Changes in the chemical composition of sugar cane (Saccharum officinarum) during storage.
An outline is given of the South African sugar industry, with particular emphasis on the unit operations which make up the industrial process for manufacturing sugar from cane. Current knowledge of the chemistry of soluble polysaccharides is reviewed and the structures of several polysaccharides, including starch, dextran, and pullulan, are discussed. It has been found that changes take place in the chemical composition of the juice in sugar cane (Saccharum officinarum) during post-harvest storage. With increasing storage time, there is a proportional decrease in the starch content of the juice, and a considerably larger proportional increase in the soluble polysaccharide content. The increased polysaccharide content was found to be due to a single glucan which, contrary to most previous publications on this subject, is definitely not a dextran. Following structural analysis, it has been established that the polysaccharide formed in stored cane had not been described before and the name "sarkaran" , derived from the Sanskrit word "Sarkara", meaning "sugar" is proposed for it. The polysaccharide was isolated from cane juice by precipitation with ethanol after the starch in the juice had been removed by centrifugation. The polysaccharide was purified by repeated dissolution in water and reprecipitation with ethanol. Analysis by gel chromatography resulted in a single symmetrical peak, indicating that the isolated polysaccharide is homogeneous. This was confirmed by hydrolysing fractions representing a section of the ascending and a section of the descending part of the peak of the chromatogram, using the enzyme pullulanase. Chromatographic separation and quantitative analysis of the isolated oligosaccharides showed that the compositions of the two enzymes digests were identical. Acid hydrolysis of the polysaccharide resulted in a single hexose. This was identified as glucose by paper chromatography, comparing the Rf value with that of pure glucose. Confirmation was obtained by comparing the osazone with that of glucose, using microscopic examination and determination of the melting points. Paper electrophoresis showed the molecule to be uncharged. Several techniques, both absolute and non absolute, were used to determine the molecular weight of the polysaccharide. A method involving viscosity determination indicated a molecular weight of 34 000 while a figure of 50 000 was obtained by gel chromatography on a Sephadex column, comparing the peak elution volume of the polysaccharide with that of dextrans of a defined molecular weight. Both these techniques are non absolute and yield rough estimates of the molecular weight. Osmometric measurement, an absolute method, showed the number average molecular weight to be 51 500. An absolute value for the weight average molecular weight of 250 000 was obtained by light scattering techniques. Data from the light scattering experiments were also used to determine a value of 200 - 250 A for the radius of gyration RG of the polysaccharide. End group analysis after exhaustive methylation resulted in a value of 24 000 for the number average molecular weight Mn. This indicates either that some degradation of the polysaccharide molecule occurs , during the methylation procedures or that there is a certain degree of association between individual molecules. Periodate oxidation showed that 32 percent of the glucosidic linkages are in ( 1 + 6 ) position. The polysaccharide was exhaustively methylated by several Haworth methylations followed by a number of Kuhn methylations. The fully methylated product was methanolysed and the methyl glucopyranosides analysed by gas liquid chromatography. The results were compared with those obtained from fully methylated starch and dextran. From the absence of disubstituted methyl derivatives in the methanolysate it was concluded that the polysaccharide is an unbranched glucan. From the quantities of Methyl 2,3,4,6 tetramethyl-O-Dglucopyranoside, Methyl 2,3,6, trimethyl-O-D-glucopyranoside and Methyl 2,3,4? trimethyl-0-D-glucopyranoside, it was concluded that the only linkages in the glucan are ( 1 + 4 ) and ( 1 + 6 ) and that these are present in the ratio 68:32. Enzymic hydrolysis, using pullulanase, was followed by paper chromatographic separation. Quantitative determination of the oligo-saccharides present in the enzyme digest resulted mainly in two oligosaccharides, maltotriose and maltotetraose, in nearly equal proportions. For this reason it was postulated that the polysaccharide is a maltotriose-maltotetraose polymer, and that the individual units are linked in ( I + 6 ) position, a linkage for which pullulanase is specific in certain configurations. The sequence of the maltotriose and maltotetraose units in the polymer has not been investigated further, although this could be carried out by partial acid hydrolysis, followed by isolation and identification of the various oligosaccharides formed. An alternate method for the determination of the sequence of the monomers is discussed. It was subsequently shown that the linkages in the polysaccharide are in the a configuration. The polysaccharide is highly dextra rotary and the magnitude of the rotation is comparable to that of other polysaccharides linked in a position, . such as starch and dextran. Infrared spectroscopy was used to confirm the configuration. The spectrogram of the polysaccharide contained an absorption peak at 840 cm-1 , which is typical of the a-anomeric absorption occurring, for example, in the IR spectrum of starch. The spectrogram exhibited no absorption peak at 891 cm-1 , the wavelength typical of the B-anomeric absorption in the IR spectrum of cellulose. In addition, it was found that all polysaccharides containing a ( 1 + 4 ) linkages show an absorption peak at 700 cm 1. This absorption peak was absent in all IR spectra obtained from various dextrans. This phenomenon has not been reported previously and it is suggested that the presence of this absorption peak in the IR spectrum of a glucan can be used to support the evidence of the presence of a( 1 + 4 ) linkages. It was not possible to correlate the formation of the polysaccharide with the occurrence of a specific micro organism. It is suggested that the formation of the polysaccharide is the result of enzymic reactions in the sugar cane after harvesting. The investigation of the composition of juices from deteriorated cane has not been confined to polysaccharides. Ethanol has been isolated from the juice of some samples of stored cane which had been burnt before harvesting. The ethanol was isolated by fractional distillation and identified by measurement of the boiling point. It was confirmed, by the formation of the molybdate-xanthate complex, that the product isolated was an alcohol. The identification was further confirmed by oxidising the ethanol to acetic acid and proving the identity of the acids by paper chromatography. It has been shown that, with the exception of two acids, the carboxylic acid composition of cane juice remains unaltered during post-harvest storage of the cane. The two exceptions , succinic and aconitic acids, were identified from their melting points and by specific spot tests. Ion exchange was used to isolate the acids from the juice. The eluate from the ion exchange column was concentrated and the acids separated by liquid-liquid chromatography, using a silica gel column. The levels of both aconitic and succinic acids were found to increase during the early period of storage but decreased again slowly thereafter. The percentage change was greater in the case of succinic acid, although aconitic acid was the most abundant carboxylic acid in the juice. Lactic acid was absent from the cane juices analysed. This is surprising, as lactic acid is a common product of the metabolism of carbohydrates by micro organisms. It is suggested that the changes in acid composition during the storage of harvested cane are caused by deactivation of enzymes of the Krebs cycle. Post-harvest deterioration of sugar cane can have serious consequences which can affect the whole Sugar Industry. Not only is crystallisable sugar lost but the products of the deterioration have adverse effects on factory processing and laboratory analysis. The problem, which will become more acute with the introduction of mechanical cane harvesting, can only be resolved through the cooperative efforts of all the parties concerned.