AOT-异辛烷反胶束萃取技术分离牛初乳IgG的研究

在一个琥珀酸二(2-乙基己基)酯磺酸钠(AOT)-异辛烷反胶束体系中，研究了水相pH值、离子强度和有机相表面活性剂AOT浓度等因素对牛初乳乳清蛋白中IgG分离效果的影响。结果表明，在以下两组优化条件：①pH值为7．668，[Na^ ]浓度为0．270mol／L，[AOT]浓度为0．088mol／L；②pH值为6524，[Na^ ]浓度为0．205mol／L，[AOT]浓度为0．175mol／L，水相IgG的残留率或纯度分别达到最大值，分别为90．23％和98．35％．RID分析表明，反胶束萃取过程对原牛初乳乳清IgG的免疫反应活性基本无影响。     

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.     

One-step separation of lysozyme by reverse micelles formed by the cationic surfactant,cetyldimethylammonium bromide

Lysozyme was selectively extracted from reconstituted freeze-dried egg-white, using reverse micelles formed by the cationic surfactant, cetyldimethylammonium bromide (CDAB). The major egg-white proteins, including ovalbumin and ovotansferrin, were solubilized into the organic phase while lysozyme was recovered in the aqueous phase. The solubilization behaviours of proteins were manipulated by processing parameters, including pH and salt concentration in the aqueous phase and concentration of surfactant in the organic phase. The optimum extraction was achieved with sodium borate buffer (50 mM, pH 9, no added KCl) and organic phase containing 50 mM CDAB. After the forward extraction, 96% of total lysozyme activity was recovered. Lysozyme was efficiently purified, more than 30-fold with only a single forward extraction. The suggested extraction procedure has advantages in terms of time and cost compared to traditional reverse micellar extraction which requires both forward and backward extraction steps.     

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.     

Refolding of cytochrome c using reversed micelles

A novel protein refolding method using reversed micelles has been developed, which could replace the conventional dilution method using a buffer solution. The novel refolding method enables efficient refolding at a high protein concentration. In the present study, denatured bovine heart cytochrome c was directly solubilized in AOT reversed micelles using the solid-liquid extraction technique. Results reveal that addition of urea in small amounts facilitates solubilization of denatured protein into the reversed micellar phase. Reversed micelles containing a high concentration of denatured cytochrome c could be easily prepared by the novel solubilization method. The nanostructural environment formed by the surfactant molecules in organic media is considered to promote the renaturation of denatured proteins because the protein molecules are isolated from each other through the solubilization step. Although the recovery of entrapping proteins from the reversed micellar phase was known to be difficult in a conventional reversed micellar extraction operation, the addition of alcohol to the recovery phase improved the efficiency of back extraction. Therefore, we succeeded in recovering renatured cytochrome c from the reversed micelles. We demonstrated that the novel protein refolding method is very useful for the renaturation of denatured proteins.     

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%。     

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.     

Thermal Stability of Bovine Milk Immunoglobulin G (IgG) and the Effect of Added Thermal Protectants on the Stability

D and z values and some thermodynamic parameters of immunoglobulin G (IgG) in phosphate buffer solution (PBS) (0.15 M NaCl/0.01 M phosphate buffer, pH 7.0) and colostral whey, with or without the presence of thermal protectants, were calculated in model systems. The D and z values for separated IgG in PBS were much lower than those for separated IgG in 20% glycerol, whey, and whey with 20% glycerol. IgG in colostral whey showed larger D and z values with the protectants. The heat denaturing rate constants at 70-82°C for separated IgG in PBS were larger than those of IgG in colostral whey; and the energies of activation for separated IgG in PBS, 0.2% glutamic acid, 10% whole milk, 20% maltose and 20% glycerol were also larger.     

Selective Solubilization of α- Lactalbumin and β-Lactoglobulin into Reversed Micelles from Their Mixtures

Phase transfer experiments were performed, involving contact between an aqueous 1:1 solution of α-lactalbumin and β-lactoglobulin and an AOT-in-isooctane reversed micellar phase. The resulting extraction and separation of the two proteins were analyzed as functions of pH, ionic strength and total protein concentration using SDS-PAGE, and compared with extractions from pure solutions. At low protein concentrations, the extent of reversed micellar solubilization of the two pure proteins predicted well the extraction from mixtures. However, at higher protein concentrations β-lactoglobulin appeared to be excluded from the micellar droplets. Because of the significantly different partitioning behavior of the two proteins, reversed micellar extraction from an initially equal weight mixture led to an effective separation of the proteins.     

Effects of extraction temperature,ionic strength and contact time on efficiency of bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) reverse micellar backward extraction of soy protein and isoflavones from soy flour

BACKGROUND: Soy protein enriched with isoflavones has been linked to various disease‐preventing and health‐promoting activities owing to the antihypertensive, hypocholesterolaemic, antiobesity and antioxidative properties of isoflavones. The isoflavone profiles of soy‐based products are known to be highly dependent on the various chemical and physical treatments to which the products have been subjected. The aim of this research was to increase the efficiency of backward extraction of soy protein and isoflavones from bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) reverse micelles by studying the effects of extraction temperature, ionic strength of the aqueous stripping solution and contact time on the amounts of soy protein and isoflavones backward extracted from an AOT/H₂O/isooctane reverse micellar system. RESULTS: By modifying the extraction temperature, ionic strength and contact time, 47.0–60.2% of protein, 43.3–68.4% of daidzin, 43.8–74.6% of genistin, 39.0–88.8% of glycitin, 20.8–92.6% of malonyl genistin, 20.2–52.0% of malonyl glycitin, 32.7–75.6% of acetyl genistin, 49.7–76.8% of daidzein and 19.6–38.1% of genistein present in the AOT reverse micellar solution were backward extracted into the aqueous stripping phase. Statistical analysis showed that there were significant linear and interactive effects of temperature and contact time on the backward extraction of daidzin, genistin, glycitin and daidzein. Significant linear and interactive effects of ionic strength and contact time were found in the backward extraction of daidzin and genistin. The backward extraction of genistein was only influenced by contact time and its interaction with temperature. CONCLUSION: This study showed the potential of reverse micelles as a protocol for extracting isoflavones from soy samples for analytical purposes. By modifying the extraction temperature, contact time and ionic strength, soy protein enriched with daidzin, genistin, daidzein and genistein could be produced from soy flour. The results represent an important contribution to current knowledge on utilising reverse micellar extraction in food technology. Copyright © 2007 Society of Chemical Industry     

Influence of milk pre-heating conditions on casein–whey protein interactions and skim milk concentrate viscosity

The viscosity of concentrates (50–55% total solids) prepared from skim milk heated (5 min at 80 or 90 °C) at pH 6.5 and 6.7 was examined. The extent of heat-induced whey protein denaturation increased with increasing temperature and pH. More denatured whey protein and κ-casein were found in the serum phase of milk heated at higher pH. The viscosity of milk concentrates increased considerably with increasing pH at concentration and increasing heating temperature, whereas the distribution of denatured whey proteins and κ-casein between the serum and micellar phase only marginally influenced concentrate viscosity. Skim milk concentrate viscosity thus appears to be governed primarily by volume fraction and interactions of particles, which are governed primarily by concentration factor, the extent of whey protein denaturation and pH. Control and optimization of these factors can facilitate control over skim milk concentrate viscosity and energy efficiency in spray-drying.     

Effects of AOT reverse micelle on properties of soy globulins

This study was focused on the influence of AOT reverse micelle on physical–chemical properties of 7S and 11S globulins from soy proteins, and compared with aqueous buffer extraction. The results showed that the contents of the surface hydrophobicity and SH groups of 7S and 11S globulins and SS bonds of 11S globulin, using AOT reverse micelle extraction, were augmented, and SS bonds of 7S globulin decreased. The thermal and rheological properties of 7S and 11S globulins extracted using the two methods were studied by differential scanning calorimetry (DSC) and rheometery. It was found that the peak denaturation temperature and heat of transition of 7S and 11S globulins with aqueous buffer extraction were different from that with AOT reverse micellar extraction. The AOT reverse micelle did not affect the gel properties of 11S globulin, while it influenced 7S globulin’s. Hardness, springiness, gumminess, adhesiveness and chewiness of 7S globulin from AOT reverse micelle were lower than that from aqueous buffer extraction, but gumminess was higher.     

FTIR spectroscopic characterization of soy proteins obtained through AOT reverse micelles

Fourier transform infrared (FTIR) method was used to study the conformations of soy protein obtained through aqueous solution and bis(2-ethylhexyl) sodium sulfosuccinate (AOT) reverse micelle extraction. The results showed that there were changes in signal intensity and/or position of IR bands at 4000–400 cm⁻¹ when the soy proteins were separated by two extraction methods. The FTIR spectral changes were subsequently assessed using the second derivative spectroscopy in the amide I region (1700–1600 cm⁻¹). The contents of α-helix, β-sheet, turn and irregular conformations for soy proteins using aqueous solution extraction were 11.6%, 32.8%, 44.3% and 11.3%, respectively; while using AOT reverse micelle extraction 13.1%, 41.6%, 32.5% and 12.9%, respectively. The amount change of these structures might affect functional properties of soy proteins.     

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。     

反胶束法萃取玉米胚芽蛋白及其功能性研究

研究了AOT/异辛烷反胶束法萃取玉米胚芽蛋白及玉米胚芽蛋白的加工功能性。在实验中分别考察了纤维素酶加酶量、AOT浓度、KCl浓度、缓冲液pH值、W0对玉米胚芽蛋白前萃率的影响,以及萃取时间、KCl浓度、缓冲液pH值对后萃率的影响,确定了前萃的最佳技术条件:加酶量为4 000 IU/g玉米胚芽、AOT浓度为3 g/50 mL异辛烷、萃取pH 6、KCl浓度0.1 mol/L、W0为25;后萃的最佳技术条件为:KCl浓度为0.5 mol/L、萃取pH 10.5,萃取时间40 min;对玉米胚芽蛋白的部分加工功能性进行研究,结果表明其吸油性(2.9 mL/g)、乳化性(54.5%)、乳化稳定性(86.5%)以及泡沫稳定性(58.3%)都较好,但吸水性和起泡性相对较差,玉米胚芽蛋白不但营养效价高,而且具有较好的加工功能特性,在食品工业中具有应用潜力。     

Isolation of bovine immunoglobulins and lactoferrin from whey proteins by gel filtration techniques

Whey is a suitable source of immunoglobulins and lactoferrin to enrich infant formulas. Gel filtration on Sephacryl S-300 and on Fractogel TSK HW-55 was used to isolate immunoglobulins from colostral whey, acid whey, and Cheddar cheese whey. The SDS-PAGE and immunoelectrophoresis techniques indicated that the purity of the fractions from fractionation on Sephacryl S-300 was better than that by fractionation on TSK HW-55 column. Biological activity of fractions from the Sephacryl S-300 column as assessed by immunochemical analysis was 99, 83.3, and 92% for colostral, acid, and sweet wheys. The well-proven antimicrobial agent, lactoferrin, was isolated from sweet whey by heparin-attached Sepharose. Lactoferrin selectively adsorbed to the column was subsequently eluted with 5 mM Veronal-HCl containing .5 M NaCl, pH 7.4. Purity of the isolated protein was confirmed by SDS-PAGE and immunoelectrophoresis.     

Clarification of Cheese Whey by Aqueous Two-Phase Systems

Aqueous two-phase systems comprising a pair of a polymer and a salt were investigated as a means to clarify Cheddar cheese whey. Fat in cheese why could partition exclusively into the bottom phase of a polyethylene glycol/KH₂PO₄ aqueous two-phase system, resulting in a clear top phase containing whey proteins. Several parameters have been studied to optimize the recovery of whey proteins in such a system. Decreasing solute concentrations to 11.7% polyethylene glycol/10% KH₂PO₄, lowering pH to 3.8, and lowering temperature to 7°C all contributed to enhance the yield. This method should be able to remove ca. 98% fat in Cheddar cheese whey and to recover > 90% whey proteins.     

黏度法对AOT/异辛烷反胶束体系的研究

利用黏度法测定AOT/异辛烷反胶束溶液的黏度,讨论了含水量对反胶束溶液的黏度、水池直径和反胶束聚集数的影响。由此解释了用AOT/异辛烷反胶束溶液萃取大豆蛋白时,萃取率变化的原因。     

Selective Enrichment of Proteins Using Formed-In-Place Membranes

A process for selective enrichment of immunoglobulins has been developed using formed-in-place membranes on sintered stainless steel tubes. The process employs both size exclusion and protein charge manipulation around the isoelectric point of proteins to selectively reject or pass components of the stream. Volume recoveries of 95% (20X concentration) could be achieved with a single filtration without loss of fractionation capabilities. The large initial volume reduction allows for small subsequent diafiltration volumes and insures optimization of fractionation while minimizing total processing volume. The process has been used to enrich proteins representing a small percentage of the total protein from a large feed volume. As an example, immunoglobulin G (IgG) from cheese whey was enriched from 8% to 20% of total whey protein with a 90% recovery of the IgG.     

反胶束提取小麦胚芽蛋白的工艺研究

以副产品小麦胚芽为原料,用反胶束法研究了小麦胚芽蛋白的提取条件,包括前萃和后萃。前萃由琥珀酸二(2-乙基己基)酯璜酸钠(AOT)-异辛烷-氯化钾缓冲溶液组成的反胶束体系从小麦胚芽中提取蛋白;后萃先回收异辛烷,少量KCl的缓冲溶液溶解剩余的固形物,最后用丙酮沉淀得小麦胚芽蛋白。通过单因素实验,考查了AOT浓度、萃取时间、加入小麦胚芽粉量、温度、KCl浓度、缓冲溶液pH、W/O对小麦胚芽蛋白前萃率的影响以及KCI浓度、缓冲液pH、缓冲液加入量对小麦胚芽蛋白后萃率的影响。