UBC Theses and Dissertations
A comparison of the carbohydrates of the coffee bean Coffea arabica L. and those of suspension-cultured coffee cells Kocal, James Thomas
Suspension cultures of coffee cells derived from the coffee plant Coffea arabica L. were grown in three media to determine the effect of hormones on extracellular polysaccharide production and composition and on the constituent sugars of the cells. Fxtracellular polysaccharide was produced in both the logarithmic and stationary phases of cell growth. Polysaccharide production paralleled cell growth in all three media. The rate of polysaccharide production decreased at the beginning of the stationary phase, so that the yield was determined by the duration of the phase of rapid cell growth. Maximum yields of extracellular polysaccharide occurred at day 8 and ranged from 131 - 142 mg/100 ml for the three media. Arabinose, xylose, mannose, glucose,^galactose and galacturonic acid were detected as components of the extracellular polysaccharide isolated from the media. The relative proportions of the monosaccharide constituents varied with hormonal treatment. The presence of 2,4-D in the medium led to the accumulation of large amounts of arabinose and lesser amounts of hexoses in the extracellular polysaccharide from the PRL-4 medium. This is believed to be due to an enhancement of the myo-inositol oxidation pathway. The extracellular polysaccharide of cells grown in the Fox-3H medium contained large amounts of hexose sugars and lesser quantities of pentose and galacturonic acid. A pool of free inositol was found within the cells, which indicates that the myo-inositol pathway was operating at a reduced level. The polysaccharide produced by cells grown in a high kinetin medium had an increased quantity of glucosyl and arabinosyl units and a decreased proportion of galactose, xylose and mannose. The change in composition in the presence of a high level of kinetin could result from suppression of the activities of enzymes involved in monosaccharide interconversions, thereby limiting the availability of galactose, xylose and mannose for incorporation. Three polysaccharides were isolated by fractionation of the mixed polysaccharide produced by Fox-3H suspension-cultured cells. The acidic fraction was selectively precipitated as a copper salt. The neutral polysaccharides were fractionated following absorption on cellulose; elution with water gave an arabinogalactomannan and elution with 7M urea and 0.5N NaOH gave two xylogalactoglucan fractions which were indistinguishable . The free sugars found within the coffee cells were sucrose, fructose, glucose and inositol. Fructose accumulated in massive quantities in the cells and spent media. These data indicate that sucrose was taken into the cells both as the disaccharide and as monosaccharides (degraded by invertase). The glucose moiety of the sucrose molecule was then preferentially used as a precursor to other metabolites. The sugar content of green and roasted coffee beans was compared to determine the effect of roasting on this component. Sucrose was calculated to be 6.5% of the green bean dry weight. Traces of fructose and glucose were also identified. Only trace amounts of sucrose were found in the roasted bean, indicating that the sucrose is almost entirely decomposed in the roasting process. Analysis of the water soluble polysaccharide isolated from the green bean indicated that it was an arabinogalactomannan (similar to that found in the extracellular polysaccharide). A water-insoluble pectin isolated as an ammonium oxalate extract of the green bean powder was found to consist of arabinose, rhamnose, mannose, galactose and galacturonic acid, resembling the acidic fraction of the extracellular polysaccharide. In an attempt to identify some of the more volatile constituents responsible for coffee aroma, 15 compounds were separated and tentatively identified. The effect of roasting on the formation of these volatile constituents was determined by analyzing the volatiles in the headspace over freshly ground coffee beans sampled at each minute of roast. In general, the concentration of volatiles increased with roasting time and temperature.
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