Sidauruk, Hotmaida (2013) Karakterisasi Asam Amino Khamir Asal Limbah Padat Pabrik Kecap sebagai Kandidat Protein Sel Tunggal (PST). Magister thesis, Universitas Brawijaya.
Abstract
Peningkatan penduduk dunia mengakibatkan meningkatnya permintaan protein untuk konsumsi manusia dan produksi hewan. Kekurangan protein menjadi masalah utama bagi manusia, sehingga sejak awal tahun 50-an telah dilakukan upaya untuk mencari sumber protein baru sebagai alternatif menggantikan protein konvensional. Sumber protein baru dapat diperoleh dari khamir, fungi, bakteri dan alga yang disebut Protein Sel Tunggal (PST). Di antara berbagai kelompok mikroorganisme yang digunakan untuk memproduksi PST, khamir adalah organisme yang paling sesuai untuk dijadikan PST karena khamir menghasilkan berbagai senyawa bioaktif seperti protein, asam amino, vitamin, polisakarida, asam lemak, fosfolipid, poliamina, astaxanthin, β -karotenoid, trehalosa, glutathion, superoksida dismutase, kitinase, amilase, phytase dan protease yang telah banyak diterima selama beberapa dekade. Penelitian ini bertujuan untuk mengetahui kandungan protein biomassa isolat khamir asal limbah padat pabrik kecap dan mengetahui karakterisik asam amino isolat khamir yang memiliki kandungan protein tertinggi dan asam nukleat terendah. Isolat khamir yang berasal dari limbah padat pabrik kecap sebagai kandidat PST diseleksi terlebih dahulu. Tahap seleksi meliputi; uji patogenitas, uji kandungan protein dan uji kandungan asam nukleat. Uji patogenitas isolat khamir dilakukan dengan menggunakan media Sheep blood agar (SBA) dan uji tabung-kecambah ( germ tube test ). Produksi biomassa isolat khamir dilakukan dalam media yeast malt broth (YMB) dengan inkubasi pada suhu 30 °C selama 48 jam. Biomassa isolat khamir disentrifugasi dengan kecepatan 5000 rpm, suhu 4 °C untuk mendapatkan pelet. Pelet khamir sebanyak satu g diekstraksi menggunakan mortar yang ditambahkan nitrogen cair dan 2 mL buffer ekstrak protein (50 mM Tris-HCl, pH 7,4, 1mM EDTA, 2 mM MgCl 2 , 2 mM DTT, 2,5 mM PMSF, 0,1% Triton –X- 100). Setelah ditambahkan buffer ekstrak, pelet khamir tersebut disentrifugasi dengan kecepatan 12.000 rpm, suhu 4 °C selama 10 menit. Supernatan berupa protein kasar diukur kadarnya menggunakan metode Bradford. Penentuan isolat khamir yang memiliki kandungan protein tinggi dilakukan produksi biomassa dengan tiga kali ulangan. Data yang diperoleh dianalisis secara statistik dengan analisis one-way Anova dan dilanjutkan dengan uji Tukey pada taraf signifikansi 5 % menggunakan software program SPSS 16.0. Kandungan asam nukleat isolat khamir diukur dengan spektrofotometer pada panjang gelombang 280 nm dan 260 nm. Data hasil analisis diintepretasikan secara kuantitatif deskriptif. Karakteristik asam amino isolat khamir dianalisis menggunakan metode separasi High Performance Liquid Chromatography (HPLC) merk Perkin-Elmer yang dilakukan di Laboratorium Sentral Ilmu Hayati Universitas Brawijaya Malang. Identifikasi biokimiawi isolat khamir menggunakan API 20 C AUX. Tujuh isolat khamir yang diuji kandungan proteinnya adalah I 2 YP 5 K 1 , I 2 YP 5 K 2 , KYP 6 K 1 , KYP 3 K 2 , AYP 6 K 1 , AYP 6 K 2 , dan AYP 5 K 4 . Analisis statistik kandungan protein hasil produksi biomassa isolat khamir menunjukkan isolat khamir KYP 3 K 2 , dan AYP 6 K 1 mempunyai kandungan protein nyata tertinggi sedangkan isolat KYP 6 K 1 mempunyai kandungan protein nyata paling terendah. Isolat AYP 6 K 2 mempunyai nilai kandungan protein diantaranya. Isolat khamir dengan kandungan protein tertinggi dan kandungan asam nukleat terendah yaitu KYP 3 K 2 dan AYP 6 K 2 dipilih untuk dianalisis profil asam aminonya dengan High Performance Liquid Chromatography (HPLC). Hasil analisis menunjukkan bahwa terdapat sembilan asam amino esensial dan tujuh asam amino non esensial dengan kadar yang berbeda-beda pada kedua isolat. Jenis asam amino esensial yang terkandung di dalam kedua isolat tersebut adalah histidin, arginin, treonin, valin, metionin, isoleusin, leusin, fenilalanin, lisin. Jenis asam amino non esensial yang terkandung di dalam kedua isolat tersebut adalah asam aspartat, asam glutamat, serin, glisin, alanin, prolin dan tirosin. Kandungan asam amino esensial yang tertinggi pada isolat khamir KYP 3 K 2 dan AYP 6 K 2 adalah lisin dengan nilai 0,953 % dan 0,924 %. Identifikasi biokimiawi menggunakan API 20 C AUX menunjukkan bahwa isolat KYP 3 K 2 merupakan Saccharomyces cerevisae dengan ID 99,7 % sedangkan AYP 6 K 2 belum teridentifikasi. Kedua isolat khamir tersebut berpotensi sebagai kandidat protein sel tunggal (PST) yang dapat digunakan untuk memenuhi kebutuhan asam amino dalam pakan ternak sebagai alternatif pengganti protein dari sumber konvensional. Isolat khamir KYP 3 K 2 merupakan Saccharomyces cerevisae sehingga isolat ini dapat dimanfaatkan sebagai feed additive untuk meningkatkan kualitas pakan unggas.
English Abstract
The increase of world population results in the increase of protein demand for human consumption and animal production. Lack of protein becomes the major problem for human beings, so since the beginning of 1950 efforts have been done to find new protein sources as alternatives to replace conventional proteins. A new protein source can be obtained from yeast, fungi, bacteria, and algae, which is called Single-Cell Protein (SCP). Among various groups of microorganisms which can be used to produce SCP, yeast is the most suitable to be made into SCP because yeast produces various bioactive compounds such as protein, amino acid, vitamins, polysaccharides, fatty acid, phospholypid, polyamines, astaxanthins, ? -carothenoid, trehalose, glutathione, superoxide dismutase, kitinase, amylase, phytase, and protease, which have been accepted for decades. This research aims at finding the protein content of yeast isolate biomass from the solid waste of ketchup factory and to know the characteristic of the amino acid of yeast isolates which have the highest protein content and the lowest nucleic acids. The yeast isolates from the solid waste of ketchup factories as SCP candidates are selected first. The selection phase includes pathogenity test, protein-content test, and nucleic acid content test. Pathogenic test of the yeast is carried out in two ways, that is using the media of Sheep Blood Agar (SBA) and germ tube test. The prodution of yeast isolate biomass is carried out in yeast malt broth (YMB) media with incubation at the temperature of 30 °C for 48 hours. The biomass of yeast isolate is centrifugated at the speed of 5000 rpm at 4 ° C to get pellets. One gram the yeast pellets is extracted by using mortar which is added liquid nitrogen and 2 mL extracted buffer of protein (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 2.5 mM PMSF, 0,1% Triton –X- 100). After the extract buffer is added, the yeast pellets are centrifugated with the speed of 12,000 rpm, at 4 oC for 10 minutes. Then content of supernatan in the form of rough protein is measured using Bradford Method. The determination of isolated yeast having the highest protein content is carried out with biomass production repeated three times. The data obtained is analyzed statisticaly with One-Way Anova Analysis and continued using Tukey test at significance degree of 5% using software program SPSS 16.0. The content of nucleic acid of yeast isolates is measured using spectrophotometer at the wavelength of 280 nm and 260 nm. The data from the analysis is interpreted in quantitative descriptive way. The characteristic of the amino acid of yeast isolates is analyzed using separation method of High Performance Liquid Chromatography (Perkin-Elmer), carried out at the Central Laboratory of Biological Sciences at Brawijaya University Malang. Biochemical identification of the yeast isolates is uses API 20 C AUX. Seven yeast isolates whose protein content are tested are I 2 YP 5 K 1 , I 2 YP 5 K 2 , KYP 6 K 1 , KYP 3 K 2 , AYP 6 K 1 , AYP 6 K 2 , and AYP 5 K 4 . Statistical analysis of the protein content from the production of yeast isolate biomass shows that yeast isolates KYP 3 K 2 and AYP 6 K 1 have the highest protein content, while isolates KYP 6 K 1 have the lowest protein content. Isolate KYP 3 K 2 has protein content between. Yeast isolate with the highest protein content and the lowest of nucleic acid content, KYP 3 K 2 and AYP 6 K 2 are chosen to be analyzed its amino acid profiles were by High Performance Liquid Chromatography (HPLC). The result of the analysis shows that there are nine essential amino acids and seven non-essential amino acids with different contents on the two isolates. The analysis of amino acid of isolated yeast KYP 3 K 2 and AYP 6 K 2 produce nine essential amino acids and seven non essential amino acids with different contents. The kinds of essential amino acid in the two isolats are histidine, arginine, treonine, valine, metionine, isoleucyne, leusine, fenilalanine, licyne. The kinds of nonessential amino acid in the two isolats are aspartat acid, glutamate acid, serine, glicyne, alanine, proline, and tyrosyne. The highest content of essential amino acid are in KYP 3 K 2 and AYP 6 K 2 with licyne of 0.953% and 0.924%. Biochemical identification using API 20 C AUX shows that KYP 3 K 2 isolates are Saccharomyces cerevisae with ID 99.7 %, while AYP 6 K 2 still unidentified. These two yeast isolates have a potency as Single-Celled Protein (SCP) which can be used to fulfil the need for amino acid in poultry feed as alternative to replace protein from conventional sources. Yeast isolate KYP 3 K 2 is Saccharomyces cerevisae , so this isolate can be utilized as feed additive to increase the quality of poultry feed.
| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | TES/572.65/SID/k/041307550 |
| Subjects: | 500 Natural sciences and mathematics > 572 Biochemistry > 572.6 Proteins |
| Divisions: | S2/S3 > Magister Biologi, Fakultas MIPA |
| Depositing User: | Endro Setyobudi |
| Date Deposited: | 13 Jan 2014 12:24 |
| Last Modified: | 13 Jan 2014 12:24 |
| URI: | http://repository.ub.ac.id/id/eprint/157685 |
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