JIA-2019-11

2544 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 cation concentration can be seen at the asymmetric and symmetric carboxylate stretching vibrations indicating a change in the intensity of interaction between alginate carboxylate groups and calcium ions. The characteristic peak for asymmetric carboxylate stretching vibrations shifts to lower wavenumber (from 1 595 to 1 589 cm –1 ) and the peak for symmetric carboxylate stretching vibrations shifts to higher wavenumber (from 1405 to 1413 cm –1 ) by exchange of sodium with calcium ions due to a change in the charge density, the increase in the radius and the atomic weight of the cations. Gradual intensity increasing and shifting of carboxylate ions stretching vibrations with increasing calcium concentrations indicated the strong electrostatic interactions between calcium ions and carboxylic groups of alginate. Similarity was observed in a previous study which suggested that bands concerning carboxylate groups can be used to follow structural changes of different alginate gels (Daemi and Barikani 2012). Influence of increasing calcium chloride concentrations on molecular interactions in microspheres loaded with T . viride is presented in Fig. 10-B. There are no significant changes in the spectrum obtained at lower calcium chloride concentration between microsphere and spectra of alginate and T . viride mixture. Stretching vibrations of O–H bonds of alginate, calcium chloride, T . viride and alginate microspheres appeared in the range of 3 000–3 600 cm –1 . At higher calcium concentrations changes occurred in the hydroxyl and carboxylate functional groups regions. The intensity of broad –OH and –NH stretching vibrations band around 3 380 cm –1 increased indicating the higher amount of water in the microsphere structure. Gradual intensity increasing and shifting of asymmetric (a shift to a lower wavenumber (from 1 595 to 1 589 cm –1 ) and at the highest calcium ions concentrations to a higher wavenumber (from 1 589 to 1 637 cm –1 ), and symmetric (from 1 405 to 1 434 cm –1 ) vibrational peak of carboxylate ions indicated difference in electrostatic interactions between calcium ions and carboxylic groups in comparison with microspheres prepared without T . viride . Microspheres prepared with 2 mol dm –3 of calcium chloride showed different bands in comparison with the spectra of the other concentrations: shifting and dividing of an asymmetric carboxylate and hydroxyl vibrational peaks into two, 1 604 and 1637 cm –1 , and 3248 and 3369 cm –1 , respectively. Changes in FTIR spectra undoubtedly show structural differences in microspheres prepared without and with T . viride . In vitro release of T. viride and calcium ions from microspheres The mechanisms by which active agent are released from microspheres are complex and involve different processes: The penetration of the surrounding solution into the microsphere, swelling of the microsphere, diffusion of the active agent through the gel layer, dissolution of the active agent in the medium and erosion of the swelled matrix (Maderuelo et al . 2011). It was shown that the most important rate-controlling release mechanisms from hydrophilic microspheres are diffusion, swelling and erosion (Sankalia et al . 2007). Korsmeyer et al . (1983) developed an empirical equation to analyze the release of the loaded agent from swelling as well as non-swelling polymeric delivery systems expressed as: f = kt n (6) where f is fraction released at time t (h), k (h –1 ) is a kinetic constant characteristic of a particular system (incorporates the overall solute diffusion coefficient and geometric characteristics of a microsphere) and n is the release exponent representing the release mechanism. In the case of the burst effect equation is modified (Lindner and Lippold 1995; Kim and Fassihi 1997): f = a + kt n (7) where a is the y -axis intercept characterizing the burst effect. According to the semiempirical model, the release exponent n can be characterized by three different mechanisms (Fickian diffusion, anomalous (non Fickian diffusion), or Type II transport). Values of n <0.43 indicate the release is controlled by classical Fickian diffusion, n >0.85 is controlled by Type II transport, involving polymer swelling and relaxation of the polymeric matrix, whereas values of n between 0.43 and 0.85 show the anomalous transport kinetics determined by a combination of the two diffusion mechanisms and Type II transport. Knowledge of kinetics and mechanism involved in the release of an active agent is important for the optimal development of formulations based on biopolymeric materials. Design of controlled delivery systems involves optimization of many parameters among which are the most important are the type and concentration of both, biopolymer and gelling cation (Sankalia et al . 2007). In this direction, the kinetics and mechanisms of T . viride and calcium cations release were studied on microspheres prepared at various calcium chloride and constant alginate concentrations. The release profiles for T . viride spores presented in Fig. 11 are characterized by rapid initial burst effect. After one week of observation, a fraction of released T . viride spores increased above 1 indicating a higher concentration of T . viride biomass then loaded in the microsphere. This can be ascribed to the germination inside microsphere and after releasing in the surrounding medium. Microscopic observations confirmed the conclusion revealing the germ tubes grow inside the microsphere matrix and protrude toward the water phase. The spores that were not germinated may be released to the surrounding media through (i) perforations made by germ tubes (microsphere