Caffeine Degradation Characteristics Of Desarmillaria Tabescens And Its Application In Tea Fermentation

Caffeine（1,3,7-trimethylxanthine）is a purine alkaloid compound synthesized by plants and is an important commercial alkaloid.Caffeine has multiple biological functions such as neurostimulation and antioxidant properties,and it is widely used in food,pharmaceuticals,and cosmetics.However,high doses of caffeine can affect sleep quality and cause adverse effects such as irritability.Moreover,the widespread use of caffeine has led to a large amount of waste containing caffeine,improper disposal of which can cause a significant environmental burden.One important direction for developing low-caffeine functional foods and harmless disposal of waste containing caffeine is a biodegradation pathway for caffeine.This study collected 20 species of edible and medicinal fungal and successfully screened a strain of Desarmillaria tabescens CGMCC 40115 with caffeine degradation function,enriching microbial resources for caffeine degradation.Through medium screening,caffeine concentration gradient,temperature,and pH optimization,the optimal fermentation medium was found to be malt extract medium,with the optimal fermentation conditions being an initial caffeine concentration of 900 mg/L,a temperature of 28℃,and a pH of 8.D.tabescens was cultured under the optimal fermentation conditions,and samples were taken at different time points（8,12,16,20,and 24 days）for HPLC and UPLC-MS/MS detection and calibration of caffeine and its metabolites.Adding caffeine in the medium was Treated group,and normal culture was Control group.According to the analysis of the degradation characteristics of D.tabescens liquid fermentation,the degradation rate of caffeine was slow in the first 10 days of fermentation;the fastest degradation rate of caffeine at 16 days in the middle fermentation stage;when the degradation rate of caffeine slowed down at 22 days later,the 6 groups of fungal at three time points were compared for transcriptome analysis.Differential gene expression was screened,and 5036,3781,and 7208 differentially expressed genes（DEGs）were obtained in the Treated₁0 d vs Control₁0 d,Treated₁6 d vs Control₁6 d,and Treated₂2 d vs Control₂2 d groups,respectively.GO analysis was performed on all DEGs,and the dominant categories relative to the entire transcriptome background were cell part and membrane part in the Cellular Component category,catalytic activity and binding in the Molecular Function category,and metabolic process and cellular process in the Biological Process category.All DEGs were searched against the KEGG database,and 115,109,and 114 KEGG pathways were annotated in the Treated₁0 d vs Control₁0d,Treated₁6 d vs Control₁6 d,and Treated₂2 d vs Control₂2 d groups,respectively.The enriched KEGG pathways were mainly related to the Metabolism,Genetic Information Processing,and Cellular Processes categories.Previous studies have shown that cytochrome P450 proteins may be involved in caffeine metabolism.Nine upregulated cytochrome P450 genes were found in the Treated₁0 d vs Control₁0 d,Treated₁6 d vs Control₁6 d,and Treated₂2 d vs Control₂2 d groups,which may be closely related to caffeine degradation by D.tabescens.D.tabescens can grow well in green tea,black tea,and tea stalk without the addition of any exogenous nutrients.The caffeine and its degradation products in the fermentation of green tea,black tea and tea stalk by D.tabescens were calibrated by HPLC,and the green tea fermented by D.tabescens was selected for metabolomic analysis.Differential metabolites were screened and annotated for KEGG enrichment analysis.The caffeine metabolism pathway was significantly affected during D.tabescens fermentation with green tea.Combined with the transcriptome sequencing analysis of D.tabescens degradation by caffeine,the biosynthesis of cofactors and purine metabolism pathways were also significantly affected.These pathways may be closely related to caffeine metabolism.Theobromine,3-methylxanthine,and 7-methylxanthine were identified as the main degradation products of successive demethylation of caffeine during fermentation.Other demethylation metabolites of caffeine（theophylline,1,7-dimethylxanthine,xanthine）were also present during the fermentation process.Oxidation products of caffeine and its metabolites such as 1,3,7-trimethyluric acid,1,3-dimethyluric acid,1,7-dimethyluric acid,3-methyluric acid,and 7-methyluric acid were also detected.These substances participate in the catabolism of caffeine.