African Journal of Microbiology Research Vol. 6(26), pp.5511-5517, 12 July, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.972 ISSN 1996-0808 ©2012 Academic Journals
Full Length Research Paper
Microencapsulation of Lactobacillus casei with calcium alginate-resistant starch and evaluation of survival and sensory properties in cream-filled cake Mohammad Ali Khosravi Zanjani1*, Babak Ghiassi Tarzi1, Anousheh Sharifan1, Nima Mohammadi2, Hossein Bakhoda3 and Mohammad Mehdi Madanipour1 1
Department of Food Science and Technology, Faculty of Agriculture, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2 Department of Food Science and Technology, Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran. 3 Department of Agricultural Mechanization, Faculty of Agriculture, Science and Research Branch, Islamic Azad University, Tehran, Iran. Accepted 8 June, 2012
Lactobacillus casei ATCC 39392 was encapsulated with calcium alginate-resistant starch via emulsion technique. The probiotics bacteria were inoculated to cream-filled cake in their free and microencapsulated forms. The survival of free and microencapsulated bacteria and pH changes in cream-filled cake were monitored during 4 weeks storage at 4 and 25°C. The morphology and size of microcapsules were measured by optical microscopy, scanning electron microscopy (SEM) technique and particle size analyzer. The results showed that the pH changes of cream-filled cake with microencapsulated L. casei were slower than product containing free L. casei during storage. The survival rate of microencapsulated L. casei was significantly higher than free bacteria (P < 0.05) due to the protective property of calcium alginate capsules and low temperature (4°C). The inoculation of probiotic culture either in encapsulated or free state had no significant effect on texture, color, flavour, taste and overall sensory characterization of cream filled cake over the storage period at 4°C (P > 0.05). The results also indicated that calcium alginate-resistant starch enhanced the survival rate of probiotic bacteria in the product. Key words: Microencapsulation, microcapsules, survival, cream-filled cake, L. casei.
INTRODUCTION Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit to the consumers (FAO/WHO, 2001). Some of these health advantages are: reduction of serum cholesterol, alleviation of lactose malabsorption symptoms, cancer suppression, resistance to infectious gastro-intestinal (GI) disease and stimulation of GI immunity (Ajmal and Nuzhat, 2009). Bifidobacteria and Lactobacillus species are commonly used as probiotic food supplements to achieve a beneficial effect. The daily
*Corresponding author.
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intake of probiotic bacteria should exceed 10 living bacteria per milliliter or per gram of the product (AragonAlegro et al., 2007; Adams, 1999). Prebiotics are non digestive food ingredients that stimulate indigenous bacteria of the host such as: resistant starch, inulin, and fructo oligosaccharides. Synbiotic is a product containing a combination of probiotic and prebiotic (Capela et al., 2006; Saminathan et al., 2011). The survival and efficiency of probiotic bacteria is influenced by several factors such as: temperature, osmotic condition, freezing, drying, processing, bile and enzymes of digestive system, and water activity (Anal and Singh, 2007; Kailasapathy, 2002, 2006; Sabikhi et al., 2010).
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Various methods are used to protect probiotics from adverse environmental conditions where they may not normally survive. Microencapsulation of the probiotic bacteria in calcium alginate as a biopolymer matrix has been widely used to provide a favorable physical barrier against adverse environmental conditions (Allan-Wojtas et al., 2008; Truelstrup-Hansen et al., 2002; Khalil and Mansour, 1998). Alginate is a natural heteropolysaccharide composed of D-mannuronic and Lguluronic acid residues joined linearly by (1-4) glycosides linkages (Mokarram et al., 2009; Allan-Wojtas et al., 2008). It is simple to handle, non toxic, low cost, efficient technique (Mokarram et al., 2009; Truelstrup-Hansen et al., 2002; Allan-Wojtas et al., 2008). Combination of calcium alginate with prebiotics such as resistant starch, does not only improve the viability of probiotics but allows creation of an integrated structures of capsules (Sultana et al., 2000; Mirzaei et al., 2012). Microencapsulation can be employed via extrusion and emulsion techniques. Emulsion technique is a method for encapsulation of probiotic bacteria in the capsules smaller than 1 mm (Kailasapathy, 2002; Chandramouli et al., 2004). Nowadays, probiotics are used in many non-dairy products such as baby foods, meats and cereal-based products (Rivera-Espinoza and Gallardo-Navarro, 2010; Weinbreck et al., 2010). Since non-dairy products are not usually a suitable carrier for probiotics in comparison with dairy products, the encapsulation technique has been applied to improve the probiotic survival (Weinbreck et al., 2010; Muthukumarasamy and Holley, 2006). The encapsulated probiotic bacteria have been incorporated in many food products such as frozen ice milk (Sheu et al., 1993), ice cream (Homayouni et al., 2009), mayonnaise (Khalil and Mansour, 1998), milk (TruelstrupHansen et al., 2002), yogurt (Kailasapathy, 2006), bread (Altamirano-Fortoul et al., 2012), cheese (Zomorodi et al., 2011; Mirzaei et al., 2012), sausage (Muthukumarasamy and Holley, 2006), infant food (Weinbreck et al., 2010), and chocolate (Possemiers et al., 2010). The inoculation of microencapsulated probiotics with calcium alginatestarch to cream-filled cake has not yet been reported. Since cakes containing cream filling are consumed by a large number of people, improving this product to a functional food has probably an important role in promoting human health. In this study Lactobacillus casei was inoculated into the filling and this probiotic filling was then injected into the cakes. The objective of this study is to evaluate the survival of free and microencapsulated L. casei in cream-filled cake over a period of 4 weeks at different storage temperature (4 and 25°C) and to determine sensorial acceptance of the product. MATERIALS AND METHODS Preparation of cell suspension L. casei ATCC 39392 (American Type Collection Culture) was purchased from Iran Scientific and Industrial Organization.
Lyophilized cells were transferred into the 5 ml de Man, Rogosa and Sharpe (MRS) broth and incubated for 24 h under aerobic condition at 37°C. The culture were then added into 100 ml MRS broth and incubated under similar condition. The cells were harvested by centrifugation at 3000 g for 5 min at 4°C and were washed twice with peptone water (0.1%, w/v).
Encapsulation procedure All glasswares and solutions used in the procedure were sterilized at 121°C for 15 min. The microencapsulation of L. casei was carried out by modified method of Sheu et al. (1993). Briefly, 3 g of sodium alginate (Sigma-Aldricht, batch number = 71238, London, UK) and 2 g of resistant starch (Hi-maize 260 national starch, London, UK) were added to 100 ml of distilled water and heated slightly (50°C) until complete dissolution. Cell suspension (0.1%, w/v) was dispersed into the solution. The mixture was transferred into 500 ml of corn oil containing 0.2% tween 80 and it was stirred (350 rpm for 30 min) until emulsification. The emulsion was broken by quickly adding calcium chloride (500 ml, 0.1 M) into the mixture while stirring (50 rpm) and the phase separation of oil/water emulsion occurred. The mixture was allowed to stand for 30 min to settle calcium alginate capsules in the bottom of beaker at the calcium chloride layer (Water phase). The oil layer was drained and capsules in calcium chloride solution were harvested by low speed centrifuge at 350 g for 10 min.
Preparation of cream-filled cake Water-based filling, was prepared by using the following formula: sugar (33%), corn starch (6%), skim milk powder (5%), sorbitol (6%), glycerin (7%), invert syrup (16%), water (17%), glucose syrup (9.4%), salt (0.3%), and flavor (0.3%). The probiotics bacteria were inoculated to the filling, individually, in three forms: nonencapsulated, encapsulated with calcium alginate, and encapsulated with calcium alginate-starch. The filling was then injected into the cakes with sterile syringe. Cakes were stored for 4 weeks at 4 and 25°C.
Size and morphology of microcapsules The size of microcapsules was determined by particle size analyzer (Master sizer 2000, Malvern, UK) with the standard deviation calculated from the cumulative distribution curve. Scanning electron microscopy (SEM) (XL30, Royal Philips Electronics, Amsterdam, Netherlands) and optical microscopy (Motic BA300, Wetzlar, Germany) were used to observe the surface and morphology of microcapsules. The capsules were placed on a specimen aluminum stub with the help of double sided sticky tape and were coated by sputter coater for 2 min. The surface morphology of the capsules was investigated by the SEM (XL30, Philips, Royal Philips Electronics, Amsterdam, Netherlands) at 10 kV.
Release of entrapped bacteria For enumeration of entrapped L. casei in product, the microencapsulated bacteria were released by buffer phosphate (Sultana et al., 2000). Ten grams of filling were resuspended in 99 ml sterile buffer phosphate (pH: 7, 0.1 M). The capsules were disintegrated by homogenizing in a stomacher (Funk Gerber, Weilheim, Germany) for 20 min at 4°C. The counts (CFU/g) were determined by plating on MRS agar plates and incubating for 48 h at 37°C. The cream-filled cake containing free bacteria were treated similarly. All samples were counted in triplicates.
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Figure 1. Optical microscope image at 40× magnification showing: (a) calcium alginate-starch, and (b) calcium alginate without starch.
a
b
Figure 2. Scanning electron photomicrograph showing: (a) calcium alginate without starch, and (b) calcium alginate-starch.
pH changes of filling and sensory evaluation of cream filled cake The pH changes of cream-filled cake samples during storage were determined according to the Institute of Standards and Industrial Research of Iran (ISIRI) regulation (ISIRI, No 2553, 2005). The pH value was determined using 10% dispersion of samples in distilled water using a pH meter (pH meter model 537, Weilheim, Germany). The pH meter was calibrated by standard buffer solutions 4 and 7. Sensory evaluation of cakes containing cream filling was performed after 4 weeks of storage. A panel consisting of 10 trained panelists, assed the appearance, texture, flavour, and taste of cream filled cake at room temperature. Scoring was carried out on a scale of 1 to 10, with 10 being most desirable.
Statistical analysis A complete randomized factorial design was used for all analysis and all results were means of three replicates. Data analysis was carried out using Statistical Package for Social Sciences (SPSS) Inc. software (20: SPSS Inc., Chicago, IL). The mean differences were analyzed by Duncan’s multiple range test.
RESULTS AND DISCUSSION Shape and size of calcium alginate microcapsules Optical microscopy and SEM showed that the shape of microcapsules was generally spherical and the surface of calcium alginate without starch was smooth (Figures 1b and 2a). SEM and optical images of calcium alginatestarch also showed that the starch granules were present in the alginate matrix (Figures 1a and 2b).The size distribution of calcium alginate-starch was analyzed by particle size analyzer as shown in Figure 3. Microcapsule diameter ranged from 70 to 500 µm and mean diameter of microcapsules was 280 µm. In this technique, the capsules are formed in micron range size and deliver smooth texture when they are added to food product, while other researchers produced millimeter size capsules which gave gritty texture (Hyndman et al., 1993; Arnaud et al., 1992). Different studies have shown that size reduction of the capsules to less than 100 µm would
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Figure 3. Particle size distribution of calcium alginate-starch.
Table 1. pH changes of cream-filled cake during storage.
Week
Control
0 1 2 3 4
6.5 ± 0.1 6.5 ± 0.1 6.5 ± 0.1 6.5 ± 0.1 6.5 ± 0.13
Cream filled cake with free bacteria 6.5 ± 0.1 6.2 ± 0.2 6.0 ± 0.2 5.88 ± 0.2 5.88 ± 0.12
not offer any significant increase in survival rate of the probiotics on the gastric secretion condition (Woraharn et al., 2010; Truelstrup-Hansen et al., 2002; Hyndman et al., 1993). Despite the fact that the micron size capsules had minor barrier effect, it created less alternation in composition of food product and further inhibit the sandy texture (Mokarram et al., 2009; Truelstrup-Hansen et al., 2002).
pH changes during the cream-filled cake storage The pH changes of the control, free and microencapsulated probiotic bacteria in cream-filled cake during 4 weeks storage are represented in Table 1. The pH of cream-filled cake inoculated with microencapsulated L.casei reached 6.38 at the end of 4 weeks storage, whereas for the samples of free bacteria, the final pH attained 5.88. This may be due to slow absorption of nutrients and sluggish release of metabolites through the calcium alginate-starch matrix (Homayouni et al., 2009; Sultana et al., 2000). It has been suggested that one of the main factor in metabolic activity of microencapsulated probiotic bacteria is the size
Cream filled cake with microencapsulated bacteria 6.5 ± 0.1 6.45 ± 0.15 6.42 ± 0.18 6.38 ± 0.2 6.38 ± 0.11
of the capsules (Mandal et al., 2006; Zomorodi et al., 2011; Larisch et al., 1994).
Survival of free and encapsulated L. casei In this experiment, L. casei was inoculated to cream-filled cake in three forms: free or non-encapsulated, encapsulated with calcium alginate, and encapsulated with calcium alginate-starch. The survival rate of L. casei in the product was monitored during 4 weeks storage period at two different temperatures: 4 and 25°C (Figure 11 4). The initial cell count of L. casei was 5 × 10 CFU/g in all three samples (Figure 4). The cell number in free L. casei samples dropped considerably (about 9.15 log numbers) after 4 weeks of storage at 25°C, whereas in microencapsulated samples with calcium alginate and calcium alginate-starch, the cell number reduction was about 5.61 and 4.18 log, respectively at the similar condition. The final count after 4 weeks storage at 4°C showed 8.09 log cycle decreases in free L. casei samples, while there was a loss of only 4.55 and 3.33 log for the calcium alginate and calcium alginate-starch samples, respectively (Figure 4). The difference between
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Figure 4. Survival of free and encapsulated L. casei (calcium alginate and calcium alginate-starch) incorporated into cream-filled cake during storage.
the final cell numbers of free and encapsulated L. casei were significant (P < 0.05). Microencapsulation can improve the survival of bacterial cells by protecting them in matrix of alginate and starch (Mirzaei et al., 2012; Sultana et al., 2000; Sheu et al., 1993; Kailasapathy, 2006). The results demonstrated the positive role of incorporation of resistant starch into the alginate matrix during microencapsulation technique (Sultana et al., 2000; Kailasapathy, 2006; Sabikhi et al., 2010; Mirzaei et al., 2012). Comparison of survival rate after 1 and 4 weeks revealed that storage time had significant effect on the viability of free cells (P < 0.05) (Figure 4). This finding is in agreement with those of Sultana et al. (2000), Kailasapathy et al. (2006), Muthukumarasamy and Holley (2006), and Krasaekoopt et al. (2004). Furthermore, our findings displayed that, encapsulated L. casei required longer time to decline 1 log cycle in live cells. Thus, the survival of bacteria in cream-filled cake at 4°C is better than at 25°C. Probiotics survived significantly greater in microencapsulated form as compared to free form. In this
study, the results determined that encapsulation with incorporation of resistant starch improved cells viability when compared with encapsulation without starch. Calcium alginate-starch can significantly increase the survival of L. casei in cream filled cake during storage (P < 0.05).
Sensory evaluation of cream-filled cake The average sensory scores obtained from 10 panelists are represented in Table 2. The results showed that there was no significant difference (P > 0.05) in the total acceptability of the cream-filled cake samples. Therefore, the panelist could not identify the difference in the appearance, color, body, texture, flavor, and taste between cream-filled cake with encapsulated L. casei, free L. casei and the control. Also, the body and texture of the cream-filled cake samples did not show significant difference (P > 0.05) between the cream-filled cake
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Table 2. Sensory properties of cream-filled cake.
Sample A B C
Color and appearance 1 - 10 9.47 ± 0.07 9.41 ± 0.1 9.40 ± 0.15
Body and texture 1 - 10 9.53 ± 0.13 9.54 ± 0.12 9.54 ± 0.14
Flavor and taste 1 - 10 9.15 ± 0.05 9.10 ± 0.06 9.14 ± 0.07
Total acceptability 1 - 10 9.38 ± 0.11 9.35 ± 0.06 9.36 ± 0.03
A: Cream-filled cake with free L. casei, B: Cream-filled cake with encapsulated L. casei, and C: Cream-filled cake without probiotic (control).
containing free bacteria and encapsulated bacteria.
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