Analysis methods differ in recovery of microbial glycogen

Abstract Text:

Microbial glycogen is an 1,4-, 1,6-alpha-glucan produced from carbohydrates and stored within bacterial and protozoal cells. Enyzmatic analysis of glycogen in bacteria requires lysis of cells to make glycogen available to enzymatic attack. Lysis is typically performed with alkaline treatment. The objective of this study was to compare the detection of corn starch and of alpha-glucan in a fermentation pellet by 3 lysis methods from the research literature: 30% KOH, boiled for 3 h (30%K), 0.2 N NaOH boiled 15 min (0.2N), and 0.31 N NaOH at room temperature for 15 min (0.31N). Fermentation pellets were prepared in an in vitro fermentation with mixed ruminal microbes in Goering and Van Soest medium, with 3 g/L of glucose, fermented for 2 h. Each fermentation vessel was quantitatively transferred into a centrifuge tube, and centrifuged at 13,000 x g for 45 min at 5°C. Supernatants were discarded. Pellets were resuspended in 0.9% saline, and recentrifuged. Supernatant was discarded and pellets frozen at -20°C. As per individual protocols, 30%K and 0.2N were performed on undried pellets transferred into 50 mL beakers; 0.31N was performed on freeze dried fermentation pellets. Samples were analyzed in duplicate in 2 analytical runs on 2 separate days in a randomized complete block design with fermentation pellet as the experimental unit. The statistical model for each substrate included method, and fermentation run as a random variable. After incubation with alkali, all samples were neutralized with acid, and acetate buffer added to bring the pH to 4.9 to 5.0. 30%K and 0.2N samples received 0.1 mL of heat stable, alpha-amylase and 200 U of amyloglucosidase and were incubated for 2 h at 50°C. 0.31N received 0.25 mL Hazyme DCL enzyme preparation and was incubated for 16 h at 55°C. After bringing samples to volume with distilled water, samples were centrifuged to clarify and the supernatant analyzed for glucose by a glucose oxidase-peroxidase assay. Alpha-glucan was expressed as glucose x 0.9. Recovery of corn starch was 96.7%, 95.8%, and 91.6% with 0.2N and 0.31N greater than 30%K, respectively (P<0.01). Alpha-glucan in fermentation pellets was 29.6, 29.8, and 26.0 mg with 0.2N and 30%K greater than 0.31N, respectively (P<0.03); 0.31N gave recoveries 87-88% of the other treatments. Pattern of starch recovery did not reflect that of microbial alpha-glucan recovery. With the alkaline lysis methods tested, a boiling treatment appears to be necessary for greatest microbial glycogen recovery.

Keywords: Glycogen, rumen, bacteria, starch