Wheat protein proteolysis in the monoculture of rumen protozoon Entodinium caudatum

Abstract

The study of the wheat protein (albumin and globulin fraction) proteolysis was performed in the culture medium of the rumen protozoon Entodinium caudatum under anaerobic conditions in vitro. The rate of the wheat protein degradation was measured by the immunological sandwich technique dot-blot. The centrifugation of the protozoal culture media was performed at 500 g for 20 min. The resulting concentration of the protozoal cells was 20,000–24,000 per ml in the experimental culture medium. The samples were taken from the culture medium at 0, 2, 4, 6 and 24 h during incubation. After centrifugation at 500 g for 20 min, the supernatants were used for the determination of: (1) the total protein concentration, (2) the nonspecific proteolytic activity (azoalbumin was used as a substrate) and (3) the wheat protein concentration in the culture medium by the immunological sandwich technique dot-blot. Subsequently, the results were used for the calculation of the degradation rate of the wheat protein. The rabbit polyclonal antiserum against the wheat protein was used as the first antibody in this method. The quantitation of this test was based on the spectrophotometric determination of the violet stain quantity on the nitrocellulose strip surfaces. The average rate of the wheat protein degradation in the culture medium E. caudatum was 0.014 mg ml −1 h −1, whereas the average rate of the azoalbumin degradation was 0.003 mg ml −1 h −1 in supernatant. Azoalbumin was more resistant to degradation in the supernatant of the culture medium in comparison with the wheat protein in the culture medium. The assay sensitivity (=the limit of detection) of the simple immunological assay dot-blot was 104 ng ml −1 (i.e., 0.52 ng wheat protein applied to nitrocellulose foil) at 88% recovery. The accuracy and reproducibility characterized as intra-assay and inter-assay CV% (coefficient of variation) were 9.75% and 14%, respectively. The screening of the samples by dot-blot procedure proved to be an efficient way for the quantitation of the proteolytic activity in the rumen protozoa monoculture cultivated in vitro.