Molecular configuration of gelatin–water suspensions at low concentration

Highlights

• Two structural configurations of gelatin suspensions were characterized at molecular level.

• Molecular configurations were generated at different temperatures and annealing times.

• At high temperature AFM showed thick structures by agglomeration single strands.

• At low temperature AFM showed network's knots with a coordination number equal 3.

• Distribution of segment size indicating triple helix's knots would be also broad in size.

Abstract

Direct assessment of gelatin molecular configuration has been difficult due the complexity of the molecule and limitations of analytical techniques. The objective of this work was the molecular characterization of bovine gelatin as a function of temperature, concentration and time. Diluted suspensions were prepared at different concentrations (7.5 × 10⁻⁵ to 1.5 g/l) and kept at temperatures above (S1) (40 °C) and below (S2) (5 °C) the gelling point as determined by Differential Scanning Calorimetry. Circular dichroism measurements showed the secondary structure of a polyproline II like spectra similar to native collagen at S1 and a denaturated configuration at S2 conditioning. Atomic Force Microscopy (AFM) at S1, using a HOPG substrate, showed single strands segments with two marked distributions in heights, ∼0.6 nm and ∼1.6 nm, indicating possible helical configurations even at high temperatures. At S2, AFM showed only one height distribution in the range of ∼0.9 nm but wider (0.3–1.6 nm range). At increasing gelatin concentration (12 g/l) and annealing time (48 h), a well-defined network was detected with narrow height distribution (∼1.0 nm) featuring aggregates and highly ordered structure zones. An analysis of gelatin strand interactions, showed a network linked by knots with a coordination number Z = 3 (strands) with a bond length of ∼50 nm. The gel network formation and aggregation was consistent with molecular size increase observed by Dynamic Light Scattering, showing variations in hydrodynamic dimensions from ∼10 nm (S1) to ∼100 nm (S2). This experimental approach has allowed to pinpoint differences in molecular configuration of gelatin, which may be applied in the study the structuring pathway of other biopolymers and the association kinetics during storage for a wide range of temperatures.

Introduction

Gelatin is one of the most used hydrocolloids in food and pharmaceutical industries due to its wide range of applications related to its well-known gelling properties. This feature has been used to improve emulsion and foam stability or to provide define mouthfeel and texture in a series of food products (Karim & Bhat, 2009). In pharmaceutical industry the use of gelatin has been related to the preparation of hard and soft capsules and in development of scaffolds for three-dimensional tissue regeneration (Acevedo et al., 2012, Karim and Bhat, 2008). More recent uses of gelatin are related to its film forming properties to extend shelf life of minimally processed foods (Antoniewski and Barringer, 2010, Dangaran et al., 2009). Due to the ability of gelatin as forming material of low moisture matrices, it has been used in the encapsulation of valuable labile bioactive compounds (eg. vitamins, probiotics, antioxidants, etc.) (Gómez-Guillén et al., 2011, Karim and Bhat, 2009, Park et al., 2007, Soper, 1999).

Gelatin is obtained from hydrolysis of collagen under controlled conditions of temperature and pH (Badii & Howell, 2006). It features a complex molecular organization based on its special aminoacid profile. The gelatin chains consists of repetitive tripeptide Glycine-X-Y sequence with iminoacids proline (Pro) and hydroxyproline (Hyp) most frequently located in the X and Y position, respectively (Gornall & Terentjev, 2007). This particular profile plays an important role in the stability of the well-known molecular configuration of random coil and helical structure (renaturation) (Bella, Brodsky, & Berman, 1995). These configurations are temperature dependent and thermo-reversible which involves a reordering of the gelatin strands in an attempt to form the native collagen structure (Mackie, Gunning, Ridout, & Morris, 1998). The highly extended nature of the triple helix, where every X and Y residue is substantially exposed to the solvent, appears to make the triple-helix domain important for self-association and for the binding of other molecules (Bella et al., 1995, Kramer et al., 1999, Persikov et al., 2000).

The high amount of Pro determines triple helix arrangement as left-handed polyproline-II-helical (PPII) conformation (Bella et al., 1995, Gornall and Terentjev, 2008a, Gornall and Terentjev, 2008b, Persikov et al., 2000). In this conformation the high content of sterically restricted iminoacids stabilizes the extended nature of the individual chains (Bella et al., 1995). Also the stereochemical restrictions of the iminoacids rings, in particular Hyp has shown to confer a greater stability than Pro in the Y position (Brodsky & Ramshaw, 1997). In the case of triple helix, it is also stabilized in part by hydrogen bonding that occurs every third residue, mainly between the backbone NH of Glycine (Gly) and the backbone CO of the residue in the X position of the adjacent chain (Gly)NH····CO(X) (Brodsky & Ramshaw, 1997). The hydroxyl groups of the Hyp residues point outward from the triple helix and therefore cannot directly hydrogen bond to any other groups within the molecule, led to the proposal that its effect is mediated through bridging water molecules (Brodsky & Ramshaw, 1997). Water molecules bridge hydrogen bonds between the hydroxyl groups of Hyp and the peptide backbone CO and NH groups both within each chain and between different chains (Bella et al., 1995) or bridge backbone carbonyl group of adjacent Pro residues (Counterman & Clemmer, 2004). The term water bridge refers to those associations of hydrogen bonded water molecules that link two different groups capable of hydrogen bonding in the triple helix (Bella et al., 1995). The number of water molecules involved in bridging two groups appears to vary along the molecule, such that two, three, four or even five water molecules may form a chain linking the two groups (Bella et al., 1995). Hence, the hydration of the chain backbone seems crucial to maintain the PPII conformation (Carvajal & Lanier, 2006).

The particular amino acid profile of gelatin also determines the map of charge distribution that helps to understand the behavior of the macromolecule in aqueous media. Gelatin is an amphoteric polyelectrolyte (also known as polyampholyte) which contains both positively and negatively charged monomers interspersed within the same linear chain (Lin et al., 2002). Hence depending of the pH of the medium, gelatin can show properties of polyacids or polybasics, the net charge of the molecule is negative or positive, respectively. At the isoelectric point (IP) the molecule maximize shelf interaction, so many polyampholytes are insoluble at IP and precipitate at this pH value. However gelatin's structure at its IP is stabilized by ionic contacts between oppositely charged units, hydrogen bonds and hydrophobic interactions, and is prevented from precipitation by hydrophilic groups, such as –COOH and –OH, on the surface of the polymer particles (Lin et al., 2002).

Literature has also suggested that gelatin chains in random coil configuration may show a significant number of β-turn structures (Guo, Colby, Lusignan, & Whitesides, 2003). This turn occurs at “hairpin” corners, where the peptide chain changes direction abruptly at which four amino acid residues often involved including Pro and Gly (Belitz, Grosch, & Shieberle, 2009, chap. 1). Moreover the persistence length of random coil gelatin has been established in ∼20 ± 3 Å (Guo et al., 2003, Joly-Duhamel et al., 2002a), therefore implying that gelatin single strands have an important degree of flexibility.

Despite the availability of chemical and configurational information of individual gelatin molecules, a direct assessment of the physical structure of the polymer chain and its network formation still a major scientific challenge. This is particular important since specific features of biomaterials at the nanoscale could be correlated to properties at the macroscale such as permeability, diffusivity, structural stability), as recently suggested for amorphous carbohydrate based systems (Roussenova et al., 2010, Townrow et al., 2010).

Thus, the nanostructural characterization of biopolymer's molecular complex organization is currently a matter of intensive research. Indeed a number of sophisticated analytical techniques have been used for this purpose, such as optical rotation (Elharfaoui et al., 2007, Haug et al., 2004, Joly-Duhamel et al., 2002b), X-ray diffraction (Bigi et al., 2004, Pinhas et al., 1996), differential scanning calorimetry (Bigi et al., 2004, D'Cruz and Bell, 2005), nuclear magnetic resonance (Pinhas et al., 1996, Traoré et al., 2000) and circular dichroism (Akbulut et al., 2008, Mohanty and Bohidar, 2005). In recent years other techniques such as asymmetrical flow field-flow fractionation coupled to multi-angle light scattering (AFIFF-MALS) (Rbii, Surel, Brambati, Buchert, & Violleau, 2011) and positron annihilation spectroscopy (Roussenova et al., 2012) has been used in gelatin–water suspension and gelatin films respectively, with the objective of describe the behavior of the system at the molecular scale. Atomic force microscopy (AFM) has also been used for biopolymer characterization. This technique has been used for the study of structural characteristics of various biopolymer-water suspensions such as gellam gum (Gunning, Kirby, Ridout, Brownsey, & Morris, 1996), γ-zein (Kogan et al., 2002) and pectin acid sugar gels (Fishman, Cooke, & Coffin, 2004). In the case of gelatin, although some studies have reported AFM images from mammalian collagen (Haugstad et al., 1993, Lin et al., 2002, Mackie et al., 1998, Mohanty and Bohidar, 2005) and most recently from gelatin obtained from marine species (Wang et al., 2008, Yang and Wang, 2009, Yang et al., 2007), in general well resolved images and studies using this technique are difficult to obtained with consequent limitations in information related to the hydrocolloid structure at the nanoscale. Moreover, a direct characterization of gelatin under the two well-known molecular configurations of the hydrocolloid (random coil and triple-helix) and its strong dependency on environmental factors temperature and concentration is still scientifically challenging.

Therefore the objective of this study was to assess the molecular structure of bovine gelatin suspended in water at different temperatures (below and above the helix to coil temperature) and concentrations by AFM, complementing this information by the well established techniques used in biopolymer characterization, differential scanning calorimetry (DSC), circular dichroism (CD) and dynamic light scattering (DLS). This work aims to provide fundamental information of this hydrocolloid that could provide new insights of the micro and macroscopic properties of the material.