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This is to certify that the

dissertation entitled

1 Antimicrobial Whey Protein Isolate-Based
Edible Casings

presented by

Arzu Cagri

has been accepted towards fulfillment
of the requirements for

Ph.D degree in Food Science

 

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Date 5/3/02

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ANTIMICROBIAL WHEY PROTEIN ISOLATE-BASED

EDIBLE CASINGS

By

ARZU CAGRI

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

2002

ABSTRACT

ANTIMICROBIAL WHEY PROTEIN ISOLATE-BASED EDIBLE CASINGS
By
ARZU CAGRI

Methods were developed to optimally produce low pH (5.2) antimicrobial whey
protein isolate (WPI) edible films containing sorbic acid (SA) or p-aminobenzoic acid
(PABA). Solutions of lactic acid and acetic acid (1:0, 1:1, and 23:1) were used to
acidify the film solution. Films containing either antimicrobial agent inhibited the
growth of Listeria monocytogenes, Escherichia coli 0157:H7 and Salmonella
Typhimurium DT104 on trypticase soy agar. Increased concentrations of PABA or SA
in WPI film increased the percent elongation (%E) and water vapor permeability
(WVP). While tensile strength (TS) of WPI films decreased with increasing levels of
SA, TS of films was not affected addition of PABA.

Antimicrobial properties of films containing PABA, SA, or PABA:SA were
tested over 21 days of refrigerated storage on slices of summer sausage and bologna
inoculated to contain 106 L. monocytogenes, E. coli 0157:H7 or S. Typhimurium
DT104 CFU/ g. These films decreased populations of the test pathogens and retarded the
growth of mesophilic aerobic bacteria, lactic acid bacteria and mold/yeast throughout
this period of storage. Contact with the slices decreased TS of the films, but increased
% E.

In the last objective, heat-cured WPI films containing PABA were heat-sealed to
form casings. A commercial-type hot dog batter was stuffed into WPI, collagen, or

natural casings. After cooking, the hot dogs were surface-inoculated with Listeria

monocytogenes, vacuum packaged, and examined for numbers of L. monocytogenes,
mesophilic aerobic bacteria (MAB), lactic acid bacteria (LAB), and yeast/mold during
42 days of storage at 4°C. Listeria populations on hot dogs prepared with WPI-PABA
casings remained relatively unchanged; however, numbers of Listeria on hot dogs
prepared with WPI, collagen, and natural casings increased during 42 days of
refrigerated storage. Populations of MAB, LAB, and mold on WPI-PABA casings were
lower compared to other casings. Physical (e.g. purge loss, color), mechanical (shear
force), chemical (e. g. moisture, fat, protein content, pH, TBA) and sensory properties of
hot dogs prepared with different casings were also analyzed. Taste panelists ranked
flavor, juiciness, and overall acceptability higher for hot dogs prepared with WPI-
PABA casings compared to hot dogs prepared with WPI, collagen or natural casings.
Hence, WPI casings containing PABA appear to be a promising means to prevent

growth of Listeria on hot dogs and extend shelf-life.

To my parents

ACKNOWLEDGMENTS

This dissertation has been made by the support and help from many people.
Foremost among them is my advisor Dr. Elliot T. Ryser. I would like to express my
sincerest and most heartfelt thanks to him. His thoughtful guidance and advice helped me
grow educationally and professionally. I cannot thank him enough for the all the support
he has provided me for the successful completion of this research project.

I would like to express my sincere appreciation to all my committee members, Dr.
James Pastka, Dr. Jack Giacin, Dr. Susan E. Selke, Dr. Wesley N. Osburn, Dr. Zeynep
Ustunol, for their guidance and support. I am grateful to Dr. Zeynep Ustunol for all
support and advice throughout my graduation program. I would also like to thank Dr
Seong-Joo Kim for her advice in film formation technique.

I owe a great deal of gratitude to Dr. Wesley N. Osburn and his research group,
Dr. Jamie Willard, Christine Ebeling, Deanna Bloom and Jeff Sindelar for their great
input and collaboration in this project. They helped me in hot dog processing and other
analysis. They also allowed me to use of the lab equipment, time, and expertise in
finishing this project. Without their help the study could not have been completed.

I would like to thank Dr. Bruce Harte for allowing me to use the labs and
equipment in the Department of Packaging. I also would like to thank teaching assistants
in Dept. of Packaging, Dr. Rujida Leepipattanawit and Krittika Tanprasert for all their
help in this project. I sincerely appreciate Dr. Janice Harte for her advice on setting up the
sensory tests.

I would like to thank Turkish Government, Center for Food and Pharmaceutical

Packaging Research and USDA FSIS for their financial support for this research. New

Zealand Milk Product, Inc. and Strahl and Pitch, Inc. are also acknowledged for
providing whey proteins and candellila wax, respectively.

I would like to acknowledge all the past and present colleagues in my laboratory
for their continues encouragement and friendship. I would also like to thank Can Mavruk
for all his support and help with data analysis. My deepest appreciation goes to my family

and friends who supported me while accomplishing this work.

vi

TABLE OF CONTENTS

LIST OF TABLES ......................................................................... xii
LIST OF FIGURES ........................................................................ xv
ABBREVIATIONS .......................................................................... xvii
INTRODUCTION ........................................................................... 1
CHAPTER 1
LITERATURE REVIEW .................................................................... 6
1.1 . Edible Films .................................................................... 6
1.1.1 Definition and historical background ........................... 6
1.1.2 Formation of edible films ......................................... 7
1.1.2.1 Components of edible films .............................. 7
1.1.2.2 Film formation techniques .............................. 8
1.1.3 Protein-based edible films ......................................... 9
1.1.3.1 Whey protein .............................................. 9
1.1.3.1.] Composition and structure ................. 9
1.1.3.1.2 Film formation ............................... 10
1.1.3.2 Corn zein .................................................. 11
1.1.3.2.] Composition and structure ................. 11
1.1.3.2.2 Film formation ............................... 13
1.1.3.3 Casein ..................................................... 16
1.1.3.3.] Composition and structure ................. 16
1.1.3.3.2 Film formation .............................. 17
1.1.3.3.3 Film properties ............................... 18
1.1.3.4 Wheat gluten .............................................. 19
1.1.3.4.1 Composition and structure ................. 19
1.1.3.4.2 Film formation ............................... 19
1.1.3.4.3 Film properties ............................... 20
1.1.3.5 Soy protein ................................................ 22
1.1.3.5.] Composition and structure ................. 22
1.1.3.5.2 Film formation ............................... 22
1.1.3.6 Collagen ................................................... 25
1.1.3.6.] Composition and structure ................. 25
1.1.3.6.2 Film formation ............................... 25
1.1.3.7 Gelatin ..................................................... 27
1.1.3.7.1 Composition and structure ................. 27
1.1.3.7.2 Film formation ............................... 28
1.1.4 Polysaccharide-based edible films ............................... 28

vii

1.1.4.1 Cellulose film .............................................. 28

1.1.4.2 Alginate .................................................. 31
1.1.4.2.] Film formation ............................... 31

1.1.4.3 Pectin ...................................................... 33

1.1.4.4 Chitosan ................................................. 34
1.1.4.4.] Film formation ................................ 34

1.1.4.5 Starch films ............................................... 35

1.1.4.6 Dextrins ................................................... 37

1.1.5 Lipid films .......................................................... 37
1.1.5.1 Wax films .................................................. 38

1.1.6 Evaluating of the properties of edible films .................... 40
1.1.6.1 Barrier properties ........................................ 4O
1.1.6.1.1 Water vapor permeability .................. 41

1.1.6.1.2 Oxygen permeability ........................ 42

1.1.6.2 Mechanical properties ................................... 43

1.1.7 Factors affecting film properties ................................. 43
1.1.8 Application of edible films ....................................... 44
1.2 Antimicrobials .................................................................... 50
1.2.1 Benzoic acid ........................................................ 51
1.2.2 Sorbic acid .......................................................... 52
1.2.3 Propionic acid ...................................................... 60
1.2.4 Mode of action of benzoate, sorbate, and propionic acid. 62
1.2.5 Parabens ............................................................. 63
1.2.6 Fatty acids ........................................................... 65
1.2.7 Organic acids ....................................................... 66
1.2.7.1 Acetic acid ................................................ 66

1.2.7.2 Lactic acid ................................................. 69

1.2.7.3 Mode of action of organic acids ...................... 70

1.2.8 Bacteriocins ......................................................... 70
1.2.8.1 Nisin ........................................................ 71

1.2.8.2 Pediocin .................................................... 74

1.2.8.3 Mode of action of bacteriocins ........................ 76

1.2.9 Natural antimicrobials ............................................. 77
1.2.9.1 Lysozyme .................................................. 77
1.2.9.1.] Mode of action .............................. 78

1.2.9.2 Spices, Herbs, and Essential oils ...................... 79

1.2.9.3 Lactoferrin ................................................. 81

1.2.9.4 Liquid smoke ............................................. 83

1.2.10 Cure agents ......................................................... 84
1.2.9.1. NaCl ....................................................... 84
1.2.9.2. Nitrite ...................................................... 85

1.3. Antimicrobial Edible Films ................................................ 87

viii

CHAPTER 2

ANTIMICROBIAL, MECHANICAL, AND MOISTURE BARRIER PROPERTIES
OF LOW pH WHEY PROTEIN-BASED EDIBLE FILMS CONTAINING

p-AMINOBENZOIC OR SORBIC ACIDS ............................................. 95
2.1 . Abstract ................................................................................... 96
2.2. Introduction ............................................................................... 96
2.3. Materials and methods .................................................................. 99

2.3.1. Film preparation .............................................................. 99
2.3.2. Bacterial strain ................................................................ 99
2.3.3. Diffusion-type assay ......................................................... 100
2.3.4. Film thickness ................................................................ 100
2.3.5. Mechanical properties ....................................................... 101
2.3.6. Water vapor permeability ................................................... 101
2.3.7. Statistical analysis ............................................................ 102
2.4. Results and discussion .................................................................. 102
2.4.1 . Antimicrobial properties .................................................... 102
2.4.2. Mechanical properties ....................................................... 108
2.4.3. Water vapor permeability ................................................... 1 12
2.5. Conclusion ................................................................................ 1 14
CHAPTER 3

INHIBITION OF LISTERIA MONOC YT OGENES, SALMONELLA TYPHIMURIUM
DT104 AND ESCHERICHIA COLI 0157:H7 ON BOLOGNA AND SUMMER
SAUSAGE SLICES USING WHEY PROTEIN ISOLATE BASED EDIBLE FILMS

CONTAINING ANTIMICROBIALS ................................................... 1 15
3.1. Abstract .......................................................................... 116
3 .2. Introduction ...................................................................... 1 16
3.3. Materials and methods ......................................................... 119

3.3.1. Products .............................................................. 119
3.3.2. Film preparation ..................................................... 119
3.3.3. Bacterial strains ...................................................... 120
3.3.4. Product Inoculation and Storage ................................... 120
3.3.5. Microbiological analysis ............................................ 121
3 .3 .6. Mechanical properties ............................................... 122
3.3.7. Statistical analysis ................................................... 123

3.4. Results and discussion ......................................................... 123

3.4.1 . Antimicrobial properties ............................................ 123
3.4.2. Mechanical properties .............................................. 137
3.5. Conclusion ...................................................................... 140
CHAPTER 4
INHIBITION OF LISTERIA MONOC YT OGENES ON HOT DOGS USING
ANTIMICROBIAL WHEY PROTEIN-BASED EDIBLE CASINGS ................ 141
4.1 . Abstract .......................................................................... 142
4.2. Introduction ...................................................................... 143
4.3. Materials and methods ......................................................... 144
4.3.1 . Experimental Design ................................................ 144
4.3.2. Casing preparation .................................................. 145
4.3.3. Culture preparation .................................................. 146
4.3.4. Hot dog processing .................................................. 149
4.3.5 Chemical analysis .................................................... 149
4.3.6. Rancidity measurement ............................................. 149
4.3.7. pH Test ................................................................ 150
4.3.8. Diffusion Test ........................................................ 150
4.3.9. Microbiological analysis ........................................... 152
4.3.10. Film thickness ...................................................... 152
4.3.1 1. Mechanical properties ............................................. 152
4.3.12. Purge ................................................................. 153
4.3.13. Shear test ............................................................ 153
4.3.14. Color measurement ................................................ 154
4.3.15. Sensory evaluation ................................................. 154
4.3.16. Statistical Analysis ................................................. 155
4.4. Results and Discussion ........................................................ 155
4.4.1 Chemical analysis ................................................... 155
4.4.2 Thiobarbutiric acid values .......................................... 155
4.4.3. pH ...................................................................... 157
4.4.4. Diffusion Coefficient ............................................... 157
4.4.5. Antimicrobial Analysis ........................................... 160
4.4.6. Mechanical properties ............................................. 169
4.4.7 Purge loss and Cook Yield ......................................... 170
4.4.8 Shear Force ........................................................... 170
4.4.9 Color .................................................................. 173
4.4.10. Sensory Analysis ................................................. 177
CONCLUSIONS ............................................................................. 181

APPENDIX I Moisture Analysis .......................................................... 184

APPENDIX II Fat Analysis ................................................................. 186
APPENDIX III Protein Analysis ............................................................ 188
APPENDIX IV TBA measurement ......................................................... 191
APPENDIX V Sensory Questionnaire ................................................... 194
APPENDIX VI Written consent form .................................................... 199
BIBLIOGRAPHY ............................................................................ 202

xi

LIST OF TABLES

Table 1.1 Water vapor permeability and oxygen permeability of edible films ........... 12
Table 1.2 Tensile strength and elongation of edible films .................................... 14
Table 1.3 Antimicrobials ......................................................................... 54

Table 2.1 Antimicrobial activities of whey protein based edible films containing
p-aminobenzoic acid (PABA) against 4 strains of L. monocytogenes ........ 103

Table 2.2 Antimicrobial activities of whey protein based edible films containing
sorbic acid (SA) against 4 strains of L. monocytogenes. ....................... 104

Table 2.3 Antimicrobial activities of whey protein based edible films containing
p-aminobenzoic acid (PABA) against 3 strains of E. coli 0157:H7 .......... 106

Table 2.4 Antimicrobial activities of whey protein based edible films containing
sorbic acid (SA) against 3 strains of E. coli 01572H7 ........................... 107

Table 2.5 Antimicrobial activities of whey protein based edible films containing
p-aminobenzoic acid (PABA) against 5 strains of S. Typhimurium DT104.. 109

Table 2.6 Antimicrobial activities of whey protein based edible films containing
sorbic acid (SA) against 5 strains of S. Typhimurium DT104 ................... 110

Table 2.5 Thickness, Tensile strength (TS), Percent elongation (%E), and Water Vapor
Permeability (WVP) of whey protein isolate-based films containing
sorbic acid (SA) and p-aminobenzoic acid (PABA) ............................. 113

Table 3.1 Population decrease (log CFU/ g) of L. monocytogenes, E. coli 01572H7, and
S. Typhimurium DT104 with WPI films containing p-aminobenzoic acid
(PABA), sorbic acid (SA), or SAzPABA on bologna (B) and fermented
summer sausage (SS) slices after 21 days of refrigerated storage ............. 132

Table 3.2 Inhibition of mesophilic aerobic bacteria (MAB) and lactic acid (LAB)
on bologna slices using whey protein isolate edible films containing
p-aminobenzoic acid (PABA) or sorbic acid (SA) at 4°C ...................... 133

Table 3.3 Inhibition of mold and yeast on bologna slices using whey protein isolate
edible films containing p-aminobenzoic acid (PABA) or sorbic acid (SA)
at 4°C ................................................................................... 134

Table 3.4 Inhibition of mesophilic aerobic bacteria (MAB) and lactic acid (LAB) on
fermented summer sausage slices using whey protein isolate edible films

xii

containing p-aminobenzoic acid (PABA) or sorbic acid (SA) at 4°C. . . . 1 35

Table 3.5 Inhibition of mold and yeast on fermented summer sausage slices using
whey protein isolate edible films containing p-aminobenzoic acid (PABA)
or sorbic acid (SA) at 4°C ............................................................ 136

Table 3.6 Thickness, percent elongation (% E) and tensile strength (TS) of whey
protein isolate edible films containing p-aminobenzoic acid (PABA)
or sorbic acid (SA) while in contact with bologna slices at 4°C ............... 138

Table 3.7 Thickness, percent elongation (% E) and tensile strength (TS) of whey
protein isolate edible films containing p-aminobenzoic acid (PABA)
or sorbic acid (SA) while in contact with fermented summer sausage
slices at 4°C ........................................................................... 139

Table 4.1 Hot Dog Smokehouse schedule .................................................... 148

Table 4.2 Moisture, fat and protein content of hot dogs prepared with WPI-p-
aminobenzoic acid (PABA), WPI, collagen and natural casings .............. 156

Table 4.3 TBA values and pH of hot dogs prepared with WPI-p-aminobenzoic acid
(PABA), WPI, collagen, and natural casings during 42 days of
refrigerated storage ................................................................... 159

Table 4.4 Analysis of Variance on the effect of easing type on microbiological,
chemical, and physical characteristics of hot dogs during 42 days of
refiigerated storage (F-values for independent variables and interactions)... 160

Table 4.5 Tensile strength (TS) and percent elongation (% E) of WPI-p-
aminobenzoic acid (PABA), WPI, collagen, and natural casings before
and after cooking and smoking of hot dogs .....................................

Table 4.6 Purge lossesof hot dogs prepared with WPI-p-aminobenzoic acid
(PABA), WPI, collagen, and natural casings during 42 days of
refrigerated storage ................................................................... 172

Table 4.7 Interior color values of hot dogs prepared with WPI-p-aminobenzoic acid
(PABA), WPI, collagen and natural casings during 42 days of refiigerated
storage .................................................................................. 174

Table 4.8 Exterior color values of hot dogs prepared with WPI-p-aminobenzoic acid
(PABA), WPI, collagen and natural casings during 42 days of refrigerated
storage ................................................................................. 178

Table 4.9 Sensory attributes and chemical content of hot dogs prepared with WPI- p-
aminobenzoic acid (PABA), WPI, collagen, and natural casings after 35

xiii

days of refrigerated storage .......................................................... 179

xiv

Figure 3.1

Figure 3.2

Figure 3.3

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

LIST OF FIGURES

Inhibition of L. monocytogenes on bologna (A) and fermented

sausage (B) slices with WPI edible films containing 0.75%, and

1.0% p-aminobenzoic acid (PABA); 0.75% and 1.0% sorbic acid

(SA) and 0.5% SA: 0.5%PABA (1 :1). Control + film (WPI edible

film without SA or PABA) and control (no film used) ......................... 122

Inhibition of E. coli 01572H7 on bologna (A) and fermented

sausage (B) slices with WPI edible films containing 0.75%,

and 1.0% p-aminobenzoic acid (PABA); 0.75% and 1.0% sorbic

acid (SA) and 0.5% SA: 0.5%PABA (1 :1). Control + film

(WPI edible film without SA or PABA) and control (no film used). . . ....123

Inhibition of S. Typhimurium DT104 on bologna (A) and fermented

sausage (B) slices with WPI edible films containing 0.75%, and

1.0% p-aminobenzoic acid (PABA); 0.75% and 1.0% sorbic acid

(SA) and 0.5% SA: 0.5%PABA (1:1). Control + film (WPI

edible film without SA or PABA) and control (no film used) ................ 124

Preliminary study. Growth of L. monocytogenes on hot dogs prepared
with WPI- 1% PABA, WPI- 1% SA, WPI- 0.5% SA: 0.5% PABA,

WPI (antimicrobial-free), and Collagen casings ................................. 163
Growth of Listeria monocytogenes on hot dogs prepared with WPI- 1%
PABA , WPI, Collagen, and Natural casings .................................... 166
Growth of mesophilic aerobic bacteria on hot dogs prepared with WPI- 1%
PABA , WPI, Collagen, and Natural casings .................................... 167
Growth of lactic acid bacteria on hot dogs prepared with WPI- 1% PABA,
WPI, Collagen, and Natural casings ............................................... 168
Growth of mold on hot dogs prepared with WPI- 1% PABA , WPI,
Collagen, and Natural casings ...................................................... 169

Cook Yield (A) and Shear force (B) of hot dogs prepared with
WPI- p-aminobenzoic acid (PABA), WPI, collagen, and natural casings....l73

XV

ASTM

CDC
CMC
Coll
% E
FDA
Gly
GRAS
HPC
HPMC
LA
LAB
MAB
MC
OP
PABA
PEG
PW

SA
SAS
SDS
Sol
SPI
SS
TBA
TS

ABBREVIATIONS

Acetic acid

American Society for Testing and Materials
Bologna slices

Centers for Disease Control and Prevention
Carboxymetylcellulose

Collagen

Percent elongation

Food and Drug Administration
Glycerol

Generaly recognized as safe
Hydroxypropyl cellulose
Hydropropionate methylcellulose
Lactic acid

Lactic acid bacteria

Mesophilic aerobic bacteria
Methylcellulose

Oxygen permeability
p-aminobenzoic acid
Polyethylene glycol

Peptone water

Relative humidity

Sorbic acid

Statistical Analysis System
Sodium dodecyl sulfate

Sorbate

Soy protein isolate

Summer sausage

2- Thiobarbutiric acid

Tensile strength

xvi

TSA
TSAYE
TSB

TS

WG
WPI

Trypticase soy agar

Trypticase soy agar + yeast extract
Trypticase soy broth

Trisodium phosphate

Wheat gluten

Whey protein isolate

Water vapor permeability

xvii

INTRODUCTION

Microbial susceptibility of the food surface is a major determinant of product
quality and safety during refiigerated storage and distribution since most Class 1 product
recalls in the US. result from post-processing contamination during subsequent handling
and packaging. The increasing rate at which ready-to-eat meat products are becoming
contaminated with Listeria monocytogenes after processing has raised new safety
concerns. From April 1998 to December 2001 over 75 Listeria-related Class I recalls
involving more than 100 million pounds of cooked ready-to-eat meats were issued
(USDA-FSIS, 2001a). In 1998, more than 35 million pounds of hot dogs and luncheon
meats contaminated with L. monocytogenes were voluntarily recalled by one Michigan
manufacturer in response to a listeriosis outbreak that resulted in 101 cases in 22 states,
including 21 fatalities (CDC, 1998). Another outbreak involving 29 cases in 10 states
(including 7 fatalities) prompted the recall of approximately 14.5 million pounds of
turkey and chicken delicatessen meat with the product again becoming contaminated with
L. monocytogenes after processing (CDC, 2000). In 2000, almost 2,300 cases of food-
borne listeriosis were reported in the United States at an estimated cost of $2.33 billion
(~$ 1 million /case), making L. monocytogenes the second most costliest food-borne
pathogen known to date after Salmonella (S 2.38 billion) (U SDA-FSIS 2001b).

Two other pathogens have emerged as major public health concerns. Escherichia
coli 0157:H7 has been highly publicized due to outbreaks of illness associated with
ground beef (Ostroff et al., 1990, Bell et al., 1994, Mead et al., 1997, Tuttle et al., 1999)
and dry-cured salami (Tilden et al., 1996). Unlike most food-borne pathogens, E. coli

0157:H7 shows high tolerance to acidic environments. Transfer of E. coli 0157:H7 from

contaminated meat or utensils to other foods such as fresh fruits and vegetables is also a
major concern (CDC, 1995). The annual cost of the 62,458 E. coli 0157:H7 infections

reported in 2000 was estimated at $659.1 million (~$10,500/case) (U SDA-F SIS, 2001b).

Salmonella is the second most frequently reported cause of foodbome illness in
the United States after Campylobacter. An estimated 1.34 million cases of salmonellosis
occur annually, resulting in 600 deaths. The annual economic cost of foodbome
salmonellosis was estimated at 2.38 billion dollars (U SDA-FSIS 2001b). The incidence
of salmonellosis appears to be rising both in the US. and other industrialized nations. S.
enteritidis isolations from humans increased dramatically during the early to mid 1990’s
particularly in the northeast United States (6-fold or more). Salmonella is most
commonly found in meat, poultry, eggs and water, with kitchen surfaces also a common

breeding ground for Salmonella.

Salmonella Typhimurium DT104, a multiantibiotic resistant strain, is also
emerging as a serious threat to public health (Glynn et al., 1998). Food-bome
transmission of S. Typhimurium DT104 has been documented for several meat-related
outbreaks; suspected vehicles included roast beef, ham, pork sausage, salami sticks,
cooked meats, frozen sausage samples, and chicken legs (Davies et al., 1996;
Anonymous, 1996). In England, 17% of 786 fresh or frozen sausage samples yielded
Salmonella spp., including S. Typhimurium DT104 (Nichols and de Louvois, 1995).
Sharma et al. (2001) also recovered S. Typhimurium DT104 at several points in pork
production. These findings indicate that meat products may pose a serious health risk if

not cooked and handled properly.

Post-processing pasteurization is one means for minimizing post-processing
contaminants on meat products. Using this strategy, packaged products are individually
pasteurized by heat or other means (e. g. high pressure, UV) to inactivate surface bacteria.
However, some of these treatments makes the products organolepticaly unacceptable to
consumers (Farber and Peterkin, 1999). Alternatively, various antimicrobial dips and
sprays have been applied to ready-to-eat foods to minimize microbial growth (Robach
and Sofos, 1982; Cunningham, 1979). However, their effectiveness over time is limited
because the preservatives diffuse into the food, allowing surface organisms to grow.
Therefore, it is important to prevent or control migration of these preservatives. Guilbert
and his research group have assessed the ability of edible films to control the diffusion of
sorbic acid and potassium sorbate (Guilbert, 1988; Giannakopoulus and Guilbert, 1986;
Guilbert et al., 1985). In another study, lactic acid-treated casein films containing sorbic
acid (Guilbert, 1988) retained 30% of the sorbic acid at the surface after 30 days of
storage at 95% RH, whereas all of the sorbic acid with no casein film application on
control samples diffused after 24 hours of storage. They confirmed that the edible film
matrix entrapped antimicrobials or other food additives and reduced diffusion dming
storage.

Research on edible films as packaging materials has increased because of two
potential that these films may improve overall food quality, extend shelf life, and
improve economic efficiency of packaging materials. In addition, environmental
concerns regarding disposal of fluid whey and increased production of non-biodegradable
packaging materials have prompted interest in the development of edible films from

whey protein isolate and whey protein concentrate as well as wheat gluten, soy protein

isolate, zein protein and others. Previously, coating foods with edible films provided
only barrier and protective functions. However, incorporating various substances into
packaging material can improve its fimctionality. In addition to acting as a barrier against
mass diffusion (moisture, gases, and volatiles), edible films can also be used as carriers
for a wide range of food additives such as antimicrobial agents, flavoring agents,
antioxidants, and colorants. Incorporating antimicrobial compounds such as nisin,
pediocin, benzoic acid, sorbic acid/propionic acid, or lysozyme into edible films or
coatings is another means of enhancing the safety and shelf-life of products subject to
surface contaminants such as ready-to-eat meats and cheeses. For example, soy protein
and corn zein films containing pediocin or nisin and calcium alginate coatings containing
organic acids were shown to inhibit the growth of pathogenic (e. g. L. monocytogenes, S.
Typhimurium and E. coli 0157:H7) and spoilage bacteria (Lactobacillus plantarum) on
meat surfaces (Sirugusa and Dickson, 1993; Dawson et al., 1997; Padget et al., 1998). In
addition, coating with an antimicrobial whey protein solution containing propionic/sorbic
acid prevented growth of L. monocytogenes on frankfiirters (McDade et al., 1999).
However, non-uniformity of the coating on frankfuiters after dipping, draining and
drying would likely produce a less effective antimicrobial barrier as compared to pre-cast
films. Whey protein isolate (WPI) edible films containing antimicrobials would be
uniform in thickness. Consequently, these films would be better suited to inhibit post-
processing surface contaminants such as L. monocytogenes.

The underlying hypothesis for this research was that low pH whey protein-based
edible films (pH 5.2) containing sorbic acid (SA) or p-aminobenzoic acid (PABA) could

be developed to inhibit L. monocytogens, E. coli 0157:H7 and S. Typhimurium DT104

both in a model system and on hot dogs when used as heat sealed tubular casings and

extend product shelf-life. The specific objectives of this research were as follows:

(1)

(2)

(3)

(4)

(5)

Develop a series of low pH whey protein isolate (WPI) edible films (pH 5.2)

containing p-aminobenzoic acid or sorbic acid and determine water vapor

permeability and mechanical (tensile strength and elongation) properties.

Test the antimicrobial properties of these films against L. monocytogenes, E.

coli 0157:H7 and S. Typhimurium DT104 on a laboratory medium.

Compare the barrier and mechanical properties of these films to commercial

collagen and cellulose films and modify the mechanical and barrier properties

if needed using heat curing.

Assess the ability of these films to retain their desirable antimicrobial and

mechanical properties while in direct contact with slices of bologna and

summer sausage during 21 days of refrigerated storage.

Develope WPI hot dog casings containing PABA, SA, and PABA:SA by

heat-sealing the aforementioned films into a tubular shape in order to:

a) Test the ability of WPI casings containing PABA, SA or PABA:SA to
inhibit L. monocytogenes on surface-inoculated hot dogs.

b) Determine the ability of the WPI casings to retain their desirable
mechanical properties during hot dog manufacture.

c) Assess the impact of different casings on storage stability of hot dogs
based on numbers of total aerobic mesophilic bacteria, lactic acid
bacteria, yeast and mold, 2-thiobarbutiric acid (TBA) values, purge loss,

shear force, color, pH, chemical analysis and sensory evaluation.

CHAPTER 1

LITERATURE REVIEW

1.1. EDIBLE FILM
1.1.1. Definition and historical background

Edible films or coatings are defined as continuous matrices that can prepared from
proteins, polysaccharides, and/or lipids. These films and coatings have the potential to
increase food quality and reduce food-packaging requirements since they act as mass
transfer barriers to moisture, oxygen, lipids and solutes and can also carry a wide range of
food additives including vitamins, colorants, flavoring agents, preservatives and
antimicrobial agents.

Historically, yuba, the first freestanding edible film, was developed in Japan from
soymilk during the 15th century and used for food preservation (Guilbert and Biquet,
1996). Edible coating for food products date back even further with waxes applied to
oranges and lemons in China to retard water loss during the twelfth century (Hardenberg,
1967). During the sixteenth century, coating food products with fat (e. g. lard) was used to
control moisture loss in foods in England (Labuza and Contrereas-Medellin, 1981). Hot
melt paraffin waxes have been used to coat citrus fruits in the United States since the
19308, and camauba wax and oil-in-water emulsions have been used for coating fresh
fruits and vegetables since the 19505 (Kaplan, 1986). Currently, edible films and coatings
find use in a variety of applications, including casings for sausages, and chocolate

coatings for nuts and fruits.

1.1.2. Formation of edible films
1.1.2.1. Components of edible films

Edible films typically contain three major compounds; proteins, polysaccharides,
and lipids. Proteins used in the edible film include wheat gluten, collagen, corn, soy,
peanut, casein and, whey protein (Kester and F ennema, 1986). Polysaccharides
incorporated in edible films have included alginate, dextrin, pectin, carrageenan and
cellulose derivatives (Greener and F ennema, 1994). Suitable lipids include waxes,
acylglycerols, and fatty acids (Greener and Fennema, 1993; Park et al., 1994; Debeaufort
and Voiley, 1995). In addition, composite films containing both lipid and hydrocolloid
components have been developed.

Plasticizers can be added to film-forming solutions to enhance properties of the
final film. Plasticizers will decrease brittleness and increase flexibility of the film, which
is important for packaging applications. Common food-grade plasticizers include
sorbitol, glycerol, mannitol, sucrose, and polyethylene glycol. Plasticizers used for
protein-based edible films decrease protein interactions, and increase both polymer chain
mobility and intermolecular spacing (Lieberman and Gilbert, 1973). Plasticizers must be
small molecules with low molecular weights and high boiling points that are highly
compatible with the polymers (Banker, 1966). The type and concentration of the
plasticizer influences the properties of protein films (Gennadios et al., 1994a; Cuq et al.,
1997; Gueguen et al., 1998). When large amounts of plasticizer are used, mechanical
strength, barrier properties, and elasticity decrease (Gontard et al., 1993; Park et al.,

1994; Cherian et al., 1995; Debeaufort et al., 1997; Galietta et al., 1998).

Crosslinking agents are used to improve water resistance, cohesiveness, rigidity,
mechanical strength and barrier properties (Guilbert, 1995; Marquie et al., 1995;
Remunan-Lopez et al., 1997). Commonly used covalent crosslinking agents include
glutaraldehyde, calcium chloride, tannic and lactic acids. Exposure to ultraviolet light
will increase the cohesiveness the protein films through the formation of crosslinks
(Brault et al., 1997). Alternatively, enzymatic crosslinking treatments with

transglutarninases or peroxidases can be used to stabilize films.

1.1.2.2. Film Formation Techniques

Many techniques have been developed for forming films. These include
coacervation, thermal gelation, solvent removal and solidification of melt. In
coacervation, two solutions of oppositely charged hydrocolloids are combined, causing
interactions and precipitation of the polymer complex. Solvent removal is another
common means of forming hydrocolloid edible films. In this process, a continuous
structure is formed and stabilized by the interactions between molecules through the
action of various chemical or physical treatments. Macromolecules in the film-forming
solution are dispersed in a solvent medium such as water, ethanol or acetic acid that
contains several additives (plasticizers, crosslinking agents, solutes). The film forming
solution is then cast in a thin layer, dried, and detached from the surface.

In preparing some types of protein films (whey protein, casein, soy protein, wheat
gluten), the macromolecule solution is heated for protein gelation and coagulation which
involves denaturation, gelification or precipitation, followed by rapid cooling of

hydrocolloid solution. Intrarnolecular and intermolecular disulfide bonds in the protein

complex are cleaved and reduced to sulfhydryl groups during protein denaturation
(Okamota, 1978). When the film-forming solution is cast, disulfide bonds are reformed
which link the polypeptide chains together to produce the film structure with hydrogen
and hydrophobic bonds also contributing to film structure.

Another common film-forming technique is melting followed by solidification.
Solidification of the melt by cooling is commonly used to prepare lipid films. Wax films
can also be formed by casting molten wax on a dried film of methylcellulose and then

dissolving away the methylcellulose film (Donhowe and Fennema, 1993).

1.1.3. Protein-Based Edible Films
The proteins that have been used most extensively for production of films and
coatings include whey protein, casein, corn zein, soy protein, gluten, collagen, and

gelatin.

1.1.3.1. Whey Protein Films
1.1.3.1.1. Composition and Structure

Whey protein is the protein that remains in milk serum after acid/rennet
coagulation of fluid milk (de Wit, 1989; Morr and Ha, 1993). Whey protein consists of
five different proteins: B-lactoglobulin, a—lactalbumin, bovine serum albumin, proteose-

peptones, and immunoglobulins (Kinsella and Whitehead, 1989).

B-Lactoglobulin (B-Lg) which comprises 50-75% of whey protein, is a globular
protein with a molecular mass of approximately 18,300 Da (Eigel, 1984). B-Lg consists

of 162 amino acid residues and contains two disulfide groups, one free sulfhydryl group,

and several hydrophobic groups located in the interior of the globular structure (Brunner,
1977). The thiol group is particularly important since it facilitates molecular thiol-
disulfate interchange reactions which allow formation of intermolecular disulfate-bonded
dimers and polymers upon heating (Kinsella, 1984). The structure of B-Lg is pH
dependent, with this protein existing as a dimer in solutions above pH 5.2. Below pH 3.5
and above 7.5, the dimer dissociates to a monomer, and between pH 3.5 and 5.2 the
dimer polymerizes to an octomer. B-Lg undergoes time and temperature dependent
denaturation reactions at temperatures above 65°C, which result in a general molecular
expansion, exposure of the internal SH group, and hydrophobic and s-NHZ groups
(Brunner, 1977; Kinsella, 1984). Structurally, B-Lg has about 15% helix, 43% B-sheet,

and 15-20% B-turns (Papiz et al., 1986).

a-Lactoalbumin (on-La) which comprises about 19% of total whey protein
(Dybing and Smith, 1991), is another globular protein that contains 123 amino acid
residues and four disulfide bonds. It has a molecular weight of 14,000 Da. The secondary
structure of this molecule is composed of 30% a- helix, 9% B-sheet, and 61% unordered
structure (Alexandrescu et al., 1993). Conformational changes occur in a-La at pH 4 with
the molecule losing Ca+2 which is tightly bound at higher pH. a-La is denatured at
65.2°C and pH 6.7 with 80 to 90% of this denaturation reversed afier cooling. This
reversibility is lost if the native S-S bonds are broken, for example, by heat-induced thiol-

disulfate interchange reactions between a-La and B-Lg (de Wit, 1989).

10

1.1.3.1.2. Film Formation

Film production requires heat denaturation since the hydrophobic and SH groups
are deep within the globular whey protein. Denaturation changes the three-dimensional
protein structure and exposes internal SH and hydrophobic groups (Shimada and Chefiel,
1998), which promote formation of new intermolecular S-S and hydrophobic bonds
during drying (McHugh and Krochta, 1994a). Film formation is favored in more alkaline
film solutions since SH reactivity increases at pH > 8 (Kella and Kinsella, 1988; Baneijee
and Chen, 1995). Glycerol, sorbitol and polyethylene glycol have been commonly used
as plasticizers to produce whey protein films that are transparent, bland, and flexible, and
have excellent oxygen, aroma and oil barrier characteristics. However these films are
poor moisture barriers due to their hydrophilic character (Table 1.1). Incorporation of
lipids, including acetylated monoglycerides, waxes, fatty alcohols and fatty acids, into
WPI film decreases water vapor permeability (WVP) by increasing hydrophobicity
(McHugh and Krochta 1994b; Sherwin et al., 1998; Kim and Ustunol, 2001). Addition of
beeswax and fatty acids to WPI film improves moisture barrier properties more than fatty
alcohols with the longer chains more effectively reducing WVP (McHugh and Krochta,
1994c). Sherwin et a1. (1998) reported that particle size in the emulsion film increased
with increasing fatty acid chain length from C12 to C22. McHugh and Krochta (1994c)
showed that incorporation of large lipid particles decreased WVP of emulsion WPI film

due to interactions at the protein-lipid interface or the dispersed phase.

WPI film properties are affected by the extent of intermolecular disulfide bonding
and plasticizers competing for protein chain-to-chain hydrogen bonding. Plasticizer

content and relative humidity (RH) both have an exponential effect on film permeability.

ll

Table 1.1. Water vapor permeability (WVP) and oxygen permeability (OP) of edible

 

 

films.
Film WVP OP References
g.mm/m2.d.kPa cm3.p.m/m2.d.kPa
Coll - <0.O4a Lieberman and Guilbert
(1973)
Zein 12-24 a 1 1.8 ° Gennadios et a1. (1993),
Parris and Coffin (1997)
WG 53 b 3.9-6.1d Gennadios et a1. (1993)
SP1 72-154 a 1.6-4.5 ° Park and Chinnan ( 1990),
Gennadios et al. (1993)
WPI 62.0- 70.2 c 18.5 —76.1° Krochta (1994)
Casein 45.2 d 1.8 a Chick and Ustunol (1998)
Alginate 42.2 e - Parris et al. (1995)
Chitosan 1.6 f 90.2 ° Caner et a1. (1998)
MC 7.7 ‘ 187.4 ° Park and Chinnan (1994)
HPMC 9.5 f 297.3 ° Park and Chinnan (1994)
Pectin 41.4 f 57.0 c Wong et al. (1992)
Starch 220 g 2.9 c Allen et al. (1963)
Beeswax 0.05 f 1.3 c Kester and Fennema (1989)
Candelilla 0.02 f 0.3 c Kester and Fennema (1989)
Camauba 0.03 f , 0.2 c Kester and Fennema (1989)
Microcrystalline 0.03 f 2.2 c Kester and Fennema (1989)
Acetylated 20.04 — 53.7 f - Lovegren and F euge (1954)
monoglycerides

 

Coll: Collagen, HPMC: Hydropropionate methylcellulose, MC: Methylcellulose, WG: Wheat gluten, WPI:
Whey protein isolate, SP1: Soy protein isolate

WVP: Temp. (°C), RH (%) a= 25°C, 50/ 100% RH, b= 21°C, 85/0% RH, c= 25°C, 0/79% RH, d= 378°C,
100/90% RH, e=30°C, 0/ 100% RH, f=25°C, 0/ 100% RH, g=38°C, 30/ 100% RH

OP: Temp. (°C), RH (%), a= 23°C, 0% RH, b = 25°C, 63% RH, c= 25°C, 0% RH, d=23-38°C, 0% RH, e=
23°C, 50% RH

At film compositions where glycerol- and sorbitol—plasticized WPI films have equal
mechanical properties, sorbitol-plasticized films have lower oxygen permeability (OP)
(McHugh and Krochta, 1994d, e). Inhibition of intermolecular disulfide bond formation
with sodium dodecyl sulfate (SDS) increases WPI film solubility, extendibility and
flexibility, while having little effect on WVP. Inhibition of sulfliydryl-disulfide
interchange with N-ethyl maleimide reduces WPI film solubility and elongation, with
little effect on other film mechanical properties or WVP. Reduction of disulfide bonds
with cysteine has no effect on WVP of WPI films.

Tensile strength of WPI films is comparably higher than most other protein-based
films; with the opposite true for elongation (Table 1.2). Heat curing is commonly used to
cross-link synthetic polymers and improve mechanical and barrier properties (El-Hibri
and Paul, 1985; Perkins, 1988). Miller et al. (1997) also showed that heat curing of
glycerol-plasticized WPI films increases film strength, while decreasing film
extendibility, flexibility and WVP. Exposing free sulfliydryl groups promotes

intermolecular disulfide bond formation to produce insoluble films.

1.1.3.2. Corn zein
1.1.3.2.1. Composition and structure

Zein, the prolamin (soluble in 70% ethanol) fraction of corn gluten, comprises
approximately 70% of corn gluten. This protein is soluble in aqueous ethanol and

insoluble in water due to higher levels of nonpolar amino acids (i.e. leucine, proline, and

13

alanine) (Shewry and Miflin, 1985). Zein is also rich in glutamine, the amide derivative
of glutamic acid, which promotes protein association by hydrogen bonding (Reiners et
al., 1973; Wall and Paulis, 1978). Corn zein can be fractionated into three distinct classes:
a-zein, B-zein, and y-zein (Esen, 1987). a-Zein and B-zein constitute 75-85 and 10-15%
of total zein and are comprised of polypeptides with molecular masses of 21,000-25,000
and 17,000-18,000, respectively. Finally, y-zein is made up of a proline-rich polypeptide
of 27,000 and constitutes 5-10% of total zein.

Zein is produced commercially by extracting corn gluten with 80-90% aqueous
isopropyl alcohol containing 0.25% sodium hydroxide at 60-70°C (Reiner, 1973). The
centrifugally clarified extract is chilled to precipitate the zein with additional extractions

and precipitation used to increase purity.

1.1.3.2.2. Film Formation

Preparation of zein films generally involves casting alcohol solutions on inert, flat
surfaces. Formed films are peeled after the solvent has evaporated (Gennadios and
Weller, 1990). Films also have been prepared from acetone solutions (Yarnada et al.,
1995). Zein films are naturally brittle, and therefore, plasticizers such as oleic acid (Kanig
and Goodman, 1962), glycerol (Mendoza, 1975; Aydt and Weller, 1988) and lactic acid
(Wu, 1995) are needed to improve their flexibility. An alternative method to prepare zein
films involves plasticization of zein by forming an emulsion with oleic acid followed by
precipitation of the protein-lipid mixture to form a soft moldable resin (Lai et al., 1997).
The plastic resin then can be stretched over rigid frames to obtain thin membranes that set

into flexible films.

14

 

Films formed upon solvent evaporation contain hydrophobic, hydrogen and
disulfide bonds and have been characterized as tough, glossy and scuff-resistant (Pomes,
1971). Cross-linking agents such as aldehydes will improve moisture barrier and tensile
properties of zein films (Szyperski and Gibbons, 1963). Epoxy resins also increase
tensile strength of zein films by crosslinking between epoxy groups and phenolic, and
aliphatic hydroxyl protein groups (Howland, 1961; Howland and Reiners, 1962; Yamada
etaL,1995)

The oxygen permeability of zein films is quite low when compared to synthetic
films such as low density polyethylene (LDPE) and polyester film; however, oxygen
permeability of zein film is greater than that of collagen (Hanlon, 1992; Krochta, 1997).
Higher plasticizer levels will increase the oxygen permeability of zein films. Films
prepared from zein have lower or similar WVP compared to methylcellulose, ethyl
cellulose, hydroxypropylmethyl cellulose, and hydroxypropyl cellulose under similar
conditions (Table 1.1).

Zein films produced using different solvents possess distinct properties. For
example, Yamada et a1. (1995) found that zein films prepared by dissolution in 30%
acetone film showed lower WVP than when dissolved in 20% ethanol. They also reported
that film strength increased linearly as film thickness increased except for films prepared
from the aqueous acetone solution with 1, 2- Epoxy-3-chlor0propane. At similar
plasticizer levels, zein film appears to have tensile strength (TS) and percent elongation
(% E) similar to collagen film (Table 1.2).

The concentration of plasticizer greatly affects the mechanical and water vapor

barrier properties of zein protein films (Aydt et al., 1991; Butler and Vergano,1994).

Table 1.2. Tensile strength (TS) and percent elongation (% E) of edible films (23°C,

 

 

50% RH).
Film TS (Mpa) % E References
Coll:Sor:Gly (3.4:0.8:l) 8.1 25 Hood (1987)
Coll:Sor:Gly(8.8:0.8:l) 9.1 38 Hood (1987)
Corn ZeinzGly (120.5) 2.7—15.7 43-198 Gennadios et al. (1993),
Aydt et al. (1991)
WGzGly (2.5:1) 4.4 194.7 Gennadios et al.(1993)
SPI:Gly (2:1) 4.3 78 Brandenburg et a1. (1993)
WPI:Gly (5.7:1) 29.1 4.1 Krochta (1994)
WPlzGly (2.3:1) 13.9 30.8 Krochta (1994)
WPlzGly (1.521) 18.2 5.0 Krochta (1994)
Casein: Gly (1 .4:1) 4.5 223 Chick and Ustunol (1998)
AlginatezGly (2:0.7) 2.5 7.9 Parris et al. (1995)
Pectin: Gly (3:0.5) 2.3 5.0 Parris et al. (1995)
Chitosan 6.3 -— 31.8 14-70 Caner et al. (1998)
MC 12.5 20 Debeaufort and Voilley
(1997)
Starch:ethylene-acrylic acid 23.9 260 Otey et al. (1977)
(2:0.8)
Pectin:Starch: Gly (1:1:0.5) 27-34 1.8-l3 Coffin and Fishman
(1994)

 

Coll: Collagen, Gly: Glycerin, MC: Methylcellulose, WG:

protein isolate, SPI: Soy protein isolate, Sor: Sorbitol

 

Wheat gluten, WPI: Whey

When Park et al. (1994) evaluated the impact of two plasticizers, glycerin (0.36 ml/g
protein) and polyethylene glycol (PEG) (0.39 ml/g protein), on the properties of zein
film, % E of corn zein films containing glycerin or PEG was 3 and 94%, respectively. TS
of zein films reportedly increased from 13.4 to 25.8 MPa during 20 days of storage;
however, percent elongation decreased from 76 to 12%. WVP of these zein films also

decreased from 0.59 to 0.41 ng.m/m2.s Pa as the ratio of glycerin to PEG decreased.

1.1.3.3. Casein
1.1.3.3.1. Composition and Structure

Casein, the major protein in milk, represents 80% of total milk protein (Dalgleish,
1989). Casein consists of three principal components, a, B, and K-casein, and a minor
component, y-casein, which together form colloidal micelles in milk containing large
numbers of casein molecules that are stabilized by calcium-phosphate bridging (Kinsella,
1984). Caseins are phosphoproteins that precipitate at pH 4.6 and 20°C.

The a-caseins have an approximate molecular weight of 23,500 and an isoelectric
point of pH 5.1 (Xiong, 1997). They are more phosphorylated than other caseins, more
calcium sensitive than B-casein and contain more charged residues and fever hydrophobic
residues than B- casein (Dalgleish, 1989).

B-casein, which comprises up 30-35% of the total casein content in milk, has a
molecular weight of 24,000 and an isoelectric point of pH 5.3 (Xiong, 1997). The last 50
residues of the terminus are charged, whereas the rest of the molecule is highly

hydrophobic (Dalgleish, 1989). B-casein shows temperature, concentration, and pH

17

dependent association-dissociation. At neutral pH and high temperatures, B-casein
associates into threadlike polymers (Dalghleish, 1989).

K-casein, which comprises approximately 15% of the total casein fraction, has a
molecular weight of 19,000 and an isoelectric point of pH 3.7 to 4.2 (Xiong, 1997). Being
calcium sensitive, K-casein associates with 011- and B-caseins in the presence of calcium
to form thermodynamically stable micelles. Apolar residues in K-casein are concentrated
at the N-terminus with the charged portion being near the C-terminus. The
hydrophobicity of K-casein is between that of a- and B-caseins (Dalgleish, 1989).

The negatively charged K-casein is cleaved by the enzyme rennin used to
coagulate milk in cheese production. When K- casein is cleaved, the micelle structure is
stabilized and the casein precipitates.

Casein contains low levels of cysteine, and thus few disulfide cross-linkages
which produces an open random structure. Caseinates function as good emulsifiers and

foaming agents because of even hydrophilic and hydrophobic amino acid distribution.

1.1.3.3.2. Film Formation

Casein films are formed from aqueous solutions of casein. No further treatment is
necessary because of their random coil structure and propensity for extensive hydrogen
bonding. Hydrophobic, ionic, and hydrogen bonding involve interactions in the film
matrix with the surfactant nature of casein making it very suitable for producing emulsion

films that are transparent, flavorless and flexible.

l8

1.1.3.3.2. Film properties

The inherently poor water barrier properties of caseinate films can be improved
by treatment with lactic or tannic acid (Guilbert, 1986). Films can also be prepared from
sodium or calcium caseinate without addition of plasticizer. Addition of plasticizers
(glycerol or sorbitol) to casein film solutions at ratios of 0.6:1, 1:1, and 1.421 decreased
tensile strength, but increased percent elongation, with glycerol being more effective than
sorbitol (Chick and Ustunol, 1998). Calcium caseinate films have lower WVP compared
to sodium caseinate films. However, when treated with calcium chloride sodium
caseinate films have even lower WVP, likely because of more effective calcium cross-
linking (Avena-Bustillos and Krochta, 1993). Incorporating various lipids, waxes, and
fatty acids into the caseinate film solution also has been examined for reduction of water
vapor permeability, with beeswax being more effective than paraffin or camauba
(Krochta et al., 1990; Ho, 1992). Caseinate film formation at the isoelectric point of
casein insolubilizes the film and also reduces water vapor permeability by about one-half
compared to films at higher pH levels (Krochta et al., 1988). Overall, caseinate films
have moisture barriers that are similar to wheat gluten and soy protein films and
somewhat greater moisture barriers than corn zein films (Table 1.1).

Additional modification techniques, including UV light and y-irradiation, also can
be used to improve the properties of casein films. The formation of bityrosine with y-
irradiation (16-64 kGy) or UV light reportedly improved tensile strength and WVP
properties of calcium caseinate film (Brault et al., 1997; Ressounany et al., 1998;

Mezhgheni et al., 1998). Furthermore, addition of CaClz increased the fluorescence signal

19

 

of bityrosine by shortening molecular distances between polypeptides, thus aiding the

formation of bityrosine.

1.1.3.4. Wheat Gluten
1.1.3.4.]. Composition and Structure

Wheat kernels contain 8-15% protein based on dry weight (Kasarda et al., 1976).
Gliadin and glutenin are the main wheat endospenn storage proteins, comprising 85% of
wheat flour protein, the remaining 15% consisting of various albumins, globulins and
smaller peptide structures (Holme, 1966). While gliadin is soluble in 70% ethanol,
glutenin is not. By hydration gliadin and glutenin form a colloidal complex known as
gluten (Pomeranz, 1988). These properties are used to produce films from gluten protein.

The amino acid composition of wheat gluten is characterized by high levels of
glutamic acid and low levels of lysine and other basic amino acids (Kasarda et al., 1976;
Lasztity, 1986). The amide group of glutamine promotes hydrogen bonding between
gluten chains. The relatively high amounts of nonpolar amino acids, such as proline and
leucine cause wheat gluten insolubility in water at neutral pH (Krull and Inglet, 1971).
Both gliadin and glutenin contain disulfate bonds. Disulfate bonds link glutenin chains
together to produce polymers of high molecular weight. The gliadin-glutenin complex

involves both covalent and noncavolent bonding in the film matrix.

1.1.3.4.2. Film Formation

Wheat gluten (WG) films are produced by deposition and drying of wheat gluten

dispersions. Aqueous ethanol is the most common solvent used in film-forming solution.

20

Mechanical mixing and heating under acidic or alkaline conditions are used to form
homogeneous gluten dispersions (Gontard et al., 1992; Gennadios et al., 1993a). Covalent
disulfide bonds are also important in wheat gluten film formation. Disulfide bonds in
gluten are reduced to sulthydryl groups in wheat gluten dispersed under alkaline
conditions (Okamoto, 1978). During casting of film-forming solutions, disulfide bonds
are reformed by reoxidation in air and sulfliydryl-disulfide interchange reactions.
Reformed disulfide bonds link polypeptide chains, to produce the film structure. Hlynka,
(1949), Beckwith et al. (1965), and Wall et a1. (1968) describe the reduction and
reoxidation of disulfide bonds while Steward and Mauritzen (1966) and McDermott et al.

(1969) describe sulthydryl-disulfide interchange reactions in wheat gluten.

1.1.3.4.3. Film Properties

Mechanical and barrier properties of cast WG films have been reported by several
investigators (Krull and Inglett, 1971; Aydt et al., 1991; Gontard et al., 1992; Gennadios
et al., 1993b; Park et al., 1994; Rayas and Ng, 1997; Rayas et al., 1998). Extensive
intermolecular interactions in WG result in quite brittle films, which require plasticizers.
Plasticizer molecules mediate between polypeptide chains, which decreases the rigidity
of the film structure (Wall and Beckwith, 1969). However, increasing film flexibility by
raising the plasticizer content (glycerin) reduces film strength and WVP (Gontard et al.,
1993)

Oxygen permeability of wheat gluten is similar to commercial nylon (Rayas et al.
1998). Unlike WVP, at comparable plasticizer content and test conditions, the oxygen

permeability of gluten films prepared from alkaline solutions is an order of magnitude

21

lower than oxygen permeability of zein films. The effects of subjecting wheat gluten
films to several treatments, including lactic acid, calcium chloride have been investigated
by Gennadios et al. (1993c). Soaking the film in calcium chloride and in buffer solution
at the isoelectric point of wheat gluten reportedly increased tensile strength (47 and 9%,
respectively) and decreased WVP (14 and 13%, respectively).

Effects of replacing a portion of the wheat gluten with keratin, soy protein, com
zein, or soy protein with cysteine on film properties was also studied (Gennadios et al.
1993a, b; Were et al., 1999). The addition of hydrolyzed keratin into a wheat-gluten
based film solution reportedly lowered oxygen permeability by 83%, WVP by 23%, and
TS by 35%, but increased % E by 32%. Replacing 30% of the wheat gluten with SP1
decreased oxygen permeability, % E, and WVP by 40, 9, and 16%, respectively, while
increasing TS by 69%. Corn zein addition had no effect on OP, but decreased WVP and
% E by 23 and 37%, while increasing TS by 58%. Adding cysteine (1%) to WG: SPI
(4:1) increased the disulfate content of the film forming solution and thus increased TS,
but had no affect on WVP and oxygen permeability (Were et al., 1999).

Properties of wheat gluten films are affected by the pH of the film-forming
solution (Gontard etal., 1992; Gennadios eta1., 1993a). Gontard et al. (1992) showed that
wheat gluten films produced at acidic conditions had significantly lower WVP than wheat
gluten films produced at alkaline conditions (Table 1.1). However, Gennadios et al.
(1993a) reported that films prepared under alkaline conditions had greater TS than films

prepared under acidic conditions with %E and WVP not affected by the pH difference.

22

1.1.3.5. Soy Protein
1.1.3.5.1. Composition and Structure

Soybeans contain 38-44% protein, most of which is insoluble in water but soluble
in dilute neutral salt solutions. Soy protein is a globular protein and is separable into four
different fractions: ZS, 78, 118 and 158 (Wolf and Smith, 1961) with the 7S and 118
fractions comprising about 37% and 31% of the total protein, respectively (Wolf et al.,
1962). Soy protein is rich in asparagine and glutamine residues. Both 7S and 118
fractions are tightly folded proteins, with the former containing two or three cystine
groups and the later containing 20 intermolecular disulfate bonds (Gennadios et al.,
1993c)

Protein in the form of meal is one of the typical end products from industrial
soybean processing. Protein meal can be further concentrated for the production of soy
protein concentrates and soy protein isolates, which contain at least 70% and 90% protein

on a dry basis, respectively.

1.1.3.5.2. Film Formation

Edible films from soybeans have been traditionally produced in the Orient on the
surface of heated soymilk. In these films, lipids and carbohydrates are incorporated with
protein. Films obtained from soymilk are known as “yuba” in Japan, “tou-fu-pi” in
China, “kong kook” in Korea, and “fu chock” in Malaysia (Snyder and Kwon, 1987).

The mechanism for forming these films involves polymerization of heat-
denatured protein followed by surface dehydration of the soymilk emulsion (Farnum et

al. 1976). Soy protein films contain a protein matrix formed by heat-catalyzed protein—

23

protein interactions that result in disulfate, hydrogen and hydrophobic bonds. Fukushima
and Van Buren (1970) showed that the mechanism of polymerization involves
intermolecular disulfate and hydrophobic bonds. Film formation is favored in more
alkaline soymilk solutions.

Soymilk used in making these films is prepared from soybeans. In one soymilk
manufacturing procedure, (Wu and Bates, 1972) soybeans that were presoaked in water
for 1 hour at 65°C were by drained, ground with water, passed through a screw expeller
and finally followed by a clarification to produce soymilk with a pH of about 6.7.

Guilbert (1986, 1988) produced films by casting aqueous solutions containing
10% soy protein isolate and 5% glycerin as a plasticizer. Films were characterized as
flexible, smooth and transparent. Production of films and coatings from soymilk and soy
protein has been reviewed by Gennadios and Weller (1991). Heating and alkaline
conditions favor soy protein polymerization by unfolding the protein secondary structure
and exposing sulfhydryl and hydrophobic groups. Sian and Ishak (1990) and Gennadios
et al. (1993) reported that film formation was possible only within pH 1-2 and pH 6-12.
The optimum pH range for producing soymilk films is between 7.0 and 8.0, with
darkening of the film occurring above pH 8.0 (Wu and Bates, 1972). Brandenberg et al.
(1993) produced film by heating alkaline solutions of soy protein isolate (5%) and
glycerin (3%) at 60°C for 10 min. These films had a smoother appearance and fewer
insoluble particles, as evidenced by scanning electron microscopy. Heat treatment of SPI
film-forming solutions at 85°C promoted intermolecular cross-linking and produced SP1
films that were smoother and more transparent, and possessed lower WVP and increased

% E, compared to those produced from unheated solutions (Stuchell and Krochta, 1994).

24

The moisture barrier properties of SPI films are similar to wheat gluten films
prepared from alkaline solutions but somewhat inferior to corn zein films (Table 1.1). SP1
films having similar TS to zein and alkaline wheat gluten films have lower E (Table 1.2).
The oxygen permeability of SPI films has been measured, but only at 0% (Brandenburg
et al., 1993, Gennadios et al., 1994). Like wheat gluten films, the oxygen permeability of
SPI films appears to be an order of magnitude lower than the oxygen permeability of zein
films at similar plasticizer levels (Table 1.2).

Soy protein films are transparent and flexible when plasticizer is added, but have
poor water barrier properties (Guilbert, 1986). When the plasticizer content was increased
from 20 to 40% the % E of soy protein films increased from 1.5 to 106%; however,
tensile strength decreased from 15.8 to 2.6 MPa (Cunningham et al., 2000).

Ionizing radiation, such as y-irradiation, cross-links soy protein films, thus
improving their functional properties. Exposing soy protein film to a y-irradiation dosage
of 5-30 ng in the presence of poly(ethylene oxide) or poly(vinyl alcohol) increased
tensile strength (Grorpade et al., 1995; Sabato et al., 2001). Gennadios et al.(1998) also
reported that UV irradiation (103.7 J/mz) improved tensile strength of soy protein films
from 3.7 to 6.1 MPa; however, percent elongation decreased from 124 to 85% with
increasing UV irradiation. Modification of SPI film properties by heat curing at 80 and
95°C was also studied by Gennadios et al. (1994), with this treatment reportedly reducing
WVP, % E, moisture content, and water solubility and increasing TS. Changes in film
properties were attributed to heat-induced cross—linking and lower moisture content

within the film, with the greatest effect observed at 95°C.

25

Cross-linking interactions of calcium chloride or calcium sulfate with SP1 can
also improve tensile strength (Park et al., 2001). In the same study, glucono-delta-lactone
(GDL) incorporation into soy protein increased elongation. In addition, both calcium salts

and GDL-treated SPI films had lower WVP than the SP1 control film.

1.1.3.6. Collagen
1.1.3.6.1. Composition and Structure

Collagen is a fibrous protein generally isolated from hides, tendon, cartilage, bone
and connective tissue (Ballian and Bowes, 1977). This protein is arranged in fibrils which
are packed together to form bundles of parallel fibers having cross-sectional diameters of
20-40 microns (Ballian and Bowes, 1977). Collagen is unique in that every third amino
acid residue throughout most of the structure is glycine, while proline and hydroxyproline
account for about one-fourth of the residues (Harrington and Von Hippel, 1961,
Ramachanran and Rarnakrishnan, 1976). Collagen fibers swell with immersion in alkali,
acid or neutral salt solutions. They are resistant to proteolytic enzymes but are easy
targets for collagenase. When heated, collagen fibers shrink and are converted to gelatin

(Harrington, 1966).

1.1.3.6.2. Film Formation

Collagen films can be produced from animal hides using a dry or wet process.
The dry process involves alkaline treatment of hide corium and acidification to pH 3.0
(Hood, 1987) followed by shredding of acid-swollen coriums to preserve maximum fiber

structure, mixing of acid-swollen fibers to produce a high-solids dough (>12%), addition

26

of plasticizing and cross-linking agents, high pressure pumping and extrusion of dough to
form tubular casings, drying, conditioning, neutralizing and/or providing additional cross-
linking. The wet process consists of acid or alkaline dehairing of hides, deacidification of
hide coriums, grinding mixing of ground collagenous material with acid to produce a
swollen slurry (4-5% solids), slurry homogenization, extrusion into tubular casings,
washing to remove salts, treatment with plasticizing and cross-linking agents and drying.

The tensile strength of collagen is less than that of methyl cellulose, corn zein,
WPI, and Chitosan-based films (Table 1.2). Several techniques have been developed to
improve the properties of collagen films and casings. The strength of collagen casings
can be improved by drying to a moisture content of 15 to 40% in the presence of a
polyhydric alcohol, such as poylethylene glycol, or a salt, preferably sodium chloride
(Kidney, 1970). Treatment with glyceraldehyde was found to promote cross-linking
which increased the strength and thermal resistance of collagen casings (Jones and
Whitmore, 1972). Alkyl diols also improved the mechanical properties of collagen
casings by reducing internal hydrogen bonding while increasing intermolecular spacing
(Lieberman and Gilbert, 1973; Boni, 1988). Furthermore, exposing edible tubular
collagen casings to ultraviolet irradiation (180-420 nm) increased casing strength (Miller
and Marder, 1998).

Exposure to proteolytic enzymes such as papain, bromelain, ficin, trypsin,
chymotrypsin, pepsin and fungal proteose can be used to partially stabilize collagen,
which will improve uniformity in diameter and wall thickness of collagen casings (Fujii,

1967; Tsuzuki and Lieberman, 1972; Miller, 1983).

27

Water vapor pemeability of collagen films can also be altered. Lieberman and
Gilbert (1973) found that formaldehyde cross-linking and chrome tanning significantly
reduced the gas permeability of collagen films. Formaldehyde reacted with collagen by
combining with free amino groups of basic amino acids, whereas chrome tanning

promoted reactions between carboxyl groups of amino acids.

1.1.3.7. Gelatin Films
1.1.3.7.1. Composition and Structure

Gelatin, a protein produced from partial hydrolysis of collagen, ranges in
molecular weight from 3000 up to 200,000 depending on production methods. Gelatin
lacks internal order with the polypeptides configured randomly in aqueous solutions
(Xiong, 1997). The process for hydrolysis of collagen to gelatin includes thermal
denaturation (40°C), which cleaves hydrogen bonds and electrostatic bonds, and
hydrolytic breakdown of covalent bonds (Eastoe and Leach, 1977). Thermally reversible
gels are produced by forming cross-links between amino and carboxyl components of
amino acid residual side groups (Glicksman, 1982).

Raw materials commercially used for gelatin manufacture include pork skin as
well as bovine hide, skin, and bones. A pretreatment or curing step is applied before
gelatin extraction of the raw material. Curing removes impurities and initiates collagen
hydrolysis. Two different curing methods, acid or alkali, produce Types A and B gelatin,
respectively. Type A gelatin with an isoelectric point between pH 7 and 9 is mostly made
from pork skin, while Type B gelatin with an isoelectric point between pH 4.6 and 5.2 is

made from bones and bovine hides (Xiong, 1997).

28

2.1.3.7.2. Film Formation

Gelatin films are prepared by casting aqueous film-forming solutions of 20%
gelatin and 0-10% glycerin (Guilbert, 1986, 1988). Such films are clear, flexible, strong
and oxygen impermeable when the aqueous solution contains plasticizer such as glycerol
or sorbitol.

Gelatin gels dry to produce films with poor moisture barrier and tensile strength
properties. Treatment with lactic or tannic acid improves water-barrier properties
although these films are less flexible and less transparent (Guilbert, 1986). Tensile
strength of gelatin films can be improved by drying 5% solutions at 20°C rather than
60°C, due to a higher degree of crystallization in films dried at the lower temperature
(Bradbury and Martin 1952). In addition, gelatin also reportedly has a configuration that

promotes interchain hydrogen bonds at temperatures less than 35°C (Robinson, 1953).

1.1.4. Polysaccharide-based edible films
Several carbohydrate-based edible films were developed and tested. This review
will address some common carbohydrate films including cellulose-, chitosan-, alginate-,

starch-, pectin-, and dextrin—based edible films.

1.1.4.1. Cellulose Films

Cellulose, composed of linear chains of B-D-glucopyranosyl units joined by
glycosidic bonds (1 —+ 4), is the most abundant organic compound on earth because it is
the principal component of plant cells. Native cellulose is insoluble in water because of

the high level of intermolecular hydrogen bonding, but can be converted into cold water—

29

soluble gums by esterification. In this process, cellulose is reacted with aqueous caustic,
then with methyl chloride, propylene oxide or sodium monochloroacetate to yield
methylcellulose (MC), hydroxypropyl methylcellulose (HPMC) and sodium
carboxymetylcellulose (CMC), respectively. MC, HPMC and hydroxypropyl cellulose
(HPC) are available commercially in powder or granular form in varying molecular
weight. Their relative hydrophilities increase in the order of HPC<MC<HPMC<CMC
(Krumel and Lindsay, 1976).

MC, HPMC and HPC are water-soluble ethers with good film-forming properties.
Dissolution of these compounds is a two-step process involving dispersion and hydration.
The powder must be added slowly to agitated water to separate and sufficiently wet their
surfaces. Another procedure is by dispersing the cellulose ethers in a water miscible
nonsolvent such as glycerin, ethanol or propylene glycol and then adding the slurry to
water.

Films prepared from MC, HPMC and HPC are tough, flexible and transparent
with linear structure (Krumel and Lindsay 1976). The first cellulose film, cellephane, was
developed by Brandenberg in 1908 (Paist, 1958), and some modified cellulose films have
been used as edible films and coating since the 19808 (Kester and Fennema, 1986). The
addition of plasticizers (polyethylene glycol 400, glycerin, propylene glycol) necessary to
improve elasticity of cellulose films (Torres, 1994) does not affect the visual appearance
at concentrations lower than 30% (Donhowe and Fennema, 1993; Park et al., 1993;
Debeaufort and Voilley, 1995a). However, addition of plasticizers during preparation
increases oxygen, water vapor and aroma transfer and sorption within the films and

decreases TS (Debeaufrt and Voiley, 1995).

30

Films based on MC, HPMC and HPC are soluble in water and insoluble in lipids,
and thus have high water vapor permeability (Table 1.1). They are very good oxygen and
hydrocarbon barriers, and their water vapor properties can be improved by adding lipids
or fatty acids (Biquet and Labuza, 1988; Kamper and Fenema, 1984a, b; Park and
Chinnan, 1990; Ayranci and Tune, 2001). Donhowe and Fennema (1992) showed that
MC and CMC prepared with beeswax or acetylated monoglycerides exhibited increased
WVP with increasing RH from 0 to 80% due to hydration and swelling across the entire
film thickness, thus facilitating water movement through the film. The addition of fatty
acids also was shown to improve water vapor barrier properties. For example, Ayranci
and Tune (2001) reported that incorporation of stearic, palmitic or lauric acids (5-40
g/ 100 g MC) in MC-based edible films decreased WVP.

The ability of hydrophobic substances to retard moisture transfer through
cellulose-based films depends on the homogeneity of their final re-partition in the matrix
and/or on the surface. Polo et al. (1992) studied the effect of paraffin oil and paraffin wax
on the moisture barrier pr0perties of three cellulose derivative-based films with different
polarity and porosity prepared using several techniques: emulsion (MC), emulsion plus
coating (MC), and dipping (porous filter paper and nonporous cellophane). Cellophane
films prepared with paraffin oil were more permeable than those prepared with wax.
Films prepared by emulsion and dipping had highly heterogeneous systems with the least
effect on movement of water. The most efficient system, in terms of water vapor
permeability, was obtained using cellophane films with homogeneous re-partition of

paraffin wax independent of thickness and RH.

31

1.1.4.2. Alginate Films

Commercial algin is a sodium salt of alginic acid, a linear polyuronic acid
obtained from brown seaweed. Alginic acid contains two monomeric units, B-D-
mannopyranosyluronic acid and a-L-gulopyranosyluronic acid. Segments containing
only B-D-mannopyranosyluronic units are called M blocks whereas those containing only

a-L-gulopyranosyluronic units are termed G blocks (Whistler and Daniel, 1990; Cottrell
and Kovacs, 1980; McDowell, 1973). These blocks determine the properties of the
polymer. For example, algins containing many G-block units produce high strength, more
brittle and less elastic film. Solutions of sodium alginates are highly viscous. The
calcium alginates are insoluble.

Alginates react with several cations including calcium and sodium to form gels
Calcium ions, the most effective gelling agent (Allen et al., 1963), pull alginate chains
together by ion interactions and interchain hydrogen bonding to produce a three-
dimensional gel network (Grant et al., 1973; King, 1983). Calcium-induced gelation of
alginate is irreversible unless treated with strong chelating agents at an alkaline pH

(Klock et al., 1994).

1.1.4.2.]. Film Formation

Alginates possess good film-forming properties, with glycerol often added as a
plasticizer to reduce brittleness (Glicksman, 1984). Films and coatings made from
sodium alginate solutions by rapid reaction with calcium in the cold contain
intermolecular associations in G-block regions. Film strength and permeability can be

altered by the concentration of calcium, the rate of its addition, pH and temperature

32

(Kester and Fennema, 1986). Homogenous release of calcium ions within an alginate
solution is necessary for uniform gelation. Alteration in pH, temperature and employment
of calcium ions with low solubility can be used to control release of calcium ions in the
alginate suspension (Deasy, 1984). Increasing the acidity of the gel solution with the
hydrolysis of glucono-S-lactone will slow the calcium ion dissociation rate from
insoluble calcium salt. Alginate films prepared under these conditions will be
homogenous, transparent and smooth (Draget et al., 1989) with the pH of the alginate
solution slowly decreasing from 5.6 to 3.8 in 6 h.

Alternatively, soluble calcium salt can be released into an alginate solution at high
temperature with gelation occurring upon cooling (Sime, 1990; Papageorgiou, 1994,
Dominic et al., 1996). Such films are homogenous, but opaque with a rough uneven
surface. Film properties depend on the gel cooling rate. Gelation occurs too rapidly (2 h)
to allow the alginate chains to align for proper cross-linking. Such films have a weak gel
structure because of irregular distribution of calcium crosslinks throughout the film
matrix (Dominic et al., 1996).

Many calcium salts can be used to form alginate gels including chloride, acetate,
lactate, tartarate, gluconate, sulfate, citrate and tri-calcium phosphate (Kester and
F ennema, 1986). Calcium chloride yields the strongest gel coatings (Allen et al., 1963).

Films made by casting an aqueous solution of alginic acid are flexible and
transparent, but water-soluble. Immersing alginate gels in aqueous solutions of
multivalent cations is an alternate method to improve film strength (Kaletunc et al.,

1990). Immersing the film in solutions containing 5 or 10% calcium and zinc for 15

33

minutes produced water insoluble films with tensile strength increasing from 5.6 to 31.0

and 51.67 MPa, respectively (Pavlath e al., 1999).

2.1.4.3. Pectin Films

Pectin, a complex group of structural polysaccharides, is found in plant cells
(Aspinall, 1970). Pectins are composed of D-galacturonic acid with various degrees of
methyl esterification. Low-methoxyl pectins dissolved in aqueous media are capable of
forming gels in the presence of calcium ions (Miers et al., 1953) which react with free
carboxyl groups on adjacent polymer molecules. Hydrogen bonding also strengthens this
association.

Low methoxyl pectin films were first developed and studied in the 1940’s (Coffin
and F ishman, 1994) with calcium or other cations used as cross-linking agents. These
films exhibited good mechanical properties, but had poor folding durability. When
Schultz et al. (1949) evaluated dried low-methoxyl pectinate films, the water vapor
permeability rate was 300 g H2O mil/m2.day.mmHg at 25°C with 50% relative humidity.

Incorporating high amylose pectin into high methoxyl pectin films modified the
mechanical properties of the pectin films (Coffin and Fishman, 1993, 1994). Films made
from high methoxyl lime pectin and high starch had very good mechanical properties.
Adding a high proportion of high amylose starch resulted in only a modest decrease in
dynamic mechanical properties (storage modulus and loss modulus) of the films, and had
a beneficial effect on surface properties.

A two-step technique was used to apply pectinate gel coatings directly to food

surfaces. This procedure is the same as that used in development of alginate coatings. An

34

 

aqueous pectin solution was applied to the surface, followed by treatment with a calcium

solution to promote gelation.

1.1.4.4. Chitosan

Chitosan is derived from chitin, the second most abundant polysaccharide after
cellulose (Lezica and Quesada-Allue, 1990). The polysaccharide chitin, which consists of
repeating units of 1,4 linked 2-deoxy-2-acetamido—B-D-glucose, occurs naturally in the
crystalline state (Minke and Blackwell, 1978). In chitin, the amino group is acetylated,
thus chitin is an amide of acetic acid. Chitosan containing a free diacetylated amino
group is soluble in aqueous acidic solutions (Muzzarelli, 1977; Rinaudo and Domard,
1989). Such acidic solutions containing positively charged chitosan are used in
agriculture as carriers for pesticides (Anon, 1987), in the food industry as adsorbents for

waste and water treatment (Knorr, 1984), and in the area of nutrition as lipid binders

(F urda, 1990).

1.1.4.4.1. Film Formation

Chitosan films can be prepared by dissolving 1% chitosan with glycerol (1 or
2.5%) in acidified aqueous solutions (1% acetic or formic acid) at room temperature
(Wong et al., 1992; Hoagland, 1996; Caner et al., 1998). After casting in petri dishes, the
solution was dried to produce freestanding chitosan films. Films prepared using HCl or
HNO3 solution are opaque and shrink due to chitosan crystallization. Adding of pectin or

starch to the film solution will produce clear and strong chitosan HCl or HN03 films that

35

are stable for at least 6 months (Hoagland, 1996). Films made from completely
diacetylated chitosan HCl will not shrink and are opaque (Samuels, 1981)

Chitosan, like many carbohydrate polymer films, tends to be a good oxygen and
carbon dioxide barrier but lacks resistance to water transmission. Incorporation of fatty
acids or lipids such as lauric or butyric acid provides hydrophobicity and decreased water
permeability (Dominic et al., 1992). Chitosan films are unsuitable foundation films
because of significant swelling. Chen et al. (1996) reported that various chitosan films
could absorb 40 to 80% of their weight in water.

Use of different acids (acetic, formic, lactic, propionic) in chitosan film
formulations distinctly affects the film properties. Caner et al. (1998) showed that acetic
acid produced the lowest WVP, followed by propionic, formic and lactic acid whereas
lowest OP was observed when lactic acid was used. Films formed with lactic acid and
formic acid solutions had uniquely high values for elongation at break and tensile
strength, respectively. J ong-Whan et al. (1998) also evaluated the effects of acetic, citric,
formic, lactic, malic, propionic, succinic, tartaric, phosphoric and hydrochloric acids on
properties of chitosan films. Films made with organic acids such as formic, acetic, malic
and propionic acids were more resistant to solubilization in water than those made with

citric, lactic, succinic and tartaric acids.

1.1.4.5. Starch Films
Starch consists of approximately 25% amylose and 75% amylopectin. Amylose is
a linear chain of D-glucose residues linked by 1,4 glycosidic bonds. Amylopectin is a

branched molecule consisting of glucose units connected by 1,4 and 1,6 linkages.

36

Starches can be derived from tubers (potato, tapioca, arrowroot and sweet potato), stems
(sago), and cereals (corn, waxy maize, wheat and rice). Although starch is insoluble in
cold water due to hydrogen bonding of polymer chains, heating will break these hydrogen
bonds allowing the starch granules to absorb water and swell.

Amylose is required for film forming and preparation of strong gels. Starches
containing 55 or 70% amylose content are commercially available, with 70% amylose
starch producing stronger, tougher and more flexible films (Jokay et al., 1967). Wolff et
al. (1951) were first to produce self-supporting films by casting aqueous solutions of
gelatinized amylose, followed by solvent evaporation. Such starch films are transparent
and have very low permeability to oxygen at low RH (Rankin et al., 1958, Coffin and
Fishman, 1993, 1994).

Addition of plasticizers and absorption of water molecules by hydrophilic
polymers can increase polymer chain mobility, which generally leads to increased gas
permeability (Banker, 1966; Gaudin et al., 1999). Starch and plasticizers such as glycerol
contain many polar chemical functions, mainly hydroxyl groups. These characteristics
can provide susceptibility to antiplasticization materials (Jackson and Caldwell, 1967).
Glycerol and water play the role of internal lubricants, preventing the rigidification of
non-crystallized macromolecular starch chains at ambient temperature (Shogren, 1993).
Dried raisins coated with an aqueous solution of corn amylose plasticizer poured freely,
and did not clump or stick together (Moore and Robinson, 1968).

Garcia et al (2000a, b) developed starch-based edible films from cold gelatinized
cornstarch and high-amylose cornstarch (amylomaize) suspensions in NaOH. Glycerol or

sorbitol used as a plasticizer decreased the crystalline/amorphous ratio and permeability

37

to CO2, 02 and water vapor, with films containing sorbitol showing lower permeability
than those containing glycerol. They also showed that amylomaize films with a higher
crystalline/amorphous ratio were less permeable to CO2, 02, or water vapor than the
corresponding cornstarch films. Addition of 2 g/L sunflower oil to the film formulation
reportedly decreases both the WVP and the amorphous/crystalline ratio (Garcia et al.

2000b)

1.1.4.6. Dextrins

Dextrin-starch hydrolysates of low dextrose equivalent also have been suggested
for use as protective coatings. Allen et al. (1963) tested the barrier properties of these
edible film materials as coatings using a cellulase acetate support. Water vapor transport
through starch films was very high, while films comprised of low dextrose equivalent
dextrin and corn syrup had WVP values that were 2- and 3-fold lower, respectively.

Dextrins are often used as film-formers and edible adhesives to replace natural
gums. Dextrin-based coatings exhibit some resistance to oxygen transmission as has been
shown by the reduction in browning of sliced apples (Murray and Lufi, 1973). A gluten-
dextrin coating was also used to coat dry roasted peanuts before application of salt
(Noznick and Bundus, 1967).
1.1.5. Lipid Films

Lipid compounds include neutral lipids of glycerides which are esters of glycerol
and fatty acids, and waxes which are esters of long-chain monohydric alcohols and fatty
acids. From this group, acetylated monocglycerides, natural waxes, and surfactants are

commonly used in edible films and coatings (Kester and Fennema, 1986).

38

1.1.5.1.Waxes

Solidification of the melted wax or lipid by cooling is a common technique for
preparing lipid films. Wax films are also formed by casting molten wax on a dried film
of methylcellulose and then dissolving away the methylcellulose film (Donhowe and
Fennema, 1993). The rate of cooling plays an important role in the predominant
polymorphic state, as well as degree of recrystallization in the solidified film. Several
waxes have been studied for film or coating preparation: paraffin, camauba, beeswax,
candellila and polyethylene wax.

Paraffin wax is derived from the distillate fraction of crude petroleum. It contains
hydrocarbon fractions ranging from eighteen to thirty-two units (Bennett, 1975). Paraffin
waxes are permitted for use as protective coatings for raw fruit and vegetables and for
cheeses. They can also be used in chewing gum as a defoamer and as a component for
microencapsulation of flavors.

Camauba wax is extruded from palm tree leaves of the Tree of Life. Refined
camauba wax, composed of saturated acid esters twenty-four hydrocarbons in length and
saturated long chain alcohols (Bennett, 1975), has a high melting point and is often added
to other waxes to increase hardness.

The surface appearance of camuaba wax films is somewhat “hilly” with small
pores disrupting the otherwise smooth contours (Donhowe and Fennema, 1993).
Camauba wax, which has low oxygen permeability probably because of its hardnesses, is
permitted for use as a coating for fresh fruits and vegetables and in both chewing gums

and sauces.

39

Beeswax is secreted by honeybees for comb building. Beeswax obtained by
melting comb wax, contains 71% long-chain fatty acid esters and alcohols, 15% long-
chain hydrocarbons and 8% free fatty acids (Tulloch, 1970). This wax is soft at room
temperature with a melting point of 61-65°C, but becomes brittle at colder temperatures.
Beeswax is partially crystalline in nature. Its crystal size is quite small, allowing for tight
packing of individual crystallites. This type of crystalline morphology accounts for
beeswax being a particularly effective barrier to water vapor transmission (Kester and
Fennema, 1989). However, beeswax has higher water vapor permeability than candellila,
camauba, and microcrystalline wax (Donhowe and F ennema, 1993) due to higher
concentrations of fatty acids, fatty alcohols and esters. Beeswax also contains low levels
of unsaturated hydrocarbons which are responsible for its flexibility (Dafler, 1977) and
high oxygen permeability. Unlike camauba wax, the micros00pic surface of beeswax
appears smooth and devoid of distinct morphological features (Greener and Fennema,
1989; Kester and F ennema, 1989; Donhowe and Fennema, 1993).

Candelilla wax is extracted from the candelilla plant, a reed-like plant that grows
in Mexico and southern Texas. Candellila wax contains 57% hydrocarbons and 29% wax
esters, with the remainder consisting mainly of fatty alcohols and fatty acids (Tolluch,
1970). Its melting point is 65-68.8°C.

Polyethylene wax is the oxidation product of polyethylene, a petroleum by-
product. Polyethylene waxes are available in several grades to provide desired emulsion
properties. These waxes which differ in molecular weight, density, and melting point

(Eastman Chemical Inc., 1990) can be used to make emulsion coatings.

4o

Acetylglycerides are produced by acetylation of glycerol monoestearate with
acetic anhydrate. Acetyglycerides exist in the or polymorphic form (Jackson and Lutton,
1952; Vicknair et al., 1954) and can be stretched up to eight times their original length.
Acetyglyceride films posses a relatively smooth surface without any well-defined
morphological characteristics (Kester and F ennema, 1989) because heterogeneous
acetyglycerides are stable in the or form (Feuge, 1955). WVP of wax-based films is lower
than most other edible films; however, oxygen permeability of beeswax is similar to
some of the protein-based films such as collagen, casein, soy protein films (Table 1.1).
Acetylated monoglycerides based-films have higher WVP values compared to other wax-
based films.

1.1.6. Evaluating of the properties of edible films
1.1.6.1. Barrier properties

Barrier properties of the aforementioned films directly impact product shelf-life
with transfer of gases (e.g. oxygen, carbon dioxide), water vapor and volatile compounds
occurring from the environment to the food product and vice versa. Edible films
possessing polymer structures can be used as barriers to these gases and water vapor.
Depending on the products, edible films or coatings may be used to prevent moisture
loss, moisture absorption, exposure to oxygen, or diffirsion of CO2. Control of the
moisture and oxygen content limits the growth of aerobic microorganisms, which provids
good microbiological characteristics as well as physicochemical and organoleptic

characteristics to intermediate moisture or dry foods.

41

2.1.6.1.1. Water Vapor Permeability

One of the most important properties of an edible film is its water vapor
permeability. Water vapor permeability (WVP) is defined as the rate of water vapor
transmission per unit area of a flat material of unit thickness.

Several techniques for measuring WVP have been described and are based on
either infrared sensors such as the Permatran-W series or the WVP tester L80-4000 series
which is a colometric method. These techniques are well-suited for high barrier
efficiency polymers such as plastics or wax-based edible films, but far less so for
hydrophilic polymers (Krochta, 1992, McHugh and Krochta, 1994). The most commonly
employed method is the “cup method” which is based on gravimetric analysis (Kester
and Fennema, 1983; Kamper and Fennema, 1985). True permeability is a process of
solubilization and diffirsion, where the vapor dissolves on one side of the film and
diffuses through to the other side.

Water vapor permeability (WVP) can be expressed as follows:

 

Where

3 = Amount of water vapor
L = Thickness

A = Film area

t = Time

Ap = Differential partial pressure of water vapor

42

The water vapor transmission rate, determined by the amount of water weight lost
or gained, depends on the material placed in the cup water salt solution or desiccant. In
the Desiccant Method, the film to be tested is sealed over the mouth of a cup containing
desiccant, and is placed in a chamber under controlled temperature and relative humidity.
In the water method, the test dish contains distilled water or a saturated salt solution. In
both cases, the rate of water transmission is determined from periodic weighings which
must be taken after a steady state rate of moisture transmission has been obtained

(ASTM, 1980).

1.1.6.1.2. Oxygen Permeability

Measurement of oxygen permeability (OP) typically involves the use of
colorimetric (e.g. Oxatran-Mocon, Modern Controls Inc., Minneapolis, MN, USA), or
manometric sensors (Lyssy L100 series, Lyssy, Zurich, Switzerland). Oxygen
transmission rates (OTR) are obtained by monitoring the permeant in the upper portion of
a diffirsion cell at specific time intervals, while passing a known amount of oxygen
through the lower portion until the steady state is reached. To obtain the OP, the
thickness of the film and the partial pressure difference must be considered as shown in
the following equation:

OP = (OTR x L)/ Ap

Where

L = thickness of film

Ap = partial pressure difference of oxygen

43

1.1.6.2. Mechanical Properties

Tensile strength and percent elongation are the most commonly tested mechanical
properties. Tensile strength, the maximum tensile stress that a material can sustain before
the onset of permanent deformation or failure, is determined by measuring the force per
unit while the film is being stretched. Percent elongation refers to the degree of
toughness and flexibility of the film, and measures the length of displacement per original
length while the film is being stretched. The established method to determine tensile
strength and elongation is ASTM standard D-882-83 which most often utilizes the Instron

machine (ASTM, 1 991).

1.1.7. Factors Affecting Film Properties

Some factors such as the chemical and structural nature of the polymer and
permeant can affect the barrier properties of edible films. Chemical composition plays a
major role in the barrier properties of edible films. For example, polar polymers such as
many proteins and polysaccharides show low gas permeability values at low RHs but
have poor moisture barrier properties. In contrast, non-polar hydrocarbon-based materials
such as lipids are excellent moisture barriers and less effective gas barriers. When added
to polymer films, low molecular weight additives can improve or reduce the barrier
properties of edible films depending on their chemical structure. Most edible film
plasticizers increase water vapor permeability by disrupting polymer chain hydrogen
bonding.

The nature of the permeant also affects its transfer through films, with smaller

molecules generally diffusing faster than larger molecules, and polar molecules diffusing

44

faster than non-polar molecules, particularly in polar films. The diffusion of gases and
water vapor though film polymers is termed permeation. During permeation, adsorption
of these gases and water vapors on the polymer surface and their desorption through the
opposite surface is also seen (Sperling, 1992). These films also can have different barrier

properties depending on their composition and method of production.

1.1.8. Application of Edible Films

Edible film barriers can serve many functions in food products. Such edible films
or coatings can provide additional nutrients, enhance sensory characteristics and serve as
a carrier for many food additives including flavoring/coloring agents, vitamins,
antioxidants and antimicrobials, the last of which is the focus of this review.

Edible films can function as oxygen and moisture barriers to retain crispiness and
inhibit oxidative and hydrolytic rancidity in fat-containing products such as nutrneats.
Although coating of hydroxypropylated starch, hydroxypropyl cellulose (Ganz, 1969,
Ramos et al., 1996), and WPI (Mate and Kroctha, 1996; Mate et al., 1996a, b; Mate and
Krochta 1997a, b) can delay rancidity in nuts, coatings based on corn zein (Andres,
1984) or low-methoxyl pectins (Swenson et al., 1953) required the addition of an
antioxidant to be effective.

Edible coatings can be used to improve the quality of fresh, frozen and processed
meat, poultry and seafood by minimizing microbial spoilage, moisture loss, lipid
oxidation and discoloration. Application of wax, mineral oil, or corn oil to whole poultry,
meats and fish before freezing will reduce moisture loss (Gennadios et al., 1997). Many

types of coatings have tested in attempt to maintain quality in frozen fish. According to

45

Anderson (1961), long chain saturated fatty alcohols and fatty acid coatings could control
moisture loss and freezer burn in frozen meats. Stuchell and Krochta (1995) studied the
effects of acetylated monoglyceride (Myvacet 9-08 and Myvacet 9-45) and acetylated
monoglyceride/whey protein isolate coatings on moisture loss and lipid oxidation in
frozen King salmon pieces stored at —— 23°C, with application of these coatings reducing
moisture loss by 42-65% during the first weeks of frozen storage. Onset of lipid oxidation
was also delayed and peroxide values were lowered. In another study, Silver salmon
fillets that were dipped in Myvacet 5-07 acetylated monoglyceride (60°C) and placed
infrozen storage at —10°C retained more moisture and had lower peroxide values than
uncoated control samples (Hirasa, 1991).

Alginate coatings have been applied to various meats such as beef and pork cuts,
poultry parts and whole lamb carcasses using a two-step procedure to reduce dehydration
(Mountney and Winter, 1961; Allen et al., 1963; Earl, 1968; Earl and McKee, 1976;
Lazarus et al., 1976; Williams et al., 1978). Meat is typically immersed in or sprayed with
an aqueous sodium alginate solution with gelation induced by application of a calcium
chloride solution. These alginate coatings reduced dehydration of meat tissue compared
to that of uncoated controls. Reduced dehydration is mainly due to the high moisture
content of alginate gels with moisture in the gel evaporating before any desiccation of the
enrobed food (Kester and Fennema, 1986). Flavor-tex®, a commercial calcium alginate
coating (D.H. McKee, Inc., 1989), is used to reduce shrinkage, oxidative rancidity,
moisture migration and oil absorption in various foods including meat, poultry, seafood,
baked goods, vegetables, extruded foods and cheese. Alginate coatings protect against

lipid oxidation in pre-cooked, ground pork patties (Wanstedt et al., 1981) with better

46

texture and absence of a warmed-over flavor (an indication of rancidity) observed in
coated compared to uncoated samples. Wu et al. (2001) evaluated edible films of starch-
alginate (SA), starch-alginate-stearic acid (SAS), SA-tocopherol, SAS-tocopherol,
tocopherol-coated SA, and tocopherol-coated SAS for their effectiveness in maintaining
quality of precooked beef patties during storage at 4°C. Patty weight loss, moisture loss,
2-thiobarbututric acid values and hexanal, pentane and total volatile content differed with
film composition. SAS-based films controlled moisture loss more effectively than lipid
oxidation, with tocopherol-treated films more effective than non-tocopherol films in
inhibiting lipid oxidation.

Application of alginate coatings can also reduce the total-surface microbial count
and some pathogenic bacteria on meat surfaces. Lazarus et al. (1976) reported that
alginate-coated lamb carcasses had lower microbial counts than uncoated samples after 7
days of refi'igerated storage. Williams et al. (1978) also observed lower microbial counts
on the surface of alginate-coated beef cuts than on uncoated controls. In addition, they
showed that coating with alginate protected the red color of beef muscle. Similarly,
Siraguas and Dickson (1992, 1993) demonstrated that calcium alginate coatings
containing organic acids (lactic acid or acetic acid) reduced the levels of L.
monocytogenes, S. Typhimurium and E. coli 0157:H7 on beef tissue.

Chitosan coatings which possess several important biological properties including
antifungal activity (Allan and Hadwiger, 1979; Hirano and Nagao, 1989) and phytoalexin
(Hadwiger and Beckman, 1980; Walker-Simons et al., 1983), have been applied to fruits
and vegetables to reduce water loss and extend shelf life (El Ghaouth et al., 1991). These

coatings also can retard ripening of tomatoes and extend storage life without adversely

47

affecting later ripening characteristics (El Ghaouth et al., 1990). Coating cucumbers and
bell peppers with chitosan also reportedly reduced respiration, color loss, wilting and
fungal infection (El Ghaouth et al., 1991). A similar delay in spoilage was also observed
in chitosan-coated strawberries and was attributed to the fimgistatic properties of chitosan
(El Ghaouth et al., 1990).

Waxes and oils such as paraffin wax or oil, beeswax, camauba wax, candelilla
wax, mineral oil, vegetable oil, acetylated monoglycerides, sucrose esters of fatty acids
and resins are generally good moisture barriers and have been used extensively on whole
fruits and vegetables, (Hagenmaier and Shaw, 1990, 1992). Protective coatings
comprised of waxes and sucrose esters of fatty acids have been developed and
commercialized for fruits and vegetables. Various waxes have been traditionaly applied
to cheese as peelable coatings to prevent mold growth. Kamper and Fennema (1984)
prepared bilayer films with HMC and beeswax, paraffin, hydrogenated palm oil, stearic
acid, or stearic-palmitic acid, with these films inhibiting water transfer from high- to low-
moisture foods.

Use of protein-based films and coatings on fresh produce has been restricted
because of high water vapor permeability. However, addition of hydrophobic materials to
protein films presents many opportunities for coating fresh fruit and vegetables. Park
(1991) investigated the effect of corn zein coatings on tomatoes and found that the shelf-
life could be extended six days by delaying color change, loss of firmness, and weight
loss. Other investigators (Krochta et al., 1989; Avena-Bustillus et al., 1993, 1997) have
designed and applied caseinate-based coatings to minimally processed fi'uits and

vegetables. Using such coatings on peeled carrots, white blush formation was eliminated

48

with decreased dehydration also observed at 25°C/70% RH (Avena-Bustillus et al.,
1993). Coating uncut celery sticks with a caseinate/acetylated monoglyceride solution
also resulted in a 75% reduction in moisture loss under the same conditions. Penicillum
spp. were not found on apple slices coated with caseinate/acetylated monoglyceride
solutions that contained included potassium sorbate (1%) (Avena-Bustillus et al., 1997).
Lerdthanangkul and Krochta (1996) coated green bell peppers with a mineral oil- or
cellulose-based coating, whey protein isolate, sodium caseinate, or a sodium caseinate
beeswax emulsion to assess the impact on respiration, internal gases, color, firmness, and
water loss during 20 days of storage at 10°C. Only the mineral oil-based coating
significantly reduced moisture loss, thus maintaining fruit firmness and freshness.
Cellulose and sodium caseinate coatings were the most effective 02 and CO2 barriers.
However, none of the coatings influenced respiration or color change. In another study,
soy protein film coatings reportedly extended the shelf-life of “Fuji” and “Golden 95
delicious” apples over 40 days at 24°C (Park, 2000) with these films retarding changes in
apple firmness, color and acidity compared to uncoated controls.

Polysaccharide-based coating also has been developed for processed fruit and nut
products. When tested on fruits, starch coatings extended the shelf-life of pitted prunes
due to O2 barrier properties (Jokay et al., 1967). Starch films are reported to be
semipermeable to CO2, but highly resistant to O2 transmission (Rankin et al., 1958). Choi
et al. (2000) showed that low pH (2.7) chitosan coatings dissolved the wax layer on apple
skin, which led to increased weight loss and a lower respiration rate.

Edible film coatings have been shown to reduce oil uptake by foods and oil

degradation during deep fat frying. Extension of frying-oil life may be accomplished

49

through appropriate selection and application of edible films to pre-fried products.
Hollownia et a1. (2000, 2001) evaluated the application of MC and HPMC edible films to
marinated whole chicken strips before and after breading. When applied before breading,
oil absorption by the chicken strips decreased during frying, with oil degradation also
reduced by 50%, due to a reduction in moisture and acetic acid migration which are
responsible for reduced free fatty acid generation in oils during frying. The potential for
gellan gum-, HC- and MC- based films to reduce absorption of fat in a fiied pastry mix
was also investigated by Williams and Mittal (1999a). All coated samples had lower
levels of fat after frying than uncoated samples, with fat absorption reduced by 50-91%.
Williams and Mittal (1999b) also developed a mathematical model for heat, moisture and
fat transfer in the film and the foods when edible films were used as coating in fried
foods (potatoes and pastry). Film diffusivities were determined for gellan gum films at a
thickness of 2.0 mm during frying. Moisture diffusivities of the food and film were 0.33 x
10'7 and 0.25 x 10'7 mz/sec., respectively. Fat diffusivities were 0.103 x 10'8 mz/sec. for
the food and 0.604 x 10'9 mz/sec. for the film. Thermal diffusivities were 0.102 x 10'6

m2/sec. for the food and 0.156 x 10'6 mzsec. for the film.

1.2. ANTIMICROBIALS

As mentioned at the outset, edible films can serve as carriers for a wide range of
food additives including various antimicrobials, which can extend product shelf-life and
reduce the risk of pathogen growth on the surface of food products. Antimicrobials
including benzoates, propionates, sorbates, parabens, acidifying agents (e.g. acetic and

lactic acids), curing agents (e.g. sodium chloride and sodium nitrite), bacteriocins and

50

 

 

natural preservatives (e.g. essential oils, lysozyme, liquid smoke) will now be addressed

individually in terms of their possible use in edible films.

1.2.1. Benzoic acid or sodium benzoate

The preservative action of benzoic acid was first described in 1875 by H. Fleck.
Sodium benzoate was the first chemical preservative permitted in foods by the FDA. In
the United States, benzoic acid and sodium benzoate are generally regarded as safe
(GRAS) preservatives (Code of Federal Regulations, 1977, Title 21, Sections 184.1021
and 184.1733) at levels up to 0.1%. In most other countries, maximum permissible levels
range between 0.15 and 0.25%.

Sodium benzoate is one of the most commonly used antimicrobials in edible
films, since it is soluble in most film solutions and remains active after fihn production.
The antimicrobial activity of benzoate is related to pH. Like many other food
antimicrobials, benzoate (pK = 4.20) is most effective in its undissociated form with 60%
of the compound undissociated at pH 4.0. Therefore, methycellulose, chitosan, and
collagen films, all of which have a relatively low pH, are good candidates for this
antimicrobial. This restriction limits the use of edible films containing benzoic acid and
its sodium salts to high-acid products such as cheese and fermented meat products
(Lueck, 1980).

Although benzoic acid acts essentially as a mold and yeast inhibitor (Balatsouras
et al., 1963), other studies have shown that sodium benzoate or benzoic acid can also
inhibit the growth of both pathogenic and psychotropic bacteria (Thomas et al., 1993,

Kasrazaden et al., 1994, El-Shenawy and Marth, 1988).

51

Use of sodium benzoate alone or in combination with various organic acids has
been proven effective for decreasing microbial populations on raw chicken. In one study
conducted by Hwang and Beuchat (1995), raw chicken wings were inoculated with
Salmonella, Campylobacter jejuni, Listeria monocyctogenes, Staphylococcus aureus or
Escherichia coli 0157:H7 and immersed in water or a solution of 0.5% lactic acid/0.05%
sodium benzoate (LB) (pH 2.64) for 30 min. Lower populations of pathogenic and
naturally occurring psychrotrophic bacteria were detected on wings immediately after
treatment with LB compared to the untreated controls. During refiigerated storage,
populations of Salmonella, C. jejuni, L. monocytogenes, and E. coli 0157:H7 decreased
significantly on lactic acid-treated wings. In another study, benzoate was more effective
than lactic acid against Staphylococcus aureus and E. coli at 35°C, especially when used
at pH <6.0 (Thomas et al., 1993). Sodium benzoate can also be used to inhibit Salmonella
in certain cheeses. Kasrazaden et al. (1999) found that addition of sodium benzoate
(0.3%) to cheese (pH 6.6) made from milk, which was acidified to pH 5.9 with propionic

acid, prevented grth of Salmonella in the final product during storage.

1.2.2. Sorbic Acid and sorbates

Sorbic acid, first isolated from the pressed unripened berry of the rowan or
mountain ash tree in 1859 by the German chemist A. W. Hoffinann (Anonymous, 1978),
is a straight chain or, B- unsaturated monocarboxylic acid and has the structure CH3CH =
CHCH = CHCOOH (Sofos, 1989). The carboxyl group reacts to form calcium, sodium or
potassium salts and esters. Potassium sorbate, the commonly used salt of sorbic acid, is

highly soluble in water (58.2% at 20°C). Like sodium benzoate, sorbic acid (pK = 4.80)

52

is most effective in acidic foods (< pH 6.5) (Cowles, 1941). At pH 4.0, 86% of
sorbic acid is undissociated (Sofos et al., 1980) with the undissociated form again most
able to penetrate the bacterial cytoplasmic membrane (Chichester and Tanner, 1972).
Increased antimicrobial activity of sorbate at low pH values has been reported for a wide
range of microorganisms (Table 1.3) (Gould, 1964; Park and Marth, 1972; Sofos et al.,
1980; Lahellec et al., 1981; Blocher et al., 1982; Elliot et al., 1982; Nose et al., 1982;
Roberts et al., 1982; Seward et al., 1982; Tsai et al., 1984; Tuncan and Martin, 1985;
Lund et al., 1987). Therefore, edible films containing sorbate only exhibit antimicrobial
activity at pH values less than 6.0.

Sorbic acid salts are the most studied antimicrobial agents in carbohydrate or
protein-based edible films such as methylcellulose, WPI and chitosan since the sorbates
are soluble in various film solutions and remain chemically active in the film matrix. The
carboxyl group, which is the active site in sorbates, forms hydrogen bonds with
carbohydrate or protein chains in films. Edible films containing sorbates have been tested
against a wide variety of microorganisms (i.e. spoilage bacteria, pathogenic bacteria,
yeast and molds) in laboratory media using diffusion-type assay (Torres et al., 1985;
Torres and Karel, 1985; Chen et al., 1996; Cagri et al., 2001).

Typical use levels range from 0.02% to 0.3% in commercial foods (Table 1.3).
These concentrations are added to bakery, dairy, fruit and meat products to inhibit a wide
range of yeasts (Lueck, 1980; Monsanto, 1987), molds (Trichosporon, Alterneria,
Aspergillus, Mucor, Fusarium) (Sofas and Busta, 1993, Chichester and Tanner, 1972;
Luck, 1976), and bacteria (E. coli 0157:H7, Clostridium botulinum, Staphylococcus

aureus, Saccharomyces italicus, pseudomonads, and colifonns) (Bradley et al., 1962;

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Gandhi et al., 1973; Kasrazadeh and Genigeorgis, 1995; Briozzo et a1, 1985) and to
prevent aflatoxin and enterotoxin production (Lueck, 1980).

Antimicrobial activity of sorbate is generally enhanced at low temperature.
Investigators showed that L. monocytogenes, Y. enterocolitica, A. hydrophila, and
Pseudomonas putida were more sensitive to potassium sorbate at refrigeration
temperature than at higher temperature (El-Shenawy and Marth, 1988; Moir and Eyles,
1992). Increased microbial inhibition by sorbate in the presence of higher solute
concentrations also has been reported in both laboratory media and foods. Sodium
chloride reportedly acts synergistically with potassium sorbate to inhibit S. aureus
(Robach and Stateler, 1980) and outgrth of C. sporagenes (PA 3679) spores (Robach
and Statelen 1980). Razavi-Rohami and Griffiths (1999) also found that the inhibitory
activity of sorbic acid increased with increasing NaCl concentration from 3 to 20% (w/v)
and lower pH (3.5) for Candida, Sporothrix, Fusarium, Penicillium, Paecilomyces and
Aspergillus spp. The inhibitory mechanism of 3% NaCl is not know but may be related to

the effects of added solutes on membrane activities as suggested by Walter et a1. (1987).

1.2.3. Propionic Acids

Propionic acid, a monocarboxylic acid (pKa = 4.87), is produced by
Propionabacterium freudenreichi subsp. shermani. Swiss cheese contains up to 1%
propionic acid from the growth of propionibacteria, which gives it a characteristic flavor
and prevents mold growth (Davidson and Branen, 1993).

Antimicrobial activity of the propionates is again pH dependent, with the

undissociated form being 45 times more inhibitory than the dissociated form (Eklund,

60

1985). Therefore, it is only effective in low pH edible films such as collagen and
chitosan.

Propionic acid or its salts are commonly used food preservatives due to their wide
spectrum of activity. While primarily active against molds, some yeasts and bacteria are
also inhibited (Table 1.3) (Chung and Goepfert, 1970; Eklund, 1985; El-Shenawy and
Marth, 1989; Cherrington et al., 1990; Cherrington et al., 1991). Bacillus cereus which
causes rope formation in bread dough, can be inhibited by the addition of propionic acid
at pH 5.6 to 6.0 (Woolford, 1975; O’Leary and Kralovec, 1986). Propionates can be
added directly to bread dough because they have no effect on the activity of baker’s
yeast. Amounts of propionate used in foods are generally less than 0.4% (Robach, 1980).

Propionic acid was most effective among the organic acids at inhibiting aflatoxin
production by Aspergillus flavus on betel nuts after 2 (62%) and 4 (85%) weeks
(Raisuddin and Misra, 1991). Kwon et al. (1998) reported that when grown in tryptic soy
broth (TSB) containing buffered propionic acid (EPA), the growth rate of S.
Typhimurium gradually decreased as the level of EPA increased and the broth pH
decreased. No growth was detected at BPA concentrations above 3% (v/v), with grth
also markedly suppressed under anaerobic conditions and at pH 5.0 compared to 7.0.

Propionic acid and the propionates (8-12%) were also effective in controlling the
growth of mold on the surface of cheese and butter (Deane and Downs, 1951). When
used at pH 4.5 at a level of 0.2%, propionic acid completely inhibited growth and

aflatoxin formation (Ghosh and Haggblom, 1985).

61

1.2.4. Mode of action of benzoate, sorbate, and propionic acid

Benzoic, sorbic and propionic acid act by inducing changes in the morphology
and appearance of microbial cells. Incorporating these acids into specific cell structures
may also inhibit specific biosynthetic pathways in the cell (Sofos et al., 1986). As one
example, sorbate prevented amino acid (L-serine and L-histidine) uptake by S.
Typhimurium at low pH (Tuncan and Martin, 1985).

These acids also have been shown to reduce the intracellular pH (pHi) in E. coli
cells (Salmond et al., 1984) and vesicles (Eklund, 1985), suggesting that this common
preservative may act as a protonophore. Ronning and Frank (1987) reported that
undissociated sorbic acid reduced the pHi in vegetative C. sporogenes PA 3679 cells, and
decreased the protonmotive force which energizes cellular activities such as amino acid
transport. They also proposed that the loss in energy and reduced uptake of essential
amino acids induced a stringent-type response that inhibited but did not kill the cells.

Past evidence has suggested that the inhibitory action of these antimicrobials on
yeasts is due to reduction of pH. However, using a novel method to measure pHi in
growing cells, little correlation was found between reduced growth rate on exposure to
sorbic acid and reduction of pH. In fact, growth inhibition correlated with an increase in
the intracellular ADP/ATP ratio due to increased ATP consumption by the cells. This was
partly attributed to the activation of protective mechanisms, such as increased proton
pumping by the membrane H+ —ATPase, which ensured that pH did not decline when the
cells were exposed to sorbic acid (Bracey et al., 1998). Thus, the inhibitory action of

sorbic acid was likely due to the induction of an energetically expensive protective

62

mechanism that compensated for any disruption of pHi homeostasis but resulted in less

available energy for normal growth.

Benzoic, sorbic and propionic acid also alter cell membrane function by
producing pores (Freese and Levin, 1978) that interfere with uptake of substrates,
electron transport and the proton-motive force involved in transport functions. In
addition, sorbic acid can bind sulfhydryl groups on various enzymes, thus leading to
disruption of vital processes involved in transport functions, cell metabolism and growth.
Sorbate also reportedly inhibits microbial growth by combining with coenzyme A (Nose
et al., 1982) with Harada et al. (1968) theorizing that sorbate may act competitively with

acetate at the site of acetyl coenzyme A.

1.2.5. Parabens

Esterification of the carboxyl group of benzoic acid produces parabens. Having
the ability to remain undissociated at pH values up to 8.5, most parabens are active at pH
3.0 to 8.0. The methyl, propyl and heptyl parabens can be used as food preservatives in
most countries, while the ethyl and buthyl esters are more restricted. Parabens can be

used effectively in a wide range of foods (Table 1.3).

Parabens with a longer alkyl chain posses more antimicrobial activity than those
having a shorter alkyl chain (Aalto et al., 1953; Shibasaki, 1969). Parabens are more
inhibitory on Gram-positive than Gram-negative bacteria due to their decreased in
polarity. Methyl, ethyl, propyl and butyl parabens completely inhibit the growth of Gram-
positive bacteria and Gram-negative bacteria at concentration levels of 40-2000 and 50-

4000 pg/ml, respectively (Table 1.3) (Aalto et al., 1953; Jurd et al., 1971; Lee, 1973;

63

Kato and Shibasaki, 1975; Robach and Pierson, 1977; Dymicky and Huhtanen, 1979;
Eklund, 1980; Lueck, 1980; Eklund, 1981; Reddy and Pierson, 1982; Reddy et al., 1982;
Payne et al., 1989; Juneja and Davidson, 1992; Moir and Eyles, 1992; Davidson, 1993).
However, parabens are generally more active against molds and yeasts than bacteria.
Using esters of p-hydroxybenzoic acid, concentrations of 32 to 1000 ug/ml are normally
needed for complete inhibition of bacteria and fungi (Table 1.3) (Aalto et al., 1953; Kato
and Shibasaki, 1975; Marwan and Nagel, 1986; Jermini and Schmidt-Lorenz, 1987;

Juneja and Davidson, 1992; Thompson, 1991)

The antimicrobial mechanism for paraben is very similar to that of phenols and
related phenolic compounds, since parabens are phenolic derivatives. Vas (1953) and
Judis (1963) both proposed that phenol physically damages the cytoplasmic membrane of
microorganisms, which causes the release of cytoplasmic compounds. Furr and Russel
(1972) detected similar leakage of intracellular RNA by Serratia marcescens in the
presence of parabens, with the amount of leakage proportional to the alkyl chain length of
the paraben. Additional studies have shown that the parabens inhibit nutrient uptake
through the cytoplasmic membrane, inhibiting both membrane transport and the electron
transport system (Freese et al., 1973; Eklund, 1980). According to Freese et al. (1973)
serine uptake by cytoplasmic membrane of B. subtilis is inhibited by parabens with
Eklund (1980) showing inhibition of alanine, serine, phenylalanine, and glucose uptake

by vesicles in E. coli, B. subtilis, and P. auruginosa.

64

1.2.6. Free Fatty Acids and Their Esters

Low concentrations of long-chain fatty acids are inhibitory to microorganisms,
especially Gram-positive bacteria and yeasts (Kabara, 1978). Saturated fatty acids
having chain lengths of C12 to C16 and Cm to C12 possess the most antimicrobially activity
against bacteria and yeasts, respectively (Kabara, 1993). Decreasing effectiveness of
longer chain fatty acids may be related to increased hydrophobicity and decreased
solubility (Wang and Johnson, 1991). Fatty acids are also more active at low pH (<5.0).
Fatty acid structure and function has been reviewed by Kabara (1982, 1993). The
inhibitory effects of unsaturated fatty acids increase as the number of double bonds
increases. For example, linoleic acid was far more inhibitory than oleic acid (Fuller and
Moore, 1967). Fatty acids and monoglycerides are inhibitory to bacterial species (Table
1.3) (Noterrnans and Dufrenne, 1981; Baker et al., 1985; Knapp and Melly, 1986).
According to Sprong et al. (1999), increased intake of bovine milk fat enhanced
gastrointestinal killing of L. monocytogenes, but had little effect on infection with
Salmonella enteritidis in rats. Free fatty acids C100, Cm) and Cm) and the
monoglycerides of Cm), Cm), and Cm) likely play a pivotal role in this enhanced
listeriacidal activity. In contrast, infection with Salmonella was not affected by milk fat

consumption.

Monoesters of glycerols and the esters of sucrose are more antimicrobial than
their corresponding free acids. Monolaurin (lauricin), the most effective of the glycerol
monoesters, is inhibitory to various Gram-positive bacteria and some fungi at 5-100 ppm
(Andrews and Grodner, 1997). Monolaurin is most effective at pH 5.0-8.0. Antimicrobial

activity of monolaurin has been demonstrated in various foods including mechanically

65

deboned chicken meat, minced fish, chicken sausage (Baker et al., 1982), soy sauce
(Kato, 1981), meat slurries (Noterrnans and Dufrenne, 1981), cottage cheese, pork
homogenate (Robach et al., 1981), skim milk (Wang and Johnson, 1992), and crawfish
tail (Oh and Marshal, 1994). However, use of monolaurin as a food preservative is
limited due to off-flavors and the loss of activity from the interaction with lipophilic
proteins, fat globules and starch. Fatty acids and poylglycerides are added to edible films
and coatings to decrease WVP properties. Long-chain alcohols (e.g. stearyl alcohol) and
fatty acids (e.g. stearic, palmitic) are commonly used as additives in edible coatings due
to their high melting point and hydrophobic chracteristic (Hagenmaier and Shaw, 1990).
Vojdani and Tores (1990) developed composite films with MC and fatty acids of
different chain lengths to decrease migration of preservatives like potassium sorbate from
the surface of foods such as cheese. The cellular membrane has been suggested as a
primary target for antimicrobial activity (Freese et al., 1973) with fatty acids impairing

cell permeability and the transport of nutrients (Greenway and Dyke, 1979).

1.2.6. Organic Acids
1.2.6.1. Acetic Acid

Acetic acid (CH3COOH), the primary component of vinegar, is produced by
Acetobacter species. Acetic acid and its salts are commonly used in variety of different
foods (Table 1.3). Acetic acid, like other organic acids, can be used to acidify edible
films prepared from chitosan, alginate, collagen and WPI. Addition of acetic acid also
increases the activity of other antimicrobial agents such as sorbic acid and benzoic acid

that can be incorporated into edible films.

66

Application of acetic acid and its salts in the meat industry has met with variable
success. Adding acetic acid to chiller water (pH 2.5) in poultry processing reportedly
extended the shelf-life of poultry (Mountney and O’Malley, 1965). Processing poultry in
scald tank water containing 0.1% acetic acid decreased the heat resistance of Salmonella
newport, S. Typhimurium, and Campylobacter jejuni (Okrend et al., 1986). However, in
another study, incorporating 0.5% acetic acid in scald water had no effect on Salmonella,
total aerobic bacteria, or members of the family Enterobactericea on poultry carcasses
(Lillard et al. 1987). Acetic acid solution dips (1 to 3%) can reduce the number of
pathogenic and spoilage microorganisms on beef and lamb tissue (Bell et al., 1986;
Anderson et al., 1988). Growth of Listeria innocua, S. Typhimurium, E. coli 0157:H7,
Clostridium sporogenes, aerobic bacteria, lactic acid bacteria, and Pseudomonads was
suppressed or eliminated in ground beef prepared from carcasses which were washed
with 2% lactic acid, 2% acetic acid, and 12% trisodium phosphate solution as compared
to the untreated control (Dorsa et al., 1998). The combination of 100 mM NaOH (pH
10.5) and 76.7 mM acetic acid (pH 5.4) applied sequentially at 55°C for 5 min was very
effective at eliminating L. monocytogenes from biofilms (~ 4.5- to 5.0 logs CPU/cm2
decrease) (Arizcun et al., 1998)

Acetic acid is variably effective as an antimicrobial when used as a spray on meat
carcasses. Washing with water (35°C) followed by a 2% acetic acid spray (55°C)
treatment was more effective at reducing the numbers of E. coli 0157:H7 and S.
Typhimurium than trimming or washing alone (Hardin et al., 1995). Similarly, a 5%
acetic acid spray decreased populations of E. coli 0157:H7 on beef tissue by 1 log/cm2 as

compared to the untreated control (Delzari et al., 1998). When used alone or in

67

combination with a pulsed electric current, another acetic acid treatment reportedly
decreased Salmonella populations 1 log on carcasses (Tinney et al., 1997).

Acetic acid caused greater inactivation of L. monocytogenes than lactic and citric
acid with inhibition increasing at lower incubation temperatures. Lactic acid
concentrations > 0.3% inhibited L. monocytogenes at 13 and 35°C (Ahamad and Marth,
1989, 1990). Addition of 0.1% acetic to TPB minimized the inhibitory effect of 100 and
200 CFU/ml SML during the first 32 h of incubation. Staphylococcus aureus was
inhibited to a lesser extent when cultured in TSB supplemented with SML alone
compared to SML with organic acids (Monk et al., 1996).

A 2% acetic acid wash reportedly killed L. monocytogenes in a model system of
fresh meat fluids within 24 h while the pathogen increased 1.0 to 2.0 logs using a nonacid
water wash (Samelis et al., 2001). Sodium diacetate is also inhibitory to L.
monocytogenes, E. coli, Pseudomonas fluorescens, Salmonella enteritidis, and
Shewanella putrefaciens (Shelef and Addala, 1994). Sodium acetate at 1% reportedly
increased the shelf life of catfish fillets by 6 days when stored at 4°C compared to the
control (Kim et al., 1995). Sodium acetate will also inhibit the rope-forming bacteria and
various molds at pH 3.5 to 4.5 in baked goods (Table 1.3) (Glabe and Maryanski, 1981)
and prevented mold growth in cheese spreads (Doores, 1993).

Vinegar vapor (4-6% acetic acid) has been used to inactivate conidia of fungal
pathogens on various fruits. Vapors of several common Vinegars containing 4.2% to 6.0%
acetic acid effectively prevented conidia of brown rot (Monilinia fiucticola), gray mold
(Botrytis cinerea), and blue mold (Penicillium expansum) from germinating and causing

decay of stone fruit, strawberries, and apples, respectively. Vapor from 1.0 ml of red

68

wine vinegar (6.0% acetic acid) eliminated decay on apricot caused by M. fructicola.
Similarly, vapors from 1.0 ml of white vinegar (5.0% acetic acid) reduced decay on
strawberries by B. cinerea from 50% to 1.4%. Eight different Vinegars, ranging from
4.2% to 6.0% acetic acid, of which 0.5 ml of each vinegar was heat-vaporized, reduced

decay by P. expansum to 1.0 % or less on apples (Sholberg et al., 2000).

1.2.6.2. Lactic Acid

Lactic acid (CH3CHOHCOOH), produced naturally by lactic acid bacteria during
food fermentation, is primarily used for improving and controlling the quality and
microbial stability of foods. Lactic acid sprays (1 to 3% solutions) often have been used
to sanitize meat surfaces as discussed in several reviews (Smulders, 1986; Cherrigton et
al., 1991; Dickson and Anderson, 1992) with psychrotrophic Gram-negative bacteria
generally being more sensitive than Gram-positive organisms to this treatment.

Lactates are generally used as humectants and flavor enhancers in meat and
poultry products (Duxbury, 1988). Levels of 2% are used in hot dogs, frankfurters and
similar products. Evans et a1 (1991) reported that injecting a 4% lactic acid solution into
beef roast before cooking increased the cooking yield (15%) and lowered the aerobic
plate count during refrigerated storage.

The antimicrobial activity of lactic acid depends on the food application and the
target microorganism. Lactic acid is more effective than malic, citric, propionic or acetic
acid in inhibiting growth of Bacillus coagulans in tomato juice (Rice and Pederson,
1954). Lactic acid is also inhibitory to spore forming bacteria as well as S. aureus and Y.

enterocolitica (Minor and Marth, 1970; Woolford, 1975; Bracket, 1987).

69

Like other organic acids, lactic acid can be used in edible film formulations such
as chitosan and collagen for acidification purposes. As mentioned earlier, lactic acid also
can be used modify both the tensile strength and antimicrobial properties of collagen

casings.

1.2.6.3. Mode of action of organic acids

Organic acids inhibit oxygen uptake and resultant ATP production in whole cells
of B. subtilus (Sheu and Freese, 1972; Freese et al., 1973). However, they do not inhibit
NADH oxidation by isolated membranes. Glycerol phosphate- or NADH-energized
uptake of serine transport in membrane vesicles yields similar results. Hence, they appear
to inhibit growth by uncoupling substrate transport and oxidative phosphorylation from
the electron transport system, which in turn inhibits uptake of metabolites by the cell.
Sheu et al. (1975), later determined that acetic acid interfered with PME, with these
organic acids also acting on cellular enzymes to reduce intracellular pH (Huang et al.,

1986).

1.2.7. Bacteriocins

Bacteriocins are antimicrobial substances that have a peptide or protein
component essential for their activity. Although most bacteriocins have a narrow
spectrum of inhibition and only inhibit closely related species, nisin and pediocin exhibit
a broader spectrum of activity and have consequently some attention as antimicrobial

additives to edible film.

70

1.2.7.1. Nisin

Nisin, the first bacteriocin to be used in the food industry, was recognized as a
safe biological food preservative by a joint F AO/WHO commission on food additives in
1968 (FAO/WHO 1969). Twenty years later, nisin was accepted by the United States
Food and Drug Administration (FDA, 1988). Nisin has been shown to be very effective
in cheese products, including processed cheeses and cold-pack cheese spreads (Delves-
Broughton, 1990). The legal precedent for use of nisin in US. foods was set with
pasteurized cheese spreads (900 IU/mg) (Food and Drug Administration, FDA, 1988).

Nisin, a protein of 34 amino acids produced by Lactococcus lactis subsp. lactis,
(Jung, 1991) possesses amphiphilic characteristics with clusters of hydrophobic and
hydrophilic residues at the N and C-terminus, respectively. Gross and Morell (1967)
identified didehydroalanyllysine and isoleucine at the C- and N-terminus of nisin,
respectively (Gross and Morel], 1970). The thioether cross-linkages and highly reactive
double bonds are likely responsible for important functional properties of nisin, including
acid tolerance, therrnostability and bactericidal activity.

One of the mostly investigated bacteriocins for antimicrobial edible film studies,
nisin can be incorporated into film solution or applied directly to the film surface after
casting. Various nisin-containing protein-based films (e.g. whey protein, corn zein-,
wheat protein-, and soy protein) have been assessed for antimicrobial activity against
Gram-positive bacteria such as L. monocytogenes and lactic acid bacteria. Since nisin is
more active in hydrophilic environments, WPI films which contain higher numbers of
hydrophilic residues than zein or wheat protein films reportedly produce larger inhibition

zones against L. monocytogenes.

71

The solubility, stability and biological activity of nisin are highly pH dependent.
Biological activity and stability decrease sharply at higher pH values (Barranova et al.,
1976; Liu and Hansen, 1990). Nisin is insoluble at neutral and alkaline conditions
(Hirsch, 1951; Hurst, 1981; Liu and Hansen, 1990) and more stable to heat at low rather
than high pH (Tramer, 1964). Stability of nisin to heat and storage depends on pH,
chemical composition of the solution and the temperature.

Nisin inhibits the majority of Gram-positive bacteria (Table 1.3) (Mattick and
Hirsch, 1947; Ogden and Tubb, 1985). The effectiveness of nisin against various bacteria
in meat and meat products has been investigated. Nisin can reportedly inhibit the growth
of Bacillus licheniformis (Bell and DeLack, 1986), CIostridium sporogenes (Rayman et
al., 1981) and lactic acid bacteria in cured and fermented meat products (Collins-
Thompson et al., 1985).

When combined with chelating agents, several reports suggest that nisin may be
effective against Gram-negative bacteria in food (Shelef et al., 1995; Mahmoud and El-
Baradei, 1998; Wells et al., 1998; Boziaris and Adams, 1999). While the outer membrane
of Gram-negative bacteria prevents penetration of nisin into the cytoplasmic membrane
(Stevens et al., 1991; Schved et al. 1994), chelating agents such as EDTA as well as
sublethal heating, low pH, or freezing disrupt the permeability barrier thus increasing the
sensitivity of Gram-negative organisms such as Salmonella enterica, S. Typhimurium, E.
coli, and Shigella to nisin (Stevens et al., 1991; Kalchayanand et al., 1992; Ganzle et al.,
1999). EDTA (0.3mM) and nisin (50ug), in combination with heating (0.3 min) at 37°C,
reduced the numbers of Erwinia carotovora, E. chrysanthemi, Pseudomonas fluorescens,

and P. viridiflava in trypticase soy broth by 2 logs, and at 49°C by 3 logs compared to the

72

unheated control at 25°C. Nisin activity against some Gram-negative bacteria can be
enhanced by trisodium phosphate (TSP). Following a 10 minute exposure to sublethal
concentrations of TSP (0.5 to 5 mM), Cameiro de Melo et al. (1998) reported that cell
suspensions of Campylobacter jejuni, Escherichia coli, Pseudomonas fluorescens and
Salmonella enteritidis showed greatly enhanced susceptibility to nisin (luM). Under
optimal conditions at 37°C, viable counts decreased up to 6 logs after 30 minutes. Pulsed-
electric field treatment, which induced sublethal injury, also enhanced the bactericidal
action of nisin against Bacillus cereus and Escherichia coli (Pol et al., 2000; Terebiznik
etaL,2000)

Activity of nisin is strongly influenced by various environmental factors including
pH, temperature and NaCl. Benkerroum and Sandini (1988) showed that L.
monocytogenes was more sensitive to nisin in tryptose soy broth at pH < 5.94 than at pH
7.0. Increased antimicrobial activity of nisin against various pathogens at low pH was
also reported by Harris et al.(1991) and Tatini (1992) with low pH increasing the
sensitivity of E. coli and S. enterica to nisin (Ganzle et al., 1999). Tolerance of vegetative
B. cereus cells to nisin increased as the pH of the broth increased from 5.53 to 6.01 and
6.57. Nisin activity is also directly related to incubation temperature. According to Tatini
(1992), the minimum concentration of nisin needed to inhibit grth of L.
monocytogenes was two to three times higher at 35 than at 4°C. Similarly, 5 and 50
ug/ml were needed to inhibit outgrowth of B. cereus spores at 8 and 15°C, respectively.
Harris et a1 and Ganzle et al. (1999) also reported that addition of 2 to 3% sodium
chloride enhanced the activity of nisin against L. monocytogenes, S. Typhimurium, and E.

coli (< 10 pg /ml) in laboratory media.

73

1.2.7.2. Pediocin

Pediocin is produced by Pediococcus acidilactici. Among the pediocins isolated
from different strains, only pediocin PA1 (Pediococcus acidilactici PAC 1.0) and
pediocin AcH (P. acidilactici LB42-923) have been well characterized. Pediocin AcH,
having a molecular mass of about 2700 Da (Bhunia et al., 1987), contains 62 amino acids
and two disulfide bonds.

The pediocins are another commonly studied group of bacteriocins for edible film
use, due to their wide spectrum of antimicrobial activity and their effectiveness over a
wide range of pH and temperature. Antimicrobial activity of pediocin is retained at
100°C, reduced at 121°C and most evident at pH 4-7 with substantial losses at pH <3 or >
9. Pediocin remains active following treatment with lipase, phospholipase C, lysozyme,
DNase or RNase, but its activity is destroyed by protease, papain and a-chymotrypsin
(Gonzalez and Konka, 1987). While pediocin activity was unaffected during 6 months of
frozen storage, over 50% of the activity was lost after 12 weeks at ambient temperature
(Ray, 1996).

Pediocin is inhibitory to a broad range of bacteria (Table 1.3). Inhibition of L.
monocytogenes with pediocin AcH in various foods has been well documented. L.
monocytogenes numbers decreased by 0.74 log within 2 h in beef wieners that were
inoculated with a pediocin-producing strain of P. acidilactici (Yousef et al., 1999).
Numbers of L. monocytogenes increased initially and then markedly decreased with
pediocin production by P. acidilactici with pediocin activity also detected in the wiener

exudate during the last stage of P. acidilactici growth. Degnan et al. (1992) investigated

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survival of L. monocytogenes in temperature-abused, vacuum-packed wieners which
contained pediocin- and non-pediocin-producing P. acidilactici. Listeria populations
increased 3.2 logs after 8 days of storage at 25°C in the absence of any Pediococcus
strain, remained unchanged in the presence of the non-pediocin-producing strain, and
decreased by 2.7 logs using the pediocin-producing strain.

Inhibition of L. monocytogenes by pediocin-producing starter cultures was
reported during fermentation of dry and semi-dry sausage (Berry et al., 1991). Using a
pediocin-producing strain of Pedioccocus and a nonbacteriocinogenic starter, L.
monocytogenes populations decreased ~2 logs and <1 log respectively, during
fermentation of semi-dry sausage. Antilisterial activity from a pediocin-producing strain
also was demonstrated during manufacture of turkey summer sausage (Luchansky et al.,
1992) with pediocin and non-pediocin—producing strains decreasing populations of L.
monocytogenes by 3.4 and 0.9 log CFU/g, respectively, after 12 h of fermentation.

Several investigators observed potential benefits of adding pediocin to dairy
products. Pucci et al. (1988) showed that incorporating a crude extract of pediocin PAl
into cottage cheese, cheese sauce and half-and-half decreased the numbers of L.
monocytogenes in all inoculated samples after the first day of refrigerated storage. The
effectiveness of pediocin AcH in controlling L. monocytogenes in dry milk, ice cream,
and cottage cheese was also studied by Motlagh et al. (1992), who found that pediocin
decreased numbers of L. monocytogenes by ~l log in sterile ground beef, sausage mix

and cottage cheese after 1 h of storage at 4°C. Pediocin (1350 AU/ml) also reduced

Listeria populations 2 to 4 logs in dry milk after 1 day of storage at 4°C.

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In addition to meat and dairy products, other foods may benefit from
biopreservation with pediocin and pediocin-producing strains. Choi and Beuchat (1994)
added a bacteriocin extract from P. acidilactici M to kimchi during fermentation. This
treatment immediately reduced numbers of L. monocytogenes in the inoculated product

and inhibited growth of the organism during 16 days of fermentation.

1.2.7.3. Mode of Action of Bacteriocins

Bacteriocins disrupt membrane activity by pore formation and may have
additional effects on electron transfer chain components. Binding of bacteriocin to the
cell membrane results in the formation of peptide pre-aggregates that induce
conformational changes and pore formation (Sahl, 1991; Benz et al., 1991; F reund et al.,
1991) which in turn decreases the membrane potential and proton gradient across the
membrane. For example, Bruno et a1 (1992) showed that the addition of 2.5 pg of
nisin/ml completely dissipated both components of the proton motive force in L.
monocytogenes. Okereke and Montville (1992) also found that nisin inhibited the growth
C. sporogenes PA 3679 by dissipating the proton motive force and possibly inhibiting
ATPase activity, thereby exhausting intracellular ATP.

Like other bacteriocins, pediocin’s bactericidal mode of action against Gram-
positive bacteria involves adsorption to lipoteichoic acid receptors on the cell surface
followed by penetration through the wall and contact with the cytoplasmic membrane
(Gonzales and Kunka, 1987; Bhunia et al., 1991). Gram-negative bacteria do not possess

lipoteichoic acid and are therefore unable to adsorb pediocin. Pediocin produces a

76

conformational change in the cell wall of Gram-positive bacteria which interferes with
proper barrier function (Jack et al., 1995)

Sublethally stressed Gram-negative bacteria can become sensitive to bacteriocins
even though the molecule is not adsorbed by the cell. In such cells, the cell-surface
barrier function is impaired, thus allowing bacteriocins to pass through the impaired wall,
come in contact with the cytoplasmic membrane, and destabilize its function leading to
cell death. Pediocin is also active against some Bacillus and Clostridium species

(Kalchayanand, 1990), preventing outgrowth of germinated spores.

1.2.8. Natural Antimicrobials
1.2.8.1. Lysozyme

Lysozyme, another popular choice for the production of antimicrobial films, is an
enzyme comprised of 129 amino acids, crosslinked by four disulfide bonds. Dried egg
white, the commercial source for lysozyme, contains about 3.5% lysozyme. However,
lysozyme is also present in mammalian milk, tears and other secretions, insects and fish.
It is heat stable (100°C) at pH <5.3, but is inactivated at lower temperatures when the pH
is increased (Smolelis and Hartsell, 1952; Matsuka et al., 1966). This remarkable stability
is attributed to the four disulfide bonds present in this small protein. Plasticizers used in
edible film such as glycerol and sorbitol help stabilize lysozyme against heat through
hydrophobic interactions that reduce the complete transfer of hydrophobic groups from
an aqueous to a non-polar environment (Yashitake and Shininichiro, 1977; Back et al.,
1979). Therefore, lysozyme is highly suited for heat processed films such as heat-pressed

corn zein-based films (Dawson et al., 1997; Padget et al., 1998).

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Lysozyme is most active against Gram-positive bacteria, most likely because
peptidoglycan of the cell wall is more exposed. Penetration through the outer membrane
can be accomplished by using either chelating agents or osmotic shock. The outer
membrane of Gram-negative organisms contains divalent cations that stabilize
lipopolysaccharide within the membrane. Chelating agents remove cations and thus
increase cell permeability of Gram-negative bacteria to lysozyme (Hancock, 1984;
Samuelson et al., 1985; Hughey et al., 1989; Payne et al., 1994; Razavi- Rohani and
Griffits, 1996).

Vedmina et al. (1979) tested the sensitivity of lysozyme against 476 strains of
Gram-negative bacteria and found high resistance to lysozyme in Vibrio cholera El Tor
and Pseudomonas. Organisms exhibiting varying sensitivity include Aeromonas and

enteropathogenic Escherichia coli.

1.2.8.1.1. Mode of Action

Lysozyme catalyzes the hydrolysis of B-1,4 glycosidic bonds between N-
acetylmuramic acid and N-acetylglucosamine of peptidoglycan in bacterial cell walls,
which leads to cell wall degradation and lysis in hypotonic solutions. Depending on the

enzyme source, chitin and certain esters are also susceptible to lysozyme.

1.2.8.2. Spices, Herbs, and Essential Oils
Essential oils are responsible for the odor, aroma, and flavor of spices and herbs.
These compounds can be added to edible film to modify flavor, aroma and odor as well

as antimicrobial properties. Films containing these ethanol-soluble compounds will also

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show activity against both Gram-negative and Gram-positive bacteria, with Gram-
positive organisms being more sensitive (Beuchat and Golden, 1989). Ting and Deibel
(1992) reported that addition of cloves, oregano, sage, rosemary and nutmeg to TSB
inhibited growth of L. monocytogenes at 24°C. According to Bahk et al.(1981), addition
of 5% cinnamon also inhibited growth of L. monocytogenes in TB at 4°C; however, 0.5%
ginger, garlic, onion and mustard as well as ginseng, saponin and mulberry extract were
noninhibitory.

Activity of garlic powder and cloves was also investigated against S.
Typhimurium and S. aureus by Teerapom (1995). Results showed that 12% garlic
powder and 0.4% clove oil led to a > 5-log reduction of S. Typhimurium, whereas 0.4%
clove oil and a much higher concentration of garlic powder (58%) were needed for
similar inhibition to S. aureus.

Antimicrobial activities have been known among various plant essential oils for
centuries. However, their use as food additives is limited by their strong flavor. These
extracts contain mostly phenolic compounds such as abietane diterpenes (Moujir et a1,
1993), camosol and ursolic acid (Collins and Charles, 1987) which are presumably
responsible for their antimicrobial action. Antimicrobial activity and composition of the
essential oils from Micromeria cristata subsp. phrygia was investigated against Gram-
negative and Gram-positive bacteria by Tabanca et al. (2001). The essential oils showed
inhibitory activity against E. coli, S. Typhimurium, S. aureus, P. aeruginosa, E.
aerogenes, P. vulgarus, and C. albicans with the major oil compenent being bomeol

(39%).

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Ethanol extracts of rosemary (Oxy’less) (100 mg/ml) effectively killed L.
monocytogenes, Leuconostoc mesenteroides, S. aureus, Streptococcus mutanss and
Bacillus cereus, whereas no activity was observed with Gram-negative bacteria (e.g. E.
coli, Salmonella enteriditis, Erwinia carotovora) or yeasts (e.g. Rhodotorula glut,
Cryptococcus laurentii) (Del Campo et al., 2000).

Elgavyar et a1. (2000) evaluated the antimicrobial activity of essential oils from
parsley, anise, angelica, carrot, cardamom, coriander, dill weed, fennel, oregano and
rosemary using a disc diffusion assay. Essential oils from cardoman, coriander, oregano,
basil, celery and parsley inhibited growth of S. Typhimurium, Y. enterocolitica, E. coli, S.
aureus, L. monocytogenes and Aspergillus niger. However, essential oils from dill weed,
fennel, carrot, rosemary and anise were noninhibitory to L. monocytogenes on standard
methods agar (SMA). Inhibition zones were observed for all essential oils except those
from carrots against S. Typhimurium, E. coli and S. aureus on SMA.

The effectiveness of some essential oils is enhanced at lower temperatures and
lower pH values. Ting and Deibel (1992) showed that refrigeration temperatures
increased the inhibitory effect of sage, but not that of cloves or oregano against L.
monocytogenes. In addition, essential oils from thyme and oregano were ten-fold more
effective against B. cereus at 8 compared to 30°C (U ltee et al., 1998).

Surface-active agents in foods such as dairy cream and albumin also were shown
to reduce the inhibitory effect of essential oils (Davidson, 1993; Tassou and Nychas,
1994; Juven et al., 1994; Del Campo et al., 2000). For example, Juven et al. (1994)
showed that serum albumin (9 mg/ml) eliminated the antimicrobial activity of thymol

against S. Typhimurium.

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Essential oils alter the fatty acid composition and phospholipid contact of the cell
membrane, which results in the release of cellular constituents that interfere with
metabolism, disrupting both electron transport and nutrient uptake. These essential oils
also have the ability to disrupt various enzyme systems, including those involved in

cellular energy and structural synthesis

1.2.8.3. Lactoferrin

Lactoferrin (lactotransferrin), an iron binding glycoprotein, is present in bovine
milk and can bind two iron atoms per molecule (Ashton and Busta, 1968; Reiter, 1983).
This protein was shown to effectively inhibit the growth of several bacteria including
Bacillus subtilis, B. stearothermophilus, L. monocytogenes, E. coli, Microccoccus spp,
and Klebsiella spp. (Oram and Reiter, 1968; Reiter, 1978; Payne et al., 1989; Payne et al.,
1990; Korhonen, 1978). Payne et al (1990) showed that lacroferrin had a bacteristatic
effect against L. monocytogenes. At 30 mg/ml Apo-lactoferrin (iron-free lactoferrin)
reduced Listeria population 10 fold. At 2.5 mg/ml, lactoferrin had no activity against S.
Typhimurium or P. fluorescens with only minimal activity against E. coli 0157:H7 and
L. monocytogenes (Payne et al., 1994). Some Gram-negative bacteria may be lactoferrin
resistant due to the presence of siderophores that aid in adaption to low-iron
environments (Crichton and Chaloteux-Wauters, 1987). In addition, bacteria with low
iron requirements such as lactic acid bacteria would not be adversely impacted by
lactoferrin (Reiter and Orarn, 1986).

Ashton and Busta (1968) reported that the inhibitory activity of lactoferrin was

likely due to chelation of iron as well as calcium and magnesium ions. Inhibition of L.

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monocytogenes by lactoferrin is directly related to iron availability in the medium, with
L. monocytogenes surviving best in iron-rich media (Payne et al. 1989). However, Arnold
et al. (1982) showed that lactoferrin inhibited many bacteria in an iron-rich environment.
Lactoferrin caused the release of anionic polysaccharides from the outer membrane of E.
coli by chelation of cations that stabilize lipopolysaccharides. Thus, lactoferrin may

increase outer membrane permeability to hydrophobic compounds.

Lactoferricin B, the active region of lactoferrin, was isolated by acid-pepsin
hydrolysis from the N-terminal region of the molecule (Bellamy et al., 1992).
Lactoferricin contains 25 amino acid residues. Bellamy et al. (1992) and Jones et al.
(1994) determined that lactoferricin was inhibitory to bacteria at concentrations of 0.3 to
150 ug/ml (Table 1.3). Pseudomonas fluorescens, Enterococcus faecalis and
Bifidobacterium biflidum strains were highly resistant to this peptide. These results
confirm and expand on earlier inhibition studies with lactoferricin B (Tomita et al.,
1992;Wakabayashi et al., 1992). According to Kumar et al. (1999), lactoferricin B (100
ug/g) also reduced E. coli 0157:H7 populations by 0.8 log CF U/g in ground beef. While
the mode of action of lactoferricin has not been fully elucidated, it is thought to alter the

permeability of the membrane because of its cationic feature (Conner, 1993; Jones et al.,

1994).

1.2.8.3. Liquid Smoke
Liquid smoke is a solution of natural wood smoke flavors prepared by burning a

wood (e.g. hickory, maple) and capturing the natural smoke flavors in water.

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Alternatively, liquid smoke can be derived from the destructive distillation of a wood - i.e
the breakdown or cracking of the wood fibers into chemical constituents which are
distilled out of the wood residue. Most liquid smokes are very acidic, although some
partially neutralized liquid smokes are also available. While some commercially available
smoke can be used at filll strength, others are normally diluted in water or another
appropriate diluent. Commercial liquid smoke products used in processed meats,
sausages and cheeses contain phenols and acetic acid, which are bactericidal at relatively
low concentrations. Liquid smoke can inactivate common food-borne pathogens
including E. coli, Salmonella, S. aureus, and L. monocytogenes.

Several investigators examined the potential of commercial liquid smokes to
inactivate L. monocytogenes in culture media and meat products. Wendorff (1989) found
that liquid smoke compounds (0.5%) were listericidal in both phosphate buffer and
processed meats. Faith et al. (1992) also reported that L. monocytogenes was inactivated
by addition of 0.2 and 0.6% liquid smoke to wiener exudate with D-values of 36 and 4.5
h, respectively; however, numbers of Listeria in the untreated exudate increased to 8 logs
after 3 days at 25°C. Among 11 individual phenols tested, only isoeugenol showed
antilisterial activity in tryptose broth (TB) during incubation at 37°C. In the presence of
isoeugenol (100 ppm), greater inhibition of the pathogen was observed in TB acidified
with acetic acid to pH 5.8 compared to pH 7.0.

In addition to meat products, antimicrobial activity of liquid smoke was also
‘ evaluated against molds on Cheddar cheese (Wendorf et al., 1993). Liquid smoke
reportedly inhibited the growth of Aspergillus oryzae and increased the lag phase of

Penicillium camemberti and P. roqueforti. Among 8 phenolic compounds tested, only

83

isoeugenol retarded the growth of all three molds. P. camemberti was slightly inhibited
by m-cresol and p-cresol, while A. oryzae was inhibited by guaiacol, 4-methylguaiacol,
m-cresol and p-cresol.

Based on these findings, liquid smokes which possess antimicrobial, antioxidant,
color, and flavor properties, have the potential to become attractive edible film additives.
Thus a, incorporation of liquid smoke has only been studied for edible collagen casings.
Liquid smoke was introduced into the acid-swollen collagen mass before extrusion as a
casing or film (Miller, 1975). Since liquid smoke is generally very acidic (pH 2.5 or less),
it is compatible with the gel system and, in fact, can replace a portion of the acid
normally added to induce swelling. The resultant edible collagen casings with uniformly
dispersed liquid smoke reportedly had increased tensile strength and improved film

clarity.

1.2.9. Curing Agents

1.2.9.1. Sodium Chloride

Sodium chloride (NaCl), recognized as a food preservative since ancient times,
can be used alone or in combination with other preservation techniques such as
pasteurization or fermentation. Bacterial food-borne pathogens are generally susceptible
to NaCl, except S. aureus, which can grow at low water activities (0.83-0.86) (McLean et
al., 1968). Another salt-tolerant pathogen is L. monocytogenes, which can grow at
concentrations up to 10% NaCl and survive for long periods of time in saturated brine
solutions. Yeasts and molds are also more tolerant to low water activity than bacteria.

with xerotolerant fungi growing at a water activity value as low as 0.61 (Corry, 1987).

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Sodium chloride inhibits microbial growth by its plasmolytic effect. The
antimicrobial activity of sodium chloride is related to its ability to reduce water activity in
the food. Microbial cells lose water when the water activity of the external environment
is reduced, which results in growth inhibition or possible death (Sperber, 1983). In
addition to this osmotic effect, NaCl limits oxygen solubility, alters pH, and is itself toxic

for microbial cells (Banward, 1979).

Incorporation of NaCl into protein-based films as an antimicrobial agent is of
limited use since physical properties of protein films decrease with increasing ionic
strength of the film solution. At high ionic strength, proteins aggregate to form turbid

opaque gels possessing a high-water holding capacity (Doi and Kitabastake, 1997).
1.2.9.2. Nitrite

Sodium nitrite (NaNOz) and potassium nitrite (KNOZ) are primarily used to
inhibit C. botulinium growth and toxin production in cured meats. Nitrite inhibits
bacterial sporeforrners by preventing outgrowth of the germinated spores (Cook and
Pierson, 1983; Duncan and Foster, 1968). Nitrite effectiveness also depends on other
environmental factors. For example, nitrite is more active at low pH and anaerobic
conditions (Buchanan and Solberg, 1972; Woods et al., 1989). When used as reducing
agents, ascorbate and isoascorbate enhance the antibotulininal action of nitrite (Tompkin
et al., 1978; Roberts et al., 1991). Nitrite is also inhibitory to other bacteria including C.
perfiingens, E. coli, Acromobacter, Enterobacter, Flavobacterium, Micrococcus, and

Pseudemonas spp. at 200 ug/g (Gibson and Roberts, 1986b). Growth of L.

monocytogenes was inhibited for 40 days at 5°C by treating smoked salmon with 200

85

ppm sodium nitrite (Pelroy et al., 1994). However, Gibson and Roberts (1986a, 1986b)
found that enteropathogenic E. coli, Salmonella spp., fecal streptococci, Lactobacillus
(Castellani and Niven, 1955) and Bacillus (Grever, 1974) were resistant to 400 ug/g

nitrite when used with 6% salt.

Incorporation of nitrite into edible films has not yet been studied, even though
nitrite appears to be a suitable antimicrobial agent for antimicrobial edible film
production. In this regard, application of the films containing nitrite on ready-to-eat-meat
products may provide a possible solution for preventing growth of L. monocytogenes and
spoilage bacteria that may contaminate such products after processing, with the potential

benefit of also improving surface color.

Nitrite works by inactivating various enzymes or enzyme systems. Using
vegetative cells of C. sporogenes, Woods et al. (1981) showed that pyruvate-ferrodoxin
oxidoreductose and ferrodoxin were susceptible to nitrite. Inhibition of these enzymes
causes a reduction in intracellular ATP and excretion of pyruvate. The phosphoroclastic
system of C. botulinium and C. pasteurianum is also inhibited by nitrite (Woods and
Wood, 1982; Carpenter et al., 1987). Rowe et al. (1979) showed that the other
antimicrobial mechanism for nitrite was inhibition of active transport, oxygen uptake, and
oxidative phosphorylation by oxidizing ferrous iron from an electron carrier (Rowe et al.,

1979, Yang, 1985)

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1.3. ANTIMICROBIAL EDIBLE FILMS

Various antimicrobial edible films have been developed to control the growth of
spoilage and pathogenic microorganisms that may contaminate the surface of foods after
processing. In most solid foods, contamination and microbial growth occurs on the food
surface, which leads to a reduction in product shelf-life. Edible films containing various
antimicrobials such as benzoic acid, sorbic acid, propionic acid, lactic acid, nisin, and
lysozyme have been investigated to retard the growth of bacteria, yeasts and molds on
different product surfaces.

The primary advantage of antimicrobial edible films is that the antimicrobial
agents in these films can be specifically targeted to post-processing contaminants on the
food surface, with the diffusion rate of the antimicrobial into the product partially
controlled by levels incorporated into the film. Guilbert and his research group
(Giannakopoulos and Guilbert, 1986; Guilbert et al., 1985; Guilbert et al., 1988)
evaluated diffusivity of sorbic acid from casein films in different model systems. Using a
cell membrane separated by casein film, their data showed that low temperatures (10°C)
decreased the diffusivity of sorbic acid; however, lower water activity had no effect.
They theorized that at higher levels, increased networking within the gel acted to restrict
the movement of sorbic acid. Vojdani and Torres (1989a, b, 1990) also evaluated the
permeability of several polysaccharide-based films prepared both with and without
various combinations of lipids and potassium sorbate. Using permeability cells
methylcellulose-palmitic acid films appeared to be most promising, with permeability of
the film to sorbic acid decreasing from 10'8 to 10'10 mg/sec.cm2 as pH increased from 3 to

7 and aw decreased from 0.8 to 0.65 (Rico-Pena and Torres, 1991).

87

Chitosan, like other polysaccharides, forms a strong film that can carry a high
amount of antimicrobials. Chitosan also has been reported to be a good choice for
antimicrobial films due to its good film-forming properties, ability to adsorb nutrients
used by bacteria (Darmadji and Izumimoto, 1994), capacity to bind water and inhibit
various bacterial enzyme systems (Young et al., 1982). However, neutralized chitosan
alone had no effect on bacterial growth when it was applied to the surface of meat
products. Antimicrobial chitosan films were subsequently prepared by dissolving
chitosan in hydrochloric, formic, acetic, lactic and citric acid solutions (Begin and
Calstren, 1999). Films made from hydrochloric, formic and acetic acid were hard and
brittle, whereas those containing lactic or citric acid were soft and could be stretched. The
same research group designed antimicrobial chitosan films containing acetic acid or
propionic acid, with or without the addition of lauric acid or cinnemaldehyde, to improve
refrigerated shelf-life of vacuum-packaged processed meats (Ouatarra et al., 2000a).
They indicated that film application delayed or completely inhibited enteric bacteria,
Lactobacillus sakei and Serratia liquefaciens, on meat products. Films containing
propionic acid were more effective than films containing acetic acid for reducing growth
of L. sakei with the opposite observed for S. liquefaciens. Diffusion of acetic acid from
the film matrix was limited by addition of lauric acid, with 2-22% of acetic acid
remaining in the chitosan after 168 h of storage at 4°C. However, propionic acid almost
totally diffused from the film after 48 h of storage. Release of organic acids from chitosan
film is dependent on many factors, including electrostatic interactions between the acid
and polymer chains (Demarger-Andre and Domad, 1994), ionic osmosis and structural

changes induced by presence of the acid (Narisawa et a1, 1996). Ouattara et al. (2000b)

88

also tested the impact of temperature (4 to 24°C) and pH (5.7 to 7.0) on diffusion of
acetic and propionic acid from chitosan films immersed in water. Whereas diffusion was
unaffected by pH, a decrease in temperature from 24 to 4°C decreased the diffusion
coefficients for acetic and propionic acid from 2.59 x 10'12 mZ/sec to 1.19 x 10"2 mz/sec
and 1.87 x 10'12 mz/sec to 0.91 x 10'12 mz/sec, respectively. The dependency of diffusion
on temperature is explained by effects on solubility of the diffusing molecule, the nature
of adhesive forces at interfaces, and molecular mobility (Vojdani and Torres, 1990;
Myint et al., 1996). Ouattara et al. (2000b) stated that addition of lauric acid (1%) or
essential oils (0.5%) (cinnemaldehyde or eugenol) decreased the diffirsion of propionic
acid, since these additives increased film hydrophobicity and modified the pore
construction/blind porosity of the film, thereby impairing water uptake and molecular

transformation.

Besides propionic acid, fatty acids, and essential oils, sorbate and benzoate also
have been tested in methylcellulose and chitosan films. For example, Chen et al. (1996)
developed antimicrobial methylcellulose, chitosan and methylcellulosezchitosan films
(3:2) containing 2, 4, or 5% sodium benzoate or potassium sorbate. Methylcellulose films
containing 2% sorbate or benzoate yielded clear inhibition zones for Rhotorula rubra and
Penicillium notatum on potato dextrose agar. Chitosan films containing 2% sorbate or
benzoate yielded no zones of inhibition, since the high affinity between chitosan and the
preservatives prevented diffusion of the antimicrobials. Incorporation of both potassium
sorbate and sodium benzoate into methylcellulose/chitosan films did not change the
tensile strength or percent elongation. In a glycerol-water model system (aw=0.8), 40%

and 50-60% of both antimicrobial agents were released from the films after 6 h at 4 and

89

25°C, respectively. Another research group from Taiwan evaluated the antimicrobial
activity of methylcellulose coatings containing benzoic and palmitic or stearic acid
against two osmophilic yeasts (Zygosaccharomyces rouxii and Zygosaccharomyces
mellis) on Taiwanese-style fruit preserves made from plums (Chen et al. 1999). Coatings
containing 50-100 ug/g benzoic acid inhibited Z. rouxii and Z mellis at room
temperature, with sensory characteristics of preserves such as flavor, texture, appearance
and overall acceptability not affected by the coating.

Protein-based edible films are also very good carriers of food additives, including
antimicrobial and flavor agents, due to their favorable encapsulated nature. Zein films
have been used in conjunction with potassium sorbate to control surface microbial
growth. The diffusion barrier properties of zein films were confirmed in microbial tests
using a model food system and S. aureus as the challenge organism. A reduced
preservative diffusion rate due to barrier properties of zein films was identified as the
mechanism for product shelf-life enhancement (Torres et al., 1985; Torres and Karel,
1985). The diffusion of sorbic acid from various wheat gluten films into a model food
was also measured and modeled. When Penicillium notatum was used as the test
organism, simple gluten-based films had no fungicidal effect. However, the gluten/lipid-
based films showed strong sorbic acid retention and marked fungicidal activity at 30 and
40°C, delaying P. notatum growth for more than 21 days.

Similarly, antimicrobial soy and corn protein-based films were developed by
Dawson and his lab group at Clemson University. When prepared to contain nisin and
lysozyme, these films were inhibitory to Gram-positive bacteria both on solid and in

liquid media (Orr et al., 1996a, b; Dawson et al., 1995; Dawson et al., 1997; Padget et al.,

90

1995; Padget et al., 1998). Addition of EDTA to these films also led to inhibition of
Gram-negative organisms (Orr et al., 1996a). Modifying the water permeability by
incorporating short chain fatty acids (lauric acid) reduced the effectiveness of nisin on
solid media, whereas films with lauric acid were as effective as nisin against Gram-
positive bacteria in liquid media (Dawson et al., 1997). Padget et al. (1998) incorporated
nisin and lysozyme into soy protein and corn zein films using the heat press and casting
methods. Both antimicrobial films containing lysozyme (10 to 133 mg/g film) or nisin
(0.1 to 6.0 mg/g film) inhibited Lactobacillus plantarum on MRS media. Orr et al. (1998)
found that com zein films containing 150 mg of nisin reduced L. monocytogenes
populations 1.3 to 2.2 logs in milk after 72 h at 4°C, with no inhibition observed with
nisin free-films. Use of zein film coatings containing nisin (1000 IU/g) also reportedly
reduced L. monocytogenes populations 1 to 3 logs on ready-to-eat chicken during 30 days
of refi'igerated storage (J anes et al., 1999).

Rodrigues and Han (2000) reported that when incorporated into WPI films,
lysozyme and nisin effectively inhibited Brochotrix thermosphacta but not L.
monocytogenes. Addition of EDTA increased the inhibitory effect of these films against
E. coli and L. monocytogenes on trypticase soy agar. Other antimicrobial WPI films
containing sorbic acid (SA) or p-aminobenzoic acid (PABA) were developed by Cagri et
al. (2001). Both of these films reportedly inhibited the growth of L. monocytogenes, E.
coli 0157:H7, and S. Typhimurium on TSAYE agar. Subsequently, these films were
tested between beef bologna and summer sausage slices that were surface inoculated with
the same pathogens at a level of 106 CFU/g (Cagri et al., 2002a). WPI films containing

SA or PABA decreased Listeria, E. coli and S. Typhimurium populations 3.4-4.1, 3.1-

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3.6, and 3.1-4.1 logs on bologna and sausage after 21 days of aerobic storage at 4°C,
respectively. Growth of mesophilic aerobic bacteria (MAB), lactic acid bacteria (LAB)
and mold/yeast on slices was also inhibited with WPI films containing SA or PABA
compared to antimicrobial-free control films. In the same study, film tensile strength
decreased while % elongation remained unchanged following 72 h of product contact.
Subsequently, heat—sealed WPI casings containing SA, PABA, or SAzPABA (1:1) were
developed for hot dog manufacture, with these casings compared to commercial collagen
and natural casings (Cagri et al., 2002b). WPI casings containing PABA inhibited L.
monocytogenes grth on hot dogs during 42 days of refrigerated storage; however,
films with SA or SAzPABA were less effective. Sensory (texture, flavor, juiciness,
overall acceptability), chemical (TBA, pH, moisture, fat, protein), physical (purge, color),
and mechanical (shear force) characteristics of hot dogs with WPI casings containing
PABA were comparable to hot dogs prepared with collagen and natural casings.
Consequently, WPI casings containing PABA may eventually prove useful in minimizing
risk of Listeria growth on hot dogs.

Ko et al. (2001) also tested the antilisterial activity of nisin (200 — 8000 IU/g film)
when incorporated into WPI, soy protein isolate, egg albumin, and wheat gluten films.
All of these films inhibited Listeria, with greatest activity observed at low pH (2.0 or
3.0). WPI films containing nisin were most effective against L. monocytogenes due to
their increased hydrophobicity, with their mechanical properties also remaining
unchanged by the addition of nisin.

Various edible antimicrobial films have also found use in vegetables to prevent

the grth of spoilage and pathogenic bacteria. Zhuang et al. (1996) investigated use of

92

antimicrobial cellulose-based edible films containing citric acid, acetic acid, or sorbic
acid (0.2 to 0.6%) on tomatoes inoculated with Salmonella montevideo. Although coating
with a hydroxypropyl methylcellulose (HPMC) solution reduced Salmonella populations
by 4.5 logs on the surface of tomatoes; a reduction of only 2.0 logs was achieved in core
tissue. S. montevideo cells penetrating into the core tissue when tomatoes were dipped in
the 30°C bacterial suspension were likely protected from inactivation during coating
(Zhuang et al.,l995). Among the antimicrobials tested in HPMC films, only 0.4% sorbic
acid enhanced the inactivation of S. montevideo (~1.0 log) on the surface of tomatoes.
However, tomatoes coated in HPMC containing 0.4% sorbic acid appeared chalky, less
firm and exhibited color changes that may limit possible commercial applications.

Most recently, Cha et al. (2001) reported that nisin-containing K-carrageenan, MC
and HPMC films prepared by either a heat press or casting method were inhibitory to
Micrococcus luteus in agar well diffusion assay. Nisin reportedly diffused faster from
MC than from K-carrageenan or HPMC films. Not surprisingly, the heat-pressed films
had lower antimicrobial activity than the cast films.

In conclusion, the use of edible films or coatings on various food products
continues to expand. The numerous benefits of edible films as carriers of antimicrobial as
well as flavors, antioxidants and color agents justifies further research in this field. Edible
films containing antimicrobial agents have been shown to effectively inhibit both
pathogenic and spoilage organisms on a wide variety of ready-to-eat food surfaces. These
films have the ability to control the diffusion rate of the antimicrobial agent and also
serve as a good barrier against oxygen and water vapor transmission. The antimicrobial

edible films appeared to be a possible solution to reduce the incidence of pathogens

93

especially L. monocytogenes on food surfaces. However, only some of these
antimicrobial edible films received commercial acceptance. Further research is needed
for development of feasible application methods for the industry such as extrusion of film

solution into tubular shaped film in various sizes for processed meat products.

94

CHAPTER 2.

ANTIMICROBIAL, MECHANICAL, AND MOISTURE BARRIER PROPERTIES

OF LOW PH WHEY PROTEIN- BASED EDIBLE FILMS CONTAINING P-

AMINOBENZOIC OR SORBIC ACIDS

Cagri, A., Ustunol, Z., Ryser, E.T.

Journal of Food Science. 2001. 66(6) : 865-870

95

2.1. ABSTRACT

Low pH (5.2) whey protein isolate—based edible films containing p-aminobenzoic
acid (PABA) or sorbic acid (SA) were developed and assessed for inhibition of Listeria
monocytogenes, Escherichia coli 0157:H7, and Salmonella Typhimurium DT104 in a
disc diffusion assay. Water vapor permeability (WVP), tensile strength (TS), and percent
elongation (% E) were also determined. Using 1.5% PABA and SA, average inhibition
zone diameters were 21.8, 14.6, 13.9 and 26.7, 10.5, 9.7 mm for L. monocytogenes, E.
coli 0157:H7 and S. Typhimurium DT104, respectively. Three strains of S. Typhimurium
DT104 were resistant to 0.5% SA. Addition of PABA and SA increased %E, but
decreased TS. WVP was not affected by 0.5 and 0.75% SA; however, PABA increased

WVP.

2.2. INTRODUCTION

Microbial stability of a food surface is a major determinant of product quality and
safety during storage and distribution since most Class I product recalls in the US. result
from post-processing contamination during subsequent handling and packaging. In
December 1998, new food safety concerns were raised when consumption of hot dogs
was traced to over 100 cases of listeriosis, including 21 fatalities in 22 states (CDC,
1999). A nationwide recall was subsequently issued ‘for 35 million pounds of
contaminated product. This pathogen continues to threaten the processed meat industry.
Sixty-three of 97 microbiologically related Class I recalls issued from January 1999 to

October 2000 involved a total of more than 3.5 million pounds of cooked/ready-to-eat

96

meats contaminated with Listeria monocoytogenes (USDA-FSIS, 2000). Two additional
foodbome pathogens, namely Escherichia coli 0157:H7 and Salmonella Typhimurium
DT104 are also raising considerable public health concerns. E. coli 0157:H7 has been
responsible for many widely publicized outbreaks involving ground beef (Bell et al.,
1994), fermented meat products (Tilden et al., 1996) and fresh produce (Besser et al.,
1993). In addition, over 30 million pounds of raw ground beef have been recalled since
1995 due to E. coli 0157:H7 contamination (USDA-FSIS, 2000). S. Typhimurium
DT104, a multiantibiotic resistant strain, is also emerging as a serious foodbome
pathogen of public health concern with 103 of 306 (34%) S. Typhimurium isolates
serotyped at CDC (Centers for Disease Control and Prevention) resistant to arnpicilin,
chloramphenical, streptomycin, sulfonomides and tetracyclines (Glynn et al. 1998).
Incorporating antimicrobial compounds into edible films or coatings provides a
novel means for enhancing the safety and shelf life of ready-to-eat foods. Dawson et al.
(1997) and Padget et al. (1998) used nisin and pediocin in soy protein and corn zein films
to inhibit Lactobacillus plantarum and E. coli on laboratory media. Antimicrobial edible
films are receiving attention as a potential pathogen intervention strategy for various
muscle foods. Sirugusa and Dickson (1993) demonstrated that calcium alginate coatings
containing organic acids were marginally effective on beef carcasses, reducing levels of
L. monocytogenes, S. Typhimurium and E. coli 0157:H7 by 1.80, 2.11, and 0.74 logs,
respectively. According to Ming et al. (1997), pediocin-coated cellulosic casings
inhibited L. monocytogenes on ham, turkey breast meat, and beef. In addition, McDade et

al. (1999) reported that dipping frankfurters in an aqueous whey protein solution (pH 5.2)

97

containing propionic/sorbic acid prevented growth of L. monocytogenes on the product
during the first 2 to 3 weeks of storage at 4 °C.

Sorbic acid, p-aminobenzoic acid, lactic acid and acetic acid have a long history
as GRAS food preservatives. The World Health Organization has set the acceptable daily
intake for sorbic and p-aminobenzoic acid at 25 and 30 ppm, respectively (Kabara et al.,
1991). When used in combination with lactic and/or acetic acid, sorbic acid can inhibit
the growth of L. monocytogenes, S. Typhimurium, and E. coli 0157:H7 in many low acid
foods including cold-pack cheese (Ryser and Marth, 1988), bologna (Wederquist et al.,
1994), beaker sausage (Hu and Shelef, 1996) and apple cider (Zhao et al., 1993; Uljas
and Ingham, 1999). p-Aminobenzoic acid reportedly exhibited greater inhibitory activity
against L. monocytogenes, E. coli and Salmonella enteritidis than formic, propionic,
acetic, lactic or citric acids (Richards et al., 1995).

Use of whey protein antimicrobial-containing films as a casing for frankfurters
appears to be a promising means of retarding surface microbial growth, thereby
enhancing product safety and extending shelf life. However, if used as a sausage casing,
the mechanical properties of such an edible film (that is tensile strength, percentage
elongation and water vapor permeability) are of equal importance if the film is to
function properly and provide adequate physical protection for the product during
production and storage. Consequently, our objectives were to (1) develop an edible film
(pH 5.2) from whey protein isolate (WPI) containing p-aminobenzoic acid (PABA) or
sorbic acid (SA) that is inhibitory to L. monocytogenes, E. coli 0157:H7 and S.
Typhimurium DT104 and (2) assess the film for water vapor permeability (WVP), tensile

strength (TS) and percentage elongation (%E) at break.

98

2.3. MATERIALS AND METHODS

2.3.1. Film Preparation

Whey protein isolate (WPI, Alacen 895) (New Zealand Milk Products, North
America, Inc., Santa Rosa, Ca., USA) (5% w/v) and glycerol (Sigma Chemical Co., St.
Louis, Mo.) (2% w/v) were dissolved in distilled water containing 0.04% CaClz (w/v)
(Sigma). After mixing and adjusting the pH to 8.0 with 1.0 N NaOH, the solution was
heated at 90 °C for 30 min in a shaking water bath (170 Marcel Drive water bath,
Precision Scientific, Winchester, VA). Following the addition of candelilla wax (Stahl
Pash, Inc., New York, NY) (0.4%, w/v) during the last 5 min of heating, the solution was
homogenized for 2 min in a SD-45 homogenizer (Tekmar Co., Cincinnati, Ohio, USA),
filtered through cheese cloth and cooled to 23 i 2 °C. After incorporating 0.5, 0.75, 1.0,
or 1.5% (w/v) sorbic acid (SA) or p-aminobenzoic acid (PABA), the pH was adjusted to
5.2 using three different solutions of lactic acid and acetic acid at ratios of 1:0, 1:1, and
7:3 lactic acid (1.0 N): acetic acid (1.0 N). Following degassing by vacuum, the whey
protein solution (40 ml / plate) was cast by pipetting the solution into sterile 17 cm-
diameter Teflon plates. The solutions were dried for approximately 24 h at 23 i 2 °C / 50
i 5% RH, after which the films were peeled from the plates and stored at 23 i 2 °C / 50 i

5% RH until used.

2.3.2. Bacterial Strains

Four strains of Listeria monocytogenes (CWD 95 and CWD 249 from silage,

CWD 201 from raw milk, and CWD 1503 from ground turkey) and three strains of

99

Escherichia coli 0157:H7 (AR, AD 305, AD 317) were obtained from C. W. Donnelly
(Dept. of Nutrition and Food Sciences, University of Vermont, Burlington, Vt., USA).
Five strains of Salmonella Typhimurium DT104 (G01074, G11601, G10931, G10601,
G10127) were obtained from B. Swaminathan (Centers for Disease Control and
Prevention, Atlanta, Ga., USA). All strains were maintained at —70 °C in trypticase soy
broth containing 10% (v/v) glycerol and subcultured twice in trypticase soy broth
containing 0.6% (w/v) yeast extract (Difco Laboratories, Detroit, Mich., USA) at 35 °C /

18-24 h before use.

2.3.3. Diffusion-type Assay

WPI films were aseptically cut into 16-mrn diameter discs using a sterile cork
borer. The discs were then aseptically transferred to pour plates containing exactly 15 ml
of either trypticase soy agar + 0.6% yeast extract (TSAYE) (pH ~6.5) or TSAYE
acidified to pH 5.2 with 1.0 N lactic acid (Difco) which had been previously seeded with
0.1 ml of an 18-24 h culture of the test organism. After 24 h of incubation at 35°C, the
diameter of the inhibition zone around the edible film disc was measured perpendicularly

to the nearest millimeter. The end result was the average of two measurements.

2.3.4. Film Thickness

A model M micrometer (Testing Machine Inc., Amityville, N.Y., USA) was used
to determine film thickness. Measurements were taken at five different locations and the
mean value was used in further calculations for moisture barrier and mechanical

properties.

100

2.3.5. Mechanical Properties

Films were cut into strips measuring 101.6 mm by 25.4 m using a Precision
Sample Cutter (Thawing Albert Instrument Co., Philadelphia, Pa., USA). All films were
conditioned for 48 hours at 23 i 2 °C / 50 i 5% RH before testing. Tensile strength (TS)
and percent elongation at break (%E) were determined according to standard D-882-91
(ASTM, 1992). The test was run using the Instron Universal Testing Machine Model
2401 (Canton, Mass, USA) at 23 i 2 °C / 50 i 5% RH with a static load cell of 1 kN and
a cross head speed of 50.8 cm/min. TS was calculated in MPa from the following
equation:

TS = load /(sample width x sample thickness)
% Elongation at break was determined by the following equation:

% E = (distance sample stretched / original length of sample) x 100

2.3.6. Water Vapor Permeability

Standard Method E96 - 80 (ASTM 1992) was used in which the film was sealed
on top of an aluminum test cup containing desiccant (calcium sulfate) and then placed in
a chamber at 37 °C / 85% RH. The area of the cup mouth was 54 cm2 and the cup well
depth was 1.1 cm. Cups were weighed at 2-h intervals during ~12 h of controlled storage.
Square edible film samples having a surface area of 9 cm2 were placed in the chamber to
examine moisture absorption. WVP was calculated from the water vapor transmission

rate through film, the partial vapor pressure difference between the two sides of the film,

and the thickness according to McHugh et al. (1993).

101

2.2.7. Statistical Analysis
All experiments were replicated three times using a complete randomized design.
Two- way analysis of variance (ANOVA) was performed using the SAS Statistical
Analysis System (SAS Institute Inc., 1990). Means were compared using the Duncan

Grouping test at p=0.05.

2.4. RESULTS AND DISCUSSION

2.4.1. Antimicrobial Properties

Increasing the concentration of PABA and SA in the film discs increased the
diameter of inhibition zones for L. monocytogenes (4 strains), E. coli 0157:H7 (3
strains), and S. Typhimurium (5 strains) on TSAYE (pH 5.2) (p<0.05). SA and PABA are
weak acids and are most effective in the undissociated form (Luck, 1980) due to their
increased ability to penetrate the cytoplasmic membrane of bacteria. (Chichester and
Tanner,1972). At pH 5.2 and 6.5, 28.48 and 1.25 % and 26.18 and 1.11 % of SA (pKa =
4.75) and PABA (pKa = 4.8) is undissociated. Hence, no inhibition was observed in
TSAYE adjusted to pH 6.5 (results not shown). Control films (pH 5.2) without
antimicrobials were non-inhibitory. Therefore, the antimicrobial containing films
developed in our study would be best suited for foods such as meats and cheeses that
have pH values 5 5.2.

All L. monocytogenes strains were inhibited using WPI film discs (pH 5.2)
containing SA or PABA at levels of 0.5, 0.75, 1.0 or 1.5% with inhibition zones ranging
from 12.0 to 32.0 and 4.0 to 27.0 mm, respectively (Table 2.1, 2.2). El-Shenawy and

Marth (1988) also showed that L. monocytogenes was inhibited when 0.2 to 0.3%

102

Table 2.1. Antimicrobial activities of whey protein based edible films containing p-
aminobenzoic acid against 4 strains of L. monocytogenes

 

Diameter of Inhibition Zone (mm)

 

 

PABA. LA:AA* CWD 95 CWD 249 CWD 201 CWD 1503
(%)(w/V)
0 1:0 0a 0a 0a 0a
7:3 0a 0a 0a 08
1:1 02‘ 0a 0a 0“
0.50 1:0 13.3 i 2.6bcd 8.7 i 2.3bc 15.0 i 1.7cd 11.7 i 2.6cd
7:3 9.3 i 1.5 b“ 8.0 i 2.2 “ 8.3 i 3.7“f 17.0 i 3.2“f
1:1 8.0 i 0.8“” 4.3 i 1.2bc 5.3 i- 0.1“ 4.0 i 0.2“b
0.75 1:0 18.0 i 0.6“f 15.0 i 1.6“ 20.0 i 0.5““ 13.0 i 12°“
7:3 13.0 i 0.4bed 12.0 i 0.9cd 17.0 8: 1.8““ 14.0 i 15°“
1:1 14.0 i 32°“ 8.7 i 0.4bcd 12.3 i 1.1“ 9.0 i 3.5“
1.00 1:0 21.0 i 0.4"f 17.0 i 5.3bc 22.3 i 0.5“f 19.3 i 2.3“f
7:3 18.3 i 2.9“" 19.3 i 4.9“ 16.0 i 0.6“ 20.3 i 3.5cf
1:1 18.7 :r. 2.3“f 13.0 i 5.7“ 18.3 i 2.1“f 17.3 i 3.8“f
1.50 1:0 24.7 i 4.6f 19.7 i 3.3“d 25.0 i 3.8f 22.3 i 4.1f
7:3 22.8 i 0.1cf 22.8 i 0.9“ 22.7 i 0.1“:f 16.7 i 3.2“f
1:1 20.3 i 2.3“f 18.1 i 1.16 27.0 .4: 0.7cf 20.3 i 5.8cf

 

Geometric mean i standard deviation (n=3). Means in same column with different superscript

are significantly different (p<0.05).
*Ratio of lactic acid (LA) to acetic acid (AA).

103

Table 2.2. Antimicrobial activities of whey protein based edible films containing sorbic
acid (SA) against 4 strains of L. monocytogenes

 

Diameter of inhibition zone (mm)

 

 

SA LA:AA* CWD 95 CWD 249 CWD 201 CWD 1503
WWW
0 1:0 0a 0° 0° 0°
7:3 08‘ 0a 0° 0°
1:1 0° 0° 0° 0°
0.50 1:0 19.0i2.3b 19.3 :1: 5.7“ 12.3 :21“ 13.7:38“
7:3 18.0i1.3b 12.3 :31“ 21.3 :37“ 127:3.2a
1:1 17.0:13b 17.0i1.5b° 15.3 :0.1°“ 18.0i1.2ab
0.75 1:0 21.0 i 1.9“ 20.0 i 0.2“ 12.0 i 0.7b 23.0 i 05°“
7:3 24.7 i 4.2“ 16.7 i 3.4“ 21.7 i 1.4“ 25.7 i 2.5“
1:1 27.3 i 0.6“ 21.7 i 3.0“ 20.3 i 1.1“ 28.3 at 25°
1.00 1:0 27.0 i 3.3“ 23.3 i 1.9“ 26.3 i 12° 27.7 i 3.3“
7:3 29.7 i 2.5"c 25.7 i 1.3“ 24.0 :r 0.8“ 28.3 i 1.5“
1:1 30.0 _+. 2.8“ 25.7 i 3.2“ 23.3 i 1.2“ 30.0 i 38°
1.50 1:0 31.3 i 25° 30.3 1 04° 22.3 i 3.8“ 30.7 i 3.1°
7:3 27.3 i 3.1“ 13.7 i 0.9“b 20.7 :r 1.1“ 30.0 i- 2.0°
1:1 25.3 i 40" 30.0 2°. 48° 27.7 i 18° 32.0 : 38°

 

Geometric mean i standard deviation (n=3). Means in same column with different superscript are
significantly different (p<0.05).

*Ratio of lactic acid (LA) to acetic acid (AA).

104

potassium sorbate was added to trypticase soy broth at pH 5.0. Films containing SA were
generally more inhibitory to L. monocytogenes than films containing PABA. McDade et
al. (1999) reported that growth of L. monocytogenes was inhibited on frankfurters during
2 to 3 weeks of storage at 4 °C by coating the frankfurters with a whey protein film-
forrning solution that contained propionic/sorbic acid (pH 5.2). However, non-uniformity
of the antimicrobial coating on frankfurters after dipping, draining and drying would
likely produce a less effective antimicrobial barrier as compared to pre-casted films. In
our study, all antimicrobial edible films were uniform in thickness. Consequently, these
films would be better suited to inhibit post-processing surface contaminants such as L.
monocytogenes.

Film discs containing SA or PABA also were inhibitory to E. coli 0157:H7 with
inhibition zones ranging from 0.7 to 13.3 mm and 5.3 to 21.3 mm, respectively (Table
2.3, 2.4). When used at concentrations of 0.5, 0.75, or 1.0%, PABA was more effective
against E. coli than SA. Richards et al., (1995) and Tsai and Chou (1996) showed similar
inhibition of E. coli 0157:H7 on laboratory media using PABA and SA, respectively.

Using SA and PABA, inhibition zones for S. Typhimurium DT104 ranged from 0
to 12.2 mm and 3.0 to 16.3 mm, respectively (Table 2.5, 2.6). Films containing PABA
inhibited all strains of S. Typhimurium DT104 on TSAYE, whereas film discs containing
0.5 and 0.75% SA and lactic acid:acetic acid (1 :0 and 7:3) failed to inhibit three strains of
S. Typhimurium DT104 (G10127, G10931 and G10601). While laboratory media
containing 0.2 or 0.5% SA is reportedly bacteriostatic to Salmonella at pH 5.5 (Restaino
et al., 1981; Elliot and Gray, 1981), the amount of SA released from our film discs

containing 0.5 or 0.75% SA was presumably too low to inhibit these three strains.

105

Table 2.3. Antimicrobial activities of whey protein based edible films containing p-

aminobenzoic acid (PABA) against 3 strains of Escherichia coli 0157:H7

 

Diameter of Inhibition zone (mm)

 

 

PABA LA: AA* AR (Acid Resistant) AD 305 AD 317
cow/v)
0 00 0a 0a 0a
23 0a 0a 0a
1 :1 0 a 0 a 0 a
050 00 57:L?° 27:0? 07:0?
23 20:0?“ 97:05“ r7:0?
01 53:1? 100:L§° 40:0?
0J5 00 73:2?“ 92:1.3b 30:1?
73 27::09 128:r?° 20:0?
n1 25:0?“ 118:09“ 27:0?
100 10 108:0?f 125:0?° 30:0?
73 103:23“cf 123:3?° 5.3:01“b
lfl 90:06“f 125:12“ 30:0?
150 10 132:2? 15.8:0?d 95:09“
73 130:10 158:12“ 103:15“
01 132:0.4f 213:L?° 100:Lw

 

Geometric Mean :2 standard deviations (n=3). Means in same column with different

superscript are significantly different (p<0.05).
*Ratio of lactic acid (LA) to acetic acid (AA).

104

Table 2.4. Antimicrobial activities of whey protein based edible films containing sorbic

acid (SA) against 4 strains of E. coli 0157:H7

 

Diameter of inhibition zone (mm)

 

 

SA(%)(w/v) LA: AR (Acid Resistant) AD 305 AD 317
AA.
0 20 0° 0° 0°
23 0° 0° 0°
11 0° 0° 0°
050 10 17:0?b 07:03° 07:0?
73 17:05°b 23:03° 17:0?
11 23:0?“ 40:0?“ 40:02°
075 10 20:0?“ 27:0.2ab 30:13°
23 43:1?“ 43:0?“ 20:02°
14 23:0?“ 17:04° 27:0?
100 10 50:1?“ 50:11“‘ 30:0?
23 53:1?“ 45:0?“ 53:0?b
11 83:0?“ 75:1?° 30:0?
150 10 90:3?“ 8.3:21°d 95:09“
73 113:1?° 100:0? 103:1?°
11 133:0? 123:0? 110:1?

 

Geometric mean i standard deviation (n=3).

superscript are significantly different (p<0.05).
*Ratio of lactic acid (LA) to acetic acid (AA).

105

Means in same column with different

In accordance with previously published data (Ahamad and Marth, 1989;
Richards et al., 1995), acetic acid is more inhibitory to L. monocytogenes, E. coli
0157:H7, and Salmonella in laboratory media at the same pH value than lactic acid.
Three ratios of lactic acid: acetic acid (1 :0, 7:3, or 1:1) were used to adjust the pH of our
film solutions. We expected that the solution containing more acetic acid (1:1) would be
most inhibitory based on the aforementioned studies. However, incorporating three
different ratios of lactic acid:acetic acid in films containing PABA or SA did not
synergistically alter inhibition of the three test pathogens on TSAYE at pH 5.2.

The antimicrobial findings in this study are based on the measurement of clear
inhibition zones surrounding film disks where growth of the pathogen was inhibited.
Diffusion of antimicrobials from the film disc depends on the size, shape, and polarity of
the diffusing molecule as well as the chemical structure of the film and the degree of
molecular cross-linking (Guilbert, 1986). According to Michaels et al., (1962), the shape
of the diffusing molecule (linear, branched or cyclic) may impact the diffusion rate.
When Chen et al., (1996) measured diffusion rates for SA and benzoic acid from chitosan
film in a water-glycerol solution (aw = 0.8), more SA (57%) was released than benzoic
acid (65%). The different interactions of SA and PABA in our WPI-based edible films

likely resulted in different diffusion rates leading to varying degrees of inhibition.

2.4.2. Mechanical properties

Average film thickness was 127.11um (i 35.39) with no significant differences
observed between films (Table 2.7). When SA and PABA concentrations increased from
0 to 1.5%, % E increased from 6.37% to 74.28 and 42.16%, respectively (Table 2.7).

While TS of WPI films significantly decreased with increasing levels of SA (p<0.05)

106

Table 2.5. Antimicrobial activities of whey protein based edible films containing p-

aminobenzoic acid (PABA) against 5 strains of Salmonella Typhimurium DT104

 

Diameter of Inhibition zone (mm)

 

 

PABA LA: (310931 010127 (301074 G10601 G11601
(%)(v/w) AA“
0 1:0 0 ° 0 ° 0 ° 0 ° 0 °
7:3 0 ° 0 ° 0 ° 0 ° 0 °
1:1 0° 0 ° 0 ° 0° 0°
0.50 1:0 6.8 : 03° 6.7 : 0.5“ 6.7 : 1.5“ 7.3 : 1.6“ 4.7 : 08°
7:3 9.2 : 0.?“ 8.3 : 04° 7.7 : 25°“ 6.8 : 24°“ 6.3 : 0.1“
1:1 7.0 : 0.6“ 3.0 : 0.5“ 4.7 : 15° 7.7 : 1.? 6.2 : 1.6“
0.75 1:0 9.7 : 15°“ 8.7 : 0.? 7.7 : 06°“ 8.5 : 20°“ 4.7 : 28°
23 11.3 : 15°“ 7.7 : 04° 8.3 : 1.5“ 10.7 : 0.5“ 8.5 : 0.5“
1:1 8.5 : 23°“ 3.0 : 0.7“ 7.5 : 29°“ 9.2 : 2.0“ 7.8 : 1.3“
1.00 1:0 12.0 : 28°“ 9.8 : 2.3“ 9.8 : 2.3“ 10.0 : 2.1“ 5.0 : 25°
7:3 13.7 : 1.2“ 10.2 : 3.6“ 10.2 : 3.6“ 11.2 : 1.3“ 10.5 : 26°
1:1 11.0: 15°“ 60:05“ 90:10“ 117:1.1“ 102:0.5°
1.50 1:0 16.3 : 10° 14.0 : 1.8d 15.3 : 2.5f 14.0 : 3.0f 14.0 : 2.4d
7:3 145:1?“ 137:3? 13.8: 1.1f 137:2.1f 13.7: 1.9“
1:1 15.0 : 18°“ 10.3 : 0.6“ 13.0 : 32°f 15.3 : 0.6°f 14.3 : 0.1d

 

Geometric mean 32 standard deviations (n=3). Means in same column with different superscript
are significantly different (p<0.05).
*Ratio of lactic acid (LA) to acetic acid (AA).

107

Table 2.6. Antimicrobial activities of whey protein based edible films containing sorbic
acid (SA) against 5 strains of Salmonella Typhimurium DT104

Diameter of Inhibition Zone (mm)

 

 

SA LA: (310931 (310127 (301074 (310601 G11601
(%)(v/w) AA"
0 10 0° 0° 0° 0° 0°
23 0° 0° 0° 0° 0°
11 0° 0° 0° 0° 0°
0.50 10 0° 0° 1.7 : 0.1“ 2.0 : 03“ 3.2 : 07“
23 0° 0° 5.3 : 07°“ 4.0 : 08°“ 0°
11 3.3 : 0.1 ° 0.7 : 0.? 1.0 : 02“ 4.5 : 15°“ 3.0 : 0.5ab
0.75 10 4.9 : 04“ 0° 1.7 : 0.5°° 2.7 : 09°“ 1.7 : 03“
7:3 6.3 : 05°“ 2.0 : 0.? 7.3 : 09°“ 7.7 : 23°“ 2.0 : 08“
1:1 5.3 : 14°“ 2.7 : 0.5°° 5.7 : 14°“ 5.7 : 0.?“ 2.0 : 0.1“
1.00 10 7.5 : 11°“ 5.0 : 0.2“ 5.3 : 03°“ 5.3 : 15°“ 6.2 : 0.4“
7:3 5.3 : 09°“ 4.7 : 0.3“ 6.2 : 14°“ 7.2 : 2.7“ 6.0 : 1.2“
1:1 8.3 : 1.5“ 8.0 : 18°“ 8.0 : 2.2“ 7.5 : 37°“ 6.5 : 24°“
1.50 10 9.3 : 06° 9.7 : 0.6“ 7.3 : 27°“ 8.3 : 31°“ 9.7 : 1.7“
7:3 8.0 : 0.3“ 9.3 : 1.2“ 9.7 : 1.4“ 9.7 : 1.9“ 8.0 : 0.8“
11 11.0:1.5° 11.3:18d 11.6:14° 122:1.8f 110:1.8“

 

Geometric mean i standard deviations (n=3). Means in same column with different superscript

are significantly different (p<0.05).

“Ratio of lactic acid (LA) to acetic acid (AA).

108

(Table 2.7), TS of films containing 1.5% PABA (5.7 MPa) was similar to the control
(5.92 MPa). Films containing SA exhibited lower TS and higher % E as compared to
films containing PABA. The reason for this phenomenon could be that the straight chain
of SA can more easily penetrate into WPI chains than PABA which has a benzene ring.
Consequently, SA may have allowed more mobility between protein chains, thereby
producing films of lower TS and greater flexibility. The various organic acid mixtures

used to adjust the pH of the film solutions did not significantly alter % E or TS.

Increasing the amount of additives other than cross-linking agents generally
produced films with lower TS and greater elongation since these molecules insert
between protein chains to form hydrogen bonds with amide groups of proteins (Guilbert,
1986; Kester and Fennema, 1986). Reduced interactions between these protein chains
lead to increased flexibility and movement. In our study, SA, PABA, acetic acid and
lactic acid might be functioning as plasticizers to increase elongation and decrease TS as

was previously suggested for lactic acid (Krull and Inglett, 1971).
CaC12 was incorporated into our film solution as a cross-linking agent to improve

the mechanical and water vapor permeability properties of the low pH films as previously
suggested by others (Guilbert, 1986; Avena-Bustillos and Krochta, 1993). As a divalent
cation, calcium cross-links between negatively charged groups on proteins, thereby
increasing cohesion between protein chains, reducing protein polymer segmental mobility
and improving both the mechanical properties and water vapor permeability (Krochta et

al., 1990). Jeyarajah and Allen (1994) reported that CaC12 induced a change in B-

lactoglobulin conformation, which facilitated polymerization during heating. Calcium

109

ions also increased the reactivity of SH groups at low pH. Although the SH — S-S
interchange reaction is not possible at pH < 6.5, aggregation of most whey proteins can
still occur in the presence of calcium (de Wit, 1981).

Whey protein films are formed by heat-catalyzed protein-protein interactions that
involve disulfide, hydrogen, and hydrophobic bonds. Heating denatures the protein and
exposes internal SH and hydrophobic groups (Watanabe and Klostermeyer, 1976;
Shimada and Cheftel, 1998) which promote intermolecular S-S and hydrophobic bonding
upon drying (McHugh and Krochta, 1994b). Film formation is favored in more alkaline
film solutions since SH reactivity increases at pH > 8 (Kella and Kinsella, 1988; Banerjee
and Chen, 1995). In the present study, the film solution was at pH 8.0 during heating at
90 °C, after which the pH was decreased to 5.2 using lactic and acetic acid. A low pH
environment would likely prevent S-S bond formation in the protein matrix, thereby
weakening the film structure. Thus, tensile strength of the low-pH film (5.92 MPa) was
substantially lower than that reported for high-pH film (13.9 MPa) (McHugh and
Krochta, 1994a). However, tensile strength of our low-pH film was higher than that
reported for corn zein (0.4 MPa) (Aydt et al., 1991), soy protein (4.5 MPa) (Gennadios
and Weller, 1991), and wheat gluten based edible films (1.9-4.4 MPa) (Gennadios et al.,

1993) when tested at 23 °C / 50%RH.

2.4.3. Water Vapor Permeability
Films containing 0, 0.5, 0.75, 1.0, or 1.5% PABA exhibited average WVP values of
27.24, 53.73, 53.90, 55.34, and 54.00 g.mm/m2.d.kPa, respectively (Table 2.7).

Increasing the concentration of PABA from 0.5 to 1.5% did not significantly alter WVP

llO

Table 2.7. Thickness, Tensile strength (TS), Percent elongation (% E), and Water Vapor
Permeability (WVP) of whey protein isolate-based films containing sorbic acid (SA) and
p-aminobenzoic acid (PABA).

 

 

Antimic. LAzAA Thickness (um) %E TS (Mpa) WVP
(%)(w/v) * (g.mm/m2.day.kPa)
Control 1:0 128.4 : 24.4° 6.4 : 33° 5.9 : 14° 27.2 : 1.?
SA (0.50) 1:0 121.1 : 366° 20.0 : 14° 4.9 : 27° 27.3 : 98°
23 126.4 : 247° 23.4 : 11° 4.5 : 03° 21.3 : 32°
11 127.6:40.2° 31.6: 19° 4.6:07b 315:2?
SA (0.75) 10 137.3 : 30.3° 26.6 : 32° 4.9 : 05° 28.6 : 6.?
23 134.6 : 231° 27.1 : 09° 4.8 : 14° 27.5 : 1.?
11 112.9 : 48.? 24.6 : 87° 4.4 : 14° 32.9 : 14°
SA (1.00) 1:0 130.5 : 404° 67.8 : 64° 3.8 : 0.? 43.5 : 53°
23 132.9 : 22.? 73.5 : 14° 3.8 : 01° 41.8 : 42°
11 120.4 : 195° 70.7 :1? 3.9 : 03° 41.5 :14°
SA(1.50) 1:0 118.9:43? 730:2.1° 31:05° 438:6.7°
23 123.7 : 235° 74.3 : 45° 2.6 : 10° 45.6 : 19°
1:1 133.4 : 27.? 73.3 : 53° 2.7 : 09° 44.1 : 21°
PABA (0.50) 10 123.7 : 22.? 18.3 : 56° 5.4 : 09° 51.8 : 2.?
7:3 145.4 : 458° 19.9 : 65° 5.4 : 0.7° 55.2 : 31°
11 120.5 : 27.3° 16.2 : 39° 4.4 : 11° 54.3 : 1.?
PABA (0.75) 10 134.1 : 235° 20.9 : 85° 5.2 : 28° 56.4 : 1.?
7:3 119.1 :45? 30.8 : 36° 52:34° 50.19:24°
11 136.3 : 56.? 28.5 : 152° 5.2 : 24° 55.08 : 16°
PABA (1.00) 10 133.2 : 431° 34.7 : 63° 4.4 : 0.? 59.79 : 35°
23 111.8 : 29.3° 33.3 : 25° 5.3 : 2.? 47.08 : 1.?
1:1 120.7 : 39.3° 30.1 : 8.? 5.8 : 18° 59.17 : 26°
PABA (1.50) 1:0 123.4 : 577° 34.9 :10.6° 5.3 : 0.? 56.11 : 21°
23 131.7: 349° 38.7 : 51° 5.8 : 14° 56.95 : 54°
11 130.0 : 431° 42.2 : 101° 6.0 :16° 48.96 : 2.?

 

Mean i standard deviation (n=3). Means in the same column with different superscript are
significantly different (p<0.05).
* Ratio of lactic acid (LA) to acetic acid (AA).

111

(p>0.05). Average WVP values for films containing 0.5 and 0.75% SA were 26.69 and

29.70 g.mm/m2.d.kPa, respectively, and were not significantly different from the control
(27.24 g.mm/m2.d.kPa) (p>0.05). However, addition of 1.0 and 1.5% SA significantly

increased WVP to 42.29 and 44.46 g.mm/m2.d.kPa, respectively (p<0.05).

WVP is a measure of the ease with which a material can be penetrated by water
vapor. WPI edible films tend to be poor moisture barriers due to abundant hydrophilic
groups in proteins. Their moisture barrier properties can be improved by adding non-
polar compounds such as lipids (McHugh and Krochta, 1994b). We incorporated
candelilla wax into the film solution to reduce WVP. In preliminary experiments,
diffusion of SA and PABA as demonstrated by inhibition zones was similar for films
prepared with and without candellila wax (results not shown). Adding SA and PABA to
the film solution increased WVP because both antimicrobials are hydrophilic compounds.
Addition of polar additives may increase the hydrophilic character and the solubility
coefficient of the film (McHugh et al., 1994). Moreover, additives such as SA or PABA
weaken chain packing in the film to produce a looser structure which increases water
mobility.

2.5. CONCLUSION

Incorporating 0.5 to 1.5% of SA or PABA into WPI films (pH 5.2) led to
inhibition of L. monocytogenes, E. coli 0157:H7, and S. Typhimurium DT104 on
TSAYE at pH 5.2. Addition of PABA and SA increased %E and WVP, but decreased
TS. Given our current work involving ready-to-eat meat products which will be reported
elsewhere, these films may prove useful for inactivating post-processing contaminants on

ready-to-eat foods such as processed meats.

112

CHAPTER 3

INHIBITION OF LIS T ERIA MONOC Y T OGENES, ESCHERICHIA C 0L1

0157:H7, AND SALMONELLA TYPHIMURIUM DT104 ON BOLOGNA AND

SUMMER SAUSAGE USING ANTIMICROBIALS EDIBLE FILMS

Cagri, A., Ustunol, Z., Ryser, E.T.

Journal of Food Science (In press)

113

3.1.ABSTRACT

Whey protein isolate (WPI) films (pH 5.2) containing 0.5 to 1.0% p-
aminobenzoic acid (PABA) and/or sorbic acid (SA) were assessed for antimicrobial
activity and mechanical properties while in contact with sliced bologna and summer
sausage that were inoculated with Listeria monocytogenes, Escherichia coli 0157:H7,
and Salmonella Typhimurium DT104. WPI films containing SA or PABA decreased
Listeria, E. coli and S. Typhimurium populations 3.4-4.1, 3.1-3.6, and 3.1-4.1 logs on
bologna and sausage after 21 days at 4°C, respectively. Background flora on slices was
inhibited compared to controls. Film tensile strength decreased while % elongation
remained unchanged following 72 h of product contact. Consequently, these films may

prove useful in extending the self-life of ready-to-eat meats.

3.2. INTRODUCTION

Post-processing contamination of ready-to-eat meat products has emerged as a
serious public health concern. In 1998, 101 cases of listeriosis in 22 states, including 21
fatalities were traced to consumption of hot dogs that became contaminated after
processing (CDC, 1998). A Class I recall was subsequently issued for 35 million pounds
of tainted product. During the second half of 2000, another outbreak involving 29 cases
in 10 states (including 7 fatalities) prompted the recall of approximately 14.5 million
pounds of turkey and chicken delicatessen meat because of probable contamination with
L. monocytogenes (CDC, 2000). Listeria continues to threaten the processed meat
industry with over 75 Class I recalls involving more than 75 million pounds of cooked

ready-to-eat meats (approximately 40 million pounds of hot dogs) issued from April 1998

114

to October 2001 (U SDA-F SIS, 2001a). In 2000, 2,298 cases of foodbome listeriosis were
reported in the United States at an estimated cost of $2.33 billion (~$ 1 million /case),
making listeriosis the costliest food-borne disease (USDA-F SIS, 2001b).

Two other meat-borne pathogens also have emerged as major public health
concerns. Escherichia coli 0157:H7 has been highly publicized due to outbreaks of
illness associated with ground beef (Ostroff et al., 1990; Bell et al., 1994, Mead et al.,
1997, Tuttle et al., 1999) and dry-cured salami (Tilden et al.,l996). Transfer of E. coli
0157:H7 from contaminated meat or utensils to other foods such as fresh fruits and
vegetables is also a major concern (CDC, 1995). The annual cost of the 62,458 E. coli
0157:H7 infections reported in 2000 was estimated at $659.1 million (~$10,500/case)
(USDA-FSIS, 2001b). Salmonella Typhimurium DT104, a multiantibiotic resistant strain,
is also emerging as a serious threat to public health (Glynn et al., 1998). Food-borne
transmission of DT104 has been documented for several meat-related outbreaks;
suspected vehicles included roast beef, ham, pork sausage, salami sticks, cooked meats,
frozen sausage samples, and chicken legs (Davies et al., 1996, Anonymous, 1996). In
England, 17% of 786 fresh or frozen sausage samples yielded Salmonella spp., including
S. Typhimurium DT104 (Nichols and de Louvois, 1995). Sharma et al. (2001) also
recovered S. Typhimurium DT104 from several points in pork production. These findings
indicate that meat products pose a serious health risk if not cooked and handled properly.

Product slicing and packaging operations are major points at which bacterial
pathogens can be introduced into cooked ready-to-eat meat products. Bologna sausage
batter contains a mixed microflora, including possible pathogens, which are destroyed

during cooking; however, vacuum-packed bologna is typically recontaminated during

115

slicing. In commercial manufacturing facilities, bacterial loads in meat reportedly
increased 0.5 to 2.0 logs CF U/g during slicing (Holley, 1997b).

Several studies have demonstrated that antimicrobial edible films can reduce
bacterial levels on meat products. Siragusa and Dickson (1992, 1993) showed that
organic acids were more effective against L. monocytogenes, S. Typhimurium, and E. coli
0157:H7 on beef carcass tissue when immobilized in calcium alginate than when used as
a spray or dip. In another study, application of edible corn starch film containing
potassium sorbate and lactic acid inhibited growth of S. Typhimurium and E. coli
0157:H7 on poultry (Baron, 1993). Antimicrobial chitosan films containing acetic or
propionic acid reportedly inhibited growth of Enterobacteriaceae and Serratia
liquefaciens on bologna, regular cooked ham and pastrami (Ouattara et al., 2000) during
168 h of storage at 5°C. Natrajan and Sheldon (2000) also incorporated nisin and
chelators into protein- and polysaccharide-based films to inhibit Salmonella on poultry
skin. These results emphasize the potential for adding antimicrobial compounds to edible
packaging materials.

In our previous study, WPI films (pH 5.2) containing 0.5 to 1.5% SA or PABA
inhibited the growth of L. monocytogenes, E. coli 0157:H7, and S. Typhimurium DT104
on acidified (pH 5.2) trypticase soy agar containing 0.6% yeast extract (Cagri et al.,
2001). In this study, WPI films containing SA or PABA were assessed for their ability to
retain their desirable antimicrobial and mechanical properties while in direct contact with

bologna and summer sausage slices.

116

3.3. MATERIALS AND METHODS

3.3.1. Products

Commercially produced all-beef bologna (dia. 9.6 cm) and fermented Thuringer
summer sausage (dia. 5.4 cm) were obtained from the delicatessen counter of a local
supermarket. Bologna was pre-sliced (~3 mm thick) while the summer sausage was
sliced approximately 3-mm thick in the laboratory using a food slicer (Chefs Mark,
Dallas, TX). All-beef bologna contained 39.3% fat, 3.6% corn syrup, 2.4% salt, 0.25%
sodium nitrite, spices and sodium erythorbate as reported by the manufacturer. Similarly,
Thuringer summer sausage contained 64% beef, 36% pork, 19.8% fat, 1.8% dextrose,
2.1% salt, 0.25% sodium nitrite, sodium erythorbate, a pediocin-producing starter culture,
spices, and flavoring. When purchased, beef bologna and summer sausage had pH values

of 6.0 and 4.6, respectively. Products used in each of three trails were from the same lot.

3.3.2. Film Preparation

Whey protein isolate (WPI, Alacen 895) (New Zealand Milk Products, North
America, Inc., Santa Rosa, CA) (5% w/v) and glycerol (Sigma Chemical Co., St. Louis,
MO) (2% w/v) were dissolved in distilled water containing 0.04% CaClz (w/v) (Sigma).
After mixing, the solution was adjusted to pH 8.0 with 1N NaOH (Sigma), and heated at
90°C for 30 min in a shaking water bath (Precision Scientific, Winchester, VA).
Following the addition of candelilla wax (Stahl Pash, Inc., New York, NY) (0.4%, w/v)
during the last 5 min of heating, the solution was homogenized for 2 min in a SD-45

homogenizer (Tekmar Co., Cincinnati, OH), filtered through cheese cloth and cooled to

117

23 1|: 2°C. After adding 0.75 or 1.0% (w/v) sorbic acid (SA) (Sigma), p-aminobenzoic
acid (PABA) (Sigma), or 0.5% SA: PABA (1.0: 1.0), the pH was adjusted to 5.2 using
1N lactic acid (Sigma). Following degassing by vacuum for 20 min, the whey protein
solution was cast by pipetting 75 ml of the solution into sterile Teflon® plates (20x30
cmz). The solutions were dried for approximately 24 h at 23 :t 2°C/50 i 5% RH, after
which the films were peeled from the plates and stored at 23 i- 2°C/50 i 5% RH until

used.

3.3.3. Bacterial Strains

Based on results from our previous WPI antimicrobial edible film work (Cagri et
al., 2001), the most resistant strain of each of the three pathogens to SA and PABA was
chosen. Strains used in this study included Listeria monocytogenes CWD 95 and
Escherichia coli 0157:H7 AR (previously obtained from C. W. Donnelly, Dept. of
Nutrition and Food Sciences, University of Vermont, Burlington, VT) and Salmonella
Typhimurium DT104 G10127 (previously obtained from B. Swaminathan, Centers for
Disease Control and Prevention, Atlanta, GA). All strains were maintained at -70°C in
trypticase soy broth (TSB) (Difco Laboratories, Detroit, MI) containing 10% (v/v)
glycerol and subcultured twice in TSB containing 0.6% (w/v) yeast extract (TSBYE)

(Difco) at 35°C/18-24 h before use.

3.3.4. Product inoculation and storage

Bologna and summer sausage slices were separately inoculated with 0.1 ml of an

appropriately diluted culture so as to contain log 6.0 (i 0.1) L. monocytogenes, E. coli

118

0157:H7 or S. Typhimurium DT104 CFU/g. The inoculum was evenly spread on both
surfaces with a sterile glass rod. The slice was then placed in a sterile 150 mm-diameter
petri dish to which a piece of edible film slightly larger than the slice was previously
added (Figure 3.1). Thereafter, an identically inoculated slice was placed on top of the
edible film with this process repeated until a stack of eight slices, each separated by a
piece of edible film, was obtained. One final piece of edible film was laid on the top slice
so that both faces of all slices were in contact with the film. These petri dishes were then
covered and stored aerobically to give a worst case scenario for film testing at 4°C. Two
controls were used - the first consisting of bologna and summer sausage slices separated
by the identical edible film prepared without antimicrobials and the second containing

slices stacked together without the use of edible film.

3.3.5. Microbiological analysis

Slices of bologna and summer sausage were examined for levels of the inoculum,
as well as mesophilic aerobic bacteria, lactic acid bacteria, and yeast/molds immediately
after inoculation and again following 4, 7, 10, 14, and 21 days of refrigerated storage.
Slices weighing 10-g each were added to 90 ml of sterile 0.1% (w/v) peptone water (PW)
(Difco) and homogenized for 3 minutes in a Stomacher 400 (Tekmar Co., Cincinnati,
OH). Appropriate dilutions in PW were surface plated on Modified Oxford Agar (Difco),
McConkey Sorbitol Agar (Difco), McConkey Agar (Difco), Plate Count Agar (Difco),
MRS Lactobaccillus Agar and Rose Bengal Agar (Difco) to quantify L. monocytogenes,

E. coli 0157:H7, S. Typhimurium DT104, mesophilic aerobic bacteria, lactic acid

119

bacteria, and yeast/molds, respectively, as outlined in the FDA Bacteriological Analytical

Manual (FDA, 1998).

3.3.6. Mechanical Properties

Edible films placed between uninoculated slices of bologna and summer sausage
were examined at 0, 24, 48, and 72 h for thickness, tensile strength and percent
elongation after conditioning the films for 24 h at 55 i 5 % RH / 23 :t 5° C.

A model M micrometer (Testing Machine Inc., Amityville, NY) was used to
determine film thickness. Measurements were taken at five different locations and the
mean value was used in further calculations for moisture barrier and mechanical
properties.

Films were cut into strips measuring 101.6 mm by 25.4 m using a Precision
Sample Cutter (Thawing Albert Instrument Co., Philadelphia, PA). All films were
conditioned for 48 h at 23 i 2°C / 50 i 5% RH before testing. Tensile strength (TS) and
percent elongation at break (% B) were determined according to standard D-882-91
(ASTM, 1992). The test was run using the Instron Universal Testing Machine Model
2401 (Canton, MA) at 23 2 2° C / 50 i 5% RH with a static load cell of lkN and a cross
head speed of 50.8 cm/min. TS was calculated in MP3 from the following equation:

TS = load /( sample width x sample thickness)

% Elongation at break was determined by the following equation:

% E = (distance sample stretched / original length of sample) x 100

120

3.3.7. Statistical Analysis

All experiments were replicated three times. All film thickness, TS, and %E data
was analyzed by one-way analysis of variance (ANOVA) using the SAS Statistical
Analysis System (SAS Institute Inc., 1990). Means were compared using the Duncan

Grouping test at p=0.05.

3.4. RESULTS AND DISCUSSION
3.4.1. Antimicrobial Properties

L. monocytogenes. Using WPI films containing 0.75% PABA, 1.0% PABA,
0.75% SA, 1.0% SA, or 0.5% SA: 0.5% PABA (1:1), populations of L. monocytogenes
decreased 1.5, 2.2, 3.0, 3.4, and 2.8 logs, respectively, on bologna slices after 21 days of
storage at 4°C (Figure 3.1A) with all films remaining intact. Listeria counts remained
relatively unchanged using films without antimicrobials; whereas in the absence of films,
populations on bologna increased 2.2 logs after 21 days of refrigerated storage. Control
WPI films without SA or PABA were acidified to pH 5.2 with lactic acid. Hence, lactic
acid present in control films would also be expected to retard the growth of L.
monocytogenes on bologna slices. L. monocytogenes was inhibited on fermented summer
sausage using WPI films containing PABA or SA (Figure 3.1B). All WPI films
containing antimicrobial agents reduced L. monocytogenes populations 3.0 to 4.1 logs on
summer sausage slices after 21 days of refrigerated storage. While Listeria populations
also decreased initially using antimicrobial-free film and slices without film, substantial

regrowth occurred following 10 days of refrigerated storage.

121

l

O—‘NO’AO’IO’NCOO
Iog1OCFU/g

 

lw‘l

   

oA-lslxlrcbtschorxloolclo
log1OCFU/g

. .’ r 0
Days 14 21 0.) 7'0 ’3‘ .7 0-6‘ o/x /
.9 '0 6‘ 6‘ A
"i, 494 4 75° ’6,
0 ‘7
4 Q?

Figure 3.1. Inhibition of L. monocytogenes on bologna (A) and fermented sausage (B) slices with WPI
edible films containing 0.75%, and 1.0% p-aminobenzoic acid (PABA); 0.75% and 1.0% sorbic acid
(SA) and 0.5% SA: 0.5%PABA (1:1). Control + film (WPI edible film without SA or PABA) and
control (no film used).

122

log 10 CFU/g

 
  

log10 CFU/g

 

7
Days

Figure 3.2. Inhibition of E. coli 0157:H7 on bologna (A) and fermented summer sausage (B) slices
with WPI edible films containing 0.75%, and 1.0% p-aminobenzoic acid (PABA); 0.75% and 1.0%
sorbic acid (SA) and 0.5% SA: 0.5%PABA (1:1). Control + film (WPI edible film without SA or

PABA) and control (no film used).

123

1
l
1

O

l
\
l
l
,3" / i;
l
L 1 L ,7

654.66.5064166454
log1OCFU/g

 

Iog1OCFU/g

    

0 . 0 Co °00
1o 7 0.2 10s '5'06 ”"04 °’
Days 14 21 0’5 '0'949584 4 84:0 £770;
A o
4194 4 484

Figure 3.3. Inhibition of S. Typhimurium on bologna (A) and fermented summer sausage (B)
slices with WPI edible films containing 0.75%, and 1.0% p-aminobenzoic acid (PABA); 0.75% and
1.0% sorbic acid (SA) and 0.5% SA: 0.5%PABA (1:1). Control + film (WPI edible film without
SA or PABA) and control (no film used).

124

Wederquist et a1. (1995) reported that potassium sorbate 0.26% (w/w) and sodium lactate
2% (w/w) were highly inhibitory to L. monocytogenes on bologna during 28 days of
storage. In our most recent study, low pH WPI-based edible films (pH 5.2) containing 0.5
to 1.5% SA or PABA also inhibited L. monocytogenes on laboratory media (Cagri et al.,
2001). Using antimicrobial films, greater inhibition of Listeria was observed on summer
sausage (pH 4.6) compared to bologna slices (pH 6.0) due to higher levels of SA and
PABA in the undissociated form. In the absence of antimicrobial WPI films, beef
bologna also allowed more growth of L. monocytogenes than summer sausage. Other
inhibitory factors in tested fermented summer sausage, such as pediocin produced by the
starter culture (Spelhaug and Harlander, 1989), nitrite, salt, low pH and low aW would
also serve to further inhibit L. monocytogenes. The combined effects of nitrite and low
pH (~4.6) were likely responsible for inhibiting L. monocytogenes on control summer
sausage slices (Buchanan and Philips, 1990), with Glass and Doyle (1989) also reporting
that L. monocytogenes was unable to multiply in vacuum-packed summer sausage (pH
4.8 - 4.9) containing 3.0 - 3.4% salt. Their results agree with those of other authors (Berry
et al., 1990; Buncic et al., 1991), who also observed a decline in numbers of L.
monocytogenes in fermented sausage during extended refrigerated storage.

E. coli 0157 :H7. Numbers of E. coli 0157:H7 on bologna slices decreased 2.7 to
3.6 logs after 21 days at 4°C using antimicrobial films containing SA or PABA (Figure
3.2A). However, the pathogen decreased only 2.1 and 1.4 logs, respectively, on control
slices with antimicrobial-free film or without film. Grth of E. coli 0157:H7 also was
inhibited on fermented summer sausage (Figure 3.2B). Using WPI films containing 0.75

and 1.0% PABA or SA or the combination of 0.5% SA and 0.5% PABA (1:1),

125

populations decreased 2.3 to 2.6, 2.7 to 3.1, and 2.9 logs, respectively. Numbers of E. coli
0157:H7 significantly decreased on both bologna and summer sausage at the end of
storage using antimicrobial films (p<0.05) (Table 3.1). In our previous study, WPI edible
films containing 0.5 to 1.5% PABA or SA inhibited E. coli 0157:H7 in laboratory media
(Cagri et al., 2001). However, numbers of E. coli 0157:H7 on summer sausage decreased
for the film-free and antimicrobial-free controls, with this inhibition likely due to the
combined effects of chemical preservatives, nitrite, salt and a pH of 4.6. Several
researchers (Pond et al., 2001, Riordan et al., 1998) also observed that 2.5% salt and 100
ppm sodium nitrite were inhibitory to E. coli 0157:H7 in fermented summer sausage.
However, other studies have claimed that this pathogen is very hardy (Benjamin and
Datta, 1995), being able to survive more than 51 days in laboratory media at 10°C
containing starter culture (107 CFU/ml), dextrose (0.8%), NaCl (2%), and NaNOz (200
ppm) (Glass et al., 1992, Tomicka et al., 1997). When initially present at 104 CFU/g, E.
coli 0157:H7 reportedly survived during manufacture and storage of fermented sausage
regardless of whether a starter culture was used (Glass et al., 1992).

S. Typhimurium DT104. Using WPI films containing 0.75 or 1.0% SA or
PABA, populations of S. Typhimurium DT104 decreased 2.7 to 3.1 logs on bologna
slices after 21 days of refrigerated storage (Figure 3.3A). In contrast, cell numbers on
antimicrobial-free film and film-free control bologna slices decreased only 1.5 and 0.5
logs after 21 days of storage, respectively. WPI films containing 0.75 or 1.0% PABA,
SA, or a combination of 0.5% PABA and 0.5% SA (1:1) decreased levels of S.
Typhimurium DT104 3.5, 4.1, 2.8, 3.6 and 3.9 logs on fermented sausage slices after 21

days, respectively (Figure 3.4B). However, a decrease of only 1.5 logs was observed for

126

control slices. Cagri et al. (2001) previously concluded that WPI edible films containing
0.75 to 1.5% PABA or SA inhibited all strains of S. Typhimurium DT104 tested on
TSAYE.

WPI films also were prepared to contain 0.5% of both SA and PABA to test their
synergistic effect against growth of the three pathogens on bologna and summer sausage.
WPI films containing SA or PABA were more inhibitory to the three test pathogens than
WPI films containing both SA and PABA, although several studies claimed that SA and
benzoic acid were more effective when used together rather than alone (Sofos and Busta,
1983, Luck, 1980)

Mesophilic Aerobic Bacteria. After 21 days of refrigerated storage, growth of
mesophilic aerobic bacteria (MAB) on bologna slices was inhibited as much as 5 .0 and
5 .8 logs using films containing 1.0% SA or PABA as compared to the antimicrobial-free
film and film-free controls, respectively (Table 3.2). However, Petaja et al.(1979) found
that incorporating 0.25% potassium sorbate into cooked sausage had no effect on levels
of aerobic bacteria when the product was stored at 7°C. Growth of MAB was generally
inhibited on summer sausage slices by all WPI films; however, MAB populations
increased 1.4 and 2.4 logs after 21 days on sausage slices with antimicrobial-free film or
film-free controls, respectively (Table 3.5). Thus, WPI films can extend the shelf life of
both bologna and summer sausage at 4°C.

Lactic Acid Bacteria. Using antimicrobial films, lactic acid bacteria (LAB)
decreased 1.0 to 3.0 logs on bologna slices after 4 days and generally remained at these
levels throughout storage; whereas no reduction in LAB populations was observed in the

controls (Table 3.2). Fermented summer sausage initially contained 6.0 log CFU/g of

127

LAB with populations remaining relatively unchanged during 21 days of storage
regardless of the type of film used (Table 3.5). However, numbers of LAB were 1.6 to
2.2 logs higher in samples stored without film. Early studies suggested that sorbate
concentrations of 1.0% were markedly inhibitory to Lactobacillus bulgaricus,
Lactobacillus acidophilus and Streptococcus lactis (Hamdan et al., 1971). LAB
predominate in bologna and are often the major species present in fully ripened
fermented sausage. According to Holley (1997a), bacteria present on bologna slices
stored at 7°C were almost exclusively LAB, with Lactobacillus sake dominating.
Bacillus thermosphacta wasabsent and coliforrns were rarely detected. Fermented
sausage also contained thermotolerant homofermentative lactobacilli which were
quantified along with pediococci. However, growth of pediococci was limited by low
temperature (<10°C) storage of the samples (Holley et al.,1988).

Yeast/Mold. As expected, mold grth on both bologna and summer sausage
was inhibited with WPI films containing SA or PABA during 21 days of refrigerated
storage (Tables 3.2 and 3.4). However, mold counts eventually reached 1.3-1.4 and 1.7-
2.2 logs in the antimicrobial-free film and film-free controls, respectively. Using WPI
films containing PABA, yeasts were not detected on bologna or summer sausage slices
until day 7. Films containing 0.75 or 1.0% SA inhibited yeasts 7 to 21 days and 21 days
on bologna and fermented summer sausage, respectively. The combination of 0.5% SA
and 0.5% PABA (1:1) prevented growth of yeasts on bologna but not on summer
sausage. In the antimicrobial-free film and film-free controls, yeast populations on
bologna and summer sausage increased 3.5-3.6 and 5.0-5.6 logs, respectively, by the end

of storage. Inhibition of yeasts and molds by sorbate is well documented (Liewen and

128

Marth; 1985, Stead, 1995). Dipping fermented sausage and raw ham in a solution of 10-
20% sorbate inhibited mold growth (Leistner et al., 1975). Application of 2% gelatin, 2%
liquid smoke, 0.2% corn starch, and 1% potassium sorbate on the inside of regenerated
cellulose casings also prevented mold growth during sausage curing (Rose and Turbark,

1969).

129

 

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3.4.2. Mechanical Properties

Films varied in thickness from 4.3 to 5.2 mil with this difference not statistically
significant (p>0.05) (Tables 3.6 and 3.7). Percent elongation at break (% E) and tensile
strength (TS) of films in contact with bologna and summer sausage slices were measured
after 0, 24, 48, and 72 hours of storage to determine their eventual suitability as sausage
casings. TS of all films decreased (p<0.05) after 24 h of contact with bologna and
summer sausage slices; whereas % E increased (p<0.05) after contact with either meat
product (Table 3.3 and 3.4). No significant increases (p>0.05) in % E were observed
between films measured after 24 and 48 or 48 and 72 h of storage at 4°C. In addition, no
significant decreases (p>0.05) in TS were observed beyond 24 h. Chemical stability of
the films was predictably altered while in contact with bologna and summer sausage
slices because of expected moisture absorption from the meat slices and release of SA,
PABA, and glycerol to reach a steady state. Moisture absorption by the film likely
weakened the polymer chain bonds since water molecules insert between protein chains
to form hydrogen bonds with amide groups of proteins, thereby reducing tensile strength
(Guilbert, 1986; Kester and Fennema, 1986). Reduced interactions between these protein
chains likely led to increased flexibility and movement, as evidenced by the marked

increase in % E.

135

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3.5. CONCLUSION

WPI films containing SA or PABA clearly inhibited growth of L. monocytogenes,
E. coli 0157:H7 and S. Typhimurium DT104 on both bologna and summer sausage
slices. These films, which retained their antimicrobial activity for 21 days, also showed
considerable promise in extending the shelf-life of sliced bologna and summer sausage.
Percent elongation of the film increased as a result of contact with bologna and summer
sausage while tensile strength sharply decreased. Eventual formulation of these
antimicrobial films into sausage casings would provide the processed meat industry with
another means of safeguarding cooked and ready—to-eat meats from post-processing

contaminants.

138

CHAPTER 4

INHIBITION OF LISTERIA MONOC YT OGENES ON HOT DOGS USING

ANTIMICROBIAL WHEY PROTEIN-BASED EDIBLE CASINGS

Cagri, A., Ustunol, Z., Ryser, E.T., Osburn, W.

139

4.1.ABSTRACT

Whey protein isolate (WPI) films (pH 5.2) containing 0.0 or 1.0% (w/v) p-
aminobenzoic acid (PABA) were heat—sealed to form casings. A commercial-type hot dog
batter was stuffed into WPI, collagen, or natural casings. Afier cooking, the hot dogs
were surface-inoculated to contain 103 Listeria monocytogenes CFU/g, vacuum packaged
and examined for numbers of L. monocytogenes, mesophilic aerobic bacteria (MAB),
lactic acid bacteria (LAB), and yeast/mold during 42 days of storage at 4°C. Tensile
strength (TS) and % elongation (% E) of casings were determined before and afier
cooking and after smoking using standard methods. Fat, protein, moisture, 2-
thiobarbuturic acid (TBA) value, purge, shear force, color, pH, and sensory attributes of
uninoculated hot dogs were also determined. Listeria populations on hot dogs prepared
with WPI-1.0% PABA casings remained relatively unchanged; however, numbers of
Listeria on hot dogs prepared with WPI-0.0% PABA, collagen, and natural casings
increased ~2.5 logs during 42 days of refrigerated storage. Populations of MAB, LAB,
and mold on WPI-1.0% PABA casings were 1-3 logs lower compared to others casings.
TS and % E of WPI casings remained unchanged after cooking and smoking; however,
TS and % E of collagen and natural casings decreased. TBA and pH values were similar
for hot dogs with different casings. Shear force of casings prepared with WPI and
collagen was lower than that for natural casings. Purge loss was higher in hot dogs
prepared with WPI than collagen or natural casings. Sensory attributes (overall
desirability, flavor, texture, juiciness) of hot dogs with WPI-1% PABA casings were

scored higher that hot dogs with commercial collagen and natural casings. WPI casing

140

 

containing 1.0% PABA may offer another potential strategy to prevent growth of Listeria

on hot dogs during extended refrigerated storage.

4.2. INTRODUCTION

Over the last decade, post-process contamination with Listeria monocytogenes has
emerged as one of the largest problems faced by the processed meat industry. In 1998,
more than 35 million pounds of hot dogs that had been linked to 101 listeriosis cases in
22 states, including 21 fatalities, were recalled by one Michigan manufacturer, with the
product presumably contaminated at the time of packaging (CDC, 1998). Two years
later, 29 listeriosis cases (7 fatalities) in 10 states prompted the recall of turkey and
chicken delicatessen meat (14.5 million pounds) that likely contained L. monocytogenes
(CDC, 2001). Listeria continues to threaten the processed meat industry with more than
74 Class I recalls involving approximately 100 million pounds of cooked ready-to-eat
(RTE) meats issued from April 1998 to December 2001 (USDA-F SIS, 2001).

In response to the Listeria problem, the processed meat industry has examined
post-processing thermal and high pressure pasteurization (Lucore et al., 1999, Murano et
al., 1999, Yuste et al., 2000) as well as various acid washes to minimize the risk of
Listeria on finished ready-to-eat (RTE) products. Antimicrobial edible films were
investigated as another possible solution to this problem. Ming et al. (1997) reported that
pediocin-coated cellulosic casings inhibited L. monocytogenes on ham, turkey breast
meat, and beef. In addition, McDade et al. (1999) found that dipping frankfurters in an
aqueous whey protein solution (pH 5.2) containing propionic/sorbic acid prevented
growth of L. monocytogenes on the product during the first 2 to 3 weeks of refrigerated

storage.

141

Consequently, we investigated the efficacy of antimicrobial edible films for
inactivating pathogens including L. monocytogenes, Escherichia coli 0157:H7 and
Salmonella Typhimurium DT104, on RTE meat products. In our initial work, whey
protein isolate (WPI)-based edible films (pH 5.2) containing 0.5 to 1.5% sorbic acid (SA)
or p-aminobenzoic acid (PABA) inhibited L. monocytogenes, E. coli 0157:H7, and S.
Typhimurium DT104 on trypticase soy agar (Cagri et al., 2001). When these WPI films
containing SA or PABA were subsequently tested between inoculated slices of
commercially produced bologna and summer sausage, significant reductions in numbers
of L. monocytogenes, E. coli 0157:H7 and S. Typhimurium DT104 were observed after
21 days of storage at 4°C (Cagri et al., 2002).

The objectives of the present study were to (a) test the ability of WPI casings
containing 1.0% PABA to inhibit L. monocytogenes on surface-inoculated hot dogs; (b)
determine the ability of the WPI casings to retain their desirable mechanical properties
during hot dog manufacture and storage; (c) examine the impact of different casings on
storage stability of hot dogs based on numbers of total aerobic mesophilic bacteria, lactic
acid bacteria, yeast and mold, 2—thiobarbutiric acid (TBA) values, purge loss, shear force,
color and pH; and (d) assess sensory attributes of hot dogs prepared with WPI casings

containing 1.0% PABA, WPI, collagen and natural casings.

4.3. MATERIALS AND METHODS

4.3.1. Experimental Design

In preliminary work, hot dogs were prepared using heat-sealed whey protein

isolate (WPI) casings containing 0.0% and 1.0% SA, PABA, 0.5% SA:0.5% PABA and

142

commercial collagen casings. Hot dogs with different casings were surface inoculated
with Listeria monocytogenes (103 CFU/g), vacuum-packaged and stored at 4°C for 42.
Listeria inhibition was examined initially and after 4, 7, 10, 14, 21, 3S, and 42 days of
storage. WPI casings containing 1% PABA which were most inhibitory against Listeria
were subsequently compared with commercial collagen and natural casings in the full-
scale study (Figure 4.1).

In latter, hot dogs prepared with WPI casing containing 0.0% or 1.0% PABA,
commercial collagen and natural casings were surface inoculated with L. monocytogenes
(103 CFU/g), vacuum-packaged and stored 4°C for 42 days. Uninoculated hot dogs
prepared with WPI, collagen and natural casings were tested for chemical (moisture, fat,
protein, pH and TBA), microbiological (mesophilic aerobic bacteria, lactic acid bacteria,
mold/yeast), mechanical (shear force, tensile strength of casings after cooking and
elongation of casings after cooking), physical (purge, cook yield) and sensory (juiciness,
texture, flavor, casing tenderness and overall desirability) characteristics.

4.3.2. Whey Protein Isolate Casing Preparation

WPI (Alacen 895, New Zealand Milk Products, North America, Inc., Santa Rosa,
CA) (5% w/v) and glycerol (Sigma Chemical Co., St. Louis, MO) (2% w/v) were
dissolved in distilled water containing 0.04% CaClz (w/v) (Sigma). After mixing and
adjusting the pH to 8.0 with 2N NaOH, the solution was heated at 90°C for 30 min in a
shaking water bath (170 Marcel Drive water bath, Precision Scientific, Winchester, VA).
Following the addition of candelilla wax (Stahl Pash, Inc., New York, NY) (0.4%, w/v)
during the last 5 min of heating, the solution was homogenized for 2 min in a SD-45

homogenizer (Tekmar Co., Cincinnati, OH), filtered through cheese cloth and cooled to

143

I!-

23 i 2°C. After incorporating 1.0% (w/v) p-aminobenzoic acid (PABA) (Sigma), sorbic
acid (SA) (Sigma), or 0.5% SA: 0.5% PABA (1:1), the pH was adjusted to 5.2 with 2N
lactic acid (Sigma). Following degassing by vacuum, the whey protein solution was cast
by pipetting 105 ml of the solution onto sterile flat Teflon plates (35 x 23 cm2) and drying
for approximately 24 h at 23 i 2°C / 50 i 5% RH after which the films were peeled from
the plates and heat-cured at 90°C in a vacuum oven for 12 h. Films were hand-cut with a
paper cutter and heat-sealed to form tubular casings measuring 2.6 cm in diameter and
12.0 cm in length, stored at 23 i 2°C / 50 i 5% RH and used within 2 days.

Commercial edible collagen (clear processed type) (dia. 2.6 cm) was provided by
Vista International Packaging, Inc. (Kenosha, WI). Commercial natural casings (lamb
casing) (dia. 2.4-2.6 cm) were obtained from Dewied International, Inc. (San Antonio,
TX).
4.3.3. Culture preparation

Listeria monocytogenes CWD 95, the most resistant strain identified from our
previous work (previously obtained from C. W. Donnelly, Dept. of Nutrition and Food
Sciences, University of Vermont, Burlington, VT), was maintained at —70°C in trypticase
soy broth (TSB) containing 10% (v/v) glycerol and subcultured twice in trypticase soy
broth containing 0.6% (w/v) yeast extract (TSBYE) (Difco Laboratories, Detroit, M1) at
35°C/18-24 h before use.
4.3.4. Hot dog processing

Beef (90% lean, 10% fat) (60%, w/w meat block) (rib lifter meat) (Food Brand

America, Lansing, MI) and pork (80% lean, 20% fat) (40%, w/w meat block) (Pork

picnic shoulder) (IBP, Inc., Lansing, M1) were coarse ground, placed in a vacuum bowl

144

chopper (Seydelmann, Frankfurt, Germany) and slowly mixed at of 80 rpm without
vacuum. Half of the ice/water mix (30%, w/w ingredients) and salt (1.8% w/w
ingredients) were then added afier which the mixture was chopped under vacuum (50 Pa)
at 4000 rpm for 2 minutes. Corn syrup solids (2.22%), potato starch (2.00%), whey
protein concentrate (1.01%), spices (0.50%), beef stock (0.48%), phosphate (0.30%),
sodium erythorbate (0.03%), and sodium nitrite (0.25%) were added. The mixture was
chopped without vacuum at 2000 rpm for 3 min and then finally chopped at 4000 rpm
under vacuum (80 Pa) for 3 min.

The meat emulsion was removed from the bowl chopper and stuffed into heat-
sealed WPI casings and commercial (collagen and natural) casings using a hand stuffer
(VOGT-deal, Chicago, IL) or a Vacuum Vemag Robot Cocyc 500 stuffer, respectively.
After stuffing, hot dogs were hung on smoke-sticks in a smoke house (Cgi Processing
Equipment, Model A 28-80101, Cicero, IL) and processed according to a standard
commercial hot dog smokehouse schedule (Table 4.1). Cellulose casings were also used
to prepare cooked batter for chemical analysis with the cellulose casings peeled from the
hot dogs before analysis. Two batches of hot dogs containing three replicates of each
casing type were preapred. The first batch of hot dogs was used for microbiological,
chemical (moisture, protein, fat content, pH, TBA), physical (purge, shear force), and
mechanical (tensile strength and percent elongation) analysis. The second batch was

examined for sensory properties as well as moisture and fat content.

Afler cooling in a chill room (0°C) for 4 h, the unpeeled hot dogs were surface-inoculated

to contain L. monocytogenes at a level of 103 CFU/g for microbial analysis. Uninoculated

145

Table 4.1. Hot dog smokehouse schedule.

 

 

 

 

Stages Time Internal Dry Bulb Wet Bulb Smoke Source
(min) Temp.(°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 ,1
Shower 32 1 min on 1 min off

146

hot dogs were used for chemical, physical and sensory analysis. All hot dogs were

individually vacuum-packaged in Cryovac® vacuum shrink bags (5 x 9 incz) (Oxygen

permeability- 3-6 cc./atm.day at 40°C) (Sealed Air Corporation, Duncan, SC) and stored

at 4°C until analysis.

4.3.5. Chemical Analysis

Hot dog samples with casings were grounded for each casing type analyzed for
moisture (AOAC 950.46B) and fat (ether extractable) (AOAC 952-473) according to
standard AOAC (1990) procedures (Appendices l and 2). Raw and cooked batter without
casings were also ground and analyzed for moisture, fat and protein content. For moisture
analysis, ~ 2.0 g-ground samples were placed in ashless wrapped filter paper and dried at
100°C in a air dry oven (Thelco model 18, GCA/Precision Scientific, Chicago, IL)
overnight with weight loss afier drying giving the moisture content (%). Dried samples
were subjected to overnight ether extraction using soxhlet apparatus with weight loss
giving the fat content. Protein content of raw and cooked batter as well as hot dogs with
casings was determined using the LECO FP-2000 Protein Analyzer (LECO Corporation,

St. Joseph, MI) (Appendix 3.).

4.3.6. Rancidity Measurement

The 2-thiobarbuturic acid (TBA) test (Appendix 4) (Tarladgis et al., 1960; Zipser
et a1, 1962) was used to assess oxidative rancidity initially and afler l, 14, 28 and 42 days
of refrigerated storage. The amount of sulfanilarnide added to samples for TBA analysis

was based on the residual amount of nitrite in each sample according to the modification

147

of Shahidi and others (1985). Absorbance was read at 538 nm using a Perkin Elmer
UV/V IS Spectrometer Lambda 20 (Perkin-Elmer Corporation, North American Organic
Division, Norwalk, CT) with a conversion factor of 7.8 used to calculate the final TBA

numbers.

4.2.7. pH test
Hot dog samples (1 g) were homogenized in 10 ml of distilled water for 1 min.
The pH of the solution was determined using a pH meter (ABIS, Fisher Scientific,

Hanover Park, IL) equipped with a standard combination electrode (Fisher).

4.3.8. Diffusion Test

Standard Calibration Curve: Standard solutions of sorbic acid or p-
aminobenzoic acid were prepared by transferring weighed samples (0.01g) to 100 ml
volumetric flasks and diluting to volume with HPLC graded water. This solution was
then serially diluted to prepare a series of standard solutions of known concentration for a
standard calibration curve.

Sample Preparation: WPI films containing SA and PABA were peeled from the
hot dogs initially and 1, 7, 14, 28, 35, and 42 days of refrigerated storage. Film samples
(0.1g) were cut into small pieces and soaked in 10 ml of methanol solution (%50 v/v) for
7 days at room temperature. The sample solution was then vortexed for 1 min, diluted
1:10 in methanol-water (1:1, v/v), filtered through a 0.45 microfilter (Fisher) and

analyzed for levels of SA and PABA using a Waters 717 HPLC system (Water

Corporate, Milford, Massachusetts). The injection volume was 10 ul. Separations were

148

performed on a stainless steel column (150x5 mm ID.) filled with 5 pm Nucleosil CN
(Waters Corporate). Methanol-water (1:1, v/v) was used as the mobile-phase. The overall
flow-rate was held at 1.0 ml/min. The levels of SA and PABA were obtained by
substitution in following equation:

Rsx C.F. x Vtotal x
Vinj x tholymer

 

%SA or PABA (wt/wt): 100

Where: R5 = detector response value for the sample
C.F. = calibration factor (g/A.U.) (from slope of standard calibration curve)
V total = total volume of solution (ml)
Vinj = volLune of unknown solution injected into the chromatograph (ml)

Wt polymer = weight of the film

The amount of SA and PABA that diffused per unit area as a function of time for
WPI edible film was plotted. Extrapolation of the steady state rate for antimicrobial
diffusion was used to determine the permeation lag time. The diffusion coefficient was

calculated using the following equation:

D = 0.049 x L2
t1/2
Where: D = Diffusion coefficient

L = Thickness of the film

t1 ,2 = Time for 50% of the initial antimicrobial diffusion

149

4.3.9. Microbiological analysis

Hot dogs were examined for numbers of L. monocytogenes immediately after
inoculation and again following 1, 7, 14, 21, 28, 35, and 42 days of storage at 4°C.
Samples weighing 10 g each were added to 90 ml of sterile 0.1% peptone water,
homogenized for 3 minutes in a Stomacher 400 (Tekmar Co., Cincinnati, OH),
appropriately diluted in sterile 0.1% peptone water and quantitatively examined for L.
monocytogenes, mesophilic aerobic bacteria, lactic acid bacteria, and yeast/mold using
Modified Oxford Agar (Difco), Plate Count Agar (Difco), MRS Lactobaccillus Agar

(Difco), and Rose Bengal Agar (Difco), respectively.

4.3.10. Film Thickness
A model M Micrometer (Testing Machine Inc., Amityville, NY) was used to
determine casing thickness. Measurements were taken at five different locations and the

mean value was used in further calculations for mechanical properties.

4.3.11. Mechanical Properties

Tensile strength (TS) and percent elongation (%E) of casings were determined
before and after cooking and afier smoking. After peeling, the casings were cut into strips
measuring 101.6 mm by 25.4 m using 3 Precision Sample Cutter (Thrawing Albert
Instrument Co., Philadelphia, PA). Samples were conditioned for 48 hours at 23 i 2°C /
50 i 5% RH before testing. Tensile strength and percent elongation at break were
determined according to standard D-882-91 (ASTM, 1992). The test was run using the

Instron Universal Testing Machine Model 2401 (Canton, MA) at 23 i 2°C / 50 i 5% RH

150

with a static load cell of lkN and a cross head speed of 50.8 cm/min. TS was calculated
in MPa from the following equation:

_ Load
Sample width x Sample thickness

 

TS

% E at break was determined by the following equation:

Distance sample stretched

%E= x100

 

Original length of sample

4.3.12. Purge loss

Purge loss of hot dogs was measured after 1, 14, 28, and 42 days of storage at
4°C. One package containing four hot dogs for each casing type was weighed and
opened. After the hot dogs and residual purge in the package were dried with a paper
towel, the hot dogs and package were reweighed. Purge loss was calculated as follow:

initial package weight — final package weight

%purge loss = x100 %

 

initial package weight

4.3.13. Shear Test

Kramer shear values were determined at room temperature using a TAHDi texture
analyzer (Texture Technologies Corp., New York, NY). The sample length measured 6
cm cut from the center of the hot dog with the casing placed perpendicular to the
multiblades of the Kramer shear attachment. The analyzer had a crosshead speed of 1.67
mm/s with a load cell of 10 kN. Peak force and area under the curve were calculated by

the system and divided by sample weight with the results recorded as kg/ g.

151

 

4.3.14. Color Measurement

Hot dogs with different casings were cut perpendicularly through the center for
color measurement. L“ (lightness), a* (redness), and b“ (yellowness) (Hunter color
system) were determined on internal and external surfaces of the hot dogs after at 1, 14,
28, and 42 days of refrigerated storage using a Minolta Chromameter (CR-300 series,
Ramsey, NJ) equipped with a granular materials attachment CR-ASO. The instrument was
standardized using a pink ceramic tile calibrated to tristimulus values. Two hot dogs per

treatment were analyzed.

4.2.15. Sensory Evaluation

An untrained, consumer panel (n=150) was used to evaluate sensory attributes of
hot dogs prepared with WPI, WPI-PABA, collagen and natural casings. Following 35
days of storage at 4°C, the packaged hot dogs were heated in hot water (82°C) for 10
minutes, removed from the package, cut into 1-inch lengths, held at 60°C, and served to
panelists within 15 min. Panelists consisting of students, faculty and staff, primarily from
the departments of Food Science and Human Nutrition and Animal Science at Michigan
State University, evaluated hot dog attributes (i.e., casing tenderness, texture, flavor,
juiciness and overall desirability) by assigning a value based on a 8 point hedonic scale
from 8 (extremely like) to l (extremely dislike). The panelists were seated in individual
lighted booths and provided with cold water and unsalted crackers to allow rinsing and
cleaning between samples. SIMS 2000 Computer Program (Sensory Computer System,
Morristown, NJ) was used for data collection in a “survey” format. Each individual
subject’s sensory scores were statistically analyzed and reported using this program in an

aggregate form for each product attribute tested.

152

 

4.2.16. Statistical Analysis
All experiments were replicated three times. Two-way analysis of variance
(ANOVA) was performed using the SAS Statistical Analysis System (SAS Institute

Inc., 1990). Means were compared using the Duncan Grouping test at p=0.05.

4.4. RESULTS AND DISCUSSIONS

 

4.4.1. Chemical Analysis

Chemical composition including moisture, fat and protein content of all four types
of hot dogs as well as the raw and cooked batter is shown in Table 4.2. Fat content was
similar for all hot dogs; whereas, moisture content was significantly lower for hot dogs
prepared with either WPI casing (p<0.05). Hot dogs prepared with WPI casings may lose
more moisture during processing and storage due to enhanced hydrophilicity of WPI
casings. The higher protein content of hot dogs with WPI 1.0% PABA casings might be
due to the composition of p-aminobenzoic acid (PABA) since casing protein was also
measured as protein by the protein analyzer. In this study, moisture fat and protein
content of hot dogs were in agreement with the composition of commercially available

hot dogs as reported by Bloukas et al. (1996).

4.3.2. Thiobarbutiric acid values
TBA values, which represent the malonaldehyde concentration in hot dogs,

decreased from 0.76, 0.81, 0.76, and 0.84 to 0.68, 0.70, 0.71, and 0.69 mg

malonaldehyde/kg for WPI-PABA, WPI, collagen, and natural casings, respectively, after

153

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154

42 days of refrigerated storage (Table 4.3). These TBA values for hot dogs with different
casings were not significantly different after 42 days (p>0.05). Using different casings for
hot dogs did not significantly change the rancidity (p>0.05). A general decrease in TBA
values was observed during storage. Addition of nitrite and phosphate to hot dog
formulations will delay rancidity because of their action as antioxidants (Zubillaga et al.,
1984). TBA values in meat are well-correlated with oxidative rancidity (Melton, 1985).
However, malonaldehyde, a highly reactive secondary product of lipid oxidation, can
react with other meat components such as amino acids and amines, forming
complexes that are not detected by TBA analysis (Gokalp et al., 1983). Therefore,
competition for malonaldehyde in a system containing protein could result in reduced
color development and incomplete quantitation of malonaldehyde. Hung and Zayas
(1989) also showed that TBA values decreased from 0.79 to 0.59 mg malonaldehyde/kg

in frankfurters during 45 days of storage.

4.3.3. pH

The pH of hot dogs decreased from ~62 to ~57 for all casings during refrigerated
storage (Table 4.3) with none of these differences significant after 42 days (p>0.05).
Paneras and Bloukas (1994) also found that the pH of frankfurters decreased from 6.4 to
5.8 during 5 weeks of storage 5°C with this pH decrease attributed to activity of

lactobacilli.

155

 

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Table 4.4. Analysis of Variance on the effect of easing type on microbiological,
chemical, and physical characteristics of hot dogs during 42 days of refrigerated storage
(P-values for independent variables and interactions)

 

Source of Variance

 

 

Casings Time Interaction
(Casings x Time)

L. monocytogenes <0.0001* * * <0.0001* * * 0.0004* *
MAB 0.019* <0.0001*** 0.31
LAB <0.0001*** <0.0001*** 0.0018“
Mold 0.64 <0.0001“ * 0.84
pH 0.95 <0.0001* ** 0.92
TBA 0.18 0.0014" 0.88
Purge 0.0015“ 0.22 0.54
Color-interior

L“ (Lightness) 0.35 <0.0001*** 0.39
a* (redness) 0.85 <0.0001“ * * 0.40
b*(Yellowness) 0.89 <0.0001* * * 0.45
Color-exterior

L* (Lightness) <0.0001"‘ * * <0.0001* * * 0.003"
a* (redness) 0.13 0.05 0.18
b*(Yellowness) 0.0008*** 0.18 0.39

 

*significant at p<0.01; ** significant at p<0.001, *" significant at p<0.0001.
MAB: Mesophilic aerobic bacteria, LAB: Lactic acid bacteria

157

4.3.4. Diffusion Coefficient
After one day of refrigerated storage, 78.7 and 75.6% of the PABA and SA
diffused from the WPI film into the hot dog with only 7.7 and 6.5 % of initial PABA and

SA remaining in the WPI casings afier 42 days of storage, respectively. For these whey

protein-based films diffusion coefficients for PABA and SA at 4°C were 1.7 x 10'13

mz/sec and 3.2x10'l3 mz/sec, respectively. Chen et a1. (1996) also studied diffusion of
SA and benzoic acid from edible film and found that about 39% of SA and benzoic acid
was released from methycellulose/chitosan films into a glycerol/water solution (aw=0.8)
within 30 minutes at 4°C. Since the storage time was only 6 hours in their study, the
maximum release of antimicrobials was only 49%. Raising the temperature to 25°C
enhanced the diffusion rate for both antimicrobials from these films; however, increasing
the pH from 3.0 to 6.0 had no effect on diffusion.
4.4.5. Antimicrobial analysis

The numbers of L. monocytogenes on hot dogs with control (WPI without
antimicrobials) and collagen casings increased 4.2 and 5.4 logs, respectively, after 42
days of storage at 4°C (Figure 4.1). Using casings containing 1.0% PABA, Listeria
populations remained relatively unchanged; however, ntunbers of Listeria on hot dogs
prepared with WPI casings containing 1.0% SA or 0.5% SA: 0.5% PABA (1:1) increased
2.8 and 2.1 logs after 42 days, respectively. WPI casings containing SA were less
effective than WPI casings containing PABA for inhibiting Listeria growth on hot dogs
even though SA-containing films decreased populations of Listeria on bologna and
summer sausage slices in our previous study (Cagri et al., 2002). Acidity of WPI casings

(pH 5.2) may decrease when used on hot dogs (pH 6.1) due to the diffusion of chemical

158

components between the hot dog batter and the casing (e.g. lactic acid). Sorbic acid (pKa
4.19) is most active at pH<6.0 since it is the undissociated form that possesses
antimicrobial activity. However, PABA (pKa 4.8) still shows some effectiveness against
L. monocytogenes at pH values as high as 6.0 (Richards et al., 1995). Overall, WPI
casings containing 1.0% PABA were most inhibitory to Listeria and were further
examined in detail.

Listeria monocytogenes: After 42 days of refrigerated storage L. monocytogenes
populations increased about 2.5 logs on hot dogs prepared with WPI-control
(antimicrobial—free), collagen, and natural casings (Figure 4.2). In contrast, WPI casings
containing 1.0% PABA inhibited the growth of L. monocytogenes throughout 42 days of
refrigerated storage. In our previous work, WPI films containing 1% PABA were more
inhibitory, decreasing numbers of L. monocytogenes 2.8 to 3.0 logs on both bologna and
summer sausage slices during 21 days of refrigerated storage (Cagri et al., 2002).
Stacking these slices in sterile petri dishes may have induced greater release of PABA
from the film to the slices compared to the hot dog casings, thereby allowing greater
inhibition. As mentioned earlier, increasing the initial pH (5.2) of WPI casings would
tend to decrease the antimicrobial activity of PABA. While phenolic compounds from the
smoking process and nitrite in the hot dogs may also enhance the inhibitory effect of
WPI-1% PABA casings against Listeria (Lou and Yousef, 1999), such effects were not
observed in our study.

Previous findings (Richards and Xing, 1992; Richard, 1995) indicated that undissociated
uncharged PABA diffuses through the cell membrane more freely and results in PABA

exerting an increased activity as the pH decreases from 7.0 to 5.0 since the pKa value of

159

 

PABA is 4.8 (Terada, 1972). Inside the cell, the dissociation of PABA leads to the
uncoupling of both substrate transport and oxidative phosphorylation from the electron
transport system with growth inhibition resulting principally from the lost the cellular
uptake of amino acids, phosphate and organic acids (Freese et al., 1973). However, even
at pH values close to neutral (pH 6.5-7.0) PABA is still reportedly inhibitory to L.
monocytogenes (Richards et al., 1995). This explains the inhibitory effect of WPI-PABA
casings against Listeria on hot dogs even at above optimal pH value in our study.
Incorporating a buffering agent in the WPI-PABA casing formulation may help stabilize
the hot dog pH at a value that would be more optimal for inhibition of Listeria.

Diffusion test results showed that while only 0.12% (w/v) PABA remained the
day after manufacturing, this concentration was still sufficient to prevent Listeria grth
during 42 days of storage. However, if diffusion of PABA could be delayed by modifying
the WPI casing preparation (e.g. tightening the protein chains with crosslinking agent),
WPI-1.0% PABA would be even more effective against L. monocytogenes.

Mesophilic Aerobic Bacteria: Populations of mesophilic bacteria increased 2.6,
3.7, 4.2, and 5.1 logs on hot dogs prepared with WPI-1.0% PABA, commercial collagen,
natural and WPI-control casings, respectively, during 42 days of storage at 4°C (Figure
4.3). Growth of mesophilic bacteria on hot dogs was suppressed 1.0 to 2.5 logs using
WPI casings containing 1% PABA. Our previous study also showed that growth of
mesophilic bacteria on bologna and summer sausage slices was retarded at 4°C using
WPI films containing 0.75 and 1.0% PABA (Cagri et al., 2002). The various microbial

reduction strategies used for processed meats typically select for specific groups of

organisms, some of which can proliferate and cause spoilage during storage. Processed

meats generally spoil due to facultative Gram-positive bacteria (lactic acid bacteria) that

160

 

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are able to grow in vacuum-packaged product at refrigeration temperatures. Common
spoilage defects during long term storage (10-12weeks) of hot dogs include various off-
odors, -flavors and -tastes, as well as color defects. These defects appear when the
bacterial count has exceeded to 108-1010 CFU/g) (Silla and Simonsen, 1985). In the
present study, none of the hot dogs exhibited any noticeable defects after 42 days of
storage. Since shelf-life of commercial hot dogs may exceed 42 days, some defects could
eventually appear on hot dogs prepared with collagen, natural and WPI-casings
(antimicrobial-free) as the storage time is increased. However, WPI casings containing
PABA may reduce the risk of spoilage by retarding grth of mesophilic bacteria even
after opening the package.

Lactic Acid Bacteria: Casings containing 1% PABA were also most effective at
retarding the growth of lactic acid bacteria on hot dogs (Figure 4.4). Populations of lactic
acid bacteria were 1.7 to 2.2 logs lower on hot dogs cased in WPI-1.0% PABA compared
to the other casings. Based on analysis of variance, casing type and storage time had an
interactive effect on growth of LAB (Table 4.4). Cagri et al. (2002) reported that WPI
films containing 1.0% PABA also partially inhibited lactic acid bacteria on bologna and
summer sausage slices. Lactic acid bacteria comprise the major group of spoilage
organisms in vacuum-packaged meat products (Borch et al., 1996; Holzapfel, 1998;
Johnston and Tompkin, 1992). In the present study, lactic acid bacteria grew to high
levels on hot dogs prepared with collagen, natural and antimicrobial-free WPI casings
with WPI casings containing PABA helping to delay spoilage and extend product shelf-

life.

162

 

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Mold/ Yeasts: Mold counts increased only 1.0 to 2.4 logs on hot dogs during 42
days due to the combination of vacuum packaging and storage at refrigeration
temperature (Figure 4.5). Mold grth was least using WPI-1.0% PABA casings.
Although benzoic acid is a well known inhibitor of yeasts and molds (Balatsouras et al.,
1963), some mold growth was observed on WPI casings containing 1.0% PABA.
Although all hot dogs were vacuum-packaged, some air diffusion through inadequateheat
seals could explain the mold growth that was detected on a limited number of samples.

No yeasts were found during 42 days of refrigerated storage.

4.4.6. Mechanical Properties

Tensile strength of WPI casings remained unchanged after cooking and smoking
(Table 4.5), whereas tensile strength of the collagen and natural casings decreased
significantly from 34.8 and 43.3 to 10.2 and 9.6 MPa, respectively (p<0.05). Percent
elongation of WPI casings containing 1.0% PABA was also unchanged afier cooking and
smoking (Table 4.5); however, percent elongation increased from 12.4 to 23.5% in WPI
casings without PABA and decreased from 41.9 and 56.7 to 24.7 and 21.4% in collagen
and natural casings, respectively.

In this study wet strength of casings were not measured. However, compared to
natural and collagen casings, WPI casings required extra care to avoid breakage during
stuffing and smoking. A hand stuffer was used for WPI casings adjusted to avoid
breaking the heat seal or rupturing. In addition, the rate of air circulation and the
humiditywere reduced during the first stage of smoking and cooking in order to keep the

hot dogs on the smoke house tract.

167

4.4.7. Cook yields and purge loss

Hot dogs prepared with WPI casings had cook yields of 88.9 -89.4% compared to
94.3 and 95.3% for hot dogs prepared with collagen and natural casings, respectively
(Figure 4.6A). Purge loss for hot dogs prepared with WPI-PABA or collagen casings did
not significantly change during 42 days (Figure 4.6). However, purge loss for hot dogs
prepared with WPI and natural casings decreased and increased after 14 days,
respectively. WPI and WPI-PABA casings exhibited higher purge losses (0.60 and
0.69%) than did hot dogs with collagen (0.24%) and natural casings (0.30%). WPI
casings are hydrophilic even after incorporation of candellila wax to decrease water vapor
permeability. This explains the higher liquid release through hydrophilic WPI casings

compared to other commercial casings.

4.3.8. Shear Force

Hot dogs are suitable for shear testing since they have a constant diameter and
homogeneous structure. The resulting shear forces for hot dogs prepared with WPI-1%
PABA (0.75 kg/g), WPI (0.80 kg/g) and collagen (0.59 kg/g) were similar (Figure 4.6B),
whereas hot dogs prepared with natural casings had a significantly higher shear force
(1.71 kg/g) (p<0.05). When consumed, hot dogs are normally first bitten through with
front teeth and then ground on the molars (Boyar and Kilcast, 1986). This biting action

was used as the basis of texture measure

168

 

Table 4.4. Tensile strength (TS) and percent elongation (% E) of WPI-p-aminobenzoic
acid (PABA), WPI, collagen, and natural casings before and afier cooking and smoking of

 

 

 

hot dogs.

Casings Before cooking and smoking After cooking and smoking

TS (MPa) WPI-1% PABA 19.5 i 02" 16.9 :I: 0.3 c
WPI 6.5 i 0.4 a 5.4 i 0.6 a
Collagen 34.8 i: 0.1d 10.2 i 0.4 b
Natural 43.3 a 0.3 c 9.6 i 0.2 b

% E WPI-1% PABA 40.7 i 0.2 c 39.8 i 0.3 c
WPI 12.4:03a 23.6:01"
Collagen 41.9 i 0.5 ° 24.7 a 0.3 b
Natural 56.7 a 0.3 d 21.4 a 0.5 b

 

Mean i standard deviation (n=3). Means with different superscript are significantly different (p<0.05).

169

98 -~ A B B

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

96 Al .
§ 94 *‘ A A ; ll l {bl
2 92 l i E
.2 90 l l l l
> as 1 l l z %
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o 841 i l l l i ?

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PABA
Type of Casings

1.6 7 B B
a. 1.4 l I
9 12 4
.2 ’ l A
3 1 't A T '_ l 1
E 0.8 1 ..=, l A , .
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‘” 0.2 l I . _ ,

0 if _ _,,, , .. . ,_.,‘H.,_ . ! __Tas - ,. . _,

WPI-1% WPI Collagen Natural
PABA
Type of Casings

Figure 4.6. Cook Yield (A) and Shear force (B) of hot dogs prepared with WPI- p-
aminobenzoic acid (PABA), WPI, collagen, and natural casings.

170

the texture meat which most closely relates to human assessment. Consequently, our
shear force results suggest that hot dogs prepared with WPI and collagen casings will be
more chewable, softer and thus more acceptable to consumers than hot dogs prepared

with natural casings.

4.3.9. Color

The color of hot dogs plays an important role in consumer acceptance. L*, a*, and
b* Hunter color values of interior and exterior hot dog surfaces are shown in Table 4.7.
L* (lightness) and a* (redness) values significantly increased in interior samples from all
hot dogs during storage (p<0.05); however, b* (yellowness) only increased for hot dogs
encased in WPI-1.0% PABA after 28 days. Exterior yellowness of hot dogs with WPI
casings was significantly higher than for hot dogs prepared with collagen and natural
casings (Table 4.8). Exterior lightness (L*) of hot dogs prepared with WPI and collagen
casings increased with storage time while lightness of hot dogs prepared with natural
casing remained unchanged during 42 days of storage. Exterior redness (a*) and
yellowness (b*) of WPI-1.0% PABA, collagen, and natural casings did not change
throughout storage, whereas both of these attributes significantly increased for WPI-0%
PABA casings. Based on analysis of variance, exterior lightness and yellowness of hot
dogs significantly changed with casing type; however, storage time and casing type
interaction only had effect on the lightness value (p<0.001) (Table 4.4).

Lightness refers to the relation between reflected and absorbed light, without

regard to a specific wavelength. Yellowness or redness results from differences

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174

in absorption of radiant energy at various wavelengths. Nitrite is the agent added to hot
dogs to produce a pink color. Nitrosylhemochrome-cured meat pigment is formed by
heating of nitric oxide and myoglobin complex. The most significant commercial
problem associated with cured meat color is rapid fading in air and light (Walch and
Rose, 1956), which can be delayed by vacuum-packaging. Light induced dissociation of
nitric oxide from heme followed by oxidation of heme and nitric oxide. Addition of
erythorbate stabilizes pigment fading (deHoll, 1981). In the present study, no fading was
observed. Although not measured, similarity in exterior color of hot dogs also suggests
that a similar amount of smoke was absorbed during smoking and cooking by the WPI
and commercial casings. These findings may indicate that the interior and exterior color
of hot dogs prepared with WPI-1% PABA would be acceptable to customers since their
color values were not significantly different from the color of hot dogs prepared with
commercial casings.
4.4.10. Sensory Analysis

Sensory characteristics of hot dogs prepared with different casings are presented
in Table 4.9. Using a hedonic scale (1-8) juiciness (6.1) and flavor (6.3) attributes of hot
dogs with WPI-1% PABA casings were preferred (p<0.05) and scored higher by panelist
than hot dogs with WPI, collagen and natural casings. Similarly, texture (5.9) and overall
desirability (5.4) attributes of hot dogs with WPI—1% PABA were not significantly
different from hot dogs with other casings (p<0.05). However, the casing tenderness
score for WPI hot dogs (4.2-4.4) was significantly less (p<0.05) than the other casings
(4.8-5.4). Collagen casings were preferred based casing tenderness. Panelists scored

tenderness by biting vertically through the casing with their front teeth. Many panelists

175

Table 4.9. Sensory attributes and chemical content of hot dogs prepared with WPI-p-
minobenzoic acid (PABA), WPI, collagen, and natural casings after 35 days of

refrigerated storage.

 

 

 

 

 

Type of Casings
Attribute WPI-1% PABA WPI Collagen Natural
Casing tenderness 4.4 i 0.1c 4.2 r. 0.2 ° 5.4 i 0.6 . 4.8 _+. 0.4 b
Juiciness 6.1 _+. 0.2a 5.9 r. 0.1ab 5.6 i 0.1 b 5.6 i 0.2 b
Texture 5.9 220.13 5.7a 0.2” 5.5.4; 0.1b 5.61:0.1ab
Flavor 6.3 a 0.1a 5.8 i 0.1b 5.6 i 0.2 b 5.8 a 0.0 b
Overall desirability 5.4 i 0.3 a 5.0 a: 0.2 b 5.3 i 0.2ab 5.2 r 0.1ab
Chemical content (%)
Moisture 71.5 i 0.3 . 70.3 :t 0.5 . 71.5 a 0.7a 71.2 i 0.5 .
Fat 6.3 i 0.3 b 6.5 i 0.2 ab 6.3 i 0.1 b 6.9 i 0.3 a

 

Mean i standard deviation (n=3). Means in same row with different superscripts are significantly
different (p<0.05). A Hedonic scale was used for sensory evaluation of hot dogs from 1
(extremely undesirable) to 8 (extremely desirable).

176

dentified all the hot dogs as being tough and chewy since most consumers prefer skinless
hot dogs. Sealed packages of hot dogs were heated in hot water to avoid flavor changes
and loss of antimicrobial with this heating likely making the hot dogs tougher. Overall,
the sensory characteristics of hot dogs prepared with WPI casings containing 1.0%
PABA were acceptable to thel 50 panelists.

Moisture content of hot dogs prepared with different casings used in the sensory
panel was not significantly different (p<0.05) (Table 4.8); however, fat content was
higher in hot dogs with natural casings. Since a low-fat frankfurter formulation was used
in this study, scores given for overall hot dog desirability were predictably low.
Reduction of fat is an important aspect of consumer acceptance because fat adds flavor to
frankfurters. Pearson et a1. (1987) suggested that acceptable low-fat frankfurters could be
produced by substituting water for fat. Park et al. (1990) reported that frankfurters with
14% fat and a high moisture content (~70%) were considered as undesirable as control
products with 29% fat.

In summary, WPI edible casings containing 1% PABA were more inhibitory
towards L. monocytogenes than other commercial casings with Listeria growth
suppressed on hot dogs during 42 days of refrigerated storage. These casings also showed
considerable promise in extending the shelf-life of hot dogs. Mechanical properties of
WPI casings remained unchanged; however, tensile strength and percent elongation of
commercial casings decreased during hot dog manufacture. Sensory panelists gave hot
dogs with WPI-PABA casings superior scores for texture, juiciness, flavor and overall

desirability compared to hot dogs prepared with collagen or natural casings. Given these

177

overall findings, WPI casings containing 1.0% PABA may provide a promising Listeria

intervention strategy to prevent growth of this post-processing contaminant on hot dogs.

178

CONCLUSION

Low pH (5.2) whey protein isolate-based edible films containing p-aminobenzoic
acid (PABA) or sorbic acid (SA) inhibited the growth of L. monocytogenes, E. coli
0157:H7 and S. Typhimurium DT104 on TSAYE. Increased concentrations of PABA or
SA in WPI film increased the percent elongation (%E) and water vapor permeability
(WVP). While tensile strength (TS) of WPI films decreased with increasing levels of SA,
TS of films was not affected addition of PABA.

Subsequently, WPI films containing 1.0% SA, PABA and 1.0% SAzPABA
(0520.5) were tested between commercially produced bologna and summer sausage slices
to observe whether the antimicrobial and mechanical properties remained constant while
being contact with slices. Populations of L. monocytogenes, E. coli and S. Typhimurium
decreased on bologna and summer sausage slices in contact with the film during 21 days
at 4°C. Mesophilic aerobic bacteria (MAB), lactic acid bacteria (LAB) and yeasts/mold
were inhibited using antimicrobial films compared to antimicrobial-free controls. Film
tensile strength decreased while % E remained unchanged following 72 h of product
contact.

After heat curing to modify mechanical properties, WPI films (pH 5.2) containing
0.0 or 1.0% (w/v) PABA were heat-sealed to form casings. A commercial-type hot dog
batter was stuffed into WPI, collagen or natural casings. After cooking, the hot dogs were
surface—inoculated to contain 103 Listeria monocytogenes CFU/ g. Listeria populations on
hot dogs prepared with WPI-1.0% PABA casings remained relatively unchanged;
however, numbers of Listeria on hot dogs prepared with WPI-0.0% PABA, collagen, and

natural casings increased ~2.5 logs during 42 days of refrigerated storage. Populations of

I78

MAB, LAB, and mold on WPI-1.0% PABA casings were 1-3 logs lower compared to the
other casings. TS and % E of WPI casings remained unchanged afier cooking and
smoking; however, TS and % E of the collagen and natural casings decreased. TBA and
pH values, which decreased during storage, were similar for hot dogs with different
casings. Shear force of hot dogs prepared with WPI and collagen was lower than that for
natural casings. Purge loss was higher in hot dogs prepared with WPI rather than collagen
or natural casings. Sensory attributes (overall desirability, flavor, texture, juiciness) of hot
dogs with WPI-1% PABA casings were scored higher than hot dogs prepared with
commercial collagen and natural casings.

WPI casings containing 1.0% PABA offer another strategy to prevent growth of
Listeria on hot dogs and extend shelf-life. However, one of the main challenges ahead
will be to develop a continuous extrusion process for WPI casing production since the
current casting method is not economically viable for commercial hot dog production.
One important hurdle to overcome will be the reduction of water activity in casing
formulation to enhance the extrusion process. In addition, the WPI casing formulation
will need to be modified for fast heat drying since heat drying is an integral step in the
extrusion process.

Antimicrobial activity of WPI casings containing PABA against Listeria and
spoilage organisms may be enhanced by minimizing the increase in pH during hot dog
manufacture. For example, addition of a buffering agent into the casing formulation may
prevent increase in the WPI casings pH from 5.2 to ~ 6.0. Since PABA is more active at

low pH, stabilizing the casing pH at 5.2 will help maintain optimal antimicrobial the

179

activity of PABA. In addition, testing different antimicrobials such as lysozyme which is
heat resistant and active over a wide pH range is highly recommended.

According to our diffusion test results, most of the PABA was released from the
WPI casing after the first day of hot dog manufacture. Diffusion of PABA from WPI
casings after hot dog processing must be somehow controlled. When protein-based edible
casings come into contact with food surface the high water activity results in absorption
of water and swelling, which in turn leads a loosen casing structure and enhanced
diffusion of any additives. In this study, the casings were heat-cured to decrease water
solubility and strengthen the casing. However, heat curing alone was insufficient to slow
the release of PABA from the casing. Incorporating of various crosslinking agents in WPI
casing may help tighten the protein chains in the casing matrix and thus decrease water
absorption and loosening of the casing structure.

Overall, this work represents only the first step towards the development of
possible commercialization WPI-based antimicrobial casings to reduce the risk of

Listeria growth on hot dogs.

I80

APPENDIX I

181

MOISTURE ANALYSIS-Air Drying Method

Sample Preparation (modified from section 983.18 Meat and Meat Products, AOAC
1990)

Hot dogs were cut into very small (<1 cm squares) pieces and were added to Tekmar
grinders filling the grinding chamber halfway, dry ice was added to fill the remaining
space the chamber. Samples were ground for 2-3 minutes into a fine powder which was
transferred to labeled whirl pack bags. The bags were loosely closed and stored in a

freezer for 2 days so that dry ice could evaporate.

Moisture Analysis (modified from section 950.46B-Air drying for determination of
moisture in meats)

Two grams (i .03 g) of a well ground hot dog sample was added to an ashless filter paper
thimble. The edge of paper thimble was fold over the top and secured with a paperclip.
Samples were placed flat on a tray in a 100°C dry-air oven (Thelco model 18,
GCA/Precision Scientific, Chicago, IL) and for 20 - 24 h. After drying, the samples were
completely cool in a dessicator before weighing. Once cool, sample weighs were
recorded. This weight was the final weight for moisture and the initial weight for fat

analysis. The following formula was used to determine percent moisture:

% Moisture : Wet sample weight — dry sample weight x 100

Wet sample weight

182

APPENDIX II

183

FAT ANALYSIS - modified from 952-47 B soxhlet ether extraction method for meat

(AOAC 1990)

Samples (approximately 2 g) were weighed in ashless filter thimble paper and
dried in an oven at 100°C for 24 h. Dried samples were weighed again after cooling to

room temperature in a dessicator.

Dried samples were placed in extraction tubes with all samples below the level
where the ether drains off (curved glass on outside of tube). Petroleum ether was added to
clean boiling flasks until about full. Two or three glass beads were placed in the boiling
flask (as a boiling aid) which was connected to the extraction'flask. Condensing units
were placed on top of extraction flasks and the Rheostat was set on high for heating.
After 24 h of heating, the cooled samples were poured onto trays in a hood and the ether
was allowed to dissipate. After 5 to 10 min in an air-dry oven to remove any possible
moisture, samples were placed in a dessicator to cool and then weighed again. Percent fat

in the sample was determined as follow:

Dry sample weight - extracted sarnple weight

 

Fat (%) = x 100

Wet sample weight

184

APPENDIX [11

185

PROTEIN ANALYSIS

Sample Preparation: A l-g sample of powdered hot dog was weighted in a
porcelain crucible. The weight and sample code were entered into the program in the
Leco computer. The sampler were then dried for 18 to 20 h in an oven to remove residual
moisture that can cause internal malfunctions with the Leco Protein Analyzer.
LECO FP-2000 Protein Analyzer

Principal: The LECO FP-ZOOO Protein Analyzer (LECO Corporation, St. Joseph,
MI) is a non-dispersive, infrared, mocrocomputer based-instrument, designed to measure
the nitrogen content in a wide variety of organic compounds. Analysis begins by
weighing a l-g-ground sample and placing it into the porcelain sample holder referred to
as a boat. When “ANALYZE” is selected and the sample enters the combustion chamber
(Temperature = 566°C), the furnace and flow of oxygen gas, cause the sample to
combust. The combustion process converts any elemental nitrogen into N2 and NO.
Combustion gases are swept down and out of the inner combustion tube and then up
between the inner and outer combustion tubes. Oxygen flow is measured by the Oxygen
Flow rotameter before it enters the combustion chamber. After the ballast tank is filled
with sample gas, the gas is permitted to equilibrate before being released through the
aliquot loop. Sample gas in the aliquot doser is swept by the carrier gas to the catalyst
heater where NOx gases are reduced to N2. Lecosorb and Anhydrone are used to remove
CO2 and H20, respectively. This leaves N2 and helium to flow through one side of the
cell. The out side of the cell contains carrier gas scrubber filters. The gases in both sides

of cell are compared, and an output voltage is generated which is fed into the computer

186

processed, displayed, and stored as the nitrogen content and divided by 6.4 to obtain the

protein concentration.

187

APPENDIX IV

188

TBA METHOD

TBA values were determined using the extraction procedure of Tarladgis et al.
(1960) and Zipser et al, (1962).

TBA reagent Preparation: Thiobarbituric acid (1.4416 g) was dissolved in
distilled water (500 ml). The flask was placed in a sonic cleaner and was shaken

occasionally until TBA was dissolved.
HCI Solution: Make volume as needed; 1:2, HCl: H2O (v/v)

Sulfonamide Reagent Preparation: Sulfanilamide (0.5 g) and concentrated HCl
(20 ml) were dissolved in a 200 ml volumetric flask after which the volume was brought
to 200 ml with distilled water.

Sample Preparation: Ten g of frankfurters were weighed directly into
homogenizer flasks. After adding 50 ml of distilled water the sample was homogenized
for 1 min using homogenizer. The homogenate was quantitatively transferred to a 500 ml
extraction flask with the volume brought up to 100 ml with distilled water. Glass beads,
2.5 ml of HCI solution, two sprays of antifoam solution (~1 ml), and 2 ml sulfanilamide
solution were added to the flask. The extraction flask was connected to distilling tubes
and tightened to heating mantles. Powerstats were turned on to line voltage and the flasks
were heated rapidly. Fifty ml of distillate was collected, transferred to culture tubes,
capped and kept refi'igerated for the TBA reaction.

TBA Reaction and Spectrophotometric Determination: Five ml of distillate
and 5 ml of TBA reagent were added to each of 3 tubes. Five ml of distilled water was

pipetted into the blank tube. After thorough mixing using a Vortex Genie shaker, the

189

tubes were immersed in a 100°C water bath for 35 min and then cooled in ice bath for 10
min. Absorbance was read at 538 nm in Perkin Elmer UV/V IS Spectrometer Lambda 20
(Perkin-Elmer Corporation, North American Organic Division, Norwalk, CT). The
reading was multiplied by 7.8 to convert absorbance to mg malonaldehyde/1000 g of

sample.

190

APPENDIX V

191

Questioner of Sensory Panel for Hot Dogs

Page Number: 1
Attribute Sequence Number: 1

Attribute Type ........... : Instruction Box

Attribute Description....: { Instruction Box }

Seen With Relative Sample: none

You will evaluate cooked hot dog samples for five characteristics- juiciness,
texture, flavor, casing tenderness, and overall acceptability. Lift the panel door and slide
the ready portion of the card under the door. You will receive a sample with a three digit
random code. Answer the questions about the sample by clicking with your mouse next

to your desirable answer. When finished, slide the empty container and the finished

portion of the card under the door and you will receive the next sample.

192

 

Page Number: 2

Attribute Sequence Number: 2
Attribute Type ........... : Hedonic
Attribute Description....: Casing tenderness
Seen With Relative Sample: none

Is the tenderness of the casing desirable, or is it too tough, too soft, or too chewy?

[] Extremely Desirable

[] Very Desirable

[] Moderately Desirable

[] Slightly Desirable

[] Slightly Undesirable

[] Moderately Undesirable
[] Very Undesirable

[] Extremely Undesirable

Attribute Sequence Number: 3
Attribute Type...........: Hedonic
Attribute Description....: J uiciness
Seen With Relative Sample: none

Is the hot dog juiciness desirable, or is it too dry?

[] Extremely Desirable

[] Very Desirable

[] Moderately Desirable

[] Slightly Desirable

[] Slightly Undesirable

[] Moderately Undesirable
[] Very Undesirable

[] Extremely Undesirable

193

Page Number: 3

Attribute Sequence Number: 4
Attribute Type...........: Hedonic
Attribute Description....: Texture
Seen With Relative Sample: none

How desirable is the texture of the hot dog? Is it too hard, too mushy, or too
coarse?

[] Extremely Desirable

[] Very Desirable

[] Moderately Desirable

[] Slightly Desirable

[] Slightly Undesirable

[] Moderately Undesirable
[] Very Undesirable

[] Extremely Undesirable

Attribute Sequence Number: 5
Attribute Type...........: Hedonic
Attribute Description....: Flavor
Seen With Relative Sample: none

Is the flavor desirable, or is it too bland, spicy, bitter, sour, salty, or is there and
after-taste?

[] Extremely Desirable

[] Very Desirable

[] Moderately Desirable

[] Slightly Desirable

[] Slightly Undesirable

[] Moderately Undesirable
[] Very Undesirable

[] Extremely Undesirable

194

Page Number: 4

Attribute Sequence Number: 6
Attribute Type ........... : Hedonic
Attribute Description....: Overall desirability
Seen With Relative Sample: none

Your reaction to the overall satisfaction derived form the consumption of the hot
dog.

[] Extremely Desirable

[] Very Desirable

[] Moderately Desirable

[] Slightly Desirable

[] Slightly Undesirable

[] Moderately Undesirable
[] Very Undesirable

[] Extremely Undesirable

Attribute Sequence Number: 7
Attribute Type...........: Comment
Attribute Description....: Comment
Seen With Relative Sample: none

Comment Type: Optional

Please write any comments about the hot dogs:

195

APPENDIX VI

196

MICHIGAN STATE

UNIVERSITY

February 8, 2062

TO: Elliot RYSER
21.08 8. Anthony

Re; map 02.031 .CATEGORY: EXEMPT 1-C,1-G
APPROVAL DATE: February a, zoo:-

? TITLE: ANTIMICROBIAL WHEY PROTEIN ISOLATE-BASED CASING FOR HOT

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