“生命营养液”中红曲菌种的分离及初步鉴定

采用传统稀释涂布法从新疆特色功能饮品"生命营养液"中分离到一种红曲菌。对该菌种进行初步分类研究,通过形态学分类方法和核酸序列系统发育分析方法将该菌种鉴定为丛毛红曲(Monascus pilosus),并将其保藏到中国工业微生物菌种中心(保藏编号为CICC5047),为进一步开发利用该菌种奠定基础。     

四种红曲菌各自固有的分子生物学和生物化学特性

对白色红曲菌、火红色红曲菌、烟灰色红曲菌和橙色红曲菌各自固有的核糖体内转录间隔区(ITS)rDNA序列和生物化学特性进行了研究。rDNA ITS测序结果显示,橙色红曲菌与火红色红曲菌相同,而白色红曲菌与灰色红曲菌相同,这两组经序列比对仅有2个碱基不同。从生物化学特性来看,作为分类指标,似乎有物种属(genus)的共性也有个体(株)质的差异或量的差异。     

红曲菌属的种之进一步研究

重温红曲菌属各家分类意见,对来自ATCC,CBS,CGMCC,IFO,IMI和NRRL的红曲菌属各个种模式菌株和可靠菌株再次进行了观察。描述了4个新种,它们是发白红曲菌,火红色红曲菌,烟灰色红曲菌和橙色红曲菌。提供了区别12种红曲菌的检索表。     

根癌农杆菌介导的定点敲除技术在红色红曲菌中的应用

红曲菌(Monascus spp.)是一种重要的丝状真菌, 广泛应用于食品和医药领域中。目前, 由于对红曲菌遗传背景了解的很少, 因而对其重要功能基因的研究报道很少。本研究采用根癌农杆菌介导的转化技术对红色红曲菌(Monascus ruber)中推断的G-蛋白信号调节子mrfA基因的RGS功能域进行定点缺失研究, 探讨基于同源重组为基础的基因定点缺失技术在红曲菌基因功能鉴定中的可行性。构建的敲除载体pC805S左右同源臂长度分别为958 bp和824 bp, 将其转入受体菌M. ruber 中, 得到的138株转化子中, 有26株转化子发生了同源重组, 重组率达到18.8%。实验结果表明该技术作为红曲菌基因功能鉴定的方法是可行的。     

红酵母属同工酶酶谱分析及其分类研究

分析了24株红酵母菌的苹果酸脱氢酶、超氧化物歧化酶和酯酶的电泳图谱,并将同工酶酶谱资料进行了聚类分析。结果发现：参试红酵母属内各种间3种同工酶酶谱差异明显,24株红酵母菌被明显分为A、B、C、D、E5群,每一群分别代表一个种（B群除外）,其中A群的18个菌株为Kreger-vanRij（1984）系统中的深红酵母（Rhodotorularubra）,又可分为2个亚群,第AI亚群相当于Lodder等人（1970）分类鉴定系统中的果蝇红酵母（Rhodotorulapilimanae),第AII亚群相当于Lodder系统中的深红酵母。本实验结果还表明,同工酶电泳方法简单易行,能精确地反映菌株之间的微小差异,适用于红酵母属的种级分类,也适用于种下分群,可以作为传统酵母菌分类的一个重要辅助手段,克服单纯依据形态特征和生理生化指标进行分类所存在的弊端。     

红曲色素的两种新结构

从中国科学院微生物研究所保藏红曲菌中,筛选得到一株高产红色素的红曲菌菌株（ＡＳ．３．４６１７）。经鉴定属于红色红曲菌。通过有机溶剂的萃取和两次硅胶柱层析,从该菌株中分离得到两种色素样品,高压液相色谱测定为纯色素样品。通过元素组成分析,核磁共振谱,快离子轰击,质谱和高分辨质谱分析确定,这两种色素与已知的六种红曲菌色素不同,为新发现的红曲菌色素,它们可能的分子式为：Ｃ２５Ｈ３１Ｏ５Ｎ和Ｃ２３Ｈ２７Ｏ５     

种间双亲灭活原生质体融合选育高产Monacolin K红曲菌

为获得高产MonacolinK的红曲菌菌株,将经农杆菌介导转化获得的携带潮霉素抗性基因且以甘油为原料液态发酵高产MonacolinK的发白红曲菌H2和以大米为原料固态发酵产Monaco-1inK的烟色红曲菌9908作为亲本,对其原生质体分别进行热灭活及紫外灭活,然后对灭活双亲用PEG作融合剂进行原生质体融合。从融合子中选出有潮霉素抗性的突变株,通过发酵与亲本对比,筛选得到一株以大米为原料固态发酵高产MonacolinK的融合株F12．11,其MonacolinK产量达到8．73mg／g;较发白红曲菌H2与烟色红曲菌9908分别提高了100．23％和48．98％;一株以甘油为原料液态发酵高产MonacolinK的融合株F13-2,其MonacolinK的产量达到1752．46mg／L,较发白红曲菌H2与烟色红曲菌9908分别提高了32．98％和1979．33％。     

福建地区94例女性阴道念珠菌感染分离株分型研究

目的 对福建地区临床阴道分泌物分离出的（94株）念珠菌菌株进行分类鉴定,并通过分子生物学及API试验分析传统科玛嘉分类方法的准确性.方法 ①通过科玛嘉显色培养基鉴定菌种,并观察菌株显微镜下形态学表现.②通过ITS区段分子生物学序列分析进行菌种鉴定.③将科玛嘉试验与ITS区段序列分析结果与化验室检验报告结果对比,将鉴定差别菌株进一步行API试验及LSU区段序列分析.结果 ①94株念珠菌经鉴定结果为白念珠菌78株、光滑念珠菌10株、近平滑念珠菌3株、热带念珠菌1株、酿酒假丝酵母菌1株及其中1株为光滑念珠菌及近平滑念珠菌混合感染.②科玛嘉试验可良好的鉴定念珠菌,但对于少见菌种（如酿酒假丝酵母菌）仍缺乏特异性.③一般化验室通过简单菌落形态学及简易科玛嘉检测鉴定仍存在一定误差率（10/94）.④分子生物学方法鉴定菌种准确性高,且可从基因序列分析中鉴定包含C.parapsilosis sensu strico,Candida metapsilosis以及Candida orthopsilosis的近平滑念珠菌复合体菌种.结论 福建地区女性外阴感染的菌种仍以白念珠菌为主,但非白念珠菌的感染也占据相当的比例（16/94）,而在检测方法上,分子生物学技术较科玛嘉试验更能准确的鉴定念珠菌菌种.     

红曲菌基因组DNA提取及RAPD反应体系优化

为了建立一套适合红曲属真菌RAPD反应的优化体系,用改进的CTAB法提取红曲菌基因组DNA,采用单因素试验探讨RAPD反应体系中模板DNA、随机引物、Taq酶、Mg^2＋、dNTPs对扩增结果的影响。结果在20μL体积中,模板DNA20 ng、随机引物0、2μmol/L、Taq酶、Mg^2＋1.5mmol/L,dNTPs 1mmol/L的反应体系可得到稳定清晰的RAPD扩增图谱,为采用RAPD技术进行红曲菌种质资源遗传多样性研究奠定基础。     

297例浅部真菌病临床及病原菌菌种分析

目的 探讨皮肤浅部真菌病致病真菌菌种的构成.方法 对297例真菌涂片阳性和培养阳性的浅部真菌病患者,取标本进行分离培养及菌种鉴定,培养阳性标本在形态学上不能准确鉴定的,进行梅里埃API 20C AUX酵母菌鉴定试剂盒或核糖体DNA （rDNA） ITS区序列测定,确切鉴定菌种.使用SPSS 17.0统计软件对于结果进行统计分析.结果 共分离培养出致病菌13种,其中红色毛癣菌86株（29.0％）,须癣毛癣菌68株（22.9％）,念珠菌属59株（19.9％）,暗色真菌属13株（4.4％）,曲霉菌属13株（4.4％）,红酵母菌12株（4.0％）,青霉菌属9株（3.0％）,毛霉菌9株（3.0％）,犬小孢子菌5株（1.7％）,浅白隐球菌3株（1.0％）,毛孢子菌属2株（0.7％）,絮状表皮癣菌1株（0.3％）,混合感染17株（5.7％）.结论 本地区浅部真菌病以甲癣为主,主要致病真菌是红色毛癣菌,但其他种类真菌感染尤其是念珠菌属有明显上升趋势.     

微藻细胞油脂含量的快速检测方法

以斜生栅藻(Scenedesmus obliquus JNU20)为实验材料,采用尼罗红(NR)荧光光谱法和傅里叶变换红外光谱法(FTIR)测定该藻细胞中的油脂含量。研究结果表明NR的最佳染色条件为:染色前微波处理40 s,二甲基亚砜体积分数1%, NR最终质量浓度1.5 μg/ml,染色时间5 min,染色温度40℃。比较了NR荧光光谱法、FTIR与传统重量法测定的该藻在不同时相的油脂积累情况,利用激光共聚焦显微镜观察了细胞中油体形成的动态过程。实验结果表明NR荧光光谱法、FTIR与传统重量法测定的结果显著相关(R²=0.9258, R²=0.9844),但NR荧光光谱法和FTIR更简便快速,研究结果为规模化筛选高含油量藻株及跟踪产油微藻油脂积累过程奠定了基础。     

Biochemical tests as an aid to the identification of Monascus species

The enzymic activity of nine strains of Monascus (Fungi, Ascomycotina), representing all four accepted species as defined on cultural and microscopical features, were compared by means of API ZYM enzyme testing strips and other tests developed for use in Penicillium. Consistent results were obtained between strains of the same species, confirming their taxonomy. Eight tests showed differential activity between the species and will therefore be of value as an aid to the identification of Monascus species. Strains of M. purpureus , used in the production of red-rice, had a strong polypectase activity at pH 6 which was not evidenced in the other three species even after six weeks incubation; those of M. ruber , which often occurs on cellulosic substrates in nature, were the only ones to exhibit cellulase activity.     

Simultaneous enrichment of deglycosylated ginsenosides and monacolin K in red ginseng by fermentation with Monascus pilosus

To improve its bioavailability and pharmacological effects in humans, red ginseng was fermented with a newly isolated fungus, Monascus pilosus KMU103. Most of the ginsenosides were converted to deglycosylated ginsenocides, such as Rh(1), Rh(2), and Rg(3). The total amount of ginsenosides Rh(1), Rh(2), and Rg(3) was 838.7 mg/kg in the red ginseng, and increased to 4,117 mg/kg after 50 L fermentation in 13% red ginseng and 2% glucose. In addition, the Monascus-fermented red ginseng contained 3,089 mg/kg of monacolin K, one of the metabolites produced by Monascus known to reduce cholesterol in the blood. This newly developed Monascus-fermented red ginseng should result in improved health effects, not only by biotransforming gisenosides to deglycosylated ones but also by creating additional bioactive compounds.     

A combined solid-state and submerged cultivation integrated with adsorptive product extraction for production of Monascus red pigments

A process taking advantages of combined solid-state and submerged cultivation of Monascus for red pigment production and integration of a product removal unit was developed. The solid-state cultivation was carried out in a 5 l fermentor, with rice being used not only as the substrate but also the support for Monascus. The inclusion of rice submergence and integration of product separation were achieved by intermittently rinsing the rice with monosodium glutamate (MSG) solutions every 12 h followed by an adsorptive extraction of the red pigment dissolved in the rinsing solution. With this new process, the Monascus red pigment production was increased by 24% as compared with that by the plain fixed-bed cultivation.     

Production of statins by filamentous fungi

Several Monascus and Aspergillus strains were screened for statins production. Lovastatin, monacolin J, pravastatin and mevastatin were produced, with higher yields from the A. terreus strains than from Monascus species. Of all the strains investigated M. paxii AM12M, an isolated spontaneous mutant, yielded 127 mg lovastatin/l and 53 mg pravastatin/l at 21 days, and 18 mg pravastatin/l at 16 days employing a whole soybean flour medium; A. terreus BST yielded 230 mg lovastatin/l and 118 mg pravastatin/l at 14 days employing a defatted soybean flour medium. Statins recovery showed that pravastatin was, in both strains, mostly found in both the mycelium and the culture filtrate, while lovastatin remained closely associated (83%) to the A. terreus mycelium or was mainly released into the culture filtrate (64%) of M. paxii culture.     

Hypolipidemic and anti-atherogenic effects of long-term Cholestin (Monascus purpureus-fermented rice,red yeast rice) in cholesterol fed rabbits

Long-term effects of Cholestin (Monascus purpureus rice; red yeast rice) on serum lipids and severity of atherosclerosis were examined in rabbits fed for 200 days on a semi-purified diet containing 0.25% cholesterol. Serum total cholesterol was 25 and 40% lower, respectively, in rabbits fed 0.4 or 1.35 g/kg/day of Cholestin (Monascus purpureus rice; red yeast rice) compared to controls. This treatment also lowered serum LDL cholesterol. This 200-day treatment significantly reduced serum triglycerides and atherosclerotic index (ratio of non-HDL-cholesterol to HDL-cholesterol). Although similar reductions of total, LDL-cholesterol and triglycerides were observed, a parallel group of rabbits fed lovastatin (0.0024 g/kg/day) failed to reduce the index significantly. Apolipoprotein A(1) was increased and apolipoprotein B was reduced in all treatment groups. Severity of atherosclerosis was reduced significantly in all treatment groups. The sudanophilic area of involvement was 80.6% in controls, and reduced significantly; to 30.1% on the low dose of Cholestin (Monascus purpureus rice; red yeast rice), and 17.2% on the high dose. Lovastatin reduced severity of lesions by 89% (sudanophilia) and 84% (visual). Visual grading of lesion severity showed reduction by 38% and 68%.     

一株高产Monacolin K紫红曲霉菌株筛选、鉴定及发酵条件优化

为从天然发酵红曲米中分离的30株红曲霉菌株中筛选高产MonacolinK的菌株,并对其产MonacolinK的发酵条件进行优化。实验采用高效液相色谱法(HPLC)筛选到9株具有产MonacolinK能力的红曲霉菌株,其中以编号ZX26的菌株产MonacolinK能力最高,发酵液中Monacolin K产量达到107.6mg/L,并且产MonacolinK能力具有良好的稳定性。微生物形态学结合ITS基因同源性分析结果表明,编号ZX26菌株为紫红曲霉。进一步采用单因素试验和正交试验法优化紫红曲霉ZX26产MonacolinK的发酵条件,结果表明在培养基组分为葡萄糖70g/L,牛肉膏15g/L,NaNO32g/L,MgSO4·7H2O0.5g/L,KH2PO41.5g/L时,其最优发酵条件为:发酵初始pH4.0,接种量为7%,培养温度30℃,发酵10天,在此条件下,紫红曲霉ZX26发酵液中MonacolinK产量达到271.36mg/L,相对于培养条件优化前MonacolinK产量提高152.19%,经验证此培养条件下MonacolinK产量最佳。     

A new process for red pigment production by submerged culture of Monascus purpureus

Formation of red pigment by Monascus purpureus via diauxic growth on glucose and ethanol in submerged culture was optimized based on inoculum preparation and culture medium. A vegetative inoculum was prepared from spores grown on ethanol. The optimized culture medium was low in phosphates, and had an initial pH?of 5.5. The characteristics of Monascus purpureus grown on glucose and on ethanol were compared: the specific consumption rate of glucose (q_G) was higher than the specific consumption rate of ethanol (q_E), whereas the specific growth rate was greatest with ethanol. The specific production rate of red pigment (p_OD) and pigment yield (Y_OD/s) with glucose was twice that with ethanol. A novel fermentation process was developed with M. purpureus initially grown with controlled ethanol formation, and consumption of the latter during pigment formation.     

Spectral monitoring of photoirradiated skin lipids: MS and IR approaches

An investigation of the effects of UV(A) irradiation on the stratum corneum lipids was carried out in vitro on films. The modifications of their conformational order were studied by FTIR and the formation of new entities was detected by mass spectroscopy. The results show not only differences in behaviour of the three lipid classes (fatty acids (FA), ceramides (CER), and cholesterol), but also variation within a class, depending on the molecules structure. Upon UV(A) irradiation, beta scission occurs on all the components, saturated and unsaturated. Moreover, unsaturated FA or CER having a double bond on their FA moiety may become saturated or may be transformed into their free radical form. Unsaturated FA are more sensitive to UV(A) and lead more easily to oxygenated components than unsaturated CER. The chemical effects are irradiation dose dependent but do not deeply influence the supramolecular organisation of these lipids. The global conformation of the lipids stays in an orthorhombic state, a decrease of the packing density however is observed.