Protection of Lactobacillus acidophilus under in vitro gastrointestinal conditions employing binary microcapsules containing inulin

In this research, microcapsules based on low acyl gellan (LAG) and sodium alginate (SA) containing inulin were developed in order to assess its protective effect on the viability of Lactobacillus acidophilus under in vitro gastrointestinal conditions.The results showed that microencapsulated cells display significantly (P<0.05) higher resistance to simulated gastrointestinal conditions (SGIC) than free cells. Besides, the incorporation of inulin into the wall matrix resulted in improved survival after 5 h incubation in SGIC. These results represent an alternative to vehiculate probiotics in food, especially in solid food due to the size of the microcapsules. Therefore, these microcapsules can contribute to possible industrial applications in the development of new alimentary products.


INTRODUCTION
Probiotics are defined as live microorganisms which when administered in appropriate concentrations provide health benefits to the host because they colonize the human gut in adequate amounts (10 6 CFU/mL) (Tripathi and Giri, 2014;WHO/FAO, 2002).These health benefits include therapeutic effects such as alleviating symptoms of lactose malabsorption, reducing the level of serum cholesterol, irritable bowel syndromes and colon cancer, besides enhancing resistance to gut infections (Kailasapathy and Chin, 2000;Sanders et al., 2013).All these effects are caused by inhibiting pathogen growth and stimulating the host's immune response (Figueroa et al., 2011).However, the incorporation and viability of these bacteria in food products still represent a technological challenge for researchers during the development of new probiotic products, because the viability of probiotics often decreases sharply during gastric transit due to the strong acidic conditions (Holkem et al., 2016).
One effective method to protect probiotic bacteria from the environmental factors encountered during the passage through the human gastrointestinal tract is the microencapsulation using various polysaccharides as wall material (that is, gellan gum and sodium alginate).Gellan gum is an anionic extracellular heteropolysaccharide produced by the bacterium Sphingomonas paucimobilis and consists of repeating units of a tetrasaccharide (1,3-β-D-glucose; 1,4-β-Dglucuronic acid; 1,4 β-Dglucose; and 1,4-α-L-rhamnose).It is available in two forms: High acyl gellan (HAG) and low acyl gellan (LAG).When HAG is exposed to strong alkali treatment at high temperature, the acyl groups are hydrolyzed and LAG is obtained.These structural differences between HAG and LAG allow great diversity of its textural properties.Therefore, HAG forms soft, elastic gels; while LAG gum forms strong gels (González et al., 2012).With regard to alginates, they are polysaccharides produced by brown algae (Laminaria digitata, Laminaria hyperborea, Ascophyllum nodosum and Macrocystis pyrifera).Alginates are widely used in the industry due to their non-toxic and gelling properties.Chemically, alginates are an anionic linear copolymer of β-D-mannuronic acid (M) and α-L-guluronic acid (G) joined by β 1-4 links and structured in blocks that can be homopolymeric (M or G) or heteropolymeric (MG) (Rosas et al., 2013).Within the most important applications of alginates in biotechnology is the ability to create stable gels through the ionic interaction between two adjacent chains with monovalent or divalent cations, forming junction zones that stabilize the gel structure (Fabich et al., 2012;Tavassoli et al., 2016).
Different methods for probiotic microencapsulation have been reported, including spray-drying, ionic gelantion, extrusion and complex coacervation (Champagne and Fustier, 2007;Martín et al., 2015).Internal ionic gelation (IIG) has been used for microorganisms microencapsulation due to its low cost, mild formulation conditions and high cellular retention making this technique one of the most promising ones (Cook et al., 2012).The microencapsulation using IIG does not require specialized equipment, complex techniques or the use of expensive reagents; moreover, IIG protects the microencapsulated cells from the acidic condition facilitating the gradual cell release in the target place (Chavarri et al., 2010;Cook et al., 2011;Guerin et al., 2003;Kanmani et al., 2011).Therefore, the aim of this study was to evaluate the Lactobacillus acidophilus survival into microcapsules containing inulin as a prebiotic compound under simulated gastrointestinal conditions.

Microencapsulation
Microcapsules w ere obtained using a technique based on the formation of a w ater-oil emulsion.The dispersion (aqueous phase) w as prepared w ith a mixture of 25SA/75LAG at 0.8% w /v, incorporating 1 mL of the cell suspensión (L.acidophilus) and 30 mM of Ca ++ .Then, the dispersion w as added into the oil phase (sunflow er oil and 0.1% v/v of surfactant) under constant agitation in a stirring plate follow ed by the incorporation of 1 mL of δgluconolactone up to pH 4 in order to start the internal ionic gelation process.The microcapsules w ere harvested by centrifugation at 5000 rpm for 5 min, and the pellets w ere w ashed tw ice w ith saline solution to remove the oil residues.

Microcapsule m orphology and size
Tw enty micro liter of the microcapsules w ere used to determine the diameter employing a Leica DM500 microscope w ith a digital camera.The samples w ere diluted in sterile saline prior to the optical analysis and the captured images w ere analyzed using the softw are Image Pro-Plus ver 5.1.The average size of microcapsules w as evaluated by measuring 100 microcapsules.

Microencapsulation efficiency
The microcapsules suspension w ere centrifuged at 5000 rpm in order to separate the free cell from microencapsulated cells.Then, the bacterial concentration in the supernatant w as determined and encapsulation efficiency (% EE) w as calculated according to Equation 1 as proposed by Gonzalez et al. (2015).
In this equation, A is the total bacterial concentration in the suspension and B is the free bacterial concentration in the supernatant.

Viability of L. acidophilus m icroencapsulated
Since the encapsulation process may affect the viability of probiotics, in the present study, the viability of L. acidophilus w as enumerated before being subjected to simulated gastrointestinal conditions.The microencapsulated bacteria w ere released from the microcapsules based on the method proposed by Sheu and Marshall (1993).The microcapsules (1 g) w ere suspended in 9 mL of phosphate buffer (pH 7, 0.1 M) and homogenized for 5 min at 14,000 rpm using a high-speed homogenizer (Ultra-Turrax, model T50) and the breaking of the microcapsules w as confirmed by optical microscopy.The enumeration of the viable cells w as carried out by the drop plate method after 48 h incubation at 37°C on MRS agar under anaerobic condition.After the incubation time, the viable probiotic cells w ere counted and expressed in log colony forming units per gram (log CFU g -1 ).

Viability of free and m icroencapsulated L. acidophilus subjected to sim ulated gastric and intestinal juices
One gram of microcapsules w as subjected during 1 h to simulated gastric juice (SGJ) w hich is prepared by adjusting the pH of 0.2% (w /v) NaCl solution to 3 through the addition of 1.0 M HCl solution in order to mimic the stomach condition (Cheow et al., 2014).Afterw ards, the same microcapsules w ere also added to simulated intestinal juice (SIJ) (6.8 g of KH2PO4 in deionized w ater at pH 7.0) for 4 h, resulting in a total simulated gastrointestinal transit time of 5 h (Graff et al., 2001).It is interesting to mention that all the tests w ere performed at 37°C in order to simulate the body temperature and the solutions employed w ere prepared on the same analysis day.The survival of free and microencapsulated Lactobacillus acidophilus w as conducted according to the aforementioned technique.

Statistical analysis
All the experimental data w ere subjected to analysis of variance (ANOVA-one w ay) using the softw are SPSS (ver.17 for Window s) follow ed by Tukey's mean comparison test at a level of 5% significance.All the tests w ere carried out in triplicate and the data expressed as the mean ± standard deviation.

Microencapsulation
The microencapsulation method employed in this work is based on the emulsion between two phases, one hydrophobic and one hydrophilic containing the anionic polysaccharide, where by agitation, a great number of drops are originated which are gelled by acidification with δ-gluconolactone, since ion calcium is released from the calcium carbonate.The obtained microcapsules showed a unimodal behavior; which may be explained by the slow release of calcium ions from calcium carbonate because of the slow disruption of the gluconolactone.Figure 1 depicts the number versus intervals of obtained microcapsules size.A unimodal behavior with particle sizes between 20 and 180 µm was observed.The microcapsule size is an important physical parameter since it can influence the sensorial attributes as aroma, texture and appearance when microcapsules are applied into food matrices.Microcapsules minor to 100 µm are desirable in liquid food, so as to avoid negative sensorial impact (Burgain et al., 2011).
Figure 2 shows the morphology of the obtained microcapsules with SA and LAG using calcium carbonate as a Ca 2+ donor which was with spherical in shape and the outside surface with regular surfaces without the presence of deformations.
In order to determine the microencapsulation efficiency of the microencapsulation process, two counts were carried out.The initial count corresponds to the number of microorganisms added to the biopolymer dispersion (aqueous phase) and the second one was determined after the microcapsules were harvested.It is worth to mention that no negative effect was observed as there was no significant difference (p < 0.05) among the obtained CFU values before and after microencapsulation process; due to that, high averages of efficiency percentages were obtained (94.32 to 95.76%).Nonetheless, the encapsulation efficiency of the microcapsules was slightly improved when the prebiotic was incorporated into the microcapsule; thus, the loss of probiotic in microencapsulation process was reduced.

Viability of L. acidophilus microencapsulated
It was noted that there was no significant difference (P < 0.05) among efficiency and viability values of L. acidophilus encapsulated in binary microcapsules before incorporation to simulated gastrointestinal juices.It means that all the microencapsulated bacteria were able to grow, thereby yielding the beneficial effect associated with the probiotic intake.This also indicates that the microorganisms did not suffer pronounced damage during the microencapsulation process, showing that IIG is a feasible and adequate technique to produce microcapsules containing probiotics.

Viability of free and microencapsulated Lactobacillus acidophilus subjected to gastric and intestinal conditions
Microcapsules containing L. acidophilus were initially exposed to SGJ for 1 h and then, the same microcapsules were transferred to SIJ for a further 4 h in order to mimic the gastrointestinal transit environment, equal procedure was realized for cells in free status. Figure 3 shows the results for the viability of L.acidophilus exposed to SGJ conditions for the free cells, microencapsulated and microencapsulated along with inulin.It was noted that the presence of inulin in the microcapsules provided the highest level of protection to the encapsulated cells, where 10.78 log CFU/ mL of the encapsulated cells survive to the SGJ during 1 h followed by cell microencapsulated alone with with 10.12 log CFU/mL, while free cells decreased sharply its viability until 5.67 log CFU/mL.Therefore, it is extremely important to protect L. acidophilus by microencapsulation These findings indicate that microcapsules based on mixture SA/LAG incorporated with inulin are stable under.acid solution likely by the interaction between biopolymer and the prebiotic.It should be clear that initial counts before the incorporation to the SGJ were 11.23 log CFU/mL for the L. acidophilus microencapsulated, 11.15 log CFU/mL for free cells and 11.20 log CFU/mL for microencapsulated cell containig the prebiotic.
After immersion in SGJ, the difference between the viable number of L. acidophilus in free status, microencapsulated alone and microencapsulated along with inulin became highly significant (P<0.05) with longer incubation time.
At the end of the exposure time of L. acidophilus (free, microencapsulated and microencapsulated along with prebiotic) to SGJ conditions, the same probiotic bacteria was subjected to SIJ conditions at pH 7.0 as can be seen in Figure 3B.After submitting the L. acidophilus microencapsulated to SIJ conditions, they showed a significant decrease (P< 0.05) of 0.45 log CFU/mL when compared to the initial count, that is before the intestinal simulation.Most likely, some microcapsules were broken or there was a penetration of gastrointestinal juices into the microcapsules killing the probiotic.With regard to L. acidophilus microencapsulated along with inulin, an increase was observed ranging from 10.78 to 11.12 log CFU/mL; this is likely by a possible consumption of inulin by the probiotic or by a controlled release from microcapsules when the environmental pH rise.Conversely, the count of L. acidophilus in free status showed a reduction (P< 0.05) of 1.55 log CFU/mL when compared to the initial count before simulation.
In general terms, the number of the microencapsulated cells (both with and without inulin) that remain viable is approximately 6.27 log CFU/mL higher than free cells after being subjected to SIJ; which means that microcapsules protected from the acidic condition found in the gastrointestinal transited to L. acidophilus at the end of incubation period (5 h).

Characterization of microcapsules loaded with L. acidophilus
All the microcapsules revealed spherical shapes, as was displayed in Figure 2. The microcapsules had an average diameter of 102.82 µm being higher than those reported by Holkem et al. (2016) Song et al. (2013), who studied microencapsulation of yeast by internal gelation and found microcapsule size with diameters between 35 and350 mm.Likewise, Wang et al. (2016) reported large size of microcapsules (1.5 mm) loaded with Lactobacillus plantarum, employing SA with or without inulin as inner layer and skim milk as outer layer.It should be noted that the diameters of microcapsules may affect the texture of the food products in which they are applied.For example, diameters about 100 µm are desired for most applications due to a better protection against acidic conditions as those found on the gastrointestinal transit (Arup et al., 2011;Champagne and Fustier, 2007).
The encapsulation efficiency (% EE) found in the present study had a mean value of 94.87%.These results are in agreement with those publised by Holkem et al. (2016) who found % EE values of 89.71% for Bifidobacterium BB-12 microencapsulated by IIG using alginate as a wall material.Likewise, Pitigraisorn et al. (2017) reported % EE values of 95.3% for L. acidophilus microencapsulated on non coated alginate beads.Nevertheless, in the current research, the efficiency was shown to be greater than that found in the studies of Zou et al. (2011) who microencapsulated Bifidobacterium bifidum F-35 obtaining values ranging from 43 to 50% using alginate microcapsules prepared by a similar technique of microencapsulation.

Figure 1 .
Figure 1.The size distributions of the microcapsules based on SA and LAG.

Figure 2 .
Figure 2. The optical micrographs at 10× of the binary microcapsules .