Professional Documents
Culture Documents
WITH CINNAMALDEHYDE
JULY 2018
i
ABSTACT
ii
ABSTRAK
iii
APPROVAL
Signature:..
Date:
iv
DECLARATION
Signature:..
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.
vi
TABLE OF CONTENT
TITLE PAGE
TITLE PAGE i
ABSTRACT ii
ABSTRAK iii
APPROVAL PAGE iv
DECLARATION v
ACKNOWLEDGEMENT vi
LIST OF FIGURE x
LIST OF TABLE X1
CHAPTER 1:INTRODUCTION
vii
2.1 edible film 4
2.5 cinnamon
2.6.1.1 antidiabetic 13
CHAPTER 3 :METHODOLOGY
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
REFFERENCES 26
APPENDIX 37
ix
LIST FO FIGURE
x
LIST OF TABLE
4.2 The clear zone surrounding the film indicates the inhibitory 21
activity of the film on Cross Streak Test.
xi
CHAPTER 1
INTRODUCTION
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.
2
1.2 Objectives
3
CHAPTER 2
LITERATURE REVIEW
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).
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).
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)
Structural formula
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
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.
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.
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%.
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).
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)
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
2.6.1.1 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
14
. CHAPTER 3
METHODOLOGY
STAGE 1:
STAGE 2:
STAGE 3:
HOHENSTEIN TESTING
15
Figure 3.1 Experimental plan
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.
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.
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
17
(%) = 100
Where,
A= no of colony of control
B= no of colony of sample
18
CHAPTER 4
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.
19
4.2 Antimicrobial activity of Cinnamaldehyde in edible film
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.
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.
MRSA 43 -
B. cereus - -
S. aureus - -
B. cerius -
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.
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
24
CHAPTER 5
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
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36
APPENDICES
a)MRSA
b) E.coli
(%) = 100%
c) P.mirabilis
(%) = 98.9%
37