Linamarase application exists in biotechnology such as potentiometric sensors for linamarin by coupling linamarase from cassava leaf with a cyanide ion-selective electrode and to measure glucose in biomedical applications. It is used in a batchwise process to detoxify fermenting cassava during ‘garri’ production. Linamarase along with its naturally occurring substrates, linamarin and lotaustralin, is found in a variety of edible plant tissues such as those of cassava from which garri is produced. However, the separation and purification of linamarase at reasonable large quantity for these applications from plants has been a challenge. In the study a miniflex Ultrafiltration (UF) Cross Flow obtained from Schleicher and Schuell (Germany) was used for linamarase isolation and purification from cassava tissues. Membranes with different pore sizes of 0.45, 0.2, 0.1 and 0.02 μm, made from polyethersulfon screnes and silicone adhensives, with surface area of 2.4 mm2, were experimented. Fluxes were observed to decrease very sharply from 0.45 to 0.02μm membrane pore sizes. No permeate was collected from 0.1 and 0.02 μm membranes due to concentration polarisation and clogging of these membranes. Permeate and retentate from 0.45 and 0.2 μm membrane contained linamarase, while the retentate of the 0.1 and 0.02 μm membranes contained linamarse and that no permeate was collected from 0.1 and 0.02 μm membranes due to the fouling and clogging of the small membrane pores. It was therefore concluded that linamarase was finally purified by the 0.2 μm membrane. A simple mathematical model derived from the Hagen-Poiseuille equation could not predict the linamarase flux data, perhaps due to the effect of concentration polarisation, which led to the proposition of the Langmuir adsorption isotherm. It was interesting to observe that the plot of 1/v versus 1/Δp from the use of the Langmuir equation gave a linear relationship from which the linamarase flux iii was predicted. The standard error between the experiment and the model was 0.011, which is a good measure of the agreement between data. The Langmuir adsorption isotherm therefore predicts the fouling and concentration polarisation of the membrane during linamarase purification from cassava tissues. This proposition was supported by the solute deposits on the pores and surface of the membrane where van der Waal forces were created between the molecules, thus resulting in the fouling and chemical polarisation.

ABSTRACT Cassava is a known source of linamarin, but difficulties associated with its isolation have prevented it from being exploited as a source. A batch adsorption process using activated carbon at the appropriate contact time proved successful in its isolation with ultrafiltration playing a pivotal role in the purification process. Result revealed that optimum purification was obtained with increasing amount of crude cassava extract (CCE) purified. 60g of CCE took 32 mins, 80 g, 34 mins while 100 g took 36 mins of contact time, where 1.7 g, 2.0 g and 2.5 g of purified product were obtained, respectively. The purification process in batch mode was also carried out at different temperatures ranging from 25 to 65oC. Results showed that purification increases with increase in temperature. In a bid to ascertain the moles of linamarin adsorbed per pore volume of activated carbon used, the composite isotherm was found to represent the measured adsorption data quite well. The adsorption of linamarin was used to study the goodness of fit criteria (R2) for the entire process. Results showed that R2 value was best with decreasing amount of CCE purified (R2=1 for 60 g) at the temperature of 45oC. Compound elucidation of purified product by Picrate paper test, IR and 1HNMR confirmed the structure of linamarin. Cytotoxic effects of linamarin on MCF-7, HT-29, and HL-60 cells were determined using the 3 - (4, 5 – dimethylthiazol-2-yl) – 2, 5 – diphenyltetrazolium bromide (MTT) assay. Cytotoxic effects were significantly increased in the presence of linamarase, which catalysed the hydrolysis of linamarin to hydrogen cyanide. A 10–fold decrease in the IC50 values obtained for linamarin or crude extract in the presence of linamarase was determined for HL-60 cells. This study thus describes a method for the isolation and purification of linamarin from cassava, as well as the potential of this compound as an anticancer agent.

faculty of Science School of Molecular and Cell Biology 9605145v vhaakuria@yahoo.co.uk / Cassava is a food crop planted in many countries in Africa. Its tubers are a major source of food and are processed to produce a variety of food products, one of which is the fermented product called gari. This research report aimed to evaluate the performance of three lactic acid bacteria for several properties with regard to the fermentation of cassava to produce gari. Three organisms were used for the evaluation, namely Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides. The organisms were evaluated for viability, biomass formation and glucose utilisation in static flasks, biomass formation and glucose utilisation in 2 L fermenters, cell viability after dehydration processes and pH and cyanide reduction in cassava substrate. In static flasks, the organisms were found to retain above 80% cell viability after cryopreservation. Maximum biomass of 108 cells/ml was formed within the first 12 hours by all the organisms. While L. fermentum, depleted glucose within 24 hours, L. plantarum formed the highest biomass of 4 x 108 cells/ml. In 2 L Braunstat B fermenters, a cell count of 109 cells/ml was obtained by L. fermentum and Leuconostoc mesenteroides within 12-15 hours. Biomass formation for L. plantarum during the same period was 1010 cells/ml. Glucose was depleted within 12 - 15 hours. The viability of cells between the dehydration processes of centrifugation, glycerol and maltodextrin addition and lyophilisation, was above 80% for all the organisms. However, this high cell viability was influenced by concentration of cells during the centrifugation step. In cassava substrate, L. fermentum, though heterofermentative, was found to be particularly acid tolerant and reduced pH to 3.98. All the organisms were able to retain good viability after lyophilisation. However, the results of cyanide reduction were inconclusive. These results show that while cultures show promise for pilot scale studies of starter culture development, further cyanide experiments need to be conducted, and synergy between the organisms investigated.

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