海藻糖对面包酵母高糖耐性的影响

通过高温刺激使面包酵母积累较高的胞内海藻糖,考察胞内海藻糖含量对面包酵母高糖发酵力和高渗存活率的影响。研究结果表明,发酵结束前2h培养温度提高至40℃后,菌株BY-6胞内海藻糖含量由4.21%提高到9.76%,高糖发酵力提高了17.2%,高渗存活率也有一定提高。另外对3株面包酵母的海藻糖积累能力和面团发酵力的比较表明,海藻糖积累能力强的酵母菌株高糖耐性较好。     

增强海藻糖胞内积累提高大肠杆菌耐受性与乙醇产率

为提高产乙醇大肠杆菌对发酵底物和乙醇的耐受性,并提高乙醇发酵性能,以大肠杆菌B0013-1031H为出发菌株,对其海藻糖代谢途径进行改造,获得了敲除海藻糖分解途径的突变株JC31和进一步加强海藻糖合成途径的突变株JC41。突变株JC31和JC41较出发菌株都具有更高的海藻糖合成与积累能力,其中JC41的胞内海藻糖含量可达出发菌株的12倍。与出发菌株相比,突变株JC31和JC41对葡萄糖和乙醇胁迫的耐受性显著提高。进一步引入乙醇合成途径,在葡萄糖质量浓度120 g/L的发酵条件下,菌株JC31-PA表现出最优的发酵性能,其最大乙醇产量为50. 6 g/L,较对照菌株提高了5. 42%;乙醇转化率为48. 72 g/100 g葡萄糖,较对照提高了12. 67%,达到理论转化率的95%。     

不同胁迫条件对鲁氏酵母胞内海藻糖积累的影响研究

为了积累鲁氏酱油酵母胞内海藻糖的含量,提高活性干酵母制作过程中的存活率。本文主要探讨了渗透压(Na Cl)、温度、乙醇和氧化(H_2O_2)条件下鲁氏酵母胞内海藻糖的积累规律。实验表明,渗透压胁迫对海藻糖积累没有明显的作用,高浓度Na Cl胁迫时海藻糖含量反而降低;添加乙醇和升高温度有利于海藻糖的积累:2%(v/v)乙醇添加9 h后海藻糖的含量达到22.32 mg/g Dw,相比对照20.46 mg/g Dw提高了9.09%;温度升高6 h后海藻糖含量从17.55 mg/g Dw升至24.41 mg/g Dw,提高了39.14%;在氧化胁迫初期,H_2O_2可明显提高鲁氏酵母胞内海藻糖的含量,0.2%(v/v)H_2O_2浓度下海藻糖含量为14.18 mg/g Dw,比对照8.17 mg/g Dw增加了73.56%,但是随着氧化胁迫延长,最终海藻糖含量没有明显区别。实验结果为提高鲁氏酵母胞内海藻糖含量从而制成耐储藏活性干酵母具有一定指导意义。     

酒精发酵过程中胁迫因子对酵母海藻糖积累的影响

针对酒精生产酵母可能面临的受迫性因子对海藻糖形成的影响进行了研究。结果表明,不同碳源如蔗糖、葡萄糖对酒精发酵液中海藻糖的含量几乎没有差别;黄曲霉毒素Afb1对酒精发酵海藻糖的累积有一定的影响;发酵起始p H值过高(6.5)或过低(3.5)都会导致发酵液中游离海藻糖含量/海藻糖总量增高,发酵起始p H值过低(3.5)还会导致酵母胞内海藻糖含量升高;发酵温度过高(38℃)会导致发酵液中游离海藻糖/胞内海藻糖/海藻糖总量升高;氮源不足会引起发酵液中海藻糖/胞内海藻糖/海藻糖总量显著升高,并且氮源对胞内海藻糖累积的影响远远大于温度。     

源于中国民间传统酿造食品的鲁氏酵母耐温特性研究

鲁氏酵母是一种重要的传统酿造应用微生物。分析了1株从中国民间传统酿造食品中筛选到的鲁氏酵母CCTCC M2013310对温度逆境的耐受能力及其影响因素。在简单营养条件如YEPD下培养的鲁氏酵母耐高温能力低,且温度越高,对鲁氏酵母的致死作用越大。然而,增加胞内海藻糖浓度后,鲁氏酵母细胞耐高温能力显著提高,但对细胞耐冷温无明显贡献,却有助于提高细胞耐冷冻水平。鲁氏酵母细胞内海藻糖积累与碳源有明显关系。甘油和果糖能较好地促进细胞积累海藻糖,葡萄糖效果最差。而且葡萄糖条件下的酵母细胞的耐热能力很弱,远低于果糖条件下。可见,营养条件、生长周期和胞内海藻糖浓度对鲁氏酵母细胞的耐逆境能力均有影响,但绝非简单的正或负线性关系。     

基于面包酵母中海藻糖提取工艺优化及菌株筛选的高密度培养工艺

确定使用酶-3,5-二硝基水杨酸（3,5-dinitrosalicylic acid,DNS）法作为酵母胞内海藻糖定量测定方法,通过微波-浸提法对活性干酵母胞内海藻糖进行提取,采用单因素试验以酵母中海藻糖含量为参考指标优化该工艺中的微波功率、微波时间、三氯乙酸浓度、三氯乙酸体积、浸提温度、浸提时间,在此基础上对微波功率、微波时间、三氯乙酸浓度、三氯乙酸体积、浸提温度5?个因素进行正交试验,优化后工艺条件为：针对1.5?g活性干酵母,微波功率231?W、微波时间40?s、三氯乙酸浓度0.7?mol/L、三氯乙酸体积40?mL、浸提温度55?℃、浸提时间150?min。使用该工艺得到的活性干酵母中的海藻糖含量为280.15?mg/g,相对于优化前提高了15.2%。同时,利用该优化后工艺在现有面包酵母菌种中筛选出了一株高产海藻糖的菌株。在50?L自吸式发酵罐中将该菌株进行流加发酵,采用糖蜜进行溶氧反馈流加时最大酵母湿质量浓度为198.34?g/L,将流加工艺进行改进,提出称质量补料的流加方法将糖蜜、尿素、磷酸二氢铵作为流加营养源,实验发现发酵后最大酵母湿质量浓度可提高到264.82?g/L,相对于原工艺提高了33.52%,达到了面包酵母高密度培养的要求。发酵过程中将乙醇体积分数控制在0.7%～1.0%之间,将有效提高酵母湿质量浓度且保证其发酵力在650?mL/h以上,且工艺稳定性良好。     

3株酿酒酵母发酵过程中有机酸含量变化分析

通过高效液相色谱法对3株酿酒酵母(Saccharomyces cerevisiae)发酵过程中葡萄糖、甘油、乙醇及有机酸含量进行测定分析。结果表明:S.cerevisiae1946发酵体系中甘油和乙醇的含量显著高于S cerevisiae P1和S.cerevisiae 32788发酵体系(P0.05);3株酿酒酵母发酵液中7种有机酸存在明显的差异性和规律性;S.cerevisiae 1946发酵体系中有机酸的含量低于S.cerevisiae P1和S.cerevisiae 32788发酵体系,S cerevisiae的产酸水平与产乙醇能力呈负相关关系。     

树干毕赤酵母和酿酒酵母耦合发酵高浓度混合糖产乙醇

研究在高浓度发酵底物下,不同木糖及葡萄糖之间的比例、P. stipitis CBS5773和S.cerevisiae的发酵先后启动顺序、两菌间的协同发酵对两茵共同发酵混合糖产乙醇的影响.结果表明,乙醇的产量主要来自于由S.cerevisiae消耗葡萄糖而产生,两菌株偶联发酵时,Scerevisiae能较快启动进入产乙醇发酵而不受P. stipitisCBS5773生长的抑制;在高浓度(200 g/L)糖浓度下,S.cerevisiaee和P.stipitis CBS5773存在相互拮抗作用,S.cerevisiae并不完全抑制P. stipitis CBS5773的生长;当木糖与葡萄糖之比为3:2,且S.cerevisiaee和P. stipitis CBS5773分开接入,发酵12 h,乙醇产量最大,达到57 g/L.     

耐高渗酿酒酵母的代谢流分析

研究了指数生长期的酿酒酵母 (Saccharomyces cerevisiae) 在高渗胁迫下的生理代谢.甘油和海藻糖是酿酒酵母的主要相容性溶质.在高渗环境下,胞外酒精质量浓度、甘油质量浓度、海藻糖质量浓度分别较对照提高100%、400%和11%.代谢流分析表明,高渗环境下,从节点6-磷酸葡萄糖合成海藻糖的碳流较常规培养增加了47.3%;从节点磷酸二羟丙酮和3-磷酸甘油醛合成甘油的碳流增加了55.7%;从节点丙酮酸流向乙醇的碳流增加了300%,而流向三羧酸 (TCA) 循环的流量减少了21.7%.     

应用细胞膜特性预测酵母质量

酵母质膜可以说是发酵性能的生物标志.对酸化势实验组成的两个独立参数--水的酸化势(WAP)和葡萄糖诱发质子流(GIPE)的修订法进行研究,GIPE准确地区分了活细胞和死细胞群体,反映了酵母泥受到的压力损伤.WAP则反映了胞内肝糖水平,从而证明了细胞在发酵中的复制能力.ale酵母团在发酵罐和贮罐中存在像饥饿,乙醇和温度变化的影响,还有受到高浓酿造时增殖和发酵初期的外加压力,像氧化的压力,WAP和GIPE适合上述这些特定压力条件下的测定.质子流变化的程度根据压力的类型和菌株的依赖而变化.     

Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex

Along the fermentation process yeasts are affected by a succession of stress conditions that affect their viability and fermentation efficiency. Among the stress conditions the most relevant are high sugar concentration and low pH in musts, temperature and, as fermentation progresses, ethanol accumulation. Nowadays, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing search for particular wine yeast strains possessing a wide range of optimized, improved or novel enological characteristics. Traditionally, the species S. cerevisiae and S. bayanus within the Saccharomyces sensu stricto species are considered some of the most important yeast species involved in fermentation processes. However, in the last years, hybrid strains between the species S. cerevisiae, S. bayanus and S. kudriavzevii have been described as yeasts conducting the alcoholic fermentations and some of them are commercially available. Our results indicate that yeasts in the Saccharomyces sensu stricto complex were not affected by low pH or high glucose content in the media; however temperature and ethanol concentration variables appreciably affected their growth. The strains pertaining to S. cerevisiae were able to tolerate high temperature stress, whereas strains within S. bayanus and S. kudriavzevii were better adapted to growth at lower temperatures. Regarding to alcohol tolerance, S. cerevisiae is tolerating alcohol better than S. bayanus or S. kudriavzevii. Surprisingly, the natural hybrids between these species have adapted to growth under ethanol and temperature stress by inheriting competitive traits from one or another parental species. These results open new perspectives in the construction of new hybrid strains with biotechnological interest, as the characteristics of the parents may result in interesting combinations in the hybrids.     

老白干酒曲中酵母菌多样性分析

采用微生物分离培养与分子鉴定相结合的方法,在对酒曲中的酵母菌进行分离、纯化和形态学鉴定基础上,进行菌株核糖体26S rDNA聚合酶链式反应-限制性片段长度多态性分析技术结合的分子鉴定。并采用非培养方法,直接提取酒曲中的总DNA进行聚合酶链式反应扩增、电泳和切胶与回收,通过聚合酶链式反应-变性梯度凝胶电泳（polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE）法鉴定酵母菌。结果表明,采用培养方式分离到57?株6?种不同形态的酵母菌,经鉴定为5?种分子类型,分别为奥默毕赤酵母（Pichia kudriavzevii）、葡萄牙棒孢酵母（Clavispora lusitaniae）、异常威克汉姆酵母（Wickerhamomyces anomalus）、胶红酵母（Rhodotorula mucilaginosa）、阿氏丝孢酵母（Trichosporon asahii）。经PCR-DGGE法鉴定得到10 株酵母菌,分别为W. anomalus、Wickerhamomyces sp.、T. asahii、热带假丝酵母（Candida tropicalis）和Candida sp.各1 株,Hyphopichia burtonii 2?株,另有uncultured Saccharomycopsis 3?株。培养与非培养两种方式共鉴定得到10 株酵母菌,分别属于8?个属,包括P. kudriavzevii、C. lusitaniae、W. anomalus、R. mucilaginosa、T. asahii、uncultured Saccharomycopsis、H. burtonii、Wickerhamomyces sp.、C. tropicalis、Candida sp.,该研究结果可为进一步研究酒曲的发酵能力提供参考。     

新疆塔城地区乳源酵母菌的分离、鉴定及其抗胁迫特性

为收集、保护和发掘西部牧区传统发酵乳制品中酵母菌资源,从新疆塔城地区牧区共采集8份牧民自制乳制品样品。采用细胞形态学、生理生化特性鉴定、5.8S rDNA序列同源性比对相结合的方法,对酵母菌进行了分离、纯化和鉴定,并考察了其耐高温、耐渗透压、耐乙醇等能力。结果表明,共分离出16株库德毕赤酵母菌(Pichia kudriavzevii)、6株戴尔有孢圆酵母(Torulaspora delbrueckii)、4株美极梅奇酵母菌(Metschnikowia pulcherrima)、2株马克思克鲁维酵母(Kluyveromyces marxianus)、2株酿酒酵母(Saccharomyces cerevisiae)、2株胶红酵母(Rhodotorula mucilaginosa)、2株乳酸克鲁维酵母(Kluyveromyces lactis)。库德毕赤酵母菌A2-1能够耐受42℃高温、2.4%高盐胁迫、50 g/100 g高糖胁迫以及12%酒精胁迫;马克思克鲁维酵母菌A2-13能够耐受42℃高温、2.4%高盐胁迫、50 g/100 g高糖胁迫以及16%酒精胁迫。可见,两株菌均能适应于工业开发需求。     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

Degradation of phytate by Pichia kudriavzevii TY13 and Hanseniaspora guilliermondii TY14 in Tanzanian togwa

The fermented cereal-based gruel togwa is used as weaning food for children in Tanzania. Togwa is rich in minerals but these are often not available for uptake in the human intestine due to natural inhibitors, such as phytate (IP(6)). The yeasts Pichia kudriavzevii TY13, Hanseniaspora guilliermondii TY14 and TY20, isolated from Tanzanian togwa, and selected for high phytase activity in complex yeast medium YPD, were now studied regarding their ability to degrade IP(6) in maize-based model togwa. A modified constitutively high-phytase producing Saccharomyces cerevisiae BY80 and commercial Aspergillus ficuum phytase were included for comparison. In addition, a strain of Lactobacillus plantarum was included in the model-togwa set-up. All yeasts in the study grew and reached final cell density 1.5-2 log units higher than the start value. S. cerevisiae BY80 degraded 85% of the IP(6) in 48 h; the same degradation level as with A. ficuum phytase (89%). Of the togwa-isolated yeasts, P. kudriavzevii TY13 and H. guilliermondii TY14 showed strong phytate degradation in the model-togwa; 95% or more of the initial IP(6) was degraded after 48 h. This corresponds to a remaining level of 0.4 and 0.3μmol IP(6)/g dw. Co-inoculation with L. plantarum did not increase IP(6) degradation. Moreover, fermentation with P. kudriavzevii TY13 yielded a successive increase in inorganic phosphate (P(i)), from 0.7 to 5.4 mM, suggesting a phytase production in TY13 which is fairly insensitive to P(i) repression. The study shows that phytate in a model togwa is available for yeast phytase enzymes, and addresses the importance of strain selection for effectively degrading the phytate. Certain yeasts originating from togwa seem to have developed a natural high phytase production, and P. kudriavzevii TY13 and H. guilliermondii TY14 seem particularly well adapted to phytate degradation in togwa, and is our choice for further studies and strain improvement.     

发酵条件对5种产酯酵母酒精发酵和产酯的影响

该实验探讨了供氧情况、发酵pH和温度对各种产酯酵母酒精发酵和产酯的影响。 结果表明,供氧情况对产酯酵母的酒精发酵和产酯均有显著影响,其中对克鲁斯假丝酵母的酒精发酵影响最大,对球拟酵母产酯影响最大。 发酵pH对产酯酵母的酒精发酵和产酯有一定影响,其中卡特多菲毕赤酵母的最适产酒pH为5.0,克鲁斯假丝酵母和汉逊酵母最适产酒pH为4.0,球拟酵母最适产酒pH 为6.0～7.0;上述4种产酯酵母的最适产酯pH均为6.7,高产酯酿酒酵母的最适产酯pH为6.0～7.0。 温度对各种酵母的酒精发酵和产酯有一定影响,其中卡特多菲毕赤酵母、克鲁斯假丝酵母、汉逊酵母和球拟酵母的最适产酒温度均为28 ℃;卡特多菲毕赤酵母的最适产酯温度为20 ℃,克鲁斯假丝酵母、汉逊酵母和高产酯酿酒酵母为24 ℃,球拟酵母为28 ℃。     

高糖胁迫对酿酒酵母抗氧化活性及代谢的影响

本文以酿酒酵母(Saccharomy cescerevisiae)为模式生物,研究了高糖培养条件对酿酒酵母的生长、抗氧化酶活性及海藻糖、甘油代谢的影响。结果表明:当葡萄糖浓度达到40%时,酿酒酵母生长的对数期延长,对酿酒酵母的生长产生了抑制作用。酿酒酵母在培养4~10 h范围内四组酿酒酵母细胞(20 g/L葡萄糖组、40 g/L葡萄糖组、60 g/L葡萄糖组和80 g/L葡萄糖组)内海藻糖的积累随着胁迫时间的增加发生显著变化(P<0.05),海藻糖的积累量呈先升高后下降的趋势,在8 h时高糖组海藻糖积累量均达到一个最高点,胞内海藻糖的浓度最高达到0.0955 mg/mL。在不同葡萄糖浓度胁迫下,酵母细胞胞内外甘油的积累随着时间增加呈现出先上升后下降的趋势,但是胞内甘油的积累量在80 g/L葡萄糖浓度时达到最高,而胞外甘油的积累量在60 g/L葡萄糖浓度时达到最高。这些结果说明在高糖胁迫下甘油和海藻糖是酿酒酵母的主要相容性溶质。另外,高糖处理后,与对照组相比,高糖组酿酒酵母胞内超氧化物歧化酶(Superoxide Dismutase,SOD)、过氧化氢酶(Catalase,CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性均显著升高(P<0.05),说明这些抗氧化物酶活性物质对维持有机体胞内正常渗透压起到关键作用。该研究结果将为今后研究酿酒酵母耐高糖渗透压方面提供理论依据。     

Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess

When chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621, grown under glucose limitation, were pulsed with excess glucose, both organisms initially exhibited similar rates of glucose and oxygen consumption. However, striking differences were apparent between the two yeasts with respect to the production of cell mass in the culture and metabolite excretion. Upon transition from glucose limitation to glucose excess, S. cerevisiae produced much ethanol but the growth rate remained close to that under glucose limitation. C. utilis, on the other hand, produced little ethanol and immediately started to accumulate cell mass at a high rate. This high production rate of cell mass was probably due to synthesis of reserve material and not caused by a high rate of protein synthesis. Upon a glucose pulse both yeasts excreted pyruvate. In contrast to C. utilis, S. cerevisiae also excreted various tricarboxylic acid cycle intermediates, both under steady-state conditions and after exposure to glucose excess. These results and those of theoretical calculations on ATP flows support the hypothesis that the ethanol production as a consequence of pyruvate accumulation in S. cerevisiae, occurring upon transition from glucose limitation to glucose excess, is caused by a limited capacity of assimilatory pathways.     

降柠檬酸菌株的筛选及鉴定

为筛选具有降柠檬酸能力的酵母菌株,对一柠檬酸厂废水废渣中的微生物进行培养分离纯化保藏,共获得具有耐柠檬酸的菌株15株。对15株菌株的降柠檬酸能力进行测试,发现5株菌的降柠檬酸能力较强,发酵5d可降解柠檬酸培养基中70%以上的柠檬酸。测定5株菌的酒精耐受性,菌株NS2、NL2的酒精耐受能力较强。观察该两株菌的形态学及生理生化特性,将NS2鉴定为毕赤酵母菌,NL2鉴定为热带假丝酵母菌。进一步通过分子生物手段进行18SrDNA PCR测序分析,将NS2鉴定为Pichia kudriavzevii,NL2鉴定为Candida tropicalis。研究成果可为今后应用生物降酸法降低山楂酒等果酒中有机酸含量提供指导。     

两种非酿酒酵母与米曲霉互作特性研究

微生物互作在白酒酿造中扮演重要角色。费比恩塞伯林德纳氏酵母（Cyberlindnera fabianii）和葡萄牙棒孢酵母（Clavispora lusitaniae）是白酒酿造中常见的非酿酒酵母,但它们与霉菌的互作关系尚不清晰。本研究围绕感官评价、挥发性风味物质含量、酵母菌生物量、酶活力和代谢产物等方面,探讨了费比恩塞伯林德纳氏酵母HY-11和葡萄牙棒孢酵母HY-21与米曲霉（Aspergillus oryzae）M-08在酿造过程中的相互作用。结果表明,两种非酿酒酵母分别与米曲霉M-08混菌培养,费比恩塞伯林德纳氏酵母HY-11和葡萄牙棒孢酵母HY-21的生物量分别比单一酵母菌发酵提高4.96倍和0.97倍,α-淀粉酶活力分别提高70.31倍和107.73倍,表明两酵母菌对米曲霉M-08有依赖性。相比葡萄牙棒孢酵母HY-21,费比恩塞伯林德纳氏酵母HY-11与米曲霉M-08混菌发酵风味更好,发酵6 d具有浓郁哈密瓜香、酒香和酯香味,挥发性风味物质总量最高1408.97 μg/g,比单一条件下发酵提升381.87倍。米曲霉M-08与费比恩塞伯林德纳氏酵母HY-11和葡萄牙棒孢酵母HY-21有较好的相互作用,混菌发酵比单一酵母菌发酵体系游离氨基酸水平分别提高105.02和3837.29 μg/g,酯化酶活力分别增加27.67和387.82 U/g,从而促进挥发性风味物质的代谢。本研究阐明了两种非酿酒酵母与米曲霉M-08的相互关系和其风味特色,为其合理应用提供了理论依据。