In the US, polysaccharides which are to be used as food additives are subject to Generally Regarded As Safe (GRAS) regulations and must be approved by the USDA. Currently only two microbial polysaccharide products have such approval - xanthan and gellan, although curdlan may also be under evaluation. Previously dextran was also an approved food additive, but it is not currently used in food manufacture. In Japan, a wider view is taken and microbial EPS are regarded as natural products. One thus finds the bacterial product curdlan being used in various foodstuffs, while pullulan (from the fungus Aureobasidium pullulans) is also acceptable. In the case of pullulan, the polysaccharide also has potential value as a food packaging material, a role which is also strictly controlled.
Xanthan, a product from the plant pathogenic bacterium Xanthomonas campestris, is typically produced copiously as an extracellular slime by the various pathovars of Xanthomonas campestris as well as by some other Xanthomonas species. Acid hydrolysates of xanthan contain D-glucose, D-mannose, and D-glucuronic acid in the molar ratio 2:2:1. Other components have been reported in polysaccharides from bacterial isolates described as Xanthomonas species, but the strains involved have generally been poorly characterized. The primary structure of the polysaccharide is a pentasaccharide repeat unit, effectively a cellulose chain to which trisaccharide side chains are attached at the C-3 position on alternate D-glucosyl residues (38,39) (Figure 9.9). The polysaccharides from several other Xanthomonas species seem to share the same composition as that from pathovars of X. campestris. Depending on the bacterial strain and on the physiological conditions for bacterial growth, the polysaccharide may carry varying amounts of O-acetyl groups on the
Typically the internal a-D-mannosyl residue is fully acetylated but only c. 30% of the mannosyl termini are ketalated.
Figure 9.9 The structure of xanthan from Xanthomonas campestris (38).
C-6 position of the internal a-D-mannosyl residue and of 4,6-carboxyethylidene (pyruvate ketal) on the side chain terminal ^-D-mannosyl residue respectively. Material from some strains (not used for food) carries two acetyl groups on the internal mannosyl residue, and there is some evidence to suggest that certain xanthan preparations may even contain in excess of 2 moles of acetate per repeat unit. However, the material used as a food additive derives from a standard strain (frequently designated NRRL B-1459) and generally contains pyruvate and acetate in approximate molar ratios of 0.3 and 1.0 per pentasaccharide repeat unit.
X. campestris will grow and produce EPS on a wide range of carbon substrates including amino acids, citric acid cycle intermediates, and carbohydrates. Either ammonium salts or amino acids can be used as nitrogen sources. Various ions are needed for bacterial growth and polysaccharide synthesis. Limitation of any of the ions required for substrate uptake or for precursor or polymer synthesis can affect the yield and properties of the EPS. Xanthan formation by X. campestris resembles many other bacterial-EPS-producing systems in that polymer production is favored by a high ratio of carbon source/limiting nutrients such as nitrogen. Typically, media for laboratory synthesis of xanthan contain 0.1-0.2% ammonium salt and 2-3% glucose or sucrose. Xanthan can even be produced in fairly good yield when the bacteria are grown in a simple synthetic medium composed of glucose, ammonium sulphate, and salts, but production is improved in the presence of organic nitrogen sources. The quality and the final yield of xanthan may be enhanced by the addition of small amounts of organic acids or of citric acid cycle intermediates such as a-ketoglutaric acid (40). Oxygen is required for growth and for xanthan production, and as the culture viscosity increases as xanthan is formed, oxygen may rapidly become limiting. For satisfactory cultivation in a fermenter, vigorous aeration was essential and fermentation vessels had to be designed to ensure minimal dead space, which would otherwise lead to stagnant areas of culture. The xanthan production process is complicated by mass transfer reactions in the high viscosity broths. Oxygen solubility decreased with increasing xanthan concentration and the diffusion constant of oxygen in dilute solutions of the polysaccharide was reduced relative to water (41). The presence of polysaccharide in the fermentation broth may also affect the availability of carbon substrates such as glucose and sucrose by interaction with these carbohydrates. Consequently, fed-batch culture is usually preferred. Nakajima et al. (42) suggested that in the design of the fermentation vessel, the volume exposed to high shear is of critical importance. The specific rate of xanthan production depended on the volume of the high shear region.
Xanthan is produced throughout growth of X. campestris and in the stationary phase. The specific rate of xanthan synthesis was closely related to the bacterial growth rate in batch culture; maximal during exponential growth and minimal during the stationary phase. In this respect, xanthan production resembled the synthesis of several other bacterial exopolysaccharides. Differences could be seen in the viscosity and the acylation of the xanthan synthesized in nutrient limited media during various phases of the growth cycle (43,44). Acetyl CoA and phosphoenolpyruvate may not be readily available during certain stages of growth to permit the complete acylation of the xanthan repeating units. Consequently, the xanthan synthesized in batch culture represents the products of all phases of growth and is possibly a mixture of several molecular types with varying degrees of acylation and varying mass.
In the laboratory, pure substrates such as glucose or sucrose are used, whereas in industrial production different substrates are employed. The substrates must be cheap, plentiful carbon sources, which frequently include starch, starch hydrolysates, corn syrup, molasses, glucose, and sucrose (derived from either sugar beet or from sugar cane). It is imperative that they also be acceptable for food use. Optimal synthesis of xanthan requires a balance between utilizable carbon and nitrogen sources. Care must therefore be taken to obtain consistency in yield and product quality when using substrates which may contain nitrogen in addition to carbohydrate. The nutritional versatility of X. campestris is clearly a major factor in favor of its use for commercial xanthan production. However, the quality of the xanthan produced from different substrates may vary considerably — the molecular weight and hence the rheological characteristics of xanthan synthesized from glucose or starch may well differ from that formed when whey or other proteinaceous material is employed. The nitrogen sources used for industrial production of xanthan may include yeast hydrolysates, soybean meal, cottonseed flour, distillers' solubles, or casein hydrolysates. In batch culture for industrial production of the polysaccharide, there must be careful control of pH and of the aeration rate. Adequate oxygen transfer may be difficult to achieve unless the fermentation vessel has been carefully designed to ensure that mixing is optimal. To minimize this problem, fed-batch processes may be used. Even so, the conditions used for production and processing must be carefully standardized to ensure that product yield and quality are consistent. In the laboratory, the polymer can be readily prepared in high yield, and then separated from the bacterial cells by high speed centrifugation or by precipitation with quaternary ammonium compounds. As an industrial product, it is manufactured in stirred tank fermenters on a large scale by batch or fed-batch fermentation by a number of commercial companies; normally fermentation proceeds for 3 days at 30°C. In this respect, Linton (45) has claimed that "exopolysaccharide production is a very efficient process" and calculations have shown that the conversion efficiency of substrate to xanthan is very high. After completion of fermentation, the product is subjected to heat treatment to eliminate viable bacteria and to destroy hydrolytic enzymes such as cellulases, amylases, pectinases, and proteases. This treatment also enhanced the rheological properties of xanthan in solution (46). Recovery of the polysaccharide from industrial cultures requires removal or destruction of the cells followed by precipitation with the polar organic solvent isopropanol. To reduce the cost of the process, the solvents are later recovered for reuse by distillation. The fibrous precipitate is dried, milled, and sieved to give material of different mesh sizes. Bacterial cells are difficult to remove from the highly viscous culture broth, although pasteurization may lead to some autolysis and degradation of cell material as well as improving the subsequent separation of cells from the polymer. Polysaccharide recovery in the presence of polar solvents may be also be increased or accelerated by the addition of electrolytes. Subsequent purification can be achieved by a variety of techniques including fractional precipitation and chromatography. Dilute solutions of xanthan may also be subjected to clarification by filtration. The purified xanthan material can then be subjected to various analytical procedures to verify the composition, and to standard techniques for the determination of polysaccharide structure. The procedures used in the production and recovery of xanthan have been reviewed by Garcia-Ochoa et al. (47) and Galindo (46).
Although it is not degraded in the human or animal body, xanthan is biodegradable. It is a substrate for a range of xanthanases (enzymes with endo-1,4-^-D-glucanase activity cleaving the main chain of the polysaccharide) and xanthan lyases. Most crude enzyme preparations contain at least two different types of enzyme activity, although there may also be associated glycosidases (Figure 9.10). Substrates are randomly cleaved to yield oligosac-charides of different sizes; there is accompanying rapid loss of solution viscosity. Most commercial cellulase preparations lack activity against xanthan unless the polysaccharide is in dilute, ion free solution (48,49). The second type of enzyme found associated with the bacterial preparations is a xanthan lyase (4, 5 transeliminase) which cleaves the ^-D-mannosyl-D-glucuronic acid linkage of the trisaccharide side chains (50). The activity of the xanthan
-ß-D-Glc (1^4) - ß-D-Glc (1^4) ß-D-Glc (1^4) ß-D-Glc (1^4) ß-D-Glc
-Glucanohydrolase II T
-Glucanohydrolase I T \ T
^ a-Mannosidase 11
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