Simpulan
Kolagen teripang gama yang diekstrak dengan metode asam memberikan hasil rendemen sebesar 16.40% basis kering (bk) dengan berat molekul 130.33 kDa. Hidrolisis kolagen dengan enzim pepsin menghasilkan 3 fraksi peptida dengan berat molekul yang bervariasi, yaitu fraksi yang pertama memiliki berat molekul antara 95.03-90.42 kDa, fraksi kedua memiliki berat molekul antara 57.6-52.11 kDa dan fraksi ketiga memiliki berat molekul antara 29.55-24.1 kDa. Persen derajat hidrolisis tertinggi terdapat pada waktu inkubasi 120 menit yaitu sebesar 54.61%. Aktivitas penghambatan ACE terbaik terdapat pada peptida kolagen yang dihasilkan dari hidrolisis selama 180 menit yaitu sebesar 82.31%. Perbedaan lama waktu hidrolisis memberikan pengaruh yang berbeda secara nyata terhadap persen penghambatan ACE. Aktivitas antioksidan terbaik terdapat pada peptida kolagen yang dihasilkan dari hidrolisis selama 120 menit dengan IC50 sebesar 1.92 mg/ml. Waktu hidrolisis yang berbeda memberikan pengaruh yang berbeda secara nyata terhadap aktivitas antioksidan.
Saran
Saran yang dapat disampaikan dari hasil penelitian ini adalah perlu dilakukan uji bioaktivitas terhadap serbuk teripang utuh tanpa hidrolisis sebagai pembanding serbuk teripang yang telah dihidrolisis. Selain itu, perlu dilakukan sekuensing peptida hasil hidrolisis untuk mengetahui urutan asam amino yang menghasilkan bioaktivitas terbaik.
Ucapan Terima Kasih
Ucapan terima kasih disampaikan kepada Balai Besar Penelitian Pengembangan Pengolahan Produk dan Bioteknologi Kelautan dan Perikanan (BBP4BKP) Jakarta Pusat atas sponsor yang telah diberikan dengan dana APBN tahun 2014/2015.
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47 Lampiran 1 Komposisi dan Pereaksi SDS-PAGE
1. Pembuatan pereaksi
Larutan A (30% b/v akrilamid, 0,8% b/v bisakrilamid 100 ml)
29.2 gram akrilamida ditambah 0.8 gram bis akrilamida lalu tambahkan aquades 100ml dan diaduk hingga merata
Larutan B (4x bufer gel pemisah 100 ml)
75 ml Tris HCl 2 M pH 8.8 ditambah 4 ml 10% SDS lalu ditambah 21 ml aquades
Larutan C (4x buffer gel penahan 100 ml)
50 ml Tris-HCl 1 M pH 6.8 ditambah 4 ml SDS 10% lalu ditambah aquades sebanyak 46 ml
Bufer Tris-HCl 1 M pH 6.8 100ml
12.1 gram Tris ditambah 50 ml aquades llau ditambah HCl 1 M hingga pH 6.8 lalu ditera hingga 100ml
Bufer Tris-HCl 2 M pH 8.8 (50 ml)
24,2 gram Tris ditambah 50 ml aquades lalu ditambah HCl 1M hingga pH 8.8. Larutan ditera hingga 100 ml dengan aquades
Gliserol 50%
50 ml gliserol 100% ditambah aquades 50 ml 10% Amonium persulfate (APS) 5 ml
0.5 gram APS ditambah 5ml aquades 10% SDS 100 ml
10 gram SDS dilarutkan dalam 100 ml aquades Bromphenol blue (BB) 1%
100 mg BB ditambah dengan 10 ml aquades Buffer elektroforesis 1 liter
3 gram Tris ditambah 14,4 gram glisin, ditambah 1 gram SDS lalu ditambah aquades hingga volume 1000ml
Buffer sampel
0.6 ml buffer Tris-HCl pH 6.8 ditambah 5 ml gliserol 50%, lalu ditambah 2 ml SDS 10% dan 0.5 ml 2-merkaptoetanol. Selanjutnya ditambah 1 ml bromphenol blue dan ditambah aquabides hingga 10 ml. Total volume larutan sebanyak 10 ml
Larutan pewarna (staining)
0,5 gram commasive brilliant blue ditambah asam asetat glacial 50 ml dan metanol p.a 200 ml. Selanjutnya ditera dengan aquades hingga 500 ml Larutan peluntur (destaining)
50 ml asam setat glasial ditambah 200 ml metanol p.a lalu ditera dengan aquades hingga 500 ml
48
2. Komposisi gel pemisah (separating gel) dan gel penahan (stacking gel)
8 % 10 % 12 % 14 % 4 % Larutan A 2,72 ml 3.4 ml 4.2 ml 4,76 ml Larutan B 2,5 ml 2,5 ml 2,5 ml 2,5 ml - Larutan C - - - - 1,25 Substrat (aq) 1 1 1 1 - Akuades 5 ml 4.17 ml 3.33 ml 2.5 ml 10 % APS 0,1 ml 0,1 ml 0,1 ml 0,1 ml 0,05 ml TEMED 0,01 ml 0,01 ml 0,01 ml 0,01 ml 0,005 ml
49 Lampiran 2 Perhitungan Berat Molekul Kolagen dan Peptida Kolagen
Berat molekul kolagen dan peptida kolagen
BM Jarak Log BM Rf kolagen utuh 130.33 10 2.11506 0.096154 Hidrolisis 30 menit 80, 42 24 1.905382 0.237624 45.59 41 1.658945 0.405941 29.55 54 1.470493 0.534653 Hidrolisis 60 menit 95.03 19 1.977864 0.188119 47.14 40 1.673441 0.39604 28.58 55 1.455996 0.544554 Hidrolisis 90 menit 95.03 19 1.977864 0.188119 44.09 42 1.644449 0.415842 27.64 56 1.4415 0.554455 Hidrolisis 120 menit 91.91 20 1.963368 0.19802 44.09 42 1.644449 0.415842 27.64 56 1.4415 0.554455 Hidrolisis 180 menit 95.03 19 1.977864 0.188119 45.59 41 1.658945 0.405941 27.64 56 1.4415 0.554455 Hidrolisis 240 menit 91.91 20 1.963368 0.19802 42.6 43 1.629952 0.425743 24.1 60 1.383515 0.594059 y = -0.675x + 1.525 R² = 0.957 -0.2 0 0.2 0.4 0.6 0.8 1 0 0.5 1 1.5 2 2.5 3 Lo g