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Title: Viability of Microencapsulated Lactobacillus acidophilus in Alginate

Matrix during exposure to Simulated Gastro - Intestinal Juice

Abstract (English)
This investigation reports the effect of microencapsulation using different concentration of sodium alginate (1, 1,5, 2%) on the tolerance of probiotic Lactobacillus acidophilus under simulated gastrointestinal environments. Microencapsulation provided better protection at simulated conditions of gastric and bile salt. Higher surviving numbers of cells in AG 2% after incubation in gastric juice stimulated more cells to survive the sequential incubation into simulated intestinal juice and showed that the microencapsulation matrix was effective in protecting the entrapped cells with levels of survivors of 6.3 log cfu mL-1 compared to levels of 3.1 log cfu mL-1 for free cells, after 2 h in simulated intestinal juice. These studies demonstrated that microencapsulation of probiotic L. acidophilus in sodium alginate is an effective technique of protection under simulated gastrointestinal environment.

Introduction
Lactic acid bacteria (LAB) are the organisms most commonly used as probiotics. Probiotic bacteria, lactic acid bacteria (LAB), which are typically associated with the human gastrointestinal tract, have been reported to suppress the growth of pathogens (Coconnier et al., 1993; Kaur, Chopra, & Saini, 2002; Lehto & Salminen, 1997; Lim, Huh, & Baek, 1993; Reid & Burton, 2002) and stabilize the digestive system by increasing intestinal barrier functions (Simon & Gorbach, 1984). Normally the stomach contains few bacteria (103 colony forming units per ml of gastric juice) whereas the bacterial concentration increases throughout the gut resulting in a final concentration in the colon of 1012 bacteria/g. Bacteria, forming the so-called resident intestinal microflora, do not normally have any acute adverse effects and some of them have been shown to be necessary for maintaining the well-being of their host. Probiotics are considered beneficial and are sometimes referred to as "friendly" bacteria. Probiotics can be found in capsule, liquid, powder, or tablet form. Once ingested, probiotics colonize the intestines and other parts of the body and can sustain themselves unless they are destroyed by
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antibiotics or other factors. There is some preliminary evidence that probiotic microorganisms can prevent or delay the onset of certain cancers (McIntosh et.al.,1999). This stems from the knowledge that members of the gut microflora can produce carcinogens such as nitrosamines. Therefore, administration of lactobacilli and bifidobacteria could theoretically modify the flora leading to decreased -glucuronidase and carcinogen levels. Lactobacillus acidophilus (LAB) has excellent acid resistance and also exerts a cholesterol-lowering effect in the host (Anderson et.al.,1999). The survival of L. acidophilus in the gastrointestinal tract is essential for exertion of its potential health benefits, such as antimicrobial activity and decrease of cholesterol level. In order to exert positive health effects, LAB have to resist gastric juice and bile salts. After the LAB pass through the stomach and upper intestinal tract, it should attach to the epithelium of the intestinal tract and grow. As a guide for positive health effect, the International Dairy Federation has recommended that the bacteria be active and be present in the product at least till the level of 107 cfu/g until the products expiration date (Ouwehand & Salminen, 1998). If probiotic bacteria have to survive and be active in the digestive tract, they should be resistant to the defense mechanisms of the host (Jonson et.al.,1992) . The gastrointestinal transit begins by exposing L. acidophilus to low pH and pepsin in stomach. When gastric juice is secreted, it has a pH of approximately 2.0 and a salt content of not less than 0.5 %. Although resistance to human gastric transit has been demonstrated in vivo for potentially probiotic lactic acid bacteria and constitutes an important in vitro selection criterion for probiotic bacteria, a satisfactory in vitro method, which closely simulates in vivo gastric transit, has not been defined. Conway et. al.,(1987) demonstrated that bacteria survive better in human gastric juice than in buffer at an equivalent pH indicating that studies using buffers probably underestimate survival potential in vivo. Microencapsulation is a process by which very tiny droplets or particles of liquid or solid material are surrounded or coated with a continuous film of polymeric material. Microencapsulation techniques have been widely employed in the food, medical and cosmetic industries (Bakan, 1973; Putney, 1998). Most microcapsules are small spheres with diameters comprised between a few micrometers and a few millimeters. Recently, several studies have shown successful application of microencapsulated LAB using various encapsulating methods (Rao et al., 1989; Sheu & Marshall, 1993; Sheu et al., 1993; Teixeira et al.,1995b; Koo et al.,

2001; Favaro-Trindade & Grosso, 2002). For the microencapsulation of LAB, polysaccharides such as starch, alginate, carrageenan and chitosan have been extensively studied (Koo et al., 2001), but only few studies have been reported on the application of functional oligosaccharide as a source of coating materials. Chandramouli et al. (2004) studied the optimal encapsulation condition for protecting LAB in artificial gastric conditions. In this study, calcium alginate was used as the wall material and they found that encapsulated LAB showed significantly higher viable cell counts compared to the non-encapsulated LAB under similar conditions. Alginate is commonly obtained from brown seaweed as a natural polysaccharide, which forms a physical hydrogel in the presence of divalent cations such as calcium or barium. Because of its biocompatibility, non-toxicity, mildness of gelation conditions, and low immunogenicity, purified alginate has been widely used in the pharmaceutical and food industries, as well as for biomedical and therapeutic purposes. Sultana et al.(2000) reported that encapsulation of probiotic bacteria in alginate beads was not able to effectively protect the organisms from high acidity. In contrast, Vodnar et al. 2010, demonstrated that alginate matrix protect the bacteria from gastric juice. In 2007, Urbanska et al.(2007) reported the survival and stability of Lactobacillus acidophilus encapsulated into chitosan-coated alginate microcapsules (CCAMs) in different pH conditions. They investigated this formulation in yogurt for therapeutic delivery of L. acidophilus. Microcapsules loaded with L. acidophilus were observed as a homogeneous spherical shape after preparation. The fixed bacterial cells loaded in each subsequent microencapsulation were kept constant in a concentration of 1010 CFU/ml. L. acidophilus loaded CCAMs were incorporated in yogurt and their survival was investigated in comparison with free cells in simulated gastric fluid (SGF) for 2 h, which was the estimated retention time of capsules in acidic stomach. Encapsulated L. acidophilus suspended in yogurt showed better survival compared with free cells in SGF. After the gastric transit, the microcapsules were exposed to simulated intestinal fluid (SIF) for 6 h, and the results showed that L. acidophilus loaded CCAMs and their incorporation in yogurt also retained their viability best compared with free cells as well as the free cells suspended in yogurt. Thus, it is obvious that in both SGF and SIF, the encapsulated bacteria can survive better compared with non-encapsulated cells and yogurtinherent cells because of the protective chitosan-coated alginate membrane. Through this study, they claimed that CCAMs and their incorporation in yogurt provided a suitable oral delivery system for Lactobacillus.
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We aimed to study the survivability of encapsulated L. acidophilus in different concentration of alginate matrix (1,1.5,2%), during exposure to simulate gastric and intestinal juice .

Methods and Materials


Microorganism preparation

Freeze-dried probiotic cultures of L. acidophilus were obtained MTC Romania.

After a

preliminary inoculation in 10 ml MRS (de Man, Rogosa, Sharpe) broth (Merck, Germany) and incubation for 24h at 37C under aerobic conditions for L. acidophilus the bacterial suspension was sub-cultured into 90 ml sterile MRS broth. The fermentations were carried out at temperature of 37C, an agitation speed of 200 rpm, with no aeration in a 200 ml Erlenmeyer flask.

Microencapsulation of Cells

Sterile AG (sodium alginate) powder (Pronova Biopolymer, Oslo, Norway) was dissolved in pure, sterile water using three different concentrations (2%, 1.5% and 1% w v). Aliquots of 120 ml from AG were mixed with 30 ml of bacterial suspension containing 109 cfu mL-1. The emulsion was dropped into a sterile hardening bath 2% (w v) solution of CaCl2 (Sigma-Aldrich) for AG using a syringe with needle (0.26 mm).The beads obtained were separated from the hardening bath, after 30 min, by filtration and washed twice with distilled water.

Gastric Experiment.

We applied the method described by Rao et al. (1989). Each type of beads (1 g) was dispersed in 10 mL of sterile simulated gastric juice (made of 0.08 m HCl containing 0.2% NaCl, pH 1.5) and incubated at 37C for 30, 60, 90 and 120 min. After each interval of incubation, the beads were removed and rinsed with 0.1% peptone solution. To check the cell viability, we disintegrated the beads in citrate solution, as mentioned earlier, and counted the cells after incubation on MRS (de
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Man, Rogosa, Sharpe) agar plates at 37C for 48 h. The viable cells were counted in triplicates and expressed as mean log cfu mL-1. In parallel, 1 mL of free, non-encapsulated cells were inoculated into 10 mL simulated gastric juice (pH 1.5), incubated at 37C and harvested at 30, 60, 90 and 120 min. The viability was determined similarly, as mentioned earlier. To compare statistically the behavior of different beads and viability, we calculated the decimal reduction time values (Dv) representing the time (min) required to destroy 90% or one log cycle of the microorganism.

Intestinal Juice Experiment..

Aliquots of each type of beads (1 g) were first incubated in 10 mL of simulated gastric juice (0.08 m HCl containing 0.2% NaCl, pH 1.5) for 60 min at 37 C. After incubation, the beads were washed in a NaOH 1 N solution and then incubated at 37 C (for 30, 60, 90 and 120 min) in 9 mL of sterile simulated intestinal juice (0.05 m KH2PO4, pH 7.25) containing 0.6% sterilised bile salt (ox gall; Sigma-Aldrich), according to the method described by Krasaekoopt et al. (2004). After incubation, we noticed the swelling of beads, resulting a suspension. One-millilitre aliquot of each suspension obtained was inoculated on MRS agar and incubated for 48 h at 37C, as mentioned earlier, to check their viability. To compare statistically the behaviour of different beads and viability, we calculated again the decimal reduction time values (Dv).

Enumeration of microencapsulated organisms A weight of 1 g from each bead type was liquefied in 99 mL 1% (w v) sterile solution of sodium citrate (Merck) at pH 6.0, at room temperature by shaking for 20 min. The bacteria released from beads were counted in triplicates. The viability of bacteria was evaluated after incubation on MRS agar plates at 37C for 48 h.

Statistical Analysis

Results for three individual experiments were used to calculate the mean of cell counts. Analysis of variance (Anova) and Duncans multiple range tests were performed to analyse the results. Significance of difference was defined at the 5% level (P < 0.05). All statistical analysis was carried out using Graph Pad Version 4.0 (Graph Pad Software Inc; San Diego, CA, USA).

Results And Discussion


Survivability of L. acidophilus during simulated gastric juice exposure

In order to determine the influence of the pH on the survival of non-encapsulated and encapsulated probiotic bacteria, in vitro system was used. Berrada et al., (1991) reported that as for acidity resistance, there was only a slight difference between in vitro and in vivo results. The initial cell population before encapsulation was in the range of 9.38-9.5 log cfu mL-1. High cell entrapping in the range of 9.39-9.49 log cfu mL-1 for beads was achieved in all variants of beads. The results reveal no significant loss of viability for strains, 99.8% of cells being successfully entrapped (Tables 1 -2). To improve viability of the strains during exposure to the low pH of the stomach, HCl solution was used to determine which matrices and matrix concentration of AG would increase survival of cells in this environment, similarly to digestive system. The survivability of strains was expressed as the destructive value (D-value), which is in the time required to destroy 90% or one log cycle of the microorganism (Tables 1-2). Initial cell population was in range of (Table 1), 3.1 0.3 x 109- 3.8 0.8 x 109 , the survival of cells in all variants of AG, beads being significant ( P<0.05 ), superior to free cells. However, AG 2% beads entrapped L. acidophilus (D-values 88.23 min) provided the best protection, followed by AG 1.5% (D-values 51.94 min) and AG 1% (D-values 39.73 min). The results are in contrast to that of Sultana et al., (2000), who found that encapsulation of bacteria in alginate beads, did not effectively protect the organisms from high acidity, but in agreement with Kim et al., (2008) who reported that at pH 1.2, non-encapsulated strain (L. acidophilus) was completely destroyed after 1h of incubation while encapsulated strains maintained above 106 cfu mL-1 after 2h.
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The results are also in agreement with Mokarram et al., (2009) who reported that at pH 1.5 after 2h of incubation the stains (L. acidophilus and L. rhamnosus) built with alginate maintained above 107-108 cfu mL-1. Chandramouli et al., (2004) reported a higher survival of LAB immobilized in alginate beads in low pH environments.
10 8

a b b c

Log cfu mL-1

6 4 2 0

1%

1. 5%

2%

Beads Type in SGJ

Figure 1. Effect of AG beads concentration on survival of L. acidophilus under simulated gastric conditions (SGJ). Values with the same letters are not significantly different (P>0.05).

Figure 1. show significant differences(P<0.05) of L. acidophilus in beads of AG 2% (8.03 log cfu mL-1) vs. rest of the beads, and no significant differences (P>0.05) between AG 1% (6.47 log cfu mL-1) vs. AG 1.5% (7.25 log cfu mL-1). Our results suggested that non-encapsulated bacteria was sensitive to the acidic environment (pH 1.5) and the ingestion of unprotected LAB might results in reduced viability (5 log reduction after 2 h). According to this study, the AG 2% beads provides the best protection in simulated gastric juice because the compactness was high, the diffusion of gastric juice into the beads may be limited. This will protect encapsulated cells from interacting with the gastric juice, as mentioned before (Murata et al., 1999).
Table.1 Survival cells of L. acidophilus after exposure to pH 1.5 solutions at different times (cfu mL-1).

Values are average standard error ( n=3 ).


A

Values with the same letters are not significantly different (P>0.05)
Time (min) 0 3.8 0.7 x 10
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Bacteria Matrix Free cell Alginate 7 1% Alginate 1.5% Alginate 2%

Fr ee

C el ls

30 2.1 0.2 x 10
7

60 1.7 0.3 x 10
6

90 1.1 0.3 x 10
5

120 3.1 0.2 x 10 3 0.1 x 106 1.8 0.3 x 107 1.7 0.3 x 108
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D-value (min) 23.62 2.7d A 39.73 6.89c 51.94 3.4b 88.23 6.55a

3.1 0.3 x 109 3.7 0.3 x 109 3.8 0.8 x 109

1.2 0.8 x 108 6.2 0.4 x 108 0.9 0.1 x 109

3.2 0.5 x 107 3.1 0.6 x 108 5.7 0.2 x 108

1.2 0.4 x 107 3.5 0.1 x 107 4.2 0.1 x 108

Survivability of L. acidophilus during simulated gastro-intestinal exposure

In order to exert positive health effects, LAB should resist the stressful conditions of the stomach and upper intestine (Chou & Weimer, 1999). To determine the tolerance of the free and encapsulated strains to the acidic pH of the stomach, an in vitro system was utilized. The culture was put into a simulated gastric juice for 60 min, followed by a further incubation in intestinal juice with 0.6% bile salt for 30, 60, 90 and 120 min. The results are shown in Table 2. The initial cell population was in range 3.1 0.1 x 109- 3.9 0.8 x 109 the survival of cells in all variants of AG, beads being significant ( P<0.05 ), superior to free cells. As indicated by D-values microencapsulated cells in alginate survived better than free cells (Table 2). The results indicate that AG 2% (D-value 407.8 min) could increase the survivability of encapsulated cells in such condition. In general the D-value of probiotic bacteria incubated in simulated gastro-intestinal juice was lower than where it was incubated in simulated gastric juice. This may be due to the fact that the environmental resistant of LAB is determined by many factors such as their medium and cytoplasmic membrane composition (Begley et al., 2005). Kim et al., (2008) demonstrated that micro encapsulation using alginate 2% may be an effective way to increase the survival of bacteria in simulated intestinal juice. The sequential transfer of the free cells and encapsulated bacteria after 60 min incubation in simulated gastric juice resulted in an initial reduction of viable cells during the first hour of exposure to simulated intestinal juice (Table 2). Overall, the sequential exposure to simulated gastric juice (60 min) and simulated intestinal juice (2h), higher numbers of bacteria surviving in the AG 2% beads (3.1 0.5 x 10 cfu mL-1) than were obtained for free cells (1.2 0.3 x 10 2 cfu mL-1).
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Log cfu mL-1

b c d

4 2 0

1%

1. 5%

2%

Beads Type in SIJ

Figure 2. Effect of AG beads concentration on survival L. acidophilus after incubation in simulated gastric and intestinal juice (SIJ). Values with the same letters are not significantly different (P>0.05).

Figure 2 shows significant differences (P<0.05) bacteria survivability in beads of AG 2% (6.2 log cfu mL-1) vs. all variants AG 1.5% (5.04 log cfu mL-1). Significant differences were noticed between all variants of beads against free cells (3.3 log cfu mL-1). The results are in agreement with previous results of Anal & Singh (2007) who showed that the formation of a hydro gel barrier by sodium alginate retards the permeation of the gastric fluid into the cells. Truelstrup et al., (2001) reported that beads of small size (less than 100m) do not significantly protect the bacteria in simulated gastric fluid, as compared to free cells. Higher surviving numbers of cells in AG 2% after incubation in gastric juice stimulated more cells to survive the sequential incubation into simulated intestinal juice and showed that the microencapsulation matrix was effective in protecting the entrapped cells with levels of survivors of 6.3 log cfu mL-1 compared to levels of 3.1 log cfu mL-1 for free cells, after 2 h in simulated intestinal juice.

Fr ee

C el ls

Table 2. Average number (mean) cfu mL-1 of survived cells and D-values of free and microencapsulated cells of L. acidophilus after incubation in simulated gastric juice (60 min) and simulated intestinal juice (pH 7.25) at 37C for 2h ( n=3)
Bacteria Matrix Free cell Alginate 1% Alginate 1.5% Alginate 2% Time (min) 0 3.6 0.6 x 10
9

30 3.2 0.4 x 10
6

60 2.5 0.6 x 10
5

90 6.4 0.5 x 10
4

120 1.2 0.3 x 10


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D-value (min) 18.51 1.2d A 22.68 4.3c 28.23 6.5b 40 7.8a

3.5 0.5 x 109 3.9 0.3 x 109 3.1 0.4 x 109

2.1 0.3 x 107 3.7 0.5 x 108 5.8 0.3 x 108

4.4 0.1 x 105 1.8 0.9 x 106 4.4 0.1 x 107

3.3 0.3 x 104 3.8 0.1 x 105 4.9 0.5 x 106

1.8 0.2 x 104 2.2 0.4 x 105 3.1 0.5 x 106

Values are average standard error ( n=3 ).


A

Values with the same letters are not significantly different (P>0.05)

Conclusion
Microencapsulation seems to be the most promising technology to protect bacterial cells from adverse environment, it appears to be a promising technology to retain the potency of probiotic bacteria cells to be delivered orally into the GI system. It appears that the chitosan-coated alginate microparticulate system has effective applications for oral delivery of probiotic bacteria because chitosan coating showed good results in terms of the survival and stability of encapsulated live cells. Alginate beads 2% microcapsules provided significant protection of entrapped L. acidophilus against the harsh acidic conditions of simulated gastric juice. As a result, significantly higher numbers of bacteria survived sequential incubation from the simulated gastric juice into the simulated intestinal fluid, where disintegration of alginate beads and release of entrapped cells occurred.

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Acknowledgements
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This work was supported by the Department of Biochemistry and Biotechnology of Food Products, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania. The author is grateful for technical support and guidance to Professor Dan Cristian Vodnar.

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