Screening of Lactic Acid Bacteria From Rumen Liquor and King Grass Silage as Well as Their Antibacterial Activities

Probiotic is a live microbial culture which has positive effect on animal by improving the natural balance of microflora in the digestive tract. This experiment aimed to screen and identify indigenous lactic acid bacteria (LAB) from rumen liquor and king grass (Pennisetum hybrid) silage as a probiotic candidate and to evaluate their resistance in low pH, and inhibitory activities against pathogenic bacteria. The LAB isolate was characterized by a clear zone formed on MRSA medium + CaCO3 0.2% (w/v) and further identified by morphological and biochemical assays. The selected isolates were evaluated for their viability in low pH, pathogenic bacterial inhibition, and lactic acid production. The experimental arrangement was a factorial block design (4 x 2) consisted of four isolates and two levels of pH value (pH 2 and 3), each treatment in 3 equal replicates. The result showed that four isolates (two isolates from the rumen liquor of fistulated cattle and two isolates from silage) were identified as lactic acid bacteria. The four isolates showed inhibition activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus and performed viability at low pH during 2 h treatment. The highest lactic acid production was obtained from isolates Sil.3 (21.42%) and followed by CR2 (19.88%), CR1 (15.40%) and Sil.9 (15.08%). Biochemical identification by standard of analytical profile index (API) 50 CHL kit showed that the selected isolates CR1 was Lactobacillus paracasei ssp. paracasei 3 (91.5%), L. paracasei ssp. paracasei 3 (76.5%), Sil.3 was Lactobacillus brevis (95.1%), and Sil.9 was Lactobacillus collinoides (92.5%). In conclusion, probiotic candidates isolated from rumen liquor are confirmed as L. paracasei ssp. paracasei (CR1 and CR2), while two other isolates from king grass silage are identified as L. brevis (Sil.3) and L. collinoides (Sil.9). L. brevis (Sil.3) and L. paracasei ssp. paracasei (CR1) has higher inhibition against pathogenic bacteria (E. coli, S. aureus, and B. subtilis) than L. paracasei ssp. paracasei (CR2) and L. collinoides (Sil.9).


IN���DU��I�N
Since antibiotic had been banned for promoting animal growth in 200�, it implied an alternative substance n alternative substance alternative substance to replace antibiotic by using organic additive based on organic additive based on on natural compound, microbial culture or their combination. Due to consumer concerns to improve health and Due to consumer concerns to improve health and safety of animal products including those resulting from organic farming (European Commission, 2012). In fact, ). In fact, supply of animal product (e.g. milk production) is only covering 30% national demands and also the ade�uacy s and also the ade�uacy and also the ade�uacy the ade�uacy ade�uacy of meat supply needs to be increased by improvement s to be increased by improvement to be increased by improvement be increased by improvement increased by improvement d by improvement by improvement of beef cattle productivity. Indonesian Ministry of Agriculture reported that milk production in 2010 was 0.90 million tones which only increased 9.94�� from 2009 d 9.94�� from 2009 (Ditjen PK�, 2012) and also meat was imported to ful��ll Ditjen PK�, 2012) and also meat was imported to ful��ll was imported to ful��ll imported to ful��ll the national demand. national demand.
Beside the nutritional problem, ruminant disease caused by pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Bacillus subtilis implied on low production and immune-suppression of animals. S.� au-au-aureus is known as a Gram positive bacteria, opportunistic is known as a Gram positive bacteria, opportunistic s known as a Gram positive bacteria, opportunistic as a Gram positive bacteria, opportunistic a Gram positive bacteria, opportunistic , opportunistic opportunistic pathogen and a major concern for both animal and human health (Charlier et al.�, 2009), and it has been found , and it has been found and it has been found associated with certain gastrointestinal disease that is responsible for mastitis in dairy herds ( Administration of direct feed additive based microbes is potential to improve feed digestibility. Probiotic contains life microbial and favorable for im-fe microbial and favorable for im-microbial and favorable for improving the digestive tract function especially in digesting forage. Furthermore, probiotic is expected to modu-to modu-modulate the immune system and inhibit pathogenic bacteria. Probiotics or direct feed microbes (DFM) are dietary es (DFM) are dietary (DFM) are dietary supplements that inhibit gastrointestinal infection and provide optimally regulated microbial environments in the digestive tract (Seo et al., 2010). Administration of probiotics in livestock in the period of growth appears to be a real impact. Lactic acid bacteria as probiotic, are able to produce lactic acid, reuterin, diacetyl, bacteriocins and other metabolic functions as an antimicrobial (Rattanachaikunsopon & Phumkhachorn, 2010), enhance ��ber and starch digestion (Fraga et al., 2013) and im-im-improve animal health (Bayatkouhsar et al., 2013). ).
Lactic acid bacteria (LAB) ferments sugars or (LAB) ferments sugars or ferments sugars or carbohydrates to produce large amounts of lactic acid. Lactic acid bacteria are naturally found in many different habitats such as fermented foods, fruits, grains, animal digestive tract or silage. In the digestive tract of cattle, , lactic acid bacteria were found in the rumen (Bureenok (Bureenok et al., 2011;Cobos et al., 2011��. Lactic acid bacteria in forage feed live as epiphytic bacteria in limited colony. live as epiphytic bacteria in limited colony. as epiphytic bacteria in limited colony. In order to optimize probiotic functions speci��c for host animal, administration of lactic acid isolated from , administration of lactic acid isolated from indigenous sources will have promise more adaptable probiotic in the ruminant digestive tract. �owever, scienti��c publication related to exploration of indigenous exploration of indigenous ation of indigenous of indigenous of indigenous microorganisms used as probiotics candidate from the candidate from the the host animal (rumen li�uor) or animal feed (forages) is still limited. Therefore it has re�uired to isolate and . Therefore it has re�uired to isolate and has re�uired to isolate and re�uired to isolate and characterize lactic acid bacteria from cattle rumen and ze lactic acid bacteria from cattle rumen and and king grass (Pennisetum hybrid) silage and determine their antibacterial activity against pathogenic bacteria.

Sample Preparation
Rumen li�uor and king grass (P.� hybrid) silage were prepared as inoculant sources. Rumen li�uor was obtained from the ��stulated cattle (�ngole Crossbred) the ��stulated cattle (�ngole Crossbred) ��stulated cattle (�ngole Crossbred) which daily consumed diet consisted of forage and conof forage and con-forage and concentrate (70:30 dry matter basis). Silage was made from dry matter basis). Silage was made from ). Silage was made from the chopped king grass which packed in a plastic bag which packed in a plastic bag packed in a plastic bag in a plastic bag plastic bag and incubated during 21 d at room temperature (25-30 at room temperature (

I��o���tio�� ����� �cr������i���� o�� L��ctic Aci� B��ct��ri��
Collected fresh rumen li�uor (100 mL) was strained through cheesecloth to obtain supernatant consisting microbial cells (rumen supernatant). Collected fresh silage (100 g) was added by 500 mL of distilled water was added by 500 mL of distilled water added by 500 mL of distilled water and then mashed in a blender, then strained through cheesecloth to obtain supernatant. An amount of 50 ml of n amount of 50 ml of supernatant was added by 1 g of d-glucose monohydrate was added by 1 g of d-glucose monohydrate d-glucose monohydrate (C 6 H 2 O 6 .H 2 �) (w/v) to stimulate the bacterial growth. Supernatant from the rumen and silage were grown on the rumen and silage were grown on rumen and silage were grown on the selective medium of MRSA (de �an Ro�osa Sharpe �an Ro�osa Sharpe �an Ro�osa Sharpe Agar) supplemented with CaC� with CaCO 3 0.2% (w/v) using a using a spread plate method and anaerobically for 24 h at 37 anaerobically for 24 h at 37 for 24 h at 37 o C in an anaerocult chamber with an atmosphere of C� 2 .

Kata kunci: isolasi, bakteri patogen, cairan rumen, probiotik, silase
Primary screening of LAB was performed by evaluating the clear zones on selective medium. LAB colony . LAB colony LAB colony with the largest clear zone diameter was chosen as the selected isolates.

Characteri�ation of Lactic Acid Bacteria
Selected isolates from rumen li�uor and king grass silage were characterized by catalase test, Gram staining, morphology, motility test, and gas production test. Motility test isolates was observed on SYP medium (sucrose-yeast extract-peptone), incubated for 24-4� h at 37 o C. Gas production test performed by growing cultures in 5 mL of li�uid SYP for 2-3 d at 37 o C with a Durham tube which placed in reverse to capture the gas h placed in reverse to capture the gas production (Damayanti et al., 2012).

Viability in Low p�
The viability of lactic acid bacteria was evaluated by evaluated by by cultivating the isolate on acidity medium (p� 2 and p� 3). Evaluation of viability was arranged a factorial block factorial block design (4 x 2) consisted of the screened isolates (four 2) consisted of the screened isolates (four ) consisted of the screened isolates (four isolates) and p� condition (p� 2 and 3), each treatment in 3 e�ual replications. Isolates were grown in MRS broth . Isolates were grown in MRS broth s were grown in MRS broth were grown in MRS broth were grown in MRS broth at 37 o C for 1� h then centrifuged using a refrigerated for 1� h then centrifuged using a refrigerated for 1� h then centrifuged using a refrigerated using a refrigerated centrifuge `Centrofriger ® BL-II series` at 4137 at 4137 4137 x � � for 5 min at 4 o C to obtain the biomass (pelleted). Pelleted were washed twice with sterile phosphate buffered saline (PBS, 0.1 M phosphate buffer with 0.�% NaCl, p� M phosphate buffer with 0.�% NaCl, p� M phosphate buffer with 0.�% NaCl, p� 7.0) referred to referred to referred to to Bakari et al. (2011). . The strain was diluted 1/100 in sterile PBS adjusted diluted 1/100 in sterile PBS adjusted diluted 1/100 in sterile PBS adjusted to p� 2 and 3 using 0.1 N �Cl. Incubation times were 0 0.1 N �Cl. Incubation times were 0 �Cl. Incubation times were 0 Incubation times were 0 Incubation times were 0 and 1 h, then continued 2 h incubation in p� 3 conditions. Serial decimal dilutions in sterile PBS were pre-s. Serial decimal dilutions in sterile PBS were pre-. Serial decimal dilutions in sterile PBS were pre-sterile PBS were pre-sterile PBS were prepared, and ali�uots (0.1 mL) from the dilutions was used was used was used for point inoculation on the surface of MRS agar plates to determine the number of surviving cells (

The Growth Curve of Lactic Acid Bacteria and Lactic Acid Production
Prior to cultivation LAB isolate into the medium, each of the four isolate was pre-cultured in MRSB, then was pre-cultured in MRSB, then was pre-cultured in MRSB, then pre-cultured in MRSB, then incubated at 37 at 37 , 9, 12, 1�, 24, 30, 3�, and rowth was evaluated at 0, 3, �, 9, 12, 1�, 24, 30, 3�, and 4� h after incubation to obtain the optical density (�D) using a spectrophotometer (Dynamica a spectrophotometer (Dynamica spectrophotometer (Dynamica (Dynamica ® �alo RB10) at at λ 625 nm . �iable cell was con��rmed by spread plate count �iable cell was con��rmed by spread plate count count method to determine the number of colony at 0, 24, to determine the number of colony at 0, 24, and 4� h after incubation. Total lactic acid and p� were . Total lactic acid and pH were Total lactic acid and pH were measured at last incubation time (4� h). Measurement of lactic acid production was conducted by method referred to A�AC (2005)  ) were performed using agar diffusion methods (Bonev Bonev et al., 2008;Cizeikiene Cizeikiene et al., 2013). The supernatant of 1� h from MRSB cultures was prepared using a refrigerated centrifuge `Centrofriger ® BL-II series` at 4137 at 4137 4137 x � � for 5 min at 4 °C. The supernatant was collected and neutralized by adding 0.5 N Na�� collected and neutralized by adding 0.5 N Na�� up to p� 7. The antibacterial activities were observed using the paper disc diffusion method. The paper the paper disc diffusion method. The paper paper disc diffusion method. The paper disc (5 mm) placed on the MRSA surface which was inoculated with 100 µL pathogenic bacteria cultures and then embedded with 50 µL of neutral LAB supernatant. Control was made by adding the antibiotic discs (15 mg Erithromycin, 10 mg Penicillin G, 10 mg Streptomycin) for each of the tested pathogenic bacteria. Observation of clear zones (inhibition zone) for 24 h at 37 at 37 o C. .

Bioc����ic��� I�����tific��tio��
Biochemical tests were performed by fermentation pattern using standard analytical pro��le index (API 50 using standard analytical pro��le index (API 50 standard analytical pro��le index (API 50 C�L, bioMérieux ® ) using the manual standard of API using the manual standard of API C�L 50 kit (Muñoz-Quezada et al., 2013). Pure culture of LAB biomass from 24 h culture was inoculated into API was inoculated into API inoculated into API 50 C�B/E medium. The result of the biochemical pro��le The result of the biochemical pro��le esult of the biochemical pro��le biochemical pro��le biochemical pro��le was used to identify the species of lactic acid bacteria using software product identi��cation e.g. APIWEB e.g. APIWEB APIWEB (bioMérieux ® , Marcy l, Etoile, France).

Data Analysis
Qualitative data consisted of cell morphology and cell morphology and morphology and biochemical characteristic were analyzed using descrip-using descripdescriptive method. Data from �uantitative parameter consisted method. Data from �uantitative parameter consisted . Data from �uantitative parameter consisted of clear zone diameter, cell viability, lactic acid and pH diameter, cell viability, lactic acid and pH , cell viability, lactic acid and pH ility, lactic acid and pH , lactic acid and pH were analyzed using analysis of variance (AN��A) and followed by Duncan�s Multiple Range Test to determine Duncan�s Multiple Range Test to determine Duncan�s Multiple Range Test to determine the effect of differences between treatments mean mean (Gomez & Gomez, 2007). The statistical analyses of parameters were performed using the CoSTAT ere performed using the CoSTAT performed using the CoSTAT the CoSTAT CoSTAT ® Statistical Software (Cohort,200�).

I��o���tio�� ����� ������ctio�� o�� L��ctic Aci� B��ct��ri��
In preliminary stage, LAB was screened by preliminary stage, LAB was screened by stage, LAB was screened by evaluating clear zone of each isolate grown on MRS agar. Initial screening were obtained 2 isolates from . Initial screening were obtained 2 isolates from Initial screening were obtained 2 isolates from Initial screening were obtained 2 isolates from were obtained 2 isolates from 2 isolates from rumen li�uor (CR), 2 isolates silage (Sil  Table 2. are shown in Table 2. shown in Table 2. n in Table 2. in Table 2. in Table 2. Resistance of lactic acid bacteria in low p� was performed by cultivation isolates on a medium that a medium that medium that was adjusted at p� 2 and 3 for 0 and 1 h. �iability of h. Viability of of four isolates were affected by p� and incubation time signi��cantly (P<0.05). �owever, there was no interaction (P>0.05) between isolates and medium acidity condition (p� value) on cell viability. Based on the viability in p� 2 and 3 indicated that the four isolates were able to survive in low p� condition (Table 3).
A number of initial colonies were similar around 5.00��.42 log cfu/mL. Incubation time for 1 h showed 00��.42 log cfu/mL. Incubation time for 1 h showed ��.42 log cfu/mL. Incubation time for 1 h showed �.42 log cfu/mL. Incubation time for 1 h showed log cfu/mL. Incubation time for 1 h showed that percentage of isolates colonies survived in p� 2 was ��% for CR1 and CR2, followed by Sil.3 and Sil.9. Meanwhile, the percentage of isolates colonies survived the percentage of isolates colonies survived d in pH 3 were 89��, 66��, 65��, and 63�� respectively for CR2, Sil.9, CR1, and Sil.3. Furthermore, incubation time for 2 h in p� 3 showed that viability of fourth isolates were not signi��cantly different. In general, the highest viability of isolates obtained from CR1, the lowest obtained from Sil.9, except for CR1 and Sil.3 were similar. It is indicated that CR2 was more able to survive survive in low p� than other isolates, conse�uently it could be other isolates, conse�uently it could be isolates, conse�uently it could be used as a probiotic candidate.
An ability bacterium to survive in acid medium was closely related to cell stability which was influenced by many factors consisted of temperature, p� and nutrient availability. Moreover, adaptability bacteria lability. Moreover, adaptability bacteria bility. Moreover, adaptability bacteria Moreover, adaptability bacteria adaptability bacteria in environmental condition were closely affected on closely affected on affected on on growth and survivability. Romero-Pérez survivability. Romero-Pérez . Romero-Pérez et al. (2011) revealed that environmental factor influenced ability bacteria to grow in acidity medium which was related to previous natural habitats. natural habitats. . In general, the pattern of LAB growth following sigmoid curve (S) was measured by turbidimetry method (�ptical Density/�D) and further con��rmed by total con��rmed by total total plate count (TPC) to evaluate the cell viability (�ogg, evaluate the cell viability (�ogg, the cell viability (�ogg, cell viability (�ogg, (�ogg, 2013�� Rehaiem et al., 2014. The growth rate was indi-  cated from absorbance of 4� h incubation at 37 o C. Based on the Figure 1, the high growth rate of LAB isolates high growth rate of LAB isolates was CR2, and Sil.3. Bacterial growth was influenced by p�, nutrient content, and temperature. The population he population population of LAB after 4� h incubation showed that CR2 and Sil.3 .3 3 were similar and higher than others (Figure 2). Cousin et al. (2012) stated that lactic acid bacteria re�uire carbon and nitrogen sources for optimizing growth and produce lactic acid as major metabolites. Characteristic of fermentability from each isolate to ferment medium (glucose and its derivatives) was explained in Table 4.
At the end of incubation (4� h), levels of lactic acid , levels of lactic acid and p� of the medium were measured to determine the type of fermentation from each isolate which obtained from the gas production test as shown in Table 2. �ogg (2013) stated that lactic acid levels are 13) stated that lactic acid levels are ) stated that lactic acid levels are different depending on the type of fermentation. A homofermentative type of lactic acid bacteria produces omofermentative type of lactic acid bacteria produces s high levels of lactic acid due to converting 95% glucose into lactic acid, a small amount of C� , a small amount of C� a small amount of C� 2 and volatile acids.
While the heterofermentative types of lactic acid bacteria produce lactic acid in low concentration and amount of in low concentration and amount of low concentration and amount of concentration and amount of and amount of CO 2 . Based on the test results of fermentation type, isolates CR2, and Sil.3 were homofermentative that had high levels of lactic acid. Isolates CR1 and Sil.9 had low levels of lactic acid and indicated heterofermentative type. Levels of lactic acid production were closely re-. Levels of lactic acid production were closely re-Levels of lactic acid production were closely re-evels of lactic acid production were closely related to p� value. König & Fröhlich (2009) stated that fermentation type of lactic acid produced dominantly lactic acid was a homofermentative however heterofermentative of LAB produce not only lactic acid but also acetic or alcohol.
Biochemical characteristic of LAB evaluated using API C�L 50 are presented in Table 5. All of the isolates All of the isolates the isolates isolates have a similar result from sugar fermentation test on API a similar result from sugar fermentation test on API similar result from sugar fermentation test on API C�L 50 except for substrates consisted of D-galactose, D-lactose, D-Saccharose, inulin and D-ra��nose. Based on analysis using software APIWEB (Table �), isolates analysis using software APIWEB (Table �), isolates (Table �), isolates CR1 was Lactobacillus paracasei ssp. paracasei 3 (91.5%), CR2 was Lactobacillus paracasei ssp. paracasei 3 (7�.5%), Sil.3 was Lactobacillus brevis (95.1%), and Sil.9 was Lactobacillus collinoides (92.5%). Lactic acid bacteria re�uire different carbohydrate sources for growth and to produce substances that can be used to defend cells from pathogenic bacteria invasions (bacteriocins).
invasions (bacteriocins). This biochemical test result supported by Gulahmadov et al. (2009) revealed that revealed that that L.� brevis, and L.� collinoides were isolated from Azerbaijan cheeses have cheeses have heeses have activity to ferment D-ribose, D-glucose, D-fructose, N-acetyglucosamine, D-Maltose, D-trehalosa, however, D-celobiose, D-lactose, sucrose and innulin were only fermented by L.� brevis. Muñoz-Quezada et al. (2013) identi��ed Lactobacillus paracasei which was able to utilize was able to utilize inulin and sucrose. Although phenotypic identi��cation identi��cation identi��cation using a biochemical test is accurate methods, molecular identi��cation need to conduct for the con��rmation of bacteria species as a probiotic candidate (�uoba et al., 2010).

I���ibitor�� A����i����t P��t�o������ic B��ct��ri�� Against Pathogenic Bacteria gainst Pathogenic Bacteria
The inhibition activity from tested isolates was s was was showed by clear zone diameter (Figure 3) by clear zone diameter (Figure 3) Figure 3) ). Antimicrobial assay results indicated that the isolates were able to the isolates were able to to inhibit the growth of pathogenic bacteria. Based on the data, all isolates inhibited B.� subtilis. LAB isolated Incubation time (h) Optical density at 625 nm    The highest inhibition of antibiotic against against S.� aureus growth was closely related to a spectrum of antibiotic activity. Based on Figure 3, Penicilin, Streptomycin and Erythromycin seem to have higher inhibition against to have higher inhibition against higher inhibition against against S.� aureus growth than LAB metabolite. Compare to Compare to chemical antibiotic, pathogenic bacteria inhibition by LAB metabolite was related to the chemical antibiotic chemical antibiotic chemical antibiotic compound in pure substance and the ability of antibiotic in pure substance and the ability of antibiotic the ability of antibiotic ability of antibiotic ability of antibiotic to penetrate pathogenic bacteria cell easily (Bonev bacteria cell easily (Bonev cell easily (Bonev easily (Bonev (Bonev et al. 200�), however antibacterial substance from lactic acid bacteria as probiotic was more safety than antibiotic due was more safety than antibiotic due more safety than antibiotic due than antibiotic due due to its lower risk of residual and has no resistance effect its lower risk of residual and has no resistance effect lower risk of residual and has no resistance effect has no resistance effect resistance effect effect on pathogenic bacteria. pathogenic bacteria.

��N�LU�I�N
Lactic acid bacteria isolated from rumen li�uor and king grass (P.� hybrid .� hybrid hybrid) could be used as probiotic. Probiotic candidates isolated from rumen li�uor Probiotic candidates isolated from rumen li�uor robiotic candidates isolated from rumen li�uor are con��rmed as as L.� paracasei ssp. paracasei (CR1 and CR2), while two other isolates from king grass silage while two other isolates from king grass silage isolates from king grass silage s from king grass silage from king grass silage are identi��ed as identi��ed as L.� brevis (Sil.3) and L.� collinoides (Sil.9). . spinach and identi��cation of antimicrobial substances spinach and identi��cation of antimicrobial substances produced by a commercial lactic acid bacteria food safety commercial lactic acid bacteria food safety commercial lactic acid bacteria food safety intervention. Food