Isolation And Identification Of Autofluorescent Microorganisms From Fu Brick Tea And Analysis Of Their Luminescence Characteristics And The Quality Of "Fahua" Tea

Autofluorescence is light reflected by structures such as mitochondria,lysosomes,and chloroplasts when they absorb excitation light at a specific wavelength.Plant chloroplasts,large fruit body fungi and various fungal microorganisms have autofluorescence,and their autofluorescence intensity varies with the type of organism,organ structure,tissue composition,development stage,etc.At the same time,exogenous conditions such as metal ions,High temperature,excitation light intensity will also affect the autofluorescence intensity.Compared with plants,due to the rapid reproduction of fungal microorganisms,the effects of exogenous conditions can be quickly obtained,At present,most researches on autofluorescence are concentrated on fungal microorganisms.In recent years,in the production and consumption of Fu Brick tea,it has been found that the autofluorescence of Hunan Fu Brick Tea is weak or non-existent,while that of Shaanxi Fu Brick Tea is relatively strong.Since "fahua" is a unique production process of Fu Brick Tea,its essence is that the high concentration of Eurotium cristatum in the Fu Brick Tea production environment grows and reproduces in the bricks under the conditions of suitable temperature and humidity,and the embryo bricks are moderately tight and forms golden flower fungus quality characteristics,so the quantity and quality of "Golden Flower" in production practice is usually used as one of the important indicators to evaluate the quality of Fu Brick Tea.According to the research background of Fu Brick Tea that belongs to post-fermented tea and autofluorescence microbes,combined with E.cristatum is a type of fungus that includes multiple subspecies,it is reasonable to speculate that the difference in autofluorescence between Hunan Fu Brick Tea and Shaanxi Fu Brick Tea may be due to the different types,quantity and metabolites of autofluorescent fungi,but it need to be further explored.this study uses the same level of Xiangyi Fu Brick Tea(Hunan Fu Brick Tea)and Jingwei Fu Brick Tea(Shaanxi Fu Brick Tea)as materials,using microbial subculture and separation and purification,morphological characteristics observation,molecular biology sequencing and autofluorescence conditions Optimizing and other methods and techniques,carrying out the separation and identification of the two fluorescent brick tea autofluorescent microorganisms and the analysis of their luminescence characteristics,with a view to clarifying whether the autofluorescence phenomenon of the Brick Tea originates from the microorganisms in the "fahua" process,and to explore the reasons for the difference in autofluorescence of Jingwei Fu Brick Tea and Xiangyi Fu Brick Tea,and it will provide a scientific basis for the development,utilization,production and consumption of autofluorescent fungus for Fu Brick Tea.The main research results and conclusions are as follows:(1)Isolation and identification of autofluorescent microorganisms from Fu Brick Tea.Isolation and purification of fungal microorganisms in Fu Brick Tea by solid plate method,it was found that six and four types of autofluorescent fungal microorganisms were separated and purified from Jingwei Fu Brick tea and Xiangyi Fu Brick tea respectively,and the colonies number of Jingwei Fu Brick Tea is more1.4 times than Xiangyi Fu Brick Tea;through further fluorescent screening of autofluorescent fungi,multiple subcultures and observation of colony morphological characteristics,and molecular biological sequencing,the autofluorescent fungus in Fu Brick Tea were identified as Eurotium cristatum and Aspergillus chevalieri,among which Eurotium cristatum is present in large quantities in Jingwei Fu Brick Tea and Xiangyi Fu Brick Tea,while Aspergillus chevalieri mainly exists in Xiangyi Fu Brick Tea in small amounts,while both tea still exist a variety of filamentous weakly fluorescent Aspergillus niger and other fungi,and Jingwei Fu Brick Tea is more than Xiangyi Fu Brick Tea in type and quantity;compared with Xiangyi Fu Brick Tea,Jingwei Fu Brick Tea has more autofluorescent colonies and colonies stacked of the density is greater.This shows that the difference in autofluorescence between Jingwei Fu Brick Tea and Xiangyi Fu Brick Tea is mainly due to the differences in the types,number,and colony stacking density of fluorescent fungi.(2)Luminescence characteristics of autofluorescent microbes in Fu Brick Tea.the study found that the Fu Brick Tea autofluorescent fungus can emit blue fluorescence and green fluorescence under excitation light with wavelengths of405 nm and 488 nm,respectively.the autofluorescence site is a closed capsule structure;there is no significant difference in the fluorescence intensity of Eurotium cristatum and Aspergillus chevalieri under the same culture conditions,but the fluorescence under short-wave excitation light is stronger than that under long-wave excitation light;When emit with long-wave fluorescence,the autofluorescence intensity is attenuated by laser excitation of fluorescent fungus,but the speed is slower and the stability is better;the autofluorescence intensity of Fu Brick Tea autofluorescent fungus is affected by pH,temperature,and dyeing concentration.pH3 acid solution can enhance the fluorescence intensity,120℃ high temperature and lactic acid Phenolic cotton staining solution can better inhibit the fluorescence intensity.(3)The quality analysis of the "Fahua" tea from the fluorescent microorganism of Fu Brick tea.According to the processing technology of Fu Brick Tea and the factors affecting the luminous intensity of the self-fluorescence of Fu Brick Tea,through screening of weak acid solution,high temperature of water bath and other treatments,the fluorescent fungus are inoculated to the same level of black tea in the same year by "Fahua".After the "Fahua",the golden flowers of the tea leaves flourish.Through further analysis of the quality of "Fahua" tea,including sensory analysis and analysis of main biochemical components,it was found that the quality of normal "Fahua" tea is related to the inoculated fluorescent fungi,and the effect of inoculation of Eurotium cristatum is better than inoculation Aspergillus chevalieri,and the change in the intensity of fluorescent microorganisms is also related to the quality of tea.Among them,the increase in fluorescence caused by high temperature,the quality of the tea inoculated with Aspergillus chevalieri is better,and the increase in fluorescence caused by acid treatment,The quality of the tea inoculation of Eurotium cristatum is better.In summary,the autofluorescent microorganisms in Fu Brick Tea are mainly Eurotium cristatum and Aspergillus chevalieri.The quantities and types of autofluorescent microorganisms in Jingwei Fu Brick Tea are more than that of Xiangyi Fu Brick Tea;the fluorescence of Fu Brick Tea with 405 nm,488nm excitation light can emit blue and green fluorescence,respectively,the fluorescence under short-wave excitation light is significantly stronger than the fluorescence under long-wave excitation light;Fu Brick Tea autofluorescence is mainly caused by Eurotium cristatum,and its location is a closed capsule structure;the fluorescence intensity of Fu Brick Tea autofluorescent microorganisms is affected by pH,temperature,and dyeing concentration.The pH3 acid solution can enhance the fluorescence intensity,120℃ high temperature,and lactic acid phenol Medan staining solution can inhibit the fluorescence intensity;Using the isolated and purified Fu Brick Tea autofluorescent microorganisms to "Fahua" with the loose tea,the tea have undergone a series of changes in sensory and physical and chemical properties.These changes are closely related to the type of inoculated microorganisms and treatment conditions.The acid treatment increased the intensity of fluorescence caused by Eurotium cristatum,which can significantly improve the quality of tea after“fahua”.The water bath treatment of Aspergillus chevalieri caused fluorescence enhancement,and the quality of “fahua” tea was better.