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Effect of pH on Proteolysis in Ensiled Legume Forage

Mckersie, B. D.
Agronomy journal 1985 v.77 no.1 pp. 81-86
Medicago sativa, Trifolium pratense, Lotus corniculatus, proteolysis, pH, silage
The hydrolysis of proteins is extensive in ensiled forages but the production practices which may influence the extent of proteolysis remain to be characterized. An in vitro assay system was developed to estimate the relative rates of proteolysis and was compared to a laboratory silo system as a means of estimating the relative extent of proteolysis in three forage legumes, alfalfa (Medicago sativa L. cv. Iroquois), red clover (Trifolium pratense L. cv. Arlington) and birdsfoot trefoil (Lotus corniculatus L. cv. Leo). Plants were harvested from field plots as first and second cuts at the same time and were in the early bud to 10% bloom stages, depending on species and sampling time. Leaf homogenates containing chloramphenicol as an antibiotic were used as in vitro systems to monitor changes in the level of free amino acids at pH values between 4.0 and 6.5 at incubation times up to 72 h. This method provided a more satisfactory estimate of the relative proteolysis in ensiled forage than previous attempts using azocasein as substrate. Proteolysis was more extensive in alfalfa than the other two species. The optimal pH of proteolysis was approximately 6.0 in alfalfa, but 6.5 or above in red clover and birdsfoot trefoil. Low pH inhibited proteolysis. In a second experiment, forage from the same field plots as the in vitro experiment was ensiled in laboratory silos with the addition of 1% Na₂CO₃, 1% formic acid, 0.7% HCl, 1% formaldehyde, or no treatment. After ensiling for 30 days, protein hydrolysis was consistently highest in the alfalfa silages. The acid additives reduced proteolysis in all species, but a significant (P≤0.01) additive ✕ species interaction was observed in the second cut. A close positive relationship between the initial pH of the forage at ensiling and proteolysis was established for each species. The amount of protein degraded to free amino acids during ensiling of these legumes appears to be dependent on the proteolytic activity in the leaves, the rate of pH decline during ensiling, and the interaction between these two factors.