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1810 40 Cyran MR and Saulnier L, Cell wall fractions isolated from outer layers of rye grain by sequential treatment with alpha-amylase and proteinase: Structural investigation of polymers in two ryes with contrasting breadmaking quality. J Agric Food Chem 53:9213–9224 (2005). www.soci.org D Dobberstein, M Bunzel 41 Dobberstein D, Steinhart H and Bunzel M, Bestimmung mono-, di- und trimerer Phenolcarbonsäuren pflanzlicher Herkunft (Determination of mono-, di- and trimeric phenolic acids from plant origin), Abstract written in German. Lebensmittelchemie 60:101–102 (2006). www.interscience.wiley.com/jsfa c○ 2010 Society of Chemical Industry J Sci Food Agric 2010; 90: 1802–1810
Research Article Received: 16 December 2009 Revised: 13 April 2010 Accepted: 26 April 2010 Published online in Wiley Interscience: 10 June 2010 (www.interscience.wiley.com) DOI 10.1002/jsfa.4019 A modified spectrophotometric assay to estimate deglycosylation of steroidal saponin to sapogenin by mixed ruminal microbes Yuxi Wang ∗ and Tim A McAllister Abstract BACKGROUND: The lack of a method for measuring deglycosylation of saponins in ruminal fluid has limited the ability to investigate the impact of these compounds on rumen microorganisms. A simple spectrophotometric assay was adapted and a protocol developed to enable measurement of steroidal saponin and sapogenin in ruminal fluid. The procedure was used for in vitro determination of deglycosylation activity of rumen bacteria obtained from cattle fed or not fed Yucca schidigera saponin, and to determine the relative deglycosylase activities of extracellular and cell-associated enzymes from ruminal content. RESULTS: Modifications to the spectrophotometric assay (i.e. heating time shortened to 10 min and 0.5 mL dH2O added to the reaction mixture) improved the stability of the optical density (425 nm) of the chromophore for up to 24 h post-reaction. Centrifugation (12 000 × g, 20 min) enabled differential estimations of steroidal saponin and sapogenin in ruminal fluid. Steroidal saponin added to defaunated ruminal fluid (dRF) or clarified ruminal fluid (cRF) was recovered completely from the mixture as saponin + sapogenin (99.1% and 100.6%, respectively), whereas saponin recovery from the supernatant of dRF was greatly reduced (P < 0.001) compared to that from supernatant of cRF (58.5 vs. 98.7%). Saponin recoveries from the supernatants of dRF and cRF did not differ between donor cattle fed or not fed Yucca schidigera saponin (59.2 vs. 57.3% and 98.4 vs. 99.3%, respectively). The majority (89–90%) of saponin added to a ruminal extracellular enzyme preparation was recoverable in supernatant after 24 h, compared with only 26–32% remaining in supernatant from incubation with a cell-associated enzymes fraction. CONCLUSION: Mixed rumen bacteria deglycosylate steroidal saponin to sapogenin, at activity levels unaffected by prior exposure to saponin, but they were unable to degrade the sapogenin core structure. Deglycosylation activity occurred primarily in the cell-associated enzyme fraction of ruminal content. Copyright c○ 2010 Crown in the right of Canada. Published by John Wiley & Sons, Ltd Keywords: assay; deglycosylation; enzymes; rumen fluid; steroidal saponins INTRODUCTION Steroidal saponins are present in a wide variety of monocotyledonous plants and less commonly in dicots and have been found to alter rumen fermentation 1–3 through direct effects on rumen microorganisms, 4–6 leading to responses that may improve ruminant productivity. 7–9 In response to the total ban on antibiotic growth promoters imposed by the European Union and increasing pressure on livestock industries worldwide to restrict sub-therapeutic use of antibiotics as growth promoters, researchers have been investigating potential alternative feed additives, among which are steroidal saponins. Although steroidal saponins may exert a desirable influence on ruminal fermentation, there is evidence that these benefits may be transient, attributable to adaptation by rumen microbial populations. 10,11 In general, two factors may contribute to rumen microbes adapting to saponins: development of tolerance or reduced sensitivity to the saponins’ antimicrobial properties, or direct microbial deactivation of the saponin via enzymatic degradation. Defining the relative importance of these two factors has been hampered by the lack of a suitable method for measuring degradation of steroidal saponins in ruminal fluid. Baccou et al. 12 described a spectrophotometric assay that accurately measured steroidal saponin and sapogenin as total steroidal sapogenin, via production of a chromophore generated from a reaction involving the aglycone core of saponin/sapogenin, p-anisaldehyde and sulfuric acid in ethyl acetate. In a preliminary study, however, we determined that non-specific reactions occurring in the reaction mixture generated an intense, unstable background color that gave rise to substantial variation in the estimation of steroidal saponin and sapogenin in ruminal fluid. Several studies have shown that saponins are deglycosylated in the rumen to form free sapogenins. 5,13,14 Segal and Schlösser 15 ∗ Correspondence to: Yuxi Wang, AAFC Research Centre, P.O. Box 3000, Lethbridge, Alberta, T1J 4B1, Canada. E-mail: yuxi.wang@agr.gc.ca Agriculture and Agri-Food Canada Research Centre, P.O. Box 3000, Lethbridge, Alberta, T1J 4B1, Canada J Sci Food Agric 2010; 90: 1811–1818 www.soci.org Copyright c○ 2010 Crown in the right of Canada. Published by John Wiley & Sons, Ltd 1811
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