Chapter 3

RESEARCH METHODOLOGY

This chapter presents an outline of the methodology

employed in the study. Specifically, it aims to discuss the research design that was

chosen for the purpose of the study, the sources of data under analysis, the

instrument used for data collection, the procedure followed to carry out the study,

the statistical treatment used to analyze the gathered data, the breakdown of cost

and expenses and lastly, the blueprint of the product under study.

Research Design

The researcher aims to use fish wastes as an alternative source of collagen

for facemask.

The researchers use the experimental method for the study. Experimental

method is a systematic approach to research in which the researchers manipulates

one or more variables, and controls and measures any change in other variables.

Using control and manipulation on the variables on the experiment aims to see if

the variables if the data that are manipulated has an effect to the results. With this,

researchers can formulate hypothesis and analyze outcomes to make new

inferences from the research. This method ensures that there will be no bias or

any mistakes in the experiment that would otherwise compromise the results of the

research. (Gottschling, 2016)

Sources of Data

Research Instrument

The data in this study were gathered by the use of laboratory experiments.

The extracted collagen sample and the produced face masks are subjected to

different experimentation using a variety of laboratory tools, equipment and

chemicals to determine the quality of the product.

 Raw Materials Tools and Equipments Chemicals

Raw Tilapia Fish  Sparkvue pH meter  0.1M NaOH

Wastes  Beakers  10% butyl alcohol

 Skin  Laboratory  NaCl

 Bones Thermometer  Distilled Water

 Scales  Test tubes  Cinammon Oil

 Fins  Test tube rack  Natural essences

 Centrifuge machine

 Petri dishes

 Watch glass

Table1
Raw materials, Tools, Equipments and Chemicals used in the Study

Data Gathering Procedure

This study aims to analyze the quality of the face mask made out of

extracted collagen from fish wastes in comparison to the commercialized face

mask products. However, before the product is made, collagen is first needed to

be extracted from the fish wastes. With that being said, it will undergo different

kinds of processes in order to attain its objective. After which Extracted collagen

will be utilized as an alternative component in the production of face masks.

A. Preparation of Collagen

The researchers will accumulate Tilapia (Oreochromis Niloticus) fish wastes

from the market and it will be used for the collagen extraction. A method on the

preparation of collagen contents from fish wastes were characterized by Cheng’s

(2016) study with minor modifications. All undergone processes were carried out

at temperatures not higher than 4°C

B. Extraction of Collagen from Tilapia Fish Wastes

B.1 Extraction of Skin Collagen

With the use of scissors, the fish skins were cut into small pieces of

0.5 cm in length. The skins were then extracted with the use of 0.1M NaOH

with a sample to alkali ratio of 1:8 (w/v) in order to withdraw the non-

collagenous proteins present in the skin. It was mixed for 6 hours; however,

the alkaline solution was replaced with a new one for every 3 hours. In order

to obtain neutral pH, the next samples were cleansed with distilled water.

Deproteinized skins were immersed in 10% butyl alcohol at sample

to alcohol ratio of 1:10 (w/v) overnight in order to remove the fats from the

fish skin. Again, the samples were cleansed repeatedly with the use of

distilled water until the skins were extracted with a variety of acetic acid

concentrations that is ranging from 0.1 to 1.0M. The remains were filtered

with the use of cotton cloth. Proteins are then retrieved with a procedure
called salting-out which uses NaCl to a final concentration of 2.5M with the

presence of 0.05M tris(hydroxymethyl) aminomethane at a 7.0 pH level.

After the process of centrifugation at 20,000xg for 30 minutes, a collection

of precipitated proteins were gained. A pellet dissolved in a 0.5M acetic acid

was then dialyzed on a 0.1M acetic acid and distilled water. The acid-

soluble collagen was done with a freeze-drying process or also known as

the lyophilization.

B.2 Extraction of Bone Collagen

Hammering of the fish head is done for the extraction of the bone

collagen. It is washed for 5 hours with the use of distilled water. It is soon

immersed in a 0.1M NaOH at a sample to alkali ratio of 1:5 (w/v) to withdraw

the non-collagenous proteins for 24 hours; the alkali solution was replaced

with a new one 4 times since it should be changed every 6 hours. In order

to achieve a neutral pH value, the remains were washed out with distilled

water.

Removing of calcium were done with the bones with the use of 0.6M

EDTA-2Na solution of pH 7.5 for 5 days, subsequently defatted with a 10%

butyl alcohol at a sample to alcohol ration of 1:10 (w/v) overnight. With the

use of distilled water, the bones were washed thoroughly; prior extraction

with a variety of acetic acid concentrations ranging from 0.1 to 1.0M for one

to five days. Re-extraction of soluble matters were conducted at a 53

sample to ratio of 1:2.5 (w/v) for 2 days. Each conducted extraction was

filtered by a cotton cloth. Now, both solutions were combined together. It is

then salted out with a NaCl to a final concentration of 2.5M in the presence

of 0.05M tris(hydroxymethyl) aminomethane. Proteins that are precipitated

were now centrifuged at 20,000xg for 30 minutes. It is dissolved in 0.5 acetic

acid and those dissolved proteins were dialysed against o.1M acetic acid

and distilled water. A freeze-drying process will then occur for the extracted

collagen content.

B.3 Extraction of Scale Collagen

The accumulated fish scales will be treated to a 0.1 M NaOH for 6

hours at a sample to alkali solution ratio 1:8 (m/v) in order to remove the

non-collagenous proteins. Afterwards, the scales will be immersed in a 0.5

M EDTA-2Na of pH 7.5 at a sample to EDTA solution ratio of 1:10 (w/v) for

24 hours to undergo the process of decalcification. The scales were then

washed with distilled water. Then, the residue was extracted with a variety

of acetic acid concentrations ranging from 0.1 to 1.0 M at sample to acid

ratio of 1:2.5 (w/v) for duration of one to five days according to the

experiment planned for optimization. Moreover, with the use of cotton cloth,

the extracted collagen from fish wastes will be acquired and the final

concentration is 2.5 M with the presence of 0.05 M tris (hydroxymethyl)

aminomethane. Centrifuging it at 20000xg will be done as well for 30

minutes to acquire the resultant pellet. Then, it will be dissolved to 0.5 M

acetic acid, and dialyzed against 0.1 M acetic acid and distilled water,

respectively. Lastly, this acid-soluble collagen from fish scales was then

lyophilized.

B.4 Extraction of Fin Collagen

In extracting the collagen content from the fish fins, the fins will be

soak to 0.1 M NaOH to remove the non-collagenous protein. It was then

washed with distilled water and will undergo lyophilized. The portion that

were insoluble was then extracted with various concentration of acetic acid

ranging from 0.1 to 1.0 M at sample to acid ratio 1:2:5 (w/v) for one to five

days. Then, the extract will undergo centrifugation for 1 hour at 20,000xg to

two fractions: acid-soluble and acid insoluble.

The first portion which is acid-soluble were salted out with NaCl to a

final concentration of 2.5 M (at neutral pH) in 0.05 M Tris-HCl (pH 7.5).

Afterwards, the proteins that were precipitated was then centrifuged again

at 20,000xg for 1 hr, and then dissolved in 0.5 M acetic acid before dialyzed

against 0.1M acetic acid. After it was lyophilized for the final process, acid-

soluble collagen was obtained.

Meanwhile, the acid-insoluble fraction was washed with distilled

water and decalcified with 0.5 M EDTA (pH 7.4) for 5 days. The EDTA

solution was changed once a day. With the use of 10 % butyl alcohol, the
 decalcified bones were washed for 1 day. Afterwards, centrifugation at

20,000xg was used to remove the extracted collagen and then washed with

distilled water and lyophilized. Lastly, it was dissolved in 0.5 M acetic acid

and dialyzed against 0.1 M acetic acid. As a result, extracted collagen were

obtained after lyophilization.

B.5 Collagen Yield

The yield of collagen extracted from the fish wastes are calculated

by measuring the percentage of the weight of collagen extracted from the

fish wastes. It is expressed as percentage of the dry weight of the extracted

collagen based on the freeze dried weight of the raw materials offish

wastes. The figure below shows the formula on calculating the collagen

yield.

𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑐𝑜𝑙𝑙𝑎𝑔𝑒𝑛 (𝑔)

𝑐𝑜𝑙𝑙𝑎𝑔𝑒𝑛 (%) = 𝑥 100%
𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑖𝑠ℎ 𝑤𝑎𝑠𝑡𝑒𝑠 (𝑔)

C. Physico-chemical Properties of the Extracted Collagen

C.1 Determination of pH Level

The concentration of hydrogen (pH) of the collagen sample was

determined using Sparkvue pH meter. The pH electrode produces a voltage

proportional to the pH of the solution that is immersed in. This voltage is

measured by the multi-sensor, which computes the pH. The pH

measurement was done by the following recommended manufacturer’s

procedure:

1. Unscrew and remove the storage bottle from the electrode (be careful not

to spill the storage solution).

2. Push the O-ring and bottle cap up the electrode handle.

3. Rinse the electrode tip with distilled water. If you see bubbles in the

electrode bulb, gently shake the electrode downward (similar to shaking

down a thermometer).

4. Start data collection. Place the tip of the electrode in the solution to be

measure and wait for the reading on to be displayed on the screen

5. Rinse the electrode with distilled water before measuring another

solution.

6. The reading at 30 Seconds is recorded.

C.2 Determination of Amino Acid Composition

Prior to doing amino acid analysis, hydrolysis is executed by simply

preparing dry collagen (100 - 200 mg) that is collected from skins, scales,

bones or fins of fish waste that were mixed with 6 N HCl to a ratio of 1:10

(w/v) at 110 °C for 24 hours. The hydrolysate was then added with 2.5 mM

of internal standard and distilled water. The mixture was then refined

through a 0.4 µm membrane before loading to the HPLC system.

 Afterwards, a total of 10 µl of sample was derivatized by adding 20 µl AccQ-

Fluor (Waters, USA) reagent and 70 µl of borate buffer. The sample was

then left to stand for 1 minute at room temperature (20 – 25 °C and 5 µl was

loaded to a AccQ Tag Column (3.9 x150 mm) in the HPLC system (Waters,

2790), with a Waters column heater and a fluorescence detector (Waters,

2475) at Eλ = 250 nm and Em = 395 nm. The total number or measure of

amino acids in the sample was estimated based on the area under the

HPLC chromatogram. An internal standard was used to estimate the

concentration of each amino acid as the internal standard method was used

to improve the precision and accuracy of the results where volume errors

are difficult to predict and control. Amino acid composition revealed from

the HPLC analysis is reported as unit residues per 1000 residues.

C.3 Determination of Odor

Odor is a subjective property that is innate in every substance. To

test the odor of the extracted collagen the researchers used the Sniff test.

The results of this test are very dependent to the observers since the

sensitivity of an individual to odor is highly variable and thus change from

time to time. Careful standardization is to be observed.

D. Product Making
 Sheet masks are face-shaped masks that are usually soaked in serum and

are made up of different materials. It generally utilizes collagen as its main

component. In this study, the extracted collagen from fish wastes is used. 1

tablespoon of extracted collagen is mixed with 4 tablespoons water and 1 teaspoon

of natural essences to make a serum. Cinnamon oil (1/4 tsp.) is added for

fragrance and additional nourishment. Compressed masks are soaked into the

serum mixture. Sheet masks are readily available in the market formed into

compressed pellets and once soaked will puff into face-shaped masks. The

mixture is left for 5 minutes. The quality of the masks is then observed and

analyzed. Afterwards, it is compared to the quality of other existing commercialized

masks.

*measurements are subjected for trials

Statistical Treatment

The statistical tools used to treat the data of this research are the following:

Percentage Difference. The trend of increase or decrease in percent is the

measure of percent change, which is the extent to which a variable gains or loses

intensity, magnitude, or value. The figures are arrived at by comparing the initial

(or before) and the final (or after) quantities according to a specific formula.

One-tailed z Test. It indicates that the Ho should be rejected when the test

value is in the critical region on one side of the mean. A one tailed z test is either

right or left, depending on the direction of the inequality of the H a.

Cost Analysis

Blueprint of the Product

 Figure 3. Research Product

The figure above shows the prospected image of the research product. The

face mask is generally white in color and is soaked in a serum with the extracted

collagen. The packaging of the product features a jumping fish (which is Tilapia)

out of water in a bluish white background. The product name Nyl is a word play of

the word Nile which is the variety of Tilapia utilized for collagen extraction in this

study.