两阶段pH控制方式提高富硒产朊假丝酵母的性能

为了提高富硒产朊假丝酵母的性能,本文在摇瓶培养和分批培养的基础上,考察了不同pH控制方式对流加培养条件下富硒产朊假丝酵母细胞生长、谷胱甘肽（GSH）合成和有机硒转化的影响,结果发现：在12 h将pH从3.5切换至5.5（pH 3.5→5.5）的两阶段pH控制方式最有利于富硒产朊假丝酵母胞内GSH和有机硒含量的提高。在pH 3.5→5.5条件下,富硒产朊假丝酵母胞内GSH含量、有机硒含量和有机硒转化率分别达到最大值13.09 mg/g、1.88 mg/g和94.69%。通过对酵母胞内GSH合成关键酶活性、氧化还原酶活性和能量代谢物质ATP水平进行测定,发现pH 3.5→5.5两阶段pH控制方式提高了富硒酵母胞内过氧化氢酶活性,降低了丙二醛含量,为酵母进行有机硒富集与转化以及GSH合成与积累提供了合适的氧化还原环境,并最终提高了富硒酵母的性能。     

高渗环境假丝酵母胞内甘油和海藻糖代谢研究

研究高渗环境中假丝酵母(T酵母)细胞内海藻糖和甘油的代谢情况。利用微波破碎法这一传统的物理破壁法来提取T酵母细胞内甘油和海藻糖,并通过高效液相色谱仪测定细胞内甘油和海藻糖的含量。经过高效液相色谱测定,可以得到不同盐浓度下T酵母胞内甘油和海藻糖含量。T酵母在高渗环境中通过对甘油和海藻糖的积累,同时阻止甘油的外排作用来抵抗外界环境对其生长的影响。     

产甘油假丝酵母两种转化方法的比较

为了研究产甘油假丝酵母高产甘油和耐高渗的机制,以Zeocin作为选择标记,考察和比较了两种转化方法,即醋酸锂法和电转化法的转化效果,以建立简单有效的产甘油假丝酵母转化体系。结果表明,细胞生长时期是影响转化率的关键因素。在OD600约为1.3时制备感受态细胞,在电压为1.5 kV时进行电击,可获得较高转化率,为每微克DNA 139个转化子。在OD600约为1.0时制备感受态细胞,醋酸锂预处理细胞1 h,获得的转化率为每微克DNA 154个转化子。综合考虑,醋酸锂法更适合于产甘油假丝酵母的转化。     

弱酸胁迫提升富硒产朊假丝酵母性能及其作用机理

以一株耐亚硒酸钠的产朊假丝酵母为研究对象,考察了该菌株在不同p H环境下的富硒和谷胱甘肽(GSH)合成能力及抗氧化性能。结果表明,在弱酸胁迫(p H3.5)条件下,富硒产朊假丝酵母胞内有机硒和GSH含量均达到最高水平,分别为1.08 mg/g和18.48 mg/g。通过对酵母胞内γ-谷氨酰半胱氨酸合成酶、过氧化氢酶、超氧化物歧化酶的活性以及丙二醛含量进行测定,发现弱酸胁迫不仅有利于增强GSH的合成能力,也有助于提高酵母细胞的抗氧化能力。该研究结果为发酵法制备高性能富硒产朊假丝酵母提供了可行的技术参考。     

产甘油假丝酵母酸性磷酸酯酶突变株的选育及其性质研究

运用化学诱变法从高产甘油的工业生产菌株产甘油假丝酵母（Ｃａｎｄｉｄａｇｌｙｃｅｒｏｌｇｅｎｅｓｉｓ）ＷＬ２００２－５（Ｈ－）筛选获得两类酸性磷酸酯酶突变株．一类为阻遏型酸性磷酸酯酶缺失突变株;另一类为对磷调节不敏感的部分去阻遏调节突变株,并研究了它们的发酵性能     

安全酵母Candida glycerinogenesis食品级甘油合成关键基因的研究

为了研究酵母甘油合成关键酶基因在食品级甘油合成中的重要作用,以产甘油假丝酵母基因组为模板,设计引物,克隆获得甘油合成关键酶基因3-磷酸-甘油脱氢酶基因(CgGPD),并在大肠杆菌中通过tac启动子串联表达来自酿酒酵母的3-磷酸甘油酯酶基因(ScGPP2)。经IPTG诱导培养后,3-磷酸-甘油脱氢酶(CgGPD)的比酶活达到了60mU/mg。在30%的葡萄糖浓度下,经过48h的摇瓶发酵,可合成甘油2.52g/L。表明3-磷酸甘油酯酶在甘油合成过程中起了关键作用,过表达CgGPD基因有助于食品级甘油大量合成,从而降低下游分离纯化的难度,可从分子水平进一步提高甘油的产量。     

镁离子浓度对杜氏盐藻生长及甘油、蛋白质积累的影响

研究了镁离子浓度对杜氏盐藻生长及甘油、蛋白质累积的影响.在28℃、pH值为8、光强为5 000 Lx-6 000 Lx、定时振荡的条件下,实验最适宜盐藻生长的镁离子的浓度为7 mmol/L;生长稳定期对盐藻胞内甘油的积累作用很小;当镁离子浓度为3 mmol/L时,盐藻胞内甘油含量最高,可达到167.449 mg/L;当镁离子浓度为7 mmoL/L时,盐藻细胞可溶性蛋白含量最高,可达到346.664 mg/L.     

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。     

酸适应海德尔堡沙门氏菌对环境胁迫耐受性分析

目的：研究海德尔堡沙门氏菌（Salmonella heidelberg）低pH值酸适应性及酸适应对细胞其他胁迫环境耐受性的影响。方法：海德尔堡沙门氏菌在pH 5.5酸性培养基中生长2 h诱导酸适应性,以pH 3.0酸胁迫培养基及不同温度、NaCl、胆盐、消毒剂环境的存活率评价其耐受性。结果：pH 5.5酸适应2 h后,海德尔堡沙门氏菌在pH 3.0酸胁迫环境的存活率大幅提高。低pH值酸适应也使海德尔堡沙门氏菌在45、50 ℃高温条件下的存活率显著提高,对4 ℃冷藏环境的耐受性显著低于非酸处理菌株,但对－20 ℃冷冻环境的生存无影响。酸适应海德尔堡沙门氏菌对高渗、胆盐胁迫也表现良好的耐受性,5～10 g/100 mL NaCl溶液处理1 h,酸适应海德尔堡沙门氏菌的存活率大于60%,在1～10 g/100 mL胆盐溶液的存活率比对照组高3.8 倍。海德尔堡沙门氏菌酸适应性也使其对消毒剂的敏感性发生变化,酸适应性海德尔堡沙门氏菌在10～25 mmol/L H2O2溶液的存活率显著高于对照菌株,最高达3.1 倍;但对NaClO、乙醇消毒剂的耐受性显著降低,高质量浓度下二者无显著差异。结论：海德尔堡沙门氏菌在低pH值酸性条件生长一段时间会诱导产生酸适应性,还会协同提高其对高温、高渗、胆盐、H2O2胁迫环境的交叉抗性;但对4、－20 ℃低温、NaClO、乙醇等杀菌、保藏措施敏感性提高。     

高表达MAL62基因对面包酵母耐高糖的影响

面包酵母(Saccharomyces cerevisiae)的抗逆性对于烘焙工业至关重要。以前期获得的耐冷冻酵母突变株B+MAL62为研究对象,测定其在高糖环境下的生长特性、形态特征、胞内海藻糖与甘油的积累以及产气的变化,并与市售高糖酵母进行对比。研究发现在质量分数为40%~60%的糖胁迫环境下,B+MAL62菌株的胞内海藻糖与甘油水平分别比对照菌株提升55. 03%~64. 27%与1. 2~1. 3倍,且高糖环境下B+MAL62具有更好的细胞形态稳定性,其产气速度以及最终产气量可优于市售高糖酵母。结果表明,麦芽糖酶编码基因MAL62高表达可增强面包酵母耐高糖能力。     

Impact of cold and cold-acid stress on poststress tolerance and virulence factor expression of Escherichia coli O157:H7

The effect of extended cold or cold-acid storage of Escherichia coli O157:H7 on subsequent acid tolerance, freeze-thaw survival, heat tolerance, and virulence factor (Shiga toxin, intimin, and hemolysin) expression was determined. Three E. coli O157:H7 strains were stressed at 4 degrees C in TSB or pH 5.5 TSB for 4 weeks. The acid (TSB [pH 2.0] or simulated gastric fluid [pH 1.5]) tolerance, freeze-thaw (-20 degrees C to 21 degrees C) survival, and heat (56 degrees C) tolerance of stressed cells were compared with those of control cells. The beta-galactosidase activities of stressed and control cells containing a lacZ gene fusion in the stx2, eaeA, or hlyA gene were determined following stress in TSB or pH 5.5 TSB at 37 degrees C and in the exponential and stationary phases. Cold and cold-acid stresses decreased acid tolerance (P < 0.05), with a larger decrease in acid tolerance being observed after cold stress than after cold-acid stress (P < 0.05). Cold stress increased freeze-thaw survival for all three strains (P < 0.05). Prior cold or cold-acid stress had no effect on virulence factor production (P > 0.05), although growth in acidic media (pH 5.5) enhanced eaeA and hlyA expression (P < 0.05). These results indicate that the prolonged storage of E. coli O157:H7 at 4 degrees C has substantial effects on freeze-thaw tolerance but does not affect subsequent virulence gene expression.     

Factors affecting the extreme acid resistance of Escherichia coli O157:H7

When stationary phase Escherichia coli O157:H7 cells were subjected to extreme acid shock (pH 2·0, 6 h, 37°C) cell survival was as great as 10%, but culture conditions greatly affected the acid resistance. Anaerobic cultures were more resistant to extreme acid shock if the glucose concentration of the growth medium was high, acids accumulated, and pH declined. By varying pH and acetate concentration, it was possible to demonstrate a high correlation (R²=0·86) between undissociated acetate and extreme acid resistance. Because dissociated acetate and extreme acid resistance were poorly correlated (R²<0·01), it appeared that the pH effects were being mediated via acetate dissociation. Propionate and butyrate were as effective as acetate, but formate, lactate, benzoate and the uncoupler, carbonylcyanide m -chlorophenylhydrazone (CCCP), were much less effective in promoting extreme acid-resistance. Acetate, propionate, butyrate, benzoate and CCCP all decreased the intracellular pH of E. coli O157:H7, but the correlation between intracellular pH and extreme acid resistance was low (R²<0·01). Cultures grown aerobically only needed half as much acetate to induce extreme acid resistance as those grown anaerobically, and the addition of the reducing agent, cysteine, to anaerobic media made the stationary phase cells less responsive to acetate. An rpoS mutant of E. coli O157:H7 was at least 100-fold more sensitive to acid shock than the wild-type, and large amounts of acetate were needed to promote even a small increase in viability.     

Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer

The URA3 gene of Candida glycerinogenes WL2002-5, an industrial glycerol producer encoding orotidine-5'-phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein.     

Induction of the acid tolerance response in Leuconostoc mesenteroides ATCC 8293 by pre-adaptation in acidic condition

Leuconostoc mesenteroides is a commercially important lactic acid bacterium which is used as a starter culture in various fermentation processes. However, because of its sensitivity to acid stress, high cell-density culture has not been accomplished yet. Therefore, we investigated the effect of preadaptation of L. mesenteroides under mildly acidic conditions for resistance to normally lethal levels of acid stress. For this, the cells grown to the early-exponential phase at pH 7.0 were incubated at pH 6.5, 6.0, and 5.0 for 1 h, and then the survival rates of acid-adapted cells in pH 4.0 solution was determined. Acid-adapted cells at pH 5.0 exhibited maximum increase in tolerance, showing an increased survival of approximately 2,500 folds compared to the control (pH 7.0).     

甘油发酵工艺的初步研究

以一种耐高渗透压的酵母为生产菌株,探讨了甘油发酵工艺对生产甘油的影响。结果表明:过低和过高的通气量都不利于甘油的生产,采用90L/h的通气量可获得较高的甘油得率;发酵液中pH的变化对甘油的生产影响显著,通过补加硫铵和自动调控pH,发酵时间大大缩短,甘油得率明显提高,达到了41.5％。     

拮抗菌罗伦隐球酵母的生长性能研究

本文研究了罗伦隐球酵母在麦芽汁中的生长性能,包括三角瓶培养及发酵罐培养的生长动态,结果表明,罗伦隐球酵母在麦芽汁培养基中能够快速生长,罗伦隐球酵母的生长没有明显的延滞期,三角瓶培养到第20h时,酵母细胞数目达到最高值,此后罗伦隐球酵母的生长进入稳定期;发酵罐培养时,从30h开始,逐步进入稳定期;罗伦隐球酵母在发酵罐中的生长会使发酵液的pH值降低。     

欧姆加热对嗜酸耐热菌的杀灭作用研究

本实验探讨了欧姆加热对嗜酸耐热菌的杀灭作用。利用自行设计的批式欧姆加热装置,对苹果汁中嗜酸耐热菌进行处理,分析了欧姆加热的电压、pH值、时间及加热体积等对杀菌效果的影响,并利用扫描电镜观察微生物细胞壁膜结构的变化,利用电导率仪和紫外吸收分光光度计测定菌悬液的成分变化。结果表明欧姆加热可以有效的杀灭苹果汁中的嗜酸耐热菌,杀菌率随电压、加热体积的升高而增大,随pH值的降低而增大。通过环境扫描电镜可观察到嗜酸耐热菌的表面出现凹陷和破损,电导率仪和紫外吸收分光光度计测定细胞内容物的溢出,据此推测欧姆加热造成了嗜酸耐热菌的"电穿孔"。     

Stress response of acid-shocked Cronobacter sakazakii against subsequent acidic pH, mild heat, and organic acids

This study was conducted to determine the resistance of acid-shocked Cronobacter sakazakii to environmental stresses. C. sakazakii pre-exposed to various pH levels was treated with acid stress (pH 3.06), heat stress (55°C), and organic acid stress, respectively. Overall, higher D-values were obtained in samples pre-exposed to acidic pH conditions (pH 3.06, 4.00, and 5.02) compared to a control (pH 7.20) when the samples were subsequently stressed. For 0.1 M acetic acid, the D-values of nonadapted C. sakazakii ATCC 29004 and ATCC 29544 were 19.69 and 15.49 h, respectively, whereas the D-values of acid-shocked C. sakazakii ATCC 29004 and ATCC 29544 by pre-exposure to pH 4.0 were 34.59 and 24.25 h, respectively. Acid adaptation of C. sakazakii by preexposure to acidic pH can enhance the resistance of cells against subsequent environmental stresses such as acidic pH, heat, and organic acids.     

Responses of Listeria monocytogenes to acid stress and glucose availability monitored by measurements of intracellular pH and viable counts

Physiological aspects of the response of Listeria monocytogenes to acidic conditions and effect of glucose availability were studied by fluorescence ratio-imaging microscopy (FRIM) as compared with traditional viable counts. Three types of experiments were conducted: (i) static with measurements of intracellular pH (pHi) at extracellular pH (pHo) values ranging from pH 3.0 to 6.0 at 0.5 pH unit intervals; (ii) kinetic with monitoring of bacterial responses to changes in the pHo from the value of 6.0 to 4.0 or 3.0; (iii) survival experiments studying bacterial recovery in response to a shift to favourable conditions after a treatment at low pH. All the experiments were performed at three levels of glucose in the medium (0, 1, and 10 mM). Both survival and pHi were greatly affected by pHo and glucose availability with the highest values for CFU and pHi at highest glucose concentration and pHo values in the medium in all trials. A high correlation (R2 = 0.995) between pHi and CFU counts was observed. The pH gradient started to collapse at pHo 4 and below for trials with glucose in the medium and at pHo 5.5 and below without glucose. A recovery step was proposed after the apparently lethal treatment to assess cell viability by FRIM.