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Evaluation of the General Antimicrobial and Antilisterial Properties of Nisin, Rosemary and EDTA on Ready-To-Eat Turkey ...

Permanent Link: http://ufdc.ufl.edu/UFE0021646/00001

Material Information

Title: Evaluation of the General Antimicrobial and Antilisterial Properties of Nisin, Rosemary and EDTA on Ready-To-Eat Turkey Ham Inoculated with Listeria monocytogenes and Stored at 4 Degrees Celsius
Physical Description: 1 online resource (121 p.)
Language: english
Creator: Ruiz Menjivar, Alba Yesenia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: antimicrobials, edta, listeria, nisin, rosemary
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Contamination of ready-to-eat (RTE) meat and poultry products with Listeria monocytogenes is a major concern for the meat processing industry and an important food safety issue. This study was divided in four separate phases, in which antimicrobial solutions were evaluated against a five-strain inoculum of L. monocytogenes. In phase one, the effectiveness of different antimicrobial solutions and their combinations were evaluated by Kirby-Bauer disc diffusion method. During phase two, the anti-Listeria and general antimicrobial properties of 0.2% nisin alone and in combination with 1% rosemary and/or 20 mM EDTA on ready-to-eat vacuum packaged turkey ham were determined. In phase three, nisin at different concentrations (0.2%, 0.3%, 0.4% and 0.5%) was evaluated on ready-to-eat vacuum packaged turkey ham. And during phase four, the antimicrobial properties of 0.5% nisin, 1% rosemary, and 20 mM EDTA were evaluated separately and in combination on ready-to-eat vacuum packaged turkey ham. The methodology used during phase one involved placing two paper-filter discs (6 mm diameter) impregnated with the corresponding antimicrobial solution on plates inoculated with L. monocytogenes. Sterile water was used as a control treatment. The plates were incubated at 35 ?C for 24 h and were observed for zones of L. monocytogenes growth inhibition. For Phases two, three and four the antimicrobial solutions were applied to the turkey ham inoculated with L. monocytogenes followed by mixing the ham and solution to ensure a proper distribution between them. The samples were then vacuum packaged, leaving the antimicrobial solution in the package. Samples were then stored at 4 ? 1?C for 28 days for Phase two and 63 days for Phases three and four. Microbiological, chemical and color analyses were conducted for all samples at one week intervals. Results from phase one indicated that nisin had a strong antibacterial activity against L. monocytogenes, yielding significantly (P < 0.05) larger inhibition zone when used alone and in combination with 1% rosemary and 20 mM EDTA. L. monocytogenes zone of inhibition increased as its concentration increased from 0.1% to 0.5%. L. monocytogenes growth was not inhibited by the treatments containing 1% and 2% of vinegar, 3% and 5% of potassium benzoate, 1% thymol, 0.15% and 0.25% sodium diacetate alone and in combination with nisin. During phase two, the antimicrobials that exhibited a greater inhibition against L. monocytogenes were further analyzed in a meat matrix. On day 0, treatments with nisin, nisin combined with rosemary, and nisin combined with rosemary and EDTA significantly (P < 0.05) reduced the population of L. monocytogenes by 3.73, 2.33, and 3.12 log CFU/g as compared to the positive control, respectively. EDTA did not inhibit the growth of L. monocytogenes throughout 28 days storage. Results obtained from Phase three demonstrated an extended lag phase of L. monocytogenes when treated with 0.5% nisin. The counts remained less than 1.95 log CFU/g for 0.5% nisin throughout 63 days. The overall mean values for all treatments revealed that nisin resulted in 1.62?3.18 log CFU/g reduction of L. monocytogenes. The results suggested that the antimicrobial effectiveness of nisin increased as its concentration increased from 0.2% to 0.5%. Results from Phase four showed that initially, treatments with nisin, nisin with rosemary, nisin with EDTA and nisin with rosemary and EDTA significantly (P < 0.05) reduced the population of L. monocytogenes by 4.42, 4.20, 3.73, and 4.11 log CFU/g as compared to the positive control, respectively. L. monocytogenes counts remained less than 2.66 log CFU/g for treatments containing nisin during the study. EDTA and rosemary alone and in combination did not inhibit the growth of L. monocytogenes throughout the 63 days. These results indicated that the observed population reductions may be attributed to the inhibitory activity of nisin rather than EDTA or rosemary. Although none of the treatments used in this study completely eliminated L. monocytogenes, the overall results indicated that ready-to-eat turkey ham will have significantly decreased numbers of L. monocytogenes when treated with nisin alone or in combination with rosemary and/or EDTA. The data suggested that nisin will function to enhance the microbial safety of ready-to-eat poultry, as well as other meat products.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Alba Yesenia Ruiz Menjivar.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Williams, Sally K.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021646:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021646/00001

Material Information

Title: Evaluation of the General Antimicrobial and Antilisterial Properties of Nisin, Rosemary and EDTA on Ready-To-Eat Turkey Ham Inoculated with Listeria monocytogenes and Stored at 4 Degrees Celsius
Physical Description: 1 online resource (121 p.)
Language: english
Creator: Ruiz Menjivar, Alba Yesenia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: antimicrobials, edta, listeria, nisin, rosemary
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Contamination of ready-to-eat (RTE) meat and poultry products with Listeria monocytogenes is a major concern for the meat processing industry and an important food safety issue. This study was divided in four separate phases, in which antimicrobial solutions were evaluated against a five-strain inoculum of L. monocytogenes. In phase one, the effectiveness of different antimicrobial solutions and their combinations were evaluated by Kirby-Bauer disc diffusion method. During phase two, the anti-Listeria and general antimicrobial properties of 0.2% nisin alone and in combination with 1% rosemary and/or 20 mM EDTA on ready-to-eat vacuum packaged turkey ham were determined. In phase three, nisin at different concentrations (0.2%, 0.3%, 0.4% and 0.5%) was evaluated on ready-to-eat vacuum packaged turkey ham. And during phase four, the antimicrobial properties of 0.5% nisin, 1% rosemary, and 20 mM EDTA were evaluated separately and in combination on ready-to-eat vacuum packaged turkey ham. The methodology used during phase one involved placing two paper-filter discs (6 mm diameter) impregnated with the corresponding antimicrobial solution on plates inoculated with L. monocytogenes. Sterile water was used as a control treatment. The plates were incubated at 35 ?C for 24 h and were observed for zones of L. monocytogenes growth inhibition. For Phases two, three and four the antimicrobial solutions were applied to the turkey ham inoculated with L. monocytogenes followed by mixing the ham and solution to ensure a proper distribution between them. The samples were then vacuum packaged, leaving the antimicrobial solution in the package. Samples were then stored at 4 ? 1?C for 28 days for Phase two and 63 days for Phases three and four. Microbiological, chemical and color analyses were conducted for all samples at one week intervals. Results from phase one indicated that nisin had a strong antibacterial activity against L. monocytogenes, yielding significantly (P < 0.05) larger inhibition zone when used alone and in combination with 1% rosemary and 20 mM EDTA. L. monocytogenes zone of inhibition increased as its concentration increased from 0.1% to 0.5%. L. monocytogenes growth was not inhibited by the treatments containing 1% and 2% of vinegar, 3% and 5% of potassium benzoate, 1% thymol, 0.15% and 0.25% sodium diacetate alone and in combination with nisin. During phase two, the antimicrobials that exhibited a greater inhibition against L. monocytogenes were further analyzed in a meat matrix. On day 0, treatments with nisin, nisin combined with rosemary, and nisin combined with rosemary and EDTA significantly (P < 0.05) reduced the population of L. monocytogenes by 3.73, 2.33, and 3.12 log CFU/g as compared to the positive control, respectively. EDTA did not inhibit the growth of L. monocytogenes throughout 28 days storage. Results obtained from Phase three demonstrated an extended lag phase of L. monocytogenes when treated with 0.5% nisin. The counts remained less than 1.95 log CFU/g for 0.5% nisin throughout 63 days. The overall mean values for all treatments revealed that nisin resulted in 1.62?3.18 log CFU/g reduction of L. monocytogenes. The results suggested that the antimicrobial effectiveness of nisin increased as its concentration increased from 0.2% to 0.5%. Results from Phase four showed that initially, treatments with nisin, nisin with rosemary, nisin with EDTA and nisin with rosemary and EDTA significantly (P < 0.05) reduced the population of L. monocytogenes by 4.42, 4.20, 3.73, and 4.11 log CFU/g as compared to the positive control, respectively. L. monocytogenes counts remained less than 2.66 log CFU/g for treatments containing nisin during the study. EDTA and rosemary alone and in combination did not inhibit the growth of L. monocytogenes throughout the 63 days. These results indicated that the observed population reductions may be attributed to the inhibitory activity of nisin rather than EDTA or rosemary. Although none of the treatments used in this study completely eliminated L. monocytogenes, the overall results indicated that ready-to-eat turkey ham will have significantly decreased numbers of L. monocytogenes when treated with nisin alone or in combination with rosemary and/or EDTA. The data suggested that nisin will function to enhance the microbial safety of ready-to-eat poultry, as well as other meat products.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Alba Yesenia Ruiz Menjivar.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Williams, Sally K.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021646:00001


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EVALUATION OF THE GENERAL ANTIMICROBIAL AND ANTILISTERIAL
PROPERTIES OF NISINT, ROSEMARY AND EDTA ON READY-TO-EAT TURKEY HAM
INOCULATED WITH Listeria monocytogenes AND STORED AT 4 DEGREES CELSIUS




















By

ALBA YESENIA RUIZ MENJIVAR


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007

































O 2007 Alba Yesenia Ruiz Menjivar





























To my mother, Alba Menjivar de Ruiz. Without her support and understanding, I certainly never
would have accomplished this work.









ACKNOWLEDGMENTS

I extend my sincere appreciation and respect to my supervisory committee chair, major

advisor, and friend, Dr. Sally K. Williams, for her invaluable guidance and patience. I also wish

to thank my supervisory committee members, Dr. Gary Rodrick and Dr. Arthur Hinton, for their

help. Special thanks go to Dr. Williams and the Department of Animal Sciences for financial

support.

I would also like to thank Frank Robbins and Doris Sartain for their advice and assistance.

The friendship, constant support and everlasting help of Noufoh Dj eri and Keawin Sarj eant are

deeply appreciated. Without their company, my days here would not have been as enjoyable.

Regards go to fellow graduate student Nicolas Lavieri for his friendship and help during my

program.

I would also like to thank Juan Carlos Rodriguez for his undying support, advice and

friendship through this time. I thank my parents, Alba Menjivar de Ruiz and Jorge Ruiz for their

support, persistence and love shown to me throughout this time and all the phases of my life.

Special thanks go to Douglas Rosales for his love, patience and support. I would like to thank to

everyone whose prayers for me were evidently heard. Thanks go to God for granting me the

heart and mind to make my visions become realities.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ........._..... ...............8..____ ......


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


1 INTRODUCTION ................. ...............13.......... ......


2 LITERATURE REVIEW ................. ...............17................


Listeria monocytogenes: Description .............. ...............17....
Route of Exposure ............ ...... .__ ...............18..
Characteristics of the Disease ............ ..... ._ ...............19...
Food Associated .............. ...............20....
Control and Prevention .............. ......_ ...............21...
Bacteriocins: Definition and Classification ....._........__..... ....._._ ............2
M ode of Action ..........._...__........ ...............24.....
Isolation and Purification .............. ...............26....
Bacteriocins Resistance ........._.....__........ ...............27.....
Toxicity................... ............ ........2
Application in Meat Products ..........._...__........ ...............28....
Natural Antimi crobi al s: Descripti on ........._..._.._ ...._._. ...............33..
Mechanism of Action .............. ...............34....

Application in the Food Industry ........._..._... .... ... ....._._ ...............34...
Characteri sti cs of EDTA (Ethyl enedi aminetetraaceti c Aci d) ................. ......................3 5
Antimicrobial Activity of EDTA. ........._..._.._ ...._._. ...._._. ...........3

3 EVALUATION OF NATURAL ANTIMICROBIAL COMPOUNDS AGAINST
Listeria monocytogenes BY KIRBY-BAUER DISC DIFFUSION METHOD .....................37

Introducti on ................. ...............37.................
Materials and Methods .............. ...............38....
Inoculum Preparation ................ ...............38...
Antimicrobial Solution Preparation............... ............ ........3
Preparation of Modified Oxford Media Agar (MOX) ........................... ...............41
Inoculation of Agar Plates ........._.___..... .___ ...............41....
Kirby-Bauer Disc Diffusion Test .............. ...............42....
Data Analysis............... ...............42
Results and Discussion .............. ...............43....












4 EVALUATION OF CONCENTRATIONS OF NISIN AGAINST Listeria
monocytogenes ON READY-TO-EAT TURKEY HAM STORED AT 4~11C FOR 63
D A Y S .............. ...............50....


Introducti on ........._.__...... ..__ ...............50....
M materials and M ethods ............... ...............51....
Inoculum Cultivation and Storage ........._._._ ...._. ...............51...
Inoculum Preparation .................. ...............52..
Antimicrobial Solutions Preparation ........._..._.._ ......._._. .........52.._.....
Sample preparation ........._..._.._ ...._._. ...............53.....
Inoculation and Treatment............... ...............5
M icrobiological Analyses.................. ..............5
Listeria monocytogenes and lactic acid bacteria analysis .............. ....................54
Aerobic bacteria analysis .............. ...............55....
pH Analysis .............. ...............55....
Data Analysis............... ...............55
Results and Discussion .............. ...............56....

pH Analysis .............. ...... .. ...............5
Listeria monocytogenes Analysis ............ .....__ ...............56..
Lactic Acid Bacteria Analysis ............ ..... ._ ...............57...
Aerobic Bacteria Analysis ............ ..... ._ ...............58...


5 EVALUATION OF THE ANTI-LISTERIAL PROPERTIES OF NISIN, ROSEMARY
AND EDTA ON READY-TO-EAT TURKEY HAM STORED AT 4~11C FOR 28
D A Y S .............. ...............64....


Introducti on ........._._ ...... .____ ...............64.....
M materials and M ethods ............... ...............66....
Inoculum Cultivation and Storage ........._._._ ....__. ...............66...
Inoculum Preparation .................. ...............66..
Antimicrobial Solutions Preparation ........._..._.._ ......._._. .........67.._.....
Sample Preparation............... ..............6
Inoculation and Treatment............... ...............6
M icrobiological Analyses............... .. .. .. ... .. .................6
Listeria monocytogenes, lactic acid bacteria and anaerobic bacteria analyses ........69
Aerobic bacteria analysis .............. ...............70....
pH Analy si s .............. ...............70....
Data Analysis............... ...............71
Results and Discussion .............. ...............71....

pH Analysis .............. ...... .. ...............7
Listeria monocytogenes Analysis ............ .....__ ...............72..
Anaerobic Bacteria Analysis .............. ...............73....
Lactic Acid Bacteria Analysi s............ ..... ._ ...............74..
Aerobic Bacteria Analysi s............ ..... ._ ...............75...












6 EVALUATION OF THE ANTI-LISTERIAL PROPERTIES OF 0.5 % NISINT, 1 %
ROSEMARY AND 20 mM EDTA ON READY-TO-EAT TURKEY HAM STORED
AT 4+10C FOR 63 DAY S ........._.___..... .___ ...............82..


Introducti on ........._.__...... ..__ ...............82....
M materials and M ethods ............... ...............84....
Inoculum Cultivation and Storage ........._._._ ...._. ...............84...
Inoculum Preparation .................. ...............85..
Antimicrobial Solutions Preparation ........._..._.._ ......._._. .........85.._.....
Sample Preparation............... ..............8
Inoculation and Treatment............... ...............8
Microbiological Analyses............... ...............87
Aerobic bacteria analysis .............. ......... .. ..............8
Listeria monocytogenes and lactic acid bacteria analysis .............. ....................88
pH Analy si s .............. ...............8 8....
Col or Analy si s ............ ..... ._ .............. 8 9....
Data Analysis............... ...............89
Results and Discussion .............. ...............89....
Aerobic Bacteria Analysis ............ ..... ._ ...............89...
Listeria monocytogenes Analysis ............ .....__ ...............90..
Lactic Acid Bacteria Analysis ............ ..... ._ ...............93...
pH A analysis .............. ...... .. .... .... .... ..... .........9
Analysis of Obj ective Color Measurement for L~a~b Values .............. ............. ..94

7 SUMMARY AND CONCLUSION ..........._.._ ....._. ....._._ ..........10


LIST OF REFERENCE S .............._ ....._. ...............110...


BIOGRAPHICAL SKETCH ..........._.._ ....._. ...............121...










LIST OF TABLES


Table page

3-1 Fifty-eight antimicrobial solutions evaluated in Phase One .............. .....................4

3-2 Seventeen antimicrobial solutions evaluated in Phase Two .............. ....................4

3-3 Mean zone of inhibition for antimicrobial solutions evaluated in Phase One ................ ..47

3-4 Mean zone of inhibition for antimicrobial solutions evaluated in Phase Two .................49

4-1 Formulation of nisin solutions for vacuum packaged ready-to-eat turkey ham stored
at 411 OC for 63 days .............. ...............59....

4-2 pH measurements on Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 4 & loC for 63 days ......___ .... ... ._ ........___.....6

4-3 Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and stored at 4 & loC for 63 days............... ...............61..

4-4 Lactic acid bacteria counts on Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 4 & loC for 63 days ......___ .... ... ._ ........___.....6

4-5 Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and analyzed prior to storage ......___ ........__ ................63

5-1 Formulation of nisin, rosemary and EDTA solutions for vacuum packaged ready-to-
eat turkey ham stored at 411 OC for 28 days............... ...............76..

5-2 Mean pH values on Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 411 OC for 28 days ......___ .... ... .___ ......._._. .....7

5-3 Mean Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and stored at 411 OC for 28 days............... ...............78..

5-4 Mean anaerobic bacteria counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and stored at 411 OC for 28 days............... ...............79..

5-5 Mean lactic acid bacteria counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and stored at 411 OC for 28 days............... ...............80..

5-6 Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes .............. ...............8 1....

6-1 Formulation of nisin, rosemary and EDTA solutions for vacuum packaged ready-to-
eat turkey ham stored at 411 OC for 63 days............... ...............96..










6-2 Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and analyzed prior to storage ......___ ........__ ................97

6-3 Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and stored at 4 & loC for 63 days............... ..................9

6-4 Lactic acid bacteria counts on Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 4 & loC for 63 days ......___ ....... .__ ........___.....9

6-5 pH measurements on Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 4 & loC for 63 days ......___ ....... .__ ........__......0

6-6 Mean for "L" values of Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 4 & loC for 63 days ......___ ....... .__ ........__......0

6-7 Mean for "a" values of Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 4 & loC for 63 days ......___ ....... .__ ........__......0

6-8 Mean for "b" values of Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 4 & loC for 63 days ......___ ....... .__ ........__......0









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EVALUATION OF THE GENERAL ANTIMICROBIAL AND ANTI-LISTERIAL
PROPERTIES OF NISINT, ROSEMARY AND EDTA ON READY-TO-EAT TURKEY HAM
INOCULATED WITH Listeria monocytogenes AND STORED AT 4 DEGREES CELSIUS

By

Alba Yesenia Ruiz Menjivar

December 2007

Chair: Sally K. Williams
Major: Animal Sciences

Contamination of ready-to-eat (RTE) meat and poultry products with Listeria

monocytogenes is a maj or concern for the meat processing industry and an important food safety

issue. This study was divided in four separate phases, in which antimicrobial solutions were

evaluated against a five-strain inoculum ofL. monocytogenes. In phase one, the effectiveness of

different antimicrobial solutions and their combinations were evaluated by Kirby-Bauer disc

diffusion method. During phase two, the anti-Listeria and general antimicrobial properties of

0.2% nisin alone and in combination with 1% rosemary and/or 20 mM EDTA on ready-to-eat

vacuum packaged turkey ham were determined. In phase three, nisin at different concentrations

(0.2%, 0.3%, 0.4% and 0.5%) was evaluated on ready-to-eat vacuum packaged turkey ham. And

during phase four, the antimicrobial properties of 0.5% nisin, 1% rosemary, and 20 mM EDTA

were evaluated separately and in combination on ready-to-eat vacuum packaged turkey ham.

The methodology used during phase one involved placing two paper-filter discs (6 mm

diameter) impregnated with the corresponding antimicrobial solution on plates inoculated with L.

monocytogenes. Sterile water was used as a control treatment. The plates were incubated at 35

oC for 24 h and were observed for zones ofL. monocytogenes growth inhibition. For Phases










two, three and four the antimicrobial solutions were applied to the turkey ham inoculated with L.

monocytogenes followed by mixing the ham and solution to ensure a proper distribution between

them. The samples were then vacuum packaged, leaving the antimicrobial solution in the

package. Samples were then stored at 4 + loC for 28 days for Phase two and 63 days for Phases

three and four. Microbiological, chemical and color analyses were conducted for all samples at

one week intervals.

Results from phase one indicated that nisin had a strong antibacterial activity against L.

monocytogenes, yielding significantly (P < 0.05) larger inhibition zone when used alone and in

combination with 1% rosemary and 20 mM EDTA. L. monocytogenes zone of inhibition

increased as its concentration increased from 0.1% to 0.5%. L. monocytogenes growth was not

inhibited by the treatments containing 1% and 2% of vinegar, 3% and 5% of potassium benzoate,

1% thymol, 0.15% and 0.25% sodium diacetate alone and in combination with nisin. During

phase two, the antimicrobials that exhibited a greater inhibition against L. monocytogenes were

further analyzed in a meat matrix. On day 0, treatments with nisin, nisin combined with

rosemary, and nisin combined with rosemary and EDTA significantly (P < 0.05) reduced the

population of L. monocytogenes by 3.73, 2.33, and 3.12 log CFU/g as compared to the positive

control, respectively. EDTA did not inhibit the growth of L. monocytogenes throughout 28 days

storage. Results obtained from Phase three demonstrated an extended lag phase of L.

monocytogenes when treated with 0.5% nisin. The counts remained less than 1.95 log CFU/g for

0.5% nisin throughout 63 days. The overall mean values for all treatments revealed that nisin

resulted in 1.62-3.18 log CFU/g reduction of L. monocytogenes. The results suggested that the

antimicrobial effectiveness of nisin increased as its concentration increased from 0.2% to 0.5%.

Results from Phase four showed that initially, treatments with nisin, nisin with rosemary, nisin









with EDTA and nisin with rosemary and EDTA significantly (P < 0.05) reduced the population

of L. monocytogenes by 4.42, 4.20, 3.73, and 4. 11 log CFU/g as compared to the positive

control, respectively. L. monocytogenes counts remained less than 2.66 log CFU/g for treatments

containing nisin during the study. EDTA and rosemary alone and in combination did not inhibit

the growth of L. monocytogenes throughout the 63 days. These results indicated that the

observed population reductions may be attributed to the inhibitory activity of nisin rather than

EDTA or rosemary. Although none of the treatments used in this study completely eliminated L.

monocytogenes, the overall results indicated that ready-to-eat turkey ham will have significantly

decreased numbers of L. monocytogenes when treated with nisin alone or in combination with

rosemary and/or EDTA. The data suggested that nisin will function to enhance the microbial

safety of ready-to-eat poultry, as well as other meat products.









CHAPTER 1
INTTRODUCTION

In spite of modern improvements in food production techniques, food safety is an

increasingly important public health issue (141). Food pathogens are ubiquitous and infect their

hosts at a local and global level. Consequently, newly introduced pathogens can spread rapidly

in susceptible hosts.

Listeria nzonocytogenes, the causative agent of listeriosis, is a widely distributed and

recognized foodborne pathogen (55). The ability ofL. nzonocytogenes to grow at temperatures

ranging from 0 to 450C (7), its high tolerance for salt (42), and its ability to initiate growth at a

relatively low pH (9) makes this pathogen particularly difficult to control in food. Recently,

numerous outbreaks have been linked to consumption of ready-to-eat (RTE) products

contaminated with L. nzonocytogenes (55). Contamination of the RTE meat products may occur

in processing plants (43) and currently represents one of the highest meat safety risks (18).

Hygienic and sanitation practices applied in meat processing facilities are often insufficient to

prevent contamination of processed meat products (21). Therefore new post-processing hurdle

technologies that control or eliminate the incidence of foodborne pathogens are needed for the

meat industry (10).

In-vitro antimicrobial susceptibility testing done in recent years has indicated that

bactericidal compounds such as organic acids, salts, and general recognized as safe (GRAS)

substances can control L. nzonocytogenes (93). The Kirby-Bauer method is a standardized filter-

paper disc-agar diffusion procedure (15) that is usually used for antimicrobial susceptibility

testing. It is recommended by the Clinical and Laboratory Standards Institute (NCCLS). This

method allows for the rapid determination of the efficacy of an antimicrobial by measuring the

diameter of the zone of inhibition that results from diffusion of the agent into the medium









surrounding the disc (15). Results obtained in these studies may help researchers in selecting

initial procedures and, antimicrobial agents that will be used for specific products.

In developing a novel post-processing antimicrobial treatment for use in the food industry

there are two constant problems: the limited range of bacteria which are sensitive to particular

agents and the high concentrations of agents that are require to inhibit growth (54). The rationale

for the increased effectiveness of combinations of antimicrobials is that simultaneous attack on

different targets in the bacterial cell is more difficult for the bacteria to overcome. The use of

antimicrobials with different mechanism can also be expected to expand the range of organisms

that may be inhibited.

Nisin, a lanthionine-containing polypeptide produced by Lactococcus lactis subsp. lactis

(5) is a bacteriocin with antimicrobial activity against L. monocytogenes (14). Nisin obtained a

GRAS status for use as a biopreservative in the United States food industry in 1988 (131). The

LD50ovalue was found to be similar to that of common salt (82). Nisin's mechanism of action is

based on the disruption of the cytoplasmic cell membrane, as evidenced by the rapid efflux of

small molecules from both whole cells and liposomes (2, 50, 138). As a result, nisin depletes the

proton motive force (PlVF) of sensitive cells and artificial liposomes (13, 49). Nisin acts through

a multistep process which includes binding of nisin to the cell, insertion into the membrane, and

pore formation (50, 105).

These antimicrobial properties have become a focus in the food science field and

researchers have found that nisin is not only effective against Gram positive bacteria (34) but

with the combination of a food grade chelator, such as EDTA, its effectiveness can be extended

to Gram negative bacteria (115). Ethylenenediaminetetraacetic acid (EDTA) is a well known

reagent used in designated foods for different functions. Those functions may include the









retardation of crystal formation, food preservative and stabilizer, antioxidant, and chelating and

sequestering agent (139). EDTA can have antimicrobial effect by limiting the availability of

cations and can act to destabilize the cell membrane of bacteria by completing divalent cations

which act as salt bridges between membrane macromolecules, such as lipopolysaccharides (118,

1 35) .

In addition, society appears to be experiencing a trend of "natural" consumerism (121,

129), demanding fewer synthetic food additives and products with a smaller impact on the

environment. Therefore, there is scope for new methods of making food safe which have a

natural image. The use of edible plants, as well as their phytochemicals, in food preservation

and improvement of organoleptic qualities of certain traditional foods has been practiced for

centuries. It has long been recognized that many plant essential oils have antimicrobial

properties (95) and the relatively recent interest in natural products by consumers has lead to a

renewal of scientific interest in these substances (92, 129). The rosemary (Rosmarinus

officinalis) extract has shown antimicrobial properties against food spoilage and foodborne

pathogenic microorganism and its antibacterial activity has been linked to a-pinene, bornyl

acetate, camphor, and 1,8-cineole (33, 97).

RTE meat and poultry products may be an excellent system in which to use nisin,

rosemary and EDTA combination treatments, since the presence of other growth restrictive

chemicals and conditions, such as nitrite and NaC1, may increase the effectiveness of

antimicrobial treatment against spoilage flora and pathogens (54).

Preliminary experiments were conducted to determine the inhibitory concentration in agar

media of different antimicrobials agents by Kirby-Bauer disc diffusion method. The

antimicrobials nisin, rosemary and EDTA were selected for further study to determine their










effectiveness in a meat matrix. This work was conducted to develop a new post-processing

hurdle technology by adding nisin, rosemary and EDTA alone or in combination into the final

RTE turkey product, leaving the antimicrobial solution into the package and during the product

storage.









CHAPTER 2
LITERATURE REVIEW

Listeria monocytogenes: Description

Listeria nzonocytogenes is particularly interesting as a food-borne pathogen in that it is

ubiquitous in nature. Animals and humans infected by L. nzonocytogenes suffer from the disease

known as listerosis. The current understanding of human listeriosis epidemiology suggests that

the organism is a common contaminant of food products. This contamination usually takes place

on the surface of the products, with up to 5% of food products harboring the organism (55). The

presence of L. nzonocytogenes in the food processing chain is evident by the widespread

distribution of the listeriae in processed products (55). The Centers for Disease Control and

Prevention estimates that approximately 2500 persons become seriously ill and 500 persons die

each year from listeriosis (18).

Listeria nzonocytogenes is a Gram-positive, motile facultative anaerobe bacterium that

inhabits a variety of environments. Using selective media, L. nzonocytogenes can be readily

isolated from soil, water, vegetation and processed products, including ready-to-eat products

designated for human consumption (58). The bacterium was named "nronocytogenes because

of its distinguished characteristic of infection in rabbits, which resulted in the production of

monocytosis in blood (120).

As a psychrophilic bacteria, L. nzonocytogenes grows at temperatures between 00C to 450C

(7), and enj oys a competitive advantage against other gram-positive and gram-negative

microorganisms in cold environments (e.g. refrigerators). Recent investigations indicate that the

organism can initiate growth at pH values as low as 4.4 (9). The maj ority of strains need a

minimum water activity of 0.93 for growth (73). However, some strains may be able to grow at









water activity values as low as 0.90 and survive for long periods of time at a water activity of

0.83 (113). The optimal water activity level is 0.97.

L. nzonocytogenes is able to grow in the presence of 10 to 12% sodium chloride; it grows

to high populations in moderate salt concentrations (6.5%) (42). The bacterium survives for long

periods in high salt concentrations (113). The survival in high-salt environments is significantly

increased by lowering the temperature.

Route of Exposure

Experts indicate that L. nzonocytogenes is present in foods such as raw and pasteurized

milk, cheeses, raw vegetables, raw and cooked poultry, raw meats, and raw and smoked fish.

The levels ofL. nzonocytogenes that could cause infection and eventual listeriosis vary with the

type of strain, as well as with the susceptibility of the host. Occurrence of sporadic listeriosis

appears to be more common in the spring and summer months (40). This could be explained by

seasonal variations in the type of food products eaten by humans, with higher-risk products eaten

in the warmer months. The occurrence of some outbreaks suggests that certain ready-to-eat

processed foods pose a high risk of contracting listeriosis for susceptible populations. These

foods are usually preserved by refrigeration and offer an appropriate environment for the

multiplication ofL. nzonocytogenes during manufacture, aging, transportation and storage.

The entry ofL. nzonocytogenes into food processing plants occurs through soil on clothing

or equipment, contaminated hides or surfaces, and possibly healthy human carriers. The

humidity and presence of nutrients support the growth of Listeria, which is commonly found in

moist areas such as processing equipment, drains, etc (21). In addition, Listeria can attach to

different types of surfaces in the form of biofilms which have been observed in meat and dairy

processing environments (61).









Post-processing contamination is the most likely route of contamination of processed foods

(53). Currently, there is no evidence to indicate that L. monocytogenes can survive heat

processing protocols. However, because it is a frequent contaminant of raw material used in

food processing plants, there are plenty opportunities for its reintroduction into food processing

facilities by cross contamination (36). If the product is contaminated post-processing, the

bacteria can survive and multiply throughout storage causing disease when it is consumed.

Epidemiologic investigations have repeatedly revealed that the consumption of

contaminated food is the primary mode of transmission of listeriosis. Food has been identified as

the vehicle of several maj or outbreaks of listeriosis investigated since 1981 (9).

Characteristics of the Disease

Listeriosis usually occurs in high risk groups who have a predisposition to the disease.

Contraction of listeriosis may lead to impairment of their T-cell mediated immunity.

Occasionally, individuals with no predisposing conditions can acquire the disease. Some of the

high risk groups may include pregnant women, newborn children, and immunocompromised

adults (57).

On average, there are 0.7 cases of listeriosis per 100,000 people, but reports show that the

disease is three times more prevalent in the elderly (>70) and 17 times higher in pregnant women

(46). A wide variety of clinical syndromes have been associated with L. monocytogenes in both

humans and animals. In healthy individuals, the disease can take the form of mild to substantial

flu-like symptoms, including: fever, fatigue, nausea, cramps, vomiting and diarrhea. More

severe complications can include: encephalitis, septicemia, mononucleosis-like syndrome,

pneumonia, endocarditis, aortic aneurysm, hepatitis, urethritis, rhombencephalitis, peritonitis,

liver abscess, febrile gastroenteritis, peritonitis, septic arthritis, etc (27). L. monocytogenes in










pregnant women can lead to an intrauterine infection, resulting in stillbirths and miscarriages.

Newborns can develop meningitis after birth via transplacental transmission (66).

The onset time for serious complications of listeriosis can be anywhere from a couple of

days to three weeks. Mortality of untreated infections is among the highest of all foodborne

illnesses (i.e. 70 percent). The infective dose ofL. monocytogenes is not yet known, since it is

related to many variables such as type of strain, susceptibility of the victim, and product type (9).

Food Associated

Some of the food products associated with L. monocytogenes include unpasteurized milk

and products prepared from unpasteurized milk, soft cheeses, frankfurters, delicatessen meats

and poultry products, and some seafood. Raw milk is a well-known source of L. monocytogenes.

Pasteurization effectively inactivates this organism. However, fluid milk that is contaminated

after pasteurization and stored under refrigeration may contain very high populations of L.

monocytogenes after one week. Furthermore, extreme temperature fluctuations may enhance the

multiplication of bacterial cells (9).

L. monocytogenes can also be found in cheese because of its ability to multiply at

refrigeration temperature and salt tolerance. During manufacturing process, L. monocytogenes is

primarily concentrated in the cheese curd, with only a very small portion of cells appearing in the

whey. During ripening of the cheese, the number of cells may increase (Camembert), decrease

gradually (Colby or cheddar), or decrease rapidly (blue cheese) and then stabilize (43).

L. monocytogenes has also been isolated from domestic and imported, fresh, frozen, and

processed seafood products, including crustaceans, molluscan shellfish and finfish (63). The

production of seafood products is done on a much smaller scale than meat and cheese

manufacture. Furthermore, consumption of seafood products is much less when compare to the

consumption of meats and cheeses. This may be the reason that large outbreaks have not been










reported and that case-control studies have not identified these foods as a maj or risk of listeriosis

(10 2) .

In addition, cooked and ready-to-eat meat and poultry products have been implicated as the

source of sporadic and epidemic listeriosis on several occasions in North America and Europe

(112). L. monocytogenes attaches strongly to the surface of raw meats and is difficult to remove

or inactivate. The multiplication in meat and poultry depends on the type of meat, pH, and the

type of cell populations of competitive flora (43). Therefore, the incidence ofL. monocytogenes

in ready-to-eat products has become a maj or concern for the meat processing industry.

According to the Center for Disease Control and Prevention, L. monocytogenes is a widely

recognized foodborne pathogen that is widespread in the environment and has the ability to

contaminate meat products during various phases of production, processing, manufacturing and

distribution (18). As mentioned previously, L. monocytogenes is highly resistant to

environmental conditions and has the ability to grow at high osmotic stress and low temperatures

(51). Most of listeriosis outbreaks have been linked to contaminated ready-to-eat meat and

poultry products (16). Experts believe that in order to prevent contamination of processed meat

products, good hygienic and sanitization practices are essential in meat industry (43).

Control and Prevention

Because of the high fatality rate and the uncertainty of the infective dose of L.

monocytogenes, the Food Safety and Inspection Service of the U.S. Department of Agriculture

(USDA-FSIS) has established a "zero tolerance" policy for this pathogen on ready to eat

products (78). In 2003, USDA-FSIS published an interim final rule addressing the control ofL.

monocytogenes on ready-to-eat meat products. The alternatives to control such pathogen involve

various levels of intervention and microbiological testing. Under the first alternative the

processor must use a post-lethality treatment that reduces or eliminates L. monocytogenes and









use an antimicrobial agent or process that suppresses or limits L. monocytogenes growth

throughout the product' s shelf life. The second alternative is for the processor to use either a

post-lethality treatment that reduces or eliminates L. monocytogenes or use an antimicrobial

agent or process that suppresses or limits L. monocytogenes growth throughout product shelf life.

A third alternative relies only on sanitation measures and testing to control the pathogen in the

post-lethality environment (130).

L. monocytogenes can be resistant to many food preservation processes. This pathogen can

increase significantly during refrigerated storage and reduced oxygen conditions (73). As a

result, hurdle technologies to inhibit growth of the pathogen are needed. Some studies show that

bactericidal compounds such as organic acids and bacteriocins can control L. monocytogenes in

meat products (108, 109). To date, the inclusion of chemical antimicrobials, such as lactates,

acetates and diacetates, in cured meat formulations remains the most effective hurdle against L.

monocytogenes (53). Different studies show that L. monocytogenes in processed meat products

may be controlled through the use of natural plant antimicrobials or chemically produced

antimicrobials (12), meat packaging materials with immobilized antimicrobials (94), thermal

pasteurization just before (24) or after packaging (103) and emerging technologies, such as

irradiation (123) or high pressure (75).

The use of antimicrobials with different mechanisms of killing can be expected to expand

the range of microorganisms that may be inhibited (54). By deliberately combining hurdles like

bacteriocins, low temperature and low pH, the microbial stability of the product can be improved

(69). Increased consumer demand for minimally processed foods with less chemical additives

makes the control of foodborne pathogens a more complicated challenge.









The Food and Drug Administration (FDA) and the FSIS advise all consumers to reduce the

risk of illness by using a refrigerator thermometer to make sure that the refrigerator always stays

at 40 OF or below and use perishable items that are precooked or ready-to-eat as soon as possible.

For high risk groups, the USDA-FSIS recommends avoid consuming hot dogs and luncheon

meats, unless they are reheated until steaming hot; avoid eating soft cheeses such as Feta, Brie,

and Camembert cheeses, unless it is labeled as made with pasteurized milk; avoid eating

refrigerated smoked seafood, unless it is contained in a cooked dish; and avoid consuming raw

(unpasteurized) milk or foods that contain unpasteurized milk (130).

Bacteriocins: Definition and Classification

Because food safety has become an important international concern, the application of

naturally occurring metabolites that target food pathogens without toxic or any other side effects

is an area of great interest. These natural inhibitors could replace the use of chemical

preservatives such as sulfur dioxide, benzoic acid, sorbic acid, nitrate, and others (20). Using

lysozyme and organic acids to extend the shelf life of food, has already been proven to be an

effective treatment (94). Many other natural antimicrobial systems may have potential for food

preservation in the future. Some studies have found that bacteriocins produced by lactic acid

bacteria may be very promising food preservatives (69). Bacteriocins are peptides or small

proteins produced by bacteria that kill or inhibit closely related species or even different strains

of the same species (50). They are specific in their antimicrobial action and offer potential

alternatives to replace antibiotics and chemical preservatives used in food (20). Their inhibitory

spectrum is restricted to Gram-positive bacteria, but several bacteriocins produced by lactic acid

bacteria are active against food spoilage and foodborne pathogenic microorganisms (84). In

addition, many bacteriocins are heat stable, making them applicable in combination with heat

treatment and appear to have a universal bactericidal and irreversible mode of action. These









bacterocins are generally food stable, biodegradable, digestible, safe to human health and active

at low concentrations (106).

Bacteriocins are a family of ribosomally synthesized peptide antibiotics, (84) generally

classified into three groups: Class I, Class II and Class III. However, this classification is

currently being revised due to better structural analyses and discoveries of new molecules. Class

I has been further subdivided into Class la and Class Ib. Class I bacteriocins, also called

lantibiotics, and are characterized by their unusual amino acids, such as lanthionine, methyl-

lanthionine, dehydrobutyrine and dehydroalanine (5). They constitute an uncommon family of

biological active peptides that are thought to exert their antimicrobial activity by formation of

transient pores in the bacterial cytoplasmic membrane (105). The only approved bacteriocin

currently being used by the food industry is nisin, which belongs to the lantibiotic family (133).

Class II contains small heat-stable, non-modified peptides, and can be further subdivided (90).

They do not usually contain posttranslationally modified amino acids as found in lantibiotics.

However, while lantibiotics have been found exclusively in gram positive bacteria, class II

bacteriocins have also been found in E. coli (colocin V and microcin 24) (45). Class III includes

large and heat labile bacteriocins for which there is much less information available. A fourth

class has been proposed, which consists of bacteriocins that form large complexes with other

macromolecules (67). However, these bacteriocins have not been purified, and there is a reason

to believe that this type of bacteriocin is an artifact due to the cationic and hydrophobic

properties of bacteriocins which results in completing with other macromolecules in the crude

extract (62).

Mode of Action

Bacteriocins are characterized by a strong bactericidal mode of action. Membrane

insertion, pore formation and simultaneous depolarization would induce a rapid and specific









efflux of cytoplasmic cell constituents of low molecular mass (potassium, hydrogen, amino acids

and nucleotides) from intact sensitive bacterial cells (77). The primary target of bacteriocins is

the cytoplasmic membrane. They initiate rearrangements in the membrane structure, which alter

the membrane permeability by generating channels in the cytoplasmic membrane (32). As a

result, the energy metabolism of the cell is destroyed, causing a deficiency of the important

metabolic intermediates and an immediate and simultaneous inhibition of the biosynthesis of

macromolecules such as DNA, RNA, proteins and polysaccharides (82). Bacteriocins do not

require a membrane receptor, but it is better if the membrane is energized (50). The amount of

negatively charged lipids in the membrane is likely to be a maj or criterion for the sensitivity of

the organism to bacteriocins.

Some experts suggest that besides disrupting membrane activity via pore formation,

bacterocins may have additional effects on electron transfer chain components and in the

inhibition of oxygen uptake (106). This may be explained either by a direct effect of the peptides

on the cytochrome c oxidase (4) or by the lack of ADP (82). Their bactericidal mode of action is

restricted to Gram-positive bacteria. However, Gram negative bacteria such as Escherichia coli

(106) and Salmonella tyiphimurium (125) become sensitive when the outer membrane is altered

osmoticc shock, disodium EDTA treatment). Bacteriocins also inhibit the outgrowth of bacterial

spores. It has been proposed that these peptides covalently modify the sulfhydryl groups of

proteins of the spore membrane of freshly germinated spores, and hence exert a profound

bacteriostatic effect resulting in inhibition of subsequent spore outgrowth (82).

One of the most studied bacteriocins is nisin, which belongs to the lactibiotic family (39).

It was demonstrated that in vitro, nisin inhibited bacterial cell wall biosynthesis (101). Nisin

forms pores in the cell membrane allowing the diffusion of small compounds. The increase in









membrane permeability results in the collapse of the proton motor force, which drives ATP

synthesis and the accumulation of ions and other metabolites (3). Failure of the proton motor

force leads to cell death through cessation of energy requiring reactions (32).

Isolation and Purification

The methods most frequently used for isolation, concentration, and purification of

bacteriocins usually involve salt precipitation of bacteriocins from culture supernatants, followed

by various combinations of gel filtration, ion-exchange chromatography, and reverse-phase high-

performance liquid chromatography (87). The purification methods are based on observations

that the bacteriocin molecules are (a) excreted by the producer cells; (b) cationic; (c)

hydrophobic; (d) adsorb to the cell surface of the producer cells; and (e) adsorb in a pH-

dependent manner, with high adsorption occurring at about pH 6.0 and low adsorption at about

pH 2.0 (26). The methods that have proven to simultaneously concentrate bacteriocins include

vacuum concentration, precipitation by salt fractionation, acid precipitation, organic solvent

precipitation, freeze drying and ultrafiltration (87). Although the purification procedures

mentioned here play an important role, they typically do not provide for a high degree of

resolution. Therefore, several methods of chromatography, including size exclusion (gel

filtration), cation exchange, and hydrophobic interaction, have been used to achieve purification

of bacteriocins. Because the methods used to purify bacteriocins can be complicated and time-

consuming, as well as very expensive a few scientists (25) have reported a simple method for

the purification involving adsorption of the bacteriocin onto the producer cells at pH 5.5

followed by extraction at pH 2.0. However, the recovery of bacteriocin activity by this method

did not exceed 10%.









Bacteriocins Resistance

Although most bacteriocins do not require specific targets in the membranes of sensitive

cells, when used together they can work synergistically or possess antagonism to each other (86).

Microbial resistance to bacteriocins could become an issue as their food-preservative use

becomes more widespread. Some experts indicate that in many cases, bacteriocins do not induce

cross-resistance (100). However, other reports show that microorganisms treated with certain

bacteriocins become resistant to not only the bacteriocin they were in contact with, but even to

unrelated bacteriocins never used against these cells (124). This observation again suggests that

bacteriocins should be used as part of hurdle technology, which allows them to act

synergistically with other food preservatives to prevent appearance of resistant bacterial forms.

Previously reported results indicate that nisin works synergistically with other preservatives, also

in food model system (100). Combining preservation techniques is a powerful tool for extending

shelf life, especially of minimally processed food (30). Hurdle technology permits the use of

less severe preservative levels and of techniques that are less damaging to the quality of the final

product (56). Because the spread of multidrug-resistant pathogenic bacteria has become a

serious issue, it is important to emphasize that bacteriocin resistance does not confer antibiotic

resistance to bacteria (22).

Toxicity

The fact that bacteriocins are produced by lactococci, the lactic acid bacteria that occurs

naturally in food, is an indication of their harmless nature. Humans and animals have ingested

bacteriocins over the past centuries, without apparent ill effect (20). From a regulatory

standpoint, it is critical in some countries to distinguish bacteriocins from antibiotics, since the

presence of antibiotics in food is often prohibited. The use of bacteriocins-producing starter

cultures as ingredients may not require special consideration in the United States if the culture









(microorganism) is considered Generally Recognized as Safe (GRAS). This is primarily due to

its history of safe use by food industries prior to the 1958 Food Additives Amendment (32). If a

purified bacteriocin is used as a food preservative, the substance might be self-affirmed as GRAS

by the company according to the Code of Federal Regulations (133).

In the United States, where antibiotics are prohibited in foods, nisin obtained a GRAS

status for use as a biopreservative in the food industry in 1988. The LD5o value was found to be

similar to that of common salt. Furthermore, consumption of nisin-containing products did not

result in an alteration of the intestinal bacterial flora, because nisin is inactivated by enzymes of

the intestinal tract (82). Several authors have outlined issues involved in the approval of new

bacteriocins for food use (98) and the USDA publishes guidelines for the safety assessment of

new preservative (132). For approval to be granted, the bacteriocin must be chemically

identified and characterized, and its use and efficacy must be shown. The manufacturing process

must be described and assays used for quantification and standardization of the peptide must be

shown. Toxicological data and fate of the molecule after ingestion are also needed (20).

Application in Meat Products

The increasing concern of foodborne listeriosis has prompted the evaluation of

bacteriocins as both bactericidal and bacteriostatic agents against L. monocytogenes. The

applications of antimicrobials produced by some specific bacteria have been studied in the meat

processing industry. In recent years, bacteriocins produced by lactic acid bacteria and

bacteriocins extracts have received great interest in the control of foodborne pathogenic L.

monocytogenes (87). Nisin is one of the most studied bacteriocins that have anti-listeria

properties (84). Nisin also, has many applications in foods and is approved for use in a variety of

foods throughout the world (131). It has been shown that this bacteriocin alters the cell

membrane of sensitive organisms resulting in leakage of low molecular weight cytoplasmic









components and the destruction of the proton motive force (13). Some studies claim that the

antimicrobial spectrum and potency of nisin can be increased when used in combination with

other antimicrobials (13, 49).

In an attempt to produce bacteriocins in situ, a bioperservation technique that has been

used is the addition of competitive microflora ofLatobacillus in ready-to-eat meat products

stored at refrigeration temperature. In a study conducted by Abee et al. (3), Lactobacillus sake

Lb674 was incorporated in the formulation of bologna-type sausage. The finished product was

sliced, vacuum packaged, and stored at 7oC for 28 days. The results showed that L. sake Lb674

produces detectable amounts of bacteriocin and delays or completely inhibits the growth of L.

monocytogenes when inoculated at levels of at least 105-106 CFU/g. Bacteriocin negative

Lactobacillus had no inhibitory effect on L. monocytogenes growth. It is important to emphasize

that the inoculation of bacteriocin-producing bacteria to produce a bacteriocin in situ would

require a strain that can grow and produce the bacteriocin at refrigeration storage temperatures

(127) .

Bacteriocins have also been applied as dipping solutions. In a study conducted by

Shanshan and Mustapha (115), fresh beef samples were inoculated with approximately 7 log

CFU/mL of L. monocytogenes Scott A or E. coli 0157:H7 and dipped in nisin or nisin combined

with EDTA solutions for 10 minutes. The results showed that treatment with nisin or with nisin

combined with EDTA reduced the population of L. monocytogenes by 2.01 and 0.99 log

CFU/cm2 TOSpectively as compared to the control, under the conditions of vacuum package and

storage at 40C for up to 30 days. However, the effect of nisin and nisin combined with EDTA

against E. coli 0157:H7 was marginal at 1.02 and 0.8 log CFU/cm2 reductions, respectively.

Similar results were reported by Tipayanate et al. (128), where in beef cubes inoculated with L.









monocytogenes Scott A were immersed in a solution of 2% polylactic acid, 2% lactic acid, 0.4%

of nisin, or combinations of each acid with nisin for 5 min and drip-dried for 15 min. Samples

were vacuum packaged and stored at refrigeration temperature for up to 42 days All treatments

showed immediate bactericidal effects on L. monocytogenes Scott A. On day 0, nisin alone and

in combination with polylactic acid and lactic acid significantly reduced (P < 0.05) L.

monocytogenes counts by 3.69, 3.76 and 3.39 log CFU/cm2, TOSpectively, when compared with

the positive control (5.33 log CFU/cm2). In a study conducted by Samelis et al. (109), dipping

solutions of nisin with or without organic acids or salts, as inhibitors of L. monocytogenes, were

evaluated on sliced cooked pork bologna before vacuum packaged and storage at 40C for 120

days. Inoculated (~103 ofu/cm2) Samples were immersed in 0.5% nisin, 1%, 3% or 5% lactic or

acetic acid, 3% or 5% of sodium acetate or diacetate, and 3% potassium benzoate or sorbate,

each combined with nisin. Nisin reduced L. monocytogenes by 1.0-1.5 log CFU/cm2 On day 0,

followed by a listeriostatic effect for 10 days. Nisin in combination with 3% or 5% acetic acid or

sodium diacetate or 3% potassium benzoate inhibited L. monocytogenes growth for 90 days. The

author concluded that nisin with 3% sodium diacetate may be the most promising combination in

dipping solutions to control L. monocytogenes on sliced, cured pork bologna.

The application of nisin-coated casing to inhibit L. monocytogenes growth is another

method that has been studied by researchers. Commercially prepared frankfurters were

formulated with and without 1.4% potassium lactate and 0. 1% sodium diacetate and were

subsequently processed in cellulose casings coated with and without nisin (~ 50,000 IU per

square inch of internal surface area) to control L. monocytogenes growth (74). The samples were

inoculated with approximately 5 log CFU/package of a five-strain inoculum ofL. monocytogenes

and then vacuum packaged and stored at 40C for up to 90 days. The results demonstrated that









potassium lactate and sodium diacetate display listeriostatic activity as an ingredient of

commercial frankfurters. These data also established that cellulose casings coated with nisin

display only moderate anti-listerial activity in vacuum packaged frankfurters stored at 40C.

In contrast, incorporation of nisin into the inner surface of sausage casings and vacuum-

packaging bags has demonstrated retention of anti-listeria activity (79). Nisin powder was

applied to plastic packaging bags at 7.75 Clg/cm:. Meat and poultry samples were inoculated with

L. nzonocytogenes. The bags coated with nisin powder completely inhibited growth of

inoculated L. nzonocytogenes samples through 12 weeks of storage at 40C.

Nisin has also been used as an antibotulinal agent for the partial replacement of nitrite in

cooked meat systems (3). Over the past three decades there has been an increasing research

interest in the development of nitrite-free meat curing systems. The main concern with the use

of nitrite for curing of meat is the eventual formation of carcinogenic N-nitrosamines. Recently,

attempts have been made to use nisin A as an altemative to nitrite. While the use of this

bacteriocin alone was not successful, promising results were obtained when it was combined

with reduced levels of nitrite: 100-250 ppm nisin A combined with 120 ppm nitrite was more

effective than the conventional 156 ppm nitrite (114).

Another application of nisin in meat systems that have been considered and investigated

includes the addition of nisin into the formulation of the meat products. Ham and bologna

sausages were prepared with or without addition of 500 mg kgl lysozyme:nisin (1:3), and 500

mg. kgl EDTA. Sausages were inoculated with L. nzonocytogenes, vacuum packed and stored

for 4 weeks at 80C. The results demonstrated an inhibition ofL. nzonocytogenes for two weeks

when lysozyme, nisin and EDTA were added (54). In another study, the effect of adding nisin to

raw meat inoculated with food-related bacteria was investigated by Chung et al. (19).The results









showed that nisin delayed the growth of L. monocytogenes and Staphylococcus aureus, but did

not inhibit Gram-negative bacteria. Thus, it can be determined that nisin performance in meat

systems is varied and that its antimicrobial properties are limited for gram positive bacteria.

On the other hand, researchers have found that use of nisin in meats could be limited by

some components which may interfere with nisin's activity. In a study conducted by Rose et al.

(104), the fate of nisin in meat products was determined by mass spectrometry. Nisin at 0.5%

was dissolved sterile water containing 0.02 N HC1. The solution was added to fresh and cooked

meat and meat juice. Samples were stored at 40C for 24 hours. Nisin was recovered from

cooked meat extract and cooked meat juice; however, only nisin bound to a food component was

detected in fresh meat extract. Mass spectra for raw meat and juice showed a signal 307 Da

greater than the mass of nisin. Results indicated that nisin was likely inactivated in raw meat by

an enzymatic reaction with glutathione. In addition, the neutral pH of the meat may interfere

with the antimicrobial properties of nisin, because it has been shown that nisin is 228 times more

soluble at pH 2 than at pH 7 (72). For this reason, other bacteriocins have also been evaluated in

meat products (20). Several researches have shown that bacteriocins, such as pediocin have

bactericidal to L. monocytogenes in meat products. The most promising outcomes were obtained

using pediocin PA-1. In a study conducted by Nielsen et al. (91), a bacteriocin produced by

Pediococcus acidilactici, known as pediocin PA-1, had an inhibitory and bactericidal effect on L.

monocytogenes. Results showed that attachment of Listeria onto the meat surface was 1.0 to 2.5

log cycles fewer when it was initially treated with the bacteriocin. The immediate antimicrobial

effect of pediocin resulted in reductions of 0.5 to 2.2 log cycles depending upon its

concentration. Similar anti-listerial properties were observed when pediocin was used alone (6)









or in combination with diacetate (111. Even though, pediocin is a very promising anti-listerial

agent, it has not yet been approved as a food additive in the United States.

Natural Antimicrobials: Description

The use of edible plants, as well as their phytochemicals, in food preservation and

improvement of organoleptic qualities of certain traditional foods has been practiced for

centuries. These antimicrobial properties derived from many plants have been empirically

recognized for centuries, but only scientifically confirmed in the last 30 years (95). The demand

for minimally processed and extended shelf life foods has further increased the interest to define

these naturally occurring bioactive ingredients.

The compounds responsible for antimicrobial activity in herbs are primarily phenolic

compounds of the essential oil fraction (11. Antimicrobial activity of cinnamon, allspice, and

cloves is attributed to eugenol (44). Oregano, thyme and savory have terpene, carvacol and

thymol, which account for their antimicrobial activity (41). The active antimicrobial fraction of

other herbs such as sage and rosemary has been suggested to also be in the terpene fraction of the

essential oils. Rosemary contains borneol, camphor and 1,8-cineole, a-pinene, camphene,

verbenone and bornyl acetate (119).

Numerous factors influence the antimicrobial activity of natural compounds. Some

suggest that chemical composition, influence by geographic origin and crop to crop variation,

and could significantly affect the activity of whole spices and essential oils (85). Furthermore,

the antimicrobial properties of these compounds may be influenced by the type of assay method

used (59). The interaction with other food components (lipid, protein) as well as surfactant,

minerals, pH, time, temperature also controls the biological activity of these natural compounds.

Unless these factors are controlled, studies on the antimicrobial activity of phytophenols from

oils, spices or herbs may vary considerably.









Mechanism of Action

Some studies have focused on the mechanism by which spices or their essentials oils

inhibit microorganism. Their mode of action is generally thought to involve interference with

functions of the cytoplasmic membrane including proton motive force and active transport (29).

Juven et al. (64) suggested that inhibition of Salmonella Typhimurium by thymol was due to a

reaction of the compound with proteins in the cytoplasmic membrane of the microorganism.

This reaction could lead to changes in the permeability of the membrane, which would result in

possible leakage and affect the proton motive force.

Application in the Food Industry

Society appears to be experiencing a trend of "natural" consumerism (121, 129), desiring

fewer synthetic food additives and products with a smaller impact on the environment.

Therefore, there is a need for new methods to make food safe which have a natural image.

Availability of information regarding the use of essential oils (EO's) in the food industry is

limited. There are approximately 3000 EO's known, of which about 300 are commercially

important, and are destined primarily for the flavors and fragrances market (136). It has long

been recognized that some EOs have antimicrobial properties (95) and the relatively recent

interest in natural consumerism has lead to a renewal of scientific interest in these substances

(92, 129). Some studies suggest that the inhibitory activity of each natural compound is different

for each pathogenic microorganism. Hammer et al. (59) found that thyme was the most essential

oil among 20 herbs, spices and plant extracts tested against E. coli, Staphylococcus aureus and

Can2dida albicans. In contrast, Hao et al. (60) found no antimicrobial activity by thyme against

Aeromona~s hydrophila or L. monocytogenes on cooked beef. In another study, Smith-Palmer et

al. (122) demonstrated that rosemary essential oil (0.02-0.05%) and sage (0.02-0.75%) were

inhibitory to L. monocytogenes and S. aureus, but not to Gram negative bacteria. Further, Pandit









et al., (95) showed that L. monocytogenes growth in refrigerated fresh pork sausage was delayed

by 0.5% ground rosemary or 1% rosemary essential oil.

Characteristics of EDTA (Ethylenediaminetetrancetic Acid)

Ethylenediaminetetraacetic acid (EDTA) is a well-known and widely used analytical

agent. The compound is a white crystalline powder. It forms water-soluble complexes with

most metal ions and is used extensively as a titrant for metal ions and a masking agent. In the

early 1940s, many metal complexes of EDTA were prepared and studied. These investigations

revealed that EDTA always formed 1:1 water-soluble complexes (99). In order for chelation to

occur, the sequestrant must have the proper steric and electronic configuration and be at the

optimal ionic strength and pH. EDTA becomes increasingly dissociated as pH rises resulting in

more metal becoming completed (28).

EDTA is available as a dried powder which is colorless, freely soluble in water, and has

only a slight taste of salinity in concentrations that are used in foods (28). The FDA has

approved EDTA as a food additive that is generally recognized as safe (GRAS) (47). EDTA's

array of biochemical properties makes it extremely valuable as a food additive. It has the ability

to bind with many metals, act synergistically with other antioxidants to stabilize fats and oils,

prevent discoloration of potato products, stabilize vitamins, prevent discoloration of fish and

shellfish, prevent flavor changes in milk, inhibit the thickening of stored condensed milk,

enhance the foaming properties of reconstituted skim milk, preserve canned products, promote

flavor retention and delay loss of carbonation in soft drinks, prevent oxidation of meat products

and prevent discoloration of canned fruits and vegetables (1.

Antimicrobial Activity of EDTA

Divalent cations such as Ca2+ and Mg2+ play specific roles in stabilizing the structure of

bacterial membranes because they form metal ions bridges between phosphate groups of









phospholipids or lipopolysaccharides and the carboxyl groups of membrane proteins (118).

Their removal leads to the disintegration of certain functional membrane proteins and the

collapse of membrane functions. It has been shown that EDTA damages outer membrane

structure by completing Ca2+ and Mg2+ which are necessary for Gram negative bacteria (70).

EDTA may act as a direct inhibitor of some microbes as well as synergistically with other

antimicrobial agents. EDTA works by disrupting the integrity of the cell wall. As a enhancer of

other agents, EDTA facilitates the entrance of other agents into the cell and chelates catios which

are essential for the repair of injured cells (28). In other cases, the opposite result occurs. For

example, EDTA combined with nisin reduced the antimicrobial effect of nisin (8). Conversely,

Stevens et al. (125) observed that the combinations of 20 mM lysozyme and 0.5% of nisin in cell

buffer resulted in a 3.2 to 6.9 log cycle reduction in Salmonella spp. and other Gram negative

bacteria. However, neither EDTA nor nisin alone showed significant inhibition against Gram

negative bacteria.

In a study conducted by Shanshan and Mustapha (115), nisin or nisin combined with

EDTA solutions were evaluated on fresh beef samples inoculated with approximately 7 log

CFU/mL of L. monocytogenes Scott A or E. coli 0157:H7. Samples were dipped in nisin or

nisin combined with EDTA for 10 minutes, vacuum package and stored at 40C for up to 30 days.

The results showed that treatment with nisin significantly (P < 0.05) reduced the population ofL.

monocytogenes by 2.01 log CFU/cm2 when compared to the control. However, when nisin was

combined with EDTA, L. monocytogenes counts were reduced by 0.99 log CFU/cm2 as

compared to the control. The results suggested that nisin alone inhibited L. monocytogenes

growth, but its anti-listerial properties were reduced when it was combined with EDTA. This

demonstrated no synergistic effects between nisin and EDTA.









CHAPTER 3
EVALUATION OF NATURAL ANTIMICROBIAL COMPOUNDS AGAINST Listeria
nzonocytogenes BY KIRBY-BAUER DISC DIFFUSION METHOD

Introduction

In spite of modern improvements in food production techniques, food safety is an

increasingly important public health issue (141). Food pathogens can travel long distances and

infect their hosts at a local and global level. Consequently, newly introduced pathogens can

spread rapidly in susceptible hosts.

Listeria nzonocytogenes is recognized as an important cause of foodborne illness, with high

hospitalization rates (88%) and fatality rates (20%) (18). Regulatory agencies have established

strict requirements for controlling L. nzonocytogenes in food products because of its widespread

distribution in nature and its ability to grow under unfavorable conditions (78). The use of

appropriate concentrations and/or combinations of antimicrobial compounds may contribute to

the safety of food products against L. nzonocytogenes (134).

In-vitro antimicrobial susceptibility testing done in recent years has indicated that

bactericidal compounds such as organic acids, salts, and general recognized as safe (GRAS)

substances can control L. nzonocytogenes (93). The results obtained in these studies may help

researchers in selecting initial procedures and antimicrobial agents that will be used for specific

products. A commonly used susceptibility test is the disc diffusion method. The antimicrobial

susceptibility testing (AST) is not only practical, but also the method of choice for the average

laboratory. All techniques used involve either the diffusion of an antimicrobial agent in agar or

its dilution in agar or broth. Some automated techniques are also variations of the above

methods (68).

The Kirby-Bauer method is a standardized filter-paper disc-agar diffusion procedure (15)

that is usually used for antimicrobial susceptibility testing. This method is recommended by the









Clinical and Laboratory Standards Institute (NCCLS), which is an international,

interdisciplinary, non-profit, non-governmental organization, composed of medical

professionals, government, industry, healthcare providers, educators, etc (89). This method

allows for the rapid determination of the efficacy of an antimicrobial by measuring the diameter

of the zone of inhibition that results from diffusion of the agent into the medium surrounding the

disc (15). The accuracy and reproducibility of the Kirby-Bauer method is dependent on

following the procedures described by the NCCLS (88).

Interpretative criteria of NCCLS are developed based on international collaborative

studies. This information is well correlated with minimum inhibitory concentration (MIC's)

results that have been corroborated with clinical data. Based on study results, the NCCLS

interpretative criteria are revised frequently. NCCLS guidelines are approved by the Food and

Drug Administration (48) and recommended by World Health Organization (140).

The obj ectives of this study were (1) to determine the anti-listerial properties of different

spices and antimicrobials alone and in combination against a Hyve strain inoculum of L.

monocytogenes using Kirby-Bauer method disc diffusion and (2) evaluate the anti-listerial

properties of nisin alone and in combination with the most effective antimicrobials identified

during preliminary evaluations.

Materials and Methods

Inoculum Preparation

Reference strains of L. monocytogenes '/ a, '/ b, 4 b, Scott A and 191 15, were obtained

from ABC Research Corporation in Gainesville, FL and used as the inoculum to evaluate the

anti-Listeria properties of different antimicrobial solutions using the Kirby-Bauer antimicrobial

susceptibility test procedure. The media and materials used for the cultivation, growth and

maintenance of the strains were purchased from Fisher Scientific (Pittsburgh, PA 15238). The









strains were transferred individually to test tubes containing 10 mL of tryptic soy broth (TSB,

Difco Laboratories, Detroit, MI 482132-7058, Cat.No. DF 0369-17-6) using a flamed sterilized

3mm inoculation loop. The broth was incubated at 350C for 24 hours. After incubation the

aliquots were poured into sterile centrifuge tubes and centrifuged (Sorvall RC-5B, Dupont

Instruments, Newton, CT 06470) at 5000g for 10 minutes. After centrifuging, the supernatants

were discarded and the pellets were re-suspended in 10 mL of sterile distilled water and

centrifuged again. The supernatants were again discarded and the pellets were re-suspended in 1

mL of 3% TSB with 30% glycerol in a 2 mL cryovial (Corning Incorporated, Corning, NY

14831, Cat.No. 03-374-21). The pellets were stored at -450C and used as the stock culture for

the inoculation studies.

Antimicrobial Solution Preparation

Natural antimicrobial solutions were selected based on results reported in previous studies

(59, 60, 71, 122). Antimicrobial solutions were prepared using distilled sterile water. The

appropriated aliquot of each antimicrobial was weighed and dissolved in sterile distilled water.

The solutions were used within 30 minutes after preparation. Sterile distilled water was used as

control treatment.

The analyses of antimicrobial solutions were conducted in two separate Phases. For Phase

one, a total of 47 antimicrobial solutions were tested (Table 3-1). These combinations were

analyzed in duplicates, and three separate trials of the study were conducted. The antimicrobial

combinations that showed the greatest inhibition of L. monocytogenes were selected for further

analysis during Phase Two of the study.

Nisaplin@~ (Danisco, Copenhagen, Denmark) is a commercial nisin product containing 106

IU nisin/g. A concentration of 0. 1%, 0.2%,and 0.3% nisin was obtained by adding 0. 1, 0.2, and

0.3 g of Nisaplin@ with 0. 17 mL of 0.02 N food grade HCL (Fisher Scientific, Pittsburgh, PA









15238, Cat. No. 7647-01-0) and 0.75g NaCl (Sigma Chemical, St. Louis, MO 63178, Cat No.

S9625-500G) into 100 mL of sterile distilled water. EDTA (Sigma Chemical, St. Louis, MO

63 178, Cat No. 59HO3 591) was prepared at a ratio of 0.75 g per 100 mL of sterile distilled water

to obtain a final concentration of 20 mM. Herbalox@ Seasoning (Kalsec, Kalamazoo, MI 49005-

0511, Code No. 41-19-02), a commercial rosemary extract, has shown antimicrobial properties

as well as inhibition of oxidative deterioration. Rosemary solution was prepared suspending 1

mL of the Herbalox@ into 100 mL of sterile distilled water. A concentration of 0. 15% and

0.25% of sodium diacetate (Sigma Chemical, St. Louis, MO 63 178, 59HO3 591) and 3% and 5%

of potassium benzoate (Versicol Chemical Co. Rosemont, IL 60018, Cat. No. 030222) was

obtained by dissolving 0.15 and 0.25 g of sodium diacetate, respectively and 3 and 5 g of

potassium benzoate, respectively, into 100 mL of sterile distilled water. Aquaresin@ thyme

(Kalsec, Kalamazoo, MI 49005-0511, Code No. 805454) is a commercial thymol extract.

Thymol solution was prepared by dissolving 1 mL of aquaresin@ thyme extract into 100 mL of

sterile distilled water to obtain a final concentration of 1%. A concentration of 1% and 2% of

vinegar (Albertson' s, Gainesville, FL 32608, UPC No. 04116310124) was obtained by adding 1

mL and 2mL of vinegar into 100 mL of sterile distilled water, respectively.

During Phase Two, 18 different combinations were analyzed (Table 3-2). These were

selected based on the results obtained in Phase one. Following the same protocol used in Phase

One, the antimicrobial combinations were analyzed in duplicates and was repeated three times to

show the reproducibility of the trials.

A concentration of 0. 1%, and 0.2% nisinwas obtained by adding 0. 1, and 0.2 g of

Nisaplin@ with 0. 17 mL of 0.02 N food grade HCL (Fisher Scientific, Pittsburgh, PA 15238,

Cat. No. 7647-01-0) and 0.75g NaCl (Sigma Chemical, St. Louis, MO 63178, Cat No. S9625-










500G) into 100 mL of sterile distilled water.20 mM EDTA (Sigma Chemical, St. Louis, MO

63 178, Cat No. 59HO3 591) was prepared by dissolving 0.75 g of EDTA into 100 mL of sterile

distilled water. Herbalox@ Seasoning (Kalsec, Kalamazoo, MI 49005-0511, Code No. 41-19-

02), a commercial rosemary extract, has shown antimicrobial properties as well as inhibition of

oxidative deterioration. 1% rosemary solution was prepared suspending 1 mL of the Herbalox@

into 100 mL of sterile distilled water. Thymol extract was obtained from the commercial product

Aquaresin@ thyme (Kalsec, Kalamazoo, MI 49005-0511, Code No. 805454). Thymol solution

was prepared by dissolving 1 mL of aquaresin@ thyme extract into 100 mL of sterile distilled

water to obtain a final concentration of 1%.

Preparation of Modified Oxford Media Agar (MOX)

Modified Oxford Media (MOX, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF

0225-17-0) and Modified Oxford antimicrobial supplement (MOX supplement, Difco

Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0218-60-5) were prepared from a

commercially available dehydrated base according to the manufacturer's instructions. Immediately

after autoclaving, the media was allowed to cool in a 45 to 500C water bath for approximately 30

minutes and 5 mL of antimicrobial supplement was added. The media was poured into plastic flat-

bottomed Petri dishes on a horizontal level surface to give a uniform depth of approximately 4 mm.

The plates were allowed to cool to room temperature and stored at 4 + 1 OC.

Inoculation of Agar Plates

Frozen L. monocytogenes inocula were allowed to thaw at room temperature for 10

minutes. A loopful of the thawed stock culture was transferred to test tubes containing 10 mL of

3% TSB and incubated at 350C for 24 hours. After incubation, the aliquots were centrifuged

(5000 rpm for 10 min at 160C) and washed with sterile 0. 1% buffered peptone water (BPW,

Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4). The aliquots were then









serially diluted with BPW to concentrations of 10-1 to 10-s. A sterile cotton swab was dipped

into the 10-s dilution. The swab was rotated several times and pressed firmly on the inside wall

of the tube above the fluid level, removing the excess inoculum from the swab. The MOX agar

plates were inoculated by streaking the swab over the entire surface. This procedure was

repeated by streaking three more times, rotating the plate approximately 600 each time to ensure

an even distribution of the inoculum. As a final step, the rim of the plate was swabbed, and the

lid was placed on the Petri dish, and plates were allowed to sit a room temperature for 2-3

minutes to allow any excess surface moisture to be absorbed before applying the antimicrobial

impregnated discs.

Kirby-Bauer Disc Diffusion Test

Paper-filter discs (6 mm diameter) (Becton & Dickinson, Sparks, MD 21152, Cat. No.

231039) were immersed into the antimicrobial solution for fifteen seconds using a flamed

sterilized forceps and placed onto the surface of the inoculated MOX agar plate. Each disc was

pressed down to ensure complete contact with the agar surface. Two discs were placed on each

150 mm plate (Figure 1). The plates were inverted and placed in an incubator set at 350C within

15 minutes after the discs were applied to the agar surfaces. After 24 hours of incubation, each

plate was examined. The diameters of the zones of complete inhibition were measured,

including the diameter of the disc. Zones were measured to the nearest millimeter using a

graduated ruler. The ruler was held on the back of the inverted Petri plate and held above a

nonreflecting background and illuminated with reflected light to take each measurement.

Data Analysis

Statistical analyses were conducted for zones of compete inhibition for a total of eight

measurements per treatment on Phase One and for a total of twelve measurements per treatment

on Phase Two. The general linear model program (PROC GLM) of SAS@ system (110) was










employed to analyze trial, treatment and treatment by trial. A total of 57 treatment combinations

were evaluated. Comparisons among means were performed using SAS@ Tukey Multiple

Range test procedure. Treatments effects and differences were considered significant when P <

0.05.

Results and Discussion

The data demonstrated L. monocytogenes was inhibited when nisin was used (Table 3-3).

The zone of inhibition for L. monocytogenes increased as the concentration of nisin increased

from 0. 1% to 0.5%. Treatments in which 0.5% nisin and 0.5% nisin combined with food grade

hydrochloric acid (HCL) were used resulted in an inhibition zone of 3.20 and 3.18 mm,

respectively. These two treatments showed the highest inhibition zone when compare to all

other treatments (P < 0.05).

No differences (P < 0.05) were found when 0.5% nisin was used alone or in combination

with food grade HCL and ethylenediaminetetraacetic acid (EDTA). The use of rosemary alone

resulted in an inhibition zone of 1.03 mm, and the zone of inhibition increased approximately

twice this size as the concentration of nisin added to rosemary was increased in 0. 1% units. L.

monocytogenes growth was not inhibited by the treatments containing 1% and 2% vinegar, 3%

and 5% potassium benzoate, 1% thymol or 0. 15% and 0.25% sodium diacetate alone and in

combination with nisin.

The results obtained in this trial suggested that nisin used alone or in combination with 1%

rosemary and 20 mM EDTA may inhibit growth ofL. monocytogenes. These findings were

useful in developing Phase Two of the study, in which, a total of 18 combinations were further

analyzed. The FDA allows the use of nisin in foods at maximum level of 0.5% (U. S. Food and

Drug Administration, 1988). During Phase Two, concentrations of nisin at 0. 1% and 0.2% were









evaluated alone and in combination with 1% rosemary, 1% thymol and 20 mM EDTA (Table 3-

4).

During Phase Two, no significant differences (P > 0.05) were found between 0.1% nisin

and 0.2% nisin when combined with 1% rosemary. The highest zone of inhibition (P < 0.05)

was achieved when 0.2% nisin was used in combination with 1% rosemary and 20 mM EDTA.

The zone of inhibition was below 0.63 mm when thymol was used alone and in combination

with nisin. There was no significant difference (P > 0.05) between the control treatment (water)

and thymol alone and in combination with 0.1% nisin.

Antimicrobial susceptibility testing procedures provide a useful proactive tool for assisting

researchers in determining the antimicrobial properties of additives prior to incorporating them

into a food matrix; thus allowing researchers to use testing procedures that are more cost

efficient and less time consuming. This study revealed the susceptibility of L. monocytogenes to

natural compounds, such as rosemary, EDTA and different concentrations of nisin. The next

step would be to evaluate these results in a food matrix and design an antimicrobial system that

would inhibit the growth of L. monocytogenes in a specific meat product.





Table 3-1. Fifty-eight antimicrobial solutions evaluated in Phase 1
List of antimicrobial solutions
1% Rosemary Extract 3% Potassiur
0. 1% Nisin 1% Rosemary Extract 3% Potassiur
0.2% Nisin 1% Rosemary Extract 3% Potassiur
0.3% Nisin 1% Rosemary Extract 3% Potassiur
0.1 % Thymol 5% Potassiur
0. 1% Thymol 0. 1% Nisin 5% Potassiur
0.1% Thymol 0.2% Nisin 5% Potassiur
0.1% Thymol 0.3% Nisin 5% Potassiur
0.15% Sodium Diacetate 0.1% Nisin
0. 15% Sodium Diacetate 0. 1% Nisin 0.2% Nisin
0.15% Sodium Diacetate 0.2% Nisin 0.3% Nisin
0.15% Sodium Diacetate 0.3% Nisin 0.4% Nisin
0.25% Sodium Diacetate 0.5% Nisin
0.25% Sodium Diacetate 0. 1% Nisin 0. 1% Nisin +
0.25% Sodium Diacetate 0.2% Nisin 0.2% Nisin +
0.25% Sodium Diacetate 0.3% Nisin 0.3% Nisin +
1% Vinegar 0.4% Nisin +
1% Vinegar 0. 1% Nisin 0.5% Nisin +
1% Vinegar 0.2% Nisin 0. 1% Nisin +
1% Vinegar 0.3% Nisin 0.2% Nisin +
2% Vinegar 0.3% Nisin +
2% Vinegar 0. 1% Nisin 0.4% Nisin +
2% Vinegar 0.2% Nisin 0.5% Nisin +
2% Vinegar 0.3% Nisin Water


m Benzoate
m Benzoate 0.
m Benzoate 0.:
m Benzoate 0.:
m Benzoate
m Benzoate 0.
m Benzoate 0.:
m Benzoate 0.:






20 mM EDTA
20 mM EDTA
20 mM EDTA
20 mM EDTA
20 mM EDTA
HCL
HCL
HCL
HCL
HCL


1% Nisin
2% Nisin
3% Nisin

1% Nisin
2% Nisin
3% Nisin










Table 3-2. Seventeen antimicrobial solutions evaluated in Phase 2
List of Antimicrobial Solutions
water
EDTA
0. 1Nisin
0.2Nisin
Thymol
Rosemary
0. 1Nisin-EDTA
0.2Nisin-EDTA
0. 1Nisin-Thymol
0. 1Nisin-Thymol-EDTA
0. 1Nisin-Rosemary
0. 1Nisin-Rosemary-EDTA
0. 1Ni sin-Rosemary-Thymol -EDTA
0.2Ni sin-Thymol
0.2Nisin-Thymol-EDTA
0.2Ni sin-Rosemary
0.2Ni sin-Rosemary-EDTA
0.2Nisin-Rosemary-Thymol-EDTA









Table 3-3. Mean zone of inhibition for antimicrobial solutions evaluated in Phase 1
Treatments Inhibition zone (mm)*
0.5% Nisin 3.20 a
0.5% Nisin + HCL 3.18 a
0.5% Nisin + HCL + 20 mM EDTA 2.95 ab
1% Rosemary + 0.3% Nisin 2.72 b
0.4% Nisin 2.63 bc
0.4% Nisin + HCL 2.38 cd
0.4% Nisin + HCL + 20 mM EDTA 2.18 de
1% Rosemary + 0.2% Nisin 2.08 def
0.2% Nisin 2.08 def
0.3% Nisin 1.95 efg
0.3% Nisin + HCL + 20 mM EDTA 1.83 fgh
0.3% Nisin + HCL 1.78 fgh
1% Thymol + 0.3% Nisin 1.70 ghi
1% Thymol + 0. 1% Nisin 1.65 ghi
0.2% Nisin + HCL + 20 mM EDTA 1.63 ghi
1% Rosemary + 0. 1% Nisin 1.53 hij
1% Thymol + 0.2% Nisin 1.40 ijk
0. 1% Nisin + HCL 1.28 jkl
0.2% Nisin + HCL 1.25 jkl
5% Potassium benzoate + 0.3% Nisin 1.13 klm"
0. 1% Nisin + HCL + 20 mM EDTA 1.10 klm"
1% Rosemary 1.03 'mn
0.1% Nisin 0.90 mo"
0.25% Sodium diacetate + 0.2% Nisin 0.88 mo"
5% Potassium benzoate + 0.2% Nisin 0.83 mo"
0.25% Sodium diacetate + 0.3% Nisin 0.75 no
3% Potassium benzoate + 0.3% Nisin 0.68 o
0.15% Sodium diacetate + 0.3% Nisin 0.65 o
1% Vinegar + 0.3% Nisin 0.63 o
2% Vinegar 0.00 P
3% Potassium benzoate 0.00P
5% Potassium benzoate 0.00P
1% Vinegar 0.00P
3% Potassium benzoate + 0. 1% Nisin 0.00P
3% Potassium benzoate + 0.2% Nisin 0.00P
5% Potassium benzoate + 0. 1% Nisin 0.00P
1% Thymol 0.00P
1% Vinegar + 0. 1% Nisin 0.00P
1% Vinegar + 0.2% Nisin 0.00P
2% Vinegar + 0. 1% Nisin 0.00P
0.15% Sodium diacetate 0.00P
0. 15% Sodium diacetate + 0. 1% Nisin 0.00P
0.15% Sodium diacetate + 0.2% Nisin 0.00P
2% Vinegar + 0.3% Nisin 0.00P









0.25% Sodium diacetate 0.00P
0.25% Sodium diacetate + 0. 1% Nisin 0.00P
2% Vinegar + 0.2% Nisin 0.00P
water 0.00 P
* Each means value represent four individual measurements
a-p values in same column with different superscripts are significantly different
(P < 0.05)









Table 3-4. Mean zone of inhibition for antimicrobial solutions evaluated in Phase 2
Treatments Inhibition zone (mm)*
0.2% Nisin + 1% Rosemary + 20 mM EDTA 1.27 a
0.2% Nisin + 1% Rosemary + 1% Thymol + 20 mM EDTA 1.22 ab
0.2% Nisin 1.17 a
0.2% Nisin + 1% Rosemary 1.17 ab
0. 1% Nisin + 1% Rosemary 1.17 ab
0. 1% Nisin 1.09 abc
0. 1% Nisin + 1% Rosemary + 1% Thymol + 20 mM EDTA 1.05 abc
0. 1% Nisin + 1% Rosemary + 20 mM EDTA 1.05 abc
20 mM EDTA 0.95 bc
0.2% Nisin + 1% Thymol + 20 mM EDTA 0.87 cd
0. 1% Rosemary 0.82 cd
0.2% Nisin + 20 mM EDTA 0.82 cd
0.2% Nisin + 1% Thymol 0.80 cd
0.1 % Nisin + 20 mM EDTA 0.80 cd
0.1 % Nisin + 20 mM EDTA + 1% Thymol 0.80 cd
1% Thymol 0.62 de
0.1 % Nisin + Thymol 0.47 e
Water 0.00f
* Each means value represent six individual measurements
a-f values in same column with different superscripts are significantly different
(P < 0.05)









CHAPTER 4
EVALUATION OF CONCENTRATIONS OF NISIN AGAINST Listeria nonocytogenes ON
READY-TO-EAT TURKEY HAM STORED AT 4+10C FOR 63 DAYS

Introduction

Listeria nzonocytogenes, the causative agent of listeriosis, has resulted in numerous maj or

foodborne outbreaks worldwide (55). The ability of L. nzonocytogenes to grow at temperatures

ranging from 0 to 450C (7), its high tolerance for salt (42), and its ability to initiate growth at a

relatively low pH (9) makes this pathogen particularly difficult to control in food. Hygienic and

sanitation practices applied in meat processing facilities are often insufficient to prevent

contamination of processed meat products (21). L. nzonocytogenes can be resistant to many food

preservation methods and can increase to high numbers during refrigerated storage (137) and

under low oxygen tension (73). Thus, effective hurdle technologies to inhibit growth of the

pathogen are needed.

A novel approach to controlling L. nzonocytogenes in foods is the use of antimicrobial

bacteriocins from lactic acid bacteria (32). Nisin, a lanthionine-containing polypeptide produced

by Lactococcus lactis subsp. lactis (5) is a bacteriocin with antimicrobial activity against L.

nzonocytogenes (14). In the United States, where antibiotics are prohibited in foods, nisin

obtained GRAS status for use as a biopreservative in the food industry in 1988 (131). The LD5o

value was found to be similar to that of common salt (82).

Nisin' s mechanism of action is based on the disruption of the cytoplasmic cell membrane,

as evidenced by the rapid efflux of small molecules from both whole cells and liposomes (4, 50,

138). As a result, nisin depletes the proton motive force (PlVF) of sensitive cells and artificial

liposomes (13, 49). Nisin acts through a multistep process which includes binding of nisin to the

cell, insertion into the membrane, and pore formation (50, 105). Anionic phospholipids play an

important role in nisin' s interaction with membranes (35). On binding to anionic phospholipids,









nisin causes a local perturbation of the lipid bilayer (38), followed by electrical potential (Auy)- or

pH gradient (ApH)- that enhance insertion into the membrane to form a wedge-like pore (80).

Nisin also, has many applications in foods and is approved for use in a variety of foods

throughout the world. Applications of nisin in meat systems that have been considered or

investigated include: the addition of nisin-producing Lactococcus lactis subsp. lactis to meat

systems in an attempt to produce nisin in situ (3); dipping solutions of nisin (109, 115, 128);

nisin- coated casing (74); addition of nisin into the formulation of the meat product (54, 108); the

use of nisin as an antibotulinal agent for the partial replacement of nitrite in cooked meat systems

(3); and the use of nisin in canned meats as a means of reducing thermal processing time (84).

The aim of the study was to evaluate the anti-listerial properties of different concentrations

of nisin (0.2%, 0.3%, 0.4% and 0.5%) on ready-to-eat (RTE) turkey ham using a novel post-

processing treatment against L. monocytogenes that would be utilized on RTE meat products.

Materials and Methods

This study was conducted in two trials during which 0.2%, 0.3%, 0.4% and 0.5% nisin

solutions were used to treat RTE turkey ham inoculated with five strains ofL. monocytogenes.

The trials were conducted at the University of Florida Meat Processing Laboratory and

Microbiology Laboratory, Gainesville, FL. The same procedure was used on both trials.

Inoculum Cultivation and Storage

Reference strains of L. monocytogenes '/ a, '/ b, 4 b, Scott A and 191 15, were obtained

from ABC Research Corporation in Gainesville, FL and were used as the inoculum to evaluate

the anti-Listeria properties of different concentrations of nisin. The media and materials used for

the cultivation, growth and maintenance of the strains were purchased from Fisher Scientific

(Pittsburgh, PA 15238). The strains were transferred individually to test tubes containing 10 mL

of tryptic soy broth (TSB, Difco Laboratories, Detroit, MI 482132-7058, Cat.No. DF 0369-17-6)









using a flamed sterilized 3mm inoculation loop. The broth was incubated at 350C for 24 hours.

After incubation the aliquots were poured into sterile centrifuge tubes and centrifuged (Sorvall

RC-5B, Dupont Instruments, Newton, CT 06470) at 5000rpm for 10 minutes. After centrifuging,

the supernatants were discarded and the pellets were re-suspended in 10 mL of sterile distilled

water and centrifuged again. The supernatants were again discarded and the pellets were re-

suspended in 1 mL of 3% TSB with 30% glycerol in a 2 mL cryovial (Corning Incorporated,

Corning, NY 14831, Cat.No. 03-374-21). The pellets were stored at -450C and used as the stock

culture for the inoculation studies.

Inoculum Preparation

Frozen L. monocytogenes strains were allowed to thaw at room temperature for 10

minutes. A loopful of the thawed stock culture was transferred to test tubes containing 10 mL of

3% TSB and incubated at 350C for 24 hours. After incubation, the aliquots were centrifuged

(5000 rpm for 10 min at 160C) and washed with sterile 0. 1% buffered peptone water (BFP, Difco

Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4). The aliquots were then

serially diluted with BPW to concentrations of 10-1 to 10-s

Antimicrobial Solutions Preparation

Formulations were developed to prepare 0.2%, 0.3%, 0.4% and 0.5% nisin solutions to

ensure that desired concentration of each solution made contact with chopped turkey ham. The

control turkey ham formulation consisted of the following (% of total weight in the formulation):

chopped turkey ham (90%) and water (10%) (Table 4-1). Each nisin concentration was

dissolved using 10% sterile water, according to each treatment formulation, which was added

into the bag containing the chopped turkey ham. Sterile deionized water was used as control.

Nisaplin@~ (Danisco, Copenhagen, Denmark) is a commercial nisin product containing 106

IU nisin/g. Nisaplin, HCL and NaCl were used to prepare the antimicrobial solutions and were









diluted in a predetermine amount of sterile deionized water (10%) (Table 4-1) A concentration

of 0.2%, 0.3%, 0.4% and 0.5% nisin was obtained by adding 0.2, 0.3, 0.4 and 0.5 g of Nisaplin@

with 0. 17 mL of 0.02 N food grade HCL (Fisher Scientific, Pittsburgh, PA 15238, Cat. No.

7647-01-0) and 0.75g NaCl (Sigma Chemical, St. Louis, MO 63178, Cat No. S9625-500G) to

the turkey ham (based on total batch weight). Nisin solutions were stored at 40C and used within

3 days.

Sample preparation

Jennie-O RTE turkey hams were purchased from Publix, Gainesville, Florida with an

expiration date of 60 days. The turkey hams were immediately transported to the University of

Florida Meat Processing Laboratory and Microbiology Laboratory, Gainesville, Florida and

stored at 40C for no longer than 24 hours before using. The hams were aseptically transferred

from the vacuum packaged bag to pre-sterilized trays (polypropylene) and chopped into

approximately 0.5 cm pieces. The ham was chopped to simulate how it may be used in ham

salad or similar foods.

Inoculation and Treatment

The turkey ham pieces were placed on pre-sterilized trays and inoculated by spraying them

with 1 mL of L. monocytogenes inoculum at 1.0 X 10s CFU/mL. Inoculated samples were left to

stand at room temperature for 20 min to allow for bacterial attachment and to ensure a final

concentration of 104 CFU/gram.

Duplicate samples of inoculated chopped turkey ham were aseptically weighted according

to its corresponding treatment (Table 1) and placed in a labeled Food Saver bag (Tilia, San

Francisco, California, FoodSaver Vacloc Roll). Corresponding treatments were applied to the

chopped ham followed by mixing the ham and treatment solution to ensure a proper distribution

between them. The bags were then vacuum packaged (Tilia, San Francisco, California,









FoodSaver Bagvac), leaving the nisin solution in the package, and stored in a 40C cooler for

subsequent microbiological and chemical analysis. Samples were analyzed after 0, 7, 14, 21, 28,

35, 42, 49, 56 and 63 days of storage.

Microbiological Analyses

Twenty-Hyve grams of chopped turkey ham were transferred aseptically from the vacuum

packaged bag into a sterile stomacher bag (Fisher Scientifie, Pittsburgh, PA 15238, Cat. No. 01-

002-44) with 225 mL of sterile 0. 1% buffered peptone water (BPW, Difco, Laboratories, Detroit,

MI 48232-7058, Cat. No. DF 01897-17-4) and shaken approximately 30 times. Further serial

dilutions were prepared by adding 1 mL of the diluted sample homogenate into 9 mL of 0. 1%

BPW until the appropriate dilutions were obtained.

Listeria monocytogenes and lactic acid bacteria analysis

A volume of 0. 1 mL of the dilutions was dispensed onto pre-poured Modified Oxford

Media (MOX, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0225-17-0) containing

Modified Oxford antimicrobic supplement (MOX supplement, Difco Laboratories, Detroit, MI

48232-7058, Cat. No. DF 0218-60-5) for L. monocytogenes identification and to lactobacilli

MRS agar (Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0882170) for the isolation

of lactic acid bacteria. The spread plate technique was used to evenly distribute the sample over

the plate. Using this technique, a flamed sterilized bacterial cell spreader (Fisher Scientifie,

Pittsburgh, PA 15238, Cat. No. 08-769-2A) was used to spread the sample over the plate as the

plate was spun on an inoculating turntable (Fisher Scientifie, Pittsburgh, PA 15238, Cat. No. 08-

758-10). All samples were plated in duplicate. The Petri plates were inverted and incubated for

24 hours at 350C for L. monocytogenes and 370C for lactic acid bacteria. Plates with 25 to 250

colonies were counted. Black colonies surrounded by a black halo and white/grayish colonies









were considered presumptively L. monocytogenes and lactic acid, respectively. Microbiological

counts were reported as Logarithmic Colonic Forming Units per gram (Log CFU/g).

Aerobic bacteria analysis

Aerobic bacteria counts were performed using 3M Petrifilm aerobic count plates (St. Paul,

Minnesota, Cat. No. 6404). The Petrifilm aerobic count plate was placed on a level surface. The

top fi1m was lifted and 1 mL of sample was dispensed to the center of the bottom of the plate.

The top film was released down onto the sample and the plastic spreader was placed on the

center of the plate, convex side down. After approximately one minute, the spreader was

removed from the plate. All samples were plated in duplicate. Plates were incubated for 24

hours at 250C in a horizontal position with the clear side up in stacks of 12 plates. Plates

containing 15 to 150 colonies were counted and recorded. Microbiological counts were

expressed as Logarithmic Colonic Forming Units per gram (Log CFU/g).

pH Analysis

The pH analysis of the chopped RTE turkey ham was performed using a pH meter

(Accumet Basic ABl15, Fisher Scientifie, Pittsburgh, PA 15238, Model No. ABl15, Serial No.

AB81210535). Twenty-Hyve grams of chopped ham was aseptically removed from the vacuum

packaged bag and placed into a sterile plastic bag (Fisher Scientifie, Pittsburgh, PA 15238, Cat.

No. 01-002-44) into which 225 mL of 0. 1% buffered peptone water (BPW, Difco, Laboratories,

Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) was added. Duplicated pH measurements

were taken from each ham homogenate.

Data Analysis

Statistical analyses were conducted for enumeration data of a total of eight measurements

per treatment of L. monocytogenes, aerobic bacteria, and lactic acid bacteria. Also, statistical

analysis was conducted for pH values of six measurements per treatment. The general linear










model program (PROC GLM) of SAS@ system (110) was employed to statistically analyze trial,

day, treatment and treatment by day. Variations in data were accounted for by four treatments

effects: trial, treatment, day, and treatment~day. Comparisons among means were performed

using SAS@ Tukey Multiple Range test procedure. Treatments effects and differences were

considered significant when P < 0.05.

Results and Discussion

pH Analysis

No difference was observed in pH values among treatments from days 0 to 49 (Table 4-2).

However, by day 56, nisin at 0.5% had significantly higher (P < 0.05) pH when compared to all

other treatments. All treatments experienced a slight decrease in pH throughout storage. This

may be attributed to the production of numerous compounds such as acidic metabolites and

carbonic acid that may decrease the pH (37). The pH values decrease as the concentration of

nisin decreased from 0.5% to 0.2%. This may have been caused by the number of lactic acid

bacteria present in the samples, which increase as the concentration of nisin decrease from 0.5%

to 0.2%.

Listeria monocytogenes Analysis

Anti-Listeria effects of nisin at different concentrations were similar (P > 0.05) for the first

two weeks (Table 4-3). Bacterial growth over time can be graphed as a cell number versus time.

This is called a growth curve (37). The cell number is plotted as the log of the cell number, since

it is an exponential function. The lag phase is the first phase observed. It is characterized by

little or no increase in cell number; however, the cells are actively metabolizing, in preparation

for cell division and depending on the growth medium, the lag phase may be short or very long

(43). The results demonstrated an extended lag phase for the 0.5% nisin treatment through 63

days storage, maintaining L. monocytogenes counts at less than 1.95 log CFU/g.









Treatment with 0.2%, 0.3%, 0.4% and 0.5% of nisin caused initial population reduction as

compared to the positive control (P < 0.05). On day 0, the initial bacterial population of the

positive control (4.97 log CFU/g) was significantly reduced (P< 0.05) to 4.00 log CFU/g, 4.37

log CFU/g, 4.37 log CFU/g, and 4.55 log CFU/g when treated with 0.2%, 0.3%, 0.4%, and 0.5%

nisin, respectively. Through out the 63 days of storage, the number ofL. monocytogenes

increased approximately 1.00 log CFU/g in the 0.5% nisin treated sample and approximately

2.00 log CFU/g in the 0.2%, 0.3% and 0.4% nisin treated samples. This suggested that these

nisin's concentrations may have an initial bacterial effect against L. monocytogenes population

and may help to maintain the L. monocytogenes counts lower during storage. The overall mean

values for all treatments revealed that nisin resulted in 1.62-3.18 log CFU/g reductions of L.

monocytogenes. Results from the study indicate that the antimicrobial effectiveness of nisin

increased as its concentration increased from 0.2% to 0.5% and that L. monocytogenes may be

controlled in vacuum package RTE turkey ham stored at 40C when treated with the

concentrations of nisin evaluated in this study.

Lactic Acid Bacteria Analysis

On day 0, lactic acid bacteria counts were significantly lower (P < 0.05) on the nisin

treated samples when compared to the positive and negative control, showing an initial

antimicrobial effect against lactic acid bacteria (Table 4-4). From days 7 to 49, no difference

was observed in pH values among treatments. However, by day 56 nisin at 0.5% had higher (P <

0.05) pH when compared to all other treatments.

Lactic acid bacteria population increased over time for all treatments under vacuum

packaged and refrigeration (40C) conditions. Lactic acid bacteria counts were significantly

lower for 0.5% nisin (P < 0.05) when compared to positive and negative controls, revealing

nisin's effectiveness against lactic acid bacteria. Results from the study indicate that lactic acid









bacteria may be restricted in vacuum package RTE turkey ham stored at 40C when treated with

at least 0.2% nisin and the effectiveness of nisin increase as its concentration increased to 0.5%.

Aerobic Bacteria Analysis

Initial counts of aerobic bacteria on ready-to-eat turkey ham inoculated with L.

monocytogenes following different treatments are shown in (Table 4-5). The initial population

of aerobic bacteria on the turkey ham was significantly higher (P < 0.05) in the positive control

when compared to all other treatments. The antimicrobial treatment that contained 0.5% nisin

was the most effective (P < 0.05) reducing the initial populations of aerobic bacteria. The initial

population of aerobic bacteria was reduced as the concentration of nisin increased from 0.2% to

0.5%. These results suggest that initial population of aerobic bacteria may be restricted when

treated with a concentration equal or greater than 0.2% nisin under vacuum package and

refrigeration (40C) conditions.










Table 4-1. Formulation of nisin solutions for vacuum packaged ready-to-eat turkey ham stored
at 4+1 OC for 63 days
Percentage (%) of ingredient in total composition
Negative Positive....
Treatment 120.2% misin 0.3% misin 0.4% msin 0.5% msin
Control Control2
Ingsre dient
Ham 90.00 90.00 88.88 88.78 88.68 88.58
Water 10.00 10.00 10.00 10.00 10.00 10.00
Nisin 0.00 0.00 0.20 0.30 0.40 0.50
NaCl 0.00 0.00 0.75 0.75 0.75 0.75
HCI 0.00 0.00 0.17 0.17 0.17 0.17
100.00 100.00 100.00 100.00 100.00 100.00
Neaiecnrl ihu .mnctgnsiouu
2 Postive control: without L. monocytogenes inoculum










Table 4-2. pH measurements on Ready-To-Eat turkey ham supplemented with various concentrations of nisin and inoculated with
Listeria monocytogenes and stored at 4 + loC for 63 days
Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63
Negative control 6.24 a~~x 5.76 a~~ 4.97 a~z 4.87 a~z 4.86 a~z 4.81 a~z 4.79 a~z 4.67 a~z 4.86 b~z 4.82 b~z
Positive control 6.14 a~~x 5.07 a~~ 4.85 a, 4.79 a~ 4.78 ay 4.82 a~ 4.77 ay 4.94 a~ 4.74 b' 4.77 b'y
0.2% nisin 6.21 a~~x 5.67 a~xY 5.46 a~xY 5.29 a~xY 5.19 a~xY 5.00 a~Y 5.02 asy 5.13 a,xy 5.01 b,Y 4.86 ab,y
0.3% nisin 6.22 a~~x 6.10 a~~x 6.11 a~x 5.72 a~x 5.43 a~x 5.41 a~x 5.40 a~x 5.29 a~x 5.10 b~x 5.24 ab,x
0.4% nisin 6.26 a~~x 5.85 a~xY 5.56 a~xY 5.41 a~xY 5.06 asY 5.15 a~Y 4.90 asy 4.76 a~Y 4.87 b'y 5.12 ab,y
0.5% nisin 6.21 a~~x 6.14 a~xY 6.14 a~xY 6.08 a~xY 5.89 a~~xyz 5.58 a~yz 5.79 a,xyz 5.67 a~z 5.80 a~xyz 5.83 a~xyz
a-b means in same column with different superscripts are significantly different
x-z means in same row with different superscripts are significantly different
(P < 0.05)













Day 42 Day 49 Day 56 Day 63


Table 4-3. Listeria monocytogenes counts on Ready-To-Eat turkey ham supplemented with various concentrations of nisin and


inoculated with Listeria monocytogenes and stored at 4 + loC for 63 days

Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35
(Loglo CFU/g)
Negative control 0.00 b 0.00 c 0.00 b 0.00 b 0.00 b 0.00 b
Positive control 4.97 asY 4.92 asY 4.95 asY 4.30 a~Y 4.23 asy 4.32 as
0.2% nisin 0.97 b z 2. 12 b yz 3.22 ab yz 2.45 ab yz 2.94 b yz 3.35 a yz
0.3% nisin 0.60 b'y 1.59 be,y 1.96 ab,y 2.16 ab,y 2.95 ab,y 2.62 ab,y
0.4% nisin 0.60 b'y 0.85 be,y 2.29 ab,y 1.35 ab,y 2.50 ab,y 2.96 ab,y
0.5% nisin 0.42 b'y 0.89 be,y 0.17 b,y 1.66 ab,y 1.90 ab,y 1.95 ab,y
a-e means in same column with different superscripts are significantly different
y-z means in same row with different superscripts are significantly different
(P < 0.05)


0.00 b y
4.18 a~Y
3.57 a~Y
2.57 a~Y
3.06 ab,y
1.67 ab,y


0.00 b y
4.55 a~Y
3.61 a~Y
2.99 ab,y
3.02 ab,y
1.89 ab,y


0.00 e Y
4.14 a~Y
2.97 b'y
1.83 ed,y
2.09 "Y
1.01 d'y


0.00 ce
4.31 a~~Y
3.52 ab,y
3.10 ab~y
2.82 ab,y
1.51 be,y










Table 4-4. Lactic acid bacteria counts on Ready-To-Eat turkey ham supplemented with various concentrations of nisin and
inoculated with Listeria monocytogenes and stored at 4 + loC for 63 days
Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63
Treatments
(Logo CFU/g
Negative control 3.25 ab~x 4.74 a~~wx 5.66 a~wx 6.86 a~w 7.10 a~w 6.84 a~w 6.66 a~w 6.71 a~~w 6.62 a~w 6.66 a~w
Positive control 4.27 a~~x 5.49 a~~wx 5.90 a~wx 6.73 a~w 6.61 a~w 6.74 a~w 6.54 a~w 6.47 a~~w 6.53 a~w 6.71 a~w
0.2% nisin 1.55 b'y 3.60 a~x 4.18 a,wx 4.65 a~wx 4.43 ab,wx 5.28 ab,w 5.43 ab,w 5.57 b~w 5.65 ab,w 5.12 ab,wx
0.3% nisin 1.99 b~w 2.44 a~w 2.79 a~w 3.46 a~w 3.94 ab,w 4.95 b~w 4.96 b~w 4.31 C~w 4.52 be~w 4.94 ab,w
0.4% nisin 1.28 b'z 2.73 ayvz 3.43 a,xy 3.86 a~wxy 4.80 ab,wxy 5.41 ab,wx 5.18 b,wx 5.66 b~w 5.29 abe,wx 5.11 ab,wx
0.5% nisin 1.32 b~wx 1.02 a"x 1.59 a~wx 3.07 a"wx 3.13 b~wx 4.20 b~w 4. 19 b~w 4.23 c"w 3.48 c~wx 3.00 b~wx
a-c means in same column with different superscripts are significantly different
w-z means in same row with different superscripts are significantly different
(P < 0.05)









Table 4-5. Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham supplemented with
various concentrations of nisin and inoculated with Listeria monocytogenes and
analyzed prior to storage
Day 0
Treatments
Logo CFU/g
Negative control 2.28b
Positive control 5.04 a
0.2% nisin 1.91 bc
0.3% nisin 1.57 bcd
0.4% nisin 1.07 cd
0.5% nisin 0.58 d
a-c values in same column with different superscripts are significantly different
(P < 0.05)









CHAPTER 5
EVALUATION OF THE ANTI-LISTERIAL PROPERTIES OF NISIN, ROSEMARY AND
EDTA ON READY-TO-EAT TURKEY HAM STORED AT 4+10C FOR 28 DAYS

Introduction

The presence of Listeria nzonocytogenes in the food processing chain is evident by its

widespread distribution in processed products (107). Post-processing contamination of cured

meat products with L. nzonocytogenes may occur in processing plants (43) and currently

represents one of the highest meat safety risks (18). Numerous sporadic and outbreak cases of

foodborne illness have been linked to consumption of ready-to-eat (RTE) products contaminated

with L. nzonocytogenes (55). Sanitizing practices and HACCP programs applied in the meat

industry are often insufficient to prevent presence or inhibit growth of L. nzonocytogenes in

processed meats; therefore, post-packaging hurdle technologies are needed for its control (10).

In developing a novel post-processing antimicrobial treatment for use in the food industry

there are two constant problems: the limited range of bacteria which is sensitive to particular

agents and the high concentration of agents required to inhibit growth (54). The rationale for the

increased effectiveness of combinations of antimicrobials is that simultaneous attack on different

targets in the bacterial cell is more difficult for the bacteria to overcome. The use of

antimicrobials with different mechanisms of action can also be expected to expand the range of

organisms that may be inhibited.

Nisin, a polypeptide bacteriocin produced by Lactococcus lactis subsp. lactis, is a

generally recognized as safe substance (131). The mechanism of nisin activity has been shown

to involve alteration of the cell membrane of sensitive organisms resulting in leakage of low

molecular weight cytoplasmic components and the destruction of the proton motive force (PMF)

(4, 50). These antimicrobial properties have become a focus in the food science field and

researchers have found that nisin is not only effective against Gram positive bacteria (34) but









with the combination of a food grade chelator, such as ethylenenediaminetetraacetate (EDTA),

its effectiveness can be extended to Gram negative bacteria (115). EDTA a chelator, can have

antimicrobial effect by limiting the availability of cations and can act to destabilize the cell

membrane of bacteria by completing divalent cations which act as salt bridges between

membrane macromolecules, such as lipopolysaccharides (118, 135).

The use of edible plants, as well as their phytochemicals, in food preservation and

improvement of organoleptic qualities of certain traditional foods has been practiced for

centuries. These antimicrobial properties derived from many plant essential oils have been

empirically recognized for centuries but only scientifically confirmed in the last 30 years (95).

The demand for minimally processed and extended shelf life foods has further increased the

interest to define these naturally occurring bioactive ingredients; however, their strong flavor

limited their use in foods. The rosemary extract has shown antimicrobial properties against food

spoilage and foodborne pathogenic microorganism and its antibacterial activity has been linked

to the compounds extracted with hexane, which are presumably phenolic diterpenoids (33).

Cured meat products may be an excellent system in which to use nisin, rosemary and

EDTA combination treatments, since the presence of other growth restrictive chemicals and

conditions, such as nitite and NaC1, may increase the effectiveness of antimicrobial treatment

against spoilage flora and pathogens (54).

Preliminary experiments were conducted to determine the inhibitory concentration in agar

media of different antimicrobial agents. The antimicrobials nisin, rosemary and EDTA were

selected for further study to determine their effectiveness when used alone or in combinations in

a meat matrix.









Materials and Methods

This study was conducted in two trials during which 0.2% nisin, 1% rosemary and 20 mM

EDTA alone or in a combination were used to treat Ready-To-Eat turkey ham inoculated with

five strains of Listeria monocytogenes. The trials were conducted at the University of Florida

Meat Processing Laboratory and Microbiology Laboratory, Gainesville, Florida. The same

procedure was used on both trials.

Inoculum Cultivation and Storage

Reference strains of Listeria monocytogenes '/ a, '/ b, 4 b, Scott A and 191 15, were

obtained from ABC Research Corporation in Gainesville, FL and used as the inoculum to

evaluate the anti-Listeria properties of nisin, rosemary and EDTA.

The media and materials used for the cultivation, growth and maintenance of the strains

were purchased from Fisher Scientific (Pittsburgh, PA 15238). The strains were transferred

individually to test tubes containing 10 mL of tryptic soy broth (TSB, Difco Laboratories,

Detroit, MI 482132-7058, Cat.No. DF 0369-17-6) using a flamed sterilized 3mm inoculation

loop. The broth was incubated at 350C for 24 hours. After incubation the aliquots were poured

into sterile centrifuge tubes and centrifuged (Sorvall RC-5B, Dupont Instruments, Newton, CT

06470) at 5000 rpm for 10 minutes. After centrifuging, the supernatants were discarded and the

pellets were re-suspended in 10 mL of sterile distilled water and centrifuged again. The

supernatants were again discarded and the pellets were re-suspended in 1 mL of 3% TSB with

30% glycerol in a 2 mL cryovial (Corning Incorporated, Corning, NY 14831, Cat.No. 03-374-

21). The pellets were stored at -450C and used as the stock culture for the inoculation studies.

Inoculum Preparation

Frozen Listeria monocytogenes strains were allowed to thaw at room temperature for 10

minutes. A loopful of the thawed stock culture was transferred to test tubes containing 10 mL of









3% TSB and incubated at 350C for 24 hours. After incubation, the aliquots were centrifuged

(5000 rpm for 10 min at 160C) and washed with sterile 0. 1% buffered peptone water (BPW,

Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4). The aliquots were then

serially diluted with BPW to concentrations of 10-1 to 10-s

Antimicrobial Solutions Preparation

Formulations were developed to prepare 0.2% Nisin, 20 mM EDTA and 1% rosemary

solutions (based on 100% total batch weight) to ensure that desired concentrations of each

solution made contact with chopped turkey ham. The control turkey ham formulation consisted

of the following (% of total weight in the formulation): chopped turkey ham (90%) and water

(10%) (Table 5-1). Each antimicrobial was dissolved using 10% sterile water, according to each

treatment formulation, which was added into the bag containing the chopped turkey ham. Sterile

deionized water was used as control.

Nisaplin@~ (Danisco, Copenhagen, Denmark) is a commercial nisin product containing 106

IU nisin/g. A concentration of 0.2% nisin was obtained by adding 0.5 g of Nisaplin@, 0.17 mL

of 0.02 N food grade HCL (Fisher Scientific, Pittsburgh, PA 1523 8, Cat. No. 7647-01-0) and

0.75g NaCl (Sigma Chemical, St. Louis, MI 63178, Cat No. S9625-500G) to the turkey ham.

EDTA (Sigma Chemical., St. Louis, MI 63 178, Cat No. 59HO3 591) was prepared at a

concentration of 20 mM by adding 0.75 g to the turkey ham total batch weight. Herbalox@

Seasoning (Kalsec, Kalamazoo, MI 49005-0511, Code No. 41-19-02) is a commercial rosemary

extract that has shown antimicrobial properties as well as inhibition of oxidative deterioration.

Rosemary solution was prepared applying 1 mL of the Herbalox@ to the turkey ham.

Combination of 0.2% nisin and 1% rosemary was prepared by adding 0.2 g of Nisaplin@

0. 17 mL of 0.02 N food grade HCL, 0.75g NaC1, and 1 mL of rosemary extract. Combination of

0.2% nisin, 1% rosemary and 20 mM EDTA was achieved by applying 0.2 g of Nisaplin@, 0.17









mL of 0.02 N food grade HCL, 0.75g NaC1, 1 mL of rosemary extract, and 0.75 g of EDTA.

The antimicrobial solutions were stored at 40C and used within 3 days

Sample Preparation

Jennie-O ready-To-Eat turkey hams were purchased from Publix, Gainesville, Florida with

an expiration date of 60 days. The turkey hams were immediately transported to the University

of Florida Meat Processing Laboratory and Microbiology Laboratory, Gainesville, Florida and

stored at 40C for no longer than 24 hours before using. The hams were aseptically removed from

the vacuum packaged bag and placed on pre-sterilized trays (polypropylene) and chopped into

approximately 0.5 cm pieces. The ham was chopped to simulate how it may be used in ham

salad or similar foods.

Inoculation and Treatment

The turkey ham pieces were placed on pre-sterilized trays and inoculated by spraying them

with 1 mL of L. monocytogenes inoculum at 1.0 X 10s CFU per gram of ham. Inoculated

samples were left to stand at room temperature for 20 min to allow for bacterial attachment and

to ensure a final concentration of 104 CFU/gram.

For each treatment, duplicate 25 grams inoculated chopped turkey ham samples were

aseptically weighed, placed in a labeled FoodSaver bag (Tilia, San Francisco, California,

FoodSaver Vacloc Roll) and vacuum packaged (Tilia, San Francisco, California, FoodSaver

Bagvac). The inoculated samples were treated with either nisin, EDTA, nisin with rosemary, or

nisin with rosemary and EDTA (Table 5-1), leaving the antimicrobial solution in the package.

Samples were stored in a 40C cooler for subsequent microbiological and chemical analysis.

Samples were analyzed after 0, 7, 14, 21, and 28 days storage.









Microbiological Analyses

Twenty-Hyve grams of chopped turkey ham were transferred aseptically from the vacuum

packaged bag into a sterile stomacher bag (Fisher Scientifie, Pittsburgh, PA 15238, Cat. No. 01-

002-44) with 225 mL of sterile 0. 1% buffered peptone water (BPW, Difco, Laboratories, Detroit,

MI 48232-7058, Cat. No. DF 01897-17-4) and shaken approximately 30 times. Further serial

dilutions were prepared by transferring 1 mL of the diluted sample homogenate into 9 mL of

0. 1% BPW until the appropriate dilutions were obtained.

Listeria monocytogenes, lactic acid bacteria and anaerobic bacteria analyses

A volume of 0. 1 mL of the dilutions was dispensed onto pre-poured Modified Oxford

Media (MOX, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0225-17-0) containing

Modified Oxford antimicrobial supplement (MOX supplement, Difco Laboratories, Detroit, MI

48232-7058, Cat. No. DF 0218-60-5) for L. monocytogenes identification, lactobacilli MRS agar

(Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0882170) for the isolation of lactic

acid bacteria, and anaerobic agar (Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF

0536-17-4) for anaerobic bacteria identification. The spread plate technique was used to evenly

distribute the sample over the plate. Using this technique, a flamed sterilized bacterial cell

spreader (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 08-769-2A) was used to spread the

sample over the plate as the plate was spun on an inoculating turntable (Fisher Scientific,

Pittsburgh, PA 15238, Cat. No. 08-758-10). All samples were plated in duplicate. The Petri

plates were inverted and incubated for 24 hours at 350C for L. monocytogenes and 370C for

lactic acid bacteria identification. For anaerobic bacteria, the petri plates were inverted and

placed in a sealed j ar (GasPak j ar system, Fisher Scientific, Pittsburgh, PA 1523 8, Cat. No. 1 1-

814-22) with an AnaeroGen sachet (Remel, Lenexa, KS 66215, Cat. No. 653 5) to create an

anaerobic environment. The anaerobic agar plates were then incubated at 350C for 48 hours. All









Plates with 25 to 250 colonies were counted. Black colonies surrounded by a black halo,

white/grayish colonies and white colonies were considered presumptively L. monocytogenes,

lactic acid bacteria and anaerobic bacteria, respectively. Microbiological counts were reported as

Logarithmic Colony Forming Units per gram (Log CFU/g).

Aerobic bacteria analysis

Aerobic bacteria counts were performed using 3M Petrifilm aerobic count plates (St. Paul,

Minnesota, Cat. No. 6404) for all treatments prior to storage on day 0. The Petrifilm aerobic

count plate was placed on a level surface. The top film was lifted and 1 mL of sample was

dispensed to the center of the bottom of the plate. The top film was released down onto the

sample and the plastic spreader was placed on the center of the plate, convex side down. After

approximately one minute, the spreader was removed from the plate. All samples were plated in

duplicate. Plates were incubated for 24 hours at 250C in a horizontal position with the clear side

up in stacks of 12 plates. Plates containing 15 to 150 colonies were counted and recorded.

Microbiological counts were expressed as Logarithmic Colony Forming Units per gram (Log

CFU/g) .

pH Analysis

The pH analysis of the chopped Ready-To-Eat turkey ham was performed using a pH

meter (Accument basic ABl15, Fisher Scientific, Pittsburgh, PA 15238, Model No. ABl15, Serial

No. AB81210535). Twenty-five grams of chopped Ready-To-Eat turkey ham was aseptically

removed from the vacuum packaged bag and placed into a sterile plastic bag (Fisher Scientific,

Pittsburgh, PA 1523 8, Cat. No. 01-002-44) into which 225 mL of 0. 1% buffered peptone water

(BPW, Difco, Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) was added.

Duplicate pH measurements were taken from each ham homogenate.









Data Analysis

Statistical analyses were conducted for enumeration data of a total of eight measurements

per treatment of L. monocytogenes, aerobic bacteria, anaerobic bacteria, and lactic acid bacteria.

Also, statistical analysis was conducted for pH values of six measurements per treatment. The

general linear model program (PROC GLM) of SAS@ system (110) was employed to

statistically analyze trial, day, treatment and treatment by day. Variations in data were accounted

for by four treatment effects: trial, treatment, day, and treatment~day. Comparisons among

means were performed using SAS@ Tukey Multiple Range test procedure. Treatments effects

and differences were considered significant when P < 0.05.

Results and Discussion

pH Analysis

On day 0, the pH of turkey ham treated with a combination of nisin, rosemary and EDTA

and EDTA alone were significantly lower (P < 0.05) than all other treatments (Table 5-2). From

days 14 to 28, the pH was similar (P > 0.05) for all treatments. Bacterial growth on turkey ham,

stored under vacuum packaging, may result in the production of numerous compounds such as

acidic metabolites and carbonic acid that may decrease the pH (37). This may explain the slight

decrease in pH of the controls and the antimicrobial treated samples over time in our study.

Also, the pH change observed with nisin, EDTA, nisin combined with rosemary and nisin

combined with rosemary and EDTA treatments may have been a comprehensive effect of pH of

the initial treatment solution, beef buffering capacity, and bacterial growth products. The overall

pH values of the treatments in this study did differ throughout the 28 days of storage. This was

in agreement with a study by Cutter and Siragusa (23) in which a significant difference was seen

in the controls and nisin treated samples between different days. Because changes in pH were









not consistent with changes in bacterial population, pH alone could not be considered as a key

factor for population reduction in this study.

Listeria monocytogenes Analysis

Throughout the 28 days, the data revealed similar (P > 0.05) L. monocytogenes counts for

the positive control and turkey ham treated with EDTA (Table 5-3). Turkey ham treated with

nisin alone or in combination with rosemary or with rosemary with EDTA, resulted in

significantly (P < 0.05) reduced L. monocytogenes counts when compared to the positive control

and hams treated with EDTA during 28 days.

On day 0, the initial bacterial population of the positive control (4.87 log CFU/g) was

reduced (P< 0.05) to 1.14 log CFU/g with 0.2% nisin, 2.54 log CFU/g with nisin combined with

rosemary, and 1.75 log CFU/g with nisin combined with rosemary and EDTA. Through out the

28 days of storage, the number ofL. monocytogenes increased to approximately 0.75 log CFU/g

in the nisin with rosemary and EDTA treated sample. The counts remain similar for nisin and

nisin with rosemary treated samples. During the 28 days of storage a significant difference (P <

0.05) existed in population reduction of nisin, nisin with rosemary and nisin with rosemary and

EDTA when compared with the positive control. By day 28, L. monocytogenes counts on turkey

ham were reduced by 2.89 log CFU/g with nisin treatment, 1.43 log CFU/g with nisin combined

rosemary treatment and 1.25 log CFU/g with nisin combined rosemary and EDTA treatment

when compared to the positive control (3.84 log CFU/g). These findings suggested that nisin,

nisin combined with rosemary and nisin combined with rosemary and EDTA may have an initial

bacterial effect against L. monocytogenes population and that these solutions may help to

maintain the L. monocytogenes counts lower during storage.

EDTA exhibited no inhibitory effect on L. monocytogenes through 28 days storage at 4+1

oC. Divalent cations such as Ca2+ and Mg2+ play specific roles in stabilizing the structure of









bacterial membranes because they form metal ion bridges between phosphate groups of

phopholipids and the carboxyl groups of membrane protein (135). In gram negative organisms,

it has been shown that EDTA damages outer membrane structure by completing these Ca2+ and

Mg2+ cations which are necessary for them to live (118). However, membrane structure of gram

negative bacteria is different from gram positive bacteria. Gram positive bacteria will have a

thick layer of peptidoglycan (a sugar-protein shell) that results in resistance to physical

disruption. Gram negative bacteria have a thin exterior peptidoglycan membrane. The outer

membrane is composed of lipid and lipid protein content which is the primary target of EDTA

(37). This may be the reason that EDTA did not inhibit the growth of L. monocytogenes under

the conditions used in this study. The fact that nisin combined with rosemary and EDTA had an

anti-listerial effect through out the 28 days of storage at 40C, indicated that the observed

population reductions were attributed to the inhibitory activity of nisin or rosemary rather than

EDTA. Similar observations have been reported (96, 115), where in EDTA lacked inhibition

against gram positive bacteria, and when EDTA combined with nisin reduced the antimicrobial

effect of nisin.

Anaerobic Bacteria Analysis

Significant differences existed among treatments (P < 0.05) in anaerobic bacteria counts

(Table 5-4). Except for samples treated with 20 mM of EDTA only, untreated turkey ham

(positive and negative controls) possessed significantly higher (P < 0.05) anaerobic bacteria

counts when compared to all treated ham samples.

Similar (P > 0.05) anaerobic bacteria counts were observed for the positive control,

negative control and samples treated with EDTA only throughout 28 days storage. Treatment

with nisin alone caused significant (P < 0.05) population reduction as compared to all other

treatments during 28 days storage at 40C. By day 28, nisin alone and in combination with









rosemary and EDTA had significantly lower (P < 0.05) anaerobic bacteria counts when

compared to all other treatments. From day 0 to day 21, nisin combined with rosemary was

significantly lower (P < 0.05) when compared to the positive control. However, by day 28 the

population of anaerobic bacteria increased and no significant differences (P > 0.05) were

observed between nisin combined with rosemary and positive control.

Results from the present study suggest that ready-to-eat turkey ham will have decreased

numbers of anaerobic bacteria when treated with any of the following: 1) 0.2% nisin, 2) nisin

combined with rosemary and EDTA. Therefore, the present study suggests that the presence of

an increased level (0.2%) of nisin alone or in combination with 1% rosemary and 20 mM EDTA,

effectively reduce the growth of anaerobes.

Lactic Acid Bacteria Analysis

The data revealed similar results for lactic acid and anaerobic bacteria. Samples treated

with nisin and nisin combined with rosemary and EDTA resulted in significantly lower (P <

0.05) lactic acid bacteria when compared to the controls and EDTA alone (Table 5-5). Samples

treated with nisin alone and in combination with rosemary and EDTA controlled the anaerobic

bacteria growth during 28 days storage when compared to all other treatments.

The number of bacteria recovered from samples treated with nisin combined with rosemary

and EDTA alone were not significantly different (P > 0.05) when compared to positive control.

This may suggest that combination of nisin with rosemary and EDTA alone had no antimicrobial

effect against gram positive bacteria.

Results from the study revealed that lactic acid bacteria may be controlled in vacuum

package ready-to-eat turkey ham stored at 40C when treated with at least 0.2% nisin or 0.2%

nisin combined with 1% rosemary and 20 mM of EDTA.









Aerobic Bacteria Analysis

Aerobic bacteria counts were significantly higher (P < 0.05) in the positive control and

EDTA treatment when compared to all other treatments (Table 6-6). This may suggest that

EDTA has no immediate antimicrobial effect against the aerobic organisms present on the turkey

ham. The antimicrobial treatment that contained 0.2% nisin was the most effective (P < 0.05)

reducing the initial populations of aerobic bacteria. In addition when combinations of nisin with

rosemary and nisin with rosemary and EDTA were used a significant (P < 0.05) reduction was

observed when compared to the positive control. These results suggest that initial population of

aerobic bacteria may be restricted when treated with nisin and combinations of nisin with

rosemary or nisin with rosemary and EDTA under vacuum package and refrigeration (40C)

conditions.










Table 5-1. Formulation of nisin, rosemary and EDTA solutions for vacuum packaged ready-to-
eat turkey ham stored at 411 OC for 28 days
Percentage (%) of ingredient in total composition
Negative Positive Nisin + EDTA Nisin +.
Treatment 12Nismn EDTA
Control' Control" Rosemary Rosemary
Ingredient
Ham 90.00 90.00 87.13 87.88 88.88 89.25
Water 10.00 10.00 10.00 10.00 10.00 10.00
Rosemary 0.00 0.00 1.00 1.00 0.00 0.00
EDTA 0.00 0.00 0.75 0.00 0.00 0.75
Nisin 0.00 0.00 0.20 0.20 0.20 0.00
NaCl 0.00 0.00 0.75 0.75 0.75 0.00
HCI 0.00 0.00 0.17 0.17 0.17 0.00
100.00 100.00 100.00 100.00 100.00 100.00
SNegative control: without L. monocytogenes inoculum
2 Positive control: with L. monocytogenes inoculum









Table 5-2. Mean pH values on Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 411 OC for 28 days
Day 0 Day 7 Day 14 Day 21 Day 28
Treatments
(Loglo CFU/g)
Negative control 6.19 a~x 5.98 a~x 5.30 ay 4.92 a~z 4.92 a~z
Positive control 5.96 a~x 5.73 ab,xy 5.42 a~x 5.18 ay 5.25 a~xy
Nisin + EDTA+ rosemary 5.53 b~x 5.67 b~x 5.80 ax" 5.71 ax" 5.72 a"x
Nisin + rosemary 6.08 a~x 5.83 ab~x 5.24 asY 5.37 asY 4.95 as~
Nisin 6.08 a~x 5.93 ab,xy 5.87 a xv 5.50 a xy 5.42 asy
EDTA 5.92 ab~x 5.73 ab~x 5.57 ax" 5.24 ax" 5.36 a"x
a-b values in same column with different superscripts are significantly different
x-z values in same row with different superscripts are significantly different
(P < 0.05)










Table 5-3. Mean Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and stored at 411 OC for 28 days
Day 0 Day 7 Day 14 Day 21 Day 28
Treatments
(Loglo CFU/g)
Negative control 0.00 ex 0.00 d"x 0.00 d~x 0.00 d~x 0.00 drx
Positive control 4.87 ax 4.30 ay 3.84 az 4. 14 ayz 3.84 az
Nisin + EDTA+ rosemary 1.75 "z 1.50 "z 1.68 ez 2.91 b'y 2.59b'y
Nisin + rosemary 2.54 bx 2.37 bx 2.45 bx 2.62 bx 2.41 bx
Nisin 1.14 d,yz 1.25 CYZ 0.50 dz 1.37 CY 0.95 CYZ
EDTA 5.19 ay 4. 10 az 3.92 az 3.84 az 4.07 az
a-e values in same column with different superscripts are significantly different
x-z values in same row with different superscripts are significantly different
(P < 0.05)










Table 5-4. Mean anaerobic plate count on Ready-To-Eat turkey ham inoculated with Listeria


Day 14
(Loglo CFU/g)
5.85ab~xy
6.34 a~x
4.36 c~x
5.05 be,xy
2.69 d,xy
6. 10 ab~x


monocytogenes and stored at 411 OC for 28 dayl
s Day 0 Day 7


Day 21 Day 28


Treatment!


Negative control
Positive control
Nisin + EDTA+ rosemary
Nisin + rosemary
Nisin
EDTA


2.69 b~z
4.81 a~z
1.77 be,y
2.24 b~z
0.29 c"z
4.85 as


4.40 ab~y
6.37 a~x
3.65 ed~x
4. 12 b~y
1.93 d'y
6.09 ab~x


5.98 ab,xy
6.61 a~x
4.87 c"x
5.30 be,x
3.33 d~x
5.89 abe"x


6. 10 a~z
5.89 a~Y
4.09 b~x
6. 10 a~x
3.55 b~x
5.27 as


a-d values in same column with different superscripts are significantly different
x-z values in same row with different superscripts are significantly different
(P < 0.05)










Table 5-5. Mean lactic acid bacteria counts on Ready-To-Eat turkey ham inoculated with Listeria
monocytogenes and stored at 411 OC for 28 days


Day 28


Day 0 Day 7 Day 14 Day 21
Treatments
(Loglo CFU/g)
Negative control 3.97abz 6.60" 7. 16" 7.20
Positive control 5.19"z 6. 19"' 6.88 a 6.63 a'
Nisin + EDTA+ rosemary 2.90 bz 4.40 b'y 4.61b'y 4.76 b'y
Nisin + rosemary 3.88 ab,z 5.65 a' 7. 14 a 7.05 "
Nisin 1.27 "z 1.39 "z 3.76 3.88 b'y
EDTA 5.20 aZ 6.01 "XY 6.46 aX 6.53 "X
a-c values in same column with different superscripts are significantly different
w-z values in same row with different superscripts are significantly different
(P < 0.05)


6.68
6. 12"
4.23 bc y
6.27 ax
3.28 7Y
5.52 ab yz









Table 5-6. Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes
Day 0
Treatments
Logo CFU/g
Negative control 3.69b
Positive control 5.43 a
Nisin + EDTA+ rosemary 2.44 b
Nisin + rosemary 3.70 b
Nisin 0.85 0
EDTA 5.67 a
a-c values in same column with different superscripts are significantly different
(P < 0.05)









CHAPTER 6
EVALUATION OF THE ANTI-LISTERIAL PROPERTIES OF 0.5 % NISIN, 1 %
ROSEMARY AND 20 MM EDTA ON READY-TO-EAT TURKEY HAM STORED AT 4+10C
FOR 63 DAYS

Introduction

Listeria monocytogenes is a foodborne pathogen which is widely distributed in nature and

whose control in food is made difficult by its ability to grow at temperatures ranging from 0 to

450C (7), its high tolerance for salt (42), and its ability to initiate growth at a relatively low pH

(9) .

Recently, numerous outbreaks have been linked to consumption of ready-to-eat (RTE)

products contaminated with L. monocytogenes (55). Contamination of the RTE meat products

may occur in processing plants. The heat treatment (cooking) that RTE meat and poultry

products undergo eliminates the pathogen; however, recontamination may occur during post-

processing exposure to the environment (e.g. peeling, slicing, and repackaging) (43). For this

reason, new post-processing hurdle technologies that control or eliminate the incidence of

foodborne pathogen are needed for the meat industry (10).

The bacteriocin, nisin, has been used as an antimicrobial in foods since the 1960's (83).

Nisin is produced by the lactic acid bacteria (LAB) Lactococcus lactis (5). The mechanism of

nisin activity has been shown to involve alteration of the cell membrane of sensitive organisms

resulting in the leakage of low molecular weight cytoplasmic components (4, 50, 138) and

destruction of the proton motive force (PMF) (13, 49).

It has been recognized that susceptibility of Gram negative organisms may be increased by

the use of membrane disrupting agents, such as detergents and chelators (115). Chelators bind

magnesium ions in the lipopolysaccharide layer of bacterial cell walls and increase susceptibility

of the cells to nisin (118). Ethylenenediaminetetraacetic acid (EDTA) is a well known reagent









used in various foods for different functions. Those functions may include the retardation of

crystal formation, food preservative and stabilizer, antioxidant, and chelating and sequestering

agent (139). EDTA can have antimicrobial effect by limiting the availability of cations and can

act to destabilize the cell membrane of bacteria by completing divalent cations which act as salt

bridges between membrane macromolecules, such as lipopolysaccharides (118, 135).

Society appears to be experiencing a trend of "natural" consumerism (121, 129), desiring

fewer synthetic food additives and products with a smaller impact on the environment.

Therefore, there is a need for new methods to make food safe which have a natural image. One

such possibility is the use of essential oils as antibacterial additives (117). Essential oils (EOs)

are aromatic oily liquids obtained from plant material (flowers, buds, seeds, leaves, twigs, bark,

herbs, wood, fruits and roots). They can be obtained by fermentation or extraction but the

method of steam distillation is most commonly used for commercial production of EOs (136).

There are approximately 3000 EOs known, of which about 300 are commercially important, and

are destined primarily for the flavors and fragrances market (136). It has long been recognized

that some EOs have antimicrobial properties (95) and the relatively recent interest in natural

consumerism has lead to a renewal of scientific interest in these substances (92, 129). The

rosemary (Rosmarinus officinalis) extract has shown antimicrobial properties against food

spoilage and foodborne pathogenic microorganisms. Rosemary's antibacterial activity has been

linked to a-pinene, bornyl acetate, camphor, and 1,8-cineole (33, 97).

Preliminary studies were conducted to determine the inhibitory concentration in agar

media of different antimicrobial agents. The antimicrobials nisin, rosemary and EDTA were

selected for further study to determine their effectiveness in a meat matrix. This work was

undertaken to develop a new post-processing hurdle technology by applying nisin, rosemary and









EDTA alone or in combination directly to the finished RTE turkey product immediately prior to

packaging.

Materials and Methods

This study was conducted in two trials during which 0.5% nisin, 1% rosemary and 20 mM

EDTA solutions were used alone and in combination to treat RTE turkey ham inoculated with

five strains of Listeria monocytogenes. The trials were conducted at the University of Florida

Meat Processing Laboratory and Microbiology Laboratory, Gainesville, Florida. The same

procedure was used on both trials.

Inoculum Cultivation and Storage

Reference strains of Listeria monocytogenes '/ a, '/ b, 4 b, Scott A and 191 15, were

obtained from ABC Research Corporation in Gainesville, FL and used as the inoculum to

evaluate the anti-Listeria properties of different concentrations of nisin. The media and materials

used for the cultivation, growth and maintenance of the strains were purchased from Fisher

Scientific (Pittsburgh, PA 15238). The strains were transferred individually to test tubes

containing 10 mL of tryptic soy broth (TSB, Difco Laboratories, Detroit, MI 482132-7058,

Cat.No. DF 0369-17-6) using a flamed sterilized 3mm inoculation loop. The broth was

incubated at 350C for 24 hours. After incubation the aliquots were poured into sterile centrifuge

tubes and centrifuged (Sorvall RC-5B, Dupont Instruments, Newton, CT 06470) at 5000rpm for

10 minutes. After centrifuging, the supernatants were discarded and the pellets were re-

suspended in 10 mL of sterile distilled water and centrifuged again. The supernatants were again

discarded and the pellets were re-suspended in 1 mL of 3% TSB with 30% glycerol in a 2 mL

cryovial (Corning Incorporated, Corning, NY 1483 1, Cat.No. 03 -3 74-21). The pellets were

stored at -450C and used as the stock culture for the inoculation studies.









Inoculum Preparation

Frozen Listeria monocytogenes strains were allowed to thaw at room temperature for 10

minutes. A loopful of the thawed stock culture was transferred to test tubes containing 10 mL of

3% TSB and incubated at 350C for 24 hours. After incubation, the aliquots were centrifuged

(5000 rpm for 10 min at 160C) and washed with sterile 0. 1% buffered peptone water (BPW,

Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4). The aliquots were then

serially diluted with BPW to concentrations of 10-1 to 10-s

Antimicrobial Solutions Preparation

Formulations were developed to prepare 0.5% Nisin, 20 mM EDTA and 1% rosemary

solutions (based on 100% total batch weight) to ensure that desired concentrations of each

solution made contact with chopped turkey ham. The control turkey ham formulation consisted

of the following (% of total weight in the formulation): chopped turkey ham (90%) and water

(10%) (Table 6-1). Each antimicrobial was dissolved using 10% sterile water, according to each

treatment formulation, which was added into the bag containing the chopped turkey ham. Sterile

deionized water was used as control.

Nisaplin@~ (Danisco, Copenhagen, Denmark) is a commercial nisin product containing 106

IU nisin/g. A concentration of 0.5% nisin was obtained by adding 0.5 g of Nisaplin@, 0.17 mL

of 0.02 N food grade HCL (Fisher Scientific, Pittsburgh, PA 1523 8, Cat. No. 7647-01-0) and

0.75g NaCl (Sigma Chemical, St. Louis, MI 63178, Cat No. S9625-500G) to the turkey ham.

EDTA (Sigma Chemical., St. Louis, MI 63 178, Cat No. 59HO3 591) was prepared at a

concentration of 20 mM by adding 0.75 g to the turkey ham total batch weight. Herbalox@

Seasoning (Kalsec, Kalamazoo, MI 49005-0511, Code No. 41-19-02) is a commercial rosemary

extract that has shown antimicrobial properties as well as inhibition of oxidative deterioration.

Rosemary solution was prepared applying 1 mL of the Herbalox@ to the turkey ham.









Combination of 0.5% nisin and 1% rosemary was prepared by adding 0.5 g of Nisaplin@

0. 17 mL of 0.02 N food grade HCL, 0.75g NaC1, and 1 mL of rosemary extract. Combination of

0.5% nisin, 1% rosemary and 20 mM EDTA was achieved by applying 0.5 g of Nisaplin@, 0.17

mL of 0.02 N food grade HCL, 0.75g NaC1, 1 mL of rosemary extract, and 0.75 g of EDTA.

Combination of 5% nisin and 20 mM EDTA was obtained by putting 0.5 g of Nisaplin@, 0. 17

mL of 0.02 N food grade HCL, 0.75g NaC1, and 0.75 g of EDTA. Combination of 1% rosemary

and 20 mM EDTA was achieved by adding 1 mL of rosemary extract and 0.75 g of EDTA. The

antimicrobial solutions were stored at 40C and used within 3 days.

Sample Preparation

Jennie-O RTE turkey hams were purchased from Publix, Gainesville, Florida with an

expiration date of 60 days. The turkey hams were immediately transported to the University of

Florida Meat Processing Laboratory and Microbiology Laboratory, Gainesville, Florida and

stored at 40C for no longer than 24 hours before using. The hams were aseptically removed from

the vacuum packaged bag and placed on pre-sterilized trays (polypropylene) and chopped into

approximately 0.5 cm pieces. The ham was chopped to simulate how it may be used in ham

salad or similar foods.

Inoculation and Treatment

The turkey ham pieces were placed on pre-sterilized trays and inoculated by spraying them

with 1 mL of L. monocytogenes inoculum at 1.0 X 10s CFU/mL. Inoculated samples were left to

stand at room temperature for 20 min to allow for bacterial attachment and to ensure a final

concentration of 104 CFU/gram.

Duplicate samples of inoculated chopped turkey ham were aseptically weighted according

to its corresponding treatment (Table 1) and placed in a labeled Food Saver bag (Tilia, San

Francisco, California, FoodSaver Vacloc Roll). Corresponding treatments were applied to the










chopped ham followed by mixing the ham and treatment solution to ensure a proper distribution

between them. The bags were then vacuum packaged (Tilia, San Francisco, California,

FoodSaver Bagvac), leaving the antimicrobial solution in the package, and stored in a 40C cooler

for subsequent microbiological, chemical and color analysis. Samples were analyzed after 0, 7,

14, 21, 28, 35, 42, 49, 56 and 63 days of storage.

Microbiological Analyses

Twenty-Hyve grams of chopped turkey ham were transferred aseptically from the vacuum

packaged bag into a sterile stomacher bag (Fisher Scientifie, Pittsburgh, PA 15238, Cat. No. 01-

002-44) with 225 mL of sterile 0. 1% buffered peptone water (BPW, Difco, Laboratories, Detroit,

MI 48232-7058, Cat. No. DF 01897-17-4) and shaken approximately 30 times. Further serial

dilutions were prepared by adding 1 mL of the diluted sample homogenate into 9 mL of 0. 1%

BPW until the appropriate dilutions were obtained.

Aerobic bacteria analysis

Aerobic bacteria counts were performed using 3M Petrifilm aerobic count plates (St. Paul,

Minnesota, Cat. No. 6404) to all treatments prior to storage on day 0. The Petrifilm aerobic

count plate was placed on a level surface. The top fi1m was lifted and 1 mL of sample was

dispensed to the center of the bottom of the plate. The top film was released down onto the

sample and the plastic spreader was placed on the center of the plate, convex side down. After

approximately one minute, the spreader was removed from the plate. All samples were plated in

duplicate. Plates were incubated for 24 hours at 250C in a horizontal position with the clear side

up in stacks of 12 plates. Plates containing 15 to 150 colonies were counted and recorded.

Microbiological counts were expressed as Logarithmic Colonic Forming Units per gram (Log

CFU/g) .









Listeria monocytogenes and lactic acid bacteria analysis

A volume of 0. 1 mL of the dilutions was dispensed onto pre-poured Modified Oxford

Media (MOX, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0225-17-0) containing

Modified Oxford antimicrobic supplement (MOX supplement, Difco Laboratories, Detroit, MI

48232-7058, Cat. No. DF 0218-60-5) for L. monocytogenes identification and onto lactobacilli

MRS agar (Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0882170) for the isolation

of lactic acid bacteria. The spread plate technique was used to evenly distribute the sample over

the plate. Using this technique, a flamed sterilized bacterial cell spreader (Fisher Scientific,

Pittsburgh, PA 15238, Cat. No. 08-769-2A) was used to spread the sample over the plate as the

plate was spun on an inoculating turntable (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 08-

758-10). All samples were plated in duplicate. The petri plates were inverted and incubated for

24 hours at 350C for L. monocytogenes and 370C for lactic acid bacteria. Plates with 25 to 250

colonies were counted. Black colonies surrounded by a black halo and white/grayish colonies

were considered presumptively L. monocytogenes and lactic acid, respectively. Microbiological

counts were reported as Logarithmic Colonic Forming Units per gram (Log CFU/g).

pH Analysis

The pH analysis of the chopped RTE turkey ham was performed using a pH meter

(Accument basic ABl15, Fisher Scientific, Pittsburgh, PA 15238, Model No. ABl15, Serial No.

AB81210535). Twenty-five grams of chopped ham was aseptically removed from the vacuum

packaged bag and placed into a sterile plastic bag (Fisher Scientific, Pittsburgh, PA 15238, Cat.

No. 01-002-44) into which 225 mL of 0. 1% buffered peptone water (BPW, Difco, Laboratories,

Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) was added. Duplicate pH measurements were

taken from each ham homogenate.










Color Analysis

A portable colorimeter (Minolta Chroma Meter CR310, Minolta, Ramsey, NJ 07446) was

used to obtain objective data for color of the RTE chopped turkey ham. Before each sampling

period, the machine was calibrated as recommended by the manufacturer. Duplicates samples

per treatment were evaluate for "L" (degree of lightness), "a" (degree of redness), and "b"

(degree of yellowness) values. Two locations were sampled from each replicate to obtain color

measurements.

Data Analysis

Statistical analyses were conducted for enumeration data of a total of eight measurements

per treatment of L. monocytogenes, aerobic bacteria, lactic acid bacteria, and color

measurements. Also, statistical analysis was conducted for pH values of six measurements per

treatment. The general linear model program (PROC GLM) of SAS@ system (110) was

employed to statistically analyze trial, day, treatment and treatment by day. Variations in data

were accounted for by four treatment effects: trial, treatment, day, and treatment~day.

Comparisons among means were performed using SAS@ Tukey Multiple Range test procedure.

Treatments effects and differences were considered significant when P < 0.05.

Results and Discussion

Aerobic Bacteria Analysis

No aerobic bacteria were detected in the negative control (Table 6-2). All samples treated

with nisin alone or in combination with EDTA or rosemary had significantly (P < 0.05) lower

aerobic bacteria counts. All treatments containing nisin achieved a reduction of approximately 3

log CFU/g, when compared to positive control. Aerobic bacteria counts were significantly

higher (P < 0.05) in the rosemary, EDTA, and combination of rosemary with EDTA treatments,









when compared to positive control. This may suggest that EDTA and rosemary had no

immediate antimicrobial effect against the aerobic organisms present in the turkey ham.

When treatments of nisin alone or combinations of nisin with rosemary, nisin with EDTA,

and nisin with rosemary and EDTA were used a significant (P < 0.05) reduction was observed

when compared to the positive control. The data revealed that EDTA and rosemary alone or in

combination had no antimicrobial effect against the aerobic bacteria growth. However, nisin

alone or combined with EDTA and/or rosemary exhibited an antimicrobial effect against the

aerobic bacteria. These findings suggested that the microbial reductions observed could be

attributed to the inhibitory activity of nisin rather than EDTA or rosemary. Results from this

study suggest that initial populations of aerobic bacteria may be controlled when treated with

nisin and combinations of nisin with rosemary and/or EDTA under vacuum package and

refrigeration (40C) conditions.

Listeria monocytogenes Analysis

L. monocytogenes counts were significantly reduced (P< 0.05) to 0.91 log CFU/g with

nisin, 1.60 log CFU/g with nisin combined with EDTA, 1.13 log CFU/g with nisin combined

with rosemary, and 1.22 log CFU/g with nisin combined with rosemary and EDTA, when

compared to positive control (5.33 log CFU/g). Treatments containing nisin alone or in

combination with rosemary and/or EDTA resulted in significant population reduction when

compared to the positive control (P < 0.05) during the 63 days storage at 4 + 1 OC. An extended

lag phase was observed for all treatments containing nisin. L. monocytogenes counts remained at

less than 2.66 log CFU/g through 63 days storage. These results suggested that the control of L.

monocytogenes by nisin containing treatments may be attributed to the initial bactericidal effect

rather than a constant effect. Similar observations have been reported (19), where L.









monocytogenes was sensitive to nisin but the effect of nisin was probably due to the initial

inhibition and not to continued activity of the nisin.

The results also revealed that on day 63, the population of L. monocytogenes on turkey

ham was reduced by 1.52 log CFU/g with nisin, by 1.92 log CFU/g with nisin combined with

EDTA, by 1.25 log CFU/g with nisin combined with rosemary, and by 1.32 log CFU/g with nisin

combined with rosemary and EDTA when compared to the positive control (3.91 log CFU/g).

These findings revealed that the use of nisin alone or in combination with EDTA and/or

rosemary had a bactericidal effect against L. monocytogenes population.

During the 63 days of storage, L. monocytogenes counts were similar (P < 0.05) in the

positive control, in ham treated with EDTA or rosemary alone, or with EDTA combined with

rosemary. Neither EDTA nor rosemary inhibited L. monocytogenes during the 63 days storage.

In this study, no increase in antimicrobial activity was observed when nisin was used with

rosemary and/or EDTA against L. monocytogenes. In contrast, a number of authors have

reported an increased in the antimicrobial activity of nisin against gram positive (65) and

negative bacteria (23, 126) in the presence of membrane disrupting agents. However, the

maj ority of these reports are based upon observations of organisms suspended in a buffer, rather

than growing in nutrient media. Since meat products provide a nutrient rich environment, test of

antimicrobials under conditions of cell starvation would appear to be of limited value in

evaluating them for application in food products. The failure to observe an enhancement in the

antimicrobial properties of nisin when combined with EDTA in this study may indicate that the

observed enhancement between these two antimicrobial agents in buffer systems (65) was a

consequence of cell starvation which prevents cell repair.










In gram negative organisms, it has been shown that EDTA can act to destabilize the cell

membrane of bacteria by completing divalent cations, such as Ca2+ and Mg2+, which act as salt

bridges between membrane macromolecules, such as lipopolysaccharide (118). However,

membrane structure differs between gram negative and gram positive bacteria. Gram negative

bacteria have an outer membrane that covers a thin layer of peptidoglycan on the outside. The

primary target of EDTA is the lipids and lipid proteins present in the outer membrane of gram

negative bacteria (37). In contrast, gram positive bacteria will have a thick layer of

peptidoglycan (a sugar-protein shell) that confers resistance to physical disruption (43). This

may be the reason that EDTA did not inhibit the growth of L. monocytogenes under the

conditions used in this study.

In this study, rosemary extract alone was not effective in controlling L. monocytogenes

growth. Del campo et al. (33) found that gram positive bacteria were the most sensitive to

rosemary extracts. However, the author states that rosemary extract should be more appropriate

in foods with low fat and protein contents. Because ready-to-eat meat products are rich in

protein content, this may explain why rosemary did not exhibit an anti-listerial effect. Another

study found that the use of encapsulated rosemary oil was much more effective than standard

rosemary essential oil extract against L. monocytogenes in pork liver sausage (95). This

suggested that the method of application of rosemary to the product may have interfered with its

antimicrobial properties.

Overall, during the 63 days storage a significant difference (P < 0.05) existed in population

reduction of nisin, nisin with rosemary, nisin with EDTA, and nisin with rosemary and EDTA

when compared with the positive control. L. monocytogenes growth was not inhibited when

EDTA and rosemary alone were used. Nevertheless, when nisin alone or combined with









rosemary and/or EDTA were used an anti-listerial effect was observed. These results showed

that L. monocytogenes inhibition could be attributed to nisin's inhibitory activity rather than

EDTA or rosemary. Similar findings have been reported (96, 115), where in EDTA exhibited no

significant inhibition against gram positive bacteria and, when combined with nisin, EDTA

reduced the antimicrobial effect of nisin.

Lactic Acid Bacteria Analysis

When nisin was used alone or in combination with EDTA and/or rosemary an immediate

population reduction (P < 0.05) of lactic acid bacteria was observed when compared to the

control (Table 6-4). On day 0, the initial bacterial population of the positive control (5.32 log

CFU/g) was reduced to 0.25 log CFU/g with nisin, to 1.26 log CFU/g with nisin combined with

EDTA, to 1.61 log CFU/g with nisin combined with rosemary, and to 0.97 log CFU/g with nisin

combined with rosemary and EDTA (P< 0.05).

Lactic acid bacteria for EDTA, rosemary and combination of EDTA with rosemary

treatments were similar (P > 0.05) to positive control during 63 days of storage. This may

suggest that EDTA and rosemary alone have a limited antimicrobial effect against gram positive

bacteria. However, when EDTA was used in combination with nisin or nisin with rosemary an

extended lag phase in the lactic acid bacteria population was observed. This may have been

caused by the buffer capacity of EDTA, which may have help to maintain the pH stable and as a

result the lactic acid bacteria growth under control. The treatments that contained nisin or

rosemary alone experienced an exponential growth of lactic acid bacteria over time. Even

though, EDTA played an important role in stabilizing the pH, which may have help controlling

lactic acid bacteria, nisin is needed to provide the initial inhibitory and killing effect.









Results from the study show that lactic acid bacteria may be controlled in vacuum package

ready-to eat turkey ham store at 40C when treated with 0.5% nisin combined with 20 mM EDTA

and/or 1% rosemary.

pH Analysis

Turkey ham treated with EDTA alone or in combination with nisin and/or rosemary had a

significantly higher (P < 0.05) pH value than all other treatments by day 63 (Table 6-5). This

probably resulted from the ability of EDTA to act as a food preservative and pH stabilizer (139).

Overall, the pH was not significantly different between turkey ham treated with nisin,

rosemary, nisin combined with rosemary, and those treated with control solutions including the

positive and negative control from days 0 to 63. Over time, there was a slight decrease in the pH

values of treatments that did not contain EDTA. This may be attributed to the production of

various compounds such as acidic metabolites and carbonic acids by spoilage bacteria (37). This

suggests that EDTA, under the conditions of this study, had an effect on the pH value due to its

buffering capacity. Because changes in pH were consistent with changes in lactic acid bacteria

population, pH alone may be considered as a key factor for lactic acid bacteria population

reduction in this study.

Overall, the pH values of the treatments in this study did differ throughout the 63 days of

storage. Similar results have been reported (23) in which a significant difference was seen in the

controls and nisin treated samples between different days.

Analysis of Objective Color Measurement for L~a~b Values

Results of L* a* b values measured on ready-to-eat vacuum packaged turkey ham stored at

40C are shown in Table 6, 7 and 8, respectively. The L~a~b values represent the colour co-

ordinates used in the Minolta Chromameter system to determine colour. The L* value is an

indication of lightness, where 100 represents perfect white and 0 black (76). Lightness increased









during storage for the positive control, EDTA, nisin with EDTA, nisin with rosemary, nisin with

rosemary and EDTA, and EDTA with rosemary treatments, showing significantly higher (P <

0.05) values by day 63, which could be due to a whitening surface observed in the turkey ham.

Nisin alone and rosemary alone did not have a significant difference (P < 0.05) in the L* value

throughout storage. The L* values oscillated in between a range of 58 to 64 for all treatments.

The a* value is an indicator of redness, where positive value for red and negative for green

(76). The redness (a* value) has been used as an indicator of colour stability in meat and meat

products (52). In this study, significant differences (P < 0.05) among treatments in each storage

time were observed. A decreasing trend (P < 0.05) on the a* value over time was observed in the

following treatments: nisin combined with EDTA, nisin combined with EDTA and rosemary,

EDTA combined with rosemary, and positive control. All other treatments experienced an

increasing trend (P < 0.05) on the a* value throughout the storage time. The decreasing trend on

the a* value may be attributed to many factors such as storage time, increased residual 02 lCVel,

increased oxygen transmission rate, increased light intensity and decreased nitrite content (81).

The b* value is an indication of yellowness, where yellow is represented by a positive

value and blue by a negative value (76). Significant differences (P < 0.05) among treatments in

each storage time were observed. All treatments showed an increasing trend on the b* value

over time (P < 0.05). However, the amount of increased storage time was very small.

Differences in b* values along the storage period could be related to the intensity of the

oxidation process that takes place during storage and might tend to increase yellowness of

samples by rancidity, although no measures of oxidation intensity are available to support this

hypothesis.










Table 6-1. Formulation of nisin, rosemary and EDTA solutions for vacuum packaged ready-to-eat turkey ham stored at 4+1 OC for 63
days
Percentage (%) of ingredient in total composition
.Nisin
N sin Nisin Rosemary
Treatment Control Nisin EDTA Rosemary Rosemary
EDTA Rosemary EDTA ET

Ingredient
Ham 90.00 88.58 89.25 89.00 87.83 87.58 86.83 88.25
Water 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00
Rosemary 0.00 0.00 0.00 1.00 0.00 1.00 1.00 1.00
EDTA 0.00 0.00 0.75 0.00 0.75 0.00 0.75 0.75
Nisin 0.00 0.50 0.00 0.00 0.50 0.50 0.50 0.00
NaCl 0.00 0.75 0.00 0.00 0.75 0.75 0.75 0.00
HCI 0.00 0.17 0.00 0.00 0.17 0.17 0.17 0.00
100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
*Two controls were prepared: negative control (without L. monocytogenes inoculum) and positive control (with L. monocytogenes
inoculum).









Table 6-2. Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with
Listeria monocytogenes and analyzed prior to storage
Day 0
Treatments
Logl0 CFU/g
Negative Control 0.00 c
Positive Control 5.20 a
0.5% Nisin 1.09 b
20 mM EDTA 5.09 a
1% Rosemary 4.79 a
0.5% Nisin + 20 mM EDTA 1.71 b
0.5% Nisin + 1% Rosemary 1.55 b
0.5% Nisin + 20 mM EDTA + 1% Rosemary 1.21 b
20 mM EDTA + 1% Rosemary 4.91 a
a-c values in same column with different superscripts are significantly different
(P < 0.05)











Table 6-3. Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 &
loC for 63 days


Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35
(Loglo CFU/g)

Negative control 0.00 "" 0.00 d~" 0.00 f 0.00 d"V 0.00 e"v 0.00 c"
Positive control 5.33 a"~ 4.78 a""V 4.87 a~wx 4.33 avyz 4.33 a~vz 4.14 ~YZ
Ni sin 0.91 bc xy 0.13 d~y 0.73 eftxy 0.76 ed~X 2.42 C~V 1.68 b xy
EDTA 4.98 a~v 4.38 a~~w 4.00 be,wx 4.14 a~w 3.93 ab,wx 3.63 a X
Rosemary 5.18 av~ 4.48 a~~wx 4.60 ab~w 4.06 a xY 3.92 ab,yz 3.70 ~YZ
Nisin+EDTA 1.60 b,wx 0.87 ed,wx 0.45 fex 0.37 d,x 2.72 d,wx 1.71 b wx
Nisin+rosemary 1.13 b~w 1.12 c"w 1.83 d"" 2. 10 b~vw 2.04 c~vw 1.99 b""
Nisin+EDTA+rosemary 1.22 b~xy 1.16 c"Y 1.43 de~xy 1.60 be~xy 2.39 c~vw 2.01 b~xy
EDTA+rosemary 4.90 a"v 3.13 blwx 3.66 c;w 3.69 a"w 3.28 b;wx 3.66 a"w
a-f values in same column with different superscripts are significantly different
v-z values in same row with different superscripts are significantly different
(P < 0.05)


Day 42 Day 49 Day 56 Day 63


0.00 d~"
3.76 az
1.00 ed xy
3.18ab~y
3.52 a"z
0.77 d~wx
1.94 c"vw
2.02 be,xy
3.24 a"wx


0.00 e"
4.44 a~xY
2.36 c"vw
3.41 b'y
3.87 ab,yz
1.65 d~wx
2.31 c~vw
2.07 ed,xy
3.41 b~wx


0.00 e"
4.26 a~Yz
2.04 d~wx
3.18 be,y
3.54 ab~z
2.71 ed~v
2.55 ed~v
2.30 d~wx
3.52 ab~w


0.00 d~v
3.91 a~~YZ
2.39 be~v
3.43 ab,y
3.89 a~~YZ
1.99 c~vw
2.6abe,v
2.9abe,v
3.22 abe"














Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35
(Loglo CFU/g)
Negative control 2.58 by 5.14 ax 5.38 awx5.31 a~x 6.54 av 6.13avw
Positive control 5.32 asV 5.64 a~v 5.93 asV 5.67 asV 5.74 ab,v 5.60 a~v
Ni sin 0.25 d~z 1.13 c"z 2.29 c~vz 3.08 ed,xy 5.08 be,vw 5.43 a,vw
EDTA 4.92 as" 4.46 a~vw 4.00 b~wx 4. 19 b~wx 4.51 c~vw 3.75 b,xy
Rosemary 5.06 a"w 5.57 a~vw 5.44 ab~vw 5.16 a"w 5.52 b~vw 5.46 a"vw
Nisin+EDTA 1.26 c"w 0.80 c~w 0.86 c"w 2.04 e~vw 1.81 e~vw 2.07 c"vw
Nisin+rosemary 1.61 be,y 3.04 b~x 4.08 b~wx 3.83 be~x 5.73 ab,v 5.72 a~v
Ni sin+EDTA+rosemary 0.97 ed~w 1.87 be~vw 2.28 c~v 2.40 de~v 2.58 e~v 2.54 c"v
EDTA+rosemary 4.87 as" 4.47 a~vw 4.20 b,xy 4.31 b~wx 3.51 d~z 4.40 b~vw
a-e values in same column with different superscripts are significantly different
v-z values in same row with different superscripts are significantly different
(P < 0.05)


Day 42 Day 49 Day 56 Day 63


Table 6-4. Lactic acid bacteria counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 & loC for
63 days


5.77 ab~wx
5.64 ab~v
6.02 as
3.49 "Y
4.96 b~w
2.94 ed~v
5.08 ab~vw
2.30 d~v
3.72 "mYz


5.77 a"wx
5.75 as
4.32 b~wx
3.56 b'y
5.53 a"vw
1.95 c"vw
5.38 as
2.34 c"v
3.67 b,yz


5.27 a"wx
5.62 as
5.46 a"vw
3.49 b'y
5.78 as
1.68 c"vw
5.10 a~vw
2.32 c"v
3.70 b,yz


6.04 a~~vw
5.36 as~
4.96 a~~vw
3.68 b,xy
5.53 a~~vw
2.15 d~vw
5.14 a~~vw
2.31 ed~v
3.37 be~z











Table 6-5. pH measurements on Readv-To-Eat turkey ham inoculated with Listeria monocytogqenes and stored at 4 & loC for 63 days


Treatments


Day 0 Day 7 Day 14 Day 21 Day 28


Day 35 Day 42 Day 49 Day 56 Day 63


5.72 bvw5.34 d"wY
5.79 b~v 5.45 edsw
5.86 ab~v 5.82 av
5.48 d'y 5.66 bc x
5.95 a~v 5.54 be vw
5.49 d'z 5.59 ab xy
5.78 b"" 5.75 av
5.46 d~z 5.73 ab~z
5.58 ed,y 5.74 ab,xy


5.29 d,wxy
5.48 ed~vw
5.74 be~v
6.10 ab~v
5.50 ed~vw
6.11 ab~v
5.56 ed~wx
6.26 as~
6.12 ab~v


5.00 "Y
5.25 be"vw
5.69 a~v
5.73 a~xY
5.39 ab~w
5.73 a~Y
5.57 ab~wx
5.80 a~Yz
5.71 axY


5.28 c~xY
5.42 c"vw
5.82 ab~v
5.85 ab~wx
5.24 c"w
5.83 ab~wx
5.52 be"wx
5.89 a~xYz
5.78 ab~wx


5.42 c"vwx
5.16 c~w
5.97 ab~v
6.03 ab~vw
5.31 c~w
6.09 a~v
5.60 be,1wx
6.03 ab,1wx
6.07 a~v


5.74 be~V
5.65 ed""
5.87 ab~v
6.11 ab~v
5.48 d~w
6.17 as
5.59 ed~wx
6.12 ab""
6.07 ab~v


5.51 be,VWX
5.36 be"vw
5.72 ab~v
5.97 a"vwx
5.31 c~w
5.97 a~wx
5.31 cxY
6.07 a"vwx
6.02 a~v


5.16 d,xy
5.18 ed~w
5.59 be"v
5.99 ab~vw
5.22 ed~w
5.99 ab~vw
5.37 ed,xy
6.01 a~wx
6.04 a~v


5.08 e~xY
5.62 be"vw
5.57 ed~v
5.99 ab~vw
5.43 de"w
6.09 a"v
5.14 ~Y
6.11 a~vwx
5.94 ab~vw


Negative control
Positive control
Nisin
EDTA
Rosemary
Nisin+EDTA
Nisin+rosemary
Nisin+EDTA+rosemary
EDTA+rosemary


a-d values in same column with different superscripts are significantly different
x-z values in same row with different superscripts are significantly different
(P < 0.05)










Table 6-6. Mean for "L" values of Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 & loC for 63
days


Treatments


Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63


62.20 bwx63.59 aw62.94 aw61.43 awx60.98 a~x60.68 awx62.98 bew61.78abw
64.65 a~w 63.32 a"wx 63.38 a~wx 62.66 ab~wx 62.92 a"wx 61.96 a~wx 61.88 d~wx 63.31 ab~wx
61.53 c"w 61.31 ab~w 61.24 a" 61.11 ab~w 61.13 a~w 60.05 ab~w 61.76 d~w 61.34 b~w
60.31 c~xY 60.42 ab~xY 61.92 a~x 64.43 a"w 61.59 a"x 61.95 a~x 61.34 d~x 61.33 ab~x
61.02 c" 61.07 ab~w 63.61 a"w 62.66 ab~w 60.39 a~w 61.36 ab~w 64.06 a~w 61.03 b~w
61.31 c~wx 60.56 ab~wx 61.42 a~wx 60.96 ab~wx 60.62 a"wx 61.02 ab~wx 58.59 e~x 63.20 ab~w
62.50 ab~wx 62.46 ab~wx 61.83 a~~xYz 59.42 b'z 62.09 a~wX 59.71 b'z 63.93 ab,w 62.85 ab,wx
64.07 ab~w 59.53 b~x 61.84 a~wx 61.32 ab~wx 61.40 a wx 60.69 ab~x 62.38 ed~wx 62.43 ab~wx
61.36 c~wxY 61.64 ab,wxy 62.69 a,wx 61.24 ab,wxy 60.22 a~X 59.28 b,yz 61.40 d,wxy 64.69 a~w


62.07 ab~wx
61.56 ab~x
61.44 ab~w
60.11 b,xy
61.44 ab~w
61.08 ab~wx
61.46 ab,xyz
60.48 b~x
63.11 a~wx


58.72 be"x
61.44 a~x
60.12 ab~w
58.61 be,y
61.25 a~w
59.10 b~x
60.28 ab,xyz
59.73 ab~x
57.10 c"z


Negative control
Positive control
Nisin
EDTA
Rosemary
Nisin+EDTA
Nisin+rosemary
Nisin+EDTA+rosemary
EDTA+rosemary


a-e values in same column with different superscripts are significantly different
w-z values in same row with different superscripts are significantly different
(P < 0.05)










Table 6-7. Mean for "a" values of Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 & loC for 63
days
Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63

Negative control 17.46 a~vz 17.10 a~z 17.96 ab~vz 17.85 a~~Yz 18.08 a~xYz 18.59 a"w" 19.56 a~w 17.59 a~Yz 18.55 abe,wxy 19.34 a,wx
Positive control 16.36 ab~wx 16.79 a~wx 18.79 a"w 17.88 a~wx 18.13 a~wx 19.03 a~w 18.29 a~wx 17.91 a~wx 19.22 a~w 13.84 abe"x
Nisin 16.59 ab~yz 17.12 as 17.00 ab~y 17.57 a"w" 18.83 a~~w 18.52 ab~wx 18.52 a~wx 15.53 b'z 17.73 ed,wxy 17.51 ab,xy
EDTA 15.82 b~wx 14.05 b~x 17.01 ab~wx 15.64 a~wx 13.47 c~x 15.28 c~wx 14.51 b~x 16.66 ab~wx 18.67 ab~w 16.03 ab~wx
Rosemary 15.51 b~z 16.51 a~Yz 18.41 ab~w 17.68 a~wx 17.62 a~wx 17.25 abc," 18.61 a~w 17.56 a,wxy 16.81 e~x 18.43 a~w
Nisin+EDTA 16.62 ab~wx 16.65 a~wx 14.34 c~wx 15.94 a~wx 14.78 be~wx 16.08 be~wx 13.62 b,wxy 12.52 C~xy 17.11 de,w 9.06 C~Y
Nisin+rosemary 16.65 ab~Y 16.50 asY 17.75 ab~wx 17.17 a~xY 18.15 a~wx 18.53 a~w 18.29 a~w 17.83 a"w 1.5 abe,w183aw
Nisin+EDTA+rosemary 15.91 b,w 15.85 a"w 16.66 b,w 17.18 a" 16.94 a,w 17.55 abe~w 16.50 a,w 17.59 a" 13.11 fex 11.88 be,x
EDTA+rosemary 16.56 awx16.99 a~x18.49 abw14.70 ax18.06 aw18.20 abw18.10 aw16.92 awx18.30 bew14.05abx
a-f values in same column with different superscripts are significantly different
w-z values in same row with different superscripts are significantly different
(P < 0.05)











Table 6-8. Mean for "b" values of Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 & loC for 63
days


Treatments Day 0 Day 7 Day 14 Day 21 Day 28
Negative control 7.05 b~wx 6.25 c" 7.97 ab~wx 7.16 d~wx 7.96 ab~wx
Positive control 7.63 b,xyz 6.73 be'z 7.77 ab,xy 8.97 abe,w 6.85 b,yz
Ni sin 7.73 b~wx 6.98 b~wx 6.65 b~x .1 abe,w 7.1awx
EDTA 7.59 b~y 7.20 ab,y 8.25 a,wxy 7.73 ed,xy 9.15 a,w
Rosemary 9.21 a"w 7.87 a~w 8.02 ab~w 9.21 abe~w 7.89 ab~w
Nisin+EDTA 7.38 b~x 7.33 ab~x 8.50 a~wx 7.6bed,wx 86 ~x
Nisin+rosemary 9.54 a~~w 7.30 ab,y 8.55 a,wxy 8.23 abe,wxy 7.52 ab,xy
Nisin+EDTA+rosemary 9.50 a~~wx 6.85 be~z 7.21 ab,yz 9.55 a,wx 8.62 a~wx
EDTA+rosemary 8.20ab~w"" 7.05 b~y 7.26 ab,xy 9.30 ab~wx 7.63 ab~wx.y
a-d values in same column with different superscripts are significantly different
w-z values in same row with different superscripts are significantly different
(P < 0.05)


Day 35 Day 42


Day 49
8.35 aew
6.99 "mYz
8.18 aew
8.61 be,y
8.16 ab"w
8.94 ab~wx
7.57 be,xy
8.53 abe,wxy
9.56 a"w


Day 56 Day 63
7.89 bwx 8.77aw
7.58 be,xyz 8.41 a~wx
7.46 c"wx 7.82 a~wx
8.01 be,w"" 9.21 a~w
8.41 ab~w 9.37 a~w
8.05 be~wx 9.68 a~w
8.12 be,w"y 8.16awxy
9.02 a"wx 9.82 a~w
7.26 C~xy 8.68 a~wxy


7.64 a"wx
6.97 a~Yz
7.08 a"wx
7.96 a~wxY
8.61 a~w
8.78 a"wx
8.86 a"wx
8.57 a~wxY
7.35 a~wxY


8.42 ab~w
7.53 b,xyz
7.62 b~wx
9.00 ab,wx
9.55 a~w
9.27 a~w
7.42 b,xy
8.19 ab~xyz
7.51 b,wxy









CHAPTER 7
SUMMARY AND CONCLUSION

It is well understood that the meat industry is in need of new post-processing hurdle

technologies that will control and inhibit L. monocytogenes growth inready-to-eat (RTE) meat

and poultry products. This investigation proposes a post-processing antimicrobial system that

includes the use of nisin alone and nisin in combination with rosemary and/or EDTA to control

the incidence ofL. monocytogenes on RTE turkey ham stored at 40C for up to 63 days.

Preliminary studies were conducted on different antimicrobial solutions using the Kirby

Bauer disc diffusion method. Results obtained in these studies revealed that when nisin was used

alone or in combination with 1% rosemary and/or 20 mM EDTA a significant inhibition in L.

monocytogenes growth (P < 0.05) was achieved. In contrast, L. monocytogenes growth was not

inhibited by treatments containing 1% and 2% of vinegar (acetic acid), 3% and 5% of potassium

benzoate, 1% thymol, or 0. 15% and 0.25% sodium diacetate alone or in combination with nisin.

These outcomes were useful in developing the research study, in which nisin, rosemary and

EDTA alone and in combination were further analyzed in a meat matrix.

Turkey ham treated with EDTA alone or in combination with nisin and/or rosemary had a

significantly higher (P < 0.05) pH value than all other treatments. This probably resulted from

the ability of EDTA to act as a food preservative and pH stabilizer (139). All treatments that did

not contain EDTA experienced a slight decrease in pH throughout storage (P < 0.05). Bacterial

growth on turkey ham, stored under vacuum packaging conditions, may result in the production

of numerous compounds such as acidic metabolites and carbonic acid that may decrease the pH

(37). This may explain the decrease in pH for the controls and the treatments without EDTA

over time in this study. This suggested that EDTA, under the conditions of this study, had an

effect on the pH value due to its buffering capacity. In addition, because changes in pH were









consistent with changes in lactic acid bacteria population, pH alone may be considered as a key

factor for lactic acid bacteria population reduction in this study. Furthermore, the results showed

that pH values decreased as the concentration of nisin decreased from 0.5% to 0.2%. This also

may be attributed to the lactic acid bacteria present in the samples, which increased as the

concentration of nisin decreased from 0.5% to 0.2%.

During the study, treatments with nisin alone and nisin combined with rosemary and/or

EDTA caused significant reduction in L. monocytogenes counts as compared to the positive

control (P < 0.05). The initial L. monocytogenes population was significantly reduced (P< 0.05)

from approximately 3.00 log CFU/g to 4.00 log CFU/g when samples were treated with nisin

alone or in combination with rosemary and/or EDTA. In addition, an extended lag phase was

demonstrated for the 0.5% nisin treatment throughout the study, maintaining L. monocytogenes

counts less than 1.95 log CFU/g. Results from the study indicated that the antimicrobial

effectiveness of nisin increased as its concentration increased from 0.2% to 0.5%. The study also

revealed that L. monocytogenes might be controlled in vacuum package RTE turkey ham stored

at 40C when treated with the concentrations of nisin alone and in combination with rosemary

and/or EDTA evaluated in this study.

In the absence of nisin, EDTA and rosemary alone and in combination did not inhibit L.

monocytogenes throughout the study under conditions of vacuum package and storage at 4oC.

Also, no increase in antimicrobial activity was observed when nisin was used with rosemary

and/or EDTA against L. monocytogenes, when compared to using nisin only. In contrast,

researchers have reported an increase in the antimicrobial activity of nisin against gram positive

(65) and negative bacteria (23, 126) in the presence of membrane disrupting agents, including

EDTA. However, the majority of these reports are based upon observation of organisms










suspended in a buffer, rather than growing in nutrient media. Since meat products provide a

nutrient rich environment, test of antimicrobials under conditions of cell starvation would appear

to be of limited value in evaluating them for application in food products. The failure to observe

an enhancement in the antimicrobial properties of nisin when combined with EDTA in this study

may indicate that the observed enhancement between these two antimicrobial agents in buffer

systems (65) is a consequence of cell starvation which prevents cell repair.

In gram negative organisms, it has been shown that EDTA can act to destabilize the cell

membrane of bacteria by completing divalent cations, such as Ca2+ and Mg2+, which act as salt

bridges between membrane macromolecules, such as lipopolysaccharides (118). However,

membrane structure differs between gram negative and gram positive bacteria. Gram negative

bacteria have an outer membrane coating a thin layer of peptidoglycan. The primary target of

EDTA is the lipids and lipid proteins present in the outer membrane of gram negative bacteria

(37). In contrast, Gram positive bacteria will have a thick layer of peptidoglycan (a sugar-

protein shell) that confers resistance to physical disruption (43). This may be the reason that

EDTA did not inhibit the growth of L. monocytogenes under the conditions used in this study.

Similar observations, that EDTA had no significant inhibition against gram positive bacteria and

that EDTA combined with nisin reduced the antimicrobial effect of nisin, have been reported by

other researchers (96, 115). In this study, rosemary extract alone was not effective controlling L.

monocytogenes growth. Del campo et al. (33) found that gram positive bacteria were the most

sensitive to rosemary extracts. However, the author states that rosemary extract should be more

appropriate in foods with low fat and protein contents. Ready-to-eat meat products are rich in

protein contents; this may be why rosemary did not exhibit an anti-listerial effect. Another study

found that the use of encapsulated rosemary oil was much more effective than standard rosemary









essential oil extract against L. monocytogenes in pork liver sausage (95). This suggests that the

method of application of rosemary to the product may have interfered with its antimicrobial

properties. The fact that EDTA and rosemary did not inhibit L. monocytogenes growth but nisin

alone or combined with rosemary and/or EDTA had an anti-listerial effect throughout the days of

storage at 40C, indicated that the observed population reductions were attributed to the inhibitory

activity of nisin rather than EDTA or rosemary.

When nisin was used alone or in combination with EDTA and/or rosemary, an immediate

population reduction (P < 0.05) of lactic acid bacteria was observed. EDTA, rosemary and

combination of EDTA with rosemary treatments were not effective (P > 0.05) when compared to

the positive control during storage. This suggested that EDTA and rosemary alone may have a

limited antimicrobial effect against gram positive bacteria. However, when EDTA was used in

combination with nisin or nisin with rosemary an extended lag phase in the lactic acid bacteria

population was observed. This may have been caused by the buffer capacity of EDTA, which

may have functioned to stabilize the pH. The treatments that contained nisin or rosemary alone

experienced an exponential growth of lactic acid bacteria over time. Even though, EDTA played

an important role in stabilizing the pH, nisin was needed to provide the initial inhibitory and

killing effect.

When nisin alone or combinations of nisin with rosemary and/or EDTA were used a

significant (P < 0.05) reduction in the initial population of aerobic bacteria was observed when

compared to the positive control. Treatments containing nisin achieved a reduction of

approximately 3 log CFU/g. In contrast, aerobic bacteria counts were significantly higher (P <

0.05) in rosemary, EDTA, and combination of rosemary with EDTA treatments when compared









to the positive control. This may suggest that EDTA has no immediate antimicrobial effect

against the aerobic organisms present in the turkey ham.

The L* value is an indication of lightness, where 100 represents perfect white and 0 black

(76). Results of L* a* b values measured on ready-to-eat vacuum packaged turkey ham revealed

that L* values (lightness) increased during storage for the positive control, EDTA, nisin with

EDTA, nisin with rosemary, nisin with rosemary and EDTA, and EDTA with rosemary

treatments, showing significantly higher (P < 0.05) values by day 63. The increase in L* value

was attibutted to a whitening surface observed in the turkey ham. L* values were similar (P <

0.05) for nisin alone and rosemary alone throughout storage. The L* values ranged between 58

to 64 for all treatments. A decreasing trend (P < 0.05) on the a* value over time was observed

for nisin combined with EDTA, nisin combined with EDTA and rosemary, EDTA combined

with rosemary, and positive control when compared to all other treatments. All other treatments

experienced an increasing trend (P < 0.05) on the a* value throughout the storage time. The

decreasing trend on the a* value may be attributed to many factors such as storage time,

increased residual 02 leVel, inCreaSed oxygen transmission rate, increased light intensity and

decreased nitrite content (81). All treatments showed an increasing trend on the b* value over

time (P < 0.05). Differences in b* values could be related to the intensity of the oxidation

process that occurs during storage and might tend to increase yellowness of samples by rancidity,

although no measures of oxidation intensity are available to support this hypothesis.

Even though, L. monocytogenes was not completely eliminated by the antimicrobial

solutions, the overall results indicate that ready-to-eat turkey ham will have decreased numbers

of L. monocytogenes, anaerobic bacteria and lactic acid bacteria when treated with nisin alone or

in combination with rosemary and/or EDTA. Nisin is a natural antimicrobial compound and










may provide a novel, environmentally safe alternative to control L. monocytogenes. This study

proposed an antimicrobial system, which consist of the addition of nisin to the finished product

before vacuum packaging, to control L. monocytogenes on ready-to-eat turkey ham stored at

4+10C. The data suggested that nisin, rosemary and EDTA will function to enhance the

microbial safety of ready-to-eat poultry, as well as meat products.

This study also raised new questions that can be further analyzed in future studies. In this

study a five strain inoculum was utilized to inoculate the turkey ham samples. By day 63, L.

monocytogenes growth was still observed. Future research will have to be conducted in order to

determine which strains were surviving and if they developed some resistance to nisin.

Furthermore, this study evaluated the antimicrobial properties of nisin against Gram

positive bacteria. Future studies can be conducted in the evaluation of antimicrobial properties

of nisin when Gram positive and Gram negative bacteria are present in a meat product.

In this study EDTA exhibited no inhibition against L. monocytogenes. Further study can

be conducted to evaluate the physical and chemical properties, mode of action and synergistic

effects of EDTA when used alone or in combination with other antimicrobials against Gram

positive and negative bacteria.










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BIOGRAPHICAL SKETCH

Alba Yesenia Ruiz was born in 1983, in San Salvador, El Salvador. She attended the Pan-

American School of Agriculture "El Zamorano" (Tegucigalpa, Honduras) where she received a

Bachelor of Science degree in agro-industrial engineering with a concentration in food

microbiology in December 2005.

In 2006, Alba began her pursuit of a Master of Science degree at the University of Florida

in the Department of Animal Sciences, Meat Science section. She was awarded an Animal

Sciences Department Assistantship to study for the Master of Science degree. She received her

Master of Science degree in December 2007. She plans to continue her studies in the Department

of Animal Sciences for the Doctor of Philosophy degree.





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1 EVALUATION OF THE GENERAL ANT IMICROBIAL AND ANTILISTERIAL PROPERTIES OF NISIN, ROSEMARY AN D EDTA ON READY-TO-EAT TURKEY HAM INOCULATED WITH Listeria monocytogenes AND STORED AT 4 DEGREES CELSIUS By ALBA YESENIA RUIZ MENJIVAR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 2007 Alba Yesenia Ruiz Menjivar

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3 To my mother, Alba Menjivar de Ruiz. Wit hout her support and understanding, I certainly never would have accomplished this work.

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4 ACKNOWLEDGMENTS I extend my sincere appreciati on and respect to my supervisory committee chair, major advisor, and friend, Dr. Sally K. Williams, for her invaluable guidance and patience. I also wish to thank my supervisory committee members, Dr. Gary Rodrick and Dr. Arthur Hinton, for their help. Special thanks go to Dr. Williams and the Department of Animal Sciences for financial support. I would also like to thank Fra nk Robbins and Doris Sartain for their advice and assistance. The friendship, constant support a nd everlasting help of Noufoh Djeri and Keawin Sarjeant are deeply appreciated. Without their company, my da ys here would not have been as enjoyable. Regards go to fellow graduate student Nicolas Lavieri for his friendshi p and help during my program. I would also like to thank Juan Carlos Rodriguez for his undying support, advice and friendship through this time. I thank my parents, Alba Menjivar de Ruiz and Jorge Ruiz for their support, persistence and love shown to me throug hout this time and all the phases of my life. Special thanks go to Douglas Rosales for his love patience and support. I would like to thank to everyone whose prayers for me were evidently heard. Thanks go to God for granting me the heart and mind to make my visions become realities.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 ABSTRACT...................................................................................................................................10 CHAPTER 1 INTRODUCTION..................................................................................................................13 2 LITERATURE REVIEW.......................................................................................................17 Listeria monocytogenes : Description..................................................................................... 17 Route of Exposure...........................................................................................................18 Characteristics of the Disease..........................................................................................19 Food Associated.............................................................................................................. 20 Control and Prevention.................................................................................................... 21 Bacteriocins: Definition and Classification............................................................................ 23 Mode of Action................................................................................................................24 Isolation and Purification................................................................................................26 Bacteriocins Resistance...................................................................................................27 Toxicity....................................................................................................................... .....27 Application in Meat Products.......................................................................................... 28 Natural Antimicrobials: Description....................................................................................... 33 Mechanism of Action...................................................................................................... 34 Application in the Food Industry..................................................................................... 34 Characteristics of EDTA (Ethylenediam ine tetraacetic Acid)................................................. 35 Antimicrobial Activity of EDTA..................................................................................... 35 3 EVALUATION OF NATURAL ANTIMI CROBI AL COMPOUNDS AGAINST Listeria monocytogenes BY KIRBY-BAUER DISC DIFFUSION METHOD..................... 37 Introduction................................................................................................................... ..........37 Materials and Methods...........................................................................................................38 Inoculum Preparation...................................................................................................... 38 Antimicrobial Solution Preparation................................................................................. 39 Preparation of Modified Oxford Media Agar (MOX)..................................................... 41 Inoculation of Agar Plates............................................................................................... 41 Kirby-Bauer Disc Diffusion Test....................................................................................42 Data Analysis...................................................................................................................42 Results and Discussion......................................................................................................... ..43

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6 4 EVALUATION OF CONCENTRAT IONS OF NISIN AGAINST Listeria monocytogenes ON READY-TO-E AT TURKEY HAM STORED AT 41C FOR 63 DAYS.....................................................................................................................................50 Introduction................................................................................................................... ..........50 Materials and Methods...........................................................................................................51 Inoculum Cultivati on and Storage ................................................................................... 51 Inoculum Preparation...................................................................................................... 52 Antimicrobial Solutions Preparation............................................................................... 52 Sample preparation..........................................................................................................53 Inoculation and Treatment............................................................................................... 53 Microbiological Analyses................................................................................................54 Listeria monocytogenes and lactic acid bacteria analysis........................................ 54 Aerobic bacteria analysis.........................................................................................55 pH Analysis.....................................................................................................................55 Data Analysis...................................................................................................................55 Results and Discussion......................................................................................................... ..56 pH Analysis.....................................................................................................................56 Listeria monocytogenes Analysis.................................................................................... 56 Lactic Acid Bacteria Analysis......................................................................................... 57 Aerobic Bacteria Analysis............................................................................................... 58 5 EVALUATION OF THE ANTI-LISTERIAL PR OPERTIES OF NISIN, ROSEMARY AND EDTA ON READY-TO-EAT TURKEY HAM STORED AT 4C FOR 28 DAYS.....................................................................................................................................64 Introduction................................................................................................................... ..........64 Materials and Methods...........................................................................................................66 Inoculum Cultivati on and Storage ................................................................................... 66 Inoculum Preparation...................................................................................................... 66 Antimicrobial Solutions Preparation............................................................................... 67 Sample Preparation..........................................................................................................68 Inoculation and Treatment............................................................................................... 68 Microbiological Analyses................................................................................................69 Listeria monocytogenes lactic acid bacteria and anaerobic bacteria analyses........ 69 Aerobic bacteria analysis.........................................................................................70 pH Analysis.....................................................................................................................70 Data Analysis...................................................................................................................71 Results and Discussion......................................................................................................... ..71 pH Analysis.....................................................................................................................71 Listeria monocytogenes Analysis.................................................................................... 72 Anaerobic Bacteria Analysis........................................................................................... 73 Lactic Acid Bacteria Analysis......................................................................................... 74 Aerobic Bacteria Analysis............................................................................................... 75

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7 6 EVALUATION OF THE ANTI-LISTERIAL PR OPERTIES OF 0.5 % NISIN, 1 % ROSEMARY AND 20 mM EDTA ON READ Y-TO-EAT TURKEY HAM STORED AT 4C FOR 63 DAYS......................................................................................................82 Introduction................................................................................................................... ..........82 Materials and Methods...........................................................................................................84 Inoculum Cultivati on and Storage ................................................................................... 84 Inoculum Preparation...................................................................................................... 85 Antimicrobial Solutions Preparation............................................................................... 85 Sample Preparation..........................................................................................................86 Inoculation and Treatment............................................................................................... 86 Microbiological Analyses................................................................................................87 Aerobic bacteria analysis.........................................................................................87 Listeria monocytogenes and lactic acid bacteria analysis........................................ 88 pH Analysis.....................................................................................................................88 Color Analysis.................................................................................................................89 Data Analysis...................................................................................................................89 Results and Discussion......................................................................................................... ..89 Aerobic Bacteria Analysis............................................................................................... 89 Listeria monocytogenes Analysis.................................................................................... 90 Lactic Acid Bacteria Analysis......................................................................................... 93 pH Analysis.....................................................................................................................94 Analysis of Objective Color Measurement for L*a*b Values........................................ 94 7 SUMMARY AND CONCLUSION..................................................................................... 104 LIST OF REFERENCES.............................................................................................................110 BIOGRAPHICAL SKETCH.......................................................................................................121

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8 LIST OF TABLES Table page 3-1 Fifty-eight antimicrobial so lutions evaluated in Phase One............................................. 45 3-2 Seventeen antimicrobial solutions evaluated in Phase Two............................................. 46 3-3 Mean zone of inhibition for antimicrobial solutions evaluated in Phase One..................47 3-4 Mean zone of inhibition for antimicrobial solutions evaluated in Phase Tw o.................49 4-1 Formulation of nisin solutions for vacuum packaged readyto-eat turkey ham stored at 4 C for 63 days.........................................................................................................59 4-2 pH measurements on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days............................................................. 60 4-3 Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days................................................ 61 4-4 Lactic acid bacteria counts on Read y-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days.............................................................62 4-5 Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and analyzed prior to storage ...................................................... 63 5-1 Formulation of nisin, rosemary and EDTA solutions for vacuum packaged ready-toeat turkey ham stored at 4 C for 28 days ...................................................................... 76 5-2 Mean pH values on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 C for 28 days ..............................................................77 5-3 Mean Listeria monocyto genes counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 C for 28 days................................................. 78 5-4 Mean anaerobic bacteria counts on R eady-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 C for 28 days................................................. 79 5-5 Mean lactic acid bacteria counts on R eady-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 C for 28 days................................................. 80 5-6 Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes .....................................................................................................81 6-1 Formulation of nisin, rosemary and EDTA solutions for vacuum packaged ready-toeat turkey ham stored at 4 C for 63 days ...................................................................... 96

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9 6-2 Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and analyzed prior to storage ...................................................... 97 6-3 Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days............................................... 98 6-4 Lactic acid bacteria counts on Read y-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days.............................................................99 6-5 pH measurements on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days........................................................... 100 6-6 Mean for L values of Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days........................................................... 101 6-7 Mean for a values of ReadyTo-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days ...........................................................102 6-8 Mean for b values of ReadyTo-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days ...........................................................103

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10 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVALUATION OF THE GENERAL ANTIMICROBIAL AND ANTI-LISTERIAL PROPERTIES OF NISIN, ROSEMARY AN D EDTA ON READY-TO-EAT TURKEY HAM INOCULATED WITH Listeria monocytogenes AND STORED AT 4 DEGREES CELSIUS By Alba Yesenia Ruiz Menjivar December 2007 Chair: Sally K. Williams Major: Animal Sciences Contamination of ready-to-eat (RTE) meat and poultry products with Listeria monocytogenes is a major concern for the meat processing industry and an important food safety issue. This study was divided in four separate phases, in whic h antimicrobial solutions were evaluated against a five -strain inoculum of L. monocytogenes In phase one, the effectiveness of different antimicrobial solutions and their combinations were evaluated by Kirby-Bauer disc diffusion method. During phase two, the antiListeria and general antimicrobial properties of 0.2% nisin alone and in combination with 1% rosemary and/or 20 mM EDTA on ready-to-eat vacuum packaged turkey ham were determined. In phase three, nisin at di fferent concentrations (0.2%, 0.3%, 0.4% and 0.5%) was evaluated on readyto-eat vacuum packaged turkey ham. And during phase four, the antimicrobial properties of 0.5% nisin, 1% rosemary, and 20 mM EDTA were evaluated separately and in combination on ready-to-eat vacuum packaged turkey ham. The methodology used during phase one invol ved placing two paper-f ilter discs (6 mm diameter) impregnated with the corresponding antimic robial solution on plates inoculated with L. monocytogenes. Sterile water was used as a control treatment. The plates were incubated at 35 C for 24 h and were observed for zones of L. monocytogenes growth inhibition. For Phases

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11 two, three and four the antimicrobial solutions we re applied to the turkey ham inoculated with L. monocytogenes followed by mixing the ham and solution to ensure a proper distribution between them. The samples were then vacuum package d, leaving the antimicrobial solution in the package. Samples were then stored at 4 1C for 28 days for Phase two and 63 days for Phases three and four. Microbiological, chemical and color analyses were conducted for all samples at one week intervals. Results from phase one indicated that nisin had a strong antibacter ial activity against L. monocytogenes, yielding significantly (P < 0.05) larger inhibition zone when used alone and in combination with 1% rosemary and 20 mM EDTA. L. monocytogenes zone of inhibition increased as its concentration increased from 0.1% to 0.5%. L. monocytogenes growth was not inhibited by the treatments containi ng 1% and 2% of vinegar, 3% and 5% of potassium benzoate, 1% thymol, 0.15% and 0.25% sodium diacetate al one and in combination with nisin. During phase two, the antimicrobials that ex hibited a greater inhibition against L. monocytogenes were further analyzed in a meat matrix. On day 0, treatments with nisin, nisin combined with rosemary, and nisin combined with rosemary and EDTA significantly (P < 0.05) reduced the population of L. monocytogenes by 3.73, 2.33, and 3.12 log CFU/g as compared to the positive control, respectively. EDTA di d not inhibit the growth of L. monocytogenes throughout 28 days storage. Results obtained from Phase thre e demonstrated an extended lag phase of L. monocytogenes when treated with 0.5% nisin. The counts remained less than 1.95 log CFU/g for 0.5% nisin throughout 63 days. The overall mean va lues for all treatments revealed that nisin resulted in 1.62.18 log CFU/g reduction of L. monocytogenes The results suggested that the antimicrobial effectiveness of ni sin increased as its concentration increased from 0.2% to 0.5%. Results from Phase four showed that initially, treatments with nisin, nisin with rosemary, nisin

PAGE 12

12 with EDTA and nisin with rosemary and EDTA significantly (P < 0.05) re duced the population of L. monocytogenes by 4.42, 4.20, 3.73, and 4.11 log CFU/g as compared to the positive control, respectively. L. monocytogenes counts remained less than 2.66 log CFU/g for treatments containing nisin during the study. EDTA and rosemary alone and in combination did not inhibit the growth of L. monocytogenes throughout the 63 days. These results indicated that the observed population reductions may be attributed to the inhibitory activity of nisin rather than EDTA or rosemary. Although none of the treatmen ts used in this study completely eliminated L. monocytogenes, the overall results indicated that ready-to -eat turkey ham will have significantly decreased numbers of L. monocytogenes when treated with nisin alone or in combination with rosemary and/or EDTA. The data suggested that nisin will function to enhance the microbial safety of ready-to-eat poultry, as well as other meat products.

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13 CHAPTER 1 INTRODUCTION In spite of modern improvements in food production techniques, food safety is an increasingly important public health issue (141) Food pathogens are ubiquitous and infect their hosts at a local and global leve l. Consequently, newly introdu ced pathogens can spread rapidly in susceptible hosts. Listeria monocytogenes the causative agent of listeriosi s, is a widely distributed and recognized foodborne pathogen (55) The ability of L. monocytogenes to grow at temperatures ranging from 0 to 45C (7) its high tolerance for salt (42) and its ability to initiate growth at a relatively low pH (9) makes this pathogen particularly diffi cult to control in food. Recently, numerous outbreaks have been linked to c onsumption of ready-to -eat (RTE) products contaminated with L. monocytogenes (55) Contamination of the R TE meat products may occur in processing plants (43) and currently represents one of the highest meat safety risks (18). Hygienic and sanitation practices applied in meat processing facilities are often insufficient to prevent contamination of processed meat products (21) Therefore new post-processing hurdle technologies that control or eliminate the incidence of foodborne pathogens are needed for the meat industry (10) In-vitro antimicrobial susceptibility tes ting done in recent years has indicated that bactericidal compounds such as organic acids, salts, and genera l recognized as safe (GRAS) substances can control L. monocytogenes (93) The Kirby-Bauer method is a standardized filterpaper disc-agar diffusion procedure (15) that is usually used for antimicrobial susceptibility testing. It is recommended by the Clinical a nd Laboratory Standards Institute (NCCLS). This method allows for the rapid determination of th e efficacy of an antimicrobial by measuring the diameter of the zone of inhibition that results from diffusion of the agent into the medium

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14 surrounding the disc (15) Results obtained in these studies may help researchers in selecting initial procedures and, antimicrobial agents that will be used for specific products. In developing a novel post-processing antimicro bial treatment for use in the food industry there are two constant problems: the limited range of bacteria which are sensitive to particular agents and the high concentrations of agen ts that are require to inhibit growth (54) The rationale for the increased effectiveness of combinations of antimicrobials is that simultaneous attack on different targets in the bacterial cell is more di fficult for the bacteria to overcome. The use of antimicrobials with different mechanism can also be expected to expand the range of organisms that may be inhibited. Nisin, a lanthionine-containing polypeptide produced by Lactococcus lactis subsp. lactis (5) is a bacteriocin with antimicrobial activity against L. monocytogenes (14) Nisin obtained a GRAS status for use as a biopreservative in the United States food industry in 1988 (131) The LD50 value was found to be simila r to that of common salt (82) Nisins mechanism of action is based on the disruption of the cytoplasmic cell membrane, as evidenced by the rapid efflux of small molecules from both whole cells and liposomes (2, 50, 138) As a result, nisin depletes the proton motive force (PMF) of sensitiv e cells and artificial liposomes (13, 49) Nisin acts through a multistep process which includes binding of nisin to the cell, insertion into the membrane, and pore formation (50, 105) These antimicrobial properti es have become a focus in the food science field and researchers have found that nisin is not only effective against Gram positive bacteria (34) but with the combination of a food grade chelator, su ch as EDTA, its effectiveness can be extended to Gram negative bacteria (115) Ethylenenediaminetetraaceti c acid (EDTA) is a well known reagent used in designated f oods for different functions. Th ose functions may include the

PAGE 15

15 retardation of crystal formation, food preservative and stabilizer, antioxidant, and chelating and sequestering agent (139) EDTA can have antimicrobial effect by limiting the availability of cations and can act to destabiliz e the cell membrane of bacteria by complexing divalent cations which act as salt bridges between membrane macromolecules, such as lipopolysaccharides (118, 135) In addition, society appears to be experi encing a trend of natural consumerism (121, 129) demanding fewer synthetic food additives and products with a smaller impact on the environment. Therefore, there is scope for new methods of making food safe which have a natural image. The use of edible plants, as well as their phytochemicals, in food preservation and improvement of organoleptic qualities of certa in traditional foods has been practiced for centuries. It has long been recognized that many plant essential oils have antimicrobial properties (95) and the relatively recent interest in na tural products by cons umers has lead to a renewal of scientific interest in these substances (92, 129) The rosemary ( Rosmarinus officinalis ) extract has shown antimic robial properties against food spoilage and foodborne pathogenic microorganism and its antibac terial activity has been linked to -pinene, bornyl acetate, camphor, and 1,8-cineole (33, 97) RTE meat and poultry products may be an excellent system in which to use nisin, rosemary and EDTA combination treatments, si nce the presence of othe r growth restrictive chemicals and conditions, such as nitrite and NaCl, may increase the effectiveness of antimicrobial treatment against spoilage flora and pathogens (54) Preliminary experiments were conducted to dete rmine the inhibitory c oncentration in agar media of different antimicrobials agents by Kirby-Bauer disc diffusion method. The antimicrobials nisin, rosemary and EDTA were selected for further study to determine their

PAGE 16

16 effectiveness in a meat matrix. This work was conducted to devel op a new post-processing hurdle technology by adding nisin, rosemary and ED TA alone or in combination into the final RTE turkey product, leaving the antimicrobial solution into the package and during the product storage.

PAGE 17

17 CHAPTER 2 LITERATURE REVIEW Listeria monocytogenes : Description Listeria monocytogenes is particularly interesting as a food-borne pathogen in that it is ubiquitous in nature. Animals and humans infected by L. monocytogenes suffer from the disease known as listerosis. The current understanding of human listeriosis epidemiology suggests that the organism is a common contaminant of food pr oducts. This contamination usually takes place on the surface of the products with up to 5% of food produc ts harboring the organism (55) The presence of L. monocytogenes in the food processing chain is evident by the widespread distribution of the listeri ae in processed products (55) The Centers for Disease Control and Prevention estimates that approximately 2500 pers ons become seriously ill and 500 persons die each year from listeriosis (18) Listeria monocytogenes is a Gram-positive, motile facultative anaerobe bacterium that inhabits a variety of environments. Using selective media, L. monocytogenes can be readily isolated from soil, water, vegetation and pro cessed products, including ready-to-eat products designated for human consumption (58) The bacterium was named monocytogenes because of its distinguished characteristic of infecti on in rabbits, which result ed in the production of monocytosis in blood (120) As a psychrophilic bacteria, L. monocytogenes grows at temperatures between 0C to 45C (7) and enjoys a competitive advantage against other gram-positive and gram-negative microorganisms in cold environments (e.g. refrigera tors). Recent investiga tions indicate that the organism can initiate growth at pH values as low as 4.4 (9) The majority of strains need a minimum water activity of 0.93 for growth (73) However, some strains may be able to grow at

PAGE 18

18 water activity values as low as 0.90 and survive for long periods of time at a water activity of 0.83 (113) The optimal water activity level is 0.97. L. monocytogenes is able to grow in th e presence of 10 to 12% sodium chloride; it grows to high populations in moderate salt concentrations (6.5%) (42) The bacterium survives for long periods in high salt concentrations (113) The survival in high-salt environments is significantly increased by lowering the temperature. Route of Exposure Experts indicate that L. monocytogenes is present in foods such as raw and pasteurized milk, cheeses, raw vegetables, raw and cooked pou ltry, raw meats, and raw and smoked fish. The levels of L. monocytogenes that could cause infection and ev entual listeriosis vary with the type of strain, as well as with the susceptibility of the host. Occurrence of sporadic listeriosis appears to be more common in the spring and summer months (40) This could be explained by seasonal variations in the type of food products eaten by humans, with higher-risk products eaten in the warmer months. The occurrence of some outbreaks suggests that certain ready-to-eat processed foods pose a high risk of contracting listeriosis for susceptible populations. These foods are usually preserved by refrigeration an d offer an appropriate environment for the multiplication of L. monocytogenes during manufacture, aging, transportation and storage. The entry of L. monocytogenes into food processing plants occurs through soil on clothing or equipment, contaminated hides or surfaces and possibly healthy human carriers. The humidity and presence of nutrients support the growth of Listeria which is commonly found in moist areas such as processing equipment, drains, etc (21) In addition, Listeria can attach to different types of surfaces in the form of biof ilms which have been observed in meat and dairy processing environments (61)

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19 Post-processing contamination is the most likel y route of contamination of processed foods (53) Currently, there is no ev idence to indicate that L. monocytogenes can survive heat processing protocols. However, because it is a frequent contaminant of raw material used in food processing plants, there are plenty opportun ities for its reintroducti on into food processing facilities by cross contamination (36) If the product is contam inated post-processing, the bacteria can survive and multiply throughout st orage causing disease when it is consumed. Epidemiologic investigations have repeat edly revealed that the consumption of contaminated food is the primary mode of transmission of listeriosis. Food has been identified as the vehicle of several major outbreaks of listeriosis inves tigated since 1981 (9) Characteristics of the Disease Listeriosis usually occurs in high risk groups who have a predisposition to the disease. Contraction of listerios is may lead to impairment of their T-cell mediated immunity. Occasionally, individuals with no predisposing conditions can acquire the disease. Some of the high risk groups may include pregnant wo men, newborn children, and immunocompromised adults (57) On average, there are 0.7 cases of listeriosis per 100,000 people, but reports show that the disease is three times more prevalent in the el derly (>70) and 17 times higher in pregnant women (46) A wide variety of clinical syndromes have been associated with L. monocytogenes in both humans and animals. In healthy individuals, the disease can take th e form of mild to substantial flu-like symptoms, including: fever, fatigue, na usea, cramps, vomiting and diarrhea. More severe complications can include: encephali tis, septicemia, mononucle osis-like syndrome, pneumonia, endocarditis, aortic aneurysm, hepatitis urethritis, rhombencephalitis, peritonitis, liver abscess, febrile gastroenteritis, peritonitis, septic arthritis, etc (27) L. monocytogenes in

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20 pregnant women can lead to an intrauterine infection, resulting in stillb irths and miscarriages. Newborns can develop meningitis after birth via transplacental transmission (66) The onset time for serious complications of lis teriosis can be anywhere from a couple of days to three weeks. Mortality of untreated infections is among the highest of all foodborne illnesses (i.e. 70 percent). The infective dose of L. monocytogenes is not yet known, since it is related to many variables such as type of strai n, susceptibility of the victim, and product type (9) Food Associated Some of the food products associated with L. monocytogenes include unpasteurized milk and products prepared from unpasteurized milk, soft cheeses, frankfurte rs, delicatessen meats and poultry products, and some seafood. Raw milk is a well-known source of L. monocytogenes Pasteurization effectively inactivat es this organism. However, fl uid milk that is contaminated after pasteurization and stored under refrigeration may cont ain very high populations of L. monocytogenes after one week. Furthermore, extreme temperature fluctuations may enhance the multiplication of bacterial cells (9) L. monocytogenes can also be found in cheese because of its ability to multiply at refrigeration temperature and salt tole rance. During manufacturing process, L. monocytogenes is primarily concentrated in the chee se curd, with only a very small portion of cells appearing in the whey. During ripening of the cheese, the number of cells may increase (C amembert), decrease gradually (Colby or cheddar), or decrease rapidly (blue cheese) and then stabilize (43) L. monocytogenes has also been isolated from dome stic and imported, fresh, frozen, and processed seafood products, including crustaceans, molluscan shellfish and finfish (63) The production of seafood products is done on a mu ch smaller scale than meat and cheese manufacture. Furthermore, consumption of s eafood products is much less when compare to the consumption of meats and cheeses. This may be the reason that large outbreaks have not been

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21 reported and that case-control studies have not identified these foods as a major risk of listeriosis (102) In addition, cooked and ready-to-e at meat and poultry products ha ve been implicated as the source of sporadic and epidemic listeriosis on several occasions in North America and Europe (112) L. monocytogenes attaches strongly to the surface of raw meats and is difficult to remove or inactivate. The multiplication in meat and poultry depends on the type of meat, pH, and the type of cell populations of competitive flora (43) Therefore, the incidence of L. monocytogenes in ready-to-eat products has become a majo r concern for the meat processing industry. According to the Center for Disease Control and Prevention, L. monocytogenes is a widely recognized foodborne pathogen that is widespread in the environment and has the ability to contaminate meat products dur ing various phases of producti on, processing, manufacturing and distribution (18) As mentioned previously, L. monocytogenes is highly resistant to environmental conditions and has the ability to gr ow at high osmotic stress and low temperatures (51) Most of listeriosis outbreaks have been linked to contaminated ready-to-eat meat and poultry products (16) Experts believe that in order to prevent contamination of processed meat products, good hygienic and sanitization prac tices are essential in meat industry (43) Control and Prevention Because of the high fatality rate and th e uncertainty of th e infective dose of L. monocytogenes, the Food Safety and Inspection Service of the U.S. Department of Agriculture (USDA-FSIS) has established a zero toleran ce policy for this pathogen on ready to eat products (78) In 2003, USDA-FSIS published an interim final rule addressi ng the control of L. monocytogenes on ready-to-eat meat products. The altern atives to control such pathogen involve various levels of intervention and microbiologi cal testing. Under the first alternative the processor must use a post-lethality treatment that reduces or eliminates L. monocytogenes and

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22 use an antimicrobial agent or proc ess that suppresses or limits L. monocytogenes growth throughout the products shelf life. The second alternative is for the processor to use either a post-lethality treatment that reduces or eliminates L. monocytogenes or use an antimicrobial agent or process that suppresses or limits L. monocytogenes growth throughout product shelf life. A third alternative relies only on sanitation measur es and testing to control the pathogen in the post-lethality environment (130) L. monocytogenes can be resistant to many food preserva tion processes. This pathogen can increase significantly during refrigera ted storage and reduced oxygen conditions (73) As a result, hurdle technologies to inhi bit growth of the pathogen are n eeded. Some studies show that bactericidal compounds such as organi c acids and bacteriocins can control L. monocytogenes in meat products (108, 109) To date, the inclusion of chemical antimicrobials, such as lactates, acetates and diacetates, in cured meat formulati ons remains the most effective hurdle against L. monocytogenes (53) Different studies show that L. monocytogenes in processed meat products may be controlled through the use of natural plant antimicrobials or chemically produced antimicrobials (12) meat packaging materials with immobilized antimicrobials (94) thermal pasteurization just before (24) or after packaging (103) and emerging technologies, such as irradiation (123) or high pressure (75) The use of antimicrobials with different mechanisms of killing can be expected to expand the range of microorganism s that may be inhibited (54) By deliberately combining hurdles like bacteriocins, low temperature a nd low pH, the microbia l stability of the product can be improved (69) Increased consumer demand for minimally pr ocessed foods with less chemical additives makes the control of foodborne pathogens a more complicated challenge.

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23 The Food and Drug Administration (FDA) and th e FSIS advise all consumers to reduce the risk of illness by using a refrigerator thermometer to make sure that the refrigerator always stays at 40 F or below and use perishable items that are precooked or ready-to-eat as soon as possible. For high risk groups, the USDA-FSIS recomme nds avoid consuming hot dogs and luncheon meats, unless they are reheated until steaming hot; avoid eating soft cheeses such as Feta, Brie, and Camembert cheeses, unless it is labeled as made with pasteurized milk; avoid eating refrigerated smoked seafood, unless it is contai ned in a cooked dish; and avoid consuming raw (unpasteurized) milk or foods that contain unpasteurized milk (130) Bacteriocins: Definition and Classification Because food safety has become an important international concer n, the application of naturally occurring metabolites that target food pathogens without toxic or any other side effects is an area of great interest. These natural inhibitors coul d replace the use of chemical preservatives such as sulfur dioxide, benzoic acid, sorbic acid, nitrate, and others (20) Using lysozyme and organic acids to ex tend the shelf life of food, has already been proven to be an effective treatment (94) Many other natural antimicrobial systems may have potential for food preservation in the future. Some studies have found that bacteriocins produced by lactic acid bacteria may be very promising food preservatives (69) Bacteriocins are peptides or small proteins produced by bacteria that kill or inhibit closely related sp ecies or even different strains of the same species (50) They are specific in their antim icrobial action and offer potential alternatives to replace an tibiotics and chemical pres ervatives used in food (20) Their inhibitory spectrum is restricted to Gram-positive bacteria, but several bacteriocins produced by lactic acid bacteria are active against food spoilage and foodborne pathogenic microorganisms (84) In addition, many bacteriocins are h eat stable, making them applicab le in combination with heat treatment and appear to have a universal bactericidal and irreversible mode of action. These

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24 bacterocins are generally food stab le, biodegradable, digestible, sa fe to human health and active at low concentrations (106) Bacteriocins are a family of ribosom ally synthesized peptide antibiotics, (84) generally classified into three groups: Class I, Class II a nd Class III. However, this classification is currently being revised due to better structural analyses and discoveries of new molecules. Class I has been further subdivided into Class Ia a nd Class Ib. Class I ba cteriocins, also called lantibiotics, and are characteri zed by their unusual amino acids, such as lanthionine, methyllanthionine, dehydrobutyrin e and dehydroalanine (5) They constitute an uncommon family of biological active peptides that are thought to ex ert their antimicrobial activity by formation of transient pores in the bacterial cytoplasmic membrane (105) The only appoved bacteriocin currently being used by the food industry is ni sin, which belongs to the lantibiotic family (133) Class II contains small heat-sta ble, non-modified peptides, and can be further subdivided (90) They do not usually contain postt ranslationally modified amino aci ds as found in lantibiotics. However, while lantibiotics have been found exclusively in gram positive bacteria, class II bacteriocins have also been found in E. coli (colocin V and microcin 24) (45). Class III includes large and heat labile bacteriocins for which ther e is much less information available. A fourth class has been proposed, which consists of bacter iocins that form large complexes with other macromolecules (67) However, these bacteriocins have not been purified, a nd there is a reason to believe that this type of bacteriocin is an artifact due to the cationic and hydrophobic properties of bacteriocins which results in co mplexing with other macromolecules in the crude extract (62) Mode of Action Bacteriocins are characteri zed by a strong bactericidal mode of action. Membrane insertion, pore formation and simultaneous depolarization would indu ce a rapid and specific

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25 efflux of cytoplasmic cell constituents of lo w molecular mass (potassium, hydrogen, amino acids and nucleotides) from intact sensitive bacterial cells (77) The primary target of bacteriocins is the cytoplasmic membrane. They initiate rearrang ements in the membrane structure, which alter the membrane permeability by generating channels in the cytoplasmic membrane (32) As a result, the energy metabolism of the cell is dest royed, causing a deficien cy of the important metabolic intermediates and an immediate and s imultaneous inhibition of the biosynthesis of macromolecules such as DNA, RNA, proteins and polysaccharides (82) Bacteriocins do not require a membrane receptor, but it is better if the membrane is energized (50) The amount of negatively charged lipids in the membrane is likely to be a major criterion for the sensitivity of the organism to bacteriocins. Some experts suggest that besides disrupting membrane activity via pore formation, bacterocins may have additional effects on elect ron transfer chain components and in the inhibition of oxygen uptake (106) This may be explained either by a direct effect of the peptides on the cytochrome c oxidase (4) or by the lack of ADP (82) Their bactericidal mode of action is restricted to Gram-positive bacteria. However, Gram negative bacteria such as Escherichia coli (106) and Salmonella typhimurium (125) become sensitive when the outer membrane is altered (osmotic shock, disodium EDTA treatment). Bacter iocins also inhibit the outgrowth of bacterial spores. It has been proposed that these peptid es covalently modify the sulfhydryl groups of proteins of the spore membrane of freshly germinated spores, and hence exert a profound bacteriostatic effect resulting in inhi bition of subsequent spore outgrowth (82) One of the most studied bacteriocins is ni sin, which belongs to the lactibiotic family (39) It was demonstrated that in vitro nisin inhibited bacteria l cell wall biosynthesis (101) Nisin forms pores in the cell membrane allowing the diffusion of small compounds. The increase in

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26 membrane permeability results in the collapse of the proton motor force, which drives ATP synthesis and the accumulation of ions and other metabolites (3) Failure of the proton motor force leads to cell death through cessation of energy requiring reactions (32) Isolation and Purification The methods most frequently used for is olation, concentration, and purification of bacteriocins usually involve salt precipitation of bacteriocins from culture supernatants, followed by various combinations of gel filtration, ion-exchange chromatography, and reverse-phase highperformance liquid chromatography (87) The purification methods are based on observations that the bacteriocin molecules are (a) excreted by the producer cells; (b) cationic; (c) hydrophobic; (d) adsorb to the cell surface of th e producer cells; and (e) adsorb in a pHdependent manner, with high adsorption occurri ng at about pH 6.0 and low adsorption at about pH 2.0 (26) The methods that have proven to simulta neously concentrate bacteriocins include vacuum concentration, precipitation by salt frac tionation, acid precipita tion, organic solvent precipitation, freeze drying and ultrafiltration (87) Although the purification procedures mentioned here play an important role, they typically do not provide for a high degree of resolution. Therefore, several methods of chromatography, including size exclusion (gel filtration), cation exchange, and hydrophobic interact ion, have been used to achieve purification of bacteriocins. Because the methods used to purify bacteriocins can be complicated and timeconsuming, as well as very expensive a few scientists (25) have reported a simple method for the purification involving adsorption of the bacteriocin onto the produc er cells at pH 5.5 followed by extraction at pH 2.0. However, the recovery of bact eriocin activity by this method did not exceed 10%.

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27 Bacteriocins Resistance Although most bacteriocins do not require specif ic targets in the membranes of sensitive cells, when used together they can work synerg istically or possess anta gonism to each other (86) Microbial resistance to bacteriocins could become an issu e as their food-preservative use becomes more widespread. Some experts indicate that in many cas es, bacteriocins do not induce cross-resistance (100) However, other reports show that microorganisms treated with certain bacteriocins become resistant to not only the bacteriocin they were in contact with, but even to unrelated bacteriocins never used against these cells (124) This observation again suggests that bacteriocins should be used as part of hurdle technology, which allows them to act synergistically with other food preservatives to pr event appearance of resistant bacterial forms. Previously reported results indicate that nisin works synergistically with other preservatives, also in food model system (100) Combining preservation techniques is a powerf ul tool for extending shelf life, especially of minimally processed food (30) Hurdle technology permits the use of less severe preservative levels a nd of techniques that are less da maging to the quality of the final product (56) Because the spread of multidrug-resist ant pathogenic bacteria has become a serious issue, it is important to emphasize that bacteriocin resistance does not confer antibiotic resistance to bacteria (22) Toxicity The fact that bacteriocins are produced by lactococci, the lactic acid bacteria that occurs naturally in food, is an indicati on of their harmless nature. Humans and animals have ingested bacteriocins over the past centuri es, without apparent ill effect (20) From a regulatory standpoint, it is critical in some countries to distinguish bacterio cins from antibiotics, since the presence of antibiotics in food is often prohib ited. The use of bacter iocins-produc ing starter cultures as ingredients may not require special consideration in the United States if the culture

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28 (microorganism) is considered Generally Recognized as Safe (GRAS). This is primarily due to its history of safe use by food industries prior to the 1958 Food Additives Amendment (32) If a purified bacteriocin is used as a food preservative the substance might be self-affirmed as GRAS by the company according to the Code of Federal Regulations (133) In the United States, where antibiotics are prohibited in foods, nisin obtained a GRAS status for use as a biopreservative in the food industry in 1988. The LD50 value was found to be similar to that of common salt. Furthermore, consumption of nisin-c ontaining products did not result in an alteration of the intestinal bacteria l flora, because nisin is inactivated by enzymes of the intestinal tract (82) Several authors have outlined issu es involved in the approval of new bacteriocins for food use (98) and the USDA publishes guidelines for the safety assessment of new preservative (132) For approval to be granted, the bacteriocin must be chemically identified and characterized, and its use and e fficacy must be shown. The manufacturing process must be described and assays used for quantifica tion and standardization of the peptide must be shown. Toxicological data and fate of the molecule after ingestion are also needed (20) Application in Meat Products The increasing concern of foodborne liste riosis has prompted the evaluation of bacteriocins as both bactericidal and bacteriostatic agents against L. monocytogenes The applications of antimicrobials produced by some sp ecific bacteria have been studied in the meat processing industry. In recent years, bacterio cins produced by lactic acid bacteria and bacteriocins extracts have r eceived great interest in the control of foodborne pathogenic L. monocytogenes (87) Nisin is one of the most studied bacteriocins that have anti-listeria properties (84) Nisin also, has many applications in foods and is approved for use in a variety of foods throughout the world (131) It has been shown that th is bacteriocin alters the cell membrane of sensitive organisms resulting in leakage of low molecular weight cytoplasmic

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29 components and the destruction of the proton motive force (13) Some studies claim that the antimicrobial spectrum and potency of nisin can be increased when used in combination with other antimicrobials (13, 49) In an attempt to produce bacteriocins in situ a bioperservation tec hnique that has been used is the addition of competitive microflora of Latobacillus in ready-to-eat meat products stored at refrigeration temperature. In a study conducted by Abee et al. (3) Lactobacillus sake Lb674 was incorporated in the formulation of bol ogna-type sausage. The finished product was sliced, vacuum packaged, and stored at 7C for 28 days. The results showed that L. sake Lb674 produces detectable amounts of bacteriocin and de lays or completely inhibits the growth of L. monocytogenes when inoculated at levels of at least 105-106 CFU/g. Bacteriocin negative Lactobacillus had no inhibitory effect on L. monocytogenes growth. It is im portant to emphasize that the inoculation of b acteriocin-producing bacteria to produce a bacteriocin in situ would require a strain that can grow and produce the ba cteriocin at refrigeration storage temperatures (127) Bacteriocins have also b een applied as dipping soluti ons. In a study conducted by Shanshan and Mustapha (115) fresh beef samples were inocul ated with approximately 7 log CFU/mL of L. monocytogenes Scott A or E. coli O157:H7 and dipped in nisin or nisin combined with EDTA solutions for 10 minutes. The results s howed that treatment with nisin or with nisin combined with EDTA reduced the population of L. monocytogenes by 2.01 and 0.99 log CFU/cm2 respectively as compared to the control, under the conditions of vacuum package and storage at 4C for up to 30 days. However, the effect of nisin and nisin combined with EDTA against E. coli O157:H7 was marginal at 1.02 and 0.8 log CFU/cm2 reductions, respectively. Similar results were report ed by Tipayanate et al. (128) where in beef cubes inoculated with L.

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30 monocytogenes Scott A were immersed in a solution of 2% polylactic acid, 2% lactic acid, 0.4% of nisin, or combinations of each acid with nisi n for 5 min and drip-dried for 15 min. Samples were vacuum packaged and stored at refrigerat ion temperature for up to 42 days All treatments showed immediate bact ericidal effects on L. monocytogenes Scott A. On day 0, nisin alone and in combiation with polylactic acid and lactic acid signi ficantly reduced (P < 0.05) L. monocytogenes counts by 3.69, 3.76 and 3.39 log CFU/cm2, respectively, when compared with the positive control (5.33 log CFU/cm2). In a study conducted by Samelis et al. (109) dipping solutions of nisin with or without orga nic acids or salts, as inhibitors of L. monocytogenes were evaluated on sliced cooked por k bologna before vacuum packaged and storage at 4C for 120 days. Inoculated (~103 cfu/cm2) samples were immersed in 0.5% nisin, 1%, 3% or 5% lactic or acetic acid, 3% or 5% of sodium acetate or diace tate, and 3% potassium benzoate or sorbate, each combined with nisin. Nisin reduced L. monocytogenes by 1.0.5 log CFU/cm2 on day 0, followed by a listeriostatic effect for 10 days. Nisin in combination with 3% or 5% acetic acid or sodium diacetate or 3% pota ssium benzoate inhibited L. monocytogenes growth for 90 days. The author concluded that nisin with 3% sodium di acetate may be the most promising combination in dipping solutions to control L. monocytogenes on sliced, cured pork bologna. The application of nisin-co ated casing to inhibit L. monocytogenes growth is another method that has been studied by researchers. Commercially prepared frankfurters were formulated with and without 1.4% potassium lactate and 0.1% sodium diacetate and were subsequently processed in cellulose cas ings coated with and without nisin ( 50,000 IU per square inch of internal surface area) to control L. monocytogenes growth (74) The samples were inoculated with approximat ely 5 log CFU/package of a five-strain inoculum of L. monocytogenes and then vacuum packaged and stored at 4C for up to 90 days. The results demonstrated that

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31 potassium lactate and sodium diacetate display li steriostatic activity as an ingredient of commercial frankfurters. These data also establ ished that cellulose casings coated with nisin display only moderate anti-listerial activity in vacuum packaged frankfurters stored at 4C. In contrast, incorporation of nisin into th e inner surface of sausage casings and vacuumpackaging bags has demonstrated re tention of anti-listeria activity (79) Nisin powder was applied to plastic p ackaging bags at 7.75 g/cm2. Meat and poultry samples were inoculated with L. monocytogenes The bags coated with nisin powd er completely inhibited growth of inoculated L. monocytogenes samples through 12 weeks of storage at 4C. Nisin has also been used as an antibotulinal agent for the partial replacement of nitrite in cooked meat systems (3). Over the past three decades there has been an increasing research interest in the development of nitrite-free meat curing systems. The main concern with the use of nitrite for curing of meat is the eventual formation of carcinogenic N-nitrosamines. Recently, attempts have been made to use nisin A as an altemative to nitrite. While the use of this bacteriocin alone was not successful, promising results were obtained when it was combined with reduced levels of nitrite: l00-250 ppm ni sin A combined with 120 ppm nitrite was more effective than the conventional 156 ppm nitrite (114) Another application of nisin in meat systems that have been consid ered and investigated includes the addition of nisin in to the formulation of the m eat products. Ham and bologna sausages were prepared with or without addition of 500 mg kg 1 lysozyme:nisin (1:3), and 500 mg. kg 1 EDTA. Sausages were inoculated with L. monocytogenes vacuum packed and stored for 4 weeks at 8C. The result s demonstrated an inhibition of L. monocytogenes for two weeks when lysozyme, nisin and EDTA were added (54) In another study, the e ffect of adding nisin to raw meat inoculated with food-relate d bacteria was investigated by Chung et al. (19) .The results

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32 showed that nisin de layed the growth of L. monocytogenes and Staphylococcus aureus, but did not inhibit Gram-negative bacteria. Thus, it can be determined that nisin performance in meat systems is varied and that its antimicrobial pr operties are limited for gram positive bacteria. On the other hand, researchers have found that use of nisin in meats could be limited by some components which may interfere with nisi ns activity. In a study conducted by Rose et al. (104) the fate of nisin in meat products was de termined by mass spectrometry. Nisin at 0.5% was dissolved sterile water containing 0.02 N HCl. The solution was added to fresh and cooked meat and meat juice. Samples were stored at 4C for 24 hours. Nisin was recovered from cooked meat extract and cooked meat juice; however, only nisin bound to a food component was detected in fresh meat extract. Mass spectra for raw meat and juice showed a signal 307 Da greater than the mass of nisin. Results indicated that nisin was likely inactivated in raw meat by an enzymatic reaction with glutathione. In addition, the neutral pH of the meat may interfere with the antimicrobial properties of nisin, because it has been show n that nisin is 228 times more soluble at pH 2 than at pH 7 (72) For this reason, other bacterioci ns have also been evaluated in meat products (20) Several researches have shown that bacteriocins, such as pediocin have bactericidal to L. monocytogenes in meat products. The most promising outcomes were obtained using pediocin PA-1. In a st udy conducted by Nielsen et al. (91) a bacteriocin produced by Pediococcus acidilactici known as pediocin PA-1, had an inhi bitory and bactericidal effect on L. monocytogenes. Results showed that attachment of Listeria onto the meat surface was 1.0 to 2.5 log cycles fewer when it was initially treated w ith the bacteriocin. The immediate antimicrobial effect of pediocin resulted in reductions of 0.5 to 2.2 log cycles depending upon its concentration. Similar anti-listerial properties were observed when pediocin was used alone (6)

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33 or in combination with diacetate (111) Even though, pediocin is a very promising anti-listerial agent, it has not yet been approved as a food additive in the United States. Natural Antimicrobials: Description The use of edible plants, as well as thei r phytochemicals, in food preservation and improvement of organoleptic qua lities of certain tr aditional foods has been practiced for centuries. These antimicrobial properties deri ved from many plants have been empirically recognized for centuries, but only scientif ically confirmed in the last 30 years (95) The demand for minimally processed and extended shelf life foods has further increased the interest to define these naturally occurring bioactive ingredients. The compounds responsible for antimicrobial activity in herbs are primarily phenolic compounds of the esse ntial oil fraction (11) Antimicrobial activity of cinnamon, allspice, and cloves is attributed to eugenol (44) Oregano, thyme and savory have terpene, carvacol and thymol, which account for their antimicrobial activity (41) The active antimicrobial fraction of other herbs such as sage and rosemary has been suggested to also be in th e terpene fraction of the essential oils. Rosemary contains borneol, camphor and 1,8-cineole, -pinene, camphene, verbenone and bornyl acetate (119) Numerous factors influence the antimicrobi al activity of natural compounds. Some suggest that chemical composition, influence by geographic origin and crop to crop variation, and could significantly affect the activity of whole spices and essential oils (85) Furthermore, the antimicrobial properties of these compounds ma y be influenced by the type of assay method used (59) The interaction with othe r food components (lipid, prot ein) as well as surfactant, minerals, pH, time, temperature also controls th e biological activity of these natural compounds. Unless these factors are controlled, studies on the antimicrobial activity of phytophenols from oils, spices or herbs may vary considerably.

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34 Mechanism of Action Some studies have focused on the mechanism by which spices or their essentials oils inhibit microorganism. Their mode of action is generally thought to in volve interference with functions of the cytoplasmic membrane incl uding proton motive force and active transport (29) Juven et al. (64) suggested that inhibition of Salmonella Typhimurium by thymol was due to a reaction of the compound with proteins in the cytoplasmic membrane of the microorganism. This reaction could lead to cha nges in the permeability of the membrane, which would result in possible leakage and affect the proton motive force. Application in the Food Industry Society appears to be experiencing a trend of natural consumerism (121, 129) desiring fewer synthetic food additives and products with a smaller impact on the environment. Therefore, there is a need for new methods to make food safe which have a natural image. Availability of information regarding the use of essential oils (EOs) in the food industry is limited. There are approximately 3000 EOs known, of which about 300 are commercially important, and are destined primarily fo r the flavors and fragrances market (136) It has long been recognized that some EO s have antimicrobial properties (95) and the relatively recent interest in natural consumerism has lead to a rene wal of scientific interest in these substances (92, 129) Some studies suggest that the inhibitory activity of each natural compound is different for each pathogenic microorganism. Hammer et al. (59) found that thyme was the most essential oil among 20 herbs, spices and plant extracts tested against E. coli, Staphylococcus aureus and Candida albicans In contrast, Hao et al. (60) found no antimicrobial activity by thyme against Aeromonas hydrophila or L. monocytogenes on cooked beef. In another study, Smith-Palmer et al. (122) demonstrated that rosemary essentia l oil (0.02-0.05%) and sage (0.02-0.75%) were inhibitory to L. monocytogenes and S. aureus, but not to Gram negative b acteria. Further, Pandit

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35 et al., (95) showed that L. monocytogenes growth in refrigerated fr esh pork sausage was delayed by 0.5% ground rosemary or 1% rosemary essential oil. Characteristics of EDTA (Ethyl enediaminetetraacetic Acid) Ethylenediaminetetraacetic acid (EDTA) is a well-known and widely used analytical agent. The compound is a white crystalline powde r. It forms water-soluble complexes with most metal ions and is used exte nsively as a titrant for metal ions and a masking agent. In the early 1940s, many metal complexes of EDTA were prepared and studied. These investigations revealed that EDTA always form ed 1:1 water-soluble complexes (99) In order for chelation to occur, the sequestrant must have the proper steric and electronic configuration and be at the optimal ionic strength and pH. EDTA becomes increasingly dissociated as pH rises resulting in more metal becoming complexed (28) EDTA is available as a dried powder which is colorless, freely soluble in water, and has only a slight taste of sali nity in concentrations that are used in foods (28) The FDA has approved EDTA as a food additive that is generally recognized as safe (GRAS) (47) EDTA's array of biochemical properties makes it extremely va luable as a food additive. It has the ability to bind with many metals, act synergistically with other antioxidants to stabilize fats and oils, prevent discoloration of potato products, stabilize vitamins, prevent discoloration of fish and shellfish, prevent flavor changes in milk, inhi bit the thickening of stored condensed milk, enhance the foaming properties of reconstituted skim milk, pr eserve canned products, promote flavor retention and delay loss of carbonation in soft drinks, prev ent oxidation of meat products and prevent discoloration of canned fruits and vegetables (1) Antimicrobial Activity of EDTA Divalent cations such as Ca2+ and Mg2+ play specific roles in st abilizing the structure of bacterial membranes because they form meta l ions bridges between phosphate groups of

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36 phospholipids or lipopolysaccharides and the carboxyl groups of membrane proteins (118) Their removal leads to the disintegration of certain functional membrane proteins and the collapse of membrane functions. It has been shown that EDTA damages outer membrane structure by complexing Ca2+ and Mg2+ which are necessary for Gram negative bacteria (70) EDTA may act as a direct inhib itor of some microbes as well as synergistically with other antimicrobial agents. EDTA works by disrupting the integrity of the cell wall. As a enhancer of other agents, EDTA facilitates th e entrance of other agents into the cell and chelates catios which are essential for the repair of injured cells (28) In other cases, the opposite result occurs. For example, EDTA combined with nisin reduc ed the antimicrobial effect of nisin (8) Conversely, Stevens et al. (125) observed that the combinations of 20 mM lysozyme and 0.5% of nisin in cell buffer resulted in a 3.2 to 6.9 log cycle reduction in Salmonella spp. and other Gram negative bacteria. However, neither EDTA nor nisin alone showed signif icant inhibition against Gram negative bacteria. In a study conducted by Shanshan and Mustapha (115) nisin or nisin combined with EDTA solutions were evaluated on fresh beef samples inoculated with approximately 7 log CFU/mL of L. monocytogenes Scott A or E. coli O157:H7. Samples were dipped in nisin or nisin combined with EDTA for 10 minutes, vacuum package and stored at 4C for up to 30 days. The results showed that treatment with nisin significantly (P < 0.05) reduced the population of L. monocytogenes by 2.01 log CFU/cm2 when compared to the contro l. However, when nisin was combined with EDTA, L. monocytogenes counts were reduced by 0.99 log CFU/cm2 as compared to the control. The result s suggested that ni sin alone inhibited L. monocytogenes growth, but its anti-listerial pr operties were reduced when it was combined with EDTA. This demonstrated no synergistic e ffects between nisin and EDTA.

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37 CHAPTER 3 EVALUATION OF NATURAL ANTIMI CROBIAL COMPOUNDS AGAINST Listeria monocytogenes BY KIRBY-BAUER DISC DIFFUSION METHOD Introduction In spite of modern improvements in food production techniques, food safety is an increasingly important public health issue (141) Food pathogens can travel long distances and infect their hosts at a local and global level. Consequently, newly introduced pathogens can spread rapidly in susceptible hosts. Listeria monocytogenes is recognized as an important cause of foodborne illness, with high hospitalization rates (88%) and fatality rates (20%) (18) Regulatory agencies have established strict requirements for controlling L. monocytogenes in food products because of its widespread distribution in nature an d its ability to grow under unfavorable conditions (78) The use of appropriate concentrations and/ or combinations of antimicrobi al compounds may contribute to the safety of food products against L. monocytogenes (134) In-vitro antimicrobial susceptibility testing done in recent years has indicated that bactericidal compounds such as organic acids, salts, and genera l recognized as safe (GRAS) substances can control L. monocytogenes (93) The results obtained in these studies may help researchers in selecting initial pr ocedures and antimicrobial agents that will be used for specific products. A commonly used susceptibility test is the disc diffusion method. The antimicrobial susceptibility testing (AST) is not only practical but also the method of choice for the average laboratory. All techniques used i nvolve either the diffusion of an antimicrobial agent in agar or its dilution in agar or broth. Some automate d techniques are also variations of the above methods (68) The Kirby-Bauer method is a standardized filter-paper disc-agar diffusion procedure (15) that is usually used for antimicrobial suscepti bility testing. This method is recommended by the

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38 Clinical and Laboratory Standards Institute (NCCLS), which is an international, interdisciplinary, non-profit, non-government al organization, composed of medical professionals, government, industry, healthcare providers, educators, etc (89) This method allows for the rapid determination of the efficacy of an antimicrobial by measuring the diameter of the zone of inhibition that results from diffusion of the ag ent into the medium surrounding the disc (15) The accuracy and reproducibility of the Kirby-Bauer method is dependent on following the procedures described by the NCCLS (88) Interpretative criteria of NCCLS are de veloped based on international collaborative studies. This information is well correlated with minimum inhibitory concentration (MICs) results that have been corroborated with clin ical data. Based on study results, the NCCLS interpretative criteria are revised frequentl y. NCCLS guidelines are approved by the Food and Drug Administration (48) and recommended by World Health Organization (140) The objectives of this study were (1) to determine the anti-lis terial properties of different spices and antimicrobials alone and in combination against a five strain inoculum of L. monocytogenes using Kirby-Bauer method disc diffusion and (2) evaluate the anti-listerial properties of nisin alone and in combination with the most effective antimicrobials identified during preliminary evaluations. Materials and Methods Inoculum Preparation Reference strains of L. monocytogenes a, b, 4 b, Scott A and 19115, were obtained from ABC Research Corporation in Gainesville, FL and used as the inoculum to evaluate the antiListeria properties of different antimicrobial solu tions using the KirbyBauer antimicrobial susceptibility test procedure. The media and ma terials used for the cultivation, growth and maintenance of the strains were purchased from Fisher Scientific (Pittsburgh, PA 15238). The

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39 strains were transferred individua lly to test tubes containing 10 mL of tryptic soy broth (TSB, Difco Laboratories, Detroit, MI 482132-7058, Cat.No. DF 0369-17-6) using a flamed sterilized 3mm inoculation loop. The broth was incubate d at 35C for 24 hours. After incubation the aliquots were poured into sterile centrifuge tubes and centrifuged (Sorvall RC-5B, Dupont Instruments, Newton, CT 06470) at 5000g for 10 mi nutes. After centrifugi ng, the supernatants were discarded and the pellets were re-suspe nded in 10 mL of sterile distilled water and centrifuged again. The supernatants were again di scarded and the pellets were re-suspended in 1 mL of 3% TSB with 30% glycerol in a 2 mL cryovial (Corning In corporated, Corning, NY 14831, Cat.No. 03-374-21). The pellets were stored at -45C and used as the stock culture for the inoculation studies. Antimicrobial Solution Preparation Natural antimicrobial solutions were selected based on results reported in previous studies (59, 60, 71, 122) Antimicrobial solutions were prepared using distilled st erile water. The appropriated aliquot of each antimicrobial was wei ghed and dissolved in sterile distilled water. The solutions were used within 30 minutes after pr eparation. Sterile dis tilled water was used as control treatment. The analyses of antimicrobial solutions were conducted in two separate Phases. For Phase one, a total of 47 antimicrobial solutions were te sted (Table 3-1). These combinations were analyzed in duplicates, and three separate trials of the study we re conducted. The antimicrobial combinations that showed the greatest inhibition of L. monocytogenes were selected for further analysis during Phase Two of the study. Nisaplin (Danisco, Copenhagen, Denmark) is a commercial nisin product containing 106 IU nisin/g. A concentration of 0.1%, 0.2%,and 0.3% nisin was obtained by adding 0.1, 0.2, and 0.3 g of Nisaplin with 0.17 mL of 0.02 N food grade HCL (Fishe r Scientific, Pittsburgh, PA

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40 15238, Cat. No. 7647-01-0) and 0.75g NaCl (Sigma Chemical, St. Louis, MO 63178, Cat No. S9625-500G) into 100 mL of sterile distilled wate r. EDTA (Sigma Chemical, St. Louis, MO 63178, Cat No. 59H03591) was prepared at a ratio of 0.75 g per 100 mL of sterile distilled water to obtain a final concentration of 20 mM. He rbalox Seasoning (Kalsec, Kalamazoo, MI 490050511, Code No. 41-19-02), a commercial rosemary extract, has shown antimicrobial properties as well as inhibition of oxidati ve deterioration. Rosemary so lution was prepared suspending 1 mL of the Herbalox into 100 mL of sterile di stilled water. A con centration of 0.15% and 0.25% of sodium diacetate (Sigma Chemical, St. Louis, MO 63178, 59H03591) and 3% and 5% of potassium benzoate (Versicol Chemical Co. Rosemont, IL 60018, Cat. No. 030222) was obtained by dissolving 0.15 and 0.25 g of sodium diacetate, respectivel y and 3 and 5 g of potassium benzoate, respectively, into 100 mL of sterile distilled water. Aquaresin thyme (Kalsec, Kalamazoo, MI 49005-0511, Code No. 8 05454) is a commercial thymol extract. Thymol solution was prepared by dissolving 1 mL of aquaresin thyme ex tract into 100 mL of sterile distilled water to obtain a final concentration of 1%. A concentration of 1% and 2% of vinegar (Albertsons, Gainesville, FL 32608, UPC No. 04116310124) was obtained by adding 1 mL and 2mL of vinegar into 100 mL of sterile distilled water, respectively. During Phase Two, 18 different combinations we re analyzed (Table 3-2). These were selected based on the results obtained in Phase one. Following the same protocol used in Phase One, the antimicrobial combinations were analyzed in duplicates and was repeated three times to show the reproducibility of the trials. A concentration of 0.1%, and 0.2% nisi nwas obtained by adding 0.1, and 0.2 g of Nisaplin with 0.17 mL of 0.02 N food grade HCL (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 7647-01-0) and 0.75g NaCl (Sigma Chemical, St. Louis, MO 63178, Cat No. S9625-

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41 500G) into 100 mL of sterile di stilled water.20 mM EDTA (Sig ma Chemical, St. Louis, MO 63178, Cat No. 59H03591) was prepared by dissolvi ng 0.75 g of EDTA into 100 mL of sterile distilled water. Herbalox Seasoning (Kal sec, Kalamazoo, MI 49005-0511, Code No. 41-1902), a commercial rosemary extract, has shown an timicrobial properties as well as inhibition of oxidative deterioration. 1% rose mary solution was prepared susp ending 1 mL of the Herbalox into 100 mL of sterile distilled water. Thymol extract was obtained from the commercial product Aquaresin thyme (Kalsec, Kalamazoo, MI 49005-0511, Code No. 805454). Thymol solution was prepared by dissolving 1 mL of aquaresin thyme extract into 100 mL of sterile distilled water to obtain a final concentration of 1%. Preparation of Modified Oxford Media Agar (MOX) Modified Oxford Media (MOX, Difco Laborat ories, Detroit, MI 48232-7058, Cat. No. DF 0225-17-0) and Modified Oxford antimicr obial supplement (MOX supplement, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0218-60-5) were prepared from a commercially available dehydrated base according to th e manufacturer's instructions. Immediately after autoclaving, the media was allowed to cool in a 45 to 50 C water bath for approximately 30 minutes and 5 mL of antimicrobial supplement was added. The media was poured into plastic flatbottomed Petri dishes on a horizontal level surface to give a uniform depth of approximately 4 mm. The plates were allowed to cool to r oom temperature and stored at 4 1 C. Inoculation of Agar Plates Frozen L. monocytogenes inocula were allowed to thaw at room temperature for 10 minutes. A loopful of the thawed stock culture was transferred to test tubes containing 10 mL of 3% TSB and incubated at 35C for 24 hours. After incubation, the aliquots were centrifuged (5000 rpm for 10 min at 16C) and washed with sterile 0.1% buffered peptone water (BPW, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF O1897-17-4). The aliquots were then

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42 serially diluted with BPW to concentrations of 10-1 to 10-8. A sterile cotton swab was dipped into the 10-8 dilution. The swab was rota ted several times and pressed firmly on the inside wall of the tube above the fluid level, removing the excess inoculum from the swab. The MOX agar plates were inoculated by streaking the swab over the entire surface This procedure was repeated by streaking three more times, rotating the plate approximate ly 60 each time to ensure an even distribution of the inoculum. As a fina l step, the rim of the plate was swabbed, and the lid was placed on the Petri dish, and plates we re allowed to sit a r oom temperature for 2-3 minutes to allow any excess surface moisture to be absorbed before applying the antimicrobial impregnated discs. Kirby-Bauer Disc Diffusion Test Paper-filter discs (6 mm diameter) (Bect on & Dickinson, Sparks, MD 21152, Cat. No. 231039) were immersed into the antimicrobial solution for fifteen seconds using a flamed sterilized forceps and placed ont o the surface of the inoculated MOX agar plate. Each disc was pressed down to ensure complete contact with th e agar surface. Two discs were placed on each 150 mm plate (Figure 1). The plates were inverted and placed in an incubator set at 35 C within 15 minutes after the discs were applied to the agar surfaces. After 24 hours of incubation, each plate was examined. The diameters of the z ones of complete inhibition were measured, including the diameter of the disc. Zones were measured to the nearest millimeter using a graduated ruler. The ruler was held on the back of the inverted Petri plate and held above a nonreflecting background and illumi nated with reflected light to take each measurement. Data Analysis Statistical analyses were conducted for zones of compete inhibition for a total of eight measurements per treatment on Phase One and for a total of twelve measurements per treatment on Phase Two. The general linear model program (PROC GLM) of SAS system (110) was

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43 employed to analyze trial, treatment and treatment by trial. A total of 57 treatment combinations were evaluated. Comparisons among means we re performed using SAS Tukey Multiple Range test procedure. Treatments effects and di fferences were considered significant when P < 0.05. Results and Discussion The data demonstrated L. monocytogenes was inhibited when nisin was used (Table 3-3). The zone of inhibition for L. monocytogenes increased as the concentration of nisin increased from 0.1% to 0.5%. Treatments in which 0.5% nisin and 0.5% nisin combined with food grade hydrochloric acid (HCL) were used resulted in an inhibi tion zone of 3.20 and 3.18 mm, respectively. These two treatments showed th e highest inhibition zone when compare to all other treatments (P < 0.05). No differences (P < 0.05) were found when 0.5% nisin was used alone or in combination with food grade HCL and ethylenediaminetetraace tic acid (EDTA). The use of rosemary alone resulted in an inhibition zone of 1.03 mm, and the zone of in hibition increased approximately twice this size as the concentration of nisin ad ded to rosemary was increased in 0.1% units. L. monocytogenes growth was not inhibited by the treatmen ts containing 1% and 2% vinegar, 3% and 5% potassium benzoate, 1% thymol or 0.15% and 0.25% sodium diacetate alone and in combination with nisin. The results obtained in this trial suggested that nisin used alone or in combination with 1% rosemary and 20 mM EDTA may inhibit growth of L. monocytogenes. These findings were useful in developing Phase Two of the study, in which, a total of 18 combinations were further analyzed. The FDA allows the use of nisin in foods at maximum level of 0.5% (U.S. Food and Drug Administration, 1988). During Phase Two, concentrations of nisin at 0.1% and 0.2% were

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44 evaluated alone and in combination with 1% ro semary, 1% thymol and 20 mM EDTA (Table 34). During Phase Two, no significant differences (P > 0.05) were found between 0.1% nisin and 0.2% nisin when combined with 1% rosemar y. The highest zone of inhibition (P < 0.05) was achieved when 0.2% nisin was used in combination with 1% rosemary and 20 mM EDTA. The zone of inhibition was below 0.63 mm when thymol was used alone and in combination with nisin. There was no signi ficant difference (P > 0.05) between the control tr eatment (water) and thymol alone and in combination with 0.1% nisin. Antimicrobial susceptibility testing procedures provide a useful proactive tool for assisting researchers in determining the antimicrobial properties of additives prior to incorporating them into a food matrix; thus allowi ng researchers to use testing procedures that are more cost efficient and less time consuming. This study revealed the susceptibility of L. monocytogenes to natural compounds, such as rosemary, EDTA and different concentrations of nisin. The next step would be to evaluate these results in a f ood matrix and design an antimicrobial system that would inhibit the growth of L. monocytogenes in a specific meat product.

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45 Table 3-1. Fifty-eight antimicrobial solutions evaluated in Phase 1 List of antimicrobial solutions 1% Rosemary Extract 3% Potassium Benzoate 0.1% Nisin 1% Rosemary Extract 3% Potassium Benzoate 0.1% Nisin 0.2% Nisin 1% Rosemary Extract 3% Potassium Benzoate 0.2% Nisin 0.3% Nisin 1% Rosemary Extract 3% Potassium Benzoate 0.3% Nisin 0.1 % Thymol 5% Potassium Benzoate 0.1% Thymol 0.1% Nisin 5% Po tassium Benzoate 0.1% Nisin 0.1% Thymol 0.2% Nisin 5% Po tassium Benzoate 0.2% Nisin 0.1% Thymol 0.3% Nisin 5% Po tassium Benzoate 0.3% Nisin 0.15% Sodium Diacetate 0.1% Nisin 0.15% Sodium Diacetate 0.1% Nisin 0.2% Nisin 0.15% Sodium Diacetate 0.2% Nisin 0.3% Nisin 0.15% Sodium Diacetate 0.3% Nisin 0.4% Nisin 0.25% Sodium Diacetate 0.5% Nisin 0.25% Sodium Diacetate 0.1% Ni sin 0.1% Nisin + 20 mM EDTA 0.25% Sodium Diacetate 0.2% Ni sin 0.2% Nisin + 20 mM EDTA 0.25% Sodium Diacetate 0.3% Ni sin 0.3% Nisin + 20 mM EDTA 1% Vinegar 0.4% Nisin + 20 mM EDTA 1% Vinegar 0.1% Nisin 0.5% Nisin + 20 mM EDTA 1% Vinegar 0.2% Ni sin 0.1% Nisin + HCL 1% Vinegar 0.3% Ni sin 0.2% Nisin + HCL 2% Vinegar 0.3% Nisin + HCL 2% Vinegar 0.1% Ni sin 0.4% Nisin + HCL 2% Vinegar 0.2% Ni sin 0.5% Nisin + HCL 2% Vinegar 0.3% Nisin Water

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46 Table 3-2. Seventeen antimicrobial solutions evaluated in Phase 2 List of Antimicrobial Solutions water EDTA 0.1Nisin 0.2Nisin Thymol Rosemary 0.1Nisin-EDTA 0.2Nisin-EDTA 0.1Nisin-Thymol 0.1Nisin-Thymol-EDTA 0.1Nisin-Rosemary 0.1Nisin-Rosemary-EDTA 0.1Nisin-Rosemary-Thymol-EDTA 0.2Nisin-Thymol 0.2Nisin-Thymol-EDTA 0.2Nisin-Rosemary 0.2Nisin-Rosemary-EDTA 0.2Nisin-Rosemary-Thymol-EDTA

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47 Table 3-3. Mean zone of inhi bition for antimicrobial solutions evaluated in Phase 1 Treatments Inhibition zone (mm)* 0.5% Nisin 3.20 a 0.5% Nisin + HCL 3.18 a 0.5% Nisin + HCL + 20 mM EDTA 2.95 ab 1% Rosemary + 0.3% Nisin 2.72 b 0.4% Nisin 2.63 bc 0.4% Nisin + HCL 2.38 cd 0.4% Nisin + HCL + 20 mM EDTA 2.18 de 1% Rosemary + 0.2% Nisin 2.08 def 0.2% Nisin 2.08 def 0.3% Nisin 1.95 efg 0.3% Nisin + HCL + 20 mM EDTA 1.83 fgh 0.3% Nisin + HCL 1.78 fgh 1% Thymol + 0.3% Nisin 1.70 ghi 1% Thymol + 0.1% Nisin 1.65 ghi 0.2% Nisin + HCL + 20 mM EDTA 1.63 ghi 1% Rosemary + 0.1% Nisin 1.53 hij 1% Thymol + 0.2% Nisin 1.40 ijk 0.1% Nisin + HCL 1.28 jkl 0.2% Nisin + HCL 1.25 jkl 5% Potassium benzoate + 0.3% Nisin 1.13 klm 0.1% Nisin + HCL + 20 mM EDTA 1.10 klm 1% Rosemary 1.03 lmn 0.1% Nisin 0.90 mno 0.25% Sodium diacetate + 0.2% Nisin 0.88 mno 5% Potassium benzoate + 0.2% Nisin 0.83 mno 0.25% Sodium diacetate + 0.3% Nisin 0.75 no 3% Potassium benzoate + 0.3% Nisin 0.68 o 0.15% Sodium diacetate + 0.3% Nisin 0.65 o 1% Vinegar + 0.3% Nisin 0.63 o 2% Vinegar 0.00 p 3% Potassium benzoate 0.00 p 5% Potassium benzoate 0.00 p 1% Vinegar 0.00 p 3% Potassium benzoate + 0.1% Nisin 0.00 p 3% Potassium benzoate + 0.2% Nisin 0.00 p 5% Potassium benzoate + 0.1% Nisin 0.00 p 1% Thymol 0.00 p 1% Vinegar + 0.1% Nisin 0.00 p 1% Vinegar + 0.2% Nisin 0.00 p 2% Vinegar + 0.1% Nisin 0.00 p 0.15% Sodium diacetate 0.00 p 0.15% Sodium diacetate + 0.1% Nisin 0.00 p 0.15% Sodium diacetate + 0.2% Nisin 0.00 p 2% Vinegar + 0.3% Nisin 0.00 p

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48 0.25% Sodium diacetate 0.00 p 0.25% Sodium diacetate + 0.1% Nisin 0.00 p 2% Vinegar + 0.2% Nisin 0.00 p water 0.00 p Each means value represent f our individual measurements a-p values in same column with different superscripts are significantly different (P < 0.05)

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49 Table 3-4. Mean zone of inhi bition for antimicrobial solutions evaluated in Phase 2 Treatments Inhibition zone (mm)* 0.2% Nisin + 1% Rosemary + 20 mM EDTA 1.27 a 0.2% Nisin + 1% Rosemary + 1% Thymol + 20 mM EDTA 1.22 ab 0.2% Nisin 1.17 ab 0.2% Nisin + 1% Rosemary 1.17 ab 0.1% Nisin + 1% Rosemary 1.17 ab 0.1% Nisin 1.09 abc 0.1% Nisin + 1% Rosemary + 1% Thymol + 20 mM EDTA 1.05 abc 0.1% Nisin + 1% Rosemary + 20 mM EDTA 1.05 abc 20 mM EDTA 0.95 bc 0.2% Nisin + 1% Thymol + 20 mM EDTA 0.87 cd 0.1% Rosemary 0.82 cd 0.2% Nisin + 20 mM EDTA 0.82 cd 0.2% Nisin + 1% Thymol 0.80 cd 0.1 % Nisin + 20 mM EDTA 0.80 cd 0.1 % Nisin + 20 mM EDTA + 1% Thymol 0.80 cd 1% Thymol 0.62 de 0.1 % Nisin + Thymol 0.47 e Water 0.00 f Each means value represent six individual measurements a-f values in same column with different superscripts are significantly different (P < 0.05)

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50 CHAPTER 4 EVALUATION OF CONCENTRAT IONS OF NISIN AGAINST Listeria monocytogenes ON READY-TO-EAT TURKEY HAM ST ORED AT 4C FOR 63 DAYS Introduction Listeria monocytogenes the causative agent of listeriosis, has resulted in numerous major foodborne outbreaks worldwide (55) The ability of L. monocytogenes to grow at temperatures ranging from 0 to 45C (7) its high tolerance for salt (42) and its ability to initiate growth at a relatively low pH (9) makes this pathogen particularly difficu lt to control in food. Hygienic and sanitation practices applied in meat processing facilities are often in sufficient to prevent contamination of processed meat products (21) L. monocytogenes can be resistant to many food preservation methods and can increase to high numbers during refrigerated storage (137) and under low oxygen tension (73) Thus, effective hurdle technol ogies to inhibit growth of the pathogen are needed. A novel approach to controlling L. monocytogenes in foods is the use of antimicrobial bacteriocins from lactic acid bacteria (32) Nisin, a lanthionine-cont aining polypeptide produced by Lactococcus lactis subsp. lactis (5) is a bacteriocin with antimicrobial activity against L. monocytogenes (14) In the United States, where antibio tics are prohibited in foods, nisin obtained GRAS status for use as a biopreservative in the food industry in 1988 (131) The LD50 value was found to be simila r to that of common salt (82) Nisins mechanism of action is based on the disruption of the cytoplasmic cell membrane, as evidenced by the rapid efflux of small mo lecules from both whole cells and liposomes (4, 50, 138) As a result, nisin depletes the proton motive force (PMF) of sensitive cells and artificial liposomes (13, 49) Nisin acts through a multistep process which includes binding of nisin to the cell, insertion into the membrane, and pore formation (50, 105) Anionic phospholipids play an important role in nisins interaction with membranes (35) On binding to anionic phospholipids,

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51 nisin causes a local pertur bation of the lipid bilayer (38) followed by electrical potential ( )or pH gradient ( pH)that enhance insertion into the membrane to form a wedge-like pore (80) Nisin also, has many applications in foods a nd is approved for use in a variety of foods throughout the world. Applications of nisin in meat systems th at have been considered or investigated include: the addition of nisin-producing Lactococcus lactis subsp. lactis to meat systems in an attempt to produce nisin in situ (3) ; dipping solutions of nisin (109, 115, 128) ; nisincoated casing (74) ; addition of nisin into the fo rmulation of the meat product (54, 108) ; the use of nisin as an antibotulinal agent for the pa rtial replacement of nitrite in cooked meat systems (3) ; and the use of nisin in canned meats as a means of reducing thermal processing time (84) The aim of the study was to evaluate the anti-lis terial properties of different concentrations of nisin (0.2%, 0.3%, 0.4% a nd 0.5%) on ready-to-eat (RTE) turkey ham using a novel postprocessing treatment against L. monocytogenes that would be utilized on RTE meat products. Materials and Methods This study was conducted in two trials dur ing which 0.2%, 0.3%, 0.4% and 0.5% nisin solutions were used to treat RTE turkey ham inoculated with five strains of L. monocytogenes. The trials were conducted at the University of Florida Meat Pr ocessing Laboratory and Microbiology Laboratory, Gainesville, FL. Th e same procedure was used on both trials. Inoculum Cultivation and Storage Reference strains of L. monocytogenes a, b, 4 b, Scott A and 19115, were obtained from ABC Research Corporation in Gainesville, FL and were used as the inoculum to evaluate the antiListeria properties of different concentrations of nisin. The media and materials used for the cultivation, growth and mainte nance of the strains were purchased from Fisher Scientific (Pittsburgh, PA 15238). The strains were transferred individually to test tubes containing 10 mL of tryptic soy broth (TSB, Difco Laboratorie s, Detroit, MI 482132-7058, Cat.No. DF 0369-17-6)

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52 using a flamed sterilized 3mm inoculation loop. The broth was incubated at 35C for 24 hours. After incubation the aliquots were poured into sterile centrifuge tubes and centrifuged (Sorvall RC-5B, Dupont Instruments, Newton, CT 06470) at 5000rpm for 10 minutes. After centrifuging, the supernatants were discarded and the pellets were re-suspended in 10 mL of sterile distilled water and centrifuged again. The supernatants we re again discarded and the pellets were resuspended in 1 mL of 3% TSB with 30% glycer ol in a 2 mL cryovial (Corning Incorporated, Corning, NY 14831, Cat.No. 03-374-21). The pellets were stored at -45C and used as the stock culture for the inoculation studies. Inoculum Preparation Frozen L. monocytogenes strains were allowed to thaw at room temperature for 10 minutes. A loopful of the thawed stock culture was transferred to test tubes containing 10 mL of 3% TSB and incubated at 35C for 24 hours. After incubation, the aliquots were centrifuged (5000 rpm for 10 min at 16C) and washed with sterile 0.1% buffered pept one water (BFP, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF O1897-17-4). The aliquots were then serially diluted with BPW to concentrations of 10-1 to 10-8. Antimicrobial Solutions Preparation Formulations were developed to prepare 0.2%, 0.3%, 0.4% and 0.5% nisin solutions to ensure that desired concentrati on of each solution made contact with chopped turkey ham. The control turkey ham formulation c onsisted of the following (% of tota l weight in the formulation): chopped turkey ham (90%) and water (10%) (Tab le 4-1). Each nisin concentration was dissolved using 10% sterile water, according to each treatment formulation, which was added into the bag containing the chopped turkey ham. Sterile deionized water was used as control. Nisaplin (Danisco, Copenhagen, Denmark) is a commercial nisin product containing 106 IU nisin/g. Nisaplin, HCL and Na Cl were used to prepare the antimicrobial solutions and were

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53 diluted in a predetermine amount of sterile deio nized water (10%) (Table 4-1) A concentration of 0.2%, 0.3%, 0.4% and 0.5% ni sin was obtained by adding 0.2, 0.3, 0.4 and 0.5 g of Nisaplin with 0.17 mL of 0.02 N food grade HCL (Fishe r Scientific, Pittsbu rgh, PA 15238, Cat. No. 7647-01-0) and 0.75g NaCl (Sigma Chemical, St. Louis, MO 63178, Cat No. S9625-500G) to the turkey ham (based on total batch weight). Nisi n solutions were stored at 4C and used within 3 days. Sample preparation JennieO RTE turkey hams were purchased fr om Publix, Gainesville, Florida with an expiration date of 60 days. The turkey hams were immediately transported to the University of Florida Meat Processing Laborat ory and Microbiology Laboratory, Gainesville, Florida and stored at 4C for no longer than 24 hours before using. The hams were aseptically transferred from the vacuum packaged bag to pre-sterilized trays (polypropylene) and chopped into approximately 0.5 cm pieces. The ham was chopped to simulate how it may be used in ham salad or similar foods. Inoculation and Treatment The turkey ham pieces were placed on pre-steril ized trays and inoculated by spraying them with 1 mL of L. monocytogenes inoculum at 1.0 X 108 CFU/mL. Inoculated samples were left to stand at room temperature for 20 min to allow for bacterial attachment and to ensure a final concentration of 104 CFU/gram. Duplicate samples of inoculat ed chopped turkey ham were aseptically weighted according to its corresponding treatment (Table 1) and placed in a labeled FoodSaver bag (Tilia, San Francisco, California, FoodSaver Vacloc Roll). Corresponding treatments were applied to the chopped ham followed by mixing the ham and treatme nt solution to ensure a proper distribution between them. The bags were then vacuum packaged (Tilia, San Francisco, California,

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54 FoodSaver Bagvac), leaving the nisin solution in the package, and stored in a 4C cooler for subsequent microbiological and ch emical analysis. Samples were analyzed after 0, 7, 14, 21, 28, 35, 42, 49, 56 and 63 days of storage. Microbiological Analyses Twenty-five grams of chopped turkey ham were transferred aseptical ly from the vacuum packaged bag into a sterile stomacher bag (F isher Scientific, Pittsburgh, PA 15238, Cat. No. 01002-44) with 225 mL of sterile 0.1% buffered pept one water (BPW, Difco, Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) and shaken approximately 30 times. Further serial dilutions were prepared by addi ng 1 mL of the diluted sample homogenate into 9 mL of 0.1% BPW until the appropriate dilutions were obtained. Listeria monocytogenes and lactic acid bacteria analysis A volume of 0.1 mL of the dilutions was di spensed onto pre-poured Modified Oxford Media (MOX, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0225-17-0) containing Modified Oxford antimicrobic supplement (MOX supplement, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0218-60-5) for L. monocytogenes identification and to lactobacilli MRS agar (Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 088 2170) for the isolation of lactic acid bacteria. The spr ead plate technique was used to evenly distribute the sample over the plate. Using this technique, a flamed sterilized bacterial cell spreader (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 08-769-2A) was used to spread the sample over the plate as the plate was spun on an inoculating turntable (Fis her Scientific, Pittsbu rgh, PA 15238, Cat. No. 08758-10). All samples were plated in duplicate. The Petri plates were inverted and incubated for 24 hours at 35C for L. monocytogenes and 37C for lactic acid bact eria. Plates with 25 to 250 colonies were counted. Black colonies surrounded by a black halo and white/grayish colonies

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55 were considered presumptively L. monocytogenes and lactic acid, respec tively. Microbiological counts were reported as Logarithmic Col onic Forming Units per gram (Log CFU/g). Aerobic bacteria analysis Aerobic bacteria counts were performed using 3M Petrifilm aerobic count plates (St. Paul, Minnesota, Cat. No. 6404). The Petrifilm aerobic count plate was placed on a level surface. The top film was lifted and 1 mL of sample was dispen sed to the center of the bottom of the plate. The top film was released down onto the sample and the plastic spreader was placed on the center of the plate, convex si de down. After approximately one minute, the spreader was removed from the plate. All samples were plat ed in duplicate. Plates were incubated for 24 hours at 25C in a horizontal posit ion with the clear side up in stacks of 12 plates. Plates containing 15 to 150 colonies were counted and recorded. Microbiological counts were expressed as Logarithmic Colonic Forming Units per gram (Log CFU/g). pH Analysis The pH analysis of the chopped RTE turk ey ham was performed using a pH meter (Accumet Basic AB15, Fisher Scientific, Pi ttsburgh, PA 15238, Model No. AB15, Serial No. AB81210535). Twenty-five grams of chopped ham was aseptically removed from the vacuum packaged bag and placed into a sterile plastic bag (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 01-002-44) into which 225 mL of 0.1% buffere d peptone water (BPW, Difco, Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) wa s added. Duplicated pH measurements were taken from each ham homogenate. Data Analysis Statistical analyses were conducted for enumera tion data of a total of eight measurements per treatment of L. monocytogenes aerobic bacteria, and lactic aci d bacteria. Also, statistical analysis was conducted for pH values of six m easurements per treatment. The general linear

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56 model program (PROC GL M) of SAS system (110) was employed to statistically analyze trial, day, treatment and treatment by day. Variations in data were accounted for by four treatments effects: trial, treatment, day, and treatment*day. Comparis ons among means were performed using SAS Tukey Multiple Range test procedur e. Treatments effects and differences were considered significant when P < 0.05. Results and Discussion pH Analysis No difference was observed in pH values among treatments from days 0 to 49 (Table 4-2). However, by day 56, nisin at 0.5% had significantly higher (P < 0.05) pH when compared to all other treatments. All treatments experienced a slight decrease in pH throughout storage. This may be attributed to the production of numer ous compounds such as acidic metabolites and carbonic acid that may decrease the pH (37) The pH values decrease as the concentration of nisin decreased from 0.5% to 0.2%. This may ha ve been caused by the number of lactic acid bacteria present in the samples, which increase as the concentration of nisin decrease from 0.5% to 0.2%. Listeria monocytogenes Analysis AntiListeria effects of nisin at different concentrat ions were similar (P > 0.05) for the first two weeks (Table 4-3). Bacterial growth over tim e can be graphed as a cell number versus time. This is called a growth curve (37) The cell number is plotted as the log of the cell number, since it is an exponential function. The lag phase is the first phase observed. It is characterized by little or no increase in cell numbe r; however, the cells are activel y metabolizing, in preparation for cell division and depending on the growth medi um, the lag phase may be short or very long (43) The results demonstrated an extended la g phase for the 0.5% nisin treatment through 63 days storage, maintaining L. monocytogenes counts at less than 1.95 log CFU/g.

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57 Treatment with 0.2%, 0.3%, 0.4% and 0.5% of nisin caused initial population reduction as compared to the positive control (P < 0.05). On day 0, the initial bacterial population of the positive control (4.97 log CFU/g) was significantly reduced (P< 0.05) to 4.00 log CFU/g, 4.37 log CFU/g, 4.37 log CFU/g, and 4.55 log CFU/g when treated with 0.2%, 0.3%, 0.4%, and 0.5% nisin, respectively. Through out the 63 days of storage, the number of L. monocytogenes increased approximately 1.00 log CFU/g in the 0. 5% nisin treated sample and approximately 2.00 log CFU/g in the 0.2%, 0.3% and 0.4% nisin tr eated samples. This suggested that these nisins concentrations may have an initial bacterial effect against L. monocytogenes population and may help to maintain the L. monocytogenes counts lower during storage. The overall mean values for all treatments revealed that nisi n resulted in 1.62.18 l og CFU/g reductions of L. monocytogenes. Results from the study indicate that th e antimicrobial effectiveness of nisin increased as its concentration incr eased from 0.2% to 0.5% and that L. monocytogenes may be controlled in vacuum package RTE turkey ham stored at 4C when treated with the concentrations of nisin evaluated in this study. Lactic Acid Bacteria Analysis On day 0, lactic acid bacteria c ounts were significantly lower (P < 0.05) on the nisin treated samples when compared to the positiv e and negative control, showing an initial antimicrobial effect against lactic acid bacteria (Table 4-4). From days 7 to 49, no difference was observed in pH values among treatments. Ho wever, by day 56 nisin at 0.5% had higher (P < 0.05) pH when compared to all other treatments. Lactic acid bacteria populat ion increased over time for all treatments under vacuum packaged and refrigeration (4C) conditions. Lactic acid bacteria counts were significantly lower for 0.5% nisin (P < 0.05) when compared to positive and negativ e controls, revealing nisins effectiveness against lactic acid bacteria. Results from the study indi cate that lactic acid

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58 bacteria may be restricted in vacuum package RTE turkey ham stored at 4C when treated with at least 0.2% nisin and th e effectiveness of nisin increase as its concentration increased to 0.5%. Aerobic Bacteria Analysis Initial counts of aerobic bact eria on ready-to-eat turkey ham inoculated with L. monocytogenes following different treatments are shown in (Table 4-5). The initial population of aerobic bacteria on the turkey ham was significantly higher (P < 0.05) in the positive control when compared to all other treatments. The an timicrobial treatment that contained 0.5% nisin was the most effective (P < 0.05) reducing the initi al populations of aerobic bacteria. The initial population of aerobic bacteria was reduced as the concentration of nisin increased from 0.2% to 0.5%. These results suggest that initial population of aerobic bacteria may be restricted when treated with a concentration equal or greater than 0.2% nisin unde r vacuum package and refrigeration (4C) conditions.

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59 Table 4-1. Formulation of nisin solutions for va cuum packaged ready-to-eat turkey ham stored at 4 C for 63 days Percentage (%) of ingred ient in total composition Treatment Negative Control1 Positive Control2 0.2% nisin 0.3% nisin 0.4% nisin 0.5% nisin Ingredient Ham 90.00 90.0088.8888.7888.68 88.58 Water 10.00 10.0010.0010.0010.00 10.00 Nisin 0.00 0.000.200.300.40 0.50 NaCl 0.00 0.000.750.750.75 0.75 HCl 0.00 0.000.170.170.17 0.17 100.00 100.00100.00100.00100.00 100.00 1 Negative control: without L. monocytogenes inoculum 2 Positive control: with L. monocytogenes inoculum

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60Table 4-2. pH measurements on Ready-To-Eat turkey ham supplemen ted with various concentrations of nisin and inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Negative control 6.24 a,x 5.76 a,y 4.97 a,z 4.87 a,z 4.86 a,z 4.81 a,z 4.79 a,z 4.67 a,z 4.86 b,z 4.82 b,z Positive control 6.14 a,x 5.07 a,y 4.85 a,y 4.79 a,y 4.78 a,y 4.82 a,y 4.77 a,y 4.94 a,y 4.74 b,y 4.77 b,y 0.2% nisin 6.21 a,x 5.67 a,xy 5.46 a,xy 5.29 a,xy 5.19 a,xy 5.00 a,y 5.02 a,y 5.13 a,xy 5.01 b,y 4.86 ab,y 0.3% nisin 6.22 a,x 6.10 a,x 6.11 a,x 5.72 a,x 5.43 a,x 5.41 a,x 5.40 a,x 5.29 a,x 5.10 b,x 5.24 ab,x 0.4% nisin 6.26 a,x 5.85 a,xy 5.56 a,xy 5.41 a,xy 5.06 a,y 5.15 a,y 4.90 a,y 4.76 a,y 4.87 b,y 5.12 ab,y 0.5% nisin 6.21 a,x 6.14 a,xy 6.14 a,xy 6.08 a,xy 5.89 a,xyz 5.58 a,yz 5.79 a,xyz 5.67 a,z 5.80 a,xyz 5.83 a,xyz a-b means in same column with different superscripts are significantly different x-z means in same row with different superscripts are significantly different (P < 0.05)

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61Table 4-3. Listeria monocytogenes counts on Ready-To-Eat turkey ham supplemente d with various concentrations of nisin and inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Treatments (Log10 CFU/g) Negative control 0.00 b,y 0.00 c,y 0.00 b,y 0.00 b,y 0.00 b,y 0.00 b,y 0.00 b,y 0.00 e,y 0.00 b,y 0.00 c,y Positive control 4.97 a,y 4.92 a,y 4.95 a,y 4.30 a,y 4.23 a,y 4.32 a,y 4.18 a,y 4.14 a,y 4.55 a,y 4.31 a,y 0.2% nisin 0.97 b,z 2.12 b,yz 3.22 ab,yz 2.45 ab,yz 2.94 b,yz 3.35 a,yz 3.57 a,y 2.97 b,y 3.61 a,y 3.52 ab,y 0.3% nisin 0.60 b,y 1.59 bc,y 1.96 ab,y 2.16 ab,y 2.95 ab,y 2.62 ab,y 2.57 a,y 1.83 cd,y 2.99 ab,y 3.10 ab,y 0.4% nisin 0.60 b,y 0.85 bc,y 2.29 ab,y 1.35 ab,y 2.50 ab,y 2.96 ab,y 3.06 ab,y 2.09 c,y 3.02 ab,y 2.82 ab,y 0.5% nisin 0.42 b,y 0.89 bc,y 0.17 b,y 1.66 ab,y 1.90 ab,y 1.95 ab,y 1.67 ab,y 1.01 d,y 1.89 ab,y 1.51 bc,y a-e means in same column with different superscripts are significantly different y-z means in same row with different superscripts are significantly different (P < 0.05)

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62Table 4-4. Lactic acid bacteria counts on Ready-To-Eat turkey ham supplemen ted with various concentrations of nisin and inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Treatments (Log10 CFU/g) Negative control 3.25 ab,x 4.74 a,wx 5.66 a,wx 6.86 a,w 7.10 a,w 6.84 a,w 6.66 a,w 6.71 a,w 6.62 a,w 6.66 a,w Positive control 4.27 a,x 5.49 a,wx 5.90 a,wx 6.73 a,w 6.61 a,w 6.74 a,w 6.54 a,w 6.47 a,w 6.53 a,w 6.71 a,w 0.2% nisin 1.55 b,y 3.60 a,x 4.18 a,wx 4.65 a,wx 4.43 ab,wx 5.28 ab,w 5.43 ab,w 5.57 b,w 5.65 ab,w 5.12 ab,wx 0.3% nisin 1.99 b,w 2.44 a,w 2.79 a,w 3.46 a,w 3.94 ab,w 4.95 b,w 4.96 b,w 4.31 c,w 4.52 bc,w 4.94 ab,w 0.4% nisin 1.28 b,z 2.73 a,yz 3.43 a,xy 3.86 a,wxy 4.80 ab,wxy 5.41 ab,wx 5.18 b,wx 5.66 b,w 5.29 abc,wx 5.11 ab,wx 0.5% nisin 1.32 b,wx 1.02 a,x 1.59 a,wx 3.07 a,wx 3.13 b,wx 4.20 b,w 4.19 b,w 4.23 c,w 3.48 c,wx 3.00 b,wx a-c means in same column with different superscripts are significantly different w-z means in same row with different superscripts are significantly different (P < 0.05)

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63 Table 4-5. Mean aerobic bacteria initial counts on R eady-To-Eat turkey ham supplemented with various concentrations of nisin and inoculated with Listeria monocytogenes and analyzed prior to storage Day 0 Treatments Log10 CFU/g Negative control 2.28 b Positive control 5.04 a 0.2% nisin 1.91 bc 0.3% nisin 1.57 bcd 0.4% nisin 1.07 cd 0.5% nisin 0.58 d a-c values in same column with different superscripts are significantly different (P < 0.05)

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64 CHAPTER 5 EVALUATION OF THE ANTI-LISTERIAL PROP ERTIES OF NISIN, ROSEMARY AND EDTA ON READY-TO-EAT TURKEY HAM STORED AT 4C FOR 28 DAYS Introduction The presence of Listeria monocytogenes in the food processing chain is evident by its widespread distribution in processed products (107) Post-processing contamination of cured meat products with L. monocytogenes may occur in processing plants (43) and currently represents one of the highest meat safety risks (18) Numerous sporadic and outbreak cases of foodborne illness have been linked to consumption of ready-to-eat (RTE) products contaminated with L. monocytogenes (55) Sanitizing practices and HACCP programs applied in the meat industry are often insufficient to prev ent presence or i nhibit growth of L. monocytogenes in processed meats; therefore, post-packaging hur dle technologies are needed for its control (10) In developing a novel post-processing antimicro bial treatment for use in the food industry there are two constant problems: the limited range of bacteria which is sensitive to particular agents and the high concentration of agents required to inhibit growth (54) The rationale for the increased effectiveness of combin ations of antimicrobials is that simultaneous attack on different targets in the bacterial cell is more difficult for the bacteria to overcome. The use of antimicrobials with different mechanisms of acti on can also be expected to expand the range of organisms that may be inhibited. Nisin, a polypeptide bacteriocin produced by Lactococcus lactis subsp. lactis is a generally recognized as safe substance (131) The mechanism of nisin activity has been shown to involve alteration of the cell membrane of sensitive organisms resulting in leakage of low molecular weight cytoplasmic components and th e destruction of the proton motive force (PMF) (4, 50) These antimicrobial properties have become a focus in the food science field and researchers have found that nisin is not only effective against Gram positive bacteria (34) but

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65 with the combination of a food grade chelator, such as ethylenenediaminetetraacetate (EDTA), its effectiveness can be extended to Gram negative bacteria (115) EDTA a chelator, can have antimicrobial effect by limiting the availability of cations and can act to destabilize the cell membrane of bacteria by complexing divalent cations which act as salt bridges between membrane macromolecules, su ch as lipopolysaccharides (118, 135) The use of edible plants, as well as thei r phytochemicals, in food preservation and improvement of organoleptic qua lities of certain tr aditional foods has been practiced for centuries. These antimicrobial properties derive d from many plant essential oils have been empirically recognized for centuries but only sc ientifically confirmed in the last 30 years (95) The demand for minimally processed and extende d shelf life foods has further increased the interest to define these naturally occurring bioa ctive ingredients; however their strong flavor limited their use in foods. The rosemary extrac t has shown antimicrobial properties against food spoilage and foodborne pathogenic microorganism and its antibacte rial activity has been linked to the compounds extracted with hexane, wh ich are presumably phenolic diterpenoids (33) Cured meat products may be an excellent system in which to use nisin, rosemary and EDTA combination treatments, si nce the presence of other grow th restrictive chemicals and conditions, such as nitite and NaCl, may increase the effectiveness of antimicrobial treatment against spoilage flora and pathogens (54) Preliminary experiments were conducted to dete rmine the inhibitory c oncentration in agar media of different antimicrobial agents. The antimicrobials nisin, rosemary and EDTA were selected for further study to dete rmine their effectiveness when used alone or in combinations in a meat matrix.

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66 Materials and Methods This study was conducted in two trials during which 0.2% nisin, 1% rosemary and 20 mM EDTA alone or in a combination were used to treat Ready-To-Eat turkey ham inoculated with five strains of Listeria monocytogenes The trials were conducted at the University of Florida Meat Processing Laboratory and Microbiology Laboratory, Gainesv ille, Florida. The same procedure was used on both trials. Inoculum Cultivation and Storage Reference strains of Listeria monocytogenes a, b, 4 b, Scott A and 19115, were obtained from ABC Research Corp oration in Gainesville, FL a nd used as the inoculum to evaluate the antiListeria properties of nisin, rosemary and EDTA. The media and materials used for the cultivat ion, growth and mainte nance of the strains were purchased from Fisher Scientific (Pitts burgh, PA 15238). The strains were transferred individually to test tubes c ontaining 10 mL of tryptic soy broth (TSB, Difco Laboratories, Detroit, MI 482132-7058, Cat.No. DF 0369-17-6) using a flamed sterilized 3mm inoculation loop. The broth was incubated at 35C for 24 hour s. After incubation th e aliquots were poured into sterile centrifuge tubes and centrifuged (Sorvall RC-5B, Dupont Instruments, Newton, CT 06470) at 5000 rpm for 10 minutes. After centrifuging, the supernatants were discarded and the pellets were re-suspended in 10 mL of steril e distilled water and centrifuged again. The supernatants were again discarded and the pellet s were re-suspended in 1 mL of 3% TSB with 30% glycerol in a 2 mL cryovial (Corni ng Incorporated, Corning, NY 14831, Cat.No. 03-37421). The pellets were stored at -45C and used as the stock cu lture for the inoculation studies. Inoculum Preparation Frozen Listeria monocytogenes strains were allowed to thaw at room temperature for 10 minutes. A loopful of the thawed stock culture was transferred to test tubes containing 10 mL of

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67 3% TSB and incubated at 35C for 24 hours. After incubation, the aliquots were centrifuged (5000 rpm for 10 min at 16C) and washed with sterile 0.1% buffered peptone water (BPW, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF O1897-17-4). The aliquots were then serially diluted with BPW to concentrations of 10-1 to 10-8. Antimicrobial Solutions Preparation Formulations were developed to prepare 0.2% Nisin, 20 mM EDTA and 1% rosemary solutions (based on 100% total batch weight) to ensure that desired concentrations of each solution made contact with chopped turkey ham. The control turkey ham formulation consisted of the following (% of total weight in the formulation): chopped turkey ham (90%) and water (10%) (Table 5-1). Each antimic robial was dissolved using 10% st erile water, according to each treatment formulation, which was ad ded into the bag containing the chopped turkey ham. Sterile deionized water was used as control. Nisaplin (Danisco, Copenhagen, Denmark) is a commercial nisin product containing 106 IU nisin/g. A concentration of 0.2% nisin was obta ined by adding 0.5 g of Nisaplin, 0.17 mL of 0.02 N food grade HCL (Fisher Scientif ic, Pittsburgh, PA 15238, Cat. No. 7647-01-0) and 0.75g NaCl (Sigma Chemical, St. Louis, MI 63 178, Cat No. S9625-500G) to the turkey ham. EDTA (Sigma Chemical., St. Louis, MI 63178, Cat No. 59H03591) was prepared at a concentration of 20 mM by addi ng 0.75 g to the turkey ham to tal batch weight. Herbalox Seasoning (Kalsec, Kalamazoo, MI 49005-0511, Code No. 41-19-02) is a commercial rosemary extract that has shown antimicrobi al properties as well as inhibiti on of oxidative deterioration. Rosemary solution was prepared applying 1 mL of the Herbalox to the turkey ham. Combination of 0.2% nisin and 1% rosemary was prepared by adding 0.2 g of Nisaplin 0.17 mL of 0.02 N food grade HCL, 0.75g NaCl, and 1 mL of rosemary extract. Combination of 0.2% nisin, 1% rosemary and 20 mM EDTA was achieved by applying 0.2 g of Nisaplin, 0.17

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68 mL of 0.02 N food grade HCL, 0.75 g NaCl, 1 mL of rosemary extract, and 0.75 g of EDTA. The antimicrobial solutions were stor ed at 4C and used within 3 days Sample Preparation JennieO ready-To-Eat turkey hams were purchased from Publix, Gainesville, Florida with an expiration date of 60 days. The turkey hams were immediately transported to the University of Florida Meat Processing Laboratory and Microbiology Laboratory, Gainesville, Florida and stored at 4C for no longer than 24 hours before us ing. The hams were as eptically removed from the vacuum packaged bag and placed on pre-ster ilized trays (polypropylene) and chopped into approximately 0.5 cm pieces. The ham was chopped to simulate how it may be used in ham salad or similar foods. Inoculation and Treatment The turkey ham pieces were placed on pre-steril ized trays and inoculated by spraying them with 1 mL of L. monocytogenes inoculum at 1.0 X 108 CFU per gram of ham. Inoculated samples were left to stand at room temperature for 20 min to allow for bacterial attachment and to ensure a final concentration of 104 CFU/gram. For each treatment, duplicate 25 grams inoculated chopped turkey ham samples were aseptically weighed, placed in a labeled FoodSaver bag (Tilia, San Francisco, California, FoodSaver Vacloc Roll) and vacuum packaged (Tilia, San Francisco, California, FoodSaver Bagvac). The inoculated samples were treated w ith either nisin, EDTA, ni sin with rosemary, or nisin with rosemary and EDTA (T able 5-1), leaving the antimicrobial solution in the package. Samples were stored in a 4C cooler for subs equent microbiological and chemical analysis. Samples were analyzed after 0, 7, 14, 21, and 28 days storage.

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69 Microbiological Analyses Twenty-five grams of chopped turkey ham were transferred aseptical ly from the vacuum packaged bag into a sterile stomacher bag (F isher Scientific, Pittsburgh, PA 15238, Cat. No. 01002-44) with 225 mL of sterile 0.1% buffered pept one water (BPW, Difco, Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) and shaken approximately 30 times. Further serial dilutions were prepared by tran sferring 1 mL of the diluted sample homogen ate into 9 mL of 0.1% BPW until the appropriate dilutions were obtained. Listeria monocytogenes lactic acid bacteria and anaerobic bacteria analyses A volume of 0.1 mL of the dilutions was di spensed onto pre-poured Modified Oxford Media (MOX, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0225-17-0) containing Modified Oxford antimicrobial supplement (MOX supplement, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0218-60-5) for L. monocytogenes identification, lactobacilli MRS agar (Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0882170) for the isolation of lactic acid bacteria, and anaerobic agar (Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0536-17-4) for anaerobic bacteria id entification. The spread plate technique was used to evenly distribute the sample over the plate. Using this technique, a flamed st erilized bacterial cell spreader (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 08-769-2A) was used to spread the sample over the plate as the plate was spun on an inoculating turntable (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 08-758-10). All samples were plated in duplicate. The Petri plates were inverted and in cubated for 24 hours at 35C for L. monocytogenes and 37C for lactic acid bacteria identificat ion. For anaerobic bacteria, the petri plates were inverted and placed in a sealed jar (GasPak jar system, Fi sher Scientific, Pittsbu rgh, PA 15238, Cat. No. 11814-22) with an AnaeroGen sachet (Remel, Lenexa, KS 66215, Cat. No. 6535) to create an anaerobic environment. The anaerobic agar plates were then incubated at 35C for 48 hours. All

PAGE 70

70 Plates with 25 to 250 colonies were counted. Black coloni es surrounded by a black halo, white/grayish colonies and white col onies were considered presumptively L. monocytogenes, lactic acid bacteria and anaerobi c bacteria, respectively. Microbi ological counts were reported as Logarithmic Colony Forming Units per gram (Log CFU/g). Aerobic bacteria analysis Aerobic bacteria counts were performed using 3M Petrifilm aerobic count plates (St. Paul, Minnesota, Cat. No. 6404) for all treatments pr ior to storage on day 0. The Petrifilm aerobic count plate was placed on a level surface. The top film was lifted and 1 mL of sample was dispensed to the center of the bottom of the pl ate. The top film was released down onto the sample and the plastic spreader was placed on th e center of the plate, convex side down. After approximately one minute, the spreader was removed from the plate. All samples were plated in duplicate. Plates were incubated for 24 hours at 25C in a horizontal positi on with the clear side up in stacks of 12 plates. Pl ates containing 15 to 150 colonies were counted and recorded. Microbiological counts were e xpressed as Logarithmic Colony Forming Units per gram (Log CFU/g). pH Analysis The pH analysis of the chopped Ready-To-E at turkey ham was performed using a pH meter (Accument basic AB15, Fisher Scientif ic, Pittsburgh, PA 15238, Model No. AB15, Serial No. AB81210535). Twenty-five grams of chopped Ready-To-Eat turkey ham was aseptically removed from the vacuum packaged bag and placed into a sterile plastic bag (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 01-002-44) into wh ich 225 mL of 0.1% buffered peptone water (BPW, Difco, Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) was added. Duplicate pH measurements were taken from each ham homogenate.

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71 Data Analysis Statistical analyses were conducted for enumera tion data of a total of eight measurements per treatment of L. monocytogenes aerobic bacteria, anaerobic bacter ia, and lactic acid bacteria. Also, statistical analysis was c onducted for pH values of six meas urements per treatment. The general linear model program (P ROC GLM) of SAS system (110) was employed to statistically analyze trial, day, tr eatment and treatment by day. Variations in data were accounted for by four treatment effects: trial, treatme nt, day, and treatment*day. Comparisons among means were performed using SAS Tukey Multiple Range test procedure. Treatments effects and differences were considered significant when P < 0.05. Results and Discussion pH Analysis On day 0, the pH of turkey ham treated with a combination of nisin, rosemary and EDTA and EDTA alone were significantly lower (P < 0.05) than all other treatments (Table 5-2). From days 14 to 28, the pH was simila r (P > 0.05) for all treatments. Bacterial growth on turkey ham, stored under vacuum packaging, may result in the production of numerous compounds such as acidic metabolites and carbonic acid that may decrease the pH (37) This may explain the slight decrease in pH of the controls and the antimicrobial treated samples over time in our study. Also, the pH change observed with nisin, EDTA, nisin combined with rosemary and nisin combined with rosemary and EDTA treatments may have been a comprehensive effect of pH of the initial treatment solution, beef buffering capac ity, and bacterial growth products. The overall pH values of the treatments in this study did diffe r throughout the 28 days of storage. This was in agreement with a st udy by Cutter and Siragusa (23) in which a significant difference was seen in the controls and nisin treated samples between different days. Becaus e changes in pH were

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72 not consistent with changes in bacterial population, pH alone coul d not be considered as a key factor for population reduction in this study. Listeria monocytogenes Analysis Throughout the 28 days, the data revealed similar (P > 0.05) L. monocytogenes counts for the positive control and turkey ham treated with EDTA (Table 5-3). Turkey ham treated with nisin alone or in combination with rosemary or with rosemary with EDTA, resulted in significantly (P < 0.05) reduced L. monocytogenes counts when compared to the positive control and hams treated with EDTA during 28 days. On day 0, the initial bacterial population of the positive control (4.87 log CFU/g) was reduced (P< 0.05) to 1.14 log CFU/g with 0.2% nisin, 2.54 log CFU/g with nisin combined with rosemary, and 1.75 log CFU/g with nisin combined with rosemary and EDTA. Through out the 28 days of storage, the number of L. monocytogenes increased to approximately 0.75 log CFU/g in the nisin with rosemary and EDTA treated sa mple. The counts remain similar for nisin and nisin with rosemary treated samples. During th e 28 days of storage a significant difference (P < 0.05) existed in population reduction of nisin, nisi n with rosemary and nisin with rosemary and EDTA when compared with the positive control. By day 28, L. monocytogenes counts on turkey ham were reduced by 2.89 log CFU/g with nisin treatment, 1.43 log CFU/g with nisin combined rosemary treatment and 1.25 log CFU/g with nisin combined rosemary and EDTA treatment when compared to the positive control (3.84 log CFU/g). These findings suggested that nisin, nisin combined with rosemary and nisin combined with rosemary and EDTA may have an initial bacterial effect against L. monocytogenes population and that these solutions may help to maintain the L. monocytogenes counts lower during storage. EDTA exhibited no inhibitory effect on L. monocytogenes through 28 days storage at 4 C. Divalent cations such as Ca2+ and Mg2+ play specific roles in st abilizing the structure of

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73 bacterial membranes because they form me tal ion bridges between phosphate groups of phopholipids and the carboxyl gr oups of membrane protein (135) In gram negative organisms, it has been shown that EDTA damages outer membrane structure by complexing these Ca2+ and Mg2+ cations which are necessary for them to live (118) However, membrane structure of gram negative bacteria is different from gram positive bacteria. Gram positive bacteria will have a thick layer of peptidoglycan (a sugar-protein shell) that re sults in resistance to physical disruption. Gram negative bacteria have a thin exterior peptidoglycan membrane. The outer membrane is composed of lipid and lipid protei n content which is the primary target of EDTA (37) This may be the reason that EDTA did not inhibit the growth of L. monocytogenes under the conditions used in this study. The fact that nisin combined with rosemary and EDTA had an anti-listerial effect through out the 28 days of storage at 4C, indica ted that the observed population reductions were attributed to the inhibito ry activity of nisin or rosemary rather than EDTA. Similar observations have been reported (96, 115) where in EDTA lacked inhibition against gram positive bacteria, and when EDTA combined with nisin reduced the antimicrobial effect of nisin. Anaerobic Bacteria Analysis Significant differences existed among treatments (P < 0.05) in anaerobic bacteria counts (Table 5-4). Except for samples treated with 20 mM of EDTA only, untreated turkey ham (positive and negative controls ) possessed significantly higher (P < 0.05) anaerobic bacteria counts when compared to all treated ham samples. Similar (P > 0.05) anaerobic bacteria counts were observed for the positive control, negative control and samples trea ted with EDTA only throughout 28 days storage. Treatment with nisin alone caused significant (P < 0.05) population reduction as compared to all other treatments during 28 days storage at 4C. By day 28, nisin alone and in combination with

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74 rosemary and EDTA had significantly lower (P < 0.05) anaerobic bacteria counts when compared to all other treatments. From day 0 to day 21, nisin combined with rosemary was significantly lower (P < 0.05) when compared to the positive control. However, by day 28 the population of anaerobic bacteria increased and no significant differenc es (P > 0.05) were observed between nisin combined with rosemary and positive control. Results from the present study suggest that r eady-to-eat turkey ham will have decreased numbers of anaerobic bacteria wh en treated with any of the fo llowing: 1) 0.2% nisin, 2) nisin combined with rosemary and EDTA. Therefore, the present study suggest s that the presence of an increased level (0.2%) of nisin alone or in combination with 1% rosemary and 20 mM EDTA, effectively reduce the growth of anaerobes. Lactic Acid Bacteria Analysis The data revealed similar results for lactic acid and anaerobic bacteria. Samples treated with nisin and nisin combined with rosemary and EDTA resulted in significantly lower (P < 0.05) lactic acid bacteria when co mpared to the controls and EDTA alone (Table 5-5). Samples treated with nisin alone and in combination wi th rosemary and EDTA controlled the anaerobic bacteria growth during 28 days storage when compared to all other treatments. The number of bacteria recovered from samples treated with nisin combined with rosemary and EDTA alone were not significan tly different (P > 0.05) when co mpared to positive control. This may suggest that combination of nisin with rosemary and EDTA alone had no antimicrobial effect against gram positive bacteria. Results from the study revealed that lactic acid bacteria may be controlled in vacuum package ready-to-eat turkey ham stored at 4C when treated with at least 0.2% nisin or 0.2% nisin combined with 1% rosemary and 20 mM of EDTA.

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75 Aerobic Bacteria Analysis Aerobic bacteria counts were significantly higher (P < 0.05) in the positive control and EDTA treatment when compared to all other trea tments (Table 6-6). This may suggest that EDTA has no immediate antimicrobial effect agai nst the aerobic organisms present on the turkey ham. The antimicrobial treatment that contai ned 0.2% nisin was the most effective (P < 0.05) reducing the initial populations of aerobic bacteria. In addition wh en combinations of nisin with rosemary and nisin with rosemary and EDTA we re used a significant (P < 0.05) reduction was observed when compared to the positive control. These results suggest th at initial population of aerobic bacteria may be restricted when treate d with nisin and combinations of nisin with rosemary or nisin with rosemary and EDTA under vacuum package and refrigeration (4C) conditions.

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76 Table 5-1. Formulation of nisin, rosemary and ED TA solutions for vacuum packaged ready-toeat turkey ham stored at 4 C for 28 days Percentage (%) of ingred ient in total composition Treatment Negative Control1 Positive Control2 Nisin + EDTA Rosemary Nisin + Rosemary Nisin EDTA Ingredient Ham 90.00 90.00 87.13 87.88 88.88 89.25 Water 10.00 10.00 10.00 10.00 10.00 10.00 Rosemary 0.00 0.00 1.00 1.00 0.00 0.00 EDTA 0.00 0.00 0.75 0.00 0.00 0.75 Nisin 0.00 0.00 0.20 0.20 0.20 0.00 NaCl 0.00 0.00 0.75 0.75 0.75 0.00 HCl 0.00 0.00 0.17 0.17 0.17 0.00 100.00 100.00 100.00 100.00 100.00 100.00 1 Negative control: without L. monocytogenes inoculum 2 Positive control: with L. monocytogenes inoculum

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77 Table 5-2. Mean pH values on Ready-To -Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 C for 28 days Day 0 Day 7 Day 14 Day 21 Day 28 Treatments (Log10 CFU/g) Negative control 6.19 a,x 5.98 a,x 5.30 a,y 4.92 a,z 4.92 a,z Positive control 5.96 a,x 5.73 ab,xy 5.42 a,xy 5.18 a,y 5.25 a,xy Nisin + EDTA+ rosemary 5.53 b,x 5.67 b,x 5.80 a,x 5.71 a,x 5.72 a,x Nisin + rosemary 6.08 a,x 5.83 ab,x 5.24 a,y 5.37 a,y 4.95 a,y Nisin 6.08 a,x 5.93 ab,xy 5.87 a,xy 5.50 a,xy 5.42 a,y EDTA 5.92 ab,x 5.73 ab,x 5.57 a,x 5.24 a,x 5.36 a,x a-b values in same column with different superscripts are significantly different x-z values in same row with different superscripts are significantly different (P < 0.05)

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78 Table 5-3. Mean Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 C for 28 days Day 0 Day 7 Day 14 Day 21 Day 28 Treatments (Log10 CFU/g) Negative control 0.00 e,x0.00 d,x 0.00 d,x 0.00 d,x 0.00 d,x Positive control 4.87 a,x4.30 a,y 3.84 a,z 4.14 a,yz 3.84 a,z Nisin + EDTA+ rosemary 1.75 c,z1.50 c,z 1.68 c,z 2.91 b,y 2.59 b,y Nisin + rosemary 2.54 b,x2.37 b,x 2.45 b,x 2.62 b,x 2.41 b,x Nisin 1.14 d,yz1.25 c,yz 0.50 d,z 1.37 c,y 0.95 c,yz EDTA 5.19 a,y4.10 a,z 3.92 a,z 3.84 a,z 4.07 a,z a-e values in same column with different superscripts are significantly different x-z values in same row with different s uperscripts are significantly different (P < 0.05)

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79 Table 5-4. Mean anaerobic plate count on R eady-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 C for 28 days Day 0 Day 7 Day 14 Day 21 Day 28 Treatments (Log10 CFU/g) Negative control 2.69 b,z4.40 abc,y 5.85 ab,xy 5.98 ab,xy 6.10 a,z Positive control 4.81 a,z6.37 a,x 6.34 a,x 6.61 a,x 5.89 a,y Nisin + EDTA+ rosemary 1.77 bc,y3.65 cd,x 4.36 c,x 4.87 c,x 4.09 b,x Nisin + rosemary 2.24 b,z4.12 bc,y 5.05 bc,xy 5.30 bc,x 6.10 a,x Nisin 0.29 c,z1.93 d,y 2.69 d,xy 3.33 d,x 3.55 b,x EDTA 4.85 a,y6.09 ab,x 6.10 ab,x 5.89 abc,x 5.27 a,y a-d values in same column with different superscripts are significantly different x-z values in same row with different superscripts are significantly different (P < 0.05)

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80 Table 5-5. Mean lactic acid ba cteria counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 C for 28 days Day 0 Day 7 Day 14 Day 21 Day 28 Treatments (Log10 CFU/g) Negative control 3.97 ab,z6.60 a,y 7.16 a,y 7.20 a,y 6.68 a,y Positive control 5.19 a,z6.19 a,y 6.88 a,y 6.63 a,y 6.12 a,y Nisin + EDTA+ rosemary 2.90 b,z4.40 b,y 4.61 b,y 4.76 b,y 4.23 bc,y Nisin + rosemary 3.88 ab,z5.65 a,y 7.14 a,w 7.05 a,w 6.27 a,x Nisin 1.27 c,z1.39 c,z 3.76 c,y 3.88 b,y 3.28 c,y EDTA 5.20 a,z6.01 a,xy 6.46 a,x 6.53 a,x 5.52 ab,yz a-c values in same column with different superscripts are significantly different w-z values in same row with different superscripts are significantly different (P < 0.05)

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81 Table 5-6. Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes Day 0 Treatments Log10 CFU/g Negative control 3.69 b Positive control 5.43 a Nisin + EDTA+ rosemary 2.44 b Nisin + rosemary 3.70 b Nisin 0.85 c EDTA 5.67 a a-c values in same column with different superscripts are significantly different (P < 0.05)

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82 CHAPTER 6 EVALUATION OF THE ANTI-LISTERIAL PR OPERTIES OF 0.5 % NISIN, 1 % ROSEMARY AND 20 MM EDTA ON READY-TO-E AT TURKEY HAM STORED AT 4C FOR 63 DAYS Introduction Listeria monocytogenes is a foodborne pathogen which is wi dely distributed in nature and whose control in food is made di fficult by its ability to grow at temperatures ranging from 0 to 45C (7), its high tolerance for salt (42) and its ability to initiate growth at a relatively low pH (9) Recently, numerous outbreaks have been linked to consumption of ready-to-eat (RTE) products contaminated with L. monocytogenes (55) Contamination of the RTE meat products may occur in processing plants. The heat treatment (cooking) that RTE meat and poultry products undergo eliminates the pathogen; how ever, recontamination may occur during postprocessing exposure to the environment (e.g. peeling, slicing, and repackaging) (43) For this reason, new post-processing hurdle technologies that control or eliminate the incidence of foodborne pathogen are needed for the meat industry (10) The bacteriocin, nisin, has been used as an antimicrobial in foods since the 1960s (83) Nisin is produced by the l actic acid bacteria (LAB) Lactococcus lactis (5) The mechanism of nisin activity has been shown to involve altera tion of the cell membrane of sensitive organisms resulting in the leakage of low mol ecular weight cytoplasmic components (4, 50, 138) and destruction of the proton motive force (PMF) (13, 49) It has been recognized that susceptibility of Gram negative organisms may be increased by the use of membrane disrupting agents such as detergents and chelators (115) Chelators bind magnesium ions in the lipopolysaccharide layer of bacterial cell walls and increase susceptibility of the cells to nisin (118) Ethylenenediaminetetraacetic acid (EDTA) is a well known reagent

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83 used in various foods for different functions. Those functions may include the retardation of crystal formation, food preservative and stabiliz er, antioxidant, and chelating and sequestering agent (139) EDTA can have antimicrobial effect by limiting the availability of cations and can act to destabilize the cell membrane of bacteria by complexing divalent cations which act as salt bridges between membrane macromolecules, such as lipopolysaccharides (118, 135) Society appears to be experiencing a trend of natural consumerism (121, 129) desiring fewer synthetic food additives and products with a smaller impact on the environment. Therefore, there is a need for new methods to ma ke food safe which have a natural image. One such possibility is the use of essential oils as antibacterial additives (117) Essential oils (EOs) are aromatic oily liquids obtained from plant mate rial (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots). They can be obtained by fermentation or extraction but the method of steam distillation is most commonl y used for commercial production of EOs (136) There are approximately 3000 EOs known, of which about 300 are commercially important, and are destined primarily for the flavors and fragrances market (136) It has long been recognized that some EOs have antimicrobial properties (95) and the relatively recent interest in natural consumerism has lead to a renewal of scientific interest in these substances (92, 129) The rosemary ( Rosmarinus officinalis ) extract has shown antimicr obial properties against food spoilage and foodborne pathogenic microorganisms. Rosemarys an tibacterial activity has been linked to -pinene, bornyl acetate, camphor, and 1,8-cineole (33, 97) Preliminary studies were conducted to determ ine the inhibitory concentration in agar media of different antimicrobial agents. The antimicrobials nisin, rosemary and EDTA were selected for further study to determine their effectiveness in a meat matrix. This work was undertaken to develop a new post-processing hurd le technology by applying nisin, rosemary and

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84 EDTA alone or in combination directly to the finished RTE turkey product immediately prior to packaging. Materials and Methods This study was conducted in two trials during which 0.5% nisin, 1% rosemary and 20 mM EDTA solutions were used alone and in combina tion to treat RTE turkey ham inoculated with five strains of Listeria monocytogenes The trials were conducted at the University of Florida Meat Processing Laboratory and Microbiology Laboratory, Gainesv ille, Florida. The same procedure was used on both trials. Inoculum Cultivation and Storage Reference strains of Listeria monocytogenes a, b, 4 b, Scott A and 19115, were obtained from ABC Research Corp oration in Gainesville, FL a nd used as the inoculum to evaluate the antiListeria properties of different concentrations of nisin. The media and materials used for the cultivation, growth and maintenance of the strain s were purchased from Fisher Scientific (Pittsburgh, PA 15238). The strains we re transferred individually to test tubes containing 10 mL of tryptic soy broth (T SB, Difco Laboratories, Detroit, MI 482132-7058, Cat.No. DF 0369-17-6) using a flamed steril ized 3mm inoculation loop. The broth was incubated at 35C for 24 hours. Af ter incubation the aliquots were poured into sterile centrifuge tubes and centrifuged (Sorvall RC-5B, Dupont In struments, Newton, CT 06470) at 5000rpm for 10 minutes. After centrifuging, th e supernatants were discarde d and the pellets were resuspended in 10 mL of sterile di stilled water and centr ifuged again. The supernatants were again discarded and the pellets were re-suspended in 1 mL of 3% TSB with 30% glycerol in a 2 mL cryovial (Corning Incorporated, Corning, NY 14831, Cat.No. 03-374-21). The pellets were stored at -45C and used as the stock culture for the inoculation studies.

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85 Inoculum Preparation Frozen Listeria monocytogenes strains were allowed to thaw at room temperature for 10 minutes. A loopful of the thawed stock culture was transferred to test tubes containing 10 mL of 3% TSB and incubated at 35C for 24 hours. After incubation, the aliquots were centrifuged (5000 rpm for 10 min at 16C) and washed with sterile 0.1% buffered peptone water (BPW, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF O1897-17-4). The aliquots were then serially diluted with BPW to concentrations of 10-1 to 10-8. Antimicrobial Solutions Preparation Formulations were developed to prepare 0.5% Nisin, 20 mM EDTA and 1% rosemary solutions (based on 100% total batch weight) to ensure that desired concentrations of each solution made contact with chopped turkey ham. The control turkey ham formulation consisted of the following (% of total weight in the formulation): chopped turkey ham (90%) and water (10%) (Table 6-1). Each antimic robial was dissolved using 10% st erile water, according to each treatment formulation, which was ad ded into the bag containing the chopped turkey ham. Sterile deionized water was used as control. Nisaplin (Danisco, Copenhagen, Denmark) is a commercial nisin product containing 106 IU nisin/g. A concentration of 0.5% nisin was obta ined by adding 0.5 g of Nisaplin, 0.17 mL of 0.02 N food grade HCL (Fisher Scientif ic, Pittsburgh, PA 15238, Cat. No. 7647-01-0) and 0.75g NaCl (Sigma Chemical, St. Louis, MI 63 178, Cat No. S9625-500G) to the turkey ham. EDTA (Sigma Chemical., St. Louis, MI 63178, Cat No. 59H03591) was prepared at a concentration of 20 mM by addi ng 0.75 g to the turkey ham to tal batch weight. Herbalox Seasoning (Kalsec, Kalamazoo, MI 49005-0511, Code No. 41-19-02) is a commercial rosemary extract that has shown antimicrobi al properties as well as inhibiti on of oxidative deterioration. Rosemary solution was prepared applying 1 mL of the Herbalox to the turkey ham.

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86 Combination of 0.5% nisin and 1% rosemary was prepared by adding 0.5 g of Nisaplin 0.17 mL of 0.02 N food grade HCL, 0.75g NaCl, and 1 mL of rosemary extract. Combination of 0.5% nisin, 1% rosemary and 20 mM EDTA was achieved by applying 0.5 g of Nisaplin, 0.17 mL of 0.02 N food grade HCL, 0.75 g NaCl, 1 mL of rosemary extract, and 0.75 g of EDTA. Combination of 5% nisin and 20 mM EDTA wa s obtained by putting 0.5 g of Nisaplin, 0.17 mL of 0.02 N food grade HCL, 0.75g NaCl, and 0.75 g of EDTA. Combination of 1% rosemary and 20 mM EDTA was achieved by adding 1 mL of rosemary extract and 0.75 g of EDTA. The antimicrobial solutions were stored at 4C and used within 3 days. Sample Preparation JennieO RTE turkey hams were purchased fr om Publix, Gainesville, Florida with an expiration date of 60 days. The turkey hams were immediately transported to the University of Florida Meat Processing Laborat ory and Microbiology Laboratory, Gainesville, Florida and stored at 4C for no longer than 24 hours before us ing. The hams were as eptically removed from the vacuum packaged bag and placed on pre-ster ilized trays (polypropylene) and chopped into approximately 0.5 cm pieces. The ham was chopped to simulate how it may be used in ham salad or similar foods. Inoculation and Treatment The turkey ham pieces were placed on pre-steril ized trays and inoculated by spraying them with 1 mL of L. monocytogenes inoculum at 1.0 X 108 CFU/mL. Inoculated samples were left to stand at room temperature for 20 min to allow for bacterial attachment and to ensure a final concentration of 104 CFU/gram. Duplicate samples of inoculat ed chopped turkey ham were aseptically weighted according to its corresponding treatment (Table 1) and placed in a labeled FoodSaver bag (Tilia, San Francisco, California, FoodSaver Vacloc Roll). Corresponding treatments were applied to the

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87 chopped ham followed by mixing the ham and treatme nt solution to ensure a proper distribution between them. The bags were then vacuum packaged (Tilia, San Francisco, California, FoodSaver Bagvac), leaving the antimicrobial soluti on in the package, and stored in a 4C cooler for subsequent microbiological, ch emical and color analysis. Samples were analyzed after 0, 7, 14, 21, 28, 35, 42, 49, 56 and 63 days of storage. Microbiological Analyses Twenty-five grams of chopped turkey ham were transferred aseptical ly from the vacuum packaged bag into a sterile stomacher bag (F isher Scientific, Pittsburgh, PA 15238, Cat. No. 01002-44) with 225 mL of sterile 0.1% buffered pept one water (BPW, Difco, Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) and shaken approximately 30 times. Further serial dilutions were prepared by addi ng 1 mL of the diluted sample homogenate into 9 mL of 0.1% BPW until the appropriate dilutions were obtained. Aerobic bacteria analysis Aerobic bacteria counts were performed using 3M Petrifilm aerobic count plates (St. Paul, Minnesota, Cat. No. 6404) to all treatments prio r to storage on day 0. The Petrifilm aerobic count plate was placed on a level surface. The top film was lifted and 1 mL of sample was dispensed to the center of the bottom of the pl ate. The top film was released down onto the sample and the plastic spreader was placed on th e center of the plate, convex side down. After approximately one minute, the spreader was removed from the plate. All samples were plated in duplicate. Plates were incubated for 24 hours at 25C in a horizontal positi on with the clear side up in stacks of 12 plates. Pl ates containing 15 to 150 colonies were counted and recorded. Microbiological counts were e xpressed as Logarithmic Colonic Forming Units per gram (Log CFU/g).

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88 Listeria monocytogenes and lactic acid bacteria analysis A volume of 0.1 mL of the dilutions was di spensed onto pre-poured Modified Oxford Media (MOX, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0225-17-0) containing Modified Oxford antimicrobic supplement (MOX supplement, Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 0218-60-5) for L. monocytogenes identification and onto lactobacilli MRS agar (Difco Laboratories, Detroit, MI 48232-7058, Cat. No. DF 088 2170) for the isolation of lactic acid bacteria. The spr ead plate technique was used to evenly distribute the sample over the plate. Using this technique, a flamed sterilized bacterial cell spreader (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 08-769-2A) was used to spread the sample over the plate as the plate was spun on an inoculating turntable (Fis her Scientific, Pittsbu rgh, PA 15238, Cat. No. 08758-10). All samples were plated in duplicate. The petri plates were inverted and incubated for 24 hours at 35C for L. monocytogenes and 37C for lactic acid bact eria. Plates with 25 to 250 colonies were counted. Black colonies surrounded by a black halo and white/grayish colonies were considered presumptively L. monocytogenes and lactic acid, respec tively. Microbiological counts were reported as Logarithmic Col onic Forming Units per gram (Log CFU/g). pH Analysis The pH analysis of the chopped RTE turk ey ham was performed using a pH meter (Accument basic AB15, Fisher Scientific, Pi ttsburgh, PA 15238, Model No. AB15, Serial No. AB81210535). Twenty-five grams of chopped ham was aseptically removed from the vacuum packaged bag and placed into a sterile plastic bag (Fisher Scientific, Pittsburgh, PA 15238, Cat. No. 01-002-44) into which 225 mL of 0.1% buffere d peptone water (BPW, Difco, Laboratories, Detroit, MI 48232-7058, Cat. No. DF 01897-17-4) was added. Duplicate pH measurements were taken from each ham homogenate.

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89 Color Analysis A portable colorimeter (Minolta Chroma Meter CR310, Minolta, Ramsey, NJ 07446) was used to obtain objective data fo r color of the RTE chopped turkey ham. Before each sampling period, the machine was calibrated as recommende d by the manufacturer. Duplicates samples per treatment were evaluate for L (degree of lightness), a (degre e of redness), and b (degree of yellowness) values. Two locations we re sampled from each replicate to obtain color measurements. Data Analysis Statistical analyses were conducted for enumera tion data of a total of eight measurements per treatment of L. monocytogenes aerobic bacteria, lactic acid bacteria, and color measurements. Also, statistical analysis was c onducted for pH values of six measurements per treatment. The general linear model program (PROC GLM) of SAS system (110) was employed to statistically analyze trial, day, treatment and treatment by day. Variations in data were accounted for by four treatment effects: trial, treatment, day, and treatment*day. Comparisons among means were performed using SA S Tukey Multiple Range test procedure. Treatments effects and differences were considered significant when P < 0.05. Results and Discussion Aerobic Bacteria Analysis No aerobic bacteria were detected in the nega tive control (Table 6-2). All samples treated with nisin alone or in combination with EDTA or rosemary had significantly (P < 0.05) lower aerobic bacteria counts. All treat ments containing nisin achieved a reduction of approximately 3 log CFU/g, when compared to positive control. Aerobic bacteria counts were significantly higher (P < 0.05) in the rosemar y, EDTA, and combination of rosemary with EDTA treatments,

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90 when compared to positive control. This may suggest that EDTA and rosemary had no immediate antimicrobial effect against the aerob ic organisms present in the turkey ham. When treatments of nisin alone or combinations of nisin with rosemary, nisin with EDTA, and nisin with rosemary and EDTA were used a significant (P < 0.05) reduction was observed when compared to the positive control. The data revealed that EDTA and rosemary alone or in combination had no antimicrobial effect against the aerobic bacteria growth. However, nisin alone or combined with EDTA a nd/or rosemary exhibited an antimicrobial effect against the aerobic bacteria. These findings suggested that the microbial reducti ons observed could be attributed to the inhibitory activity of nisin rath er than EDTA or rosemary. Results from this study suggest that initial populations of aerobic bacteria may be controlled when treated with nisin and combinations of ni sin with rosemary and/or ED TA under vacuum package and refrigeration (4C) conditions. Listeria monocytogenes Analysis L. monocytogenes counts were significantly reduced (P< 0.05) to 0.91 log CFU/g with nisin, 1.60 log CFU/g with nisin combined with EDTA, 1.13 log CFU/g with nisin combined with rosemary, and 1.22 log CFU/g with nisin combined with rosemary and EDTA, when compared to positive control (5.33 log CFU/g). Treatments containing nisin alone or in combination with rosemary and/or EDTA resulted in significant population reduction when compared to the positive control (P < 0.05) during the 63 days storage at 4 1 C. An extended lag phase was observed for all treatments containing nisin. L. monocytogenes counts remained at less than 2.66 log CFU/g through 63 days storage. These results suggested that the control of L. monocytogenes by nisin containing treatments ma y be attributed to the in itial bactericidal effect rather than a constant effect. Si milar observations have been reported (19) where L.

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91 monocytogenes was sensitive to nisin but the effect of nisin was probably due to the initial inhibition and not to conti nued activity of the nisin. The results also revealed that on day 63, the population of L. monocytogenes on turkey ham was reduced by 1.52 log CFU/g with nisin, by 1.92 log CFU/g with nisin combined with EDTA, by 1.25 log CFU/g with nisin combined w ith rosemary, and by 1.32 log CFU/g with nisin combined with rosemary and EDTA when compared to the positive control (3.91 log CFU/g). These findings revealed that the use of nisin alone or in combination with EDTA and/or rosemary had a bactericidal effect against L. monocytogenes population. During the 63 days of storage, L. monocytogenes counts were similar (P < 0.05) in the positive control, in ham treated with EDTA or rosemary alone, or with EDTA combined with rosemary. Neither EDTA nor rosemary inhibited L. monocytogenes during the 63 days storage. In this study, no increase in antimicrobial ac tivity was observed when nisin was used with rosemary and/or EDTA against L. monocytogenes In contrast, a number of authors have reported an increased in the antimicrobial activity of nisin against gram positive (65) and negative bacteria (23, 126) in the presence of membrane disrupting agents. However, the majority of these reports are based upon observati ons of organisms suspended in a buffer, rather than growing in nutrient media. Since meat produc ts provide a nutrient rich environment, test of antimicrobials under conditions of cell starvation would appear to be of limited value in evaluating them for application in food products. The failure to observe an enhancement in the antimicrobial properties of nisin when combined w ith EDTA in this study may indicate that the observed enhancement between these two an timicrobial agents in buffer systems (65) was a consequence of cell starvation which prevents cell repair.

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92 In gram negative organisms, it has been show n that EDTA can act to destabilize the cell membrane of bacteria by complexing divalent cations, such as Ca2+ and Mg2+, which act as salt bridges between membrane macromolecules, such as lipopolysaccharide (118) However, membrane structure differs between gram negative and gram positive bacteria. Gram negative bacteria have an outer membrane that covers a thin layer of peptidoglycan on the outside. The primary target of EDTA is the lipids and lipid pr oteins present in the outer membrane of gram negative bacteria (37) In contrast, gram positive bacteria will have a thick layer of peptidoglycan (a sugar-protein shell) that confers resistance to physical disruption (43) This may be the reason that EDTA did not inhibit the growth of L. monocytogenes under the conditions used in this study. In this study, rosemary extract alon e was not effective in controlling L. monocytogenes growth. Del campo et al. (33) found that gram positive bacteria were the most sensitive to rosemary extracts. However, the author states th at rosemary extract should be more appropriate in foods with low fat and protein contents. Be cause ready-to-eat meat products are rich in protein content, this may explain why rosemary di d not exhibit an anti-lis terial effect. Another study found that the use of encapsulated rosemary oil was much more effective than standard rosemary essential oil extract against L. monocytogenes in pork liver sausage (95) This suggested that the method of application of rosema ry to the product may have interfered with its antimicrobial properties. Overall, during the 63 days storage a significant difference (P < 0.05) existed in population reduction of nisin, nisin with rosemary, nisin with EDTA, and nisin with rosemary and EDTA when compared with the positive control. L. monocytogenes growth was not inhibited when EDTA and rosemary alone were used. Nevert heless, when nisin alone or combined with

PAGE 93

93 rosemary and/or EDTA were used an anti-liste rial effect was observed. These results showed that L. monocytogenes inhibition could be attributed to nisins inhibitory activity rather than EDTA or rosemary. Similar findings have been reported (96, 115) where in EDTA exhibited no significant inhibition against gram positive bact eria and, when combined with nisin, EDTA reduced the antimicrobial effect of nisin. Lactic Acid Bacteria Analysis When nisin was used alone or in combinati on with EDTA and/or rosemary an immediate population reduction (P < 0.05) of lactic acid bacteria was obse rved when compared to the control (Table 6-4). On day 0, the initial bacterial population of the positive control (5.32 log CFU/g) was reduced to 0.25 log CFU/g with nisi n, to 1.26 log CFU/g with nisin combined with EDTA, to 1.61 log CFU/g with nisin combined w ith rosemary, and to 0.97 log CFU/g with nisin combined with rosemary and EDTA (P< 0.05). Lactic acid bacteria for EDTA, rosemary and combination of EDTA with rosemary treatments were similar (P > 0.05) to positive co ntrol during 63 days of storage. This may suggest that EDTA and rosemary alone have a limited antimicrobial effect against gram positive bacteria. However, when EDTA was used in comb ination with nisin or nisin with rosemary an extended lag phase in the lactic acid bacteria population was observed. This may have been caused by the buffer capacity of EDTA, which may have help to maintain the pH stable and as a result the lactic acid bacteria growth under c ontrol. The treatments that contained nisin or rosemary alone experienced an exponential grow th of lactic acid bacteria over time. Even though, EDTA played an important role in stabil izing the pH, which may have help controlling lactic acid bacteria, nisin is needed to provi de the initial inhibitory and killing effect.

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94 Results from the study show that lactic acid b acteria may be controlled in vacuum package ready-to eat turkey ham store at 4C when trea ted with 0.5% nisin comb ined with 20 mM EDTA and/or 1% rosemary. pH Analysis Turkey ham treated with EDTA alone or in co mbination with nisin and/or rosemary had a significantly higher (P < 0.05) pH va lue than all other treatments by day 63 (Table 6-5). This probably resulted from the ability of EDTA to act as a food preservativ e and pH stabilizer (139) Overall, the pH was not signi ficantly different between turk ey ham treated with nisin, rosemary, nisin combined with rosemary, and t hose treated with control solutions including the positive and negative control from days 0 to 63. Over time, there was a slight decrease in the pH values of treatments that did not contain EDTA This may be attribut ed to the production of various compounds such as acidic metabolite s and carbonic acids by spoilage bacteria (37) This suggests that EDTA, under the conditions of this study, had an effect on the pH value due to its buffering capacity. Because changes in pH were cons istent with changes in lactic acid bacteria population, pH alone may be considered as a ke y factor for lactic acid bacteria population reduction in this study. Overall, the pH values of the treatments in this study did differ throughout the 63 days of storage. Similar results have been reported (23) in which a significant difference was seen in the controls and nisin treated samples between different days. Analysis of Objective Color Measurement for L*a*b Values Results of L* a* b values measured on ready-to -eat vacuum packaged turkey ham stored at 4C are shown in Table 6, 7 and 8, respectively. The L*a*b values represent the colour coordinates used in the Minolta Chromameter system to determine colour. The L* value is an indication of lightness, where 100 repr esents perfect white and 0 black (76) Lightness increased

PAGE 95

95 during storage for the positive control, EDTA, nisin with EDTA, nisin with rosemary, nisin with rosemary and EDTA, and EDTA w ith rosemary treatments, showing significantly higher (P < 0.05) values by day 63, which could be due to a whitening surface observed in the turkey ham. Nisin alone and rosemary alone did not have a significant difference (P < 0.05) in the L* value throughout storage. The L* values oscillated in between a range of 58 to 64 for all treatments. The a* value is an indicator of redness, where positive value for red and negative for green (76) The redness (a* value) has been used as an i ndicator of colour stability in meat and meat products (52) In this study, significant differences (P < 0.05) among treatments in each storage time were observed. A decreasing trend (P < 0.05) on the a* value over time was observed in the following treatments: nisin combined with EDTA, nisin combined with EDTA and rosemary, EDTA combined with rosemary, and positive cont rol. All other treatments experienced an increasing trend (P < 0.05) on the a* value throughout the storage time. The decreasing trend on the a* value may be attributed to many factor s such as storage time, increased residual O2 level, increased oxygen transmission rate, increased lig ht intensity and decreased nitrite content (81) The b* value is an indication of yellowness, where yellow is represented by a positive value and blue by a negative value (76) Significant differences (P < 0.05) among treatments in each storage time were observed. All treatments showed an increasing trend on the b* value over time (P < 0.05). However, the amount of increased storage time was very small. Differences in b* values along the storage peri od could be related to the intensity of the oxidation process that takes place during stor age and might tend to increase yellowness of samples by rancidity, although no measures of oxidat ion intensity are availa ble to support this hypothesis.

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96 Table 6-1. Formulation of nisin, rosemary a nd EDTA solutions for vacuum packaged readyto-eat turkey ham stored at 4 C for 63 days Percentage (%) of ingred ient in total composition Treatment Control Nisin EDTA Rosemary Nisin EDTA Nisin Rosemary Nisin Rosemary EDTA Rosemary EDTA Ingredient Ham 90.00 88.58 89.25 89.00 87.83 87.58 86.83 88.25 Water 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 Rosemary 0.00 0.00 0.00 1.00 0.00 1.00 1.00 1.00 EDTA 0.00 0.00 0.75 0.00 0.75 0.00 0.75 0.75 Nisin 0.00 0.50 0.00 0.00 0.50 0.50 0.50 0.00 NaCl 0.00 0.75 0.00 0.00 0.75 0.75 0.75 0.00 HCl 0.00 0.17 0.00 0.00 0.17 0.17 0.17 0.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 *Two controls were prepared: negative control (without L. monocytogenes inoculum) and positive control (with L. monocytogenes inoculum).

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97 Table 6-2. Mean aerobic bacteria initial counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and analyzed prior to storage Day 0 Treatments Log10 CFU/g Negative Control 0.00 c Positive Control 5.20 a 0.5% Nisin 1.09 b 20 mM EDTA 5.09 a 1% Rosemary 4.79 a 0.5% Nisin + 20 mM EDTA 1.71 b 0.5% Nisin + 1% Rosemary 1.55 b 0.5% Nisin + 20 mM EDTA + 1% Rosemary 1.21 b 20 mM EDTA + 1% Rosemary 4.91 a a-c values in same column with different superscripts are significantly different (P < 0.05)

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98 Table 6-3. Listeria monocytogenes counts on Ready-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Treatments (Log10 CFU/g) Negative control 0.00 c,v 0.00 d,v 0.00 f,v 0.00 d,v 0.00 e,v 0.00 c,v 0.00 d,v 0.00 e,v 0.00 e,v 0.00 d,v Positive control 5.33 a,v 4.78 a,vw 4.87 a,wx 4.33 a,yz 4.33 a,yz 4.14 a,yz 3.76 a,z 4.44 a,xy 4.26 a,yz 3.91 a,yz Nisin 0.91 bc,xy 0.13 d,y 0.73 ef,xy 0.76 cd,xy 2.42 c,v 1.68 b,xy 1.00 cd,xy 2.36 c,vw 2.04 d,wx 2.39 bc,v EDTA 4.98 a,v 4.38 a,w 4.00 bc,wx 4.14 a,w 3.93 ab,wx 3.63 a,xy 3.18 ab,y 3.41 b,y 3.18 bc,y 3.43 ab,y Rosemary 5.18 a,v 4.48 a,wx 4.60 ab,w 4.06 a,xy 3.92 ab,yz 3.70 a,yz 3.52 a,z 3.87 ab,yz 3.54 ab,z 3.89 a,yz Nisin+EDTA 1.60 b,wx 0.87 cd,wx 0.45 f,x 0.37 d,x 2.72 d,wx 1.71 b,wx 0.77 d,wx 1.65 d,wx 2.71 cd,v 1.99 c,vw Nisin+rosemary 1.13 b,w 1.12 c,w 1.83 d,vw 2.10 b,vw 2.04 c,vw 1.99 b,vw 1.94 c,vw 2.31 c,vw 2.55 cd,v 2.66 abc,v Nisin+EDTA+rosemary 1.22 b,xy 1.16 c,y 1.43 de,xy 1.60 bc,xy 2.39 c,vw 2.01 b,xy 2.02 bc,xy 2.07 cd,xy 2.30 d,wx 2.59 abc,v EDTA+rosemary 4.90 a,v 3.13 b,wx 3.66 c,w 3.69 a,w 3.28 b,wx 3.66 a,w 3.24 a,wx 3.41 b,wx 3.52 ab,w 3.22 abc,x a-f values in same column with different superscripts are significantly different v-z values in same row with different superscripts are significantly different (P < 0.05)

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99 Table 6-4. Lactic acid bacter ia counts on Ready-To-Eat tu rkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Treatments (Log10 CFU/g) Negative control 2.58 b,y 5.14 a,x 5.38 ab,wx 5.31 a,wx 6.54 a,v 6.13 a,vw 5.77 a,wx 5.27 a,wx 5.77 ab,wx 6.04 a,vw Positive control 5.32 a,v 5.64 a,v 5.93 a,v 5.67 a,v 5.74 ab,v 5.60 a,v 5.75 a,v 5.62 a,v 5.64 ab,v 5.36 a,v Nisin 0.25 d,z 1.13 c,z 2.29 c,yz 3.08 cd,xy 5.08 bc,vw 5.43 a,vw 4.32 b,wx 5.46 a,vw 6.02 a,v 4.96 a,vw EDTA 4.92 a,v 4.46 a,vw 4.00 b,wx 4.19 b,wx 4.51 c,vw 3.75 b,xy 3.56 b,y 3.49 b,y 3.49 c,y 3.68 b,xy Rosemary 5.06 a,w 5.57 a,vw 5.44 ab,vw 5.16 a,w 5.52 b,vw 5.46 a,vw 5.53 a,vw 5.78 a,v 4.96 b,w 5.53 a,vw Nisin+EDTA 1.26 c,w 0.80 c,w 0.86 c,w 2.04 e,vw 1.81 e,vw 2.07 c,vw 1.95 c,vw 1.68 c,vw 2.94 cd,v 2.15 d,vw Nisin+rosemary 1.61 bc,y 3.04 b,x 4.08 b,wx 3.83 bc,x 5.73 ab,v 5.72 a,v 5.38 a,v 5.10 a,vw 5.08 ab,vw 5.14 a,vw Nisin+EDTA+rosemary 0.97 cd,w 1.87 bc,vw 2.28 c,v 2.40 de,v 2.58 e,v 2.54 c,v 2.34 c,v 2.32 c,v 2.30 d,v 2.31 cd,v EDTA+rosemary 4.87 a,v 4.47 a,vw 4.20 b,xy 4.31 b,wx 3.51 d,z 4.40 b,vw 3.67 b,yz 3.70 b,yz 3.72 c,yz 3.37 bc,z a-e values in same column with different superscripts are significantly different v-z values in same row with different superscripts are significantly different (P < 0.05)

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100 Table 6-5. pH measurements on ReadyTo-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Negative control 5.72 bc,vw 5.34 d,wxy 5.00 c,y 5.28 c,xy 5.42 c,vwx 5.74 bc,v 5.51 bc,vwx 5.16 d,xy 5.08 e,xy 5.29 d,wxy Positive control 5.79 b,v 5.45 cd,vw 5.25 bc,vw 5.42 c,vw 5.16 c,w 5.65 cd,vw 5.36 bc,vw 5.18 cd,w 5.62 bc,vw 5.48 cd,vw Nisin 5.86 ab,v 5.82 a,v 5.69 a,v 5.82 ab,v 5.97 ab,v 5.87 ab,v 5.72 ab,v 5.59 bc,v 5.57 cd,v 5.74 bc,v EDTA 5.48 d,y 5.66 bc,xy 5.73 a,xy 5.85 ab,wx 6.03 ab,vw 6.11 ab,v 5.97 a,vwx 5.99 ab,vw 5.99 ab,vw 6.10 ab,v Rosemary 5.95 a,v 5.54 bc,vw 5.39 ab,w 5.24 c,w 5.31 c,w 5.48 d,w 5.31 c,w 5.22 cd,w 5.43 de,w 5.50 cd,vw Nisin+EDTA 5.49 d,z 5.59 ab,xy 5.73 a,y 5.83 ab,wx 6.09 a,v 6.17 a,v 5.97 a,wx 5.99 ab,vw 6.09 a,v 6.11 ab,v Nisin+rosemary 5.78 b,vw 5.75 a,v 5.57 ab,wx 5.52 bc,wx 5.60 bc,vwx 5.59 cd,wx 5.31 c,xy 5.37 cd,xy 5.14 e,y 5.56 cd,wx Nisin+EDTA+rosemary 5.46 d,z 5.73 ab,z 5.80 a,yz 5.89 a,xyz 6.03 ab,vwx 6.12 ab,vw 6.07 a,vwx 6.01 a,wxy 6.11 a,vwx 6.26 a,v EDTA+rosemary 5.58 cd,y 5.74 ab,xy 5.71 a,xy 5.78 ab,wx 6.07 a,v 6.07 ab,v 6.02 a,v 6.04 a,v 5.94 ab,vw 6.12 ab,v a-d values in same column with different superscripts are significantly different x-z values in same row with different superscripts are significantly different (P < 0.05)

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101 Table 6-6. Mean for L values of Rea dy-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Negative control 62.07 ab,wx 58.72 bc,x 62.20 bc,wx 63.59 a,w 62.94 a,w 61.43 ab,wx 60.98 a,wx 60.68 ab,wx 62.98 bc,w 61.78 ab,wx Positive control 61.56 ab,x 61.44 a,x 64.65 a,w 63.32 a,wx 63.38 a,wx 62.66 ab,wx 62.92 a,wx 61.96 a,wx 61.88 d,wx 63.31 ab,wx Nisin 61.44 ab,w 60.12 ab,w 61.53 c,w 61.31 ab,w 61.24 a,w 61.11 ab,w 61.13 a,w 60.05 ab,w 61.76 d,w 61.34 b,w EDTA 60.11 b,xy 58.61 bc,y 60.31 c,xy 60.42 ab,xy 61.92 a,x 64.43 a,w 61.59 a,x 61.95 a,x 61.34 d,x 61.33 ab,x Rosemary 61.44 ab,w 61.25 a,w 61.02 c,w 61.07 ab,w 63.61 a,w 62.66 ab,w 60.39 a,w 61.36 ab,w 64.06 a,w 61.03 b,w Nisin+EDTA 61.08 ab,wx 59.10 b,x 61.31 c,wx 60.56 ab,wx 61.42 a,wx 60.96 ab,wx 60.62 a,wx 61.02 ab,wx 58.59 e,x 63.20 ab,w Nisin+rosemary 61.46 ab,xyz 60.28 ab,xyz 62.50 ab,wx 62.46 ab,wx 61.83 a,xyz 59.42 b,z 62.09 a,wxy 59.71 b,z 63.93 ab,w 62.85 ab,wx Nisin+EDTA+rosemary 60.48 b,x 59.73 ab,x 64.07 ab,w 59.53 b,x 61.84 a,wx 61.32 ab,wx 61.40 a,wx 60.69 ab,x 62.38 cd,wx 62.43 ab,wx EDTA+rosemary 63.11 a,wx 57.10 c,z 61.36 c,wxy 61.64 ab,wxy 62.69 a,wx 61.24 ab,wxy 60.22 a,xy 59.28 b,yz 61.40 d,wxy 64.69 a,w a-e values in same column with different superscripts are significantly different w-z values in same row with different superscripts are significantly different (P < 0.05)

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102 Table 6-7. Mean for a values of Rea dy-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Negative control 17.46 a,yz 17.10 a,z 17.96 ab,yz 17.85 a,yz 18.08 a,xyz 18.59 a,wxy 19.56 a,w 17.59 a,yz 18.55 abc,wxy 19.34 a,wx Positive control 16.36 ab,wx 16.79 a,wx 18.79 a,w 17.88 a,wx 18.13 a,wx 19.03 a,w 18.29 a,wx 17.91 a,wx 19.22 a,w 13.84 abc,x Nisin 16.59 ab,yz 17.12 a,y 17.00 ab,y 17.57 a,wxy 18.83 a,w 18.52 ab,wx 18.52 a,wx 15.53 b,z 17.73 cd,wxy 17.51 ab,xy EDTA 15.82 b,wx 14.05 b,x 17.01 ab,wx 15.64 a,wx 13.47 c,x 15.28 c,wx 14.51 b,x 16.66 ab,wx 18.67 ab,w 16.03 ab,wx Rosemary 15.51 b,z 16.51 a,yz 18.41 ab,w 17.68 a,wx 17.62 a,wx 17.25 abc,xy 18.61 a,w 17.56 a,wxy 16.81 e,xy 18.43 a,w Nisin+EDTA 16.62 ab,wx 16.65 a,wx 14.34 c,wx 15.94 a,wx 14.78 bc,wx 16.08 bc,wx 13.62 b,wxy 12.52 c,xy 17.11 de,w 9.06 c,y Nisin+rosemary 16.65 ab,y 16.50 a,y 17.75 ab,wx 17.17 a,xy 18.15 a,wx 18.53 a,w 18.29 a,w 17.83 a,wx 18.51 abc,w 18.30 a,w Nisin+EDTA+rosemary 15.91 b,w 15.85 a,w 16.66 b,w 17.18 a,w 16.94 ab,w 17.55 abc,w 16.50 ab,w 17.59 a,w 13.11 f,x 11.88 bc,x EDTA+rosemary 16.56 ab,wx 16.99 a,wx 18.49 ab,w 14.70 a,x 18.06 a,w 18.20 ab,w 18.10 a,w 16.92 ab,wx 18.30 bc,w 14.05 abc,x a-f values in same column with different superscripts are significantly different w-z values in same row with different superscripts are significantly different (P < 0.05)

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103 Table 6-8. Mean for b values of Rea dy-To-Eat turkey ham inoculated with Listeria monocytogenes and stored at 4 1C for 63 days Treatments Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Negative control 7.05 b,wx 6.25 c,x 7.97 ab,wx 7.16 d,wx 7.96 ab,wx 7.64 a,wx 8.42 ab,w 8.35 abc,w 7.89 bc,wx 8.77 a,w Positive control 7.63 b,xyz 6.73 bc,z 7.77 ab,xy 8.97 abc,w 6.85 b,yz 6.97 a,yz 7.53 b,xyz 6.99 c,yz 7.58 bc,xyz 8.41 a,wx Nisin 7.73 b,wx 6.98 b,wx 6.65 b,x 8.10 abc,w 7.81 ab,wx 7.08 a,wx 7.62 b,wx 8.18 abc,w 7.46 c,wx 7.82 a,wx EDTA 7.59 b,y 7.20 ab,y 8.25 a,wxy 7.73 cd,xy 9.15 a,w 7.96 a,wxy 9.00 ab,wx 8.61 bc,y 8.01 bc,wxy 9.21 a,w Rosemary 9.21 a,w 7.87 a,w 8.02 ab,w 9.21 abc,w 7.89 ab,w 8.61 a,w 9.55 a,w 8.16 abc,w 8.41 ab,w 9.37 a,w Nisin+EDTA 7.38 b,x 7.33 ab,x 8.50 a,wx 7.96 bcd,wx 8.68 a,wx 8.78 a,wx 9.27 a,w 8.94 ab,wx 8.05 bc,wx 9.68 a,w Nisin+rosemary 9.54 a,w 7.30 ab,y 8.55 a,wxy 8.23 abc,wxy 7.52 ab,xy 8.86 a,wx 7.42 b,xy 7.57 bc,xy 8.12 bc,wxy 8.16 a,wxy Nisin+EDTA+rosemary 9.50 a,wx 6.85 bc,z 7.21 ab,yz 9.55 a,wx 8.62 a,wx 8.57 a,wxy 8.19 ab,xyz 8.53 abc,wxy 9.02 a,wx 9.82 a,w EDTA+rosemary 8.20 ab,wxy 7.05 b,y 7.26 ab,xy 9.30 ab,wx 7.63 ab,wxy 7.35 a,wxy 7.51 b,wxy 9.56 a,w 7.26 c,xy 8.68 a,wxy a-d values in same column with different superscripts are significantly different w-z values in same row with different superscripts are significantly different (P < 0.05)

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104 CHAPTER 7 SUMMARY AND CONCLUSION It is well understood that th e meat industry is in need of new post-processing hurdle technologies that will control and inhibit L. monocytogenes growth inready-to -eat (RTE) meat and poultry products. This inves tigation proposes a post-processi ng antimicrobial system that includes the use of nisin alone a nd nisin in combination with rosemary and/or EDTA to control the incidence of L. monocytogenes on RTE turkey ham stored at 4C for up to 63 days. Preliminary studies were conducted on differe nt antimicrobial solutions using the Kirby Bauer disc diffusion method. Result s obtained in these studies reveal ed that when nisin was used alone or in combination with 1% rosemary and/or 20 mM EDTA a significant inhibition in L. monocytogenes growth (P < 0.05) was achieved. In contrast, L. monocytogenes growth was not inhibited by treatments containing 1% and 2% of vinegar (acetic acid), 3% and 5% of potassium benzoate, 1% thymol, or 0.15% and 0.25% sodium di acetate alone or in comb ination with nisin. These outcomes were useful in developing the research study, in which nisin, rosemary and EDTA alone and in combination were further analyzed in a meat matrix. Turkey ham treated with EDTA alone or in co mbination with nisin and/or rosemary had a significantly higher (P < 0.05) pH va lue than all other treatments. This probably resulted from the ability of EDTA to act as a f ood preservative and pH stabilizer (139) All treatments that did not contain EDTA experienced a slight decrease in pH throughout storage (P < 0.05). Bacterial growth on turkey ham, stored under vacuum p ackaging conditions, may result in the production of numerous compounds such as acidic metabolite s and carbonic acid that may decrease the pH (37) This may explain the decrease in pH for the controls and the treatments without EDTA over time in this study. This suggested that EDTA, under the conditions of this study, had an effect on the pH value due to its buffering capaci ty. In addition, because changes in pH were

PAGE 105

105 consistent with changes in lactic acid bacteria population, pH alone may be considered as a key factor for lactic acid bacteria population reduction in this study. Furthermore, the results showed that pH values decreased as th e concentration of nisi n decreased from 0.5% to 0.2%. This also may be attributed to the lactic acid bacteria present in the samples, which increased as the concentration of nisin de creased from 0.5% to 0.2%. During the study, treatments with nisin alone an d nisin combined with rosemary and/or EDTA caused significant reduction in L. monocytogenes counts as compared to the positive control (P < 0.05). The initial L. monocytogenes population was signifi cantly reduced (P< 0.05) from approximately 3.00 log CFU/g to 4.00 log CFU/g when samples were treated with nisin alone or in combination with rosemary and/or EDTA. In addition, an extended lag phase was demonstrated for the 0.5% nisin treat ment throughout the study, maintaining L. monocytogenes counts less than 1.95 log CFU/g. Results from the study indicated that the antimicrobial effectiveness of nisin increased as its concentration increased from 0.2% to 0.5%. The study also revealed that L. monocytogenes might be controlled in vacuum package RTE turkey ham stored at 4C when treated with the concentrations of nisin alone and in comb ination with rosemary and/or EDTA evaluated in this study. In the absence of nisin, EDTA and rosemary alone and in combination did not inhibit L. monocytogenes throughout the study under conditions of vacuum package and storage at 4 C. Also, no increase in antimicrobial activity was ob served when nisin was used with rosemary and/or EDTA against L. monocytogenes when compared to using nisin only. In contrast, researchers have reported an increase in the an timicrobial activity of nisin against gram positive (65) and negative bacteria (23, 126) in the presence of membrane disrupting agents, including EDTA. However, the majority of these re ports are based upon obs ervation of organisms

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106 suspended in a buffer, rather than growing in nutrient media. Since meat products provide a nutrient rich environment, test of antimicrobial s under conditions of cell starvation would appear to be of limited value in evalua ting them for application in food products. The failure to observe an enhancement in the antimicrobial properties of nisin when combined with EDTA in this study may indicate that the observed enhancement betw een these two antimicrobial agents in buffer systems (65) is a consequence of cell starva tion which prevents cell repair. In gram negative organisms, it has been show n that EDTA can act to destabilize the cell membrane of bacteria by complexing divalent cations, such as Ca2+ and Mg2+, which act as salt bridges between membrane macromolecules, such as lipopolysaccharides (118) However, membrane structure differs between gram negative and gram positive bacteria. Gram negative bacteria have an outer membrane coating a thin layer of peptidoglycan. The primary target of EDTA is the lipids and lipid prot eins present in the outer membra ne of gram negative bacteria (37) In contrast, Gram positive bacteria will have a thick layer of peptidoglycan (a sugarprotein shell) that confers resistance to physical disruption (43) This may be the reason that EDTA did not inhib it the growth of L. monocytogenes under the conditions used in this study. Similar observations, that EDTA had no significant inhibition against gram positive bacteria and that EDTA combined with nisin reduced the antimicrobial effect of nisin, have been reported by other researchers (96, 115) In this study, rosemary extract alone was not effective controlling L. monocytogenes growth. Del campo et al. (33) found that gram positive bacteria were the most sensitive to rosemary extracts. However, the auth or states that rosemary extract should be more appropriate in foods with low fa t and protein contents. Ready-to -eat meat products are rich in protein contents; this may be why rosemary did not exhibit an anti-listerial effect. Another study found that the use of encapsulated rosemary oil wa s much more effective than standard rosemary

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107 essential oil extract against L. monocytogenes in pork liver sausage (95) This suggests that the method of application of rosemary to the produc t may have interfered with its antimicrobial properties. The fact that EDTA and rosemary did not inhibit L. monocytogenes growth but nisin alone or combined with rosemary and/or EDTA had an anti-listeri al effect throughout the days of storage at 4C, indicated that the observed population reductions were attributed to the inhibitory activity of nisin rather than EDTA or rosemary. When nisin was used alone or in combinati on with EDTA and/or rosemary, an immediate population reduction (P < 0.05) of lactic acid bacteria was obse rved. EDTA, rosemary and combination of EDTA with rosemary treatments were not effective (P > 0.05) when compared to the positive control during storage. This suggested that EDTA and rosemary alone may have a limited antimicrobial effect against gram positive bacteria. However, when EDTA was used in combination with nisin or nisin with rosemary an extended lag phase in the lactic acid bacteria population was observed. This may have been caused by the buffer capacity of EDTA, which may have functioned to stabilize the pH. The treatments that c ontained nisin or rosemary alone experienced an exponential growth of lactic aci d bacteria over time. Even though, EDTA played an important role in stabilizi ng the pH, nisin was needed to pr ovide the initial inhibitory and killing effect. When nisin alone or combina tions of nisin with rosemary and/or EDTA were used a significant (P < 0.05) reduction in the initial populati on of aerobic bacteria was observed when compared to the positive control. Treatments containing nisin ach ieved a reduction of approximately 3 log CFU/g. In contrast, aerobic bacteria counts were significantly higher (P < 0.05) in rosemary, EDTA, and combination of ro semary with EDTA treatments when compared

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108 to the positive control. This may suggest that EDTA has no immediate antimicrobial effect against the aerobic organisms present in the turkey ham. The L* value is an indication of lightness, where 100 represents perfect white and 0 black (76) Results of L* a* b values measured on read y-to-eat vacuum packaged turkey ham revealed that L* values (lightness) increased during st orage for the positive control, EDTA, nisin with EDTA, nisin with rosemary, nisin with rose mary and EDTA, and EDTA with rosemary treatments, showing significantly higher (P < 0.05) values by day 63. The increase in L* value was attibutted to a whitening surface observed in the turkey ham. L* values were similar (P < 0.05) for nisin alone and rosemary alone throughout storage. The L* values ranged between 58 to 64 for all treatments. A d ecreasing trend (P < 0.05) on the a* value over time was observed for nisin combined with EDTA, nisin combined with EDTA and rose mary, EDTA combined with rosemary, and positive control when compared to all other treatments. All other treatments experienced an increasing tre nd (P < 0.05) on the a* value th roughout the storage time. The decreasing trend on the a* value may be attrib uted to many factors such as storage time, increased residual O2 level, increased oxygen transmission rate, increased light intensity and decreased nitrite content (81) All treatments showed an increasing trend on the b* value over time (P < 0.05). Differences in b* values could be related to the intensity of the oxidation process that occurs during storag e and might tend to increase ye llowness of samples by rancidity, although no measures of oxidation intensity ar e available to suppor t this hypothesis. Even though, L. monocytogenes was not completely eliminated by the antimicrobial solutions, the overall results indicate that readyto-eat turkey ham will have decreased numbers of L. monocytogenes, anaerobic bacteria and lact ic acid bacteria when treated with nisin alone or in combination with rosemary and/or EDTA. Nisin is a natural antimicrobial compound and

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109 may provide a novel, environmentally safe alternative to control L. monocytogenes This study proposed an antimicrobial system, which consist of the addition of nisin to the finished product before vacuum packaging, to control L. monocytogenes on ready-to-eat turkey ham stored at 4C. The data suggested that nisin, rosemary and EDTA will function to enhance the microbial safety of ready-to-eat poultry, as well as meat products. This study also raised new questions that can be further analyzed in futu re studies. In this study a five strain inoculum was utilized to in oculate the turkey ham samples. By day 63, L. monocytogenes growth was still observed. Future resear ch will have to be conducted in order to determine which strains were surviving and if they developed some resistance to nisin. Furthermore, this study evaluated the antimic robial properties of nisin against Gram positive bacteria. Future studies can be conducte d in the evaluation of antimicrobial properties of nisin when Gram positive and Gram negativ e bacteria are present in a meat product. In this study EDTA exhi bited no inhibition against L. monocytogenes Further study can be conducted to evaluate the physical and chemi cal properties, mode of action and synergistic effects of EDTA when used alone or in comb ination with other antimicrobials against Gram positive and negative bacteria.

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121 BIOGRAPHICAL SKETCH Alba Yesenia Ruiz was born in 1983, in San Salvador, El Salvador. She attended the PanAmerican School of Agriculture El Zamorano (Tegucigalpa, Honduras) where she received a Bachelor of Science degree in agro-industria l engineering with a concentration in food microbiology in December 2005. In 2006, Alba began her pursuit of a Master of Sc ience degree at the University of Florida in the Department of Animal Sciences, Meat Science section. She was awarded an Animal Sciences Department Assistantship to study for th e Master of Science degree. She received her Master of Science degree in December 2007. She plan s to continue her studies in the Department of Animal Sciences for the Doctor of Philosophy degree.