OPTIMIZATION AND CHARACTERIZATION OF

GELATIN EXTRACT FROM CAMEL HIDE (Camelus

dromedarus) FOR APPLICATION IN HALAL INDUSTRY

BY

SLIMANE ABOULALA

A dissertation submitted in fulfillment of the requirement for

the degree of Master of Science Biotechnology Engineering

Kulliyyah of Engineering

International Islamic University Malaysia

AUGUST, 2017

ii

ABSTRACT

This research aimed to optimize the pre-treatment and the extraction conditions of

camel hide gelatin as well as different characterization analysis was performed.

Firstly, this research investigate the effect of pretreatment on Camel Hide (CH) using

two different type of alkaline chemicals, namely, Sodium hydroxide " NaOH", and

Calcium hydroxide "Ca(OH)2 ." This treatment was done with the help of Sodium

sulfide as an unhearing agent. The combination of different set was done by using

Design of Expert (DOE) software. The total of 20 experiments runs was performed

following the suggested mix combinations of parameters: time of pre-treatment

(days), NaOH/ Ca(OH)2 concentration (g/l), Sodium sulfide concentration (g/l), and

response was chosen to be the Camel Hid thickness. The model was significant only

with Calcium hydroxide. The results of the study on the preference of calcium

hydroxide 40 g / l, sodium Sulfide 5 g / l, and duration of 8 days and has reached the

skin thickness of 5.2 ml. As for the second goal was a study of the effect of pH on the

process of gelatin extraction, which has been using the same software and install both

the temperature and time of extraction, where it was a constant in six phases until total

dissolve of the camel hid. The optimal pH was 3.5 for a yield of gelatin extracted

estimated (28.52g). The gelatin extracted dried in two ways: freeze-drying and heat-

dried. The research concluded with the study of physical and chemical properties of

camel gelatin. It was found that the gel strength was high (260 g), but for Amino acids

using HPLC device was found just like any other types of gelatin. As for the study of

functional groups using FTIR detect the presence of each of the amines 1,2 and 3 and

amides A and B. As for the study of thermal transitions using DSC gave similar

results compared to the buffalo gelatin as a reference.

iii

خلاصة البحثABSTRACT IN ARABIC

الكيميائة للجيلاتين راسة الخواص الفيزيائية و د ودراسة طرق الدعالجة والإستخلاص الأمثلين لجيلاتين جلود الجمال سعى هذا البحث إلى الدقارنة. فكان الذدف التحليل و قصد بالدستخلص مقارنة بالأنواع الأخرى. كان منهج الدراسة تجريبيا مخبريا باستخدام العديد من الأجهزة

الأول هو دراسة الدعالجة الدثلى لجلود الجمال قبل استخلاص الجيلاتين منها. قارنت الدراسة بين عاملين قاعديين و هما هيدروكسيد م(، و ة)ييازمن فترة الدعالج و كان كل من: ”Design of Expert“الصوديوم و هيدروكسيد الكالسيوم )الجير( بالإستعانة ببرنامج

كعوامل و سمك الجلد)مم( كنتيجة ،ل( /جم)الصوديوم كبريتاتو تركيز ،( جم/لهيدروكسيد الكالسيوم ) تركيز هيدروكسيد الصوديوم/ات الصوديوم كبريت/ل، جم 04كعامل كيميائي للمعالجة بتركيز قدره هيدروكسيد الكالسيومالتصميم. يسفرت نتائج الدراسة عن يفضلية

pH. يما عن الذدف الثاني فكان دراسة تأثير درجة الحموضة مم 5.5ييام كمدة مثلى للمعالجة. و قد بلغ سمك الجلد 8جم/ل، و 5على عملية استخلاص الجيلاتين، حيث تم باستخدام نفس البرنامج و تثبيت كل من درجة الحرارة و زمن الإستخلاص حيث كان مستمرا

) بعد تعديل %58.55بعائد للجيلاتين الدستخلص قدر بـ 5.5لد. و كانت درجة الحموضة الدثلى في ست مراحل حتى تلاشي الجالمجفف الحراري. في الجلد و الجيلاتين الدستخلص(. تم تجفيف الجيلاتين الدستخلص بطريقتين : التجفيد و % 04على نسبة الرطوبة

الكيميائية للجيلاتين الدستخلص، حيث تبين ين خاصية قوة تماسك الذلام خلص الذدف الأخير إلى دراسة الخواص الفيزيائية و باستعمال فتم دراستها يما عن الأحماض الآمنية Texsture anlyser جرام( باستخدام جهاز 564)البلوم(كانت عالية )

كشف FTIRفكانت موجودة كباقي الأنواع الأخرى من الجيلاتين. يما عن دراسة المجاميع الوظيفية باستعمال جهاز HPLCجهازيث ح DSCالتحولات الحرارية باستخدام . فيما كانت دراسة(ب)و (ي)و الأمينات 5و 5و 0 عن وجود كل من الأميدات

مرجع. كيعطت نتائج مماثلة مقارنة بجيلاتين الجاموس

iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms

to acceptable standards of scholarly presentation and is fully adequate, in scope and

quality, as a dissertation for the degree of Master of Science (Biotechnology

Engineering)

…………………………………..

Irwandi Jaswir

Supervisor

…………………………………..

Mohammed Elwathig Saeed

Mirghani

Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable

standards of scholarly presentation and is fully adequate, in scope and quality, as a

dissertation for the degree of Master of Science (Biotechnology Engineering).

…………………………………..

Nassereldeen A Kabbashi

Internal Examiner

…………………………………..

Parveen Jamal

Internal Examiner

This thesis was submitted to the Department of Biotechnology Engineering and is

accepted as a fulfilment of the requirement for the degree of Master of Science

(Biotechnology Engineering)

…………………………………..

Faridah Youcef

Head, Department of

Biotechnology Engineering

This thesis was submitted to the Kulliyyah of Engineering and is accepted as a

fulfilment of the requirement for the degree of Master of Science (Biotechnology

Engineering)

…………………………………..

Erry Yulian Triblas Adesta

Dean, Kulliyyah of Engineering

v

DECLARATION

I hereby declare that this dissertation is the result of my own investigations, except

where otherwise stated. I also declare that it has not been previously or concurrently

submitted as a whole for any other degrees at IIUM or other institutions.

Slimane Aboulala

Signature........................................................... Date.........................................

vi

COPYRIGHT PAGE

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF

FAIR USE OF UNPUBLISHED RESEARCH

OPTIMIZATION AND CHARACTERIZATION OF GELATIN

EXTRACT FROM CAMEL HIDE (Camelus dromedarus) FOR

APPLICATION IN HALAL INDUSTRY

I declare that the copyright holders of this dissertation are jointly owned by the student

and IIUM.

Copyright © 2016 Slimane Aboulala and International Islamic University Malaysia. All rights reserved.

No part of this unpublished research may be reproduced, stored in a retrieval system,

or transmitted, in any form or by any means, electronic, mechanical, photocopying,

recording or otherwise without prior written permission of the copyright holder

except as provided below

1. Any material contained in or derived from this unpublished research may

be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print

or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieved system

and supply copies of this unpublished research if requested by other

universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM

Intellectual Property Right and Commercialization policy.

Affirmed by Slimane Aboulala

……..…………………….. ………………………..

Signature Date

vii

ACKNOWLEDGEMENTS

Alhamdolillah, all prise is due to Allah, the lord of the universe for making the

completion of this thesis and research a reality and may his blessing upon his prophet

Muhammed, (PBUH) the last and the final messenger.

I wish to express my appreciation and thanks to those who provided their time,

effort and support for this project. To my supervisor Prof.Dr.Irwandi Jaswir and my

co-supervisor Associate Professor. Dr.Mohammed Elwathig Saeed Mirghani, thanks

for your supervision, motivation, your wise advice and encouragements.

Thanks, for the partial financial support during my research as Research

Assistant (RA) from the fund of: Camel Gelatine Project from King Saud University

(KSU), Saudi Arabia.

My sincere appreciations go to department of Biotechnology Engineering,

IIUM, staff and technicians. To the International Halal Research and Training

(INHART) staff and technicians for their support and help.

To my senior friends in the Biotech department, Hammed, Saheed, Nureddin,

Mohammed Suleiman, Harmen, Deni, Norlina, for their support during my research.

Thanks for your friendship and happy souvenir.

To all my friends in Algerian Student Community IN IIUM, and Tawat

Malaysia for their emotional and guidance support. Especially Ahmed Hamid Aissa

(BASHA)

viii

DEDICATE

It is my utmost pleasure to dedicate this work to my dear parents: Hammou and

Fatimah, and to all my family, who granted me the gift of their unwavering belief in

my ability to accomplish this goal: thank you for your support and patience, as well as

financial support to fulfil the fees of my study.

A special dedication to my wife for her continuous support, encouragement and

advices, and for that, I will be forever grateful.

To my two sons: El- Yakdan Hadj Ibrahim, and Moussa AL –Amin, I took from their

time to my study. To my new baby Mohammed Ifaw which he born after my thesis

examination by two weeks 24/05/2017.

To the memoir of ALLAH yarham: Prof. Dr.Yakoob Che Man, the founding director of

Halal Institute Research Product in UPM, Malaysia.

To all whom their aim is to realise the project of HALAL GELATIN INDUSTRY to

provide the alternative to Muslim consumers. And for the sincere hard working in the

field of Halal Research and development.

ix

TABLE OF CONTENTS

Abstract .................................................................................................................... ii abstract in arabic ...................................................................................................... iii Approval Page .......................................................................................................... iv

declaration ................................................................................................................ v Copyright Page ......................................................................................................... vi acknowledgements ................................................................................................... vii

Dedice.. ……………….…………….……………………………..……….…….. xii

Tables of Contents………………………………………………………..………. ixii

list of tables .............................................................................................................. xii list of figures ............................................................................................................ xiv

List of Abbreviations……………………………………… ……………………. xiiv

CHAPTER ONE: INTRODUCTION ................................................................. 1 1.1 Background of the Study ........................................................................ 1 1.2 Problem Statement .................................................................................. 2

1.3 Research Objectives................................................................................ 3 1.4 Research Methodology Outline .............................................................. 3

1.5 Research Scope ....................................................................................... 4 1.6 Outline of the Thesis ............................................................................... 5

CHAPTER TWO: LITERATURE REVIEW .................................................... 7 2.1 Introduction............................................................................................. 7

2.2 History of gelatin .................................................................................... 7

2.3 Overviw of collagen ............................................................................... 7

2.4 From collagen to gelatin ......................................................................... 8

2.5 The structure and composition of collagen and gelatin .......................... 9

2.6 Raw material for gelatin production ....................................................... 04

2.6.1 Preparation of bones ...................................................................... 00

2.6.1.1 Assorting and size reduction .............................................. 10

2.6.1.2 Degreasing the bones ........................................................ 15

2.6.1.3 Deminiralization .............................................................. 15

2.6.2 Preparation of hides ...................................................................... 05

2.6.2.1 Alkaline Process for Bovine Hides ................................... 13

2.6.2.1.1 Liming process ................................................. 13

2.6.2.2.2 Neutralization .................................................. 16

2.6.2.2 Acid Process for Porcine skin ......................................... 16

2.6.2.3 Enzymatic Process ......................................................... 17

2.7 Fish and Poultry Gelatin ........................................................................ 18

2.8 Physico-chemical Properties of Gelatin.................................................. 18

2.8.1 Physical Properties ....................................................................... 18

2.8.1.1 Gel strength /Bloom .......................................................... 19

2.8.1.2 Viscosity ............................................................................. 19

2.8.1.3 Setting point ....................................................................... 20

2.8.1.4 Melting point ..................................................................... 20

2.8.1.5 Color and clarity ............................................................... 21

x

2.8.2 Chemical propertie .................................................................... 21

2.8.1.1 Isoelectric point ........................................................... 21

2.8.3 Functional properties ................................................................ 22

2.8.4 Nutritional featurs ..................................................................... 24

2.9 Applications of gelatin ..................................................................... 24

2.9.1 Gelatin in food industry ......................................................... 24

2.9.2 Gelatin in non food industry ................................................... 24

2.10 Camel as new source of gelatin ........................................................ 25

2.10.1 Camel hide and bons ............................................................ 26

2.10.2 Camel gelatin as value added products ............................... 27

2.11 Chapter Summary ................................................................................. 28

CHAPTER THREE: MATERIALS AND METHODS .................................... 29 3.1 Introduction............................................................................................ 29 3.2 Materials ................................................................................................ 29

3.2.1 Chemical reagent and laboratory equipments.............................. 29 3.2.2 Camel hide collection .................................................................. 29 3.3 Experimental methods ........................................................................... 30 3.3.1 Sample preparation protocol ......................................................... 30

3.4 Optimization of Camel Hide Processing .............................................. 33 3.4.1 Experimental Designe for Pre-treatment Study ............................ 33

3.4.2 Experimental Designe for Gealtin Extraction step .................... 34 3.4.3 Statistical Analysis ....................................................................... 35 3.5 Characterization of camel hide Gelatin ................................................. 36

3.5.1 Gel yield calculation of extracted Gelatin ................................... 36

3.5.1.1 Moisture content for hide and gelatin ............................. 36

3.5.1.2 Ash content for hide and gelatin ..................................... 37

3.5.2 Gel Strength analysis(Bloom test) ............................................... 37

3.5.3 Amino acid Analysis (HPLC) ....................................................... 38

3.5.4 Fourier Transform Infrared Spectra analysis (FTIR).................... 38

3.5.5 Differential Scanning Calorimetry (DSC) ................................... 39

3.6 Summary of Chapter Three .................................................................... 39

CHAPTER FOUR: RESULTS AND DISCUSSION ......................................... 40

4.1 Introduction............................................................................................ 40 4.2 Characterization of Crude Salted Camel Hide: Moisture, Ash

Content and Thickness ........................................................................... 40 4.3 Experimental Design of camel hide gelatin (CHG) ............................... 42

4.3.1 Experimental Designe for pre-treatments steps ............................ 42

4.3.1.1 Results of Optimization of Pre-treatment using Lime

and NaOH as Alkali Agent .................................................................... 42

4.3.1.1.1 Results of Optimization of Pre-treatments

Using Lime ............................................................................................. 42

4.3.1.1.2 Results of Optimization of Pre-treatments

Using NaOH ........................................................................................... 44

4.3.1.2 Comparative Result of Analyzing the Pre-treatment by

using Lime and NaOH ............................................................................ 51

4.3.1.3 Validation of Pre-treatment Test ..................................... 52

4.3.2 Optimization of Camel Hide Gelatin Extraction ........................ 53

xi

4.3.2.1 Study of The Effect of pH on Camel Hide Gelatin

Extraction (OFAT Analysis) .................................................................. 53

4.3.2.2 Analysis of Variance (ANOVA) .................................. 56

4.3.2.3 Gelatin Extractability Calculation ................................. 57

4.4 Camel Hide Gelatin Characterization .................................................... 58

4.4.1 Proximate Analysis: Ash and Moisture ....................................... 58

4.4.1.1 Ash Analysis .................................................................... 58

4.4.1.2 Moisture Analysis ........................................................... 58

4.4.2 Gel Strength or Bloom ................................................................ 58

4.4.3 Amino Acid Analysis ................................................................. 60

4.4.4 FTIR Spectroscopy of Extracted Camel Hide Gelatins .............. 62

4.4.5 DSC of Extracted CHG .............................................................. 66

4.5 Summary ................................................................................................ 69

CHAPTER FIVE: CONCLUSION AND RECOMENDATIONS ................... 70

5.1 Conclusion .............................................................................................. 70

5.2 Recommendations................................................................................... 70

REFERENCES ....................................................................................................... 72

APPENDIX K: RESEARCH ACHIEVEMNTS ............................................... 78

xii

LIST OF TABLES

Table 2.1 Some Techniques used for Hide liming process. 15

Table 2.2 Viscosity and Bloom Ranges in Commercial Gelatin 20

Table 2.3 Arabian camels’ numbers in some Arabic countries 25

Table 2.4 Characteristic of Camels Hide. Adopted from (Salahi, et al, 2013) 27

Table 3.1 Experimental designs for camel hide pretreatments conditions 33

Table 3.2 The Experiment Design Matrix for The FCCCD Using DOE for Pre-

treatment Optimization 34

Table 3.3 Experimental design for extractions step (OFAT) 35

Table 3.4 The Illustrated Design for The 6 Steps of Camel Hide Gelatin

Extraction 35

Table 4.1 Proximate analysis of salted camel hides 41

Table 4.2 Face Central Composit Design (FCCD) of Pre-treatment Analysis of

CH Using Lime Ca (OH)2 as Liming Agent 43

Table 4.3 Face Central Composite Design (FCCD) of Pre-treatment Analysis of

C.H using NaOH as Liming Agent 44

Table 4.4 Analysis of Variance of the Pre-treatment of Camel Hide using Lime as

Alkali Agent 47

Table 4.5 Analysis of Variance of the Pre-treatment of Camel Hides using NaOH

as Alkali Agent 49

Table 4.6 Validation of pre-Treatments Results 52

Table 4.7 Result of Extraction Condition by using the pH as OFAT 53

Table 4.8 Camel Hide Multiple Extraction at pH 3.5(Run3) 54

Table 4.9 Analysis of variance of pH on CHG Yield 56

xiii

4.10 Extractability of CH Gelatin in Each Step of Extraction 57

4.11 Moisture and Ash Content in Camel Hide Gelatin 58

4.12 Amino Acid Profiling of Camel Hide Gelatin (300 unit) 62

xiv

LIST OF FIGURES

2.1 Gelatin Market Shared by Raw Material, 2013. Adopted from (Grand view research,

2014) 10

2.2 Types of Bones Used in Gelatin Industry 11

3.1 Pretreatment of salted Camel hides 31

3.2 Extraction steps of Camel hide gelatin 32

4.1 (a), (b), (c) 3D contour plot showing the interactions of operating

parameters. (a) The interaction between SS and lime, (b) The interaction

between time and SS (c) Interaction between time and lime 48

4.2 (a), (b), (c) 3D contour plot showing the interactions of operating parameters.

(a) The interaction between sodium sulfide and NaOH. (b) The interaction

between time and sodium sulfide (c) Interaction between time and NaOH 50

4.4 Design Expert Plot of Camel Gelatin Yield 55

4.5 Effect of the pH on the Yield of Extracted Camel Gelatin 55

4.6 Gel Strength of the First Step of Camel Gelatin Extraction at pH 3.5 dried in

Freeze-Dryer 59

4.7 Gel strength of the First Step of Camel Gelatin Extraction at pH 3.5 dried in

Oven-dryer 60

4.8 FTIR Spectra for CHG Extracted in Different pH Comparing with Buffalo

Gelatin as Standard 64

4.9 DSC analysis of Buffalo gelatin as standard 67

4.10 DSC analysis for CHG extracted on pH 3.5 68

xv

LIST OF ABBREVIATIONS

ANOVA Analysis of variance

Ca(OH)2 Calcium Hydroxide

CH Camel hides

CHG Camel Hide Gelatin

DSC Deferential Scanning Calorimetric

FAO Food and Agriculture Organization

FCH Fresh camel Hide

FTIR Fourier Transmission Infra-red Spectroscopy

HCl Hydrochloric acid

HPLC High Performance Liquid Chromatography

IEP Iso Electric Point

Kg Kilogram

kcal Kilo-calorie

mm millimeter

NaOH Sodium Hydroxide

Na2S Sodium sulfide

OFAT One Factor At Time

SCH Salted camel hide

ºC Degree Celsius

1

CHAPTER ONE

INTRODUCTION

1.1 BACKROUND OF THE STUDY

Gelatin (Gelatine) is a protein which is originally derived from partial hydrolysis of

Collagen. This collagen is obtained from skins / hides, bones, and connective tissues

of land animal, especially mammalian, as well as from sea animals like fish. The most

important physical properties of gelatin are gel strength and viscosity. The quality of

gelatin is measured by the gel strength or Bloom value, classified as low (150g),

medium (150g-220g) to high Bloom (220g-300) (Johnston-Banks 1990).

Because gelatin is animal by product, a question regards to it Halal status is

raised. Halal is the terminology given to some animal base products follows specific

criteria according to Sharia “Islamic legislation”. Pork is not allowed to be consumed

according to Islamic teaching. It is used specific terminology which is Haram. Haram,

or Non Halal, which means prohibited either to be consumed (for food or

pharmaceutical product) or to be used (nonfood products such as cosmetic). However,

other ruminant animals like, cows, gates, sheep, and camels are permeated to consume

its meat and other derivatives only when it is slaughtered according to Islamic

guidance.

Furthermore, some disease was seriously highlighted, Bovine Spongiform

Encephalopathy (BSE) for cows, and swine flu for pork by products such meat and

meat derivatives, is considered as non-safe product. The interest on alternative Halal

source is because of the growing concern on the Halal gelatin among Muslim

manufacturer as well as consumer. Camels serve an interesting new source of gelatin

which is not exploited yet.

2

1.2 PROBLEM STATEMENT

Halal is an Arabic term which means permissible, wholesome, and hygiene.

ALLAH the GOD states clearly in the Quran about Halal related matters and what we

should eat and what we should forbid. In chapter “Surah” AL-IMRAN:

“O men! Eat the lawful and good things out of what is in the earth, and

do not follow the footsteps of the shaitan; surely he is your open enemy”

Quran: Chapter: 2, Verse: 168.

In addition, according to hadith, saying of the prophet Mohammed (Peace be

upon Him)

“That which is lawful is clear and that which is unlawful is clear, and between

the two of them are doubtful matters about which many people do not know…”

[Narrated by al-Bukhaari (52) and Muslim (1599).]

The growing demand for Halal gelatin among Muslim consumer, and the

rejection of Haram sources of gelatin mainly pork gelatin, makes scientist and

researcher looking for other alternative sources, one of them is fish gelatin. Fish

gelatin has been widely studied last 10 years by many institutions cross the world.

Fish gelatin has been given a great concern due its wide use in food, cosmetic, and

even in technical applications. Gómez-Guillén et al (2009), reported that both, warm

and cold water fish species represent an alternative competitive to mammalian gelatin

in the aspect of religious related issues (HALAL and COSHER) as well as disease-

free contaminations. However, it needs more research to overcome the weaknesses

such as low bloom, and yield (Gómez-Guillén et al ,2009).

In 2012, more than US 1Billion were the total trade of gelatin globally

(www.gtis.com, 2013), and it is expected to reach USD 2.79 Billion in 2018. In

3

addition, 19.78% was the change between 2011 and 2012. (www.gtis.com, 2013). The

same report mentioned about the global gelatin production was 348.9 kilo tons in 2011

and is expected to reach 450.7 kilo tons in 2018. This high demand on gelatin lets the

investor to look for other sources for raw materials. Camel hides and boon promise to

be new source not explored yet, however, the biggest challenge it could be the

sustainability of this kind of raw material.

1.3 RESEARCH OBJECTIVES

The general objective of the research is to produce (extract) gelatin from camel hide

using chemical approach and subsequent characterization of the gelatin produced.

The specific objectives devised to achieve the main objective are:

1. To optimize the pretreatment of camel hide (hair removing).

2. To optimize the extraction of camel hide gelatin, and

3. To characterize the extracted camel hide gelatin and to compare it with

commercial gelatin.

1.4 RESEARCH METHODOLOGY OUTLINE

The study was a laboratory-based experimental works in shake flask using water-bath

thermos controlled. The research commenced by pretreatment of camel hide selected

for the study. The determined variables were used to optimize the hide hair removal.

Subject to findings in the literature, the extraction of the gelatin was carried out

by considering three important factors (Temperature, Time, and pH) which determine

the yield.

The operating parameters were then evaluated for optimization of both the

pretreatment and the extraction process using face centered central composite design

4

(FCCCD) and One Factor At Time (OFAT), of Design-Experts® 6.0.8 (Stat East,

Inc).

Conclusively, the gelatin produced was purified and dried. For the

characterization, six (6) type of analytical instruments and methods, in accordance

with the official method for gelatin tests was conducted. The results obtained were

compared with the international standards of the gelatin, Gelatin Manufacturers of

America, GMA.

1.5 RESEARCH SCOPE

The research investigated the production of gelatin by hydro-extraction approach

using chemicals for its pretreatments. This research is important considering the

benefits it offers in the propensity of Halal gelatin to replace Haram gelatin. The use

of conventional (Porcine and Bovine) gelatin has posed some religious and health

concern, because of the cause of some disease like BSE and swine flu. Besides, the

growing concern of Halal gelatin is another constraint as compared to fish gelatin

which is available and can be produced from renewable raw materials and as such

considered sustainable.

To achieve this proposition, camel hide was selected to investigate its potential

for gelatin production. Camel hide was originally obtained in dried form (NaCl was

used to preserve it) from local slaughter house in Algeria, and Kingdom of Saudi

Arabia. The research utilized chemicals which is an established research in the

Biotechnology Engineering Department, Kulliyah of Engineering, IIUM.

Operating conditions of pretreatment and extraction such as Chemicals like :

calcium hydroxide, Ca(OH)2), sodium sulfide (Na2S), Hydrochloric acid (HCl), and

5

parameters like: pH, extraction temperature, and extraction time were studied in

details. The production rate and yield was studied using Design-Expert (6.0.8.).

Furthermore, optimization of the parameters was carried out to determine the

optimum production yield of the gelatin in relation to the selected operating

parameters that determine the yield. The yield of extraction was studied and

parameters such as moisture and ash content were evaluated. Other tests which

include gel strength, FTIR spectroscopy, Deferential Scanning Calorimetry, and

Amino Acid Analysis, was conducted too.

Finally, the results derived from the optimization procedure of the production

process were validated using statistical conditions suggested by the Design of

experiment software “Design Expert (6.0.8.)” The gelatin composition was analyzed

for the determination of the quality of the product and its comparable parameters with

the commercial gelatin.

1.6 OUTLINE OF THE THESIS

CHAPTER ONE of the thesis presents the historical developments and the

background of the research work while also discussing the objectives devised for

achievement of the research methodology and also the scope of the research was

highlighted. CHAPTER TWO presents the review of the findings based on available

materials which were found on gelatin, the various approaches used over time to

produce gelatin utilizing both skin / hide and bone as raw material and the various

findings on the considered variables which affect the yield of the product. In

CHAPTER THREE, the methodology employed in the study was presented and

discussed in detail including the materials used, and the analytical procedure adopted

for the results analysis. CHAPTER FOUR is a presentation of the experimental results

6

and analysis of the results including statistical approach used to achieve the targeted

objectives in the method adopted. In CHAPTER FIVE, a concise conclusion based on

the results and analysis presented was discussed and then some recommendations

were highlighted.

7

CHAPTER TWO

LITERATURE REVIEW

2.1 INTRODUCTION

This chapter illustrate some of the latest researches that have been done on

mammalian gelatin especially, and its usage and application in different industries. It

is also discussed multiple methods of extraction and its physio-chemical

characterization as well as its functional properties.

2.2 HISTORY OF GELATIN:

The usage of gelatin goes back to ancient Egypt era; however, it did not get its actual

name until the 1700s. In England, the first gelatin patent was registered in 1754 for

producing glue. During the early 20th

century, booming research was investigated the

properties and the gelatin as molecule. Namely by Eastman Kodak, as photography

industry. In the 1950th

, most of the advancement of technical application was

achieved, when the optimum standard in gelatin and its derivatives was in focus.

(Ward and Courts, 1977). Thus, in this period, the significant characteristics of gelatin

were well-defined.

2.3 OVERVIEW OF COLLAGEN

Colagen is the major structural protein found in the skin and bones of animals .

collagen is the most aboundant protein of animal origin, comprising aproximatly 30 %

of totla animal protien. Being a major constituent of the connective tissues, collagen

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plays an important part in creating mechanical strangt, integrity and rheological

properties of the muscles and fillets (Sikorski & Borderies, 1994). Collagen molecule

composed of three α-chains intertwined in the so-colled collagen triple-helix, adapt a

3D structure that provide an ideal geometry for inter-chain hydrogen bonding (Te

Nijenhuis, 1997). Each chain in the helix rotates counter clochwise a molecular wieght

of appximetly 105 kDa (Papon, et al., 2007). The collagen molecule is stabilized by

interchain hydrogen bounds and by interaction of the radially extending amino acids

residues with water molecules. The rod like molecules is aggreagated into fibrile

(Bailey & Etherlington, 1980). Ther are at least 19 variant of collagen, named type I –

XIX (Bailey et al., 1998). Type I, II, III, and V are the fibrose collagens. Type I

collagen is found in all connective tissue, including bones and skins. It is a

heteropolymer of two α-1 chains and α-2chain. It consistes of one third glycine,

conten. It does not contain tryptophan, nor cysteine. The tyrosine and histidine

represented in very low quantity. The stability of the triple helices is secured by the

inter-chain hydrogen bounds. Regardless of the type of animal, in each α chain there

are twenty different amino acids which is repeated in specific pattern. The majority of

amino acid represented by glycine (30%), in which it is sequentially repeated with

every two other amino acids. The typical representation is looks as Glycine-x-y, in

which the possibility of X to be proline and Y to be hydroxyl-proline in not unusual.

(Bailey et al., 1998).

2.4 FROM COLLAGEN TO GELATIN

In order to get gelatin, the skin or hide of animal, firstly, undergoes into chemical or

enzymatic treatment and then finally obtained through thermal hydro-extraction. This

treatment can vary from one animal to other. In case of mammals, the treatment was

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usually performed using chemical agents. Alkali treatments that mostly involved

NaOH, or Ca(OH)2 (lime), forms type B gelatin while, type A gelatin was formed as a

result of acidic treatment of the hide performed by using strong acid like HCl, or weak

acid like organic acid such as; citric and acetic acid. (Ward and Courts, 1977).

2.5 THE STRUCTURE AND THE COMPOSITION OF COLLAGEN AND

GELATIN

The gelatin products are highly influenced by the time of liming (Ames, 1949)

whereof the longer time of liming not only causes progressive chemical changes in the

collagen but also leads to destruction and removal of non-collagen components.

One of the methods of preparation of gelatin involved the extraction of alkali

pretreated ossein (crushed bone) at low temperature and in the presence of hydrogen

bond breaking reagents that fetched gelatin with a chain weight of 62,000 (Courts,

1958). Therefore, the drop-in chain weight must have occurred in course of alkali

treatment and not during extraction, since reagents that cause breakage of hydrogen

bond works at 20°C and are unlikely to cause peptide bond cleavage. (Johns & Courts,

1977).

Conversion was found to be far more extensive in a type of gelatin produced

by the Eastman Kodak Company whereby calf skin was limed for 4-8 months.

Hamilton and Anderson (1954) performed it for another sample of atypical gelatin.

The content of arginine and tyrosine was lower in comparison to the other gelatins

processed either using alkali or acid and was apparent in both analyses. In case of calf

skin gelatin, the values of aforesaid amino acids were diminished and thus led

Neuman (1949) to conclude that gelatins show broad variation in composition and

could be considered reliable for the study of collagen composition. Hence, it is not