Ellen M. Hoffmann

A modified dot-blot method of protein determination applied in the tannin-protein precipitation assay to facilitate the evaluation of tannin activity in animal feeds.

Tannins have received considerable attention from animal nutritionists as potential agents for modifying ruminal fermentation patterns, or for exploring new feed resources. This group of secondary plant compounds is defined by their ability to form complexes with proteins. A widely accepted method for assaying the biological activity of extracted tannins is the precipitation of bovine serum albumin. The protein carries a radioactive label (125I) to allow direct quantification from the precipitate. Tannin-protein complexes dissolve in sodium dodecylsulfate solution. A dot-blot assay for protein determination, which is based on the reversible binding of a fluorochrome, benzoxanthene yellow, to the protein spots and is not disturbed by the presence of detergents, can replace the radioactive method by a fluorimetric measurement. A novel alternative to the last part of the dot-blot assay is to scan the stained protein spots in situ using a video camera and computer image analysis. Several filter sets were tested and, within a concentration range of 0.1-2.0 mg protein/ml, each of them yielded results identical to the original method while the time required was only 30 % of the working time consumed by the original procedure. The modified dot-blot assay should be applicable to the evaluation of tannin activity in all shrub and tree foliages considered as animal feed.

Effects of Moringa oleifera seed extract on rumen fermentation in vitro.

Moringa oleifera is a pantropical tree of the family Moringaceae. A previously undescribed property of an aqueous extract from the seeds of this plant is the modulation of ruminal fermentation patterns, especially protein degradation, as demonstrated in a short-term batch incubation system. Gas, short chain fatty acids (SCFA) and cellulolytic enzyme activities were determined as general fermentation parameters. A dot blot assay able to directly detect true protein in rumen fluid samples was used to quantify protein degradation. For complex substrates the interpretation of protein degradation profiles was amended by polyacrylamide gel electrophoresis (PAGE) of the samples. When incubated with pure carbohydrates at a concentration of 1 mg ml–1, the extract reduced microbial degradation of the model protein, bovine serum albumin (BSA), such that its concentration was at least 40% above the control after 12 h of incubation. Total protein degradation was thus delayed by approximately 9 h. When fermented along with wheat straw, leaf protein (Rubisco) was almost entirely protected during 12 h of fermentation. The degradation of soy proteins was retarded by at least 4- 6 h, depending on the protein band. There were strong side effects on the fermentation of pure cellulose (SCFA yield -60% after 12 h), whereas cellobiose and starch fermentation were less affected (-18 and -8%, respectively). When the complex substrates were fermented, SCFA yield was reduced by approximately 30% after 12 h. In our work we clearly demonstrate the efficacy of the new substance, which is neither a tannin nor a saponin, in an in vitro system, using pure as well as complex substrates. The properties shown in vitro for the crude extract suggest that it could have a positive effect on the protein metabolism of ruminants under intensive management and that negative side effects can be overcome by an optimized dosage. If the chemical nature of the active substance and its mechanism of action can be clarified, it may provide an alternative to replace critical synthetic feed additives (such as antibiotics) for high yielding dairy cows.

The fermentation of soybean meal by rumen microbes in vitro reveals different kinetic features for the inactivation and the degradation of trypsin inhibitor protein

Soybean meal is a widely used protein supplement in animal feeds. When fed to monogastrics, thermal pretreatment (roasting) is needed to destroy antinutritional components. The major antinutritive compound of soybeans is trypsin inhibitor, a protein of 21 kDa molecular mass. Ruminants can tolerate this compound due to the microbial fermentation in the rumen rendering the inhibitor ineffective. This process was monitored under controlled conditions in an in vitro fermentation system. Raw soybean meal was added at different levels to a basal roughage and incubated with bovine rumen fluid. Samples were taken at regular time intervals up to 24 h of incubation. The samples were assayed enzymatically for trypsin inhibitor activity, and parallel to this the proteolytic degradation of trypsin inhibitor was documented qualitatively and quantitatively through polyacrylamide gel electrophoresis and densitometric methods. It was shown that trypsin inhibitor is both inactivated and degraded in the rumen, but with different kinetic characteristics. Its concentration in the soluble fraction showed a more pronounced initial increase than the apparent activity, and the time of complete degradation lagged 1–2 h behind the complete loss of activity. Protein concentration and activity were related to each other by calculating specific trypsin inhibitor activity. As this parameter showed a rapid, presumably exponential decay with the onset of fermentation, inactivation must be due to a mechanism preceding and not directly related to proteolysis.

Screening for Anti-proteolytic Compounds

Dietary protein entering the rumen is broken down in an apparently uncontrolled way, resulting in ammonia formation and subsequent loss of N in the urine. The low efficiency of nitrogen retention represents a major economic loss, causes metabolic stress in the animal, and places a burden on the environment, by way of nitrogen-rich wastes. If a means of slowing the breakdown process at any of the individual steps can be identified, these problems would be decreased.