Effect of different coating materials on the biological characteristics and stability of microencapsulated Lactobacillus acidophilus

Abstract

The effect of different coating materials on the biological characteristics and stability of microencapsulated Lactobacillus acidophilus was investigated. Results indicated that the surface and microstructure of microcapsules were significantly affected by the type of coating material. A complex carrier could provide protection for L. acidophilus cells against simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Cell survivals give higher counts with 2.1 and 3.72 logarithmic cycle reduction found in microencapsulated L. acidophilus with complex wall materials and free cells after exposure to SIF for 180 min, respectively. Furthermore, at the high temperatures investigated, a higher cell survival rate in microcapsules embedded with complex materials was found than for free cells and those with other materials. Cell counts were reduced to 8.16, 7.17, and 6.42 log CFU mL⁻¹ and 5.86, 4.29, and 2.32 log CFU mL⁻¹ for microcapsules with complex materials and free cells treated at 50, 60 or 70 °C for 20 min, respectively. Stability was also improved compared to free cells at refrigerated temperatures. For the cells that were released from microcapsules, the counts increased with a prolonged incubation time. Moreover, the survival rate of cells with microencapsulation was better than that of free cells at high concentrations of bile salt. Results showed that for improving protection against deleterious factors, complex materials might be a better choice for the preparation of microcapsules.

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1. Introduction

Lactobacillus acidophilus, as an important probiotic microorganism, could confer beneficial effects on the human gastrointestinal tract, and it has become increasingly popular all over the world in recent years.^1,2 However, the live cell count of probiotics, including L. acidophilus, is required to be at least 10⁶ to 10⁷ CFU g⁻¹ product at the end of storage and at the time of consumption.^2–6 More importantly, high survival numbers of bacteria are not easily observed during processing and application of dairy products.^4–6 On the other hand, a high survival rate of probiotics is critical during processing and for their application in products.⁶ This is because they need to retain higher viable activity during the time of passage through the stomach and intestine, since the viability and activity of probiotics are needed at their site of action.^2,7–9 However, many factors have been reported to influence the viability of L. acidophilus cells, such as the type and concentration of coating materials, hydrogen peroxide production, oxygen toxicity, stability in dried or frozen form, storage temperature, microenvironment pH, bile concentration, and the high temperature of dairy processing.^10–12 Therefore, in order to increase the survival rate of probiotic cells, further research needs to be carried out. In recent years, microencapsulation has been recognized as a useful technology to maintain higher viability and stability of cells during their storage and application in foods.^7–9

Microencapsulation could provide a better barrier and protection for free cells against harsh environmental conditions such as heat treatment, pH and freezing.^2,7,9 It has been used as one important technique and has been investigated for improving cell viability by many researchers.^13–16 As reported by Picot and Lacroix, compared to free cells, microencapsulated B. breve with whey protein as coating material significantly improved the viable count of bacterial cells.⁴ On the other hand, the type of coating material is critical for the preparation of microcapsules and could affect their microbiological characteristics.^14,17–20 Carbohydrates, such as hydrolyzed starches, porous starches and starches/alginate, are the most common carrier materials for probiotic cells.^18,20–24 Porous starch could form strong complexes with alginates, which are stable in the presence of Ca²⁺ chelators and reduce the porosity of the gel.²² Microencapsulation of cardamom oleoresin was achieved by spray-drying using modified starch as wall material.²¹ As reported by Xing et al., porous starch with the optimum concentration mixed with sodium alginate as a carrier can be considered an innovative technology to improve the stability of L. acidophilus.² On the other hand, a protective agent including mannitol, glycerol and sodium alginate in the preparation solution might provide a better function for the survival of cells in microencapsulation.^2,18 Furthermore, the investigation conducted by Mokarram et al. evaluated the influence of alginate coating on the survivability of probiotic bacteria in simulated gastric and intestinal juice.²³ More importantly, the biological characteristics and stability of cells in microencapsulation under different conditions are quite important for their application and were significantly influenced by the type of wall material. However, no papers have been found to report the effect of different coating materials on the biological characteristics and stability of L. acidophilus in microencapsulation.

Therefore, the objective of this study was to understand the effect of different coating materials on the biological characteristics and stability of microencapsulated L. acidophilus. Morphological observation and thermogravimetry/derivative thermogravimetry were conducted. The influence of different coating materials on the survival rate of cells was also evaluated during exposure to artificial gastrointestinal fluid and storage at different temperatures. The release of cells in simulated colonic pH solution was analyzed. Moreover, survival of cells on exposure to bile salt solution was evaluated in order to understand their application properties.

2. Materials and methods

2.1 Materials

L. acidophilus (CICC 6075), used as the active core material, was purchased from the China Center of Industrial Culture Collection (Beijing, China). Porous starch was purchased from Liaoning Lida Bio-Technology Co. Ltd. (Jinzhou, China). Sodium alginate (240 ± 20 mPa s) was purchased from Chengdu Ruifeng Lier Technology Co. Ltd. (Chengdu, China). All the other chemicals used were of analytical grade.

2.2 Preparation of microcapsules

L. acidophilus in a freeze-dried ampoule was activated according to the method reported by Liserre et al. and Xing et al., with some modifications.^2,25 L. acidophilus was activated in chalk litmus milk at 37 °C for 24 h, and the cultures were maintained in a refrigerator (7 ± 1 °C). The culture was reactivated in MRS broth by transferring three times and the cells were harvested by centrifugation (3000g) at 4 °C for 10 min and then washed twice with 0.85% (w/v) NaCl solution. The pellet was resuspended in saline solution to obtain a suspension with a cell concentration of 10⁹ to 10¹⁰ CFU mL⁻¹.

L. acidophilus, as core material, was encapsulated in different coating materials as described by Mandal et al., Liserre et al. and Xing et al., with some modifications.^2,6,25 A total of 50 mL L. acidophilus cell suspension with 10⁹ to 10¹⁰ CFU mL⁻¹ was transferred into a sterilized beaker, to which were added mannitol (10%), glycerol (10%), porous starch (10%) and complex wall material (porous starch (10%) + mannitol (3%) + glycerol (2%)) as wall materials, respectively. Then this solution was shocked by ultrasonic waves for 20 min in order to coat the free cells with wall material. The pH value of this solution was adjusted to 6.0 using NaOH and HCl solutions. After absorption, sodium alginate (1.5%) and Tween 80 (0.2%) were added dropwise into the solution with stirring. The obtained uniform emulsion was treated by adding 0.1 M calcium chloride (100 mL) dropwise for hardening the microcapsules. The microcapsules were harvested by centrifuging at 4 °C at 500g for 10 min and washed twice with distilled water in order to remove the residual liquid of calcium chloride, mannitol and/or glycerol on the surface of the microcapsules. Furthermore, the residual bacteria on the surface were also washed twice with sterilized saline solution (0.85%). The beads were separated by filtration using filter paper and precooled in a refrigerator (−40 °C) for 4 h. Then the obtained material was freeze-dried at −58 °C for 72 h using a freeze-drying machine (Lyolab3000, Heto-Holten Co., Denmark) at 5 mmHg. The frozen state was maintained throughout the freeze-drying procedure. The obtained microcapsules were transferred to a sterile Petri dish and stored in a refrigerator (7 °C).

2.3 Enumeration of L. acidophilus cells

Viable numbers of L. acidophilus were counted on MRS agar according to Grosso and Fávaro-Trindade, Pedroso et al. and Xing et al., with some modifications.^2,26,27 MRS agar was supplemented with lithium chloride (0.1%), L-cysteine (0.05%) and aniline blue (0.01%) for the enumeration of L. acidophilus cells. Serial dilutions were prepared with 2% sodium citrate solution. The plates were incubated in an anaerobic system at 37 °C for 72 h.

2.4 Morphological observation and thermogravimetric analysis

The morphology of microcapsules with different coating materials was observed with an SEM (scanning electron microscope). Microcapsules on a piece of adhesive paper were coated with gold with a vacuum sputtering coater before observing with an SEM (JSM 6390 LV, Jeol, Tokyo, Japan) at an accelerating voltage of 15 kV.^2,7,28

Thermogravimetry/derivative thermogravimetry (TGA/DrTGA) of microcapsules with different coating materials were performed using a TG/DTA-6300 thermobalance (Shimadzu model DTA-6300, Kyoto, Japan).²⁹ Samples were placed in aluminum pans and heated from 30 °C to 350 °C at a rate of 10 °C min⁻¹ under a dynamic synthetic N₂ atmosphere (100 mL min⁻¹). The equipment was preliminarily calibrated with a standard reference of calcium oxalate.

2.5 Resistance to simulated gastric fluid and intestinal fluid

Resistance to simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was determined as described by Gbassi et al. and Xing et al.^2,30 SGF consisted of 9 g L⁻¹ sodium chloride and 3 g L⁻¹ pepsin, and the pH was adjusted to 1.5 with hydrochloric acid. SIF consisted of 9 g L⁻¹ sodium chloride, 10 g L⁻¹ each of pancreatin and trypsin and 3 g L⁻¹ bile salts, and the pH was adjusted to 6.5 with sodium hydroxide. Survey assays were conducted at 37 °C for 60, 120 and 180 min after incubation of free and encapsulated cells in SGF and SIF, respectively.

2.6 Stability of microcapsules under different temperatures

The stability of microencapsulated L. acidophilus under heat treatment (50, 60 or 70 °C for 20 min) and low temperature (4 °C for 14 weeks) was investigated according to the method reported by Mandal et al. and Xing et al.^2,6 A total of 1 mL free cell suspension or one gram of microcapsules (10⁹ to 10¹⁰ CFU mL⁻¹) was transferred into test tubes with 10 mL distilled water. The content was cooled to room temperature (30 °C) after the different temperature treatment and the total number of viable cells was enumerated as described in Section 2.3.

2.7 Release of microencapsulated cells

In vitro release of encapsulated L. acidophilus in simulated colonic pH solution was investigated according to the method described by Rao et al., Mokarram et al. and Mandal et al.^6,23,31 A total of 1 g microcapsules was transferred into 10 mL simulated colonic pH solution (0.1 M KH₂PO₄, pH 7.4 ± 0.2), followed by homogenization with a magnetic stirrer for 10 min. Then the mixed solution was incubated at 37 °C for 3 h. The count of viable probiotic cells was carried out as described in Section 2.3 during the incubation period.

2.8 Survival of microencapsulated cells in bile salt solution

Tolerance to a bile salt concentration simulating the human small intestine was investigated as reported by Lee et al., Mandal et al. and Xing et al.^2,6,32 Similarly to low pH tolerance, 1 g microcapsules or 1 mL free cell suspension (10⁹ to 10¹⁰ CFU mL⁻¹) were transferred into test tubes containing 10 mL of 1% or 2% bile salt and incubated at 37 °C. The viable cells of each bacterium were then enumerated using methods described in Section 2.3.³³

2.9 Statistical analysis

The tests in this investigation were carried out in triplicate and the obtained test data were analyzed using SPSS 13.0 software (SPSS Inc.). The results were expressed as mean ± S.D. The one-way analysis of variance procedure followed by the Student–Newman–Keuls test was used to determine a significant difference (p < 0.05) between treatment methods.

3. Results and discussion

3.1 Morphological characterization

Morphological observation of microencapsulated L. acidophilus prepared with different coating materials was carried out by SEM. Photomicrographs of microcapsules with mannitol, glycerol, porous starch and complex wall materials are shown in Fig. 1A–D, respectively. As can be seen in Fig. 1A and B, the SEM photos reveal that many L. acidophilus cells were found on the surface of the wall materials. These photos indicate that the wall materials mannitol and glycerol could provide a carrier function as protective agents for free cells. However, L. acidophilus cells could not be embedded completely with mannitol and glycerol during the processing period. Furthermore, the presence of mannitol and glycerol could provide better protection as components of complex coating materials for L. acidophilus cells. As shown in Fig. 1C and D, these photos reveal that, although the microparticles prepared with porous starch and complex wall materials containing porous starch were nearly spherical in shape, the walls of the particles were irregular. However, the SEM photos in Fig. 1C also reveal the absence of agglomeration was also observed among the microparticles of porous starch. On the other hand, for the microcapsules prepared with complex materials, these photos also reveal the absence of various sizes of micropores in the surface of modified starches, confirming the formation of microcapsules and protection provided for L. acidophilus cells. Moreover, the modified starches in Fig. 1D dispersed better than those in Fig. 1C, but some broken starch particles were also observed.

Fig. 1 Morphology of microencapsulated L. acidophilus with different coating materials observed by SEM ((A) mannitol (10%), (B) glycerol (10%), (C) porous starch, (D) complex wall material; (a) 500×, (b) 3000×).

These results reveal that the application of these wall materials could sufficiently guarantee the integrity of free cells and provide a sufficient cell number for the application of microencapsulated L. acidophilus. For the preparation of microcapsules, the choice of wall material is very important for their stability and application.^7,20,34 Similar results were observed by Krishnan et al. and Rodríguez-Huezo et al.^21,35 Moreover, investigations about microencapsulated flavors also demonstrated a similar pattern for microcapsules prepared with modified starch as wall materials.^20,21 The investigation of Krishnan et al. showed that microcapsules of cardamom oleoresin which used modified starches as coating material were broken and not complete.²¹ These results might be due to the reason that the microstructure of a biopolymer as a coating material could be influenced by the composition and integrity of wall materials. This might be due to micropores in the modified starch and the protective agents mannitol, glycerol and sodium alginate as carriers, as shown in Fig. 1. The micropores in the starch could act as important protective carriers and mannitol, glycerol and sodium alginate as a protective membrane adhering to the surface of porous starches, as shown in Fig. 1D. This combined action might permit a continued reduction in cell death and improve the stability of cells.^2,18,20 These results might be due to widespread surface growth and cells released from the gel beads, which could lead to a decreasing cell population in the beads, and hence a higher cell release from a microcapsule resulted in a lower cell number inside the beads.³⁶ In the present investigation, there were different cells loaded among different kinds of beads because they had a different type of coating material. In addition to differences in chemical characteristics, the capsule materials also possessed different physical properties.²⁸ Coating materials with more protection could result in lower permeability for external and internal mass transfer and lead to a smaller amount of cells being loaded. The complex materials as the coating showed less permeability for external and internal mass transfer compared to the other biopolymers. On the other hand, interaction between the different coating materials and L. acidophilus could induce a change in the structure and accordingly the physical properties of biopolymers. Porous starch is one kind of modified starch and has been used as a carrier material successfully in microcapsule preparation.^4,37 It is a denatured starch with a suitable pore size on the surface and excellent biocompatibility. The honeycomb structure of porous starch could improve the adhesive property and absorbability of the core materials.^4,37,38 In the process of microencapsulation, mannitol could be used as an anti-caking agent, filler and quality improver and improve the stability and storage properties of particle products.³⁹ Glycerol has gained considerable attention because it can be used as a softener, desiccant, lubricant and plasticizer for the preparation of microcapsules.⁴⁰ Moreover, glucose with a lower diffusion property was also reported in concentrated alginate gels, due to a decreased number and length of pores rather than a decrease in pore diameter.^12,41 Furthermore, alginate could also form a gel when in contact with calcium and multivalent cations. This crosslinked alginate matrix system could cause faster degradation and release of active ingredients.^2,6,32 More importantly, the viscosity of prepared solutions during emulsification could also influence the interaction between different coating materials and the preparation of microcapsules. During stirring in the preparation process, the viscosity of the coating solutions was evaluated by a digital rotational viscometer (NDJ-5S, Shanghai Jingxi Instrument Co., Ltd., Shanghai, China), and was about 3320 mPa s, 2040 mPa s, 12 600 mPa s and 12 080 mPa s for mannitol, glycerol, porous starch and complex wall material as coating materials, respectively. This interaction could provide a better function for protection of the complex carrier for cell loading. This starch complex capsule coating possesses an interphasic membrane and has the possibility to encapsulate L. acidophilus without loss of viability.^2,5,6 SEM photos also indicate the suitability of porous starches compared with other materials as a wall material for encapsulation of L. acidophilus. Furthermore, the stability and other characteristics of microcapsules coated with different types of materials need to be investigated for their extensive application.

3.2 TG analysis of microencapsulated L. acidophilus

Thermogravimetric analysis was conducted in order to determine the thermal stability of complex materials as the carrier in the preparation of microcapsules.²⁹ The weight loss process for many kinds of materials exhibits a relatively wide temperature range and indicates different thermogravimetric properties.^29,42 The thermal behavior of microencapsulated L. acidophilus embedded with different materials can be observed in the TG curves shown in Fig. 2. As shown in Fig. 2a, the thermal behavior of microcapsules with mannitol as coating material shows that two representative stages are found in the thermogravimetric curves. The first mass loss of the TG curves refers to moisture loss (between 41 °C and 85.8 °C). Breakdown of the fructose chains in microcapsules may have occurred between 249 °C and 305.6 °C. For the thermal behavior of microcapsules with glycerol, the result shown in Fig. 2b indicates that three representative stages are found. The first mass loss of the TG curves refers to moisture loss, which occurred at a temperature between 53.3 °C and 92.7 °C. The other two stages also indicate that breakdown of the fructose chains in microcapsules might have occurred between 170.9 °C and 197.7 °C and between 271.3 °C and 307.6 °C. More importantly, the thermal behavior of microcapsules with porous starch and complex wall materials are shown in Fig. 2c and d, respectively. Two representative stages are found in the TG analysis curves. The first mass loss of the TG curves refers to moisture loss, which took place between 50.2 °C and 115.3 °C for the microcapsules prepared with porous starch. For microcapsules coated with complex wall materials, the first stage of mass loss is not observed very clearly in the curves of Fig. 2d. These might refer to two stages of moisture loss, which took place between 50.0 °C and 111.3 °C and between 173.0 °C and 193.9 °C, respectively. Furthermore, breakdown of the fructose chains in microcapsules may have occurred between 223.7 °C and 259.4 °C and between 233.8 °C and 271.8 °C for microcapsules coated with porous starch and complex wall materials, respectively. Above this temperature range, the second stage of mass loss corresponds to a decomposition process.

Fig. 2 TG/DTG analysis of microencapsulated L. acidophilus with different coating materials ((a) mannitol (10%), (b) glycerol (10%), (c) porous starch, (d) complex wall material).

The thermogravimetric analysis in this investigation is one of the criteria needed in the design and manufacture of microcapsules.²⁹ In the results of thermogravimetric analysis, TG curves (red curves) express the relation between the weight reduction rates of the tested samples and the specified heating rates at the tested temperature.^29,43 It can be found from a curve that the weight of the sample decreased most at a certain temperature, which indicates that this temperature might be the decomposition temperature of the sample. DTG curves (blue curves) indicate the functional relationship between the weight change rate with time and the temperature.^29,44 They might indicate that a reaction occurred between different groups of the coating materials with the formation of a new structure within the composite process observed.^29,43,44 The results above indicate that the thermal stability of microcapsules with different coating materials is described as having a limit temperature, which implies that the material could be used with no damage to its usable properties as a carrier. According to Macêdo et al., in this step decomposition reactions can occur among the constituents of the microcapsules, i.e., proteins and carbohydrates.⁴² Moreover, Bohm et al. reported that thermal degradation of inulin has been described as being a consequence of the breakdown of fructose chains and, as was noted in this study, breakdown of the fructose chains in microcapsules with different coating materials might have occurred between 227 °C and 308 °C.⁴³ The analysis results also show that more than one temperature was found at which the decomposition process occurred at the maximum rate for microcapsules obtained from different carriers.²⁹ Moreover, with an increase in the heating rate, a shift of the decomposition process towards higher temperatures was also observed.^29,43,44 Two or three distinct areas of mass loss and two or three maxima on the DTG curves are observed in the course of changes to the TG and DTG curves of microcapsules. This result indicates that this is a complex heterogeneous two-phase or three-stage process.²⁹ During this period, the first stage of thermal decomposition is connected with the degradation of hard segments and the second or third stages might be connected with the decomposition of soft segments.^29,44 It was noted that the course of thermal decomposition in the microcapsule decomposition process depends on the structure and type of the complex materials.^29,44 However, further research needs to be performed in order to understand the fundamental mechanism of the different thermal behavior of microcapsules coated with different materials.

3.3 Resistance to simulated gastric and intestinal fluids

In order to determine the survival rate of microencapsulated L. acidophilus during passage through the stomach, its stability in SGF and SIF needed to be investigated. The effects of different coating materials on resistance to SGF and SIF are shown in Fig. 3a and b, respectively. The initial population found in encapsulated L. acidophilus was about 10⁹ to 10¹⁰ CFU mL⁻¹. As shown in Fig. 3a, after exposure to SGF for 180 min, the survival counts of cells in microcapsules with mannitol, glycerol, porous starch and complex wall material as the carrier remained at 98.76%, 99.12%, 99.26% and 99.72% of the initial population found in encapsulated L. acidophilus, respectively. Moreover, for free cells after exposure to SGF for 180 min, the survival number of L. acidophilus remains with a count of 0.25 logarithmic cycle reduction. The higher survival rate of cells in microparticles indicates that the SGF and exposure time did not significantly affect the survival of Lactobacillus cells. On the other hand, after exposure to SIF for 60 min and 180 min, cell survivals remain with counts of 2.24, 2.38, 2.19, and 1.84 logarithmic cycle reduction and 3.03, 3.09, 2.66, and 2.1 logarithmic cycle reduction from the initial populations found in microencapsulated L. acidophilus with mannitol, glycerol, porous starch and complex wall material, respectively. Cell survivals remain with counts of 2.55 and 3.72 logarithmic cycle reduction for free cells as a control after exposure to SIF for 60 min and 180 min, respectively. The counts of free cells decreased significantly with exposure to pH 1.5 for 180 min. After 1 h of incubation, the survival of microencapsulated cells was significantly lower in the microcapsules with mannitol and glycerol as the carrier compared to porous starch and complex wall material beads, but it was still higher than for free cells. Moreover, the highest viability of cells was observed in the microparticles with complex wall material after 180 min exposure to SIF.

Fig. 3 Effect of pH on viable counts of free and microencapsulated L. acidophilus with different coating materials (log CFU mL⁻¹ for free cells and log CFU g⁻¹ for microencapsulated cells; (a) pH 6.5 and (b) pH 1.5). Mean bars with different letters (a–c) for the same coating material with different incubation times differ significantly (p < 0.05). Mean bars with different letters (p–t) for the same incubation time with different coating materials differ significantly (p < 0.05).

For the application of microencapsulated bacteria cells, one of the major problems is the low survival rate of L. acidophilus at gastric pH in the intestinal system.⁴⁵ Results showed that microencapsulation using a complex material containing porous starch as the carrier provided better protection for L. acidophilus against SGF and SIF. The resistance of microencapsulated L. acidophilus cells differed with the different coating materials. The highest and lowest resistance to simulated gastric fluid were found in the microcapsules prepared with mannitol and glycerol and with complex materials, respectively. However, no significant reduction in viable count was observed in microcapsules with different coating materials, as well as free cells in distilled water (pH 6.5), on incubation for up to 180 min. There was also no significant difference in the viability of free cells in distilled water. In the present study, results showing an increase in resistance to simulated gastric and intestinal fluids were observed compared to the survival rate of free cells.^23,45–47 A large variation in the ability of L. acidophilus to resist acid has been reported by other researchers.^20,33,48 According to the investigation conducted by Corcoran et al., they reported that the presence of glucose could enhance the viability of probiotic Lactobacilli during gastric transit.⁴⁹ As reported by Krasaekoopt et al., microencapsulated cells of L. acidophilus in alginate beads survived better after sequential incubation in simulated gastric and intestinal juices.¹⁷ This was in agreement with Kim et al., who reported that microencapsulated L. acidophilus still remained above 10⁶ CFU mL⁻¹ at pH 1.5 after 2 h, but free L. acidophilus cells were completely destroyed after 1 h.⁴⁷ Chandramouli et al. reported that a higher survival rate of L. acidophilus immobilized in alginate beads was found in low pH environments.⁵⁰ The results reported by Chandramouli et al. and Iyer et al. also indicated that microencapsulation could maintain high viability in gastrointestinal conditions.^{17,18,32,48–53} According to the results reported by Wang et al., the stability of allicin microcapsules against pH improved when porous starch was used as a supplementary wall material.³⁷ In their investigation, porous starch with β-cyclodextrin could act as a cell carrier against many negative factors including high temperature, acid and light; thus cell stability might be improved.^2,54 These results indicated that the protection of L. acidophilus cells against SGF and SIF might be explained by the combined function of porous starch at the appropriate concentration and mannitol, glycerol, or sodium alginate as a protective agent. The added coating afforded better protection to probiotic organisms compared to uncoated microcapsules at the same time points.⁵³ This combined action could provide protection of the cellular integrity of L. acidophilus and improve its stability. Therefore, probiotic cells loaded in microcapsules of starches could pass through gastric transit and be released in the vicinity of their site of action.

3.4 Stability of microcapsules under different temperatures

Loss of activity could occur in microcapsules under heat treatment and cold storage conditions. Therefore, the effects of different coating materials on the stability of L. acidophilus microcapsules during storage at different temperatures are shown in Fig. 4 and 5. As shown in Fig. 4a, free cells in distilled water were drastically reduced to 5.86, 4.29 and 2.32 log CFU mL⁻¹ on heat treatment at 50, 60 or 70 °C for 20 min, respectively. Microencapsulated L. acidophilus with different coating materials exhibited higher survival for a period of up to 20 min of storage at these high temperatures than that of free cells. Cell counts in the microcapsules were drastically reduced from 9.10 log CFU mL⁻¹ (in distilled water) to 7.35, 6.37, and 5.53 log CFU mL⁻¹ for mannitol, 7.73, 6.69 and 5.81 log CFU mL⁻¹ for glycerol, 7.98, 6.94 and 5.85 log CFU mL⁻¹ for porous starch, and 8.16, 7.17 and 6.42 log CFU mL⁻¹ for complex materials on heat treatment at 50, 60 or 70 °C for 20 min, respectively. Microencapsulation with complex wall materials improved the stability of L. acidophilus and exhibited a logarithmic cycle reduction of 2.69 (70 °C), 1.99 (60 °C) and 0.98 log CFU g⁻¹ (50 °C) during the storage period, respectively. Furthermore, the survival rate of Lactobacilli was found to decrease with an increase in the treatment time. On the other hand, Fig. 5 shows the stability of free and encapsulated probiotic bacteria during 12 weeks of storage in a refrigerator at 4 °C. The viability of microencapsulated cells demonstrated different stability between microcapsules with different coating materials in the same storage conditions. After 12 weeks, the survival of L. acidophilus in microcapsule mannitol, glycerol, porous starch and complex materials decreased to 4.12, 4.06, 4.66 and 4.99 log CFU g⁻¹, respectively. However, the number of free cells decreased from 9.08 to 4.12 log CFU g⁻¹ after 12 weeks, and low survival counts were noted at the end of storage. The decrease rate was significantly different between the microcapsules with different materials and was also significantly influenced by the storage time.

Fig. 4 Effect of heat treatments on viable counts of microencapsulated L. acidophilus (log CFU g⁻¹) with different coating materials ((a): free cells, (b): mannitol (10%), (c): glycerol (10%), (d): porous starch, (e): complex wall material). Mean bars with different letters (a–c) at the same temperature for different heating times differ significantly (p < 0.05). Mean bars with different letters (p–r) for same heating time at different temperatures differ significantly (p < 0.05).

Fig. 5 Effect of low temperature on viable counts of free cells and microencapsulated L. acidophilus (log CFU g⁻¹) with different coating materials ((a) free cells; (b) microencapsulated L. acidophilus with different coating materials). Mean bars with different letters (a–g) with the same coating material for different incubation times differ significantly (p < 0.05). Mean bars with different letters (p–r) at the same incubation time for different coating materials differ significantly (p < 0.05).

Results showed that the immobilized cells in the micropores of modified starches as complex materials exhibited the lowest loss of cell viability and good stability at the end of the time under the different temperature conditions. Improving the stability of L. acidophilus cells with complex wall materials during storage could reduce the loss of cells to a medium during their application. As reported by Krasaekoopt et al., it was confirmed that starch mixed with cationic polymers could improve the stability of the microcapsules.¹⁸ They also reported that the stability of cells could increase with a decrease in the treatment temperature and low temperatures could prevent exposure of the active ingredient and promote a longer shelf life of microcapsules. It is well known that heat treatment could influence the survival of lactic acid bacteria.^47,54 This observation on the survival of L. acidophilus at high temperatures agrees with the findings of Jeffery et al. and Kim et al.^47,54 Moreover, L. acidophilus might die quickly during storage at a refrigerated temperature. Therefore, one of the main aims of the present study was to check the viability of microencapsulated L. acidophilus over a period of time under refrigeration. Several investigations also showed that the survival of microencapsulated bacteria was higher than that of free cells during storage time.^18,48 Koo et al. reported that L. bulgaricus loaded in chitosan-coated alginate exhibited higher stability than free cell culture.⁵⁵ Furthermore, the results of Medina and Jordano demonstrated that viable cells of L. acidophilus decreased rapidly during storage time under refrigeration at 7 °C, especially between days 10 and 17.⁵⁶ For microencapsulated products stored under refrigeration, one of the main focuses should be on the minimum viable level which is required for the bacterium to be beneficial to health. The results of this study indicate a great variability after storage at 4 °C for 12 weeks in the survival ability of microencapsulated L. acidophilus with complex wall materials. The inactivation of cells in the microparticles can be related to many factors, such as fatty acid oxidation, DNA damage and the formation of free radicals under high temperature.⁵⁷ The release of cells under heated conditions might be due to the collapse of beads. More importantly, survival mechanisms exhibited by bacteria are generally referred to as adaptive stress response.⁵⁸ The higher viability of microencapsulated cells under heat treatment could be explained by this theory. However, probiotic cells were injured and killed by an increase in osmotic pressure.⁵⁸ On the other hand, the variability is also highly dependent on the kinds of capsule materials. The complex wall material including porous starch is a substance that is accumulated within the cells to reduce the osmotic difference from the external environment or a substance that surrounds cells to improve their heat and cold tolerance.⁵⁹ The wall materials mannitol and glycerol might not be able to protect cells from injury completely, which could induce a reduction in probiotic viability. It is interesting to note that mixtures of porous starch, mannitol and glycerol as capsule materials provided the best viability and led to better viability than the individual wall materials. Therefore, our results suggest that the complex materials containing porous starches used as a wall material had a positive effect on the protection of L. acidophilus during heating and refrigeration periods. For this reason, changes in the population of viable bacteria during the expected shelf life of a product should be known to some extent and taken as a basis for the selection of a coating material.

3.5 Release of microencapsulated cells in simulated colonic pH solution

An in vitro system was utilized in order to determine the effect of the acidic pH of the stomach on the survival of encapsulated probiotics. The release of cells from microencapsulation in simulated colonic pH solution at 37 °C was investigated and the results are shown in Fig. 6. The released cell counts were between 8.34 and 8.76 log CFU g⁻¹ at the end of the storage period upon immediate exposure to a solution with simulated colonic pH. With an increase in incubation time, the release of cells increased. After 1.0 h and 2.0 h in the medium, the cell counts released from microencapsulation with mannitol, glycerol, porous starch and complex materials as wall material were 7.32, 6.91, 6.74, and 6.46 log CFU g⁻¹ and 8.76, 8.63, 8.53, and 8.34 log CFU g⁻¹, respectively. The results also indicate that the total counts of cells released from microcapsules prepared with different coating materials increased significantly before the end of the first 1.5 h. During this period, the type of wall material affected the released cell counts. However, after an exposure time of 1.5 h, no significant change in the count of released cells was observed. As shown in Fig. 6, free L. acidophilus cells were sensitive to an acidic environment and ingestion of unprotected cells might result in a reduced viability of 5 log reduction after 2 h.

Fig. 6 Effect of different coating materials on the release characteristics of microencapsulated L. acidophilus (log CFU g⁻¹). Mean bars with different letters (a–e) for the same coating materials at different incubation times differ significantly (p < 0.05). Mean bars with different letters (p–s) at the same incubation time for different coating materials differ significantly (p < 0.05).

Release of L. acidophilus cells from microcapsules is essential for the growth and colonization of probiotics in the vicinity of the site of action.⁶ The effect of different coating materials on the release of L. acidophilus needed to be investigated.⁶⁰ This study was conducted to compare the release performance of probiotic Lactobacillus from microcapsules with different coating materials in simulated colonic pH solution during storage at 37 °C. The above result also indicates that the type and structure of the coating materials also affected the counts of cells released from microcapsules in the first period of 1.5 h. However, there was no significant change in the count of released cells between microcapsules with different coating materials at the end of the exposure time. This demonstrates that no effect of different coating materials on cell release was observed after exposure to the medium solution for 2.5 h. This action could promote longer shelf life of the microcapsules and provide a better release performance of active cells in the microcapsules. The result obtained in this investigation was consistent with that of Rao et al., who reported that microencapsulated B. pseudolongum resisted simulated gastric and intestinal juices for up to 60 min.³¹ This implies that different coating materials could control the release of cells from microcapsules during the first period after inoculation. These results also indicate that release of cells from the product will depend on many factors such as pH, the presence of preservatives and the structure of different coating materials.^2,61 Zhang et al. have shown that the survival ability of L. acidophilus was significantly affected when subjected to low pH.⁶² This indicated that acid injury was responsible for the inhibitory effect, as reported by Vinderola et al. on the survival of B. bifidum and L. acidophilus during refrigerated storage at 5 °C in milk.⁶⁰ Their results also proved that product acidity has a major impact on microbial viability during its shelf life.⁶⁰ In order to provide successful candidates for functional food applications, the release characteristic of active cells in the intestine is one of the aims of microencapsulation.^4,60,61 Picot and Lacroix also reported progressive release of viable cells from whey protein-based microcapsules in simulated intestinal conditions.⁴ This indicates that a coating of complex beads provided the best protection in simulated gastric juice because a protected internal space forms in the double-layer membrane and, as a result, diffusion of gastric juice into the beads may be limited.^6,23,31 This microstructure will protect cells from interacting with the gastric juice.⁶³ Microencapsulated cells survived better than did free cells after sequential incubation in simulated gastric and intestinal juices, and the complex coating enhanced the survivability of cells more than other coating materials. Porous starch with a honeycomb structure can provide stable release characteristics of cells from the network formed from porous starch and sodium alginate.³⁷ Porous capsules suitable for immobilizing L. acidophilus cells were coated with sodium alginate, which served both to position the microorganisms in the capsule pores and to form spaces for cell release.^37,38,64 These results allow us to conclude that microcapsules prepared with complex materials as the wall material could provide satisfactory release properties of L. acidophilus cells.

3.6 Survival rate of microencapsulated cells in bile salt solutions

The effect of different coating materials on the survival rate of L. acidophilus in microcapsules exposed to solutions of 1% and 2% bile salt was investigated. As the results show in Fig. 7a, free cells decreased from 9.10 to 6.59 log CFU mL⁻¹ and from 9.13 to 4.85 log CFU mL⁻¹ after exposure to 1% and 2% bile salt solutions for 12 h, respectively. However, according to the results shown in Fig. 7b–e, the survival rate of cells in microcapsules with different coating materials improved first and then the viability of cells was reduced at similar bile salt concentrations. Microencapsulated cells embedded with mannitol decreased from 9.1 to 7.63 log CFU g⁻¹ and from 9.14 to 6.60 log CFU g⁻¹, and with glycerol from 9.10 to 7.85 log CFU g⁻¹ and from 9.12 to 7.10 log CFU g⁻¹, on exposure to 1% and 2% bile salt after 12 h, respectively. However, after exposure to 2% bile salt for 12 h, cells from microcapsules with porous starch and complex material decreased from 9.14 to 6.95 log CFU g⁻¹ and from 9.12 to 7.10 log CFU g⁻¹, respectively. This indicates that the viability of L. acidophilus decreased proportionately with concentration and time of exposure to bile salt and that the highest survival of cells among these microparticles was obtained by encapsulation in the complex wall material containing porous starch.

Fig. 7 Effect of bile salt on viable counts of microencapsulated L. acidophilus (log CFU g⁻¹) with different coating materials ((a): free cells, (b): mannitol (10%), (c): glycerol (10%), (d): porous starch, (e): complex wall material). Mean bars with different letters (a–c) in the same bile salt concentration at different incubation times differ significantly (p < 0.05). Mean bars with different letters (p–r) at the same incubation time in different bile salt concentrations differ significantly (p < 0.05).

The survival of microencapsulated L. acidophilus was better at high bile salt concentrations than that of free cells. The obtained results indicate that the protection of L. acidophilus cells against the bile salt solution might be explained by the combined function of porous starch and sodium alginate in the preparation solution. According to the investigation conducted by Trindade and Grosso, immobilization of L. acidophilus in alginate beads was not effective in protecting the cells from 2% and 4% bile salt.⁶⁵ Chandramouli et al. reported that encapsulation of L. acidophilus in alginate significantly increased the viability in 1% bile salt.⁵⁰ As reported by Murata et al., a chitosan coating provides the best protection in bile salt solution because an ion exchange reaction takes place when the beads absorb bile salt.⁶³ This is consistent with the results of Koo et al., who reported that L. casei entrapped in alginate beads containing chitosan had higher viability than in alginate without chitosan.⁵⁵ Bile salt solutions were always used to determine whether coating materials would increase the survival of cells in this environment. This is because bile tolerance is often used as a criterion for selection of probiotic strains, which are similar to those of the digestive system. As reported by Sultana et al., microencapsulated L. acidophilus decreased by two log cycles compared to the initial cell count in 1% and 2% bile salt solutions.⁵² Moreover, the survival rate decreased proportionately with the time exposed to bile salt solutions.³² The combined action could provide protection of the cellular integrity of L. acidophilus and improve its stability. This might be due to the shell and net structures that were formed from porous starch and sodium alginate, which could act as a physical and permeable barrier to negative factors.^2,37,38,64 A similar result was also reported by Corcoran et al.; in their investigation, the presence of glucose could also enhance the viability of probiotic Lactobacilli during gastric transit.⁴⁹ The protection afforded was dependent to some extent on the type and chemical characteristics of the media.^2,6,66 The polysaccharide-containing matrix with micropores provided more protection to the probiotic when porous starch was used in combination with sodium alginate for the preparation of microencapsulated L. acidophilus. This feature might be due to interaction with the role of porous starch in protecting probiotics, which could provide enough space and maintain the integrity of cell membranes.^66,67 Therefore, our results suggest that the complex material containing porous starches and alginate beads used as the wall material has a positive effect on the resistance of L. acidophilus to bile salt solution.

4. Conclusions

New methods to produce applicable coating materials appear an important task. This investigation was conducted about the effect of different coating materials on the biological characteristics and stability of microencapsulated L. acidophilus. In fact, the type of coating material significantly affected the surface and microstructure of microcapsules. Complex materials as a coating could provide better protection for probiotic cells against passage through gastric and intestinal fluids. They provided a higher survival rate of L. acidophilus among the different coating materials at the different temperatures evaluated. With an increase in incubation time, the release of cells increased, and there was no significant change that could indicate an effect of different coating materials on release of the cells from microcapsules. The viability and stability of cells in the microcapsules at refrigerated storage temperatures were also improved. These results demonstrate that a complex wall material containing porous starch might be the best choice for the preparation of microencapsulated L. acidophilus in order to increase protection against environmental deleterious factors.

Acknowledgements

This study was supported by Chunhui Program Research Project from Ministry of Education of China (Z2010104 and Z2011094), which was also financially supported by the Open Research Fund of Key Laboratory of Food Biotechnology, Xihua University (SZJJ2012-005), the Key Project Postgraduate Innovation Fund of Xihua University (ycjj201346), the Key Research Foundation Program of Xihua University (Z1120539) and Xihua University Young Scholars Training Program (01201413).

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