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EXTRACTION OF COLLAGEN FROM THE SKIN OF RED

SNAPPER (Lutjanus erythropterus)

DG NURDAYANA AZMAN (2012276608)


HANNA HAREEZA HANAFIAH (2012813192)
MARLIA ABDUL GHANIE (2012874832)
ZUL LIYANA ZULKIFLI (2012634324)

DIPLOMA IN SCIENCE
FACULTY OF APPLIED SCIENCES
UNIVERSITI TEKNOLOGI MARA

AUGUST 2014
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DECLARATION

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

Universiti Teknologi MARA or other institutions.

Dg Nurdayana Azman

Hanna Hareeza Hanafiah

Marlia Abdul Ghanie

Zul Liyana Zulkifli

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ACKNOWLEDGEMENTS

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

all the staffs of KOMSAT for helping us in chemical preparations, providing us

laboratory’s apparatus and assisting us on the usage of laboratory machine. The

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

helping us throughout the entire process of finishing this project.

Dg. Nurdayana Azman


Hanna Hareeza Hanafiah
Marlia Abd Ghanie
Zul Liyana Zulkifli

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4.4. Effect of temperature and pH value of water on collagen 16
4.5. Red Snapper skin as an alternative source of collagen 17
4.6. Application of collagen 18

CHAPTER 5 CONCLUSION 19

REFERENCES 20
ATTACHMENT 24
CURRICULUM VITAE

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LIST OF FIGURES

Figure Caption Page

2.1 Collagen in powdered form 2

2.5 Red Snapper (Lutjanus erythropterus) 4

4.1 Result of collagen yield from first and second method 12

3.1 Schematic diagram of collagen extraction from skin of 24-25

Lutjanus erythropterus for first method

3.2 Schematic diagram of collagen extraction from skin of 26-28

Lutjanus erythropterus for second method

3.3 Qualitative test for collagen 28

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ABSTRACT

COLLAGEN EXTRACION FROM THE SKIN OF RED SNAPPER

(Lutjanus erythropterus)

Collagen is the most abundant protein in human body. It is a part of the

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 24oC temperature and water pH is 8. The second

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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CHAPTER 1
INTRODUCTION

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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2.2 IMPORTANCE OF COLLAGEN

Collagen is important in strengthening vessels and most commonly known to give


skin its elasticity and strength. As people grow older, the production of collagen
will start to deteriorate. As a result, a sagging skin will be more noticeable as
people age. (Nordqvist, 2013). Additionally, collagen also works hand-in-hand
with elastin in supporting the body’s tissues. Elastin gives the body much of
flexibility. This combination of collagen and elastin is very important in many
parts of the body. Often, collagen is discussed in relation to the skin. It works with
keratin to provide the skin with strength, flexibility, and resilience. (Blum, 2011)

2.3 USAGE OF COLLAGEN

As people age, however, collagen degradation occurs, leading to wrinkles. As


such, it is an important substance for those looking for ways to fight the visible
effects of aging on the skin. Some skincare professionals actually advise people on
ways to stimulate the production of collagen in skin cells. (Blum, 2011). Collagen
supplement can and may be taken to as another alternative in having collagen in
body. As collagen made up most of our fibrous tissues, collagen plays an
important role in maintaining skin’s elasticity. According to a study published in
Archives of Dermatology, injections with dermal fillers contain hyaluronic acid,
which is thought to stimulate the production of collagen, restoring the structure of
damaged skin. (Wang, Garza, Kang, Varani, Orringer, Fisher & Voorhees, 2013)

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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erythropterus species. (Poey. 1860). The adult species lives offshore on the
continental shelf, over deep reefs, banks, and rocky bottoms. Adults of this
species are found at depths of 33-620 feet (10 to 190m) with older, larger fish
tending to prefer the cooler, deeper spots while the young species live over sandy
and muddy bottoms that provide a rich food supply of shellfish for the foraging
juvenile fish. As the youngs mature, they seek cover in the form of ledges, rocky
outcroppings, and wrecks. During winter red snapper move offshore to avoid the
cooler, shallow inshore waters. (Bester. C. n.d.)

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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In addition, most of them contained two or three distinct α chains while
mammal commonly contain only two different α chains. As fish collagen has
some unique characters in its composition and contents of amino acid, it can be a
useful substitute for cattle and pig collagen (Liu et al., 2006). There are many
reasons to substitute cattle and pig collagen with fish collagen. For example, 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 (Ogawa et al., 2003,
Kittiphattanabawon et al., 2004).

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3.1.3 SOLUBILIZING COLLAGEN

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.

3.1.4 CENTRIFUGING AND PERCIPITATION

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

3.2.1 SKIN PREPARATION

50g of Red Snapper fish skin obtained from WanWan Restaurant


Bundusan, Kota Kinabalu and place in a box full of ice. The skin was
cleaned in the laboratory using laboratory knife to remove scales; present
of scale will affect the yield of collagen extracted.Then, it is store in the
fridge at temperature of -15oC until used.

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were then inserted into a refrigerated centrifuge machine and the solution
was centrifuged at 18000rpm and 4oC for 30 mins. After 30mins, the
precipitates were collected and transferred into a weighed petri dish. The
collected precipitates were then put into an oven at the temperature of 30
⁰C for a few days until it was completely dried and constant mass was
obtained. Subsequently, the dried precipitates in the petri dish were
scrapped using a spatula. The mass of the powder was taken using an
analytical balance.

3.3 QUALITATIVE TEST

Presence of collagen was qualitatively tested using Fehling’s Test. Firstly,


2 ml of sample obtained and placed in an empty test tube. 1 ml of
Fehling’s A solution and 1 ml of Fehling’s B solution obtained and placed
into the test tube. Observation for the colour change was taken amd
negative indicator was preprared by using the same method with distilled
water.

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Methods Mass of sample Mass of Percentage (%)
(g) collagen yield(g)
1 100 0.0014 0.0014
2 50 6.8161 13.63
Table 4.1.1: Comparison of collagen yields between the two methods.

COMPARISON OF COLLAGEN YIELD


BETWEEN TRIAL 1 AND TRIAL 2
14
13.5
13
12.5
12
11.5
11
10.5
10
9.5
9
percentage yield (%)

8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Method 1 Method 2 Method

Graph 4.1: Comparison of collagen yield between method 1 and method 2

Types of fish Percentage yield References


(%)
Sole Fish 1.66 Al-Zahrani (n.d)
Unicorn Leatherjacket 4.19 Ahmad (2010)
Striped Catfish 9.41 Nurul Amilin (2012)
Table 4.1.2: Comparison of collagen yield from the skin of different types of fish

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Modification done in second method has increased the yield of collagen. Low
temperature keeps the collagen from denaturing. When the experiment was
conducted using the first method, time taken to complete the experiment is 14
days. Compared to the second method, the time taken to complete the experiment
is only 10 days. The next step of the experiment, which is to agitate the solution
containing fish skin, the fish skin were not completely disintegrated and the
solution is not so cloudy whereas in the second method, the fish skin was
completely disintegrated after agitating it and the solution is cloudier compared to
the solution in the first method. Increasing agitation speed and time will increase
the disintegration of fish skin, thus allowing collagen to break out from the skin.
Concentration of NaCl was increased on the second method; increasing the
concentration of NaCl will decrease protein solubility thus result in precipitation
of collagen. The uses of acidic water or water with neutral pH increase the
solubility of collagen. As for the condition of the fish, the fish used in the first
method has a foul odor while the fish used in the second method has better and
less putrid odor. Lastly, at the end of the experiment, the yield obtained in second
method is higher than the yield obtained in first method.

4.3 CHEMICAL USED

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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at 4 and 10 °C were 39.5 and 37.5°C, respectively as determined by thermal
analysis using differential scanning calorimetry (Aukkanit N, Garnjanagoonchorn
W, 2010). Chemical used for pre-treatment as well as extraction condition such as
temperature and time can influence the length of polypeptide chains and the
functional properties of the collagen. (Kolodziejska et al., 2004)

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.

4.5 RED SNAPPER SKIN AS AN ALTERNATIVE SOURCE OF


COLLAGEN

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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CHAPTER 5
CONCLUSION

In conclusion, 0.00014g and 6.8161g of collagen was extracted from red


snapper fish skin based on both trials respectively. Through this study, the
optimum temperature and pH of water was determined which are 4oC and pH 6-7.
Therefore, skin of red snapper fish can be used as an alternative source of collagen
so that religious issues can be avoided and consumers’ safety are assured.
However, some modification can be made to improve the amount of collagen
yield for future extraction. For instance, use acidic water to wash the skin after
every treatment. The concentration of NaCl can be increase to obtain maximum
precipitation of collagen. Time taken and speed for agitation can be increase to
ensure that collagen completely solubilize in acetic acid. Other than that,
researcher can use different types of fish for future study.

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Moran. D (1988). Species Profiles: Life Histories and Environmental
Requirements of Coastal Fishes and Invertebrates (Gulf of Mexico).
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J. Ratanavaraporn, S. kanokpanont, Y. Tabata, S. Damrongsakkul. (2007) Effects
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