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THESIS

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This is to certify that the

thesis entitled

DEVELOPMENT AND CHARACTERIZATION
OF CASEIN-WAX-BASED EDIBLE FILMS

presented by
Jay L. Chick

has been accepted towards fulfillment
of the requirements for

M - S - degree in mm

 

 

 

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MAR Ii 702004.»

 

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DEVELOPMENT AND CHARACTERIZATION OF CASEIN-WAX EDIBLE FILMS

Jay Lyle Chick

A THESIS

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

MASTER OF SCIENCE

School of Packaging

1998

ABSTRACT
DEVELOPMENT AND CHARACTERIZATION OF CASEIN-WAX EDIBLE FILMS
By
Jay Lyle Chick

This study was conducted to develop and characterize edible ﬁlms produced from
lactic acid casein and either camauba or candelilla wax. Films were produced by mixing
lactic acid casein and sorbitol in distilled water at a pH of 10, then adding either melted
carnauba or candelilla wax, followed by homogenization. Film microstructure was viewed
using scanning electron microscopy, and differential scanning calorimetry was used to
evaluate thermal properties of the ﬁlms and ﬁlm forming components. Water and oxygen
barrier properties, as well as the mechanical properties of these ﬁlms were evaluated over
various wax contents and relative humidities.

Films were found to be continuous and homogeneous with an increasing separation
of wax within the ﬁlm structure as it’s content increased. Films with candelilla wax
incorporated were found to provide the best water barrier. However, water bam'er
properties of these ﬁlms were still poor compared to synthetic polymers. These ﬁlms
provided a good oxygen barrier, and were found not to be affected by the incorporation of
either camauba or candelilla wax. Incorporation of wax increased tensile strength while
decreasing elongation compared to ﬁlms without wax, these properties also being lower
than synthetic polymers. A good, but weak, heat seal was able to be made around the

melt temperatures of lactic acid casein and sorbitol.

Dedicated to the memory of my Grandparents,
Dr. OB. and Bernice McGillicuddy and Herman and Leeta Chick,
for showing me that hard work, education, and kindness are keys to success,
and to my ﬁancee Heather for all of her love and support

ACKNOWLEDGMENTS

I would like to give a special thanks to Dr. Ruben J. Hernandez for all the help and
guidance he has given me throughout my degree program. His tutelage has helped change
my way of thinking, making me look at and solve problems in many diﬂ‘erent ways.

I would like to thank my committee members Dr. Zeynep Ustunol and Dr. Bruce
Harte for their continued help and guidance. I would also like to thank Center for Food
and Pharmaceutical Packaging Research for their ﬁnancial support of this research. New
Zealand Milk Products Inc., and Strahl and Pitsch Inc,. are also acknowledged for
providing me with the lactic acid casein and waxes respectively.

A special thanks goes out to my family for their continued support. I also want to
thank Dr. Flegler, Carol Flegler, and Shirley Owens in the Pesticide Research Center at
Michigan State University for their work on the Scanning Electron Microscope. Mike
Rich in the Composite Materials and Structure Center at Michigan State University for his
help on the Differential Scanning Calorimetry. Dr. Virginia Vega-Wamer for her help
with lab techniques. Thanks to my lab mates Han, Kim, Jong, Jeong, and Julie. A big
thanks to my friends in packaging Bill Green, Laura Bix, DeLynne Vail, Jill Warnick,
Krittika, Shannon Adams, John Jackson, Derek McDowell, Stephane Fayoux, Yoon Suk

Lee, Aaron F itchko, and all my other friends from graduate school.

iv

TABLE OF CONTENTS

 

LIST OF TABLES .............................................................................. viii
LIST OF FIGURES ............................................................................. x
INTRODUCTION .............................................................................. l
1.0 LITERATURE REVIEW ................................................................. 4
1.1 Formation of Edible Films and Coatings ..................................... 4
1.1.1 Components of Edible Films ........................................... 4
1.1.2 Processes of Edible Film a_nd Coating Formation .................... 6

1.2 Composition anleroperties of Components Used in Edible Films 7

1.2.1 Casein ..................................................................... 7
1.2.1.1 a.1- and eta-casein ............................................. 9
1.2.1.2 B- and y-casein ................................................ 10
1.2.1.3 K-casein ......................................................... 11
1.2.2 Mes ...................................................................... 12
1.2.2.1 Candelilla Wax ................................................. 12
1.2.2.2 Carnauba Wax . ................................................ 13
1.2.3 Plasticizers ................................................................ 13
1.3 Structure and Characterization of Edible Films ............................ 15
1.4 Intrinsic Bulk Properties ........................................................ 17
1.5 Properties of Films Important to Food Applications ....................... 18
1.5.1 _B_ar_ri_q ..................................................................... 18
1.5.1.1 Water Vapor Permeability (WVP) of Edible Films ....... 21

1.5.1.2 Oxygen Permeability (OP) of Edible Films ................ 26

1.5.2 Mgﬂm’gﬂ Properties of Edible Films ................................ 30

2.0 MATERIALS AND METHODS ........................................................ 34
2.1 Film Development ................................................................. 34
2.2 Film Microstructure .............................................................. 38
2.3 Thermal Characteristics ........................................................ 39
2.4 Moisture Sorption Isotherm .................................................... 39
2.5 Barrier Properties ................................................................. 42
2.5.1 _W_VP_; ...................................................................... 42

2.5.2 _O_l_’ ......................................................................... 42

2.6 Mechanical Properties ........................................................... 44
2.6.1 Strength and Elongation ............................................... 44

2.6.2 Heat Sealing ............................................................. 45

2.7 Thickness ............................................................................ 46
2.8 Statistical Analysis ............................................................... 46
3.0 RESULTS AND DISCUSSION .......................................................... 47
3.1 Film Development ................................................................. 47
3.2 Microstructure ..................................................................... 47
3.3 Thermal Characteristics ......................................................... 57
3.4 Moisture Sorption Isotherm ................................................... 61
3.5 Water Vapor Permeability ...................................................... 64
3.5.1 Wax Content ............................................................. 64

3.5.2 Wax Type ............................................................... 70

 

 

3.5.3 RH Effect ................................................................. 70
3.6 Oxygen Permeability ............................................................ 72
3.6.1 Wax Content ............................................................. 72
3.6.2 Wax Type ................................................................ 75
3.6.3 RH Effect ................................................................ 77
3.7 Mechanical Properties ........................................................... 77
3.7.1 Terﬂle Strengt_h ......................................................... 77
3.7.2 Elongation ................................................................ 85
3.7.3 Comparison to Other Polymers ........................................ 91
3.7.4 Heat Sealing ...................................... ~ ........................ 91
4.0 CONCLUSIONS ........................................................................... 100
4.1 Film Development and Characterization .................................... 100
4.2 Water Vapor Permeability ...................................................... 100
4.3 Oxygen Permeability ............................................................. 101
4.4 Mechanical Properties .......................................................... 102
5.0 APPENDICES .............................................................................. 103
Appendix A ............................................................................. 104
Appendix B ............................................................................. 106
6.0 LIST OF REFERENCES ................................................................ 107

vii

LIST OF TABLES

Table l. Casein Composition .................................................................. 8
Table 2. Composition of Candelilla and Carnauba Wax .................................... 14
Table 3. Water Vapor Permeability (WVP) of Selected Edible Films .................... 22
Table 4. Oxygen Permeability (OP) of Selected Edible Films ............................. 27
Table 5. Mechanical Properties of Selected Edible Films .................................. 31
Table 6. Composition of Lactic Acid Casein Powder ....................................... 35
Table 7. Composition of Lactic Acid-Based Edible Films ................................. 37
Table 8. Saturated Salt Solutions Used to Create RH Conditions in Moisture

Sorption Isotherm Study ...................................................... 41
Table 9. Diﬂ‘erential Scanning Calorimetry Results of Primary Film Forming

Components and Casein-Based Edible Films ................................ 59
Table 10. Experimental Data for Moisture Sorption Isotherm .............................. 63

Table 11. Water Vapor Permeabilityl of Lactic Acid Casein (50%)-Sorbitol-Wax
Edible Films ...................................................................... 66

Table 12. Water Vapor Permeability (WVP) Comparison of Lactic Acid
Casein (50%)-Sorbitol-Wax Edible Films to Other Films

(Edible and Synthetic) ......................................................... 69
Table 13. Oxygen Permeability (09)1 of Lactic Acid Casein (50%)-Sorbitol-
Wax Edible Films ............................................................... 74

Table 14. Oxygen Permeability (OP) Comparison of Lactic Acid Casein (50%)-
Sorbitol-Wax Films to other Films (Edible and Synthetic) ................ 76

Table 15. Oxygen Permeability (OP)l of Lactic Acid Casein (50%)-Sorbitol (3 5%)-
Wax (15%) Edible Films2 (dry basis) as Effected by Relative

Humidity (RH) .................................................................. 79
Table 16. Tensile Strength (TS)l of Lactic Acid Casein (50%)-Sorbitol-Wax Edible
Films ............................................................................. 82

Table 17. Elongation1 of Lactic Acid Casein (SO%)-Sorbitol-Wax Edible Films ........ 86

Table 18. Comparison of Lactic Acid Casein-Based Edible Films Mechanical
Properties ........................................................................ 92

Table 19. Seal Strength of Lactic Acid Casein (50%)-Sorbitol (50%) Edible Films .... 94

Table 20. Seal Strength of Lactic Acid Casein (50°/o)-Sorbitol (3 5%)-Carnauba

Wax (15%) Edible Films ...................................................... 95
Table 21. Seal Strength of Lactic Acid Casein (SO%)-Sorbitol (3 5%)-Carnauba

Wax (15%) Edible Films ...................................................... 96
Appendix B. Amino Acid Composition of Casein Fractions .............................. 106

ix

LIST OF FIGURES

Figure l. The Diffusion Cell for Permeability Testing ...................................... 20
Figure 2. Schematic Diagram of the Film Forming Process ............................... 36
Figure 3. Modiﬁed Oxtran 200 Schematic ................................................... 43

Figure 4. SEM of the Bottom Surface of Lactic Acid Casein (50%)-Sorbitol (50%)
Edible Film ....................................................................... 48

Figure 5. SEM of the Bottom Surface of Lactic Acid Casein (50%)-Sorbitol (3 5%)-
Carnauba Wax (15%) and Lactic Acid Casein (50%)-Sorbitol (3 5%)-
Candelilla Wax (15%) Edible F ilrns .......................................... 49

Figure 6. SEM of the Top surface of Lactic Acid Casein (50%)-Sorbitol (50%)
Edible Film ....................................................................... 50

Figure 7. SEM of the Top Surface of Lactic Acid Casein (50%)-Sorbitol (45%)—
Carnauba Wax (5%)(top) and Lactic Acid Casein (50%)-Sorbitol
(3 5%)-Carnauba Wax (15%)(bottom) Edible Films ........................ 52

Figure 8. SEM of the Top Surface of Lactic Acid Casein (50%)—Sorbitol (45%)-
Candelilla Wax (5%)(top) and Lactic Acid Casein (50%)-Sorbitol
(3 5%)-Candelilla Wax (15%)(bottom) Edible Films ........................ 53

Figure 9. SEM of the Cross Sectional Surface of Lactic Acid Casein (50%)-Sorbitol
(50%) Edible Film ............................................................... 54

Figure 10. SEM of the Cross Sectional Surface of Lactic Acid Casein (50%)-Sorbitol
(45%)-Carnauba Wax (5%)(top) and Lactic Acid Casein (50%)-
Sorbitol (3 5%)-Carnauba Wax (15%)(bottom) Edible Films .............. 55

Figure 11. SEM of the Cross Sectional Surface of Lactic Acid Casein (50%)-Sorbitol
(45%)-Candelilla Wax (5%)(top) and Lactic Acid Casein (50%)-

Sorbitol (3 S%)~Candelilla Wax (15%)(bottom) Edible Films ............. 56
Figure 12. Diﬂ‘erential Scanning Calorimetry of Primary Film Forming

Components ...................................................................... 58
Figure 13. Diﬂ‘erential Scanning Calorimetry of Casein-Based Films ....................... 60

Figure 14. Moisture Sorption Isotherm of Lactic Acid Casein (50%)-

X

Figure 15.

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21 .

Figure 22.

Figure 23.

Figure 24.

Figure 25.

Figure 26.

Figure 27.

Figure 28.

Sorbitol (50%) Edible Films .................................................... 62

Water Vapor Permeability (WVP) of Lactic Acid Casein (50%)-Sorbitol-
Carnauba Wax Edible Films .................................................... 65

Water Vapor Permeability (WVP) of Lactic Acid Casein (50%)-Sorbitol-
Candelilla Wax Edible Films .................................................... 67

Water Vapor Permeability (WVP) Comparison of Lactic Acid Casein (50%)~
Sorbitol-Carnauba Wax and Lactic Acid Casein (50%)-Sorbitol (3 5%)-
Candelilla Wax (15%) Edible Films ........................................... 71

Water Vapor Permeability (WVP) of Lactic Acid Casein (50%)-Sorbitol
(50%) Edible Films as Effected by Relative Humidity (RH) ............... 73

Oxygen Permeability (OP) of Lactiic Acid Casein (50%)-Sorbitol-Wax
Edible Films ... .................................................................... 78

Tensile Strength (TS) of Lactic Acid Casein (50°/o)-Sorbitol-Carnauba
Wax Edible Films ................................................................ 80

Tensile Strength (TS) of Lactic Acid Casein (50%)-Sorbitol-
Candelilla Wax Edible Films .................................................... 81

Tensile Strength (TS) Comparison of Lactic Acid Casein (50%)-Sorbitol-
Camauba Wax and Lactic Acid Casein (50%)-Sorbitol-Candelilla Wax
Edible Films ....................................................................... 84

Elongation of Lactic Acid Casein (50%)-Sorbitol-Carnauba Wax
Edible Films ....................................................................... 88

Elongation of Lactic Acid Casein (50%)-Sorbitol-Candelilla Wax Edible
Films ............................................................................... 89

Elongation Comparison of Lactic Acid Casein (50%)-Sorbitol-Carnauba
Wax and Lactic Acid Casein (50%)-Sorbitol-Candelilla Wax
Edible Films ....................................................................... 90

SEM of Lactic Acid Casein (50%)-Sorbitol (50%) Edible Film Heat Seal
Cross Sectional Area ............................................................ 97

SEM of Lactic Acid Casein (50%)-Sorbitol (3 5%)«Carnauba Wax (15%)
Edible Film Heat Seal Cross Sectional Area ................................. 98

SEM of Lactic Acid Casein (50%)-Sorbitol (3 5%)-Candelilla Wax (15%)

xi

Edible Film Heat Seal Cross Sectional Area ................................. 99

Appendix A. Structures of Various Compounds found in Lactic Acid Casein-Sorbitol
Edible Films ..................................................................... 104

xii

INTRODUCTION

In 1909 Dr. Leo Hendrick Baekland reacted phenol and formaldehyde together
to form the ﬁrst synthetic plastic called Bakelite. Soon to follow was the development of
many other new synthetic polymers, like cellulose acetate and polyvinyl chloride in 1927.
Polyethylene, today’s major food packaging plastic, was developed in 1935 by Imperial
Chemical Industries in England (Hanlon, 1992). Synthetic polymers now have been
developed that provide good barriers against vapor migration, possess good mechanical
properties, are transparent or opaque, and can be printed upon. As these properties have
been developed to perform useﬁrl functions for packaging their use has become
widespread. Although, natural plastics have been always used in some beneﬁcial way,
recently edible natural polymers have gained increased attention in the food and
packaging industries, due to their value added properties they can impart.

Edible ﬁlms have been used for centuries for longer food preservation, such as
the coating of ﬁuits with wax or coating foods with lard. Edible ﬁlms have a number of
advantages. They can enhance ﬁmctional and nutritional properties of a food, they can
be used to protect small pieces or portions of food, they can be used inside
heterogeneous foods to separate components, since they are edible there is the
opportunity for little to no waste being generated by allowing for the change of a
multilayer plastic to a single, recyclable layer, for example (Krochta and DeMulder-
Johnston, 1997; Guilbert, 1986).

Natural edible ﬁlms properties differ from those of synthetic materials, especially
mechanically, where they are weaker. However, edible ﬁlms show other properties that

1

make them useful materials. The advantages of natural edible ﬁlms are associated with
lowering the migration of moisture, fats, and oils from food. Edible ﬁlms also decrease
the transport of gases like oxygen and carbon dioxide, while forming a barrier against the
contamination from outside microorganisms (Kester and Fennema, 1986; Donhowe and
Fennema, 1994).

The formation of edible ﬁlms has been accomplished using naturally occurring
high molecular weight molecules, these provide a polymer matrix with enough cohesive
strength. The types of high molecular weight polymers used in the making of edible
ﬁlms fall into three categories: hydrocolloids, lipids, and a composite of both a
hydrocolloid and lipid. Composite ﬁlms are either found as a bilayer (laminate) structure
or as an emulsion. Hydrocolloids can be either proteins or polysaccharides, like corn,
soy, wheat, and milk proteins, or pectin, starch, and cellulose derivatives (Kester and
Fennema, 1986). Lipids used are fatty acids, fatty alcohols, or materials consisting of a
combination of these, such as acetoglycerides, surfactants, and waxes. Edible ﬁlms from
protein are good oxygen barriers but poor water barriers, while lipid ﬁlms have been
shown to be good water barriers and poor oxygen barriers.

This research was to develop emulsiﬁed edible ﬁlms using lactic acid casein,
either carnauba wax or candelilla wax, and sorbitol as a plasticizer. The intention was to
combine the positive qualities that the protein and lipid materials possess into one ﬁlm.
Lactic acid casein produces transparent ﬁlms with very good oxygen barrier properties
(Chick, 1996), while carnauba and candelilla wax are good water barriers and also have a
GRAS status (Donhowe and Fennema, 1993). Lactic acid casein, carnauba wax, and

candelilla wax were chosen for this study based on past studies showing that ﬁhns

produced from these components displayed better than ﬁlms produced ﬁ'om rennet

casein, beeswax, or acetylated monoglyceride (Chick, 1996; Donhowe and Fennema,

1993). The objectives of this research were:

1. To develop optimal formulations and processes in the formation of lactic acid casein,
and carnauba or candelilla wax emulsion ﬁlms.

2. To study some of the basic characteristics of these ﬁlms, including their
rnicrostructure and thermal properties.

3. To study the water and oxygen barrier properties of these emulsion ﬁlms.

4. To study the mechanical properties (tensile strength, elongation, and heat sealing

qualities) of these emulsion ﬁlms.

1.0 LITERATURE REVIEW

1.1 Formation of Edible Films and Coatings

There are three basic steps usually followed in the formation of a protein edible
ﬁlm or coating. Mixing of the ﬁlm forming constituents in a solvent is the ﬁrst step, to
obtain a dispersion of the high molecular weight polymer. This is followed by casting or
spreading a thin layer of the ﬁlm forming solution onto a smooth level surface or the food
item. A drying process then takes place to allow the solvent to evaporate, allowing the
protein to form a matrix and subsequently the free standing ﬁlm or coating (Cuq er a]. ,

1995).

1.1.1 Components of Edible Films

A high molecular weight polymer is the one basic requirement for the formation of
an edible ﬁlm or coating. This is needed because ﬁlms with enough cohesive strength
require long chain polymeric structures (Banker, 1966). Although the molecular weight of
casein (220,000 daltons) is lower than other polymers, like wheat gluten (50,000-100,000
daltonsXGennadios and Weller, 1990) or high density polyethylene (100,000 - 250,000
daltonsXSnrith, 1986), it is still high enough to form a polymer matrix. Two types of high
molecular weight polymers are used in the formation of edible ﬁlms and coatings,
hydrocolloids and lipids. There are two categories of hydrocolloids used, polysaccharides
and proteins. The polysaccharides consist of starches, gums, and modiﬁed starches.

These include alginate, carrageenan, arnylose, and cellulose derivatives. Proteins used in

edible ﬁlms and coatings include com, soy, wheat, collagen, peanut, and milk protein

4

among others (Donhowe and F ennema, 1994; Kester and Fennema, 1986). The lipids fall
into two categories, neutral lipids of glycerides which are esters of glycerol and fatty acids,
and waxes which are esters of long chain monohydric alcohols and fatty acids (Hernandez,
1994). Acetylated monoglycerides, natural waxes, and surfactants are the common lipids
used in the manufacturing of edible ﬁlms and coatings (Kester and F ennema, 1986;
Hernandez, 1994). These have included lauric, oleic, and stearic acid, carnauba,
candelilla, and beeswax, and corn, soybean, and palm oil.

A solvent system is often used in the formation of edible ﬁhns and coatings, usually
when a hydrocolloid or a composite ﬁhn is being produced. This makes it possible to
solubilize and spread the high molecular weight polymer into a thin layer. Water and
ethanol have been the two primary solvents used (Kester and Fennema, 1986).

A number of additives can also be incorporated into the ﬁlm or ﬁlm forming
solution that alter the properties of the edible ﬁlm or coating. Their purpose is to impart
more desirable properties to the ﬁlm or coating, or to give an added value to the food
system. These additives can include plasticizers, crosslinkers, vitamins, antioxidants,
ﬂavors, colors, and antimicrobials (Donhowe and F ennema, 1994; Guilbert, 1986).

Plasticizers are widely used in hydrocolloid and emulsion ﬁlms. These reduce
brittleness and increase ﬂexibility by interfering with intermolecular bonding between
adjacent polymer chains (Koelsch, 1994; Guilbert, 1986; Kester and Fennema, 1986).
Conunon plasticizers used in edible ﬁlms and coatings are glycerol, polyethylene glycol,

' and sorbitol. Crosslinkers have been used to impart an increase in cohesive strength by
enhancing intermolecular bonding. These have included such things as transglutarninase,

tannic acid, and formaldehyde. Formaldehyde, not being edible imposes some limits on

it’s use for edible packaging. Antioxidants are incorporated to delay the degradation of
the food product by way of oxidation. Ascorbic acid, citric acid, butylated hydroxyanisole
(BHA), and butylated hydroxytoluene (BHT) are commonly used food antioxidants.
Antimicrobials are used to hinder the growth of microbes, that can cause spoilage of the

food product. These have included sorbic acid and potassium sorbate among others.

1.1.2 Propesses of Edible Film apd Coating Formation

One of several processes can be used to form an edible ﬁlm or coating. These
include solidiﬁcation of melt, coacervation, and solvent removal. Solidiﬁcation of melt is
the common process by which lipid ﬁlms and coatings are produced. This involves the
melting of the lipid followed by it’s subsequent cooling to resolidify. Coacervation is the
separation of the ﬁlm forming material from solution by heating, changing pH, adding
solvents, or changing the charge. This can be simple, where only one high molecular
weight polymer is involved, or complex, where two oppositely charged high molecular
weight polymers are used. The most common process used to form hydrocolloid edible
ﬁlms is by solvent removal. In this process the ﬁlm forming constituents are dispersed in
an aqueous phase, which then undergoes a drying process to remove the solvent
(Donhowe and F ennema, 1994; Kester and Fennema, 1986).

Casting involves spreading the ﬁlm forming solution in a thin layer, so evaporation
of the solvent and formation of the ﬁlm can occur. Numerous surface types have been
used to cast edible ﬁlms. The requirements for these surfaces is that it be smooth and
level, it is able to contain the ﬁlm forming solution during drying, and that the ﬁlm is able

to be peeled intact from it’s surface after drying. Materials used for casting protein ﬁlms

have included glass, teﬂon (polytetraﬂuoroethylene), polystyrene, plexiglass

(polymethacrylate), polyethylene (PE), and polyvinyl chloride (PVC).

1.2 Composition and Properties of Components Used in Edible Films

The composition, structure, and characteristics of the components that make up
the edible ﬁlm will effect the ultimate properties of that ﬁlm. Casein and waxes have been
used as high molecular weight polymers in edible ﬁlms, while sorbitol has been a common

plasticizer.

1.2.1 Mr;

Natural liquid milk contains 33-39% protein, of which 20% are whey proteins
and 80% are casein proteins. The casein proteins are comprised of our, our, B-, rc-, and
y-casein (Table 1). Casein is generally characterized as being mainly random coil in
structure, amphiphilic (containing both hydrophobic and hydrophilic regions), with the
presence of phosphoseryl groups able to bind calcium. Two of the major casein types,
lactic acid casein and rennet casein, are precipitated from milk by the use of lactic acid or
rennet (chymosin). Lactic acid casein is characterized by an isoelectric point (pI) of 4.6,
while rennet casein has a pI near 7.0 (Southward and Walker, 1980). Primary active
groups in casein consist of the peptide, amido, and carboxyl groups present at a 832, 114,
and 99 mole equivalence per 100 kg (Appendix A)(Mark et al., 1989a). Casein has been
commonly used as adhesives, sizing agents, and as stabilizers/emulsiﬁers in foods or
paints. The properties of casein have been reviewed by Leman and Kinsella (1989),

Swaisgood (1985), Kinsella (1984), Dalgleish (1982), and Fox and Mulvihill (1982).

7

Table 1. Casein Composition

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Casein % Total Amino Acids Phosphoseryl Molecular Wt.
Fraction Casein Groups (Daltons)
01,,- 34-45 199 8-9 23,000
£12- 10-12 207 10-13 23 poo—25,000
[3- 3 1-36 209 5 23,900-23,980
y- 3-5
7,- 181 1 20,520
72- 104 0 1 1,822
73- 102 0 11,557
K- 1013 169 1 19,000

 

 

1.2.1.1 or.1- and (1.2-casein

On average, 38-45% of casein is comprised of out-casein. This protein has been
determined to be 199 amino acid units long (Appendix B), with a calculated molecular
weight of about 23,000 daltons, depending on the variant. Amino acids are monomers of
the protein molecule, consisting of at least one primary amino group (-NH2) and one
carboxyl group (-COOH), with a side chain (R) of variable composition (Appendix A).
There are 20 amino acid types commonly found in proteins.

The C-terminal of cal-casein, the end of the amino acid chain with the carboxyl
group, is comprised of some or—helices, B-sheets, and B-turns, with the rest being random
coil in structure. The or-helix is a stable structure with 3.6 amino acids per helical turn, the
side chains point outward. B-sheets are zigzag structures, where chains of amino acids are
linked together by interchain hydrogen bonds forming the sheet. B-turns occur when a
polypeptide chain bends over itself forming the turn, with it’s stability provided by a
hydrogen bond. The N-terminal, the end of the amino acid chain with the amino group, is
mainly random coil in structure. Secondary structure is limited due to the presence of
proline residues, making up about 8.5% of the residues. Proline, because of it’s
electrostatic and steric properties, interrupts helices, giving rise to bending, and cannot be
integrated into B structures (Appendix A).

There are two variants of this protein, the difference being in the number of
phosphoseryl groups, either 8 or 9. These phosphoseryl groups allow the protein to bind
Ca2+. This calcium binding can aid in strengthening the protein matrix. (1.1-casein can
bind up to 8-10 moles Cay/mole protein under normal circumstances and up to 20 mole

Cay/mole protein at high Ca2+ concentrations. All but one of the phosphoseryl groups, in
9

both variants, are found between amino acids 41 and 80, closer to the N-terminal,
contributing to it’s random coil nature. Three hydrOphobic regions are present in out-
casein, from amino acid units 1 to 40, 90 to 110, and 130 to 199. The overall charge
being -21mV at pH 6.8, with a hydrophobicity of 1172 cal/residue.

The aa-caseins comprise 10-12% of the casein proteins. It consists of a 207
amino acid unit chain (Appendix B), with a calculated molecular weight of 23,000-25,000
daltons. Again with the presence of proline, about the same amount as out-casein, any
secondary structure is limited.

There are 4 variants of org-casein, again differing in the number of phosphoseryl
groups, ranging from 10-13. These phosphoseryl groups are fairly evenly distributed
throughout the protein. A hydrophobic region is found at the C-terminal, between amino
acid units 160 and 207 . There are two cysteine residues in the protein, which could
participate in disulﬁde bonds. It is the most hydrophillic of the casein proteins, with a
hydrophobicity of 1111 cal/residue, and thus the most ionic strength dependent. The

overall charge ranges from -16 to -22mV at pH 6.8, depending on the variant.

1.2.1.2 B- and y—casein

B-casein makes up 31-36% of the casein proteins. This protein is 209 amino acid
in length (Appendix B), with a molecular weight of 23,900-23,980 daltons. B—casein is
comprised of 10% a-helix, 13% B-sheet, and 77% unordered structure (Andrews er a1. ,
1979). This random structure is again the result of the presence of 16% proline residues.
There are ﬁve phosphoseryl groups which are all located in the N-terminal. It can bind 4-

5 mole Ca2+/mole protein. B-casein is the most hydrophobic of the casein proteins, with an

10

overall hydrophobicity of 1334 cal/residue. The hydrophobic C-terminal, between amino
acid residues 30 to 209, has a hydrophobicity of 1408 cal/residue, and carries a net charge
of-12mV at pH 6.8. .
B-casein can undergo proteolysis at 1 of 3 lysine residues, 28, 104, and 106,
producing a pair of polypeptide chains, of which one halfis lost into the milk serum, with
the other halfmaking up y-casein, representing 3-5% of the total casein fraction. The
variants of this protein have amino chain lengths of 181, 104, and 102 residues (Appendix
B), with molecular weights of 20,520, 11,822, and 11,557 daltons respectively. Of these

only the largest contains a phosphoseryl group.

1.2.1.3 x-casein

The ﬁnal casein protein is K-casein, making up 10-13% of the total. It contains
169 amino acid units (Appendix B), with a molecular weight of 19,000 daltons.
Loucheaux-Lefebvre et al. (1978) determined that x-casein consisted of 26% or-helix,
31% B-sheet, and 24% B-turns. They determined that amino acid residues 105 and 106
form either an or-helix or B-sheet between two stable B-turns and another B-tum at
residues 113 to 116, making that linkage accessible to proteolysis by rennet (or chymosin).
There is only one phosphoseryl group and two cysteine residues. lc-casein can contain
from 0-3 oligosaccharide chains. These oligosaccharides can attach at the threonine
residues located at amino acid chain units 131, 133, and 135. The primary initial point of
attachment being at threonine 133. Due to the presence of only 1 phosphoseryl group 1:-
casein is able to bind only 1-2 mole Caz’lmole protein. The overall hydrophobicity is 1224

cal/residue, with an overall charge of -4 mV, at pH 6.8.
l l

1.2.2 ms

Waxes are described as being simple lipids comprised of the combination of a long
chain alcohol and a long chain fatty acid (Nawar, 1984). Carnauba and candelilla wax are
two waxes with a Generally Regarded As Safe (GRAS) status. They both are derived
from plant sources. Properties of carnauba and candelilla wax have been reviewed by

Mark et al. (1989b), Howe-Grant (1984), Bennett (1975), and Warth (1956).

1.2.2.1 Candelilla Wax

Candelilla wax is obtained from either of two small reed-like plants, the Euphorbia
celifera Alcocer or the E. antiwhilitica Zuccarini, found in Mexico and Southwest
Texas. It has been commonly used in products like chewing gum and cosmetics.

Candelilla wax is comprised of 28-29% wax esters, these being simple or
hydroxylated. In simple wax esters the side chains are comprised of a ethyl groups. It
contains 12-14% alcohols, sterols, and resins, 49-51% hydrocarbons (29,31, or 33 carbon
units long). Straight chain or cyclic free acids constitute 7-9%, with a moisture content of
0.5 - 3% and 0.7-1% inorganic residue (Table 2).

Candelilla wax has a melting point ranging from 68.5-72.5°C and a ﬂash point of
241°C. Candelilla wax is reported as being a fairly hard material. It’s saponiﬁcation
number ranges ﬁ'om 43-65, this being mg’s of potassium hydroxide (KOH) per gram of
sample to neutralize any free acids and saponify the ester linkages. The acid number, the
amount (mg’s) of KOH per gram of sample to neutralize any ﬁee acids, ranges ﬁom 12-

22, with the ester number ranging from 31-43, this being the difference between the

12

saponiﬁcation number and acid number. The iodine value is reported to range from 15-44,

this being a measure of unsaturated linkages, reported as percent iodine absorbed.

1.2.2.2 Carnauba Wax

Carnauba wax is obtained from a palm tree, Copernica cerifera Martins, found in
Brazil and the northern parts of Argentina and Paraguay. It has been commonly used for
polishes, candles, candies, and cosmetics. Carnauba wax is comprised of 84-85% alkyl
esters of wax acids, these being simple, acid, hydroxylated acid esters or diesters. It
contains 2-3% polyhydric or oxy-alcohols, 4-6% resins, and 2-3% lactides. 1.5-3% is
comprised of hydrocarbons (27, 29, and 31 carbons in length), 3-3.5% free wax acids
(carnaubic or cerotic), and 0.5-1% moisture and mineral matter (Table 2).

It has a melting point of 82.5-86°C, and a ﬂash point ranging ﬁom 298-310°C.
Carnauba wax is also considered a hard material. The saponiﬁcation number ranged
from 70-88, with an acid number ranging from 2-10, and and ester number between 75

and 88. Carnauba wax has an iodine value between 7 and 14.

1.2.3 Plasticizers

Sorbitol is a naturally occurring carbon backboned polyhydric alcohol. Besides
use as a plasticizer it is oﬁen used as a sweetener, humectant, and pharrnaceutic aid.
Properties of sorbitol have been reviewed by Merck (1989), Sicard and Leroy (1983), and
Sicard (1982).

Sorbitol was discovered in 1872 in the berries of mountain ash (Sorbus aucuparia

l3

Table 2. Composition of Candelilla and Carnauba Wax

 

Composition of Waxes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Kirk and Othmer, 1984; Bennet, 1975; Warth, 1956.

14

Structure Candelilla Wax Carnauba Wax
Wax esters 0 28-29 84-85
R-C-O-C-C-R
simple 20-21 5-6
acid 5-6
hydroxylated 6-8 53-55
diesters 19-21
Alcohols R-OH ? 2-3
Hydrocarbons C’s and H’s 49-51 1.5-3
Sterols tetracyclic rings 7-8
Resins 5-6 4-6
Free Acids 0 7-9 3-3.5
R-C-OH
Lactides - 2-3
Moisture, 0.5-3 0.5-1
Minerals, and
InoEganic Residue

 

L.) and now is produced by high pressure hydrogenation or electrolytic reduction of D-
glucose, or by catalytic hydrogenation of dextrose. It has a molecular

weight of 182 daltons, consisting of a 6 carbon chain with 6 hydroxyl groups
(CeHuOsXAppendix A). It is mainly found in the stable crystalline (y-) form, with a
melting point of 96°C. It is highly soluble in water (solubility of 234g/ 100g water at
25°C), but insoluble in most other organic solvents. It is stable and chemically unreactive.
At equal RH’s the water content of sorbitol will be lower than that of glycerol. Sorbitol is

also more resistant to changes in water content as relative humidity changes.

1.3 Structure and Characterization of Edible Films

Structure and characterization of edible ﬁlms have been used to explain the
ﬁrnctional properties which they possess. Methods commonly used to determine
microstructure are x-ray diffraction, scanning electron microscopy (SEM), and
transmission electron microscopy (TEM).

Carnauba and candelilla wax ﬁlms were found to have a high degree of
orthorhombic packing, with side-to-side hydrocarbon chain packing. The three most
common types of hydrocarbon packing are triclinic, orthorhombic, and hexagonal.
Hexagonal packing is the least dense with the lowest melting point, while triclinic is the
most dense and has the highest melting point. Candelilla wax had a higher relative
intensity (RI) ratio (0.65), ﬁom x-ray diffraction, compared to carnauba wax (0.41). This
value being stated as the R1 at 3.74 Angstroms/RI at 4.13 Angstroms. As RI increases the

amount of hexagonal packing decreases.

15

In SEM’s of the top surface, that exposed to the environment during drying,
neither had penetrating imperfections. Candelilla wax ﬁlms were shown to be relatively
smooth, while carnauba wax ﬁlms possessed a ‘hilly’ appearance (Donhowe and Fennema,
1993)

Beeswax (BW) ﬁlms are similar in composition to carnauba and candelilla wax.
They were also found to be highly orthorhombically packed (RI being 0.34), with side-to-
side hydrocarbon chain orientation. They were shown to be smooth and free of
imperfections (Donhowe and Fennema, 1993; Kester and F ennema, 1989a). Greener and
Fennema (1989) found similar morphology in a BW laminate ﬁlm. Where the BW was
formed onto the surface of a methylcellulose ( MC) ﬁlm. SEM showed a continuous and
homogeneous MC and lipid layer present in cross sectional analysis of MC/lipid laminate
ﬁlms (Park et al., 1996).

In MC and paraﬁ'rn wax emulsion ﬁlms wax droplets could be seen dispersed
throughout the protein matrix. These droplets causing a ‘hilly’ appearance to the surface
(Martin-Polo et al., 1992; Debeaufort er al., 1993).

Milk protein and fatty alcohol or fatty acid emulsion ﬁlms showed the presence of
crystalline platelets on the top surface (McHugh and Krochta, 1994b & d; Ho, 1992). The
size and number irregular structures increased as chain length decreased and concentration
of the lipid increased. More surface irregularities appeared in caseinate/fatty acid ﬁlms
compared to caseinate/fatty alcohol ﬁlms, with lipids of equal carbon backbone length
(Ho, 1992).

Whey protein isolate (WPI)/BW emulsion ﬁlms were also reported as having

discrete crystalline particles present (McHugh and Krochta, 1994d). Further studies

16

showed that increased homogenization of the ﬁlm fornring solution resulted in a smaller
lipid particle size and a more even distribution throughout the protein matrix (McHugh

and Krochta, 1994b). Caseinate/BW showed the appearance of wax droplets dispersed
uniformly throughout the protein matrix. Similar results were found for

caseinate/carnauba wax ﬁhns (Ho, 1992).

1.4 Intrinsic Bulk Properties

Intrinsic bulk properties are inﬂuenced by the molecular mass and distribution, and
how the polymer chain is structured, either linear, branched, or crosslinked (Hernandez,
1997). Some important intrinsic bulk properties for edible ﬁlms include the melt range
(T...) and the heat seal temperature range. With a higher melting temperature (T...) a ﬁlm
or material generally possesses less low melting components, which can correspond to a
stiffer less permeable ﬁlm. This is a result ﬁom stronger packing and more secondary
bonding, thus less chain mobility (Birley, Haworth, and Batchelor, 1992). Diﬁ’erential
scanning calorimetry (DSC) is the general method used in testing thermal properties.

Donhowe and F ennema (1993) performed thermal studies on microcrystalline wax,
BW, carnauba wax, and candelilla wax. They found that microcrystalline wax to have a
broad melting range, with a T... of 71 .7°C. BW and candelilla wax were similar in that
their melting points were 62.0 and 640°C, respectively, with each having a solid-solid (s-

3) transition. Carnauba wax had a melting point of 81 .7°C, with two s-s transitions.

17

1.5 Properties of Films Important for Food Applications
1.5.1 13,431;

The quality of many food products is dependent on their loss of or exposure to
gases and vapors. These include water (H20), oxygen (02), carbon dioxide (C02), and
volatiles (ﬂavors, antioxidants, etc.) One of the roles a polymer used in packaging plays
is to control the migration of these gases and/or vapors (mass transport) into or out of the
package.

The rate at which these gases or vapors pass through a polymer is known as it’s
permeation. There are three steps involved in the action of permeation, absorption of the
gas or vapor into the polymers surface, followed by it’s diﬂ‘usion through the polymer, and
ﬁnally by it’s desorption through the opposite surface (Sperling, 1992; Birley er al., 1992).
This is expressed by the equation:

P = D x S (1)
where P = permeability coefﬁcient, D = diffusion coefﬁcient, and S = solubility constant.
Solubility is based on the fact that like dissolves like, so gases and vapors with similar
solubility parameters to the particular polymer will dissolve more easily into the polymer.

Henry’s law expresses this action as:

CD = Sp (2)
where CD = dissolved equilibrium concentration, and Sp = gas solubility constant. This
expression holds true if Sp is a linear function of the volumetric proportion of the

amorphous phase, but a temperature dependence and the presence of polymer crystallirrity

l8

can aﬂ’ect sorption (Sperling, 1992; Birley et al., 1992). To compensate for this a dual
sorption model was developed, based on Henry’s law. This expression states:

C. = C1) + CH (3)
where C. = total effective gas concentration, and CH = gas concentration assumed to be
adsorbed into the holes of the polymer (Birley et al., 1992).

Diﬂ’usion is the transport of the gas or vapor molecules through the polymer. This
occurs in a direction from high concentration to that of low. This process is expressed by
Fick’s laws, Fick’s ﬁrst law of steady state transfer states:

J = -D (SC/6x) (4)
where J = ﬂux, the rate of transfer per unit area, D = diffusion coefﬁcient, and 8C/8x =
concentration gradient of the permeant in the x-direction. The diffusion coeﬂicient is very
temperature dependent. For unsteady state diffusion Fick’s second law states:

8C/8t =D(82C/6x2) (5)
where the change in rate is proportional to the change in concentration gradient with
permeant penetration depth (62C/8x2 = O at steady state) (Sperling, 1992; Birley er al. ,
1992)

There are two basic processes for determining the permeation of the gas or vapor,
the isostatic or quasi-isostatic method. In both cases you have the permeant ﬂowing over
one side of the ﬁlm, permeating through the ﬁlm and collected on the other side of the
ﬁlm. The difference in the two methods is that with the isostatic method the permeant is
constantly swept out of a diﬂilsion cell (Figure 1) and carried to a quantitating sensor,

while with the quasi-isostatic method the permeant is allowed to collect in the diﬁirsion

l9

 

 

Carrier gas in
+ Upper cell half:
collection of permeant
Carrier gas out

Permeation through material

A
___>
/
Lower cell half: permeant source

Figure l. The Diffusion Cell for Permeability Testing

 

 

 

 

 

 

 

\

20

cell and then sampled over a period of time. The test is generally run until steady state is

reached.

1.5.1.1 Water Vapor Permeability (WVP) of Edible Films

Lipid ﬁlms have been found to provide a water barrier similar to that of high
density polyethylene (HDPE), which is considered to be an excellent barrier to water.
Protein ﬁlms do not provide good barriers to water, due to their hydrophilic nature (Table
3).

Donhowe and F ennema (1993) studied the WVP of wax ﬁlms and found candelilla
wax ﬁlms displayed the best water barrier properties, similar to HDPE. Microcrystalline
and carnauba wax had WVP values almost double that of candelilla wax, with BW being
almost three times larger. The higher WVP in microcrystalline wax was attributed to the
absence of polar lipids, causing a less rigid structure. The larger concentration of polar
compounds (fatty acids, fatty alcohols, and esters) in BW was attributed to it’s higher
WVP. Carnauba wax, while having a high concentration of polar compounds, possessed
the highest melting point (less low melting fraction) providing it with less molecular
mobility. The superior barrier properties of candelilla wax were found to be due to the
low concentration of polar compounds, the large concentration of alkanes, and being the
most orthorhombically packed of the waxes (Donhowe and Fennema, 1993).

Kester and Fennema (1989b) reported the water barrier properties of their ﬁlms in
terms of the resistance to water vapor transmission. This was calculated according to:

r=Ac/J (6)

21

Table 3. Water Vapor Permeability (WVP) of Selected Edible Films.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Film Typel WVP x10“ Testing Conditions Reference
(ljjgm/m2 3 Pa)

Carnauba Wax 0.033 21°C, 0%/85% RH 309:2?6 and Fennema
Candelilla Wax 0.018 21°C, O%/85% “
Beeswax 0.058 21°C, 0%/85% “
CZ(83)-G(17) 8.9-13.3 21°C, 0%/85% ParkandChinnaM1995)
SPI(63)-G(37) 179-304 25°C, 0%/50% Brandenburg er a1. (1993)
WG(71.4)-G(28.6) 125.4 26°C, 50%/100% Aydt etal. (1991)
NFDM(75)-G(25) 93.7 21°C, O%/85% Maynes and Krochta (1994)
WPI(50)-G(50) 7.5 25°C, 0%/77% McHughandKrochta (1994)
WPI(50)-S(50) 4.1 25°C, 0%/77% “
WPI(62.5)-S(37.5) 3.0 25°C, 0%/77% “
WPI(67)<3(33) 336.5 23°C, 55%/100% Banerjee and Chen (1995)
WPC(67)-G(33) 295.5 23°C, 55%/100% “
CC(67)<3(33) 219.6 23°C, 55%/100% “
scum-003) 358.2 23°C, 55%/100% “
PC(67)-G(33) 349.0 23°C, 55%/100% “
RC(50)-S(50) 57.4 378°C, 0%/90% Chick (1996)
LAC(50)-S(50) 52.1 378°C, 0%/90% “
LAC(50)-G(50) 68.6 378°C, 0%/90%
LAC<58)-S(42) 39.3 378°C, 0%/90%
WPI<67)-G(33)£R(40) 38.2 25°C, 0%/100% (Sgélxmmerandmhta
WPI(67)-G(33)-CN(40) 35.9 25°C, 0%/100% “
WPI(67)-G(33)-BW(40) 12.5 25°C, 0%/100% “

 

 

 

 

r CZ - corn zein, SPI- soy protein isolate, WG - wheat gluten, NFDM - non fat dry milk, WPI - whey

protien isolate, WPC - whey protein concentrate, CC - calcium caseinate, SC - sodium easeinate, PC -
potassium caseinate, RC - rennet casein, LAC - lactic acid casein, G - glycerol, S - sorbitol, CR - carnauba
wax, CN-eandelillawax, BW-beeswax. Numbersinparenthesesare%ofﬁlmcomposition

22

 

where r = resistance to vapor or gas transmission, Ac = driving force for vapor or gas
transmission in terms of concentration gradient across the ﬁlm, I = steady state ﬂux of the
vapor or gas.

BW ﬁlms formed on Whatman No. 50 ﬁlter paper BW provided resistance to
water transmission similar to that of PET. These properties were attributed to the
smooth, defect ﬁ'ee surface, and it’s orthorhombic packing. Hydrocarbon chains present
on the surface mask the polar groups of wax esters. Esters will not spread on the surface
of water (Kester and Fennema, 1989b).

In protein ﬁlms it was found that formation of ﬁlms at a pH away ﬁom the
isoelectric point (pI) provided them with better water barrier properties (Stuchell and
Krochta, 1994; Gennadios et al., 1993 a). In preformed ﬁlms, however, it was found that
water barrier properties were enhanced by adjusting the ﬁhns pH to the pI of the protein
(Avena-Bustillos and Krochta, 1993). This allowed for more protein-protein
interactions.

The type and the concentration of plasticizer has also been found to effect the
water barrier properties of ﬁlms (Chick, 1996; McHugh er al., 1994a; Gennadios et a1. ,
1993a). As plasticizer concentration decreased, an increase in overall protein
concentration, a decrease in WVP has been witnessed. Sorbitol was found to provide a
ﬁlm with better water barrier properties then either glycerol or polyethylene glycol. This
was attributed in part to the ability of glycerol to absorb more water than sorbitol (Sicard
and Leroy, 1983). Protein concentration was shown not to effect water barrier properties
in casein ﬁlms (Chick, 1996). The high plasticizer content in these ﬁlms, 42% minimum,

most likely being the main factor in these results.

23

Different variations of the same protein have been found to eﬂ‘ect water barrier
properties of protein ﬁlms (Chick, 1996; Banerjee and Chen, 1995; Avena-Bustillos and
Krochta, 1993). Whey protein concentrate (WPC) ﬁlms promoted a better water barrier
than WPI ﬁlms (Banerjee and Chen, 1995). A higher residual fat content present in WPC
was cited as the main reason for the better properties. Calcium caseinate (CC) ﬁlms were
shown to provide a better water barrier than other caseinate ﬁlms (Banerjee and Chen,
1995; Avena-Bustillos and Krochta, 1993). Stronger ionic crosslinking was present in CC
ﬁlms, decreasing segmental rotation. Chick (1996) found no difference in water barrier
properties of lactic acid casein and rennet casein ﬁlms. The lactic acid casein in this study
contained a higher protein and fat content than the rennet casein, 87.3, 1.2% and 80.6,
0.5% respectively. Rennet casein, however, does not have the hydrophilic macropeptide
(in x-casein) associated with it, and contained a higher ash content, 7.8% compared to
1.8%. Calcium, 8 portion of the overall ash content, present in rennet casein ﬁlms could
have provided for more calcium crosslinking.

In wax emulsion ﬁlms, as lipid concentration was increased in emulsion ﬁlms the
water barrier properties improved (Kim and Ustunol, 1997; Baker, 1996; McHugh and
Krochta, 1994d; Avena-Bustillos and Krochta, 1993; Ho, 1992). This improvement
would continue until a critical lipid content was reached, at which point no further
reduction in WVP would occur.

As the chain length of the lipid increases the water barrier properties were
improved in emulsion ﬁlms, due to more chain mobility associated with the shorter carbon
chains (McHugh and Krochta, 1994d; Kamper and Fennema, 1984). Fatty alcohol

emulsion ﬁlms provide poorer water barriers than fatty acid emulsion ﬁlms of the same

24

chain length. The polar hydroxyl group in the fatty alcohol provided more interaction with
water. BW emulsion ﬁlms were found to provide a better water barrier than stearic acid
emulsion ﬁlms (Avena-Bustillos and Krochta, 1993). BW emulsion ﬁlms also provided a
better water barrier than candelilla wax emulsion ﬁlms, which in turn were found to be
better than carnauba wax emulsion ﬁlms (Shellharnmer and Krochta, 1997; Gontard et al. ,
1994; Ho, 1992). In emulsion ﬁlms, carnauba wax was found to be diﬁcult to mix into
solution.

Changes in RH have been found to effect the water barrier properties of edible
ﬁlms. F ennema, Donhowe, and Kester (1994) observed no change in the water resistance
of BW ﬁlms at O and 50% RH (100% RH on the other side of the ﬁlm), while a decrease
in resistance occurred at 65 and 80% RH (100% RH on the other side of the ﬁlm). These
results coincided with moisture sorption results for BW.

McHugh er al. (1994a) found similar results for WPI ﬁlms made with sorbitol or
glycerol. An exponential increase in WVP was observed at about 30% RH, with a more
pronounced RH effect on ﬁlms containing glycerol. In SC ﬁlms an exponential increase in
WVP was witnessed at around a 65% RH (McHugh, Avena-Bustillos, and Krochta,
1993)

WVP of cellophane ﬁlms dipped in paramn wax were equivalent at 22 and 44%
RH, then increased at 57% RH (Martin-Polo, Manguin, and Voilley, 1992). In WPI-BW
emulsion ﬁlms an exponential increase was observed at around 50% RH, this same
increase was seen at around 30% RH in ﬁlms without BW (McHugh and Krochta, 1994d).

BW was also seen to decrease the effect of RH on WVP. Kim and Ustunol (1997)

25

observed WVP’s approximately ten times greater at 80% RH compared to that at 50% in

ﬁlms comprised of WPC or WPI, with or without candelilla wax.

1.5.1.2 Oxygen Permeability (OP) of Edible Films

Lipid ﬁlms have been found to be rather poor oxygen barriers. Most of which
falling in the permeability range of high density polyethylene (HDPE) to polyethylene
terephthalate (PET), both considered poor oxygen barriers (Donhowe and Fennema,
1993; Kester and Fennema, 1989a). Protein ﬁlms, however, have generally possessed
good oxygen barrier properties, comparable to that of nylon (Table 4).

Donhowe and Fennema (1993) found that candelilla and carnauba wax ﬁlms
displayed much better oxygen barrier properties than microcrystalline wax or BW ﬁlms.
Microcrystalline wax ﬁlms were found to have the highest OP of the waxes. They
attributed this to the microscopic sized crystals creating intercrystalline paths for oxygen
permeation. They also suggest the presence of irregular crystal sizes and lattice
distortions, leading to these intercrystalline pathways. Microcrystalline wax also contains
some distillate oil, creating a less rigid structure.

The OP of BW ﬁlms were found to be two times higher than HDPE. The presence
of unsaturated hydrocarbons was cited as the reason for higher permeability. Kester and
Fennema (1989a), however, found BW ﬁlms to be a much better 02 barrier than HDPE.
The diﬂ‘erence being that these ﬁlms were formed on Whatman No. 50 paper, which could
have aided in their barrier properties. Candelilla wax, being the most orthorhombically
packed created it’s better oxygen barrier ﬁlm properties. Carnauba wax ﬁlms had similar

OP properties to candelilla wax, with it’s smaller low melting point fraction making up for

26

Table 4. Oxygen Permeability (OP) of Selected Edible Films.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Film Type’ or x 10 Testing Reference
(m3 m/rn2 s Pa) Conditions

Carnauba Wax 180 21°C, 0% RH Donhowe and Fennema
(1993)

Candelilla Wax 200 21°C, 0% “

Beeswax 1080 21°C,0% “

CZ(83)-G(l7) 15.0-51.9 21°C, 0% Park 311d Chinnan (1995)

SPI(63)-G(37) 5_5 25°C, 0% Brandenburg er a1. (1993)

WG(71.4)-G(28.6) 4_4 23°C, 0% Gennadios (1993)

WPI(70)-G(30) 33,0 23 cc, 0% McHugh and Krochta
(1994)

WPI(70)-S(30) 5.0 23°C, 0% “

WPI(50)-S(50) 9.6 23°C, 0% “

Ream-sow 1.18 23°C, 0% Chick (1996)

LAC(50)-S(50) 0.85 23°C, 0% “

LAC(50)-G(50) 2.52 23°C, 0% “

LAC(58)-S(42) 0.94 23°C, 0% “

WPI(55.6)-S(28.5)-BW(16) 13.4 23cc, 0% McHugh and Krochta
(1994)

 

 

 

 

1 Cl - corn zein, SPI - soy protein isolate, WG - wheat gluten, WPI - whey protien isolate, RC - rennet
casein, LAC - lactic acid casein, G - glycerol, S - sorbitol, BW - beeswax. Numbers in parentheses are %

of ﬁlm composition.

27

 

it being less orthorhombically packed than candelilla wax.

WG ﬁlms were found to provide a better oxygen barrier than corn zein (CZ) ﬁlms.
This was attributed to a high or-helical content in CZ ﬁlms (Gennadios, Weller, and Testin,
1993c). ). WG properties were derived from it’s tight and complex structure formed by
extensive covalent and non-covalent bonding, with inter- and intramolecular disulﬁde
bonding (Gennadios er al., 1993 c).

Similar to WVP an increasing pH above the p1 was found to enhance the oxygen
barrier properties (Rayas, 1996; Brandenburg et al., 1993). The eﬂ’ect that plasticizer type
and concentration have on the OP of WPI-based ﬁlms was studied by McHugh and
Krochta (1994c). They found that ﬁlms containing sorbitol will provide a better oxygen
barrier than ﬁlms containing glycerol, at equal concentrations. They also showed that as
plasticizer concentration increased OP increased.

OP was found to be between EVOH and Nylon 6 in ﬁlms made with lactic acid or
rennet casein (Chick, 1996). Protein concentration was found to have no signiﬁcant effect
on OP. Protein or plasticizer type also showed no signiﬁcant eﬂ‘ect on OP, except with
rennet casein ﬁlms plasticized with glycerol, which had higher OP values.

In emulsion ﬁlms made with WG and AM there was no signiﬁcant difference in
OP’s compared to ﬁlms without lipid (Gennadios, Weller, and Testin, 1993 d). However,
lipid content was below 3%. Kim and Ustunol (1997) reported a signiﬁcant decrease in
OP in emulsion ﬁlms made ﬁom WPI or WPC and candelilla wax compared to ﬁlms
without wax (wax content greater than 2%). They also reported no signiﬁcant diﬂ‘erences

in OP among ﬁlms containing 2, 4, or 8% wax.

28

The eﬂ‘ect of RH on OP was studied by Rayas (1996). No signiﬁcant eﬂ’ect was
observed until about 30% RH, here a signiﬁcant increase in OP was seen. This data
correlated fairly closely with moisture sorption isotherms of WC ﬁlms. This same effect
was seen in WPI ﬁlms (McHugh and Krochta, 1994c). WPI-glycerol ﬁlms witnessed no
signiﬁcant RH eﬂ‘ect until 30 or 40% when an exponential increase in OP was observed,
with maximal OP occurring at about 75% RH. WPI-sorbitol ﬁlms were similar with the
exponential increase occurring at around 50% RH and the maximum OP being witnessed
also at 7 5%. These results were similar to the eﬁ‘ects of RH on WVP properties of these
WPI ﬁlms.

Rico-Pena and Torres (1990) observed no RH eﬂ‘ect in the 0 to 57% range in MC-
palmitic acid ﬁlms. However, a doubling of OP was observed at 79% RH. These results
also coincided with moisture sorption isotherms. Hagenmaier and Baker (1996) reported
OP values of 60% gelatin and 40% candelilla wax-oleic acid emulsion ﬁlms to be
equivalent at 40 and 55% RH, with and increase at 75%. In ﬁlms comprised of 20%
gelatin and 80% candelilla wax-oleic acid OP’s at 40 and 55% were equivalent, however,
a decrease was observed at 75%.

Hernandez (1994) and Gavara and Hernandez ( 1994) studied the eﬂ‘ects of RH on
nylon 6 and amorphous nylon ﬁlms tested at 23°C. In amporphous nylon they observed a
sharp decrease in OP from 0 to 20% RH reaching an asymptotic value after that. This was
explained by the combination of an initial increase in diﬂirsion from the O to 5% RH region
followed by a slow increase in diffusion and a sharp decrease in oxygen solubility in the 0
to 20% RH region which then leveled oﬂ’. The sharp decrease in solubility was explained

by the competition for chemi-active sites between water and oxygen, with the interaction

29

between water and amophous nylon being stronger in this region (Hernandez, 1994). In
semicrystalline nylon 6 ﬁlms OP decreased ﬁom 0% RH to a minimum reached in the
range of 30 to 50% RH followed by an increasing OP with an increasing RH (Hernandez,
1994; Gavara and Hernandez, 1994). Diﬂirsion was similar to that of amorphous nylon
(Gavara and Hernandez, 1994), while solubility acted similar to it’s OP, with a minimum
in the 30 to 40% RH range (Hernandez, 1994). Here, formation of water clusters may
have disrupted the polymer crystalline regions instead of ﬁlling void regions as with the

amorphous nylon (Hernandez, 1994).

1.5.2 Mechanical Prpperties of Edible Films

The mechanical properties a polymer possesses can be determined by it’s stress-
strain tensile characteristics. Stress is measured as the force/area, while strain is the
dimensionless fractional length increase (Hernandez, 1997). The ultimate tensile strength
(TS) is the maximum tensile stress a material can sustain. Ultimate elongation (E%) is the
strain at which the sample ruptures. Strength increases with an increase in molecular
mass and an increase in intermolecular forces, but decreases with the presence of
plasticizers. Generally, the TS and E% of edible ﬁlms have not been as high as synthetic
polymers (Table 5).

TS was found to increase as pH increased (Brandenburg et al., 1993; Gennadios er
al., 1993 a). The E% was also found to peak at higher pH’s, however, differences were
not signiﬁcant compared to other pH values. The use of crosslinking agents was found to

increase the tensile strength, with no effect to the E% (Rayas, 1997; Motoki et al., 1987).

30

Table 5. Mechanical Properties of Selected Edible Films.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Film 'rypel TS (MPa) E (“/19 Reference
SPI(63)-G(3 7) 3.1-52 66.5-90.3 Brandenbmg et a1. (1993)
WG(71.4)-G(28.6) 1,8 25 Aydt et al. (1991)
NFDM(75)-G(25) 9,1 5,2 Maynes and Krochta (199)
WP1(70)-G(30) 13 ,9 30, 8 McHugh and Krochta (1994)
WPI(70)-S(30) 14.0 1.6 “
WPI(50)-S(50) 14.7 8.7 “
WPC(67)-G(33) 3 ,4 20,8 Banerjee and Chen (1995)
WPI<67)-G(33) 5.9 22.7 “
CC(67)-G(33) 4.3 1.5 “
scam-C(33) 3.0 29.9 “
PC(67)-G(33) 3.0 42.8 “
RC(50)-S(50) .9, 5 77 ,6 Chick (1996)
LAC<50)-S(50) 7.5 156.0 “
LAC(50)-G(50) 1.2 253.6 “
LAC<58)-S(42) 1 1.6 150.6 “
WPI(67)-G(33)-CR(40) E 23 £15 Shellhammer and Krochta

(1997)
WPI<67)-G(33)-CN(40) 5 16 z 1.0 “
WPI(67HS(33)-BW(40) a 18 a 2.0 “

 

 

 

 

’ CZ - corn zein, SP1 - soy protein isolate, WG - wheat gluten, WPI - whey protien isolate, RC - rennet
casein, LAC - lactic acid casein, G - glycerol, S - sorbitol. Numbers in parentheses are % of ﬁlm

composition.

31

 

With an increase in plasticizer content (a decrease in protein content) a decrease in TS is
observed while E% increased (Maynes and Krochta, 1994; Stuchell and Krochta, 1994).

Banjeree and Chen (1995) demonstrated that WPI produced stronger ﬁlms than
WPC. E% was found to be similar between the WP1 and WPC ﬁlms. CC ﬁlms produced
stronger ﬁlms than SC or PC. However, the reverse of this was true pertaining to E%.
Due to the stronger ionic crosslinking in CC, creating a less extensible ﬁlm.

Gennadios et al. (1993b) found TS to decrease as RH increased in both CZ and
WG ﬁlms. RH was shown to have more of an eﬂ’ect on WG ﬁlms, due to WG being more
hydrophilic than CZ. Isotherms of CZ and WG ﬁlms showed this to be true (Gennadios
and Weller, 1994).

In emulsion ﬁlms the addition of lipids to protein ﬁlms has produced mixed results
in terms of the ﬁlms TS. However, E% has either remained unchanged or decreased with
the addition of lipids (Shellhammer and Krochta, 1997; Kim and Ustunol, 1996; Banerjee
and Chen, 1995; Debeaufort and Voilley, 1995; Gennadios et al., 1993).

Shellharnmer and Krochta (1997) found a fairly linear decrease in TS with the
addition of wax from 10 to 90%, in ﬁhns comprised of WPI and candelilla wax, carnauba
wax, or BW. Films containing carnauba wax were always found to be the strongest, while
candelilla wax always produced the weakest ﬁlms. The addition of BW did not have much
of an effect on E%, while the addition of candelilla and carnauba wax produced a slight
reduction in E%. Kim and Ustunol (1996) found a signiﬁcant increase in TS in WPI-
candelilla wax emulsion ﬁlms as wax content increased, from 0 to 8%. In WPC-candelilla
wax emulsion ﬁlms a signiﬁcant increase in TS was observed with the addition of wax,

but did not increase with increasing wax content, from 2 to 8%. The addition of candelilla

32

wax to WPC ﬁlms was found to decrease E%, but that decrease remained unchanged with
an increasing wax content, from 2 to 8%. In WPI-candelilla wax emulsion ﬁlms a

signiﬁcant decrease in E% was not observed until the addition of 4% wax.

33

2.0 MATERIALS AND METHODS

2.1 Film Development

The primary ﬁlm components were lactic acid casein powder, carnauba or
candelilla wax, and sorbitol. Lactic acid casein powder (Alacid 710, 30 mesh),which will
be referred to as lactic acid casein, was obtained from New Zealand Milk Products (N.
America) Inc, (Santa Rosa, CA)(Table 6). Carnauba and candelilla wax were obtained
from Strahl and Pitsch Inc. (West Babylon, NY). Sorbitol was purchased from Sigma
Chemical Co., (St. Louis, MO).

Figure 2 shows a schematic diagram of the ﬁlm forming process. Emulsion
solutions, 150 g ﬁnal weight, were prepared by ﬁrst mixing lactic acid casein (5% w/w)
with sorbitol (3.5, 4, 4.5, or 5% w/w), distilled water, and 1M NaOH (to adjust pH to
10.0). Solutions were heated and stirred, using the “Magna-4” magnetic stirrer and hot
plate, model 4820-4 (Cole-Parmer, Chicago, IL), for 30 minutes to a ﬁnal temperature of
65.5 :1: 25°C, and then held at that temperature for 15 minutes. This process solublized
the lactic acid casein and sorbitol in accordance with that developed by Chick (1996).

Solutions were then heated to the melting temperature of the wax, 75 i 2°C for
solutions where no wax or candelilla wax were to be added, and 85 :1: 2°C for solutions
where carnauba wax was to be added. Melted wax (0.5, 1, or 1.5% w/w) was then added
to the solutions, with the total primary ﬁlm components of the ﬁnal solutions (casein,
sorbitol, and wax) being maintained at 10% w/w (Table 7).

This was followed by homogenization for 1 min with a Polytron PT 10/35
(Brinkmann Instruments, Switzerland) using a PTA 20 TS generator (homogenizing

34

Table 6. Composition of Lactic Acid Casein Powderl

% found in
Protein x 87.3
Ash 1 .8

Moisture 9.6

Fat 1.2
Lactose O. l
1 Values based on company speciﬁcations with pH (5% at 20°C) being 4.6 (New Zealand Milk Products
(N. America) Inc.)

 

35

Mix ﬁlm forming components
lactic acid casein, sorbitol,
distilled water, and 1M NaOH

Heat / Stir
45 min total
(to 656°C, then hold for 15 min)

Melt wax (carnauba or candelilla wax)

1

Heat protein solution to the melting point of the wax

1

Add melted wax to the protein solution

1

Homogenize
(l min)

i

Filter solution
(cheeTcloth)

Equilibrate
(4 hr a1 23°C)

Vacuum
(30 min)

Cast solution
(polystyrene petri dish)

Dry
(36 hr)

Figure 2. Schematic Diagram of the Film Forming Process

36

Table 7. Composition of Lactic Acid Casein-Based Edible Films

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primary Film Components
Lactic Acid Casein Sorbitol Wax
Powder (Carnauba or
Candelilla)
Treatment % W/W % total % WIW % total % w/w % total
aqueous primary aqueous primary aqueous primary
solution components solution components solution components
1 5 50 5.0 50 0 0
2 5 50 4.5 45 0.5 5
3 5 50 4.0 40 1.0 10
4 5 50 3 .5 3 5 1 .5 l 5

 

37

 

head) and a PCU 11 power control unit at setting 5 (settings ranging from 0 tolO). This
was performed in order to produce more uniform was particles within the ﬁlm structure,
as was shown by McHugh and Krochta (1994b).

Solutions were maintained at 23 i 20°C for 4 hours to allow any foaming created
during the mixing/homogenization process to settle. Next, samples were ﬁltered through
1 layer of cheesecloth. A vacuum was applied to the solutions for 30 minutes, using a
hydrometric vacuum system, to remove any residual air in the solution. The ﬁlm forming
solution (19 i 2mL) was then cast on to a 150 x 15mm polystyrene petri plate. Casting
was performed using these petri plates as compared to teﬂon coated pans previously used
(Chick, 1996), in an attempt to better control thickness. These petri plates provided a
smaller and more level casting surface than that provided by the larger teﬂon coated pans.
Drying was carried out at 23 at 2°C and 50 j: 5% RH for 36 :1: 5 hours, at which point
ﬁlms were able to be peeled intact from the casting surface. Previously, ﬁlms were dried
in an oven to reduce drying times (Chick, 1996), however, again to better control
thickness ﬁlms were dried in room conditions on a smooth level surface. The oven racks
were found not to be completely level. Once peeled from the casting surface, ﬁlms were

kept at 23 i 2°C until testing was performed.

2.2 Film Microstructure

Microstructure of ﬁlm samples were determined using a scanning electron
microscope, JOEL 6400V SEM (Japan Electron Optics Limited, Japan). Samples were
prepared using standard techniques, mounted on aluminum stubs and sputter coated with
gold (20nm). Samples were observed using an accelerating voltage of 10 to 15kV. SEM

38

pictures were taken of the ﬁlms top and bottom surface, the top surface being that
exposed to the air when cast, and the cross section. SEM pictures of heat sealed ﬁlms
were taken of the cross section of the seal area, using an accelerating voltage of 13kV.
For surface analysis a magniﬁcation of 500X was used, while for cross sectional analysis
magniﬁcation ranged between 350 to 500x, ﬁtting the entire cross section into the picture.

A representative samme of each ﬁlm type was used for SEM studies.

2.3 Thermal Characteristics

Thermal characteristics were determined by differential scanning calorimetry
(DSC), DSC 2920 Modulated‘DSC (TA Instruments). Samples, 6 to 12mg, were sealed
in a standard aluminum dish, with a sealed empty aluminum dish used as the reference
sample for testing. Tests were conducted from -25 to 150°C with an increase in
temperature of 10°C/min. Lactic acid casein, carnauba wax, candelilla wax, and sorbitol,
and ﬁlm samples of lactic acid casein (50%)-sorbitol (50%), lactic acid casein (50%)-
sorbitol (3 5%)-carnauba wax (15%), and lactic acid casein (50%)-sorbitol (3 5%)-
candelilla wax (15%)(the composition is reported on a primary ﬁlm component basis,

unless otherwise speciﬁed) all tests were carried out in triplicate.

2.4 Moisture Sorption Isotherm

A moisture sorption isotherm curve was determined for lactic acid casein (50%)-
sorbitol (50%) ﬁlm. Five to six grams of ﬁlm, out into small pieces, were placed into
aluminum dishes then allowed to reach equilibrium (when weight gain of sample ceased) at

23°C and RH’s of 9. 1, 34.8, 46.3, 54.0, 75.5, and 80.8%. Proper RH conditions were

39

obtained by use of salt solutions placed in sealed 5 gallon HDPE buckets (Table 8). Once
equilibrium was established, when weight gain ceased, moisture content was determined
by drying ﬁhn samples in a vacuum oven (Precision Scientiﬁc model 524) at 100°C for 24
hours (Banerjee and Chen, 1995), according to the equation:

MC = [1 - (wtf)/(Wti)] x 100 (7)
where MC = percent moisture in the ﬁlm sample, Wtf = ﬁnal weight of the ﬁlm sample,
wt; = initial weight of the ﬁlm sample. MC values are reported as the average of 3 values
at each RH condition.

The experimented moisture sorption isotherm values were ﬁt by the Guggenheim-
Anderson-DeBoer model (GAB):

EMC=(WmC kaw)/[(1-kaw)(1-kaw+Ckaw)] (8)

where EMC = equilibrium moisture content on a dry basis, Wm = monolayer in the BET.
theory, C = Guggenheim constant, k = factor correcting properties of the multilayer
molecules with respect to bulk liquid, aw = water activity (Bizot, 1983). Equation (8) can

then be transformed to:
atJEMC=aaw2+Baw+Y (9)
where:
or = k/Wm[1/C - 1] (10)
B =1/Wm[1- 2/C] (11)
y = 1/Wm [C x k] , (12)

where a quadratic equation is performed on experimental values to ﬁnd or, B, and y.

40

Table 8. Saturated Salt Solutions Used to Create RH Conditions in Moisture Sorption
Isotherm Study

 

 

 

 

 

 

 

 

 

 

Sat. Salt Solution Formula RH% Q3°Q
Lithium Chloride LiCl H20 9.1
Magnesium Chloride MgClz 6H20 34.8
Potassium Carbonate K2C03 2H20 46.3
Magnesium Nitrate Mg (N03); 6H20 54.0
Sodium Chloride NaCl 75.5
Ammonium Sulfate (NI-la)2SOa 80.8

 

 

41

2.5 Barrier Properties
2.5.1 W

Water vapor transmission rate (WVTR) was tested at 37.8 at 05°C and at 50 i 3
and 70 i 3% RH. according to ASTM standard F 1249-90, by using the Permatran-W
3/31 (MoCon Inc, Minneapolis, MN). The testing surface area of ﬁlm samples were
reduced ﬁom 50 to 5 cm2 with the use of a foil backing, to stay within sensor range.
Samples were conditioned for 6 hours at the RH conditions before testing was conducted.
The WVTR was measured until steady state was reached. Steady state occurs when
permeation of the gas or vapor is constant. Calibration of the system was executed with
0.025 mm (1 mil) thick standard polyester (PET) ﬁlm. WVP was then calculated
according to:

WVP = WVTR x t/ Ap (13)
where t = thickness of ﬁlm, Ap = pressure differential acting on the ﬁlm. WVP values
were the average of at least three ﬁlm samples produced from a diﬂ‘erent ﬁlm forming

solution batch.

2.5.2 Q2

Oxygen transmission rate (OTR) was evaluated according to ASTM standard D-
3985-81, using a modiﬁed Oxtran 200 (MoCon Inc., Minneapolis, MN)(Figure 3)(Gavara
and Hernandez, 1994). Tests were performed at 23 :1: 0.5°C and 0, 20, 35, 50, and 70%
RH. Humidity of the gas streams was monitored using wide-range hygrosensors (10-90 i
2.0% RH) with a Hydrodynamics digital hygrometer indicator and controller, model 3088

(Newport Scientiﬁc, Inc., Jessup, MD). Only ﬁhn samples with no wax and 15% wax

42

  

 
   

detector

 

diffusion
cell
chamber
N2 nitrogen tank X needle valve i
0, oxygen tank ® hygrometer
wb washing bottle E ﬂow meter

 

 

 

Figure 3. Modiﬁed Oxtran 200 Schematic

43

were evaluated at RH’s above 0%. Samples with 15% wax content, instead of 5 and
10%, were chosen because they possessed the better water barrier properties.

A 50 cm2 diffusion cell was used, with compressed air (1 atm) being the permeant
and nitrogen the carrier gas. Samples were conditioned for at least 4 hours at the testing
conditions before testing was conducted. The ﬂow was measured until steady state was
reached. Calibration of the system at 23 °C and 0% RH was executed with 0.025 mm (1
mil) standard PET ﬁlm. This same PET ﬁlm was also tested at all the elevated RH
conditions. OP was then calculated according to:

OP=OTRxl/Ap _ (14)
where t = thickness of ﬁlm, Ap == pressure differential acting on the ﬁlm. OP values were
the average of at least three ﬁlm samples, each produced from a diﬂ’erent ﬁlm forming

solution batch.

2.6 Mechanical Properties
2.6.1 Strgngth app Elongation
Tensile strength (TS) and elongation were tested according to ASTM standard D-
882-3, using the Instron Universal Testing Machine model 4201 (Instron Corp, Canton,
MA). TS and elongation tests were performed at 23 :1: 2°C and either 50 or 75 i 5% RH.
Temperature conditions were maintained by the use of a conditioned room at 23°C and
50% RH, while the 7 5% RH was maintained by the use of a saturated salt solution (NaCl)
in a sealed bucket HDPE stored in the conditioned room. Samples stored at 75% RH
were tested at room conditions, however, the exposure time to these conditions was kept

at an absolute minimum, the time needed to measure thickness and test the sample. Heat

44

seal strength was tested at 23°C and 50% RH. Sample sizes for all tests were 2.54 cm
wide and at least 8 cm long, with a jaw separation of 5.08 cm and a jaw speed of 50.8
cm/min (20 in./min). TS and elongation were calculated according to:

TS=ld/( lxw) (15)
where id = load (lbs), I = material thickness, w = material width.

Elongation = (AL / L.) x 100 (16)
where AL = change in length, L, = original length.
All mechanical property values reported are the average of at least three ﬁlm samples
tested, each from a different ﬁlm forming solution batch. The values of each individual
ﬁlm sample in each ﬁlm forming solution batch was an average of at least two ﬁlm samples

from that batch.

2.6.2 Heat Sealing

Heat sealing of ﬁlm samples was accomplished using a heat sealer, model 12ASL
(Sencorp Systems, Inc., Hyannis, MA). Two strips of ﬁlm, 2.54 cm wide and
approximately 5.08 cm long, were cut, measured for thickness, then sealed widthwise.
The seal area was approximately 2.54 cm x 1.5 cm. Seal temperatures ranged ﬁom 93 to
121°C, based on DSC and preliminary results. Dwell time was either 2 or 4 seconds, with
a seal pressure of 40 or 60 psi. Seal strength was derived from the same test procedure
and calculations as TS (equation 15). The seal was located midway between the jaw grips

when tested. Heat seal testing was performed on ﬁlm samples with no wax and 15% wax.

45

2.7 Thickness

Thickness was measured using a micrometer, TMI model 549M micrometer
(Testing Machines Inc; Amittyville, NY). For water and oxygen barrier testing thickness
was the average of 5 measurements taken over the testing surface. Average thickness for
WVP and OP were 0.104 :t 0.007 mm and 0.111 i 0.009 mm respectively. For TS and
elongation thickness was the average of three measurements taken over the length the test
specimen. For heat seal strength testing thickness was the average measurement taken

from each ﬁlm layer in the area that the seal was formed in.

2.8 Statistical Analysis

Statistical analysis and appropriate comparisons as related to the effect of wax
type, wax content, and the effect of RH on WVP, OP, TS, elongation, and the eﬂ‘ect of
sealing parameters (time, pressure, and temperature) on seal strength were made using
Sigma Stat 1.0 (Jandel Corp, San Rafael, CA) performing multiple comparisons with the
Student-Newman-Keuls method for multiple comparisons or analysis on ranks when equal

variance was not observed. A randomized block design was used for all analyses.

3.0 RESULTS AND DISCUSSION

3.1 Film Development

Films formed without wax were smooth, transparent in appearance, and ﬂexible.
Films made with wax were smooth and ﬂexible, however, they showed a hazy or opaque
appearance. Wax could not be incorporated above 15%, at a protein powder
concentration of 50%, because higher wax contents produced ﬁlms that could not be
separated from the casting surface. These results are similar to those found by Ho (1992).
Carnauba wax was found to be more difﬁcult to mix into solution than candelilla
wax. This is shown by a non-consistent coloring in ﬁlms made with carnauba wax, versus
a consistent coloring in ﬁlms with candelilla wax. This most likely occurs due to the
higher melting point of carnauba wax, causing it to solidify more rapidly upon cooling.
Homogenization was performed at room temperature, causing the quicker cooling of

carnauba wax. Similar results were found in ﬁlms produced by Gontard et al. (1994).

3.2 Microstructure

SEM’s of the bottom surface, adjacent to the casting surface when drying, of ﬁlms
with no wax, 15% carnauba wax, and 15% candelilla wax showed no diﬂ‘erences in
appearance (Figures 4 and 5). In ﬁhns withor‘rt wax the top surface, open to the
environment when drying, appeared to be similar to that of the bottom surface (Figure 6).
Films without wax appeared to be smooth and defect ﬁ'ee, except for occasional tiny air

bubbles appearing on the surface.

47

 

Figure 4. SEM of the Bottom Surface of a Lactic Acid Casein (50%)-Sorbitol (50%)
Edible Film

48

 

Figure 5. SEM of the Bottom Surface of a Lactic Acid Casein (50%)-Sorbitol (3 5%)-
Carnauba Wax (15%)(top) and a Lactic Acid Casein (50%)-Sorbitol
(3 5%)-Candelilla Wax (15%)(bottom) Edible Film

49

 

Figure 6. SEM of the Top Surface of a Lactic Acid Casein (50%)-Sorbitol (50%) Edible
Film

50

Top surface microstructure of ﬁlms containing wax, 5 or 15% carnauba wax and 5
or 15% candelilla wax, showed the appearance of wax droplets dispersed throughout the
ﬁlm giving the ﬁlms a ‘hilly appearance’ (Figures 7 and 8). These results correspond well
with those found by Ho (1992) in caseinate-BW and caseinate-camauba wax ﬁlms. Wax
droplet size appeared to be fairly consistent due to the homogenization process. McHugh
and Krochta (1994b) showed this to occur in WPI-BW-sorbitol emulsion ﬁlms. When
comparing SEM’s of the top surface to those of the bottom surface in ﬁlms containing
wax, a separation of the wax, with the wax being mainly dispersed at or near the top
surface is seen. No signiﬁcant morphological differences could be seen among the surface
of ﬁlms containing carnauba wax or candelilla wax. The increased wax content can be
observed when comparing SEM’s of ﬁlms containing 5% wax to those with 15% wax,
coverage of the ﬁlms surface with wax is more complete in ﬁlms containing 15% wax
(Figures 7 and 8).

Cross sectional analysis showed that the ﬁlms were continuous and homogeneous
(Figures 9, 10, and 11). No defects, like air bubbles, were found to go through the entire
thickness of the ﬁlms. In ﬁlms without wax a layering structure is present (Figure 9). One
layer shows up at the bottom surface, and is what we believe to be unsolubilized sorbitol
(Figure 9). This layer also appears in ﬁlms containing wax, most prevalent in those
containing candelilla wax (Figure 11). This is assumed to be sorbitol because it’s relative
thickness appeared to decrease as the sorbitol content decreased, protein content remained
constant. A thin layer is also seen near the top surface of ﬁlms without wax. This is

believed to be a drying layer, because of the increase in cracking evident (Figure 9). This

51

 

Figure 7. SEM of the Top Surface of a Lactic Acid Casein (50%)-Sorbitol (45%)-
Carnauba Wax (5%)(top) and a Lactic Acid Casein (50%)-Sorbitol (3 5%)-
Camauba Wax (15%)(bottom) Edible Film

52

 

 

Figure 8. SEM of the Top Surface of a Lactic Acid Casein (50%)-Sorbitol (45%)-
Candelilla Wax (5%)(top) and a Lactic Acid Casein (50%)-Sorbitol (35%)-
Candelilla Wax (15%)(bottom) Edible Film

53

 

 

Figure 9. SEM of the Cross Sectional Surface of a Lactic Acid Casein (50%)-Sorbitol
(50%) Edible Film (arrow represents perceived sorbitol layer)

54

 

Figure 10. SEM of the Cross Sectional Surface of a Lactic Acid Casein (50%)-Sorbitol
(45%)-Camauba Wax (5%)(top) and a Lactic Acid Casein (50%)-
Sorbitol (3 5%)-Carnauba Wax (15%)(bottom) Edible Film

55

 

Figure 11. SEM of the Cross Sectional Surface of a Lactic Acid Casein (50%)-Sorbitol
(45%)-Candelilla Wax (5%)(top) and a Lactic Acid Casein (50%)-Sorbitol
(3 5%)-Candelilla Wax (15%)(bottom) Edible Film

56

layer does not show up in ﬁlms containing wax, possibly due to the wax content at the top
surface, not allowing this cracking to occur.

In cross sections of ﬁlms containing wax, less of a layering structure was evident in
ﬁlms containing 5% wax as compared to ﬁlms with 15% wax. A deﬁnite change in
appearance occurs near the midpoint of the cross section, in ﬁlms containing 15% wax,
that is not as apparent in ﬁlms containing 5% wax. One possible explanation could be the
result of the higher wax content altering the drying of the ﬁlm, producing this eﬂ’ect.
Another possibility is that 15% wax is a high enough content to show a separation of

phasesbeginning to occur.

3.3 Thermal Characteristics

DSC results of the lactic acid casein powder showed a rather broad endothermic
peak, with it’s melt occurring between 100 and 110°C (Figure 12, Table 9). The change
in heat ﬂow was relatively low, with a maximum around -0.82 W/ g. Sorbitol possessed a
narrow endothermic peak around 100°C (Figure 12, Table 9). However, it’s change in
heat ﬂow was higher, around -4.5 W/g. DSC analysis of carnauba wax and candelilla wax
where similar to that of Donhowe and F ennema (1993 ), with carnauba wax and candelilla
wax having fairly narrow endothermic peaks between 82 and 86°C and 68 and 73°C
respectively (Figure 12, Table 9). The change in heat ﬂow for the waxes were around -3.1
and -2.4 W/g for carnauba and candelilla wax respectively.

Thermal analysis of lactic acid casein (50%)-sorbitol (50%) edible ﬁlms showed
one broad endothermic peak in the temperature range of 100 to 110°C and a change in
heat ﬂow of -O.61 W/g (Figure 13, Table 9), corresponding more closely to that of

57

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1 1 me“!

17 m. Fl

IF-

1. m6.

(ﬁlm) Mot-.1 12911

58

Table 9. Diﬂ'erential Scanning Calorimetry Results of Primary Film Forming Components

 

 

 

 

 

 

 

 

 

 

 

 

 

and Casein-Based Edible Films
Approximate Approximate Change
Melt Range in Heat Flow
(°CL (we:

Film Components

Lactic Acid Casein 100-110 -0.82

Sorbitol 98-102 -4.5

Carnauba Wax 82-86 -3.1

Candelilla Wax 65-7 3 -2.4
Films

Lactic Acid Casein (50%)- 100-110 -0.61

Sorbitol (50%)

Lactic Acid Casein (50%)- 85, 105-110 -0.8, -0.55

Sorbitol (3 5%)-Carnauba Wax (15%)

Lactic Acid Casein (50%)- 65, 110-115 10.7, -055

Sorbitol (3 5%)-Candelilla Wax (15%)

 

 

59

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60

lactic acid casein powder than sorbitol. However, ﬁlm samples containing wax showed
what appears to be two endothermic peaks. Lactic acid casein (50%)-sorbitol (3 5%)-
carnauba wax (15%) ﬁlms showed one deﬁnite narrow endothermic peak in the
temperature range similar to that of carnauba wax, and one possible broad peak in the
range similar to the lactic acid casein-sorbitol ﬁlms (Figure 13, Table 9). The change in
heat ﬂow in both peaks are similar to that of lactic acid casein, being -.08 and -O.55 W/g
for the ﬁrst and second peak respectively. The same is shown for lactic acid casein
(50%)-sorbitol (3 5%)-candelilla wax (15%) ﬁlms, where two endothermic peaks appear
(Figure 13, Table 9), one similar to that of candelilla wax and one similar to the lactic acid
casein-sorbitol ﬁlm. Again, with the change in heat ﬂow for both being similar to lactic
acid casein, -0.7 and -0.55 for the ﬁrst and second peak respectively. These results
coincide closely with those from the SEM studies, where a partial separation of the two
phases may have occurred. Partial in these DSC studies because we do not see a change

in heat ﬂow of the ﬁlms containing wax to be similar to the waxes on their own.

3.4 Moisture Sorption Isotherm

The GAB model curve corresponded closely with experimental data for the lactic
acid casein (50%)-sorbitol (50%) ﬁlms (Figure 14, Table 10). The GAB model constants
for the lactic acid casein ﬁlm were 4.778, 22.739, and 1.034 for Wu, C, and k respectively.
From these results we see a slow increase in the equilibrium moisture content of the lactic
acid casein-sorbitol ﬁlms from 0 to about 7 0% RH, followed by exponential growth
peaking at around 85% RH. From this you can predict an increase in permeability and

elongation properties, and a decrease in tensile strength will occur in these ﬁlms near the

61

EMC (9 1120/1110 9 solids)

 

0 0.2 0.4 0.6 0.8

Water Activity

 

 

 

—GAB Model Curve
I Experimental Data

 

 

Figure 14. Moisture Sorption Isotherm of Lactic Acid Casein

(50%)-Sorbitol (50%) Edible Films
(Tested at 23°C)

62

 

Table 10. Experimental Data for Moisture Sorption Isotherm

 

Lactic acid casein powder

Rennet casein powder

 

 

 

 

 

 

 

 

 

 

 

(50%)-sorbitol (50%) (58%)-glycerol (42%)
RH (7.) EMC(g 1120/1003 solid) EMng H20/100g solid)
0 o o
9.1 2.5 16.4
34.8 6.7 25.9
46.3 9.4 27.4
54.0 11.1 28.3
75.5 22.6 39.2
80.8 26.5 45.9

 

63

 

70% RH point. The added moisture present in these ﬁlms above this level would act to

further plasticize the ﬁlms.

3.5 Water Vapor Permeability
3.5.1 Wax Content

It was found that as the content of carnauba wax was increased ﬁ'om 5 to 15% in
lactic acid casein (50%)-sorbitol-wax edible ﬁlms (sorbitol and wax comprise the other
50%) there was a signiﬁcant decrease (p < 0.05) in WVP at both 50 and 70% RH (Figure
15, Table 11). This can be explained by the increased surface area of the ﬁlm covered by
the carnauba wax, with less of the casein exposed, as seen in the SEM’s (Figure 7). This
same was also seen with the increase in candelilla wax content (Figure 16, Table l 1). At
50% RH, no signiﬁcant difference (p < 0.05) in WVP was observed between the lactic
acid casein (50%)-sorbitol (50%) ﬁlms and those containing up to 10% carnauba wax
(Figure 15, Table 11). However, WVP was better in ﬁlms containing 15% carnauba wax.
It was expected that WVP would decrease as carnauba wax content increased because of
the good water barrier properties carnauba wax possesses, however, this did not occur.
This is most likely a result of the difﬁculty in the incorporation of carnauba wax into the
ﬁlms, due to it’s high melting point (Section 3.1). Due to this, more disruption in the ﬁlm
matrix occurred, creating an increased number of void spaces at the protein-wax interface.
This is ﬁrrther seen at 70% RH, a higher moisture driving force, where the WVP was
signiﬁcantly higher (p < 0.05) in ﬁlms containing carnauba wax compared to ﬁlms with no

wax.

18

 

 

16

14

 

 

.5
N

 

a
O

 

WVP x 10“ (kg mint2 8 Pa)
or

 

El 50% RH.
.7096 R.H.

 

 

 

 

'16 Carnauba Wax
(prinary carponant basis)

Figure 15. Water Vapor Permeability (WVP) of Lactic Acid
Casein (50%)-Sorbitol-Camauba Wax Edible Films
(Tested at 318°C, thickness = 0.1mm)

65

Table 11. Water Vapor Permeabilityl of Lactic Acid Casein (50%)-Sorbitol-Wax Edible

 

 

 

 

 

 

 

 

 

 

 

 

 

Films2
Wax Type(%) WVP x 10“ (kg m/m2 s Pa) WVP x 10“ (kg mm2 s Pa)
@ 50% RH @ 70% RH
No Wax (0) 2.23 x 060'” 8.54 :t 0.82’
Carnauba (5) 2.45 a 0.60' 17.91 i 1.02”
(10) 2.26 i 074‘” 18.32 a 0.30”
(15) 1.08 :1: 006° 13.76 i 0.66c
Candelilla (5) 1.42 a 0.26“ 6.78 :1: 0.61‘1
(10) 1.01 :1; 022°" 6.00 :132“
(15) 0.68 i016“ 5.14:1:088‘
"° Different letter columnwise denotes signiﬁcant diﬂerence (p < 0.05)
‘ All WVP tests done at 378°C

2 Sorbitol and Wax comprised the remaining 50% of the ﬁlms (on a dry basis), thickness of ﬁlms
were 0.104 :1: 0.007 mm (4.09 i 0.03 mils)

 

10

 

 

 

 

 

WVP x 10“(ltg mlm’ s Pa)
01

El 50% RH.
.7096 R.H.

 

  

  

 

 

 

 

 

 

 

 

 

II t
5 10
% Candelilla Wax
(primary cosmonaut basis)

Figure 16. Water Vapor Permeability (WVP) of Lactic Acid
Casein(50%)-Sorbitol-Candelilla Wax Edible Films

(Tested at 878°C, thickness = 0.1mm)

67

At both 50 and 70% RH, with the addition of candelilla wax to ﬁlms a signiﬁcant
decrease (p < 0.05) in WVP occurred versus those without wax, (Figure 16, Table 11).
This further supports carnauba wax being more diﬁcult to incorporate into ﬁlms, causing
disruptions in the ﬁlm matrix.

These results show that these ﬁlms are more inﬂuenced by the properties of lactic
acid casein. This is most likely due to the relatively low concentration of wax able to be
incorporated into the ﬁlms. With the low wax content and the high percentage of the
lactic acid casein, with it’s hydrophilic characteristics, these ﬁlms are able to adsorb
moisture readily. Another factor in the high WVP is the large concentration of plasticizer
required in these ﬁlms, breaking up some of the intermolecular bonding, allowing for the
easier diffusion of water.

These results help to support SEM and DSC ﬁndings where a partial, not
complete, phase separation of the wax occurred. If a complete separation had occurred
the water barrier properties should have been closer to that of wax ﬁlms, because of a
solid wax layer. However, these show WVP properties more similar to those of the lactic
acid casein-sorbitol ﬁlms, meaning many of the polar reactive groups present in lactic acid
casein (peptide, amido, and carboxyl groups) are found at the top surface of the ﬁlms
commingled with wax. WVP of these ﬁlms still are much higher than ﬁlms considered to
be good water barriers like HDPE, carnauba wax, and candelilla wax (Table 12). When
comparing WVP of diﬁ‘erent ﬁlms the RH and temperature of testing must be taken into

consideration.

68

Table 12. Water Vapor Permeability (WVP) Comparison of Lactic Acid Casein (50%)-
Sorbitol-Wax Edible Films to Other Films (Edible and Synthetic)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Film Type1 WVP x 10“ Test Conditions Reference
(Itrgm/m2 s Pa)
LAC-S(45)-CR(5) 2.45 378°C, 50% RH Present
LAC-S(45)-CR(5) 17.91 378°C, 70% RH Present
LAC-S(35)-CR(15) 1.08 378°C, 50% RH Present
LAC-S(35)-CR(15) 13.76 378°C, 70% RH Present
LAC-S(45)-CN(5) 1.42 378°C, 50% RH Present
LAC-S(45)-CN(5) 6.78 37 .8°C, 70% RH Present
LAC-S(35)-CN(15) 0.68 378°C, 50% RH Present
LAC-S(35)-CN(15) 5.14 378°C, 70% RH Present
LAC-S(50) 2.23 378°C, 50% RH Present
LAC-S(50) 8.54 378°C, 70% RH Present
LAC-S(50) . 52.07 378°C, 90% RH Chick (1996)
Carnauba Wax 0.033 25°C, 100% RH Donhowe and Fennema
(1993)
Candelilla Wax 0.018 25°C, 100% RH Donhowe and Fennema
(1993)
HDPE 0.02 37 .83 90% RH Smith (1986)
Nylon 6 8.21 378°C, 90% RH Tubrity & Foster (1986)

 

 

 

 

 

 

' LAC - Lactic Acid Casein, s - Sorbitol, CR - Carnauba Wax, CN - Candelilla Wax, HDPE - High
Density Polyethylene. Sorbitol and Wax comprised remaining 50% of ﬁlm (dry basis), number in
parenthesis is that percentage.

69

3.5.2 Wax Type

At 50% RH, candelilla wax incorporated into lactic acid casein ﬁlms provided a
signiﬁcantly better (p < 0.05) water barrier than ﬁlms made with carnauba wax at 5 and
10% wax, however, no signiﬁcant difference was observed at 15% wax (Figure 17, Table
11). At 70% RH, the WVP of ﬁlms containing candelilla wax were signiﬁcantly lower (p
< 0.05) than ﬁlms containing carnauba wax, at all wax levels (Figure 17, Table 11). This
can be explained by the higher hydrocarbon content in candelilla wax (about 50% in
candelilla wax and 1.5 to 3% in carnauba wax) that form on the surface, while carnauba
wax is predominantly made up of wax esters (about 85% compared to about 29% in
candelilla wax), which contain a polar group. Also, the diﬂiculty of incorporating
carnauba wax into the lactic acid casein ﬁlms probably plays a more signiﬁcant role,
increasing the void spaces. WVP was close to two times lower in ﬁlms containing
candelilla wax at 50% RH compared to ﬁlms with carnauba wax, while it was almost three
times lower at 70% RH. Which again, could be due to increased sorption of water by the
ester groups in carnauba wax and a more efﬁcient barrier created by the hydrocarbon
chains in candelilla wax, but most likely by the increased void spaces in the ﬁlms
containing carnauba wax. These are consistent with ﬁndings by Donhowe and F ennema
(1993) in wax ﬁlms and Shellhammer and Krochta (1997) in emulsion ﬁlms when

comparing carnauba to candelilla wax.

3.5.3 RH Effect
With an increase in RH from 50 to 70%, the WVP In lactic acid casein (50%)-

sorbitol (50%) ﬁlms increased 3.8 times, from 2.23 to 8.54 kg m/m2 8 Pa. Chick (1996)

70

.. _-.-._ -.__.__.L..“_..r"

 
    

Carnauba (70)

JCandelilla (70)

Wax Type (% RH)

 

Carnauba (50)

WVP x 10“ (kg mlm’ s Pa)

Candelilla (50)

Figure 17. Water Vapor Permeability (WVP) Comparison of Lactic Acid
Casein (50%)-SorbitoI-Camauba Wax and Lactic Acid Casein (50%)-
Sorbitol-Candelilla Wax Edible Films
(Tested at 318°C, thickness = 0.1 mm)

71

found the WVP in lactic acid casein (50%)-sorbitol (50%) edible ﬁlms (thickness 0.203
mm) to be 52.07 kg m/m2 5 Pa, tested at 23°C and 90% RH. By adding this result to the
ﬁndings ﬁ'om this study we observe an exponential rise in WVP of these ﬁlms (Figure 18).
These results correspond closely to the isotherm model of these ﬁlms.

As RH was increased from 50 to 70%, the WVP of lactic acid casein (50%)-
sorbitol-camauba wax edible ﬁlms increased 7.3, 8.1, and 12.7 times for ﬁlms containing
5, 10, and 15% carnauba wax respectively. In ﬁlms containing candelilla wax WVP
increased 4.8, 5.9, and 7.6 times for 5, 10, and 15% candelilla wax respectively. This
shows that the increasing RH had more of an effect on WVP in ﬁlms containing carnauba
wax. Again, because of the incorporation problems. Increasing RH having more of an
eﬂ’ect on ﬁlms as the wax content increased. This is probably the result of increased

disruptions in the ﬁlm matrix, due to the presence of wax.

3.6 Oxygen Permeability
3.6.1 Wax Qpntent

no statistical difference (p < 0.05) was witnessed in OP with the addition of wax,
either candelilla or carnauba wax, to lactic acid casein-sorbitol ﬁlms (Table 13). This is
similar to results found by Gennadios er al. (1993) with WG-AM edible ﬁlms. Kim and
Ustunol (1997 ) witnessed a decrease in OP with the addition of candelilla wax in WPI and
WPC-based edible ﬁlms. As wax content increased ﬁom 5 to 15%, either carnauba or
candelilla wax, no signiﬁcant difference (p < 0.05) in OP was witnessed. This is similar to

results found by Kim and Ustunol (1997) with WPI and WPC-candelilla wax edible ﬁlms.

72

501

WVP x 10“ (kg tin/m2 s Pa)
8

10 ~—

 

 

 

O C r i i e i 1 r t
O 10 20 a) 4) 50 w 70 80
RH (96)

Figure 18. Water Vapor Permeability (WVP) of Lactic Acid
Casein (50%)-Sorbitol (50%) Edible Films as Effected by
Relative Humidity (RH)

(Tested at 378°C, thickness = 0.1mm)

73

Table 13. Oxygen Permeability (OP)l of Lactic Acid Casein (50%)-Sorbitol-Wax Edible

 

 

 

 

 

 

 

 

 

 

 

Films2
OP x 105(m3 m/m2 s Pa)
Wax Content (%) Carnauba Wax Candelilla Wax

0 0.48 i 0.16' 0.48 i 0.16'
5 0.31 i 0.09' 0.35 :1: 0.07'
10 0.38 :1: 0.12' 0.44 :1: 0.04'
15 0.63 i 0.24' 0.71 :i: 0.31'

' Diﬂ‘erent letter throughout table denotes signiﬁcant dilference (p < 0.05)

’ All OP tests done at 23°C

2 Sorbitol and Wax comprised the remaining 50% of the ﬁlms (on a dry basis), thickness of ﬁlms
were 0.111 :1: 0.009 mm (4.37 :1: 0.04 mils)

74

It was expected that OP would increase with the addition of wax , because of their
poor oxygen barrier properties. Although, there may be more void areas near the surface
of ﬁlms containing wax, where the wax is present in larger concentrations (ﬁ'om the SEM
studies), a predominantly protein, defect ﬁ'ee matrix could seen throughout the rest of the
ﬁlms’ thickness. These results coincide with results from SEM studies, which show that
the bottom portion of these ﬁlms probably consist of a lactic acid casein-sorbitol layer,
creating the good oxygen barrier. While the wax is found predominantly near the top
portion of the ﬁhn in perhaps a partially separated emulsion form. Since, OP is mostly
dependent on diffusion, because oxygen is less compressible then water (Banker, 1966),
these results also support the presence of a good matrix system.

The OP properties of these ﬁlms fall in between those possessed by EVOH,
considered an excellent oxygen barrier, and nylon 6, a very good oxygen barrier. While
they proved to be much better barriers than carnauba and candelilla wax on their own

(Table 14).

3.6.2 Wax Type

Wax type, carnauba or candelilla wax, did not eﬂ’ect the oxygen barrier properties
of lactic acid casein-sorbitol-wax edible ﬁlms. Since the lactic acid casein matrix found
near the bottom surface is thought to have been the predominant inﬂuence on OP, the wax
did not play a signiﬁcant role in the oxygen barrier properties. Even if they did play a role
in the OP, differences between the waxes would not be expected because of the ﬁndings of
Donhowe and Fennema (1993 ), showing no signiﬁcant diﬁ‘erences between carnauba wax

and candelilla wax ﬁlms.

75

Table 14. Oxygen Permeability (OP) Comparison of Lactic Acid Casein (50%)-Sorbitol-
Wax Fihns to other Films (Edible and Synthetic)

 

 

 

 

 

 

 

 

 

 

 

 

 

Film Type1 O x 10’” Test Conditions Reference
(m3 m/m2 s Pa)

LAC(50)-S(50) 0.475 23°C, 0% RH Present
LAC-S(35)-CR(15) 0.625 23°C, 0% RH Present
LAC-S(35)-CN(15) 0.706 23°C, 0% RH Present

Carnauba Wax 180 23°C, 0% RH Donhowe and

Fennema (1993)

Candelilla Wax 200 23 °C, 0% RH Donhowe and

Fennema (1993)
HDPE 494 23°C, 50% RH Smith (1986)
EVOH 0.076 23°C, 0% RH Foster (1986)
Nylon 6 11.7 23°C, 0% RH Tubrity & Sibilia
(1986)

 

’LAC-LacticAcidCasein, S-Sorbitol,CR-CarnaubaWax,CN-CandelillaWax, HDPE-High
Density Polyethylene. Sorbitol and Wax comprised remaining 50% of ﬁlm (dry basis), number in

parenthesis is that percentage.

76

 

3.6.3 RH Effect

As RH increase from 0 to 50%, no signiﬁcant difference (p < 0.05) in OP was
observed in any of the edible ﬁlm samples, with or without wax. At 70% RH, however, a
signiﬁcant increase (p < 0.05) in OP occurred in all samples (Figure 19, Table 15). In
general, a minimum OP occurred at 35% RH for the ﬁlm samples. Vifrth PET there was a
decreasing trend, with a minimum at 50% RH, followed by an increase at 70% RH.
However, there was no signiﬁcant difference (p < 0.05) in OP as RH increased from 0 to
70% with the PET.

Lactic acid casein-sorbitol and lactic acid casein-sorbitol-wax edible ﬁlms acted
like nylon 6 (Gavara and Hernandez, 1994; Hernandez, 1994), where solubility is
decreasing until around the 35% RH region. Then, around the 70% RH region the matrix
is probably being disrupted by clustering water allowing for the greater OP. This also
coincides with the moisture sorption isotherm results, where a large increase in moisture
content occurred around 70% RH. This same result was witnessed in PET ﬁlms tested,
with a minimum around 50%. However, the increase was only slight following this point

and more studies at higher RH’s should be done to conclude this increasing phenomenon.

3.7 Mechanical Properties
3.7.1 Tgsile Strgrgth

At a 50% RH, the addition of 5 to 15% wax, either carnauba or candelilla, was
found to have no signiﬁcant effect (p < 0.05) on the TS of lactic acid casein (50%)-
sorbitol-wax ﬁlms compared to ﬁlms without wax (Figures 20 and 21, Table 16). At 7 5%

RH, with the addition of carnauba wax, a signiﬁcant increase in TS was observed at 10

77

3.5 -.

2.5 4'

OP x 102" (m’ mlm2 s Pa)

 

 

 

 

 

+ no wax

+15% carnauba wax
+15% candelilla wax
+ polyester

 

 

o 20 4o 60 so
Relative Humidity (‘16)

1G)

Figure 19. Oxygen Permeability (OP) of Lactic Acid Casein

(50%)-Sorbitol-Wax Edible Films

(Tested at 23°C, sorbitol and wax comprise 50% of ﬁlm)

78

 

Table 15. Oxygen Permeability (OP)l of Lactic Acid Casein (50%)-Sorbitol (3 5%)-Wax

(15%) Edible Films2 (dry basis) as Effected by Relative Humidity (RH)

 

 

 

 

 

 

 

 

 

 

 

 

OP x 107“(m3 m/m2 s Pa)
RH (%) N o Wax Carnauba Wax Candelilla Wax Polyester
0 0.48 :1: 0.16‘ 0.63 :1: 024' 0.71 i 0.31‘” 3.84 i 020'
20 0.46 a 0.13' 0.61 a 023' 0.73 «.1- 0.01‘ 3.50 a 0.10'
35 0.37 a 0.09' 0.60 i 0.23' 0.72 r. 0.06’ 3.37 i: 0.09'
50 0.43 i 0.07' 0.63 a 0.28’ 0.76 a. 0.07' 3.24 a 0.08'
70 1.29 a: 029" 2.21 i 0.60" 1.12 i 026" 3.63 i 0.09'

 

‘b Different letter columnwise denotes signiﬁcant diﬁ'erence (p < 0.05)

‘ All OP tests done at 23°C

2 Thickness ofﬁlms were 0.111 a 0.009 mm (4.37 a 0.04 mils)

79

 

TS (MP8)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

0 5 10

'16 Carnauba Wax
(primary component basis)

 

 

U 50% RH.
I75% R.l-l.

 

 

Figure 20. Tensile Strength (TS) of Lactic Acid Casein (50%)-

SorbitoI-Camauba Wax Edible Films
(‘l'ested at 23°C)

 

12

 

11

 

 

10

 

 

 

 

 

 

 

 

TS (MP8)
|
|

 

 

 

 

 

 

 

 

 

 

 

4 .. __ 050% RH.
" I75% R.H.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

tj:_l -

0 5 10

96 Candelilla Wax
(primary component basis)

 

 

 

 

Figure 21. Tensile Strength (T8) of Lactic Acid Casein (50%)-
Sorbitol-Candelilla Wax Edible Films
(Tested at 23°C)

81

Table 16. Tensile Strength (TS)l of Lactic Acid Casein (50%)-Sorbitol-Wax Edible

 

 

 

 

 

 

 

 

 

 

Films2

Wax Type(%) TS (MPa) TS (MPa)
@ 50% RH @ 75% RH

No Wax (0) 6.2 a 2.2"” 1.1 i: 0.2'
Carnauba (5) 4.4 a 01' 1.2 :1: 0.2“

(10) 6.0 :1: 0.9" 1.5 a 02°

(15) 7.9 i 04° 1.9 :1: 02°

No Wax (0) 6.2 a 2.2' 1.1 x 02‘

Candelilla (5) 4.5 i 0.8‘ 1.1 i 01‘
(10) 5.221;].4’ 14:01“

(15) 8.3 21:2.5' 1.8 :04"

 

 

 

 

"b Different letters in columnwise wax groups denote signiﬁcant diﬂerence (p < 0.05)

' All TS tests done at 23°C

2 Sorbitol and Wax comprised the remaining 50% of the ﬁlms (on a dry basis)

82

 

and 15% wax, compared to ﬁlms without wax. A signiﬁcant increase (p < 0.05) in TS
was also observed in ﬁlms containing candelilla wax at a 15% level, and a RH of 7 5%.
Kim and Ustunol (1997 ) found and increase in TS with the addition of candelilla wax to
WPI and WPC edible ﬁlms, at 23°C and 50% RH.

A signiﬁcant increase (p < 0.05) in TS occurred with an increasing concentration
of wax at both RH’s, except with candelilla wax at 50% RH (Figures 20 and 21, Table
16). However, an increasing trend was present in this case. In the lactic acid casein
(50%)-sorbitol (50%) ﬁlms TS is created by the protein matrix. The addition of wax at
low levels caused a disruption in the protein matrix causing a decrease in TS, at 50% RH.
As wax content increased a more uniform layer of wax was established, providing the
ﬁlms with an increased TS. At 70% RH, wax did not play as large a role in T8 in these
ﬁlms due to the plasticization of the ﬁlms by the increased moisture content. These results
are contrary to those found by Shellhammer and Krochta (1997) who found a linear
decrease in TS as either carnauba or candelilla wax content increased, in WPI-glycerol-
wax emulsion ﬁlms. Kim and Ustunol (1997), on the other hand, found no change in TS
as candelilla wax content increased from 2 to 8%, in WPI and WPC-candelilla wax ’
emulsion ﬁlms. Differences in these results could be due to diﬂ‘erences in the way the
protein and waxes interact in emulsion or to differences in the protein. These results
correspond well to those showing the possibility of a partial phase separation of the
emulsion as wax content increases.

There were no signiﬁcant diﬂ‘erences (p < 0.05) in TS values between lactic acid
casein (50%)-sorbitol-carnauba wax edible ﬁlms and lactic acid casein (50%)-sorbitol-

candelilla wax edible ﬁlms at equivalent wax levels (Figure 22, Table 16). This is most

83

Ts (M98)

 

Figure 22. Tensile Strength (TS) Comparison of Lactic Acid
Casein (50%)-Sorbitol-Camauba Wax and Lactic Acid Casein
(50%)-Sorbitol-Candelilla Wax Edible Films
(Tested at 23°C)

likely due to the relatively small concentration of wax within the ﬁlms, 15% or less.
Showing that differences between the waxes are not present at these relatively small
concentrations, compared to protein. This is contrary to results found by Shellhammer
and Krochta (1997), where WPI-glycerol-candelilla wax ﬁlms were found to be weaker
than WPI-glycerol-carnauba wax ﬁlms. However, wax content in these ﬁlms was much
higher, ranging from 20 to 40%.

An increase in relative humidity, ﬁom 50 to 75%, produced a signiﬁcant decrease
(p < 0.05) in TS (Figure 22). This is again due to the increased moisture content in ﬁlms
at the higher RI-I, as seen by the moisture sorption isotherm results (Figure 14), further
plasticizing the ﬁlms. TS decreased around 4 times for ﬁhns containing wax and 5.6 times
for lactic acid casein (50%)-sorbitol (50%) edible ﬁlms, with the increase in RH. This
decrease shows the more hydrophobic properties that wax ﬁlms possess, compared to

lactic acid casein-sorbitol ﬁlms alone.

3.7.2 Elam

At 50% RH, ﬁlms containing carnauba wax, at 10 and 15%, and candelilla wax,
from 5 to 15%, signiﬁcantly decreased (p < 0.05) the elongation of lactic acid casein
(50%)-sorbitol ﬁlms compared to ﬁlms without wax. While at 75% RH, a signiﬁcant
decrease (p < 0.05) in elongation was witnessed with the addition of 10 and 15% of either
wax (Table 17). This is most likely due to the wax disrupting the protein matrix,
producing a less extensible ﬁlm. At a 75% RH, the increase in moisture present in the
ﬁlms, further plasticizing the ﬁlms, probably helps to counteract the addition of wax at the

smaller concentrations. Similar results were reported by Shellhammer and Krochta (1997)

85

Table 17 . Elongation’ of Lactic Acid Casein (50%)-Sorbitol-Wax Edible

 

 

 

 

 

 

 

 

 

 

Films2
Wax Type(%) Elongation(%) Elongation(%)
@ 50% RH @ 75% RH
No Wax (0) 156:1:6' 167i16'
Carnauba (5) 159 a 18' 146 :1: 20'
(10) 55 :21" 96:1:4"
(15) 31:5" 88:9"
No Wax (0) 156 :1: 6’ 167 i16‘
Candelilla (5) 93 :1: 30" 156 i 14'
(10) 71 :26"c 116d: 17"
(15) 37: 12° 74i21°

 

 

 

 

"° Different letters in columnwise wax gruops denote signiﬁcant difference (p < 0.05)
‘ All Elongation tests done at 23 °C
2 Sorbitol and Wax comprised the remaining 50% of the ﬁlms (on a dry basis)

86

 

and Kim and Ustunol (1997), where they found elongation to decrease with the addition
of wax in whey protein ﬁlms.

As wax content increased, carnauba or candelilla wax, at both 50 and 75% RH, a
signiﬁcant decrease in elongation was witnessed (Figures 23 and 24, Table 17). This again
being due to the relative brittleness of wax and ﬁrrther disruption of the protein matrix.

As has been seen, with the larger concentrations of wax a further separation of the wax is
occurring. This would also cause the earlier ﬁlm break, with the wax layer breaking,
initiating the ﬁlms failure. Kim and Ustunol (1997) found no change in elongation as wax
content increased and WPC and WPI-candelilla wax ﬁlms, however, the concentration of
wax was relatively low, '2 to 8%.

No signiﬁcant difference (p < 0.05) was observed in elongation between ﬁlms
containing carnauba wax and those containing candelilla wax, at equal wax levels and
RH’s, except at the 5% wax level and 50% RH (Figure 25). These results are most likely
due to the waxes disrupting the protein matrix to similar degrees, and the relatively low
wax concentration with the protein matrix giving the ﬁlms their extension characteristics.

A signiﬁcant increase (p < 0.05) in elongation was witnessed with an increase in
RH from 50 to 70%, except those ﬁlms without wax and with the addition of carnauba
wax at 5% where no signiﬁcant change occurred (Figure 25). An increase of around two
times was witnessed in those ﬁlms where a signiﬁcant increase was noticed. This increase
again being due to ﬁrrther plasticization of the ﬁhns by the increased moisture at the higher

RH.

87

Elongation (%)

1&3

1m

10‘

120-

100-

so

 

 

 

 

 

 

 

.1 —d l

1

 

 

 

 

0 5

 

 

 

 

 

 

 

 

96 Carnauba Wax

(primary component basis)

Figure 23. Elongation of Lactic Acid Casein (50%)-Sorbitol-
Camauba Wax Edible Films

(rested at 23°C)

88

 

 

D 5096 RH.
.7096 R.H.

 

 

 

 

Elongation (%)

180

1a)

10

120

100

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T
l.
.1.
1.
El 50% RH.
I 70% RH.
o 5 10 15
‘lb Candelilla Wax
(primary component basis)

Figure 24. Elongation of Lactic Acid Casein(50%)-Sorbitol-
Candelilla Wax Edible Films
(Tested at 23°C)

89

 

Elongation (%)

   
 
 
 

Candelilla (50)
Wax Type (lit. R.H.)

Carnauba (50)

Figure 25. Elongation Comparison of Lactic Acid Casein
(50%)-SorbitoI-Wax Edible Films
(Tested at 23°C)

3.7.3 Comparison to ﬁber Polmers

As has been the case with edible ﬁlms, they have generally been found to be much weaker
than synthetic ﬁlms (Table 18). Although the incorporation of wax did add some strength
to these lactic acid casein-based ﬁlms, the strongest are only comparable to the weaker of
the LDPE ﬁlms, and are much weaker than HDPE and nylon ﬁlms. This strength could be
increased with a decrease in plasticization levels and/or by the ﬁrrther incorporation of
wax, as seen with the Shellhammer and Krochta (1997), the strength of casein-wax ﬁlms
could be comparable to some of the synthetic polymers. However, with an increase in
wax content a large drop in elongation would likely occur. Lactic acid casein-based ﬁlms
without wax are shown to have slightly lower elongation properties than HDPE and
EVOH. However, with the incorporation of wax we see these properties decrease

drastically.

3.7.3 Hg; Sealing

From preliminary testing it was found that no heat seal could be made at the melt
temperatures of either wax, but a seal was formed around the melting temperatures around
those of lactic acid casein and sorbitol, 100 and 110°C. A better heat seal was produced
when sealing the bottom surfaces together. From the microstructure studies this showed a
mostly protein-plasticizer structure. When sealing the top surfaces, in ﬁlms containing
wax, a weak seal that easily delaminated was produced. From this it can be concluded
that either the lactic acid casein, the sorbitol, or a combination of both were responsible
for the melt ﬂow characteristics to obtain a good seal.

Although seal strengths were found to be rather weak, an acceptable heat seal

91

Table 18. Comparison of Lactic Acid Casein-Based Edible Films Mechanical Properties

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Film 'I‘ypel TS Elongation Test Reference
(MPa) (%) Conditions
LAC(50)-S(50) 6.2 210 23°C, 50% RH Present
LAC-S(35)-CR(15) 7.9 31 23°C, 50%) RH Present
LAC-S(35)-CN(15) 8.3 37 23°C, 50% RH Present
WPI(60)-CN(40) 23 1.5 23°C, 50% RH Shellhammer &
Krochta (1997)
WPI(60)-CR(40) 17 1.0 23°C, 50% RH Shellhammer &
. Krochta (1997)
LDPE 8.6-]7 500 23°C, 50°/o RH Salame (i986)
HDPE 17-35 300 23°C, 50% RH Smith (1986)
EVOH 39-69 23 5-325 23°C, 50°/o RH Foster (1986)
Nylon 6 69-83 400-500 23°C, 50°/o RH Tubrity & Sibilia
(1986)

 

 

‘LAC-LacticAcidCasein, S-Sorbitol, CR-CarnaubaWax, CN-CandelillaWax, WPI-WheyProtcin
Isolate Solution, LDPE - Low Density Polyethylene, HDPE - High Density Polyethylene. Number in
parenthesis is the percentage of that material in ﬁlm (dry basis).

92

between the two ﬁlm layers was able to be accomplished (Tables 19, 20, and 21). With an
increasing seal temperature seal strengths tended to increase. Below 8 temperature of
93°C a good seal was not formed, and at temperatures greater than 121°C degradation of
the ﬁlms would start to occur, especially with a 4 second dwell time. This degradation did
occur at 121°C in ﬁlms without wax. Degradation in this study was noted as excessive
burning/melting of the ﬁlm, usually resulting in it sticking to the heat platens. These
results correspond to DSC results where we observe ﬁlm degradation beginning to occur
around 120°C.

Seal strength also tended to increase with an increasing dwell time, ﬁ'om 2 to 4
seconds. This allowed the heat to penetrate ﬁrrther into the ﬁlm thickness, creating more
ﬂow and interaction at the interfaces. Seal pressure, 40 or 60 psi, did not produce
differences in the overall strength of the ﬁlms.

A good seal, where a break in the seal would occur rather than delarnination, was
found to occur with a seal strength at around 0.6 MPa or greater. SEM pictures taken of
ﬁlms with strengths higher than this showed that good adhesion between the two ﬁlm.
layers had occurred (Figures 26, 27, and 28). This was concluded because a clear line of
separation between the two ﬁhn layers is diﬂ'rcult to differentiate, that is the two ﬁlms

layers appeared to have melted into one ﬁlm.

93

Table 19. Seal Strength of Lactic Acid Casein (50%)-Sorbitol (50%) Edible Films

 

 

 

 

 

 

 

 

 

 

 

 

94

Seal Strength (MPa)
Seal Parameters
Seal 2sec-40psi 2sec-60psi 4sec-40psi 4sec-60psi
Temparature -
(°C)
200 0.22 i 0.13’ 0.38 i 0.29“ 0.99 i 0.64’ 0.66 i 0.15'I
212 0.24 :t 0.17’ 0.67 d: 0.19’ 0.71 i 0.31' 0.62 d: 0.19’
225 0.90 a 054‘" 0.43 :1: 0.18‘ 1.33 i 0.52" 0.94 :1: 054'
250 1.37 x 0.32" 1.81 :t .34" - -
'" Different letter columnwise denotes signiﬁcant diﬂ’erence (p < 0.05)

 

Table 20. Seal Strength of Lactic Acid Casein (50%)-Sorbitol (35%)-Carnauba Wax

 

 

 

 

 

 

(15%) Edible Films
Seal Strength (MPa)
Seal Parameters
Seal 2sec-40psi 28ec-60psi 4sec-40psi 4sec-60psi
Temparature

(°C)

200 0.28 i 0.05‘ 0.37 i 0.12’ 0.80 :1: 0.43" 0.70 :1: 0.15'
212 0.43 d: 009' 0.31 i 0.09' 0.61 i 0.10" 0.79 a 035'
225 0.98 i 0.43' 0.57 i 0.33’ 1.23 i: 0.14’ 0.79 i 0.39’
250 0.68 :1: 0.30‘ 1.29 :1: 0.62" 1.19 :1: 0.36' 1.63 i 0.69‘I

 

 

 

 

 

 

95

‘ Different letter columnwise denotes signiﬁcant diﬂ‘erence (p < 0.05)

 

Table 21. Seal Strength of Lactic Acid Casein (50%)-Sorbitol (3 5%)-Carnauba Wax

 

 

 

 

 

 

(15%) Edible Films
Seal Strength (MPa)
Seal Parameters

Seal 2sec-40psi 28ec-60psi 4sec-40psi 4sec-60psi
Temparature

(°C)

200 0.13 a 0.02" 0.21 i 0.08’ 0.65 r. 0.50" 0.60 :1: 0.40"

212 0.43 x 026‘" 0.22 i 0.08' 1.58 i 080‘" 1.89 a 072'

225 1.23 a 0.63" 1.14 a 0.74" 1.53 a 052'" 1.90 a 0.86’

250 0.68 :1: 030'" 1.19 x 0.85" 2.47 :1: 0.18" 1.01 :1; 0.43"

 

 

 

 

 

 

96

‘ Diﬂ‘erent letter columnwise denotes signiﬁcant diﬂ‘crence (p < 0.05)

 

 

Figure 26. SEM of the Cross Sectional Surface of the Seal Area of Lactic Acid Casein
, (50%)-Sorbitol (50%) Edible Films

97

 

Figure 27 . SEM of the Cross Sectional Surface of the Seal Area of Lactic Acid Casein
(50%)-Sorbitol (3 5%)-Carnauba Wax (15%) Edible Films

98

 

Figure 28. SEM of the Cross Sectional Surface of the Seal Area of Lactic Acid Casein
(50%)-Sorbitol (3 5%)-Candelilla Wax Edible F ilrns

99

4.0 CONCLUSIONS

4.1 Film Development and Characterization

1.

Films with wax incorporated into them were less transparent than ﬁlms without wax,
but were still smooth and ﬂexible.

Wax concentrations above 15% (on a primary component basis) could not be
incorporated into ﬁlms containing 50% lactic acid casein powder and sorbitol.

Films produced were found to be continuous and homogeneous.

. Partial phase separation is believed to have occurred, due to a layering structure that

was found in ﬁlms containing wax, with the wax being found mainly at the top surface,
and the presence of two endothermic peaks from thermal studies.
Moisture sorption isotherm data correlated well with theoretical values from the GAB

model for lactic acid casein (50%)-sorbitol (50%) edible ﬁlms.

4.2 Water Vapor Permeability

l.

WVP signiﬁcantly decreased (p < 0.05) as wax content (carnauba or candelilla wax)
was increased ﬁ'om 5 to 15% in lactic acid casein (50%)-sorbitol-wax edible ﬁlms at
both 50 and 70% RH.

WVP of lactic acid casein (50°/o)-sorbitol-carnauba wax edible ﬁlms were not
signiﬁcantly different (p < 0.05) at 50% RH then these ﬁlms with no wax
incorporated, while WVP was signiﬁcantly higher (p < 0.05) at 70% RH.

WVP of lactic acid casein (50°/o)-sorbitol-candelilla wax edible ﬁlms were signiﬁcantly

lower (p < 0.05) then these ﬁlms with no wax incorporated at both 50 and 70% RH.

100

4. Lactic acid casein (50%)-sorbitol edible ﬁlms made with candelilla wax had a

signiﬁcantly lower (p < 0.05) WVP then ﬁlms made with carnauba wax, at both 50 and
70% RH and equal wax content.

WVP of lactic acid casein (50%)-sorbitol edible ﬁlms increased as RH increased (Horn
50 to 70%), with and without wax (carnauba and candelilla wax, 5 to 15% wax)
added.

Lactic acid casein (50%)-sorbitol edible ﬁlms were found to be poor water barriers,
with or without wax (carnauba or candelilla wax) incorporated compared to synthetic

and wax polymers.

4.3 Oxygen Permeability

1.

There was no signiﬁcant change (p < 0.05) in OP in lactic acid casein (50%)-sorbitol
(50%) edible ﬁlms with the addition of wax, carnauba or candelilla wax, or as wax
content increased ﬁom 5 to 15% (dry basis), with protein content constant.

There was no signiﬁcant diﬂ‘erence (p < 0.05) in OP between lactic acid casein-sorbitol

edible ﬁlms with carnauba wax compared to those with candelilla wax.

. OP in lactic acid casein(50°/o)-sorbitol(50°/o) and lactic acid casein (50%)-sorbitol

(3 5%)-wax (15%) edible ﬁlms, carnauba or candelilla wax, tended to decrease as RH
increased to 35%, followed by a signiﬁcant (p < 0.05), sharp increase at 70% RH.
Lactic acid casein(50°/o)-sorbitol(50°/o) and lactic acid casein (50%)-sorbitol (3 5%)-
wax edible ﬁlms, carnauba or candelilla wax, were found to be good oxygen barriers

as compared to synthetic polymers.

101

4.4 Mechanical Properties

1.

The addition of wax, carnauba or candelilla wax, signiﬁcantly increases (p < 0.05) TS
and signiﬁcantly decreases elongation in lactic acid casein (50%)-sorbitol edible ﬁlms.
TS signiﬁcantly increases (p < 0.05) and elongation signiﬁcantly decreases as wax
content, carnauba or candelilla wax, increases in lactic acid casein (50%)-sorbitol
edible ﬁlms.

There is no signiﬁcant diﬁ‘erence (p < 0.05) in tensile or elongation properties
between lactic acid casein (50%)-sorbitol edible ﬁlms with carnauba or candelilla wax
added to them.

A signiﬁcant decrease (p < 0.05) in TS and a signiﬁcant increase in elongation were
observed in lactic acid (50%)-sorbitol-wax edible ﬁlms when RH was increased ﬁ'om
50 to 7 5%.

Tensile and elongation properties of lactic acid casein (50%)-sorbitol-wax edible ﬁlms
are lower than those of synthetic polymers.

A good heat seal, although weak, of lactic acid casein-based edible ﬁlms was obtained
in the melting range near that of lactic acid casein and sorbitol, between 100 and

110°C (212 and 230°F).

102

5.0 APPENDICES

103

5.1 APPENDIX A

Appendix A. Structures of Various Compounds Found in Lactic Acid Casein-Sorbitol

Edible Films
A1. Peptide
O H
U - Ill - CH2 -
A2. Amido
- C - NH;
A3. Carboxyl
IOI
- C - OH
A4. Amino Acid
H
l
R - C - COOH
l
N H;

R = side chain of variable composition

104

A5. Proline
COOH
I-IN-C-H

HzC CH2

\ér

A6. Sorbitol
C H2 OH
H C OH
HO C H
H C OH
H C OH

C H2 OH

105

5.2 APPENDIX B

Appendix B. Amino Acid Composition of Casein Fractions

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Casein Type
Amino Acid (1,1- 01,2- B- '11- y,— 7,. 1r-
1/2 Cysteine 0 2 0 0 0 0 2
Alanine 9 8 5 5 2 2 15
Argenine 6 6 4 2 2 2 5
Asn 8 l4 5 3 l l 7
Aspartic Acid 7 4 4 4 2 2 4
Gin 15 15 21 21 ll ll 14
Glutamic Acid 24 25 18 ll 4 4 12
Glycine 9 2 5 4 2 2 2
Histidine 5 3 5 5 4 3 3
Isoleucine l l l l 10 7 3 3 l3
Leucine 17 ' 13 22 19 14 14 8
Lysine 14 24 11 10 4 3 9
Methionine 5 4 6 6 4 4 2
Phenylalanine 8 6 9 9 5 5 4
Proline 17 10 35 34 21 21 20
Serine l6 l7 l6 1 l 7 7 l3
Threonine 5 15 9 8 4 4 14
Tryptophan 2 2 1 1 1 1 1
Tyrosine 10 12 4 4 3 ‘ 3 9
Valine l l l4 l9 17 10 10 l 1
Total 199 207 209 181 104 102 169

 

 

 

 

 

 

 

106

 

6.0 REFERENCES

107

6.0 REFERENCES

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