Isolation And Cultivation Of Eurotium Cristatum And Its Polysaccharides And Applied Enzymology Of Fermentative Broth From Fuzhuan Tea

Fuzhuan tea has its unique quality and the distinct function in adjusting digestion, obesity and hyperlipidemia E.cristatum is the predominant microbes in Fuzhuan Tea.In order to identify the relationship between E.cristatum and function of Fuzhuan tea,a systematic study with the technique of liquid fermentation,contemporary technology of isolation and purification,high-throughput screening(HTS)and qualitative analysis technology of LC-MS, were applied at E.cristatum from Fuzhuan tea.All results were as follows:1.Isolation and purification of E.cristatum and its liquid fermentation technologyAfter direct selecting yellow sealed shell from Fuzhuan tea,a colony like E.cristatum was isolated with PDA medium and plate lineation method.With microscope and scanning electric microscope analysis,we found its characteristics of colony,mycelium,ascocarp,structure of ascus spore and secretion type are same with that of typical E.cristatum.Then this colony we isolated was identified as E.cristatum with 18S rDNA method,because it has 98%similarity with E.cristatum,its mycelium was conglobated tightly,and growing bigger with culture time in liquid fermentation,but melted partly at late phase of fermentation.Single factor and orthogonal experiments were used to analyze the main material and environmental factors which affect the liquid fermentation of E.cristatum.E.cristatum can use sucrose,fructose,maltose,soluble starch and lactose as carbon source,and it can grow at pH4-6,the juice of black tea can accelerate its growth,and the best culture medium for liquid fermentation is 0.8%black tea juice with 6%sucrose.The extend of environmental factors which affect the fermentation of E.cristatum was the primary pH value>culture temperature>inoculation quantity>rotation speed.The best culture condition with the liquid fermentation of E.cristatum was that the primary pH value 5.5,culture temperature at 30℃, inoculation quantity 1%(the concentration of spore was 5.0～6.0×10⁸CFU/mL)and rotation speed 120r/min.2.Dynamic changes of biochemical components during the fermentation of E.cristatumIt has been observed that the color of the fermentation solution of E.cristatum was becoming darker and darker as the time of fermentation,from orange to brown,and clear to thick.But the viscosity was becoming little.With the time of fermentation longer,its solution flavor smells thicker and thicker.The following are the results in more details.(1)The growth curve of E.cristatum was the shape of "s" during liquid fermentation and its simulative equation of Logistic is y=0.371/(l+11.341e^-0.051t(R²=0.93607),and its growth curve includes lag phase(0-21h),logarithmic phase(21-72h),stationary phase(72-96h)and decline phase(>96h).Its mycelium growth rate continuously reaches to the maximum quantity of 0.3754g/100mL until 84 hours,then it tend to descend with culture time.(2)The pH value of the fermentation solution during fermentation descended with culture time.During first 0-84h period,the pH value decreased from 5.51 to 4.05,and reached its minimum value of 4.05 at 84h,then increased slowly until 96h,kept at about 5.0 at stage of 96h-120h.(3)During liquid fermentation of E.cristatum,the content of the amino acid in fermentation broth decreased early but increased lately.From 0 to 84 hours fermentation,the total content of the amino acid reduced to 78.7%of original content,and then increased to 2.76mg/100mL at 120 hours,reduced to 66.95%.This showed that the main reason for the decline of the amino acid content in the processing of Fuzhuan tea is that the amino acid was used as the nitrogen source for E.cristatum growth.(4)During liquid fermentation of E.cristatum,the content of sucrose in fermentation broth gradually decreased from 5.95%at fermentation start to 2.57%at fermentation end.(5)The total content of tea polyphenols and catechins reduced obviously during the liquid culture of E.Cristatum.After fermented with 120 hours,60.45%tea polyphenols and 73.68%catechins had been lost.Content of catechins and tea flavin(TF)are unstable in different fermentation stage,but it was certain that catechins and the TF decreased,while teabrownine(TB)increased largely to 39.44%greater than that of original content,after 120-hour fermentation,lead to the change of(TF+TR)/TB ratio first up and then down.This means that the decrease of catechins and others was mainly caused by polyhphenol oxidase excreted from E.cristatum.3.Studies on extraction and purification of extracellular polysaccharide of E.cristatum and its chemical and biological activitiesThe best technological parameters of the extraction of extracellular polysaccharide from E.cristatum was obtained by orthogonal experiments,whose concentrated ratio was one-fourth of the original culture,precipitated for 10 hours with 95%ethanol.Under this condition,the extraction concentration of extracellular polysaccharide in the ECP(extracellular polysaccharide fermented with element culture)and ECP(extracellular polysaccharide fermented with black green tea juice)were 195.2μg/mL and 136.8μg/mL respectively,and the total suger in its rude polysaccharide were 62.2%and 70.1%,respectively.The separation and purification of ECP and ECP^# were studied by DEAE-52 chromatogram and Sephadex G-100 gel chromatogram.Four compositions were obtained by DEAE-52 chromatogram from ECP,ECP-A rinsed with 1.0 mol/L NaCl is the main composition among that four.ECP-A1 and ECP-A2 were obtained through Sephadex G-100 from ECP-A,and ECP^#-A1 and ECP^#-A2 from ECP^#-A.No absorbing peak of protein and nucleic acid were found in polysaccharide samples through ultraviolet radiation scanning. those molecular weight determined by the gel column chromatography were 8.61×10⁴Da,1.75×10⁴Da,1.18×0⁵Da and 2.6×10⁴Da,respectively.The components of the polysaccharides hydrolyzed TFA in ECP-A1,ECP-A2,ECP ^#-A1 and ECP^#-A2 were analyzed with gas chromatogram(GC),The molar ratio of rhamnose,arabinose,mannose,glucose and galactose in those four admixture were 12.73:2.47:23.75:1:1.79;0.97:3.13:2.76:1:0.81; 0.18:0.93:0.05:1:0.23 and 1.43:6.31:0.73:1:0.85,respectively.All results of IR(Infrared ray) spectrum,periodate oxidation and Smith reaction showed that the four polysaccharides have the same primary structure,belong to neutral hetero polysaccharide excepted uronic acid,all monosaccharide residue were pyranose format,and the main chain were jointed with a(1→3),α(1→6),α(1→2)orα(1→4)glycosidic bond.The activities of reducing weight and fat of extracellular polysaccharide of E.cristatum was screened through HTS method,and three sorts of PPAR(peroxisome proliferator-activated receptors)subisomers(PPARa,PPARγand PPAR5)used as receptors.Our results showed that the extracellular polysaccharide of E.cristatum had activity on these three receptors,the highest activity is on PPARγ.The activation value of ECP and ECP^# on PPARy were 1.63 and 1.41,respectively.The effect of extracellular polysaccharide of E.cristatum on restrained cancer cells was also screened with cell strains of HepG2 and K562.And the results showed that ECP^# had not restrained on the cell strains of HepG2 and K562.ECP had not on HepG2 also,but had restrained on K562.4.Studies on applied enzymology of fermentation broth of E.CristatumBy simulated gastric and intestinal solution in vitro,the fermentation broth of E. cristatum affected the activities of a-amylase,proteinase,pancreatic proteinase and lipase as well were studied,compared with the function of the green tea,black tea,Fuzhuan tea and the compound of tea polyphenols.All results showed that all samples have the function of activation to the activities of a-amylase,gastric proteinase,pancreatic proteinase.The activations of the fermentation broth of E.cristatum and the juice of Fuzhuan tea were obviously higher than that of the juice of black tea,green tea and the compound of the tea polyphenols,and the fermentation broth of E.cristatum is the most active one,whose activation ability on a-amylase,gastric proteinase,pancreatic proteinase were 1.6,3.98 and 3.61 times those of the control,respectively.The activation effect of tea polyphenols compound on a-amylase existed the quality-effecting relationship,and all experimental samples restrained the activity of lipase to some extent.5.Qualitative analysis of the chlorogenic acids derivatives in Fuzhuan teaThe existence and change of the chlorogenic acids(CA)derivatives in Fuzhuan tea were studied with LC-MS qualitative analysis.It was the first time to find 1,4-diCQA(l,4-dicoumaroylquinic acids)and 4,5-diCQA(4,5-dicoumaroylquinic acids)of CA derivatives existed in the finished product of Fuzhuan tea,and 4-pCoQA(4-pcoumaroylquinic acids)of CA derivatives existed in the raw and processed materials of Fuzhuan tea.