Journal of Food Engineering 103 (2011) 123128

Contents lists available at ScienceDirect

Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Effect of microencapsulation on survival of Lactobacillus plantarum in simulated gastrointestinal conditions, refrigeration, and yogurt
Graziela Brusch Brinques, Marco Antnio Zchia Ayub
Food Science and Technology Institute, Federal University of Rio Grande do Sul State, Av. Bento Gonalves, 9500, PO Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil

a r t i c l e

i n f o

a b s t r a c t
In the present research the survival of free and microencapsulated cells of a new strain of Lactobacillus plantarum BL011 under stress conditions was tested in sodium alginate or pectin, coated with sodium alginate or chitosan. Results for the simulated gastrointestinal medium (SGT) showed no change in viability of cells in relation to the control. However, the simulated gastric medium (GM) drastically reduced the viability under the tested conditions, with no signicant differences between free and immobilized cells. Under refrigerated storage viability of immobilized cells were greatly enhanced compared to the free microorganisms, and the treatments showing the lowest loss of viability were those of 4% (w/v) pectin, 3% (w/v) sodium alginate coated with chitosan and a mixture of 2% (w/v) sodium alginate and 2% (w/ v) pectin, respectively. Loss of viability of immobilized L. plantarum in 3% alginate coated with chitosan in yogurt was of 0.55 log cycles during 38 days of storage. The results of this study suggest the efciency of immobilization techniques to increase the survival of lactobacilli in yogurt under refrigerated storage. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 5 May 2010 Received in revised form 20 September 2010 Accepted 10 October 2010 Available online 14 October 2010 Keywords: Lactobacillus plantarum Microencapsulation Viability Probiotics

1. Introduction Lactobacillus plantarum is one of the most widely used lactic acid bacterium, showing a homofermentative metabolism, moderate acid tolerance, and is considered as a GRAS (Generally Regarded as Safe) organism. Many strains of L. plantarum are marketed as probiotics (De Vries et al., 2006). When a new strain of probiotic bacterium is being tested to be used in foods, viability test to gastrointestinal conditions and to conditions of processing and storage should be performed (FAO/WHO, 2001). The benets promoted by probiotic bacteria are increasingly explored in different uses in various types of foods (Kailasapathy et al., 2008; Nazzaro et al., 2008; Prado et al., 2008; Souza and Saad, 2009; Vasiljevic and Shah, 2008). However, cell viability in these products is often low and the ability to survive and multiply in the digestive tract strongly inuences the benets that probiotics can produce (zer et al., 2009). Probiotics have to be metabolically stable and active in the product, survive the passage of the upper digestive tract in large numbers, and show the ability to adhere and colonize the intestine system (Champagne et al., 2005; Mattila-Sandholm et al., 2002; Stanton et al., 2005). Microencapsulation is a promising technique for bacterial cell protection and several studies have been carried out investigating the protective role of this technique against adverse conditions to

Corresponding author. Tel.: +55 51 3308 6685; fax: +55 51 3308 7048.
E-mail address: mazayub@ufrgs.br (M.A.Z. Ayub). 0260-8774/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2010.10.006

which probiotics can be exposed (Capela et al., 2006; Chen et al., 2006; Heidebach et al., 2010; Mandal et al., 2006; Michida et al., 2006; zer et al., 2008; Sheu and Marshall, 1993; Sultana et al., 2000). One advantage of microencapsulation with hydrocolloids is that cells are entrapped within the matrix during the formation of the spheres, while in other techniques such as spray drying, freeze drying and uidized bed drying, the microorganisms are completely released into the product (Krasaekoopt et al., 2003). The most widely used matrix for microencapsulation is alginate. However, alginate gels are susceptible to disintegration in the presence of excess monovalent ions, Ca2+ chelating agents, and harsh chemical environments, such as those of low pH (Gserd et al., 1999). Although some works have reported that the presence of fermentable carbohydrates in the gastrointestinal medium signicantly enhances survival of probiotic cells to the gastric environment (Corcoran et al., 2005), so far no studies were carried out using the technique of microencapsulation by emulsication in order to verify the possibility of increasing the viability of L. plantarum towards gastric medium. Other technique investigated for increased stability of the microcapsules made with polymers is the application of coating with polyelectrolytes such as chitosan and polylysine (Huguet et al., 1996; Krasaekoopt et al., 2004, 2006; Mokarram et al., 2009). The aims of the present work were to study the viability of free and microencapsulated L. plantarum under simulated conditions of gastrointestinal transit, refrigerated storage, and in model yogurt systems in order to know whether these techniques could improve the availability of probiotics in foods.

124

G.B. Brinques, M.A.Z. Ayub / Journal of Food Engineering 103 (2011) 123128

2. Materials and methods 2.1. Reagents All reagents used in this work were of analytical grade and purchased from SigmaAldrich (St. Louis, USA). 2.2. Microorganism A strain of Lactobacillus plantarum, isolated by our group from Serrano cheese and described elsewhere (De Souza et al., 2003), was used in this study. This strain was identied as Lactobacillus plantarum BL011 and it is kept as a certied stock at Microbiology Culture Collection of BiotecLab (UFRGS, Brazil). Working stocks of cultures were maintained in 20% glycerol suspension frozen at 18 C. 2.3. Microencapsulation of microorganisms

(50 mL) was added dropwise to ve parts of vegetable soybean oil (Soya, Bunge, Gaspar, Brazil) (250 mL in asks of 1000 mL) containing Tween 80 (0.2%, w/v), and stirred for 10 min at 200 rpm by magnetic stirring. After this, 500 mL of calcium chloride (0.05 M for ALG or 0.10 M for PEC and ALPE) was quickly and gently added (20 mL s1, Masterex Peristaltic Pump L/S, Ultrapump2, Model 7520-47, Cole-Parmer Instrument Company, Illinois, USA) down the side of the ask until the water/oil emulsion was broken. The beads were formed within 10 min and were collected by centrifugation (350g, 10 min, 4 C). The spheres were washed twice and recovered under the same centrifugation conditions with 0.9% (w/v) sodium chloride solution and re-suspended in 50 mL of 0.9% (w/v) sodium chloride solution for further assays. The average cell concentration in the nal solution of the encapsulation process was 1.5 109 CFU mL1. The whole procedure was performed using autoclaved (121 C, 15 min) materials and under sterile conditions in a horizontal laminar air-ow cabinet. 2.6. Coating with chitosan and sodium alginate

The method described by Sheu and Marshall (1993) was adopted for encapsulation of L. plantarum cells. Microencapsulation was carried out using sodium alginate (A; A1089.01.AF, Synth, Diadema, Brazil), low-methoxyl citric pectin (P; GENU BTM LM102-AS, Plury Qumica, Diadema, Brazil), and blends of A and P (Table 1). It was also tested the coating of beads with sodium alginate and chitosan (448869, Chitosan low molecular weight, Aldrich, St. Louis, USA) using the methodology described by Krasaekoopt et al. (2004). The treatments used and their respective abbreviations are shown in Table 1. 2.4. Concentrated cell culture preparation Erlenmeyer asks (1000 mL) containing 200 mL of the MRS broth (De Man et al., 1960), were inoculated with 1.5 mL of a glycerol stock culture and incubated at 37 C 1 C in a rotatory shaker (MA 830, Marconi, Piracicaba, Brazil) at 180 rpm and grown to optical density (OD, 600 nm) of 1.0. The cells were harvested by centrifugation (Hitachi, Himac CR 21E, Tokio, Japan) at 5000g for 10 min at 4 C. Cell pellets were washed twice with 0.9% (w/v) sodium chloride solution and were nally re-suspended in 10 mL of 0.9% (w/v) sodium chloride solution. These cell suspensions were used as free cells or were aseptically mixed with 40 mL of polymer solution, according Table 1, and were applied to the immobilization system. The average cell concentrations of these polymer-probiotic mixtures were approximately 5 1012 CFU mL1. 2.5. Preparation of microencapsulated cell Cells were microencapsulated by mixing one part culture concentrated with four parts polymer solution. The treatments used for the immobilization of L. plantarum and their respective abbreviations are shown in Table 1. One part of the mixture
Table 1 Treatments used in the immobilization of L. plantarum. Trial Free cells Beads prepared Beads prepared Beads prepared Beads prepared Beads prepared Beads prepared Beads prepared Beads prepared Beads prepared with with with with with with with with with

The chitosan and sodium alginate solutions were prepared according to Krasaekoopt et al. (2004). Briey, the beads of immobilized L. plantarum BL011, prepared as described in item 2.5, were immersed in 100 mL of chitosan solution and shaken at 100 rpm, 37 C for 40 min on an orbital shaker for coating. The chitosancoated beads were collected by centrifugation (350g, 10 min, 4 C). The pellet was washed twice with 0.9% (w/v) sodium chloride solution and ressuspended in 50 mL of 0.9% (w/v) sodium chloride solution. For the sodium alginate coating, the washed immobilized L. plantarum BL011 beads were mixed with 100 mL of a 0.17% (w/v) sodium alginate solution and placed in a rotatory shaker at 100 rpm, 37 C for 20 min. The same procedures described for the coating with chitosan were used here for beads recover. 2.7. Resistance to gastrointestinal media 2.7.1. Preparation of simulated gastric and intestinal juices The simulated juices were prepared according to Charteris et al. (1998) and Michida et al. (2006). Simulated gastric juices (SGJ) were prepared by suspending pepsin (P7000, 1:10,000) in sterile sodium chloride solution (0.5%, w/v) to a nal concentration of 3 g L1 (1038 U mL1) and adjusting the pH to 2.0 with concentrated HCl or sterile 0.1 mol L1 NaOH. Simulated intestinal juices (SIJ) were prepared by suspending pancreatin USP (P-1500) in sterile sodium chloride solution (0.5%, w/v) to a nal concentration of 1 g L1, with 4.5% bile salts (Oxoid, Basingstoke, UK) and adjusting the pH to 8.0 with sterile 0.1 mol L1 NaOH. Both solutions were ltered for sterilization through a 0.22 lm membrane. 2.7.2. Cell tolerance to gastrointestinal The tolerance of free and immobilized cells of L. plantarum on simulated gastric and intestinal juices was determined using the

Abbreviation sodium alginate 3% (w/v) citric pectin 4% (w/v) mixture of sodium alginate 2% (w/v) and citric pectin 2% (w/v) sodium alginate 3% (w/v) coating with sodium alginate solution 0.17% (w/v) citric pectin 4% (w/v) coating with sodium alginate solution 0.17% (w/v) mixture of sodium alginate 2% (w/v) and citric pectin 2% (w/v) coating with sodium alginate solution 0.17% (w/v) sodium alginate 3% (w/v) coating with chitosan solution 0.4% (w/v) citric pectin 4% (w/v) coating with chitosan solution 0.4% (w/v) mixture of sodium alginate 2% (w/v) and citric pectin 2% (w/v) coating with solution 0.4% (w/v) SIMO ALG PEC ALPE ALGA PECA ALPEA ALGQ PECQ ALPEQ

G.B. Brinques, M.A.Z. Ayub / Journal of Food Engineering 103 (2011) 123128

125

adapted method described by Charteris et al. (1998). The tests were performed using a series of sterile Falcon tubes of 15 mL, one for each sampling point (see times of sampling bellow). Two different conditions were tested: in the rst and second tests, 0.4 mL of the suspension of either immobilized or free cells were mixed with 1.8 mL of SGJ or SIJ, gently mixed and incubated for 120 min at 37 C 1 C. The control for these tests (CONT) was done by incubating 0.4 mL of either free or immobilized cells in 1.8 mL sterile sodium chloride solution (0.5 %, w/v) for 120 min at 37 C 1 C. After the addition of free cells or immobilized to SGJ and SIJ, the pH of these were corrected to 2.0 and 8.0, respectively, with sterile 0.1 mol L1 NaOH or concentrated HCl. Aliquots of 1 mL were removed at 0, 30, 60, and 120 min (for all trials) for the determination of total viable counts. 2.8. Resistance to refrigerated storage The viability of L. plantarum under refrigeration was evaluated by incubating 0.4 mL (approximately 1.5 109 cells mL1) of free or immobilized cells suspension added of 1.8 mL of sterile sodium chloride solution (0.5%, w/v) and kept in the refrigerator at 4 1 C. Aliquots of 1 mL were taken every other day for 38 days to determine the total number of viable cells. The immobilized beads were dissolved in the appropriate buffer solution and they were used to determine the total number of viable cells. 2.9. Production of yogurt supplemented with L. plantarum immobilized in calcium alginate beads coated with chitosan The model yogurt used in this work was produced as shown in Fig. 1, using as starter cultures Yo-Flex YF-L812 (Christian Hansen, Hoersholm, Denmark) composed of Streptococcus thermophilus and L. delbrueckii subsp. bulgaricus. A volume of 1.8 mL of yogurt was placed in sterile Falcon tubes of 15 mL and added of 0.4 mL of L. plantarum immobilized in ALGQ. The tubes were stored under refrigeration at 4 1 C for 38 days. The immobilized beads were dissolved and used to determine the total number of viable cells on selective differential medium for L. plantarum (LPSM) described by Bujalance et al. (2006), and composed of (in g L1): bacterial peptone (10), beef extract (10), yeast extract (5), D-sorbitol (20), ciprooxacin (0.0004), sodium acetate (5), ammonium citrate (2), potassium phosphate (2), magnesium sulfate (0.1), manganese sulfate (0.05), bromocresol purple (0.02) and agar (15). Ciprooxacin

was sterilized by ltration through a membrane lter of 0.22 lm before being added to the cooled medium. The pH of the medium was 6.0 0.1. When solidied, LPSM was a purple color and when L. plantarum grows in this medium yellow color develops around the colonies due to acidication. 2.10. Solubilization of polymer beads The encapsulated cells on ALG, PEC, ALPE, ALGA, PECA, and ALPEA were released dissolving 1 mL of bead suspensions in 9 mL 0.1 M phosphate buffer, pH 7.5, followed by shaking for 10 min on a rotatory shaker at 37 C and 180 rpm. The chitosan coating beads (ALGQ, PECQ, ALPEQ) were dissolved using 1 mL of the incubated material added of 9 mL of sodium citrate buffer (0.1 M), pH 6.0, with 10 glass beads, diameter of 0.45 mm, used as an aid in the physical dissolution of the microcapsules, for 10 min on a rotatory shaker at 37 C, 180 rpm. The formed solution was then used to determine the number of viable cells. 2.11. Determination of total viable counts Total viable counts of L. plantarum were determined by a pour plate method using MRS agar (except to yogurt trials) after serial 10-fold dilutions in peptone water. Plates were incubated at 37 C for 48 h. 2.12. Data analysis ANOVA and Tukey0 s mean comparison tests (p 6 0.05) were used to evaluate the data obtained from the test using the Statistica 7.0 (Statsoft, Tulsa, USA). All experiments and analyses were run in triplicates. 3. Results and discussion 3.1. Resistance to gastrointestinal media The results showing the in vitro viability of probiotics when exposed to SGJ and SIJ are presented in Figs. 2 and 3. These experiments aimed at evaluating their tolerance towards models of stomach pH, bile salts, and enzymes present in the upper gastrointestinal tract (Champagne et al., 2005; Stanton et al., 2005). Fig. 2 shows the survival curves of free and immobilized L. plantarum

Fig. 1. Flow diagram for the model yogurt production used in this work.

Fig. 2. Variation in the number of viable cells of L. plantarum BL011 subjected to the SIJ and the respective controls. (j) SIMO; (d) SIJ without immobilization; (N) control ALG; () SIJ ALG; (s) control ALPE; (e) SIJ ALPE.

126

G.B. Brinques, M.A.Z. Ayub / Journal of Food Engineering 103 (2011) 123128

between cells with the gastric juice. Martoni et al. (2007) observed small losses of cell viability at pH 2.5 and 3.0, with 1.09 and 0.6 log CFU mL1 reductions, respectively, when L. plantarum 80 BSH + was exposed to simulated stomach conditions for 4 h. The same authors reported a linear decrease in viability of cells when exposed at pH 2.0, with 8.98 log CFU mL1 reduction after 4 h, while at pH 1.5, after 30 min of exposure, cells completely lost their viability. It is important to note that the juices used in simulated tests reported in the literature differ widely among them. For the simulated gastric environment, most authors only use sodium chloride solutions with adjusted pH to the desired value (Lee and Heo, 2000), with few reports with the addition of enzymes (Charteris et al., 1998; Michida et al., 2006). The same happens for simulated intestinal juice, with many studies showing the use of sodium chloride solution with different concentrations of bile salts (Mandal et al., 2006), but seldom with the addition of pancreatin to these solutions (Michida et al., 2006).
Fig. 3. Change in the number of viable cells of L. plantarum BL011 subjected to SGJ between 0 and 120 min. (white bar) initial number of cells; (black bars) nal number of cells.

3.2. Resistance to refrigerated storage Experiments were performed in order to evaluate the efciency of immobilization to reduce the losses in viability of probiotics under refrigeration and the results showing the survival of L. plantarum BL011 under 38 days of incubation at 4 oC are presented in Table 2. Results show that at the end of this time, the immobilized cells in PEC and ALGQ had the lowest loss of viability. Even with the release of some cells during storage due to the collapse of beads, treatment with PEC showed better viability under refrigeration. Thus, improving the stability of the beads during storage could reduce the loss of cells to the medium and positively affect viability. The ALGQ system showed good stability and there was little loss of viability of L. plantarum BL011 during the storage period. Krasaekoopt et al. (2003) commented that special treatments, such as coating of beads, are used to improve the properties of it. In these cases, the cross linking with cationic polymers, coating with other polymers, mixed with starch and incorporation of additives could improve the stability of the spheres. One of the properties for a given microorganism to be considered probiotic is its capacity to survive storage as a formulated product (FAO/WHO, 2001; Kosin and Rakshit, 2006). In general, fermented products containing added probiotics should be stored under refrigeration at 4 C. Our results, therefore, suggest that immobilization is promising technology to improve viability of probiotics in formulated products.

BL011, in ALG and ALPE when they were incubated with the SIJ. SIJ did not shown any negative inuence over cell viability compared to the controls except for the SIMO trial (95% condence level), where there was a decrease of 0.2 and 0.4 log for control and SIJ, respectively. Michida et al. (2006) found that free cells of L. plantarum resisted to exposure to SIJ. Mandal et al. (2006) showed decreased viability after 12 h of free cells of Lactobacillus casei NCDC-298 from 9.45 to 7.29 log CFU mL1 and from 9.34 to 5.60 log CFU mL1 when they were exposed to 1% and 2% bile salts, respectively. After immobilization in alginate, these authors reported improved viability, which was proportional to the concentration of alginate used. Martoni et al. (2007) veried an increase in viability of 0.91.0 log CFU mL1 when L. plantarum 80 BSH + strain was exposed to simulated intestinal conditions for a 10 h incubation time. Since our results showed that SIJ did not impaired L. plantarum BL011 viability, this test was not used for the other immobilization techniques. Fig. 3 shows the variation in the number of viable cells of L. plantarum BL011 subjected to the SGJ. Exposure to SGJ resulted in a drastic decrease in the total number of survivors and there was no signicant difference between treatments at 95% condence. The encapsulation on the tested materials was not effective in protecting the microorganisms. Sultana et al. (2000) found a signicant drop in viability when probiotics were exposed to pH 2, and immobilization failed to protect the microorganisms from these adverse conditions. Gbassi et al. (2009) reported that, after 90 min of incubation, three different strains of L. plantarum encapsulated in calcium alginate showed a total loss of viability. However, when the same authors used alginate matrix coating with whey protein, there was an increase in survival, demonstrating that the technique was effective for the protection of the probiotic strains. Mandal et al. (2006) observed large decrease in the viability of free cells when subjected to pH 1.5, with only a small increase in viability after immobilization. Krasaekoopt et al. (2004) found that encapsulation with alginate coated with chitosan was the best treatment to protect studied Lactobacillus for all conditions tested, but no treatment promoted the protection of B. bidum to acidic conditions. Mokarram et al. (2009) showed that L. acidophilus and L. rhamnosus exposed to SGJ had higher viability when encapsulated in calcium alginate with double coating sodium alginate, suggesting that there were a reduction of pore size and distribution of gastric juice in the spheres, thus hindering the interaction

Table 2 Reduction of the number of viable cells of L. plantarum during 38 days of refrigerated storage at 4 C. Trial PEC ALGQ ALPE ALPEQ PECQ ALPEA ALG ALGA PECA SIMO Log10 (N0/N) 1.95 0.08 2.17 0.49 2.96 0.20,b 3.62 0.22b,c 3.64 0.10b,c 3.85 0.10b,c 4.35 0.33c,d 5.28 0.92d 5.40 0.71d 6.73 0.13e

N0 and N mean CFU mL1 on rst and 38th day, respectively. a,b,c,d,e Equal letters indicates no statistical difference at 95% of condence.

G.B. Brinques, M.A.Z. Ayub / Journal of Food Engineering 103 (2011) 123128

127

used in yogurt, the viability was approximately four times higher when compared to cells kept in saline suspension. This work demonstrated the potential for using the isolated strain of L. plantarum BL011 since it showed to be stable and keep viability in presence of the simulated intestinal juice and storage under refrigeration. However, it is important to note that we tested high concentrations of immobilized cells in the model yogurt and lower concentrations should be tested, specially considering the sensorial aspects of the product. These are two interesting characteristics that must be met for the commercial implementation of probiotics in dairy and other food products. Acknowledgements The authors wish to thank CNPq (Brazilian Bureau for Science & Technology Research) for the nancial support of this work. References
Fig. 4. Viability of L. plantarum BL011 under refrigerated storage (4 1 C). N0 is the initial number of cells and N is the number of for each time t (CFU mL1). -d- free cells; -j- ALGQ in sodium chloride solution (0.5%, w/v); -N- ALGQ in yogurt. Adhikari, K., Mustapha, A., Grn, I.U., Fernando, L., 2000. Viability of microencapsulated bidobacteria in set yogurt during refrigerated storage. Journal of Dairy Science 83 (9), 19461951. ANVISA, 2008. Agncia Nacional de Vigilncia Sanitria. Alimentos com Alegaes de Propriedades Funcionais e ou de Sade, Novos Alimentos/Ingredientes, Substncias Bioativas e Probiticos. <http://www.anvisa.gov.br/alimentos/ comissoes/tecno_lista_alega.htm>. Bujalance, C., Jimnez-Valera, M., Moreno, E., Ruiz-Bravo, A., 2006. A selective differential medium for L. plantarum. Journal of Microbiological Methods 66 (3), 572575. Capela, P., Hay, T.K.C., Shah, N.P., 2006. Effect of cryoprotectants, prebiotics and microencapsulation on survival of probiotic organisms in yoghurt and freezedried yoghurt. Food Research International 39 (2), 203211. Champagne, C.P., Gardner, N.J., Roy, D., 2005. Challenges in the addition of probiotic cultures to foods. Critical Reviews in Food Science and Nutrition 45 (1), 6184. Charteris, W.P., Kelly, P.M., Morelli, L., Collins, J.K., 1998. Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bidobacterium species in the upper human gastrointestinal tract. Journal of Applied Microbiology 84 (5), 759768. Chen, K.N., Chen, M.J., Lin, C.W., 2006. Optimal combination of the encapsulating materials for probiotic microcapsules and its experimental verication (R1). Journal of Food Engineering 76 (3), 313320. Corcoran, B.M., Stanton, C., Fitzgerald, G.F., Ross, R.P., 2005. Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Applied and Environmental Microbiology 71 (6), 30603067. De Man, J.C., Rogosa, M., Sharpe, M.E., 1960. A medium for the cultivation of lactobacilli. Journal of Applied Microbiology 23 (1), 130135. De Souza, C.F.V., Dalla Rosa, T., Ayub, M.A.Z., 2003. Changes in the microbiological and physicochemical characteristics of Serrano cheese during manufacture and ripening. Brazilian Journal of Microbiology 34 (3), 260266. De Vries, M.C., Vanghan, E.E., Leerebezem, M., De Vos, W.M., 2006. L. plantarum survival, functional and potential probiotic properties in the human intestinal tract. International Dairy Journal 16 (9), 10181028. FAO/WHO, 2001. Report on joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. <http://www.who.int/foodsafety/publications/ fs_management/en/probiotics.pdf>. Gserd, O., Sannes, A., Skjk-Brk, G., 1999. Microcapsules of alginatechitosan. II. A study of capsule stability and permeability. Biomaterials 20 (8), 773783. Gbassi, G.K., Vandamme, T., Ennahar, S., Marchioni, E., 2009. Microencapsulation of L. plantarum spp in an alginate matrix coated with whey proteins. International Journal of Food Microbiology 129 (1), 103105. Heidebach, T., Frst, P., Kulozik, U., 2010. Inuence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. Journal of Food Engineering 98 (3), 309316. Huguet, M.L., Neufeld, R.J., Dellacherie, E., 1996. Calciumalginate beads coated with polycationic polymers: comparison of chitosan and DEAE-Dextran. Process Biochemistry 31 (4), 347353. Kailasapathy, K., 2006. Survival of free and encapsulated probiotic bacteria and their effect on the sensory properties of yoghurt. LWT Food Science and Technology 39 (10), 12211227. Kailasapathy, K., Harmstorf, I., Phillips, M., 2008. Survival of L. acidophilus and B. animalis ssp. Lactis in stirred fruit yogurts. LWT Food Science and Technology 41 (7), 13171322. Kosin, B., Rakshit, S.K., 2006. Microbial and processing criteria for production of probiotics: a review. Food Technology and Biotechnology 44 (3), 371379. Krasaekoopt, W., Bhandari, B., Deeth, H., 2003. Review: evaluation of encapsulation techniques of probiotics for yoghurt. International Dairy Journal 13 (1), 313. Krasaekoopt, W., Bhandari, B., Deeth, H., 2004. The inuence of coating materials on some properties of alginate beads and survivability of microencapsulated probiotic bacteria. International Dairy Journal 14 (8), 737743.

3.3. Resistance of L. plantarum BL011 immobilized in calcium alginate beads coated with chitosan in yogurt ALGQ immobilization was chosen to verify the viability of L. plantarum BL011 in yogurt under refrigeration and results can be seen in Fig. 4. Each curve in Fig. 4 represents the change in the population of L. plantarum BL011 with the storage time related to the initial population. Results show that ALGQ in yogurt presented good cell viabilities, with losses of only 0.55 log cycles, compared to 6.73 log cycles for the free cells and 2.17 log cycles for ALGQ, both in sodium chloride solution. Cell count of the product remained above 109 CFU mL1, indicating that this product would conform to the recommended minimum requirements of several countries that dairy products should contain at least 107 109 CFU of probiotic cultures per product portion for packingclaiming of probiotic qualities (ANVISA, 2008; Pagano, 1998; Stanton et al., 2005). Although the amount of spheres used in this work were well above the acceptable quantities for real yogurt formulations, these results suggest the efciency of the technique in order to increase survival of lactobacilli in refrigerated yogurt. Moreover, the Lactobacillus plantarum BL011 strain compared very well with other probiotic bacteria reported in the literature concerning their viability under refrigeration in yogurt (Kailasapathy, 2006; Picot and Lacroix, 2004). Adhikari et al. (2000) found that non-encapsulated Bidobacterium longum in yogurt showed reductions of viability of 7870% for the two strains tested when stored for 30 days under refrigeration, while for the encapsulated cells with c-carrageenan showed no difference in bacterial population during refrigerated storage. Finally, Krasaekoopt et al. (2006) found an increase of 1 log in the viability of encapsulated alginate chitosan-coated cells of L. acidophilus 547 and L. casei 01 compared against free cells in yogurt under refrigeration at 4 C for 28 days. 4. Conclusions The viability of both free and immobilized L. plantarum BL011 was not affected by the SIJ. However, the SGJ drastically reduces the viability of L. plantarum under all tested conditions, and while resistance to simulated gastric juice was low, a viable population remained in the samples, showing a good resistance to the acid conditions tested. The microencapsulation in ALGQ was effective in maintaining the stability of the probiotic under storage at refrigeration temperature and when this method of encapsulation was

128

G.B. Brinques, M.A.Z. Ayub / Journal of Food Engineering 103 (2011) 123128 zer, B., Uzun, Y.S., Kirmaci, H.A., 2008. Effect of microencapsulation on viability of L. acidophilus LA-5 and B. bidum BB-12 during kasar cheese ripening. International Journal of Dairy Technology 61 (3), 237244. zer, B., Kirmaci, H.A., Senel, E., Atamer, M., Hayaloglu, A., 2009. Improving the viability of B. bidum BB-12 and L. acidophilus LA-5 in white-brined cheese by microencapsulation. International Dairy Journal 19 (1), 2229. Pagano, J.C., 1998. Nueva legislacion del MERCOSUR para leches fermentadas. Industria Lechera 7 (13), 813. Picot, A., Lacroix, C., 2004. Encapsulation of bidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. International Dairy Journal 14 (6), 505515. Prado, F.C., Parada, J.L., Pandey, A., Soccol, C.R., 2008. Review: trends in non-dairy probiotic beverages. Food Research International 41 (2), 111123. Sheu, T.Y., Marshall, R.T., 1993. Microentrapment of lactobacilli in alginate gels. Journal of Food Science 58 (3), 557561. Souza, C.H.B., Saad, S.M.I., 2009. Viability of L. acidophilus La-5 added solely or in coculture with a yoghurt starter culture and implications on physico-chemical and related properties of Minas fresh cheese during storage. LWT Food Science and Technology 42 (2), 633640. Stanton, C., Ross, R.P., Fitzgerald, G.F., Van Sinderen, D., 2005. Fermented functional foods based on probiotics and their biogenic metabolites. Current Opinion in Biotechnology 16 (2), 198203. Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., Kailasapathy, K., 2000. Encapsulation of probiotic bacteria with alginatestarch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt. International Journal of Food Microbiology 62 (12), 4755. Vasiljevic, T., Shah, N.P., 2008. Review: probiotics from metchnikoff to bioactives. International Dairy Journal 18 (7), 717728.

Krasaekoopt, W., Bhandari, B., Deeth, H., 2006. Survival of probiotics encapsulated in chitosan-coated alginate beads in yoghurt from UHT- and conventionally treated milk during storage. LWT Food Science and Technology 39 (2), 177 183. Lee, K.Y., Heo, T.R., 2000. Survival of bidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution. Applied and Environmental Microbiology 66 (2), 869873. Mandal, S., Puniya, A.K., Singh, K., 2006. Effect of alginate concentrations on survival of microencapsulated L. casei NCDC-298. International Dairy Journal 16 (10), 11901195. Martoni, C., Bhathena, J., Jones, M.L., Urbanska, A.M., Chen, H., Prakash, S., 2007. Investigation of microencapsulated BSH active Lactobacillus in the simulated human GI tract. Journal of Biomedicine and Biotechnology 2007, Article no. 13684, 9p. Mattila-Sandholm, T., Myllrinen, P., Crittenden, R., Mogensen, G., Fondn, R., Saarela, M., 2002. Technological challenges for future probiotic foods. International Dairy Journal 12 (23), 173182. Michida, H., Tamalampudi, S., Pandiella, S., Webb, C., Fukuda, H., Kondo, A., 2006. Effect of cereal extracts and cereal ber on viability of L. plantarum under gastrointestinal tract conditions. Biochemical Engineering Journal 28 (1), 73 78. Mokarram, R.R., Mortazavi, S.A., Naja, M.B.H., Shahidi, F., 2009. The inuence of multi stage alginate coating on survivability of potential probiotic bacteria in simulated gastric and intestinal juice. Food Research International 42 (8), 1040 1045. Nazzaro, F., Fratianni, F., Sada, A., Orlando, P., 2008. Symbiotic potential of carrot juice supplemented with Lactobacillus spp. And inulin or fructooligosaccharides. Journal of the Science of Food and Agriculture 88 (13), 22712276.