‫ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﺣﻤﻦ ﺍﻟﺮﺣﻴﻢ‬
FRACTIONATION; PHYSICOCHEMICAL
AND FUNCTIONAL PROPERTIES OF
ACACIA POLYACANTHA GUM.
By
Ahmed Adam Hassan Mohammed Elnour
B.Sc. Biochemistry and Food Science
Faculty of Natural Resources and Environmental Studies
University of Kordofan
Septermber 2003
Thesis submitted to the University of Khartoum in Partial
fulfillment of the Requirements for the Degree of Master in
Agriculture (Food Science)
Supervisor
Dr: Khogali Elnour Ahmed Ishag
Co-Supervisor
Dr: Abdullah Abdualsamad Abuallah
Department of Food Science and Technology
Faculty of Agriculture
University of Khartoum
November 2007
Dedication
In the name of Allah, the Passionate and the Merciful
To all members of my family;
My father Adam who has inculcated in me the love of
learning and knowledge and who was an endless help support;
My mother Hawa who taught me the rudiments of the
alphabet, who surrounded me with her love, care and tenderness;
To the soul of my grand father Gebial.
My dear, wife Rasha who gave her full continuous
support, encouragement and blessing from the very beginning of
this work;
and to my sons, daughters and dear beloved friends;
With my respect and love I forward this work.
Ahmed
i
ACKNOWLEDGEMENTS
My special praise and thanks be to Allah, the Almighty, most
Gracious and most Merciful who gave me the health, strength and
patience to conduct this research.
Also I would like to express my deepest gratitude to my
supervisor Dr. Khogali A. Ishag for his persistent encouragement,
continuous support, advice and indispensable help throughout this work.
His ever pleasant, realistic, elegant and respectful style of sighted
guidance has always been appreciated.
I would like to express my deep thanks and sincere gratitude and
indebtedness to my co-supervisor Dr. Abdallah A. Alsamad for his
guidance, encouragement, suggestions and continuous assistance
throughout this work.
Special thanks are due to Dr. M. Elmobark (Khartoum Gum
Arabic processing company Ltd.) for his keen guidance, suggestions and
advice throughout this study.
Thanks are also extended to the Department of Biochemistry,
Nutrition and Toxicology Veterinary Research Corporation especially
Dr. A. Shamat, Dr. S. Elbasheer, Rawda, Elfatih and all staff for helping
me to do some analytical measurements
I wish to thank teaching staff and members in the Department of
Food Science and Technology (Faculty of Agriculture, Khartoum
ii
University) for their continuous assistance, which went beyond the
official call. I wish to thank staff and members of the Central Lab
instruments especially Dr. El Fatih A. El Hassan, Nadia and Safa for
allowing me to use the UV and IR instruments.
I also wish to acknowledge with great appreciation the members
of the Industrial Research and Consultancy Centre department of
Nutrition especially Ustaza Magda, Abubaker, Yagub, Muawia and
Boles.
I am, most grateful for the members of the Pharmacy Department
(Industrial Research and Consultancy Centre) especially Ustaz Mubarak
Elsiddig, Ustaz M. Abbas Elkadro, Ustaz Rania Yossif and A. Hassan for
their Keen assistance during the practical phase of fractional
chromatographical techniques during this study. Special thanks also are
due to Dr. A. Mohamdein the head Department of Biochemical Society
for his help and all the staff of food quality control, National Laboratory
Centre (STAK) for carrying out HPLC analysis.
Thanks are also extended to staff of Wahag computer centre
especially Mr. Omer Elfadel for typing the manuscript and also to Ustaz
Iman and Mr. Ali for their help.
The financial support of Ustaz. Elsadig A. Makkein, Mr. A.
Abdulbast, Mr. A. H. Mohager, Mr. Elzaki, Mr.Salih H. Makkein, Mr.A.
Abdelkrim and Eastern Kordofan Corporation for Development, and
Kordofan University is acknowledged and greatfully appreciated.
iii
LIST OF CONTENTS
DEDICATION
ACKNOWLEDGEMENT
LIST OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
ABSTRACT
ARABIC ABSTRACT
CHAPTER ONE: GENERAL INTRODUCTION
CHAPTER TWO: LITERATURE REVIEW
2.1 Plant gums
2.2 Gum from Acacia polyacantha tree
2.2.1 Classification of Acacia polyacantha
2.2.2 General distribution
2.2.3 Description
2.2.4 Uses
2.3 Structure of plant gums
2.4 Application of plant gums
2.4.1 Application in the food industry
2.4.2 Pharmaceutical and cosmetic applications
2.4.3 Paints and coating composition application
2.4.4 Other industrial uses
2.5 Physicochemical properties of gums
2.5.1 Solubility
2.5.2 Colour
2.5.3 Shape
2.5.4 Moisture
2.5.5 Ash
2.5.6 Nitrogen
2.5.7 Specific rotation
2.5.8 Viscosity
2.5.9 Acidity and pH measurements
2.5.10 Equivalent weight and uronic acid anhydride
2.5.11 Molecular weight determination
2.6 Functional properties
2.6.1 Emulsifying properties
2.6.2 Water holding capacity (WHC)
2.7 Analytical studies of plant gums
2.7.1 Purification of gums
2.7.2 Fractionation
2.7.2.1 Preparative fraction
2.7.2.2 Analytical fractionation
2.7.2.3 Enzymatic fractionation
2.7.2.4 Immunological fractions
iv
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2.7.3
2.7.4
Electrophoresis
Hydrolytic methods of analysis
2.7.4.1 Partial acid hydrolysis
2.7.4.2 Auto hydrolysis
2.7.5 Chromatographic methods for Analysis of Polysaccharides
2.7.5.1 Paper chromatography
2.7.5.2 Thin layer chromatography
2.7.5.3 Column chromatography (CC)
2.7.5.4 Gas liquid chromatography (GLC)
2.7.5.5 High performance liquid chromatography (HPLC)
2.7.6 Spectrophotometery and spectroscopy
2.7.6.1 Absorption spectroscopy
2.7.6.2 Ultraviolet (UV)
2.7.6.3 Infra red Spectroscopy (IR).
CHAPTER THREE: MATERIALS AND METHODS
3.1 Material
3.2 Preparation of samples
3.3 Analytical methods
3.3.1 Moisture content
3.3.2 Total ash
3.3.3 Nitrogen and Protein contents
3.3.4 Specific optical rotation
3.3.5 Determination of viscosity
3.3.6 Molecular weight
3.3.7 pH measurement
3.3.8 Apparent equivalent weight
3.3.9 Uronic acid anhydride
3.3.10 Determination of reducing sugars content
3.3.11 UV Absorption spectra
3.3.12 Analysis for inorganic matter
3.4 Functional properties
3.4.1 Emulsifying stability
3.4.2 Water holding capacity (WHC)
3.5 Structural analysis
3.5.1 Acid hydrolysis
3.5.2 Chromatographic method
3.5.2.1 Paper chromatography (PC)
3.5.2.2 Analysis of sugar composition using high
performance liquid chromatography (HPLC).
3.5.3 Fractionation
3.5.4 Infrared
3.6 Conductivity
3.7 Statistical analysis
v
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CHAPTER FOUR: RESULTS AND DISSCUSSION
4.1 Physical properties of A. polyacantha gum
4.1.1 Shape and colour
4.1.2 Specific optical rotation
4.1.3 Intrinsic viscosity
4.1.4 Refractive index
4.2 Functionality
4.2.1 Emulsifying stability (ES)
4.2.2 Water holding capacity (WHC)
4.3 Chemical properties of A. polyacantha gum
4.3.1 Moisture content
4.3.2 Ash content
4.3.3 Nitrogen and protein contents
4.3.4 Reducing sugars
4.3.5 Uronic acid content
4.3.6 pH value
4.3.7 Equivalent weight
4.3.8 Molecular weight
4.4 Minerals
4.4.1 Sodium
4.4.2 Potassium
4.4.3 Calcium
4.4.4 Magnesium
4.4.5 Iron
4.5 Characterization of A. polyacantha gum fractions
4.6 Sugar composition of A. polyacantha gum
4.7 U.V. absorption
4.8 Infra-red (IR) spectral analysis
CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS
5.1 Conclusions
5.2 Recommendations
REFERENCES
APPENDICES
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LIST OF TABLES
Table
Page
1
Physical properties of A.polyacantha gum
46
2
Chemical properties of A.polyacantha gum
50
3
Minerals content (µ g/g) of A. polyacantha gum
55
4
Physicochemical properties of A. polyacantha gum
fractions
58
Some physical properties of A.polyacantha gum
fractions
60
Sugars content of A. polyacantha gum (mg/100gl).
63
5
6
vii
LIST OF FIGURES
Figure
Page
1
The “wattle blossom” model of the arabinogalacto-protein
21
2
Molecular weight distribution of Acacia polyacantha (S4)
22
3
Molecular weight distribution of Acacia polyacantha (S5)
22
4
Molecular weight distribution of Acacia polyacantha (S14)
22
5
Molecular weight distribution of F1 Acacia polyacantha (S14)
23
6
Molecular weight distribution of F2 Acacia polyacantha (S14)
23
7
Molecular weight distribution of F3 Acacia polyacantha (S14)
23
8
FT.IR spectrum of Sterculia setigera gum sample collected from
Southern Kordofan
31
FT.IR spectrum of Acacia seyal sample collected from Southern
Kordofan
32
FT.IR spectrum of A. senegal gum obtained from Gum Arabic
Company.
33
UV absorption spectra of A. polyacantha gum collected from
Kadogli
65
UV absorption spectra of A. polyacantha gum collected from
Edamazine
66
13
UV absorption spectra of fraction 1 of A. polyacantha gum
67
14
UV absorption spectra of fraction 2 of A. polyacantha gum
68
15
FT.IR spectrum of A. polyacantha gum sample collected from
Kadogli.
69
FT.IR spectrum of A. Polyacantha gum sample collected from
Eldamazine
70
17
FT.IR spectrum of fraction 1 of A. Polyacantha gum sample
71
18
FT.IR spectrum of fraction 2 of A. polyacantha gum sample
72
9
10
11
12
16
.
viii
ABSTRACT
Fourty samples of A. polyacantha gum were collected as natural
exudate the inform nodules from A. polyacantha trees from Kadogli
forests (South Kordofan State) and Eldamazine (Blue Nile State) during
the season 2005/2006. Twenty samples were collected from each site.
The gum samples were subjected for physicochemical and functional
tests.
The mean specific rotation of Kadogli samples was -19.6°, while
that of Eldmazine was -14°, intrinsic viscosities were 9.9 and 10.2 ml/g
for Kadogli and Eldamazine samples, respectively. Refractive indices of
all samples from the two different locations showed the same value of
1.3354.
The mean value of the emulsifying stability of samples from
Kadogli was 0.99200 while that from Eldamazine was higher (1.06540).
The water holding capacities of all tested samples were in the range
63.0 - 63.9%.
The two sources of samples gave approximately the same average
moisture (10.5%) and ash (3.4%) contents. Nitrogen content of Kadogli
samples was 0.30 to 0.42% (1.88 to 2.63 % protein), while that of
Eldamazine samples was 0.36 to 0.48% (2.30 to 2.90% protein). The
mean concentration of reducing sugars was 0.23 and 0.16% for Kadogli
and Eldamazine samples, respectively. Uronic acid contents of Kadogli
samples ranged from 12.02% to 17.30 % and that of Eldamazine samples
ranged from 12.10% to 19.48%. Location significantly affected uronic
ix
acid content. The mean pH value for Kadogli samples (4.96) was lower
than that for Eldamazine samples (5.23).
The mean value of the equivalent weight for gum from Kadogli
(1397.90) was found to be higher than the value of 1280.20 for
Eldamazine samples. On the other hand, Eldamazine samples gave lower
average molecular weight of 3.20×103 compared to value of 3.29×103 for
Kadogli samples.
With respect to the mineral contents, gum samples from the two
sources showed the same range of 35 to 70 µg/g sodium. Whereas gum
samples from Kadogli recorded higher potassium in the range of 0.075 to
0.135 µg/g, compared to the range of 0.025 to 0.095µg/g related to gum
samples from Eldamazine. Calcium content was 0.330 - 0.666 µg/g
without significant differences between the two locations. Gum samples
from Kadogli and Eldamazine were found to contain 0.165 - 0.291 and
0.176 - 0.263 µg/g Mg, 20.3 to 28.1 and 20.1 - 56.2 µg/g Fe, respectively.
Solutions of fractions 1 and 2 showed -22o and -16o specific
rotation, 1345.11 and 1530.20 equivalent weight, 3.37×103 and 2.77×103
molecular weight, 10.5 and 9.43 ml/g Intrinsic viscosity, respectively.
Interestingly the two fractions gave the same refractive index of 1.3354.
Fraction 1 showed significantly (p ≤ 0.05) lower moisture, ash and
nitrogen (6.6%, 2.67 and 0.35%, respectively) compared to Fraction 2
(15.45%, 3.75 and 0.38%, respectively). On the other hand Fraction 2
showed significantly (P≤ 0.05) lower uronic acid, pH, Emulsifying
stability and conductivity (12.11%, 2.9, 0.83432 and 0.728µ/s,
x
respectively) compared to Fraction 1 (14.2%, 4.7, 1.11211 and 151.21
µ/s, respectively).
The sugar components of the whole gum and its fractions were
determined in acid hydrolysated by chromatography. L+arabinose,
L+rhaminose
and
D-galacturonic
observations
were
confirmed
acid
by
were
high
detectable.
These
performance
liquid
chromatography (HPLC) and showed that A. polyacantha gum contained
1.5 and 0.49 mg/100g L+Rhaminose and L+Arabinose, respectively. On
the other hand, L+Rhaminose was found to be 7.98 and 8.1 mg/100g for
Fraction1 and Fraction2, respectively. The two fractions showed negative
result for L+Arabinose, D-Glucose and D-Mannose.
The spectral measurements of ultra violet (UV) and infra red (IR)
were scanned for gum samples and their fractions.
xi
‫ﺍﻟﺨﻼﺼﺔ‬
‫ﺘﻡ ﺠﻤﻊ ‪ 40‬ﻋﻴﻨﺔ ﻤﻥ ﺼﻤﻎ ﺍﻟﻜﺎﻜﻤﻭﺕ ﺍﻟﻨﺎﺘﺞ ﺒﺼﻭﺭﺓ ﻁﺒﻴﻌﻴﺔ ﻤﻥ ﻤﻨﻁﻘﺘﻴﻥ ﻤﺨﺘﻠﻔﺘﻴﻥ‬
‫ﻓﻲ ﺍﻟﺴﻭﺩﺍﻥ ﻫﻤﺎ ﻜﺎﺩﻭﻗﻠﻲ ﻭﺍﻟﺩﻤﺎﺯﻴﻥ )ﻤﻭﺴﻡ ‪ (2006/2005‬ﻋﺸﺭﻭﻥ ﻋﻴﻨـﺔ ﻟﻜـل ﻭﺃﺠﺭﻴـﺕ‬
‫ﺩﺭﺍﺴﺔ ﺘﺤﻠﻴﻠﻴﺔ ﻋﻠﻰ ﺍﻟﻌﻴﻨﺎﺕ ﻟﺘﻭﻀﻴﺢ ﺍﻟﺨﻭﺍﺹ ﺍﻟﻌﺎﻤﺔ ﺍﻟﻔﻴﺯﻴﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﻭﻜﺎﻥ ﻤﺘﻭﺴﻁ ﺍﻟﺩﻭﺭﺍﻥ‬
‫ﺍﻟﻨﻭﻋﻲ ﻟﻠﻀﻭﺀ ﺍﻟﻤﺴﺘﻘﻁﺏ‪ 19.6°-‬ﻭ‪ 14 °-‬ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ ﻭ ﺍﻟﻠﺯﻭﺠﺔ ﺍﻟـﻀﻤﻨﻴﺔ ‪ 9.9‬ﻭ ‪10.2‬‬
‫ﻤل‪/‬ﺠﺭﺍﻡ ﻟﻜل ﻤﻥ ﻜﺎﺩﻭﻗﻠﻲ ﻭﺍﻟﺩﻤﺎﺯﻴﻥ ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ ﺒﻴﻨﻤﺎ ﻤﻌﺎﻤل ﺍﻻﻨﻜﺴﺎﺭ ﺃﻋﻁﻲ ﻗﻴﻤـﺔ ﺜﺎﺒﺘـﺔ‬
‫‪ 1.3354‬ﻟﻜل ﺍﻟﻌﻴﻨﺎﺕ‪ .‬ﻤﺘﻭﺴﻁ ﺍﻟﻘﺩﺭﺓ ﺍﻻﺴﺘﺤﻼﺒﻴﺔ ﻓﻲ ﻋﻴﻨﺎﺕ ﻜﺎﺩﻗﻠﻲ ﻜﺎﻥ ‪ 1.06540,‬ﺒﻴﻨﻤـﺎ‬
‫ﻜﺎﻥ ﻋﺎﻟﻴﹰﺎ ﻓﻲ ﻋﻴﻨﺎﺕ ﺍﻟﺩﻤﺎﺯﻴﻥ )‪ .(0.99200‬ﻗﺩﺭﺓ ﺍﻻﺤﺘﻔﺎﻅ ﺒﺎﻟﻤﺎﺀ ﻟﻜل ﺍﻟﻌﻴﻨﺎﺕ ﺘﺤﺕ ﺍﻟﺩﺭﺍﺴـﺔ‬
‫ﻜﺎﻥ ﻓﻲ ﺍﻟﻤﺩﻯ ‪ %63.9-63.0‬ﻟﻜل ﻤﻥ ﻜﺎﺩﻭﻗﻠﻲ ﻭﺍﻟﺩﻤﺎﺯﻴﻥ ﻋﻠـﻲ ﺍﻟﺘـﻭﺍﻟﻲ‪ .‬ﺍﻟﻌﻴﻨـﺎﺕ ﻤـﻥ‬
‫ﺍﻟﻤﺼﺩﺭﻴﻥ ﺃﻅﻬﺭﺕ ﺘﻘﺭﻴﺒﹰﺎ ﻨﻔﺱ ﻤﺘﻭﺴـﻁ ﺍﻟﺭﻁﻭﺒـﺔ )‪ (%10.5‬ﻭﺍﻟﺭﻤـﺎﺩ )‪ ،(%3.4‬ﻤﺤﺘـﻭﻯ‬
‫ﺍﻟﻨﺘﺭﻭﺠﻴﻥ ﻜﺎﻥ ‪ 0.30‬ﺇﻟﻰ ‪ 1.88) %0.42‬ﺇﻟﻲ ‪ %2.63‬ﺒﺭﻭﺘﻴﻥ( ﻟﻜﺎﺩﻭﻗﻠﻲ ﺒﻴﻨﻤﺎ ﻜـﺎﻥ ‪0.36‬‬
‫ﺇﻟﻲ ‪ 2.30) %0.48‬ﺇﻟﻲ ‪ %2.90‬ﺒﺭﻭﺘﻴﻥ( ﻟﻠﺩﻤﺎﺯﻴﻥ‪ .‬ﻤﺘﻭﺴﻁ ﺘﺭﻜﻴﺯ ﺍﻟﺴﻜﺭﻴﺎﺕ ﺍﻟﻤﺨﺘﺯﻟﺔ ﻟﻜل‬
‫ﻤﻥ ﻜﺎﺩﻭﻗﻠﻲ ﻭﺍﻟﺩﻤﺎﺯﻴﻥ ﻜﺎﻥ ‪ 0.23‬ﻭ‪ %0.16‬ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ‪ .‬ﺤﻤﺽ ﺍﻟﻴﻭﺭﻨﻴﻙ ﻴﺘﺭﺍﻭﺡ ﺒـﻴﻥ‬
‫‪ %12.02‬ﺇﻟﻰ ‪ %17.30‬ﻭ‪ 12.10‬ﺇﻟﻲ ‪ %9.48‬ﻟﻜل ﻤﻥ ﻜﺎﺩﻭﻗﻠﻲ ﻭﺍﻟﺩﻤﺎﺯﻴﻥ ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ‪.‬‬
‫ﻤﺘﻭﺴﻁ ﺍﻷﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ ﻟﻌﻴﻨﺎﺕ ﻜﺎﺩﻭﻗﻠﻲ‪ 4.96‬ﻜﺎﻥ ﺃﻗل ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻟﺩﻤﺎﺯﻴﻥ ‪،5.23‬‬
‫ﺃﻅﻬﺭﺕ ﻋﻴﻨﺎﺕ ﻜﺎﺩﻭﻗﻠﻲ ﻭﺯﻥ ﻤﻜﺎﻓﺊ ‪ 1397.90‬ﺃﻋﻠﻲ ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻟﻘﻴﻤـﺔ ‪ 1280.20‬ﻟﻌﻴﻨـﺎﺕ‬
‫ﺍﻟﺩﻤﺎﺯﻴﻥ ﺒﻴﻨﻤﺎ ﻜﺎﻥ ﻤﺘﻭﺴﻁ ﺍﻟﻭﺯﻥ ﺍﻟﺠﺯﺌﻲ ﻟﻌﻴﻨﺎﺕ ﺍﻟﺩﻤﺎﺯﻴﻥ ﺃﻗل ﻤـﻥ ﺍﻟﻘﻴﻤـﺔ‬
‫‪3‬‬
‫‪3.29 × 10‬‬
‫ﻟﻌﻴﻨﺎﺕ ﻜﺎﺩﻭﻗﻠﻲ‪.‬‬
‫ﺒﺎﻟﻨﺴﺒﺔ ﻟﻠﻌﻨﺎﺼﺭ ﺍﻟﻤﻌﺩﻨﻴﺔ ﺃﻅﻬﺭﺕ ﺍﻟﻌﻴﻨﺎﺕ ﻤـﻥ ﺍﻟﻤـﺼﺩﺭﻴﻥ ﻨﻔـﺱ ﺍﻟﻤـﺩﻱ ‪70-35‬‬
‫ﻤﻴﻜﺭﻭﺠﺭﺍﻡ‪/‬ﺠﺭﺍﻡ ﺼﻭﺩﻴﻭﻡ ﺒﻴﻨﻤﺎ ﻋﻴﻨﺎﺕ ﺍﻟﺼﻤﻎ ﻤﻥ ﻜﺎﺩﻭﻗﻠﻲ ﺃﻅﻬﺭﺕ ﻤـﺴﺘﻭﻱ ﻋـﺎﻟﻲ ﻤـﻥ‬
‫ﺍﻟﺒﻭﺘﺎﺴﻴﻭﻡ ﺘﺘﺭﺍﻭﺡ ﺒﻴﻥ ‪ 0.135 – 0.075‬ﻤﻴﻜﺭﻭﺠﺭﺍﻡ‪/‬ﺠﺭﺍﻡ ﻤﻘﺎﺭﻨـﺔ ﻤـﻊ ﺍﻟﻤـﺩﻱ ‪– 0.025‬‬
‫‪ 0.095‬ﻤﻴﻜﺭﻭﺠﺭﺍﻡ‪/‬ﺠﺭﺍﻡ ﻟﻌﻴﻨﺎﺕ ﺍﻟﺩﻤﺎﺯﻴﻥ‪.‬‬
‫ﻴﺘﺭﺍﻭﺡ ﻤﺴﺘﻭﻱ ﺍﻟﻜﺎﻟﺴﻴﻭﻡ ﻟﺠﻤﻴﻊ ﺍﻟﻌﻴﻨﺎﺕ ﺒﻴﻥ ‪ 0.330‬ﺇﻟﻲ ‪ 0.666‬ﻤﻴﻜﺭﻭﺠﺭﺍﻡ‪/‬ﺤـﺭﺍﻡ‬
‫ﺒﺩﻭﻥ ﻭﺠﻭﺩ ﻓﺭﻭﻗﺎﺕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺍﻟﻤﻭﻗﻌﻴﻥ‪ .‬ﻭﺠﺩ ﺃﻥ ﻋﻴﻨﺎﺕ ﺍﻟﺼﻤﻎ ﻤﻥ ﻜـﺎﺩﻭﻗﻠﻲ ﻭﺍﻟـﺩﻤﺎﺯﻴﻥ‬
‫ﺘﺤﺘﻭﻱ ﻋﻠﻰ ‪ 0.291 – 0.165‬ﻭ‪ 0.263 – 0.176‬ﻤﻴﻜﺭﻭﺠﺭﺍﻡ‪/‬ﺤـﺭﺍﻡ ﻤـﺎﻏﻨﺯﻴﻭﻡ‪-20.3 ،‬‬
‫‪ 28.1‬ﻭ‪ 56.2-20.1‬ﻤﻴﻜﺭﻭﺠﺭﺍﻡ‪/‬ﺤﺭﺍﻡ ﺤﺩﻴﺩ‪ ،‬ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ‪.‬‬
‫‪xii‬‬
‫ﻋﻨﺩ ﺘﺠﺯﺌﺔ ﺍﻟﺼﻤﻎ ﺇﻟﻲ ﻤﺠﺯﺌﻴﻥ ‪ 1‬ﻭ‪ 2‬ﺃﻅﻬﺭ ﺍﻟﻤﺠﺯﺌﻴﻥ ‪ 22-‬ﻭ‪ o16-‬ﺩﻭﺭﺍﻥ ﻨـﻭﻋﻲ‪،‬‬
‫‪ 1345.11‬ﻭ‪ 1153.20‬ﻭﺯﻥ ﻤﻜــﺎﻓﺊ‪ 310 × 3.37 ،‬ﻭ ‪ 310 × 2.77‬ﻭﺯﻥ ﺠﺯﻴﺌــﻲ‪10.5 ،‬‬
‫ﻭ‪ 9.43‬ﻤل‪/‬ﺠﺭﺍﻡ ﻟﺯﻭﺠﺔ ﻀﻤﻨﻴﺔ‪ ،‬ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ‪.‬‬
‫ﺃﻅﻬﺭ ﺍﻟﻤﺠﺯﺌﻴﻥ ﻨﻔﺱ ﻗﻴﻤﺔ ﻤﻌﺎﻤل ﺍﻻﻨﻜﺴﺎﺭ ‪ .1.3354‬ﻤﺠﺯﺃ ‪ 1‬ﻭﺠﺩ ﺃﻨﻪ ﻴﺤﺘﻭﻱ ﻋﻠـﻰ‬
‫ﺭﻁﻭﺒﺔ ﻭﺭﻤﺎﺩ ﻭﻨﻴﺘﺭﻭﺠﻴﻥ )‪ 2.67 ،6.6‬ﻭ‪ (%0.35‬ﻋﻠﻰ ﺍﻟﺘـﻭﺍﻟﻲ ﺃﻗـل ﻤﻌﻨﻭﻴـﹰﺎ )‪(P<0.05‬‬
‫ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻟﻤﺠﺯﺃ ‪ 3.75 ،15.45) 2‬ﻭ‪ (%0.38‬ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ‪ .‬ﻤﻥ ﻨﺎﺤﻴﺔ ﺃﺨﺭﻱ ﺃﻅﻬﺭ ﺍﻟﻤﺠﺯﺃ‬
‫‪ 2‬ﻤﺤﺘﻭﻱ ﺤﻤﺽ ﻴﻭﺭﻨﻙ‪ ،‬ﺃﺱ ﻫﻴﺩﺭﻭﺠﻴﻨﻲ‪ ،‬ﺜﺒﺎﺕ ﺃﺴﺘﺤﻼﺒﻲ ﻭﻤﻭﺼﻠﻴﺔ ﻜﻬﺭﺒﻴـﺔ )‪،%12.11‬‬
‫‪ 0.83432 ،2.9‬ﻭ‪ 0.728‬ﻤﻴﻜﺭﻭﺴﻤﻨﺱ‪ ،‬ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ(‪.‬‬
‫ﺘﻡ ﺘﺤﺩﻴﺩ ﺍﻟﺴﻜﺭﻴﺎﺕ ﺍﻟﻤﻭﺠﻭﺩﺓ ﻓﻲ ﺍﻟﺼﻤﻎ ﻭﺍﻟﻤﺠﺯﺁﺀﺕ ﺒﺎﻟﻜﻤﻭﺘﻐﺭﺍﻓﻴﺎ ﻭﺃﻅﻬﺭ ﻭﺠﻭﺩ ﻜل‬
‫ﻤﻥ ل‪-‬ﺃﺭﺍﺒﻴﻨﻭﺯ‪ ،‬ل‪-‬ﺭﺍﻤﻴﻨﻭﺯ ﻭﺩﻱ – ﺤﻤﺽ ﺠﻼﻜﺘﻭﺭﻭﻨﻙ‪ .‬ﻫﺫﻩ ﺍﻟﻨﺘﺎﺌﺞ ﺘﻡ ﺍﻟﺘﺄﻜﺩ ﻤﻨﻬﺎ ﺒﺎﺴﺘﺨﺩﺍﻡ‬
‫ﻜﺭﻭﻤﺎﺘﻐﺭﺍﻓﻴﺎ ﺍﻟﺴﺎﺌل ﻋﺎﻟﻰ ﺍﻷﺩﺍﺀ )‪ (HPLC‬ﻭﺍﻟﺫﻱ ﺃﻭﻀﺢ ﺃﻥ ﺼﻤﻎ ﺍﻟﻜﺎﻜﻤﻭﺕ ﻴﺤﺘﻭﻱ ﻋﻠـﻰ‬
‫‪ 1.5‬ﻭ‪ 0.49‬ﻤﻠﺠﺭﺍﻡ‪ 100/‬ﺠﺭﺍﻡ ل‪-‬ﺭﺍﺒﻴﻨﻭﺯ ﻭل‪-‬ﺭﺍﻤﻴﻨﻭﺯ ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ‪.‬‬
‫ﻤﻥ ﻨﺎﺤﻴﺔ ﺃﺨﺭﻱ ﻜﺎﻥ ﻤﺴﺘﻭﻱ ل‪-‬ﺭﺍﻤﻴﻨﻭﺯ ﻓﻲ ﺍﻟﻤﺠﺯﺃ ‪ 1‬ﻭ‪ 7.98) 2‬ﻭ ‪ 8.1‬ﻤﻠـﻡ‪100/‬‬
‫ﺠﺭﺍﻡ ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ(‪ .‬ﻜﻤﺎ ﺃﻥ ﺍﻟﻤﺠﺯﺌﻴﻥ ﺃﻅﻬﺭﺍ ﻨﺘﻴﺠﺔ ﺴﺎﻟﺒﺔ ﻟﻠﺴﻜﺭ ل‪-‬ﺍﺭﺍﺒﻴﻨﻭﺯ‪.‬‬
‫ﻓﻲ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻴﻀﹰﺎ ﺘﻡ ﺍﻟﻘﻴﺎﺱ ﺍﻟﻁﻴﻔﻲ ﻟﻁﻴﻑ ﺍﻷﺸﻌﺔ ﻓﻭﻕ ﺍﻟﺒﻨﺴﻔﺠﻴﺔ )‪ (UV‬ﻭﺘﺤـﺕ‬
‫ﺍﻟﺤﻤﺭﺍﺀ )‪ (IR‬ﺒﺎﻟﻨﺴﺒﺔ ﻟﻠﺼﻤﻎ ﻭﺍﻟﻤﺠﺯﺁﺀﺍﺕ‪.‬‬
‫‪xiii‬‬
CHAPTER ONE
GENERAL INTRODUCTION
Although there are more than 1100 species of Acacias botanically,
known distributed throughout the tropical and subtropical areas of the
world, most commercial gum Arabic is derived from Acacia senegal
locally known as hashab gum (in the Sudan) and as Kordofan gum in the
world. Gum Arabic has been known for many thousands of years and
there are no artificial substitutes that match it for quality or cost of the
production (Gabb, 1997). Chemically, gum Arabic consists mainly of
high-molecular weight polysaccharides made up of rhamnose, arabinose,
and galactose, glucuronic and 4-o-methoxylglucuronic acid, and the salts
of calcium, magnesium, potassium, and sodium of the two acids (Gabb,
1997).
The Sudanese, major gums of economic importance are gum
Arabic, gum talha and Acacia polyacantha gum. The source of gum
Arabic is Acacia senegal var senegal. A. polyacantha exudates are
closely related to, and can hardly be distinguished from Acacia senegal
exudates unless recognized by acknowledged gum expert or by studying
the physico-chemical characteristics. The two species, Acacia senegal
and Acacia polyacantha belong to the same group known as Acacia
senegal complex. All gum exudates, from this group of Acacia species,
have a laevorotatory (-ve) specific rotation in contrast to the Acacia seyal
complex which produce gum exudates, that have a dextrorotary (+ve)
specific rotation, other structural, botanical characteristics are noticeable
even with in the same species. Most of the research work is directed
1
towards gum Arabic and to a lesser extent towards gum talha.
Regrettably the A.polyacantha gum and all other gum resource from
Acacia species received very little attention. The objectives of this study
are:
1.
Determination of the physico-chemical properties of the gums
from A. polyacantha trees.
2.
Study the functional properties of the whole gum and different
fractions of the gum.
2
CHAPTER TWO
LITERATURE REVIW
2.1 Plant gums
Plant gums are organic substances obtained as an exudation from
trunks, or branches of trees, spontaneously or after mechanical injury of
the plant by incision of the bark, or by the removal of a branch, or after
invasion by bacteria or fungi (Smith,1949).The term gum often describes
materials which affect sense of touch, taste and sight in measure summed
up as property of gummosis” which is difficult to define but visual and
manual examination of the material may cause the observer, to call it
gum (Mantell, 1947). Gum refers to any polysaccharide that is
dispersible in water to give viscous solutions, gels or colloidal
dispersions. Generally gums are long chain high molecular weight
polymers that dissolve or disperse in water to give thickening or gelling
effect and exhibit related secondary functional properties, such as
emulsification, stabilization, and encapsulation (Sharma, 1981). Gums or
hydrocolloids are mainly long–chain, straight to branched polysaccharides that contain hydroxyl groups that can bond to water
molecules. These chains consist of 2×10³ to 1084 monosaccharide units.
The sugar monomers can contain linked side unite, or substituent groups,
such as sulphate, methyl, ether, ester and acetals (Kuntz, 1990). Gums
composed mainly of C, H, O, and N elements; and acidic gums (eg.gum
Arabic) contain mainly Ca, Mg, Na and Fe as cations (Jones et al., 1958).
3
2.2 Gum from Acacia Polyacantha Tree.
2.2.1 Classification of Acacia polyacantha tree
Family:
Leguminosae
Subfamily:
Mimosaceae
Genus:
Acacia
Species:
Polyacantha
English name:
Flacons claw acacia
Arabic name:
Kakamut, Umsinina (Amin, 1977 and Voget, 1995).
2.2.2 General distribution.
Kakamut is dispersed through out tropical Africa. In Sudan there
are several regional varieties, which usually occur along rivers and
valleys where the water table is fairly high, and soils are good (Voget,
1995).
2.2.3 Description
The tree, occasionally, reaches 20 m in height and the trunk can be
70 cm in diameter. A knobbly bark and paired thorns are it's most
conspicuous features. The bark is yellowish with brown scale and thorn.
Thorns occur in pairs and are sharply curved; they are brown with black
tips. Leaves may reach 25 cm in length, biparipinnate with 10-40 pairs of
pinnate and 35-60 of leaflets each. A prominent gland is present at the
leave base. Flowers occur in pairs or 3 spicate racemes from the leaf
axial and are cream colored and strongly secented. Fruits consist of pods
up to 15cm long, which each contain 5-9 seeds (Voget, 1995).
4
2.2.4 Uses
The wood is used mainly in fuel and charcoal of good quality,
fence posts, farm implements, and railway, sleeper, beams and; rafters.
The gum is edible and is used as adhesive in the treatment of textile
fibers. The roots are used to act as general health tonic as antidote for
snake bite, and cure for venereal diseases. A preparation from the bark is
used for general stomach disorders (Voget, 1995).
2.3 Structure of plant gums
Gum nodules contain polysaccharide material of complex nature
usually contaminated with impurities such as bark fragments, entrapped
dust and insects. Inert pertinacious material and a few amounts of
terpenoid resins can also be present. Gums are polyuronides; the uronic
acid residues may carry acetyl or methyl groups and, generally, occur at
least in part as methyl groups and generally occur, at least in part, as
metallic salts. The hexose residues are present in the pyranose
configuration, while the pentose residues occur in the furanose (Stephen
et al., 1955 and 1957) Beside the foregoing gums, others have been
studied; khaya senegalese gum contains galactose, rhaminose and
probably 4-O-methyl, D-glucuronic acid and galactouronic acid (Aspinal
et al., 1956). Sterculia termentosa gum contains rhminose, galactose and
probably galacturonic acid, Olibanum gum (Boswellia carterii) was
found to be of an arabino-galactan and a polysaccharide containing
galactose and galactouronic acid (Elkhatem et al., 1956). It was noted
that the gum was very heterogeneous and it has been described as
heteropolymolecular, i.e. having either a variation in monomer
5
composition and/or a variation in the mode of linking and branching of
the monomer unites, in addition to distribution in molecular weight
(Lewis and Smith, 1957; Dermyn, 1962 and Stoddart, 1966). According
to Philips (1988) and Williams (1989), fractionation by hydrophilic
affinity chromatography revealed that Acacia Senegal gum consists of at
least three distinct components. Fraction 1 AG (arabino galactan),
fraction 2 AGP (arabino galactan-protein) and fraction 3 GP
(galactoprotein). But even those contain a range of different molecular
weight components revealing the polydiverse nature of the gum (Osman,
1994). Fraction 1 containing 88% of the total has only small amount of
protein content. Fraction 2 represents 10% of the total and had 12%
protein content. Fraction 3 resembles 1.24% of the total but contains
almost 50% of protein AGP is responsible for the emulsifying properties
of gum Arabic (Williams, 1989, and Phillips, 1988). No mention has
been made to detailed comparison between the structures of gums from
different species of trees, but is believed that D-galactose and uronic acid
residues generally constitute the backbone of gum polysaccharide with 13 and 1-6 linkages predominating side chain are characterized by the
presence of D-xylopyranose, L-arabinose, and L-arabino-furanose
linkage.
2.4 Applications of plant gums
The solubility and viscosity of gum are the most fundamental
properties, which make it unique among polysaccharides, the majority of
gums dissolve in water at different concentrations, and such properties
are exploited in many applications.
6
2.4.1 Applications in the food industry
Gums for their high viscosity in solutions and inability to
crystallize, are particularly suited to serve in foodstuff such as: thickeners
for beverages, stabilizers for oil and water emulsions and as wider
application where function is to prevent agglomeration and setting of
minute particles. They are also used to incorporate flavors in
confectionery such as pastilles and gum drops, and the preparation of
lozenges. The role of gum Arabic in confectionary products is usually
either to prevent crystallization of sugar or to act as an emulsifier
(Glicksman et al., 1973).
2.4.2 Pharmaceutical and cosmetic applications
Gums are used as a suspending and emulsifying or binding agents
in pharmaceutical industries, it has been used in tablet manufacturing,
where it functions as a binding agent or as a coating prior to sugar
coating, some times in combination with other gums .A. polyacantha
gum used to act as general health tonic as antidote for snake bite, and
cure for venereal diseases. A preparation from the bark is used for
general stomach disorders (Voget, 1995).
2.4.3 Paints and coating composition application.
The hydrophilic colloids and modified cellulose find application in
paint industry because of their stabilizing effect on paint emulsions,
waxes and numerous others products. Gamble and Grady (1938) treated
pigments with water soluble hydrocolloids such as gum Arabic to add
controllable chemotropic properties to paints. The gum also finds
application in coating composition Horne et al. (1953) developed non7
glare coating based on a water soluble dye dissolved in gum Arabic
solutions.
2.4.4 Other industrial uses
Due to its adhesive properties gum have been used in the
manufacturing of adhesives for postage stamps and also in the
formulations of paints and inks. Gum may serve as a source of
monosaccharide, as e.g. mesquite gum (family prosopis) serve as a
source of L-arabinose (51%) because of its easier hydrolysis, and
availability of the gum in large quantities. The mesquite gum can be
dialyzed by addition of ethanol (White, 1947 and Hudson, 1951), or
alternatively, isolated by crystallization from methanol after removal of
acidic oligosaccharides on ion exchange resin or precipitated by barium
salts. Gums are widely used in textile industries to impart luster to certain
materials (silk), as thickeners for colors and mordant in calico printing
(Omer, 2004).
2.5 Physicochemical properties of gums
The physical properties of the natural gum are most important in
determining their commercial value and their use. These properties vary
with gums different botanical source, and even substantial differences in
gum from the same species when collected from plants growing under
different climatic conditions or even when collected from the plant at
different season of the year (Hirst et al., 1958). The physical properties
may also be affected by the age of the tree and treatment of the gum after
collection such as washing, drying, sun bleaching and storage
temperature.
8
2.5.1 Solubility
Gums can be classified into three categories with regard to their
solubilities:
1.
Entirely soluble gums: e.g. A. senegal, A. seyal.
2.
Partially soluble gums: e.g. Gatti gum.
3.
Insoluble gums: e.g. Tragacanth gum (Omer, 2004).
2.5.2 Colour
The colours of gums vary from water- white (colourless) through
shades of yellow to black. The best grades of gum are almost colourless
with slight traces of yellow; some possess pink likes (Siddig, 2003). On
the other hand dark or even black gums some times occur e.g. mesquite
gum. There are also the pale rose pinks, darker pink and yellowish gums.
The pink colour is probably due to the presence of different quantities of
tannin materials (Omer, 2004).
2.5.3 Shape
Natural gums are exuded in a variety of shapes and forms: usually
the fragments are irregularly globular or tear globular or tear shaped. The
best known being the tear or drop shape of various grades of gum Arabic.
Other shapes are flakes or threat like ribbons with gum tragacanth. The
surface is perfectly smooth when fresh but may become rough or crusty,
covered with small cracks (Omer, 2004).
2.5.4 Moisture
The hardness of gum would be determined by moisture content.
The moisture content of good quality gum dose not exceed 15 and 10%
9
for granular and spray dried material respectively (FAO, 1999). The
moisture content is weight lost due to the evaporation of water (Person,
1970). It shows the hardness of the gum and hence variability of
densities, the amount of densities, and the amount of the air entrapped
during formation. Omer (2004) recently, reported that the moisture
content of A.polyacantha gum to be around 8.2%.
2.5.5 Ash
Ash content is a measure of inorganic residue remaining after
organic mater has been burnt. The inorganic residues exist as elements.
Siddig (1996) explained that the type of the soil (clay or sand) affected
the ash content significantly; previously the ash content for A.
polyacantha gum was determined as 2.929 ash% (Anderson, 1985).
Recently (FAO, 1999), reported that the ash content of A. senegal gum is
not more than 4%.
2.5.6 Nitrogen
The role of nitrogen and nitrogenous component in the structure,
physicochemical properties and functionality of gum Arabic was recently
subjected to intensive investigation (Anderson. 1985; Gammon
et al., 1968). Dickinson (1988) studied the emulsifying behavior of gum
Arabic and concluded that there is strong correlation between the
proportion of protein in the gum and it is emulsifying stability. Idris
(1989) showed that the protein contents of fresh samples were fairly
constant (2%) irrespective of the age of the tree. Siddig (1996) reported
that the average value of nitrogen content of commercial samples of
Acacia senegal gum and 80% of authenticated samples analyzed were in
10
the range (0.27-0.39%). Recently Omer (2004) reported that the mean of
nitrogen content of A. polayacantha gum samples was 0.35%.
2.5.7 Specific rotation
The optical activity of organic molecules (saccharides and
carbohydrates) is related to their structure and a characteristic property of
the substance (Stevens et al., 1987). The gum of natural origin, e.g.
A. senegal gum, has the property of rotating the plane of the polarized
light. The direction of the rotation, as well as the magnitude is considered
as a diagnostic parameter (Biswas et al., 2000). Acacia senegal gum
gives a negative optical rotation ranging between -20˚to -34˚. The optical
rotation is used to differentiate between A. senegal and other botanically
related Acacia gums. Anderson and Stoddart, (1966b) reported that the
specific rotation for electrodialysed Acacia senegal gum was -31, 5˚.
Pure gum from A. senegal has specific optical rotation of -27˚ to -30˚
(Tioback, 1922). Certain variation in the degree of the optical rotation
(-27˚to-32˚) has been noticed by (Anderson, 1968). Karamalla et al.
(1998) found that the mean of the specific optical rotation of commercial
A. senegal gum was -30.54˚. The optical rotation is not affected by both
auto hydrolysis and variation, while mild acidic hydrolysis has a
significant effect on optical rotation (Barron, 1991). Omer (2004)
reported that the mean of specific rotation of authenticated samples of
A. polyacantha gum was -16.6˚.
2.5.8 Viscosity
The viscosity of liquid is its resistance to shearing, to stirring or to
flow through a capillary tube (Bancraft, 1932). Studies of flow of gum
11
solutions play an important role in identification and characterization of
their molecular structure. Since viscosity involves the size and the shape
of the macromolecule, it was considered as one of the most important
analytical and commercial parameter (Anderson et al., 1969). The
viscosity of a solution may have a complicated variation with
composition, due to the possibility of hydrogen bonding among the
solute and solvent molecules (Pimentel et al., 1960). More hydroxyl
groups makes high viscosities, because a network of hydrogen bonds is
formed between the molecules, this net work extends throughout the
liquid, thus making flow difficult. The viscosity of gum solutions is
inversely proportional to temperature. They also found that the viscosity
of gum Arabic solutions changes with pH, but they found a maximum
viscosity at pH 6-7. Viscosity can be explained in different terms such as
relative viscosity, specific viscosity, reduced viscosity, inherent viscosity
and intrinsic; it is also represented as kinematics or dynamic viscosity.
Anderson (1978) reported that the intrinsic viscosity of A. polyacantha
gum was 15.8 ml/g. Omer (2004) recently reported 10.34 ml/g intrinsic
viscosity for A. polyacantha gum.
2.5.9 Acidity and pH measurements
The hydrogen ion concentration is very important in chemistry and
industry of gums, therefore functional properties of gum are affected by
changes in pH e.g. viscosity, emulsifying power. Arabic acid substance is
the major component of commercial gum Arabic and when decomposed,
it gives arabinose, so that the gum Arabic is called Arabic acid and hence
the gum solution is moderately acidic (pH= 4.5). Concerning the pH of
12
A. polyacantha gum Siddig (2003) reported a value of (4.9) and Omer
(2004) reported 4.7-5.7 with a mean value of 5.2.
2.5.10 Equivalent weight and uronic acid anhydride
Titrable acidity, which is the number of mls of 0.02N sodium
hydroxide that neutralize 10 mls of 3% gum solution, represents the acid
equivalent weight of gum, from which the uronic acid content can be
determined (Karamalla, 1965; Jurasick, 1993). Uronic acids constitute a
major component of many natural polysaccharides. They are widely
distributed in animal and plant tissues. A number of methods have been
developed for determination of uronic acids. They include colorimetric,
decarboxylation and acid base titimetric methods. Gums differ widely in
their equivalent weight and uronic acid content (Karamalla, 1965).
Anderson (1965) reported that the equivalent weight and uronic acid
content of A. polyacantha gum are1900 and 9%, respectively.
2.5.11 Molecular weight
The molecular weight of the polymers can be determined from
physical measurement or by application of chemical methods. The
applications of chemical methods require that the structure of the
polymer should contain well known number of functional groups per
molecule and they invariably occur as end groups. The end group
analysis method gives an approximately number of molecules in a given
weight of sample; they yield the average number of molecules for
polymeric materials. This method becomes insensitive at high molecular
weight, as the fraction of end groups becomes too small to be measured
with precision (Meyer, 1971). This is due to the fact that fraudulent
13
sources of the end groups not considered in the assumed reaction
mechanism steadily become consequential as the molecular weight
increases and the number of end groups diminishes to such an extent
their quantities determination is not feasible. Those reactions confine
frequent application of chemical methods to condensation polymers with
average molecular weight seldom exceeding 2.5 ×10³ (Flory, 1953).
Physical methods frequently used for establishing polymer molecular
weight are osmometry, polymer viscosity, measurement of coefficient of
diffusion, ultra centrifugation and light scattering. One of the most recent
advanced methods is light scattering (LS), which provides an absolute
method for polymer molecular weight and size measurement. LS are
rapid, accurate and requires small amount of sample. The molecular
weight of gums varies greatly in values due to gum heterogeneity as well
as variation in techniques used to separate, purify and determine the
molecular weight. A 3.0 × 10³ was reported by Saverbon (1953) using
centrifugal method. Using the light scattering technique gave higher
values Veil and Eggenberger (1954) reported a Mw =1.0×106; Mukherjee
and Deb (1962) reported Mw up to 5.8 × 105 and Fenyo (1988) reported a
range of 4.0 × 106 to 2.2 × 106. Recently GPC coupled on line to multi
angle laser light scattering (MALLS) has been demonstrated to be a very
powerful method for characterizing highly polydisperse polymer systems
and the molecular weight of A. senegal gum was found to be equivalent
to 5.4 × 105 (Picton, 2000). The weight average molecular weight for A.
polyacantha gum using the GPC-MALLS was reported to range between
2.94 ×105 to 7.346 × 105 Omer (2004).The most recent E 414
specification of the (European Commission, 1996) has now re-focused
14
attention on the problem of molecular weight, since it has included in the
product definition, the requirement that the molecular weight should be
approximately 3.5 × 105 is far below the reported value for A. senegal var
senegal.
2.6 Functional properties
2.6.1 Emulsifying properties
Emulsions are chemical mixtures of liquids that are immiscible
under ordinary conditions, and which may be separated into two layers
on, standing, heating and freezing, by agitation and the addition of other
chemicals (Encyclopedia, 1966). The emulsifying agents act as surface –
active agents, which when added to an emulsion it would increase its
stability by interfacial action, each emulsifying agent depends on its
action on different principle to achieve stable product. Gum Arabic is
used to stabilize flavor and oil emulsions in dried food mixes (such as
soup, cakes,…etc) and the soft drinks industry, where the gum is required
to stabilize a concentrated oil emulsion (about 20%) for long periods and
also to continue to stabilize following dilution prior to bottling (Islam
et al., 1997). An emulsifying agent is, usually a long – chain organic
compound that has producing chains that are soluble in oil (lipophilic) as
well as side chains or groups that are soluble in water (hydrophilic). Thus
one portion of each molecule dissolve in the water phase while another
portion dissolves in the oil phase and the main chain forms a link or
bridge to keep both phases in position and there by emulsified. Gum
Arabic produces highly stable emulsions making it very useful in the
preparation of oil in water food flavour emulsions particularly for citrus
15
oils (Randall et al., 1988). Some believe that gums are not true
emulsifiers. That is, they do not act by means of hydrophilic chemical
functionality; they perform as emulsion stabilizers or protectors. Their
function is essentially to increase the viscosity of the aqueous phase by
thickening it so that it approximates or slightly exceeds that of the oil. In
this way the tendency of the dispersed phase to slip or coalesce is
minimized, and the emulsion, is so to be, stabilized. Such stabilization is
a protective effect based on thickening properties of the gums. (Randall
et al., 1988) studied the effect of heat on the emulsification action,
stability of the gum followed by changes in the GPC profile of the gum.
He concluded that heating at 100ºC for 3 hrs, results in a decreases in the
intensity of the high molecular mass peak with a corresponding increase
in the intensity of the lower molecular mass peaks. Continuous heating
leads to further loss of the high molecular mass fraction and loss in the
emulsifying stability of the gum. Chikamai et al. (1993) reported that
heating solutions at 100ºC for more than six hours causes significant loss
of emulsification properties; where as, heating at 60ºC for 24 hrs has only
a minor effect. Dickinson et al. (1988) studied the surface and
emulsifying properties of six Acacia gum samples and stated that the
relationship between nitrogen content and emulsifying properties of the
gum samples, depend not only on their total protein content, but also on
the distribution of the protein/peptide between the low and high
molecular weight fractions, and on the molecular accessibility of the
protein/peptide for adsorption. Dickinson
et al. (1991) studied the
influence of the nature of the oil phase on emulsifying behavior of
gum Arabic. They found that gum lowered the surface tension at the
16
n-hexadecane-water interface also it gave the most stable n-hexadecane –
water emulsion is smallest droplets with all three oils (n-hexadecane and
orange oil). They also concluded that a high molecular weight fraction
(0.87 nitrogen ) corresponding to 10% of a natural gum (0.38% nitrogen)
gives initially larger droplets but better emulsion stability than the lowmolecular weight fraction (0.35%). In common with most emulsifiers,
the AGP complex has a hydrophilic region (protein) and hydrophilic
region (carbohydrate). During the formation of oil in water emulsions the
protein (arabinogalactan) protein products in to the water phase. The bulk
of gum Arabic in the form of free AG can improve stability by increasing
viscosity of the water (Islam et al., 1997). The relatively low protein
content of gum Arabic requires high concentration of gum in most
emulsification systems (Imerson, 1997). In this study a comparative
emulsifying property of A. polyacantha, A. senegal, and A. seyal gum
were carried out, effect of concentration, pH, addition of protein,
blending, temperature and time of stirring investigated.
2.6.2 Water holding capacity (WHC)
The water holding capacity is the ability of the material to hold
water against gravity (Hansen, 1978). Elamin (1972) reported that the
main value of A. senegal was 65.59%. Eltayeb (1999) reported a range of
63.77 – 65.6 % for A. senegal gum.
2.7. Analytical studies of plants gum
2.7.1 Purification of gums
Pure polysaccharides can be obtained from crude sodium
hydroxide solution. The free acidic polysaccharide can then be obtained
17
by passing through cation ion exchange resin (Harris, 1953), or more
generally by precipitation in glacial acetic acid (Bell, 1934), or acidified
methanol or ethanol. The polysaccharide is then obtained in purified form
the repeated solution in water and repreciption or by electro dialysis
Thomas et al. (1928). Different purification methods do not appear to
alter significantly the physical properties of the gum. However
Balabanova and Kristova (1982) reported that purification of gum
material by different solvents exhibited certain differences in fraction at
the same treatment.
2.7.2 Fractionation
Fractionation is one of the most important methods of analysis; the
techniques for fractionating polydispersed polymer are divided into
preparative and analytical methods (Tager, 1972).
2.7.2.1 Preparative fractionation
This method is the simplest; it involves the addition of precipitants
to an aqueous solution of fractions having different solubilities. Coprecipitation may occur (Meer, 1980). Different fractions of different
solubilities of the gum can be collected, Anderson et al. (1969). The gum
can also be fractionated by careful precipitation with near saturated
sodium sulphate solution (Randal, 1989) and complex formation using
complexing agents e.g. cupric acetate. Gum Tragacanth suffers
methylation and is fractionated by addition of ethanol and subsequently
acetone to yield acidic and neutral menthylated polysaccharides and
glycosides. The fractions obtained by fractional precipitation of gum
Arabic using hydrophobic affinity chromatography have similar branched
18
carbohydrate structures based on a-ß-1,3-Linked galactan core (Randal,
1989).
2.7.2.2 Analytical fractionation
Gel permeation chromatography (GPC) is widely used to
determine the molecular mass distribution of macromolecules. GPC
coupled on line with absolute molecular weight determining device such
as a laser light scattering photometer and a concentration sensitive
detector such as refractive index or ultraviolet are currently the best
available techniques for the quick and absolute determination of
polymers molecular weights and their distribution. The typical elution
profile Acacia polyacantha gum and its fractions were shown by (Omer,
2004) the figures 2, 3, 4, 5, 6; and 7 showed follow this page. The light
scattering response showed two distinctive peaks. The first peak had high
response (AGP) content. The second peak was broader with lower
response and it accounted for the rest of the gum (90%). The refractive
index (RI) response also showed two peaks but the response was
opposite to that in light scattering. This is because it is a concentration
detector and since the AGP is only 10% of the total its peak was smaller
than of AG and GP. The UV response showed three peaks. The first peak
was for AGP, which had the protein core, and the carbohydrate attached
to it. The second peak appeared as a shoulder immediately after the AGP
and corresponds to AG. Finally the third peak eluted just before the total
volume and it corresponded to the GP. The GP peak is not detected on
the light scattering (mass detector) since it had low molecular weight.
Also it can not be seen on the refractive index (concentration detector).
AGP could be degraded by proteolytic enzymes, to give molecules with
19
molecular mass similar to the bulk of the gum, and hence, it has been
suggested that this fraction has a Wattle- blossom structure Fig.1, where,
approximately, five blocks of carbohydrate are attached to a common
polypeptide chain (Fenyo et al, 1988; Osman et al., 1993). Qi et al.
(1991) isolated the molecular species of gum Arabic corresponding to
AGP by GPC fractionation, and following hydrogen fluoride
deglycosylation, they concluded that the polypeptide chain consisted of
about 400 amino acid residues with a simple empirical formula [Lyp4,
Ser 2, Ther, Gly, Leu, His]. This finding was also consistent with
Williams et al. (2000) findings. Qi (1991) suggested that the molecules
were rod like and resembled a twisted hairy rope with small blocks of
polysaccharide about 30 residues attached to the peptide chain.
2.7.2.3 Enzymatic fractionation
The degradation of viscous polysaccharides by faced bacteria was
studied by measuring viscosity, pH and short chain fatty acids (SCFA).
Guar gum, locust been gum and gum tragacanth were completely
degraded as their viscosities deminished and SCFA produced. The extent
of degradation and Pattern of end products may be related to the
chemical linkages and the tertiary structure of the molecule (Omer,
2004). Fractionation studies showed that the degree of hydrolysis of
polysaccharides (gum Arabic) to reducing sugars depends upon the strain
of bacteria (Sampah, 1988).
20
Arabinogalactane subunit
Polypeptide backbone
Fig. 1 the “wattle blossom” model of the arabinogalacto-protein
(Fincher et al., 1983).
21
2
3
4
Molecular weight distribution of different fractions of A.
polyacantha gum (Omer, 2004).
22
5
Molecular weight distribution of different fractions of
6
A.polyacantha gum (Omer, 2004)
7
23
2.7.2.4 Immunological fractionation
Of useful tool, which can be used for separation of polysaccharide
of gum Arabic, is the Arabino-galactan protein (AGP). This anti AGP
showed fractionation to contain epitopes characteristic of an arabinogalactan protein complex. (Osman etal., 1993).
2.7.3 Electrophoresis
Electrophoresis may be defined as the migration of charged
species in solution under influence of an electric field. This method
offers some criterion of hetero-homogeneity. Joubert (1954) reported that
gum Arabic and cyanophylla posse's different moieties. However, the
electrophoretic technique employing polysaccharides solution in molar
potassium hydroxide is capable of resolving certain mixture of
structurally different polysaccharides. That technique might be applicable
to characterization and fractionation of limited class of polysaccharides
but can not be used with confidence as general criterion of purity of
polysaccharides. The main advantages of such methods are, in assessing
polymer homogeneity, its speed and also the fact that very little material
is used.
2.7.4 Hydrolytic methods of analysis
Acid hydrolysis is a key step in the analysis of polysaccharides
because it is used to determine the nature and relative proportions of the
monosaccharide molecules by hydrolyzing the polysaccharide into
simpler
components,
which
can
be
analyzed
by
analytical
chromatographic technique (Samuelson, 1967). The conditions for
hydrolysis must be controlled such that, complete hydrolysis is achieved
with little or no degradation of the mono-saccharide units. Appropriate
24
conditions for hydrolysis of hexoses containing polysaccharides are 1M
and 2M aqueous H2SO4 solutions at 100 C˚, for different interval of time,
using 0.5 N H2SO4 may result in partial hydrolysis (Munro,1970).
According to Harris et al. (1984) hydrolysis of polysaccharides can be
done by trifluro acetic acid at different concentrations. Usually the
hydrolysis process is followed by neutralization of the hydrolysate,
deionization
and
examination
by
chromatographic
techniques
(Eggenberger, 1954).
2.7.4.1 Partial acid hydrolysis
This method is a useful technique employed for determination of
some of the sequences of monosaccharide constituents, by the isolation
of oligosaccharides formed during the break down of polysaccharide
molecule. According to Whistler and Miller (1958) the mixture of
oligosaccharides can be separated from monosaccharide and from one
another by chromatography or charcoal or celite cellulose (Wadman,
1952), resin column (Pitte, 1960) or by thick paper chromatography,
partial acid hydrolysis also gives information about the model of linkage
of each sugar unit and the ratio of non reducing end groups.
2.7.4.2 Autohydrolysis
The whole polysaccharide can be subjected to mild hydrolysis
and to a sample from which acid- labile side chains had been removed by
partial acid hydrolysis with objectives of gaining further information,
about the molecule core units ascertaining the probability of the
sequences of subunits in the molecule (Stephen, 1983). The acidity of a
hot solution of a gum may be sufficient to strip off arabinose end –
25
groups which usually exit in the furanoside form. This process known as
autohydrolysis produces a degraded polysaccharide having a simple
structure than the original molecule (Karamalla, 1965).
2.7.5 Chromatographic methods for analysis of polysaccharides
According to JECFA (1990) chromatography defined as
analytical technique where by mixture of chemicals may be separated by
virtue of their deferential affinities for two immiscible phases; one of
these the stationary phase, consist of a fixed bed of a small particles with
surface area, while the other, the mobile phase or "eluent" is a fluid that
moves constantly or over the surface of fixed phase. Chromatography has
been used for separating mixture into constituents for qualitative and
quantitative analysis, preparation of pure substances, and the purification
of the products of reaction, it proofs the homogeneity of substance,
identification of substances, and monitoring for other separation
processes and reactions (Denman, 1973).
2.7.5.1 Paper chromatography
Paper chromatography used for qualitative and quantitative
analysis. The separation of sugars depends upon the relative case with
which sugar can pass through the stationary phase. The ratio of the
distance moved by the sugar to distance moved by the solvent front
(measured from the point of application) is the Rf value. The movement
will be fairly constant under a given set of conditions e.g. temperature,
pH and type of paper. Using this technique, sugars encountered in the
hydrolysate of gum can be readily separated and qualitatively identified
by comparing the Rf values of standard sugars (Chalmers, 1966).
26
Paper chromatography revealed the presence of L-arabinose, Dgalactouronic
acid,
L-rhamnose
and
D-glucuronic
acid
in
A.
polyaxcantha gum (Omer, 2004). It also indicated that Oleo-gum
obtained from B.papyrifrea contain D (-) glucuronic acid D (-) galactose
and L (-) arabinose (Mustafa, 1997).
2.7.5.2 Thin layer chromatography
Thin layer chromatography (TLC) is a simple rapid technique,
which is very useful for the preliminary examination of carbohydrate
mixtures. The stationary phase is a thin layer of silica gel or cellulose
powder bound on an inert plate (glass, aluminum or plastic).
Elamin (2001) reported that the TLC showed three kinds of sugars
(arabinose, galactose and rhamnose) liberated from A.senegal gum. Omer
(2004) reported that the TLC chromatogram of A .polyacantha gum
confirmed the presence of arabinose, galactose, and rhamnose. Ahmed
(2003) using TLC reported that hydrolysis of Anogeissus liocarpus gum
gave L- arabinose, D-galactose, L-rhamnose and D-glucuronic acid.
Mustafa (1994) stated that sugar components of B.papyrifera deresinified
gum were D-galactose, L-arabinose, D-glucuronic acid, and D-arabinose.
2.7.5.3 Column chromatography (CC).
The conventional procedure is to collect the elute as a series of
fractions usually with an automatic fraction collector and to examine
each fraction by UV/VIS spectrophotometer. Such separation depends on
distribution of the components of the sugar mixture to different ratios
between the mobile phase and the stationary phase. Those components
27
that interact more with the stationary phase are retarded with respect to
ones that interact less with the stationary phase (Poter, 1941).
2.7.5.4 Gas liquid chromatography (GLC)
In GLC the mobile phase is a gas (helium, nitrogen or argon). The
stationary phases are packed into a long coiled column. GLC separates
the mixture using chromatographic distribution between a moving gas
and vapors phase (the developer) and the stationary liquid phase
(Chalmers, 1966). A prerequisite of the method is the sugars are
converting into volatile derivatives. Since GLC separation is dependant
upon the differential extractive distillation, of the components in the
mixture, several types of volatile derivative have been used (Munro,
1970). The trimethyl silyl derivatives of hydrosylates obtained from B
.payrifera and C .africana gums were found to contain 18.72 and 19.69%
D-galactose, 12.74 and 20.20 L-arabinose, 15.84 and 19.69% Lrhamnose, 15.66 and 17.62% L-fucose, 25.27 and 22.79% D-glucuronic
acid, respectively (Mustafa, 1997).
2.7.5.5 High performance liquid chromatography (HPLC)
HPLC separates compounds (monosaccharide mixture and oligosaccharides) easier and gives more accurate quantification (Geyer, 1982).
There are a number of HPLC processes for separating carbohydrates,
which depends on chemical and physical properties for resolution. The
typical run time is 50-60 minutes and a high pressure is to be provided to
maintain solvent flow. Columns used in HPLC are, usually, ready
packed. All solvents to be passed through the column should be as pure
as possible. In some HPLC methods, the composition of the solvent
gradually, changes during the run, this is known as gradient (elution)
28
chromatography using borate buffer or increasing normality or wateracetonitrile mixture. Monosaccharides may be separated in all these
process using aqueous acetonitrile solvents. The selection of water –
acetonitrile ratios and flow rate must be done properly (Omer, 2004).
Other methods used continuous delivery of simple solvent e.g. water or
borate buffer (Kenedy, 1986). Detection methods generally applicable to
carbohydrates make use of change in refractive index. The refractive
index detector is, inherently, as sensitive absorbance measurements in the
far UV. This method is not suitable for separation involving gradient
elution.
Eltayeb (1999) using HPLC found that crude gum samples of
Anogeissus leiocarpus contain 6.82% L-rhaminose, 48.08% L-arabinose,
11.26% D-galactose and two unknown oligosaccharides having values
of 0.22% and 32.16%, whereas A.senegal gum showed 10% L-rhamnose,
14% L-arabinose, and 15% D-galactose.
2.7.6 Spectrophotometery and spectroscopy
2.7.6.1 Absorption spectroscopy
It is the measurement of selective absorption by atoms, molecules,
or ions of electromagnetic radiations at definite and narrow wave length
range, approximating monochromatic light. Absorption spectrophotometry encompasses the following wave length regions: ultraviolet (185 to
380 nm), visible (380 to 780 nm), near infra-red (780 to3000 nm) and far
infra-red (500 to 40000 nm).
2.7.6.2 Ultraviolet (UV)
Ultra violet is not used primarily to show the presence of individual
groups, but rather to the relationship between functional groups chiefly
29
conjugated either between carbon or carbon oxygen double bonds,
between double bonds, and in aromatic ring and even in the presence of
the presence of aromatic ring it self. It can additionally reveal the number
and location of constituents attached to the carbons of the conjugated
system (Boyd, 1978.)
2.7.6.3 Infra red spectroscopy (IR).
Infrared spectrophotometery is one of the most powerful tools
available to the chemist for identifying pure organic and inorganic
compounds because each molecular species has a unique infrared
absorption spectrum. Thus, an exact match between the spectrum of a
compound of known structure and that of an analyte unambiguously
identifies the latter (Daly et al., 1990). FTIR spectrometrical measurements for gum of Sterculia setigera collected from South Kordofan
showed eight peaks with abroad one at about 3445.65 cm-1 most likely
for hydroxyl groups (OH), and two at 1700-1600 cm-1 probably for
carboxyl, aldehyde or ketone groups, the remaining peaks were specific
for the gum sample (Fig. 8). Similarly the FTIR spectra for A. senegal
gum sample collected from gum Arabic Company and A. seyal gum
sample collected from South Kordofan (Fig 9 and 10) showed clear peaks
with abroad one at about 3401.60 cm-1 and 3398.29 cm-1 most likely for
hydroxyl groups (OH), respectively. In addition to two another peaks at
1700-1600cm-1 probably for carboxyl, aldehyde or ketone groups, the
remaining peaks (at 600-700cm-1) were specific for the gum type
(Mohammed, 2006).
30
Fig: 8 FT.IR spectrum of Sterculia setigera gum sample collected from Southern Kordofan (Elabssyia) area
(Mohammed, 2006)
31
Fig: 9 FT.IR spectrum of Acacia seyal sample collected from Southern Kordofan (Mohammed 2006)
32
Fig: 10 FT.IR spectrum of Acacia senegal collected from Gum Arabic Company Ltd Elobied branch (Mohammed, 2006)
33
CHAPTER THREE
MAERIALS AND METHODS
3.1 Materials
Authenticated samples of A .polyacantha gum were collected from
A. polyacantha trees and identified by experts from the Forestry
Department of Ministry of Agriculture and Forestry, and Khartoum Gum
Arabic Processing Company. The gum samples were collected during the
season 2005 /2006 from Kadogli forest (South Kordofan State) and
Eldamazine (Blue Nile State).Twenty samples were collected from each
site.
3.2 Preparation of samples
Gum nodules were dried at room temperature, and then hand
cleaned by hand to insure freedom from sand, dust and bark impurities,
then ground using a mortar and pestile, sieved through sieve No.16 and
kept in a labeled container for analysis.
3.3 Analytical Methods
The following analytical methods were adopted in this study:
3.3.1 Moisture content
The determination was conducted according to AOAC (1990).
Crucibles were dried in Hearus oven at 105oC for 30 minutes, cooled in a
desiccator and then weighed (M1). About two grams of sample were
placed in the crucible and weighed accurately (M2). Contents were
heated in an oven for 5 hours at 105oC cooled in desiccator and re
weighed (M3). Loss percentage on drying was calculated as follows:
M2 – M3
M2 – M1
X 100
34
Where:
M1:
M2:
M3:
Weight of the empty crucible
Weight of crucible +sample
Weight of crucible +sample after drying
3.3.2 Total ash
Total ash was determined according to AOAC (1990). Crucibles
were heated in an oven for 30 minutes cooled in a desiccator and then
weighed (W1). About two grams of sample were placed in the crucible
and accurately weighed (W2), then ignited at 550oC in a Heracus
electronic muffle furnace for 2 hours, cooled in a desiccater and weighed
(W3). Total ash% was calculated as follows:
W3 – W1
X 100
W2 – W1
Where:
W1:
W2:
W3:
Weight of the empty crucible
Weight of crucible +sample
Weight of crucible +sample after drying
3.3.3 Nitrogen and protein contents
Nitrogen was determined using semi-micro khjeldahal method as
described by Anderson (1986). Accurately weighed 0.2 gram of gum
samples were taken in triplicates in khjeldahal digestion flasks then
khjeldahal tablet (Copper sulphate-potassium sulphate) along with 3.5
mls of concentrated nitrogen free euphoric acid were added to each flask,
The flasks and contents were then heated over an electric heater until the
solution attained a clear blue color and the walls of the flask were free
from carbonized materials. The contents of the flask were then
35
transferred to a steam distillation unit (BÜCHI, B.323.SWITZERLAND),
and 15 mls of 40% sodium hydroxide solution were added, and
distillations were carried out. The distillate was then collected in 10 mls
of 2% boric acid solution with three drops of methyl red indicator, and
titrated against 0.01 N HCl. The same procedure was carried out for
blank (distilled water).
N %=
(M1 – M2) × N× 14.01
S × 1000
× 100
Where:
M1:
M2:
N :
S :
mls of HCl that neutralized the sample distillate
mls of HCl that neutralized the blank distillate
Normality of HCl titrate (0.01).
Sample weight (0.2g).
The reactions involved in these steps are as follow:
Sample +H2SO4 (conc.) + Catalyst +heat → (NH4)2SO4.
(NH4)2SO4 +2NaOH → 2NH3 + Na2SO4 +2H2O.
NH3+H3BO3→ NH4 + H2BO3
The protein content was determined by multiplying nitrogen
percent by the factor 6.6 (Anderson, 1986).
3.3.4 Specific optical rotation
The specific rotation was determined according to FAO (1991). A
1.0% solution (on dry weight basis) was measured at room temperature,
using an optical activity Polarimeter type AA-10 automatic polarimeter
(ENGLAND) fitted with a sodium lamp with a cell path length of 20 cm.
The solution was passed through a No.42 filter paper before carrying out
measurements at room temperature. Triplicate readings were taken and
36
averaged. The specific rotation for gum solution was calculated
according to the relationship:
Specific rotation =
α × 100
C×L
Where:
α=
Observed angular rotation
L=
Length of the polarimeter tube in decimeters
C=
Concentration of the solution expressed as number of grams
substance in 100cm³ of solution
3.3.5 Determination of viscosity
Viscosity was measured using U- tube viscometer (type Volac
/BS.UC, serial No.1094) with the time for 1% aqueous solution of
sample at room temperature (25 oC). The relative viscosity (‫ן‬r) was then
calculated using the following equation:
[‫ן‬r]=
T- T◦
T▫
Where:
Specific Viscosity =
Reduced Viscosity =
‫ן‬r -1
‫ן‬sp/C%
T▫-T▫
T▫ c % Reduced > 0
Flow time of sample solution expressed in seconds
Flow time of solvent (Distilled water) expressed in seconds
Intrinsic Viscosity =
T=
T◦=
The reduced viscosity (‫ן‬rd) was determined for different
concentrations of gum solution, 0.12, 0.25, 0.50 and 1% and was then
calculated from the above equation.
The intrinsic viscosity was determined by extrapolation of reduced
viscosity against concentrations back to zero concentration. The
interception on Y-axis gives (‫)ן‬.
37
3.3.6 Molecular weight
The molecular weight was calculated from the intrinsic viscosity
using Mark-Houwink equation. (Mark, 1938; Howink, 1940).
[‫]ן‬
= KMwª
Where:
Mw = Molecular weight.
[‫= ]ן‬Intrinsic viscosity.
K and a = Marck –Houwink constant.
Based on (Anderson and Rahman, 1967b), the values of K and a
were determined for A.sensgal gum as follows:
K=
a=
1.3×10¯²
0.54
3.3.7 pH measurement
The pH value was determined for 1% aqueous solution at room
temperature, using a microprocessor pH meter, HANNA, (ROMANIA)
Type 211.
3.3.8 Apparent equivalent weight
Apparent equivalent weight was determined according to the
method
reported
in
the
encyclopedia
of
chemical
technology
(Encyclopedia, 1966).With some modification, the aqueous gum solution
(3%) was treated with acid washed Amberlite Resin 120 (H+) [2gms per
10mls gum solution] for an hour and then titrated against 0.02 N Sodium
hydroxide solution using phenolphthalein as an indictor. The equivalent
weight was calculated as follows:
Equivalent weight=
weight of sample × 1000
Volume of titer× molarity of alkali
38
3.3.9 Uronic acid
Uronic acid percentage was determined according to Elamin (2001)
by multiplying the molecular weight of uronic acid (194) by 100 and
dividing by apparent equivalent weight of the sample as follow:
Uronic acid =
194
=
194 x 100
Equivalent weigh
Molar mass of uronic acid.
3.3.10 Determination of reducing sugars content
Reducing sugars content were determined according to the
procedure of Robyte and White (1987) using the alkaline ferricyanide
method. The procedure uses a single reagent composed of 0.34gm of
potassium ferricyanide, 5gm of potassium cyanide and 20gm of sodium
carbonate dissolved in one liter of water. A 0.1ml of 1%gum solution
was added to 4.0 ml of reagent, then heated in a boiled water bath for 10
minutes and cooled. The absorbance was measured at 420 nm using
JENWAY, MODEL 6300,SERIAL NO.7914 U.K spectrophotometer
.Standard curve of different arabinose concentrations was plotted against
absorbance in order to calculate the reducing sugar content as arabinose.
3.3.11 UV Absorption spectra
Maximum, absorption spectra of 1% gum solution were determine
using a JEN WAY, MODEL 6300, SERIAL NO.7914 U.K,UV/VIS
spectrophotometer, according to (Eltayeb, 1999).
3.3.12 Analysis for inorganic matter.
Accurately weighed 2 grams of dry sample were ignited to ash in a
muffle furnace at 550oC for 4 hours. Ten ml of 50% HCl and 5 ml of
39
33% HNO3 were added to each crucible then allowed to warm for one
hour to dissolve the minerals and cooled, then 10 ml of HCl and 10 ml
distilled water were added and allowed to stand for 15-20 minutes. The
mixture was filtered with ashless filter paper No.41mm and distilled
water was added to complete the volume to 100 ml in volumetric flask.
Sodium (Na+) and potassium (K+) were determined using flame
photometer. While calcium (Ca+), iron (Fe+) and magnesium (Mg+), were
determined using UNICAM 8625 UV/VIS spectrophotometer.
3.4 Functional properties
Analyses for Functional properties of A. polyacantha gum in the
present study were carried out as follows:
3.4.1 Emulsifying stability
According to the method reported by Eltayeb (1999), a
concentrated solution of gum sample 20% w/v in distilled water was
prepared. Then it was mixed with oil (peanut oil) in ratio of 80:20 w/w
respectively. The suspension was mixed using an emulsifier for a minute
at 1800 rpm. Then the mixture was diluted in the ratio of 1:1000 and was
read at ‫ ג‬max 520 nm. Using JENWAY UV/VIS spectrophotometer,
MODEL 6300, SERIAL NO.7914 U.K, another reading was taken after
one hour. The readings represented emulsifying index. Emulsifying
stability was calculated using the following formula:
Emulsifying stability =
First reading
Reading after1hour
40
3.4.2 Water holding capacity (W.H.C)
The method was according to Elamin (2001). One gram of A.
polyacantha gum was accurately weighed in a Petri-dish, which was
transferred to a desiccator half-filled with distilled water and was
incubated for certain length of time: 24, 48, 96,120; and 144 hours. The
Petri dishes containing the sample were reweighed (g/g). The increase in
weight was expressed as percentage to explain the water holding capacity
of the sample and finally expressed as follows:
WHC% =
WHC =
weight of water × 100
Weight of sample
Water holding capacity
3.5 Structural analysis
The following analytical methods were adopted in this study to
show the structural features of A.polyacantha gum:
3.5.1 Acid hydrolysis
Acid hydrolysis was carried out according to Munro (1970)
method. One gm of gum (composite sample) dissolved in 100 mls
sulphuric acid with different concentrations (0.5 and 1N). The solutions
were then heated on a boiling water bath for eight hours, cooled and
neutralized by adding barium hydroxide, then barium carbonate, filtered
and stirred with Amberlite IR (H+) resin to remove the cations. The
deioniozed hydrolysates were filtered from the resin and concentrated
under vacuum at 4oC then examined by paper chromatography and
HPLC.
41
3.5.2 Chromatographic method.
3.5.2.1 Paper chromatography (PC)
Qualitative paper chromatography was carried by ascending and
desending techniques using what man No. 1 paper. The solvent system
used was n-Butanol-ethanol-water (4:1:5). The locating reagent used was
β-naphthylamine.
3.5.2.2 Analysis of sugar composition using high performance liquid
chromatography (HPLC).
The L (+) arabinose, L (+) rhaminose, D (+) glucose and mannose
content of the whole gum sample (A) and the fractions (F1, F2) of A.
polyacantha gum was determined by high performance liquid
chromatography (HPLC). The hydrolyzed samples were subjected to the
following procedure described by Randall et al. (1989). Gum samples of
0.03 g were weighed accurately into stoppered Pyrex test tubes (15cm3)
and 10 ml of 4.0%w/w Sulphuric acid were added to each. The tubes
were placed in water bath at 100oC for 4 hours. The hydrolysed solutions
were neutralized by adding barium carbonate (2.0 gram) and left to mix
over night at room temperature. The hydrolysates were filtered using
0.45 Whatman filter paper and analyzed using an HPLC system
SHIMADZU, Japan, Auto sample controller, linked to LC column
No.6158614 shin – pack clc, NH2-(6.0×150), with refractive index
detector (RID-10A). The sample (10 ml hydrolysate) was injected into
the column and eluted using a mobile phase 75:25 v/v acetonitrile: water
(filtered and degassed before use). Analysis were performed at ambient
room temperature (30oC) and the flow rate maintained at 1.0 ml min-1
42
The retention times of the monosaccharides were monitored using
a differential refractometer (RID-10A, water). The retention times
obtained were compared to those determined using L (+) arabinose, L (+)
rhaminose, D (+) glucose, and D (+) Mannose as standards. The area
percent of each peak was calculated by millinum program, which was
connected with RI-10A detector. Then the sugar (percent) was calculated
as follows:
Component Sugar (i) =
Area percent of component (i)
Total area
X 100
3.5.3 Fractionation
It was done according to Abdelrahim (2006). Gum solution (30%)
was prepared in distilled water and the solution was then blown with air
using an aerating pump in a water bath at 50-60oC till foaming stops. The
foam was collected on a Petri dish and exposed to air to be dried. The
foam was Fraction 1. The remaining solution was treated with 4 volumes
of ethanol and few drops of HCl so as to form precipitate; the precipitate
was Fraction 2, which was then dried from ethanol by exposing it to air.
3.5.4 Infrared
Twenty five mg of gum samples were mixed with 75 mg KBr and
then pressed in the disc to form proper kits. The kits were then
transferred to the measuring compartment and subjected to Floro
transmissions-infra red analysis (F.T-IR) using Thermo-Ni coblet-IR300
spectrophtometer (stanely, 1980).
43
3.6 Conductivity:
Conductivity was determined according to FAO (1991). A 1%
solution on dry weight basis was measured at room temperature (30oC).
Using conductivity meter, Jenway Type Model 4320, No. 1726.
3.7 Statistical analysis
Each sample was analyzed chemically in triplicate then averaged.
Data was assessed by analysis of variance (ANOVA), the mean
separation was carried out by Duncan’s multiple range tests with
probability of 0.05 levels.
44
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Physical properties of A. polyacantha gum
The physical properties of A. polyacantha gum are shown in Table
(1).
4.1.1 Shape and colour
The shape of the gum nodules, as exuded naturally, are irregularly
globular or tear shaped. The colour varies from water-white (colourless)
through shades of yellow (Plate 1). There are no noticble variations in
either shape or color between samples from Kadogli and Eldamazine.
4.1.2 Specific optical rotation.
Aqueous solutions of all samples were found to be optically active
(laevorotatory). The specific rotation of Kadogli samples were found to
range from -8.7° to -25.0° with mean value of -19.6°, while that of
Eldmazine ranged from -12° to -25° with mean value of -14°Table 1.
These findings were within the range of the recent results of Omer (2004)
who reported -13.5ْ to -26.0ْ specific rotation for A. polyacantha gum, but
were higher than the values of -10.3° and -7 to -13° given by Biswas
(2000) and Siddig (2003), respectively. Within each location, analysis of
variance showed no significant differences (P≤0.05) between the
samples. Relevant to specific rotation, the two locations proved to be
insignificantly (P≤0.05) different.
45
Table (1): Physical properties of A.polyacantha gum
Sample
No.
Specific rotation
Intrinsic viscosity
(ml/g)
Kadogli Eldamazine
11.1
10.0
Kadogli
Eldamazine
1
-17.5°
-15°
2
-14.0°
-12°
10.5
3
-16.0°
-13°
4
-13.0°
5
Emulsifying stability
Water holding
capacity (%)
Kadogli Eldamazine
63.0
63.0
Kadogli
1.11211
Eldamazine
1.0102
10.5
1.12101
1.0112
63.0
63.0
10.7
10.4
1.10010
1.1112
63.0
63.1
-14°
10.0
10.4
1.01112
1.1112
63.1
63.5
-23.0°
-13°
10.3
10.4
1.00110
1.0251
62.9
63.1
6
-14.0°
-14°
10.3
10.3
1.02110
1.0141
62.8
63.5
7
-15.0°
-15°
10.3
10.4
1.02110
1.0113
63.2
63.6
8
-13.0°
-15°
10.5
10.2
1.02110
1.0123
63.7
63.8
9
-14.0°
-13°
10.7
10.9
1.02110
1.0212
63.9
63.8
10
-12.0°
-12°
10.0
9.0
1.01110
1.1100
63.8
63.1
11
-14.0°
-12°
9.9
9.0
1.11110
1.2110
63.0
63.1
12
-12.0°
-17°
10.3
9.2
1.11210
1.2110
63.0
63.1
13
-23.0°
-13°
10.4
9.9
1.21210
1.2113
63.5
63.0
14
-22.0°
-25°
10.4
10.1
1.01011
1.0111
63.0
63.0
15
-22.5°
-24°
10.3
10.4
1.01110
1.0211
63.1
63.0
16
-24.5°
-13°
10.3
10.5
1.01110
1.0111
63.0
63.0
17
-21.6°
-13°
10.2
10.7
1.01110
1.0211
63.0
63.2
18
-25.0°
-12°
10.4
10.8
0.99100
1.0311
63.1
63.7
19
-8.7°
-13°
10.4
10.5
0.11110
1.0312
63.1
63.5
20
-14.0°
-14°
10.3
10.7
1.01110
1.1112
63.0
63.9
Mean
-19.6°
0.41
-14°
0.39
9.9
10.2
0.99200
1.0654
63.1
63.3
0.10
0.096
0.049
0.036
Each value in the table is a mean of three replicates.
0.104
0.07
SE±
46
Plat 1. A.polyacantha gum nodules (color and shape).
47
4.1.3 Intrinsic viscosity
Aqueous solutions of all samples of A. Polyacantha gum showed
low viscosity. As presented in Table (1), the intrinsic viscosity of
Kadogli samples ranged from 9.9 to 11.1 ml/g with average value of 10.0
ml/g, while Eldamazine samples showed 9.0 to 10.9 ml/g intrinsic
viscosity with mean value of 10.2 ml/g. Present results were in a good
agreement with the value of 10.3 ml/g reported by Omer (2004), however
Siddig (2003) recorded slightly higher value of 12.7 ml/g. These values
were markedly lower compared to the 14 ml/g intrinsic viscosity reported
for A. seyal gum (Elkhatim, 2001).
Within each location, mean separation revealed no significant
differences (p≤0.05) between the samples. Also location insignificantly
(P≤0.05) affected the intrinsic viscosity of A. Polyacantha gum.
4.1.4 Refractive index
Refractive indices of all samples from the two different locations
were found to be similar having the same value of 1.3354. Omer (2004)
and Elkhatim (2001) reported fairly similar values of 1.3337 and 1.3339,
respectively. It is clear that location has no effect on the refractive index.
4.2 Functionality
The functional properties (emulsifying stability and water holding
capacity) of A. polyacantha gum are shown in Table 1.
4.2.1 Emulsifying stability (ES)
The data shown in Table 1 demonstrates that the mean value of the
emulsifying stability of samples from Kadogli location was 0.99200 with
48
a minimum value of 1.1110 and a maximum value of 1.21210. On the
other hand, the samples obtained from Eldamazine location showed
average value of 1.06540 with a range from 1.0102 to 1.21130. Results
obtained were in accordance with the values of 0.95454 - 1.11111 for A.
polyacantha gum and 0.980792 - 1.017857 for A. senegal gum (Omer,
2004). No significant differences (P≤ 0.05) were observed among each
location or between the two locations.
4.2.2 Water holding capacity (WHC)
As shown in Table 1, the water holding capacity (WHC) of all
tested samples had been found to range between 63.0 and 63.9%. The
mean value was 63.1 and 63.3 g/100g for Kadogli and Eldamazine
location, respectively.
Statistically, location exerted no significant (P≤ 0.05) effect on water
holding capacity.
4.3 Chemical properties of A. polyacantha gum
The chemical properties of A. polyacantha gum are presented in
Table 2.
4.3.1 Moisture content
The moisture content of A. polyacantha gum from both Kadogli
and Eldamazine was found to be in the range of 9.5 – 11.5% with a mean
value of 10.5% for Kadogli and 10.3% for Eldamazine samples (Table
2). Abdelsalam (1998) recorded fairly similar range of 9.4 –11.6%.
However, the mean values reported here were slightly higher than the
mean value of 8.2% indicated by Omer (2004).
49
Table (2): Chemical properties of A.polyacantha gum.
Sample
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Mean
S.E±
Moisture (%)
Kadogli Eldamazine
10.0
10.0
11.5
11.0
10.0
11.0
11.0
10.5
9.5
10.5
10.5
10.5
10.0
9.5
11.5
11.0
11.0
10.5
10.5
10.5
10.5
0.13
10.5
9.5
10.0
10.0
10.0
10.5
10.5
11.5
10.0
10.0
11.0
9.5
10.5
10.0
10.5
10.0
10.0
9.5
10.5
10.5
10.3
0.123
Ash (%)
Kadogli Eldamazine
3.5
4.0
3.0
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.3
3.0
3.1
3.2
3.4
3.0
3.2
3.1
3.4
3.7
3.3
0.012
3.0
3.5
3.5
3.7
3.1
3.2
3.5
3.7
3.0
3.0
3.1
3.2
3.5
3.5
3.5
3.5
3.5
3.7
3.5
3.5
3.4
0.056
Nitrogen (%)*
Kadogli Eldamazine
0.36
0.35
0.34
0.36
0.41
0.37
0.37
0.38
0.30
0.30
0.34
0.37
0.42
0.35
0.37
0.39
0.36
0.36
0.37
0.36
0.36
0.036
0.45
0.43
0.40
0.42
0.40
0.40
0.48
0.41
0.45
0.41
0.38
0.42
0.36
0.40
0.42
0.43
0.42
0.38
0.39
0.39
0.41
0.006
Protein (%)*
Kadogli
Eldamazine
2.38
2.28
2.14
2.23
2.13
2.32
2.32
2.36
1.88
1.88
2.14
2.32
2.63
2.19
2.32
2.40
2.13
2.38
2.32
2.13
2.24
0.224
* The two locations are significantly (P≤ 0.05) different.
50
2.80
2.70
2.40
2.60
2.50
2.40
2.90
2.60
2.80
2.50
2.40
2.60
2.30
2.50
2.60
2.70
2.60
2.40
2.40
2.50
2.60
0.037
Reducing sugars (%)
Kadogli Eldamazine
0.14
0.12
0.14
0.14
0.15
0.13
0.36
0.36
0.10
0.31
0.31
0.14
0.14
0.36
0.37
0.36
0.36
0.31
0.31
0.27
0.23
0.058
0.12
0.13
0.13
0.13
0.10
0.13
0.14
0.13
0.14
0.12
0.13
0.12
0.13
0.13
0.14
0.12
0.15
0.21
0.37
0.35
0.16
0.016
Sample
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Mean
S.E±
Equivalent wieght*
Kadogli
Eldamazine
1530.41
1490.30
1381.75
1613.97
1585.61
1407.30
1523.16
1482.31
1497.71
1376.81
1292.67
1370.37
1155.04
1148.27
1121.11
1470.12
1320.19
1430.19
1530.21
1230.31
1397.90
28.80
Molecular wieght
Kadogli
Eldamazine
pH*
Kadogli Eldamazine
1325.99
3.72×103
3.90×103
5.33
5.55
3
3
1282.63
3.12×10
3.38×10
5.35
5.71
3
3
1300.70
3.49×10
3.32×10
4.84
5.05
1340.01
3.08×103
3.32×103
4.84
5.35
3
3
1394.66
3.23×10
3.32×10
5.05
5.17
1301.33
3.23×103
3.23×103
4.71
5.38
3
3
1247.53
3.23×10
3.32×10
4.77
5.06
1376.89
3.12×103
3.19×103
4.86
5.00
3
3
1139.71
3.49×10
3.62×10
4.91
5.25
3
3
1256.35
3.08×10
2.54×10
5.09
5.99
1144.74
3.03×103
2.57×103
5.17
4.86
3
3
1243.75
3.64×10
2.56×10
4.81
4.90
1602.70
3.32×103
3.02×103
4.93
4.97
3
3
1507.30
3.32×10
3.14×10
4.36
4.75
3
3
1120.61
3.32×10
3.32×10
4.91
5.32
1240.71
3.32×103
3.38×103
5.05
5.37
3
3
995.620
3.17×10
3.49×10
5.06
5.08
1121.12
3.32×103
3.55×103
5.05
5.48
3
3
1360.01
3.32×10
3.38×10
5.50
5.25
1313.43
3.32×103
3.49×103
5.01
5.04
3
3
1280.80
3.29×10
3.20×10
4.96
5.23
31.95
83.8
81.9
0.19
0.06
Table (2): Contd.
* The two locations are significantly (P≤ 0.05) different
51
Uronic acid (%)
Kadogli
Eldamazine
12.67
13.02
14.04
12.02
12.23
13.78
12.74
13.09
12.95
14.09
14.16
14.15
16.79
16.89
17.30
13.19
14.69
13.56
12.68
15.77
13.99
0.252
14.63
15.12
14.91
14.48
13.91
14.90
15.55
14.09
17.02
15.44
16.95
15.59
12.10
12.87
17.31
15.64
19.48
17.30
14.26
14.77
15.32
0.241
Statistical analysis showed no significant difference (P≤0.05) in
moisture content within or between the two locations.
4.3.2 Ash content
The average ash content was found to be 3.3% and 3.4% for
Kadogli and Eldamazine samples, respectively (Table 2). Results
obtained were within the range 3.06-3.67% reported by Siddig (1996) for
A. sensgal gum, but higher if compared to the value of 2.9% obtained by
Abdelsalam (1998) and Omer (2004) for A. polyacantha gum.
Within each location, mean values revealed no significant
differences (P≤0.05) between the samples. Also location insignificantly
(P≤0.05) affected the ash content.
4.3.3 Nitrogen and protein contents
Results indicate that the nitrogen content of Kadogli samples was
0.30 to 0.42% (1.88 to 2.63 % protein), while that of Eldamazine samples
was 0.36 to 0.48% (2.30 to 2.90% protein). Nitrogen as well as protein
levels in the two locations were found to be significantly (P≤ 0.05)
different.
In this study, the average nitrogen content (0.36% for Kadogli and
0.41% for Eldamazine) agreed with the earlier findings: 0.35%
(Karamalla, 1995); 0.33 % - 0.36% (Siddig, 1996); 0.34% (Ishag, 1977);
0.38 to 0.40% (Omer, 2004). The average protein content in the two
locations was 2.24 and 2.56%, respectively which is comparable to the
values varying from 2.2 to 2.6% previously reported by Ishag (1977),
Karamalla (1995), Siddig (1996) and Omer (2004).
52
4.3.4 Reducing sugars
Reducing sugars content (calculated as arabinose) of A.
polyacantha gum procured from the two locations was in the range of
0.10 to 0.37% without significant difference (P≤0.05) between them. The
mean values were 0.23 and 0.16% for Kadogli and Eldamazine samples,
respectively. However, Omer (2004) reported a mean value of 0.19%.
Karamalla, (1965) reported similar results for A. senegal gum which may
explain the similarity of the physico-chemical properties of the two
different types of gum. Also the presence of reducing sugars gives
evidence to the reducing power (free reducing groups) of this type of
gum.
4.3.5 Uronic acid content
The presence of uronic acids in all samples of A. polyacantha gum
indicated that all samples have acidic sugar (glucuronic acids). Uronic
acid contents of Kadogli samples ranged from 12.02% to 17.30 % and
that of Eldamazine samples ranged from 12.10% to 19.48% (Table 2).
Analysis of variance indicated significant differences (P≤0.05)
among all samples. Table 2 shows that location significantly affected
uronic acid content. The two locations showed a mean value of 14.66%,
which was in conformity with the value of 14.5% obtained by Omer
(2004) for A. polyacantha gum. Results indicated here were comparable
to the values ranging from 10.34% to 23.32% reported for A .senegal
gum (Siddig, 1996), and the value of 14.3% reported by Elatyeb (1998)
for Anogesissus leiocapus gum.
53
4.3.6 pH value
The pH of A. polyacantha gum aqueous solution was found to be
slightly acidic. Table 3 shows that the pH of Kadogli samples ranged
between 4.36 and 5.35, while Eladamazine samples were in the range of
4.75 – 5.99. Significant differences (P≤0.05) in the pH were observed
among the samples of each location. The mean pH value for Kadogli
samples (4.96) was significantly (P≤0.05) lower compared to that for
Eldamazine samples (5.23). Siddig (2003) and Omer (2004) reported pH
values of 4.9 and 4.7-5.7 for A. polyacantha gum, respectively.
4.3.7 Equivalent weight
Concerning the equivalent weight, samples from Kadogli showed a
minimum value of 1121.11 and a maximum value of 1613.97 with
significant (P≤ 0.05) variations among the twenty different samples of
the same source. Whereas Eldamazine samples showed 999.5 to 1602.7
equivalent weight with significant (P≤0.05) differences between the
samples (Table 2). The mean value for A. polyacantha gum from
Eldamazine (1280.20) was proved to be significantly (P≤0.05) lower
compared to the value of 1397.90 for Kadogli samples. This value is
relatively similar to the result 1367.46 for A. polyacantha gum samples
obtained by Omer (2004). Also matching with the range of 1136 to 1875
reported by Siddig (1996) for A. senegal gum.
4.3.8 Molecular weight
As illustrated in Table 2, the molecular weight of Kadogli samples
varied from 3.03×103 to 3.72×103 with average value of 3.29×103 and that
for Eldamazin samples ranged from 3.02×103 to 3.90×103 with average
54
Table (3): Minerals content (µ g/g) of A. polyacantha gum.
Samples
No.
Kadogli
Na
Eldamazine
Kadogli
K
Eldamazine
1
35.0
70.0
0.125
0.075
2
70.0
35.0
0.075
0.04
3
52.5
35.0
0.11
0.05
4
35.0
70.0
0.08
0.095
5
55.0
52.5
0.10
0.075
6
35.0
52.5
0.10
0.08
7
35.0
52.5
0.12
0.075
8
70.0
70.0
0.11
0.095
9
35.0
35.0
0.075
0.055
10
52.5
35.0
0.08
0.035
11
35.0
35.0
0.11
0.05
12
35.0
52.0
0.135
0.025
13
35.0
52.5
0.18
0.055
14
35.0
52.5
0.10
0.055
15
35.0
52.5
0.075
0.045
16
52.5
35.0
0.08
0.07
17
35.0
35.0
0.075
0.075
18
35.0
35.0
0.09
0.05
19
35.0
35.0
0.95
0.04
20
35.0
52.5
0.125
0.05
Mean
41.98
47.23
0.151
0.0595
S.E±
0.060
0.030
3.69
2.79
Each value in the table is a mean of three replicates.
Kadogli
0.458
0.666
0.330
0.451
0.332
0.452
0.611
0.352
0.369
0.482
0.405
0.490
0.398
0.415
0.450
0.361
0.442
0.386
0.392
0.402
0.432
0.015
55
Ca
Eldamazine
0.362
0.462
0.433
0.398
0.444
0.451
0.622
0.431
0.422
0.433
0.621
0.555
0.433
0.621
0.529
0.449
0.421
0.391
0.388
0.405
0.464
0.004
Kadogli
0.206
0.200
0.185
0.201
0.214
0.185
0.215
0.203
0.203
0.183
0.165
0.184
0.203
0.213
0.219
0.185
0.203
0.166
0.203
0.191
0.196
4.72
Mg
Eldamazine
0.263
0.225
0.201
0.224
0.214
0.220
0.225
0.262
0.204
0.22
0.218
0.209
0.214
0.232
0.238
0.176
0.190
0.202
0.209
0.216
0.207
2.43
Kadogli
Fe
Eldamazine
24.8
25.2
28.1
26.2
21.8
22.3
23.4
25.5
25.2
23.9
22.1
20.5
20.3
26.6
25.5
27.3
21.1
22.8
23.4
24.5
24.025
0.022
25.3
26.6
27.7
28.1
29.1
23.4
22.2
20.1
21.9
22.3
24.4
25.5
26.6
56.2
52.1
48.2
42.5
43.4
50.2
52.1
31.795
0.059
value of 3.20×103. Analysis of variance revealed no significant
differences (P≤0.05) between the two the locations. The present findings
could fairly be related to the mean value of 4.165×105 mentioned by
Siddig (2003) for A. polyacantha gum and also with the mean value of
2.5×103 reported for A. Senegal gum (Flory, 1953). On the other hand,
these results were greater than the mean value of 1.0 ×106 reported by
Eggenberger (1954) for A.senegal gum, but lower if compared to the
range of 2.2×105 to 4.4×10 6 reported for A.senegal gum (Fenyo, 1988).
4.4 Minerals
Table 3 shows the mineral composition of A. polyacantha gum
from two different locations (Kadogli and Eldamazine).
4.4.1 Sodium
Sodium content of both Kadogli and Eldamazine gum samples
showed same range of 35 to 70 µg/g, with average value of 44.61 µg/g,
which was related to the value of 43 µg/g reported by Omer (2004) for A.
polyacantha gum. It is also close to the range of 6.54 - 49.55 µg/g
reported for A. seyal gum (Anderson, 1992).
4.4.2 Potassium
Potassium content of gum samples from Kadogli ranged from
0.075 to 0.135 µg/g, which was insignificantly (P≤0.05) higher compared
to the range of 0.025 to 0.095 µg/g related to gum samples from
Eldamazine. However, Omer (2004) reported slightly higher mean value
of 0.311µg/g.
56
4.4.3 Calcium
As demonstrated in Table 4, A.polyacantha gum was found to
contain 0.330 - 0.666 µg/g Calcium, which was in line with the range of
0.451 to 2.165 µg/g reported by Omer (2004). The mean values for
Kadogli and Eldamazine (0.432 and 0.364, respectively) were found to
be insignificantly (P≤0.05) different.
4.4.4 Magnesium
Gum samples from Kadogli were found to contain 0.165 - 0.291
µg/g Mg with average value of 0.196 µg/g, while Eldamazine samples
showed 0.176 - 0.263 µg/g Mg with average value of 0.206 µg/g.
4.4.5 Iron
Iron content of Kadogli gum samples was found to range from
20.3 to 28.1µg/g, while Eldamazine samples showed 20.1 - 56.2 µg/g Fe.
Analysis of variance revealed no significant differences (P≤0.05)
between the two locations. In this study the overall mean (27.91 µg/g)
fairy similar to the mean value of 28.17µg/g reported for A.polyacantha
gum (Omer, 2004).
4.5 Characterization of A. polyacantha gum fractions
As shown in Table 4, the moisture content of Fraction 1 and
Fraction 2 were 6.6% and 15.45%, respectively. Statistically, the two
fractions were significantly (P≤0.05) different.
Ash content was 2.67% for fraction 1 and 3.75% for fraction 2.
Results showed significant (P≤0.05) difference between the two
fractions. Nitrogen content of Fraction 1 was 0.35% (2.19% protein),
which was significantly (P≤0.05) lower compared to the value of 0.38%
57
Table (4): physicochemical properties of A. polyacantha gum
fractions
Sample
Moisture
(%)
Ash
(%)
N
(%)
Protein
(%)
Uronic acid
(%)
pH
E.w*
Mw*
E.S
F1
6.6
2.67
0.35
2.51
14.2
4.7
1345.11
3.37×103
1.11211
F2
15.4
3.75
0.38
2.31
12.11
2.9
1530.20
2.77×103
0.83432
S.E±
4.41
0.54
0.035
0.23
1.04
0.9
92.55
0.3
0.139
* The two locations are insignificantly (p ≤ 0.05) different.
F 1 = Fraction 1
F 2 = Fraction 2
E.W=Equivalent weight
M.W=Molecular weight
E S =Emulsifying stability
58
(2.38% protein) reported for fraction 2. However, Omer (2004) reported
a mean value of 0.37% nitrogen for A.polyacantha gum fractions.
Uronic acid content of Fraction 1 (14.2%) was found to be
significantly (P≤0.05) higher compared to that of Fraction 2 (12.11%).
Omer (2004) reported slightly lower mean value of 12.26% uronic acid
for five fractions of A. polyacantha gum.
The pH value of Fraction1 and Fraction 2 were 4.7 and 2.9,
respectively. Analysis of variance showed a significant (P≤0.05)
difference between the two fractions. Omer (2004) reported an average
pH value of 4.7 of A. polyacantha gum fractions.
Fractions 1 and 2 sequentially showed Equivalent weight of
1345.11 and 1530.20 without significant (P≤0.05) difference between
them. The present values were comparable to the mean value of 1583.20
previously reported for five fractions of A.polyacantha gum (Omer,
2004). The molecular weight was 3.37×103 and 2.77×103 for Fraction
1and fraction 2, respectively. Analysis of variance showed no significant
(P≤0.05) difference between the two fractions. The two fractions showed
significantly (P≤0.05) different emulsifying stability. Values obtained
were 1.11211 and 0.83432 for Fraction 1 and Fraction 2, respectively.
Relevant to the physical properties (Table 5), A. polyacantha gum
Fraction 1 and 2 showed -22◦ and -16◦ specific rotation, respectively.
Interestingly the two fractions gave the same refractive index of 1.3354,
which in turn typical to that of the whole gum. Recently, Omer (2004)
reported refractive indice of 1.3337 for five A. polyacantha gum
fractions.
59
Table (5): Some physical properties of A. polyacantha gum fractions
Sample
Specific
Rotationْ
Refractive
Index*
Intrinsic
.viscosity
(ml/g)*
Conductivity
(µ/s)
F1
-22
1.3354
10.5
151.7
F2
-16
1.3354
9.43
0.728
S.E±
0.49
0.000
0.54
75.5
* The two locations are insignificantly (p ≤ 0.05) different.
F 1 = Fraction 1
F 2 = Fraction 2
60
Plate 2. Paper chromatogram (acid hydrolysis, 0.5 N H2SO4 8hr.)
A : Whole gum
Ar: Arabinose
MI: Maltose
F1 :
Rh:
Ga:
Fraction 1
F2 : Fraction 2
Rhaminose
Mn: Manose
Galactorunic acid
61
Intrinsic viscosity was 10.5 ml/g for Fraction 1 and 9.43 ml/g for
Fraction 2 without significant (P≤0.05) difference between them. Results
obtained were closely similar to the mean of 10.99 ml/g reported for A.
polyacantha gum fractions (Omer. 2004).
The conductivity of Fraction 1 was found to be 151.21µ/s. On the
other hand Fraction 2 showed a significantly (P≤0.05) lower conductivity
value of 0.728 µ/s.
4.6 Sugars composition of A. polyacantha gum
Whole A. polyacantha gum sample along with its two fractions were
hydrolyzed using sulphuric acid at different concentrations. The
separation of monosaccharide was done at the acid concentration of 0.5N
and hydrolysis time was 8 hours. The separation is shown in Plate 2.
Qualitative paper chromatography indicated the presence of the
following
monosaccharides:
L+arabinose,
L+rhaminose
and
D-
galacturonic acid. Chromatogram also indicates that A.polyacantha gum
was free from mannose and maltose. These observations were confirmed
by HPLC (Table 6). High performance liquid chromatography (HPLC)
showed that A. polyacantha gum contain 1.5 and 0.49 mg/100g
L+Rhaminose and L+Arabinose, respectively (Table 6). On the other
hand, L+Rhaminose presented at concentrations of 7.98, and 8.1
mg/100g for Fraction1 and Fraction2, respectively. The two fractions
showed negative result to L+Arabinose.
62
Table (6): Sugars content of A. polyacantha gum (mg/100gl).
Sample L+Rhaminose
L+Arabinose
D-Glucose D-Mannose
A
1.5 ± 0.1
0.49 ± 0.1
-
-
F1
7.98 ± 0.1
-
-
-
F2
8.1 ± 0.1
-
-
-
A=
Whole sample of A. polycacantha gum
F1= Fraction 1
F2= Fraction 2
63
4.7 UV Absorption
Fig.11, 12, 13; and 14 show UV absorption of A. polyacantha gum
obtained from Kadogli and Eldamazin in addition to Fraction 1 and 2,
respectively. It has been observed that maximum was approximately the same
(280 nm), and this may prove to be a diagnostic feature and therefore an
apparent analytical parameter for A. polyacantha gum. Omer (2004)
investigated A. polyacantha gum reported that the absorbance approximately
the same (280nm).
4.8 Infra-red (IR) spectral analysis.
The infra red (IR) spectra for samples of A.polyacantha gum collected
from kadogli and Eldamazine are shown in Fig 15, 16, 17 and 18 respectively.
The IR spectra showed six peaks with abroad one at about 3274.44cm-1 most
likely for hydroxyl groups (OH); and two at 2922.99 cm-1 probably for
aliphatic groups (C-H), and two peaks also at 1617.96cm-1and 1420.40 cm-1
probably for ketone (C=O) groups, the remaining peaks (at 1066.34 – 599.13
cm-1) were specific for A. polyacantha gum. Similarly the FT.IR spectra for A.
senegal gum sample donated by Gum Arabic Company and A. seyal gum
sample collected from South Kordofan showed clear peaks with abroad one at
about 3401.60 cm-1 and 3398.29 cm-1 most likely for hydroxyl groups (OH),
respectively.
64
Fig.11. UV absorption spectra of A. polyacantha gum collected from
Kadogli.
65
Fig. 12. UV absorption spectra of A. polyacantha gum collected from Edamazine.
66
Fig. 13.UV absorption spectra of fraction 1 of A. polyacantha gum.
67
Fig. 14. UV absorption spectra of fraction 2 of A. polyacantha gum.
68
Fig. 15. FT.IR spectrum of A.polyacantha gum sample collected from Kadogli.
69
Fig. 16. FT.IR spectrum of A. Polyacantha gum sample collected from Eldamazine.
70
Fig. 17. FT. IR spectrum of fraction 1 of A. Polyacantha gum sample.
71
Fig. 18: FT.IR spectrum of fraction 2 of A.polyacantha gum
sample.
72
In addition to two another peaks at 1700-1600cm-1 probably for
carboxyl, aldehyde or ketone groups, the remaining peaks (at 600700cm-1) were specific for the gum type (Mohammed, 2006).
IR spectra for fraction 1 and 2 (Fig. 17, 18), showed seven
peaks typically with abroad one at 3739.35cm-1 mostlikly for hydroxyl
groups (OH), aliphatic (C-H) and two other peaks at 29.22.99cm-1
probably for aliphatic groups (C-H), and anther two peaks at 16001300cm-1 probably for, alkene groups, the remaining peaks ranged
between 1042.85 and 606.24 cm-1.
73
CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusions:
On the basis of results obtained, it could be concluded that:
1-
A.polyacantha gum samples from the two locations showed
similar refractive index value and the same moisture, ash and
Na levels.
2-
Kadogli samples were found to have insignificantly (P≤0.05)
higher specific optical rotation, molecular weight, reducing
sugars and K compared to Eldamazine gum samples.
3-
Eldamazine samples showed insignificantly (P≤ 0.05) higher
intrinsic viscosity, emulsifying stability, water holding capacity,
Ca, Mg and Fe compared to Kadogli gum samples.
4-
Gum samples obtained from Eldamazine showed significantly
(P≤0.05) higher nitrogen, uronic acid and pH levels, whereas
that from Kadogli showed significantly (p ≤ 0.05) higher
equivalent weight.
5-
The two fractions obtained from A.polyacantha whole gum
gave approximately similar refractive index value, specific
optical rotation, intrinsic viscosity, equivalent weight and
molecular weight.
6-
Fraction
1
showed
significantly
(P≤0.05)
higher
pH,
emulsifying stability, uronic acid and conductivity compared to
Fraction 2.
7-
Fraction 2 showed significantly (P≤0.05) higher moisture, ash
and nitrogen contents compared to Fraction 1.
74
8-
High performance liquid chromatography (HPLC) showed that
A.polyacantha whole gum contain 1.5 and 0.49 mg/100g
L+rhaminose and L+arabinose, respectively. On the other hand,
L+rhaminose concentrations were 7.98 and 8.1 mg/100g for
Fraction1 and Fraction 2, respectively. The two fractions
showed negative result to L+arabinose.
9-
The infra red spectra showed functional group peaks and special
peaks for A.polyacantha gum.
5.2 Recommendations:
1-
Further research efforts should be directed to elucidate the
molecular structure of A.polyacantha gum.
2-
More investigations are needed to study the functional
properties of A.polyacantha gum as well as its different
fractions.
3-
The data obtained showed promising value of the gum
compared to A. senegal gum especially for the functional
properties, thus the potential uses of A.polyacantha gum in food
and pharmaceutical industries should be examined.
4-
The gum has high iron content, so that it can be used for
nutritive values.
75
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