MATERIALS AND METHODS Isolates Bacteria

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1. INTRODUCTION

The development of feeding system based on the local resources is the pillars supporting the development of sustainable animal production in Indonesian Ginting, 2004. Utilization of agricultural by-productwaste as feed will reduce cost animal production. However, research results Mudita et al. 2009 and 2012 and Putri et al. 2009 revealed the use of local resources waste as fed without the application of processing technology will decrease the productivity of livestock and increase pollutant emissions due to high crude fiber content in particular lignocellulose compounds and very difficult to be utilized by animals. Degradation of lignocellulosic substrates requires the cooperative action of family of lignocellulolytic enzyme that have been classified into three complex enzyme groups: i.e lignolytic enzyme lignase, cellulolytic enzim cellulase and hemicellulolytic enzyme hemicellulose. Lignocellulase complex enzyme can produce by certain microbe or corporative of lignocellulolytic bacteria consortium that work synergistically. Formulated the probiotic lignocelulolytic bacteria consortium used by selected bacteria isolate from the Bali cattle colon and organic waste landfills based research result Mudita et al 2014 is a strategic thing to be implemented. Besides that, the utilization of bacterial consortium will increase the overall digestibility, good digestibility in the rumen and post-rumen, improving health throughout the gastrointestinal tract as well as the balance of micro flora in the rumen and post-rumen. The research results Mudita et al 2014 showed that from the bali cattle colon and organic waste respectively selected two 2 superior probiotic lignocellulolytic bacteria isolates with coded BCC 4 LC and BCC 12.1 LC Bali Cattle Colon and BW 1 LC and BW 4 LC organic waste of landfill which has higher the enzyme activity and the ability of lignocellulosic substrates degradation. Formulation of selected bacteria isolates of bali cattle colon and organic waste of landfill as bacteria consortium inoculant is expected to produce a synergistic activity so that the process of degradation of lignocellulosic compounds will be more easily implemented. The c ombination of twoany isolates from different sources is likely to produce bacteria consortium inoculant that are potentially capable to improving the productivity of ruminants including Bali cattle fed agricultural waste.

2. MATERIALS AND METHODS Isolates Bacteria

Bacterial isolates are utilized in the production of probiotic lignoselulolitik bacteria consortium inoculant is elected superior isolates of the First Year of the research Mudita et al 2014 coded BCC 4 LC and BCC 12.1 LC isolated from the bali cattle colon and code BW 1 LC and BW 4 LC isolated from organic waste of landfill. Production of bacterial cultures done by inoculating bacteria isolates of the pure stocks into fluid thioglicollate medium with 1 lignocellulose substrates combination of 50 CMC, 30 Xylan and 20 tannic acid at 0,1 600 nm. Going further cultures incubated under anaerobic conditions for 5 days at 39 o C. Culture that has grown further exploited in the production inoculant. Medium Inoculant Medium inoculant is made from a combination of natural ingredients and chemical materials such as Table 1. Mixing medium using vortex for 30 minutes at 80-100 o C. Medium inoculant that has been mixed homogeneously subsequently sterilized in an autoclave for 15 www.ijcas.net 86 minutes T 121 o C. After the medium inoculant begin cooling T ± 40°C, medium ready to be used for the production inoculant. Tabel 1. Composition of Inoculant Medium on 1 liter No Material Composition 1 Thioglicollate Medium g 0.1 2 Sugarcane ml 50 3 Urea g 1 4 Tannic Acid g 0,025 5 CMC g 0,025 6 Xylan g 0,025 7 Rice Straw g 0.25 8 Rice Brand g 0.25 9 Cassava 0.25 10 Multy vitamin-mineral“Pignox”g 0.15 11 Water until volume 1 liter Bacteria Consortium Inoculant Bacteria consortium inoculants were produced by inoculating 10 a combination of bacterial culture according to treatment on the inoculant medium aseptically under anaerobic conditions. The formulated inoculant are presented in Table 2. Table 2. Formulated of bacteria consortium inoculant on 1 liter No Bacteria Consortium Inoculant Bacteria Isolate Culture from Landfill Waste ml Bacteria Isolate Culture fromBali Cattle Colon ml Medium Inoculant ml BW 1 LC 1 BW 4 LC 2 BCC 12.1 LC 1 BCC 4 LC 2 1 IW 12 5 5 - - 990 2 IC 12 - - 5 5 990 3 IW 1 C 1 5 - 5 - 990 4 IW 1 C 2 5 - - 5 990 5 IW 2 C 1 - 5 5 - 990 6 IW 2 C 2 - 5 - 5 990 7 IW 12 C 1 2.5 2.5 5 - 990 8 IW 12 C 2 2.5 2.5 - 5 990 9 IW 1 C 12 5 - 2.5 2.5 990 10 IW 2 C 12 - 5 2.5 2.5 990 11 IW 12 C 12 2.5 2.5 2.5 2.5 990 Evaluation of Lignocellulosic Substrates Degradation Ability The ability of lignocellulosic substrates degradation is determined from clear or diffusion zone formed by bacteria isolates tested Ogimoto and Imai, 1981. The substrate used: CMC for cellulose, xylan for hemicellulose, tannic acid for lignin and combination of 50 CMC, 30 Xylan and 20 tannic acid for lignocellulose substrate. Each pure bacteria isolate15lwasinoculatedbyspotmethod using paper disc blank 0.6 cm and placed on www.ijcas.net 87 the selective medium solid growth medium containing 1 substrate test for experimentation Subbarao, 1993. Diffusion and clear zone wide were measured after 24 hours of anaerobic incubation. Evaluation of Lignocellulolytic enzyme activity Enzyme extract was collected from centrifuged liquid media culture in 12.000 rpm for 15 minutes at 4 o C. Extracts enzyme were tested in three kinds of substrates that contained 1 CMC powderxylanTannic Acid as source of lignin in 50 mM acetate buffer pH 5.5. Each substrate liquid in buffer was taken 8 ml, added 1 ml enzymes source, and 1ml aquadest. The mixture then were shaken by shaking bath, enzyme activity was measured in 30, 60, 180 and 360 minutes durations. Reduction sugar glucose from CMC and xylose from xylan, or vanillin from tannic acid lignin produced from the reaction were the cellulasexylanase or lignase enzyme activities Efiok, 1996. For sugar reduction:1 ml of sample was added to 3 ml DNS reagent and 1 ml aquadest Miller, 1959, for vanillin: 1ml of sample was added to 4 ml methanol, then measured the absorbent by spectrophotometer in wavelength 508,5 nm for glucose, 509 nm for xilosa and 279 nm for vanillin. Lignasecellulasexylanase enzyme activities was estimated by using vanillinglucosexylose calibration curve Adney and Baker, 2008; Ghose, 1987. One unit U of enzyme activity was dened as 1 mol of vanillinglucosexylose equivalent released per minute under standard assay condition Irfan et al., 2012; Lo et al., 2009.

3. RESULTS AND DISCUSSION Ability of lignocellulosic compounds degradation