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3.1.
Size and encapsulation yield of silica-alginate beads

In
our study, the size of the beads increased with the addition of silica coating.
The mean diameter of alginate beads without silica coating was 1.54 ± 0.07 mm,
which was significantly (P<0.05) lower than beads coated with silica (Table 1). Researchers reported similar results in studies involving very large calcium alginate beads (>1 mm) (Arnaud et al., 1992; Hyndman et al., 1993; Klemmer et
al., 2011). Alginate beads obtained by the dripping process usually
exhibit a diameter within the range of 0.5-3.5 mm (Coradin
et al., 2003). Microcapsules below these sizes can be obtained by
reduction of dripping-needle diameter, alginate concentration, or flow rate (Willaert and Baron, 1996). The results showed
that the beads were globular in shape (Fig. 1, 2)
and addition of silica did not affect it. Moreover, there were no significant
differences (P<0.05) between beads in microencapsulation yield (Table 1). Moreover, it was reported that the shape of the beads did not change when the layer was added to alginate beads (Koo et al., 2001; Annan et al., 2008; Mokarram et al., 2009). 3.2. Formation of alginate-silica shells The IR spectra (4000-400 cm-1) of alginate and alginate-silica are shown in Figure 3. As can be seen, the alginate FT-IR spectrum showed the characteristic peaks at 1620 and 1416 cm?1 (COO? asymmetric and symmetric stretching), 1030 cm?1 (C-O-C stretching), and 3453 cm?1 (OH? stretching) (Xu et al., 2007). In the FT-IR spectrum of alginate-silica the peaks appearing at 1633 and 1435 cm?1 could be assigned to COO? asymmetric and symmetric stretching, while the peak at 3444 cm?1 could be attributed to OH stretching. This suggests that the hydroxyl and carboxyl of the alginate interacted with the hydroxyl groups in silica film through hydrogen bonding, resulting in the shift of position and shape of its characteristic peaks. Generally, at pH 7, monomeric silica is found mainly as silicic acid Si(OH)4. However, as silica oligomers are formed during the polymerization process, their silanol groups (Si-OH) become increasingly acidic, resulting in a negative charge being borne. Since alginate is also negatively charged at this pH, no electrostatic attractive interaction is expected between the two components. Therefore, hydrogen bonding takes place between the polysaccharide chain (alginate) and the silanol groups (silica), as illustrated by Figure 3. 3.3. Survival of microencapsulated cells in simulated gastric juice Table 2 shows the viability of free and encapsulated probiotic bacteria during 120 minutes of incubation in simulated gastric conditions. The survivability of L. casei ATCC 39392 was expressed as the destructive value (D-value), which is the time, required destroying 90% or one log cycle of the organism. The results showed that the survival of cells in coated beads was significantly (P<0.05) better than that of free cells. However, with increasing concentrations of silica, survival increased and beads made from alginate with 15% Sio2 (D-values 500 min) provided the best protection, followed by alginate with 10% Sio2 (D-values 166.67 min), alginate with 5% (D-values 111.12 min), alginate (D-values 52.63 min) and free cells (D-values 34.48 min). This result is in agreement with Chandramouli et al., (2004) who reported a higher survival of L. acidophilus CSCC 2400 and CSCC 2409 immobilized in alginate bead in low pH environments. Moreover, Krasaekoopt et al. (2004) showed that for L. acidophilus, with initial cell counts within the range of 2.171-1.970 × 109, the survival of cells in coated beads was significantly (P<0.05) better than that of uncoated beads. Kim et al. (2008) reported that at pH 1.2, non-encapsulated L. acidophilus were completely destroyed after one hour of incubation while encapsulated L. acidophilus maintained above 106 cfu mL-1 at pH 1.5 after two hours. Mokarram et al. (2009) demonstrated that the alginate coat prevented the acid-induced reduction of the strains in simulated gastric juice (pH 1.5, 2 h), resulting in significantly (P<0.05) higher numbers of survivors. Moreover, our results suggested that non-encapsulated L. casei ATCC 39392 was sensitive to an acidic environment (pH 2.4). Furthermore the ingestion of unprotected bacteria might result in reduced viability 3-log reduction after 2 hours, thus suggesting that the coating of beads provide the best protection in simulated gastric juice (Fig. 4). The formation of silica-coated alginate beads for this study was prepared as follows. First, the alginate solution was added drop-wise into the CaCl2 solution and was then cross-linked with Ca2+ ions to form the Ca-alginate gel beads. Then, the Ca-alginate gel beads were taken out and dissolved in Sio2 solution. Xu et al. (2007) reported that the hydrolysis and condensation of silica could be accelerated by an alginate molecule at ambient temperature and neutral pH, without additional catalysts such as an acid, alkali or fluoride salt. When these alginate beads were put in contact with the Sio2 solution, the guluronic acid and mannuronic acid of alginate interacted with Sio2 and promoted its hydrolysis to silanol. Thus, the polycondensation of silanol occurred and the silica particles began to deposit on the surface of alginate beads, thereby inducing the formation of silica film. 3.4. Survival of free and microencapsulated bacteria in simulated intestinal juice The effect of simulated intestinal juice on the viability of the microencapsulated and free probiotic bacteria is presented in Table 3. The number of probiotics declined significantly as the incubation time increased. The rate of decrease was significantly greater in the free cells (P<0.05). In case of free L. casei ATCC 39392, the cell number was reduced to 0 CFU/mL after 120 minutes (Table 3). The most susceptible cell to intestinal juice was 15% Sio2-coated samples; the D value was about 303 minutes (Table 3). Our results indicated that alginate microcapsules with Sio2 coating were the most effective in protecting probiotic bacteria from simulated intestinal juice (P<0.05). This is in good agreement with the results of Krasaekoopt et al. in 2004, who indicated that the survival of probiotic bacteria was highly enhanced in gastro-intestinal conditions when encapsulated with alginate-chitosan or poly-L-lysine. The protective effect of chitosan on bile acid tolerance was measured by Khosravi Zanjani et al. (2014). They found that microencapsulation with alginate-gelatinized starch coated with chitosan could successfully and significantly protect probiotic bacteria against adverse condition of simulated human gastro-intestinal condition. 4. Conclusion Calcium alginate capsules can be easily produced by extruding a sodium alginate solution into an aqueous solution of calcium chloride, which enables it to preserve the biological activity of entrapped living microorganisms. However, calcium alginate capsules show poor mechanical stability. It is known that alginate is a swelling component that over time leads to leakage of entrapped constituents like living cells. These cells are released gradually and proliferate in the external medium. Therefore, alginate capsules may not be a suitable host matrix for the encapsulation of components like living cells. In our study, silica has a positive effect on the viability of probiotics. The combination of calcium alginate with silica not only improves the viability of probiotics, but also facilitates the formation of integrated structures of capsules.

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