Reversed Micellar Extraction of Hen Egg Lysozyme

Egg white was diluted to 10 times its original volume with 50 mM phosphate buffer (pH 9.2) containing 0.1 M potassium chloride. The aqueous solution was mixed with an equal volume of isooctane containing 50 mM bis-(2-ethylhexyl) sodium sulfosuccinate at 10°C for 50 min. After extraction, the organic phase containing lysozyme was separated from the aqueous phase and mixed with an equal volume of 50 mM phosphate buffer (pH 11.8) containing 1M potassium chloride. Backward extraction was then performed at 30°C for 45 min. The procedures recovered 90% lysozyme from the eggwhite. The specific activity of the extract was near 73,000 units/mg.     

混合型反相微乳体系萃取茶渣蛋白的工艺优化

使用双（2-乙基己基）磺基琥珀酸钠（AOT）、十六烷基三甲基溴化铵（CTAB）、十二烷基硫酸钠（SDS）分别和吐温80（Tween80）混合制备了三种反相微乳体系,并通过单因素实验研究了表面活性剂浓度、离子型表面活性剂含量、水分含量（W₀）、水相pH、萃取温度和萃取时间等因素对蛋白质前萃率和KCl浓度、水相pH和萃取温度对蛋白质后萃率的影响,然后通过正交试验得到了最佳萃取条件。结果表明,Tween80-CTAB微乳体系对茶渣蛋白的提取效果较好,在表面活性剂浓度0.10 mol/L,离子型表面活性剂含量70%,水相pH13.0,W₀ 25,萃取温度40 ℃,萃取时间为40 min的最佳条件下,茶渣蛋白的前萃率达到最大值16.17%,其后萃率在KCl浓度为1.2 mol/L,pH7.0,提取温度40 ℃的最佳条件下可达到94.78%。SDS-PAGE电泳图的结果表明,反相微乳萃取得到的茶渣蛋白分子条带较小,即能够选择性的萃取小分子蛋白。     

Convenient partial purification of polyphenol oxidase from apple skin by cationic reversed micellar extraction

Polyphenol oxidase (PPO) was selectively extracted from reconstituted freeze-dried apple skin using reverse micelles formed by a cationic surfactant, dodecyl trimethyl ammonium bromide (DTAB). An optimum forward extraction was achieved with sodium phosphate buffer (pH 6, 100 mM, no added KCl) and an organic phase (isooctane:hexanol at a ratio of 5:1) containing 100 mM DTAB. The solubilised PPO was efficiently recovered by a stripping solution (pH 6, 1 M KCl) containing 10% ethanol. Under the optimised conditions, the purification fold and recovered activity of PPO were 12.6% and 71%, respectively. This purification fold and recovery were maintained when the extraction volume increased from 10–200 ml. Overall, reversed micellar extraction can be used as an efficient first step for the purification of PPO from apple skin.     

Extraction of immunoglobulin-G from colostral whey by reverse micelles

Separation of immunoglobulin G (IgG) from the other colostral whey proteins was carried out by reversed micellar extraction. The colostral whey was diluted to 5 times its original volume with 50 mM phosphate buffer at pH 6.35 containing 100 mM of sodium chloride. The aqueous solution was then mixed with an equal volume of isooctane containing 50 mM bis-(2-ethylhexyl) sodium sulfosuccinate (AOT), and shaken at 200 rpm and 25 degrees C for 10 min. After extraction, the mixture was separated to the aqueous phase and the reversed micellar phase by centrifugation. This procedure extracted most of the non-IgG proteins to the reversed micellar phase and recovered more than 90% of the IgG in the aqueous phase. The IgG in the aqueous phase had a purity of 90%, and still possessed immunological activity. AOT was not detectable in the aqueous phase.     

Selective Precipitation of Lysozyme from Egg White Using AOT

ABSTRACT: Lysozyme from egg white was purified using the anionic surfactant, di-(2-ethylhexyl) sodium sulfosuccinate (Aerosol-OT or AOT), as a precipitating ligand. Upon addition, AOT formed an insoluble lysozyme-surfactant complex with lysozyme and precipitated from the egg white solution. The lysozyme was then recovered as a solid from the insoluble complex using acetone, while AOT remained in the acetone phase. The precipitation efficiency was highest when the pH of the egg white solution was between 6 and 9. A molar ratio of 15 between surfactant and lysozyme provided 80% precipitation efficiency. The recovered lysozyme product was free of surfactant, and retained its original enzymatic activity.     

Separation of gamma-globulins from porcine plasma by reversed micellar extraction

Factors affecting the separation of gamma-globulins from porcine plasma using reversed micelles were screened based on a fractional factorial design. The optimal processing conditions for obtaining the maximum yield of gamma-globulins under various constraints of product purity were determined using response surface methodology (RSM) and nonlinear programming. Results showed that the pH and sodium chloride concentration of the aqueous phase, and the concentration of surfactant (bis-(2-ethylhexyl) sulfosuccinate sodium salt, AOT) of the organic phase were the most important factors affecting the extraction performance. An eighty-five percent product purity and ninety-seven percent yield were obtained under the extraction conditions of 400 mM NaCl, 350 mM AOT, and pH 7.0. The extract exhibited immunological reactivity against anti-pig IgG.     

Reverse micellar extraction of bovine serum albumin – A comparison between the effects of gemini surfactant and its corresponding monomeric surfactant

Gemini surfactant displayed distinct advantages over monomeric surfactant in the liquid–liquid reverse micellar extraction process. First, less amount of gemini surfactant than monomeric surfactant was needed for transferring almost complete bovine serum albumin (BSA) into organic phase from aqueous phase. Second, the loading capacity of gemini surfactant reverse micelle phase was much higher than that of the corresponding monomeric surfactant reverse micelle. Third, efficient backward extraction (75–92%) of BSA could be effected in a wide pH range from 4 to 9 with gemini surfactant reverse micelle while a pH of ca. 4.3 is prerequisite to the recovery of BSA from monomeric surfactant reverse micelle. So far, the reports about the effect of surfactant structure on protein extraction have been limited. This study indicates the important role of the spacer of gemini surfactant in protein extraction process and may provide more knowledge on how to optimise surfactant structure.     

反胶团萃取分离葡萄酒下脚料中葡萄皮蛋白质

采用阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)反胶团萃取葡萄皮中的蛋白质。分别考察水相pH值、表面活性剂浓度、离子强度、蛋白质浓度、有机溶剂与助剂种类和比例、萃取温度对前萃取率的影响,并采用二次正交旋转组合的试验方法,考察前萃取过程中离子强度、表面活性剂浓度、水相pH值对前萃取率的影响。结果表明:32.68 mmol/L CTAB/三氯甲烷-正丁醇(体积比4∶1)的反胶团体系用于前萃取水相pH为6.4,包含0.4 mol/L NaCl的葡萄皮中蛋白质的浸提液,前萃取率可达56.54%。理想的前萃取条件是:pH 6.4、NaCl浓度0.4 mol/L、CTAB浓度32.68 mmol/L、蛋白质浓度1 mg/mL、萃取温度30℃。在该条件下,采用pH 3.8、后萃取温度20℃、0.8 mol/L NaCl水相进行后萃取,后萃取率可达71.29%。     

Production of corn zein microparticles with loaded lysozyme directly extracted from hen egg white using spray drying: Extraction studies

Feasibility of achieving sustained release of lysozyme by encapsulation in corn zein using spray drying was examined. To reduce the materials cost, this part of work focused on partially purifying lysozyme from hen egg white using 30–90% (v/v) aqueous ethanol, adjusted to pH 3.0–9.24. After extraction for up to 24 h and centrifugation, the purification performance was evaluated for the supernatant. Extraction was insignificantly affected by kinetics (P = 0.6186) but was inefficient at pH above 5.0 and above 60% ethanol. The optimal extraction was achieved at 50% ethanol and pH 3.5. Further, most of the lysozyme precipitated from the 50% ethanol (at pH 3.5) extract after increasing ethanol concentration to 90% but was completely recovered after diluting the precipitate back to 50% ethanol. Findings from this work may lead to low-cost encapsulation technologies using partially-purified lysozyme, such as spray drying.     

混合反胶团提取α-淀粉酶

以十六烷基三甲基溴化铵(CTAB)和脱水山梨醇单硬脂酸酯聚氧乙烯醚(Tween-60)为混合表面活性剂溶于正丁醇-异辛烷中构成反胶团系统,萃取纯化α-淀粉酶。研究不同萃取条件下,α-淀粉酶的萃取率。其中反胶团相组成为：ρ(CTAB+Tween-60)＝4g/L;n(CTAB):n(Tween-60)＝2.0:1.0;V(正丁醇):V(异辛烷)＝1.0:1.0。水相组成为：α-淀粉酶配制的粗酶液,此时c(NaCl)＝0.04mol/L,水相pH11.04。结果表明：萃取温度40℃、V(有机相):V(水相) ＝2.0:1.0、振荡时间10min时,α-淀粉酶萃取率可达91%;反萃取水相组成为c(NaCl)＝2.5mol/L、pH4.5、V(水相):V(有机相)＝1.0:2.0,反萃取振荡时间10min、温度50℃时,α-淀粉酶反萃取率可达65%。反胶团相可重复使用,当V(水相):V(有机相)＝1.0:2.75时,反胶团第2次α-淀粉酶萃取率达到71%。     

反胶束萃取蛋白质的动力学研究进展

介绍了反胶束的特点及其溶解和萃取蛋白质的原理,详述了影响反胶束萃取的主要因素,包括水相的pH值、离子强度和阳离子种类、蛋白质的分子量和浓度以及表面活性剂的类型和浓度,综述了国内外对反胶束萃取蛋白质的动力学研究进展、反胶束萃取蛋白质的推动力、蛋白质在反胶束中的三种增溶模型以及研究中存在的一些需亟待解决的问题。     

新型离子液体亲和萃取鸡蛋清中的溶菌酶

建立并优化利用辛巴蓝(Cibacron Blue F-3GA,CB)修饰[C₄MIM]Cl的新型离子液体[C₄MIM]₃CB特异性萃取分离鸡蛋清中溶菌酶的方法,并采用AutoDock Vina分析[C₄MIM]₃CB与蛋白的结合能,研究溶菌酶与[C₄MIM]₃CB相结合的能力。通过亲和萃取和反萃取并进一步脱盐、浓缩、冷冻干燥,得到溶菌酶产品。结果表明,通过对影响因素的研究,优化出最佳的萃取分离条件:蛋清粉溶液pH为7,新型离子液体中[C₄MIM]₃[CB]浓度为8 mg/mL,反应震荡时间为15 min,反萃取KCl溶液pH为7.0,浓度为1.5 mol/L。最终250 mg蛋清粉可得12.3 mg的溶菌酶产品,纯度为97.56%,酶活力为35000 U/mg,纯化倍数达到37.39,酶活回收率89.74%,且与其他蛋白达到完全分离。该方法具有选择性高、操作简便、产物纯度和活性高等优点。     

Gemini型阳离子表面活性剂反胶束体系萃取纤维素酶的研究

本文研究了Gemini型阳离子酯季铵盐表面活性剂Ⅱ-14-3反胶束萃取纤维素酶的性能,以探索新型表面活性剂在反胶束萃取酶蛋白中的应用。考察了水相pH、离子强度、离子种类、酶浓度、表面活性剂浓度、助溶剂浓度、溶剂比和助表面活性剂(卵磷脂)等因素对萃取率的影响,确定了萃取纤维素酶的最佳条件:[NaCl]=50mmol/L,[Ⅱ-14-3]=0.3mmol/L,pH6.4,C0=0.14,溶剂比S=1.0,萃取率E接近80%,其酶活达到原来的93.38%;若加入适量的卵磷脂([Ⅱ-14-3]/[PC]=36:1)可提高萃取率,萃取率E达90%以上,且酶活达到121.41%。并且从反胶束微观结构给予解释。     

蛋清溶菌酶提取工艺研究

采用盐析法从鸡蛋清中提取溶菌酶,探讨不同条件对溶菌酶收率等的影响,确定了最优操作参数。最佳提取条件为:蛋清浓度100%,氯化钠浓度5%,盐析pH10.5,盐析时间120h,提取收率在0.37%。采用比浊法对提取的溶菌酶进行酶活力的测定,酶活为13500U/mg。     

One-step separation of β-galactosidase from β-lactoglobulin using water-in-oil microemulsions

Solubilisation of β-galactosidase from Kluyveromyces lactis in Aerosol-OT water-in-isooctane microemulsions was measured as a function of buffer type and concentration, pH, and protein concentration. For buffer concentrations above ∼40 mM, the enzyme was largely excluded from the droplets. Based on these results, a one-step separation was developed. A protein-containing aqueous feed was injected into an AOT/isooctane solution, with the feed volume slightly in excess of the predicted water solubility. Impurity proteins were entrapped inside the microemulsion droplets that then formed in the organic phase, while the high MW target protein was excluded and entered a newly formed, excess aqueous phase. The separation of β-galactosidase from the test protein β-lactoglobulin was most complete at 100 mM KCl salt concentration, where the droplets were large enough to carry β-lactoglobulin but too small for β-galactosidase. At 100 mM [KCl], 92% of the total enzyme activity was recovered in a concentrated and virtually pure form.     

Purification,characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39.3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56.5% homology with bovine milk lysozyme and 30.4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.     

Kinetics of Protein Extraction in Reverse Micelle

Sodium bis (2-ethylhexyl) sulfosuccinate (AOT)-sodium dodecyl sulfate (SDS)/isooctane-octanol reverse micelle extraction was tested an efficient and effective approach to separate peanut protein from full-fat peanut powder. Here, important kinetic factors including pH, ion strength, and temperature were studied during reverse micelle backward extraction. The extraction conditions were obtained by response surface experiments as follows: pH 7.5, ion concentration 1.1 mol/L at temperature 35°C. Under these optimum extraction conditions, the extraction rate of protein reached 79.03%. A model on the kinetic partitioning of peanut protein was also developed. The backward extraction in this reverse micelle system was controlled by interfacial resistance instead of diffusion resistance in reverse micelle and aqueous phase with the total mass transfer rate of 0.8×10^?5 m³·s^?1. A two-film theory may be the mechanism for flat interface. Results of mass transfer process are helpful for creating an reverse micelle extraction process, and used for purification of peanut proteins, promoting the development of food industry.     

CTAB反微团萃取大豆蛋白

本文考察了CTAB-反微团从水溶液中萃取大豆蛋白质的影响因素，因素包括：萃取时间、离子强度、有机相中表面活性剂(CTAB)浓度、助溶剂配比、水相pH等，并提出了最佳的萃取条件。最后从反微团微观结构给予解释。     

Removal of ionic dyes from water by solvent extraction using reverse micelles

Several methods (e.g., UV/H2O2 oxidation, adsorption, flocculation-precipitation) are normally employed to remove dye from water. A new technique based on liquid/liquid extraction using reverse micelles is proposed whereby recovery of solvent and reuse of dye is possible. Experiments were conducted by mixing a known quantity of dye in aqueous phase and solvent-containing surfactants in a simple mixer. The separation of solvent phase, containing encapsulated dye in reverse micelles, from aqueous phase due to gravity results in separation of dye from water. The removal of different ionic dyes (e.g., eosin yellow, methylene blue, malachite green, methyl orange, orange G) from aqueous phase in the presence of different cationic and anionic surfactants [e.g., sodium dodecylbenzene sulfonate, sodium bis(2-ethylhexyl) sulfosuccinate, hexadecyltrimethylammonium bromide, and cetyl pyridinium chloride] in different solvents (e.g., amyl alcohol, benzyl alcohol, methyl benzoate, and isooctane) were studied by conducting experiments. The percentage removal of dye from aqueous phase increases with the decrease in dye concentration or with the increase in surfactants concentration. Furthermore, the percentage COD removal of dye is increased with the increase in surfactant concentration. The nature of solvent has minimal effect on percentage removal of dye. The ratio of solventto aqueous phase volume required for the removal of dye decreases with the increase in surfactant concentration. It is possible to back-extract dye into aqueous phase and recover solvent by using counterionic surfactants. The separation of aqueous phase from the aqueous-phase solvent dispersion is faster for amyl alcohol as compared to benzyl alcohol and methyl benzoate. A theoretical model based on ion-exchange reaction between surfactants and dye is used to analyze the experimental data.     

Heat stability and reactivation of mare milk lysozyme.

Mare milk and aqueous solution of mare milk lysozyme were incubated for variable times between 30 C and 100 C at pH 3, 6, or 9. Lysozyme activity was stable at acid and neutral pH and labile at alkaline pH. Some of the results show the existence of a reactivation process in mare's milk and in aqueous solution. reaching 30 to 40% after incubation of the aqueous solution at 4 C for 20 days at pH 3 or 6.