Size Changes of Reverse Micelles after Extraction of Peanut Protein and Their Forward Extraction Rates

The aim of this study was to detect the size changes of reverse micelles after extraction of peanut protein and their forward extraction rates. Factors that affect the size of reverse micelles and the extraction of peanut protein were also investigated. The size of reverse micelles and the size changes were measured according to the theory of dynamic light scattering under different conditions such as different sodium bis(2-ethylhexyl) sulfosuccinate(AOT) concentrations, p H values, ion concentrations, and salt species.With the increase of AOT surfactant concentrations in a certain range, the size of empty and full reverse micelles increased and the forward extraction rate decreased. The effect of pH on empty reverse micelles was not significant. However, the effect of pH on the full reverse micelle size and forward extraction rate were significant. Its forward extraction rate increased to the maximum39.6% at pH 7.5. The increase of the salt concentration of a buffer solution in a certain range decreased the size of empty and full reverse micelles and reduced the forward extraction rate of peanut protein. Ionic species had important effects on reverse micelles and peanut protein extraction. An increase in the amount of buffer solution enlarged the empty reverse micelle size in 0.03%-0.11%(V/V). However, it did not translate to a larger reverse micelle size. The size of the empty reverse micelles containing K_2SO_2 reached 24.1 nm with a 0.19%(V/V) buffer solution added. The sizes of the full reverse micelles were larger than those of the empty reverse micelles after forward extraction. However, maximum sizes were achieved with the addition of a 0.03%(V/V) buffer solution. The amount of 0.03%(V/V) buffer solution added was appropriate for extracting peanut protein.

Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM

In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning turmeling microscope（STM） was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic sodium （AOT）/iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of extraction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol·L^-1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca^2＋, Na^＋, K^＋ which were in the range of 0.2-1.0mol.L^-1, and influence of concentration of Ca^2＋ is greater than that of Na^＋, and K^＋ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were： AOT reversed micelles 0.05mol·L^-1, trypsin concentration in crude pancreatin solution 3mg·ml^-1, pH 5.2-- 5.3, ratio （by volume） of extraction phase to strip-extraction phase 1 ： 1, and time of 5min. The corresponding percentage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginlne ethyl ester （BAEE） U·mg^-1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol·L^-1 ceryl-trimethyl-ammonium bromide （CTAB）, total extraction percentage of trypsin was 74.18% and specific activity was 3148.3 BAEE U·mg^-1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sulfate-polyacryl amide gel electrophoresis （SDS-PAGE） and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.

Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM

In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning tunneling microscope(STM) was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic so- dium (AOT)/ iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of ex- traction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol·L-1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca2+, Na+, K+ which were in the range of 0.2—1.0mol·L-1, and influence of concentration of Ca2+ is greater than that of Na+, and K+ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were: AOT reversed micelles 0.05mol·L-1, trypsin concentration in crude pancreatin solution 3mg·ml-1, pH 5.2— 5.3, ratio (by volume)of extraction phase to strip-extraction phase 1︰1, and time of 5min. The corresponding per- centage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginine ethyl ester (BAEE) U·mg -1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol·L -1 ceryl-trimethyl-ammonium bromide (CTAB), total extraction percentage of trypsin was 74.18% and specific activ- ity was 3148.3 BAEE U·mg-1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sul- fate-polyacryl amide gel electrophoresis (SDS-PAGE) and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.

Mass transfer process of peanut protein extracted by bis(2-ethylhexyl)sodium sulfosuccinate reverse micelles

The liquid-liquid extraction method using reverse micelles can simultaneously extract lipid and protein of oilseeds,which have become increasingly popular in recent years.However,there are few studies on mass transfer processes and models,which are helpful to better control the extraction process of oils and proteins.In this paper,mass transfer process of peanut protein extracted by bis(2-ethylhexyl)sodium sulfosuccinate(AOT)/isooctane reverse micelles was investigated.The effects of stirring speed(0,70,140,and 210 r/min),temperature of extraction(30,35,40,45,and 50℃),peanut flour particle size(0.355,0.450,0.600,and 0.900 mm)and solidliquid ratio(0.010,0.0125,0.015,0.0175,and 0.020 g/mL)on extraction rate were examined.The results showed that extraction rate increased with temperature rising,particle size reduction as well as solid-liquid ratio increase respectively,while little effect of stirring speed(P>0.05)was observed.The apparent activation energy of extraction process was calculated as 10.02 kJ/mol and Arrhenius constant(A)was 1.91 by Arrhenius equation.There was a linear relationship between reaction rate constant and the square of the inverse of initial particle radius(1/r_(0)^(2))(P<0.05).This phenomenon and this shrinking core model were anastomosed.In brief,the extraction process was controlled by the diffusion of protein from the virgin zone interface of particle through the reacted zone and it was in line with the first order reaction.Mass transfer kinetics of peanut protein extracted by reverse micelles was established and it was verified by experimental results.The results provide an important theoretical guidance for industrial production of peanut protein separation and purification.

New Development of Reverse Micelles and Applications in Protein Separation and Refolding

Reverse micelles bring mild and effective microenvironments in organic solvent that contain bitmolecules, which have attracted immense attention for application in the isolation of proteins, protein refolding, and enzymatic reaction. In this review, the application of reverse micelles for protein separation and refolding has been briefly summarized and various reverse micellar systems composed of different surfactants, including ionic, non- ionic, mixed, and affinity-based reverse micelles, have been highlighted. It illustrates especially the potential application of the novel affinity-based reverse micelles consisting of biocompatible surfactant coupled with affinity ligands. Moreover, the importance to develop universal affinity-based reverse micelles for protein separation and refolding in the downstream processing of biotechnology has been pointed out.

Mechanisms of Cytochrome C Extraction by Reverse Micelles

The extraction of cytochrome C was carried out by means of phase transfer technique with three different reverse micellar systems, i.e. , a CTAB micellar solution in n butyl alcohol chloroform(volume ratio 4∶1), an AOT micellar solution in isooctane and a SDSS D 2EHPA micellar solution in isooctane. The extraction mechanisms were studied. The results show that the extraction mechanisms for the same proteins with different types of reverse micellar systems can be distinct. The extraction of cytochrome C with CTAB and SDSS D 2EHPA reverse micellar systems are carried out according to the mechanism of electrostatic interaction. However, in the extraction of cytochrome C with the AOT reverse micellar system, the electrostatic interaction between the protein and the surfactant is not important.

Preparation of Highly Concentrated Silver Nanoparticles in Reverse Micelles of Sucrose Fatty Acid Esters through Solid-Liquid Extraction Method

Silver nanoparticles were synthesized in reverse micelles consisting of sucrose fatty acid esters by dissolving reactant powder in the water pool of reverse micelles through the solid-liquid extraction. Silver nanoparticles having various sizes and shapes were obtained at high concentration. The size of silver nanoparticles was controlled by reaction temperature. Moreover, the size of silver nanoparticles was dependent upon the average esterification degree of sucrose fatty acid esters forming reverse micelles. The wavelength in the peaks, which corresponded upon the localized surface plasmon resonance of resultant silver nanoparticles, was correlated with their sizes.

LIQUID-LIQUID EXTRACTION OF BSA BY USING CTAB-HEXANOL-OCTANE REVERSED MICELLAR SYSTEM

1 INTRODUCTIONIn biotechnology there is a need for new protein recovery process,which combines a highselectivity for the desired product with substantial concentration increased and easy to scale-up.In this context,liquid-liquid extraction with reversed micellar phase might serve this purpose.Reversed micelles are aggregates of surfactant molecules containing an inner core of water mole-cules,dispersed in a continuous organic solvent medium.These systems are opticallytransparent and thermodynamically stable.It has beendemonstrated [1,2]that under certainconditions proteins can be transferred from an aqueous phase towards a reversed micellarphase or vice versa(Fig.1)

Effect of Dissolved CO_2 on Protein Solubilization in Reverse Micelle

Effect of dissolved CO2 on bovine serum albumin (BSA) solubilization in the reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in 2,2,4-trimethylpentane (iso-octane) has been studied at 308.2 K. It was found that BSA can be precipitated completely by CO2 while the AOT and water remain in the iso-octane continuous phase. This opens up a possibility for recovery of pure protein solids directly from reverse micellar solutions.

Determination of Teicoplanin A2's Partition Coefficient in Different Liquid-Liquid Extraction Systems

Teicoplanin is one of the macrocyclic glycopeptide antibiotics, which is active against Gram-positive bacteria, and has attracted a lot of attention in the field of chiral separation recently. In this work, the partition coefficients and extraction ratio of teicoplanin in three different solvent systems were studied: conventional extraction, reactive extraction and reverse micelle extraction. With conventional solvent extraction, n-butanol demonstrated high partition coefficient for teicoplanin, but low extraction ratio because of its high solubility in water. Reactive extraction of teicoplanin showed the highest partition coefficient with almost 100% recovery in organic phase when tri-n-octylmethyl ammonium chloride (TOMAC) was used as extractant and pH value was above 5.0. A reverse micelle system, consisted of isooctane +10mmol-L-1 TOMAC +1% n-octanol, also offered high separation factor for teicoplanin. The results are beneficial for the design of teicoplanin separation and purification process.

Phase separation in solvent extraction of cobalt from acidic sulfate solution using synergistic mixture containing dinonylnaphthalene sulfonic acid and 2-ethylhexyl 4-pyridinecarboxylate ester

Phase separation rate is a critical character in determining the usefulness of a solvent extraction system in hydrometallurgy. A survey of the synergistic mixture containing dinonylnaphthalene sulfonic acid (HDNNS) and 2-ethylhexly 4-pyridinecarboxylate ester (4PC) for the extraction of cobalt from acidic single metal sulfate solution was carried out to suggest how the physicochemical properties and the morphology of the reverse micelles in the loaded organic phase affect the phase separation. The results show that effective parameters affecting the phase separation are the viscosity and the excess water uptake of the loaded organic phase. It is obvious that the specific settling rate (SSR) decreases with the apparent increase of these two parameters. The measurement of small angle X-ray scattering (SAXS) proves that the morphology of the reversed micelles in the loaded organic phase changes evidently with the change of the specific settling rate (SSR).

Recent Advances in Protein Extraction and Chiral Separation of Biomolecules

Reverse micelles create unique environment in organic media. They are capable of solubilizing hydrophilic biomolecules （e.g., proteins, peptides, amino acids, and DNAs） in their aqueous interior. This feature brings about the practical use of biomaterials in organic media because reverse micelles solubilize them with the intrinsic activity. In this paper, we focus on recent two topics concerning protein extraction and chiral separation of biomolecules using liquid membranes. In the first topic, we present recent attempts to extract proteins from an aqueous solution into isooctane using reverse micelles, and some important operational parameters to achieve an efficient protein transfer are discussed. Furthermore, novel function of reverse micelles as a protein activation medium is introduced. In the reverse micellar phase, denatured proteins were completely reactivated in the reverse micellar solution. The reverse micellar technique is found to be a useful tool not only for protein separation but also for protein refolding. Furthermore, we found that a cyclic ligand carixarene has an extraction ability to set up optimum conditions for protein transfer. In the second topic, we have found that a supported liquid membrane （SLM） encapsulating enzymes shows high enantioselectivity （enantioselective excess value is over 96%） in the transport of racemic pharmaceutical compound ibuprofen. A different experiment also suggests that the α-chymotrypsin-catalyzed reactions droved the enantioselective transport of L-phenylalanine based on the enantioselectivity of the enzyme. The SLM encapsulating the surfactant-enzyme complex enabled the highly enantioselective separation of racemic mixtures. It can be envisioned that arrangement of appropriate enzymes in the SLM system will allow enantioselective separation of various useful organic compounds.

Reverse micelles extraction ofnattokinase:From model system to real system

Nattokinase is a novel fibrinolytic en- zyme, which is homologous to Subtilisin Carlsberg. In this paper, Subtilisin Carlsberg was taken as a model protein of nattokinase. Effects of pH, ionic strength, concentration of isopropanol on the extraction of Subtilisin Carlsberg with AOT/isooctane reverse mi- celles system were investigated. Further, the process of reverse micelles extraction of nattokinase from fermentation broth was studied. By taking the reverse micelles of AOT/isooctane as extractant to perform a full extraction cycle, it was found that about eighty percent of the total activity of nattokinase in the fer- mentation broth could be recovered and the purifica- tion factor was about 2.5. Homologous protein could be reasonably used as model protein of a target protein.

DTAB反胶束萃取花椒籽中蛋白质和油脂研究

利用十二烷基三甲基溴化铵(dodecyl trimethyl ammonium bromide,DTAB)-异辛烷-正己醇反胶束体系,对花椒籽中的蛋白质和油脂同步萃取进行了探究。结果表明,前萃过程油脂直接溶解于有机相中,蛋白质增溶于反胶束极性核中,蛋白质的前萃率受DTAB浓度、萃取时间、缓冲溶液pH值、氯化钠浓度、料液比的影响。随后,反胶束溶液通过加入等体积含有一定离子强度和pH值的水相构筑WinsorⅡ微乳体系来实现蛋白质转移。其蛋白质后萃率和油脂萃取率受后萃水相的酸度和离子强度等因素的影响。

水飞蓟蛋白提取工艺优化及理化性质

目的优化反胶束法萃取水飞蓟蛋白工艺,探究该方法对水飞蓟蛋白理化性质的影响。方法通过单因素试验和响应面试验优化反胶束法萃取水飞蓟蛋白工艺,分析提取因素对蛋白提取率的影响;对比碱提酸沉法,分析反胶束法水飞蓟蛋白溶解度、持油性、起泡性、起泡稳定性、乳化性及乳化稳定性。结果反胶束法萃取水飞蓟蛋白最佳提取工艺参数为含水量20、液固比201(mL g)、前萃取pH 8.0、前萃取温度40℃、后萃取时间90 m in、后萃取pH 8.0,该条件下水飞蓟蛋白提取率为(60.44±0.20)%;反胶束法萃取的水飞蓟蛋白溶解度、持油性、起泡性、乳化性和乳化稳定性均高于碱提酸沉法提取的水飞蓟蛋白。结论反胶束法提取水飞蓟蛋白较传统方法更具优势,在水飞蓟蛋白开发中具有较好的应用前景。

湿法磷酸萃取技术发展现状与研究进展

磷酸作为一种重要的化工原料,在化工业中占有极其重要的地位.磷酸的应用主要由磷酸的纯度决定,低纯度磷酸可用于工业和农业领域,而高纯度磷酸则可用于医药、食品和电子等行业.我国磷矿以低品位磷矿为主,生产磷酸主要采用湿法工艺.相比热法生产,湿法工艺更加清洁环保,但产品杂质含量多、种类复杂,故发展磷酸净化技术尤为重要.本文从湿法磷酸纯化技术中的萃取法出发,综述了萃取法的主要研究进展.重点介绍了溶剂萃取法、双水相萃取法、反胶团萃取法、超声协助萃取法和超临界流体萃取法的基本原理和发展趋势.分析了不同萃取方法的优缺点、分离效果和适用条件.突出介绍了溶剂萃取法,梳理了磷酸除杂的主要萃取剂,特别强调了复合萃取剂和新型萃取剂在磷酸纯化方面的显著优势,最后,对磷酸的萃取技术做出了前景展望.

Selective Affinity Separation of Yeast Alcohol Dehydrogenase by Reverse Micelles with Unbound Triazine Dye

The reversed micelles were formed with cationic cetyltrimethylammonium bromide (CTAB) as surfactant and n-hexanol as cosolvent in the CTAB (50mmol.L-1)/hexanol (15% by volume)/hexane system. Cibacron Blue 3GA (CB) as an affinity ligand in the aqueous phase was directly introduced to the reversed micelles with electrostatic interaction between anionic CB and cationic surfactant. High molecular weight (Mr) protein, yeast alcohol dehydrogenase (YADH, Mr = 141000) from baker's yeast, has been purified using the affinity reversed micelles by the phase transfer method. Various parameters, such as CB concentration, pH and ionic strength, on YADH forward and backward transfer were studied. YADH can be transferred into and out from the reversed micelles under mild conditions (only by regulation of solution pH and salt concentration) with the successful recovery of most YADH activity. Both forward and backward extractions occurred when the aqueous phase pH＞pI with electrostatic attraction between YADH and CTAB. The recovery of YADH activity and purification factor have been improved with addition of a small amount of affinity CB. The recovery of YADH activity obtained was ～99% and the purification factor was about 4.0-fold after one cycle of full forward and backward extraction. The low ionic strength in the initial aqueous phase might be responsible for the YADH transfer into the reversed micellar phase.

反胶束提取扁桃仁蛋白工艺优化及理化特性比较

以AOT为表面活性剂构建的反胶束体系对扁桃仁蛋白的提取。对前、后提取分别进行单因素试验并阐述其影响作用,在此基础上对前提取进行响应面优化试验。最终前提取的最佳工艺参数分别为料液质量比1:15、AOT质量浓度0.11 g/mL、温度41℃、pH值为7.0和后提取的KCl浓度0.5 mol/L、温度45℃、新水相pH值为8.0。该条件下扁桃仁蛋白的前提取率为67.37%,后提取率为66.50%。其理化特性的试验结果显示乳化性和起泡性在20~100℃范围内先升高后降低,在pH值3.0~9.0范围内缓慢下降后持续升高,在40℃下和pH值为9.0时的乳化性和起泡性较好,分别为乳化性:48.93 m^(2)/g、60.22 m^(2)/g;起泡性:75.70%、88.05%。这结果与温度对表面疏水性的影响有较大差异(80℃时蛋白表面疏水性最好)。抗氧化活性试验结果显示扁桃仁蛋白对ABTS+.的清除能力强于DPPH.。其他如溶解性(74.84%)、持水性(4.34 g/g)和持油性(5.77 g/g)等特性均较理想。综上所述,反胶束法用于扁桃仁蛋白的提取有助于得到高质量的扁桃仁蛋白,为反胶束法应用于扁桃仁蛋白的提取提供科学依据。

Fabrication of Macroporous Protein-containing Films through the Reverse Emulsions Approach Featuringβ-Cyclodextrin-conjugated PEG-PLGA Copolymers

A series of β-cyclodextrin-conjugated 4-arm poly（ethylene glycol）-poly（lactide-co-glycolide） （4-arm PEG-PLGA） copolymers were synthesized by a ring-opening polymerization of D,L-lactide and glycolide using 4-arm PEG as initiator, and then conjugated with mono（6-ethylenediamine-6-deoxy）- β-cyclodextrin （CDen） or ethylenediamino-bridged bis- β-CD （BCDen）. The chemical structures of copolymers were confirmed by IH-NMR and FTIR spectroscopy. The , β-CD-conjugated PEG-PLGA formed stable reverse micelles due to the formation of fl-CD and bovine serum albumin （BSA） inclusion complexation, which could accommodate BSA in the organic solvent with improved encapsulation efficiency. Moreover, we demonstrated a one-step approach to construct macroporous protein-containing films using these reverse micelles. The films with ordered pore arrays were directly prepared from reverse micelles. Interestingly, the protein was totally located in the whole matrix except for the pores.

Application of Reverse Micelles of Alkyl Glucosides to Synthesis of Silver Nanoparticles

Silver nanoparticles were prepared in reverse micelles of alkyl glucosides by the injection method. The TEM image showed that the obtained silver nanoparticles displayed a wide variety of shapes. The size of silver nanoparticles was strongly dependent upon the kind of reducing agents, and tended to slightly increase with an increase in reaction temperature. The size of silver nanoparticles was hardly dependent upon the molar ratio of water to surfactant and the alkyl chain length of surfactants. Silver nanoparticles having various shapes were obtained at high concentration by the solid-liquid extraction method. The resultant silver colloid could be preserved for at least one month without precipitation.