短梗霉(Aureobasidium pullulans)与短梗霉多糖发酵

短梗霉多糖系短梗霉发酵产物,它具有巨大的经济价值。短梗霉是一种多形态真菌,具有复杂的生活史,并且其形态学变化与其多糖合成之间关系密切。本文就短梗霉及其发酵特点进行较详细的综述。

出芽短梗霉(Aureobasidium pullulans)生物合成普鲁兰多糖的研究进展

普鲁兰多糖是由出芽短梗霉(Aureobasidium pullulans)发酵产生的一种胞外多糖,其特殊的结构赋予了它许多优良的理化性质,广泛应用于食品、制药、化工、石油及其他制造业。在普鲁兰多糖生物合成过程中,普鲁兰多糖的化学结构、产率及颜色的深浅等受到多种营养条件和环境因素的影响。介绍了普鲁兰多糖的结构、性质,重点综述了国内外学者对发酵条件优化的研究现状,最后总结了普鲁兰多糖生物合成研究的发展方向。

Amylase Production by the Marine Yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identi-fication methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 ℃, aeration rate 6 Lmin-1 and agitation speed 250 rmin-1. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.

Herbicidal activity of Aureobasidium pullulans PA-2 on weeds and optimization of its solid-state fermentation conditions

Fungal strain PA-2 was isolated from infected poplar leaves from the Ping’an District, Haidong City of Qinghai Province, China. Based on the culture characteristics and the internal transcribed spacer(ITS) sequence of its 16S r DNA, the strain was identified as Aureobasidium pullulans. The culture and metabolites of strain PA-2 showed high herbicidal potential to five tested weeds Galium aparine var. tenerum, Chenopodium album, Malva crispa, Polygonum lapathifolium and Avena fatua. For the in vitro test, 5 days after the detached leaves were inoculated with PA-2 culture, all leaves infected by the hyphae and became black and rotten. For the in vivo test, a metabolite filtrate of PA-2 culture was sprayed over the living weed plants, and five days after inoculation, the weed plants became withered and necrotic. Seven days after inoculation, the fresh weight reductions of G. aparine var. tenerum, C. album, M. crispa, P. lapathifolium and A. fatua were 87.25, 78.46, 82.25, 62.11, and 80.27%, respectively. Galium aparine var. tenerum and M. crispa exhibited significant reductions in fresh weight. The bio-safety test on the five crops showed no significant plant height reductions, which was also observed for wheat(Triticum aestivum), faba bean(Vicia faba), and barley(Hordeum vulgare). By contrast, oilseed rape(Brassica napus) and pea(Pisum sativum) exhibited light spots but no significant reductions in plant height. These results indicated that A. pullulans could be a potential microbial herbicide for the control of the target weeds in crops. Optimization of the carbon and nitrogen sources for cultural media and substances for solid-state fermentation indicated that PA-2 had better colony growth and spore production with the optimal carbon source of glucose(C4), nitrogen source of soybean flour(N2), and the optimal substance was wheat bran. The results in this study provide useful information for the development ofA. pulluans PA-2 as an herbicide for bio-control of the weed.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase(CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U(mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 k Da. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other fungi. The optimal p H of the enzyme was 5.6, and the activity profile was stable in a range of acidity(p H 5.0–6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. Km and Vmax values of the purified enzyme were 4.7 mg m L-1 and 0.57 μmol L-1 min-1(mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose(CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp(EU978473). The protein deduced contained the conserved domain of cellulase superfamily(glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

负载互叶白千层精油的Zein/Pullulan复合膜乳液对芒果保鲜贮藏品质的影响

为研究新型复合乳液对芒果贮藏品质的影响,制备了负载0、1%、2%(v/v)互叶白千层精油的玉米醇溶蛋白/普鲁兰多糖复合乳液,并对芒果进行涂膜处理,于20℃、RH 65%贮藏,定期测定指标。结果表明:3种复合乳液涂膜处理均能减缓芒果失重率的升高,且维持更高的果肉Vc含量,但对果肉TA和TSS含量影响不大;而负载互叶白千层精油的复合乳液还能有效地抑制芒果果实的呼吸作用,延缓果皮叶绿素的降解和类胡萝卜素含量的升高,其中负载1%互叶白千层精油的玉米醇溶蛋白/普鲁兰多糖乳液处理在延缓果实叶绿素的降解、减缓失重率的上升和维持Vc含量方面的效果更好。综上,负载1%互叶白千层精油的玉米醇溶蛋白/普鲁兰多糖乳液更利于维持芒果的贮藏品质。

Intraspecific Diversity of Aureobasidium pullulans Strains from Different Marine Environments

Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nine strains among these marine yeasts belong to Aureobasidium pullulans, although the morphologies of their colonies are very different. The marine yeasts isolated from different marine environments indicate that A. pullulans is widely distributed in different environmental conditions. These Aureo-basidium pullulans strains include A. pullulans 4#2, A. pullulans N13d, A. pullulans HN3-11, A. pullulans HN2-3, A. pullulans JHSc, A. pullulans HN4.7, A. pullulans HN5.3, A. pullulans HN6.2 and A. pullulans W13a. A. pullulans 4#2 could produce cellulase and single cell protein. A. pullulans N13d could produce protease, lipase, amylase and cellulase. Both A. pullulans HN3-11 and A. pullulans HN2-3 were able to produce protease, lipase and cellulase. A. pullulans JHSc could secrete cellulase and killer toxin. Both A. pullulans HN4.7 and A. pullulans HN5.3 could yield lipase and cellulase. A. pullulans W13a was able to secrete extracellular amylase and cellulase while A. pullulans HN4.7 and A. pullulans N13d could produce siderophores. This means that different A. pullulans strains from different marine environments have different physiological characteristics, which may be applied in many different biotechnological industries.

Studies on the optimal culture conditions of <i>Aureobasidium pullulans</i>to produce exopolysaccharides

In the current study, in order to change the permeability of cell membrane and solve the problem of linked group of fungi mycelium, the method of adjusting osmotic pressure of medium and adding tween-80 was established. The utilized strain with relatively high exopolysaccharide (EPS) yield and low pigment level was obtained after the rejuvenation and sifting of long-preserved Aureobasidium pullulans strain. The optimal proportion of substrate was determined by means of orthogonal test. The transformation ratio of EPS was increased by 10% - 20% and the pigment content was greatly reduced. The fermenting liquor is between creamy white and pale yellow, and the white primary product can be gained without decolourization step. Furthermore, to magnify to 5 L bioreactor can get the similar result.

The Synergism of Chemical Herbicides and Aureobasidium pullulans for Control Cleavers(Galium aparine L.) in Wheat

Aureobasidium pullulans, a biocontrol agent for the annual weed Galium aparine L. was evaluated in vitro for its compatibility with commercial formulation of five herbicides at 1X(recommended field rate), 0.5X, 0.2X, 0.1X, 0.067 X, and 0.05 X concentrations. Germination of A. pullulans with paraquat, 2, 4-D, quizalofop-p, and clethodim treatment appeared reduced compared with germination of A. pullulans with fluroxypyr treatment at all concentrations. Stunted and shorter germ tubes in comparison with the control were observed with 2, 4-D, quizalofop-p, and clethodim at 0.2X. All concentration of paraquat, 2, 4-D, quizalofop-p, and clethodim except0.05 X, significantly decreased radial growth of A. pullulans compared with its growth on the untreated PDA medium. Field trials to further develop A. pullulans as biocontrol agent for control G. aparine L. was conducted to test the effectiveness of this fungus in wheat plots for 2 years at the same location in Xining. Treatments included spore suspensions of A. pullulans alone, a mixture of both fungus and fluroxypyr in wheat. Biocontrol agent effectiveness was estimated at approximately 7and 14 days after treatment, as disease incidence, percent weed control, and weed biomass reduction. Significant reduction in weed biomass occurred in combination treatments, and potential exists to tank mix A. pullulans with fluroxypyr. Leaf surface moisture and air temperatures following application may account for inconsistencies in field results between years. This fungal organisms show potential as bioherbicides for weeds in G. aparine L.

共表达分子伴侣蛋白提高III型普鲁兰水解酶在短小芽孢杆菌中的分泌表达及发酵优化

本研究旨在通过共表达分子伴侣蛋白以及优化发酵条件来提高III型普鲁兰水解酶(TK-PUL)在短小芽孢杆菌中的分泌表达水平。通过构建共表达TK-PUL和分子伴侣蛋白的多种重组短小芽孢杆菌,并以胞外酶活为指标对其进行筛选,确定最有利于TK-PUL分泌表达的分子伴侣蛋白及对应的重组短小芽孢杆菌。在此基础上采用单因素实验和响应面法优化重组短小芽孢杆菌的发酵条件。结果表明,共表达分子伴侣蛋白PrsA^(Ba)的重组短小芽孢杆菌的胞外酶活达到98.79 U/mL,提高了0.31倍。该重组短小芽孢杆菌的最佳培养基包括19.65 g/L葡萄糖、21.46 g/L酵母提取物、12.01 g/L MgCl_(2)·6H_(2)O、9.02 g/L脯氨酸、0.01 g/L FeSO4·7H_(2)O、0.01 g/L MnSO4·4H_(2)O、0.001 g/L ZnSO4·7H_(2)O。在发酵温度为35℃、起始发酵pH为7.0的条件下,该重组短小芽孢杆菌于最佳培养基中培养66 h时,其胞外酶活达到192.68 U/mL,提高了1.56倍。本研究通过共表达分子伴侣蛋白和发酵优化实现了TK-PUL在短小芽孢杆菌的高效分泌表达,为TK-PUL的应用提供了基础。

生物絮凝剂Pullulan絮凝Pb^(2+)的性能研究

进行了生物絮凝剂Pullulan絮凝水中Pb2+的试验,探讨了Pullulan与电解质A lC l3的复合比、絮凝溶液pH值以及初始Pb2+浓度对絮凝效果的影响。结果表明:在Pullulan与A lC l3用量比为4∶1.1、溶液pH值为6.5~7.0、Pb2+初始浓度为10、25、60、100 mg/L时,分别投加8、25、40、80 mg/L的Pullulan,对Pb2+的去除率可达最高,分别为73.86%、76.30%、77.07%、81.19%。6次重复性试验表明Pullulan的絮凝效果具有较高的稳定性。

一株高产普鲁兰多糖短梗霉菌株的基因组分析及产糖机理探究

从分子水平解析微生物的基因组变化,从而对其进行遗传改造以提高普鲁兰多糖的产量是目前亟待解决的问题。通过比较基因组学方法,对一株高产普鲁兰多糖的出芽短梗霉(TN-31)与其他短梗霉菌株进行了基因组比较。结果显示,该菌株发生了基因组加倍,其基因组中存在大量的遗传变异,这可能是其高产普鲁兰多糖的重要原因。此外,该菌株的基因组大小高于其他短梗霉菌株,其特有基因和功能基因启动子区的特殊调控序列可能赋予其不同于其他短梗霉菌株的特性。这种特殊表型可能是适应性进化的结果,通过遗传变异和适应性选择,能够适应其所处的环境。

一种新型负载淫羊藿苷的普鲁兰纳米粒制备及其性能研究

目的 设计一种新型淫羊藿苷修饰的普鲁兰聚合物,通过化学连接和物理包埋的方式高效装载淫羊藿苷,制备一种具有缓释性的纳米球形载体,并对其性能进行初步探讨。方法 淫羊藿苷通过丁二酸酐作为连接臂接枝亲水性普鲁兰形成淫羊藿苷修饰的普鲁兰聚合物,通过傅里叶红外光谱对终产物鉴定。采用透析法制备负载淫羊藿苷的普鲁兰纳米粒。测量空白纳米粒和载药纳米粒的粒径和Zeta电位,利用透射电镜观察其形貌,并测定其在不同pH条件下的体外药物释放行为。结果 纳米粒具有规则的形态、均匀的尺寸。空白纳米粒电位为+2.23 mV,载药纳米粒电位为+3.98 mV;空白纳米粒平均粒径为122 nm,载药纳米粒粒径为190 nm。纳米粒能明显延长淫羊藿苷的缓释作用时间,在pH 6.8的酸性条件下更有利于淫羊藿苷的释放。结论 通过两亲性聚合物自组装行为,成功制备了尺寸均匀的纳米粒。通过聚合物化学连接方式丁二酸将中药单体淫羊藿苷连接普鲁兰制备为纳米递送系统,淫羊藿苷的普鲁兰纳米递送系统该系统能使疏水性药物成为纳米型的水溶性药物,药物具有缓释性,并在弱酸度环境下可以定向释放。

Pullulan分子结构的研究

利用 GC- MS、IR和 13 C- NMR对 Pullulan的连接碳位和键型进行了研究 ,结果表明 ,Pullulan分子的主链由 β(1→ 3)糖苷链相连 ,主链上每 2～ 3个葡萄糖带有一个葡萄糖残基分枝 ,与主链以 β(1→ 6 )链相连 。

基于壳聚糖的抗菌可注射自愈性水凝胶的制备及其生物相容性研究

为了解决传统水凝胶作为敷料或支架在生物体内应用时所表现出的抑菌活性低、生物相溶性差和机械性能差等问题,本文以氧化普鲁兰多糖(OP)为交联剂,分别同硫醇化季铵盐壳聚糖(NAC-QCS)和己二酸二酰肼(ADH)发生动态化学反应,首次制备出一种可注射的自愈性抗菌的水凝胶——NQC-OP-ADH水凝胶,并对水凝胶的物理化学性质、抑菌活性和生物相容性进行研究。实验表明:NQC-OP-ADH对Escherichia coli和Staphlococcu aureus均表现出较高的抑菌活性,抑菌率分别为84%和99%;溶血率小于2%,具有良好的血液相容性;对培养24、48和72 h的L929细胞显示出较低的细胞毒性;在NQC-OP-ADH水凝胶表面和内部的培养至第5天的L929细胞仍具有良好的生长状态,特别是表面培养过程中有较多细胞迁移到NQC-OP-ADH水凝胶内部,表明NQC-OP-ADH水凝胶具有良好的细胞相容性。成胶后的NQC-OP-ADH可顺利通过注射器,无堵塞现象发生,且具有良好的自愈能力。

产聚苹果酸出芽短梗霉的筛选、鉴定及发酵培养基优化

从自然土壤样本中筛选得到一株产聚苹果酸(PMLA)菌株,该菌株经形态学观察和分子进化分析鉴定为出芽短梗霉(Aureobasidium pullulans)。筛选菌株同时具有聚苹果酸、普鲁兰多糖和liamocin胞外聚合物的合成能力。通过单因素和响应面试验对该菌种产PMLA的发酵培养基组分进行优化,获得较优培养基组分为葡萄糖130 g/L、硝酸钠5 g/L、玉米浆1.22 g/L、氯化钾0.5 g/L、磷酸二氢钾0.1 g/L、七水硫酸镁0.2 g/L、碳酸钙30 g/L,采用该培养基获得聚苹果酸的最大产量为25.82 g/L,比优化前提高了70.72%。

胞外多糖Pullulan处理养猪场污水的研究

对 Pullulan处理养猪场污水进行了研究 .结果表明 :污水的稀释倍数与 Pullulan的添加量对处理效果有极显著的影响 ,当 Pullulan在 2倍稀释污水中的添加量达到 3mg/ kg时 ,污水的 BOD、COD、总 P、N的去除率达到 90 %以上 ;大肠杆菌的去除率达到 95 %以上 ,透过率也从 8%上升到 92 % .

复合生物保鲜剂对微冻大口黑鲈鱼保鲜效果的优化配比

【目的】为延长大口黑鲈鱼的货架期,以期为大口黑鲈鱼的贮藏保鲜提供理论参考依据。【方法】选取乳酸链球菌素(Nisin,NI)、普鲁兰多糖(Pullulan,LP)和山梨酸钾(Potassium sorbate,PS)进行单因素试验,利用响应面Box-Behnken试验设计对复合生物保鲜剂进行优化,筛选出了0.22%NI、1.61%LP、4.14%PS为最佳配比。使用最佳配比的复合保鲜剂对微冻贮藏期间大口黑鲈鱼肉的pH值、菌落总数(total viable count,TVC)、TVB-N(total volatile base nitrogen,TVB-N)值、肌动球蛋白含量、巯基含量、Ca^(2+)-ATPase活性、持水力(Water holding capacity,WHC)进行测定。【结果】复合生物保鲜剂组(NLP组)在贮藏期结束时(10 d),pH值仍小于7.0;TVB-N值未超过12 mg/100g;菌落总数未超过5.0 lg(CFU/g);鱼肉持水力、肌动球蛋白含量、巯基含量和Ca^(2+)-ATPase活性下降了7.08%、11.01%、22.07%和28.33%。对照组(CK组)在贮藏期结束时,pH值大于7.0;TVB-N值已达到12.55 mg/100g;菌落总数已超过5.0 lg(CFU/g);持水力、肌动球蛋白含量、巯基含量和Ca^(2+)-ATPase活性分别下降了10.75%、22.62%、34.78%和40.48%。【结论】复合生物保鲜剂能有效减缓大口黑鲈鱼肉微生物指标及各项理化指标的变化,对延缓鱼肉腐败变质、延长货架期具有较好的效果。

pH响应型磁性纳米复合材料的合成及其载药性能

采用改进的Stöber法制备了介孔硅包覆的磁性纳米材料,然后通过枝接的方法对材料表面进行了氨基化处理(NH_(2)-MMNPs)。利用安全无毒的氧化普鲁兰作为门控材料,通过纳米材料表面的氨基与氧化普鲁兰的醛基之间形成的对酸敏感的亚胺键构建门控开关,并将负载的盐酸阿霉素封装,形成了智能给药系统(oMMNPs/DOX)。通过透射电子显微镜(SEM)、傅里叶变换红外光谱仪(FT-IR)、比表面积测试仪、X射线衍射仪(XRD)和振动样品磁强仪等技术手段对复合材料的结构、性能和形貌进行了表征。结果显示,复合材料具有良好的形貌、单分散性以及超顺磁性。载药和释药实验表明,NH_(2)-MMNPs对阿霉素的载药率为51.65%;oMMNPs/DOX在药物释放过程中表现出pH响应性和药物缓释性。细胞毒性和荧光成像实验结果显示,复合材料具有良好的生物兼容性,能够成功地载药并被细胞摄取。

胞外多糖pullulan处理养猪场污水效果分析

用pullulan处理养猪场污水 ,结果表明 :污水的稀释倍数与pullulan的添加量对处理效果有极显著的影响。当Pullulan在 2倍稀释污水中的添加量达到 3mg/kg时 ,污水的生物需氧量 (BOD)、化学耗氧量 (COD)、总P、N的去除率达到 90 %以上 ;大肠杆菌的去除率达到 95 %以上 ,透过率也从 8%上升到 92 %。