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Effect of calcium alginate and resistant starch microencapsulation on the survival rate of Lactobacillus acidophilus La5 and sensory properties in Iranian white brined cheese
Food Chemistry 132 (2012) 1966–1970
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of calcium alginate and resistant starch microencapsulation on the survival rate of Lactobacillus acidophilus La5 and sensory properties in Iranian white brined cheese H. Mirzaei a, H. Pourjafar b,⇑, A. Homayouni c,⇑ a b c
Department of Food Hygiene, Tabriz Branch, Islamic Azad University, Tabriz, Iran Tabriz Branch, Islamic Azad University, Tabriz, Iran Department of Food Science and Technology, Faculty of Health and Nutrition, Tabriz University of Medical Science, Tabriz, Iran
a r t i c l e
i n f o
Article history: Received 4 August 2011 Received in revised form 3 November 2011 Accepted 8 December 2011 Available online 17 December 2011 Keywords: Calcium alginate Resistant starch Microencapsulation Lactobacillus acidophilus Survival Iranian white brined cheese
a b s t r a c t Two types of probiotic cheese, with free and microencapsulated bacteria, were manufactured in triplicate under the same conditions. The number of viable cells during 182 days of storage in refrigerated conditions was evaluated. The number of viable cells of Lactobacillus acidophilus was reduced significantly from day 28 to day 182 of storage period in both types of cheese, but reduction in the cheese containing free cells (5.1 ± 0.67 log cfu g 1) was significantly p < 0.05 higher than the cheese containing microencapsulated cells (11.00 ± 0.58 log cfu g 1). The results showed that, microencapsulation in calcium alginate gel and resistant starch was able to increase the survival rate of L. acidophilus La5 in Iranian white brined cheese after 6 months of storage. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fuller defined probiotics as ‘‘a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance’’ (Klaenhammer, 2001). Probiotic (which means for life) bacteria have long been considered to influence general health and safety with their commensal relationship with the gastrointestinal tract and its normal micro flora (Klaenhammer, 2001). Due to their health benefits, probiotic bacteria have been incorporated into dairy products, including soft and hard cheeses, ice cream, yoghurt, and frozen dairy desserts (Anal & Singh, 2007). Foods containing such bacteria fall within the ‘‘functional foods’’ class and these foods should contain at least 107 cfu g 1 probiotic bacteria and consumed at levels higher than 100 g/day to have helpful effects on health (Anal & Singh, 2007; Homayouni, 2008; Klaenhammer, 2001). Nevertheless, there are still some problems with respect to the low survival of probiotic bacteria in dairy foods as well as gastrointestinal conditions (Homayouni, Ehsani, Azizi, Razavi, & Yarmand, 2008b). This subject encouraged researchers to investigate different techniques for survival improvement of probiotics ⇑ Corresponding authors. Tel.: +98 411 2804828 (H. Pourjafar), tel.: +98 411 3357581; fax: +98 411 3340634 (A. Homayouni). E-mail addresses: [email protected] (H. Pourjafar), homayounia@tbzmed. ac.ir (A. Homayouni). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.12.033
(Pourjafar, Mirzaei, & Manafi, 2007). Microencapsulation of the probiotic cells is one of the newest and most efficient techniques. Microencapsulation via extrusion and emulsion techniques has been employed for the protection of probiotic bacteria against hostile environmental conditions (Anal & Singh, 2007; Homayouni, Azizi, Ehsani, Yarmand, & Razavi, 2008a; Pourjafar, 2010, 2011; Özer, Kirrmaci, Sßenel, Atamer, & Hayalog˘lu, 2009). In addition microencapsulation via calcium alginate has been used extensively for immobilization of lactic acid bacteria (LAB) because of its simplicity of handling, its non-toxic nature, and its low cost (Anal & Singh, 2007; Hansen, Allan-Wojtas, Jin, & Paulson, 2001; Homayouni et al., 2008a; Mandal, Puniya, & Singh, 2006; Pourjafar, 2010, 2011; Simpson, Stabler, Simpson, Sambanis, & Canstantinidis, 2004; Sultana et al., 2000). Combining alginate with starch leads to better efficiency of different bacterial cells particularly LAB. Also combination of calcium alginate with starch produces beads with a good integrated structures and prebiotic effect of the bead shell (Homayouni et al., 2008a). Lactobacillus acidophilus is a probiotic microorganism existing in usual fermented foods and nutritional supplements. Cheese could proffer certain benefits over yoghurt-type products to carry viable probiotics to gastrointestinal tract (GIT). Higher pH of cheese, higher fat content, and more total solids of cheese may offer protection for the probiotic cells against the harsh gastrointestinal environment (Corbo, Albenzio, de Angelis, Sevi, & Gobbetti, 2001;
H. Mirzaei et al. / Food Chemistry 132 (2012) 1966–1970
Kasimog˘lu, Göncüog˘lu, & Akgün, 2004; Pourjafar, 2010; Vinderola, Prosello, Ghiberto, & Reinheimer, 2000). White cheese is a semi-hard cheese and is manufactured in many regions around the world, especially, in the Middle East. It has different names, for example, Telema, Feta, Danni, Jibnah and Bulgarian white cheese. Iranian white brined cheese is a brine-ripened cheese which mainly is produced in Lighvan Valley (Tabriz, Iran) and is famous for Lighvan cheese. Lighvan cheese is one of the most consumed types of cheese in Iran and its probiotic viability is very important. The aim of this study was to evaluate the survival rate of free and microencapsulated L. acidophilus La5 by calcium alginate and resistant starch in Iranian white cheese during manufacture and storage time. 2. Material and methods
1967
was carried out at pilot plant, adapting industrial technology for white-brined Iranian cheese. Milk was pasteurized at 72 °C for 15 s and then cooled down to the coagulating temperature (32 °C). Then calcium chloride was added as a solution (40%, w/v) at a level of 0.02% (v/v). To incorporate probiotic strain into milk, first, free bacteria of L. acidophilus La5 was incorporated into sample A containing 15 L milk at a level of 1 1011 cfu ml 1. Then, microencapsulated bacteria (see Section 2.2) were incorporated into sample B. The counts of probiotic cells in this type of cheese were approximately 1 1012 cfu ml 1. The inoculated milk was then left undisturbed for an hour to allow acidity development and was coagulated via rennet within 120 min. The coagulated milk was then cut into small cubes (5 5 5 cm3). The cubes were brine-salted (12% w/w, NaCl) at 5 °C for 182 days (Pourjafar, 2010). Cheese samples for analyses were taken at time 0 and during 182 days with two weeks as interval time.
2.1. Preparation and enumeration of free and encapsulated probiotics 3. Statistical analyses Pure probiotic culture of L. acidophilus La5 was obtained from CHR-Hansen (Harsholm, Denmark) and inoculated into MRS-broth (de Man-Rogosa-Sharpe) and incubated at 37 °C for 24 h under aerobic conditions. The probiotic biomass in late-log phase was collected by centrifugation (Centrion Centrifuge, Model 2010, West Sussex, BNI8OHY, UK) at 10,000 rpm for 10 min., and then it was washed twice in sterile saline before using in the microencapsulation procedure. Bacterial counts were determined immediately after manufacture of cheese (at time 0) and during 182 days at 5 °C. The samples of cheese (10 g) were diluted in 100 ml sterile peptone water (0.1%) and 1 ml aliquot dilutions were poured onto plates of the MRS-Salicin-agar. Salicin (10 ml solution at 10% w/v) was added into 90 ml of sterilized MRS agar (Vinderola & Reinheimer, 2000; Shah, 2000; Sultana et al., 2000). Enumeration of the probiotic bacteria was achieved as described by Shah (2000) and Sultana et al. (2000). All enumerating plates of L. acidophilus were incubated at 37 °C for 72 h under aerobic condition. The averages were expressed as colony-forming units per gram of sample (cfu g 1). To count the microencapsulated bacteria in cheese, the entrapped bacteria were released from the beads according to the method of Sheu and Marshall (1993). Ten grams of cheese were re-suspended in 100 ml of phosphate buffer (0.1 M, pH 7.0) followed by 15 min shaking on a shaker (IKA-Model Janke & Kunkel GMBH. Type VX5-Germany). The cheese sample containing free bacteria was treated in a similar way so as to maintain the same treatment conditions. All experiments were done in triplicate. 2.2. Microencapsulation procedure In this study extrusion technique was performed for microencapsulation process described earlier by Sheu and Marshall (1993), Sultana et al. (2000), Kailasapathy (2002), Krasaekoopt, Bhandari, and Deeth (2004). A 2% Na–alginate mixture in distilled water containing 2% Hi-maize resistant starch (Merck, Darmstadt, Germany) and 0.1% culture was prepared. Then, the mixture of cell suspension and Na–alginate and resistant starch were injected into a 0.1 M CaCl2 solution through a sterile insulin syringe. The droplets formed gel spheres immediately. The distance between the syringe and CaCl2 solution was 25 cm. Diameter of the resultant beads was 50–80 lm (Pourjafar, 2011). 2.3. Probiotic cheese manufacture Raw bovine milk (30 L) was obtained from Pegah Dairy Co., (Tabriz, Iran). The milk was divided into two parts. Two types of probiotic cheese, with free and microencapsulated probiotic bacteria, were manufactured under the same conditions. Cheese making
All statistical analysis were performed by SPSS 11.5 (SPSS Inc., Chicago, IL) software. Data were presented as mean (SD) or median (Quartile1–Quartile3). The normality of the total acceptability score was tested and confirmed by Kolmogorov–Smirnov test. The mean of Total acceptability score were compared between two cheese type using independent samples t-test and this was compared between 60 days and 182 days for each cheese type using paired samples t-test. These comparisons were carried out for colours and appearance, for body and texture and for flavour and taste by Mann–Whitney and Sign test, respectively. p < 0.05 considered to be as significant. 4. Results and discussion 4.1. Shape, size and internal appearance of alginate starch beads It is possible to determine the distribution size of the beads by optical microscopy. In this study the diameters of 40 randomly selected beads were measured with an eyepiece micrometre on an optical microscope at a magnification of 40. Also, the surface morphology of calcium alginate beads was investigated by optical microscope (Homayouni, Ehsani, Azizi, Yarmand, & Razavi, 2007). Photographs in Fig. 1(a and b) shows the shape and size. Also, the internal appearance of coated alginate-resistant starch beads is shown in Fig. 2. Optical microscopic image of bead at 40 magnification showed that the bead shape was spherical with a mean diameter of about 50–80 lm, and cross-section and internal appearance of coated alginate-resistant starch bead at 100 magnification showed that bacterial cells were located inside of beads. The distribution of beads in cheese texture was uniform (Fig. 3). Similar shapes of beads were also shown by Sheu and Marshall (1993), Sultana et al. (2000), Krasaekoopt et al. (2004) and Homayouni et al. (2007). 4.2. Survival of free and microencapsulated L. acidophilus La5 in cheese during storage time L. acidophilus La5 was added as adjunct strain during whitebrined cheese manufacture in free (sample A) or microencapsulated (sample B) form into the vats. Variations in counts of free and microencapsulated L. acidophilus La5 during 182 days storage period at 5 °C as (cfu gr 1) are shown in Figs. 4.1 and 4.2. Live cell counts in cheese samples were determined from 0 until 182 days with two weeks of intervals (Table 1). In day zero, the average number of viable cells in cheese containing free bacteria (10 ± 0.58 log cfu g 1) was less than cheese containing the
1968
H. Mirzaei et al. / Food Chemistry 132 (2012) 1966–1970
Fig. 1. Optical microscope image of coated alginate–resistant starch bead (a and b) at 40 magnification (diameter: 50–80 lm).
14
Fig. 2. Vertical cross-section and internal appearance of coated alginate–resistant starch bead at 100 magnification.
Counts of free La5 (Log cfu/g)
13 12 11 10 9 8 7 6 5 4 3
Free La5
2
Linear (Free La5)
1 0 0
14
28
42
56
70
84
98 112 126 140 154 168 182
Storage time (days)
Fig. 3. Optical microscope image of coated alginate–resistant starch beads at 40 magnification inside cheese matrix.
microencapsulated bacteria (12.32 ± 0.35 log cfu g 1); however, during 28 days of storage period, the number of viable cells in both types of cheese increased their average rate in cheese containing free cells (12.09 ± 0.59 log cfu g 1) to a level higher than the cheese containing microencapsulated cells (12.59 ± 0.23 log cfu g 1). Number of viable cells of Lactobacillus acidophilus reduced significantly from days 28 to 182 of storage period in both types of cheese, but reduction rate in cheese containing free cells (5.1 ± 0.67 log cfu g 1) was significantly (p < 0.01) higher than the cheese containing microencapsulated cells (11 ± 0.58 log cfu g 1). Similar results were reported by Kailasapathy and Masondole (2005), Özer, Uzun, and Kirrmaci (2008), Özer et al. (2009) in brine ripened cheese. This study showed that microencapsulation is able
Counts of microencapsulated La5 (Log cfu/g)
Fig. 4.1. Survival of free L. acidophilus (La5) cells in Iranian white brined cheese during 182 days of storage at 5 °C.
14 13 12 11 10 9 8 7 6 5
microencapsulated La 5
4
Linear (microencapsulated La 5)
3 2 1 0 0
14
28
42
56
70
84
98 112 126 140 154 168 182
Storage time (days) Fig. 4.2. Survival of microencapsulated L. acidophilus (La5) cells in Iranian white brined cheese during 182 days of storage at 5 °C.
1969
182
5.10 ± 0.66 11.00 ± 0.58
168
6.12 ± 0.59 10.08 ± 1.04
154
6.22 ± 0.62 10.83 ± 1.48
140
7.06 ± 0.58 10.84 ± 0.93 8.01 ± 0.59 11.26 ± 0.63
6.19 ± 0.74 10.23 ± 0.79
112
8.04 ± 0.59 10.95 ± 0.98 8.76 ± 0.39 10.62 ± 0.62 9.12 ± 0.59 11.32 ± 0.66
70
11.00 ± 0.58 12.01 ± 0.58
56 42
12.08 ± 0.58 12.58 ± 0.22 11.71 ± 0.36 13.04 ± 0.55 10.00 ± 0.58 12.31 ± 0.34
28 14 0
A B
Live probiotic cell counts (Mean log ± SE) during storage at days:
Cheese samples
Table 1 Live probiotic cell counts in cheese samples during 182 days with two week intervals.
10.30 ± 0.65 12.00 ± 0.58
84
98
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H. Mirzaei et al. / Food Chemistry 132 (2012) 1966–1970
Table 2 Sensory scores of 60-day-old cheeses and 182-day-old cheeses: sample A (containing probiotic bacteria in the free state); sample B (containing probiotic bacteria in the microencapsulated state). Samples
Colour and appearance (1–5)
Body and texture (1–5)
60 days A B
5 (4–5) 5 (4–5)
5 (4–5) 5 (4–5)
182 days A B
5 (4–5) 5 (4–5)
4 (3–4) 4 (4–5)
Flavour and taste (1–10) 9 (8.50–9) 10 (8.50–10) 9 (8–9) 9 (9–9.50)
Total acceptability (1–20) 18.00 ± 0.707 18.80 ± 0.837 17.40 ± 0.894 18.40 ± 0.894
to keep the number of probiotic bacteria above the threshold level for therapeutic minimum (P107 cfu g 1) in cheese. 4.3. Sensory evaluations The sensory scores of 60-day-old and 182-day-old probiotic cheese samples are given in Table 2. The points allocated for colour, texture and taste showed that the addition of free and encapsulated probiotics had no significant effect on sensory properties of probiotic Iranian white brined cheese. Total evaluation in terms of colour, texture and taste of all samples were good and did not have any marked off flavour during the storage period and overall acceptability of cheese samples containing microencapsulated La5 was better than samples with free one. 5. Conclusion According to the results of this study, microencapsulation of L. acidophilus (La5) cells with calcium alginate gel and resistant starch can successfully keep the count of this probiotic bacterium high enough for the therapeutic minimum in the Iranian white brined cheese and can serve as a good carrier for delivering the probiotic bacterial cells into human gut. Higher pH of cheese, higher fat content and more total solid of cheese may offer a protection for the probiotic cells. In this study, at the end of four months of storage, the number of viable probiotic bacteria in two cheese types (see Table 1) were higher than that recommended by the International Dairy Federation (P107 cfu g 1). After four months, until the end of six months of storage period, the number of viable probiotic bacteria, only in cheese samples containing encapsulated cells (see Table 1), was higher than that recommended by IDF. However, further studies are needed to evaluate the survival of other probiotic strains in Iranian white brined cheese as well as investigate the protection effect of microencapsulation on the probiotic survival in animal models and human gastrointestinal tract. Acknowledgement The authors would like to express their thanks Dr. M. Asghari-Jafarabadi for his assistance in data analysis.
to
References Anal, A. K., & Singh, H. (2007). Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Food Science & Technology, 18, 240–251. Corbo, M. R., Albenzio, M., de Angelis, M., Sevi, A., & Gobbetti, M. (2001). Microbiological and biochemical properties of Canestrato pugliese hard cheese supplemented with Bifidobacteria. Journal of Dairy Science, 84, 551–561. Hansen, L. T., Allan-Wojtas, D. M., Jin, Y. L., & Paulson, A. T. (2001). Survival of Caalginate microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal conditions. Food Microbiology, 19, 35–45.
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Homayouni, A. (2008). Therapeutical effects of functional probiotic prebiotic and synbiotic food (1st ed.). Tabriz: Tabriz University of Medical Sciences, pp. 156. Homayouni, A., Azizi, A., Ehsani, M. R., Yarmand, M. S., & Razavi, S. H. (2008a). Effect of microencapsulation and resistant starch on the probiotic survival and sensory properties of synbiotic ice cream. Food Chemistry, 111, 50–55. Homayouni, A., Ehsani, M. R., Azizi, A., Razavi, S. H., & Yarmand, M. S. (2008b). Growth and survival of some probiotic strains in simulated ice cream conditions. Journal of Applied Sciences, 8, 379–382. Homayouni, A., Ehsani, M. R., Azizi, A., Yarmand, M. S., & Razavi, S. H. (2007). Effect of lecithin and calcium chloride solution on the microencapsulation process yield of calcium alginate beads. Iranian Polymer Journal, 16, 597–606. Kailasapathy, K. (2002). Microencapsulation of Probiotc bacteria: Technology and potential applications. Current Issues in Intestinal Microbiology, 3, 39–48. Kailasapathy, K., & Masondole, L. (2005). Survival of free and microencapsulated Lactobacillus acidophilus and Bifidobacterium lactis and their effect on texture of feta cheese. Australian Journal of Dairy Technology, 60, 252–258. Kasimog˘lu, A., Göncüog˘lu, M., & Akgün, S. (2004). Probiotic white cheese with Lactobacillus acidophilus. International Dairy Journal, 14, 1067–1073. Klaenhammer, T. R. (2001). Food microbiology: Fundamentals and frontiers (2nd ed.). Washington, DC: ASM Press. Krasaekoopt, W., Bhandari, B., & Deeth, H. (2004). The influence of coating materials on some properties of alginate beads and survivability of microencapsulated probiotic bacteria. International Dairy Journal, 14, 737–743. Mandal, S., Puniya, A. K., & Singh, K. (2006). Effect of alginate concentrations on survival of microencapsulated Lactobacillus casei NCDC-298. International Dairy Journal, 16, 1190–1195. Özer, B., Kirrmaci, H. A., S ß enel, E., Atamer, M., & Hayalog˘lu, A. (2009). Improving the viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in white-brined cheese by microencapsulation. International Dairy Journal, 19, 22–29.
Özer, B., Uzun, Y. S., & Kirrmaci, H. A. (2008). Effect of microencapsulation on viability of Lactobacillus acidophilus LA-5 and Bifidobacterium bifidum BB-12 during Kasar cheese ripening. International Journal of Dairy Technology, 61, 237–244. Pourjafar, H. (2010). Probiotic cheese process deploying probiotics microencapsulation technology. IR. Patent: 63525, Inventory record. Pourjafar, H. (2011). Multi nozzle extrusion Microencapsulator device, in order to probiotic microorganism microencapsulation. IR. Patent: 68678, Inventory record. Pourjafar, H., Mirzaei, H. & Manafi, M. (2007). A survey on some effective factors on survival of probiotic organisms in milk products. In Proceedings of the 5th National Biotechnology Congress of Iran (pp. 623). Vinderola, C. G., & Reinheimer, J. A. (2000). Enumeration of Lactobacillus casei in the presence of L. acidophilus, Bifidobacteria and lactic starter bacteria in fermented dairy products. International Dairy Journal, 10, 271–275. Shah, N. P. (2000). Probiotic bacteria: Selective enumeration and survival in dairy foods. Journal of Dairy Science, 83, 894–907. Sheu, T. Y., & Marshall, R. T. (1993). Microentrapment of Lactobacilli in calcium alginate gels. Journal of Food Science, 54, 557–561. Simpson, N. E., Stabler, C. L., Simpson, C. P., Sambanis, A., & Canstantinidis, I. (2004). The role of the CaCl2–guluronic acid interaction on alginate encapsulated bTC3 cells. Biomaterial, 25, 2603–2610. Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., & Kailasapathy, K. (2000). Encapsulation of probiotic bacteria with alginate–starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt. International Journal of Food Microbiology, 62, 47–55. Vinderola, C. G., Prosello, W., Ghiberto, D., & Reinheimer, J. A. (2000). Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and nonprobiotic microflora in Argentinean Fresco cheese. Journal of Dairy Science, 83, 1905–1911.
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of calcium alginate and resistant starch microencapsulation on the survival rate of Lactobacillus acidophilus La5 and sensory properties in Iranian white brined cheese H. Mirzaei a, H. Pourjafar b,⇑, A. Homayouni c,⇑ a b c
Department of Food Hygiene, Tabriz Branch, Islamic Azad University, Tabriz, Iran Tabriz Branch, Islamic Azad University, Tabriz, Iran Department of Food Science and Technology, Faculty of Health and Nutrition, Tabriz University of Medical Science, Tabriz, Iran
a r t i c l e
i n f o
Article history: Received 4 August 2011 Received in revised form 3 November 2011 Accepted 8 December 2011 Available online 17 December 2011 Keywords: Calcium alginate Resistant starch Microencapsulation Lactobacillus acidophilus Survival Iranian white brined cheese
a b s t r a c t Two types of probiotic cheese, with free and microencapsulated bacteria, were manufactured in triplicate under the same conditions. The number of viable cells during 182 days of storage in refrigerated conditions was evaluated. The number of viable cells of Lactobacillus acidophilus was reduced significantly from day 28 to day 182 of storage period in both types of cheese, but reduction in the cheese containing free cells (5.1 ± 0.67 log cfu g 1) was significantly p < 0.05 higher than the cheese containing microencapsulated cells (11.00 ± 0.58 log cfu g 1). The results showed that, microencapsulation in calcium alginate gel and resistant starch was able to increase the survival rate of L. acidophilus La5 in Iranian white brined cheese after 6 months of storage. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fuller defined probiotics as ‘‘a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance’’ (Klaenhammer, 2001). Probiotic (which means for life) bacteria have long been considered to influence general health and safety with their commensal relationship with the gastrointestinal tract and its normal micro flora (Klaenhammer, 2001). Due to their health benefits, probiotic bacteria have been incorporated into dairy products, including soft and hard cheeses, ice cream, yoghurt, and frozen dairy desserts (Anal & Singh, 2007). Foods containing such bacteria fall within the ‘‘functional foods’’ class and these foods should contain at least 107 cfu g 1 probiotic bacteria and consumed at levels higher than 100 g/day to have helpful effects on health (Anal & Singh, 2007; Homayouni, 2008; Klaenhammer, 2001). Nevertheless, there are still some problems with respect to the low survival of probiotic bacteria in dairy foods as well as gastrointestinal conditions (Homayouni, Ehsani, Azizi, Razavi, & Yarmand, 2008b). This subject encouraged researchers to investigate different techniques for survival improvement of probiotics ⇑ Corresponding authors. Tel.: +98 411 2804828 (H. Pourjafar), tel.: +98 411 3357581; fax: +98 411 3340634 (A. Homayouni). E-mail addresses: [email protected] (H. Pourjafar), homayounia@tbzmed. ac.ir (A. Homayouni). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.12.033
(Pourjafar, Mirzaei, & Manafi, 2007). Microencapsulation of the probiotic cells is one of the newest and most efficient techniques. Microencapsulation via extrusion and emulsion techniques has been employed for the protection of probiotic bacteria against hostile environmental conditions (Anal & Singh, 2007; Homayouni, Azizi, Ehsani, Yarmand, & Razavi, 2008a; Pourjafar, 2010, 2011; Özer, Kirrmaci, Sßenel, Atamer, & Hayalog˘lu, 2009). In addition microencapsulation via calcium alginate has been used extensively for immobilization of lactic acid bacteria (LAB) because of its simplicity of handling, its non-toxic nature, and its low cost (Anal & Singh, 2007; Hansen, Allan-Wojtas, Jin, & Paulson, 2001; Homayouni et al., 2008a; Mandal, Puniya, & Singh, 2006; Pourjafar, 2010, 2011; Simpson, Stabler, Simpson, Sambanis, & Canstantinidis, 2004; Sultana et al., 2000). Combining alginate with starch leads to better efficiency of different bacterial cells particularly LAB. Also combination of calcium alginate with starch produces beads with a good integrated structures and prebiotic effect of the bead shell (Homayouni et al., 2008a). Lactobacillus acidophilus is a probiotic microorganism existing in usual fermented foods and nutritional supplements. Cheese could proffer certain benefits over yoghurt-type products to carry viable probiotics to gastrointestinal tract (GIT). Higher pH of cheese, higher fat content, and more total solids of cheese may offer protection for the probiotic cells against the harsh gastrointestinal environment (Corbo, Albenzio, de Angelis, Sevi, & Gobbetti, 2001;
H. Mirzaei et al. / Food Chemistry 132 (2012) 1966–1970
Kasimog˘lu, Göncüog˘lu, & Akgün, 2004; Pourjafar, 2010; Vinderola, Prosello, Ghiberto, & Reinheimer, 2000). White cheese is a semi-hard cheese and is manufactured in many regions around the world, especially, in the Middle East. It has different names, for example, Telema, Feta, Danni, Jibnah and Bulgarian white cheese. Iranian white brined cheese is a brine-ripened cheese which mainly is produced in Lighvan Valley (Tabriz, Iran) and is famous for Lighvan cheese. Lighvan cheese is one of the most consumed types of cheese in Iran and its probiotic viability is very important. The aim of this study was to evaluate the survival rate of free and microencapsulated L. acidophilus La5 by calcium alginate and resistant starch in Iranian white cheese during manufacture and storage time. 2. Material and methods
1967
was carried out at pilot plant, adapting industrial technology for white-brined Iranian cheese. Milk was pasteurized at 72 °C for 15 s and then cooled down to the coagulating temperature (32 °C). Then calcium chloride was added as a solution (40%, w/v) at a level of 0.02% (v/v). To incorporate probiotic strain into milk, first, free bacteria of L. acidophilus La5 was incorporated into sample A containing 15 L milk at a level of 1 1011 cfu ml 1. Then, microencapsulated bacteria (see Section 2.2) were incorporated into sample B. The counts of probiotic cells in this type of cheese were approximately 1 1012 cfu ml 1. The inoculated milk was then left undisturbed for an hour to allow acidity development and was coagulated via rennet within 120 min. The coagulated milk was then cut into small cubes (5 5 5 cm3). The cubes were brine-salted (12% w/w, NaCl) at 5 °C for 182 days (Pourjafar, 2010). Cheese samples for analyses were taken at time 0 and during 182 days with two weeks as interval time.
2.1. Preparation and enumeration of free and encapsulated probiotics 3. Statistical analyses Pure probiotic culture of L. acidophilus La5 was obtained from CHR-Hansen (Harsholm, Denmark) and inoculated into MRS-broth (de Man-Rogosa-Sharpe) and incubated at 37 °C for 24 h under aerobic conditions. The probiotic biomass in late-log phase was collected by centrifugation (Centrion Centrifuge, Model 2010, West Sussex, BNI8OHY, UK) at 10,000 rpm for 10 min., and then it was washed twice in sterile saline before using in the microencapsulation procedure. Bacterial counts were determined immediately after manufacture of cheese (at time 0) and during 182 days at 5 °C. The samples of cheese (10 g) were diluted in 100 ml sterile peptone water (0.1%) and 1 ml aliquot dilutions were poured onto plates of the MRS-Salicin-agar. Salicin (10 ml solution at 10% w/v) was added into 90 ml of sterilized MRS agar (Vinderola & Reinheimer, 2000; Shah, 2000; Sultana et al., 2000). Enumeration of the probiotic bacteria was achieved as described by Shah (2000) and Sultana et al. (2000). All enumerating plates of L. acidophilus were incubated at 37 °C for 72 h under aerobic condition. The averages were expressed as colony-forming units per gram of sample (cfu g 1). To count the microencapsulated bacteria in cheese, the entrapped bacteria were released from the beads according to the method of Sheu and Marshall (1993). Ten grams of cheese were re-suspended in 100 ml of phosphate buffer (0.1 M, pH 7.0) followed by 15 min shaking on a shaker (IKA-Model Janke & Kunkel GMBH. Type VX5-Germany). The cheese sample containing free bacteria was treated in a similar way so as to maintain the same treatment conditions. All experiments were done in triplicate. 2.2. Microencapsulation procedure In this study extrusion technique was performed for microencapsulation process described earlier by Sheu and Marshall (1993), Sultana et al. (2000), Kailasapathy (2002), Krasaekoopt, Bhandari, and Deeth (2004). A 2% Na–alginate mixture in distilled water containing 2% Hi-maize resistant starch (Merck, Darmstadt, Germany) and 0.1% culture was prepared. Then, the mixture of cell suspension and Na–alginate and resistant starch were injected into a 0.1 M CaCl2 solution through a sterile insulin syringe. The droplets formed gel spheres immediately. The distance between the syringe and CaCl2 solution was 25 cm. Diameter of the resultant beads was 50–80 lm (Pourjafar, 2011). 2.3. Probiotic cheese manufacture Raw bovine milk (30 L) was obtained from Pegah Dairy Co., (Tabriz, Iran). The milk was divided into two parts. Two types of probiotic cheese, with free and microencapsulated probiotic bacteria, were manufactured under the same conditions. Cheese making
All statistical analysis were performed by SPSS 11.5 (SPSS Inc., Chicago, IL) software. Data were presented as mean (SD) or median (Quartile1–Quartile3). The normality of the total acceptability score was tested and confirmed by Kolmogorov–Smirnov test. The mean of Total acceptability score were compared between two cheese type using independent samples t-test and this was compared between 60 days and 182 days for each cheese type using paired samples t-test. These comparisons were carried out for colours and appearance, for body and texture and for flavour and taste by Mann–Whitney and Sign test, respectively. p < 0.05 considered to be as significant. 4. Results and discussion 4.1. Shape, size and internal appearance of alginate starch beads It is possible to determine the distribution size of the beads by optical microscopy. In this study the diameters of 40 randomly selected beads were measured with an eyepiece micrometre on an optical microscope at a magnification of 40. Also, the surface morphology of calcium alginate beads was investigated by optical microscope (Homayouni, Ehsani, Azizi, Yarmand, & Razavi, 2007). Photographs in Fig. 1(a and b) shows the shape and size. Also, the internal appearance of coated alginate-resistant starch beads is shown in Fig. 2. Optical microscopic image of bead at 40 magnification showed that the bead shape was spherical with a mean diameter of about 50–80 lm, and cross-section and internal appearance of coated alginate-resistant starch bead at 100 magnification showed that bacterial cells were located inside of beads. The distribution of beads in cheese texture was uniform (Fig. 3). Similar shapes of beads were also shown by Sheu and Marshall (1993), Sultana et al. (2000), Krasaekoopt et al. (2004) and Homayouni et al. (2007). 4.2. Survival of free and microencapsulated L. acidophilus La5 in cheese during storage time L. acidophilus La5 was added as adjunct strain during whitebrined cheese manufacture in free (sample A) or microencapsulated (sample B) form into the vats. Variations in counts of free and microencapsulated L. acidophilus La5 during 182 days storage period at 5 °C as (cfu gr 1) are shown in Figs. 4.1 and 4.2. Live cell counts in cheese samples were determined from 0 until 182 days with two weeks of intervals (Table 1). In day zero, the average number of viable cells in cheese containing free bacteria (10 ± 0.58 log cfu g 1) was less than cheese containing the
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Fig. 1. Optical microscope image of coated alginate–resistant starch bead (a and b) at 40 magnification (diameter: 50–80 lm).
14
Fig. 2. Vertical cross-section and internal appearance of coated alginate–resistant starch bead at 100 magnification.
Counts of free La5 (Log cfu/g)
13 12 11 10 9 8 7 6 5 4 3
Free La5
2
Linear (Free La5)
1 0 0
14
28
42
56
70
84
98 112 126 140 154 168 182
Storage time (days)
Fig. 3. Optical microscope image of coated alginate–resistant starch beads at 40 magnification inside cheese matrix.
microencapsulated bacteria (12.32 ± 0.35 log cfu g 1); however, during 28 days of storage period, the number of viable cells in both types of cheese increased their average rate in cheese containing free cells (12.09 ± 0.59 log cfu g 1) to a level higher than the cheese containing microencapsulated cells (12.59 ± 0.23 log cfu g 1). Number of viable cells of Lactobacillus acidophilus reduced significantly from days 28 to 182 of storage period in both types of cheese, but reduction rate in cheese containing free cells (5.1 ± 0.67 log cfu g 1) was significantly (p < 0.01) higher than the cheese containing microencapsulated cells (11 ± 0.58 log cfu g 1). Similar results were reported by Kailasapathy and Masondole (2005), Özer, Uzun, and Kirrmaci (2008), Özer et al. (2009) in brine ripened cheese. This study showed that microencapsulation is able
Counts of microencapsulated La5 (Log cfu/g)
Fig. 4.1. Survival of free L. acidophilus (La5) cells in Iranian white brined cheese during 182 days of storage at 5 °C.
14 13 12 11 10 9 8 7 6 5
microencapsulated La 5
4
Linear (microencapsulated La 5)
3 2 1 0 0
14
28
42
56
70
84
98 112 126 140 154 168 182
Storage time (days) Fig. 4.2. Survival of microencapsulated L. acidophilus (La5) cells in Iranian white brined cheese during 182 days of storage at 5 °C.
1969
182
5.10 ± 0.66 11.00 ± 0.58
168
6.12 ± 0.59 10.08 ± 1.04
154
6.22 ± 0.62 10.83 ± 1.48
140
7.06 ± 0.58 10.84 ± 0.93 8.01 ± 0.59 11.26 ± 0.63
6.19 ± 0.74 10.23 ± 0.79
112
8.04 ± 0.59 10.95 ± 0.98 8.76 ± 0.39 10.62 ± 0.62 9.12 ± 0.59 11.32 ± 0.66
70
11.00 ± 0.58 12.01 ± 0.58
56 42
12.08 ± 0.58 12.58 ± 0.22 11.71 ± 0.36 13.04 ± 0.55 10.00 ± 0.58 12.31 ± 0.34
28 14 0
A B
Live probiotic cell counts (Mean log ± SE) during storage at days:
Cheese samples
Table 1 Live probiotic cell counts in cheese samples during 182 days with two week intervals.
10.30 ± 0.65 12.00 ± 0.58
84
98
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Table 2 Sensory scores of 60-day-old cheeses and 182-day-old cheeses: sample A (containing probiotic bacteria in the free state); sample B (containing probiotic bacteria in the microencapsulated state). Samples
Colour and appearance (1–5)
Body and texture (1–5)
60 days A B
5 (4–5) 5 (4–5)
5 (4–5) 5 (4–5)
182 days A B
5 (4–5) 5 (4–5)
4 (3–4) 4 (4–5)
Flavour and taste (1–10) 9 (8.50–9) 10 (8.50–10) 9 (8–9) 9 (9–9.50)
Total acceptability (1–20) 18.00 ± 0.707 18.80 ± 0.837 17.40 ± 0.894 18.40 ± 0.894
to keep the number of probiotic bacteria above the threshold level for therapeutic minimum (P107 cfu g 1) in cheese. 4.3. Sensory evaluations The sensory scores of 60-day-old and 182-day-old probiotic cheese samples are given in Table 2. The points allocated for colour, texture and taste showed that the addition of free and encapsulated probiotics had no significant effect on sensory properties of probiotic Iranian white brined cheese. Total evaluation in terms of colour, texture and taste of all samples were good and did not have any marked off flavour during the storage period and overall acceptability of cheese samples containing microencapsulated La5 was better than samples with free one. 5. Conclusion According to the results of this study, microencapsulation of L. acidophilus (La5) cells with calcium alginate gel and resistant starch can successfully keep the count of this probiotic bacterium high enough for the therapeutic minimum in the Iranian white brined cheese and can serve as a good carrier for delivering the probiotic bacterial cells into human gut. Higher pH of cheese, higher fat content and more total solid of cheese may offer a protection for the probiotic cells. In this study, at the end of four months of storage, the number of viable probiotic bacteria in two cheese types (see Table 1) were higher than that recommended by the International Dairy Federation (P107 cfu g 1). After four months, until the end of six months of storage period, the number of viable probiotic bacteria, only in cheese samples containing encapsulated cells (see Table 1), was higher than that recommended by IDF. However, further studies are needed to evaluate the survival of other probiotic strains in Iranian white brined cheese as well as investigate the protection effect of microencapsulation on the probiotic survival in animal models and human gastrointestinal tract. Acknowledgement The authors would like to express their thanks Dr. M. Asghari-Jafarabadi for his assistance in data analysis.
to
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