Bacillus subtilis spores in probiotic feed quantified via bacterial metabolite using planar chromatography

Interest in probiotics in animal production has increased due to the European ban on antibiotic growth promoters in 2006. Bacillus subtilis DSM 29784 (B. s. 29784) is one such probiotic feed additive used in poultry. Cell counting has been the most common tool for feed analysis, besides flow cytometry and quantitative polymerase chain reaction. However, quantification of the active probiotic in feed is challenging, since results are influenced by cultivation conditions, viable but non-culturable bacteria and the high contents of feed ingredients. This study presents the first quantitative analysis of a metabolite generated by B. s. 29784 spores in feed to draw conclusions on the amount of active dried spores in the feed. Thus, it is the first quantification of active probiotic bacteria at the trace level in feed based on metabolite production but not cell counting. To generate the calibration standards, solutions with different amounts of dried B. s. 29784 spores were cultured under the same conditions as the feed sample, ensuring independence from growth performance. Upstream cultivation, metabolite extraction and high-performance thin-layer chromatography analysis were proven to be highly reliable and reproducible. The repeatability of the method (RSD 1.9%) and the recovery (111% ± 21% in feed additive matrix, 96% ± 13% in ionized feed matrix) were excellent. The variations during cultivation occurred due to the complex spore germination process and presence of other microbes in the feed. This new procedure, detecting only those cells that produced the metabolite of interest, has several advantages as it takes into account bacterial viability, cultivation conditions, spore germination process, growth behavior and the influence of the nutrient-rich feed matrix. It truly reflects the activity of the probiotic in the feed product, allows side-by-side comparison of characteristic metabolite patterns and nutrient consumptions to understand the metabolism of dried spores in matrix.