STUDI PEPTIDA BIOAKTIF DARI PROTEIN DAGING TERNAK LOKAL INDONESIA SEBAGAI AGENSIA ANTIHIPERTENSI
JAMHARI, Prof. Dr. Ir. Lies Mira Yusiati, S.U.; Ir. Edi Suryanto, M.Sc., Ph.D.; Dr. Ir. Muhammad Nur Cahyanto, M.Sc.
2014 | Disertasi | S3 Ilmu PeternakanPenelitian ini bertujuan untuk mengetahui adanya potensi angiotensin converting enzyme inhibitor (ACE) dari hidrolisat protein daging ternak lokal Indonesia, dan mempurifikasi peptida potensial sebagai angiotensin converting enzyme inhibitor dari hidrolisat protein daging ternak lokal Indonesia. Materi yang digunakan dalam penelitian ini adalah daging Sapi Bali, Kambing Kacang, Ayam Kampung dan Itik Lokal. Penelitian ini terdiri dari 2 tahap, yaitu tahap pertama mengetahui potensi ACE inhibitor dari Daging Sapi Bali, Kambing Kacang, Ayam Kampung dan Itik Lokal, dan tahap kedua mempurifikasi peptida ACE inhibitor dari hidrolisat protein yang mempunyai aktivitas ACE inhibitor paling tinggi. Tahap pertama: daging digiling dibuat ekstrak daging, kemudian diuji kadar protein terlarut dan aktivitas ACE inhibitor. Ekstrak daging kemudian dihidrolisis dengan enzim pepsin, trypsin dan chymotrypsin. Hidrolisis dengan enzim pepsin dilakukan pada pH 2 dan suhu 37,5oC selama 2 jam, sedangkan hidrolisis dengan enzim trypsin dan chymotrypsin dilakukan pada pH 7 dan suhu 37,5oC selama 2 jam. Hidrolisat protein daging kemudian disaring dengan filter Advantec Dismic-25ES. Filtrat hidrolisat protein daging kemudian diuji kadar protein terlarut dan aktivitas ACE inhibitor. Profil protein ekstrak daging dan hidrolisat protein juga diuji melalui SDS-PAGE. Tahap kedua: peptida ACE inhibitor dari hidrolisat protein daging Kambing Kacang dipurifikasi dengan dengan reverse phase high performance liquid chromatography (RP-HPLC). Hasil penelitian tahap pertama menunjukkan bahwa konsentrasi protein terlarut hidrolisat daging lebih tinggi daripada konsentrasi protein terlarut ekstrak daging. Konsentrasi protein terlarut ekstrak daging Sapi Bali, Kambing Kacang, Ayam Kampung dan Itik Lokal masing-masing adalah 14,00, 17,13, 18,20, dan 12,07 mg/mL dan konsentrasi protein terlarut hidrolisat daging masing-masing adalah 32,44, 31,57, 35,57, dan 33,09 mg/mL. Berat molekul (BM) protein ekstrak daging berada pada kisaran antara 21 sampai 200 kDa, dan sebagian besar, berada pada berat molekul sekitar 45 sampai 65 kDa dan setelah dihidrolisis berada pada kisaran di bawah 6,5 sampai 65 kDa, dan sebagian besar berada pada berat kisaran di bawah 6,5 sampai 21 kDa. Aktivitas ACE inhibitor (%) hidrolisat protein daging Sapi Bali, Kambing Kacang, Ayam Kampung, dan Itik Lokal yang telah dihidrolisis dengan enzim protease masing-masing adalah 79,43, 80,58, 77,67, dan 73,32% dan IC50 masing-masing adalah 341, 316, 333, dan 380 ïg/mL. Hasil penelitian tahap kedua menunjukkan bahwa peptida ACE inhibitor mempunyai sekuen asam amino leu-thr-glu-ala-pro-leu-asn-pro-lys-ala-asn-arg-glu-lys (LTEAPLNPKANREK) dan mempunyai berat molekul sebesar 1581 (1,6 kDa). Peptida tersebut mempunyai aktivitas ACE inhibitor (IC50) sebesar 190 ïg/mL (120 ïM).
The study was done to investigate the potency of angiotensin converting enzyme (ACE) inhibitor derived from protein hydrolysate, and to identify the potent ACE inhibitory peptide of Indonesian Local animals. The study used Bali cattle, Kacang goat, Native chicken, and Local duck meat. The study consisted of two stages. First, to investigate the potency of ACE inhibitory activity of protein hydrolysate of Bali cattle, Kacang goat, Native chicken, and Local duck meat. Meat extract was made by grinding and adding water to respective meat with meat to water ratio of 1:2. Meat extract was then tested on water soluble protein and ACE inhibitory activity. Meat extract was then hydrolyzed by pepsin at pH 2 and temperature of 37.5oC for 2 hours, and was continued by typsin and chymotrypsin at pH 7 and temperature of 37.5oC for 2 hours. Hydrolizate of meat protein was then filtered using Advanced Dismic-25ES filter. Filtrate of protein hydrolysate was then tested on water soluble protein and ACE inhibitory activity. The protein profile of meat extract and protein hydrolysate were also identified by SDS-PAGE.The second stage was done to identify the highest potent ACE inhibitory peptide. ACE inhibitory peptide of Kacang goat was purified by reverse phase high performance liquid chromatography (RP-HPLC). The results of the first stage showed that water soluble protein concentration of protein hydrolysate was higher than meat extract. The water soluble protein concentration of meat extract of Bali cattle, Kacang goat, Native chicken and Local duck were 14.00; 17.13; 18.20; and 12.07 mg/mL, respectively, and the water soluble protein concentration of protein hydrolysate were 32.44; 31.57; 35.57; and 33.09 mg/mL, respectively. Molecular weight of meat extract ranged from 21 to 200 kDa, mostly they were at 45 to 65 kDa before being hydrolysed, the molecular weight of protein after being hydrolyzed by pepsin, trypsin and chymotrypsin ranged from under 6.5 to 65 kDa, and mostly they were under 6.5 to 21 kDa. ACE inhibibitory activity of protein hydrolysate of Bali cattle, Kacang goat, Native chicken and Local duck were 79.43; 80.58; 77.67; and 73.32%, respectively, and the IC50 were 341; 316; 333; and 380 ïg/mL, respectively. The results of second research stage showed that ACE inhibitory peptide had been identified. The ACE inhibitory peptide had amino acid sequence as leu-thr-glu-ala-pro-leu-asn-pro-lys-ala-arg-asn-glu-lys (LTEAPLNPKANREK) and occured at the position of 20th to 33rd in the ï¢-actin protein of goat meat (Capra hircus). The ACE inhibitory peptide had a molecular weight (MW) of 1581 (1.6 kDa) and ACE inhibitory activity (IC50) of of 190 ïg/mL (120 ïM).
Kata Kunci : Ternak lokal Indonesia, Hidrolisat protein, Peptida, Angiotensin converting enzyme, IC50.
