单宁酶发酵生产的研究进展

单宁酶可水解没食子酸单宁中的酯键和缩酚羧键,生成没食子酸和葡萄糖。单宁酶广泛应用于制革、酿酒、医药、饮料等领域。文中综述了国内外有关单宁酶发酵生产的研究进展,包括产单宁酶菌种的选育、发酵生产方法、底物选择和培养基中添加剂对产酶的影响。     

单宁酶的性质与应用研究进展

单宁酶,又称单宁酯酰水解酶(Tannase,EC 3.1.1.20),可水解没食子酸单宁中的酯键和缩酚羧键,生成没食子酸和葡萄糖,单宁酶在自然界中分布广泛,主要用于食品工业、精细化工等领域.并综述了单宁酶研究与应用的最新进展.     

单宁酶及其应用研究进展

单宁酶（Tannase EC3.1.1.20）全称单宁酰基水解酶（Tannin Acyl Hydrolase）是一种细胞膜结合酶,可水解没食子酸单宁中的酯键和缩酚键,生成没食子酸和葡萄糖。是酶解转溶＂茶乳酪＂的专一酶类,近年来伴随着茶饮料工业的迅猛发展而得到深入地研究。美国食品与药物管理局已确定单宁酶为安全产品,在日本单宁酶已通过FAD批准应用。本文就单宁酶的研究情况作一综述。单宁酸广泛应用于制革、酿酒、医药、饮料等领域[2].     

黑曲霉利用五倍子生料固体发酵产单宁酶优化发酵条件研究

单宁酶全称是单宁酯酰水解酶(Tannase,EC3.1.1.20),它可以水解没食子酸单宁中的酯键和缩酚羧键,生成没食子酸和葡萄糖.主要来源于微生物,存在于细胞内和细胞外,目前研究得较多较为深入的是青霉和黑曲霉.本实验对五倍子生料固体发酵产单宁酶进行了研究,发酵条件的单因素优化后得到:初始水分含量80%,接种量1%,20%五倍子,温度30℃为最佳产酶条件.在此基础上,利用正交实验对显著影响产单宁酶的培养基及培养条件因素进行优化,得到固体发酵黑曲霉诱变菌株最佳产单宁酶条件为:湿度80%、接种量1%、发酵温度33℃、五倍子含量15%.其中温度对黑曲霉产单宁酶酶活性的影响是显著的,并测得在此最佳发酵条件下产单宁酶活性达57.32U/gds.     

单宁酶制备研究进展

单宁酶主要由曲霉属等微生物发酵制备。它能够水解单宁中的酯键和缩酚羧键,生成重要的工业原料没食子酸等化合物。单宁酶已广泛应用于食品饮料、化妆品工业和饲料工业等领域。文章着重综述国内外在单宁酶菌种选育、培养基发酵体系、发酵生产、提取与分离纯化、固定化技术等制备方法方面研究的新进展,分析单宁酶制备研究的发展趋势。     

单宁酶的分离纯化及其在软饮料工业中的应用

单宁酶是一种广泛存在于自然界中的水解酶,可将没食子酸单宁中的酯键和缩酚羧键水解,生成没食子酸和葡萄糖。由于单宁等多酚类物质可与蛋白质、咖啡碱等通过疏水作用及氢键形成络合物进而形成沉淀,所以单宁酶不仅在饮料澄清领域得到了广泛应用,也在皮革制造、饲料及化妆品生产中发挥着极大作用。目前,单宁酶主要由真菌类的曲霉属和青霉属发酵制得,少数细菌、酵母也可产生。美国FDA已确认单宁酶为安全的食品添加剂,我国卫生部也将单宁酶加入食品添加剂目录。文中综述了单宁酶的主要性质、提取与纯化方法及其在软饮料工业中的应用,分析了单宁酶在软饮料工业中的应用现状及存在的问题,并结合实际展望了单宁酶的未来发展和应用。     

单宁酶及其在茶饮料中的应用

单宁酶可水解没食子酸单宁中的酯键和缩酚羧键,主要由曲霉属等微生物发酵生产。单宁酶防止冷混浊、提高提取率、保持色泽和减轻苦涩味的作用在茶饮料工业中有广泛的应用。     

交联酶聚集体法制备单宁酶及固定化酶性质研究

交联酶聚集体法(cross-linked enzyme aggregates,CLEAs)是一种新型的无载体酶固定化方法。使用该法固定化黑曲霉产单宁酶,制备无载体固定化单宁酶(Cross-linkedenzyme aggregates-tannase,CLEAs-TA),并对其制备条件、结构特征、酶学性质进行分析。结果表明,最优制备条件为单宁酶游离粗酶液在冰水浴中经80%的硫酸铵沉淀30min后,以1.5%戊二醛水溶液进行酶聚集体交联反应,可获得较高活性、较高稳定性的交联酶聚集体,酶活回收率达47.33%,对酯键水解作用的最适温度、最适pH值分别为50℃、4.5,与游离酶相比,表现更高的pH及温度稳定性,重复使用6次后,活力剩余32.86%,其良好的操作稳定性有利于没食子酸丙酯高效转化为没食子酸及广泛应用于水解茶饮料中单宁的反应。     

产单宁酶海洋短梗霉的分离筛选及酶学性质的研究

单宁酶能水解单宁物质中的酯键,在食品和饲料工业中有重要应用价值。 该研究从海洋微生物中获得了几株高产单宁酶 菌株,其中菌株95水解圈与菌落（直径）大小的比值最大,对其进行生理生化鉴定,并扩增内转录间隔区（ITS）序列作进化树分析, 鉴定菌株95号为Aureobasidium subglaciale,其在发酵培养基中发酵72 h达到最大酶活力323.2 U/mL。 考察了温度和pH对酶促反应 影响,同时考察了粗酶的热稳定性和pH稳定性。 结果表明,菌株95号所产单宁酶最适反应温度为50 ℃,该酶在30 ℃、40 ℃、50 ℃水浴 保温4 h,相对酶活80%以上,具有较好的热稳定性;最适反应和最稳定pH均为6.0,在pH 3.0～7.0之间均有较高的酶活,表示其具有 较广的pH值稳定性。     

1 株产单宁酶嗜热真菌的鉴定、酶学性质分析及碳水化合物活性酶表达

对产单宁酶的1 株嗜热真菌HBHF5进行鉴定,开展单宁酶酶学性质的分析及碳水化合物活性酶（carbohydrate?active enzymes,CAZymes）的转录组学研究,探究嗜热真菌HBHF5在食品酶制剂开发中的潜力。经对菌株的菌落、孢子形态观察及ITS序列比对分析,最终鉴定嗜热真菌HBHF5为烟曲霉（Aspergillus fumigatus）。经分析,菌株HBHF5在固态发酵培养时不产单宁酶,而液态诱导培养时,菌株HBHF5胞内和胞外均检测到单宁酶活性,且以胞外酶为主（94%）,酶活力最高达136?U/mL。单宁酶最适反应温度为60?℃,在60?℃处理30?min,能够维持酶原活力的90%以上。该酶最适反应pH值为6.0,在pH?5.0～9.0范围内,能够维持60%以上的酶活力。不同金属离子对酶活力的影响存在差异,Cu2＋、Fe3＋、Mn2＋和Zn2＋对该单宁酶活性抑制较强。经转录组学分析,该菌以麸皮为唯一碳源时,共有淀粉酶、纤维素酶和果胶酶等239?个CAZymes基因表达,其中糖苷水解酶类最为丰富,约占CAZymes表达总数的70%。A. fumigatus HBHF5是1 株优良产酶菌株,为具有食品酶制剂开发潜力的嗜热真菌。     

产单宁酶菌株的筛选及选育

为得到高产单宁酶的菌株,从多年种植的茶园、柿子园以及掩埋柿子、五倍子和茶叶的花园土壤中通过平板初筛和三角瓶固体发酵复筛筛选到一株产单宁酶的曲霉菌株DNM1-39,固体培养其产单宁酶活力为1.58U/g。以DNM1-39为出发菌株,经紫外线照射、硫酸二乙酯单独处理和紫外线照射-硫酸二乙酯复合诱变处理,获得突变株UD-6,其产酶活力达2.80U/g。连续传代实验结果表明,该菌株产酶活力不变,性能稳定。     

Enzymatic treatment to improve the quality of black tea extracts

Enzymatic extraction was investigated to improve the quality of black tea extracts with pretreatment of pectinase and tannase independently, successively and simultaneously. Pectinase improved the extractable-solids-yield (ESY) up to 11.5%, without much of an improvement in polyphenols recovery, while tannase pre-treatment showed a significant improvement in polyphenols recovery (14.3%) along with an 11.1% improvement in ESY. Among the four treatments, tannase-alone treatment showed the maximum improvement in tea quality, with higher polyphenols-in-extracted solids. Treatments involving tannase resulted in the significant release of gallic acid, due to its hydrolytic activity, leading to greater solubility besides favourably improving TF/TR ratio. The results suggested that employing a single enzyme, tannase, for the pre-treatment of black tea is desirable. Enzymatic extraction may be preferred over enzymatic clarification as it not only displayed reduction in tea cream and turbidity but also improved the recovery of polyphenols and ESY in the extract, as well as maintaining a good balance of tea quality.     

香蕉及茶叶中单宁降解菌株的筛选

本实验从香蕉茎叶、茶叶及香蕉土壤、茶土壤环境中经初筛和复筛分离纯化出16株产单宁酸酶的菌株,对其菌株的外在特性和产酶活力进行了研究和测定。结果表明,从培养基来看,产酶菌株以从牛肉膏蛋白胨中性培养基里分离出来的居多（11株）;从原料来源看,以从茎叶分离出的菌株居多（14株）;菌株多为棕或绿色霉菌（15株）;水解圈以透明的居多（10株）,且16株菌株的水解圈与菌落直径比均在1．0．2．0之间。产酶菌株的酶活和水解圈的透明状态基本相符;其中以牛中香茎处10^0-1、牛中香茎处10^0-2、牛中香茎处10^0-6等3株的酶活值最大,为3．4。     

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

The tannase‐encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587‐amino acid protein, preceded by an N‐terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (–Gly–X–Ser–X–Gly–) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild‐type and recombinant strains were essentially indistinguishable. A molecular mass of ～320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35–40 °C and a pH optimum at ～6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wild‐type strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks. Copyright © 2009 John Wiley & Sons, Ltd.     

利用黑曲霉B1401发酵废弃茶末浸提液产单宁酶的工艺优化

以废弃茶末为原料,接种黑曲霉,以液态摇瓶的形式进行单宁酶生产的研究。通过单因素实验、Plackett-Burman实验及Box-Behnken响应面实验优化黑曲霉B1401液态发酵产单宁酶的最佳工艺条件。结果表明,诱导物为6%单宁酸+3%茶多酚,碳源为复配碳源,即0.746%可溶性淀粉:0.254%蔗糖,氮源为复配氮源,即0.3%尿素:0.7%硝酸钠,接种量为1%,装液量为90 mL/250 mL,转速为140 r·min-1,温度为30 ℃,发酵时间为72 h,此条件下单宁酶酶活可达104.82 U/g。     

Effect of tannase treatment on protein-tannin aggregation and sensory attributes of green tea infusion

Effect of tannase enzymatic treatment on protein-tannin aggregation and sensory attributes of green tea infusion was investigated. Green tea leaves were extracted with hot water at 85 °C for 20 min, the tea infusion was then treated with tannase. Results showed that both EGCG and ECG of the tea catechins were hydrolyzed by tannase into EGC and EC, respectively, accompanied by production of gallic acid. The tannase-treated tea infusion had a relatively lower binding ability with protein. Changes in the content of tea catechins, formation of tea cream, and turbidity of tea infusion with or without tannase treatment were measured after 4 weeks. Content of catechins in the tannase-modified tea remained almost unchanged, while those without tannase treated (control) decreased significantly (p < 0.05). Meanwhile, better color appearance and less tea cream formation were observed for the tannase-treated green tea, and tea cream formed for the control after storage. Results of the sensory evaluation showed that mouth feeling, taste and the overall acceptance of the tannase-treated green tea infusion were all better than those of the control.     

利用微生物酶两步法生产没食子酸的初步研究

没食子酸及其衍生物是食品、医药等工业的一类重要原料。以黑曲霉B0201为菌种,以五倍子为原料,直接生料固体发酵法不能生成没食子酸,但是,先用生料固体发酵生产单宁酶,再用酶法制备没食子酸是可行的。研究表明,分两步制备没食子酸反应4h时,100mL单宁酶酶液中积累了0.77g没食子酸,建立了一种黑曲霉单宁酶两步法生产没食子酸的制备工艺。     

Bacillus sphaericus: the highest bacterial tannase producer with potential for gallic acid synthesis

An indigenously isolated strain of Bacillus sphaericus was found to produce 1.21 IU/ml of tannase under unoptimized conditions. Optimizing the process one variable at a time resulted in the production of 7.6 IU/ml of tannase in 48 h in the presence of 1.5% tannic acid. A 9.26-fold increase in tannase production was achieved upon further optimization using response surface methodology (RSM), a statistical approach. This increase led to a production level of 11.2I U/ml in medium containing 2.0% tannic acid, 2.5% galactose, 0.25% ammonium chloride, and 0.1% MgSO(4) pH 6.0 incubated at 37°C and 100 rpm for 48 h with a 2.0% inoculum level. Scaling up tannase production in a 30-l bioreactor resulted in the production of 16.54 IU/ml after 36 h. Thus far, this tannase production is the highest reported in this bacterial strain. Partially purified tannase exhibited an optimum pH of 5.0 with activity in the pH range of 3 to 8; 50°C was the optimal temperature for activity. Efficient conversion of tannic acid to purified gallic acid (90.80%) was achieved through crystallization.     

微生物源单宁酶的研究进展

单宁酶可将单宁水解成没食子酸和葡萄糖，是一种重要的工业用酶。植物、动物和微生物中都含单宁酶，其中微生物是单宁酶的主要来源。基于单宁酶的国内外研究成果，该文归纳总结了不同微生物来源的单宁酶、酶学性质、作用机制，阐述了单宁酶在食品、饲料、制革、精细化工等实际生产中的应用，并对单宁酶今后的研究方向和应用前景进行了展望。