均匀设计优化酿酒酵母液态发酵豆渣工艺及其风味分析

为提高豆渣的利用价值,以豆渣为研究对象,通过均匀设计试验优化酿酒酵母(Saccharomyces cerevisiae)液态发酵豆渣工艺,并测定发酵豆渣中的品质指标及挥发性风味物质含量。结果表明,酿酒酵母液态发酵豆渣的最佳工艺条件为：接种量2%、白砂糖0.3%、K₂HPO₄0.08%、MgSO₄0.03%、含水量95%、发酵时间28 h。在此最佳发酵条件下,发酵豆渣的氨基酸态氮、可溶性膳食纤维含量及感官评分分别为0.174 5 g/100 g、6.49 g/100 g、27.50分,分别是发酵前的1.51倍、3.57倍、1.42倍。从发酵豆渣中共鉴定出72种挥发性风味物质,包括醇类16种、醛类4种、酮类5种、酯类18种、烷烯烃类19种、呋喃类3种和其他7种,相较于发酵前增加了大量醇、酯类物质,使豆渣具有花草香、果香等独特风味,同时醛类物质下降,削减了豆渣中的豆腥味。     

响应面法优化酿酒酵母产孢培养基及单倍体的鉴定

采用响应面法对酿酒酵母4608菌种的产孢培养基进行了优化。用Plackett-Burman方法对影响培养各因素的效应进行评价,筛选出有显著效应的3个因素：酵母膏、蛋白胨和葡萄糖;通过中心组合实验及响应面分析优化此3个主要因素。采用优化后的条件,酵母膏2.8 g/L,蛋白胨1.2 g/L,葡萄糖0.48 g/L,经培养验证,实测值与预测值间有-0.58%的偏差,实际酵母产孢率为44.12%,比优化前的产孢率提高了60.4%。利用PCR技术检验酵母的单倍体,验证了酵母的交配型,方法准确、迅速。     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16–1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M₁ replication defects of mak16–1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1·2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20°C, 30°C, or 37°C, but could not grow at 8°C. The deleted region is thus essential only at 8°C, and we name this gene LTE1 (low temperature essential).     

Role of OGG1 and NTG2 in the repair of oxidative DNA damage and mutagenesis induced by hydrogen peroxide in Saccharomyces cerevisiae: relationships with transition metals iron and copper

The base excision repair pathway of Saccharomyces cerevisiae possesses three DNA N-glycosylases, viz. Ogg1p, Ngt1p and Ntg2p, involved in the repair of oxidative DNA damage. It was previously reported that inactivation of any of these activities, in most cases, did not generate a sensitive mutant phenotype to a variety of oxidative agents. Only the ntg1 mutant appeared to be more sensitive to hydrogen peroxide (H2O2) than a wild-type (WT) strain. In the present study we evaluated the role of S. cerevisiae OGG1 and NTG2 genes in the repair of oxidative lesions induced by high H2O2 concentrations (5-100 mM for 20 min), followed by catalase treatment (500 IU/ml). In these conditions, the ogg1 mutant was more sensitive than the WT strain to H2O2 (concentration 40-60 mM). Unexpectedly, the inactivation of NTG2 in an ogg1 background was able to suppress both sensitivity and mutagenesis induced by H2O2. Indeed, even the ntg2 single mutant was more resistant than the WT (60-100 mM H2O2). The use of metal ion chelators dipyridyl and neocuproine allowed us to evaluate the participation of iron and copper ions in the production of lethal and mutagenic lesions during H2O2 treatment in different DNA repair-deficient S. cerevisiae strains. The roles of OGG1 and NTG2 genes in the repair of lethal and mutagenic oxidative lesions induced by H2O2 and their relationships with iron and copper ions are discussed.     

Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae.

A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl-coenzyme A (acetyl-CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromosome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79.2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons downstream, is in a more conventional context. Possible implications of two alternative translational start sites for the cellular localization of the enzyme are discussed. A stable mutant of this gene, obtained by the gene disruption technique, had the same low basal activity of acetyl-CoA synthetase as wild-type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetyl-CoA synthetase (ACS1) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction of isocitrate lyase on acetate media, indicating that activity of acetyl-CoA synthetase is dispensable for induction of the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACS1 gene did not affect growth on media containing ethanol as the sole carbon source, demonstrating that there are alternative pathways leading to acetyl-CoA under these conditions.     

Role in anaerobiosis of the isoenzymes for Saccharomyces cerevisiae fumarate reductase encoded by OSM1 and FRDS1

Saccharomyces cerevisiae possesses both a cytoplasmic and a mitochondrial fumarate reductase, encoded by FRDS1 and OSM1, respectively. While previous studies have shown that mutants lacking FRDS1 and OSM1 cannot grow under anaerobiosis (Arikawa et al., 1998), the physiological role of fumarate reductase (FR) remains poorly understood. Here, we report that an osm1 frds1 mutant is unable to grow anaerobically, even with glutamate as a sole nitrogen source, when succinate can be produced by the TCA oxidative branch. We also show that the growth of the mutant is not restored by adding acetoin, an alternative sink for NADH oxidation, but it is at least partly restored by the addition of oxygen or menadione, which can oxidize FADH(2) in addition to NADH. These data indicate that the growth inhibition of the mutant is due to an inability to reoxidize FAD, rather than an indirect effect on NADH or an inability to produce succinate per se. During anaerobic growth, FRDS1 expression was two to eight times higher than that of OSM1, and fumarate reductase activity was higher in the osm1 mutant than in the frds1 mutant. FRDS1 expression was induced by anaerobiosis, and this induction was abolished in a rox1 mutant. We conclude that the formation of succinate is strictly required for the reoxidation of FADH(2) during anaerobiosis, and that it is regulated through the control of FRDS1 expression when oxygen is limiting. Based on these data, we discuss the potential role of fumarate reductase in the regeneration of the FAD-prosthetic group of essential flavoproteins.     

The HTL1 gene (YCR020W-b) of Saccharomyces cerevisiae is necessary for growth at 37 degrees C, and for the conservation of chromosome stability and fertility

A small 78 codon ORF, named HTL1 (Chen et al., unpublished results), situated between loci MAK31 and HSP30 on chromosome III of Saccharomyces cerevisiae, is required for growth at 37 degrees C. In this communication, we characterize the ORF and show that disruption of HTL1, besides preventing growth at 37 degrees C, causes genetic and/or epigenetic instability at 26 degrees C: ploidy increases in about 10% of cells grown from individual disruptants and a fraction of disruptant clones are predestined to a rapid and progressive loss of fertility during growth at 26 degrees C.     

响应面优化酿酒酵母与窖泥酯化细菌协同发酵产丁酸乙酯和己酸乙酯

为提高浓香型白酒发酵过程丁酸乙酯和己酸乙酯的生成量,以酿酒酵母和3 株窖泥酯化细菌为菌源,利用单因素试验和响应面法中的Box-Behnken试验设计模型优化己酸乙酯和丁酸乙酯的固态发酵条件。结果表明,水分质量分数、酸度和接种量对己酸乙酯和丁酸乙酯的影响极显著（P＜0.01）,而温度不显著（P＞0.05）。最佳发酵条件为温度32 ℃、水分质量分数78.00%、酸度2.60 mmol/100 g、接种量4.60%。在此条件下,己酸乙酯和丁酸乙酯的产量分别为9.55 mg/100 g和1.87 mg/100 g,分别达模型预测值的99.07%和100%。经反复验证,工艺合理可行,可为进一步提高浓香型白酒质量和窖泥功能微生物在生产实际中的应用提供理论基础。     

Optimised methodology for carboxymethylation of (1→3)‐β‐d‐glucan from Yeast (Saccharomyces cerevisiae) and promotion of mechanical activation

With a view to utilise yeast (1→3)‐β‐d ‐glucan as biological response modifiers with better water solubility, carboxymethylation was carried out by a two‐step alkalisation and etherification with monochloroacetic acid. Four technological parameters of carboxymethylation were investigated by orthogonal experiments for obtaining the maximum degree of substitution (DS), apparent viscosity (η) and solubility of carboxymethyl derivatives. In view of the orthogonal analysis, the optimal technological parameters were reaction temperature 50 °C, total reaction time 5 h, 3 mL of 50% sodium hydroxide as the second alkali dosage and 15 mL of 4 m chloroacetic acid. In addition, it was found that ball milling pretreatment for original (1→3)‐β‐d ‐glucan can be an advantage for carboxymethylation. By contrast, DS, η and solubility of carboxymethyl product increased 24%, 6% and 22%, respectively, suggesting the effect of ball milling pretreatment could not be neglected on improvement of DS, η and solubility for carboxymethyl products.