The Bio-synthetic Pathway Of 2-ethyl-3,5/3,6-dimethylpyrazine,the Valuable Flavor Compounds

Pyrazines are a class of six-membered heterocyclic compounds containing N atoms at the1 and 4 positions and usually has important flavor values.Among pyrazines,compounds of 2-ethyl-3,5/3,6-dimethylpyrazine（EDMPs）are isomers,both of these compounds have typical chocolate、roasted and nuts aroma with extremely lower threshold.Moreover,EDMPs as one of the top five pyrazine compounds have widely used as aroma fragrances and it were proved safely to eat.At present,mainly EDMPs used in the food industry are produced chemically.However,food additives produced biologically are more acceptable by consumers.With the consumers’preference of natural aromatic substances,microbial methods for producing EDMPs compounds are receiving attention.However,the knowledge about what microorganisms can produce EDMPs,and what is the microbial synthesis pathway of EDMPs is limited,which brings great challenges to the efficient synthesis of EDMPs industrially.Traditional fermented foods have various flavor compounds which were produced by complex microbial community by degrading diverse substrates.Meanwhile,pyrazine is an important class of flavor compounds in traditional fermented food,including Chinese baijiu and soy sauce.It is suggested that microorganisms in traditional fermented food samples might have capability of pyrazine-producing.The main research contents of this study include:（1）Strain screening for EDMPs production:As the concentrations of pyrazines in fermented samples are always extremely low(ng-μg·L^-1).In this study we initially optimized the detected methods and parameters of GC-MS,then 242 traditional brewing samples were tested for volatile compounds.A relatively high of EDMPs was detected from a soy-sauce flavored fermented grains and then a strain with EDMPs-producing capacility were successfully screened from this positive samples.After the biochemical experiments and molecular biological identification,the EDMPs-producing strain was identified as B.subtilis species and named as Bacillus subtilis Nr.5（2）Biosynthesis pathway analysis of EDMPs:Based on the structural characteristics of EDMPs and the existed biosynthesis pathway of other alkylpyrazines.This study proposed that D-glucose and L-threonine might be the synthesis substrates for EDMPs,and metabolic precursors,aminoacetone and 2,3-pentanedione,are involved in the synthesis of EDMPs.Therefore,through substrate addition experiments this study proves that D-glucose and L-threonine can significantly enhance the synthesis of EDMPs.In addition,we added the isotope labelled substrates D-[¹³C₆]-glucose and L-[¹³C₄,¹⁵N]-threonine in culture solution with different combination.Then,we analyzed the mass spectral of the producing EDMPs and the proposed intermediates-aminoacetone and 2,3-pentanedione.The results show that D-glucose and L-threonine are substrates for EDMPs synthesis,in which aminoacetone（from L-threonine）and2,3-pentanedione（from D-glucose and L-threonine）are intermediate metabolites synthesized by EDMPs.（3）The model fermentative system of soy sauce:EDMPs are the important flavor compounds in brewing soy sauce.Exploring the synthetic route of EDMPs in the process of soy sauce brewing will help achieve accurate flavor-oriented fermentation.So,this study firstly constructed a soy sauce fermented system,by adding different concentrations of D-glucose or L-threonine in the soy sauce brewing system,and the content of EDMPs in soy sauce samples which inoculated/uninoculated the EDMPs-producing strain B.subtilis Nr.5 were analyzed.Results showed that the inoculation of B.subtilis Nr.5 could significantly improve the production of EDMPs in soy sauce brewing system.In addition,the system which added higher concentration of D-glucose or L-threonine have higher concentration of EDMPs.Therefore,we speculate that the synthesis pathway of EDMPs in this study is one of the synthesis pathways of EDMPs in the traditional fermentation process of soy sauce.