ORIGINAL PAPER Probiotic potential of lactic acid bacteria isolated from chicken gastrointestinal digestive tract H. Musikasang Æ A. Tani Æ A. H-kittikun Æ S. Maneerat Received: 28 October 2008 / Accepted: 16 March 2009 / Published online: 31 March 2009 Ó Springer Science+Business Media B.V. 2009 Abstract This study was conducted in order to evaluate the probiotic properties of lactic acid bacteria (LAB) iso- lated from intestinal tract of broilers and Thai indigenous chickens. The major properties, including the gastric juice and bile salts tolerance, starch, protein and lipid digesting capabilities, and the inhibition on certain pathogenic bac- teria were investigated. Three-hundred and twenty-two and 226 LAB strains were isolated from ten broilers and eight Thai indigenous chickens, respectively. The gastrointesti- nal transit tolerance of these 548 isolates was determined by exposing washed cell suspension at 41°C to simulated gastric juice (pH 2.5) containing pepsin (3 mg ml -1 ), and to simulated small intestinal juice (pH 8.0) in the presence of pancreatin (1 mg ml -1 ) and 7% fresh chicken bile, mimicking the gastrointestinal environment. The survival of 20 isolates was found after passing through the gastroin- testinal conditions. The survival rates of six strains; KT3L20, KT2CR5, KT10L22, KT5S19, KT4S13 and PM1L12 from the sequential study were 43.68, 37.56, 33.84, 32.89, 31.37 and 27.19%, respectively. Twelve isolates exhibited protein digestion on agar plate but no isolates showed the ability to digest starch and lipid. All 20 LAB showed the antimicrobial activity against Salmonella sp., Staphylococcus aureus and Escherichia coli except one strain which did not show the inhibitory activity toward E. coli. Accordingly, five isolates of selected LAB (KT2L24, KT3L20, KT4S13, KT3CE27 and KT8S16) can be classified as the best probiotics and were identified as Enterococcus faecalis, Enterococcus durans, Enterococcus faecium, Pediococcus pentosaceus, and Enterococcus faecium, respectively. The survival rate of microencapsulation of E. durans KT3L20 under simulated small intestine juice after sequential of simulated gastric juice was also investigated. An extrusion technique exhib- ited a higher survival rate than emulsion technique and free cell, respectively. Keywords Probiotic Á Lactic acid bacteria Á Chicken intestinal tract Á Broiler Á Thai indigenous chicken Introduction In recent years considerable interest has been shown in using some probiotic microorganisms and organic acids as an alternative to the use of antibiotics in feeds (Guerra et al. 2007). Probiotics are a live microbial feed supplements which positively affects the health of the host animal by improving its intestinal balance (Fuller 1989). LAB is one of the probiotic groups which make up a large group of microorganism in gastrointestinal tract of all human and animals. The basic requirements for an LAB strain which is to be used as probiotic have been described as follows. They should be tolerant to acid and bile and be able to: adhere to the intestinal epithelium of the hosts; show an antagonistic activity against pathogenic bacteria and; keep their viability during processing and storage (Lin et al. 2007). The most probiotic microorganisms used are: Lac- tobacillus (e.g. L. bulgaricus, L. acidophilus, L. casei, L. helveticus, L. lactis, L. salivarius, L. plantarum); Bifi- dobacterium; Bacillus; Streptococcus; Pediococcus; H. Musikasang Á A. H-kittikun Á S. Maneerat (&) Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90112, Thailand e-mail: suppasil.m@psu.ac.th A. Tani Research Institute for Bioresources, Okayama University, Okayama, Japan 123 World J Microbiol Biotechnol (2009) 25:1337–1345 DOI 10.1007/s11274-009-0020-8 Enterococcus; and yeasts such as Saccharomyces cerevi- siae and S. boulardii (Fuller 1989; Hyronimus et al. 2000). The use of probiotic bacteria and their metabolites has many beneficial effects on cattle, pigs and chickens. These include the improvement of: general health; feed conver- sion ratios; growth rates; resistance to diseases; promoting body weight and increase in milk yield; quality and egg production (Ahmad 2006; Guerra et al. 2007; Hyronimus et al. 2000). They have been used as a substitute of anti- biotics in considerable amounts and as growth promoters in broilers production (Ahmad 2006). When selecting LAB for use as dietary adjuncts, a number of factors should be considered. While the func- tionality of probiotics depends on their ability to survive and colonize the gastrointestinal tract, the resistance of cells to bile acids is a property that is necessary (Taranto et al. 2006). In order to be effective the bacteria must therefore survive when exposed to the acid in the stomach and bile in the intestine (Shah 2000). However, many studies have indicated that probiotic bacteria may not survive in sufficient numbers when they pass through the gastrointestinal tract in in vitro test (Lin et al. 2006; Maragkoudakis et al. 2006). The encapsulation technique is an approach which is currently receiving significant inter- est for resisting environmental conditions that are adverse to probiotics. Entrapment in calcium alginate beads has frequently been used for the immobilization of LAB. Alginate has the benefits of being non-toxic to the cells being immobilized, and is an accepted food additive. The reversibility of encapsulation, i.e. solubilizing alginate gel by sequestering calcium ions, and the possible release of entrapped cells in the human or animal intestine is another advantage (Chandramouli et al. 2004; Kailasapathy 2002; Sheu and Marshall 1993). Therefore, this study was carried out in order to isolate and screen LAB as probiotics from gastrointestinal diges- tive tracts of marketable broilers and Thai indigenous chickens. The enhancement of LAB survival through the application of microencapsulation to use as feed supple- ment for broilers was also studied. Materials and methods Lactic acid bacteria isolation The gastrointestinal digestive tracts (crop, small intestine, large intestine and cecum) of ten marketable broilers and eight Thai indigenous chickens were used as LAB sources. Each part of the intestinal tract was washed in 70% ethanol and washed twice with sterile distilled water. Twenty-five grams of washed section gut was homogenized in 225 ml phosphate-buffered saline (PBS : 50 mM potassium di-hydrgenphosphate, 50 mM di-potassium hydrogen phosphate trihydrate, 0.85% sodium chloride, pH 7.0) for 5 min using a stomacher (Stomacher Ò 400 Circulator, Seward Ltd., UK). Appropriate serial dilutions were plated onto Man, Rogosa and Sharpe (MRS) agar (HiMedia Laboratories, Pvt. Ltd., India) supplemented with 0.02% bromocresol purple (Ajax Finechem, Australia) and incu- bated anaerobically for 24 h at 41°C. Colonies which exhibited a clear halo were randomly selected from the highest dilutions of each MRS agar plate. Bacterial colo- nies were then purified by re-streaking on MRS agar 2–3 times. The pure cultures were characterized using Gram stain, cell morphology and catalase reaction tests. Gram- positive and catalase-negative isolates were stored at -20°C in MRS broth supplemented with 25% (v/v) glycerol. For routine analysis, the strains were subcultured twice in MRS broth for 24 h at 41°C. The selected isolates were further identified along full length of 16S rRNA sequence based on the methods of Gonza ´ lez et al. (2007). Resistance to simulated intestinal juice after sequential incubation in simulated gastric juice of isolated LAB A simulated gastric juice was prepared by suspending 3mgml -1 pepsin (Fluka, Biochemika, Japan) in sterile saline (0.85% NaCl, w/v) and adjusted the pH to 3.0 with 1.0 M HCl. Twenty-four hour 1.0 ml cultures of the strains were subjected to centrifugation in an Eppendrof centrifuge (Eppendorf Centrifuge 5415R, Hamberg, Germany) at 10,000 rev min -1 for 10 min and washed twice with sterile saline before being re-suspended in simulated gastric juice. Resistance was assessed in terms of the viable colony count and enumerated after incubation at 41°C for 2 h. After 120 min of gastric digestion, cells were harvested and suspended in simulated intestinal fluid which contained 1mgml -1 pancreatin (Sigma, Germany) and 7% fresh chicken bile at pH 8.0. The suspension was incubated at 41°C for 6 h and the viable count was determined (modi- fied from Madureira et al. 2005). Starch, protein and lipid digesting capabilities Modified MRS agar containing skimmed milk (HiMedia Laboratories Pvt. Ltd., India), tributyrin (Fluka, USA) and soluble starch (Labchem, Ajax Finechem, Australia) was used for detecting the protein, lipid and starch digesting capabilities of selected LAB strains, respectively. The overnight cultures of LAB (10 ll) were dropped on the modified MRS agar and incubated at 41°C for 24 h. The diameters of the holo zone on the agar plate were then measured. The digesting capability of the tested strains was classified as positive when the diameters of clear zone were 1338 World J Microbiol Biotechnol (2009) 25:1337–1345 123 more than 1 mm. Each assay was performed in triplicate (Thongsom 2004). Antibacterial activity Antibacterial activity was studied using the agar diffusion method (Makras and Vuyst 2006). The indicator strains used in this study were gram-negative strains as the main pathogenic microorganisms in the intestinal tract of chicken such as Escherichia coli, and Salmonella sp. In addition, some bacterial species potentially pathogenic to humans, Staphylococcus aureus, was also used. All strains were obtained from Songklanagarind Hospital, Prince of Songkla University, Thailand. Indicator strains were cultivated in nutrient broth (HiMedia Laboratories Pvt. Ltd., India) at 41°C for 18 h. To measure the antibacterial activity, LAB were cultivated in MRS broth at 41°C for 18 h. The culture containing 10 ll of LAB (10 8 cfu ml -1 ) was dropped on MRS agar and incubated at 41°C under anaerobic condition for 18 h. The LAB on MRS agar plate were overlaid with 9 ml of soft nutrient agar with 1 ml of culture of indicator strains activated overnight (10 6 cfu ml -1 ). The agar plates were incubated at 41°C for 18 h and diameters of inhibition zone on the agar plate were measured. Each assay was performed in triplicate. The antibacterial activity was cal- culated as follows: The antibacterial activity ðmmÞ ¼ Diameter of inhibition zone À Diameter of LAB colony Cell preparation for microencapsulation The 20% starter cultures of selected LAB were inoculated in 50 ml MRS broth and incubated at 41°C for 24 h to obtain a cell density of about 10 9 cfu ml -1 . Harvesting of cells was done by centrifugation at 8,500 rev min -1 for 20 min at 4°C. Cell pellet was washed twice with sterile saline. Washed cells were then suspended in 1 ml of sterile saline and stored at 4°C until use. Microencapsulation and enumeration of microencapsulated LAB Washed cells were prepared for encapsulation by extrusion and emulsion techniques. For the extrusion technique, probiotic capsules were prepared by mixing the 1 ml of LAB suspension with 20 ml of 3% sodium alginate (Fluka, Switzerland). The cell suspension was extruded through dropping with a 24G syringe needle into 0.1 M calcium chloride solution (the distance between the syringe and the calcium chloride collecting solution was 5 cm). The beads were allowed to stand for 30 min to ensure complete gelification (Krasaekoopt et al. 2003, 2004; Muthukumar- asamy and Holley 2007). For the emulsion technique, 1 ml of washed cell sus- pension was added to 20 ml of 3% sodium alginate and the mixture was then emulsified into palm oil (Morakot, Morakot industry Co. Ltd., Thailand) with the ratio 1:5. The emulsion was produced by stirring for 20 min at a constant speed (900 rev min -1 ) until it was creamy. A solution of 0.1 M calcium chloride was then added quickly along the side of the beaker. The mixture was allowed to stand for 30 min. The oil layer was then removed (Annan et al. 2008; Krasaekoopt et al. 2003; Sultana et al. 2000). The beads from the two encapsulation techniques were harvested by filtration (Whatman No. 4, filter paper, Fisher Scientific) then rinsed and stored in peptone saline (1 g l -1 peptone, 8.5 g l -1 sodium chloride) containing 0.05 M calcium chloride pending further analysis. The microencapsulated LAB were enumerated as described by Kailasapathy (2006) and Annan et al. (2008). The encapsulated bacteria in the microcapsules were released by using 1.0 g of a filtered microcapsule and were re-suspended in 9.0 ml of PBS buffer (pH 7.5) in a plastic bag. It was homogenized for 10 min to allow complete release of the bacteria from alginate capsules by using a stomacher. The homogenized samples were diluted to appropriate concentrations and drop-plated on MRS agar. The plates were incubated anaerobically for 24 h at 41°C and the encapsulated bacteria were enumerated as cfu ml -1 . Survival of encapsulated probiotic in simulated small intestinal juice after sequential incubation in simulated gastric juice One gram of the encapsulated probiotic and 1 ml of non- encapsulated probiotic samples of individual treatments were incubated in 9 ml of simulated gastric juice (3 mg ml -1 pepsin, pH 2.5) at 41°C for 2 h. Microencap- sulated beads in simulated gastric juice were then centrifuged at 8,500 rev min -1 at 4°C for 20 min and washed with 0.85% sodium chloride. The obtained cap- sules were re-suspended in 9 ml of simulated small intestinal juice (1 mg ml -1 pancreatin, 7% fresh chicken bile, pH 8.0) at 41°C for 6 h. The survivals of free cell and encapsulated probiotic before and after exposure to simu- lated small intestinal juice for 6 h after sequential incubation in simulated gastric juice for 2 h were deter- mined by plating in MRS agar containing 0.02% bromocresol purple. Plates were incubated anaerobically at 41°C for 24 h using the anaerobic jar (modified from Madureira et al. 2005). World J Microbiol Biotechnol (2009) 25:1337–1345 1339 123 Results and discussion Lactic acid bacteria isolation Three-hundred and twenty-two and 226 LAB strains were isolated from 10 marketable broilers and eight marketable Thai indigenous chickens, respectively (Table 1). The 548 isolates of LAB were isolated from crop, small intestine, large intestine and caecum of chicken intestinal tracts. The number of LAB isolated from each organ of broilers or Thai indigenous chicken was almost similar. In addition, observation under light microscopic revealed that about 85% of isolated LAB was rod shape and 15% was cocci (data not shown). The result was in accordance with a previous study that major LAB in native chicken intestinal tract was rod shape (Sonplang et al. 2007). Studies on microbiota of the alimentary tract in animals show the complex of bacteria. Base on their roles, the intestinal bacteria may be divided into two groups: LAB and putrefactive bacteria. LAB are evaluated as beneficial bacteria by their product of acids (lactic acid), bacteriocin or bacteriocin-like substances. Putrefactive bacteria are regarded as harmful bacteria in that they decompose pro- teins, produce foul-smelling substances and some cause of diarrhea or produce toxins. For these reasons, LAB are paid great attention for use as probiotics for animal produce. For the chicken, the intestinal LAB are mainly Lactobacillus and Enterococcus (Lan et al. 2003). Resistance to simulated small intestinal juice after sequential incubation in simulated gastric juice of isolated LAB High acidity in the stomach and the high concentration of bile components in the proximal intestine of the host influence probiotic strain selection (Hyronimus et al. 2000). In this study, the 20 isolates of LAB were selected after the passage through the simulated gastric juice (pH 3.0) containing pepsin (3 mg ml -1 ) for 120 min at 41° C. Then there was sequential incubation with simulated small intestinal juice (pH 8.0) containing pancreatin (1 mg ml -1 ) and 7% of fresh chicken bile for 6 h. Amoung these 20 isolates there was only one strain, PM1L12, isolated from Thai indigenous chickens. However, isolates were mostly isolated from the small intestine. There were only six strains (KT3L20, KT2CR5, KT10L22, KT5S19, KT4S13 and PM1L12) that could survive in the sequential study by showing the survival rate of 43.68, 37.56, 33.84, 32.89, 31.37 and 27.19%, respectively (Fig. 1). Results from this study showed that a few strains are acid and bile tolerant. The viable LAB cell numbers initially decreased approxi- mately 1–2 log cfu ml -1 for most strains. In general, the acid tolerance of LAB depends on the pH profile of H ? -ATPase and on the composition of the cytoplasmic membrane. This is largely influenced by the type of bac- terium, the type of growth medium and the incubation conditions (Hood and Zotolla 1988; Madureira et al. 2005). However, all 20 isolates could survive higher than 10 6 cfu ml -1 even after 2 h of exposure to the simulated Table 1 Number of LAB from broilers and Thai indigenous chicken gastrointestinal tracts Organ Thai indigenous chickens (isolate) Broilers (isolate) Total (isolate) Crop 60 86 146 Small intestine 54 78 132 Large intestine 57 76 133 Cecum 55 82 137 Total 226 322 548 27 .19 7 .73 33.84 1.68 12.42 0.54 32.89 8.64 2.43 10.47 31 . 73 10.63 15 .13 43 .68 0.23 0.92 1.98 3.74 37.56 1.85 0 2 4 6 8 10 12 KT2 CR 3 KT 2 CR5 KT 2S11 KT 2S 15 KT 2 L 24 KT 3 CR 2 KT3 L 20 KT 3 CE 27 KT 3 CE 28 KT 4S13 KT 5S13 KT 5S15 KT 5 S 16 KT 5S19 KT 8 CR 4 KT 8S 16 KT 10 L19 KT 10 L 22 KT 10CE 33 PM 1 L 12 0 5 10 15 20 25 30 35 40 45 50 0h 6h %survival Viable counts (log cfu ml -1 ) Survival (%) Lactic acid bacteria Fig. 1 Survival rates of selected LAB in the presence of 7% fresh chicken bile and pancreatin (1 mg ml -1 )atpH 8.0 (4 h) after sequential incubation in simulated gastric juice (2 h) 1340 World J Microbiol Biotechnol (2009) 25:1337–1345 123 gastric juice (pH 2.5) containing pepsin (3 mg ml -1 ) (data not shown). In comparison to the acid tolerance of the Lactobacillus species isolated from the gastrointestinal tracts of swine and chicken, Lin et al. (2007) found that L. acidophilus and L. bulgaricus from chicken were less stable in the chicken gizzard extract (pH 2.6). However, some L. acidophilus strains isolated from other origins such as human digestive tract showed acid tolerance in the pH 2.5 gastric juice environment. In order to describe selected isolates from the probiotic point of view, resistance to pH and bile salts is of great importance in survival and growth of bacteria in animal gastrointestinal tracts. The second important criterion is the resistance against bile salts that is a prerequisite for the colonization and metabolic activity of probiotic bacteria in the small intestine of the host (Strompfova ´ and Laukova ´ 2007). The current study was based on the approach of Madureira et al. (2005). This combined the effect of exposure to gastric juice, followed by the effect of expo- sure to bile salts on the viability of probiotic strains. After 120 min of exposure to artificial gastric juice, 0.3% (w/v) bile salts was added to the homogenates and the incubation was extended for a further 2 h. This approach simulated two situations that prevailed during transit through the gastrointestinal tract: passage through the stomach, fol- lowed by release of bile salts in the small intestine. The pH levels of gastric juice may vary from 2.0 to 3.5 depending on the feeding time, the growing stage or the kind of ani- mal (Yu and Tsen 1993). The pH in chicken proventriculus and gizzard ranges from 2.5 to 4.74 (Malaipuang 2001) and food ingestion can take up to 1–3 h depending on feed size. The combined effect of a pepsin-pH solution aims at simulating the gastric juice. However, it is not clear whe- ther the decrease of viability conferred by the pepsin solution at pH 2 was due to the enzyme alone, or in synergy with low acidity (Maragkoudakis et al. 2006). In contrast to pepsin, most strains examined in this study could survive well in a pancreatin solution at pH 8.0 or in the presence of fresh chicken bile (7%, v/v), simulating the chicken small intestine environment. Bile salts at high concentrations can rapidly dissolve membrane lipids and cause dissociation of integral mem- brane proteins resulting in the leakage of cell contents and cell death (Begley et al. 2005). It has been suggested that the major effect of bile acids would be the disaggregation of the lipid bilayer structure of the cell membrane. Con- jugated bile acids are less inhibitory than free bile acids (cholic and deoxycholic acid, DCA) toward intestinal aerobic and anaerobic bacteria. Taurine-conjugated deoxycholic acid (TDCA) was less toxic than DCA. The tolerance to bile salts was initially associated with the presence of bile salt hydrolase activity (Moser and Savage 2001; Taranto et al. 2006). In the small intestine of chicken, the total concentration of bile salts is about 10–11 mmol kg -1 digesta and the proportion of conjugated to unconjugated bile varies according diet. However, the conjugated forms of chenodeoxycholic and cholic acid dominate most frequently (Knarreborg et al. 2003; Strom- pfova ´ and Laukova ´ 2007). Lactobacillus casei NCDC 63, L. casei VT and L. casei C1 could survive after being treated with 2% (ox bile) for 2 h of incubation (Mishra and Prasad 2005). Lactobacillus sp. exhibited survival to bile salt and the presence of 0.3 mg l -1 pancreatin (Maragkoudakis et al. 2006). Starch, protein and lipid digesting capabilities The agar plate assays were used to study digesting capability of the 20 isolates of LAB. In this study, sterilized skimmed milk, tributyrin and soluble starch were used for detecting protein, lipid and starch digestion capabilities, respectively. There were 12 isolates (KT2CR3, KT2CR5, KT2S11, KT2L15, KT2L24, KT4S13, KT5S13, KT5S15, KT5S16, KT5S19, KT8CR4 and KT10CE33) which exhibited protein digestion. However, neither starch nor lipid digestions was detected. Some strains of LAB are able to utilize protein, starch and lipid (Duangchitchareon 2006; Kawai et al. 1999; Thongsom 2004). LAB, which are able to digest starch, protein and lipid, could enhance the health of aquatic animals (Austin et al. 1995). Antibacterial activity The agar diffusion method was used to study antimicrobial activity of the 20 isolates of LAB. All 20 isolates showed the antimicrobial activity against Escherichia coli (with an inhibition zone 8–25 mm in diameter), Salmonella sp. (13– 40 mm) and Staphylococcus aureus (6–24 mm). However, one strain; PM1L12 did not show the inhibitory activity towards E. coli (Table 2). Lan et al. (2003) reported that the two selected probiotic strains (L. agillis JCM 1048 and L. salivarius subsp. salicinius JCM 1230) which were isolated from chicken, were able to inhibit growth of Sal- monella spp. (with an inhibition zone 16–18 mm in diameter). They were less effective for Escherichia coli (7– 8 mm) in the agar spot test. It was shown in this study that most of the selected LAB showed high antimicrobial activity against Salmonella sp. The antibacterial activity of LAB may often be due to the production of organic acids, with a consequent reduction in pH, or to the production of hydrogen peroxide (Gonza ´ lez et al. 2007). LAB could produce various compounds such as organic acids, diacetyl, hydrogen peroxide, and bacte- riocin or bactericidal proteins during lactic fermentations. World J Microbiol Biotechnol (2009) 25:1337–1345 1341 123 Levels and types of organic acids produced during the fermentation process depended on LAB species or strains, culture compositions and growth conditions (Lindgren and Dobrogosz 1990). Lactic acid is the major organic acid in LAB fermentation where it is in equilibrium with its undissociated and dissociated forms, and the extent of the dissociation depends on pH. The antimicrobial effect of organic acids lies in the reduction of pH, as well as the undissociated form of the molecules. It has been proposed that the low external pH causes acidification of the cell cytoplasm, while the undissociated acid, being lipophilic, can diffuse passively across the membrane. The undisso- ciated acid acts by collapsing the electrochemical proton gradient, or by altering the cell membrane permeability, which results in disruption of substrate transport systems (Ammor et al. 2006). In general, organic acids have a strong inhibitory activity against gram-negative bacteria (Makras and Vuyst 2006). The bacteriocins, generally recognized as safe LAB by the GRAS, have generated a great deal of attention as a novel approach to control pathogens in food-stuffs (Savadogo et al. 2004). Hydrogen peroxide is produced by LAB in the presence of oxygen as a result of the action of flavoprotein oxidases or nicotin- amide adenine dinucleotide (NADH) peroxidase. The antimicrobial effect of hydrogen peroxide may result from the oxidation of sulfhydryl groups causing denaturing of a number of enzymes, and from the peroxidation of mem- brane lipids, thus increasing membrane permeability (Condon 1987; Kong and Davison 1980). LAB strains were reported to inhibit the growth of pathogenic bacteria in many studies (Ammor et al. 2006; Bernbom et al. 2006; Collado et al. 2005; Olkowski et al. 2008; Santos et al. 2003). It may also be due to the production of bacteriocins or bacteriocin-like compounds (Gonza ´ lez et al. 2007). In this research, it has been demonstrated that isolated LAB had probiotic properties against both gram-negative and gram-positive pathogenic bacteria but the mode of inhibition is not exactly known. Additional investigations have to be performed to examine for the mode of action of these LAB toward pathogens. Strains identification From the over all testing, there were only five isolations of LAB that showed the best preliminary probiotic properties, including resistance to simulated gastric and intestinal fluids, digesting capability and antibacterial activity. There were KT2L24, KT3L20, KT4S13, KT3CE27 and KT8S16. These five LAB strains were isolated from the lower intestinal tracts of broilers. These LAB were then identified by comparing the full length of 16s rRNA sequences. The results indicated that they were identified as Enterococcus faecalis (100%), Enterococcus durans (99.73%), Entero- coccus faecium (99.93%), Pediococcus pentosaceus (99.93%) and Enterococcus faecium (99.67%), respec- tively. The rRNA sequences were deposited in DDBJ/ EMBL/GenBank as accession numbers AB481105, AB481101, AB481104, AB481102 and AB481103, respectively. Enterococci constitute part of the natural gut microflora in mammals (De Fa’tima Silva Lopes et al. 2005; Devriese et al. 1992). They also have good microbiologic features such as short generation time and bacteriocin production, but there is concern about the transmission of antimicrobial resistance gene (Mombelli and Gismondo 2000). However, Enterococcus such as E. faecium and Pediococcus such as P. acidilactici are mainly bacterial strains of gram-positive bacteria which were used in animal feed in the European Union (EU) (Anado ´ n et al. 2006). Pediococci are gram-positive LAB that is being used as starters in the industrial fermentation of meat and vegeta- bles. Some strains of the Pediococcus species produce antimicrobial peptides that inhibit closely related LAB and gram-positive spoilage and pathogenic bacteria (Gurira and Buys 2005). Table 2 Antibacterial activity of selected LAB against pathogenic bacteria Strains Antibacterial activity (mm) Escherichia coli Salmonella sp. Staphylococcus aureus KT2CR3 17.0 ± 1.0 19.7 ± 0.6 11.7 ± 1.2 KT2CR5 18.3 ± 2.1 26.7 ± 1.2 15.3 ± 1.5 KT2S11 24.3 ± 0.6 15.0 ± 1.7 16.3 ± 2.1 KT2S15 17.0 ± 2.0 25.7 ± 2.1 16.7 ± 2.3 KT2L24 16.3 ± 1.2 17.0 ± 1.0 11.0 ± 1.0 KT3CR2 11.7 ± 0.6 36.3 ± 1.2 20.7 ± 1.2 KT3L20 25.7 ± 1.5 25.3 ± 1.5 16.7 ± 1.5 KT3CE27 17.0 ± 1.0 28.0 ± 0.0 21.0 ± 1.0 KT3CE28 08.3 ± 0.6 13.7 ± 0.6 06.7 ± 0.6 KT4S13 25.3 ± 1.2 26.7 ± 0.6 13.7 ± 0.6 KT5S13 14.0 ± 1.0 29.3 ± 2.3 14.7 ± 0.6 KT5S15 15.3 ± 1.5 29.7 ± 2.3 15.7 ± 0.6 KT5S16 18.0 ± 1.0 28.3 ± 2.1 14.3 ± 1.2 KT5S19 18.0 ± 1.0 24.7 ± 0.6 15.7 ± 1.2 KT8CR4 18.0 ± 2.0 40.0 ± 2.6 24.0 ± 1.0 KT8S16 21.0 ± 1.0 26.3 ± 0.6 14.0 ± 2.0 KT10L19 13.3 ± 1.5 26.0 ± 2.0 15.3 ± 0.6 KT10L22 15.3 ± 0.6 35.7 ± 1.2 17.0 ± 1.0 KT10CE33 08.7 ± 0.6 26.0 ± 1.7 18.7 ± 1.5 PM1L12 – 22.3 ± 1.5 09.3 ± 0.6 Each value in the table is the mean ± standard deviation of three trials ‘–’ Represents the absence of an inhibition efficiency 1342 World J Microbiol Biotechnol (2009) 25:1337–1345 123 Survival of free and encapsulated probiotic LAB during sequential incubation in simulated gastric and intestinal juices The survival rate of free cell and microencapsulated E. durans KT3L20 using simulated small intestine juice after sequential use of simulated gastric juice were inves- tigated. The extrusion technique exhibited higher survival rates than the emulsion technique and free cell, respec- tively (Table 3). The encapsulation techniques could protect these E. durans KT3L20 effectively from high acid and bile conditions. Muthukumarasamy et al. (2006) found that microencapsulation using alginated solutions by extrusion or emulsion techniques provided greater protec- tion against gastric juice for L. reuteri. The extrusion technique was better able to protect cells. In this study there were 1.4 log decreases in viable cells of encapsulated E. durans KT3L20 after 2 h of incubation with simulated gastric juice (pH 2.5) compared to 3 log decrease in the free cells. Chandramouli et al. (2004) reported the survival of L. acidophilus CSCC 2409. There was a two log decrease in encapsulated cells after 3 h incubation at pH 2, compared to a four log decrease in the free cells under similar conditions. Microencapsulation of L. casei NCDC-298 in alginate beads resulted in better survival than free cells after incubation in a simulated gastric and intestinal bile salt solution (Mandal et al. 2006). Microencapsulation is able to protect the bacterial cell against harsh environments such as during passage through the acidic pH of the stomach (Muthukumarasamy et al. 2006). This study indicated that the immobilization of this culture with alginate could enhance bacterial survival under simulated intestinal condition. Higher survival was also reported when probiotic immobilized in alginate beads were incubated in simulated gastric and bile salt solution (Chandramouli et al. 2004; Krasaekoopt et al. 2004; Lee and Heo 2000; Mandal et al. 2006). Alginate gels are stable in low pH solutions but swell in weakly basic solutions. While the ionotropic alginate gel formed by Ca 2? crosslinking of carboxylate groups is insoluble at low pH, exposure to neutral pH or higher solubilizes the alginate (Annan et al. 2008). Alginate cap- sules and microspheres can be used to protect cells from the acidity of gastric juices while allowing subsequent release in the basic environment of intestinal fluids. Conclusions Enterococcus faecalis KT2L24, Enterococcus durans KT3L20, Enterococcus faecium KT4S13, Pediococcus pentosaceus KT3CE27 and Enterococcus faecium KT8S16 were found in vitro to possess desirable probiotic properties. These strains are good candidates for further investigation in vivo studies to elucidate their potential heath benefits and their application as promising probiotic strains in the feed industry. Acknowledgments This work was financially supported by Prince of Songkla University through Contract No. AGR5122020037S, Faculty of Agro-Industry and Graduate school, Prince of Songkla University. 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(2007) found that L. acidophilus and L. bulgaricus from chicken. B.V. 2009 Abstract This study was conducted in order to evaluate the probiotic properties of lactic acid bacteria (LAB) iso- lated from intestinal tract of broilers and Thai indigenous chickens.