Elsevier

Carbohydrate Polymers

Volume 179, 1 January 2018, Pages 402-407
Carbohydrate Polymers

Research paper
Gums induced microstructure stability in Ca(II)-alginate beads containing lactase analyzed by SAXS

https://doi.org/10.1016/j.carbpol.2017.09.096Get rights and content

Highlights

  • Structure at different scales of Ca(II)-alginate beads were assessed by SAXS.

  • Gums as second excipients induce a stabilization in the microstructure at rod scale.

  • Gums stabilize the size and density of the dimers and the interconnection of rods.

  • Guar gum improves the lactase activity during operation conditions.

Abstract

Previous works show that the addition of trehalose and gums in β-galactosidase (lactase) Ca(II)-alginate encapsulation systems improved its intrinsic stability against freezing and dehydration processes in the pristine state. However, there is no available information on the evolution in microstructure due to the constraints imposed by the operational conditions. The aim of this research is to study the time course of microstructural changes of Ca(II)-alginate matrices driven by the presence of trehalose, arabic and guar gums as excipients and to discuss how these changes influence the diffusional transport (assessed by LF-NMR) and the enzymatic activity of the encapsulated lactase.

The structural modifications at different scales were assessed by SAXS. The incorporation of gums as second excipients induces a significant stabilization in the microstructure not only at the rod scale, but also in the characteristic size and density of alginate dimers (basic units of construction of rods) and the degree of interconnection of rods at a larger scale, improving the performance in terms of lactase activity.

Introduction

In recent years, the encapsulation of β-galactosidase (lactase) in order to control its release and improve its stability against thermal and mechanical effects has been widely investigated (Estevinho, Damas, Martins, & Rocha, 2014; Traffano-Schiffo, Castro-Giraldez, Fito, & Santagapita, 2017; Zhang, Zhang, Chen, & McClements, 2016; Zhang, Zhang, & McClements, 2017).

Alginate is one of the most used anionic polyelectrolytes for the encapsulation of bioactive compounds (Santagapita, Mazzobre, & Buera, 2012). It consists of (1  4)- linked residues of β-d-mannuronate (M) and α-l-guluronate (G), and in solution it generates a hydrogel matrix through the complexation of G-blocks with di- or trivalent cations such as Ca2+, Mn2+, Zn2+, Al3+, Cr3+, and Ce3+ (Santagapita, Mazzobre, & Buera, 2011), embodying them into the cavities formed by a cooperative pairing of contiguous G-blocks (Stokke et al., 2000) forming the structure commonly known as “egg-box” (He, Liu, Li, & Li, 2016). Additionally, the sodium alginate solution can be gelled at acidic pH. Once a critical fraction of carboxylate residues are protonated, the decrease in the polymer charge density allows for chain–chain interactions leading to gelation. The inherent pH for the consolidation of H–alginate hydrogel is around 3.5, depending on the mannuronic (pKa = 3.38) to guluronic acid (pKa = 3.65) relative content of the employed alginate. Moreover, the Ca(II)–alginate structure can be obtained by cation exchange from a parent H–alginate hydrogel (Sonego, Santagapita, Perullini, & Jobbágy, 2016), indicating a higher affinity of alginate polymer for Ca2+. It has been demonstrated that the combined use of alginate with sugars and/or other biopolymers such as arabic and guar gums allows to improve the stability of enzymes within the hydrogel (Traffano-Schiffo, Castro-Giraldez et al., 2017).

The alginate microstructure depends on several factors such as the alginate’s concentration, average molecular weight and monomer composition (M/G ratio), as well as the presence of secondary excipients and the synthesis conditions (mainly the Ca2+ concentration and pH). On the other hand, the alginate bead constitutes an intrinsically inhomogeneous system, determined by the dropping method followed in conventional synthesis procedures. This method generates a Ca2+ gradient established from the surface to the core of the forming bead (Thu et al., 2000). All these parameters are related to the capacity of the hydrogel network to interact with the encapsulated biomolecule and the surrounding medium (Gombotz and Wee, 2012, Santagapita et al., 2011). Cross-linking and gelation of alginate was widely used for immobilization of bio-entities, from macromolecules to metazoans (Perullini, Orias, Durrieu, Jobbágy, & Bilmes, 2014; Santagapita et al., 2011). Beyond its high biocompatibility, by selection of the type of alginate and synthesis conditions, the pore size, degradation rate, and ultimately release kinetics can be finely controlled, allowing the design of controlled release systems (Tønnesen & Karlsen, 2002). Proteins immobilized in alginate matrices are released by two mechanisms: (i) diffusion of the protein through the pores of the polymer network and (ii) degradation of the polymer network. Analysis of Ca(II)-alginate gels by electron microscopies has shown that a very broad range of pore sizes (from 5 nm to 300 nm in diameter) can be obtained (Agulhon, Robitzer, David, & Quignard, 2012; Andresen, Skipnes, Smidsrød, Ostgaard, & Hemmer, 1977). In a different approach, the porosity evaluated from size-exclusion chromatography, smaller cut-off values in the order of 8–16 nm were obtained (Klein, Stock, & Vorlop, 1983; Stewart & Swaisgood, 1993). It is worth noting that in the latter approach a relatively high compactness of the biopolymer network is expected from the synthesis conditions employed (homogeneous method and long- term aging in the crosslinking cation solution). Regardless of the initial pore size, the leakage of immobilized species in bare alginate matrices is well documented, not only for macromolecules but even for much higher species as whole-cells (Perullini et al., 2005; Zhang, Wang, Charles, Rooke & Su, 2016).

One of the most precise and powerful techniques to evaluate the microstructure of hydrogels is the small-angle X-ray scattering (SAXS). The SAXS method is able to reveal subtle differences in electron density within hydrogels cross-linked networks in the range 1–100 nm, providing information on the supramolecular structure formed by biopolymers (Waters et al., 2010). SAXS patterns are indicative of rod like objects formed as the accumulation of cross-linked alginate chain dimers randomly orientated, establishing junction zones of different multiplicity (Agulhon et al., 2012). Previous works of lactase encapsulation in Ca(II)-alginate beads (Traffano-Schiffo, Castro-Giraldez et al., 2017; Traffano-Schiffo, Aguirre Calvo, Castro-Giraldez, Fito, & Santagapita, 2017) revealed that the remaining lactase activity after storage, freezing and freeze/thaw cycles, and dehydration, preserved by trehalose, was even improved by the presence of gums. The microstructure of the beads generated at pH 3.8 showed rods with smaller cross-sectional radius and with lower compactness when gums were used as additives. However, there is no available information on the evolution of the microstructure due to the constraints imposed by the operational conditions. On the other hand, information regarding the structure of the polymer chain dimers by analyzing SAXS curves at values of q higher than 1.5 nm−1 is presented for the first time. These “egg-box” structures were traditionally regarded as invariant blocks of construction of rods, and rods’ structural changes were evaluated in terms of the multiplicity of the junction zones (i.e. the number of alginate dimers in the cross-section of the rod) (Stokke et al., 2000). Thus, the aim of this research is to study the time course of microstructural changes of Ca(II)-alginate matrices driven by the presence of trehalose, arabic and guar gums as excipients and to discuss how these changes influence the diffusional transport (assessed by LF-NMR) and the enzymatic activity of the encapsulated lactase in operation conditions.

Section snippets

Materials

The employed materials are listed below: sodium alginate (Algogel 5540) from Cargill S.A. (San Isidro, Buenos Aires, Argentina), molecular weight of 1.97·105 g/mol and mannuronate/guluronate ratio of 0.6; d-trehalose dihydrate (Hayashibara Co., Ltd., Shimoishii, Okayama, Japan/Cargill Inc., Minneapolis, Minnesota, USA), molecular weight of 378 g/mol; guar gum (Cordis S.A., Villa Luzuriaga, Buenos Aires, Argentina), molecular weight of 220.000 g/mol and a mannose/galactose ratio of 1.8; arabic gum

Results and discussion

Fig. 1 shows the scattering intensity as a function of the scattering vector for a representative sample of Ca(II)-alginate hydrogel containing lactase (EA), incubated in 0.1 M acetate buffer pH 4.5 for 20 min. A schematic representation of the scale of different structural parameters derived from SAXS scattering experiments is included. SAXS scattering curves can be divided in three regions: at low, intermediate and high q values. From the slope of the log–log plot at low q, α1 parameter is

Conclusions

Though being a key in preserving enzymatic activity toward freezing and dehydration processes, the addition of trehalose as additive in Ca(II)-alginate lactase encapsulation systems prompts important structural changes leading to a loss of enzymatic activity during operation conditions. Trehalose drastically reduces the water self-diffusion coefficient in the starting hydrogel system (synthesized at pH 3.8), concomitant with the reduction in size and compactness of the structure at the scale of

Acknowledgements

This work was supported by the Brazilian Synchrotron Light Laboratory (LNLS, Brazil, proposal SAXS1-20160278), Universidad de Buenos Aires (UBACyT 20020130100610BA), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT PICT 2013 0434 and 2013 1331), CIN–CONICET (PDTS 2015 n° 196), and Consejo Nacional de Investigaciones Científicas y Técnicas. The author María Victoria Traffano-Schiffo wants to thank “Programa para la Formación de Personal Investigador (FPI)” Pre-doctoral Program of

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