Activity of encapsulated Lactobacillus bulgaricus in alginate-whey protein microspheres

Abstract

In this work, alginate-whey protein was used as wall materials for encapsulating Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The characteristics of encapsulated and free L. bulgaricus showed that the free L. bulgaricus lost viability after 1 min exposure to simulated gastric fluid (SGF) at pH 2.0 and 2.5. However, the viability of encapsulated L. bulgaricus did not decrease in SGF at pH 2.5 for 2 h incubation. The viable numbers of encapsulated L. bulgaricus decreased less than 1.0 log unit for 2 h incubation in SGF at pH 2.0. For bile stability, only 1.2 log units and 2.0 log units viability of the encapsulated L. bulgaricus was lost in 1 and 2% bile for 1 h exposure, respectively, compared with no survival of free L. bulgaricus under the same conditions. Encapsulated L. bulgaricus was completely released from the microspheres in simulated intestinal fluid (SIF, pH 6.8) in 3 h. The viability of the encapsulated L. bulgaricus retained more 8.0 log CFU/g after stored at 4°C for four weeks. However, for free L. bulgaricus, only around 3.0 log CFU/mL was found at the same storage conditions. Results showed that the encapsulation could improve the stability of L. bulgaricus.

Encapsulation; Alginate; Whey protein; Probiotics; Stability

INTRODUCTION

Several studies have shown that the probiotics play an important role in human health; they can prevent diarrhea, balance the intestinal microflora, stimulate of the immune system, have anticarcinogenic properties and lactose intolerance (Shah 2007). FAO has recommended that the least viability of probiotics should be at 10⁷ CFU/g of product at the time of consumption. The biggest problem in probiotics application is its sensitivity to environment conditions (Carvalho et al. 2004). Hence, protecting the viability of probiotics is one of the premises of its application.

Encapsulation is one of most effective methods that improve the viability and stability of probiotic bacteria (Gbassi et al. 2009; Heidebach et al. 2010; Dolly et al. 2011). Alginate is by far the most widely used wall material for encapsulation. However, encapsulation of probiotic bacteria in alginate microspheres was not able to protect the organisms effectively from high acidic environment (Sultana et al. 2000; Krasaekoopt et al. 2003; Dong et al. 2013; Pan et al. 2013; Shi et al. 2013 a, b). Whey protein or denatured whey protein is another extensively used as wall material for the encapsulation of probiotics. Denatured whey protein can be obtained through heating whey protein solution (Anandharamakrishnan et al. 2007; Tang et al. 2013). The application of polysaccharide-protein matrix for probiotics encapsulation is a relatively new strategy and can be seen as a promising alternative approach developed for probiotic encapsulation. In this study, extrusion technique was employed for the encapsulation of L. bulgaricus with wall material combinations of denatured whey protein and sodium alginate. Characteristics of encapsulated L. bulgaricus were investigated and compared with those of free L. bulgaricus.

MATERIAL AND METHODS

Cell culture Preparation

Lactobacillus bulgaricus was provided by Hangzhou Wahaha Group Co Ltd. (ZhejiangChina) as a 25% glycerol stock in MRS broth (Difco, Michigan, USA) at -80°C. All the glass-wares used in this study were sterilized at 121°C for 15 min. The culture was activated in MRS broth at 37°C and propagated two times prior to use in the experimental trials. The fresh culture was prepared by adding 1% inoculum to MRS broth and incubated at 37°C for overnight. The cells were harvested by centrifugation at 3,000 g at 4°C for 10 min. After the removal of supernatant, the cells were washed with sterile 0.85% (w/v) sodium chloride solution.

Encapsulation of L. bulgaricus

Whey protein powder was mixed with sterile distilled water. The solution was stirred gently using a magnetic stirrer to dissolve the whey protein prior to heat treatment. In order to denature the whey protein, the solution was kept at 80°C for 30 min in a water bath (Tang et al. 2013). Alginate-whey protein microspheres were made using an Inotech Encapsulator IER-50 (Inotech Biosystems Intl. Inc.) by extruding a mixture containing L. bulgaricus suspended in sodium alginate solution and de-natured whey protein into 0.1 M CaCl₂ solution while gently stirring with a magnetic bar. The microspheres were prepared using a 450 µm nozzle. The resulting microspheres were allowed to harden in the CaCl₂ solution for 30 min, rinsed with distilled water, filtered, and sealed in sterilized conical tubes for storage at 4°C.

Survival assay and numeration of encapsulated and free L. bulgaricus

pH stability: Microspheres or free L. bulgaricus were added to test tubes containing SGF (0.2%, w/v, NaCl at pH 2.5 or pH 2.0) and incubated at 37°C for 0, 10, 30, 60, 90 and 120 min. At pre-determined time, wet microspheres were removed from the SGF, washed with saline solution, dispersed and broken down in SIF (50 mm KH₂PO₄) pH 6.8. The survival of encapsulated or free L. bulgaricus was immediately assayed (Pan et al. 2013; Shi et al. 2013 a, b).

Bile stability: Microspheres or free L. bulgaricus suspension were added to test tubes containing simulated bile (1 or 2%, w/v) porcine bile extract (Sigma-Aldrich) and incubated at 37°C for 1 and 2 h. After pre-determined time, the beads were washed with saline solution. Microspheres were dissolved in SIF (pH 6.8). The viability of encapsulated or free L. bulgaricus was immediately assayed as above.

Release profile: Microsphere samples were added to SIF solution and incubated at 37°C with shaking at 100 rpm/min. At predetermined time points, 100 μL of this solution was taken out and immediately assayed for the activity. The same volume of the fresh medium was added to replace the volume of the withdrawn samples. The cumulative amount of released L. bulgaricus was plotted against the time (Pan et al. 2013; Shi et al. 2013 a, b).

Storage stability: Microspheres or free L. bulgaricus were stored at 4°C. At pre-determined time points, samples were taken out for activity measurement. Total viable counts of L. bulgaricus were determined by a pour plate method using MRS agar after serial 10-fold dilutions in saline solution. Plates were incubated at 37°C for 48 h (Pan et al. 2013; Shi et al. 2013 a, b).

Statistical analysis

All the experiments were repeated at least three times and the results were presented in mean value ± standard deviation (SD). Statistical analysis was performed using Original 8.0 for Windows. Student's t test was used to compare the significance of differences. The differences were considered significant at the level of P < 0.05.

RESULTS AND DISCUSSIONS

Preparation of encapsulated L. bulgaricus microspheres

One of necessary pre-requisite for encapsulation method is high encapsulation yield. The initial number of viable L. bulgaricus in aqueous suspension used to prepare the microspheres was about 10.01 log CFU/mL. After encapsulation, the number of viable cells recovered in pH 6.8 SIF was 9.98 log CFU/g. The encapsulation yield of L. bulgaricus in alginate-whey protein microspheres was close to 100%. It indicated that this encapsulation process led to a high encapsulation yield. High encapsulation efficiency of 96% was also reported for the encapsulation of probiotic yeasts in alginate-whey protein microspheres (Hébrard et al. 2010). Therefore, it appeared that alginate-whey protein matrices had a good compatibility with functional biological substances, including L. bulgaricus.

The size of alginate-based microspheres can be affected by various factors such as nozzle size, polymer concentration, hardening time in calcium chloride, initial cell concentration, etc (Chandramouli et al. 2004; Dong et al. 2013; Shi et al. 2013 a, b). In this work, the, nozzle size was 450 μm. Alginate-whey protein microspheres with diameter around 800 μm and a spherical shape were obtained (results not shown). In the case of encapsulation for food application, the size of the microspheres is an important factor. Large microspheres can adversely affect the texture of food after supplementation by causing coarseness; small microspheres may not give sufficient protection. Alginate-based microspheres often with between 1.0 to 3.0 mm are obtained, which are too large for many food applications (Sheu and Marshall 1993; Hansen et al. 2002; Muthukumarasamy et al. 2006; Dong et al. 2013; Shi et al. 2013 a, b). In the present study, alginate-whey protein microspheres were below 1.0 mm. It might not produce enough detrimental effect on the texture of food. Smaller microspheres could be obtained when smaller nozzle size was used.

Stability of encapsulated L. bulgaricus in SGF

One of major problems in efficacy of probiotic food is low survival of the cells in the gastric pH. Encapsulated and free L. bulgaricus were tested for the survival under simulated gastric pH of 2.0 and 2.5 (Fig. 1). It was found that the encapsulation could improve the pH tolerance of L. bulgaricus. The numbers of encapsulated cells did not decrease in SGF pH at 2.5 for 2 h incubation. The viable count of the encapsulated L. bulgaricus decreased less than 1.0 log unit for 2 h incubation in pH 2.0 SGF. However, free cells of L. bulgaricus were extremely sensitive to acidic environments and were not detectable after 1 min exposure to SGF at pH 2.5 and 2.0 (results not shown). These results agreed with several earlier reports. Chandramouli et al. (2004) found that the viable numbers of L. acidophilus encapsulated in alginate microspheres was significant increased in SGF at pH 2.0. Encapsulated L. acidophilus in alginate microspheres survived better after sequential incubation in simulated gastric and intestinal juices (Krasaekoopt et al. 2004). Higher survival was also reported when the encapsulated lactobacilli in alginate microspheres were incubated in SGF (Lee et al. 2004).

Figure 1
Survival of encapsulated Lactobacillus bulgaricus in pH 2.0 SGF and pH 2.5 SGF.

Stability of encapsulated L. bulgaricus in bile salt stability

The results of free and encapsulated L. bulgaricus subjected to different concentrations of bile salts (1 and 2%) are shown in Table 1. It indicated a similar trend for viability of encapsulated and free L. bulgaricus in bile salt. From the initial count of 10.01 log CFU/mL, no survival was found in 1 and 2% bile salt after 1 h exposure. The decrease rate was lower for the encapsulated L. bulgaricus.

Table 1
Survival of free and encapsulated L. bulgaricus after treatment in simulated bile concentrations of 1 and 2% for 1 h and 2 h (Log 10 CFU/g microspheres).

Condition/time	Bile concentration (%)
	0	1	2
Free L. bulgaricus(1 h)	10.01±0.13	0	0
Encapsulated L. bulgaricus(1 h)	9.98±0.12	8.78±0.03	7.98±0.08
Free L. bulgaricus(2 h)	10.01±0.08	0	0
Encapsulated L. bulgaricus(2 h)	9.96±0.14	7.69±0.07	7.03±0.15

• Values shown are means ±standard deviations (n =3). Means in the table differ significantly (P < 0.05).

Table 1 illustrated around 2.0 log and 3.0 log reduction of L. bulgaricus after 2 h incubation in 1 and 2% bile salt concentrations, respectively. The numbers declined more as the bile concentration and incubation time increased. The sensitivity of many probiotics strains to bile concentrations encountered in the human gastro-intestinal tract has been reported by several authors (Chung et al. 1999; Amor et al. 2002; Picot & Lacroix 2004). Clark and Martin (1994) observed 5.0 log reductions in viable cell counts of B. adolescentis in 2% bile solution after 12 h incubation at 37°C. Amor et al. (2002) found that the viability of B. lactis and B. adolescentis was more susceptible to the damaging effects of bile salts. In the present study, higher survival rate of the cells in alginate-whey protein microspheres after bile solution treatment was pre-requisite for providing the host with a beneficial health effect.

Release of encapsulated L. bulgaricus in SIF

The release of the cells from microcapsules in colon is essential for the growth and colonization of probiotics. Figure 2 illustrates the release of cells of the encapsulated L. bulgaricus in pH 6.8 SIF. When encapsulated L. bulgaricus was placed into SIF at pH 6.8, the microspheres began to swell and eventually disintegrated. For alginate-whey protein microspheres, the cells of L. bulgaricus released within 3 h and 70% of cell had already been released within 1 h. Gunsaekaran et al. (2007) encapsulated caffeine using whey protein hydrogel and observed similar release profiles, in which a burst release of caffeine happened within 1 h at pH 7.5. At pH 6.8, whey proteins are negatively charged, which may interact with the carboxyl group from the alginate molecules. This electrostatic repulsion also favors the dissociation of the gel network. It is well known that alginate gel formed through calcium ions is very rigid and only swells slowly at neutral pH. Mandal et al. (2006) found that the release of cells progressed with increased incubation. A progressive release of viable cells from whey protein-based microcapsules in SIF was also reported by Picot and Lacroix (2004).

Figure 2
Release profile of encapsulated Lactobacillus bulgaricus in pH 6.8 SIF.

Storage stability of encapsulated L. bulgaricus

The storage stability of free and encapsulated L. bulgaricus during four weeks at 4°C is shown in Figure 3. The viability of the encapsulated L. bulgaricus was higher compared to free L. bulgaricus. The viability of free L. bulgaricus was from 10.01 log CFU/mL to 9.93 log CFU/mL after one week, and around 3.0 log CFU/mL after four weeks. However, the number of encapsulated L. bulgaricus cells only decreased from 9.97 log CFU/mL to 8.37 after four weeks. The viability of encapsulated L. bulgaricus cells was fully preserved in one week. Therefore, encapsulation of L. bulgaricus in alginate-whey protein microspheres could improve the storage stability of free L. bulgaricus cells. Several studies that encapsulated bacteria in alginate-based microspheres reported better survival ability than non-encapsulated bacteria during the storage period (Truelstrup Hansen et al. 2002; Krasaekoopt et al. 2003; Pan et al. 2013; Shi et al. 2013 a, b).

Figure 3
Storage stability of free and encapsulated Lactobacillus bulgaricus at 4°C.

CONCLUSIONS

Alginate-whey protein microspheres encapsulating model L. bulgaricus were produced by extrusion method. Encapsulated microspheres had better survival ability after bile salt concentrations, and at low pH, long storage time as compared to free cells. Encapsulated L. bulgaricus was released from the microspheres within a reasonable time so as to exert their beneficial effects. These findings demonstrated alginate-whey protein microspheres were effective wall carriers for probiotics encapsulation.

ACKNOWLEDGEMENTS

This study was financially supported by the Xinmiao Talent Program of Zhejiang Province (2013R421006).

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Publication Dates

• Publication in this collection
  Sep-Oct 2014

History

• Received
  09 Sept 2013
• Accepted
  13 Jan 2014