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Chapter 11. Proposing Studies with Manipulating Affinity of Peptides/Amino Acids for Microbes in Protein Cell Synthesis in Rumen Digestion.
The Problem and the Potential.
The author has had decades involvement in this problem and especially with silage as a theoretical possibility although until recently it has evaded notice until so-called timed release capsules for various bioactive peptides such as vasoactive ones for regulating blood pressure was developed by Japanese scientists and now it has come to the author's attention from a Swiss commercial concern, Osmopharm SA, that develops resin polymers with ion-exchange properties that can have an affinity for charged amino acids/peptides/proteins as in the usual ion-exchange chromatograph procedures of separating out amino acids, peptides and proteins.
Resin-based Slow-Release Peptides/Amino Acids for Ruminal Applications in Livestock.
The ion-exchange resins will be designed from biopolymers with attached amino acids (acidic to basic group attached) with pectin pelleting as the backbone which is digestible at a given rate giving it a characteristic binding constant and therefore a release rate. The ion-exchange matrix in the micropellet particles on which microbial cells can attach to uptake amino acid/peptides is via microbial chemotaxis. Concentrations/dosing levels in the volume compartment of the stomach will be calculated and measured rate of uptake of both amino acid/peptide mixture ratio calculated after recovery to measure isotope labels on amino acids from of peptides/amino acids. The nature of the chemotaxis will be observed by direct confocal and electron microscopy examination with time after sampling particulates from the harvested biopolymeric pellet fraction. Osmopharm SA is a company in southern Switzerland or nr. northern Italy that believes it has technology at hand by the protein-utilizing microbial rumen to actually test and document the use of time-release capsules using their unique gel matrix (the exact biopolymer involved is not known to the author) with ion-exchange movement of H2O in the process of defragmentation.
The Proposed Experimental Design.
In vivo studies utilizing rumen cannulated animals for rumen samplings can now be carried out using materials as pectins designed with ion-exchange resin groups concentrated varied accordingly and of a pKa according to the neutral point of the peptide's amino acid profile loaded into the rumen as substrate in pectin using peptide dosed with 15N stable isotope label to populate rumen contents with mixing; time of mixing established and then random sampling carried out to 15% of pooled samples, 'killed' or stopped at time of sampling, and the total pool further subsampled; the microbial cell protein (MCP) fraction is separated and measured for 15N content via 15N-NMR using differential centrifugation for microbial cells and then hydrolyzed to separate by prep liquid-liquid HPLC chromatography before recovery quantitatively of the amino acid 15N load in ug/g MCP. Comparative study is made between peptide amino acid in the pectin gel matrix and 15N content in microbial cell protein to measure a so-called "Km" of the gel matrix binding capacity generating an "S" curve to saturation of 15N concentration in MCP vs. peptide amino acid concentration in the gel matrix with the "Km" determined by drawing a line at 0.5 of the "Y" axis, the range to saturation across to the "S" curve and down to the "X" axis.
To determine the parameters to calculate experimentally a "Km," "in range," an in vitro study will be carried out first with an artificial rumen machine or Rusitec(R): a) concentration of 15N-peptide used in the gel, b) time of samplings given the mixing time in the volume of the Rusitec(R), c) the peptide compositional profile in amino acids to be synthesized and provided in bulk by the manufacturer, d) the manufacture of the gelled pellets by the pelleter machine of a given design, e) the current cost estimate of commonly available 15N amino acids labelled for making the peptides in the pellets, and f) the load of gel pellets into the Rusitec(R) machine. Analytics will be performed on the processed and treated subsamples from Rusitec(R) as per well-established technique in the laboratory as was referred to.
No doubt, this is the first time new technology with ion-echange type resins used for slow or time released drug agents in pharma now in use has been borrowed for use to studying microbial cell studies and MCP synthesis as one of 2 factors that was originally proposed to limit the efficiency of MCP synthes in rumen digestion in gMCP/kgOMD in the rumen: peptides/amino acids and water-soluble carbohydrates (WSC) especially where silage is involved in ruminant diets. Apart from critically measuring the rate of transport of peptides during uptake into cells other factors will also play a part in determining efficiencies. These will be discussed further elsewhere (see: Chapter 12).
Last update of this entry: July 27, 2020