Enhanced deacidification activity in Schizosaccharomyces pombe by genome shuffling

A problem frequently occurring in making some kinds of wines, particularly Vitis quinquangularis Rehd wine, is the presence of malic acid at high concentrations, which is detrimental to the quality of wines. Thus, there is a need of the ways for effectively reducing the malic acid levels in wine. This study aimed to generate shuffled fusants of Schizosaccharomyces pombe with enhanced deacidification activity for reducing the excessive malic acid content in wine. Sz. pombe CGMCC 2.1628 was used as the original strain. The starting mutant population was generated by UV treatment. The mutants with higher deacidification activity were selected and subjected to recursive protoplast fusion. The resulting fusants were screened by using the indicator of malic acid concentration of fermentation supernatants on 96‐well microtitre plates, measured with bromocresol green. After three rounds of genome shuffling, the best‐performing fusant, named GS3‐1, was obtained. Its deacidification activity (consumed 4.78 g/l malic acid within 10 days) was increased by 225.2% as compared to that of original strain. In the Vitis quinquangularis Rehd wine fermentation test, GS3‐1 consumed 4.0 g/l malic acid during the whole cycle of fermentation, providing up to 185.7% improvement in malic acid consumption compared with that of the original strain. This study shows that GS3‐1 has great potential for improving the quality of Vitis quinquangularis Rehd wine. Copyright © 2014 John Wiley & Sons, Ltd.     

紫外诱变法选育酒酒球菌乙醇胁迫耐受菌株及其发酵性能研究

该研究以酒酒球菌（Oenococcus oeni）SD-2a为研究对象,采用紫外诱变法选育乙醇胁迫耐受突变菌株,评价其在乙醇胁迫条件下的生长能力,并测定其产β-葡萄糖苷酶活性及其在模拟酒中苹果酸、乳酸含量及活菌数的变化。结果表明,分离筛选到3株乙醇胁迫耐受性好的突变菌株,分别编号为UVe1、UVe2、UVe3,在高体积分数乙醇（12%和14%）胁迫环境下,3株突变菌株的生长速度均显著高于出发菌株SD-2a（P＜0.05）;突变菌株UVe2的β-葡萄糖苷酶活性显著高于出发菌株SD-2a和对照菌株L-450（P＜0.05）;在模拟酒中,3株突变菌株的苹果酸降解与乳酸生成速度均显著高于出发菌株SD-2a（P＜0.05）,且突变菌株UVe1和UVe2的存活率显著高于出发菌株SD-2a（P＜0.05）;综上,突变菌株UVe2具有优良商业酒酒球菌的潜力。     

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

高效降解OTA的发酵工业用菌株筛选及鉴定

为筛选一株产L-苹果酸能力强的黑曲霉菌株,通过紫外诱变与高浓度放线菌酮迭代诱变的方法,将野生型菌株诱变为一株产L-苹果酸能力强的黑曲霉菌株,并对其种子或发酵培养基成分进行优化。结果表明,诱变所得一株产L-苹果酸能力强的黑曲霉CGMCC NO. 40550菌株,其摇瓶发酵时种子培养基最适葡萄糖浓度为60 g/L、最适氮源为（NH₄）₂SO₄ 4.95 g/L,黑曲霉菌球形成最优转速220 r/min;发酵培养基最适葡萄糖和最适（NH₄）₂SO₄浓度分别为180 g/L和4.95 g/L,CuSO₄·5H₂O为其生产苹果酸最佳微量元素,最适添加量为 0.065 g/L。通过单因素实验的优化,优化后的培养基更有利于L-苹果酸的合成,发酵96 h时L-苹果酸产酸量达到18.15 g/L,显著提高了245%,L-苹果酸占总酸的百分比达到71.69%。本研究为L-苹果酸的工业化生产奠定基础。

     

Isolation and characterization of Kluyveromyces marxianus mutants deficient in malate transport

In malic acid-grown cells of the strains ATCC 10022 and KMS3 of Kluyveromyces marxianus the transport of malic acid occurred by a malate-proton symport, which accepted l-malic, d-malic, succinic and fumaric acids, but not tartaric, malonic or maleic acids. The system was inducible and subjected to glucose repression. Mutants of the strain KMS3, unable to grow in a medium with malic acid, were isolated and checked for their capacity to utilize several carbon sources and to transport dicarboxylic acids by the malate-proton symport. Two distinct clones affected on malate transport were obtained. Both were able to grow on a medium with glycerol or ethanol but not with dl-malic, succinic, oxoglutaric and oxaloacetic acids as the sole carbon and energy sources. However, while one of the mutants (Mal7) displayed activity levels for the enzymes malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase similar to those of the wild strain, in the other mutant type (Mal6) the activities for the same enzymes were significantly reduced. Plasma membranes from derepressed cells of the wild strain and of the mutants Mal6 and Mal7 were isolated and the protein analysed by SDS–PAGE. The electrophoretic patterns of these preparations differed in a polypeptide with an apparent molecular mass of about 28 kDa, which was absent only in the mutant Mal7. The results indicated that Mal7 can be affected in a gene that encodes a malate carrier in K. marxianus. © 1998 John Wiley & Sons, Ltd.     

利用玉米芯水解液产苹果酸的戴尔根霉突变菌株选育及代谢分析

诱变筛选发酵玉米芯水解液高产苹果酸的根霉属菌株,并对其进行代谢分析,研究其高产苹果酸的机理。对分离得到的菌株进行ITS序列鉴定,进一步利用软X射线辐射对菌种进行诱变筛选、对出发菌株和突变菌株的相关酶活力进行测定及代谢通量分析。利用软X射线辐射结合丙烯醇平板筛选乙醇脱氢酶缺陷型菌株,得到突变株-1,发现其乙醇脱氢酶基因的3 处密码子突变为“TAA”,阻断了突变菌株的乙醇代谢途径。进而利用软X射线辐射结合氟乙酸平板筛选乙醛酸循环缺陷型菌株,得到复合突变株-2,降低了副产物富马酸及琥珀酸的产量。复合突变株-2的葡萄糖-6-磷酸脱氢酶中几处NADP（H）结合位点发生突变,增加了糖酵解和磷酸戊糖两种途径的相互作用,促进了其戊糖代谢。经过两步分离筛选,得到的复合突变株-2能发酵玉米芯水解液高产苹果酸,且减少了副产物乙醇、富马酸、琥珀酸的生成。复合突变株-2的苹果酸产量占代谢物总产量的比例由出发菌株的71%增加到91%,苹果酸产量增大1 倍,研究成果对工业化利用突变菌株生产苹果酸具有重要意义。     

荔枝果酒加工过程中有机酸的变化研究

该文研究了荔枝果酒在加工过程中有机酸的变化,结果表明,荔枝酒中主要的有机酸为乳酸、苹果酸、酒石酸、乙酸和琥珀酸.荔枝果酒的口感不佳,是由于苹果酸、乙酸、酒石酸及琥珀酸含量过高而引起.荔枝汁加工成荔枝酒的过程中,苹果酸、酒石酸及琥珀酸的含量分别增加2.80g/L～2.27g/L、0.77g/L和0.27g/L;乙酸含量减少9.44g/L～9.38g/L.要调整相关有机酸的含量和比例以达到改善荔枝酒口感的目的,可以通过控制酶解、调整成分及酒精发酵等工艺过程而实现.     

Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis

Cyclodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. B1001 catalyzed the production predominantly of alpha-cyclodextrin (CD) from starch (Tachibana, Y. et al., Appl. Environ. Microbiol., 65, 1991-1997, 1999). The CGTase gene (cgtA) from this strain was cloned and sequenced. It was composed of 2217 nucleotides, and encoded a protein (739 amino acids) with a molecular mass of 83,240 Da. Recombinant CgtA expressed in Escherichia coli also catalyzed the production predominantly of alpha-CD from starch, as did native CgtA from strain B1001. Based on a substrate binding model of Bacillus circulans no. 8 CGTase, Tyr100, Trp191 and Tyr267 were specified to locate the spiral amylose and to minimize the size of the CD by saccharide aromatics interaction. In order to determine the critical residue for catalyzing production predominantly of alpha-CD, site-directed mutations were introduced in CgtA (Y100W, Tyr100-->Trp; W191Y, Trp191-->Tyr; W191F, Trp191-->Phe; Y267W, Tyr267-->Trp; Y267F, Tyr267-->Phe). Analysis of the reaction products by HPLC revealed that the mutant enzyme Y267W produced more beta- and gamma-CD than the wild-type enzyme. However, the other mutants still produced high levels of alpha-CD, suggesting that Tyr267 plays a critical role in alpha-CD production catalyzed by B1001 CGTase.     

Respiration-deficient mutants of Zymomonas mobilis show improved growth and ethanol fermentation under aerobic and high temperature conditions

Respiration-deficient mutant (RDM) strains of Zymomonas mobilis were isolated from antibiotic-resistant mutants. These RDM strains showed various degrees of respiratory deficiency. All RDM strains exhibited much higher ethanol fermentation capacity than the wild-type strain under aerobic conditions. The strains also gained thermotolerance and exhibited greater ethanol production at high temperature (39°C), under both non-aerobic and aerobic conditions, compared with the wild-type strain. Microarray and subsequent quantitative PCR analyses suggest that enhanced gene expression involved in the metabolism of glucose to ethanol resulted in the high ethanol production of RDM strains under aerobic growth conditions. Reduction of intracellular oxidative stress may also result in improved ethanol fermentation by RDM strains at high temperatures.     

紫外氦氖诱变选育高产聚苹果酸菌株

以出芽短梗霉(Aureobasidium Pullulans) TKPM00006为出发菌株,用紫外和氦氖激光结合诱变,通过溴甲酚绿变色指示平板和分别以丁二酸、柠檬酸为唯一碳源的摇瓶初筛得到90株产酸明显的菌株,再经过摇床产量测定,复筛得到1株高产聚苹果酸( PMLA)的菌株.其摇瓶产量为12.62 g/L,较原菌聚苹...     

Developmental changes in pulp organic acid concentration and activities of acid-metabolising enzymes during the fruit development of two loquat (Eriobotrya japonica Lindl.) cultivars differing in fruit acidity

Changes in organic acid concentration and related enzyme activities in loquat (Eriobotrya japonica Lindl.) pulp were studied, using low-acid ‘Changhong 3’ and high-acid ‘Jiefangzhong’ cultivars. Both titratable acidity (TA) and malic acid concentration increased during the early stages of fruit development and decreased at the later stages. The difference in TA between the two cultivars could be explained by the difference in malic acid concentration, which could result from a difference in NAD-malate dehydrogenase (NAD-MDH) and NADP-malic enzyme (NADP-ME) activities. Although the difference in malic acid concentration between the two cultivars could not result from a difference in phosphoenolpyruvate carboxylase (PEPC) activity, malic acid concentration in both ‘Changhong 3’ throughout fruit development and ‘Jiefangzhong’ at the early stages increased linearly and curvilinearly with increasing PEPC activity, respectively. Therefore, NAD-MDH, NADP-ME and PEPC activities may play significant roles in malic acid biosynthesis and degradation.     

Coculture-inducible bacteriocin activity of Lactobacillus plantarum strain J23 isolated from grape must

Detection and characterization of bacteriocin production by Lactobacillus plantarum strain J23, recovered from a grape must sample in Spain, have been carried out. Bacteriocin activity was degraded by proteolytic enzymes (trypsin, alfa-chymotrypsin, papaine, protease, proteinase K and acid proteases), and it was stable at high temperatures (121 degrees C, 20min), in a wide range of pH (1-12), and after treatment with organic solvents. L. plantarum J23 showed antimicrobial activity against Oenococcus oeni, and a range of Lactobacillus and Pediococcus species. Bacteriocin production was detected in liquid media only when J23 was cocultivated with some inducing bacteria, and induction took place when intact cells or 55 degrees C heated cells of the inducer were cocultivated with J23, but not with their autoclaved cells. Bacteriocin activity of J23 was not induced by high initial J23 inocula, and it was detected in cocultures during the exponential phase. The presence of ethanol or acidic pH in the media reduced bacteriocin production in the cocultures of J23 with the inducing bacteria. The presence of plantaricin-related plnEF and plnJ genes was detected by PCR and sequencing. Nevertheless, negative results were obtained for plnA, plnK, plNC8, plS and plW genes.     

Hyaluronic acid production by recombinant Streptococcus thermophilus

Generally recognized as safe, Streptococcus thermophilus was transformed using a plasmid expressing endogenous hyaluronic acid (HA) synthase genes. A single expression of hyaluronic acid synthase (hasA), uridine diphosphate-glucose dehydrogenase gene (hasB), or pyrophosphorylase gene (glmU) and double expression of hasA and hasB were attempted. A streptococcus-Escherichia coli shuttle vector, pBE31, was successfully transfected in S. thermophilus. The single expression of hasA or hasB allowed S. thermophilus to produce about 0.5-1.0 g/l HA. The strains coexpressing of hasA and hasB showed a markedly increased HA production (1.2g/l) which was six-fold increase compared with the wild-type strain. The maximum cell concentration and specific growth rate of each recombinant strain were lower than those of the wild-type strain; however, the specific production rate was more than 100-fold higher. Galactose concentration decreased in the coexpressing strain after depletion of lactose. The bacterial metabolism would be altered in order to achieve a higher production by changing the intracellular metabolism. The average molecular weight of HA (1.0 × 10(6) Da) was not affected by the expression of hasA and hasB. HA produced from recombinant strain could be an alternative material for medical, cosmetic and food utilization instead of HA from conventional pathogenic streptococci.     

INTERACTIONS BETWEEN pH AND MALIC ACID CONCENTRATION ON THE INACTIVATION OF LISTERIA MONOCYTOGENES1

The effects and interactions of malic acid concentration and pH on the inactivation kinetics of a three strain mixture of Listeria monocytogenes was studied in brain heart infusion broth (BHI). The medium was supplemented with malic acid and monosodium malate to achieve pH levels of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0 in conjunction with concentrations of 0.0, 0.1, 0.5, 1.0 and 2.0 M. Duplicate 20-mL portions of each pH/malate level were inoculated (ca. 10⁸ cfu/mL), stored aerobically at 28C, and assayed periodically for viable counts by plating on BHI agar. Survivor curves were generated by fitting data to a linear model that includes a lag term and used to calculate D-values and “times to 4-D inactivation”. Inactivation rates were dependent on both the pH and malic acid concentration. At the higher pH levels, malic acid appeared to provide some degree of protection compared to control cultures where the pH was adjusted with HCl. At lower pH values and at higher malic acid concentrations, a concentration-dependent anion effect was observed. The results indicate that malic acid is a relatively benign organic acid. Its antimicrobial characteristics are similar to those of citric acid and is substantially less bactericidal than lactic or acetic acids.     

Physiology of pre-cut mango II. Evolution of organic acids

The effect of a 30-min osmotic dehydration (OD) treatment under vacuum and storage temperature on the organic acid content in slices prepared from Haden and Kent mangoes was evaluated. Additionally, respiration rate (RR), titratable acidity, pH and soluble solids were monitored. Greater RR was detected in osmotically dehydrated slices. Citric, ascorbic and fumaric acids were measured in greater concentrations in Haden slices, while malic acid was predominant in Kent slices. No succinic acid was found in any sample. Low malate concentrations suggest that the observed increase in CO₂ production caused by the mechanical stress may be due to malic acid decarboxylation by the malic enzyme. OD caused lesser alterations in oganic acid balance than cutting and possibly allowed an extended operation of the tricarboxylic acid cycle.     

过量表达苹果酸酶对E.coli脂肪酸合成能力的影响

为了考察苹果酸酶对原核生物脂肪酸合成能力的影响,本研究从E.coliK-12中克隆了苹果酸酶基因（NADPME,EC1.1.1.40）MaeB,并插入质粒pET30a-T,构建了重组质粒pMX20,经IPTG诱导在E.coli BL2（IDE3）获得了大量表达。摇瓶发酵结果表明,过量表达苹果酸酶基因会改变大肠杆菌脂肪酸合成能力,并在添加适宜底物苹果酸（15mmol/L）时,胞内总酯含量比受体菌提高了4倍,约197.74mg/g,产率为1.89%。本研究为脂肪酸生产提供了优良菌株,具有一定的应用开发前景。      

一株高产乳酸菌株的鉴定及其发酵特性研究

采用MRS培养基从果醋醋醅中分离具有强产酸能力的乳酸菌,通过形态观察、生理生化试验及分子生物学技术对其进行鉴定,并研究其乙醇耐受能力、产酸能力和抗生素敏感性。结果表明,从果醋醋醅中分离得到一株产酸能力较高的菌株,编号为D2,并鉴定其为鼠李糖乳杆菌（Lactobacillus rhamnosus）,该菌株在体积分数4%乙醇条件下能够正常生长,在体积分数14%乙醇条件下能够存活,具有较强的乙醇耐受力;在MRS肉汤培养基中发酵48 h时,总酸含量达到20.03 g/L,其中乳酸含量增加最多,其次是苹果酸和琥珀酸;对四环素、氯霉素、红霉素和吉他霉素敏感,对链霉素、卡那霉素、青霉素、苯唑西林和复方新诺明具有耐药性。综上,该菌株具有较强的乙醇耐受能力和产乳酸能力,安全性高,有作为发酵菌剂改善果醋风味和口感的潜在应用价值。     

同步糖化淀粉质原料高产L-苹果酸菌株的选育

本研究选育了一株高糖化酶活性且传代稳定的突变株,在不添加商品糖化酶的情况下,可以通过对脱胚玉米粉的糖化发酵生产苹果酸。以Aspergillus oryzae(米曲霉)ME303为出发菌株,经ARTP诱变,2-脱氧葡萄糖(2-DG)筛选压力诱变处理,并通过比较原始菌株和诱变菌株的糖化酶活力和代谢特征,获得一株性能稳定的糖化酶活力较高的L-苹果酸生产菌株A. oryzae SS3,当粗淀粉质原料——脱胚玉米粉作为单一碳源时,30 ℃、150 r/min摇瓶发酵96 h后,糖化酶表现出较高的活力,为320.0 U/mL,与原始菌株相比(220.0 U/mL),酶活增加了45.45%,发酵后191 h,A. oryzae SS3菌株同步糖化发酵产苹果酸达到41.39 g/L,与原始菌株相比(33.98 g/L)提高了21.80%,为目前报道的利用淀粉质原料生产苹果酸且酶活性较高的米曲霉突变菌株,可进一步降低生产成本。