JIA-2019-11

2541 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 cm –1 corresponds to C–O group, whereas bands situated around 1026 cm –1 are attributed toALG saccharide structure (1125 cm –1 to medium sharp C–C stretching, 1081 cm –1 to medium-sharp C–O and C–O–C stretching, 1 025 cm –1 to C–O stretching vibrations, 950 cm –1 to C–O stretching of uronic acids, 881 and 813 cm –1 to mannuronic acid residues, respectively) (Sartori et al . 1997). The spectrum of T . viride and alginate mixture shows broadening and more intense stretching vibrations around 3 321 cm –1 suggesting enhanced intermolecular hydrogen bonds. Disappearance of T . viride peaks at 2 921 and 2854 cm –1 , small peaks between 1500 and 1200 cm –1 and bands attributed to the ALG saccharide structure (from 1 125 to 813 cm –1 ) as well as shifting and smaller intensity of alginate bands corresponding to asymmetric (1 595 to 1 630 cm –1 ) and symmetric (1 405 to 1 415 cm –1 ) are an indication of at least interactions with hydroxyl, amine, carboxylate and C–O groups. 3.2. Physico-chemical characterization of microspheres Loading efficiency, loading capacity and swelling degree of microspheres Loading efficiency and loading capacity determination were performed to obtain information on the content of calcium and T . viride in microspheres. Results on calcium loading efficiency are listed in Table 1. The increase in calcium concentration decreased loading efficiency for both types of microspheres, what is more, pronounced for ALG/(Ca+ Tv ) microspheres. The decrease in encapsulation efficiency is a consequence of almost constant loading capacity of microsphere (Table 2), that is, microspheres reached almost maximum od loading capacity even at the lowest calcium cations concentration. Smaller calcium loading efficiency inALG/Ca microspheres is consistent with somewhat lower values of calcium loading capacity (Table 2). No T. viride spores presence in filtrate after microspheres preparation indicated loading efficiency is 100%. Table 2 displays almost constant calcium content inALG/ Ca and T . viride content in ALG/(Ca+ Tv ) microspheres prepared at various calcium concentrations. In comparison withALG/Ca, microspheres with T . viride showed somewhat higher calcium loading capacity. This may be attributed to the ability of calcium binding to T . viride . Trichoderma viride content in microspheres is also almost constant (within the method error) showing only somewhat lower value at 1.5 mol dm –3 of calcium chloride. When dispersed in solution, microspheres swell thus changing many properties, such as mechanical strength, permeability, the release of active agents, stability and the rate of disintegration. The swelling process involves two underlying molecular processes: (i) penetration of the solution into the matrix and (ii) polymer stress relaxation (transition of glassy structure to a rubbery state) (Siepmann and Siepmann 2012). The effect of increasing calcium concentration on swelling degree is presented in Fig. 7. It can be seen that the increase in calcium concentration decreased the degree of swelling of both types of microspheres, without and with T . viride spores. This is in accordance with studies showing that an increase in the calcium concentration reduces the degree of swelling of alginate microcapsules (Liu et al . 2004; Davidovich-Pinhas and Bianco-Peled 2010). During gelation calcium cations cooperatively interact with a block of L-guluronic acid groups forming ionic crosslinks between different polymer chains. The increase in calcium cation concentrations alter the number of alginate strands held together in the three-dimensional network and thus change its strength (Simpson et al . 2014) as well as the size of cavities inside Table 1 Variation of calcium cation loading efficiency, LE Ca , in ALG/Ca and ALG/(Ca+ Tv ) microspheres prepared at various initial calcium chloride concentrations, c i (CaCl 2 ) 1) c i (CaCl 2 ) (mol dm –3 ) LE Ca (ALG/(Ca+ Tv ), %) SD LC Ca LE Ca (ALG/Ca, %) SD LE Ca 0.5 57.61 3.56 50.31 4.37 1.0 49.38 1.28 50.40 1.45 1.5 41.46 0.38 41.19 0.36 2.0 32.64 0.84 37.53 0.92 1) ALG/(Ca+ Tv ), calcium alginate microspheres containing Trichoderma viride spores; ALG/Ca, calcium alginate microspheres; SD, the standard deviation of the means. Table 2 Variation of calcium loading capacity, LC Ca , in ALG/Ca and ALG/(Ca+ Tv ) microspheres, and Trichoderma viride loading capacity, LC Tv , in ALG/(Ca+ Tv ) prepared at various initial calcium chloride concentrations, c i (CaCl 2 ) 1) c i (CaCl 2 ) (mol dm –3 ) LC Ca (ALG/Ca, mmol g –1 ) SD LC Ca LC Ca (ALG/(Ca+ Tv ), mmol g –1 ) SD LC Ca LC Tv (ALG/(Ca+ Tv ), NS g –1 ) SD LC Tv 0.5 4.12E–02 3.74E–03 5.00E–02 1.33E–03 4.150E+3 42 1.0 4.16E–02 3.31E–03 4.68E–02 1.58E–03 3.970E+3 40 1.5 4.11E–02 2.93E–03 5.12E–02 2.41E–03 3.890E+3 39 2.0 4.20E–02 2.39E–03 4.50E–02 2.90E–03 4.140E+3 41 1) ALG/Ca, calcium alginate microspheres; ALG/(Ca+ Tv ), calcium alginate microspheres containing T. viride spores; SD, the standard deviation of the means.