啤酒酵母海藻糖提取工艺研究

[目的]研究优化啤酒酵母海藻糖提取工艺.[方法]以啤酒废酵母为原料,使用各种不同的方法（微波破壁法、高温处理破壁、煮沸提取法）提取海藻糖,以海藻糖得率以及工艺的效益性为指标考察料液比、浸提时间、浸提温度、辅助因素处理等单因素对海藻糖提取的影响,从而确定从废酵母中提取海藻糖的较佳工艺.[结果]试验得出,微波破碎法耗时少,但不易工业化且提取率不高,比较发现煮沸提取的工艺方法相对最佳.以水作提取剂从废酵母中提取海藻糖的最佳条件是：煮沸80 min,海藻糖提取率为8.147 mg/g干酵母.[结论]研究可为海藻糖的提取及进一步开发利用提供参考.     

Utilization of trehalose, benzoate, valerate, and seed and root exudates by genotypes of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens

Isolates of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are effective biocontrol agents against soilborne pathogens. A previous study showed that the superior (“premier”) root colonizer P. fluorescens Q8r1-96 (genotype D) utilized trehalose, benzoate and valerate as sole carbon sources but average colonizers Q2-87 (genotype B) and 1M1-96 (genotype L) did not. We tested the utilization of these three carbon sources by a collection of 55 2,4-DAPG-producing P. fluorescens strains from 17 genotypes and found no correlation between a strain's ability to utilize these carbon sources and superior rhizosphere competence on wheat and pea. Of the strains tested, 73%, 48% and 69% were able to utilize trehalose, benzoate and valerate as sole carbon sources, respectively. With some exceptions, we found a correlation between the utilization of these compounds and previous groupings of these strains by BOX-PCR; genotype D strains utilized all three compounds. Twenty-three strains grew efficiently on root and seed exudates from wheat and pea, with doubling times between 0.9 and 1.6 h generation⁻¹ and lag phases between 5 and 8 h, comparable to growth on glucose as a sole carbon source. Only 10 strains, including those with “premier” (Q8r1-96) and “average” (Q2-87) rhizosphere competence, showed slower growth in wheat root exudates, with lag phases between 16 and 22 h. Results were the same when soil was added to the culture medium. Growth of four strains in media containing glucose or wheat or pea seed exudates as a sole carbon source was not influenced by whether the bacterial cells used as inoculum were harvested from wheat seeds or broth culture. We conclude that the superior ability of some strains to colonize the roots of certain crops cannot be explained by the utilization of the carbon sources tested in our study.     

海藻糖和甘油相互协同提高绒山羊精液冷冻保存品质

本实验旨在研究不同浓度的海藻糖和甘油相互协同对绒山羊精液冷冻保存的影响。将0 mmol/L (T0)、50mmol/L(T50)的海藻糖和0%(G0)、1%(G1)、2%(G2)、3%(G3)的甘油分别组合为T0G0、T0G1、T0G2、T0G3、T50G0、T50G1、T50G2和T50G3。以Tris-柠檬酸-葡萄糖(TCG)为基础稀释液,分析不同组合对绒山羊精液冷冻保存的影响。检测解冻后精子活力、运动参数、DNA完整率、顶体、质膜完整率、抗氧化水平。结果表明:海藻糖和甘油联合添加时精子冷冻保存效果显著高于单独添加海藻糖或甘油组,其中T50G1组作用最显著。在基础稀释液中添加50 mmol/L(T50)的海藻糖时,精子冷冻解冻后的活力、质膜完整率随着甘油浓度的升高而降低,而在基础稀释液中不添加海藻糖时,精子冷冻解冻后的活力、质膜完整率随着甘油浓度的升高而升高。综上表明,海藻糖和甘油通过协同作用提高精子冷冻保存效果,两者发挥作用的最适浓度为50 mmol/L海藻糖和1%甘油,并且海藻糖和甘油协同作用对精子活力以及质膜完整性具有浓度依赖性。     

Trehalose Synthase Gene Transfer Mediated by Agrobacterium tumefaciens Enhances Resistance to Osmotic Stress in Sugarcane

Trehalose synthase gene (TSase) from Grifola frondosa was transferred into sugarcane (Sac-charum hybrid L. ) using Agrobacterium-mediated method to improve sugarcane drought-tolerance. The re-sults indicated that embryogenic callus of sugarcane was sensitive to A. tumefaciens EHA105 strain in thetransformation system employed. The high frequency PPT-resistant plants were obtained from transformatedwith 3 weeks callus after incubation, which reached 4.5 % on average. The transgenic plants were confirmedby PCR and Southern analysis. Some transgenic plants showed multiple phenotypic alterations and some plantsdemonstrated improvement tolerance to osmotic stress.     

耐铝大豆BX10铝胁迫下基因表达差异初步分析

以耐铝大豆种质BX10为材料,以0或50 μmol/L铝处理48h的根尖RNA为样品,反转录成cDNA第一链后,利用cDNA-RAPD技术研究铝处理下大豆基因表达差异的情况.结果表明:从72条随机引物中筛选出9条能产生稳定差异的引物,多态性比率为12.5％;9条随机引物共扩增得到19个差异表达的基因,其中,50μmol/L铝处理组中6个基因表达下调,2个基因不表达,10个基因表达上调,1个基因受铝诱导特异性表达.此结果可为进一步挖掘大豆抗铝基因提供候选靶标及耐铝机理的研究奠定一定的理论基础.     

响应面法优化海藻糖合酶产菌的发酵条件

为了优化响应面法海藻糖合酶产生菌pET-30a(+)/TreS的发酵培养基。用Plackett-Burman设计法评价发酵培养基的8个成分对海藻糖合酶的影响,然后用最陡爬坡实验确定重要成分在中心复合设计中的取值变化范围,最后用中心复合设计响应面法求出了重要成分的最佳浓度。结果表明：麦芽糖、蛋白胨和牛肉膏对海藻糖合酶酶活力影响显著(P＜0.05),是重要成分,其最佳浓度分别为：麦芽糖22.07 g/L、蛋白胨5.46 g/L、牛肉膏2.16 g/L。优化后的培养基酶活力达到0.826 U/mL,比基础发酵培养基培养重组菌的酶活力提高了2倍。说明数理统计实验设计及分析的方法成功地用于了海藻糖合酶基因工程菌pET-30a(+)/TreS的发酵培养基优化,为工业化发酵生产海藻糖合酶奠定了理论基础。     

外施海藻糖对高温胁迫下小麦幼苗膜脂过氧化的影响

在非生物胁迫条件下,海藻糖可以提高植物的抗逆性,减少逆境胁迫对植物组织的伤害,维持植物的相对正常生长。为了解其具体的作用机制,以小麦为材料,通过检测高温胁迫及室温恢复过程中丙二醛(MDA)含量、过氧化氢(H2O2)含量、超氧自由基(O·-2)含量、抗氧化物质[抗坏血酸(AsA)、还原型谷胱甘肽(GSH)]含量、抗氧化酶[超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)]活性及其基因转录的表达调控,以探究外源海藻糖对小麦幼苗抗氧化系统的影响。结果表明,在高温胁迫下外源海藻糖处理主要提高了AsA含量,增强了CAT和APX活性,同时上调了Mn-SOD、Cu/ZnSOD、CAT、POD和APX的相对表达量,从而降低了MDA及H2O2的产生。而随后的室温恢复过程基本与高温胁迫的结果一致,也提高了这些酶的基因转录水平以及AsA含量,主要差别是对抗氧化酶活性的影响,即室温恢复阶段外源海藻糖主要提高了POD和APX活性。综合来看,外源海藻糖在小麦幼苗的高温胁迫及室温恢复过程中,通过促进抗氧化酶基因的上调表达,提高抗氧化酶活性和抗氧化物质含量,以酶促和非酶促两种机制共同清除高温胁迫产生的活性氧,减少氧化胁迫的损伤,维持小麦幼苗的生长。     

干旱诱导的海藻糖和脱落酸对苹果品质的影响

为了研究在适度干旱条件下,苹果果实中海藻糖和脱落酸对果实品质提升所起的生理作用。以7～8a生的盆栽嘎啦Gala/M26苹果树为试材,采用高效液相色谱法和酶学测定法,测定不同程度干旱胁迫(田间最大持水量的75%～80%、60%～65%、50%～55%)和果心注射脱落酸(ABA)[0(蒸馏水)和1mmol/L]的果实的果糖、葡萄糖、蔗糖、山梨醇和海藻糖质量分数,内源ABA质量分数,海藻糖代谢相关酶[6-磷酸海藻糖合成酶(TPS)、6-磷酸海藻糖磷酸酯酶(TPP)和海藻糖酶(TRE)]活性。结果显示,干旱处理和ABA处理均使果实内源ABA质量分数增加,有利于果实内糖分的积累,促进TPS和TPP活性,但抑制海藻糖酶的活性。适度干旱条件下所产生的ABA影响海藻糖代谢途径的改变,促进海藻糖的积累,有利于果实中糖分的积累,提升果实品质。     

超高浓度酒精发酵条件下酵母菌生理代谢的变化

随着葡萄糖浓度的增加,酒精产率不断增加,当葡萄糖浓度为30%(W/V)时,终点酒精浓度最大,达到13.4%(V/V)。胞内海藻糖的积累与起始葡萄糖浓度成正比,在葡萄糖浓度为30%和36%(W/V)发酵液中的菌体的胞内海藻糖含量分别是20%(W/V)的2倍和2.4倍。海藻糖积累量一般在葡萄糖利用率达到48%时达到稳定,此时的残糖浓度仍然较高,且酒精浓度仍在增加。