【作者】 李健；

【导师】 李平；

【作者基本信息】 安徽农业大学 ， 农产品加工及贮藏工程， 2007， 硕士

【摘要】 茶氨酸是茶树中一种特殊的非蛋白质游离氨基酸,对绿茶的良好滋味起到积极作用。近年来,大量研究表明茶氨酸具有重要的生理和药理作用,可作为食品滋味增强剂和人体保健添加剂,在食品、医药行业具有广阔的应用前景,人们对茶氨酸的需求量也将越来越大。因此,为了获得大量的茶氨酸,很多学者研究了不同的茶氨酸生产方法,主要包括茶叶或其富集液提取法、化学合成法、茶愈伤组织培养法和微生物酶促合成法。众所周知,茶氨酸主要存在于茶树(Camellia sinensis)等山茶科植物中,此外,在一种牛肝菌(褐绒盖牛肝菌,Xerocomus badius)中也有微量茶氨酸存在。但是否与茶氨酸存在于山茶科类植物中类似,茶氨酸也存在于牛肝菌科中的其他牛肝菌中,目前未见有相关研究报道。同时,能否利用相关牛肝菌液态发酵生成茶氨酸,也未见报道。本论文围绕上述问题,主要进行了以下研究:不同牛肝菌子实体中茶氨酸的分析检测;牛肝菌液态发酵生成茶氨酸的实现及条件优化;牛肝菌液态发酵合成茶氨酸关键酶的初步推断。首先,论文运用毛细管电泳技术对褐绒盖牛肝菌(Xerocomus badius)、乳牛肝菌(Suillus bovinus)、黄牛肝菌(Boletus auripes)和黑牛肝菌(Boletus aereus)子实体干品中的茶氨酸及其他游离氨基酸同时进行了分析定量。结果表明,褐绒盖牛肝菌、乳牛肝菌、黄牛肝菌和黑牛肝菌中茶氨酸的含量(占干重)分别为29.86mg/100g、27.35mg/100g、25.61mg/100g和15.60mg/100g;四种牛肝菌中总游离氨基酸的含量(占干重)分别为2458.08mg/100g、3064.38mg/100g、2400.44mg/100g和1531.32mg/100g。其次,论文首次实现了褐绒盖牛肝菌和乳牛肝菌的液态发酵,用于生成茶氨酸,并通过响应曲面法优化了液态发酵条件。结果表明,褐绒盖牛肝菌液态发酵的最佳关键因子值分别为葡萄糖29.17g/L,酵母膏6.38g/L,装液量61.43mL,起始pH 5.63时,茶氨酸产量可达17.22mg/L;乳牛肝菌液态发酵的最佳关键因子值分别为葡萄糖30.12g/L,酵母膏6.77g/L,装液量60.10mL,起始pH 5.58时,茶氨酸产量可达15.15mg/L。最后,论文对牛肝菌液态发酵合成茶氨酸关键酶进行了初步推断。结果表明,谷氨酰胺合成酶催化反应体系中检测到了茶氨酸的生成,初步推断牛肝菌体内合成茶氨酸的关键酶可能为谷氨酰胺合成酶。本论文利用牛肝菌液态发酵直接生物合成天然茶氨酸,不同于传统方法,这将为开辟一条新的茶氨酸生物合成的绿色生产途径奠定理论基础。更多还原

【Abstract】 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.更多还原