【作者】 王波；

【导师】 于汉寿；

【作者基本信息】 南京农业大学 ， 微生物学， 2009， 硕士

【摘要】 青砖茶是砖茶中重要的一大类茶,在我国内地不常见,主要供应边疆少数民族饮用。关于砖茶中的微生物研究较多的是茯砖茶,而对青砖茶、康砖茶等砖茶及其中微生物的研究很少。本文开展了对青砖茶中微生物种类及数量的调查以及其中主要真菌对其品质和风味影响和散囊菌黄色素的研究,主要研究内容和结果如下：1、2007年1月,利用稀释平板法对青砖茶原料、不同制作阶段茶样和成品茶共二十个材料中的微生物进行分离,调查整个青砖茶加工阶段真菌的种类及其各种微生物数量变化的规律。渥堆过程中微生物的种类和数量最多,渥堆前后分别存在增加和减少的过程。共分离到真菌156株,占70.58%；酵母14株,占6.33%；细菌30株,占13.57%；放线菌21株,占9.50%。表明青砖茶中的微生物以丝状真菌占优势,细菌次之,放线菌第三,酵母最低。鉴于在青砖茶微生物的调查过程中,无论从种类和数量上,真菌都是最多的,因此本研究重点对茶样中的真菌菌株进行纯化、培养和鉴定。2、经典分类法仍然是被广泛用来鉴定真菌的主要方法。为了解青砖茶加工过程中存在的主要真菌种类和后续研究,本研究主要对这156株真菌分离菌株的形态学特征进行调查,包括菌落特征和产孢结构等特征的观察。结果表明,青砖茶加工过程中存在曲霉属、青霉属和散囊菌属等近10个属的真菌。在丝状真菌的调查中,曲霉属真菌的分离频率最高；其次是青霉属真菌；曲霉中尤以黑曲霉(Aspergillus niger)分离频率最高,为57.8%；其次是灰绿曲霉(A. glaucus),分离频率为17.02%。3、对相关基因序列进行系统发育学分析是近年来真菌分类鉴定的重要依据。本研究以分离自不同年代、不同产地的茯砖、康砖和青砖茶中的散囊菌(Eurotium spp.)以及5个标准菌株共计16个Eurotium属菌株为材料,对它们的tubB和ITS序列进行克隆测序,并结合该属内所有已经建立的种进行系统发育学分析。结果表明：分离自我国砖茶中的散囊菌菌株间的tubB和ITS序列相似度很高；由tubB和ITS序列构建的系统发育树表明,分离自青砖茶成品中的Qa-30406-1,形态学和分子水平的鉴定结果均证实为E. cristatum。分离自茯砖茶中的Fa-20719-3、F4-20608-1、F5-20608-1、F2-20608-2和F3-20608-2均鉴定为E. cristatum,除了分离自康砖茶中的K8-30721-6为E. chevalieri,其它分离自康砖茶中的菌株也为E. cristatum。4、为进一步了解青砖茶中的真菌对其品质的影响,本研究通过人工接种从青砖茶中分离到的主要真菌(包括曲霉属、青霉属和散囊菌属)到青砖原料中,25℃培养一定时间,研究了不同菌株对茶样生化成分的影响。青砖原料接种真菌分离菌株后,与对照相比除了接种22F10 (Aspergillus sp.)菌株的茶样中茶多酚略有下降外,接种菌株35F06 (Penicilliumsp.)、AF09 (Eurotium cristatum)、21F06 (Aspergillus sp.)和35F03 (Penicillium sp.)的四个处理都有所增加；氨基酸含量与对照相比均减少,咖啡碱含量变化很少；以上结果表明青砖茶中的主要真菌分离菌株对青砖茶的品质和风味有一定的影响。5、为寻找优质黄色素生产资源,本研究以黄色的冠突散囊菌(Eurotium cristatum)为材料,利用有机溶剂辅助超声波等方法从固体平板和液体培养的菌丝体中均提取出黄色素,并研究了光、温度及一些化学成分对该色素稳定性的影响。采用有机溶剂辅助超声波等方法能成功提取出该色素；根据吸收光谱特征,初步推断该色素含有类胡萝卜素。并且该色素耐受灯光、室内散射光、不耐阳光和紫外光；耐酸,不耐碱；对温度敏感。供试的11种金属离子对该色素有不同程度的影响；磷酸的破坏性显著；H2O2、NaHSO3对该色素有较强的破坏；柠檬酸对该色素有一定的破坏作用；同等条件下,苯甲酸钠对该色素的破坏作用大于山梨酸钾；EDTA-Na2和Vc有一定的护色作用；糖类、NaCl、草酸和乳酸对该色素的稳定性几乎无影响。更多还原

【Abstract】 Green brick tea is a kind of tea which is mainly consumed by the minority who live in western China. Currently, more attentions have been paid in microbes in Fuzhuan other than microbes in green brick tea, Kangzhuan and other teas.In January,2007, we investigated microbes in 20 various samples such as the materials made in the process or in different stages of the process.156 fungal strains were obtained with a ration of 70.58%, together with 30 bacterial strains (13.57%),14 strains of yeasts (6.33%) and 21 strains of actinomycetes (9.50%). Meanwhile, methods such as plate diluting and colony counting were used to get the exact amount communities of microbes in each process stage. In summary, fungi in green brick tea were superior to bacteria, antinomies, yeasts in number. The fungal isolates from green brick tea were purified to investigate the population and importance.Morphological characteristics such as colony and the sizes of cleistothecia, ascus, ascospore, conidia were investigated for these 156 fungi strains. During the producing process of tea, ten types of fungi such as Aspergillus, Penicillium and other fungi species were check out. Aspergillus niger was dominant with a ratio of 57.8%, secondly, A. glaucus was with a ratio of 17.02%. Meanwhile, the morphologies and the sizes of cleistothecia, ascus, ascospore conidia and other fungi of Eurotium were found out too.Sequences of tubB and ITS of 16 strains including 5 type Eurotium strains and 11 strains isolated from different stages and places were analyzed. tubB and ITS squences among these isolates exhibited high similarities. In addition, phylogentic tree based on tubB and ITS sequences supported that the isolates were Eurotium cristatum.In order to investigate whether the fungi isolates play a role in the development of the quality of green brick tea or not, raw materials of green brick tea were inoculated with the fungal isolates and then incubated at 25℃for about 5 days. Through analyses of the content of water extracted, tea polyphenols, amino acids, caffeine and flavone glycosides, the role of the fungal isolates inoculated played was investigated. The content of caffeine was almost constant during the treatments, while other substances listed above decreased significantly. This was also observed on green brick tea. These indicated that the fragrance of green brick tea was possibly resulted by the fungi isolated. The resource and stability of yellow pigment were also investigated. Yellow pigment was successfully extracted from plates and liquid cultures of Eurotium cristatum through the method of organic solutions and supersonic waves. Furthermore, lights, temperatures and other factors influenced on this pigment were studied. The results showed that high temperatures affected yellow pigment’s stability, and so do sunlight and UV. The yellow pigment was more stable in acidic condition. Many kinds of metal ions had different influences on this pigment. Phosphate affected the stabilization of the pigment greatly, while H2O2, NaHSO3 and citric acid destroyed it less obviously. Sodium benzoate’s destruction against this pigment was stronger than potassium sorbate; EDTA-Na2 and Vc might protect the pigment because EDTA-Na2 might bind proteins that would, perhaps, disrupt the stability of the pigment. And, carbohydrates, sodium chloride, oxalate and lactic acid had no evident effect on the pigment.更多还原