KARAKTERISASI PEDIOSIN PaF-11 DARI Pediococcus acidilactici F-11 DAN GEN PENYANDINYA

TRI MARWATI, Prof. Dr. Ir. Endang Sutriswati Rahayu, MS.,

2013 | Disertasi | S3 Ilmu Pangan

Pediosin PaF-11 dari Pediococcus acidilactici F-11 berpotensi sebagai pengawet pangan karena kemampuannya dalam mengendalikan pertumbuhan bakteri pembusuk dan patogen pangan, akan tetapi penggunaannya masih terbatas. Guna mendukung pengembangan aplikasi pediosin PaF-11 maka dilakukan penelitian yang bertujuan untuk (1) meningkatkan efektivitas purifikasi pediosin PaF-11 melalui perlakuan variasi pH adsorpsi desorpsi dan konsentrasi penambahan massa sel mati P. acidilactici F-11 pada proses adsorpsi (2) mengetahui karakter pediosin PaF-11 meliputi stabilitas terhadap pH, suhu tinggi dan selama penyimpanan (3) mengetahui karakter molekuler pediosin PaF-11 meliputi lokasi dan sekuen nukleotida gen penyandi, serta sekuen asam amino dan berat molekul dan (4) mengetahui mekanisme awal penghambatan pediosin PaF-11 sebagai antibakteri. Pada penelitian ini digunakan P. acidilactici F-11 dan Lactobacillus pentosus LB42 berturut turut sebagai bakteri penghasil dan indikator uji aktivitas pediosin PaF-11, yang keduanya diperoleh dari Food and Nutrition Culture Collection (FNCC), Pusat Studi Pangan dan Gizi Universitas Gadjah Mada Yogyakarta. Produksi pediosin PaF-11 dilakukan menumbuhkan P. acidilactici F-11 pada media TGE cair (trypticase [1%], glucose [1%],yeast extract [1%], tween 80 [0.2%], Mn2+ [0.033 mM], Mg2+ [0.02 mM] pH 6,5 dan inkubasi selama 18 jam pada suhu 37°C. Purifikasi parsial pediosin PaF-11 dilakukan dengan metode adsorpsi desorpsi dan aktivitasnya diuji dengan metode difusi agar sumur. Penelitian ini dilakukan dalam empat percobaan: (1) Purifikasi parsial pediosin PaF-11 dengan perlakuan variasi pH adsorpsi desorpsi dan konsentrasi massa sel mati. Penentuan hasil terbaiknya dengan menguji aktivitas pediosin PaF-11 yang dihasilkan, (2) Karakterisasi pediosin PaF-11 terhadap parameter yang mengarah pada aplikasinya sebagai pengawet pangan, meliputi stabilitas terhadap suhu, pH dan penyimpanan, (3) Karakterisasi molekuler, meliputi penentuan lokasi dan sekuen nukleotida gen penyandi pediosin PaF-11, sekuen asam amino dan berat molekul pediosin PaF-11, (4). Studi mekanisme awal penghambatan padiosin PaF-11 sebagai antibakteri. Pada percobaan ini dipelajari pengaruh gadolinium terhadap aktivitas pediosin PaF-11 dan kadarnya pada dinding sel target serta pengaruhnya terhadap morfologi sel target. Hasil penelitian ini menunjukkan bahwa efektivitas purifikasi pediosin PaF11 dipengaruhi oleh pH. Aktivitas pediosin PaF-11 hasil purifikasi pada pH adsorpsi 6,5 dan pH desorpsi 2,0 mempunyai nilai 1500AU/ml. Penambahan massa sel mati pada proses adsorpsi sebanyak 3 kali konsentrasi awal dapat meningkatkan efektivitas purifikasi, yaitu dengan nilai aktivitas 3000AU/ml. Akan tetapi apabila dilihat dari aktivitas total pediosin yang terdapat pada kultur yaitu 6000 AU/ml, maka purifikasi dengan kedua cara tersebut masih dapat dioptimalkan. Pediosin PaF-11 dengan aktivitas 1500 AU/ml memiliki stabilitas terhadap pemanasan pada suhu 100C selama 30 menit maupun suhu 121C selama 15 menit dan selama 11 dan 13 minggu penyimpanan pada suhu ruang (30 o C) dan lemari pendingin (4 o C). Selain itu, aktivitas pediosin PAF-11 stabil pada kisaran pH yang cukup luas yaitu dari pH 3 sampai 8. Karakteristik tersebut membuat pediosin PaF11 sesuai digunakan sebagai pengawet pangan terutama produk yang melibatkan proses pengolahan pada pH yang beragam seperti dalam proses fermentasi dan pengolahan pada suhu tinggi serta penyimpanan. Pediosin PaF-11 memiliki gen penyandi yang berada dalam plasmid. Hal ini ditunjukkan oleh hilangnya aktivitas pediosin PaF-11 dan tidak terdeteksinya DNA aplikon setelah P. acidilactici F11 mengalami proses curing. Hasil elektroforesis agarose DNA plasmid P. acidilactici F-11 dengan marker λDNA/HindIII menunjukkan bahwa gen P. acidilactici F-11 terbawa dalam plasmid 12 kb. Amplifikasi gen pediosin PaF-11 dari P. acidilactici F-11 menunjukkan bahwa kultur P. acidilactici F-11 mengandung DNA plasmid, diindikasikan dengan gen papA (256 bp). Sekuen nukleotida gen penyandi pediosin PaF-11 tersebut sebagai berikut : atgaaaaaaa ttgaaaaatt aactgaaaaa gaaatggcca atatcattgg tggtaaatac tacggtaatg gggttacttg tggcaaacat tcctgctctg ttgactgggg taaggctacc acttgcataa tcaataatgg agctatggca tgggctactg gtggacatca aggtaatcat aaatgctag. Penjajaran sekuen nukleotida tersebut dengan sekuen nukleotida gen penyandi pediosin PA.1 (pedA) pada P. acidilactici PAC1.0; prepediosin pada P. acidilactici K10; prepediosin CP2 pada P. acidilactici MTCC 5101; pediosin AcH (papA) pada P. acidilactici H plasmid pSMB74 dan P. acidilactici LB 42-923 menunjukkan homologi yang tinggi (100%). Dari nukelotida tersebut dihasilkan pediosin PaF-11 dengan sekuen sebagai berikut : MKKIEKLTEKEMANIIGGKYYGNGVTCGKHSCSVDWGKATTCIINNG AMAWATGGHQGNHKC. Dari sekuen tersebut terlihat bahwa pediosin PaF-11 berada dalam konsensus sekuen YYGNGVXCXXXXCXVXXXXA yang mengindikasikan bahwa pediosin PaF-11 merupakan bakteriosin klas IIa. Sekuen tersebut memiliki homologi yang tinggi (100%) dengan pediosin PA-1 (dari P. acidilactici PAC 1.0; P. pentosaceus IE-3); prepediosin PA-1 (dari P. acidilactici K10); pediosin AcH (dari P. acidilactici H; P. acidilactici LB42-923 ; P. parvulus ATO77; P. pentosaceus S34) dan prepediosin CP2 dari P. acidilactici MTCC 5101 dan pediosin dari P. acidilactici CFR K7 dan P. Acidilactici PED 01. Dari sekuen asam amino tersebut, diketahui bahwa pediosin PaF-11 memiliki berat molekul 6,65 kDa. Berdasarkan densitas optik dan morfologi sel indikator serta kadar ion gadolinium (Gd3+) pada dinding sel diketahui bahwa gadolinium berpengaruh terhadap aktivitas pediosin. Ion gadolinium (Gd3+) diduga mengikat muatan negatif pada reseptor untuk pediosin PaF-11. Adanya kontak tersebut menyebabkan terbentuknya tonjolan pada dinding sel bakteri yang dihambat Dengan demikian maka diduga mekanisme awal penghambatan pediosin PaF-11 sebagai antibakteri yaitu melalui kontak molekul pediosin PaF-11 dengan reseptor asam teikoat dan asam lipoteikoat yang bermuatan negatif.

Pediocin PaF-11 produced by Pediococcus acidilactici F-11 has potency as a food preservative because of its ability in controlling the growth of food spoilage and pathogene bacteria, but its use is still limited. To support the application development of pediocin PaF-11, research that aims to (1) increase the effectiveness of pediocin PaF-11 purification through pH adsorption and desorption treatment and the addition of biomass of heat killed cells of P. acidilactici F-11 during adsoprtion (2) know the characters of pediocin PaF-11 include the stability of the pH, temperature and storage conditions (3) know the molecular character of pediocin PaF-11 includes a nucleotide sequence and the location of pediocin PaF-11 encoding gene, as well as the sequence of amino acids and the molecular weight and (4) find out the initially mechanism of pediocin PaF-11 inhibitation as an antibacterial, were conducted. P. acidilactici F-11 and Lactobacillus pentosus LB42 were used as pediocin PaF-11 producer and indicator bacteria respectivelly, both of which were obtained from the Food and Nutrition Culture Collection (FNCC), Center for Food and Nutrition Studies of Gadjah Mada University in Yogyakarta. P. acidilactici F-11 produced pediocin PaF-11 in the TGE broth (trypticase [1%], glucose [1%], yeast extract [1%], tween 80 [0.2%], Mn2+ [0.033 mM], Mg2+ [0.02 mM] of pH 6.5 within 18 hours incubation at 37 °C. Partial purification of the pediocin PaF-11 was performed by the adsoprtion desorption methods and its activity was determined by well diffusion agar methods. This research was conducted in four experimens: (1) Partial purification of pediocin PaF-11 with treatment variation in pH adsorption desorption and concentarion of heat killed cells (2) Characterization of pediocin PaF-11 parameters that lead to its application as a food preservative, include stability in temperature, pH and storage (3) Molecular characterization, including determination of the nucleotide sequence and the location of pediocin PaF-11 encoding gene, the amino acid sequence and molecular weight of pediocin PaF-11. (4) Study the initially mechanism of pediosin PaF-11 inhibition as an antibacterial, by analysis the influence of gadolinium toward activity of pediocin PaF-11 and the content of Gd3+ on the cell wall as well as its influence on the morphology of the target cell. Pediocin PaF-11 activity produced by P. acidilactici F-11 and purified at pH adsorption 6,5 and desorption 2,0 was 1500AU/ml. Pediocin PaF-11 activity obtained from purification without addition of heat killed cells was 1500AU/ml, while by addition of heat killed cells of 3, 6 and 11 times of the original concentrations were 3000AU/ml. Therefore it was suggested that addition of heat killed cells of P. acidilactici F-11during adsorption with 3 times of original concentration was able to increase the pediocin PaF-11 obtained. Total activity of the pediocin PaF-11 found in the culture was 6000 AU/ml. This means that the effectiveness of the pediocin PaF-11 purification with the both methods can still be optimized. Pediocin PaF-11 with activity of 1500 AU/ml, was stable after heated at 100°C for 30 minutes and 121°C for 15 minutes, and during 11 and 13 weeks storage at room temperatur (30 o C) and refrigerator (4 o C). Pediocin PaF-11 was found to be stable over a wide pH range between 3 and 8. These characteristics make pediocin PaF-11 suitable as a food preservative, especially products that involve processing on pH such as in the process of fermentation and processing at high temperatures as well as storage. Pediocin PaF-11 from the cured cell of P. acidilactici F-11 loss the activity, suggested that the pediocin PaF-11 gene was carried in the plasmid. Agarose gel electrophoresis of P. acidilactici F-11 plasmid DNA with marker λDNA/HindIII showed that pediocin PaF-11 gene was carried in 12 kb plasmid. Amplification pediocin PaF-11 gene from P. acidilactici F-11 showed that uncured P. acidilactici F-11 culture contain plasmid DNA, indicated by amplification of the papA gene (256 bp). Cured P. acidilactici F-11 culture, plasmid eliminated, indicted by no aplicon DNA detected. This result also suggested that pediocin PaF-11 gene in P. acidilactici F-11 was carried in plasmid. Nucleotide of pediocin PaF-11 encoding gene was sequenced : atgaaaaaaa ttgaaaaatt aactgaaaaa gaaatggcca atatcattgg tggtaaatac tacggtaatg gggttacttg tggcaaacat tcctgctctg ttgactgggg taaggctacc acttgcataa tcaataatgg agctatggca tgggctactg gtggacatca aggtaatcat aaatgctag. The alignment of that sequence with the nucleotide sequence of pediocin PA.1 encoding gene (pedA) in P. acidilactici PAC1.0; prepediocin encoding gene in P. acidilactici K10; prepediocin CP2 encoding gene in P. acidilactici MTCC 5101; and pediocin AcH encoding genes (papA) in P. acidilactici H and P. acidilactici LB 42-923 showed the similarity percentage of 100%. From that nucleotide sequence can be generated pediocin PaF-11 with the following sequence : MKKIEKLTEKEMANIIGGKYYGNGVTCGKHSCSVDWGKATTCIINNGA MAWATGGHQGNHKC. The pediocin PaF-11 is in the consensus sequence YYGNGVXCXXXXCXVXXXXA which indicates that pediocin PaF-11 is belong to class IIa bacteriocin. The sequence of pediocin PaF-11 has a high homology (100%) with pediocin PA-1 (from P. acidilactici PAC 1.0 and P. pentosaceus IE-3); prepediocin PA-1 (from P. acidilactici K10); pediocin AcH (from P. acidilactici H; P. acidilactici LB42-923; P. parvulus ATO77 and P. pentosaceus S34) and prepediocin CP2 from P. acidilactici MTCC 5101 and pediocin from P. acidilactici CFR K7 and P. acidilactici PED 01. Based on that sequence, the pediocin PaF-11 has a molecular weight of around 6.65. kDa. Based on the optical density and the morphology of cell indicators as well as the content of gadolinium ion (Gd3+) on the cell wall revealed that gadolinium effect on the activity of pediocin PaF-11. Ion gadolinium (Gd3+) is thought to bind to receptors on the negative charge of pediocin PaF-11. The existence of these contacts led to the formation of the blebs on the cell walls of bacteria are inhibited thus suspected initially inhibitory mechanism pediocin PaF-11 as antibacterial i.e. through contact of the molecules of pediocin PaF-11 with teichoic acid receptors and lipoteichoic acids which are negatively charged.

Kata Kunci : pengawet makanan, antibakteri