Inherent limitations to the problem of reducing the lysine microbiological assay time

A kinetic approach is proposed to shorten the microbiological assay time for the determination of unbound L-lysine. The present lysine bacterial assay takes from 16 to 24 h using Pediococcus cerevisiae P-60 ATCC 8042 (formerly Leuconostoc mesenteroides P-60 ATCC 8042) and uses a medium in which lysine is the limiting substance. Measurements of the final cell concentration are linearly correlated with the initial concentration of lysine, S, to provide an indirect estimate of S. We propose to understand the limitations inherent to the reduction of the assay time to 4 h by focusing in our analysis on the bacterial late lag or early growth transient phases, rather than the stationary phase of growth. Generally, the Monod equation is expected to describe a hyperbolically increasing correlation between the bacterial specific growth rate at about 2-4 h and the initial lysine concentration. A hyperbolic correlation is obtained by 3 h, but the lysine region of interest falls in the saturated portion of the curve. Discriminations between different initial lysine levels are therefore difficult with this nearly flat curve. On the other hand, when the initial inoculum level is lowered, so that substrate inhibition becomes effective, a correlation with a large negative slope is obtained by 4 h. Limitations to using absorbance measurements for the rapid assay turn up in a lack of reproducibility and, hence, a large variance associated with the measurements. Alternative microbial measuring techniques, such as impedance methods, need to be examined in order to reduce that large variance.