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Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal conditions
International Journal of Food Microbiology 142 (2010) 185–189
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal conditions María Chávarri a,⁎, Izaskun Marañón a, Raquel Ares a, Francisco C. Ibáñez b, Florencio Marzo b, María del Carmen Villarán a a b
Food Area, Fundación LEIA Technological Development Centre, Parque Tecnológico de Álava, c/Leonardo Da Vinci n 11, 01510 Miñano, Spain Laboratory of Animal Physiology and Nutrition, School of Agronomy, Public University of Navarra, Campus Arrosadia, 31006, Pamplona, Spain
a r t i c l e
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Article history: Received 25 November 2009 Received in revised form 13 May 2010 Accepted 22 June 2010 Keywords: Lactobacillus gasseri Bifidobacterium bifidum Quercetin Microencapsulation Gastro-intestinal conditions
a b s t r a c t Chitosan was used as a coating material to improve encapsulation of a probiotic and prebiotic in calcium alginate beads. Chitosan-coated alginate microspheres were produced to encapsulate Lactobacillus gasseri (L) and Bifidobacterium bifidum (B) as probiotics and the prebiotic quercetin (Q) with the objective of enhancing survival of the probiotic bacteria and keeping intact the prebiotic during exposure to the adverse conditions of the gastro-intestinal tract. The encapsulation yield for viable cells for chitosan-coated alginate microspheres with quercetin (L + Q and B + Q) was very low. These results, together with the study about the survival of microspheres with quercetin during storage at 4 °C, demonstrated that probiotic bacteria microencapsulated with quercetin did not survive. Owing to this, quercetin and L. gasseri or B. bifidum were microencapsulated separately. Microencapsulated L. gasseri and microencapsulated B. bifidum were resistant to simulated gastric conditions (pH 2.0, 2 h) and bile solution (3%, 2 h), resulting in significantly (p b 0.05) improved survival when compared with free bacteria. This work showed that the microencapsulation of L. gasseri and B. bifidum with alginate and a chitosan coating offers an effective means of delivery of viable bacterial cells to the colon and maintaining their survival during simulated gastric and intestinal juice. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In order to provide health benefits for probiotic bacteria (ColbèreGarapin et al., 2007; de Vrese and Marteau, 2007; Orrhage and Nord, 2000; Reid et al., 2003; Shah, 2007; Vaughan et al., 2005) it has been recommended that they must be present at a minimum level of 106 CFU/g of food product (Doleyres and Lacroix, 2005) or 107 CFU/g at point of delivery (Lee and Salminen, 1995) or be eaten in sufficient amounts to yield a daily intake of 108 CFU (Lopez-Rubio et al., 2006). Several studies have shown that certain strain of lactic acid bacteria, such as Bifidobacterium bifidum (You et al., 2004) and Lactobacillus gasseri (Carroll et al., 2007), prevent some diseases linked to the gastro-intestinal tract. The prebiotics, nondigestible food ingredients, affects the host by selectively stimulating the growth, activity, or both of one or a limited number of bacterial species already resident in the colon (Gibson et al., 2004; McCartney and Gibson, 2006). Quercetin is the most abundant flavonoid in the diet of humans (Boyer et al., 2004) and it is known that it has nutraceutic potential to reduce the levels of cholesterolemia. The consumption of prebiotic quercetin is beneficial for controlling serum cholesterol (Ishikawa et al., 1997) and has antioxidant, anticarcinogenic,
⁎ Corresponding author. Tel.: + 34 945 298144; fax: + 34 945 298217. E-mail address: [email protected] (M. Chávarri). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.022
anti-inflammatory, and cardioprotective properties (Erlund, 2004; Lien et al., 1999; Middleton et al., 2000; Scarfato et al., 2008). The benefit of prebiotics in combination with probiotics has given rise to the concept of symbiotics (Gibson and Roberfroid, 1995; Reid et al., 2003). The addition of quercetin and modulating packaging conditions has improved the microecological balance of the gut microflora (Gibson et al., 2004). However, a high percentage of ingested bifidobacteria lose their viability during delivery through the gastro-intestinal tract (Marteau et al., 1997). Providing probiotic living cells with a physical barrier against adverse environmental conditions is an approach currently receiving considerable interest (Doleyres and Lacroix, 2005; Kailasapathy, 2002; Krasaekoopt et al., 2003). Microencapsulation technologies are hypothesized to be a promising prospect for introducing viable probiotic bacteria in foods because the encapsulation matrix can provide a physical barrier against harsh environmental conditions such as freezing and those encountered during gastric juice passage (Boh, 2007; Capela et al., 2006; Champagne and Kailasapathy, 2008; Kailasapathy, 2002; Shah, 2000). Although studies have used cellulose acetate phthalate (Rao et al., 1989) gelatin, vegetable gum (Shah, 2000), fats (Siuta-Cruce and Goulet, 2001) or κ — carrageenan (Adhikari et al., 2000; Krasaekoopt et al., 2003) as encapsulating agents, alginate remains the most commonly used bio polymer for microencapsulation. The advantages of using alginate as an encapsulating agent include: non-toxicity, formation of
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gentle matrices with calcium chloride to trap sensitive materials such as living microbial cells, simplicity in entrapping living microbial cells and low cost (Krasaekoopt et al., 2003; Sheu and Marshall, 1993). Alginate is also an accepted food additive and can be safely used in foods (Dinakar and Mistry, 1994; Kim et al., 1996; Sheu and Marshall, 1993). The use of alginate is limited due to its low stability in the presence of chelating agents and in acidic conditions below pH 2.0 (Adhikari et al., 2000; Ding and Shah, 2007; Gombotz and Wee, 1998; Hussein and Kebary, 1999; Sultana et al., 2000). The coating of alginate beads and its effectiveness in protecting probiotic bacteria has been extensively studied. Previous researchers have reported that coating alginate microcapsules with chitosan had improved the stability of the alginate beads and thus improved the viability of the encapsulated probiotic organisms (Krasaekoopt et al., 2004). It was suggested that degradation of chitosan occurs by the microflora that are available in the colon and solubilizing alginate gel by sequestering calcium ions (Hejazi and Amiji, 2003). Furthermore, having in mind bio/mucoadhesive properties of natural biopolymers, chitosan-alginate microparticulated systems should have potential for colon targeting (Hejazi and Amiji, 2003; Senel and McClure, 2004; Simonoska et al., 2008). The aim of the study was to enhance alginate microencapsulation by chitosan-coating and to assess their ability to improve the survival of B. bifidum and L. gasseri, in combination with quercetin, during exposure to simulated conditions of the gastro-intestinal tract. 2. Materials and methods 2.1. Bacterial strain and culture condition L. gasseri and B. bifidum were purchased in lyophilized form from the Spanish Type Culture Collection (CECT). Bacteria were routinely grown in MRS broth. Freeze-dried cells were rehydrated in 5 mL MRS broth and incubated, for L. gasseri at 37 °C for 24 h under aerobic conditions and for B. bifidum at 39 °C for 72 h under anaerobic conditions using the Gas Pak Plus system. The cultures were transferred into MRS broth and incubated under the same conditions as before to obtain a cell density of about 1010 CFU/mL. Harvesting of cells was done by centrifugation at 1500 ×g for 5 min at low temperature (4 °C) and the cell pellet was washed twice with sterile saline solution. The cell suspension of each probiotic bacterium was divided in four parts: one part was used for microencapsulation of L. gasseri and B. bifidum (L+ B) together, the second part was used for microencapsulation of L. gasseri and quercetin (L+ Q) together and B. bifidum and quercetin (B+ Q) together, the third part was used for microencapsulation of L. gasseri (L) and B. bifidum (B) and the fourth was used as free cells. Fresh cell suspensions were prepared for each experiment and numerated by pour plating in MRS agar. Plates were incubated under the same conditions as before. 2.2. Microencapsulation and coating procedures Probiotic organisms and quercetin were incorporated into 10 mL of 20 g/L of sodium alginate. Chitosan aqueous solution was prepared. In brief, chitosan was dissolved in 100 mL distilled water acidified with glacial acetic acid to achieve a final chitosan concentration of 0.4% (w/v). This solution was filtered through a nylon cloth to remove any remaining insoluble material. CaCl2 0.1 M was added to the chitosan solution (Zhou et al., 1998). The extrusion technique of microencapsulation was derived from Krasaekoopt et al. (2004) by using alginate as the supporting matrix. To form beads, the sodium alginate solution was extruded into a previous sterile chitosan solution and stirred. The beads were sieved off from the chitosan solution and washed with sterile distilled water.
The capsulates were suspended in cryoprotectant agent and then frozen at −20 °C. The frozen samples were desiccated under vacuum, at a condenser temperature of −40 °C for about 18 h with a freeze-drier (Freezedryer Lyobeta25). Dried cells were stored in closed containers at 4 °C, under darkness. 2.3. Survival assay and numeration of microencapsulated bacteria Entrapped bacteria in uncoated alginate microspheres were released by homogenizing 0.1 g of filtered microsphere slurry in 10 mL of sodium citrate 0.1 M for 10 min and stirred. The homogenized samples were diluted to appropriate concentrations and pour plated in MRS agar. The plates were incubated for 2 days at 37 °C and the encapsulated bacteria enumerated as CFU/mL. The encapsulation yield (EY), which is a combined measurement of the efficacy of entrapment and survival of viable cells during the microencapsulation procedure, was calculated as: EY = ðN = NoÞ × 100: Where N is the number of viable entrapped cells released from the microspheres, and No is the number of free cells added to the biopolymer mix during the production of the microspheres. The particles sizes and formation of microspheres were measured with a light microscope (Axioscop40, Carl Zeiss). The data analysis was performed using the software Ellix 5.0 (Microvision Instruments, France). The mean diameters of microspheres were calculated from 100 beads. Simulated gastric juice (SGJ) consisted of 9 g/L of sodium chloride containing 3.0 g/L of pepsin with pH adjusted to 2.0 with hydrochloric acid (Altman, 1961). 0.2 g of microspheres with entrapped bacteria (B or L) or 0.2 mL of cell suspensions of B or L were mixed in 10 mL of SGJ and incubated for 5, 30, 60 and 120 min at 37 °C with constant agitation at 50 rpm. Simulated intestinal juice (SIJ) was prepared by dissolving bile salts in intestinal solution (6.5 g/L NaCl, 0.835 g/L KCl, 0.22 g/L CaCl2 and 1.386 g/L NaHCO3) pH 7.5 to final concentrations of 3.0 g/L. Triplicate samples were mixed, incubated at 37 °C and sampled 60, 90 and 120 min after addition of the beads with bacteria (B or L) or cell suspensions of B or L. Surviving bacteria were numerated by pour plate counts in MRS agar aerobically incubated at 37 °C for L and in MRS agar anaerobically incubated at 39 °C for B, for 2 days. 2.4. Statistical analysis Results are presented as mean ± standard deviation (SD) of replicated determinations. Data were subjected to one-way analysis of variance (ANOVA) and multiple comparisons were performed by Duncan's test. Statistical significance was set at p b 0.05. All analyses were performed using SPSS version 17.0 for Windows (SPSS, Chicago, Illinois, USA). 3. Results and discussion 3.1. Microsphere characteristics. Size and entrapment efficiency Table 1 shows results for diameters and encapsulation yields of chitosan-coated alginate microspheres containing bacteria B or L in combination with or without quercetin. The mean diameters of microspheres Q, Q+ B and Q +L generated were significantly (p b 0.05) higher than B and L. The mean diameters of chitosan-coated alginate microspheres B and L were 345.43 and 362.05 μm and of Q, Q+ B and Q +L were between 542 and 518 μm. Interestingly, there was a large variation in capsule size depending of the probiotic strain and prebiotic contents.
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Table 1 Size and encapsulation yields of different microspheres. Microsphere type
Microsphere size (μm; n = 100)
Encapsulation yield (%; n = 10)
Viability (log CFU/g; n = 3)
Quercetin Quercetin B. bifidum Quercetin L. gasseri B. bifidum L. gasseri
518.53 ± 3.05c 542.74 ± 4.33d
22.2 ± 2.8a
2.99 ± 0.97a
523.09 ± 3.95c
19.5 ± 9.6a
3.98 ± 0.86b
345.43 ± 1.65a 362.05 ± 1.42b
40.2 ± 2.1b 39.2 ± 2.3b
9.38 ± 0.92c 9.61 ± 1.14c
Means with different letter in a column are significantly different (p b 0.05).
EY for viable cells were significantly higher (p b 0.05) for microspheres without quercetin (B 40.2% and L 39.2%) as compared to microspheres with quercetin (Q + B 22.2% and Q + L 19.5%). On the other hand, EY for viable cells were similar in each group with or without quercetin. Calcium alginate microencapsulation can be affected by various factors such as capsule size, alginate concentration, probiotic cell load, and hardening time in calcium chloride. In the present study, although it used the same concentration of alginate, microspheres with quercetin needed higher microsphere size for coencapsulation of a probiotic and prebiotic. Furthermore, there were different microsphere sizes among different kinds of beads because they had a different amount of probiotic inside (Chandramouli et al., 2004). Other studies showed that the coencapsulation of different probiotic bacteria with Hi-maize starch (prebiotic) and further coating with chitosan significantly enhanced the survival of encapsulated probiotic bacteria (Iyer & Kailasapathy, 2005). In addition, coencapsulated probiotic bacteria with Hi-maize also survived better than the encapsulated bacteria without the prebiotic. Our study tried to perform coencapsulation of probiotic with prebiotic (quercetin). However, poor encapsulation efficiency and viability of cells in quercetin beads were observed due to interaction of the flavonoid with the probiotic. These results, together with the study about the survival of symbiotic during storage at 4 °C, demonstrated that Q + B and Q + L microencapsulateds did not survive. Owing to this, the probiotics and the prebiotic were microencapsulated separately. 3.2. Stability of microencapsulated probiotic bacteria during storage at 4 °C Fig. 1 shows the stability of free and encapsulated probiotic bacteria with or without quercetin during 4 weeks of storage in the refrigerator at 4 °C. The viability of microencapsulated cells showed different stability between microcapsules with or without quercetin in the same storage conditions. After 28 days, the survival of L. gasseri and B. bifidum in separated microcapsule without quercetin decreased from 4.07 × 109 to 3.09 × 107 CFU/mL and from 2.40 × 109 to 1.38 × 107 CFU/ mL respectively. However, the numbers of microencapsulated L. gasseri with quercetin and B. bifidum with quercetin decreased from 2.63 × 105 to 1.19 × 102 and 1.48 × 106 to 1.14 × 102 CFU/mL respectively after 11 days, and no survival was noted after 14 days. The rate of decrease was significantly different (p b 0.05) between the microencapsulated with and without quercetin. Several studies showed that the survival of microencapsulated bacteria was improved in alginate microparticles over that of nonencapsulated bacteria during the storage period (Adhikari et al., 2000; Krasaekoopt et al., 2003; Sultana et al., 2000; Truelstrup Hansen et al., 2002). Koo et al. (2001) reported that L. bulgaricus loaded in chitosancoated alginate microparticles showed higher storage stability than free cell culture. We also observed a similar effect in our study. Preparation in a dry form is necessary for both prolonged storage and application of microencapsulated bacteria. The process of drying
Fig. 1. Stability of free and microencapsulated L. gasseri and B. bifidum with or without quercetin during 4 weeks of storage at 4 °C. Symbols: (▼) free L. gasseri, (○) free B. bifidum, (■) chitosan-coated alginate capsule L. gasseri, (▲) chitosan-coated alginate capsule B. bifidum, (♦) chitosan-coated alginate capsule L. gasseri with quercetin and (◀) chitosan-coated alginate capsule B. bifidum with quercetin. Means (n = 5) ± SD.
without protective agents resulted in an almost complete inactivation of bacteria. Probiotic organisms are sensitive to freeze-drying due to deterioration of the physiological state of the cells. A cryoprotectant is a substance that is accumulated within the cells to reduce the osmotic difference with the external environment (Kets et al., 1996) or a substance that surrounds cells to improve cold tolerance. The extent to which cryoprotection is provided may vary between cultures (Das et al., 1976). The skim milk used as a cryoprotectant agent is expected to prevent cellular injury by stabilizing the cell membrane constituents (Castro et al., 1995; Kearney et al., 1990; Valdez et al., 1983). The viability of the probiotic organisms decreased over a 4 day storage when the probiotic and prebiotic are coencapsulated together. This finding was unexpected. The explanation may be that the prebiotic present outside the cells was concentrated during freezing. Consequently, as osmotic pressure increased, probiotic cells were injured and killed (Mazur, 1984). According to Baati et al. (2000), lactic acid bacteria also suffer from stresses caused by a changing environment. The quercetin may not have been able to protect cells from injury, which may have contributed to a reduction in probiotic viability. 3.3. Survival of free and encapsulated B. bifidum and L. gasseri in SGJ Chandramouli et al. (2004) and Iyer et al. (2005) have shown that only the microencapsulated probiotics were able to maintain viability in gastro-intestinal conditions. Microencapsulation of probiotics in alginate beads has previously been tested for improving viability of probiotic bacteria in simulated gastric conditions (Ding and Shah, 2009; Krasaekoopt et al., 2003). Moreover, Yu et al. (2001) found that the survival rate of bifidobacteria in alginate beads containing chitosan was higher than that of alginate beads. However, Sultana et al. (2000) found that encapsulation of bacteria in alginate beads did not effectively protect the organisms from high acidity. Although some authors have reported the effect of alginate encapsulation on survival of lactic acid bacteria in simulated gastro-intestinal conditions (Lee and Heo, 2000; Shah, 2000; Sheu and Marshall, 1993; Sultana et al., 2000; Truelstrup Hansen et al., 2002), there is no uniformity in the reported encapsulation procedure. To determinate the likelihood of microencapsulated and free probiotic bacteria surviving passage through the stomach following oral administration, microencapsulated and free probiotic bacteria were tested for stability in simulated gastric fluid (Fig. 2). Encapsulation in chitosan-coated alginate microspheres significantly (p b 0.05) improved survival of bifidobacteria and lactobacilli. Cell survival after
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M. Chávarri et al. / International Journal of Food Microbiology 142 (2010) 185–189 Table 2 Number of surviving cells (log CFU/mL) for free and encapsulated L. gasseri and B. bifidum during sequential incubation (37 °C) in simulated intestinal juices (pH 6.0). Treatment
Free L. gasseri Free B. bifidum L. gasseri capsule B. bifidum capsule
Simulated intestinal juices (SIJ) 0 min
60 min
90 min
120 min
7.58 ± 0.01b 8.08 ± 0.02a 7.03 ± 0.01c 7.01 ± 0.01c
4.48 ± 0.01c 2.05 ± 0.01d 7.07 ± 0.01a 6.85 ± 0.01b
2.24 ± 0.02c b 1.00 ± 0d 6.90 ± 0.01b 7.09 ± 0.01a
b1.00 ± 0c b1.00 ± 0c 6.95 ± 0.01a 6.78 ± 0.01b
10 CFU/mL is the detection limit. Means with different letter in a column are significantly different (p b 0.05).
Fig. 2. Survival of free and encapsulated B. bifidum and L. gasseri during exposure to simulated gastric juice (pH 2.0) at 37 °C. Symbols: (▼) free L. gasseri, (●) free B. bifidum, (■) chitosan-coated alginate capsule L. gasseri and (▲) chitosan-coated alginate capsule B. bifidum. Survival (%) represents the percentage of cells surviving relative to the initial population. Means (n = 10) ± SD.
exposure to SGJ for 5 min was 95%, 94%, 78% and 66% of the initial population found in chitosan-coated alginate microspheres with B. bifidum, with L. gasseri, free L. gasseri and free B. bifidum, respectively. Microencapsulated B. bifidum and microencapsulated L. gasseri were resistant to simulated gastric conditions. More than 107 CFU/mL B. bifidum capsules and L. gasseri capsules survived after 120 min, indicating that B. bifidum capsule decreased by less than one logarithm unit and L. gasseri capsule decreased a little bit more than one logarithm unit. It is estimated that 107 CFU/mL of live probiotic cells are needed to confer health benefits to the consumer (Ouwehand and Salminen, 1998). However, there was a rapid loss of free probiotic bacteria in SGJ at pH 2.0, initial numbers of 109 CFU/mL for free L. gasseri rapidly decreased to less than 10 CFU/mL (the detection limit) after exposure of 2 h, but in only 1 h, free B. bifidum decreased more than 10 CFU/mL. Several reports have indicated differences among strains of probiotic bacteria with respect to their survival in acid environment (Kailasapathy, 2005; Shah and Jelen, 1990; Truelstrup Hansen et al., 2002). Specifically, Ding and Shah (2009) showed that the Bifidobacterium strains were the most acid-sensitive strains, which was observed in this study. Specifically free L. gasseri had greater viable numbers than free B. bifidum possibly due to its higher tolerance to acids. Overall, the added coating afforded better protection to probiotic organisms compared to uncoated microcapsules in the same time points (Ding and Shah, 2009). Encapsulation of probiotic bacteria in coated chitosan microspheres improved the survival over that of free cells (Fig. 2).
Chitosan-coated alginate microspheres were the most effective in protecting probiotic bacteria from bile salt. The chitosan coating provides the best protection in bile salt solution because an ionexchange reaction takes place when the beads absorb bile salt (Murata et al., 1999). Therefore, the diffusion of bile salt into the beads may be limited. This will protect encapsulated probiotics from interacting with the bile salt. Koo et al. (2001) and Yu et al. (2001) also reported that bifidobacteria and Lactobacillus casei entrapped in alginate beads containing chitosan had higher viability than in alginate without chitosan. Chitosan, a positively charged polyamine, forms a semipermeable membrane around a negatively charged polymer such as alginate. This membrane does not dissolve in the presence of Ca2+ chelators or antigelling agents and thus enhances stability of the gel (Smidsrod and Skjak-Braek, 1990), and provides a barrier to cell release. Our results concur with other studies that used similar concentrations of bile salts. For instance Chandramouli et al. (2004) and Kailasapathy (2005), showed that encapsulated probiotic bacteria can survive better than free probiotic cells. 4. Conclusions The present study has shown that chitosan-coated technique enhance the stability of alginate microencapsulation in adverse conditions. Also, significantly improve the bacterial survival in simulated gastric environment, and allow viable cells to reach a beneficial level as probiotic. The results shown here support the data obtained by other authors (Adhikari et al., 2000; Sun and Griffiths, 2000). Coating on alginate beads with chitosan develops a complexation of chitosan with alginate. This complex reduces the porosity of alginate beads and decreases the leakage of the encapsulated probiotic and, moreover it is stable at broad pH ranges. According to this, chitosan-coated alginate microspheres could be a good way to administer these beneficial microorganisms orally, but quercetin and probiotic bacteria ought to be microencapsulated separately. In conclusion, the microencapsulation of L. gasseri and B. bifidum with alginate and a chitosan coating offers an effective means of delivery of viable bacterial cells in levels appropriate to the colon and helps in maintaining their survival during simulated gastric and intestinal juice.
3.4. Survival of microencapsulated probiotic bacteria in SIJ
Acknowledgments
The effect of the bile salt on the viability of the microencapsulated and free probiotic bacteria is presented in Table 2. In the case of free L. gasseri, the initial average viable count of 7.58 log CFU/mL was reduced to 2.24 log CFU/mL after 90 min and the average viable number was further reduced to more than 10 CFU/mL after 120 min. The most susceptible cell to bile salt solution was free B. bifidum with an initial viability of 8.08 log CFU/mL, which was reduced to more than 10 CFU/mL after 90 min of incubation. Microencapsulated bacteria survival after exposure to SGI for 120 min was 98.86% and 96.72% of the initial population found in encapsulated L. gasseri and B. bifidum respectively.
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal conditions María Chávarri a,⁎, Izaskun Marañón a, Raquel Ares a, Francisco C. Ibáñez b, Florencio Marzo b, María del Carmen Villarán a a b
Food Area, Fundación LEIA Technological Development Centre, Parque Tecnológico de Álava, c/Leonardo Da Vinci n 11, 01510 Miñano, Spain Laboratory of Animal Physiology and Nutrition, School of Agronomy, Public University of Navarra, Campus Arrosadia, 31006, Pamplona, Spain
a r t i c l e
i n f o
Article history: Received 25 November 2009 Received in revised form 13 May 2010 Accepted 22 June 2010 Keywords: Lactobacillus gasseri Bifidobacterium bifidum Quercetin Microencapsulation Gastro-intestinal conditions
a b s t r a c t Chitosan was used as a coating material to improve encapsulation of a probiotic and prebiotic in calcium alginate beads. Chitosan-coated alginate microspheres were produced to encapsulate Lactobacillus gasseri (L) and Bifidobacterium bifidum (B) as probiotics and the prebiotic quercetin (Q) with the objective of enhancing survival of the probiotic bacteria and keeping intact the prebiotic during exposure to the adverse conditions of the gastro-intestinal tract. The encapsulation yield for viable cells for chitosan-coated alginate microspheres with quercetin (L + Q and B + Q) was very low. These results, together with the study about the survival of microspheres with quercetin during storage at 4 °C, demonstrated that probiotic bacteria microencapsulated with quercetin did not survive. Owing to this, quercetin and L. gasseri or B. bifidum were microencapsulated separately. Microencapsulated L. gasseri and microencapsulated B. bifidum were resistant to simulated gastric conditions (pH 2.0, 2 h) and bile solution (3%, 2 h), resulting in significantly (p b 0.05) improved survival when compared with free bacteria. This work showed that the microencapsulation of L. gasseri and B. bifidum with alginate and a chitosan coating offers an effective means of delivery of viable bacterial cells to the colon and maintaining their survival during simulated gastric and intestinal juice. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In order to provide health benefits for probiotic bacteria (ColbèreGarapin et al., 2007; de Vrese and Marteau, 2007; Orrhage and Nord, 2000; Reid et al., 2003; Shah, 2007; Vaughan et al., 2005) it has been recommended that they must be present at a minimum level of 106 CFU/g of food product (Doleyres and Lacroix, 2005) or 107 CFU/g at point of delivery (Lee and Salminen, 1995) or be eaten in sufficient amounts to yield a daily intake of 108 CFU (Lopez-Rubio et al., 2006). Several studies have shown that certain strain of lactic acid bacteria, such as Bifidobacterium bifidum (You et al., 2004) and Lactobacillus gasseri (Carroll et al., 2007), prevent some diseases linked to the gastro-intestinal tract. The prebiotics, nondigestible food ingredients, affects the host by selectively stimulating the growth, activity, or both of one or a limited number of bacterial species already resident in the colon (Gibson et al., 2004; McCartney and Gibson, 2006). Quercetin is the most abundant flavonoid in the diet of humans (Boyer et al., 2004) and it is known that it has nutraceutic potential to reduce the levels of cholesterolemia. The consumption of prebiotic quercetin is beneficial for controlling serum cholesterol (Ishikawa et al., 1997) and has antioxidant, anticarcinogenic,
⁎ Corresponding author. Tel.: + 34 945 298144; fax: + 34 945 298217. E-mail address: [email protected] (M. Chávarri). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.022
anti-inflammatory, and cardioprotective properties (Erlund, 2004; Lien et al., 1999; Middleton et al., 2000; Scarfato et al., 2008). The benefit of prebiotics in combination with probiotics has given rise to the concept of symbiotics (Gibson and Roberfroid, 1995; Reid et al., 2003). The addition of quercetin and modulating packaging conditions has improved the microecological balance of the gut microflora (Gibson et al., 2004). However, a high percentage of ingested bifidobacteria lose their viability during delivery through the gastro-intestinal tract (Marteau et al., 1997). Providing probiotic living cells with a physical barrier against adverse environmental conditions is an approach currently receiving considerable interest (Doleyres and Lacroix, 2005; Kailasapathy, 2002; Krasaekoopt et al., 2003). Microencapsulation technologies are hypothesized to be a promising prospect for introducing viable probiotic bacteria in foods because the encapsulation matrix can provide a physical barrier against harsh environmental conditions such as freezing and those encountered during gastric juice passage (Boh, 2007; Capela et al., 2006; Champagne and Kailasapathy, 2008; Kailasapathy, 2002; Shah, 2000). Although studies have used cellulose acetate phthalate (Rao et al., 1989) gelatin, vegetable gum (Shah, 2000), fats (Siuta-Cruce and Goulet, 2001) or κ — carrageenan (Adhikari et al., 2000; Krasaekoopt et al., 2003) as encapsulating agents, alginate remains the most commonly used bio polymer for microencapsulation. The advantages of using alginate as an encapsulating agent include: non-toxicity, formation of
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gentle matrices with calcium chloride to trap sensitive materials such as living microbial cells, simplicity in entrapping living microbial cells and low cost (Krasaekoopt et al., 2003; Sheu and Marshall, 1993). Alginate is also an accepted food additive and can be safely used in foods (Dinakar and Mistry, 1994; Kim et al., 1996; Sheu and Marshall, 1993). The use of alginate is limited due to its low stability in the presence of chelating agents and in acidic conditions below pH 2.0 (Adhikari et al., 2000; Ding and Shah, 2007; Gombotz and Wee, 1998; Hussein and Kebary, 1999; Sultana et al., 2000). The coating of alginate beads and its effectiveness in protecting probiotic bacteria has been extensively studied. Previous researchers have reported that coating alginate microcapsules with chitosan had improved the stability of the alginate beads and thus improved the viability of the encapsulated probiotic organisms (Krasaekoopt et al., 2004). It was suggested that degradation of chitosan occurs by the microflora that are available in the colon and solubilizing alginate gel by sequestering calcium ions (Hejazi and Amiji, 2003). Furthermore, having in mind bio/mucoadhesive properties of natural biopolymers, chitosan-alginate microparticulated systems should have potential for colon targeting (Hejazi and Amiji, 2003; Senel and McClure, 2004; Simonoska et al., 2008). The aim of the study was to enhance alginate microencapsulation by chitosan-coating and to assess their ability to improve the survival of B. bifidum and L. gasseri, in combination with quercetin, during exposure to simulated conditions of the gastro-intestinal tract. 2. Materials and methods 2.1. Bacterial strain and culture condition L. gasseri and B. bifidum were purchased in lyophilized form from the Spanish Type Culture Collection (CECT). Bacteria were routinely grown in MRS broth. Freeze-dried cells were rehydrated in 5 mL MRS broth and incubated, for L. gasseri at 37 °C for 24 h under aerobic conditions and for B. bifidum at 39 °C for 72 h under anaerobic conditions using the Gas Pak Plus system. The cultures were transferred into MRS broth and incubated under the same conditions as before to obtain a cell density of about 1010 CFU/mL. Harvesting of cells was done by centrifugation at 1500 ×g for 5 min at low temperature (4 °C) and the cell pellet was washed twice with sterile saline solution. The cell suspension of each probiotic bacterium was divided in four parts: one part was used for microencapsulation of L. gasseri and B. bifidum (L+ B) together, the second part was used for microencapsulation of L. gasseri and quercetin (L+ Q) together and B. bifidum and quercetin (B+ Q) together, the third part was used for microencapsulation of L. gasseri (L) and B. bifidum (B) and the fourth was used as free cells. Fresh cell suspensions were prepared for each experiment and numerated by pour plating in MRS agar. Plates were incubated under the same conditions as before. 2.2. Microencapsulation and coating procedures Probiotic organisms and quercetin were incorporated into 10 mL of 20 g/L of sodium alginate. Chitosan aqueous solution was prepared. In brief, chitosan was dissolved in 100 mL distilled water acidified with glacial acetic acid to achieve a final chitosan concentration of 0.4% (w/v). This solution was filtered through a nylon cloth to remove any remaining insoluble material. CaCl2 0.1 M was added to the chitosan solution (Zhou et al., 1998). The extrusion technique of microencapsulation was derived from Krasaekoopt et al. (2004) by using alginate as the supporting matrix. To form beads, the sodium alginate solution was extruded into a previous sterile chitosan solution and stirred. The beads were sieved off from the chitosan solution and washed with sterile distilled water.
The capsulates were suspended in cryoprotectant agent and then frozen at −20 °C. The frozen samples were desiccated under vacuum, at a condenser temperature of −40 °C for about 18 h with a freeze-drier (Freezedryer Lyobeta25). Dried cells were stored in closed containers at 4 °C, under darkness. 2.3. Survival assay and numeration of microencapsulated bacteria Entrapped bacteria in uncoated alginate microspheres were released by homogenizing 0.1 g of filtered microsphere slurry in 10 mL of sodium citrate 0.1 M for 10 min and stirred. The homogenized samples were diluted to appropriate concentrations and pour plated in MRS agar. The plates were incubated for 2 days at 37 °C and the encapsulated bacteria enumerated as CFU/mL. The encapsulation yield (EY), which is a combined measurement of the efficacy of entrapment and survival of viable cells during the microencapsulation procedure, was calculated as: EY = ðN = NoÞ × 100: Where N is the number of viable entrapped cells released from the microspheres, and No is the number of free cells added to the biopolymer mix during the production of the microspheres. The particles sizes and formation of microspheres were measured with a light microscope (Axioscop40, Carl Zeiss). The data analysis was performed using the software Ellix 5.0 (Microvision Instruments, France). The mean diameters of microspheres were calculated from 100 beads. Simulated gastric juice (SGJ) consisted of 9 g/L of sodium chloride containing 3.0 g/L of pepsin with pH adjusted to 2.0 with hydrochloric acid (Altman, 1961). 0.2 g of microspheres with entrapped bacteria (B or L) or 0.2 mL of cell suspensions of B or L were mixed in 10 mL of SGJ and incubated for 5, 30, 60 and 120 min at 37 °C with constant agitation at 50 rpm. Simulated intestinal juice (SIJ) was prepared by dissolving bile salts in intestinal solution (6.5 g/L NaCl, 0.835 g/L KCl, 0.22 g/L CaCl2 and 1.386 g/L NaHCO3) pH 7.5 to final concentrations of 3.0 g/L. Triplicate samples were mixed, incubated at 37 °C and sampled 60, 90 and 120 min after addition of the beads with bacteria (B or L) or cell suspensions of B or L. Surviving bacteria were numerated by pour plate counts in MRS agar aerobically incubated at 37 °C for L and in MRS agar anaerobically incubated at 39 °C for B, for 2 days. 2.4. Statistical analysis Results are presented as mean ± standard deviation (SD) of replicated determinations. Data were subjected to one-way analysis of variance (ANOVA) and multiple comparisons were performed by Duncan's test. Statistical significance was set at p b 0.05. All analyses were performed using SPSS version 17.0 for Windows (SPSS, Chicago, Illinois, USA). 3. Results and discussion 3.1. Microsphere characteristics. Size and entrapment efficiency Table 1 shows results for diameters and encapsulation yields of chitosan-coated alginate microspheres containing bacteria B or L in combination with or without quercetin. The mean diameters of microspheres Q, Q+ B and Q +L generated were significantly (p b 0.05) higher than B and L. The mean diameters of chitosan-coated alginate microspheres B and L were 345.43 and 362.05 μm and of Q, Q+ B and Q +L were between 542 and 518 μm. Interestingly, there was a large variation in capsule size depending of the probiotic strain and prebiotic contents.
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Table 1 Size and encapsulation yields of different microspheres. Microsphere type
Microsphere size (μm; n = 100)
Encapsulation yield (%; n = 10)
Viability (log CFU/g; n = 3)
Quercetin Quercetin B. bifidum Quercetin L. gasseri B. bifidum L. gasseri
518.53 ± 3.05c 542.74 ± 4.33d
22.2 ± 2.8a
2.99 ± 0.97a
523.09 ± 3.95c
19.5 ± 9.6a
3.98 ± 0.86b
345.43 ± 1.65a 362.05 ± 1.42b
40.2 ± 2.1b 39.2 ± 2.3b
9.38 ± 0.92c 9.61 ± 1.14c
Means with different letter in a column are significantly different (p b 0.05).
EY for viable cells were significantly higher (p b 0.05) for microspheres without quercetin (B 40.2% and L 39.2%) as compared to microspheres with quercetin (Q + B 22.2% and Q + L 19.5%). On the other hand, EY for viable cells were similar in each group with or without quercetin. Calcium alginate microencapsulation can be affected by various factors such as capsule size, alginate concentration, probiotic cell load, and hardening time in calcium chloride. In the present study, although it used the same concentration of alginate, microspheres with quercetin needed higher microsphere size for coencapsulation of a probiotic and prebiotic. Furthermore, there were different microsphere sizes among different kinds of beads because they had a different amount of probiotic inside (Chandramouli et al., 2004). Other studies showed that the coencapsulation of different probiotic bacteria with Hi-maize starch (prebiotic) and further coating with chitosan significantly enhanced the survival of encapsulated probiotic bacteria (Iyer & Kailasapathy, 2005). In addition, coencapsulated probiotic bacteria with Hi-maize also survived better than the encapsulated bacteria without the prebiotic. Our study tried to perform coencapsulation of probiotic with prebiotic (quercetin). However, poor encapsulation efficiency and viability of cells in quercetin beads were observed due to interaction of the flavonoid with the probiotic. These results, together with the study about the survival of symbiotic during storage at 4 °C, demonstrated that Q + B and Q + L microencapsulateds did not survive. Owing to this, the probiotics and the prebiotic were microencapsulated separately. 3.2. Stability of microencapsulated probiotic bacteria during storage at 4 °C Fig. 1 shows the stability of free and encapsulated probiotic bacteria with or without quercetin during 4 weeks of storage in the refrigerator at 4 °C. The viability of microencapsulated cells showed different stability between microcapsules with or without quercetin in the same storage conditions. After 28 days, the survival of L. gasseri and B. bifidum in separated microcapsule without quercetin decreased from 4.07 × 109 to 3.09 × 107 CFU/mL and from 2.40 × 109 to 1.38 × 107 CFU/ mL respectively. However, the numbers of microencapsulated L. gasseri with quercetin and B. bifidum with quercetin decreased from 2.63 × 105 to 1.19 × 102 and 1.48 × 106 to 1.14 × 102 CFU/mL respectively after 11 days, and no survival was noted after 14 days. The rate of decrease was significantly different (p b 0.05) between the microencapsulated with and without quercetin. Several studies showed that the survival of microencapsulated bacteria was improved in alginate microparticles over that of nonencapsulated bacteria during the storage period (Adhikari et al., 2000; Krasaekoopt et al., 2003; Sultana et al., 2000; Truelstrup Hansen et al., 2002). Koo et al. (2001) reported that L. bulgaricus loaded in chitosancoated alginate microparticles showed higher storage stability than free cell culture. We also observed a similar effect in our study. Preparation in a dry form is necessary for both prolonged storage and application of microencapsulated bacteria. The process of drying
Fig. 1. Stability of free and microencapsulated L. gasseri and B. bifidum with or without quercetin during 4 weeks of storage at 4 °C. Symbols: (▼) free L. gasseri, (○) free B. bifidum, (■) chitosan-coated alginate capsule L. gasseri, (▲) chitosan-coated alginate capsule B. bifidum, (♦) chitosan-coated alginate capsule L. gasseri with quercetin and (◀) chitosan-coated alginate capsule B. bifidum with quercetin. Means (n = 5) ± SD.
without protective agents resulted in an almost complete inactivation of bacteria. Probiotic organisms are sensitive to freeze-drying due to deterioration of the physiological state of the cells. A cryoprotectant is a substance that is accumulated within the cells to reduce the osmotic difference with the external environment (Kets et al., 1996) or a substance that surrounds cells to improve cold tolerance. The extent to which cryoprotection is provided may vary between cultures (Das et al., 1976). The skim milk used as a cryoprotectant agent is expected to prevent cellular injury by stabilizing the cell membrane constituents (Castro et al., 1995; Kearney et al., 1990; Valdez et al., 1983). The viability of the probiotic organisms decreased over a 4 day storage when the probiotic and prebiotic are coencapsulated together. This finding was unexpected. The explanation may be that the prebiotic present outside the cells was concentrated during freezing. Consequently, as osmotic pressure increased, probiotic cells were injured and killed (Mazur, 1984). According to Baati et al. (2000), lactic acid bacteria also suffer from stresses caused by a changing environment. The quercetin may not have been able to protect cells from injury, which may have contributed to a reduction in probiotic viability. 3.3. Survival of free and encapsulated B. bifidum and L. gasseri in SGJ Chandramouli et al. (2004) and Iyer et al. (2005) have shown that only the microencapsulated probiotics were able to maintain viability in gastro-intestinal conditions. Microencapsulation of probiotics in alginate beads has previously been tested for improving viability of probiotic bacteria in simulated gastric conditions (Ding and Shah, 2009; Krasaekoopt et al., 2003). Moreover, Yu et al. (2001) found that the survival rate of bifidobacteria in alginate beads containing chitosan was higher than that of alginate beads. However, Sultana et al. (2000) found that encapsulation of bacteria in alginate beads did not effectively protect the organisms from high acidity. Although some authors have reported the effect of alginate encapsulation on survival of lactic acid bacteria in simulated gastro-intestinal conditions (Lee and Heo, 2000; Shah, 2000; Sheu and Marshall, 1993; Sultana et al., 2000; Truelstrup Hansen et al., 2002), there is no uniformity in the reported encapsulation procedure. To determinate the likelihood of microencapsulated and free probiotic bacteria surviving passage through the stomach following oral administration, microencapsulated and free probiotic bacteria were tested for stability in simulated gastric fluid (Fig. 2). Encapsulation in chitosan-coated alginate microspheres significantly (p b 0.05) improved survival of bifidobacteria and lactobacilli. Cell survival after
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M. Chávarri et al. / International Journal of Food Microbiology 142 (2010) 185–189 Table 2 Number of surviving cells (log CFU/mL) for free and encapsulated L. gasseri and B. bifidum during sequential incubation (37 °C) in simulated intestinal juices (pH 6.0). Treatment
Free L. gasseri Free B. bifidum L. gasseri capsule B. bifidum capsule
Simulated intestinal juices (SIJ) 0 min
60 min
90 min
120 min
7.58 ± 0.01b 8.08 ± 0.02a 7.03 ± 0.01c 7.01 ± 0.01c
4.48 ± 0.01c 2.05 ± 0.01d 7.07 ± 0.01a 6.85 ± 0.01b
2.24 ± 0.02c b 1.00 ± 0d 6.90 ± 0.01b 7.09 ± 0.01a
b1.00 ± 0c b1.00 ± 0c 6.95 ± 0.01a 6.78 ± 0.01b
10 CFU/mL is the detection limit. Means with different letter in a column are significantly different (p b 0.05).
Fig. 2. Survival of free and encapsulated B. bifidum and L. gasseri during exposure to simulated gastric juice (pH 2.0) at 37 °C. Symbols: (▼) free L. gasseri, (●) free B. bifidum, (■) chitosan-coated alginate capsule L. gasseri and (▲) chitosan-coated alginate capsule B. bifidum. Survival (%) represents the percentage of cells surviving relative to the initial population. Means (n = 10) ± SD.
exposure to SGJ for 5 min was 95%, 94%, 78% and 66% of the initial population found in chitosan-coated alginate microspheres with B. bifidum, with L. gasseri, free L. gasseri and free B. bifidum, respectively. Microencapsulated B. bifidum and microencapsulated L. gasseri were resistant to simulated gastric conditions. More than 107 CFU/mL B. bifidum capsules and L. gasseri capsules survived after 120 min, indicating that B. bifidum capsule decreased by less than one logarithm unit and L. gasseri capsule decreased a little bit more than one logarithm unit. It is estimated that 107 CFU/mL of live probiotic cells are needed to confer health benefits to the consumer (Ouwehand and Salminen, 1998). However, there was a rapid loss of free probiotic bacteria in SGJ at pH 2.0, initial numbers of 109 CFU/mL for free L. gasseri rapidly decreased to less than 10 CFU/mL (the detection limit) after exposure of 2 h, but in only 1 h, free B. bifidum decreased more than 10 CFU/mL. Several reports have indicated differences among strains of probiotic bacteria with respect to their survival in acid environment (Kailasapathy, 2005; Shah and Jelen, 1990; Truelstrup Hansen et al., 2002). Specifically, Ding and Shah (2009) showed that the Bifidobacterium strains were the most acid-sensitive strains, which was observed in this study. Specifically free L. gasseri had greater viable numbers than free B. bifidum possibly due to its higher tolerance to acids. Overall, the added coating afforded better protection to probiotic organisms compared to uncoated microcapsules in the same time points (Ding and Shah, 2009). Encapsulation of probiotic bacteria in coated chitosan microspheres improved the survival over that of free cells (Fig. 2).
Chitosan-coated alginate microspheres were the most effective in protecting probiotic bacteria from bile salt. The chitosan coating provides the best protection in bile salt solution because an ionexchange reaction takes place when the beads absorb bile salt (Murata et al., 1999). Therefore, the diffusion of bile salt into the beads may be limited. This will protect encapsulated probiotics from interacting with the bile salt. Koo et al. (2001) and Yu et al. (2001) also reported that bifidobacteria and Lactobacillus casei entrapped in alginate beads containing chitosan had higher viability than in alginate without chitosan. Chitosan, a positively charged polyamine, forms a semipermeable membrane around a negatively charged polymer such as alginate. This membrane does not dissolve in the presence of Ca2+ chelators or antigelling agents and thus enhances stability of the gel (Smidsrod and Skjak-Braek, 1990), and provides a barrier to cell release. Our results concur with other studies that used similar concentrations of bile salts. For instance Chandramouli et al. (2004) and Kailasapathy (2005), showed that encapsulated probiotic bacteria can survive better than free probiotic cells. 4. Conclusions The present study has shown that chitosan-coated technique enhance the stability of alginate microencapsulation in adverse conditions. Also, significantly improve the bacterial survival in simulated gastric environment, and allow viable cells to reach a beneficial level as probiotic. The results shown here support the data obtained by other authors (Adhikari et al., 2000; Sun and Griffiths, 2000). Coating on alginate beads with chitosan develops a complexation of chitosan with alginate. This complex reduces the porosity of alginate beads and decreases the leakage of the encapsulated probiotic and, moreover it is stable at broad pH ranges. According to this, chitosan-coated alginate microspheres could be a good way to administer these beneficial microorganisms orally, but quercetin and probiotic bacteria ought to be microencapsulated separately. In conclusion, the microencapsulation of L. gasseri and B. bifidum with alginate and a chitosan coating offers an effective means of delivery of viable bacterial cells in levels appropriate to the colon and helps in maintaining their survival during simulated gastric and intestinal juice.
3.4. Survival of microencapsulated probiotic bacteria in SIJ
Acknowledgments
The effect of the bile salt on the viability of the microencapsulated and free probiotic bacteria is presented in Table 2. In the case of free L. gasseri, the initial average viable count of 7.58 log CFU/mL was reduced to 2.24 log CFU/mL after 90 min and the average viable number was further reduced to more than 10 CFU/mL after 120 min. The most susceptible cell to bile salt solution was free B. bifidum with an initial viability of 8.08 log CFU/mL, which was reduced to more than 10 CFU/mL after 90 min of incubation. Microencapsulated bacteria survival after exposure to SGI for 120 min was 98.86% and 96.72% of the initial population found in encapsulated L. gasseri and B. bifidum respectively.
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