Extraction of hyperoside from Hypericum perforatum L. using CTAB reversed micelles

In this paper, the extraction of hyperosides from Hypericum perforatum L. using Cetyl‐trimethyl‐ammonium bromite (CTAB) reversed micelles was studied and the affecting factors, that is, pH value, CTAB concentration, and existence of anions in the system were comprehensively investigated. The result showed that extraction using CTAB reversed micelles is a very effective method to separate hyperoside from Hypericum perforatum L. This founding is very significant because it demonstrated that extraction using reversed micelles is a promising way to separate and purify materials of small molecules.     

Extraction of aminoglycoside antibiotics with reverse micelles

The reverse micelle system of sodium di-2-ethylhexyl phosphate was used to extract aminoglycoside antibiotics, neomycin and gentamicin. The aminoglycosides can be efficiently extracted into a reverse micelle solution, and the antibiotics extracted into the micelle phase can readily be recovered back to a divalent cation aqueous solution, such as Ca²⁺. The transfer efficiency, %E, is heavily dependent on pH and salt concentration in the aqueous feed solution. %E decreases drastically with pH in the pH range 8·5–11, and declines with increasing (NH₄)₂SO₄ concentration. A simple transfer mechanism was proposed which suggests that the antibiotic molecules were extracted into the inner water cores of reverse micelles through attractive electrostatic interaction during forward transfer. In backward transfer, the antibiotics loaded in the micelle phase are released back to an aqueous phase through breaking up of the reverse micelles by using divalent cation solutions. The model is supported by the results of dynamic light scattering and infra-red spectroscopy study.     

Extraction of yeast alcohol dehydrogenase using reversed micelles formed with CTAB

A large molecular weight protein, alcohol dehydrogenase (ADH, 141 kDa), has been purified from baker's yeast using reversed micelles formed with a cationic surfactant (cetyltrimethylammonium bromide, CTAB) using the phase transfer method. Various parameters, such as pH, ionic strength and contact time of the two phases in ADH forward and backward transfer were studied. The active ADH was successfully recovered after a full forward and backward extraction cycle. The recovery of ADH activity obtained was ∼90% and the purification factor was 3.1 for the overall process. © 2000 Society of Chemical Industry     

基于表面活性剂的反胶团萃取技术

基于表面活性剂的反胶团萃取技术是一种新型的有发展前途的生物分离技术。介绍了反胶团萃取技术的驱动力、表面活性剂选择和影响因素,提出了反胶团萃取技术目前存在的问题,探讨了反胶团萃取技术在其酶促反应和纳米材料制备方面的应用及其发展方向。     

Liquid–liquid extraction of a recombinant protein with reverse micelles

Recombinant cytochrome b₅ was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase. The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein. This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis. The back-extraction of cytochrome b₅ to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1–2 mol dm^?3 NaCl) and low temperatures (4°C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances.     

Selective reverse micellar extraction of penicillin acylase from E coli

The recovery of penicillin acylase from E coli by a new reverse micellar treatment is described. The results are compared with the cell disruption by ultrasound followed by reverse micellar extraction. The process gave selective extraction of penicillin acylase directly from the periplasmic space of E coli without disrupting the cells. Unlike ultrasonication which breaks open the cells entirely, making subsequent processing difficult and expensive, reverse micellar treatment of cells gave a moderate recovery of 60% of enzyme activity in a highly pure form. © 2001 Society of Chemical Industry     

Polyphenol oxidase in reverse micelles of AOT/cyclohexane: A thermostability study

Polyphenol oxidase catalysing the oxidation of 4‐methylcatechol in reverse micelles of AOT/cyclohexane had an optimum temperature 15 °C higher than in aqueous medium. However, the enzyme lost stability when it was preincubated in reverse micelles in the absence of substrate regardless of the temperature although the effect was more pronounced at higher temperatures. The thermostability of polyphenol oxidase is higher when it is injected in reverse micelles containing buffer than when injected in initially empty micelles. Moreover, the thermostability of polyphenol oxidase in reverse micelles is strongly dependent on the size of the micelles, the bigger the micelle the greater the stability. The thermoinactivation of the enzyme follows a monomolecular process characteristic of a conformational change so the protein is protected by ligands towards inactivation. p‐Nitrophenol as competitive inhibitor and acetyl tyrosine ethyl ester as alternate substrate increase the half‐life of the polyphenol oxidase by about 2.5 and 4 times respectively. This finding may allow the use of the enzyme at higher temperatures with a gain in its stability. © 2001 Society of Chemical Industry     

CTAB逆胶束体系中Lipex脂肪酶催化合成油酸乙酯

研究了CTAB逆胶束体系的适宜构建条件以及Lipex脂肪酶催化合成油酸乙酯的适宜反应条件。探讨了有机溶剂、底物配比、pH值、水含量W₀、酸浓度、反应时间等因素对反应的影响。研究表明,在以CTAB为表面活性剂时,以异辛烷为溶剂,正己醇作助溶剂条件下,能得到较为稳定的逆胶束体系,更有利于Lipex脂肪酶的活性发挥;在该反应体系下反应温度25 ℃,缓冲液pH7. 0,W₀10,酸醇比1∶4（摩尔比）,油酸浓度0. 01 mol·L^-1,摇床转速150 r·min^-1,反应36 h,油酸乙酯产率达到79%。并建立了逆胶束体系中双底物作用的动力学模型,经验证模拟计算结果与实验结果吻合良好。     

Purification of recombinant human interferon gamma from fermentation broth using reverse micellar extraction: A process optimization study

In the present investigation, we have shown a single step purification of IFN-γ from the fermentation broth of recombinant Kluyveromyces lactis (K. lactis) strain, using cationic surfactant based reverse micellar extraction (RME) system. The forward and back extraction processes were optimized in order to improve the overall extraction process. 78%, 93% and 98% forward extraction efficiencies (FEE) were obtained after the process by using aqueous phase pH 12, 150 mM CTAB and 0.2 M NaCl respectively. Afterwards, back extraction efficiency (BEE) was maximized by the optimization of stripping phase pH, isopropyl alcohol (IPA) and potassium chloride (KCl) concentration by employing Taguchi’s method of orthogonal array. Stripping phase pH 7, 15% IPA & 0.8 M KCl were found to be the best possible parameters producing 83% BEE. The usage of IPA has proven to reduce the effect of micellar-micellar based interaction, which was reflected as an improvement in BEE. This study demonstrates that the reverse micellar system is a highly promising and efficient tool for the direct extraction of recombinant proteins from fermentation broth. Moreover, this system holds a high potential to be an integral part of the downstream process used in biopharmaceutical industry.     

Reverse Micellar Extraction of Bromelain from Pineapple (Ananas comosus L. Merryl) Waste: Scale-up,Reverse Micelles Characterization and Mass Transfer Studies

Scale-up studies for phase transfer mode of reverse micellar extraction are attempted for the separation and primary purification of bromelain (EC 3.4.22.33) from pineapple (Ananas comosus L. Merryl) waste. Characterization of reverse micelles and mass transfer studies for the real system has been attempted for the first time. Scale-up of the extraction process employing commercial grade surfactant cetyltrimethylammonium bromide (CTAB) and solvent isooctane resulted in purification of 2.43 fold with an activity recovery 81.3%. The reverse micellar size estimated using empirical and geometrical models indicated that the reverse micelles are large enough (R_m = 7.2–9.6 nm) to host bromelain molecules that are relatively smaller in size (～1.67 nm). The studies on the kinetics of mass transfer indicated a relatively slower rate (by ～34%) of mass transfer in case of back extraction compared to forward extraction. Process scale-up did not significantly affect the extraction efficiency whereas purity of phase components played a major role. The mass transfer across the phases was high in the initial period of mixing for both forward and back extractions.     

贯叶连翘中金丝桃素提取分离方法研究

研究了从贯叶连翘中提取分离金丝桃素,探讨了提取剂、料液比、提取次数、提取温度、提取时间等因素对金丝桃素提取率的影响,并通过正交实验对提取工艺进行了优化。实验表明:提取最佳条件为将原料在45℃水浸泡2 h,以75%的甲醇为提取剂,超声强化20 m in(功率1000 W),按料液比1∶6和1∶4各提取1次,每次3 h,LSI-106型大孔树脂分离,以甲醇洗脱(洗脱速度1~2 mL/m in),提取率为96.36%,得率为5.33 mg/10 g,纯度达到94.71%,同时测得金丝桃素的稳定性(RSD=0.586%)良好。     

Synthesis of macroporous poly(styrene-divinyl benzene) microspheres by surfactant reverse micelles swelling method

Macroporous poly(styrene-divinyl benzene) microspheres with pore size of about 500 nm were prepared by a new method, surfactant reverse micelles swelling method. The macroporous microspheres were prepared by convenient suspension polymerization. The difference from conventional suspension polymerization was that a higher concentration of surfactant was added in the oil phase. The effects of the amount and type of surfactants on the morphology of microspheres were investigated, and the formation mechanism was also discussed. Macropores were formed when the concentration of surfactant was much higher than critical micelle concentration (cmc). It was proposed that a large amount of reverse micelles formed by adding a large amount of surfactant in the oil droplet phase, and the reverse micelles could absorb water from the external aqueous phase. The water in the oil phase formed macropores after polymerization. The method developed in this study was convenient to prepare microspheres with larger pore size than the conventional method such as agglomeration method of nanoparticles.     

大孔树脂对贯叶连翘中金丝桃素的静态吸附

考察了18种不同类型的大孔树脂对贯叶连翘中金丝桃素的静态吸附和解吸效果,并对3种效果较好的树脂进行了吸附动力学研究;比较了上样液pH对吸附效果的影响;探讨了NKA-9树脂在25℃等温吸附过程,并应用Langmuir方程进行了拟合。结果表明,弱极性树脂NKA-9对金丝桃素吸附解吸作用最好,到达吸附平衡时间为1h左右,吸附量可达12.45mg/g干树脂。上样液最佳pH=4,上样液pH对树脂的吸附有很大影响。NKA-9树脂对金丝桃素的吸附为单分子吸附,符合Langmuir吸附理论。     

Reverse micellar extraction of bromelain from Ananas comosus L. Merryl

BACKGROUND: A reverse micellar system (RMS) of ionic surfactants is used for the first time for the extraction and primary purification of fruit bromelain (EC 3.4.22.33) from the aqueous extract of pineapple (Ananas comosus L. Merryl). The effect of various process parameters on both forward and back extraction of bromelain is studied to improve the extraction efficiency of RMS. Most of the reverse micellar extraction (RME) studies reported so far are on model systems and its application to enzyme extraction from a natural source is rarely reported. RESULTS: Studies carried out with ionic surfactants sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) and cetyltrimethylammonium bromide (CTAB) confirmed that electrostatic interaction was the main driving force for the extraction of fruit bromelain. Among the two surfactants studied, CTAB was found to be the most suitable for the extraction of fruit bromelain with respect to activity recovery (97.56%) and degree of purification (4.54 fold) when employed as a 150 mmol L^?1 CTAB/iso‐octane/5% (v/v) hexanol/15% (v/v) butanol system. Activity recovery with a counterionic system is higher (94.30%) in comparison with isopropyl alcohol added system (85.35%). CONCLUSION: RME could be used as an efficient primary purification step for the recovery of bromelain from pineapple juice. Reverse micellar phase components can easily be recovered and efficiently reused for fresh or subsequent extraction, which contributes favorably to the process economics and environmental issues. Copyright © 2007 Society of Chemical Industry     

分子模拟十六烷基三甲基溴化铵对石油污染物在粗糙石英砂表面脱附的影响

溶剂萃取法广泛应用于石油污染土壤的修复,萃取前后土壤微观结构的改变会影响石油污染物在土壤中的迁移和传递。本文利用分子动力学模拟方法,通过在石英砂表面上构造不同的凹槽代表矿物表面粗糙的结构,研究十六烷基三甲基溴化铵（CTAB）水溶液萃取石油污染物（正十二烷）的过程。结果表明：①脱附过程的主要驱动力为凹槽外的正十二烷和CTAB分子与凹槽内正十二烷分子之间的相互作用;②凹槽的宽度减小或者凹槽的深度增加,均会增加槽内正十二烷分子的脱附难度,同时还会影响CTAB分子的促进脱附效果;③CTAB分子对石油脱附的促进效果随其浓度的增加而减弱,当持续增加体系中CTAB的含量时（不同模拟体系中的CTAB分子数量分别为0、16、32）,会抑制其促进脱附的效果。     

Preparation of albumin–ferrite superparamagnetic nanoparticles using reverse micelles

BACKGROUND: Micellar systems are widely applied as reactors to encapsulate inorganic nanoparticles in polymeric materials. However, only low inorganic contents and microscale dimensions are often achieved. RESULTS: The adsorption of albumin protein on ferrite nanoparticles permits to increase the stability of inorganic dispersions in water by inhibiting particle flocculation. Subsequent glutaraldehyde addition induces protein crosslinking and ferrite entrapment. Polymer–ferrite composite nanoparticles were obtained in this way. The magnetic albumin nanoparticles (25 nm mean diameter) obtained contain about 40 wt% of ferrite and show superparamagnetic behaviour. The composite powder was successfully functionalized with a model drug and the biological activity was evaluated. CONCLUSION: Using a reverse micelle approach, ferrite–albumin composite nanoparticles with a high inorganic content were obtained. The method permits the formulation of biocompatible magnetic particles of nanoscale dimensions. The exhibited superparamagnetic behaviour permits to hypothesize an application of the powder composite as a carrier in biomedical technologies. Copyright © 2009 Society of Chemical Industry     

Solvent extraction of bacteriocins from model solutions and fermentation broths

A novel method for the extraction and purification of bacteriocins (small peptides with antimicrobial properties) from fermentation broths is described. Model solutions of nisin were prepared from Nisaplin®, a commercial whey‐based powder containing about 2.5% nisin and sold as a food preservative. These model solutions and fermentation broths from a culture of nisin‐producing Lactococcus lactis subsp lactis were cross‐flow filtered and stirred with toluene to obtain a white suspension in the toluene layer, which was back extracted with 10 mmol dm⁻³ HCl. Yields of the order of 90% for a single stage extraction were obtained followed by similar yields on back extraction. Yields dropped with decreasing pH, going through a minimum at pH 2. In larger scale experiments (11 dm³) at pH 6, a gel layer was formed which, on centrifugation and drying, gave a white solid containing about 50% nisin, the remainder being water and salt. Further process development would almost certainly bring this figure closer to the small‐scale value. The phase transfer technique was also applied to solutions containing variacin and carnocin (produced by Micrococcus varians and Carnobacterium piscicola, respectively). The mechanism of bacteriocin extraction probably involves concentration of the peptides, which are surface active, at the interface until their solubility is exceeded and they form reverse submicelles, which aggregate into reverse micelles to give a gel. © 2000 Society of Chemical Industry     

Enzymatic interesterification of triolein with tripalmitin in canola lecithin-hexane reverse micelles

Lipase-catalyzed interesterification of tripalmitin with triolein in canola lecithin-hexane reverse micelles allowed for the successful modification of triolein and tripalmitin to yield a fat of intermediate properties between the two initial substrates. Acetone-insoluble canola lecithin (AIL) reverse micelles containingRhizopus arrhizus lipase in buffer, or plain 0.1M sodium phosphate buffer of pH 7.0, formed readily in hexane. Both had an average Stokes’ radius of approximately 40Å, as determined by quasielastic light-scattering determinations. The reverse micelle system was stable and did not form higher-order micelle oligomers or aggregates. Biotransformation of the triglycerides was performed at 47°C in a 50-mM AIL-hexane reverse micelle system containing 50% (w/w) oil at a water-to-surfactant ratio (w_o) of 5.5. Dynamic (oscillatory) mechanical analysis indicated that the crystallization temperature of the fat dropped from 47.7 to 37.5°C as judged by the storage (G′) and loss (G″) modulusvs. temperature profiles after 48 h of reaction. Differential scanning calorimetric studies showed that the melting point of the fat dropped from 61 to 57°C after 48 h of reaction. Triglyceride analysis of the fat mixture by gas-liquid chromatography (GLC) indicated that, after 48 h of reaction, the tripalmitin content dropped from 34.5 to 29% (w/w), the triolein content dropped from 64.5 to 52.1% (w/w) and the 1-oleyl-2,3-dipalmitin content reached 7.5% (w/w) while the 1-palmitoyl-2,3-diolein content reached 7.2% (w/w). 1,2-Dipalmitoyldiglyceride and 1,2-dioleyldiglyceride contents reached 1.6 and 2.4% (w/w), respectively, after 48 h. Free fatty acid analysis of the fat mixture by GLC revealed that the free palmitic acid content increased from 0.28 to 2.4% (w/w) while the free oleic acid content increased from 1.4 to 5.4% (w/w) in the initial 24 h, after which the levels remained constant. The relatively high initial free fatty acid content of the mixture was due to free fatty acids present in the canola lecithin and not in the oils. This enzymatic interesterification protocol utilizes, for the first time, an organic solvent commonly used in food processing operations and a food-grade and inexpensive surfactant that readily forms reverse micelles and yields a modified fat with improved rheological properties for use as an edible plastic fat.     

Kinetic behavior of yeast alcohol dehydrogenase in AOT/isooctane reverse micelles

The kinetic behavior of yeast alcohol dehydrogenase (YADH) in sodium di-2-ethylhexylsulfosuccinate (AOT) isooctane reverse micelles has been studied using methyl ethyl ketone (MEK), NADH and Tris as the substrate, coenzyme and buffer, respectively. The solubility diagrams of aqueous buffer in the isooctane solution of AOT were established as a function of temperature and molar ratio of water of surfactant (ω₀) at various Tris and AOT concentrations. The dependence of enzyme activity on enzyme concentration, pH, ω₀ and Tris concentration was determined. The optimal ω₀ was at 10–15, increasing slightly with an increase in Tris concentration. The YADH entrapped in reverse micelles exhibited minimum activity at a Tris concentration of 0·1 mol dm^?3, while in aqueous buffer enzyme activity was not significantly affected by Tris concentration. Comparing the rate equation of the reduction of MEK by YADH in reverse micelles with that in aqueous buffer, the association of YADH and NADH could apparently have proceeded with an irreversible reaction before the substrate was bound, when performed in a reverse micellar system. Although the YADH entrapped in reverse micelles was less stable than when dissolved in aqueous buffer, the enzyme retained at least 20% activity after 21 h at 25°C and ω₀ = 20. This result was an improvement over previously reported data.     

SDSS-D2EHPA/异辛烷混合反胶团体系萃取细胞色素C

用SDSS－D2EHPA／异辛烷混合反胶团体系提取蛋白酶细胞色素C,研究了水相pH值、离子强度、SDSS浓度、提取时间等对提取率的影响,结果表明,提取率可达100％,并且提取条件温和、简便,可在中性介质和室温下进行,提取量大,提取时间短,5min即可完成。