A simple method for purification of bromelain in a thermosensitive triblock copolymer-based protection system and recycling of phase components

In the work, we chose stem bromelain as a model to investigate the storage and purification of bromelain from pineapple peel. Extraction of bromelain from pineapple peel using a two-stage aqueous two-phase extraction system composed of a thermoseparating copolymer EOPOEO and K₂HPO₄. Bromelain predominantly partitioned to the EOPOEO-rich phase and then re-extract to the top dilute phase. The recovery of enzyme activity (68.6%) and purification factor (6.53) were determined under optimum conditions. The EOPOEO-rich phase and salt were recycled, and the recovery of enzyme activity could reach up to 60%. This method has been proved to obtain highly purified and stable bromelain.     

Chelatometric salting-out extraction and characteristics of flavonoids from Folium nelumbinis based on an ethanol/K2HPO4 system

A new salting-out extraction method, namely chelatometric salting-out extraction, was established for rapidly extracting flavonoids from Folium nelumbinis. Borax was used as a chelating agent and an ethanol/K₂HPO₄ system as the salting-out extraction system. The maximum flavonoid concentration in the top phase was observed at an ethanol concentration of 43.0% and an amount of salt of 2.45 g under the experimental conditions. The pH value of the system and extraction time had no obvious influence on the extraction efficiency of flavonoids. The results also showed that the extraction efficiency of flavonoids exceeds 90.4% at the first time extraction. Moreover, the antioxidant activities of flavonoids were evaluated by DPPH? radical assays.     

Salting‐out of acetone, 1‐butanol,and ethanol from dilute aqueous solutions

The salting‐out phase equilibria for acetone, 1‐butanol, and ethanol (ABE) from dilute aqueous solutions using potassium carbonate (K₂CO₃) and dipotassium hydrogen phosphate trihydrate (K₂HPO₄?3H₂O) as outstanding salting‐out agents were investigated. Increasing the salt concentration strengthened the salting‐out effects and improved the distribution coefficients of all three solvents (ABE) significantly. Temperature had a slight effect on the phase equilibria. The K₂HPO₄ solution (69 wt %) showed a stronger salting‐out effect than the K₂CO₃ solution (56 wt %) on recovering ABE from dilute aqueous solutions. Dilute aqueous solutions containing more solvents increased the recoveries of acetone and 1‐butanol, while the results showed a negligible effect on the solubility of ABE. The solubility of ABE was also correlated well with the molar number of salt per gram of water in the aqueous phase. A new equation demonstrated this satisfactorily. © 2015 American Institute of Chemical Engineers AIChE J, 61: 3470–3478, 2015     

Separation of α‐Lactalbumin and β‐Lactoglobulin by a Convenient Liquid‐Solid Extraction System: Application to Milk Whey

A liquid‐solid extraction system based on Tween 80/phosphate was developed. Under the optimized conditions (9 wt % Tween 80, 1.6 : 1 (molar ratio) K₂HPO₄ : NaH₂PO₄, 1.25 mol/L total phosphate, pH = 7.4), α‐Lactalbumin (α‐La) and β‐Lactoglobulin (β‐Lg) were separated with recovery rates of 87.6 % (in the solid polymeric phase) and 98.2 % (in the salt aqueous phase), respectively. Under the effects of water and salt, the solid phase had the ability to form a new liquid‐solid extraction system, and 85.1 % of α‐La could be reversely extracted into the new salt aqueous phase. Following dialysis against water, proteins obtained through extraction and reverse extraction, were analyzed by polyacrylamide gel electrophoresis (PAGE) and thin‐layer scanning. The method was applied successfully to separate α‐La and β‐Lg from milk whey.     

Selective partition of caffeine from coffee bean and guaraná seed extracts using alcohol–salt aqueous two-phase systems

This work proposes the application of flexible alcoholic aqueous two-phase systems to manipulate the partition of caffeine. An optimization study was performed the caffeine partitioning towards top phase (60.0 wt% 2-propanol + 20.0 wt% K₃PO₄ at 303 K – K_caf = 3.4 and R_T = 89.1%) and bottom phase (60.0 wt% methanol + 17.5 wt% K₂HPO₄/KH₂PO₄ at 278 K – K_caf = 0.06 and R_B = 81.1%). These processes were evaluated by applying them to real biomass as guaraná seeds (purification factor (PF) = 6.59-fold) and coffee beans (PF = 3.24-fold) for the 2-propanol and K₂HPO₄/KH₂PO₄ system.     

Application of Water-Miscible Alcohol-Based Aqueous Two-Phase Systems for Extraction of Dyes

The water-miscible alcohols (ethanol, 1-propanpol, 2-propanol) were used to form aqueous two-phase systems with K₂HPO₄ and Na₂HPO₄. Meanwhile, the organic citrates were also investigated as the phase-separation salts. The effects of the types and concentrations of phase-forming substances as well as temperature on the formation of aqueous two-phase systems were discussed. The alcohol (ethanol, 1-propanpol, 2-propanol)–salt (K₂HPO₄, K₃C₆H₅O₇, (NH₄)₃C₆H₅O₇) aqueous two-phase systems were used to extract Rhodamine B and methyl orange in aqueous solution. It proved that the water-miscible alcohol–salt (inorganic salt, organic salt) aqueous two-phase systems can be both used to extract hydrophilic targets directly without adding any reaction reagents. The aqueous two-phase extraction process was also discussed.     

Enrichment of Aromatic Compounds Using Ionic Liquid and Ionic Liquid-Based Aqueous Biphasic Systems

Both hydrophibic [C₈mim][PF₆] and [C₄mim]Cl/(K₂CO₃, K₂HPO₄ or K₃PO₄) aqueous biphasic system (ABS) were employed for the enrichment of aromatic compounds including nitrobenzene, 4-nitrophenol, phenol and aniline from aqueous solutions. In [C₈mim][PF₆] enrichment system, the distribution ratio (D) of nitrobenzene and aniline increase with the increasing of pH values of aqueous phase from 2 to 8, but there is no obvious change for 4-nitrophenol and phenol. And the D values of the aromatic compounds decrease with the phase ratio change from 1/5 to 1/30 (IL phase volume/aqueous phase volume). In [C₄mim]Cl/(K₂CO₃, K₂HPO₄ or K₃PO₄), the distribution ratio increase with the increasing of the salts concentrations. The distribution ratio order in [C₈mim][PF₆] and [C₄mim]Cl/kosmotropic salt ABS is nitrobenzene > phenol > aniline > 4-nitrophenol. It is expected that these results are promising for the removal of the aromatic compounds from water, especially for sample enrichment in analytical science.     

Simultaneous recovery of glycyrrhizic acid and liquiritin from Chinese licorice root (Glycyrrhiza uralensis Fisch) by aqueous two-phase system and evaluation biological activities of extracts

Aqueous two-phase system (ATPS) was used for simultaneous purification of glycyrrhizic acid (GA) and liquiritin (LQ) from crude extract of Chinese licorice root. It was revealed that 87% GA and 94% LQ were retrieved in the ATPS top phase, under the optimum conditions of 25% (w/w) ethanol, 30% (w/w) K₂HPO₄ and 4% (w/w) loading sample at 10–40°C. Compared with crude extract, the ATPS top-phase extract exhibited the highest antioxidative activity, but no tyrosinase inhibitory effect. Whereas, the ATPS bottom-phase extract was proved to be effective ABTS radical scavenger and tyrosinase inhibitor, suggesting the potency of the alcohol-salt ATPS purification for the different medicinal purposes.     

Optimal culture condition for the production of phenyalanine ammonia lyase from <Emphasis Type="Italic">E. coli</Emphasis>

The effects of culturing conditions on phenylalanine ammonia lyase production by a recombinant E. coli strain were investigated by using a controlled fed-batch fermentation system. In a 5 L fermentor, the optimal composition of the batch medium was 2% glucose, 1% yeast extract, 0.7% K₂HPO₄, 0.8% KH₂PO₄, 0.5% (NH₄)₂SO₄, 0.1% MgSO₄·7H₂O. The optimal feed glucose solution was 50%. Glucose concentration and pH of the culture broth were maintained at about 2.0 g/L and 7.0 during the fed-batch phase, respectively. Following 24-h cultivation, 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) was added and temperature was shifted from 37°C to 42°C to induce pal gene expression. Under optimal conditions, a high productivity of 300 U/g could be achieved after 48 h culture, and a cell density of OD₆₀₀ about 82 was obtained at 52 h culture at 500 r/m stirrer speed and 1 vvm, respectively.     

Studies on cultivation and biological activities ofPleurotus nebrodensis inzenga

Environmental factors affecting mycelial growth and exo-polysaccharide production fromPleurotus nebrodensis Inzenga (PN) and biological activities of PN extractsin vitro were studied. The culture conditions for effective mycelial growth and exo-polysaccharide production were found to be 25 ‡C, 5% of inoculum size, and an initial pH from 6.5 to 7.0. When 5% of glucose was used, the maximum mycelial growth and exo-polysaccharide concentrations were 8.3 and 3.07 g/L, respectively. Among the various nitrogen sources, the mycelial growth and exo-polysaccharide production were very strong when polypeptone was used. In the case of the minerals sources, K₂HPO₄ and MgSO₄·7H₂O were found to best support for mycelial growth and exo-polysaccharide production. Under optimal conditions and methods, the maximum mycelial growth and exo-polysaccharide production were obtained after 10 days of culture, 12.84 and 4.85 g/L, respectively, in a jar fermentor. The effects of the PN extracts on the viability of three human cancer cell lines and antioxidant activity were investigatedin vitro. When cancer cells of the lung (A549), cervical region (HeLa) and colon (KM12C) were incubated at 6 mg/mL of the PN ethanol extracts, the viabilities of the HeLa and KM12C cells were slightly decreased. However, the growth of the A549 cells was remarkably inhibited when the PN ethanol extract was over 4 mg/mL. The antioxidant activity showed 46.2% at 40 μL, which was about 3.2 fold higher than that of the PN methanol extract.     

Extraction of Puerarin using Ionic Liquid Based Aqueous Two-Phase Systems

Various ionic liquid-based aqueous two-phase systems (IL-ATPS) were evaluated to extract puerarin. The results indicated that the nature of ILs, the salting-out ability of salt, and the acidity and basicity of the salt-rich solution had important influence on the extraction efficiency. Various factors were optimized systematically, that is, the amount of IL, salt, puerarin, and short-chain alcohol. Under the optimal experimental conditions (the amount of K₂HPO₄ 0.30–0.42 g/mL, [Bmim]Br 0.30–0.36 g/mL and 1.0 mL puerarin stock solution), the extraction efficiency of puerarin was over 99% by a single-step extraction, which indicated that the evaluated IL-ATPS was a prospective extraction medium. Finally, the IL-ATPS was successfully used to extract puerarin from Radix Puerariae Lobatae extracts.     

Optimization of salts in medium for production of carboxymethylcellulase by a psychrophilic marine bacterium, <Emphasis Type="Italic">Psychrobacter aquimaris</Emphasis> LBH-10 using two statistical methods

The concentration of four mineral salts in the medium for the production of carboxymethylcellulase (CMCase) by Psychrobacter aquimaris LBH-10 were optimized using orthogonal array method (OAM) and response surface method (RSM) and their results from two statistical methods were compared. The analysis of variance (ANOVA) of data from central composite design (CCD) based on OAM indicated that potassium phosphate gave the highest percentage participation for cell growth as well as production of CMCase. However, their relative participations of four salts for cell growth were different from those for production of CMCase. The ANOVA of results from RSM indicated that highly significant factors (“probe>F” less than 0.0001) for cell growth were K₂HPO₄ and (NH₄)₂SO₄, whereas those for production of CMCase were K₂HPO₄, NaCl, and MgSO₄·7H₂O. The optimal concentration of K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄ for cell growth extracted by Design Expert Software based on RSM were 7.10, 0.84, 0.24, and 0.33 g/L, respectively, whereas those for production of CMCase were 3.00, 0.52, 0.34, and 0.45 g/L. The optimal concentrations of salts for cell growth and production of CMCase using RSM basically coincided with those using OAM as well as those from ‘one-factor-at-a-time’ method. The production of CMCase by P. aquimaris LBH-10 with optimized concentrations of salts was 273.0 U/mL, which was enhanced by 1.27 times higher than the previous report.     

Deep eutectic solvents aqueous two-phase system based ultrasonically assisted extraction of ursolic acid (UA) from Cynomorium songaricum Rupr.

Deep eutectic solvents (DESs) are new green solvents that have attracted the attention of the scientific community mainly due to their unique properties and special characteristics, which are different from those of traditional solvents. A method based on ultrasonically assisted deep eutectic solvent aqueous two-phase systems (UAE-DES-ATPS) was developed for extracting ursolic acid (UA) from Cynomorium songaricum Rupr. Four different types of choline chloride-based DESs were prepared. Choline chloride-glucose (ChCl-Glu) exhibited good selective extraction ability. An optimum DES-ATPS of 36% (w/w) ChCl-Glu and 25% (w/w) K₂HPO₄ was considered to be a satisfactory system for extracting UA. Response surface methodology (RSM) method was utilized to optimize the extraction of UA using UAE-DES-ATPS. The quadratic models were extremely significant (p?<?0.01) for all the parameters studied with high correlation coefficients (R²) of 0.9593. The optimum ultrasound-assisted conditions were as follows: solvent to solid ratio of 15:1 (g/g), ultrasound power density of 4.7?W·mL^?1, and extraction time of 54?min. Compared with the conventional UAE method, the yields were basically the same, but the presented method had higher purity. The structure of UA did not change between pure UA and UA in the upper phase using UV–vis and FT-IR. This approach using ChCl-based DES-ATPS as a novel extraction system and ultrasound as a source of energy provided a better choice for the separation of active components from other natural products.     

Supercritical Carbon Dioxide Extraction of Flavonoids from Pomelo (Citrus grandis (L.) Osbeck) Peel and Their Antioxidant Activity

Supercritical carbon dioxide (SC-CO₂) extraction of flavonoids from pomelo (Citrus grandis (L.) Osbeck) peel and their antioxidant activity were investigated. Box-Behnken design combined with response surface methodology was employed to maximize the extraction yield of flavonoids. Correlation analysis of the mathematical-regression model indicated that a quadratic polynomial model could be used to optimize the SC-CO₂ extraction of flavonoids. The optimal conditions for obtaining the highest extraction yield of flavonoids from pomelo peel were a temperature of 80 °C, a pressure of 39 MPa and a static extraction time of 49 min in the presence of 85% ethanol as modifier. Under these conditions, the experimental yield was 2.37%, which matched positively with the value predicted by the model. Furthermore, flavonoids obtained by SC-CO₂ extraction showed a higher scavenging activity on hydroxyl, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals than those obtained by conventional solvent extraction (CSE). Therefore, SC-CO₂ extraction can be considered as a suitable technique for the obtainment of flavonoids from pomelo peel.     

Reactive Extraction of Pyridine Carboxylic Acids with N,N-Dioctyloctan-1-Amine: Experimental and Theoretical Studies

The paper represents the equilibrium study on reactive extraction of pyridine-3-carboxylic acid (NA) and pyridine-4-carboxylic (iNA) acid from aqueous solution by N, N-dioctyloctan-1-amine (TOA) dissolved in five different diluents [dodecane, methyl benzene, decan-1-ol, 4-methylpentan-2-one (MIBK), and chloroform] at constant temperature of 298 ± 1 K. According to an experimental study, the extraction ability of diluents with TOA is found to be in the order of chloroform > decan-1-ol > MIBK > methyl benzene > dodecane for both acids. The highest extraction efficiency in terms of the distribution coefficient (K _D) is found to be 45.15 and 25.79 for NA (0.12 mol · dm^?3) and iNA (0.03 mol · dm^?3), respectively. The values of loading ratio, Z (between 0.194 and 0.512) for both acids indicate the formation of 1:1 acid-TOA complexes in the organic phase. The values of the equilibrium constants (K ₁₁) are determined from the experimental data using mass action law. These estimated values of K ₁₁ are compared with the predicted values of K ₁₁ from relative basicity and linear solvation energy relationship (LSER) models. The LSER model predicts the K ₁₁ with an error limit of ±3% for NA and ±2% for iNA.     

The fabrication and characterization of bioactive Akermanite/Octacalcium phosphate glass-ceramic scaffolds produced via PDC method

In the present study, a bioactive silicate-phosphate glass-ceramic scaffold was fabricated via the polymer-derived ceramics (PDC) method. K₂HPO₄ phosphate salt was used as the P₂O₅ precursor in this method. The effect of K₂HPO₄ wt% and heat treatment temperatures (900–1100 °C) was evaluated. It was observed that although increasing the wt% of K₂HPO₄ led to the formation of scaffolds with higher densities and strengths, it could also increase the formation of the calcium phase, which could result in improper release behavior of scaffolds. On the other hand, higher heat treatment temperatures enhanced the strength of the scaffolds but eliminated the bioactive octacalcium phosphate (OCP) phase. X-ray diffraction (XRD) analysis showed that the dissolution of the OCP phase in simulated body fluid (SBF) resulted in precipitation of hydroxyapatite (HA) on the scaffold surface which enhanced the bioactivity. Furthermore, based on microstructural studies by Scanning Electron Microscopy (SEM), the fabricated scaffold possessed a wide range of pore sizes, appropriate for osteointegration and bone formation. The optimum wt% of phosphate salt was less than 6 wt% and the optimum heat treatment temperature was 1000 °C. After the optimization of compositions and processing, Alamar Blue Assay was used to evaluate HOb cell cultures, showing a continuous proliferation for the optimized samples.     

Application of statistical experimental design for optimization of physiological factors and their influences on production of pullulan by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> HP-2001 using an orthogonal array method

Physiological factors for the production of pullulan by A. pullulans HP-2001 were optimized using orthogonal array method and their influences were compared using Qualitek-4 software. The analysis of variance (ANOVA) indicated that the most important factor for cell growth was yeast extract, whereas that for production of pullulan was glucose. The optimal conditions for cell growth were found to be 100.0 g/L glucose, 10.0 g/L yeast extract, and initial pH of 6.0, whereas those for the production of pullulan were 100.0 g/L glucose, 2.5 g/L yeast extract, and initial pH of 5.5. Among four mineral salts in the medium, potassium phosphate (K₂HPO₄) was found to be the most important factor for cell growth as well as production of pullulan. Next important salt for cell growth was (NH₄)₂SO₄, whereas that for production of pullulan was NaCl. The optimal concentrations of K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄ for cell growth were 7.5, 1.00, 0.1, and 1.20 g/L, respectively, whereas those for production of pullulan were 2.5, 0.25, 0.8, and 0.30 g/L. The expected cell growth and the production of pullulan by A. pullulans HP-2001 under these optimized conditions were 12.61 and 11.49 g/L, respectively.     

Fatty Imidazolines: A Novel Extractant for the Recovery of Palladium(II) from Hydrochloric Acid Solutions

The extraction of palladium(II) from hydrochloric acid solutions with a novel highly basic extractant, a mixture of homologous 1-[2-(alkanoylamino)ethyl]-2-alkyl-2-imidazolines (AAI) in toluene with 15% (v/v) of n-octanol was studied. Palladium(II) is rapidly and most effectively extracted with AAI hydrochloride at the low hydrochloric acid (chloride ions) concentration (up to 1 M) and can be completely separated from Fe(III), Cu(II), and Co(II). The palladium(II) extraction at the optimum acidity occurs via an anion-exchange mechanism with the formation of ionic associates (LH)₂PdCl₄ (K _ex = (1.5 ± 0.2) · 10⁴ at 0.5 M HCl) and is accompanied by the dimerization of palladium(II) in the organic phase with the formation of ionic associates (LH)₂Pd₂Cl₆ (K _dim = (3.9 ± 0.4) · 10^?4 at 0.5 M HCl). The anion-exchange extraction of palladium(II) at the acidity of 0.5 M HCl is temperature independent in the range 20–49°C. Complete stripping of palladium(II) can be performed using a 5% solution of thiourea in 0.1 M HCl.     

Supercritical fluid extraction from dried banana peel (Musa spp., genomic group AAB): Extraction yield, mathematical modeling, economical analysis and phase equilibria

Supercritical fluid extraction from dried banana peel (Musa spp., subgroup Prata, genomic group AAB, popularly known in Brazil as Enxerto) was studied. The aspects investigated were: overall extraction curve (OEC), mass transfer modeling of the yield curves, economical analysis of the process and phase equilibrium data for the pseudo-ternary system of banana peel extract, carbon dioxide and ethanol. The extraction operating conditions evaluated were: pressure ranging from 100 bar to 300 bar, temperature from 40 to 50 °C and constant solvent flow rate of 5.0 gCO₂/min. Experimental extraction data were correlated using three kinetic models based on mass transfer equations (logistic, diffusion and Esquível models). Phase equilibrium measurements were performed using pressure from 64.9 bar to 239.9 bar and mass fraction of supercritical extract from 0.52 to 3.55 wt%. Yield results ranged from 0.6 to 6.9% d.b. (dry basis). The lowest deviation between experimental and correlated data was obtained by the Logistic model, except for the curve at 300 bar and 40 °C which was best represented by the Esquível model. The economical analysis identified the possibility to apply the supercritical fluids to obtain extracts from banana peel in an industrial scale. Phase equilibrium for the supercritical extract from banana peel with carbon dioxide modified by ethanol exhibited liquid-liquid, vapor-liquid (bubble point) and vapor-liquid-liquid phase transitions. A crossover phenomenon for the systems evaluated was observed for pressures between 200 bar and 240 bar, for both groups of assays, i.e., supercritical extraction and phase equilibrium.