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United States Department of Agriculture

Agricultural Research Service

Title: Mechanism of Polyphenol Oxidase Action in Reducing Lipolysis and Proteolysis in Red Clover During Batch Culture Incubation

Authors
item Lee, Michael - INST GRASSLAND ENVIR RES
item Tweed, J - INST GRASSLAND ENVIR RES
item Scollan, N - INST GRASSLAND ENVIR RES
item Sullivan, Michael

Submitted to: Symposium Proceedings
Publication Type: Proceedings
Publication Acceptance Date: December 11, 2007
Publication Date: March 31, 2008
Citation: Lee, M.R., Tweed, J.K., Scollan, N.D., Sullivan, M.L. 2008. Mechanism of polyphenol oxidase action in reducing lipolysis and proteolysis in red clover during batch culture incubation. In: Proceedings of the British Society of Animal Science, March 31-April 2, 2008, Scarborough, United Kingdom. p. 31.

Technical Abstract: Introduction: We previously showed that red clover, with the PPO1 gene silenced (Sullivan and Hatfield, 2006), exhibited higher levels of lipolysis than the wild type in the presence of rumen micro-organisms. This questioned the hypothetical mode of action of polyphenol oxidase (PPO) being solely the deactivation of the plant enzymes, and implied some level of protection of the red clover lipid. It was hypothesized that this may be a result of protein-phenol complexes, formed by the action of PPO, complexing around lipid micelles and so offering a level of protection. This study investigated whether red clover lipid is protected in the absence of such protein matrixes. At the same time, levels of free amino acids were monitored in cultures with the protein matrixes formed to determine whether these offer any form of protection from microbial degradation. Materials and Methods: PPO1 gene silenced (PPO-) and wild type (PPO+) plants grown in controlled conditions were harvested at 5 cm above soil level, crushed, cut into 5-mm strips, and wilted for 1 h. Half of the PPO- and PPO+ material was frozen with liquid nitrogen and stored at –20 degrees C. This was used as the protein complex treatment (PC). The other half of the material was freeze-dried, ground, and 1 g weighed out into 12 extraction tubes. The lipid was extracted with 3 X 5 ml of chloroform:methanol (2:1, v/v), and the extract was dried down under nitrogen at 50 degrees C. Once dried, 0.19 g zein, 0.1 g glucose, and 0.33 g cellulose were added to each tube; these acted as the free lipid treatment (FL). The PC material was defrosted and approximately 5 g (to account for 1 g DM) were weighed into 12 extraction tubes for both PPO- and PPO+. This gave 48 tubes in total and four treatments (FL PPO+; FL PPO-; PC PPO+; PC PPO-). Into each tube, 7.5 ml of anaerobic buffer, 0.35 ml reducing agent, and 2.5 ml of strained rumen liquor were added before being purged with carbon dioxide and incubated at 39 degrees C with a set of three tubes for each treatment allocated to each of the sampling time points of: 0, 2, 6, and 24 h. At these time points for the PC tubes only, the supernatant was sub-sampled for free amino acid analyses. The tubes (PC and FL) were then frozen with liquid nitrogen and freeze-dried. The lipid was extracted as before and fractionated by thin-layer chromatography. Lipid fractions were bimethylated and run on GC. Lipolysis was calculated as the proportional loss of membrane lipid, and the rise in free amino acids was used as a predictor of proteolysis. Results: In the PC treatment, PPO had a significant effect in reducing the extent of lipolysis, whereas in the FL treatment, there was no effect of PPO. Proteolysis was shown to be significantly reduced by the action of PPO. Conclusions: Red clover PPO significantly reduces proteolysis by the formation of bound-phenol protein complexes. As the protection of forage lipid was lost with the removal of the protein complexes, it appears that the reduction in lipolysis by PPO in red clover is due to the protection of lipid micelles within bound-phenol protein complexes.

Last Modified: 7/30/2014
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