Beer with Reduced Ethanol Content Produced Using Saccharomyces cerevisiae Yeasts Deficient in Various Tricarboxylic Acid Cycle Enzymes

A collection of Saccharomyces cerevisiae strains deficient in the tricarboxylic acid cycle enzymes activities has been examined for the production of beer with reduced ethanol content. Strains deficient in fumarase and α‐ketoglutarate dehydrogenase encoded by the genes FUM1 (0.48%), KGD1 (0.42%) and KGD2 (0.48%) made non‐alcoholic beers with an alcohol content lower than 0.5% (v/v). The rest of the yeast mutants also gave rise to low‐alcoholic beers but with a slightly elevated ethanol concentration (mostly in the range of 0.57‐0.84% and 1.64% for the lip5 mutant). Low ethanol content was compensated by the considerable increase of organic acids (citrate succinate, fumarate, and malate). In addition, some of the mutants released high levels of lactic acid (144 fum1), 622 (kgd1) and 495 (kgd2) mg/L). Lactic acid protects beers against contamination and masks an unacceptable worty off‐flavour.     

Thermal Resistance of Saccharomyces cerevisiae in Pilsen Beer

The thermal resistance of S. cerevisiae (CMOJ896) was determined in Pilsen beer (pH = 4.28 ± 0.05; extract of original wort (EOW %) = 11.30 ± 0.08; percentage of alcohol by volume (% at 20°C) = 4.97 ± 0.05; total nitrogen content (mg/L) = 590 ± 37; bitterness units (BU) = 20.5 ± 1.3; carbonation of the beer (Vol. CO₂) = 2.89 ± 0.09; color (SRCM) = 3.3 ± 0.4). The flask method was used for an initial population of 1 × 10⁴ cells/mL. Decimal reduction times of D_47°C = 3.16 min, D_48°C = 2.65 min, D_49°C = 1.74 min and D_50°C = 0.68 min were obtained at the temperatures studied. Values of D_60°C = 0.01 min and z = 4.6° were obtained for this microorganism.     

螺旋藻啤酒生产工艺的研究

本文以大麦芽为主要原料，螺旋藻提取液为添加荆，研究优化螺旋藻啤酒的生产工艺，感官评定最佳工艺得到的酒体为优。     

固定化后期添加啤酒酵母菌降低啤酒中双乙酰含量的探讨

为探索用后期添加固定化酵母菌降低啤酒中双乙酰的工艺,通过单因素和正交试验,发现后期接入固定化啤酒酵母菌降低双乙酰含量的最佳工艺为:发酵温度为12℃,接入时间为发酵第16d,发酵再次接入固定化酵母菌的接种量为1.0%,此时双乙酰的量为0.083mg/L,啤酒口感达到9.2分。固定化的酵母菌可以重复利用3次,啤酒的双乙酰值和口感维持稳定。该研究表明利用固定化细胞的后期添加可以降低啤酒中的双乙酰含量。     

上面发酵高粱啤酒的工艺研究

高粱啤酒作为一种营养丰富、口感独特的无麸质酒精饮料,具有广阔的发展前景。为研制品质优良的高粱啤酒,本研究以高粱芽为原料,分别对糖化工艺对高粱汁中总还原糖和α-氨基氮量的影响;发酵工艺对高级醇含量的影响以及发酵后不同澄清剂对高粱啤酒的澄清效果和色度的影响进行了研究。结果表明:高粱汁制备的最佳糖化方法为倾出糖化法,糖化条件为:浸酶温度35℃,浸酶时间20 min,糊化温度90℃,糊化时间30 min,糖化温度65℃,糖化时间1 h,糖化后高粱芽汁的总还原糖含量为83.23 g/L,α-氨基氮含量适中,为180.8 mg/L。高粱啤酒的最佳发酵条件为:高粱芽汁浓度12°P、酵母菌接种量2.0×107个/m L、发酵温度16℃,发酵后啤酒中的高级醇含量为109.09 mg/L,该条件可有效降低高级醇含量。最佳的澄清剂为皂土,当添加比例为0.9%时,透光率达到90%,高粱啤酒的澄清效果最佳。在此工艺条件下,酿造出的上面发酵高粱啤酒澄清透明、色泽鲜亮、口感独特,高级醇含量适宜,是一种风味独特的新型酒精饮品。     

葡萄柚种子提取物对酿酒酵母抑制机理的研究

通过揭示葡萄柚种子提取物(GSE)诱导酿酒酵母凋亡的现象,探讨其对酵母抑制的机理。结果表明：GSE有效抑制酿酒酵母的最低质量浓度为0.13mg/mL。DAPI 和TUNEL 核酸荧光染色表明GSE 能使酿酒酵母细胞核浓缩并导致核裂及DNA 断链。ROS 检测表明GSE 能引起酿酒酵母细胞内活性氧大幅增加。     

乙醇脱氢酶I基因敲除的酿酒酵母重组菌构建的初步研究

本实验根据酿酒酵母乙醇代谢途径,构建一株低乙醇产量的酿酒酵母基因工程菌株,以满足人们对低醇啤酒的需要。利用抗性基因筛选基因敲除突变体的方法,通过引物L1和L2扩增潮霉素B基因(两翼与酿酒酵母同源),按常规醋酸锂法转化酵母细胞后,筛选标记与酵母adhI基因发生同源重组,得到一株ADHI酶活性降低的工程菌株。发酵实验结果表明,转化菌株乙醇含量平均值为1.8%(V/V),较原始菌株低了65%。说明转化菌株体内乙醇生成途径受到干扰。     

A vector system for efficient and economical switching of C-terminal epitope tags in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, one-step PCR-mediated modification of chromosomal genes allows fast and efficient tagging of yeast proteins with various epitopes at the C- or N-terminus. For many purposes, C-terminal tagging is advantageous in that the expression pattern of epitope tag is comparable to that of the authentic protein and the possibility for the tag to affect normal folding of polypeptide chain during translation is minimized. As experiments are getting complicated, it is often necessary to construct several fusion proteins tagged with various kinds of epitopes. Here, we describe development of a series of plasmids that allow efficient and economical switching of C-terminally tagged epitopes, using just one set of universal oligonucleotide primers. Containing a variety of epitopes (GFP, TAP, GST, Myc, HA and FLAG tag) and Kluyveromyces lactis URA3 gene as a selectable marker, the plasmids can be used to replace any MX6 module-based C-terminal epitope tag with one of the six epitopes. Furthermore, the plasmids also allow additional C-terminal epitope tagging of proteins in yeast cells that already carry MX6 module-based gene deletion or C-terminal epitope tag.     

酿酒酵母发酵生产S-腺苷甲硫氨酸工艺的优化

目的 优化酿酒酵母LS101发酵生产S-腺苷甲硫氨酸(SAM)的工艺.方法 用单因素实验法,通过测定SAM浓度、细胞浓度、胞内SAM含量以及胞外SAM浓度等参数来确定较为适合的工艺.结果 得到适合的培养基组成为:蔗糖10%～12%,L-甲硫氨酸0.4%,尿素1.5%～2%,酵母粉3%,甘氨酸0.1%,生物素4 mg/L.8 L发酵罐间歇分批补料发酵,培养54 h后SAM产量为3.9 g/L,细胞浓度达到42.2 g/L.结论 得到了优化的酿酒酵母LS101生产SAM发酵工艺,提高了SAM发酵水平.     

酿酒酵母工业菌株胁迫条件耐受性分析

对酿酒酵母（Saccharomyces cerevisiae）工业菌株胁迫条件，包括高浓度酒精、高渗透压、高温、营养饥饿、氧化胁迫、糠醛毒性的耐受性进行了分析，同时测定了抗生素G418对这些菌株的最低抑菌浓度。结果表明，所测定的酵母菌株对这些逆境条件的耐受性有明显差别，表现出良好耐受性的是6508和安琪酵母菌株，同时多倍性的酿酒酵母工业菌株的耐受性均比单倍性实验室菌株高。     

啤酒发酵中代谢工程的初步研究

将代谢工程理论应用于啤酒的发酵过程,通过构建啤酒发酵过程中酵母的代谢网络模型,对啤酒发酵过程进行代谢通量分析。研究不同压力下啤酒酵母代谢能力的变化,并对其进行了代谢通量分析,结果表明,高压可对酵母在啤酒发酵过程中的代谢产生一定的抑制作用。     

A Review of Phenotypes in Saccharomyces cerevisiae

A summary of previously defined phenotypes in the yeast Saccharomyces cerevisae is presented. The purpose of this review is to provide a compendium of phenotypes that can be readily screened to identify pleiotropic phenotypes associated with primary or suppressor mutations. Many of these phenotypes provide a convenient alternative to the primary phenotype for following a gene, or as a marker for cloning a gene by genetic complementation. In many cases a particular phenotype or set of phenotypes can suggest a function for the product of the mutated gene. © 1997 John Wiley & Sons, Ltd.     

低双乙酰酿酒酵母的DNA标记研究

对15株酿酒酵母菌株的外观发酵度、双乙酰生成和还原能力进行了比较,通过随机扩增多态性DNA(RAPD)和HSP150编码基因的长度多态性对不同酿酒酵母菌株的基因组DNA分子差异进行了分析。结果显示,菌株YZU-APK和SS-05的外观发酵度分别为77.2%和76.8%,双乙酰峰值分别为0.26mg/L和0.25mg/L,并在发酵第8d均降至0.09 mg/L。在46条随机引物中筛选出的2条引物P07和P42能够扩增出具有较好多态性的RAPD指纹图谱,可以将15个酵母菌株分成9个遗传型;对细胞壁热休克蛋白HSP150编码基因的PCR扩增,并根据扩增条带长度的不同可以将15株酵母分成6个遗传型。综合2种分析结果,并对低双乙酰酿酒酵母菌株YZU-APK和SS-05进行DNA分子标记,确定了其基因型分别是(P07:1,P42:1,hsp150:1)和(P07:1,P42:5,hsp150:2)。     

沙城产区酿酒酵母多样性研究

对沙城产区龙眼葡萄相关环境中分离筛选的酿酒酵母进行多样性研究。在连续3年(2008、2009、2010年)的实验中,共从葡萄园土壤、葡萄酒厂设备表面、接触过葡萄酒厂设备的葡萄汁和自然发酵过程中采集菌源样品227份,共分离得到1358株酵母菌。用5.8S-ITS区域RFLP方法进行分子水平的分类鉴定及赖氨酸培养基复筛,得到了270株酿酒酵母。再利用Interdelta PCR指纹图谱法将酿酒酵母区分为16类,其中土壤5类,自然发酵过程中第2、3、4期分别得到4类、10类和11类;酒厂设备表面3类;接触酒厂设备的葡萄汁3类。酿酒酵母的种类因样品采集时间、采集地点等不同有明显区别。自然发酵过程中得到的酿酒酵母被认为是本土酵母的可能性最大。     

PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae

Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra- or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat-generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5-fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome.     

酿造水对低度啤酒质量的影响——诠释红石梁的酿造用水

近来低浓度啤酒在中国较受欢迎,尤其在南方地区更为突出。但是随着原麦汁浓度的降低,啤酒的各种异杂味易露头,因此酿造用水的质量对低度啤酒的口感起着非常重要的作用。通过对酿造水的改良,可以解决低度啤酒质量中遇到的口感酸涩、非生物稳定性差等一些技术难题,提高啤酒质量。     

低产尿素黄酒酵母工程菌的酿造特性

为考察酵母工程菌在黄酒酿造过程中的发酵性能及其降低发酵液中尿素和氨基甲酸乙酯(ethyl carba-mate,EC)的能力,以前期构建的降低黄酒中尿素和EC效果最好的酵母工程菌N85^DUR1,2-c为研究对象,利用单因素试验考察黄酒发酵工艺对其降低发酵液中尿素和EC能力的影响,并对其在生产试验过程中的发酵性能进行研究。结果表明,酵母接种量、发酵温度以及麦曲添加量等工艺参数对工程菌N85^DUR1,2-c低产尿素和EC的性能没有明显的影响,且含量低于亲本菌株。50 kL生产试验表明,工程菌N85^DUR1,2-c所酿黄酒中理化指标含量正常,符合黄酒国标的要求。而N85^DUR1,2-c发酵液中尿素和EC的含量分别为(2.4±0.2)mg/L和(14.9±0.6)μg/L,较亲本菌株分别降低了90.7%和54.6%,且贮存过程中EC含量增加缓慢。说明酵母工程菌N85^DUR1,2-c在不改变黄酒优良品质的前提下,能够显著地降低发酵液中尿素的含量,可以从根源上减少黄酒中EC的积累,提高饮用...     

构建具有大麦脂转移蛋白1分泌能力的酿酒酵母

为了增强纯生啤酒的泡沫性能,从酿酒酵母表达质粒YEplac181出发,将大麦脂转移蛋白1(LTP1)成熟肽的编码序列置于酿酒酵母ADH1启动子(alcohol dehydrogenase promoter)和CYC1终止子(cytochrome C terminator)的调控下,构建大麦脂转移蛋白1的酿酒酵母表达质粒YEp181-KAMLC。通过酿酒酵母α-信息素信号肽的引导分泌,酿酒酵母表达的成熟大麦LTP1被分泌到发酵液中。对发酵液的检测表明,在发酵132h后LTP1的产量可达到29.45mg/L。     

酿酒酵母产乙醇脱氢酶发酵条件的研究

选择了一株酿酒酵母菌(Saccharomyces cerevisiae)As2.399,通过单因素和正交实验得到酿酒酵母产乙醇脱氢酶(Alcohol Dehydrogenase,ADH)的最优发酵条件。结果表明:产ADH的最优培养条件是温度为25℃、pH为5、通气量为250mL、接种量为7%,液体发酵培养中pH对ADH活性的影响最大。