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Acacia Senegal) (i) DEGRADATIVE AND ANALYTICAL STUDIES OF PLANT GUM EXUDATES WITH PARTICULAR REFERENCE TO GUM ARABIC (ACACIA SENEGAL) (ii) THE MECHANISM OF INTERACTION BETWEEN UNLIKE CELLULOSIC ETHERS AND GALACTOMANNANS IN SOLUTION. by N.A.MORRISON B.Sc. Thesis presented for the degree of Doctor of Philosophy University of Edinburgh Aug 1993 The research in this thesis comprises of two sections. The first section of the research (Chapters I-IV) investigates structural degradations and analytical studies of plant gum exudates, carried out at Edinburgh University. The second section (Chapters V-Vu) investigates and develops a mechanism of interaction which exists between various water-soluble cellulose ethers and galactomannan gums in solution. The thesis was sponsored by Courtaulds Fine Chemicals, who produce several commercial cellulose ethers. I hereby declare that this thesis was composed by myself and that it is based upon results of original research experiments, carried out by me (unless otherwise stated) within the Chemistry Department, University of Edinburgh, and Courtaulds Fine Chemicals Research Department, Coventry, from October 1988 to September 1991. None of the work included in this thesis has been submitted for any other degree or professional qualification. Some of the analytical data reported in Chapters III and IV have been published or are in press; e.g. (a)D.M .W .Anderson, D.M.Brown-Douglas, N.A.Norrison and W.Wang, Food AddiJ. Contam; 1990, 1, (3), 303. (b)D.M.W.Anderson and N.A.Morrison, Food Addit. Contain; 1990, 1, (2), 181. (c)D.M.W.Anderson and N.A.Morrison, Food Addit. Contain; 1990, 1, (2), 175. (d)D.M.W.Anderson and N.A.Morrison, Fond Hvdrocolloids; 1989, a, (1), 57. The research findings from Chapter VII have been presented at the Cellucon 1993 Conference in Lund, Sweden and a publication is currently in press. Paste CHAPTER I GENERAL INTRODUCTION CHAPTER IT EXPERIMENTAL METHODS 11.1 GENERAL METHODS 14 YI.2 PHYSICAL METHODS 19 11.3 CHEMICAL METHODS 20 CHAPTER III DEGRADATIVE STUDIES ON GUM ARABIC III INTRODUCTION 23 111.1 EFFECT OF IRRADIATION ON THE STRUCTURE AND FUNCTIONALITY OF GUM ARABIC 111.1(i) Introduction 28 111.1(u) Materials and methods 32 III.1(iii) Results and discussion 33 111.1 Conclusion 47 111.2 MILD SEQUENTIAL DEGRADATIONS OF GUM ARABIC 111.2(i) Introduction 48 111.2(11) Experimental Methods 51 III.2(iii) Results and discussion 52 111.2 Conclusion 65 111.3 DEPROTEINATION AND FRACTIONATION OF GUM ARABIC 111.3(i) Introduction 67 111.3(u) Materials and methods 71 III.3(iii) Results and discussion 74 111.4 FRACTIONATION OF GUM ARABIC BY EXTRACTION WITH LIMONENE 111.4(i) Introduction 90 111.4(u) Materials and methods 92 III.4(iii) Results and discussion 93 111.4 Conclusion 97 CHAPTER IV AN ANALYTICAL STUDY OF GUM EXUDATES. IV.I VARIATION IN GUM ARABIC SAMPLES COLLECTED BETWEEN 1958-1988 IV.I(i) Introduction 104 IV.I(ii) Materials and methods 106 IV.I(iii) Results and discussion 108 IV.I(iv) Conclusion 124 IV. II AN ANALYTICAL STUDY OF SIX COMBRETUM GUM EXUDATES IV. II( i) Introduction 126 IV. II(ii) Materials and methods 129 IV. II(iii) Results 130 IV. II(iv) Discussion 130 IV.III AN ANALYTICAL STUDY OF FOUR PROTEINACEOUS ACACIA GUM EXUDATES IV.III(i) Introduction 139 IV.III(ii) Materials and methods 141 IV.III(iii) Results and discussion 142 IV.IV AN ANALYTICAL STUDY OF SEVEN ALBIZIA GUM EXUDATES IV.IV(i) Introduction 149 IV.IV(ii) Materials and methods 150 IV.IV(iii) Results and discussion 151 THE MECHANISM OF INTERACTION BETWEEN WATER-SOLUBLE CELLULOSIC POLYMERS IN SOLUTION. CHAPTER V. INTRODUCTION 166 CHAPTER VI. EXPERIMENTAL METHODS General methods 187 Analytical methods 189 Abbreviations and terminology 193 CHAPTER VII. RESULTS AND DISCUSSION VII(i) Introduction to synergistic and 195 antagonistic polymer blends. VII(ii) Hydrodynamic volume study on 202 polymer blends. VII(iii) Effect of modifying CMC structure 211 by varying free carboxyl content. VII(iv) Mechanism 2 - 222 Competitive dehydration. VII(v) Effect of variation in degree 232 of substitution. VII(vi) Variation in the fine structure 245 of guar gum. VII(vii) Solid state N.M.R on a synergistic polymer blend. 253 VII(viii) Rheological properties of polymer 257 blends. VII(ix) Competitive inhibition in polymer 264 blends. VII(x) Effect of gamma irradiation of guar on polymer interaction 266 VII(xi) Effect of substituent on non- 268 ionic cellulosic polymer. VII(xii) Effect of temperature and 276 electrolytes on blend viscosity. VII(xiii) An example of antagonism in a blend of an anionic and non-ionic 280 cellulosic polymer. VII Conclusion. 284 PUBLICATIONS REFERENCES ARE TO BE FOUND AT THE END OF EACH CHAPTER. I would like to thank the Chemistry Department, University of Edinburgh, for the use of its laboratory facilities and for helpful assistance rendered by various members of staff, in particular Mrs. E. McDougal, Departmental Analyst for determining amino acid analysis, and Mr J. Millar for running 13C N.M.R spectras, and members of the Analytical Department in Courtaulds Research, Coventry, in particular Dr. R.Ibbet for N.M..R spectroscopy analysis. My sincere thanks go to my supervisor, Dr. D.M.W. Anderson for his help and encouragement throughout this period of study, and to Mr A.J.Fowler (Technical manager) of Courtaulds Fine Chemicals Research, Coventry. I am indebted to Courtaulds Fine Chemicals for financial support of this study. 1 The technological (i.e. non-food) use of water-soluble polymers dates back thousands of years. Polysaccharide gums are hydrocolloids, and a wide range of applications in the construction, detergent, mining, oil well drilling, food and pharmaceutical industries has been developed since World War II. The natural gums can be classified into three distinct categories according to raw material source; plant gum exudates, seaweed extracts and seed endosperm gums (1). The polysaccharide gums of interest in this section of the thesis are the plant exudate gums. Exudate gums are of considerable importance (2) commercially and as the products of the specific wound response (3) gummosis, are also of biological interest. The ability of these exudate gums to dissolve readily in water to give viscous solutions makes them attractive commercially. They are widely used in cosmetics, tablet coatings, adhesives and paints (4). They are commonly used as thickeners, suspending and stabilising agents. Other uses include film forming properties, lubricating agents, and binding agents (5). However their main use is in the food industry as a food additive or as an ingredient in confectionery. Unlike many of the polysaccharides which are used in foods to alter (6) the rheology of the solution by thickening, ie. as a viscosity modifier or 2 as gelling agents at 1-2% polymer concentrations, some exudate gums are soluble up to 50% solution concentrations and are surface active, that is they can stabilise an oil in water emulsion (7). The term "gum' designates a wide range of natural products in the form of tears, flakes, or angular fragments, but most commonly as clear amber oval nodules, which are sticky in nature and are exuded by certain tropical trees. This section of the thesis investigates degradative studies of Acacia senea1 (gum arabic) and an analytical study of gum exudates, from the Acacia (8), Combretum (9), and Albizia genera (10). No Combretuin or Albizia gum exudates are permitted as food additives (11), although they have reportedly been, and still may be, sold in blends as adulterants or as substitutes for gum arabic. Almost all the worlds output of gum arabic is from the sub-Sahara or Sahel regions of Africa. However other geographical sources of gum exudates for technological uses include; Australia, India, and South America from the stems of sub-tropical shrubs and bushes. Despite much structural elucidation of these gums in recent decades, the precise mechanism of gum biosynthesis is not totally understood (12), exudation usually following mechanical injury or microbiological infestation of the bark. Possible mechanisms of gum formation have been postulated (3) including; enzymatic modification of starches or 3 cellulosic material, bacterial action or by direct synthesis. It is interesting to note that the formation of a specific gum e.g. gum arabic, by a certain species of tree e.g. Acacia senegal, has been remarkably constant over several decades during which the variation in its analytical parameters has been studied Functionally, the plant gums may act to seal off wounds to prevent further injury and inhibit tissue dehydration in the hot arid climate. Chemically the plant gum exudates have previously been regarded as complex, partially neutralised salts of acidic hetero-polysaccharides The major neutral sugars which occur commonly in the gums in varying proportions are D-galactose, L-arabinose, L-rhamnose, whilst smaller quantities of D-xylose and D-mannose occur in certain species. The acidity of the gums commonly arises from the presence of D-glucuronic acid and its 4-0-Methyl derivative, but small proportions of D-galacturonic acid occur in certain genera. Although some studies had reported a small protein content in certain gums as early as 1954 (15,16), other earlier investigators ignored, or failed to analyse for, the presence of nitrogen in plant gums (17). Although it was shown to be functionally important and not entirely isolatable, the precise location of the protein in certain gum exudates is still under considerable debate. Certain gums although essentially polysaccharide in nature are now more 4 correctly referred to as proteoglycans with a protein content reported as high as 53% in Acacia difficilis gum (18,19). Chapter III of this thesis presents results of degradative studies on gum arabic, which is the gum exuded by Acacia senegal.
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