二价阳离子短梗霉多糖发酵的影响

就二价阳离子对短梗霉多糖和黑色素的影响进行了分析和研究。结果表明,二价阳离子对短梗霉多糖的合成和黑色素的形成均有较大的影响。通过对培养基中二价阳离子含量和种类的控制不仅可以抑制细胞黑色素的形成,而且还保持了很高的多糖发酵水平,在３０Ｌ生物反应器中短梗霉多糖的产量和转化率分别达到了５９．８ｇ／Ｌ和６１．５％。     

几株出芽短梗霉在不同发酵条件下产生多糖的比较

将已有的４株出芽短梗霉在摇瓶中于不同发酵条件下进行比较,考察了它们的生长情况,不同的碳源、氮源、磷酸盐、初始ｐＨ和通气量等对短梗霉多糖合成的影响,获得一株产短梗霉多糖的高产菌株,为以后工作打下良好基础。     

短梗霉多糖研究进展

短梗霉多糖是一种微生物发酵产物,具有极好的成膜性,无色、无味,且不透气,易生物降解,对人体和环境无毒无害,受到国际上广泛关注,是一种极具研究潜力和经济价值的新型生物环保材料。该文综合了国内外众多学者的研究成果,从发酵底物(包括碳源、氮源、二价离子等)、发酵条件(包括pH、温度、通气量、接种量、种龄等)、多糖性质、应用研究(包括传统应用和最新应用成果)等方面进行阐述,为短梗霉多糖的进一步研究和应用提供参考。     

发酵条件对短梗霉多糖产量的影响

对短梗霉发酵培养基的初始pH,初始蔗糖浓度,酵母膏浓度,NH4^ 浓度,接种量和装液量等发酵条件对短梗霉多糖发酵影响进行了研究。结果表明,发酵条件对多糖发酵有显著的影响,当初始pH,初始蔗糖浓度,NH4^ 浓度,酵母膏浓度和装液量分别为6.5,5.0%,0.5g/L,0.2%和25ml时多糖的产量达到最大值;但接种量在2.0%-7.0%之间对多糖的产量影响不大,可见,通过对培养条件的调整,有助于短梗霉多糖的产量的提高。     

短梗霉黑色素的分离纯化及结构的初步分析

采用热碱提取、水煮酸沉法从短梗霉发酵液中提取得到黑色素粗品,再经DMSO萃取、酸性甲醇(pH=2)沉淀得到不含多糖和蛋白的短梗霉黑色素。此黑色素不溶于水及常规有机溶剂,可溶于碱性溶液和DMSO;离子交换色谱分析表明黑色素组分均一,出峰时间26±0.5 m in;紫外光谱谱图最大吸收峰为215 nm左右,未见蛋白(280 nm)与核酸(260 nm)的特征吸收峰;红外光谱谱图具有黑色素3μm和6μm的特征吸收峰,并含大量的羟基、氨基,与核磁共振和液质联机谱图结合分析推出短梗霉黑色素可能含有酚羟基、羧基和吲哚等官能团,主要结构骨架为5,6-二羟基吲哚-羧酸和多巴醌,推断该黑色素为酪氨酸酶控制合成的真黑素。     

短梗霉多糖超滤器的计算

超滤浓缩是短梗霉多糖分离纯化过程中的重要工序。本文研究了短梗霉多糖溶液的浓度每超滤速率的关系,推导出新的超滤器计算公式。根据预定的生产规模及工艺条件,可以对超滤器进行设计计算。实验结果表明用这种方法计算的结果是可靠的。     

响应面法优化普鲁兰多糖发酵培养基

以出芽短梗霉IFO 4464为实验菌种,采用响应面法(RSM)优化了出芽短梗霉IFO 4464产普鲁兰多糖的发酵培养基。通过实验得到出芽短梗霉最佳发酵培养基为蔗糖59.8g/L,硫酸铵0.7 g/L,硫酸镁0.3 g/L,磷酸二氢钾5.0g/L,氯化钾0.5g/L,氯化钠1.5g/L,酵母浸膏2.5 g/L,多糖产量可达21.92 g/L。     

短梗霉多糖在水果保鲜方面的应用研究

短梗霉多糖具有极佳的成膜性,其水溶液可以在水果表面形成一层透明有光泽的薄膜。该薄膜对氧气、氮气、二氧化碳、永分和有关香气有很好的阻隔作用,而且有较强的硬度及对温度变化的稳定性。因此,在水果表面挂上短梗霉多糖薄膜后,能有效地阻止水分、氧、氮、和二氧化碳等气体在果品内外的交换,从而抑制了水果的呼吸作用,减少了营养的储藏性损耗,进而达到了良好的保鲜目的。     

聚苹果酸生产菌出芽短梗霉CCTCC M2012223的全基因组测序及序列分析

【目的】解析出芽短梗霉CCTCC M2012223的基因组序列信息,分析其代谢产物聚苹果酸、黑色素、普鲁兰多糖合成相关基因,为深入研究遗传多样性和代谢工程改造提供序列背景信息。【方法】使用Illumina Hi Seq高通量测序平台对出芽短梗霉CCTCC M2012223菌株进行全基因组测序,并对测序数据进行序列拼接,基因预测与功能注释,COG/GO聚类分析,比较基因组学分析等。下载其他5株出芽短梗霉基因组序列,比较分析6株菌的种内同源基因、全基因组进化以及代谢产物合成相关基因。【结果】出芽短梗霉CCTCC M2012223基因组序列全长30756831 bp,GC含量47.49%,编码9452个基因。比较基因组分析表明出芽短梗霉CCTCC M2012223的基因组组装长度最长,6株菌的同源基因数达到7092个,普鲁兰多糖和聚苹果酸合成相关基因的蛋白序列有很高的保守性。出芽短梗霉CCTCC M2012223和Aureobasidium pullulans var.melanogenum亲缘关系最近,而这2株菌的黑色素合成相关基因的蛋白序列有一些插入和突变。【结论】本研究解析了出芽短梗霉CCTCC M2012223的基因组序列信息,获得黑色素、普鲁兰多糖和聚苹果酸合成相关基因,为后续的代谢机制解析和改造提供相关依据。     

短梗霉细胞内黑色素的分离提取

采用单因素试验和正交试验对短梗霉细胞内的黑色素提取条件进行了优化,结果表明:在pH为2～3、温度为70℃、NaOH浓度为1.5mol/L、发酵液与浸提液之比1:1(V)的条件下黑色素的提取量可达2.2g/L。对提取的黑色素进行了纯化,研究了其紫外和红外光谱学性质,紫外光谱图显示随波长的减少其吸收值增大,在215nm处有特征吸收峰;红外光谱图在3和6μm处有吸收峰,证实短梗霉黑色提取物是一种以芳香环为结构主体的异聚体黑色素。     

Discovering the role of the apolipoprotein gene and the genes in the putative pullulan biosynthesis pathway on the synthesis of pullulan,heavy oil and melanin in <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Pullulan produced by Aureobasidium pullulans presents various applications in food manufacturing and pharmaceutical industry. However, the pullulan biosynthesis mechanism remains unclear. This work proposed a pathway suggesting that heavy oil and melanin may correlate with pullulan production. The effects of overexpression or deletion of genes encoding apolipoprotein, UDPG-pyrophosphorylase, glucosyltransferase, and α-phosphoglucose mutase on the production of pullulan, heavy oil, and melanin were examined. Pullulan production increased by 16.93 and 8.52% with the overexpression of UDPG-pyrophosphorylase and apolipoprotein genes, respectively. Nevertheless, the overexpression or deletion of other genes exerted little effect on pullulan biosynthesis. Heavy oil production increased by 146.30, 64.81, and 33.33% with the overexpression of UDPG-pyrophosphorylase, α-phosphoglucose mutase, and apolipoprotein genes, respectively. Furthermore, the syntheses of pullulan, heavy oil, and melanin can compete with one another. This work may provide new guidance to improve the production of pullulan, heavy oil, and melanin through genetic approach.     

Effects of melanin on the accumulation of exopolysaccharides by Aureobasidium pullulans grown on nitrate

Aureobasidium pullulans produced pullulan and melanin when grown in medium containing low nitrate levels. With high nitrate concentrations, however, this fungus produced a mixture of exopolysaccharides (EPS) without melanin synthesis. At 0.78 g l(-1) N as nitrate, where no melanin synthesis occurred, maximum EPS yields reached 6.92 g l(-1) and then decreased to the final yield of 2.36 g l(-1). Following melanin addition (0.1 g l(-1)), yields reached 7.02 g l(-1) at 48 h and fell to a final yield of 5.21 g l(-1). The EPS produced in high nitrate medium contained both pullulan and (1-->3)-beta-glucan, but only pullulan was produced with melanin-supplementation. With melanin addition a doubling of (1-->3)-beta-glucanase activity was observed in high nitrate medium compared to that without supplementation. On the other hand amylolytic activities disappeared in medium with melanin production or addition. Culture filtrates sustained a higher reducing capacity (RC) when melanin was present. Low RC appeared to reduce (1-->3)-beta-glucanase activity and increase amylolytic activities. Thus, higher RC appears to inhibit production/activity of amylose-degrading enzymes capable of degrading pullulan, and stimulates (1-->3)-beta-glucanase synthesis/activity, leading to a preferential accumulation of pullulan.     

Production of the fungal exopolysaccharide pullulan by batch-wise and continuous fermentation

A mutant strain of the deuteromycete Aureobasidium pullulans deficient in melanin synthesis was used to investigate the production of the exopolysaccharide pullulan and biomass, respectively. Shake-flask experiments with different carbon sources showed significant differences in pullulan elaboration. Sucrose was most suitable for pullulan synthesis among the carbon sources examined. Fermentations were carried out both batch-wise and continuously in a stirred vessel fermentator. In batch fermentations about 45% of the glucose offered was converted into pullulan at maximum formation rates of 0.16 g/l per hour using standard medium. The yield of polysaccharide could be maintained at 45% in continuous fermentations. At a dilution rate of 0.05 l/h, the formation rate of polysaccharide increased up to 0.35 g/l per hour. Alterations in the nitrogen content of the feed significantly affected the consumption rate of glucose and the production rate of polysaccharide, but final concentrations of biomass were hardly affected. Correspondence to: R. Schuster     

出芽短梗霉发酵产生普罗蓝糖的研究

对一株野生型的出芽短梗霉(Aureobasidium pullulans)Ft1和从Ft1出发经原生质体再生筛选出的菌株R45进行了摇瓶发酵产普罗蓝糖的比较研究,结果表明R45无论从形态,菌体生长情况,还是从普罗蓝糖的产量,黑色素的产生等方面都与亲株Ft1有明显的区别,说明R45是一株具有一定生产价值的变异菌株.     

Pullulan-hyperproducing color variant strain of Aureobasidium pullulans FB-1 newly isolated from phylloplane of Ficus sp

The studies were carried out for the isolation of efficient pullulan producing strains of Aureobasidium pullulans. Five strains were isolated from phylloplane of different plants. Amongst these, three were producing black pigment melanin, while the remaining two produced pink pigment. These two color variant isolates of A. pullulans were designated as FB-1 and FG-1, and obtained from phylloplane of Ficus benjamina and Ficus glometa, respectively. The parameters employed for the identification of the isolates included morphology, nutritional assimilation patterns and exopolysaccharide (EPS) production. Isolates were compared with standard cultures for EPS production. A. pullulans FB-1 was the best producer of pullulan giving up to 1.9, 1.4 and 1.7 times more pullulan than the control of A. pullulans NCIM 976, NCIM 1048 and NCIM 1049, respectively. The IR spectra of the isolates and standard strains revealed that the polysaccharide was pullulan, but not aubasidan. The study also supported the fact that A. pullulans is a ubiquitous organism and phylloplane being the important niche of the organism.     

Oxygen requirement in pullulan fermentation

Summary Oxygen was essential for the biosynthesis of pullulan by Aureobasidium pullulans. In a growth medium, pullulan yield and synthesis rate were proportional to the oxygen availability. However, under controlled oxygen environment in a non-growth medium, the synthesis rate and the yield of pullulan were inversely proportional to the oxygen tension. A relationship between melanin production and oxygen transfer conditions was also observed. The elapsed time prior to the appearance of the pigment was dependent upon the degree of oxygen availability.     

Optimization of physico‐chemical and nutritional parameters for a novel pullulan‐producing fungus,Eurotium chevalieri

Aims: To isolate the novel nonmelanin pullulan‐producing fungi from soil and to optimize the physico‐chemical and nutritional parameters for pullulan production. Methods and Results: A selective enrichment method was followed for the isolation, along with development of a suitable medium for pullulan production, using shake flask experiments. Pullulan content was confirmed using pure pullulan and pullulanase hydrolysate. Eurotium chevalieri was able to produce maximum pullulan (38 ± 1·0 g l^?1) at 35°C, pH 5·5, 2·5% sucrose, 0·3% ammonium sulfate and 0·2% yeast extract in a shake flash culture medium with an agitation rate of 30 rev min^?1 for 65 h. Conclusions: The novel pullulan‐producing fungus was identified as E. chevalieri (MTCC no. 9614), which was able to produce nonmelanin pullulan at from poorer carbon and nitrogen sources than Aureobasidium pullulans and may therefore be useful for the commercial production of pullulan. Significance and Impact of the Study: Eurotium chevalieri could produce pullulan in similar amounts to A. pullulans. Therefore, in future, this fungus could also be used for commercial pullulan production, because it is neither polymorphic nor melanin producing, hence its handling during pullulan fermentation will be easier and more economical.     

Production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82 with pH control and DL-dithiothreitol addition

Xylose, the second most abundant sugar in lignocellulosic materials, is not efficiently utilized in current lignocellulose biotransformation processes, such as cellulosic ethanol production. The bioconversion of xylose to value-added products, such as pullulan, is an alternative strategy for efficient lignocellulose biotransformation. This paper reports the production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82. The effects of DL-dithiothreitol (DTT) and pH on pullulan production from xylose were also intensively investigated. A maximal increase of 17.55% of pullulan production was observed in flasks added with 1.0 mM DTT. Batch fermentations with controlled pH were also conducted, and the optimal pH for cell growth and pullulan synthesis was 3.0 and 5.0, respectively. Based on these findings, two-stage pH control fermentations were performed, in which the pH of the medium was first adjusted to 3.0 for cell growth, and then changed to 5.0 for pullulan synthesis. However, the earlier the pH was changed to 5.0, the more pullulan was produced. Fermentation with controlled pH of 5.0 acquired the highest pullulan production. Under the optimized conditions (with the addition of 1.0 mM DTT and controlled pH of 5.0), the maximal pullulan production obtained from xylose was 17.63 g/L. A. pullulans AY82 also readily fermented hemicellulose hydrolysate under these optimized conditions, but with lower pullulan production (12.65 g/L). Fourier transform infrared spectroscopy and high-performance liquid chromatography showed that the structure of the pullulan obtained in this study was identical to that of the pullulan standard.     

Metal ion-induced activation of molecular oxygen in pigmented polymers

Diamagnetic and paramagnetic metal ions enhanced the rate of production of hydrogen peroxide during autoxidation of melanin pigments, as measured using an oxidase electrode. However, redox-active metal ions, such as Fe3+ and Cu2+, caused a marked decrease in H2O2 production. Evidence for redox-active metal ion-dependent formation of hydroxyl radicals during autoxidation of melanin pigments has been obtained using the electron spin resonance-spin trapping method. Evidence for direct reduction of Fe3+ by melanin polymers also has been obtained using optical spectroscopy. Mechanisms of molecular activation of oxygen induced by metal ions on melanin polymers are discussed.