Study On Isolated Cultrue And Artificial Inoculation Of E.Cristatum To Ferment Summer And Autumn Tea

Fuzhuan brick tea is one of the special Chinese dark tea,it has become more and more popular with its unique quality and health function been discovered in recent years.At the same time,problem such as the summer and autumn tea are not effectively used has been existing in our country.Therefore,it’s necessary to get a pure strain of Eurotium Cristatum which plays a key role during the formation of Fuzhuan brick tea,also it’s important to explore the physiological and biochemical characteristics of Eurotium Cristatum in the process of liquid fermentation and try to get the high quality fermented tea using summer and autumn tea as raw materials.The main results were as follows:1.A strain was isolated from the Fuzhuan brick tea and identifiedThe "golden flower fungus" were isolated by streak plate method on CZ medium and purified by three point inoculation method on PDA medium.Observing the morphology of strain during the progress of culture,it was found that the surrounding of colonies were white or pale yellow while the middle part were close to olive yellow.At the beginning of culture,there were some small transparent oil drop were generated,and it turned purple black while pigment diffusing in the matrix gradually as times went by.The strain had a colorless mycelium and no branchs,the yellow cleistothecium was spherical or nearly spherical with a diameter of 45-65μm.Universal primer ITS1 and ITS4 were used for PCR amplification of the isolated strain,and a 500 bp sequence was obtained.A homology search was performed on Genbank database.The result indicated that the isolated strain revealed the highest homology(99%)with that of Eurotium amstelodami(GU723274.1).According to the colony morphology and microscopic characteristics,we can confirm that the isolated strain is the Eurotium Cristatum.2.The optimal conditions for liquid fermentation of Eurotium Cristatum and the dynamic changes of physical and chemical componentsOn the basis of CZ medium,sucrose was the best source of carbon and two ammonium hydrogen phosphate was the best source of nitrogen were obtained by single factor experiment of the liquid fermentation of Eurotium Cristatum.In addition,the result of the experiment showed that the sucrose concentration of 2%-4%,two ammonium hydrogen phosphate concentration of 0.15%-0.2%was the most suitable for the growth of stain.Under the optimum conditions of liquid fermentation culture,designing response surface methodology(RSM)with the pH value,inoculation volume and rotation speed as the independent variable,mycelial dry weight as the response value and analysing the data using Design-expert.The results showed that pH value of 5.55,inoculation volume of 0.96%,rotation speed of 125.98 r/min was the optimal conditions for liquid fermentation,in which the mycelial dry weight can reach 0.323 g/100mL.During the liquid fermentation of Eurotium Cristatum,the mycelial pellets became more and bigger while the color of fermentation liquor darker gradually with the extension of fermentation time.At last,the fermentation liquor became nigger-brown and turbid.The pH value of the fermentation liquor was first decreased and then increased along with the fermentation,and it reached the lowest point of 4.32 at 60 h.The content of polyphenol,flavonoids and polysaccharide decreased during fermentation.The polyphenol content was reduced from 91.56 mg/100mL to 40.01 mg/100mL,and it decreased rapidly in the early stage of the fermentation.The descending trend of flavonoids was slow as the polysaccharide content was reduced from 28.32 mg/mL to 14.98 mg/mL.The total amino acids in the fermentation liquid decreased first and then increased with the prolonging of fermentation time,and it reached the minimum value of 4.55 mg/100mL at 84 h.3.Using Eurotium Cristatum to ferment summer and autumn tea and the effects of fermentation on tea under the conditions of laboratoryAdding quantity of water,intensity of sterilization,addition of fermentation liquid,packaging modes were selected as affecting factors to design orthogonal test tabulation in a laboratory environment,which was aim to explore the conditions of artificial fermentation of summer and autumn tea.The results showed that the optimal condition of fermentation was:adding quantity of water:10%;intensity of sterilization:560 W,60 s;addition of fermentation liquid:5%;packaging modes:completely open.The tea obtained through the condition above were most popular among volunteers.Determining the conventional components of the fermented tea,and compared with summer and autumn tea,Hunan brick tea and dark tea of Jiangsu Province.The results showed that there was no significant difference among the four kinds of tea samples in the respect of water content,catechins content and protein content.Compared with the summer and autumn tea,the water extract content of fermented tea increased significantly,while the content of polysaccharide,tea polyphenol and free amino acid decreased significantly.The content of all kinds of phenolic acid changed and there were significant difference between summer and autumn tea and the fermented tea.The content of gallic acid and ferulic acid decreased,and the content of 4-hydroxy benzoic acid and(+)-catechin significantly reduced.The content of coffee acid had increased,and the content of chlorogenic acid had increased significantly.The content of(+)-catechin and ferulic acid in fermented tea and dark tea of Jiangsu provice had no significant difference.The research also measured the antioxidant capacity of different tea in order to explore the effect offermentation deeply.The antioxidant capacity of several tea samples were determined by the ability of scavenging ABTS free radical,the ability of scavenging hydroxyl radical and the ability of metal chelating.The results showed that the ability of scavenging ABTS free radical and scavenging hydroxyl radical were decreased significantly,the ability of metal chelating also decreased but not significantly.