Growth responses of lysine auxotrophic mutants of Escherichia coli have been used as a measurement of bioavailable lysine in protein sources and animal feeds. Sterilizing feed samples by autoclaving to eliminate non‐specific background growth of indigenous feed micro‐organisms prior to conducting the bacterial assay may introduce chemical and physical alterations to the feeds, influencing the estimation of available feed lysine. In this study, an antibiotic‐ and antifungal‐supplemented medium was constructed to support growth of an E. coli lysine auxotroph assay organism, and was tested for its ability to repress indigenous bacterial and fungal growth in feed samples. To determine which antibiotics to include, an ampicillin‐sensitive E. coli lysine mutant strain (ATCC no. 23812) was screened for antibiotic resistance and transformed with a plasmid carrying an ampicillin resistance gene. Maximum optical density quantitative response of the E. coli auxotroph to lysine was not altered by the antibiotic medium amendments (ampicillin, novobiocin and cycloheximide). Indigenous microfloral growth in a variety of typical animal feeds was suppressed in the presence of the antistatic agents. The estimated lysine recovery was 91·6% and 98·1% when the medium was used in an assay of available lysine in a lysine‐supplemented feed. This indicates that the antibiotic‐amended basal medium can be used for the E. coli‐determined lysine availability of a variety of animal feeds without prior sterilization of the feed sources.

As an essential amino acid, lysine is an important component of animal and human diets and its bioavailability can depend on a variety of factors. Therefore, an accurate pre-determination of bioavailable lysine in foods and feeds is important. In this study a whole cell fluorescent biosensor for the quantification of lysine in protein sources was constructed. A gene encoding for green fluorescent protein (GFPmut3) was introduced into an E. coli lysine auxotroph genome as a part of a mini-Tn5-Km transposon. The location of the transposon was determined and the growth kinetics of the newly constructed biosensor were examined. The transposon disrupted the ybhM gene, which encodes for the synthesis of a protein with an unknown function. No effect of the transposon's location in the genome or the expression of gfp on bacterial growth rates was observed. Based on the fluorescence emitted by GFPmut3, a standard curve after 6-h growth of the strain was generated. A correlation coefficient of 0.95 was observed when the fluorescence method was compared to the conventional optical density (OD) growth-based lysine assay. Using the newly developed lysine fluorescent whole cell sensor we determined the total lysine in casein acid hydrolyzate (7.13 ± 0.34%). When lysine added to 12 lg/ml and 30 lg/ml of casein acid hydrolyzate was quantified, recoveries of 97 ± 1.65% and 103 ± 4.66% respectively were detected. The results suggest that the microbial assay using GFP fluorescence represents a promising alternative method for the potential estimation of lysine in protein sources.

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