Microbial and enzymatic methods for the removal of caffeine
Introduction
Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6 dione), a purine alkaloid, is a key component in most popular drinks especially tea and coffee. Caffeine has been purified and was found to be responsible for the stimulant action of coffee. Because of its stimulatory nature, it was used as a cardiotonic till the end of 19th century [1]. In the first half of the 20th century, it was used as a stimulant of respiration and circulation in Dutch medicine. In modern medicine, caffeine is used as an adjuvant to the analgesic actions of aspirin and paracetamol.
Coffee and tea plants are the major sources of natural caffeine. The caffeine content in coffee plant varies between 1 and 4% by dry weight [2], [3]. The availability of nutrients in soil affects caffeine content in coffee plants. For example, depletion of phosphorus and potassium caused a 20% decrease and 12% increase, respectively, in caffeine content [4]. Caffeine is also present in Cocoa, Herrania, Cola, Ilex, Paullinia, Citrus flowers and Calviceps [5], [6]. Several confectionary food products like chocolates contain 2.3–3.6 mg of caffeine and 20–49 mg of theobromine, a biological derivative of caffeine [7]. Caffeine content in regular and instant coffee ranges from 0.43 to 0.85 and from 0.61 to 0.82 mg/ml, respectively [8].
Section snippets
Why caffeine needs to be degraded or removed?
The main mode of entry of caffeine, theophylline, theobromine and other natural xanthines in human systems are through coffee, tea, caffeinated cola drinks, cocoa-derived beverages and chocolates. Average consumption of caffeine in humans ranges from 80–400 mg/(person day), which would result in a plasma level of 5–20 μM. Even low doses of caffeine affect the quality and quantity of sleep [9], [10]. The withdrawal effects of caffeine in humans are headache, fatigue, apathy and drowsiness [11], [12]
Caffeine removal by conventional methods
Conventional methods of caffeine removal are water decaffeination, solvent extraction and supercritical carbon dioxide extraction (Fig. 1). In water decaffeination method, first caffeine is extracted in water and then removed from water by solvent extraction process. Sorbent trapping with HPLC has been used to remove caffeine from water [23]. Caffeine laden solvent has been made free from caffeine by various processes which include evaporation, charcoal addition, filtration, centrifugation and
Caffeine degradation in eukaryotes and prokaryotes
Information on different enzymes involved in the degradation of caffeine in different organisms could help in developing an enzymatic process for caffeine removal. The overall pathway of caffeine degradation in eukaryotes and prokaryotes is given in Fig. 2.
Microbial degradation of caffeine
Studies on caffeine degradation by microorganisms were not reported till 1970 probably because caffeine was regarded as toxic to bacteria [46], [47], [48]. Caffeine concentration greater than 2.5 mg/ml in the growth medium has been found to inhibit the growth of many bacterial species. Synergistic effect has been observed when caffeine is added to antimicrobial agents like chloramphenicol [46]. First report on caffeine degradation by microorganisms was in early 1970 [49]. Since then progress has
Enzymatic methods of caffeine degradation
The most studied enzymes are plant caffeine anabolic enzymes which are aimed at producing decaffeinated plants. The caffeine synthesis in plants comprises of sequential methylations at N-7, N-3 and N-1 of xanthosine ring which are catalyzed by different N-methyl transferases (NMT), viz., 7-methyl transferase, 3-methyl transferase and 1-methyl transferase [67], [68], [69]. Caffeine synthesis in plants can be stopped if xanthosine synthesis in plants is inhibited since caffeine is synthesized
Conclusion and future perspectives
Though the search for caffeine degrading microorganism began nearly 35 years ago, studies conducted in this area are inadequate. In bacteria, Pseudomonas species and in fungi Aspergillus and Penicillium species are efficient in degradation of caffeine. Degradation in bacteria occurred predominantly through demethylation route but oxidative route occurs predominantly in mammals. However, the degradation pathway in fungi is not clearly known. Even in microbial systems, studies have to be
Acknowledgements
The authors acknowledge Dr. Manoj for critical reading of manuscript. Gummadi acknowledge Ram and Shyam for their support.
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