Effect of physicochemical parameters on enzymatic biodecaffeination during tea fermentation.

We report for the first time the development of a biodecaffeination process for tea synchronised with tea fermentation process using enzymes isolated from Pseudomonas alcaligenes. Cell-free extract was used for biodecaffeination of tea during fermentation of tea and 80% of the caffeine in the tea dhool was degraded within 90 min of incubation. Several factors that tend to effect the biodecaffeination during this stage, like moisture, aeration, intermittent enzyme addition and mixing, were optimized, and inhibitory interactions of proteins with polyphenols, caffeine-polyphenol interactions, which directly influence the biodecaffeination process were prevented by the use of glycine (5% w/w) in the dhool. Tea decaffeinated through the enzymatic route retained the original flavor and aroma, and there was an increase in the total polyphenol content of the tea.

Development of a biosensor for caffeine.

We have utilized a microbe, which can degrade caffeine to develop an Amperometric biosensor for determination of caffeine in solutions. Whole cells of Pseudomonas alcaligenes MTCC 5264 having the capability to degrade caffeine were immobilized on a cellophane membrane with a molecular weight cut off (MWCO) of 3000-6000 by covalent crosslinking method using glutaraledhyde as the bifunctional crosslinking agent and gelatin as the protein based stabilizing agent (PBSA). The biosensor system was able to detect caffeine in solution over a concentration range of 0.1 to 1 mg mL(-1). With read-times as short as 3 min, this caffeine biosensor acts as a rapid analysis system for caffeine in solutions. Interestingly, successful isolation and immobilization of caffeine degrading bacteria for the analysis of caffeine described here was enabled by a novel selection strategy that incorporated isolation of caffeine degrading bacteria capable of utilizing caffeine as the sole source of carbon and nitrogen from soils and induction of caffeine degrading capacity in bacteria for the development of the biosensor. This biosensor is highly specific for caffeine and response to interfering compounds such as theophylline, theobromine, paraxanthine, other methyl xanthines and sugars was found to be negligible. Although a few biosensing methods for caffeine are reported, they have limitations in application for commercial samples. The development and application of new caffeine detection methods remains an active area of investigation, particularly in food and clinical chemistry. The optimum pH and temperature of measurement were 6.8 and 30+/-2 degrees C, respectively. Interference in analysis of caffeine due to different substrates was observed but was not considerable. Caffeine content of commercial samples of instant tea and coffee was analyzed by the biosensor and the results compared well with HPLC analysis.