ANTIMICROBIAL ACTIVITY OF STAR ANISE, ANISEED AND …umkeprints.umk.edu.my/6632/1/THESIS NIK NOR...

2016

ANTIMICROBIAL ACTIVITY OF STAR ANISE,

ANISEED AND CINNAMON AGAINST FOODBORNE

BACTERIA

NIK NOR FATIN BINTI ROZIK

MASTER OF SCIENCE

2016

Antimicrobial Activity of Star Anise, Aniseed and

Cinnamon Against Foodborne Bacteria

by

Nik Nor Fatin Binti Rozik

A thesis submitted in fulfillment of the requirements for the degree of

Master of Science

Faculty of Veterinary Medicine

UNIVERSITI MALAYSIA KELANTAN

i

THESIS DECLARATION

I hereby certify that the work embodied in this thesis is the result of the original

research and has not been submitted for a higher degree to any other University or

Institution.

OPEN ACCESS

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Graduate Committee.

Dated from _ until

(Contains confidential information under the office

Official Secret Act 1972)*

(Contains restricted information as specified by the

organization where research was done)*

I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.

1. The thesis is the property of Universiti Malaysia Kelantan.

2. The library of Universiti Malaysia Kelantan has the right to make copies for the

purpose of research only.

3. The library has the right to make copies of the thesis for academic exchange.

SIGNATURE SIGNATURE OF SUPERVISOR

IC/ PASSPORT NO. NAME OF SUPERVISOR

Date: Date:

ii

ACKNOWLEDGEMENTS

In the name of Allah, the Most Gracious and Most Merciful. Alhamdulillah, all praises

to Allah S.W.T for giving me strength to face all challenges and problems in

accomplishing my thesis project succesfully.

First and foremost, special thanks to my parents (Rozik Abas & Tuan Norizan Tuan

Soh), my beloved husband (Nik Mohd Hafizuddin Mohd Zuhdi) and family for giving

me their continuos support, pray and encouragement. To my dedicated supervisor, AP

Dr. Mohd Mokhtar Arshad, thank you for the advised and guidance in completing my

thesis project. I would like to show my gratitute to my co-supervisor, Datin Prof. Dr.

Kalthum Hashim, FPV lecturers especially to Prof. Dr. Wan Zahari Bin Mohamed and

Dr. Erkihun Aklilu Woldegiorgis and committee for the helps and guidancesv. Special

appreciations goes to Miss Nani Izreen, Miss Siti Rokiah, Miss Ain Basirah and other

laboratory staffs, without them, this study would not have been successful.

Last but not least, I would like to thank my beloved friends (Nurul Afiqah, Nur

Fadhilah, Suhaily Suhana, Ain Auzureen and Nadiah) for giving me help and support

without any hesitation. Lastly, thanks to those who have directly or indirectly involved

in helping me to finish my thesis project succesfully.

iii

TABLE OF CONTENTS

PAGE

THESIS DECLARATION i

ACKNOWLEDGEMENT ii

TABLE OF CONTENT iii

LIST OF TABLES vi

LIST OF FIGURES viii

LIST OF ABBREVIATIONS ix

LIST OF SYMBOLS x

ABSTRAK xi

ABSTRACT xii

CHAPTER 1 INTRODUCTION 1

1.1 General Introduction 1

1.2 Problem Statement 4

1.3 Research Question 5

1.4 Hypothesis 5

1.5 Objectives 5

1.6 Significance of Research 6

CHAPTER 2 LITERATURE REVIEW 7

2.1 Spices 7

2.1.1 Star anise 8

iv

2.1.2 Aniseed 11

2.1.3 Cinnamon 13

2.2 Foodborne Illness 15

2.3 Foodborne Bacteria 19

2.4 Extraction of Essential Oil From Spices 24

CHAPTER 3 MATERIALS AND METHODS 27

3.1 Spices 27

3.2 Preparation of Crude Extract 27

3.3 Tested and Control Bacteria 28

3.4 Antimicrobial Activity of Crude Extraction by Agar Diffusion Method 28

3.5 Statistical Analysis 29

3.6 Extraction of Essential Oils 29

3.7 Antimicrobial Activity of Essential Oils using Hole Plate Agar 30

Diffusion Method

3.8 Minimal Inhibitory Concentration of Essential Oil 31

3.9 Decontaminating Chicken Carcasses 31

CHAPTER 4 RESULTS 33

4.1 Result of Antimicrobial Activity of Crude Extract by Agar Diffusion 33

Method

4.2 Result of Antimicobial Activity of Essential Oils Extract using Hole 42

Plate Agar Method

4.3 Result of Minimal Inhibitory Concentration of Essential Oils 49

4.4 Result of Decontaminating Chicken Carcasses 50

v

CHAPTER 5 DISCUSSION 51

5.1 Crude Extract 51

5.2 Essential Oils Extract 54

CHAPTER 6 CONCLUSION AND RECOMMENDATION 59

REFERENCES 61

APPENDIX A 69

APPENDIX B 73

vi

LIST OF TABLES

NO. PAGE

3.1 Diameter of zone of inhibition according sensitivity 29

against bacteria

4.1.1 Mean diameter of inhibition zone of different concentration 34

of extracts and method of extraction of star anise against

Salmonella Typhimurium



Staphylococcus aureus



Escherichia coli

4.1.4 Mean diameter of inhibition of different concentration of extracts 37

and method of extraction of star anise against S. aureus

ATCC 6538


and method of extraction of star anise against

Escherichia coli ATCC 25922


and method of extraction of star anise against

Salmonella Typhimurium ATCC 14028


and method of extraction of cinnamon against S.aureus


and method of extraction of cinnamon against S. aureus

ATCC 6538

4.2.1 Mean diameter of zone of inhibition of essential oil of different 43

spices against E. Coli


spices against E. coli ATCC 25922


spices against S. aureus


spices against S. aureus ATCC 6538

vii


spices against S. Enteritidis


spices against S.Typhimurium ATCC 14028


spices against S.Typhimurium

4.3 Mnimal Inhibitory Concentration of essential oil of different 49

spices against different bacteria

4.4 Mean total plate count for decontamination of chicken meat 50

viii

LIST OF FIGURES

NO.

2.1

Star anise (Illicium verum)

PAGE

9

2.2 Aniseed (Pimpinella anisum) 11

2.3 Cinnamon (Cinnamomun verum) 14

ix

LIST OF ABBREVIATIONS

µ g Microgram

µ L Microlitre

ANOVA Analysis of Variance

ATCC American Type Culture Collection

CFU Colony Form Unit

mL Mililitre

mg Miligram

MIC Minimal Inhibitory Concentration

SD Standard deviation

DIZ Diameter of inhibition zone

mm Milimetre

HIV Human immunodeficiency virus

EO Essential oils

g Gram

mbar Milibars

MHA Mueller hinton agar

DMSO Dimethyl sulfoxide

TPC Total plate count

Eh Oxidation-reduction potential

h Hour

GRAS Generally recognized as safe

x

LIST OF SYMBOLS

ºC Degree Celsius

% Percentage

xi

Aktiviti Antimikrob Bunga Lawang, Jintan Manis dan Kulit Kayu Manis

Terhadap Bakteria Bawaan Makanan

ABSTRAK

Salmonella spp, Escherichia coli and Staphylococcus aureus adalah bakteria yang

kebiasaannya terlibat dalam pencemaran daging ayam, sehingga menyebabkan wabak

keracunan makanan. Bahan kimia sering digunakan sebagai bahan pengawet untuk

mengurangkan pencemaran bakteria. Objektif kajian ini adalah untuk mengetahui

aktiviti antimokrob ekstrak mentah dan pati minyak dari jintan manis, bunga lawang

dan kayu manis terhadap bakteria bawaan makanan dan menialai potensi dekontaminasi

pati minyak ke atas daging ayam. Air, etanol dan metanol digunakan untuk

menghasilkan ekstrak mentah jintan manis, bunga lawang dan kayu manis. Pati minyak

dihasilkan menggunakan radas ‘Soxhlet’. Aktiviti antimikrob ditentukan dengan

menguji ekstrak mentah dan pati minyak ke atas Salmonella spp, E. coli and S. aureus

menggunakan “agar well diffusion assay”. “Minimal inhibitory concentration (MIC)”

pati minyak ditentukan menggunakan kaedah “microtitre broth dilution”. Aktiviti

dekontaminasi pati minyak ditentukan dengan menjalankan ujian “total plate count

(TPC)” ke atas daging ayam sebelum dan selepas dirawat dengan 0.1%, 0.5% and 1.0%

pati minyak. Ekstrak air dari bunga lawang menghasilkan diameter zon perencatan

paling panjang ke atas S. aureus (12.33 mm) berbanding dengan ekstrak etanol dan

metanol pada kepekatan 200 mg/mL. Ekstrak etanol dan metanol kayu manis

menghasilkan diameter zon perencatan yang sama ke atas S. aureus (15.67mm). Pati

minyak kayu manis menghalang pertumbuhan E. coli pada semua kepekatan yang

digunakan. Pati minyak bunga lawang menghalang pertumbuhan E.coli pada kepekatan

80% (purata diameter zon perencatan = 9.33 mm) and 100% (purata diameter zon

perencatan = 11.67 mm). Pati minyak jintan manis tidak menghalang pertumbuhan S.

Enteritidis pada semua kepekatan yang digunakan. Pati minyak kayu manis menghalang

pertumbuhan S. Enteritidis pada semua kepekatan yang digunkan. Pati minyak bunga

lawang menghalang pertumbuhan S. Enteritidis pada kepekatan 80% and 100%. MIC

jintan manis ke atas S. aureus dan S. aureus ATCC 6538 adalah 0.6 µ g/µ l. MIC paling

rendah pati minyak bunga lawang adalah 0.08 µ g/µ l ke atas S. aureus dan yang paling

tinggi adalah 0.8 µ g/µ l ke atas E. coli, S. Typhimurium dan E. coli ATCC 25922. MIC

kayu manis mempunyai MIC yang rendah (0.02 µ g/µ l) ke atas S. aureus dan S. aureus

ATCC 6538 dan MIC yang tinggi ke atas S. Enteritidis (0.1 µ g/µ l). Selepas dirawat

dengan 1.0% pati minyak selama 24 jam “TPC” daging ayam berkurangan dari 6.14

log CFU/mL kepada 5.28 log CFU/mL bagi jintan manis, kepada 5.16 log CFU/mL

bagi bunga lawang dan kepada 5.04 log CFU/mL bagi kayu manis. Kesimpulan nya

kajian ini menunjukkan pati minyak dari rempah yang digunakan mempunyai aktiviti

antimikrob yang lebih baik dari ekstrak mentah.

xii

Antimicrobial Activity of Star Anise, Aniseed and Cinnamon against Foodborne

Bacteria

ABSTRACT

Salmonella spp, Escherichia coli and Staphylococcus aureus frequently contaminate

chicken meat and result in outbreaks of food poisoning. Chemical preservatives were

commonly used to reduce bacterial contamination.The objectives of this study wereto

determine the antimicrobial activity of the crude extracts and essential oil of aniseed,

cinnamon and star anise against foodborne bacteria and to evaluate the decontamination

potential of the essential oil of the spices on chicken meat. Crude extract of star anise,

aniseed and cinnamon were extracted using water, ethanol and methanol. Essential oils

were extracted using Sohxlet apparatus. Antimicrobial activity was determine by testing

the crude extracts and essential oils against Salmonella spp, E. coli and S. aureus using

agar well diffusion assay. Minimal inhibitory concentration (MIC) of essential oil was

determined by microtitre broth dilution method. Decontamination activity of the

essential oil was determined using total plate count (TPC) of chicken meat before and

after treatment with the 0.1%, 0.5% and 1.0% essential oils. Aqueous extract of star

anise had the highest mean of inhibition zone against S. aureus (12.33 mm) compared to

ethanol and methanol extract. Ethanol and methanol extracts of cinnamon gave the same

mean of inhibition zone (15.67 mm) against S. aureus. Essential oil of cinnamon

inhibited the growth of E. coli at all the tested concentration and essential oil of star

anise inhibited E. coli at the concentration of 80% (mean diameter of zone of

inhibition= 9.33 mm) and 100% (mean diameter of inhibition zone = 11.67 mm).

Aniseed oil did not inhibited growth S. Enteritidis at all concentration but cinnamon oil

inhibited the growth of S. Enteritidis at all concentration tested while star anise oil

inhibited S. Enteritidis at the concentration of 80% and 100% respectively. MIC of

aniseed against S. aureus and S. aureus ATCC 6538 was 0.6 µ g/µ l. The lowest MIC of

essential oil of star anise was 0.08 µ g/µ l against S. aureus and the highest was 0.8 µ g/µ l

against E. coli, S. Typhimurium and E. coli ATCC 25922. Cinnamon oil had the lowest

MIC which is 0.02 µ g/µ l against S. aureus and S. aureus ATCC 6538 where its highest

value was against S. Enteritidis which is 0.1 µ g/µ l. After being treated with 1.0%

essential oil for 24h the TPC of the chicken meat reduced from 6.14 log CFU/mL to

5.28 log CFU/mL for aniseed, to 5.16 log CFU/mL for star anise and to 5.04 log

CFU/mL for cinnamon. In conclusion, this study showed that the essential oils of the

spices had better antimicrobial activity compared to the crude extracts.

1

CHAPTER 1

INTRODUCTION

1.1 General Introduction

Consumer demand for food rises yearly. In order to meet the demand,

many food are locally produced and processed locally under very intensive

production and processing systems. In addition, food are also imported from

foreign countries. During the production, processing and importation, the food

can be contaminated with microorganisms, resulting in foodborne illness in

humans, if they are not stored, processed and cooked properly (Beuchat & Ryu,

1997).

The globalization of the food industry has resulted in increased cases of

foodborne illness due to the unhygienic condition present during processing and

handling of food, along with workers involved in the production line. People

who are busy and live a hectic lifestyle often dine at fast food outlets or

restaurants. These eateries generally have many people involved in preparing

and handling the food, thus increasing the chance of contamination resulting in

foodborne illness (Abdul-Mutalib, Syafinaz, Sakai & Syirai, 2015).

Microorganisms which are already known to cause foodborne disease can appear

in a new way, for example, new form of infection associated with new food, or

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found in a new geographical location, as well as changes in microbial genotypes.

(Altekruse, Cohen & Swerdlow, 1997).

About 76 million cases of foodborne illness occur every year in the

United States. Roughly, about 30% from 13.8 million cases occurred due to

bacterial contamination (McEntire, 2004). In 2002, more than 100 possible

foodborne and waterborne outbreaks have been investigated by the Illinois

Department of Public Health (IDPH) and local health departments. Ninety-one

were classified as foodborne outbreaks; 24 outbreaks were laboratory confirmed;

38 outbreaks were characteristic of a particular foodborne pathogen; and 29

outbreaks were of unknown origin. Salmonella was the cause of six outbreaks

while Escherichia coli O157:H7 accounted for three outbreaks (Austin, 2004).

Consumption of chicken and chicken products caused a high number of acute

Campylobacter enteritis or campylobacteriosis in humans (CDC, 2005).

Malaysia had a lower number of cases of foodborne diseases compared to

other countries owing to unreported cases as the affected persons had a minor

bacterial infection therefore had not sought treatment at the hospitals (Abdul-

Mutalib et al, 2015). Foodborne illness occured in schools were reported

because the students were treated at hospitals while incidences that involve

people who ate at food stores or restaurants were hardly detected as some of

them received treatment from private clinics and hospitals (Lim, 2002).

About 50% of foodborne cases occurred in Malaysia are due to

unhygienic food handling procedures (Abdul-Mutalib et al, 2015; Sharifa Ezat,

2013). Bacterial pathogens commonly isolated were non-typhoidal Salmonella

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(57%), Aeromonas spp. (4%), Shigella spp. (11%), enteropathogenic E. coli

(EPEC) (14%) and Campylobacter spp. (5%) (Lim, 2002).

Acute food poisoning symptoms caused by foodborne bacteria include

nausea, vomiting, fever, abdominal pain, and diarrhea or bloody diarrhea. The

foodborne illness may also lead to chronic or long- lasting health problem such

as dehydration, Guillain-Barre syndrome, Reiter’s syndrome and Hemolytic

uremic syndrome (Marler, 2011).

Nowadays, consumers demand for more ready-to- cook and ready- to-eat

foods that are of high quality, long shelf-life and safe. Controlling microbial

growth can reduce the occurrence of food spoilage and food poisoning. One of

the methods is by using chemical preservatives on ready-to-cook and ready-to-

eat food. Pathogenic bacteria are inhibited by these chemical preservatives.

However, consumers nowadays are more inclined to use natural preservatives

instead of chemical ones. This is because continuous use of chemical

preservatives may result in residual toxicity and microbial resistance and may be

carcinogenic, which are health risks to consumers. Food were introduced with

synthetic chemicals in order to control deterioration caused by bacteria and also

used to alter the palatability of the food (Ozcan & Erkmen, 2001)

Natural sources of antimicrobial compound have been tested such as

essential oils from herbs or spices to replace the use of chemicals in food

preservatives. Many researchers have found natural substances that have a good

antimicrobial activity (Ead, Jangchud, Chonhenchob & Suppakul, 2006; Bin, Yi,

Brooks & Corke, 2007). Spices and herbs are some natural substances that

4

posses antimicrobial activity. Microbial resistance do not exist due to differences

of mechanism of actions in essential oils. They were also considered as

Generally Recognized as Safe (GRAS) by U. S. Food and Drug Administration

and safe for human consumption (Dobre, Gagiu & Petru, 2011).

Extracts of the spices exhibited antimicrobial activity towards

microorganism due to their secondary metabolites and can be utilised in food

industries to replace the chemical preservatives used in reducing food poisoning

and spoilage during food production. In this research, antimicrobial effect of

extracts from star anise, aniseed and cinnamon were evaluated against

Staphylococcus aureus, Salmonella Typhimurium, Salmonella Enteritidis, and

Escherichia coli.

1.2 Problem Statement

Foodborne bacteria such as Salmonella spp. and Campylobacter jejuni

are the common causes of food poisoning. Chicken meats are frequently

reported as the source of outbreak caused by bacterial contamination. Chemical

preservatives had been included in ready-to-cook and ready-to-eat food in order

to inhibit the growth of the undesirable bacteria. However, consumers nowadays

are more discerning to use natural preservatives instead of chemical. This is

because the continuous use of chemical preservatives may result in residual

toxicity and microbial resistance and may be carcinogenic, which are health

risks to consumers. Antimicrobial compound from natural sources such as

5

essential oils from herbs or spices can be used as natural preservatives in ready-

to-cook or ready-to-eat food.

In Malaysia, spices such as star anise, aniseed and cinnamon are

frequently used for cooking and are easily available in the market. However,

their antimicrobial activities against foodborne bacteria such as Salmonella spp,

Staphylococcus aureus and Escherichia coli have not been investigated.

1.3 Research Question

Do star anise, aniseed and cinnamon possess antimicrobial activity against

foodborne bacteria?

1.4 Hypothesis

Crude extracts and essential oil of star anise, aniseed and cinnamon possess

antimicrobial activity against foodborne bacteria such as Salmonella spp,

Staphylococcus aureus and Escherichia coli.

1.5 Objectives

To determine the antimicrobial activity of star anise, aniseed and cinnamon

extracts against foodborne bacteria.

6

To determine in vitro the minimal inhibitory concentration (MIC) of the

essential oil extracts against foodborne bacteria.

To evaluate the decontamination potential of essential oil from star anise on

chicken meat.

1.6 Significance of Research

The results of the research can help food producers to choose natural

preservatives to preserve ready-to-cook and ready-to-eat foods that they

manufacture in order to inhibit the growth of foodborne bacteria.

7

CHAPTER 2

LITERATURE REVIEW

2.1 Spices

Spices are plants that possess antimicrobial properties, medicative

properties and have stimulating effects in the animal digestive system (Rahman,

Parveez, Islam & Khan, 2011). The spices have been used in Europe, the Middle

East and Asia for thousands of years. Shelf life of vegetables, fishes, breads and

meats were prolonged using spices before refrigeration methods came to

existance. Spices were used in foods as seasonings, preservatives, food

additives, flavor enhancer and aroma and appetizing effects. Deterioration and

spoilage of food can be reduced by using spices together with pickling smoking,

and salting in order to extend shelf life of food (Bin et al., 2007).

Previous studies confirmed that growth of Gram-positive and Gram-

negative foodborne and food spoilage bacteria were inhibited by cinnamon, sage,

onion, cloves, garlic, thyme, and other spices (Hoque, Bari, Juneja &

Kawamoto, 2008). Ertuk (2006) reported that extracts of the spices were

effective against bacteria compared to fungi. Powders, extracts and essential oils

of various herbs and spices had antimicrobial activity and inhibited aflatoxin

formation by fungus ( Thanaboripat, Suvathi, Srilohasin & Sripakdee, 2007).

8

Antimicrobial activity of spices depends on the type of spice, and on the

condition of the spices when added upon, such as fresh, dried or extracted form.

It may also vary between strains within the same species and also depends on

storage conditions, geographical sources, processing and harvesting season, its

composition and concentration used (Hoque et al., 2008; Rahman et al., 2011).

Antimicrobial activity of spices is affected by the type and concentrations of the

targeted bacteria and its substrate composition. Bioactive compound that posses

antimicrobial potency in spices are essential oils, phenolic compounds, tannins,

saponins and flavonoids (Rahman et al., 2011).

2.1.1 Star Anise (Illicium verum)

Star Anise or also known as Illicium verum is native spice of China,

which is the main ingredient in Chinese five-spice blend, and possess important

roles in food flavoring. Star anise is an uneven star-shaped fruit with a central

core joining the eight carpels with a seed in each carpel. Star anise are used

either in pieces, dried whole shaped and in powdered form. Star anise are sun

dried since their harvesting was done before the star anise ripens and hence

green in color (Chempakam & Balaji, 2008). Compared to fennel and anise, the

hard and reddish brown carpels of star anise have more sweet, licorice-like and

strong in flavor. Used in a high concentration can lead to a bitter aftertaste.

Spicy and sweet flavors are strongly detected during the cooking process. Star

9

anise pods contain more flavors compared to their seeds but the broken pieces of

pods are less aromatic compared to the original star anise whole shape.

Figure 2.1: Star anise (Illicium verum)

Chemical components of fresh fruit pod or pericarp has 5% to 8%

essential oil, and the dried fruit pericarp has 2.5% to 3.5%, mainly anethole

(85% to 90%), limonene, phellandrene, d-terpineol, ρ-cymene, α-pinene, and

1,4-cineole. Anethole has a higher antibacterial activity compared to anisyl

acetone, anisyl alcohol, and anisyl aldehyde against Acinetobacter baumannii,

with Minimal Inhibitory Concentration (MIC) value of 0.11 mg/mL (Yang et al.,

2010). MIC values indicate that the isolated anethole is as effective as the

standard anethole in not only inhibiting bacterial growth, but also fungal and

yeast growth (De M, De K, Mukhopadhyay, Miro & Anarjee, 2001).

Yang, Hong, Li, Dong & Xing (2010) reported that 67 clinical drug-

resistant isolates, including 27 A. baumannii, 20 Pseudomonas aeruginosa, and

20 strains of methicillin-resistant Staphylococcus aureus have been affected by

extract of star anise. (Singh, Maurya, deLampasona & Catalan, 2006) stated that

both essential oil and the acetone extract of star anise exhibited a broad spectrum

10

of antifungal and antibacterial activity, including inhibitory activity against S.

aureus , P. viridicatum, Bacillus spp., Fusarium moniliforme and P. citrinum.

Tamil-flu (oseltamivir phosphate) was the antiviral drug produced from star

anise to cure the illness caused by avian influenza H5N1 strain of virus (Li &

Liu, 2000).

Confectionery industries use essential oils of star anise as a flavor in

licorice and candies while in baking industries it is used to enhance flavor of

cakes, cookies and biscuits. Heath (1981) indicated that star anise had a volatile

oil content of 8-9% in dried material and 2.5-3.5% in the fresh fruit. It has about

20% fixed oil (Dang & Sarath, 1997;Raghavan, 2007). East Asians use star anise

to relieve colic and stomach pains. They chew it after a meal to promote

digestion and fresh breath. Star anise is also used in tea to cure sore throats and

coughs. Its oil is used as an ingredient in cough lozenges (Raghavan, 2007). Star

anise are used in its original shape and also its essential oils. East Asian people

used star anise to facilitate digestion, freshen the breath by chewing the star

anise, while sore throats and coughs can be reduced by adding some star anise in

tea.

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2.1.2 Aniseed (Pimpinella anisum)

Aniseed is originally from India and southwest Asia. It is a flowering

plant in the family Apiaceae and can grow up to 1m tall. The leaves of this

herbaceous plant are 2- 5cm long and shallowly lobed located at the base of the

plant whereas the leaves at the higher stem of the plant are divided. Aniseed has

white flowers, 3 mm diameter and is formed in thick umbels. The fruit is 3- 5

mm long and has oblong shaped dry schizocarp. Each carpel of the aniseed fruit

splits apart to form two or more parts during maturity stages (Akhtar,

Deshmukh, Bhonsle, Kshirsagar & Kolekar, 2008). Anise seeds have a sweet

licorice- like taste and is warm, fruity, and camphoraceous. Aniseed’s flavour is

more camphor-like and delicate compared to star anise and fennel.

Figure 2.2: Aniseed (Pimpinella anisum)

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