REAGENT KIT FORMULATION BASED C-PHYCOCYANIN FROM SPIRULINA PLANTESIS FOR DETERMINATION OF PANCREATIN PROTEASE ACTIVITY

• Main Author: Puspita Anggraeni, Yulia
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/85396
• Institution: Institut Teknologi Bandung

C-Phycocyanin (C-PC) is a protein that has a characteristic blue color and is contained in Spirulina plantesis. Based on research that has been carried out, C-Phycocyanin can be used as a substrate in determining pancreatin protease activity. However, there are drawbacks to using C-PC as a substrate, related to the stability of C-Phycocyanin during storage which can be influenced by various factors such as pH, temperature, light exposure, oxidation processes, and microbes. This research aims to increase the stability of C-Phycocyanin through the addition of several preservatives and antioxidants. C-Phycocyanin was isolated from dry biomass of Spirulina plantesis and purified through a combination of ammonium sulfate precipitant fractionation and aqueous two-phase system (ATPS) methods. The isolation results were characterized using UV-Vis spectrophotometry to determine the UV-Vis spectrum, C-PC content, and purity; ATR-FTIR for determining concentration of polyethylene glycol (PEG) 6000; and the Bradford method for total protein determination. The general formula used in this study was 0.3 mg/ml C-Phycocyanin, 15% PEG 6000, and 0.05 M phosphate buffer at pH 7.2. Formula screening was carried out using several preservatives such as fructose, benzoic acid, sodium azide, sodium chloride, and polyethylene glycol 6000; as well as antioxidants such as ascorbic acid at certain concentrations. The first stage of the stability test was carried out at a temperature of 50oC for 30 minutes, then the second stage was carried out at refrigerator temperature (4oC) and room temperature (25oC) for 7 days which was determined based on the relative concentration value (Cr). The results of the formula screening showed that sodium chloride at a concentration of 5% can provide the most optimal protection in maintaining the stability of the phycocyanin structure and sodium azide at concentrations of 0.55% and 1% is the best preservative in preventing the appearance of bad odors in C-PC after storage. at room temperature. The concentration of PEG 6000 that can maintain the color of phycocyanin when stored at room temperature for 7 days is at concentrations of 35%, 45% and 55%. Protease activity was also determined for each formula by determining changes in substrate levels per time (?g/ml/minute). Formula selection was carried out through a combination of 1% sodium azide and 35% PEG 6000 with the results of relative concentration values and changes in substrate levels per time in the pancreatin protease activity test being higher compared to the general formula. Therefore, a reagent kit formulation with a combination of 1% sodium azide and 35% PEG 6000 can be used to increase the stability of the C-Phycocyanin substrate.