Ferulic Acid Esterase Produced By Eurotium Cristatum And Its Application In The Preparation Of Dark Tea

Dark tea is a unique microbial fermented tea.Microorganisms play an important role in the process of dark tea,which is an important factor affecting the quality and flavor of dark tea.And Eurotium cristatum plays an important role in the fermentation process of“Fu Zhuang”dark tea.Eurotium cristatum can not only improve the quality of tea,but also synthesize the essential amino acids of the animal body,secrete water-soluble pigments,produce a rich aroma,regulate and maintain the balance of the intestinal flora.Feruloyl esterase?EC 3.1.1.73,abbreviated as FAE?belongs to the subclass of carboxylate hydrolase and is an extracellular enzyme that hydrolyzes phenolic acids such as FA,p-coumaric acid and XOS in plant cell walls the ester bond formed by hemicellulose and lignin,which releases antioxidants such as FA,p-coumaric acid,etc.These substances are widely used in food,medicine,cosmetics,bioethanol and other industries,and have a broad market prospect.In this paper we selected FAE-producing Eurotium cristatum,dark tea with high FA and XOS content and rich in active substances was prepared by using FAE produced during solid-state fermentation of Eurotium cristatum and xylanase added to synergistically degrade wood fiber in tea.The research idea of this dark tea has not been reported.The experimental results are as follows:?1?A strain of Eurotium cristatum from FAE was screened.The FA content of 15 kinds of commercial dark tea was determined by liquid chromatography.A strain ec-hqu4 with high yield of ec-FAE was selected from 3 kinds of teas with high FA content and used for tea fermentation and identified as Eurotium cristatum.After fermentation,the content of FA in the tea reached 28.77?g/g;ec-FAE activity up to 433.78 mU/g.?2?A response surface design method was prepared to optimize ec-FAE fermentation conditions for ec-hqu4 strain.The results showed that the feed-water ratio was 1?1?W/W?,MgSO4·7H2O was 0.04%,KH2PO4 was 0.20%,the inoculation amount was 3.2 mL of spore suspension?5?6×10⁶ CFU/mL?,and the fermentation time was 6 d,ec-FAE enzyme activity can reach 1318.8 mU/g.Separated and purified by ammonium sulfate salting out,anion exchange chromatography,and molecular sieve chromatography.The experimental results show that the apparent molecular weight of ec-FAE is about 28 kD.?3?The physical and chemical properties and kinetic constants of ec-FAE were studied.The ec-FAE optimum reaction temperature is 50?,and the optimum reaction pH is5.0.Significant substrate specificity.Na⁺,Zn²⁺has a certain promotion effect,Mn²⁺,Mg²⁺,Ca²⁺has a slight promotion effect,and Fe²⁺,K⁺,Cu²⁺has a slight inhibition effect.The ec-FAE K_m of ec-FAE is 4.95 mg/mL,V_max is 11.16?mol/mL/s,k_cat is 5.84s^-1,and k_cat/K_m is 1.18mg/mL/s.?4?A leavening agent for sun-dried green tea was prepared.Optimized the sporulation culture conditions of the starter through orthogonal experiments.The medium of raw malt?tea powder 1?1?W/W?is added with 30%water content,inoculum volume is 3 mL?5?6×10⁶ CFU/mL?,the temperature is 28?,and the fermentation time is 5 d.A fermentation agent with an ec-hqu4 spore number of 1.79×10⁸ CFU/g can be prepared.?5?The orthogonal experiment was used to optimize the fermentation conditions of sun-dried tea,and the results showed that at a water content of 20%,a fermentation time of8 days,a fermentation agent inoculation amount of 0.90 g?1.79×10⁸ CFU/g?,and the content of xylanase added was at 2.25 U/g,the ec-FAE enzyme activity,FA content,and XOS content in tea products reached 1734.57 mU/g,131.56?g/g,and 140.97?g/g,respectively.All the physical and chemical properties of the produced dark tea are in accordance with the GB/T9833.3-2013"Fuzhuan Tea Pressed Tea"standard and evaluated by a tea appraiser.?6?The headspace solid-phase microextraction method was used to determine the volatile components in tea by gas chromatography-mass spectrometry.It was found that the contents of alcohols and acids in dark tea products prepared by adding a starter and a xylanase at the same time were significantly reduced,heterocyclic materials were significantly increased,and esters were increased by 26?30%compared with sun-dried tea.And the salicylic acid methyl ester with holly oil aroma increased by 20.40%compared with sun-dried tea,and the linalool with floral aroma accounted for 62.81%of the alcohol content,which were the linalool in sun-dried tea and commercial dark tea,respectively 1.58 times and1.53 times.Analysis by ultra-high performance liquid chromatography-mass spectrometry combined with fermenting agent and xylanase fermentation showed that dark tea fermented with simultaneous addition of fermenting agent and xylanase has fewer types of amino acids,glycosides,catechins,flavones,and flavonols compared with sun-green tea There was no significant difference in alkaloids.In summary this article shows that fermenting sun-dried tea by synergistic action of a fermentation agent containing Eurotium cristatum and xylanase can significantly increase the content of FA and XOS in tea,increase the content of active substances in tea soup,and make the internal quality of tea more rich,long-lasting tea aroma.