Advances in Physicochemical Properties of Biopolymers (Part 1)

In this chapter, molecular weight (M), and molecular weight distribution (MWD), of polymers with emphasis on M and MWD of biopolymers, e.g., carbohydrate polymers, proteins, deoxyribonucleic acid, DNA, and ribonucleic acid, RNA, are reviewed. The M and MWD of biopolymers are compared with those of synthetic polymers. The following conclusions are drawn. (1) Unlike simple low molecular substances, most polymers do not have unique molecular weights. Practically, no polymer exists whose molecules are all strictly of the same size or have the same degree of polymerization. Thus, polymers are more or less heterogeneous with respect to their molecular weights. (2) The concept of average molecular weight is used for polymers. (3) Different numerical values for molecular weights of polymers have already been defined as average molecular weights (Mn, Mw, Mz, and Mv), depending on the methods by which they are measured. (4) The average values vary in the following order: Mn < Mv < Mw < Mz < Mz+1. The disparity between average molecular weights provides a measure of the degree of heterogeneity, i.e. dispersity, in the molecular weight distribution. (5) The constitution of a polymer as well as the MWD may be described either by a set of different average molecular weights, the ratios of two different types of average molecular weights, or by the distribution functions via graphical presentation and (6) Polysaccharides in a similar way to synthetic polymers are polydisperse polymers, whereas proteins, DNA, and RNA, are mostly monodisperse macromolecules.

Bovine serum albumin (BSA) in aqueous solution is scarcely studied, and the Mark-Houwink parameters from the intrinsic viscosity measurements have not been reported at different temperatures. This work discusses these with a simple calculation of the Mark-Houwink parameters of BSA in aqueous solution when the concentration ranged from 0.2 to 1.0% wt., and the temperature ranged from 20 to 45°C. The relationship between the concentration and intrinsic viscosity was determined according to different methods. It is well known that when the temperature increases, the intrinsic viscosity decreases. This is reflected in the stiffer chain curve with d(ln[ɳ])/d(1/T) of -398.97 for A zone from 20-30ºC (gel zone), -2759.1 for B zone from 35-40ºC (active zone) and 5604.5 for C zone from 41-45ºC (denatured protein zone), the point of intersection between the zones A and B is 34.6ºC. The linear relation between the logarithmic of viscosity and reverse temperature is ΔEavf with a value of 680 Cal/mol. Furthermore, this work proposes the determination of M-H parameters of a protein-water system and their thermodynamic implications in conformational changes.

Small-angle scattering of X-rays (SAXS) or neutrons (SANS) is a wellestablished method to study the overall structure and structural transitions of biopolymers in solution. Determination of overall structural parameters such as the radius of gyration Rg, the molecular mass Mw or the hydrated volume V is now easily computed using appropriate free Softwares. In addition, increasingly sophisticated ab initio methods were developed over the last two decades for building structural models of biopolymers from x-ray and neutron solution scattering data. Even if most of the developments were focused on protein in solution, folded or intrinsically disordered, protein complexes or protein fibrils, ab initio methods were also used to calculate three-dimensional shape models of polysaccharides or nucleic acids. The models derived from experimental scattering pattern up to a resolution of 0.5 nm are classified as low resolution models compared to atomistic resolution structures using X-ray crystallography. The efficiency of the methods is illustrated in this chapter by focusing mainly on proteins or polysaccharides with unknown crystal structures and for which the ab initio reconstruction of three-dimensional models may help to define, in combination or not with other structural techniques, such as microscopy, overall dimensions and global shape. These methods improved the resolution and reliability of models derived from scattering data substantially and has made solution scattering, in combination with recent developments using size-exclusion chromatography, robotic sample changer or microfluidics, and a useful technique in high-throughput large-scale structural characterization of biopolymers.

High-Performance Size-Exclusion Chromatography (HPSEC) is widely used for the determination of the molar mass and size distribution of biopolymers in aqueous or organic solvents. Elements of the theory of fractionation by HPSEC, column calibration and online light scattering detection are given, showing that coupling HPSEC with multi-angle laser light scattering detection makes it easier to obtain molar mass distributions since light (MALLS) scattering gives the weight-average molar mass at each elution volume of the chromatogram. Some applications of HPSEC-MALLS for the macromolecular characterization of starches, glycogens, dextrans, celluloses, hemicelluloses, β-glucans, pectins, gums, alginates, carrageenans, chitosans, lignins, proteins and peptides are also presented.

Field-Flow Fractionation techniques (FFF) are size-based separation methods first described in 1966 by Giddings. They belong to the family of liquid chromatographic techniques, but they are operated without any stationary phase.Yet, they have the unique ability to separate an extremely broad range of molecules, macromolecules and particles, and in particular very large particles, with a high resolution. FFF are versatile: by varying the experimental conditions, the range, speed, and power of the separation could be optimized. FFF techniques can succeed when Size-Exclusion Chromatography (SEC) methods fails, and they have a broad range of applications. In this chapter the theory of FFF is approached together with calibration and determination of some structural parameters such as size and molar mass, the instrumentation and detection of various classic FFF types is described and we show the added value of FFF techniques for the characterization of various biopolymers including polysaccharides, proteins and natural rubber.

In this chapter, the rheology of polysaccharides extracted from cactus and agave plants is summarized. These vegetal sources of polysaccharides (Opuntia mucilage, Opuntia pectin, and agave fructans) were selected for their functional properties (thickening, gelling, or emulsifying) and their bioactive role in the the prevention and/or treatment of disease. The source, production, extraction processes, structure, and functional properties (related to conformation) of these polysaccharides are briefly described before the rheology of these biopolymers is discussed, and recommended uses are suggested.

One of the major by-products of bioethanol production is distillers dried grains with solubles (DDGS). Maize is one of the main sources for the production of this biofuel. In this way, dietary fiber represents the principal fraction of DDGS, which could be a potential source of added-value biomolecules such as ferulated arabinoxylans (AX). In this chapter, ferulated arabinoxylans extracted from DDGS (DDGAX) were gelled and the gels were studied in terms of rheology, structural parameters and microstructural characteristics. The DDGAX formed gels at 2% (w/v) induced by laccase. The mechanical spectrum and strain sweep of DDGAX gel presented the typical behavior of a solid-like material. The gels swelling ratio (43 g water/g DDGAX) suggested the formation of a compact polymeric network which decreased the water uptake of the gel. DDGAX gels presented an average mesh size value of 96 nm. The surface of the gels was analyzed by scanning electron microscopy revealing a heterogeneous microstructure resembling an imperfect honeycomb. These results indicate that ferulated arabinoxylans from DDGS form elastic and macroporous gels, presenting a microstructure with irregular pore sizes.

The objective of this chapter is to describe the main techniques adapted to characterize ionic or neutral polysaccharides, especially when they are water soluble. The steric exclusion chromatography (SEC), rheology and NMR spectroscopy are, in our opinion, the most important techniques to study the main characteristics of polysaccharides. The same techniques and approaches may also be valid for other water soluble biopolymers such as proteins and nucleic acids. The main characteristics of polyelectrolytes are recalled and it is shown that the ionic behavior of the polymers can be used to establish the nature of the polymer conformation (coil, helix). In this case, it is necessary to combine thermodynamic characteristics with optical rotary power (or differential scanning calorimetry DSC and circular dichroism CD) and molar mass determinationss. The semi-rigid characterization of the polysaccharide chains depending on their conformation is introduced. The persistence length Lp which characterizes the local stiffness of the chain determined by steric exclusion chromatography in application of the worm-like chain model and validated by molecular modelling. Rheology of polysaccharides is of great interest due to fundamental and applied point of views (especially in food, cosmetic or biomedical applications). The solutions even at low polymer concentration are often non Newtonian due to the stiffness of the polysaccharides which also depresses the critical overlap concentration. The main relationships relating viscosity to molar mass and/or polymer concentration are given; the influence of the shear rate for measurement is pointed out; and, the flow and dynamic measurements are described for sol systems. Originality of polysaccharides is that they may associate to form 3-D network, or physical network. This gel is controlled by the thermodynamic conditions and environmental conditions (ionic concentration, nature of ions, temperature, pH… ). The main mechanisms of physical gelation are recalled and characterization of the sol-gel transition is introduced. The techniques to follow this transition as well as those for gel rheology are also introduced.

The mesquite tree (Prosopis spp.) is a native plant species in arid zones that has been documented as a potential source of biopolymers, including polysaccharides, and more recently, proteins. The native flora of the Sonoran Desert offers considerable potential for the recovery of such compounds. Mesquite trees produce a water-soluble exudate known as mesquite gum (MG). Pods contain seeds with high levels of protein, and the endosperm yields a novel gum of the galactomannan family. MG and galactomannan (GM) are not yet considered food additives in the international market due to lack of FDA approval. However, structural, physicochemical and functional studies have shown that MG and GM are potential substitutes for commercial Arabic and guar gums, respectively. In addition, it was recently documented that mesquite seeds are a rich source of protein, with considerable quantities of essential amino acids. This chapter presents information about the structural and chemical characterization and applications of the biopolymers that can be obtained from P. velutina.

There has been a long history of theoretical and experimental papers which are concerned with the development of the free volume concept to explain transport and diffusion in polymer systems. Several theoretical efforts also took place to clinch the mechanism of the glass transition on the basis of free volume concept. In spite of the strong presence of free volume concept in scientific arena, it carries ambiguity in terms of definition. Many consider free volume concept as nothing but a guide to discussion since it has no physical existence. Past papers on theory and experiments dealt with the development of free volume concept to explain transport and diffusion in polymers. Many models based on free volume concept, show a number of unsatisfactory features as they stand at present. However, a number of theoretical approaches emerged to achieve a unanimous definition of free volume concept. Since the last few decades, Positron Annihilation Lifetime Spectroscopy (PALS) has become a well-established experimental technique for gathering information on the microstructure of free volume in polymeric materials. A brief discussion is presented in this chapter on free volume models along with PALS effectiveness in investigating free volume properties.

Sulfated polysaccharides are widely distributed in nature. The aim of this chapter is to give a brief description of methods of structural characterization of sulfated galactans, fucans, mannans, and arabinans from seaweeds, and sulfated polysaccharides rich in uronic acids (glycosaminoglycans and polysaccharides from green seaweeds), among other sulfated biopolymers. These polysaccharides are heterogeneous with respect to chain length and sulfate content and must be purified to homogeneity before structural analysis is carried out. Structural analysis of sulfated polysaccharides may be carried out by chemical methods: carbohydrate and sulfate content, monosaccharide composition, methylation/ ethylation and desulfation-methylation/ethylation, Smith degradation, etc. Herein, the application of matrix-assisted laser-desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR) in some of their wide variety of experimental methods is described. 1H and 13C NMR spectroscopy, together with 2D NMR techniques, provide very important information about sequence, interresidue linkage position and substitution pattern. MALDI-MS and ESI-MS have become important tools in the last years, however, they have limitations due to the labile nature of the sulfate group. In MALDIMS, desorption difficulties with increasing molecular weight were also found. Thus, before application of mass spectrometry, depolymerization in controlled conditions is often required.

Currently, there is great interest in the study of new ecological technologies searching a harmonious lifestyle with the environment. In this sense, numerous investigations on thermoplastic materials have been conducted as a significant alternative due to their promising applications. In this regard, thermoplastics materials from different biodegradable and edible polymer sources, which can be used in food packaging, films or coatings, have been evaluated as an alternative to replace synthetic materials to contribute to environmental pollution. The aim of this chapter was to study the physicochemical, mechanical and structural properties of biopolymer films for use in the food industry. In this chapter, different components used for the production of biodegradable and edible coatings: biopolymers, plasticizers, antimicrobials and antioxidants are reviewed.

The design and engineering of innovative bionanocomposites for a wide variety of applications should be based on the understanding of the relationship between their nanoscale structure and their chemical and mechanical properties. The understanding of the chemistry and structure at the nanometric level imply an essential knowledge for future developments of novel materials based on polysaccharides and nanostructured materials toward several applications (for instance bioremediation, medical applications, biotechnology, food industry, etc.). The study of the physicochemical characteristics of a material would imply not only its basic description but could be significantly relevant in the optimization of its synthesis toward the obtaining of tailored properties or the control of the material behavior in a particular application. Herein, we present the introduction to the most used techniques in the characterization of bionanocomposites, such as electron microscopies, FTIR, Raman spectroscopy, NMR, Rheology behavior, DSC, TGA, SAXS and SANS. The basics of these techniques and key approaches in the characterization of bionanocomposites are presented for the understanding of the relevance of the characterization step in a material evaluation.

Evaluation of reliable experimental quantities characterizing the isolated strong polyelectrolyte chain in solution at low ionic strengths is a challenge for researchers working in polymer science and biophysics. A simple method for estimating the intrinsic viscosity as initial slopes of the lnηr and ηsp vs. c dependencies is discussed. Using several examples, we have demonstrated the adequacy of this estimated intrinsic viscosity. The results determined in salt free water solutions for homologous series of sodium poly(styrene-4-sulfonate) in 30-fold range of molar mass, as well as for the polyions with the persistence length of the corresponding bare chains differing 30 times are discussed. The “apparent intrinsic viscosities” of poly(styrene-4- sulfonate) samples at non-zero polymer concentration in salt-free solutions are compared with the directly measured intrinsic viscosities at different ionic strengths.

Interactions between polysaccharides and surfactants in aqueous solutions are the topic of this chapter. These components are often found in foods as well as in other systems like cosmetics, paints, detergents, textiles and many more. Therefore, investigation of the interactions between polysaccharides and surfactants has a great practical and fundamental interest. For the sake of clarity, we have divided the discussion into three sections: polysaccharides-nonionic surfactants; polysaccharidescationic surfactants and polysaccharides-anionic surfactants. The behavior of polysaccharide-surfactant systems depends on many factors, which mainly include: 1) physical properties of both components, like molecular weight, degree of branching, number, and type of charge groups, backbone rigidity, 2) polysaccharide concentrations, 3) surfactant type; ionic or nonionic, polar head, chain length, and concentration. Environmental conditions like pH, ionic strength and presence of salt can partially screen electrostatic interactions, and there are also other factors needed to be considered when working with this type of mixtures. All these factors make it difficult to predict the behavior of polysaccharide-surfactant mixtures. However, in almost all cases, addition of surfactant modifies the behavior of the polysaccharide, regardless of polysaccharide and surfactant, but the modification is particular to the chemical nature of the polysaccharide and surfactant.

Theoretical models play a very important role in experimental practice and are essential for the development of current science and technology. They have been accessible for many years, but their development in recent decades, in parallel with the improvements in computation, has allowed the emergence of an important number of methodologies to complement experimental measurements. In this chapter, we present a brief overview of these methodologies. Among them, bead modeling has put a significant step forward. For rigid molecules, programs like those contained in the HYDRO suite have become very popular tools. For flexible molecules, computer simulation of their behavior in solution, either with a detailed atomic description (molecular dynamics) or based on coarse grain models for longer time scales (Brownian dynamics), is now a usual tool with a variety of accessible programs for use by scientists. Finally, a few examples of recent applications of these methodologies for proteins and polysaccharides are included. With this, we seek to illustrate to nonspecialized readers the possibilities available today and, hopefully, to encourage them to find ways to incorporate theoretical models to complement their work.