Aqueous Two-phase Extraction Research Articles

Microwave-assisted aqueous two-phase extraction of l-Citrulline from watermelon rind: optimization, antioxidant potential, and microstructural insight

Microwave-assisted aqueous two-phase extraction of l-Citrulline from watermelon rind: optimization, antioxidant potential, and microstructural insight

Hexafluoroisopropanol-based deep eutectic solvents for efficient aqueous two-phase extraction of flavonoids from Flos Sophorae Immaturus

Aqueous two-phase systems (ATPSs) based on deep eutectic solvents (DESs) are green extraction and separation media and have demonstrated remarkable potential for extracting bioactive substances from natural matrices. However, thus far, flavonoid extraction using DES-based ATPSs has rarely been reported. In this study, the state-of-the-art technique using DES-based ATPSs was employed for developing an effective method for extracting flavonoids from Flos Sophorae Immaturus (FSI). DESs prepared with hexafluoroisopropanol (HFIP) and five quaternary ammonium salts, N111xCl (x = 1, 4, 8, 12, and 16), were used as the hydrogen bond donor and hydrogen bond acceptors, respectively, for constructing ATPSs with K2HPO4 as a salting-out agent and utilized for extracting flavonoids from FSI. Fourier-transform infrared, 1H nuclear magnetic resonance, 1H–1H nuclear Overhauser effect spectroscopy analyses, and density functional theory calculations were implemented to demonstrate the successful preparation of DESs. The DES containing dodecyltrimethylammonium chloride and HFIP with a molar ratio of 1:2 was screened as the optimal DES, showing a higher extraction efficiency and yield of flavonoids compared to those of other DESs and the traditional solvent ethanol. Subsequently, various experimental parameters (amount of DES, salt concentration, liquid-to-solid ratio, extraction temperature, and ultrasonic time) for the extraction process were studied comprehensively and optimized using response surface methodology. The yield of flavonoids from FSI was 21.05 % under optimal extraction conditions (amount of DES = 2 g, salt concentration = 0.21 g/mL, liquid-to-solid ratio = 81 mL/g, and extraction temperature = 60 ℃), which is close to the predicted value of 20.82 %. Extraction mechanisms underlying the performance of DES-based ATPSs during the extraction of bioactive substances are yet to be fully understood. Therefore, molecular dynamics (MD) simulations were employed with rutin as the target compound to probe the extraction mechanism under DES-based ATPS conditions, and the results revealed that hydrogen-bonded frameworks are pivotal for the extraction process. Moreover, the existence of K2HPO4 was found to promote not only the increase in hydrogen bonding networks and π–π stacking interactions within rutin molecules but also the increase in solvent flexibility, facilitating the extraction process. The present work offers a promising approach for flavonoid extraction using DES-based ATPSs. Interpreting the extraction mechanism at the molecular level by adopting the MD simulation technique provides new insights that enable a deeper understanding and aid in the design of the extraction process for bioactive substances under DES-based ATPS circumstances.

Ultrasonic-assisted aqueous two-phase extraction and purification of polyphenols from hawk tea (Litsea coreana var. lanuginose): Investigating its impact on starch digestion

Hawk tea, a conventional herbal beverage, is renowned for its beneficial properties in enhancing digestion and mitigating hyperglycemic tendencies. However, the extraction methodology for hawk tea polyphenols (HTP) has been understudied thus far, impeding its progress and broader application. To develop an efficient approach for HTP extraction, the present study introduced and optimized the application of ultrasonic-assisted aqueous two-phase extraction. Under optimal extraction conditions, the extraction yield of polyphenols from raw HTP was 7.86 %. During purification, LX-B14 was selected due to the highest adsorption and desorption abilities. Then in an in vitro simulated digestion system, HTP significantly reduced the expected glycemic index, raised the content of resistant starch, and decreased the activities of α-amylase and α-glucosidase, indicating its potential for alleviating starch digestion. Accordingly, the results provide an alternative approach for efficiently obtaining phenolic compounds from hawk tea, facilitating the advanced utilization of HTP within the food industry.

Aqueous Two-Phase Extraction of Single-Chirality Carbon Nanotubes with Functionalized ssDNA for Biomarker-Specific Antibody Conjugation

Multiplexed detection of biomarkers for cancer and chronic inflammatory diseases is necessary to improve diagnostic sensitivity and specificity. Our prior studies and that of others has used near-infrared fluorescent single-walled carbon nanotubes (SWCNT) as sensor transducers because of their sensitivity to the local environment, photostability, and emission in the tissue-transparent window. That work engineered the SWCNT surface through non-covalent interactions with short ssDNA oligonucleotides containing a functional group to which a biomarker-specific antibody was conjugated. Further, previous work by other groups has demonstrated the ability to obtain single SWCNT chiralities using the aqueous two-phase extraction (ATPE) technique with non-functionalized ssDNA sequences. Here, we have incorporated primary amine-functionalized ssDNA sequences into the ATPE separation technique to obtain individual SWCNT chiralities with functionalized ssDNA. To those primary amine-functionalized ssDNA-SWCNT complexes, we conjugated antibodies against two inflammatory cytokines. We investigated the sensitivity and specificity of these individual SWCNT chirality sensors specific to two biomarkers together in buffer and serum, finding that each chirality responds specifically only to its cognate biomarker and not the other. This is the first demonstration of single-chirality, molecularly-specific multiplexed SWCNT sensors. It forms the basis of ongoing work to obtain additional chiral species with other functional groups, as well as to develop multiplexed cytokine sensors for in vivo diagnostics.

The Effect of Carbon Nanotube Enantiomers in Macrophages

The inherent asymmetry within our bodies enables remarkable selectivity to chiral molecules. This selectivity is crucial because one stereoisomer may induce undesired effects, remain inactive, or, in the worst case, exhibit toxicity. Even though the knowledge of the interactions of stereoisomer, including enantiomer, with biological systems has been accumulated to a certain extent, our understanding of the body's response to single-wall carbon nanotube (SWCNT) enantiomers is very limited. In this study, we aimed to provide evidence of how macrophages respond to (6,5) enantiomers, finding the importance of their helicity at the nanometer scale. To achieve this, we first separated the left-handed (M) and right-handed (P) enantiomers of (6,5) using aqueous two-phase extraction (ATPE) and conducted a surfactant displacement procedure to coat them with single-stranded DNA. Following the separation and displacement processes, we quantified reactive oxygen species (ROS) as a crucial criterion for assessing the potential influence of the (6,5) enantiomers on macrophages. Considering ROS's role as a signaling molecule at a physiological level, quantifying ROS allows us to determine which enantiomer is more related to oxidative stress and, subsequently, nanotoxicity. To corroborate our findings, we conducted parallel assessments of cells, focusing on metabolic activity, cellular uptake, and proliferation. However, the complexity of the SWCNTs including the coating and aggregation states makes the observed cellular behaviors difficult to explain, even though the SWCNTs have been purified to the enantiomer level. In this regard, we probed the fluorescence signal to evaluate the aggregation states of SWCNTs, hoping to extract the sole enantiomer effects within biological environments. Our results improve the overall comprehension of the relation between SWCNT enantiomeric properties and macrophage responses, emphasizing the distinction between the left and right forms of SWCNTs and providing valuable insights for future biomedical applications of SWCNTs.

Impact of Surfactant to DNA Exchange on Carbon Nanotube Emission for Biosensing Applications

Synthesis methods of single walled carbon nanotubes (SWCNTs) result in mixtures of different chiralites. Chirality affects SWCNT diameter as well electronic and optical properties. For use in optical sensors, mono-chirality SWCNTs offer better signal compared to chirality mixtures. A common chirality separation method is aqueous two-phase extraction, where solutions of surfactants are used to partition SWCNTs based on chirality. The process results in a mono-chiral solution of SWCNTs wrapped in surfactant, however for sensing applications SWCNTs need to be exchanged from this surfactant wrapping to single stranded DNA wrapping. Concerns have been raised about residual surfactant remaining on the SWCNT surface after the exchange process and the effect this would have on the sensing capabilities of the SWCNT. To investigate this, we compared the emission spectra of covalently modified SWCNTs sonicated directly in DNA to that of SWCNTs exchanged from surfactant into DNA. We observed that emission from exchanged SWCNTs was red shifted compared to direct DNA sonicated SWCNTs, however exchanged SWCNTs had similar environmental responsivity to direct DNA sonicated SWCNTs.

(Invited) Purification of Enantiomeric Pairs of DNA-Wrapped Carbon Nanotubes and Their Use in Bilateral Chiral Sensing

This presentation will focus on our recent progress in purifying enantiomeric pairs of DNA-wrapped carbon nanotubes (DNA-CNTs) via the aqueous two-phase extraction method. We will also discuss a potential application of enantiomeric pairs of DNA-CNTs in bilateral chiral sensing.

Application of a kosmotrope (Na2CO3) and chaotrope (NaCl) in chemometric optimization of aqueous two-phase extraction of bioactive compounds in Hypoxis iridifolia.

Enterolactone, coumaric acid and vitexin are polyphenolic compounds present in a variety of fruits, vegetables, cereals and plants. These bioactive compounds are in high demand due to their antioxidant property in various tissues and organs. The purpose of this study was to develop a simultaneous extraction method, an aqueous two-phase extraction (ATPE) method, that would enable the extraction of these compounds from Hypoxis iridifolia. This environmentally friendly extraction method only applied water and ethanol as extraction solvents for these analytes from the plant matrix. After phase separation, the analytes were salted-out from the aqueous phase into the organic phase with the aid of a chaotrope (NaCl) or kosmotrope (Na2CO3). Thereafter, the analytes were withdrawn by a micro-pipette for analysis on the high-performance liquid chromatography-photodiode array detector. Optimization was conducted using a central composite design, where three parameters were examined which involved percentage ethanol, centrifugation time and salt type. Generally, the optimized conditions for extraction were an ethanol percentage of 100% and a centrifugation time of 10min, which yielded concentrations of 2942, 23,823 and 8881mg kg-1 for enterolactone, vitexin and coumaric acid, respectively, in the presence of a kosmotrope. The optimized conditions of extraction in the presence of chaotrope were an ethanol percentage of 66% and a centrifugation time of 10min with concentrations of 6727, 20,833 and 8618mg kg-1 for enterolactone, vitexin and coumaric acid, respectively. The ATPE method involving Na2CO3 was a better extractant of all the compounds studied relative to that of NaCl. The superior extraction capability of Na2CO3 in ATPE could serve as a prototype for the development of efficient extraction methods to meet the high demand for medicinal compounds derived from natural products.

Orthogonal Determination of Competing Surfactant Adsorption onto Single-Wall Carbon Nanotubes During Aqueous Two-Polymer Phase Extraction via Fluorescence Spectroscopy and Analytical Ultracentrifugation.

A combination of analytical ultracentrifugation (AUC) and fluorescence spectroscopy are utilized to orthogonally probe compositions of adsorbed surfactant layers on the surface of (7,5) species single-wall carbon nanotubes (SWCNTs) under conditions known to achieve differential partitioning in aqueous two-phase extraction (ATPE) separations. Fluorescence emission intensity and AUC anhydrous particle density measurements independently probe and can discriminate between adsorbed surfactant layers on a (7,5) nanotube comprised of either of two common nanotube dispersants, the anionic surfactants sodium deoxycholate and sodium dodecyl sulfate. Measurements on dispersions containing mixtures of both surfactants indicate near total direct exchange of the dominant surfactant species adsorbed to the carbon nanotube at a critical concentration ratio consistent with the ratio leading to partitioning change in the ATPE separation. By conducting these orthogonal measurements in a complex environment reflective of an ATPE separation, including multiple surfactant and polymer solution components, the results provide direct evidence for the hypothesis that it is the nature of the adsorbed surfactant layer that primarily controls partitioning behavior in selective ATPE separations of SWCNTs.

Digital Twin for Centrifugal Extractors Exemplified for pDNA Clarification Process after Lysis.

Plasmid DNA is an important substance for the pharmaceutical industry. A major challenge in its production is the clarification of the lysate after harvesting. In this work, a novel process for this is demonstrated in an annular centrifugal contactor (ACC) with an aqueous two-phase extraction. The ACC can increase the space-time yield of the clarification step by at least a factor of 2 compared to the horizontal continuous separator. A model for describing and predicting the conditions in the rotor is created for the ACC and calibrated with an accuracy of R 2 = 0.94, which is sufficient for prediction in process design and operation. A digital twin (DT), which determines the purity of the phases at the outlet of the ACC, is calibrated with an accuracy of 95% for the light phase and 97% for the heavy phase, which defines in manufacturing operation the safety margin for both phases below the theoretical optimal operation point. By using the DT, an additional increase in productivity of 25% can be achieved in the ACC with model-predictive control.

Investigation of europium(III) uptake from highly sulphate solution via cloud point extraction by mono-Schiff base combined with Triton X-100

ABSTRACT Owing to its unique physico-chemical properties, europium is one of the most precious and sought-after rare earth elements in the field of high technology. The major economic and commercial importance of such an element, combined with the pollution risks associated with its intensive use, require the development of efficient and eco-compatible recovery and recycling processes. This study focuses on the recovery of europium from highly saline sulphate media (0.5 mol/L) using an environmentally friendly two-phase aqueous extraction technique (known as cloud point extraction (CPE)), using 2((phenylimino)methyl)phenol mono-Schiff base (HPIMP) as the extractant and Triton X-100 as the non-ionic surfactant. The influence of key experimental parameters such as pH, extractant concentration, surfactant concentration and separation temperature on the europium extraction process was systematically studied and optimized. Under optimum experimental conditions, a quasi-quantitative extraction with a minimal volume fraction of surfactant-rich phase (φs = 0.025), and concentration factor of (CF = 38) was achieved at pH 9.8, in one stage. The analysis of the extraction data revealed that the CPE of europium(III) takes place by a cation exchange-solvation mechanism. The stoichiometry of the complex extracted into the surfactant-rich phase was ascertained to have a composition of 1:2 [Eu:HPIMP] with the slope analysis method. A higher extraction constant was obtained for CPE compared with conventional solvent extraction, confirming the feasibility and usefulness of CPE for Eu(III) recovery. On the other hand, this new HPIMP/Triton X-100 chelating system showed superior extractability for Eu(III) in the CPE process relative to other systems reported previously.

Continuous flow aqueous two-phase extraction of betalains in millifluidic channel

Aqueous Two-Phase System (ATPS) has a proven ability for the selective partitioning and purification of bioactives. This work explores the application of ATPS for the continuous flow extraction of bio-active solute (betalains) from an actual crude extract i.e. red beetroot extract. ATPS composed of Polyethylene Glycol (PEG) 6000 and ammonium sulphate is used for the differential partitioning of betalains and sugars. The continuous flow experiments were performed in millichannel (ID = 1.2 mm) with T-junction. The effect of residence time, multiphase hydrodynamics, and flow-rate ratios on the extraction performance in the continuous flow is studied. The effectiveness of the continuous flow extraction is compared with batch mode extraction of betalains. The continuous flow extraction offers 72.01 % extraction and 89.27 % equilibrium extraction efficiency for betalains with a residence time of 3.38 min. In comparison, batch extraction offered lower performance, with 60.35 % extraction and 74.81 % equilibrium extraction efficiency in 5 min. The improved performance for the continuous flow extraction is attributed to the uniform slug flow in the millichannel. Inclusively, this work develops a continuous flow process for the intensification of extraction of bioactives from the crude extracts of natural sources.

Synergized enzyme-ultrasound-assisted aqueous two-phase extraction and antioxidant activity validation of polysaccharides from tobacco waste

A powerful and innovative technique was developed to efficiently extract and refine polysaccharides from tobacco waste. This was achieved through a combination of enzyme and ultrasound-assisted extraction assisted aqueous two-phase extraction (EUAATPE). The formation of an aqueous two-phase system (ATPS) using various salts and ethanol was investigated. ATPS of 18.0 % (NH4)2SO4 (w/w) and 25.0 % ethanol (w/w) was chosen due to its favorable selection extraction ability and high recovery of 93.68 ± 0.40 %. By utilizing both single-factor experiments and response surface method (RSM), the key factors involved in the EUAATPE process were optimized. The optimal conditions were as follows: ultrasonic power 180 W, ultrasonic time 9 min, cellulase concentration 2.0 %, pectinase concentration 1.0 %, extraction temperature 50 °C, extraction time 1.5 h, and liquid-to-solid ratio 30:1 (g/g). Under the optimal conditions, the extraction yield of polysaccharides was 6.72 ± 0.11 (mg/g), and the purity of the polysaccharides from the bottom phase was 78.73 ± 3.17 %. EUAATPE resulted in a significant enhancement in the extraction yield and purity of polysaccharides from tobacco waste compared to traditional methods such as hot water extraction (HWE), ultrasound-assisted extraction (UAE) and enzyme-assisted extraction (EAE). Polysaccharides of tobacco waste obtained from various methods had analogous monosaccharide compositions, different molar ratio compositions and surface structure characteristics. Moreover, all polysaccharides exhibited a certain extent antioxidant activities and polysaccharides from the bottom phase (PBP) extracted by EUAATPE showed superior antioxidant activity. Hence, EUAATPE, being an innovative and eco-friendly extraction technique, presents a productive substitute for extracting and refining polysaccharides from discarded tobacco resources.

Efficient Separation of Re (VII) and Mo (VI) by Extraction Using E-1006–Ammonium Sulfate Aqueous Two-Phase System

Aqueous two-phase extraction (APTE) stands out as an environmentally friendly technique for the separation of metal ions. The separation of Re (VII) and Mo (VI) in an aqueous solution was investigated using a novel aqueous two-phase system (ATPS) consisting of isodecanol polyoxyethylene ether (E-1006), ammonium sulfate, and water. A phase diagram of this system was developed, and the effects of pH, temperature, extraction time, the concentrations of E-1006 and (NH4)2SO4, and metal ions on the separation of Re (VII) and Mo (VI) were examined. The results show that at pH 7.0, Mo (VI) had almost transformed into the (NH4)2SO4-rich phase, while Re (VI) was extracted into the E-1006-rich phase. The increase in temperature induces a transition of Mo (VI) to the salt-rich phase, which is unfavorable for the extraction of Re (VII). The increase in the concentrations of E-1006 and (NH4)2SO4 has a positive effect on the separation of rhenium and molybdenum. Overall, the ATPS consisting of 200 g/L of E-1006, 200 g/L of (NH4)2SO4, and water yields an extraction efficiency of 97.2% for Re and a high separation factor of 2700 for Re (VII) and Mo (VI) from a mixture of 0.1 g/L of Re (VII) and 5 g/L of Mo (VI) at pH 7.0 and 323.15 K. Separation studies of the simulated leaching solution show that the extraction efficiency for Re (VI) is 99.1% and the separation factor of Re (VII) and Mo (VI) is 5100.

Practical deployment of automation to expedite aqueous two-phase extraction

The feasibility of bioprocess development relies heavily on the successful application of primary recovery and purification techniques. Aqueous two-phase extraction (ATPE) disrupts the definition of "unit operation" by serving as an integrative and intensive technique that combines different objectives such as the removal of biomass and integrated recovery and purification of the product of interest. The relative simplicity of processing large samples renders this technique an attractive alternative for industrial bioprocessing applications. However, process development is hindered by the lack of easily predictable partition behaviours, the elucidation of which necessitates a large number of experiments to be conducted. Liquid handling devices can assist to address this problem; however, they are configured to operate using low viscosity fluids such as water and water-based solutions as opposed to highly viscous polymeric solutions, which are typically required in ATPE. In this work, an automated high throughput ATPE process development framework is presented by constructing phase diagrams and identifying the binodal curves for PEG6000, PEG3000, and PEG2000. Models were built to determine viscosity- and volume-independent transfer parameters. The framework provided an appropriate strategy to develop a very precise and accurate operation by exploiting the relationship between different liquid transfer parameters and process error. Process accuracy, measured by mean absolute error, and device precision, evaluated by the coefficient of variation, were both shown to be affected by the mechanical properties, particularly viscosity, of the fluids employed. For PEG6000, the mean absolute error improved by six-fold (from 4.82% to 0.75%) and the coefficient of variation improved by three-fold (from 0.027 to 0.008) upon optimisation of the liquid transfer parameters accounting for the viscosity effect on the PEG-salt buffer utilising ATPE operations. As demonstrated here, automated liquid handling devices can serve to streamline process development for APTE enabling wide adoption of this technique in large scale bioprocess applications.

Purification, characterization of a plant esterase from red kidney bean and its feasibility for pesticide residue analysis in foods

Plant-derived esterase is a promising alternative enzyme source for pesticide residue analysis. The present study aimed to obtain purified red kidney bean esterase (RKBE), investigate its biochemical properties and application potential in pesticide residue assay. RKBE was extracted and purified by simple aqueous two-phase extraction combined with dextran gel filtration chromatography, with specific activity of 11.845 U/mg and purification fold of 18.952. SDS-PAGE revealed that RKBE has a molecular weight of 33 kDa. Preliminary structural characterization showed the amino acid sequence of RKBE was similar to that of α/β-hydrolase (ESW18998) derived from Phaseolus vulgaris with 19.5% sequence coverage, and the relative contents of β-sheet, β-turn, α-helix, and random coil were 22.69%, 24.19%, 30.12%, and 23.00%, respectively. The optimal pH and temperature of RKBE were 6.5 and 40 °C. Ca2+, Mg2+, Zn2+, and low concentrations of methanol and ethanol stimulated the enzyme activity. The half-life was 138.6 days at −20 °C. Kinetic studies showed that RKBE exhibited a high affinity and catalytic activity towards 1-naphthyl acetate, with a Km of 0.277 mM and a Kcat/Km of 0.455 s−1 mM−1. Enzyme-specific inhibition assay indicated RKBE to be a carboxylesterase. Finally, pesticide susceptibility experiments confirmed that organophosphorus and carbamate pesticides exerted inhibitory effects on RKBE catalytic activity. For methamidophos, methomyl, dichlorvos, and carbaryl, the limit of detection (IC10) values in cabbage were 0.0031, 0.0045, 0.001, and 0.0065 mg/kg, respectively, suggesting that RKBE can serve as a new, widely available and cost-effective enzyme source for pesticide residual assay in foods.

Partitioning of SWCNT mixtures using amphiphilic carbohydrate-based surfactants

Single-walled carbon nanotubes (SWCNTs) have substantial application potential in many branches of technology. Considering that the spatial order of carbon atoms in SWCNTs determines their properties, the characteristics of a material can be adjusted for a specific application by using only selected types of SWCNTs. Unfortunately, SWCNT purification is challenging as the mechanisms of the currently available sorting techniques are not fully understood. The aqueous two-phase extraction (ATPE) method is a promising approach in this context as it offers SWCNT separation in an aqueous environment, so elucidation of its workings is essential. To come closer to achieving this goal, we synthesized a new range of surfactants based on ionic liquids equipped with amphiphilic carbohydrate moieties. We then examined how the modification of their structure influences SWCNT partitioning in two-phase systems composed of poly(ethylene glycol) and dextran. We concluded that the capacity of the ATPE method for purification of SWCNTs depended upon establishing a sufficient degree of order or disorder in the aqueous medium caused by the introduction of chemical compounds of a kosmotropic and chaotropic nature, respectively.

Separation and purification of phycobiliproteins from Thermosynechococcus PCC 6715 by foam fractionation and aqueous twophase extraction

The use of foam fractionation followed by aqueous two-phase extraction has emerged as a potential alternative to traditional liquid chromatography, hitherto irreplaceable in the purification of phycobiliproteins. The crude extracts of C-phycocyanin and allophycocyanin were obtained after Thermosynechococcus PCC 6715 biomass disintegration. The FF process with air flow of 2.4 L·h -1 resulted in purification factors up to 1.47 and partitioning coefficients of about 39, and did not require the addition of surfactants. A temperature of 35˚C allowed for the highest partitioning coefficient of 67.6 and yield of 76%; however, the purity of C-PC in condensate at this temperature was lower than at 25˚C. ATPE was tested in 20 different systems consisting of polyethylene glycol and phosphate or citrate salts, of which PEG1500-citrate gave the highest purification factor value of 2.31. Conversely, a partitioning coefficient of 2416 and 1094 were obtained for the PEG1500-phosphate and PEG3000-phosphate systems, respectively. Interestingly, the use of FF condensate in subsequent ATPE step resulted, for the first time, in the separation of the polymer phase into two fractions, one contained C-phycocyanin and the other allophycocyanin. It can be concluded that the use of a two-step system of FF and ATPE is a viable way to separate phycobiliproteins.

From 3G biofuels to high value-added bioproducts

The paper focused on the co-production of high-value-added product thermostable C-phycocyanin (C-PC) and biomass, further utilized in pyrolysis. The photobiosynthesis of CPC was carried out by the thermophilic cyanobacteria Synechococcus PCC6715 cultivated in the helical and flat panel photobioreactors (PBR). Despite the application of different inorganic carbon sources, both PBRs were characterized by the same growth efficiency and similar C-PC concentration in biomass. To release the intracellular C-PC the biomass was concentrated and disintegrated by the freeze-thaw method. The crude C-PC was then further purified by foam fractionation (FF), aqueous two-phase extraction (ATPE), membrane techniques (UF) and fast protein liquid chromatography (FPLC). Each of the tested methods can be used separately; however, from a practical and economic point of view, a three-stage purification system (FF, FPLC and UF) was proposed. The purity ratio of the final C-PC was about 3.9, which allows it to be classified as a reactive grade. To improve the profitability of 3G biorefinery, the solid biomass residue was used as a substrate to pyrolysis process, which leads to production of additional chemicals in the form of oils, gas (containing e.g. H 2) and biochar.

Ultrasound-assisted Aqueous Two-Phase Extraction of Flavonoids from Scutellariae Radix and Evaluation of their Bioactivities In Vitro

Background: Scutellariae Radix, one of the most widely used herbs in Traditional Chinese Medicine, exhibits various biological activities due to its chemical components, which stand out for a number of flavonoids. In this study, Ultrasound-assisted aqueous two-phase extraction (UAATPE) was employed for the first time to obtain a high extraction rate and high purity of flavonoids from Scutellariae Radix. Methods: The Box-Behnken response surface method (RSM) was utilized to optimize the extraction conditions with the application of the new aqueous two-phase system (ATPS) composed of ethanol and ammonium sulfate. The major influence factors, including ethanol concentration, ammonium sulfate concentration, liquid-to-solid ratio, sonication time, and extraction temperature, were investigated by the single-factor experiment. The compositional characterization of flavonoids was characterized with HPLC-UV. Scanning electron microscopy (SEM) was applied to research the surface morphology of raw material. Furthermore, the bioactivities of the extract obtained by UA-ATPE were studied in vitro. Results: The optimal extraction conditions were as follows: the ethanol content was 26.12% (w/w), the ammonium sulfate content was 20.02% (w/w), the liquid-to-solid ratio was 40 mL/g, the sonication time was 5 min with the ultrasonic power of 250 W, and the operating process was performed at room temperature. Compared with the traditional extraction methods, UA-ATPE exhibited higher extraction efficiency and better extraction selectivity. The DPPH and ABTS radical scavenging tests showed that enriched products possessed strong antioxidant activity. Conclusion: The study confirmed that the developed method of UA-ATPE could be used as an efficient, eco-friendly, and low-consumption method for the extraction and purification of flavonoids from Scutellariae Radix.