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DIPLOMA IN SCIENCE
FACULTY OF APPLIED SCIENCES
UNIVERSITI TEKNOLOGI MARA
AUGUST 2014
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We hereby declare that the thesis is based on our original work except for
quotations and citations which have been duty acknowledged. We also declare
that it has not been previously or concurrently submitted for any other report at
Dg Nurdayana Azman
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In the name of Allah s.w.t the Most Beneficent and the Most Merciful, who gave
us wisdom and ease everything during the entire process of this study. Our highest
gratitude to our supervisor, Madam Farnidah Binti Hj. Jasnie, for guiding and
providing knowledge related to our study and for all the support and motivation
that she gave us during the whole progression of this project. To Sir Makdi
Masnoddin, our BIO300 lecturer, thank you for briefing us about the projection of
BIO300 Mini Project and guiding us in writing our thesis. We also want to convey
our high appreciations to En. Mohd Ruzaleh Nurdik, coordinator KOMSAT, and
great hospitality and support they gave to us are very much appreciated. Last but
not least, our gratitude also goes to all that have been directly and indirectly
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CHAPTER 5 CONCLUSION 19
REFERENCES 20
ATTACHMENT 24
CURRICULUM VITAE
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(Lutjanus erythropterus)
connective tissues that function in firmness and elasticity. The primary objectives
of this study are to extract collagen from the skin of red snapper fish and to
identify the effect of different parameters (temperature and pH) on the collagen
yield. Studies were conducted based on the method of Al-Zahrani (n.d) with
slightly modification. Collagen was solubilized with acetic acid and was
precipitated by using NaCl. Two trials were conducted for this study. The first
method was conducted in 4 oC temperature and water pH is 6-7. The yields for
both trials are 0.0014% and 13.63% respectively. Yield of collagen obtained is
compared with the yield from previous studies with different types of fish used.
The present of collagen was qualitatively tested using Fehling’s Test, and the
result is positive. In conclusion, collagen can be extract from red snapper fish skin
and the optimum temperature and pH of collagen is 4oC and pH 6-7 respectively.
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In mammals, the most abundant protein is the collagen accounting for around 30%
of the protein content of the human body. Collagen can be found in fibrous tissues
for example, ligaments and tendons. Collagen is also present in all the smooth
muscle tissues, blood vessels digestive tract, heart, gallbladder, kidneys and
bladder holding the cells and tissues together. As collagen is found exclusively in
animals, especially in the flesh and connective tissues of mammals, collagen can
be extracted in the fish skin. Collagen is a part of the connective tissue in the skin
that helps in firmness, elasticity and even renewal of skin cells.
In food industry, a large amount of fish skin left as the by-product. The disposal of
abandoned fish waste can cause pollution as it emits defensive odour. In addition,
the existence of mad cow disease makes the consumer feel unsafe to consume
collagen from cattle. Besides that, the uses of pig collagen have been prohibited
due to religious constraints. Therefore, a demand for alternative safe and
permissible source of collagen is increasing.
As a solution, skins that left as the by-product can be utilized and processed into
something for example collagen. It will save large amount of money that is used
for extraction of collagen from other animal. This study also aims to use fish skin
as an alternative to mammalian source in the production of collagen.
The objective of this study is to extract collagen from red snapper fish skin and to
identify the effect of different parameter (temperature and pH) on the yield of
collagen.
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As a matter of fact, collagen has been widely used in various branches of industry.
Not only useful in pharmaceutical, biomedical, cosmetics and food process,
collagen also used in leather and film industries. For an instance, collagen can be
derived and made into roll film and drug capsules and also be used as a
biomaterial in biomedical applications, such as in drug delivery system which are
very different from the traditional capsule (Einersona, N.J., K.R. Stevensa and
W.J. Kao, 2002).
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The red snapper contain a body and fins that are pinkish red in colour. The size of
the fish is less than 14 inches (35cm) to differentiate this type of snapper and other
type of snapper, is through the lack of distinctive black. Red snapper lacks the
distinctive black spot found on the pectoral fins of the blackfin snapper. Young
red snapper may also exhibit bluish stripes on their sides. (Bester. C. n.d.)
Red snapper has long pectoral fins and a truncate caudal fin. The first and second
dorsal fins are continuous with a slight notch in between the two and the anal fin
tapers to a point posterior. The pectoral fins are long and reach the anus when
pressed against the body. They have a large head with small red eyes and a
somewhat pointed snout. (Bester. C. n.d.)
The red snapper reaches an average length of 24 inches (60cm), with a maximum
length of 39 inches (100cm) and may weigh up to 20 pounds. After 2 years, it will
sexually mature, which the red snapper are typically 12-16 inches (30-40cm) in
length. Its maximum age is estimated at 40-50 years. This snapper grows
approximately 4 inches per year for the first 6 years followed by a generally
declining growth rate thereafter. (Bester. C. n.d.)
Both young and adult northern red snapper are carnivorous, and adults are bottom-
oriented predators. Young red snapper commonly feed on zooplankton. But as they
mature, their diet switches over to larger prey including shrimp, squid, and
octopus. Adult red snapper feed on a variety of smaller fishes, crustaceans, and
molluscs, which they find in flat bottom areas adjacent to the reefs. (Bester. C.
n.d.)
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After 24 hours, by using a vacuum pump, solution in the 1 litre beaker was
removed by filtering using 125 mm filter paper. 12.3 g of fish skin was put
into seven different conical flasks. The fish skin in each flask was treated
with 250 mL of 0.5M acetic acid and were then placed in a shaker and
agitated at 170 rpm for 3 days.
After 3 days, the solution was filtered using double filter paper.by using
vacuum pump throughout the filtration process. The filtered solution was
inserted into vials where each of it held 2mL of the solution. The vials
were then inserted into a refrigerated centrifuge machine and the solution
was centrifuged at 18000rpm for 60 minutes.After 60 minutes, 2.77 mL of
0.9M NaCl was added into the centrifuged solution and was centrifuged
again for another 60 minutes at the same speed.After 60mins, the
precipitates were collected and transferred into a weighed petri dish.. The
precipitate was let to completely dried and the mass of the precipitate was
taken.
3.2 METHOD 2
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8.5
8
7.5
7
6.5
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5.5
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4.5
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3.5
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2.5
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1.5
1
0.5
0
Method 1 Method 2 Method
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In this method, NaOH solution was used to remove non-collagen protein and
subcutaneous tissues of skin (Wangtueai & Noomhorn, 2008). The purpose of
using alkaline, such as NaOH is to remove non-collagenous proteins without
removing collagenous proteins. The acid pre-treatments could cause the loss of
collagen even by using the weakest acid with a low H+ concentration (Zhou &
Regenstein, 2005). Then, n-butyl alcohol was used to remove fat tissues. Butyl
alcohol having at most 4 carbon atoms is the most suitable solvents extracting fat
tissues because of its low polarity (Knutsen & Osterman). High polarity alcohol
may cause collagen to denature. Based on the study, acetic acid was used as
solvent to solubilize the collage. Since fish skin is a type I collagen, glycine,
proline and hydroxyproline are the most dominant amino acids. To dissolve
collagen, it must be treated in acid with pH 3 or lower. Collagen from animal
tissues can be isolated by direct extraction with organic acids, like acetic, citric
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On the first method, the pH of the fish skin was more to basic pH which is 8
compare to our second method which is around 6 to 7. Both acid-soluble
collagens and pepsin-soluble collagens had the highest solubility at acidic pH.
(Prabjeet Singh, Soottawat Benjakul, 2011). Fish collagen is more soluble in the
acidic pH ranges while in alkaline pH it will show sharp decrease in solubility.
The pH value is in charge of the charge density of protein, which modifies the
electrostatic interaction and structure of proteins (Verheul et al. 1998). However,
solubility of collagen slightly decreased at extremely acidic pH. Therefore the best
pH value of water used should be around pH 5 to pH7.
In this study, the skin of Lutjanus erythropterus or also known as red snapper was
used as sample because it is treated as a waste and its skin is thick. Fish with thick
and tough skin may be associated with the collagen cross-links, especially cross-
linking caused by hydroxylysine (Nurul Amilin, 2012). Fish solid wastes constitute
50 – 70 % of the original raw material, depending on the processes used, and types
of product. These wastes have been used as high-protein human foods, instead of
as pet foods (Shahidi, 1994; Montero et al., 1991). Type I collagen is the fibrous
collagen and is found as the major type in fish waste materials. (Kimura, 1992;
Nagai and Suzuki, 2000a; Nagai et al., 2002; Sato et al., 1989, Kimura, 1983,
Ikoma et al., 2003). Nevertheless, fish skin contains a large amount of collagen.
Nagai and Suzuki (2000) reported that the collagen contents in the fish skin waste
of Japanese sea-bass, chub mackerel and bullhead shark were 51.4%, 49.8% and
50.1% (dry basis), respectively. Collagen is a major structural protein in the
connective tissue of animal skin and bone (Foegeding et al., 1996, Liu et al., 2007
and Ogawa et al., 2003). Therefore, fish skin, being a by-product
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Nurul Amilin Binti Hussin (2012). Final Year Project Report: Extraction and
Characterization of collagen extracted from the skin of Striped Catfish
(Pangasianodon Hypophthalmus). UiTM
http://eprints.uitm.edu.my/4981/1/NURUL_AMILIN_BINTI_HUSSIN_12_2
4.pdf
Einersona, N.J., K.R. Stevensa and W.J. Kao (2002). Synthesis and
Physicochemical Analysis of Gelatin-based Hydrogels for Drug Carrier
Matrices. Biomaterials, 24: 509 – 523.
IMoler, E., Bahnweg, G., & Sandermann, H. (1992). A simple and eficient
protocol for isolation of high molecular weight DNA from filamentous fungi,
fruit bodies, and infected plant tissues. 2-2. Retrieved September 1, 2014,
from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC334490/pdf/nar00233-
0230.pdf
Raed Al Zahrani (n.d). Extraction and Isolation of Collagen Type I from Fish Skin.
University of Otago, Dunedin, New Zealand.
Lingzhao Wang, Bao Yang, Xiuqiao Du. (December 18, 2007). EXTRACTION
OF ACID-SOLUBLE COLLAGEN FROM GRASS CARP
(CTENOPHARYNGODON IDELLA) SKIN. Retrieved from
http://web.b.ebscohost.com.ezaccess.library.uitm.edu.my/ehost/pdfviewer/
pdfviewer?sid=91ada03f-9803-48ef-9ef4-
f4656000f672%40sessionmgr113&vid=1&hid=112
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