Enzymatic Preparation Of Theaflavins And Its Health-promoting Activity On Oral Microbiota

Black tea is the most popular consumed type of all teas worldwide,which is one of also the most important source of dietary polyphenols for humans.Theaflavins(TFs)is one of representative polyphenols known to synthesize by fermentation during the process of black tea production.Tooth decay is a dynamic disease induced by dental plaque,in which predominant pathogens(such as S.mutans)are widely recognized to play pioneering roles in the process.As far as the field of stomatology is concerned,only a few reports described the effects of TFs on oral health care.In this thesis,the heterogeneous enzymatic biosynthesis conditions of TFs were systematically optimized,and the multiple antibacterial properties of S.mutans were studied simultaneously.The animal safety,toxicology testing of TFs was completed,which filled the gap of related research.The anti-carious effects of TFs on dental caries and its regulatory mechanism were explored based on transcriptomics and oral microbiota firstly.On the basis of the multiple antibacterial activity against S.mutans in vitro,the function of health-promoting and molecular mechanism by TFs on oral microecological system were clarified,which laid a solid theoretical foundation for the development and application of TFs.The heterogeneous reaction conditions of TFs were optimized from the factors affecting enzymatic oxidation.The catalytic properties of PPO from withered-frozen,fresh tea leaves were carried out,based on which,the reaction kinetics of PPO was investigated.The dynamic enzymatic oxidation technology for TFs was established,and the process parameters of biosynthesis were determined meanwhile.Then the separating enrichment conditions were screened and optimized,and the technical parameters of dynamic gradient purification were obtained.The green preparation path of TFs was accomplished,which provided a means to a large-scale production of TFs.Biosynthesized TFs have no obvious toxicity and good food safety.The safety of TFs was systematically evaluated by acute oral toxicity test,Ames test,mouse bone marrow cell micronucleus test,sperm abnormality test and 30 d feeding test in rats.The results showed that Ames test,mouse bone marrow micronucleus test and sperm abnormality test were negative.The 30-day feeding experiment showed that there were no significant differences in body weight,food intake,food utilization rate,hematological indexes,end-stage blood biochemical indexes(except blood glucose),and visceral-body ratio between the rats and the control group(P > 0.05).The Organ tissue is normal in pathology,and there was no toxic damage induced among various dosage groups.The overall results revealed that TFs had no subacute toxicity.In vitro antibacterial evaluation showed that TFs had multiple inhibitory activities against S.mutans.Under the experimental conditions,the MIC value of TFs against S.mutans planktonic cells was 2.5 mg / m L.TFs could significantly reduce the survival rate of persisters,which was lower than that of the positive drug group significantly.In addition,TFs at the sub-inhibitory concentration level could exert antibacterial and inhibitory effects on virulence factors(acid production,adhesion,glucosyltransferase activity,extracellular water insoluble polysaccharide and biofilm synthesis)of S.mutans in a dose-dependent manner.Genomewide transcriptome analysis showed that TFs may weaken the virulence factors and biological functions of S.mutans by weakening the ability of cells to resist oxidative stress through multiple pathways such as cell signal transduction,cell membrane stability,glycolysis,meanwhile,inhibiting DNA repair system and disrupting protein homeostasis.In contrast,the comparative experiments showed that using the toothpaste containing TFs brushing could significantly increase the microbial abundance in saliva,and homeostatically regulate the oral microbiota communities of saliva and supragingival plaque.Furthermore,the dominant species in the core microbiome remained stable,the abundance of dominant bacteria at the phylum level accounted for 97.0 %,and the abundance of core microbiome was more than 80 %,which indicated that TFs did not result in a collapse in the structure of oral core microbiota.In turn,at the genus level,the abundance of biomarker bacteria related to oral health including Streptococcus,Rothia,Fusobacterium,Corynebacterium and Actinomyces were increased significantly,and some opportunistic pathogens such as Veillonella,Prevotella,Selenomonas,Atopobium,Granulicatella,Leptotrichia and Alloprevotella were reduced in saliva and supragingival plaque drastically.PICRUSt functional analysis showed that the fascinating shifts in the mentioned ecological balance were tightly related to the down-regulation of metabolism associated with extracellular polymer synthesis,and the up-regulation of ABC transporters,two-component systems and amino acid metabolism,simultaneously.The results provide new insights into the oral health related with tea consumption,and provide a theoretical basis for the development of TFs-containing oral care products as well.