Study On The Forming Mechanism Of The Quality Of Dark Tea Fermented With Eurotium Cristatum PW-1

Post-fermented dark tea is indispensable to the dietary structure of ethnic minorities dwelling in the border regions of China.Distinct from other dark teas,Fuzhuan brick tea(FBT)is favored by consumers for its unique quality and health-promoting functions,which is formed during the key “golden flower blossoming” fermentation process.The Eurotium cristatum is considered to be the predominant fungus in the “golden flower blossoming” fermentation process,and plays a decisive role in the quality formation of FBT.Generally,the quantity of E.cristatum reached during the “golden flower blossoming” process is considered to be the direct criterion to evaluate the FBT quality.However,the “golden flower blossoming” fermentation process is hard to control due to the open fermentation environment,which greatly affects the FBT quality.In addition,the traditional brick-formed FBT is not convenient to brew,which restricts the development of FBT industry.In this regard,it is of great importance to study the effect of E.cristatum on the FBT quality,and to explore the forming mechanism of the quality of dark loose tea fermented with E.cristatum.Herein,in the present study,we isolated and identified the E.cristatum strain PW-1 in local Pingwu FBT,and studied the effect and its mechanism of PW-1 inoculated fermentation on the quality of FBT and dark loose tea.The results can provide insights into the application of E.cristatum in high-quality dark loose tea,and this approach can promote the development of dark tea industry.The main research contents and results are as follows.Firstly,the E.cristatum strain PW-1 in Pingwu FBT was isolated and identified,and its genome information and functional genes were analyzed through the whole-genome sequencing technology.The morphological characteristics of strain PW-1 were similar to the previous reports of E.cristatum,and the phylogenetic tree using neighbor-joining method based on 18 S r DNA partial sequence of PW-1showed that the strain PW-1 had 99% homology with Aspergillus cristatus(AB002073.1).Based on the morphological and molecular characteristics,PW-1 was identified as E.cristatum.The whole-genome sequencing of E.cristatum PW-1showed that the total sequence length of PW-1 genome assembly was 35.4 Mb with a GC% of 49.48%.After gene annotation,a total of 11,411 protein-coding sequences of PW-1 genome were identified along with 59 r RNAs and 228 t RNAs.PW-1functional genes belonging to KOG categories were mostly involved in the(R)general function prediction only;(O)posttranslational modification,protein turnover,chaperones;and(I)lipid transport and metabolism.On the other hand,functional genes assigned to GO database were enriched in the(i)biological process,mainly including the cellular process,metabolic process,and the single-organism process;(ⅱ)cellular component,mainly including the cell,cell part,and the organelle;(ⅲ)molecular function,mainly including the catalytic activity and the binding categories.Metabolic and regulatory pathways of the strain PW-1 were constructed based on KEGG Orthology,and results showed that functional genes of PW-1 were mostly involved in the metabolism process,especially the carbohydrate metabolism and the amino acid metabolism.Then,the quality characteristics of PW-1 inoculated fermentation(IF)/natural fermentation(NF)FBT samples at different fermentation stages were analyzed to explore the effect of E.cristatum PW-1 inoculated fermentation on the quality of FBT.It was found that a total of 82 volatile compounds were identified in all FBT samples,which mainly included the alcohols,aldehydes and unsaturated hydrocarbons.The content of alcohols was increased with deeper fermentation degree,which accounted for 70.22% and 66.99% in NF16 d and IF16 d FBT samples,respectively.The aldehydes(stale aroma)and ketones(woody aroma)were also relatively abundant in NF16 d and IF16 d samples,which worked with alcohols to contribute to the characteristic FBT aroma of NF16 d and IF16 d samples.Compared with the natural fermentation FBT samples,it was found that the E.cristatum PW-1inoculated fermentation accelerated the biotransformation of polyphenols,flavonoids,water-soluble carbohydrates,catechins and free amino acids in FBT,which resulted in the quality improvement of PW-1 fermented FBT samples.In addition,the OPLS-DA and HCA analysis results showed that the PW-1 inoculated fermentation promoted the quality of FBT,as well as shortened the fermentation time.It indicates the potential of E.cristatum PW-1 inoculated fermentation in the dark tea production.Based on the positive effect of PW-1 inoculated fermentation on the FBT quality,we further applied the PW-1 into the fermentation of autumn green tea(AT)to investigate its effect on the astringency and quality of AT.The GC-MS analysis results showed that the PW-1 inoculated fermentation increased the content of alcohols in PW-1 fermented autumn green tea(FT),especially the linalool,(Z)-linalool oxide and(E)-linalool oxide(furanoid).Compared with the AT,the contents of polyphenols,flavonoids and water-soluble carbohydrates were significantly decreased in FT.In addition,PW-1 inoculated fermentation greatly promoted the biotransformation of astringent compounds EGCG,ECG,EGC and EC,of which the contents decreased from 47.48 mg/g,36.01 mg/g,21.49 mg/g and 11.21mg/g to 0.37 mg/g,4.99 mg/g,0.40 mg/g and 1.14 mg/g,respectively.Caffeine was found to be the most abundant alkaloid in AT and FT,and its content significantly decreased after PW-1 fermentation.The contents of free amino acids in AT and FT were 11.65 mg/g and 0.97 mg/g,respectively,indicating the amino acids had little contribution to the taste of FT.However,the content of theabrownins was significantly increased after PW-1 fermentation.Based on the E-tongue data,the astringency along with its aftertaste of AT was significantly reduced after PW-1fermentation,while the umami intensity was comparable between AT and FT.The results were considered to be closely related to the increased theabrownins in FT.Therefore,the taste characteristics of theabrownins were analyzed by E-tongue,and results confirmed that theabrownins exhibited strong umami taste,not astringent taste.The above results showed that the quality of AT can be improved by PW-1 inoculated fermentation,which was mainly attributed to the increased theabrownins,linalool and linalool oxides.Furthermore,pathways including the aminoacyl-t RNA biosynthesis;alanine,aspartate and glutamate metabolism;glycine,serine and threonine metabolism;arginine biosynthesis;and butanoate metabolism were closely related to the fermentation of AT by PW-1.So,it was revealed the mechanism of reducing astringency and improving quality of AT by strain PW-1 inoculated fermentation,as well as provides insights into the utilization of summer and autumn green teas.In order to study the potential of PW-1 in producing high-quality dark tea,we prepared the PW-1 fermented loose tea and analyzed its quality characteristics and lipid-lowering activity in vivo.GC-MS analysis revealed that hydrocarbons and alcohols were abundant in loose tea with a proportion of 60.57% and 36.59%,respectively.Among them,the high content of D-limonene,β-myrcene,(Z)-linalool oxide and 2-methyl-1-octanol in loose tea largely contributed to its flowery and fruity aroma.As same as the traditional FBT,the contents of catechins,phenolic compounds and amino acids were low in loose tea,while the theabrownins was abundant.In addition,the tea infusion of PW-1 fermented loose tea was reddish-brown,indicating the good quality of PW-1 fermented loose tea.On the other hand,it was found that the loose tea was effective in lowering the lipid levels of high-fat diet induced obesity zebrafish.The aqueous extract of loose tea at 100μg/m L,300μg/m L,500 μg/m L and 700 μg/m L significantly reduced the lipid levels to 82.31%,57.88%,51.49% and 52.55% level of high-fat zebrafish,respectively.It revealed the potent lipid-lowering activity of PW-1 fermented loose tea.So,the above results provide insights into the production of high-quality dark loose tea by E.cristatum PW-1 inoculated fermentation.Based on that theabrownins was found to play an important role in the quality formation of dark tea,the lipid-lowering activity and structural characteristics of theabrownins were analyzed.According to the chemical composition analysis,the extracted theabrownins from PW-1 fermented loose tea contained 24.62% of flavonoids,20.74% of phenols,16.10% of carbohydrates and 5.62% of protein.Four fractions of theabrownins with different molecular weights,TBs-LT3 k,TBs-3-10 k,TBs-10-30 k,and TBs-GT30 k,accounted for 49.31%,32.46 %,16.18 %,and 2.05 %in TBs,respectively.The TBs,TBs-LT3 k,TBs-3-10 k,and TBs-10-30 k all showed lipid-lowering activity in vivo,and the TBs-10-30 k showed better lipid-lowering activity.Furthermore,UV-Vis and FT-IR data revealed that all the theabrownin fractions were similar in functional groups,and all of these contained a large number of hydroxyl and carboxyl groups.Therefore,the structural characteristics of TBs-10-30 k with better lipid-lowering activity were further studied.XRD analysis showed that TBs-10-30 k was an amorphous polymer.SEM and AFM observation showed that TBs-10-30 k had irregular slice shape with a relatively smooth and dense surface.TG/DTG showed that TBs-10-30 k exhibited two-stage weight loss,one occurred at the temperature range of 30 ℃ to 138 ℃,while the other occurred at the temperature range of 138 ℃ to 500 ℃.The DSC curve of TBs-10-30 k showed an obvious endothermic peak at 81.94 ℃ with an energy requirement of 132.16 J/g.The results of TG/DTG and DSC revealed the good thermal stability of TBs-10-30 k.Furthermore,the Py-GC-MS analysis showed that TBs-10-30 k was mainly composed of phenols,saccharides and proteins.The results revealed that theabrownins was amorphous macromolecular phenolic polymers with abundant hydroxyl and carboxyl groups,and has good thermal stability.Finally,the lipid-lowering mechanism of theabrownins was explored.The UV-Vis analysis result showed that the microenvironment of cholesterol chromophore was changed by different concentrations of theabrownins solution,and the characteristic absorption wavelength of cholesterol was redshifted,indicating that theabrownins could bind to cholesterol.In addition,theabrownins had significant inhibitory effects on pancreatic lipase and cholesterol esterase,and the optimal inhibitory rates were 87.95% and 40.27% at experimental concentrations,respectively.Theabrownins had a certain chelating ability to bile acids,and the best binding efficiency was 22.77%.The effect of theabrownins on micelles was studied.It was found that theabrownins significantly increased the size of micelles,reduced the solubility of cholesterol in micelles,and significantly reduced the stability of micelles.The results revealed that theabrownins exhibited lipid-lowering activity by inhibiting the absorption of lipids in intestinal tract.In summary,this study revealed the biological characteristics of E.cristatum strain PW-1 from a genome-wide perspective for the first time.The quality improvement of dark tea by PW-1 inoculated fermentation was closely related to that PW-1 promoted the biotransformation of polyphenols,catechins and amino acids;increased the contents of alcohols and unsaturated hydrocarbons;and increased the content of theabrownins in dark tea.The contribution of theabrownins to the taste of dark tea was confirmed by E-tongue for the first time.In addition,the structural characteristics,lipid-lowering activity,and lipid-lowering mechanism of theabrownins were revealed.This study could provide theoretical foundation for the quality formation mechanism of dark tea and the application of PW-1 in high-quality dark tea,which is of great significance for quality improvement of dark tea and the development of dark tea industry.