VIABILITY OF ENCAPSULATED BIFIDOBACTERIUM LACTIS (BB-12) IN SYNBIOTIC UF CHEESE AND IT S SURVIVAL UNDER IN VITRO SIMULATED GASTROINTESTINAL CONDITIONS

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1 International Journal of Probiotics and Prebiotics Vol. 6, No.3/4, pp , 2011 ISSN print, Copyright 2011 by New Century Health Publishers, LLC All rights of reproduction in any form reserved VIABILITY OF ENCAPSULATED BIFIDOBACTERIUM LACTIS (BB-12) IN SYNBIOTIC UF CHEESE AND IT S SURVIVAL UNDER IN VITRO SIMULATED GASTROINTESTINAL CONDITIONS Roghayeh Nejati 1, Hamidreza Gheisari 1, Saeid Hosseinzadeh 1, and Hosein Amin² 1 Department of Food Hygiene and Technology, School of Veterinary Medicine, Shiraz, Iran and 2 Reaserch and Development unit, Ramak Dairy Company, Shiraz, Iran [Received May 15, 2011; Accepted August 10, 2011] ABSTRACT: In the current study, effects of inulin and microencapsulation on the survival time of Bifidobacterium lactis (Bb-12) (B. lactis (Bb-12)) and its resistance to the gastric and enteric simulated conditions in the synbiotic Ultra Filterated (UF) cheese were investigated. The survival of B.lactis (Bb-12) was monitored during the storage for 60 days at 4 ± 1 C and also under an in vitro gastro-intestinal model. Results showed the significant decrease in B.lactis (Bb-12) survival during storage and in vitro gastrointestinal simulation for all the samples. However, survival time of microencapsulated probiotic bacteria was enhanced during storage and in vitro gastrointestinal simulation, but addition of inulin did not improve the viability of it during shelf life and in vitro gastrointestinal simulation. Encapsulated probiotic bacteria exhibited a satisfactory resistance to low ph values and high concentrations of bile salts. In general, the results indicated that encapsulation can significantly increase the survival rate of probiotic bacteria in UF-cheese over its shelf life and in vitro simulated gastrointestinal conditions. KEY WORDS: Bifidobacterium lactis (Bb-12), Gastrointestinal simulation, Inulin, UF-cheese Corresponding Author: Dr. Hamidreza Gheisari, Department of Food Hygiene and Technology, School of Veterinary Medicine, Shiraz, Iran; FAX: ; ghaisari@shirazu.ac.ir KEY WORDS: Lactobacillus salivarius, Probiotic, Poultry, Salmonella typhimurium INTRODUCTION Manipulation of human gastrointestinal microbiota is currently attempted as a means of introducing new microorganisms into the digestive tract that are beneficial to the human host. Microorganisms that have been used to achieve these goals have been called probiotics. Lactobacillus and Bifidobacterium species constitute a significant proportion of probiotic cultures that extensively used in the developed countries. In order to exert the health benefits, probiotic bacteria are expected to be at the level of 10 7 CFU of live microorganisms per milliliter of product at the time of consumption. Therefore, it is important to ensure a high survival rate of these bacteria during the product shelf life to maintain the consumer confidence (Saxelin et al., 1999). Dairy products are recognized as the ideal food systems for the delivery of probiotic bacteria to the human gut. At present, bifidobacteria are increasingly incorporated into the fermented dairy products. However, due to the acidic nature of these products, probiotics show low viability in yogurt and fermented milk (Vinderola et al., 2000; Gardini et al., 1999). On the other hand, cheese may offer certain advantages over fermented milk products in terms of delivery of the viable probiotics. But, cheese may offer certain advantages over fermented milk products in terms of delivery of the viable probiotics. This may be resulted from the fact that cheese have a higher ph, a higher fat content and a more solid consistency, all of which should afford added protection to the probiotics in the gastrointestinal tract (Kasimoglu et al., 2004; Corbo et al., 2001). Since the viability and activity of probiotics are needed at the lower digestive tract, these organisms should withstand the adverse conditions encountered in the host s upper gastrointestinal tract (GIT). However, many probiotic bacteria lack the ability to survive the harsh acidity and bile concentration commonly encountered in the GIT (Gardiner et al., 2000). Bile acids inhibit many Gram-positive probiotic organisms such as Bifidobacterium and Lactobacillus genera. This information has been known for decades and is actually incorporated into the taxonomic identification methods. The immobilization of probiotics using microencapsulation may improve the survival of these microorganisms in products, both during processing and storage, and digestion (Sultana et al., 2000; Capela et al., 2005).

2 198 Viability of encapsulated Bifidobacterium lactis Microencapsulation protects the probiotics from the adverse environmental conditions. There is a need for microencapsulation not only to help the probiotic bacteria to survive in the food product but also during the passage through the human stomach, where the ph can be reached as low as 2. Alginate, as a polymer extracted from seaweed, has been widely used as an encapsulant material. Alginate is a favored encapsulation agent because it is nontoxic, biocompatible, and inexpensive. It has been reported that microcapsules made of alginate polymers increased the survival and viability of probiotic bacteria in acidic food products during cold storage (Picot and Lacroix, 2004). Recently, researchers have focused on natural ingredients such as fructopolysaccharides (FPS) like inulin that called prebiotics. Prebiotics are non-digestible dietary ingredients that benefit the host by selectively stimulating the growth and/ or activity of beneficial bacteria in the colon. The most frequently studied examples are inulin-type fructans and fructooligosaccharides. The positive effect of combining probiotic and prebiotic agents is termed synbiotics. Such combinations improve the survival of probiotic bacteria in the upper gastrointestinal tract and enhance their effect in the large bowel (Gilliland, 2001). Incorporating both prebiotics and calcium alginate in coating materials may improve the protection of probiotics in food systems and the gastrointestinal tract due to their synbiosis characteristics (Chen et al., 2005). Many researchers have been undertaken to improve the growth of probiotic bacteria in yogurt during its fermentation process, its survival in yogurt during the storage, and, hence their viability in the gastrointestinal conditions. No published data exist on microencapsulation and effects of inulin on the survival of Bifidobacterium lactis (BB-12) in synbiotic UFcheese. Thus, our objectives were to compare the effects of inulin and microencapsulation on the survival of Bifidobacterium lactis (BB-12) during storage and also in the gastrointestinal tract in synbiotic UF- cheese. MATERIALS AND METHODS Probiotic bacteria Pure DVS probiotic cultures of B. lactis (Bb-12) were obtained from CHR-Hansen (Horsholm, Denmark). Before use, the probiotic organisms were maintained in -20 C. 0.02% w/v of free probiotics were added to retentate milk that subsequently used for UF-cheese preparation. Microencapsulation of probiotics All glassware and solutions used in the protocols were sterilized at 121 C for 15 min. Alginate beads were produced using encapsulation method originally reported by Sheu and Marshall (1993) and Sultana et al. (2000). Briefly, a 100 ml volume of 2% alginate mixture in distilled water was prepared containing probiotic culture (DVS). The mixture was added into 200 ml canola oil (Familla, Behshahr Oil Industry, Behshahr, Iran) containing 0.2% lecithin (sigma p3644). The mixture was stirred vigorously (400 rpm for 20 min, Heidolph Stirrer, Germany) until it was fully emulsified. Then 200 ml of 0.1 M calcium chloride solution were added. The mixture was allowed to stand for 30 min in order to separate the prepared calcium-alginate beads in the bottom of beaker at the calcium chloride layer. The oil layer was drained and beads were collected in the calcium chloride solution that was then washed with 0.9% saline containing 5% glycerol and immediately used in the formulation. Acidification kinetics of microencapsulated bacteria: Free and encapsulated cells of B.lactis (Bb-12) were cultivated in MRS-broth to determine if the encapsulated cells were still metabolically active and if nutrients and metabolites could permeate bead wall. To obtain the time needed for free and encapsulated cells to acidify MRS-broth medium, equal logs of free cells and of encapsulated cells (in beads) were inoculated into the 100 ml MRS-broth medium and incubated at 37 C for 56 h. The ph values and optical density (OD) at nm were measured every 4 h (Homayouni et al., 2008). Cheese manufacture Synbiotic UF-cheese was manufactured at a cheese factory in Ramak Dairy Company, Shiraz, Iran. A total of 1000 L volume of raw milk containing a high microbial quality was standardized to % fat, and after bactofugation, the preparation was pasteurized using a high temperature short time process (18 s at 74 C) and cooled. The pasteurized milk was ultrafiltered to reach the final concentration of 35 37% of total solids at 45 C before the retentate being homogenized and pasteurized again. Then the retentate milk was divided into four equal batches and cheese batches were processed at the same time. After addition of mixed-strain inoculums % w/v of starter culture FRC 65 (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Streptococcus thermophilus and Lactobacillus delbruckii subsp. bulgaricus) from Chr. Hansen (Denmark) and CHOOZIT Feta B LYO 100 DCU(Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Streptococcus thermophilus, Lactobacillusdelbruckii subsp. Bulgaricus, Lactobacillus helventicus) from Danisco Cuilturs compony] and 0.002% w/v rennet (Fromase TL granulate, P2200 IMCU g-1) as microbial coagulant from Rhizomucor miehei supplied by DSM Food Specialities (Seclin, France) was added to each of the retentate, four different formulations of UF- cheese, denoted as T1, T2, T3, and T4, were produced. T1 was supplemented with 1% gr beads/v encapsulated probiotic culture and 1.5% Native inulin (Frutafit IQ) was supplied by Sensus (Roosendaal, Netherlands). T2 was produced with addition of 1% gr beads/ v encapsulated probiotic culture and without addition of inulin.t3 was produced with addition of 0.2%w/v free probiotic culture and addition of 1.5% inulin. T4 was prepared with 0.2%w/v free probiotic culture and without adding any inulin. In the sealing machine, 3% (w/v) salt was

3 199 Viability of encapsulated Bifidobacterium lactis added onto the parchment paper on the top of cheese and then by using aluminum foil, the container was sealed. In the ripening stage cheeses were stored at 37 C. After dropping the cheese ph to 5, cheeses stored at (4 ±1) C for 60 days. Enumeration of probiotic bacteria The samples of cheese (10 g) were transferred into a sterile bag under aseptic conditions and homogenized in 90 ml of sterile normal saline solution for 2 min using a lab blender 400 stomacher (Seward, Model NO BA6021,UK). Serial dilutions were prepared by adding l ml to 9 ml sterile peptone water (0.1 g 100 ml 1 ). These samples were then pour plated on the ABC-MRS agar (containing 0.5 mg/l dicloxacillin, 1.1 g/l lithium chloride and 0.5 g/l cystein) in triplicate. Cheeses from each trial were subjected to analysis following 1, 15, 30, 45 and 60 days of refrigerated storage and the evaluation of probiotic survival in each trial submitted to the gastric and enteric simulated conditions on the days 1 and 60 of the storage. All the plates were incubated at 42 C for 72 h under anaerobic condition. Results are presented as log cfu/g of the samples. Survival of B. lactis (Bb-12) under simulated gastrointestinal conditions The evaluation of probiotic survival submitted to the gastric and enteric simulated conditions was performed according to the method described by Buriti et al. (2010) with some modifications. At each sampling day, as described previously for refrigerated Cheese, 10 gr from each triplicate dilution of cheese in 0.5% NaCl solution was transferred into 4 sterilized flasks, with the final numbers of 12 flasks containing the samples (3 dilutions 4 sampling periods), and ph was adjusted to using 1 N HCl. Pepsin (from porcine stomach mucosa, Sigma p7125) and lipase (Fluka 62305), were subsequently added to the samples to reach the concentrations of 3 g/l and of 0.9 mg/l, respectively. Flasks were incubated at 37 C, with agitation of approximately 110 rpm (Shaker incubator Lab Tech), during 2 h (gastric phase). In the next step, the ph of samples was increased to using an alkaline solution (150 ml of 1 N NaOH, 14 g of PO4H2Na.2H20 and distilled water up to one 1 L). Bile (bovine bile, Sigma B8381) and pancreatin (pancreatin from porcine pancreas, Sigmap1750) were added to reach a concentration of 10 g/l and of 1 g/l, respectively. Samples were incubated at 37 C for 2 h under agitation (enteric phase 1). In the last step, the ph was increased to using the same alkaline solution, bile and pancreatin concentrations were adjusted (10 g/l and 1 g/l, respectively), and samples were re-incubated at 37 C for 2 h under agitation (enteric phase 2), until 6 h of the assay. Enumeration of B. lactis (Bb-12) was performed in aliquots collected from triplicate samples after 30 min, 2 h, 4 h and 6 h (three different flasks of the same trial for each time), using proper volume (varying from 0.01 to 1 ml). Aliquots of 0.01 ml and 0.1 ml were pour plated in ABC- MRS agar modified by the containing 0.5 mg/l dicloxacillin, 1.1 g/l lithium chloride and 0.5g/l cystein as described by Lima et al.(2009) and incubated at 42 C for 72 h under anaerobic condition. Statistical analysis Analysis of variance (ANOVA) and independent-samples T- Test were applied on the microbiological data using the SPSS program. Differences among means were re-tested by Duncan s multiple range tests. The level of significance was set at p< RESULTS The viability of B. lactis (Bb-12) obtained for all trials is shown in Table 1. The results of this study showed that the counts of B. lactis (Bb-12) in all the samples were significantly increased during the first 15 th days of the storage. However, after 60 days, the viability of B. lactis (Bb-12) was significantly reduced all the trials (Fig. 1). This reduction was lower than 1 log cycle just for the samples containing encapsulated cells of B.lactis (Bb-12). Results indicated that the survivals of the microencapsulated probiotic bacteria were dramatically enhanced during the storage period (Table 1). No significant differences were found in the number of B. lactis ( Bb-12) among the samples treated or untreated with inulin in the same days during storage. Table 1. Viability (log cfu/g) of B. lactis (Bb-12) in different preparations of cheese stored at 4±1 C. Temperature treatment Time (days) of storage ±1 C T1 7.52±.08 ca 8.17±.01 aa 7.88±.01 ba 7.56±.18 ca 7.31±.11 ca T2 7.45±.03 ca 7.97±.03 ab 7.83±.05 ba 7.43±.03 ca 7.12±.06 da T3 7.15±.10 bb 7.81±.05 ad 7.19±.01 ba 6.48±.17 cb 6.23±.08 cb T4 7.24±.05 bb 7.72±.06 ad 7.27±.06 bb 6.79±.07 cb 6.33±.04 db A-D Within a column, different superscript capital letters denoted the significant differences between different trials (P<0.05); a-d Within a row, different lowercase superscript letters denoted the significant differences during the storage periods (P<0.05). FIGURE 1. Survival of B. lactis (Bb-12) during storage at 4 ± 1 C

4 200 Viability of encapsulated Bifidobacterium lactis The survival of B.lactis (Bb-12) in samples T1 T4 submitted for the gastric and enteric simulation are presented in Fig. 2. In all samples, a steady loss in the survival of probiotics was shown at the time of exposure to the acidic conditions. 0n the first day, Initially there was an average of 7.34 log CFU/ml of viable probiotic bacteria, but after 30 min of exposure the average viability of cells was reduced to 4.03-log CFU/ml and the viability was further reduced to an average of 3.77-log CFU/ml after 2 h of exposure. Results s h o w e d microencapsulated probiotic bacteria survived better than free probiotic bacteria in low ph (Fig. 2). However samples containing microencapsulated probiotic bacteria and inulin had the highest probiotic survival in gastric phase but inulin had no significant effect in the probiotic survival for all trials during the whole gastric phase. Similar results were also obtained in the samples on the last day of storage. These data indicate that Bifidobacterium species may survive through passage of the human stomach, particularly when supported with alginate beads but not inulin. All samples showed a loss of viability when exposed to bile. However the effect of enteric phase 1 and 2 on the viability of probiotic bacteria was severely less than gastric phase. Samples containing encapsulated probiotic presented the highest probiotic survival after 6 h of assay on both days 1 and 60, without any significant differences between on days of storage (Table 2). However addition of inulin could not increase the viability of B. lactis (Bb-12) enteric phase 1 and 2, which was found statistically significant according to the mean value of storage. days of storage FIGURE 2. Time course of survival of B.lactis (Bb-12) exposed to simulated gastrointestinal conditions measured on day 1 and 60 of storage at 4 ± 1 C. For the same storage period, different superscript in capital letters denote significant differences between trials for the same sampling period of the in vitro assay where as different lowercase superscript letters denote significant differences between different sampling periods of the in vitro assay for the same trial. Table 2. Bacterial counts after 1 and 60 days of storage samples T1 T2 T3 T ±0.01 a 3.64± 0.01 b 4.88±0.11 c 4.86±0.11 c ±0.18 a 3.07±0.14 a 3.77±0.04 b 3.87±0.11 b Within a row, different lowercase superscript letters denoted significant differences during the storage periods (P<0.05). DISCUSSION Survival of B.lactis (Bb-12) in the specimens during the Storage times The decline in bacterial counts throughout the storage was most likely due to the temperature that eventually led to the cells death. Furthermore, the incorporation of ph may have resulted in an additional decrease in viable cell counts. No data are available in the literature on the viability of encapsulated probiotic bacteria in UF- cheese. However, Ozer et al. (2008) detected the positive effects of microencapsulation on viable counts of B. bifidum (Bb-12) in Kasar cheese which were similar to our results except for T1 and T2 experimental groups which were contrary to what we have observed, here. Kailasapathy and Masondole (2005) reported that microencapsulation did not offer any protection to the Lactobacillus acidophilus (DD 910) and B.lactis (DO 920) probiotic bacteria, over a 7-weeks period in Feta cheese. Our study showed that there was approximately one log decrease in the survived cells of B.lactis (Bb-12) after 60 th days of storage at (4±1) C in all samples which was much higher than that what It was reported before. On the other hand, this study indicated that UF-cheese is a good vehicle that had a protective effect on the probiotic viability during refrigerated storage. Moreover, within the first 15 days of storage a significant increased (approximately half log) in the number of viable cells of

5 201 Viability of encapsulated Bifidobacterium lactis B.lactis (Bb-12) was observed in all samples. The results obtained in this work speculated that probiotic UF-cheese would be a food matrix allows the growth of B.lactis (Bb-12) during certain conditions of cold storage. Similar results about the survival of bifidobacteria in cheese can be found in the literature. Fritzen-Freire et al. (2010) tested the use of Bifidobacterium Bb-12 in Minas Frescal cheese and they observed even higher counts after 28 days of storage (5 ± 1) C. In this study, addition of inulin did not improve the viability of this probiotic microorganism during shelf life. This observation confirms the results presented by Rodrigues et al. (2011) who showed that inulin did not significantly affect the growth and/or viability of Bifidobacterium lactis LAFTI in curdled milk matrices. Although there are many studies detected the positive effect of inulin on viable counts of bifidobacteriume species in dairy products (Buriti et al., 2007). Inulin is a mixture of oligo- and polysaccharides which are composed of fructose units connected by ß (2-1) links Fructooligosaccharides (FOS) and types of fructo-polysaccharides, comprised of (glucose-fructose)- subunits. In fact, the growth and viability of bifidobacteria in the presence of fructopolysaccharides (FPS) are varied depending on the chain length or DP of FPS and concentrations of FPS used. FPS, which has a DP of 4, had a higher bifidogenic effect than larger molecular weight carbohydrates like inulin (Gibson and Wang, 1994). The utilization of FOS by bifidobacteria was enhanced when it was applied accompany to a DP below 6 units according to Kaplan and Hutkins (2000) and below 10 according to Roberfroid et al. (1998). The HP inulin was employed in order to avoid any undesirable sweetening changes in the tastes of products. Additionally, due to the sensible delicate structure of the UF cheese, a 1.5% concentration of inulin was used. This result was in agreement with Shin et al. (2000) who observed that viabilities in some strains of Bifidobacteria were higher when they were grown and stored in the presence of oligosaccharides and inulin. They have also shown that the effects of oligosaccharides and inulin increased with increasing carbohydrate concentration with a maximum level of 5% w/v. Eleva et al. (2009) indicated that 2% inulin was stimulated the growth of B. lactis and increased its counts gradually in the UF-white soft cheese following the 20th days of storage, that was in contrary to what we have observed in the present study. Survival of B.lactis (Bb-12) in specimens under exposure to an in vitro gastrointestinal simulation Microencapsulation of probiotics in alginate beads has previously been tested for improving the viability of probiotic bacteria in the simulated gastric conditions. The results of our study concur with other similar work where microencapsultaion in alginate beads has been found to increase the survival of probiotic bacteria in acidic conditions. However, a high susceptibility of B.lactis (Bb-12) to HCl containing pepsin was observed in all samples. Nchez et al. (2010) speculated that encapsulation was significantly improved the survival of bifidobacteria during the exposure to nisin, in the simulated gastric juice. However, there are examples of studies that encapsulation with alginate, carrageenan, and modified starch have generally failed to substantially improve the survival of acid-sensitive bifidobacteria in models simulating the acidic conditions in the stomach (Shah and Ravula, 2000; Sultana et al., 2000). Truelstrup Hansen et al. (2002) reported that the alginate microspheres retained their integrity at ph 2 and microencapsulation of bifidobacteria did not significantly improve the survival of free cells when exposed to simulated gastric juice. Ritter et al. (2009) reported that B. animalis subsp. lactis had the highest survival rate (10%) of approximately cfu ml -1 compared to the other tested bifidobacteria strains in the bioreactor model of the stomach-intestine passage. They showed that B. animalis subsp. lactis was more resistant up to 40 min at ph 2.0, but was decreased by about 3 log when incubated for 120 minutes. In the present study, maximum reductions in the viability of B.lactis (Bb-12) during 2 h of the in vitro assay were around 2 log cycles for all of the trials. This resistance could be depended on the components of the food matrix were important to protect the probiotic microorganism throughout the simulated enteric conditions. In addition, the protective effects of milk proteins in the digestive system have already been described in the literature (Charteris et al.,1998).our result showed that microencapsulation was improved the probiotic survival time treated under in simulated intestinal conditions. Other studies using concentrated bile salts between 1% and 3% have shown that encapsulation can improve the survival time of probiotic bacteria (Chandramouli et al., 2004; Kailasapathy, 2005). Results presented by Dring and Shah (2009) also show that the survival time of probiotic bacteria was enhanced in encapsulated in alginate (P < 0.05), when exposed to the acidic condition and bile salts. Ritter et al. (2009) observed an 85% (2 log) decrease in the viability of B.lactis in the presence of bile salts and pancreatic juice after 7h. which are similar to our results of in vitro stimulated enteric conditions. The present study has shown that the addition of inulin, did not offer any significant protection to maintain the viability of probiotic bacteria. Although, there are no reports on the effects of inulin on bifidobacteria survival vitro gastrointestinal conditions but researchers have investigated the use of inulin in dairy products caused significant protective in other probiotics. Buriti et al. (2010) reported that the addition of inulin in refrigerated guava mousses enhanced the L. acidophilus survival during the in vitro gastrointestinal conditions in the first week of storage. CONCLUSION Microencapsulation offers significant benefits to food technology and production. The present study showed that the survival time of the microencapsulated cells was remarkably improved under simulated gastrointestinal

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