Peptida Bioaktif Hasil Hidrolisis Enzimatis dari Kolagen Kulit Tuna (Thunnus obesus): Kombinasi Studi In Vitro dan In silico

Abstract

Kulit tuna bigeye (Thunnus obesus) dimanfaatkan dalam penelitian ini dalam memperoleh kolagen dan peptida kolagen. Penelitian ini bertujuan mengkaji peptida bioaktif hasil hidrolisis enzimatis kolagen kulit tuna bigeye melalui metode in vitro dan in silico. Penelitian ini terdiri atas empat tahap. Tahap 1 yaitu ekstraksi kolagen dari kulit tuna bigeye menggunakan asam asetat encer dengan dan tanpa protease (bromelin, papain, pepsin, tripsin) sampai diperoleh kolagen kering. Rendemen kolagen dihitung berdasarkan basis kering untuk acid soluble collagen (ASC), bromelain soluble collagen (BSC), papain soluble collagen (PaSC), pepsin soluble collagen (PSC), dan trypsin soluble collagen (TSC). Kolagen dengan dua rendemen tertinggi dipelajari lebih lanjut karakteristik kimia dan aktivitas antioksidannya. Kulit sebagai sumber kolagen dianalisis terlebih dahulu. Hasil menunjukkan bahwa, kulit memiliki kandungan protein yang tinggi (65,42% (bk) dari total kadar kulit), bebas dari bahaya logam berat (As, Cd, Co, Cr, Cu, Pb) dan tidak ada kandungan histamin. Kulit yang diekstrak kolagennya adalah kulit liofilisasi selama 14 jam. Rasio kulit liofilisasi adalah sebesar 50,06% dari bobot kulit basah tertentu. Hasil ekstraksi kolagen menunjukkan PSC (yakni 52,02%, bk) dan BSC (yakni 42,76%, bk) adalah kolagen dengan rendemen tertinggi. PSC dan BSC ditemukan sebagai kolagen tipe I berdasarkan analisis SDS-PAGE dan FTIR. Mereka larut sangat baik dalam dimetil sulfoksida, yakni 73,48 dan 76,30 %, masing-msaing untuk BSC dan PSC. Kisaran pH adalah 4,60–4,70 dan 4,30–4,40 masing-masing untuk PSC dan BSC. PSC dan BSC bebas dari As, Cd, Co, Cr, Cu, dan Pb. Keduanya menunjukkan aktivitas antioksidan dengan metode DPPH dan metode daya reduksi. Tahap 2 yaitu preparasi dan uji aktivitas antioksidan dari hidrolisat dan fraksi peptida. Hidrolisat diperoleh melalui hidrolisis lanjut kolagen rendemen tertinggi (PSC dan BSC) menggunakan enzim alcalase, neutrase, dan savinase. Hasil hidrolisis adalah enam hidrolisat, yaitu: HBA, HBN, dan HBS, dari hidrolisis BSC masing-masing dengan alcalase, neutrase, dan savinase; serta HPA, HPN, dan HPS, dari hidrolisis PSC masing-masing dengan alcalase, neutrase, dan savinase. Fraksi peptida diperoleh melalui semi-purifikasi hidrolisat menggunakan tabung ultrafiltrasi 10 dan 3 kDa. Tiga fraksi peptida (fraksi peptida > 10, 3−10, dan < 3 kDa) diperoleh untuk setiap hidrolisat, sehingga diperoleh 18 fraksi peptida. Fraksi peptida tersebut adalah fraksi PBA, PBN, dan PBS masing-masing merupakan fraksi ultrafiltrasi dari HBA, HBN, dan HBS; serta fraksi PPA, PPN, dan PPS masing-masing merupakan fraksi ultrafiltrasi dari HPA, HPN, dan HPS. Semua hidrolisat dan fraksi peptida memperlihatkan aktivitas antioksidan menggunakan metode scavenging terhadap radikal DPPH. Dua aktivitas antioksidan tertinggi diperoleh dari fraksi PBN dan PPS < 3 kDa, masing-masingnya yaitu 23,95 dan 17,65 mg AAE/ g protein, atau 135,40 dan 99,78 µmol AAE/ g protein. Aktivitas antioksidan hidrolisat dan fraksi peptida yang diperoleh dikaji lebih lanjut dalam Tahap 3 dan Tahap 4. Tahap 3, aktivitas antioksidan dipelajari sebagai pengaruh dari jenis kolagen (BSC, PSC), enzim penghidrolisis kolagen (alcalase, neutrase, savinase), dan ukuran molekul (hidrolisat, fraksi peptida > 10 kDa, 3−10 kDa, dan < 3 kDa). Pengaruh faktor-faktor ini pada aktivitas antioksidan dinyatakan sebagai profile plots. Terlihat bahwa dalam menghasilkan aktivitas antioksidan: PSC lebih baik dibandingkan dengan BSC; savinase paling baik dibandingkan dengan neutrase dan alcalase; dan untuk fraksi peptida, fraksi peptida kecil lebih baik daripada fraksi peptida besar. Tahap 4, yaitu analisis sekuens asam amino, prediksi bioaktivitas dan penentuan sifat penciri dari peptida dalam fraksi PBN dan PPS < 3 kDa. Analisis sekuens dilakukan mengunakan LC-MS/MS. Prediksi sifat bioaktif dipelajari menggunakan aplikasi yang tersedia pada biopep website (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep). Penentuan sifat penciri dipelajari menggunakan aplikasi yang tersedia pada PepDraw website (http://www.tulane.edu/~biochem/WW/PepDraw/). Satu peptida dengan panjang residu 23 asam amino teridentifikasi dalam fraksi PBN < 3 kDa, dan 12 peptida dengan panjang residu 10-20 asam amino teridentifikasi dalam fraksi PPS < 3 kDa, menurut hasil analisis LC-MS/MS. Hasil in silico dengan program Biopep menghasilkan 16 peptida bioaktif untuk fraksi PBN < 3 kDa dan 83 peptida bioaktif untuk fraksi PPS < 3 kDa. Peptida bioaktif yang teridentifikasi dalam fraksi PBN < 3 kDa adalah peptida antidiabetes 81,25%, peptida antihipertensi 56,25% dan peptida antioksidan 6,25%. Serta peptida bioaktif yang teridentifikasi dalam fraksi PPS < 3 kDa adalah peptida antidiabetes 73,49%, peptida antihipertensi 61,45%, peptida antioksidan 6,02% dan 15,66% peptida lainnya (chemotactic, antithrombotic, antiamnestic). Peptida antidiabetes yang teridentifikasi memiliki aktivitas sebagai inhibitor alfa-glucosidase, inhibitor dipeptidyl peptidase III, dan inhibitor dipeptidyl peptidase IV. Peptida antihipertensi teridentifikasi sebagai inhibitor ACE. Peptida-peptida bioaktif dalam fraksi PBN dan PPS < 3 kDa umumnya mempunyai ujung N dari asam amino non polar (hidrofobik). Beberapa kebaharuan (novelty) dihasilkan dalam penelitian ini. Yakni: (1) ekstraksi kolagen kulit tuna bigeye dapat dilakukan secara efektif dengan menambahkan bromelin ke dalam asam asetat encer; (2) hidrolisat kolagen kulit tuna bigeye hasil hidrolisis enzimatis menggunakan savinase, dan neutrase dapat meningkatkan aktivitas antioksidan; (3) jenis kolagen, enzim penghidrolisis kolagen, dan ukuran molekul mempengaruhi aktivitas antioksidan yang dihasilkan; (4) sekuens asam amino, bioaktivitas, dan sifat penciri dari peptida bioaktif dalam fraksi ultrafiltrasi dari kolagen kulit tuna bigeye diketahui. Kesimpulan dari penelitian ini adalah: (1) penggunaan kombinasi asam asetat dan enzim protease meningkatkan hasil ekstraksi kolagen kulit tuna bigeye, dengan rendemen tertinggi diperoleh dengan penambahan pepsin dan bromelin; (2) PSC dan BSC dikonfirmasi sebagai kolagen tipe I; (3) Hidrolisis lanjut menggunakan alcalase, neutrase dan savinase, serta fraksinasinya menghasilkan antioksidan yang lebih baik, dan terbaik pada fraksi PBN dan PPS < 3 kDa; (4) Jenis kolagen, enzim, dan ukuran molekul berpengaruh terhadap aktivitas antioksidan; (5) Uji in silico menunjukkan peptida dalam fraksi PBN dan PPS < 3 kDa memiliki potensi beberapa bioaktivitas, termasuk sebagai antioksidan. Peptida bioaktif umumnya mempunyai ujung N dari asam amino non polar (hidrofobik).

The skin of bigeye tuna (Thunnus obesus) was used in this study to obtain collagen and collagen peptides. The objectives of this research were to study the bioactive peptides resulting from enzymatic hydrolysis of bigeye tuna skin collagen through in vitro and in silico methods. This research consisted of four steps. Step 1 was collagen extraction from bigeye tuna skin using dilute acetic acid with and without protease (bromelain, papain, pepsin, trypsin) until dry collagens were obtained. Collagen yields were calculated on a dry basis for acid soluble collagen (ASC), bromelain soluble collagen (BSC), papain soluble collagen (PaSC), pepsin soluble collagen (PSC), and trypsin soluble collagen (TSC). Collagens with the two highest yields were further studied for their chemical properties and antioxidant activities. The skin as a source of collagen was analyzed first. The results showed that, the skin had a high protein content (65.42% (db) of the total skin content), was free from the dangers of heavy metals (As, Cd, Co, Cr, Cu, Pb) and no histamine content was detected. The skin used was 14 h lyophilized skin. The lyophilized skin ratio was 50.06% from the certain weights of wet skin. The results of collagen extraction showed that PSC (namely 52.02%, db) and BSC (namely 42,76%, db)were the collagen with the highest yield. PSC and BSC were found as type I collagens based on SDS-PAGE and FTIR analysis. They dissolved very well in dimethyl sulfoxide, ie 73.48 and 76.30%, for BSC and PSC, respectively. The pH ranges were 4.60–4.70 and 4.30–4.40 for PSC and BSC, respectively. PSC and BSC were free from As, Cd, Co, Cr, Cu, and Pb. Both showed antioxidant activity with the DPPH method and the reducing power method. Step 2 was the preparation and the antioxidant activity test of hydrolysate and peptide fraction. The hydrolysates were obtained through further hydrolysis of the highest yield collagen (PSC and BSC) using the enzymes alcalase, neutrase, and savinase. The results of hydrolysis were six hydrolysates, namely: HBA, HBN, and HBS, from the hydrolysis of BSC with alcalase, neutrase, and savinase, respectively; and HPA, HPN, and HPS, from PSC hydrolysis with alcalase, neutrase, and savinase, respectively. The peptide fractions were prepared by semi-purification of hydrolysate using 10 and 3 kDa of ultrafiltration tubes. Three peptide fractions (peptide fractions > 10, 3–10, and < 3 kDa) were obtained for each hydrolysate, so that 18 peptide fractions were obtained. The peptide fractions were the PBA, PBN, and PBS fractions, respectively, the ultrafiltration fractions of HBA, HBN, and HBS; as well as the PPA, PPN, and PPS fractions, respectively, the ultrafiltration fractions of HPA, HPN, and HPS. All hydrolysates and peptide fractions showed antioxidant activities by the scavenging method against DPPH radicals.. The two highest antioxidant activities were obtained from PBN and PPS fractions < 3 kDa, namely 23.95 and 17.65 mg AAE / g protein, or 135.40 and 99.78 µmol AAE / g protein, respectively. The antioxidant activities of the hydrolysates and peptide fractions obtained were studied further in Step 3 and Step 4. Step 3, antioxidant activities were studied as the influence of collagen type (BSC, PSC), enzymes used in hydrolyzing collagen (alcalase, neutrase, and savinase), and molecular size (hydrolysate, peptide fractions > 10 kDa, 3–10 kDa, and < 3 kDa). The effect of these factors on antioxidant activities was expressed as profile plots. It appeared that in producing antioxidant activities: PSC was better than BSC; savinase had the best result compared to neutrase and alcalase; and for peptide fractions, small peptide fraction was better than large peptide fraction. Step 4, namely the analysis of amino acid sequences, prediction of bioactivity and determination of the characteristics of peptides in the PBN and PPS fractions < 3 kDa. Sequence analysis was performed using LC-MS/MS. The characteristics were studied using an application available on the PepDraw website (http://www.tulane.edu/~biochem/WW/PepDraw/). The prediction of bioactive properties was studied using an application available on the biopep website (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep). One peptide with a residue length of 23 amino acids was identified in the PBN fraction < 3 kDa, and 12 peptides with a residue length of 10-20 amino acids were identified in the PPS fraction < 3 kDa, according to the results of LC-MS/MS analysis. The in silico results with the Biopep program produced 16 bioactive peptides for the PBN < 3 kDa fraction and 83 bioactive peptides for the PPS < 3 kDa fractions. The bioactive peptides identified in the PBN < 3 kDa fraction were 81.25% antidiabetic peptide, 56.25% antihypertensive peptide and 6.25% antioxidant peptide. The bioactive peptides identified in the PPS < 3 kDa fractions were 73.49% antidiabetic peptides, 61.45% antihypertensive peptides, 6.02% antioxidant peptides and 15.66% other peptides (chemotactic, antithrombotic, antiamnestic). The identified antidiabetic peptides had activities as alpha-glucosidase inhibitors, dipeptidyl peptidase III inhibitors, and dipeptidyl peptidase IV inhibitors. Antihypertensive peptides were identified as ACE inhibitors. The bioactive peptides in the PBN and PPS fractions < 3 kDa generally had the N terminal of a non-polar (hydrophobic) amino acid. Several novelties were produced in this study. Namely: (1) bigeye tuna skin collagen extraction can be done effectively by adding bromelain to dilute acetic acid (2) collagen hydrolysates of bigeye tuna skin resulted from enzymatic hydrolysis using savinase and neutrase can increase antioxidant activity; 3 collagen type, collagen-hydrolyzing enzymes and molecular sizes affect the antioxidant activity produced; (4) amino acid sequences, bioactivity, and characterizing properties of bioactive peptides in the ultrafiltration fraction of bigeye tuna skin collagen were known. The conclusions of this research were: (1) the use of a combination of acetic acid and protease enzymes increased the yield of bigeye tuna skin collagen extraction, with the highest yield obtained by the addition of pepsin and bromelin; (2) PSC and BSC were confirmed as collagen type I; (3) Further hydrolysis using alcalase, neutrase and savinase, and their fractionation produced better antioxidants, and the best at the PBN and PPS fractions < 3 kDa; (4) The type of collagen, enzymes, and molecular size had effects on antioxidant activities; (5) The in silico test showed that the peptides in the PBN and PPS fractions < 3 kDa had the potential for several bioactivities, including as antioxidative. Bioactive peptides generally had the N terminal of a non-polar (hydrophobic) amino acid.