbitol showed a narrow endothermic peak (96-
106°C) as with crystalline polymers. The T9 of sorbitol was 101°C, which was close to
the melting temperature of anhydrous sorbitol, 110-112°C (Budavari et al., 1989).
Glycerol showed a rather broad endothermic peak (165-220°C) with the heat ﬂow change
of 66.65 J/g. Since its melting temperature is 17.8°C, the Tp (178°C) of glycerol may be
due to its decomposition, which corresponds to the degradation temperature of 182.2°C at
20mmHg reported by Budavari et al. (1989). BF showed two low Tp at 10 and 30°C,
which were within a melting temperature range (0 - 40°C) of BF (Lane, 1992). BF had
one more distinctive peak at 101°C, which may correspond to the decomposition of BF.
CW showed Tp at 68°C with heat ﬂow change of 21 1.2 J/g. This temperature was similar
to the melting temperature of CW, 64.0°C, reported by Donhowe and Fennema (1993).

All ﬁlms showed broad endothermic peaks in the temperature range of 108-221°C
(Table 4.4). The T0 of glycerol-plasticized WPI ﬁlms were slightly lower than T0 of
sorbitol-plasticized WPI ﬁlms, 108-122°C and 126-127°C, respectively, probably due to
the diﬁ'erences in the plasticizing effect of glycerol and sorbitol. A plasticizer’s ﬁmctional

efﬁcacy is often estimated by examining the reduction caused in the glass transition

temperature or melting temperature of polymers (Karlsson and Singh, 1998). Glycerol

with its lower melting temperature (178°C) may be more effective than sorbitol with
higher melting temperature (101°C) in lowering thermal transition temperatures (T0) of

whey protein-based ﬁlms. However, this effect of the plasticizer was not as great as

110

anticipated. A possible explanation may be the lack of pre-conditioning (i.e., drying) for
WPI powder prior to DSC analysis. To conﬁrm this, TGA was used to determine mass
changes of WPI powder during heating under conditions close to that of the DSC
experiment. Indeed, a 6.4% weight loss was observed (Figure 4.4) due to the moisture in
the WPI powder. Water is known to also act as a plasticizer, and any water molecules
present in WPI powder could reduce thermal transition temperatures and conformed the
effects observed due to the diﬁerences in the plasticizer (Pouplin et al., 1999). Further
studies are needed with regards to the inﬂuence of the plasticizer in thermal transition
temperatures of protein-based edible ﬁlms.

Film samples containing CW showed what appeared to be two endothermic peaks
(Figures 4.2, 4.3), and a more deﬁnite narrow endothermic peak at 66°C, corresponding
to Tp of CW, 68°C (Tables 4.3, 4.4). All ﬁlms with the exceptions of BF-added ﬁlms
showed multiple peaks around 175-212°C, which appeared to be the degradation

temperatures of ﬁlms (Figures 4.2, 4.3).

4.4.2 Heat scalability and seal strength of the ﬁlms

Films were heat sealed only on the non-lipids oriented side of the ﬁlm because
lipid-oriented sides did not seal or formed seals that easily delaminated. Heat-sealing was
conducted near the onset thermal transition temperatures (T0) of whey protein/lipid
emulsion ﬁlms. The thermal transition temperature sets the application, such as heat-
sealing, temperature range of a polymer (Hernandez, 1997).

The seal strength measurements of sorbitol- and glycerol-plasticized whey protein/

lipid emulsion ﬁlms are shown in Table 4.5 and Table 4.6, respectively. All ﬁlms were

111

 

 

 

 

 

 

 

 

 

 

 

Table 4.5. Seal strength (N/m) of whey protein/lipid emulsion edible ﬁlms plasticized
withsorbitol.
Parameter Treatments 1
Temp. Pressure Dwell
(°C) (psi) time IS IS-BF4% IS-CW16%
(sec)
110 40 1 110116“ 115114“ 10519“
3 14719c 12718“ 11914“
60 1 116110“ 112119“ 108115“
3 158111“c 124112“ 120120“
120 40 1 160115 ““ 15015 “ 15218“
3 191126“ 191124“ 186112c
60 1 162110“c 150113“ 15415“
3 188118“ 17818“ 184121“
130 40 1 293112“ 215113““ 248117“
3 298128“ 268116“ 285115“
60 1 284128“ 239117“ 236111“
3 301119“ 261129“ 29616“

 

 

 

 

 

 

 

 

 

”7' Different letters columnwise denotes signiﬁcant difference (p < 0.05), n = 3 for all treatments.
1 I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax

112

Table 4.6. Seal strength (N/m) of whey protein/lipid emulsion edible ﬁlms plasticized

 

 

 

 

 

 

 

 

 

 

 

 

 

 

with glycerol.
Parameter Treatments 1
Temp. Pressure Dwell
(°C) (psi) time IG IG-BF4% IG-CW16%
(sec)
110 40 1 2851 23“ 2651 9“ 2611 10“
3 3231 42“ 288119“ 297115“
60 1 2821 10“ 2751 40“ 2631 8“
3 2961 37“ 2911 38“ 291117“
120 40 1 2111 17c 2161 13“ 2131 21“
3 2571 12“ 2691 25“ 2601 20“
6o 1 2251 8“ 217111“ 202116““
3 2631 28““ 2621 16“ 2571 18“
130 40 1 1711 20“ 1471 57“ 1411 36“
3 1871 22“ 1691 10c 1731 9“
60 1 1731 5“ 1591 28“ 1561 21“
3 1981 15““ 1651 21 “ 1681 27“

 

 

 

 

 

 

“1 Different letters columnwise denotes signiﬁcant difference (p < 0.05), n = 3 for all treatments.

1 I = whey protein isolate; G = glycerol; BF = butter fat; CW = candelilla wax

113

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114

heat scalable. The seal strength of sorbitol-plasticized ﬁlms ranged from 105 to 301 N/m,
while glycerol-plasticized ﬁlms showed seal strength between 141 to 323 N/m. Overall,
the seal pressure and dwell time variation did not produce signiﬁcant diﬁ‘erences in the seal
strengths of the ﬁlms. However, heat-sealing temperature signiﬁcantly (p < 0.05)
inﬂuenced seal strength of the ﬁlms. The highest seal strengths (p < 0.05) were observed
at temperature 130°C for sorbitol-plasticized WPI ﬁlms (Table 4.5), and 110°C for
glycerol-plasticized WPI ﬁlms (Table 4.6). These heat-sealing temperatures corresponded
with the T0 of the ﬁlms ﬁ'om DSC analysis (Table 4.4). Sorbitol-plasticized WPI ﬁlms had
a T0 of 126-127°C, and optimum seal strength was obtained when ﬁlms were sealed at
130°C. In case of glycerol-plasticized ﬁlms, they had a T0 of 108-122°C, and optimum
seal strength was obtained at 110°C. These results indicate that the T0 may be used to
determine thermal processing temperatures for protein-based edible ﬁlms.

Lower seal strength of glycerol-plasticized ﬁlms at 130°C may be due to the
excessive temperature resulting in distorted and weakened seals. According to Martin
(1986), when the heat required to produce a seal exceeds the heat-sealing temperature
range for that material, it induces a distorted or nonfunctional seal. In our study, slight
deformation of seal structure was observed with glycerol-plasticized ﬁlms at 130°C
indicating that degradation of the materials started to occur at this temperature, thus,
reducing seal strength It is recommended that heat-sealing temperature should not
exceed 130°C for glycerol-plasticized WPI ﬁlms.

The highest seal strength obtained with sorbitol- and glycerol-plasticized ﬁlms
were 301 and 323 N/m, respectively. These results were lower than the seal strengths of

most synthetic polymers, but comparable to the seal strength obtained with lactic acid

115

 

casein- or carageenan-based edible ﬁlms (Table 4.7). However, these are very general
comparisons, as stated previously caution must be taken in comparing data of this nature
since different experiments were conducted under different heat-sealing and testing

conditions.

4.4.3 Surface elemental compositions of the ﬁlms determined by ESCA survey

spectra.

Figures 4.5 and 4.6 show survey spectra of sorbitol- or glycerol-plasticized WPI
ﬁlms containing BF or CW before and alter heat-sealing. Surface elemental compositions
of the ﬁlms were calculated and are shown in Table 4.8. All treatments showed distinctive
peaks in Ols, N13 and Cls regions except no le spectra were observed for the unsealed
sorbitol-plasticized ﬁlms containing BF and CW. The absence of N15 spectra is probably
due to low N ratio and/or poor quality of the spectra. Thus it was diﬁicult to determine
relative changes of elemental composition upon heat-sealing of the sorbitol-plasticized
WPI ﬁlms containing BF and CW. Except for these two ﬁlms mentioned, the other ﬁlms
(sorbitol-plasticized WPI ﬁlms, glycerol-plasticized WPI ﬁlms, and glycerol-plasticized
ﬁlms containing BF and CW) showed distinctive changes in their surface elemental
compositions upon heat-sealing.

Carbon was the main element of the ﬁlms, and oxygen was the second most
prominent element. Nitrogen was less than 8.3% in all ﬁlms. Carbon comprised 71.5-
77.4% of the total surface composition of the unsealed ﬁhns. After heat-sealing, carbon
compositions declined approximately 1.4-6.5% depending on the ﬁlm Oxygen and

nitrogen compositions, on the other hand, increased upon heat-sealing. Approximately 1-

Ols
A. (a) ' (b) Ols Cls
Cls -

le

 

B. (a) (b)

 

 

 

 

 

 

Ols
C. (a) Cls
Ols
77.3.3.3..Soséozéo-éo, domain-Soaaioaéo,
Binding energy (eV) Binding energy (eV)

Figure 4.5. Electron Spectroscopy for Chemical Analysis survey spectra of whey
protein/lipid emulsion edible ﬁlms plasticized with sorbitol.

A. (a) IS ﬁlm unsealed, (b) IS ﬁlm sealed ;

B. (a) IS-BF4% ﬁlm unsealed, (b) IS-BF4% ﬁlm sealed ;

C. (a) IS-CW16% ﬁlm unsealed, (b) IS-CW16% ﬁlm sealed

I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax

117

Ols Cls
A. (a)
Cls
B. (a)
C. (a) Ols

 

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Binding energy (eV)

 

 

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le
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(b)
(b)

Cls
Ols

 

 

1 J

l I I l A
roosmmaoo’soomroo 0

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Figure 4.6. Electron Spectroscopy for Chemical Analysis survey spectra of whey
protein/lipid emulsion edible ﬁlms plasticized with glycerol.

A. (a) 16 ﬁlm unsealed, (b) 10 ﬁlm sealed ;

B. (a) IG-BF4% ﬁlm unsealed, (b) IG-BF4% ﬁlm sealed ;
C. (a) IG-CWl6% ﬁlm unsealed, (b) IG-CW16% ﬁlm sealed
I = whey protein isolate; G = glycerol; BF = butter fat; CW = candelilla wax

118

Table 4.8. Surface elemental compositions of whey protein/lipid emulsion edible ﬁlms
before and after heat-sealing determined with survey spectra of Electron Spectroscopy for
Chemical Analysis.

 

 

 

 

% C % O % N
Treatmentsl unsealed sealed unsealed sealed unsealed sealed
IS 71.5 68.9 24.4 25.8 4.0 5.3
IS-BF4% 66.5 66.9 33.3 25.6 0.2 7.5
IS-CW16% 59.6 72.7 40.4 21.4 0.0 5.9
IG 77.4 70.9 18.1 20.8 4.5 8.3
IG-BF4% 76.7 73.1 18.1 20.9 5.1 6.0
IG-CW16% 76.5 70.8 18.2 21.3 5.3 7.9

 

 

 

 

' I = whey protein isolate; S = sorbitol; G = glycerol; BF = butter fat; CW = candelilla wax

119

2% increases occurred for the oxygen components on the surfaces of the sealed ﬁlms,
whereas nitrogen increased by 1-4% again depending on the ﬁlm (Table 4.8), indicating

that some oxygen and nitrogen components have formed on the surfaces of sealed ﬁlms

upon heat-sealing.

4.4.4 Surface compositional changes of the ﬁlms upon heat-sealing

To further understand the mechanism of sealing, high resolution ESCA peaks were
obtained from the unsealed and sealed WPI ﬁlms. Figure 4.7 showed the high-resolution
Cls, Ols and N15 spectra of the sorbitol-plasticized WPI ﬁlms before and after heat-
sealing. Since all ﬁlms showed similar spectra, only this spectrum (Figure 4.7) was
selected to be presented here. Each peak was assigned according to their binding energy
(eV). The assignments were made by comparing observed binding energies to comparable
data from the literature (Briggs and Seah, 1990; Buchwalter, 1995; Ratner and Castner,
1997; Pleul et al., 1998). Peak positions and assignments are given in Table 4.9. Surface
compositions of the ﬁlms before and after heat-sealing are presented in Tables 4.10, 4.11
and 4.12.

The Cls spectra (Figures 4.7a, d) was composed of four components and the
components appeared at 284.6eV (C-H), 286.36V (C-O), 287.6eV (O=C) and 289.5eV
(O-C=O; Table 4.9). The relative concentrations of the components are listed in Table
4.10. Main components of Cls spectra appeared to be OH and 00. A comparison of
the C1 3 spectra for all unsealed and sealed ﬁlms indicated that the relative intensities of
diﬂ‘erent carbon components were essentially the same for the unsealed and the sealed

surfaces of the ﬁlms.

120

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125

In Figures 4.7b and 4.7e, the Ols spectra of the sorbitol-plasticized WPI ﬁlms
showed three components, C-O-H at 534.3eV, O=C at 532.3eV and O-N at 530.4eV
(Table 4.9). The relative concentrations of these components for unsealed and sealed
ﬁlms are shown in Table 4.11. Main components of Ols spectra were O=C and C-O-H.
Overall, O=C percentage was higher for the unsealed ﬁlms and C-O—H amount was higher
in the sealed ﬁlms. In Figures 4.7b and 4.7e, shitting of O=C to C-O-H was observed
indicating disappearance of O=C component and formation of C-O—H component upon
heat-sealing. Formation of C-O-H may be responsible for the sealing of whey protein-
based ﬁlms.

The N13 spectra for the unsealed and sealed ﬁhns appeared at 399.7eV (N-C) and
401.8eV (N=C; Figures 4.7c, f). Similar trends were observed as with the results of Ols
spectra. The relative intensity of the N=C component was higher in the unsealed sorbitol-
plasticized WPI ﬁlms, while the N-C intensity was higher in the sealed ﬁlms (Figures 4.7e,
1). Similar trends were observed with other ﬁlms (Table 4.12). Based on the spectra
intensity changes, it may be concluded that C-O-H (534.3eV) and N-C (399.7eV)
components may be responsible for heat-sealing mechanism of whey protein-based ﬁlms.

Wu et al. (1995) employed ESCA to investigate adhesive bondings on the surface
of annnonia treated polyoleﬁns. Reportedly, C-O-H components on the surfaces
participated in hydrogen bonding formation across the interfaces of polyoleﬁns, also amine
groups on the surfaces formed covalent bonds at the interface. In the adhesion mechanism
of polymers, hydrogen bonds and covalent bond are known to play critical roles in

interfacial interactions to achieve sufﬁcient bond strength (Allen, 1987; Urban, 1993;

Misra, 1994).

126

In proteins, hydrogen bonds may appear between oxygen of the . , =0
(carbonyl groups: hydrogen acceptors) of a peptide bond and hydrogen of -NH, or -0H
(imino groups, or hydroxyl groups: hydrogen donor) of another peptide bond (Cheﬁel et
al.,1985): ‘ C=o-- H-N
Hydrogen bonds are one of the main forces that are involved in structural formation of
protein-based ﬁlms. Plasticizers are typically added to increase ﬂexibility of protein ﬁlm,
which also provides for additional hydrogen bonding. For example, upon addition of
glycerol as a plasticizer:

H-O(Glycerol) + C=OH H-N . —> / C=O°°H-O(Glycerol) + H-N \
Plasticizers, such as glycerol, have been reported to act as heat-sealing promoters
(Georgevits, 1967). Figure 4.8 provides proposed models for nature of hydrogen bonding
between plasticizer and plasticizer, plasticizer and protein, and protein and protein upon
heat-sealing of whey protein-based ﬁlms. It is assumed that glycerol and sorbitol will
behave in a similar manner with regards to hydrogen bonding. Therefore, only one
example is provided with glycerol and one with sorbitol in Figure 4.8.

B-Lg is the major whey protein, and aspartic acid (11%) and glutamic acid (16%)
are the most abundant amino acids in B-Lg. These two amino acids are also abundant in
a-La, 9 and 8%, respectively (Appendix I). Carboxyl groups in aspartic acid and glutamic
acid residues my be involved in the formation of hydrogen bonds (Figure 4.8). Another
major amino acid both in B-Lg (15%) and a—La (12%) is lysine (Appendix I). Lysine has
a reactive s-NH; group that is available for covalent bond formation with glutamine or
asparagine amino acid residues, upon heat treatments (Cheﬁel et al., 1985). A proposed
model for these covalent bond formations on the surface of heat sealed whey protein-

127

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129

based ﬁlms are illustrated in Figure 4.9. These proposed models in Figure 4.8 and 4.9
would account for the increases in C-O-H and NC observed with ESCA due to heat-
sealing of the whey protein-based ﬁlms. Although not illustrated in this study, other
possible interfacial bondings include van der Waals forces and electrostatic interaction
(Urban, 1993).

In surmnary, thermal analysis of whey protein/lipid emulsion ﬁlms was an emcient
tool to obtain information with regards to heat processing, i.e., heat-sealing and
degradation of the ﬁlms. All ﬁlms were heat scalable and showed good seal strength that
were comparable to the seal strength of lactic acid casein- and carageenan-based edible
polymers. Optimum heat-sealing temperatures (that provided the highest seal strength)
were 130 and 110°C for sorbitol- and glycerol-plasticized ﬁlms, respectively. Formation
of C-O-H and N-C bonds appeared to be important in obtaining a sealed joint for the whey

protein-based edible ﬁlms.

130

CHAPTER 5

SUITABILITY OF WHEY PROTEIN/ LIPID EMULSION EDIBLE FILMS AS

FOOD PACKAGING MATERIALS.

5.1 ABSTRACT

Whey protein isolate (WPI; 5%, w/v) ﬁlms plasticized with sorbitol (4.2-5%, w/v)
or glycerol (2.7-3.5%, w/v) were prepared with butter fat (BF; 4%, w/w of protein) or
candelilla wax (CW; 0.8%, w/v) to produce whey protein/lipid emulsion edible ﬁlms.
Moisture contents, water solubilities, moisture sorption isotherm, and sensory attributes of
the ﬁlms were evaluated to determine their suitability as food packaging materials. Heat
sealed edible pouches were manufactured using the glycerol-plasticized WPI ﬁlms
containing CW16%. The pouches were used to package and store powder cocoa mix.
Results of these studies showed that solubility of the ﬁlms in water were aﬂ‘ected by
plasticizer type; the higher the plasticizer amount, the greater was the solubility. Moisture
contents of the ﬁlms were also inﬂuenced by plasticizer type. Lower moisture content of
the ﬁlms resulted in lower equilibrium moisture contents at all aw levels. Sensory
evaluation of the ﬁlms revealed that WPI ﬁlms had no distinctive milk odor. However, the
ﬁlms were perceived to be slightly sweet and adhesive by the trained panelists. Results
ﬁom the storage study with powder cocoa mix packaged in edible whey protein-based
pouches showed that these ﬁlms were suitable for packaging powder cocoa mix and may

be suitable for packaging of non-hygroscopic foods.

131

5.2 INTRODUCTION

Various applications for free-standing protein-based edible ﬁlms have been
proposed (Debeaufort et al., 1998). These proposed applications include packaging
individual portions of foods using edible ﬁlms, dividing components within one food using
these ﬁlms, and soluble packages for pre-measure food ingredients or additives. However,
so far not many of these proposed applications have been investigated.

Water solubility, moisture sorption isothemr, sensory attributes and stability during
storage are all important attributes when considering protein-based ﬁlms as packaging
materials. Edible ﬁlms with high water solubility may be required for a pouch packaging
containing pre-measured portions which should be released into the water quickly. Also,
instant dried food preparation (as with individual beverage mixes or soups) requires quick
dispersion, thus very soluble materials (Gontard and Guilbert, 1994). Conversely, the
insolubility of edible ﬁlms needs to be considered for other speciﬁc applications, such as
when the ﬁlm has to be in contact with water during processing without releasing its
contents or if controlled release is desirable (Gontard et aL, 1994).

Typically, protein-based ﬁlms are sensitive to humidity changes (Frederick, 1996).
Moisture migration in food can induce adverse effects on the stability of food products.
The microbial and physical stability, sensory quality, and enzymatic reaction in foods are
greatly inﬂuenced by moisture content, which can change drastically by the loss or gain of
moisture during processing and/or storage (Fennema, 1985). Thus, water sorption is of
practical interest when considering particular materials for food packaging use.

Sensory attributes of edible ﬁlms, on the other hand, are important for consumer
acceptance. Food preferences by human often are based on sensory attributes, such as

132

appearance, color, ﬂavor, texture and mouth feel (Damodaran, 1996). When consumed
with other food products, it is desirable that edible ﬁlms be as tasteless as possible in
consideration for consumer acceptance (Gontard and Guilbert, 1994). There is very little
published on the sensory properties of edible ﬁlms. So far no study has been reported on
the sensory attributes of whey protein-based ﬁlms.

We have developed whey protein/lipid emulsion ﬁlms containing butter fat or
candelilla wax, and reported their barrier and mechanical properties in chapter 2. Also, we
reported on the thermal properties, heat scalability and seal strength of these ﬁlms in
chapter 4. The signiﬁcance of research of this nature is to demonstrate its possible
application. Thus, the intent of this particular study was to determine moisture contents,
solubilities in water, moisture sorption properties and sensory attributes of whey
protein/lipid emulsion ﬁlms to establish their suitability as food packaging materials. The
ﬁlms’ performances as pouch packaging materials were investigated for powder cocoa

mix.

5.3 MATERIALS AND METHODS

5.3.1 Materials

Sodium azide was purchased from Sigma Chemical Co. (St. Louis, MO). For the
determination of moisture sorption isotherm of the ﬁlms, KC2H302 and NaNOz were
obtained ﬁ'om J .T. Baker Co. (Phillipsburg, NJ). NaCl and KC] were supplied by
Mallinckrodt Specialty Chemical Co. (Paris, KY). LiCl'HZO and K2C03'2H20 were

purchased ﬁom Sigma Chemical Co. (St. Louis, MO), and CaClz-ZHZO was from Fisher

133

Scientiﬁc Co. (Fair Lawn, NJ). Mg(N03)2°6H20 was provided by EM Science (Cherry
Hill, NJ).

For the sensory evaluation, whey protein-based ﬁlms were prepared with all food
grade ingredients. D-Sorbitol and glycerol were donated by Lonza, Inc. (Fair Lawn, NJ).
NaOH was purchased from Mallinckrodt Specialty Chemical Co. and was also food grade.

Powder cocoa mix was obtained ﬁom Nestle, Inc. (Glendale, CA)

5.3.2 Film preparation

Whey protein/lipid emulsion ﬁlms were prepared as described in section 2.3.2 of
chapter 2 (Figure 2.1). Sorbitol- or glycerol-plasticized whey protein isolate (WPI) ﬁlms
were prepared without, or with butter fat (BF; 4%, w/w of protein) or candelilla wax
(CW; 16%, w/w of protein). Compositions of the ﬁlms produced are shown in Table 3.2
of chapter 3. All food grade ingredients were used to prepare the ﬁlms used for the
sensory evaluation. BF-added ﬁlms were excluded ﬁom sensory evaluation due to their

poor acceptability (they appeared and felt greasy) based on the preliminary test results.

5.3.3 Moisture content of the ﬁlms

Aluminum dishes were weighed and approximately 3 grams of the ﬁlm was added
to each dish. All ﬁlm samples were weighed to the nearest 0.0001 gram before and after
drying. Samples were dried in a drying oven (Precision Scientiﬁc model 524, Chicago, IL)
at 100 :1: 2°C for 24 hr. After drying, samples were cooled in desiccator for 30 min to
equilibrate to room temperature then re-weighed. Moisture contents of the ﬁlms were
calculated as follows:

134

Moisture (%) = [ (Wi - Wt) / Wi ] x 100 (20)
where, Wi = initial wt. of sample

Wf= ﬁnal wt. of sample after drying

5.3.4 Solubility of the films in water

A method modiﬁed from Gontard et.al. (1992) was used to measure ﬁlm’s water
solubility. The water solubility was reported as the percentage of soluble matter to initial
dry matter of the ﬁlm. Approximately 3 grams, of a all ﬁlm sample was weighed to the
nearest 0.0001 gram before and after drying. The ﬁlm was weighed and dried in a drying
oven (100 :t 2°C, 24 hr) to determine its initial dry matter weight. Another 3grams of ﬁlm
was immersed into 50 ml of water containing trace of sodium azide (0.02 % w/v; to
prevent microbial growth), and gently agitated for 24 hr at 20 i 2°C. Undissolved ﬁlm
was then taken out and dried (100 i 2°C for 24hr) to determine the weight of dry nutter
which was not solubilized in water. The weight of dry matter solubilized was calculated
by subtracting the weight of dry matter not solubilized from the weight of initial dry matter
and was reported on an initial dry weight basis as follows:

Water solubility (%) =

(wt. of initial m matter - wt. of dry matter not solubilized) x 100
wt. of initial dry matter

 

(21)

5.3.5 Moisture sorption isotherm of the films
All ﬁlms were conditioned at 25 i- 0.1°C for 48 hr in hermetically sealed glass jars

containing desiccant. First, initial moisture content (IMC) of ﬁlms were determined.

135

Aluminum dishes were weighed and approximately 3 grams of the ﬁlm was added to each
dish. All ﬁlm samples were weighed to the nearest 0.0001gram before and after drying.
Samples were dried in a drying oven at 100 i 2°C for 24 hr. Atter drying, samples were
cooled in desiccator for 30 min to equilibrate to room temperature then weighed to
determine weight loss of the samples due to moisture loss. IMC was determined as the
percentage of moisture based on the oven dry weight using the following equation:
IMC (%) = [(Wi - Wt) / Wt] x 100 (22)
where, Wi = initial wt. of sample
Wf= ﬁnal wt. ofsample aﬁer drying
To determine moisture sorption isotherm (MSI) of samples, temperature was set at 25 :t
0.1°C. Different humidity conditions were prepared by setting saturated salt solution in
desiccators. Saturated salt solutions were obtained according to ASTM standard E104
“Standard practice for maintaining constant relative humidity by means of aqueous
solutions” (ASTM, 1997). Eight diﬂ‘erent humidity conditions 18 :t 0.5%, 23¢ 0.5%,
34: 0.5%, 46: 0.5%, 54i- 0.5%, 64: 0.5%, 73: 0.5%, and 90¢ 0.5% were obtained by
using the following chemicals: LiC1°H20, KC2H302, CaC12°2H20, K2C03°2H20,
Mg(NO3)2°6H20, NaNOz, NaCl, and KC]. Aluminum dishes were ﬁrst weighed;
approximately 3 grams of the sample was added to each dish. The dishes were then placed
in the desiccator, and allowed to equilibrate. The samples were weighed every 2 days
until they reached a constant mass (taken as a smaller than 1% change in the mass of the
sample). Equilibrium moisture contents (EMC) were calculated as following:
EMC (%) = [{Pf(1 + IMC) / Pi} - 1] x 100 (23)

where, IMC = gHzO/g dry wt. product

136

Pf = ﬁnal wt. of sample
Pi =initial wt. of sample
The Guggenheim-Anderson—de Boer (GAB) equation was used to ﬁt the moisture

sorption data.

5.3.6 Sensory evaluation of the ﬁlms

Sensory evaluation was conducted using a lS-member trained sensory panel
consisting of faculty and graduate students (8 female, 7 male, age 20-55) at Michigan
State University. They were selected through a screening process for their ability and
reliability to distinguish the tested ﬁlm’s attributes. The panelists participated in one
orientation and one training session. Panelists were trained to discriminate and score
consistently for the attributes being tested; turbidity, odor, sweetness, and adhesiveness.
The training involved sampling several samples of varying intensities for each attributes
being investigated (Appendix IV). Panelists also practiced using the structured rating
scale to quantify tested attributes. Panelists were provided with feedback on their ratings.
Data collection sessions were held once a day in three consecutive days. All testing and
training sessions were conducted in a climate-controlled, sensory analysis laboratory
equipped with individual testing booths. Panelists were provided water at room
temperature (~23°C) for rinsing between samples.

Whey protein-based edible ﬁlms, stored at ambient condition (23 i 2°C, 50 i 5%
RH), were cut into 7.62 cm x 2.54 cm strips before testing. Two strips of each treatment
were presented in randomized group of four. The panel was instructed to evaluate the

ﬁlms for turbidity, odor, sweetness, and adhesiveness. Turbidity was evaluated by

137

observing the sample. To determine odor, the panelists were advised to sniﬁ‘ the sample
and allow time to rest between samples. For sweetness, the panelists were advised to taste
the sample by taking the entire sample in their mouth To determine adhesiveness,
panelists were advised to place the sample between the molars and chew ﬁve times, and
evaluate the forces required to remove the sample ﬁom the teeth aﬁer mastication.
Panelists evaluated each characteristic using a structured 9-point intensity scale,
where 9 indicated the highest and 1 the lowest intensity of an attribute. Each attribute was
rated on a separate ballot. Sensory scores were averaged for 15 judges for each treatment
(for all three replicates) and attributes tested. A space for written comments was included
at the bottom of the questionnaire. Sensory evaluation was conducted as approved by

MSU UCRIHS for use of human subjects (Appendix IV).

5.3.7 Whey protein-based pouches for powder cocoa mix

Glycerol-plasticized WPI ﬁlms without and with CW16% were selected to
investigate their effectiveness as pouch packaging materials for powder cocoa mix. A
pouch was manufactured from two pieces of ﬁlms (cut 7.62cm x 5.08cm), and heat sealed
(110°C, 40psi, 3sec) on the sides using a thenml heat sealer Model-12ASL (Sencorp
system Inc., Hyannis, MA). Approximately 10 gram of powder cocoa mix was placed
inside each pouch and the open end was sealed. The samples were stored at ambient
condition (23 i- 2°C, 50 i 5 % RH). Control samples included unpackaged powder cocoa
mix and powder cocoa mix in its original (LDPE/foil/LDPE/paper/LDPE) package. All

samples were stored and tested simultaneously.

138

Moisture content of the powder cocoa mix and whey protein-based ﬁlm pouches
were determined initially at 0 day and then every 10 days for 40 days. Powder cocoa mix
was removed ﬁom the packages by cutting one end of the pouch. Approximately 3 grams
of powder cocoa mix was placed in an aluminum weighing dish and weighed to obtain
initial weight of samples, then dried at 100 i: 2°C for 3 h in a drying oven. After drying,
samples were cooled in a desiccator for 30 min then weighed. All ﬁlm samples were
weighed to the nearest 0.0001gram before and after drying- Moisture contents of the
whey protein-based ﬁlm pouches were also determined by drying them at 100 i 3°C for
24 h. Moisture contents of the powder cocoa mix and the whey protein-based ﬁlm
pouches during storage were calculated using the equation (20) in section 5.3.3 of chapter

5.

5.3.8 Statistical analysis

The moisture content, solubility in water, equilibrium moisture content, sensory
attributes of the whey protein/lipid emulsion ﬁlms, and the moisture content changes
determination of powder cocoa mix and whey protein-based ﬁlm pouches were replicated
three times in a randomized block experiment. A new ﬁlm forming solution and new set
of ﬁlms were prepared for each replicate. Statistical analysis were made using Sigma Stat
2.0 (Jandel Corp., San Rafael, CA) and appropriate comparisons were done using

Student-Newman-Keuls method for multiple comparisons.

139

5.4 RESULTS & DISCUSSION

5.4.1 Moisture content of the films

Table 5.1 shows moisture contents of sorbitol— or glycerol-plasticized whey
protein/lipid emulsion ﬁlms. Overall, moisture contents of sorbitol-plasticized ﬁlms were
lower than those of glycerol-plasticized ﬁlms, 10.1-11.4 and 13.3-16.7 %, respectively,
although statistical comparisons were made only within the same plasticizer type. Non-
lipid containing ﬁlms had the highest moisture contents among treatments within each
plasticizer type. Addition of BF4% and CW16% to ﬁlms decreased (p < 0.05) moisture
contents of sorbitol- and glycerol-plasticized WPI ﬁlms (Table 5.1). However, this may
be also due to the lower plasticizer content of these ﬁlms since plasticizer content was
subtracted from the total dry weight of ﬁhns to accormnodate lipids incorporation (Table
4.2).

Sorbitol and glycerol (polyhydric alcohols) are carbohydrate derivatives containing
hydroxyl groups as functional groups (Appendix H). Hydroxyl groups interact with water
molecules by hydrogen bonding, thus their structural differences greatly affect the rate of
water bonding and the amount of water bound (Lindsay, 1985; Whistler and Daniel,
1985). Thus, it was expected that sorbitol-plasticized ﬁlms would have higher moisture
contents because sorbitol is a larger molecule and has more hydroxyl groups available for
hydrogen bonding than glycerol. However, this was not case. The higher moisture
contents of glycerol-plasticized ﬁlms observed here might also be responsible for lower
thernml transition temperatures (108-122°C) compared to sorbitol-plasticized ﬁlms (Table
4.4) reported previously.

140

Table 5.1. Moisture content of whey protein/lipid emulsion edible ﬁlms.

 

 

 

Treatments ' Moisture (%)
IS 11.4 :1: 0.1'
IS-BF4% 10.3 :t 0.4 b
lS-CW16% 10.1 :t 0.3b
IG 16.7 i 0.28
IG-BF4% 14.3 :1: 0.5b
IG-CW16% 13.3 :i: 0.2c

 

 

*° Comparisons are made only within the same plasticizer type and different letters columnwise
denotes signiﬁcant difference (p < 0.05), n = 3 for all treatments.
' I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; G = glycerol

141

5.4.2. Solubility of the ﬁlms in water

Water solubilities of whey protein/lipid emulsion ﬁlms are shown in Table 5.2.
Statistical comparisons were made only within the ﬁlms containing the same plasticizer.
Sorbitol-plasticized ﬁlms were dissolved after 7, 15 and 17 h depending on their
compositions (Table 5.2). Time to dissolve these ﬁlms in water increased (p < 0.05) with
decreased sorbitol contents. After 24 h at 20°C, all sorbitol-plasticized ﬁlms were soluble
in water. On the other hand, glycerol-plasticized ﬁlms were not dispersed alter 24 hr
immersion in water and showed no visual loss of integrity. Glycerol-plasticized ﬁlms
without lipids showed the highest water solubility (p < 0.05) while the glycerol-plasticized
ﬁlms with CW16% exhibited the lowest water solubility (p < 0.05) among the glycerol-
plasticized ﬁlms. These results are consistent with Cuq et al. (1997b). They reported a
strong relationship between the solubilities of the myoﬁbrillar protein-based ﬁlms and their
plasticizer contents: increased plasticizer contents increased solubilities of the ﬁlms in
water. However, addition of lipids may have also reduced ﬁlms’ solubility in water,
because incorporation of lipids reduces hydrophilicity of polymers (McHugh and Krochta,
1994c). Less hydrophilic ﬁlms are likely to interact less with water thus be less soluble.
The results of this present study indicate that whey protein-based ﬁlms with varying water
solubilities for different applications can be obtained by monitoring plasticizer contents in
ﬁlm composition.

Interestingly, moisture contents (Table 5.1) of the ﬁlms were not relevant to the
ﬁlm’s solubilities in water. Glycerol-plasticized ﬁlms had higher (p < 0.05) moisture
content of 13.3-16.7 %, but showed better resistance to water than sorbitol-plasticized

ﬁlms, whose moisture contents ranged ﬁom 10.1 to 11.4 %-
1 4 2

Table 5.2. Water solubility and elapsed time to solubilize whey protein/lipid emulsion

 

 

 

edible ﬁlms (at 20° for 24hr).

Treatments 1 Water solubility (%) Time (hr)
IS 100:0.0' 71.0.o°
IS-BF4% 100 i 0.0 ' 15 3: 0.3 "
IS-CW16% 100 i: 0.0 ‘ 17 :t 0.3 '
IG 31.6 :07“ 2410.0'
IG-BF4% 27.6 i 0.5 b 24 i 0.0 '
IG-CW16% 24.7 1: 0.3 ° 24 1; 0.0 '

 

 

 

*° Comparisons are made only within the same plasticizer type and different letters columnwise

denote signiﬁcant difference (p < 0.05), n=3 for all treatments.

1I = whey protein isolate; S = sorbitol; BF = butter fat; CW = candelilla wax; G = glycerol

143

5.4.3 Moisture sorption isotherm of the ﬁlms

The moisture sorption isotherms (MSI) of sorbitol- and glycerol-plasticized ﬁlms
are shown in Figures 5.1 and 5.2. A slow increase in the equilibrium moisture content
(EMC) of the sorbitol-plasticized WPI ﬁlms ﬁom 0 to 0.7 aw was observed followed by
exponential growth peaking at around 85% RH (Figure 5.1). A similar trend was
observed in glycerol-plasticized WPI ﬁlms (Figure 5.2).

CW16%-added ﬁlms showed the lowest EMC values in both sorbitol- and
glycerol-plasticized ﬁhns, while ﬁlms without lipids showed the highest EMC values at all
aw levels. EMC of BF4%-added ﬁlms fell between the values of the ﬁlms without lipids
and with CW. This indicates that incorporation of lipids (BF and CW) lowered water
adsorption by WPI ﬁhns, while CW being more effective than BF.

Overall, EMC values were higher for glycerol-plasticized ﬁlms. This was probably
due to the more hygroscopic characteristic of glycerol thus glycerol-plasticized ﬁlms
compared to sorbitol and sorbitol-plasticized ﬁlms. This was consistent with the
discussion earlier (section 5.4.1) that glycerol retained more water molecules in the ﬁlms
than sorbitol. The MSI of foods represents the hygroscopic properties of the components
(Iglesias and Chirife, 1982). Thus, the MSI of edible ﬁlms reﬂect hydrophilic nature of
ﬁlm-forming components, the plasticizer and the protein (Jangchud and Chinnan, 1999).
Plasticizers are generally added during ﬁlm formation to decrease intermolecular forces
among polymer chains and to increase ﬂexibility of ﬁlms, however, depending on the
plasticizer used this may increase hydrophilicity of the ﬁlms.

The GAB model curve corresponded closely with the experimental data for the

ﬁlms. The GAB model parameters are shown in Table 5.3. The C and k are factors that

144

 

% Mo'sture content (dry basis)

90

80‘

70*

60+

50

40 —«

30-

20*

10

 

 

0 IS
I IS-BF4%
A IS-CW16%

 

 

 

 

 

 

 

l r r j 1 1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0. 1

8w

Figure 5.1. Moisture sorption isotherm of whey protein/lipid

emulsion edible ﬁlms plasticized with sorbitol (tested at 25°C).
I = whey protein isolate; S = sorbitol; BF = butter fat;
CW = candelilla wax.

145

% Moisture content (dry basis)

90

 

 

OIG

3° 1 - IG-BF4%

a IG—CW16%

 

 

 

 

 

 

 

O I T I T T Y T j I
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

8w

Figure 5.2. Moisture sorption isotherm of whey protein/lipid

emulsion edible ﬁlms plasticized with glycerol (tested at 25°C).
I = whey protein isolate; G = glycerol; BF = butter fat;
CW = candelilla wax.

146

Table 5.3. Parameters of ﬁtted Guggenheim-Anderson—de Boer equation for whey
protein/lipid emulsion edible ﬁlms‘.

 

 

 

Treatments 2 Wm C k

IS 7.88 21.92 0.95
IS-BF4% 6.77 23.83 0.98
IS-CW16% 4.54 24.01 1.01
IG 1 1.26 23.73 0.96
IG-BF4% 9.83 25.68 0.97
IG-CW16% 9.24 28.39 0.99

 

 

I Wm = monolayer moisture content;
C = a factor correcting enthalpy difference between monolayer and multilayer water;
k = a factor correcting enthalpy difference between ﬁ'ee water and multilayer water.
2 I = whey protein isolate; S = sorbitol; G = glycerol; CW = candelilla wax

147

 

correct for differences in enthalpy of monolayer and free water compared to that of
multilayer water, respectively. Wm is the monolayer moisture content (Lim et al., 1999).
Monolayer moisture is the amount of water needed to form a monolayer over the
accessible polar groups of the dry matter (Fennema, 1985). The C and k parameters were
increased and Wm was decreased upon decreased concentration of sorbitol or glycerol in
the ﬁlms. Wm values of glycerol-plasticized ﬁlms were higher than those of sorbitol-

plasticized ﬁlms.

5.4.4 Sensory evaluation of the ﬁlms

Sorbitol- and glycerol-plasticized ﬁlms with BF4% felt too greasy, thus, they were
left out of the sensory evaluation. Only sorbitol- and glycerol-plasticized ﬁlms with and
without CW16% were used for sensory evaluation by the trained sensory panels. Results
of sensory evaluation: turbidity, odor, sweetness and adhesiveness, are shown in Table
5.4, and comments by the panelists are provided in Appendix IV.

In comparing turbidity (structured scale of 1-9) of the ﬁlms, the trained sensory
panel assigned 8.42 and 8.46 to sorbitol- and glycerol-plasticized ﬁlms, respectively, with
CW16%, indicating similarity of these ﬁlms to wax paper (panel training scale 9) in
appearance. Sorbitol- and glycerol-plasticized ﬁlms without lipids received lowest scores
of 2.02 and 2.07, respectively, these ﬁlms possessed transparencies close to that of LDPE
(panel training scale 1). Rhim et al. (1999a) also observed increased opacity of soy
protein-based ﬁlms upon addition of lipids (fatty acids) and reported increased whiteness
of the ﬁlms as determined by HunterLab Colorimeter L-value. According to Hernandez

(1997), transparency or opacity of polymers is due to the morphology of the polymer, and

148

Table 5.4. Sensory characteristics of whey protein/lipid emulsion edible ﬁlms.

 

Treatments 1 Turbidity Odor Sweetness Adhesiveness

 

IS 2.02 :1: 0.79b 1.38 i 0.67at 4.60 i 1.661' 3.96 :1: 1.44'
IS-CW16% 8.42 i 0.57 a 1.64 d: 0.67“ 3.78 :1: 1.60ll 3.40 :1: 1.68 '
IG 2.07:1:0.99b 1.64:1.01a 5.58:1:2.10' 2.42:1: 1.19b

IG-CW16% 8.64 :1: 0.56 ' 2.00 :1: 1.27 a 3.93 i 1.85 ' 1.76 :l: 0.80b

 

 

 

 

 

8'” Comparisons are made within the same column means, with different superscripts are
signiﬁcantly different (P < 0.05), n = 45 (3 reps x 15 judges).
1 I = whey protein isolate; S = sorbitol; G = glycerol; CW = candelilla wax

149

not related with their chemical structure or molecular mass. Morphological
inhomogeneities of CW-added ﬁlms may have caused visible light to scatter through the
thus resulting in their opaqueness.

The sensory panel did not detect any speciﬁc milk odor from the ﬁlms, and scored
them ﬁom 1.38 to 2.0 for milk odor. These values were close to the score for puriﬁed
water (panel training scale 1). These results are contrary to Morr and Ha (1993) who’s
reported that commercial whey protein products often have off-ﬂavors that limit their
uses. However, based on the data from the present study, I don’t anticipate milk odor will
be a limitation in the use of whey protein-based ﬁlms as edible packaging materials.

The panel scored sweetness of the ﬁlms as slightly less than 2.5% (w/v) of sugar
solution (panel training scale 5), and there were no signiﬁcant differences in sweetness of
the ﬁlms depending on the treatments. Relative sweetness of both sorbitol and glycerol is
approximately 60% that of sucrose (Budavari et al., 1989), which have contributed
somewhat high sweetness scores of these ﬁlms.

The adhesiveness of sorbitol-plasticized ﬁlms were higher than that of glycerol-
plasticized ﬁlms (p < 0.05). Addition of CW16% did not affect adhesiveness. Although it
was not statistically signiﬁcant, CW16%-added ﬁlms were less adhesive compared to ﬁlms
without lipids added in both sorbitol- and glycerol-plasticized ﬁlms. This may also be due
to the reduction of plasticizers in these ﬁlms. CW16%-added ﬁlms had less plasticizer
content than ﬁlms without lipid added in both plasticizer types. The glycerol-plasticized
ﬁlms with CW16% were the least adhesive among all treatments (Table 4.2).

Overall, whey protein/lipid emulsion edible ﬁlms were an acceptable in odor,

sweetness, and adhesiveness characteristics as determined by a trained sensory panel.

150

 

These qualiﬁcations may extend use of whey protein-based edible ﬁlms on foods as
coatings, wrappings, and casings, since these indistinctive sensory characteristics will not

interfere with food’s taste, ﬂavor or texture.

5.4.5 Whey protein-based pouches for powder cocoa mix

Moisture uptakes (%) of unpackaged and packaged (in commercial packages, and
in glycerol-plasticized WPI pouches with and without CW16%) powder cocoa mix
samples are compared in Table 5.5. Statistical comparisons were made within the same
storage day of powder cocoa mix stored in different packages. Glycerol-plasticized WPI
pouches with and without CW were effective in lowering (p < 0.05) moisture adsorption
by the powder cocoa mix up to 30 days compared to that of unpackaged powder cocoa
mix, however, they were not as effective as the commercial package. No differences in
moisture contents were detected in powder cocoa mix packaged in the commercial
package throughout the storage period of 40 days. This was expected since the
commercial package was a hermetically sealed bag that can maintain integrity of the food
with a long shelf life.

No signiﬁcant differences were observed between the moisture contents of powder
cocoa mix packaged in glycerol-plasticized WPI pouches with and without CW
throughout the 40 days storage period. At 40 days of storage, moisture contents of
powder cocoa mix packaged in glycerol-plasticized WPI pouches with and without CW
showed no differences compared to that of unpackaged powder cocoa mix (~3%; Table
5.5). The whey protein-based pouches started cracking when stored for 40 days. These

edible pouches were effective (P < 0.05), however, up to 30 days of storage indicating

151

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that whey protein-based ﬁlms could provide protection to a food product like powder
cocoa mix after the primary packaging is opened until this time.

Moisture content changes of pouches used to store powder cocoa mix for 40 days
are shown in Table 5.6. The moisture content of glycerol-plasticized WPI pouches
decreased signiﬁcantly (p < 0.05) after 10 days (ﬁ'om 16.7 to 11.5%) and then remained
the same throughout the storage period. A similar trend was observed for glycerol-
plasticized WPI pouches with CW, however this decrease was observed after 20 days
instead. Cracking of these pouches probably occurred due to moisture loss ﬁom the
pouch materials, thus made them brittle and crack. Moisture contents of both pouches
were ~10% after 40 days storage period. This value was lower than what was expected at
the 50% RH storage condition

Powder cocoa mix probably absorbed moisture ﬁ'om the WPI ﬁlms as well as ﬁ'om
the environment. Powder cocoa mix used in this present study contains approximately
68% sugar (w/w of serving size; value provided by Nestlé Inc.), which makes it fairly
hygroscopic (Whistler and Daniel, 1985). Better results probably would have been
obtained if these ﬁlms were used for packaging of non-hygroscopic foods such as pasta,
oatmeal, mashed potatoes ﬂake, etc. (Figure 5.3). Their stability when packaged in whey
protein-based pouches have not yet been determined.

Studies were also conducted to investigate the possible use of whey protein-based
ﬁlms as a wrap for processed cheese slices. These experiments and results are presented
in Appendix V. Overall, whey protein-based ﬁlms were ineffective as wraps for processed

cheese slices.

154

 

Figure 5.3. Various food products in pouches manufactured
using whey protein-based edible ﬁlms.

155

In summary, moisture contents of the whey protein-based ﬁlms were mainly
inﬂuenced by plasticizer type, but within the same plasticizer type, concentration of the
plasticizer affected the moisture contents of the ﬁlms. Solubilities of the ﬁlms were also
inﬂuenced primarily by plasticizer type and contents. Although no statistical comparisons
were made, glycerol-plasticized ﬁlms appeared to be less soluble than sorbitol-plasticized
ﬁlms. Incorporation of lipids effectively lowered water adsorption of the ﬁlms and formed
less hygroscopic ﬁlms as determined by the MSI. Whey protein-based ﬁlms were slightly
sweet and adhesive but lacking in milk odor. Incorporation of CW resulted in opaque
ﬁlms but did not affect odor, taste or adhesiveness of the ﬁlms as determined by a trained
sensory panel. Whey protein/lipid emulsion edible ﬁlms were suitable for packaging of
powder cocoa mix up to 30 days and may be more suitable for packaging of non-

hygroscopic foods.

156

CONCLUSIONS

. Whey protein/lipid emulsion ﬁlms containing butter fat (BF) and candelilla wax (CW)
showed better water vapor permeability (WVP) and oxygen permeability (OP) than
non-lipid containing ﬁlms.

. Lipid concentration affected WVP of the ﬁlms. Overall, WVP decreased with increase
in lipid concentration. Most desirable WVP was observed in WPI ﬁlms containing
CW16%.

. OP of the ﬁlms were signiﬁcantly inﬂuenced by lipid type, rather than lipid

concentration or whey protein type.

. Mechanical properties of whey protein/lipid emulsion ﬁlms were inﬂuenced by protein
and lipid types as well as lipid concentration. Overall, whey protein isolate (WPI)
ﬁlms with CW showed better tensile strength than whey protein concentrate (WPC)
ﬁlms with BF.

. Scanning Electron Micro graphs elucidated relationship between microstructure, and
barrier and mechanical properties of the ﬁlms.

. The main forces involved in the formation and stability of whey protein and whey
protein/lipid emulsion ﬁlms were disulﬁde and hydrogen bonds. Contribution of
hydrophobic interactions to their formation and stability was minimal.

. Protein types affected free sulthydryl group contents of the ﬁlms: 2.5 to 3.0 urnol/g of
ﬁlm for WPI ﬁlms, and 1.39 to 1.44 mng of ﬁlm for WPC ﬁlms. Disulﬁde bond
contents, although not statistically different were lower in WPC ﬁlms than in WPI
ﬁlms. Lower disulﬁde contents of WPC ﬁlms coincided with lower TS values of the

157

ﬁlms, while higher disulﬁdc contents of WPI ﬁlms agreed with higher TS values of the
ﬁlms.

8. Hydrophobicity of the ﬁlms increased with incorporation of lipids. Although CW
containing ﬁlms were more hydrophobic than BF containing ﬁlms, these differences
were not statistically signiﬁcant. Hydrophobicity of the ﬁlms coincided with reduced
WVP of the ﬁlms upon addition of lipids. Films that showed the lowest WVP had the
highest hydrophobicity values.

9. Differential Scanning Calorimetry showed onset transition temperature (T0) of 108-
122°C for the glycerol-plasticized WPI ﬁlms, and 126-127°C for the sorbitol-
plasticized WPI ﬁlms.

10. All ﬁlms were heat scalable. The seal strengths of the ﬁlms ranged ﬁ'om 110 to 323
N/m. Pressure and dwell time variation did not affect seal strength of the ﬁlms.
However, the plasticizer type inﬂuenced optimum heat-sealing temperature of the
ﬁlms, 130°C for sorbitol-plasticized and 110°C for glycerol-plasticized WPI ﬁlms.

11. Electron Spectroscopy for Chemical Analysis (ESCA) was used to study the nature of
the interfacial interactions in heat sealed and unsealed whey protein/lipid emulsion
edible ﬁlms. Main interfacial interactions on the surfaces of heat sealed whey protein-
based ﬁlms appeared to be hydrogen and covalent bonds involving C-O-H and N-C
components, respectively.

12. Moisture contents of the whey protein/lipid emulsion ﬁlms were mainly inﬂuenced by
plasticizer type, but within the same plasticizer type, concentration of plasticizer
affected moisture contents of the ﬁhns. Overall, glycerol-plasticized ﬁlms showed
higher moisture content compared to those of sorbitol-plasticized ﬁlms.

158

13. Solubilities of the ﬁlms in water were primarily inﬂuenced by the plasticizer content.
The higher the plasticizer content, the greater was the solubility of the ﬁlms in water.

14. Incorporation of lipids effectively lowered water adsorption of the ﬁlms and formed

less hygroscopic ﬁlms as determined by moisture sorption isotherm.

15. Sensory evaluation of the ﬁlms revealed that no distinctive milk odor existed in whey
protein and whey protein/lipid emulsion ﬁlms. However, the ﬁlms were perceived to
be slightly sweet and adhesive by the panelists. Addition of CW resulted in opaque
ﬁlms.

16. Whey protein/lipid emulsion edible ﬁlms may be suitable for packaging of powder
cocoa mix and should be suitable for packaging of non-hygroscopic foods.

159

APPENDIX I

160

Appendix 1. Amino acid composition of B-lactoglobulin and or-lactoalbumin
(Swaisgood, 1985).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Amino acid B-Lactoglobulin A a-Lactoalbumin B
Alanine (Ala, A) 14 3
Isoleucine (Ile, I)* 10 8
Leucine (Leu, L)* 22 13
Methionine (Met, M)“ 4 1
Phenylalanine (Phe, F)* 4 4
Proline (Pro, P) 8 2
Tryptophan (Trp, W)* 2 4
Valine (Val, V)‘ 10 6
Asparagine (Asn, N) 5 12
Cysteine (Cys, C) 5 8
Glutamine (Gln, Q) 9 5
Glycine (Gly, G) 3 6
Histidine (His, H)‘ 2 3
Serine (Ser, S) 7 7
Threonine (Thr, T)’ 8 7
Tyrosine (Tyr, Y) 4 4
Aspartic acid (Asp, D) 11 9
Glutamic acid (Glu, E) 16 8
Arginine (Arg, R)’ 3 1
Lysine (Lys, K)"I 15 12
Total residues 162 123

 

 

 

 

 

* Essential amino acids

161

APPENDIX H

162

Appendix II. Structures of polyol plasticizers.
A. Sorbitol (hexahydric alcohol); B. Glycerol (trihydric alcohol).

A. Sorbitol
ICHz-OH
H-C-OH
HO-C-H
H-C-OH
H-C-OH

CH2 - OH

B. Glycerol
ICH2 - OH
H - C - OH

CH2 - OH

163

APPENDIX III

164

 

/ Electron gun

Condenser lens

 

 

 

 

Objective

 

 

 

 

 

 

 

 

 

Final /
aperture
ﬂ

 

Electron Cathode-ray tube
beam

 

 

 

1

Detector ]

 

 

 

 

 

 

 

 

Appendix III. Schematic diagram of scanning electron microscope (SEM)
(F lcgler et al., 1993).

165

APPENDIX IV

166

 

Written consent form

Department of Food Science and [human Nutrition
Michigan State University

Edible ﬁlms prepared from whey protein isolate, sorbitol, glycerol, candelilla wax and ,
water. i

l . have read the above list of
ingredientsandﬁndnonethatIamallergictthavealsobeeninfomiedonthenauueot‘
the research (including experimental materials and procedures) which will be used during
thetasﬁngseesionlundastandtlmdietastepandwilltakeappmxinmdy 10-15
minutes.IagreetOserveonthetaste’paneLwhichwillbecouductedon'

, 1999.1understandthatlamfreetowithdrawmyconsentandto'
discontinue participation in the panel at any time without penalty.

 

UCRIHS APPROVAL FOR
THIS protect EXPIRES:

5’3“” ' FEB 1 7 zone

 

 

Date ' ‘ ' PRIOR
ABovemrEwcomivoue

167

Advertisement

Need
Whey protein-based edible ﬁlms
Sensory panel

Monday, Feb. 22, 1999
1:00 pm. - 2:00 pm.

and

Monday, March 1, 1999
1:00 pm. - 2:00 pm.

Take 5-10 minutes out of your day
to try a new product and earn a treat for helping us out.
Just stop by at any time during the above listed times.

168

Trained panel prescrecning questionnaire

Name

Phone (Day) (Evening)

Gender M or F

 

 

Age 18-25 _, 26—35 _, 36-55 _, >55 _

Time

1.

Do you plan to be on campus during this semester? _

2. Are there any weekdays that you will not be available on a regular basis?

3.

What part of the day are you normally available?
Morning (8-11) _
Early afternoon (1 1-2) _
Afternoon (2-5) __

Health

1.

2.

Do you have any food allergies (speciﬁcally to milk proteins) ?

Do you take any medications which affect your senses?

. Are you currently on a restricted diet? If yes, please explain.

What foods (speciﬁcally dairy foods) can you not eat?

What foods (speciﬁcally dairy foods) do you not like to eat?

Thank you for your time!

169

Questionnaire for panel training
1/4

Name : Date :

Characteristics studied : Turbidity

 

Please evaluate two samples by observing turbidity. Place an X next to the value which
best describe the turbidity of the samples.

Sample : __ Sample : _
Turbidity Turbidity
_9 Opaque _9 Opaque
_8 _8

__7 _7

_6 _6

_5 Moderate ___5 Moderate
_4 _4

_3 _3

0.02 _2

_1 Transparent _1 Transparent

170

2/4
Questionnaire for panel training

Name : Date :

Characteristics studied : Odor

 

Please evaluate the two samples by sniﬂing. Allow time to rest between samples. Place an
X next to the value which best describe the odor of the samples.

Sample : Sample :
Odor Odor
__9 Strong ____9 Strong
_8 _8
_7 _7
_6 __6
_5 Moderate _5 Moderate
_4 _4
_3 _3
2 2

171

3/4
Questionnaire for panel training

Name : Date :

 

 

Characteristics studied : Sweetness

Please rinse your mouth with water before starting. There are two samples for you to
evaluate. Taste each of the coded samples by taking the entire sample in your mouth.
Rinse your mouth with water between samples and expectoratc all samples and water.
Place an X next to the value which best describe the sweetness of the samples.

Sample : Sample :
Sweetness Sweetness
__9 Sweet _9 Sweet
_8 _8

_7 _7

_6 _6

_5 Moderate _5 Moderate
_4 _4

_3 __3

_2 _2

_1 Not sweet _1 Not sweet

172

4/4
Questionnaire for panel training

Name : Date :

 

Characteristics studied : Adhesiveness

Please rinse your mouth with water before starting. There are two samples for you to
evaluate. Place sample between molars and chew ﬁve times. Evaluate the force required
to remove the sample ﬁom the teeth alter mastication of the product. Rinse your mouth
with water between samples and expectoratc all samples and water. Place an X next to the
value which best describe the adhesiveness of the samples.

Sample : Sample :
Adhesiveness Adhesiveness
_9 Very adhesive __9 Very adhesive
_8 _8

_7 _7

_6 _6

_5 Moderate _5 Moderate
_4 _4

_3 _3

_2 _2

_1 Not adhesive ___1 Not adhesive

173

Trained panel Questionnaire

Product: Whey protein-based edible ﬁlm

Name :

Date :

 

1/2

 

1. Please evaluate the whey protein-based edible ﬁlms by observing and tasting each
sample in the following order. Taste the sample and rinse with water between tasting.
Place an X next to the value which best describes the characteristic intensity of the sample.

Sample number:

 

Turbidity Odor Sweetness Adhesiveness
__9 Opaque _9 Strong _9 Sweet _9 Very adhesive
_8 _8 _8 _8
_7 _7 _7 _7
_6 _6 _6 __6
_5 Moderate _5 Moderate _5 Moderate _5 Moderate
_4 _4 _4 _4
_3 _3 _3 __3
_2 _2 _2 _2
_1 Transparent _1 Weak _1 Not sweet __1 Not adhesive
Connnents :

Sample number: _

Turbidity Odor Sweetness Adhesiveness
_9 Opaque _9 Strong _9 Sweet _9 Very adhesive
_8 _8 _8 _8
_7 _7 _7 _7
_6 _6 ___6 _6
_5 Moderate _5 Moderate _5 Moderate _5 Moderate
_4 _4 _4 _4
_3 _3 _3 _3
_2 _2 _2 _2
___1 Transparent __1 Weak __1 Not sweet ___1 Not adhesive

Comments :

 

174

2/2

 

Sample number: _

Turbidity Odor Sweetness Adhesiveness
__9 Opaque _9 Strong _9 Sweet _9 Very adhesive
_8 _8 _8 _8
_7 _7 _7 __7
_6 _6 _6 _6
_5 Moderate _5 Moderate _5 Moderate _5 Moderate
__4 _4 _4 _4
_3 _3 _3 _3
_2 _2 _2 _2
_1 Transparent __1 Weak _1 Not sweet _1 Not adhesive
Comments :

Sample number: _

Turbidity Odor Sweetness Adhesiveness
_9 Opaque _9 Strong _9 Sweet _9 Very adhesive
_8 _8 _8 _8
_7 _7 _7 _7
_6 _6 __6 __6
_5 Moderate _5 Moderate _5 Moderate __5 Moderate
_4 _4 _4 _4
_3 _3 _3 _3
_2 _2 _2 _2
_1 Transparent ___1 Weak _1 Not sweet __1 Not adhesive
Comments :

 

175

Attributes Hedonic scale

Panel training samples codes

Samples

Wax paper
IS ﬁlm
LDPE

8% WPI soln.
4% WPI soln.
Puriﬁed water

5% Sugar soln.
2.5% Sugar soln.
Puriﬁed water

Caramel
Jelly bean
Gummi-bear

Trained panel treatments codes

Turbidity 9
3
1
Odor 9
5
1
Sweetness 9
5
1
Adhesiveness 9
6
1
Treatment
IS
IS-CWl 6
16
IG-CWl 6

Rep #1
code

435
1 22
644
893

Rep #2
code

585
151
974
628

176

Codes

285
516
949

491
149
981

620
352
778

257
185
564

Rep #3
code

885
117
394
931

 

Trained panel comments

Mini
Rep #1 - Slightly sweet when ﬁrst tasted
Rep #2 - Stronger whey taste.
- Sweet taste initially
- Milky taste
Rep #3 - More adhesive than other samples

- Dissolve relatively easily

m
Rep #1 - Slightly sweet at ﬁrst, not really adhesive
Rep #2 - Sweet taste initially
- Milky taste, breaks upon chewing

Rep #3 - Sweetest sample tasted.

IS-CW16 ﬁlm
Rep #1 - A little bitterness tasted after chewing
- Slightly sweet after a while
- It has some milky ﬂavor.
Rep #2 - Bad after taste.
Rep #3 - Sweet taste later on.

IG-CW16 ﬁlm
Rep #1 - Doesn’t dissolve well in the mouth
- No taste, not really adhesive
- After chewing, it disintegrate, diﬁ‘erent texture
Rep #2 - Disintegrate easily

177

APPENDIX V

178

WHEY PROTEIN-BASED EDIBLE FILMS AS A CHEESE WRAP FOR
PROCESSED CHEESE SLICES
Materials

Whey protein isolate (WPI; ALACEN 895) was provided by New Zealand Milk
Products (North America) Inc. (Santa Rosa, CA). Candelilla wax (CW) was purchased
form Strahl and Pitch Inc. (West Babylon, NY). D-Sorbitol was obtained ﬁom Sigma
Chemical Co. (St. Louis, MO), and NaOH was purchased from Mallinkrodt Specialty
Chemical Co. (Paris, KY). Kraft singles" American processed cheese slices (Kraft Foods
Inc., Glenview, IL), and Ziploc“ ﬁeezer bags (Dow Chemical Co., Indianapolis, IN) were

purchased at a local retail outlet (E. Lansing, MI). -

Methods

Film preparation The sorbitol-plasticized WPI ﬁlms and CW16%-added ﬁlms,
showed the best moisture barrier property (Table 2.3 in chapter 2), thus were selected as
cheese wraps to investigate their effectiveness on moisture loss from processed cheese
slices. Both whey protein ﬁlms were produced as described in Figure 2.1 in chapter 2.,
and composition of the ﬁlm produced were shown in Table 2.2 of chapter 2.

Preparation of cheese wraps Cheese slices (8.26cm x 7.87cm) were placed
between two layers of sorbitol-plasticized WPI ﬁlms and CW16%-added ﬁlms, then the
edge of the ﬁlms were sealed using a thermal heat sealer Model-12ASL (Sencorp System
Inc., Hyannis, MA) at 130°C, 60psi for 3scc. This heat sealing condition was selected
based on the results ﬁom the seal strength study (Table 3.5 in chapter 3). Cheese

wrapped with sorbitol-plasticized WPI ﬁlms and CW16%-added ﬁlms were placed

179

individually inside Ziplocup polyethylene bags (17.78 cm x 20.32 cm) and stored in the
chamber with ambient conditions. Effectiveness of whey protein ﬁlms as cheese wraps
were determined at 4.0 :1: 10°C in two different RH conditions of 10 :1: 3 and 88 i 5%.
Another set of cheeses wrapped in whey protein ﬁlms but without being packed in
polyethylene bags were stored in both RH. Control samples included unwrapped cheeses
and cheeses in their original package, LDPE, were stored and tested simultaneously. The
samples stored in 10% RH condition was tested for moisture loss and color change every
3 days for 9 days. The samples in 88% RH storage condition were tested every 5 days for
15 days.

Moisture content “Standard Methods for the Examination of Dairy Products” were
used to determine the moisture content of the cheese slices (Marshall, 1992). Shredded
3.0 i 0.5 g of cheese was placed in an aluminum weighing dish and dried for 16 hrs at 80
i 3°C using gravity convention oven (Precision Instruments, Chicago, IL), until a constant
weight was reached. After the drying, samples were placed in a dessicator for 30 min to
equilibrate to room temperature and weighed. Moisture content of the whey protein ﬁlms
were determined by drying 3.0 i 0.5 g of the ﬁlms for 24 hrs at 100 i 3°C. Moisture
content (% Moisture) was calculated using the equation (12) in chapter 5.

Color test HunterLab colorimeter (Hunter Associates Laboratory, Inc., Reston, VA)
were used to test color change of the cheeses. A black and a white standard tile were
used for calibration, and the black tile as the background when testing samples. Values of

L (black to white), a (green to red), and b (blue to yellow) were determined.

180

Statistical analysis

Experiments for moisture loss of cheeses, color change, and moisture contents of
ﬁhns were replicated three times in a randomized block experiment. A new ﬁlm forming
solution and new set of ﬁlms were prepare for each replicate. Statistical analysis were
made using Sigma Stat 2.0 (Jandel Corp., San Rafael, CA) and the appropriate

comparisons were done using the Student-Newman-Keuls method for multiple

comparisons.

181

 

Table V.l. Effect of wrap type on moisture content of processed cheese slices packaged

in various wraps (4°C).

 

 

 

 

 

 

 

 

 

 

 

a)

Moisture (%) at 10% RH
Wrap types ' 0 day 3 days 6 days 9 days
Unwrapped 40.3 r 0.3 a 16.2 :r 1.1 c 10.7 r 0.4 d 10.3 r 0.3 ‘
Commercial package 40.3 i 0.3 ' 39.9 :1: 0.6 ' 39.6 :1: 0.4 ' 39.1 :1: 0.9 '
IS ﬁlrnwrap 40.3 :03 ‘ 16.0:1:0.1c 10.821201" 10.3 r03d
IS-CW ﬁlm wrap 40.3 r 0.3 ° 13.2 r 0.3 d 10.2 :1: 0.3 d 10.8 r 0.5 d
IS ﬁlm wrap/ PE 40.3 r 0.3 ' 30.9 :1: 0.6 b 31.5 r 0.3 b 29.6 r 0.3 °
IS-CW ﬁlm wrap/ PE 40.3 r 0.3 a 31.5 r 0.2 b 30.4 r 0.4 ° 30.4 r 0.7 b
b)

Moisture (%) at 88% RH
Wrap types 1 0 day 5 days 10 days 15 days
Unwrapped 40.3 r 0.3 ‘ 35.9 :1: 0.7 b 34.3 :1: 0.6 " 32.7 i 0.3 "
Commercial package 40.3 a 0.3 ' 38.8 a: 0.5 r 39.2 r 0.5 ' 39.3 r 1.0 °
IS ﬁlm wrap 40.3 r. 0.3 ' 32.4 r 0.5 ° 33.1 :r 0.3 c 31.7 r 0.7 ”
IS-CW ﬁlm wrap 40.3 r 0.3 a 31.0 r 0.1 d 30.1 r 0.4 " 33.0 a 0.7 b
IS ﬁlm wrap/ PE 40.3 :t 0.3 ‘ 29.7 r 0.8 ° 29.0 :1: 0.5 c 30.2 r 0.8 °
IS-CW ﬁlm wrap/ PE 40.3 r 0.3 ' 29.1 r 0.7 ° 29.6 r 1.0 “‘ 31.1 r 0.9 “

 

 

 

 

 

"° Comparisons are made within the same column means with different superscripts are

signiﬁcantly different (P < 0.05), n = 3 for all treatments.

1 I = whey protein isolate; S = sorbitol; CW = candelilla wax; PE = polyethylene.

182

Table v.2. Effect of storage time on moisture content of edible ﬁlms used as processed

 

 

 

 

 

 

 

 

 

 

 

 

 

 

cheese slice wraps (4°C).
a)

Moisture (%) at 10% RH
Wrap types ' 0 day 3 days 6 days 9 days
IS ﬁlrnwrap 11.4101d 21.7i0.8' 15.9mm" 13.2ro.1°
IS-CWﬁlmwrap 10.1103c 19.1:02' 15.5101 b 13.7:1: 18"
IS ﬁlm wrap/ PE 11.4 r 0.1 b 42.7 r 0.2 ' 42.9 r 0.5 ' 41.9 r 0.4 '
IS-CW ﬁlm wrap/ PE 10.1 r 0.3 ° 42.7 :r 0.6 ' 42.0 r 0.4 ' 39.0 :t 0.6 b
b)

Moisture (%) at 88% RH
Wrap types 1 0 day 5 days 10 days 15 days
IS ﬁlmwrap 114:0.1d 47.210.6" 48.0:1:0.l‘ 44.3103c
IS-CW ﬁlm wrap 10.1 r 0.3 ° 41.3 r 0.2 ' 40.5 :r 0.3 b 40.6 :1: 0.2 "
IS ﬁlm wrap/ PE 11.4 i 0.1 ° 40.0 r 0.7 b 42.4 3: 0.6 ‘ 43.6 r 1.2 '
IS-CW ﬁlm wrap/ PE 10.1 r 0.3 ° 39.5 r 0.3 b 39.8 :1: 0.5 b 40.5 r 0.3 '

 

 

“i Comparisons are made within the same raw means with different superscripts are
signiﬁcantly different (P < 0.05), n = 3 for all treatments.

1 I = whey protein isolate; S = sorbitol; CW = candelilla wax; PE = polyethylene.

183

 

Table V.3. Effect of wrap type on color changes of processed cheese slices during

storage (4°C, 10% RH).

a) L-value (0 black to 100 white)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Wrap types I 0 day 3 days 6 days 9 days
Unwrapped 64.7 :1: 0.6 ‘ 52.5 i: 1.0 ° 56.0 :1: 1.3 ° 56.9 :1: 0.8 °
Conunercial package 64.7 i 0.6 ‘ 65.2 i 0.7 ' 64.1 i: 0.4 ' 64.9 :1: 0.5 '
IS ﬁlm wrap/ PE 64.7 i 0.6 ’ 60.9 :t 0.6 b 60.7 :1: 0.7 b 59.2 :1: 0.7 ”
IS-CW ﬁlm wrap/ PE 64.7 :1: 0.6 ' 60.6 i 0.7 b 60.7 :1: 0.4 b 60.2 :t 0.3 b
b) a-value (- green to + red)

Wrap types ' 0 day 3 days 6 days 9 days
Unwrapped 5.43 i 0.41 ' 9.63 :1: 0.34 ' 9.56 :t 0.81 ' 9.60 :1: 0.54 ’
Commercial package 5.43 :1: 0.41 ‘ 5.68 i: 0.41 ° 5.23 :1: 0.36 ° 5.37 i 0.15 °
IS ﬁlm wrap/ PE 5.43 :1: 0.41 ' 7.30 :1: 0.32 b 6.65 i 0.41 " 6.53 :1: 0.37 °
IS-CW ﬁlm wrap/ PE 5.43 :t 0.41 ' 7.00 :1: 0.41 b 6.51 i 0.31 b 6.38 :1: 0.31 "
c) b-value (- blue to + yellow)

Wrap types 1 0 day 3 days 6 days 9 days
Unwrapped 30.1 :1: 0.2 ‘ 26.4 i: 0.6 ° 27.4 d: 0.5 ° 27.5 1: 0.6 °
Commercial package 30.1 :t 0.2 ‘ 29.0 i 0.4 ' 29.3 i 0.4 ' 29.4 :1: 0.3 '
IS ﬁlm wrap/ PE 30.1 i 0.2 ‘ 28.1 :1: 0.6 b 28.6 :1: 0.4 b 28.3 r 0.4 "
IS-CW ﬁlm wrap/ PE 30.1 i 0.2 ' 27.9 :1: 0.4 b 28.5 i 0.2 b 28.3 t 0.3 "

 

 

 

 

 

” Comparisons are made within the same column means, with different superscripts are

signiﬁcantly different (P < 0.05), n = 3 for all treatments.

1 I = whey protein isolate; S = sorbitol; CW = candelilla wax; PE = polyethylene.

184

Table v.4. Effect of wrap type on color changes of processed cheese slices during

storage (4°C, 88% RH).

a) L-value (0 black to 100 white)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Wrap types ‘ 0 day 5 days 10 days 15 days
Unwrapped 64.7 t 0.6 ' 63.0 i 1.3 b 61.4 i 1.0 b 59.7 :1: 0.6 "
Conunercial package 64.7 i 0.6 ' 64.3 i 0.9 ' 65.0 :t 0.7 ' 65.4 i 0.8 '
IS ﬁlm wrap 64.7 :1: 0.6 ' 59.7 :1: 0.7 ° 60.3 :1: 0.4 ° 58.8 :t 0.5 "
IS-CW ﬁlm wrap 64.7 :1: 0.6 ‘ 58.8 r 0.5 ° 56.3 r 0.4 d 56.6 r 0.8 °
IS ﬁlm wrap/ PE 64.7 :1: 0.6 ' 57.2 r 0.5 d 56.7 :1: 0.3 d 57.3 :1: 0.6 °
IS-CW ﬁlm wrap/ PE 64.7 i 0.6 ' 57.4 :1: 0.5 d 57.1 i 0.7 " 56.8 :1: 0.3 °
b) a-value (- green to + red)
Wrap types ' 0 day 5 days 10 days 15 days
Unwrapped 5.43 r 0.41 ' 6.34 :t 0.69 'b 5.57 :1: 0.72 “b 5.28 :1: 0.49 '
Commercial package 5.43 :t 0.41 ‘ 5.97 i 0.51 'b 4.05 :1: 0.57 ° 3.93 i 0.35 "
IS ﬁlm wrap 5.43 i 0.41 a 6.52 i 0.26 ' 4.93 :1: 0.31 b 5.56 r 0.51 ‘
IS-CW ﬁlm wrap 5.43 :1: 0.41 ' 5.80 r 0.37 b 5.87 :1: 0.19 a g 5.68 r 0.57 '
IS ﬁlm wrap/ PE 5.43 i 0.41 ' 6.25 :1: 0.35 'b 4.97 i 0.16 " 5.42 i 0.40 '
IS-CW ﬁlm wrap/ PE 5.43 :1: 0.41 ' 5.62 i 0.23 b 5.30 t 0.18 ‘b 5.20 :1: 0.82 '
c) b-value (- blue to + yellow)
Wrap types ' 0 day 5 days 10 days 15 days
Unwrapped 30.1 :1: 0.2 ' 30.7 i 0.4 ' 31.2 i 0.5 ' 31.6 :1: 0.7 '
Commercial package 30.1 :1: 0.2 " 30.0 :1: 0.6 b 31.4 :1: 0.4 ‘ 31.5 i 0.6 '
IS ﬁlm wrap 30.1 i 0.2 ' 30.6 :t 0.6 ' 31.0 i 0.4 ' 30.7 t 0.5 "
IS-CW ﬁlm wrap 30.1 :1: 0.2 ' 29.8 :1: 0.3 b 29.7 :1: 0.4 " 30.8 r 0.2 “b
. IS ﬁlm wrap/ PE 30.1 i 0.2 ' 29.9 :t 0.4 b 29.8 :1; 0.3 b 30.3 :I: 0.6 "
IS-CW ﬁlm wrap/ PE 30.1 :1: 0.2 ° 29.6 :1: 0.3 b 29.9 i 0.5 b 30.1 i 0.3 "

 

“I Comparisons are made within the same column means, with different superscripts are

signiﬁcantly different (P < 0.05), n = 3 for all treatments.

1 I = whey protein isolate; S = sorbitol; CW = candelilla wax; PE = polyethylene.

185

 

Table V.5. Effect of storage time on color changes of whey protein ﬁlms as processed
cheese slice wraps (4°C, 88% RH).

a) L-value (0 transparent to 100 translucent)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Wrap types ' 0 day 5 days 10 days 15 days

IS ﬁlmwrap 6.83 i025 " 18.2 i1.6 ‘ 19.1:1:0.5 ' 19.3 10.8 ‘
IS-CWﬁlmwrap 29.3 i 1.3 b 42.5 i 1.1 ' 42.8i 1.3 ' 43.9:1:1.1’
IS ﬁlm wrap/ PE 6.83 t 0.25 b 19.7 :1: 1.7 ' 20.9 :1: 0.8 ' 20.1 i 1.1 ‘
IS-CW ﬁlm wrap/ PE 29.3 :1: 1.3 b 42.4 t 1.0 ' 43.2 r 0.6 ° 43.3 i: 1.2 '

b) a-value (- green to + red)

Wrap types ' 0 day 5 days 10 days 15 days

18 ﬁlrnwrap - 0.90 i 0.43 ' - 0.83 t 0.24 ' - 1.60 :1: 0.20 b - 1.88 3: 0.31b
IS-CW ﬁlm wrap - 1.47 r 0.50 ' - 3.32 :1: 0.22 " - 4.10 :1: 0.67 ° - 4.34 :1: 0.17 °
IS ﬁlm wrap/ PE - 0.90 :1: 0.43 ' - 1.28 :I: 0.15 ' - 2.25 :1: 0.27 b - 2.08 :1: 0.57 "
IS-CW ﬁlm wrap/ PE .147 :1: 0.50 ' - 3.67 i 0.24 b - 4.46 :1: 0.11 ° - 4.57 i 0.19 °
c) b-value (- blue to + yellow)

Wrap types ' 0 day 5 days 10 days 15 days

IS ﬁlm wrap 0.57 :1: 0.29 b 0.65 :1: 0.44 b 1.52 :1: 0.22 ' 1.92 i 0.26 '
IS-CW ﬁlm wrap 0.53 t 0.55 ° 3.89 :1: 0.29 b 4.37 i 0.30 ' 5.38 t 0.62 '
IS ﬁlm wrap/ PE 0.57 t 0.29 ° 1.52 :1: 0.28 b 2.31 :1: 0.13 b 2.33 :t 0.35 ‘
IS-CW ﬁlm wrap/ PE 0.53 d: 0.55 ° 3.95 :1: 0.29 b 4.56 :1: 0.57 a 4.98 :1: 0.47 '

 

 

"° Comparisons are made within the same column means, with different superscripts are

signiﬁcantly diﬂ‘erent (P < 0.05), n = 3 for all treatments.

' I = whey protein isolate; S = sorbitol; CW = candelilla wax; PE = polyethylene.

186

 

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187

 

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