viability of microencapsulated lactobacillus acidophilus in alginate matrix during exposure to...
DESCRIPTION
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.TRANSCRIPT
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 β-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 product’s 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.,
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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 yogurt-
inherent 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 MaterialsMicroorganism 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 37°C 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 37°C, 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 (0.2ˣ6 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 37°C 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 37°C 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 37°C 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 37°C,
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 37°C for 48 h.
Statistical Analysis
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Results for three individual experiments were used to calculate the mean of cell counts. Analysis
of variance (Anova) and Duncan’s 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.
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).
Bacteria
Matrix
Time (min)
D-value (min)0 30 60 90 120
Free cell 3.8 ± 0.7 x 109 2.1 ± 0.2 x 107 1.7 ± 0.3 x 106 1.1 ± 0.3 x 105 3.1 ± 0.2 x 104 23.62 ± 2.7d A
Alginate 1% 3.1 ± 0.3 x 109 1.2 ± 0.8 x 108 3.2 ± 0.5 x 107 1.2 ± 0.4 x 107 3 ± 0.1 x 106 39.73 ± 6.89c
Alginate 1.5% 3.7 ± 0.3 x 109 6.2 ± 0.4 x 108 3.1 ± 0.6 x 108 3.5 ± 0.1 x 107 1.8 ± 0.3 x 107 51.94 ± 3.4b
Alginate 2% 3.8 ± 0.8 x 109 0.9 ± 0.1 x 109 5.7± 0.2 x 108 4.2 ± 0.1 x 108 1.7 ± 0.3 x 108 88.23 ± 6.55a
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).
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Values are average ± standard error ( n=3 ).A Values with the same letters are not significantly different (P>0.05)
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 40±7.8 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 6
cfu mL-1) than were obtained for free cells (1.2 ± 0.3 x 10 2 cfu mL-1).
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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 100µm) 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.
Bacteria
Matrix
Time (min)
D-value (min)0 30 60 90 120
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Free cell 3.6 ± 0.6 x 109 3.2 ± 0.4 x 106 2.5 ± 0.6 x 105 6.4 ± 0.5 x 104 1.2 ± 0.3 x 103 18.51± 1.2d A
Alginate 1% 3.5 ± 0.5 x 109 2.1 ± 0.3 x 107 4.4 ± 0.1 x 105 3.3 ± 0.3 x 104 1.8 ± 0.2 x 104 22.68 ± 4.3c
Alginate 1.5% 3.9 ± 0.3 x 109 3.7 ± 0.5 x 108 1.8 ± 0.9 x 106 3.8 ± 0.1 x 105 2.2 ± 0.4 x 105 28.23 ± 6.5b
Alginate 2% 3.1 ± 0.4 x 109 5.8 ± 0.3 x 108 4.4 ± 0.1 x 107 4.9 ± 0.5 x 106 3.1 ± 0.5 x 106 40 ± 7.8a
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 37°C for 2h ( n=3)
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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