The effects of freezing and freeze-drying of Oenococcus oeni upon induction of malolactic fermentation in red wine

Summary The use of Oenococcus oeni starter cultures for the induction of malolactic fermentation (MLF) in wine permits control over the timing of the process and the quality of the wine. Successful inoculation of bacterial starter cultures into wine depends on the selection of suitable strains and on the preparation and conservation of those cultures. Medium for Leuconostoc oenos (MLO) is the best medium for easy and rapid growth of O. oeni cultures under laboratory controlled conditions for isolation and identification. However, this study showed that O. oeni cells inoculated in MLO failed to induce MLF in wine while cells grown in Medium of Preculture (MP) or wine, stored at −20 °C or freeze-dried retained the ability to induce MLF when inoculated in wine. Our results suggest that the use of freeze-dried cultures of O. oeni previously grown in MP is the best choice for industrial application.     

采用膜生物反应器进行连续苹果酸-乳酸发酵的研究

采用膜生物反应器进行连续的苹果酸-乳酸发酵,研究了酿酒酒球菌的苹果酸降解活力稳定性。试验结果表明,采用错流微滤膜生物反应器,添加高密度酿酒酒球菌（〉10^8cfu／mL）可以成功地实现连续高效的苹果酸．乳酸发酵;在连续72h运行期间,控制流速0．48L／h,苹果酸降解率〉95％;果酒的酒精浓度是影响酿酒酒球菌苹果酸降解活力稳定性的最主要因素,而果酒pH值及营养状况通常情况下不影响其苹果酸降解活力稳定性：经酒精胁迫培养的酿酒酒球菌表现出更好的苹果酸降解活力稳定性。     

Use of a continuous multistage bioreactor to mimic winemaking fermentation

Continuous fermentation set-ups are of great interest for studying the physiology of microorganisms. In winemaking conditions, yeasts go through a growth phase and a stationary phase during which more than half of the sugar is fermented. A comprehensive study of wine-yeast physiology must therefore include yeasts in a non-growing phase. This condition is impossible to achieve within a chemostat, which led us to design a multi-stage fermentation device. In this study, we evaluated the ability of such a device to reproduce, in a series of steady states, the conditions of batch fermentation. Two-stage and four-stage fermentations were carried out with two different strains of Saccharomyces cerevisiae. The main characteristics of the fermentation process (biomass growth, by-product content of the medium) were compared with those observed in batch mode at the same stage of fermentation, which was defined by glucose uptake. The four-stage configuration showed a better ability to reproduce batch fermentation characteristics than the two-stage set-up. It also allowed to uncouple the variations of environmental parameters and proved to be a promising tool to gain new insights into yeast metabolism during alcoholic fermentation.     

膜生物反应器用于乙醇连续发酵菌株的适应性培养

将一种耐高温酿酒干酵母在体积为5L、膜而积为0.08m2的硅橡胶膜生物反应器乙醇连续发酵系统中进行适应性培养,选育出适应该环境的优势菌种.三轮乙醇连续发酵实验的结果表明,糖的转化率差别不大,但单位酵母细胞代谢产生乙醇的能力有了提高,说明酵母细胞在长期封闭循环发酵环境条件下,原种菌经过几百代繁殖后其后代遗传特征发生了改变,能逐渐适应所处的不利环境.表明通过膜生物反应器长期封闭循环发酵对酵母细胞进行适应性培养,可以获得具有某种遗传优势的新菌株.     

野生酵母菌在膜生物反应器中的适应性培养与发酵动力学研究

进行了野生酵母菌在硅橡胶膜生物反应器长期封闭循环连续发酵环境中的多轮发酵-优势菌株筛选-转移培养的适应性进化实验,研究了菌株的发酵性能变化和发酵动力学.发酵实验共进行了5轮,每轮发酵持续时间500h,且采用前一轮发酵残液中筛选出的优势菌株作为发酵菌种.第4轮、第5轮发酵实验在乙醇产率、乙醇生成比速率、细胞比生长率等发酵性能上较第一轮有显著提高.多轮适应性培养有效增强了酵母细胞对乙醇的耐受力以及缩短了发酵适应期.     

膜生物反应器封闭循环发酵系统对野生酵母的驯化

自然界中选育的野生酵母菌S3在PDMS膜生物反应器长期封闭循环系统中进行驯化,在经过3轮连续的封闭循环发酵适应性驯化实验后,对第四轮实验和第一轮实验的酵母发酵性能进行比较,分析野生酵母的适应性进化行为,乙醇-细胞平均比产率、葡萄糖转化率分别提高42.6%、2.1%,细胞平均死亡率减少11.04%;与商用的安琪酵母(ADY)相比,发酵性能也更优秀。结果表明在膜生物反应器封闭循环发酵系统中可以使野生酵母菌得到驯化。     

膜生物反应器处理印染废水

采用膜生物反应器处理印染废水,探讨膜产水量与抽吸压力的关系,膜的安装方式对减缓膜污染的影响,以及在中试运行过程中所存在的问题;分析该系统的可靠性、连续性和稳定性.试验结果表明,用膜生物反应器法处理印染废水是可行的,出水满足DB 61-234-2006地方一级排放标准.     

New applications for Schizosaccharomyces pombe in the alcoholic fermentation of red wines

The fermentation of grape must using non‐Saccharomyces yeasts with particular metabolic and biochemical properties is of growing interest. In the present work, red grape must was fermented using four strains of Schizosaccharomyces pombe (935, 936, 938 and 2139), Saccharomyces cerevisiae 7VA and Saccharomyces uvarum S6U, and comparisons were made over the fermentation period in terms of must sugar (glucose + fructose), malic acid, acetic acid, ammonia, primary amino nitrogen, lactic acid, urea (a possible fermentation activator or precursor of other metabolites) and pyruvic acid (a molecule affecting vitisin formation and therefore colour stability) concentration. The colour intensity of the fermenting musts was also recorded. The Schizosaccharomyces strains consumed less primary amino nitrogen and produced less urea and more pyruvic acid than other Saccharomyces species. Further, three of the four Schizosaccharomyces strains completed the breakdown of malic acid by day 4 of fermentation. The main negative effect of the use of Schizosaccharomyces was strong acetic acid production. The Schizosaccharomyces strains that produced most pyruvic acid (938 and 936) were associated with better ‘wine’ colour than the remaining yeasts. The studied Schizosaccharomyces could therefore be of oenological interest.     

立式静态固态发酵罐的研制

介绍了一种新型立式静态固态物料发酵罐的结构特点及工作原理，对灭菌、接种、通气等关键生产工序进行了探讨。经多批次纤维素酶的培养试验，结果表明，立式静态固态物料发酵罐实现了产生蒸气，直接对固态物料进行蒸气灭菌、冷却、接种、发酵、无菌空气的供给，杜绝杂菌污染，降低了设备造价，提高了性价比，提高了产品的产量和质量。     

The use of malolactic Oenococcus oeni (ATCC 39401) for deacidification of media containing glucose, malic acid and citric acid

 Malolactic fermentation is widely used to reduce the acidity of grape juices in wine production. However, application of the same technology to the production of wine from berries of the northern regions is not straightforward. Unlike grapes, these berries are rich in citric and malic acid while the sugar content is low. An ideal deacidification process for the northern berries would be the microbial degradation of these acids with minimal loss of sugars. Therefore, the co-metabolism of citric acid and glucose was studied under different conditions of malic acid degradation by Oenococcus oeni. At low pH values (pH <4.5) degradation of malic acid always proceeded first to completion with practically no consumption of glucose or citric acid. After the exhaustion of malic acid the degradation of both citric acid and glucose were initiated simultaneously. Following the exhaustion of malic acid and citric acid the remaining glucose remained nonfermentable. Thus, it is concluded that, by maintaining the culture in a resting state by the control of pH, selective degradation of acids can be achieved without subsequent loss of glucose. Received: 20 January 2000     

生物反应器操作参数对秸秆固态发酵酒精的影响

在转鼓式生物反应器中，研究了搅拌频率、通气速率对秸秆发酵产纤维素酶和酒精的影响，试验的结果表明：搅拌频率为10min／4h能保证生物量和产酶达到最大，而搅拌对生物量和酵母酒精产量影响很小；通气速率达到3．0vvh时，生物量最大达0．31g／g，酶活达到170U／g(干基)；对转轴及转鼓式反应器的效果进行了初步的比较。     

野生猕猴桃酒苹果酸-乳酸发酵优良乳酸菌的筛选与耐受性研究

该研究通过形态观察及生理生化试验,采用酸性番茄（ATB）培养基从自然启动苹果酸-乳酸发酵（MLF）的野生猕猴桃酒中筛选MLF优良乳酸菌,并对其SO2、酒精、pH耐受性进行测定。结果表明,经初筛得到6株乳酸菌,分别编号为R6、R7、R11、R14、R15、R18。其中,菌株R6、R15可耐受SO2 80 mg/L,菌株R7可耐受SO2 100 mg/L;菌株R6、R7可耐受酒精度14%vol,菌株R15可耐受酒精度12%vol;菌株R6、R7可耐受pH 3.2,菌株R15可耐受pH 3.0。综合分析,菌株R6、R7和R15具有较强的SO2、酒精及pH耐受能力,为MLF优良乳酸菌。     

苹果酸-乳酸发酵细菌乙醇胁迫应答机制研究进展

由乳酸菌引起的苹果酸-乳酸发酵是生产优质葡萄酒的一个重要工艺过程。在这个过程中,乙醇是乳酸菌活性的主要抑制因子,特别是随着全球气候变暖带来的葡萄含糖量的增加,乙醇对乳酸菌的抑制作用越来越成为人们的研究热点。乙醇胁迫使得乳酸菌细胞结构发生变化,影响苹果酸-乳酸发酵的顺利进行。因此,乳酸菌在乙醇胁迫下的生长、代谢对葡萄酒生产是非常重要的。该文对苹果酸-乳酸发酵过程中常见乳酸细菌在乙醇胁迫下的应答机制进行了阐述,为乳酸菌乙醇胁迫耐受性机制的研究和优良菌株的选育提供参考。     

发酵法制备右旋糖酐菌种培养

选择中国工业微生物菌种保藏中心的肠膜明串珠菌20074,通过直接发酵法制备右旋糖酐,以培养液浊度和pH值作为考察指标,对基础培养基的各组分含量进行优化,得到最优组成为:蔗糖10%、蛋白胨0.2%、磷酸氢二钠0.2%、磷酸二氢钾0.03%、酵母浸膏0.5%。实验所选的范围内,金属离子中Mn2+对菌体生长的影响较大。肠膜明串珠菌20074的生长曲线表明,0～8h是生长延迟期,8～24h为对数期,24～30h为稳定期,30h之后为衰亡期。     

Biogenic amines and the importance of starter cultures for malolactic fermentation

Biogenic amines are compounds produced mainly by microorganisms from the decarboxylation of some amino acids which may cause several health issues in susceptible people. In the winemaking process, one of the factors with the greatest impact on the final concentration of biogenic amines in wine is the malolactic fermentation, which mainly involves the action of lactic acid bacteria. This article gathers scientific literature on the importance of the use of inoculation in winemaking, especially for the potential formation of biogenic amines in spontaneous fermentations, with emphasis on malolactic fermentation. It establishes the set of attributes that influence the formation of biogenic amines in winemaking and aspects related to their toxicity, especially histamine and tyramine. With the growing interest of the market in products with less chemical appeal, the safety of wines, whether from non‐conventional or traditional winemaking, must increasingly be observed. Therefore, the importance of using cultivated starter cultures is discussed, along with the safety of organic wines and the presentation of articles that seek to reconcile the production of organic wine with the preservation of the ‘microbiological terroir’ in a safe way, using selected wild yeasts and lactic acid bacteria.     

Molecular analysis of Oenococcus oeni population dynamics and the effect of aeration and temperature during alcoholic fermentation on malolactic fermentation

The effects of aeration and temperature during alcoholic fermentation (AF) on spontaneous and inoculated malolactic fermentation (MLF) of wine have been analysed by following the population dynamics of Oenococcus oeni strains with multiplex random amplified polymorphic DNA‐polymerase chain reaction. It has enabled us to follow precisely the proportion of different autochthonous strains and the starter strain through fermentations. Lack of aeration led to delays in AF, which meant that autochthonous lactic acid bacteria could develop earlier and prevented the starter strain from developing correctly. Temperature was not found to lead to any differences. Two strains were isolated in the same spontaneous MLF, suggesting that, in some cases, multiple strains might be responsible for the degradation of malic acid in wine. It can be concluded that delays in the AF can negatively affect the control of MLF and that this can be studied by following the development of the different strains of O. oeni.     

DFA III production from inulin with inulin fructotransferase in ultrafiltration membrane bioreactor

An ultrafiltration membrane bioreactor was used for the production of DFA III from enzymatic conversion of inulin. Compared with the traditional batch reactor, the productivity and purity of DFA III could be markedly enhanced and product inhibition was removed and IFTase could be continuously used for six runs in the UF membrane bioreactor. When the substrate concentration was 100 g/L, the concentration of DFA III was about 78.4 g/L, while the productivity and purity of DFA III could attain about 2385 and 92%, respectively.     

浸没式膜生物反应器协同活性炭处理海产养殖废水效果

该文结合海产养殖废水的盐度效应特点,开展了浸没式膜生物反应器(membrane bioreactor,MBR)协同粉末活性炭(powder activated carbon,PAC)处理含盐废水的试验。考察了投加PAC对于MBR污染物去除性能及膜污染的影响;盐度变化过程中(0~35 g/L)MBR对化学需氧量(chemical oxygen demand , COD)、氨氮(ammonia nitrogen,NH+4-N)、亚硝酸盐氮(nitrite nitrogen,NO₂-N)处理效果;以及含盐废水长期作用下微生物性能、膜通量、絮体粒径的变化情况。重点分析0~5 g/L的盐度变化,本体溶液中的溶解性有机物(soluble microbile products,SMP)和污泥絮体中胞外聚合物(extracellular polymeric substances,EPS)组成及含量的变化情况。结果表明:MBR PAC对COD的去除效率比MBR高7.3%,对NH+4-N、NO₂-N去除的稳定性优于MBR;两工艺条件下膜通量随盐度变化呈现类似的趋势,即敏感期衰减,稳定期得到一定程度的恢复。养殖废水长期作用下,MBR PAC膜通量是MBR的1.5倍,MBR PAC的污泥粒径相对于MBR增加了52 μm。盐度变化过程中,PAC由于其吸附性能及絮凝能力,能吸附本体溶液中的溶解性微生物代谢产物,相对于MBR,蛋白质的含量减少了34.0%。MBR PAC适用于海产养殖废水的处理。     

Evolution of red wine anthocyanins during malolactic fermentation,postfermentative treatments and ageing with lees

A comparative study was conducted on nine batches of wine, from the same initial wine, subjected to malolactic fermentation and ageing in barrels, under different technological conditions: Malolactic fermentation in barrel or in tank, with or without wine clarification, ageing with or without lees and stirring or no stirring of the lees. Samples were taken of the initial wine, of the wine at the end of malolactic fermentation, of the wines after clarifying treatments, and after 3, 6, 9, 12 and 14 months of ageing in the barrel, making a total of 48 wines. As a result of the anthocyanin analysis of all the wines studied, a total of 21 different anthocyanin compounds were detected, which can be classified into four groups: simple glucosides, acetyl glucosides, cinnamoyl glucosides and pyroanthocyanins. During MLF, it was shown that the effect of the container used seems to be more important than the metabolic activity of the bacteria responsible for the process. From application of the LSD test, significant differences were found in the concentrations of all the anthocyanin compounds identified due to ageing time and significant differences were also revealed for most anthocyanin compounds in relation to the manufacturing method, especially the presence or absence of lees.     

重组大肠杆菌E. coli BL21/pET28a(+)-mle产苹果酸乳酸酶发酵条件的优化

该研究以实验室构建的重组大肠杆菌E. coli BL21/pET28a(+)-mle为研究对象,以L-乳酸量为评价指标,通过单因素和正交试 验研究了诱导温度、培养基起始pH值、异丙基-β-D-硫代吡喃半乳糖苷（IPTG）浓度和诱导时间对重组大肠杆菌E. coli BL21/pET28a (+)-mle分泌表达苹果酸乳酸酶（MLE）的影响。 结果表明,重组大肠杆菌E. coli BL21/pET28a(+)-mle分泌表达苹乳酶的最佳培养条件 为培养基起始pH值为5.9,诱导温度为28 ℃,IPTG 浓度0.6 mmol/L,诱导时间3 h。 在此优化条件下,L-乳酸产量为2.37 g/L,较优化前 提高9.48倍。