Investigation of the fruit juice clarification potential of immobilized multi enzyme systems

In this thesis, it is aimed to immobilize and reuse multiple enzyme systems such as pectinase (PG) and α-amylase (A), which are used in the removal of polysaccharides in the clarification stage of fruit juice processing and are denatured and discarded by precipitation after pasteurization. In this thesis, the potential of multi-enzyme system for fruit juice clarification was investigated by binding pectinase and α-amylase together on cryogel. With the obtained multi-enzyme system, it was aimed to reduce the use of enzymes, reduce the cost and reduce the amount of waste and thus contribute to sustainability. In this thesis, single and dual immobilization of these enzymes on cryogels was carried out in order to remove pectin and starch during fruit juice production in the food industry and continuous system applications were performed with the use of cryogels. In this study, 2-hydroxyethylmethacrylate (HEMA) and 1,2-epoxy-5-hexene (EH), two biocompatible monomers, were used for cryogel synthesis. The kinetic parameters (Km and Vmax) and optimum working conditions of the immobilized and free pectinase and α-amylase enzymes were determined. Applications were made in fruit juice samples to demonstrate the industrial potential of the immobilized enzyme. In this thesis, a multi-enzyme system poly(HEMA-EH)-A-PG cryogel was synthesized for co-immobilized pectinase and α-amylase enzymes intended to be used in fruit juice clarification. Thermal stability was found to increase at high temperature (55 °C) with immobilization for pectinase enzyme. This supported the reason to prefer immobilized enzyme for enzymes denatured at high temperature. The Vmax and Km values of immobilized pectinase in poly(HEMA-EH)-A-PG cryogel were 11.3% and 75.7% times of free pectinase, respectively. The decrease in Vmax indicated a decrease in enzyme velocity. The decrease in Km indicated an increase in the enzyme's affinity for the substrate. Since the substrate molecules were large molecules, they could not meet the enzyme, which was thought to cause diffusional limitations. The reusability of immobilized pectinase was studied at 25 °C for 15 min repetitions. It was found that pectinase enzyme activity in poly(HEMA-EH)-A-PG cryogel could maintain 70% activity after 10 uses. In the orange juice clarification application, a better result was obtained than the substrate and it was determined that the poly(HEMA-EH)-A-PG cryogel retained 97% pectinase activity at the end of the 7th use, retained over 70% pectinase activity at the end of 10 uses and retained over 56% activity at the end of the 13th use. For the reusability of immobilized α-amylase at 25 °C with 15 min repetitions, it was determined that poly(HEMA-EH)-A-PG cryogel could maintain 70% activity up to 14 uses. In apple juice treatment at 25 °C with 15 min repetitions, it was determined that poly(HEMA-EH)-A-PG cryogel retained 81% activity until the 9th use and 57% activity at the end of the 13th use. According to the results of the 15-minute clarification process of orange and apple juice with poly(HEMA-EH)-A-PG cryogel, it was determined that the turbidity of both juices decreased after the clarification process with visual and % clarification amounts. The storage stability of immobilized pectinase enzyme in cryogels stored at +4 °C was 35 days (90%) in poly(HEMA-EH)-A-PG cryogel and 28 days (85%) in poly(HEMA-EH)-PG cryogel. It was determined that co-immobilization with α-amylase increased the storage stability of immobilized pectinase. The storage stability of immobilized α-amylase enzyme in cryogels stored at +4 °C was 21 days (46%) in poly(HEMA-EH)-A-PG cryogel and 21 days (52%) in poly(HEMA-EH)-A cryogel. The cryogels synthesized in the thesis can be used in solid waste reduction, textile and paper industry, except for fruit juice clarification processes. Although the rate of the synthesized poly(HEMA-EH)-A-PG multi-enzyme system is lower than that of the free enzyme, it is thought that the reaction yield can be increased by extending the incubation time and that it is an example for immobilized multi-enzyme systems.