SC-CO2和乙酸乙酯萃取芸香中活性成分的比较分析

为了探讨超临界二氧化碳(supercritical carbon dioxide, SC-CO2)技术与提取物的分级分离在萃取芸香活性成分的应用价值,本研究采用SC-CO2和乙酸乙酯萃取芸香中植物蜡和活性成分,并调查粒径和CO2流量对提取产量的影响。在250 bar、40℃条件下提取,并使第一个分离器冷却到-10℃,可获得较好的提取效率。当粒径较小时,提取过程更快,即内部传质控制该过程。分级分离可选择性去除表皮植物蜡,约占由SC-CO2处理产生的总提取物的77.5%W/W。第二分离器中的获得的提取物中活性化合物可达86.3%W/W。随后采用气相色谱-质谱联用仪(gas chromatography-mass spectrometry, GC-MS)分析表明,乙酸乙酯提取物低于SC-CO2提取物的萃取效率,主要是由于提取物中含有大量的植物蜡。本研究为超临界二氧化碳技术在萃取芸香活性成分方面的提供技术参考。     

The lipase-catalyzed hydrolysis of lutein diesters in non-aqueous media is favored at extremely low water activities

The enzymatic hydrolysis of a mixture of lutein diesters from Marigold flower (Tagetes erecta) was performed both in organic solvents and supercritical CO(2) (SC-CO(2)) using two commercial lipases: lipase B from Candida antarctica (Novozym 435) and the lipase from Mucor miehei (Lipozyme RM IM). Both lipases showed an unexpected dependence of initial reaction rate with the initial water activity (a(wi)) in hexane, with the highest rates of hydrolysis taking place at the lowest a(wi) of the biocatalyst particles. The same result was observed using isooctane, toluene, or SC-CO(2). It is proposed that an increase in a(wi) generates a hydrophilic microenvironment that prevents efficient partitioning of the highly hydrophobic lutein diesters to the enzyme. The critical role of water in this system has not been reported for other hydrolytic reactions in low water media. Calculations of water available for hydrolysis from isotherm analysis, Karl-Fischer titration, and substrate conversion at a(wi) = 0.13, indicate that the extent of reaction is not limited by the amount of available water. Accordingly, the enzyme that holds the largest amount of water after prehydration at the same a(wi) (0.13) will yield the greatest substrate conversion and concentration of the free lutein product. The highest conversion occurred in SC-CO(2), which opens up new opportunities to develop a combined extraction-reaction process for the environmentally benign synthesis of lutein, an important nutraceutical compound.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

益智的抗氧化作用

本文探讨了益智(Alpinia oxyphylla Miquel)超临界CO2提取物及其渣的水提物、正丙醇提取物和乙酸乙酯提取物的抗氧化作用,测定了总酚含量、黄酮含量、抗氧化力、还原能力、DPPH清除率.结果表明,益智超临界CO2提取物和正丙醇提取物的总酚含量最高,均为5.53%,乙酸乙酯提取物的总酚含量为4.04%,水提物总酚含量最低,为O.89%.抗氧化力与酚含量相关(R2=0.703).四种提取物中黄酮含量顺序为:乙酸乙酯提取物(6.29%)＞丙醇提取物(5.81%)＞水提取物(4.85%)＞超临界CO2提取物(4.70%).在还原能力、清除DPPH自由基和羟自由基方面,乙酸乙酯提取物表现出了很强的抗氧化能力,呈现剂量依赖关系.     

Recovery of phenolic antioxidants from wine industry by-products

The recovery process of the phenolic compounds contained in the wine industry by-products and their antioxidant activity were examined in this work. To this purpose, a combined process of liquid and supercritical solvent extraction was employed. At first the effect of various process parameters of the liquid solvent extraction--the type of solvent and the pretreatment of the raw material (composition (skins, seeds, stems) and crushing)--on the antioxidant activity of the extract was examined. It was shown that an extract of a higher antioxidant activity was obtained by using ethyl acetate as solvent and raw material free of stems. These extracts were further treated with supercritical carbon dioxide (SC CO(2)) at various extraction pressures, which resulted in their significant enrichment in phenolic compounds and the improvement of their antioxidant and organoleptic properties, especially at pressures higher than 100 bar. The antioxidant activity of the products was determined by using the Rancimat method, as well as a simple and not time-consuming free radical method. It was proved that both the ethyl acetate extracts and those treated with SC CO(2) had antioxidant activity comparable to that of antioxidants commonly used in industry, that is butylhydroxytoluene (BHT), a synthetic antioxidant, and Rosemary extract, a widely known natural one.     

Selective separation of cis-trans geometrical isomers of beta-carotene via CO2 supercritical fluid extraction

We investigated a novel method for the selective separation of beta-carotene isomers from a freeze-dried powder of the algae Dunaliella bardawil using supercritical fluid extraction. The separation method relies on the different dissolution rate of the 9Z and all-E isomers of beta-carotene in SC-CO(2). At first, the equilibrium solubility of the two isomers in SC-CO(2) was determined at the extraction conditions of 44.8 MPa and 40 degrees C. The solubility of the 9Z isomer was found to be nearly 4 times higher than that of the all-E isomer (1.92 x 10(-5) g all-E isomer/g CO(2) compared to 7.64 x 10(-5) g 9Z isomer/g CO(2)). When supercritical fluid extraction was applied to a carotenoid concentrate from the algae (29 wt% beta-carotene) or a freeze-dried powder of the algae (3.1% beta-carotene), a selective separation of the 9Z/all-E isomers of beta-carotene was obtained. Thirty-nine percent recovery of beta-carotene with 80% purity of 9Z isomer was achieved at the initial stages of extraction (40 mL CO(2)). The extraction rate of beta-carotene from the freeze-dried algae powder was slower than that from the carotenoid concentrate, resulting in a reduction in the recovery and purity of the 9Z isomer. This indicates that even at the initial stage of the extraction the internal mass resistance is significant. Isomer purity and recovery could be enhanced upon grinding of the algae powder.     

Extraction conditions affecting supercritical fluid extraction (SFE) of lycopene from watermelon

Lycopene, a carotenoid linked to protection against certain forms of cancer, is found in produce such as papaya, red-fleshed tomatoes, grapefruit and watermelon. The preparation of a supercritical CO2 (SC-CO2) watermelon-lycopene extract could serve as a food grade source of this carotenoid. This study established preliminary conditions for enhancing SC-CO2 extraction of lycopene from watermelon. Freeze-dried watermelon was extracted with SC-CO2 and ethanol as an organic co-solvent. The lycopene concentration was determined by HPLC, with absorbance measured at 503 nm. In an initial set of experiments, the effects of extraction temperature (70-90 degrees C), pressure (20.7-41.4 MPa) and co-solvent ethanol addition (10-15%) were evaluated. A lycopene yield of 38 microg per gram of wet weight was obtained at 70 degrees C, 20.7 MPa, and 15% by volume ethanol. The extraction of fresh (non-freeze-dried) watermelon yielded 103+/-6 microg lycopene per gram fresh fruit weight. Of the parameters tested, temperature had the most effect on lycopene yield. Thus, in another set of experiments, the temperature was varied from 60-75 degrees C at an extraction pressure of 20.7 MPa in the presence of 15% ethanol. Studies showed that freeze-dried watermelon flesh loses lycopene in storage. In accounting for lycopene storage losses, lycopene yields at 60 degrees C extraction temperature were 14% greater than those obtained at 70 degrees C.     

超临界CO2流体对纤维素酶催化反应的影响

超临界二氧化碳流体预处理对纤维素超分子结构及纤维素酶催化反应有重要影响。一定含水量的微晶纤维素用SC-CO2在10MPa,50℃处理30min,其结构发生了有利于进一步被酶解的变化。上述超临界条件单独作用于纤维素酶时,并未造成酶催化活力的降低;但与纤维素共同进行SC—CO2处理时,纤维素酶则失去催化活性,但这种处理却能提高纤维素进一步被酶解的效率。一定范围内处理时的酶用量与酶解效率的增加正相关。纤维素的含水量对SC-CO2处理后的酶解效率有显影响。     

Supercritical CO2 pretreatment of lignocellulose enhances enzymatic cellulose hydrolysis

The supercritical carbon dioxide (SC-CO2) pretreatment of lignocellulose for enzymatic hydrolysis of cellulose was investigated. Aspen (hardwood) and southern yellow pine (softwood) with moisture contents in the range of 0-73% (w/w) were pretreated with SC-CO2 at 3100 and 4000 psi and at 112-165 degrees C for 10-60 min. Each pretreated lignocellulose was hydrolyzed with commercial cellulase to assess its enzymatic digestibility. Untreated aspen and southern yellow pine (SYP) gave final reducing sugar yields of 14.5 +/- 2.3 and 12.8 +/- 2.7% of theoretical maximum, respectively. When no moisture was present in lignocellulose to be pretreated, the final reducing sugar yield from hydrolysis of SC-CO2-pretreated lignocellulose was similar to that of untreated aspen. When the moisture content of lignocellulose was increased, particularly in aspen, significantly increased final sugar yields were obtained from enzymatic hydrolysis of SC-CO2-pretreated lignocellulose. When the moisture content of lignocellulose was 73% (w/w) before pretreatment, the sugar yields from the enzymatic hydrolysis of aspen and southern yellow pine pretreated with SC-CO2 at 3100 psi and 165 degrees C for 30 min were 84.7 +/- 2.6 and 27.3 +/- 3.8% of theoretical maximum, respectively. The SC-CO2 pretreatments of both aspen and SYP with moisture contents of 40, 57, and 73% (w/w) showed significantly higher final sugar yields compared to the thermal pretreatments without SC-CO2.     

Alcalase-catalyzed, kinetically controlled synthesis of a precursor dipeptide of RGDS in organic solvents

The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide of RGDS, Z-Asp-Ser-NH2 in organic solvents was studied. Alcalase, an industrial alkaline protease, was used to catalyze the synthesis of the target dipeptide in water-organic cosolvents systems with Z-Asp-OMe as the acyl donor and Ser-NH2 as the nucleophile. Acetonitrile was selected as the organic solvent from acetonitrile, ethanol, methanol, DMF, DMSO, ethyl acetate, 2-methyl-2-propanol, and chloroform tested under the experimental conditions. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including water content, temperature, pH, and reaction time on the Z-Asp-Ser-NH2 yields. The optimum conditions are pH 10.0, 35 degrees C, in acetonitrile/Na2CO3-NaHCO3 buffer system (85:15, v/v), 6 h, with a dipeptide yield of 75.5%.     

Enzymatic polymerization catalyzed by surfactant-coated lipases in organic media

Structural ring-opening of lactones driven by enzymatic polymerization has been performed using low concentration dosages of surfactant-coated lipases in organic media. By comparison, enzymatic polymerization rate with coated lipase proceeded at a rate 100-fold better than native powder. Similarly a higher polymeric molecular weight (21,300), narrow dispersity (Mw/Mn=1.9) and better conversion (100%) were obtained following polyesterification tests with surfactant-coated lipase.     

Effect of supercritical fluids on C11beta-hydroxylation activity of Absidia coerulea

The viability and C11beta-hydroxylation activity of Absidia coerulea were measured after treatment with compressed or supercritical CO2 and C2H4 under various initial pH and temperature conditions. The specific activity of A. coerulea on C11beta-hydroxylation can reach 23% and 75%, respectively, after treatment with 7.5 MPa of CO2 and C2H4, leading to the feasibility of enhancing both the solubility of the reactants and the beta-hydrocortisone yield for the hydroxylation of Reichsterin's substance acetate by using supercritical C2H4 as an alternative to the organic solvent.     

Nanostructured microspheres produced by supercritical fluid extraction of emulsions

The system poly(lactic-co-glycolic) acid/ piroxicam (PLGA/PX) was selected, as a model system, to evaluate the effectiveness of supercritical carbon dioxide (SC-CO(2)) extraction of the oily phase (ethyl acetate) from oil-in-water emulsions used in the production of polymer/drug microspheres for sustained drug release applications. The influence of process parameters like operating pressure and temperature, flow rate and contacting time between the emulsion and SC-CO(2) was studied with respect to the microsphere size, distribution and solvent residue. Different polymer concentrations in the oily phase were also tested in emulsions formulation to monitor their effects on droplets and microspheres size distribution at fixed mixing conditions. Spherical PLGA microspheres loaded with PX (10% w/w) with mean sizes ranging between 1 and 3 microm and very narrow size distributions were obtained due to the short supercritical processing time (30 min) that prevents the aggregation phenomena typically occurring during conventional solvent evaporation process. A solvent residue smaller than 40 ppm was also obtained at optimized operating conditions. DSC and SEM-EDX analyses confirmed that the produced microparticles are formed by a solid solution of PLGA and PX and that the drug is entrapped in an amorphous state into the polymeric matrix with an encapsulation efficiency in the range of 90-95%. Drug release rate studies showed very uniform drug concentration profiles, without any burst effect, confirming a good dispersion of the drug into the polymer particles.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

Evaluation of CO2-based cold sterilization of a model hydrogel

The purpose of the present work is to evaluate a novel CO(2)-based cold sterilization process in terms of both its killing efficiency and its effects on the physical properties of a model hydrogel, poly(acrylic acid-co-acrylamide) potassium salt. Suspensions of Staphylococcus aureus and Escherichia coli were prepared for hydration and inoculation of the gel. The hydrogels were treated with supercritical CO(2) (40 degrees C, 27.6 MPa). The amount of bacteria was quantified before and after treatment. With pure CO(2), complete killing of S. aureus and E. coli was achieved for treatment times as low as 60 min. After treatment with CO(2) plus trace amounts of H(2)O(2) at the same experimental conditions, complete bacteria kill was also achieved. For times less than 30 min, incomplete kill was noted. Several physical properties of the gel were evaluated before and after SC-CO(2) treatment. These were largely unaffected by the CO(2) process. Drying curves showed no significant change between treated (pure CO(2) and CO(2) plus 30% H(2)O(2)) and untreated samples. The average equilibrium swelling ratios were also very similar. No changes in the dry hydrogel particle structure were evident from SEM micrographs.     

The effect of stirring and seeding on the AcPheLeuNH(2) synthesis and crystallization in a reversed micellar system

The present work describes the enzymatic synthesis and simultaneous crystallization of the dipeptide AcPheLeuNH(2) by alpha-chymotrypsin in a reversed micellar system of tetradecyltrimethylammonium bromide (TTAB)/heptane/octanol/carbonate buffer. The low solubility of the dipeptide in the micellar solution led to the formation and growth of needle-like crystals during the synthesis as soon as supersaturation was achieved. The crystallization process then followed a typical pattern, proceeding in three phases: nucleation, de-supersaturation, and re-equilibrium of saturation. Crystallization was followed by visual observation with an optical microscope, and the increase of crystal number and size was confirmed. Experiments showed that the supersaturation concentration decreases with the addition of AcPheLeuNH(2) seeds before the reaction, and also with a decrease of the stirring speed. It was also observed that the increase of both seed concentration and stirring advances the start of crystallization, so that the dipeptide is more quickly removed from solution. The consequent decrease in its loss through hydrolysis causes an increase in its yield. Both stirring and seeding could constitute important generic strategies for promoting crystallization of more soluble dipeptides during their synthesis in similar reversed micellar systems.     

Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media

Several surfactant-coated enzymes have been prepared by coating lipases of various origins with a nonionic surfactant, glutamic acid dioleylester ribitol (2C(18)Delta(9)GE). Enzymatic interesterification of tripalmitin with oleic acid using the surfactant-coated lipase was carried out in organic media. The surfactant-coated lipases could effectively catalyze the interesterification of glycerides better than did the powder lipases. A suitable organic solvent was an aliphatic hydrocarbon such as isooctane. The enzymatic activity for the interesterification strongly depended on the origin of the lipase. The surfactant-coated lipase prepared by Mucor javanicus showed the highest enzymatic activity for the interesterification of glycerides, although its powder lipase did not show enzymatic activity. Selective interesterification of glycerides could be performed by adjusting the concentration ratio of oleic acid to tripalmitin in isooctane. Di-substituted glyceride could be selectively produced when the concentration ratio of carboxylic acid to glycerides was 7. (c) 1995 John Wiley & Sons, Inc.     

Comparative Study between Gas Chromatography and Ion-exchange Chromatography on Amino Acid Analysis of Foods

Lactobacillus brevis and Saccharomyces cerevisiae were completely sterilized by the supercritical (SC) CO2 micro-bubble method. Gaseous (G) and liquid (LQ) CO2 were used in a similar manner to compare the sterilizing effect. Among the three treatments, the microorganisms were only effectively sterilized by the SC CO2 treatment at 25 MPa and 35°C.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Peptide synthesis using proteases dissolved in organic solvents

Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of k_cat/K_m for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH₂ to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH₂ that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.