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.

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.

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.

Absorption Complex between Porphyrin and Phenothiazine in Reverse Micelles

The interaction between amphiphilic porphyrin and phenothiazine in AOT/isooctane/water reverse micelle was investigated by UV-Vis spectra. A new absorption complex between the two species is formed in such circumstances, which is ascribed to the enrichment of the components by the reverse micelle. The fluorescence quenching of CHTTP by PTH becomes more efficient after the formation of the absorption complex.

Porphyrins in Reverse Micelles: the Side-chain Length and the Triplet-state Lifetime

Using bis(2-ethylhexyl) sodium sulfosuccinate (AOT) as surfactant, two amphiphilic porphyrin terminated with imidazole were studied in AOT/iso-octane/water reverse micelles, intending to mimic the relationship between microenvironments in organism and the amphiphilic properties of porphyrins for photodynamic therapy drugs.

Terahertz spectroscopy of water in nonionic reverse micelles

The dynamics of water within a nanopool of a reverse micelle is heavily affected by the amphiphilic interface.In this work,the terahertz(THz)spectra of cyclohexane/Igepal/water nonionic reverse micelle mixture are measured by THz timedomain spectroscopy and analyzed with two Debye models and complex permittivity of background with volume ratios.Based on the fitted parameters of bulk and fast water,the molar concentration of all kinds of water molecules and hydration water molecule number per Igepal molecule are calculated.We find that slow hydration water has the highest proportion in water when the radius parameterω_(0)<10,while bulk water becomes the main component whenω_(0)≥10.The feature radius ratio of nonhydrated and hydrated water to total water nanopool is roughly obtained from 0.39 to 0.85 with increasingω_(0).

Influence of Capping Ligands on the Self-organization of Gold Nanoparticles into Superlattices from CTAB Reverse Micelles

Gold nanoparticles with size 3-10 nm (diameter) were prepared by thereduction of HAuCl_4 in a CTAB/octane + 1-butanol/H_2O reverse micelle system using NaBH_4 as thereducing agent. The as-formed gold nanoparticle colloid was characterized by UV/vis absorptionspectrum and transmission electron microscopy (TEM). Various capping ligands, such as alkyl-thiolswith different chain length and shape, trioctylphosphine (TOP), and pyridine are used to passivatethe gold nanoparticles for the purpose of self-organization into superstructures. It is shown thatthe ligands have a great influence on the self-organization of gold nanoparticles intosuper-lattices, and do-decanethiol C_(12)H_(25)SH is confirmed to be the best ligand for theserf-organization. Self-organization of C_(12)H_(25)SH-capped gold nanoparticles into 1D, 2D and 3Dsuperlattices has been observed on the carbon-coated copper grid by TEM without using any selectiveprecipitation process.

Aggregation Behaviors of Tricarbocyanine Dye in Water and in AOT Reverse Micelles

The photophysical property of the tricarbocyanine dye IR144 has been extensively studied in non-aqueous solvents. However, as a potential near-infrared biomedical imaging probe, the photophysical property of IR144 in water is still little known. So, the aggregation behaviors of IR144 in water with steady-state absorption spectroscopy and integrated polarization dependent femtosecond pump-probe spectroscopy were investigated. Through comparing the absorption spectral bandshape of IR144 in water and in water pool of AOT reverse micelles, It is found that IR144 form dimer aggregates in water even at very low concentration （〈 1.0× 10^- 7 moloL 1）. And the absorption spectrum of the IR144 aggregates always displays a bimodal feature, which is independent of the dye concentration ranging from 1.0 × 10^-7 to 1.0 × 10^-4 mol·L^-1. For better understanding the aggregation behaviors of IR144 in water, we measured the ground state recovery kinetics and the reorientation kinetics of IR144 in water and in water pool of AOT reverse micelles （W0= [H2O]/[AOT], W0=40）. It is found that the fluorescence quantum yield of 1R144 in water is lower than that in water pool of AOT reverse micelles, and the reorientation time of IR144 in water is slower than that in water pool of AOT reverse micelles. Those kinetic measurements also verify that IR144 exists as dimer aggregates in water.

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.

Kinetic Studies on of Veratryl the Lignin Peroxidase Catalyzed Oxidation Alcohol by H2O2 in AOT/Isooctane/ Toluene/Water Reverse Micelles

The steady state kinetics of the lignin peroxidase （LIP） catalyzed oxidation of veratryl alcohol （VA） by H2O2 in a sodium bis（2-ethylhexyl） sulfosuccinate （AOT）/isooctane/toluene/water reverse micellar medium was studied and a comparison with the corresponding aqueous medium was made to understand the effect of the reverse micellar medium on the catalytic mechanism and kinetic parameters. Results indicated that the model reaction in the AOT reverse micelle followed the ping-pong mechanism with true kcat, Km,VA and KmH2O2 being 59.6min^-1, 13.9 mmol· L^-1 and 94.8 μmol·L^-1, respectively; inhibition of high level of H2O2 on LiP followed the reversible competitive pattern with Ki being 0.140 mmol·L^-1. The reaction mechanism and inhibition pattern in the AOT reverse micellar medium were the same as those in bulk aqueous medium, but the kinetic parameters except KmH2O2 were greatly different in the two media. The kcat and Ki values in the reverse micelle were approximately 2 and 20 times smaller than the corresponding values in the aqueous solution, but the Michaelis constant of VA was approximately 100 times greater than that in the aqueous solution. The above mentioned differences in the kinetic parameters were caused by the microheterogeneity and the interface of the AOT reverse micelle, which resulted in the partitioning of VA and H2O2, and by the changes of the conformation of LiP and the reactivity of the substrates.

Enzymatic reaction of ethanol and oleic acid by lipase and lignin peroxidase in rhamnolipid(RL) reversed micelles

An environment friendly bio-surfactant of rhamnolipid(RL) was used as a solvent. The enzymatic reaction of oleic acid catalyzed by lipase and lignin peroxidase(lip) was evaluated. The optimum conditions of enzymatic reaction catalyzed by lipase(lip) were water to amphiphile molar ratio of 30(20), RL of 60(60) critical micelle concentration(CMC), pH of 7.0(3.0) and temperature of 40(30) °C, respectively. The change of enzyme conformation indicates that, for catalytic of lipase, water content is the most important factor of the enzymatic reaction of oleic acid, and p H for lip. With individual optimum conditions, the enzymatic efficiency of oleic acid catalyzed by lipase is higher than that by lip. In the presence of ethanol, the enzymatic reaction of oleic acid catalyzed by lipase suits Ping-Pong Bi-Bi mechanism. As an alternative to chemical reversed micelles, the RL reversed micelles are promising methods to enzymatic reaction of oleic acid.

Formation of reverse micelles in supercritical carbon dioxide and its thermodynamics

The solubilization behavior of methyl orange as a solvation probe in multiple systems composed of super-critical carbon dioxide,surfactants and co-solvents,is studied.It is concluded that some surfactants,such as sodium bis-(2-ethylhexyl)sulfosuccinate(AOT)and isooctyl phenol polyethoxylate(TX-10),could form reverse micelles in supercritical carbon dioxide under the action of butanol.The formation of reverse micelles is a spontaneous process thermodynamically.Specifically for the nonionic surfactant TX-10,the formation of reverse micelles is dependent on the entropy increase in the system,while for the anionic sur-factant AOT,the micellization is mainly dominated by the increase in enthalpy at higher temperatures,but by the increase in entropy at lower temperatures.

Supramolecular Micelles and Reverse Micelles Based on Cyclodextrin Polyrotaxanes

A new supramolecular self-assembly approach to construct micelles and reverse micelles was reported.Double-hydrophilic block copolymers poly(ethylene oxide)-b-poly(N-isopropylacrylamide) (PEO-b-PNIPAAm) was synthesized via atom transfer radical polymerization (ATRP) using PEO macroinitiator.Because of the lower critical solution temperature (LCST) phase behavior of PNIPAAm,PEO-b-PNIPAAm block copolymers self-assembled to form PNIPAAm-core micelles at 40 ℃.The PNIPAAm-core micelles were disassembled to unimers when the temperature was decreased to 25 ℃.But the addition of α-cyclodextrins (α-CDs) could induce the formation of PNIPAAm-shell micelles because of the 'channel-type' crystallities induced by PEO/α-CDs polyrotaxanes.The assembly and disassembly procedure of micelles and reverse micelles were investigated by dynamic light scattering (DLS),X-ray diffraction (XRD),1H NMR and transmission electron microscopy (TEM).

Fluorescence quenching of eosin upon adsorption by colloidal silver mediated in aqueous solution and reverse micelles

The optical effects of the adsorption of eosin on the colloidal sliver particle have been investigated in aqueous solutions. It was found that upon adsorption the fluorescence of eosin was effectively quenched. This was explained as the photoinduced interfacial electron transfer from the excited singlet state of eosin to the silver particle. Decreasing pH of the solution favors the adsorption of eosin and so enhances the fluorescence quenching. For comparison, the fluorescence quenching in reverse micelles was also investigated. The quenching behavior was much different from that mediated in aqueous solution. This was attributed to the unique microenvironment of reverse micelles.

TR-ESR Investigation on Reaction of Vitamin C with Excited Triplet of 9,10-phenanthrenequinone in Reversed Micelle Solutions

Time-resolved electron spin resonance has been used to study quenching reactions between the antioxidant Vitamin C （VC） and the triplet excited states of 9,10-phenanthrenequinone （PAQ） in ethylene glycol-water （EG-H2O） homogeneous and inhomogeneous reversed micelle solutions. Reversed micelle solutions were used to be the models of physiological environment of biological cell and tissue. In PAQ/EG-H2O homogeneous solution, the excited triplet of PAQ （3PAQ＊） abstracts hydrogen atom from solvent EG. In PAQ/VC/EG-H2O solution, 3pAQ＊ abstracts hydrogen atom not only from solvent EG but also from VC. The quenching rate constant of 3pAQ＊ by VC is close to the diffusion-controlled value of 1.41 × 108 L/（mol.s）. In hexadecyltrimethylammonium bromide （CTAB）/EG-H2O and aerosol OT （AOT）/EG- H2O reversed micelle solutions, 3pAQ＊ and VC react around the water-oil interface of the reversed micelle. Exit of 3pAQ＊ from the lipid phase slows down the quenching reaction. For Triton X-100 （TX-100）/EG-H2O reversed micelle solution, PAQ and VC coexist inside the hydrophilic polyethylene glycol core, and the quenching rate constant of 3pAQ＊ by VC is larger than those in AOT/EG-H2O and CTAB/EG-H2O reversed micelle solutions, even a little larger than that in EG-H2O homogeneous solution. The strong emissive chemically induced dynamic electron polarization of As＇- resulted from the effective TM spin polarization transfer in hydrogen abstraction of 3pAQ＊ from VC.

Activity Regulation of Lignin Peroxidase from Phanerochaete chrysosporium in Nonionic Reversed Micellar Medium

The activity of lignin peroxidase (LiP) in reversed micelles of polyoxyethylene lauryl ether (Brij30) changed with the molar ratio of water to the surfactant and the denaturant concentration of guanidinium chloride. At low water contents the activity of LiP could be enhanced by the denaturant at moderate concentration. This phenomenon, together with the spectral characteristics of the intrinsic fluorescence of LiP, suggested that the conformation of the active center of LiP was flexible.

Polymerization of o-Phenylenediamine Catalyzed byHemeproteins Encapsulated in Reversed Micelle

Hemeproteins encapsulated in reversed micelle formulated with di-2-ethylhexyl sulfosuccinate(AOT) was found to catalyze the polymerization of o-phenylenediamine(o-PDA) with hydrogen peroxide, whereas o-PDA catalyzed by hemeproteins dissolved in water could only form its trimers. As the nanostructural environment in reversed micelle acts as a certain orientation surrounding medium, it offers a strong electrostatic field that alters the reductive potential of Fe 3+/Fe 2+(E m7) in the heme of hemeproteins and thus increases the catalytic activity of peroxidase accordingly. According to the results of UV-Vis, 1H NMR and FTIR, the polymer catalyzed by hemoglobin(Hb) in reversed micelle was presumed to be constructed of lines and trapeziforms alternatively.

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.