NUTRITIONAL ANALYSIS AND EFFECTS OF VARIABLES ON PROTEIN EXTRACTION FROM PERAH SEED

HAMIMAH SATIRAH BT MAZLAN

A dissertation submitted in partial fulfillment of the

requirements for the award of the degree of

Master of Engineering (Chemical)

Faculty of Chemical Engineering

Universiti Teknologi Malaysia

APRIL 2011

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To my beloved mother, Zainon Mamat and my supported brothers.

iv

ACKNOWLEDGEMENT

All praises to God for the opportunity to finish this project. I would like to

express my appreciation to both my supervisors, PM Dr. Ida Iadyu Muhamad and Dr.

Mohd Azizi Che Yunus for the guidance, encouragements and also criticism throughout

this project. Special thanks are also dedicated to the laboratory technicians, Pn. Siti

Zalita, Encik Yaakop and En. Mior Ramli for their advice and unreserved assistance

while conducting my experimental works. Last but not least, I would like to express my

heartiest appreciation towards my mother, siblings and friends for all the help, support

and special care given during this period.

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ABSTRACT

The nutritional content of fermented and non-fermented Perah seed

(Elateriospermum tapos) was investigated. The proximate analyses such as moisture, fat,

fiber, protein and ash contents from fermented seed were lower compared to non-

fermented seed. However, only the fat content of fermented seed was higher than non-

fermented seed. From that, maximum protein extractions from fermented and non-

fermented Perah seed were further investigated using response surface methodology. A

central composite design with three independent variables i.e. NaOH concentration (6, 8

and 10 %), extraction time (10, 20 and 30 minute) and solvent/meal ratio (50:1, 100:1

and 150:1, v/w) was used to study the response of protein yield. The experimental values

of protein yield ranged between 13.8 and 0.05 g/100 g seed meal. A second-degree

equation for independent and response variables was produced from simulation to obtain

the contour plot graphs. From the graph and mathematical solutions, the best protein

extraction protocol from both fermented and non-fermented Perah seed was obtained at

5.5 percent of solvent extraction, 40:1 ratio of solvent/meal and 32 minutes of reaction

time. Surface concentration and ratio of solvent/meal were found to influence the protein

yield from fermented seed; meanwhile only solvent concentration influenced protein

yield from non-fermented seed. The maximum protein yields for both fermented and

non-fermented perah seed were 18.0 g/100g and 5.0 g/100g seed meal respectively.

.

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ABSTRAK

Kandungan nutrisi dalam biji buah perah (Elateriospermum tapos) yang diperam

dan segar tanpa peraman telah dikaji. Analisis hampiran seperti kandungan kelembapan,

lemak, serat, protin dan abu daripada biji diperam adalah kurang berbanding dengan biji

segar. Walau bagaimanapun, hanya kandungan lemak daripada biji diperam adalah lebih

tinggi berbanding biji segar. Selain itu, pengekstrakan maksimum protin daripada biji

perah diperam dan biji segar dikaji menggunakan metodologi sambutan permukaan.

Eksperimen penggabungan dengan tiga pembolehubah tak bersandar iaitu kepekatan

NaOH (6, 8 and 10 %); masa pengekstrakan (10, 20 and 30 minit) dan nisbah

larutan/tepung (50:1, 100:1 and 150:1, v/w) telah digunakan untuk mengkaji jumlah

sambutan protin. Julat jumlah sambutan protin bagi ujikaji yang dijalankan adalah antara

13.8 dan 0.05 g/100 g tepung biji. Persamaan tertib kedua bagi sambutan pembolehubah

tak bersandar akan diperolehi menerusi simulasi bagi mendapatkan plot bentuk graf.

Menerusi graf dan penyelesaian matematik, tahap terbaik bagi pengekstrakan protin

daripada biji perah akan diperolehi pada 5.5 peratus kepekatan pelarut, 40:1 ratio larutan

per tepung dan 32 minit masa pengekstrakan. Kepekatan larutan dan nisbah larutan per

tepung mempengaruhi jumlah protin daripada biji diperam manakala hanya kepekatan

larutan mempengaruhi jumlah protin bagi biji segar. Jumlah protin maksimum bagi

kedua-dua biji diperam dan segar adalah masing-masing 18.0 g/100g and 5.0 g/100g

kisaran biji.

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TABLE OF CONTENTS

CHAPTER TITLE PAGE DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES xii

LIST OF FIGURES xiv

LIST OF ABBREVIATIONS xvi

LIST OF SYMBOLS xvii

LIST OF APPENDICES xviii

1 INTRODUCTION 1

1.1 Background 1

1.2 Problem Statement 3

1.3 Objective 3

1.4 Significant of Result 4

1.5 Scope of Study 4

1.6 Project Outline 5

1.7 Summary 5

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2 LITERATURE REVIEW 6 2.1 Introduction 6

2.2 Perah Seed Background 6

2.2.1 Background 7

2.2.2 Habitat and Ecology 8

2.2.3 Growth 8

2.2.4 Perah Fruit and Seed 9

2.2.5 Leaves 11

2.2.6 Perah Utilization 12

2.3 Protein 13

2.3.1 Chemical structure 13

2.3.2 Protein in Seed 15

2.3.3 Protein Utilization 19

2.4 Nutritional Contents of Seed 20

2.4.1 Previous Study of Nutritional 20

Perah Seed Contents

2.4.2 Previous Study of Nutritional 23

Other Seed Contents

2.5 A Review of Extraction Process 24

2.5.1 Definition 25

2.5.2 Inter-phase Extraction Method 25

2.5.3 Solid-liquid Extraction 26

2.5.4 Advantages of Using Solid-liquid Extraction 27

2.5.5 Extraction Techniques 28

2.6 Effects of Operating Variables on Extraction Process 29

2.6.1 Reaction Time 30

2.6.2 Selectivity of pH 31

2.6.3 Type of Solvent 32

2.6.4 Ratio of Solvent/meal 33

2.6.5 Solvent Concentration 35

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2.6.6 Temperature 35

2.7 Previous Study of Non Fermented and 36

Fermented Seed

2.7.1 Comparison of Nutrient Analysis 37

from Perah Seed

2.7.2 Comparison of Nutrient Analysis 38

from Other Seed

2.8 Response Surface Methodology 39

2.8.1 Determination of Response Surface 40

Methodology

2.8.2 Advantages of Response Surface 40

Methodology

2.8.3 Analysis of Variance (ANOVA) 41

2.9 Summary 43

3 METHODOLOGY 44 3.1 Introduction 44

3.2 Preparation of Seed Powder 44

3.3 Physicochemical Properties Analysis 45

3.4 Proximate Composition 45

3.4.1 Moisture Content 46

3.4.2 Crude Fat 46

3.4.3 Crude Fiber 47

3.4.4 Ash 48

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3.4.5 Crude Protein 49

3.5 Protein Extraction 50

3.5.1 Design of Experiment 50

3.5.2 Protein Analysis 53

3.5.2.1 Equipment and Materials 53

3.5.2.3 Procedure 53

3.5.3 Model and Statistical Analysis 56

4 RESULTS AND DISCUSSIONS 57

4.1 Introduction 57

4.2 Physicochemical Properties of Perah Seed 58

4.3 Proximate Analysis on Non-Fermented and 58

Fermented Perah Seed

4.4 Analysis of Protein Extraction Process 60

4.5 Statistical Analysis 62

4.5.1 Analysis of Variance (ANOVA) 62

4.5.2 Observed and Predicted Value 64

4.6 Response Surface Modeling to Yield of 66

Protein Extract

4.6.1 Mathematical Model and Analysis of 66

Central Composite

4.6.2 Effects of the Process Parameters on 67

Fermented Seed

4.6.2.1 Pareto Chart of Effects on 71

Fermented Seed

4.6.3 Effects of the Process Parameters on 72

Non Fermented Seed

4.6.3.1 Pareto Chart of Effects on Non 76

Fermented Seed

4.7 Comparison of Protein Extraction Response 77

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between Fermented and Non Fermented

Perah Seed

4.7.1 The Effects of Use for Fermented and 78

Non Fermented Perah Seed

5 CONCLUSION AND RECOMMENDATIONS 80

5.1 Conclusion 80

5.2 Recommendations 83 REFERENCES 84

Appendices A-D 92-100

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LIST OF TABLES TABLE NO. TITTLE PAGE 2.1 Scientific classification of Perah tree 7 2.2 Structural of protein 14 2.3 Comparison of Perah seed nutritional content to Soyabean 21 2.4 Mineral analysis of Perah seed 22 2.5 The proximate analysis of Soybean. Result expressed 23

as g/100g 2.6 Proximate analysis of Watermelon seed 24 2.7 Polar and non-polar description 33 2.8 Comparison of proximate analysis in non-fermented 37

and fermented Perah seed 2.9 Comparison of proximate analysis in non-fermented 38

and fermented Soybean 2.10 Comparison of proximate analysis in non-fermented 39

and fermented Pumpkin seed 2.11 ANOVA data analysis 42 3.1 Independent variable values of the process and 50

their corresponding levels 3.2 Number of experimental design 52

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4.1 Physical characteristic of Perah seed 58 4.2 Chemical properties of Perah seed 59 4.3 Protein yield extraction 61 4.4 ANOVA results for fermented seed 63 4.5 ANOVA results for non-fermented seed 63 4.6 Comparison of protein extraction between non-fermented 80

and Fermented Seed

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LIST OF FIGURES FIGURE NO. TITTLE PAGE

2.1 Perah seed tree 9 2.2 Physical condition of Perah seed in coating 10 2.3 Physical condition of Perah seed without coating 10 2.4 Old Perah leaves 11 2.5 Young Perah leaves 12 2.6 Identification of protein in Soybean seed 16 3.1 Flow diagram of the experimental procedure 54 4.1 Observed and predicted values of the protein yield for 65

fermented seed 4.2 Observed and predicted values of the protein yield 65

for non-fermented seed 4.3 Response surface effects of solvent concentration to reaction 67

time on yield of protein extraction with solvent/meal ratio of 100v/w

4.4 Response surface effects of solvent concentration to 69

solvent/meal ratio on yield of protein extraction with reaction time at 20 minutes

4.5 Response reaction time to solvent/meal ratio on yield of protein 70

extraction with solvent concentration at 8 Percent.

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4.6 Pareto chart for fermented Perah seed 72 4.7 Response surface effects of solvent concentration to reaction 73

time on yield of protein extraction with solvent/meal Ratio of 100v/w

4.8 Response surface Effects of solvent concentration to solvent/meal 75

ratio on yield of protein extraction with reaction time at 20 minutes

4.9 Response reaction time to solvent/meal ratio on yield of protein 76 extraction with solvent concentration at 8 Percent

4.10 Pareto chart for non-fermented Perah seed 77

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LIST OF ABBREVIATIONS

ANOVA analysis of variance

RSM response surface methodology

xvii

LIST OF SYMBOLS

k number of independent variable

n0 number of replication

N number of test

p probability

X process variable (independent variable)

Y response

Z coded value of the independent variable

� significance level

� regression coefficient

R2 coefficient of determination

w/v weight to volume ratio

xviii

LIST OF APPENDICES APPENDIX TITTLE PAGE A1 Data for Proximate Analysis 92 B1 Standard Curve for BSA 93 C1 F-Value Table 94 D1 ANOVA Table 95 D2 Data for Observed and Predicted Analysis 97 D3 Contour Plot for Fermented Seed 98 D4 Contour Plot for Non Fermented Seed 100

CHAPTER I

INTRODUCTION 1.1 Background

Through generations, protein has become widely used in many sectors such as

pharmaceutical, cosmetics and many more. Protein is one of the important nutrients in a

human diet and many studies have been developed in order to get a good, high value and

loads of protein extract. These studies have given many advantages to human lives and

one of them has been to help the third world countries to get a sufficient diet. Extracted

protein also helps human get faster nutrients during disasters or troubles. In addition,

extracted proteins are very useful for medical teams and to help people.

Many studies have also been carried out to obtain materials that give a good and

high volume of protein extracts. Perah seed or scientifically called elateriospermum

tapos has been introduced as a protein source. Perah is a local wild tree that is large and

has red colored young leaves that can be easily spotted. In Malaysia, it can be found in

Sabah and Sarawak and also in the east peninsular states such as Kelantan and

Terengganu. It is used by locals as a dish where the seed is cooked and fermented.

However, not everyone knows that Perah seed actually has a higher protein nutrient

compared to soya bean and other seeds such as watermelon seed. There is scarce

literature on Perah seed except for its oil content.

2

In this study, the nutritional compositions for both the non- fermented and the

fermented Perah seed were determined. These nutritional compositions were then

compared to other seed in terms of the volume. The nutritional content can be

determined according to the previous experimental methods such as moisture content by

Ezeagu et al. (2002), crude fat and ash by Chopra and Kanwar (1976) and crude fiber by

CEPP (UTM), (2007).

The study is also carried out to get the best condition of protein extraction. Many

extraction methods have been considered, such as ultrasonic extraction and supercritical

extraction but liquid-solid extraction was chosen due to its less expensive method and

convenience for a lab scale. Liquid-solid extraction is also less hazardous and minimal

effect to the nutrient components after several cycles of process.

Protein extraction by using solvents is very important in order to get the best

variable conditions. The variables used are the solvent/seed ratio, reaction time and

solvent concentration. At best, the solvent concentration gives a high quality of protein

extract. The Perah seed is prepared in powder form to remove the moisture content first,

before the protein can be extracted. Different reaction times give different values of

protein extracted. Sufficient reaction time is a must to make sure all the protein content

can be extracted successfully by the solvent. The high volume of protein extraction is

also proportional to the volume of the solvent per seed. It is believed that the highest

protein extracted comes from a sufficient volume of solvent per seed supply.

When it comes to optimization experiments, the study must consider influential

variables and how they can relate to or influence the results. The use of Response

Surface Methodology (RSM) is one of the experimental strategies to obtain optimization

experiments. The RSM is widely used as it can account for interactions in several

influential variables by using central composite design. In RSM use, there are three main

stages involved in this study, which are designing the experiments, building the

3

mathematical models and determining the optimum conditions according to the variables

that have been chosen.

1.2 Problem Statement

The insufficient supply of protein in human diet, especially in the growth of

human population and also due to malnutrition in third world countries, has resulted in

continuous efforts in exploring new resources such as plants that have higher protein

contents. Perah seed has been chosen in this study since it is plentiful and easily found in

the east coast of Malaysia, and easily grown. The problem of this study is whether Perah

seed has the desired nutritional content. The problems are also in regard to which

potential variables must be considered and what are the best conditions in order to get

higher protein extraction.

1.3 Objective

i) To determine physical characteristics of fermented and non-fermented Perah

seed.

ii) To determine potential nutritional contents of fermented and non-fermented

Perah seed.

iii) To investigate the effect of independent variables (solvent concentration,

reaction time and solvent/meal ratio) on the production of protein from

fermented and non-fermented Perah seed by using solid liquid extraction.

iv) To determine the best condition of process variables for both fermented and non-

fermented seed.

4

1.4 Significance of Study The potentials of nutrition from Perah seed can be used as a new source for

human needs. Protein extracted from Perah seed can be an alternative source as

compared to animal protein, as it is less expensive and safe to use.

1.5 Scope of Study The scope of this study is based on the experimental works and the analysis of

both fermented and non-fermented Perah seed. There are several experimental works

that need to be developed and done to achieve the objective of this study. The scope is

divided into several sections:

i) Preparation of the fermentation and powder seed.

Perah seed was being fermented, dried at certain temperature and grinded until at

desired and homogenized size.

ii) Nutritional content of Perah seed

Analyses of nutritional contents consist of moisture, crude fat, crude protein, ash,

crude protein and crude fiber content. Powdered Perah seed has followed by

referred methodologies in order to get desired proximate analysis.

iii) Protein extraction from fermented and non-fermented Perah seed.

Liquid-solid extraction method was used to extract protein from both fermented

and non-fermented powdered Perah seed.

5

iv) Analysis of Perah seed.

The powdered Perah seed from the protein extraction process was analyzed by

the protein contents or protein yield. A spectrophotometer was used to determine

the protein yield as being compared to a standard curve.

v) Optimization of protein extraction

Response Surface Methodology (RSM) is used to get the relationship between

the responses of each independent variable by the contour plots. An analysis of

variance showed the adequacy of the model that has been developed and Pareto

Chart showed the most significant independent variables.

1.6 Project Outline

This report contains five chapters. First chapter outlines the background of the

research, problem statement, objectives, significant results and scope of the study.

Second chapter discusses the Perah seed background, literatures of process extraction

and variables involved and response surface methodology. Next is the third chapter,

which covers the experimental methods for nutritional content determination, protein

extraction and obtaining best condition by using response surface methodology. The

fourth chapter covers the results and discussion from this study. Finally, the fifth

chapter presents the conclusion and recommendations.

1.7 Summary

Perah seed nutritional content is determined by biological processes. Protein

extraction from Perah seed is also determined by considering several variables such as

solvent concentration, liquid-solid ratio from the seed soluble and reaction time. This

study optimizes condition of extraction process by using response surface methodology.

84

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