.L. .2 .
, gnaw»
933...

 

£052., ,4 .
. .{x‘u 9.3.3917» .a
1:. r n 4

 

 

“WINS

540lé‘é33

 

LIBRARY
Michigan State
University

 

 

 

This is to certify that the

dissertation entitled

DEVELOPMENT OF AN ANTIMICROBIAL FILM
FOR FOOD PACKAGING

presented by

Paweena Limjaroen

has been accepted towards fulﬁllment
of the requirements for

Ph . D . degree in Packaging

 

 

 

X27166 ﬁg {/é;

Major professor

Date 12(41;',.-,n,,b:£ k 91 3‘ QC) 0 a)

 

MSU is an Afﬁrmative Action/Equal Opportunity Institution 0-12771

PLACE IN RETURN Box to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

JAN 3 1 2005

 

1‘1"“ 0 .
lr‘ir‘ln‘ﬁﬁl 210,3

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 cJCIRC/DateDuepes-sz

 

DEVELOPMENT OF AN ANTIMICROBIAL FILM FOR FOOD PACKAGING

By

Paweena Limjaroen

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
School of Packaging

2002

ABSTRACT
DEVELOPMENT OF AN ANTIMICROBLAL FILM FOR FOOD PACKAGING
By
PAWEENA LIMJAROEN

Polyvinylidene chloride (PVDC) copolymer ﬁlms containing nisin, potassium
sorbate, lactoferrin, sodium diacetate or sorbic acid were developed to have antimicrobial
activity against four strains of Listeria monocytogenes (CWD 95, CWD 246, CWD 201
and CWD 1503). Only ﬁlms containing nisin, potassium sorbate and sorbic acid had
antimicrobial activity. The minimum concentrations of nisin, sorbic acid and potassium
sorbate that had antimicrobial activity using a disc diffusion assay were 1.0%, 1.5% and
2.0% (w/v), respectively. Films containing sorbic acid had the most antimicrobial
activity, best barrier and mechanical properties, and greatest distribution of sorbic acid in
the polymer structure. The polyvinylidene chloride copolymer coating containing 3.0%
(w/v) sorbic acid in a 0.75 mil coating thickness on polyethylene terephthalate (PET) film

had antimicrobial activity against L. monocytogenes.
PVDC ﬁlms containing 1.5% and 3.0% (w/v) sorbic acid were selected to verify

their antimicrobial activity on Cheddar cheese and bologna, which were previously

surface inoculated with L. monocytogenes (CWD 95) at inoculum levels of 105 or 103
CFU/g. Both' products were examined at selected intervals for numbers of L.
monocytogenes, mesophilic aerobic bacteria, lactic acid bacteria, and yeast/mold. Films
containing 1.5 and 3.0% (w/v) sorbic acid decreased L. monocytogenes populations 0.1-1
log and 4.0-7.0 logs on cheese and bologna after 35 and 28 days refrigerated storage,

respectively. Potential reduction in the number of mesophilic and lactic acid bacteria was

also found on both cheese and bologna using film containing sorbic acid. Mold growth
was found only on cheese wrapped with sorbic acid-free film.

The migration of sorbic acid from PVDC antimicrobial films into Cheddar cheese
and beef bologna was determined using high performance liquid chromatography
(HPLC). At the end of refrigerated storage, 40 and 93% of the sorbic acid migrated from
the ﬁlm into Cheddar cheese and bologna, respectively. The rate constant for Cheddar

cheese was 0.007 per day, and for bologna was 0.040 per day.

To my mother

Panadda Amomjarusiri

iv

ACKNOWLEDGEMENTS

I would like to express my deepest heartfelt thanks to Dr. Bruce Harte and Dr.
Hugh Lockhart, my co-advisors, for their educational and professional guidance and
advice. I could not successfully complete this research project without all their support. I
cannot thank them enough for providing ﬁnancial support throughout my educational
program.

I also would like to express my sincere appreciation to my committee members,
Dr. Elliot Ryser and Dr. Susan Selke for their research guidance. I am grateful to Dr.
Elliot Ryser for all the great input and collaboration in this project. I would like to thank
him for allowing me to use the lab equipment to ﬁnish the project. I also owe a great deal
of gratitude to Dr. Susan Selke for all her support and great advice through my graduate
program.

I would like to thank Mike Mounts at Dow Chemical Company for his technical
support and for providing on unlimited supply of plastic resins and his arrangement for
me to complete part of the research at the Dow Chemical facility. I owe my success to
Joseph Leykam, the director of the Molecular Structure Facility, Department of
Biochemistry for allowing me to use the HPLC machine and for supplying valuable
technical knowledge. I sincerely appreciate Dr. Richard Schalek at the Composite Center
for his expertise and advice on scanning electron microscopy.

I owe a great deal of gratitude to Dr. Gregory Zeikus and his research group in the
Department of Biochemistry for allowing me to work on my research in his laboratory.

Without their support the study could not have been completed.

I would like to thank the School of Packaging and the Center for Food and
Pharmaceutical Packaging Research for their ﬁnancial support for this research.

I would like to thank Arzu Cagri for sharing her expertise and supporting me
throughout my research. I sincerely appreciate Emily Smith at the Statistics Consulting
Center at MSU for her expertise in data analysis. I also would like to acknowledge all the
past and present colleagues in the School of Packaging and Dr. Ryser’s laboratory for
their continued encouragement and friendship. ‘

I am most grateful to my best friend Dinlaka 'Sriprapundh for his dedication and
encouragement. I could not have undertaken this " endeavor without his help and
understanding. My deepest appreciation goes to my family, my mother Ms. Panadda
Amomjarusiri and younger sisters Ms. Suchada and Viraya Limjaroen who supported me

through the difﬁcult times and the good times while accomplishing my education.

vi

TABLE OF CONTENTS

(A page
LIST OF TABLES ........................................................ z ......................... x
LIST OF FIGURES ....................... xii
INTRODUCTION .................................................................................. 1
CHAPTER 1
LITERATURE REVIEW ........................................................................... 4
1.1. Antimicrobial Film .................................................................. 4
1.2. Antimicrobial substances .......................................................... 7
1.2.1. Bacteriocin.............................;;.‘ .................................. 7
1.2.2. Organic acids ................. ‘..' ............................................ 9
1.2.3. Parabens ................................................................... 10
1.2.4. Curing agents ............................................................. 11
1.2.5. Natural preservatives ..................................................... 12
1.2.6. Lactoferrin ................................................................. 12
1.2.7. Silver ion .................................................................. 13
1.3. Listeria monocytogenes and foodborne disease ............................... 14
CHAPTER 2
DEVELOPMENT OF A FOOD PACKAGING FILM WITH ANTIMICROBIAL
ACTIVITY ........................................................................................... 17
2.1. Abstract ............................................................................. 18
2.2. Introduction ........................................................................ 18
2.3. Materials and methods ............................................................ 21
2.3.1. Film preparation .......................................................... 21
2.3.2. Coating ﬁlm preparation ................................................ 21
2.3.3. Culture preparation ...................................................... 22
2.3.4 Disc diffusion-type assay ................................................ 22
2.3.5. Mechanical properties ................................................... 26
2.3.6. Seal strength testing ...................................................... 26
2.3.7. Water vapor permeability ............................................... 26
2.3.8. Oxygen permeability .................................................... 27

vii

2.3.9. Surface energy ............................................................ 27
2.3.10. Scanning electron microscope .......................................... 28
2.3.1 1. Differential scanning calorimetry (DSC) .............................. 28
2.4. Results and discussion ........................................................... 29
2.4.1. Antimicrobial properties ................................................ 29
2.4.2. Antimicrobial properties of a coating ﬁlm ............................ 38
2.4.3. Mechanical properties ................................................... 40
2.4.4. Seal strength ............................................................... 43
2.4.5. Water vapor permeability ............................................... 44
2.4.6. Oxygen permeability ..................................................... 47
2.4.7. Surface energy ............................................................ 49
2.4.8. Scanning Electron Microscope ......................................... 49
2.4.9. Differential Scanning Calorimeter .................................... 54
2.5. Conclusion ......................................................................... 54

CHAPTER 3

INACTIVATION OF LIST ERIA MONOCYTOGENES ON BEEF BOLOGNA
AND CHEDDAR CHEESE USING DEVELOPED ANTIMICROBIAL

POLYVINYLIDENE CHLORIDE FILM ....................................................... 58
3.1. Abstract ............................................................................. 59
3.2. Introduction ........................................................................ 60
3.3. Materials and methods ............................................................ 61

3.3.1. Target organism .......................................................... 61
3.3.2. Products .................................................................... 62
3.3.3. Film preparation .......................................................... 62
3.3.4. Product inoculation of Cheddar cheese and beef bologna
and storage ................................................................ 62
3.3.5. Microbiological analysis ................................................ 63
3.3.5.1. Cheddar cheese ................................................. 63
3.3.5.2. Bologna ......................................................... 63
3.3.6 Statistical analysis ........................................................ 64
3.4. Results and discussion ............................................................ 64
3.4.1. Antimicrobial activity on Cheddar cheese inoculated to
contain 105 and 103 L. monocytogenes cfu/g ......................... 64
3.4.1 . l. L .monocytogenes .............................................. 64
3.4.1.2. Mesophilic aerobic bacteria .................................. 70
3.4.1.3. Lactic acid bacteria ............................................. 73
3.4.1.4. Mold and yeast .................................................. 79

viii

3.4.2. Antimicrobial activity on bologna inoculated to contain

105 and 103 L. monocytogenes cfu/ g ................................... 80

3.4.2.1. L .monocytogenes ............................................. 80

3.4.2.2. Mesophilic aerobic bacteria ................................... 87

3.4.2.3. Lactic acid bacteria ............................................ 87

3.4.2.4. Mold and yeast .................................................. 92

3.5. Conclusion .......................................................................... 93

CHAPTER 4

MIGRATION OF SORBIC ACID FROM POLYVINYLIDENE CHLORIDE

ANTIMICROBIAL FILM TO CHEDDAR CHEESE AND BOLOGNA .................. 94

4.1. Abstract ............................................................................. 95

4.2. Introduction ................................. . ...................................... 95

4.3. Materials and methods ............................................................ 96

4.3.1. Products .................................................................... 96

4.3.2 Film preparation ........................................................... 96

4.3.3 Sample preparation ...................................................... 97

4.3.4. Migration test .............................................................. 97

4.3.4.1. Standard calibration curve .................................... 97

4.3.4.2. Extraction procedure and HPLC evaluation ............... 97

4.3.4.3. Calculation of migration/releasing rate ..................... 98

4.4. Results and discussion ............................................................ 101

4.5. Conclusion .......................................................................... 119

CONCLUSION ................................................................................... 120

APPENDIX I ...................................................................................... 122

APPENDIX [1 ..................................................................................... 181

ix

Table 1.1

Table 2.1

Table 2.2

Table 2.3

Table 2.4

Table 2.5

Table 2.6

Table 2.7

Table 2.8

Table 3.1

Table 3.2

LIST OF TABLES

page
Antimicrobial agents used in food packaging ....................................... 5

Antimicrobial activity of SaranR F-310 containing nisin against
4 strains of L. monocytogenes ........................................................ 30

Antimicrobial activity of SaranR F-310 containing potassium
sorbate against 4 strains of L. monocytogenes .................................... 31

Antimicrobial activity of SaranR F-310 containing sorbic acid
against 4 strains of L. monocytogenes ............................................... 32

Antimicrobial activities of polyvinylidene copolymer containing
sorbic acid, potassium sorbate and nisin against 4 strains of
L. monocytogenes ...................................................................... 34

Tensile strength, Percent elongation and toughness of SaranR F-310
control ﬁlm and ﬁlms containing sorbic acid, nisin and potassium
sorbate ................................................................................... 41

Seal strength of SaranR F -3 10 ﬁlm and ﬁlms containing 1.5%,
2.0% or 3.0% (w/v) sorbic acid ...................................................... 45

Water vapor transmission rate and water vapor permeability of
SaranR F-310 control ﬁlm and SaranR F-3lO ﬁlms containing
sorbic acid, nisin, and potassium sorbate ........................................... 46

Oxygen transmission rate (OTR) and oxygen permeability of
SaranR F-310 control ﬁlm and SaranR F -310 ﬁlms containing
sorbic acid, nisin, and potassium sorbate ........................................... 48

Inhibition of L. monocytogenes, mesophilic bacteria and lactic acid

bacteria on Cheddar cheeses with an initial inoculum of 105 CFU/ g

using PVDC copolymer ﬁlms containing 0%, 1.5% and 3% (w/v)

sorbic acid .............................................................................. 65

Inhibition of L. monocytogenes, mesophilic bacteria and lactic acid

bacteria on Cheddar cheeses with an initial inoculum of 103 CFU/g

using PVDC copolymer ﬁlms containing 0%, 1.5% and 3.0%

sorbic acid .............................................................................. 67

Table 3.3 Population change (log CFU/ g) of L. monocytogenes due to PVDC
copolymer ﬁlms containing 0%, 1.5% or 3.0% (w/v) sorbic acid on
Cheddar cheese after 35 days, and bologna slices after 28 days of
reﬁ'igerated storage ................................................................... 68

Table 3.4 Population change (log CFU/ g) of mesophilic aerobic bacteria
due to PVDC copolymer ﬁlms containing 0%, 1.5% or 3.0% (w/v)
sorbic acid on Cheddar cheese after 35 days, and bologna slices
after 28 days of refrigerated storage ................................................ 74

Table 3.5 Population change (log CFU/ g) of lactic acid bacteria (LAB)
due to PVDC copolymer ﬁlms containing 0%, 1.5% or 3.0% (w/v)
sorbic acid on Cheddar cheese after 35 days, and bologna slices
after 28 days of reﬁ'igerated storage ................................................ 77

Table 3.6 Inhibition of L. monocytogenes, mesophilic bacteria and lactic acid
bacteria on bologna which were inoculated with 10s CFU/ g using
PVDC copolymer ﬁlms containing 0%, 1.5% and 3% (w/v) sorbic
acid ...................................................................................... 81

Table 3.7 Inhibition of L. monocytogenes, mesophilic bacteria and lactic acid
bacteria on bologna which were inoculated with 103 CFU/ g using
PVDC copolymer ﬁlms containing 0%, 1.5% and 3% (w/v) sorbic
acid ...................................................................................... 83

Table 4.1 Loss of sorbic acid from polyvinylidene chloride antimicrobial
ﬁlm wrapped around Cheddar cheese during storage at 4°C .................. 102

Table 4.2 Loss of sorbic acid from polyvinylidene chloride antimicrobial ﬁlm
sandwiched between beef bologna during storage at 4 °C .................... 103

Table 4.3 Loss of sorbic acid from polyvinylidene chloride antimicrobial ﬁlm
(no food contact) during storage at 4 °C .......................................... 104

Table 4.4 The rate constants of releasing/loss of sorbic acid from PVDC
antimicrobial ﬁlms .................................................................. 118

xi

Figure 1.1

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 2.9

Figure 2.10

Figure 2.1 1

LIST OF FIGURES

page

Electron micrograph of Listeria monocytogenes (From Listeria,
Listeriosis and Food Safety; Ryser and Marth, 1991) ........................ 15

Listeria monocytogenes culture preparation for using as Listeria
stock culture for disc diffusion-type assay ..................................... 23

Disc diffusion-type assay for measuring antimicrobial activity
of inhibition zone of the ﬁlms against Listeria monocytogenes ............ 24

Inhibition zone of the foodborne pathogen Listeria monocytogenes
around the disc of polyvinylidene copolymer ﬁlm containing
sorbic acid after 48 hours at 35 °C ....... _. ...................................... 25

Comparison of the inhibition zones of ﬁlms containing 1%, 2%
or 2.5% (w/v) nisin ............................................................... 35

Comparison of the inhibition zones of ﬁlms containing 2% or
3% (w/v) potassium sorbate ...................................................... 36

Comparison of the inhibition zones of ﬁlms containing 1.5%, 2%
or 3% (w/v) sorbic acid with and without adjusted to pH 5.2 .............. 37

Comparison of the inhibition zones of ﬁlms containing sorbic

acid with and without pH adjusted to 5.2, potassium sorbate

or nisin against four strains of Listeria monocytogenes.

Mean i standard deviation ...................................................... 39
SEM micrographs of the surface structure of polyvinylidene

chloride copolymer ﬁlms containing no antimicrobial

(control ﬁlm) ........................................................................ 50

SEM micrographs of the surface structure of polyvinylidene
chloride copolymer ﬁlms (Top) containing 1.5% (w/v) sorbic
acid, (Bottom) 3% (w/v) sorbic acid ............................................ 51

SEM micrographs of the surface structure of polyvinylidene
chloride copolymer ﬁlms containing potassium sorbate ..................... 52

Scanning electron microscopy photomicrograph of the structure

of polyvinylidene chloride copolymer ﬁlms containing nisin

(Top) nisin spread in ﬁlm (Bottom) interphase between ﬁlm

matrix and nisin .................................................................... 53

xii

Figure 2.12

Figure 2.13

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

Differential Scanning Calorimeter (DSC) proﬁle of
polyvinylidene chloride copolymer (Saran F-310) ﬁlm
containing no antimicrobial ...................................................... 55

Differential Scanning Calorimeter (DSC) proﬁle of
polyvinylidene chloride copolymer (Saran F -3 10) ﬁlm
containing 3.0% sorbic acid ...................................................... 56

L. monocytogenes on Cheddar cheese with an
initial inoculum of 105 CFU/ g L. monocytogenes ............................ 69

L. monocytogenes on 3Cheddar cheese with an
initial inoculum of 10 CFU/ g L. monocytogenes ............................. 71

Mesophilic aerobic bacteria on Cheddarcheese
with an initial inoculum of 105 CF U/ g L. monocytogenes .................. 72

Mesophilic aerobic bacteria or; Cheddar cheese
with an initial inoculum of 10 CFU/ g L. monocytogenes ................... 75

Lactic acid bacteria on Cheddar cheese with an
initial inoculum of 105 CFU/ g L. monocytogenes ............................ 76

Lactic acid bacteria or; Cheddar cheese with an
initial inoculum of 10 CFU/g L. monocytogenes ............................. 78

L. monocytogenes on bologna with an initial inoculum
of 105 CF U/ g L. monocytogenes using different ﬁlm types ................. 85

L. monocytogenes on bologna with an initial inoculum
of 103 CFU/ g L. monocytogenes using different ﬁlm types ................. 86

Mesophilic aerobic bacteria on bologna with an initial

inoculum of 105 CFU/ g L. monocytogenes using
different ﬁlm types ................................................................ 88

Mesophilic aerobic bacteria on bologna with an initial

inoculum of 103 CFU/ g L. monocytogenes using
different ﬁlm types ................................................................ 89

Lactic: acid bacteria on bologna with an initial inoculum
of 10 CFU/ g L. monocytogenes using different ﬁlm types .................. 90

xiii

Figure 3.12

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.1 1

Figure 4.12

Lactig: acid bacteria on bologna with an initial inoculum
of 10 CFU/g L. monocytogenes using different ﬁlm types .................. 91

Chromatogram of sorbic acid from the polyvinylidene
chloride antimicrobial ﬁlm ...................................................... 99

Standard curve of sorbic acid concentration in a methanol
solution ............................................................................ 100

The change in concentration of sorbic acid as a function of
storage time ﬁ'om PVDC antimicrobial ﬁlm wrapped around
Cheddar cheese at 4°C .......................................................... 105

The relative concentration of sorbic acid as a function of
storage time from PVDC antimicrobial ﬁlm wrapped around
Cheddarcheeseat4°C.............. ....... . ..................................... 106

The change in concentration of sorbic acid as a function of
storage time from PVDC antimicrobial ﬁlm sandwiched
between bologna at 4°C ......................................................... 107

The relative concentration of sorbic acid as a ﬁmction of
storage time from PVDC antimicrobial ﬁlm sandwiched
between bologna at 4°C ......................................................... 108

The change in concentration of sorbic acid of PVDC
antimicrobial ﬁlm (control ﬁlm) as a function of storage
time at 4°C ........................................................................ 109

A ﬁrst order rate plot of the loss of sorbic acid of PVDC
antimicrobial ﬁlm (control ﬁlm) at 4°C ....................................... 110

Comparison of the change in concentration of sorbic acid
between PVDC antimicrobial ﬁlm wrapped cheese and
bologna as a function of storage time at 4°C ................................. 1 12

Comparison of the relative concentration of sorbic acid as
a function of storage time between PVDC antimicrobial ﬁlm
wrapped Cheddar cheese and bologna at 4°C ................................ 1 13

A ﬁrst order rate plot of the loss of sorbic acid from PVDC
antimicrobial ﬁlm wrapped Cheddar cheese at 4°C ........................ l 14

A ﬁrst order rate plot of the loss of sorbic acid from PVDC
antimicrobial ﬁlm wrapped bologna at 4°C .................................. 115

xiv

Figure 4.13

Figure 4.14

A ﬁrst order rate plot of the loss of sorbic acid from PVDC
control ﬁlm (without wrapping food) at 4°C ................................. 116

A ﬁrst order rate plot of the loss of sorbic acid from PVDC
antimicrobial ﬁlm wrapped cheese and bologna as a ﬁmction

of storage time at 4°C ........................................................... 117

XV

INTRODUCTION

Contamination of food by foodborne pathogens and spoilage microorganisms is of
great concern to the food industry. Microbial contamination of food may occur in post-
processing during packaging and distribution. Listeria monocytogenes continues to pose a
major threat to the food industry as a post-processing contaminant. Commonly found in
home refrigerators (Ziney and Debevere, 1998), this psychotropic pathogen can readily
contaminate reﬁigerated foods and grow to potentially hazardous levels. L.
monocytogenes has remained the leading cause of Class I microbiologically related
recalls for more than 15 years. Class I is a health hazard situation where there is a
reasonable probability that the use of the product will cause serious, adverse health
consequence or death. A multistate outbreak involving at least 100 cases of listeriosis in
22 states from consumption of hot dogs during August 1998 to February 1999 (which
caused 21 fatalities) heightened concerns regarding foodborne listeriosis (CDC 1999).
From April 1998 to October 2001 there were more than 75 million pounds of cooked
ready-to-eat meats from over 75 Class I recalls (USDA-FSIS, 2001).

L. monocytogenes can cause mastitis leading to excretion of the organism in milk
from infected animals (Gitter et al, 1980). Ryser and Marth (1987) reported that L.
monocytogenes can survive more than 1 year in Cheddar cheese. In cottage cheese L.
monocytogenes survived during fermentation and manufacture (Hicks and Lund 1991,
Ryser et al., 1985) with viable cells recovered from refrigerated cheese (Piccin and
Shelef, 1995). The outbreaks originated in both domestic and imported cheese (Ryser

and Marth, 1988). In 2000, there were 2,298 cases of listeriosis reported in the US,

which cost approximately $1 million per case, thus it is the costliest foodborne disease on
a per-case basis (USDA-PISS, 2001).

Antimicrobial ﬁlms may be an effective approach to inhibit foodborne pathogens
or spoilage microorganisms, to enhance food safety and decrease product spoilage.
Incorporation of chemical preservatives or antimicrobial agents into a ﬁlm may provide a
way to enhance microbial safety. An antimicrobial agent in ﬁlms can diffuse into the
food to inactivate target microorganisms. Weng and Hotchkiss (1992) developed a
polyethylene-based antimycotic ﬁlm using the antimycotic substance imazalil to inhibit
mold growth on the surface of cheese. Han and Floros (1997) incorporated potassium
sorbate into low-density polyethylene ﬁlm to inhibit the growth of yeast (Saccharomyces
cerevisiae). Nisin and lysozyrne in combination with EDTA can be used in corn zein and
soy protein ﬁlms to inhibit Escherichia coli 0157: H7 (Padgett et al., 1998).

The research hypothesis of this study is that an antimicrobial food packaging
polyvinylidene chloride (PVDC) ﬁlm can be made by incorporating some well-known
antimicrobial agents in the PVDC. In order to prove the hypothesis the objectives of this
work were:

(1) To develop antimicrobial packaging ﬁlms using nisin, lactoferrin, sorbic acid,

potassium sorbate or sodium diacetate.

(2) To determine the antimicrobial activity of the ﬁlms against the Listeria

monocytogenes in laboratory media.

(3) To verify the ﬁlm’s antimicrobial activity against L. monocytogenes on two

food products, Cheddar cheese and beef bologna.

(4) To assess the barrier, water and oxygen permeability, and mechanical
properties, and surface energy of the antimicrobial ﬁlm. Electron microscopy
is used to assess the three-dimensional structure of the ﬁlm.

(5) To verify the antimicrobial activity of the ﬁlms coated on base ﬁlm,
polyethylene terephthalate (PET), against the foodborne pathogen Listeria
monocytogenes in laboratory media.

(6) To determine the migration rates of the antimicrobial agent (sorbic acid) from

the ﬁlm into the food, Cheddar cheese and beef bologna.

CHAPTER 1

LITERATURE REVIEW

1.1. ANTIMICROBIAL FILM

Antimicrobial ﬁlms have been in continuing development since the 19903 (Rice,
1995). The potential application of antimicrobial ﬁlms is mostly in food packaging to
reduce surface contamination of solid or semisolid food. When antimicrobial agents are
incorporated into a ﬁlm, the ﬁlm may then have the ability to prevent or inhibit microbial
growth (Han, 2000). Antimicrobial ﬁlms can be classiﬁed into two types. The ﬁrst type
is ﬁlm containing antimicrobial agents that migrate to the surface of the packaging
material, and then migrate to the food inhibiting food spoilage organism or foodborne
pathogen. The second type is ﬁlm containing antimicrobial agents that move to the
A surface of the ﬁlm, and inhibit microbial growth on food surfaces without migration of
the active agent into the food. Several synthetic and naturally occurring compounds have
been proposed as antimicrobial agents in packaging ﬁlm (Table 1.1). Brody et a1. (2001)
listed the citeria that should be considered when developing antimicrobial ﬁlms:

0 Consideration of the spectrum of microorganisms against which the
package system might be effective. Films may inhibit food spoilage
organisms without affecting the growth of pathogenic microorganisms,
which will raise safety questions similar to those arising from technologies

such as modiﬁed atmosphere packaging.

Table 1.1. Antimicrobial agents used in food packaging (Hotchkiss, 1995)

 

Class

Examples

 

Organic acids
Bacteriocins
Spice extracts
Thiosulﬁnates
Enzymes
Proteins
Isothiocyanates
Antibiotics
Fungicides
Chelating agents
Metals

Parabens

Propionic, benzoic, sorbic acid
Nisin

Thymol, p-cymene, wasabi
Allicin

Peroxidase, lysozyme
Conalbumin
Allylisothiocyanate
Irnazalil

Benomyl

EDTA

Silver

Heptylparaben

 

0 Consideration of the effects the antimicrobial additive may have on the
mechanical and physical properties of the plastic packaging material
structure.

0 Does the antimicrobial activity cause a reduction in growth rate, while
there is still an increase in cell numbers, or does it cause cell death, with a
decline in cell number?

0 Consideration of the migration of the antimicrobial agent into the food,
and any toxicological and regulatory concern that may exist.

0 Consideration of the effect of food characteristics such as pH and water
activity (Aw). Some antimicrobial agents are effective only at a speciﬁc
pH.

For food packaging applications or direct contact with humans, safety must be
ensured. The antimicrobial agents used in ﬁlms have to be approved by the Food and
Drug Administration (FDA). Ben-Yehoshua et a1. (1987) incorporated Irnazalil (a
commercial antimycotic) into low density polyethylene (LDPE) ﬁlm for wrapping fruits
and vegetables. This ﬁlm also effectively prevented mold growth on cheese surfaces.
Imazalil is not, however, approved for use with cheese in the United States. Metallic ions
of silver and copper are regarded in Japan (Brody et al., 2001) and in the US as safe
antimicrobial agents.

Antimicrobial ﬁlms may include antimicrobial packaging ﬁlms and antimicrobial
edible ﬁlms. An example of an antimicrobial packaging ﬁlm is LDPE impregnated with
benzoic anhydride. This ﬁlm exhibited antimycotic activity on laboratory media and

cheese (Hotchkiss, 1995). Nisin and lysozyme in combination with EDTA in corn zein

and soy protein ﬁlms inhibited Escherichia coli 0157: H7 (Padgett et al., 1998), and is an

example of an antimicrobial edible ﬁlm.

1.2. ANTIMICROBIAL SUBSTANCES

A chemical preservative can be incorporated in a packaging material to add
antimicrobial activity. Common antimicrobial chemicals for food products include
bacteriocins (e.g. nisin), organic acids (such as sorbic acid and its salts, benzoic acid and
sorbate), parabens, curing agents (e. g. sodium chloride), natural preservatives (e. g.
wasabi used in Japan), lactoferrin, and metals (e.g. Silver ions). Antimicrobial agents
which may have possible use in antimicrobial packaging ﬁlms will be addressed
individually.
1.2.1. Bacteriocins
Nisin

Nisin, an antimicrobial agent, belongs to a unique group called bacteriocins.
Bacteriocins are deﬁned as a group of microbially synthesized proteinaceous
antimicrobial substances, which have a narrow spectrum of inhibition and only inhibit
bacteria closely related to the producer organism. Nisin is a small peptide bacteriocin
produced by several Lactococcus lactis strains (Sahl et al., 1995). It is produced during
the exponential phase of bacterial growth (Buchanan et al., 1988). The complete structure
was elucidated by Gross and Morell (1971) and conﬁrmed by chemical synthesis (Fukase
et al., 1988), DNA sequencing and NMR resonance (Chan et al., 1989). Nisin is
composed of 34 amino acids (AA) (Jung, 1991) and possesses amphiphilic characteristics

with clusters of hydrophilic and hydrophobic residues at the C and N-terminus,

respectively. Gross and Morell (1967; 1971) identiﬁed didehydroalanyllysine at the C-
terminal and isoleucine as the N-terminal AA. Nisin contains a number of uncommon
amino acids, which are formed in a post-translational modiﬁcation process including co
and B unsaturated amino acids, dehydroalanine (Dha), B-methyldidehydroalanine or
didehydrobutyrine (Dhb) and thioether amino acids lanthionine and B-methyllanthionine
(Rollema et al., 1996). It is suggested that the unusual amino acids might be responsible
for important properties of the nisin molecule such as acid tolerance, thermostability, and
its bactericidal mode of action.

Nisin was the ﬁrst bacteriocin to attain GRAS (General Recognized As Safe)
status by FDA. Nisin is active against a broad range of gram-positive microorganisms
(e. g. L. monocytogenes and Clostridium botulinum). It has been used as a preservative in
cold-pack cheeses (Delves-Broughton, 1990).

The exact molecular mechanism by which nisin inhibits microbial growth is still
not fully understood, although several studies have found that nisin interacts with the
bacterial cell membrane forming pores ultimately leading to cell lysis (Hurst, 1981; Gao
et al., 1991; Driessen et al., 1995; Sahl et al., 1995). When combined with Chelating
agents such as EDTA, nisin effectively inhibits Gram-negative bacteria such as
Salmonella and Escherichia coli (Shelef et al., 1995; Wells et al., 1998). The Chelating
agent reacts with the outer membrane (lipopolysaccharide) of gram-negative bacteria to
allow nisin to penetrate the cell membrane, forming pores which induces cell lysis

(Stevens etal., 1991).

1.2.2. Organic acids
Sorbic acid and sorbate

Sorbic acid is a well-known food preservative 'and is classiﬁed as GRAS. It is
used in bakery, meat products, fruit and dairy products to inhibit a wide range of yeasts
(Lueck, 1980), molds such as F usarium, Aspergillus, Mucor (Sofas and Busta, 1981), and
bacteria including E. coli 0157:H7, Staphylococcus aureus, C. botulinum and coliforrn
(Kasrazadeh and Genigeorgis, 1995; Gandhi et al., 1973; Briozzo et al., 1985). It can also
inhibit aﬂatoxin and enterotoxin production (Luck, 1980). The typical usage level in
food is from 0.02% to 0.3%. Sorbic acid (CH3CH=CHCH=CHCOOH) is a straight chain
or, B-unsaturated monocarboxylic acid (Windholdz et al., 1976).

Potassium sorbate is the well-known salt of sorbic acid, which is highly soluble in
water. Sorbic acid is most effective in its undissociated form (pKa = 4.8). Sorbic acid
exists in its undissociated (86%) form at pH 4.75 (Sofos et al., 1980) which is able to
penetrate the bacterial cytoplasmic membrane (Chichester and Tanner, 1972). Sorbic
acid is most effective in acidic foods (Cowles, 1941).

Sorbic acid induces changes in the morphology and appearance of microbial cells.
Sorbic acid can inhibit speciﬁc biosynthesis pathways (Sofos et al., 1986). For example,
sorbate prevents the amino acids, L-serine and L-histidine ﬁom being taken up by
Salmonella Typhimerium at low pH (Tuncan and Martin, 1985). Sorbic acid also
produced pores on cell membrane (Freese and Levin, 1978). Induction of an energy-
expensive protective membrane has been reported as one mode of action. Proton

pumping increases (H‘i, ATPase) to ensure that the pH will not decline or compensate for

any disruption of intracellular pH to maintain homeostasis, which results in less available
energy for normal growth.
Benzoic acid and benzoate

Benzoic acid and its sodium salt (sodium benzoate) are also GRAS preservatives.
Benzoic acid is well known as a mold and yeast inhibitor, and it can also inhibit
pathogenic bacteria. The antimicrobial activity of benzoic acid against foodborne
pathogen increases when combined with organic acids. Huwang et a1. (1995) showed that
the population of L. monocytogenes, Escherichia coli 0157:H7, Salmonella,
Campylobacterjejuni and Staphylococcus aureus decreased signiﬁcantly on raw chicken
wings treated with 0.05% sodium benzoate/0.5% lactic acid solution (pH 2.6) for 30
minutes during storage at 4°C. The typical use level is up to 0.1% in commercial foods.
The antimicrobial activity of benzoic acid is related to pH. The pKa of benzoic acid is
4.2. It is most effective in its undissociated form with 60% undissociated at pH 4.0.
Benzoic acid induces change in the morphology and appearance of microbial cells.
Benzoic acid also reduces the intracellular pH. Salmond et a1. (1984) reported on the
reduction in intracellular pH in E. coli. Benzoic acid also alters cell membrane function
by producing pores that interfere with the uptake of substrate, electron transport and
proton-motive forces.
1.2.3. Parabens

Parabens are made by esteriﬁcation of the carboxyl group of benzoic acid
(phenolic derivatives). Parabens in the undissociated form (active form) exist at pH 3.0-
8.0. Parabens are used as food preservatives in the methyl, propyl and heptyl forms.

Parabens with longer alkyl chains possess more antimicrobial activity than those with

10

shorter alkyl chain (Shibasaki, 1969). Parabens are mostly used in butter, margarine,
maple syrup and meat products. Parabens are generally more active against molds and
yeasts than against bacteria. Parabens are more effective against gram-positive bacteria
than gram-negative bacteria.

Parabens damage the cytoplasmic membrane of microorganisms leading to the
release of cell cytoplasmic compounds (Judis, 1963). Furr and Russel (1972) reported
that parabens caused leakage of RNA in Serratia marcescens, with the extent of leakage
proportional to their alkyl chain length. Parabens were also reported to inhibit membrane
transport, electron transport, and nutrient uptake through the cytoplasmic membrane
(Freese et al., 1973; Eklund, 1980).

’ 1.2.4. Curing agent
Sodium chloride

Sodium chloride (N aCl) is a well-known curing agent used as a food preservative
since ancient times. The antimicrobial activity of sodium chloride is related to its ability
to reduce water activity in food (plasmolytic effect). Microbial cells lose water when the
water activity of the external environment is reduced, which results in growth inhibition
or cell death (Sperber, 1983). Sodium chloride also limits oxygen solubility (osmotic
effect) and alters pH (Banward, 1979). While most foodborne pathogens are susceptible
to sodium chloride, Staphylococcus aureus, can grow at low water activity, and Listeria
monocytogenes, is a salt-tolerant pathogen that can grow at concentrations of up to 10%

NaCl.

11

1.2.5. Natural preservative
Wasabi

Wasabi is a compound extracted from Japanese horseradish. Japan’s Sekisui
Jushi has developed an antimicrobial food packaging material by incorporation of a
wasabi derivative, allylisothiocyanate (AIT), into polyethylene ﬁlm (Brody et al., 2001).
This packaging material is claimed to inhibit the proliferation of bacteria and fungi on
food surfaces, thus extending product shelf life. This antimicrobial ﬁlm is intended to be
placed between‘ food layers such as in a lunch box or ready-to-eat foods. It is now
available to consumers in Japan.

An allylisothiocyanate (AIT) substance has received approval to be used as a
food additive to deliver wasabi ﬂavor in food (Brody et al., 2001). It is known to have
antimicrobial activity against bacteria and fungi. However, it has a strong odor and
pungent taste. Therefore, when used as an antimicrobial, this substance is generally used
indirectly in the packaging material.

1.2.6. Lactoferrin

Lactoferrin is a newly isolated antimicrobial peptide derived from bovine
lactoferrin present in cow’s milk (Wakabayashi et a1, 1992). It is an iron binding
glycoprotein, which can bind two iron atoms per molecule. Lactoferrin has antimicrobial
activity against many bacteria including Listeria monocytogenes, Bacillus subtilis, B.
stearothermophilus, Micrococcus spp., and E. coli (Hutchens et a1, 1994; Payne et al.,
1990; Reiter, 1978; Cram and Reiter, 1968). Lactoferricin B is the active component of

lactoferrin, containing 25 amino acid residues, and can be isolated by acid-pepsin

12

hydrolysis from the N-terminal region of the molecule (Bellamy et al., 1992;
Wakabayashi et al, 1992).

Lactoferrin chelates iron, calcium and magnesium ions, causing cell dealth.
Payne et a1. (1989) reported that the inhibition of L. monocytogenes using lactoferrin was
directly related to iron availability in the medium because L. monocytogenes survives
best in iron-rich media. Arnold et a1. (1982) reported that lactofenin inhibited several
bacteria in an iron-rich environment. For gram-negative bacteria such as E. coli,
lactoferrin caused the chelation of cations that stabilize lipopolysaccharides, which
increased permeability of the outer membrane to hydrophobic compounds.
1.2.7 Silver ion

Among metallic ions, silver ion has the strongest antimicrobial activity (Brody et
al., 2001). Metallic silver does not release the ion easily, compared with other metallic
ions. Therefore, its antimicrobial activity is not as strong in its metallic state, and its
greatest potential appears to be as releasable silver salts such as silver nitrate and Ag-
zeolite. Silver nitrate, which forms silver ions in a water solution, has strong
antimicrobial activity. Microbial cells absorb the silver ion by active transportation,
inhibiting a range of metabolic enzymes which inhibit metabolic processes necessary for
sustaining life (Brody et al., 2001). Ag-zeolite maintains Ag+ ions in a stable and
effective condition. It has antimicrobial activity against bacteria (no effect against spores
of heat-resistant bacteria), yeast and fungi (Brody et al., 2001). For application of Ag-
zeolite to packaging, it is usually laminated as a thin coextruded layer (3-6 pm) because

of its expense.

13

1.3. LISTERIA MONOCY T OGENES AND FOODBORNE DISEASE

Listeria monocytogenes is a foodborne pathogen, and is of major concern to the
food industry. L. monocytogenes is a gram-positive, non-spore-forming, rod-shaped
bacterium (Figure 1.1). L. monocytogenes grows at temperatures of 1-45 °C, with the
optimum growth at 30-37 °C. L. monocytogenes can cause the human disease called
listeriosis. Listeria monocytogenes is ubiquitous in nature, being commonly found in soil,
water, silage and plants. Consequently, this pathogen is frequently found on raw
materials used in food processing. Cox et a1. (1989) demonstrated that Listeria spp.
could be found in all types of food production environments. L. monocytogenes also has
the ability to attach to various food contact surfaces such as stainless steel,
polypropylene, glass and rubber (Herald and Zottola, 1988; Mafu et al., 1990; Blackrnan
and Frank, 1996). Once present in the processing environment, control of L.
monocytogenes has proven to be difﬁcult. L. monocytogenes is present in various foods.
Dairy foods have received the most scrutiny as vehicles for listeriosis. Dairy products can
be particularly susceptible to contamination by Listeria because cows can shed the
organism in the milk. Thus, contaminated raw milk could serve to introduce the
bacterium into dairy plants or foods made from raw milk. Contamination of L.
monocytogenes can be found in poultry and meat products including processed meat
products such as fermented sausage. During August 1998 to February 1999, one
listeriosis outbreak from hot dogs resulted in 101 cases of listeriosis (including 21
fatalities) in 22 states and greatly heightened concerns regarding the presence of L.

monocytogenes in ready-to-eat foods (CDC 1999). L. monocytogenes can be found in

14

 

Figure 1.1. Electron micrograph of Listeria monocytogenes (From Listeria, Listeriosis
and Food Safety; Ryser and Marth, 1991)

slaughterhouse and meat packaging areas. Fruits and vegetables can be contaminated
with L. monocytogenes. In 1981, there was an outbreak involving cabbage (Schlech et al.,
1983). It was concluded that the sheep manure used to fertilize the cabbage was the
source of the contamination. Anyone can become infected with L. monocytogenes.
However, some groups of people are more susceptible than others, including pregnant
women, newborns, infants, and adults with a compromised immune system such as
cancer patients. For listeriosis in pregnant women, infection commonly is manifested by
fever, chills, headache, backache, and discolored urine. The ﬂu-like symptoms are the
expression of Listeria bacteremia, and Listeria monocytogenes can be isolated from
blood. Infection in pregnant women leads to infection of the fetus either via the
transplacental route or during delivery (Ryser and Marth, 1991). Furthermore, in some
cases abortion or stillbirth occurred immediately after the mother experienced ﬂu-like
symptom (Ryser and Marth, 1991). Neonatal listeriosis is now among the most
dangerous forms of listeriosis, and is a major cause of fetal damage and infant death. The
symptoms of neonatal listeriosis include vomiting, refusal to drink, respiratory distress,
heart failure and forced respiratory (Ryser and Marth, 1991). L. monocytogenes can also

cause meningitis in healthy adults.

16

CHAPTER 2.

DEVELOPMENT OF A FOOD PACKAGING FILM WITH ANT IIVIICROBIAL

ACTIVITY

l7

2.1. ABSTRACT

Polyvinylidene chloride copolymer ﬁlms containing nisin or potassium sorbate or
sorbic acid were developed to have antimicrobial properties against four strains of
Listeria monocytogenes (CWD 95, CWD 246, CWD 201 and CWD 1503). The minimum
inhibitory concentrations of nisin, sorbic acid and potassium sorbate were 1%, 1.5% and
2%, respectively. Films containing l%-2.5% nisin yielded average inhibition zones of
18.7 to 26.7 mm. Films containing 2% and 3% potassium sorbate had average inhibition
zones of 18.3 and 22.0 mm, respectively, whereas ﬁlms containing 1.5%-3%sorbic acid
yielded average inhibition zones of 20.5-32.7 mm. Incorporating nisin, potassium
sorbate or sorbic acid increased moisture and oxygen permeability (except for ﬁlms
containing sorbic acid), decreased tensile strength, and toughness of the ﬁlms, and
signiﬁcantly altered elongation at break. Surface energy of the ﬁlms remained unchanged
after incorporating these antimicrobial agents. The three-dimensional structure of the
ﬁlms was also observed using an environmental scanning electron microscope (ESEM).
Films containing sorbic acid were partly homogenous, while ﬁlms containing nisin had
nisin particles distributed throughout the ﬁlm. Adding potassium sorbate caused

pits/minuscule holes throughout the ﬁlm structure.

2.2. INTRODUCTION

Contamination of food by foodborne pathogens and spoilage microorganisms is of
great concern in the food industry. Microbial contamination of food may occur during
post-process handling, packaging and distribution. Listeria monocytogenes, which causes

listeriosis, is the major cause of class I microbiologically related recalls. Microbial

l8

growth on food surfaces is the key determinant of spoilage and safety for many food
products including refrigerated meats and intermediate moisture foods (IMF) (Torres et
a1, 1985; Vojdani and Torres, 1989a, b; Rico-Pena and Torres, 1991; Roth and Loncin,
1985). Several approaches have been developed to reduce the risk of surface
contamination.

Antimicrobial ﬁlms are a new and effective approach to inhibit foodborne
pathogens or spoilage microorganisms, to enhance food safety and decrease product
spoilage. Incorporating of chemical preservatives or antimicrobial agents into the ﬁlm
may provide a way to enhance microbial safety. Antimicrobial agents in ﬁlms can diffuse
into the food to inactivate target microorganisms. Weng and Hotchkiss (1992) developed
a polyethylene-based antimycotic ﬁlm using imazalil to inhibit mold growth on the
surface of cheese. Han and Floros (1997) incorporated potassium sorbate into low-density
polyethylene ﬁlm to inhibit the growth of yeast (Saccharomyces cerevisiae). Nisin and
lysozyme in combination with EDTA were similarly used in corn zein and soy protein
ﬁlms to inhibit Escherichia coli 0157: H7 (Padgett et al., 1998).

Common antimicrobial agents include organic acids such as sorbic, lactic, acetic
and benzoic acids and bacteriocins such as nisin. Nisin, the best-known antimicrobial
peptide and most widely studied bacteriocin, is a secondary metabolite from Lactococcus
lactis subsp. lactis. This natural compound has been well characterized as a food
preservative and has attained GRAS status. Nisin inhibits a broad spectrum of gram-
positive pathogenic bacteria such as L. monocytogenes and Clostridium botulinum
(Shefet et a1, 1995), as well as bacterial spores (Stevens et a1, 1991). Hansen (1994)

stated that nisin was a model food preservative.

l9

Lactoferrin is an antimicrobial peptide derived from bovine lactoferrin present in
cow’s milk (Wakabayashi et a1, 1992). The susceptibility of L. monocytogenes to bovine
lactoferrin has been studied on laboratory media (Hutchens et a1, 1994). Sodium diacetate
has been studied for its antimicrobial activity against foodborne pathogens and spoilage
microorganisms. Sodium diacetate had antimicrobial activity against L. monocytogenes
in brain heart infusion broth (Shelef and Addala, 1994) and turkey breast meat (Schlyter
et al., 1993). Sorbic acid and potassium salts (potassium sorbate) are well-known food
preservatives that have attained GRAS status. They are effective inhibitors of most
molds, yeasts and some bacteria. Sorbic acid in combination with lactic acid or acetic
acid can be successfully used to inhibit L. monocytogenes in cold-pack cheese and many
low acid foods (Ryser and Marth, 1988). Potassium sorbate has been used to preserve
meat products including reﬁigerated packaged beef (Zamora and Zaritzky, 1987a, b).

The objectives of this work were to (1) develop an antimicrobial ﬁlm wrap for
foods by incorporating nisin, lactoferrin, sodium diacetate, potassium sorbate or sorbic
acid into a polyvinylidene chloride copolymer ﬁlm, with targeted inhibition of L.
monocytogenes, (2) assess the effect of ﬁlm casting technique on mechanical and barrier
properties including water vapor and oxygen permeability, tensile strength, and surface
energy of the test ﬁlms, and (3) examine the morphological characteristics of the ﬁlms
using scanning electron microscopy, and (4) verify the antimicrobial activity of ﬁlm

coated on base ﬁlm (PET) against L. monocytogenes.

20

2.3. MATERIALS AND METHODS
2.3.1. Film preparation

To make ﬁlm, polyvinylidene chloride (PVDC) copolymer resin (SaranR F-310,
Dow Chemical, Midland, MI) (18% w/v) was added to methyl ethyl ketone (J .T Baker,
Phillipsburg, NJ) at room temperature and continuously agitated until completely
dissolved. Then, 0.5%, 1%, 1.5%, 2% or 3% (w/v) of either nisin, lactoferrin, sodium
diacetate, potassium sorbate, or sorbic acid (Sigma Chemical Co., St. Louis, MO) was
added. For ﬁlm with pH 5.2, ﬁlm solution was adjusted to pH 5.2 using lactic acid (1 N)
and 2 M sodium hydroxide (Sigma). The original "pH of ﬁlm was 3.5. PVDC ﬁlm
solutions (14 ml) were cast in a glass petri dish (150 mm diameterx 15 mm height) or 40
ml solutions were cast to a glass plate (9 x 13 inches). Films containing sorbic acid,
potassium sorbate or sodium diacetate were dried in a hot air oven at 86 i 0.5 °C for 5
minutes and peeled off from the plates. Films containing nisin or lactoferrin were dried
at room temperature (23°C) in a chemical hood with airﬂow of 45 ft/min, for 2-3 hours or
until dried, and peeled from the plates. The thickness of the ﬁlm was determined using a
micrometer (model 549, Testing Machines Inc., Amityville, NY). The average thickness
was obtained from 10 measurements from different locations on each sample.
2.3.2. Coating film preparation

To coat ﬁlms, SaranR F-310 (Dow Chemical, Midland, MI) at a concentration of
18% w/v was dissolved gradually in methyl ethyl ketone (J .T Baker, Phillipsburg, NJ) at
room temperature with continuous agitation until completely dissolved. Sorbic acid
concentration 1.5%, 2.0% or 3.0% (w/v) (Sigma Chemical Co., St. Louis, MO) was

incorporated into the coating solution. This solution was coated on the base ﬁlm, 0.5 mil

21

polyethylene terephthalate (PET) ﬁlm (Dow Chemical, Midland, MI), using a wire-rod
and dried at 86 °C. The wire-rods (Dow Chemical, Midland, M1) were fabricated by the
Saran Barrier Division at Dow Chemical (Midland, MI). The coating thicknesses were
0.2, 0.5 and 0.75 mil :1: 0.03 mil.
2.3.3. Culture preparation
Four strains of Listeria monocytogenes (CWD 95 and CWD 246 from silage,

CWD 201 from raw milk, and CWD 1503 from ground turkey) were obtained from MSU
culture collection (Dept. of Food Sciences and Nutrition, Michigan State University, East
Lansing, MI). The cultures were maintained at ~70°C in trypticase soy broth (TSB)
(Difco Laboratories, Detroit, MI) containing 10% (v/v) glycerol (J .T. Baker, Phillipsburg,
NJ) and subcultured twice in T88 containing 0.6% (w/v) yeast extract (Difco) at 35 °C/
18-24 hours before use (Figure 2.1).
2.3.4. Disc diffusion-type assay

Antimicrobial ﬁlms were cut aseptically into l6-mm diameter discs using a sterile
cork borer and sterilized using UV light for 20 minutes. These discs were then aseptically
placed on 20 ml melted trypticase soy agar (TSA) containing 0.6% yeast extract (TSA-
YE)(Difco) (acidiﬁed to pH 5.2 using 1 N hydrochloric acid) which was previously
inoculated with 0.2 ml of an 18-24 h L. monocytogenes culture. Following incubation at
35 °C for 48 hours, the diameter of inhibition zone around each antimicrobial ﬁlm disc
was measured to the nearest millimeter (Figure 2.2-2.3). An average of two

measurements was used as the result. All experiments were replicated three times.

22

Listeria monocytogenes (CWD 95, 246, 201 or 1503)

(frozen culture/ keep at - 70 °C)
Inoculate in 9 ml of trypticase soy broth + 0.6% yeast extract (TSB-YE)
35 °C 22 h
Inoculate in 9 m1 of TSB-YE

35°C lZZh

Listeria culture (contain ~ 109 CFU/ml)

Figure 2.1. Listeria monocytogenes culture preparation for using as Listeria stock

culture for disc diffusion—type assay.

23

Listeria culture Antimicrobial ﬁlm
(contain ~ 109 CPU/m1)

l

0.2 ml culture Cut intol6 mm-mm diameter disc
+
20 ml Trypticase Soy Agar + 0.6% Yeast Extract

(acidiﬁed to pH 5 .2)

 

V
Sterilize (20 minutes under UV light)

 

 

 

 

Put antimicrobial disc on TSA-YE (contain ~ 107 CFU/ml)

35 °C 48h

 

V
Measure inhibition zone around the ﬁlm disc

(Use the average of two measurements of the diameter of the zone, 90° apart)

Figure 2.2. Disc diffusion-type assay for measuring antimicrobial activity of inhibition

zone of the ﬁlms against Listeria monocytogenes.

24

 

Growth of Listeria Inhibition zone

Figure 2.3. Inhibition zone of the foodborne pathogen Listeria monocytogenes around

the disc of polyvinylidene copolymer ﬁlm containing sorbic acid after 48 hours at 35 °C

25

2.3.5. Mechanical properties
Tensile properties of the ﬁlms were determined according to ASTM D 882-91

using the IN STRON Universal Testing Machine Model 4201 equipped with an
INSTRON Chart Recorder (INSTRON Corporation, Canton, MA) with a load cell of 1
kN and crosshead speed of 50.8 cm/min. Films were cut into strips (2.54 cm width)
using a Precision Sample Cutter (Thawing Albert Instrument Co., Philadelphia, PA), and
conditioned according to ASTM D 618 at 23 i 2 °C/ 50% i 5% RH. All tests were run at
23 i 2°C/50% i 5% RH. Tensile strength, % elongation and toughness were determined.
2.3.6. Seal strength testing

Films containing 1.5%, 2% or 3% sorbic acid (without pH adjust to 5.2) were cut
into strips 2.54 centimeters wide and 25.4 centimeters long using a Precision Sample ‘
Cutter (Thawing Albert Instrument Co., Philadelphia, PA). Each strip was folded in half
(lengthwise) and sealed approximately 0.5 cm from the fold with a heat sealer (Sencorp
Systems Inc., Hyannis, MA). The sealing condition was set at 250 °F for 1 second with
the pressure at 35 psi (lb/inz). The seals were slit using scissors along the fold line of each
sample. The samples were conditioned according to ASTM D 618 at 23 i: 2°C/50% 2|:
5% RH not less than 40 hours before testing. Seal strength was determined according to
ASTM D 882-91 using an INSTRON Universal Testing Machine Model 2401 (Canton,
MA) with a load cell of 1 kN and crosshead speed of 50.8 cm/min. The test conditions
were at 23 i 2 °C/ 50% i- 5% RH.
2.3.7. Water vapor permeability

The MOCON PERMATRAN-W 3/31 system (MOCON/Modem Controls, Inc.,

Minneapolis, MN) was used to determine the water vapor transmission rate. The rate of

26

permeation was calculated at steady state (no further change in the permeation rate). To
test ﬁlms, the ﬁlm sample was prepared by putting the sample on an aluminum mask
(MOCON/Modem Controls, Inc., Minneapolis, MN) having a test area of 5 cm2. The
aluminum mask with the ﬁlm sample was placed into the cell chamber. The sample was
calibrated at the test condition for two hours before starting the test process. The test
condition was 37.8 °C (100 °F) and 90% relative humidity (RH) on one side and 0% on
the other side, which is in accordance with ASTM D 895, Standard Test Method for
Water Vapor Permeability of Packages. The test process was automatically started after
two hours of calibration and the test was ended after steady state was reached.
2.3.8. Oxygen permeability

An Oxtran 100 (MOCON/Modern Controls, Inc., Minneapolis, MN) was used to
measure oxygen gas permeability. The test was performed at 23°C and 0% relative
humidity (RH). The ﬁlm sample was put on the aluminum mask (MOCON/Modern
Controls, Inc., Minneapolis, MN) having a test area of 5 cm2. The ﬁlm was calibrated on
the machine for 2 — 3 hours before testing. The test ended after steady state was reached.
The result was obtained from the chart recorder (MOCON/Modem Controls, Inc.,
Minneapolis, MN).
2.3.9. Surface energy

ACCU DYNE TEST marker pens (Diversiﬁed Enterprises, Claremont, NH) were
used to measure the surface energy of ﬁlms. This test parallels ASTM D 2578-84. The

ﬁlms were conditioned at 23 i 2°C/50% i- 5% RH according to ASTM 618-81 for at least
40 hours before testing. The test was also performed at 23 :1: 2°C/50% i 5% RH. First,

the sample was placed on a clean and smooth area. Then the selected ACCU DYNE

27

TEST marker pen was pressed ﬁrmly down against the sample in three parallel passes
and only the result from the third pass was observed (the ﬁrst and second passes were
ignored). If the ink swath beaded up or tore apart or shrank into a thin line within one
second or less, it meant that the dyne level (interpreted as surface energy) used was
reading higher surface energy than the actual surface energy of the ﬁlm sample. Then,
the test would be repeated with a next lower dyne level marker (retesting was conducted
at a different location on the sample). If the ink held or stood for one to three seconds
before losing its integrity, that indicated that the dyne level of the marker closely matched
the surface energy of the ﬁlm sample.

2.3.10. Scanning electron microscopy (SEM)

The environmental scanning electron microscope (ESEM), model 2020
conﬁgured with a lanthium hexaboride (LaB6) ﬁlament, manufactured by ElectroScan
(FEI company, Hillsboro, Oregon) was used to observe the ﬁlms three-dimensional
structure. The acceleration voltage ranged between 10 and 20 kV, while the water vapor
pressure ranged between 2 and 3 Torr. The specimens were examined in their natural
state (no conductive coating).

2.3.11. Differential scanning calorimetry (DSC)

Differential Scanning Calorimeter (TA Instruments, New Castle, DE) was used to
determine the heat seal temperature range of SaranR F-310 without antimicrobial agents
(control ﬁlm) and ﬁlm containing 3% w/v sorbic acid. The heating rate was used 10 °C

/min and the weight of the samples was 3 mg. Nitrogen gas ﬂow rate was 50 cc/min.

28

2.4. RESULTS AND DISCUSSION
2.4.1. Antimicrobial properties

Antimicrobial-free ﬁlm (control ﬁlm; no antimicrobials) showed no antimicrobial
activity. Films containing nisin, and adjusted to pH 5.2 showed antimicrobial activity at a
minimum concentration of 1% w/v (Table 2.1). Films containing 0.5% to 2.5% (w/v)
lactoferrin either with or without a pH adjusted to 5.2 showed no antimicrobial activity.
Films containing potassium sorbate in solutions of 0.01 N lactic acid and in distilled
water showed antimicrobial activity. Films containing potassium sorbate solution in 0.01
N lactic acid showed antimicrobial activity at a concentration of 2% w/v, while ﬁlm
containing potassium sorbate solution in distilled water had antimicrobial activity at
minimum concentration of 5% w/v (Table 2.2). Therefore, a potassium sorbate solution
of 0.01 N‘ lactic acid was selected for all remaining experiments with potassium sorbate.
Film containing potassium sorbate could be made only with adjustment to pH 5.2,
otherwise the ﬁlm could not be cast and peeled off. Films containing sodium diacetate in
solution of 0.01 N lactic acid or in distilled water, with or without adjustment to pH 5 .2
showed no antimicrobial activity. Film containing lactoferrin either with or without
adjustment to pH 5.2 showed no antimicrobial activity. Film containing sorbic acid with
and without adjustment to pH 5.2 showed antimicrobial activity at a minimum
concentration of 1.5% (w/v) (Table 2.3). Sorbic acid appeared to be the most compatible
with the SaranR F-310 solution among the ﬁve antimicrobial agents considered, based on
the apparent solubility of the substances in the Saran F-310 solution. In addition, the
SaranR F-310 ﬁlms containing sorbic acid had the best physical appearance. PVDC ﬁlms

containing nisin, potassium sorbate or sorbic acid had the most antimicrobial activity.

29

Table 2.1. Antimicrobial activity of SaranR F-310 containing nisin against 4 strains of

L. monocytogenes

 

Diameter oflnhibition Zone (mm)

 

 

Film / Concentration CWD 95 CWD 1503 CWD201 CWD 246
(w/v)
Nisin
(adjusted to pH 5.2)
0.5% 0 0 g 0 0
1% 21.8 :1: 0.3a 22.3 i 0.63 18.67 i 0.6a 19.33 i 1.2a
2% 23.7 :1: 0.6a 23.0 i' 1.02‘ 19.83 i 0.33‘b 21.67 i 1.23‘b
25% 26.5 i 0.5b 26.7 i 1.5b 21.00 i 1.03 23.67 i 0.6b

 

Mean i standard deviation. Means in the same column with diﬁ‘erent superscripts were

signiﬁcantly different (p < 0.05).

30

Table 2.2. Antimicrobial activity of SaranR F-310 containing potassium sorbate against 4

strains of L. monocytogenes

 

Diameter of Inhibition Zone (mm)

 

 

 

Film CWD 95 CWD 1503 CWD201 CWD 246
Potassium sorbate
(in 0.01 N lactic &
adjusted to pH 5.2)
0.5% w/v 0 0 0 0
1% w/v 0 0 0 0
1.5% w/v 0 0 0 0
2% w/v 20.3 i- 0.63 20.7 i 06“ 18.3 i 0.6a 18.7 i 0.6a
3% “W 21.7 1: 06° 22.0 i 1.0a 19.0 i 1.02‘ 20.3 i- 06"
Potassium sorbate
(in distilled water
&
adjusted to pH 5.2)
0.5% w/v 0 0 0 0
1% w/v 0 0 0 0
2% w/v 0 0 0 0
3% w/v 0 0 0 0
5% w/v 24.3 i 1.2 24.3 i 0.6 21.3 i 0.6 23.0 i 1.0

 

Mean i standard deviation. Means in the same column with different superscripts were

signiﬁcantly different (p < 0.05) for the same type of ﬁlms.

31

Table 2.3. Antimicrobial activity of SaranR F-310 containing sorbic acid against 4 strains

of L. monocytogenes

 

Diameter ofInhibition Zone (mm)

 

 

 

Film / CWD 95 CWD 1503 CWD201 CWD 246
Concentration (w/v)
Sorbic acid
(unadjusted pH)
0.5% 0 0 0 0
1% 0 0 0 0
1.5% 24.8 i: 0.3“ 22.3 i 0.6“ 20.3 i 1.2“ 20.3 i 0.3“
2% 29.0 :1: 1.0b 29.2 i 1.0b 21.7 :1: 0.6“ 24.7 :t 0.6b
3% 32.8 i 08‘ 32.3 i 1.2c 25.7 i 1.2b 25.8 i 1.0b
Sorbic acid
(adjusted to pH 5.2)
0.5% 0 0 0 0
1% 0 0 0 0
1.5% 22.2 :1: 0.3“ 21.7 i 0.6“ 20.8 a 0.3“ 20.5 _+_ 0.5“
2% 26.7 i 1.5b 26.7 i 0.6b 21.7 :I: 1.2“ 23.0 i 1.0“
3% 32.2 i 1.0c 32.7 i 0.6“ 25.2 i 0.3b 26.7 i 1.5b

 

Mean i standard deviation. Means in the same column with different superscripts were

signiﬁcantly different (p < 0.05).

32

Increasing the concentration of nisin, potassium sorbate or sorbic acid in the ﬁlm
increased the diameter of the L. monocytogenes inhibition zones for for all 4 strains (p <
0.05) (Table 2.4). Films containing nisin had antimicrobial activity at a concentration of
1% w/v (Figure 2.4). Films containing 1%, 2% and 2.5% nisin had inhibition zone
diameters ranging from 18.7 to 26.7 mm. Increasing the concentration of nisin in the ﬁlm
disc increased the diameter of the inhibition zones (p<0.05). Ko et a1. (2001) reported that
incorporation of nisin in whey protein isolates, soy protein isolates, egg albtunin or wheat
gluten ﬁlms inhibited the growth of L. monocytogenes, and the greater the concentration
of nisin, the greater the level of inhibition in all ﬁlms tested. Hoffman et a1. (2001)
showed that com zein ﬁlm containing nisin and lauric acid decreased the number of
Listeria monocytogenes by more than 4 logs in 48 hours.

Films containing potassium sorbate had antimicrobial activity at a minimum
concentration of 2% (w/v), with the inhibition zones ranging from 18.33 to 22 mm for
2% and 3% (w/v) respectively (Figure 2.5). Increasing the concentration of potassium
sorbate did not result in increased level of inhibition for any of the four strains of L.
monocytogenes (p > 0.05). El-Shenawy and Marth (1988) showed that adding 0.2-0.3%
(w/v) potassium sorbate to trypticase soy broth (pH 5) inhibited the growth of L.
monocytogenes. Han (1996) developed-an antimicrobial ﬁlm by incorporating potassium
sorbate into low density polyethylene (LDPE) during the extrusion method and veriﬁed
the antimicrobial activity against Saccharomyces cerevisiae.

PVDC copolymer ﬁlms containing 1.5-3.0% (w/v) sorbic acid adjusted to pH 5.2,
or unadjusted (pH 3.5) were also inhibitory to all four L. monocytogenes strains (Figure

2.6), producing inhibition zones measuring 205-32.? and 20.3- 32.8 mm in diameter,

33

Table 2.4. Antimicrobial activities of polyvinylidene copolymer containing sorbic acid,

potassium sorbate and nisin against 4 strains of Listeria monocytogenes

 

 

 

 

Film Type Conc Diameter of Inhibition Zone (mm)
(% w/v)
Strains
CWD 95 CWD 1503 CWD201 CWD 246
Control
(antimicrobial free) 0 0a 0a 0a 0"
Sorbic acid 1.5% 22.2 i 0.3bf 21.7 i 0.6b 20.8 :t 0.3”: 20.5 d: 0.5"(1
(pH 5.2) 2% 26.7 :1: 1.5c 26.7 : 0.6c 21.7 i 1.2bf 123.0 i 1.0““
3% 32.2 i 1.0d 32.7 :t 0.6d 25.2 a 0.3“ 26.7 i 1.5““f
Sorbic acid 1.5% 24.8 i 0.3“f 22.3 i 0.6b 20.3 i 1.2bdc 20.3 :1: 0.3“8
(unadjusted pH) 2% 29.0 i 1.0“ 29.7 :t 1.0c 21.7 i 0.6bf 24.7 i 0.6““
3% 32.8 i 0.8“l 32.3 i 1.2“l 25.7 i 1.2c 25.8 :1; 1.0fh
Potassium sorbate 2% 20.3 i 0.6“ 20.7 i 0.6“ 18.3 1: co“ 18.7 i 0.6“
3% 21.7 a 06“" 22.0 i 1.0b 19.0 i 1.0““ 20.3 i 0.6“lg
Nisin 1% 21.8 i 0.3bf 22.3 i 0.6b 18.7 i 0.6“ 19.3 i 1.2“lg
2% 23.7 : 0.6f 23.0 i 1.0b 19.8 i 0.3cf 21.7 :1: 1.2bgh
25% 26.5 :t 0.5c 26.7 :1: 1.5c 21.0 i 1.0bfg 23.7 i: 0.6h

 

Mean i standard deviation. Means in the same column with different superscripts were
signiﬁcantly different (p < 0.05).

34

Inhibition zone of nisin

El Nisin 1%

\\\\\\\\\\\\\\\\\\\\

I len 2%
I Nisin 2.5%

 

 

 

 

 

 

 

 

 

8
2

d _ q _
6 4 2 o 8 6
2 2 2 2 1 1

AEEV OCON £033.59: hOLQuQEuRu

CW D201 CW0246

CWD1503

CW095

L monocytogenes strains

35

Figure 2.4. Comparison of the inhibition zones of ﬁlms containing 1%, 2% or 2.5%

(w/v) nisin.

Inhibition Zone of potassium sorbate
(solution in 0.01 N lactic acid)

 

 

 

 

 

 

 

 

 

 

 

 

24 _ 132%P. Sorbate
l3% P. Sorbate

0
8
N 22 —
C
.9
I§ .
5 E 20 .
f e
o V
E
0
E 18 -
.9
o

16 I T ,T

CWD95 CWD1503 CWD201 CWD246
L monocytogenes strains

Figure 2.5. Comparison of the inhibition zones of ﬁlms containing 2% or 3% (w/v)
potassium sorbate.

36

Inhibition Zone of Sorbic acid l-5°/°S°Ibi°
I 1.5% sorbic (pH 5.2)

 

 

 

 

 

 

 

 

 

El 2%sorbic
2%sorbic(pH 5.2
A 34 — .
E I 3%sorb1c
E 32 - .
v E1 3%sorb1c (pH 5.2
o
c 30
2.
S 28 .
E 26 ~
:2 \;
g 24 I \.
‘5 22 ~
5 20 ~
0
E 18, ~
.9.
a 16 _ 1:15;:
CWD95 CWD1503 CWD201 CWD246
L. monocytogenes strains

Figure 2.6. Comparison of the inhibition zones of ﬁlms containing 1.5%, 2% or 3%

(w/v) sorbic acid with and without adjusted to pH 5.2.

37

respectively. pH 5.2 was selected due to sorbic acid is most effective in the undissociated
form (pKa 4.75) because its increased ability to penetrate the cytoplasmic membrane of
bacteria. Film containing sorbic acid had the best antimicrobial activity against four
strains of L. monocyrogenes (Figure 2.7). McDade et a1. (1999) reported that growth of
L. monocytogenes was inhibited on frankfurters, which were coated with whey protein
ﬁlm-forming solution containing sorbic acid/propionic acid (pH 5.2) and stored at 4 °C.
Cagri at al. (2001) reported that whey protein isolate-based edible ﬁlms containing 0.5-
1.5% (w/v) sorbic acid had antimicrobial activity against Listeria monocytogenes on
trypticase soy agar pH 5.2, and also inhibited L. monocytogenes on luncheon meats
(Cagri et al., 2002a) and hot dogs (Cagri et al., 2002b).

According to Guilbert (1986), diffusion of antimicrobial agents from ﬁlm discs
depends on the shape, size, polarity of the molecule, chemical structure of the ﬁlm, and
degree of molecule cross-linking. The shape of the molecule, linear, branched or cyclic,
may impact the diffusion rate (Micheals et al., 1962).

2.4.2 Antimicrobial properties of a coating ﬁlm

The SaranR F-310 solution containing sorbic acid as an antimicrobial agent was
coated on PET ﬁlms (0.5 mil), and tested for its antimicrobial activity against Listeria
monocytogenes. Only the ﬁlms containing 3% (w/v) sorbic acid with a 0.75 mil coating
thickness had antimicrobial activity against L. monocytogenes CWD 95, with a hair line
around the ﬁlm disc. Films containing 3% (w/v) sorbic acid with coating thicknesses of
0.2 and 0.5 mil, and ﬁlms containing 1.5% and 2% (w/v) sorbic acid with coating

thicknesses ranging from 0.2 - 0.75 mil did not have antimicrobial activity.

38

Comparison of Inhibition Zone

36 —
Ml I CWD95
ICWDISOS
32: cw0201
30 A UCWD246

 

 

 

 

28i
261
24.

 

20*
18~
164

1

Diameter of inhibition zone (mm)

 

 

 

S '7
‘ a
i \

I

Nisin Nisin Nisin 2% 3% 1.5% 1.5% 2% 2% 3% 3%
1% 2% 2.5% Sorbate Sorbate sorbic sorbic sorbic sorbic sorbic sorbic
(PH) (11H) (pH)

Antimicrobial Agents

Figure 2.7. Comparison of the inhibition zones of ﬁlms containing sorbic acid with and
without pH adjusted to 5.2, potassium sorbate or nisin against four strains of Listeria

monocytogenes. Mean i standard deviation.

39

One of possibility could be the thickness of the coating. The thicker the coating of the
ﬁlm is, the greater the amount of antimicrobial agent would be in the ﬁlm. Guilbert
(1986) stated that measurement of antimicrobial activity using clear inhibition zones
surrounding ﬁlm discs (where the growth of the pathogen was inhibited) depended on the
diffusion of antimicrobials from the ﬁlm discs, which also depended on the size and
shape of the ﬁlms.
2.4.3 Mechanical properties

The average tensile strength of ﬁlms containing sorbic acid with and without pH
adjustment to 5.2 decreased from 3010 to 2843 lb/inz'and 4076 to 2389 1b/in2 when the
concentration of sorbic acid in the ﬁlm was increased from 1.5% to 3% (w/v),
respectively (Table 2.5). Increasing the sorbic acid concentration showed no signiﬁcant
difference in the tensile strength for ﬁlms with pH adjusted to 5.2, and no signiﬁcant
difference for ﬁlms without pH adjustment to 5.2 up to 2.0% (w/v)(p > 0.05). Tensile
strength values of ﬁlms containing 1.5 and 2.0% (w/v) sorbic acid without pH adjustment
to 5.2 were not signiﬁcantly different from the tensile values of the control ﬁlm (p >
0.05), while the other ﬁlms containing sorbic acid were signiﬁcantly different from the
control ﬁlm (p < 0.05). When the concentration of potassium sorbate increased ﬁ'om 2%
to 3% (w/v) and the concentration of nisin increased ﬁom 1% to 2.5% (w/v), tensile
strength values decreased ﬁ'om 1922 to 1258 lb/in2 and 1043 to 796 lb/inz, respectively.
Both were signiﬁcantly different from the control ﬁlm (p < 0.05). Increasing the
potassium sorbate concentration made a signiﬁcant difference in the tensile strength (p <

0.05), while increasing the concentration of nisin did not signiﬁcantly alter tensile

strength (p > 0.05).

40

Table 2.5. Tensile strength, percent elongation and toughness of SaranR F-310 control

ﬁlm and ﬁlms containing sorbic acid, nisin and potassium sorbate

 

 

 

 

 

 

Film/Concentration (% w/v) Tensile strength Elongation Toughness
(lb/in“) (%) (lb/in“)
Control 4936.4 i 809.8“ 266.7 1 20.8“ 11069 i: 1688“
Sorbic acid (pH 5.2)
1.5% 3010.3 i 458.5” 153.3 3: 11.5”“ 4413 i 794”
2% 2985.5 i 380.9” 146.7 i 70.2”“ 3624 i 949””
3% 2842.5 :1: 150.7” "2867 i 23.1“ 6613 i 185“
Sorbic acid (unadjusted pH)
1.5% 4075.9 i 138.4“ 206.7 x 61.6“”“ 661011: 1161“
2% 4010.5 :1: 347.1“ 66.7 i 5.8““ 2095 :I: 113““
3% 2389.0 i 2343”“ 31.7 i 2.9“ 1103 i 168“
Potassium sorbate
2% 1922.0 1- 84.7“ 26.7 i: 5.8“1 538 i 27f
3% 1258.2 i 110.5“ 146.7 E 40.4““ 1780 :1: 89“
Nisin
1% 1042.9 i 22.8““ 240.0 r. 17.3““ 2135 j: 83““
2% 952.1 1 16.0“ 395.0 a 22.0“ 3025 i 162”8
2.5% 796.2 i 18.1“ 426.7 i 6.03 2811 i 18“”

 

Mean i standard deviation. Means in the same column with different superscripts were
signiﬁcantly different (p < 0.05).

41

Films containing 1.5%, 2% or 3% (w/v) sorbic acid with pH adjusted to 5.2
exhibited average elongation at break values of 153%, 147% and 287% respectively, and
207%, 67% and 32%, respectively, for ﬁlms without pH adjustment (Table 2.5). Both
were signiﬁcantly different from the control ﬁlm (elongation value 267%) (p < 0.05)
except for ﬁlms containing 3% and 1.5%. (w/v) sorbic acid with pH adjusted to 5.2 and
without pH adjustment, respectively. Films containing 2% and 3% (w/v) potassium
sorbate had percent elongations of 27% and 147%, respectively, which was signiﬁcantly
different from the control ﬁlm (p < 0.05). Films containing nisin at concentrations of
1%, 2% or 2.5% (w/v) had average percent elongation values of 240%, 395% and
426.7% respectively. Only ﬁlms containing 2.0 or 2.5% (w/v) nisin were signiﬁcantly
different from the control ﬁlm (p < 0.05). Increasing the concentration of nisin from 2%
to 2.5% (w/v) did not signiﬁcantly change the percent elongation (p > 0.05).

The toughness of the ﬁlms was also evaluated (Table 2.5). The toughness of the
control ﬁlm was 11069 lb/inz. Films adjusted to pH 5.2 and containing 1.5%, 2% or 3%
(w/v) sorbic acid exhibited average toughness of 4413, 3624 and 6613 lb/in“,
respectively, and 6610, 2095 and 1103 lb/inz, respectively for unadjusted pH ﬁlm. Films
containing 2% or 3% (w/v) potassium sorbate had average toughneSs values of 538 and
1780 lb/in2 respectively. For nisin ﬁlms containing 1%, 2% or 2.5% (w/v) nisin, average
toughness was 2135, 3025 and 2811 lb/inz, respectively. The toughness of all ﬁlms
containing sorbic acid, potassium sorbate or nisin was signiﬁcantly different from their
antimicrobial-ﬂee counterparts (p < 0.05).

Han and Floros (1997) showed that antimicrobial low-density polyethylene

(LDPE) ﬁlms containing potassium sorbate up to 3% (w/w) did not affect the tensile

42

strength, elongation, and modulus properties of LDPE ﬁlms because the incorporated
potassium sorbate was considered to be captured in the void volume of the amorphous
polymer structure, and therefore should not affect the polymer structure. However, if the
amorphous area of LDPE was saturated with potassium sorbate, the tensile strength of the
antimicrobial LDPE could be affected adversely. In this study, the antimicrobial agents
nisin, potassium sorbate or sorbic acid were considered to be held in the void volume of
the polymer structure (See Fig 2.8-2.11 micrographs by scanning electron microscopy).
These may cause concentration stress points in the polymer structure, and result in
decreasing the tensile strength and toughness of the ﬁlms. Antimicrobials may function
as plasticizers to increase the ﬂexibility and movement of the polymer chains, yet
decrease the tensile strength and toughness of the ﬁlm. Chen et a1. (1996) reported that
incorporation of 4% potassium sorbate in methycellulose/chitosan antimicrobial ﬁlms did
not signiﬁcantly change ﬁlm tensile strength, but the ﬁlms had poor elongation compared
to ﬁlms containing no antimicrobial substance. Incorporation of antimicrobial agents in
whey protein isolate-based edible ﬁlms generally produced ﬁlms with lower tensile
strength and greater elongation (Kester and Fennema, 1986). Addition of antimicrobials
into the ﬁlms in order to produce antimicrobial ﬁlms generally produced ﬁlms with lower
tensile strength compared to ﬁlms without antimicrobials for corn zein ﬁlm (Aydt et al.,
1991), soy protein ﬁlm (Gennadios and Weller, 1991) and whey protein-based edible ﬁlm
(Cagri et al., 2001).
2.4.4 Seal strength

Polyvinylidene copolymer ﬁlms containing antimicrobials (sorbic acid) could be

sealed using the heat-sealing machine the same as the control ﬁlm (no antimicrobial).

43

Both ﬁlm with and without antimicrobials could be sealed at the same temperature (Table
2.6). Films containing 1.5%, 2% or 3% (w/v) sorbic acid exhibited average seal strength
values ranging from 1293 to 670 lb/inz, respectively. The antimicrobial-free ﬁlm had
average seal strength of 1757 lb/inz.

Incorporation of an antimicrobial agent, sorbic acid, into the ﬁlm caused a
reduction in the seal strength of the seal. The higher the amount of sorbic acid added into
the ﬁlm, the weaker the seal was.

2.4.5 Water vapor permeability:

Water vapor permeability (WVP) is an important property of plastic ﬁlms,
polyvinylidene chloride ﬁlm is a good moisture barrier. Films containing 1.5%, 2% or
3% (w/v) sorbic acid with pH adjusted to 5.2 exhibited average WVP values of 1.39, 1.45
and 2.00 cc.mil/m2.day.mmHg respectively (Table 2.7), and were signiﬁcantly different
(p < 0.05) from the control ﬁlm. A WVP value of 0.54 cc.mil/m2.day.mmHg was
determined for the control ﬁlm. Increasing the concentration of sorbic acid from 1.5% to
3% (w/v) did not signiﬁcantly alter water vapor permeability (p > 0.05). Average WVP
of ﬁlms containing 1.5%, 2.0% or 3.0% (w/v) sorbic acid (unadjusted pH) were 0.71,
0.50, 0.62 cc.mil/m2.day.mmHg respectively, and were not signiﬁcantly different ﬁom
the control ﬁlm (p > 0.05). Increasing the concentration of sorbic acid from 1.5% to 3%
(w/v) did not signiﬁcantly alter water vapor permeability G) > 0.05). Films containing 2%
or 3% (w/v) potassium sorbate had average WVP values of 3.69 and 3.83
cc.mil/m2.day.mmHg respectively, signiﬁcantly different from the control (p < 0.05).
Increasing the concentration of potassium sorbate from 2% to 3% (w/v) did not

signiﬁcantly change the WVP (p > 0.05).

44

Table 2.6. Seal strength of SaranR F-310 ﬁlm and ﬁlms containing 1.5%, 2.0% or 3.0%

(w/v) sorbic acid

 

 

 

Film/Concentration (% w/v) Seal strength
' (lb/in“)
Control 1756.6 i 446.4a
Sorbic acid
1.5% 1292.5 i 187.83'
2% 963.5 :1: 139.9”
3% 670.2 i l49.2°

 

45

Table 2.7. Water vapor transmission rate and water vapor permeability of SaranR F -3 10

control ﬁlm and SaranR F-310 ﬁlms containing sorbic acid, nisin, and potassium sorbate

 

 

 

 

 

 

Films/Concentration WVTR Permeability Permeability
(% w/v) (gm/100 in“/day> (snail/100 in“.day.mmHg) (g.mil/m2.day.mmHg)
Control 1.37 0.04 a 0.01“ 0.54 r. 0.01“
sorbic acid (pH 5.2)
1.5%
3.03 0.09 :0.01”““ 1.39 a 0.14”““
2% 3.52 0.09: 0.01”c 1.45 i: 0.19”“
3% 3.59 0.13 i 0.02” 2.00 i 0.25”
sorbic acid
(unadjusted pH)
1.5% 2.06 0.05 i 0.01“c 0.71 i 0.02““
2% 1.63 0.03 i 0.01““ 0.50 i 0.05““
3% 1.42 0.04 a 0.01“ 0.62 :I: 0.05“
Potassium sorbate
2% 3.03 0.24 a 0.03“ 3.69 i 0.42“
3% 3.07 0.25 a 0.04“ 3.83 i 0.62“
Nisin N/A N/A N/A

 

Mean i standard deviation. Means in the same column with different superscripts were

signiﬁcantly different (p < 0.05).

46

Adding potassium sorbate to the ﬁlm solution increased water vapor permeability (WVP)
because both are hydrophilic compounds, which may increase the solubility of water in
the ﬁlms. Films containing nisin could not be tested for their water vapor permeability.
Films failed during the calibration process during testing. This could be because the nisin
crystals inside the ﬁlm caused stress concentration and resulted in micro voids when the
ﬁlm was exposed to high pressure during sample conditioning in the permeability testing
(see Fig 2.11 scanning electron micrograph). McHugh et a1. (1994) stated that adding
sorbic acid to ﬁlm might increase the hydrophilic character and the water solubility
coefﬁcient of the ﬁlm. In this study, ﬁlms containingpotassium sorbate had the highest
water vapor permeability. This could be because potassium sorbate created pits in the
polymer structure, increasing the permeability of moisture (see Fig 2.10 scanning
electron micrograph). Antimicrobials may function as plasticizers to increase the
distance between polymer chains or weaken chain packing resulting in a looser polymer
structure, which increases water permeability of ﬁlm.

2.4.6 Oxygen permeability:

Films containing 1.5, 2.0 or 3.0% (w/v) sorbic acid with pH adjusted to 5.2
showed average oxygen permeability values of 33.5, 57.4 and 86.2 cc.mil/ m2.day.atm
respectively (Table 2.8). Film containing 1.5% (w/v) sorbic acid had lower oxygen
permeability (OP) than the control ﬁlm (53.3 cc.mil/ m2.day.atrn), and ﬁlm'containing
2.0% (w/v) sorbic acid was not signiﬁcantly different from the control ﬁlm (p > 0.05).
Average 02 penneabilities of non-pH adjusted ﬁlms containing 1.5, 2.0 or 3.0% (w/v)
sorbic acid were much higher: 171, 210, and 522 cc.mil/ m2.day.atm, and were

signiﬁcantly different from the control ﬁlm (p < 0.05). Increasing the concentration of

47

Table 2.8. Oxygen transmission rate (OTR) and oxygen permeability of SaranR F-310

control ﬁlm and SaranR F-310 ﬁlms containing sorbic acid, nisin, and potassium sorbate

 

 

 

 

 

F ilms/ Concentration (% w/v) OTR Oxygen Permeability
(cc/mz/day) (cc.mil/ m“.day.atm)
Control 61.3 i 16.2 53.3 i 2.6“
sorbic acid (pH 5.2)
1.5% 33.8 it 0.3 33.5 :t 2.1”
2% 44.2 i: 0.9 ,, 57.4 i 1.2“
3% 66.3 i 1.1 86.2 :I: 1.5“
sorbic acid (unadjusted pH)
1.5% 76.7 i 5.8 171.0 i 8.7“
2% , 103.3 a 5.8 210.0 :1: 10.0“
3% 270.0 i 10.0 521.7 :1: 9.6f
Potassium sorbate
2% 21001 45i0u
3% 2000 i 0.01 4600 :1: 0.01”
Nisin N/A N/A

 

 

Mean 1' standard deviation. Means in the same column with different superscripts were

signiﬁcantly different (p < 0.05).

48

sorbic acid from 1.5% to 3.0% (w/v) resulted in signiﬁcantly different oxygen
permeability (p < 0.05). PVDC ﬁlms containing 2.0% (w/v) potassium sorbate had an
average OP value of 4.53 cc.mil/ m2.day.atm, with ﬁlms prepared with 3.0% potassium
sorbate exhibiting an average that was about 1000-fold higher. Again ﬁlms containing
nisin could not be tested for 02 permeability due to the aforementioned problem. The
results showed that the ﬁlms containing antimicrobial agents had higher oxygen
permeability than control ﬁlms, except for ﬁlms containing 1.5% (w/v) sorbic acid
adjusted to pH 5.2 and ﬁlms containing 2% (w/v) potassium sorbate. Statistical analysis
showed that incorporation of different antimicrobial agents and increased concentration
resulted in statistically signiﬁcant differences in oxygen permeability (p < 0.05).
2.4.7 Surface energy

The ability of a ﬁlm to anchor inks, coating or adhesives is directly related to its
surface energy. If the substrate surface energy does not signiﬁcantly exceed the surface
tension of the ﬂuid that is to cover it, wetting will be impeded and a poor bond will result.
All ﬁlms containing sorbic acid, potassium sorbate or nisin (at all concentrations)
exhibited the same surface energy, 38 dynes/cm, which was the same as the control ﬁlm
(without the antimicrobial agents). Therefore, the surface energy of the ﬁlms was not
affected by addition of antimicrobial agents.
2.4.8 Scanning electron microscopy:

The three-dimensional structures of the ﬁlms were determined using scanning
electron microscopy. These are shown in Figures 2.8 - 2.11. Films without
antimicrobial agents (control ﬁlms) had a homogeneous structure (Figure 2.8). For ﬁlm

containing sorbic acid (Figure 2.9 A, B) the sorbic acid was distributed in the ﬁlm

49

_ 45”““1 —

 

Figure 2.8. SEM micrographs of the surface structure of polyvinylidene chloride

copolymer ﬁlms containing no antimicrobial (control ﬁlm).

50

451111111

W"W __._..._._.-.

_ WWI" _

 

Figure 2.9. SEM micrographs of the surface structure of polyvinylidene chloride

copolymer ﬁlms (Top) containing 1.5% (w/v) sorbic acid, (Bottom) 3% (w/v) sorbic acid

51

11“"

I)”
I 11111 — .-

 

Figure 2.10. SEM micrographs of the surface structure of polyvinylidene chloride

copolymer ﬁlms containing potassium sorbate.

52

 

Figure 2.11. Scanning electron microscopy photomicrograph of the structure of
polyvinylidene chloride copolymer ﬁlms containing nisin (Top) nisin spread in ﬁlm

(Bottom) interphase between ﬁlm matrix and nisin

53

structure, and created a non-homogeneous interphase (between the polyvinylidene
copolymer phase and the sorbic acid phase) in the polymer structure, which resulted in
decreased mechanical properties. Addition of potassium sorbate created sieves/holes
(widely distributed) in the ﬁlm structure (Figure 2.10 A, B). This porous morphology
caused reduction in tensile strength and toughness of the ﬁlm, and also produced higher
permeability of the ﬁlms. Incorporation of nisin into the polyvinylidene chloride
copolymer also created a non-homogeneous structure (Figure 2.11 A, B). The nisin
crystals, which are much larger than sorbic acid, resulted in decreased in tensile strength,
decreased toughness and failure of water vapor and oxygen permeability testing.

2.4.9. Differential scanning calorimeter (DSC)

DSC was used to determine the sealing temperature of ﬁhns with and without
sorbic acid. For ﬁlm without sorbic acid, the sealing temperature started from
approximately at 125 °C (Figure 2.12). The sealing temperature for ﬁlm containing
sorbic acid started approximately at 120 °C (Figure 2.13). Thus, adding sorbic acid to the

ﬁlm slightly shifted the heat seal conditions for the ﬁlm.

2.5. CONCLUSION

Polyvinylidene chloride copolymer ﬁlms containing 1.5 to 3.0% (w/v) sorbic
acid, 2.0 to 3.0% (w/v) potassium sorbate, or 1.0 to 2.5% (w/v) nisin inhibited the growth
of L. monocytogenes on TSAYE in a disc diffusion assay. Films containing lactoferrin or
sodium diacetate did not show inhibition against L. monocytogenes. Water vapor and
oxygen barrier properties decreased when antimicrobials other than sorbic acid were

added. Tensile strength of the antimicrobial ﬁlms decreased except for ﬁlm containing

54

Heat Flow (VV/g)

 

-0.15

-O.20 -

-0.25 -

0.30 -

0.35“

 

 

 

 

 

0.40

Temperature (‘C)

Figure 2.12. Differential Scanning Calorimetry (DSC) proﬁle of polyvinylidene

chloride copolymer (Saran F -3 10) ﬁlm containing no antimicrobial

55

Heat F low (VWg)

 

0.0

 

 

0.1-
.02:
0.3-
0.4“
0.5 _ r ,w I T a I I
60 1m 120 140 160 1%
TernpenanC)

 

Figure 2.13. Differential Scanning Calorimetry (DSC) proﬁle of polyvinylidene

chloride copolymer (Saran F-310) ﬁlm containing 3.0% sorbic acid

56

1.5% or 2.0% sorbic acid and toughness decreased with incorporation of antimicrobial
agents. Films containing sorbic acid exhibited the most favorable antimicrobial, barrier
and mechanical properties with sorbic acid relatively evenly distributed in the polymer
structure. Films containing sorbic acid either without pH adjustment (pH 3.5) or with pH
adjusted to 5.2 were most antimicrobial effective against L. monocytogenes. PVDC
(SaranR F-310) containing 3% (w/v) sorbic acid at a 0.75 mil (0.00075 inch) coating
thickness on polyethylene terephthalate ﬁlm (PET) also had antimicrobial activity against
L. monocytogenes.

These antimicrobial packaging ﬁlms may eventually prove to be useful in
inhibiting both pathogenic and spoilage organisms on the surface of cooked/ready-to-eat

or processed foods that may be exposed to contamination after processing.

57

CHAPTER 3.

INACTIVIATION OF LISTERIA MONOCYTOGENES ,ON BEEF BOLOGNA

AND CHEDDAR CHEESE USING ANTIMICROBIAL POLYVINYLIDENE

CHLORIDE FILM

58

3.1 ABSTRACT

This study investigated the ability of an antimicrobial polyvinylidene chloride
(PVDC) copolymer ﬁlm to inactivate Listeria monocytogenes and extend the shelf life of
beef bologna and Cheddar cheese. Sorbic acid-containing PVDC (SaranR F-310)
copolymer ﬁlms were made by a solvent-casting method using methyl ethyl ketone.
PVDC copolymer ﬁlm solutions containing 0, 1.5 and 3.0% (w/v) sorbic acid were cast
on glass plates and dried at 86°C for 5 minutes. These ﬁlms were aseptically cut and
placed between 3-mm thick slices of commercially produced beef bologna which were
previously surface inoculated with L monocytogenes at a level of 105 or 103 CFU/g.
Locally produced Cheddar cheese was cut into slices measuring 3 x 3 x 2.5 cm, surface
inoculated to contain 105 or 103 L. monocytogenes CFU/g, wrapped with 0, 1.5 or 3.0%
(w/v) sorbic acid ﬁlms and stored at 4°C. Both products were examined at appropriate
intervals for numbers of L. monocytogenes, mesophilic aerobic bacteria, lactic acid
bacteria, and yeast/mold using Modiﬁed Oxford Agar, Plate Count Agar, MRS
Lactobacillus Agar, and Rose Bengal Agar, reSpectively.

Films containing 1.5 and 3.0% (w/v) sorbic acid decreased L. monocytogenes
populations 4.4 and 4.5 logs, respectively on bologna slices initially inoculated to contain
105 CPU/g, and 6.5 and 7.2 logs on bologna initially inoculated to contain 103 CFU/g,
after 28 days of refrigerated storage; whereas numbers of Listeria increased 3.8 and 5.8
logs using antimicrobial-free ﬁlm on bologna initially inoculated with 105 and 103 CFU/ g
of Listeria, respectively. When Cheddar cheese was stored for 35 days, ﬁlms containing
1.5 and 3.0% (w/v) sorbic acid decreased Listeria population 0.8 and 1.3 logs on cheese

initially containing 105 CFU/g, and 0.6 and 1.2 logs on cheese initially containing 103

59

CF U/ g. Listeria p0pulations remained constant on Cheddar cheese wrapped in sorbic
acid-ﬁee ﬁlms. These ﬁlms, which also inhibited mesophilic bacteria, lactic acid bacteria
and yeast/mold, may be useful in enhancing the safety and shelf life of refrigerated ready-

to-eat foods.

3.2. INTRODUCTION

Post-processing contamination of ready-to-eat (RTE) foods by foodborne
pathogens and spoilage microorganisms is of great concern in the food industry. Ryser
and Marth (1987) reported that L. monocytogenes Can survive more than 1 year in
Cheddar cheese and persist during manufacture and storage in cottage cheese (Hicks and
Lund 1991, Ryser et al., 1985, Piccin and Shelef, 1995). Outbreaks have been reported in
both domestic and imported cheese (Ryser and Marth, 1988).

In 2000, there were 2,298 cases of listeriosis reported in US, which cost
approximately 8 1 million per case, making listeriosis the costliest foodborne disease per
case (U SDA-FISS, 2001). Use of antimicrobial ﬁlms may provide manufactures another
means of reducing bacterial pathogens on foods. Weng and Chen (1997) incorporated
antimicrobial agents within packaging materials to minimize growth of surface
contaminants. Antimicrobial additives such as sorbic acid are increasingly used as one
means to improve food preservation (Gianakopoulos and Guilbert, 1986).

Sorbic acid and its salt (such as potassium sorbate) are two primary food
preservatives (Sofos and Busta, 1981; Liewen and Marth, 1985; Luck, 1990; Weng and
Chen, 1997) in many food products including intermediate moisture foods (Troller and

Christian, 1978; Torres and Karel, 1985), poultry products (Robach and Ivey, 1978;

60

Cunningham, 1979), cheese and meat products (Zamora and Zaritzky, 1987a, b). Sorbic
anhydride was used as antimicrobial additive in polyethylene ﬁlm to produce an
antimicrobial food packaging ﬁlm (Weng and Chen, 1997, Weng and Hotchkiss, 1993).
Whey protein isolate ﬁlms containing sorbic acid or p-aminobenzoic acid reportedly
inhibited L. monocytogenes as well as Escherichia coli 0157:H7 and Salmonella
Typhimerium DT104 on aerobically packaged slices of bologna and summer sausage
(Cagri et al., 2002).

In a previous study, polyvinylidene chloride (PVDC) copolymer ﬁlms containing
1.5 to 3% (w/v) sorbic acid were found to inhibit the growth of L. monocytogenes on
acidiﬁed (pH 5.2) trypticase soy agar containing 0.6% yeast extract (Limjaroen et al.,
2002). The objective of this study was to assess the antimicrobial activity of sorbic acid-
containing PVDC ﬁlms for activity against L. monocytogenes and several groups of

spoilage organisms while in direct contact with bologna and Cheddar cheese.

3.3. MATERIALS AND METHODS
3.3.1. Target Organism

Listeria monocytogenes strain CWD 95 D Gallup, the most sorbic acid resistant of
four strains previously tested by Cagri et a1. (2001), was obtained from Michigan State
University Food Microbiology culture collection. The strain was maintained at — 70 °C in
trypticase soy broth containing 10% (v/v) glycerol (Difco Laboratories, Detroit, MI).
Cultures of L. monocytogenes were prepared by transferring the Listeria strain from —70
°C storage into TSB + 0.6% yeast extract (TSB-YE) (Difco Laboratories, Detroit, MI).

Following 18 - 24 hours of incubation at 35 °C, a second transfer was similarly prepared

61

prior to use. The initial culture had a population of 109 CFU/ml and was serially diluted
in 0.1% peptone water to inoculate beef bologna and Cheddar cheese.
3.3.2. Products

Beef bologna (diameter ~ 9.6 cm) and Cheddar cheese (dimension ~ 3 x 5 cm)
were obtained from a local supermarket and the MSU dairy store, respectively. Beef
bologna contained beef, salt, sodium lactate, ﬂavor, dextrose, hydrolyzed yeast, sodium
phosphate, sodium diacetate, sodium erythorbate, sodium nitrite, and extracts of paprika
as reported by the manufacture. Cheddar cheese contained pasteurized milk, cheese
culture, salt, enzymes and color. The pre-sliced bologna (~3 mm thick) (pH value of
6.12) was cut into 10-g squares shapes (7.5 X7.5 cm). Cheddar cheese (pH~5.0) was cut
into 25-g pieces measuring 3 x 3 x 2.5 cm.
3.3.3. Film preparation

Polyvinylidene chloride (PVDC) copolymer (Dow Chemical, Midland, MI) resin
(18%lw/v) was slowly dissolved in methyl ethyl ketone (J .T Baker, Phillipsburg, NJ) at
room temperature with continuous agitation. Thereafter, 0%, 1.5% or 3% (w/v) of sorbic
acid (Sigma Chemical Co., St. Louis, MO) was added to the solution (pH 3.5), which was
cast on glass plate (9 x 13 inches) and dried in an Oven (Fisher Scientiﬁc, Pittsburgh,
PA) at 86 i 0.5 °C for 5 minutes. After drying, the ﬁlms were peeled from the plates.
3.3.4. Product inoculation of Cheddar cheese and beef bologna and storage

Listeria monocytogenes (0.1 ml) was spread on the top and bottom surface of each
piece of Cheddar cheese, bologna using a sterile glass rod to obtain inoculum level of 10S
CFU/ g and 103 CFU/ g. The inoculated cheese and bologna slices were air dried. Cheddar

cheese samples were wrapped in ﬁlm (8.75 x 12.5 cm) containing 0% (without

62

antimicrobial agent), 1.5% or 3% (w/v) sorbic acid, which was previously sterilized by
exposure to UV light for 20 minutes. For bologna, the inoculated slices were placed
between PVDC ﬁlms (3.5 x 3.5 inch) containing 0, 1.5 or 3.0% (w/v) sorbic acid and
stacked 4 slices high in 150 mm-diameter sterile Petri dishes. The samples were stored
aerobically at 4 °C. All experiments were replicated three times.
3.3.5. Microbiological analysis
3.3.5.1. Cheddar cheese

Samples were taken for microbiological analysis immediately after inoculation
and again following 4, 7, 10, 14, 21 and 35 days of refrigerated storage. For analysis, a
25-g sample was diluted in 225 ml of warm 2% trisodium citrate (Sigma), homogenized
in a stomacher (Tekmar Co., Cincinnati, OH) for 3 minutes, and serially diluted in 0.1%
peptone water. The population of L. monocytogenes, mesophilic aerobic bacteria, lactic
acid bacteria, molds and yeast were determined by plating appropriate dilutions on
Modiﬁed Oxford Agar (Difco), Plate Count Agar (Difco), MRS Lactobacillus Agar
(Difco) and Rose Bengal Agar for both mold and yeast (Difco), respectively, as outlined
in the FDA Bacteriological Analytical Manual (FDA 1998). All experiments were
replicated three times.
3.3.5.2. Bologna

Samples were taken for microbiological analysis immediately after inoculation
and again following 4, 7, 10, 14, 21 and 28 days of refrigerated storage. A 10-g sample
was diluted in 90 ml of 0.1% peptone water, homogenized in a stomacher (Tekmar Co.,

Cincinnati, OH) and serially diluted in 0.1% peptone water. Appropriate dilutions were

63

plated to determine the numbers of L. monocytogenes, mesophilic aerobic bacteria, lactic
acid bacteria, molds and yeast. All experiments were replicated three times.
3.3.6. Statistical analysis

Two-way analysis of variance (ANOVA) using SAS Statistical Analysis System
(SAS Institute Inc., 1990) was performed to analyze the results at the 95% conﬁdence

level (p = 0.05) using the Tukey-Kramer adjustment.

3.4. RESULTS AND DISCUSSION

3.4.1. Antimicrobial activity on Cheddar cheese inoCulated to contain 105 and 103 L.
monocytogenes CFU/g.
3.4.1.1. L. monocytogenes

Cheddar cheese was initially inoculated to contain 105 L. monocytogenes CFU/g
and examined during 35 days of storage. The population distributions of L.
monocytogenes during 35 day were shown in Table 3.1 and 3.2. Populations of L.
monocytogenes decreased 0.78 and 1.31 logs (Table 3.3) on cheese wrapped with PVDC
ﬁlm containing 1.5% and 3.0% (w/v) sorbic acid, respectively (Figure 3.1) after 35 days
of stored at 4 °C. In contrast, cell numbers on cheese wrapped with antimicrobial-free
ﬁlm remained unchanged throughout 35 days storage (Table 3.3). The reduction in
number of L. monocytogenes using PVDC ﬁlms containing 1.5% sorbic acid was not
signiﬁcantly different (p > 0.05) from 3.0% (w/v) sorbic acid. Both ﬁlms containing
1.5% or 3.0% (w/v) sorbic acid were not signiﬁcantly different (p > 0.05) ﬁ'om the
control ﬁlm for Cheddar cheese initially inoculated to contain 105 L. monocytogenes

CFU/g on the surface of the cheese. For cheese inoculated to contain 103 CFU/g

64

 

 

 

3 n 3 v ed n 3 v ammo 4. 8 ate n 2. .mod n on 2
ed a 3 v od H 3 v aid n E an; n 2 :86 a an a

Qo n 3 v 30 .1. 3 v ammo n 2 cats 0 2 8.85 n ca 2

3 n 3 v 3 n 3 v 33o 4. 5 sand n 3 an; 0.. ca 2

ed a S v 2 n 3 v 8:3 n M: 3.26 n 3 386 n S 5

ed a 3 v as n S v .485 n E ammo n so .336 n we 4

ed .1. 3 v ed A 3 v as; n we sea a we age A E o 28 636... s2
3 n 3 v ed a 3 v aid n me 826 n 5 28d n S mm

30.3 v can? v seen 5 32.3 2 35.33 a

can? v 33.. v each 2 32.3 2 28.32 E

33;: v 31.3 v 32.3 5 82.33 33.33 2

ed 6.. S v ed a 3 v he a 2 .86 n we :86 n S e

3 n 3 v ed 0 3 v .25 a S .26 n as 2.26 n on 3.

ed .4 3 v ed a S v .23 n 3 same 0 we .85 u an o 28 63.8 :5

Ease
ammo.» 202 3.583 200 03004 «€203 022332 monomgncozoﬁ d ED 33828::

 

.23 658... 35 am as $2 .§ 353:8 use 8:388 005 mean $20 .2

no 839005 BEE an 525 880:0 320050 :0 «.5203 30m 0:02 98 «C063 2338.08 .BzomeOogE 4:0 003335 .~.m 030,—.

65

Amodvmv 0:000.“an 3:805:me 0003 30.53095 Eobbﬁ 5:5 55:00 088 2: E 0002 4028300 08988 H :82 .395 m2 M «ED

 

 

30H? v 933 v NEHS 59.33 HHSH: mm
ASHE v 30H? v €033.” ”MESH S ”8.3 2 a
od H 3 v 3 H 3 v H80 H 2 ammo H 2 03.0 H E E
3 H 3 v 0.0 H 3 v good H 3 :80 H 2 0.080 H «H 2
3 H 3 v 3 H 3 v Head H E :86 H 3 0.03.0 H mm H
o.o H 3 v o..o H 3 v £80 H 3 :25 H 3 .230 H 3 H
0.0 H 3 v 3 H S v an _ .c H 3 and H 3 280 H 5. o 28 038m :3
33.5
.000.» 202 30803 200 20004 £00003 3::0802 mmzmwgnuozoﬁ 4 Hana ﬁEEﬂEné

 

Chevy Axe. 035—.

6
6

.Amodvnc 000000.06 >350anme 0003 030008090 0:000bm0 5:3 55:00 0800 0.: E .802 0850300 200520 H :82 $5an me. u :03

 

 

 

 

0.0 H 00 v 0.0 H 00 v 0000 H on 000.0 H 0.0 ".000 H 00 mm

0.0 H 00 v 0.0 H 00 v 02.0 H 00 3800 H 00 32.0 H E a

0.0 H 0._ v 0.0 H 00 v 0.300 H 0.0 .000 H 00 a: _.0 H 00 0_

0.0 H 00 v 0.0 H 00 v 38.0 H 0.0 08.0 H 00 .030 H 00 0

0.0 H 00 v 0.0 H 0.. v -.00.0 H 0.0 0000 H 00 .0000 H 00 0 0000 0508 $00

0.0 H 00 v 0.0 H 00 v 300.0 H 00 3000 H 0.0 on.000 H 00 mm

0.0 H 00 v 0.0 H 0._ v n000.0 H E .000 H 00 2000 H 00 a

0.0 H 00 v 0.0 H 0._ v 02000 H 0.0 0000 H 00 .0000 H 00 00

0.0 H 00 v 0.0 H 0._ v 300.0 H E .000 H 00 .000 H 00 0

0.0 H 00 v 0.0 H 00 v .000 H 0.0 -“00.0 H 00 a; _.0 H 00 0 060 05.5.". $2

0.0 H 00 v :0 H ~00 300.0 H E “.000 H 00 2080 H 2“ mm

0.0 H 0._ v 0.0 H 00 v 00000 H 00 0000 H 2 .0000 H 00 a

0.0 H 00 v 0.0 H 00 v 22.0 H 0.0 00.00 H 00 a~00 H 00 00

0.0 H 0.0 v 0.0 H 00 v .0000 H E 0.80 H 00 a~00 H 00 0

0.0 H 00 v 0.0 H 00 v 0000 H m0 .000 H 00 a; 0.0 H 00 0 200 05.8 .00
umm0> U—Cz atom—0&3 ﬁmow omuomd QCOHOQQ Om=£n~0m02 ”NthngﬁUQtQE {N HAND ~mm£OuOmEUG<

 

.200 2080 x00 000 $2 .00 055908 800 083308 00>0 05% 0500 H00

00 82:00.: BEE :0 5:» 00000.00 330:0 co 3.00003 200 0:02 0:0 3000000 05.30008 00:0MSA0020E d Mo 0051:? .N.m 030,—.

67

. 00003000 00000000 000000000 3 000:2

.Awodvmv 000000.00 300000300 0003 00000000000 00000000 003 08200 00000 05 E 00002 0000300 00000000 H 0002

 

ommd H 2.0-

 

 

nomd H 3.0- 0000 05000 $0.0”
0m _ .o H 00.0- 00— .o H 90.0- 060 03.000 00m;
09.0 H mud 0w _ .o H ww.m 060 05000 $0 00w0_0m
omdemZ- «EdH 3.7 0000 03000 0\0o.m
03.0 H mod- amod H mud- 0000 05000 00m;
0m _ .o H 3.0. «00.0 H mod- 0000 05000 $0 000000
@300 02 @300 03 00gb 0.0—E 008? 000m

 

0000M0§00=05 .H

 

.0w0080 000000w£00 .00 000 mm 0000 000:0 003—00 000 .0000 mm 0000 000000 0000000 00 0000 03000

903 000.0“ 00 $00 000 000000000 0000 00050000 DEE 0. 000 0000000000005 :20 85.00 wos 0m0000 0000—0000 .m.m 030,—.

68

0000060000005 .0 33,00 we («0 830008 0008 00 .005 000000 0000000 00 0.000.080.0865 .0 .3” 000mg

908 0u003m
N0 0 m mm 0 m 3 0 o

_[ f p _ L _ o

a: 00.00 0.0.0 Ii!

000 00:8 .00 no: T 0
000 03.8 0.0.0:... - m

M

I m 3

3

.d

- 0 m

90

 

 

 

 

69

L. monocytogenes, Listeria was inhibited as much as 0.62 and 1.15 logs when the cheeses
were wrapped with PVDC ﬁlms containing 1.5% and 3.0% (w/v) sorbic acid,
respectively (Figure 3.2), while the antimicrobial-free ﬁlm wrapped cheese had a slightly
decreased level after 35 days of refrigerated storage. The log reduction of Listeria of
PVDC copolymer ﬁlms containing 1.5% (w/v) was signiﬁcantly different (p < 0.05) from
ﬁlm containing 3.0% (w/v) sorbic acid and signiﬁcantly different (p < 0.05) from the
control ﬁlm for Cheddar cheese initially inoculated to contain 103 L. monocytogenes
CFU/ g. The low pH of the cheese (pH ~ 5.09) may have cause consistent number of L.
monocytogenes in cheese for the ﬁlm without antimicrobial agent during 35 days.
Although sorbic acid is primarily used as a mold inhibitor in cheese products, it also
possesses antibacterial activity against Listeria spp (Ryser and Marth, 1988). Piccinin
and Shelef (1995) reported that potassium sorbate in cheese reduced the growth of L.
monocytogenes after 24 days of storage at 5 °C. Larson et a1. (1999) found that addition
of 1.0% potassium sorbate or 1.0% sodium benzoate decreased survival of L.
monocytogenes in cheese brines Number of L. monocytogenes decreased in cheese
containing 0.3% sorbic acid or 0.3% sodium propionate, which was acidiﬁed to pH 5.0 to
5.1 using lactic and/or acetic acid (Ryser and Marth, 1988). More recently, PVDC
copolymer ﬁlms containing 1.5% to 3.0% (w/v) sorbic acid inhibited L. monocytogenes
on laboratory media (Paweena et al., 2002).
3.4.1.2. Mesophilic Aerobic Bacteria (MAB)

The population of mesophilic aerobic bacteria (MAB), decreased 0.8 and 0.9 logs

after 35 days storage (Figure 3.3) using a PVDC copolymer ﬁlm containingl.5% and

70

0000M§000=0E d @300 02 00 820002 022 00 505 000020 0000000 00 0000800000005 .4 .Nd 0.5th

 

€000 0w003m
00 mm om mm om m _ S m o
0000 03.80 $90 Iii.
E00 03000 £0: II I -
0000 00.000 009m . . 9 . . I
r

 

 

 

o.o

We

o;

m2

ON

Wm

o.m

Wm

0.0

3mm) 301

71

60:0MSA0805 :N m5m0 mom mo 829005 3:5 :0 5?» 800:0 002025 :0 300003 05800 02200002 .m.m 0.53m

NV mm mm ﬂ

F F _ f

303 03.85
N

 

 

3

b

h

_

a: 03.8 $3 LT
E: 05.3 .00 u- ..

a: 0308 $3” . . o -.

 

I

o.o

o;

ON

72

3.0% (w/v) sorbic acid, respectively compared to the antimicrobial—free ﬁlm (Table 3.4).
For cheeses initially containing 103 L. monocytogenes CFU/ g, MAB decreased about 0.05
and 0.4 logs (Figure 3.4) using PVDC copolymer ﬁlms containing 1.5% and 3.0% (w/v)
sorbic acid, respectively as compared to cheeses wrapped with antimicrobial-free ﬁlm. In
contrast, the number of MAB on cheeses wrapped with ﬁlms containing no antimicrobial
agent increased in cheeses initially inoculated to contain 105 or 103 L. monocytogenes
CFU/g after 35 days of storage at 4 °C. The reduction in mesophilic aerobic bacteria due
to contact with PVDC copolymer ﬁlms containing 1.5% sorbic acid was not signiﬁcantly
different (p > 0.05) from 3.0% (w/v) sorbic acid ﬁlm, and both were signiﬁcantly
different (p < 0.05) from the control ﬁlm for cheese initially inoculated to contain 105 or
103 L. monocytogenes CFU/g. The polyvinylidene chloride copolymer showed the
potential to extend the shelf life of Cheddar cheese.
3.4.1.3. Lactic Acid Bacteria (LAB)

The populations of lactic acid bacteria on cheese inoculated to contain 105 or 103
L. monocytogenes CFU/ g were also examined during 35 days of refrigerated storage. The
number of lactic acid bacteria decreased slightly, 0.2 and 0.5 log using ﬁlms containing
1.5% and 3.0% (w/v) sorbic acid, respectively, on cheese initial inoculated to contain L.
monocytogenes 105 CFU/g compared to the control ﬁlm (Figure 3.5, Table 3.5). The
reduction in LAB for PVDC copolymer ﬁlms containing 1.5% (w/v) sorbic acid was not
signiﬁcantly different (p > 0.05) from 3.0% (w/v) sorbic acid, and was also not
signiﬁcantly different (p > 0.05) from the antimicrobial-free ﬁlm. A 0.2 and 2 log
reduction (Figure 3.6) in LAB was observed after 35 days for cheeses that were surface

inoculated with 103 L. monocytogenes CFU/g, and wrapped with ﬁlms containing 1.5

73

douﬂsaoq 00000000 00000500 3 0:52
Amodvav 000000.03 b0000£~§0 0003 0005000030 0:80.006 5:5 5:28 0800 05 E 0002 0030300 E00530 H 5002

 

 

 

ammd H 3.0- nmmd H 3.? 200 03000 food

nomd H $3.. an _ .c H mmé- 200 05.80 3m;

«mod H Kw «mod H 09m 200 03000 .xé Sac—om
0:3 med- nomd H mod- 200 05000 :65

03.0 H mod- A~mmd H mwd- 200 05000 RE;

amod H Sam ammd H mm.“ 200 03000 £5 00025
m5m0 02 - 3:05.02 0093. .EE 0003. coon

 

0t0000m 038010 05030002

 

.0380 00000030000 00 009% mm 00% 00020 Ewe—0n 000 .0030 mm 00.00 00005 00200.5 :0 200 03.80 A35 £60

00 $2 .Xo mew—5:00 085 00530000 09>; 9 0:0 £00000m 038000 05000002 .00 $505 was 0m00€ 0020300; in 030,—.

74

0020000300208 .4 meU 2 .00 830008 338 00 £3, 00005 002025 00 800000; 050000 02200002 in Rama
0

$03 09205
N0 mm mm a E n o _

_ b b P h r O I o

03.80 .x.m._ .LTI
03.80 03000 X... ll I

03.80 03000 3m . . O . . r m

 

 

 

75

.0020MSA00208 d 33.00 mg .00 820008 328 :0 505 00005 0000000 :0 3000000 200 0000..— .m.m 0.53m

Ne mm mm mm

P _ P h

903 09205
E

—

 

 

h

h

000 05.8 30.0 If
nuanuﬂ Qua—ha; $6 till
23 03.80 0.0.0:...

 

 

3mm 301

76

80303800 000000000 80000000 3 0082

Amodvﬁ 80000.30 b80038w0 0003 00800000000 8000.300 .303 08300 0800 05 8 0002 8030300 00000000 H 0002

 

 

 

02.0 H 00.0- .000 H 031 28 0508 0.0.0

0006 H 00 .0- 0006 H 00.0- 0000 05000 fem.—

00o.o H mum awed H 3.x 0000 05000 o\co 000w20m

on 0 .o H o0;- Kod H 006- 0000 05000 $66

ammd H omd- L 3.0 H 0N6- 0000 05000 0X00;

awed H m0.o 926 H 006- 0000 05000 0\oo 000050
0500 02 0500 02 025 SE 880. 080

 

030000m 0000. 0300-0

.0w00000 0000009300 .00 0x00 mm 0000 00030 0&205 000 .0030 mm 0000 000030 0000030 00 0000 05000 €05 $06

00 0x00; xxco 903000800 083-0 0080:0000 UQ>A 00 0:0 Am<d 030000m 000< 0300..— .«0 REED mos 09850 0030500; .m.m 050,—.

77

00200000000008 4 33050 no. .00 820008 3308 00 0303 000020 0000050 00 0300000 0000 0300A .0.m 0.8me

3000 0w0000m
m0 m m mm 3 00 0 o

.i 0 _ L _ 0

Human Own—.2; .X.m.~ LII.-
000 000.80 $0 - n -
000 00003. 0.0.0 . . o ..

 

 

 

Rama 301

78

and 3.0% (w/v) sorbic acid, respectively. In contrast, cheese wrapped with the
antimicrobial-tree ﬁlm had a 0.6 log increase (Table 3.5). Defects in cheese, such as
undesirable ﬂavors, gas formation, or white surface haze can result from growth of
nonstarter lactic acid bacteria (Somers et al., 2001). Swearingen et al. (2001) reported
that nonstarter lactic acid bacteria, Lactobacillus spp., Streptococcus thermophillus and
Lactococcus spp., found in 3 month ripened Cheddar cheese affected the ﬂavor and
quality. During Cheddar cheese ripening, nonstarter lactic acid bacteria levels normally
exceed 107 CFU/g, which in some instances can be signiﬁcantly higher than the starter
levels in cheese (Peterson and Marshall, 1990, Fox et al., 1993, Ryan et al., 1996), and
can play a signiﬁcant role in proteolysis and ﬂavor development during ripening
(McSweeney et al., 1994 and Ryan et al., 1996). .
3.4.1.4. Mold and Yeast

No mold or yeast grth on Cheddar cheese was found, for either L.
monocytogenes inoculum level. When cheese wrapped in ﬁlms containing either 1.5% or
3.0% (w/v) sorbic acid was refrigerated for 35 days. Mold growth appeared only on
cheeses initially surface inoculated to contain 103 L. monocytogenes CFU/ g, when
wrapped with antimicrobial-free ﬁlms at day 35 of storage. Sorbic acid is well known as
a yeast and mold inhibitor in cheese food (Marth and Ryser, 1988). Weng and Chen
(1997) also reported that polyethylene containing sorbic anhydride inhibited Penicillium

spp. and Aspergillus niger on laboratory media.

79

3.4.2. Antimicrobial activity on bologna inoculated to contain 105 and 103 L.
monocytogenes CF U/g.
3.4.2.1. L. monocytogenes

The populations of L. monocytogenes on bologna for both inoculum levels were
examined immediately after inoculation and again following 4, 7, 10, 14, 21 and 28 days
of storage (Table 3.6 and 3.7). The number of L. monocytogenes on bologna slices,
which was initially surface inoculated to contain 105 L. monocytogenes CFU/ g, decreased
4.4 and 4.5 logs using PVDC copolymer containing 1.5% and 3.0% (w/v) sorbic acid,
respectively, after 28 days at reﬁigerated storage; whereas Listeria increased 3.9 logs
using ﬁlm without sorbic acid (Figure 3.7). The log reduction of Listeria due to contact
with PVDC copolyrrier ﬁlms containing 1.5% sorbic acid was signiﬁcantly different (p <
0.05) from ﬁlm containing 3.0% (w/v) sorbic acid, and both were signiﬁcantly different
(p < 0.05) from the antimicrobial-free ﬁlm. For bologna slices with an initial inoculum
level of 103 CFU/g of L. monocytogenes, Listeria was inhibited up to 6.5 and 7.2 logs
(Table 3.7) using ﬁlms containing 1.5°/-i and 3.0% (w/v) sorbic acid, respectively
compared to the control ﬁlm (Figure 3.8). Listeria on bologna in contact with the
antimicrobial-free ﬁlms increased 5.8 logs after 28 days storage at 4 °C. The log
reduction in numbers of L. monocytogenes for ﬁlm containing 1.5% sorbic acid was not
signiﬁcantly different (p > 0.05) from 3.0% (w/v) sorbic acid, and both were signiﬁcantly
different (p < 0.05) from the ﬁlm containing 0% sorbic acid. In a previous study, PVDC
copolymer ﬁlms containing 1.5% to 3.0% (w/v) sorbic acid inhibited the growth of L.
monocytogenes on laboratory media (Limjaroen et al., 2002). Inhibition of L.

monocytogenes was found on sliced bologna using 0.26% potassium sorbate during 28

80

 

 

 

000.0 H 2. 0000 H 0.0. .20 0.0 H 0.0 00
02.0 H 0.0 000.0 H 5. 0000.0 H 0.0 0.0.

000.0 H 0.0 .0000 H S. .0000 H 0.... 2

00.0 H 0.0 0.000 H 0.0. 00 0.0 H 0.0 00

.000 H 0.0 ..00.0 H 0.0 0000.0 H 0.... 0

0.00.0 H 0.0 0.00.0 H 0.0 .000 H 0.0 0

.00 H 0.0 v .000 H .00 0.: .0 H 0.0 0 000 20000 0.0.0
.000 H 0.0 .000 H 0.0 .000 H 0.0 00

—00.0 H 0.0 .000 H 0.0 .000 H 0.0 00

0.00.0 H E .000 H 0.0 .000 H 0.0 E

.30 H 0.0 0.000 H 0.0 .000 H 0.0 00

.000 H 0.0 .000 H 0.0 .000 H 0.0 0

.00 0.0 H 0.0 00.0 H 0.0 ..000 H 0.0 0.

.00 H 0.0 v .000 H 0.0 0.0 0 .0 H 0.0 0 000 00008 0.0

330.0
0.000002 0000 03002 0300002 03500002 00000000000005 4 0002 20500008380

 

.002. 00000... 300 0.0 0:0 0.0.0 .00 0000008 0800 00800008 00?. 0:00 0500

03 283 00020008 0003 2053 00w205 00 2000002 0000 0300— 000 00000002 03500008 .0000M00A0000E :20 003558 .06 050,—.

81

Amodqu 0:80.008 30:02.2:me 0003 065000000 000000000 023 08200 0800 05 E 0002 0030300 0000030 H 0002 .3050 wax u :03

 

 

_ 000.0 H 0.0 00.0 H 0.0 u.000 H 0.0 00
.20. H 000 0026 H Ev 00026 H :0 3.
086 H wé 00m ﬁo H mé 00.3.0 H wé E
08.0 H mé ﬁﬁmd H ed 0036 H ad 9
036 H 5v 0086 H m6 0036 H 0.0. 0
000.0 H 0.0 0000 H. 00 .0000 H 0.0 0
be H o._ v 002.0 H ﬁm 32.0 H ﬁn o 200 05.80 0\0o.m
$3.00
3000002 200 05004 £00.00: 02300002 «mammgnuezoﬁ .H END 103000083900

 

0.0000 .00 0.00.0

82

 

 

 

02.0 H 0.0 00.0 H 0.0 000 H 0.0 00

0: .0 H 0.0 00.0 H 0.0 .0: .0 H 0.0 00

00_.0H0.0 0_.0H0.0 .0000H00 E

.000 H 0.0 00.0 H 0.0 00.0 H 0.0 0_

00.0 H 0._ v 00.0 H 0.0 0000 H 0.0 0

00.0H0._ v 0_.0H0.0 0_.0H0.0 0.

00.0 H 0.. v 00.0 H 0.0 L .0 H 0.0 0 000 20.00 0000
“.000 H 0.0 :0 H 0.0 30 H 0.0 00

000.0 H 0.0 1.0 H 0.0 :0 H 0.0 00

o:.0H0.0 "._.0H _.0 00.0H0.0 E

0000 H 0.0 a .0 H 0.0 00.0 H 0.0 00

.000 H 0.0 00.0 H 0.0 00.0 H 0.0 0

«0.0 H 0._ v 0.00 H 0.0 a .0 H 0.0 0

00.0 H 0._ v 0000 H 0.0 0.00 H 0.0 0 000 20.50 000

$3.00
0303005 200 03004 0303005 03500002 00=0MS0000=05 .H >0Q 30500030350

 

.200 05.000 903 0\0m 300 gm; .0\0o $053308 0033 0050—0000 DEA $300 3305

03 3:3 30.03.0003 0003 5053 000M055 :0 0300005 300 0300— 300 0300005 03500005 .0000m0000000005 Q .30 003555 ﬁn 050,—.

83

.Amodvmv 00000.35 003000330 0003 00030000000 0000.330 533 000500 0800 05 E 0002 00303003 30030030 H 0002 QED m0. n 30D

 

 

0000 H 0.0 00.0 H 0.0 00.0 H E 00
00.0 H 0.0 _00 H 00 0.00 H 00 _0
0: .0 H 0.0 00.0 H 0.0 00.0 H 0.0 3
020 H 0.. 00.0 H 0.0 00.0 H 0.0 0_
00.0 H 0._ v 00.0 H 0.0 0000 H 0.0 0
00.0H0._ v 00.0H0.0 0._.0H0.0 0
“0.0 H 0.0 v 00.0 H 0.0 0; .0 H _.0 0 000 20.50. 00.0
$3.00
0300005 500 03004 0300005 03500002 00000600000005 .0 0009 350003030000

 

3.008 sum 050,—.

84

.0005 053 3000.35 w0m00 00000000000205 d 3300 we .30 0050005 50333 00 0335 003—05 00 820M00000=0E .H gum 0.5”?—

00 00 00 A003 00230 E 0 0
r . . 0 . _ 0
000 03.50 00.0 LT
000 03.00 .00 an ..
000 05.80. 000.0 . . o : I 0

 

 

 

 

3mm 301

 

 

85

.0080 053 30000330 m0m00 00000000000005 4 @300 me .00 0003005 30333 00 0335 00m0_05 00 00:0MSA00005 d .w.m 000mm,.—

00 00 _0 0003 00.030 3 0

 

 

x! x 000 00.30 000.0 IT.
\ x x x 000 00.8 .00 In ..
000 03000 000.0 . . o ..

7|

 

I

3

3mm 301

86

days of refrigerated storage (Wederquist et a1, 1994). Cagri et al. (2002) reported that
low pH whey protein isolate ﬁlms containing 0.5% to 1.0% (w/v) p-aminobenzoic acid
and/or Sorbic acid inhibited growth of L. monocytogenes about 3.4-4.1 logs on bologna
and summer sausage slices during 21 days of reﬁ'igerated storage.

3.4.2.2. Mesophilic Aerobic Bacteria (MAB)

Numbers of mesophilic aerobic bacteria (MAB) after 28 days of storage on
bologna slices, which were initially surface inoculated with 105 L. monocytogenes CFU/ g
decreased 4.3 and 4.3 logs (Figure 3.9) due to contact with ﬁlms containing 1.5% and
3.0% (w/v) sorbic acid, respectively, compared to a 3.6 log increase for the antimicrobial-
free ﬁlm. Population of MAB decreased by 6.4 and 6.9 (Figure 3.10) logs on bologna
slices initially contaminated with 103 L. monocytogenes CFU/g due to contact with
PVDC copolymer ﬁlms containing 1.5% and 3.0% (w/v) sorbic acid, respectively.
Numbers of MAB on bologna increased 5.7 logs after 28 days of storage using sorbic
acid-free ﬁlm (Figure 3.10). Log reductions in MAB on bologna inoculated with 105 or
103 L. monocytogenes CFU/g due to ﬁlms containing 1.5% or 3.0% (w/v) sorbic acid
were signiﬁcantly different from the antimicrobial-free ﬁlm (p < 0.05). Cagri et al.
(2002) reported that whey protein isolate ﬁlm containing sorbic acid reduced MAB
populations about 5.0 logs on bologna slices aﬁer 21 days of storage at 4 °C.
3.4.2.3. Lactic Acid Bacteria (LAB)

Populations of lactic acid bacteria were also examined during 28 days of
refrigerated storage (Figure 3.11 and 3.12). Lactic acid bacteria decreased 4.4 and 4.5
logs on bologna slices initially surface inoculated with 105 L. monocytogenes CFU/ g

using ﬁlms containing 1.5% and 3.0% (w/v), respectively, compared to the control ﬁlm,

87

.0005

053 30000330 w0m00 0000M00000=0£ .4 3300 we .30 0050005 50333 00 533 00w0_05 00 0300005 050000 03500002 5.0.“ 0.0—ME

00 . 00 00 00000 00230 E 0 0
F _ _ r . 0
000 03.8 000.0 IT.
000 03.00 .00 in ..
000 03000 000.0 . . o .. I

 

3/1103 301

 

 

OOOOFWVKVMN

88

.0005

SE 00000.35 w0m0= 0000M0§00=05 .H 3300 03 .00 00500003 3333 00 0335 00m055 00 0303005 050000 03500002 .34” 0.0-ME

00 00 00 00000 000030 E 0 0

 

 

 

 

iiinx rv
xxx-1K0. 0m
-0 m
xxx .0 0W
\\
\x .0
xix
xxxx 000 0008.00.01.01 - 0
Ix\\\ 33.0 05.000 £0: 1!!
,0

000 30.8 000.0 . . o ..

 

89

.0005 033 0000.35 w0_00 00000000000005 d $300 03 .00 0050005 50333 00 .335 00w205 00 0300005 500 03004 .:.m 0.5mm...—

00 S E 303 0w003m 0 o
_ o

 

 

 

3mm) 30']

 

\ \ l \ x 500 05.000 0\0m.~ Iii
It i 500 05.000 0\0o II I I
500 05.000 $3.0” . - 9 . .

 

OONOOFOWVMN

9O

.893

SE EBabE m5? «mammSAucnoE .4 3P5 «.2 mo 82305 RES 5 ”EB «$23 :o 2.583 Boa 283 .26 char.—

 

 

 

 

om mm om 2355 $3on 3 m o
F l o
I _
w m
T m
r V cm.
a m D
g M
\\\\ Boa 93.8w ..\om.—‘|LTI I n
\\\\\\I\\\ EuaoEucmﬁ... Ill 1w
ﬁxxxxx 2933555.... To
I 2

91

where the number of LAB increased 8.9 logs. The log reduction in LAB using PVDC
00polymer ﬁlms containing 1.5% sorbic acid was signiﬁcantly different (p < 0.05) from
ﬁlm containing 3.0% (w/v) sorbic acid and both were signiﬁcantly different (p < 0.05)
ﬁ'om the antimicrobial-free ﬁlm. For bologna, which initially contained 103 L.
monocytogenes CPU/g, the population of LAB decreased 6.2 and 6.5 logs due to PVDC
copolymer ﬁlms containing 1.5% and 3.0% (w/v) sorbic acid, respectively. LAB
populations increased 8.7 logs on bologna slices after 28 days when wrapped with
antimicrobial-free ﬁlm. The log reduction in LAB due to contact with PVDC copolymer
ﬁlm containing 1.5% sorbic acid was not signiﬁcantly different (p > 0.05) from 3.0%
(w/v) sorbic acid but both were signiﬁcantly different (p < 0.05) from the control ﬁlm.
Holly (1997) reported that for bologna slices stored at 7 °C lactic acid bacteria (LAB),
dominated were most plentiﬁll with Lactobacillus sake dominating.
3.4.2.4. Mold and Yeast

No mold or yeast growth (< 1.0 i 0.0 log CPU/g) occurred on bologna slices, for
either inoculum level of L. monocytogenes CFU/ g, regardless of the level of sorbic acid
aﬁer 28 days of refrigerated storage. Matamoros et al. (1999) reported that sorbic acid
salt and potassium sorbate inhibited the growth of Penicillium digitatum, Penicillium
glabrum and Penicillium italium in potato dextrose agar. Cagri et al. (2002) reported that
whey protein isolate ﬁlms containing sorbic acid inhibited mold growth on bologna

during 21 days storage at 4 °C.

92

3.5. CONCLUSION

PVDC copolymer ﬁlms containing 1.5 or 3.0% sorbic acid reduced L.
monocytogenes population by 0.1 — 1 on Cheddar cheese and 4 - 7 logs on bologna slices
after 35 and 28 days of refrigerated storage, respectively. Minimal reductions in the
numbers of mesophilic and lactic acid bacteria were observed for Cheddar cheese,
whereas these same groups of potential spoilage organisms decreased 4—6 logs on

bologna slices wrapped in PVDC ﬁlm containing 1.5 — 3.0 % sorbic acid.

93

CHAPTER 4.

MIGRATION OF SORBIC ACID FROM POLYVINYLIDENE CHLORIDE

ANTIMICROBIAL FILM TO CHEDDAR CHEESE AND BOLOGNA

94

4.1. ABSTRACT

The migration of sorbic acid from the polyvinylidene antimicrobial ﬁlms to
Cheddar cheese and beef bologna was determined using High Performance Liquid
Chromatography (HPLC). The sorbic acid migration to Cheddar cheese and bologna was
monitored over a 28 day storage period, and the sorbic acid concentration remaining in
the ﬁlm was determined. The migration rate constant was also determined using linear
regression with experimental data based on a ﬁrst order reaction. The migration of sorbic
acid at the end of 28 days of storage to Cheddar cheese was 6.5% WM, and to bologna
was 15% wt/wt. The rate constant for Cheddar cheese was 0.007 per day, and for
bologna was 0.040 per day. This indicates that sorbic acid could migrate from ﬁlms to

Cheddar cheese and bologna to inhibit microorganisms.

4.2. INTRODUCTION

Sorbic acid is widely used as a preservative in food products such as cheese and
meat products. Sorbic acid inhibits the grth of mold, yeast and some bacteria. Sorbic
acid is commonly used as an antimicrobial agent in antimicrobial ﬁlms. A polyvinylidene
chloride (PVDC) copolymer ﬁlm containing sorbic acid was shown to inhibit the growth
of Listeria monocytogenes on laboratory media (Limj aroen et al., 2002a), and on Cheddar
cheese and beef bologna (Limjaroen et al., 2002b). The antimicrobial ﬁlm was positioned
in direct contact with the food. The antimicrobial agent migrates to the surface of the
packaging material and then to the food to inhibit microbial growth. The release rates
and migration amounts of the antimicrobial agents from the packaging material to food is
very important (Han, 2000). Difﬁlsion between the packaging material and the food, and

partitioning at the interface are the main migration phenomena involved. In other cases,

95

the antimicrobial is effective against microorganisms on the food surface without
migration of active agents to the food.

In this chapter, the migration/release of sorbic acid from the PVDC antimicrobial
ﬁlm to food over storage time at 4°C was determined. The effect of food on
migration/release of sorbic acid was also determined. Two types of food were used in this
study, Cheddar cheese and beef bologna. The migration or loss of sorbic acid from the

PVDC ﬁlm was veriﬁed as well.

4.3. MATERIALS AND METHODS
4.3.1. Products

Beef bologna (diameter ~ 9.6 cm) and Cheddar cheese (dimensions ~ 3 x 5 cm)
commercially produced were obtained ﬁ'om a local supermarket and the MSU dairy store,
respectively. The pre—sliced bologna (~3 mm thick), which had a pH value of 6.1 when
purchased, was cut into 10-g squares (3 X 3 inches). Cheddar cheese (pH~5.0) was cut
into 25-g pieces measuring 3 x 3 x 2.5 cm.
4.3.2. Film preparation

Polyvinylidene chloride (PVDC) copolymer (Saran F-310, Dow Chemical,
Midland, MI) resin was slowly dissolved in methyl ethyl ketone (J .T Baker, Phillipsburg,
NJ) (18% w/v) at room temperature with continuous agitation until completely dissolved.
3% (w/v) sorbic acid (Sigma Chemical Co., St. Louis, MO) was then incorporated into

the solution. The ﬁlm solution (pH 3.5) was cast onto a glass plate (9 X 13 inches) and

dried in a Oven (Fisher Scientiﬁc, Pittsburgh, PA) at 86 i 0.5 °C for 5 minutes. After

96

drying, the ﬁlms were peeled from the plates. The ﬁlms were then positioned in direct
contact with the food or without food for the control.
4.3.3. Sample preparation

For cheese, 25-g pieces were wrapped with ﬁlm (3.5 x 5 inch) containing 3%
(w/v) sorbic acid. For bologna, 10-g slices were placed between PVDC ﬁlms (3.5 x 3.5
inch) containing 3% (w/v) sorbic acid and stacked 3 slices high in 150 min-diameter
sterile Petri dishes. The samples were stored at 4°C for 28 days. The samples were
(evaluated after 0, 1, 2, 3, 4, 5, 6, 7, 14, 21 and 28 days of refrigerated storage. PVDC
ﬁlms containing 3.0% (w/v) sorbic acid samples (control ﬁlm, no food contact) were also
analyzed for sorbic acid content over this storage period. The samples were stored at 4°C.
All experiments were replicated three times.
4.3.4. Migration test
4.3.4.1. Standard calibration curve

Standard solutions of sorbic acid were ﬁrst prepared by dissolving 0.1 gram in
100 ml HPLC grade methanol in a volumetric ﬂask. The solution was then serially
diluted to 1, 2.5, 5, 10, 25, 50 and 80 parts per million (ppm) to prepare a series of
standard solutions of known sorbic acid concentration for a standard calibration curve.
The standard curve is shown in Figure 4.1.
4.3.4.2. Extraction procedure and HPLC evaluation

The ﬁlm samples, unwrapped from Cheddar cheese and un-stacked from bologna,
and the control ﬁlm (no food contact) were cut into small pieces. 0.5 grams of the
samples were immersed in 50 m1 HPLC grade methanol and vortexed thoroughly, and

stored at room temperature for 7 days. During the 7 days storage, the sample solutions

97

were thoroughly vortexed once a day. For analysis, the sample solution was ﬁltered
through a 0.45 pm microﬁlter (Fisher Scientiﬁc, Pittsburgh, PA), and then serially diluted
to 1:100 using HPLC grade methanol. Sorbic acid was analyzed using a Waters’ HPLC
system (Waters Corporate, Milford, MA) with UV detector, and C 18 column with inside
dimension of 150 X 5 mm (Waters). The mobile-phase solution was methanol-water
(1:1), injection volume 10 u], ﬂow rate 1.0 ml/min. The sorbic acid concentration was
determined using a UV detector at 254 nm. The retention time was 1.4 minutes (see
Figure 4.2). The concentration of sorbic acid was determined by the following equation:
% sorbic acid (wt/wt) = RsX C.F. X Vm
Vinj X thoiymer
where R9, = detector response value for the sample (area unit)
C. F. = calibration factor from slope of standard calibration curve (g/Au)
le = total volume containing analyte (ml)
Vinj = injection volume of unknown sample solution
tholyme, = weight of the polymer sample used for analysis (g)
4.3.4.3. Calculation of migration/release rate
The migration/release rate of sorbic acid can be calculated from the kinetic curve,
which followed a ﬁrst rate order relationship. A ﬁrst order reaction is one in which the
rate of the reaction is proportional to the concentration of only one of the reacting

substances (Benson, 1960 and Han, 1984).

98

200-

180 ~

160 -

140 -

120 .

Area
8

40*

20*

 

 

o I I r I I I I I I
0 1 0 20 30 40 50 60 70 80 90

concentration (ppm)

Figure 4.1. Standard curve of sorbic acid concentration in a methanol solution.

99

Absorbancc
1.35

0.05 . _

 

 

 

 

.71 . J . f

0.00 2.00 4.00 5.99

Time (min)

 

 

Figure 4.2. Chromatogram of sorbic acid from the polyvinylidene chloride antimicrobial

ﬁlm

100

g = -kC (1)
dt

Integrated to; In C/Co = -kt

Where: Co, C = Initial and ﬁnal concentration of sorbic acid in the ﬁlm sample,
% (w/w), respectively
k = rate constant, l/day

t = time interval, day

4.4 RESULTS AND DISCUSSION

The migration of antimicrobial agent from ﬁlm to the food is an important key to
inhibit the growth of pathogens, and spoilage organisms. In this study, the
migration/release of sorbic acid from polyvinylidene chloride copolymer ﬁlms to food
products, Cheddar cheese and beef bologna, was determined. Antimicrobial activity on
food products was evaluated in the previous chapter. Film with no food contact (control
ﬁlm) was also evaluated for loss of antimicrobial agent during 28 days of storage. The
results are shown in Tables 4.1, 4.2 and 4.3, respectively. For better illustration, the
results are presented graphically in Figures 4.3 and 4.4 for ﬁlms with cheese, Figures 4.5
and 4.6 for ﬁlms with bologna, and Figures 4.7 and 4.8 for control ﬁlms.

After 7 days storage at 4 °C, the remaining sorbic acid content in ﬁlm wrapped
around cheese and bologna, and the control ﬁlm relative to the initial control ﬁlm were
64%, 50% and 92% sorbic acid, respectively. About 36% of sorbic acid migrated/loss
from ﬁlm to cheese and 50% sorbic acid from ﬁlm to the bologna after 7 days of storage.

The control ﬁlm showed 8% loss (or volatilization) of sorbic acid. After 28 days of

101

.EE 5 E685 Eon 03.5w mo countenance 325 mo “:8qu 03.32 35858 33> *

 

 

8 So mm
8 3o 2
8 2.2 _ a
on 92 o 8.2
a 8.2 m
on 2.2 N
a 8.2 _
.25 Eng 3.. EE
82 x SQ ex. 353
Eon 388 me 0353* O 4 0:5 Baa cocwbcoocoo Eon 658w . H .3283 08:. o0 .couabcooaoo Eon 038m 325

 

.Oo
w 8 owmcoum macaw 385 .8325 95on BESS SE 3583835 octoEo ecoEEEKEQ Sow Eon 05.8 .«o 33 .mé 93:.

102

.EE 5 Samoa Eon 038m mo coup—250:8 BEE mo E859 3338 8:829: 025/ *

 

 

a :._ mm
: m: a
2 42 2
cm 8.x 5
9. as m 8.2 -
S no a.
on as m
a o; N
Q 3.: _
.25 3.. SE: 933 § $3

82 x SQ

Eon 052% we 03.20% D J 68: Loan Samba—cocoa Eon 658m . .3225 6:5. 00 .coumbcoocoo Eon 038m REE

Uo v a
.0303 maize «amazon «no: 502509 Bang/can» SE 3582835 02.820 05213338 80¢ Eon 038m mo 33 .n6 033—.

103

.65 E .585 Eon 0328 .«o 83.52828 325 .«o :8ch 033—8 mucomoae 33> *

 

 

mm 8.2 mm
3 8.2 a
.8 :2 E
8 8.2 5
mm 8.2 o
8 8.3 m 8.2
a 8.: a.
8 8.2 m
8 8.: _
82 8.2 8
85 3. $83 983 Q. EEC

82 x oobv
Box 052% Co 3333* O J 08: “can countenance Eon 038m B .3>.§E 08:. co .coumbcoocoa Eon 058m 33::

.0o v 3 omﬁoum maize 968:8 meow o5 SE 3382833 02.830 25213333 88% Bow 3&8 we 9.3 .m.v 2:5.

104

cm

0% 3 88:0 335:0 9:52

3&8? SE 3582835 Ua>m 89¢ 08: omﬁoum mo coco—.3 a ma Eon 058m mo nomuabcooaoo E omega 2P .m.v charm

 

 

 

 

 

A3233:
mN ON mP O—. m D
_ _ _ _ _ _ Ix‘lrl‘r c

I N S

m

I Q m.

u

I m .0...

O

O

1 Q We

a

T 1T m.
m

.o..

U

low

 

 

105

0% a 885 3306

9398 coughs SE BEBEEEa 0Q>m 80¢ 08: owEBm mo 5:05 a 3 How 398 .«o 223.3588 0358 oi .vé 95w:—

 

 

$338.:
on mu cm mp or m o

_ _ _ P _ , _ O
12 a
W
.2 m.
m
r on S
V
- cu m
U
Ion m
T 18 m.
0 O
U
1 Oh Na
0 o 1 cm Mg
m

1 cm

 

c 2:.

106

0% 3 sumo—on 5053

Baogcamm SE 3383835 0Q>m Bob 08: “9meon mo 550:3 a ma Eon 058m mo countenance 5 $55 2:. .m.v 0.53%

363 25

on ma cm mp or m o

_ _ _ _ _ r

 

I
V
F

(°/. 1M/1M) uogmnuaauoo plan oquos

 

107

 

0% a 328

ﬂockﬁoﬁ ﬂoaomaﬁgm E—w BMDOHOMEUGN Ugh 80¢ 08% OWN—Cum wo :0303 n ma ﬁmuw OMDHOm m0 Gomuwbﬁvodoo 0>Uﬂ~0h Oﬁ .wdu Dun-Mar.—

EEB 9::

on mm on mw cw m o

T _ _ _ p _ i [EEJI O

 

 

(% 03/3) uonenuaauoo vs annelau

I

am
%,cop

 

108

0% S 2:: omﬁoum mo gouge a 8 A55 .0983 SE 3383835 DD>m mo Eon "item we 53.5588 5 omﬁﬁo 2F .56 charm

on

mm

ON

@223 08¢
2

_

cw

 

low

-NF

[3.

Av S

 

[mp

(°/. mam) uouenuaauoa pgoe agqms

109

 

on

0% 8 ASE ﬁobcoov 8E HBobmﬁzcm DEA mo Eon 05.8 90 m8. 2: mo 83 8E BBQ 65 < .m... 25w:—

 

 

 

E633:
mm on mp op m o
F _ r _ _ L O
- cu m.
m
m.
, ow w
0
0
w
I co m
m
m.
r 8 m,
. I»! o o m
0 LI, 0 O ”/9
_ e 2: (
O

 

[GNP

110

storage, there was 60% sorbic acid remaining in the ﬁlm wrapped around cheese, and 7%
in the ﬁlm wrapped around bologna. There was 85% remaining in the control ﬁlm. The
migration/loss of sorbic acid at the end of storage from ﬁlms wrapped around cheese,
bologna and the control ﬁlms were 40%, 93% and 15%, respectively. Comparison of
sorbic acid migrated/loss from the ﬁlms wrapped around cheese and bologna and the
control ﬁlm is shown in Figures 4.9 and 4.10. The results showed that the migration of
sorbic acid to bologna was higher than to cheese. Chen et al. (1996) reported that
methylcellulose/chitosan ﬁlms (antimicrobial ﬁlm) released 39% of the sorbic acid into a
glycerol/water solution in 30 minutes at 4 °C. Aﬁer 6 hours of storage time, 49% of the
sorbic acid was released from the antimicrobial ﬁlm. Weng et al. (1998) developed a
poly(ethylene—co-methacrylic acid) ﬁlm (PEMA ﬁlm) containing sorbic acid as an
antimicrobial ﬁlm for food packaging. PEMA ﬁlm with NaOH or HCl treatment released
55 mg (0.49 mmol) and 0.5 mg (0.004 mmol), respectively, and PEMA ﬁlm without
treatment released 0.5 mg (0.004 mmol) from the initial sorbic acid concentration of 0.5
mol.

The migration/loss of sorbic acid from PVDC ﬁlms can be presented on a semi-
logarithmic plot where the coordinates of log C/Co vs. time gave a straight line
relationship (Figures 4.11, 4.12 and 4.13). The rate constants for the migration/loss of
sorbic acid were determined from their gradient, calculated using Equation (1), and are
presented in Table 4.4. Migration/release rate of sorbic acid from ﬁhn wrapped around
cheese was 0.007 per day while ﬁlm wrapped around bologna was 0.040 per day. The

loss rate of control ﬁlm without food contact was 0.001 per day. The comparison among

ﬁlms wrapped with cheese and bologna, and control ﬁlm is presented in Figure 4.14.

111

.Uov an 08m..— Dmmuon MO COmuoda a ma

«amazon ES omoono woman; 8E 3583835“ Data 5033: Eon 058m mo coumbcoocoo E omega 2: mo acmEmEoU .mé 0.53m

ESE 25»

mm mu 5 E x. o
_ _ b _ k C

 

 

mcmoﬁn

mmoozo
T

 

T

(% nit/1M) uonenuaouoo pgoa ogqms

 

Ezw _O.=COO

 

112

0% “a memo—on was omoono 325:0 3383

SE 3583835 0Q>m 5353 DE: owns; .«o 850:3 a ma Eon 058m mo 5:838:00 3:28 05 mo somtwanU .S.v 95$...—

 

 

 

 

 

 

 

Am>uuv 05:.
_ i r _ F _ o
oi
H
«.5202 m.
m.
a
S
v
O
O
U
0
T m
a
30050 W.
U
1 m
a w
EE .2200 4 2: low
4

 

l
O
N
F

113

cm

0% 8 38:0 533:0 3&8? BE 3582835 0Q>m Eat Bus 288 we 82 05 .«o 83 88 BBQ “mum < .36 Paw:—

Eas 25»
mm ca 2 S m o

 

 

uonenuaouoo vs lenpgsai annals: °/. 601

 

114

on

0% an «@015 38.83 85 3588885 Ont/m 88% Box 880.... mo 32 05 .«o 83 88 .880 BE < .N—é PSME

 

£33 25
mm cm 2 8 m c
. _ _ _ _ r r o
- «d
- ed
- ed

 

.. ad

- N._.

T T
“2 ‘1:
1- 1-

 

(cc/o)
uonenuaouoo vs lenpgsar aAnelaJ °/. 60']

115

on

 

.Uov E €08 8888380513 85 38:8 UD>m 88m Boa 888 .«o 82 05 mo 83 88 88o 8% < .2..“ 8st.9

85 2::

 

 

 

mm 8 2 S m c 1
_ _ r _ _ _ I O m
- «d w.

a

I .3 m

.. S. m

- a... ) m.

D.

- F Mm

, 3 w

T 3 m

U

I 3 w

- 3 m

Li 1! . n . . _ N m.

U

116

.Uov “a 2:: omESm mo

someone a ma ammo—on 98 83:0 3&8? 8E 3582835 Ont/m Soc Boa 038m mo 32 05 mo 63 88 89o Em < 6:. ensure

@63 95h

 

     

 

 

 

mm an E er 5 c
F _ h _ _ °.°
.I
1 N6 .m
/o
r to w
. mu...
acmoﬁm 1 m c w...
. 1 m-c ) MSW
. m m
r c P m m.
I a; W.
m
I v.9 w
a
33:0 7 m... m.
1 .p...
o o I a; w.
.2250 1

O.N

117

Table 4.4. The rate constants of releasing/loss of sorbic acid from PVDC antimicrobial

 

 

ﬁlms
Food types Rate constant of releasing/loss
K (l/day)
Cheddar cheese 0.007
Beef bologna 0.040
Control ﬁlm (without food) 0.001

 

118

.el

4.5. CONCLUSION
The study showed that the sorbic acid was released from the ﬁlms to food for both
Cheddar cheese and beef bologna. There was 40% sorbic acid release from ﬁlm wrapped
around cheese at the end of 28 days of storage with a rate of 0.007 per day, 93% was
released ﬁom ﬁlm sandwiched between bologna with a rate of 0.040 per day. About 15%

of the sorbic acid in the control ﬁlm was lost at the end of 28 days of storage.

119

CONCLUSION

Polyvinylidene chloride copolymer (SaranR F-310) ﬁlms containing 1.5 to 3.0%
(w/v) sorbic acid, 2.0 to 3.0% (w/v) potassium sorbate, or 1.0 to 2.5% (w/v) nisin showed
inhibition against Listeria monocytogenes (CWD 95, CWD 246, CWD 201 and CWD
1503). Films containing lactofen'in or sodium diacetate did not show inhibition against L.
monocytogenes in laboratory media, trypticase soy agar containing 0.6% yeast extract.
Films containing sorbic acid had the best antimicrobial activity, banier and mechanical
properties, and distribution of the antimicrobial in the polymer structure. Polyvinylidene
chloride ﬁlm containing 3% sorbic acid coated on polyethylene terephthalate (PET) ﬁlm
at a minimum thickness of 0.75 mil or 0.00075 inch had antimicrobial activity against
Listeria monocytogenes in laboratory media.

Subsequently, polyvinylidene chloride (PVDC) copolymer ﬁlms containing 1.5 or
3.0% sorbic acid were tested on Cheddar cheese and beef bologna, with an initial surface
inoculation of 105 or 103 L. monocytogenes CFU/g of product. On Cheddar cheese the
number of L. monocytogenes was reduced by 0.1 — 1 log, and 4 — 7 logs on bologna slices
after 35 and 28 days refrigerated storage for Cheddar cheese and bologna, respectively.
Reduction in the numbers of mesophilic aerobic bacteria and lactic acid bacteria were
also observed on Cheddar cheese and bologna slices.

Migration/loss of sorbic acid from PVDC copolymer ﬁlm showed that 40% of the
impregnated sorbic acid migrated/lost from the ﬁlm wrapped around the Cheddar cheese
at the end of 28 days storage with the rate of 0.007 per day. For bologna, 93%

migrated/lost from the ﬁlm sandwiched between bologna, at a rate of 0.040 per day.

120

Polyvinylidene chloride ﬁlm containing sorbic acid not in contact with any food had a
loss about 15% at the end of 28 days of storage.

This work shows that it may be pOssible to use a ﬁlm such as polyvinylidene
chloride polymer containing antimicrobial agent as a food wrap to reduce the risk of
Listeria spp. contamination, while extending the shelf life of food products.

The directions of future research should include;

1) Detailed study of role(s) of the distribution of sorbic acid in the ﬁlm
chemical structure, the polymer chain orientation of the ﬁlm and
barrier property.

2) Detailed study of the relationship of the thickness of antimicrobial ﬁlm
and efﬁcacy against L. monocytogenes. According to the study, it
showed that increasing ﬁlm thickness resulted in an increase in
antimicrobial activity. It would be interesting to address why it

behaves such way and what is responsible for such event.

121

APPENDIX I

122

STATISTIC ANALYSIS OF STORAGE FOR CHEESE AND BOLOGNA
A. L. monocytogenes on Cheddar cheese during storage at 4 °C for 35 days

(Initially inoculated L. monocytogenes 103 CFU lg)

Num Den

Effect DF DF F Value Pr > F

Chemical 2 30 28.41 <.0001

time 4 30 57.29 <.0001

Chemical*time 8 30 5.92 0.0001
Effect Chemical time Chemical time Adjustment Adj P
Chemical C S Tukey 0.0005
Chemical C SS Tukey <.0001
Chemical S SS Tukey 0.0075
time 0 7 Tukey 0.9412
time 0 14 Tukey 0.9850
time 0 2 1 Tukey 0.0002
time 0 35 Tukey <.0001
time 7 14 Tukey 0.7095
time 7 21 Tukey <.0001
time 7 35 Tukey <.0001
time 14 21 Tukey 0.0010
time 14 35 Tukey <.0001
time 2 1 3 5 Tukey <.0001
Chemical*time C 0 C 7 Tukey 1.0000
Chemical*time C 0 C 14 Tukey 1.0000
Chemical*time C 0 C 21 Tukey 1.0000
Chemical*time C 0 C 35 Tukey 0.1822
Chernical*time C 0 S 0 Tukey 1.0000
Chemical*time C 0 S 7 Tukey 1.0000
Chemical*time C 0 S 14 Tukey 0.9998
Chemica1*time C 0 S 21 Tukey 0.1151
Chernical*time C 0 S 35 Tukey <.0001
Chemical*time C 0 SS 0 Tukey 1.0000
Chemical*time C 0 SS 7 Tukey 1.0000
Chemical*time C 0 SS 14 Tukey 0.9975
Chemical*time C 0 SS 21 Tukey 0.0001
Chemica1*time C 0 SS 35 Tukey <.0001
Chemica1*tirne C 7 C 14 Tukey 1.0000
Chemical*time C 7 C 21 Tukey 1.0000
Chemical*time C 7 C 35 Tukey 0.0541
Chemical*time C 7 S O Tukey 0.9998
Chemical*time C 7 S 7 Tukey 1.0000

123

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S

wwwwmwmwmmNNNwammmmwu—--H---
OOOOOMMMMMMMMMM--—--—v---—-—~4>AAAAAAAAAAAQQQVQQQQ

UJUJUJMUJ

14
21
35

14
21
35
21
35

14
21
35

14
21
35
35

14
21
35

14
21
35

14
21
35

14
21
35

14
21
35

124

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.9750
0.0316
<.0001
1.0000
0.9996
0.9173
<.0001
<.0001
1.0000
0.0541
0.9998
1.0000
0.97 50
0.0316
<.0001
1.0000
0.9996
0.9173
<.0001
<.0001
0.1389
1.0000
1.0000
0.9992
0.0858
<.0001
1.0000
1.0000
0.9923
0.0001
<.0001
0.3216
0.0541
0.6599
1.0000 -
0.01 13
0.2654
0.3459
0.8092
0.2761
<.0001
0.9998
1.0000
0.2163
<.0001
1.0000

Chemical*time S 0 SS 7 Tukey 1.0000
Chemical*time S 0 SS 14 Tukey 0.9999
Chemical*time S 0 SS 21 Tukey 0.0004
Chemical*time S 0 SS 35 Tukey <.0001
Chemical*time S 7 S 14 Tukey 0.9750
Chemical*time S 7 S 21 Tukey 0.0316
Chemical*time S 7 S 35 Tukey <.0001
Chemical*time S 7 SS 0 Tukey 1.0000
Chemical*time S 7 SS 7 Tukey 0.9996
Chemical*time S 7 SS 14 Tukey 0.9173
Chemical*time S 7 SS 21 Tukey <.0001
Chemical*time S 7 SS 35 Tukey <.0001
Chemical*time S 14 S 21 Tukey 0.5109
Chemical*time S 14 S 35 Tukey <.0001
Chemical*time S 14 SS 0 Tukey 1.0000
Chemical*time S 14 SS 7 Tukey 1.0000
Chemical*time S 14 SS 14 Tukey 1.0000
Chemical*time S 14 SS 21 Tukey 0.0016
Chemical*time S 14 SS 35 Tukey <.0001
Chemical*time S 21 S 35 Tukey 0.0202
Chemical*time S 21 SS 0 Tukey 0.1743
Chemical*time S 21 SS 7 Tukey 0.2351
Chemical*time S 21 SS 14 Tukey 0.6746
Chemical*time S 21 SS 21 Tukey 0.3977
Chemical*time S 21 SS 35 Tukey <.0001
Chemical*time S 35 SS 0 Tukey <.0001
Chemical*time S 35 SS 7 Tukey <.0001
Chemical*time S 35 SS 14 Tukey <.0001
Chemical*time S 35 SS 21 Tukey 0.9750
Chemical*time S 35 SS 35 Tukey 0.0601
Chemical*time SS 0 SS 7 Tukey 1.0000
Chemical*time SS 0 SS 14 Tukey 0.9997
Chemical*time SS 0 SS 21 Tukey 0.0003
Chemical*time SS 0 SS 35 Tukey <.0001
Chemical*time SS 7 SS 14 Tukey 1.0000
Chemical*time SS 7 SS 21 Tukey 0.0004
Chemical*time SS 7 SS 35 Tukey <.0001
Chemical*time SS 14 SS 21 Tukey 0.0030
Chemical*time SS 14 SS 35 Tukey <.0001
Chemical*time SS 21 SS 35 Tukey 0.0016

 

B. L. monocytogenes on Cheddar cheese during storage at 4 °C for 35 days

(Initially inoculated L. monocytogenes 105 CFU/g)

125

Num Den

Effect DF DF F Value Pr > F

Chemical 2 105 304.50 <.0001

time 6 105 167.61 <.0001

Chemical*time 12 105 53.89 <.0001
Effect Chemical time Chemical time Adjustment Adj P
Chemical C S Tukey <.0001
Chemical C SS Tukey <.0001
Chemical S SS Tukey <.0001
time 0 4 Tukey <.0001
time 0 7 Tukey 0.0008
time 0 1 0 Tukey <.0001
time 0 14 Tukey <.0001
time 0 2 1 Tukey <.0001
time 0 35 Tukey <.0001
time 4 7 Tukey 0.0688
time 4 10 Tukey 0.6178
time 4 14 Tukey 0.9840
time 4 2 l Tukey <.0001
time 4 35 Tukey <.0001
time 7 10 Tukey 0.8982
time 7 14 Tukey 0.3722
time 7 2 1 Tukey <.0001
time 7 35 Tukey <.0001
time 10 14 Tukey 0.9727
time 1 0 2 1 Tukey <.0001
time 10 35 Tukey <.0001
time 14 21 Tukey <.0001
time 14 35 Tukey <.0001
time 2 l 3 5 Tukey <.0001
Chemical*time C 0 C 4 Tukey 0.0377
Chemical*time C 0 C 7 Tukey 0.9996
Chemical*time C 0 C 10 Tukey 0.9987
Chemical*time C 0 C 14 Tukey 0.9996
Chemical*time C 0 C 21 Tukey 0.9999
Chemical*time C 0 C 35 Tukey 1.0000
Chemical*time C 0 S 0 Tukey 1.0000
Chemical*time C 0 S 4 Tukey 0.0003
Chemical*time C 0 S 7 Tukey 0.9991
Chemical*time C 0 S 10 Tukey 0.2772
Chemical*time C 0 S 14 Tukey 0.0071
Chemical*time C 0 S 21 Tukey <.0001
Chemical*time C 0 S 35 Tukey <.0001

126

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

SSQQQQQQQQQQQQQ\lexlxlxl-bAA-hAhAA-bA-bA-hhhhbh-ﬁOOOOOOO

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35
10
14
21
35

10
14
21
35

10
14
21
35
14
21

127

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.9994
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.5968
0.6818
0.5968
0.5389
0.2357
0.2772
0.9987
0.6539
1.0000
1.0000
0.0667
<.0001
0.6255
0.9518
0.1817
0.0919
0.0296
<.0001
<.0001
1.0000
1.0000
1.0000
1.0000
1.0000
0.0262
1.0000
0.9744
0.2559
<.0001
<.0001
1.0000
0.0047
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
14
14
14
14
14
14
14
14
14
14
14
14
l4
l4
l4
14
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21

mmmmmmmn

35

10
14
21
35

10
14
21
35
21
35

10
14
21
35

10
14
21
35
35

10
14
21
35

10
14
21
35

128

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

1.0000
1.0000
0.0377
1.0000
0.9878
0.3230
<.0001
<.0001
1.0000
0.0071
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
1.0000
0.0262
1.0000
0.9744
0.2559
<.0001
<.0001
1.0000
0.0047
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
0.0205
1.0000
0.9605
0.2166
<.0001
<.0001
1.0000
0.0035
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S

qqqqqqqpaAAAAAAAAAAooooooooooooog

wwwwwwwwwmmmw
MMMMMMMMMMMMM

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35
10
14
21
35

129

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

1.0000
0.0041
1.0000
0.7609
0.0667
<.0001
<.0001
1.0000
0.0006
<.0001
<.0001
<.0001
<.0001
<.0001
0.0054
1.0000
0.8084
0.0827
<.0001
<.0001
1.0000
0.0008
<.0001
<.0001
<.0001
<.0001
<.0001
0.0334
0.8506
1.0000
0.8084
<.0001
0.0296
1.0000
0.9605
0.8696
0.6255
<.0001
<.0001
0.9842
0.2996
<.0001
<.0001
1.0000
0.0062
<.0001

 

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

\l\l\)\.l

10
10
10
10
10
10
10
10
10
l4
l4
14
14
14
14
14
14
14
21
21
21
21
21
21
21
21
35
35
35
35
35
35
35

AAOOOOOO

SS
SS
SS
SS

S

S

SS
SS
SS
SS
SS
SS
SS

SS
SS
SS
SS
SS
SS
SS

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS

10
14
21
35
14
21
35

10
14
21
35
21
35

10
14
21
35
35

10
14
21
35

10
14
21
35

10
14
21
35

10

130

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
0.9994
0.0054
<.0001
0.9797
0.5100
0.0205
0.0082
0.0020
<.0001
<.0001
0.2357
<.0001
0.2772
0.9987
0.4815
0.2996
0.1246
<.0001
<.0001
<.0001
<.0001
0.9797
1.0000
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0180
<.0001
0.0054
<.0001
<.0001
<.0001
<.0001
<.0001
0.9991
0.9907

 

Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS

QQQQ-h-h-h

10
10
10
14
14
21

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS

14
21
35
10
14
21
35
14
21
35
21
35
35

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.9174
<.0001
<.0001
1.0000
1.0000
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001

C. L. monocytogenes on beef bologna during storage at 4 °C for 28 days

(Initially inoculated L. monocyto enes 103 CFU/g)
g

Effect

Chemical
time
Chemical*time

Effect

Chemical C
Chemical C
Chemical S
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time

Sqqquhhbhhoooooo

S

SS
SS

Num Den
DF DF
2 42

6 42
12 42

4 .
7

10
14
21

28
7

10
14
21
28
10
14
21
28
14

Chemical time Chemical time

131

F Value

2189.83

Adjustment

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey

Pr>F

<.0001
<.0001
<.0001

Adj P

<.0001
<.0001
<.0001
0.9321
0.9994
1.0000
0.0912
<.0001
<.0001
0.7388
0.8828
0.0056
<.0001
<.0001
0.9999
0.2217
<.0001
<.0001
0.1249

time 1 0
time 10
time 14
time 14
time 2 1

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

\lﬂb-h-A-h-bhkA-b-h-h-h«#43h-k-b##OOOOOOOOOOOOOOOOOOOO

NNNNN
OOOOr-‘OOH

mmmmmmmoonnﬁﬁ

mmmmmmmmmmmmmm
onmmmmmmm 00000

4

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28
10
14

132

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey

<.0001
<.0001
<.0001
<.0001
<.0001
0.9966
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
0.9795
0.0863
0.1 190
0.0023
<.0001
0.0002
1.0000
0.8747
0.0004
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.8747
0.2519
0.0017
0.0026
<.0001
<.0001
<.0001
0.9995
0.1 1 18
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

v—il—‘t—‘U—‘I—‘D—Hh—‘r—it—‘H—Il—‘I—‘D—‘I—di—‘I—‘h—‘HHt—‘I—‘t—dl—lh—II—ﬁb—i
3'34:A4:4:-AAAAAAhooooooooooooooooo\’\‘\’\'\‘“VQVVQQV\IVN

‘mmmmmmmon

21
28

10
14
21
28

10
14
21
28
14
21
28

10
14
21
28

10
14
21
28
21
28

10
14
21
28

10

133

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

wmmmmmmmmmmmmm

NNNNNNNNNNNNNNNNNNNNNNNNNNNNHU—‘I—I
mmmmmmmmmmmmmv—r—v—ir—nn—tu—nv—Iu—nu—ou—an—nu—‘r—nt—an—tAbh

Aoooooooooooocgg

14
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14

21

28

10
14
21
28
7

134

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
0.3238
0.4064
0.0152
0.0001
0.0015
1.0000
0.9966
0.0030
0.0003
<.0001
<.0001
<.0001
0.9267

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical *time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *tirne
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm

QQQQQQQQNQﬂ-b-h-hh-hhhA-bhh

10
14
21
28

10
14
21
28
10
14
21
28

10
14
21
28
14
21
28

10
14
21
28
21
28

10
14
21
28
28

10

135

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.9618
0.1918
0.0036
0.0302
0.9427
1.0000
0.0537
0.0064
0.0020
<.0001
<.0001
1.0000
0.9988
0.3891
0.8623
0.0537
0.9919
0.9427
0.5162
0.2793
<.0001
<.0001
0.9957
0.3085
0.7903
0.0756
0.9973
0.8976
0.4240
0.2145
<.0001
<.0001
0.9919
1.0000
0.0013
0.3891
1.0000
0.9984
0.9717
0.0015
<.0001
1.0000
<.0001
0.01 1 1
1.0000
1.0000

 

Chemical*time S 21 SS 14 Tukey 1.0000
Chemical*time S 21 SS 21 Tukey 0.1049
Chemical*time S 21 SS 28 Tukey 0.0014
Chemical*time S 28 SS 0 Tukey 0.0001
Chemical*time S 28 SS 4 Tukey 0.0808
Chemical*time S 28 SS 7 Tukey 1.0000
Chemical*time S 28 SS 10 Tukey 1.0000
Chemical*time S 28 SS 14 Tukey 1.0000
Chemical*time S 28 SS 21 Tukey 0.0152
Chemical*time S 28 SS 28 Tukey 0.0001
Chemical*time SS 0 SS 4 Tukey 0.7742
Chemical*time SS 0 SS 7 Tukey 0.0002
Chemical*time SS 0 SS 10 Tukey <.0001
Chemical*time SS 0 SS 14 Tukey <.0001
Chemical*time SS 0 SS 21 Tukey <.0001
Chemical*time SS 0 SS 28 Tukey <.0001
Chemical*time SS 4 SS 7 Tukey 0.1347
Chemical*time SS 4 SS 10 Tukey 0.0192
Chemical*time SS 4 SS 14 Tukey 0.0064
Chemical*time SS 4 SS 21 Tukey <.0001
Chemical*time SS 4 SS 28 Tukey <.0001
Chemical*time SS 7 SS 10 Tukey 1.0000
Chemical*time SS 7 SS 14 Tukey 0.9997
Chemical*time SS 7 SS 21 Tukey 0.0081
Chemical*time SS 7 SS 28 Tukey <.0001
Chemical*time SS 10 SS 14 Tukey 1.0000
Chemical*time SS 10 SS 21 Tukey 0.0660
Chemical*time SS 10 SS 28 Tukey 0.0008
Chemical*time SS 14 SS 21 Tukey 0.1613
Chemical*time SS 14 SS 28 Tukey 0.0026
Chemical*time SS 21 SS 28 Tukey 0.9901

D. L. monocytogenes on beef bologna during storage at 4 °C for 28 days

(Initially inoculated L. monocytogenes 105 CFU/g)

Num Den
Effect DF DF F Value Pr > F
Chemical 2 105 3234.61 <.0001
time 6 105 98.94 <.0001
Chemical*time 12 105 228.69 <.0001
Effect Chemical time Chemical time Adjustment Adj P
Chemical C S Tukey < .000 1

136

Chemical
Chemical
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time

\lﬂﬂﬂ-bAAAAOOOOOO

21

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

bk-ﬁOOOOOOOOOOOOOOOOOOOO

NNNNNH ““44;
coco 0° NNv—H—NN—u—xllom
4) ~43 A0 mm

mmmmmmmoonoon

10
14
21
28

10
14
21
28

10
14
21
28

10
14

137

Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
0.0001
<.0001
0.0002
<.0001
<.0001
<.0001
<.0001
0.9565
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.9015
<.0001
0.0002
<.0001
<.0001
0.7887
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
0.0880
0.1072
0.8776 '
0.4993
0.0010
<.0001
1.0000
0.3687
0.0034
0.5406
0.0087
0.0010
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

mmmmmmmno

t—nt—tv—Il—tl—tv—tv—a
gagsgoooooocqqqqqq\quqqqqqqqqqaAAAAAAAAAAAAhA-b

mmmmmmwmmnoo

mm

21
28

10
14‘
21
28

10
14
21
28
10
14
21
28

10
14
21
28

10
14
21
28
14
21
28

10
14
21
28

138

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey

<.0001
<.0001
<.0001
0.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

. <.0001

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001 ‘
<.0001
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

10
10
10
10
10
14
14
14
14
14
14
14
l4
14
14
14
14
14
14
14
14
21
21
21
21
21
21
21
21
21

'21

21
21
21
21
21
28
28
28
28
28
28
28
28
28
28

10
14
21
28
21
28

10
14
21
28

10
14
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

139

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

C
C
C
C

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

28
28
28

N
00

gsgqqqqquqqqqqa-Jx-bhA-bA-bcs-h-b-hooooooooooooo

10
10
10

SS
SS
SS

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28
10
14
21
28

10
14
21
28
14
21
28
0

4

7

140

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey

<.0001
<.0001
<.0001
<.0001
0.1856
0.0468
0.7036
0.2991
0.0003
<.0001
1.0000
0.2020
0.001 1
0.3329
0.0030
0.0003
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0128
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.9991
1.0000
0.9982
0.5130
0.4192
1.0000
1.0000
1.0000
1.0000
0.9982
0.0551
1.0000
0.4587
0.0209
0.9971
1.0000
0.6906

 

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmmmmmmmmmmmmmmmmmmm

l—sl—al—nl—t
OOOO

t—‘r—ir-‘t—‘t—‘r—‘l-d
AhA-h-b-h-b

NNNNNNNNNNNNNN
mmmmmmmn—Ar—n—v—tl—Iu—u—n

SS
SS
SS
SS

l—nl—t
JK-h
CDC/J

SS
SS
SS
SS
SS
SS
SS

N
(I)

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
10 SS
10 SS
10 SS

\IQQQ-b-bA-b-D-OOOOOO

10
14
21
28
21
28

10
14
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28
10
14
21
28
14
21
28

141

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

1.0000
0.8509
0.4587
0.0005
0.8509
0.1 125
0.9012
1.0000
0.9616
1.0000
0.9927
0.8509
0.0048
0.9996
0.0106
0.9277
1.0000
0.8212
1.0000
1.0000
0.7887
<.0001
0.1778
0.9927
0.0972
0.9616
0.9996
1.0000
0.8107
0.0297
0.9216
0.0646
0.0106
<.0001
0.9875
1.0000
0.9985
0.9277
0.0093
0.9490
1.0000
1.0000
0.5683
0.9890
0.8212
0.0039

 

Chemical*time SS 14 SS 21 Tukey 1.0000
Chemical*time SS 14 SS 28 Tukey 0.3811
Chemical*time SS 21 SS 28 Tukey 0.7887

E. Mesophilic bacteria on Cheddar cheese during storage at 4 °C for 35 days

(Initially inoculated L. monocytogenes 103 CFU/g)

 

Num Den

Effect DF DF F Value Pr > F

Chemical 2 75 88.82 <.0001

time 4 75 1417.30 <.0001

Chemical*time 8 75 17.1 1 <.0001
Effect Chemical time Chemical time Adjustment Adj P
Chemical C S Tukey <.0001
Chemical C SS Tukey <.0001
Chemical S SS Tukey <.0001
time 0 7 Tukey <.0001
time 0 14 Tukey <.0001
time 0 21 Tukey <.0001
time 0 35 Tukey <.0001
time 7 14 Tukey 0.0084
time 7 2 1 Tukey <.0001
time 7 35 Tukey <.0001
time 14 21 Tukey <.0001
time 14 35 Tukey <.0001
time 2 1 3 5 Tukey < .0001
Chemical*time C 0 C 7 Tukey <.0001
Chemical*time C 0 C 14 Tukey <.0001
Chemical*time C 0 C 21 Tukey <.0001
Chemical*time C 0 C 35 Tukey <.0001
Chemical*time C 0 S 0 Tukey 1.0000
Chemical*time C 0 S 7 Tukey <.0001
Chemical*time C 0 S 14 Tukey <.0001
Chemical*time C 0 S 21 Tukey <.0001
Chemical*time C 0 S 35 Tukey <.0001.
Chemical*time C 0 SS 0 Tukey 1.0000
Chemical*time C 0 SS 7 Tukey <.0001
Chemical*time C 0 SS 14 Tukey <.0001
Chemical*time C 0 SS 21 Tukey <.0001
Chemical*time C 0 SS 35 Tukey <.0001
Chemical*time C 7 C 14 Tukey <.0001
Chemical*time C 7 C 21 Tukey <.0001

142

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chernical’time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time S
Chemical*time S

mmmmmmmmmmmmmmm mmmmmmmmmm mmmmmmmmmmo
mmmmm Ommmmm OOmmmmm

SS
SS
SS
SS
SS
0 S

0 S

wwuwwwwwwwNNNNNNNNNwmwu—u—-H~HH-~
V'U‘UIU‘U‘UIU‘MUIUI—‘HH—‘F‘Hr—v—H—F-hA-h-hA-bAA-bAkhqqqqqqqqqqq

35

14
21
35

14
21
35
21
35

14

21'

35

14
21
35
35

14
21
35

14
21
35

14
21
35

14
21
35

14

143

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
0.5798
0.6762
<.0001
<.0001
<.0001
0.6762
0.3609
<.0001
0.7795
<.0001
0.9989
<.0001
<.0001
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
0.9989
0.0050
<.0001
<.0001
<.0001
<.0001
0.0002
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001

 

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

S
S

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS

QQQQQQQQOOOOOOO
U)

\IQQOOOO
(I)
U)

14 SS

21 SS

21
35

14
21
35
14
21
35

14
21
35
21
35

14
21
35
35

14
21
35

14
21
35

14
21
35
14
21
35
21
35
35

144

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
<.0001
l .0000
<.0001
<.0001
<.0001
1.0000
1 .0000
<.0001
0.0037
<.0001
<.0001
<.0001
1 .0000
1.0000
<.0001
0.0059
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
0.0017
<.0001
<.0001
<.0001
<.0001
1 .0000
<.0001
0.0059
<.0001
0.0012
<.0001

F. Mesophilic bacteria on Cheddar cheese during storage at 4 °C for 35 days

(Initially inoculated L. monocytogenes 105 CFU/g)

Effect

Chemical
time
Chemical *time

Effect

Chemical C
Chemical C
Chemical S
time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C

ﬁﬂﬂﬁbh-ﬁthOOOOO

Nv—Il—Iu—ar—or—n
v—AAOOO

ooooooooo
mmmoooono

Num Den

DF DF
105
105

12 105

S

SS
SS

4
7
10
14

21
35
7
10
14

21
35
10
14

21
35
14
21
35

21
35
35

Chemical time Chemical time

10
14
21
35

145

F Value

Pr>F

<.0001
<.0001
<.0001

Adjustment

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

Adj P

<.0001
<.0001
0.2751
1.0000
0.9999
<.0001
<.0001
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.7970
0.0232
0.0208
0.5041
0.4796
1.0000
1.0000
0.9099
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
0.9952

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

\IQQ\1QQQQQQQQQQﬂﬂ-§Ah#¢sb###-§A&Ah#A-§A-§OOOOOOOOOOO

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35
10
14
21
35

10
14
21
35

10
14

146

T ukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
0.0044
0.0361
0.0295
1.0000
1.0000
1.0000
<.0001
<.0001
0.1932
0.2363
1.0000
<.0001
<.0001
<.0001
<.0001
0.9985
0.9985
0.5508
<.0001
<.0001
0.0008
0.0006
1.0000
1.0000
1.0000
<.0001
<.0001
0.0081
0.01 1 1
<.0001
<.0001
<.0001
<.0001
0.7553
0.7553
0.0797
<.0001
<.0001
<.0001
<.0001
0.9999
0.9668
0.9978
<.0001
<.0001

 

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

7 SS
7 SS
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
14
14
14 S
14 S
14 S
14 S
14 S
14 S
14 S
14 SS

SS

SS

SS

SS

mmmmmmmmmmmmmm
onmmmmmmm 000

l4

14

14

14

14 SS
14 SS
21 C
21 S
21 S
21 S
21 S
21 S
21 S
21 S
21 SS
21 SS
21 SS

21
35
14
21
35

10
14
21
35

10
14
21
35
21
35

10
14
21
35

10
14
21
35
35

10
14
21
35

147

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

. Tukey

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.0002
0.0003
0.9990
1 .0000
1.0000
<.0001
<.0001
<.0001
0.5702
0.0005
<.0001
<.0001
<.0001
<.0001
<.0001
0.5799
0.0240
<.0001
<.0001
1.0000
1.0000
<.0001
<.0001
<.0001
0.0250
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0261
0.0002
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
0.0641
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

 

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmmmmmmmmmmmmmm

wmwwwwwwwwwuwwm MN
\l\lxlb-h-b41hAAA43-h-hAOOOOOOOOOOOOOMU‘U‘MMMU‘U‘mummmM_B__‘

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35
10
14
21

148

Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.0665
0.0006
<.0001
<.0001
<.0001
<.0001
<.0001
0.0716
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0742
0.0007
<.0001
<.0001
1.0000
0.9998
<.0001
0.0126
0.0856
0.0716
0.9998
1.0000
1.0000
<.0001
0.0002
0.3571
0.4184
0.9998
<.0001
0.0126
0.0856
0.0716
0.9998
1.0000
1.0000
<.0001
0.0002
0.3571
0.4184
0.0001
0.3322
0.7719

Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmwant/2mmmmmmmmmmmmmmmmmmmmmmmmmmmmm

\)\l\l\l\l\)\l\l

10
10
10
10
10
10
10
10
10
14
14
14
14
14
14
14
14
14
21
21
21
21
21
21
21
21
35
35
35
35
35
35

COCO

SS
SS
SS
SS
SS
SS
SS

8

S

SS
SS
SS
SS
SS
SS
SS

SS
SS
SS
SS
SS
SS
SS

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS

35

10
14
21
35
14
21
35

10
14

21'

35
21
35

10
14
21
35
35

10
14
21
35

10
14
21
35

10
14

149

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

- Tukey

Tukey
Tukey
Tukey
Tukey

0.7295
0.7028
0.9789
0.8719
0.0001
0.0203
0.9861
0.9927
0.6753
0.2498
0.2855
<.0001
<.0001
<.0001
1.0000
0.9988
0.0512
0.0390
1.0000
1.0000
0.0001
0.0020
0.0005
0.6659
1.0000
0.9996
0.9990
1.0000
0.0018
0.0187
0.0051
0.2430
0.9820
1.0000
1.0000
0.0014
0.0151
0.0040
0.2781
0.9883
1.0000
1.0000
1.0000
1.0000
<.0001
<.0001

1

150

Chemical*time SS 0 SS 21 Tukey 0.0164
Chemical*time SS 0 SS 35 Tukey 0.0221
Chemical*time SS 4 SS 7 Tukey 1.0000
Chemical*time SS 4 SS 10 Tukey <.0001
Chemical*time SS 4 SS 14 Tukey <.0001
Chemical*time SS 4 SS 21 Tukey 0.1166
Chemical*time SS 4 SS 35 Tukey 0.1465
Chemical*time SS 7 SS 10 Tukey <.0001
Chemical*time SS 7 SS 14 Tukey <.0001
Chemical*time SS 7 SS 21 Tukey 0.0406
Chemical*time SS 7 SS 35 Tukey 0.0532
Chemical*time SS 10 SS 14 Tukey 0.9986
Chemical*time SS 10 SS 21 Tukey 0.0493
Chemical*time SS 10 SS 35 Tukey 0.0375
Chemical*time SS 14 SS 21 Tukey 0.7468
Chemical*time SS 14 SS 35 Tukey 0.6845
Chemical*time SS 21 SS 35 Tukey 1.0000
G. Mesophilic bacteria on beef bologna during storage at 4 °C for 28 days
(Initially inoculated L. monocytogenes 103 CFU/g)
Num Den

Effect DF DF P Value Pr > F

Chemical 2 42 1230.97 <.0001

time 6 42 51.86 <.0001

Chemical*time 12 42 128.55 <.0001
Effect Chemical time Chemical time Adjustment Adj P
Chemical C S Tukey <.0001
Chemical C SS Tukey <.0001
Chemical S SS Tukey 0.0001
time 0 4 Tukey 0.0117
time 0 7 Tukey 1.0000
time 0 10 Tukey 0.9987
time 0 14 Tukey 0.0678
time 0 21 Tukey <.0001
time 0 28 Tukey <.0001
time 4 7 Tukey 0.0191
time 4 10 Tukey 0.0440
time 4 14 Tukey 0.9929
time 4 21 Tukey <.0001
time 4 28 Tukey <.0001
time 7 10 Tukey 0.9999

time

time

time

time

time

time

time

time

time

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

10
10
10
14
14
21

h-b-hA-hA-hAhab-b45-h#k-k-bOOOOOCOOOOOOOOOOOOOO

mmmmmmmnonooo

14
21
28
14
21
28
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14

151

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.1018
<.0001
<.0001
0.1986
<.0001
<.0001
<.0001
<.0001
0.0071
0.0004
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
0.1329
0.5034
0.6497
0.3107
0.0145
1.0000
1.0000
0.0852
0.0008
0.0012
<.0001
<.0001
0.9992
0.4515
<.0001
<.0001
<.0001
0.0001
0.0009
<.0001
<.0001
<.0001
<.0001
<.0001
0.0003
0.0005
<.0001
<.0001
<.0001

 

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

\l\l\l\l\l\l\l\l\l\l\l\l\l\l\l\l\l\lA-§

mm
(AC/J

mmmmmmmnnno

mmmmmmmoo

21
28
10
14
21
28

10
14
21
28

10
14
21
28
14
21
28

10
14
21
28

10
14
21
28
21
28

10
14
21
28

152

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
0.9946
0.0005
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0374
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S

NNNNNNNNNNNNNNNNNNNNNNNNNNNNHHb-dh—‘I—tr—‘t—I
mmmmmmmm'mmmmm—h—‘O—‘D—h—‘l—lh—iI—II—It—‘h—Ib-III—Ih—II—I.h.§AhbhA

oooooooooo§

10
14
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10

153

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001-
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
0.3214
0.7964
0.8983
0.5966
0.0480
1.0000
1.0000
0.2248
0.0031

Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S

ﬁdﬂﬂﬂﬂﬂﬂﬂﬂﬂ-ﬁhAh-khhAh-A-bhOOO

21

S

14
21
28

10
14
21
28

10
14
21
28
10
14
21
28

10
14
21
28
14
21
28

10
14
21
28
21
28

10

14
21
28
28

154

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.0047
<.0001
<.0001
0.0777
0.3546
0.4902
0.2003
0.0075
1 .0000
1.0000
0.0480

~ 0.0004

0.0006
<.0001
<.0001
1.0000
1.0000
1.0000
1.0000
0.1851
0.1 167
1.0000
0.9473
0.9740
0.0584
0.1329
1.0000
1.0000
0.9856
0.6100
0.4643
1.0000
0.5565
0.6497
0.0079
0.021 1
1.0000
0.9520
0.7506
0.6100
0.9998
0.4139
0.5034
0.0042
0.01 16
0.9989

 

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

Effect

Chemical
time

Chemical*time

mmmmmmmmmmmmmm

SS
SS
SS
SS
SS
SS
SS
SS
SS

88‘

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS

NNNNNNNNNNNNNN
OOOOOOOOOOOOOOv—t-‘v—t—v—r—v—

Egzgggqqqqsshthoooooo

SS 0 Tukey
SS 4 Tukey
SS 7 Tukey
SS 1 0 Tukey
SS 14 Tukey
SS 2 1 Tukey
SS 28 Tukey
SS 0 Tukey
SS 4 Tukey
SS 7 Tukey
SS 10 Tukey
SS 14 Tukey
SS 2 1 Tukey
SS 28 Tukey
SS 4 Tukey
SS 7 Tukey
SS 10 Tukey
SS 14 Tukey
SS 2 1 Tukey
SS 28 Tukey
SS 7 Tukey
SS 10 Tukey
SS 14 Tukey
SS 21 Tukey
SS 28 Tukey
SS 10 Tukey
SS 14 Tukey
SS 2 1 Tukey
SS 28 Tukey

SS 14 ' Tukey

SS 2 1 Tukey

SS 28 Tukey

SS 21 Tukey

SS 28 Tukey

SS 28 Tukey

(Initially inoculated L. monocytogenes 105 CFU/g)

Num Den

DF DF F Value

2 105 1727.65
105 71.35

12 105 106.42

155

Pr>F

<.0001
<.0001
<.0001

0.4017
0.2800
1.0000
0.7624
0.8378
0.0190
0.0480
0.0223
0.0123
1.0000
1.0000
1.0000
0.3661
0.5966
1.0000
0.1219
0.0013
0.0020
<.0001
<.0001
0.0741
0.0007
0.0010
<.0001
<.0001
0.9816
0.9927
0.0934
0.2003
1.0000
0.9314
0.9903
0.8827
0.9767
1.0000

H. Mesophilic bacteria on beef bologna during storage at 4 °C for 28 days

Effect

Chemical
Chemical
Chemical
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time
time

\lﬂﬂﬂA-h-h-b-hOOOOOO

10
10
10
14
14
21

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

OOOOOOOOOOOOOOOOOOO

NNv—Ir—nNNt—t—t NNt—nt—n
g§§g§jgoo~hooo—Ao‘loo~ho\lh

mmmmmmmnnaono

Chemical time Chemical time

SS
SS

10
14
21
28

10
14
21
28

10
14
21

156

Adjustment

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

Adj P

<.0001
<.0001
0.0010
0.0072
0.0185
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.3245
0.0121
0.1895
0.8363
1.0000
0.9421
0.0012
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
0.9995
0.9997
0.7574
0.0182
0.0092
0.0015
1.0000
<.0001
0.2343
0.9883
0.1 137
0.0489

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

h—‘l—‘h-‘I-dt-dh-‘h—‘H
ooooooooﬂﬂﬂﬂﬂﬁﬂﬂﬂﬁﬂﬂﬂQQQQQ-hA-bA-hAkAh-kA-ﬁ-ﬁ-ﬁbbh-ﬁAC

U)
U)

mmmmmmwooooo

28

10
14
21
28

10
14
21
28

10
14'
21
28
10
14
21
28

10
14
21
28

10
14
21
28
14
21
28

10
14

157

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.0003
0.0010
<.0001
<.0001
<.0001
<.0001
0.0006
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0002
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time *C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
' Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

Chemical*time C 1

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

10 S
10 S
10 SS
10 SS
10 SS
10 SS
10 SS
10 SS
10 SS
14 C
14 C
14 S
14 S
14 S
14 S
14 S
14 S
14 S
14 SS
14 SS
14 SS
14 SS
14 SS
14 SS
14 SS
21 C
21 S
21 S
21 S
21 S
21 S
21 S
21 S
21 SS
21 SS
21 SS
21 SS
21 SS
21 SS
21 SS
28 S
28 S
28 S
28 S
28 S
28 S

21
28

10
14
21
28
21
28

10
14
21
28

10
14
21
28
28

10
14
21
28

10
14
21
28

10
14
21

158

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.3847
0.3194
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm

28

NNNNNN
000000000000

SS

28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28
10
14
21
28

10
14
21
28
14
21

159

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.9999
1.0000
0.8527
0.0308
0.0160
0.0028
1.0000
<.0001
0.3272
0.9968
0.1710
0.0783 '
0.0006
1.0000
1.0000
0.4464
0.31 17
0.0996
1.0000
<.0001
0.9651
1.0000
0.8652
0.6795
0.0308
1.0000
0.4195
0.2892
0.0900
1.0000
<.0001
0.9573
1.0000
0.8463
0.6521
0.0274
0.9807
0.9416

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical *time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS

10 S

SS
SS
SS
SS
SS
SS
SS

I—‘t—‘U-‘h—‘HD—tt—IHO—dh—d
##AOOOOOOO
MU)

SS
SS
SS
SS
SS
SS
SS

NNu—nl—nl—Ih-ov—‘l—o
-——-AAAJ>AA
U2

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS

NNNNNNNNNNNNN
OOOOOOOOOOOOOOh-‘F—F-‘F-‘F-‘ﬂ

\IQQAA-ﬁAhOOOOOO

28

10
14
21
28
21
28

10
14
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28
10
14
21

160

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

- Tukey

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.6885
0.9483
<.0001
1.0000
1.0000
1.0000
0.9984
0.3933
1.0000
1.0000
0.0656
<.0001
1.0000
0.7063
1.0000
1.0000
0.9999
1.0000
0.0361
<.0001
0.9999
0.5583
1.0000
1.0000
1.0000
0.0070
<.0001
0.9883
0.2343
0.9991
1.0000
1.0000
<.0001
0.5017
0.9998
0.2966
0.1514
0.0016
<.0001
<.0001
<.0001
<.0001
0.0001
0.9971
1.0000
1.0000

 

Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS

7

10
10
10
14
14
21

SS
SS
SS
SS
SS
SS
SS

28
14
21
28
21
28
28

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.8987
0.9738
0.8881
0.0869
1.0000
0.9775
0.9977

1. Lactic acid bacteria on Cheddar cheese during storage at 4 °C for 35 days

(Initially inoculated L. monocytogenes 103 CFU/g)

Effect

Chemical
time
Chemical*time

Effect

Chemical C
Chemical C
Chemical S
time

time

time

time

time

time

time

time

time

time
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

OOOOOOOOOOOO

Num Den
DF DF
2 75

4 75

8 75

S
SS
SS

mmmmmooon

Chemical time Chemical time

161

F Value

Adjustment

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

. Tukey

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

Adj P

0.0360
<.0001
<.0001
<.0001
<.0001
0.7649
0.9372
0.6787
<.0001
<.0001
<.0001
<.0001
0.3004
<.0001
<.0001
0.1535
0.0232
1.0000
<.0001
<.0001
0.3665
0.4186
1.0000
<.0001
0.3007

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

:qqqqqqqqqqqqqco

WWWMWWWWNNNNNNNNNNNHF‘F‘HF-‘HI-It-‘v—Ir—u—t
MMMMMMMMH~HH---~¥k##«F-b##-§Ab

mmmmmmmmmm mm
Oman/2mm 000mm

mmmmmmmmmmmmmmmommmmmmmmmmo
CDUJCDCDUJ UJC/JUJUJUJ

SS
SS
SS

21
35
14
21
35

14
21
35

14
21
35
21
35

14
21
35

14
21
35
35

14
21
35

14
21
35

14
21
35

14

162

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.0155
<.0001
0.8927
0.0055
0.0483
<.0001
1.0000
0.9043
0.0012
0.0009
<.0001
0.8621
0.0018
<.0001

<.0001 .

<.0001
0.0001
<.0001
0.6923
0.0431
<.0001
<.0001
<.0001
0.0323
<.0001
<.0001
<.0001
1.0000
0.1302
0.0176
0.4803
1.0000
1.0000
0.1019
0.5513
1.0000
<.0001
<.0001
0.0187
0.1241
0.9081
0.9980
0.9959
0.0137
0.9400
0.9993

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

C
C
S

SS
SS
SS
SS
SS
SS
SS
SS
SS

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm

WU)
MM
mm
mm

UJUJUJUJMUJUJUJ
mmmm

33312385325333;Ezgzzzuquqqqquooooooooo
mmmmmmmmmmmmmmmmmmmmmgﬂggammmm
mmmmmmmmmmmmm mmmmm m

U)
(I)

\IQQOOOO
U) m
U) U)

U)
(A

14 SS
14 SS

21
35

14
21
35

14
21
35
14
21
35

14
21
35
21
35

14
21
35
35

14
21
35

14
21
35

14
21
35
14
21
35
21
35

163

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey.
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
0.3238
0.3729
1.0000
<.0001
0.2627
0.0193
<.0001
0.9847
0.0043
0.0033

. <.0001

0.9721
0.0062
<.0001
<.0001
0.2232
0.1880
<.0001 .
1.0000
0.2786
<.0001
<.0001
1.0000
0.2679
0.2732
1.0000
<.0001
<.0001
0.3121 .

02327

1.0000
<.0001
<.0001
<.0001
0.2140
0.0262
<.0001
0.3357
<.0001
<.0001
<.0001
<.0001

Chemical*time SS

J. Lactic acid bacteria on Cheddar cheese during storage at 4 °C for 35 days

(Initially inoculated L. monocytogenes 105 CFU/g)

Effect

Chemical
time
Chemical*time

Effect

Chemical C
Chemical C
Chemical S
time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time

time
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

\lﬂﬂﬂ-ﬁA-h-h-hOOOOOO

21

ooooooo
03000000

SS

Num Den
DF DF

2 105
6 105
12 105

S
SS
SS

35

Chemical time Chemical time

10
14
21
35

10
14
21
35
10
14
21
35
14
21
35
21
35
35

10
14
21
35

164

Tukey

F Value

Pr>F

<.0001
<.0001
<.0001

Adjustment

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

0.0113

Adj P

<.0001
0.0009
0.0016
0.0010
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.9987
0.2492
<.0001
0.0821
<.0001
<.0001
0.4697
<.0001
<.0001
<.0001
<.0001
<.0001
0.3633

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

QQQQQQNQQQQQQﬂbh-kJk-h-b-ﬁs-h-hcli-bA-bA-h##b-ﬁOOOOOOOOOOOOO

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35
10
14
21
35

10
14
21
35

165

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
0.0083
<.0001
<.0001
<.0001
<.0001
<.0001
0.8556
<.0001
<.0001
<.0001
<.0001
<.0001
0.1015
0.8084
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.1075
<.0001
0.9958
0.9867
<.0001
<.0001
<.0001
0.7253

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

\l\l\l

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
14
l4
14
14
l4
14
14
14
l4
14

14‘

14
14
14
14
14
21
21
21
21
21
21
21
21
21

mmmwmmmm

U)

10
14
21
35
14
21
35

10
14
21
35

10
14
21
35
21
35

10
14
21
35

10
14
21
35
35

10
14
21
35

166

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

‘ Tukey

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
0.6158
1.0000
<.0001
<.0001
<.0001
0.0427
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
0.001 1
0.9916
0.5343
1.0000
<.0001
0.9822
<.0001
<.0001
<.0001
<.0001
0.7400
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0140
1.0000
0.1579
<.0001
<.0001
<.0001
<.0001
0.0027
1.0000
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S

{ADJUJUJUJUJWWMUJWUJWNNNNNN
MMMMMMMMMMMMMl—au—nu—au—a—ap—t

qAAAAAhAAAA-tussooooooooooooog

4
7
10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35

10
14
21
35
10

167

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
T ukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
0.6951
0.9660
0.9929
<.0001
0.1939
<.0001
<.0001
<.0001
<.0001
<.0001
0.0801
0.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.1015
<.0001
<.0001
<.0001
<.0001
<.0001
0.8556
0.1272
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.9993
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
0.0248

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time ‘
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm

\)\l\l\l\l\l\l\)\)\l

UJUJUJ

14
21
35

10
14
21
35
14
21
35

10'

14
21
35
21
35

10
14
21
35
35

10
14

'21

35

10
14
21

35'

168

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

Tukey ‘

0.9406
0.9735
<.0001
<.0001
<.0001
1.0000
0.8445
<.0001
0.2826
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0005
0.9735
0.6639
1.0000
<.0001
1.0000
<.0001
<.0001
<.0001
1.0000
0.0150
<.0001
0.0007
<.0001
<.0001
<.0001
<.0001
1.0000
0.0248
<.0001
0.0013
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.4228
0.9998
<.0001

169

Chemical*time SS 0 SS 10 Tukey <.0001
Chemical*time SS 0 SS 14 Tukey <.0001
Chemical*time SS 0 SS 21 Tukey <.0001
Chemical*time SS 0 SS 35 Tukey <.0001
Chemical*time SS 4 SS 7 T ukey <.0001
Chemical*time SS 4 SS 10 Tukey <.0001
Chemical*time SS 4 SS 14 Tukey <.0001
Chemical*time SS 4 SS 21 Tukey <.0001
Chemical*time SS 4 SS 35 Tukey <.0001
Chemical*time SS 7 SS 10 Tukey 0.1752
Chemical*time SS 7 SS 14 Tukey <.0001
Chemical*time SS 7 SS 21 Tukey 0.0162
Chemical*time SS 7 SS 35 Tukey <.0001
Chemical*time SS 10 SS 14 Tukey 0.0096
Chemical*time SS 10 SS 21 Tukey 1.0000
Chemical*time SS 10 SS 35 Tukey <.0001
Chemical*time SS 14 SS 21 Tukey 0.1204
Chemical*time SS 14 SS 35 Tukey <.0001
Chemical*time SS 21 SS 35 Tukey <.0001
K. Lactic acid bacteria on beef bologna during storage at 4 °C for 28 days
(Initially inoculated L. monocytogenes 103 CFU/g)
Num Den

Effect DF DF F Value Pr > F

Chemical 2 42 1000.97 <.0001

time 6 42 465.06 <.0001

Chemical*time 12 42 98.5 1 <.0001
Effect Chemical time Chemical time Adjustment Adj P
Chemical C S Tukey <.0001
Chemical C SS Tukey <.0001
Chemical S SS Tukey 0.0585
time 0 4 Tukey 1.0000
time 0 7 Tukey <.0001
time 0 10 Tukey <.0001
time 0 14 Tukey <.0001
time 0 2 1 Tukey <.0001
time 0 28 Tukey <.0001
time 4 7 Tukey <.0001
time 4 10 Tukey <.0001
time 4 14 Tukey <.0001
time 4 2 1 Tukey <.0001

time

time

time

time

time

time

time

time

time

time

time

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C

\l\l\)~§

10

10
14
14
21

$43-h-h-bubAA##A-ﬁ«hr-h-hOOOOOOOOOOOOOOOOOOOO

mmmmmmmnnonnn

28
10
14
21
28
14
21
28
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

170

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
0.0040
<.0001
<.0001
0.0397
<.0001
0.0003
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
1.0000

Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

wH—bHo—at—dt—or—v—Iu—‘p—tt—np—IH—‘Hr—tt—It—ot—HHI—Ir—
a-AAAA-n4sooooooooooooooooo““““QQQQ‘1“\‘VﬂﬂﬁﬂﬂﬂhhhA

10
14
21
28
10
14
21
28

10
14
21
28

10
14
21
28
14
21
28

10
14
21
28

10
14
21
28
21
28

10
14

171

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001 ‘
<.0001
<.0001
0.6518
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

Chemical*time C'

Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical‘time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time S
Chemical*time S
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

UJUJMUJCDUJ

14 S
14 S
14 SS
14 SS
14 SS
14 SS
14 SS
14‘ SS
14 SS
21 C
21 S
21 S
21 S
21 S
21 S
21 S
21 S
21 SS
21 SS
21 SS
21 SS
21 SS
21 SS
21 SS
28 S
28 S
28 S
28 S
28 S
28 S
28 S
28 SS
28 SS
28 SS
28 SS
28 SS
28 SS

oooooooog
mmmmmmmm

21
28

10
14
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

172

Tukey
T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

Tukey ‘

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0465
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

<.0001

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

<.0001

<.0001
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmmwmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm

\)\l\)\)\l\)\l\l\l\)\)h-¥>J§JBA&A&J§h-h-bOOOOO
(I)
m

10 S

10 SS
10 SS
10 SS
10 SS
10 SS
10 SS
10 SS
14 S

14 S

14 SS
14 SS
14 SS
14 SS
14 SS
14 SS

7

10
14
21
28

10
14
21
28

10
14
21
28
10
14
21
28

10
14
21
28
14
21
28

10
14
21
28
21
28

10
14
21

173

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

1.0000
<.0001
<.0001
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
0.9999
0.9992
1.0000
<.0001
<.0001
<.0001
0.8771
1.0000
0.0061
0.9964
1.0000
0.9992
<.0001
<.0001
<.0001
1.0000
1.0000
0.1074

Chemical*time S 14 SS 28 Tukey 1.0000
Chemical*time S 21 S 28 Tukey 0.9953
Chemical*time S 21 SS 0 Tukey <.0001
Chemical*time S 21 SS 4 Tukey ' <.0001
Chemical*time S 21 SS 7 Tukey <.0001
Chemical*time S 21 SS 10 Tukey 1.0000
Chemical*time S 21 SS 14 Tukey 1.0000
Chemical*time S 21 SS 21 Tukey 0.1625
Chemical*time S 21 SS 28 Tukey 1.0000
Chemical*time S 28 SS 0 Tukey <.0001
Chemical*time S 28 SS 4 Tukey <.0001
Chemical*time S 28 SS 7 Tukey <.0001
Chemical*time S 28 SS 10 Tukey 0.7813
Chemical*time S 28 SS 14 Tukey 1.0000
Chemical*time S 28 SS 21 Tukey 0.0035
Chemical*time S 28 SS 28 Tukey 0.9854
Chemical*time SS 0 SS 4 Tukey 1.0000
Chemical*time SS '0 SS 7 Tukey 1.0000
Chemical*time SS 0 SS 10 Tukey <.0001
Chemical*time SS 0 SS 14 Tukey <.0001
Chemical*time SS 0 SS 21 Tukey <.0001
Chemical*time SS 0 SS 28 Tukey <.0001
Chemical*time SS 4 SS 7 Tukey 1.0000
Chemical*time SS 4 SS 10 Tukey <.0001
Chemical*time SS 4 SS 14 Tukey <.0001
Chemical*time SS 4 SS 21 Tukey <.0001
Chemical*time SS 4 SS 28 Tukey <.0001
Chemical*time SS 7 SS 10 Tukey <.0001
Chemical*time SS 7 SS 14 Tukey <.0001
Chemical*time SS 7 SS 21 Tukey <.0001
Chemical*time SS 7 SS 28 Tukey <.0001
Chemical*time SS 10 SS 14 Tukey 0.9764
Chemical*time SS 10 SS 21 Tukey 0.5983
Chemical*time SS 10 SS 28 Tukey 1.0000
Chemical*time SS 14 SS 21 Tukey 0.0171
Chemical*time SS 14 SS 28 Tukey 0.9999
Chemical*time SS 21 SS 28 Tukey 0.2235

L. Lactic acid bacteria on beef bologna during storage at 4 °C for 28 days

(Initially inoculated L. monocytogenes 105 CFU/g)

Num Den
Effect DF DF F Value Pr > F
Chemical 2 105 419.31' <.0001

174

time 6 105 705 .04 <.0001
Chemical*time 12 105 34.34 <.0001
Effect Chemical time Chemical time Adjustment Adj P
Chemical C S Tukey <.0001
Chemical C SS Tukey <.0001
Chemical S SS Tukey 0.0074
time 0 4 Tukey <.0001
time 0 7 Tukey <.0001
time 0 10 Tukey <.0001
time 0 14 Tukey <.0001
time 0 21 Tukey <.0001
time 0 28 Tukey <.0001
time 4 7 Tukey <.0001
time 4 10 Tukey <.0001
time 4 14 Tukey <.0001
time 4 21 Tukey <.0001
time 4 28 Tukey <.0001
time 7 10 Tukey 0.5696
time 7 l4 Tukey 0.4601
time 7 21 Tukey 0.6629
time 7 28 Tukey <.0001
time 10 14 Tukey 1.0000
time 10 2 1 Tukey l .0000
time 10 28 Tukey 0.0185
time 14 21 Tukey 0.9999
time 14 28 Tukey 0.0302
time 21 28 Tukey 0.0120
Chemical*time C 0 C 4 Tukey <.0001
Chemical*time C 0 C 7 Tukey <.0001
Chemical*time C 0 C 10 Tukey <.0001
Chemical*time C 0 C 14 Tukey <.0001
Chemical*time C 0 C 21 Tukey <.0001
Chemical*time C 0 C 28 Tukey <.0001
Chemical*time 'C 0 S 0 Tukey 1.0000
Chemical*time C 0 S 4 T ukey <.0001
Chemical*time C 0 S 7 Tukey <.0001
Chemical*time C 0 S 10 Tukey <.0001
Chemical*time C 0 S 14 Tukey <.0001
Chemical*time C 0 S 21 Tukey <.0001
Chemical*time C 0 S 28 Tukey <.0001
Chemical*time C 0 SS 0 Tukey . 1.0000
Chemical*time C 0 SS 4 Tukey <.0001
Chemical*time C 0 SS 7 Tukey <.0001
Chemical*time C 0 SS‘ 10 Tukey <.0001

175

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical‘time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

SSSSSSQ\IQQQQQQQQQQQQQQQQAhA-bAAAA-bA-h-bA-hA-AAAAOOO

14
21
28

10
14
21
28

10
14
21
28

10
14
21
28
10
14
21
28

10
14
21
28

10
14
21
28
14
21
28

176

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.9574
0.0007
<.0001
<.0001
<.0001
0. 1384
<.0001
<.0001
0.0021
0.0006
0.0006
0.0121
0.2953
0.9627
0.9996
0.3693
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1 .0000
l .0000
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical *time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C

10 S
10 S
10 S
10 S
10 SS
10 SS
10 SS
10 SS
10 SS
10 SS
10 SS
14 C
14 . C
14 S
14 S
14 S
14 S
14 S
14 S
14 S
14 SS
14 SS
14 SS
14 SS
14 SS
14 SS
14 SS
21 C
21 S
21 S
21 S
21 S
21 S
21 S
21 S
21 SS
21 SS
21 SS
21 SS
21 SS

21 ss’

21 SS
28 S
28 S
28 S
28 S

10
14
21
28

10
14
21
28
21
28

10
14
21
28

10
14
21
28
28

10
14
21
28

10
14
21
28
0
4
7
10

177

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.9906
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001 ‘
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001

Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time C
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S
Chemical*time S

NNNNNNNNN
OOOOOOOOOOOOOOOOOO

qqquqqqqqqqxs-nAAA-AAAcpbbooooooooooooog

14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28
10
14
21
28

10
14
21
28

178

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

, Tukey

T ukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0003
<.0001
0.0515
<.0001
<.0001
<.0001
<.0001
0.0010
1.0000
0.9691
1.0000
0.9900
<.0001
<.0001
1.0000
1.0000
1.0000
1.0000
0.9304

Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time
Chemical*time

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm

10 S
10 S
10 S
SS
SS
SS
SS
SS
SS
SS

v—ot—ot—ov—aU—tt—nr—nr—ou—th—n
A-hAOOOOOOO
mm

SS
SS
SS
SS
SS
SS
SS

NNv—nu—nv—nu—tI—or—t
-AA4>AA4>
U)

SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS

NNNNNNNNNNNNN
OOOOOOOOOOOOOOI-‘t-‘O-‘t-‘P-Av—

\l-bA-h-b-FOOOOOO

14
21
28

10
14
21
28
21
28

10
14
21
28
28

10
14
21
28

10
14
21
28

10
14
21
28

10
14
21
28
10

179

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

1.0000
1.0000
0.5952
<.0001
<.0001
0.9997
1.0000
1.0000
0.9803
0.3527
0.9993
0.1 1 16
<.0001
<.0001
0.8788
0.9758
0.9758
0.5699
0.0432
0.8788
<.0001
<.0001
1.0000
1.0000
1.0000
0.9996
0.6758
<.0001
<.0001
0.9993
0.9866
0.9866
1.0000
1.0000
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
1.0000

Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS
Chemical*time SS

7 SS
7 SS
7 SS
10 SS
10 SS
10 SS
14 SS
14 SS
21 SS

14
21
28
14
21
28
21
28
28

180

Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey
Tukey

1.0000
1.0000
0.9866
1.0000
1.0000
0.9162
1.0000
0.9162
0.9999

APPENDIX II

181

BIBLIOGRAPHY

Arnold, R. R., Russell, J. E., Champion, W. J ., Brewer, M. and Authier, J. J. 1982.
Bactericidal activity of human lactoferrin: differentiation from the stasis of iron
deprivation. Infect. Immun. 35: 792-797. '

ASTM D882. 1991. Standard Test Methods for Tensile Properties of Thin Plastic
Sheeting. American Society for Testing Materials, Philadelphia, PA.

ASTM D618. 1981. Practice for conditioning Plastics and Electrical Insulating Materials
for testing. American Society for Testing Materials, Philadelphia, PA.

ASTM D985. 1984. Standard Test Methods for Water Vapor Permeability of Packages.
American Society for Testing Materials, Philadelphia, PA.

ASTM D2578. 1984.
American Society for Testing Materials, Philadelphia, PA

Aydt, T. P., Weller, C. L. and Testin, R. F. 1991. Mechanical and barrier properties of
edible corn and wheat protein ﬁlms. Am. Soc. Agric. Eng. 34: 207-301.

Bellamy, W., Takase, M., Wakabayashi, H., Kawase, K. and Tomita, M. 1992.
Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived
from the N-terrninal region of bovine lactoferrin. J. Appl. Bacteriol. 73: 472-479.

Ben-Yehoshua, S., Shapiro, B. Y., Gutter, Y. and Barak, E. 1987. Comparative effects of
applying imazalil by dipping or by incorporation into the plastic ﬁlm on decay
control, distribution and persistence of this fungicide in shamouti oranges
individually seal-packaged. J. Plastic Film and Sheet. 3(1): 9-22.

Blackman, I. C. and Frank, J. F. 1996. Growth of Listeria monocytogenes as a bioﬁlrn on
various food-processing surface. J. Food Prot. 59: 827-831.

Brody, A. L., Strupinsky, E. R. and Kline, L. R. 2001. Active packaging for food
application: Antimicrobial packaging. Technomic Publishing Company, Inc.
Lancaster, PA. P. 131-194

Buchman, G. W., Banerjee, S. and Hansen, J. N. 1988. Structure, expression and
evolution of a gene encoding the precursor of nisin, a small protein antibiotic. J.
Biol. Chem. 263: 16260-16266.

Cagri, A., Ustunol, Z. and Ryser, E. T. 2001. Antimicrobial, mechanical, and moisture

barrier properties of low pH whey protein-based edible ﬁlms containing p-
arninobenzoic or sorbic acids. J. Food Sci. 66: 865-870.

182

 

Cagri, A., Ustunol, Z. and Ryser, E. T. 2002. Inhibition of Listeria monocytogenes,
Escherichia coli 0157:H7, and Salmonella T yphimerium DT 104 on bologna and
summer sausage slices using whey protein isolated-based edible ﬁlms containing
antimicrobials. J. Food Sci. (In press)

Center for Disease Control and Prevention. 1999. Multi-state outbreak of listeriosis.
MMWR. 49:1129-1130

Chan, W. C., Bycroft, B. W., Lian, L-Y. and Roberts, G. C. K. 1989. Isolation and
characterization of twodegradation products derived from the peptide antibiotic
nisin, FEBS Lett. 252: 29-36.

Chen, M., Yeh, G. H. and Chiang, B. 1996. Antimicrobial and physicochemical
properties of methylcellulose and chitosan ﬁlms containing a preservative. J.
Food Process. Preserv. 20: 379-390.

Chichester, D. F. and tanner, f. W. 1972. Antimicrobial food additives. Handbook of food
additives. T.E. Furia (Ed.) CRC Press. Cleveland, OH

Cox, L. J ., Kleiss, T., Cordier, J. L., Cordellana, C., Konkel, P., Pedrazzini, C., Beumer,
R. and Siebenga, A. 1989. Listeria spp. in food processing, non-food and
domestic environments. Food Microbiol. 6: 49-61.

Cowles, ‘P. B. 1941. The germicidal action of the hydrogen ion and of the lower fatty
acids. Yale J. Biol. Med. 13: 571-578.

Cunningham, F. E. 1979. Shelf-life and quality characteristics of poultry parts dipped in
potassium sorbate. J. Food Sci. 44: 863.

Delves-Broughton, J. 1990. Nisin and its uses as a food preservative. Food Technol. 44:
100-1 17.

Driessen, A. J. M., Van den Hooven, H. W., Kuiper, W., Van de Kamp, M., Sahl, H. G.,
Konnings, R. N. H. and Konings, w. N. 1995. Mechanistic studies of lantibiotic-
induced perrneabilization of phospholipid vesicles. Biochemistry. 34: 1606-1614. ‘

Eklund, T. 1980. Inhibition of growth and uptake processes in bacterial by some chemical
food preservatives. J. Appl. Bacteriol. 48: 423-427.

El-Shenawy, M. A. and Marth, E. H. 1988. Inhibition and inactivation of Listeria
monocytogenes by sorbic acid. J. Food Prot. 51: 842-847.

Fox, P. E., Law, J ., McSweeney, P. L. H. and Wallance, J. 1993. Biochemistry of cheese

ripening. P. 389-438. In P. F. Fox (ed.), cheese-chemistry, physics and
microbiology, 2 nd ed., vol 1. Chapman and Hall, London.

183

Freese, E., Sheu, C. W. and Galliers, E. 1973. Function of lipophilic acids as
antimicrobial food additives. Nature. 241: 321-327.

Freese, E. and Levin, B. C. 1978. Action mechanism of preservatives and antiseptics, in
Developments in Industrial Microbiology. Underkoﬂer, L. A. (Ed.)., Society of
Industrial Microbiology, Washington D. C. p. 207-218.

Furr, J. R. and Russell, A. D. 1972. Some factors inﬂuencing the activity of esters of p-
hydroxybenzoic acid against Serratia marcescens. Microbiol. 12: 153-159.

Gao, F. H., Abee, T. and Konings, W. N. 1991. Mechanism of action of the peptide
antibiotic nisin in liposomes and cytochrome c oxidase-containing
proteoliposomes. Appl. Environ. Microbiol. 57: 2164-2170.

Gennadios, A. and Weller, C. L. 1991. Edible ﬁlms and coatings from soy milk and soy
protein. Cereal Foods World. 36: 1004-1007.

Gianakopoulos, A. and Guilbert, S. 1986. Determination of sorbic acid diffusivity in
model food gels. J. Food Technol. 21:399.

Gitter, M., Bradley, R. and Blampied, P. H. 1980. Listeria monocytogenes infection in
bovine mastitis. Vet. Rec. 10: 390-393.

Gross, E. and Morell, J. L. 1971. The structure of nisin. J. Am. Chem. Soc. 93: 4634-
4635.

Gross, E. and Morell, J. L. 1967. The presence of dehydroalanine in the antibiotic nisin
and its relationship to activity. J. Am. Chem. Soc. 89: 2791-2792.

Guilbert, S. 1986. Technology and application of edible protective ﬁlms. In Food
Packaging and Preservation-Theory and Practice. M. mathlouthi, (Ed), Elsevier
Applied Science Publisher. New York, NY. P. 371-394.

Han, J. H. 1996. Modeling the inhibition kinetics and the mass transfer of controlled
releasing potassium sorbate to develop an antimicrobial polymer for food
packaging. Ph.D dissertation, Purdue Univ., West Lafayette, IN

Han, J. H. and Floros, J. D. 1997. Casting antimicrobial packaging ﬁlm and measuring
their physical properties and antimicrobial activity. J. Plastic Film Sheeting. 13:
287 — 298.

Han, J. H. 2000. Antimicrobial food packaging. Food Technol. 54(3): 56-65.

Hansen, J. N. 1994. Nisin as a model food preservative. Critical Reviews in Food
Science and Nutrition. 34: 69-93.

184

Herald, P. J. and Zottola, E. A. 1988. Effect of various agents on the attachment of
Pseudomanas fragi to stainless steel. J. Food Sci. 54: 461-464.

Hicks, S. J. and Lund, B. M. 1991. The survival of Listeria monocytogenes in cottage
cheese. J. Appl. Bacteriol. 70: 308-314.

Hoffman, K. L., Han, I. Y., Dawson, P. L. 2001. Antimicrobial effects of corn zein ﬁlms
impregnated with nisin, lauric acid and EDTA. J. Food Prot. 64: 885-889.

Hotchkiss, J. W. 1995. Inﬂuence of new packaging technologies on the microbial safety
of muscle foods. The annual meeting of Institute of Food Technologists,
Anaheim, CA

Hurst, A. 1981. Nisin. Adv. Appl. Microbiol. 27: 85-123.

Hutchens, T. W., Rumball, S. V. and Lonnerdall, B. 1994. Lactoferrin; structure and
function. 4 th. edition. Plenum Press, New York

J udis, J. 1963. Studies on the mechanism of action of phenolic disictants. 11. Patterns of
release of radioactivity from Escherichia coli labeled by growth on various
compounds. J. Pharm. Sci. 52: 126-130.

Jung, G. 1991 . Lantibiotics-ribosomaly synthesized biologically active polypeptides
containing sulphide bridges and or, B-didehydro amino acids. Angew. Chem. Int.
Ed. Engly. 30: 1051-1068.

Kester, j. J. and Fennema, O. R. 1986. Edible ﬁlms and coatings: A review. Food
Technol. 40: 47-59.

K0, 8., lanes, M.E., Hettiarachchy, N. S., Johnson, M. G. 2001. Physical and chemical
pr0perties of edible ﬁlms containing nisin and their action against Listeria
monocytogenes. J. Food Sci. 66: 1006-1011.

Liewen, M. B. and Marth, E. H. 1985. Growth and inhibition of microorganisms in the
presence of sorbic acid: A review. J. Food Prot. 48: 364-375.

Luck, E. 1990. Application of sorbic acid and its salt. Food Additives and Contaminants.
7: 711-715.

Lueck, E. 1980. Antimicrobial food additives. New York: Springer-Verlag. p. 183-199.
Mafu, A. A., Roy, D., Goulet, J. and Magney, P. 1990. Attachment of Listeria
monocytogenes to stainless steel, glass, polypropylene, and rubber surfaces after

short contact times. J. Food Prot. 53: 742-746.

Marth, E. H. 1988. Disease characteristics of Listeria monocytogenes. Food Technol.

185

165-168

Matamoros, L. B., Argaiz, A. and Lopez-malo, A. 1999. Individual and combined effects
of vanillin and potassium sorbate on Penicillium digitatum, Penicillium glabrum
and Penicillium italium growth. J. Food Prot. 62: 540-542.

McDade, C. R., Zutara, S. M., Ryser, E. T., Donnelly, C. W. and Chen, H. 1999. Use of
whey-based edible ﬁlm containing antimicrobial agents to inhibit Listeria
monocytogenes in frankfurters. Program & Abstract book. T10. Annual Meeting
of International Association for Food Protection; 1999. Dearborn, MI.

McHugh, T. H., Aujard, J. F ., Krochta, J. M. 1994. Plasticized whey protein edible ﬁlms:
water vapor permeability properties. J. Food Sci. 59: 416-419, 423.

McSweeney, P. L. H., Walsh, E. M., Fox, P. F., Cogan, T. M., Driman, F. D. and
Castelo-Gonzalez, M. 1994. A procedure for the manufacture of Cheddar cheese

under controlled bacteriological conditions and the effect of adjunct lactobacilli
on cheese quality. Ir. J. Agric. Food Res. 33: 183-192.

Oram, J. D. and Reiter, B. 1968. Inhibition of bacteria by lactoferrin and other iron
Chelating agents, Biochem. Biophys. Acta. 170: 351-365.

Padgett, T., Han, I. Y. and Dawson, P. L. 1998. Incorporation of food-grade antimicrobial
compounds into biodegradable packaging ﬁlms. J. Food Prot. 61: 1330 — 1335.

Payne, K. D., Rico-Munoz, E. and Davidson, P. M. 1989. The antimicrobial activity of
phenolic compounds against Listeria monocytogenes and their effectiveness in a
model milk system. J. Food Prot. 52: 151-156.

Payne, K. D., Davidson, P. M., Oliver, S. P. and Christen, G. L. 1990. Inﬂuence of
bovine lactoferrin on the growth of Listeria monocytogenes. J. Food Prot. 53:
468-471.

Peterson, 8. D. and Marshall, R. T. 1990. Non-starter lactobacilli in Cheddar cheese: a
review. J. Dairy Sci. 73: 1395-1410.

Piccin, D. M. and Shelef, L. A. 1995. Survival of Listeria monocytogenes in cottage
cheese. J. Food Prot. 58: 128-131.

Reiter, B. 1978. Review of the progress of dairy science: Antimicrobial systems in milk.
J. Dairy Res. 45: 131-140.

Rice, J. 1995. Antimicrobial polymer food packaging. Food Proc. 56(4): 56-58.

Rico-Pena, D. C. and Torres, J. A. 1991. Sorbic acid and potassium sorbate permeability

186

of an edible methylcellulose-palmitic acid ﬁlm: water activity and pH effects. J.
Food Sci. 56: 497-499.

Robach, M. C. and Ivey, F. J. 1978. Antimicrobial efﬁcacy of potassium dip on freshly
processed poultry. J. Food Prot. 45: 374.

Rollema, H. S., Metzger, J. W., Both, P., kuipers, O. P. and Siezen, R. J. 1996. Structure
and biological activity of chemically modiﬁed nisin A species. Eur. J. Biochem.
241: 716-722.

Roth, T. and Loncin, M. 1985. Fundamentals of diffusion of water and rate of approach
to equilibrium aw. In Properties of Water in Foods in Relation to Quality and
Stability, D. Simatos and J. L. Multon ( Ed.), p.335. Martinus Nijhoff Publishers,
Dordrech, Netherland.

Ryan, M. P., Rea, M. G, Hill, C. and Ross, R. P. 1996. An application in Cheddar cheese
manufacture for a strain of Lactococcus lactis producing a novel broad-spectrurn
bacteriocin, lacticin 3147. Appl. Environ. Microbiol. 62: 612-619.

Ryser E. T., Marth, E. H. and Doyle, M. P. 1985 . Survival of Listeria monocytogenes
during manufacture and storage of cottage cheese. J. Food Prot. 48: 746-750.

Ryser E. T. and Marth, E. H. 1987. Behavior of Listeria monocytogenes during the
manufacture and ripening of Cheddar cheese. J. Food Prot. 50: 7-13.

Ryser E. T. and Marth, E. H. 1988. Survival of Listeria monocytogenes in cold-pack
cheese food during refrigerated storage. J. Food Prot. 51: 615-621.

Sahl, H. G., Jack, R. W. and Bierbaum, G. 1995. Lantibiotics: biosynthesis and biological
activities of peptides with unique post-translation modiﬁcations. Eur. J. Biochem.
230: 827-853.

Salmond, C. V., Kroll, R. G. and Booth, 1. R. 1984. The effect of food preservatives on
pH homoestosis in Esherichia coli. J. Gen. Microbiol. 130: 2845-2849.

Schlech, W. F ., Lavigne, p. M., Bortolussi, R. A., Allen, A. C., Haldane, E. V.,
hightower, A. W., Johnson, S. E., King, S. H., Nicholls, E. S. and Broome, C. V.
1983. Epidemic listeriosis-evidence for transmission by food. New Eng. J. Med.
308: 203. '

Schlyter, J. H., Degnan, A.J., Loeffelholz, J ., Glass, K.A. and Lunchansky, J .B. 1993.
Evaluation of sodium diacetate and ALTA 2341 on viability of Listeria
monocytogenes in turkey slurries. J. Food Prot. 56: 808—810.

Seeliger, H. P. R. and Finger, H. 1961. Listeriosis Haﬁrer Pub. Co., New York.

187

Shefet, S. M., Sheldon, B. W. and Klaenharnmer, T. R. 1995. Efﬁcacy of optimized
nisin-based treatments to inhibit Salmonella typhimurium and extend shelf life of
broiler carcasses. J. Food Prot. 58: 1077-1082.

Shelef, LA. and Addala, L. 1994. Inhibition of Listeria monocytogenes and other
bacteria by sodium diacetate. J. Food Saf. 14: 103-115

Shibasaki, I. 1969. Antimicrobial activity of alkyl esters of p-hydroxybenzoic acid. J.
Ferment. Technol. 47: 167-177.

Sofos, J. N. and Busta, F. F. 1981. Antimicrobial activity of sorbate. J. Food Prot. 44:
614-622.

Sperber, W. H. 1983. Inﬂuence of water activity on foodborne bacteria-a review. J. Food
Prot. 46: 142-150.

Stevens, K. A., Sheldon, B. W., Klapes, N. A. and Klaenharnmer, T. R. 1991. Nisin
treatment for inactivation of Salmonella species and other gram-negative bacteria.
Appl. Environ. Microbiol. 57: 3613-3615.

Swearingen, P. A, O’ Sullivan, D. J. and Warthesen, J. J. 2001. Isolation,
characterization, and inﬂuence of native, nonstarter lactic acid bacteria on
Cheddar cheese quality. J. Dairy Sci. 84: 50-59.

Somers, e. B., Johnson, M. E. and Wong, A. C. L. 2001. Bioﬁlrn formation and
contamination of cheese by nonstarter lactic acid bacteria in the dairy
environment. 84: 1926-1 936.

Torres, J. A., Motoki, M. and Karel, M. 1985. Microbial stabilization of intermediate
moisture food surfaces. 1. Control of surface preservative concentration. J. Food
Proc. Preserv. 9: 75-92.

Torres, J. A. and Karel, M. 1985. Microbial stabilization of intermediate moisture food
surfaces. 111. Effect of surface preservative concentration and surface pH control
on microbial stability of an intermediate moisture cheese analog. J. Food Proc.
Preserv. 9: 75.

Troller, J. A. and Christian, J. H. B. 1978. Control of water activity and moisture. In
Water Activity and Food, Ch. 9, p. 187. Academic Press, New York.

Tuncan, E. U. and Martin, S. E. 1985. Effect of pH, temperature of potassium sorbate on
amino acid uptake in Salmonella T yphimurium 7136. Appl Environ. Microbiol.
49: 505-508.

USDA-FSIS. 1999-2000. Recall Information Center.

188

USDA-FSIS. 2001. Recall Information Center.

Vas, K. 1953. Mechanism of antimicrobial action. Interference with the cytoplasmic
membrane.

Vojdani, F. and Torres, J. A. 19893. Potassium sorbate permeability of polysaccharide
ﬁlms: chitosan, methylcellulose and hydroxypropyl methylcellulose. J. Food
Proc. Eng. 12:33-48.

Vojdani, F. and Torres, J. A. 1989b. Potassium sorbate permeability of edible cellulose
ether multi-layer ﬁlms J. Food Proc. Preserv. 13: 417-430.

Wakabayashi, H., bellamy, W., Takase, M. and Tomita, M. 1992. Inactivation of
Listeria monocytogenes by lactoferrin, a potent antimicrobial peptide derived
from cow’s milk. J. Food Prot. 55: 238-240

Wells, J. M., Liao, C. H. and Hotchkiss, A. t. 1998. In vitro inhibition of soft-rotting
bacteria by EDTA and nisin and in vivo response on inoculated fresh cut carrots.
Plant Dis. 82: 491-495.

Weng, Y.M. and Hotchkiss, J. H. 1992. Inhibition of surface molds on cheese by
polyethylene ﬁlm containing the antimycotic imazalil. J. Food Prot. 55: 367—369.

Weng, Y.M. and Hotchkiss, J. H. 1993. Anhydrides as antimycotic agents added to
polyethylene ﬁlm for food packaging. Packaging Technol. Sci. 6: 123-128.

Zamora, M. C. and Zaritzky, N. E. 1987a. Potassium sorbate inhibition of
microorganisms growing on reﬁigerated packaged beef. J. Food Sci. 52: 257.

Zamora, M. C. and Zaritzky, N. E. 1987b. Antimicrobial activity of undissociated sorbic
acid in vacuum packaged beef. J. Food Sci. 52: 1449.
Ziney, M. G. and Debevere, J. M. 1998. The effect of Reuterin on Listeria

monocytogenes and Escherichia coli 0157:H7 in milk and cottage cheese. J. Food
Prot. 61 : 1275-1280.

189

 

69479