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Antimicrobial Properties of Selected Asian Herbs

Permanent Link: http://ufdc.ufl.edu/UFE0022001/00001

Material Information

Title: Antimicrobial Properties of Selected Asian Herbs
Physical Description: 1 online resource (107 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antimicrobials, asian, freeze, herbs, lemongrass, oven, solvents, turmeric
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Many pathogens including Salmonella spp, Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 are commonly implicated in foodborne illness outbreaks in the United States. Increasing antibiotic resistance of bacterial foodborne pathogens has raised concerns in the scientific community. Many herbs such as lemongrass and turmeric have been considered medicinal plants that can be potentially used for controlling foodborne pathogens in place of chemicals or antibiotics. Objective of this study was to systematically evaluate the antimicrobial properties of lemongrass Cymbopogon citratus (C. Nees) Stapf (Poaceae) and turmeric Curcuma longa (Zingiberaceae) as affected by drying methods (oven and freeze drying) and different extraction solvents (water, acetone, hexane, and ethanol). Antimicrobial assay was done using an agar disc diffusion method against Staphylococcus aureus (ATCC 29247, 12600U, and 35548), Escherichia coli O157:H7 (204P, 301C and 505B) and Salmonella serotypes Enteritidis, Typhimurium and Thompson (ATCC 8391) strains. Minimum inhibitory concentration (MIC) was measured using the multiple tube dilution method. Minimum bactericidal concentration (MBC) was measured using the colony-forming assay. Results showed that among bacterial strains tested, Staphylococcus aureus strains were sensitive to the hexane and acetone extracts from lemongrass stems; and to hexane, acetone and ethanol extracts from turmeric. No antimicrobial activity was observed in any lemongrass leaf samples regardless of solvents used in the extraction or drying methods. Water and ethanol extracts of lemongrass stem did not show any antimicrobial activity against any of the test bacteria. Inhibitory zone from lemongrass stem extracts ranged from 6.5 to 21 mm. For lemongrass stem, oven dried methods yielded significantly higher (P < 0.0001) antimicrobial activity than that of stems prepared from the freeze dried method. Hexane extract yielded significantly higher (P < 0.0001) antimicrobial activity (MIC) than that of the acetone extracts. Seasonal variation was observed with respect to antimicrobial activity for lemongrass stem; plants harvested in November showed a higher activity (P < 0.0001) than plants which were harvested in October. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) range for lemongrass stem was 0.8 to? 17.4 mg/mL. Water extracts of turmeric did not show any antimicrobial activity against any test bacterial strains. Turmeric had a significantly lower (P < 0.0001) antimicrobial activity against Staphylococcus aureus strains tested than that of lemongrass stem. However the antimicrobial activity of turmeric was observed in extracts of the other solvents (acetone, hexane and ethanol) than that of lemongrass (acetone and hexane). Inhibitory zone of turmeric extracts ranged from 6.5 to 11 mm. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) range for dried and fresh turmeric were 12.5 to 40.0 mg/mL. Little variation in antimicrobial activity was observed for dried and fresh turmeric extracts. Results of the study demonstrated that hexane and acetone extracts from lemongrass stem exhibit antimicrobial activity against Staphylococcus aureus, and hexane, acetone, and ethanol extracts of dried powder and fresh turmeric rhizomes, exhibited antimicrobial activity against Staphylococcus aureus. Both lemongrass stem and turmeric extracts could be potentially used for controlling Staphylococcus aureus.
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.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Simonne, Amarat H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022001:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022001/00001

Material Information

Title: Antimicrobial Properties of Selected Asian Herbs
Physical Description: 1 online resource (107 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antimicrobials, asian, freeze, herbs, lemongrass, oven, solvents, turmeric
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Many pathogens including Salmonella spp, Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 are commonly implicated in foodborne illness outbreaks in the United States. Increasing antibiotic resistance of bacterial foodborne pathogens has raised concerns in the scientific community. Many herbs such as lemongrass and turmeric have been considered medicinal plants that can be potentially used for controlling foodborne pathogens in place of chemicals or antibiotics. Objective of this study was to systematically evaluate the antimicrobial properties of lemongrass Cymbopogon citratus (C. Nees) Stapf (Poaceae) and turmeric Curcuma longa (Zingiberaceae) as affected by drying methods (oven and freeze drying) and different extraction solvents (water, acetone, hexane, and ethanol). Antimicrobial assay was done using an agar disc diffusion method against Staphylococcus aureus (ATCC 29247, 12600U, and 35548), Escherichia coli O157:H7 (204P, 301C and 505B) and Salmonella serotypes Enteritidis, Typhimurium and Thompson (ATCC 8391) strains. Minimum inhibitory concentration (MIC) was measured using the multiple tube dilution method. Minimum bactericidal concentration (MBC) was measured using the colony-forming assay. Results showed that among bacterial strains tested, Staphylococcus aureus strains were sensitive to the hexane and acetone extracts from lemongrass stems; and to hexane, acetone and ethanol extracts from turmeric. No antimicrobial activity was observed in any lemongrass leaf samples regardless of solvents used in the extraction or drying methods. Water and ethanol extracts of lemongrass stem did not show any antimicrobial activity against any of the test bacteria. Inhibitory zone from lemongrass stem extracts ranged from 6.5 to 21 mm. For lemongrass stem, oven dried methods yielded significantly higher (P < 0.0001) antimicrobial activity than that of stems prepared from the freeze dried method. Hexane extract yielded significantly higher (P < 0.0001) antimicrobial activity (MIC) than that of the acetone extracts. Seasonal variation was observed with respect to antimicrobial activity for lemongrass stem; plants harvested in November showed a higher activity (P < 0.0001) than plants which were harvested in October. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) range for lemongrass stem was 0.8 to? 17.4 mg/mL. Water extracts of turmeric did not show any antimicrobial activity against any test bacterial strains. Turmeric had a significantly lower (P < 0.0001) antimicrobial activity against Staphylococcus aureus strains tested than that of lemongrass stem. However the antimicrobial activity of turmeric was observed in extracts of the other solvents (acetone, hexane and ethanol) than that of lemongrass (acetone and hexane). Inhibitory zone of turmeric extracts ranged from 6.5 to 11 mm. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) range for dried and fresh turmeric were 12.5 to 40.0 mg/mL. Little variation in antimicrobial activity was observed for dried and fresh turmeric extracts. Results of the study demonstrated that hexane and acetone extracts from lemongrass stem exhibit antimicrobial activity against Staphylococcus aureus, and hexane, acetone, and ethanol extracts of dried powder and fresh turmeric rhizomes, exhibited antimicrobial activity against Staphylococcus aureus. Both lemongrass stem and turmeric extracts could be potentially used for controlling Staphylococcus aureus.
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.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Simonne, Amarat H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022001:00001


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2bc3557ab72d63af60e8b8514d0ad192c29ff4d9







ANTIMICROBIAL PROPERTIES OF SELECTED ASIAN HERBS


By

THABILE PRECIOUS NKAMBULE

















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

2008


































2008 Thabile Precious Nkambule


































To my Family and Friends









ACKNOWLEDGMENTS

I would like to express my special appreciation to my advisor Dr. Amarat H. Simonne (my

supervisory committee chair) and Dr. Charles Sims, Dr. Samuel R. Farrah, and Dr. Maurice R.

Marshall, Jr., for serving on my committee. I would also like to thank Rob M. Pelick, Kimberly

Evans, and Wei-Yea Hsu for their assistance and support. I would also like to thank the faculty

and staff of Food Science and Human Nutrition department, University of Florida. I would like

the express much appreciation to my family for their love, encouragement, and support.










TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

LIST OF FIGURES .................................. .. .... ..... ................. 10

L IST O F A B B R E V IA T IO N S ......... ...................................... ....................................... 11

A B S T R A C T ............ ................... ............................................................ 12

CHAPTER

1 INTRODUCTION ............... ................. ........... ......................... .... 15

2 L ITE R A TU R E R E V IE W ........................................................................ .. ....................... 19

M ost Com m on A sian H erbs and Spices ..................................................................... ......20
L em o n g ra ss............................. ................................................................ ............... 2 1
T u rm eric ........................................... ....... ............ ........... .....................2 2
Antimicrobial Activities of Lemongrass (Cymbopogon citratus)............................23
Antimicrobial Activities of Turmeric (Curcuma long) ............. ....................30
D trying M methods ............. .......................... ..................... ........... ...... 33
Oven-D trying ............. .............................................. ...... ... ...... 34
Freeze-Drying ......... ......... .....................................................35
Extraction Solvents ......... ............. ...................................................................39
D im ethyl Sulfoxide (C 2H 60 S) ........................................ ..................... .....................40
W ate r (H 20 ) ............................................................................................................... 4 0
E th an o l (C 2H 60 ) .............................................................................................4 1
H exane (C 6H 14) .............................................................................................4 1
A cetone (C3H 60 ) ....................... ... ......... ..... ... ........... ......................... 41
Effect of Extraction Solvents on the Antimicrobial Properties of Herbs ........................41
E v ap oration T ech n iqu es ................................................................................................... 4 3
Rotary Rotavapor .............................................. 43

3 M A TER IA L S A N D M ETH O D S ..................................................................................... 51

Bacterial Strains and Cultivation ............................................................... ..............51
P lan t M materials .........................................................................................5 1
Preparation of Plant M material ............................................................................ 52
Preparation of Plant Extracts ............................................................................ 52
Antimicrobial Assay ........................................ ......... ... 54
Minimum Inhibitory Concentration (MIC) Determination ..................................... 55
Minimum Bactericidal Concentration (MBC) Determination.............. .. ......56
Experimental Design and Statistical Analysis................................ .... ......... 56

4 RESULTS AND DISCUSSION ...................................... .........60









Antimicrobial Activity of Lemongrass....................................................... .. ................ .... 60
Inhibitory Zone of Lemongrass Extracts....................................... ...............60
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC) of Lemongrass Stem Extracts.........................................64
Antim icrobial A activity of Turm eric Extracts ........................................... ............... .... 66
Inhibitory Zone of Turmeric (Fresh Rhizomes and Dried Powder) Extracts................66
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC) of Turmeric (Fresh Rhizomes and Dried Powder) Extracts ....68

5 SUMMARY AND CONCLUSSION.............. ......... ..................... 81

APPENDIX

A ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY
O F L EM O N G R A SS STE M S ........................................................................ ...................83

B ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY
OF TURM ERIC (FRESH RHIZOM ES)..................................................... ..... .......... 91

C ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY
OF TURMERIC (DRIED POWDER) ........... ..... ................................... 96

L IST O F R E F E R E N C E S ......... .. ............... ................. ..........................................................100

B IO G R A PH IC A L SK E T C H ........................................... ......... ................... .......................... 107









LIST OF TABLES


Table page

2-1 Reported and estimated cases, number of outbreaks and deaths caused by known
foodborne bacterial pathogens from 1983 to 1997. ................................ .................45

2-2 Most common pathogens associated with foodborne illness outbreaks ..........................46

2-3 Com m on A sian herbs used for cooking............................................................ ........... 48

2-4 Properties of different solvents ...................................................................... 49

4-1 Inhibitory zone (mm) of lemongrass stems extracts (solvent extracts) on bacterial
p ath o g e n s .............................................................................. 7 1

4-2 Inhibitory zone (mm) of turmeric (fresh rhizome) extracts (solvent extracts) on
bacterial pathogens..................... ........... .. .... ..... ..... .......... 72

4-3 Inhibitory zone (mm) of turmeric (dried powder) extracts (solvent extracts) on
b acterial pathogen s........ ............................................................................ ...... .. .... 73

4-4 Overall comparison of solvents, drying methods, and harvest-time for lemongrass
stem.................... ........................................ 74

4-5 Sensitivity of each Staphylococcus aureus strain to lemongrass stem ..............................75

4-6 Antimicrobial activities of fresh rhizome turmeric extracts ................... ..................76

4-7 Sensitivity of each Staphylococcus aureus strain to fresh rhizome turmeric extracts.......77

4-8 Antimicrobial activities of dried powder turmeric extracts..................... .............. 78

4-9 Sensitivity of each Staphylococcus aureus strain to dried powder turmeric extracts........79

4-10 Comparison oflemongrass stem and turmeric (fresh rhizomes) extracts..........................80

4-11 Comparison of fresh rhizomes and dried powder turmeric extracts...............................80

A -i Inhibitory zone of lem ongrass stem s ........................................... .......................... 83

A-2 Minimum Inhibitory Concentration (MIC) of lemongrass stem........................................83

A-3 Minimum Bactericidal Concentration (MBC) of lemongrass stem...............................84

A-4 Inhibitory zone oflemongrass stems against Staphylococcus aureus ATCC 12600-U ....85

A-5 Minimum Inhibitory Concentration (MIC) of lemongrass stem against
Staphylococcus aureus ATCC 12600-U ...................................................................... 86









A-6 Minimum Bactericidal Concentration (MBC) of lemongrass stem against
Staphylococcus aureus ATCC 12600-U ................................................. ............... 86

A-7 Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC 29247.........87

A-8 Minimum Inhibitory Concentration (MIC) of lemongrass stem against
Staphylococcus aureus ATCC 29247 ........................................... ......................... 87

A-9 Minimum Bactericidal Concentration (MBC) of lemongrass stem against
Staphylococcus aureus A TCC 29247 ........................................... ......................... 88

A-10 Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC A35548......89

A-11 Minimum Inhibitory Concentration (MIC) of lemongrass stem against
Staphylococcus aureus ATCC A35548 ........................................ ......................... 89

A-12 Minimum Bactericidal Concentration (MBC) of lemongrass stem against
Staphylococcus aureus ATCC A35548 ........................................ ......................... 90

B-1 Inhibitory zone of turmeric (fresh rhizomes) ......................................... ...............91

B-2 Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes) .........................91

B-3 Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes)...................91

B-4 Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC
S A 12 6 0 0 -U ...................................... ................................................... 9 2

B-5 Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes) against
Staphylococcus aureus ATCC SA12600-U................................................ 92

B-6 Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes) against
Staphylococcus aureus ATCC SA12600-U................................................ 92

B-7 Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC
S A 2 92 4 7 .................................................................................9 3

B-8 Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes)
Staphylococcus aureus ATCC SA29247................................................ ............... 93

B-9 Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes)
Staphylococcus aureus ATCC SA29247 ................................................ ............... 94

B-10 Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC
35548.......... ... ........................ .............................................. ....... 95

B-11 Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes) against
Staphylococcus aureus A TCC 35548 ........................................... ......................... 95









B-12 Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes) against
Staphylococcus aureus A TCC 35548 ........................................... ......................... 95

C-1 Inhibitory zone of turmeric (dried powder) ............................... ...............96

C-2 Minimum Inhibitory Concentration (MIC) of turmeric (dried powder)..........................96

C-3 Minimum Bactericidal Concentration (MBC) of turmeric (dried powder) .....................96

C-4 Inhibitory zone of turmeric (dried powder) against Staphylococcus aureus ATCC
12 6 0 0 -U ............................................................................... 9 7

C-5 Minimum Inhibitory Concentration (MIC) of turmeric (dried powder) against
Staphylococcus aureus ATCC 12600-U ................................................. ....... ........ 97

C-6 Minimum Bactericidal Concentration (MBC) of turmeric (dried powder) against
Staphylococcus aureus ATCC 12600-U ................................ ........................ ....... 97

C-7 Inhibitory zone of turmeric (dried powder) against Staphylococcus aureus ATCC
29247................. ...................... ..................................................... 98

C-8 Minimum Inhibitory Concentration (MIC) of turmeric (dried powder)
Staphylococcus aureus ATCC 29247 ........................................... ......................... 98

C-9 Minimum Bactericidal Concentration (MBC) of turmeric (dried powder)
Staphylococcus aureus ATCC 29247 ........................................... ......................... 98

C-10 Inhibitory zone of turmeric (dried powder) against Staphylococcus aureus ATCC
35548.......... ... ........................ .............................................. ....... 99

C-11 Minimum Inhibitory Concentration (MIC) of turmeric (dried powder) against
Staphylococcus aureus A TCC 35548 ........................................... ......................... 99

C-12 Minimum Bactericidal Concentration (MBC) of turmeric (dried powder) against
Staphylococcus aureus A TCC 35548 ........................................... ......................... 99









LIST OF FIGURES

Figure page

2-1 Chemical structure of Citral (C10H160) (3, 7-dimethyl-2, 6-octadienal)...........................50

2-2 Chemical structure of Curcumin (C21H2006 ).............. .......................................... 50

3-1 Lemongrass (Cymbopogon citratus) plants. ........................................... ............... 57

3-2 Turm eric (Curcuma long) rhizom es ........................................ .......................... 57

3-3 Lem ongrass leaves ......... ............. ........... ........ ........... ...... .. .......... 58

3-4 Lem ongrass stem ................................. .... .. ............ .. ........... 58

3-5 Inhibitory zone of lemongrass stem oven dried, hexane extracts on Staphylococcus
aureus ATCC 29247.............................................. .......... 59




































10









LIST OF ABBREVIATIONS


Antimicrobials


MBC


MIC


A large variety of chemical compounds or substances that are used to
destroy (kill) microorganisms or to prevent (or slow) their growth

Minimum bactericidal concentration is the lowest concentration of an
antimicrobial agent needed to kill 99.9% of the initial organism inoculums

The minimum inhibitory concentration is as the lowest concentration of an
antimicrobial that prevents the growth of microorganism after a specific
incubation time









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

ANTIMICROBIAL PROPERTIES OF SELECTED ASIAN HERBS

By

Thabile Precious Nkambule

May 2008

Chair: Amarat H. Simonne
Major: Food Science and Human Nutrition

Many pathogens including Salmonella spp, Listeria monocytogenes, Staphylococcus

aureus and Escherichia coli 0157:H7 are commonly implicated in foodborne illness outbreaks in

the United States. Increasing antibiotic resistance of bacterial foodborne pathogens has raised

concerns in the scientific community. Many herbs such as lemongrass and turmeric have been

considered medicinal plants that can be potentially used for controlling foodborne pathogens in

place of chemicals or antibiotics. Objective of this study was to systematically evaluate the

antimicrobial properties of lemongrass [Cymbopogon citratus (C. Nees) Stapf (Poaceae)] and

turmeric [Curcuma long (Zingiberaceae)] as affected by drying methods (oven and freeze

drying) and different extraction solvents (water, acetone, hexane and ethanol). Antimicrobial

assay was done using an agar disc diffusion method against Staphylococcus aureus (ATCC

29247, 12600U, and 35548), Escherichia coli 0157:H7 (204P, 301C and 505B) and Salmonella

serotypes Enteritidis, Typhimurium and Thompson (ATCC 8391) strains. Minimum inhibitory

concentration (MIC) was measured using the multiple tube dilution method. Minimum

bactericidal concentration (MBC) was measured using the colony-forming assay. Results showed

that among bacterial strains tested, Staphylococcus aureus strains were sensitive to the hexane

and acetone extracts from lemongrass stems; and to hexane, acetone and ethanol extracts from









turmeric. No antimicrobial activity was observed in any lemongrass leaf samples regardless of

solvents used in the extraction or drying methods. Water and ethanol extracts of lemongrass stem

did not show any antimicrobial activity against any of the test bacteria. Inhibitory zone from

lemongrass stem extracts ranged from 6.5 to 21 mm. For lemongrass stem, oven dried methods

yielded significantly higher (P < 0.0001) antimicrobial activity than that of stems prepared from

the freeze dried method. Hexane extract yielded significantly higher (P < 0.0001) antimicrobial

activity (MIC) than that of the acetone extracts. Seasonal variation was observed with respect to

antimicrobial activity for lemongrass stem; plants harvested in November showed a higher

activity (P < 0.0001) than plants which were harvested in October. Minimum inhibitory

concentration (MIC) and Minimum bactericidal concentration (MBC) range for lemongrass stem

was 0.8 17.4 mg/mL.

Water extracts of turmeric did not show any antimicrobial activity against any test bacterial

strains. Turmeric had a significantly lower (P < 0.0001) antimicrobial activity against

Staphylococcus aureus strains tested than that of lemongrass stem. However the antimicrobial

activity of turmeric was observed in extracts of the other solvents (acetone, hexane and ethanol)

than that of lemongrass (acetone and hexane). Inhibitory zone of turmeric extracts ranged from

6.5-11 mm.

Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC)

range for dried and fresh turmeric were 12.5 40.0 mg/mL. Little variation in antimicrobial

activity was observed for dried and fresh turmeric extracts.

Results of the study demonstrated that hexane and acetone extracts from lemongrass stem

exhibit antimicrobial activity against Staphylococcus aureus, and hexane, acetone, and ethanol

extracts of dried powder and fresh turmeric rhizomes, exhibited antimicrobial activity against









Staphylococcus aureus. Both lemongrass stem and turmeric extracts could be potentially used for

controlling Staphylococcus aureus.









CHAPTER 1
INTRODUCTION

Foodborne diseases are still a major problem in the United States. Many microorganisms

such as Salmonella spp, Listeria monocytogenes, Escherichia coli 0157:H7 and Staphylococcus

aureus are often reported as causative agents of foodborne diseases. Many of the pathogens of

greatest concern today (e.g., Campylobacterjejuni, Escherichia coli 0157:H7, Listeria

monocytogenes, Cyclospora cayetanensis) were not recognized as causes of foodborne illness 20

years ago (Mead and others 1999). Thus, at present, many interventions including chemical

preservatives must be used to kill or prevent the growth of foodborne pathogens as well as

spoilage microorganisms in the food industry (Sagdic and Ozcan 2003). Due to consumer

concerns about the safety of food containing synthetic chemicals as preservatives and the

increasing antibiotic resistance of bacterial foodborne pathogens, there is a growing interest in

the use of natural antibacterial compounds such as herb and spice extracts because they have

both characteristic flavor as well as a potential antimicrobial activity (Smid and Gorris 1999).

Many herbs, such as lemongrass and turmeric, have been considered medicinal plants which can

be potentially used for controlling foodborne pathogens in place of chemicals or antibiotics.

The word herb is derived from the Latin 'herba,' meaning grass or, by extension, green

crop (Gerald 1975). Herb was originally applied to a wide range of leafy vegetables. Herbs are

seed plants that do not produce woody stems like a tree and will live long enough to develop and

produce flowers and seeds (Gerald 1975). For thousands of years herbs have been used for their

flavor, medicinal, antimicrobial, and antioxidative properties. Use of spices and herbs in cooking

is the oldest form of aromatherapy. Active components of spices and herbs are considered as

powerful tools to create a state of wellness such as stimulate production of enzymes that detoxify

carcinogens, inhibit cholesterol synthesis, block estrogen, lower blood pressure, and prevent









blood clotting (Uhl 2000). Early cultures recognized that certain herbs had healing powers;

therefore, some herbs were thought to have magical properties. In the ancient civilizations,

people began to associate less magic with the treatment of diseases. They understood that illness

was natural and not supernatural, and medicine should be given without magic. Chinese

herbalism is widely regarded as the oldest because it has the longest unbroken recorded history.

Chinese have practiced herbal use for 5000 years.

Early use of antimicrobial plant extracts (i.e. herbs and oils) was well documented in

ancient Egypt. Ancient Egyptians used plant extracts for food preservation as well as for

embalming the dead. Pliny, Virgil, and Hippocrates mentioned garlic as a treatment for a variety

of ailments, including indigestion, pneumonia, wounds, and infections (Conner 1993). Although

ancient civilizations recognized the antiseptic or antimicrobial potential of many plant extracts, it

was only in the eighteenth century that the scientific documentation of the preservative effects of

spices and spice-type extracts were developed or recorded (Conner 1993).

Many plant tissues contain a variety of compounds called "secondary" plant compounds

metabolitess) grouped as glucosides, saponins, tannins, alkaloids, essential oils, organic acids and

others (Fraenkel 1959). Phytoalexins another group of compounds of low molecular weight are

typically synthesized in response to environmental stress. Many of these phytoalexins have both

antimicrobial and antifungal properties. They are produced after infection as a defense

mechanism against microbial infection. These compounds interfere with the cell membrane

associated functions (Beuchat and Golden 1989). Major antimicrobial components of spices are

in their essential oils (Shibasaki 1982). Each compound in spices and herbs exhibits different

biological activity (Duke 1994; Webb 1997).









Amount of spices and herbs extracts used in food systems range from 0.05 to 0.1% (500 to

1000 ppm) (Salzer 1982). Billing and Sherman (1998) calculated that meat recipes contained

roughly 0.25-3.0 g/kg of spices (250-3000 ppm). Although many essential oils from spices and

herbs have antimicrobial activity against bacteria at less than 1000 ppm, some spices require

higher concentrations to exhibit antimicrobial activity (Zaika 1988). Antimicrobial activity of

herbs and spices varies widely, depending on the type of spice or herb, test medium, and

microorganism. Zaika (1988) summarized the factors that could affect the antimicrobial activity

of herbs and spices. Microorganisms differ in their resistance to a given spice or herb, and a

given microorganism differs in its resistance to many types of spices and herbs. In general,

bacteria are more resistant to antimicrobials than fungi. Furthermore, antimicrobial action on

spores may be different from that of vegetative cells. Gram-negative bacteria are more resistant

than Gram-positive bacteria. Effect of a spice or herb may either be inhibitory or germicidal.

Spices and herbs harbor microbial contaminants, and may serve as substrates for microbial

growth and toxin production. Generally, the amounts of spices and herbs added to foods are

generally too low to prevent spoilage by microorganisms. Active components of spices/herbs at

low concentrations may interact synergistically with other factors (NaC1, acids, and

preservatives) to increase preservation. Nutrients present in spices/herbs may stimulate growth

and/or biochemical activities of microorganisms. Lastly, the extent to which spices and herbs

inhibit microbial growth depends on their source and processing.

For these reasons, effectiveness of the antimicrobial properties of herbs and spice might be

questionable considering the concentration at which they are used in the food systems.

Therefore, the objective of this study was to systematically evaluate the antimicrobial activity of

Asian herbs (lemongrass [Cymbopogon citratus (C. Nees) Stapf (Poaceae)] and turmeric









[Cucuma long (Zingiberaceae)]) using different extraction solvents (water, ethanol, hexane and

acetone) and drying methods (oven and freeze drying). Lemongrass extracts, from different

sources, were evaluated to compare results. Antimicrobial activity of commercial (dried powder)

turmeric was also evaluated. Hypothesis was that Asian herbs may have some antimicrobial

properties that may impact growth and survival of microbial pathogens in Asian foods. In order

to validate this hypothesis, Asian herbs were systematically evaluated for antimicrobial

properties using a non-polar solvent, slightly polar and highly polar solvents. Results of the study

could potentially be used in controlling some pathogens by using some Asian herbs.









CHAPTER 2
LITERATURE REVIEW

More than 200 known diseases are transmitted through food (Bryan 1982). In the United

States, foodborne diseases have been estimated to cause 6 million to 81 million illnesses and up

to 9,000 deaths each year (Archer and Kvenberg 1985; Todd 1989). Mead and others (1999)

estimated food-related illnesses, hospitalizations, and deaths from known pathogens to be

13,814, 924; 60, 854; and 1,809, respectively. Bacteria, parasites, and viruses accounted for 72%,

21%, and 7%, respectively including Salmonella (31%), Listeria (28%), Toxoplasma (21%),

Norwalk-like viruses (7%), Campylobacter (5%), and Escherichia coli 0157:H7 (3%). Bennet

and others (1987) estimated that there are 2,100; 1,200; and 1,000 deaths each year due to

campylobacteriosis, staphylococcal food poisoning, and trichinosis, respectively; however, Mead

and others (1999) had a total estimate for all three diseases as 101 deaths. Severe illnesses caused

by Salmonella and Campylobacter accounted for 26% and 17% hospitalizations, respectively.

Bacteria causing the most reported foodborne outbreaks from 1983 to 1997 include Salmonella

(3,640), .\/nge//l spp (1,476), Clostridium perfringens (654), Escherichia coli 0157:H7 (500),

Staphylococcus aureus (487), Escherichia coli, enterotoxigenic (209), and Campylobacter spp

(146) (Mead and others 1999). Table 2-1 gives reported and estimated cases, number of

outbreaks, and deaths caused by known foodborne bacterial pathogens (Mead and others 1999).

Table 2-2 summarizes the pathogens most commonly implicated in foodborne illnesses in the

United States. It is important to note that these numbers tend to under estimate the actual

numbers because foodborne illnesses are often, not reported.

Even though the United States has one of the safest food supplies in the world, there are

still millions of cases of foodborne illnesses each year, and this may relate to the diversity of

ethnic foods (Beattie 2006). Simonne and Others (2004) examined the CDC foodborne illness









data (1990-2000) and they revealed that Asian foods had few numbers of foodborne illness

outbreaks compared to other ethnic foods. In all, only 79 total outbreaks were reported in Asian

foods compared to Italian and Mexican foods, which accounted for 152 and 147 outbreaks,

respectively. Asian foods had a different profile of microorganisms, which included Vibrio

parahaelymoticus, associated with Asian seafood dish. Major microorganism involved in the

outbreaks in Asian foods between 1990-2000 was Bacillus cereus (50%). Difference in the

profile of microorganisms may be attributed to the ingredients, cooking, and preparation

methods, and how food is served (Simonne and others 2004). Asian food usually contains many

herb ingredients. Among other factors, the lower foodborne outbreak incident in Asian foods

could be due to the spices and herbs that are used in this cuisine. Most distinctive feature of

Asian cuisines is the blending of seasonings either fresh herbs, spices or a host of unusual

aromatics to produce some of the world's most flavorful food (Hutton 2003). For centuries,

people from different cultures around the world have embraced herbs, not only for their flavors

and aromas in their cuisines but also for the medicinal qualities of those herbs. In Thailand,

cooks love to throw cupfuls of basil (Ocimum basilicum) into their stir-fry and soups, treating

them no differently than vegetables. In Laos and Cambodia, herbs are used to garnish and flavor

noodle soups, broth soups, and salads (Pham 2001).

Most Common Asian Herbs and Spices

Most common herbs used in Asian dishes include but are not limited to lemongrass

(Cymbopogon citratus), turmeric (Curcuma longa, coriander (Coriandrum sativum), Chinese

chives (Allium ramosum-L), Thai basil (Ocimum basilicum), cress (Lepidium sativum L), chili

pepper (Capsicum frutescens), piper Chaba (Piper sarmentosum), and finger root (Boesenbergia

pandurata) (Table 2-3). These herbs add a unique flavor to many Asian dishes (Dudman 2005).

For this study, lemongrass (Cymbopogon citratus) and turmeric (Curcuma long) were used for









the evaluation of their antimicrobial properties. They are also used in main Asian dishes to create

different flavors and color in these cuisines.

Lemongrass

Lemongrass (also known as citronella, fever grass, serai, sereh, takrai) is a perennial herb

widely cultivated in the tropics and subtropics with two different designated species, East Indian,

Cymbopogonflexuosus and West Indian, Cymbopogon citratus (Simon and others 1984). West

Indian lemongrass (Cymbopogon citratus) consists of many organic compounds such as

terpenoids, but the major component is citral. Other terpenoids in this species include nerol,

limonene, linaloale, 0-caryophyllene, and a very low content of myrcene (Kasumov and Babaev,

1983). East Indian lemongrass (Cymbopogonflexuosus) consists of alcohols (20 to 30%

citronellol, geraniol) and aldehydes (15% geranial, 10% neral, 5% citronellal). This species of

lemongrass is dominantly used in the perfume industry and has a longer shelf life (Kasumov

and Babaev 1983).

Lemongrass, in general, grows in dense clumps up to 2 meters in diameter and has leaves

up to one meter long. Lemongrass is a very pungent herb and is normally used in small amounts.

Entire stalk of the grass can be used. Lemongrass is widely used in herbal teas, non-alcoholic

beverages, baked goods, and confectionary products. Oil from lemongrass is widely used as a

fragrance in perfumes and cosmetics products such as soaps and creams. Citral, (Figure 2-1),

extracted from lemongrass, is used in flavoring soft drinks, in scenting soaps and detergents, as a

fragrance in perfumes and cosmetics, and as a mask for disagreeable odors in several industrial

products (Simon and others 1984). Citral is a mixture of two geometric isomers of compounds in

lemongrass oil. Geranial is the trans isomer of citral in lemongrass, which accounts for 40-62%,

and neral is the cis isomer of citral in lemongrass, which accounts for 25-38% (Simon and others

1984).









Use of fresh lemongrass is typical for Southeast Asia and Sri Lanka. Lemongrass is most

popular in Thailand, Vietnam, Cambodia and Indonesia. In Thailand, finely ground fresh

lemongrass is added to curry pastes. Its fine fragrance goes well with poultry, fish and sea food.

In Vietnamese cooking, being much less spicy, makes use of lemongrass in several ways

including Vietnamese curries. Lemongrass is added to a popular Vietnamese meal bo nhung dam

[bo nhung dam], often translated "vinegar beef" or "Vietnamese fondue". Lemongrass plant is

generally recognized as safe (GRAS) for human consumption and as a plant extract/essential oil

(21 CFR section 182.20) (Simon and others 1984).

Turmeric

Curcuma long (Zingiberaceae) is a native plant of southern Asia and is cultivated

extensively throughout the warmer parts of the world. It is a member of the ginger family,

derived from an old Arabic name for the kurkum plant known as saffron (Crocus sativus L.).

It is the rhizome of turmeric plant that is used as a spice, usually in the dried form.

However, in some areas of the Far East, fresh turmeric is used and stored much like ginger

(Pulford 2003). Turmeric is used as an herb in Asian cooking such as curry dishes. It can also be

added to chutneys, pickles and mustards for its color. Rhizomes contain 3-4% volatile oil with

unique aromatic characteristics. Curcumin is the main biologically active phytochemical

compound of turmeric (Figure 2-2). Curcumin is the principal curcuminoid of turmeric.

Chemical formula of curcumin is C12H2006. It has a molecular weight of 368.38 g/mol. These

curcuminoids are responsible for the yellow color of the root. In fact, it is the curcuminoids that

posses all the bio-protective properties in turmeric (Badmaev and others 2004). Turmeric has

long been used as a powerful anti-inflammatory in both the Chinese and Indian systems of

medicine (Gescher and others 2005). Turmeric is also documented to have wound healing









capacity (Biswas and Mukherjee 2003). Turmeric is taken in some Asian countries as a dietary

supplement, which allegedly helps with stomach problems and other ailments (Pulford 2003).

Due to negative consumer perceptions of artificial preservatives in foods, attention of the

scientific community worldwide is shifting towards spices and herbs to harness their

antimicrobial properties for use as natural food preservatives (Sagdic and Ozcan 2003). Large

variety of these including lemongrass and turmeric, have been examined for their inhibitory

action (antimicrobial activity) against the microorganisms responsible for food spoilage and

foodborne illnesses (Grag and Menon 2003). However, gaps of knowledge still exist. In this

study, lemongrass stem and leaves were evaluated separately; furthermore, dried and fresh

turmeric was also evaluated. In this study, several factors were considered when doing the

research; different sources (two local growers, for comparison purposes), different solvents

(hexane, acetone, ethanol and water), drying methods (oven drying and freeze drying) and

different bacterial pathogens (Gram positive and Gram negative). This was done to evaluate the

results obtained from all these different treatments and come up with the best combination that

could enhance the antimicrobial activity of lemongrass and turmeric extracts.

Antimicrobial Activities of Lemongrass (Cymbopogon citratus)

Kim and others (1995) evaluated the antimicrobial activity of citral (lemongrass oil),

carvacrol (thyme oil), geraniol (rose oil), and other oils against five foodborne pathogens; E. coli,

E. coli 0157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Vibrio vulnificus.

Carvacrol was to found to have the most inhibitory action on the bacteria tested, while citral and

geranial had potent antibacterial action against the five test bacteria. Antimicrobial activity of the

components was different for each bacterial culture and the response of these cultures varied

toward each component. Carvacrol (at 250 iL/mL), and citral and geraniol (at 500 iL/mL)

completely inhibited the growth of Salmonella Typhimurium. Lis-Balchin and Deans (1997)









studied 93 commercial essential oils against 20 Listeria monocytogenes strains. Lemongrass was

among the oils that exhibited antibacterial activity against all the Listeria strains tested. Same

essential oil had variable antibacterial activity against different strains of Listeria. Williams and

others (1998) evaluated essential oils with high antimicrobial properties for therapeutic use by

comparing the antimicrobial activity of the essential oils of Australian tea tree oil (Melaleuca

alternifolia), Australian lavender (Lavandula angustifolia), New Zealand manuka

(Leptospermum scoparium), lemongrass oil (Cymbopogon citratus), and eucalyptus oil

(Eucalyptus cinerea). Lemongrass oil had the highest antimicrobial activity (inhibition zones)

against Candida albicans (30 mm) and Stapyhlococcus aureus (38 mm). Baratta and others

(1998) evaluated antimicrobial and antioxidant properties of some commercial essential oils;

ylang-ylang (Cananga odorata), frankincense (Boswellia thurifera), lemongrass (Cymbopogon

citratus), marjoram (Marjorana hortensis), basil (Ocimum basilicum), rosemary (Rosmarinus

officinalis), cinnamon (Cinnamomum zeylanicum), and lemon (Citrus limon). Twenty-five

different genera of bacteria (spoilage and foodborne pathogens) and one fungal species

(Aspergillus niger) were tested in this study. These microorganisms included animal and plant

pathogens, food poisoning and spoilage bacteria and the spoilage fungus Aspergillus niger.

Volatile oils exhibited considerable inhibitory action against all the tested organisms. Oils also

demonstrated antioxidant capacities.

Hammer and others (1999) investigated 52 plant oils and extracts for activity against

Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans, Enterococcus

faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella

enterica subsp. enterica serotype Typhimurium, Serratia marcescens and Staphylococcus

aureus, using an agar dilution method. Lemongrass, oregano and bay inhibited all organisms at









concentrations of 2.0% (v/v). Twenty of the plant oils and extracts were investigated, using a

broth microdilution method, for activity against C. albicans, S. aureus and E. coli. Lowest

minimum inhibitory concentrations were 0.03% (v/v) for thyme oil against C. albicans and E.

coli and 0.008% (v/v) for vetiver oil against S. aureus. These results supported the notion that

plant essential oils and extracts may have a role as pharmaceuticals and preservatives.

Chao and others (2000) evaluated the inhibitory action of essential oils against a broad

spectrum of microorganisms including bacteria, yeast, molds, and two bacteriophages. Inhibitory

action of 45 essential oils on eight bacteria (four Gram positive and four Gram negative), two

fungi, and one yeast were examined using the disc assay method. All the oils that were tested

exhibited antimicrobial activity. However, several of the plant essential oils exhibited only weak

inhibition against several Gram positive bacteria. Gram negative bacteria were generally more

resistant than Gram positive bacteria to essential oil treatment with Pseudomonas aeruginosa

being the most resistant bacteria. Only cinnamon bark (Cinnamomum zeylanicum) and tea tree

(Melaleuca alternifolia) oils showed inhibitory action against all the test organisms and

bacteriophages. Coriander oil (Coriandrum sativum) highly inhibited Gram positive bacteria and

fungi. Lemongrass (Cymbopogonflexuosus) and Roman chamomile (Chamaemelum nobile) oils

showed a high degree of inhibition against both phage types, while some oils showed no

inhibition against either phage. Angelica (Angelica archangelica) and pine (Pinus sylvestris) oils

inhibited the bacteria, but had no effect on any fungi. Oils that exhibited high antimicrobial

properties and the broadest range of inhibition included cinnamon bark (Cinnamomum

zeylanicum), lemongrass (Cymbopogonflexuosus), savory (Satureja montana), Roman

chamomile (Chamaemelum nobile), rosewood (Aniba rosaeodora), spearmint (Mentha spicata)









and tea tree (Melaleuca alternifolia). Results of the study showed that lemongrass had a high

antimicrobial activity.

Mejlholm and Dalgaard (2002) evaluated the antimicrobial action of nine essential oils

(EO) on P. phosphoreum and also determined the action of oregano oil on the shelf-life of

modified atmosphere-packed (MAP) cod fillets. Oils of oregano and cinnamon had strongest

antimicrobial activity, followed by lemongrass, thyme, clove, bay, marjoram, sage, and basil oils.

Results of the study suggested that herbs essential oils, such as lemongrass, may be used in

modified atmosphere-packaged foods to reduce P. phosphoreum.

Siripongvutikorn and others (2005) evaluated the antimicrobial and antioxidant action of

Thai seasoning, Tom-Yum. Garlic, shallot, kaffir lime leaves, chili, galangal, and lemongrass are

the spices used in Tom-Yum. These spices were extracted using water. Fresh garlic had the

highest antimicrobial properties of the spices examined in this study. Shallot, lemongrass,

galangal, and chili had no antimicrobial action. This could be because only water was used for

extracting the herbs. Water only extracts the water soluble component and leaves other important

substances, which could be responsible for the antimicrobial activity of the plants.

Sacchetti and others (2005) did a comparative evaluation of 11 selected essential oils,

namely, ylang-ylang (Cananga odorata), Italian Cypress (Cupressus sempervirens), turmeric

(Curcuma longa, lemongrass (Cymbopogon citratus), Tasmanian blue gum (Eucalyptus

globules), Monterey pine (Pinus radiata), Piper crassinervium (no common name), guava

(Psidium guayava), rosemary (Rosmarinus officinalis), lemon thyme (Thymus x citriodorus), and

ginger (Zingiber officinale). Among the essential oils tested, lemongrass oil was the most

effective against five food-spoilage yeasts: Candida albicans ATCC 48274, Rhodotorula glutinis

ATCC 16740, Schizosaccharomyces pombe ATCC 60232, Saccharomyces cerevisiae ATCC









2365, and Yarrowia lypolitica ATCC 16617. This study demonstrated that not only lemongrass

has antimicrobial properties but also antifungal properties.

Raybaudi-Massilia and others (2006) evaluated the antimicrobial activity of essential oils

(lemongrass, girasol, and cinnamon) on Salmonella Enteritidis, Escherichia coli, and Listeria

innocula in fruit juices. Lemongrass, cinnamon, and girasol at concentration of 2 [tL/mL

inactivated the pathogens in apple and pear juice. However in melon juice and tryptone soy broth

medium, concentrations of 8, and 10 [tL/mL for cinnamon, were needed respectively. For

girasol, 6 itL/mL was necessary to eliminate the three microorganisms; whereas lemongrass

required only 5 itL/mL to inactivate them. Results of the study showed that lemongrass essential

oil had strong antimicrobial activity than cinnamon and girasol essential oils.

Kakuta and others (2006) tested the antimicrobial action of essential oils on four oral

pathogenic microorganisms. Pure essential oils; tea tree (Camellia sinensis), lemon

(Citrus limonium), peppermint (Mentha Xpiperita), sonnerat (Ravensara aromatic), lemongrass

(Cymbopogon citratus), lemon eucalyptus (Eucalyptus citriodora) and geranium bourbon

(Pelargonium graveolens), and one blended oil (consisted of tea tree, lemon and peppermint oils)

were tested against Porphyromonas gingivalis, Fusobacterium nuclatum, Streptococcus mutans

and Candida albicans. Minimum inhibitory concentration (MIC) of essential oils was measured

by liquid dilution assay, and the minimum bactericidal concentration (MBC) of essential oils was

measured by colony forming assay. Results indicated that MIC of lemongrass oil was the lowest

among the eight essential oils, concentration ranging from 0.26 to 0.84% (v/v). Minimum

bactericidal concentration of lemongrass oil was also the lowest with concentrations ranging

from 0.31 to 1.25% (v/v). On the other hand, Ravensara aromatica oil showed the highest MIC









(more than 5%) among the eight essential oils. Blended essential oil showed a lower MBC than

any of the pure essential oils, against all test microorganisms except F. nuclatum.

Schwiertz and others (2006) evaluated antibacterial and antifungal activity often essential

oils against a range of vaginal bacterial and fungal strains isolated from existing vaginal

infections including Atopobium vaginae, Gardnerella vaginalis, Bacteroides vulgatus,

Streptococcus agalactiae, H202-producing lactobacilli and non H202-producing lactobacilli,

Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. Results

revealed that lemongrass, tea tree, and lavender exhibited the lowest minimum inhibitory

concentration (MIC) and minimum bactericidal concentration (MBC) at 1-2.5 tL/mL, thus being

the most potent essential oils against the tested bacteria. Interestingly, the MIC and MBC values

of palmarosa, neroli, manuka, rose-scented geranium, rosemary, common thyme and clary sage

were at >7.5 [tL/mL for protective H202-lactobacilli but lower for pathogenic bacteria. Overall,

lemongrass, palmarosa, lavender, and rose scented geranium were the most potent oils in the

inhibition of pathogenic bacteria and fungi.

Oussalah and others (2006) evaluated the antimicrobial action of selected plant essential

oils on the growth of Pseudomonasputida strain isolated from meat. Inhibitory action on 60

plants was examined. Results of the study showed that some plants have strong antimicrobial

activity while others had less antimicrobial activity. Lemongrass [stem (bulb) and leaf used

together] presented an MIC (0.8%) against Pseudomonas. Rojas-Grau and others (2006)

evaluated the mechanical barrier and antimicrobial properties of apple puree edible films

containing plant essential oils against Escherichia coli 0157:H7. Effect of oregano, cinnamon,

and lemongrass in apple puree containing film was investigated along with mechanical and

physical properties of the film. Results indicated that the order of antimicrobial activities was:









oregano oil > lemongrass oil > cinnamon oil. Rojas-Grau and others (2007) evaluated the action

of plant essential oil compounds on mechanical, barrier and antimicrobial properties of alginate-

apple puree edible films against the foodborne pathogen Escherichia coli 0157:H7. Results

indicated that the antimicrobial activities were in the following order: carvacrol > oregano oil >

citral > lemongrass oil > cinnamaldehyde > cinnamon oil. Results of the two studies above

indicated that lemongrass essential oil had considerable amount of antimicrobial activity. Results

also showed that citral, a main component of lemongrass, had stronger antimicrobial activity

than the lemongrass essential oil.

Akin-Osanaiye and others (2007) studied the antimicrobial activity of essential oil and

extracts of lemongrass and two other herbs. Freeze dried water extracts were used. Results of the

study showed that lemongrass oil had high antimicrobial activity against all microorganisms

tested (Salmonella typhi, Staphylococcus aureus and Escherichia coli). Maizura and others

(2007) evaluated the antimicrobial activity and mechanical properties of partially hydrolyzed

sago starch-alginate edible film containing lemongrass oil. Results indicated that the film

containing lemongrass was effective against Escherichia coli 0157:H7 at all levels [0.1-0.4%

(v/v)] based on the inhibition zone assay.

Kotzekidou and others (2008) studied the antimicrobial activity of plant extracts and

essential oils against foodborne pathogens in vitro and against the inoculated pathogens in

chocolate. Plant extracts and essential oils used extensively as flavor ingredients in confectionery

products were used as antimicrobials in laboratory media against the following microorganisms:

Escherichia coli 0157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus

aureus, Listeria monocytogenes, and Bacillus cereus. Using the disc diffusion method, inhibition

zones in diameter (> 20 mm) were observed by adding 10 tL of each antimicrobial substance on









the following microorganisms: lemon flavor applied on E. coli 0157:H7, lemongrass essences

against S. aureus, plum flavor on B. cereus strain, and strawberry flavor on L. monocytogenes

strain. E. coli 0157:H7 strains were the most susceptible microorganisms inhibited by 18

extracts, followed by S. Typhimurium and S. aureus, which were inhibited by 17 extracts.

Lemon flavor, lemongrass essences, pineapple, and strawberry flavor inhibited the foodborne

pathogens at the lowest concentration (5 mL/100 mL). Plant extracts and essential oils with

potent antimicrobial activities were tested in chocolate held at different temperatures (7 and

200C) in dry or humidified environment, which resulted in different aw values of the product (i.e.

0.340, 0.450, and 0.822), in order to determine their efficacy on the fate of the inoculated

pathogens. Most inhibition was observed in lemon flavor applied on chocolate inoculated with E.

coli cocktail culture after storage at 200C for 9 days. Plant extracts tested on chocolate showed

an enhanced inhibitory action during storage at 200C indicating that their application may

provide protection for storage at the above temperature or even higher.

Antimicrobial Activities of Turmeric (Curcuma long)

Huhtanen (1980) tested 33 spices against Clostridium botulinum, nutmeg, bay leaf, and

white and black pepper (125 ppm) were the most inhibitory while paprika, rosemary, cloves,

oregano, turmeric and thyme (500 ppm) only showed small inhibitory action against Clostridium

botulinum. Results of the study showed that turmeric had a weak antimicrobial activity against

Clostridium botulinum.

Packiyasothy and Kyle (2002) studied the antimicrobial properties of some herb essential

oils: sage, turmeric, thyme, mustard, and fenugreek. These essential oils exhibited antimicrobial

activity against Salmonella Typhimurium, Bacillus cereus, Escherichia coli, and Aspergillus

flavus. Turmeric exhibited a high potency for antimicrobial activity, while fenugreek gave a

weak antimicrobial activity. Turmeric exhibited a strong antibacterial activity at concentration of









20 mg/mL. Mandeel and others (2003) evaluated the antibacterial activity of seventeen selected

spices including turmeric. Ethanol extracts were evaluated against six Gram positive and Gram

negative bacteria using a well-diffusion assay. All the spice extracts, except black cardamom,

possess biological activity on one or more of the test bacteria. Clove extracts showed the highest

antimicrobial activity (19.5 mm) against Escherichia coli, followed by bay leaf extracts (19 mm)

against the same bacteria, and cumin extracts (19 mm) against Pseudomonas aeruginosa, at 1000

Gg/100 [iL. Galangal, turmeric, and fennel extracts also exhibited a broad spectrum of

antimicrobial activity. Results of the two studies above demonstrated that turmeric, essential oils

and extracts, at a high concentration were necessary to exhibit strong antimicrobial activity

against test organisms.

Goel (2005) evaluated the antimicrobial properties of turmeric. Turmeric showed

considerable amount of antimicrobial activity measured by a standard MIC assay. Pathogens

were inhibited at 20-100 [g/mL. Among the pathogens tested, no evidence of inhibition was

observed on Campylobacterjejuni for up to 100 tg/mL. Kim and others (2005) investigated the

antimicrobial activity of ethyl acetate, methanol and water extracts of Curcuma long L. (C.

long) against methicillin-resistant Staphylococcus aureus. Ethyl acetate extract of C. long

demonstrated highest antibacterial activity than that of methanol or water extracts. Results of the

study suggested that the ethyl acetate extract of C. long may have antibacterial activity.

Shivani and Ravishankar (2005) studied the antimicrobial action of garlic, ginger, carrot,

and turmeric pastes against Escherichia coli 0157:H7 in laboratory buffer and a model food

system. Turmeric paste, fresh carrot, ginger and garlic pastes from roots, and commercial ginger

and garlic paste were heated alone or with buffered peptone water (BPW) or ground beef at 700C

for 7 min. All samples were inoculated with a three-strain cocktail of overnight cultures of E.









coli 0157: H7 and stored at 40C and 80C for 2 weeks. Each paste exhibited different

antimicrobial actions alone or in ground beef or BPW at 40C and 80C for 2 weeks. Commercial

ginger paste and fresh garlic paste showed the strongest antimicrobial activity with complete

inhibition of E. coli 0157:H7 in the paste at 3 days fort 4 and 80C. Carrot and turmeric pastes did

not show any antimicrobial activity at either at 4 or 80C. Commercial garlic showed

antimicrobial activity at both 4 and 80C (about 1 log cfu/g reduction) in the paste. However,

fresh ginger paste showed antimicrobial activity only at 80C. Only commercial ginger paste had

antimicrobial activity in BPW at 40C for 2 weeks. However, commercial ginger paste showed

antimicrobial activity in ground beef at 3 days and after (about 1-2 log cfu/g) compared to

control samples at 80C for 2 weeks. Fresh garlic paste showed antimicrobial activity only in

BPW (1.3 log cfu/g) at 80C. These results indicated that the antimicrobial activity of these pastes

is decreased in ground beef and laboratory buffer. This might be because lipids in foods reduce

the antimicrobial properties of herbs.

Oonmetta-aree and others (2006) evaluated ethanol extracts of Zingiberaceae family

(galangal, ginger, turmeric and krachai) for antimicrobial action on Staphylococcus aureus 209P

and Escherichia coli NIHJ JC-2 by using agar disc diffusion method. Galangal extract had the

strongest inhibitory action against Staphylococcus aureus. Results showed that Staphylococcus

aureus (Gram positive bacteria) was more sensitive than Escherichia coli (Gram negative

bacteria). Turmeric showed less growth inhibition for both Staphylococcus aureus and

Escherichia coli. Galangal, krachai and ginger had no effect on Escherichia coli.

All results from the above studies show different antimicrobial activity when different

plants were used against many groups of microorganisms. Difference in results depends on

factors including extraction solvents, type of microorganism studied, plant parts, type of herbs









used, oils or plant extracts, and the concentration of the oil or extract. Therefore, this study

focused on a systematic evaluation of the antimicrobial activity of lemongrass and turmeric

extracts against the following bacteria strains (Salmonella serotypes, Staphylococcus aureus, and

Escherichia coli 0157:H7) using four extraction solvents (water, ethanol, hexane and acetone),

and two drying methods (freeze drying and oven drying).

Drying Methods

Many herbs and spices are often used in dried form. This is because drying preserves the

herbs and spices, prolonging their shelf life (Reynolds and Williams 1993). Drying also slows

down enzymatic reactions but does not eliminate them (Ozcan and others 2005). When the food

is ready for use, the water maybe added back and the food returns to its original shape. Foods can

be dried in the sun, in an oven or in a food dehydrator by using the right combination of

temperatures, low humidity and air current (Ho and others 2002).

Optimum temperature for drying food is 1400F (Reynolds and Williams 1993). If higher

temperatures are used, the food will be cooked instead of drying. When the food is cooked on the

outside and the moisture cannot escape, "case hardening" can occur. Food will eventually be

moldy. Lowering humidity aids the drying process and the water must move from the food to the

surrounding air. If the surrounding air is humid, then drying will be slowed down. Furthermore,

an increase of the air current speeds up the drying process by moving the surrounding moist air

away from the food. To reduce the drying time, the air flow should be increased.

Most foods can be dried indoors using modern food dehydrators, counter-top convection

ovens or conventional ovens. Microwave ovens are recommended only for drying herbs, because

there is no way to create enough air flow to dry denser foods (Venskutonis 1997). There are

many existing methods for drying foods including herbs and spices. Initially, salting and drying

in the sun, an open room or on stove tops were the accepted methods. It wasn't until 1795 that the









first dehydrator was introduced in France for drying fruits and vegetables. Today a variety of

dried foods in the marketplace has created a multimillion dollar industry (Hughes and others

1994). Currently oven-drying, freeze-drying, dehydrator-drying (solar and electric) and

microwave vacuum drying are among commonly used drying methods (Hughes and others

1994). This study employed oven-drying and freeze-drying methods to dry the lemongrass and

turmeric plants. These drying methods were selected based on different operating principles as

well as on the interest to establish the method that could better preserve the important

antimicrobial components of the plants.

Oven-Drying

By combining the factors of heat, low humidity and air current, an oven can be used as a

dehydrator. An oven is ideal for occasional drying of meat jerky, fruit leathers, and banana chips

or for preserving excess produce like celery or mushrooms (Reynolds and Williams 1993).

Principle of oven-drying consists of two distinct phases, an initial fast rate of moisture loss

followed by a slower second phase. Initially, when the food surface is wet, water evaporates

from the food forming a thin boundary layer of high-humidity air. Thickness of this layer

determines the rate of drying in the first phase of drying. Positive air movement over the fruit

surface reduces the thickness of the high-humidity layer, which increases the evaporation rate.

During the second phase of drying, the rate of moisture loss decreases. Second phase

begins when the rate of moisture movement to the surface of the food is less than the rate of

evaporation from the surface that is, the speed of drying is limited by the rate at which moisture

can move through the food tissue. Circulation can be improved by placing a fan outside the oven

near the door. Oven thermometer placed near the food gives an accurate reading. Temperature

dial should be adjusted to achieve the needed temperature of 1400F. Trays should be narrow

enough to clear the sides of the oven and should be 3 to 4 inches shorter than the oven from front









to back. Oven racks, holding the trays, should be 2 to 3 inches apart for air circulation (Ho and

others 2002).

Freeze-Drying

Freeze-dying is a technique that forms a vacuum while the food is freezing. Water in food

specimen is frozen and then removed by a process of sublimation, from solid to vapor phase,

maintaining the structure and chemical composition of the food. This avoids the use of chemical

dehydration and can be conveniently used to prepare bulk specimens. Boiling or hot-air

dehydration frequently impairs the chemical composition of a medicinal product. Freeze-drying

is frequently used in the commercial manufacture of antibiotics, vaccines, nutritional

supplements, and food products, because the technology employs very low temperatures and

high vacuum, and thus, it avoids overheating and perhaps deteriorating the organic product

(Brennan 1994).

Freeze-drying is based on a principle of quickly extracting humidity from a product with

a minimum of alteration of its molecular structure. If a product contains frozen water and is

exposed to a very low atmospheric pressure a virtual vacuum the water vaporizes directly. As

it does not go through the liquid state, steam requires a minimum of space to get out. If the

freezing process is done adequately, the structure of the organic matter is not altered. When a

food product is frozen very quickly, the water forms small ice crystals, and when heat is applied

in vacuum, the tiny water crystals are sublimated. Ice water crystals are converted directly to

steam, without breaking the molecular structure. (Brennan 1994).

Freeze-drying process is classically split into three stages: freezing, primary-drying, and

secondary-drying (Cameron 1997). Primary-drying is the removal of the free moisture that has

been frozen and the secondary-drying phase is the desorption of bound moisture. However, the









boundary between the primary and the free drying is not a clear-cut process. Freeze-drier

operates in a similar way during both stages of freeze drying (Cameron 1997).

There is a need for technology development in process monitoring, particularly in

developing a way to measure the status of the product during freezing and freeze-drying without

placing temperature measurement probes in individual vials of product. Current practice of

placing thermocouples in vials is uncertain with respect to reliability of the data, inconsistent

with elimination of personnel in close proximity to open vials of product in an aseptic

environment, and incompatible with technology for automatic material handling in freeze-drying.

In addition, a method for controlling the degree of super cooling during freezing would allow

better control of freezing rate and would, in many cases, result in more consistent product quality

(Nail and others 2002).

Botanical samples are often freeze-dried lyophilizedd) for use in research studies, and a

variety of freeze-dried botanicals are marketed to the public. In both instances, there is an

underlying assumption that freeze-drying properly preserves the medicinal qualities of plants,

and is superior to other preservation methods (Abascal and others 2005). In fact, little systematic

research has been done to verify this assumption. Review of the existing research, done primarily

by the food and spice industry, indicates that freeze-drying has unanticipated and significant

effects on the constituent profiles of medicinal plants that puts into question whether freeze-

drying necessarily is the best method to preserve botanical medicines. Lerici (1976) performed a

research study to investigate the retention of volatile organic compounds in a freeze-dried model

food gel and the data from the study suggested a theory about the different mechanisms, which

influence the retention of volatiles during freeze-drying of foods. Van Sumere and others (1983)

concluded that freeze-drying of biological material, which is to be quantitatively analyzed









(micro-amount level) for compounds of low or intermediate molecular weight, should be either

omitted or handled under a strict control. This is because compounds such as amino acids,

sugars, flavonoids, glycosides, coenzymes, peptides, etc., might be removed from concentrates

and (or) the ground biological material by the high vacuum. Study by Asami and others (2003),

aimed at comparing the total phenolic and ascorbic acid content of freeze-dried and air-dried

marionberry, strawberry, and corn grown using conventional, organic, and sustainable

agricultural practices, showed that freeze-drying preserved higher levels of total phenolics in

comparison with air-drying. Research review by Abascal and others (2005) found there is

insufficient information to conclude that freeze-drying has negative effects on the medicinal

qualities of plants. However, because existing research indicated that freeze-drying imperfectly

preserves important classes of medicinal compounds (such as volatiles, phenolics and

carotenoids), may increase the mutation rate in unicellular organisms, and may diminish some

medicinal plant actions, researchers and practitioners should carefully consider how the use of

freeze-dried material may affect pharmacological and clinical study results.

Several research studies on the effect of drying methods show different results depending

on several factors such as the type of food and the compounds of interest studied. In a research

aimed at studying the effect of drying on the volatile constituents of thyme and sage, two

methods were used to study the effect of drying on aroma constituents of the widely used herbs

thyme (Thymus vulgaris L.) and sage (Salvia officinalis L.). Volatile constituents of herbs (fresh,

freeze-dried and oven dried at 300C and 600C) were isolated by dynamic headspace and

simultaneous distillation-extraction methods and analyzed by capillary gas chromatography and

coupled gas chromatography-mass spectrometry. In total, 68 compounds were identified in

thyme and 44 in sage, and more than 100 components were screened quantitatively. Significant









reduction in the amount of extracted volatiles was found only in the case of oven drying at 600C,

mainly as a result of the loss of non-oxygenated monoterpenes. Character of the changes in the

headspace volatiles was more complex, especially for thyme, and the content of aroma

compounds were the highest when the herb was dried at 600C (Venskutonis 1997). Mahanon and

others (1999) evaluated the effect of oven drying at 500C for 9 hours, 70C for 5 hours and freeze

drying on retention of chlorophyll, riboflavin, niacin, ascorbic acid and carotenoids in herbal

preparations consisting of eight medicinal plants. Medicinal plants were leaves of Apium

graveolens (saderi), Averrhoa bilimbi (belimbing buluh), Centella asiatica (pegaga), Mentha

arvensis (pudina), Psidium guajava (jambu batu), Sauropus androgynous (cekor manis),

Solanum nigrum (terung meranti) and Polygonum minus (kesum). Results of the study revealed

that both type and conditions of the drying treatments affected retention of all phytochemicals

analyzed. Herbal preparations developed using oven-drying was found to have inferior

phytochemical content compared to those obtained by freeze-drying method. However, herbal

preparations developed using all treatments still retained appreciable amount of phytochemicals

studied especially carotenoids, ascorbic acid, niacin and riboflavin, and thus, have potential for

commercial purposes.

Julkunen-Tiitto and Sorsa (2001) conducted a study to test the effects of drying methods

on willow flavonoids, tannins, and salicylates. They compared the effects of several preservation

methods on the secondary phenols of the mature leaves of purple willow (Salix purpurea L.,

salicaceae) with those in fresh leaves. Conventional freeze-drying, in which the leaves were first

frozen with liquid nitrogen and then placed in a freeze-dryer, produced substantial qualitative

and quantitative changes on willow flavonoids and salicylates. Modified freeze-drying, in which

leaves were put into a freeze-dryer without being pre-frozen, gave comparable concentrations of









most secondary components in fresh leaves. Reducing the freeze-dryer chamber temperature

delayed the decomposition of phenolics in prefrozen leaves and in leaves dried without

prefreezing. Heat drying induced substantial changes in the composition of all phenolics, except

for apigenin-7glucoside. Vacuum drying at room temperature gave the highest concentrations for

nearly all phenolics, while room temperature-drying with desiccation gave results that were

comparable with those obtained by fresh leaf analyses. Effect of different drying treatments on

the volatiles in bay leaf (Laurus nobilis L.) was studied by Perez-Coello and others (2002). Four

drying treatments were employed: air-drying at ambient temperature, oven-drying at 450C,

freezing, and freeze-drying. Oven-drying at 450 C and air-drying at ambient temperature

produced quite similar results and caused very little loss in volatiles as compared to the fresh

herb; whereas, freezing and freeze-drying brought about substantial losses in bay leaf aroma and

led to increases in the concentration levels of certain components, e.g., eugenol, elemicin (3,4,5-

trimethoxyallylbenzene) and spathulenol.

Among the performed studies on the effects of drying methods, none showed the effects of

drying methods on the antimicrobial properties of herbs, thus this study seeks to evaluate oven

drying and freeze drying method in order to determine the drying method that results in a better

retention of the antimicrobial properties of lemongrass and turmeric plants.

Extraction Solvents

Solvents differ in their extraction capabilities depending on their own chemical properties

and the solute's chemical structures. Other factors affecting solvent selection are boiling point,

density, surface tension, viscosity, corrosiveness, flammability, toxicity, stability, compatibility

with product, availability, and cost (Cowan 1999). Many types of solvents are available for

extracting plant materials including water, ethanol, acetone, hexane, dimethyl sulfoxide, and

petroleum ether. Most widely used solvents are water and ethanol. Water is often referred to as a









universal solvent, (while ethanol is widely used in alcoholic beverages). For this study, water,

ethanol, hexane, and acetone were used. This is because all the solvents have different

properties; therefore they differ in the substances they extract. It is very important to utilize a

variety of solvents to be able to determine the one that gives the highest antimicrobial activity in

lemongrass and turmeric, as well as other herbs. Table 2-4 summarizes the properties of the

solvents that are discussed below.

Dimethyl Sulfoxide (C2H60S)

Dimethyl sulfoxide (DMSO) is an important polar solvent. It is less toxic than other

members of its class such as dimethylformamide. It is a colorless liquid with slight odor. It has

an excellent solvating power that dissolves both polar and non-polar compounds and is miscible

in a wide range of organic solvents as well as water. Main problem with DMSO as a solvent is its

high boiling point (1890C), thus its solutions are not typically evaporated but instead diluted to

isolate the reaction product (Cheremisinoff 2003).

Water (H20)

Water is referred to as the universal solvent, dissolving many types of substances.

Hydrophilic (water-loving) substances mix and dissolve well with water (Cowan 1999).

Properties and nature of water are due largely to the strong intramolecular hydrogen bonding

within the water molecule and intermolecular hydrogen bonding between water molecules. It

expands upon freezing. Water has a melting point of OC and a boiling point of 100C. Water is a

highly polar solvent with polarity index value of 9 (Cheremisinoff 2003). In the presence of

miscible organic solvents, water might display less polarity and hydrogen bonding character. In

the home, dried plants can be ingested as teas (plants steeped in hot water) or, rarely, tinctures

(plants in alcoholic solutions) or inhaled via steam from boiling suspensions of the plant parts.

Initial screenings of plants for possible antimicrobial activities typically begins by using crude









aqueous or alcohol extractions, and then followed by various organic solvent extraction methods

(Cowan 1999).

Ethanol (C2H60)

Ethanol, also known as ethyl alcohol, drinking alcohol or grain alcohol is a flammable,

colorless, slightly toxic chemical compound. Ethanol is best known as the alcohol found in

alcoholic beverages (Cheremisinoff 2003). In a common usage, ethanol is often referred to

simply as alcohol. Ethanol has a melting point of -114.30C (158.8 K) and a boiling point of

78.40C (351.6 K). It is volatile, and has a burning taste. Ethanol has no residual odor. Ethanol is

commonly used for dissolving medicines, food flavorings and coloring agents, which are not

water soluble. Ethanol can dissolve both polar and non-polar substances. Hydrophilic OH group

in ethanol helps dissolve both polar molecules and ionic substances (Scheflan and Jacobs 1953).

Hexane (C6H14)

Hexane is frequently used as an inert solvent in organic reactions because it is very non-

polar. Hexane is a colorless liquid with a melting point of -95C (178 K) and a boiling point of

69C (342 K). It is volatile and has a faint peculiar odor (Scheflan and Jacobs 1953). Among its

many uses, hexane is a solvent for extracting cooking oils (Cheremisinoff 2003).

Acetone (C3H60)

Acetone is the strongest solvent available. Acetone is a colorless, flammable liquid with a

freezing point of -94.60C and boiling point of 56.530C. It has a relative density of 0.819 (at

0C). It is readily soluble in water, ethanol, and ether. In laboratory, acetone is used as a polar

solvent in a variety of organic reactions (Scheflan and Jacobs 1953).

Effect of Extraction Solvents on the Antimicrobial Properties of Herbs

Very few studies have evaluated the ability of various solvents to solubilize antimicrobials

from plants. None of the studies have used water, acetone, hexane and ethanol together in one









study to determine a solvent that yields the highest antimicrobial activity of herbs. Therefore, this

study used all four solvents to determine the best solvent to extract antimicrobial compounds

from lemongrass and turmeric.

Elloff (1998) examined different solvents to determine which solvent should be used for

the screening and isolation of antimicrobial components from plants. Focus of this study was to

provide a more standardized extraction method for the wide variety of researchers working on

diverse settings. Freeze dried and finely ground leaves of two plants with known antimicrobial

activity, Aii4th,, leivia grandiflora and Combretum erythrophyllum were extracted with acetone,

ethanol, methanol, methylene dichloride, methanol/chloroform/water and water at a 1 to 10 (v/v)

ratio in each case. Acetone gave the best results with these plants followed by

methanol/chloroform/water, methylene dichloride, methanol, ethanol, and water. Afolayan and

Aliero (2006) examined a variety of solvents for their ability to solubilize antimicrobials from

plants. This study used acetone, methanol, and water for extraction. Results showed that acetone

and methanol extracts were active against the Gram positive and Gram negative bacteria at a

concentration of 5 mg/ml. Antimicrobial activities of acetone extracts were stronger on Gram

negative bacteria than on Gram positive bacteria, while the methanol extract displayed more

antimicrobial activity on Gram positive bacteria. There was little or no antimicrobial activity

from any water extracts. Rojas and others (2006) examined the antimicrobial activity of ten

medicinal plants used in Colombian folk medicine by using ethanol, hexane and water to extract

the plant materials. Results showed that the water extracts ofBidenspilosa L., Jacaranda

mimosifolia D.Don, and Piperpulchrum showed a higher antimicrobial activity against Bacillus

cereus and Escherichia coli than that of gentamycin sulfate (antibiotics). Similarly, the ethanol

extracts of most species were active against Staphylococcus aureus, except for Justicia secunda.









This means that different plants have different chemical components and some of which are

polar and other are non-polar compounds. Water extracts water soluble components (polar

components) while ethanol extracts both polar and non-polar substances. This means they will

produce different results on the antimicrobial properties of the plants based on the chemical

constituents of that particular plant.

Laohakunjit and others (2007) evaluated the antibacterial action of Zingiberaceae essential

oils using water, ethanol, and petroleum ether. Turmeric oil was among the oils that

demonstrated antibacterial activity. Ethanol extracts gave the highest antimicrobial activity

against the Gram positive bacteria. Gram negative bacteria were resistant to Turmeric oil (no

inhibition zone).

In this study; water, hexane, ethanol, and acetone were used for the extraction because they

have different properties (Table 2-4). This study was aimed to determine, which solvent yielded

the highest antimicrobial activity in lemongrass and turmeric plant extracts.

Evaporation Techniques

During the extraction of the plant materials, different solvents are used. These solvents are

then evaporated in a variety of ways. There are many types of evaporation techniques. These

include stationary evaporation and rotational type evaporation. Evaporation using a rotary

evaporator (rotavapor) is the most common method used to separate a solvent from plant

extracts. This study used the rotavapor evaporation technique because it evaporates solvents

efficiently without overheating the plant extracts.

Rotary Rotavapor

Rotary evaporator (rotavapor) is essentially an evaporation unit with a rotating evaporation

flask, which was invented by Buchi. Vacuum evaporation is the most frequently used method

because it is more efficient. Rotavapor will evaporate solvent at a much faster rate than the









stationary evaporation flasks. Its rotating flask produces a very efficient transfer of heat, whilst

ensuring thorough mixing and avoiding local overheating of the contents (Buchi 2008).

Rotation transfers a thin film of the liquid sample to the whole inner surface of the flask,

markedly increasing evaporation rate and assisting heat transfer from the heating bath. Rotating

flask and vapor duct have a sealing system, which allows operation under vacuum, further

accelerating the evaporation process because of the reduction in boiling point of the solvent and

efficient removal of the vapor phase. Vacuum operation also permits heat-labile materials to be

successfully concentrated without degradation (Buchi 2008).









Table 2-1. Reported and estimated cases, number of outbreaks and deaths caused by known
foodborne bacterial pathogens from 1983 to 1997.
Pathogens Reported Cases Outbreaks Deaths %
Salmonella spp 1,412,498 3,640 31
h/nge//I spp 448,240 1,476 0.8
Clostridium perfringens 248,520 654 0.4
Escherichia coli 0157:H7 73,480 500 3
Staphylococcus aureus 18,5060 487 0.1
Listeria monocytogenes 2,518 373 28
Escherichia coli enterotoxigenic 79,420 209 0
Campylobacter spp 2,453,926 146 5.5
This table demonstrates the reported and estimated cases, outbreaks and deaths caused by
known foodborne bacterial pathogens by Mead and others (1999). Bacteria are arranged
according to the number of reported and estimated outbreaks, starting with the bacteria with
the highest outbreak. Numbers of outbreak -related cases were calculated as the average
annual number of cases reported to CDC from 1983 to 1997.









Table 2-2. Most common pathogens associated with foodborne illness outbreaks


Reservoirs


Salmonella spp.








/nge//,t spp






Clostridium
perfringens





E. coli 0157:H7


Pathogens


Symptoms/Incubation period


Humans and domestic or wild
animals







Humans are the most significant
source. People may carry this
pathogen for several weeks and
excrete it in their feces.


The bacteria can be found in soil,
dust, sewage, and intestinal tracts of
animals and humans. The organism
grows in little or no oxygen.



Humans and animals


Associated foods

Meat, poultry, egg
or milk products







Ready to eat
foods: salads, raw
vegetables, dairy
products, and
poultry

Foods that have
experienced
temperature abuse
such as meat
dishes (gravies and
beef stew)

Hamburger and
other ground meat
products


Headache, abdominal pain, diarrhea, nausea,
vomiting, dehydration, fever and loss of appetite.
Symptoms appear within 6-72 hours after the
ingestion of the organism and may persist for as
long as 2-3 days.
Death is uncommon, except for the very young,
very old and the immuno-compromised.

This illness is usually characterized by diarrhea,
cramps and chills, often accompanied by fever.
Symptoms usually appear within 12-96 hours, but
can take as long as one week. Duration of illness is
usually 4-7 days.

Diarrhea and gas pains about 8 to 24 hours after
eating.
The illness usually lasts 1 day, but some symptoms
may last 1 to 2 weeks for the elderly or very
young.


Severe abdominal pain, cramps, nausea, vomiting,
diarrhea and occasionally fever.
Hemolytic Uremic Syndrome (HUS) is a serious
consequence of this disease and is the leading
cause of kidney failure in children.









Table 2-2. Continued
Pathogens Reservoirs


Staphylococcus
aureus










Listeria
monocytogenes


Humans are the most common
source, but cows, dogs and fowl also
can be a source.









The organism is frequently found in
environment such as soil, water and
plant matter that animals ingest and
excrete, allowing further
transmission.


Associated foods

Contaminated
ready-to-eat foods,
Custard- or cream-
filled baked goods,
ham, tongue,
poultry, dressing,
gravy, eggs, potato
salad, cream
sauces, sandwich
fillings

Ready to eta foods


Symptoms/Incubation period


Nausea, vomiting, diarrhea, dehydration, cramps,
subnormal temperatures and lowered blood
pressure. Symptoms appear within 30 minutes to 7
hours (2-4 hours is most common) after eating the
contaminated food, and may last for up to 24-48
hours.






Nausea, vomiting, headaches, delirium, coma,
collapse, shock and lesions on vital organs.
In pregnant women, the illness can cause a
miscarriage or result in stillbirths.
Listeriosis may also cause severe retardation,
meningitis and death in newborns.


Campylobacter Healthy chicken carry this bacteria contaminated Initial symptoms include fever, headache, and
jejuni in their intestinal tracts sometimes water, raw milk, muscle pain followed by diarrhea, abdominal pai
causing the contamination of raw and raw and and nausea. These symptoms may appear 2 to 5
poultry. Raw milk can also be a undercooked meat, days after eating and may last up to 7 to 10 days.
source; the bacteria are carried by poultry or shellfish
healthy cattle and by flies on farms.
Non chlorinated water may also be
a source for the infection.
This table gives a brief description of the bacterial pathogens mostly associated with foodborne outbreaks. This table outlines the
reservoirs, foods associated with pathogens and the symptoms/incubation period associated caused by bacterial pathogens.
Sources: Beattie Sam (2006), FDA Bad bug book (2007), CDC (2005)


in,









Table 2-3. Common Asian herbs used for cooking
Botanical Name Common name Parts used in cooking
Cymbopogon citratus Lemon grass stem, leaf
Poaceae or Gramineae
Curcuma long Turmeric Rhizome
Zingiberaceae, or the Ginger
family
Coriandrum sativum Corienda (Cilantro leaves) Leaves
Apiaceae or Umbelliferae
Allium ramosum L. Chinese chives Stem
Alliaceae
Ocimum basilicum Thai Basil Leaf
Lamiaceae or Labiatae
Lepidium sativum L. Cress Leaf
Brassicaceae
Capsicum frutescens Chili pepper Whole plant
Solanaceae
Piper sarmentosum Piper Chaba Leaf
Piperaceae
Boesenbergiapandurata Finger root Rhizome, finger root
Zingiberaceae
This table presents some of the Asian herbs used for cooking. It gives the botanical name,
common name and the different parts that are used in cooking.









Table 2-4. Properties of different solvents
Solvents Chemical Boiling Melting
formula point point
(C) (C)


Molecular
weight
(g/mol)


Dielectric
constant
(Debye)


Dipole Index
moment Polarity


Dimethyl C2H60S 189 18.45 78.14 47.2 3.96 7.2
Sulfoxide
Water H20 100 0 18.02 80 1.85 9
Ethanol C2H60 78.4 -114.3 46.07 24.3 1.69 5.2
Hexane C6H14 69 -95 86.18 2.02 ---- 0
Acetone C3H60 56.5 -94 58.08 20.7 2.88 5.1
This table above presents the different properties of dimethyl sulfoxide, water, ethanol, hexane
and acetone. Solvents are arranged according to their boiling point, starting from the highest to
the lowest boiling point.
















H30 0


Figure 2-1. Chemical structure of Citral (C10H160) (3, 7-dimethyl-2, 6-octadienal)








HO Curcumin OH
0 0

Figure 2-2. Chemical structure of Curcumin (C21H2o006)









CHAPTER 3
MATERIALS AND METHODS

Bacterial Strains and Cultivation

Three strains of E. coli 0157:H7 [204P, 301C, 505B] from Dr. Tun-Shi Haung (Auburn

University, Auburn, Ala), three strains of Salmonella species [Salmonella Thompson, Salmonella

Enteritidis, Salmonella Typhimurium], and three stains of Staphylococcus aureus [ATCC 29247,

ATCC 12600, and ATCC 35548] were used as tested organisms in all antibacterial assays. Both

Salmonella and Staphylococcus aureus strains were obtained from American Type Culture

Collection (ATCC). These organisms were selected because they are among many pathogens

often implicated in foodborne outbreaks in the United States. Different strains of bacteria were

streaked onto Tryptone Soy agar (TSA) (Becton, Dickinson and Company Sparks MD) to obtain

pure isolated colonies, following a standard aseptic technique and the four-way streak plate

inoculation (Cappuccino and Sherman 2005). Once the isolated colonies were obtained, the

bacterial strains were enumerated with Mueller Hinton Broth (MHB) for the next step of the

experiment, the antimicrobial assay.

Plant Materials

Lemongrass (Cymbopogon citratus (C. Nees) Stapf (Poaceae)) samples were obtained

from two local growers in Gainesville, FL. First harvest was in October 2007 and second harvest

was in November 2007. Figure 3-1 displays the lemongrass plant (Cymbopogon citratus)

Turmeric (Curcuma long) samples were obtained from a major produce market in

Atlanta, GA (DeKalb Farmer's Market). Two forms (fresh rhizomes and dried powder from) of

turmeric were obtained. Figure 3-2 displays fresh rhizome turmeric samples.









Preparation of Plant Material

Lemongrass (Cymbopogon citratus (C. Nees) Stapf (Poaceae)) leaf (Figure 3-3) and stem

(Figure 3-4) and turmeric (Curcuma long) were used for examining the antimicrobial activity

against all nine bacterial strains. Plant samples were washed and sanitized in a 6% sodium

hypochlorite solution (50 ppm), (Clorox company; Oakland, CA) (Ritenour and others 2002).

After washing, the plant materials were left in the biological hood (LABGARD Laminar Flow

Biological Safety Cabinet; nuAire, Inc) to get rid of excess water, and then they were thinly

sliced (approximately 25 mm) and flatly spread in oven trays (13 in x 9 in) and freeze drier

containers (melamine organizer; 15 in x 6 in x 2 in). Plant materials in ovens trays were dried in

an oven-drier (Scientific Fisher Isotemp oven Standard 600) set at 600C for 48 hours. For

samples to be freeze-dried, deionized water was added to the same level of the plants in the

freeze-drying containers and was first frozen in a regular freezer (Kenmore 19) set at -20C

overnight, and then taken to the freeze-drier (Model 25 SRC Virtis Company Gardiner, NY) for

72 hrs. After drying, the plants were grounded using a 12-speed blender (Hamilton Beach;

Michael Graves Design) for 5 minutes.

Preparation of Plant Extracts

Ten grams of dried ground plant material (weighed by Fisher Scientific scale) was added to

500 mL conical flasks, and 150 mL of each solvent (water, acetone, hexane, and ethanol) was

added to each flask. Ratio (1 to 15) of the plant materials and solvents was determined following

a standard method for evaluation of antimicrobial in foods (Parish and Davidson 1993). Each

mixture was placed on a shaker (Excella El platform, New Brunswick Scientific) and left to

extract for 18 hours at a speed of about 133 rpm at room temperature (25C). Extract was then

filtered using a conical flask with side arm, a filter funnel (size 2), and a 90 mm diameter filter

paper (Whatman #1). Filtered extract was then poured in a weighed 500 mL round bottom flask.









Solvent was evaporated with a rotary evaporator (Buchi). Temperature of the water bath in the

rotavapor was set at 400C. This temperature was used because the evaporation under reduced

pressure makes it possible to evaporate at much lower temperatures. Evaporation time ranged

from 10 to 30 minutes depending on the solvent type. After evaporation of solvent, the flask was

weighed again. To determine the weight of the sample, the weight of flask was subtracted from

the weight of the flask and sample. Dimethyl sulfoxide (DMSO) and water was used to

reconstitute the extracts in order to make a stock solution using volumetric flasks. Extracts were

then sterilized by filtration using 0.45im aqua membrane nylon filter disk (Becton, Dickinson

Company). Concentration was recorded on a weight by volume (w/v) basis. Stock solution was

stored it the freezer of a regular refrigerator (General Electric; Association of Home Appliance)

set at 200C. Working solutions were made from the stock solution. Concentration of the

working solution for lemongrass was 125 mg/mL, and 200 mg/mL for turmeric extracts.

Lemongrass has a strong flavor, and therefore, is being used in very small amounts to add flavor

in foods. Turmeric on the other hand is being used mainly to enhance color and to add a unique

taste to the food. Flavor of turmeric is not as strong as lemongrass, thus it is being used at

slightly high concentrations than lemongrass extract. Filtered and reconstituted extracts were

then prepared for the evaluation for antimicrobial properties against the nine bacterial strains.

This was done by taking 20 [tL of each plant extract and impregnating a 6 mm blank paper disc

(Becton, Dickinson Company) and leaving them in the biological hood (LABCONCO Purifier

Class II biosafety cabinet; Kansas, Missouri) to dry. Concentration of the plants in the discs was

2.5 mg/disc for lemongrass and 4 mg/disc for turmeric. Negative controls were prepared the

same way as the extract impregnated discs but, using 95% ethanol (number: 61511-0040;

ACROS, New Jersey), to make the discs sterile. Antibiotics (imipenem and ampicillin 10









.g/disc) (Becton, Dickinson and Company Sparks, MD) were used as positive reference

standards or control.

Antimicrobial Assay

Five isolated colonies of each bacterial strain was inoculated in 5 mL of Mueller Hinton

Broth (MHB) (Becton Dickinson Microbiology Systems) using a sterile wood stick. The 5 mL

tubes were then incubated at 370C for approximately 2 hours or until the bacterial density

reached 1.5x 108 cfu/mL (Casimiri and Burstein 1998). Bacterial density was determined by

using a spectrophotometer (Spectronic 20+ series, Thermo Electron Corporation) at wavelength

of 600 nm. Blank of MHB alone was used to calibrate the spectrophotometer. After calibrating

the spectrometer, the inoculated tubes were put in the spectrophotometer and the reading was

taken. When the bacterial density was at the desirable level, the reading was 1.32. When the

reading was below that level, the tubes were incubated a little longer; and when the tubes reading

was above that level, the bacterial suspension was adjusted by diluting the inoculated bacteria

with MHB until it reached the desirable level. When the bacterial density reached a desirable

level (1.5 x 108 cfu/mL), 100 [iL bacterial suspension for each bacterial strain were spread on to

the surface of the Mueller Hinton Agar (MHA) (Difco Becton, Dickinson and Company Sparks

DM) using sterile swabs (Casimiri and Burstein 1998).

The 6 mm diameter filter paper discs (containing 20 [iL of the particular plant extract)

were applied onto the surface of the spread MHA. Antibiotics; imipenem and ampicillin (10

[g/disc) (Becton, Dickinson and Company Sparks, MD) were used as positive reference

standards or control. Negative control (95% ethanol) was also used to make sure that the discs

were sterile. Plates were incubated at 370C for 24 hours and the inhibition zones were measured

to determine the effectiveness of the extract against each organism. Results were expressed as









the diameter (in millimeters) of inhibition for the paper disk (6 mm) with each extract (Figure 3-

5) (Parish and Davidson 1993).

Minimum Inhibitory Concentration (MIC) Determination

Minimum inhibitory concentration (MIC) is defined as the lowest concentration of an

antimicrobial that prevents the growth of microorganism after a specific incubation time. The

MIC was studied for the bacterial strains, being sensitive to the plant extracts in the disc

diffusion assay (Parish and Davidson 1993). The MIC was determined by using the 48 well

sterile plates (Costar Corning Incorporated, NY) which has a maximum volume capacity of 2

mL. For each well, 0.5 mL of MHB was added. Then the -prepared discs containing various

concentrations of plant extracts were added to achieve the desirable concentrations from the

highest to the lowest as described by previous literature (Duke and others, 2003). For

lemongrass, the concentrations used were 20, 10, 5, 2.5, 1.25, 0.625, 0.32, and 0.16 mg/mL. For

turmeric, the concentrations were 80, 40, 20, 10, 5, 2.5, and 1.25 mg/mL. Concentrations used

were based on previous research; lemongrass is said to be effective at lower concentrations than

turmeric (Duke and others, 2003). Bacterial innocula was prepared from MHB cultures and the

suspensions density were adjusted to standard turbidity (7.5 x 105 cfu/mL) (Casimiri and

Burstein 1998). Bacterial density was measured in the same manner as the antibacterial assay

using a spectrophotometer. When the bacterial innocula had reached the required density (7.5 x

105 cfu/mL), 5 [tL of the bacterial suspension was added to each well from the lowest to highest

extract concentration. Plates were then incubated at 370C for 24 hours. Color reaction with p-

iodonitrotetrazolium violet (Bio Medicals, Inc.) was used in lieu of traditional turbidity

measurement to eliminate difficulties of observing turbidity for each 48 wells. One hundred

micro liters of p-iodonitrotetrazolium violet (0.2 mg/mL) in water was added to each well in the

48 well plates and the plates were incubated for 30 minutes at 370C. Color changes were









observed thereafter. Wells showing red color indicated the bacterial growth (presence of

turbidity) and a well with the lowest concentration of plant extract without any red color was

determined as the minimum inhibitory concentration (MIC). Wells with no color change were

used for the determination of minimum bactericidal concentration (MBC).

Minimum Bactericidal Concentration (MBC) Determination

The MBC is defined as the lowest concentration of an antimicrobial agent needed to kill

99.9% of the initial inoculums. Minimum bactericidal concentration was determined on the

selected extracts in the 48 well plates that had the highest antimicrobial activities (i.e. those that

did not have any turbidity on the MIC determination) (Parish and Davidson 1993). Ten aIL from

each well that showed no bacterial growth was taken and inoculated in Tryptone Soy agar (TSA)

plates that were divided into quadrants. Plates were incubated at 370C for 24 hours, and then

bacterial growth was evaluated. Last quadrant (i.e. with the lowest concentration of plant extract)

showing no growth was taken as the MBC. Values were recorded as mg/mL.

Experimental Design and Statistical Analysis

A factorial design was used to design the experiment. Lemongrass factors were harvest

time x 2, trial x 2, pathogen x 3, solvent x 2, and drying method x 2. Fresh rhizome turmeric

factors were trial x 2, pathogen x 3, solvent x 3, and drying method x 2. Dried powder turmeric

factors were trial x 2, pathogen x 3, and solvent x 3. Microbial testing was done in triplicates for

each extract for each bacteria strain. General liner model procedure (PROG GLM) of the

Statistical Analyses System was employed and the significance among the different observed

antimicrobial activities were evaluated using Duncan's multiple range test (SAS Institute, Inc.

1991, Cary, NC) at a = 0.05 level. Data generated from the procedures described in the

experimental sections were tabulated and discussed in the next chapters.





























Figure 3-1. Lemongrass (Cymbopogon citratus) plants. Source:
http://www.paintedflowerfarm. cor


Figure 3-2. Turmeric (Curcuma long) rhizomes





























Figure 3-3. Lemongrass leaves


Figure 3-4. Lemongrass stem






























Figure 3-5. Inhibitory zone of lemongrass stem oven dried, hexane extracts on Staphylococcus
aureus ATCC 29247.









CHAPTER 4
RESULTS AND DISCUSSION

Antimicrobial Activity of Lemongrass

Inhibitory Zone of Lemongrass Extracts

Lemongrass leaves extracts (water, ethanol, acetone, and hexane) demonstrated no

antimicrobial activity (data not shown). This may be due to the fact that leaves of lemongrass are

exposed to the light. Although the lemongrass leaves contain citral, it is highly volatile and

instable to air, light and alkalis, thus citral hardly sustaining its activity in the leave part of the

lemongrass (United States Patent 2003; European Patent 2006).

Water and ethanol extracts of lemongrass stem (Table 4-1) did not show any antimicrobial

activity against any test bacterial strains. Neither water nor ethanol extract of lemongrass stem

yielded any antimicrobial activity (inhibition zone). Water is considered to have large dipole

molecules and a high dielectric constant. Thus, water is very polar and only miscible in itself.

However, the main compounds of the lemongrass are oily substances. Water has been used by

other researchers as an extract solvent, most of their results indicated that water extracts gave

little or no inhibitory effect (Siropngvutikom and others 2005; Afolayan and Allerozo 2006).

One important factor of solvent effectiveness is the active components of the plants. If most of

the compounds contained in the plants are miscible in water, then an antimicrobial activity can

be observed. According to a study by Rojas and others (2006), some of the water extracts of

some plants such as Bidenspilosa L., Jacaranda mimosifolia D. Don and Piperpulchrum had

antimicrobial activity, but others such as Justica secunda did not. This means based on the

compounds' miscibility in water, some will have antimicrobial action and some will not.

Unfortunately, in the home water is the major medium in food and drink preparation.









Ethanol is also classified as a polar solvent, although not very polar as water. This means

that this solvent is miscible in water and it will extract mostly the ionic compounds from

lemongrass. Ethanol has better dissolving capabilities than water because it has a slightly low

dipole and dielectric constant than water, thus it is slightly polar (Scheflan and Jacobs 1953).

Same reason that water did not have an effect as an extraction solvent could be the same reason

that ethanol was not effective since they are both said to be polar solvents (Cheremisinoff 2003).

These results from this study revealed that the benefit of antimicrobial properties from

lemongrass stem may not be achieved under a typical use of the lemongrass in water or ethanol

medium.

Hexane and acetone extracts of lemongrass stem showed antimicrobial activity against

Staphylococcus aureus strains (Table 4-1). Inhibitory zone of lemongrass stem extracts ranged

from 6.5 to 21 mm (also shown in Table 4-1).

Lemongrass samples prepared by the oven-dried methods yielded significantly higher (p <

0.0001) inhibition zone (Table 4-4) than those prepared by the freeze-dried method (with up to

21 mm inhibition zone). Results showed that the oven-dried lemongrass stem gave a better

overall antimicrobial activity (inhibition zone) than the freeze-dried lemongrass stem. These

results are in accordance with one study that demonstrated that oven-drying produced quite

similar results and caused hardly any loss in volatiles as compared to the fresh herb; whereas,

freezing and freeze-drying brought about substantial losses of aroma in bay leaf and led to

increases in the concentration levels of certain components (Perez-Coello and others 2002).

However, other studies showed that freeze drying method retains compounds better (Mahanon

and others 1999; Julkunen-Titto and Sorsa 2001). Results may vary depending on the type of

plant and the compounds of interest. There is an underlying assumption that freeze-drying









properly preserves the medicinal qualities of plants, and is superior to other drying preservation

methods (Abascal and others 2005). However, little systematic research has been done to verify

this assumption (Abascal and others 2005). Our results had proved that the assumption that

freeze-drying is better than other drying methods might not always be true.

There was no significant difference (P > 0.05) on the level of antimicrobial activity

(inhibitory zone) of the lemongrass stem against the three Staphylococcus aureus strains.

Lemongrass stem extracts showed a considerable amount of inhibition zone against

Staphylococcus aureus strains tested in this study. As citral is the main component of

lemongrass, some researchers have found that this compound is the one responsible for the

antimicrobial activity of lemongrass (Kim and others 2005; Rojas-Grau and others 2007; and

Onawunmi 1989). Results showed that Gram positive bacteria are more sensitive to turmeric and

lemongrass stem extracts. These results are in agreements with previous reports by Zaika (1988);

Consentino and others (1999), and Karaman and others (2003). Staphylococcus aureus strains

are considered the most sensitive bacteria (Oonmetta-aree and others 2006); whereas, Gram

negative bacteria showed resistance (i.e. no inhibition zone) towards the plant extracts. Varying

degrees of sensitivity of the test bacteria may be due to both the intrinsic tolerance of

microorganisms and the nature and combinations of phytochemical compounds present in the

essential oil portion of the plants. Outer cell membrane of Gram-negative organisms has several

lipid compounds that protect the cells from antimicrobial agents (Shelef and others 1980; Russel

1991) and thus, protecting them from the action of the essential oils.

Seasonal variation in lemongrass stem extracts was observed with respect to the

antimicrobial activity (inhibition zone); plants harvested in November showed a higher activity

(p < 0.0001) than plants harvested in October. Inhibition zone of lemongrass stems were by the









harvesting time. Lemongrass harvested in November showed a higher antimicrobial activity than

the lemongrass harvested in October. This is in accordance with previous research that a

composition of essential oils could be influenced by time of harvest (Senatore 1996).

There was no significant difference (P > 0.05) in the inhibition zone of hexane and acetone

extracts of lemongrass stem. Both hexane and acetone extracts yielded the same inhibition zone

(13.7 mm). Acetone and hexane extracts of lemongrass stem displayed a considerable amount of

antimicrobial activity. This could be because acetone is considered to be a dipolar aprotic

solvent. This means that acetone is of medium polarity; therefore it can dissolve a wide range of

compounds (Scheflan and Jacobs 1953). It has a slightly lower dielectric constant than ethanol,

which could aid in not only dissolving water soluble compounds but also other compounds,

which dissolve in other less polar solvents (Cheremisinoff 2003). For acetone, having an extra

carbon than ethanol adds to its capability to extract other compounds other than what ethanol can

extract. Hexane has low dielectric constants and is not miscible with water. Chemical formula of

hexane (Table 2-3) shows it is the least polar solvent. This means because hexane has 100%

hydrocarbons, it has the capability to extract most of the hydrocarbon compounds that are in

lemongrass or at least the most abundant compound of lemongrass, citral.

Result of the inhibition zone (Table 4-5) by pathogens show a same trend of

antimicrobial activity against the Staphylococcus aureus strains tested. Inhibition zone of the

Staphylococcus aureus strains showed that oven-drying had a significantly higher (P < 0.0001)

inhibition zone than freeze-drying. Inhibition zone of oven-dried lemongrass extracts against

Staphylococcus aureus strains: ATCC 35548, ATCC 12600-U, and ATCC 29247 were 15.3,

16.1, and 17.4 mm, respectively; while the inhibition zone of freeze-dried lemongrass extracts

against the same strains were 11.1, 12.1, and 10.3 mm, respectively. Similarly, the









Staphylococcus aureus strains were more sensitive to lemongrass stem extracts harvested in

November than those that were harvested in October. Inhibitory zone of November harvested

extracts against Staphylococcus aureus strains: ATCC 35548, ATCC 12600-U, and ATCC

29247 were 14.9, 16.0, and 14.6 mm, respectively; while the inhibition zone of October

harvested extracts against the same strains were 11.5, 12.2, and 13.1 mm, respectively. Inhibition

zone of hexane and acetone extracts of lemongrass stem show a different trend against the

Staphylococcus aureus strains that were tested. Staphylococcus aureus ATCC 12600-U was

more sensitive to hexane extracts with an inhibition zone of 14.9 mm, than to acetone extracts

which had an inhibition zone of 13.3 mm. Interestingly, the Staphylococcus aureus ATCC 29247

and 35548 were more sensitive to acetone extracts. Both had an inhibition zone of 13.9 mm,

while hexane extracts had an inhibition zone of 12.8 and 12.5 mm, respectively. Even though

there was a variation observed among the solvent extracts, they both seemed to exhibit a

reasonable amount of antimicrobial activity (inhibition zone).

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration
(MBC) of Lemongrass Stem Extracts

All other pathogens strains tested were not tested for MIC and MBC as they did not show

positive results on the antibacterial assay. The MIC and MBC values of lemongrass stem extracts

are presented in Tables 4-4 and 4-5. Results demonstrate a wide range of activities of the

different extracts against Staphylococcus aureus strains. Oven-dried lemongrass stem extracts

had lower MIC and MBC: 2.2 and 3.3 mg/mL, respectively than those of freeze-dried

lemongrass stem which had MIC and MBC of 3.1 and 7.1 mg/mL, respectively. Results

demonstrated that, overall, hexane extracts of lemongrass stem gave the lowest MIC (2.3

mg/mL) than acetone extracts (3.6 mg/mL), however there was no significant difference

demonstrated by the MBC between the hexane and acetone extracts in lemongrass stem. Hexane









and acetone extracts gave an MBC of 4.9 and 5.4 mg/ml, respectively. The MIC of lemongrass

stem did not demonstrate a statistical difference in the sensitivity of the Staphylococcus aureus

strains. Their sensitivity to the lemongrass stem was not statistically different; however for MBC

it was observed that the sensitivity of Staphylococcus aureus strain (ATCC 29247), was

statistically lower (p < 0.0001) MBC (4.2 mg/mL) than the two other Staphylococcus aureus

strains (ATCC 12600-U and 35548) which presented an MBC of 5.9 and 5.6 mg/mL

respectively. Results showed that the Staphylococcus aureus strain (ATCC 29247) may be more

sensitive to lemongrass stem extracts than the other Staphylococcus aureus strains used in the

study.

Results of the MIC and MBC of lemongrass stem extracts when analyzed against each

Staphylococcus aureus strain showed the same trend as the results discussed above. The MIC

and MBC of oven-dried lemongrass stem was significantly lower against all the Staphylococcus

aureus strains than MIC and MBC of freeze-dried lemongrass stem extracts. Oven-dried

lemongrass stem extracts presented an MIC and MBC of 1.9 and 3.9 mg/mL against both

Staphylococcus aureus strains: ATCC 12600-U and 35548; and 0.8 and 2.1 mg/mL against

Staphylococcus aureus strain (ATCC 29247). Freeze-dried lemongrass stem extracts presented

an MIC and MBC of 4.6 and 8.0 mg/mL; 4.4 and 6.3 mg/mL; and 4.4 and 7.1mg/mL, against

Staphylococcus aureus ATCC 1600-U, 29247, and 35548, respectively. These results show that

oven-dried lemongrass stem extracts had a stronger antimicrobial activity against all the

Staphylococcus aureus strains that were tested. Similar to the results obtained in the inhibition

zone, hexane extracts of lemongrass showed a lower MBC (5.2 mg/mL) against Staphylococcus

aureus ATCC 1600-U than acetone extracts which showed an MBC of 6.7 mg/mL. Both

Staphylococcus aureus ATCC 29247 and 35548 had a higher sensitivity to acetone extracts with









an MIC and MBC of 1.6 and 3.3 mg/mL; and 2.1 and 4.8 mg/mL, respectively, while hexane

extracts showed less sensitivity with a higher MIC and MBC of 3.5 and 5.0 mg/mL; and 4.0 and

6.2 mg/mL. Difference in the solvent extracts may be due to the previously suggested theory of

different mechanisms, which influence the retention of compounds during freeze drying (Lerici

1976).

Sensitivity of all the Staphylococcus aureus strains that were tested showed similar results

to those obtained in the inhibition zone assay. All strains were more sensitive to lemongrass stem

extracts from November harvest that those extracts from October harvest. Lower MIC and MBC

from the November harvest was expected because, as seen in the antimicrobial activity

(inhibition zone), antimicrobial properties of lemongrass were affected by the harvesting time in

accordance with previous research (Senatore 1996).

Antimicrobial Activity of Turmeric Extracts

Inhibitory Zone of Turmeric (Fresh Rhizomes and Dried Powder) Extracts

Water extracts of turmeric (prepared from fresh rhizomes) were observed to have no

antimicrobial activity against any of the test bacterial strains (Table 4-2). Water is very polar and

is only miscible in itself. This means that water cannot extract non-polar compounds in turmeric

Turmeric extracted by ethanol, acetone and hexane demonstrated antimicrobial activity

against Staphylococcus aureus strains (Table 4-2). This might be due to the difference in

chemical compounds found in lemongrass and turmeric. Differences were observed in their

chemical structures presented in Figure 2-1 and Figure 2-2. First figure, which shows the

chemical structure of citral, lacks the hydrogen (O-H) bond but contains a multiple bond between

the hydrocarbon and oxygen; and yet the chemical structure of curcumin contains two hydroxyl

groups and O-H bond. This means turmeric has polar components, which can be easily dissolved

in ethanol because ethanol also has a hydroxyl group. Another study showed that ethanol gave a









better antibacterial activity on turmeric plants compared to petroleum ether and water

(Laohakunjit and others 2007). Inhibitory zone of turmeric extracts against the Staphylococcus

aureus strains ranged from 6.5 mm 11 mm.

E. coli 0157:H7 and Salmonella strains were not affected by any turmeric extracts no

matter which solvent or drying method was used.

Staphylococcus aureus strain (ATCC 12600U) demonstrated a significantly higher

susceptibility (p < 0.0001) to turmeric extracts than the other Staphylococcus aureus strains

(Table 4-6). Turmeric extracts from samples prepared by freeze drying yielded a significantly

higher antimicrobial activity (inhibition zone) than those prepared from the oven drying method.

No significant difference (p> 0.05) was observed among different solvents (hexane, acetone and

ethanol).

Turmeric extracts (prepared from dried powder) demonstrated the same pattern as the

turmeric extracts from fresh rhizomes (Table 4-3). However a statistical difference was observed

in the Staphylococcus aureus strains' susceptibility to the turmeric (dried powder) extracts

(Table 4-8). Staphylococcus aureus ATCC 35548 was the most sensitive (P < 0.0001) to the

turmeric extract prepared from dried turmeric powder, followed by the Staphylococcus aureus

ATCC 12600, and Staphylococcus aureus ATCC 29247, respectively. Neither E. coli 0157:H7

or Salmonella test strains used in this study were sensitive to the dried powder turmeric extracts.

This is because Gram-negative bacteria are more resistant to antimicrobials than Gram-positive

bacteria (Shelef and others 1980; Russel 1991).

Acetone extracts of fresh turmeric rhizomes gave a significantly higher (P < 0.0001)

inhibition zone (8.0 mm) than the extracts prepared from hexane (8.0 mm), however acetone

extracts were not statistically different from inhibition zone of ethanol extracts (8.8 mm).









Acetone is a solvent of medium polarity and thus it can dissolve a wide range of compounds

(Scheflan and Jacobs 1953). Acetone dissolves water soluble compounds as well as other

compounds which dissolve in less polar solvents (Cheremisinoff 1953). Ethanol extracts of

turmeric also showed to have a higher inhibition zone. Results were in accordance with one

study which demonstrated that ethanol extracts of turmeric gave the highest antimicrobial

activity against Gram-positive bacteria (Laohakunjit and others 2007).

There was a statistical difference in the antimicrobial activity (inhibition zone) (p <0.

0001) between the turmeric extracts prepared from fresh rhizomes and the turmeric extracts

prepared from dried-powder (Table 4-11). Dried powder turmeric had a significantly higher

inhibition zone (8.6 mm) than fresh rhizome's turmeric (7.7 mm); however the observed

difference was small.

Inhibitory zone of turmeric (fresh rhizomes) extracts (7.7 mm) was significantly lower

(p<0.0001) than that of lemongrass stem (13.7 mm) extracts (Table 4-10). This is in accordance

with previous research, which showed that turmeric only gave low inhibition compared to other

plant extracts against Clostridium botulinum (Huhtanen 1980).

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration
(MBC) of Turmeric (Fresh Rhizomes and Dried Powder) Extracts

The MIC and MBC results of turmeric (prepared from fresh rhizomes) demonstrated no

significant difference (P > 0.05) in either solvents, drying methods or among the Staphylococcus

aureus strains. Results showed that all the solvent extracts and drying methods gave statistically

similar antimicrobial activity against Staphylococcus aureus strains. Sensitivity of the

Staphylococcus aureus strains to the fresh rhizome turmeric extracts was also similar. Very little

variations were observed (among trials, solvents and drying methods), demonstrating the









sensitivity of each Staphylococcus aureus strain to the fresh rhizome turmeric extracts (Table 4-

7).

Results of dried powder turmeric (Table 4-8) demonstrated that the MIC and MBC of

acetone extracts were significantly lower (P < 0.0001) than the extracts of ethanol and hexane.

Acetone extracts presented an MIC and MBC of 12.5 and 20.0 mg/mL, respectively, while the

MIC and MBC of ethanol and hexane were 19.2 and 33.0 mg/mL; and 26.6 and 40.0 mg/mL,

respectively. These results showed that acetone extracts of turmeric (dried powder) had a higher

antimicrobial activity than that of ethanol and hexane.

Turmeric extracts showed a higher antimicrobial activity against Staphylococcus aureus

ATCC 29247 with a low MIC of 18.3mg/mL. The MBC of turmeric (dried powder) extracts

against all the Staphylococcus aureus strains tested were not significantly different (P > 0.05).

Staphylococcus aureus ATCC 12600-U and 35548 both had an MBC of 31.7 mg/mL; and

Staphylococcus aureus ATCC 29247 had an MBC of 30.0 mg/mL. There was very little

variation observed (among trials and solvents) demonstrating the sensitivity of each

Staphylococcus aureus strain to the dried powder turmeric extracts (Table 4-9). Results showed

to be consistent with those presented in Table 4-8, which showed that acetone and ethanol

extracts had a higher antimicrobial activity than that of hexane extracts.

Lemongrass extracts had a significantly lower MIC and MBC than that of turmeric (Table

4-10). The MIC and MBC for lemongrass were 3.0 mg/mL and 5.2 mg/mL, respectively, yet for

turmeric; the MIC and MBC were observed to be 19.4 mg/mL and 29.3 mg/mL, respectively.

These results showed that the extracts of lemongrass stems were more powerful against

Staphylococcus aureus than turmeric extracts.









All the results on the MIC and MBC of lemongrass and turmeric are consistent with the

initial results showing the antimicrobial activity (inhibition zone) against the test pathogens. As

with inhibition zone, lemongrass was observed to have a lower MIC and MBC than turmeric

against Staphylococcus aureus strains. This was expected because the bigger the inhibition zones

the lower the MIC and MBC will be. These results are in accordance with most research studies

that have been done where the MIC and MBC of lemongrass are considerably lower ranging

from 0.16-20 iL/mL for most bacteria and 0.25- 10 iL/mL for most fungi (Duke and others

2003; Oussalah and others 2006; Raydaudi-Massilla and others 2006; Shwiertz and others 2006).

Results obtained in this study were in accordance with other research, which shows turmeric start

exhibiting antibacterial activity at 20 mg/mL (Packiyasothy and Kyle 2002). Another study

found that turmeric inhibited some pathogens at a wide range 20-100 [g/mL. This means that the

antimicrobial activity of herbs and spices varies widely, depending on the type of spice or herb,

test medium, and microorganism (Zaika 1988).










Table 4-1. Inhibitory zone (mm) of lemongrass stems extracts (solvent extracts) on bacterial pathogens
Solvents and Drying methods
Hexane Acetone Ethanol Water


Pathogens OD FD OD
Staphylococcus aureus ATCC
12600-U 19 3 12 2 14 4
Staphylococcus aureus ATCC
29247 18 3 10 2 14 3
Staphylococcus aureus ATCC
35548 18 3 8 1.5 15+2
Salmonella Typhimurium 0 0 0
Salmonella Thompson 0 0 0
Salmonella Enteritidis 0 0 0
E. coli 0157:H7 204P 0 0 0
E. coli 0157:H7 301C 0 0 0
E. coli 0157:H7 505B 0 0 0
Values presented are averages of replicates of each bacterial strain. OD


FD OD FD OD FD


10
+1 0
11
+1 0


0 0 0

0 0 0


2 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
- Oven dried; FD -
O O-


0
0
0
0
0
0
0
Freeze dried











Table 4-2. Inhibitory zone (mm) of turmeric (fresh rhizome) extracts (solvent extracts) on bacterial pathogens
Solvents and Drying methods
Hexane Acetone Ethanol Water
Pathogens OD FD OD FD OD FD OD FD


Staphylococcus aureus ATCC
12600-U
Staphylococcus aureus ATCC
29247
Staphylococcus aureus ATCC
35548
Salmonella Typhimurium
Salmonella Thompson
Salmonella Enteritidis
E. coli 0157:H7 204P
E. coli 157:H7 301C
E. coli 0157:H7 505B
Values presented are averages c


7 0.5 7 0.5 7 0.5 8 0.5


7.5 0.5 9 0.5

7 0.5 7 0.5
0 0


7 0.5 7.5 0.5 0


7 0.5 8.5 0.5 7 0.5 7 0.5

7 0.5 8.5 0.5 7 0.5 7 0.5
0 0 0 0


0 0


0 0


0 0 0 0 0 0 0 0
,f replicates of each bacterial strain. OD Oven dried; FD Freeze dried









Table 4-3. Inhibitory zone (mm)
bacterial pathogens


of turmeric (dried powder) extracts (solvent extracts) on


Solvents
Pathogens Hexane
Staphylococcus aureus ATCC
12600-U 8.5 0.5
Staphylococcus aureus ATCC
29247 7.5 0.5
Staphylococcus aureus ATCC
35548 8.5 + 0.5
Salmonella Typhimurium 0
Salmonella Thompson 0
Salmonella Enteritidis 0
E. coli 0157:H7 204P 0
E. coli 0157:H7 301C 0
E. coli 0157:H7 505B 0
Values presented are averages of replicates of each


Acetone Ethanol Water

8.5 0.5 9 0.5 0

9.5 0.5 8.5 0.5 0


9 0.5 9.5 0.5
0 0
0 0
0 0
0 0
0 0
0 0
bacterial strain.










Table 4-4. Overall comparison of solvents, drying methods, and harvest-time for lemongrass
stem.
Variables Lemongrass
MIC
Zone (mm) (mg/mL) MBC (mg/mL)
Harvest time
1 (October) 12.3 B 4.0 A 6.9 A
2 (November) 15.2 A 2.0 B 3.5 B
Pathogens
Staphylococcus aureus ATCC 12600-U 14.1 A 3.3 A 5.9 A
Staphylococcus aureus ATCC 29247 13.8 A 2.6 A 4.2 B
Staphylococcus aureus ATCC 35548 13.2 A 3.1 A 5.6 A
Solvent
Hexane 13.7 A 2.3 B 4.9 A
Acetone 13.7 A 3.6 A 5.4 A
Drying methods
Oven drying 16.3 A 2.2 B 3.3 B
Freeze drying 11.2 B 3.0 A 7.1 A
Values presented are averages of three replicates. Values in the same column within each
category following different uppercase letters designate a significant difference (Duncan's
multiple range test, P < 0.05).









Table 4-5. Sensitivity of each Staphylococcus aureus strain to lemongrass stem
Staphylococcus aureus ATCC Staphylococcus aureus ATCC
12600-U 29247 Staphylococcus aureus ATCC 35548
Zone MIC MBC Zone MBC MIC MBC
Variables (mm) (mg/mL) (mg/mL) (mm) MIC (mg/mL) (mg/mL) Zone (mm) (mg/mL) (mg/mL)


1 (October)
2
(November)

Acetone
Hexane


Oven
drying
SFreeze
drying


12.2 B 4.7 A

16.0A 1.9B

13.3 B 3.3 A
14.9 A 3.3 A



16.1 A 1.9B

12.1 B 4.6 A


8.1 A

3.7 B

6.7 A
5.2 B



3.9B

8.0 A


Harvest time
13.1 B 3.4 A

14.6 A 1.7 B
Solvents
13.9 A 1.6 B
12.8 B 3.5 A
Drying
methods

17.4 A 0.8 B

10.3 B 4.4 A


Values presented are averages of three replicates. Values in the same column within each category following different uppercase
letters designate a significant difference (Duncan's multiple range test, P < 0.05).


5.2 A

3.1B

3.3 B
5.0 A



2.1B

6.3 A


11.5B

14.9 A

13.9 A
12.5 B



15.3 A

11.1 B


3.8 A

2.4 B

2.1B
4.0 A



1.9B

4.3 A


7.3 A

3.7B

4.8 B
6.2 A



3.9B

7.1 A









Table 4-6. Antimicrobial activities of fresh rhizome turmeric extracts
Variables Zone (mm) MIC (mg/mL) MBC (mg/mL)
Pathogens
Staphylococcus aureus ATCC 12600-U 8.1 A 18. 5 A 29.2 A
Staphylococcus aureus ATCC 29247 7.4 B 20.3 A 30.0 A
Staphylococcus aureus ATCC 35548 7.5 B 19.3 A 28.6 A
Solvents
Hexane 7.5 A 19.3 A 30.6 A
Acetone 8.0 A 18.9 A 28.1 A
Ethanol 7.6 A 19.9 A 29.2 A
Drying methods
Oven drying 7.3 B 19.5 A 30.6 A
Freeze drying 8.0 A 19.3 A 28.0 A
Values presented are averages of three replicates. Values in the same column within each
category following different uppercase letters designate a significant difference (Duncan's
multiple range test, P < 0.05).









Table 4-7. Sensitivity of each Staphylococcus aureus strain to fresh rhizome turmeric extracts
Staphylococcus aureus ATCC Staphylococcus aureus ATCC Staphylococcus aureus ATCC
12600 29247 35548
Zone MIC MBC Zone MIC MBC Zone MIC MBC
Variables (mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL)


7.8B
8.4 A

8.0 AB
8.5 A
7.8 B


19.4 A
17.5 B

18.8 AB
17.1 B
19.6 A


32.2 A
26.1 B

30.8 A
25. OB
31.7A


7.7 B 18.9 A 31.1 A

8.5 A 18.1 A 27.2 B


7.1B
7.7 A

7.8 A
7.1B
7.3 B


Trial
21.7A
18.9B
Solvents
19.2 B
22.5 A
19.2 B
Drying
methods


31.7A
28.3 B

28.3 B
32.5 A
29.2 B


7.0 B 20.0 B 30.0 A

7.8 A 20.6 A 30.0 A


7.1B
7.8 A

8.1 A
7.3 B
7.1B


20.0 A
18.6B

18.8B
20.0 A
19.2 AB


29.4 A
27.8 B

25.0 B
30.0 A
30.8 A


7.2 B 19.4 A 30.6 A

7.8 A 19.2 A 26.7 B


Values presented are averages of three replicates. Values in the same column within each category following different uppercase
letters designate a significant difference (Duncan's multiple range test, P < 0.05).


1st Trial
2nd Trial

Acetone
Hexane
Ethanol


Oven
drying
S Freeze
drying









Table 4-8. Antimicrobial activities of dried powder turmeric extracts
Variables Zone (mm) MIC (mg/mL) MBC (mg/mL)
Pathogens
Staphylococcus aureus ATCC 12600-U 8.7 B 19.7 A 31.7 A
Staphylococcus aureus ATCC 29247 8.3 C 18.3 B 30.0 A
Staphylococcus aureus ATCC 35548 9.0 A 20.3 A 31.7 A
Solvents
Hexane 8.0 B 26.6 A 40.0 A
Acetone 9.1 A 12.5 C 20.0 C
Ethanol 8.8 A 19.2 B 33.0 B
Values presented are averages of three replicates. Values in the same column within each
category following different uppercase letters designate a significant difference (Duncan's
multiple range test, P < 0.05).









Table 4-9. Sensitivity of each Staphylococcus aureus strain to dried powder turmeric extracts
Staphylococcus aureus ATCC Staphylococcus aureus ATCC Staphylococcus aureus ATCC
12600 29247 35548
Zone MIC MBC Zone MIC MBC Zone MIC MBC
Variables (mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL) (mm) (mg/mL) (mg/mL)
Trial
1st Trial 8.5 B 20.6 A 33.3 A 8.0 B 20.0 A 30.0 A 8.8 B 20.6 A 33.3 A
2nd Trial 8.8 A 18.9 A 30.0 B 8.6 A 16.7 B 30.0 A 9.2 A 20.0 B 30.0 B
Solvents
Acetone 8.8 A 20.0 B 20.0 C 9.3 A 17.5 B 20.0 C 9.3 A 20.0 B 20.0 C
Hexane 8.3 A 26.7 A 40.0 A 7.2 C 25.0 A 40.0 A 8.6 B 28.3 A 40.0 A
Ethanol 8.9 A 12.5 C 35.0 B 8.3 B 15.0 C 30.0 B 9.2 A 12.5 C 35.0 B
Values presented are averages of three replicates. Values in the same column within each category following different uppercase
letters designate a significant difference (Duncan's multiple range test, P < 0.05).









Table 4-10. Comparison of lemongrass stem and turmeric (fresh rhizomes) extracts
Herb Zone (mm) MIC (mg/mL) MBC (mg/mL)
Lemongrass 13.7 A 3.0 B 5.2 B
Turmeric 7.7 B 19.4 A 29.3 A
Values presented are averages of three replicates. Values in the same column within each
category following different uppercase letters designate a significant difference (Duncan's
multiple range test, P < 0.05).

Table 4-11. Comparison of fresh rhizomes and dried powder turmeric extracts.
Herb Zone (mm) MIC (mg/mL) MBC (mg/mL)
Turmeric (dried powder) 8.6 A 19.4 A 31.1 A
Turmeric (fresh rhizomes) 7.7 B 19.4 A 29.3 A
Values presented are averages of three replicates. Values in the same column within each
category following different uppercase letters designate a significant difference (Duncan's
multiple range test, P < 0.05).









CHAPTER 5
SUMMARY AND CONCLUSION

Lemongrass stem and turmeric (fresh rhizomes and dried powder) extracts showed

antimicrobial properties against Staphylococcus aureus strains used in this study. Lemongrass

stems showed antimicrobial properties only when acetone and hexane were used as extraction

solvents. Oven dried extracts of lemongrass stems gave a greater antimicrobial activity than that

of the freeze dried extracts. Both hexane and acetone extracts of lemongrass stem had a

considerable amount of antimicrobial activity. Lemongrass harvested in November had higher

antimicrobial properties than that from October harvest.

Turmeric showed antimicrobial properties when acetone, hexane and ethanol were used as

solvents for the extraction. Difference in the solvents performance could be attributable to their

properties. Dried powder and fresh rhizome turmeric extracts did not show any differences on

the antimicrobial activity regardless of the solvents and drying methods used in this study.

Turmeric had less antimicrobial action compared to lemongrass. This could be due to the

differences in compounds and the properties in both plants.

Antimicrobial activity of lemongrass and turmeric could be greatly affected by the solvents

used to extracts the plants. Drying methods could also affect the antimicrobial activity of plants

such as lemongrass. Turmeric whether purchased in fresh or dry form could provide the same

effect of antimicrobial activity against Staphylococcus aureus, therefore obtaining either one of

them would not make a difference. Lemongrass and turmeric could be potentially used for

controlling Staphylococcus aureus. Even though lemongrass leaves are widely used to make tea,

it was found that they did not have any antimicrobial properties.

Increasing the concentrations of lemongrass and turmeric extracts may be necessary for

antimicrobial activity to be observed in other bacterial pathogens. It might be of interest to study









other Gram positive bacteria such as Bacillus cereus to determine if the antimicrobial action will

be similar. Studying the compounds extracted from plants by using the different solvents could

also be of interest to determine the important compounds responsible for the antimicrobial

activity of each extract.











APPENDIX A
ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY OF
LEMONGRASS STEMS


Table A-1. Inhibitory zone of lemongrass stems


Source
Model
Error
Corrected Total


Source
Harvest time
Trial
Pathogen
Solvent
Dry method
Solvent*Dry method


Sum of
Squares Mean Square F Value
1507.96 215.42 28.34
1033.83 7.60
2541.78


Type I SS
296.13
28.00
21.00
0.01
927.71
235.11


Mean Square
296.13
28.00
10.50
0.01
927.71
235.11


Table A-2. Minimum Inhibitory Concentration (MIC) of lemongrass stem


Source
Model
Error
Corrected Total


Source
Harvest time
Trial
Pathogen
Solvent
Dry method


Sum of
DF Squares Mean Square F Value Pr > F


7 784.37
136 480.68
143 1265.06


112.05
3.53


DF Type I SS Mean Square
1 138.56 138.56


1 13.69
2 11.99
1 58.86


13.69
5.99
58.86


1 297.37 297.37


Solvent*Dry method 1


31.70 <.0001


F Value Pr>F
39.20 <.0001
3.87 0.0511
1.70 0.1871
16.65 <.0001
84.14 <.0001
74.66 <.0001


Pr> F
<.0001


F Value
38.96
3.68
1.38
0.00
122.04
30.93


Pr> F
<.0001
0.0570
0.2547
0.9759
<.0001
<.0001


263.88 263.88












Table A-3. Minimum Bactericidal Concentration (MBC) of lemongrass stem
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 7 1259.70 179.95 43.65 <.0001
Error 136 560.65 4.12
Corrected Total 143 1820.36


Source DF Type I SS Mean Square F Value Pr> F


Harvest time 1
Trial 1
Pathogen 2
Solvent 1
Dry method 1
Solvent*Dry method 1


408.05
0.28
80.73
10.88
532.47
227.26


408.05
0.28
40.36
10.88
532.47
227.26


98.98
0.07
9.79
2.64
129.16
55.13


<.0001
0.7927
0.0001
0.1065
<.0001
<.0001











Table A-4. Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC 12600-U
Sum of
Source DF Squares Mean Square F Value Pr > F


Model
Error
Corrected Total


5 521.53 104.30
42 265.46 6.32
47 786.99


16.50 <.0001


Source I
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


Type I SS
167.81
25.15
31.28
193.00
104.28


Mean Square
167.81
25.15
31.28
193.00
104.28


F Value
26.55
3.98
4.95
30.54
16.50


Pr > F
<.0001
0.0525
0.0315
<.0001
0.0002











Table A-5. Minimum Inhibitory Concentration (MIC) of lemongrass stem against
Staphylococcus aureus ATCC 12600-U
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 5 322.91 64.5827367 21.96 <.0001
Error 42 123.49 2.9403678
Corrected Total 47 446.40


Source
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


Type I SS
93.99
14.10
0.00
89.51
125.29


Mean Square
93.99
14.10
0.00
89.51
125.29


F Value
31.97
4.80
0.00
30.44
42.61


Pr > F
<.0001
0.0341
0.9700
<.0001
<.0001


Table A-6. Minimum Bactericidal Concentration (MBC) of lemongrass stem against
Staphylococcus aureus ATCC 12600-U


Source
Model
Error
Corrected Total


Source
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


Sum of
Squares
568.41
127.16
695.58


Mean Square F Value Pr > F
113.68 37.55 <.0001
3.02


Type I SS
235.89
0.75
24.66
203.81
103.28


Mean Square
235.89
0.75
24.66
203.81
103.28


F Value
77.91
0.25
8.15
67.32
34.11


Pr > F
<.0001
0.6213
0.0067
<.0001
<.0001











Table A-7. Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC 29247


Source
Model
Error
Corrected Total


Sum of
Squares
686.25
139.41
825.66


Mean Square F Value Pr > F
137.25 41.35 <.0001
3.31


Source
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


Type I SS
25.52
0.19
0.19
595.02
65.33


Mean Square
25.52
0.19
0.19
595.02
65.33


FValue Pr>F
7.69 0.0083
0.06 0.8133
0.06 0.8133
179.25 <.0001
19.68 <.0001


Table A-8. Minimum Inhibitory Concentration (MIC) of lemongrass stem against
Staphylococcus aureus ATCC 29247


Source
Model
Error
Corrected Total

Source
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


Sum of
Squares
343.14
152.30
495.45

Type I SS
33.85
2.40
43.56
151.47
111.84


Mean Square
68.62
3.62


FValue Pr>F
18.93 <.0001


Mean Square
33.85
2.40
43.56
151.48
111.84


F Value Pr>F
9.33 0.0039
0.66 0.4198
12.01 0.0012
41.77 <.0001
30.84 <.0001












Table A-9. Minimum Bactericidal Concentration (MBC) of lemongrass stem against
Staphylococcus aureus ATCC 29247
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 5 393.90 78.78 27.03 <.0001
Error 42 122.42 2.914
Corrected Total 47 516.33


Source
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


DF Type I SS
1 51.57
1 5.64
1 33.74
1 211.49
1 91.45


Mean Square
51.57
5.64
33.74
211.49
91.45


F Value
17.69
1.94
11.58
72.56
31.37


Pr>F
0.0001
0.1713
0.0015
<.0001
<.0001











Table A-10. Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC A35548


Source
Model
Error
Corrected Total

Source
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


Sum of
Squares
463.04
445.07
908.12

Type I SS
139.23
21.00
25.15
209.37
68.28


Mean Square F Value Pr> F
92.6096354 8.74 <.0001
10.5970362


Mean Square F Value Pr> F


139.23
21.00
25.15
209.37
68.28


13.14
1.98
2.37
19.76
6.44


0.0008
0.1666
0.1309
<.0001
0.0149


Table A- 1. Minimum Inhibitory Concentration (MIC) of lemongrass
Staphylococcus aureus ATCC A35548


stem against


Source
Model
Error
Corrected Total


Source
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


Sum of
DF Squares Mean Square
5 191.45 38.29
42 119.73 2.85
47 311.19


DF Type I SS
1 23.76
1 17.68
1 43.86
1 65.60
1 40.54


FValue Pr>F
13.43 <.0001


Mean Square F Value Pr> F
23.76 8.34 0.0061
17.68 6.20 0.0168
43.86 15.39 0.0003
65.60 23.01 <.0001
40.54 14.22 0.0005












Table A-12. Minimum Bactericidal Concentration (MBC) of lemongrass stem against
Staphylococcus aureus ATCC A35548
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 5 349.69 69.93 16.50 <.0001
Error 42 178.02 4.23
Corrected Total 47 527.71


Source
Harvest time
Trial
Solvent
Dry method
Solvent*Dry method


Type I SS
154.94
5.93
23.74
124.29
40.77


Mean Square
154.94
5.93
23.74
124.29
40.77


F Value
36.55
1.40
5.60
29.32
9.62


Pr > F
<.0001
0.2433
0.0226
<.0001
0.0034











APPENDIX B
ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY OF
TURMERIC (FRESH RHIZOMES)

Table B-1. Inhibitory zone of turmeric (fresh rhizomes)


Source
Model
Error
Corrected Total


Source
Trial
Pathogen
Solvent
Dry method
Solvent*dry method


Sum of
DF Squares Mean Square F Value
8 90.99 11.37 39.11
207 60.21 0.29
215 151.20


DF Type I SS Mean Square
1 19.56 19.56
2 21.67 10.83
2 10.64 5.32
1 29.62 29.62
2 9.48 4.74


Table B-2. Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes)


Source
Model
Error
Corrected Total


Sum of
DF Squares
8 462.03
207 1347.22
215 1809.25


Source DF
Trial 1 224.07
Pathogen 2 117.59
Solvent 2
Dry method 1
Solvent*dry method 2


Mean Square F Value
57.754630 8.87
6.508320


Type I SS
224.07
58.79
34.25
1.85
84.25


Pr> F
<.0001


Mean Square F Value Pr > F


17.12
1.8518
42.12


34.43 <.0001
9.03 0.0002
2.63
0.28
6.47


Table B-3. Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes)


Source
Model
Error
Corrected Total


Mean Square
244.90
18.94


Source
Trial
Pathogen
Solvent
Dry method
Solvent*dry method


DF Type I SS
1 740.74
2 70.370
2 225.92
1 362.96
2 559.25


Mean Square F Value Pr > F


740.74
35.18
112.96
362.96
279.62


39.09
1.86
5.96
19.16
14.76


<.0001
0.1587
0.0030
<.0001
<.0001


Pr> F
<.0001


F Value
67.25
37.26
18.30
101.86
16.31


Pr > F
<.0001
<.0001
<.0001
<.0001
<.0001


0.0743
0.5943
0.0019


DF
8
207
215


Sum of
Squares
1959.25
3922.22
5881.48


F Value
12.93


Pr> F
<.0001











Table B-4. Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC
SA12600-U


Source
Model
Error
Corrected Total


Sum of
DF Squares
6 13.055
29 10.97
35 24.03


Mean Square F Value Pr > F
2.17 5.75 0.0005
0.37


Source DF Type I SS Mean Square F Value Pr > F
Trial 1 3.06 3.06 8.09 0.0081
Solvent 2 3.67 1.83 4.85 0.0153
Dry method 1 5.84 5.84 15.43 0.0005
Solvent*Dry method 2 0.48 0.24 0.64 0.5359

Table B-5. Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes) against
Staphylococcus aureus ATCC SA12600-U
Sum of
Source DF Squares Mean Square F Value Pr > F


Model
Error
Corrected Total


Source
Trial
Solvent
Dry method
Solvent*Dry method


6 79.16
29 211.80
35 290.97

DF Type I SS
1 34.02
2 38.88
1 6.25
2 0.00


13.19
7.30


1.81 0.1327


Mean Square F Value Pr > F


34.02
19.44
6.25
0.00


4.66
2.66
0.86
0.00


0.0393
0.0868
0.3626
1.0000


Table B-6. Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes) against
Staphylococcus aureus ATCC SA12600-U


Source
Model
Error
Corrected Total

Source
Trial
Solvent
Dry method
Solvent*Dry method


Sum of
DF Squares
6 861.11
29 613.88
35 1475.00


Mean Square F Value Pr > F
143.51 6.78 0.0001
21.16


DF Type I SS Mean Square F Value Pr > F


1 336.11
2 316.66
1 136.11
2 72.22


336.11
158.33
136.11
36.11


15.88
7.48
6.43
1.71


0.0004
0.0024
0.0169
0.1993











Table B-7. Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC
SA29247


Source I
Model
Error
Corrected Total

Source
Trial
Solvent
Dry method
Solvent*Dry method


Sum of
Squares
12.83
4.47
17.31

Type I SS
2.64
3.14
6.46
0.58


Mean Square F Value Pr > F
2.14 13.87 <.0001
0.15


Mean Square
2.64062500
1.57465278
6.46006944
0.29340278


F Value
17.12
10.21
41.87
1.90


Pr > F
0.0003
0.0004
<.0001
0.1675


Table B-8. Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes)
Staphylococcus aureus ATCC SA29247
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 6 250.00 41.66 6.13 0.0003
Error 29 197.22 6.80
Corrected Total 35 447.22


Source
Trial
Solvent
Dry method
Solvent*Dry method


Type I SS
69.44
88.88
2.77
88.88


Mean Square F Value Pr > F
69.44 10.21 0.0034
44.44 6.54 0.0045
2.77 0.41 0.5278
44.44 6.54 0.0045












Table B-9. Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes)
Staphylococcus aureus ATCC SA29247


Source
Model
Error
Corrected Total


Sum of
Squares
333.33
266.66
600.00


Mean Square
55.55
9.19


F Value
6.04


Pr> F
0.0003


Source
Trial
Solvent
Dry method
Solvent*Dry method


DF Type I SS
1 100.00
2 116.66
1 0.00
2 116.66


Mean Square
100.00
58.33
0.00
58.33


F Value
10.88
6.34
0.00
6.34


Pr> F
0.0026
0.0052
1.0000
0.0052











Table B-10. Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC
35548


Source DF
Model 6
Error 29
Corrected Total 35


Source
Trial
Solvent
Dry method
Solvent*Dry method


Sum of
Squares
19.51
3.90
23.42


Mean Square
3.25
0.13


F Value
24.16


DF Type I SS Mean Square F Value Pr>F
1 4.16 4.16 30.96 <.0001
2 6.50 3.25 24.14 <.0001
1 2.91 2.91 21.68 <.0001
2 5.93 2.96 22.02 <.0001


Table B-11. Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes) against
Staphylococcus aureus ATCC 35548
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 6 54.16 9.03 4.90 0.0014
Error 29 53.47 1.84
Corrected Total 35 107.63


Source
Trial
Solvent
Dry method
Solvent*Dry method


DF Type I SS
1 17.36
2 9.72
1 0.69
2 26.38


Table B-12. Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes) against
Staphylococcus aureus ATCC 35548
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 6 572.22 95.37 10.71 <.0001
Error 29 258.33 8.91
Corrected Total 35 830.55


Source DF
Trial 1
Solvent 2
Dry method 1
Solvent*Dry method 2


Type I SS
25.00
238.88
136.11
172.22


Mean Square
25.00
119.44
136.11
86.11


F Value
2.81
13.41
15.28
9.67


Pr > F
0.1046
<.0001
0.0005
0.0006


Pr>F
<.0001


Mean Square
17.36
4.86
0.69
13.19


F Value
9.42
2.64
0.38
7.16


Pr > F
0.0046
0.0887
0.5442
0.0030











APPENDIX C
ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY OF
TURMERIC (DRIED POWDER)


Table C-1. Inhibitory zone of turmeric (dried powder)


Source
Model
Error
Corrected Total


Sum of
Squares
38.78
17.62
56.41


Mean Square
7.75
0.17


Type I SS Mean Square
4.89 4.89
10.16 5.08
23.72 11.86


F Value
28.34
29.41
68.62


FValue Pr>F
44.88 <.0001


Pr > F
<.0001
<.0001
<.0001


Table C-2. Minimum Inhibitory Concentration (MIC) of turmeric (dried powder)
Sum of


Source
Model
Error
Corrected Total


DF Squares
5 3781.48
102 985.18
107 4766.67


Mean Square F Value Pr> F
756.29 78.30 <.0001
9.6586


Type 1 SS
92.59
72.22
3616.66


Mean Square
92.59
36.11
1808.33


Table C-3. Minimum Bactericidal Concentration (MBC) of turmeric (dried powder)


Source
Model
Error
Corrected Total


Source
Trial
Pathogen
Solvent


Sum of
DF Squares Mean Square F Value Pr > F


5 7666.67
102 600.00
107 8266.67

DF Type I SS
1 133.33
2 66.67
2 7466.67


1533.33
5.88


Mean Square
133.33
33.33
3733.33


260.67 <.0001


F Value
22.67
5.67
634.67


Pr> F
<.0001
0.0046
<.0001


Source
Trial
Pathogen
Solvent


Source
Trial
Pathogen
Solvent


F Value
9.59
3.74
187.22


Pr > F
0.0025
0.0271
<.0001










Table C-4. Inhibitory zone of turmeric (dried powder) against Staphylococcus aureus ATCC
12600-U


Source
Model
Error
Corrected Total


Sum of
DF Squares
3 4.17
32 2.83
35 7.00


Mean Square F Value Pr > F
1.38 15.69 <.0001
0.08


Source DF Type I SS Mean Square F Value Pr> F
Trial 1 1.00 1.00 11.29 0.0020
Solvent 2 3.17 1.58 17.88 <.0001

Table C-5. Minimum Inhibitory Concentration (MIC) of turmeric (dried powder) against
Staphylococcus aureus ATCC 12600-U
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 3 1230.55 410.18 41.45 <.0001
Error 32 316.66 9.89
Corrected Total 35 1547.22

Source DF Type I SS Mean Square F Value Pr > F
Trial 1 25.00 25.00 2.53 0.1218
Solvent 2 1205.55 602.77 60.91 <.0001

Table C-6. Minimum Bactericidal Concentration (MBC) of turmeric (dried powder) against
Staphylococcus aureus ATCC 12600-U
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 3 2700.00 900.00 144.00 <.0001
Error 32 200.00 6.25
Corrected Total 35 2900.00

Source DF Type I SS Mean Square F Value Pr > F


Trial
Solvent


1 100.00
2 2600.00


100.00 16.00 0.0004
1300.00 208.00 <.0001











Table C-7. Inhibitory zone of turmeric (dried powder) against Staphylococcus aureus ATCC
29247


Source
Model
Error
Corrected Total

Source
Trial
Solvent


Sum of
Squares
29.52
2.72
32.25


DF Type I SS
1 3.36
2 26.17


Mean Square F Value Pr > F
9.84 115.70 <.0001
0.08


Mean Square
3.36
13.08


FValue Pr>F
39.51 <.0001
153.80 <.0001


Table C-8. Minimum Inhibitory Concentration (MIC) of turmeric (dried powder) Staphylococcus
aureus ATCC 29247


Source
Model
Error
Corrected Total


Sum of
Squares
1050.00
350.00
1400.00


DF Type I SS
1 100.00
2 950.00


Mean Square F Value
350.00 32.00
10.93


Pr> F
<.0001


Mean Square F Value Pr > F
100.00 9.14 0.0049
475.00 43.43 <.0001


Table C-9. Minimum Bactericidal Concentration (MBC) of turmeric (dried powder)
Staphylococcus aureus ATCC 29247


Source
Model
Error
Corrected Total


Sum of
Squares
2400.00
0.00
2400.00


DF Type I SS
1 0.00
2 2400.00


Mean Square
800.00
0.00


Mean Square
0.00
1200.00


FValue Pr>F
Infty <.0001


F Value Pr>F

Infty <.0001


Source
Trial
Solvent


Source
Trial
Solvent











Table C-10. Inhibitory zone of turmeric (dried powder) against Staphylococcus aureus ATCC
35548


Source
Model
Error
Corrected Total


Sum of
Squares
4.16
2.83
7.00


Mean Square
1.38
0.08


FValue Pr>F
15.69 <.0001


Source DF Type I SS Mean Square F Value Pr > F
Trial 1 1.00 1.00 11.29 0.0020
Solvent 2 3.16 1.58 17.88 <.0001

Table C-11. Minimum Inhibitory Concentration (MIC) of turmeric (dried powder) against
Staphylococcus aureus ATCC 35548


Source
Model
Error
Corrected Total


Source
Trial
Solvent


Sum of
Squares
1508.33
238.88
1747.22

Type I SS
2.77
1505.55


Mean Square
502.77
7.46


Mean Square
2.77
752.77


F Value Pr>F
67.35 <.0001



F Value Pr>F
0.37 0.5462
100.84 <.0001


Table C-12. Minimum Bactericidal Concentration (MBC) of turmeric (dried powder) against
Staphylococcus aureus ATCC 35548
Sum of
Source DF Squares Mean Square F Value Pr > F
Model 3 2700.00 900.00 144.00 <.0001
Error 32 200.00 6.25
Corrected Total 35 2900.00

Source DF Type I SS Mean Square F Value Pr > F


100.00
2600.00


100.00
1300.00


16.00 0.0004
208.00 <.0001


Trial
Solvent









LIST OF REFERENCES

Abascal K, Ganoral L, Yarnell E. 2005. The effect of freeze-drying and its implications for
botanical medicine: A review. Phytother. Res.; 19(8):655-660

Akin-Osanaiye BC, Agbaji AS, Dakare MA. 2007. Antimicrobial activity of oils and extracts of
Cymbopogon citratus (Lemon grass), Eucalyptus citriodora and Eucalyptus
camaldulensis, J. Med Sci., 7(4): 694-697.

Afolayan AJ and Aliero AA. 2006. Antimicrobial activity of Solanum tomentosum. Afri. J.
Biotechnol., 5(4): 369-372

Archer DL, Kvenberg JE. 1985. Incidence and cost of foodborne diarrheal disease in the United
States. J. Food Protect., 48: 887-894.

Asami DK, Hong YJ, Barrett DM, and Mitchell AE.2003. Comparison of the total phenolic and
ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn
grown using conventional, organic, and sustainable agricultural practices. J. Agric. Food
Chem. 51: 1237-1241

Badmaev V, Majeed M, Prakash L. 2004. Curcuminoids: bioactive compounds from turmeric.
Sabinsa Corporation, 2.

Baratta MT, Dorman HJD, Deans SD, Figueiredo AC, Barroso JG, Ruberto G. 1998.
Antimicrobial and antioxidant properties of some commercial essential oils. J. Flavor and
Fragar., 13(4): 235-244.

Beattie S. 2006. Common foodborne pathogens. Consumer and Processing Food Safety and
Science, Iowa State University, USA
http://www.extension.iastate.edu/foodsafety/pathogens/index.cfm

Bennet J, Holmberg S, Rogers M, Solomon S. 1987. Infectious and parasitic diseases. In: Almer
R, Dull H, Ed., Closing the gap: the burden of unnecessary illness. New York: Oxford
Univ. Press; p 102-114.

Beuchat LR, Golden DA. 1989. Antimicrobial occurring naturally in foods. Food Technol., 43:
134-143.

Billing J, Sherman PW. 1998. Antimicrobial function of spices: why some like it hot. Q. Rev.
Biol., 73: 3-49.

Biswas TK, Mukherjee B. 2003. Plant medicines of Indian origin for wound healing activity: A
healing review. Int. J. Low. Extrem. Wounds, 2(1): 25-39.

Brennan JG. 1994. Food dehydration a dictionary and guide. CRC Press Oxford: Butterworth-
Heinemann, p 137-182.









Bryan FL. 1982. Diseases transmitted by foods. Sec. Ed. Atlanta, GA, Centers for Disease
Control. http://chppm-www.apgea.army.mil/tsunami/Diseasesfromfood(2).pdf

Buchi Labortechnik AG. 2008. Rotary Evaporators. Buchi Corporation. www.buchi.com

Cameron P, Butler DL, Kinnary K, Monger P, Murgatroyd. 1997. Good Pharmaceutical Freeze-
Drying Practice. Interpharm/CRC, Boca, Raton, London New York, Washington D.C.
P1-269.

Cappuccino JG, Sherman N. 2005. Microbiology: A laboratory Manual, 7th Ed., Pearson
Benjamin Community College, New York, NY, p 13-20.

Casimiri V, Burstein C. 1998.Biosensor for L -lactate determination as an index of E. coli
number in crude culture medium. J. Analytica Chimica Acta, 361 (1-2): 45-53

Centers for Disease Control and Prevention (CDC). 2005. Foodborne illnesses. Division of
Bacterial and Mycotic Diseases. Atlanta, GA.

Chao SC, Young DG, Oberg CJ. 2000. Screening for inhibitory activity of essential oils on
selected bacteria, fungi and viruses. J. Essent. Oil. Res.,12(5): 639-649.

Cheremisinoff NP. 2003. Industrial solvents Handbook. Second Edition. Marcel Dekker, Inc.
New York.

Conner DE. 1993. Naturally Occurring Compounds. Editors: Davidson PM, Branen AL.
Antimicrobials in foods. Second Ed.Marcel Dekker, New York, p 441-468

Cosentino S, Tuberoso CIG, Pisano B, Satta M, Mascia V, Arzedi E, Palmas F. 1999. In-vitro
antimicrobial activity and chemical and chemical composition of Sardinian Thymus
essential oils. Lett. Appl. Microbiol., 29: 130-135.

Cowan MM. 1999. Plant products as microbial agents. Clinical Microbiol. Rev., 12 (4), 564-582.

Dudman P. 2005. Growing Asian herbs and Spices. ABC North Coast.

Duke JA. 1994. Biologically active compounds in important spices. In spices, herbs and edible
fungi, (G. Charalambous, ed.), Elservier, Mew York, p 225-250.

Duke JA, Bogenschutz-Gogwin MJ, duCellier J, Duke PK. 2003. CRC Handbook of medicinal
spices. Health and Fitness, p 240-245.

Elloff JN. 1998. Which extract should be used for the screening and isolation of antimicrobial
components from plants? J. Entropharmacol., 60(1): 1-8

European Patent. 2006. Microbial formulation comprising essential oils or their derivatives. EP,
1434486.

Food and Drug Administration (FDA/CFSAN). 2007. Bad bug book: Foodborne Pathogenic
Microorganisms and natural Toxins Handbook.

101









Fraenkel GS. 1959. The raison d'Etre of secondary plant substances. Science, 129: 1466-1470.

Gescher A, Sharma R, Steward W. 2005. Curcumin: the story so far. Europ. J. Cancer, 41(13):
1955-1968.

Gerald J. 1975. The herbal or general history of plants. Arco Pub. Co., New York, pl-13.

Goel S. 2005. Bioprotective properties of turmeric: An investigation of the antioxidant and
antimicrobial activities. J. Young Invest., 16(17): 1-6.

Grag SR, Menon KV. 2003. Spices and herbs- promising antimicrobial agents and natural food
preservatives. Haryana Agricultural University Journal of Research: 3(1), 1-6.

Hammer KA, Carson CF, Riley TV. 1999. Antimicrobial activity of essential oils and other plant
extracts. Journal of Applied Microbiology: 86(6): 985-990.

Ho JC, S. Chou SK, Chua KJ, Mujumdar AS and Hawlader MNA. 2002. Analytical study of
cyclic temperature drying: effect on drying kinetics and product quality. J. Food
Engineering, 51 (1): 65-75.

Hughes KV, Wellenberg BJ. 1994. Quality for keeps: Drying foods. DMCA, MU Extension,
Columbia, MO.

Hutton W. 2003. Handy pocket Guide to Asian herbs and spices. Tuttle Publishing, Singapore. p
3-64.

Huhtanen CN. 1980. Inhibition of Clostridium botulinum by spice extracts and aliphatic alcohol.
J. Food Prot., 43: 195-196.

Julkunen-Titto R, Sorsa S. 2001. Testing the effects of drying methods on willow flavonoids,
tannins, and salicylates. J. Chem. Ecol. 27: 779-789.

Kakuta E, Ohshima T, Maeda N. 2006. The antimicrobial effects of essential oils against oral
pathogenic microorganisms. Microbiol. Immunol. Infect. Control System, Preliminary
Program for IADR General Session and Exhibition.

Karaman I, Sahin F, Gulluce M, Qgutcu H, Sengul M, Adiguzel A. 2003. Antimicrobial activity
of aqueous and methanol extracts ofJuniperous oxycedrus L. J. Ethnopharmacol, 85:
231-235.

Kasumov and Babaev RI. 1983. Components of the essential oil of lemongrass. Chem. Natural
Comp., 19(1): 108.

Kim JM, Marshall MR, Cornell JA, Preston JF, Wei CI. 1995. Antibacterial activity of some
essential oil components against five foodborne pathogens. J. Agric. Food Chem., 43:
2839-2845.









Kim KJ, Yu HH, Cha JD, Seo SJ, Choi NY, You YO. 2005. Antibacterial activity of Curcuma
long L. against methicillin resistant Staphylococcus aureus. Phytotherapy Res., 19(7):
599-604.

Kotzekidou P, Giannakidis P, Boulamastis A. 2008. Antimicrobial activity of some plant extracts
and essential oils against foodborne pathogens in vitro and on the fate of inoculated
pathogens in chocolate. J. Food Sci. Technol., 41(1): 119-127.

Laohakunjit N, Kerdchoechuen O, Norajit K. 2007. Antibacterial effect of five Zingiberaceae
essential oils. Molecules, 12: 2047-2060.

Lerici CR. 1976. Retention of volatile compounds in a freeze dried model food gel. S TA NU.,
6(1): 27-32.

Lis-Balchin M, Deans SG. 1997. Bioactivity of selected plant essential oils against Listeria
monocytogenes. J. Appl. Microbiol., 82: 759-762.

Maizura M, Fazilah A, Norziah MH, Karim AA. 2007. Antibacterial activity and mechanical
properties of partially hydrogenated sago starch-alginate edible film containing
lemongrass oil. J. Food Sci., 72(6): 324-330.

Mahanon H, Azizah AH, Dzulkifly MH. 1999. Effect of drying methods on concentrations of
several phytochemicals in herbal preparation of 8 medicinal plants leaves. Mal. J. Nutr.,
5: 47-54.

Mandeel Q, Hassan A, Isa Z. 2003. Antibacterial activity of certain spice extracts. J. Spice Arom.
Crops., 12(2): 146-153.

Mead PS, Slutsker L, Dietz V, McCaig LF, Breese JS, Shapiro C, Griffin PM, Tauxe RV. 1999.
Food related illness and death in the United States. Emerg. Infect. Dis. 5: 607-625.

Mejlholm O, Dalgaard P. 2002. Antimicrobial effect of essential oils on the seafood spoilage
micro-organism Photobacteriumphosphoreum in liquid media and fish products. Lett.
Appl. Microbiol., 34 (1): 27-31.

Nail SL, Jiang S, Chongprasert S, Knopp SA. 2002. Fundamentals of freeze drying. Pharm.
Biotechnol., 14: 281-360.

Onawunmi, GO. 1989. Evaluation of the antimicrobial activity of citral. Lett. Appl. Microbial.,
9: 105-108.

Oonmetta-aree J, Suzuki T, Gasaluck P, Eemkeb G. 2006. Antimicrobial properties and action of
galangal (Alpinia galangal Linn.) on Staphylococcus aureus. J. Food Sci. and Technol.,
39(10): 1214-1220.









Oussalah M, Caillet S, Saucier L, Lacroix M. 2006. Antimicrobial effects of selected essential
oils on the growth of a Psuedomonasputida strain isolated from meat. Meat Sci., 73(2):
236-244.

Ozcan M, Arslan D, Unver A. 2005. Effect of drying methods on the mineral content of basil
(Ocimum basilicum L.). J. Food Engin., 69(3): 375-379.

Packiyasothy EV, Kyle S. 2002. Antimicrobial properties of some herb essential oils. J. Food
Aust., 54(9): 384-387.

Parish ME, Davidson PM. 1993. Methods for evaluation: In Antimicrobials in foods, 2nd Ed.
Marcel Dekker, Inc., New York, p 597-615.

Perez-Coello MS, Diaz-Maroto MC, Cabezudo MD. 2002. Effect of drying method on the
volatiles in bay leaf(Laurus nobilis L.). J. Agric. Food Chem., 50 (16): 4520-4524.

Pham M. 2001. Asian herbs: For flavor and for health. HarperCollons Publishers.

Pulford A. 2003. Turmeric root. Health and Fitness: In Homeopathic material medical of
graphical drug pictures, p 149-150.

Raybaudi-Massilia RM, Mosqueda-Melgar J, Martin-Belloso 0. 2006. Antimicrobial activity of
essential oils on Salmonella Enteritidis, Escherichia coli, and Listeria innocula in fruit
juices. J. Food Prot., 69(7): 1579-1586.

Reynolds S, Williams P. 1993. So Easy to Preserve. Cooperative Extension Service, The
University of Georgia. Revised by Judy Harrison.

Ritenour MA, Sargent SA, bartz JA. 2002. Chlorine use in produce packing lines. HS-761, IFAS
Extension, Gainesville, FL; pl-4.

Rojas-Grau M, Avena Bustillos R, Friedman M, Henika P, Martin-Belloso O, McHugh T. 2006.
Mechanical barrier and antimicrobial properties of apple puree edible films containing
plant essential oils. J. Agric and Food Chem., 54: 9263-9267.

Rojas-Grau M, Olsen C, Avena Bustillos R, Friedman M, Henika P, Martin-Belloso O, Pan Z,
McHugh T. 2007. Effects of plant essential oils and oil compounds on mechanical,
barrier and antimicrobial properties of alginate-apple purees edible films. J. Food Engin.,
81(3): 634-641.

Rojas JJ, Ochoa VJ, Ocampo SA, Mufioz JF. 2006. Screening for antimicrobial activity often
medicinal plants used in Colombian folkloric medicine: A possible alternative in the
treatment of non-nosocomial infections. BMC Complementary and Alternative Medicine,
6(2): 53-108.









Russell AD. 1991. Mechanisms of bacterial resistance to non-antibiotics: food additives and food
and pharmaceutical preservatives. J. Appl. Microbiol., 71: 191-201.

Sacchetti G, Maietti S. Muzzoli M, Scaglianti M, Manfredini S, Radice M, Bruni R. 2005.
Comparative evaluation of 11 essential oils of different origin as functional antioxidants,
antiradicals and antimicrobials in foods. J. Food Chem., 91(4): 621-632.

Sagdic O, Ozcan M. 2003. Antibacterial activity of Turkish spice hydrosols. Food Control, 14:
141-143.

Salzer UJ. 1982. Antimicrobial action of some spice extracts and mixtures. Fleischwirtschaft, 62:
885-887.

SAS. 1991. SAS/STAT User's Guide. 6.04 Ed. SAS Institute, Inc., Cary, NC.

Scheflan L, Jacobs MB. 1953. The handbook of solvents. First Edition. Robert E. Krieger
Publishing Co., New York.

Schwiertz A, Duttke C, Hild J, Muller HJ. 2006. In vitro activity of essential oils on
microorganisms isolated from vaginal infection. Int. J. Aromath., 16(3-4): 169-174.

Shelef LA, Naglik OA, Bogen DW. 1980. Sensitivity of some common foodborne bacteria to the
spices sage, rosemary, and all spice. J. Food Sci., 45: 1042-1044.

Senatore F. 1996. Influence of harvesting time on yield and composition of essential oil of thyme
(Thymuspulegioides L.) growing wild in Campania (Southern Italy). J. Agric. Food
Chem., 44: 1327-1332.

Shibasaki I. 1982. Food preservation with nontraditional antimicrobial agents. J. Food Saf, 4:
35-58.

Shivani G, Ravishankar S. 2005. Foodborne Pathogens and Disease. A Comparison of the
antimicrobial activity of garlic, ginger, carrot, and turmeric pastes against Escherichia
coli 0157:H7 in laboratory buffer and ground beef. Food Saf. Technol., 2(4): 330-340.

Simon, J.E., A.F. Chadwick and L.E. Craker. 1984. Herbs: An Indexed Bibliography. 1971-
1980. The Scientific Literature on Selected Herbs, and Aromatic and Medicinal Plants of
the Temperate Zone. Archon Books, 770 pp., Hamden, CT.

Simonne AH, Nille A, Evans K, Marshall, Jr MR. 2004. Ethnic foods safety trends in the United
States based on CDC foodborne illness data. Food Prot. Tre., 24(8): 590-604.

Siripongvutikorn S, Thummaratwasik P, Huang Y. 2005. Antimicrobial and antioxidant effects
of Thai seasoning, Tom-Yum. J. Food Sci. and Technol., 38(4): 347-352.









Smid EJ, Gorris LGM. 1999. Natural antimicrobials for food preservation. In handbook of food
preservation; Rahman, MS, Ed.; Marcel Dekker: New York, p285-308.

Todd ECD. 1989. Preliminary estimates of costs of foodborne disease in the United States. J.
Food Protect., 52: 595-601.

Uhl S. 2000. Spices: tools for alternative or complementary medicine. Food Technol., 54(5): 61-
65.

United States Patent. 2003. Citral acetal. USP, 6506793.

Van Sumere C, Geiger H, Bral D, Fockenier G, Vande Casteele K, Martens M, Hanselaer R,
Gevaert L. 1983. Freeze drying and analysis of plant and other biological material. Anal.
Biochem., 131(2): 530-532.

Venskutonis PR. 1997. Effect of drying on the volatile constituents of thyme (Thymus vulgaris
L.) and sage (Salvia officinalis L.). J. Food Chem.; 59(2):219-227.

Webb G. 1997. Herbs and spices with antioxidant and/or antimicrobial compounds. J. Amer.
Botan. Council: 39 p20.

Williams LR, Stockley JK, Yan W, Home VN. 1998. Essential oils with high antimicrobial
activity for therapeutic use. Int. J. Aromath., 8(4): 30-40.

Zaika LL. 1988. Spices and Herbs: their antimicrobial activity and its determination. J. Food
Saf., 9: 97- 118.









BIOGRAPHICAL SKETCH

Thabile Precious Nkambule, daughter of Annah S. Magagula, was born in Mbabane,

Swaziland. She completed her bachelor of science in home economics with a Dean's Award at

the University of Swaziland (Faculty of Agriculture). Before attending the University of Florida

as a Fulbright Scholar, Thabile worked at UNICEF and REDI (Regional Excellence &

Development Initiative). Thabile also worked as a teacher at Dwalile Central High School in

Swaziland for three years. She taught food and nutrition, home economics, mathematics, and

fashion and fabrics. After finishing her master's degree in food science, she plans to return to

Swaziland to resume a teaching position at the University of Swaziland (Faculty of Agriculture).





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ANTIMICROBIAL PROPERTIES OF SELECTED ASIAN HERBS By THABILE PRECIOUS NKAMBULE 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 2008 1

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2008 Thabile Precious Nkambule 2

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To my Family and Friends 3

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ACKNOWLEDGMENTS I would like to express my speci al appreciation to my adviso r Dr. Amarat H. Simonne (my supervisory committee chair) and Dr. Charles Sims Dr. Samuel R. Farrah, and Dr. Maurice R. Marshall, Jr., for serving on my committee. I would also like to thank Rob M. Pelick, Kimberly Evans, and Wei-Yea Hsu for their assistance and support. I would also lik e to thank the faculty and staff of Food Science and Hu man Nutrition department, University of Florida. I would like the express much appreciation to my family fo r their love, encouragement, and support. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF FIGURES.......................................................................................................................10LIST OF ABBREVIATIONS........................................................................................................11ABSTRACT...................................................................................................................................12 CHAPTER 1 INTRODUCTION................................................................................................................. .152 LITERATURE REVIEW.......................................................................................................19Most Common Asian Herbs and Spices.................................................................................20Lemongrass......................................................................................................................21Turmeric....................................................................................................................... ...22Antimicrobial Activities of Lemongrass ( Cymbopogon citratus)............................23Antimicrobial Activities of Turmeric ( Curcuma longa)..........................................30Drying Methods......................................................................................................................33Oven-Drying....................................................................................................................34Freeze-Drying..................................................................................................................35Extraction Solvents............................................................................................................ .....39Dimethyl Sulfoxide (C2H6OS)........................................................................................40Water (H2O).....................................................................................................................40Ethanol (C2H6O)..............................................................................................................41Hexane (C6H14)................................................................................................................41Acetone (C3H6O).............................................................................................................41Effect of Extraction Solvents on the Antimicrobial Properties of Herbs........................41Evaporation Techniques.........................................................................................................43Rotary Rotavapor....................................................................................................................433 MATERIALS AND METHODS...........................................................................................51Bacterial Strains and Cultivation............................................................................................51Plant Materials........................................................................................................................51Preparation of Plant Material.................................................................................................. 52Preparation of Plant Extracts.................................................................................................. 52Antimicrobial Assay............................................................................................................ ...54Minimum Inhibitory Concentration (MIC) Determination....................................................55Minimum Bactericidal Concentration (MBC) Determination................................................56Experimental Design and Statistical Analysis........................................................................564 RESULTS AND DISCUSSION.............................................................................................60 5

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Antimicrobial Activity of Lemongrass...................................................................................60Inhibitory Zone of Lemongrass Extracts.........................................................................60Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Lemongrass Stem Extracts..................................................64Antimicrobial Activity of Turmeric Extracts.........................................................................66Inhibitory Zone of Turmeric (Fresh Rhizomes and Dried Powder) Extracts..................66Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Turmeric (Fresh Rhizomes and Dried Powder) Extracts....685 SUMMARY AND CONCLUSSION.....................................................................................81 APPENDIX A ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY OF LEMONGRASS STEMS.................................................................................................83B ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY OF TURMERIC (FRESH RHIZOMES)................................................................................91C ANALYSIS OF VARIANCE (ANOVA) TABLES: ANTIMICROBIAL ACTIVITY OF TURMERIC (DRIED POWDER)....................................................................................96LIST OF REFERENCES.............................................................................................................100BIOGRAPHICAL SKETCH.......................................................................................................107 6

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LIST OF TABLES Table page 2-1 Reported and estimated cases, number of outbreaks and deaths caused by known foodborne bacterial pathogens from 1983 to 1997............................................................452-2 Most common pathogens associated with foodborne illness outbreaks............................462-3 Common Asian herbs used for cooking.............................................................................482-4 Properties of different solvents.......................................................................................... 494-1 Inhibitory zone (mm) of lemongrass st ems extracts (solvent extracts) on bacterial pathogens...........................................................................................................................714-2 Inhibitory zone (mm) of turmeric ( fresh rhizome) extracts (solvent extracts) on bacterial pathogens.............................................................................................................724-3 Inhibitory zone (mm) of turmeric (d ried powder) extracts (solvent extracts) on bacterial pathogens.............................................................................................................734-4 Overall comparison of solvents, dryi ng methods, and harvest-time for lemongrass stem....................................................................................................................................744-5 Sensitivity of each Staphylococcus aureus strain to lemongrass stem..............................754-6 Antimicrobial activities of fresh rhizome turmeric extracts..............................................764-7 Sensitivity of each Staphylococcus aureus strain to fresh rhizome turmeric extracts.......774-8 Antimicrobial activities of dried powder turmeric extracts...............................................784-9 Sensitivity of each Staphylococcus aureus strain to dried powder turmeric extracts........794-10 Comparison of lemongrass stem and turmeric (fresh rhizomes) extracts..........................804-11 Comparison of fresh rhizomes a nd dried powder turmeric extracts..................................80A-1 Inhibitory zone of lemongrass stems.................................................................................83A-2 Minimum Inhibitory Concentr ation (MIC) of lemongrass stem........................................83A-3 Minimum Bactericidal Concen tration (MBC) of lemongrass stem...................................84A-4 Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC 12600-U....85A-5 Minimum Inhibitory Concentrati on (MIC) of lemongrass stem against Staphylococcus aureus ATCC 12600-U............................................................................86 7

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A-6 Minimum Bactericidal Concentra tion (MBC) of lemongrass stem against Staphylococcus aureus ATCC 12600-U............................................................................86A-7 Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC 29247.........87A-8 Minimum Inhibitory Concentrati on (MIC) of lemongrass stem against Staphylococcus aureus ATCC 29247................................................................................87A-9 Minimum Bactericidal Concentra tion (MBC) of lemongrass stem against Staphylococcus aureus ATCC 29247................................................................................88A-10 Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC A35548......89A-11 Minimum Inhibitory Concentrati on (MIC) of lemongrass stem against Staphylococcus aureus ATCC A35548.............................................................................89A-12 Minimum Bactericidal Concentra tion (MBC) of lemongrass stem against Staphylococcus aureus ATCC A35548.............................................................................90B-1 Inhibitory zone of turmeric (fresh rhizomes).....................................................................91B-2 Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes).........................91B-3 Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes).....................91B-4 Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC SA12600-U........................................................................................................................92B-5 Minimum Inhibitory Concentration (MIC ) of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC SA12600-U.......................................................................92B-6 Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC SA12600-U.......................................................................92B-7 Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC SA29247............................................................................................................................93B-8 Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes) Staphylococcus aureus ATCC SA29247...........................................................................93B-9 Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes) Staphylococcus aureus ATCC SA29247...........................................................................94B-10 Inhibitory zone of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC 35548..................................................................................................................................95B-11 Minimum Inhibitory Concentration (MIC ) of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC 35548................................................................................95 8

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B-12 Minimum Bactericidal Concentration (M BC) of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC 35548................................................................................95C-1 Inhibitory zone of turmeric (dried powder).......................................................................96C-2 Minimum Inhibitory Concentrati on (MIC) of turmeric (dried powder)............................96C-3 Minimum Bactericidal Concentratio n (MBC) of turmeric (dried powder).......................96C-4 Inhibitory zone of turm eric (dried powder) against Staphylococcus aureus ATCC 12600-U.............................................................................................................................97C-5 Minimum Inhibitory Concentration (M IC) of turmeric (dried powder) against Staphylococcus aureus ATCC 12600-U............................................................................97C-6 Minimum Bactericidal Concentration (MBC) of turmeric (d ried powder) against Staphylococcus aureus ATCC 12600-U............................................................................97C-7 Inhibitory zone of turm eric (dried powder) against Staphylococcus aureus ATCC 29247..................................................................................................................................98C-8 Minimum Inhibitory Concentrati on (MIC) of turmeric (dried powder) Staphylococcus aureus ATCC 29247................................................................................98C-9 Minimum Bactericidal Concentration (MBC) of turmeric (dried powder) Staphylococcus aureus ATCC 29247................................................................................98C-10 Inhibitory zone of turm eric (dried powder) against Staphylococcus aureus ATCC 35548..................................................................................................................................99C-11 Minimum Inhibitory Concentration (M IC) of turmeric (dried powder) against Staphylococcus aureus ATCC 35548................................................................................99C-12 Minimum Bactericidal Concentration (MBC) of turmeric (d ried powder) against Staphylococcus aureus ATCC 35548................................................................................99 9

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LIST OF FIGURES Figure page 2-1 Chemical structure of Citral (C10H16O) (3, 7-dimethyl-2, 6-octadienal)...........................502-2 Chemical structure of Curcumin (C21H20O6 )....................................................................503-1 Lemongrass ( Cymbopogon citratus ) plants.......................................................................573-2 Turmeric ( Curcuma longa) rhizomes................................................................................573-3 Lemongrass leaves......................................................................................................... ....583-4 Lemongrass stem........................................................................................................... ....583-5 Inhibitory zone of lemongrass stem oven dried, hexane extracts on Staphylococcus aureus ATCC 29247..........................................................................................................59 10

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LIST OF ABBREVIATIONS Antimicrobials A large variety of chemical compounds or substances that are used to destroy (kill) microorganisms or to prevent (or slow) their growth MBC Minimum bactericidal concentration is the lowest concentration of an antimicrobial agent needed to kill 99.9% of the in itial organism inoculums MIC The minimum inhibitory concentration is as the lowest concentration of an antimicrobial that prevents the grow th of microorganism after a specific incubation time 11

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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 ANTIMICROBIAL PROPERTIES OF SELECTED ASIAN HERBS By Thabile Precious Nkambule May 2008 Chair: Amarat H. Simonne Major: Food Science and Human Nutrition Many pathogens including Salmonella spp, Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 are commonly implicated in f oodborne illnes s outbreaks in the United States. Increasing antibiotic resistan ce of bacterial foodborne pathogens has raised concerns in the scientific community. Many herb s such as lemongrass and turmeric have been considered medicinal plants that can be potentially used for c ontrolling foodborne pathogens in place of chemicals or antibiotics. Objective of this study was to systematically evaluate the antimicrobial properties of lemongrass [ Cymbopogon citratus (C. Nees) Stapf (Poaceae)] and turmeric [ Curcuma longa (Zingiberaceae)] as affected by drying methods (oven and freeze drying) and different extraction solvents (wat er, acetone, hexane and ethanol). Antimicrobial assay was done using an agar disc diffusion method against Staphylococcus aureus (ATCC 29247, 12600U, and 35548) Escherichia coli O157:H7 (204P, 301C and 505B) and Salmonella serotypes Enteritidis, Typhimurium and Thomps on (ATCC 8391) strains. Minimum inhibitory concentration (MIC) was measured using the multiple tube dilution method. Minimum bactericidal concentration (MBC) was measured using the colony-forming assay. Results showed that among bacterial strains tested, Staphylococcus aureus strains were sensitive to the hexane and acetone extracts from lemongrass stems; a nd to hexane, acetone and ethanol extracts from 12

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turmeric. No antimicrobial activity was observed in any lemongrass leaf samples regardless of solvents used in the extraction or drying methods Water and ethanol extracts of lemongrass stem did not show any antimicrobial activity against a ny of the test bacteria. Inhibitory zone from lemongrass stem extracts ranged from 6.5 to 21 mm. For lemongrass stem, oven dried methods yielded significantly higher (P < 0.0001) antimicrobial activity than that of stems prepared from the freeze dried method. Hexane extract yielded significantly higher (P < 0.0001) antimicrobial activity (MIC) than that of the acetone extracts. Seasonal variation was observed with respect to antimicrobial activity for lemongrass stem; plan ts harvested in November showed a higher activity (P < 0.0001) than plants which were harvested in October. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) range for lemongrass stem was 0.8 17.4 mg/mL. Water extracts of turmeric did not show any an timicrobial activity agai nst any test bacterial strains. Turmeric had a significantly lowe r (P < 0.0001) antimicrobial activity against Staphylococcus aureus strains tested than that of lemongr ass stem. However the antimicrobial activity of turmeric was observed in extracts of the other solvents (acetone, hexane and ethanol) than that of lemongrass (acetone and hexane). Inhibitory zone of turmeric extracts ranged from 6.5-11 mm. Minimum inhibitory concentra tion (MIC) and Minimum bacter icidal concentration (MBC) range for dried and fresh turmeric were 12.5 40.0 mg/mL. Little variation in antimicrobial activity was observed for dried and fresh turmeric extracts. Results of the study demonstrated that hexane and acetone extracts from lemongrass stem exhibit antimicrobial activity against Staphylococcus aureus, and hexane, acetone, and ethanol extracts of dried powder and fresh turmeric rhizomes, exhibited antimicrobial activity against 13

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14 Staphylococcus aureus. Both lemongrass stem and turmeric extracts could be potentially used for controlling Staphylococcus aureus.

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CHAPTER 1 INTRODUCTION Foodborne diseases are still a major problem in the United States. Many microorganisms such as Salmonella spp, Listeria m onocytogenes, Escherichia coli O157:H7 and Staphylococcus aureus are often reported as causativ e agents of foodborne diseases. Many of the pathogens of greatest concern today (e.g., Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Cyclospora cayetanensis ) were not recognized as causes of foodborne illness 20 years ago (Mead and others 1999). Thus, at pres ent, many interventions including chemical preservatives must be used to kill or prevent the growth of foodborne pathogens as well as spoilage microorganisms in the food industry (Sagdic and Ozcan 2003). Due to consumer concerns about the safety of food containing synthetic chemi cals as preservatives and the increasing antibiotic resistance of bacterial foodborne pa thogens, there is a growing interest in the use of natural antibacterial compounds such as herb and spice extracts because they have both characteristic flavor as we ll as a potential antimicrobial activity (Smid and Gorris 1999). Many herbs, such as lemongrass and turmeric, have been considered medicinal plants which can be potentially used for controlling foodborne pa thogens in place of chemicals or antibiotics. The word herb is derived from the Latin h erba, meaning grass or, by extension, green crop (Gerald 1975). Herb was originally applied to a wide range of leafy vegetables. Herbs are seed plants that do not produce woody stems like a tree and will live long enough to develop and produce flowers and seeds (Gerald 1975). For thousands of years herbs have been used for their flavor, medicinal, antimicrobial, and antioxidative properties. Use of spices and herbs in cooking is the oldest form of aromatherapy. Active com ponents of spices and herbs are considered as powerful tools to create a state of wellness such as stimulate production of enzymes that detoxify carcinogens, inhibit cholesterol synthesis, bl ock estrogen, lower blood pressure, and prevent 15

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blood clotting (Uhl 2000). Early cultures recogni zed that certain herbs had healing powers; therefore, some herbs were t hought to have magical properties. In the ancient civilizations, people began to associate less magic with the tr eatment of diseases. They understood that illness was natural and not supernatural, and medici ne should be given without magic. Chinese herbalism is widely regarded as the oldest be cause it has the longest unbroken recorded history. Chinese have practiced herbal use for 5000 years. Early use of antimicrobial plant extracts (i.e. herbs and oils) was well documented in ancient Egypt. Ancient Egyptians used plant extracts for food preservation as well as for embalming the dead. Pliny, Virgil, and Hippocrates mentioned garlic as a treatment for a variety of ailments, including indigestion, pneumoni a, wounds, and infecti ons (Conner 1993). Although ancient civilizations recognized the antiseptic or antimicrobial potential of many plant extracts, it was only in the eighteenth century that the scient ific documentation of the preservative effects of spices and spice-type extracts were developed or recorded (Conner 1993). Many plant tissues contain a variety of compounds called secondary plant compounds (metabolites) grouped as glucosides, saponins, tannins, alkaloids, essential oils, organic acids and others (Fraenkel 1959). Phytoale xins another group of compounds of low molecular weight are typically synthesized in response to environmen tal stress. Many of these phytoalexins have both antimicrobial and antifungal properties. They are produced after infection as a defense mechanism against microbial infection. These compounds interfere with the cell membrane associated functions (Beuchat and Golden 1989). Major antimicrobial components of spices are in their essential oils (Shibasaki 1982). Each compound in spices and herbs exhibits different biological activity (Duke 1994; Webb 1997). 16

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Amount of spices and herbs extracts used in food systems range from 0.05 to 0.1% (500 to 1000 ppm) (Salzer 1982). Billing and Sherman (1998) calculated that meat recipes contained roughly 0.25-3.0 g/kg of spices (250-3000 ppm). Although many essential oils from spices and herbs have antimicrobial activity against bacteria at less than 1000 ppm, some spices require higher concentrations to exhibi t antimicrobial activity (Zaika 1988). Antimicrobial activity of herbs and spices varies widel y, depending on the type of spic e or herb, test medium, and microorganism. Zaika (1988) summarized the factor s that could affect the antimicrobial activity of herbs and spices. Microorganisms differ in th eir resistance to a give n spice or herb, and a given microorganism differs in its resistance to many types of spices and herbs. In general, bacteria are more resistant to antimicrobials than fungi. Furthermore, antimicrobial action on spores may be different from that of vegetative cells. Gram-negative bacteria are more resistant than Gram-positive bacteria. Effect of a spice or herb may either be inhibitory or germicidal. Spices and herbs harbor microbial contaminants and may serve as substrates for microbial growth and toxin production. Generally, the amount s of spices and herbs added to foods are generally too low to prevent spoilage by microor ganisms. Active components of spices/herbs at low concentrations may interact synergistica lly with other factors (NaCl, acids, and preservatives) to increase pres ervation. Nutrients present in spi ces/herbs may stimulate growth and/or biochemical activities of microorganisms. Lastly, the extent to which spices and herbs inhibit microbial growth depends on their source and processing. For these reasons, effectiveness of the antimicrobial properties of herbs and spice might be questionable considering the concentration at which they are used in the food systems. Therefore, the objective of this st udy was to systematically evaluate the antimicrobial activity of Asian herbs (lemongrass [ Cymbopogon citratus (C. Nees) Stapf (Poaceae)] and turmeric 17

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[ Cucuma longa (Zingiberaceae)]) using di fferent extraction solvents (water, ethanol, hexane and acetone) and drying methods (oven and freeze drying). Lemongrass extracts, from different sources, were evaluated to compare results. Antim icrobial activity of commercial (dried powder) turmeric was also evaluated. Hypothesis was th at Asian herbs may have some antimicrobial properties that may impact growth and survival of microbial pat hogens in Asian foods. In order to validate this hypothesis, Asian herbs were systematically evaluated for antimicrobial properties using a non-polar solvent, slightly polar and highly polar solvents. Results of the study could potentially be used in controlling some pathogens by using some Asian herbs. 18

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CHAPTER 2 LITERATURE REVIEW More than 200 known diseases are transm itted through food (Bryan 1982). In the United States, foodborne diseases have been estimated to cause 6 million to 81 million illnesses and up to 9,000 deaths each year (Archer and Kvenbe rg 1985; Todd 1989). Mead and others (1999) estimated food-related illnesse s, hospitalizations, and deaths from known pathogens to be 13,814, 924; 60, 854; and 1,809, respectively. Bacteria, pa rasites, and viruses accounted for 72%, 21%, and 7%, respectively including Salmonella (31%), Listeria (28%), Toxoplasma (21%), Norwalk-like viruses (7%), Campylobacter (5%), and Escherichia coli O157:H7 (3%). Bennet and others (1987) estimated that there ar e 2,100; 1,200; and 1,000 deaths each year due to campylobacteriosis, staphylococcal food poisoning, and trichinosis, respectively; however, Mead and others (1999) had a total estimate for all thre e diseases as 101 deaths. Severe illnesses caused by Salmonella and Campylobacter accounted for 26% and 17% hospitalizations, respectively. Bacteria causing the most reported fo odborne outbreaks from 1983 to 1997 include Salmonella (3,640), Shigella spp (1,476), Clostridium perfringens (654), Escherichia coli O157:H7 (500), Staphylococcus aureus (487), Escherichia coli enterotoxigenic (209), and Campylobacter spp (146) (Mead and others 1999). Table 2-1 gives reported and estimated cases, number of outbreaks, and deaths caused by known foodborne bacterial pathogens (Mead and others 1999). Table 2-2 summarizes the pathogens most commonly implicated in foodborne illnesses in the United States. It is important to note that these numbers tend to under estimate the actual numbers because foodborne illnesses are often, not reported. Even though the United States has one of the safest food supplies in the world, there are still millions of cases of foodborne illnesses each year, and this may relate to the diversity of ethnic foods ( Beattie 2006). Simonne and Others (2004) examined the CDC foodborne illness 19

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data (1990-2000) and they revealed that Asia n foods had few numbers of foodborne illness outbreaks compared to other ethnic foods. In all, only 79 total outbreaks were reported in Asian foods compared to Italian and Mexican f oods, which accounted for 152 and 147 outbreaks, respectively. Asian foods had a different profile of microorganisms, which included Vibrio parahaelymoticus, associated with Asian seafood dis h. Major microorganism involved in the outbreaks in Asian foods between 1990-2000 was Bacillus cereus (50%). Difference in the profile of microorganisms may be attributed to the ingredients, cooking, and preparation methods, and how food is served (Simonne and ot hers 2004). Asian food usually contains many herb ingredients. Among other factors, the lo wer foodborne outbreak incident in Asian foods could be due to the spices and herbs that are used in this cuisine. Most distinctive feature of Asian cuisines is the blending of seasonings either fresh herbs, spices or a host of unusual aromatics to produce some of the worlds most flavorful food (Hutton 2003). For centuries, people from different cultures around the world have embraced herbs, not only for their flavors and aromas in their cuisines but also for the medicinal qualities of those herbs. In Thailand, cooks love to throw cupfuls of basil ( Ocimum basilicum ) into their stir-fry and soups, treating them no differently than vegetables. In Laos and Cambodia, herbs are used to garnish and flavor noodle soups, broth soups, and salads (Pham 2001). Most Common Asian Herbs and Spices Most common herbs used in Asian dishes include but are not limited to lemongrass ( Cymbopogon citratus ), turmeric ( Curcuma longa), coriander ( Coriandrum sativum ), Chinese chives ( Allium ramosum -L), Thai basil (Ocimum basilicum ), cress ( Lepidium sativum L), chili pepper (Capsicum frutescens), piper Chaba ( Piper sarmentosum ), and finger root ( Boesenbergia pandurata ) (Table 2-3). These herbs add a unique fl avor to many Asian dishes (Dudman 2005). For this study, lemongrass (Cymbopogon citratus ) and turmeric ( Curcuma longa) were used for 20

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the evaluation of their antimicrobial properties. They are also used in main Asian dishes to create different flavors and color in these cuisines. Lemongrass Lemongrass (also known as citronella, fever grass, serai, sereh, takrai) is a perennial herb widely cultivated in the tropics and subtropics with two different designated species, East Indian, Cymbopogon flexuosus and West Indian, Cymbopogon citratus (Simon and others 1984). West Indian lemongrass ( Cymbopogon citratus) consists of many organic compounds such as terpenoids, but the major component is citral. Ot her terpenoids in this species include nerol, limonene, linaloale, -caryophyllene, and a very low content of myrcene (Kasumov and Babaev, 1983). East Indian lemongrass ( Cymbopogon flexuosus) consists of alcohols (20 to 30% citronellol, geraniol) and aldehydes (15% geranial, 10% neral, 5% citronellal). This species of lemongrass is dominantly used in the perfum e industry and has a l onger shelf life (Kasumov and Babaev 1983). Lemongrass, in general, grows in dense clumps up to 2 meters in diameter and has leaves up to one meter long. Lemongrass is a very pungent herb and is normally used in small amounts. Entire stalk of the grass can be used. Lemongrass is widely used in herbal teas, non-alcoholic beverages, baked goods, and confectionary products. Oil from lemongrass is widely used as a fragrance in perfumes and cosmetics products su ch as soaps and creams. Citral, (Figure 2-1), extracted from lemongrass, is used in flavoring soft dr inks, in scenting soaps and detergents, as a fragrance in perfumes and cosmetics, and as a mask for disagreeable odors in several industrial products (Simon and others 1984). Citral is a mixt ure of two geometric isomers of compounds in lemongrass oil. Geranial is the trans isomer of citral in lem ongrass, which accounts for 40-62%, and neral is the cis isomer of citral in lemongrass, wh ich accounts for 25-38% (Simon and others 1984). 21

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Use of fresh lemongrass is typical for Southeast Asia and Sri Lanka. Lemongrass is most popular in Thailand, Vietnam, Cambodia and In donesia. In Thailand, finely ground fresh lemongrass is added to curry pastes Its fine fragrance goes well with poultry, fish and sea food. In Vietnamese cooking, being much less spicy, makes use of lemongrass in several ways including Vietnamese curries. Lemongrass is added to a popular Vietnamese meal bo nhung dam [b nhng d m], often translated vinegar beef or Vietnamese fondue. Lemongrass plant is generally recognized as safe (GRAS) for human consumption and as a plant extract/essential oil (21 CFR section 182.20) (Simon and others 1984). Turmeric Curcuma longa (Zingiberaceae) is a native plant of southern Asia and is cultivated extensively throughout the warmer parts of the world. It is a member of the ginger family, derived from an old Arabic name fo r the kurkum plant known as saffron ( Crocus sativus L.). It is the rhizome of turmeric plant that is used as a spi ce, usually in the dried form. However, in some areas of the Far East, fresh turmeric is used and stored much like ginger (Pulford 2003). Turmeric is used as an herb in Asian cooking such as curry dishes. It can also be added to chutneys, pickles and mustards for its color. Rhizomes contain 3-4% volatile oil with unique aromatic characteristics. Curcumin is the main biologically active phytochemical compound of turmeric (Figure 2-2). Curcumin is the principal curcuminoid of turmeric. Chemical formula of curcumin is C12H20O6. It has a molecular weight of 368.38 g/mol. These curcuminoids are responsible for th e yellow color of the root. In fact it is the curcuminoids that posses all the bio-protective pr operties in turmeric (Badmaev and others 2004). Turmeric has long been used as a powerful anti-inflammator y in both the Chinese and Indian systems of medicine (Gescher and others 2005). Turmeric is also documented to have wound healing 22

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capacity (Biswas and Mukherjee 2003). Turmeric is taken in some Asian countries as a dietary supplement, which allegedly helps with stomach problems and other ailments (Pulford 2003). Due to negative consumer perceptions of artif icial preservatives in foods, attention of the scientific community worldwide is shifting towards spices and herbs to harness their antimicrobial properties for use as natural f ood preservatives (Sagdic and Ozcan 2003). Large variety of these including lemongrass and turmer ic, have been examined for their inhibitory action (antimicrobial activity) against the micr oorganisms responsible for food spoilage and foodborne illnesses (Grag and Me non 2003). However, gaps of know ledge still exist. In this study, lemongrass stem and leaves were evaluated separately; furthermore, dried and fresh turmeric was also evaluated. In this study, se veral factors were considered when doing the research; different sources (two local growers, for comparison purposes), different solvents (hexane, acetone, ethanol and water), drying methods (oven drying and freeze drying) and different bacterial pathogens (Gram positive and Gram negative). This was done to evaluate the results obtained from all these different treatment s and come up with the best combination that could enhance the antimicrobial activity of lemongrass and turmeric extracts. Antimicrobial Activities of Lemongrass (Cymbopogon citratus ) Kim and others (1995) evaluated the antimicr obial activity of citr al (lemongrass oil), carvacrol (thyme oil), geraniol (rose oil), and other oils against five foodborne pathogens; E. coli E. coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Vibrio vulnificus Carvacrol was to found to have the most inhibitory action on the bacteria tested, while citral and geranial had potent antibacterial action against the fi ve test bacteria. Antimicrobial activity of the components was different for each bacterial cultur e and the response of these cultures varied toward each component. Carvacrol (at 250 L/mL) and citral and geraniol (at 500 L/mL) completely inhibited the growth of Salmonella Typhimurium. Lis-Balchin and Deans (1997) 23

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studied 93 commercial e ssential oils against 20 Listeria monocytogenes strains. Lemongrass was among the oils that exhibited antib acterial activity against all the Listeria strains tested. Same essential oil had variable antibacterial activity against different strains of Listeria Williams and others (1998) evaluated essentia l oils with high antimicrobial properties for therapeutic use by comparing the antimicrobial activity of the essential oils of Australian tea tree oil ( Melaleuca alternifolia ) Australian lavender (Lavandula angustifolia ), New Zealand manuka ( Leptospermum scoparium ), lemongrass oil ( Cymbopogon citratus ), and eucalyptus oil ( Eucalyptus cinerea ). Lemongrass oil had the highest antimicrobial activity (inhibition zones) against Candida albicans (30 mm) and Stapyhlococcus aureus (38 mm). Baratta and others (1998) evaluated antimicrobial a nd antioxidant properties of some commercial es sential oils; ylang-ylang ( Cananga odorata ), frankincense (Boswellia thurifera ), lemongrass ( Cymbopogon citratus), marjoram ( Marjorana hortensis ), basil ( Ocimum basilicum ), rosemary (Rosmarinus officinalis ), cinnamon ( Cinnamomum zeylanicum ), and lemon ( Citrus limon) Twenty-five different genera of bacteria (spoilage and foodborne pathogens) and one fungal species ( Aspergillus niger ) were tested in this study. These microorganisms included animal and plant pathogens, food poisoning and spoilage bacteria and the spoilage fungus Aspergillus niger. Volatile oils exhibited considerab le inhibitory action against all th e tested organisms. Oils also demonstrated antioxidant capacities. Hammer and others (1999) inve stigated 52 plant oils and extracts for activity against Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans Enterococcus faecalis Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype Typhimurium, Serratia marcescens and Staphylococcus aureus using an agar dilution method. Lemongrass, oregano and bay inhibited all organisms at 24

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concentrations of 2.0% (v/v). Twenty of the plan t oils and extracts were investigated, using a broth microdilution method, for activity against C. albicans S. aureus and E. coli. Lowest minimum inhibitory concentrations we re 0.03% (v/v) for thyme oil against C. albicans and E. coli and 0.008% (v/v) for vetiver oil against S. aureus. These results supported the notion that plant essential oils and extracts may have a role as pharmaceuticals and preservatives. Chao and others (2000) evaluate d the inhibitory action of es sential oils against a broad spectrum of microorganisms including bacteria, yeas t, molds, and two bacteriophages. Inhibitory action of 45 essential oils on eight bacteria (f our Gram positive and four Gram negative), two fungi, and one yeast were examined using the disc assay method. All the o ils that were tested exhibited antimicrobial activity. Ho wever, several of the plant esse ntial oils exhibited only weak inhibition against several Gram positive bacteria. Gram negative bacteria were generally more resistant than Gram positive bacteria to essential oil treatment with Pseudomonas aeruginosa being the most resistant bacteria. Only cinnamon bark ( Cinnamomum zeylanicum ) and tea tree ( Melaleuca alternifolia ) oils showed inhibitory action against all the test organisms and bacteriophages. Coriander oil ( Coriandrum sativum ) highly inhibited Gram positive bacteria and fungi. Lemongrass ( Cymbopogon flexuosus) and Roman chamomile ( Chamaemelum nobile ) oils showed a high degree of inhibition against both phage types, while some oils showed no inhibition against eith er phage. Angelica ( Angelica archangelica ) and pine ( Pinus sylvestris ) oils inhibited the bacteria, but had no effect on any fungi. Oils that exhibited high antimicrobial properties and the broadest range of inhibition included cinnamon bark ( Cinnamomum zeylanicum ), lemongrass ( Cymbopogon flexuosus ), savory ( Satureja montana ), Roman chamomile ( Chamaemelum nobile ), rosewood ( Aniba rosaeodora ), spearmint (Mentha spicata ) 25

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and tea tree ( Melaleuca alternifolia ). Results of the study showed that lemongrass had a high antimicrobial activity. Mejlholm and Dalgaard (2002) evaluated the anti microbial action of nine essential oils (EO) on P. phosphoreum and also determined the action of oregano oil on the shelf-life of modified atmosphere-packed (MAP) cod fillets. Oils of oregano and cinnamon had strongest antimicrobial activity, followed by lemongrass, thym e, clove, bay, marjoram, sage, and basil oils. Results of the study suggested that herbs essential oils, such as lemongrass, may be used in modified atmosphere-packaged foods to reduce P. phosphoreum. Siripongvutikorn and others (2005) evaluated th e antimicrobial and antioxidant action of Thai seasoning, Tom-Yum Garlic, shallot, kaffir lime leaves chili, galangal, and lemongrass are the spices used in Tom-Yum These spices were extracted us ing water. Fresh garlic had the highest antimicrobial properties of the spices examined in this study. Shallot, lemongrass, galangal, and chili had no antimicrobial action. Th is could be because only water was used for extracting the herbs. Water only ex tracts the water soluble component and leaves other important substances, which could be responsible for the antimicrobial activity of the plants. Sacchetti and others (2005) did a comparativ e evaluation of 11 selected essential oils, namely, ylang-ylang ( Cananga odorata), Italian Cypress ( Cupressus sempervirens) turmeric ( Curcuma longa), lemongrass ( Cymbopogon citratus), Tasmanian blue gum (Eucalyptus globules ), Monterey pine ( Pinus radiata), Piper crassinervium (no common name), guava ( Psidium guayava ), rosemary ( Rosmarinus officinalis ), lemon thyme ( Thymus x citriodorus ), and ginger ( Zingiber officinale ). Among the essential oils test ed, lemongrass oil was the most effective against five food-spoilage yeasts: Candida albicans ATCC 48274, Rhodotorula glutinis ATCC 16740, Schizosaccharomyces pombe ATCC 60232, Saccharomyces cerevisiae ATCC 26

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2365, and Yarrowia lypolitica ATCC 16617. This study demonstrated that not only lemongrass has antimicrobial properties bu t also antifungal properties. Raybaudi-Massilia and others ( 2006) evaluated the antimicrobial activity of essential oils (lemongrass, girasol, and cinnamon) on Salmonella Enteritidis, Escherichia coli and Listeria innocula in fruit juices. Lemongrass, cinnam on, and girasol at concentration of 2 L/mL inactivated the pathogens in appl e and pear juice. Howe ver in melon juice and tryptone soy broth medium, concentrations of 8, and 10 L/mL fo r cinnamon, were needed respectively. For girasol, 6 L/mL was necessary to eliminate the three microorganisms; whereas lemongrass required only 5 L/mL to inactivate them. Results of the study showed th at lemongrass essential oil had strong antimicrobial activity than cinnamon and girasol essential oils. Kakuta and others (2006) tested the antimicrobi al action of essential oils on four oral pathogenic microorganisms. Pure essential oils; tea tree ( Camellia sinensis ), lemon ( Citrus limonium ), peppermint ( Mentha X piperita) sonnerat (Ravensara aromatic), lemongrass ( Cymbopogon citratus ), lemon eucalyptus ( Eucalyptus citriodora ) and geranium bourbon ( Pelargonium graveolens ), and one blended oil (consisted of tea tree, lemon and peppermint oils) were tested against Porphyromonas gingivalis Fusobacterium nuclatum Streptococcus mutans and Candida albicans Minimum inhibitory concentration (MIC) of essen tial oils was measured by liquid dilution assay, and the minimum bactericidal concentrati on (MBC) of essential oils was measured by colony forming assay. Results indicated that MIC of lemongrass oil was the lowest among the eight essential oils, concentration ranging from 0.26 to 0.84% (v/v). Minimum bactericidal concentration of lemongrass oil was also the lowest with concentrations ranging from 0.31 to 1.25% (v/v). On the other hand, Ravensara aromatica oil showed the highest MIC 27

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(more than 5%) among the eight essential oils. Bl ended essential oil showed a lower MBC than any of the pure essential oils, agai nst all test microorganisms except F. nuclatum Schwiertz and others (2006) ev aluated antibacterial and antif ungal activity of ten essential oils against a range of vagina l bacterial and fungal strains isolated from existing vaginal infections including Atopobium vaginae, Gardnerella vaginalis Bacteroides vulgatus Streptococcus agalactiae H2O2-producing lactobacilli and non H2O2-producing lactobacilli, Candida albicans Candida glabrata, Candida parapsilosis, and Candida tropicalis Results revealed that lemongrass, tea tree, and lave nder exhibited the lowest minimum inhibitory concentration (MIC) and minimum bacter icidal concentration (MBC) at 1.5 L/mL, thus being the most potent essential oils against the tested bacteria. Interestingly, the MIC and MBC values of palmarosa, neroli, manuka, rose-scented gera nium, rosemary, common thyme and clary sage were at 7.5 L/mL for protective H2O2-lactobacilli but lower for pathogenic bacteria. Overall, lemongrass, palmarosa, lavender, and rose scente d geranium were the most potent oils in the inhibition of pathogeni c bacteria and fungi. Oussalah and others (2006) evaluated the anti microbial action of sel ected plant essential oils on the growth of Pseudomonas putida strain isolated from m eat. Inhibitory action on 60 plants was examined. Results of the study showed that some plants have strong antimicrobial activity while others had less antimicrobial ac tivity. Lemongrass [stem (bulb) and leaf used together] presented an MIC (0.8%) against Pseudomonas. Rojas-Grau and others (2006) evaluated the mechanical barrier and antimicr obial properties of apple puree edible films containing plant essential oils against Escherichia coli O157:H7. Effect of oregano, cinnamon, and lemongrass in apple puree containing film was investigated along with mechanical and physical properties of the film. Results indicated that the order of antimicrobial activities was: 28

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oregano oil > lemongrass oil > cinnamon oil. Roja s-Grau and others (2007) evaluated the action of plant essential oil compounds on mechanical, barrier and an timicrobial properties of alginateapple puree edible films ag ainst the foodborne pathogen Escherichia coli O157:H7. Results indicated that the antimicrobial activities were in the following order: carvacrol > oregano oil > citral > lemongrass oil > cinnamaldehyde > cinna mon oil. Results of the two studies above indicated that lemongrass essentia l oil had considerable amount of antimicrobial activity. Results also showed that citral, a main component of lemongrass, had stronger antimicrobial activity than the lemongra ss essential oil. Akin-Osanaiye and others (2007) studied the antimicrobial activity of essential oil and extracts of lemongrass and two other herbs. Freeze dried water extracts were used. Results of the study showed that lemongrass oil had high anti microbial activity agai nst all microorganisms tested ( Salmonella typhi S taphylococcus aureus and Escherichia coli ). Maizura and others (2007) evaluated the antimicrobial activity and mechanical properties of partially hydrolyzed sago starch-alginate edible film containing lemo ngrass oil. Results indicated that the film containing lemongrass was effective against Escherichia coli O157:H7 at all levels [0.1-0.4% (v/v)] based on the inhibition zone assay. Kotzekidou and others (2008) studied the antimicrobial activity of plant extracts and essential oils against foodborne pathogens in vitro and against the inoculated pathogens in chocolate. Plant extracts and essential oils used extensively as flavor ingr edients in confectionery products were used as antimicrobials in laborato ry media against the following microorganisms: Escherichia coli O157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus Listeria monocytogenes and Bacillus cereus. Using the disc diffu sion method, inhibition zones in diameter ( > 20 mm) were observed by adding 10 L of each antimicrobial substance on 29

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the following microorganisms: lemon flavor applied on E. coli O157:H7, lemongrass essences against S. aureus, plum flavor on B. cereus strain, and strawberry flavor on L. monocytogenes strain. E. coli O157:H7 strains were the most sus ceptible microorganisms inhibited by 18 extracts, followed by S. Typhimurium and S. aureus, which were inhibited by 17 extracts. Lemon flavor, lemongrass essences, pineapple, and strawberry flavor inhibited the foodborne pathogens at the lowest concentration (5 mL/100 mL). Plant extracts a nd essential oils with potent antimicrobial activities were tested in ch ocolate held at different temperatures (7 and 20C) in dry or humidified environment, which resulted in different aw values of the product (i.e. 0.340, 0.450, and 0.822), in order to determine their efficacy on the fate of the inoculated pathogens. Most inhibition was observed in lemon flavor applied on chocolate inoculated with E. coli cocktail culture after storage at 20C for 9 days. Plant extracts test ed on chocolate showed an enhanced inhibitory action during storage at 20C indicating that th eir application may provide protection for storage at the above temperature or even higher. Antimicrobial Activities of Turmeric ( Curcuma longa ) Huhtanen (1980) tested 33 spices against Clostridium botulinum nutmeg, bay leaf, and white and black pepper (125 ppm) were the most inhibitory while paprika, rosemary, cloves, oregano, turmeric and thyme (500 ppm) only showed small inhib itory action against Clostridium botulinum Results of the study showed that turmeric had a weak an timicrobial activity against Clostridium botulinum. Packiyasothy and Kyle (2002) studied the antimi crobial properties of some herb essential oils: sage, turmeric, thyme, mustard, and fenugree k. These essential oils exhibited antimicrobial activity against Salmonella Typhimurium, Bacillus cereus, Escherichia coli, and Aspergillus flavus Turmeric exhibited a high potency for antimicrobial activity, while fenugreek gave a weak antimicrobial activity. Turmeric exhibited a strong antibacterial activity at concentration of 30

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20 mg/mL. Mandeel and others (20 03) evaluated the antibacterial ac tivity of seventeen selected spices including turmeric. Ethanol extracts we re evaluated against six Gram positive and Gram negative bacteria using a well-diffusion assay. All the spice extracts, except black cardamom, possess biological activity on one or more of the test bacteria. Clove extracts showed the highest antimicrobial activity (19.5 mm) against Escherichia coli followed by bay leaf extracts (19 mm) against the same bacteria, and cumin extracts (19 mm) against Pseudomonas aeruginosa, at 1000 g/100 L. Galangal, turmeric, and fennel extr acts also exhibited a broad spectrum of antimicrobial activity. Results of the two studies above demonstrated that turmeric, essential oils and extracts, at a high concentration were neces sary to exhibit strong antimicrobial activity against test organisms. Goel (2005) evaluated the antimicrobial pr operties of turmeric. Turmeric showed considerable amount of antimic robial activity measured by a standard MIC assay. Pathogens were inhibited at 20-100 g/mL. Among the path ogens tested, no evidence of inhibition was observed on Campylobacter jejuni for up to 100 g/mL. Kim and ot hers (2005) investigated the antimicrobial activity of ethyl acetate, methanol and water extracts of Curcuma longa L. ( C. longa) against methicillin-resistant Staphylococcus aureus Ethyl acetate extract of C. longa demonstrated highest antibacterial activity than that of methanol or water extracts. Results of the study suggested that the et hyl acetate extract of C. longa may have antibacterial activity. Shivani and Ravishankar (2005) studied the antim icrobial action of garlic, ginger, carrot, and turmeric pastes against Escherichia coli O157:H7 in laboratory buffer and a model food system. Turmeric paste, fresh carrot, ginger and garlic pastes from roots, and commercial ginger and garlic paste were heated alone or with bu ffered peptone water (BPW ) or ground beef at 70C for 7 min. All samples were inoculated with a th ree-strain cocktail of overnight cultures of E. 31

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coli O157: H7 and stored at 4C and 8C for 2 weeks. Each paste exhibited different antimicrobial actions alone or in ground beef or BPW at 4C and 8C for 2 weeks. Commercial ginger paste and fresh garlic paste showed the strongest antimicrobial activity with complete inhibition of E. coli O157:H7 in the paste at 3 days fort 4 and 8C. Carrot and turmeric pastes did not show any antimicrobial activity at either at 4 or 8C. Commer cial garlic showed antimicrobial activity at both 4 and 8C (about 1 log cfu/g reduction) in the paste. However, fresh ginger paste showed antimicrobial activity on ly at 8C. Only commercial ginger paste had antimicrobial activity in BPW at 4C for 2 week s. However, commercial ginger paste showed antimicrobial activity in ground beef at 3 days and after (about 1 log cfu/g) compared to control samples at 8C for 2 weeks. Fresh garlic paste showed antimicrobial activity only in BPW (1.3 log cfu/g) at 8C. These results indicated that the antimic robial activity of these pastes is decreased in ground beef and laboratory buffer. This might be because lipids in foods reduce the antimicrobial properties of herbs. Oonmetta-aree and others (2006) evaluated ethanol extracts of Zingiberaceae family (galangal, ginger, turmeric and krachai) for antimicrobial action on Staphylococcus aureus 209P and Escherichia coli NIHJ JC-2 by using agar disc diffu sion method. Galangal extract had the strongest inhibito ry action against Staphylococcus aureus. Results showed that Staphylococcus aureus (Gram positive bacteria) was more sensitive than Escherichia coli (Gram negative bacteria). Turmeric showed less growth inhibition for both Staphylococcus aureus and Escherichia coli Galangal, krachai and ginger had no effect on Escherichia coli All results from the above studies show diffe rent antimicrobial activity when different plants were used against many groups of micr oorganisms. Difference in results depends on factors including extraction solvents, type of mi croorganism studied, plant parts, type of herbs 32

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used, oils or plant extracts, and the concentrati on of the oil or extract. Therefore, this study focused on a systematic evaluation of the antim icrobial activity of lemongrass and turmeric extracts against the following bacteria strains ( Salmonella serotypes Staphylococcus aureus and Escherichia coli O157:H7) using four extraction solvents (water, ethanol, hexane and acetone), and two drying methods (freeze drying and oven drying). Drying Methods Many herbs and spices are often used in dried form. This is because drying preserves the herbs and spices, prolonging their shelf life (R eynolds and Williams 1993). Drying also slows down enzymatic reactions but does not eliminate them (zcan and others 2005 ) When the food is ready for use, the water maybe added back and the food returns to its or iginal shape. Foods can be dried in the sun, in an oven or in a f ood dehydrator by using the right combination of temperatures, low humidity and air current (Ho and others 2002). Optimum temperature for drying food is 140 F (Reynolds and Williams 1993). If higher temperatures are used, the food will be cooked in stead of drying. When the food is cooked on the outside and the moisture cannot escape, "case ha rdening" can occur. Food will eventually be moldy. Lowering humidity aids the drying process a nd the water must move from the food to the surrounding air. If the surrounding air is humid, th en drying will be slowed down. Furthermore, an increase of the air current speeds up the drying process by moving the surrounding moist air away from the food. To reduce the drying time, the air flow should be increased. Most foods can be dried indoors using mode rn food dehydrators, counter-top convection ovens or conventional ovens. Microwave ovens are recommended only for drying herbs, because there is no way to create enough air flow to dry denser foods (Venskutonis 1997). There are many existing methods for drying foods including herbs and spices. Initia lly, salting and drying in the sun, an open room or on stove tops were the accepted methods. It wasn't until 1795 that the 33

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first dehydrator was introduced in France for dr ying fruits and vegetables. Today a variety of dried foods in the marketplace has created a multimillion dollar industry (Hughes and others 1994). Currently oven-drying, freeze-drying, dehy drator-drying (solar and electric) and microwave vacuum drying are among commonly used drying methods (Hughes and others 1994). This study employed oven-drying and freezedrying methods to dry the lemongrass and turmeric plants. These drying methods were select ed based on different op erating principles as well as on the interest to esta blish the method that could be tter preserve the important antimicrobial components of the plants. Oven-Drying By combining the factors of heat, low humidity and air current, an oven can be used as a dehydrator. An oven is ideal for occasional drying of meat jerky, fr uit leathers, and banana chips or for preserving excess produce like celery or mushrooms (Reynolds and Williams 1993). Principle of oven-drying consists of two distinct phases, an initial fast rate of moisture loss followed by a slower second phase. Initially, when the food surface is wet, water evaporates from the food forming a thin boundary layer of high-humidity air. Thickness of this layer determines the rate of drying in the first phas e of drying. Positive air movement over the fruit surface reduces the thickness of the high-humidity layer, which increases the evaporation rate. During the second phase of drying, the rate of moisture loss decreases. Second phase begins when the rate of moisture movement to the surface of the food is less than the rate of evaporation from the surface that is, the speed of drying is limited by the rate at which moisture can move through the food tissue. Circulation can be improved by placing a fan outside the oven near the door. Oven thermometer placed near the food gives an accurate reading. Temperature dial should be adjusted to achieve the need ed temperature of 140F. Trays should be narrow enough to clear the sides of the oven and should be 3 to 4 inches shorter than the oven from front 34

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to back. Oven racks, holding the trays, should be 2 to 3 inches apart for air circulation (Ho and others 2002). Freeze-Drying Freeze-dying is a technique that forms a vacu um while the food is freezing. Water in food specimen is frozen and then removed by a process of sublimation, from solid to vapor phase, maintaining the structure and chemical composition of the food. This avoids the use of chemical dehydration and can be conveniently used to prepare bulk specimens. Boiling or hot-air dehydration frequently impairs the chemical composition of a medicinal product. Freeze-drying is frequently used in the commercial manufacture of antibiotics, vaccines, nutritional supplements, and food products, because the t echnology employs very low temperatures and high vacuum, and thus, it avoids overheating and perhaps deteriorating the organic product (Brennan 1994). Freeze-drying is based on a principle of quick ly extracting humidity from a product with a minimum of alteration of its molecular structur e. If a product contains frozen water and is exposed to a very low atmospheric pressure a vi rtual vacuum the water vaporizes directly. As it does not go through the liquid state, steam requires a minimum of space to get out. If the freezing process is done adequately, the structure of the organic matter is not altered. When a food product is frozen very quickly, the water form s small ice crystals, and when heat is applied in vacuum, the tiny water crystals are sublimate d. Ice water crystals are converted directly to steam, without breaking the mol ecular structure. (Brennan 1994). Freeze-drying process is classi cally split into three stages : freezing, primary-drying, and secondary-drying (Cameron 1997). Primary-drying is the removal of the free moisture that has been frozen and the secondary-d rying phase is the de sorption of bound moisture. However, the 35

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boundary between the primary and the free drying is not a clear-cut process. Freeze-drier operates in a similar way during both stages of freeze drying (Cameron 1997). There is a need for technology development in process monitoring, particularly in developing a way to measure the status of th e product during freezing and freeze-drying without placing temperature measurement probes in indi vidual vials of product. Current practice of placing thermocouples in vials is uncertain with respect to reliab ility of the data, inconsistent with elimination of personnel in close proxi mity to open vials of product in an aseptic environment, and incompatible with technology fo r automatic material ha ndling in freeze-drying. In addition, a method for controlling the degree of super cooling during freezing would allow better control of freezing rate and would, in many cases, result in more consistent product quality (Nail and others 2002). Botanical samples are often freeze-dried (lyophi lized) for use in research studies, and a variety of freeze-dried botanicals are marketed to the public. In both instances, there is an underlying assumption that freeze-drying properly pr eserves the medicinal qualities of plants, and is superior to other preserva tion methods (Abascal a nd others 2005). In fact little systematic research has been done to verify this assumpti on. Review of the existing research, done primarily by the food and spice industry, indicates that fr eeze-drying has unanticipated and significant effects on the constituent profiles of medicinal plants that puts into question whether freezedrying necessarily is the best me thod to preserve botanical medici nes. Lerici (1976) performed a research study to investigate the retention of volatile organic compounds in a freeze-dried model food gel and the data from the study suggested a theory about the different mechanisms, which influence the retention of volatiles during freez e-drying of foods. Van Sumere and others (1983) concluded that freeze-drying of biological material, which is to be quantitatively analyzed 36

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(micro-amount level) for compounds of low or inte rmediate molecular weight, should be either omitted or handled under a strict control. This is because compounds such as amino acids, sugars, flavonoids, glycosides, coenzymes, peptid es, etc., might be removed from concentrates and (or) the ground biological material by the hi gh vacuum. Study by Asami and others (2003), aimed at comparing the total pheno lic and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn grown usi ng conventional, organi c, and sustainable agricultural practices, showed that freeze-drying preserved higher levels of total phenolics in comparison with air-drying. Research review by Abascal and others (2005) found there is insufficient information to conc lude that freeze-drying has ne gative effects on the medicinal qualities of plants. However, because existing re search indicated that freeze-drying imperfectly preserves important classes of medicinal co mpounds (such as volatiles, phenolics and carotenoids), may increase the mu tation rate in unicellular orga nisms, and may diminish some medicinal plant actions, research ers and practitioners should caref ully consider how the use of freeze-dried material may affect pharm acological and clinical study results. Several research studies on th e effect of drying methods show different results depending on several factors such as the type of food and the compounds of in terest studied. In a research aimed at studying the effect of drying on the volatile constituents of thyme and sage, two methods were used to study the effect of drying on aroma constituents of the widely used herbs thyme ( Thymus vulgaris L.) and sage (Salvia officinalis L.). Volatile constituents of herbs (fresh, freeze-dried and oven dried at 30C and 60C) were isolated by dynamic headspace and simultaneous distillation-extract ion methods and analyzed by capillary gas chromatography and coupled gas chromatography-mass spectrometry. In total, 68 compounds were identified in thyme and 44 in sage, and more than 100 compon ents were screened quantitatively. Significant 37

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reduction in the amount of extract ed volatiles was found only in the case of oven drying at 60C, mainly as a result of the loss of non-oxygenated monoterpenes. Character of the changes in the headspace volatiles was more complex, especially for thyme, and the content of aroma compounds were the highest when the herb wa s dried at 60C (Venskutonis 1997). Mahanon and others (1999) evaluated the effect of oven drying at 50oC for 9 hours, 70oC for 5 hours and freeze drying on retention of chlorophyll, riboflavin, niacin, ascorbic acid and ca rotenoids in herbal preparations consisting of eight medicinal plants. Medicinal plan ts were leaves of Apium graveolens (saderi), Averrhoa bilimbi (belimbing buluh), Centella asiatica (pegaga), Mentha arvensis (pudina), Psidium guajava (jambu batu), Sauropus androgynous (cekor manis), Solanum nigrum (terung meranti) and Polygonum minus (kesum). Results of the study revealed that both type and conditions of the drying trea tments affected retention of all phytochemicals analyzed. Herbal preparations developed us ing oven-drying was found to have inferior phytochemical content compared to those obt ained by freeze-drying method. However, herbal preparations developed using all treatments still retained appreciable amount of phytochemicals studied especially carotenoids, as corbic acid, niacin and ribofla vin, and thus, have potential for commercial purposes. Julkunen-Tiitto and Sorsa (2001) conducted a st udy to test the effects of drying methods on willow flavonoids, tannins, and salicylates. They compared the effects of several preservation methods on the secondary phenols of the mature leaves of purple willow ( Salix purpurea L., salicaceae) with those in fresh l eaves. Conventional freeze-drying, in which the leaves were first frozen with liquid nitrogen and then placed in a freeze-dryer, produced substantial qualitative and quantitative changes on willow flavonoids and sa licylates. Modified freeze-drying, in which leaves were put into a freeze-dryer without being pre-frozen, gave comparable concentrations of 38

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most secondary components in fresh leaves. Reducing the freeze-dryer chamber temperature delayed the decomposition of phenolics in pref rozen leaves and in leaves dried without prefreezing. Heat drying induced substantial changes in the com position of all phenolics, except for apigenin-7glucoside. Vacuum drying at room temperature gave the highest concentrations for nearly all phenolics, while room temperaturedrying with desiccation ga ve results that were comparable with those obtained by fresh leaf analyses. Effect of different drying treatments on the volatiles in bay leaf ( Laurus nobilis L.) was studied by Prez-Coel lo and others (2002). Four drying treatments were employed: air-drying at ambient temperature, oven-drying at 45oC, freezing, and freeze-drying. Oven-drying at 45o C and air-drying at ambient temperature produced quite similar results and caused very little loss in volatiles as compared to the fresh herb; whereas, freezing and freeze-drying brought about substantial losses in bay leaf aroma and led to increases in the concentr ation levels of certain compon ents, e.g., eugenol, elemicin (3,4,5trimethoxyallylbenzene) and spathulenol. Among the performed studies on th e effects of drying methods, none showed the effects of drying methods on the antimicrobial properties of herbs, thus this study seeks to evaluate oven drying and freeze drying method in order to determ ine the drying method that results in a better retention of the antimicrobial propertie s of lemongrass and turmeric plants. Extraction Solvents Solvents differ in their extraction capabilities depending on their own chemical properties and the solutes chemical structures. Other factors affec ting solvent selection are boiling point, density, surface tension, viscosity, corrosiveness, flammability, toxicity, stability, compatibility with product, availability, and cost (Cowan 1999). Many types of solvents are available for extracting plant materials including water, etha nol, acetone, hexane, dimethyl sulfoxide, and petroleum ether. Most widely used solvents are wa ter and ethanol. Water is often referred to as a 39

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universal solvent, (while ethanol is widely used in alcoholic beverage s). For this study, water, ethanol, hexane, and acetone were used. This is because all the solvents have different properties; therefore they differ in the substances they extract. It is very important to utilize a variety of solvents to be able to determine the one that gives the highest antimicrobial activity in lemongrass and turmeric, as well as other herb s. Table 2-4 summarizes the properties of the solvents that are discussed below. Dimethyl Sulfoxide (C2H6OS) Dimethyl sulfoxide (DMSO) is an important polar solvent. It is less toxic than other members of its class such as dimethylformamide. It is a colorless liquid with slight odor. It has an excellent solvating power th at dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as wa ter. Main problem with DMSO as a solvent is its high boiling point (189oC), thus its solutions are not typically evaporated but instead diluted to isolate the reaction product (Cheremisinoff 2003). Water (H2O) Water is referred to as the universal solvent dissolving many types of substances. Hydrophilic (water-lovin g) substances mix and dissolve well with water (Cowan 1999). Properties and nature of wate r are due largely to the str ong intramolecular hydrogen bonding within the water molecule and intermolecula r hydrogen bonding between water molecules. It expands upon freezing. Water has a melting point of 0oC and a boiling point of 100oC. Water is a highly polar solvent with polarity index value of 9 (Cheremisinoff 2003). In the presence of miscible organic solvents, water might display less polarity and hydroge n bonding character. In the home, dried plants can be ingested as teas (p lants steeped in hot wate r) or, rarely, tinctures (plants in alcoholic solutions) or inhaled via st eam from boiling suspensions of the plant parts. Initial screenings of plants for possible antimi crobial activities typically begins by using crude 40

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aqueous or alcohol extractions, and then followed by various organic solvent extraction methods (Cowan 1999). Ethanol (C2H6O) Ethanol also known as ethyl alcohol, drinking alcohol or grain alcohol is a flammable, colorless, slightly toxic chemical compound Ethanol is best known as the alcohol found in alcoholic beverages (Cheremisinoff 2003). In a common usag e, ethanol is often referred to simply as alcohol Ethanol has a melting point of 114.3C (158.8 K) and a boiling point of 78.4C (351.6 K). It is volatile, and has a burning taste. Ethanol has no residual odor. Ethanol is commonly used for dissolving medicines, food fl avorings and coloring agents, which are not water soluble. Ethanol can dissolve both polar and non-polar substances. Hydrophilic OH group in ethanol helps dissolve both polar molecules and ionic substances (Scheflan and Jacobs 1953). Hexane (C6H14) Hexane is frequently used as an inert solven t in organic reactions because it is very nonpolar. Hexane is a colorless liquid with a melting point of 95C (178 K) and a boiling point of 69C (342 K). It is volatile and has a faint p eculiar odor (Scheflan and Jacobs 1953). Among its many uses, hexane is a solvent for extracting cooking oils (Cheremisinoff 2003). Acetone (C3H6O) Acetone is the strongest solven t available. Acetone is a colo rless, flammable liquid with a freezing point of 94.6C and boiling point of 56.53C. It has a relative density of 0.819 (at 0C). It is readily soluble in water ethanol and ether In laboratory, acetone is used as a polar solvent in a variety of organic r eactions (Scheflan and Jacobs 1953). Effect of Extraction Solvents on the Antimicrobial Properties of Herbs Very few studies have evaluated the ability of various solvents to solubilize antimicrobials from plants. None of the studies have used wa ter, acetone, hexane and ethanol together in one 41

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study to determine a solvent that yields the highest antimicrobial activity of herbs. Therefore, this study used all four solvents to determine the best solvent to extrac t antimicrobial compounds from lemongrass and turmeric. Elloff (1998) examined different solvents to determine which solvent should be used for the screening and isolation of antimicrobial com ponents from plants. Focus of this study was to provide a more standardized ex traction method for the wide vari ety of researchers working on diverse settings. Freeze dried and finely ground leaves of two plants with known antimicrobial activity, Anthocleista grandiflora and Combretum erythrophyllum were extracted with acetone, ethanol, methanol, methylene dichloride, methano l/chloroform/water and water at a 1 to 10 (v/v) ratio in each case. Acetone gave the be st results with these plants followed by methanol/chloroform/water, methylene dichloride methanol, ethanol, and water. Afolayan and Aliero (2006) examined a variety of solvents fo r their ability to solubi lize antimicrobials from plants. This study used acetone, methanol, and wa ter for extraction. Result s showed that acetone and methanol extracts were active against the Gram positive and Gram negative bacteria at a concentration of 5 mg/ml. Antimicrobial activit ies of acetone extracts were stronger on Gram negative bacteria than on Gram positive bacteria while the methanol extract displayed more antimicrobial activity on Gram positive bacteria. There was little or no antimicrobial activity from any water extracts. Rojas and others (2006 ) examined the antimicrobial activity of ten medicinal plants used in Colombian folk medici ne by using ethanol, hexane and water to extract the plant materials. Results show ed that the water extracts of Bidens pilosa L., Jacaranda mimosifolia D.Do n, and Piper pulchrum showed a higher antimicr obial activity against Bacillus cereus and Escherichia coli than that of gentamycin sulfate (antibiotics). Similarly, the ethanol extracts of most species were active against Staphylococcus aureus, except for Justicia secunda 42

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This means that different plants have differe nt chemical components and some of which are polar and other are non-polar compounds. Water extracts water soluble components (polar components) while ethanol extract s both polar and non-polar substa nces. This means they will produce different results on the an timicrobial properties of the plants based on the chemical constituents of that particular plant. Laohakunjit and others (2007) ev aluated the antibacterial action of Zingiberaceae essential oils using water, ethanol, and petroleum ether. Turmeric oil was among the oils that demonstrated antibacterial activity. Ethanol extr acts gave the highest antimicrobial activity against the Gram positive bacteria. Gram negative bacteria were resistant to Turmeric oil (no inhibition zone). In this study; water, hexane, ethanol, and acet one were used for the extraction because they have different properties (Table 2-4). This study was aimed to determine, which solvent yielded the highest antimicrobial activity in le mongrass and turmeric plant extracts. Evaporation Techniques During the extraction of the plant materials, di fferent solvents are used. These solvents are then evaporated in a variety of ways. There are many types of evaporation techniques. These include stationary evaporation and rotational type evaporation. Evaporation using a rotary evaporator (rotavapor) is the most common method used to separate a solvent from plant extracts. This study used the rotavapor evaporat ion technique because it evaporates solvents efficiently without overhea ting the plant extracts. Rotary Rotavapor Rotary evaporator (rotavapor) is essentially an evaporation unit with a rotating evaporation flask, which was invented by Buchi. Vacuum eva poration is the most fr equently used method because it is more efficient. Rotavapor will eva porate solvent at a much faster rate than the 43

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stationary evaporation flasks. It s rotating flask produces a very efficient transfer of heat, whilst ensuring thorough mixing and avoiding local ov erheating of the contents (Buchi 2008). Rotation transfers a thin film of the liquid sa mple to the whole inner surface of the flask, markedly increasing evaporation rate and assisting heat transfer from the heating bath. Rotating flask and vapor duct have a sealing system, which allows operation under vacuum, further accelerating the evaporation proces s because of the reduction in boiling point of the solvent and efficient removal of the vapor pha se. Vacuum operation also permits heat-labile materials to be successfully concentrated without degradation (Buchi 2008). 44

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45 Table 2-1. Reported and estimated cases, num ber of outbreaks and deaths caused by known foodborne bacterial pathogens from 1983 to 1997. Pathogens Reported Cases Outbreaks Deaths % Salmonella spp 1,412,498 3,640 31 Shigella spp 448,240 1,476 0.8 Clostridium perfringens 248,520 654 0.4 Escherichia coli O157:H7 73,480 500 3 Staphylococcus aureus 18,5060 487 0.1 Listeria monocytogenes 2,518 373 28 Escherichia coli enterotoxigenic 79,420 209 0 Campylobacter spp 2,453,926 146 5.5 This table demonstrates the reported and es timated cases, outbreaks and deaths caused by known foodborne bacterial pathogens by Mead and others (1999). Bact eria are arranged according to the number of reported and estimated outbreaks, starting with the bacteria with the highest outbreak. Numbers of outbreak rel ated cases were calculated as the average annual number of cases reporte d to CDC from 1983 to 1997.

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Table 2-2. Most common pathogens associ ated with foodborne illness outbreaks Pathogens Reservoirs Associated foods Symptoms/Incubation period Salmonella spp. Humans and domestic or wild animals Meat, poultry, egg or milk products Headache, abdominal pa in, diarrhea, nausea, vomiting, dehydration, fever and loss of appetite. Symptoms appear with in 6-72 hours after the ingestion of the organism and may persist for as long as 2-3 days. Death is uncommon, except for the very young, very old and the immuno-compromised. Shigella spp Humans are the most significant source. People may carry this pathogen for several weeks and excrete it in their feces. Ready to eat foods: salads, raw vegetables, dairy products, and poultry This illness is usually characterized by diarrhea, cramps and chills, often accompanied by fever. Symptoms usually appear within 12-96 hours, but can take as long as one week. Duration of illness is usually 4-7 days. Clostridium perfringens The bacteria can be found in soil, dust, sewage, and intestinal tracts of animals and humans. The organism grows in little or no oxygen. Foods that have experienced temperature abuse such as meat dishes (gravies and beef stew) Diarrhea and gas pains about 8 to 24 hours after eating. The illness usually lasts 1 day, but some symptoms may last 1 to 2 weeks for the elderly or very young. E. coli O157:H7 Humans and animals Hamburger and other ground meat products Severe abdominal pain, cramps, nausea, vomiting, diarrhea and occasionally fever. Hemolytic Uremic Syndrome (HUS) is a serious consequence of this disease and is the leading cause of kidney failure in children. 46

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47Table 2-2. Continued Pathogens Reservoirs Associated foods Symptoms/Incubation period Staphylococcus aureus Humans are the most common source, but cows, dogs and fowl also can be a source. Contaminated ready-to-eat foods, Custardor creamfilled baked goods, ham, tongue, poultry, dressing, gravy, eggs, potato salad, cream sauces, sandwich fillings Nausea, vomiting, diarrhea, dehydration, cramps, subnormal temperatures and lowered blood pressure. Symptoms appear within 30 minutes to 7 hours (2-4 hours is most common) after eating the contaminated food, and may last for up to 24-48 hours. Listeria monocytogenes The organism is frequently found in environment such as soil, water and plant matter that animals ingest and excrete, allowing further transmission. Ready to eta foods Nausea, vomiting, headaches, delirium, coma, collapse, shock and lesions on vital organs. In pregnant women, the illness can cause a miscarriage or result in stillbirths. Listeriosis may also ca use severe retardation, meningitis and death in newborns. Campylobacter jejuni Healthy chicken carry this bacteria in their intestinal tracts sometimes causing the contamination of raw poultry. Raw milk can also be a source; the bacteria are carried by healthy cattle and by flies on farms. Non chlorinated water may also be a source for the infection. contaminated water, raw milk, and raw and undercooked meat, poultry or shellfish Initial symptoms include fever, headache, and muscle pain followed by diarrhea, abdominal pain, and nausea. These symptoms may appear 2 to 5 days after eating and may last up to 7 to 10 days. This table gives a brief description of th e bacterial pathogens mostly associated w ith foodborne outbreaks. This table outlines the reservoirs, foods associated with pathogens and the symptoms/incubation period associated caused by bacterial pathogens. Sources: Beattie Sam (2006), FDA Bad bug book (2007), CDC (2005)

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Table 2-3. Common Asian herbs used for cooking Botanical Name Common name Parts used in cooking Cymbopogon citratus Poaceae or Gramineae Lemon grass stem, leaf Curcuma longa Zingiberaceae, or the Ginger family Turmeric Rhizome Coriandrum sativum Apiaceae or Umbelliferae Corienda (Cilantro leaves) Leaves Allium ramosum L. Alliaceae Chinese chives Stem Ocimum basilicum Lamiaceae or Labiatae Thai Basil Leaf Lepidium sativum L. Brassicaceae Cress Leaf Capsicum frutescens Solanaceae Chili pepper Whole plant Piper sarmentosum Piperaceae Piper Chaba Leaf Boesenbergia pandurata Zingiberaceae Finger root Rhizome, finger root This table presents some of the Asian herbs used for cooking. It gives the botanical name, common name and the different parts that are used in cooking. 48

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49 Table 2-4. Properties of different solvents Solvents Chemical formula Boiling point (oC) Melting point (oC) Molecular weight (g/mol) Dielectric constant (Debye) Dipole moment Index Polarity Dimethyl Sulfoxide C2H6OS 189 18.45 78.14 47.2 3.96 7.2 Water H2O 100 0 18.02 80 1.85 9 Ethanol C2H6O 78.4 -114.3 46.07 24.3 1.69 5.2 Hexane C6H14 69 -95 86.18 2.02 ---0 Acetone C3H6O 56.5 -94 58.08 20.7 2.88 5.1 This table above presents the di fferent properties of dimethyl sulfoxide, water, ethanol, hexane and acetone. Solvents are arranged according to their boiling point, starting from the highest to the lowest boiling point.

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Figure 2-1. Chemical st ructure of Citral (C10H16O) (3, 7-dimethyl-2, 6-octadienal) Figure 2-2. Chemical structure of Curcumin (C21H20O6 ) 50

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CHAPTER 3 MATERIALS AND METHODS Bacterial Strains and Cultivation Three strains of E. coli O157:H7 [204P, 301C, 505B] from Dr. Tun-Shi Haung (Auburn University, Auburn, Ala), three strains of Salmonella species [ Salmonella Thompson Salmonella Enteritidis, Salmonella Typhimurium], and three stains of Staphylococcus aureus [ATCC 29247, ATCC 12600, and ATCC 35548] were used as tested organisms in all antibacterial assays. Both Salmonella and Staphylococcus aureus strains were obtained from American Type Culture Collection (ATCC). These organisms were sel ected because they are among many pathogens often implicated in foodborne outbreaks in the Un ited States. Different strains of bacteria were streaked onto Tryptone Soy agar (TSA) (Becton, Dickinson and Company Sparks MD) to obtain pure isolated colonies, following a standard asep tic technique and the fo ur-way streak plate inoculation (Cappuccino and Sherman 2005). Once the isolated colonies were obtained, the bacterial strains were enumerat ed with Mueller Hinton Broth (M HB) for the next step of the experiment, the antimicrobial assay. Plant Materials Lemongrass ( Cymbopogon citratus (C. Nees) Stapf (Poaceae)) samples were obtained from two local growers in Gainesville, FL. Firs t harvest was in October 2007 and second harvest was in November 2007. Figure 3-1 di splays the lemongrass plant ( Cymbopogon citratus) Turmeric (Curcuma longa) samples were obtained from a major produce market in Atlanta, GA (DeKalb Farmers Market). Two form s (fresh rhizomes and dried powder from) of turmeric were obtained. Figure 3-2 displa ys fresh rhizome turmeric samples. 51

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Preparation of Plant Material Lemongrass ( Cymbopogon citratus (C. Nees) Stapf (Poaceae)) l eaf (Figure 3-3) and stem (Figure 3-4) and turmeric ( Curcuma longa) were used for examining the antimicrobial activity against all nine bacterial strains. Plant samples were washed and sanitized in a 6% sodium hypochlorite solution (50 ppm), (Clorox compan y; Oakland, CA) (Ritenour and others 2002). After washing, the plant materials were left in the biological hood (LABGARD Laminar Flow Biological Safety Cabinet; nuAire, Inc) to get rid of excess water, and then they were thinly sliced (approximately 25 mm) a nd flatly spread in oven trays (13 in x 9 in) and freeze drier containers (melamine organizer; 15 in x 6 in x 2 in). Plant materials in ovens trays were dried in an oven-drier (Scientific Fisher Isotemp oven St andard 600) set at 60oC for 48 hours. For samples to be freeze-dried, deionized water was a dded to the same level of the plants in the freeze-drying containers and was first frozen in a regular freezer (Kenmore 19) set at -20oC overnight, and then taken to th e freeze-drier (Model 25 SRC Vir tis Company Gardiner, NY) for 72 hrs. After drying, the plants were gr ounded using a 12-speed blender (Hamilton Beach; Michael Graves Design) for 5 minutes. Preparation of Plant Extracts Ten grams of dried ground plant material (weigh ed by Fisher Scientific scale) was added to 500 mL conical flasks, and 150 mL of each solv ent (water, acetone, hexane, and ethanol) was added to each flask. Ratio (1 to 15) of the plan t materials and solvents was determined following a standard method for evaluation of antimicrobi al in foods (Parish and Davidson 1993). Each mixture was placed on a shaker (Excella E1 plat form, New Brunswick Scientific) and left to extract for 18 hours at a speed of about 133 rpm at room temperature (25oC). Extract was then filtered using a conical flask with side arm, a f ilter funnel (size 2), and a 90 mm diameter filter paper (Whatman #1). Filtered extract was then poured in a weighed 500 mL round bottom flask. 52

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Solvent was evaporated with a rotary evaporator (Buchi). Temperature of the water bath in the rotavapor was set at 40oC. This temperature was used because the evaporation under reduced pressure makes it possible to evaporate at mu ch lower temperatures. Evaporation time ranged from 10 to 30 minutes depending on the solvent type After evaporation of solvent, the flask was weighed again. To determine the weight of the sa mple, the weight of flask was subtracted from the weight of the flask and sample. Dimethyl sulfoxide (DMSO) and water was used to reconstitute the extracts in order to make a st ock solution using volumetric flasks. Extracts were then sterilized by filtration using 0.45m aqua membrane nylon filter disk (Becton, Dickinson Company). Concentration was recorded on a wei ght by volume (w/v) basis. Stock solution was stored it the freezer of a regular refrigerator (General Electric; A ssociation of Home Appliance) set at 20oC. Working solutions were made from the stock solution. C oncentration of the working solution for lemongrass was 125 mg/m L, and 200 mg/mL for turmeric extracts. Lemongrass has a strong flavor, and therefore, is being used in very small amounts to add flavor in foods. Turmeric on the other hand is being used mainly to enhance color and to add a unique taste to the food. Flavor of turmer ic is not as strong as lemongrass, thus it is being used at slightly high concentrations than lemongrass ex tract. Filtered and recons tituted extracts were then prepared for the evaluation for antimicrobial properties against the nine bacterial strains This was done by taking 20 L of each plant ex tract and impregnating a 6 mm blank paper disc (Becton, Dickinson Company) and leaving them in the biological hood (LABCONCO Purifier Class II biosafety cabinet; Kansas, Missouri) to dr y. Concentration of the plants in the discs was 2.5 mg/disc for lemongrass and 4 mg/disc for turm eric. Negative controls were prepared the same way as the extract impregnated discs but, using 95% ethanol (number: 61511-0040; ACROS, New Jersey), to make the discs ster ile. Antibiotics (imipenem and ampicillin 10 53

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g/disc) (Becton, Dickinson and Company Spar ks, MD) were used as positive reference standards or control. Antimicrobial Assay Five isolated colonies of each bacterial stra in was inoculated in 5 mL of Mueller Hinton Broth (MHB) (Becton Dickinson Microbiology Syst ems) using a sterile wood stick. The 5 mL tubes were then incubated at 37oC for approximately 2 hours or until the bacterial density reached 1.5x 108 cfu/mL (Casimiri and Burstein 1998 ) Bacterial density was determined by using a spectrophotometer (Spectronic 20+ series, Thermo Electron Co rporation) at wavelength of 600 nm. Blank of MHB alone was used to ca librate the spectrophotome ter. After calibrating the spectrometer, the inoculated tubes were put in the spectrophotomet er and the reading was taken. When the bacterial density was at the desirable level, the r eading was 1.32. When the reading was below that level, the tubes were incubated a little longer; and when the tubes reading was above that level, the bacterial suspension wa s adjusted by diluting the inoculated bacteria with MHB until it reached the desirable level. Wh en the bacterial density reached a desirable level (1.5 x 108 cfu/mL), 100 L bacterial suspension for each bacterial strain were spread on to the surface of the Mueller Hinton Agar (MHA) (Difco Becton, Dickinson and Company Sparks DM) using sterile swabs (Casimiri and Burstein 1998 ) The 6 mm diameter filter paper discs (containing 20 L of the particular plant extract) were applied onto the surface of the spread MH A. Antibiotics; imipenem and ampicillin (10 g/disc) (Becton, Dickinson and Company Spar ks, MD) were used as positive reference standards or control. Negative cont rol (95% ethanol) was also used to make sure that the discs were sterile. Plates were inc ubated at 37C for 24 hours and the inhibition zones were measured to determine the effectiveness of the extract against each organism. Results were expressed as 54

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the diameter (in millimeters) of inhibition for th e paper disk (6 mm) with each extract (Figure 35) (Parish and Davidson 1993). Minimum Inhibitory Concentration (MIC) Determination Minimum inhibitory concentra tion (MIC) is defined as the lowest concentr ation of an antimicrobial that prevents the growth of mi croorganism after a specific incubation time. The MIC was studied for the bacterial strains, bei ng sensitive to the plant extracts in the disc diffusion assay (Parish and Davidson 1993). The MIC was determined by using the 48 well sterile plates (Costar Corning Incorporated, NY) which has a maximum volume capacity of 2 mL. For each well, 0.5 mL of MHB was added. Th en the -prepared discs containing various concentrations of plant extracts were added to achieve the desi rable concentrations from the highest to the lowest as de scribed by previous literatu re (Duke and others, 2003). For lemongrass, the concentrations used were 20, 10, 5, 2.5, 1.25, 0.625, 0.32, and 0.16 mg/mL. For turmeric, the concentrations were 80, 40, 20, 10, 5, 2.5, and 1.25 mg/mL. Concentrations used were based on previous research; le mongrass is said to be effectiv e at lower concentrations than turmeric (Duke and others, 2003). Bacterial inno cula was prepared from MHB cultures and the suspensions density were adjusted to standard turbidity (7.5 x 105 cfu/mL) (Casimiri and Burstein 1998) Bacterial density was measured in the same manner as the antibacterial assay using a spectrophotometer. When the bacterial i nnocula had reached the required density (7.5 x 105 cfu/mL), 5 L of the bacterial suspension was added to each well from the lowest to highest extract concentration. Plates were then incubated at 37oC for 24 hours. Color reaction with piodonitrotetrazolium violet (Bio Medicals, Inc.) was used in lieu of traditional turbidity measurement to eliminate difficulties of observing turbidity for each 48 wells. One hundred micro liters of p-iodonitrotetrazoli um violet (0.2 mg/mL) in water was added to each well in the 48 well plates and the plates were incubated for 30 minutes at 37oC. Color changes were 55

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observed thereafter. Wells show ing red color indicated the b acterial growth (presence of turbidity) and a well with the lowest concentration of plant extract without any red color was determined as the minimum inhibitory concentr ation (MIC). Wells with no color change were used for the determination of minimum bactericidal concentration (MBC). Minimum Bactericidal Concentration (MBC) Determination The MBC is defined as the lowest concentrati on of an antimicrobial agent needed to kill 99.9% of the initial inoculums. Minimum bacter icidal concentration was determined on the selected extracts in the 48 well plates that had th e highest antimicrobial ac tivities (i.e. those that did not have any turbidity on the MIC dete rmination) (Parish and Davidson 1993). Ten L from each well that showed no bacterial growth was ta ken and inoculated in Tryptone Soy agar (TSA) plates that were divided into quadrants. Plates were incubated at 37oC for 24 hours, and then bacterial growth was evaluated. Last quadrant (i.e. with the lowest concentration of plant extract) showing no growth was taken as the MBC. Values were recorded as mg/mL. Experimental Design and Statistical Analysis A factorial design was used to design the e xperiment. Lemongrass f actors were harvest time x 2, trial x 2, pathogen x 3, solvent x 2, and drying method x 2. Fresh rhizome turmeric factors were trial x 2, pathogen x 3, solvent x 3, and drying method x 2. Dried powder turmeric factors were trial x 2, pathogen x 3, and solvent x 3. Microbial testing was done in triplicates for each extract for each bacteria strain. Genera l liner model procedure (PROG GLM) of the Statistical Analyses System was employed and the significance among the different observed antimicrobial activities were evaluated using Duncans multiple range test (SAS Institute, Inc. 1991, Cary, NC) at = 0.05 level. Data generated from the procedures described in the experimental sections were tabulated and discussed in the next chapters. 56

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Figure 3-1. Lemongrass (Cymbopogon citratus ) plants. Source: http://www.paintedflowerfarm.com Figure 3-2. Turmeric (Curcuma longa) rhizomes 57

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Figure 3-3. Lemongrass leaves Figure 3-4. Lemongrass stem 58

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59 Figure 3-5. Inhibitory zone of lemongrass stem oven dried, hexane extracts on Staphylococcus aureus ATCC 29247.

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CHAPTER 4 RESULTS AND DISCUSSION Antimicrobial Activity of Lemongrass Inhibitory Zone of Lemongrass Extracts Lemongrass leaves extracts (water, ethanol acetone, and hexane) demonstrated no antimicrobial activity (data not shown). This may be due to the fact that leaves of lemongrass are exposed to the light. Although the lemongrass leav es contain citral, it is highly volatile and instable to air, light and alkalis, thus citral hardly sustaining its activity in the leave part of the lemongrass (United States Patent 2003; European Patent 2006). Water and ethanol extracts of lemongrass stem (Table 4-1) did not show any antimicrobial activity against any test bacteria l strains. Neither water nor etha nol extract of lemongrass stem yielded any antimicrobial activity (inhibition zone). Water is considered to have large dipole molecules and a high dielectric c onstant. Thus, water is very polar and only miscible in itself. However, the main compounds of the lemongrass are oily substances. Water has been used by other researchers as an extract solvent, most of their results indicated that water extracts gave little or no inhibitory effect (Siropngvutikorn and others 2005 ; Afolayan and Allerozo 2006). One important factor of solvent effectiveness is the active components of the plants. If most of the compounds contained in the plants are miscible in water, then an antimicrobial activity can be observed. According to a study by Rojas and ot hers (2006), some of the water extracts of some plants such as Bidens pilosa L., Jacaranda mimosifolia D. Do n and Piper pulchrum had antimicrobial activity, but others such as Justica secunda did not. This means based on the compounds miscibility in water, some will ha ve antimicrobial action and some will not. Unfortunately, in the home water is the majo r medium in food and drink preparation. 60

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Ethanol is also classified as a polar solvent, although not very polar as water. This means that this solvent is miscible in water and it will extr act mostly the ionic compounds from lemongrass. Ethanol has better di ssolving capabilities than water because it has a slightly low dipole and dielectric constant than water, thus it is slightly polar (Scheflan and Jacobs 1953). Same reason that water did not have an effect as an extraction solvent could be the same reason that ethanol was not effective si nce they are both said to be polar solvents (Cheremisinoff 2003). These results from this study revealed that the benefit of antimicr obial properties from lemongrass stem may not be achie ved under a typical use of the le mongrass in water or ethanol medium. Hexane and acetone extracts of lemongrass st em showed antimicrobi al activity against Staphylococcus aureus strains (Table 4-1). Inhibitory zone of lemongrass stem extracts ranged from 6.5 to 21 mm (also shown in Table 4-1). Lemongrass samples prepared by the oven-drie d methods yielded significantly higher (p < 0.0001) inhibition zone (Table 4-4) than those prepared by the freeze-dried method (with up to 21 mm inhibition zone). Results showed that the oven-dried lemongrass stem gave a better overall antimicrobial activity (inhibition zone) than the freeze-dried lemongrass stem. These results are in accordance with one study that demonstrated that oven-drying produced quite similar results and caused hardly any loss in vola tiles as compared to the fresh herb; whereas, freezing and freeze-drying brought about substantial losses of aroma in bay leaf and led to increases in the concentration levels of certa in components (Prez-Coe llo and others 2002). However, other studies showed that freeze dryi ng method retains compounds better (Mahanon and others 1999; Julkunen-Titto and Sorsa 2001) Results may vary depending on the type of plant and the compounds of interest. There is an underlying assumption that freeze-drying 61

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properly preserves the medicinal qua lities of plants, and is superior to othe r drying preservation methods (Abascal and others 2005). However, little systematic research has been done to verify this assumption (Abascal and others 2005). Our results had proved that the assumption that freeze-drying is better than other dryi ng methods might not always be true. There was no significant difference (P > 0.05) on the level of antimicrobial activity (inhibitory zone) of the lem ongrass stem against the three Staphylococcus aureus strains. Lemongrass stem extracts showed a consider able amount of inhi bition zone against Staphylococcus aureus strains tested in this study. As citral is the main component of lemongrass, some researchers have found that this compound is the on e responsible for the antimicrobial activity of lemongrass (Kim and others 2005; Rojas-Grau and others 2007; and Onawunmi 1989). Results showed that Gram positive bacteria are more sensitive to turmeric and lemongrass stem extracts. These results are in ag reements with previous reports by Zaika (1988); Consentino and others (1999), a nd Karaman and others (2003). Staphylococcus aureus strains are considered the most sensitive bacteria (Oonmetta-aree and others 2006); whereas, Gram negative bacteria showed resistance (i.e. no inhi bition zone) towards the plant extracts. Varying degrees of sensitivity of the test bacteria may be due to both the intrinsic tolerance of microorganisms and the nature and combinati ons of phytochemical compounds present in the essential oil portion of the plants. Outer cell membrane of Gram-negative organisms has several lipid compounds that protect the cells from antimicrobial agents (Shelef and others 1980; Russel 1991) and thus, protecting them from the action of the essential oils. Seasonal variation in lemongrass stem ex tracts was observed with respect to the antimicrobial activity (inhibition zone); plants harvested in November showed a higher activity (p < 0.0001) than plants harvested in October. Inhibition zone of lemongrass stems were by the 62

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harvesting time. Lemongrass harvested in November showed a higher antimicrobial activity than the lemongrass harvested in October. This is in accordance with prev ious research that a composition of essential oils could be in fluenced by time of harvest (Senatore 1996). There was no significant difference (P > 0.05) in the inhibition zone of hexane and acetone extracts of lemongrass stem. Both hexane and a cetone extracts yielded the same inhibition zone (13.7 mm). Acetone and hexane extracts of lemongrass stem displayed a considerable amount of antimicrobial activity. This could be because acetone is considered to be a dipolar aprotic solvent. This means that acetone is of medium po larity; therefore it can dissolve a wide range of compounds (Scheflan and Jacobs 1953) It has a slightly lower diel ectric constant than ethanol, which could aid in not only dissolving wate r soluble compounds but also other compounds, which dissolve in other less polar solvents (C heremisinoff 2003). For acetone, having an extra carbon than ethanol adds to its capability to extr act other compounds other than what ethanol can extract. Hexane has low dielectric constants and is not miscible w ith water. Chemical formula of hexane (Table 2-3) shows it is the least polar solvent. This means because hexane has 100% hydrocarbons, it has the capability to extract most of the hydrocarbon compounds that are in lemongrass or at least the most abunda nt compound of lemongrass, citral. Result of the inhibition zone (Table 4-5) by pathogens show a same trend of antimicrobial activity against the Staphylococcus aureus strains tested. Inhibition zone of the Staphylococcus aureus strains showed that oven-drying had a significantly higher (P < 0.0001) inhibition zone than freeze-dryi ng. Inhibition zone of oven-drie d lemongrass extracts against Staphylococcus aureus strains: ATCC 35548, ATCC 12600-U, and ATCC 29247 were 15.3, 16.1, and 17.4 mm, respectively; while the inhibiti on zone of freeze-dried lemongrass extracts against the same strains were 11.1, 12.1, and 10.3 mm, respectively. Similarly, the 63

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Staphylococcus aureus strains were more sensitive to lemongrass stem extracts harvested in November than those that were harvested in October. Inhibitory zone of November harvested extracts against Staphylococcus aureus strains: ATCC 35548, ATCC 12600-U, and ATCC 29247 were 14.9, 16.0, and 14.6 mm, respectively; wh ile the inhibition zone of October harvested extracts against the same strains were 11.5, 12.2, and 13.1 mm, re spectively. Inhibition zone of hexane and acetone extracts of lemongr ass stem show a different trend against the Staphylococcus aureus strains that were tested. Staphylococcus aureus ATCC 12600-U was more sensitive to hexane extracts with an inhi bition zone of 14.9 mm, than to acetone extracts which had an inhibition zone of 13.3 mm. Interestingly, the Staphylococcus aureus ATCC 29247 and 35548 were more sensitive to acetone extrac ts. Both had an inhibition zone of 13.9 mm, while hexane extracts had an inhibition z one of 12.8 and 12.5 mm, respectively. Even though there was a variation observed among the solvent extracts, they both seemed to exhibit a reasonable amount of antimicrobial activity (inhibition zone). Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Lemongrass Stem Extracts All other pathogens strains tested were not te sted for MIC and MBC as they did not show positive results on the antibacterial assay. The MIC and MBC values of lemongrass stem extracts are presented in Tables 4-4 and 4-5. Results de monstrate a wide range of activities of the different extracts against Staphylococcus aureus strains. Oven-dried lemongrass stem extracts had lower MIC and MBC: 2.2 and 3.3 mg/mL, respectively than those of freeze-dried lemongrass stem which had MIC and MBC of 3.1 and 7.1 mg/mL, respectively. Results demonstrated that, overall, hexane extracts of lemongrass stem gave the lowest MIC (2.3 mg/mL) than acetone extracts (3.6 mg/mL), however there was no significant difference demonstrated by the MBC between the hexane a nd acetone extracts in le mongrass stem. Hexane 64

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and acetone extracts gave an MBC of 4.9 and 5.4 mg/ml, respectively. The MIC of lemongrass stem did not demonstrate a statistical difference in the sensitivity of the Staphylococcus aureus strains. Their sensitivity to th e lemongrass stem was not statistic ally different; however for MBC it was observed that the sensitivity of Staphylococcus aureus strain (ATCC 29247), was statistically lower (p < 0.0001) M BC (4.2 mg/mL) than the two other Staphylococcus aureus strains (ATCC 12600-U and 35548) which pr esented an MBC of 5.9 and 5.6 mg/mL respectively. Results showed that the Staphylococcus aureus strain (ATCC 29247) may be more sensitive to lemongrass stem extracts than the other Staphylococcus aureus strains used in the study. Results of the MIC and MBC of lemongrass st em extracts when analyzed against each Staphylococcus aureus strain showed the same trend as the results discussed above. The MIC and MBC of oven-dried lemongrass stem was significantly lower against all the Staphylococcus aureus strains than MIC and MBC of freeze-dried lemongrass stem extracts. Oven-dried lemongrass stem extracts presented an MIC and MBC of 1.9 and 3.9 mg/mL against both Staphylococcus aureus strains: ATCC 12600-U and 35548; and 0.8 and 2.1 mg/mL against Staphylococcus aureus strain (ATCC 29247). Freeze-dried le mongrass stem extracts presented an MIC and MBC of 4.6 and 8.0 mg/mL; 4.4 a nd 6.3 mg/mL; and 4.4 and 7.1mg/mL, against Staphylococcus aureus ATCC 1600-U, 29247, and 35548, respectivel y. These results show that oven-dried lemongrass stem extracts had a str onger antimicrobial ac tivity agains t all the Staphylococcus aureus strains that were tested. Similar to the results obtained in the inhibition zone, hexane extracts of lemongrass s howed a lower MBC (5.2 mg/mL) against Staphylococcus aureus ATCC 1600-U than acetone extracts which s howed an MBC of 6.7 mg/mL. Both Staphylococcus aureus ATCC 29247 and 35548 had a higher sensitiv ity to acetone extracts with 65

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an MIC and MBC of 1.6 and 3.3 mg/mL; and 2.1 and 4.8 mg/mL, respectively, while hexane extracts showed less sensitivity with a highe r MIC and MBC of 3.5 and 5.0 mg/mL; and 4.0 and 6.2 mg/mL. Difference in the solvent extracts may be due to the previously suggested theory of different mechanisms, which influence the rete ntion of compounds during freeze drying (Lerici 1976). Sensitivity of all the Staphylococcus aureus strains that were tested showed similar results to those obtained in the inhibition zone assay. Al l strains were more sensitive to lemongrass stem extracts from November harvest that those extr acts from October harvest. Lower MIC and MBC from the November harvest was expected because, as seen in the antimicrobial activity (inhibition zone), antimicrobial properties of lemongrass were affected by the harvesting time in accordance with previous research (Senatore 1996). Antimicrobial Activity of Turmeric Extracts Inhibitory Zone of Turmeric (Fresh Rhizomes and Dried Powder) Extracts Water extracts of turmeric (prepared from fresh rhizomes) were observed to have no antimicrobial activity against any of the test bacterial strains (Table 4-2). Water is very polar and is only miscible in itself. This means that wa ter cannot extract non-polar compounds in turmeric Turmeric extracted by ethanol, acetone and he xane demonstrated antimicrobial activity against Staphylococcus aureus strains (Table 4-2). This might be due to the difference in chemical compounds found in lemongrass and turm eric. Differences were observed in their chemical structures presented in Figure 21 and Figure 2-2. First fi gure, which shows the chemical structure of citral, lacks the hydrogen (O-H) bond but contains a multiple bond between the hydrocarbon and oxygen; and yet the chemical structure of curcumin contains two hydroxyl groups and O-H bond. This means turmeric has polar components, which can be easily dissolved in ethanol because ethanol also has a hydroxyl group. Another study showed that ethanol gave a 66

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better antibacterial activity on turmeric plan ts compared to petroleum ether and water (Laohakunjit and others 2007). Inhibitory zone of turmeric extracts against the Staphylococcus aureus strains ranged from 6.5 mm 11 mm. E. coli O157:H7 and Salmonella strains were not affected by any turmeric extracts no matter which solvent or drying method was used. Staphylococcus aureus strain (ATCC 12600U) demonstrat ed a significantly higher susceptibility (p < 0.0001) to tu rmeric extracts than the other Staphylococcus aureus strains (Table 4-6). Turmeric extracts from samples prepared by freeze drying yielded a significantly higher antimicrobial activity (inhibition zone) than those prepared from the oven drying method. No significant difference (p> 0.05) was observed among different solvents (hexane, acetone and ethanol). Turmeric extracts (prepared from dried pow der) demonstrated the same pattern as the turmeric extracts from fresh rhizomes (Table 43). However a statistical difference was observed in the Staphylococcus aureus strains susceptibility to the tu rmeric (dried powder) extracts (Table 4-8). Staphylococcus aureus ATCC 35548 was the most se nsitive (P < 0.0001) to the turmeric extract prepared from dried turmeric powder, followed by the Staphylococcus aureus ATCC 12600, and Staphylococcus aureus ATCC 29247, respectively. Neither E. coli O157:H7 or Salmonella test strains used in this study were sensit ive to the dried powder turmeric extracts. This is because Gram-negative bacteria are more resistant to antimicrobials than Gram-positive bacteria (Shelef and others 1980; Russel 1991). Acetone extracts of fresh turmeric rhizomes gave a significantly higher (P < 0.0001) inhibition zone (8.0 mm) than the extracts prep ared from hexane (8.0 mm), however acetone extracts were not statistically different from inhibition zone of et hanol extracts (8.8 mm). 67

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Acetone is a solvent of medium polarity and th us it can dissolve a wide range of compounds (Scheflan and Jacobs 1953). A cetone dissolves water solubl e compounds as well as other compounds which dissolve in less polar solvents (Cheremisinoff 1953). Ethanol extracts of turmeric also showed to have a higher inhibition zone. Results were in accordance with one study which demonstrated that ethanol extracts of turmeric gave the highest antimicrobial activity against Gram-positive bacteria (Laohakunjit and others 2007). There was a statistical difference in the an timicrobial activity (inhibition zone) (p <0. 0001) between the turmeric extracts prepared fr om fresh rhizomes and the turmeric extracts prepared from dried-powder (Table 4-11). Dr ied powder turmeric had a significantly higher inhibition zone (8.6 mm) than fresh rhizom es turmeric (7.7 mm); however the observed difference was small. Inhibitory zone of turmeric (fresh rhiz omes) extracts (7.7 mm) was significantly lower (p<0.0001) than that of lemongrass stem (13.7 mm) ex tracts (Table 4-10). Th is is in accordance with previous research, which showed that turmer ic only gave low inhibition compared to other plant extracts against Clostridium botulinum (Huhtanen 1980). Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Turmeric (Fresh Rhizomes and Dried Powder) Extracts The MIC and MBC results of turmeric (prepa red from fresh rhizomes) demonstrated no significant difference (P > 0.05) in either solvents, drying methods or among the Staphylococcus aureus strains. Results showed that all the solvent extr acts and drying methods gave statistically similar antimicrobial activity against Staphylococcus aureus strains. Sensitivity of the Staphylococcus aureus strains to the fresh rhizome turmeric extracts was also similar. Very little variations were observed (among trials, solv ents and drying methods), demonstrating the 68

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sensitivity of each Staphylococcus aureus strain to the fresh rhizom e turmeric extracts (Table 47). Results of dried powder turmeric (Table 48) demonstrated that the MIC and MBC of acetone extracts were significantly lower (P < 0. 0001) than the extracts of ethanol and hexane. Acetone extracts presented an MIC and MBC of 12.5 and 20.0 mg/mL, respectively, while the MIC and MBC of ethanol and hexane were 19.2 and 33.0 mg/mL; and 26.6 and 40.0 mg/mL, respectively. These results showed that acetone extracts of turmeric (dried powder) had a higher antimicrobial activity than that of ethanol and hexane. Turmeric extracts showed a higher antimicrobial activity against Staphylococcus aureus ATCC 29247 with a low MIC of 18.3mg/mL. The MBC of turmeric (dried powder) extracts against all the Staphylococcus aureus strains tested were not si gnificantly different (P > 0.05). Staphylococcus aureus ATCC 12600-U and 35548 both had an MBC of 31.7 mg/mL; and Staphylococcus aureus ATCC 29247 had an MBC of 30.0 mg /mL. There was very little variation observed (among trials and solven ts) demonstrating the sensitivity of each Staphylococcus aureus strain to the dried powder turmeric extracts (Table 4-9). Results showed to be consistent with those presented in Table 4-8, which showed that acetone and ethanol extracts had a higher antimicrobial activ ity than that of hexane extracts. Lemongrass extracts had a significantly lower MIC and MBC than that of turmeric (Table 4-10). The MIC and MBC for lemongrass were 3.0 mg/mL and 5.2 mg/mL, respectively, yet for turmeric; the MIC and MBC were observed to be 19.4 mg/mL and 29.3 mg/mL, respectively. These results showed that th e extracts of lemongrass stem s were more powerful against Staphylococcus aureus than turmeric extracts. 69

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70 All the results on the MIC and MBC of lemongr ass and turmeric are consistent with the initial results showing the antimicrobial activity (inhibition zone) against the test pathogens. As with inhibition zone, lemongrass was observed to have a lower MIC and MBC than turmeric against Staphylococcus aureus strains. This was expected becau se the bigger the inhibition zones the lower the MIC and MBC will be. These results are in accordance with most research studies that have been done where the MIC and MBC of lemongrass are considerably lower ranging from 0.16-20 L/mL for most bacteria and 0.2510 L/mL for most fungi (Duke and others 2003; Oussalah and others 2006; Raydaudi-Massilla and others 2006; Shwiertz and others 2006). Results obtained in this study were in accordance with other research, which shows turmeric start exhibiting antibacterial activity at 20 mg /mL (Packiyasothy and Kyle 2002). Another study found that turmeric inhibited some pathogens at a wide range 20-100 g/mL. This means that the antimicrobial activity of herbs and spices varies widely, depending on the t ype of spice or herb, test medium, and microorganism (Zaika 1988).

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Table 4-1. Inhibitory zone (mm) of lemongrass stems extracts (sol vent extracts) on bacterial pathogens Solvents and Drying methods Hexane Acetone Ethanol Water Pathogens OD FD OD FD OD FD OD FD Staphylococcus aureus ATCC 12600-U 19 3 12 2 14 4 10 1 0 0 0 0 Staphylococcus aureus ATCC 29247 18 3 10 2 14 3 11 1 0 0 0 0 Staphylococcus aureus ATCC 35548 18 3 8 1.5 15 2 11 2 0 0 0 0 Salmonella Typhimurium 0 0 0 0 0 0 0 0 Salmonella Thompson 0 0 0 0 0 0 0 0 Salmonella Enteritidis 0 0 0 0 0 0 0 0 E. coli O157:H7 204P 0 0 0 0 0 0 0 0 E. coli O157:H7 301C 0 0 0 0 0 0 0 0 E. coli O157:H7 505B 0 0 0 0 0 0 0 0 71 Values presented are averages of replicates of each bacterial strain. OD Oven dried; FD Freeze dried

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72 Table 4-2. Inhibitory zone (m m) of turmeric (fresh rhizome) extracts (solvent extracts) on bacterial pathogens Solvents and Drying methods Hexane Acetone Ethanol Water Pathogens OD FD OD FD OD FD OD FD Staphylococcus aureus ATCC 12600-U 7 0.5 7 0.5 7 0.5 8 0.5 7 0.5 7.5 0.5 0 0 Staphylococcus aureus ATCC 29247 7.5 0.5 9 0.5 7 0.5 8.5 0.5 7 0.5 7 0.5 0 0 Staphylococcus aureus ATCC 35548 7 0.5 7 0.5 7 0.5 8.5 0.5 7 0.5 7 0.5 0 0 Salmonella Typhimurium 0 0 0 0 0 0 0 0 Salmonella Thompson 0 0 0 0 0 0 0 0 Salmonella Enteritidis 0 0 0 0 0 0 0 0 E. coli O157:H7 204P 0 0 0 0 0 0 0 0 E. coli O157:H7 301C 0 0 0 0 0 0 0 0 E. coli O157:H7 505B 0 0 0 0 0 0 0 0 Values presented are averages of replicates of each bacterial strain. OD Oven dried; FD Freeze dried

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Table 4-3. Inhibitory zo ne (mm) of turmeric (dried powder) extracts (solvent extracts) on bacterial pathogens Solvents Pathogens Hexane Acetone Ethanol Water Staphylococcus aureus ATCC 12600-U 8.5 0.5 8.5 0.5 9 0.5 0 Staphylococcus aureus ATCC 29247 7.5 0.5 9.5 0.5 8.5 0.5 0 Staphylococcus aureus ATCC 35548 8.5 0.5 9 0.5 9.5 0.5 0 Salmonella Typhimurium 0 0 0 0 Salmonella Thompson 0 0 0 0 Salmonella Enteritidis 0 0 0 0 E. coli O157:H7 204P 0 0 0 0 E. coli O157:H7 301C 0 0 0 0 E. coli O157:H7 505B 0 0 0 0 Values presented are averages of rep licates of each bacterial strain. 73

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74 Table 4-4. Overall comparison of solvents, dr ying methods, and harvest-time for lemongrass stem. Variables Lemongrass Zone (mm) MIC (mg/mL) MBC (mg/mL) Harvest time 1 (October) 12.3 B 4.0 A 6.9 A 2 (November) 15.2 A 2.0 B 3.5 B Pathogens Staphylococcus aureus ATCC 12600-U 14.1 A 3.3 A 5.9 A Staphylococcus aureus ATCC 29247 13.8 A 2.6 A 4.2 B Staphylococcus aureus ATCC 35548 13.2 A 3.1 A 5.6 A Solvent Hexane 13.7 A 2.3 B 4.9 A Acetone 13.7 A 3.6 A 5.4 A Drying methods Oven drying 16.3 A 2.2 B 3.3 B Freeze drying 11.2 B 3.0 A 7.1 A Values presented are averages of three replicat es. Values in the same column within each category following different uppercase letters designate a significant difference (Duncans multiple range test, P < 0.05).

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Table 4-5. Sensitivity of each Staphylococcus aureus strain to lemongrass stem Staphylococcus aureus ATCC 12600-U Staphylococcus aureus ATCC 29247 Staphylococcus aureus ATCC 35548 Variables Zone (mm) MIC (mg/mL) MBC (mg/mL) Zone (mm) MIC (mg/mL) MBC (mg/mL) Zone (mm) MIC (mg/mL) MBC (mg/mL) Harvest time 1 (October) 12.2 B 4.7 A 8.1 A 13.1 B 3.4 A 5.2 A 11.5 B 3.8 A 7.3 A 2 (November) 16.0 A 1.9 B 3.7 B 14.6 A 1.7 B 3.1 B 14.9 A 2.4 B 3.7 B Solvents Acetone 13.3 B 3.3 A 6.7 A 13.9 A 1.6 B 3.3 B 13.9 A 2.1 B 4.8 B Hexane 14.9 A 3.3 A 5.2 B 12.8 B 3.5 A 5.0 A 12.5 B 4.0 A 6.2 A Drying methods Oven drying 16.1 A 1.9 B 3.9 B 17.4 A 0.8 B 2.1 B 15.3 A 1.9 B 3.9 B Freeze drying 12.1 B 4.6 A 8.0 A 10.3 B 4.4 A 6.3 A 11.1 B 4.3 A 7.1 A 75 Values presented are averages of three replicates. Values in the same column within each category following different uppercase letters designate a significant difference (Duncans multiple range test, P < 0.05).

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Table 4-6. Antimicrobial activities of fresh rhizome turmeric extracts Variables Zone (mm) MIC (mg/mL) MBC (mg/mL) Pathogens Staphylococcus aureus ATCC 12600-U 8.1 A 18. 5 A 29.2 A Staphylococcus aureus ATCC 29247 7.4 B 20.3 A 30.0 A Staphylococcus aureus ATCC 35548 7.5 B 19.3 A 28.6 A Solvents Hexane 7.5 A 19.3 A 30.6 A Acetone 8.0 A 18.9 A 28.1 A Ethanol 7.6 A 19.9 A 29.2 A Drying methods Oven drying 7.3 B 19.5 A 30.6 A Freeze drying 8.0 A 19.3 A 28.0 A Values presented are averages of three replicat es. Values in the same column within each category following different uppercase letters designate a significant difference (Duncans multiple range test, P < 0.05). 76

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Table 4-7. Sensitivity of each Staphylococcus aureus strain to fresh rhizome turmeric extracts Staphylococcus aureus ATCC 12600 Staphylococcus aureus ATCC 29247 Staphylococcus aureus ATCC 35548 Variables Zone (mm) MIC (mg/mL) MBC (mg/mL) Zone (mm) MIC (mg/mL) MBC (mg/mL) Zone (mm) MIC (mg/mL) MBC (mg/mL) Trial 1st Trial 7.8 B 19.4 A 32.2 A 7.1 B 21.7 A 31.7 A 7.1 B 20.0 A 29.4 A 2nd Trial 8.4 A 17.5 B 26.1 B 7.7 A 18.9 B 28.3 B 7.8 A 18.6 B 27.8 B Solvents Acetone 8.0 AB 18.8 AB 30.8 A 7.8 A 19.2 B 28.3 B 8.1 A 18.8 B 25.0 B Hexane 8.5 A 17.1 B 25. 0 B 7.1 B 22.5 A 32.5 A 7.3 B 20.0 A 30.0 A Ethanol 7.8 B 19.6 A 31.7 A 7.3 B 19.2 B 29.2 B 7.1 B 19.2 AB 30.8 A Drying methods Oven drying 7.7 B 18.9 A 31.1 A 7.0 B 20.0 B 30.0 A 7.2 B 19.4 A 30.6 A Freeze drying 8.5 A 18.1 A 27.2 B 7.8 A 20.6 A 30.0 A 7.8 A 19.2 A 26.7 B 77 Values presented are averages of three replicates. Values in the same column within each category following different uppercase letters designate a significant difference (Duncans multiple range test, P < 0.05).

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Table 4-8. Antimicrobial activities of dried powder turmeric extracts Variables Zone (mm) MIC (mg/mL) MBC (mg/mL) Pathogens Staphylococcus aureus ATCC 12600-U 8.7 B 19.7 A 31.7 A Staphylococcus aureus ATCC 29247 8.3 C 18.3 B 30.0 A Staphylococcus aureus ATCC 35548 9.0 A 20.3 A 31.7 A Solvents Hexane 8.0 B 26.6 A 40.0 A Acetone 9.1 A 12.5 C 20.0 C Ethanol 8.8 A 19.2 B 33.0 B Values presented are averages of three replic ates. Values in the same column within each category following different uppercase letters designate a significant difference (Duncans multiple range test, P < 0.05). 78

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Table 4-9. Sensitivity of each Staphylococcus aureus strain to dried powder turmeric extracts Staphylococcus aureus ATCC 12600 Staphylococcus aureus ATCC 29247 Staphylococcus aureus ATCC 35548 Variables Zone (mm) MIC (mg/mL) MBC (mg/mL) Zone (mm) MIC (mg/mL) MBC (mg/mL) Zone (mm) MIC (mg/mL) MBC (mg/mL) Trial 1st Trial 8.5 B 20.6 A 33.3 A 8.0 B 20.0 A 30.0 A 8.8 B 20.6 A 33.3 A 2nd Trial 8.8 A 18.9 A 30.0 B 8.6 A 16.7 B 30.0 A 9.2 A 20.0 B 30.0 B Solvents Acetone 8.8 A 20.0 B 20.0 C 9.3 A 17.5 B 20.0 C 9.3 A 20.0 B 20.0 C Hexane 8.3 A 26.7 A 40.0 A 7.2 C 25.0 A 40.0 A 8.6 B 28.3 A 40.0 A Ethanol 8.9 A 12.5 C 35.0 B 8.3 B 15.0 C 30.0 B 9.2 A 12.5 C 35.0 B Values presented are averages of three replicates. Values in the same column within each category following different uppercase letters designate a significant difference (Duncans multiple range test, P < 0.05). 79

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Table 4-10. Comparison of lemongrass stem and turmeric (fresh rhizomes) extracts Herb Zone (mm) MIC (mg/mL) MBC (mg/mL) Lemongrass 13.7 A 3.0 B 5.2 B Turmeric 7.7 B 19.4 A 29.3 A Values presented are averages of three replicat es. Values in the same column within each category following different uppercase letters designate a significant difference (Duncans multiple range test, P < 0.05). Table 4-11. Comparison of fresh rhizomes and dried powder turmeric extracts. Herb Zone (mm) MIC (mg/mL) MBC (mg/mL) Turmeric (dried powder) 8.6 A 19.4 A 31.1 A Turmeric (fresh rhizomes) 7.7 B 19.4 A 29.3 A Values presented are averages of three replicat es. Values in the same column within each category following different uppercase letters designate a significant difference (Duncans multiple range test, P < 0.05). 80

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CHAPTER 5 SUMMARY AND CONCLUSSION Lemongrass stem and turmeric (fresh rhizomes and dried powder) extracts showed antimicrobial properties against Staphylococcus aureus strains used in this study. Lemongrass stems showed antimicrobial properties only when acetone and hexane were used as extraction solvents. Oven dried extracts of lemongrass stems gave a greater antimicrobial activity than that of the freeze dried extracts. Both hexane a nd acetone extracts of lemongrass stem had a considerable amount of antimic robial activity. Lemongrass harv ested in November had higher antimicrobial properties than that from October harvest. Turmeric showed antimicrobial properties wh en acetone, hexane and ethanol were used as solvents for the extraction. Difference in the solven ts performance could be attributable to their properties. Dried powder and fresh rhizome turmer ic extracts did not show any differences on the antimicrobial activity regard less of the solvents and dryi ng methods used in this study. Turmeric had less antimicrobial action compared to lemongrass. This could be due to the differences in compounds and the properties in both plants. Antimicrobial activity of lemongrass and turmeric could be greatly affected by the solvents used to extracts the plants. Drying methods could also affect the antimicro bial activity of plants such as lemongrass. Turmeric whether purchased in fresh or dry form could provide the same effect of antimicrobial activity against Staphylococcus aureus, therefore obtaining either one of them would not make a difference. Lemongrass and turmeric could be potentially used for controlling Staphylococcus aureus. Even though lemongrass leaves ar e widely used to make tea, it was found that they did not have any antimicrobial properties. Increasing the concentrations of lemongrass and turmeric extracts may be necessary for antimicrobial activity to be observe d in other bacterial pa thogens. It might be of interest to study 81

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82 other Gram positive bacteria such as Bacillus cereus to determine if the antimicrobial action will be similar. Studying the compounds extracted from plants by using the different solvents could also be of interest to determine the importa nt compounds responsible for the antimicrobial activity of each extract.

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APPENDIX A ANALYSIS OF VARIANCE (ANOVA) TABL ES: ANTIMICROBIAL ACTIVITY OF LEMONGRASS STEMS Table A-1. Inhibitory zone of lemongrass stems Sum of Source DF Squares Mean Square F Value Pr > F Model 7 1507.96 215.42 28.34 <.0001 Error 136 1033.83 7.60 Corrected Total 143 2541.78 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 296.13 296.13 38.96 <.0001 Trial 1 28.00 28.00 3.68 0.0570 Pathogen 2 21.00 10.50 1.38 0.2547 Solvent 1 0.01 0.01 0.00 0.9759 Dry method 1 927.71 927.71 122.04 <.0001 Solvent*Dry method 1 235.11 235.11 30.93 <.0001 Table A-2. Minimum Inhibitory Concen tration (MIC) of lemongrass stem Sum of Source DF Squares Mean Square F Value Pr > F Model 7 784.37 112.05 31.70 <.0001 Error 136 480.68 3.53 Corrected Total 143 1265.06 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 138.56 138.56 39.20 <.0001 Trial 1 13.69 13.69 3.87 0.0511 Pathogen 2 11.99 5.99 1.70 0.1871 Solvent 1 58.86 58.86 16.65 <.0001 Dry method 1 297.37 297.37 84.14 < .0001 Solvent*Dry method 1 263.88 263.88 74.66 <.0001 83

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Table A-3. Minimum Bactericidal Con centration (MBC) of lemongrass stem Sum of Source DF Squares Mean Square F Value Pr > F Model 7 1259.70 179.95 43.65 <.0001 Error 136 560.65 4.12 Corrected Total 143 1820.36 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 408.05 408.05 98.98 <.0001 Trial 1 0.28 0.28 0.07 0.7927 Pathogen 2 80.73 40.36 9.79 0.0001 Solvent 1 10.88 10.88 2.64 0.1065 Dry method 1 532.47 532.47 129.16 <.0001 Solvent*Dry method 1 227.26 227.26 55.13 <.0001 84

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Table A-4. Inhibitory zone of lemongrass st ems against Staphylococcus aureus ATCC 12600-U Sum of Source DF Squares Mean Square F Value Pr > F Model 5 521.53 104.30 16.50 <.0001 Error 42 265.46 6.32 Corrected Total 47 786.99 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 167.81 167.81 26.55 <.0001 Trial 1 25.15 25.15 3.98 0.0525 Solvent 1 31.28 31.28 4.95 0.0315 Dry method 1 193.00 193.00 30.54 <.0001 Solvent*Dry method 1 104.28 104.28 16.50 0.0002 85

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Table A-5. Minimum Inhibitory Concentration (MIC) of lemongrass stem against Staphylococcus aureus ATCC 12600-U Sum of Source DF Sq uares Mean Square F Value Pr > F Model 5 322.91 64.5827367 21.96 <.0001 Error 42 123.49 2.9403678 Corrected Total 47 446.40 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 93.99 93.99 31.97 <.0001 Trial 1 14.10 14.10 4.80 0.0341 Solvent 1 0.00 0.00 0.00 0.9700 Dry method 1 89.51 89.51 30.44 <.0001 Solvent*Dry method 1 125.29 125.29 42.61 <.0001 Table A-6. Minimum Bactericidal Concentr ation (MBC) of lemongrass stem against Staphylococcus aureus ATCC 12600-U Sum of Source DF Squares Mean Square F Value Pr > F Model 5 568.41 113.68 37.55 <.0001 Error 42 127.16 3.02 Corrected Total 47 695.58 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 235.89 235.89 77.91 <.0001 Trial 1 0.75 0.75 0.25 0.6213 Solvent 1 24.66 24.66 8.15 0.0067 Dry method 1 203.81 203.81 67.32 <.0001 Solvent*Dry method 1 103.28 103.28 34.11 <.0001 86

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Table A-7. Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC 29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 5 686.25 137.25 41.35 <.0001 Error 42 139.41 3.31 Corrected Total 47 825.66 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 25.52 25.52 7.69 0.0083 Trial 1 0.19 0.19 0.06 0.8133 Solvent 1 0.19 0.19 0.06 0.8133 Dry method 1 595.02 595.02 179.25 <.0001 Solvent*Dry method 1 65.33 65.33 19.68 <.0001 Table A-8. Minimum Inhibitory Concentra tion (MIC) of lemongrass stem against Staphylococcus aureus ATCC 29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 5 343.14 68.62 18.93 <.0001 Error 42 152.30 3.62 Corrected Total 47 495.45 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 33.85 33.85 9.33 0.0039 Trial 1 2.40 2.40 0.66 0.4198 Solvent 1 43.56 43.56 12.01 0.0012 Dry method 1 151.47 151.48 41.77 <.0001 Solvent*Dry method 1 111.84 111.84 30.84 <.0001 87

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Table A-9. Minimum Bactericidal Concentr ation (MBC) of lemongrass stem against Staphylococcus aureus ATCC 29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 5 393.90 78.78 27.03 <.0001 Error 42 122.42 2.914 Corrected Total 47 516.33 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 51.57 51.57 17.69 0.0001 Trial 1 5.64 5.64 1.94 0.1713 Solvent 1 33.74 33.74 11.58 0.0015 Dry method 1 211.49 211.49 72.56 <.0001 Solvent*Dry method 1 91.45 91.45 31.37 <.0001 88

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Table A-10. Inhibitory zone of lemongrass stems against Staphylococcus aureus ATCC A35548 Sum of Source DF Squares Mean Square F Value Pr > F Model 5 463.04 92.6096354 8.74 <.0001 Error 42 445.07 10.5970362 Corrected Total 47 908.12 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 139.23 139.23 13.14 0.0008 Trial 1 21.00 21.00 1.98 0.1666 Solvent 1 25.15 25.15 2.37 0.1309 Dry method 1 209.37 209.37 19.76 <.0001 Solvent*Dry method 1 68.28 68.28 6.44 0.0149 Table A-11. Minimum Inhibitory Concentra tion (MIC) of lemongrass stem against Staphylococcus aureus ATCC A35548 Sum of Source DF Squares Mean Square F Value Pr > F Model 5 191.45 38.29 13.43 <.0001 Error 42 119.73 2.85 Corrected Total 47 311.19 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 23.76 23.76 8.34 0.0061 Trial 1 17.68 17.68 6.20 0.0168 Solvent 1 43.86 43.86 15.39 0.0003 Dry method 1 65.60 65.60 23.01 <.0001 Solvent*Dry method 1 40 .54 40.54 14.22 0.0005 89

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90 Table A-12. Minimum Bactericidal Concentration (MBC) of lemongrass stem against Staphylococcus aureus ATCC A35548 Sum of Source DF Squares Mean Square F Value Pr > F Model 5 349.69 69.93 16.50 <.0001 Error 42 178.02 4.23 Corrected Total 47 527.71 Source DF Type I SS Mean Square F Value Pr > F Harvest time 1 154.94 154.94 36.55 <.0001 Trial 1 5.93 5.93 1.40 0.2433 Solvent 1 23.74 23.74 5.60 0.0226 Dry method 1 124.29 124.29 29.32 <.0001 Solvent*Dry method 1 40.77 40.77 9.62 0.0034

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APPENDIX B ANALYSIS OF VARIANCE (ANOVA) TABL ES: ANTIMICROBIAL ACTIVITY OF TURMERIC (FRESH RHIZOMES) Table B-1. Inhibitory zone of turmeric (fresh rhizomes) Sum of Source DF Squares Mean Square F Value Pr > F Model 8 90 .99 11.37 39.11 <.0001 Error 207 60.21 0.29 Corrected Total 215 151.20 Source DF Type I SS Mean Square F Value Pr > F Trial 1 19.56 19.56 67.25 <.0001 Pathogen 2 21.67 10.83 37.26 <.0001 Solvent 2 10.64 5.32 18.30 <.0001 Dry method 1 29.62 29.62 101.86 <.0001 Solvent*dry method 2 9.48 4.74 16.31 <.0001 Table B-2. Minimum Inhibitory Concentrati on (MIC) of turmeric (fresh rhizomes) Sum of Source DF Squares Mean Square F Value Pr > F Model 8 462.03 57.754630 8.87 <.0001 Error 207 1347.22 6.508320 Corrected Total 215 1809.25 Source DF Type I SS Mean Square F Value Pr > F Trial 1 224.07 224.07 34.43 <.0001 Pathogen 2 117.59 58.79 9.03 0.0002 Solvent 2 34.25 17.12 2.63 0.0743 Dry method 1 1.85 1.8518 0.28 0.5943 Solvent*dry method 2 84.25 42.12 6.47 0.0019 Table B-3. Minimum Bactericidal Concentra tion (MBC) of turmeric (fresh rhizomes) Sum of Source DF Squares Mean Square F Value Pr > F Model 8 1959.25 244.90 12.93 <.0001 Error 207 3922.22 18.94 Corrected Total 215 5881.48 Source DF Type I SS Mean Square F Value Pr > F Trial 1 740.74 740.74 39.09 <.0001 Pathogen 2 70.370 35.18 1.86 0.1587 Solvent 2 225.92 112.96 5.96 0.0030 Dry method 1 362.96 362.96 19.16 <.0001 Solvent*dry method 2 559.25 279.62 14.76 <.0001 91

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Table B-4. Inhibitory zone of tu rmeric (fresh rhizomes) against Staphylococcus aureus ATCC SA12600-U Sum of Source DF Squares Mean Square F Value Pr > F Model 6 13.055 2.17 5.75 0.0005 Error 29 10.97 0.37 Corrected Total 35 24.03 Source DF Type I SS Mean Square F Value Pr > F Trial 1 3.06 3.06 8.09 0.0081 Solvent 2 3.67 1.83 4.85 0.0153 Dry method 1 5.84 5.84 15.43 0.0005 Solvent*Dry method 2 0.48 0.24 0.64 0.5359 Table B-5. Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC SA12600-U Sum of Source DF Squares Mean Square F Value Pr > F Model 6 79.16 13.19 1.81 0.1327 Error 29 211.80 7.30 Corrected Total 35 290.97 Source DF Type I SS Mean Square F Value Pr > F Trial 1 34.02 34.02 4.66 0.0393 Solvent 2 38.88 19.44 2.66 0.0868 Dry method 1 6.25 6.25 0.86 0.3626 Solvent*Dry method 2 0.00 0.00 0.00 1.0000 Table B-6. Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC SA12600-U Sum of Source DF Squares Mean Square F Value Pr > F Model 6 861.11 143.51 6.78 0.0001 Error 29 613.88 21.16 Corrected Total 35 1475.00 Source DF Type I SS Mean Square F Value Pr > F Trial 1 336.11 336.11 15.88 0.0004 Solvent 2 316.66 158.33 7.48 0.0024 Dry method 1 136.11 136.11 6.43 0.0169 Solvent*Dry method 2 72.22 36.11 1.71 0.1993 92

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Table B-7. Inhibitory zone of tu rmeric (fresh rhizomes) against Staphylococcus aureus ATCC SA29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 6 12.83 2.14 13.87 <.0001 Error 29 4.47 0.15 Corrected Total 35 17.31 Source DF Type I SS Mean Square F Value Pr > F Trial 1 2.64 2.64062500 17.12 0.0003 Solvent 2 3.14 1.57465278 10.21 0.0004 Dry method 1 6.46 6.46006944 41.87 <.0001 Solvent*Dry method 2 0.58 0.29340278 1.90 0.1675 Table B-8. Minimum Inhibitory Concentrati on (MIC) of turmeric (fresh rhizomes) Staphylococcus aureus ATCC SA29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 6 250.00 41.66 6.13 0.0003 Error 29 197.22 6.80 Corrected Total 35 447.22 Source DF Type I SS Mean Square F Value Pr > F Trial 1 69.44 69.44 10.21 0.0034 Solvent 2 88 .88 44.44 6.54 0.0045 Dry method 1 2.77 2.77 0.41 0.5278 Solvent*Dry method 2 88.88 44.44 6.54 0.0045 93

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Table B-9. Minimum Bactericidal Concentra tion (MBC) of turmeric (fresh rhizomes) Staphylococcus aureus ATCC SA29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 6 333.33 55.55 6.04 0.0003 Error 29 266.66 9.19 Corrected Total 35 600.00 Source DF Type I SS Mean Square F Value Pr > F Trial 1 100.00 100.00 10.88 0.0026 Solvent 2 116.66 58.33 6.34 0.0052 Dry method 1 0.00 0.00 0.00 1.0000 Solvent*Dry method 2 116.66 58.33 6.34 0.0052 94

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Table B-10. Inhibitory zone of tu rmeric (fresh rhizomes) against Staphylococcus aureus ATCC 35548 Sum of Source DF Squares Mean Square F Value Pr > F Model 6 19.51 3.25 24.16 <.0001 Error 29 3.90 0.13 Corrected Total 35 23.42 Source DF Type I SS Mean Square F Value Pr > F Trial 1 4.16 4.16 30.96 <.0001 Solvent 2 6.50 3.25 24.14 <.0001 Dry method 1 2.91 2.91 21.68 <.0001 Solvent*Dry method 2 5.93 2.96 22.02 <.0001 Table B-11. Minimum Inhibitory Concentration (MIC) of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC 35548 Sum of Source DF Squares Mean Square F Value Pr > F Model 6 54.16 9.03 4.90 0.0014 Error 29 53.47 1.84 Corrected Total 35 107.63 Source DF Type I SS Mean Square F Value Pr > F Trial 1 17.36 17.36 9.42 0.0046 Solvent 2 9.72 4.86 2.64 0.0887 Dry method 1 0.69 0.69 0.38 0.5442 Solvent*Dry method 2 26.38 13.19 7.16 0.0030 Table B-12. Minimum Bactericidal Concentration (MBC) of turmeric (fresh rhizomes) against Staphylococcus aureus ATCC 35548 Sum of Source DF Sq uares Mean Square F Value Pr > F Model 6 572.22 95.37 10.71 <.0001 Error 29 258.33 8.91 Corrected Total 35 830.55 Source DF Type I SS Mean Square F Value Pr > F Trial 1 25.00 25.00 2.81 0.1046 Solvent 2 238.88 119.44 13.41 <.0001 Dry method 1 136.11 136.11 15.28 0.0005 Solvent*Dry method 2 172.22 86.11 9.67 0.0006 95

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APPENDIX C ANALYSIS OF VARIANCE (ANOVA) TABL ES: ANTIMICROBIAL ACTIVITY OF TURMERIC (DRIED POWDER) Table C-1. Inhibitory zone of turmeric (dried powder) Sum of Source DF Squares Mean Square F Value Pr > F Model 5 38.78 7.75 44.88 <.0001 Error 102 17.62 0.17 Corrected Total 107 56.41 Source DF Type I SS Mean Square F Value Pr > F Trial 1 4.89 4.89 28.34 <.0001 Pathogen 2 10.1 6 5.08 29.41 <.0001 Solvent 2 23.72 11.86 68.62 <.0001 Table C-2. Minimum Inhibitory Concentra tion (MIC) of turmeric (dried powder) Sum of Source DF Squares Mean Square F Value Pr > F Model 5 3781.48 756.29 78.30 <.0001 Error 102 985.18 9.6586 Corrected Total 107 4766.67 Source DF Type I SS Mean Square F Value Pr > F Trial 1 92.59 92.59 9.59 0.0025 Pathogen 2 72.22 36.11 3.74 0.0271 Solvent 2 3616.66 1808.33 187.22 <.0001 Table C-3. Minimum Bactericidal Concentr ation (MBC) of turmeric (dried powder) Sum of Source DF Squares Mean Square F Value Pr > F Model 5 7666.67 1533.33 260.67 <.0001 Error 102 600.00 5.88 Corrected Total 107 8266.67 Source DF Type I SS Mean Square F Value Pr > F Trial 1 133.33 133.33 22.67 <.0001 Pathogen 2 66.67 33.33 5.67 0.0046 Solvent 2 7466.67 3733.33 634.67 <.0001 96

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Table C-4. Inhibitory zone of tu rmeric (dried powder) against Staphylococcus aureus ATCC 12600-U Sum of Source DF Squares Mean Square F Value Pr > F Model 3 4.17 1.38 15.69 <.0001 Error 32 2.83 0.08 Corrected Total 35 7.00 Source DF Type I SS Mean Square F Value Pr > F Trial 1 1.00 1.00 11.29 0.0020 Solvent 2 3.17 1.58 17.88 <.0001 Table C-5. Minimum Inhibitory Concentration (MIC) of turmeric (dried powder) against Staphylococcus aureus ATCC 12600-U Sum of Source DF Squares Mean Square F Value Pr > F Model 3 1230.55 410.18 41.45 <.0001 Error 32 316.66 9.89 Corrected Total 35 1547.22 Source DF Type I SS Mean Square F Value Pr > F Trial 1 25.00 25.00 2.53 0.1218 Solvent 2 1205.55 602.77 60.91 <.0001 Table C-6. Minimum Bactericidal Concentratio n (MBC) of turmeric (dried powder) against Staphylococcus aureus ATCC 12600-U Sum of Source DF Squares Mean Square F Value Pr > F Model 3 2700.00 900.00 144.00 <.0001 Error 32 200.00 6.25 Corrected Total 35 2900.00 Source DF Type I SS Mean Square F Value Pr > F Trial 1 100.00 100.00 16.00 0.0004 Solvent 2 2600.00 1300.00 208.00 <.0001 97

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Table C-7. Inhibitory zone of tu rmeric (dried powder) against Staphylococcus aureus ATCC 29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 3 29.52 9.84 115.70 <.0001 Error 32 2.72 0.08 Corrected Total 35 32.25 Source DF Type I SS Mean Square F Value Pr > F Trial 1 3.36 3.36 39.51 <.0001 Solvent 2 26.17 13.08 153.80 <.0001 Table C-8. Minimum Inhibitory Concentra tion (MIC) of turmeric (dried powder) Staphylococcus aureus ATCC 29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 3 1050.00 350.00 32.00 <.0001 Error 32 350.00 10.93 Corrected Total 35 1400.00 Source DF Type I SS Mean Square F Value Pr > F Trial 1 100.00 100.00 9.14 0.0049 Solvent 2 950.00 475.00 43.43 <.0001 Table C-9. Minimum Bactericidal Concentr ation (MBC) of turmeric (dried powder) Staphylococcus aureus ATCC 29247 Sum of Source DF Squares Mean Square F Value Pr > F Model 3 2400.00 800.00 Infty <.0001 Error 32 0.00 0.00 Corrected Total 35 2400.00 Source DF Type I SS Mean Square F Value Pr > F Trial 1 0.00 0.00 Solvent 2 2400.00 1200.00 Infty <.0001 98

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99 Table C-10. Inhibitory zone of tu rmeric (dried powder) against Staphylococcus aureus ATCC 35548 Sum of Source DF Squares Mean Square F Value Pr > F Model 3 4.16 1.38 15.69 <.0001 Error 32 2.83 0.08 Corrected Total 35 7.00 Source DF Type I SS Mean Square F Value Pr > F Trial 1 1.00 1.00 11.29 0.0020 Solvent 2 3.16 1.58 17.88 <.0001 Table C-11. Minimum Inhibitory Concentration (MIC) of turmeric (dried powder) against Staphylococcus aureus ATCC 35548 Sum of Source DF Squares Mean Square F Value Pr > F Model 3 1508.33 502.77 67.35 <.0001 Error 32 238.88 7.46 Corrected Total 35 1747.22 Source DF Type I SS Mean Square F Value Pr > F Trial 1 2.77 2.77 0.37 0.5462 Solvent 2 1505.55 752.77 100.84 <.0001 Table C-12. Minimum Bactericidal Concentratio n (MBC) of turmeric (dried powder) against Staphylococcus aureus ATCC 35548 Sum of Source DF Squares Mean Square F Value Pr > F Model 3 2700.00 900.00 144.00 <.0001 Error 32 200.00 6.25 Corrected Total 35 2900.00 Source DF Type I SS Mean Square F Value Pr > F Trial 1 100.00 100.00 16.00 0.0004 Solvent 2 2600.00 1300.00 208.00 <.0001

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LIST OF REFERENCES Abascal K, Ganoral L, Yarnell E. 2005. The eff ect of freeze-drying and its implications for botanical medicine: A review. Phytother. Res. ; 19(8):655-660 Akin-Osanaiye BC, Agbaji AS, Dakare MA. 2007. Antim icrobial activity of o ils and extracts of Cymbopogon c itratus (Lemon grass), Eucalyptus citriodora and Eucalyptus camaldulensis J. Med Sci., 7(4): 694-697. Afolayan AJ and Aliero AA. 2006. Antimicrobial activity of Solanum tomentosum. Afri. J. Biotechnol., 5(4): 369-372 Archer DL, Kvenberg JE. 1985. Incidence and cost of foodborne diarrheal disease in the United States. J. Food Protect., 48: 887-894. Asami DK, Hong YJ, Barrett DM, and Mitchell AE .2003. Comparison of the total phenolic and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn grown using conventional, organi c, and sustainable agricultur al practices. J. Agric. Food Chem. 51: 1237-1241 Badmaev V, Majeed M, Prakash L. 2004. Curc uminoids: bioactive compounds from turmeric. Sabinsa Corporation, 2. Baratta MT, Dorman HJD, Deans SD, Figueiredo AC, Barroso JG, Ruberto G. 1998. Antimicrobial and antioxidant properties of some commercial essential oils. J. Flavor and Fragar., 13(4): 235-244. Beattie S. 2006. Common foodborne pathogens. Consumer and Processing Food Safety and Science, Iowa State University, USA http://www.extension.iastate.e du/foodsafety/pathogens/index.cfm Bennet J, Holmberg S, Rogers M, Solomon S. 1 987. Infectious and parasitic diseases. In: Almer R, Dull H, Ed., Closing the gap: the burden of unnecessary illness. New York: Oxford Univ. Press; p 102-114. Beuchat LR, Golden DA. 1989. Antimicrobial oc curring naturally in foods. Food Technol., 43: 134-143. Billing J, Sherman PW. 1998. Antimic robial function of spices: why some like it hot. Q. Rev. Biol., 73: 3-49. Biswas TK, Mukherjee B. 2003. Pl ant medicines of Indian origin for wound healing activity: A healing review. Int. J. Low. Extrem. Wounds, 2(1): 25-39. Brennan JG. 1994. Food dehydration a dictionary and guide. CRC Press Oxford: ButterworthHeinemann, p 137-182. 100

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Bryan FL. 1982. Diseases transmitted by foods. Sec. Ed. Atlanta, GA, Centers for Disease Control. http://chppm-www.apgea.army.mil/tsunami/Diseasesfromfood(2).pdf Buchi Labortechnik AG. 2008. Rotary Evaporators. Buchi Corporation. www.buchi.com Cameron P, Butler DL, Kinnary K, Monger P, Murgatroyd. 1997. Good Pharmaceutical FreezeDrying Practice. Interpharm/CRC, Boca, Raton, London New York, Washington D.C. P1-269. Cappuccino JG, Sherman N. 2005. Microb iology: A laboratory Manual, 7th Ed., Pearson Benjamin Community College, New York, NY, p 13-20. Casimiri V, Burstein C. 1998.Biosensor for -lactate determination as an index of E. coli number in crude culture medium. J. Analytica Chimica Acta 361 (1-2 ): 45-53 Centers for Disease Control and Prevention (CDC). 2005. Foodborne illn esses. Division of Bacterial and Mycotic Diseases. Atlanta, GA. Chao SC, Young DG, Oberg CJ. 2000. Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses. J. Essent. Oil. Res.,12(5): 639-649. Cheremisinoff NP. 2003. Industrial solvents Handbook. Second Edition. Marcel Dekker, Inc. New York. Conner DE. 1993. Naturally Occurring Compou nds. Editors: Davidson PM, Branen AL. Antimicrobials in foods. Second Ed.M arcel Dekker, New York, p 441-468 Cosentino S, Tuberoso CIG, Pisano B, Satta M, Mascia V, Arzedi E, Palmas F. 1999. In-vitro antimicrobial activity and chemical and chemical composition of Sardinian Thymus essential oils. Lett. Appl. Microbiol., 29: 130-135. Cowan MM. 1999. Plant products as microbial ag ents. Clinical Microbiol. Rev., 12 (4), 564-582. Dudman P. 2005. Growing Asian herbs and Spices. ABC North Coast. Duke JA. 1994. Biologically active compounds in impor tant spices. In spices, herbs and edible fungi, (G. Charalambous, ed.), El servier, Mew York, p 225-250. Duke JA, Bogenschutz-Gogwin MJ, duCellier J, Duke PK. 2003. CRC Handbook of medicinal spices. Health and Fitness, p 240-245. Elloff JN. 1998. Which extract should be used fo r the screening and isol ation of antimicrobial components from plants? J. Entropharmacol., 60(1): 1-8 European Patent. 2006. Microbial fo rmulation comprising essential oi ls or their derivatives. EP, 1434486. Food and Drug Administration (FDA/CFSAN) 2007. Bad bug book: Foodborne Pathogenic Microorganisms and natural Toxins Handbook. 101

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Fraenkel GS. 1959. The raison dEtre of seconda ry plant substances. Science, 129: 1466-1470. Gescher A, Sharma R, Steward W. 2005. Curcumin : the story so far. Europ. J. Cancer, 41(13): 1955-1968. Gerald J. 1975. The herbal or general history of plants. Arco Pub. Co., New York, p1-13. Goel S. 2005. Bioprotective properties of turmeric : An investigation of the antioxidant and antimicrobial activities. J. Young Invest., 16(17): 1-6. Grag SR, Menon KV. 2003. Spices and herbspromising antimicrobial agents and natural food preservatives. Haryana Agricultural Univ ersity Journal of Research: 3(1), 1-6. Hammer KA, Carson CF, Riley TV. 1999. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology: 86(6): 985-990. Ho JC, S. Chou SK, Chua KJ, Mujumdar AS and Hawlader MNA. 2002. Analytical study of cyclic temperature drying: effect on dryi ng kinetics and product quality. J. Food Engineering, 51 (1): 65-75. Hughes KV, Wellenberg BJ. 1994. Quality for ke eps: Drying foods. DMCA, MU Extension, Columbia, MO. Hutton W. 2003. Handy pocket Guide to Asian herbs and spices. Tuttle Publishing, Singapore. p 3-64. Huhtanen CN. 1980. Inhibition of Clostridium botulinum by spice extracts and aliphatic alcohol. J. Food Prot., 43: 195-196. Julkunen-Titto R, Sorsa S. 2001. Testing the e ffects of drying methods on willow flavonoids, tannins, and salicylates. J. Chem. Ecol. 27: 779. Kakuta E, Ohshima T, Maeda N. 2006. The antimicrobi al effects of essentia l oils against oral pathogenic microorganisms. Microbiol. Immuno l. Infect. Control System, Preliminary Program for IADR General Session and Exhibition. Karaman I, Sahin F, Gulluce M, Qgutcu H, Sengul M, Adiguzel A. 2003. Antimicrobial activity of aqueous and methanol extracts of Juniperous oxycedrus L. J. Ethnopharmacol, 85: 231-235. Kasumov and Babaev RI. 1983. Components of the essential oil of lemo ngrass. Chem. Natural Comp., 19(1): 108. Kim JM, Marshall MR, Cornell JA, Preston JF Wei CI. 1995. Antibacteria l activity of some essential oil components agai nst five foodborne pathogens. J. Agric. Food Chem., 43: 2839-2845. 102

PAGE 103

Kim KJ, Yu HH, Cha JD, Seo SJ, Choi NY, You YO. 2005. Antibacterial activity of Curcuma longa L. against methicillin resistant Staphylococcus aureus. Phytotherapy Res., 19(7): 599-604. Kotzekidou P, Giannakidis P, Boul amastis A. 2008. Antimicrobial activity of some plant extracts and essential oils against foodborne pathogens in vitro and on the fate of inoculated pathogens in chocolate. J. F ood Sci. Technol., 41(1): 119-127. Laohakunjit N, Kerdchoechuen O, Norajit K. 2007 Antibacterial effect of five Zingiberaceae essential oils. Molecules, 12: 2047-2060. Lerici CR. 1976. Retention of volatile compounds in a freeze dried model food gel. S TA NU., 6(1): 27-32. Lis-Balchin M, Deans SG. 1997. Bio activity of selected plant esse ntial oils agai nst Listeria monocytogenes. J. Appl. Microbiol., 82: 759-762. Maizura M, Fazilah A, Norziah MH, Karim AA 2007. Antibacterial activity and mechanical properties of partially hydrogenated sago starch-alginate edible film containing lemongrass oil. J. Food Sci., 72(6): 324-330. Mahanon H, Azizah AH, Dzulkifly MH. 1999. Effect of drying methods on concentrations of several phytochemicals in herbal preparation of 8 medicinal plants leaves. Mal. J. Nutr., 5: 47-54. Mandeel Q, Hassan A, Isa Z. 2003. Antibacterial activ ity of certain spice extracts. J. Spice Arom. Crops., 12(2): 146-153. Mead PS, Slutsker L, Dietz V, McCaig LF, Breese JS, Shapiro C, Griffin PM, Tauxe RV. 1999. Food related illness and death in the Unite d States. Emerg. Infect. Dis. 5: 607-625. Mejlholm O, Dalgaard P. 2002. Antimicrobial effect of essential oils on the seafood spoilage micro-organism Photobacterium phosphoreum in liquid media and fish products. Lett. Appl. Microbiol., 34 (1): 27. Nail SL, Jiang S, Chongprasert S, Knopp SA 2002. Fundamentals of freeze drying. Pharm. Biotechnol., 14: 281-360. Onawunmi, GO. 1989. Evaluation of the antimicrobial activity of citral Lett. Appl. Microbial., 9: 105-108. Oonmetta-aree J, Suzuki T, Gasaluck P, Eemk eb G. 2006. Antimicrobial properties and action of galangal ( Alpinia galangal Linn.) on Staphylococcus aureus. J. Food Sci. and Technol., 39(10): 1214-1220. 103

PAGE 104

Oussalah M, Caillet S, Saucier L, Lacroix M. 20 06. Antimicrobial effects of selected essential oils on the growth of a Psuedomonas putida strain isolated from m eat. Meat Sci., 73(2): 236-244. zcan M, Arslan D, nver A. 2005. Effect of drying methods on the mineral content of basil ( Ocimum basilicum L.). J. Food Engin., 69(3): 375-379. Packiyasothy EV, Kyle S. 2002. Antimicrobial prope rties of some herb essential oils. J. Food Aust., 54(9): 384-387. Parish ME, Davidson PM. 1993. Methods for ev aluation: In Antimicrobials in foods, 2nd Ed. Marcel Dekker, Inc., New York, p 597-615. Prez-Coello MS, Daz-Maroto MC, Cabez udo MD. 2002. Effect of drying method on the volatiles in bay leaf ( Laurus nobilis L.). J. Agric. Food Chem ., 50 (16): 4520-4524. Pham M. 2001. Asian herbs: For flavor and for health. HarperCollons Publishers. Pulford A. 2003. Turmeric root. Health and F itness: In Homeopathic material medica of graphical drug pictures, p 149-150. Raybaudi-Massilia RM, Mosqueda-Melgar J, Mart in-Belloso O. 2006. Antimicrobial activity of essential oils on Salmonella Enteritidis, Escherichia coli and Listeria innocula in fruit juices. J. Food Prot., 69(7): 1579-1586. Reynolds S, Williams P. 1993. So Easy to Preserve Cooperative Extension Service, The University of Georgia. Revised by Judy Harrison. Ritenour MA, Sargent SA, bartz JA. 2002. Chlorine use in produce packing lines. HS-761, IFAS Extension, Gainesville, FL; p1-4. Rojas-Grau M, Avena Bustillos R, Friedman M, Henika P, Martin-Belloso O, McHugh T. 2006. Mechanical barrier and antimicrobial propert ies of apple puree edible films containing plant essential oils. J. Agric and Food Chem., 54: 9263-9267. Rojas-Grau M, Olsen C, Avena Bustillos R, Friedm an M, Henika P, Martin-Belloso O, Pan Z, McHugh T. 2007. Effects of plant essentia l oils and oil compounds on mechanical, barrier and antimicrobial properties of algina te-apple purees edible films. J. Food Engin., 81(3): 634-641. Rojas JJ, Ochoa VJ, Ocampo SA, Muoz JF. 2006. Sc reening for antimicrobial activity of ten medicinal plants used in Colombian folklo ric medicine: A possible alternative in the treatment of non-nosocomial in fections. BMC Complementary and Alternative Medicine, 6(2): 53-108. 104

PAGE 105

Russell AD. 1991. Mechanisms of ba cterial resistance to non-antib iotics: food additives and food and pharmaceutical preservatives. J. Appl. Microbiol., 71: 191-201. Sacchetti G, Maietti S. Muzzoli M, Scaglianti M, Manfredini S, Radice M, Bruni R. 2005. Comparative evaluation of 11 esse ntial oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. J. Food Chem., 91(4): 621-632. Sagdic O, Ozcan M. 2003. Antibacterial activity of Turkish spice hydrosols. Food Control, 14: 141-143. Salzer UJ. 1982. Antimicrobial action of some spi ce extracts and mixtures. Fleischwirtschaft, 62: 885-887. SAS. 1991. SAS/STAT User's Guide. 6.04 Ed. SAS Institute, Inc., Cary, NC. Scheflan L, Jacobs MB. 1953. The handbook of so lvents. First Edition. Robert E. Krieger Publishing Co., New York. Schwiertz A, Duttke C, Hild J, Muller HJ. 2006. In vitro activity of essential oils on microorganisms isolated from vaginal in fection. Int. J. Aromath., 16(3-4): 169-174. Shelef LA, Naglik OA, Bogen DW. 1980. Sensitivity of some common foodborne bacteria to the spices sage, rosemary, and all spice. J. Food Sci., 45: 1042-1044. Senatore F. 1996. Influence of harvesting time on yield and composition of essential oil of thyme ( Thymus pulegioides L.) growing wild in Campania (Southern Italy). J. Agric. Food Chem., 44: 1327-1332. Shibasaki I. 1982. Food preservation with nontraditio nal antimicrobial agents. J. Food Saf., 4: 35-58. Shivani G, Ravishankar S. 2005. Foodborne Pat hogens and Disease. A Comparison of the antimicrobial activity of garlic, ginger, carrot, and turmeric pastes against Escherichia coli O157:H7 in laboratory buffer and ground beef. Food Saf. Technol., 2(4): 330-340. Simon, J.E., A.F. Chadwick and L.E. Crak er. 1984. Herbs: An Indexed Bibliography. 19711980. The Scientific Literature on Selected Herb s, and Aromatic and Medicinal Plants of the Temperate Zone. Archon Books, 770 pp., Hamden, CT. Simonne AH, Nille A, Evans K, Marshall, Jr MR 2004. Ethnic foods safety trends in the United States based on CDC foodbor ne illness data. Food Pr ot. Tre., 24(8): 590-604. Siripongvutikorn S, Thummaratwasik P, Huang Y. 2005. Antimicrobial and antioxidant effects of Thai seasoning, Tom-Yum J. Food Sci. and Technol., 38(4): 347-352. 105

PAGE 106

Smid EJ, Gorris LGM. 1999. Natural antimicrobi als for food preservation. In handbook of food preservation; Rahman, MS, Ed.; Marcel Dekker: New York, p285-308. Todd ECD. 1989. Preliminary estimates of costs of foodborne disease in the United States. J. Food Protect., 52: 595-601. Uhl S. 2000. Spices: tools for alternative or co mplementary medicine. Food Technol., 54(5): 6165. United States Patent. 2003. Citral acetal. USP, 6506793. Van Sumere C, Geiger H, Bral D, Fockenier G, Vande Casteele K, Martens M, Hanselaer R, Gevaert L. 1983. Freeze drying and analysis of plant and other biological material. Anal. Biochem., 131(2): 530-532. Venskutonis PR. 1997. Effect of drying on th e volatile constituents of thyme (Thymus vulgaris L.) and sage (Salvia officinalis L.). J. Food Chem.; 59(2):219-227. Webb G. 1997. Herbs and spices with antioxidant and/or antimicrobial compounds. J. Amer. Botan. Council: 39 p20. Williams LR, Stockley JK, Yan W, Home VN. 1998. Essential oils with high antimicrobial activity for therapeutic use. Int. J. Aromath., 8(4): 30-40. Zaika LL. 1988. Spices and Herbs: their antimicr obial activity and its determination. J. Food Saf., 9: 97 118. 106

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107 BIOGRAPHICAL SKETCH Thabile Precious Nkambule, daughter of Annah S. Magagula, was born in Mbabane, Swaziland. She completed her bachelor of scienc e in home economics with a Deans Award at the University of Swaziland (Faculty of Agricultur e). Before attending the University of Florida as a Fulbright Scholar, Thabile worked at UNICEF and REDI (R egional Excellence & Development Initiative). Thabile also worked as a teacher at Dwalile Central High School in Swaziland for three years. She taught food and nutrition, home economics, mathematics, and fashion and fabrics. After finishing her masters degree in food science, she plans to return to Swaziland to resume a teaching position at the Univ ersity of Swaziland (Facu lty of Agriculture).