Optimasi hasil seleksi bisa dilakukan mnggunakan ELISA kit dengan konjugat yang telah diverifikasi terlebih dahulu dan hasil yang diperoleh langsung dikonfirmasi dengan metode HPLC. Media yang digunakan sebaiknya sudah menjalani uji potensi untuk memastikan bahwa media tersebut mendukung produksi aflatoksin optimum termasuk pengaruh pH awal dan nutrien yang diberikan. Penggunaan instrumen seperti HPLC maupun GC-MS bisa dijadikan alternatif metode TLC untuk menghindari adanya data yang membutuhkan pengukuran subjektif. Proses purifikasi lebih lanjut juga dapat dilakukan untuk mencapai kemurnian yang memenuhi persyaratan untuk dijadikan standar acuan dalam analisis aflatoksin.
44
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Lampiran 1. Tabel Hasil Seleksi dengan ELISA
Sampel Kadar aflatoksin terukur (ppb) Faktor Pengenceran (FP) Kadar aflatoksin (ppb) S3 (A)d 6/8/09 0,5 65,61 31,4 (B) 3/8/09 - n.d (C) 31/7/09 12,3 40 491,4 (D) 29/7/09 6,7 40 267 (E) 27/7/09 13,4 40 534 S5 (A) 6/8/09 10,8 40 433,7 (B) 3/8/09 2,5 40 101,2 (C) 31/7/09 5,5 40 219,9 (D)d 29/7/09 3,6 65,61 234,2 (E) 27/7/09 5,5 40 219,9 S9 (A) 6/8/09 4,5 40 178,6 (B) 3/8/09 7,6 40 302,5 (C) 31/7/09 5,6 40 223 (D) 29/7/09 4,5 40 178,6 (E) 27/7/09 6,7 40 267 S11 (A) 6/8/09 6,2 40 249 (B) 3/8/09 2,7 40 108,4 (C) 31/7/09 0,5 40 18,1 (D) 29/7/09 3,4 40 135, 4 (E) 27/7/09 1,0 40 38,352 Sampel Kadar aflatoksin terukur (ppb) Faktor Pengenceran (FP) Kadar aflatoksin (ppb) S14 (A) 27/7/09 2,8 10 27,7 (B) 29/7/09 2,9 10 29,4 (C) 31/7/09 87,9 10 878,6 (D) 3/8/09 14,6 10 145,7 (E) 6/8/09 8,0 10 79,9 S17 (A) 27/7/09 n.d - n.d (B) 29/7/09 n.d - n.d (C) 31/7/09 n.d - n.d (D) 3/8/09 n.d - n.d (E) 6/8/09 n.d - n.d S19 (A) 6/8/09 n.d - n.d (B) 3/8/09 n.d - n.d (C) 31/7/09 n.d - n.d (D) 29/7/09 n.d - n.d (E) 27/7/09 n.d - n.d S23 (A) 6/8/09 n.d - n.d (B) 3/8/09 n.d - n.d (C) 31/7/09 n.d - n.d (D) 29/7/09 n.d - n.d (E) 27/7/09 n.d - n.d S26 (A) 6/8/09 3,6 40 143,1 (B) 3/8/09 3,6 40 145,1 (C)d,e 31/7/09 18,5 65,61 1212,3 (D) 29/7/09 2,7 40 108,41 (E) 27/7/09 8,0 40 319,7 F-0213 (A) 6/8/09 1,6 40 64,9 (B)f 3/8/09 3,9 65,61 252,8 (C) 31/7/09 0,6 40 25,6 (D) 29/7/09 2,1 40 84,5 (E) 27/7/09 0,4 40 14,7
Contoh perhitungan: Sampel JCM (D) 3/8/09
Kadar aflatoksin (ppb) = Kadar aflatoksin terukur (ppb) x FP = 20,2 x 40
= 809,4
Lampiran 2. Gambar microplate yang digunakan dalam uji ELISA
Lampiran 3. Microplate Spectrophotometer yang digunakan dalam uji ELISA
Sampel Kadar aflatoksin terukur (ppb) Faktor Pengenceran (FP) Kadar aflatoksin (ppb) JCM (A) 27/7/09 n.d - n.d (B) 29/7/09 18,4 40 734,6 (C) 31/7/09 13,9 40 556,7 (D) 3/8/09 20,2 40 809,4 (E) 6/8/09 11,1 40 445,9
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Lampiran 4. Pertumbuhan isolat S.26 dalam media GAN termodifikasi
Sampel S.26 Media GAN H-9 Sampel S.26 Media GAN H-12
Sampel S.26 Media GAN H-14 Sampel S.26 Media GAN H-16
Lampiran 5. Pertumbuhan isolat S.26 dalam media PDB
Sampel S.26 Media PDB H-9 Sampel S.26 Media PDB H-12
Sampel S.26 Media PDB H-14 Sampel S.26 Media PDB H-16
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Lampiran 6. Pertumbuhan isolat JCM dalam media GAN termodifikasi
Sampel JCM Media GAN H-9 Sampel JCM Media GAN H-12
Sampel JCM Media GAN H-14 Sampel JCM Media GAN H-16
Lampiran 7. Pertumbuhan isolat JCM dalam media PDB
Sampel JCM Media PDB H-9 Sampel JCM Media PDB H-12
Sampel JCM Media PDB H-14 Sampel JCM Media PDB H-16
58
Lampiran 8. Tabel Hasil Analisis dengan TLC pada media PDB
Sampling
Pembacaan TLC (µL)a
Ulangan 1 Ulangan 2 Ulangan 3 Ulangan 4 Ulangan 5 Rataan (+)b S.26c (+)b S.26c (+)b S.26c (+)b S.26c (+)b S.26c (+)b S.26c H0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 H2 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 H5 2 2 0,0 0,0 2 2 0,0 0,0 0,0 0,0 0,8 0,8 H7 2 5 5 5 3 2 1 1 2 2 2,6 3 H9 3 9 6 10 4 4 4 4 3 3 4 5,8 H12 4 6 8 6 4 3 5 4 3 4 4,8 4,6 H14 4 2 5 4 4 3 2 2 3 2 3,6 2,6 H16 2 2 2 3 3 3 2 1 2 3 2,2 2,4 H19 0,0 2 1 3 2 1 1 1 2 2 1,2 1,6 H21 0,0 0,0 1 1 1 0,0 0,5 0,5 1 1 0,7 0,5 a
Semua sampel dianalisis menggunakan standar 0,2 ppm, volume pengenceran 500 µL, dan volume spotting 2 µL.
Contoh perhitungan: S.26 ulangan 1 H9
Diketahui: S = 9 µL Y = 0,2 µg/mL V = 500 µL FP = 1 W = 1 mL Z = 2 µL Kandungan aflatoksin (ppm) = 9 µL x 0,2 µg/mL x 500 µL x 1 1 mL x 2 µL = 0,450 µg/mL = 0,450 ppm Kandungan aflatoksin = 450 ppb
Lampiran 9. Gambar peralatan TLC yang digunakan
60
Lampiran 11. Tabel Hasil Analisis HPLC pada media PDB
Sampel Kadar Aflatoksin (ppb)
Faktor Pengenceran
Ulangan Jenis Sampel Kode Sampel B1 B2 G1 G2
Isolat S.26 H9c HPLC 3 H9 44 1,1 --nd-- --nd-- 50 ULANG AN 2 Isolat JCM PDB (+) H7 7,4 -nd- --nd-- 0,1 50 PDB (+) H9 13,1 -nd- --nd-- 0,2 50 PDB (+) H12 16,9 0,4 --nd-- --nd-- 50 PDB (+) H14 8,9 0,2 --nd-- --nd-- 50 PDB (+) H16 14,9 -nd- --nd-- --nd-- 5 PDB (+) H19 0,0 --nd-- --nd-- --nd-- - PDB (+) H21 0,0 --nd-- --nd-- --nd-- - Isolat Aspergillusflavus lokal S.26 PDB H7 5,8 --nd-- --nd-- --nd-- 50 PDB H9 18,7 --nd-- --nd-- --nd-- 50 PDB H12 11,9 --nd-- --nd-- --nd-- 50 PDB H14 29,7 0,9 60,8 --nd-- 5 PDB H16 40,3 16,5 354,5 --nd-- 5 PDB H19 0,0 --nd-- --nd-- --nd-- - PDB H21 0,0 --nd-- --nd-- --nd-- -
Contoh perhitungan: Sampel S.26 PDB H7
/
Diketahui: Ch = 5,8 ppb FP = 50 Bc = 1 mL Cp = 5,8 ppb x 50 1 mL = 290,2 ppb62
