白酒酿造中产正丙醇的微生物溯源研究

利用生物信息数据资源,对白酒酿造过程中产正丙醇微生物进行定向补充筛查,并对筛查后的菌株进行了发酵验证。结果表明,基于KEGG数据库明确了正丙醇的产生途径为丙酸代谢途径,其催化的关键酶为丙二醇脱水酶、1,3-丙二醇脱氢酶和1-丙醇脱氢酶;通过NCBI数据库确定了表达该3种关键酶的微生物种类信息,结合白酒发酵过程中的微生物群落构成,确定乳杆菌属（Lactobacillus）具有代谢产生正丙醇的潜在特性;发酵结果显示,面包乳杆菌（Lactobacillus panis）代谢生成正丙醇的能力相对较强,产量为263.7 mg/L。该研究结果表明,乳酸杆菌是白酒酿造中产正丙醇的重要微生物来源之一。该研究为白酒酿造过程正丙醇的调控提供新的研究方向,同时该方法也可以为其他食品发酵过程中目标风味物质的微生物溯源提供新思路。     

产乳酸乙酯酿酒酵母菌株的构建

乳酸乙酯是白酒中重要的呈香物质,并影响着白酒质量和风格。 为提高产乳酸乙酯的能力,以植物乳杆菌（Lactobacillus plantarum）为出发菌株,利用同源重组技术,获得高产L-乳酸的重组菌株。 并分别模拟液态白酒发酵和外源添加乳酸进行发酵,研究 产乳酸乙酯菌株P与出发菌AY12-α株外源添加乳酸发酵产乳酸乙酯的差异。 结果表明,用植物乳杆菌的乳酸脱氢酶基因ldhL1替换 酿酒酵母丙酮酸脱羧酶基因PDC1,得到重组菌株P。 模拟液态白酒发酵过程中,重组菌株P的乳酸和乳酸乙酯产量分别达12.64 g/L和 162.75 mg/L;出发菌株AY12-α中外源添加相同浓度乳酸进行发酵,乳酸乙酯的产量为115.47 mg/L,仅为重组菌株P的71%。 产乳酸乙 酯酵母菌株的构建,初步为豉香型等特定香型白酒的清洁化和机械化酿造奠定了基础。     

高粱两步法厌氧发酵产己酸研究

研究以富含淀粉的高粱为原料,通过两步法厌氧发酵提高己酸生成效率,即第一步分别接种己酸复合菌液、接种嗜酸乳杆菌TYCA06、添加酿酒酵母制得丁酸发酵液、乳酸发酵液、乙醇发酵液,第二步将丁酸发酵液、乳酸发酵液、乙醇发酵液等3种发酵液混合,并接种己酸复合菌液,厌氧发酵制得己酸。研究发现：通过优化丁酸发酵液、乳酸发酵液、乙醇发酵液的发酵工艺,丁酸发酵液中的丁酸、乙酸产量达到20.16 g/L和8.47 g/L,淀粉转化率达到76.02%;乳酸发酵液中的乳酸产量和乳酸产率分别达到11.41 g/L和42.48%;乙醇发酵液中的乙醇产率可达95.06%。继续优化己酸发酵工艺,发现当乙醇发酵液和乳酸发酵液同时作为电子供体时,己酸产量和产率均显著提升,分别达到4.15 g/L和75.2 mg/g,且当乙醇发酵液和乳酸发酵液中乙醇与乳酸添加的质量浓度比为2∶1时,己酸产量和产率均达到最高,分别为6.65 g/L和99.78 mg/g。对己酸发酵过程中微生物与己酸的相关性分析发现,发酵过程中和己酸含量呈正相关的微生物有产己酸菌属(Caproiciproducens)、Clostridium＿sensu＿s...     

代谢工程改造谷氨酸棒杆菌合成透明质酸

透明质酸是一种被广泛应用于医药、化妆品和食品等领域的糖胺聚糖。透明质酸的主要工业生产菌株为兽疫链球菌,由于该细菌具有致病性,因此迫切需要构建安全的透明质酸生产菌株。通过在食品级表达宿主谷氨酸棒杆菌中异源表达透明质酸合酶基因(pmHasA),实现了透明质酸的合成,摇瓶中产量达到0.35 g/L。通过强化代谢途径中尿苷二磷酸-葡萄糖脱氢酶基因(ugdA2)和磷酸氨基转移酶基因(glmS)的表达以及敲除乳酸脱氢酶基因(ldh),透明质酸产量提高至0.81 g/L。在此基础上优化诱导条件,确定异丙基-β-D硫代半乳糖苷浓度为0.8 mmol/L,诱导剂添加时间为2 h时,摇瓶中透明质酸产量最高达到1 g/L。经过3 L发酵罐分批补料发酵,透明质酸产量达到4.8 g/L。该文通过调控谷氨酸棒杆菌代谢途径实现了透明质酸安全、高效的合成,为食品级透明质酸的生产奠定了基础。     

高耐性拜耳接合酵母在酱香型白酒中的应用

传统白酒酿造是开放式的发酵过程,酿造微生物菌群演替及代谢活动极易受到外界环境影响。因此,分离筛选高耐性的核心功能微生物菌株,并将其应用于生产中是解决这些问题的有效手段。该研究在高温、高酸双胁迫条件下从酱香型白酒酒醅中筛选获得9 株具有高耐性拜耳接合酵母（Zygosaccharomyces bailii）,并通过模拟白酒发酵表征其发酵能力和风味物质合成能力。结果表明,菌株BM09 在37 ℃、16 g∕L 乙酸、110 g∕L 乳酸和14%乙醇溶液条件下生长性能均较为突出,且具有良好的产乙醇和高产酯能力,故将其作为强化菌株,应用于酱香型白酒实际生产中。原位发酵试验表明强化菌剂BM09 的应用使得酱香型白酒第7 轮次的原料利用率提高29.80%,乙醇产量提升63.10%,同时乙酸乙酯含量提高85.17%,乙醛含量降低70.37%。     

全细胞催化合成L-苯基乳酸重组大肠杆菌的构建

苯基乳酸是一种新型天然广谱抑菌物质,L-乳酸脱氢酶是微生物转化苯丙酮酸合成L-苯基乳酸的关键酶,通过构建过量表达L-乳酸脱氢酶的重组大肠杆菌,提高大肠杆菌合成L-苯基乳酸(苯基乳酸,phenyllactic acid,PLA)的能力。以Bacillus megaterium Z2013513基因组为模板,经PCR扩增得到L-乳酸脱氢酶基因,连接表达载体p ET-28a(+)并导入到大肠杆菌BL21(DE3),获得重组大肠杆菌BL21(DE3)/p ET-28a-ldh L。SDS-PAGE电泳和酶活分析表明,约在40 ku处出现显著特异性条带,粗酶液酶活力达3.4 U/mg。在37℃、200 r/min条件下,25 g/L(干重)重组大肠杆菌经60 min将70.32 mmol/L苯丙酮酸全细胞转化合成50.59 mmol/L L-苯基乳酸。由于具有较高的产物光学纯度(96.98%e.e.)和底物摩尔转化率(71.94%),表明一步生物转化法能高效地合成L-苯基乳酸。     

重组大肠杆菌全细胞合成苯基乳酸的研究

在E.coli BL21（DE3）中过量表达D-乳酸脱氢酶基因（D-ldh）,并优化该重组菌全细胞转化苯丙酮酸钠合成苯基乳酸的条件。通过单因素实验和正交实验优化诱导表达条件,并在此基础上对全细胞转化苯丙酮酸钠合成苯基乳酸进行了优化。结果表明,菌体OD600为1.2时添加IPTG至终浓度0.2mmol/L,25℃诱导4h后收集菌体具有最佳转化活性;最优分批转化条件:p H7.0,8.0g/L苯丙酮酸钠,20g/L葡萄糖,1%（v/v）吐温-80,菌体浓度20g/L（干重）,37℃,转速200r/min转化0.5h,苯基乳酸产量和转化率分别达到4.91g/L,56%。在上述优化条件上通过流加苯丙酮酸钠和葡萄糖,经6h转化,苯基乳酸最终产量达到17.23g/L,转化率为54%。研究结果表明该重组大肠杆菌成功转化苯丙酮酸合成苯基乳酸,具有较好的应用前景,为系统化代谢工程改造大肠杆菌生物合成苯基乳酸的进一步研究和应用提供了有用的技术参数。      

重组大肠杆菌全细胞合成苯基乳酸的研究

在E.coli BL21(DE3)中过量表达D-乳酸脱氢酶基因(D-ldh),并优化该重组菌全细胞转化苯丙酮酸钠合成苯基乳酸的条件。通过单因素实验和正交实验优化诱导表达条件,并在此基础上对全细胞转化苯丙酮酸钠合成苯基乳酸进行了优化。结果表明,菌体OD600为1.2时添加IPTG至终浓度0.2mmol/L,25℃诱导4h后收集菌体具有最佳转化活性;最优分批转化条件:p H7.0,8.0g/L苯丙酮酸钠,20g/L葡萄糖,1%(v/v)吐温-80,菌体浓度20g/L(干重),37℃,转速200r/min转化0.5h,苯基乳酸产量和转化率分别达到4.91g/L,56%。在上述优化条件上通过流加苯丙酮酸钠和葡萄糖,经6h转化,苯基乳酸最终产量达到17.23g/L,转化率为54%。研究结果表明该重组大肠杆菌成功转化苯丙酮酸合成苯基乳酸,具有较好的应用前景,为系统化代谢工程改造大肠杆菌生物合成苯基乳酸的进一步研究和应用提供了有用的技术参数。     

调控辅因子水平减少酿酒酵母积累副产物乙醇

C4二元羧酸广泛用于食品、医药和化学等行业,市场潜在需求量巨大。酿酒酵母被认为是发酵生产C4二元羧酸的潜在最适微生物,却产生大量的乙醇,导致了碳流的损失。通过敲除硫胺素合成途径中的调控基因THI2,阻断了硫胺素的合成,使得副产物乙醇产量从5.27±0.23 g/L下降到0.53±0.12 g/L,但影响了葡萄糖消耗和菌体的生长。在此基础上,通过外源添加0.04μmol/L的硫胺素二磷酸,促进了葡萄糖的消耗和菌体生长;进一步通过外源添加1 000μg/L的NAD+,使得葡萄糖的消耗量和菌体的生长分别提高了48.6%和47.2%,而乙醇产量仅增加了0.56 g/L。通过调控辅因子水平(硫胺素和NAD+)可以有效减少副产物乙醇的积累,为解决利用酿酒酵母生产C4二元羧酸中副产物乙醇积累这一普遍性问题提供了一个新的策略。     

高产琥珀酸重组大肠杆菌的构建及厌氧发酵

以野生型大肠杆菌Escherichia coli W为出发菌株,利用Red同源重组系统分别敲除了乳酸脱氢酶基因(ldhA)、乙醇脱氢酶基因(adhE)、丙酮酸甲酸裂解酶基因(pflB)、丙酮酸氧化酶基因(poxB)和乙酸激酶基因(ackA),再通过无氧生长进化筛选过程,构建得到在厌氧条件下能有效生长,并以琥珀酸为主要发酵产物的重组大肠杆菌WS100(△ldhA,△adhE,△pflB,△poxB,△ackA)。利用15 L发酵罐进行厌氧发酵测定显示,经72 h发酵,菌体密度OD600最大值可提高至6.48,琥珀酸产量达到70.13 g/L,琥珀酸的生产强度为0.98 g/(L.h),葡萄糖-琥珀酸转化率为76%。发酵液中副产物含量低,乙酸含量为5.34 g/L,乳酸产量仅为0.15 g/L,未检测到甲酸和乙醇生成。结果表明,厌氧条件下,该工程菌可有效利用低营养成分的无机盐培养基,在不表达任何外源基因的条件下可稳定高产琥珀酸,具有极大的工业化开发前景。     

Identification of microorganisms producing lactic acid during solid‐state fermentation of Maotai flavour liquor

Lactic acid is the main acid produced during the Maotai liquor brewing process, influencing the quality of the base liquor and fermentation process. However, the microorganisms responsible for lactic acid production have not been identified. In this work, the dynamic changes in bacterial community structure in the Zaosha round (second sorghum feeding and fermentation) of the brewing process were analysed by 16S rRNA high‐throughput sequencing. Results show that lactic acid bacteria (LAB) and Bacillus spp. are the dominant bacteria in the brewing process, where Bacillus spp. are found in the early stage, whilst LAB are found throughout the brewing process. Furthermore, 10 types of LAB and five Bacillus spp. were isolated from Zaopei (a mixture of fermented grains including sorghum and wheat) by a culture‐dependent method. Lactobacillus panis accounts for 68% of the LAB, and Bacillus amyloliquefaciens for 54% of Bacillus spp. Solid‐state fermentation experiments were performed with L. panis and B. amyloliquefaciens and lactic acid production was consistent with the accumulation of lactic acid in Zaosha. The results showed that L. panis was the main producer of lactic acid in pits, while B. amyloliquefaciens plays an important role in the production of lactic acid in the early stages of fermentation. The approach used in this study may facilitate the identification of key microorganisms with specific functionality involved in other food and beverage fermentation processes. © 2018 The Institute of Brewing & Distilling     

Co-metabolism of citrate and maltose byLactobacillus brevis subsp.lindneri CB1 citrate-negative strain: effect on growth, end-products and sourdough fermentation

Growth, substrates and end-product formation of the maltose and citrate co-metabolization byLactobacillus brevis subsp.lindneri CB1 citrate-negative strain were initially studied in synthetic medium. Compared to maltose (19 g/l) fermentation, the co-metabolization of maltose (10 g/l) plus citrate (9 g/l) caused faster cell growth, increased the concentrations of lactic acid and especially of acetic acid (from 0.7 g/l to 2.9 g/l), produced succinic acid (0.5 g/l) and reduced ethanol synthesis. Highest activities of acetate kinase, the same of lactate dehydrogenase and a reduced alcohol dehydrogenase activity were detected in cytoplasmic extracts of cells growing on maltose plus citrate. The breakdown of citrate depended upon the continuous presence of maltose in the growth medium. Upon depletion of citrate, the cells continued through the normal maltose fermentation, having a diauxic metabolic curve as shown by impedance measurements. Concentrations of citrate from 3 g/l to 15 g/l led to increases of acetic acid from 1.25 g/l to 5.55 g/l. Since maltose was naturally present during sourdough fermentation, the addition of 9 g citrate per kg wheat dough enabled the co-metabolization of maltose and citrate byL. brevis subsp.lindneri CB1. Compared with traditional sourdough fermentation, faster cell growth, a higher acetic acid concentration and a reduced quotient of fermentation were obtained by co-metabolism.     

Efficient production of L‐lactic acid by Crabtree‐negative yeast Candida boidinii

Industrial production of L ‐lactic acid, which in polymerized form as poly‐lactic acid is widely used as a biodegradable plastic, has been attracting world‐wide attention. By genetic engineering we constructed a strain of the Crabtree‐negative yeast Candida boidinii that efficiently produced a large amount of L ‐lactic acid. The alcohol fermentation pathway of C. boidinii was altered by disruption of the PDC1 gene encoding pyruvate decarboxylase, resulting in an ethanol production that was reduced to 17% of the wild‐type strain. The alcohol fermentation pathway of the PDC1 deletion strain was then successfully utilized for the synthesis of L ‐lactic acid by placing the bovine L ‐lactate dehydrogenase‐encoding gene under the control of the PDC1 promoter by targeted integration. Optimizing the conditions for batch culture in a 5 l jar‐fermenter resulted in an L ‐lactic acid production reaching 85.9 g/l within 48 h. This productivity (1.79 g/l/h) is the highest thus far reported for L ‐lactic acid‐producing yeasts. DDBJ/EMBL/GenBank nucleotide database with Accession Nos. AB440630 and AB440631. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of Inhibitory Environmental Factors on Growth of Oenococcus oeni CCSYU2068 for Malolactic Fermentation of Cider Production

The effects of several inhibitory factors (sulfur dioxide, pH and ethanol) on the growth of lactic acid bacteria and the subsequent malolactic fermentation (MLF) were studied by inoculation of different culture strains of Oenococcus oeni, the major lactic acid bacteria (LAB) in cider production. After comparing their organoleptic properties, three strains of Oenococcus oeni were selected from indigenous and commercial sources and their inhibitory effects on cell growth and MLF examined. The malolactic bacteria expressed variations in tolerance to the environmental conditions of pH, sulfur and ethanol concentration. Isolated from an indigenous cider production facility, O. oeni L4 had a better capacity with constant growth even when the concentration of SO₂ was 50 ppm, ethanol 10% (v/v) and pH 3.0. O. oeni L4 showed better properties for metabolizing the major acids: malic, lactic and acetic acid. The decomposition mean rate of malic acid was as high as 228.52 mg/L per day with a low acetic acid concentration of 101.78 mg/L under the stress conditions of cider production.     

Improvement of L(+)‐lactic acid production from cassava wastewater by Lactobacillus rhamnosus B 103

BACKGROUND: L (+)‐Lactic acid is used in the pharmaceutical, textile and food industries as well as in the synthesis of biodegradable plastics. The aim of this study was to investigate the effects of different medium components added in cassava wastewater for the production of L (+)‐lactic acid by Lactobacillus rhamnosus B 103. RESULTS: The use of cassava wastewater (50 g L^?1 of reducing sugar) with Tween 80 and corn steep liquor, at concentrations (v/v) of 1.27 mL L^?1 and 65.4 mL L^?1 respectively led to a lactic acid concentration of 41.65 g L^?1 after 48 h of fermentation. The maximum lactic acid concentration produced in the reactor after 36 h of fermentation was 39.00 g L^?1 using the same medium, but the pH was controlled by addition of 10 mol L^?1 NaOH. CONCLUSION: The use of cassava wastewater for cultivation of L. rhamnosus is feasible, with a considerable production of lactic acid. Furthermore, it is an innovative proposal, as no references were found in the scientific literature on the use of this substrate for lactic acid production. Copyright © 2010 Society of Chemical Industry     

Simultaneous lactic acid and xylitol production from vine trimming wastes

Hemicellulosic hydrolyzates from vineshoot trimmings obtained by dilute sulfuric acid hydrolysis were evaluated for xylitol production by Debaryomyces hansenii NRRL Y‐7426. Bioconversion was not efficient, however, since a mixture of products (mainly ethanol) was achieved. Taking into account that hexoses (such as glucose or mannose) can inhibit xylose metabolism by repression and inactivation of the xylose transport system or catabolic enzymes and that these hemicellulosic hydrolyzates are characterized by a high glucose concentration, a novel technology was developed, sequentially transforming glucose into lactic acid by Lactobacillus rhamnosus followed by fermentation of xylose into xylitol by Debaryomyces hansenii after L. rhamnosus removal by microfiltration. Optimal conditions were achieved using detoxified concentrated hemicellulosic hydrolyzates, after CaCO₃ addition in both stages of fermentation and using nitrogen purges after sampling in order to reduce the oxygen dissolved. Under these conditions 31.5 g lactic acid L^?1 (Q_LA = 1.312 g L^?1 h^?1 and Y_LA/S = 0.93 g g^?1) and 27.5 g xylitol L^?1 (Q_Xylitol = 0.458 g L^?1 h^?1 and Y_Xylitol/S = 0.53 g g^?1) were produced. Finally, lactic acid was selectively recovered using the resin Amberlite IRA 400 (0.0374 g of lactic acid g^?1 of dry resin), allowing a further recovery of xylitol by sequential stages of adsorption, concentration, ethanol precipitation, concentration and crystallization to obtain food‐grade xylitol according to a developed process. Copyright © 2007 Society of Chemical Industry     

克雷伯氏菌产1,3-丙二醇aldH基因缺失菌株的构建

该研究利用λRed重组技术对克雷伯氏菌（Klebsiella pneumonia）中的乙醛脱氢酶（aldH）基因进行敲除,成功构建缺失菌株K.pneumonia2-1ΔaldH,与原始菌株相比,缺失菌株K.pneumonia2-1ΔaldH发酵液中乙醇产量由原来的7.24 g/L降为0.47 g/L,降低了93.51%;1,3-丙二醇产量由原来的78.83 g/L增长至82.55 g/L,提高了4.72.%;甘油转化率由60.64%增长至63.50%,提高了2.86%。缺失菌株K.pneumonia2-1ΔaldH 50 L发酵罐小试试验中,1,3-丙二醇的产量为78.13 g/L,乙醇产量为0.50 g/L。     

植物乳杆菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响

在高粱汁培养基内同时接种植物乳杆菌（Lactobacillus plantarum）和高产酯酿酒酵母（Saccharomyces cerevisiae）,探究植物乳杆菌及其代谢产物对高产酯酿酒酵母酒精发酵及酯醇代谢的影响。结果表明：植物乳杆菌对高产酯酿酒酵母生长及酒精发酵的影响不大,发酵结束后残糖量均＜5 g/L,乙醇含量为74～78 g/L;植物乳杆菌使高产酯酿酒酵母乙酸乙酯和高级醇产量下降,分别最多下降了15.31%、36.14%;培养基内不同乳酸质量浓度使酿酒酵母乙酸乙酯产量提高,乳酸质量浓度为7.1 g/L时,乙酸乙酯最多提高了39.84%;培养基内乳酸质量浓度在1.8～7.1 g/L时,高产酯酿酒酵母高级醇的产量明显降低,特别是苯乙醇的产量显著下降,下降了25.05%～75.64%。     

乳酸菌发酵香菇对肠道菌群的调节作用及其产品研制

将香菇经乳酸菌发酵后,通过小鼠灌胃实验和16S rRNA高通量测序,研究其对肠道菌群结构和多样性的影响,并研制调味即食产品。结果表明,中等剂量（10 g/kg,以小鼠质量计）的发酵香菇改变了小鼠肠道菌群的物种多样性,其特有物种和稀有物种数量增加,且个体差异减小,物种分布更均匀。发酵香菇可不同程度地增加小鼠肠道内有益菌群,如Muribaculaceae、乳杆菌属（Lactobacillus）、Dubosiella、Lachnospiraceae等的定殖,降低螺杆菌属（Helicobacter）、支原体（Mycoplasma）等致病菌的相对丰度,从而改善肠道健康。通过单因素和响应面优化实验,获得乳酸菌发酵香菇调味即食产品的最佳配方为：发酵香菇块81.5%、甜面酱1.0%、豆瓣酱1.5%、白砂糖6.0%、藤椒油3.0%、芝麻2.0%、辣椒油4.0%、姜汁1.0%（均为质量分数）。为乳酸菌发酵技术在香菇深加工中的应用提供了有益参考。     

副产物对运动发酵单胞菌酒精发酵的影响

研究了乙酸、乳酸、糠醛作为酒精发酵副产物 ,对细菌酒精发酵的影响。在发酵培养基中 ,当乙酸浓度为 1 5 g/L ,糠醛浓度为 4g/L时 ,细菌酒精产量开始减少。在乳酸浓度为 0～ 2 g/L的范围内 ,没有观察到其对细菌酒精发酵的抑制作用。运用化学渗透假说 ,对酸在细菌酒精发酵中的抑制作用进行了解释 ,同时对糠醛在细菌酒精发酵中的抑制作用进行了理论探讨