Enzymatic Hydrolysis of Flaxseed Oil to Produce Free Fatty Acids

Abstract

Microorganisms with the potential to secrete extracellular enzymes such as Candia rugosa and Aspergillus niger have the potential for industrial applications. The primary application of interest in this study includes the development of biocatalysts for the bioprocessing industry. Both microbial species secrete enzymes including lipase which has been used to catalyse the hydrolysis of glycerols. Saskatchewan is the largest producer of flaxseed worldwide accounting for 40% of worldwide flaxseed production. Flaxseed can be further processed to acquire nutrient-rich flaxseed oil. Linolenic and linoleic acids account for 58 wt. % and 15 wt. % of the total free fatty acids content of flaxseed oil. The free fatty acid concentrations can be increased via the process of enzymatic hydrolysis. α- linoleic and linolenic acids are high-value nutritional supplements in great demand in the pharmaceutical and health industries. In this study, the potential of lipase from Candida rugosa and Aspergillus niger to catalyse the hydrolysis of naturally occurring glycerols in flaxseed oil into free fatty acids was investigated. The research work was divided into 2 phases. The first, optimization of reaction conditions. In this phase, the optimal reaction conditions for the hydrolysis of flaxseed oil catalyzed by lipase were determined. The reaction conditions pH, temperature and flaxseed oil concentration were set as the reaction parameters for optimization. A central composite design with three center points was used as the experimental design for optimization. The optimal reaction conditions for lipase from Aspergillus niger and Candida rugosa were determined independently. The second phase studied the enzyme kinetics of the hydrolysis of flaxseed oil using lipase. Kinetic analyses were performed using Michaelis-Menten modelling for enzyme kinetics. The reaction conditions such as pH, reaction temperature, and oil to buffer ratio in a homogenous mixture (mL flaxseed oil to mL TRIS-HCl buffer) were investigated to optimize free fatty acid production. Optimum reaction conditions for Aspergillus niger lipase, ANL, were found to occur at a pH of 5 and a temperature of 35 C with a 15% flaxseed oil concentration. These conditions yielded an average of 78% increase in the free fatty acid concentration when compared to the FFA concentration of pure flaxseed oil. Optimum reaction conditions for Candida rugosa lipase, CRL occurred at a pH of 6.5 and a temperature of 30 C with a 10% flaxseed oil concentration. These conditions yielded a 95% increase on average in the free fatty acid concentration compared to pure flaxseed oil. Brunaurer-Emmett-Teller, BET, analysis of the surface characteristics of lipase showed CRL to have the greatest surface area of 11.7±0.1 (m2/g) and a pore diameter of 7.6 nm. The surface area and pore diameter for ANL were undetectable by BET characterization. Analyses of the kinetic parameters were conducted to determine the Michaelis-Menten constant, Km, maximum reaction velocity, Vm, and, the turnover number, kcat, respectively. The results are as follows; 4.25x10-5g/mL-sec, 1.06g/mL, 6.61x10-2/sec using CRL, 6.46x10-7, 1.62, 3.45x10-2/sec using ANL. The biomedical imaging and therapy facility beamline, BMIT, beamline imaging taken at the Canadian light source confirmed that the hydrolysis reaction takes place at the flaxseed oil-water interface as lipase becomes active and migrates to the interface. The results of this study showed CRL is the superior source of lipase compared to ANL based on the experimental findings, kinetic analyses, and enzyme characterizations.