Isolation And Identification Of “Jinhua” Fungi And Quality Analysis Of Raw Dark Tea And “Jinhua” Fermentation Product

Eurotium Cristatum is popular named "Jinhua",which is the important dominant prebiotic in Fu brick tea.This fungus grows and propagates on the basis of tea,which changes the physical and chemical properties of tea,improves the quality of Fu brick tea,and produces active metabolites beneficial to human body.The marketing Fu brick tea,there are obvious presenting differences in fungi colony size and colony color expression as golden yellow,light yellow and green yellow in its splitting section.It is the different subspecies or the same strain? There is no consistent conclusion.In order to identification the differences of phenotype and genotype among these "Jinhua" fungi,distinct morphological differences of 14 strains of "Jinhua" fungi were isolated from Fu brick tea of Hunan,Sichuan and Shanxi by fungi purification and cultivation techniques.Plate culture method combining with morphological observation and molecular identification,14 different strains of "Jinhua" fungi owned different morphological characteristics were carried out cluster identification.Using primary dark tea as culture medium and cultivating these 14 strains of "Jinhua" fungi for obtaining different tea products,then tea taste quality components were analyzed.It will provide scientific support for further exploration of“Jinhua”fungi resources and guide the development of Fu brick tea products in the future.The results are as follows:According to the observation of colony morphology and optical microscopic characteristics for each fungus,14 strains of "Jinhua" fungi were preliminary identified as Eurotium;there were existing obviously different colony morphology on PDA culture medium;14 strains of "Jinhua" fungi represented as different growth rates,strains 11,9,6and 3 grew faster in the early stage;strains 1,2,12 and 10 grew faster in the later stag;strains 7,5,13 and 14 grew slower in the whole culture stage;Under optical microscope observation,some difference in micro morphology;strains 1,4,5,7 and 12 which ascocarps were mixed in mycelium nets,and the hyphae between the ascocarp were connected in bundles,strains 2-3,6,8-11 and 13-14 which mycelium around ascocarps were irregular distributed,and the hyphae between the ascocarp were no fascicular.Using sequence analysis for 18 S r DNA,internal transcribed spacer(ITS),beta tubulin gene(Ben A),calmodulin gene(CAM)and RNA polymerase ? gene(RPB2),14 strains "Jinhua" fungi were classified.18 S Sequence analysis for r DNA and ITS region could identify 14 strains "Jinhua" fungi belonging to ascomycetes and Aspergillus,but it could not effectively distinguish the species and below genus.Sequence analysis of Bena gene has a certain role in distinguishing genera and species;sequence analysis of Ca M gene and RPB2 gene could accurately identify strains to species level,however,the research on identification of subspecies or varieties could not meet the very accurate requirements.Comprehensive analysis found that,14 strains of "Jinhua" fungi were all belonged to Aspergillus cristatus also called Eurotium Cristatum.These strains may be different Eurotium Cristatum for them genetic diversity and heteromorphy and have subspecies or varieties,which is consistent with the diversity of colony morphological characteristics.Primary dark tea inoculated of different strains "Jinhua" fungi,which contents of water extract,tea polyphenol and free amino acid all decreased and the p H values increased in totality;contents of soluble sugar in tea samples from strain 5 and 6 increased significantly,but the other tea samples decreased;contents of non-ester catechins increased,but contents of ester catechins and gallic acid decreased as a whole;contents of alkaloids was basically unchanged.The effect of different “Jinhua” fungi strains on quercetin and kaempferol contents in dark tea is basically the same,the content of quercetin and kaempferol in tea samples from strains 1 and 5 was relatively high,while that from strains 9 and 10 was relatively low.Compared with the unfermented tea sample by Eurotium cristatum,the color of the “Jinhua” tea sample soup becomes darker,increasing red and orange parts;its taste becomes sweet and mellow,reducing acidity,increasing sweetness,and the color of the leaf bottom becomes darker and brighter.