In vitro protein breakdown by enzyme extracts of rumen origin: comparison with methods in situ and proteases of Streptomyces griseus

Abstract

A. Velásquez, and G. Pichard. 2010. In vitro protein breakdown by enzyme extracts of rumen origin: comparison with methods in situ and proteases of Streptomyces griseus. Cien. Inv. Agr. 37(3): 57-70. Proteolytic activity of enzymatic extracts generated from rumen microorganisms cultivated in vitro was evaluated. The incubation of rumen fluid used different substrates to generate a higher enzyme concentration and promote a broad spectrum of hydrolytic activity. The composition of the substrates used in the cultivation of the fluid was enriched in protein, starch or cell wall. Enzyme preparations were evaluated by incubating in 30 mL of buffer 50 mM Tris-HCl (pH 6.5) at 39 ºC during 48 hours, 100 mg of crude protein from feeds soybean meal, canola meal, sunflower meal, gluten feed, dehydrated alfalfa meal, berseem clover, oat forage and perennial ryegrass. Enzyme extracts from cultivated rumen fluid showed an average protein breakdown of 75.5%, in eight feed samples tested. This value was very close to that measured with the technique of proteases from Streptomyces griseus (74.6% CP), but significantly lower (P≤0.05) than the one obtained by the in situ methodology (84.8% CP). The technique with extracted rumen enzymes showed higher level of proteolysis in the early hours of incubation (6 H) compared to the other techniques. These results suggest that the enzyme preparations of ruminal origin have the ability to predict degradability of feed proteins in the rumen, particularly in the first phase when most of proteins are hydrolyzed and become available for microbial utilization.

RUMINAL DEGRADATION OF AERIAL BIOMASS AND SEEDS OF WILD SPECIES OF Lupinus

“…or particle losses (Velásquez and Pichard, 2010). In contrast, the treatment applied for extracting oil from soybeans protects the paste from rapid degradation (Solanas et al, 2005).…”

Empirical Attributes and Limitations of Methodologies for Predicting the Degradability of Ruminal Protein

“…Despite the strengths of this technique, some aspects of the procedure have been criticized, mainly its methodological rigidity and the lack of representation of the biological phenomena that occur in the rumen (Stern et al, 1997;Mustafa et al, 2001). Moreover, some authors have reported large differences in the sizes of the different nitrogenous fractions between samples of a single type of feed, suggesting that local conditions and the method itself are major sources of variation in the characterization of these chemical fractions of proteins (Mullahey et al, 1992;Jones et al, 1995;Reed, 1995;Velásquez and Pichard, 2010b). Additionally, this technique does not allow for the hydrolysis rate (kd) to be calculated as it does not yield different degradation levels for different kinetic periods.…”

“…Another technique used for studies of proteolytic degradation in the rumen is based on commercial enzymes, basically the fungus Streptomyces griseus (Pichard and Van Soest, 1977;Mahadevan et al, 1987;Assoumani et al, 1992;Velásquez and Pichard, 2010b). Previous authors achieved high levels of protein degradation for various feed substrates using fungal proteases.…”

“…Then, there is the possibility of using the isotopes of some chemical elements as microbial markers, such as N 15 , S 35 or P 32 (Robinson et al, 1996;D'Mello, 2000), which can be used for evaluations both in situ and in vitro. One of the reported limitations of this method is that enrichment with these isotopes is not homogeneous for among different groups of bacteria and protozoa, leading to large skews in the final measurements of microbial N. A simpler technique for correcting microbial contamination of in situ experiments is that used by Velásquez and Pichard (2010b), which consists of incubating bags similar to those used in the in situ assay and containing 500 mg of sterile cotton wool. It is hypothesized that the microorganisms will adhere to the cellulose fibers of the cotton wool (free of N) simulating the colonization of feed particles.…”

“…It has been criticized, however, for the presence of various disturbing effects, the most important of which are microbial and N-compound contamination in the residue bags, the escape of particles from the bags into the ruminal medium, and the rarefication of the environment inside the bags (Mathis et al, 2001;Cone et al, 2004). One possibility for correcting particle loss from the bags into the ruminal medium is the method developed by Velásquez and Pichard (2010b) and modified from Weisbjerg et al (1996), which states that the corrected b fraction (bc) = b + P (b (1-(P + SN)) -1 ), where b is the nitrogenous fraction enzymatically degraded in situ (corrected, in turn, for microbial contamination); P is the particle loss from the bag into the ruminal medium; and SN is the soluble nitrogen. The particle loss is determined by the difference between the total material escaping from the bag after washing and the fraction of soluble N at time zero, so the total in situ protein degradation is equal to the sum of pool A + pool B 1 + pool bc.…”