Encapsulation of Lactobacillus acidophilus and different prebiotic agents by external ionic gelation followed by freeze-drying Encapsulação de Lactobacillus acidophilus e diferentes agentes prebióticos por gelificação iônica externa seguida de liofilização

The aim of this study was to investigate the influence of the addition of prebiotics rice bran, inulin and hi-maize, on the survival of Lactobacillus acidophilus in alginate microparticles obtained by external ionic gelation followed by freeze-drying. The microparticles size ranged from 127.5μm to 234.6μm. Microparticles added from the different prebiotics demonstrated an increase in the protection of the microorganism, which presented greater viability against the gastrointestinal simulation. As for storage under different conditions, rice bran treatment at 25oC kept probiotics viable for 30 days. Under storage conditions -18°C and 7°C, treatments containing prebiotics hi-maize and rice bran maintained viable probiotic microorganisms for a period of 60 days.


INTRODUCTION
Functional foods are those that, in addition to the basic nutritional functions, when consumed as part of the usual diet, produce metabolic, physiological and/or beneficial effects on health, preventing chronic, degenerative and cardiovascular diseases, among others (MARTIROSYAN & SINGH, 2015).Thus, consumption of these foods has been growing worldwide, with emphasis on probiotics.
Probiotics are defined as live microbial food ingredients that, when ingested in adequate amounts, alter intestinal microbiota and confer benefits on host health (HILL et al., 2014).However, a great challenge in the incorporation of probiotic bacteria in food is the maintenance of its viability during processing, packaging, storage conditions and passage through gastrointestinal tract (POP et al., 2015;BARBOSA & TEIXEIRA, 2017).Thus, microencapsulation appears as a viable alternative for the protection of probiotics, conferring greater viability to strains and greater effectiveness of probiotic action due to controlled release (ARGIN et al., 2014).
Among techniques of microencapsulation of probiotics,external ionic gelation/extrusion is the most popularly used, since it presents simple and accessible conditions for microparticles production, since it does not use temperature and solvent (RIBEIRO et al., 2014).Drying of microparticles is an important factor in improving storage properties of capsules due to water removal and ease of use and homogeneous distribution throughout the product (MORTAZAVIAN &SOHRABVANDI, 2007).Use of prebiotics in capsule may increase viability of probiotic microorganisms, since they may stimulate probiotic multiplication and/or activity during processing and storage (JANTARATHIN et al., 2017).
Therefore, the aim of this study was to evaluate the viability of Lactobacillus acidophilus in alginate microparticles containing different prebiotic sources (rice bran, inulin and hi-maize) produced by external ionic gelation technique followed by freeze-drying when exposed to simulated gastrointestinal conditions and storage at different temperatures.

Microencapsulation by the external ionic gelation and freeze-drying process
Microparticles were produced according to the extrusion technology developed by ETCHEPARE et al., (2016).For this, an airbrush (nozzle size: 0.3mm) model EW 110 was coupled to an air compressor model MB24/BV, at the air pressure of 2.72kgf/cm 2 , using the height of 30cm between atomization nozzle and CaCl 2 solution.

Morphology and particle size distribution
Morphology of dried microparticles was evaluated using a Scanning Electron Microscope (JEOL, model JM6360).Microparticles were fixed with a double-sided tape in aluminum beads and covered with a thin layer of gold.
Particle size distribution was performed by laser diffraction after dispersion in water of the dried microparticles in Mastersizer 2000 equipment (Malvern, Germany).The particle size results reflected the mean diameter of the microparticles analyzed (D [4: 3]), resulting from ten replicates made by the apparatus.

Enumeration of microencapsulated Latobacillus acidophilus
Viable cell count was performed according to the methodology proposed by SHEU, MARSHALL (1993) with modifications,where 0.1gram of dried microparticles was added in 9ml of phosphate buffer solution (0.1M, pH 7,5) followed by homogenization.Then samples were diluted in series with peptone water (0.1g 100g -1 ), plated on MRS agar (Himedia Curitiba, Paraná, Brazil) and incubated at 37°C for 72h.After incubation, viable probiotic cells were enumerated and results expressed in colony forming units per gram (log CFUg -1 ).

Viability of free and microencapsulated Lactobacillus acidophilus exposed to simulated gastrointestinal conditions
Simulation of gastrointestinal conditions was performed according to the method proposed by MADUREIRA et al. ( 2011) with modifications.Therefore, the microparticles were submitted to different pHs, simulating sections of gastrointestinal tract, such as esophagus/stomach (addition of pepsin, pH adjusted to 2.0 for 90 minutes), duodenum (addition of pancreatin and bile salts, pH adjusted to 5.0 for 20min.)and ileum (pH adjusted to 7.5 for 90min.).The analysis was carried out in a shaking refrigerated incubator (TE-421, Tecnal, Piracicaba, Brazil) at 37°C to simulate body temperature at agitation conditions (130rpm for esophageal/ stomach assay, and 45rpm for duodenum and ileum assay) to simulate the peristaltic movements of digestive tract.During the procedure, 1ml aliquots were collected from the fluid after 0, 90min.(esophagus/stomach), 110min.(duodenum), and 200min.(ileum), and survival of free and microencapsulated Lactobacillus acidophilus was analysed by enumeration on MRS agar.

Statistical analysis
The analyzes were performed in triplicate and results were evaluated by an analysis of variance (ANOVA); followed by Tukey's means comparison test at a level of 5% significance (P<0.05), using the Statistica 7 software.

Morphology and particle size distribution
It is observed in figure 1, through Scanning Electron Microscopy, that the microparticles presented a rough surface, which can be explained, since the dehydration of gels of freeze-dried polysaccharides can lead to the formation of a porous matrix, similar to a sponge.In the freeze-drying process, microparticles are subjected to low temperatures, leading to the formation of ice crystals and sublimation of ice crystals under reduced pressure, resulting in a porous dry product (DOLLY et al., 2013).
In this study, mean diameter of microparticles of different matrices ranged from 127.5±1.58-234.6±2.79μm and the smallest particle size was obtained in AHM treatment while the treatment with the largest mean diameter was AIN.The AIN and ALG treatments did not present significant differences (P<0.05)among themselves since the size of the alginate microparticles was 233.5±6.43μm.The ARB treatment had a particle size of 224.2±2.57μm.Microparticles within the size range 100-200μm provide an ideal balance, since the larger the microparticles, the better the protection for probiotics, but the worse the sensory effect (NAG et al., 2011).Ciência Rural, v.49, n.2, 2019.
Poletto et al.

Viability of free and microencapsulated Lactobacillus acidophilus exposed to simulated gastrointestinal conditions
Results obtained regarding the survival of the microorganisms under the simulated gastrointestinal conditions are shown in table 1.After 90min. of incubation, the hydrogenation potential was gradually adjusted with a pepsin solution to a value of 2.0 (esophagus/stomach portion).Survival of probiotics was higher in the alginate microparticles added hi-maize (AHM) compared to the other treatments, since they presented lower release (1.76 log cycles) in relation to their initial count.In another study, CHAN (2011) observed a 4 log reduction in the viability of the freeze-dried Lactobacillus casei microparticles obtained by extrusion with alginate and a reduction of 1.5 to 2 log CFUg -1 when starch was used in conjunction with alginate, under the same conditions.
After exposure of the free cells to acidic conditions (esophagus/stomach), there was a decrease of 5.35 log CFUg -1 (p<0.05) in free cells compared to the initial count due to the low pH in this step process, resulting in low viability of probiotic bacteria.These results corroborated those of DIMITRELLOU et al. ( 2016), since when the acidic conditions were exposed, Lactobacillus casei ATCC 393 continuously lost its viability, with viable cell counts falling 1.46 log CFUg -1 at pH 3 and 4.03 log CFUg -1 at pH 2, clearly indicating the need for protection through microencapsulation.
After exposure to simulated duodenal conditions (bile salts, pancreatin and pH 5.0), the free cells presented an even more significant drop (P<0.05) in the viable cell population (6.21 log CFUg -1 ) when compared to the initial count.In relation to the microencapsulated microorganism, the treatments did not present a significant difference in comparison with results obtained after the simulation of the acidic conditions, however, there was no total release of the microorganism, since the microparticles promoted its protection.
At the end of the analysis, after exposure to the simulated ileum conditions (pH 6.5), the free microorganism presented reduction of 6.69 log CFUg -1 relative to its initial count.This was due to the variation in pH during the simulation of the gastrointestinal tract, which caused a significant reduction in viability.Conversely, it was observed that the microencapsulation of Lactobacillus acidophilus in alginate, as well as associated with the different prebiotics, gave greater protection to the probiotic during the exposure to the simulation of the gastrointestinal tract, once, after the passage through the ileum, treatments containing alginate, rice bran, inulin and hi-maize presented log reductions of 1.37, 4.14, 1.47 and 0.72 respectively, when compared with time zero, with significant differences between treatments (P<0.05).
Results presented in this research corroborated other studies demonstrating the efficiency of microencapsulation in protection of probiotics during exposure to gastrointestinal conditions.PANKASEMSUK et al. ( 2016) reported that L. casei 01 microencapsulated with 2% alginate and 1% maize starch allowed optimal survival in both gastric and biliary fluids.In another study, HALIM et al. (2017) compared the viability of free and microencapsulated Pediococcus acidilactici ATCC 8042 in calcium alginate and co-coating with calcium alginate and chitosan by extrusion technique with subsequent freeze-drying using 10% (w/w) of skimmed milk as cryoprotectant, compared to simulated gastric juice and bile tolerance assays and reported that probiotic cell mortality significantly reduced when microencapsulated probiotics were compared to free cells.

Viability of Lactobacillus acidophilus at different storage conditions
Table 2 shows the stability of microencapsulated Lactobacillus acidophilus for 75 days of storage under temperatures of 25°C, 7°C and -18°C, simulating different environmental conditions.At 25°C, viability of microencapsulated bacteria was maintained until the 15 days of storage for alginate and inulin treatments.Microparticles containing the prebiotics hi-maize and rice bran maintained viability for 15 and 30 days, with counts of 6.02±0.01log CFUg -1 and 6.05±0.03log CFUg -1 , respectively.After this period, there was a significant reduction (p<0.05)1.5±0.01log CFUg -1 to ARB and 3.09±0.03log CFUg -1 to AHM in viability of microorganisms until 75 days of storage.OKURO et al. (2013) also did not obtain positive results at 22°C when evaluating solid lipid microparticles in which Lactobacillus acidophilus was co-encapsulated with prebiotic inulin or polydextrose using spray chilling technology, since its stability was evaluated for 120 days and the pre-biotic treatment remained viable for a period of between 0 and 7 days while the treatment containing inulin remained viable between 7 and 30 days and the treatment containing polydextrose remained viable for about 60 days.

CONCLUSION
Alginate microparticles as well as those added from the prebiotics produced by the external extrusion/gelation technique followed by freezedrying promoted the resistance of Lactobacillus acidophilus during the simulation passage through the gastrointestinal tract.In relation to storage conditions, at the temperatures of -18ºC and 7ºC, the best results were obtained in the microparticles containing the prebiotics hi-maize and rice bran; although, rice bran deserves attention, since it is a by product little studied at present and presented excellent results in the protection to maintain probiotic viability.Thus, it is concluded that the himaize and rice bran compounds besides attributing to the microparticles health-promoting prebiotic effects are also capable of increasing the resistance of the L. acidophilus-containing alginate microparticles obtained by external gelation, conferring to these a protective effect and may be alternative means for the development of novel functional foods.

Table 1 -
Survival of free and microencapsulated Lactobacillus acidophilus in different treatments after each step of simulated gastrointestinal conditions.
* Averages followed by the same letter, lowercase in the column and upper case in the row, do not differ statistically from each other by the Tukey test with a significance of 5%.Averages found in triplicate.