Proanthocyanidins inhibit hydrolysis of leaf proteins by rumen microflora in vitro

Proanthocyanidins (condensed tannins; PA) purified from the leaves of forage legumes Trifolium arvense, Lotus pedunculatus, Lotus corniculatus, Dorycnium rectum, Coronilla varia, Onobrychis viciifolia, or Hedysarum coronarium, were added to soluble lucerne (Medicago sativa) leaf protein and incubated with strained rumen fluid in vitro. Fractions were collected and frozen immediately. Denatured proteins were fractionated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), stained, and relative levels were quantified by densitometry. In the absence of PA the large subunit (LSU) of ribulose bisphosphate carboxylase (EC 4.1.1.39) was susceptible to proteolysis by rumen microflora but the small subunit (SSU) resisted breakdown. PA purified from Onobrychis was added to soluble leaf protein, at PA: protein ratios between 1:1 and 1:20. The rate of proteolysis of LSU was significantly reduced at PA: protein ratios of 1:2 and 1:1 (P < 0.001) and the rate of digestion was reduced by between 3- and 21-fold. In separate experiments PA isolated from the range of species described was added to rumen fluid to give PA: protein ratios of 1:5. The addition of PA significantly reduced the rate of proteolysis of LSU, when compared with PA-free control. There were only small differences between PA from different species. The inhibitory effect of PA may have been due to PA binding to the dietary protein or to the rumen proteases, interfering with the action of proteases on susceptible sites within the substrate.