Influence of the Chelation Process on the Stability of Organic Trace Mineral Supplements Used in Animal Nutrition.

The effect of the chelation process on the pH-dependent stability of organic trace minerals (OTMs) used as mineral supplements in animal nutrition was assessed using analytical techniques such as potentiometry, Fourier Transform Infrared Spectroscopy (FTIRS) and amino acid profiling. The aim was to understand the influence and relative importance of the manufacturing conditions on mineral chelation and the subsequent pH stability of OTMs. A selection of OTMs were assessed over a wide pH range to account for the typical environmental changes encountered in the gastrointestinal (GI) tract. In the case of proteinate type products, the potentiometric assessment of free mineral concentration indicated that the hydrolysis procedure used to generate the chelating peptides was the major influencer of the pH stability of the products. Many products are available under the umbrella term "OTMs", including amino acid complexes, amino acid chelates, polysaccharide complexes and proteinates. Significant differences in the pH-dependent stability of a range of commercially available OTMs were observed.

Tracking Yeast Metabolism and the Crabtree Effect in Real Time via CO2 Production using Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS).

In this study, a new approach to measure metabolic activity of yeast via the Crabtree effect is described. BARDS is an analytical technique developed to aid powder and tablet characterisation by monitoring changes in the compressibility of a solvent during solute dissolution. It is a rapid and simple method which utilises a magnetic stir bar to mix added solute and induce the acoustic resonance of a vessel containing a fixed volume of solvent. In this study it is shown that initiation of fermentation in a yeast suspension, in aqueous buffer, is accompanied by reproducible changes in the frequency of induced acoustic resonance. These changes signify increased compressibility of the suspension due to CO2 release by the yeast. A simple standardised BARDS protocol reveals yeast carbon source preferences and can generate quantitative kinetic data on carbon source metabolism which are characteristic of each yeast strain. The Crawford-Woods equation can be used to quantify total gaseous CO2 produced by a given number of viable yeast when supplied with a fixed amount of carbon source. This allows for a value to be calculated for the amount of gaseous CO2 produced by each yeast cell. The approach has the potential to transform the way in which yeast metabolism is tracked and potentially provide an orthogonal or surrogate method to determining viability, vitality and attenuation measurements in the future.

Quantitative assessment of copper proteinates used as animal feed additives using ATR-FTIR spectroscopy and powder X-ray diffraction (PXRD) analysis.

The aim of the present work was to establish a reliable analytical method to determine the degree of complexation in commercial metal proteinates used as feed additives in the solid state. Two complementary techniques were developed. Firstly, a quantitative attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopic method investigated modifications in vibrational absorption bands of the ligand on complex formation. Secondly, a powder X-ray diffraction (PXRD) method to quantify the amount of crystalline material in the proteinate product was developed. These methods were developed in tandem and cross-validated with each other. Multivariate analysis (MVA) was used to develop validated calibration and prediction models. The FTIR and PXRD calibrations showed excellent linearity (R2 > 0.99). The diagnostic model parameters showed that the FTIR and PXRD methods were robust with a root mean square error of calibration RMSEC ≤3.39% and a root mean square error of prediction RMSEP ≤7.17% respectively. Comparative statistics show excellent agreement between the MVA packages assessed and between the FTIR and PXRD methods. The methods can be used to determine the degree of complexation in complexes of both protein hydrolysates and pure amino acids.

Biochemical Comparison of Commercial Selenium Yeast Preparations.

The trace mineral selenium (Se) is an essential element for human and animal nutrition. The addition of Se to the diet through dietary supplements or fortified food/feed is increasingly common owing to the often sub-optimal content of standard diets of many countries. Se supplements commercially available include the inorganic mineral salts such as sodium selenite or selenate, and organic forms such as Se-enriched yeast. Today, Se yeast is produced by several manufacturers and has become the most widely used source of Se for human supplementation and is also widely employed in animal nutrition where approval in all species has been granted by regulatory bodies such as the European Food Safety Authority (EFSA). Characterisation and comparison of Se-enriched yeast products has traditionally been made by quantifying total selenomethionine (SeMet) content. A disadvantage of this approach, however, is that it does not consider the effects of Se deposition on subsequent digestive availability. In this study, an assessment was made of the water-soluble extracts of commercially available Se-enriched yeast samples for free, peptide-bound and total water-soluble SeMet. Using LC-MS/MS, a total of 62 Se-containing proteins were identified across four Se yeast products, displaying quantitative/qualitative changes in abundance relative to the certified reference material, SELM-1 (P value <0.05; fold change ≥2). Overall, the study indicates that significant differences exist between Se yeast products in terms of SeMet content, Se-containing protein abundance and associated metabolic pathways.

Validation of method for total selenium determination in yeast by flame atomic absorption spectrometry.

A procedure using open digestion followed by flame atomic absorption spectrometry is described for measuring the total selenium content of Se-enriched yeast. The limits of detection and quantitation were 2.5 mg/L and 5 mg/L Se, respectively. The signal response was linear over the range of 5-50 mg/L Se, and the average recovery from spiked samples was 98.9%. The validated method was used to measure the Se content of Se-enriched yeast reference material and produced a result of 2145 +/- 38 mg/kg (n = 3), which is in good agreement with the certified level of 2125 +/- 65 mg/kg.

Accelerated determination of selenomethionine in selenized yeast: validation of analytical method.

The purpose of this study was to reduce the extraction time, to hours instead of days, for quantification of the selenomethionine (SeMet) content of selenized yeast. An accelerated method using microwave-assisted enzymatic extraction and ultrasonication was optimized and applied to certified reference material (selenized yeast reference material (SELM)-1). Quantitation of SeMet in the extracts was performed by liquid chromatography with inductively coupled plasma mass spectrometry. The limits of detection and quantitation were 5 ppb SeMet and 15 ppb SeMet respectively and the signal response was linear up to 1,500 ppb SeMet. The average recovery of spiked SeMet from the selenized yeast matrix was 97.7 %. Analysis of an SELM-1 using this method resulted in 100.9 % recovery of the certified value (3448 ± 146 ppm SeMet). This method is suitable for fast reliable determination of SeMet in selenized yeast.

Characterization of metal-peptide complexes in feed supplements of essential trace elements.

Metal chelates with biomolecules are increasingly used in animal supplementation to increase the bioavailability of essential trace elements. However, the transfer of the chelates is not well understood and speciation studies may bring a comprehensive insight to further investigate the biological uptake mechanism(s) implicated. An analytical method was developed for the characterization of the water-soluble metal complexes in animal feed supplements obtained by reaction of a metal salt with a non-GMO soybean enzymatic digest. The method was based on fractionation of the extract by size-exclusion chromatography followed by the analysis of the metal-containing fraction by reversed-phase nanoHPLC with parallel ICP MS and electrospray MS/MS detection. The metal complexes were identified in the mass spectra owing to the Cu characteristic isotopic pattern; the complexation was corroborated by the presence of a peak corresponding to the non-metallated peptide. The study demonstrated the feasibility of SEC-ICP MS to produce characteristic metal (Cu, Zn, Mn, Fe) distribution patterns, which can be of interest to test batch-to-batch reproducibility and to determine the origin of the supplement. The use of the method could be extended to animal feeds prepared using the metal-chelated complexes. Electrospray MS/MS allowed the identification of a number of Cu complexes with peptides. Four different structure conformations were modeled by means of molecular mechanics investigations to assess the chelation stability.