effect of encapsulation of selected probiotic cell on...
TRANSCRIPT
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Effect of encapsulation of selected probiotic cell on survival in simulated 1
gastrointestinal tract condition 2
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Submitted to Songklanakarin Journal of Science and Technology 6
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Hasiah Ayama, Punnanee Sumpavapol* and Suphitchaya Chanthachum
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Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla 14
University, Hat Yai, Songkhla 90112, Thailand 15
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*Corresponding author: Tel: +66-7428-6366, Fax: +66-7455-8866 23
E-mail address: [email protected] 24
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Abstract 25
The health benefits of probiotic bacteria have led to their increasing use in foods. 26
Encapsulation has been investigated to improve their survival. In this study, the 27
selection, encapsulation and viability of lactic acid bacteria (LAB) with probiotic 28
properties in simulated gastrointestinal tract (GIT) condition were investigated. 150 29
isolates of LAB were obtained from 30 samples of raw cow and goat milk and some 30
fermented foods. 9 isolates could survive under GIT condition and only 3 isolates 31
exhibited an antimicrobial activity against all food-borne pathogenic bacteria. Among 32
them, 2 isolates (CM21 and CM53) exhibited bile salt hydrolase activity on 33
glycocholate and glycodeoxycholate agar plates and were identified as Lactobacillus 34
plantarum. CM53 was selected for encapsulation using 1-3% alginate and 2% Hi-maize 35
resistant starch by emulsion system. Viability and releasing ability of encapsulated 36
CM53 in simulated GIT condition were increased in accordance to the alginate 37
concentration and incubation time, respectively. 38
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Keywords: Encapsulation; Probiotic; Lactic acid bacteria; Simulated gastrointestinal 40
tract, Survival 41
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1. Introduction 51
The consumption of definite species of probiotic microorganisms is beneficial 52
for reducing the duration and severity of diarrhea symptoms. Supplementing a diet with 53
food containing beneficial bacteria can be used as a strategy for preventing diarrhea. 54
Probiotics are defined as living microorganisms, which upon ingestion in certain 55
number exert health benefits beyond inherent basic nutrition. For obtaining health 56
benefits, a minimum of one million probiotic organisms per gram of food is 57
recommended (Capela et al., 2007). The most common commercial bacteria are of the 58
genera Lactobacillus spp. These bacteria are typically saccharolytic (capable of 59
metabolizing sugars), gram-positive, rod shaped and reside in the large bowel (Cook et 60
al., 2012). The key criteria involved in the selection of probiotic microorganisms 61
include the origin and biosafety of the strains, survival during passage through the 62
gastrointestinal tract (GIT), ability to adhere and colonize on the epithelial cell surface 63
of the host GIT, and inhibitory activity against enteric pathogens (Petsuriyawong and 64
Khunajakr, 2011) 65
For processing and storage of foods, probiotic microorganisms may be exposed 66
to low pH, high osmotic pressure and high levels of oxygen. These factors may have 67
deleterious effects on probiotics. The survival of viability probiotic is also affected by 68
acid encountered in the stomach and the bile salts in the intestine tract. 69
Microencapsulation is the process of applying a shell to sensitive probiotic to protect 70
them from their external environment (Capela et al., 2007) and the probiotic cells are 71
retained within an encapsulating matrix. Microencapsulation of probiotics has been 72
examined for increasing their viability in food products and the intestinal tract. The 73
most widely used encapsulating material is alginate, a linear heteropolysaccharide of D-74
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mannuronic and L-guluronic acid extracted from various species of algae. Alginate 75
beads can be formed by extrusion and emulsion methods. The use of alginate is 76
privileged because of its simplicity, biocompatibility and inexpensive (Krasaekoopt et 77
al., 2004). Shah and Ravula (2000) found that calcium alginate beads formed by the 78
emulsion technique can increased the viability of probiotic during processing and 79
storage of frozen yoghurt. However, there are several disadvantages of using the 80
emulsion technique including a wide distribution of bead size, difficulty in the 81
automating technique and exceedingly the large beads with diameters ranging from 200 82
to 1000 µm (Poncelet et al., 1992). 83
The aim of this work was to screen lactic acid bacteria (LAB) with probiotic 84
potential from raw cow and goat milk and some fermented foods. Encapsulation of 85
probiotic with alginate by emulsion technique, the survival of encapsulated cell in 86
simulated GIT condition, the efficiency of encapsulation and morphology of 87
encapsulated cell were then evaluated. 88
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2. Materials and Methods 90
2.1 Screening of LAB with probiotic potential 91
2.1.1 Isolation of LAB 92
Ten raw cow milk, ten raw goat milk and ten fermented food samples were 93
collected from five markets in Singhanakhon, Am-per meuang, Jana, Hatyai and Sadao 94
districts in Sonkhla province, Thailand. To isolate the LAB, 25 g of each sample were 95
taken aseptically, transferred to sterile 0.85% NaCl (J.T. Baker, USA) and then 96
homogenized using the stomacher (Seward, England). One ml of homogenized samples 97
was removed for diluted by ten-fold dilution with 0.85% NaCl. Appropriate 98
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concentrations were pour plate in MRS agar containing 0.5% CaCO3 (Finechem, 99
Australia) and incubated at 37°C for 48 h. Difference colonies with clear zone from agar 100
plate were randomly selected from countable plates and then streaked onto MRS agar 101
containing 0.5% CaCO3. Strains with Gram positive and catalase-negative were selected 102
and stored at -20°C in MRS broths containing 20% glycerol (VWR International, 103
England). All strains were subcultured twice prior use. 104
2.1.2 Simulated GIT condition tolerance 105
This analysis was based on the method described by Krasaekoopt et al (2004). 106
Freshly prepared cells were placed in a tube containing sterile simulated gastric juice 107
(phosphate-buffer saline (PBS) pH 2.0 containing 3 mg/ml pepsin (Sigma, USA)) and 108
incubated at 37ºC for 3 h. After incubation, the cells were removed for counting the 109
survival cell and placed in sterile simulated intestinal juice (PBS pH 8.0 containing 3 110
mg/ml pancreatin and 1% bile salt (Sigma, USA)). The tubes were then incubated at 111
37ºC for 4 h. After incubation, a 1 ml aliquot of each isolates was removed and survival 112
rate was counted by drop plate on MRS agar containing 0.5% CaCO3. Isolates which 113
showed survival rate higher than 50% were selected for subsequence tests. Survival rate 114
was calculated by the following expression: 115
Survival (%) =
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2.1.3 Adhesion to hydrophobic solvent 117
Selected isolates were measured for cell surface hydrophobicity by measuring 118
microbial adhesion to hydrocarbons as described by Ocaña et al. (1999). Cells were 119
harvested by refrigerated centrifugation at 8500 rpm for 4 min, washed twice and 120
suspended in 2 ml of PBS pH 7.2 with the optical density at 600 nm of 0.4-0.6. Then 1 121
ml of n-hexadecane (Merk, Germany) was added to the cell suspensions, mixed by 122
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vortex for 3 minutes and allowed to separate at room temperature for 20 min. After the 123
separation, the optical density of the aqueous phase (lower phases) was determined at 124
600 nm. Isolates which showed high hydrophobicity were selected for subsequence test. 125
The index of hydrophobicity was the result of the decrease of turbidity of the aqueous 126
phase calculated by the following expression: 127
Hydrophobicity (%)
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2.1.4 Antimicrobial activity 129
Bacteria used as tested organism in this study consist of 4 pathogenic bacteria 130
(Escherichia coli DMST 4212, Listeria monocytogenes DMST 1327, Salmonella 131
Typhymurium DMST 562 and Staphylococcus aureus DMST 8840). They were 132
obtained from Department of Medical Sciences, Ministry of Public Health, Nonthaburi, 133
Thailand (DMST). The antimicrobial activity of selected isolates was determined by 134
agar spot test as described by Buntin el al (2008). Briefly, LAB were grown in MRS 135
broth then spotted on MRS agar. Plates were incubated at 37ºC for 18h. Pathogenic 136
bacteria were grown in Brain heart infusion (BHI) (Difco, USA) at 37ºC for 6 h (to 137
obtain approximately 6 log cfu/ml). Then 1 ml of growth culture was suspended in 9 ml 138
BHI soft agar (0.75%, w/v) and overlaid on MRS agar which LAB were grown. The 139
plates were incubated at 37°C for 24 h. The antimicrobial activity was recorded as 140
growth-free inhibition zones (diameter) around the cell spot. 141
2.1.5 Bile salt hydrolase (BSH) activity 142
The ability of strains to deconjugate primary and secondary bile salts was 143
determined according to Taranto et al. (1996). Bile salt plates were prepared by adding 144
0.5% (w/v) sodium salts of taurocholate (TC), taurodeoxycholate (TDC), glycocholate 145
(GC) and glycodeoxycholate (GDC) (Sigma, Germany) to MRS agar. All selected 146
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isolates were grown in MRS broth for 18 h. The liquid cultures of bacteria strains (10 147
µl) were spotted on agar plates and incubated at 37ºC for 72 h in anaerobic condition. 148
The presence of precipitated bile acid around colonies (opaque halo) was considered as 149
positive result. 150
2.1.6 Identification of LAB 151
Selected isolates with probiotic potential were identified by 16s rDNA 152
sequencing method as described by Chen et al. (2008). Briefly, LAB were cultured for 153
12 h in 1.5 ml MRS broth at 37°C and cells were collected by centrifugation at 5000 154
rpm for 3 min at 4°C. MRS broth was removed from the tube and genomic DNA was 155
extracted from the cell. The DNA extracted was amplified by forward Primer 28 F and 156
reverse primer 519 R. The amplification was done in 50 μl reaction volumes. Each PCR 157
reaction consisted of 11 μl water, 5 μl 28F (10 pmole), 5 μl 519R (10 pmole), 25 μl 158
master mix (Econo Taq®PlusGreen, Lucigen) and 4 μl DNA sample. The thermal 159
cycling included an initial denaturation step at 94°C for 5 min; 35 cycles of a 160
denaturation step at 95°C for 30 s, an annealing step at 52°C for 1 min, an extension 161
step at 72°C for 1 min; and a final extension at 72°C for 7 min. PCR products were 162
purified and then submitted to sequence. The sequence identities were determined by 163
BLAST program with the GenBank database. 164
2.2 Encapsulation of probiotic bacteria 165
2.2.1 Preparation of probiotic LAB for encapsulation 166
Selected probiotic LAB was subcultured twice in MRS broth. Cells were 167
harvested by centrifugation at 8500 rpm for 15 min at 4°C. Cell pellets were washed 168
twice with 0.85% NaCl and were finally re-suspended in 5 ml UHT milk with initial 169
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load of approximately 9-10 log cfu/ml, and the initial population of probiotic were 170
counted by pour plate in MRS agar containing 0.5% CaCO3. 171
2.2.2 Encapsulation procedure 172
All glassware and solutions used in the protocols were controlled in an aseptic 173
condition. Alginate beads were produced using a modified encapsulation method 174
originally reported by Homayouni et al. (2008). 1, 2 and 3% alginate (Fluka, USA) 175
mixture in distilled water containing 2% Hi-maize resistant starch (Merck, Germany) 176
and probiotic culture were prepared. Each mixture was added into 200 ml of soybean oil 177
(Morakot, Thailand) containing 0.2% lecithin (MEGA Lifesciences Ltd., Australia). 178
The mixture was stirred vigorously at 400 rpm for 20 min until it was fully emulsified. 179
Then 200 ml of 0.1 M calcium chloride solution were added. The mixture was allowed 180
to stand for 30 min to separate the prepared calcium-alginate beads in the bottom of 181
beaker as in calcium chloride layer. The oil layer was drained and beads were collected 182
in calcium chloride solution which then washed with 0.9% NaCl containing 5% glycerol 183
and stored at 4°C. 184
2.2.3 Alginate bead morphology 185
The shape of alginate beads was examined according to the method of 186
Allan-Wojtas et al. (2008). Samples were chemically stabilized by immerse fixation in 187
0.1 M PBS pH 5.0 containing 2.5% glutaraldehyde at 4ºC for 4 h then dehydrated 188
through an ethanol series as follows: 50% (1×15 min), 70% (1×15 min), 90% (1×15 189
min) and 100% (3×15 min). The alginate bead was then dried in a critical point drier 190
(Quorum Technologies Ltd, UK.). Dried samples were mounted on aluminium supports 191
using silver cement, sputter coated with gold/ palladium using a Hummer VII sputter 192
coater (Anatech Ltd., USA) and observed in a FEI Quanta 400 SEM (FEI Company, 193
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USA) operated at 20 kV at ambient temperature. Images were captured on Polaroid 194
Type 55 positive/ negative film (Polaroid Corporation, USA). 195
2.2.4 Determination of encapsulation efficiency 196
Alginate beads (1 g) were added to 9 ml of PBS pH 7.2 and then incubated at 197
37ºC for 2 h. 1 ml of homogenized samples was removed, diluted to appropriate 198
concentrations and pour plated on MRS agar containing 0.5% CaCO3. The 199
encapsulation efficiency (EE) was calculated by the following expression: 200
EE (%) = (Xt / Xi) ×100. 201
Where Xt is the total amount of probiotic loaded in alginate beads and Xi 202
represents the initial amount of probiotic added in the preparation process. 203
2.2.5 Simulated GIT condition tolerance 204
Alginate beads (1 g) were placed in a sterile tube and assayed using method 205
described in section 2.1.2. 206
2.2.6 Release of encapsulated cells 207
Alginate beads (1g) were transferred into 9 ml PBS pH 7.2, mixed gently and 208
then incubated at 37ºC for 6 h. Samples were kept after exposed to PBS pH 7.2 at 0, 1, 209
2, 3, 4, 5 and 6 h (Sheu and Marshall, 1993). Released bacteria were counted using pour 210
plate technique in MRS agar containing 0.5% CaCO3. The index of cell release was 211
calculated by the following expression: 212
Cell release (%)
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Statistical analysis 214
All experiments and analyses were run in triplicates. Data were recorded as 215
mean±standard deviation (S.D.) and were subjected to one-way analysis of variance 216
(ANOVA). Multiple comparisons were performed by Duncan's test. Statistical 217
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significance was set at P < 0.05. All analyses were performed using SPSS (SPSS Inc, 218
USA). 219
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3. Result and discussion 221
3.1 Screening of LAB with probiotic potential 222
3.1.1 Isolation of LAB 223
To isolate LAB, bacteria which showed clear zone on MRS agar containing 224
CaCO3 were collected. Three hundred isolated bacteria obtain from the different sources 225
were then identified on the basis of Gram-staining morphology and catalase test. The 226
isolates with Gram-positive and catalase-negative were collected. Table 1 shows the 227
morphological classification of isolated LAB. These 150 isolates were classified into 228
two groups based on cell morphology, including 76 cocci and 74 rod isolates. 229
3.1.2 Simulated GIT condition tolerance 230
In order to act as probiotic in the GIT and to exert their beneficial effect on the 231
host, the bacteria must be able to survive the acidic conditions in the stomach and resist 232
bile acids at the beginning of the small intestine. Approximately 2.5 l of gastric juice 233
and 1 l of bile are secreted into the human digestive tract every day. Thus, it is essential 234
for the bacteria to have protection systems to withstand the low pH in the stomach, 235
digestive enzymes and bile in the small intestine (Argyri et al., 2012). Table 2 shows 236
the survival of selected LAB isolates under simulated gastric juice condition and further 237
simulated small intestinal condition. Among 150 LAB isolates, only 9 isolates were able 238
to survive more than 50% in simulated GIT condition. These 9 isolates were composed 239
of 7 rods and 2 cocci bacteria. Argyri et al. (2012) reported the majority of L. plantarum 240
and L. pentosus strains showed high resistance to low pH. 241
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3.1.3 Adhesion to hydrophobic solvent 242
Hydrophobicity has been considered as one of the main physical interactions 243
during bacterial adhesion to epithelial cells. The hydrophobicity of a bacterial surface 244
can been determined by direct methods, such as measuring the contact angle and 245
indirect method such as the microbial adhesion to n-hexadecane (MATH) assay, which 246
was used in this work. 247
MATH assay was employed to evaluate the hydrophobic character of the 248
bacterial cell surface as shows in Figure 1. CM21, a rod-shape LAB, showed the highest 249
hydrophobicity among other strains. However, it can be observed that adhesion to n-250
hexadecane of all isolates was lower than 50%. 251
The adhesion ability of LAB has been linked with their surface properties, which 252
in turn is reflected by the composition, structure and organization of the cell wall. The 253
Gram-positive cell wall of lactobacilli consists of a thick peptidoglycan layer, which is 254
decorated with various surface components, including (lipo-)teichoic acids, 255
polysaccharides, covalently bound proteins and S-layer proteins. Several studies have 256
shown that these components are likely to contribute to the surface properties of a 257
bacterium (Deepika et al., 2009). 258
3.1.4 Antimicrobial activity 259
The bacteria used as indicators in this study included Gram-positive bacteria (S. 260
aureus and L. monocytogenes) and Gram-negative bacteria (E. coli and S. 261
Typhimurium). Only 3 isolates, CM21, CM47 and CM53 showed inhibition effect 262
against all phothogenic bacteria (Table 3). Among them, CM53 showed the highest 263
inhibitory effect against all pathogenic bacteria. Maldonado et al. (2012) reported that 264
natural inhibitor substances of LAB can be organic acids, hydrogen peroxide or 265
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bacteriocin. The lactic acid produced by LAB and the low pH is inhibitory to 266
susceptible microorganisms such as Enterobacteriaceae. The results indicated that LAB 267
isolates may inhibited the pathogens mainly by production of lactic acid. 268
3.1.5 BSH activity 269
BSH is an enzyme that catalyzes the hydrolysis of conjugated bile salts resulting 270
in free bile acid and amino acid. The white precipitates around colonies and the clearing 271
of the medium are indicative of BSH activity. Moreover, BSH activity has been 272
reported to correlate with cholesterol lowering. 273
Out of the 3 selected LAB isolates, based on antimicrobial activity against all 274
pathogens, two isolates (CM21 and CM53) displayed the precipitation zones on GC and 275
GDC agar plate as shows in Table 4. Dashkevicz and Feighner (1989) reported that L. 276
plantarum showed the BSH activity on GDC-MRS agar after incubated anaerobically 277
for 48 h. 278
3.1.6 Identification of LAB 279
In this work, 3 LAB isolates (CM21, CM47 and CM 53) presented probiotic 280
properties and showed antimicrobial activity against all tested bacteria. Thus they were 281
selected to identify by 16s rDNA sequencing and were determined by BLAST 282
comparison of the obtained sequences with the nucleotide database in the GenBank 283
database. 16S rDNA sequences of CM21, CM47 and CM53 identified as the species in 284
the Genus Lactobacillus with the similarity between CM21, CM47 and CM53 and L. 285
plantarum subsp. plantarum ATCC 14917T
of 99.17, 99.79 and 98.91%, respectively. In 286
addition, CM21, CM47 and CM53 showed most closely related to L. pentosus JCM 287
1558T, L. plantarum subsp. argentoratensis DKO 22
T and L. paraplantarum DSM 288
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10667T, L. fabifermentans DSM 21115
T and L. xiangfangensis 3.1.1
T with similarity 289
higher than 97% as shown in Figure 2. 290
In this study, L. plantarum CM53 which showed the greater antimicrobial effect 291
than CM21 and CM47 was selected for study the effect of encapsulation on survival in 292
simulated GIT condition. 293
3.2 Encapsulation of probiotic bacteria 294
3.2.1 Size, morphology and encapsulation efficiency of alginate bead 295
In this study, L. plantarum CM53 was selected for encapsulated by emulsion 296
technique. A small volume of the cell CM53-alginate suspension (discontinuous phase) 297
is added to a large volume of a soy bean oil (continuous phase). The mixture is 298
homogenized to form a water-in-oil emulsion. Once the water-in-oil emulsion is 299
formed, the water soluble polymer must be insolubilized (cross-linked) to form tiny gel 300
particles within the oil phase. The mean diameters of beads were measured, calculated 301
and presented in Table 5. The diameter of 1% alginate beads containing L. plantarum 302
CM53 was none determined by microscopy. While, mean diameter of 2 and 3% alginate 303
beads were determined and showed the different average diameters between the ranged 304
of 65-127 µm and 146-228 µm, respectively. The size and spherical of the bead depend 305
mainly on the viscosity of the sodium alginate solution, calcium chloride collecting 306
solution and speed of agitation (Krasaekoopt et al., 2004). Moreover, EEs for viable cell 307
of L. plantarum CM53 at 1-3% alginate were not significantly different (P > 0.05) 308
(Table 5). 309
SEM micrograph of the alginate bead containing L. plantarum CM53 showed 310
the spherical shape and the exterior surface of the bead was covered with a network of 311
small cracks and fissures (Figure 3A). The Figure 2D showed the internal structure of 312
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alginate bead. The bacteria were distributed randomly in the alginates matrix (Figure 313
3C) while the Figure 3D indicated that matrix alginate entrapped L. plantarum CM53. 314
3.2.2 Survival of free and encapsulated L. plantarum CM53 in simulated 315
GIT condition 316
Encapsulation in alginate containing 2% Hi-maize resistant starch bead improve 317
the survival of L. plantarum CM53 in simulated GIT significantly (P < 0.05) (Figure 4). 318
The addition of Hi-maize resistant starch at 2% concentration in the encapsulation 319
procedure was to provide prebiotic to the encapsulated probiotic bacteria (Sultana et al., 320
2000). Cell survival after exposure to stomach gastric juice (SGJ) for 3 h was 92.97, 321
89.55, 77.55, and 69.82% of the initial population found in alginate beads produced by 322
3%, 2% and 1% alginate and free cells, respectively. Overall, the sequential exposure to 323
SGJ (3 h) followed by simulated small intestine (4 h) resulted in significantly (P<0.05) 324
higher number of L. plantarum CM53 surviving in the 2 and 3% alginate bead than 325
were obtained for cells entrapped in 1% alginate bead and free cell. The cell survival 326
after exposure to simulated small intestine for 4 h was 91.18, 89.09, 74.30 and 69.09% 327
of the initial population found in alginate beads produced by 3, 2 and 1% alginate and 328
free cells, respectively. After the initial losses, the populations of L. plantarum CM53 329
declined at the same rate for all treatments at the 7 h incubation period with final 330
decreases of 2.96 log cfu/g for free cells and 0.70, 0.87 and 2 log cfu/g for 3, 2 and 1% 331
alginate beads, respectively. The survival rate of L. plantarum CM53 in GIT was 332
influenced by the concentration of alginate. Guérin et al. (2003) also found that an 333
initial immobilized Bifidobacteria bifidum population of 10 log cfu/g in mixed alginate, 334
pectin and whey protein matrix could reach the small intestine in numbers of 7.5 log 335
cfu/g and hence provide the host with a beneficial health effect. 336
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3.2.3 Release of encapsulated cells 337
While the ionotropic alginate gel formed by Ca2+
cross linking of carboxylate 338
groups is insoluble in low pH, exposure to neutral pH or higher solubilises the alginate 339
(Annan et al., 2008). In our study, this pH-dependent behavior of the biopolymer was 340
used to control the degradation of alginate bead and release of the microencapsulated 341
cell load under the natural conditions found in the small intestine. The ability to release 342
of L. plantarum CM53 from alginate beads in PBS pH 7.2 at the beginning of 343
incubation (0 h) was 48.15±0.06, 45.70±0.85 and 45.25±1.90% for 1, 2 and 3% alginate 344
beads, respectively as shown in Figure 5. When incubation time increased the release of 345
cells was increased. Ability to release L. plantarum CM53 after exposure to PBS pH 7.2 346
for 2 h were 97.00, 90.25 and 87.81% of the initial population found in alginate beads 347
produced by 1, 2 and 3% alginate, respectively. At 5-6 h of incubation, there were no 348
significant change (P > 0.05) indicating no effect of alginate concentrations on the 349
release of cells from microcapsules after 4 h. An efficient release of viable and 350
metabolically active cells in the intestine is one of the aims of microencapsulation 351
(Mandal et al., 2006). 352
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4. Conclusion 354
In conclusion, the results of this study showed that 3 strains of L. plantarum 355
(CM21, CM47 and CM53), were found to possess desirable in vitro probiotic 356
properties. These strains are good candidates for further investigation with in vivo 357
studies to elucidate their potential health benefits as well as in fermentation studies to 358
assess their technological characteristics for application as novel probiotic starters. 359
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Encapsulation of L. plantarum CM53 with alginate provided significant 360
protection for viable cell from harsh acidic condition of simulated GIT condition. As a 361
result, significantly higher number of bacteria survived sequential incubation from the 362
simulated gastric juice into the simulated small intestine where in accordance with the 363
soluble of alginate beads in an appropriate condition. 364
365
Acknowledgements 366
This work was supported by the Higher Education Research Promotion and the 367
National Research University Project of Thailand, Office of the Higher Education 368
Commission, the TRF senior research scholar program and the Graduate School, Prince 369
of Songkla University. 370
371
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19
Sheu, T.Y. and Masshall, R.T. 1993. Microencapsulation of lactobacilli in calcium 429
alginate gel. Journal of Food Science. 54, 557-561. 430
Sultana, K. Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P. and 431
Kailasapathy, K. 2000. Encapsulation of probiotic bacteria with alginate-starch 432
and evaluation of survival in simulated gastrointestinal conditions and in 433
yoghurt. International Journal of Food Microbiology. 62, 47-55. 434
Taranto, M. P., de Llano, D.G., Rodriguez, A., de Ruiz Holgado, A.P., and Font de 435
Valdez, G. 1996. Bile tolerance and cholesterol reduction by Enterococcus 436
faecium, a candidate microorganism for the use as a dietary adjunct in milk 437
products. Milchwissenschaft. 51, 383–385. 438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
20
Figure Legends 453
Figure 1. Cell surface hydrophobicity of selected lactic acid bacteria. The error bars 454
represent standard deviations. Different superscript indicate significant difference 455
(p<0.05). 456
Figure 2. Neighbor-joining tree comprising 16S rDNA sequences of CM21, CM53, 457
CM47 and related Lactobacillus species. Bootstap values (expressed as percentages of 458
1000 replications) greater than 60% are shown at the branch points. Bar, 0.01 459
substitution per nucleotide position. 460
Figure 3. Scanning electron microscopy of calcium alginate beads. (a) Alginate bead 461
(b) Fractured microcapsules without bacteria (an air pocket in dark area is visible) (c) 462
Fractured microcapsule loaded with bacteria (d) Higher magnification view of 463
microencapsulated bacteria. Magnification is indicated individually on data bar at 464
bottom at the bottom of micrograph 465
Figure 4. Survival of free and encapsulated L. plantarum CM53 during exposure to 466
simulated gastrointestinal tract condition at 37ºC for 7 h. Symbols: ( ) free cells, 467
( ) 1% alginate, ( ) 2% alginate and ( ) 3% alginate. Survival (%) represents 468
the percentage of cell surviving relative to the initial population. Different superscript in 469
the same time indicate significant difference (p<0.05). 470
Figure 5. Release of L. plantarum CM53 from alginate microcapsules during exposure 471
to simulated small intestine juice at different time intervals (0–6 h). Symbols: ( ) 1% 472
alginate, ( ) 2% alginate and ( ) 3% alginate. Cell release (%) represents the 473
percentage of cell surviving relative to the initial population. Different superscript in the 474
same time indicate significant difference (p<0.05). 475
476
477
21
Fig. 1 478
479
480
a
bc
ab
a
bc
d
bc bc
c
0
2
4
6
8
10
12
14
16
GM41 CM7 CM15 CM21 CM46 CM47 CM53 JJ4 KJ9
Hydro
phobic
ity (
%)
Isolate
22
Fig. 2 481
482
483
Lactobacillus koreensis DCY50T (FJ904277)
Lactobacillus senmaizukei L13T (AB297927)
Lactobacillus odoratitofui YIT 11304T (AB365975)
Lactobacillus similis JCM 2765T (AB282889)
Lactobacillus paracollinoides DSM 15502T (AJ786665)
Lactobacillus siliginis M1-212T (DQ168027)
Lactobacillus rossiae DSM 15814T (AKZK01000036)
Lactobacillus rapi YIT 11204T (AB366389)
Lactobacillus diolivorans JKD6T (AF264701)
Lactobacillus senioris YIT 12364T (AB602570)
Lactobacillus lindneri DSM 20690T (X95421)
Pediococcus parvulus JCM 5889T (D88528)
Pediococcus inopinatus DSM 20285T (AJ271383)
Pediococcus ethanolidurans Z-9T (AY956789)
Pediococcus cellicola Z-8T (AY956788)
Pediococcus lolii NGRI 0510QT (AB362985)
Lactobacillus tucceti R 19cT (AJ576006)
Lactobacillus versmoldensis KCTC 3814T (BACR01000055)
Lactobacillus bobalius 203T (AY681134)
Lactobacillus kimchii MT-1077T (AF183558)
Lactobacillus alimentarius DSM 20249T (M58804)
Lactobacillus kimchiensis L133T (HQ906500)
Lactobacillus nantensis LP33T (AY690834)
Lactobacillus xiangfangensis 3.1.1T (HM443954)
Lactobacillus fabifermentans DSM 21115T (AM905388)
Lactobacillus paraplantarum DSM 10667T (AJ306297)
CM21
CM53
CM47
Lactobacillus plantarum subsp. plantarum ATCC 14917T (ACGZ01000098)
Lactobacillus pentosus JCM 1558T (D79211)
Lactobacillus plantarum subsp. argentoratensis DKO 22T (AJ640078)
98
98
97
87
100
99
82
78
60
100
61
87
99
99
75
99
100
69
64
89
62
61
0.005
23
484
485
486
487
488
489
B A
C D
Fig. 3
24
Fig. 4 490
491
492
493
d c
c b b a
a a
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7
Surv
ival
(%
)
Time (h)
25
Fig. 5 494
495
Table Legends 496
Table 1. The morphological characteristic of isolated LAB 497
Table 2. Survival rate of selected LAB strains under simulated gastric juice conditions 498
at pH 2.0 for 3 h and further in simulated small intestinal tract condition at pH 8.0 for 4 499
h. 500
Table 3. The antimicrobial activity of selected LAB strains against food-borne 501
pathogenic bacteria 502
Table 4. The bile salt hydrolase activity of selected LAB strains 503
Table 5. Size and encapsulation efficiency of alginate bead at difference concentrate of 504
alginate 505
506
a
a a a a a
b
b b a
b
c b b
a
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6
Cel
l re
leas
e (%
)
Time (h)
26
Table 1. The morphological characteristic of isolated LAB 507
Samples
Number of LAB strains
Cocci Rod Total strains
Cow milk 1 53 54
Goat milk 43 11 54
Pla-Som 16 2 18
Fermented Jing-Jang Fish 6 2 8
Kimji 10 6 16
Total strains 76 74 150
508
509
27
Table 2. Survival rate of selected LAB strains under simulated gastric juice conditions 510
at pH 2.0 for 3 h and further in simulated small intestinal tract condition at pH 8.0 for 4 511
h. 512
Isolates
0 h
(log cfu/ml)
pH 2 for 3 h
(log cfu/ml)
Survival
(%)
pH 8 for 4 h
(log cfu/ml)
Survival
(%)
GF41 9.2 ± 0.7 6.1 ± 0.3 65.9 5.6 ± 0.2 60.1
CM7 8.9 ± 0.0 5.4 ± 0.1 61.0 4.5 ± 0.1 50.4
CM15 8.8 ± 0.1 5.3 ± 0.2 59.8 5.1 ± 0.2 58.3
CM21 9.6 ± 0.1 6.0 ± 0.1 62.7 5.9 ± 0.1 61.2
CM46 9.2 ± 0.9 6.1 ± 0.1 65.5 5.5 ± 0.3 59.3
CM47 8.6 ± 0.0 5.6 ± 0.4 64.8 5.2 ± 0.2 59.8
CM53 9.6 ± 0.1 5.8 ± 0.1 60.1 5.4 ± 0.3 55.9
JJ4 8.6 ± 0.1 5.8 ± 0.5 67.8 5.5 ± 0.1 64.7
KJ9 8.8 ± 0.0 5.4 ± 0.1 59.2 5.0 ± 0.0 56.9
Values are given as mean ± SD from triplicate determinations (n=3). 513
514
515
28
Table 3. The antimicrobial activity of selected LAB strains against food-borne 516
pathogenic bacteria 517
Isolates
Radius of inhibition zone around colony (mm)
E. coli Sal. Typhimurium S. aureus L.
momocytogenes
GF41 5.7 ± 0.4b _ 2.46 ± 0.22
c _
CM7 4.1 ± 0.5b _ _ _
CM15 6.3 ± 1.2b _ 2.50 ± 0.49
c _
CM21 6.4 ± 0.9b 3.49 ± 0.44
b 5.23 ± 0.32
b 3.38 ± 0.88
a
CM46 5.2 ± 1.2b _ 2.42 ± 0.20
c 1.19 ± 0.30
c
CM47 8.6 ± 0.3b 5.44 ± 0.13
a 4.59 ± 0.72
b 3.56 ± 0.02
a
CM53 9.7 ± 0.8a 5.05 ± 0.02
a 7.61 ± 0.18
a 3.06 ± 0.02
ab
JJ4 4.3 ± 0.2b _ 2.32 ± 0.62
c _
KJ9 5.7 ± 1.0b _ 2.58 ± 0.11
c 2.11 ± 0.02
bc
Values are given as mean ± SD from triplicate determinations (n=3). 518
Different superscripts in the same column indicate significant difference (p<0.05). 519
520
29
Table 4. The bile salt hydrolase activity of selected LAB strains 521
Isolates GC GDC TC TCD
CM21 + +
CM47 - +
CM53 + +
+ : the present of opaque halo around colonies, - : the absent of opaque halo around 522
colonies. 523
GC: glycocholate, GCD: glycodeoxycholate, TC: taurocholate and TCD: 524
taurodeoxycholate. 525
526
30
Table 5. Size and encapsulation efficiency of alginate bead at difference concentrate of 527
alginate 528
Treatments Alginate beads size (µm) Encapsulation efficiency (%)
1% Alginate - 81.10ns
2% Alginate 95.4 82.58 ns
3% Alginate 187.4 80.55 ns
ns No significant difference (p<0.05). 529
530
531