Profil asam amino kecambah kedelai :: Keterkaitannya dengan jumlah insulin pancreas islet tikus normal dan diabetes

KANETRO, Bayu, Promotor Prof. Dr. Ir. Zuheid Noor, M.Sc

2009 | Disertasi |

Kurangnya ketersediaan energi dalam sel β pankreas untuk sekresi insulin ditengarai menjadi salah satu penyebab diabetes mellitus tipe 2 yang ditimbulkan oleh adanya gangguan sekresi insulin. Penderita diabetes mellitus tipe 2 dihadapkan pada ketergantungan OHG (Oral Hipoglycemic Drugs ) yang justru bisa memperparah penderita. Beberapa macam asam amino spesifik telah diketahui kemampuannya menstimulasi sekresi insulin atau dikenal sebagai asam amino penstimulasi insulin, yaitu meliputi leusin (Leu), arginin (Arg), alanin (Ala), fenilalanin (Phe), isoleusin (Ile), lisin (Lys), dan metionin (Met) yang dapat meningkatkan metabolisme di mitokondria pada sel β pankreas melalui jalur siklus TCA ( tricarboxylic acid). Biji kedelai telah diketahui sebagai makanan fungsional bagi penderita diabetes karena mengandung asam amino tertentu tersebut dan trypsin inhibitor , namun asam amino pada biji kedelai masih saling berikatan satu dengan yang lain membentuk makromolekul protein dan konsumsi biji kedelai yang mengandung trypsin inhibitor kemungkinan bisa mengakibatkan pembengkakan pankreas. Asam-asam amino bebas yang tidak saling berikatan diduga lebih cepat dimanfaatkan oleh sel β pankreas dalam menstimulasi sekresi insulin. Perkecambahan biji terbukti dapat meningkatkan aktivitas protease yang mendegradasi protein, sehingga kecambah kedelai yang lazim dikonsumsi segar sebagai sayur atau ”lalapan” diduga mengandung asam amino bebas penstimulasi insulin yang lebih tinggi daripada biji kedelai. Oleh karenanya penelitian ini merupakan langkah awal untuk membuktikan bahwa kecambah kedelai merupakan sumber asam amino bebas penstimulasi insulin sehingga berpotensi sebagai makanan fungsional pengganti OHG pada masa mendatang. Secara umum penelitian ini bertujuan mempelajari hubungan profil asam amino kecambah kedelai yang lazim dikonsumsi segar, dengan jumlah insulin pancreas islet tikus normal dan diabetes. Kecambah kedelai yang lazim dikonsumsi segar diperoleh melalui perkecambahan biji kedelai Varietas Sinabung selama 36 jam. Protein kecambah dan biji kedelai sebagai kontrol diisolasi dengan dua metode untuk mendapatkan presipitat protein yang mampu memerangkap asam amino bebas dalam jumlah banyak. Pertama protein diekstraksi pada pH 4 kemudian dipresipitasi pada pH 3 dan dikeringkan dengan freeze dryer sehingga diperoleh sampel presipitat protein biji dan kecambah kedelai dari metode 1. Kedua, protein diekstraksi pada pH 9 kemudian dipresipitasi pada pH 4 dan dikeringkan dengan freeze dryer sehingga diperoleh sampel presipitat protein biji dan kecambah kedelai dari metode 2. Sampel -sampel presipitat protein selanjutnya diuji aktivitas trypsin inhibitor, kadar protein total, asam amino bebas, profil asam amino total, profil asam amino bebas, dan kemampuannya menstimulasi sekresi insulin melalui pengujian biologis secara in vitro dan pengukuran jumlah insulin pancreas islet tikus normal dan diabetes dalam berbagai media sampel presipitat protein dengan Metode ELISA (Enzyme Linked Immunosorbent Assay ). Hasil penelitian ini menunjukkan bahwa asam amino bebas pada kecambah kedelai dapat diperoleh melalui isolasi protein dengan metode 2. P erkecambahan biji kedelai terbukti mampu menurunkan aktivitas trypsin inhibitor. Perkecambahan biji kedelai selama 36 jam berpengaruh terhadap profil asam amino kecambah kedelai, yang ditunjukkan dengan kenaikan dan penurunan macam serta jumlah asam amino bebas. Beberapa macam asam amino bebas yang naik adalah asam amino spesifik yang telah diketahui kemampuannya menstimulasi sekresi insulin, yaitu Ala, Leu, Lys, dan Phe dengan kenaikan berturut -turut sebesar 8,5; 3,5; 2,8; dan 1,7 kali terhadap jumlahnya pada biji kedelai. Kenaikan asam amino spesifik tersebut diikuti dengan kenaikan jumlah insulin pancreas islet tikus normal dan diabetes pada media presipitat protein kecambah yang lebih tinggi daripada media presipitat protein biji kedelai. Kenaikan jumlah in sulin dalam media presipitat protein kecambah kedelai pada pancreas islet tikus normal dan diabetes ditunjukkan dengan kenaikan absorbansi, yaitu berturut-turut mencapai 1,56 dan 1,62 kali terhadap media glukosa (kontrol). Sementara jika dibandingkan deng an media presipitat protein biji kedelai, maka kenaikan absorbansi pada media presipitat protein kecambah sebesar 1,22 kali pada pancreas islet tikus normal maupun diabetes. Kenaikan jumlah insulin ini lebih disebabkan oleh kenaikan kadar asam amino bebas penstimulasi insulin daripada asam amino total penstimulasi insulin, yang dibu ktikan pada jumlah asam amino bebas penstimulasi insulin dalam media presipitat protein kecambah naik secara nyata sebesar 2,19 kali terhadap media presipitat protein biji kedelai. Sedangkan jumlah asam amino total penstimulasi insulin -nya tidak berbeda nyata. Hal ini menunjukkan bahwa potensi asam amino bebas pada kecambah kedelai, khususnya Ala, Leu, Lys, dan Phe untuk menstimulasi sekresi insulin terbukti lebih tinggi dari pada asam amino yang masih saling berikatan satu dengan yang lain membentuk makro molekul protein.

There are many causes of type 2 diabetes, among them is impaired –insulin secretion. It is due to the lack of energy for insulin secretion in pancreatic β-cell. The patients must control their blood glucose by OHG (Oral Hipoglycemic Drugs ), but they get harmful effects for the body. The specific amino acids (leucine/Leu, arginine/Arg, alanine/Ala, phenylalanine/Phe, isoleucine/Ile, lysine/Lys, and methionine/Met), that were known as insulin stimulation, could activate mitochondrial metabolism in pancreatic β-cell via TCA (tricarboxylic acids) cycle. Soybeans had been known as functional food for diabetic, because they had the specific amino aci ds and trypsin inhibitor. However the availability of amino acids in soybean naturally combined with each other to make long chains in the form of protein -macromolecule complex, and the consumption of soybeans that contain trypsin inhibitor may cause panc reatic hyperthropy. The acceleration of amino acids in the form of macromolecule to be utilized by pancreatic β-cell may be lower than freeamino acids. In fact, seed germination increased protease activity that could degrade protein. The free amino acids particularly the specific amino acids as insulin stimulation of soybean sprouts, that are usually consumed as fresh vegetables (”lalapan”), may be higher than the original seed. Therefore, this research is preliminary study to prove that the soybean sprout is potential as functional food and for the future it will be consumed by diabetes patients in o rder to stop using of OHG. The aim of this research was generally to study a relationship between the amino acids profile of soybean sprout, that are ussually consumed as fresh vegetables, with the amount of insulin of pancreas islet of normal dan diabetic rats. The soybean sprouts , that are ussually consumed as fresh vegetables, were obtained through seed germination of Sinabung -Soybean Variety for 36 h. The protein of soybean sprout and soybean seed as a control were isolated by 2 methods to get protein precipitate that could trap a lot of free amino acids. First, protein was extracted at pH 4 and then precipitated at pH 3 and dried by freeze dryer that resulted samples of seed and sprout protein precipitate from 1 st method. Second, protein was extract ed at pH 9 and then precipitated at pH 4 and dried by freeze dryer that resulted samples of seed and sprout protein precipitate from 2 nd method. Subsequently, the samples were analyzed for trypsin inhibitor activity, total protein, free amino acids, total amino acids profile, free amino acids profile, and the potency of stimulation of insulin secretion by in vitro bioassay and the insulin was determined by ELISA method after incubation of pancreas islet of normal and diabetes rats in various media of prote in precipitates. The results showed that free amino acids in soybean sprouts could be got by 2 nd method of protein isolation. Soybean seed germination could decrease trypsin inhibitor activity. The analysis of amino acids profile showed that some kind o f free amino acids in 1 soybean sprout increased, and some kind of amino acids decreased significantly, compared to the original seeds. Soybean seed germination for 36 h increased the specific free amino acids, particularly Ala, Leu, Lys, and Phe that was known as stimulation of insulin secretion, which were 8.5; 3,5; 2.8; and 1.7 times respectively compared to the specific free amino acids of the original seeds. The increases in the specific amino acids were followed by increasing of insulin of pancreas islet of normal and diabetes rats in the media of protein precipitate from soybean sprout, which were higher than the media of protein precipitate from soybean seed. The insulin of pancreas islet of normal and diabetes rats in the media of protein precipi tate from soybean sprout increased that were indicated by increasing the absorbance 1.56 and 1.62 times respectively compared to the media of glucose (control). On the other hand, the insulin of pancreas islet of normal and diabetes rats in the media of protein precipitate from soybean sprout increased that was indicated by increasing the absorbance 1.22 times compared to the media of protein precipitate from soybean seed. There was a relationship between the increase in the insulin with the increases in the free amino acids as insulin stimulation, that was indicated by the free amino acids as insulin stimulation in the media of protein precipitate from soybean sprout increased significantly, that was 2.19 times compared to the media of protein precipita te from soybean seed. While, the total amino acids were no significant different between soybean sprout and seed. These result indicated that the potency of free amino acids in soybean sprout particularly Ala, Leu, Lys, and Phe for the stimulation of insul in secretion were proven higher than amino acids in the form of protein macromolecule.

Kata Kunci : Kecambah kedelai,Asam amino bebas,Asam amino penstimulasi insulin,Pancreas islet