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Penelitian ini bertujuan untuk melakukan ekstraksi gelatin dari tulang ikan patin menggunakan asam sitrat dan menganalisis karakteristik fisiko-kimianya. Ekstraksi gelatin melalui dua tahap yaitu tahap pre-treatment dengan asam sitrat dan ekstraksi utama dengan aquadest. Tahap pre-treatment dilakukan dengan variasi waktu yakni 24, 36, 48 dan 56 jam. Tahapan ekstraksi utama dilakukan pada suhu 45, 55, 65, dan 75°C. Hasil ekstraksi dilanjutkan dengan analisis keberadaan protein gelatin dengan metode SDS-PAGE. Analisis fisiko-kimia gelatin meliputi derajat keasaman (pH), rendemen, kekuatan gel, profil tekstur, viskositas, kadar air, kadar abu, kadar protein dan kadar lemak juga dilakukan. Hasil SDS-PGE gelatin perlakuan terbaik (yakni dengan pre-treatment 48 jam dan ekstraksi utama pada suhu 75°C) diketahui memiliki bobot molekul 162 kDa. Gelatin hasil ekstraksi terbaik memiliki rendemen sebesar 6,14%. Gelatin tulang ikan patin memiliki pH 4,46, kekuatan gel 364,19 bloom, daya kunyah sebesar 261,76 g dan viskositas 3,83 cP. Kadar proksimat gelatin tulang ikan patin meliputi kadar air, kadar abu, kadar protein dan kadar lemak masing-masing 7,72; 0,38; 58,70, dan 2,79%. Hasil penelitian juga menunjukan bahwa semakin tinggi suhu ekstraksi dan lamanya proses pre-treatment yang digunakan, maka bobot molekul protein menjadi semakin tinggi pula. Kesimpulannya, pre-treatment asam sitrat selama 48 jam dan ekstraksi utama pada suhu 75°C telah berhasil menunjukkan hasil yang paling baik dan dapat terkarakterisasi sifat fisik serta kimianya. Physical and Chemical Characteristics of Gelatin from Pangasius Catfish Bone with Pre-Treatment of Citric Acid This research aimed to extract gelatin from Pangasius catfish bone using citric acid and then analyse its physicochemical characteristics. The extraction of gelatin was done in two stages soaking i.e pre-treatment with citric acid and main extraction by aquadest. Pre-treatment was done by soaking bones at 24, 36, 48 and 56 h, then the main extraction was done by soaked leached bones in temperature 45, 55, 65, and 75°C. The gelatin from catfish bone then was analyzed the presence of protein by SDS-PAGE and its physico-chemical including pH, yield, gel strength, texture profile, viscosity, moisture content, ash content, protein content, and fat content. Based on SDS-PAGE, fish bone gelatin had molecular weight of 162 kDa. The best extraction treatment (pre-treatment 48 h and main extraction at 75°C) produced 6.14% of gelatin yield. Gelatin of pangasius catfish bone had pH 4.46, gel strength of 353.76 bloom, chewing power of 261.76 g, and viscosity of 3.83 cP. Water, ash, protein and fat content was 7.72, 0.38, 58.70, and 2.79%, respectively. The higher extraction temperature and the longer time for pre-treatment process, the higher molecule weight of protein. As conclusion, the best treatment that was found in the sample with the pre-treatment at 48 h and the main extraction at 75˚C, was successfully characterized on its physicochemicals. •|•|•|•