Immobilization Of Lipase-producing Cell Of Candida Cylindracea For Preparation Of Glyceride Fish Oil

In this paper, glyceride fish oil was prepared by enzymatic studies with immobilized Candida Cylindracea cell. The main contents included the development of lipase activity determination, optimization of Candida Cylindracea growth and optimal conditions for enzyme production, the exploration of Candida Cylindracea cell fix with polyurethane foam and the hydrolysis of crude fish oil with immobilized Candida Cylindracea and so on.The traditional olive oil emulsion method was with shortcomings of poor reproducibility and low accuracy, so the parameters and reaction conditions were optimized: the 4mL, 25% olive oil emulsion instead of 5mL, 20% olive oil emulsion was used and the 3ml phosphate buffer instead of 5ml phosphate buffer was used. Meanwhile, the homogenization speed and time from high-speed (> 10000r/min), 6min reduced to 5000r/min, 2min; the reaction temperature decreased from 40℃to 35℃; the static reaction environment was changed to 100r/min oscillation; 1ml, 0.025mol / L phosphate buffer at pH7.5 was added in the control group instead of 1ml enzyme solution. Under optimum conditions, lipase activity was increased by 35% than the activity of before optimization. The method is with high precision and good repeatability and can be a reference for related study.The Candida Cylindracea growth conditions were explored by single factor test and orthogonal test. The results showed that the optimum temperature for Candida Cylindracea growth was 25℃, the living environment was acidic, and when pH was 5.2 and rotation speed was 150r/min, it had the strongest enzyme production ability. 10h after inoculation, it was in logarithmic growth phase and 25h after inoculation was the stable phase and after 45h was the decline phase; The best medium was olive oil concentration of 6.0 g·L^-1, yeast extract concentration of 9.0 g·L^-1, Tween-80 1%, Ca^{2 +}0.5 g·L^-1, K₂HPO₄ 3.0 g·L^-1, MgSO₄·7H₂O 1.0 g·L^-1. Under the optimum conditions for enzyme production, after 5 times of repeated experiments, the average enzyme activity was around 15.00U/ml and RSD was 2.34% with good reproducibility. The Candida Cylindracea can use cylindrical oil as single carbon source and the enzyme activity was better than other carbon source tests. With oil induction, lipase was produced with cell growth, but in the presence of other carbon sources, yeast uses other carbon sources first. The living environment for the Candida was acid and enzyme production was the best when pH was 5.2, suggesting that it produced acid lipase. Fat hydrolysis on free fatty acids will be resistant to a certain extent and it has clear research meaning.With commercial polyurethane foam as a carrier, the impact of five kinds of pretreatment patterns on cell fixation and enzyme production were investigated and the most effective treatment was first with 1% NaOH solution treatment for 24h, and then 1% HCl solution treatment for 24h; the impact of six different carrier sizes on cells fixation and enzyme production was also investigated and the best carrier size was 2.0cm×2.0cm×0.5cm; rotation speed influenced the dissolved oxygen amount and cell fixation and in the test, fixed cells were more and broth activity was also higher under150r/min; considered from the cost of test and fermentation activity, the best carrier was to put 12 carriers with size 2.0cm×2.0cm×0.5cm in 150ml .after Immobilized cells were washed 6 times, the dry cell weight in the carrier was of 42.1% of the pre-washed state, slightly lower than reported in the literature. Immobilized cell fermentation maintains a certain advantage either in total biomass, or in the activity of the fermentation broth.Immobilized yeast cells was used in refined fish oil to enrich glyceride DHA/EPA .when the rotation speed was 110r/min, ratio of oil to water was 3:40 and reaction time was 30h, the total DHA / EPA content can reach 28.55%.