Antony SP, Philip R. 2006. Biomerediation in shrimp culture system. NAGA Worldfish Center Quarter. 29(3&4): 62-66.
Aslamyah S. 2006. Penggunaan mikroflora saluran pencernaan sebagai probiotik untuk meningkatkan pertumbuhan dan kelangsungan hidup ikan Bandeng [disertasi]. Bogor (ID) : Institut Pertanian Bogor.
Aygan A, Arikan B, Korkmaz H, Dincer S, Colak O. 2008. Highly thermostable
and alkaline α-amylase from a halotolerant alkaliphilic Bacillus sp. AB68.
Braz J Microbiol. 39:547-553.
Bairagi A, Shakar Ghosh K, Sen SK, Ray AK. 2002. Enzyme producing bacterial flora isolated from fish digestive tracts. Aquacult Int 10: 109-21.
Bernfeld P. 1955. Amylases a and ß: Method Enzymol 1:149-58.
Brashear MM, Jaroni D, Trimble J. 2003. Isolation, selection and characterization of lactic acid bacteria as competitive exclusion product to reduced Escherichia coli 0157:H7 in cattle. J food Protec 66:355-363.
[CLSI] Clinical and Laboratory Standards Institute. 2007. Performance Standars for Antimicrobial Susceptibility Testing; Twenty-Second Informational Supplement. Pennsylavania (US): CLSI.
Cordeiro CAM, Martin MLL, Luciano AB. 2002. Production and properties of α -amylase from thermophilic Bacillus sp.. Braz J Microbiol. 33:57-61.
Das S, Surendran PK, Thampuran N. 2009. PCR-based detection of enterotoxigenic isolate of Bacillus cereus from tropical seafood. Indian J Med Res. 129:316-320.
Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM. 2004. Characterization of Bacillus probiotic available for human use. Appl Environ Microbiol. 70(4):2161-2171.
Essakiraj P, Immanuel G, Sowmya, Iyaparaj P, Palevesam A. 2009. Evaluation of protease-producing ability of fish gut isolate Bacillus cereus for aqua feed. J. Food. Bioproc. Technol. 2: 383-390.
Farzanfar A. 2006. The use of probiotic in shrimp aquaculture. FEMS. 20:1-10. Fardiaz S. 1992. Mikrobiologi Pangan 1. Jakarta (ID): PT. Gramedia.
Fatoni A, Zusfahair, Lestari P. 2008. Isolasi dan karakterisasi protease ekstraseluler dari bakteri dalam limbah cair tahu. J Natur Indones. 10(2):83-88.
Faturrahman. 2012. Potensi bakteri agarolitik sebagai penyedia enzim agarase untuk memperbaiki pertumbuhan juvenil abalon (Haliotis asinina Linn. 1758) [disertasi]. Bogor (ID) : Institut Pertanian Bogor.
Feliatra, Efendi I, Suryadi E. 2004. Isolasi dan identifikasi bakteri probiotik dari ikan kerapu Macan (Ephinephelus fuscogatus) dalam upaya efisiensi pakan ikan. J Natur Indonesia. 6(2): 75-80.
Fleming AB, Tangney M, Jorgensen PL, Diderrichsen B, Priest FG. 1995. Extracellular enzymes synthesis in a sporulation-deficient strain of Bacillus licheniformis. J Appl Environ Mirobiol. 61: 3775-3780.
Gatesoupe FJ. 1999. The Use of Probiotics in Aquaculture. Aquaculture. 180:147-165.
20
Gomez-Gill B, Roque A, Trunbull JF. 2000. The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquacult. 191:259-270.
Gou JJ, Liu KF, Cheng SH, Chang C, Lay JJ, Hsu YO, Yang JY, Chen TY. 2009. Selection of probiotic bacteria for use in shrimp larvi culture. Aquacult Research. 40:609-618.
Gullian M, Thompson F, Rodriguez J. 2004. Selection of probiotic bacteria and study of their immunostimulatory effect in penaeus vanname. Aquaculture. 233:1-14.
Hadioetomo RS. 1993. Mikrobiologi Dasar dalam Praktek: Teori dan Praktek. Jakarta (ID): PT. Gramedia.
Hong HA, Duc LH, Cutting SM. 2005. The use of bacterial spore formers as probiotics. FEMS Microb Rev 29:813-835.
Irianto A. 2003. Probiotik Akuakultur. Yogyakarta (ID): Gadjah Mada University Press.
Jamilah I, Meryandini A, Rusmana I, Suwanto A, Mubarik NR. 2009. Activity of proteolytic and amylolytic enzymes from Bacillus spp. isolated from shrimp ponds. Microbiol Indones. 3(2):67-71.
Joshi GK, Kumar S, Sharma V. 2007. Production of moderately Halotoleran, SDS stable, alkaline protease from Bacillus cereus MTCC 6840 isolated from lake Nainital Uttarachal State, India. Braz J Microbiol. 38:773-779.
Jusadi D, Gandara E, Mokoginta I. 2004. Pengaruh penambahan probiotik Bacillus sp. pada pakan komersil terhadap konversi pakan dan pertumbuhan ikan patin (Pangasius hypophthalamus). J Akuakul Indones. 3(1):15-18. Kementerian Kelautan dan Perikanan. 2010. Kelautan dan Perikanan dalam
Angka 2010. Kementerian Kelautan dan Perikanan. Jakarta (ID).
Kesarcodi-Watson A, Kaspan H, Lategan J, Gibson L. 2008. Probiotics in aquaculture: the need principles and mechanismes of action and screening processes. Aquaculture. 274: 1-14.
Lin LL, Chyau CC, Hsu WH. 1998. Production and properties of a raw-starch-degrading amylase from the thermophilic alkaliphilic Bacillus sp. TS-23. Biotechnol Appl Biochem. 28:61-68.
Lim G, Tan TK, Rahim NA. 1987. Variations in amylase and protease activities among Rhizopus isolates. Mircen J. 3:319-322.
Lisal JS. 2005. Konsep probiotik dan prebiotik untuk modulasi mikrobiota usus besar. Medical Nusantara. 26: Oktober-Desember.
Madigan MT. Matinko JM, Dunlap PV, Clark DP. 2009. Brock Biology of Microorganisms. Edisi 12. San Francisco (US): Pearson Benjamin Cummings.
Marchesi J.R, Sato T, Weightman A.J, Martin T.A, Fry J.C, Hiom S.J, Wade W.G.1998. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S-rRNA. J Appl Environ Microbiol. 64(2):795-799.
Mesalhy SA, Abd- El- Rahman, John G, Mohamed. 2008. Characterization of some Bacteria isolated from Oreochromis niloticus and their potential use as probiotics. Aquaculture. 277:1-6.
21
Mohapatra S, Chakraborty T, Prusty AK, Das P, Paniprasad K, Mohanta KN. 2012. Use of different microbial probiotics in the diet of rohu, Labeo rohita fingelings: effects on growth, nutrient digestibility and retention, digestive enzyme activities and intestinal microflora. Aquacult Nutr. 18:1-11.
Moriarty DJW. 1999. Disease control in shrimp aquaculture with probiotic bacteria. Di dalam: Bell CR, Brylinski M, Johnson-Green P, editor. Microbiol Biosystem: New Frontiers. Proceding of 8 th, International Symposium on Microbial Ecology; Halifax: Atlantic Canada Society for Microbiol Ecology. 237-243.
Moriarty D. 1998. Control of luminous Vibrio species in penaeid aquaculture ponds. Aquaculture. 164:351-358.
Murni. 2004. Pengaruh penambahan bakteri probiotik Bacillus sp. dalam pakan buatan terhadap aktivitas enzim pencernaan, efisiensi pakan dan pertumbuhan ikan gurame [tesis]. Bogor (ID): Institut Pertanian Bogor. Pangastuti A. 2006. Definisi spesies prokaryota berdasarkan urutan basa gen
penyandi 16S-rRNA dan gen penyandi protein [Ulasan]. Biodiversitas. 7(3):292-296.
Pertsemlidis A, Fondon III JW. 2002. Having a BLAST with bioinformatics (and avoiding BLAST phemy). Gen Biol. 2:1-10.
Putra AN. 2010. Kajian probiotik, prebiotik dan sinbiotik untuk meningkatkan kinerja pertumbuhan ikan Nila (Oreochromis niloticus) [tesis]. Bogor (ID): Institut Pertanian Bogor.
Ray AK, Bairagi A, Ghosh KS, Sen SK. 2007. Optimazation of fermentation condition for cellulase production by Bacillus subtilis CY5 and Bacillus circulans TB3 isolated from fish gut. Acta Ichthyol Piscat. 37(1):47-53. Rengpipat S, Rukpratanporn S, Piyatitiratitivorakul S, Menasaveta P. 2000.
Immunity enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium (Bacillus S11). J. Aquacult. 167:301-313.
Singh SK, Tripathi VR, Jain RK, Vikram S. 2010. An antibiotic, heavy metal resistent and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease. Microb Cell Fac. 9:59-64.
Sivaramakrishnan S, Gangadharan D, Madhavan K, Nampothiri, Soccol CR, Pandet A. 2006. α-amylase from microbiol sources an overview on recent development. Food Technol Biotechnol. 44(2):173-184.
Solano O, Olmos S. 2006. The functional property of Bacillus for shrim feeds. Aquaculture. 23:519-525.
Sonenschein AL, Losick R, Hoch JA. 1993. Bacillus subtilis and Other Gram Positive Bacteria. Biochemistry Physiology and Moleculer Genetics Washington DC (US): American Society for Microbiology.
Sun YZ, Yang HL, Ma RL, Lin WY. 2010. Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune response of grouper Epinephelus coioides. J Fish Shellfish Immunol. 29:803-809.
Suryanto D, Irmayanti, Lubis, S. 2007. Karakterisasi dan uji kepekaan antibiotik beberapa isolat Staphylococcus aureus dari Sumatera Utara. Majalah Kedokteran Nusantara. 40(2):104-107.
Toldar K. 2009. The genus Bacillus. Toldar’s online texbook of bacteriology.
22
Verschuere L, Rombaut G, Sorgeloos P, Verstraete W. 2000. Probiotic bacteria as biological control agents in aquaculture. J Mircobiol Mol Biol Rev. 64(4): 655-671.
Walter HE. 1984. Method with haemoglobin, casein, and azocoll as substrate. Di dalam: Bergmeyer, editor. Methods of Enzymatic Analysis. Ed ke-3. Weinheim (JE): Verlag Chemie. hlm. 270 – 278.
Wang H, Liu Y, Cheng C, Ma O, Zhang Y. 2007. Screening and mutagenesis of a novel Bacillus pumilis strain producing alkaline protease for dehairing. Lent Appl Microb. 44: 1-6.
Wang YB, Li JR, Lin J. 2008. Probiotics in aquaculture: Challenges and outlook. J Aquacult. 281: 1-4.
Widanarni, Sukenda, Setiawati M. 2008. Bakteri probiotik dalam budidaya udang: seleksi, mekanisme aksi, karakterisasi, dan aplikasinya sebagai agen biokontrol. JIPI. 13(2): 80-89.
Zhou XX, Wang YF, Li WF. 2009. Effect of probiotic on larva shrimp (Penaeus vannamei) based on water quality, survival rate and digestive enzyme activities. J Aquacult. 287: 349-353.
Ziaei-Nejad S, Rezaei MH, Takami GA, Lovett DL, Mirvaghefi A-R, Shakouri M. 2006. The effect of Bacillus spp. bacteria used as probiotic on digestive enzyme activitiy, survival and growth in the Indian white shrimp Fenneropenaeus indicus. Aquaculture 252: 516-524.
23
Lampiran 1 Prosedur uji aktivitas protease (Walter 1984)
Pereaksi Blanko (μl) Kontrol (μl) Sampel (μl)
Kasein 1% 500 500 500
Bufer fospat 50 - -
Tirosin 5 mM - 50 -
Enzim - - 50
Diinkubasi 10 menit pada suhu optimum
TCA 0,3 M 500 500 500
Enzim 50 50 -
Bufer fosfat - - 50
Diinkubasi 10 menit suhu ruang, sentrifugasi kecepatan 14000 rpm; 10 menit
Na2CO3 0,4 M 2000 2000 2000
Filtrat 600 600 600
Folin Ciocalteu (1:2) 400 400 400
Diinkubasi 20 menit = 578 nm
Aktivitas enzim dihitung menurut rumus: x P x 1/T
Keterangan :
UA : Jumlah enzim yang dapat menghasilkan 1 mikromol produk tirosin permenit
A Sampel : Absorbansi sampel A Standar : Absorbansi standar A Blanko : Absorbansi Blanko
P : Faktor pengenceran
24
Lampiran 2 Prosedur uji aktivitas amilase (Bernfeld 1959)
Pereaksi Blanko (mL) Kontrol (mL) Sampel (mL)
Substrat (pati terlarut 0.3 %) 1 1 1 Enzim (EEK) - - 1 DNS - 1 - Aquades 1 - -
Dikocok dan diinkubasi 10 menit pada suhu ruang DNS 1 - 1 Enzim (EEK) - 1 -
dikocok dan dipanaskan pada 100 oC selama 5 menit, kemudian didinginkan dalam air selama 20 menit, diukur absorbansi pada = 550 nm
Keterangan :
UA : Unit aktivitas enzim
FP : Faktor pengenceran
BM : Berat molekul (BM maltosa = 360.32 Da)
25
Kurva standar maltosa
Persamaan regresi linear y = 1,4165x – 0,0428 Konsentrasi Maltosa ( mg/mL) Absorban rata-rata ( =550 nm) Absorbansi terkoreksi ( =550 nm) 0 0.052 0 0.15 0.180 0.128 0.25 0.341 0.289 0.35 0.511 0.459 0.45 0.645 0.593 0.55 0.793 0.741 0.65 0.942 0.890 y = 1,4165x - 0,0428 R² = 0,9932 -0,2 0 0,2 0,4 0,6 0,8 1 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 Abs orba sni (λ =5 50 nm ) Konsentrasi Maltosa
26
Lampiran 3 Komposisi reagen yang digunakan
1. Phosphate Buffered saline (PBS): 10 mM K2PO4-KH2PO4, 0,14 M NaCl pH 7.2
2. Reagen Dinitrosalisilic acid (DNS) (g/L):
-NaOH padat : 10
- KNa –Tartarat : 182
- Na2SO3 : 0.5
- DNS : 10
-Aquades : sampai dengan 1000 mL
Sebanyak 10 g NaOH, 182 g KNa-Tartarat, 0.5 g Na2SO3 dimasukkan dalam erlenmeyer 1000 mL dan dilarutkan dengan akuades sedikit demi sedikit. Setelah larut ditambahkan DNS sambil dihomogenkan perlahan-lahan. Larutan disaring dengan kertas saring dan disimpan dalam botol gelap pada suhu dingin. 3. Lugol’s Iodine (100 mL)μ - KI : 2 g - Akuades : 20 mL - I2 : 1 g - Akuades : 80 mL
27
Lampiran 4 Karakteristik morfologi isolat dari usus ikan lele
No Kode
Isolat Karateristik morfologi
1. PTB 1.1 Batang, putih susu, licin, disekitar tepian melengkung
2. PTB 1.2 Batang, putih susu, tepian bergerigi, permukaan sedikit kasar 3. PTB 1.3 Batang, putih susu, tepian licin, permukaan licin/halus (ada
bulatan kecil ditengah)
4. PTB 1.4 Batang, putih susu, tepian licin, bulat, permukaan halus
5. PTB 1.5 Batang, krem, tepian bergerigi, bulat kecil, permukaan mengkerut
6. PTB 1.6 Batang, krem, tepian bergerigi, bulat kecil, permukaan kasar/mengkerut
7. PTB 1.7 Batang, kuning, tepian licin, permukaan licin
8. PTB 2.0 Batang, krem, tepian bergerigi, permukaan kasar, bulat besar 9. STB 1.1 Batang, putih susu, tepian bergerigi, permukaan kasar/tidak
licin
10. STB 1.5 Batang, putih susu, tepian licin, permukaan licin 11. STB 1.6 Batang, krem, tepian licin, bulat, permukaan licin 12. STB 2.1 Batang, krem, tepian bergerigi, permukaan kasar 13. STB 2.2 Batang, krem, tepian bergerigi, permukaan kasar 14. STB 2.3 Batang, krem, tepian licin, permukaan licin
15. STB 2.4 Bundar, tepian bergerigi, bening, permukaan kasar/mengkerut 16. STB 2.5 Bundar, tepian licin, krem, permukaan licin
28
Lampiran 5 Hasil contig gen 16S-rRNA isolat PTB 1.4
GGCCGCGGCATGCTGATCCGCGATTACTAGCGATTCAGCTTCATGTAGGCGAGTTGCA GCCTACAATCCGAACTGAGAATGGTTTTATGGGATTGGCTTGACCTCGCGGTCTTGCA GCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGA TTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAA CTAAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATC TCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCCGAAGGGG AACGCTCTATCTCTAGAGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTT GCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAG TTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACT AAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGG GTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAAA AAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGG AATTCCGCTTTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCACGGTTG AGCCGTGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCCAATA ATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCG TGGCTTTCTGGTTAGGTACCGTCAAGGTACGAGCAGTTACTCTCGTACTTGTTCTTCCC TAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGCGTTGCTCCGTCA GACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAATCTGGGCCCG GGCCTCAGTCCCAGTTGGGGCCGATCACCCCTCTCAGGTCNGGCAATGCATCGTTTGC CTTGGTGAAGCCGTTACCCTCACCAACTAAGCTAATGGAACCGCGGGGCCCATTCTGG AAAGTGATAGCCGAAAACCATCTTTTCAATCCATCTCCCCATTGAAGAGAAGAAGAA CCCTATCCCGGTATATACGTTTCCGGTTTCCCGAAAATTATCCCCAGTTNTTACAGGGC AGGTTGTGCCAGCTGTTGAACTCAACCCGGTCCCGCCGCTAAACGCTATAAGAAANC AGATTTCTTATAAGAATT
Lampiran 6 Hasil Blast sekuen basa nukleotida isolat PTB 1.4 berdasarkan data bank gen koleksi NCBI (www.ncbi.nlm.nih.gov)
29