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Technical note: A method for isolating glycogen granules from ruminal protozoa for further characterization1

Mary Beth Hall
Journal of dairy science 2016 v.99 no.3 pp. 1956-1958
Protozoa, detergents, enzymatic hydrolysis, fermentation, freeze drying, glucose, glycogen, granules, inoculum, isolation techniques, rumen, rumen fermentation, rumen protozoa, starch granules
Evaluation of physical, chemical, and enzymatic hydrolysis characteristics of protozoal glycogen is best performed on a pure substrate to avoid interference from other cell components. A method for isolating protozoal glycogen granules without use of detergents or other potentially contaminating chemicals was developed. Rumen inoculum was incubated anerobically in vitro with glucose. Glycogen-laden protozoa produced in the fermentation, primarily isotrichids, were allowed to sediment in a separatory funnel and were dispensed. The protozoa were processed through repeated centrifugations and sonication to isolate glycogen granules largely free of feed and cellular debris. The final water-insoluble lyophilized product analyzed as 98.3% α-glucan with very rare starch granules and 1.9% protein. Observed losses of glycogen granules during the clean-up process indicate that this procedure should not be used for quantitative assessment of protozoal glycogen from fermentations. Further optimization of this procedure to enhance the amount of glycogen obtained per fermentation may be possible.