Comparison of methods for glycogen analysis of in vitro fermentation pellets produced with strained ruminal inoculum

Microbial glycogen measurement is used to account for fates of carbohydrate substrates. It is commonly applied to washed cells or pure cultures which can be accurately subsampled, allowing the use of smaller sample sizes. However, the nonhomogeneous fermentation pellets produced with strained rumen inoculum cannot be accurately subsampled, requiring analysis of the entire pellet. In this study, two microbial glycogen methods were compared for analysis of such fermentation pellets: boiling samples for 3h in 30% KOH (KOH) or for 15min in 0.2M NaOH (NaOH), followed by enzymatic hydrolysis with α-amylase and amyloglucosidase, and detection of released glucose. Total α-glucan was calculated as glucose×0.9. KOH and NaOH did not differ in the α-glucan detected in fermentation pellets (29.9 and 29.6mg, respectively; P=0.61). Recovery of different control α-glucans was also tested using KOH, NaOH, and a method employing 45min of bead beating (BB). For purified beef liver glycogen (water-soluble) recovery, BB (95.0%)>KOH (91.4%)>NaOH (87.4%; P<0.05), and for wheat starch (water-insoluble granules) recovery, NaOH (96.9%)>BB (93.8%)>KOH (91.0%; P<0.05). Recovery of isolated protozoal glycogen (water-insoluble granules) did not differ among KOH (87.0%), NaOH (87.6%), and BB (86.0%; P=0.81), but recoveries for all were below 90%. Differences among substrates in the need for gelatinization and susceptibility to destruction by alkali likely affected the results. In conclusion, KOH and NaOH glycogen methods provided comparable determinations of fermentation pellet α-glucan. The tests on purified α-glucans indicated that assessment of recovery in glycogen methods can differ by the control α-glucan selected.