Effects of phyto-phenolic compounds on ammonia production by select amino acid fermenting bacteria

Abstract

Bacteria that ferment amino acids to ammonia can be categorized as generalists or specialist hyper-ammonia-producing bacteria. In the rumens of ruminant animals, most of the ammonia produced is eventually excreted as urea in urine. This process can be controlled with off-label use of antibiotics, but the practice can lead to antibiotic resistance; therefore, discovery of antibiotic alternatives is pertinent. Plant-derived phenolic compounds have demonstrated antimicrobial efficacy for such purposes. This study investigated the antimicrobial and metabolic suppressive potential of six phenolic compounds on five amino acid fermenting bacteria: Clostridium sporogenes MD1, C. aminophilum F, Acetoanaerobium sticklandii SR, Peptostreptococcus sp. BG1, and Prevotella bryantii B14. Inhibitory action of the compounds was determined using a 10% v/v serial dilution method in basal media. Carvacrol (1 mM), thymol (1 mM), and eugenol (10 mM) demonstrated the greatest antimicrobial potential, where carvacrol and eugenol inhibited growth of all five species and thymol four species except BG1. The cinnamic acids (trans and hydro) demonstrated variable activity against all organisms. Suppression of metabolic activity was determined via colorimetric assay quantifying ammonia in washed stationary phase culture supernatant after 24 h of metabolism on fresh substrate. Carvacrol and eugenol yielded the greatest reduction of ammonia by all organisms except B14, which produced no ammonia under the growth conditions. Thymol greatly reduced ammonia production of four organisms except F. These data demonstrate that eugenol, carvacrol, and thymol may be worthy antimicrobial candidates for the control of ammonia-producing organisms.

Keywords: amino acid fermentation; generalists; hyper-ammonia producing bacteria; phenolic compounds; phytochemicals.

Figure 1.

Molecular structures of the six phyto-phenolic phytochemicals investigated for antimicrobial and ammonia suppressive activity in this study.

Figure 2.

Effect of phenolic phytochemical exposure on ammonia production (mM) by cell suspensions of amino acid fermenting bacteria after 24 h. (a) Ammonia produced by A. sticklandii SR after 24 h in the presence or absence (control) of carvacrol (1 mM; Carv), eugenol (10 mM; Eug), hydro- or trans-cinnamic acid (10 mM; H-Cin, and T-Cin, respectively), and thymol (1 mM; Thym). (b) Ammonia production of Peptostreptococcus sp. BG1 after 24 h of incubation without phytochemical (control) or with 1 mM carvacrol, 10 mM eugenol, 10 mM hydro- or trans-cinnamic acid, or 10 mM thymol. (c). Ammonia produced by C. sporogenes MD1 unamended (control) or amended with 1 mM carvacrol, 10 mM eugenol, 10 mM trans-cinnamic acid, or 1 mM thymol after 24 h. (d). Ammonia produced after 24 h by cell suspensions of C. aminophilum F unamended (control) or amended with 1 mM carvacrol, 10 mM eugenol, or 1 mM thymol. (e). Ammonia concentrations detected in cell suspensions of P. bryantii B₁4 after 24 h of incubation without (control) or with 1 mM carvacrol, 10 mM eugenol, or 1 mM thymol. Significant differences (P < .05) are represented as those with different alphanumeric designations within each panel, e.g. “a” and “b” are statistically different. Data are represented as the average of three replicates with error bars ± the standard deviation.