PRODUKSI SERBUK FITASE HASIL TEKNOLOGI REKOMBINAN DAN APLIKASINYA UNTUK

MENINGKATKAN KUALITAS PAKAN DAN KINERJA AYAM BROILER

Disertasi

Oleh:

Adi Magna Patriadi Nuhriawangsa 05/1676/PS

PROGRAM PASCASARAJANA FAKULTAS PETERNAKAN

UNIVERSITAS GADJAH MADA YOGYAKARTA

2012

ii

PRODUKSI SERBUK FITASE HASIL TEKNOLOGI REKOMBINAN DAN APLIKASINYA UNTUK

MENINGKATKAN KUALITAS PAKAN DAN KINERJA AYAM BROILER

Disertasi

Oleh:

Adi Magna Patriadi Nuhriawangsa 05/1676/PS

PROGRAM PASCASARAJANA FAKULTAS PETERNAKAN

UNIVERSITAS GADJAH MADA YOGYAKARTA

2012

146

RINGKASAN

Asam fitat mengikat sekitar 80% P dalam biji-bijian, tidak dapat dicerna dalam

saluran pencernaan unggas dan menurunkan nilai nutrien bahan pakan yang

berasal dari tanaman pertanian (Saryska et al., 2005). Senyawa ini mampu

mengikat ion mineral seperti: Mg++, Fe++, Zn++, Mn++, Ca++ (Pallauf et al., 1998),

fosfat dan protein yang berguna bagi pertumbuhan ternak

(Deshpande dan Cheryan, 1984). Ternak nonruminansia tidak mempunyai fitase

pada saluran pencernaannya, sehingga kandungan senyawa fitat tidak bisa

dicerna. Hal ini disebabkan karena sifat chelating, sehingga senyawa fitat

terbuang bersama kotoran dan mencemari lingkungan (Shin et al., 2001).

Menurut Phillippy dan Mullaney (1997) mikrobia asli menghasilkan fitase

dengan jumlah lebih rendah dibanding mikrobia rekombinan. Selain itu mikrobia

rekombinan terbukti lebih efektif memproduksi fitase pada skala besar (over

production). Native phytase kurang stabil terhadap pH dan temperatur, tetapi

fitase yang dihasilkan oleh bakteri rekombinan mempunyai keunggulan lebih

stabil terhadap pH dan temperatur (Pasamontes et al., 1997). Fitase bakteri

rekombinan mempunyai kelebihan dibanding fitase bakteri asli, yaitu: aktivitas

spesifik yang tinggi (sekitar 1.000 kali lebih tinggi dibanding yang dihasilkan dari

bakteri asli), mampu menghidrolisis senyawa fitat sampai Inositol (2) monofosfat

dan mampu merombak senyawa fosfat organik dan anorganik lainnya

(Sajidan, 2007).

Fitase mengalami degradasi terutama oleh faktor temperatur dan kadar air,

sehingga memerlukan teknologi penyimpanan agar aktivitasnya stabil

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(Anselme, 2006). Beberapa syarat yang penting untuk diperhatikan pada aplikasi

terapan produk enzim pada ternak unggas, yaitu enzim harus tahan panas, tahan

pH asam dan tahan terhadap aktivitas protease (Abondano, 2009).

Fitase dari bakteri Klebsiella pneumoniae strain ASR1 telah dapat dimurnikan,

dikarakterisasi dan disekuensi gennya. Gen phyK dari bakteri tersebut telah

dapat dikloning ke dalam plasmid pET22b(+) produk Novagen dan diekspresikan

dalam sel kompeten E. coli strain C41 (DE3), sehingga dapat dihasilkan fitase

dari bakteri rekombinan yang mempunyai karakter tertentu

(Sajidan et al., 2004b).

Enzim hasil produksi dari bakteri asli mempunyai kelemahan dalam

aplikasinya, sehingga diperlukan teknologi rekombinan untuk memperbaiki dan

meningkatkan produktivitas enzim tersebut. Secara rekombinan enzim dapat

diproduksi dengan teknologi kloning gene. Gen phyK telah dapat disisipkan

dalam plasmid pET22b(+) sehingga dihasilkan pET22b(+)+pEAS1. Produksi

bakkteri rekombinan dengan cara transformasi pET22b(+)+pEAS1 ke dalam dua

jenis sel kompeten, sehingga dihasilkan bakteri rekombinan E. coli

BL21+pET22b(+) dan E. coli BL21(DE3) penghasil fitase baik secara ekstra dan

intraseluler. Produksi enzim pada bakteri rekombinan membutuhkan induktor

yaitu IPTG dan pemurnian enzim lebih mudah dengan adanya His-Tag yang

akan terikat pada kolom Ni-NTA agarosa dan mudah dielusi dengan Imidazole.

Optimasi pada bakteri rekombinan dan enzim yang dihasilkan dapat

mengoptimalkan aktivitas dan produksi dari bakteri rekombinan dan enzim

tersebut.

Sistem pencernaan ayam broiler mempunyai variasi pH dari rendah sampai

basa dan beberapa enzim pencernaan. Proses pembuatan pakan ternak berupa

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crumble melalui tahap pemanasan pada temperatur 80 sampai 90oC. Kedua

faktor tersebut dapat menurunkan aktivitas fitase. Beberapa metode telah

digunakan untuk menguji aktivitas hidrolisis dan stabilitas fitase secara in vitro.

Enkapsulasi kitosan berguna untuk imobilisasi dan melindungi fitase dari

cekaman selama penyimpanan, pemanasan dan aplikasi pada pencernaan ayam

broiler. Pakan tambahan dihasilkan dari fitase bakteri rekombinan yang dicampur

dengan bahan pembawa (bekatul jagung, sekam padi) dan dikeringkan (kering

matahari, kering oven, kering beku). Proses tersebut bermanfaat untuk

mempertahankan stabilitas fitase selama penyimpanan dan mempermudah

pencampuran dalam pakan. Pakan tambahan diberikan pada ayam broiler

dengan tujuan tertentu dan keberhasilan pemanfaatan pakan tambahan yang

mengandung fitase dapat dilihat dengan menggunakan metode in vivo dengan

melihat kecernaan ekskretal semu nutrien, profil darah, kinerja produksi fitase

dan kinerja produksi.

Asam fitat merupakan antinutrisi bagi ternak ayam, karena unggas tidak

memiliki fitase pada sistem pencernaannya. Asam fitat mengikat beberapa

nutrien yang dapat dimanfaatkan oleh ayam broiler diantaranya mineral

(Ca, P, Mg, Zn, Fe, Cu), fosfat, glukosa dan protein. Fitase dapat menghidrolisis

fitat pada C3 atau C6 dari bentuk mio-inositol heksakisfosfat menjadi bentuk lebih

sederhana, yaitu: D-inositol(1,2,4,5,6)P5 menjadi inositol(2,4,5,6)P4 menjadi

inositol(2,4,6)P3 atau inositol(2,4,5)P3 atau inositol(1,2,6)P3 dan akhirnya menjadi

inositol-2-P, sehingga nutrien yang terikat pada asam fitat terhidrolisis dan lepas

dari ikatannya.

Kalsium, P dan fosfat merupakan mineral penyusun tulang, Ca berperan

dalam pembentukan ikatan miofilamen dalam pembentukan otot, Mg berperan

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sebagai prekusor dan aktivator pada biosintesis asam amino dan arginin

berperan dalam pembentukan kreatinin yang berfungsi sebagai buffer energi

pada otot skeletal. Fosfat mempunyai pernan penting dalam pembentukan DNA

dan RNA. DNA dan RNA berperan penting dalam pembentukan asam amino dan

juga protein yang berperan pada pertumbuhan tubuh ternak, dengan demikian

juga berpengaruh terhadap pembentukan otot.

Penelitian Tahap ke-1 mengembangkan plasmid EAS1 ditransformasi ke

dalam sel kompeten E. coli BL21 dan E. coli BL21 (DE3) untuk digunakan

dalam produksi fitase dari bakteri rekombinan (Sajidan et al., 2004a). Produksi

enzim menggunakan induktor 1,5 mM IPTG, ekstraksi menggunakan kolom Ni-

NTA agarosa dan elusi menggunakan imidazole. Transformasi, produksi bakteri

dan produksi fitase menggunakan metode menurut Sajidan (2002) dan Novagen

(2005b). Pengukuran aktivitas enzim fitase dengan metode dari Sajidan (2002).

Karakterisasi pH, temperatur, grafik pertumbuhan dan konsentrasi IPTG bakteri

rekombinan menggunakan metode Sajidan (2002) dan Novagen (2005b)

berdasarkan aktivitas relatif dengan melihat aras parameter tersebut.

Karakterisasi pH, temperatur, waktu inkubasi, konsentrasi subtrat dan kofaktor

ion logam fitase dengan melihat aktivitas relatifnya pada berbagai

aras parameter tersebut (Greiner et al., 1997; Sajidan, 2002). Karakterisasi Km

dan Vm menggunakan metode menurut Robyt dan White (1997) dengan melihat

produk yang dihasilkan berdasarkan grafik standar KH2PO4 sebagai nilai V. BM

protein menggunakan poliakrilamid gel elektroforesis (Sulandari dan Zein, 2003)

dengan protein standar Proteina Marker (Bio-Rad katalog 161-0318) pada

sekuensi protein 36,2 dan 52,2 kDa.

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Transformasi pEAS1 ke dalam sel kompeten E. Coli BL21 (DE3)

menghasilkan bakteri rekombinan penghasil fitase dengan karakteristik kondisi

optimum pada pH 7, temperatur 40oC, induktor IPTG konsentrasi 1,5 mM yang

diberikan pada waktu 94 menit setelah inkubasi. Fitase kasar ekstraseluler dari

bakteri rekombinan pEAS1/AMP mempunyai kondisi optimum pada pH 5,

temperatur 40oC, waktu inkubasi 60 menit, konsentrasi substrat 1%, Km = 12,33

µM, Vm = 1,37 µM/detik, Mg2+ (10-4 M) bersifat sebagai kofaktor, Pb2+ dan Fe3+

(10-4 M) sebagai inhibitor. Fitase murni ekstraseluler dari bakteri rekombinan

pEAS1/AMP mempunyai kondisi optimum pada pH 5, temperatur 45oC, waktu

inkubasi 60 menit, konsentrasi substrat 3%, berat molekul 47,3 kDa, Km = 54,82

µM, Vm = 5,05 µM/detik dan Mg2+ (10-4 M) bersifat sebagai kofaktor. Fitase

kasar intraseluler dari bakteri rekombinan mempunyai kondisi optimum pada

pH 5, temperatur 40oC, waktu inkubasi 60 menit, konsentrasi substrat 2%, Km =

15,91 µM, Vm = 2,41 µM/detik, Mg2+ (10-4 M) bersifat sebagai kofaktor, dan

Fe3+ (10-4 M) sebagai inhibitor. Fitase murni intraseluler dari bakteri rekombinan

mempunyai kondisi optimum pada pH 5, temperatur 45oC, waktu inkubasi 60

menit, konsentrasi substrat 4%, berat molekul 46,4 kDa, Km = 34,82 µM,

Vm = 6,39 µM/detik dan Mg2+ (10-4 M) bersifat sebagai kofaktor.

Pada penelitian Tahap ke-2 menguji stabilitas pH dan temperatur

menggunakan metode menurut Sajidan (2002) dengan melihat aktivitas relatif

fitase pada aras pH dan temperatur. Hidrolisis abu, Ca dan P diuji dengan

metode in vitro menurut Wu et al. (2004) dengan menggunakan bekatul padi.

Bekatul padi dianalisis uji Ca (Barrow et al., 1962), P (Jackson, 1985) dan

abu (AOAC, 1978) dan nilai hidrolisis dengan melihat selisih Ca, P dan abu

sebelum dan sesudah uji in vitro dalam BK (Tillman et al., 1986).

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Fitase mempunyai stabilitas aktivitas waktu simpan selama enam hari pada

temperatur refrigerasi pada kondisi cair, pH antara pH 2 sampai 8 dan mencapai

optimum pada pH 5, temperatur antara 0 sampai 65oC dan mengalami

penurunan dengan meningkatnya temperatur. Ketersediaan kandungan Ca, P

dan abu bekatul padi meningkat dengan adanya hidrolisis oleh fitase, stabil

terhadap pepsin tetapi kurang stabil terhadap pankreas secara in vitro.

Pada penelitian Tahap ke-3 fitase terkarakterisasi diimobilisasi dan

dipertahankan stabilitasnya dengan enkapsulasi kitosan. Optimasi fitase

terhadap kitosan dilakukan untuk mengetahui konsentrasi asam asetat, kitosan

dan perbandingan fitase dengan larutan kitosan dengan melihat aktivitas

relatif fitase (Sajidan, 2002) pada aras parameter tersebut. Kitosan dilarutkan

dengan menggunakan asam asetat (No et al., 2006). Fitase dan kitosan

dilarutkan pada bahan pembawa dengan cara disemprotkan menggunakan

spayer dan diaduk sampai rata (Anselme, 2006). Bahan pembawa yang

digunakan bekatul jagung dan sekam padi dengan ukuran 1 mm. Campuran

fitase-kitosan dengan masing-masing bahan pembawa kemudian dikeringkan

secara kering matahari selama 72 jam (Yunus et al., 2006), kering oven

menggunakan alat pengering merek Memmert pada temperatur 39oC

selama 48 jam (Stanbury dan Whitaker, 1987) dan kering beku menggunakan

alat freez dryer (Mujumdar, 2006) selama 6 jam. Serbuk fitase hasil teknologi

rekombinan disimpan dan diuji stabilitasnya setiap minggu dengan melihat

aktivitas relatif enzim secara in vitro.

Fitase optimum pada konsentrasi asam asetat 0,2 %, kitosan 0,8% dan

perbandingan fitase dan kitosan 1:1. Aktivitas fitase stabil dengan menggunakan

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bahan pembawa bekatul jagung, pengeringan dengan kering beku dan tahan

selama penyimpanan 5 minggu.

Penelitian Tahap ke-4 menguji serbuk fitase secara in vivo menggunakan

ayam pedaging New Lohmann MB202 sebanyak 100 ekor. Kecernaan yang

dianalisis adalah kecernaan secara ekskretal (Leeme et al., 2004). Ransum yang

digunakan untuk fase Starter adalah BR1 produksi PT. Japfa Comfeed Indonesia

dan fase Grower (15 sampai 23 hari) menggunakan ransum berbasis jagung dan

kedelai. Perlakuan ransum berupa ransum kontrol (R0), ransum rendah P tanpa

fitase (R1) dan ransum rendah P dengan fitase (R2). Pemeliharaan ayam DOC

sampai 14 hari dalam kandang postal secara kelompok dan setelah berumur 15

hari dalam kandang baterai secara individu masing-masing perlakuan 12 ekor.

Manajemen pemeliharaan meliputi pemberian pakan dan minum, vaksinasi dan

obat-obatan dan kebutuhan nutrien ransum didasarkan pada buku pedoman New

Lohmann (MB202) Broiler Management Programme dari PT. Multibreeder

Adirama Indonesia Tbk. Pengukuran konsumsi pakan dalam g/ekor/hari, konversi

pakan dan kenaikan bobot badan dalam g/ekor/hari (Tillman et al., 1989).

Preparasi dan pengambilan ekskreta pada umur 19 dan 20 hari. Sampel ekskreta

dimasukan dalam kantong plastik dan disimpan pada temperatur -20oC dan diuji

kecernaan bahan kering, Ca, P, total protein dan glukosa

ekskretal semu (El-Hakim et al., 2009). Analisis kandungan Ca menggunakan

metode titrasi dengan larutan EDTA (Barrow et al., 1962), P menggunakan

spektrofotometer pada panjang gelombang 415 nm (Jackson, 1985), protein

kasar dengan metode Kjehldahl (AOAC, 1975) dan glukosa dengan

metode GOD-PAP (DiaSys, 2008a). Pengambilan plasma darah pada umur 20

hari dan diambil pada vena sayap kanan. Darah dianalisis kadar Ca

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(DiaSys, 2007a), P (DiaSys, 2007b), glukosa (DiaSys, 2008a) dan total protein

(DiaSys, 2008b). Pemotongan pada umur 24 hari dengan jumlah ayam 24

ekor (masing-masing 8 ekor dari R0, R1 dan R2). Ayam dipotong menggunakan

metode dzabah (Nuhriawangsa, 1999). Peubah yang dianalisis pada tulang tibia

kiri dengan melihat berat, panjang (Kocabagli, 2001), kandungan P (Jackson,

1985), Ca (Barrow et al., 1962) dan abu (AOAC, 1975). Bobot karkas, bobot

dada dan bobot daging diperoleh dengan menimbang dalam satuan

gram (Soeparno, 1992).

Kecernaan ekskretal semu (bahan kering, P, kalsium, protein kasar, glukosa),

kandungan P dan protein dalam darah, bobot daging dada, panjang tibia,

kandungan Ca dan abu tibia, pertambahan bobot badan dan konversi pakan

meningkat dengan penggunaan serbuk fitase hasil teknologi rekombinan pada

ransum rendah P dengan fitase dibanding ransum rendah P tanpa fitase.

Kandungan Ca darah, bobot tulang tibia, kandungan P tulang tibia, bobot karkas

dan bobot dada antara ransum kontrol, ransum rendah P dan ransum rendah P

ditambah serbuk fitase hasil teknologi rekombinan tidak menunjukkan perbedaan

nilai. Kandungan P dan protein dalam darah mengalami penurunan pada ransum

rendah P dibanding ransum kontrol, tetapi kandungan glukosa darah tidak

mempunyai nilai yang berbeda. Panjang, kandungan Ca dan abu tibia pada

ransum kontrol mempunyai nilai lebih tinggi dibanding ransum rendah P

ditambah serbuk fitase hasil teknologi rekombinan.

Secara umum dapat dihasilkan bakteri rekombinan dengan karakter optimum

dapat diproduksi dan dapat menghasilkan fitase yang mempunyai karakter

optimum. Aktivitas fitase mempunyai stabilitas dalam pencernaan ayam broiler

secara in vitro. Fitase dapat dienkapsulasi dengan kitosan dan dihasilkan serbuk

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fitase yang siap diaplikasikan sebagai bahan tambahan pakan ayam broiler.

Serbuk fitase dapat digunakan sebagai bahan tambahan untuk meningkatkan

kecernaan pakan, profil darah, kinerja produksi fitase dan kinerja produksi.

Secara keseluruhan dapat disimpulkan bahwa serbuk fitase dapat dihasilkan dari

bakteri rekombinan AMP yang stabil selama kondisi penyimpanan dan saluran

pencernaan ayam broiler dan dapat diaplikasikan pada pakan.

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