CHAPTER 3

RESEARCH METHODOLOGY

In this chapter of the study, a representation of the design, research methods and

research techniques that are to be used will be presented. This part will state how the

collagen is extracted and subjected to physicochemical analyses.

Research Design

According to Michael, Lewis,  Bryman & Liao (2004), the major aim of

comparative research is to identify similarities and differences between subject entities by

comparing and contrasting their point of comparisons. The main objective of this study is

to evaluate the physicochemical properties of chicken feet and bones collagen and

compare results with the commercial collagen.

Research Method

Chicken feet and bones are considered as waste products because people normally

do not consume feet parts. It is highly available here in the Philippines and thus has the

high potential to be the alternative source to produce collagen other than fish, bovine or

porcine. To obtain collagen, which is the main ingredient for this study, bones, and feet

of chicken broiler will be collected from a local market of Dagupan City, Pangasinan.

After collection, chicken bones and feet are then washed to remove any contaminants that

may affect collagen isolation. The collected chicken bones and feet are then stored in a

cold temp at -20*C.

 Pre Formulation of Powdered Collagen
from Chicken feet and bones

After washing, pre-treatment was done for the removal of non-collagenous tissue.

After which, pre-treated chicken bones and feet are subjected to acid extraction method to

obtain the acid solubilized collagen. The collagen yield will be physically and chemically

measured. As for the reference standard, Genacol will be analyzed for the same

physicochemical measure. From that, we can determine the similarities & 18

difference between chicken-derived collagen and commercial collagen used in the

pharmaceutical industry.

Duration and Locale of the study

The study was conducted in the University of Pangasinan, Dagupan City, during

the second semester of the academic year 2018-2019. The laboratory scale procedures for

collagen characterization of its physical and chemical properties are common knowledge

to experimentations with immediate results provided upon action. Thus, the results are

practically attainable in a short period.

Population/Subjects of the study

As for the subject of this study, we have randomly selected and bought broiler

chicken parts from the market. All characterizations were conducted directly to the yield

collagen. Percentage yield is influenced by the raw material, treatment and extraction

procedures. The quantity of the sample required to complete all physicochemical tests

must be considered in the target percentage yield. The extraction process was done

appropriately to have a sufficient sample of collagen. The composite sample of the

extracted collagen should not be less than twice the amount required for one complete

test. The physicochemical tests were conducted in three trials.

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 Pre Formulation of Powdered Collagen
from Chicken feet and bones

Materials, Methods, and Procedures

The main raw material which is the chicken feet and bones were collected from

the market. Different chemical reagents for the pre-treatment and extraction are prepared,

standardized and assured that are in good conditions, as well as the standardization and

calibration of instruments that will be used in the process.

I. Extraction of Collagen Process

Collagen can be obtained by chemical hydrolysis and enzymatic hydrolysis

(Zavareze 2009). All the preparative procedures were performed at 30°C. The methods

for extraction of collagen from broiler chicken feet waste consisted of the following

steps: sample collection & preparation, removing noncollagenous tissue, solubilizing

collagen, centrifuging and precipitating collagen, concentration measurement and

characterizations of collagen.

Sample Collection and Preparation

Broiler Chicken feet was used as the raw material. This is thrown as waste in

every butcher shops in the local market. It is collected and brought to the laboratory in an

ice bag. Chicken feet were cleaned under running tap water to remove blood strains and

dirt using sterilized double distilled water at least three times. Then it was cut into small

pieces (0.5x 0.5 cm) using a sterile knife and kept on ice before collagen extraction.

(Kaewdang 2014)

Pre-treatment: Removing Non-Collagenous Tissue

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 Pre Formulation of Powdered Collagen
from Chicken feet and bones

To remove noncollagenous substance and to make the tissue very loose, the

chicken feet waste was treated with 0.1 N of NaOH for 24 hours. Then, the solution was

placed for gentle stirred. The solution was changed every 8 hours, and then washed with

distilled water (Kaewdang et al. 2014).

Extraction of Collagen Procedure: Acid Solubilized Collagen (ASC)

Acid hydrolysis is usually performed either with organic acids such as acetic acid,

citric acid, and lactic acid, or inorganic acids such as hydrochloric acid (HCl). However,

organic acids are more efficient than inorganic acids (Wang et al., 2008). Organic acids

are capable of solubilizing non-crosslinked collagens and also of breaking some of the

inter-strand cross-links in collagen, which leads to a higher solubility of collagen during

the extraction process (Liu et al., 2015). Therefore, acidic solutions, especially acetic

acid, are commonly used to extract collagen. To extract acid-solubilized collagen, the

pretreated collagen sample was soaked in 0.5 M acetic acid with a sample per solution

ratio of 1: 20 (W/V) for 48 h at 25°C. This acetic acid suspension was filtered by using

cheese cloth to remove the bone residue. To precipitate the acid solubilized collagen, it

was salted out by adding NaCl to 0.9 M and centrifuged at 30,000 × g for 60 m. The

pellet was collected and then dissolved in 0.5 M acetic acid, then dialyzed against 0.1M

acetic acid and deionized water in a dialysis membrane and air dried. The dialysate

referred to acid- solubilized collagen. (Nagai et al. 2015)

II. Physical and Chemical Testing

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 Pre Formulation of Powdered Collagen
from Chicken feet and bones

Extracted collagen will be subjected to physicochemical analyses to assess if it

confirm to specifications and suggested characteristics. The following parameters and

procedures are to be observed and measured.

Physical Properties:

o Organoleptic properties. The extracted collagen was evaluated by using organs of

sense and included the macroscopic appearance such as its color, odor, taste, and

texture.

o Solubility. One gram of the crude sample was dissolved in different parts and

types of solvent, polar and nonpolar, mainly those solvents indicated in the USP

monograph for hydrolyzed collagen. Solubility will be defined, according to the

USP standard, as parts of solvent in milliliters required to dissolve 1g of the

solute. The following table provides for the description of their solubility.

o Figure
Particle2. Solubility Chart. Values are basedsize.
on USP & IP Solubility Criteria Use

After mechanical shaking, weigh the retained sample for every sieve to obtain

their particle size distribution.

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 Pre Formulation of Powdered Collagen
from Chicken feet and bones

Figure 3. Mesh Sieve Analysis. Values are based on

American National Standard for Industrial Wire Cloth
(American Standard ASTM – E11)

Chemical Properties:

o Moisture Content

Five grams of the sample was weighted and placed to the dish and spread

uniformly. The sample was placed in an oven for 3h at 105ºC. Then transferred the

sample to the desiccator to cool and reweighed the sample, (AOAC 2000). The formula

for calculating the moisture % is:

% Moisture Content = Loss in weight x 100

Weight of powdered collagen after
3 hrs of heating

o pH

The pH meter is an electrical device that will determine the acidity or alkalinity of

powdered dried collagen dissolved in water to form an aqueous solution of 1:2

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 Pre Formulation of Powdered Collagen
from Chicken feet and bones

concentration ratio. To use a pH meter, the pH’s electrode is first calibrated with standard

buffer solutions with known pH values that span the range being measured. And to make

a pH measurement, the electrode is immersed in the sample solution until a steady

reading is reached.

III. Percentage Yield of Collagen:

The yield of the collagen isolated was reported on a dry weight basis according to

the method of (B. Jamilah K., 2011) as stated:

% Yield (dry weight) of collagen= Dry weight of collagen x 100

Wet weight of sample – moisture content of the sample

Treatment of data

The results of physicochemical testing and extraction yield of collagen from

chicken feet and bones will be compared from the commercial collagen, Genacol.

Comparative results would suggest the significance of chicken feet and bones as an

alternative source of collagen.

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