酵母菌的分离培养及发酵实验方法

通过分离培养得到纯种酵母菌并对其进行观察区分;用几种酵母菌进行糖发酵实验,比较酵母菌对不同糖的利用能力;利用培养所得到的酿酒酵母和假丝酵母酿造葡萄酒,测量葡萄酒的酒精度数,对比确定哪一种酵母菌的酿酒效果更好、产酒率更高。通过平板划线法及稀释涂布法获得纯种的酵母菌种,同时结合菌落形态观察法初步区分酵母菌的种类;通过压滴法区别酵母菌的细胞形态;通过美蓝染色法鉴别酵母菌的死活;采用穿刺接种法分别将酵母接种在带有指示剂的半固体培养基上。3种实验酵母都不发酵乳糖,酿酒酵母的发酵蔗糖和葡萄糖的能力远不及红酵母和假丝酵母。酿酒酵母所酿造出的葡萄酒酒精度数高于假丝酵母。     

酿酒酵母和异常毕赤酵母混菌发酵对白酒液态发酵效率和风味物质的影响

[目的]通过酿酒酵母(Saccharomyces cerevisiae)和异常毕赤酵母(Pichia anomala)在麸皮汁培养基中的混菌发酵,以增加发酵液的风味酯含量并保证发酵效率.[方法]采用两种酵母混合接种、顺序接种混菌发酵方式,以酵母单独接种发酵作对照,测定酵母的发酵性能和发酵液中乙酸乙酯含量,并对发酵结束时风味物质进行半定量;利用无细胞系统,分析两种酵母之间的相互作用.[结果]采用顺序接种混菌发酵方式,避免S.cerevisiae 对P.anomala的生长竞争性抑制,使两种酵母均能获得较高的生物量;发酵结束时,乙醇浓度为20.17 g/L,比酿酒酵母单菌种发酵时降低了9.14％;但乙酸乙酯含量达到0.74 g/L,比异常毕赤酵母单菌种发酵时提高了80％;发酵液风味物质的测定结果表明,酿酒酵母与异常毕赤酵母的混合发酵能够形成更多的酯类物质,总酸和高级醇含量却相对较低,有效改善了发酵液的风味特性;在混菌发酵时,碳源是影响酿酒酵母繁殖的重要因素,但酵母的代谢物对异常毕赤酵母产生明显的抑制作用.[结论]混菌发酵,为丰富发酵产物的风味复杂性和增强风格的独特性提供了一条有效的途径.     

利用食品厂的废料发酵生产饲用微生物添加剂

利用枯草芽胞杆菌 ,以玉米废渣为原料发酵生产饲用微生物添加剂 ,结果发酵产品的活菌数为 1.76× 10 1 1 个 /kg,粗蛋白质含量为 5 2 % ,比原料的粗蛋白质含量提高了 2 8% ;用均匀设计的方法设计四种酵母菌混合发酵模式 ,以啤酒糟为原料 ,生产饲用微生物添加剂 ,结果四种酵母菌的最佳接种量比例为 :酿酒酵母 :红酵母 :热带假丝酵母 :白地霉 =5 :0 :0 :5 ;发酵产品的最高活菌数为 2 .77× 10 1 1个 /kg,最高粗蛋白含量为 6 2 .81%。     

芝麻香型白酒微生物多样性及其与胁迫因子相关性

【背景】近年来芝麻香型白酒的生产工艺日臻成熟,然而相应的科学研究却没有同步发展起来。高通量测序技术越来越多地应用于物种多样性的研究,但偏重于研究物种的相对丰度,没有关注物种的生物数量。【目的】深度解析芝麻香型白酒发酵过程微生物群落结构变化及其与胁迫因子相关性,并研究主要酵母菌与细菌的相关性,为揭示芝麻香型白酒发酵机理和控制发酵质量提供理论支撑。【方法】使用Thermofisher的Ion S5~(TM)XL测序平台进行16S rDNA和ITS rDNA扩增子高通量测序,结合微生物传统的定量方法,测定芝麻香型白酒发酵过程微生物群落结构的变化,同时监测发酵过程乳酸、乙酸、乙醇的含量变化,通过样品复杂度分析、多样品比较分析、环境因子关联性分析探究发酵过程微生物群落及其与胁迫因子的关系。通过Pearson相关性分析酵母菌与细菌的相关性。【结果】发酵前期纤维素菌、魏斯氏菌和芽孢杆菌占主要优势,发酵中后期乳杆菌占绝对优势,其次是纤维素菌、魏斯氏菌和芽孢杆菌。整个发酵过程伊萨酵母占绝对优势,其次是维克霉菌、酿酒酵母、假丝酵母。大部分微生物与胁迫因子呈负相关,只有乳杆菌与乙酸呈极显著性正相关。酵母菌与部分细菌呈正相关性。【结论】白酒发酵过程胁迫因子和微生物间的相互作用促进了群落演替过程,发酵后期乳杆菌和芽孢杆菌发酵产酸抑制了大部分不耐酸菌,有机酸是影响群落结构变化的主要胁迫因子。微生物数量结合相对丰度揭示了发酵过程群落结构演替及其与环境因子相关性的更多信息。     

三株酵母菌子囊孢子形成条件及染色方法的比较

本文对八孢裂殖酵母、酿酒酵母和异常汉逊酵母等三株酵母菌的子囊孢子形成条件及染色方法进行了对比试验。结果认为,八孢裂殖酵母在麦芽汁琼脂培养基、马铃薯琼脂培养基和醋酸钠琼脂培养基上均能产孢,而且在接种8小时以后就能产生孢子。酿酒酵母和异常汉逊酵母只在醋酸钠琼脂培养基上产孢,而且在接种24小时以后才能产生孢子。三种酵母菌的子囊孢子用碘液浸片法染色后着色效果不同,对八孢裂殖酵母的孢子染色是成功的,对酿酒酵母和异常汉逊酵母则不理想。     

高氨氮利用酵母菌的筛选及相关酶活性

【背景】蛋白饲料的缺乏,促进了蛋白含量高、安全性能好的酵母类单细胞蛋白的研究与应用。【目的】筛选氨氮利用能力强的菌株,为单细胞蛋白的发酵提供优良菌株。【方法】从土壤、奶制品、水果采集样品分离酵母菌,根据形态学和分子生物学鉴定菌株,然后以硫酸铵为唯一氮源培养基,测定菌落大小、菌体干重、蛋白质含量,复筛氨氮利用率高的酵母菌,并对复筛菌株氨同化相关酶活性进行测定。【结果】经过形态学、分子生物学鉴定和氨氮利用能力评价,获得3株高氨氮利用的酵母菌,分别是胶红酵母(Rhodotorula mucilaginosa)、酿酒酵母(Saccharomyces cerevisiae)和戴尔有孢圆酵母(Torulaspora delbrueckii)。通过比较3株酵母菌的谷氨酸脱氢酶、谷氨酸合成酶、谷氨酰胺合成酶活性,酿酒酵母的3种酶活性最高,其次是胶红酵母。【结论】从奶酪和西瓜中分离的胶红酵母N5和酿酒酵母J1具有较强的氨氮利用能力以及酶活性,可为单细胞蛋白发酵提供优良菌株。     

一种简单高效的酵母单菌落 PCR 方法

目的:为快速简便地挑选出酿酒酵母重组克隆,探索建立一种经济、直接、高效的酵母单菌落 PCR 方法.方法:以 Leu2MX6基同重组或重组质粒转化得到的酵母突变菌为材料,分别采用传统的提取基组或质粒的方法、煮沸法及化学试剂处理法等制备 PCR 模板进行重组克隆鉴定,并对6种 PCR 模板制备方法的效果进行比较与分析;对加热提取法进行优化并进行重组子的提取和验证.结果与结论:直接以1 mm2单克隆菌株95℃处理5 min 后的酵母菌落水悬浮液为模板进行单菌落 PCR,是一种简单高效的酵母重组克隆鉴定方法.该方法能弥补传统方法的不足,且简便快速、结果稳定,可作为筛选和鉴定阳性克隆的有效手段.同时,这种单菌落 PCR 法也可应用重组毕赤酵母的阳性克隆筛选.     

中条山野生葡萄产香酵母的筛选及其混菌发酵对葡萄酒香气成分的影响

【背景】产香酵母可赋予葡萄酒独特的香气,因此,分离筛选优良产香酵母对酿造具有地域风味的特色葡萄酒具有重要意义。【目的】从中条山野生葡萄中筛选产香酵母,进行种群鉴定和生理生化特性研究,并将其应用于葡萄酒发酵过程,研究其对葡萄酒香气成分的影响。【方法】采用稀释涂布平板法从中条山野葡萄中分离筛选酵母菌,对其进行分子生物学鉴定。优选其中具有显著香气的产香酵母,与酿酒酵母F15进行混合发酵,采用气相色谱质谱联用(gas chromatograph-mass spectrometer,GC-MS)对香气成分进行分析,采用半定量法测定香气成分含量。【结果】共分离获得各种菌株13株,26S rRNA基因D1/D2区序列分析表明它们分布于Issatchenkia、Torulaspora、Pichia、Saccharomyces和Rhodotorula等5个不同属内。优选其中一株香气较为浓郁的酵母菌株Issatchenkia orientalis strain XS-6开展研究,结果发现该菌株最高耐受乙醇浓度为8%,最高耐受NaCl浓度为6%,最适生长温度为38℃。与酿酒酵母F15混菌发酵的葡萄酒中共检测出31种香气成分。香气物质总含量较单菌发酵增加19.8%,其中11种香气成分含量增加明显,尤其是具有玫瑰香气的苯乙醇。醇类与酯类物质含量较单菌发酵增加19.6%,并发现了香草酸乙酯(ethyl vanillate)、邻苯二甲酸二丁酯(dibutyl phthalate)等7种新的酯类物质。【结论】产香酵母XS-6对乙醇、NaCl、温度等具有良好的耐受性,而且与酿酒酵母F15混菌发酵对西拉葡萄酒香气成分具有明显的影响,可能在改善葡萄酒风味方面具有潜在的应用价值。     

芝麻香型白酒大曲中酵母菌的分离与鉴定

为了开发利用白酒大曲中的酵母菌资源,采用稀释涂布平板法,从芝麻香型白酒大曲中分离得到15株酵母菌株。利用26S rRNA基因序列分析技术对其进行分类鉴定。结果表明,其中6株酵母菌为酿酒酵母（Saccharomyces cerevisiae）,6株为库德里阿兹威氏毕赤酵母（Pichia kudriavzevii）,3株为热带假丝酵母（Candida tropicalis）;并对代表菌株Y1、Y2及Y4进行了形态观察;最后通过随机扩增多态性DNA标记（random amplified polymorphic DNA, RAPD）对分离得到的酵母菌进行分型,表明6株酿酒酵母分属于5个株型,6株库德里阿兹威氏毕赤酵母分属于5个株型,3株热带假丝酵母分属于3个株型。     

侗族传统发酵肉的微生物特性

目的：对侗族传统发酵肉的微生物生态系的构成进行分析。方法：稀释平板法,结果：乳酸细菌在发酵30d达到最大值,4个处理的logCFU/g值均在8.2以上;280个MRS平板分离物中,米酒乳杆菌占37.91%,片球菌占20.71%。MSA平板培养物在发酵全过程中,均呈上升趋势;60d后,4个处理的logCFU/g值平均为5.5,增加了2.0;腐生葡萄球菌、肉葡萄球菌和木糖葡萄球菌是其优势菌群。酵母菌的数量在发酵过程中未见明显增加,发酵完毕,菌数的logCFU/g值在5.7左右,鉴定结果表明主要是德巴利酵母和球拟酵母。革兰阴性的肠细菌群发酵50d后,其logCFU/g值在2.0以下,结论：对发酵肉品微生态系的不断了解和肉品发酵技术不断完善,将为人们提供新颖、营养、安全的食品。     

Molecular events associated with acquisition of heat tolerance by the yeast Saccharomyces cerevisiae

Abstract: The heat shock response is an inducible protective system of all living cells. It simultaneously induces both heat shock proteins and an increased capacity for the cell to wisthstand potentially lethal temperatures (an increased thermotolerance). This has lead to the suspicion that these two phenomena must be inexorably linked. However, analysis of heat shock protein function in Saccharomyces cerevisiae by molecular genetic techniques has revealed only a minority of the heat shock proteins of this organism having appreciable influences on thermotolerance. Instead, physiological perturbations and the accumulation of trehalose with heat stress may be more important in the development of thermotolerance during a preconditioning heat shock. Vegetative S. cerevisiae also acquires thermotolerance through osmotic dehydration, through treatment with certain chemical agents and when, due to nutrient limitation, it arrests growth in the GI phase of the cell cycle. There is evidence for the activities of the cAMP-dependent protein kinase and plasma membrane ATPase being very important in thermotolerance determination. Also, intracellular water activity and trehalose probably exert a strong influence over thermotolerance through their effects on stabilisation of membranes and intracellular assemblies. Future investigations should address the unresolved issue of whether the different routes to thermotolerance induction cause a common change to the physical state of the intracellular environment, a change that may result in an increased stabilisation of cellular structures through more stable hydrogen bonding and hydrophobic interactions.     

Effect of sodium azide on heat-shock resistance in Saccharomyces cerevisiae and Debaryomyces vanriji yeasts]

The pretreatment of Saccharomyces cerevisiae and Debaryomyces vanriji with sodium azide was found to induce thermotolerance in both yeasts, whereas sodium azide used in combination with heat shock enhanced the thermotolerance of S. cerevisiae and substantially decreased the thermotolerance of D. vanriji. It is suggested that the different responses of the yeasts to sodium azide during heat shock are due to the different functional organizations of their mitochondrial apparatus.     

Thermotolerance is independent of induction of the full spectrum of heat shock proteins and of cell cycle blockage in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae are known to acquire thermotolerance in response to the stresses of starvation or heat shock. We show here through the use of cell cycle inhibitors that blockage of yeast cells in the G1, S, or G2 phases of the mitotic cell cycle is not a stress that induces thermotolerance; arrested cells remained as sensitive to thermal killing as proliferating cells. These G1- or S-phase-arrested cells were unimpaired in the acquisition of thermotolerance when subjected to a mild heat shock by incubation at 37 degrees C. One cell cycle inhibitor, o-phenanthroline, did in fact cause cells to become thermotolerant but without induction of the characteristic pattern of heat shock proteins. Thermal induction of heat shock protein synthesis was unaffected; the o-phenanthroline-treated cells could still synthesize heat shock proteins upon transfer to 37 degrees C. Use of a novel mutant conditionally defective only for the resumption of proliferation from stationary phase (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987) indicated that o-phenanthroline inhibition produces a stationary-phase arrest, a finding which is consistent with the increased thermotolerance and regulated cessation of proliferation exhibited by the inhibited cells. These findings show that the acquired thermotolerance of cells is unrelated to blockage of the mitotic cell cycle or to the rapid synthesis of the characteristic spectrum of heat shock proteins.     

The absence of direct relationship between the ability of yeasts to grow at elevated temperatures and their survival after lethal heat shock

The study of the growth of the yeasts Rhodotorula rubra, Saccharomyces cerevisiae, and Debaryomyces vanriji at elevated temperatures and their survival after transient lethal heat shock showed that the ability of these yeasts to grow at supraoptimal temperatures (i.e., their thermoresistance) and their ability to tolerate lethal heat shocks (i.e., their thermotolerance) are determined by different mechanisms. The thermotolerance of the yeasts is suggested to be mainly determined by the division rate of cells before their exposure to heat shock.     

Induction of barotolerance by heat shock treatment in yeast

In Saccharomyces cerevisiae, heat shock treatment provides protection against subsequent hydrostatic pressure damage. Such an induced hydrostatic pressure resistance (barotolerance) closely resembles the thermotolerance similarly induced by heat shock treatment. The parallel induction of barotolerance and thermotolerance by heat shock suggests that hydrostatic pressure and high temperature effects in yeast may be tightly linked physiologically.     

Arsenic oxide-induced thermotolerance in Saccharomyces cerevisiae.

The growth response of Saccharomyces cerevisiae to arsenite and arsenate and the relationship between the enhancement of heat shock protein (hsp) synthesis caused by these arsenic oxides and thermotolerance are reported. Arsenite and arsenate transiently inhibited cell growth and overall protein synthesis; arsenate enhanced the synthesis of the 42-, 74-, 84-, and 100-kilodalton hsps, whereas arsenite enhanced synthesis of only the 74-kilodalton hsp. The induction of these hsps reached a maximum 45 min following metal oxide treatment and then declined. A delayed thermotolerance peaked 4 h after metal oxide addition, at which time cell growth and protein synthesis were recovering. These data show that the arsenate- and arsenite-induced thermotolerance in S. cerevisiae cells does not appear to be causally related to either hsp synthesis or cell cycle arrest.     

Chemical Induction of Stress Proteins Does Not Induce Splicing Thermotolerance under Conditions Producing Survival Thermotolerance

Cell survival during a severe heat stress can be enhanced when heat shock proteins are induced prior to the severe heat treatment. Induction can be accomplished either by heat or chemical treatments. The increase in survival at these severe elevated temperatures after pretreatment has been referred to as thermotolerance, which we now refer to as survival thermotolerance. It has also been shown previously that mild heat treatment allows splicing in cells subjected to a severe heat treatment, now referred to as splicing thermotolerance. The experiments shown here demonstrate that even though chemical induction of the heat shock proteins leads to survival thermotolerance, this same treatment does not induce splicing thermotolerance. These are the first results that demonstrate at least two distinct aspects of thermotolerance.     

Thermotolerance is developmentally dependent in germinating wheat seed

During the initial 9 to 12 hours of imbibition, the imbibing wheat (Triticum aestivum L.) seed was found to exhibit substantial tolerance to high temperature relative to later times of imbibition. Tolerance was assessed by seed viability and seedling growth. This initial high temperature tolerance gradually declines with increasing time of seed imbibition. A range of 2 hour heat pretreatments (38-42°C) prior to imposition of a 2 hour heat shock (51-53°C) during this same 9 to 12 hour interval was unable to increase survival or seedling growth over that of seed that did not receive a pretreatment. However, after 9 to 12 hours of imbibition the pretreatment provided both increased survival and increased seedling growth, measured 120 hours later, i.e., classical thermotolerance could be acquired. This response is called a `thermotolerance transition.' Isolated embryos responded in a similar manner using a 2,3,5-triphenyltetrazolium chloride assay for viability determination following heat treatments. The high temperature tolerance during early imbibition indicates that the thermotolerance transition involves the loss of an existing thermotolerance coincident with acquiring the ability to become thermotolerant following heat pretreatment. Despite the inability to acquire thermotolerance, heat shock protein synthesis was induced by heat shock immediately upon imbibition of wheat seed or isolated embryos. Developmentally regulated heat shock proteins of 58 to 60, 46, 40, and 14 kilodaltons were detected at 1.5 hours of imbibition following heat shock, but were absent or greatly reduced by 12 hours. Constitutive synthesis of 70 and 90 kilodalton hsp groups appeared to be greater at 1.5 hours of imbibition than at 12 hours of imbibition.     

Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp 104 and in the absence of protein synthesis.

Acquisition of thermotolerance in response to a preconditioning heat treatment at 40 degrees C was studied in mutants of the yeast Saccharomyces cerevisiae lacking a specific heat shock protein or the ability to synthesize proteins at 40 degrees C. A mutant carrying a deletion of heat shock protein hsp 104 and the corresponding wildtype strain were both highly sensitive to heat stress at 50.4 degrees C without preconditioning but both acquired almost the same level of thermotolerance after 60 min of preconditioning. Both strains showed equal induction of trehalose-6-phosphate synthase and accumulated equal levels of trehalose during the treatment. The conditional mutant ts--187 synthesized no proteins during the preconditioning heat treatment but nevertheless acquired thermotolerance, albeit to a lesser degree than the corresponding wildtype strain. Induction of trehalose-6-phosphate synthase and accumulation of trehalose were reduced to a similar extent. These results show that acquisition of thermotolerance and accumulation of trehalose are closely correlated during heat preconditioning and are modulated by protein synthesis but do not require it.