普洱茶后发酵过程中微生物的研究进展

对有关普洱茶后发酵生产与微生物的关系、微生物的种类、微生物对普洱茶品质的影响以及主要微生物的生长特性等方面的研究工作进行综述。指出加强普洱茶后发酵过程中微生物基础研究的必要性,提出建立普洱茶后发酵菌物库,重视对影响普洱茶品质的菌物开展系统研究的建议。     

普洱茶中功能性微生物的筛选及其对普洱茶感官品质的影响

普洱茶的渥堆发酵过程是以晒青毛茶的内含成分为基础,在微生物分泌的胞外酶及湿热作用下,发生一系列化学变化,最终形成普洱茶独特的风味。采用稀释涂布法,根据产酶微生物的特性,经过固体平板初筛和液体茶汤培养基复筛,从普洱茶中分离得到若干株产酶菌株,并从中挑选优良菌株接种普洱茶固体发酵,考察其对普洱茶感官品质的影响。经分子生物学鉴定,D13-16为米曲霉(Aspergillus oryzae),相似度为98.62%;GJ-02为米根霉(Rhizopus cryzae),相似度为98.57%;XW-10和DF-03均为黑曲霉(Aspergillus niger),相似度分别为99.10%和99.92%。结果表明:普洱茶香气的形成主要与蛋白酶产生菌有关,DB-16发酵后香气评分达到31分(对照28,总分40);汤色主要受多酚氧化酶产生菌的影响,DF-03发酵后汤色评分达到19分(对照12,总分20);而这四种功能性微生物均在不同程度上促进了普洱茶滋味的形成。     

普洱茶后发酵中咖啡碱变化研究进展(综述)

普洱茶（熟茶）的后发酵加工工艺,是形成其独特品质特征的关键工序。咖啡碱是一种甲基黄嘌呤,是茶叶中含量比较丰富的生化成分之一,也是茶叶主要呈味物质和生理活性成分,具有促进兴奋、强心、解毒、利尿、助消化等功效。咖啡碱在普洱茶后发酵过程中呈增加趋势。文章对普洱茶后发酵过程中咖啡碱变化的相关研究进行系统综述,为普洱茶咖啡碱理论研究提供科学依据。     

曲霉属真菌在普洱茶后发酵中的作用

为阐明微生物在普洱茶后发酵过程中的作用及其机制,本文进行了普洱茶温湿度试验,并利用从普洱茶后发酵堆中分离鉴定的优势曲霉属真菌,进行接种试验和专用菌剂的发酵生产试验.结果提示,只有在微生物的作用下,普洱茶才具特有的感官特性,曲霉属真菌能控制后发酵的过程,改变茶叶的感官特性.曲霉属真菌的种类及组合对普洱熟茶的茶多酚组成与含量,以及没食子酸、茶氨酸、咖啡因等特征成分的含量均有显著的影响.因此,应用专用菌剂进行后发酵生产,能加快普洱茶的熟化速度,提高发酵成功率,保证产品质量的稳定性,使普洱熟茶的规范化生产成为可能.     

普洱茶发酵前后对PTPs靶标抑制效果的比较

用不同品种无性系茶树良种制成普洱生茶和普洱熟茶,进行PTP1B、TCPTP、SHP-1、SHP-2、HePTP、YopH的靶标对比试验,结果表明经自然渥堆发酵的普洱熟茶对六个靶标的的抑制效果明显好于普洱生茶,说明普洱茶的发酵工艺是形成其特有保健功效的关键所在;另外,普洱熟茶对靶标的抑制效果还与其原料有关,在研究的3个大叶种茶中,对六个的靶标的平均半数抑制浓度(IC50)为云抗10号<云抗14号<大黑茶。     

普洱茶渥堆发酵与堆肥渥堆发酵的比较研究

普洱茶的自然渥堆发酵工艺与农业及环保中的堆肥发酵工艺在堆垛方法、堆垛形状和大小、自然升温过程、高温发酵、翻堆工艺等方面有许多相似之处,主要差别是原料和含水量的不同。对不同原料堆肥已有详细的研究及其动态菌群和多种发酵剂的研究和应用报道,明确了高温菌在渥堆发酵过程中起重要作用,对其翻堆工艺也有较详细的参数及其机械化的应用,但普洱茶渥堆发酵基本上还停留在自然发酵、人工翻堆、常温菌的研究及老茶头作发酵剂的应用等水平上。堆肥的许多研究手段和方法甚至物质转化、脱毒、高温菌的研究等都值得普洱茶渥堆发酵研究加以借鉴。     

黑曲霉B0201利用五倍子固体发酵产单宁酶的条件研究

通过比较常规灭菌发酵和生料发酵, 研究黑曲霉B0201利用五倍子固体发酵产单宁酶的条件。结果表明, 在生料发酵过程中, 采用20%五倍子并且以(NH4)2SO4为氮源制备单宁酶的最佳条件为: 液固比=1.6:1、温度30°C、初始pH 6.0。在该条件下通过96 h的培养, 单宁酶的活力可达51.2 U/gds, 是常规灭菌发酵的3.6倍。以上结果显示, 生料发酵生产单宁酶是一种高效可行的方法。     

从黑茶中分离和筛选产单宁酶菌株

茶叶中富含单宁化合物。从分离自黑茶的真菌菌株中,筛选高产单宁酶的菌株;进而分离纯化单宁酶,分析单宁酶对茶汤的转溶效果。从不同产地的3个黑茶样品中,共分离获得44个真菌分离物;经初步鉴定,这些真菌分离物以曲霉属（Aspergillus）、青霉属（Penicillium）和散囊菌属（Eurotium）的真菌居多。以单宁酸为底物的鉴别培养基初筛表明,其中26个真菌分离物在鉴别平板上产生透明圈,显示单宁水解酶活性;通过固体发酵复筛,筛选到1株产单宁酶活性较高的菌株,初步鉴定为青霉属（Penicillium）菌株,命名为青霉MP-24菌株。青霉MP-24可以以茶叶、茶梗和麸皮等农副产品作为原料固体发酵产生单宁酶。以麸皮为原料的发酵产物经过硫酸铵分级沉淀、DEAE阴离子交换层析和葡聚糖G-150凝胶层析等分离纯化步骤,得到分子量为70 kDa的单一蛋白质条带,单宁酶活力达到603.68 U/mg。纯化获得的单宁酶对茶汤有良好的转溶效果。研究结果表明,在黑茶相关微生物中含有丰富的产单宁酶菌株,是工业酶制剂的重要资源。     

普洱熟茶后发酵加工过程中曲霉菌的分离和鉴定

从云南省易武、基诺山、普洱和昆明后发酵加工生产的普洱熟茶堆中分离到7种曲霉菌,分别鉴定为：温特曲霉烟色变种,帚状曲霉,具黄曲霉,埃及曲霉,臭曲霉,日本曲霉原变种,以及局限灰曲霉。发现不同产地普洱熟茶中曲霉菌菌群的组成存在差异,对曲霉菌在普洱熟茶生产中的作用与意义进行了初步的讨论。     

黑曲霉及其与普洱茶品质关系研究进展

近年来,黑曲霉菌的研究受到了国内外大量学者的重视,并取得了一系列新进展,这些进展主要集中在:黑曲霉的分离鉴定方法;黑曲霉发酵生产多酚氧化酶、果胶酶和纤维素酶等酶类的机理;黑曲霉对普洱茶色泽、滋味和香气的影响等方面。文章集中对近年来黑曲霉及其与普洱茶品质形成相关的研究进展作简要综述,以期为黑曲霉在普洱茶中研究利用提供一定的参考。     

Antibacterial property and mechanism of a novel Pu-erh tea nanofibrous membrane

Pu-erh tea is made via a natural fermentation process. In this study, Pu-erh tea was used as a raw material for nanomaterials preparation and as an antibacterial agent. Antibacterial activities on Escherichia coli of Pu-erh tea, Pu-erh tea powder (PTP) of different sizes, and Pu-erh tea residual powder were firstly determined, respectively. With polyvinyl alcohol as the carrier, through an electrospinning technique, different kinds of nanofibrous membranes were obtained from the extract of Pu-erh tea and nano-PTP (NPTP), and their antibacterial properties and mechanism against E. coli were evaluated. The results showed better antibacterial activity with smaller PTP particles, the nano-sized particles had the best effects, and the MIC of NPTP was 13.5 mg/mL. When NPTP was in nanofibrous membranes, the antibacterial activity decreased slightly, but increased with modification by ZnO. Pu-erh tea in nanofibrous membranes damaged the E. coli cell membranes and caused leakage of K⁺ and enzymes. What is more is that damage of the cell walls led to the leakage of fluorescent proteins from enhanced green fluorescence protein-expressing E. coli. These results indicate that the Pu-erh tea nanofibrous membranes had good antibacterial activities against E. coli, which may provide a promising application of novel antibacterial materials.     

Diversity of lactic acid bacteria from Miang,a traditional fermented tea leaf in northern Thailand and their tannin-tolerant ability in tea extract

The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041^T. Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.     

Bioconversion of tea polyphenols to bioactive theabrownins by Aspergillus fumigatus

Theabrownins (TB) are water-soluble phenolic compounds associated with the various health benefits of Pu-erh tea, a post-fermented Chinese dark tea. This work reports on the production of theabrownins from infusions of sun-dried green tea leaves using a pure culture of Aspergillus fumigatus isolated from a solid-state Pu-erh tea fermentation. A theabrownins yield of 158 g kg^?1 sun-dried green tea leaves was obtained in 6 days at 45 °C in an aerobic fermentation. In a 2 l fermenter, the yield of theabrownins was 151 g kg^?1 sun-dried green tea leaves in 48 h of aerobic culture (45 °C, 1 vvm aeration rate, 250 rpm agitation speed). Extracellular polyphenol oxidase and peroxidase of A. fumigatus contributed to this bioconversion. Repeated batch fermentation process was used for producing theabrownins but was less productive than the batch process.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.     

普洱茶中微生物的筛选及其安全性初步评价

目的在对普洱茶中微生物筛选和鉴定基础上对蜡样芽胞杆菌毒素基因的分布、普洱茶下调毒素基因的表达和改善肠上皮细胞的损伤进行研究。方法分别采用无菌水和沸水泡制普洱茶,获得分离株,通过16SrDNA测序以及生理生化试验确定其归属;对所筛选的菌株进行耐模拟胃肠液能力评价和毒力基因的检测;采用细胞实验和荧光定量PCR技术,研究普洱茶对蜡样芽胞杆菌毒素的抑制作用。结果无菌水浸泡普洱茶获得的45株菌中44株为芽胞杆菌属,沸水浸泡获得的7株菌均为蜡样芽胞杆菌(FBCE01、FBCE06、FBCE10、FBCE14、FBCE20、FBCE26和FBCE29),多重PCR技术结果表明其分别含有毒力基因cytK、nheA和hblD的2种或3种。耐模拟胃肠液实验表明,7株菌均具有很强的耐模拟胃肠液消化能力;细胞实验结果发现,普洱茶汤能显著降低蜡样芽胞杆菌对Caco-2细胞的粘附(P0.05);MTT实验结果显示,普洱茶能有效降低蜡样芽胞杆菌对细胞的损伤;荧光定量PCR技术结果进一步说明,普洱茶使蜡样芽胞杆菌肠毒素的mRNA表达水平下调。结论普洱茶具有抑制蜡样芽胞杆菌毒素的作用。     

Characterization of a bacterial tannase from Streptococcus gallolyticus UCN34 suitable for tannin biodegradation

The gene in the locus GALLO_1609 from Streptococcus gallolyticus UCN34 was cloned and expressed as an active protein in Escherichia coli BL21 (DE3). The protein was named TanSg1 since it shows similarity to bacterial tannases previously described. The recombinant strain produced His-tagged TanSg1 which was purified by affinity chromatography. Purified TanSg1 protein showed tannase activity, having a specific activity of 577 U/mg which is 41 % higher than the activity of Lactobacillus plantarum tannase. Remarkably, TanSg1 displayed optimum catalytic activity at pH 6–8 and 50–70 °C and showed high stability over a broad range of temperatures. It retained 25 % of its relative activity after prolonged incubation at 45 °C. The specific activity of TanSg1 is enhanced by the divalent cation Ca²⁺ and is dramatically reduced by Zn²⁺ and Hg²⁺. The enzyme was highly specific for gallate and protocatechuate esters and showed no catalytic activity against other phenolic esters. The protein TanSg1 hydrolyzes efficiently tannic acid, a complex and polymeric gallotanin, allowing its complete conversion to gallic acid, a potent antioxidant. From its biochemical properties, TanSg1 is a tannase with potential industrial interest regarding the biodegradation of tannin waste or its bioconversion into biologically active products.     

青霉单宁酶高活性菌株的诱变选育

利用塔拉单宁诱导丝状真菌产生单宁酶的原理,通过富集培养,从天然源分离得到30株具有较高单宁酶活性的青霉菌;经二级发酵程序,对这30株菌进行了生物转化复筛实验,选择出能水解塔拉单宁,且生物催化活性较高的青霉野生株Penicilliumsp.No.23,对No.23进行经紫外诱变处理,诱变株经筛选,最后得到1株具有稳定遗传性的单宁酶高活性菌株,其单宁酶活性比出发菌株提高了35%。     

Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.