Pembentukan Fibril Amiloid dari Isolat Protein, Globulin 7S dan 11S Kacang Bogor [Vigna subterranea (L.) Verdc.]

Abstract

One of the alternatives to enhance the benefits of Bogor nut which had examined in this dissertation is modifying nut protein to be functioned as a food ingredient. The globular structure of nut protein is modified into fibrillar structure by fibrillation. The fibrillation process of nut protein is carried out by giving special treatment in vitro. These treatments include giving temperature above denaturation temperature, low pH, and low ionic strength. This condition causes the peptide fragments resulting from protein hydrolysis will aggregate into length and forming semi flexible fibrils, hereinafter is referred to as amyloid fibrils. The changes of proteins conformation from globular to fibrillar will cause physical and chemical properties changes on the fibrils surface, thus providing different functions than its globular proteins. These structural changes resulted in an increase of the fibril solution viscosity, changes of flow behavior (rheology), and also surface properties of the fibrils. Thus, fibrillation can increase the protein functional characteristics to be developed as food ingredient according to the needs for processed food. The first stage of the research was preparing the beans and flour of Bogor nut. After 120 days of planting, fresh Bogor nut pods are harvested and then to be processed into Bogor nut flour. From 100 kg of fresh pods, it was obtained about 18.65 kg of Bogor nut flour, or from 100 kg of dry beans, 91.7 kg of Bogor nut flour was obtained. The protein content of nut flour is 21.66% (w/w) and the fat content is around 8.76%. The low-fat content is very beneficial to the fat extraction process, making it more economical. Thus, Bogor bean flour becomes a potential protein source to be isolated and fractionated. The second stage, through gradual pH adjustment starting from pH 6.4 to precipitate the 11S globulin, then followed by pH adjustment on the supernatant to pH 4.8 to precipitate the 7S globulin, this research succeeded in obtaining the protein isolate of Bogor nut, as well as the globulin fraction 11S and 7S which are purer as an ingredient for the formation of amyloid fibrils. In relation to the characteristics of protein sedimentation (the term S = Svedberg Unit), this research also studied the effect of globulin deposition at different centrifuge speeds, on 10,000 ×g and 19,000 ×g. The results showed that 10,000 ×g centrifuge speed was more economical and capable of harvesting isolates and globulin (PI-10, 7SG-10, 11SG-10) with higher protein yield and protein content compared to 19,000 ×g centrifuge speed (PI-19, 7SG -19, 11SG-19). The third stage, the six types of isolates and globulins that were extracted from the protein of Bogor Nut (PI-10, 7SG-10, 11SG-10, PI-19, 7SG-19, 11SG-19) has succeeded in forming amyloid fibril. Through various treatments such as the temperature of 65, 75, 85 oC, and heating duration of 6, 12, 18, 24, 30 hours, has obtained process conditions that produce a significant number of fibrils, which are on temperature 85 oC, 24 hours of heating duration for 1% concentration, 21 hours heating duration for 2% concentration, medium with pH 2, and medium agitation of 150 stokes per minute. The forming of amyloid fibril is monitored and identified integrately through the viscosity-increasing of the fibril solution with rheometer, morphology analysis of the fibril with TEM (Transmission Electron Microscopy), presence of beta-sheet on the fibril structure with FTIR (Fourier Transform Infrared spectroscopy) test, and the ability of the fibrils to bind specific dye of congo red with spectroscopy assay. Integrated identification has proven that fibrils from globulin 7S and 11S of Bogor Nut are a kind of fibril that has amyloid fibril characteristics. From the research result datas, it is proposed a formation mechanism of amyloid fibril globulin 11S from Bogor Nut through the way: 1) globular structures opening by heat; 2) hydrolysis process by low pH, so the polypeptide is cut into peptide fragments; 3) peptide aggregation process by low ionic strength medium, so among the peptide fragments aggregate to form an aggregate/fibril; 4) adequacy of the initial protein concentration or adequacy of the heating duration, the fibrillation process continues into amyloid fibrils. The fourth stage is directed to isolate 11SG-10 fibrils through filtration method using microfilter with 3kDa molecular weight cut-off, centrifugation on 3500 ×g speed for 15 minutes at 4 oC temperature, fractionation of fibrils using gel filtration chromatography of Sephadex G-100-120 and spectroscopy at 214 nm and 280 nm wavelengths. The obtained fibril fractions were characterized by analysis of the hydrolysis profile with SDS PAGE (Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis) and identification of the presence of ß-sheets with FTIR. The results of the amyloid fibrils isolation-fractionation from the 11S globulin produced four fibril fractions. Through FTIR spectra analysis and SDS PAGE analysis, it is predicted that the formation of amyloid fibrils of 11S globulin will occur gradually, beginning with the formation of ß -sheets from the F4 fraction with a 9 kDa polypeptide molecular weight. Then followed by ß-sheets from the F3 fraction with ß-strands of 9, 13, and 18 kDa molecular weight. This is followed by the formation of ß-sheets from the F2 fraction and from the F1 fraction, which each of them is composed of ß-strands with a molecular weight of about 9 kDa. Some novelties of this dissertation research include: 1) Bogor nut as local wisdom, has never been used as a source of 7S and 11S globulin for further modification and development; 2) proving the formation of amyloid fibrils globulin Bogor nut was carried out by integrating several analytical methods (analysis of the fibril solution viscosity, analysis of the fibril morphology, analysis of the hydrolysis profile, identification of the ß-sheet presence on the fibril structure, and identification of the amyloid fibrils properties); 3) the formation mechanism of amyloid fibrils globulin Bogor nut; 4) the method of amyloid fibrils formation in vitro from protein isolates, 7S globulin and 11S globulin of Bogor nut; 5) the modification approach of the proteins functional characteristics that have been carried out using chemistry conventionally such as cross-linking, is replaced by a new approach that is amyloid fibrils formation (fibrillation). Therefore, this research provides new prospects in an effort to modify protein components that have characteristics in accordance with the processed food needs.