Condensed tannins from Lotus corniculatus and Lotus pedunculatus exert different effects on the in vitro rumen degradation of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) protein

Condensed tannins (CT) or proanthocyanidins (PA), which occur in a restricted range of forages, have the ability to interact with proteins and enzymes and can influence the digestion of plant protein in the rumen. We compared the effects of CT extracts from Lotus corniculatus and pedunculatus on degradation of the principal leaf protein, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), by rumen microorganisms. Total soluble leaf protein extracted from white clover (Trifolium repens ) was incubated with fresh rumen fluid from sheep and a range of concentrations of each CT extract. The rate of degradation of the large (LSU) and small subunit (SSU) of Rubisco was quantified by fractionating the proteins in samples taken from in vitro rumen incubations using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and imaging densitometry. To deduce the effects of the CT extracts, experiments were performed in the presence (CT inactive) and absence (CT active) of polyethylene glycol (PEG; MW 3350). The two CT extracts differed markedly in their effects on the degradation of the LSU and SSU of Rubisco. At concentrations of 0.89 and 1.79 mg CT mg ⁻¹ total soluble leaf protein, the CT extract from L pedunculatus was more effective at preventing the degradation of the LSU and SSU by rumen microorganisms than the CT extract from L corniculatus. At a concentration of 1.79 mg CT mg ⁻¹ total soluble leaf protein, the CT extracts from L corniculatus and pedunculatus prevented about 0.75 and 0.83 of the LSU and about 0.69 and 0.86 of the SSU, respectively, from being degraded. Addition of PEG removed the inhibition and almost complete degradation of these proteins occurred, as was the case in incubations without CT extracts. The results of this study suggest that the concentration of CT in the diet and the chemical structure which affects the activity of the CT needs to be considered when assessing the effects of CT on protein metabolism in ruminants. © 1999 Society of Chemical Industry     

In-vitro rates of rumen proteolysis of ribulose-1,5-bisphosphate carboxylase (rubisco) from lucerne leaves,and of ovalbumin,vicilin and sunflower albumin 8 storage proteins

Laboratory systems were developed, based upon in-vitro incubations with rumen fluid, to examine the rate of proteolysis (ie degradation) of sunflower albumin 8 (SFA 8; 24% sulphur amino acids; SAA) from sunflower seeds, ovalbumin (6% SAA) from chicken egg white, ribulose-1,5-bisphosphate carboxylase (Rubisco; 3% SAA) from lucerne leaves, and vicilin (0% SAA) from pea seeds. After fractionation by SDS-PAGE, proteins were analysed by either Western blotting, using specific antibodies (SFA 8, vicilin and ovalbumin) or by Coo-massie blue staining (Rubisco). Proteolysis of the large subunit of Rubisco occurred very quickly and as two components, with half-lives of 11.6 and 1.5 h. The small subunit of Rubisco was more resistant to degradation, with a half-life of 17.3 h. Vicilin was degraded extremely rapidly (half-life 0.16 h). SFA 8 and ovalbumin both showed resistance to degradation, but by two different mechanisms. Ovalbumin was not degraded at all during the initial 16 h of incubation, but then degraded with a half-life of 8.7 h. SFA 8 (mol wt 12 100) disappeared very rapidly, with a half-life of 3.0 h. The degradation of SFA 8 was associated with the appearance of a polypeptide (mol wt 8000), which was extremely resistant to degradation, with a half-life of 69.3 h. It was concluded that both the number of disulphide linkages and tertiary structure were important in determining resistance of proteins to rumen degradation, and that incorporation of SFA 8 and ovalbumin proteins into forages using genetic engineering techniques would be likely to increase the quantity of SAA by passing rumen fermentation.