酵母菌辅酶Q提取方法的改进及在假丝酵母属分类学研究中的应用*

本文根据普通实验室的条件,对酵母菌辅酶Q（CoQ）的提取方法进行了改进,既有效地节约了试剂,又避免了一些非常规仪器的使用,使得CoQ分子类型的测定简便易行。用改进的方法,测定了37株假丝酵母属（CandidaBerkhout）菌株的CoQ类型,并据此解决了一些根据常规的形态和生理生化性状难以作出精确鉴定的疑难菌株的分类学问题。     

微生物CoQ合成途径及CoQ10生产菌株的分子生物学改造

CoQ10具有呼吸链电子传递者、抗氧化性、调控基因表达等多种生理生化功能,目前不仅用作药物也用作食品添加剂。微生物发酵法是目前生产CoQ10最有效的方法。本文就有关微生物CoQ合成途径及基于CoQ合成途径的CoQ10生产菌株分子生物学改造的策略与研究进展进行了综述和展望。     

辅酶Q10的生物合成及其高表达研究进展

辅酶Q10(CoQ10)不仅是呼吸链上的电子传递体,同时也具有抗氧化功能。目前全球市场上的CoQ10正处于一种供不应求的状态。我们简要论述了CoQ10的结构、性质、功能及其生物合成过程,同时概括总结了现阶段为提高CoQ10产量而采用的新型技术手段。     

大鼠心肌细胞线粒体辅酶Q含量的动态分析

用高效液相色谱法(HPLC)测定了大鼠心肌组织和心肌线粒体CoQ同系物的含量,并动态地分析了心肌线粒体摄取外源性CoQ₁₀后的代谢变化。结果表明,线粒体中CoQ同系物总量约占心肌全匀浆的19.49％;外源性的CoQ₁₀能够为线粒体所摄取。     

辅酶Q10补充对青少年运动员肝线粒体功能和有氧运动能力的影响

目的：观察CoQ10补充对青少年运动员肝线粒体功能和有氧运动能力的影响。方法：18名进行耐力训练的男性青少年游泳运动员单盲随机分为Q组及P组,分别补充CoQ10 100mg／d或安慰剂28d。结果：①补充后Q组血浆CoQ10浓度显著增高,且显著高于P组;②补充后Q组安静状态的血浆MDA水平无显著改变,且显著低于P组;③首次恒定负荷运动后总体血浆CoQ10浓度较安静状态显著降低;④总体血浆CoQ10基础浓度与首次递增负荷运动中测定的VO2max显著正相关;⑤补充前后在1h耐力运动中动脉血酮体比的改变程度Q组与P组无组间差异;⑥Q组与P组VO2 max、个体乳酸阈和运动节省化的改变程度无组间差异。结论：尽管急性耐力运动中机体对CoQ10的需求增加,CoQ10补充也可降低血浆脂质过氧化水平,外源性CoQ10不能改善青少年运动员的肝线粒体功能和有氧运动能力。     

对沼泽红假单胞菌生产CoQ10添加前体物的优化

CoQ10因其具有多种生理生化功能而不仅用于药物也用作食品添加剂.本研究采用全因析中心复合设计及响应面法对茄尼醇、对羟基苯甲酸及甲硫氨酸三种CoQ10前体物的添加量进行了优化,以达到沼泽红假单胞菌J001最大量地生产CoQ10的目的.结果表明:经过对所得模型进行响应曲面法分析,当茄尼醇、对羟基苯甲酸及甲硫氨酸最佳添加量分别为124.8mg1-1,267.7mg1-1,130.2mg1-1时,得最大CoQ10产量预测值40.6[(mgCoQ10)(g干细胞)-1].对上述最佳组合进行试验验证得40.5±0.2[(mg CoQ10)(g干细胞)-1],很接近预测值,比对照(未加三种CoQ10前体物)CoQ10产量提高了109.8%.     

几种营养物质对烟草细胞生长和CoQ_(10)含量的影响

研究了蔗糖、KH2PO4、NH 4/NO3比对烟草细胞生长和CoQ10含量的影响,结果表明,当蔗糖浓度为30g/L时,CoQ10总量最高,此时细胞产量、CoQ10含量和总量分别为198g/L、4147μg/g(dwt)、8212μg/L。在上述蔗糖浓度下,当KH2PO4起始浓度为340mg/L、NH 4/NO-3为12时,细胞产量、CoQ10含量和总量分别最高,高于此比例时有利于CoQ10形成,但不利于细胞生长。     

代谢工程在微生物法产辅酶Q_(10)中的应用

辅酶Q10作为细胞呼吸链上的重要组成部分在电子传递过程中发挥着重要的作用。辅酶Q10的抗氧化、抗衰老功能使其广泛应用于医药、食品和化妆品等行业。CoQ10市场需求不断增加,这使得大规模提高CoQ10工业化生产的产量显得十分必要。目前,主要依靠从自然界中筛选到的各种微生物作为生产菌种发酵生产CoQ10,但这些原始生产菌种由于产量低、营养要求高等各种原因很难实现大规模发酵生产。随着对CoQ10生物合成途径以及代谢调控机制的了解清楚,通过对易于商业化生产的优良宿主细胞(如大肠杆菌)进行代谢工程的改造,有助于促进代谢工程菌的CoQ10工业化生产发展。     

离子束诱变粟酒裂殖酵母产辅酶Q_(10)的初步研究

辅酶Q10(coenzyme Q10,CoQ)对心脏充血性病人有较好的疗效,是临床常用药物之一。实验研究了离子束诱变粟酒裂殖酵母对提高CoQ10的产量的影响与作用。实验筛选出六株突变菌株,研究了突变株生理生化特性。结果表明:突变菌株的CoQ10产量都有不同程度的提高,其中编号为N1菌株产量达6.9344mg/L,是对照菌株的10倍多,最低的N2菌株的产量也是对照菌株的1.3倍。     

离子束诱变粟酒裂殖酵母产辅酶Q10的初步研究

辅酶Q10(coenzyme Q10,CoQ)对心脏充血性病人有较好的疗效,是临床常用药物之一.实验研究了离子束诱变粟酒裂殖酵母对提高CoQ10的产量的影响与作用.实验筛选出六株突变菌株,研究了突变株生理生化特性.结果表明:突变菌株的CoQ10产量都有不同程度的提高,其中编号为N1菌株产量达6.9344 mg/L,是对照菌株的10倍多,最低的N2菌株的产量也是对照菌株的1.3倍.     

Antimicrobial activity of fatty acids from fruits of Peucedanum cervaria and P. alsaticum

Plants of the genus Peucedanum have been used in traditional medicine for a long time to treat different diseases including infectious diseases. The hexane fruits extracts of Peucedanum cervaria and P. alsaticum were examined for antimicrobial activity and analyzed for their fatty acid content. Fatty acid composition of oils were analyzed by GC/FID in methyl ester form. Minimal inhibitory concentrations (MICs) of fatty acid fractions against twelve reference bacterial and yeast strains were performed by the twofold serial microdilution broth method. Fourteen fatty acids were identified. Oleic and linoleic acids were found to be dominant. The extracts from both plants examined exhibited inhibitory effects against Gram‐positive strains tested with different MIC values (0.25–2 mg/ml); however, extract from P. alsaticum possessed stronger antibacterial properties and a broader spectrum. The growth of Gram‐negative bacteria and Candida spp. strains was not inhibited even at the highest extract concentration used (MIC>4 mg/ml). Standard fatty acids exhibited inhibitory effects towards all bacterial and yeast strains used in this study; however, the majority of bacteria were more sensitive to linoleic than to oleic acid. These results revealed, for the first time, that hexane extracts obtained from fruits of P. alsaticum and P. cervaria possess moderate in vitro antibacterial activity against Gram‐positive bacteria including staphylococci. Linoleic and oleic acids appear to be the compounds responsible for this effect, and a synergistic antimicrobial effect between these two fatty acids was indicated.     

Comparative study on the identification of food-borne yeasts

Morphologically distinct yeast colonies from partially and fully processed fruits and vegetables were isolated over a 3-year period. Identification of 239 strains was achieved by using standard methods, commercial identification kits (API 20C and API YEAST-IDENT), and a simplified system for food-borne yeasts. The identified strains of fruit origin represented 36 species belonging to 19 genera. Among strains of vegetable origin, 34 species representing 17 genera were identified. The simplified identification system and the conventional method provided the same results in 80% of the cases. The commercial identification kits were easy to use but were not appropriate for food-borne yeast species. Computer-assisted identification was helpful.     

Comparative study on the identification of food-borne yeasts.

Morphologically distinct yeast colonies from partially and fully processed fruits and vegetables were isolated over a 3-year period. Identification of 239 strains was achieved by using standard methods, commercial identification kits (API 20C and API YEAST-IDENT), and a simplified system for food-borne yeasts. The identified strains of fruit origin represented 36 species belonging to 19 genera. Among strains of vegetable origin, 34 species representing 17 genera were identified. The simplified identification system and the conventional method provided the same results in 80% of the cases. The commercial identification kits were easy to use but were not appropriate for food-borne yeast species. Computer-assisted identification was helpful.     

The use of yeast cultures for the detection of environmental mutagens using a fluctuation test.

A microbial fluctuation test, modified for the detection of environmental mutagens has been evaluated using a number of strains of the yeast Saccharomyces cerevisiae. Auxotrophic diploid cultures of yeast which produce prototrophic colonies by both mitotic gene conversion and mutation have been extensively utilized for the detection and evaluation of chemicals showing genetic activity. A number of the yeast strains utilized were shown to be suitable for use in the fluctuation test although the time scales of the experiments were considerably extended (up to 16 days) compared to those involving bacteria. The yeast strains respond to doses of mutagens at least a 100-fold lower than that required in a conventional short exposure treat and plate experiment. In experiments involving the induction of mitotic gene conversion at the tryptophan-5 and histidine-4 loci in the fluctuation test significant increases in prototrophic cells were produced in the presence of the insecticide Lindex (0.05 microng/ml), the preservative Thiomersal (0.0001 microng/ml), a mahogany hair dye (0.01 microng/ml), the herbicide Paraquat (0.02 microng/ml) and the alkylating agent ethyl methane sulphonate (0.1 microng/ml). The results demonstrate that the fluctuation test provides an extremely sensitive assay for the detection of chemicals which show genetic activity in yeast at non-toxic concentrations.     

Antifungal Activities of Different Extracts of Marine Macroalgae Against Dermatophytes and Candida Species

Algae are bioactive natural resources, and due to the medical importance of superficial mycoses, we focused the action of macroalgae extracts against dermatophytes and Candida species. Seaweed obtained from the Riacho Doce beach, Alagoas (Brazil), were screened for the antifungal activity, through crude extracts using dichloromethane, chloroform, methanol, ethanol, water and chloroform and hexane fractions of green, brown and red algae in assays with standard strains of the dermatophytes Trichophyton rubrum, T. tonsurans, T. mentagrophytes, Microsporum canis, M. gypseum and yeasts Candida albicans, C. krusei, C. guilliermondi and C. parapsilosis. The M44-A and M27-A2/M38A manuals by CLSI were followed, and the minimum inhibitory concentration (MIC) ranged from 0.03 to 16.00 μg ml(-1), and an inhibition halo of 10.00-25.00 mm was observed for dermatophytes, while for yeast, it was from 8.00 to 16.00 μg ml(-1) and 10.00-15.00 mm. M. canis showed MIC of 0.03 μg ml(-1) and the largest inhibition halo in T. rubrum (25.00 mm) through the use of the methanol extract. For C. albicans, dichloromethane, methanol and ethanol extracts formed the largest inhibition halo. The ethanol extract was shown to be the best inhibiting fungi growth, and chloroform and hexane fractions of H. musciformis inhibited the growth of all dermatophytes and C. albicans, yielding the conclusion that apolar extracts obtained from algae presented the best activity against important pathogenic fungi.     

Ochratoxin A adsorption phenotype: An inheritable yeast trait

This study aimed to evaluate the inheritance of the trait ochratoxin A adsorption in two wine strains of Saccharomyces cerevisiae and their 46 descendants. Each strain was inoculated in triplicate in test tubes containing 10 ml of must obtained from the Calabrian Zibibbo white grape variety, artificially contaminated with ochratoxin A to reach a total content of 4.10 ng/ml. The microvinification trials were performed at 25°C. After 30 days, ochratoxin A values ranged from 0.74 to 3.18 ng/ml, from 0.01 to 2.69 ng/ml, and from 0.60 to 2.95 ng/ml respectively in wines, in lees after washing, and in the saline solution used to wash the lees. The analysis of OTA in wines was performed to find the residual toxin content after yeast activity, thus obtaining technological evidence of yeast influence on wine detoxification. The analysis of OTA in lees after washing was performed to distinguish the OTA linked to cells. The analysis of OTA in the saline solution used to wash the lees was performed to distinguish the OTA adsorbed on yeast cell walls and removed by washing, thus focusing on the adsorption activity of wine yeast through electrostatic and ionic interactions between parietal mannoproteins and OTA. Ploidy of the two parental strains was controlled by flow cytometry. Results demonstrated that the ochratoxin A adsorption is genetically controlled and is a polygenic inheritable trait of wine yeasts. The majority of the descendants are characterized by a great and significant diversity compared to their parents. Both the parental strains had genome sizes consistent with their being diploid, so validating the observed results. These findings constitute an initial step to demonstrate the mechanisms of inheritance and establish breeding strategies to improve the ochratoxin A adsorption trait in wine yeasts. This will allow a decrease in the ochratoxin A content of contaminated musts during winemaking, by using genetically improved wine yeasts.     

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and its nutritive analysis

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

Ethanol from Sugar Cane: Flask Experiments Using the EX-FERM Technique

Alcohol production at the laboratory scale from sugar cane pieces by the EX-FERM technique was studied with 37 strains of Saccharomyces spp. The EX-FERM process is novel in that it employs the simultaneous extraction and fermentation of the sucrose in a cane-water suspension. Two types of cane treatments were used: chips and shredded pith, either fresh or dried. A mother culture of the yeast was prepared in enriched cane juice and then added to the cane-water mixture. After static fermentation for 40 h at 30°C, the cane was removed, and fresh cane was added to the yeast-alcohol broth. After an additional 24 h, the cane was again removed and the liquor was analyzed. After the first 40-h cycle, sugar consumption was above 99% with 10 of the 37 yeast strains tested, and ethanol reached levels of 1.29 to 4.00 g per 100 ml, depending on the yeast strain. The final ethanol concentration reached 4.27 to 5.37 g per 100 ml, and sugar consumption was above 98% in three cases during a second EX-FERM cycle employing previously air-dried chips and pith. Product yields were within accepted values. Cane treatment did not appear to affect the results at this level.