Enzymatic Synthesis Of ?-[Glu]_(n?1)-Arg Using High Solid Concentrations And Its Application

?-glutamyl peptides are widely distributed in animal,plant and fermented foods,which have attracted widespread attention because of their strong taste characteristics and rich physiological activities.In addition,?-glutamyl peptides can be synthesized by enzymatic method,which has the advantages of simple,rapid,safe and non-toxic,and can be applied to food without separation and purification.Although researchers have established a variety of enzymatic synthesis methods of?-glutamyl peptides and explored the characteristics of them,a large number of functional?-glutamyl peptides still need to be further developed.This project aims to use glutamine(Gln)and arginine(Arg)as substrates to synthesize a new type of?-glutamyl peptides,?-[Glu](n?1₎-Arg,which have a strong kokumi taste and salty taste enhancement effect,through enzymatic method,and to explore its functional properties in inhibiting fat cells,the specific results are as follows:(1)A glutaminase aggregate was prepared by ethanol precipitation.When the addition ratio of ethanol was 50%(v/v),the hydrolysis activity and transpeptidation activity of the aggregate were 91.27%and 292.34%that of the initial glutaminase.The optimum temperature of glutaminase before and after treatment is 45°C,and both can exert maximum catalytic activity under alkaline conditions(p H 9-11),but the glutaminase aggregate intolerant to high temperature.In addition,ethanol treatment could make glutaminase aggregate obtain a significantly increased particle size but Zeta potential and PDI are not significantly different from those before treatment,and increase the?-helix of glutaminase protein,decrease the antiparallel?-sheets.(2)It was determined that glutaminase from Bacillus amyloliquefaciens could be used to synthesize?-[Glu](n?1)-Arg,and the effect of different solid concentrations(5%-40%)on the enzymatic synthesis reaction has been explored.Studies showed that high solid concentrations could not only speed up the reaction,but also increase the content of?-[Glu](n?1)-Arg in the enzymatic product;at the same time,when the molar ratio of Gln to Arg was 2:1,only?-[Glu](n?1)-Arg but no by-product?-[Glu](n?1)-Gln was detected in the reaction product.(3)The methods for qualitative and quantitative detection of?-[Glu]_(n=1,2,3,4)-Arg by UPLC-MS/MS and HPLC have been established and the products of enzymatic synthesis according to these methods have been detected.The results showed that the contents of?-[Glu]_(n=1)-Arg and?-[Glu]_(n=2)-Arg were the highest at 30%solid concentration,which were16.61%and 10.50%respectively;while the contents of?-[Glu]_(n=3)-Arg and?-[Glu]_(n=4)-Arg were the highest at 20%solid concentration,which were 9.95%and 5.92%respectively;overall,?-[Glu]_(n=1,2,3,4)-Arg had the highest content at 30%solids concentration,up to42.19%.(4)The kokumi and salty enhancement properties of?-[Glu](n?1)-Arg were studied,and the taste enhancement effects of?-[Glu](n?1)-Arg on basic taste substances,simulated broth and Na Cl solution were measured.Studies have proved that both the enzymatic reaction mixture and the standard?-[Glu]_(n=1,2,3,4)-Arg only showed a light bitter and astringency taste in aqueous solution,but they could enhance the basic taste when added to the basic taste substance solutions,of which the umami taste was the most obvious,the salty taste was the second,and the sweetness was the least.The enzymatic product synthesized with 30%solid concentration had the most obvious enhancement effect on the basic taste substances.When the addition amount was 20 mg/m L,the product could also increase the kokumi and salty taste of simulated broth by 2 times and 1.7 times respectively,and increase the salty taste of Na Cl solution by 1.5 times.(5)Using 3T3-L1 preadipocytes as a model,the effects of Arg,?-Glu-Arg and?-Glu-Glu-Arg on its proliferation and differentiation were initially explored.Studies showed that the above three samples could not only inhibit the proliferation of 3T3-L1 preadipocytes,but also reduce the accumulation of lipid droplets and triglycerides during the differentiation of 3T3-L1 preadipocytes,and enhance the release of glycerol.In this process,Arg,?-Glu-Arg or?-Glu-Glu-Arg participated in at least one m RNA expression regulation of genes related to3T3-L1 preadipocyte differentiation.