3.1.Size and encapsulation yield of silica-alginate beadsInour 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 instudies involving very large calcium alginate beads (>1 mm) (Arnaud et al., 1992; Hyndman et al., 1993; Klemmer etal.
, 2011). Alginate beads obtained by the dripping process usuallyexhibit a diameter within the range of 0.5-3.5 mm (Coradinet al., 2003). Microcapsules below these sizes can be obtained byreduction of dripping-needle diameter, alginate concentration, or flow rate (Willaert and Baron, 1996).
The results showedthat the beads were globular in shape (Fig. 1, 2)and addition of silica did not affect it. Moreover, there were no significantdifferences (P<0.05) between beads in microencapsulation yield (Table 1). Moreover, it was reported that the shape ofthe 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 shellsTheIR spectra (4000-400 cm-1) of alginate and alginate-silica are shownin Figure 3. As can be seen, the alginate FT-IRspectrum showed the characteristic peaks at 1620 and 1416 cm?1 (COO?asymmetric and symmetric stretching), 1030 cm?1 (C-O-C stretching), and3453 cm?1 (OH? stretching) (Xuet al., 2007). In the FT-IR spectrum of alginate-silica the peaksappearing at 1633 and 1435 cm?1 could be assigned to COO?asymmetric and symmetric stretching, while the peak at 3444 cm?1 couldbe attributed to OH stretching. This suggests that the hydroxyl and carboxyl ofthe alginate interacted with the hydroxyl groups in silica film throughhydrogen bonding, resulting in the shift of position and shape of itscharacteristic peaks. Generally, at pH 7, monomeric silica is found mainly assilicic acid Si(OH)4.
However, as silica oligomers are formed duringthe polymerization process, their silanol groups (Si-OH) become increasinglyacidic, resulting in a negative charge being borne. Since alginate is alsonegatively charged at this pH, no electrostatic attractive interaction isexpected between the two components. Therefore, hydrogen bonding takes placebetween the polysaccharide chain (alginate) and the silanol groups (silica), asillustrated by Figure 3.3.3.
Survival of microencapsulated cells in simulated gastric juiceTable 2shows the viability of free and encapsulated probiotic bacteria during 120 minutesof incubation in simulated gastric conditions. The survivability of L. caseiATCC 39392 was expressed as the destructive value (D-value), which isthe time, required destroying 90% or one log cycle of the organism. The resultsshowed that the survival of cells in coated beads was significantly (P<0.05)better than that of free cells. However, with increasing concentrations ofsilica, survival increased and beads made from alginate with 15% Sio2(D-values 500 min) provided the best protection, followed by alginatewith 10% Sio2 (D-values 166.
67 min), alginate with 5% (D-values111.12 min), alginate (D-values 52.63 min) and free cells (D-values34.48 min). This result is in agreement with Chandramouli et al., (2004) who reported a highersurvival of L. acidophilus CSCC 2400 and CSCC 2409 immobilized inalginate bead in low pH environments.
Moreover, Krasaekooptet al. (2004) showed that for L. acidophilus, with initial cellcounts within the range of 2.171-1.970 × 109, the survival of cellsin coated beads was significantly (P<0.05) better than that ofuncoated beads.
Kim et al. (2008) reportedthat at pH 1.2, non-encapsulated L. acidophilus were completelydestroyed after one hour of incubation while encapsulated L.
acidophilusmaintained above 106 cfu mL-1 at pH 1.5 after two hours. Mokarram et al. (2009) demonstrated that thealginate coat prevented the acid-induced reduction of the strains in simulatedgastric 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 wassensitive to an acidic environment (pH 2.
4). Furthermore the ingestion ofunprotected bacteria might result in reduced viability 3-log reduction after 2 hours,thus suggesting that the coating of beads provide the best protection insimulated gastric juice (Fig. 4). The formationof silica-coated alginate beads for this study was prepared as follows. First,the alginate solution was added drop-wise into the CaCl2 solutionand was then cross-linked with Ca2+ ions to form the Ca-alginate gelbeads. Then, the Ca-alginate gel beads were taken out and dissolved in Sio2solution.
Xu et al. (2007) reported thatthe hydrolysis and condensation of silica could be accelerated by an alginatemolecule at ambient temperature and neutral pH, without additional catalystssuch as an acid, alkali or fluoride salt. When these alginate beads were put incontact with the Sio2 solution, the guluronic acid and mannuronicacid of alginate interacted with Sio2 and promoted its hydrolysis tosilanol. Thus, the polycondensation of silanol occurred and the silicaparticles began to deposit on the surface of alginate beads, thereby inducing theformation of silica film.3.4.
Survival of free and microencapsulated bacteria in simulated intestinal juiceTheeffect of simulated intestinal juice on the viability of the microencapsulatedand free probiotic bacteria is presented in Table 3.The number of probiotics declined significantly as the incubation timeincreased. 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 0CFU/mL after 120 minutes (Table 3).
The mostsusceptible 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 coatingwere the most effective in protecting probiotic bacteria from simulatedintestinal juice (P<0.05). This is in good agreement with the resultsof Krasaekoopt et al. in 2004, who indicatedthat the survival of probiotic bacteria was highly enhanced in gastro-intestinalconditions when encapsulated with alginate-chitosan or poly-L-lysine. Theprotective effect of chitosan on bile acid tolerance was measured by Khosravi Zanjani et al. (2014).
They found that microencapsulationwith alginate-gelatinized starch coated with chitosan could successfully andsignificantly protect probiotic bacteria against adverse condition of simulatedhuman gastro-intestinal condition. 4.Conclusion Calcium alginate capsules can be easily producedby extruding a sodium alginate solution into an aqueous solution of calciumchloride, which enables it to preserve the biological activity of entrappedliving microorganisms. However, calcium alginate capsules show poor mechanicalstability. It is known that alginate is a swelling component that over time leadsto leakage of entrapped constituents like living cells.
These cells are releasedgradually and proliferate in the external medium. Therefore, alginate capsules maynot be a suitable host matrix for the encapsulation of components like livingcells. In our study, silica has a positive effect on the viability ofprobiotics. The combination of calcium alginate with silica not only improves the viability of probiotics, but alsofacilitates the formation of integrated structures of capsules.