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MECHANICAL PROPERTIES OF WHEY PROTEIN ISOLATE
BASED EDIBLE FILMS AS AFFECTED BY MEAT
PROCESSING CONDITIONS AND OPTIMIZATION OF
THESE PROPERTIES

presented by
Sindisiwe N. Simelane

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MECHANICAL PROPERTIES OF WHEY PROTEIN ISOLATE BASED EDIBLE
FILMS AS AFFECTED BY MEAT PROCESSING CONDITIONS AND
OPTIMIZATION OF THESE PROPERTIES

By

Sindisiwe N. Simelane

A THESIS

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

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

2003

 

 

 

 

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ABSTRACT
MECHANICAL PROPERTIES OF WHEY PROTEIN ISOLATE BASED EDIBLE

FILMS AS AFFECTED BY MEAT PROCESSING CONDITIONS AND
OPTIMIZATION OF THESE PROPERTIES

By
Sindisiwe N. Simelane

This study was conducted to test the hypothesis that whey protein isolate (WPI)-
based edible ﬁlms could be used as sausage casings comparable to collagen casings.
Heat cured (80°C, 24 h or 90°C, 12 h) WPI-based ﬁlms containing glycerol and candelilla
wax were treated under meat processing conditions; the effects of temperature, time and
relative humidity on the tensile strength (TS) and percent elongation (%E) of the ﬁlms
were determined. Tensile strength of WPI-based ﬁlms was lower while %E was similar
to that of collagen ﬁlms. To optimize the mechanical properties of the WPI-based ﬁlms,
different ﬁlm forming solutions (5.8% or 6.4% w/v solids content), casting on different
surfaces (anodized or non-anodized Teﬂon®), heat curing (90°C, 12 h) and compression
molding (single- and double-stage; and varying temperature, pressure and time) were
studied. Heat curing and compression molding increased TS and had no effect on %E.
In a separate double-stage compression molding experiment, temperature, pressure and
time conditions were optimized. No optimum combination of these conditions was
obtained. Finally, seal strengths and surface chemistry (using electron spectroscopy for
chemical analysis, ESCA) of unsealed and sealed uncured, heat cured and compression
molded WPI-based ﬁlms were studied. Uncured films had the highest seal strengths.
Sealed, heat cured and compression molded ﬁlms had a preponderance of C-O-H and C-

O-C bonds, which were not detected in uncured ﬁlms.

Copyright by
Sindisiwe N. Simelane

2003

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
INTRODUCTION

CHAPTER 1
LITERATURE REVIEW
1.1. EDIBLE FILMS
1.1.1. Deﬁnition
1.2. FORMATION AND STABILITY OF PROTEIN BASED EDIBLE
FILMS
1.3. PROPERTIES OF PROTEHNI BASED FILMS
1.3.1. Barrier properties
1.3. 1.1. Water vapor permeability
1.3.1.2. Oxygen permeability
1.3.2. Mechanical properties
1.3.2.1. Factors affecting mechanical properties
1.3.2.2. Means of improving mechanical properties
1.3.3. Sorption and sorption isotherms
1.3.4. Thermal and scaling properties
1.4. ELECTRON SPECTROSCOPY FOR CHEMICAL ANALYSIS
1.5. COMPRESSION MOLDING
1.6. COLLAGEN CASINGS AND SAUSAGE MANUFACTURE

CHAPTER 2
EFFECT OF MEAT PROCESSING CONDITIONS ON MECHANICAL
PROPERTIES OF HEAT CURED WHEY PROTEIN-BASED EDIBLE
FILMS: A COMPARISON TO COMMERCIAL COLLAGEN CASINGS
2.1. ABSTRACT
2.2. INTRODUCTION
2.3. MATERIALS AND METHODS
2.3.1. Materials
2.3.2. Proximate analysis
2.3.2.1. Moisture content
2.3.2.2. Pretein content
2.3.2.3. Ash
2.3.2.4. Lactose content
2.3.3. Whey protein isolate based ﬁlm preparation
2.3.4. Collagen ﬁlms
2.3.5. ﬁlm thickness
2.3.6. Film treatment

iv

vi

vii

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30
31
34
34
34
34
35
35
35
36
37
37
37

2.3.8. Statistical analysis
2.4. RESULTS AND DISCUSSION
2.4.1. Whey protein isolate composition
2.4.2. Film thickness
2.4.3. Mechanical properties
2.4.3.1. Tensile strength
2.4.3.1. Elongation
2.5. CONCLUSIONS

CHAPTER 3
OPTIMIZATION OF MECHANICAL PROPERTIES OF WHEY
PROTEIN ISOLATE BASED EDIBLE FILMS FOR USE IN
SAUSAGE CASING MANUFACTURE
3.1. ABSTRACT
3.2. INTRODUCTION
3.3. MATERIALS AND METHODS
3.3.1. Materials
3.3.2. Film formation
3.3.3. Collagen
3.3.4. Film thickness
3.3.5. Compression molding
3.3.6. Mechanical properties
3.3.7. Statistical analysis
3.4. RESULTS AND DISCUSSION
3.4.1. Mechanical properties
3.4.1.1. Effects of heat curing, solids content of casting
solution and casting surface
3.4.1.2. Compression molding
3.5. CONCLUSIONS

CHAPTER 4
OPTIMIZATION OF SEAL PROPERTIES OF WHEY PROTEIN
ISOLATE BASED EDIBLE FILMS
4.1. ABSTRACT
4.2. INTRODUCTION
4.3. MATERIALS AND METHODS
4.3.1. Materials
4.3.2. Whey protein isolate ﬁlm formation
4.3.3. Compression molding
4.3.4. Seal strength determination
4.3.5. Electron spectroscopy for chemical analysis
4.3.6. Statistical analysis
4.4. RESULTS AND DISCUSSION
4.4.1. Seal strengths
4.4.2. Electron spectroscopy for chemical analysis
4.5. CONCLUSIONS

4O
4O
40
4O
4 l
4 l

47

48
49
51
51
51
53
53
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54
55
56
56
56

61
7O

72

72
73
75
75
75
77
77
78
79
79
79
83

SUMMARY 91

FUTURE RESEARCH 92
APPENDIX I 93
APPENDIX II 99

REFERENCES 105

v1

Table 2.1.

Table 3.1.

Table 3.2.

Table 3.3.

Table 3.4

Table 4.1.

Table 4.2.

Table 4.3.

Table 4.4.

LIST OF TABLES

Compositional analysis of the whey protein isolate powder
utilized for whey protein isolate-based ﬁlms.

Surface response methodology design for optimization of
two-stage compression molding conditions for heat cured whey
protein isolate based ﬁlms.

Signiﬁcance level of main effects and their interactions for
tensile strength

Signiﬁcance level of main effects and their interactions for
Elongation.

Mechanical properties of uncured and cured whey protein isolate
based ﬁlms cast on non-anodized and anodized surfaces, from
solutions varying in solids content

Influence of casting surface and solids content on seal strengths
of whey protein isolate-based ﬁlms

Inﬂuence of casting surface and solids content on seal strengths
of uncured, cured and sealed before curing whey protein
isolate-based ﬁlms

Effect of wetting the seal area before sealing and compression
molding on seal strength of whey protein isolate-based ﬁlms

Bond composition of unsealed and sealed whey protein isolate-

based edible ﬁlms determined with Electron Spectroscopy for
Chemical Analysis

v11

41

55

57

57

59

81

81

82

84

Figure 2.1.
Figure 2.2.

Figure 2.3.

Figure 2.4.

Figure 2.5.

Figure 2.6.

Figure 3.1

Figure 3.2.

Figure 3.3.

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

LIST OF FIGURES

Schematic diagram of ﬁlm forming procedure
Schematic diagram of meat processing simulation

Tensile strength of whey protein isolate-based ﬁlms heat
cured at 80°C for 24 hours and collagen ﬁlms subjected to
Polish sausage manufacturing conditions

Tensile strength of whey protein isolate-based ﬁlms heat
cured at 90°C for 12hours and collagen ﬁlms subjected to
Polish sausage manufacturing conditions

Elongation of whey protein isolate-based ﬁlms heat cured
at 80°C for 24 hours and collagen ﬁlms subjected to
Polish sausage manufacturing conditions

Elongation of whey protein isolate-based ﬁlms heat-cured
at 90°C for 24 hours and collagen ﬁlms subjected to
Polish sausage manufacturing conditions

Schematic diagram of the ﬁlm optimization process for
whey protein isolate-based ﬁlms

Tensile Strength of compression molded whey protein
isolate-based ﬁlms

Elongation of compression molded whey protein isolate-
based ﬁlms

Effect of temperature and pressure on tensile strength (T8) of
2-stage compression molded whey protein isolate-based ﬁlms as
determined by response surface methodology

Effect of temperature and time on tensile strength (TS) of 2-stage
compression molded whey protein isolate based ﬁlms as
determined by response surface methodology

Effect of temperature and time on elongation (%) of 2-stage
compression molded whey protein isolate based ﬁlms as
determined by response surface methodology

Effect of temperature and time on elongation (%) of 2-stage

compression molded whey protein isolate based ﬁlms as
determined by response surface methodology

viii

38

39

42

43

45

45

52

62

65

67

68

69

7O

Figure 4.1. Schematic diagram of the seal optimization process for whey 76
protein isolate ﬁlms

Figure 4.2a. Representative Electron Spectroscopy for Chemical Analysis 85
spectra of whey protein isolate based ﬁlms

Figure 4.2b. Representative Electron Spectroscopy for Chemical Analysis 86
spectra of whey protein isolate based ﬁlms

Viv

INTRODUCTION

Edible films have been developed and characterized by several researchers.
These ﬁlms have been developed from various proteins including whey proteins
(Mahmoud and Savello 1990; McHugh and Krochta 1994a; Galietta and others 1998;
Kim and Ustunol 2001a), casein (lactic-acid and rennet; Chick and Ustunol 1998, Chick
and Hernandez 2002), wheat gluten (Micard and others 2000; Larré and others 2000),
gelatin (Lim and others 1999), soy protein (Gennadios and Weller 1991; Gennadios and
others 1996), egg whites (Gennadios and others 1998a), pea protein (Choi and Han 2001,
2002) and others.

The growing interest in the development and application of edible ﬁlms has
resulted in extensive research focused on the development and characterization of whey
protein isolate (WPI)-based ﬁlms (Kim and Ustunol 2001 a,b; McHugh and Krochta
1994 a,b,c,d; Shaw and others 2002; Sothomvit and Krochta 2001; Sothomvit and others
2003). These ﬁlms have been found to have good mechanical properties when compared
to synthetic polymers such as high density polyethylene and low density polyethylene as
well as to other protein based ﬁlms including soy protein isolate and wheat gluten ﬁlms.
Furthermore, whey protein based ﬁlms were found to be transparent, bland and flexible,
having good aroma, lipid and oxygen barrier properties (McHugh and Krochta 1994
a,b,c,d). Their disadvantage, as with many biopolymers, has been high moisture
sensitivity resulting from the hydrophilic nature of the native and denatured proteins.

Applications for whey protein isolate (WPI)-based ﬁlms have been investigated

and suggested including pouches for soups, cocoa powder and coverings for cheese slices

(Amin and others 2001; Kim 2000) as well as anti-microbial casings for hot-dogs (Cagri
and others 2003). Kim and Ustunol (2001b) reported that WPI-based ﬁlms had no
distinct milk flavor or odor and were thus acceptable with respect to their sensory
attributes. Cagri and others (2003) further conﬁrmed the organoleptic acceptability of the
ﬁlms by demonstrating that hot-dogs made with WPI-based ﬁlms were as acceptable as
hot-dogs made with commercial collagen and natural casings. Findings of the above
researchers indicate the potential for WPI-based ﬁlms to be used in food applications
such as sausage casings.

In order for WPI-based ﬁlms to be used as sausage casings, their mechanical
properties need to be comparable to the properties of commercial casings currently used
for this purpose. Collagen casings are currently the most widely used edible sausage
casings as an alternative to natural casings (Osbum 2002). Brown, caramel and other
food grade colors are added to collagen casings to provide products with distinct colors
that are marketed as distinctive smoked or spicy specialty products. This practice is often
employed because non-colored casings tend to appear gray after steam cooking (Osbum
2002). Heat cured WPI-based ﬁlms provide the beneﬁt of a natural pigmented
appearance similar to caramel or smoked colored casings without the addition of food
coloring. Increased yellowness has been reported following heat curing of these ﬁlms
(Kim and others 2002; Cagri and others 2003). These ﬁndings further indicate the
potential for WPI-based ﬁlms to be used as sausage casings.

Previous research from our lab has shown that heat-cured WPI—based ﬁlms have

mechanical properties similar to collagen films (Amin and others 2003). A WPI-based

film that can withstand meat process conditions may provide the meat industry with an
alternative to collagen ﬁlms.
The speciﬁc objectives of the study were:

(i) To determine if heat cured whey protein isolate (WPI) based edible
ﬁlms could withstand the temperature, time and relative humidity
conditions encountered in a meat cooking process scheme used for
Polish sausage

(ii) To reformulate the films and optimize their mechanical properties for
use as sausage casings.

(iii) To optimize and determine the scaling properties of the WPI—based

ﬁlms developed in the optimized ﬁlm protocol.

CHAPTER 1

LITERATURE REVIEW

1.1. EDIBLE FILMS
1.1.1. Deﬁnition

Edible ﬁlms are deﬁned as thin layers of edible material able to inhibit migration
of moisture, lipids, oxygen, carbon dioxide, and flavor volatiles (Krochta and De Mulder-
Johnston 1997). Edible films may be an essential component to quality and stability
control of many foods by providing a barrier against mass transfer in foods (Miller and

Krochta 1997).

1.2. FORMATION AND STABILITY OF PROTEIN BASED EDIBLE FILMS

Formation and stability of protein based ﬁlms mostly depends on the formation of
cross-links. High temperature, pH extremes — particularly alkaline pH and exposure to
oxidizing conditions and enzymatic activity can result in the introduction of protein
cross-links or hydrolytic fragments thereby inducing substantial changes in the structure
of the proteins. The cross-links are organized according to the amino acids that react to
form the cross-links. It is important to note, however, that no matter how extreme the
processing conditions may be, not all amino acids will participate in cross-linking
(Gerrard 2002).

Some cross-links found in foods are disulfide bonds (S-S), links derived from the

Maillard reaction and cross-links formed through transglutarrrinase catalysis. The

oxidative coupling of two adjacent cysteine residues within a protein forms S-S bonds,
the most common and well-characterized cross-links. These bonds are linked together to
form polypeptide chains that contribute to a protein’s tertiary structure and are important
in ﬁlm formation. A heating process is used to denature the protein, thus unfolding it to
reveal free sulfhydryl (S-H) groups. In whey proteins, heating exposes free S-H groups
in B-lactoglobulin allowing S-H or S-S interchange reactions to occur (Krochta 1998).
Hydrolysis with acid or alkali may also be used to denature proteins. Alkaline conditions
aid ﬁlm formation because S-S bonds are cleaved and reduced to free S-H groups when
dispersed in alkaline conditions. Di-sulﬁde bonds are then reformed during drying of the
film forming solutions (McHugh and Krochta 1994c). The SS bonds resulting from heat
denaturation of proteins have been hypothesized to be partly responsible for ﬁlm
structure (Fairley and others, 1996a). In another study Fairley and others (1996b)
investigated the mechanical properties and WVP of edible ﬁlms from WPI treated with
N-Ethylmaleimide or cysteine. This study indicated that SH/S-S interchange did not
have a signiﬁcant role in deterrrrining the functional properties of WPI-based ﬁlms. This
was evidenced by ﬁlm retention of WVP and mechanical properties after the blocking of
SH/S-S interchange.

Hydrogen bonds are important in stabilization of protein networks. Hydrogen
bonds are the electrostatic interaction forces between charged groups of protein
molecules and between charged groups on a protein molecule and surrounding water
molecules when water is present (Rasco and Zhong 2000). The intermolecular
association between the proteins results in brittle ﬁlms. This is the reason that

plasticizers are added to these ﬁlms as they disrupt some of these associations and thus

decrease the rigidity of the protein structures (Gerrard 2002). Hydrophobic interactions
establish another set of interactions between protein groups that are important for the
formation of protein networks. These are deﬁned as the attractive forces between non-
polar molecules or groups of molecules that induce association of these molecules in an
aqueous environment (Stenesh 1989). The hydrophobic groups mainly exist on the inside
of the molecules in native proteins; for example, during heating, whey proteins are
denatured, hydrophobic groups are exposed and during drying they are drawn closer and
intermolecular hydrophobic interactions are established (Cheftel and others 1985).

Since formation and stability of protein-based ﬁlms depends on the protein used
and its amino acid composition, proteins high in cysteine residues will produce ﬁlms
higher in S-S bonds, while those having low cysteine content will have more hydrophobic
and hydrogen types of linkages. For example, ﬁlms derived from corn zein possess more
hydrophobic structures than ﬁlms derived from soy proteins (Krochta 1998). Disulﬁde
bonds have been reported to be responsible for cross-linking in soy-protein based ﬁlms.
These S—S bonds are suggested to predominate in whey protein based ﬁlms. Native whey
proteins are globular proteins containing mostly hydrophobic and S-H groups in the
interior of the molecule where they are not readily exposed. Heat denaturation of whey
proteins in an aqueous solution has been used for the formation of whey protein ﬁlms.
The three dimensional structure of the protein is altered during heating, exposing the
internal SH and hydrophobic groups (Shimada and Cheftel 1998) this further promotes S-
S bonding and hydrophobic interaction when the ﬁlm is subsequently dried (McHugh and

Krochta 1994b).

Protein based ﬁlms are dependent on the protein network formed during protein-
protein and protein-solvent interactions as well as the balance between the attractive and
repulsive forces between and among polypeptide chains. These structures are referred to
as “cross-links” and occur within a protein (intramolecular) and between proteins
(intermolecular) (Feeney and Whitaker 1988). Cross-links are vital for maintaining the
correct conformation of certain proteins and may control the degree of ﬂexibility of the
polypeptide chains (Gerrard 2002). Crosslinking not only stabilizes and aids in ﬁlm
formation but is also important for improving ﬁlm insolubility properties and tensile
strength (Pérez-Gago and Krochta 2001). Functional characteristics of protein-based
films are determined by protein-protein interactions. All protein conformational
properties may be dictated by the primary structure of the native protein (arrrino acid
sequence). Thus, ﬁlm forming ability may be inﬂuenced by amino-acid composition,
distribution and polarity; conditions affecting formation of ionic cross-links between
amino and carboxyl groups; presence of hydrogen-bonding groups and intramolecular

and intermolecular S-S bonds (Gennadios and Weller 1991).

1.3. PROPERTIES OF PROTEIN BASED FILMS

Edible ﬁlms are often studied to deterrrrine their ability to protect foods from the
environment and adjacent ingredients. Film water vapor permeability, oxygen
permeability, aroma and oil permeability are important for many foods. Mechanical
properties including tensile strength (TS) and elongation (%E) are most commonly
investigated to assess the ﬁlm’s ability to protect foods from mechanical abuse (Krochta

1997).

1.3.1. Barrier properties
1.3.1.1. Water vapor permeability

This is the most commonly studied property of edible polymer ﬁlms. Due to the
hydrophilic nature of proteins, their moisture barrier properties tend to be relatively low.
The water vapor permeability (WVP) of edible ﬁlms is affected by relative humidity
(RH) and plasticizer type and amount as well as lipid type and amount when lipids are
added (McHugh and Krochta 1994 a,c; Shellhammer and Krochta 1997). When
compared to synthetic materials or polymers such as cellophane and low density
polyethylene (LDPE), plasticized whey protein ﬁlms have a WVP ranging from 1-4
orders of magnitude greater than cellophane and LDPE, respectively (Morillon and others
2000). This indicates that WPI-based ﬁlms need to be optimized so that their WVP can

be reduced to increase their potential for use in increased RH conditions.

To decrease WVP, the hydrophobicity of the ﬁlms can be increased. This has
been done by either: 1) addition of a lipid to the ﬁlm forming solution to produce
emulsion composite or 2) laminating the ﬁlm with a lipid layer. Lamination processed
ﬁlms were found to have signiﬁcantly improved water vapor barrier prOperties compared
to emulsion formed ﬁlms (Weller and others 1998; Cho and others 2002). Laminating,
however, requires more steps than forming a composite ﬁlm in which both hydrophilic
and hydrophobic components are dispersed in the film forming suspension. Furthermore,
delamination of these ﬁlms due to the high surface energy existing between the polar and
nonpolar materials can be a problem (Kamper and Fennema 1984; Shellhammer and

Krochta 1997). McHugh and Krochta (1994c) further reported that decreasing the mean

droplet diameter of ﬁlm forming emulsions correlated well with a linear decrease in

WVP.

More success has been found in homogenizing. the lipid into the protein ﬁlm
forming solution to form emulsion-composite ﬁlms. This is intricately enhanced by the
emulsifying ability of whey proteins (McHugh and Krochta 1994a; Shellhammer and
Krochta 1997; Perez-Gago and Krochta 1999). Lipids such as fatty alcohols and
acetylated monoacylglycerols increase WVP of whey protein ﬁlms while beeswax,
camauba wax, candelilla wax (CW) and parafﬁn wax, which all possess hydrophobic
characteristics, contribute to improved resistance of ﬁlms to water vapor permeability.
Studies by Gontard and others (1994) concluded that the use of lipids with higher melting
points resulted in decreased WVP of wheat gluten (WG)-lipid ﬁlms. Chick and _
Hernandez (2002) studied the effect of increasing wax (camauba or candelilla) content
on the WVP of lactic-acid casein based ﬁlms. Wax content was increased from 5 to 15%
(w/w of protein) and WVP was tested for all wax levels at two RH conditions: 50% and
70%. At 70% RH camauba wax did not effectively decrease WVP despite increases in
wax content. They indicated that CW was more effective than camauba wax in reducing

WVP of lactic acid casein-based ﬁlms at increased RH.

1.3.1.2. Oxygen permeability

Oxygen permeability (OP) for protein ﬁlms such as whey protein isolate (WPI),
soy protein isolate (SP1) and lactic-acid casein has been reported to be relatively high and
comparable to Nylon 6 ﬁlms (Chick 1998). The addition of lipids such as beeswax,
acetylated monoacylglycerol and rrricrocrystalline wax has been shown to increase the

OP of protein ﬁlms (Gennadios and others 1993b; Donhowe and Fennema 1993). The

increase in OP after the addition of microcrystalline wax was attributed to the
microscopic sized crystals creating intercrystalline pathways for oxygen permeation. The
presence of irregular crystal sizes and lattice distortions were given as the reason that lead

to these intercrystalline pathways (Donhowe and Fennema, 1993).

Studies by McHugh and Krochta (l994d) on the effect of plasticizer type and
concentration (IS-30% w/w of protein for glycerol and 30-50% w/w of protein for
sorbitol) on OP of WPI-based ﬁlms showed that ﬁlms containing sorbitol provided better
oxygen barrier properties than those containing glycerol at equal concentrations (30%).
They also showed that as plasticizer concentration increased, OP increased for both
plasticizers (4.3 to 11.6 cm3um/m2.d.kPa and 18.5 to 76.1 cm3.um/m2.d.kPa for sorbitol
and glycerol plasticized ﬁlms respectively). Plasticizer content increased from 35-50%
(glycerol) and 15-30% (sorbitol). These results were for ﬁlms tested at 50% RH. When
WPI-based films were compared to the synthetic polymers high density polyethylene
(HDPE) and ethylene vinyl alcohol (EVOH), they showed lower OP than HDPE and
compared favorably with EVOH, making the use of these ﬁlms (WPI-based) in control of
oxidation and respiration in food systems extremely promising. In experiments
conducted by Chick and Hernandez (2002) on lactic-acid-casein-based ﬁlms, the addition
of lipid (camauba or candelilla wax) and lipid concentration (5-15% w/w of protein) were
found to have no signiﬁcant effect on OP. Increase in RH from 0-50% had no signiﬁcant
effect on OP, but as RH increased from 50% to 70%, signiﬁcant increases in OP were
observed. These were: 0.63 to 2.21 MPa and 0.76 to 1.12 m°.m/mzs.Pa for camauba and

candelilla wax (CW) respectively. These researchers made comparisons to polyester and

10

showed that protein films had better OP than polyester (0.63 and 0.76 m°.m/mz.s.Pa for

camauba and CW containing films vs. 3.24 m3.ni/m2.s.Pa for polyester ﬁlms at 50% RH).

Kim (2000) reported a signiﬁcant decrease in OP in emulsion ﬁlms made with
WPI or whey protein concentrate (WPC) and CW compared to ﬁlms without wax.
Increasing wax content, however, did not further decrease OP. Films containing 2, 4 or
8% wax (w/w of protein) were reported as having no signiﬁcant differences in OP.
Unlike the relationship observed for WVP, OP did not conform to predictable trends

associated with formulation or ﬁlm formation procedures.

1.3.2. Mechanical properties

Mechanical properties possessed by a polymeric material are determined by the
polymer’s stress-strain tensile characteristics. Stress is measured as the force/area and _
strain is the dimensionless fractional increase in length (Hernandez 1997). The ultimate
tensile strength (TS) is the maximum tensile stress a material can sustain prior to rupture.
Ultimate elongation (%E) is the strain at which the sample breaks (Hernandez 1997).
1.3.2.1. Factors affecting mechanical properties

There are several factors that affect mechanical properties of edible protein ﬁlms.
Gennadios and others (1993a) found that TS of W6 and soy protein isolate (SP1) ﬁlms
increased as pH increased above the isoelectric point (pl) of the proteins. For soy protein
ﬁlms the optimum pH range was 6-11; both TS and %E of SPI-based ﬁlms were found to
peak within this pH range. At acidic pH (1-3), TS and %E were signiﬁcantly lower than
at pH 6-11. For WG ﬁlms however, differences in %E at high and low pH were not
signiﬁcant. Since S-H reactivity increases at pH > 8, ﬁlm formation is favored by more

alkaline pH (Banerjee and Chen 1995). Extreme pH (acidic or alkaline), however, may

11

result in reduced TS and %E (Gennadios and others 1993a). The reduced mechanical
properties would be due to the fact that as pH shifts away from the pI, protein molecules
repel one another and protein-protein interactions are reduced as the protein becomes
more soluble. For the ﬁlm to have adequate mechanical properties, protein-protein
interactions are required that become reduced when the protein is very soluble.

McHugh and Krochta (l994d) studied the effect of plasticizer type and amount on
mechanical properties of WPI-based edible ﬁlms. They reported that increasing glycerol
and sorbitol signiﬁcantly decreased TS while increasing %E. Levels of glycerol studied
were 15% and 30% (w/w of protein), TS was found to be the same for equal sorbitol and
glycerol concentration (30%). As plasticizer concentration increased, TS of glycerol
plasticized ﬁlms decreased more than that of sorbitol plasticized ﬁlms. However, %E of
glycerol plasticized ﬁlms exhibited higher elongation than sorbitol plasticized ﬁlms
prepared at 30% plasticizer concentration. Sothomvit and Krochta (2001) also reported
that plasticizer type and amount affected mechanical properties of B-lactoglobulin (B-lg)
ﬁlms. These researchers studied the effect of glycerol, polyethylene glycol (with
molecular weights of 200 = PEG 200 and 400 = PEG 400), sorbitol, sucrose and
propylene glycol at 0.34, 0.44, 0.54 and 0.64 M concentrations. Glycerol and PEG 200
were the most effective plasticizers providing the best mechanical properties. A linear
increasing relationship was found between %E and plasticizer concentration; these
increases in plasticizer concentration, however, reduced TS.

Experiments by Fang and others (2002) showed that the effect of protein
concentration on mechanical properties of WPI ﬁlms was not as signiﬁcant as that of

plasticizer concentration. They studied the effect of increasing B-lactoglobulin (B-lg)

12

from 40% to 90% while keeping the total weight of protein constant at 12% w/w of the
solution. Glycerol was added at a concentration of 20% or 40% to all protein solutions
(protein to glycerol rations of 4:1 or 3:2, respectively). Films prepared with 20% glycerol
had higher TS than ﬁlms possessing 40% glycerol. Increase in protein concentration did
not increase TS. The %E of ﬁlms plasticized with 20% glycerol was lower than that of
ﬁlms plasticized with 40% glycerol; again the protein concentration had no effect on %E.
Possible increases in T8 resulting from increased protein content may not have been
observed due to the high plasticizer concentrations used.

The effects of beeswax, CW, camauba wax and a high temperature melting
fraction of anhydrous milk fat on TS and %E of whey protein based ﬁlms were
investigated by Shellhammer and Krochta (1997). They found that increasing lipid levels
decreased TS for all lipid types. Films formulated with carnauba wax were the strongest
of all the ﬁlms at all lipid levels tested. Elongation was not signiﬁcantly affected by the
lipid type or concentration except for milk-fat-added ﬁlms. Increase in the concentration
of milk fat increased %E signiﬁcantly, this could have been due to plasticizing effects of
unsaturated and low molecular weight triacylglycerols in the milk fat. Lipid type and
concentration were important factors affecting mechanical properties of the protein-lipid
emulsion ﬁlms. Contrary to these ﬁndings, Kim (2000) found a signiﬁcant increase in
the TS of WPI-CW emulsion ﬁlms as wax content increased from 0 to 8% (w/w of
protein). Whey protein concentrate (WPC)-CW emulsion films showed signiﬁcant
increase in T8 with the addition of wax, but, as wax content increased from 2 to 8% (w/w
of protein), no further increases in TS were observed. The addition of CW to WPC ﬁlms

decreased %E, however this decrease remained unchanged as wax content increased.

13

1.3.2.2. Means of improving mechanical properties

Mechanical properties of protein-based ﬁlms can be improved by cross-linking of
proteins. Cross-links confer elastomeric properties due to the formation of intermolecular
covalent linkages (branched chains). These branches increase the rigidity of a material.
When the cross-linking density is sufﬁciently high, it increases the water resistance of the
film (Gontard and others 1994). There are several ways to achieve this including
enzymatic, physical and chemical means. Transglutaminase is an enzyme that has been
used in the cross-linking of proteins (Mahmoud and Savello 1990, 1993; Lim and others
1999). Transglutaminase catalyzes acyl-transfer reactions that cause formation of
glutaminyl-lysine intra and intermolecular cross-links in protein (Nielsen 1995). Larré
and others (2000) reported that transglutarrrinase increased both TS and %E in wheat
gluten ﬁlms. Enzymes, however, are generally costly and this limits their application on
large scale (Vachon and others 2000).

Gamma and ultraviolet (UV) irradiation have been used as physical means to
cross-link protein ﬁlms (Ressouany and others 1998; Brault and others 1997). Brault and
others (1997) reported increased resistance to puncture and moisture due to irradiation
treatment of caseinate ﬁlms. Maximum mechanical strength of irradiated caseinate ﬁlms
was obtained at 64 kGy (Ressouany and others 1998). Vachon and others (2000) also
reported that y—irradiation was efﬁcient in inducing cross-linking of WPI-caseinate ﬁlms.
WPI (5% w/w) ﬁlm forming solutions were irradiated at a total dose of 32 kGy following
heating of the solutions at 90°C for 30 min. Evidence of the cross-linking was shown by
an increase in molecular mass from 0.2 x 103 to >10 x 104 kDa when compared to a

native protein solution. The lower dosage required for these ﬁlms was attributed to a

14

higher dose rate [38.1 kGy/h vs. 1.5 kGy/h used by Ressouany and others (1998)].
Ultraviolet (UV) radiation treatment has also been used in cross-linking of sodium
caseinate ﬁlms (Rhim and others 1999) and SPI-based ﬁlms (Gennadios and others
1998b; Rhim and others 2000). In both studies, UV treatment resulted in decreased
puncture strength (PS), increased TS and decreased %E. The researchers suggested that
UV treatment resulted in the occurrence of other covalent bonds other than S-S bonds.
Although UV treatment decreased the solubility of these of ﬁlms, there were no
differences in TS compared to the untreated control. The effect of UV irradiation in
WPI-based edible ﬁlms was reported by Lim and others (1999). They demonstrated that
UV radiation reduced water solubility and increased TS of these ﬁlms.

Another physical means of improving properties of protein based edible ﬁlms is
heat curing. Heat curing involves application of heat to polymers to induce cross-linking.
Gennadios and others (1996) reported that heat curing soy protein ﬁlms at 80 or 90°C
resulted in ﬁlms with increased TS, reduced %E, increased moisture content and WVP.
Miller and others (1997) reported similar results with heat cured whey protein ﬁlms.
They suggested that heat curing might elicit additional cross-linking of proteins. Rhim
and others (2000) also investigated heat-curing of SPI ﬁlms. Film strips were heat-cured
at 90°C for 24 h in an air-circulating oven. Heat-curing increased TS from 8.2 1- 0.2 MPa
to 14.7 t 0.4 MPa. %E was reduced from 30 t 3.3 to 6.1 t 0.7%.

Chemical agents used for covalent cross-linking of proteins include
gluteraldehyde, glyceraldehydes, formaldehyde, glyoxal and lactic acid. Galietta and
others (1998) reported that cross-linking of whey proteins using formaldehyde enhanced

mechanical properties and decreased solubility of whey protein ﬁlms. Gluteraldehyde is

15

commonly used as a cross-linking agent in the production of collagen casings (Osbum
2002). Although the gluteraldehyde is chemically reacted with the collagen so that it
should not be available in its free form, the toxicity of gluteraldehyde in these materials is
still of concern as they may have residual gluteraldehyde. Dialdehyde starch and l-
l’carbonyldiimidazole have been investigated as less toxic alternatives to the toxic
aldehyde cross-linkers mentioned above (Gennadios and others 19983). Furthermore,
cross-linking of casein with gluteraldehyde has been effectively used to create a high
definition matrix suitable for controlled drug release applications (Latha and
Jayakrishnan, 1994; Lanaerts and others 1991).

Fang and others (2002) also studied the effect of calcium chloride (Ca2+) content
on the mechanical properties of WPl-based ﬁlms. Films containing 20% glycerol (4:1
proteinzglycerol) to ﬁlm forming solutions demonstrated increased TS following the
addition of 10 mM Ca2+. The higher ionic strength due to Ca2+ at this concentration may
have shielded electrostatic repulsions encouraging protein-protein interactions, which
resulted in greater TS. However, TS of WPI ﬁlms prepared with 40% glycerol (3:2
protein:glycerol) only slightly increased after the addition of 5mM Ca2+ to ﬁlm forming
solutions. It is noteworthy that the addition of lOmM Ca2+ resulted in fragile ﬁlms that
could not be tested for mechanical properties.

The addition of calcium chloride (CaClz; 0.04% w/v)) into WPI ﬁlm forming
solution as a cross-linking agent used to improve mechanical and water vapor barrier
properties was also reported by Cagri and others (2001). The rationale used was the
phenomenon that the divalent cation, Ca2+, cross-links between negatively charged

groups on proteins and thus increases cohesion between protein chains, reducing protein

16

polymer segmental mobility and improving both mechanical properties and WVP
(Krochta and others 1990). It was also stated that the addition of Ca2+ enhanced protein
aggregation at low pH (<6.5).

Park and others (2001) investigated the effect of calcium salts and glucano-S—
lactone (GDL) on mechanical and moisture barrier properties of soy protein isolate (SP1)
based ﬁlms. The levels of calcium salts and GDL used were 0.1, 0.2 or 0.3% (w/w of
SPI). Their results showed that CaSO4 improved TS and puncture strength (PS) while
CaClz did not result in a signiﬁcant improvement in these properties. Protein solubility
enabled divalent Ca2+ to bind strongly with polar groups on protein to form denser 3-
dimensional networks in SP1 ﬁlms. Calcium salts also improved WVP of the SPI-based
ﬁlms. CaSO4 reacted with protein constituents to yield insoluble protein fragments and
altered ﬁlm consistency; calcium bridges maximized interactions between negatively
charged molecules and improved protein network and stability. GDL was also effective
in improving mechanical and moisture barrier properties as it increased hydrophobicity
and decreased solubility of the unfolded protein and thus promoted its aggregation.

The addition of calcium to whey proteins in ﬁlm forming solutions presents a
possibility for developing cross-links through the addition of negatively charged ions.
However, no studies were found in the literature that investigated this hypothesis.

1.3.3. Moisture sorption isotherms

Moisture sorption isotherms (MSI) are plots of correspondent equilibrium
moisture content (EMC) of a material with its water activity (aw) at a constant
temperature. Water activity is also used in reference to RH. Moisture sorption isotherms

provide a valid indicator of a food’s storage stability in reduced moisture environments;

17

they also characterize the status of water (bound or free) in the food (Coupland and others
2000). Moisture sorption isotherms of food products must be determined to understand
the behavior of the food product in different moisture or RH environments. They are
especially important for edible ﬁlms if they are to be used to protect foods from moisture
transfer.

Coupland and others (2000) studied the effect of glycerol on moisture sorption of
WPI-based ﬁlms. Glycerol was added in the following ratio: [glycerol (G):non-volatile
material (NVM)] where NVM = glycerol and WPI. The ratio of GINVM was expressed
within the range of G = 005. They found that the moisture sorption of WPl-based ﬁlms
increased slowly as RH increased up to aW ~ 0.75 after which it increased rapidly. Films
higher in glycerol content had higher moisture sorption; native WPI powder had the same
sorption characteristics as WPI-based ﬁlms without glycerol. These results showed that
denaturation and ﬁlm casting had no effect on the protein’s capacity to bind water.

Chick and Hernandez (2002) reported on the MSI of lactic-acid-casein ﬁlms
plasticized with sorbitol (3.5, 4.0, 4.5, or 5.0% w/w) and containing CW or camauba wax
(5, 10 or 15% w/w of protein). The ﬁlms had low moisture uptake up to 70% RH but this
rapidly increased after 85% RH. RH was shown to decrease TS of the ﬁlms; at 75% RH,
ﬁlms possessed 5-fold less strength than at 50% RH. The results also showed an increase
in %E following an increase in RH within the same wax level.

Similar results were reported by Cho and Rhee (2002) for SP1 ﬁlms plasticized
with glycerol, sorbitol and a 1:1 mixture of glycerol and sorbitol. Plasticizer was added
at three levels (0.3, 0.5 and 0.7 plasticizer/g SPI). _ They found that ﬁlms with higher

plasticizer content had higher EMC and ﬁlms higher in glycerol ratio absorbed more

18

moisture. It was suggested that this phenomenon resulted because glycerol has higher
moisture afﬁnity than sorbitol. Yoshida and others (2002) weighed pieces of ﬁlms placed
at 75% RH over a ﬁxed period to determine weight gain: They showed that adsorption of
moisture for whey protein ﬁlms followed a linear diffusion model by.

Investigations by Kim and Ustunol (2001a) showed that EMC of WPI-based ﬁlms
plasticized with glycerol increased slowly up to aw of 0.65 after which EMC rapidly
increased. The presence of CW reduced the amount of moisture uptake. Films were
compared to WPI-based ﬁlms plasticized with sorbitol. Glycerol-plasticized ﬁlms
resulted in a higher EMC than sorbitol plasticized ﬁlms; this was attributed to the
hydroscopicity of glycerol. Their results indicated that EMC appeared to be a function of
the plasticizer hydroscopicity. Further research is required to determine how WPI-based
ﬁlms would function in a high RH environment via practical tests under these
environmental conditions. Additionally, the ﬁlms tested by Kim and Ustunol (2001a)
were uncured ﬁlms; the effect of RH on cured WPI-based ﬁlms requires investigation.
1.3.4. Thermal and sealing properties

Thermal characteristics provide information necessary for determining processing
parameters for edible ﬁlms. Three temperatures are frequently reported with respect to
polymers: the glass transition temperature (T3), the temperature at which reversible
change occurs in amorphous polymers or in amorphous regions of a partially crystalline
polymer (Hernandez 1997); the melting temperature (Tm) and the degradation-
temperature (T d). T.u is the solid to liquid phase change temperature while Ta is the point

of thermal disruption of bonds in the absence of oxygen

19

Using therrnogravimetric analysis, Ogale and others (2000) studied the thermal
characteristics of SPI films plasticized with glycerol (30 w/w). They determined the
upper thermal processing limit to be 150°C. Rapid degradation was observed at
temperatures above 180°C for plasticized films. Non-plasticized ﬁlms degraded at
200°C. Similar processing temperatures were reported by Cunningham and others (2000)
for SP1 ﬁlms.

Thermal properties of lactic-acid casein were studied by Chick and Hernandez
(2002). Differential scanning calorimetry (DSC) results showed that lactic-acid-casein
powder melts between 100 and 110°C. Candelilla wax, a component often used to
increase hydrophobicity of edible ﬁlms, had a narrow endothermic peak (68-73°C).
Lactic-acid-casein-sorbitol ﬁlms had a peak in the temperature range loo-110°C.
Decomposition of lactic-acid-casein ﬁlms started to occur at 120°C.

WPI-based edible ﬁlm thermal characteristics were determined by Kim and
Ustunol (2001c). The composite ﬁlm (WPI-glycerol-CW) had a transition onset
temperature of 108°C and transition peak at 145°C. These characteristics were used to
determine optimal sealing conditions for WPI-based composite ﬁlms. Stehling and Meka
(1994) reported on the effect of melting distribution on heat-sealing properties of
polyoleﬁns. Melting distribution was important for seal strength determination and they
reported that DSC analysis could be used to accurately determine the melting distribution
and hence establish the heat sealing curve of the ﬁlm.

Sealability of edible ﬁlms is an important attribute as it determines the possibility
of using these ﬁlms in food applications such as casings for sausages. Sealing involves

melting of speciﬁed polymer regions by the application of heat and bringing them

20

together while they are in a partially molten state to form integrated bonds between the
two polymer surfaces. Pressure is applied causing polymer chain segments on opposite
sides of the interface to diffuse across the interface. Thus, molecular entanglements are
induced between the melted surfaces and fused upon cooling (Meka and Stehling 1994).

Seal formation depends on the sealing temperature, applied pressure (the force
applied during sealing) and dwell time (the length of time the sealing bars are kept in
contact during sealing). Optimization of dwell time is an important control point. Dwell
times need to be carefully monitored because sealing at high temperatures for a short time
may result in poor temperature control, conversely long dwell times should also be
avoided as they may result in distorted seals. The surface must be pressed together an
adequate length of time and with adequate pressure to induce diffusion across the
interface and the formation of bridged bonds (Mueller and others 1998).

Appropriate seal temperature is determined by examining thermal properties of
the polymer to be sealed. Thermal properties and heat-sealing conditions of WPI-CW
edible ﬁlms were investigated by Kim and Ustunol (2001c). Whey protein isolate based
edible ﬁlms were heat-sealed on the non-lipid oriented side of the ﬁlm because the lipid-
oriented sides did not adhere well. Heat sealing temperatures were based on onset
thermal temperature (T0) of WPI-CW emulsion ﬁlms as determined by DSC analysis.
The seal strengths for glycerol and sorbitol plasticized ﬁlms containing CW ranged from
141 3; 36 to 297 :t 15 N/m for glycerol-plasticized ﬁlms and 105:0.9 to 296 10.6 N/m for
sorbitol-plasticized ﬁlms. The highest seal strength corresponded with the To (108-
122°C) for glycerol-plasticized ﬁlms and (126-127°C) for sorbitol plasticized ﬁlms. It

was suggested that these results might be used to determine thermal processing

21

temperatures for WPI-based edible films. The lowest seal strengths for glycerol and
sorbitol plasticized ﬁlms were obtained when the ﬁlms were heat-sealed at 130°C and
110°C respectively. The lower seal strengths at higher temperatures for the glycerol-
plasticized ﬁlms were explained as having resulted from excessive temperature that
caused distorted and weaker seals. According to Martin (1986), when the heat required
to produce a seal exceeds the heat-sealing temperature range for that material, it induces
distorted or non-functional seals. Based on the results of Kim and Ustunol (2001c) it was
recommended that heat-sealing temperatures should not exceed 130°C for glycerol
plasticized WPI-based ﬁlms.

Results obtained by Kim and Ustunol (2001c) for sorbitol plasticized ﬁlms were
comparable to seal strengths obtained by Chick (1998) for lactic acid casein ﬁlms
plasticized with sorbitol (153-247 N/m). Experimental conditions for these studies,
however, were not identical even though both researchers used a thermal sealer. Seal
temperatures used by Chick ranged from 93°C to 121°C at a pressure of 410 kPa. Seals
formed using a voltage based impulse sealer have not been reported. Because of the
broad surface area required to form seals with the thermal sealer, which may range up to
1.5 cm seal widths, voltage-based impulse sealing might be more appropriate in food
applications since more narrow seal widths can be obtained (0.25 cm). This would be an

advantage since less area and total ﬁlm would be used.

1.4. ELECTRON SPECTROSCOPY FOR CHEMICAL ANALYSIS
Seal strength values provide general information about the strength of the bond;

this information, however, does not provide insight into the chemical characteristics of

22

the bond. By conducting a surface analysis, surface speciﬁc information is obtained that
can be valuable for determining chemical changes due to sealing. This information can
also be useful when developing new materials and applications (Rindlav-Westling and
Gatenholm 2003). Electron Spectroscopy for Chemical Analysis (ESCA), also referred
to as XPS (X-ray photoelectron spectroscopy) provides insight into the surface chemistry
of the material tested and has been used as the principal technique for deﬁning interfacial
molecular properties associated with adhesive behavior of materials. The use of ESCA
allows identiﬁcation and quantiﬁcation of all elements except hydrogen and helium
(Ratner and Castner 1997). It is an ideal technique to study surfaces of scaled ﬁlms
because of its surface sensitivity (Gerenser 1993). .

Surface composition of starch, amylose and amylopectin ﬁlms was investigated
by Rindlav-Westling and Gatenholm (2003) using ESCA. Resolved Cls spectra showed
the presence of nitrogen which was believed to have been derived from protein, bonds
observed were O-C=O, N-C=O, O-C-O and GO These researchers stated that C-C
bonds did not originate from the starch in the polymer but from proteins, lipids and
possibly contaminants on the sample surface. Kim and Ustunol (2001c) conducted a
unique study where seals of uncured whey protein based ﬁlms were investigated.
Findings of this research showed that carbon was the main element of the ﬁlms followed
sequentially by oxygen and nitrogen. After heat-sealing, there was a reduction in carbon
by 1.4 - 6.5% depending on the ﬁlm tested. However, oxygen and nitrogen increased by
1-2% and 1-4% respectively under these sealing conditions. Kim and Ustunol (2001c)
suggested that that some oxygen and nitrogen components had formed on the surface. It

was, however, not explained how this might have happened. Possibly, by virtue of the

23

decrease in C and moisture loss there was a subsequent or apparent increase in these
elements. High-resolution ESCA peaks showed Cls, Ols and N15 spectra. Kim and
Ustunol (2001c) suggested that formation of C-O-H and N-C bonds upon heat sealing
might be responsible for the mechanism of seal formation. These data provided by these
researchers focused on uncured WPI-based ﬁlms; properties of cured WPI-based ﬁlms

have not been investigated using this method.

1.5. COMPRESSION MOLDING

Compression molding is a thermal process that was originally developed by the
automobile industry for replacement of metal components with composite parts. The
molding process can be carried out for either thermoset materials or thermoplastics. Most
applications today use thermoset polymers, and compression molding has become the '
most common processing method employed (Davis and others 2003).

The advantages of compression molding include short cycle times, typically
running at 1-6 minutes, high volume production and high quality surfaces.
Disadvantages include high capital investment, labor intensiveness, and secondary
operations that are at times required (Davis and others 2003). The advantage of short
cycle times involved in using compression molding has been maximized in developing
edible WPI-based ﬁlms. Using compression molding, Sothomvit and others (2003)
developed WPI-based ﬁlms within only 2-3 minutes. This proved to be an advantage
over the solvent casting method that takes 18-24 hours, excluding preparation time.

More recently, compression molding has been applied to the production of plastic
sheets including biodegradable sheets made from soy and wheat protein (Cunningham

and others 2000; Mo and others 1999; Mo and Sun 2000; Zhang and others 2001).

24

Production of SPI plastic sheets has received commercial interest due to the abundance
and relative low cost of the soy bean, and particularly due to environmental
considerations (biodegradability/renewable resource) (Wu and Zhang 2001). Wu and
Zhang (2001) applied compression molding to the development of SPI ﬁlms in which the
properties and structure of SP1-ethylene glycol (EG) sheets were investigated. Films
were compression molded at 150°C with 15 MPa of pressure for 1 min. The sheets
formed had good TS, %E, water resistance and therrnostability (films containing 50%
g/100g of EG) had a T3 of 4.23 MPa, %E of 220% and water resistance was higher than
that of thermoplastic starch or cellulose ﬁlm.

Cunningham and others (2000) studied the tensile properties of SPI ﬁlms formed
through compression molding. Films were made with various plasticizer amounts (20,
25, 30, 35, 40% w/w of protein) and variations in mixing procedures and aging (1 week
or 4 weeks) of the ﬁlms prior to testing were employed. Aging consisted of keeping the
protein mixture for 1 or 4 weeks before developing ﬁlms. Aging conditions (temperature
and relative humidity), however, were not speciﬁed. Thickness and tensile strength
values ranged from 0.16 mm and 1.6 MPa at the highest plasticizer level (40 w/w) to
about 0.35mm and 15.8 MPa at the lowest plasticizer content (20 w/w). Compression
molding conditions were established as: 150°C, 10 MPa for 2 min. Elongation (%)
ranged from 1.5% to 106% and increased with increasing plasticizer content. In a
companion study, Ogale and others (2000) determined the processing temperature for SP1
based compression molded ﬁlms to be about 150°C; thermal degradation temperatures

were determined to occur above 180°C.

25

Very limited research has been conducted on the compression molding of WPI
ﬁlms; yet, like soy proteins, these proteins have received much interest for potential use
in food industry applications. Sothomvit and others (2003) determined the effects of
moisture and glycerol content on ﬁlm WVP and protein solubility, using film obtained by
compression molding. The pressures studied ranged from 0.81MPa — 2.25 MPa.
Compression molding was conducted for 2 minutes because at high temperatures films
degraded after 2 minutes. According to Sothomvit and others (2003), the feasible
temperature range for compression molding of WPI-based ﬁlms was determined to be
104°C — 140°C. Film formation without degradation was obtained at temperatures lower
than 140°C. Temperatures within this range should provide ideal conditions for studying
combinations of time, temperature and pressure for WPI ﬁlms as they fall within the
range of temperatures recommended by Kim and Ustunol (2001c) for thermal processing
of WPI ﬁlms. No research has been conducted using compression molding as a post
treatment for solvent cast ﬁlms. Research into the possibility of this application is

required.

1.6. COLLAGEN CASINGS AND SAUSAGE MANUFACTURE

Casings are important in the sausage manufacturing industry and are used for
forming sausage products into a speciﬁc shape and or portion control. Different types of
casings are used and these include natural casings, reconstituted or regenerated collagen
casings and regenerated cellulose casings (Wang 1986). Traditionally, natural casings
from beef, pork and lamb were stuffed with comminuted meat. However, high bacterial

loads, preservation requirements (salting or salting and refrigeration), absence of

26

uniformity (thickness, color and diameter) and breakage limited utility of these casings.
These are inherent problems with the use of natural gut casings, which are further
complicated by the labor intensive process of cleaning these casings. Furthermore, the
performance and supply of natural casings hindered the processed meat industry’s ability
to move forward into modern automated sausage manufacture at higher production
capacities These factors led to the development of regenerated collagen casings. (Osbum

2002).

Collagen casings are primarily produced from cattle hides. The hides are split in
half and collagen is obtained from the corium (ﬂesh side) of the skin. They may be
manufactured in both edible and non-edible varieties. The edible varieties are smaller,
thin, and chewable and solubilize during cooking. Hood (1987) and Courts (1977)
describe two different methods used for the industrial manufacture of collagen casings,
the ‘dry’ and ‘wet’ methods. The ‘dry process’, also called “dry spinning technology”
was developed in Europe during the 19305 and results in a high solid content suitable for

extrusion.

The ‘wet process' also called “wet spinning technology” consists of using the hide
corium from acid- or alkaline-dehaired cattle hides which are decalciﬁed, ground into
small pieces and then mixed with acid to produce a viscous suspension (4-5% solids)
which is pumped through high shear homogenization. Cellulose and
carboxymethylcellulose may be added to improve mechanical properties of the casing.
The acidiﬁed collagen slurry is then extruded to form a woven ﬁber structure, passed
through a coagulation bath of brine and then formed into a tubular casing shape. The

high salt concentration and acid neutralization shrinks the collagen ﬁbers to form a strong

27

casing. The casings are then washed to remove the salt and treated with plasticizing
(glycerol and sorbitol) and cross-linking (gluteraldehyde) agents to improve casing
strength and pliability. The plasticized collagen casings are then collapsed in an
“accordion—like fashion” (shirred) so that they can ﬁt over various sized sausage-stufﬁng
horns. The collagen casings are then dried under special temperature and humidity
conditions, to 13-18% moisture content, before they are sealed in plastic bags and packed
in boxes. The shelf-life of these products depends upon maintaining favorable (cool and

dry) storage conditions (Hood 1987).

Collagen casings must possess a variety of product attributes to aid in the
manufacturing of various sausage products with numerous types of sausage
manufacturing equipment. Casing strength is therefore important for withstanding the _
rigors of high speed ﬁlling and linking operations. The strength of casings is affected by
solids content (ﬁber content), drying conditions, and addition of cross-linking agents such
as salts and enzymes. Another means for strengthening collagen casings is UV radiation.
Collagen casings may be colored to enhance the external color and appeal of various
sausage products. Colors available include blush, brown, red and smoke. Smoke colored
collagen casings may be used to replace natural or liquid smoke application or reduce
quantities necessary for uniform smoke uptake. Smoke-colored casings provide a richer

uniform smoke-colored product surface (Osbum 2002).

The term sausage refers to chopped or minced meat preserved by salting.
Traditionally, the mixture would be encased in animal intestines or stomachs. As a result,
the characteristic shape of the sausage was cylindrical. The deﬁnition of sausage is

derived from these properties; that is, sausages are salted, seasoned and chopped meat

28

products that are generally cylindrical in shape (Pearson 1984). Sausages are normally
manufactured from the less expensive cuts of meat and from byproducts; hence they are
economical. Process variables associated with ﬁnal product quality can be classiﬁed into
several groups: 1) degree of chopping and added water; 2) amount of cooking, smoking

and curing; and 3) fermentation time and ﬁnal moisture of the product.

Cooked sausages are comminuted, semi-solid sausages prepared from one or
more kinds of raw skeletal meat and/or poultry meat. Generally, they contain no more
than 30% fat and 10% added water and may or may not be smoked. There are three basic
types of smoking/cooking methods: 1) natural air circulation 2) air-conditioned or forced
air and 3) continuous. When cooking is carried out simultaneously with smoking,
temperature and RH are very important. The cooking is often achieved by gas or steam
heat. The RH is usually highest at the highest temperature because heat transfer is more
efﬁcient at higher RHs. Target temperatures in the ﬁnal processing stage are expressed as

internal temperature reached by the product (Pearson 1996).

Smoke uptake, also referred to as deposition of the smoke on the meat, is
dependent upon smoke density, smokehouse air velocity, smokehouse RH and the surface
area of the product. RH affects the rate of deposition as well as the nature of the deposit.
Higher RH results in more smoke deposition but reduced color development. The
smoking/cooking process must therefore be carefully monitored to produce required or

desired results (Pearson 1996).

29

CHAPTER 2

EFFECT OF MEAT PROCESSING CONDITIONS ON MECHANICAL
PROPERTIES OF HEAT CURED WHEY PROTEIN-BASED EDIBLE FILMS: A

COMPARISON TO COMMERCIAL COLLAGEN CASINGS

2.1. ABSTRACT

Edible ﬁlms were produced using WPI (5%, w/w), glycerol (3.3%, w/w) and
candelilla wax (CW; 0.8%, w/w). One set of ﬁlms was heat cured at 90°C for 12 h and
another at 80°C for 24 h. The WPI-based ﬁlms together with collagen films were put
through a meat processing scheme typical of Polish sausage manufacture. The meat
processing conditions were stage 1: 57°C/60min/36%RH; stage 2: 65°C/90min/60%RH;
and stage 3: 77°C/30min/80%RH. The effects of meat processing conditions on
mechanical properties: tensile strength (TS) and elongation (%E) were determined. All
ﬁlms remained intact throughout the process. Initially TS of WPI-based ﬁlms heat cured
at 90°C for 12 hours was similar to that of collagen ﬁlms (p<0.05) but decreased during
the second stage of the cooking process. The TS of ﬁlms heat cured at 80°C for 24 hours
was lower (p<0.05) than that of collagen ﬁlms before processing and also decreased
during the cooking process. The %E of collagen ﬁlms and WPI-based ﬁlms were similar

before and after processing.

30

2.2. INTRODUCTION

Various applications of whey protein isolate (WPI)-based ﬁlms have been
proposed, such as wrapping for cheese slices, individual serving pouches for cocoa mix
(Kim 2000) and antimicrobial sausage casings for hot-dogs (Cagri and others 2003).
Sensory evaluation of WPl-based ﬁlms showed that the ﬁlms had acceptable taste, and no
distinct milk ﬂavor or odor was detected in them (Kim 2000). Cagri and others (2003)
developed antimicrobial WPI-based films with which they made hotdog casings. They
conducted a sensory evaluation of the hotdogs and demonstrated that the casings were of
acceptable taste and color in comparison to commercial collagen and natural casings.
Their ﬁndings indicate the potential for WPI-based ﬁlms to be used in food applications
such as sausage casings.

Sausage manufacture involves the use of high relative humidity (RH) for the
purpose of heat transfer to the core of the sausage product (Ockerman 1989). The meat
product in contact with the casing is also high in moisture, beef and pork contain between
70 and 73% moisture content and sausage formulations have water added that my
increase the moisture content to ~99%. The hydrophilic nature of proteins presents a
challenge in using the WPI-based ﬁlms in high moisture environments. Several
researchers have shown that with increased relative humidity (RH), the moisture
absorption of protein-based ﬁlms, speciﬁcally WPI-based ﬁlms dramatically increases.
This increase results in increased elongation (%E) and decreased tensile strength (TS)
(Coupland and others 2000; Kim and Ustunol 2001a; Chick and Hernandez 2002). The
mechanical properties (TS and %E) of edible ﬁlms are important in determining their

application to foods because these properties provide an indication of how much stress

31

and strain the film can withstand prior to fracture (Hernandez 1997). This is important
because sausage casings must be tender enough to be pliable during stufﬁng and yet be
strong enough to hold the meat batter during cooking (Osbum 2002). The
hydrophobicity of ﬁlms has been increased by the addition of lipids. Kim and Ustunol
(2001a) studied the effect of candelilla wax (CW) and butterfat on ﬁlm properties and
showed reduced moisture sorption and increased TS with the addition of CW. Chick and
Hernandez (2002) studied the effect of camauba wax and CW on the barrier and
mechanical properties of lactic-acid-casein ﬁlms. Their ﬁndings revealed that the
addition of wax, particularly CW, not only decreased water vapor permeability of the
ﬁlms but also increased TS compared to ﬁlms without wax. To further improve the
tensile properties of protein-based ﬁlms, various methods have been used. Tensile
strength has been optimized through heat curing, among other physical or chemical
means. Heat curing of WPI-based ﬁlms was reported to increase covalent cross-linking
and thus cause increase in TS and reduce %E (Miller and others 1997). Similar results
were reported for soy protein isolate (SP1) ﬁlms (Gennadios and others 1996; Rhim and
others 2000).

In order for WPI-based ﬁlms to be used as sausage casings, their mechanical
properties need to be comparable to the properties of commercial casings currently used
for this purpose. Collagen casings are currently the most widely used edible sausage
casings as alternatives to natural casings. Sausage casings enable comnrinuted meat
batters to be shaped into speciﬁc shapes as they undergo thermal processing (Osbum
2002). Collagen casings have numerous advantages over natural casings, which include

1) more uniform size, strength and ﬂexibility required to withstand a variety of

32

processing environments; 2) a cleaner, more sanitary product and 3) greater consistency
in net product weight (Kutas 1987). To enhance the appeal of sausage products encased
in collagen casings, brown, caramel and other food grade colors are often added to
provide products with distinct colors that are marketed as distinctive, smoked, or spicy
specialty products. This practice is often employed because non-colored casings tend to
appear gray after steam cooking (Osbum 2002). Heat cured WPI-based ﬁlms provide the
beneﬁt of a natural pigmented appearance similar to caramel or smoked colored casings
without the addition of food coloring. Increased yellowness (demonstrated by increased
Hunter Lab b/yellowness values) has been reported following heat curing of these ﬁlms
(Kim and others 2002; Cagri and others 2003). These ﬁndings further indicate the
potential for WPI-based films to be used as sausage casings.

Previous research from our lab has shown that heat-cured (80, 90 and 100°C for
12, 24, 48 and 72 h) WPI-based ﬁlms plasticized with glycerol and containing candelilla
wax have mechanical properties similar to collagen ﬁlms (Amin and others 2003). A
WPI-based ﬁlm that can withstand meat process conditions may provide the meat
industry with an alternative to collagen ﬁlms. Therefore, the objective of this research
was to determine if heat-cured WPI-based edible ﬁlms could withstand the temperature,
time and RH processing conditions encountered in a comminuted meat cooking

processing scheme typically used for Polish sausage manufacture.

33

2.3. MATERIALS AND METHODS

2.3.1. Materials

Whey protein isolate (WPI, Provon 190) was supplied by Glanbia ingredients
(Monroe, WI). Glycerol was purchased from IT Baker Co. (Phillipsburg, NJ). Sodium
hydroxide (2N, NaOH) was purchased from Mallinckrodt Specialty Chemical Co. (Paris,
KY). Candelilla wax (CW) was purchased from Strahl and Pitsch Inc. (West Babylon,
NY). Edible collagen casings (inside diameter, 32mm) were obtained from The
Brechteen Co. (Chesterfield, MI). Magnesium nitrate, lactose, boric acid, hydrochloric
acid, methylene blue and sodium hydroxide were purchased from Sigma Chemical Co.
(St Louis, MO) and preweighed Kjeldahl catalyst tablets were purchased from Fisher

Scientiﬁc (Pittsburgh, PA).
2.3.2. Proximate analysis

Proximate analysis was conducted to verify the composition of the whey protein
isolate (WPI). The values were compared to speciﬁcations provided by Glanbia

Products.
2.3. 2.1 . Moisture content

Moisture content was determined according to the AOAC gravimetric method
927.05 (2000). A 1.0 — 1.5g sample of WPI was weighed into a ﬂat-bottomed metal dish
and dried in a convection oven at 130°C until equilibrium was reached. Equilibrium was
reached when the sample was at constant weight and this was determined by weighing
the sample at regular time intervals. Samples were removed from the oven, allowed to

cool in a dessicator and weighed. Moisture content was determined as loss of sample

34

weight during drying and expressed as a percentage of initial weight of sample [(initial
sample weight — dried weight)/(initial weight of sample)] x 100).

2.3.2.2. Protein content

Protein content was determined according to the AOAC micro Kjeldahl method
930.29 (2000). A 0.06g sample of WPI was weighed and portioned into a Kjeldahl
digestion ﬂask with I/2 a catalyst tablet [(3.5g K2804, 3.5mg Se) or (3.6 x 10‘4 M K2S04,
3.6 x 104 mM Se)]. The sample was heated to digest the WP1 until the solution was clear,
~6 h. Approximately 20 ml of 30% NaOH was added to the digested sample and this was
distilled into a ﬂask containing 10ml boric acid plus 2 drops methylene blue indicator.
The digest was then diluted with water (5ml) and titrated with standardized 0.0501 N
HCl. Percent protein was calculated using the equation: %protein = (vol. HCl) x (1.4007) _
x (6.38) x (normality of HCl)/sample weight in g; where 6.38 is the protein conversion
factor.

2.3.2.3. Ash

Ash content was determined using the AOAC gravimetric method 930.30 (2000).
A 2.0g WPI sample was weighed into a crucible and ashed in a mufﬂe furnace at 600°C
until it was carbon-free (2'/2 hours). The crucible was then removed from the furnace,
allowed to cool in a dessicator and weighed. Ash content was determined as the
(residue/initial weight of sample) x 100.

2.3.2.4. Lactose content

Lactose content was determined using high pressure liquid chromatography
(HPLC). The WPI sample was prepared according to the method described in Standard

Methods for the Examination of Dairy Products (Marshall 1992) with the following

35

changes: a 5.0g sample of WPI was dissolved in 20 ml of deionized H20. From this
solution, a 10.0g sample was obtained to which 1 ml of 0.9N H2804 was added; water
was then added to yield 100 ml and the solution was shaken for 20 s. This solution was
allowed to stand for 1 h, then ﬁltered through a Whatman #41 filter. Samples were
separated on‘ a Waters HPLC system using a Rezex RNM carbohydrate column
(Phenomenex, Torrance CA). Lactose in the sample was quantiﬁed using a Waters
refractive index detector (Waters, Milford MA). The areas under the sample and
standard (0.5% w/v) lactose peaks were determined (in cmz). These areas were used to
determine the lactose levels. The reportable sample lactose value (%) was calculated

from the equation shown below.

% recovery = [Y(a) — 4.75)/X(a)] x 100, where Y(a) = standard lactose level, X(a) _

= sample lactose level and 4.75 is a constant.
2.3.3. Whey protein isolate based film preparation

WPI (5% w/v) was dissolved in distilled water, glycerol (3.3% w/v) was added
and pH was adjusted to 8 with 2N NaOH. The solution was heated to 90 i 2°C while
being stirred continuously to denature the whey protein. Candelilla wax (0.8%, w/v) was
added during heating and allowed to melt into the solutions to provide a ﬁnal solids
content of 5.8% w/v for the ﬁlm forming solution. The solution was homogenized for 2
minutes using a Polytron PI‘ 10/35 homogenizer with a PI‘A 20 T8 homogenizing head
(Tekmar Co., Cincinnati, OH) at speed 5 (1350 rpm). The ﬁlm forming solution was
ﬁltered through a layer of cheesecloth, equilibrated to room temperature for 1.5 h and
degassed using a vacuum pump at room temperature for 30 rrrin. Films were prepared by

casting the ﬁlm forming solution (27.5 ml) onto 18.5 cm circular Teﬂon® plates (Kim

36

2000). The ﬁlms were dried at room temperature: 23 i 2°C and 30 j; 5% relative
humidity (RH) for 18 i 3 h. Dried ﬁlms were peeled, wrapped in foil and heat cured in a
vacuum (30mm Hg) oven maintained at 1) 80°C for 24h or 2) 90°C for 12 h. The ﬁlms
were removed from the oven and peeled from the foil while still warm for ease of
removal. The ﬁlms were subsequently conditioned at 50% RH, 23°C in a dessicator
containing saturated magnesium nitrate for 48 h prior to testing (Figure 2.1). Standards
for the use of aqueous salt solutions for maintaining constant RH are well documented

(ASTM 1997).
2.3.4. Collagen films

Collagen ﬁlms were conditioned in a similar way to WPI-based ﬁlms. Collagen
ﬁlm thickness and mechanical properties: TS and %E were evaluated for comparisons
with heat cured WPI-based ﬁlms. These analyses were conducted simultaneously to

assure direct and meaningful comparisons.
2.3.5. Film thickness

Films were cut into strips (101.6mm x 25.4mm) using 3 Precision Sample Cutter
(Thawing Albert Instrument Co., Philadelphia, PA). Film thickness was determined
using a TM] model 549M micrometer (Testing Machines, Inc. Amityville, NY). Five
different measurements were taken in random locations within the ﬁlmstrip test area and

the mean values were used for calculations of mechanical properties.

2.3.6. Film treatment

The ﬁlm strips were hung in sausage sticks using ﬂexible wire and put through a

cooking process typical for Polish sausage manufacture in a smokehouse; Enviro-Pak,

37

Dissolve WPI (5%w/v) + Glycerol (3.3 w/v) in Deionized H20

11

Adjust to pH 8 with 2 N NaOH

8

Heat solution, stirring (90°C, 45 min), add candelilla wax during last 5 min

11

Homogenize for 2 min

0

Strain & equilibrate to room temperature (~ 23°C, 1.5 h)

[I

Degas under vacuum (30 min)

11

Cast onto Teﬂon® plates

I1

Dry at room temperature (~ 23°C, 24 h; 30 j; 5%RH)

11

Peel ﬁlms from casting surface, wrap in aluminum foil

11

Place in vacuum oven (80°C, 24 h or 90°C, 12h)

ll

Peel ﬁlms from foil before cooling

3

Condition samples before testing (~ 23°C, 48 h; 50 RH)

Figure 2.1 Schematic diagram of ﬁlm forming procedure
WPI = whey protein isolate, RH = relative humidity

38

CHU-lSOE; Micro-Pak series MP 500 (Clackamas, OR). The meat process conditions
were stage 1: 57°C/60 min/36%RH; this is the initial drying step in the cooking process.
It is done to create a uniformly dry surface to regulate the smoke absorbed by the
product; stage 2: 65°C/90min/60%RH; this simulates the cooking step and stage 3:
77°C/30min/80%RH; the ﬁnishing step where the product is cooked to target internal
temperature (72°C). The intent of the high RH in stage 3 is to increase heat transfer to
the center of the product. Processing began with six samples and two samples were taken
for testing at the end of each stage. Samples in stage 3 underwent the entire cooking

process, whereas, control samples did not undergo any processing (Figure 2.2).

 

 

 

Stage 1
57°C/60 min/36%RH

Stage2 Stage3
65°C/90min/60%RH ‘W 77°C/30min/80%RH

 

 

 

 

 

 

 

 

 

Z

 

 

Z

 

 

N

 

 

 

1 Y

Analyze for mechanical properties: tensile strength (TS) and
elongation (%E)

 

 

 

 

Figure 2.2 Schematic diagram of meat processing simulation, each square represents a
ﬁlm sample. Stages 1, 2 and 3 represent sausage cooking stages.

39

2.3.7. Mechanical properties

Film strips were evaluated immediately in a room maintained at 50% RH and
23°C after removal from the smokehouse. TS and %E were determined according to
standard D882-91 (ASTM 1995) using the Instron Universal Testing Machine Model
2401 (Canton, MA) at 23 i 2°C and 50 i 5% RH. A lkN static load cell with a

crosshead speed of 50.8 cm/min was used.
2.3.8. Statistical analysis

All the experiments were replicated three times in a randomized design. A new ﬁlm
forming solution and new set of ﬁlms was prepared for each replicate. Comparisons
between means were determined using the Student-Newman-Keuls method for multiple
comparisons and statistical significance was tested at (p<0.05). Sigma Stat (J andel Corp.,

San Rafael, CA) was used to conduct statistical analysis and for mean comparisons.

2.4. RESULTS AND DISCUSSION
2.4.1. Whey protein isolate composition

Table 2.1 shows the composition of the WPI powder used for the production of
WPI ﬁlms. The WPI composition determined was consistent with the speciﬁcations

provided by Glanbia products (Monroe, WI).

2.4.2. Film thickness

Thickness of the ﬁlms was 0.14 t 0.02 mm for ﬁlms heat-cured at 80°C for 24
hours, 0.14 1 0.02 mm for ﬁlms heat-cured at 90°C for 12 hours and 0.11 :1: 0.02 mm for

collagen ﬁlms. There was no statistical difference in thickness between any of the ﬁlms.

40

The WPI-based film thickness values were consistent with range of film thickness
previously reported by Kim (2000) which were 0.14 t 0.02 mm. They were, however,
higher than those found by Vachon and others (2000), who reported thicknesses of 0.05-
0.06 1- 0.002 mm for WPI-based ﬁlms cross-linked by heating and irradiation. The
difference may have resulted from differences in ﬁlm forming solution formulations, that
is, in this study, CW was incorporated in to the ﬁlms while Vachon and others (2000) did

not incorporate lipids.

Table 2.1 Compositional analysis of the whey protein isolate powder utilized for whey
protein isolate based ﬁlms.

 

 

Experimental Mean Valuesl (%) Supplier’s Speciﬁcations (%)
Protein 92.08 i 0.65 92.0-95.6r
Lactose 0.012 t 0.004 <1.0
Ash 2.64 1 0.01 <3.0
Moisture 4.11 :I: 0.33 <5.0

 

 

 

' =3 for all components tested.
2 Protein was determined on a dry basis and a nitrogen conversion factor of 6.38

2.4.3. Mechanical properties
2.4. 3. I . Tensile Strength

Before the meat processing treatment, WPI-based ﬁlms heat-cured at 80°C for 24
h had lower TS than collagen ﬁlms (p<0.05). Although the TS declined during the
cooking process, this decrease was not statistically signiﬁcant (Figure 2.3). The initial
T8 of WPI-based ﬁlms heat-cured at 90°C for 12 h was similar to that of collagen ﬁlms;

during the second cooking stage, however, the TS of the WPI-based ﬁlms declined

41

(p<0.05; Figure 2.4). Tensile strength of collagen films did not decrease at any time
during the multi-stage cooking process

Several researchers have reported decline in TS as RH increases. Cho and Rhee
(2002) reported on the effect of RH on mechanical properties of SP1-based ﬁlms
plasticized with glycerol (0.3-0.7 g/g SP1). These ﬁlms had 3 TS of 14-32 MPa at 11%
RH, which decreased to 14 MPa when the ﬁlms were stored at 75% RH. They reported
that at higher RH, the ﬁlms absorbed moisture that resulted in decreased TS. Moisture
sorption isotherm (MSI) data has also indicated the sensitivity of WPI-based ﬁlms to
increases in RH. Coupland and others (2000) reported that the moisture content of WPI-
based ﬁlms increased slowly as RH increased up to aW ~ 0.75 after which it increased

very rapidly.

 

18.00 __

16,00 - IWPI IColIagen
E 14.00 ~
5: 12.00 4
310.00 -
8.00 4 a
6.00 a
4.00 .

  
 
 
 
   
 
 

 

Tensile Stre

 

 

Control , Stage 1: Stage 2: Stage 3:
57/60/36 65/90/60 77/30/80
Cooking Stages

Figure 2.3 Tensile strength of whey protein isolate based ﬁlms heat-cured at 80°C for 24
hours and collagen ﬁlms subjected to Polish sausage manufacturing conditions. n = 3 for
all treatments. WPI = whey protein isolate, control ﬁlms did not undergo processing;

a't’Different letters denote signiﬁcant differences; cooking stage conditions: l°C/rrrin/%RH

42

 

16.00 W

‘ IWPI ICollagen

 

g 14.00 ‘ a

E_ 12.00 ‘

.:

g, 10.00 t

I:

g 8.004.

m I

2 6.00 4

g 4.00i

'- 2.00 41

0.00 4—
Control | Stage 1: Stage 2: Stage 3:

57/60/36 65/90/60 77/30/80
Cooking Stages

Figure 2.4 Tensile strength of whey protein isolate based ﬁlms heat-cured at 90°C for 12
hours and collagen ﬁlms subjected to Polish sausage manufacturing conditions. n = 3 for
all treatments. WPI = whey protein isolate, control films did not undergo processing;

a'l’Different letters denote signiﬁcant difference; cooking stage conditions: l°C/rnin/%RH
Similar ﬁndings were reported by Kim and Ustunol (2001a) for WPI-based ﬁlms ‘
plasticized with glycerol or sorbitol and containing butterfat or CW. Equilibrium
moisture content (EMC) of their ﬁlms increased slowly up to aw 3 0.65, further increase
in aw increased EMC dramatically. Glycerol-plasticized ﬁlms had higher EMC than
sorbitol-plasticized ﬁlms, this was attributed to the higher hygroscopicity of
glycerolcompared to sorbitol. The presence of butterfat and CW reduced the EMC for
both plasticizer types. Chick and Hernandez (2002) reported on the MSI of lactic-acid
casein ﬁlms plasticized with sorbitol and containing CW or camauba wax. The ﬁlms had
low moisture uptake up to 70% RH but this rapidly increased after 85% RH. Relative
humidity was shown to decrease TS of the ﬁlms, at 75% RH, ﬁlms were 1/5 of the
strength of those maintained at 50% RH. The decline in TS observed in this study is

consistent with the ﬁndings of Chick and Hernandez (2002). Based on their results, we

43

can conclude that it was the increase in RH during the cooking process that lowered the
T8 of the ﬁlms.

The higher TS of ﬁlms heat cured at 90°C for 12h compared to films heat-cured at
80°C for 24 h was consistent with ﬁndings by Kim and others (2002) who reported that as
curing temperature increased, T8 of SPI ﬁlms increased. They showed that ﬁlms heat-
cured at 85°C had higher TS than ﬁlms heat-cured at 60 or 725°C. Kim and others
(2002) standardized heat curing of ﬁlms at 24 h for all curing temperatures. Increases in
TS from 8.2 to 15.8 MPa following heat-curing (90°C for 4h) was also reported by Rhim
and others (2000) for SP1 ﬁlms. The increase in heat-curing temperature is reported to
increase the formation of covalent bonds between protein chains and as water is
evaporated, closer interaction occurs between the protein chains resulting in increased

TS.
2.4.3.2. Elongation

There was no difference between the %E of WPI-based ﬁlms and collagen ﬁlms both
before and after processing at both heat curing conditions of the ﬁlms for all cooking
stages (Figures 2.5 and 2.6). With the increase in RH, an increase in %E was expected as
it was previously reported by Chick and Hernandez (2002). They reported that as RH
increased from 50% to 75%, %E of lactic-acid-casein ﬁlms containing 10% CW
increased from 71 i: 26% to 116 i 17%. Such an increase was not observed in the
current study (Figures 2.5, 2.6). Kim and others (2002) reported that heat-curing SPI-
based ﬁlms resulted in decreased %E. Similar results were reported by Rhim and others
(2000) for SP1 heat cured at 90°C for 24h. Heat curing also decreases solubility of ﬁlms

in water. Kim and others (2002) reported that as curing temperature increased, solubility

o 'mw—.=~

100
90w " IWPI IaCollagen

 

 

\\\\\\"2
\ Y \\

Elongation, %

 

 

 

.\.\

Control Stage 1: Stage 2: Stage 3:
' 57/60/36 65/90/60 77/30/80
Cooking Stages

Figure 2.5 Elongation of whey protein isolate based ﬁlms heat cured at 80°C for 24 hours
and collagen ﬁlms subjected to Polish sausage manufacturing conditions. n = 3 for all
treatments, WPI = whey protein isolate, control ﬁlms did not undergo processing;

aSame letter denotes no signiﬁcant differences; cooking stage conditions: l°C/rnin/%RH

100

 

90 « a IWPI ICollagen

Elongation, %

. \p
\ .

,\ \‘ _. \

\\

. \

 

 

 

Control Stage 1: Stage 2: Stage 3:
‘ 57/60/36 65/90/60 77/30/80
Cooking Stages

Figure 2.6 Elongation of whey protein isolate based ﬁlms heat cured at 90°C for 12 hours
and collagen ﬁlms subjected to Polish sausage manufacturing conditions. n = 3 for all
treatments, WPI = whey protein isolate, control ﬁlms did not undergo processing;

aSame letter denotes no signiﬁcant differences; cooking stage conditions: l°C/min/%RH

45

of SPI films decreased; Rhim and others (2000) reported a 12-fold decline in solubility of
SPI ﬁlms heat cured at 90°C for 24 h. The reduction in solubility was attributed to cross-
linking resulting from the heat-curing treatment (Gennadios and others 1996; Perez-Gago
and Krochta 1999). Reduced solubility also suggests reduced hydrophilicity in the ﬁlms;
a reduction in hydrophilicity would be expected to reduce elasticity (%E) because the
plasticizing effect of adsorbed water would be lost. It is possible that no increase was
seen in %E because the films were heat cured. Heat curing induces protein cross-linking,
which in turn reduces water adsorption (Perez-Gago and Krochta 2001). This may
explain why %E in this study did not increase with RH during processing as would be
expected. Because the films were heat cured, their water adsorption was reduced; had

adsorption not been reduced %E might have increased with increasing RH.

Decreases in %E resulting from heat-curing were also reported by Miller and
others (1997) for WPI-based ﬁlms heat cured at 60, 70 and 80°C. They suggested that
the moisture content of a ﬁlm determines its mechanical properties. Since moisture
content is decreased during heat curing, this may result in decreased %E since water has a
plasticizing effect on protein ﬁlms. This, however, was not the case with the ﬁlms
observed in this study (Figures 2.5, 2.6). It has also been reported that effective
plasticizers will generally resemble most closely the structure of the polymer they
plasticize. Protein ﬁlms are, therefore, best plasticized by hydroxyl compounds like
glycerol and glycol (Banker 1966). Water being a hydroxyl compound would, therefore,
have plasticizing effects on protein ﬁlms. Lim and others (1999) reported that absorbed
water and glycerol plasticize protein ﬁlms synergistically resulting in films that are more

ﬂexible. The stabilization of the %E of the ﬁlms in this study may also be attributed to

46

the added glycerol used to plasticize the ﬁlms. This would suggest that glycerol was an
effective plasticizer for the WPI-based ﬁlms. The addition of CW to the WPI-based
films may be yet another reason the %E was not affected by the cooking process. The
CW added to the ﬁlms may have increased their hydrophobicity making them more

resistant to the effect of RH.

2.5. CONCLUSIONS

Heat curing temperature was effective in increasing TS of WPI-based ﬁlms,
evidenced by the similarity between ﬁlms heat cured at 90°C for 12 h to collagen ﬁlms.
The TS of WPI-based ﬁlms decreased with the increased temperature, time and RH of the
cooking process. Even though the statistics showed similarity between WPI-based ﬁlms
and collagen ﬁlms, the TS of WPI-based ﬁlms was lower (p<0.05) than that of collagen
ﬁlms (6 MPa vs. 12 MPa for control ﬁlms) respectively. The lower T8 of WPI-based
edible ﬁlms demonstrated the need to improve their mechanical properties to enable their
use in the high RH sausage cooking conditions. The similarity between the %E of WPI-
based ﬁlms and collagen ﬁlms indicates a potential for WPI-based ﬁlms to be used as
sausage casings. The RH increase directly affected the TS of WPI-based ﬁlms, however,
there was sufﬁcient hydrophobicity and plasticizer within the protein-ﬁlm matrix to

retain the %E properties.

47

CHAPTER 3

OPTIMIZATION OF MECHANICAL PROPERTIES OF WHEY
PROTEIN ISOLATE BASED EDIBLE FILMS FOR USE IN SAUSAGE

MANUFACTURE

3.1. ABSTRACT

Edible ﬁlms were produced using WPI (5 or 5.5% w/v), glycerol (3.3 or 3.6%
w/v) and candelilla wax (CW; 0.8 or 0.9% w/v) to provide ﬁlm forming solutions of
5.8% w/v and 6.4% w/v solids content. Films were cast on anodized and non-anodized
Teﬂon® surfaces. Film treatments included heat curing at 90°C for 12 h and
compression molding at single-stage (110°C, 1.24 MPa, 1 h or 2 h) and double stage
(110°C, 0.99 MPa, 25 min and 110°C, 1.24 MPa, 12 min) conditions. A separate
compression molding experiment was conducted to optimize temperature (90, 98, 110
and 121°C), pressure (0.99, 1.10, 1.24 and 1.38 MPa) and time (2, 12 and 22 min) using
response surface methodology. All ﬁlms were evaluated for mechanical properties:
tensile strength (TS) and elongation (%E). Film forming solution solids content and
casting surface affected %E but not TS. The %E of WPI-based ﬁlms cast on anodized
surface after heat curing was higher than that of uncured ﬁlms; %E of 5.8% w/v solids
ﬁlms was lower than that of 6.4% w/v solids ﬁlms on non-anodized surfaces. Heat
curing and compression molding had similar effects on TS and %E of uncured WPI-

based ﬁlms; they increased TS and did not change %E.

48

3.2. INTRODUCTION

Mechanical properties such as tensile strength (TS) and elongation (%E) are
important properties in casings to be used in high speed automated processes like sausage
manufacture. Sausage casings must be able to shrink and stretch to allow contraction and
expansion of meat batter during processing. They must also be strong enough to hold the
meat batter during cooking and yet tender enough to be pliable (Osbum 2002).
Processing of sausage includes stufﬁng, linking, cooking, packaging and storing (Osbum
2002). For whey protein isolate (WPD-based ﬁlms to be used in food applications like
sausage manufacture, their mechanical properties need to be optimized so that they are
comparable to commercial casings currently used in the manufacture of sausage.
Mechanical properties of protein edible ﬁlms have been optimized using different cross-
linking means including heat denaturation (Rhim and others 2000; Perez-Gago and
Krochta 2001), heat curing (Rhim and others 2000; Kim and others 2002) and
compression molding (Mo and others 1999; Mo and Sun 2000; Micard and others 2000).
Gennadios and others (1996) reported that heat curing soy protein ﬁlms at 80 or 90°C
resulted in ﬁlms with increased TS and reduced %E. Miller and others (1997) reported
similar results with heat cured whey protein ﬁlms. They suggested that heat curing elicits
additional cross-linking of proteins. Rhim and others (2000) also investigated heat curing
of soy protein isolate (SP1) ﬁlms. In compression molding studies, Mo and others (1999)
investigated the effect of molding temperature, pressure and time on TS and %E. Their
ﬁndings showed that at low temperature, it took a longer time to reach maximum TS and
%E. Increase in temperature resulted in decreased mechanical properties. They reported

similar effects with increasing pressure. Micard and others (2000) investigated the effect

49

of molding temperatures without the application of pressure on wheat gluten (WG). They
reported that as temperature increased, TS increased while %E decreased. Sothomvit and
others (2003) have conducted a unique study on the compression molding of WPI-based
ﬁlms. Their results showed that compression molding was a viable method for
development of WPI-based edible ﬁlms.

Other means used for improving protein films mechanical properties include: the
use of enzymes (Larre’ and others 2000), irradiation (gamma and ultraviolet) (Rhim and
others 1999; Micard and others 2000; Vachon and others 2000), formaldehyde (Micard
and others 2000), calcium salts (Galietta and others 1998; Fang and others 2002; Cagri
and others 2002), protein concentration (Chick and Ustunol 1998; Fang and others 2002)
and pH (Gennadios and others 19933). To increase the ﬂexibility of protein ﬁlms,
plasticizers are added to the ﬁlm forming solution; by increasing the free volume within
the protein matrix, they allow for mobility and in this way increase %E (Banker 1966;
McHugh and Krochta 1994d; Sothomvit and Krochta 2001). Flexibility in WPI-based
films to be used as casings is important because they need to be pliable enough for
stufﬁng and linking. To increase the hydrophobicity of protein ﬁlms, lipids or waxes
such as butterfat, beeswax, camauba and candelilla wax (CW) are added to the ﬁlm
forming solution. Increasing the hydrophobicity of protein ﬁlms reduces their moisture
uptake when placed in high relative humidity (RH) (Kim and Ustunol 2001a, Chick and
Hernandez 2002). Low moisture uptake with increased RH is necessary for WPI-based
ﬁlms to be used in sausage manufacturing because proteins are hydrophilic and the
cooking process uses elevated RH. Candelilla wax was also reported to increase TS of

protein ﬁlms (Kim 2000). The current study investigated ways of improving the

50

mechanical properties (TS and %E) of WPI—based ﬁlms for the possibility of using the
ﬁlms as sausage casings. Heat curing and compression molding were used as physical
means to improve the properties while other methods like increasing solids content and

casting surface were also investigated.

3.3. MATERIALS AND METHODS
3.3.1. Materials

Whey Protein Isolate (WPI, Provon 190) was supplied by Glanbia ingredients
(Monroe, WI). Glycerol was purchased from J .T. Baker Co. (Phillipsburg, NJ). Sodium
hydroxide (2N, NaOH) was purchased from Mallinckrodt Specialty Chemical Co. (Paris,
KY). Candelilla wax (CW) was purchased from Strahl and Pitsch Inc. (West Babylon,
NY). Edible collagen casings (inside diameter, 32mm) were obtained from The
Brechteen Co. (Chesterﬁeld, MI). Magnesium nitrate was purchased from Sigma
Chemical Co. (St Louis, MO) and phosphate solution was obtained from Butcher and

Packer Supply Company (Detroit, MI).
3.3.2. Film formation

Figure 3.1 shows a schematic diagram of the experiments conducted under this
objective designed to optimize the mechanical properties of the ﬁlms. Whey protein
isolate (WPI) (5 or 5.5% w/v) was dissolved in distilled water and glycerol was added
(3.3 or 3.6% w/v). Then, pH was adjusted to 8.0 with 2 N NaOH. The solution was

heated to 90 ;I-_ 2°C while being stirred continuously to denature the whey protein.

51

 

Film Forming Solution
1. 5.8% w/v Solids
2. 6.4 % w/v Solids

I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Casting Surface
Optimization
I
V V
Anodized Non-Anodized
I I
f V Y V V
UC C CM: UC C
(UC. C)
—-h 1 h-SSM
_’ 2h-SSM
—u 2-SM
_. Tested for: 1)Thrckness 2) TS 3) %E <—

 

 

 

 

 

Figure 3.1. Schematic diagram of the ﬁlm optimization process for whey protein isolate
based ﬁlms .
UC = uncured, C = cured, CM = compression molded, NCM = non-compression molded,
SSM = Single stage compression molded (110°C, 1.24 MPa 1 h or 2 h), 2-SM = 2-stage
compression molded (90-110°C, 0.99-1.38 MPa, 2-25min), TS = Tensile Strength (MPa),
% E = Elongation (%).

52

Candelilla wax (0.80 or 0.88 %, w/v) was added during heating and allowed to melt into
the solutions to provide ﬁnal solids content of 5.8% or 6.4%w/v for the ﬁlm forming
solution. The solutions were homogenized for 2 rrrinutes using a Polytron PT 10/35
homogenizer with a PTA 20 T8 homogenizing head (Tekmar Co., Cincinnati, 01-1) at
speed 5 (1350 rpm). The homogenized ﬁlm forming solution was ﬁltered through a layer
of cheesecloth, equilibrated to room temperature for 1.5 h and degassed using a vacuum
pump at room temperature for 30 min. Films were cast by pouring ﬁlm forming solution
(172.6 mi) onto anodized (aluminum) Teﬂon® or non-anodized (aluminum) Teﬂon®
plates (33.0 cm x 45.7 cm). The process of anodizing turns the aluminum into aluminum
oxide making it heavier and corrosion resistant. The ﬁlms were dried at 23 i 2°C and 30
i 5% relative humidity (RH) for 18 i 3 h. Dried ﬁlms were peeled, wrapped in foil and
heat cured in a vacuum (30mm Hg) oven maintained at 90°C for 12 h. The ﬁlms were
removed from the oven and peeled from the foil while still warm for ease of removal.
The ﬁlms were subsequently conditioned at 50% RH, 23°C in a dessicator containing

saturated magnesium nitrate for 48 h prior to testing.
3.3.3. Collagen films

Collagen ﬁlms were conditioned in a similar way to WPI-based ﬁlms. Collagen
film thickness and mechanical properties: TS and %E were evaluated for comparisons
with heat cured WPI-based ﬁlms. These analyses were conducted simultaneously to

assure direct and meaningful comparisons.

3.3.4. Film thickness

Films were preconditioned at 23°C, 50% RH for a minimum of 8 h to enable ease

of handling and cutting cut into strips (101.6mm x 25.4mm) using a Precision Sample

53

Cutter (Thawing Albert Instrument Co., Philadelphia, PA). Film thickness was
determined using a TMI model 549M micrometer (Testing Machines, Inc. Amityville,
NY). Five different measurements were taken in random locations within the ﬁlmstrip
test area and the mean values were used for calculations of mechanical properties.

Thickness measurements ranged from 0.11-0.16 : 0.02 mm.
3.3.5. Compression molding

The films were compression molded using a Carver Laboratory Press (Fred
Carver, Inc.; Wabash IN). Film sheets (130 x 200 mm) were sandwiched between
Teﬂon® sheets and placed between 2 stainless steel molding plates (220mm x 220mm).
Single and double stage molding conditions were: 1) single-stage (110°C, 1.24 MPa, 1 h
or 2 h) and 2) two-stage (110°C, 0.99 MPa, 25 min and 110°C, 1.24 MPa, 12 min). In a
separate experiment, response surface methodology was used to determine the best
conditions for two-stage compression molding of heat cured ﬁlms. Stage one conditions
were kept constant (110°C, 0.99MPa, 25 minutes) while stage 2 conditions were altered
as follows: temperature: 90, 98, 110, 121°C; pressure: 0.99, 1.10, 1.24, 1.38 MPa and
time: 2, 12, 22 min (Table 3.1). Temperatures were controlled using a temperature dial

that showed temperatures of the molding plates.
3.3.6. Mechanical properties

Film strips (101.6mm x 25.4mm) conditioned at 50% RH, 23°C for 48 h prior to
testing were used to determine mechanical properties. Tensile strength (TS, force at
failure/original cross-sectional area) and percent elongation at break (%E, increase in
length/initial length) were determined according to standard D882-9l (ASTM 1995)

using the Instron Universal Testing Machine Model 2401 (Canton, MA) at 23 _-_I-_ 2°C and

54

50% RH. A lkN static load cell with a crosshead speed of 50.8 cm/min was used.

Thickness measurements were used to calculate TS.

Table 3.1. Surface response methodology design for optimization of two-stage
compression molding conditions for heat cured whey protein isolate based ﬁlms.

 

 

 

 

Day Temperature (°C) Time (minutes) Pressure (MPa)

93 i 5 2 1.10

98 z 5 22 1.38

1 121 t 5 2 1.38
121: 5 22 1.10

110 1 5 12 1.24

110 z 5 12 1.24

98 i 5 2 1.38

98 :t 5 22 1.10

+ 121 i 5 2 1-10

2 121: 5 22 1.38
110 i 5 12 1.24

110:5 12 1.24

90 i 5 12 1.24

130 i- 5 12 1.24

110 i- 5 2 1.24

110:5 22 1.24

3 110 i 5 12 0.99
110: 5 12 1.38

110 :i: 5 12 1.24

110 i 5 12 1.24

 

3.3.7. Statistical analysis

The effect of heat curing (curing), casting surface (surface), total solids content of
casting solution (solution) and compression molding (molding) on TS and %E of WPI-

based ﬁlms were determined using the models shown below:
Y = function (curing; surface; solution) (1)

Y: function (curing; compression molding) (2)

55

Where Y = T8 or %E, curing = uncured or heat cured, surface = anodized or non-
anodized, solution = 5.8 or 6.4% w/v solids and compression molding = single or 2-stage
as described in section (3.3.4). All comparisons were also made to collagen ﬁlms. The
rrrixed procedure of SAS (SAS 1996) was used to analyze the data. Treatment means
were compared using the least squares means (LSM) and the means were based on 3
replicates. Response surface methodology (SAS 1996) was employed to optimize the
compression molding conditions. The effects of temperature, pressure and time on TS
and %E were evaluated using the conditions shown in Table 3.1. A second degree
polynomial model was used for ﬁtting TS and %E values:

Y = Bo + Brxr + Bzxz + B3X3 + ﬂirxr2 + Bzzxz2 + I333X32 + BIZXIXZ + 513X1X3

4‘ Bz3X2X3 + C (3)
Where Y = TS or %E; I30. Br. [32. I33. I311. 522. I333. I312. I313. 823 = ngI'CSSiOD

constants; X1, X2, X 3 are the coded independent variables for temperature, pressure and
time and e = the error term. This experiment was repeated over three days; statistical
analysis was done using the RSREG procedure of SAS (SAS 1996). Statistical

signiﬁcance was tested at p<0.05.

3.4. RESULTS AND DISCUSSION

3.4.1. Mechanical properties

3.4.1.1. Eﬂect of heat curing, solids content of the casting solution and casting surface
Tensile strength is the maximum tensile stress a material can sustain while %E is

the strain under elastic behavior at which the sample breaks (Hernandez 1997). Initially

the ﬁlms were cast on round non-anodized Teﬂon® plates, to make larger ﬁlms required

56

to make adequate sausage casings, the ﬁlms were cast on similar plates with a larger

surface area. These, however, were not successful as the solution coalesced to one side.

This was possibly due to the shiny ﬁnish on the non-anodized surface compared to the

rougher ﬁnish on the anodized surface.

Data in Tables 3.2 and 3.3 show the signiﬁcance levels of the main effects

(surface, solution and heat curing) on TS and %E. Statistical analysis of these effects

showed that the casting surface and solids content of the ﬁlm forming solution were not

signiﬁcant with respect to TS; heat curing, however, was signiﬁcant (p<0.05).

Table 3.2 Signiﬁcance level of main effects and their interactions for tensile strength

 

 

 

 

 

 

Effect N umI DenI F-value Signiﬁcance
DF DF

Solution 1 16 0.53 ns
Surface 1 16 1.02 ns
Curing l 16 108.01 p<0.05
Solution x Surface 1 16 0.59 ns
Solution x Curing 1 16 1.92 ns
Surface x Curing 1 16 4.31 p<0.05
Surface x Solution x Curing l 16 6.46 p<0.05

 

1 Num DF = numerator degrees of freedom,
2Den DF = denominator degrees of freedom

ns = not signiﬁcant

Table 3.3 Signiﬁcance levels of main effects and their interactions for elongation

 

 

 

 

 

 

Effect NumI Den2 F-value Signiﬁcance
DF DF

Solution 1 16 1.81 ns
Surface 1 16 0.27 ns
Curing 1 16 0.01 ns
Solution x Surface 1 16 7.28 p<0.05
Solution x Curing 1 16 0.04 ns
Surface x Curing 1 16 5.25 p<0.05
Surface x Solution x Curing 1 16 0.24 ns

 

' Num DF = numerator degrees of freedom,
2Den DF = denominator degrees of freedom

ns = not signiﬁcant

57

The two-way and three-way interactions: (surface x curing) and (surface x solution x
curing) were signiﬁcant (p<0.05). With respect to %E, only the interaction effects
(solution x surface) and (surface x curing) were signiﬁcant (p<0.05). Even though the
three-way interaction between surface, solution and curing appeared signiﬁcant, there
were no differences between mean TS values among uncured or heat cured ﬁlms from
different casting solutions and surfaces. Two-way interactions are discussed under the
TS and %E discussions. Table 3.4 shows the mechanical properties (TS and %E) of
uncured and heat cured WPI-based ﬁlms cast on non-anodized and anodized Teﬂon®
surfaces from solutions with two levels of solids content. Heat cured ﬁlms had higher TS
than uncured ﬁlms; for example, 2.00 MPa vs. 8.57 MPa for 5.6% w/v solids ﬁlm
(p<0.05). This trend was consistent with the remainder of the data. These results are in
agreement with those reported by Rhim and others (2000) for SP1 ﬁlms. Soy protein
isolate (SP1) ﬁlms were prepared from heated aqueous SPI solutions (70°C for 20 min)
and heat cured at 90°C for 24 h. Increases from 8.2 to 14.7 MPa were reported for
uncured vs. cured ﬁlms. Gennadios and others (1996) had previously showed that heat
curing of SPI ﬁlms at 80 or 95°C for 2, 6, 14 and 24 h increased TS and this increase was
positively correlated to increase in heat curing time and temperature. These data provide
adequate basis for heat curing of WPI-based ﬁlms for the purpose of improving their
mechanical properties. WPI-based ﬁlms in this study were heat-cured at 90°C for 12 h;
the increase in TS resulting from heat curing was, however, not sufﬁcient to make the
ﬁlms comparable to collagen ﬁlms. Tensile strength of all WPI-based ﬁlms was lower

(p<0.05) than that of collagen ﬁlms (5.13-8.57 MPa vs. 13.29 MPa).

58

Table 3.4. Mechanical properties of uncured and cured whey protein isolate-based ﬁlms
cast on non-anodized and anodized surface, from solutions varying in solids content

 

 

 

 

 

 

Casting Film TS (MPa) %E
Surface Solution Uncured Cured Uncured Cured
5.8% '
w/v 2.00 3.0.16“ 8.57 11.12b 35.81 : 9.37a 30.16 19.25a
Non- Solids
Anodized 6.4%
w/v 2.40 x 0.213 5.13 : 0.57b 42.73 : 1.50b 37.64 i 15.2b
Solidsl
5.8%
w/v 2.43 i 0.342‘ 5.85 : 0.44b 43.81: 2183" 56.64 :1; 16.33b
Anodized Solids
6.4%
w/v 2.89 : 0.58a 7.11 i 0.24” 36.30 1 10.662' 54.09 x 19.10b
Solidsz
Collagen 13.29 : 3.47c 31.78 1 4.88“”

 

 

 

”Means with the same superscript are not signiﬁcantly different from each other
(p<0.05). Comparisons are made within the same column and row for each mechanical
property, n=3 for all treatments. TS = Tensile strength, %E = Elongation (%)

These results indicate that WPI-based ﬁlms did not have the kind of strength required to
withstand sausage manufacturing conditions. Preliminary experiments to test the
performance of WPI-based ﬁlms in sausage manufacturing conditions are shown in
Appendix [1.

Increased solids content (6.4% w/v) and non-anodized surface resulted in ﬁlms
with higher %E compared to 5.8% w/v solids ﬁlms (p<0.05). Films cast on anodized
surfaces had higher %E than their counterparts formed on non-anodized surfaces
(p<0.05). The two-way interaction between casting surface and heat curing is clearly

seen in the higher %E for heat cured ﬁlm on the non-anodized surface, for example

59

30.16% vs. 56.64% for 5.8% w/v solids ﬁlms cast on non-anodized and anodized
surfaces respectively. Similar results were seen for 6.4% w/v solids ﬁlms. The higher
%E values observed for ﬁlms cast on the anodized the surface suggest that casting
surfaces affect mechanical properties of ﬁlms cast on them. To my knowledge, no
previous research has been conducted to determine the effect of casting surface on
protein ﬁlm properties. Various casting surfaces have been used by different researchers,
however, no explanations for the use of one surface over another have been provided.
Some surfaces that have been used include: Teﬂon®~coated glass plates (Gennadios and
others 1993a, b; Cho and Rhee 2002), polystyrene Petri dishes (Choi and Han 2001) and
ultra high molecular weight, high density polyethylene (Fairley and others 1996a, b).
The main difference between the surfaces used in this study was that the anodized
Teﬂon® surface was heavier and allowed better spreading of the ﬁlm-fornring solution.
This may have contributed to a conformation that allowed better mobility of the
molecules within the protein ﬁlm than the non-anodized surface since the ﬁlm-forming
solution tended to coalesce more on the non-anodized surface. Further research is
required to determine the cause of this effect.

The %E values obtained in this study are higher than those reported by Kim
(2000) for similar uncured ﬁlms plasticized with sorbitol. Kim studied properties of
WPI-based ﬁlms plasticized with glycerol and sorbitol. Mechanical properties were,
however, reported only for sorbitol-plasticized ﬁlms. This researcher reported %E of
15.6 :1: 3.4% for these ﬁlms while in this study a %E of 35.81 i 9.37% was obtained. The
%E of ﬁlms in this study did not change following heat curing which is contrary to

ﬁndings of other researchers who have reported that heat curing increased TS but

decreased %E (Cuq and others 2000; Rhim and others 2000; Micard and others 2000). In
the study conducted by Rhim and others (2000), ﬁlm strips were heat cured at 90°C for
24 h in an air-circulating oven. Heat curing decreased %E from 30 t 3.3% for uncured
ﬁlms to 6.1 i 0.7% for heat cured ﬁlms.

The purpose of increasing solids content in the ﬁlm forming solution was to
induce changes to optimize the mechanical properties of the ﬁlms. Mechanical properties
are largely dependent on the structure of the ﬁlm. According to Anker and others (2001)
the microstructure of WPI-based ﬁlms is dependent on protein concentration. Increased
protein concentration results in a more aggregated structure that has a denser protein
network. Chick and Ustunol (1998) studied the effect of increasing proteinzplasticizer
ratios (0.6:1, 1:1, 1.4:1) on lactic-acid-casein and rennet-casein-based edible ﬁlms.
Plasticizers studied were glycerol and sorbitol. For lactic-acid—casein ﬁlms, increase in
protein concentration decreased %E; for rennet casein ﬁlms, however, %E increased as
protein concentration increased. The results obtained for rennet-casein ﬁlms are
consistent with the results seen for ﬁlms cast on non-anodized surfaces in this study. The
difference between the two studies is that total solids in this study were increased while
maintaining the proteinzplasticizer ratio, whereas, proteinzplasticizer ratio was altered in
the study by Chick and Ustunol (1998). The results presented by Chick and Ustunol
(1998) indicate that a higher increase in the protein concentration in the current study I
may have yielded more signiﬁcant effects on the mechanical properties of the WPI-based
ﬁlms.

3.4.1.2. Compression molding

Figure 3.2 shows T8 of compression molded ﬁlms from the initial compression

molding experiments. Compression molding resulted in increased TS of uncured ﬁlms

61

(p<0.05). This increase was similar to that caused by heat curing, this is shown by the
heat cured unmolded ﬁlm on the same ﬁgure (Figure 3.2). Single stage compression
molding conditions yielded similar results and there was no difference between uncured

and heat cured ﬁlms compression molded under these conditions.

 

8.00 ~ IUncured ICured

Tensile Strength (MPa)

 

 

 

Unmolded Molded 2 Molded 1h Molded 2h

stages
Treatments

Figure 3.2. Tensile Strength of compression molded whey protein isolate based ﬁlms.
“Different letters denote signiﬁcant differences; n = 3 for all treatments;

Molded 2 stages = 2 stage molding: 110°C, 0.99 MPa, 25 minutes and 110°C, 1.24 MPa,
12 minutes, Molded lh or 2h = single stage molding: 110°C, 1.24 MPa, 1 or 2 hours
Two-stage compression molding signiﬁcantly increased (p<0.05) the TS of heat cured
ﬁlms compared to uncured ﬁlms. Sothomvit and others (2003) reported that a feasible
temperature range for compression molding of WPI-based ﬁlms was 104°C — 140°C.
Adequate ﬁlm formation without degradation was obtained at temperatures lower than
140°C. The pressures they studied ranged from 0.81MPa — 2.25 MPa. Compression
molding was conducted for 2 rrrinutes because it was observed that at high temperature

(140°C), ﬁlms degraded after 2 rrrinutes. Film degradation was evidenced by the

darkening of the ﬁlm color. The ﬁlm testing conditions used in this study were similar to

62

the conditions used by Sothomvit and others (2003). These researchers, however, did not
report mechanical properties and thus it was not possible to compare the effect of
compression molding on TS and %E of WPI-based ﬁlms. The increased TS resulting
from heat application to free-standing ﬁlms is consistent with results reported by Micard
and others (2000) for WG ﬁlms. They heat treated WG ﬁlms by placing them between
two plates heated at 80, 95, 110 and 125°C for 15 rrrin and at 140°C for 1.5 or 15 rrrin
without the application of pressure. The TS increased from 1.7 MPa for non-treated ﬁlms
to 3.1 MPa at 110°C and 7.3 MPa at 140°C. A two-fold increase in TS was reported as
temperature increased from 110°C to 125°C. A similar trend was seen in this study with
the WPI-based ﬁlms since uncured, un-compression molded ﬁlms’ TS increased from
1.65 MPa to 4.31 MPa after 2-stage compression molding at 110°C (Figure 3.2).

The larger increase in the TS of the WPI-based films could be due to the longer I
compression time, 25 and 12 min compared to 15 min used by Micard and others (2000)
as well as the application of pressure, which they did not use. The results obtained by
Micard and others (2000) suggest that increased TS for WPI-based ﬁlms may be merely
due to the increase in temperature since these results are consistent with the results
reported for heat curing of protein ﬁlms and heat cured ﬁlms in this study. Gennadios
and others (1996) showed that increased heat curing temperature and time caused
increases in TS of SPI ﬁlms. Similar results were reported by Kim and others (2002) for
SP1 ﬁlms heat cured at 60-85°C under 61.32-101.3 KPa. The TS of the SP1 ﬁlms was
reported to increase as curing temperature and pressure increased. Mo and others (1999)
studied the effect of molding temperature (100, 120, 140, 150, 160°C), pressure (5, 10,

20, 40, 60 MPa) and time (3, 5, 10, 15 min) on compression molded SP1 polymers, they

63

reported that as pressure increased from 5 to 10 MPa, TS increased to 42.2 MPa but as
pressure was increased to 20 MPa, TS decreased to 39 MPa and remained constant as
pressure continued to increase.

Results of this current study were consistent with those of Mo and others (1999)
for 2-stage compression molded heat cured ﬁlms. Increasing pressure from 0.99 to 1.24
MPa increased TS. Cuq and others (2000) studied the effect of molding temperature (80,
100, 110, 125, 135, and 150°C) on WG ﬁlms, pressure and time were held constant at 20
MPa and 10 min respectively. As with Mo and others (1999), they showed that increases
in temperature caused increase in TS (0.26 to 2.04 MPa) as temperature increased from
80 to 135°C. The similarity in the effect of compression molding and heat curing
suggests that compression molding can be used as an alternative to heat curing under
vacuum. Heat curing free-standing ﬁlms (Rhim and others 2000; Kim and others 2002)
increases covalent cross-linking between protein polymer chains that increases water
insolubility and increases tensile properties.

Compression molding had no signiﬁcant effect on %E for all compression
molding conditions that were investigated (Figure 3.3). The differences in %E between
uncured and cured ﬁlms were also not signiﬁcant. These results are contrary to the
ﬁndings of Mo and others (1999) and Cuq and others (2000). Mo and others (1999)
reported an increase in %E that increased from 0.9 to 5.4% as pressure increased from 5
to 10 MPa and remained constant at 4.5% regardless of increase in pressure. The effect
of time and temperature was studied at constant pressure (20 MPa). Mo and others
(1999) found that at high temperature (150°C) it took 3 min to reach optimum curing

quality (max TS and %E) and up to 10 min at low temperature (120°C). Cuq and others

(2000) reported that as molding temperature increased from 80 to 135°C, %E decreased
from 468 to 236%. The difference between the films in this study compared to ﬁlms

studied by the researchers mentioned above is the presence of CW, which may have

 

Elongation (%)

 

 

 

Unmolded Molded 2 Molded 1h Molded 2h
stages
Treatments

Figure 3.3. Elongation of compression molded whey protein isolate-based ﬁlms.
aSame letter denotes no signiﬁcant differences between means; n = 3 for all treatments,
Molded 2 stages = 2 stage molded: 110°C, 0.99 MPa, 25 minutes and 110°C, 1.24 MPa,
12 minutes, Molded 1h or 2h = single stage molded: 110°C, 1.24 MPa, 1 hour or 2 hours
allowed mobility within the polymer matrix by reducing protein-protein interactions.
Callegarin and others (1997) reported that the presence of lipids might increase ﬂexibility
of films by weakening the intermolecular forces between adjacent polymer chains.
Figures 3.4-3.7 show mechanical properties (TS and %E) of WPI-based ﬁlms
compression molded in two stages (stage 1: 110°C, 0.99 MPa, 25 min and stage 2: 90, 98,
110,121°C; 0.99, 1.10, 1.24; 1.38 MPa, 2, 12, 22 min) as determined by response surface
methodology. The effects of temperature, pressure and time on TS and %E can be seen
on the response surface plots generated by equations (4) and (5) (Figures 3.4-3.7). The

equations show the values of the regression coefficients.

65

TS = 26.63 - 0.22 xl - 0.13 x2 + 0.04 x3 + 0.006 x,2 + 0.0002 x.2 + 0.0005 x.2
+ 0.0006 x12 + 0.0002 xl3 — 0.0002 x23 (4)

%E = 10.45 — 0.22 xl + 0.06 x2 - 0.10 x3 + 0.008 x,2 - 0.0002 x.2 + 0.0004 x.2
+ 0.0002 x12 + 0.0002 x., — 0.002 x23 (5)

Figure 3.4 shows the effect of temperature and pressure on TS and Figure 3.5 show the
effect of temperature and time on TS. The effect of temperature and pressure on %E is
shown in Figure 3.6 and Figure 3.7 shows the effect of temperature and time on %E.
Statistical analysis of the data indicated that the regression coefﬁcients for both
TS and %E were not statistically signiﬁcant. Statistical insigniﬁcance of the regression
coefﬁcients suggests that increases or decreases in the variables (temperature, pressure
and time) did not result in signiﬁcant changes in the dependent variables (TS and %E).
As a result, no combination of temperature, pressure and time was found to be optimal or
to yield signiﬁcant increases or decrease in TS or %E under the conditions investigated.
The relatively ﬂat nature of the response surface graphs demonstrates these results. Since
the regression coefﬁcients were not statistically signiﬁcant, a reduced linear model
without the quadratic and interaction terms was used to ﬁt the TS and %E. Using the
reduced model, the regression coefﬁcient corresponding to temperature (the X. term) was
statistically signiﬁcant (p<0.05) and positive, indicating that an increase in temperature
resulted in an increase in TS; pressure and time were not signiﬁcant. Equation (6) shows
the TS model. For %E, the regression coefﬁcients of temperature, pressure and time
remained statistically insigniﬁcant. This indicates that changes in these variables did not
increase or decrease %E of WPI-based ﬁlms. Equation (7) shows the %E model. As

previously discussed, these ﬁndings were contrary to those obtained by other researchers

66

who have investigated similar processes (Mo and Sun 1999; Cuq and others 2000;

Micard and others 2001).
TS = 3.19 + 0.02 X. - 0.01 X2 + 0.01 X3 (6)
%E = 18.65 - 0.96 X. - 0.02 X2 + 0.31 X3 (7)

6.0 ‘

1

‘2'- / / / / ,..:

\\
TStkPa)
\...' .

 

 

 

 

 

 

 

 

3.4- ‘... - 122 to
-- .. 114 $3
3.0 -..I- 1060514»

Pressure (MPa)

Figure 3.4. Effect of temperature and pressure on tensile strength (TS) of 2-stage
compression molded whey protein isolate-based films as determined by response surface
methodology.

Compression molding conditions: Stage 1: 110°C, 0.99 MPa, 25 min; Stage 2: 90, 98,
110, 121°C; 0.99, 1.10, 1.24, 1.38 MPa; 2, 12, 22 min

11 = 3 for all treatments; No signiﬁcant differences were found between treatment means.

67

 

 

 

Figure 3.5. Effect of temperature and time on tensile strength (TS) of 2-stage _
compression molded whey protein isolate-based ﬁlms as determined by response surface

methodology.

Compression molding conditions: Stage 1: 110°C, 0.99 MPa, 25 rrrin; Stage 2: 90, 98,

110, 121°C; 0.99, 1.10, 1.24 1.38 MPa; 2, 12, 22 min.

11 = 3 for all treatments; No signiﬁcant differences were found between treatment means.

68

I

5W“ W 4:53.
71.46 - '5‘“. &.
. .. Q
67.76- 15;“. ...;”..

Ix
$1
q
11'.
A
6

I
I
1’
19
1,1
{6
’
:‘o
’9

44.07 ‘

I/
i
6

 

 

.3 3.
1:1
11
It
,1

130

ii

1
:1
1
’1

114

P11. 1'24 106 01

s 1146 ... .

safe (ma) 1m m “963566 K
0.990 90 “1°

Figure 3.6. Effect of temperature and pressure on elongation (%) of 2-stage compression
molded whey protein isolate-based ﬁlms.

Compression molding conditions: Stage 1: 110°C, 0.99 MPa, 25 min; Stage 2: 90, 98,
110, 121°C; 0.99, 1.10, 1.24, 1.38 MPa; 2, 12, 22 min.

11 = 3 for all treatments; No signiﬁcant differences were found between treatment means.

69

52.?“ °’ I\‘x

’ \ «er-raw
\\—’-’.

67.14 - ” ”\\\>’-
"I-r.‘ .4-

s1.11~ ’ 1’

35.07- W
W, W

m. ’ .‘.>
22 “‘4‘“

 

 

7}: 10 -
”Io 0%) 6 Q 106 13°C)
2 90 Teﬁ‘Pe‘a‘M

Figure 3.7. Effect of temperature and time on elongation (%) of 2-stage compression
molded whey protein isolate-based ﬁlms. ‘
Compression molding conditions: Stage 1: 110°C, 0.99 MPa, 25 min; Stage 2: 90, 98,
110, 121°C; 0.99, 1.10, 1.24, 1.38 MPa; 2, 12, 22 min. ,
n = 3 for all treatments, No signiﬁcant differences were found between treatment means.
3.5. CONCLUSIONS

Casting surface and ﬁlm forming solution solids content were signiﬁcant factors
affecting %E but not TS of WPI-based ﬁlms. Heat curing and compression molding had
similar effects on T8 and %E of uncured WPI-based ﬁlms, they increased TS and did not
change %E. Compression molding can therefore be used as an alternative to heat curing.
This would be advantageous because compression molding takes less time (> 1 h vs. 12 h
for heat curing), has less equipment requirements (hydraulic press vs. vacuum pump and
oven) and less total energy requirements and thus more economic. WPI-based edible

ﬁlms had lower TS than collagen ﬁlms. The lower TS of WPI-based ﬁlms compared to

collagen ﬁlms indicated that even though the TS was increased with heat curing and

70

compression molding, the increase was not sufﬁcient to enable use of these ﬁlms as
sausage casings. An additional experiment to optimize mechanical properties WPI-based

ﬁlms was conducted using potassium phosphate; this experiment is shown in Appendix I.

71

CHAPTER 4

OPTIMIZATION OF SEAL PROPERTIES OF WHEY PROTEIN

ISOLATE EDIBLE FILMS.

4.1. ABSTRACT

Edible ﬁlms were produced using WPI (5 or 5.5% w/v), glycerol (3.3 or 3.6%
w/v) and candelilla wax (CW; 0.8 or 0.9% w/v) to provide ﬁlm forming solutions of
5.8% w/v and 6.4% w/v solids content. Films were cast on anodized and non-anodized
Teﬂon® surfaces. Film treatments included heat curing at 90°C for 12 h and
compression molding at single-stage (110°C, 1.24 MPa, 1 h or 2 h) and double stage
(110°C, 0.99 MPa, 25 min and 110°C, 1.24 MPa, 12 min) conditions. The effect of heat-
sealing prior to heat curing and wetting the seal area of uncured and heat cured ﬁlms
before heat sealing were investigated. All ﬁlms were evaluated for seal strengths.
Electron Spectroscopy for Chemical Analysis (ESCA) was used to determine surface
chemistry of the unsealed and sealed ﬁlms. Uncured ﬁlms had higher (p<0.05) seal
strengths than cured ﬁlms. Seal strengths of uncured and heat cured compression molded
ﬁlms were similar. Neither heat-sealing prior to heat curing nor wetting the seal area of
uncured and heat cured ﬁlms before sealing improved seal strengths. ESCA indicated
that cured, compression molded and sealed ﬁlms had higher concentrations of C~O-H and
C-O-C bonds on their surface and that sealing of the ﬁlms was likely a result of the

formation of C-O-H or C-O-C bonds.

72

4.2. INTRODUCTION

Heat scaling is very widely used in the packaging industry for joining polymers
(Meka and Stehling 1994). Adequate scaling is obtained when two polymer surfaces are
pressed together with sufﬁcient pressure and time to cause the polymer chains to diffuse
across the interface and form bridges (Mueller and others 1998). Meka and Stehling
(1994) showed that the dwell time had a relatively small inﬂuence on seal strength
compared to temperature and pressure. Mueller and others (1998), however, stated that
factors affecting seal strength were temperature and dwell time.

Scalability of edible ﬁlms is an important attribute in the development and
applications of edible ﬁlms. Their scalability determines the possibility of using the ﬁlms
in such applications as pouches and sachets for soups and other products (Amin and
others 2001; Kim 2000). Heat scalability of whey protein isolate (WPI)-based ﬁlms -
plasticized with glycerol or sorbitol and containing candelilla wax was reported by Kim
and Ustunol (2001c). They formed seals using a thermal heat sealer and reported that the
optimum sealing temperatures for glycerol- and sorbitol-plasticized ﬁlms were 110°C and
130°C, respectively. The optimum sealing temperatures corresponded to the transition
onset temperature of the ﬁlms as determined by differential scanning calorimetry (DSC).
To my knowledge, voltage-based impulse sealers have not been used in sealing
bi0polymers. Due to the surface area required to form seals with the thermal sealer,
1.50m seal width, impulse sealing with a voltage based sealer rrright be more appropriate
for food applications since a narrower seal width can be obtained (0.25cm). The

advantage of a narrow seal is that less total ﬁlm would be used were casings to be

73

casings to be produced in high volumes, furthermore, the narrower seal would be visually
more appealing.

Seal strength values provide general information about the strength of the bond
formed between polymer layers during sealing; this information, however, does not
provide insight into the chemical characteristics of the bond. By conducting a surface
analysis, surface-speciﬁc information is obtained that can be valuable for determining
bonding mechanisms; this information can also be useful when developing new materials
and applications (Rindlav-Westling and Gatenholm 2003). Electron Spectroscopy for
Chemical Analysis (ESCA) also referred to as XPS (X—ray photoelectron spectroscopy)
provides insight into the surface chemistry of the material tested and has been used as the
principal technique for deﬁning interfacial molecular properties associated with adhesive
behavior of materials. The use of ESCA allows identiﬁcation and quantiﬁcation of all
elements except hydrogen and helium (Ratner and Castner, 1997).

Kim and Ustunol (2001c) conducted a unique study where surface chemistry of
seals of uncured whey protein based ﬁlms was investigated. They reported that carbon
was the main element of the ﬁlms followed sequentially by oxygen and nitrogen. Their
results indicated that formation of C-O-H and N-C bonds upon heat sealing might be
responsible for the mechanism of seal formation. These data provided by Kim and
Ustunol (2001c) focused on uncured WPI-based ﬁlms. Surface chemistry of heat cured
WPI-based ﬁlms have not been investigated using this method. The objectives of this
study were to optimize seal strengths of WPI-based ﬁlms for sausage casing manufacture

and to determine their surface chemistry using ESCA.

74

4.3. MATERIALS AND METHODS
4.3.1. Materials

Whey protein isolate (WPI, Provon 190) was supplied by Glanbia ingredients
(Monroe, WI). Glycerol was purchased from IT. Baker Co. (Phillipsburg, NJ). Sodium
hydroxide (2N, NaOH) was purchased from Mallinckrodt Specialty Chemical Co. (Paris,
KY). Candelilla wax (CW) was purchased from Strahl and Pitsch Inc. (West Babylon,

NY) and magnesium nitrate was purchased from Sigma Chemical Co. (St Louis, MO).
4.3.2. Whey protein isolate ﬁlm preparation

Figure 4.1 shows a schematic diagram of the experiments conducted under this
objective designed to optimize the ﬁlms for sausage casing manufacture. The levels of
the ﬁlm constituents used were: WPI (5.0% or 5.5% w/v), glycerol (3.3% or 3.6% w/v) I
and CW (0.8% or 0. 9%, w/v). Two ﬁlm forming solutions having 5.8% w/v and 6.4%
w/v solids contents were developed by dissolving whey protein isolate (WPI) in distilled
water, glycerol was added and pH was adjusted to 8.0 using 2N NaOH. The solutions
were heated to 90 -_1-_ 2°C with continuous stirring to effectively denature the whey protein.
Candelilla wax (CW) was added during heating and allowed to melt into the solutions.
The solutions were homogenized for 2 rrrinutes using a Polytron PI‘ 10/35 homogenizer
with a PT A 20 TS homogenizing head (Tekmar Co., Cincinnati, OH) at speed 5 (1350
rpm); ﬁltered through a layer of cheesecloth, equilibrated to room temperature for 1.5 h
and vacuum degassed at room temperature for 30 min. Films were prepared by pipetting
the emulsion (172.6 ml) onto either anodized or non—anodized Teﬂon® plates (33.0 cm x

45.7 cm). The process of anodizing turns the aluminum into aluminum oxide making it

75

 

 

Film Forming Solution
1. 5.8% w/v Solids
2. 6.4% w/v Solids

 

 

I

Casting Surface

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I
V j
Anodized Non-Anodized
I
V V T V V I j
UC C SBC CM: SBC UC C
(UC. C)
l h-SSM
—H 2 h-SSM
SAWBS
—-> 2-SJM
1 I1 1
Test for Seal Strength

Figure 4.1. Schematic diagram of the seal optimization process for whey protein isolate

based ﬁlms

UC = uncured, C = cured, CM = compression molded, NCM = non-compression molded,
SSM = Single stage compression molded (110°C, 1.24 MPa 1 h or 2 h), 2-SM = 2-stage
compression molded (90-110°C, 0.99-1.38 MPa, 2-25min), P1“ = phosphate treatment,

 

 

 

 

SBC = Sealed before curing, SAWBS = seal area wet before sealing

heavier and corrosion resistant. The ﬁlms were dried at room temperature (23 i 2°C) and
30 i 5% relative humidity (RH) for 18 i 3 h. Dried ﬁlms were peeled, wrapped in foil
and heat cured in a vacuum (30mm Hg) oven maintained at 90°C for 12 h. The ﬁlms

were removed from the oven and peeled from the foil while still warm for ease of

76

III

I ..L .i.

 

 

removal. The films were subsequently conditioned at 50% RH, 23°C using magnesium

nitrate for 48 h prior to testing.
4.3.3. Compression molding

Whey protein isolate (WPD-based ﬁlms were compression molded using a Carver
Laboratory Press (Fred Carver, Inc.; Wabash IN). Film sheets (130 x 200 mm) were
sandwiched (one at a time) between Teﬂon® sheets, placed between 2 stainless steel
molding plates and inserted between heated plates of the compression compartment of the
compression molder. Molding conditions were: 1) single-stage (110°C, 1.24 MPa, 1 h or
2 h) and 2) two-stage compression molding (110°C, 0.99 MPa, 25 min and 110°C, 1.24

MPa, 12 min). 5.8% w/v solids ﬁlms were used for the compression molded ﬁlm studies.
4.3.4. Seal strength determination

Two strips (101.6mm x 25.4mm) cut using a Precision Sample Cutter (Thawing
Albert Instrument Co., Philadelphia, PA) were sealed together using a voltage based
impulse sealer Model-12ASL (Sencorp System Inc., Hyannis, MA). The impulse sealer
settings were as follows: pressure: 300kPa, voltage: 22 amps, impulse time: 1.8 seconds
and dwell time: 1.4 seconds. Seal strength tests were conducted using an Instron
Universal Testing Machine, Model 2401 (Instron Corp., Canton, MA) at 23 :1: 2°C and
50% i 5% RH according to standard ASTM F—88 (ASTM 1997). Each end of the sealed
specimen was clamped to the machine and the sealed ﬁlm was held perpendicularly to the
direction of the pull as the seal was tested. The distance between the clamps was 5.08cm
and a 1kN static load cell with a speed of 50.8cm/min was used. Seal strength was
reported as the maximum force required to cause seal failure and expressed as load

(N)/width (m).

77

4.3.5. Electron spectroscopy for chemical analysis

Surface chemical analysis of uncured, heat cured and compression molded films
was conducted using Electron Spectroscopy for Chemical Analysis (ESCA). The
unsealed ﬁlm and the outer and inner seal of scaled ﬁlms were examined. This was
performed to determine the type of bonding that took place during seal formation in the
films. The outer seal was observed to make adequate comparisons with the inside of the
seal. A PH] 5400 ESCA lab workstation was used (Physical Electronics, Eden Prairie,
MN). A 15 mm diameter circular ﬁlm (sealed or unsealed) was placed in a sample holder
(non-lipid oriented side up) and monochromatic X-rays were used as the radiation source.
A magnesium anode operated at 300 W with an analyzer pass energy of 33eV was used
to collect all spectra and the takeoff angle from the surface to the detector was 45°. An
electron kinetic energy analyzer plotted the intensity of the emitted photoelectrons and

optimum spot size for the conditions used in the experiments was 1mm diameter aperture.

. The bonding scale was calibrated to 284.6eV for the main carbon ls (Cls; C-H)
spectra. Spectra were run at both high and low resolution (wide/survey scan) modes.
Curve ﬁtting was used to resolve the ESCA spectra into separate peaks to determine the
carbon chemistry detected within the spectral envelope. The spectra were ﬁt with a
Lorentzian-Gaussian mix Voigt proﬁle function using a nonlinear least-square program
PHI PC Explorer Software multipack (Physical Electronics, Eden Prairie, MN). The
resulting curve-ﬁts had an experimental error of approximately 5%. The area under each
peak was used to quantify bonds and elemental compositions were obtained from the
wide/survey scan spectra, the element and bond quantities were reported in percent of

total elements and bonds present.

78

4.3.6. Statistical analysis

The effect of heat curing (curing), casting surface (surface), total solids content of
casting solution (solution) and compression molding (molding) on TS and %E of WPI-

based films were determined using the models shown below:
Y = function (curing; surface; solution) (1)
Y: function (curing; compression molding) (2)

Where Y = seal strength, curing = uncured or heat cured, surface = anodized or
non-anodized, solution = 5.8 or 6.4% w/v solids and compression molding = single or 2-
stage as described in section (4.3.3). The mixed procedure of SAS was used to analyze
the data. Treatment means were compared using the least squares means (LSM) and the
means were based on 3 replicates (SAS, 1996). ESCA results provided are for only one

replicate.

4.4. RESULTS AND DISCUSSION
4.4.1. Seal strengths

Table 4.1 shows the seal strengths of uncured and heat cured ﬁlms cast from 5.8%
and 6.4% w/v solids ﬁlm forming solutions and cast on non-anodized and anodized
surfaces. With the exception of the 6.4% w/v solids solution film cast on a non-anodized
surface, uncured ﬁlms had higher seal strengths than cured ﬁlms (p<0.05). The highest
seal strength was observed in uncured 6.4% w/v solids ﬁlms (173.62 N/m). Because
higher seal strengths were observed for uncured ﬁlms compared to cured ﬁlms, two

additional experiments were conducted to test if seal strength of heat cured ﬁlms could be

79

increased. These were: sealing ﬁlms prior to heat curing and wetting the seal area of
uncured and heat cured films prior to sealing.

It was hypothesized that by sealing the ﬁlms prior to heat-curing, the seal
strengths of the heat-cured ﬁlms would improve. Table 4.2 shows the seal strengths of
ﬁlms that were uncured, heat cured and sealed before curing. Even though the seal
strength values of the ﬁlms sealed prior to heat curing are higher than those of cured
ﬁlms, no statistical difference was detected. The high variation (evidenced by the wide
standard deviations) in the seal strengths could explain the non-detection of statistical
differences. This variability was seen consistently among the seal strength values of the
ﬁlms. The experiment in which the seal area was wet prior to scaling was based on the
hypothesis that the higher seal strengths of uncured ﬁlms were a result of the higher
moisture in these ﬁlms. Wetting the seal area of uncured and cured WPI-based ﬁlms did
not increase the seal strength of the ﬁlms (Table 4.3). This treatment, however,
decreased the seal strength of uncured ﬁlms making it similar to that of the cured ﬁlms.
It is possible that the water molecules between the layers being sealed prevented close
interaction required to form strong seals. Furthermore, the water had to be evaporated
ﬁrst before bond formation could take place; this too could have affected the strength of
the seal formed. Table 4.3 also shows seal strengths of compression molded ﬁlms. Films
compression molded for one hour had the highest seal strength (148.9 N/m). The lowest
seal strength was observed for cured ﬁlms compression molded for two hours (32.09
N/m). Compared to the heat cured ﬁlms, seal strengths of compression molded ﬁlms
were not different. This indicates that compression molding had a similar effect to heat

curing on WPI-based ﬁlms.

80

Table 4.1. Inﬂuence of casting surface and solids content on seal strengths of whey
protein isolate based ﬁlms

 

 

 

 

Casting Solution Seal Strength (N/m)
Casting Surface Solids Content
Uncured Cured
(w/v)
5.80/ a b
Non-Anodized 0 115.331 77.39 78.18 1 37.25
64% 79.97 1 28.68” 92.28 1 29.90”
5.8% a b
Anodized 92.18 1 39.71 61.56 1 36.19
64% 173.62 1 91.43” 80.91 1 51.29”

 

 

 

 

”Means with different superscripts are signiﬁcantly different (p<0.05); means :1: standard
deviations; n=3 for all treatments; comparisons are made within each row.

Table 4.2. Inﬂuence of casting surface and solids content on seal strengths of uncured,
heat cured and sealed before curing whey protein isolate based ﬁlms.

 

 

 

 

Casting Seal Strength (N/m)
Casting
Solution Solids Sealed Before
Surface Uncured Cured
Content (w/v) Curing
5.87 a a a
Non- Anodized 0 115.331 77.39 78.18 1 37.25 80.28 1 37.75
64% 79.97 1: 28.68” 92.28 1 29.90” 96.70 :1: 30.44”
5.8% a a 1*
Anodized 92.18 1 39.71 61.56 1 36.19 54.29 1 21.56
64% 173.62 1 91.43” 80.91 1; 51.29” 142.79 1 32.46”

 

 

 

 

 

”Means with different superscripts are signiﬁcantly different (p<0.05); means 1 standard
deviations; n=3 for all treatments; comparisons are column-wise within each surface

treatment.

81

Table 4.3. Effect of wetting the seal area before sealing and compression molding on seal
strength of whey protein isolate ﬁlms

 

 

 

Seal Strength (N/m)
Casting Solution
Film Treatment
Solids Content (w/v)
Uncured Cured
A“°°‘z°° 5.8% 92.18 1 39.713 61.56 1 36.198
(control)
seal Area W“ 5.8% 67.87 1 26.70” 68.70 1 6324“
Before Sealing
2-Stage
Compression 5.8% 93.86 1 54.012‘ 82.18 1 71.69“
Molded
l-Hour
Compression 5.8% 101.75 1 42.043 148.90 1 49.818
Molded
2- Hour
Compression 5.8% 91.86 1 30.96a 32.09 1 22.87”
Molded

 

 

 

 

”Means with different superscripts are signiﬁcantly different (p<0.05); means 2!: standard
deviations; n=3 for all treatments; comparisons are made within each row; compression
molding conditions were: single-stage (110°C, 1.24 MPa, 1 h or 110°C, 1.24MPa, 2 h)
and two-stage (110°C, 0.99 MPa, 25 min and 110°C, 1.24 MPa, 12 min)

The seal strengths of the ﬁlms in this study varied from 32.09 N/m to 173.62 N/m.
The latter value, obtained for uncured ﬁlms was in agreement with seal strength values
obtained by Kim and Ustunol (2001c) for WPI-glycerol-CW ﬁlms heat sealed at 130°C
for 1 min. Kim and Ustunol (2001c) tested ﬁlms sealed with a thermal sealer over a
temperature range of 110 to 130°C and dwell times of 1 or 3 sec and a pressure of 296 or
445 kPa. The results of this current study indicate that such values are typical and can be

expected for uncured WPI-based ﬁlms since the ﬁlms tested by Kim and Ustunol (2001c)

were also uncured.

82

One of the possible reasons for the variation in the seal strengths observed in this
study may have been the failure mode of the ﬁlm. Failure mode refers to the manner in
which the seal breaks during testing. Meka and Stehling (1997) described three types of
seal failure modes; these were “peeling failure”, “tearing failure” and a combination of
“peeling and tearing failure”. During testing of the ﬁlms in this current study, all three
failure modes were observed for different samples as each leg of the sealed ﬁlm was
pulled to determine seal strength. The seal strength measured depended in part on the
predominant qualitative failure mode. Some seals peeled open, less strength was
obtained for these; others tore, these demonstrated higher seal strength. The way in
which the seal opened (failed) however, could not be controlled.

4.4.2. Electron spectroscopy for chemical analysis

Table 4.4 shows the chemical bond composition and concentration of unsealed,
outer and inner seals of uncured, heat cured and compression molded WPI-based ﬁlms as
determined by ESCA. Figure 4.2 shows the resolved Cls spectra for the same ﬁlms.
Carbon was the most abundant element and was followed by oxygen. Nitrogen was
detected only in unsealed and inner seals of uncured ﬁlms. These results suggest that
during sealing or heat curing, molecular conformations within the protein ﬁlm change
causing certain groups to be less detectable on the surface. This may be due to the groups
being buried below the penetration depth of ESCA, which ranges from 5-10 nm. Such
changes can be expected because new or additional bonds develop as a result of cross-

linking due to heat. These ﬁndings are different from those obtained by Kim (2001c)

who reported detection of N for both unsealed and sealed uncured WPI-based ﬁlms.

83

 

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84

3)

 

 

 

 

 

C-H, C-C
C-N
C=O
/ A\ _ \
220 238 256 2B4 252 220 2&8 2B6 2B4 2B2
Binding Energy (eV) Binding Energy (eV)

 

 

 

 

290 268 286 284 282
Binding Energy (eV)

Figure 4.2a. Representative Electron Spectroscopy for Chemical Analysis spectra of
whey protein isolate based ﬁlms. a) uncured b) uncured outer seal 0) uncured inner seal

85

law

 

 

 

 

 

 

 

290 288 286 284 282 290 288 286 284 282
Binding Energy (eV) Binding Energy (eV)

 

l 1 ‘ V L 1
290 288 286 284 282

Binding Energy (eV)

Figure 4.2b. Representative Electron Spectroscopy for Chemical Analysis spectra of
whey protein isolate based ﬁlms.d) cured e) cured outer seal f) cured inner seal

86

The abundance of C can be attributed to the protein, wax and glycerol used to make the
ﬁlms and is expected due to the total organic matter inherent in these ﬁlms. The Cls
spectra consisted of three components for heat cured, sealed and compression molded
films whose binding energies ranged from 284.8eV to 288.0eV. These were CR or C-C
(285.0eV), C-O-H or C-O-C (286.5eV) and C=O (288.0eV). Uncured films had an
additional component (C-N), which had a binding energy of 286.0 eV. Resolving of the
Cls spectra revealed peaks representing bonds present within each ﬁlm (Figure 4.2).
Once the Cls spectra was resolved, bond concentrations were quantiﬁed based on the
area under the curve for each peak and the results showed that the hydrocarbon OH and
C-C bonds constituted the bulk of the bonds in these ﬁlms and they increased with heat
curing and compression molding (45.34% in uncured ﬁlms vs. 87.03% in heat cured
ﬁlms). In surface studies of starch, amylose and amylopectin ﬁlms, Rindlav-Westling
and Gatenholm (2003) attributed the presence of this bond to the presence of proteins and
lipids contained within potatoes. They tested starch ﬁlms prepared by solvent casting of
starch-water solutions made of potato starch, amylose and amylopectin. Examinations of
the ﬁlms using ESCA showed the presence of the CC bond, which they reported, did not
originate from starch but from proteins and lipids. Russel and others (1987) stated that
low binding energy (285 eV), indicates the presence of lipids or proteins within the
matrix of ﬁlms tested. This is consistent with the formulation protocol of ﬁlms used in
this study, which consisted of WPI and CW.

It was also apparent that compression molding had a similar effect to heat curing.
Similar bonding was found between ﬁlms put through both of these treatments. Uncured

unsealed ﬁlms did not contain the preponderance of C-O-H or C-O-C bonds compared to

87

 

 

the cured sealed films and uncured sealed film. These bonds were also present in all
heat-cured and compression molded ﬁlms. This indicates that the C-O-H or C-O-C
bonds are important for adequate seal formation, as shown by the uncured sealed film. It
also demonstrates that these bonds are formed during heat curing and compression
molding. These results are consistent with adhesion reports by Wu and others (1995)
who reported that C-O-H components on the surfaces of polyoleﬁns participated in
hydrogen bonding across the interfaces of these polymers. Hydrogen and covalent bonds
are reported to play important roles in interfacial interactions required for adequate bond
strengths (Urban 1993, Misra 1994). Hydrogen bonding in proteins is well documented
(Cheftel and others 1985, Rasco and Zhong, 2000). Hydrogen bonds appear between
carbonyl (C=O), amine (NH) or hydroxyl (OH) groups of the polypeptide chains.
Together with hydrophobic, disulﬁde, electrostatic and dipole interactions, hydrogen
bonds help to stabilize protein structures (Cheftel and others 1985, Rasco and Zhong,
2000). The ﬁndings of this study are also consistent with those of Kim and Ustunol
(2001c) who reported that formation of C-O-H bonds was important for obtaining
adequate seals in WPI-based ﬁlms. Kim and Ustunol (2001c) proposed a model for the
nature of the hydrogen bonding that takes place between plasticizer and plasticizer;
plasticizer and protein; as well as between protein and protein upon heat-sealing of WPI-
based ﬁlms.

In this study nitrogen bonding was only detected in uncured ﬁlms and there was a
signiﬁcant decrease in this bond after sealing, this is clearly seen in the inner seal bond
composition of the uncured ﬁlm (Table 4.4). A possible explanation for the non-

detection of C-N bonds after sealing may be the formation of new bonds or changes in

88

 

 

molecular conformation that may result in these groups becoming bun’ed below ESCA
penetration levels. This phenomenon, however, requires further research. An assessment
of carbonyl (C=O) bonds demonstrated that uncured ﬁlms had the highest concentration
of C=O bonds (2.09%) among all the samples tested. Inner seal of uncured ﬁlms had the
second highest concentration of this bond (0.40 %); these bonds were not detected in
outer seals of cured ﬁlms. The reduction of the C=O bond in sealed, cured and
compression molded ﬁlms may possibly be due to the participation of these bonds in the
formation of hydrogen bonds. The model proposed by Kim and Ustunol (2001c) shows
hydrogen bonding taking place between carbonyl and amine groups. Formation of the
hydrogen bonds would cause reduction of the carbonyl (C=O) bonds and an increase in
C-O-H and C-O-C bonds. This would explain the increase observed in the concentration
of these bonds. This would also be consistent with the hydrogen bonding mechanism
presented by Cheftel and others (1985).

The presence of N and the C-N bond in the unsealed and inner seal of the uncured
ﬁlms suggests the possibility of protein cross-linking taking place between these ﬁlms
upon sealing. This would be consistent with the protein-protein model proposed by Kim
(2001c) for bonding between lysine and asparagine and; lysine and glutamine during heat
sealing. These amino acids are abundant in B-lactoglobulin and a—lactalbumin, the main
components of whey proteins. This may explain the higher seal strengths observed for
uncured ﬁlms compared to those of heat cured ﬁlms, for example 173.62 N/m vs. 80.91
N/m for 6.4% w/v solids ﬁlms (Table 4.1). The absence of N and abundance of
hydrocarbon groups on the heat-cured and compression molded ﬁlms suggests that

bonding in these ﬁlms is likely taking place between hydrophobic groups associated with

89

wax molecules; whose relative concentration may have increased on the ﬁlm surface
during the heating processes. This phenomenon would not be unlikely as lipids have a

lower density than proteins.

 

4.4. CONCLUSIONS

Seal strengths of uncured WPI-based ﬁlms were higher than those of heat cured
and compression molded ﬁlms. Seal strengths could not be optimized by sealing films-
prior to heat curing or wetting the seal area of uncured and heat cured ﬁlms prior to

sealing. Additional experiments were conducted to test the performance of scaled tubular

 

WPI-based casings under sausage manufacturing conditions. Results of these
preliminary experiments are shown in Appendix II. C-O-H and C-O-C bonds were
prevalent in heat cured, sealed and compression molded ﬁlms. C-N bonds were only

detected in uncured ﬁlms.

SUMMARY

In this research, experiments were conducted to investigate the potential of
utilizing whey protein isolate (WPI)-based ﬁlms in a sausage manufacturing process and
thereafter optimizing the mechanical and sealing properties of the ﬁlms for development
of a sausage casing. Further experiments were conducted to determine the surface and
interfacial chemical properties of unsealed and sealed ﬁlms.

The results showed that although WPI-based ﬁlms remained intact, the TS of the
films decreased with increasing temperature, time and relative humidity of the sausage
manufacturing process. Sausage manufacturing conditions did not affect %E. Compared
to commercial collagen casings, WPI-based ﬁlms had similar %E but lower TS.
Mechanical property optimization experiments showed the following:

o Heat-curing increased TS of uncured ﬁlms and did not decrease %E, the presence of
wax in the ﬁlm matrix may have contributed to mobility within the protein film
structure, thus causing the ﬁlms to retain their ﬂexibility.

0 Compression molding increased TS of uncured ﬁlms and the TS values were similar
to those obtained by heat-curing. These results indicate that compression molding
can be used to heat-cure ﬁlms for a lesser time than vacuum heat-curing.

o The increase in T8 resulting from heat curing and compression molding was not
sufﬁcient to give WPI-based ﬁlms TS equivalent to collagen films. This indicates
that T8 of WPI-based ﬁlms needs to be increased further to enable these films to be

used in sausage manufacture

91

 

 

0 Through ESCA we discovered that heat curing, sealing and compression molding
increased carbon concentration on the surface of the ﬁlms and N became

undetectable.

FUTURE RESEARCH

In order to manufacture sausage casings with the WPI-based edible films, further
work needs to be done to improve their mechanical properties, particularly tensile
strength under increased relative humidity (RH) conditions. Reformulation of the film
forming solution by incorporating materials such as cellulose might be one way to
increase the strength of these ﬁlms while increasing their resistance to increased RH.
Furthermore, I suggest that instead of using solvent casting for ﬁlm formation, it might be
better to use compression molding, because variables like thickness can be controlled
during the ﬁlm formation process. In order to reduce problems that may be encountered
with sealing when developing tubular casings from the WPI-based ﬁlms, I recommend
that investigation be made into forming the sausage casings using extrusion; in this way a
continuous casing would be obtained. Research is also required to precisely determine
the cause of the non-detection of C-N bonds after sealing, heat curing and compression

molding of WPI-based ﬁlms.

92

 

 

APPENDIX I

93

 

PRELIMINARY STUDIES ON POST-TREATMENT OF WHEY PROTEIN

ISOLATE-BASED FILMS WITH PHOSPHATE SOLUTIONS

Materials

Whey Protein Isolate (WPI, Provon 190) was supplied by Glanbia ingredients
(Monroe, WI). Glycerol was purchased from IT Baker Co. (Phillipsburg, NJ). Sodium
hydroxide (2N, NaOH) was purchased from Mallinckrodt Specialty Chemical Co. (Paris,
KY). Candelilla wax (CW) was purchased from Strahl and Pitsch Inc. (West Babylon,
NY) and magnesium nitrate was purchased from Sigma Chemical Co. (St Louis, MO)
and phosphate solution was obtained from Butcher and Packer Supply Company (Detroit,

MI).

METHODS
Whey protein isolate based ﬁlm preparation

Two film forming solutions having 5.8% w/v and 6.4% w/v solids contents were
developed by dissolving whey protein isolate (WPI) in distilled water, glycerol was
added and pH was adjusted to 8 using 2N NaOH. The solutions were heated to 90 j; 2°C
with continuous stirring to effectively denature the whey protein. Candelilla wax was
added during heating and allowed to melt into the solutions. The levels of the ﬁlm
constituents used were: WPI (5.0% or 5.5% w/v), glycerol (3.3% or 3.6% w/v) and CW
(0.8% or 0. 9%, w/v). The solutions were homogenized for 2 minutes using a Polytron

PT 10/35 homogenizer with a PTA 20 TS homogenizing head (Tekmar Co., Cincinnati,

94

 

 

OH) at speed 5; filtered through a layer of cheesecloth, equilibrated to room temperature
for 1.5 h and vacuum degassed at room temperature for 30 min. Films were prepared by
pipetting the emulsion onto either anodized or non-anodized Teflon® plates. The ﬁlms
were dried at room temperature (23 i 2°C) and 30 i 5% relative humidity (RH) for 18 -_i-_
3 h. Dried ﬁlms were peeled, wrapped in foil and heat cured in a vacuum (30mm Hg)
oven maintained at l) 80°C for 24h or 2) 90°C for 12 h. The ﬁlms were removed from
the oven and peeled from the foil while still warm for ease of removal. The ﬁlms were
subsequently conditioned at 50% RH, 23°C using magnesium nitrate for 48 h prior to

testing.
Film thickness

Films were preconditioned at 23°C, 50% RH using magnesium nitrate for a ,
minimum of 8 h to enable ease of handling and cutting into strips (101.6mm x 25.4mm)
using a Precision Sample Cutter (Thawing Albert Instrument Co., Philadelphia, PA).
Film thickness was determined using a M model 549M micrometer (Testing Machines,
Inc. Amityville, NY). Five different measurements were taken in random locations
within the ﬁlmstrip test area and the mean values were used for calculations of

mechanical properties.
Phosphate treatment

Uncured and cured ﬁlm strips were post-treated by dipping them in a potassium
phosphate solution for 10 3. Food grade potassium phosphate solution obtained from
Butcher and Packer Supply Company (Detroit, MI) was used. The treatment
concentrations used were: 0%, 12.5%, 25% and 50% (v/v). Following dipping in the

phosphate solution, the ﬁlms became brittle and cracked easily. The phenomenon

95

 

 

causing the ﬁlm brittleness was investigated as follows: 1) ethyleneglycol-bis-(B-amino-
ethyl-ether)-N,N’-tetra acetic acid (EGTA) was added to the ﬁlms forming solutions after
WPI and glycerol had been dissolved. The concentrations of EGTA were 2.5, 5, 10, 15,
20 or 25 mM in the ﬁlm forming solutions 2) potassium phosphate solution (9.2% v/v)
was added to the ﬁlm forming solutions after the addition of EGTA 3) the solutions were

then heated as described above.
Statistical analysis

The effect of heat curing (curing), total solids content of casting solution
(solution) and phosphate post-treatment (phosphate post-treatment) on TS and %E of

WPI-based ﬁlms were determined using the models shown below:
Y = a linear function (heat curing; solution; phosphate post-treatment) (7)

Where Y = TS or %E, curing = uncured or heat cured, solution = 5.8 or 6.4% w/v
solids and phosphate post-treatment = 12.5, 25, 50% w/v phosphate concentration. All
comparisons were also made to collagen ﬁlms. The mixed procedure (PROC MIXED) of
SAS was used to analyze the data. Treatment means were compared using the least

squares means (LSM) and the means were based on 3 replicates.

RESULTS
Table 1.1 shows the mechanical properties (TS and %E) of ﬁlms post-treated with

potassium phosphate solution (0, 12.5, 25 and 50% v/v). Uncured ﬁlms had lower TS
and %E than cured ﬁlms for all treatments, comparisons to untreated ﬁlms showed that

TS of phosphate treated ﬁlms were similar to non-treated ﬁlms; however, %E was

96

 

 

signiﬁcantly reduced (p<0.05). Phosphate treated ﬁlms became brittle and, thus, no
sealing tests could be performed on these ﬁlms. These results are different from those of
Otaigbe and Adams (1997) who showed that addition of polyphosphate ﬁllers to SP1
ﬁlms improved TS while reducing water absorption. Polyphosphate ﬁllers were added
prior to compression molding at 10, 20 and 30% (wt of SP1) and were effective up to
20% w/w. Results of Otaigbe and others (1997) showed that increases in polyphosphate
concentration were not effective in increasing the TS. The successful use of
polyphosphate ﬁllers by these researchers suggests that similar treatments may be applied

to WPI-based ﬁlms and may yield more successful results instead of the post—treatment

method used in the current study.

Table 1.1. Mechanical properties of ﬁlms post-treated with potassium phosphate solution

 

 

 

 

 

 

 

Film TS (MPa) %E
Solution Phosphate
Solids Solution
(%w/v) (%) Uncured Cured Uncured Cured
0 2.71 :t 1.31“ 7.16 i 2.33 b 20.52 3; 18.66b 36.89 :i: 10.72d
5.8 12.5 2.88 3; 1.16“ 5.19 1 3.42 b 24.59 1 9.47“ 30.66 3; 5.48 d
25-0 2.91 t 1.34“ 5.35 : 3.21b 10.21:. 4.35“ 21.59 :t: 13.15d
50-0 2.28 :i: 1.26“ 4.45 3; 2.74b 7.58 1- 3.56c 20.47 3; 13.74‘1
0 3.01 :i: 0.62“ 3.90 t 1.71 b 21.62 :1: 10.44“ 9.63 3; 8.70d
6.4 12.5 2.50 3; 1.51 “ 5.38 :t 2.83 b 27.78 3. 7.35“ 20.44 :i: 9.18 “'
25-0 2.79 3: 1.68“ 4.86 :1: 2.52 b 10.77 :1; 3.81“ 18.05 : 5.42d
50-0 2.36 3; 1.14“ 4.63 t 1.92 b 6.33 t 3.16“ 31.90 3; 19.356
Collagen 13.29 3; 3.47“ 31.78 :t 4.88“

 

 

 

a"’Means followed by the same superscript are not signiﬁcantly different (p<0.05), comparisons

are made within the same column, n=3 for all treatments,

Elongation (%).

97

TS = Tensile strength, %E =

 

The presence of some bonding interaction between the phosphate and the protein
was demonstrated by the phenomenon of the ﬁlms becoming brittle. The behavior of the
phosphate in the ﬁlm-forming solution was thereafter investigated. Addition of the
potassium phosphate solution to the film-forming solution resulted in gel formation upon
heating of the solution. One possible mechanism of this gel structure is the formation of
calcium-phosphate bridges. To test this hypothesis, ethyleneglycol-bis-(B-amino-ethyl-
ether)-N,N’-tetra acetic acid (EGTA) was added to the film forming solution to chelate
calcium. The addition of EGTA, however, was not successful in reducing or preventing
gelation. This indicated that the interaction between the whey proteins and phosphates
was not a result of calcium-phosphate bridges and suggests that there may be a different
interaction taking place between whey proteins and phosphates that we are not aware of.
Further studies are required to determine precisely the nature of the interaction between

phosphates and WPI.

98

 

 

 

APPENDIX 11

99

PRELIMINARY STUDIES OF POLISH SAUSAGE MANUFACTURE USING

WHEY PROTEIN ISOLAT E BASED FILMS AND COLLAGEN CASINGS

Materials

Whey Protein Isolate (WPI, Provon 190) was supplied by Glanbia ingredients
(Monroe, WI). Glycerol was purchased from IT. Baker Co. (Phillipsburg, NJ). Sodium
hydroxide (2N, NaOH) was purchased from Mallinckrodt Specialty Chemical Co. (Paris,
KY). Candelilla wax (CW) was purchased from Strahl and Pitsch Inc. (West Babylon,
NY) and edible collagen casings (inside diameter, 32mm) were obtained from The

Brechteen Co. (Chesterfield, MI).

METHODS
Whey protein isolate based ﬁlm preparation

Figure 4.1 shows a schematic diagram of the experiments conducted under this
objective designed to optimize the ﬁlms for sausage casing manufacture. Two ﬁlm
forming solutions having 5.8% w/v and 6.4% w/v solids contents were developed by
dissolving whey protein isolate (WPI) in distilled water, glycerol was added and pH was
adjusted to 8 using 2N NaOH. The solutions were heated to 90 i 2°C with continuous
stirring to effectively denature the whey protein. CW was added during heating and
allowed to melt into the solutions. The levels of the ﬁlm constituents used were: WPI
(5.0% or 5.5% w/v), glycerol (3.3% or 3.6% w/v) and CW (0.8% or 0. 9%, w/v). The

solutions were homogenized for 2 minutes using a Polytron PT 10/35 homogenizer with a

100

 

PT A 20 TS homogenizing head (Tekmar Co., Cincinnati, OH) at speed 5; ﬁltered through
a layer of cheesecloth, equilibrated to room temperature for 1.5 h and vacuum degassed at
room temperature for 30 min. Films were prepared by pipetting the emulsion onto either
anodized or non-anodized Teﬂon® plates. The ﬁlms were dried at room temperature (23
i 2°C) and 30 i 5% relative humidity (RH) for 18 i 3 h. Dried ﬁlms were peeled,
wrapped in foil and heat cured in a vacuum (30mm Hg) oven maintained at l) 80°C for
24h or 2) 90°C for 12 h. The ﬁlms were removed from the oven and peeled from the foil
while still warm for ease of removal. The ﬁlms were subsequently conditioned at 50%

RH, 23°C using magnesium nitrate for 48 h prior to testing.
Compression molding

Whey protein isolate-based ﬁlms were compression molded using a Carver _
Laboratory Press (Fred Carver, Inc.; Wabash IN). Film sheets (130 x 200 mm) were
sandwiched (one at a time) between Teﬂon® sheets, placed between 2 stainless steel
molding plates and inserted between heated plates of the compression compartment of the
compression molder. Molding conditions were: 1) single-stage (110°C, 1.24 MPa, 1 h or
2 h) and 2) two-stage compression molding (110°C, 0.99 MPa, 25 min and 110°C, 1.24

MPa, 12 min). 5.8% w/v solids ﬁlms were used for compression molding.
Manufacturing of tubular whey protein-based casings

Figure [1.1 shows tubular WPI-based casings developed by heat-sealing of the
films. Heat-sealed tubular casings were manufactured to resemble sausage casings and
commercial collagen casing dimensions were used (32m). A sheet of whey protein ﬁlm
(35mm x 200mm) was rolled and heat-sealed on two sides using an impulse sealer

Model-12ASL(Sencorp System Inc., Hyannis, MA). The impulse sealer settings were as

101

 

 

Figure 11.1. Whey protein isolate based edible ﬁlms heat-sealed to form sausage casings
1 = uncured ﬁlm, 2 = compression molded ﬁlm, 3 = ﬁlm sealed before heat curing, 4 =
heat cured ﬁlm

follows: pressure: 300kPa, voltage: 22 amps, impulse time: 1.8 seconds and dwell time:

1.4 seconds. Casings were made with uncured, heat cured and compression molded ﬁlms
Sausage manufacturing

Figures [1.2 and 112 show stufﬁng of the Polish sausage and the smokehouse in
which the sausages were cooked, respectively. Sausage was made using turkey meat and
seasoned with Polish sausage seasoning (Legg’s sausage seasoning, modemcure
(156ppm) with salt and erythorbate (500ppm)). The mixture was stuffed into the casings
using a hand stuffer (VOGT-deal, Chicago IL, USA). The cooking schedule used is

shown in Table 11.1.

Table 11.1. Polish sausage smokehouse schedule

 

 

 

Stages Time, Internal Dry Bulb, Wet Bulb, Smoke Source,
min Temperature, °C °C °C Natural
1 20 0 60 43 off
2 20 0 71 52 on
3 15 0 77 57 on
4 90 72 82 77 off
Shower 32 l min on 1 min off

 

102

 

    

Figure 11.3. Sausage stuffed in whey protein isolate based edible casings (ﬂat on rack) and
collagen casings (hanging on sausage stick) in smokehouse

RESULTS
Figure 11.4 shows the external appearance of sausages stuffed in experimental

WPI-based edible casings and commercial collagen casings used as control. Although
sausage was successfully stuffed into the casings, the casings were very delicate,

indicating that the sausage casings still needed further improvement to enable them to

103

 

withstand sausage manufacturing conditions. The inadequacy of WPI-based casings was
demonstrated by tearing of the seals due to slight increases in the stufﬁng pressure or
ﬁlling speed. The best-stuffed casings developed in this experiment were the heat cured
ﬁlms: 5.8%w/v solids, 6.4% w/v solids and the 5.8%w/v ﬁlm sealed-before heat curing.
The uncured and compression molded ﬁlms did not perform well as they took up a lot of
moisture ﬁom the meat. This was evidenced by moisture observed on the surface of the
ﬁlm immediately after stufﬁng. Aﬁer cooking, the casings that were well stuffed had
acceptable appearance while loosely stuffed casings were shriveled and not appealing due
to air pockets between the meat and the casing. Uncured casings were shriveled and

unappealing; they were also very pale in color. The collagen-cased sausage remained the

best-stuffed and best-looking casings.

 

Figure 11.4. Sausages made from various stuffed whey protein isolate based edible
casings

l = Uncured ﬁlm 2 = 5.8% w/v solids ﬁlm 3 = 6.4% w/v ﬁlm 4 2 ﬁlm sealed before
curing 5 = compression molded 6 = collagen casing

104

 

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