野生酿酒酵母耐受性分析

对野生酿酒酵母(Saccharomyces cerevisiae)的工业胁迫条件,包括高浓度酒精、高渗透压、高温、高糖浓度和营养饥饿的耐受性进行了分析.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C242和G2257,耐受性差的有C190和N5254.     

酿酒酵母SOD1、SOD2基因缺失对胁迫耐受性的影响

以sod1Δ、sod2Δ、sod1Δsod2Δ酿酒酵母基因缺失菌株为遗传材料,采用休止细胞梯度生长法,分析SOD1和SOD2基因缺失对高温、乙醇毒性、高渗透压、高盐、乙酸毒性及营养饥饿胁迫条件耐受性的影响。结果显示,与野生型菌株相比,sod1Δ菌株对高温、高渗透压和乙酸胁迫的耐受性降低;sod2Δ菌株耐受性无明显变化;sod1Δsod2Δ双缺失菌株对高温、乙醇毒性、乙酸毒性、高渗透压和高盐的耐受性均下降,表明酵母超氧化物歧化酶基因与多种胁迫耐受性密切相关。     

香格里拉本土葡萄酒酵母的筛选及其胁迫耐受性分析

本文对香格里拉葡萄酒产区葡萄果实及土壤中分离获得76株酵母菌株进行序列测定和生理特征分析。结果表明,这些菌株在亲缘关系上可归类于9个属10个种,其中优势菌株为酿酒酵母(Saccharomyces cerevisiae),占5 2.6%,其次是巴布维红酵母(Rhodosporidium babjevae)和葡萄酒有孢汉生酵母(Hanseniaspora vineae),分别占22.4%和7.9%。利用改良模拟葡萄汁培养基对菌株进行胁迫耐受性分析发现,酿酒酵母菌株CZ11和BZL12具有较高的酒精度、高糖和低温耐受性,具有冰酒发酵应用潜力。     

高产多糖干白葡萄酒酵母的初步筛选及其发酵特性研究

试验采用苯酚-硫酸法对120株酿酒酵母属（Saccharomyces cerevisiae）进行了菌株多糖产率的初步筛选，得到了3株多糖产率高于工业对照菌株的酿酒酵母属酵母，通过小型发酵实验对3株酿酒酵母的发酵力、耐低温能力、耐S02能力、耐乙醇的能力进行了测定，其中NE-18菌株符合干白葡萄酒生产的需求，而W-15和NX-4在发酵能力或耐受性上不能满足白葡萄酒生产的需求。     

TOR1基因缺失对酿酒酵母耐受性的影响

酿酒酵母是最常见且应用最为广泛的酵母菌种,是以糖质和淀粉质为原料的乙醇发酵最经典的菌株。在发酵过程中,有很多不可避免的胁迫环境如高温条件、高渗条件等出现,这些胁迫会阻碍细胞生长并降低细胞的发酵能力,给发酵行业带来一定的经济损失。因此,为改善菌种的耐受性,该研究主要以实验室现有菌株AY12a为亲本菌株,URA3基因作筛选标记,通过胞内同源重组,实现TOR1基因的敲除,最终成功构建突变株AY12a-tor1Δ。对酵母进行耐受性的测定,发现AY12a-tor 1Δ具有一定的耐高温性能,在高渗条件下也有一定的耐受性,同时具有一定的氧化环境耐受性。同时将突变株与AY12a进行模拟白酒发酵(玉米浓醪发酵),并对发酵完成后的酒度、残糖、48 h细胞存活率、CO 2失重及发酵时间进行测定。发酵数据显示突变株AY12a-tor 1Δ乙醇产量有所上升,残糖含量下降,48 h细胞存活率没有下降,发酵时间有所延长。     

适应性进化提高酿酒酵母糠醛耐受性及其机理研究

燃料乙醇的原材料多样、对环境污染小,是一种真正意义上的可再生绿色能源。为了加大生物质能源的工业化进程,并克服预处理过程中产生的抑制剂对酵母的抑制作用,本研究以酿酒酵母（Saccharomyces cerevisiae） CEN. PK 1B-7D为出发菌株进行适应性进化培养,得到了对糠醛耐受性高且生长延滞期明显缩短的进化菌株CEN. PK 1B-7D 385。进一步比较原始菌株和进化菌株在4 g/L糠醛条件下细胞膜的通透性、膜电位变化情况和细胞内活性氧含量（ROS）。结果发现,相对于原始菌株,碘化丙啶（PI）染色细胞数降低了45.2%,去极化细胞数量减少了66.7%,说明在糠醛胁迫下进化菌株的细胞膜更加完整。同时,进化菌株胞内ROS含量从原始菌株的9.1%降低为6.5%,说明在糠醛胁迫下进化菌株胞内ROS有所下降。结合生物量、乙醇产量、细胞膜通透性及膜电位等诸多因素,进化菌株CEN. PK 1B-7D 385对糠醛的耐受性较原始菌株有明显提升。     

葡萄酒发酵优良酵母的筛选、鉴定及应用

该研究以商业酿酒酵母（Saccharomyces cerevisiae）F15为对照,通过测定产乙醇性能、产酯性能及絮凝特性从宁夏葡萄酒产区的土壤、葡萄中分离筛选优良酵母菌株,采用分子生物学技术对其进行菌种鉴定,并对其耐受性及其在葡萄汁中的发酵性能进行研究。结果表明,筛选得到两株优良酵母菌株,编号为J-1、J-3,经鉴定,均为酿酒酵母（Saccharomyces cerevisiae）。这2株菌株生长特性良好,均具有较好的耐受性,均可耐受pH值3.0、体积分数14%的乙醇,300 mg/L SO2,400 g/L葡萄糖,且菌株J-1的耐受性更好。菌株J-1的发酵速度与商业酿酒酵母F15相当;与商业酿酒酵母F15发酵酒样相比,酿酒酵母J-1发酵酒样中残糖含量较低（4.48 g/L）,挥发性香气物质增加2种,且酯类物质含量较高,花香和果香比较浓郁,口感较好。综上,酿酒酵母J-1具有较好的酿造特性,在葡萄酒工业上具有一定的应用潜力。     

优良酿酒酵母菌的发酵性能研究

从甘肃武威产区美乐葡萄中筛选出3株优良酿酒酵母菌,对其发酵性能进行了比较.测定了3株酿酒酵母菌和对照菌株在葡萄汁小瓶发酵时的CO2失重量、产酒精能力和残糖量,并对其在酒精耐受性、SO2耐受性、高糖耐受性、酸耐受性、受温度变化的影响等各方面的指标进行了综合比较.结果显示菌株NJZGM1的综合性能最佳,具有应用于我国地区特色葡萄酒生产的潜能.     

一株有氧条件下高产乙醇酵母的筛选、鉴定及其生物学特性研究

采用平板涂布法从老白干香型大曲中获得一株有氧条件下高产乙醇酵母菌株,命名为YF1914。通过菌落形态、细胞显微结构、生理生化特性和26S rDNA D1/D2区方法对其进行鉴定,借助液体培养方式考察其乙醇耐受性、葡萄糖耐受性、乙酸耐受性、生长温度和pH等生物学特性。结果表明,该菌株为酿酒酵母(Saccharomyces cerevisiae)属于乙醇高耐受性酵母,耐受乙醇最高体积分数为18%,同时还具有较高的NaCl和葡萄糖耐受性,最高耐受质量分数分别为15%和80%,在温度为20~50 ℃和pH1~10能够生长,具有宽广的温度和pH适应性。综上,该酵母这些优良特性有利于其在未来白酒酿造方式变化中发挥作用。     

TPK3基因敲除对酿酒酵母耐受性的影响

酿酒酵母是乙醇发酵时常用的微生物,在发酵过程中会受到高温、乙醇、渗透压、乙酸等胁迫环境的影响。为增强酵母对胁迫环境的耐受,该试验以AY12a为出发菌株,利用胞内同源重组,敲除编码蛋白激酶A（protein kinase A,PKA）催化亚基的TPK3基因,构建TPK3基因敲除菌株AY12a-2。利用敲除TPK3基因的分子操作来调控PKA活性,以此来提升酵母耐受性。耐受性测定结果表明,该菌株的耐热击性能、耐乙酸性能及耐盐性明显优于出发菌株。发酵数据表明,AY12a-2的葡萄糖消耗量增加及CO2的失重量提升34%,乙醇产量增长了17.8%。     

Development of a “Stress Model” Fermentation System for Fuel Ethanol Yeast Strains

During industrial scale fuel ethanol fermentations, yeast encounters a multitude of stress factors that impose constraints on growth and fermentative metabolism. These stresses include high sugar concentration, elevated temperature, high ethanol concentrations, low external pH and the weak organic acids lactic and acetic. Yeast strains which are tolerant to these stresses and able to synthesize high ethanol concentrations in their presence would be most desirable for use in industrial scale fuel ethanol production. In this study, a “stress model” fermentation system was developed as a tool to screen candidate yeast strains for relative stress resistance. The stress model was designed on the basis that the degree of ethanol produced by a particular strain would be indicative of the stress resistance of that particular strain. Eight strains of Saccharomyces cerevisiae, each with different backgrounds and fermentative capabilities, were screened for relative stress resistance using the stress model. The results obtained indicate that the sum of the stress factors in the stress model exceeded the tolerance level of most of the strains screened (approximately 40%). Two strains in particular, J006 and A007, displayed superior fermentative performance and produced significantly (P > 0.01) higher final ethanol concentrations when compared to the other strains.     

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。     

Isolation of vancomycin-resistant Enterococcus faecium from Italian cheeses

Several industrial and artisan Italian cheeses were screened for the presence of vancomycin-resistant enterococci (VRE). One hundred and two strains, mostly belonging to the species Enterococcus faecium after species-specific PCR identification, were tested for antibiotic susceptibility by standard disc diffusion method and for resistance to vancomycin in liquid medium. About 25% of strains were resistant to both vancomycin and teicoplanin. PCR experiments showed that all resistant isolates harboured the vanA gene. Minimum inhibitory concentration (MIC) values of vancomycin for most of the vancomycin resistant E. faecium (VREF) ranged from 128 to 512 μg ml⁻¹(high level resistance). PCR fingerprinting of VREF indicated that multiple isolation of the same strains did not occur. The importance of finding VRE in cheeses is discussed.     

Lactococcus lactis, a bacterial model for stress responses and survival

The dairy organism, Lactococcus lactis, is continuously exposed to stress conditions generated during industrial processes. To identify the mechanisms that confer resistance to the lethal effects of oxygen and thermal stress, we isolated resistant strains by insertional mutagenesis. Mutated genes were identified and mutations were shown to confer resistance to multiple stresses (including non-selected stresses such as carbon starvation). Our results revealed that metabolic flux plays an important role in L. lactis stress response, and suggested that phosphate and guanine pools may be intracellular stress sensors. As previously shown, we also observed an increase of stress resistance during the stationary phase. We have evidence that stationary phase actually initiates very early during growth. Taken together, these data show that the stationary phase is a very complex system with multiple participants interacting altogether. These results reinforce the idea of the interdependence of stress response and the intimate relation between metabolic flux and stress responses in L. lactis.     

Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress

Many of the considerable abilities of Listeria monocytogenes to persist and grow in a wide range of adverse environmental conditions are thought to be at least partly under the control of the alternative sigma factor (sigmaB), encoded by the sigB gene. However, little is known about the role of this master regulon in the impressive ability of Listeria to persist and grow under conditions of alkaline pH. In this study, Northern blot analysis of parent Listeria mRNA revealed that alkali adaptation (pH 9.5 for 1 h) significantly increased the expression of sigB-derived mRNA. The study included a comparison of the relative survival of mid-exponential populations of adapted and nonadapted parent type (sigmaB expressing) and mutant (not sigmaB expressing, deltasigB) Listeria strains during subsequent alkaline (pH 12.0), osmotic (25% NaCl, wt/vol), or ethanol (16.5%) stress. Alkali-adapted parent strains were more resistant to pH 12.0 than were adapted deltasigB type strains, but both alkali-adapted parent and deltasigB strains were more resistant to pH 12.0 than were nonadapted strains. Alkali-adapted parent strains were more resistant to osmotic stress than were adapted deltasigB type strains. No significant differences in viability were observed between alkali-adapted parent and deltasigB strains after ethanol stress, suggesting that cross-protection against osmotic stress is mediated by sigmaB whereas cross-protection against ethanol is sigmaB independent. Overall, alkali-induced cross-protection against osmotic and ethanol challenges may have serious implications for food safety and human health because such stress conditions are routinely used as part of food preservation and surface cleaning processes.     

Application of wild starter cultures for flavour development in pilot plant cheese making

A number of wild lactococci of dairy and non-dairy origin which have the ability to produce unusual new flavours in model systems were studied with regard to various characteristics important for cheese making. All strains were found to be non-lysogenic and resistant to phages affecting strains present in commercial starters. Since the overall acidifying activity of many potentially interesting strains is rather low, they were used in combination with commercial starters. Defined-strain starter cultures (DSS) were prepared, composed of a combination of wild strains together with industrial strains, and tested in real cheese making (Gouda-type) experiments. The population dynamics of DSS were studied to understand the behaviour of the selected wild strains in the cheese environment. Wild strains showed various interactions with industrial strains in a defined-strain starter culture. Some wild strains, which were able to grow well together with industrial strains could be used relatively easily for practical applications. Other strains appeared to inhibit the growth of the industrial strains, due to the production of bacteriocins. In many cases the bacteriocin appeared to be nisin. Sensory evaluation revealed that the selected wild strains also produced typical flavours in a real cheese environment which corroborated the results obtained in model systems. GC/MS data confirmed the results of sensory evaluations.     

Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses

Trehalose is believed to play an important role in stress tolerance in the yeast Saccharomyces cerevisiae. In this research, the responses to various environmental stresses, such as high ethanol concentration, heat, oxidative, and freezing stresses, were investigated in a strain with deletion of the NTH1, NTH2, and ATH1 genes encoding trehalases that are involved in trehalose degradation and the triple deletion strains overexpressing TPS1 or TPS2, both of which encode trehalose biosynthesis enzymes in S. cerevisiae. The contents of trehalose constitutively accumulated in the TPS1- and TPS2-overexpressing triple deletion strains were higher than that in the original triple deletion strain. High trehalose accumulation and growth activity were observed in the TPS2-overexpressing triple deletion strain after ethanol stress induction. The same was also observed in the triple deletion and the TPS1- and TPS2-overexpressing triple deletion strains after heat stress induction. In case of freezing stress, all the recombinant strains with high constitutive trehalose content showed high tolerance. However, in case of oxidative stress, trehalose accumulation could not make the yeast cells tolerant. Our results indicated that high trehalose accumulation can make yeast cells resistant to multiple stresses, but the importance of this accumulation before or after stress induction is varied depending on the type of stress.     

高产γ-氨基丁酸的双歧杆菌的筛选及对抑郁细胞模型的改善作用

为了筛选出具有缓解抑郁作用的菌株,本研究以94株双歧杆菌为研究对象,以胆盐水解酶活性、抗生素耐性、抗逆性、粘附性作为益生菌评价的基本指标;以高产γ-氨基丁酸(gamma-aminobutyric acid,GABA)作为潜在抗抑郁症菌株的筛选指标;最后通过体外抑郁细胞模型试验,研究菌株对抑郁细胞模型的改善作用,进而评价菌株改善抑郁的潜力。结果显示:13株双歧杆菌的胆盐水解酶活性较高,且都不具有种属特异性耐性之外的抗生素耐性。耐酸耐胆盐实验证明:双歧杆菌ZT3、84和95都具有较高的抗逆性能;粘附结果表明:双歧杆菌91、92、95和98具有较强的粘附能力,粘附能力分别为:19.34、33.77、23.23、20.46 CFU/Cell。13株双歧杆菌产GABA能力检测结果显示,双歧杆菌98和95的GABA的产量较高,分别为285.0和232.8 mg/L。抑郁细胞模型试验结果显示:高、中、低剂量高产GABA的双歧杆菌98都具有缓解CORT诱导的细胞损伤的能力,而只有高剂量的双岐杆菌95可以缓解CORT诱导的细胞损伤。由于双歧杆菌98、95具有安全的抗生素耐受范围,较高的酸、胆盐耐受性和粘附能力以及高产GABA和缓解细胞模型损伤的能力,因此,其可以作为潜在的改善抑郁症状的菌株。     

Growth, survival and inactivation of Pseudomonas aeruginosa and Staphylococcus aureus strains of various origin in the presence of ethanol

The influence of ethanol on the behaviour of Pseudomonas aeruginosa and Staphylococcus aureus strains was evaluated throughout this study. Strains of different origin were used: collection, clinical and industrial strains were selected. Concentrations of ethanol from 0 to 20% (v/v) were evaluated by automated optical density measurements and by enumeration. When growth conditions were observed, predictive microbiology models were used to assess quantitatively for the ethanol effect. Primary modelling of kinetics was performed to determine growth rate values; secondary modelling was performed on these growth rates as influenced by ethanol, and minimum inhibitory concentrations of ethanol were determined for each strain. Staphylococcus aureus strains were more resistant to ethanol than P. aeruginosa strains, in growth conditions as well as in inactivation conditions. Furthermore, clinical S. aureus strains were more resistant than the collection strain. The method was promising for management of microbiological safety in cosmetics.     

Drug resistance, plasmids, biotypes and susceptibility to bacteriophages of Salmonella isolated from poultry in Canada

Salmonella isolates from 295 layer and 294 broiler flocks in Canada were examined to determine resistance to antimicrobial agents, plasmid profiles, biochemical properties, and susceptibility to polyvalent bacteriophages. Except for the high number of strains resistant to spectinomycin (97.8%), the frequency of drug resistance of Salmonella isolates from layer flocks was low. None of 457 isolates from layer flocks was resistant to amikacin or ciprofloxacin, and less than 2% of the strains were resistant to cephalothin, chloramphenicol, cotrimoxazole, gentamicin, kanamycin, neomycin, nitrofurantoin, and/or polymyxin B. About 3% of the strains were resistant to ampicillin, carbenicillin and/or tetracycline, whereas 8% of the strains were resistant to sulfisoxazole. Salmonella anatum var. O15 + and S. typhimurium var. copenhagen strains were resistant to multiple antimicrobial agents. None of 1159 Salmonella strains from broiler flocks was resistant to amikacin, cephalothin, ciprofloxacin or polymyxin B, less than 1% of the strains were resistant to chloramphenicol, 2% were resistant to ampicillin, carbenicillin and/or chloramphenicol; 5–7% were resistant to the aminoglycosides gentamicin, kanamycin and/or neomycin; 6% were resistant to nitrofurantoin; 10% to tetracycline; 14% to sulfisoxazole; and 99% to spectinomycin. A high percentage of S. binza, S. anatum var. O15 +, S. schwarzengrund and S. heidelberg strains were resistant to antimicrobial agents. Some of the single or multiple resistances were encoded by conjugative plasmids or by plasmids that were thermosensitive for transfer. Eight percent of S. heigelberg strains did not produce hydrogen sulfide. Ninety-seven percent of the Salmonella strains were susceptible to the lytic effect of polyvalent bacteriophages.