Oenococcusoeni苹果酸-乳酸酶基因克隆及其在酿酒酵母中的表达

苹果酸-乳酸酶是苹果酸-乳酸发酵过程中负责苹果酸转化为乳酸的功能酶。在进行酒酒球菌SD2a的苹果酸-乳酸酶基因(mleA)克隆测序基础上,以PGK1强启动子和ADH1终止子为调控元件,以大肠杆菌-酵母菌穿梭质粒YEp352为载体,构建了重组表达质粒并转化酿酒酵母YS58。酵母转化子用SD/Ura平板筛选鉴定。斑点杂交检测表明目的基因mleA转化到受体菌中,SDSPAGE检测表明获得的转化子表达了约60kDa的目标蛋白。获得的转化子在添加了L苹果酸的培养基中培养4d;取培养液上清用HPLC检测L苹果酸及L乳酸含量,采用t检验进行差异显著性分析,结果表明mleA基因进行了功能性的表达,将L苹果酸转化成L乳酸,L苹果酸和L乳酸含量分别与对照差异极显著和显著,苹果酸的相对降低率平均为20.95%。在有选择压力条件下,重组质粒相对稳定,而在无选择压力条件下,传代培养10d后大约有65%的重组质粒丢失。     

酒类酒球菌mleP基因的克隆及其在酿酒酵母中的表达

苹果酸通透酶具有协助苹果酸 乳酸发酵 (MLF)的重要功能。以酒类酒球菌 (Oenococcusoeni)优良菌系Oenococcus Lee SD 2a的总DNA为模板 ,用PCR方法克隆到苹果酸通透酶基因mleP ,构建了重组质粒pBMmleP。序列分析表明克隆到的基因序列与已报道的序列同源性为 99%。为使目的基因在酿酒酵母中表达 ,以大肠杆菌 酿酒酵母穿梭质粒YEp35 2为载体 ,以PGK1强启动子和ADH1终止子为调控元件 ,构建了重组表达质粒YEpmleP ,并转化酿酒酵母 (Saccharomycescerevisiae)YS5 8。酵母转化子用含有亮氨酸、组氨酸和色氨酸的YNB平板筛选鉴定。获得的转化子在添加了L 苹果酸 (5g L)的培养基中培养 4d ;取培养液上清用HPLC检测 ,结果显示重组转化子YSP的培养液中L 苹果酸剩余含量均低于空载体转化子YS35 2 ,因此所得酵母重组转化子对苹果酸的转运能力有所提高     

Influence of phenolic acids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii

Aims: To determine the effect of several wine-associated, phenolic acids on the growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Methods and Results: Growth was monitored in ethanol-containing medium supplemented with varying concentrations of hydroxybenzoic acids (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acids) and hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids). Progressive inactivation was monitored in ethanol-containing phosphate buffer supplemented in a similar manner to the growth experiments. Hydroxycinnamic acids proved to be more inhibitory to the growth of O. oeni than hydroxybenzoic acids. On the other hand, some acids showed a beneficial effect on growth of Lact. hilgardii. p-Coumaric acid showed the strongest inhibitory effect on growth and survival of both bacteria. Conclusions: Most phenolic acids had a negative effect on growth of O. oeni, for Lact. hilgardii this effect was only noted for p-coumaric acid. Generally, O. oeni was more sensitive to phenolic acid inactivation than Lact. hilgardii. Significance and Impact of the Study: Eight wine-derived, phenolic acids were compared for their effects on wine lactic acid bacteria. Results indicate that phenolic acids have the capacity to influence growth and survival parameters. The differences found between phenolic compounds could be related to their different chemical structures.     

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni

AIMS: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions. METHODS AND RESULTS: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast-malolactic bacteria (MLB) coupling. Heat and protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that the inhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts either bacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grape juice and wine. CONCLUSIONS: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth and malic acid fermentation was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The individuation, characterization and exploitation of yeast proteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management of malolactic fermentation.     

葡萄酒生境对乳酸菌代谢的影响

在葡萄酒酿造中,为了提高其稳定性及质量,经常利用乳酸菌进行苹果酸.乳酸发酵.苹果酸一乳酸发酵一般自发进行,也可以接种乳酸菌.本文从酿酒酵母与乳酸菌的交互作用及酚类物质和酿酒工艺对乳酸菌的作用等方面进行了综述,讨论了葡萄酒生态环境对乳酸菌代谢的影响,为苹果酸一乳酸发酵的有效控制提供一些参考.     

不同培养基对酒酒球菌SD-2a存活率及膜脂肪酸组分的影响

[目的] 为获得高效的葡萄酒乳酸菌发酵剂,本文研究了3种具有不同pH缓冲能力的培养基对酒酒球菌接种存活率、冻干存活率及细胞膜脂肪酸组分的影响.[方法]采用平板计数法测定菌体的接种存活率、冻干存活率;并采用GC/MS色谱方法测定收获菌体细胞膜脂肪酸组分.[结果]实验结果表明,没有添加苹果酸的ATB培养基,其pH缓冲能力弱.分别与FMATB和MATB培养基相比,ATB培养基培养获得的菌体,其接种模拟酒培养基后的存活率提高了20.3%和40.2%,其冷冻干燥存活率提高了48.5%和68.3%,其细胞膜中C19cyc11的相对含量提高了10.0%和36.8%,其细胞膜U/S值提高了20.4%和45.2%.[结论]本文推测ATB培养基培养所得菌体,由于自我酸胁迫反应,增强了其对葡萄酒胁迫因素及冷冻干燥的抗性,而该反应与菌体细胞膜脂肪酸组分的变化密切相关.故ATB培养基更适合于酒酒球菌SD-2a发酵剂的制备.     

Purification and partial characterization of Oenococcus oeni exoprotease

The exoprotease from Oenococcus oeni produced in stress conditions was purified to homogeneity in two steps, a 14-fold increase of specific activity and a 44% recovery of proteinase activity. The molecular mass was estimated to be 33.1 kDa by gel filtration and 17 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggest that the enzyme is a dimer consisting of two identical subunits. Optimal conditions for activity on grape juice were 25 degrees C and a pH of 4.5. Incubation at 70 degrees C, 15 min, destroyed proteolytic activity. The SDS-PAGE profile shows that the enzyme was able to degrade the grape juice proteins at a significantly high rate. The activity at low pH and pepstatin A inhibition indicate that this enzyme is an aspartic protease. The protease activity increases at acidic pH suggesting that it could be involved in the wine elaboration.     

Lysis of yeast cells by Oenococcus oeni enzymes

Oenococcus oeni exhibited extracellular β (1→3) glucanase activity. This activity increased when cells were cultivated with glycosidic cell-wall macromolecules. In addition, the culture supernatant of the organism effectively lysed viable or dead cells of Saccharomyces cerevisiae. This lytic activity appeared in the early stationary phase of bacterial growth. Yeast cells at the end of the log phase of growth were the most sensitive. The optimum temperature for lysis of viable yeast cells was 40°C, which is very different from the temperatures observed in enological conditions (15–20°C). Moreover, the rate of the lytic activity was significantly lower in comparison with yeast cell wall-degrading activities previously measured in various other microorganisms. Therefore, yeast cell death that is sometimes observed during the alcoholic fermentation could hardly be attributed to the lytic activity of O. oeni. Journal of Industrial Microbiology & Biotechnology (2000) 25, 193–197. Received 27 December 1999/ Accepted in revised form 14 July 2000     

酒酒球菌苹果酸-乳酸酶基因的测序及分析

苹果酸乳酸酶是乳酸菌进行苹果酸乳酸发酵(MLF)的关键酶。以携带酒酒球菌(Oenococcusoeni)优良菌系OenococcusoeniSD2a的苹果酸乳酸酶基因mleA的重组质粒pLmleA作为测序质粒,进行测序分析。测序结果表明,克隆到的mleA基因序列与已报道的序列同源性为99%。mleA基因序列中有2个碱基与报道不同,其中1614碱基的改变导致错意突变,编码的氨基酸由报道的Asp变为Glu,这一改变使得原有的BamHI位点不再存在。     

Influence of oxygen on the biosynthesis of cellular fatty acids,sterols and phospholipids during alcoholic fermentation by Saccharomyces cerevisiae and Torulaspora delbrueckii

Saccharomyces cerevisiae and Torulaspora delbrueckii were grown under different O₂ availabilities on grape must. Oxygen requirements for the two yeasts were different: under anaerobic conditions, S. cerevisiae produced a higher percentage of unsaturated fatty acids, and had a greater cell yield and fermentation activity than T. delbrueckii. Addition of ergosterol (25mg/l) and oleic acid (31mg/l) caused total recovery of cellular growth and the fermentation activity of S. cerevisiae in anaerobiosis, but not of T. delbrueckii. However a short period of aeration to a 48 h culture in anaerobiosis, led to total recovery of the cellular growth and fermentation activity in both yeasts. Likewise, the effect of a short aeration period on unsaturated fatty acid biosynthesis was similar for both species.     

The effects of selenium,vitamin E and their combination on the composition of fatty acids and proteins in Saccharomyces cerevisiae

The aim of our studies was to test the effect and role of vitamin E and selenium supplements on yeast cell. In this study, the effects of selenium (Se), vitamin E (Vit. E), and their combination (Se plus Vit. E) on the composition of fatty acids and proteins were examined in Saccharomyces cerevisiae strains WET136 and 522. S. cerevisiae cells were grown up in YEPD medium supplemented with Se, Vit. E or their combination. It was found that the level of stearic acid was increased in all supplemented groups (p<0·05; p<0·001). The content of saturated and unsaturated fatty acids was decreased (p<0·05; p<0·01; p<0·001) in Vit. E and Vit. E plus Se supplemented S. cerevisiae. On the other hand, Se alone caused an increase (p<0·001) in the saturated fatty acids but a decrease (p<0·05; p<0·001) in the unsaturated fatty acids. Total proteins in S. cerevisiae were significantly increased (p<0·001) by Vit. E supplement. There was no significant change observed in S. cerevisiae supplemented with Se. These findings indicate that membrane composition of S. cerevisiae is affected by both Vit. E and Se supplements. © 1997 John Wiley & Sons, Ltd.     

Influence of epigallocatechin gallate and phenolic compounds from green tea on the growth of Oenococcus oeni

Aims: To investigate the effect of phenolic compounds on the growth of Oenococcus oeni. Methods and Results: Oenococci are usually grown in media often supplemented with complex additives such as tomato juice. In order to improve our knowledge about the growth requirements of oenococci, we added several juices and leaf extracts such as green tea to the culture media and screened them for growth‐stimulating substances to substitute complex supplements such as juices by more defined components. We found that also green tea could cause a growth stimulation of Oenococcus oeni strain B2. Conclusions: Further experiments showed that the stimulating effect was as a result of the phenolic compounds of green tea, especially epigallocatechin gallate (EGCG). On the other hand, EGCG could also inhibit the growth of O. oeni strain B2 just depending on its concentration. Significance and Impact of the Study: Individual catechins should have a minor influence on the growth of oenococci during wine making as their concentration in grapes is <30 mg kg^?1 grape. Whether there is a synergistic effect of the different catechins in wine has to be investigated.     

Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni

Malolactic fermentation (MLF) is carried out by Oenococcus oeni under very harsh conditions. This paper shows that stress compounds in wine such as SO(2), fatty acids and copper have an inhibitory effect on cell growth and MLF duration, and relates this effect to an inhibition of ATPase activity. Of the stress compounds, SO(2) and dodecanoic acid had the strongest effect, decreasing the ATPase specific activity to 37% and 58%, respectively. It can be concluded that ATPase is a good indicator of the physiological state of the cells and their ability to lead MLF.     

酒酒球菌液氮超低温保存

【目地】为安全、长期的保藏酒酒球菌,本文研究了菌体生长时间、冷冻方法、解冻温度、菌密度以及保护剂等对酒酒球菌细胞冷冻存活率的影响,找到最优液氮超低温保存方法。【方法】采用平板计数法测定冷冻存活率。【结果】实验结果表明酒酒球菌的最佳保存方法为:首先在稳定期前期离心收集菌体;其次加入保护剂(20 g/L酵母浸提物,40V/V甘油,20 g/L蔗糖,30 g/L谷氨酸钠)稀释菌体,使菌密度为109CFU/mL;然后直接投入液氮冷冻;最后在37℃温水浴中迅速解冻。保存6个月后,其中21株酒酒球菌的冷冻存活率达到99%以上。【结论】初步研究表明酵母浸提物,甘油,蔗糖,谷氨酸钠复合保护剂对酒酒球菌的保护效果较好,液氮超低温保存可用于酒酒球菌的长期保存。     

Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures

AIMS: To study the effect of sugars and sugar mixtures on the growth kinetics of Oenococcus oeni NCIMB 11648 in batch culture with the aim of producing a high cell productivity system for starter cultures. METHODS AND RESULTS: The growth of O. oeni was investigated on single sugars (glucose, fructose or sucrose) and their mixtures (glucose-fructose, glucose-sucrose or fructose-sucrose). Better growth was obtained on sugar mixtures compared with growth on a single sugar. The production system of O. oeni biomass was investigated in batch culture with or without pH control with respect to kinetics, specific growth rate and biomass yield. The effect of pH and substrate concentration on fermentation balances and ATP yield were determined. The optimal growth of O. oeni was achieved on the glucose-fructose mixture (9 g l(-1), 1 : 1) at pH 4.5 and 25 degrees C with pH control, with highest cell volumetric productivity (7.9 mg cell l(-1) h(-1)), biomass yield (0.041 g cell g(-1) sugar) and specific growth rate (0.066 h(-1)). CONCLUSIONS: The limitations to the growth of O. oeni were pH and inhibition by end product resulting in poor utilization of the medium with low cell yields. The cell productivity of the system can be improved by the appropriate use of mixed sugar growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uniquely showed that appropriate sugar mixtures with the correct environmental conditions can significantly improve the productivity of O. oeni cultures.     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

Initiation of anaerobic growth of Saccharomyces cerevisiae by amino acids or nucleic acid bases: ergosterol and unsaturated fatty acids cannot replace oxygen in minimal media

Nine out of ten industrially important strains of Saccharomyces cerevisiae did not grow in minimal media under anaerobic conditions even when ergosterol and unsaturated fatty acids were provided. Anaerobiosis was maintained either by flushing the culture flasks with prepurified nitrogen or by incubating the flasks in an anaerobic chamber. Traces of oxygen present in ‘prepurified nitrogen gas’ were sufficient to initiate yeast growth and on removal of the oxygen by catalytic means the yeasts failed to grow. The yeast grew very well anaerobically if the medium was supplemented with a mixture of amino acids or with a mixture of purines and pyrimidines. The growth initiated by including a mixture of amino acids was further enhanced when the medium was supplemented with ergosterol and an unsaturated fatty acid. Since no oxygen requirement for the synthesis of amino acids or purines and pyrimidines has been demonstrated, growth promotion by these compounds under anaerobic conditions is most likely not by eliminating the need for oxygen for their synthesis. We suggest that the amino acids and the nucleic acid bases yielded, through some hitherto unknown reactions, small amounts of a molecular or usable form of oxygen which allowed key reactions essential for ‘anaerobic’ growth to proceed. Received 16 July 1998/ Accepted in revised form 8 October 1998