Antimicrobial Activity of Thai Traditional Medicinal Plants Extract Incorporated Alginate-Tapioca

370 Pages·2009·1.63 MB·English

Institute for Environmental and Animal Hygiene
and Veterinary Medicine (with Animal Clinic)
Faculty of Agricultural Sciences
University of Hohenheim

Department: Environmental and Animal Health
Prof. Dr. Reinhard Böhm

Antimicrobial Activity of Thai Traditional Medicinal Plants Extract
Incorporated Alginate-Tapioca Starch Based Edible Films
against Food Related Bacteria Including Foodborne Pathogens

Dissertation
Submitted in fulfillment of the requirements for the degree
“Doktor der Agrarwissenschaften”
(Dr.sc.agr. / Ph.D. in Agricultural Sciences)

to the
Faculty of Agricultural Sciences

Presented by
Racha Tepsorn
B.Sc., M.Sc. Food Science and Technology
Pattani, Thailand
Hohenheim 2009
This thesis was accepted as a doctoral dissertation in fulfillment of the requirements
for the degree “Doktor der Agrarwissenschaften” by the Faculty of Agricultural
Sciences at University of Hohenheim on 23 March 2009.

Date of oral examination: 20 April 2009

Examination Committee

Supervisor and Review Prof. Dr. Reinhard Böhm

Co-Reviewer Prof. Dr. Herbert Schmidt

Additional examiners Prof. Dr. Werner Bessei

Vice-Dean and Head of Committee Prof. Dr. Werner Bessei
Für die bedingungslose Liebe meiner Eltern und
meines Bruders, für mein geliebtes Heimatland und
dem thailändischen Volk, dem wirklich echten Besitzer
der verwendeten Pflanzen in dieser Arbeit.

แด ความรักอันยิ่งใหญของ คุณพอ คุณแม และพี่ชาย
แด แผนดินไทย บานเกิดอันเปนที่รัก และชาวไทยทุกทาน
ผูเปนเจาของภูมิปญญา และพืชพรรณไม
ที่ใชในการทดลองนี้ อยางแทจริง
Acknowledgement i
ACKNOWLEDGEMENT
I would sincerealy like to express my most profound thanks to my advisors, Prof. Dr.
Reinhard Böhm, for his dedicative continuous support, guidance, encouragement, expertise,
and invaluable advice throughout the study period at University of Hohenheim, Germany.
Without him the work would never have been undertaken. Grateful thanks are also extended
to Prof. Dr. Herbert Schmidt, my co-reviewer and Prof. Dr. Werner Bessei, my additional
examiner for their helps and suggestions to complete this dissertation.

I would also like to express my sincere and heartfelt gratitude to Dr. Birgit Hunsinger for her
guidance, friendship and support throughout the research and writing of this dissertation. Her
patience and attention to the many details have helped me to remain on the path during the
long and arduous course of my study. Without her, there would be no this dissertation.

Honestly thanks to Ven. Prasit Nanthasittho for his unconditioned and unlimited spirit and his
Buddhism’s dharmic principle, leading me constantly to the right and bright way. Sincerely
remember to the 19th monk group of Phradhammagaya Temple, Thailand for their goodwill
and wish to make me warm during the serious winter in Germany.

I also thank my colleagues at the Institute for Environmental and Animal Hygiene, University
of Hohenheim for their supports in various ways. Sincere thanks in particular to Angela Ortiz;
Beate Filohn; Petra Veit; and Slyvia Speck for their invaluable helps and technical supports;
Mrs. Kinzler for her administration suports and also to my thanksworthly friends, Mumi,
Karen, Saskia, Philipp, Greta, Phon, Mint and all Thai students in Stuttgart, especially, Tik,
Jay, Suwit, June, Tor, Auddy, Noi, Hope, Nop, Kae, Jeab, Beer, Eye, Am and Dreas for their
invaluable friendships.

This work had been funded by a grant from the German Accademic Exchange Service
(DAAD). Without this grant, this work would have been very difficult unless imposible.

I will be ever grateful for the unconditioned love of my heroic parents, Mr. Pakorn Tepsorn
and Mrs. Angkana Tepsorn, my brother, Mr. Noppasoon Tepsorn, and all the member of my
kindred. Without you all being there, this dissertation would never been completed.

Racha Tepsorn
Stuttgart-Hohenheim, January 2009
Table of contents ii
TABLE OF CONTENTS
ACKNOWLEDGEMENT............................................................................................i

TABLE OF CONTENTS............................................................................................ii

LIST OF USED ABBREVIATIONS.........................................................................ix

1 INTRODUCTION AND OBJECTIVE................................................................1
1.1 Introduction..........................................................................................................1
1.2 Objective..............................................................................................................3

2 REVIEW OF LITERATURE...............................................................................4
2.1 Medicinal Plants (Herbs).....................................................................................4
2.1.1 Andrographis paniculata (Burm.f.) Wall. ex Nees (King of bitters).........5
2.1.2 Curcuma zedoaria (Christm.) Roscoe (Zedoary)......................................9
2.1.3 Gracinia mangostana L. (Mangosteen)...................................................12
2.1.4 Hibiscus sabdariffa L. (Roselle)..............................................................14
2.1.5 Musa sapientum L. (Banana)...................................................................17
2.1.6 Nelumbo nucifera Gaertn. (Lotus)...........................................................19
2.1.7 Piper betle L. (Betel leaf or Betelvine)....................................................25
2.1.8 Punica granatum L. (Pomegranate).........................................................27
2.1.9 Psidium guajava L. (Guava)....................................................................31
2.2 Antimicrobial Activity of Medicinal Plants, Spices, and their Essential
Oil......................................................................................................................34
2.2.1 In Vitro Test of Antimicrobial Activity...................................................34
2.2.2 Tests of Antibacterial Activity of Medicinal Plants, Spices and their
Essential Oils in Food System.................................................................37
2.2.3 The Studies of Antimicrobial Activity of Medicinal Plants, Spices
and their Extracts in Thailand against Foodborne Phathogens................46
2.3 Methods for Evaluation the Efficacy of Food Antimicrobial............................47
2.3.1 In vitro Methods.......................................................................................47
2.3.2 Application Methods................................................................................56
2.3.3 Combination Studies................................................................................57
Table of contents iii
2.4 Active Packaging Technologies and its Applications........................................60
2.4.1 Oxygen Scavenging System....................................................................60
2.4.2 Moisture-Absorbing and Controlling System..........................................61
2.4.3 Ethanol Generating Systems....................................................................62
2.4.4 Antimicrobial Migrating and Non-migrating Systems............................63
2.5 Antimicrobial Biodegradable Films and Coating..............................................65
2.5.1 Edible Films and Coating History............................................................65
2.5.2 Developing the Antimicrobial Packaging Systems..................................66

3 MATERIALS AND METHODS........................................................................69
3.1 Material..............................................................................................................69
3.1.1 Medicinal Plants.......................................................................................69
3.1.2 Bacterial Strains.......................................................................................69
3.1.3 Culture Media..........................................................................................70
3.1.4 Chemical Agents......................................................................................70
3.1.5 Equipment................................................................................................71
3.2 Methods..............................................................................................................72
3.2.1 Stock Culture Preparation and Condition................................................72
3.2.2 Preparation of Medicinal Plants...............................................................73
3.2.3 Preparation of Crude Extracts..................................................................74
3.2.4 Determination of the Antimicrobial Activity...........................................75
3.2.5 Application Study....................................................................................79
3.2.6 Studying the Cytotoxicity of the Ethanolic Extracts................................82
3.2.7 Studying the Antimicrobial Properties of Alginate-Tapioca Based
Edible Films and Coating Incorporated Thai Traditional Medicinal
Plant Extracts in a Model Food System...................................................83

4 RESULTS.............................................................................................................90
4.1 Determination of the Minimum Inhibitory Concentration and the
Minimum Bactericidal Concentration of Thai Traditional Medicinal
Plants..................................................................................................................90
4.1.1 Antimicrobial properties of Andrographis paniculata (Burm.f.) Wall.
ex Nees.....................................................................................................90
Table of contents iv
4.1.2 Antimicrobial properties of Curcuma zedoaria (Christm) Roscoe..........92
4.1.3 Antimicrobial properties of Garcina mangostana L...............................92
4.1.4 Antimicrobial properties of Hibiscus sabdariffa L..................................95
4.1.5 Antimicrobial properties of Musa sapientum L.......................................97
4.1.6 Antimicrobial properties of Nelumbo nucifera Gaertn............................97
4.1.7 Antimicrobial properties of Piper betle L..............................................100
4.1.8 Antimicrobial properties of Psidium guajava L....................................102
4.1.9 Antimicrobial properties of Punica granatum L...................................102
4.2 Determination of the Antimicrobial Effect of Thai Traditional
Medicinal Ethanolic Extracts by Studying the Time Killing Analysis
without Interfering Substances by Time-Killing Analysis..............................105
4.2.1 Time Killing Analysis of Andrographis paniculata (Burm.f.) Wall.
ex Nees...................................................................................................105
4.2.2 Time Killing Analysis of Curcuma zedoaria (Christm.) Roscoe..........109
4.2.3 Time Killing Analysis of Garcinia mangostana L................................112
4.2.4 Time Killing Analysis of Hibiscus sabdariffa L....................................116
4.2.5 Time Killing Analysis of Musa sapientum L.........................................120
4.2.6 Time Killing Analysis of Nelumbo nucifera Gaertn..............................123
4.2.7 Time Killing Analysis of Piper betle L.................................................127
4.2.8 Time Killing Analysis of Psidium guajava L........................................131
4.2.9 Time Killing Analysis of Punica granatum L.......................................135
4.3 Determination of the Antimicrobial Activity of Thai Traditional
Medicinal Ethanolic Extracts by Studying the Time Killing Analysis
with Interfering Substances..............................................................................139
4.3.1 Antimicrobial property of Curcuma zedoaria (Christm.) Roscoe-
ethanolic extract in the presence of interfering substances....................140
4.3.2 Antimicrobial property of Garcinia mangostana L.-ethanolic extract
in the presence of interfering substances...............................................142
4.3.3 Antimicrobial property of Hibiscus sabdariffa L.-ethanolic extract in
the presence of interfering substances...................................................144
4.3.4 Antimicrobial property of Piper betle L.-ethanolic extract in the
presence of interfering substances.........................................................146
Table of contents v
4.3.5 Antimicrobial property of Punica granatum L.-ethanolic extract in
the presence of interfering substances...................................................148
4.3.6 The effect of interfering substances on the growth of test organisms...150
4.4 Application Study: Antimicrobial Activity of the Artificial Coated
Surface.............................................................................................................151
4.5 Application Study: Antimicrobial Activity of Thai Traditional
Medicinal Plant Ethanolic Extract-Incorporated Alginate-Tapioca
Starch based edible films.................................................................................161
4.5.1 Antimicrobial activity of Andrographis paniculata (Burm.f.) Wall. ex
Nees-ethanolic extract-incorporated Alginate-Tapioca starch based
edible film..............................................................................................163
4.5.2 Antimicrobial activity of Curcuma zedoaria (Christm.) Roscoe -
ethanolic extract-incorporated Alginate-Tapioca starch based edible
film.........................................................................................................168
4.5.3 Antimicrobial activity of Garcinia mangostana L. -ethanolic extract-
incorporated Alginate-Tapioca starch based edible film.......................174
4.5.4 Antimicrobial activity of Hibiscus sabdariffa L. -ethanolic extract-
incorporated Alginate-Tapioca starch based edible film.......................179
4.5.5 Antimicrobial activity of Musa sapientum L. -ethanolic extract-
incorporated Alginate-Tapioca starch based edible film.......................185
4.5.6 Antimicrobial activity of Nelumbo nucifera Gaertn. -ethanolic
extract-incorporated Alginate-Tapioca starch based edible film...........190
4.5.7 Antimicrobial activity of Piper betle L. -ethanolic extract-
incorporated Alginate-Tapioca starch based edible film.......................194
4.5.8 Antimicrobial activity of Psidium guajava L. -ethanolic extract-
incorporated Alginate-Tapioca starch based edible film.......................200
4.5.9 Antimicrobial activity of Punica granatum L. -ethanolic extract-
incorporated Alginate-Tapioca starch based edible film.......................205
4.6 Application Study: Cytotoxicity Test of Thai Traditional Ethanolic
Extract..............................................................................................................210
Table of contents vi
4.7 Application Study: Antimicrobial Activity of Alginate-Tapioca Starch
Based Edible Films and Coating Incorporated Ethanolic Extract of
Thai Traditional Medicinal Plants in Model Food System..............................221
4.7.1 Antimicrobial activity of Hibiscus sabdariffa L.-, Piper betle L.-and
Punica granatum L.-ethanolic extract incorporated Alginate-Tapioca
starch based edible coating against pre-contaminated Bacillus cereus
and Salmonella Typhimurium contaminated on carrot..........................222
4.7.2 Antimicrobial activity of Hibiscus sabdariffa L.-, Piper betle L.-and
Punica granatum L.-ethanolic extract incorporated Alginate-Tapioca
starch based edible coating against post-contaminated Bacillus cereus
and Salmonella Typhimurium contaminated on carrot..........................226
4.7.3 Antimicrobial activity of Hibiscus sabdariffa L.-, Piper betle L.-and
Punica granatum L.-ethanolic extract incorporated Alginate-Tapioca
starch based edible coating against pre-contaminated Bacillus cereus
and Salmonella Typhimurium contaminated on chicken meat..............231
4.7.4 Antimicrobial activity of Hibiscus sabdariffa L.-, Piper betle L.-and
Punica granatum L.-ethanolic extract incorporated Alginate-Tapioca
starch based edible coating against post-contaminated Bacillus cereus
and Salmonella Typhimurium contaminated on chicken meat..............234
4.7.5 Antimicrobial activity of Hibiscus sabdariffa L.-, Piper betle L.-and
Punica granatum L.-ethanolic extract incorporated Alginate-Tapioca
starch based edible films against pre-contaminated Bacillus cereus
and Salmonella Typhimurium contaminated on carrot..........................238
4.7.6 Antimicrobial activity of Hibiscus sabdariffa L.-, Piper betle L.-and
Punica granatum L.-ethanolic extract incorporated Alginate-Tapioca
starch based edible films against post-contaminated Bacillus cereus
and Salmonella Typhimurium contaminated on carrot..........................243
Table of contents vii
4.7.7 Antimicrobial activity of Hibiscus sabdariffa L.-, Piper betle L.-and
Punica granatum L.-ethanolic extract incorporated Alginate-Tapioca
starch based edible films against pre-contaminated Bacillus cereus
and Salmonella Typhimurium contaminated on chicken meat..............247
4.7.8 Antimicrobial activity of Hibiscus sabdariffa L.-, Piper betle L.-and
Punica granatum L.-ethanolic extract incorporated Alginate-Tapioca
starch based edible films against post-contaminated Bacillus cereus
and Salmonella Typhimurium contaminated on chicken meat..............252

5 DISCUSSION.....................................................................................................257
5.1 Antimicrobial Properties of Nine Thai Traditional Medicinal Plants
Determined by the Minimum Inhibitory Concentration and the
Minimum Bactericidal Concentration..............................................................257
5.1.1 Dried leaves of Andrographis paniculata (Burm.f.) Wall. ex Nees......259
5.1.2 Dried rhizomes Curcuma zedoaria (Christm) Roscoe...........................261
5.1.3 Dried pericarp of Garcinia mangostana L.............................................262
5.1.4 Dried flowers of Hibiscus sabdarifa L..................................................264
5.1.5 Dried blossom of Musa sapientum L.....................................................265
5.1.6 Dried rhizome of Nelumbo nucifera Gaertn..........................................266
5.1.7 Dried leaves of Piper betle L.................................................................267
5.1.8 Dried leaves of Psidium guajava L........................................................269
5.1.9 Dried pericap of Punica granatum L.....................................................271
5.2 Effect of the Extraction Method on the Antimicrobial Activity......................272
5.3 Influence of the Gram type on the Antimicrobial Activity..............................273
5.4 Antimicrobial Activity of the Ethanolic Extract of the Medicinal Plants
Determined by Time Killing Analysis.............................................................275
5.5 Influence of Interfering Substances on the Antimicrobial Activity of
the Ethanolic Extracts of Thai Traditional Medicinal Plants
Determined by the Time Killing Analysis.......................................................282
5.6 Artificial Antimicrobial Coated Surface..........................................................284
5.7 Antimicrobial Activity of Medicinal Plant Ethanolic Extract-
Incorporated Alginate-Tapioca Starch Based Edible Films.............................286