Co-fermentation of grape must by Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid content in wine

Grape must was fermented by a mixed culture of Saccharomyces cerevisiae W-3 (a wine yeast) and Issatchenkia orientalis KMBL 5774 (a malic acid-degrading yeast). Co-fermentation with 1:1 (v/v) inoculum ratio of W-3 and KMBL 5774 decreased malic acid to 0.33 mg/ml from 1.1 mg ml with W-3 alone. Ethanol production was the same in both cases (7.8%, v/v). Acetaldehyde, 1-propanol, 2-butanol and isoamyl alcohol all decreased, with an increase in methanol, in the co-fermented wine. Sensory evaluation showed a higher score in the wine fermented with 1:1 (v/v) inoculum ratio than those obtained by 4:1 (v/v) inoculum ratio or W-3 alone.     

木质纤维素水解液副产物对东方伊萨酵母乙醇发酵的影响

为了客观评判耐高温东方伊萨酵母HN-1利用木质纤维素水解液生产燃料乙醇的潜力,本文采用单因素试验和响应面中心组合试验研究了木质纤维素水解液有毒副产物甲酸钠(1.0-5.0 g/L)、乙酸钠(2.5-8.0 g/L)、糠醛(0.2-2.0 g/L)、5-羟甲基糠醛(0.1-1.0 g/L)和香草醛(0.5-2.0 g/L)对其乙醇发酵的影响。结果表明,木质纤维素水解液有毒副产物对东方伊萨酵母HN-1乙醇发酵的影响较小,除添加2 g/L香草醛或添加1 g/L 5-羟甲基糠醛可使乙醇产量分别降低20.38%和11.2%外,其他抑制物的添加对乙醇的生成未有显著影响。但是,当副产物浓度较高时,可以显著抑制菌体生长,添加1-5 g/L甲酸钠、2.5-8.0 g/L乙酸钠、0.4-2 g/L糠醛或0.5-2 g/L香草醛,发酵36 h时菌体细胞干重分别较对照下降了25.04%-37.02%、28.83%-43.82%、20.06%-37.60%和26.39%-52.64%。中心组合试验结果表明各抑制物交互作用对乙醇的生成影响不显著。该研究表明木质纤维素水解液副产物对东方伊萨酵母HN-1乙醇发酵的影响较小,适合用于纤维乙醇发酵。     

竹炭固定化微生物对水中壬基酚的降解效率

竹炭是一种优质生物质炭,不仅比表面积大,孔隙发达,而且机械强度高,是微生物固定化载体的最佳选择之一.本文采用正交试验确定了竹炭固定化微生物的最佳制备条件,对比了竹炭固定菌和游离菌对水中类雌激素壬基酚的降解效果,并考察了竹炭固定菌的重复利用性.结果表明:固定化后降解菌大量地附着在竹炭表面及内部孔隙中,其最佳制备条件为温度30℃、pH=7、竹炭粒径35目.壬基酚的降解符合一级动力学方程,在不同的壬基酚初始浓度下(30、50、80、100 mg·L^-1),竹炭固定菌对壬基酚的7 d降解率分别为100%、75.3%、67.3%和78.7%,显著优于游离菌(54.2%、51.5%、30.6%和23.5%).经过8轮重复利用后,竹炭固定菌对壬基酚的降解率仍可达到36.5%,而此时游离菌的降解率仅为8.9%,说明竹炭固定菌具有长期可重复利用性,在去除废水有机污染物中具有较好的工程应用前景.     

Degradation of malic acid by Issatchenkia orientalis KMBL 5774, an acidophilic yeast strain isolated from Korean grape wine pomace

Several yeast strains degrading malic acid as a sole carbon and energy source were isolated from Korean wine pomace after enrichment culture in the presence of malic acid. Among them, the strain designated as KMBL 5774 showed the highest malic acid degrading ability. It was identified as Issatchenkia orientalis based on its morphological and physiological characteristics as well as the nucleotide sequences of the internal transcribed spacer (ITS) I-5.8S rDNA-ITS II region. Phylogenetic analysis of the ITS I-5.8S rDNAITS II sequences showed that the KMBL 5774 is the closest to I. orientalis zhuan 192. Identity of the sequences of the KMBL 5774 was 99.5% with those of I. orientalis zhuan 192. The optimal pH of the media for the growth and malic acid degradation by the yeast was between 2.0 and 3.0, suggesting that the strain is an acidophile. Under the optimized conditions, the yeast could degrade 95.5% of the malic acid after 24 h of incubation at 30 degrees in YNB media containing 2% malic acid as a sole carbon and energy source.     

Production of polymalic acid and malic acid by Aureobasidium pullulans fermentation and acid hydrolysis

Malic acid is a dicarboxylic acid widely used in the food industry and also a potential C4 platform chemical that can be produced from biomass. However, microbial fermentation for direct malic acid production is limited by low product yield, titer, and productivity due to end‐product inhibition. In this work, a novel process for malic acid production from polymalic acid (PMA) fermentation followed by acid hydrolysis was developed. First, a PMA‐producing Aureobasidium pullulans strain ZX‐10 was screened and isolated. This microbe produced PMA as the major fermentation product at a high‐titer equivalent to 87.6 g/L of malic acid and high‐productivity of 0.61 g/L h in free‐cell fermentation in a stirred‐tank bioreactor. Fed‐batch fermentations with cells immobilized in a fibrous‐bed bioreactor (FBB) achieved the highest product titer of 144.2 g/L and productivity of 0.74 g/L h. The fermentation produced PMA was purified by adsorption with IRA‐900 anion‐exchange resins, achieving a ～100% purity and a high recovery rate of 84%. Pure malic acid was then produced from PMA by hydrolysis with 2 M sulfuric acid at 85°C, which followed the first‐order reaction kinetics. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application. Biotechnol. Bioeng. 2013; 110: 2105–2113. © 2013 Wiley Periodicals, Inc.     

玉米芯、竹炭及油枯吸附-海藻酸钠包埋施氏假单胞菌PFS-4对二氯喹啉酸的降解

采用玉米芯、竹炭及油枯吸附-海藻酸钠包埋对分离到的施氏假单胞菌PFS-4进行复合固定.采用正交试验对固定化条件进行优化,研究了固定化菌剂及游离菌体对二氯喹啉酸的降解效果.结果表明: 固定化菌剂制备的最佳条件为:海藻酸钠质量分数为4%、吸附载体比例(玉米芯∶竹炭∶油枯)为1∶2∶1、CaCl₂质量分数为3%、交联时间4 h.固定化菌剂在温度为30 ℃、初始pH=7的条件下,经6 d培养后,对浓度为800 mg·L^-1的二氯喹啉酸降解率为91.4%,而游离菌体的降解率为72.8%.将游离菌体和固定化菌剂用于实际污水及土壤处理时,固定化菌剂对水中及土壤中二氯喹啉酸去除率仍能分别达到84.2%和74.3%.研究结果表明,载体及其联结方式对土壤中二氯喹啉酸去除产生显著影响,翻动频率与土壤中二氯喹啉酸的去除率呈显著正相关.因此,玉米芯、竹炭及油枯吸附-海藻酸钠复合固定施氏假单胞菌PFS-4对不良环境具有较好的缓冲性能,对二氯喹啉酸污染水体及土壤原位生态修复具有潜力.     

过表达羧化途径及失活苹果酸酶基因对大肠杆菌好氧发酵产苹果酸的影响

苹果酸是一种重要的C4二羧酸,在食品、医药、化工等领域有广泛的应用。本文主要研究羧化途径强化及苹果酸酶失活对大肠杆菌好氧发酵生产苹果酸的影响。首先在大肠杆菌E2中过表达了磷酸烯醇式丙酮酸羧化酶基因ppc,得到菌株E21,苹果酸积累量从0.57 g/L提高到3.83 g/L。随后,分别过表达来自谷氨酸棒杆菌的丙酮酸羧化酶基因pyc和来自琥珀酸放线杆菌的磷酸烯醇式丙酮酸激酶pck基因,相应的工程菌株E21(pTrcpyc)和E21(pTrc-A-pck)分别产6.04和5.01 g/L苹果酸,得率分别达到0.79和0.65 mol/mol葡萄糖。敲除E21中的苹果酸酶基因mae A和mae B,苹果酸产量也显著提高了36%,达到5.21 g/L,得率为0.62 mol/mol。然而,在过表达pyc的基础上敲除苹果酸酶基因并不能进一步提高苹果酸的产量。经过摇瓶发酵条件的初步优化,菌株E21(pTrcpyc)生产12.45 g/L苹果酸,得率为0.84 mol/mol,达到理论得率的63.2%。     

Changes in gluconic acid, polyols and major volatile compounds in sherry wine during aging with submerged flor yeast cultures

The traditional biological process by which sherry wines are aged can be accelerated by using submerged Saccharomyces cerevisiae var. capensis (G1) strain cultures previously grown in glycerol. The used controlled shaking conditions raise the acetaldehyde, acetoin, and meso 2,3-butanediol contents in the wine, and increases the consumption of gluconic acid by flor yeast relative to traditional biological aging under flor yeast velum.     

Degradation of free and sulfur-dioxide-bound acetaldehyde by malolactic lactic acid bacteria in white wine

AIMS: Acetaldehyde is the major carbonyl compound formed during winemaking and has implications for sensory and colour qualities of wines as well as for the use of the wine preservative SO(2). The current work investigated the degradation of acetaldehyde and SO(2)-bound acetaldehyde by two commercial Oenococcus oeni starters in white wine. METHODS AND RESULTS: Wines were produced by alcoholic fermentation with commercial yeast and adjusted to pH 3.3 and 3.6. While acetaldehyde was degraded rapidly and concurrently with malic acid at both pH values, SO(2)-bound acetaldehyde caused sluggish bacterial growth. Strain differences were small. CONCLUSIONS: Efficient degradation of acetaldehyde can be achieved by commercial starters of O. oeni. According to the results, the degradation of acetaldehyde could not be separated from malolactic conversion by oenococci. While this may be desirable in white winemaking, it may be necessary to delay malolactic fermentation (MLF) in order to allow for colour development in red wines. SO(2)-bound acetaldehyde itself maybe responsible for the sluggish or stuck MLF, and thus bound SO(2) should be considered next to free SO(2) in order to evaluate malolactic fermentability. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides new results regarding the metabolism of acetaldehyde and SO(2)-bound acetaldehyde during the MLF in white wine. The information is of significance to the wine industry and may contribute to reducing the concentration of wine preservative SO(2).     

重组酸性脲酶对黄酒中尿素和氨基甲酸乙酯的降解应用

氨基甲酸乙酯(Ethyl carbamate,EC)作为一种潜在致癌物质普遍存在于传统发酵食品中。利用酸性脲酶消除EC前体物质尿素是一种具有潜在重要应用价值的策略。本研究在前期成功实现食品级耐乙醇酸性脲酶高效表达制备的基础上,系统研究了重组酸性脲酶对尿素和EC的水解过程。重组酸性脲酶对模拟体系以及黄酒体系中的尿素具有很好的降解能力(60mg/L的尿素在25h内完全被降解),表明该重组酸性脲酶适用于黄酒中尿素的消除。虽然重组酸性脲酶也具有降解EC的催化活性,但在黄酒中添加重组酸性脲酶对EC的浓度无明显影响。进一步研究发现重组酸性脲酶对尿素和EC的Km值分别为0.714 7mmol/L和41.32mmol/L,研究结果为应用定向进化策略改造重组酸性脲酶实现同时水解尿素和EC提供了理论依据。     

Growth of yeasts, lactic and acetic acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana

AIM: To investigate the microbiological and biochemical changes which occur in palm wine during the tapping of felled oil palm trees. METHODS AND RESUlts: Microbiological and biochemical contents of palm wine were determined during the tapping of felled oil palm trees for 5 weeks and also during the storage. Saccharomyces cerevisiae dominated the yeast biota and was the only species isolated in the mature samples. Lactobacillus plantarum and Leuconostoc mesenteroides were the dominated lactic acid bacteria, whilst acetic acid bacteria were isolated only after the third day when levels of alcohol had become substantial. The pH, lactic and acetic acid concentrations during the tapping were among 3.5-4.0%, 0.1-0.3% and 0.2-0.4% respectively, whilst the alcohol contents of samples collected within the day were between 1.4% and 2.82%; palm wine which had accumulated over night, 3.24% to 4.75%; and palm wine held for 24 h, over 7.0%. CONCLUSION: Accumulation of alcohol in palm wine occurs in three stages during the tapping and marketing with the concurrent lactic and acetic acid fermentation taking place as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts, lactic and acetic acid bacteria are all important in the fermentation of palm wine and influence the composition of the product.     

Continuous deacidification of wine by immobilized Schizosaccharomyces pombe cells: evaluation of malic acid degradation rate and analytical profiles

Schizosaccharomyces pombe cells entrapped in Ca alginate gel were used under continuous fermentation conditions to evaluate the extent of malic acid degradation at different dilution rates ( D , h⁻¹) and the analytical profiles of wines obtained. Gel entrapped cells caused the deacidification of wine without affecting the analytical profiles. Maximum deacidification rate was obtained at 0.076 h⁻¹ D while higher dilution rates (up to 0.100 h⁻¹ D ) resulted in a clear reduction of activity. At D -value of 0.055 h⁻¹, the deacidification activity remained constant during the observation period of 360 h.     

Measurement of catechin and gallic acid in tea wine with HPLC

First a kind of fermented tea wine was prepared from Dancong tea. The content of four major catechins and gallic acid (EC, EGC, EGCG and ECG) in tea wine was measured with HPLC. The results showed that the content of EC, EGC, EGCG, ECG and catechins in tea wine decreased when compared with that before fermentation. The content of EC decreased the most, reaching 26.79%, while the content of GA changed the least with a decrease of only 13.56%. Nevertheless, tea wine still contains a relatively large amount of catechins, thus proper consumption of tea wine may be salubrious.     

Changes in volatile compounds and aromatic series in sherry wine with high gluconic acid levels subjected to aging by submerged flor yeast cultures

Volatile compounds of sherry wine containing gluconic acid under aging by submerged flor yeast cultures were analyzed. The aroma profile was obtained by grouping the compounds in nine aromatic series. The balsamic, fatty, herbaceous and empyreumatic series increased significantly as consequence of the increase of pantolactone, acids (butanoic, 2-methylbutanoic and 3-methylbutanoic), methionol and gamma-butyrolactone compounds, respectively. The decrease of higher alcohols promoted solvent series diminished. These changes are consistent with those observed in the production of commercial sherry wine using traditional biological aging.     

Lactic acid bacteria in the quality improvement and depreciation of wine

The winemaking process includes two main steps: lactic acid bacteria are responsible for the malolactic fermentation which follows the alcoholic fermentation by yeasts. Both types of microorganisms are present on grapes and on cellar equipment. Yeasts are better adapted to growth in grape must than lactic acid bacteria, so the alcoholic fermentation starts quickly. In must, up to ten lactic acid bacteria species can be identified. They belong to the Lactobacillus, Pediococcus, Leuconostoc and Oenococcus genera. Throughout alcoholic fermentation, a natural selection occurs and finally the dominant species is O. oeni, due to interactions between yeasts and bacteria and between bacteria themselves. After bacterial growth, when the population is over 10⁶CFU/ml, malolactic transformation is the obvious change in wine composition. However, many other substrates can be metabolized. Some like remaining sugars and citric acid are always assimilated by lactic acid bacteri a, thus providing them with energy and carbon. Other substrates such as some amino acids may be used following pathways restricted to strains carrying the adequate enzymes. Some strains can also produce exopolysaccharides. All these transformations greatly influence the sensory and hygienic quality of wine. Malic acid transformation is encouraged because it induces deacidification. Diacetyl produced from citric acid is also helpful to some extent. Sensory analyses show that many other reactions change the aromas and make malolactic fermentation beneficial, but they are as yet unknown. On the contrary, an excess of acetic acid, the synthesis of glucane, biogenic amines and precursors of ethylcarbamate are undesirable. Fortunately, lactic acid bacteria normally multiply in dry wines; moreover some of these activities are not widespread. Moreover, the most striking trait of wine lactic acid bacteria is their capacity to adapt to a hostile environment. The mechanisms for this are not yet c ompletely elucidated . Molecular biology has provided some explanations for the behaviour and the metabolism of bacteria in wine. New tools are now available to detect the presence of desirable and undesirable strains. Even if much remains unknown, winemakers and oenologists can nowadays better control the process. By acting upon the diverse microflora and grape musts, they are more able to produce healthy and pleasant wines.     

Malolactic fermentation in wine with high densities of non-proliferating Oenococcus oeni

Five strains of Oenococcus oeni (syn. Leuconostoc oenos) under non-proliferating conditions were assessed for the performance of the malolactic fermentation in wine at various initial pH values, malic acid concentration and densities of cells. We succeeded in inducing the malolactic fermentation after inoculation of high densities of O. oeni G6 even in recalcitrant wines where the traditional malolactic fermentation was inhibited by adverse environmental conditions (low pH and high concentration of malic acid). Optimal degrading conditions in wine, under different physico-chemical environments, were determined in order to achieve rapid depletion of malic acid in red wine. Off-odour compounds were not formed under these conditions, suggesting an attractive alternative for wine production. This revised version was published online in November 2006 with corrections to the Cover Date.     

Enhanced production of succinic acid by metabolically engineered<Emphasis Type="Italic">Escherichia coli</Emphasis> with amplified activities of malic enzyme and fumarase

Apfl ldhA double mutantEscherichia coli strain NZN111 was used to produce succinic acid by overexpressing theE. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of malic acid as well as succinic acid. After the analyses of the metabolic pathways, thefumB gene encoding the anaerobic fumarase ofE. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducibletrc promoter. From the batch culture of recombinantE. coli NZN111 harboring pTrcMLFu, 7 g/L of succinic acid was produced from 20 g/L of glucose, with no accumulation of malic acid. From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes.     

Effects of malic acid on rumen fermentation, urinary excretion of purine derivatives and feed digestibility in steers

The objective of this study was to evaluate the effects of malic acid (MA) supplementation on rumen fermentation, urinary excretion of purine derivatives (PDs) and whole gastro-intestinal tract feed digestibility in steers. Eight ruminally cannulated Simmental steers (465 ± 13 kg) were used in a replicated 4 × 4 Latin square design. The treatments were: control (without MA), LMA (MA-low), MMA (MA-medium) and HMA (MA-high) with 0.0, 7.8, 15.6 and 23.4 g MA per kg dry matter (DM), respectively. Diets consisted of corn stover and concentrate (60/40, DM basis). DM intake was approximately 9 kg per day, which was 90% of ad libitum intake including 5.4 kg corn stover and 3.6 kg concentrate. Ruminal pH (range of 6.91 to 6.56), ratio of acetate to propionate (range of 3.88 to 3.25), ammonia N (range of 9.03 to 6.42 mg/100 ml) and lactate (range of 91.25 to 76.31 mg/100 ml) decreased linearly as MA supplementation increased, whereas total volatile fatty acid (VFA) concentration (range of 55.68 to 61.49 mM) linearly (P < 0.05) increased with increase in MA supplementation. In situ ruminal neutral detergent fiber (aNDF) degradation of corn stover was improved but the crude protein (CP) degradability of concentrate mix was decreased with increasing the dose of MA. Urinary excretion of PDs was quadratically (P < 0.01) changed with altering MA supplementation (67.88, 72.74, 75.81 and 73.78 mmol/day for control, LMA, MMA and HMA, respectively). Similarly, digestibilities of DM, organic matter (OM), NDF and acid detergent fiber (ADF) in the total tract were also quadratically increased with increasing MA, and no differences in terms of CP and ether extract digestibility were observed. The results indicate that MA supplementation has the potential to improve rumen fermentation and feed digestion in beef cattle. The MA stimulates the digestive microorganisms or enzymes in a quadratic response. In the experimental conditions of this trial, the optimum MA dose was 15.6 g MA per kg DM.