Aqueous Two-phase Extraction Research Articles

Flavonoids from Fig (Ficus carica Linn.) Leaves: The Development of a New Extraction Method and Identification by UPLC-QTOF-MS/MS

Flavonoid-rich leaves of the Ficus carica L. plant are usually discarded as waste. In this work, ultrasonic enzyme-assisted aqueous two-phase extraction (UEAATPE) was proposed as an innovative method to estimate the total flavonoids present in F. carica L. leaves. Total flavonoids were analyzed qualitatively and quantitatively by UPLC-QTOF-MS. At 38% (w/w) ethanol/18% (w/w) ammonium sulfate, we achieved the optimum conditions in which to establish an easy-to-form aqueous two-phase extraction (ATPE) as the final system. The optimal UEAATPE conditions were set at an enzymatic concentration of 0.4 U/g, 150 min enzymolysis time, an enzymolysis temperature of 50 °C, a liquid–solid ratio of 20:1 (mL/g), and 30 min ultrasonic time. The yields of the total flavonoids, i.e., 60.22 mg/g, obtained by UEAATPE were found to be 1.13-fold, 1.21-fold, 1.27-fold, and 2.43-fold higher than those obtained by enzyme-assisted ATPE (EAATPE), ultrasonic-assisted ATPE (UAATPE), ATPE, and soxhlet extraction (SE) methods, respectively. Furthermore, eleven flavonoids from the leaves of the F. carica L. plant were completely identified and fully characterized. Among them, ten flavonoids have been identified for the first time from the leaves of the F. carica L. plant. These flavonoids are quercetin 3-O-hexobioside-7-O-hexoside, 2-carboxyl-1,4-naphthohydroquinone-4-O-hexoside, luteolin 6-C-hexoside, 8-C-pentoside, kaempferol 6-C-hexoside-8-C-hexoside, quercetin 6-C-hexobioside, kaempferol 6-C-hexoside-8-C-hexoside, apigenin 2″-O-pentoside, apigenin 6-C-hexoside, quercetin 3-O-hexoside, and kaempferol 3-O-hexobioside. Therefore, F. carica L. leaves contain new kinds of unidentified natural flavonoids and are a rich source of biological activity. Therefore, this research has potential applications and great value in waste handling and utilization.

Comparison of three methods for thermostable C-phycocyanin separation and purification

The efficient separation and purification methods are the major obstacles in the production and further use of bioproducts. One such valuable bioproduct is the thermostable C-phycocyanin (C-PC). There is a great commercial need to develop C-PC production with an appropriate degree of purity. However, the selected methods and their sequences are also depended on the quality of crude extract obtained from biomass. In this paper three down-stream processing methods, such as foam fractionation (FF), which according to our knowledge has not been used for separation of C-PC, aqueous two-phase extraction (ATPE) and conventional ultrafiltration were investigated for purification of thermostable C-PC from two crude extracts with lower and higher protein concentration and purity obtained from biomass of Synechococcus sp. PCC6715. The FF process resulted in a recovery yield of 49% and a purification factor of 1.472 without the addition of surfactants or pH adjustment. The most effective ATPSystem consists of polyethylene glycol 6000 and phosphate salt with a recovery yield of 97%, partitioning coefficient of about 28 and purification factor of 1.466. Ultrafiltration gave a recovery yield of 92% and purification factor of 1.123. Two possible ways of C-PC purification depending on the crude extract purity and concentration was proposed.

Intensifying aqueous two-phase extraction by adding decisive excipients for enhancement of stability and solubility of biomolecules

Despite the benefits of aqueous two-phase extraction (ATPE) for purification of high-value biomolecules, phase-formers that are used to generate the aqueous two-phase system (ATPS) often reduce the stability and solubility of the biomolecule. Within this work, we thus evaluated the use of suitable excipients to increase the biomolecule stability and solubility during ATPE, whilst not negatively affecting yield and selectivity. Applying this new physically sound approach enabled a rapid selection of L-arginine as suitable excipient for the ATPE of Immunoglobulin G. It was demonstrated that both the (conformational and colloidal) stability and solubility of Immunoglobulin G in the ATPS were increased, thus maximizing yield, minimizing precipitation, and improving the extraction performance of the ATPS. The applicability and transferability of the approach was confirmed by optimizing the ATPE of an industrial relevant amine dehydrogenase. The optimized ATPS enabled an extraction yield of 79.17 wt% (impurities < 7 wt%) directly from crude extract. Furthermore, using the excipients broadened the ATPE process window, thus simplifying process handling during ATPE and further downstream unit operations. Prospectively, the use of excipients can support the establishment of ATPE as downstream technology.

High yield recovery of 2,3-butanediol from fermented broth accumulated on xylose rich sugarcane bagasse hydrolysate using aqueous two-phase extraction system

Downstream processing of chemicals obtained from fermentative route is challenging and cost-determining factor of any bioprocess. 2,3-Butanediol (BDO) is a promising chemical building block with myriad applications in the polymer, food, pharmaceuticals, and fuel sector. The current study focuses on the recovery and purification of BDO produced (68.2 g/L) from detoxified xylose-rich sugarcane bagasse hydrolysate by a mutant strain of Enterobacter ludwigii. Studies involving screening and optimization of aqueous-two phase system (ATPS) revealed that 30% w/v (NH4)2SO4 addition to clarified fermented broth facilitated BDO extraction in isopropanol (0.5 v/v), with maximum recovery and partition coefficient being 97.9 ± 4.6% and 45.5 ± 3.5, respectively. The optimized protocol was repeated with unfiltered broth containing 68.2 g/L BDO, cell biomass, and unspent protein, which led to the partitioning of 66.7 g/L BDO, 2.0 g/L xylose and 9.0 g/L acetic acid into organic phase with similar BDO recovery (97%) and partition coefficient (45).

English

Quinoa (Chenopodium quinoa Willd.) is a functional and ideal food for human nutrition and an Andean seed-producing crop. In this study, ultrasonic-assisted extraction of total flavonoids in Quinoa with ethanol (C2H5OH)-ammonium sulfate ((NH4)2SO4) aqueous two-phase system was performed based on the Box-Behnken experimental design principle. The highest extraction rate of TFQ under the condition of 28% C2H5OH -14% (NH4)2SO4 aqueous two-phase extraction system was used to analyze the variance of TFQ extraction rate as the response value. The multiple quadratic linear regression equation was obtained by a three-factor three-level response surface method. The extraction rate= 74.28+1.78 A+0.10 B+0.38 C+0.20 AB+0.05 AC+0.05 BC+1.000E-002 A2-0.94 B2-0.69 C2. The response surface analysis showed that the best extraction conditions of aqueous two-phase were the crude TFQ mass fraction 20.6%, pH 7.18, NaCl mass fraction 2.23% and the maximum value predicted by the extraction rate model was 75.929 3% (P=0.994). The average extraction rate of TFQ was 75.3%, according to the optimal two-aqueous phase extraction conditions. The ETFQ has varying degree of scavenging effect on hydroxyl radical, oxygen free radicals, nitrite and ·ABTS+ compared with vitamin C. Among them, the scavenging effect of the ETFQ on hydroxyl radical, oxygen free radicals and ·ABTS+ was greater than vitamin C, except nitrite. Also, the ETFQ has the strongest inhibitory effect on E. coli and Bacillus subtilis, and the inhibitory rate can reach up to high dose 97.59 and 98.44%, MIC is 1.56 mg/mL; the second is the inhibition of S. aureus, MIC is 6.25 mg/mL. It has the weakest inhibitory effect on Salmonella. The antibacterial rate was positively correlated with the ETFQ mass concentration. The results help to discover the medicinal effects of quinoa in addition to nutrition to carry out more in-depth research and increase economic value. © 2021 Friends Science Publishers

(Invited) Stereoselective Photoluminescent Properties of DNA-Carbon Nanotubes: A Primer for Molecular Perceptron

Single-walled carbon nanotubes (SWCNTs) have emerged to be useful tools in molecular recognition in recent years. Due to their well-defined optical properties, SWCNTs offer unique characteristics that most small-molecule sensors lack. For example, SWCNTs exhibit a near-IR (NIR) photoluminescence which makes them great candidates for sensing biomolecules. Typically, the synthesis of SWCNTs yields over 30 distinct tube chiralities, with two-thirds of the mixture comprising semiconducting and the remaining third consisting of quasi-metallic or true-metallic tubes. To add to the mixture’s structural complexity, each chirality that has the same helicity could also have different handedness – species that are enantiomeric in nature. In our lab at NIST, we have recently developed techniques based on aqueous two-phase extraction (ATPE) to separate enantiomers of SWCNTs wrapped with specific DNA sequences. Most of the sensors that have been developed in this field, whether they are coated and dispersed by surfactants or DNA, take advantage of mixtures of various SWCNT helicities to elicit an optical response from interactions of small molecules. However, the responses obtained from each enantiomeric species of SWCNT is largely ignored, or at best, assumed to be similar. In this presentation, we discuss the differences in photoluminescence responses observed when SWCNTs wrapped with various DNA sequences interact with enantiopure small molecules. With this study, we give proof that specific DNA sequences allow for specific optical responses between the mixtures of diastereomeric DNA-SWCNT nanohybrids and single-chirality enriched DNA-SWCNTs, and enantiomeric amino acids. This study is the first proof-of-concept that small chiral molecules interact with chirality enriched DNA-SWCNTs to different extents and give rise to specific optical signatures that could be exploited for future developments of sensing arrays for complex biological mixtures.

(Invited) Sensing with Chirality Pure Near Infrared Fluorescent Carbon Nanotubes

Semiconducting single wall carbon nanotubes (SWCNTs) fluoresce in the near infrared (NIR) and the emission wavelength depends on their chirality (n,m). Interactions with the environment affect the fluorescence and can be tailored by functionalizing SWCNTs with biopolymers such as DNA, which is the basis for fluorescent biosensors. So far, such biosensors were mainly assembled from mixtures of SWCNT chiralities with large spectral overlap, which affects sensitivity as well as selectivity and prevents multiplexed sensing. The main challenge to gain chirality pure sensors has been to combine approaches to isolate specific SWCNTs and generic (bio)functionalization approaches. Here, we created chirality pure SWCNT-based NIR biosensors for important analytes such as neurotransmitters and investigated the impact of SWCNT chirality/handedness as well as long-term stability and sensitivity. For this purpose, we used aqueous two-phase extraction (ATPE) to gain chirality pure (6,5)-, (7,5)-, (9,4)- and (7,6)-SWCNTs (emission at ~ 990, 1040, 1115 and 1130 nm). Exchange of the surfactant sodium deoxycholate (DOC) to specific single-stranded (ss)DNA sequences yielded monochiral sensors for small analytes (dopamine, riboflavin, ascorbic acid, pH). DOC residues impaired sensitivity and therefore sufficient removal was necessary. The assembled monochiral sensors were up to 10 times brighter than their non-purified counterparts and the ssDNA sequence determined absolute fluorescence intensity as well as colloidal (long-term) stability and selectivity for the analytes. (GT)40-(6,5)-SWCNTs displayed the maximum fluorescence response to the neurotransmitter dopamine (+140 %, Kd = 1.9 x10-7 M) and a long-term stability > 14 days. The specific ssDNA sequences imparted selectivity to the analytes in majority of the cases independent of SWCNT chirality and handedness of (+/-) (6,5)-SWCNTs, which allows a predictable design. Finally, multiple monochiral/single-color SWCNTs were combined to achieve ratiometric/multiplexed sensing of the important analytes dopamine, riboflavin, H2O2 and pH. In summary, we demonstrated the assembly, characteristics and potential of monochiral (single-color) SWCNTs for multiple NIR fluorescent sensing applications.

En route to single-step, two-phase purification of carbon nanotubes facilitated by high-throughput spectroscopy

Chirality purification of single-walled carbon nanotubes (SWCNTs) is desirable for applications in many fields, but general utility is currently hampered by low throughput. We discovered a method to obtain single-chirality SWCNT enrichment by the aqueous two-phase extraction (ATPE) method in a single step. To achieve appropriate resolution, a biphasic system of non-ionic tri-block copolymer surfactant is varied with an ionic surfactant. A nearly-monochiral fraction of SWCNTs can then be harvested from the top phase. We also found, via high-throughput, near-infrared excitation-emission photoluminescence spectroscopy, that the parameter space of ATPE can be mapped to probe the mechanics of the separation process. Finally, we found that optimized conditions can be used for sorting of SWCNTs wrapped with ssDNA as well. Elimination of the need for surfactant exchange and simplicity of the separation process make the approach promising for high-yield generation of purified single-chirality SWCNT preparations.

Recovery of chlorogenic acid from haskap leaves (Lonicera caerulea) using aqueous two-phase extraction

The concept of a biorefinery can be applied to the haskap plant (Lonicera caerulea), where the berries are harvested but the leaves are an under-utilized resource. In this study, aqueous two-phase extraction (ATPE) was used to extract chlorogenic acid (CGA) from haskap leaves. The consumption of CGA is associated with health benefits and it is sold as a natural health supplement. Two different aqueous two-phase systems (ATPSs), consisting of ethanol/sodium dihydrogen phosphate (NaH2PO4) and ethanol/potassium phosphate dibasic (K2HPO4), were compared. The factors investigated were salt/ethanol composition and extraction time. The ethanol/NaH2PO4 system had higher yield of 85.03 mg CGA/g leaves, partition coefficient of 40.41, and 92.97% extraction efficiency. The yield from conventional Soxhlet extraction was 109.74 mg CGA/g leaves; however, this required a much longer extraction time of 24 h. LC-HRMS also showed the presence of CGA, and possibly lonicerin, rutin, and diosmin isomers in the leaf extracts.

A Doubly Green Separation Process: Merging Aqueous Two-Phase Extraction and Supercritical Fluid Extraction

Aqueous two-phase extraction (ATPE) is a green separation technique which uses mixtures of water and environmentally benign polymers such as polyethylene glycol (PEG) as solvents. One of the challenges in implementing this extraction on an industrial scale is finding a suitable method for the isolation of target compounds from water-polymer solutions after the extraction, without diminishing ecological benefits of the method. In this paper, we propose using another green separation technique, supercritical fluid extraction (SFE), for the back-extraction of low molecular weight medium polarity compounds from ATPE solutions. Experiments with two model compounds, caffeine and benzoic acid, showed principal applicability of SFE for this task. Pressure (100–300 bar) and temperature (35–75 °C) of supercritical carbon dioxide play a major role in defining extraction capability. Extraction ratios of 35% for caffeine and 42% for benzoic acid were obtained at high fluid pressure and moderate temperature at 1:6 volume phase ratio. That gives an estimation of 10–20 theoretical steps required for complete exhaustive extraction from the ATPE solution, which is readily achievable in standard counter-current column SFE. Combining these two green methods together not only serves as an environmentally friendly method for the isolation of valuable low molecular weight compounds from diluted water solutions, but also allows for simple, energy effective recuperation of ATPE solvents.

Vapor-phase epitaxial re-growth of large diameter single-walled carbon nanotubes

Long single-wall carbon nanotubes (SWCNTs) with a controlled conductivity type or chirality are interesting for fundamental study and are promising in many different technological applications, such as nanoelectronics, optoelectronics, and also upon utilizing them as nanoscale reactors to produce nanomaterials. In this study, the long aligned large diameter SWCNTs and the large diameter nanotube dense networks were synthesized via a vapor-phase epitaxial re-growth method. The nanotubes were re-grown on ST (stable temperature)-cut quartz substrates from short SWCNT seeds using the mixture of ethanol and acetylene as a precursor. The efficient nanotube re-growth was achieved using unsorted SWCNTs with diameters of 1.2–2.0 nm and semiconducting SWCNTs, sorted by an aqueous two-phase extraction method, as seeds. According to our study, the re-grown nanotubes in an array have an average length of 5.5 μm, while the individual re-grown nanotubes can reach up to 20–30 μm. The extensive optical study confirms the preservation of SWCNTs diameter during the re-growth and signifies the high quality of produced nanotubes. We demonstrate the SWCNT chirality selective efficiency of the re-growth, which leads to predominance of the metallic nanotubes.

Sensing with Chirality-Pure Near-Infrared Fluorescent Carbon Nanotubes.

Semiconducting single-wall carbon nanotubes (SWCNTs) fluoresce in the near-infrared (NIR) region, and the emission wavelength depends on their chirality (n,m). Interactions with the environment affect the fluorescence and can be tailored by functionalizing SWCNTs with biopolymers such as DNA, which is the basis for fluorescent biosensors. So far, such biosensors have been mainly assembled from mixtures of SWCNT chiralities with large spectral overlap, which affects sensitivity as well as selectivity and prevents multiplexed sensing. The main challenge to gain chirality-pure sensors has been to combine approaches to isolate specific SWCNTs and generic (bio)functionalization approaches. Here, we created chirality-pure SWCNT-based NIR biosensors for important analytes such as neurotransmitters and investigated the effect of SWCNT chirality/handedness as well as long-term stability and sensitivity. For this purpose, we used aqueous two-phase extraction (ATPE) to gain chirality-pure (6,5)-, (7,5)-, (9,4)-, and (7,6)-SWCNTs (emission at ∼990, 1040, 1115, and 1130 nm, respectively). An exchange of the surfactant sodium deoxycholate (DOC) to specific single-stranded (ss)DNA sequences yielded monochiral sensors for small analytes (dopamine, riboflavin, ascorbic acid, pH). DOC residues impaired sensitivity, and therefore substantial removal was necessary. The assembled monochiral (6,5)-SWCNTs were up to 10 times brighter than their nonpurified counterparts, and the ssDNA sequence determined the absolute fluorescence intensity as well as colloidal (long-term) stability and selectivity for the analytes. (GT)40-(6,5)-SWCNTs displayed the maximum fluorescence response to the neurotransmitter dopamine (+140%, Kd = 1.9 × 10-7 M) and a long-term stability of >14 days. The specific ssDNA sequences imparted selectivity to the analytes mostly independent of SWCNT chirality and handedness of (±) (6,5)-SWCNTs, which allowed a predictable design. Finally, multiple monochiral/single-color SWCNTs were combined to achieve ratiometric/multiplexed sensing of the important analytes dopamine, riboflavin, H2O2, and pH. In summary, we demonstrated the assembly, characteristics, and potential of monochiral (single-color) SWCNTs for NIR fluorescence sensing applications.

Study on Extraction of Procyanidins from Grape Seeds by Aqueous Two-Phase System

Discussed with the method of the aqueous two-phase extraction of grape seed procyanidins optimum extraction conditions and extraction efficiency. Methods: Procyanidins extraction experimental conditions were optimized with the single, calculate the content and extraction rate of procyanidins from grape seeds with catechin standard curve comparison method. Results: Experiments show that: aqueous two-phase systems are made up of water 3.0 mL, ethanol 3.5 mL, ammonium sulfate 2.5 g, grape seed 0.5 g, centrifuged 10 min at 3600 r/min speed, placed 120 minutes, procyanidins at a rate of 0.23%, extraction rate reached 97.86%. Conclusions: This aqueous two-phase systems extraction rate, simple operation, environmental conservation, conditions required in practical applications easily met.

Deciphering the effects of kosmotrope and chaotrope salts during aqueous two phase extraction (ATPE) of polyphenolic compounds and glycoalkaloids from the leaves of a nutraceutical plant, Solanum retroflexum, with the aid of UPLC-QTOF-MS

Solanum plants (Solanaceae) are renowned source of nutraceuticals and have widely been explored for their phytochemical constituents. This work investigated the effects of kosmotropic and chaotropic salts on the number of phytochemicals extracted from the leaves of a nutraceutical plant, Solanum retroflexum, and analyzed on the ultra-performance liquid chromatography hyphenated to a quadrupole time of flight mass spectrometer (UPLC-QTOF-MS) detector. Here, a total of 20 different compounds were putatively characterized. The majority of the identified compounds were polyphenols and glycoalkaloids. Another compound, caffeoyl malate was identified for the first time in this plant. Glycoalkaloids such as solanelagnin, solamargine, solasonine, β-solanine (I) and β-solanine (II) were found to be extracted by almost all the salts used herein. Kosmotrope salts, overall, were more efficient in extracting polar compounds with 4 more polyphenolic compounds extracted compared to the chaotropes. Chaotropes were generally more selective for the extraction of less polar compounds (glycoalkaloids) with 3 more extracted than the kosmotropes. The chaotrope and the kosmotrope that extracted the most metabolites were NaCl and Na2SO4, respectively, with 12 metabolites extracted for each salt. This work demonstrated that a comprehensive metabolome of S. retroflexum, more than what was previously reported on the same plant, can be achieved by application of kosmotropes and chaotropes as extractants with the aid of the Aqueous Two Phase Extraction approach. The best-performing salts, Na2SO4 or NaCl, could potentially be applied on a commercial scale, to meet the ever-growing demand of the studied metabolites. The Aqueous Two Phase Extraction technique was found to be efficient in simultaneous extraction of multiple metabolites which can be applied in metabolomics.

Aqueous two-phase extraction of lead from contaminated aqueous solutions

Aqueous two-phase extraction is an ecologically safe method for heavy metal recovery and regeneration from contaminated aqueous solutions. Investigation has been carried out experimentally for the extraction of lead from aqueous solutions using Polyethylene Glycol 1500-Ammonium sulfate two-phase system. The extracting agent used is sodium chloride. Aqueous two-phase extraction experiments for Pb(II) have been performed in batch mode according to the central composite design under Response Surface Methodology. The effect of independent parameters as to the extractant concentration, initial salt solution pH, and initial salt phase metal concentration on the extraction efficiency has been studied experimentally. The results indicate that all the three independent parameters studied have significant effect on extraction efficiency. A second-order polynomial model has been used to fit the dependent variable in the experimental range of parameters. The maximum extraction efficiency obtained from the model is about 51% atinitial salt solution pH of 2.8, NaCl concentration of 49.9mmol/Land initial salt phaselead concentration of 9.8 mg/L. This is in agreement with the results obtained from the experiment conducted at optimum conditions.

Optimization studies on aqueous two-phase extraction of hexavalent chromium from contaminated aqueous solutions

Chromium has wide applications in industry and its resources are limited. The total chromium discharge to the environment needs to be monitoredand regulated. The extraction of chromium to PEG offers its reuse in pure form. The present study focuses on the optimization of hexavalent chromium extraction to PEG 1500 using ammonium salt. The effect of independent parameters as to the initial salt solution pH and initial salt phase metal concentration on the extraction efficiency has been studied experimentally. Second-order polynomial models have been used to fit the dependent variables in the experimental range of parameters.Central composite rotatable design in Minitabstatistical software version 16is used as the optimization tool. The maximum efficiency that is achieved through the optimization studies is 29.8%at an initialsalt solution pH of 6.5 and initial metal concentration 24.4 ppm. The corresponding distribution coefficient at the optimum conditionshas been found to be 1.14. The results of the optimization studies have been validated by conducting experiments at the optimum conditions.

Towards Autonomous Operation by Advanced Process Control—Process Analytical Technology for Continuous Biologics Antibody Manufacturing

Continuous manufacturing opens up new operation windows with improved product quality in contrast to documented lot deviations in batch or fed-batch operations. A more sophisticated process control strategy is needed to adjust operation parameters and keep product quality constant during long-term operations. In the present study, the applicability of a combination of spectroscopic methods was evaluated to enable Advanced Process Control (APC) in continuous manufacturing by Process Analytical Technology (PAT). In upstream processing (USP) and aqueous two-phase extraction (ATPE), Raman-, Fourier-transformed infrared (FTIR), fluorescence- and ultraviolet/visible- (UV/Vis) spectroscopy have been successfully applied for titer and purity prediction. Raman spectroscopy was the most versatile and robust method in USP, ATPE, and precipitation and is therefore recommended as primary PAT. In later process stages, the combination of UV/Vis and fluorescence spectroscopy was able to overcome difficulties in titer and purity prediction induced by overlapping side component spectra. Based on the developed spectroscopic predictions, dynamic control of unit operations was demonstrated in sophisticated simulation studies. A PAT development workflow for holistic process development was proposed.

Correction: Determination of sulfonamides in blood using acetonitrile–salt aqueous two-phase extraction coupled with high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry

Correction for ‘Determination of sulfonamides in blood using acetonitrile-salt aqueous two-phase extraction coupled with high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry’ by Wei Yu et al., Anal. Methods, 2013, 5, 5983–5989, DOI: 10.1039/C3AY40902C.

Validation of aqueous two-phase extraction method

Nowadays, consumer interest in food with natural ingredients has increased. This need has led to the research of new sources and green extraction methods. Betalains are compounds responsible for giving color to cacti fruits. The aim is to obtain low-sugar betacyanins extracts from jiotilla Escontria chiotilla using aqueous two-phase systems (ATPS) to color food with the extract. The effect of principal parameters of ATPS (Ethyl alcohol- KH2PO4/K2HPO4) as tie-line length (TL;40,50 and 70), phase volume ratios (Vr; 1 and 3) on the partitioning of betacyanins, betaxanthins, total sugars, reducing sugars, and antioxidant activity in the extract was evaluated. The yields were determined from the top and bottom phases of the aforementioned parameters. Multivariate analysis of variance (MANOVA, α = 0.05) showed that TLL and Vr were statistically significant (P < 0.05). The lowest bottom sugar yield (25.78 ± 3.14%) corresponds to TLL = 40, Vr = 3. Under these conditions, the corresponding value for betacyanins yield is 62.98±4.52%. For the first time, the ATPS was used to extract betacyanins from cactus fruit.•Escontria chiotilla, as a biological source, contained a high percent of betalains•Aqueous two-phase systems (ATPS) was statistically optimized•The developed method enriches the valorization of environmentally related plants waste materials

Separation and purification of horseradish peroxidase from horseradish roots using a novel integrated method

The aim of the present work was to develop a novel method integrating two-step aqueous two-phase extraction and temperature-controlled affinity precipitation for the separation and purification of horseradish peroxidase (HRP) from horseradish roots.