Study On Content Of Folate In Wheat And Its Interaction With Trypsin

Folate,a kind of water-soluble vitamin B,is a transport carrier of one carbon unit in the body.Inadequate folate intake will lead to a series of physiological diseases.Pregnant women lacked of folate prone to megaloblastic anemia,and it is easy to lead to fetal neural tube malformation and increase atherosclerosis and cardiovascular diseases.Plants and microorganisms make folate themselves,but humans in common with all other vertebrates cannot synthesize folate on their own and depend solely on nutritional sources.Wheat is the most widely distributed grain crop in the world and it is an effective method for biofortification of folate.In this study,the representative Chinese wheat microcore germplasm was selected as the research material,and germplasm resources with high folate content were screened.The interaction between folates and trypsin was investigated through fluorescence spectroscopy and molecular docking.Comprehensively,the main results of the present study are as follows:(1)The elution conditions of the mobile phase in HPLC were optimized by selecting Linfen 6308 and Fa 52.The results showed that the liquid phase conditions were as follows:injection volume 20?L,flow rate 0.3 m L/min,UV detection wavelength 280 nm,column temperature 25?.The mobile phase consisted of phase A(aqueous solution containing 0.1%formic acid)and phase B(acetonitrile solution containing 0.1%formic acid).The elution conditions were as follows:phase B linearly increased from 5%to 9%in the first 2 min,then increased to 9.6%after 6 min,and then returned to the initial proportion of 5%within 0.2 min,and finally maintained equilibrium.The peak times of THF,5-CH₃-THF,5-CHO-THF,5,10-CH₂-THF were 1.686 min,2.302 min,5.751 min and 6.496 min,respectively.Single factor experiment was used to optimize the extraction process of folate.The optimal extraction conditions were determined by single factor experiment as follows:p H value of folate extract was 7.0,ratio of solid to liquid was 1:10,extraction temperature was 37?,and water bath time was 120 min.(2)The folate content of Chinese wheat microcore germplasm under three different environments was determined.The results showed that genotype was the main factor affecting the variation of folate content of wheat varieties,and the interaction between genes and environment also had a certain influence.Total folate ranged from 22.68±14.19-98.87±14.55?g/100g,with an average of 50.49±14.45?g/100g.The highest content of total folate was Wumangchunmai(94.37?g/100g),followed by Geerhongmai(86.30?g/100g),Xiaobaimai(85.23?g/100g),Fuyanghong(84.92?g/100g)and Lovrin 10(84.00?g/100g).The results showed that the content of four folate derivatives was not significantly different among different cultivar types(Local,Breeding and Introduced).It was found that the contents of5-CH₃-THF and 5-CHO-THF in Chinese wheat microcore germplasm are relatively high and they contributed to more than 70%of the total folates quantified in this study.It can be seen5-CH₃-THF and 5-CHO-THF were the two major folate vitamers present in wheat accessions.In addition,the content of four folate derivatives and total folate in different germplasm had no significant difference with the character of winterness and springness and grain color,but had significant negative correlation with grain characters of wheat(grain length,grain width,grain thickness and 1000-grain weight).The results provided high content germplasm resources and cultivation guidance for the genetic improvement of folate in wheat.(3)The interaction between four folate derivatives and trypsin was studied by fluorescence spectroscopy and molecular docking.The results showed that the endogenous fluorescence of trypsin was quenched by four folate derivatives,but the quenching types were different.Among them,THF and 5-CH₃-THF are static quenching.5-CHO-THF and 5,10-CH₂-THF are combined dynamic and static quenching.THF,5-CH₃-THF,5-CHO-THF and 5,10-CH₂-THF all redshifted the maximum emission peaks of trypsin from 333 nm-337.5nm,333 nm-339.5 nm,334 nm-343 nm and 333 nm-338.5 nm,respectively.5-CHO-THF caused the maximum emission peak of Trypsin to have the greatest degree of red shift,which was about 9 nm.At 37?,the binding constants(K_a)of THF,5-CH₃-THF,5-CHO-THF,5,10-CH₂-THF and trypsin were 5.61×10⁵ L·mol^-1,1.25×10⁶ L·mol^-1,2.76×10⁷ L·mol^-1 and2.82×10⁶ L·mol^-1,respectively,and the number of binding sites(n)were 0.9646,1.0002,1.4847 and 1.2474,respectively.The thermodynamic parameters of THF,5-CH₃-THF and Trypsin,?H>0,?S>0,which implies that hydrophobic forces were the main force.The thermodynamic parameters of 5-CHO-THF,5,10-CH₂-THF and trypsin,?H<0,?S<0.It showed the interactions between 5-CHO-THF and trypsin are mainly hydrogen bond force and van der Waals force.In addition,the distance between donor and acceptor were evaluated to be 2.01 nm(THF)?2.07 nm(5-CH₃-THF)?2.17 nm(5-CHO-THF)and 2.53 nm(5,10-CH₂-THF).Molecular docking results further proved that the interaction between different folate derivatives and trypsin included hydrogen bonding,van der Waals force and hydrophobic force.This study provides an important reference for understanding the binding mode and interaction mechanism of folate and protein.