Size-exclusion Chromatography Coupled With Multi-angle Laser Light Scattering Research Articles

Exploring the structural, functional, and biocompatibility aspects of marine bacterial extracellular polysaccharides for biopharmaceutical applications

Marine bacterial extracellular polysaccharides (EPS) represent a unique class of biopolymers with vast potential in biopharmaceutical research due to their diverse structural characteristics, functional properties, and biocompatibility. This study delves into the comprehensive analysis of the monosaccharide composition, molecular weight distribution, and conformational dynamics of marine bacterial EPS, employing advanced analytical techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy, Mass Spectrometry (MS), and Size-Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering (SEC-MALLS). Furthermore, we explore the functional applications of these EPS in biofilm inhibition, immunomodulation, and as innovative drug delivery systems, highlighting their role in enhancing antibiotic efficacy and mitigating resistance. Through quantitative analysis and mathematical modeling, including rheological studies and kinetic modeling of biodegradation, this work assesses the EPSs biocompatibility and biodegradability, essential for their safe use in biomedicine. Our findings underscore the potential of marine bacterial EPS in developing novel biopharmaceutical formulations that are not only effective but also environmentally sustainable and biocompatible, paving the way for future advancements in medical and therapeutic applications.

Structural evolution of pea-derived albumins during pH and heat treatment studied with light and X-ray scattering

Pea albumins are found in the side stream during the isolation of pea proteins. They are soluble at acidic pH and have functional properties which differ from their globulin counterparts. In this study, we have investigated the aggregation and structural changes occurring to pea albumins under different environmental conditions, using a combination of size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and small-angle X-ray scattering (SAXS). Albumins were extracted from a dry fractionated pea protein concentrate by precipitating the globulin fraction at acidic pH. The albumins were then studied at different pH (3, 4, 4.5, 7, 7.5, and 8) values. The effect of heating at 90 °C for 1, 3, and 5 min on their structural changes was investigated using SAXS. In addition, size exclusion of the albumins showed 4 distinct populations, depending on pH and heating conditions, with two large aggregates peaks (∼250 kDa): a dimer peak (∼24 kDa) containing predominantly pea albumin 2 (PA2), and a monomer peak of a molar mass of about 12 kDa (PA1). X-ray scattering intensities as a function of q were modeled as polydisperse spheres, and their aggregation was followed as a function of heating time. Albumins was most stable at pH 3, showing no aggregation during heat treatment. While albumins at pH 7.5 and 8 showed aggregation after heating, solutions at pH 4, 4.5, and 7 already contained aggregates even before heating. This work provides new knowledge on the overall structural development of albumins under different environmental conditions, improving our ability to employ these as future ingredients in foods.

The exopolysaccharides produced by Leuconostoc mesenteroides XR1 and its effect on silk drawing phenomenon of yoghurt

Yoghurt fermented by Leuconostoc mesenteroides XR1 from Kefir grains was found to produce a unique silk drawing phenomenon. This property was found to be associated with the exopolysaccharides (EPS), X-EPS, produced by strain XR1. In order to better understand the mechanism that produced this phenomenon, the X-EPS was extracted, purified and characterized. The molecular weight and monosaccharide composition were determined by size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) and ion chromatography (IC) analysis, respectively. The results showed that its molecular weight was 4.183 × 106 g/mol and its monosaccharide composition was glucose, and glucuronic acid, with the contents of 567.6148 and 0.2096 μg/mg, respectively. FT-IR and NMR analyses showed that X-EPS was an α-pyranose polysaccharide and was composed of 92.22 % α-(1 → 6) linked d-glucopyranose units and 7.77 % α-(1 → 3) branching. Furthermore, it showed a chain-like microstructure with branches in atomic force microscopy (AFM) and scanning electron microscopy (SEM) experiments. These results suggested that the unique structure of X-EPS, gave the yoghurt a strong viscosity and cohesiveness, which resulted in the silk drawing phenomenon. This work suggested that X-EPS holds the potential for food and industrial applications.

Preparation and Characterization of High-Melt-Strength Polylactide with Long-Chain Branched Structure through γ-Radiation-Induced Chemical Reactions

An easy procedure was applied to prepare high-melt-strength polylactide (PLA) that involves γ-radiation-induced free-radical reactions to introduce a long-chain branched structure onto a linear PLA precursor with addition of a trifunctional monomer, trimethylolpropane triacrylate (TMPTA). The results from size-exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) detection indicate that the resultant long-chain branched PLA (LCB PLA) samples have an increased molecular mass and an elevated branching degree with increasing amount of TMPTA incorporated during the irradiation process. Various rheological plots including viscosity, storage modulus, loss tangent, Cole–Cole plots, and weighted relaxation spectra were used to distinguish the improved melt strength for LCB PLA samples. The effect of LCB structure on elongational rheological properties was further investigated. The LCB PLA samples exhibited an enhancement of strain-hardening under elongational flow. The enhanced melt ...

Functional Characterization of SbmA, a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35)

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95 ± 0.89 μM peptide and a Vmax of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.

Stability and mechanism of whey protein soluble aggregates thermally treated with salts

The formation of whey protein aggregates, often termed soluble aggregates, with specific physicochemical properties has been shown to result in improved functionality in gels, foams, emulsions, encapsulation, films and coatings. This work evaluated the potential of whey protein soluble aggregates to improve thermal stability in the presence of salts and determine the mechanism of improved thermal stability. Solutions of whey protein isolate (WPI) or β-lactoglobulin (β-lg) (7% w/w, pH 6.8) were heated for 10 min at 90 °C to form soluble aggregates. Native proteins and soluble aggregates were diluted to 3% w/w in solutions containing 0–108 mM NaCl and thermally treated (90 °C, 5 min). Turbidity, solubility, and viscosity were evaluated, in addition to ζ-potential and S o (surface hydrophobicity). Size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) and dynamic light scattering were used to determine aggregate size and transmission electron microscopy (TEM) was used to evaluate aggregate shape. Use of soluble aggregates improved thermal stability due to their altered aggregate shape and higher charge, and resulted in final aggregates that were smaller and less dense, leading to reduced viscosity and turbidity, and increased solubility compared to native proteins. It is concluded that soluble aggregates formed under the appropriate conditions to produce the desirable physicochemical properties can be used to improve whey protein thermal stability with a possible application in beverages.

Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-I digest patterns

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M(w)) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A(2)) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M(w) of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M(w) values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M(w) results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.

Composition and Molecular Weight Distribution of Carob Germ Protein Fractions

Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC) coupled with multiangle laser light scattering (SEC-MALS), and electrophoretic analysis. Using a modified Osborne extraction procedure, carob germ flour proteins were found to contain approximately 32% albumin and globulin and approximately 68% glutelin with no prolamins detected. The albumin and globulin fraction was found to contain low amounts of disulfide-bonded polymers with relatively low M(w) ranging up to 5 x 10(6) Da. The glutelin fraction, however, was found to contain large amounts of high molecular weight disulfide-bonded polymers with M(w) up to 8 x 10(7) Da. When extracted under nonreducing conditions and divided into soluble and insoluble proteins as typically done for wheat gluten, carob germ proteins were found to be almost entirely ( approximately 95%) in the soluble fraction with only ( approximately 5%) in the insoluble fraction. As in wheat, SEC-MALS analysis showed that the insoluble proteins had a greater M(w) than the soluble proteins and ranged up to 8 x 10(7) Da. The lower M(w) distribution of the polymeric proteins of carob germ flour may account for differences in functionality between wheat and carob germ flour.

Isolation and characterization of water-soluble prebiotic compounds from Australian and New Zealand plants

The water-soluble carbohydrates (WSCs) extracted from the underground parts (rhizome) of Arthropodium cirratum (Rengarenga lily extract); third order branches of Cordyline australis (Cabbage tree extract); a seaweed, Undaria pinnatifida (Undaria extract), and exudates from Acacia pycnantha (Acacia extract) were investigated. Extracts of Rengarenga lily, Cabbage tree, Undaria, and Acacia contained 576, 250, 275 and 794 g/kg DM WSCs, respectively. Constituent sugar analysis by gas–liquid chromatography (GLC) showed that extracts of Rengarenga lily and Cabbage tree contained predominantly fructose and glucose (82–95%). The analysis also revealed that Acacia extract contained mainly galactose (78%) and arabinose (22%) while Undaria extract, contained fucose (55%) and galactose (44%). Thin-layer chromatography (TLC) showed that, on the basis of R F values, fructan composition of Rengarenga lily extract and Cabbage tree extract was different. Cabbage tree extract contained 45% (w/w) fructans while Rengarenga lily extract contained 65% (w/w) fructans. High performance size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) showed that the extracts had varying weight average molecular weight due to differences in the average chain length of the major carbohydrates. Data for the amino acid compositions differed considerably depending on the type of extract. Water-soluble carbohydrate extracts prepared from the four plant sources gave a wide range of WSC (250–794 g/kg DM) due to the different proportions of structural material in different species. It is not known how these differences will impact on animal production, if diets are supplemented with the extracts.

Preparation and Characterization of Enzyme-Modified Konjac Glucomannan/Xanthan Blend Films

Enzyme-modified konjac glucomannan (KGM) and xanthan blend films have been prepared and characterized. Enzymatic hydrolysis of purified KGM by β-mannanase yielded samples of various weight-average molecular weight (M w) that were determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) and calculated using the established Mark–Houwink–Sakurada equation [η] = 4.07 × 10−4 M w 0.733. KGM degradation products were blended with xanthan for preparation of films that were used in orthogonal-designed experiments. These films were characterized in terms of their structures, thermal stability, crystallinities and mechanical properties. The results of Fourier transform infrared spectroscopy indicated that, after blending, there was strong intermolecular interaction caused by hydrogen bonds between xanthan and KGM hydrolysates. This phenomenon was more marked at lower M w of KGM. The thermal stability of the blend films determined by differential scanning calorimetry was higher with increasing KGM molecular weight. The degrees of crystallinity characterized by X-ray diffraction (XRD) decreased with the increasing content of non-degraded KGM. Mechanical properties of the polymer blends including the tensile strength and elongation rate were also correlated to M w of KGM. The tensile strength achieved a maximum at 10.25 MPa with KGM of M w = 4.25 × 105 and the elongation rate reached a maximum value of 89.5% with KGM of M w = 8.95 × 105. These results were directly attributed to intermolecular interactions between xanthan and KGM, and this synergistic behavior led to good physicochemical and mechanical performance of the blended films.

Multilevel structural nature and interactions of bovine serum albumin during heat-induced aggregation process

To study the changes in secondary, tertiary and quaternary structures and the alteration in protein–protein interactions during the protein aggregation upon heating, bovine serum albumin (BSA) was incubated over a range of temperature, and its structure was monitored by various biophysical techniques including circular dichroism (CD), second-derivative UV absorption spectroscopy, dynamic light scattering (DLS) and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS). The experimental data demonstrated that the size of BSA was relatively stable up to 60 °C. Above 65 °C, however, BSA underwent the obvious heat-induced aggregation (a dramatic growth of hydrodynamic radius R h), accompanied with great unfolding of its secondary structure (a sigmoidal reduction of α-helix with the transition midpoint at 67.6 °C), with significant expansion of its tertiary structure (an increased solvent exposure of some aromatic residues with the transition midpoint at 65.5 °C), with distinct screening of electrostatic repulsion (a great reduction of second virial coefficient A 2 above 65 °C) and with remarkable formation of aggregates (an evident increase of molecular weight above 65 °C).

Determination of Modification Degree of Succinylated Gelatin by Size Exclusion Chromatography Coupled with Multi Angle Laser Light Scattering

The clinical effectiveness of succinylated gelatin as a plasma substitute depends on its molar mass and modification degree of its free amino group. In this study, a novel method was developed to determine the modification degree of succinylated gelatin. Succinylated gelatin was derivatized with trinitrobenzenesulfonic acid (TNBS). The molar masses of gelatin and succinylated gelatin were determined before and after derivatization with TNBS using size-exclusion chromatography coupled with multi angle laser light scattering (SEC-MALLS). The modification degree was obtained by estimating the free amino groups of succinylated gelatin before and after succinylation. Human serum albumin was used as a model protein to validate the method. The determined value was consistent with the predicted value. Actual samples were analyzed with the developed method; the mean modification degree of succinylated gelatins was 38%. This research indicates that size-exclusion chromatography multi angle laser light scattering coupled with the trinitrobenzenesulfonic acid derivatization is a feasible method for the determination of the modification degree of succinylated gelatin.

Isolation of an Exopolysaccharide-producing Bacterium,Sphingomonassp. CS101, Which Forms an Unusual Type of Sphingan

An exopolysaccharide-producing Gram negative bacterium was isolated and determined to be a Sphingomonas sp. (CS101). A sugar composition analysis of an exopolysaccharide indicated that the Sphingomonas sp. CS101 secreted an exopolysaccharide composed of glucose, mannose, fucose, and rhamnose in the ratio of 2.1:1.1:1.0:0.1, suggesting that this exoplysaccharide is an unusual type of sphingan family. The mean molecular weight of the exopolysaccharide was determined to be 4.2x10(5) Da by size exclusion chromatography coupled with multi-angle laser-light scattering (SEC/MALLS) analysis. An exopolysaccharide was produced up to 17 g/l (pH 7; 30 degrees C) with the optimal medium condition over 4 days of cultivation.

Production and characterization of exopolysaccharides from submerged culture of Phellinus linteus KCTC 6190

The optimal temperature and initial pH for both mycelial growth and exopolysaccharide (EPS) production in shake flask cultures of Phellinus linteus KCTC 6190 were found to be 30 °C and pH 4.0, respectively. Optimal medium composition was determined to be sucrose 50 g/l, corn steep powder 3 g/l, KH 2PO 4 0.68 g/l, and CaCl 2 0.55 g/l. Under optimal culture conditions, the maximum mycelial biomass and EPS achieved in a 5-l stirred-tank fermenter indicated 11 and 3.3 g/l, respectively. To the best of our knowledge, this is the highest polysaccharide yield amongst liquid cultures of P. linteus reported in the literature. The three groups of EPSs (designated as Fr-I, -II, and -III) were obtained from the culture filtrates by a gel filtration chromatography on Sepharose CL-4B and the different fractions were analyzed directly using the size exclusion chromatography coupled with multi-angle laser-light scattering (SEC/MALLS) analysis. It was revealed that all fractions of EPS were polysaccharides consisted of mainly mannose, galactose, and glucose. The molecular weights of Fr-I, -II, and -III of EPS were determined to be 433,400 (±7801), 31,470 (±283), and 12,950 (±90) g/mol, respectively. The SEC/MALLS analysis revealed that molecular dimension of the Fr-I was a spherical form, whereas the Fr-II was a random coil in an aqueous solution.

An experiment on the succinylation of wheat gluten and its consequences for pH-solubility

The clinical effectiveness of succinylated gelatin as a plasma substitute depends on its molar mass and modification degree of its free amino group. In this study, a novel method was developed to determine the modification degree of succinylated gelatin. Succinylated gelatin was derivatized with trinitrobenzenesulfonic acid (TNBS). The molar masses of gelatin and succinylated gelatin were determined before and after derivatization with TNBS using size-exclusion chromatography coupled with multi angle laser light scattering (SEC-MALLS). The modification degree was obtained by estimating the free amino groups of succinylated gelatin before and after succinylation. Human serum albumin was used as a model protein to validate the method. The determined value was consistent with the predicted value. Actual samples were analyzed with the developed method; the mean modification degree of succinylated gelatins was 38%. This research indicates that size-exclusion chromatography multi angle laser light scattering coupled with the trinitrobenzenesulfonic acid derivatization is a feasible method for the determination of the modification degree of succinylated gelatin.