Development of multiple probe cryo-concentration system for progressive freeze concentration of lysozyme aqueous solution

Abstract

A new crystalliser to concentrate lysozyme aqueous solution through freeze concentration was designed in this study to overcome the shortcomings of the currently available methods in concentrating protein. A new compatible, simple, reliable and low maintenance design was developed in this study based on progressive freeze concentration principles called multiple probe cryo-concentration system (MPCC). Progressive freeze concentration process is a process which rejects all impurities or solute by generating ice crystal lattice from the mother liquor, thus the remaining solution is more concentrated. The aim of this research is to observe the possibility of the new design system in producing high concentration lysozyme aqueous solution according to the four-effect parametric condition which includes coolant temperature, stirrer speed, operation time and initial concentration. The complete design of MPCC system consists of a solution tank with outer tubular cooling jacket and insulator, containing the protein solution and the cooled multiple probes immersed in it. The concentration process began when the temperature of the lysozyme aqueous solution dropped until the ice crystal formed on the wall of the probe while assisted by the stirrer. The concentrated protein solution was then separated from the ice crystal layer formed and collected as product. In order to evaluate the capability of the design, effective partition constant (K), solute yield of lysozyme (Y), concentration index (CI) and average ice growth rate (?ice) were analysed using UV-Vis spectrophotometer to determine the solute concentration. The findings revealed that coolant temperature at -12 °C, stirrer speed at 350 rpm, operation time at 40 minutes and initial concentration at 10 mg/ml gave the best result of K, Y and Cl and ?ice. Meanwhile, the determination of optimum condition by response surface methodology indicated that coolant temperature is the most significant parameter followed by stirrer speed and operation time but initial concentration was found to be not significant in affecting the process for both responses of K-value and Y. A thermodynamic prediction model was also built and its validity for ice crystal growth rate prediction was found to be adequately accurate compared to the actual experimental result based on the error analysis obtaining R-squared of 0.98 and absolute average relative deviation of 8.08 %. The heat transfer analysis discovered that the overall heat transfer coefficient, U? and heat remover, Q are quite similar for stirrer speed and operation time where increased stirrer speed and lower operation time resulted in lower U? and Q while increased initial concentration would increase the U? and Q. Despite, U? and Q are slightly different and contradict with each other when coolant temperature was increased where U? would increase but Q was decreased. The results from the analysis and investigation shed light on the theory behind the concentration method of protein using freezing method with the newly designed cryo-concentration device, which has never been investigated, tested and discussed specifically for protein concentration.