【作者】 许爱清；

【导师】 李宗军；

【作者基本信息】 湖南农业大学 ， 微生物学， 2011， 博士

【摘要】 作为微生物发酵茶之一,茯砖茶是湖南黑茶中边销茶的主导品种,是我国西北边疆少数民族生活必需的日常饮料和常用民间医药。在茯砖茶的生产加工利用过程中仍存在一些生产商和消费者所关注的问题：(1)建立在传统自然发酵基础上的茯砖茶加工过程中微生物种类多而杂,产品品质不稳定,发酵周期长而使发花房周转慢；(2)目前虽然建立了现代化洁净的茯砖茶加工生产线和发花房,但没有适用的发酵剂及实用的发酵剂施用技术,它成为该装备的技术瓶颈；(3)茯砖茶产品及发酵功能菌对人体生理功能影响的研究不够全面透彻,消费者心存疑虑,这制约着产品的市场开发。解决这些问题的关键是继续深入研究茯砖茶发酵菌群特性和产品的生理功能,为茯砖茶产业提供强力的科技支撑,并为消费者解密茯砖茶所蕴涵的神奇奥妙。本研究的主要目标包括：茯砖茶发酵过程和产品样品中的真菌菌群结构分析,优势真菌的菌种鉴定,茯砖茶相关样品中蒽醌类衍生物的测定,茯砖茶干预肠出血性大肠杆菌0157：H7感染作用和冠突散囊菌对肠道菌群影响作用等。取得的研究结果分述如下：1茯砖茶中真菌菌群结构的解析从国内最大的茯砖茶生产商加工生产线上提取了茯砖茶渥堆样品、间隔两天的发酵样品,以及收集了黑毛茶、茯砖茶成品样品。运用传统的分离培养技术和新兴的PCR-DGGE技术研究这些样品中的真菌菌群结构。理化特性分析表明茯砖茶工业发酵过程为耐旱微生物生长提供了最适宜的环境条件。平板培养计数法表明散囊菌属是茯砖茶加工过程中的优势真菌。采用3对以真菌rDNA为靶标的通用引物,即NS1//GCfung5, su-0817-GC//ssu-1196和NL1F-GC//LS2R, PCR扩增样品的总DNA,再经DGGE电泳分离。结果比较显示以引物NL1F-GC//LS2R的PCR-DGGE图谱解析样品中的真菌菌群结构效果最好。通过PCR-DGGE分析发现至少有8个属的真菌：散囊菌属(Eurotium)、德巴利酵母属(Debaryomyces)、曲霉属(Aspergillus)、轮枝孢属(Verticillium)、毕赤酵母属(Pichia)、拟盘多孢霉属(Pestalotiopsis)、根毛霉属(Rhizomucor)和白僵菌属(Beauvera),存在于所检测的茯砖茶相关的样品中。除了根毛霉属和白僵菌属,在茯砖茶发酵过程的样品中都能检测到其它6个属的DGGE印迹,其中散囊菌属、德巴利酵母属和曲霉属持续可见。耐旱真菌散囊菌属和德巴利酵母属是茯砖茶发酵过程中的优势真菌,曲霉属是次要的伴生菌。首次检测到德巴利酵母属参与茯砖茶发酵过程。首次显示轮枝孢属和根毛霉属在黑毛茶原料中大量存在。以及首次检测到白僵菌属存在于贮藏的茯砖茶成品中。白僵菌的存在可能与茯砖茶的长期保存有关,可以考虑利用安全微生物白僵菌防控茯砖茶成品的虫害。本研究获得了工业生产规模上茯砖茶发酵过程的真菌菌群结构PCR-DGGE指纹图谱,并为筛选用作茯砖茶发酵剂的功能菌指引了方向。2茯砖茶中分离的功能菌株的鉴定及系统发育分析菌种鉴定工作是工业菌种开发研究的重要基础。本实验针对从茯砖茶中分离纯化的菌株Eurotium sp. FZ,获得了其在培养基CZA30、CZA200和M40Y上生长的形态学特征,及其有性繁殖结构的显微特征和扫描电镜下子囊孢子的特征。依据形态学特征鉴定所测菌株的形态学种名为冠突散囊菌(Eurotium cristatum)。多位点序列分型(MLST)测定了4个基因位点,即ID区(rDNA中ITS1-5.8SrDNA-ITS2-28S rDNA D1/D2区)、RPB2(RNA聚合酶Ⅱ)、BT2(p-微管蛋白)和CF(钙调素)的DNA序列,提交到GenBank数据库中登记号分别为HQ148160、HQ148161、HQ148162和HQ148163。MLST技术鉴定所测菌株的系统发育种名为冠突散囊菌(Eurotium cristatum)。形态学特征鉴定方法和多位点序列分型方法的鉴定结果一致。对酵母菌株测定了ID区DNA序列,提交到GenBank数据库中登记号为JF710627。酵母菌株鉴定为汉逊德巴利酵母(Debaryomyces hansenii)。该种酵母菌具有QPS身份,可用作开发茯砖茶混菌发酵剂的候选微生物。3茯砖茶相关样品中蒽醌类物质组分分析运用HPTLC方法检测了普洱茶、黑毛茶、茯砖茶、冠突散囊菌接种发酵的黑毛茶和冠突散囊菌菌体等5种样品中蒽醌类衍生物(大黄素、大黄素甲醚和大黄酚)的存在情况。在HPTLC实验条件下,三种标准样品即大黄素、大黄素甲醚和大黄酚得到有效地分离,其迁移率(Rf)分别是0.28、0.43和0.51。在430 nm下扫描条带的吸收峰和峰面积,表明所检样品中都存在大黄素,按相对含量递减排序是黑毛茶、普洱茶、冠突散囊菌接种发酵的黑毛茶、茯砖茶和冠突散囊菌菌体。可见大黄素在黑茶产品样品中普遍存在。所有样品中没有检出大黄酚。大黄素甲醚仅能从冠突散囊菌菌体和冠突散囊菌接种发酵的黑毛茶中检出。黑茶(包括茯砖茶)中蒽醌类物质(如大黄素)是茶叶消费者摄取膳食性蒽醌类物质的重要来源。4茯砖茶水提物对肠出血性大肠杆菌感染小鼠的干预通过给SPF级成年KM小鼠灌胃OD600=1.0左右新制的0157：H7细胞悬液,建立了受0157：H7感染的小鼠模型,新鲜粪便中0157：H7检测呈阳性,在行为活动上表现出疾病体征。实验设计5组小鼠：正常组、0157：H7感染模型组和茶汁干预治疗的低(日灌胃量1.0 g/kg)、中(10.0 g/kg)、高剂量组(20.0 g/kg),相当于成人(60kg)正常饮茶量(6.6g/d)及其10倍和20倍。从组织病理学、血液学、免疫学和肠道微生态学方面对实验小鼠进行了比较分析,结果发现：模型小鼠表现出典型的0157：H7感染疾病症状。对其组织病理学检查表明其胃、十二指肠、回肠、结肠和肾脏组织细胞结构都遭受损伤；血液学指标反映其肝、肾功能都受损害；免疫学指标反映其免疫系统受损,免疫力低下。低剂量茶汁对0157：H7感染有一定程度的康复促进作用。灌胃茶汁中、高剂量两组小鼠的所检测指标较模型组有所好转,恢复到正常组的水平甚至更优于正常组,表明合适剂量茯砖茶水提物对0157：H7感染机体引发的损害具有防护和修复作用。茯砖茶汁的抗0157：H7感染功能表现在：(1)维护肠道粘膜的完整性,(2)调整肠道菌群,(3)增强机体的免疫力和(4)保护肝脏和肾脏免受损害,保障其正常生理功能。由于中、高剂量组茶汁用量相当于成人(60kg)日饮茶量(6.6 g)的10倍和20倍剂量。外推到人体,提示日常饮用茯砖茶的10-20倍浓缩茶汤可能具有治疗0157：H7感染性腹泻的作用。研究结果为运用茯砖茶治疗0157：H7感染性腹泻提供了实验依据和理论参考。5冠突散囊菌菌体对小鼠结肠菌群的影响运用SPF级KM小鼠检验了冠突散囊菌的急性毒性,发现在最大耐受剂量10000 mg/kg灌胃情况下,小鼠未发生死亡病例。表明受试物冠突散囊菌对KM小鼠的经口急性毒性最大耐受剂量大于10000mg/kg。根据化合物经口急性毒性分级标准,小鼠一次经口LD50范围5001-15000mg/kg时属于2级毒性,为实际无毒物。实验设计4组小鼠：正常组、灌胃冠突散囊菌菌体水悬液的低(日灌胃量50mg/kg)、中(200 mg/kg)、高剂量组(1000 mg/kg)。灌胃组相当于成人(60 kg)每天消耗冠突散囊菌菌体0.33 g、1.32 g和6.6 g。运用PCR-DGGE技术检测了小鼠结肠中总体细菌、乳酸杆菌属、拟杆菌属和梭菌属Ⅳ簇的菌群结构,发现小鼠的结肠菌群结构上存在个体差异性。低剂量冠突散囊菌悬液引起小鼠结肠内的总体细菌、乳酸杆菌属和梭菌属Ⅳ簇的种群多样性增加,但对拟杆菌属的作用不明显。考虑到乳酸杆菌和梭菌属Ⅳ簇细菌具有健康促进作用,提示低剂量冠突散囊菌悬液可能具有调节肠道菌群功能。外推到人体,提示日常消耗0.33 g冠突散囊菌可能对人体肠道菌群具有调节作用。研究结果为冠突散囊菌菌剂制品的开发利用提供了实验依据。更多还原

【Abstract】 Fuzhuan brick-tea is a kind of microbial-fermented tea and is the leading product amongst the Hunan dark teas that provided for the border tea market in China. It is an essential beverage in daily life for the minorities living in northwestern China and is a common folk medicine as well. Nevertheless, several open problems troubling the manufacturers and consumers that were encountered in the Fuzhuan brick-tea product processing and utilization, id est,1) The classical processing of Fuzhuan brick-tea based on natural fermentation that induced the complex diversity of microorganisms existing in the tea product, unstable product quality, slow rate of turnover of fermentation room since longer fermentation periods.2) Although modern uncontaminated Fuzhuan brick-tea processing lines and fermentation rooms were constructed, there had not any applicable fermentation starters and practicable employment techniques for starters. Those became technique bottlenecks of the equipments.3) The effects of Fuzhuan brick-tea products and the functional fermentation fungi on the physiological functions in human body should be studied thoroughly and intensively, since the consumers had some concerns about that. It constrained the product-market development. The perfect solution to those problems was to deeply characterize the fermentation microbial communities and to study the physiological functions of the tea products. Consequently, powerful technical support would be provided for the Fuzhuan brick-tea product industry, and the mysteries of Fuzhuan brick-tea would be disclosed for the consumers.The main objectives of present study include analysis of the fungal communities in the Fuzhuan brick-tea processing and product samples, identification of the predominant fungi, determination of the anthraquinone derivatives in the Fuzhuan brick-tea related samples, the effects of Fuzhuan brick-tea intervention into EHEC O157:H7 infection, and the effects of Eurotium cristatum on colonic microbiota. The results are stated as follows:1. Analysis of the fungal community structure in Fuzhuan brick-teaThe piling tea samples and fermenting tea samples in the fermentation room at 2-day intervals were carried out in a major Fuzhuan brick-tea production factory in Hunan province, China. The samples of raw dark green tea and ready made Fuzhuan brick-tea were collected. The fungal communities in these samples were characterized with classical culture-depended methods and the most recent culture-independed approach such as polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE).Physicochemical analysis showed that the optimal growth conditions for xerophilic fungi were provided during the fermentation processing of Fuzhuan brick-tea. The genus Eurotium is the predominant fungi during the processing of Fuzhuan brick-tea detected with culture-based method.Three sets of universal primers targeting at fungal rDNA, such as NS1//GCfung5, su-0817-GC//ssu-1196 and NL1F-GC//LS2R, were adopted to amplify total DNA extracted from each tea sample. After PCR, the amplicons were separated by DGGE. Aimed at analysis of fungal community structure in samples, a comparison of the resulted three PCR-DGGE profiles showed that the best effect was obtained with the primer set of NL1F-GC//LS2R.In present study, at least eight genera of fungi, Eurotium, Debaryomyces, Aspergillus, Verticillium, Pichia, Pestalotiopsis, Rhizomucor and Beauveria in the Fuzhuan brick-tea related samples were identified by PCR-DGGE analysis. Except for genera Rhizomucor and Beauveria, fingerprints of the remaining six genera were detected in the fermenting tea samples. Fingerprints of Eurotium, Debaryomyces and Aspergillus were consistently visible, with the genus Debaryomyces not previously reported in Fuzhuan brick-tea. Xerophilic fungi such as genera Eurotium and Debaryomyces were predominant fungi during the fermentation processing of Fuzhuan brick-tea, and the genus Aspergillus acted as a subordinate partner under normal conditions.The genera Rhizomucor and Verticillium which were dominant fungi in raw dark green tea had never been detected previously. This is the first report of the genus Beauveria in stored ready made Fuzhuan brick-tea. The presence of genus Beauveria may be related to long-term preservation of the stored Fuzhuan brick-tea. It might be considered to utilize this safe microorganism to help protect tea products from insect spoilage.The PCR-DGGE fingerprints of fungal communities in industrial scale fermentation processing of Fuzhuan brick-tea were achieved in the present study. The results gave directions to screening of functional fungi used as fermentation starters for Fuzhuan brick-tea.2. Identification and phylogenetic analysis of the functional strains isolated from Fuzhuan brick-teaIdentification of the microbial species is the most importance for developing an industrial microorganism. For the isolated strain of Eurotium sp. FZ, the morphological features of colonies growing on media CZA30, CZA200 and M40Y, the microscopic features of sexual reproduction structures, the sub-microscopic features of ascospores under scanning electron microscope, were investigated. The morphological species name of the strain was identified as Eurotium cristatum relied on those typical morphological features.When applying multilocus sequence typing (MLST) approach to identify the strain of Eurotium sp. FZ, we determined the sequences from the four loci, ID region (ITS 1-5.8S rDNA-ITS2 and D1/D2 divergences of 28S rDNA), RPB2 (RNA polymerase II), BT2 (β-tubulin) and CF (calmodulin) and deposited the resulted sequences in GenBank under the accession numbers HQ148160, HQ148161, HQ148162 and HQ148163, respectively. The phylogenetic species name of the strain was identified as Eurotium cristatum based on the MLST approach.The identification results based on the morphological attributes and on the MLST approach were in agreement with each other.The ID region sequence of the yeast strain was determined and submitted in GenBank under the accession number of JF710627. The yeast was identified as Debaryomyces hansenii. This yeast is proposed to grant QPS (Qualified Presumption of Safety) status along the list of units referred to EFSA (European Food Safety Authority). The yeast strain of Debaryomyces hansenii would be a candidate microorganism for developing mixture fermentation starters for production of Fuzhuan brick-tea.3. Analysis of anthraquinone compounds in the Fuzhuan brick-tea related samplesThe presence of three anthraquinone derivatives (emodin, physcion and chrysophanol) in five Fuzhuan brick-tea related samples (Pu’er tea, raw dark green tea, Fuzhuan brick-tea, tea fermented by E. cristatum, biomass of E. cristatum) was detected by HPTLC (High performance thin layer chromatography). Under the experimental conditions for HPTLC, the three standards of emodin, physcion and chrysophanol were separated effectively with the Rf values 0.28,0.43 and 0.51, respectively. The bands’absorption peak and average peak areas were detected at 430 rm. The results indicated that emodin was present in all five samples in descending order of peak areas:raw dark green tea, Pu’er tea, tea fermented by E. cristatum, Fuzhuan brick-tea and biomass of E. cristatum. The physcion contained in the biomass of E. cristatum far exceeded that of tea fermented by E. cristatum, with no physcion found in the remaining three samples. Chrysophanol was not detectable in any of the samples. The emodin was present in all the dark tea samples detected which implied that the anthraquinone compounds (e.g. emodin) existing in dark teas (including Fuzhuan brick-tea) were important sources of dietary anthraquinones for tea consumers.4. Intervention of EHEC O157:H7 infected mice with water extracts from Fuzhuan brick-teaThe EHEC O157:H7 infected murine models were established via intragastric administration with fresh EHEC O157:H7 cells suspension (OD600=1.0) to the mature SPF grade KM mice. The detection of EHEC O157:H7 in their fresh feces was positive. The mice behaved with disease symptoms.Five groups of mice were designed, including normal control, EHEC O157:H7 infected model, intervention with low-dosage water extract (ig.1.0 g/kg daily), middle-dosage (ig.10.0 g/kg daily) and high-dosage (ig.20.0 g/kg daily). The dosages corresponded to normal consumption of Fuzhuan brick-tea (6.6 g/d) for a mature human being (60 kg) and by a factor of 10 and 20, respectively.A comparison analysis was conducted for the five groups of experimental mice in aspects of histopathology, haematology, immunology and intestinal microecology. The results showed that the infected mice model displayed typical symptoms of EHEC O157:H7 infectious disease. The histopathological examination indicated that severe lesions were observed in their gastric, duodenal, ileal, colonic and renal tissue and cell structures. The haematological indices reflected their impaired liver function and renal function. The immunological indices approved their compromised immune system.Low-dosage tea extracts showed rehabilitation-promoting effects on EHEC O157:H7 infected mice to some extent. Moreover, for the middle- and high-dosage groups, the detected indices were improved in comparison with that of infected models, recovered to levels of normal controls and even become superior to that. The results indicated appropriate dosage of water extracts from Fuzhuan brick-tea would exhibit protection and recovery effects on the detriments induced by EHEC O157:H7 infection. The extracts of Fuzhuan brick-tea showed anti-infective effects against EHEC O157:H7 as following:1) maintaining the integration of intestinal epithelium, 2) regulation of the intestinal microbiota,3) improving the body immunity, and 4) protecting liver and kidney against impairment, and ensuring the normal functions of these organs. Considering the dose administered to middle- and high- dosage groups matched with the 10- and 20-times of normal consumption of Fuzhuan brick-tea (6.6 g) for mature human being (60 kg), extrapolating to human, it was suggested that the 10- 20 times concentrated extracts from daily consumed Fuzhuan brick-tea would have therapeutic effect on EHEC 0157:H7 caused infectious diarrhea.The results provided experimental basis and theoretical references for applying Fuzhuan brick-tea to treat EHEC O157:H7 caused infectious diarrhea.5. Effects of Eurotium cristatum on mice colonic microbiotaThe acute toxicity of E. cristatum to SPF grade KM mice was determined. Under the maximal tolerance dose (10000 mg/kg) of suspension of E. cristatum after intragastric administration, zero mortality of the experimental mice was evident. The result indicated that the maximal tolerance dose of the trial (ig), E. cristatum, was greater than 10000 mg/kg. According to the grades of acute toxicity (ig) of chemical compounds, the LD50 range of 5001-15000 mg/kg for mice (ig) indicates the trial belonging in toxicity grade II, that is, an actual non-toxic substance.Four groups of mice were designed, including normal control, intragastric administration with low-dosage water suspension of E. cristatum (50 mg/kg, daily), middle-dosage (200 mg/kg, daily) and high-dosage (1000 mg/kg, daily). The dosages corresponded to daily consumption of E. cristatum 0.33 g,1.32g and 6.6g for a mature human being (60 kg), respectively.The general bacteria, Lactobacillus, Bacteroides and Clostridium cluster IV population structure were detected by PCR-DGGE method. The results showed the colonic bacterial structures in mice were different individually. Low-dosage suspension of E. cristatum increased the population diversity of general bacteria, Lactobacillus and Clostridium cluster IV, but had not obvious effects on Bacteroides population. Considering the health-promoting benefits of Lactobacillus and Clostridium cluster IV, the results implicated that low-dosage suspension of E. cristatum would have up-regulation function to intestinal microbiota. Extrapolating to human, it was implicated that daily consumption of E. cristatum 0.33 g for a mature human being (60 kg) would have up-regulation function to human intestinal microbiota.The results provided experimental basis for developing E. cristatum preparation.更多还原