The objectives of this research were to validate the sensitivity and precision of an in vitro assay for evaluating the efficacy of antimicrobials, to evaluate the ability of natural animal proteins/peptides to kill in vitro antibiotic-resistant, as well as, -susceptible bacteria, and to determine the effects of key components of animal digesta (e.g., pH, mineral content, and proteolytic digestive enzymes) on the estimated antimicrobial activity of these proteins/peptides.

The minimum inhibitory concentrations (MIC) for polymyxin B (control antibiotic) were determined to be .76, .76, and .90 µg/mL for Escherichia coli, Escherichia coli (nalidixic acid-resistant), and Staphylococcus aureus, respectively. The intra- and inter-assay variation for MIC determination was .18 and .2%, respectively.

The natural animal proteins and peptides (lactoferrin, lactoferricin B, hen egg lysozyme, and alpha-lactalbumin LDT2) were determined in in vitro (acetic acid medium) to kill selected bacteria. Each of the tested proteins/peptides was active against an antibiotic-resistant (nalidixic acid) strain of E. coli; however, the required concentrations for antimicrobial activity were 10 to 15 times higher than that of the nonantibiotic-resistant strain. The antimicrobial activity of each protein/peptide in animal digesta fluid was 130 to 300% greater than that in the acetic acid media. Overall, the intra- and inter-assay variation values for the tested proteins/peptides was 3 and 3.4%, respectively.

The antimicrobial activity of two of the three proteins/peptides was not affected by the presence of cationic minerals. The change in pH (digesta fluid and acetic acid media) from 7 to 2 resulted in a loss of antimicrobial activity of 33% for all proteins/peptides. Therefore, the increase in antimicrobial activity associated with the digesta fluid is not related to change in H or the mineral concentration of the digesta. Based on these data, natural proteins/peptides represent potential antibiotic substitutes.