Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry.

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75-85% of the soluble sugars generated are monomeric sugars, while the remaining 15-25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC-MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4-20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

Characterizing the Diffusion and Rheological Properties of Aged Asphalt Binder Rejuvenated with Bio-Oil Based on Molecular Dynamic Simulations and Laboratory Experimentations.

Soybean-derived bio-oil is one of the vegetable-based oils that is gaining the most interest for potential use in the rejuvenation of aged asphalt binders. This laboratory study was conducted to characterize and quantify the diffusion and rheological properties of bio-oil-rejuvenated aged asphalt binder (BRAA) using soybean oil. In the study, the chemical structure of the soybean oil was comparatively characterized using an element analyzer (EA), gel permeation chromatography (GPC), and a Fourier infrared (FTIR) spectrometer, respectively. Based on the chemical structure of the bio-oil, BRAA molecular models were built for computing the diffusion parameters using molecular dynamic simulations. Likewise, a dynamic shear rheometer (DSR) test device was used for measuring and quantifying the rheological properties of the aged asphalt binder rejuvenated with 0%, 1%, 2%, 3%, 4%, and 5% soybean oil, respectively. The laboratory test results indicate that bio-oil could potentially improve the diffusion coefficients and phase angle of the aged asphalt binder. Similarly, the corresponding decrease in the complex shear modulus has a positive effect on the low-temperature properties of BRAA. For a bio-oil dosage 4.0%, the diffusion coefficients of the BRAA components are 1.52 × 10-8, 1.33 × 10-8, 3.47 × 10-8, 4.82 × 10-8 and 3.92 × 10-8, respectively. Similarly, the corresponding reduction in the complex shear modulus from 1.27 × 107 Pa to 4.0 × 105 Pa suggests an improvement in the low-temperature properties of BRAA. Overall, the study contributes to the literature on the potential use of soybean-derived bio-oil as a rejuvenator of aged asphalt binders.

Structural characterization and osteogenic activity in vitro of novel polysaccharides from the rhizome of Polygonatum sibiricum.

Polygonatum sibiricum Red., a kind of edible and medicinal plant, has a long history of use in traditional Chinese medicine and as a nutritional food additive. Water-soluble crude polysaccharide (PS50) was obtained from P. sibiricum rhizome. After deproteinization and dialysis, PS50 was isolated and purified via DEAE-52 cellulose and Sephadex G-75 gel filtration chromatography to obtain two novel polysaccharides (PSP50-2-1 and PSP50-2-2). Chemical analysis and nuclear magnetic resonance (NMR) studies indicated that PSP50-2-1 was composed of ß-d-Glcp-(1→, →2)-ß-d-Galp-(1→, →2,6)-ß-d-Galp-(1→, ß-d-Fruf-(2→, and PSP50-2-2 was composed of ß-d-Glcp-(1→, ß-d-Galp-(1→, →2)-ß-d-Galp-(1→, →6)-ß-d-Galp-(1→, →2,6)-ß-d-Galp-(1→, ß-d-Fruf-(2→. The morphological analysis suggested that PSP50-2-1 and PSP50-2-2 both had a rough surface with cracks. Pharmacological studies showed that PSP50-2-2 at concentrations of 2.59 and 5.19 µM significantly promoted the differentiation and mineralization of MC3T3-E1 cells in vitro, which indicated that PSP50-2-2 had a function of osteogenic activity. Above all, this study provides evidence that PSP50-2-2 may be used as an anti-osteoporotic agent, with applications in health-care and pharmaceutical industries.

Isolation of sesquiterpenoids from Matricaria chamomilla by means of solvent assisted flavor evaporation and centrifugal partition chromatography.

The (semi)volatile fraction of Matricaria chamomilla L., an annual herbal plant from the family of Asteraceae, contains high quantities of sesquiterpenes and sesquiterpenoids. A method was developed to achieve isolation and separation of these compounds, using a combination of solvent assisted flavor evaporation (SAFE) and solid support-free liquid-liquid chromatography. The biphasic liquid solvent system n-heptane/ethyl acetate/methanol/water, 5/2/5/2 v/v/v/v (Arizona S) was elaborated as a suitable solvent system for the simultaneous separation of the target compounds. The lab-scale liquid-liquid chromatography separation performed in a countercurrent chromatography (CCC) column was successfully transferred to a semi-preparative centrifugal partition chromatography (CPC) column, which enabled the isolation of artemisia ketone, artemisia alcohol, α-bisabolone oxide A, and (E)-en-yn-dicycloether. α-Bisabolol oxide A and (Z)-en-yn-dicycloether co-eluted, but were successfully separated by subsequent size-exclusion chromatography (SEC). Similarly, spathulenol and α-bisabolol oxide B were obtained as a mixture, and were separated by means of column chromatography using silica gel as stationary phase. The isolated compounds were characterized by means of nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS).

Sephadex® LH-20, Isolation, and Purification of Flavonoids from Plant Species: A Comprehensive Review.

Flavonoids are considered one of the most diverse phenolic compounds possessing several valuable health benefits. The present study aimed at gathering all correlated reports, in which Sephadex® LH-20 (SLH) has been utilized as the final step to isolate or purify of flavonoid derivatives among all plant families. Overall, 189 flavonoids have been documented, while the majority were identified from the Asteraceae, Moraceae, and Poaceae families. Application of SLH has led to isolate 79 flavonols, 63 flavones, and 18 flavanones. Homoisoflavanoids, and proanthocyanidins have only been isolated from the Asparagaceae and Lauraceae families, respectively, while the Asteraceae was the richest in flavones possessing 22 derivatives. Six flavones, four flavonols, three homoisoflavonoids, one flavanone, a flavanol, and an isoflavanol have been isolated as the new secondary metabolites. This technique has been able to isolate quercetin from 19 plant species, along with its 31 derivatives. Pure methanol and in combination with water, chloroform, and dichloromethane have generally been used as eluents. This comprehensive review provides significant information regarding to remarkably use of SLH in isolation and purification of flavonoids from all the plant families; thus, it might be considered an appreciable guideline for further phytochemical investigation of these compounds.

Combining online size exclusion chromatography and electrospray ionization mass spectrometry to characterize plant polysaccharides.

Characterizing polysaccharides with large molecular weights and isomeric heterogeneity with mass spectrometry (MS) is generally difficult. In this work, we demonstrate how coupling size exclusion chromatography (SEC) and high-resolution MS with source-induced dissociation (SID) can be used for the separation and direct structural evaluation of intact polysaccharides. The analytical method was successfully developed using dextran standards up to 3755 kDa. This method was used to separate naturally occurring plant polysaccharides based on size, after which numerous polysaccharide fragments were identified from the resulting MS spectra. The results provided strong evidence for structural diversity, complexity, and heterogeneity among polysaccharides. MS showed superior sensitivity and reliability for the polysaccharides in eluted fractions when compared to a refractive index detector. Putative compositions for the fragments were proposed based on exact mass values. The work demonstrated that SEC-SID-MS is a feasible alternative for obtaining valuable structural information from the analysis of intact polysaccharides.

Effects of extraction methods on immunology activity and chemical profiles of Lycium barbarum polysaccharides.

It has been proven that polysaccharides have bioactivities and are beneficial to cure many diseases. Lycium barbarum fruit is widely used as a functional food all over the world, which main active component is L. barbarum polysaccharides (LBPs). In this study, classical hot water extraction (HWE), microwave assisted extraction (MAE), ultrasonic assisted extraction (UAE) and pressurized liquid extraction (PLE) were used to extracted LBP. The chemical properties of LBPs were evaluated in terms of total polysaccharide contents, uronic acid contents and protein contents. High performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detector was applied to measure the characters such as molecular weight, radius of gyration and polydispersity index. Then the immunomodulatory activity of LBPs was evaluated through RAW 264.7 cells. The results showed that HWE was the best method to get the highest total sugar and acidic polysaccharides, MAE was preferable to extract polysaccharide-protein complex, but PLE, UAE and HWE could get better immunomodulatory activity polysaccharides than MAE. Besides, the peak 3 in chromatogram of MAE extracted LBPs was obviously higher than those of LBPs produced by other 3 extraction methods, which suggested that peak 1 and peak 2 might be biologically active polysaccharides fractions in LBPs. Therefore, effect of different extraction methods on structure and composition of LBPs attributed to their variance of immunological activities.

Qualitative and quantitative characterization of carbohydrate profiles in three different parts of Poria cocos.

Poria cocos (Schw.) Wolf has been widely used in traditional Chinese medicine (TCM) for centuries. Its three medicinal parts are Poria Cutis, the epidermis or fulingpi in Chinese; White Poria, the middle part or baifuling; and Poria cum Radix Pini, the sclerotium with some part of host pine root or fushen. The hostwood in fushen is the inner part, known as fushenmu. The epidermis, middle part and middle-plus-inner part have different clinical applications, but the differences in their chemistry have not been well determined. Previous studies only concentrated on the differences in secondary metabolites in different parts of P. cocos; however, in this study, we focused on the carbohydrates, another major type of bioactive chemicals in P. cocos, which is also different from most of the other TCM researches. The carbohydrates (polysaccharides, oligosaccharides and monosaccharides) in three parts (epidermis, middle and inner part) of P. cocos were qualitatively and quantitatively characterized by high performance gel permeation chromatography coupled with charged aerosol detector (HPGPC-CAD) and ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). The obtained data were further processed by principal component analysis (PCA) and supervised orthogonal partial least squared discriminant analysis (OPLS-DA). The results showed that the epidermis contained more polysaccharides with larger molecular weight and higher amount of glucose residue than that of the middle and inner parts, indicating the epidermis as the key site of accumulation of P. cocos polysaccharides. When compared with the epidermis and inner part, the middle part contained the highest glucose molar ratio greater than 92 % in the three types of carbohydrates, whereas the inner part possessed the greatest molar ratio of mannose, xylose, arabinose, rhamnose, glucuronic acid, and galacturonic acid in all kinds of carbohydrates. Furthermore, PCA and OPLS-DA clearly demonstrated that arabinose, glucose, galacturonic acid, and ribose played key roles in the clusters between the epidermis, middle and inner parts. The observed differences in the chemical components in the three parts could provide some explanation for the discriminative clinical applications of Poria Cutis, White Poria, and Poria cum Radix Pini. These findings also provided a chemical basis for quality assessment of P. cocos.

Characterization and in vitro evaluation of chitosan/konjac glucomannan bilayer film as a wound dressing.

A novel bilayer film of chitosan and konjac glucomannan were prepared by the two-step casting technique. Blend films were also prepared to investigate the interactions between the two polymers in the interfacial region of the bilayer structure. Scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction analysis showed that, unlike in the blends, the physicochemical properties of each biopolymer were preserved in the bilayer film. Differential scanning calorimetry and thermogravimetric analysis also indicated a good thermostability and miscibility for both polymers, probably due to strong hydrogen bonds between their polymer chains. Biological, mechanical and water vapor transmission tests showed a high biocompatibility, low cytotoxicity, and suitable mechanical and barrier properties of the bilayer films for wound dressing applications.

A new SE-HPLC method for simultaneous quantification of proteins and main phenolic compounds from sunflower meal aqueous extracts.

The aim of this research was to develop a method for simultaneous quantification of proteins and main polyphenolic compounds extracted from oleaginous meal by aqueous media. Size exclusion chromatography with a Biosep column (exclusion range from 1 to 300 kDa) and acetonitrile/water/formic acid (10:89.9:0.1 v/v) eluent at 0.6 mL min-1 yielded the most efficient separation of sunflower proteins and chlorogenic acid monoisomers (3-caffeoylquinic acid, 5-caffeoylquinic acid, and 4-caffeoylquinic acid). After a study of the stability of the extract components, the incorporation of a stabilization buffer (0.5 mol L-1 tris(hydroxymethyl)aminomethane-hydrochloric acid/1.0 mol L-1 sodium chloride at pH 7) was proposed to avoid polyphenol-protein interactions and/or isomeric transformation. The use of 214 nm as the wavelength for protein quantification was also included to minimize the effect of interference from polyphenol-protein interactions on the quantification. Under the used experimental conditions, the protein and chlorogenic acid monoisomer signals remained stable during 300 min at 20 °C (95-125% of the starting value). The developed method was validated and parameters such as specificity, sensitivity, precision, and accuracy were determined. The results from size exclusion chromatography correlated well with the results of protein determination by the reference Kjeldahl method. The proposed method was successfully applied for rapeseed extract analysis making simultaneous quantification of proteins and major rapeseed polyphenols (sinapine and sinapic acid) possible. Graphical abstract.

Speciation of zinc in fish feed by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry - using fractional factorial design for method optimisation and mild extraction conditions.

Zinc (Zn) is an element essential to all living organisms and it has an important role as a cofactor of several enzymes. In fish, Zn deficiency has been associated with impaired growth, cataracts, skeletal abnormalities and reduced activity of various Zn metalloenzymes. Fish meal and fish oil traditionally used in salmon feed preparation are being replaced by plant-based ingredients. Zinc additives are supplemented to salmon feed to ensure adequate Zn levels, promoting good health and welfare in Atlantic salmon (Salmo salar). The main objective of the present study was to evaluate Zn species found in an Atlantic salmon feed. This work describes a Zn extraction method that was optimized using a fractional factorial design (FFD), whereby the effect of six factors could be studied by performing only eight experiments. The effects of the type of extraction solution and its molar concentration, pH, presence of sodium dodecyl sulphate, temperature and extraction time on Zn extraction were investigated. Mild extraction conditions were chosen in order to keep the Zn species intact. Total Zn (soluble fractions and non-soluble fractions) was determined by inductively coupled plasma mass spectrometry (ICP-MS). The highest Zn recovery was obtained using 100 mM Tris-HCl, pH 8.5 at a temperature of 4 °C for 24 h where the total Zn in soluble fraction and non-soluble fraction was 9.9 ±â€¯0.2% and 98 ±â€¯6%, respectively. Zinc speciation analysis (on the soluble fractions) was further conducted by size exclusion inductively coupled plasma mass spectroscopy (SEC-ICP-MS). The SEC-ICP-MS method provided qualitative and semi-quantitative information regarding Zn species present in the soluble fractions of the feed. Four Zn-containing peaks were found, each with different molecular weights: Peak 1 (high molecular weight - ≥600 kDa), peak 2 and peak 3 (medium molecular weight - 32 to 17 kDa) were the least abundant (1-6%), while peak 4 (low molecular weight - 17 to 1.36 kDa) was the most abundant (84-95%).

Simultaneous Preparation of Salidroside and p-Tyrosol from Rhodiola crenulata by DIAION HP-20 Macroporous Resin Chromatography Combined with Silica Gel Chromatography.

The Rhodiola species have a long history of utilization in traditional medicine and have been considered as a source of adaptation to environmental challenges; salidroside and p-tyrosol are the major responsible compounds. Here we propose a novel UPLC-guided two-step method consisting of a DIAION HP-20 adsorption and silica gel column chromatographies, which can simultaneously prepare high purities of salidroside and p-tyrosol with noticeable yields from the rhizome of Rhodiola crenulata. Results demonstrated that DIAION HP-20 could successfully remove all impurities except crenulatin during a gradient elution with 5⁻20% ethanol, which could achieve an optimal purification of salidroside and p-tyrosol with increasing rates of 29.19% and 33.44%, respectively. Furthermore, chloroform was selected as an ideal solvent for separating p-tyrosol with salidroside, and thus crenulatin was subsequently applied in the silica gel chromatography, and the separation of salidroside with crenulatin could be achieved using silica gel chromatography with a mixture of chloroform and methanol at a volume ratio of 4:1. High purity rates of 94.17% and 97.29% and overall yields of 39.09% and 43.73% for salidroside and p-tyrosol were simultaneously achieved. Our method provides a new way to simultaneously obtain salidroside and p-tyrosol from R. Crenulata, as well as other related plant species.

Characterization of plant polysaccharides from Dendrobium officinale by multiple chromatographic and mass spectrometric techniques.

Plant polysaccharides have numerous medicinal functions. Due to the differences in their origins, regions of production, and cultivation conditions, the quality and the functions of polysaccharides can vary significantly. They are macromolecules with large molecular weight (MW) and complex structure, and pose great challenge for the analytical technology used. Taking Dendrobium officinale (DO) from various origins and locations as model samples. In this investigation, mechanochemical extraction method was used to successfully extract polysaccharides from DO using water as solvent, the process is simple, fast (40 s) and with high yield. The MWs of the intact saccharides from calibration curve and light scattering measurement were determined and compared after separation with size exclusion chromatography (SEC). The large polysaccharide was acid hydrolyzed to oligosaccharides and the products were efficiently separated and identified using liquid chromatography coupled to a high resolution tandem mass spectrometry (LC-MS2). Obvious differences were observed among LC-MS2 chromatograms of digested products, and the chemical structures for the products were proposed based on accurate mass values. More importantly, isomeric digested carbohydrate compounds were explored and characterized. All the chromatographic and mass spectrometric results in this study provided a multi-dimensional characterization, fingerprint analysis, and molecular structure level assessment of plant polysaccharides.

Pectin characterisation using size exclusion chromatography: A comparison of ELS and RI detection.

A high-performance size-exclusion chromatography (HPSEC) method coupled to Evaporative Light Scattering (ELS) and Refractive Index (RI) detectors were evaluated and compared for the molecular mass (Mw) estimation of pectin in a wide range (0.342-805 kDa). Instrumental parameters of the ELSD were optimised by Response Surface Methodology (RSM) being 73 °C the evaporator temperature and 0.9 mL/min the air flow rate. The linear range for the ELSD concentration response was wider (10-2250 mg/L) and better (R2 = 0.985) than RID (10-1500 mg/L; R2 = 0.875). The limits of detection (LOD) and quantitation (LOQ) for all pullulans hardly changed in ELSD (LOD: 1.22-1.99 mg/L; LOQ: 4.07-6.63 mg/L); however, RID showed huge variations (LOD: 0.49-10.41 mg/L; LOQ: 1.64-34.70 mg/L), which increased with the Mw. In general, responses of both detectors were similar for the Mw estimation, although pectin characterisation with HPSEC-ELSD exhibited better results in the lowest Mw compounds.

Amylase Zymography.

Amylase zymography was carried out for the detection of amylases produced by a Geobacillus stearothermophilus strain isolated from the Thermal Center "Las Trincheras" in Venezuela. Zymography is an electrophoretic technique used to study hydrolases by means of thin gels containing copolymerized-specific substrates, under nonreducing conditions. In this study, 0.1% starch was incorporated into the gel as substrate. The formation of clear zones against a dark background in the gel stained with iodine indicated the presence of amylolytic activity. The thermophilic bacteria released several extracellular amylases to a selective growth medium supplemented with 1% soluble starch at 55 °C after 40 h incubation. The amylolytic enzymes showed an optimum temperature of 60 °C and an optimum pH at 6.0. The amylases were partially purified by cold acetone precipitation followed by two chromatographic techniques. These purified amylases showed different molecular masses which were determined by sodium dodecyl sulfate gel electrophoresis and confirmed by zymography.

Purification and Biochemical and Kinetic Properties of an Endo-Polygalacturonase from the Industrial Fungus Aspergillus sojae.

An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.

Efficacy of soluble glycoprotein fraction from Allium sativum purified by size exclusion chromatography on murine Schistosomiasis mansoni.

In this work, the efficiency of crude MeOH extracts and soluble glycoprotein fraction of Allium sativum purified by size-exclusion chromatography (SEC) on parasitological, histopathological and some biochemical parameters in Schistosoma mansoni infected mice were investigated. Animals were infected by tail immersion with 100 cercariae/each mouse and divided into five groups in addition to the normal control. The results revealed a significant decrease in mean worm burden in all treated mice especially in the group treated with soluble glycoprotein fraction of A. sativum as compared to infected non-treated control with the disappearance of female worms. Administration of the studied extracts revealed remarkable amelioration in the levels of all the measured parameters in S. mansoni infected mice. In addition, treatment of mice with crude A. sativum MeOH extract and soluble glycoprotein fraction of A. sativum decreased significantly the activities of studied enzymes as compared to the infected untreated group. The highest degrees of enhancement in pathological changes was observed in the treated one with soluble glycoprotein fraction of A. sativum compared to the infected group represented by small sized, late fibro-cellular granuloma, the decrease in cellular constituents and degenerative changes in eggs. In conclusion, A. sativum treatment had effective schistosomicidal activities, through reduction of worm burden and tissue eggs, especially when it was given in purified glycoprotein fraction. Moreover, the soluble glycoprotein fraction of A. sativum largely modulates both the size and the number of granulomas.

Production of Polyhydroxyalkanoates from Sludge Palm Oil Using Pseudomonas putida S12.

Polyhydroxyalkanoates (PHAs) are biodegradable plastics produced by bacteria, but their use in diverse applications is prohibited by high production costs. To reduce these costs, the conversion by Pseudomonas strains of P HAs from crude s ludge p alm oil ( SPO) a s an inexpensive renewable raw material was tested. Pseudomonas putida S12 was found to produce the highest yield (~41%) of elastomeric medium-chain-length (MCL)-PHAs from SPO. The MCL-PHA characteristics were analyzed by gas-chromatography/mass spectrometry, gel permeation chromatography, and differential scanning calorimetry. These findings may contribute to more widespread use of PHAs by reducing PHA production costs.

Understanding the Effects of Roasting on Antioxidant Components of Coffee Brews by Coupling On-line ABTS Assay to High Performance Size Exclusion Chromatography.

INTRODUCTION: Coffee is a widely consumed beverage containing antioxidant active compounds. During roasting the phytochemical composition of the coffee bean changes dramatically and highly polymeric substances are produced. Besides chlorogenic acids that are already present in green coffee beans, melanoidins show antioxidant capacity as well. OBJECTIVE: To employ post-column derivatisation by coupling high performance size exclusion chromatography (HPSEC) to an antioxidant assay to investigate the effect of roasting on the properties of antioxidant active compounds in coffee brews. METHODOLOGY: We have investigated the antioxidant capacity of Coffea arabica (Arabica) and C. canephora (Robusta) beans that were roasted over the full spectrum of roast conditions (four roasting speeds to three roast degrees) by comparing the results from HPSEC coupled on-line to the ABTS assay with those from two batch assays, Folin Ciocalteu (FC) and oxygen radical absorbance capacity (ORAC) assay. RESULTS: The antioxidant capacity showed a general decrease towards slower and darker roasted coffee for all three assays, indicative of heat degradation of active compounds. Hence, low molecular weight (LMW) compounds such as chlorogenic acids (CGAs) decreased progressively already from relatively mild roasting conditions. In contrast, high molecular weight (HMW) compounds (e.g. melanoidins) increased from light to dark roast degrees with lowering magnitude towards slower roasting profiles. CONCLUSION: By coupling HPSEC on-line to the ABTS assay we were able to separately quantify the contribution of HMW and LMW compounds to the total antioxidant capacity, increasing our understanding of the roast process. © 2016 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd.

Purification and Characterization of a Thermostable ß-Mannanase from Bacillus subtilis BE-91: Potential Application in Inflammatory Diseases.

ß-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the ß-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a "two-step" method comprising ultrafiltration and gel chromatography. The purified ß-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5-7.0. The ß-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+ and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 µmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, ß-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the ß-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.