Lactic Acid Bacterial Screening From Gastrointestinal Digestive Tract of Native and Broiler Chicken for Probiotic Candidate Purposes

The aim of his research was to obtain lactic acid bacteria (LAB) from gastrointestinal digestivetract (GIT) of chickens for probiotic candidate purposes. LAB was isolated from GIT of broiler andnative chickens on selective medium (MRS+0.2% CaCO3). Screening method based on microbiologicaland biochemical characteristics, antibacterial properties, growth on various temperature, aeration, andagitation, antibiotic sensitivity, and viability on acid pH, gastric juice and bile salt. Thirty nine of LABisolates was selected from native chicken and 18 isolates from broiler chicken. The selected LABinhibited Escherichia coli FNCC 0091 growth and grown on 30, 39 and 45oC of temperature, aerobic,anaerobic and agitation conditions. Biochemical identification using API 50 CHL kit revealed that I72from native chicken ileum as Lactobacillus salivarius and Db9 from broiler chicken duodenum asPediococcus pentosaceus. All LAB were resistant to Erythromicin, Penicillin G and Streptomycin astested antibiotics. Both of them have non significantly different of viability on acid pH (1, 2 and 3),gastric juice pH 2 and bile salt which were 91.78% for L. salivarius I72 and 94.48% for P. pentosaceusDb9 (P<0.05). Based on characteristics, both the selected LAB have potentiality as chicken probioticcandidates.


INTRODUCTION
The history of the Indonesian poultry industry illustrates significant contributions to the national meat supply. Indonesian consumers preference for chicken meat creates a large domestic market (Helinna, 2001). Indonesian people consumption rates only 6.1 kg per capita per year, it is still lower than others leading countries (Bond et al., 2007). One of factor which caused less production of chicken's meat is the diseases problem (Patterson and Burkholder, 2003). Animal enteric pathogens are a direct source for food contamination. The prohibition of antibiotics as growth promoters (AGPs) use has been a challenge for animal nutrition therefore need to find alternative methods to control and prevent pathogenic bacterial colonization. The modulation of the gut microbiota with new feed additives such as probiotics against hostprotecting functions to support animal health, is a topical issue in animal breeding and creates fascinating possibilities (Gaggia et al., 2010).
Currently, probiotics are used as health supplements in food and feeds and they are replacing the use of antibiotic growth promotors or chemical supplements (Kosin and Rakshit, 2006). Others, probiotic is a natural organic matter that could not leave the residual effect on animal product so it will not cause pathogen bacterial resistance effect. Fortunately, consumers are taking very serious attention on the food availability with the beneficial addition for their healthiness and also diseases prevention (de Lima and Filho, 2005).
Some of related research which contained lactic acid bacteria isolation and probiotic were L. reuteri, L. salivarius, or Lactobacillus spp use that could inhibited the pathogen bacterial such as Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, and Salmonella spp. Some of Lactobacillus isolates could produce anti microbe's peptide or bacteriosin (Lima et al., 2007;Pilasombut et al., 2006). L. salivarius CTC2197 already know had inhibitory effect on S. enteritidis C-114 colonization by in vivo on GIT tract of chicken after single doses addition on feed mixture (Pascual et al., 1999).
The ideal requirements for probiotic agent purposes of microbes are animal host origin, nonpathogenic, withstand processing and storage, resist on gastric acid and bile, adhere to epithelium or mucus, persist in the intestial tract, produce inhibitory compounds and modulate immune respons (Pattershon and Burkholder, 2003). The objectives of this research was to select LAB from native and broiler chicken which have ideal probiotic characteristics.

Lactic
Acid Bacteria  Isolation  and  Identification LAB isolated from chicken's GIT tract of 5 month old of native chicken and 35 days old of broiler chicken (Cobb strain) using Torshizi et al. (2008) method. GIT sampling location was gizzard, duodenum, jejunum, ileum, and caecum. Samples were cutted, washed, and diluted in steril peptone water (Oxoid) and made up to 10 5 dilution. Each serials dilution was plated in de Man Rogosa Sharpe (MRS) Agar media (Oxoid) pH 6.2 then was added by 0.2% CaCO 3 (Merck) and incubated at 37 o C for 24 h. The LAB colonies was detected by clearing zone appearance. LAB identification procedures consist of morphology, catalase, gas production, Gram staining, and motility tests. LAB isolates were maintained on microbank (Pro-lab) containing 15% glycerol.

Antibacterial Activity Assay
The selected LAB isolates grown on MRS Broth media at 37 o C for 24 h. Cell free supernatant was obtained by centrifugation at 12,500 g for 20 min at 4 o C. Supernatant were neutralized using 5 N NaOH (Merck), and sterilized using miliphore filter 0.20 µ. Antibacterial activities against E. coli FNCC 0091 in Nutrient Broth (NB) (Merck) medium were observed using turbidimetric method with incubation time for 48 h at 37 o C. Sterile supernatants were mixed with double strength of NB about 1:1 (v/v) comparison and inoculated with 2% (v/v) of bacterial test. NB media without supernatant which had been inoculated with E. coli was used as a control. The optical density (OD) were observed at 0, 2, 4, 6, 8, 10, 12, 18, 24, 36 and 48 h incubation time using a spectrophotometer at λ 600 nm (Seeley et al., 2001).

Optimization of the Growth Temperature and Condition
A total of 1% (v/v) new cultures of the selected LAB inoculated on MRSB in Hungate tube then incubated at 30, 49 and 45 o C, anerobic with CO 2 addition, aerobic without and aerobic with 100 rpm agitation. The control tube contained MRSB without LAB culture addition. The OD were measured using a spectrophotometer at λ 600 nm at 12 and 24 h incubation time.

Biochemical Identification
Biochemical identification of the selected LAB were observed by API 50 CHL kit (bioMērieux). The test procedure using the manual standard of API 50 CH kit. The observation data was analyzed by API web software (bioMērieux).

Antibiotic Sensitivity Test
The antibiotic sensitivity test were measured using Kirby Bouer method (Cappuccino and Natalie, 1986) with Erithromycin 15 µg, Penicillin G 10 µg and Streptomycin 10 µg as antibiotics. The 100 µL of LAB isolates were inoculated on MRSA plate. The antibiotic paper discs were put on MRSA surface and then incubated at 37 o C for 24 h. Diameter of clear zone (mm) around paper disc was observed using calipers.

Acid Tolerance
Acid tolerance test refers to modified methods of Torshizi et al. (2008). LAB cultures on MRS Broth were centrifuged at 5000 rpm for 10 min at 4ºC. Pellet were washed two times by sterile phosphat buffered saline (PBS) and diluted in sterile PBS before inoculated on MRS Broth (pH 2, with 1 M HCl addition). Cell viability were calculated by the total plate count (TPC) method on MRS Agar media.

Gastric Juice Tolerance
Gastric juice tolerance were observed according to modified gastric juice simulation (Thorsizhi et al., 2008). The selected LAB isolates were incubated on MRS Broth at 37 ºC for 18 h. A total of 1 ml culture was centrifuged at 5000 g, 10 min, 4ºC then it washed in two times using steril PBS and diluted on 0.3 ml steril PBS. A total of 0.2 ml dilution was taken and then mixed with 1 ml of artificial gastric juice. The mixture liquid was homogenized and incubated at 37ºC for 2 h and then sampled after 0, 1 and 2 h. Serial dilutions of samples was made on sterile PBS and then inoculated on MRS Agar media for cell viability observation. The artificial gastric juice was made from pepsin (Sigma) (3 g/l) dilution at pH 2.

Bile Salt Tolerance
Bile salt tolerance was determined by modified method of Torshizi et al. (2008). A total of 1 ml LAB culture was centrifuged at 5000 g for 10 min at 4ºC and washed in two times by using strile PBS. The cells were diluted in 0.3 ml of PBS then mixed 0.2 ml of dilution and 1 ml PBS containing 0.3% (w/v) bile salt (Merck). The mixture was incubated at 37ºC for 3 h and sampled after 0, 1 and 3 h. The cell viability was calculated using serial dilution and plated on MRS Agar media. Three replicates were used for each treatment.

Data Analysis
The quantitative data was analyzed by using One-way analysis of variance (ANOVA) with post hoc test (Duncan multiple F-test (P<0.05)) to distinguish the treatments means. The total of bacteria cell (cfu/ml) from viability test was converted to the logatimic value before statistically analysis.

RESULTS AND DISCUSSION
Lactic acid bacteria (LAB) which obtained from gastrointestinal digestive tract (GIT) were 39 isolates from native chicken and 18 isolates from broiler chicken. All LAB isolates had negative catalase, non gas production, non motile, Gram positive, rod and coccus shape characteristics. The LAB isolates characteristic and the optical density (OD) of E. coli on NB media containing extracelluler metabolite of LAB isolates are shown in Table 1. The E. coli growth on media with extracelluler metabolite of LAB isolates was lower than media without extracelluler metabolite and they were significantly different (P<0.05) from control. Antibacterial activities of LAB isolates against E. coli was came from bacteriocins compound of metabolite extracelluler during grow in media. Extracelluler metabolite which have antibacterial activities such as antimicrobial peptide or bacteriosin of L. reuteri, L. salivarius, and Lactobacillus spp. were isolated from gizzard and caecum of poultry and inhibitted Enterococcus, Listeria, and Salmonella (Lima et al., 2007). Alpha and beta bacteriosin Abp 118 produced by L. salivarius isolated from digestive tract of poultry and showed inhibition activities against B. coagulans JCM 2257T (Pilasombut et al., 2006). Bacteriocin from L. salivarius NRRL B-30514 could reduced Campilobacter jejuni population in digestive tracts of poultry (Stern et al., 2006). Lactobacillus sp. isolated from silage feed had antibacterial activities against E. coli and S. aureus (Damayanti et al., 2009). L. plantarum fed to broiler chicken showed terapeutic effect of bacteriocin against E. coli infection in broiler chickens (Ogunbanwo et al., 2004).
Inhibition mechanism of bacteriocin occured in two phases. First phase was bacteriocin absorption on specific and nonspesific receptor on target bacterial membrane cells. During this phase, the bacteriocine became sensitive especially to proteolitic enzyme. Second phase was irreversibel and involves lethal changes in the sensitive strains. The idea that bacteriocins act on the cell membrane has been well accepted (de Lima and Filho, 2005).
Based on data in Table 1, LAB isolates which had the highest inhibition against E. coli were Db9, D1, I72, Db2, Db5, Db1, D2, Ib1, T4 and D23, respectively. The differences of antibacterial activities in each isolates were based on the differences of LAB species and the ability to produce antibacterial compounds. Previous result from Torshizi et al. (2008) showed that P. pentosaceus TMU457 significantly had higher inhibition activity than L. fermentum TMU121 and L. rhamnosus TMU094 against E. coli and S. pullorum. Tatsadjieu et al. (2009) had reported that free cell supernatant from several strains of Lactobacillus had clear zone difference against E. coli.
One of expected characteristics of probiotic LAB was the stability during industrial processing, storage and delivery and had viability at high population (Gaggia et al., 2010). Beside its able to produce the antibacterial compounds, LAB as probiotic agent must be able to survive in host intestinal (Patterson and Burkholder, 2003).
Probiotic survival in agitation condition became essensial factor because after entering the gastrointestine of the host, the probiotic strains have to attach to the brush border of microvilli or adhere to the mucus layer to prevent sweep from the colon by peristalsis (Kim et al., 2007). All of LAB isolates had ability to growth at anaerobic and aerobic conditions whereas at aerobic condition with 100 rpm agitation, both I72 and Db9 isolates had higher growing ability than D1 and Db2 isolates. Differences in species level were effect on physiology and biochemical characteristic especially in growth optimum temperature which shown in Table 2. According to the growth curves of LAB during 24 h, the fourth of LAB isolates had the best growth at 39ºC. The lowest growth for I72, Db9 and D1 occured at 45ºC, except for Db1 which occured at 30ºC.
Identification result by API 50 CHL kit are presented in Table 3. Two selected strains were identified as L. salivarius I72 and P. pentosaceus Db9. Both of LAB isolates had different ability to ferment carbohydrate, but they had similar ability to ferment monosacharide such as glucose and fructose, and the other carbohydrate like Nacetylglucosamine and D-Trehalose. Several studies had found LAB isolate from digestive  Temoin Raffinose  -+  11  D-galactose  -+  36  Amidon  --12 D-glucose  +  +  37  Glycogen  --13 D-fructose  +  +  38  Xylitol  --14 D-mannose  +  -39 Gentibiose  tract of poultry such as L. salivarius from gizard and caecum of broiler breeder (Cobb strain) in 56 weeks of age (Lima et al., 2007), L. salivarius TMU121 and P. pentosaceus TMU457 from digestive tract of broiler in 42-50 days of age (Thorshizi et al., 2008), L. salivarius K7 from poultry intestine (Pilasombut et al., 2006), L. salivarius NRRL B-30514 from feces of intestine broiler (Stern et al., 2006), L. acidophilus, L. salivarius, and L. brevis from broiler feces (Kizerwetter-Świda and Binek, 2006). The result of antibiotic sensitivity test showed that both of the selected strains had the same tolerance and not significantly different (P>0.05) to the other antibiotics (Table 4). Several poultry feeds contained some antibiotic in certain amount. The resistance characteristic of two LAB isolates to the broad spectrum of antibiotic (Erithromycin 15 µg), as well as Gram negative specify antibiotic (Penicillin G 10 µg) and Gram positive specify antibiotic (Streptomycin 10 µg) caused both of LAB isolates had possibility to survive in digestive tract of poultry which have exposed antibiotics. In a previous report, Torshizi et al. (2008) reported that all three selected LAB isolated from broiler chicken had some degree of antibiotic resistance againts several of the tested antibiotic.
One of the essential characteristic of probiotic in order to give beneficial health for individual host was resistance to the effect of gastrointestinal enviroment such as acid and bile salt in digestive tract (Kosin and Rakshit, 2006). The result of pH acid, gastric juice and bile salt tolerance test of the two selected LAB isolates were shown on Table 5. This study showed that both of LAB isolates had ability to survive on pH 1, 2 and 3 after 1 hour incubation. Based on cell viability percentage, L. salivarius I72 and P. pentosaceus Db9 had the higher viability on the higher pH but not significantly different (P<0.05) to others. In the gastric juice tolerance test, both of LAB isolates also showed had viability after 1-2 hours of incubation. The cell viability of two LAB isolates were decreased at second hour of incubation but not significantly different to others (P<0.05). Similar to the result in gastric juice tolerance test, both of LAB had higher cell viability on 0.3% (b/v) bile salt at one hour of incubation than three hours of incubation. However, both of LAB isolates were categorized had ability to survive in bile salt condition after 3 hours of incubation with 102.43-105.62% of cell viability.
LAB as an intestinal bacteria could experience a wide number of stresses in the intestinal tract including those caused by low pH and presence of bile. In this case, bile salt tolerances was though to be an important aspect of survival for bacteria which inhabit the intestinal tract. Bile salt tolerance in intestinal lactobacilli assosiated with bile salt hydrolase (BSH) activity (O'Sullivan et al., 2009). BSH split the peptide linkage of bile acids, which results in removal of the amino acid group from the steroid core. The resulting unconjugated bile acids precipitate at low pH (Begley et al., 2006). On the basis of the results of molecular screening, both of selected LAB L. salivarius and P. pentosaceus had a genetic equipment for their survival at low pH (such as groEL gene for heat shock protein 60) and in the presence of bile salt (such as bsh gene for conjugated bile salt acid hydrolase) (Turpin et al., 2011). Based on the average of cell viability, both of the selected LAB had an equal viability on all treatments and had characteristic as probiotic bacteria.