Bio‐based extraction and stabilization of anthocyanins

This work reports a novel method of recovering anthocyanin compounds from highly‐pigmented grapes via a fermentation based approach. It was hypothesized that batch growth of Zymomonas mobilis on simple medium would produce both ethanol and enzymes/biomass‐acting materials, the combination of which will provide a superior extraction when compared to simple alcohol extraction. To examine this hypothesis, Z. mobilis was fermented in a batch consisting of mashed Vitis vinifera and glucose, and the recovered anthocyanin pool was compared to that recovered via extraction with ethanol. Data indicated higher amounts of anthocyanins were recovered when compared to simple solvent addition. Additionally, the percent polymeric form of the anthocyanins could be manipulated by the level of aeration maintained in the fermentation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:601–605, 2016     

Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues

The increasing use of high‐throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low‐salt CTAB (MoLSC) extraction protocol to accommodate contaminant‐rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in‐house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuitable for some applications of massively parallel sequencing. Our open‐source MoLSC protocol provides a cost‐effective, scalable, alternative DNA extraction method that can be easily optimized and adapted for sequencing applications in other contaminant‐rich samples.     

Protein extraction by Winsor‐III microemulsion systems

Proteins (bovine serum albumin (BSA), α‐chymotrypsin, cytochrome c, and lysozyme) were extracted from 0.5 to 2.0 g L^?1 aqueous solution by adding an equal volume of isooctane solution that contained a surfactant mixture (Aerosol‐OT, or AOT, and a 1,3‐dioxolane (or cyclic ketal) alkyl ethoxylate, CK‐2,13‐E_5.6), producing a three‐phase (Winsor‐III) microemulsion with a middle, bicontinuous microemulsion, phase highly concentrated in protein (5–13 g L^?1) and small in volume (12–20% of entire volume). Greater than 90% forward extraction was achieved within a few minutes. Robust W‐III microemulsion systems were formulated at 40°C, or at 25°C by including a surfactant with shorter ethoxylate length, CK‐2,13‐E₃, or 1.5% NaCl (aq). Successful forward extraction correlated with high partitioning of AOT in the middle phase (>95%). The driving force for forward extraction was mainly electrostatic attractions imposed by the anionic surfactant AOT, with the exception of BSA at high ionic strength, which interacted via hydrophobic interactions. Through use of aqueous stripping solutions of high ionic strength (5.0 wt %) and/or pH 12.0 (to negate the electrostatic attractive driving force), cytochrome c and α‐chymotrypsin were back extracted from the middle phase at >75% by mass, with the specific activity of recovered α‐chymotrypsin being >90% of its original value. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Separation of bio‐based chemicals from fermentation broths by salting‐out extraction

The possibility of creating a biorefinery using inexpensive biomass has attracted a great deal of attention, which is mainly focused on the improvement of strains and fermentation, whereas few resources have been spent on downstream processing. Bio‐based chemical downstream processing can become a bottleneck in industrial production because so many impurities are introduced into the fermentation broth. This review introduces a technique referred to as salting‐out extraction, which is based on the partition difference between chemicals in two phases consisting of salts and polymers or hydrophilic solvents, hydrophobic solvents, and amphipathic chemicals. The effects of solvents and salts on the formation of two phases were discussed, as was the use of this method to recover bio‐based chemicals. This review focused on the separation of hydrophilic chemicals (1,3‐propanediol, 2,3‐butanediol, acetoin, and lactic acid) from fermentation broths. Diols could be recovered at a high yield from fermentation broths without pretreatment especially with a hydrophilic solvent‐based system, whereas the recovery of organic acids was slightly lower. Most of the impurities (cells and proteins) were removed during the same step. Extractive fermentations were also used for polymer‐based aqueous two‐phase systems.     

Microwave‐assisted water extraction of green tea polyphenols

Introduction – Green tea, a popular drink with beneficial health properties, is a rich source of specific flavanols (polyphenols). There is a special interest in the water extraction of green tea polyphenols since the composition of the corresponding extracts is expected to reflect the one of green tea infusions consumed worldwide. Objective – To develop a microwave‐assisted water extraction (MWE) of green tea polyphenols. Methodology – MWE of green tea polyphenols has been investigated as an alternative to water extraction under conventional heating (CWE). The experimental conditions were selected after consideration of both temperature and extraction time. The efficiency and selectivity of the process were determined in terms of extraction time, total phenolic content, chemical composition (HPLC‐MS analysis) and antioxidant activity of the extracts. Results – By MWE (80°C, 30 min), the flavanol content of the extract reached 97.46 (± 0.08) mg of catechin equivalent/g of green tea extract, vs. only 83.06 (± 0.08) by CWE (80°C, 45 min). In particular, the concentration of the most bioactive flavanol EGCG was 77.14 (± 0.26) mg of catechin equivalent/g of green tea extract obtained by MWE, vs 64.18 (± 0.26) mg/g by CWE. Conclusion – MWE appears more efficient than CWE at both 80 and 100°C, particularly for the extraction of flavanols and hydroxycinnamic acids. Although MWE at 100°C typically affords higher yields in total phenols, MWE at 80°C appears more convenient for the extraction of the green tea‐specific and chemically sensitive flavanols. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioselective resolution of 4‐chloromandelic acid by liquid–liquid extraction using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid

A liquid–liquid extraction resolution of 4‐chloro‐mandelic acid (4‐ClMA) was studied by using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐Cl‐Z‐AA) as a chiral extractant. Important factors affecting the extraction efficiency were investigated, including the type of chiral extractant, pH value of aqueous phase, initial concentration of chiral extractant in organic phase, initial concentration of 4‐ClMA in aqueous phase, and resolution temperature. It was observed that the concentration of (R)‐4‐ClMA was much higher than that of (S)‐4‐ClMA in organic phase due to a higher stability of the complex formed between (R)‐4‐ClMA and 2‐Cl‐Z‐AA. A separation factor (α) of 3.05 was obtained at 0.02 mol/L 2‐Cl‐Z‐Valine dissolved in dichloromethane, pH of 2.0, concentration of 4‐ClMA of 0.11 mmol/Land T of 296.7K.     

His‐tagged protein purification by metal‐chelate affinity extraction with nickel‐chelate reverse micelles

Di(2‐ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X‐45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R₂. The Ni(II)‐chelate reverse micelles were characterized for the purification of recombinant hexahistidine‐tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R₂ was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni²⁺ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two‐stage purification by the metal‐chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Downstream antibody purification using aqueous two‐phase extraction

The extraction of antibodies using a polyethylene glycol (PEG)‐citrate aqueous two‐phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6‐fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product‐related impurities (aggregates and low molecular product fragments) from ～40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale‐up model for the use of counter‐current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Enantioselective liquid‐liquid extraction of 3‐chloro‐phenylglycine enantiomers using (S,S)‐DIOP as extractant

(S,S)‐DIOP, a common catalyst used in asymmetric reaction, was adopted as chiral extractant to separate 3‐chloro‐phenylglycine enantiomers in liquid‐liquid extraction. The factors affecting extraction efficiency were studied, including metal precursors, organic solvents, extraction temperature, chiral extractant concentration, and pH of aqueous phase. (S,S)‐DIOP‐Pd exhibited good ability to recognize 3‐chloro‐phenylglycine enantiomers, and the operational enantioselectivity (α) is 1.836. The highest performance factor (pf) was obtained under the condition of extraction temperature of 9.1°C, (S,S)‐DIOP‐Pd concentration of 1.7 mmol/L, and pH of aqueous phase of 7.0. In addition, the possible recognition mechanism of (S,S)‐DIOP‐Pd towards 3‐chloro‐phenylglycine enantiomers was discussed.     

Automated extraction of bank angles from bird‐borne video footage using open‐source software

The use of miniaturized video cameras to study the at‐sea behavior of flying seabirds has increased in recent years. These cameras allow researchers to record several behaviors that were not previously possible to observe. However, video recorders produce large amounts of data and videos can often be time‐consuming to analyze. We present a new technique using open‐source software to extract bank angles from bird‐borne video footage. Bank angle is a key facet of dynamic soaring, which allows albatrosses and petrels to efficiently search vast areas of ocean for food. Miniaturized video cameras were deployed on 28 Wandering Albatrosses (Diomedea exulans) on Marion Island (one of the two Prince Edward Islands) from 2016 to 2018. The OpenCV library for the Python programming language was used to extract the angle of the horizon relative to the bird’s body (= bank angle) from footage when the birds were flying using a series of steps focused on edge detection. The extracted angles were not significantly different from angles measured manually by three independent observers, thus being a valid method to measure bank angles. Image quality, high wind speeds, and sunlight all influenced the accuracy of angle estimates, but post‐processing eliminated most of these errors. Birds flew most often with cross‐winds (58%) and tailwinds (39%), resulting in skewed distributions of bank angles when birds turned into the wind more often. Higher wind speeds resulted in extreme bank angles (maximum observed was 94°). We present a novel method for measuring postural data from seabirds that can be used to describe the fine‐scale movements of the dynamic‐soaring cycle. Birds appeared to alter their bank angle in response to varying wind conditions to counter wind drift associated with the prevailing westerly winds in the Southern Ocean. These data, in combination with fine‐scale positional data, may lead to new insights into dynamic‐soaring flight.     

Protein recovery from enzyme‐assisted aqueous extraction of soybean

Enzyme‐assisted aqueous oil extraction from soybean is a “green” alternative to hexane extraction that must realize potential revenues from a value‐added protein co‐product. Three technologies were investigated to recover protein from the skim fraction of an aqueous extraction process. Ultrafiltration achieved overall protein yields between 60% and 64%, with solids protein content of 70%, and was effective in reducing stachyose content, with fluxes between 4 and 10 L/m² hr. Protein content was limited because of high retention of lipids and the loss of polypeptides below 13.6 kDa. Isoelectric precipitation was effective in recovering the minimally hydrolyzed proteins of skim, with a protein content of 70%, again limited by lipid content. However, protein recovery was only 30% because of the greater solubility of the hydrolyzed proteins. Recovery by the alternative of protein capture on dextran‐grafted agarose quaternary‐amine expanded bed adsorption resins decreased with decreasing polypeptide molecular weight. Proteins with molecular mass greater than 30 kDa exhibited slow adsorption rates. Expanded bed adsorption was most effective for recovery of proteins with molecular weight between 30 and 12 kDa. Overall, adsorption protein yields were between 14% and 17%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Enantioselective liquid‐liquid extraction of DL‐mandelic acid using chiral diphosphine ligands as extractants

In order to expand the application range of chiral diphosphine ligands, (S)‐BINAP, (S)‐SEGPHOS, and (S)‐MeO‐BIPHEP were employed as extractants to recognize DL‐mandelic acid. The results indicated that (S)‐SEGPHOS‐Cu exhibited considerable ability to recognize DL‐mandelic acid with operational enantioselectivity (α) of 2.677. The process of extraction of DL‐mandelic acid using (S)‐SEGPHOS‐Cu as extractant was systematically investigated. Performance factor (pf) was adopted to comprehensively evaluate the extraction. After optimization by response surface methodology (RSM), the optimal extraction condition is temperature of 5.5°C, (S)‐SEGPHOS‐Cu concentration of 3.0 mmol/L, and pH of 8.0. And the predicted and experimental maximum values of pf were 0.26374 and 0.26839, respectively.     

Land‐sharing versus land‐sparing logging: reconciling timber extraction with biodiversity conservation

Selective logging is a major driver of rainforest degradation across the tropics. Two competing logging strategies are proposed to meet timber demands with the least impact on biodiversity: land sharing, which combines timber extraction with biodiversity protection across the concession; and land sparing, in which higher intensity logging is combined with the protection of intact primary forest reserves. We evaluate these strategies by comparing the abundances and species richness of birds, dung beetles and ants in Borneo, using a protocol that allows us to control for both timber yield and net profit across strategies. Within each taxonomic group, more species had higher abundances with land‐sparing than land‐sharing logging, and this translated into significantly higher species richness within land‐sparing concessions. Our results are similar when focusing only on species found in primary forest and restricted in range to Sundaland, and they are independent of the scale of sampling. For each taxonomic group, land‐sparing logging was the most promising strategy for maximizing the biological value of logging operations.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Analysis of marker compounds with anti‐platelet aggregation effects in mailuoning injection using platelet binding assay combined with HPLC‐DAD‐ESI‐MS and solid‐phase extraction technique

Introduction – Mailuoning is prepared from a traditional formula of Chinese medicines and widely used as an antithrombotic agent. In this study, the platelet binding assay was used as a novel biospecific separation and analysis method to explore its active constituents, which could be considered as marker compounds for quality control. Objective – To establish a rapid and simple method to predict marker compounds in herbal medicine injection and evaluate the effects of those compounds. Material and methods – Platelets were used to bind and separate constituents. Binding constituents were analysed and taken as potential active compounds for further evaluation. Solid‐phase‐extraction was adopted to improve sensitivity. HPLC‐DAD and ESI‐MS were used to determine the binding constituents. Results – Five compounds were extracted through the platelet binding process and identified as neochlorogenic acid, caffeic acid, isochlorogenic acid and their isomers. Caffeic acid was selected for the flow cytometric assay to test its effect on platelets activation, which was determined by CD62P (P‐selectin) expression. The results indicated that caffeic acid could significantly inhibit platelet activation while chlorogenic acid did not. Conclusion – Caffeic acid could be considered as a marker compound of Mailuoning injection due to its anti‐platelet effect. The study also suggested that platelet binding assay combined with some preconcentration technique could be efficiently used to predict anti‐platelet compounds in complicated herbal medicines. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of microwave‐assisted extraction coupled with high‐speed counter‐current chromatography for separation and purification of Dehydrocavidine from Corydalis saxicola Bunting

Introduction – Dehydrocavidine is a major component of Corydalis saxicola Bunting with sedative, analgesic, anticonvulsive and antibacterial activities. Conventional methods have disadvantages in extracting, separating and purifying dehydrocavidine from C. saxicola. Hence, an efficient method should be established. Objective – To develop a suitable preparative method in order to isolate dehydrocavidine from a complex C. saxicola extract by preparative HSCCC. Methodology – The methanol extract of C. saxicola was prepared by optimised microwave‐assisted extraction (MAE). The analytical HSCCC was used for the exploration of suitable solvent systems and the preparative HSCCC was used for larger scale separation and purification. Dehydrocavidine was analysed by high‐performance liquid chromatography (HPLC) and further identified by ESI‐MS and 1H NMR. Results – The optimised MAE experimental conditions were as follows: extraction temperature, 60°C; ratio of liquid to solid, 20; extraction time, 15 min; and microwave power, 700 W. In less than 4 h, 42.1 mg of dehydrocavidine (98.9% purity) was obtained from 900 mg crude extract in a one‐step separation, using a two‐phase solvent system composed of chloroform–methanol–0.3 m hydrochloric acid (4 : 0.5 : 2, v/v/v). Conclusion – Microwave‐assisted extraction coupled with high‐speed counter‐current chromatography is a powerful tool for extraction, separation and purification of dehydrocavidine from C. saxicola. Copyright © 2009 John Wiley & Sons, Ltd.