Christodoulou, Speroulla (1998) Enzymatic degradation of egg yolk cholesterol. Research Master thesis, Victoria University of Technology.

Abstract

A rapid, reliable and accurate gas chromatographic method for the determination cholesterol in egg yolk and processed foods was developed from a method of Kovacs (1990). A sample is heated in a sealed tube with a solution of potassium hydroxide in ethanol containing the internal standard, dihydrocholesterol. The sterols are then isolated by solvent extraction, converted to trimethylsilyl (TMS) derivatives and subjected to gas chromatography. The method was used to determine the cholesterol content of a number of foods in order to demonstrate its application to more complex matrixes. Among egg-based products, Italian egg spaghetti had a lower cholesterol content (0.14-0.74mg/g) than egg noodles (0.87-1.33mg/g). Some egg rolls contained cholesterol levels as high as 1.72-3.6lmg/g. Among meat-based foods, ham products had cholesterol levels similar to pork cakes or pork loafs (0.59-0.67mg/g and 0.66-0.8lmg/g respectively). Fish balls and cheddar cheese contained almost the same level of cholesterol (0.93mg/g and 0.95mg/g, respectively). Of all foods investigated, chicken liver, chicken paste and prawn contained the highest levels of cholesterol. The capacity of four cholesterol oxidases, from Pseudomonas fluorescens (Pf), Nocardia erythropolis (Ne), Brevibacterium species (Bs) and Streptomyces species (Ss), and cholesterol reductase isolated from cucumber leaves to degrade cholesterol in egg yolk was investigated. Incubation of egg yolk with a cholesterol reductase for 72h at 37°C resulted in minimal (3.6%) cholesterol reduction. In contrast, up to 93.4% of the total available cholesterol was degraded by cholesterol oxidases from Pseudomonas fluorescens and Streptomyces species under similar conditions. At 4°C, cholesterol oxidase from Pseudomonas fluorescens could degrade up to 64.9% cholesterol after 48h; cholesterol lowering was barely evident at 4°C with the other cholesterol oxidases. The most effective temperature was 37°C for Bs cholesterol oxidase, and 45°C for the other cholesterol oxidases. The enzyme derived from Pseudomonas fluorescens had an effective temperature range from 15 to 60°C, and from 5 to 37°C it was clearly superior to the other three oxidases. The capacity of the cholesterol oxidases to degrade cholesterol in egg yolk followed the sequence Pf > N e > Ss > Bs. The optimal enzyme concentration for cholesterol degradation was the lowest for Pf cholesterol oxidase at 0.5U/µmole cholesterol. Even at the low concentration of 0.125U/µmole cholesterol, Pf cholesterol oxidase degraded up to a third of the egg cholesterol in 2h. The cholesterol degrading activities of Rhodococcus equi, Rhodococcus erythropolis and two other unspeciated isolates of Rhodococcus were investigated in egg yolk and milk. These microorganisms, when grown on a cholesterol containing medium, resulted in halo formation around colonies of the Rhodococcus species consistent with the production of cholesterol degrading enzymes. The ability of Rhodococcus equi No. 23 to degrade cholesterol in whole homogenised UHT milk or preparations diluted to half strength was minimal. However, R. equi No. 23 degraded up to 52.7% of cholesterol in a 1/8 strength preparation of UHT milk. In these preparations cholesterol degradation was further enhanced by growth of R. equi No. 23 cells used as the inoculum in UHT milk rather than in an artificial medium, prior to determination of cholesterol degradation. The cholesterol degrading system of R. equi No. 23 was equally effective in reducing the concentrations of cholesterol in egg yolk. The rate of cholesterol degradation was also enhanced by pre-incubations of R. equi No. 23 cells in yolk compared with artificial medium. Cholesterol degradation in egg yolk was almost complete after growth of R. equi No. 23 for 7d at 37°C.

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