ANTIMICROBIAL ACTIVITY OF ALGINATE-BASED FILM INCORPARATED

WITH CINNAMALDEHYDE

NUR HUMAIRA BINTI MOHAMMAD HAMID

A thesis submitted in partial fulfilment of the

requirements for the award of the degree of
Diploma of Chemical Engineering Technology in Bioprocess

University Kuala Lumpur

Malaysian Institute Chemical & Bioengineering Technology

JULY 2018

i
 ABSTACT

Food spoilage is the process of contamination of food leading to loss of color,

texture and their nutritive values. Antimicrobial packaging can be developed by
incorporating antimicrobial substances to polymer film to inhibit the growth of food
spoilage microorganisms. However, some of these antimicrobial substances are
prohibited due to toxicological reasons. Hence, this study was conducted to develop
alginate based film incorporated with a natural compound, cinnamaldehyde.
Cinnamaldehyde is an aromatic compound that gives cinnamon its flavor and odor. To
develop the polymer film, cinnamaldehyde was mixed with the sodium alginate and
Tween 80. The antimicrobial activity of the developed film was evaluated on food borne
bacteria. On cross streak test, the film exhibited antimicrobial activity on both Gram
positive and Gram negative bacteria. 3 out of 6 bacteria were inhibited by the developed
film. The largest inhibition zone was observed on methicillin resistant Staphylococcus
aureus (MRSA). On Hohenstein Challenge Test, the film incorporated with
cinnamaldehyde showed 100% growth reduction on both MRSA and Proteus mirabilis.
98.9% growth reduction was reported for Escherichia coli relative to control. In
conclusion, the alginate-based film exhibited significant antimicrobial activity on food
borne bacteria.

ii
 ABSTRAK

Kerosakan makanan adalah proses pencemaran makanan yang menyebabkan

kehilangan warna, tekstur dan nilai nutrisi mereka. Pembungkusan antimikrobial boleh
dibangunkan dengan memasukkan bahan antimikrobial ke filem polimer untuk
menghalang pertumbuhan mikroorganisma yang rosak makanan. Walau bagaimanapun,
beberapa bahan antimikrob ini dilarang kerana sebab toksikologi. Oleh itu, kajian ini
dijalankan untuk membangunkan filem berasaskan alginate yang digabungkan dengan
sebatian semula jadi, cinnamaldehyde. Cinnamaldehyde adalah sebatian aromatik yang
memberikan cinnamon rasa dan bau. Untuk membangunkan filem polimer,
cinnamaldehyde telah dicampur dengan natrium alginat dan Tween 80. Aktiviti
antimikrob filem maju telah dinilai pada bakteria yang ditanggung makanan. Pada ujian
coretan silang, filem itu mempamerkan aktiviti antimikrobial pada bakteria Gram positif
dan Gram negatif. 3 daripada 6 bakteria telah dihalang oleh filem yang maju. Zon
perencatan terbesar diperhatikan pada Staphylococcus aureus (MRSA) tahan
methicillin. Mengenai Ujian Cabaran Hohenstein, filem yang digabungkan dengan
cinnamaldehyde menunjukkan pengurangan pertumbuhan 100% pada kedua-dua
MRSA dan Proteus mirabilis. Pengurangan pertumbuhan 98.9% dilaporkan untuk
Escherichia coli relatif terhadap kawalan. Kesimpulannya, filem berasaskan alginat
menunjukkan aktiviti antimikrob penting pada bakteria makanan.

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 APPROVAL

I certify that I have supervised and read this research; as in my opinion, it

conforms to acceptable standard of scholarly presentation and is fully adequate, in
scope and quality, as a thesis for the Diploma of Chemical Engineering Technology in
Bioprocess

Signature:..

Name: DR. TONG WOEI YENN

Date:

iv
 DECLARATION

I declare that this thesis entitled Antimicrobial activity of Alginate-Based film

incorporated with cinnamaldehyde is the result of my own research except as cited in the
references. The thesis has been accepted and is not concurrently submitted in
candidature of any other diploma.

Signature:..

Name: NUR HUMAIRA BINTI MOHAMMAD HAMID

Date:

v
 ACKNOWLEDGEMENT

Firstly, I would like to highlight my thankful and grateful to Allah S.W.T because
gave me this opportunity and healthy to complete my final year project successfully.
Special appreciation to my respected supervisor Dr Tong Woei Yenn for his valuable
guidance, advices, tolerances and concern during this project.

Besides, I also like to thank to the technicians, Mr. Yusuf, Mr. Nizam, and Mr.
Syukri for their kindness and cooperation for helping me to provide the apparatus that I
needed.

Last but not least, I would also like to express a deep sense of gratitude to my
beloved family especially my parents, Mr. Hamid and Mrs. Maziah for their prayers,
advices, supports, and their endless love with valuable guidance in this study period.
The blessing help and guidance was a deep inspiration to me. I am also proud and thak
you for myself for the spirit, attitude and patience until the end of this study.

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 TABLE OF CONTENT

TITLE PAGE

TITLE PAGE i

ABSTRACT ii

ABSTRAK iii

APPROVAL PAGE iv

DECLARATION v

ACKNOWLEDGEMENT vi

TABLE OF CONTENT Vii

LIST OF FIGURE x

LIST OF TABLE X1

CHAPTER 1:INTRODUCTION

1.1. background of study 1

1.2. problem statement
2
1.3. objectives

CHAPTER 2:LITERATURE REVIEW

vii
2.1 edible film 4

2.2 antimicrobial packaging 5

2.3 sodium alginate 7

2.4 alginate base film 8

2.5 cinnamon

2.5.1 history of cinnamon 9

2.5.2 benefit of cinnamon 10

2.6 cinnamon aldehyde 11

2.6.1 biological activity of cinnamaldehyde 13

2.6.1.1 antidiabetic 13

2.6.1.2 antioxidant activity 13

2.6.1.3 antimicrobial activity 14

CHAPTER 3 :METHODOLOGY

1.1 Flowchart of the procedure 15

3.2 antimicrobial activity of Alginate-based incorporating with
16
cinnamaldehyde

16
3.3.1 test microorganisms

16
3.3.2 Preparation of inoculum

17
3.3.3 cross streak test

viii
 3.3.4 Hoheinstein Challenge test 17

CHAPTER 4: RESULT AND DISCUSSION

4.1 Development of Alginate-Based film incorporating with 19

cinnamaldehyde
4.2 Antimicrobial activity of Alginate-Based film incorporating
with cinnamaldehyde 20
4.2.1 cross streak assay
4.2.1 Hoheinstein Challenge test 23

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 25

5.0 Conclusion and recommendations 25

REFFERENCES 26

APPENDIX 37

ix
 LIST FO FIGURE

FIGURE TITLE PAGE

2.1 The bark of cinnamon 8
2.2 The chemical structure of cinnamaldehyde 11
4.1 Alginate-based film produced 18

x
 LIST OF TABLE

FIGURE TITLE PAGE

2.1 Physical and chemical properties of sodium alginate 6

2.2 The physical can chemical propertiest of cinnamadehyde 12

4.1 Antimicrobial activity of alginate based film incorporated with 20

cinnamaldehyde on Cross Streak Test

4.2 The clear zone surrounding the film indicates the inhibitory 21
activity of the film on Cross Streak Test.

4.3 The antimicrobial efficacy of alginate based film incorporated 22

with cinnamaldehyde on Hohenstein Challenge Test.

xi
 CHAPTER 1

INTRODUCTION

1.1 background of study

Antimicrobial resistance is refer to the ability of microorganism to stop by

antimicrobial agent from working against. The definition of antibiotics is refer to
substances produced by microorganisms that act against another
microorganisms.Ineffective standards treatment may prolong the illness, disability and
even death. Infection with resistance is hard to treat due to longtime used of
antimicrobial agent as the infectious organisms have adapts to surrounding environment.
This issue may require cost and sometime toxic alternatives. Resistance bacteria can
spread through many routes. Effective treatment of infectious diseases in humans can
be compromise by the antimicrobial resistance occurs in zoonotic bacteria that presence
in animal and food. In the field of food safety, the best control options to reduce risk may
be establish where to the consumers from risks related the food chain. At least 23,000
people die each year cause by the infectious of bacteria reported by Centre for Disease
Prevention and Control (CDC).

In this project, new sources of antimicrobial agents were evaluated. The

cinnamalaldehyde was studied on its potential as antimicrobial agent. Plants have a long
story of clinical relevance as source of potential bacteria chemotherapeutic agents
(Cushnie, 2005). The antimicrobial activity of different type of spice has been known a
long time ago and traditionally added to food as seasoning due to it aromatic properties

1
(Valdes, A., 2015). The uses of plant extract on medical treatments led to investigation
of the microbial activity of the medicinal plant ( Kadi et al., 2011).

The edible film that incorporated with antimicrobial compound can prolong the shelf
life of food. Next, the antimicrobial compound was help in preventing the growth of food
borne microorganism. In this study, the cinnamaldehyde was used as antimicrobial
compound that presented in the bark of cinnamon tree. The cinnamaldehyde had the
pungent smell which prevent the rate growth of microorganisms.

1.2 Problem statement

Consumers demand for microbiologically safer foods, convenience, smaller

packages, and longer product shelf life is increasing which forcing the industry to
develop new food-processing, handling, and packaging strategies. . Earlier, the food
was preserved by smoking or kept in the iceboxes. The citrus fruits was preserved in
the boxes with pouring the molten wax over them to send to the North (hardenburg,
1967). Consumers now days choose to spend less time in the kitchen in preparing
meals. The most material used for food packaging based on polymers as they are
low cost with large availability in large quantity and good functionality characteristics
(Ttharanathan, 2003).therefore, some of the polymers may bring damage on the
resource and environment. These phenomena can leads to serious ecological
problem. Furthermore, the harmful toxin and chemical that contain in the food
packaging may contaminate the food. The use of a biopolymer such sodium alginate
is interesting solution as it is inexpensive, abundant, biodegradable, and edible (Mali
and Grossmann, 2003). This application was limited due to lack of knowledge about
the effectiveness to the product, and no specific regulations for active packaging.

2
1.2 Objectives

To determine the antimicrobial activities of alginate-based film incorporated

with cinnamaldehyde.

To develop alginate-based film incorporated with cinnamaldehyde.

3
 CHAPTER 2

LITERATURE REVIEW

2.1 Edible film

Edible film can be considered as any type of material that used for coating and
wrapping various foods to extend the shelf life of the product. A film may provide surface
sterility which can prevent loss of other important compound in the food. Generally, the
thickness of the film is less than 0.3 mm. Protein, lipids, polysaccharide or any
combination are different type of biopolymer where they have been used as carrier to
produce edible coating with antimicrobial properties (tavassoli-kafrani, 2016). These
biopolymer films can be used as host additives and nutrients to be released for
controlled rate of food and forming the basis of active packaging systems (Campos et al.
2011). Alginate that presence in the film will prolong the shelf life of product without
causing anaerobic respiration (Olivas et al. 2007).

Antimicrobial addictive can be addictive into film to protect food product from
microbial spoilage which can extending their shelf-life and safety. Bioactive edible
coatings can be applied to food surfaces by dipping, coating, and spraying on food. and
they have been tested in meat, fish, dairy products or minimally-processed fruit and
vegetables. Polymer with 1.0% of oregano essential oil was act as active material to
maintain the safety and quality of fresh Brussels sprouts stored at 16 C for 14 days
(Krasniewska et. al., 2016).

4
 Esseintial oil is example of the antimicrobial addictive into food packaging which
composed of aromatic and volatile oil extract that obtain from aromatic and medicinal
plant and show strong antimicrobial activities (Sanchez et. al., 2011). The antimicrobial
activity of essential oils is consistently link to presence of hydroxyl group in their phenolic
constituents (Kuorwell et. al., 2014). The combination of cinnamaldehye and wheat
gliadins in the film provide effectiveness against food contaminating fungi in the design
of active food packaging systems (Balaguer et. al., 2013). Nest, the organic acid also act
as antimicrobial addictive in the food packaging. The organic acids such acetic, benzoic,
citric and lactic are used to inhibit microbial growth. The addition of organic acid in the
edible film was proposed to minimized microbial contamination surface (Kuorwel et. al.,
2011). Presence of lactic acid in the whey protein-based film effect the molds and
bacteria (Ramos et. al., 2012).

2.2 Antimicrobial packaging

Antimicrobial packaging refers to the packaging system where able to inhibit

spoilage and pathogenic and pathogenic microorganisms (Ahvenainen, 2003). Active
packaging is the packaging systems by adding active ingredients and active functional
polymers in the packaging systems (Han, Rooney, 2002). By adding antimicrobial agent,
the packaging system limits and reduces the microbial growth rate (Han, 2000). The
antimicrobial activity acquire in the packaging system decreasing the live count of
microorganisms by extending the lag period (Han, 2000). Different type of antimicrobials
can be apply in food product ( Campos et. Al., 2010). Antimicrobial packaging based on
active agent released from the surface of the packaging material without migrate to the
product itself (Quantavalla 2002).

Antimicrobial packaging promising active packaging system controlling spoilage

and pathogenic microorganisms (Vacini 2002). Specific microbial pathogens were
identified to contribute agents of many diseases. Therefore, immediate action must be
done to start to fine effective which is kills the microorganisms. Thus, antimicrobial

5
agents that are used in the treatment of disease include synthetic chemicals as well as
chemical substances or metabolic products made by microorganisms and chemical
substances derived from plants ( Halimi, 2017). Antimicrobial edible film and coating is
able to protect the food from spoilage which can decrease the risk of pathogens growth
by controlling the diffusion of antimicrobial agents onto the food surface ( Nithya,
Murthy,& Halimi, 2013). The food safety, quality and extending the shelf life of food can
be improved by incorporating antimicrobial compound in the food packaging material
(Campos et. at., 2010).

Active agent incorporated in the packaging material in multilayer structures

associated packaging itself (Hotckiss, 2002). There are some advantage of this type of
packaging system compared to direct addition like spraying and immersion of active
compound to product. This type of packaging can control the migration of the bacterial to
maintain the concentration of functional on the surface (Appendini, 2002). Next, avoid
the loss of effectiveness of packaging by food matrix component due to dilution below
active compound (Cagri et. al.,2004). There are widely active compound that can be
added such as nature of material, bacteriocins, natural extract, organic acids and
enzyme (Bastarachia et. al., 2011). The nature of material can be seen as Edible and
nonedible material. Example of edible material is polysaccharide, protein and lipid while
non edible of material is paper and plastic ( Ponce et. al., 2008).

2.3 sodium alginate

Sodium alginate is widely use as stabilizer and emulsifier. These are very
popular in textile printing and manufacturing industries. Alginate was named base on
:alga as the alginate is discovered by E.C.C. Stanford, a Scottish chemist, from British
kelp in the 1880s. Alginate is the natural saccharide with dietary properties. Not all large
brown algae have large quantity of alginate like Sargassum muticum where there only

6
16% - 18% of alginate present in it as it is the adventive species come from japan .The
alginic acid is produce in the seaweed as the jelly bodies after combining with mineral
from sea water. Therefore, the structure of the seaweed is provided by alginate. Figure 1
shows that the chemical structure of sodium alginate.

Table 1: The physical and chemical properties of sodium alginate (Adopted from:
http://www.fao.org/docrep/w6355e/w6355e0x.htm)

Molecular formula C5H7O4COONa

Structural formula

Non property names Alginic acid, sodium salt

Color White to pale yellowish-brown color powder

Functional uses Stabilizer, thickener and gelling agent

Solubility Dissolve slowly in water; insoluble in ethanol and ether

7
 There are many uses of the alginate as it can act as food additives, textile
manufacturing, in pharmaceutical product and cosmetic. Now, there are a few new
products from pharmaceutical industry where the alginate is combine with calcium
bicarbonate to protect the layer at the top of stomach to inhibit reflux. Furthermore, it
promotes healing in medical product including skin wound dressing. The sodium alginate
also used in the textile screen-printing to thickener the reactive dyes. Lastly, it also
waterproofing and fireproofing fabrics and thickening agent in food industries

2. 4 Alginate-Based film

The alginate was a polysaccharide that extracted from brown algae

(phaeophyceae). In food industries, the alginate was used as a common type gelling
agent (Mancini F., Hugh T. H., 2000). The alginate was improved the barrier and tensile
properties of fruit based film as it was hydrocolloids (Mancini F., Hugh T. H., 2000).
Alginate is able to react with trivalent cations in film formation (Anna P., Alicja M., 2003).
The alginate act as antimicrobial agent in film (Kamph N., 2000). The sodium alginate
was naturally non-toxic polysaccharide where it was soluble in water (Anna P., Alicja M.,
2003). The alginate-based film have high potential to carry active ingredient like colorant,
nutrient, falvours, and anti-browning agent (Pranoto et. al., 2005). The alginate-based
film with incorporating with cinnamon, clove or lemon grass oil increase the antimicrobial
effect and extend the microbiology shelf life of food.

8
2.5 Cinnamon

2.5.1 History

Cinnamon has been widely used as a spice. It can be obtained from inner bark of
several trees with genus Cinnamomum for centuries by different type of cultural group of
people around the world. Cinnamon refers to the brownish- reddish of eternal tree of
tropical medicine. This plant needs 2 to 3 years to bear. The mature height of this plant
is 40 to 60 fts. The size of the leaves is 4 to 7 inches where it is thick oval shape. It is
native to South India and Srilanka. Cinnamon is known as Ceylonzeim in German,
Dalchini in Hindi, Cannella in Italian, Yookgway in Chinese and Kurunda in Sinhalese
(Peter, 2006). The figure 1 shows that the type of cinnamon.

Figure 2.1 bark of cinnamon (Adopted from:

http://www.fao.org/docrep/w6355e/w6355e0x.htm)

Early 2000, the Egyptians was employed cinnamon as a perfume agent

especially during embalming process. In the middle age, the cinnamon has been used
as status symbol in Europe as it was expensive supply and limited. The cinnamon was
used in preservation of meat during winter. In 1518, the cinnamon was discovered by

9
Portuguese traders at Ceylon (Sri Lanka). Therefore, there were two type of commercial
cinnamon which Ceylon cinnamon and cassia cinnamon. The cassia cinnamon was
produced in Indonesia with strongest smell and flavor. The presence of aromatic
essential oil gave the flavor of the cinnamon (Robeiro-santos, et ., 2017). But, Ceylon
cinnamon was more expensive than cassia cinnamon. This is because, this type of
cinnamon have sweeter flavor that popular for baking and flavoring hot drinks.

2.5.2 Benefit of cinnamon

On early of the century, the cinnamon had been used as a spice and herbs in
cook as it was delicate in flavor.it is widely used in bake recipe like cake, churros,
chocolate dishes and fruit dessert. It also used as spiced where it was putted in Indian
curry and form of garam masala. Based on the journal of medicinal food, the research
found that the blood sugar reduction was in 3% to 5%.

Firstly, the cinnamaon can be anti-inflammatory depends on the

cinnamamdehyde content (Mercola, 2017). Mostly, the chronic inflammation is role in
development of Alzheimers disease, parkinsonss disease, and mengitis (Shampa C.,
Wolfgang J., 2017). The inflammatory pathways target to help the prevention on
neurodegenerative disease (Kannapan R., et al., 2017).

Other than that, the cinnamon help to boost brain function. According to the
cinnamon boosts brain acticle, who participating in smelling the cinnamon, the scored
can be improved due to task that related to process of attention, the recognition memory,
and response speed. The scents of the cinnamon give better result than jasmine and
peppermint to enhance cognitive function (Mercola, 2016).

Cinnamon act as relieve Symptoms of Attention Deficit Hyperactivity Disorder

(ADHD). The cinnamon aromatherapy had significantly reduce the symptoms of ADHD.
Normally, the children easily had the symptom of ADHD. The children that had the

10
symptoms of ADHD may increase the oxidative stress (Mercola, 2016). Therefore, the
cinnamon is the best antioxidants that help to counteract of this problem.

The cinnamons also help to improve the glycemic status among people in two
type of diabetes (Vafa M., 2017). The cinnamons act as bioactive component with
insulin-like effect which indicate as potential insulin for the diabetes patient (Vafa M.,
mohammadi F., 2012). By consuming the cinnamon, the lipid profile can be control
(Akilen R. E., 2017).

Therefore, by consuming half teaspoon of the spices may help reducing blood
sugar and cholesterol. Other than that, a small pinch of powdered cinnamon help in
treating cough bye chewing or swallowing it.

2.6 Cinnamaldehyde

Cinnamon aldehyde is refer to aldehyde that give the flavor and odor of
cinnamon itself. Cinnamaldeyde occurs naturally from the genus of cinnamomum like
cassia and camphor. The essential oil of cinnamon bark is 90% of cinnamaldehyde.
These essential oil is an effective insecticide with the scent to repel the animal. The
large doses of chemical may be a toxic and skin irritant on concentrated
cinnamaldehyde but, there is agencies that suspect the compound for long term health
hazard. Figure 2 shows that the chemical structure of the cinnamaldehyde.

11
 Figure 2.2 the chemical structure of cinnamaldehyde.( Adopted from:
http://www.fao.org/docrep/w6355e/w6355e0x.htm)

The table 1 shows the physical properties of the cinnamaldehyde like boiling
point, melting point and the color of the cinnamaldehyde. In industry, the
cinnamaldehyde is used as the odor agent, photosensitive chemicals, and solid
preparation agents. But, the consumer used this essential oil as adhesives and sealants,
personal care product, paints and coating. In india, the cinnamon is used widely as
medicines to treat diabetes in traditional system. Cinnamaldehyde restored and altered
the plasma enzyme to near normal (Subash B. P., 2007).

12
 Table 1 the physical and chemical properties of cinnamaldehyde (Adopted from:
http://www.fao.org/docrep/w6355e/w6355e0x.htm)

Color Yellowish oily liquid

odor Pungent and spicy note

taste Sweet taste

Boiling point and 2530C of boiling point and -7.50C of melting point
melting point

Solubility Dissolve in 700 part of water and 60% of alcohol, sol in ether

2.6.1 Biological activity of cinnamaldehyde

2.6.1.1 Antidiabetic

Antidiabetic agent is a substance that control the levels of glucose in blood which
help the person with diabetes. The antidiabetic agent can be include as insulin and oral
hypoglycemic agents ( ). There were antidiabetic drugs to stabilize and control the levels
of blood glucose in body. Cinnamaldehyde alter the plasma enzyme like alanine
aminotransferase and lactate dehydrogenase levels to near normal (Subash B. P.,
2006). Cinnamaldehyde was moderated of reducing concentration of plasma glucose in
both diabetic patients and animals (Kim et. al.,2006).

13
2.6.1.2 Antioxidant activity

Antioxidant is refers to compound that able to delay or prevent oxidation process

process by reacting free radicals (Mancini-Filho J., 1998). They also act as oxygen
scavangers. The natural antioxidant occurs in all part of the plant. Usually, they are
phenolic or poly phenol compound (Mancini-Filho, 1998). Examples of molecule of
antioxidant are derivatives or isomeric forms of polyphenolics, phosphatides, and
polyfunctional organic acids (Marino E. M., 1994). The active compound that presence in
cinnamon possible to be acting concurrently, serially, and additively with other substance
(Nakati, 1992). The cinnamaldehyde helps against the formation of ulcer in stomach
(Blumenthal M., 2004).

2.6.1.3 Antimicrobial activity

The antimicrobial activity of cinnamon is due to its essential oil

(cinnamaldehyde). The cinnamaldehyde mainly used as pain killer in dental surgueries
and aromatherapy that required in digestive desease (Balch, James., 2000). When the
cinnamaldehyde is added to food, it help to inhibit the growth of mold, yeast and bacteria
(Matan N. et. al., 2006).

14
 . CHAPTER 3

METHODOLOGY

3.1 Flow chart of the methodology

STAGE 1:

PREPARATION OF THE EDIBLE FILM

STAGE 2:

CROSS STREAK TESTING

STAGE 3:

HOHENSTEIN TESTING

15
 Figure 3.1 Experimental plan

3.2 Preparation of film

3.2.1 Film with cinnamaldehyde (treated film)

The film was made by mixing the 5 g of sodium alginate, 20 l of Tween 80, 2 ml
of glycerol and 3 ml of cinnamaldehyde with 100 ml of water. The mixture was heated on
the hot plate. Then, 10 ml of the mixture was poured into each petri dish. The mixture
was dried in the 400C oven for 4 hours.

3.2.2 Film with no cinnamaldehyde (control)

The film was made by mixing the 5 g of sodium alginate, 20 l of Tween 80, 2 ml
of glycerol with 200 ml of water. The mixture was heated on the hot plate. Then, 10 ml of
the mixture was poured into each petri dish. The mixture was dried in the 400C oven for
4 hours.

3.3 Antimicrobial activity of Alginate-based film incorporated with

cinnamaldehyde

3.3.1 Test Microorganisms

The test bacteria used in this study were four Gram positive bacteria (B. cereus,
B. subtilis,S. areus and MRSA ), two Gram negative bacteria (E.coli and P. mirabilis).
The stated bacterial were provided from Industrial Biotechnology Research Laboratory
and Plant Pathology Research Laboratory Universiti Sains Malaysia (USM). The test
microorganisms were sub-cultured on nutrient agar. This sub-culture was done every
two weeks to maintain microorganism viability.

16
3.3.2 Preparation of inoculums

The bacterial inoculums were prepared by transferring the microorganism

colonies using wire loop into a universal bottle containing 10 ml of sterile distilled water.
Then, the inoculums were mixed by using vortex. Turbidity of the suspensions was
adjusted according to 0.5 Mc Farland standards. If the turbidity of the suspension was
higher compared to 0.5 Mc Farland standards, the sterile distilled water was added to
inoculum. Add some colonies of microorganisms to suspension when the turbidity of the
suspension was lower than 0.5 Mc Farland.

3.3.3 Cross streak test

The bacterial inoculum prepared in section 3.3.2.was streaked on the nutrient

agar plate. The streaking was done on the surface of nutrient agar by making 7.5 cm
long parallel streak with 1 cm apart at the center of the plate. The film was gently
pressed on the five inoculum of streak with size 2 cm x 2 cm. The plate was incubated
for 24 hours at 370C. After incubation period, a streak of interrupted growth underneath
and the side of the film indicated antibacterial effectiveness of the film.

3.3.4 Hohestein challenge test

The 2 cm x 2 cm of film was transfer into fresh culture of microorganism by 0.1

ml of that was inoculated to strerile nutrient broth. The culture was incubated at 370C for
24 hours in incubator shaker at 150 rpm. After the incubation, the antibacterial activity
was determined by comparing the concentration of the sample with the control. The
serial dilution was done to compare the concentration of sample and the control. The
spread plate was done for 10-3 and 10-4 dilution for the treated film. For the untreated
film (control), the 0.1 ml dilution of 10-7 and 10-8 was spread in nutrient agar plate, the
The percentage of reduction in standard time was expressed by using this equation:

17

(%) = 100

Where,

A= no of colony of control

B= no of colony of sample

18
 CHAPTER 4

RESULT AND DICUSSION

4.1 development of antimicrobial Alginate based-film incorporated with

cinnamaldehyde

The alginate was the polysaccharide that used in this study. The polysaccharide
was very hydrophilic where it was poor water vapour and had gas barrier. In this study,
the alginate-based film had thickness about 0.3 mm. Based on the figure 5.1, the film
had a smooth surface where it can easily remove from the plate. The film became more
plasticizer as there was added glycerol in the mixture. The film had a sweet and pungent
smell where it came from the cinnamaladehyde. The cinnamaldehyde had the pungent
smell in the concentrated state. The color of the film was colorless.

Table 4.1 the Alginate based film

19
4.2 Antimicrobial activity of Cinnamaldehyde in edible film

4.2.1 Cross streak assay

Table 4.1 shows that the antimicrobial activity of the cinnamaldehyde are tested
on cross streak method. The inhibition zone was observed on the plate by measuring the
diameter on the surface and expressed in unit mm (Ramos O. L., Silva, Fernandes J. C.,
2012). The cross streak is easy and rapid method to establish the inhibition properties of
any bacterial and mold that grow on agar plate (Williston et. al., 1947). This method
showed the higher inhibition zone compared to agar well diffusion method (willston
et.al.,1947).

In this study, there were six type of bacterial tested where two from gram
negative bacteria (E.coli, and P. mirabilis) and four from gram positive bacteria (MRSA,
B. cereus, S. aureus, B. cerius). But, only three of them were had positives result and
other had negative result. Two of them were resulted from gram negative bacteria where
it was E. coli and P. mirabilis and one of them from gram positive, MRSA .

Control is used in this experiment was to provide the baseline to compare the
result of the main experiment or test where the independent variable is really responsible
for the observation. Therefore there was no inhibition zone at the control film test. For
treated film, the inhibition zone of the cinnamaldehyde on the E. coli is 34 mm while on
the P. mirabilis is 40 mm. The size of inhibition zone of the MRSA is 43 mm. The largest
clear zone was shown by MRSA where there was the higher the antimicrobial activity.
MRSA was a Gram positive bacteria.

The size of inhibition zone was affected by susceptible of microorganisms to

extract. This is because, the bigger the size of inhibition zone, the more the susceptible
of microorganisms to the film (Kaushik et al. 2016). Based on the previous study, the
Gram negative bacteria had a lower antimicrobial activity compared to Gram positive
bacteria ( Mahadevamurthy, 2016).Gram negative bacteria have peptidoglycan cell wall
where its contains of lipopolysaccharide that makes them more resistant to antimicrobial

20
 agents (Silhavy et al., 2010). Therefore, E. coli and P. mirabilis had the shorter diameter
of inhibition zone while the MRSA have the bigger size of inhibition zone. The gram
negative bacteria do not have peptidoglycan cell wall.

Table 4.1: Antimicrobial activity of Cinnamaldehyde on cross streak assay.

Test Microorganisms Diameter of Inhibition Zone (mm) zone (mm)

Film with Cinnamaldehyde Control Film

Gram Positive Bacteria

MRSA 43 -

B. cereus - -

S. aureus - -

B. cerius -

Gram Negative Bacteria

E. coli 34 -

P. mirabilis 40 -

21
Table 4.2 The clear zone surrounding the film indicates the inhibitory activity of the film
on Cross Streak Test.

Test microorganisms sample control

E. coli

P. mirabilis

22
4.2 Hohenstein Challenge test

The antimicrobial activity of the treated film with cinnamaldehyde was tested with
Hoheinstein Challenge test. Hoheinstein Challenge test was used to evaluate
antimicrobial activity in the film by calculating percentage reduction of bacterial. This
method was expressed by calculating the number of colony based o diffenfence
concentration. This antimicrobial activity was comparing the higher of the culture
concentration with treated film with cinnamaldehyde and lower culture concentration with
control. There were three types of microorganisms that were used in this test based on
the positive result of the cross streak test. There was P. mirabilis, E.coli and MRSA.

Based on the result stated in Table 4.2, the reduction of bacterial can be seen
with resulting 100% of growth of P. mirabilis and MRSA where the E. coli had 98.9% of
percentage of reduction. The percentage of reduction was affected by treated fabric
against test microorganism. Therefore, the higher the percentage of reduction of tested
microorganisms, the increases the effectiveness of the film. The treated textile had
antimicrobial property as the extract was attached with the fabric as antimicrobial
compound (Jothi, 2009).

23
 Table 4.3 : Hoheinstein Challenge test result

Test microbial Load (cfu/ml) percentage reduction

microorganisms %
treated film Untreated film

P. mirabilis 8.40 x 106 2.40 x 109 100

MRSA 2.40 x 105 1.05 x 109 100

E. coli 1.44 x x107 1.35 x 109 98.9

24
 CHAPTER 5

CONCLUSION AND RECOMMENDATION

5.0 Conclusion And Rcommendation

As the conclusion, the cinnamaldehye was proven as antimicrobial agent on E.

coli, P. mirabilis, and MRSA. The Hoheinstein test showed significant reduction of
bacteria was followed as followed to the other journal and theory. For cross streak test,
the inhibition size was measured. The largest inhibition zone was MRSA that is 43 mm.
The Hoheinstein Challenge test of the film incorporated with cinnamaldehyde showed
100% of growth reduction on MRSA and P.mirbilis. Therefore, the alginate-based film
exhibited significant antimicrobial activity on food borne bacteria.

As recommendation, the binding of the film must be improve to make sure the
result observed is excellent. Besides, this film can be innovated for developing the health
care product food packaging and industries. Further investigation must be done for
mechanical properties of the film.

25
 REFERENCES

Adnyana, I. K., Setiawan, F. & Isanu, M. (2013). From ethnopharmacology to clinical

study of orthosiphon stamineus benth. International Journal of Pharmacy
and Pharmaceutical Sciences, 5(3),66-73.

Ahmad, M., Benjakul, S., Prodpran, T, Agustini, T. W., Physico-mechanical and

antimicrobial properties of gelatin film from the skin of unicorn leatherjacket
incorporated with essential oils. Food Hydrocolloids. 2012; 28: 189-199.

Akilen, R., (2017). Glycated haemoglobin and blood pressure-lowering effect of

cinnamon in multi-ethnic Type 2 diabetic patients in the UK: a randomized,
placebo-contr... - PubMed - NCBI. Ncbi.nlm.nih.gov.

Alqareer. A., Alyahya, A., Andersson, L., The effect of clove and benzocaine versus
placebo as topical anesthetics. Journal of dentistry 2012; 34

Ameer, O.Z., Salman, I. M., Asmawi, M. Z, Ibraheem, Z.O. & Yam, M.F. (2012).
Orthosiphon stamineus: traditional uses, phytochemistry, pharmacology,
and toxicology. Journal of Medicinal Food, 15(8), 678-690.

antimicrobial agent | pharmacology. (2017). Encyclopedia Britannica. Retrieved 20

November 2017, from https://www.britannica.com/science/antimicrobial-agent

Antimicrobial brass coatings prepared on poly(ethylene terephthalate) textile by high

power impulse magnetron sputtering. Materials Science and Engineering:
C, 48, 41-47. Chin,Y.H., Abbas, H.H. & Sabariah. (2007). Toxicity study of
orthosiphon stamineus benth (misai kucing) on sprague dawley rats.
Tropical Biomedicine, 25(1), 9-16.

26
Antimicrobial Resistance | European Food Safety Authority. (2017). Efsa.europa.eu.
Retrieved 21 November 2017, from
https://www.efsa.europa.eu/en/topics/topic/antimicrobial-resistance

Antimicrobials: An Introduction Antimicrobial Resistance Learning Site For Veterinary

Students. (2017). Amrls.cvm.msu.edu. Retrieved 20 November 2017, from
https://amrls.cvm.msu.edu/pharmacology/antimicrobials/antimicrobials-an-
introduction

Balch, Phyllis and Balch, James. Prescription for Nutritional Healing. Avery
Publishing, 2000: 3, 94

Blumenthal M., The Complete Commission E Monographs, Therapeutic Guide Herbal

Medicines. Boston, Mass: Integrative Medicine Communications; 1998:110.

Carballo, J. L., Hernndez-Inda, Z. L., Prez, P. & Garca-Grvalos, M. D. (2002). A

comparison beTween two brine shrimp assays to detect in vitro cytotoxicity
in marine natural products. BMC Biotechnology,2,17. Chen, J.H,. Wu, G.W.
& He, J. L. (2015).

Chatterjee, S., Jungraithmayr, W., & Bagchi, D. (2018). Preface. Immunity and
Inflammation in Health and Disease, Xv-Xvi. doi:10.1016/b978-0-12-
805417-8.00041

Cinnamon boosts brain activity. (2017). nutraingredients.com. Retrieved 30 November

2017, from http://www.nutraingredients.com/Research/Cinnamon-boosts-
brain-activity

Cinnamon, ground. (2017). Whfoods.com. Retrieved 30 November 2017, from

http://www.whfoods.com/genpage.php?tname=foodspice&dbid=68

Deshmukh, S. K., Verekar, S. A. & Bhave, S. V. (2014). Endophytic fungi: a reservoir

of antibacterials. Front. Microbiol. 5, 715.

27
Deshmukh, S.K., Mishra, P.D., Kulkarni-Almeida, A., Verekar, S., Sahoo, M.R.,
Periyasamy, G., Goswami, H., Khanna, A., Balakrishnan, A. &
Vishwakarma, R. (2009). anti-inflammatory and anticancer activity of
ergoflavin isolated from an endophytic fungus. Chem Biodivers, 6(5), 784-
789.

Devi, P., Rodrigues, C., Naik, C. G. & DSouza, L. (2012). Isolation and
characterization of antibacterial compound from a mangrove-endophytic
fungus, Penicillium chrysogenum MTCC 5108. Indian Journal of
Microbiology, 52(4), 617623.

Ding D., Wang, F., Liu, X., & Han, C. (2015). Antimicrobial Effects of Cinnamon and
Rosemary Essential Oils. Applied Mechanics And Materials, 731, 385-388.
http://dx.doi.org/10.4028/www.scientific.net/amm.731.385

El-tahlawy, K.F., El-bendary, M.A., Elhendawy, A.G. & Hudson, S.M. (2005). The
antimicrobial activity of cotton fabrics treated with different crosslinking
agents and chitosan. Carbohydrate Polymer, 60, 421430.

Fenaroli's Handbook of Flavor Ingredients. Volume 2. Edited, translated, and revised by

T.E. Furia and N. Bellanca. 2nd ed. Cleveland: The Chemical Rubber Co.,
1975., p. 91

Fouda, M. M. (2014). Antibacterial modification of textiles using nanotechnology. A

Search for Antibacterial Agents ,3:50-51. Frisvad, J. C., Yilmaz, N., Thrane,
U., Rasmussen, K. B., Houbraken, J. & Samson, R. A. (2013).
Talaromyces atroroseus, a new species efficiently producing industrially
relevant red pigments, 8(12). 50

Fu, Y., Xie, B., Ben, D. F., Lv, K.Y., Zhu, S. H., Lu, W., Tang, H. T., Cheng, D. S., Ma,
B., Wang, G. Y., Xiao, S. C., Wang, G. Q., Xia, Z. F.(2012). Pathogenic
alteration in severe burn wounds, Burns, 38(1), 90-94.

28
Garcia, A., Rhoden, S.A., Bernardi-Wenzel, J., Orlandelli, R.C., Azevedo, J.L. &
Pamphile, J.A. (2012). Antimicrobial activity of crude extracts of endophytic
fungi isolated from medicinal plant sapindus saponaria L. Journal of
Applied Pharmaceutical Science, 2(10), 35-40

Gimnez, B., Gmez-Guilln., MC, Lpez-Caballero., ME, Gmez-Estaca, J., Montero,

P.Role of sepiolite in the release of active compo unds from gelatin e egg
white films. Food Hydrocolloids. 2012 ; 27: 475- 486.

HerbClip: The Importance of Cinnamon as Spice and Medicine.

(2017). Cms.herbalgram.org. Retrieved 30 November 2017, from
http://cms.herbalgram.org/herbclip/291/review44247.html

Iqbal., Mohammed (1993). International trade in non-wood forest products: An

overview.Food and Agriculture Organization of the United Nations.
Retrieved November 12, 2012.

Jorgensen, J. H., & Ferraro, M. J., (2009). Antimicrobial susceptibility testing: a review of
general principles and contemporary practices. Clinical Infectious Diseases,
49(11), 1749-1755.

Jorgensen, J.H. & Ferraro, M.J (2009). Antimicrobial susceptibility testing: a review of
general principles and contemporary practices. Clinical Infectious Diseases,
49(11), 1749-1755.

Joseph, S., Shanmughapriya, S., Gandhimathi, R., Kiran S. G., Ravji R. T.,
Natarajaseenivasan, K., Optimization and production of novel antimicrobial
agents from sponge associated marineactinomycetes Nocardiopsis
dassonvillei MAD08. Appl Microbiol Biotechnol 2009;83:435,45.9.

Joshi, M., Ali, S. W. & Purwar, R. (2009). Ecofriendly antimicrobial finishing of textile
using bioactive agents based on natural products. Indian Journal of Fibre and
Textile Research, (30), 295-304.

29
Kang, C.-G., Hah, D.-S., Kim, C.-H., Kim, Y.-H., Kim, E. & Kim, J.-S. (2011). Evaluation
of antimicrobial activity of the methanol extracts from 8 traditional medicinal
plants. Toxicological Research, 27(1), 3136.

Kannappan, R., Gupta, S., Kim, J., Reuter, S., & Aggarwal, B.
(2017). Neuroprotection by Spice-Derived Nutraceuticals: You Are What
You Eat!. Retrieved 30 November 2017, from

Kaushik, V., Chaudhary, G., Ahmad, S. & Saini, V. (2016). Evaluation of antimicrobial
potential of Anemone obtusiloba D. Don. Der Pharmacia Lettre, 8(4), 273-
276.

Kaushik, V., Chaudhary, G., Ahmad, S. & Saini, V. (2016). Evaluation of antimicrobial
potential of Anemone obtusiloba D. Don. Der Pharmacia Lettre, 8(4), 273-
276.

Khokhar, I., Mukhtar, I. & Mushtaq, S. (2011). Antifungal effect of penicillium metabolites
against some fungi . Archives of Phytopathology and Plant Protection, 44(14),
1347-1351.

Krasniewska, K.; Gniewosz, M.; Kosakowska, O.; Cis, A. Preservation of brussels

sprouts by pullulan coating containing oregano essential oil. J. Food
Protect. 2016, 79, 493500

Lertcanawanichakul M, Sawangnop S. A Comparison of Two Methods Used for

Measuring theAntagonistic Activity of Bacillus Species. Walailak J Sci Tech
2008;5:161-71.10.

M. Balouiri., M. Sadiki. & Ibnsouda, S.K. (2016). Methods for in vitro evaluating
antimicrobial activity: A review, Journal of Pharmaceutical Analysis, 6 ,71
79.

30
M. Lertcanawanichakul, S. Sawangnop A comparison of two methods used for
measuring the antagonistic activity of Bacillus species Walailak J. Sci.
Tech., 5 (2008), pp. 161-171

Mahadevamurthy, M., Puttaswamy, H., Channappa, TM., Sidappa, M., Madegowda, P.,
Chikkamanchegowda, J.S. & Nagaraj, A.K (2016). Antibacterial potential of
fungal endophytes isolated from boerhaavia diffusa L, Journal of Applied
Pharmaceutical Science, 6(10), 216-221.

Malarvizhi, G., (2015). Development of herbal finished baby diapers with bamboo
fiber, International Journal of Humanities, Arts, Medicine and Sciences,
3(2), 42-46. Morse, S.S. (1995). Factors in the emergence of infectious
diseases, Emerging Infectious Diseases, 1(1),7-15.

Matan, N., Rimkeeree, H., Mawson A. J., Chompreeda. P., Haruthaithanasan V,

Parker M: Antimicrobial activity of cinnamon and clove oils under modified
atmosphere conditions. Int J Food Microbiol 2006, 107:180-185.

Mishra, A., Jaitly, A.K. & Srivastava, A.K (2009). Antihyperglycaemic activity of six
edible plants in validated animal models of diabetes mellitus, Indian
Journal of Science and Technology, 2(4), 80-86.

Mohamed, S. A., & Janson, J. C. (2012). Hydrolytic and chromatographic studies on

the pegylation of dextranase from penicillium sp. Journal of Genetic
Engineering and Biotechnology, 10(1), 145-150.

Muhammad, A. H., & Mizanur R., S.M. (2015). Isolation and characterisation of
flavonoids from the leaves of medicinal plant Orthosiphon stamineus.
Arabian Journal of Chemistry, 8(2),:218-221.

Nicoletti, R., Buommino, E., & Tufanno, M.A. (2012). Patenting penicillium Strains,
Recent Patents on Biotechnology, 6(2), 81-96. Ogunbodede, E.O. (2004).
HIV/AIDS situation in Africa. International Dental Journal, 54(6), 352 -360.

31
Nisa, H., Kamili, A.N., Nawchoo, I.A., Shafi, S., Shameem, N. & Bandh. S.A. (2015).
Fungal endophytes as prolific source of phytochemicals and other bioactive
natural products: A review. Microbial Pathogenesis, 82, 50-59.

Nithya, E., Radhai, R., Rajendran, R., Shalini, S., Rajendran, V. & Jayakumar, S.
(2011). Synergetic effect of DC air plasma and cellulase enzyme treatment
on the hydrophilicity of cotton fabric. Carbohydrate Polymers, 83(4), 1652-
1658.

Nithya, V., Murthy, P., & Halami, P. (2013). Development and application of active films
for food packaging using antibacterial peptide ofBacillus
licheniformisMe1. Journal Of Applied Microbiology, 115(2), 475-483.
http://dx.doi.org/10.1111/jam.12258

O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and
Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 383

Park, H. J. 1991. Edible coatings for fruit and vegetables: Determination of gas
diffusivities, prediction of internal gas composition and effects of coating on
shelf life. Georgia, USA: University of Georgia, Ph.D. Thesis.

Park, H. J., Bunn, J. M., Weller, C. L., Vergano, P. J. and Testin, R. F. 1994a. Water
vapor permeability and mechanical properties of grain protein-based films
as affected by mixtures of polyethylene glycol and glycerin plasticizers.
Transaction of the American Society of Agricultural Engineers 37: 1281-
1285.

Perelshtein, L., Perkas, N. & Gedanken, A. (2016). Making the hospital a safer place
by the sonochemical coating of textiles by antibacterial nanoparticles. In
Surface Chemistry of Nanobiomaterials,71-105.

Peter, K. V. (Ed.). (2006). Handbook of herbs and spices (Vol. 3). Woodhead publishing

32
Pranoto, Y., Salokhe, V. , & Rakshit, K. S. (2005). Physical and antibacterial
properties of alginate-based edible film incorporated with garlic oil. Food
Research International, 38, 267-272.

Raju, P. S., & Bawa, A. S. (2006). Food additives in fruit processing. In Y. H. Hui
(Ed.), Handbook of fruits and fruit processing (pp. 145- 170).

Raybaudi-Massilia, R. M., Mosqueda-Melgar, & Martin-Belloso, 0 . (2008). Edible

alginate-based coating as carrier of antimicrobials to improve shelf-life and
safety of fresh-cut melon. International journal of Food Microbiology, 121,
313-327.

Raybaudi-Massilia, R. M., Rojas-Grau, M. A., Mosqueda-Melgar, ]., & Martin-Belloso,

0. (2008). Comparative study on essential oils incorporated into an
alginate-based edible coating to assure the safety and quality of fresh-cut
Fuji apples.

Ribeiro-Santos, R., Andrade, M., Madella, D., Martinazzo, A., de Aquino Garcia Moura,
L., de Melo, N. and Sanches-Silva, A. (2017). Revisiting an ancient spice with
medicinal purposes: Cinnamon. Trends in Food Science & Technology, 62,
pp.154-169.

Sahgal, G., Ramanathan, S., Sasidharan, S., Mordi, M. N., Ismail, S. & Mansor, S. M.
(2010). Brine shrimp lethality and acute oral toxicity studies on Swietenia
mahagoni (Linn.) Jacq. seed methanolic extract. Pharmacognosy
Research, 2(4), 215220.

Sathianarayanan, M.P., Bhat, N.V., Kokate, S.S. & Walunj, V.E. (2010). Antibacterial
finish for cotton fabric from herbal products. Indian Journal of Fibre and
Textile Research, 35, 50-58.

Shahidi, S. & Wiener, J. (2012). Antibacterial agents in textile industry. Antimicrobial

Agents, 387-406, INTECH Open Access Publidher: Rijeka, Croatia. Sheila,

33
 P. (2007).Understanding wound infection and colonisation. Wound
Essentials, 2, 132-142.

Silhavy, T. J., Kahne, D. & Walker, S. (2010). The bacterial cell envelope. Cold Spring
Harbor Perspectives in Biology, 2(5), 1-17.

Simic, A., Sokovic, M. D., Ristic, M., Grujic-Jovanovic, S., Vukojevic J., Marin PD: The
chemical composition of some Lauraceae essential oils and their antifungal
activities. Phytother Res 2004, 18:713- 717

Subash, B. P. Prabuseenivasan, S., Ignacimuthu, S., Phytomedicine, 14 (1): 15-22

(2007). Available from, as of July 14, 2009:

Thilagavathi, G. & Rajendrakumar, K. (2005). Development of ecofriendly

antimicrobial textile finish using herbs. Indian Journal of Fibre and Textiles
Research, 30, 431-436.

Tomme, M., Aime, M. C., (2012). Kingdom fungi. Biological Science Fundamentals
and Systematic, EOLSS Publisher. Windler, L,. Height. M., & Nowack. B.
(2013).Comparative evaluation of antimicrobials for textile applications .
Environment International, 53, 62-73. 57

Ustunol, Z. Edible films and coatings for meat and poultry. In Edible Films and
Coatings for Food Applications; Embuscado, M.E., Huber, K.C., Eds.;
Springer: New York, NY, USA, 2009; pp. 245268.

Vafa, M., e. (2017). Effects of cinnamon consumption on glycemic status, lipid profile
and body composition in type 2 diabetic patients. - PubMed -
NCBI. Ncbi.nlm.nih.gov. Retrieved 30 November 2017, from
http://www.ncbi.nlm.nih.gov/pubmed/22973482

34
Williston, E. H., Zia-Walrath, P., Youmans. G. P., Plate methods for testing antibiotic
activity ofactinomycetes against Virulent human type Tubercle Bacilli. J
Bacteriol 1947;54:563,68

World Union of Wound Healing Societies (WUWHS)., (2008). Principles of best

practice : Wound infection in clinical practice. An International Consensus.
London: MEP Ltd,. Available from www.mepltd.co.uk Wu, C. (2014).

Yam M.F., Zaini A., & Rusliza B., 2008. An investigation of anti- inflammatory and
analgesic effects of orthosiphon stamineus leaf extract, Medicinal food,
11(2), 362-368.

Yam, M.F., Ang, L.F., Salman, I.M., Ameer, O.Z., Lim, V., Ong, L.M., Ahmad, V.,
Asmawil, M.Z., & Basir, R. (20009). Orthosiphon stamineus leaf extract
protects against ethanol-induced gastropathy in rats. Medicinal food, 12(5),
1089-1097.

Yang, M. H., Li T.X., Wang, Y., Liu R.H., Luo, J., & Kong, L.Y., (2016). Antimicrobial
metabolites from the plant endophytic fungus penicillium Sp. Fitoterapia.

Yenn, T.W., Ang S.N., Darah, I. & Latiffah, Z. (2014). Antimicrobial activity of
penicillium minioluteum, an endophytic fungus residing in orthosiphon
stamineus benth, World Journal Of Pharmacy And Pharmaceutical
Sciences, 3(3), 121-132.

Yilmaz N., Houbraken J., Hoekstra E. S., Frisvad J. C., Visagie C. M., Samson R.,
2012. Delimitation and characterisation of Talaromyces purpurogenus and
related species. Persoonia, 29, 39-54.

Yilmaz, N., Hagen, F., Meis, J.F., Houbraken, J., & Samson, R. A. (2016). Discovery
of a sexual cycle in Talaromyces amestolkiae. Mycologia, 108,70-79. 58

35
Yilmaz, N., Visagie, C. M., Houbraken, J., Frisvad, J. C., & Samson, R. A. (2014).
Polyphasic taxonomy of the genus Talaromyces. Studies in Mycology, 78,
175341.

Zabelgogeazcoa, I., (2008). Fungal endophytes and their reaction with plant
pathogens. Spanish Journal of Agriculture Research. 6, 138-146.

36
 APPENDICES

APPENDIX A: Percentage of reduction (%)

a)MRSA

2.4 x109 -8.40x106

(%) = 100
2.40x109
(%) = 100%

b) E.coli

1.05 X109 -2.40x105

(%) = 100
1.05X109

(%) = 100%

c) P.mirabilis

1.35 X109 -1.44x107

(%) = 100
1.35X109

(%) = 98.9%

37