Research On The Formation Of High Content Gallic Acid And Degradation Of Tea Protein Promoting The Release Of Active Peptides In Tea Anaerobic Fermentation

Gallic acid(GA)is an important active ingredient for its biological activities,and its naturally little in tea leaves(about 0.20-1.00 mg/g).However,a pickled tea rich in more than 20 mg/g GA was processed from summer and autumn fresh leaves through an anaerobic fermentation by us,but its mechanism is still unclear.Gallated catechin,especially EGCG,can be hydrolyzed to form GA,which is the main pathway for GA biosynthesis in aerobic fermented tea.Interestingly,the bioconversion of EGCG into GA was not predominant in anaerobic fermentation,which means that it possibly has other pathways to form GA during anaerobic fermentation.As is well known,tea leaves is fairly rich in protein,up to 21-28% of dry weight,and tea protein also has different bioactivities.Since most of its water insoluble protein,its nutritional value requires enzymatic hydrolysis to form small proteins,peptides or amino acids.Microbial fermentation was considered to be one of the effective ways to degrade tea protein.Unfortunately,the changes of tea proteome during fermentation and its effect on the release of peptides and amino acids are less studied.Here,pickled tea samples were collected at 0,6,12,18,24,and 30 day of anaerobic fermentation,and the contents of GA and catechins were tested in these samples.Meanwhile,the key precursors of GA formation were identified by metabolomics analysis,and the key microorganisms involved in GA production were studied by microbiomics;in addition,the microbial enzyme protein responsible for the bioconversion of precursor into GA were initially obtained.On the other hand,the change of tea proteins during fermentation were analyzed by label-free quantification proteomics,and the important tea protein degraded in this process,as well as the microorganisms that can promote the tea protein degradation were preliminarily obtained.Moreover,the active peptides,especially the potential antioxidant peptides,from tea protein degradation during this process were also understood.It will provide a theoretical basis to clarify the formation of quality and function in tea and develop special and functional tea products,and will promote the utilization and benefits of Chinese tea resources,and the sustainable and healthy development of Chinese tea industry.The main findings are as follows:(1)GA was significantly increased from 1.72 mg/g(0 d)to 24.26 mg/g after 18 d of anaerobic fermentation,which was accompanied by the reducing levels of ECG,EAG,and 7-GC.However,EGCG was basically stable during the whole fermentation process.It suggested that EGCG contributes little to GA formation,while ECG,EAG,and 7-GC should be the key precursors to form GA in fermentation.(2)The carboxylesterase secreted by A.phoenicis ATCC 13157 and A.tubingensis strain CBS 134.48 may be the driving force for the conversion of ECG,EAG and 7-GC to GA.(3)GA from bacteria is another source of increasing GA content in pickled tea.Bacillus and other bacteria are considered to be the key microorganisms to produce GA.(4)There was a slow degradation process of tea protein in anaerobic fermentation,and hydrophilic tea protein may be the main target of hydrolysis in this process,such as tubulin and rubisco family protein.(5)The active physiological metabolism of bacteria,such as Lactobacillus,Staphylococcus,and Shewanella,as well as the hydrolytic enzymes from Aspergillus may promote the degradation of tea protein.(6)Acidity and total amino acids(FAAs)were gradually increased during anaerobic fermentation.18 free amino acids had no significant effect on the improvement of FAAs,while a large number of short peptides was released which may be the main reason for the increase of FAAs in pickled tea.(7)In pickled tea,26 peptides were predicted to have potential antioxidant activity,among which 7 key peptides(I L W,W L R,W Y,Y V,E L W,F Y,and P Y V)were considered to be responsible for the improvement of antioxidant(DPPH and hydroxyl radical scavenging power)in pickled tea.Some soluble proteins were degraded in this process,especially tubulin and rubisco family proteins,may be the key proteins that lead to the release of these potential antioxidant peptides.