Effect of extraction method on activities of polysaccharide-depolymerase enzymes in the microbial population from the solid phase in the rumen

Samples of rumen digesta were collected from 2 cows fed with a 100:0 or 40:60 forage/concentrate diet 1 h before and 2 h after feeding. Enzymes from particle-associated microorganisms were extracted by 5 different methods, including physical (freezing, grinding and sonication) or chemical and enzymatic (autolysis in a buffer, lysozyme and CCl4) treatments. Hydrolytic enzyme activities were measured on 3 polysaccharidic substrates: xylan, carboxymethylcellulose and Avicel cellulose. Contamination of enzyme preparations by feed proteins was evaluated. For the 3 substrates, the effect of feeding (sampling time and diet) on enzyme activities was independent of extraction method. Maximal activities were obtained with treatments including lysozyme and CCl4 or grinding in liquid nitrogen. Contamination of enzyme preparations by feed proteins was higher with chemical and enzymatic methods than with physical treatments.