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Title:   Author  item
                                            Hall, Mary Bethitem
                                            Hatfield, Ronald
  Submitted to: Journal of Microbiological Methods
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/15/2015
                                    Publication  Date: 9/18/2015
                                    Publication URL: http://handle.nal.usda.gov/10113/62087Citation: Hall, M., Hatfield, R.D. 2015. Comparison of methods for glycogen analysis of in vitro fermentation pellets produced with strained ruminal inoculum. Journal of Microbiological Methods. 118:147-151.Interpretive Summary: Rumen microbes largely determine the profile of nutrients that a cow receives from her diet because they have the first opportunity to ferment or process feed in the cow’s first stomach. To understand how microbes process feed and predict the nutrients a cow receives, it is essential that we accurately measure the products the rumen microbes produce. One of those products is microbial glycogen, a starch-like carbohydrate. We found that a recently developed assay was as accurate as a long-established approach for measuring glycogen, but took one-twelfth the time, only 15 minutes, to perform the processing step. The improved assay will allow researchers to process microbial samples more rapidly and efficiently as they work to gain insights into how microbes process feeds. Ultimately, this will enhance our ability to balance diets for cattle to more closely meet their nutrient requirements and support their production.
                                Technical Abstract: 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, and it uses smaller sample sizes. However, the nonhomogenous fermentation pellets produced with strained rumen inoculum cannot be accurately subsampled, and so require analysis of the entire pellet. In this study, two microbial glycogen methods were compared for analysis of such fermentation pellets: boiling samples for 3 h in 30% KOH (tKOH) or for 15 min in 0.2 M NaOH (tNaOH), followed by enzymatic hydrolysis with alpha-amylase and amyloglucosidase, and detection of released glucose. Total alpha-glucan was calculated as glucose x 0.9. tKOH and tNaOH did not differ in the alpha-glucan detected in fermentation pellets (29.9 and 29.6 mg, respectively; P = 0.61). Recovery of different control alpha-glucans was also tested using tKOH, tNaOH, and a method employing 45 min of bead beating (tBB). For purified beef liver glycogen (water-soluble) recovery, tBB (95.0%) > tKOH (91.4%) > tNaOH (87.4%; P < 0.05). For wheat starch (water-insoluble granules) recovery, tNaOH (96.9%) > tBB (93.8%) > tKOH (91.0%; P < 0.05). Recovery of isolated protozoal glycogen (water-insoluble granules) did not differ among tKOH (87.0%), tNaOH (87.6%), and tBB (86.0%; P = 0.81), but recoveries for all were below 90%. Differences among substrates in need for gelatinization and susceptibility to destruction by alkali likely affected the results. In conclusion, tKOH and tNaOH glycogen methods provided comparable determinations of fermentation pellet alpha-glucan. The tests on purified alpha-glucans indicated that assessment of recovery in glycogen methods can differ by the control alpha-glucan selected.


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