双水相系统纯化山楂叶中黄酮类物质的研究

应用水、乙醇和硫酸铵三者形成双水相系统,并利用黄酮在乙醇中的溶解度要大于水中的溶解度,以脱除其中一些易溶于水和在高浓度乙醇中溶解度低的物质。尝试了不同盐、不同体积比的乙醇和水、以及盐加入量对分相的影响并测定了其在不同情况下的纯化效果。发现在硫酸铵中的分相情况最好,在加入量到2．5g时既可以达到分相完全。并测定：（1）在5mL乙醇＋5mL水＋5g硫酸铵＋0．15g粗体物（粗体物中黄酮的百分含量为25％）的双水相中,黄酮类物质的回收率为89．2％,而黄酮百分含量提升为47．6％;（2）在4mL乙醇＋5mL水＋5g硫酸铵＋0．15g粗体物（粗体物中黄酮的百分含量为25％）的双水相中,黄酮类物质的回收率为81．9％,而黄酮含量提升为50．9％。水、乙醇和硫酸铵三者形成双水相系统应用于山楂叶中黄酮类物质的纯化在文献中少有报道,而本文应用此法可以将黄酮含量从25％提升到将近47．6％,并且回收率为89％。     

脉冲回波法研究微乳液体系的稳定性

本文主要以正庚炕（C71116）、水乳浊液（以span-80为表面活性剂）为模型体系,研究了脉冲回波法探测微乳体系结构稳定性的可能性,并进行了初步讨论。实验结果表明,超声衰减系数（a）与微乳体系配比、表面活性剂含量及微乳液制备时搅拌转速密切相关。微乳液体系在实验当中均出现了由油和水两相混合的状态到油和水分相的状态。微乳液体系的超声衰减系数在分相前后均保持着相对平稳的变化,但对应于不同条件下制备的溶液,分相时间都有所不同。且一般而言,当微乳液开始两相（水相和油相）分离时,超声衰减系数将发生明显变化。因此预计脉冲回波法有可能发展成为一种简单、可靠、非破环性的微乳体系稳定性检测新方法。     

脱皮与不脱皮青稞酿造营养黄酒风味差异的研究

以青稞为原料酿造新型黄酒,为研究原料脱皮与否对新型黄酒风味的影响,利用气相色谱质谱联用(GC-MS)测定脱皮青稞黄酒、不脱皮青稞黄酒及青稞麸皮中挥发性和半挥发性风味物质。结果表明未脱皮青稞黄酒中香气物质总量为71 134.67μg/L;脱皮青稞酿造黄酒香气物质含量较少为39 208.99μg/L。挥发性香气成分在含量上差异主要来源于,3-甲基丁醇、苯乙醇、2-甲基丙醇。通过香气活力值OVA分析发现,对两种青稞黄酒香气贡献的较大的物质种类均为辛酸乙酯、乙酸异戊酯、苯乙醇和苯甲醛,贡献值的大小有差异。对麸皮挥发性风味成分分析发现,青稞黄酒中香气成分均是通过微生物作用产生,麸皮中富含的苯丙氨酸、缬氨酸和亮氨酸通过代谢产生苯乙醇、2-甲基丙醇、3-甲基丁醇,是造成两种青稞黄酒中挥发性风味物质差异的主要原因。研究表明青稞麸皮对黄酒风味的形成有积极意义。     

岩溶干旱胁迫下青冈栎水分参数变化

根据岩溶生境特点设计表层岩溶水-岩石(灰岩)-土壤水分供应分层模拟柱,对青冈栎(Cyclobalanopsis glauca)进行水分控制试验;运用压力容积(P-V)技术,研究了在水分胁迫下青冈栎的日水势(P)、饱和含水量时的最大渗透势(ψsats)、初始质壁分离时的渗透势(ψtlps)、初始质壁分离时渗透水相对含量(ROWCtlp)、初始质壁分离时相对含水量(RWCtlp)、质外体水的相对含量(AWC),以及饱和含水量时最大渗透势与初始质壁分离时的渗透势之差(△P)的变化.结果表明,在无表层岩溶水-岩石-土壤处理中,随着土壤干旱胁迫程度的增加,ROWCtlp、RWCtlp、ψsats、ψtlp呈明显下降,而AWC值上升;在有表层岩溶水-岩石-土壤处理下,青冈栎受土壤水分胁迫不明显,水分参数变化不显著.表层岩溶缺水时,土壤水分含量是影响青冈栎水分参数变化的主要因素,在一定的干旱胁迫范围内,随着干旱程度的提高,青冈栎耐旱性不断增加.     

四个桂花品种挥发油成分研究(英文)

采用顶空固相微萃取结合气质联用技术首次同时对桂花四大栽培品系代表品种白洁、败育丹桂、佛顶珠和金桂的挥发油成分进行对比研究。从四个代表品种中共分离鉴定了72个化学成分,其中,21个是共有成分,但这四个品系中还存在着不同。结果发现,α-甲基--α[4-甲基-3-戊烯基]环氧丙醇存在于4个品种中,是白洁、败育丹桂和佛顶珠的主要成分之一,含量高达14~16%,但在金桂中仅有3.94%;芳樟醇存在于四个样品中,在佛顶珠中含量最高(9.83%);3-乙烯基-3-甲基-环己烷酮(7.89%)仅存在于佛顶珠中,邻苯二甲酸二异丁酯仅存在于金桂中,并且含量最高(23.99%)。     

菜心中SDS-PAGE向负极泳动的富含苯丙氨酸的蛋白的发现

在提取乙醇酸氧化酶同工酶的过程中, 发现了一种在SDS变性下向负极泳动的富含苯丙氨酸的碱性蛋白.在3次重复实验中, 均可以从SDS-PAGE后的负极缓冲液中获得这种蛋白, 其碱性与酸性氨基酸残基的比例分别高达1.46、1.28和0.89,而苯丙氨酸含量分别高达为60.00%、65.62%和62.30%.其余氨基酸残基的含量则和普通蛋白的相近似.由于该富含苯丙氨酸的碱性蛋白是水溶性的, 碱性氨基酸残基应主要分布在蛋白的表面,而苯丙氨酸则组成一个强疏水内核.     

萃取破碎法提取酵母醇脱氢酶的研究

将双水相萃取同细胞破碎结合在一起,以水平搅拌式珠磨机为设备,以聚乙二醇和硫酸铵为成相体系,从酿酒酵母中以边破碎边萃取的方式提取醇脱氢酶。节省了萃取设备和时间,并利用双水相对蛋白质的保护作用提高了醇脱氢酶的活性。经过萃取破碎的酵母匀浆离心分相后,细胞碎片滞留在下相,90％以上的醇脱氢酶分配在上相,分配系数大于10,纯化倍数大于2。     

基于文献分析的中国猕猴桃属植物亚麻酸资源研究

通过调查和文献整理,对中国猕猴桃属植物资源中含α-亚麻酸的猕猴桃种类、分布特点以及α-亚麻酸含量、提取方法进行统计分析。结果表明,我国已报道富含亚麻酸的猕猴桃属共有11个物种、10个栽培品种;富含α-亚麻酸的猕猴桃植物资源主要分布在长江流域,少量分布在东北和西南地区;猕猴桃籽油中α-亚麻酸含量总体集中在40%~70%之间,属于高含量亚麻酸资源植物;不同提取方法对猕猴桃亚麻酸的提取效果有较大影响,以尿素包合法和分子蒸馏技术分离纯化的提取效果较好,提取的α-亚麻酸含量高达87.20%。     

利用大孔树脂同时制备穿心莲内酯和脱水穿心莲内酯

采用大孔吸附树脂色谱法,以不同体积分数乙醇水溶液进行洗脱,分离富集穿心莲内酯和脱水穿心莲内酯,并配以HPLC进行同步监控.结果表明,穿心莲中这2种主要有效成分分别得到了富集.40%乙醇水溶液洗去目标物以外的杂质后,45%乙醇洗脱液中富含穿心莲内酯,含量为46.99%,55%乙醇洗脱液中富含脱水穿心莲内酯,含量为79.74%.     

蓝细菌Anacystis nidulans R-2藻胆体中43 Kd蛋白细胞定位的初步研究

高盐浓度条件下分离了蓝细菌Anacystis nidulans R-2的藻胆体,藻胆体中存在一种43kD的蛋白。Western blotting分析表明,该蛋白能与蓝细菌Fd：NADP氧还酶中FNRE占构域的抗体发生反应,解聚的藻胆体具有FNR黄递酶的活性,初步证明该43kD蛋白就是Fd：NADP氧还酶。Triton X-114分相实验表明,这种43kD的蛋白不能进入Triton X-114相。对藻胆体的部分解聚合实验表明,富含外周杆的组分中不存在43kD的蛋白。     

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: II. Recovery and purification of pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae

The novel approach of using aqueous two-phase systems for the elution of protein from ligand-coupled particles is investigated using pyruvate kinase and alcohol dehydrogenase from recombinant Saccharomyces cerevisiae and Cibacron blue F3G-A-coupled Sepharose CL6B (Blue-Sepharose) particles as a model system. The ligand-coupled particles distribute quantitatively to the polyethylene glycol-(PEG-) rich top phase and the recovered enzymes partition selectively to the dextran-(DEX-) rich bottom phase. An effective recovery and partial purification of pyruvate kinase and alcohol dehydrogenase from Blue-Sepharose particles using PEG8000-DEXT500 aqueous two-phase systems are demonstrated through a modest increase of salt concentration. The bioselective eluting agent, MgADP, which is useful in chromatographic operations, is not required for the process using aqueous two-phase systems. Recovery of pyruvate kinase, which is bound to ligand-coupled particles, in the DEX-rich bottom phase of aqueous two-phase systems can be up to 95% in one-step operations. The mixing time of ligand-coupled particles with aqueous two-phase systems is a major controlling variable. The salt concentration, the molecular weight of polymer, and the total volume of aqueous two-phase systems also influence the recovery of pyruvate kinase from ligand-coupled particles. The recovered enzymes in the DEX-rich bottom phase remain biologically stable over a long period of storage time. The concentration of product protein in a reduced volume and the easy separation from ligand-coupled particles are added advantages of the process using aqueous two-phase systems. Preliminary studies with goat polyclonal anti-pyruvate kinase-coupled Sepharose particles indicate that the process also may be applicable when a high-affinity ligand such as antibody is used. The experimental results and a theoretical derivation based on equilibrium models for binding/dissociation of ligands and proteins show that the process results in better recovery as compared to that of conventional bulk elution techniques.     

Purification of plasma membranes by aqueous two-phase affinity partitioning.

A rapid method for purifying rat liver plasma membranes of high purity and yield is described. Squashed liver was homogenized in an aqueous polyethylene glycol-dextran two-phase system. After phase separation and reextraction of the bottom phase with fresh top phase, the combined polyethylene glycol-rich top phases were affinity partitioned in the presence of borate buffer with new bottom phase containing dextran-linked wheat-germ agglutinin. Under these conditions the lectin selectively pulled plasma membranes into the dextran-rich bottom phase, while other membranes preferentially distributed in the top phase. The lectin-containing bottom phase was reextracted with fresh top phase before collecting the purified plasma membranes by centrifugation. This protocol resulted in a preparation that was 30- to 40-fold enriched compared to the homogenate in plasma membrane markers for both the apical and basolateral domains and had yields of 55-70%. The contamination by other membranes was low. The entire procedure was completed within 90 min. The method should be useful for purifying plasma membranes also from other sources.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable alpha-amylase with temperature optimum at 80 degrees C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000-20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000-20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of alpha-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60-75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na2SO4.     

Ucon-benzoyl dextran aqueous two-phase systems: protein purification with phase component recycling

Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride. A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed. The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature. This two-phase system has been used to purify 3-phosphoglycerate kinase from bakers' yeast. The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature. The bottom-phase polymer (benzyol dextran) could be recovered by addition of salt. Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system. After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase. The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na₂SO₄.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable a-amylase with temperature optimum at 80 °C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000—20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000—20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of a-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60—75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na₂SO₄.     

Application of the response surface methodology for optimization of whey protein partitioning in PEG/phosphate aqueous two-phase system

In order to develop a new strategy for β-lactoglobulin (β-lg) removal from whey protein, partitioning of α-lactalbumin (α-la), β-lg and glycomacropeptide (Gmp) was studied using aqueous two phase systems (ATPS). A system composed of 13% (w/w) polyethylene glycol (PEG, average molar mass 2000 g/mol) and 13% (w/w) potassium phosphate was used at 25°C. A central composite rotatable design (CCRD) associated to the response surface methodology (RSM) was applied to investigate the effects of NaCl concentration and pH on the partition of these proteins. It was found that α-la and Gmp partitioned to the top phase rich in PEG, whereas β-lg partitioned to the bottom phase rich in salt. According to the RSM, optimal conditions for β-lg removal where found where pH was equal to 6.7 and salt concentration was 0.35 mol/L. Under these conditions, the partition coefficient K(α) was 0.48 and K(Gmp) was 0.92. On the other hand, the partition coefficient K(β) was only 0.01. In such conditions β-lg preferentially concentrates in the bottom phase, while the top phase exclusively contains the proteins α-la and Gmp. Fractionation of the proteins from fresh whey was performed in a three stage cross-flow extraction system. The extraction yield for β-lg in the bottom phase was 97.3%, while the yields for α-la and Gmp in the top phase were 81.1% and 97.8%, respectively.     

Enzyme purification using temperature-induced phase formation.

A new type of aqueous two-phase system composed of an ethylene oxide and propylene oxide random co-polymer, UCON 50-HB-5100, as the upper phase polymer and either dextran or hydroxypropyl starch as the lower phase polymer has been characterized and used to purify 3-phosphoglycerate kinase (EC 2.7.2.3) and hexokinase (EC 2.7.1.1) from bakers' yeast. The UCON 50-HB-5100 polymer has a cloud point of 55 degrees C at which temperature it phase separates from water. This cloud point can be lowered to 40 degrees C by the addition of 0.2 M sodium sulfate salt. The low cloud point of this UCON polymer makes it possible to obtain the target enzymes in a water and buffer solution, and to recover and recycle the UCON 50-HB-5100 polymer. The phase diagrams for the systems UCON 50-HB-5100/Dextran T500 and UCON 50-HB-5100/hydroxypropyl starch have been determined. Yeast homogenate was first partitioned in a system composed of a top phase containing UCON 50-HB-5100 and a bottom phase containing either dextran or hydroxypropyl starch. The top phase containing the enzyme free of cell debris was removed and the temperature increased above the cloud point of the UCON until a new two phase system composed of water as the top phase and a concentrated liquid UCON 50-HB-5100 bottom phase was formed. The water phase containing the enzyme was removed and the bottom phase containing the UCON 50-HB-5100 could be recycled to perform a second extraction.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Lipid-DNA and lipid-polyelectrolyte mesophases: structure and exchange kinetics.

Cationic lipid-DNA complexes are used as gene transfer vehicles in molecular biology and potentially in human gene therapy. In recent synchrotron X-ray scattering studies the molecular structure of such self-assembling aggregates was elucidated. A rich polymorphism of lamellar, hexagonal, lamellar-columnar and micellar mesophases was found. In this article we describe composite phases of cationic lipid mixed with hyaluronic acid and dextran sulfate which likewise form intercalated lamellar complexes. Heterogeneous phases of lipid/dextran sulfate mixed with lipid/DNA exhibit macroscopic phase separation. When dextran sulfate is added to preformed cationic lipid DNA complexes the latter are dissolved in favor of the lipid-polyelectrolyte phases. We investigated the kinetics of the DNA replacement by dextran sulfate. The experiments are intended to mimic the interaction of cationic lipid gene delivery complexes with highly charged extracellular matrix components.     

Phase separation rates of aqueous two-phase systems: correlation with system properties

The kinetics of phase separation in aqueous two-phase systems have been investigated as a function of the physical properties of the system. Two distinct situations for the settling velocities were found, one in which the light, organic-rich (PEG) phase is continuous and the other in which the heavier, salt-rich (phosphate) phase is continuous. The settling rate of a particular system is a crucial parameter for equipment design, and it was studied as a function of measured viscosity and density of each of the phases as well as the interfacial tension between the phases. Interfacial tension increases with increasing tie line length. A correlation that describes the rate of phase separation was investigated. This correlation, which is a function of the system parameters mentioned above, described the behavior of the system successfully. Different values of the parameters in the correlation were fitted for bottom-phase-continuous and top-phase-continuous systems. These parameters showed that density and viscosity play a role in the rate of separation in both top continuous- and bottom continuous-phase regions but are more dominant in the continuous top-phase region. The composition of the two-phase system was characterized by the tie line length. The rate of separation increased with increasing tie line length in both cases but at a faster rate when the bottom (less viscous) phase was the continuous phase. These results show that working in a continuous bottom-phase region is advantageous to ensure fast separation.