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Braz. J. Microbiol. vol.34 suppl.1 São Paulo Nov. 2003
Degradação de cafeína por Rhizopus delemar em biorreator de colunas usando casca de café como substrato
Cristiane Vanessa TagliariI,II; Raquel K. SansonII; André ZanetteII; Telma Teixeira FrancoI; Carlos Ricardo SoccolII
ILaboratório de Engenharia Bioquímica, Faculdade de Engenharia Química, Universidade Estadual de Campinas, Campinas, SP, Brasil
IILaboratório de Processos Biotecnológicos, Departamento de Engenharia Química, Universidade Federal do Paraná, Curitiba, PR, Brasil
Various microorganisms including bacteria, yeast and fungi can degrade caffeine. There are few publications about caffeine degradation pathway in filamentous fungi, mainly by solid-state fermentation (SSF). Studies were carried out on degradation of caffeine and their metabolites by filamentous fungi in SSF using coffee husk as substrate. The purpose of this work was to investigate the caffeine degradation pathway by Rhizopus delemar in packed bed column fermenter and to compare this degradation metabolism with glass flasks fermentation. The methylxanthines were quantified by HPLC analysis. The experiments were realized with the optimized conditions in previous experiments: pH 6.5, 28ºC, inoculation rate 106 spores/g substrate, aeration rate 60 mL/min and initial moisture 73%. Under these conditions, after 72 hous of fermentation was achieved only 0.19% of caffeine and 0.014% of theophylline in the coffee husk. The strain proved to be able for caffeine and theophylline degradation by SSF in packed bed column bioreactor.
Key words: decaffeination, fermentation, caffeine, theophylline, bioreactor, filamentous fungi.
Diversos microrganismos incluindo bactérias, fungos e leveduras são capazes de assimilar a cafeína de meios sintéticos ou de resíduos de café. Existem poucos trabalhos sobre a via de degradação da cafeína em fungos filamentosos, principalmente por fermentação no estado sólido (FES). Estudos de degradação da cafeína por fungos filamentosos em FES usando casca de café como substrato vêm sendo realizados. O objetivo deste trabalho foi investigar a via de degradação da cafeína por Rhizopus delemar em biorreator de colunas aeradas e comparar este metabolismo de degradação com o da fermentação em frascos de vidro. As metilxantinas foram quantificadas por análises em HPLC. Os experimentos foram realizados com as condições otimizadas previamente: pH 6,5, 28ºC, 106 espores/g substrato, vazão de ar 60 mL/min e 73% de umidade inicial. Após 90 horas de fermentação, 65% da cafeína foi reduzida, resultando 0,19% de cafeína e 0,014% de teofilina na casca de café. Esta cepa provou ter habilidade para degradar cafeína e teofilina por FES em biorreator de colunas.
Palavras chave: descafeinação, fermentação, cafeína, teofilina, biorreator, fungos filamentosos.
The caffeine (1,3,7-trimethylxanthine) is an alkaloid found in more than 60 plant species. Caffeine is, present with significant levels in coffee, tea, cocoa and Cola genera. The pharmacological effects of caffeine are well known: stimulation of central nervous system, toxicity when fed excessively, and mutagenicity on microorganisms (3).
Caffeine is considered toxic for many microorganisms; however, some microorganisms have the ability to grow in the presence of caffeine and the capacity to degrade the alkaloid. Several studies were carried out to investigate the use of caffeine, as a source of energy for microorganism growth (8). Penicillium and Aspergillus is the more frequent caffeine-degradation genera for fungi and Pseudomonas for bacteria (1,5).
Kurtzman and Shwimmer (7) first studied the degradation of this alkaloid using strains of Penicillium roqueforti and Stemphylum sp. The strains were able to degrade caffeine in a liquid medium containing caffeine concentrations below 19 g/L (7).
Roussos et al. (10) isolated strains from coffee and byproducts, which were mainly identified as Penicillium and Aspergillus. Eight of these strains showed great capacity to degrade caffeine in synthetic liquid medium without additional nitrogen source (10).
Brand et al. (2) isolated from coffee husk one strain of Aspergillus niger which is able to degraded 90% of caffeine and 57% of tannin by SSF (2).
These authors showed that the total degradation of caffeine by SSF was possible. However, in all fermentation studies involving caffeine degradation, no particular attention was given to its degradation products.
According to Hakil (4), theophylline is the major degradation product of caffeine by various filamentous fungi. The toxicity of theophylline in the system cardio-vascular and gastrointestinal is higher than caffeine. Lethal doses (LD50) of caffeine and theophylline have been reported to be 200 mg/kg and 206 mg/kg respectively in rat (4).
The purpose of this work was to investigate the caffeine degradation pathway by filamentous fungi using SSF in packed bed column bioreactor.
MATERIALS AND METHODS
Microorganism and Substrate
Rhizopus delemar LPB 34 was maintained on coffee husk extract agar medium (CHEAM). Spores suspension was prepared after 10 days of culture on CHEAM at 32ºC.
Coffee husk used as substrate contained approximately 7% proteins, 60% fibers, 2% fats, 10% minerals, 8% total sugars and 0.6% of caffeine.
Solid State Fermentation
The experiments were carried out with the previously optimized conditions: pH 6.5, 28ºC, inoculation rate 106 espores/g substrate, airflow 60 mL/min and initial moisture 73%.
The packed bed column bioreactor (Fig. 1) consists of a glass column with lids at both the ends, which allows air flow control at the exit of the reactor (9). Each column had the capacity for 40 g of the substrate (dry weight basis). Two replicates of samples were collected for analysis every 24 h.
The fermented was submitted to mechanical agitation with water for 15 minutes and then was filtrated and centrifuged.
The analyses of methylxanthines were performed on a Varian system with photodiode array detector (PDA) and Microsorb C18 column (4.6 x 250 mm). The eluents were methanol and water (24:76, v/v) with a flow rate of 1 mL/min, by a isocratic system.
RESULTS AND DISCUSSION
For better evaluation of the degradation pathway of caffeine by Rhizopus delemar in coffee husk, the evolution of pH, moisture contends and the appearance of methylxanthines was monitored. The evolution of the methylxanthines quantified to more than 0.01% of dry material is shown in the Fig. 2.
During the experiment was observed that the pH increased from 6.0 to 9.0 after caffeine degradation, possibly due urea formation. The principal caffeine degradationproducts identified in the samples were the theophylline and 3-methylxanthine. This result is similar that observed in glass flasks and by Hakil (4) and Ina (6). First a 7-demethylation, giving theophylline from caffeine followed by a 1-demethylation leading to 3-methylxanthine from theophylline (Fig. 3).
We can conclude that the first steps of caffeine degradation by Rhizopus delemar LPB 34 by solid state fermentation of coffee husk consist of demethylation reactions. This result is the same showed by others filamentous fungi but differs from observations made in the bacterial and human metabolism.
We thank FAPESP, European Union and CNPQ for the financial support.
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Cristiane Vanessa Tagliari
Laboratório de Processos Biotecnológicos
Departamento de Engenharia Química, Universidade Federal do Paraná
81531-970, Curitiba, PR, Brasil
Fax: (+5541) 361-3195
This paper corresponds to an "extended abstract" selected for oral presentation in the 22nd Brazilian Congress of Microbiology, held in Florianópolis, SC, Brazil, in November 17-20, 2003