高效液相色谱法测定酵母中麦角固醇含量

本文建立了酵母中麦角固醇含量高效液相色谱测定方法。其色谱条件为,色谱柱HYPersilBDSC185u反相柱,流动相为甲醇:水(97∶3),紫外检测波长为283nm。酵母样加碱乙醇皂化、提取、洗涤、蒸干、定量测定。结果表明:标准曲线范围是002—08mg/ml线性良好,最低限量为001mg/ml;日内及日间RSD(n=4)分别在21～40%和24～48%,回收率为960～980%。本方法操作简便、准确、可靠。     

高生物量富铁酵母菌的选育及其发酵条件的研究

对402株不同种属的酵母菌株进行初筛、复筛,筛选到一株生物量较高的二倍体菌株ZY-46(Saccharomyces cerevisiae)和一株铁富集量较高的二倍体菌株ZY-173(Saccharomyces kluyveri)。然后以它们为出发菌,分别进行单倍体分离、硫酸二乙酯(DES)诱变,并通过原生质体融合,得到一株高生物量富铁酵母融合菌株ZYF-15。在优化的发酵条件下,该融合菌株生物量可达11．2g／L,细胞铁含量达24．5mg／g干细胞,细胞总铁含量分别比原始亲株ZY-46和ZY-173提高了2．6倍和1．9倍。     

Efficient use of DNA molecular markers to construct industrial yeast strains

Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure.     

啤酒酵母工业菌株单倍体的诱导、分离和鉴定

【目的】探索适宜的方法进行啤酒酵母工业菌株单倍体的诱导、分离和鉴定,为啤酒酵母改良和遗传学研究提供便利。【方法】首先,选择产孢效果最好的培养基进行产孢诱导,诱导产生的孢子在YPD培养基上形成菌落后,用流式细胞技术检测其DNA含量,进而判断其倍性;单倍体菌株的交配型通过MAT-PCR和杂交实验确定。【结果】啤酒酵母工业菌G-03通过产孢诱导和孢子分离、富集后得到26株菌,最终通过流式细胞技术确定了其中4株为单倍体,MATa和MATα型各2株。通过扫描电镜法观察4株单倍体菌株及出发菌G-03的细胞形态,发现单倍体菌株的形态和出发菌有较大区别,单倍体菌株长期培养没有假丝生长的现象发生。【结论】啤酒酵母工业菌单倍体育种较为困难,严格的单倍体筛选、鉴定尤其具有挑战性。     

离子注入麦角甾醇酵母选育研究

用10 keV、剂量为2.6×1013N+/cm2～8.0×1014N+/cm2的氮离子注入产麦角甾醇酵母,可产生可遗传的变异.离子注入产麦角甾醇酵母的存活率与注入剂量呈负相关,在存活率为25%～45%,即注入剂量为1.3～2.3×1014N+/cm2时菌种有较高的正变率.最终筛选到的高产菌株YA1和YA2,麦角甾醇得率较出发菌株分别提高了60%和55%.经复筛及传代实验表明高产菌株遗传性能稳定.     

高产辅酶Q10结构类似物抗性突变株的选育

以土壤杆菌(Agrobacteriumsp.)TLY-4为出发菌株,采用70%致死剂量的NTG进行诱变处理,通过筛选抗辅酶Q10结构类似物维生素K3突变株,定向选育到了两株辅酶Q10高产突变株,编号为R-122和R-015,其摇瓶发酵72h时的辅酶Q10产量分别为57.3 mg/L和59.9 mg/L,较出发菌株提高了35.7%和41.6%。通过连续传代实验,表明突变株高产辅酶Q10的遗传性状稳定。实验以有机溶剂DMF和吐温-80共同增溶的方法,解决了维生素K3在培养基中易析出的问题,并确定了平板培养基中维生素K3的最小抑菌浓度为0.15 mg/mL。     

Breeding of a cordycepin-resistant and adenosine kinase-deficient sake yeast strain that accumulates high levels of S-adenosylmethionine

ABSTRACT

Adenosine kinase (ADO1)-deficient mutants can be obtained from cordycepin-resistant strains, and the disruption of ADO1 causes S-adenosylmethionine (SAM) accumulation. To breed a high-SAM-accumulating yeast strain without genetic manipulation for industrial purposes, we bred a cordycepin-resistant strain using sake yeast kyokai No. 9 as the parent strain with a mutation in adenosine kinase (ADO1) and acquired high-SAM-accumulating strain. In the bred strain (NY9-10), a single mutation (T258I) was present in the ADO1, and this mutation site is an ATP binding site and is highly conserved during evolution. Moreover, it was suggested that high accumulation of SAM and cordycepin resistance in NY9-10 was due to functional deficiency of ADO1 by this mutation. This strain is not a genetically-modified organism and can be employed for use in the food and medicine industry such as mass production and sake making.     

产赤藓糖醇菌种的诱变育种

以耐高渗酵母为出发菌株,采用紫外线诱变处理,得到了一株变异株C1。其摇瓶发酵产赤藓糖醇24．9g／L左右。通过摇瓶发酵试验,研究了菌株C1的遗传稳定性。研究结果表明,经多次传代实验,菌株C1的发酵产糖醇能力没有改变,表明其是稳定的变异株。赤藓糖醇的产量由诱变前的12、594g／L提高到24.943g／L,提高了98.1％。关键词：     

Selection of a high-biomass, chromium-rich yeast strain and optimization of cultivation conditions

Saccharomyces cerevisiae LZ-53 was selected from 240 primary yeast strains from different genera and species, whose chromium (Cr) resistance and biomass were higher than others were. The highest biomass and Cr content of the strain was obtained in 30 h at 28°C and 200 rpm, when 20 ml of the culture in 250-ml shake flasks was grown in wort containing 1200 μg/ml Cr. The initial pH was adjusted to 6.0. The optimal inoculum volume was 10% (v/v). The Cr content of the cells was determined by neutron activation analysis. Under the optimized cultivation conditions, the Cr content reached 3248 μg/g. Journal of Industrial Microbiology & Biotechnology (2001) 27, 195–198. Received 28 January 2001/ Accepted in revised form 11 June 2001     

Activity of different ''killer'' yeasts on strains of yeast species undesirable in the food industry

Killer strains of the genera Saccharomyces, Hansenula and Kluyveromyces were tested for killing activity against yeasts that cause trouble in the food industry (in the genera Zygosaccharomyces, Kloeckera, Saccharomycodes and Schizosaccharomyces). Saccharomyces strains killed only Zygosaccharomyces rouxii strains, while non-Saccharomyces strains showed a wider anti-yeast spectrum. The Kluyveromyces phaffii killer strain was of particular interest because of its killer action against Kloeckera apiculata, Saccharomycodes ludwigii and Zygosaccharomyces rouxii.     

高发酵度酵母的筛选及鉴定

主要研究了Saccharomyces cerevisiae wx、Saccharomyces cerevisiae qp、Saccharomycescerevisiae nq、Saccharomyces cerevisiae cx、Saccharomyces cerevisiae hs和Saccharomyces cerevisiae3322(以下简称S.C.wx、S.C.qp、S.C.nq、S.C.cx、S.C.hs和S.C.3322)6株酵母菌的形态、同化氮源、发酵力、α-葡萄糖苷酶活力等生理特性,通过实验比较发现S.C.nq和S.C.cx两株菌种在利用糖的种类、同化氮源、真正发酵度及α-葡萄糖苷酶活力等方面较高,因此对这两株菌种进行了还原双乙酰、糖代谢两方面性质的比较,结果表明:两株菌种在还原双乙酰、糖代谢两方面效果显著,筛选出两株优良的高发酵度啤酒酵母菌种。     

Characterization of yeast strains by fluorescence lifetime imaging microscopy

The results of fluorescence lifetime imaging microscopy of selected yeast strains were presented and the fact that the lifetime distributions can be successfully used for strain characterization and differentiation was demonstrated. Four strains of industrially relevant yeast Saccharomyces were excited at 405 nm and the autofluorescence observed within 440-540 nm. Using statistical tools such as empirical cumulative distribution functions with Kolmogorov-Smirnov testing, the four studied strains were categorized into three different groups for normal sample size of 70 cells slide(-1) at a significance level of 5%. The differentiation of all of the examined strains from one another was shown to be possible by increasing the sample size to 420 cells, which is achievable by taking the lifetime data at six different positions in the slide.     

基因组重排技术在酵母菌育种中的应用

基因组重排(genome shuffling)技术是在传统诱变育种的基础上与细胞原生质体融合技术相结合一种新兴微生物菌种改良手段,由于该技术高效的正向突变效率和频率,近年来被广泛应用于酵母菌种的选育和改良。本文主要对基因组重排技术在酵母菌育种中的应用进行了综述。     

高生物量富硒酵母的选育及培养条件初步优化

通过筛选、单倍体分离、诱变和原生质体融合,从融合子中选育了一株高生物量富硒酵母菌株（编号为ZFF-28）,其细胞硒总含量分别是原始亲株ZY-67和ZY-198的2.8倍和2.0倍。通过单因素实验和正交试验设计,确定了优化培养条件：6%糖浓度的蔗糖糖蜜,添加0.5% (NH4)2SO4、0.1% H3PO4、60μg/mL Se,pH60～6.5,装液量50mL/250mL三角瓶,接种量10%,培养时间25h。在优化培养条件下,菌株ZFF_28的生物量可达8.2g/L,细胞中硒的含量达2050μg/g,硒总含量达到了16810μg/L,是培养条件优化前的1.3倍。细胞硒含量的91%为有机硒。     

The selection of yeast strains for the production of premium quality South African brandy base products

One hundred seven yeast strains were screened for their ability to produce a brandy base wine of exceptional sensory quality. Volatile acids, esters and higher alcohols were quantified and the results were interpreted using a multivariate analysis of variance (MANOVA) and an average linkage cluster analysis. Significant differences between yeast strains for higher alcohol, fatty acid ester and acetate concentrations were observed. On the basis of their chemical profiles, 16 strains were selected and re-evaluated in larger-scale fermentations and subsequent double distillations. Results show that the yeast lees can have an important effect on the final concentration of higher alcohols and esters in the distillate. Highly elevated levels of ethyl acetate and iso-amyl acetate were found to be undesirable. Elevated levels of all the esters present contributed positively to the overall potential quality of the brandy base product. Too low higher alcohol concentrations were also not desirable. Sensory evaluations showed that, since the panel was composed of representatives of the three largest brandy-producing companies, each company preferred a different yeast strain most suitable for their style of brandy. For these reasons, three strains, B7, LL2 and 20-2, warranted further evaluation on a semi-commercial scale for each of the respective companies. Journal of Industrial Microbiology & Biotechnology (2000) 24, 431–440. Received 17 November 1999/ Accepted in revised form 07 March 2000     

甾醇C-22去饱和酶高表达对酵母细胞麦角甾醇合成的影响

通过PCR扩增克隆到酵母菌甾醇C-22去饱和酶基因(ERG5)的编码序列及其终止子序列,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pYPE5。以铜离子螯合蛋白基因CUP1替换ERG5基因内部序列获得ERG5破坏菌株YSE5,其中麦角甾醇的合成被阻断,而积累了甾醇中间体Ergosta-5,7-dien-3β-ol。表达质粒pYPE5转化破坏菌株后使细胞恢复了合成麦角甾醇的能力。说明表达质粒上的ERG5基因得到了功能性的表达。将表达质粒pYPE5转化酿酒酵母单倍体菌株YS58,通过营养缺陷互补筛选到重组菌株YS58(pYPE5)。对重组菌株、破坏菌株和互补菌株细胞甾醇组分和含量进行测定,发现重组菌株和互补菌株的麦角甾醇和总甾醇含量明显低于对照菌YS58(YEp352)。测定不同培养时间细胞的麦角甾醇含量,发现重组菌株的麦角甾醇含量始终低于对照菌YS58(YEp352)。可见,ERG5在酵母中的高表达导致细胞麦角甾醇含量降低。     

Production of ethanol from guava pulp by yeast strains

Guava pulp used for ethanol production by three yeast strains contained 10% (w/v) total sugars and was pH 4.1. Ethanol production at the optimum sugar concentration of 10%, at pH 4.1 and 30°C was 1.5%, 3.6% and 3.9% (w/v) by Saccharomyces cerevisiae MTCC 1972, Isolate-1 and Isolate-2, respectively, at 60 h fermentation. Higher sugar concentrations at 15 and 20% were inhibitory for ethanol production by all test cultures. The maximum production of ethanol at optimum natural sugar concentration (10%) of guava pulp, was 5.8% (w/v) at pH 5.0 by Isolate-2 over 36 h fermentation, which was only slightly more than the quantity of ethanol produced by Saccharomyces cerevisiae (5.0%) and Isolate-1 (5.3%) over 36 and 60h fermentation, respectively.     

赤霉素产生菌的激光,化学复合诱变育种研究

采用激光和氯化锂复合诱变赤霉菌,对其产生的赤霉素中活性最高的ＧＡ３含量作为效价测定。结果表明与出发菌株相比,激光诱变,ＬｉＣｌ诱变,复合诱变效价分别提高为１１３．７％,４０．２％,１８９．６％。展示激光复合诱变是遗传育种的一种有效方法。     

Studies of on-line viable yeast biomass with a capacitance biomass monitor

A commercially available biomass monitor has been employed in a number of applications. For capacitance monitors, a relationship between capacitance measurement and cell counts or colony forming units has been reported in the literature. However, for use as an online instrument, a more practical correlation with the biomass concentration is needed. In this study, we followed the batch growth of brewer's yeast and a correlation with viable biomass concentration (g DW/L) was demonstrated. This correlation was utilized with the capacitance biomass monitor in a control loop to maintain setpoint biomass levels in a cyclic reactor under perturbations. Not only did the system demonstrate the capability of the biomass monitor to control biomass in such a system, but it also confirmed the correlation reported in our earlier work. (c) 1994 John Wiley & Sons, Inc.