Biochemical and microbiological changes in sugarcane stalks during a simulated harvest-to-crush delay.
Post-harvest cane deterioration in the South African sugar industry results in significant revenue loss that is estimated to be in the region of ZAR 60 million per annum. Despite these large losses, precise biological data relating to the process of cane deterioration under South African conditions is limited. Severity of deterioration is influenced by a number of factors, including the length of the harvest-to-crush delay (HTCD), ambient temperature and harvesting practices. For example, burning of cane prior to harvest may result in rind splitting, which provides entry for microbes, particularly Leuconostoc mesenteroides that may exacerbate deterioration. The effect of these factors on deterioration was examined by quantifying the biochemical and microbiological changes that occur in sugarcane stalks after harvest, with the influence of length of HTCD, degree of L. mesenteroides infection and ambient temperature receiving attention. The primary novelty of the work resides in the analysis of deterioration under tightly regulated temperatures, which were designed to reflect diurnal variations typically experienced during summer and winter in the South African sugar belt. In addition, inoculation of mature internodes with a consistent titre of L. mesenteroides was used as a means to mimic a consistent level of infection of harvested stalks by the bacterium. Metabolites selected for analysis were those both native to the stalk and produced as by-products of microbial metabolism, viz. sucrose, glucose, fructose, ethanol, lactic acid, dextran and mannitol. Simulated HTCDs under summer temperatures resulted in increasing glucose and fructose levels with time, which contrasted to the approximately constant levels of these hexose sugars under winter conditions. Commonly referred to as ‘purity’ in an industrial context, precise determination of the concentration of these hexoses in cane consignments could potentially indicate the extent of deterioration. Despite the detection of a basal concentration of lactic acid in unspoiled cane, the observed increase in concentration of this organic acid over the simulated summer HTCD suggests that this metabolite could also potentially serve as an indicator for postharvest deterioration. In contrast, the investigation indicated that ethanol was an unsuitable biochemical marker for deterioration of L. mesenteroides infected cane. An inability to detect dextran and mannitol in the samples, combined with consistent sucrose levels and variable mill room data, suggest that extreme proliferation of L. mesenteroides is facilitated primarily by in-field practices, particularly the manner in which cane is prepared prior to harvest and transport to the mill. Bacterial proliferation and infection by L. mesenteroides of inoculated stalks were monitored by standard selective culturing techniques. Despite the limited detection of L. mesenteroides-associated metabolites, culture-based analyses revealed that the bacterium was the dominant bacterial species within the samples. A number of other bacterial species were isolated and identified, however the extent to which the total number of microorganisms proliferated was limited to a maximum of 1 x 105 colony forming units per gram of fresh tissue. In conjunction with these analyses, a molecular approach known as Polymerase Chain Reaction-Mediated Denaturing Gradient Gel Electrophoresis (PCR-DGGE) was undertaken to investigate the bacterial diversity patterns associated with deteriorating sugarcane stalks throughout the delay period. In contrast to the results obtained by means of the culture-based assays, PCR-DGGE revealed that L. mesenteroides was not the dominant bacterial population, and showed that the level of bacterial diversity was relatively consistent across the differing treatments and with time. The use of complimentary culture-dependent and cultureindependent analyses thus permitted the detection of this discrepancy and indicated the utility of PCR-DGGE in the determination of bacterial community structure of postharvest sugarcane tissue. The biology of post-harvest deterioration of green sugarcane stalks is highly complex, even under rigorously controlled temperature and infection regimens. The results of this study emphasize the important effects that harvest method and environmental conditions have on post-harvest sugarcane deterioration. Towards the formulation of industry-relevant recommendations for combating post-harvest deterioration, future work will strive to mimic the effects that harsh harvesting and transport practices have on the severity of the problem.