Submerged Fermentation For Production Of Theanine By The Bolete Species (Mushrooms)

Theanine is a specific non-proteinic free amino acid in tea plants(Camellia sinensis).It contributes highly and positively to the good taste of green tea infusion.In recent years, many studies have shown that theanine plays important roles in physiological and pharmaceutical functions.Due to its good taste and favorable physiological effects,the demand for theanine could increase not only as a taste-enhancing food additive but also as a supplement for improving and/or maintaining human health.Consequently,in order to obtain large amounts of theanine,several different methods were investigated to produce theanine by many researchers in the past years.The methods for theanine production include extraction from tea leaves,chemical synthesis,tea callus cultivation and enzymatic synthesis.It is well known that theanine is mostly confined to the genus Camellia(mainly in C. sinensis).Besides,it was also separated and identified from a bolete(mushroom) Xerocomus badius.To date,however,there is no report on whether theanine also exists in other boletes as well as theanine could be produced by the related boletes using submerged fermentation.In order to investigate the two points mentioned above,we focus on the following studies in this thesis.Analysis and determination of theanine from different bolete species;establish and optimization of submerged fermentation conditions for theanine production by the boletes;identification of the key enzyme involving in theanine biosynthesis in the boletes.Firstly,theanine and other free amino acids were simultaneously analyzed and determined in the fruiting bodies of Xerocomus badius,Suillus bovinus,Boletus auripes, and BoIetus aereus using micellar electrokinetic capillary chromatography(MEKC).The results indicated that the contents of theanine in the fruiting bodies of the four boletes were determined to be 29.86mg/100g,27.35mg/100g,25.61mg/100g,and 15.60mg/100g(dry weight),respectively.The sum of free amino acids in the above boletes was 2458.08mg/100g,3064.38mg/100g,2400.44mg/100g,and 1531.32mg/100g(dry weight), respectively.Secondly,the boletes(mushrooms)X.badius and S.bovinus were first used for theanine production by submerged fermentation.The response surface methodology(RSM)was employed to optimize various parameters in the fermentation processes.The results suggested that the maximum theanine yield of X badius(17.22mg/L)could be achieved when the key factors glucose,yeast extract,broth content and initial pH were set at 29.17g/L,6.38g/L,61.43mL and 5.63,respectively.While,for S.bovinus,the maximum theanine yield(15.15mg/L)was obtained when the parameters were as follows,glucose 30.12g/L,yeast extract 6.77g/L,broth content 60.10mL,and initial pH 5.58.Lastly,identification of the key enzyme responding for theanine biosynthesis in the fermentation processes of the boletes was preliminarily studied in this thesis.The results shown that theanine was synthesized in the glutamine synthetase reaction system.Thus,we conclude from the evidence that the key enzyme is the glutamine synthetase.In this thesis,theanine is successfully produced by the related boletes(mushrooms) using submerged fermentation,which is rather different from the traditional methods.More importantly,our results could provide a novel and promising way for the mass theanine production in the future.